Tumor specific glycoprotein antibody G11 and application thereof

Abstract

The invention discloses a tumor specific glycoprotein antibody G11 and application thereof. The amino acid sequence of the CDR1 of the tumor specific glycoprotein antibody G11 comprises a sequence as shown in SEQ ID NO. 1; the amino acid sequence of the CDR2 of the tumor specific glycoprotein antibody G11 comprises a sequence as shown in SEQ ID NO. 2; the amino acid sequence of the CDR3 of the tumor specific glycoprotein antibody G11 comprises a sequence as shown in SEQ ID NO. 3. The tumor specific glycoprotein antibody G11 has affinity to GPC3 and is smaller in molecular weight, and engineered immune cells developed on the basis of the tumor specific glycoprotein antibody G11 are higher in exogenous gene expression level, better in effectiveness, better in drug effect and lower in modification difficulty.

Description

Technical Field

[0001] The invention belongs to the field of antibody biotechnology and relates to a tumor-specific glycoprotein antibody G11 and an application thereof. Background Art

[0002] GPC3 (Glypican-3) is a carcinoembryonic antigen that is involved in cell proliferation, differentiation, migration and apoptosis. It is a biomarker for a variety of malignant tumors, especially expressed in up to 70-80% of hepatocellular carcinoma, but almost not expressed in normal tissues. GPC3 is considered to be a "golden" target that can accurately attack liver cancer cells due to its tumor specificity. In addition, GPC3 is moderately expressed in some melanoma, ovarian cancer, and lung cancer cells. Therefore, GPC3 can also be used as a target for the above indications.

[0003] The existing technologies mostly adopt the methods of developing therapeutic monoclonal antibodies, bispecific antibodies, chimeric antigen receptor T cells, etc. GPC3 protein is easily cut into N-terminal soluble protein and C-terminal membrane protein, and then released from cancer cells into the microenvironment. Therefore, in the process of antibody screening, it is necessary to obtain specific antibodies that bind to the 30KDa C-terminal membrane protein. Steric hindrance and other reasons will also affect the efficiency of ScFv antibody binding to GPC3. In addition, the existing technologies mostly use mouse / humanized antibodies, which are easily limited by molecular weight when used, which is not conducive to achieving high permeability.

[0004] Therefore, there is an urgent need to provide a GPC3 antibody with small molecular weight, high affinity and high specificity. 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 G11 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 G11, wherein the amino acid sequence of CDR1 of the tumor-specific glycoprotein antibody G11 includes the sequence shown in SEQ ID NO.1; the amino acid sequence of CDR2 of the tumor-specific glycoprotein antibody G11 includes the sequence shown in SEQ ID NO.2; and the amino acid sequence of CDR3 of the tumor-specific glycoprotein antibody G11 includes the sequence shown in SEQ ID NO.3.

[0008] SEQ ID NO.1: GPTFSSYA.

[0009] SEQ ID NO. 2: QFVAAISRGATYT.

[0010] SEQ ID NO. 3: AAGPDTVAQRTGRGEYDY.

[0011] The tumor-specific glycoprotein antibody G11 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 G11 includes the sequence shown in SEQ ID NO.4.

[0015] SEQ ID NO.4:

[0016] DVQLVESGGGLVQAGGSLRLSCAASGPTFSSYAVGWFRQAPGKERQFVAAISRGATYT YYADSVKGRFTISSRDNAKNTSYLQMNSLKLEDTAVYYCAAGPDTVAQRTGRGEYDYWGQ GTQVTVSS.

[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 G11 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 is the sequence shown in SEQ ID NO.6.

[0021] SEQ ID NO.5:

[0022] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0023] SEQ ID NO.6:

[0024] DVQLVESGGGLVQAGGSLRLSCAASGPTFSSYAVGWFRQAPGKERQFVAAISRGATYTYYADSVKGRFTISRDNAKNTSYLQMNSLKLEDTAVYYCAAGPDTVAQRTGRGEYDYWGQGTQVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[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 G11 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 G11 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] K.

[0040] SEQ ID NO.9: CDIYIWAPLAGTCGVLLLSLVITLYCNHRNR.

[0041] SEQ ID NO.10:

[0042] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0043] SEQ ID NO.11:

[0044] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[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] MALPVTALLLPLALLLHAARPSDVQLVESGGGLVQAGGSLRLSCAASGPTFSSYAVGWFRQAPGKERQFVAAISRGATYTYYADSVKGRFTISRDNAKNTSYLQMNSLKLEDT AVYYCAAGPDTVAQRTGRGEYDYWGQGTQVTVSSESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKCDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0049] In a fifth aspect, the present invention provides a nucleic acid molecule encoding the tumor-specific glycoprotein antibody G11 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 or an adeno-associated viral vector containing the gene encoding the chimeric antigen receptor described in the fourth aspect.

[0052] In a seventh 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.

[0053] Preferably, the chimeric antigen receptor immune cell comprises the expression vector described in the sixth aspect.

[0054] 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.

[0055] In an eighth aspect, the present invention provides the use of the tumor-specific glycoprotein antibody G11 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 seventh aspect in the preparation of a product that can prevent and / or treat cancer or tumors.

[0056] Preferably, the tumor comprises a GPC3-positive solid tumor; and the cancer comprises any one of liver cancer, ovarian cancer, lung cancer or melanoma.

[0057] In a ninth aspect, the present invention provides the use of the tumor-specific glycoprotein antibody G11 described in the first aspect or the chimeric antigen receptor described in the fourth aspect in the preparation of a preparation for killing NK cells, macrophages or neutrophils.

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

[0059] (1) The tumor-specific glycoprotein antibody G11 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 G11 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 G11 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 G11 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 BVHGC3-015 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 BVHGC3-015 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;

[0071] Figure 5B IncuCyte real-time quantitative live cell imaging analysis of the killing function and specificity of CAR-T cells targeting GPC3;

[0072] Figure 5C IncuCyte real-time quantitative live cell imaging analysis of the killing function and specificity of CAR-T cells targeting GPC3;

[0073] Figure 6 The figure shows the result of 74-G3-1-G11 antibody-mediated NK cell ADCC killing of tumor cells.

[0074] Reagents used in the following examples:

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

[0076] SK-Hep-1 cells: ATCC, HTB-52;

[0077] Huh7 cells: Cell Bank of Chinese Academy of Sciences, SCSP-526;

[0078] FITC-labeled Rabbit anti-Llama IgG (H+L) antibody: Invitrogen, A16155;

[0079] Anti-M13-HRP antibody: SinoBiological, 11973-MM05T-H;

[0080] PE-labeled anti-human IgG antibody: Biolegend, 410708;

[0081] AF647-labeled Anti-human IgG antibody: Jackson ImmunoResearch, 109-606-170;

[0082] Anti-VHH antibody: GenScript, A02017;

[0083] AF647-labeled anti-human GPC3 antibody: R&D system, FAB2119A. DETAILED DESCRIPTION

[0084] 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.

[0085] 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.

[0086] Example 1

[0087] Alpaca immunization and serum titer testing.

[0088] (1) Construction of GPC3 overexpressing cells

[0089] 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.

[0090] (2) Alpaca immunization and serum titer testing

[0091] 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.

[0092] 5 mL of peripheral blood was collected before immunization, two weeks after the second immunization, the third and the fourth immunization, respectively. The centrifuge tubes containing the blood samples were placed in a centrifuge and centrifuged at 800 × g for 10 min to collect the upper serum. Use sterile PBS 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 solution, wash 5 times with PBST (containing 0.05% Tween 20); add 200 μL / well of blocking solution and block at 37°C for 2h; shake off the blocking buffer, wash the plate 5 times with PBST; use PBS to gradient 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; shake off the liquid in the well and wash 5 times with PBST; add 100 μL HRP anti-LlamaIgG ​​(H+L) antibody (1:50000 dilution), incubated at room temperature for 1h; after shaking off the liquid in the well, wash the well plate 5 times with PBST; add 100μL / well TMB colorimetric solution; incubate at room temperature in the dark for 10min; add 50μL / well stop solution; use a microplate reader to read the OD450 value in the well. The results are shown in Tables 1, 2 and 3. The binding titers of the sera of the three alpacas before immunization with the GPC3 protein were all lower than 1:2000 (the antibody titer is expressed in the form of 1:X, X is the maximum dilution multiple at which the antibody can be detected; the higher the dilution multiple, the higher the antibody titer), indicating that the content of anti-GPC3 specific antibodies contained therein is extremely low. On the contrary, the binding titers of the serum of 73# and 74# alpacas after the fourth immunization to GPC3 protein were both over 1:32K, and the binding titer of the serum of 194# alpacas after the fourth immunization to GPC3 protein was also higher than 1:8K, indicating that the content of anti-GPC3 specific antibodies in the sera of these three alpacas was significantly increased.

[0093] Table 1

[0094]

[0095] Table 2

[0096]

[0097] Table 3

[0098]

[0099] Example 2

[0100] Construction of single domain antibody phage display library.

[0101] (1) VHH antibody fragment cloning

[0102] After confirming that the serum of the two alpacas contained GPC3-specific antibodies, peripheral blood was collected, RNA was extracted, and cDNA was obtained by reverse transcription. 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 products were analyzed by electrophoresis using 1% agarose, and the target fragment with a molecular weight of about 750bp was recovered and separated ( Figure 1A ). Then, using the first round PCR product as a template, 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 region, and the 5' end contains the SfiI restriction site GGCCACGAAGGCC) were used to amplify the heavy chain antibody VHH fragment. The PCR product was analyzed by electrophoresis using 1% agarose, and the target fragment with a molecular weight of about 400 bp was recovered and separated ( Figure 1B ).

[0103] (2) Electroporation of library vectors and detection of library capacity and diversity

[0104] The phage surface display vector pComf and the VHH fragment library obtained above were digested with SfiI endonuclease. The linearized pComf vector and the VHH fragment library were connected at 16°C overnight with T4 ligase. The connection product was frozen. After the connection product was transformed into E. coli, the bacteria were resuspended and revived. In addition, the bacterial solution was diluted and spread on an LB plate containing ampicillin and cultured 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. The results showed that the library capacity of the single-domain antibody phage surface display library obtained from 73# alpaca PBMC was 1.60×10 9 The single-domain antibody phage display library obtained from 74# alpaca PBMC has a library capacity of 1.32×10 9. Pick 20 monoclones on the plate. The single domain antibody phage display library obtained from 194# alpaca PBMC has a library capacity of 1.15×10 9 . Twenty 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.

[0105] (3) Enrichment of phage display libraries

[0106] Take the above electroporation and resuscitation culture solution and culture it to OD 600 When the pH is 0.6, add M13KO7 helper phage, shake well and let stand at 37℃ for 30min, then culture on a shaker for 1h. After the helper phage completes the infection of the target strain, centrifuge and discard the supernatant, resuspend and culture overnight. Centrifuge the bacterial solution, let the supernatant containing phage particles stand at 4℃ for 2h, centrifuge to collect the phage precipitate, resuspend to remove insoluble impurities, and obtain the original library of single-domain antibody phage surface display.

[0107] Take 10 μL of the precipitate and perform 10-fold gradient dilutions. Add 200 μL 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 an LB plate. After overnight culture at 37°C, the plaques were counted to obtain the titer of the phage surface display library.

[0108] Example 3

[0109] Target antibody library screening.

[0110] (1) First round of panning and product amplification

[0111] Dilute GPC3 antigen and add it to a 96-well solid phase plate and coat it at 4°C overnight. 7 CHO-S-GPC3 cells were washed 3 times with PBS, resuspended in PBSA and blocked for 1h. At the same time, the precipitated product of the original library displayed on the surface of single-domain antibody phage was taken and blocked at 4℃ for 1h as a premix. The blocked CHO-S cells were centrifuged at 500×g to remove the supernatant, and incubated at 4℃ to remove the phage clones that non-specifically bound to CHO-S. The GPC3 protein in the 96-well plate was discarded, and the buffer was added and allowed to stand at room temperature. After blocking, the CHO-S-GPC3 cells were centrifuged, the supernatant was taken and added to the GPC3 protein wells respectively, and incubated at room temperature for 1h. The phage supernatant in the GPC3 protein wells was discarded, washed, and Gly-HCl elution solution was added to each well, incubated with shaking, and the specifically bound phages were eluted twice, and the eluted products were stored in pre-sealed centrifuge tubes at 4℃.

[0112] Take 2YT medium, add tetracycline and Escherichia coli ER2738 and culture in an incubator until OD600 The eluted phage product was added to the ER2738 bacterial solution until the OD 600 When the concentration is 0.5, add M13KO7 helper phage, shake well and let stand, centrifuge to discard the supernatant and collect the bacteria, resuspend, and culture overnight. Centrifuge the phage suspension that has been infected and amplified overnight, and divide the supernatant into centrifuge tubes. Add 10mL PEG / NaCl to each tube, mix well and place on ice, let stand for 1h to precipitate the phage, and centrifuge for 30min. Discard the supernatant and resuspend the phage with 1mL sterile PBS, centrifuge to remove insoluble impurities, this is the phage product obtained in the first round of panning amplification.

[0113] (2) Second to fourth rounds of panning and product amplification

[0114] 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.

[0115] (3) ELISA detection and sequencing of monoclonal phage

[0116] The single-domain antibody phage surface display library after multiple rounds of panning was used to infect ER2738 Escherichia coli, mixed and placed in a 37°C water bath for 10 minutes, then spread on an LB plate and incubated at 37°C overnight. 2YT-A medium was added to a 96-well deep-well plate at 200 μL per well, and a single clone on the plate was picked and incubated at 37°C and 225 rpm overnight. 2YT-A medium was added to a 96-well deep-well plate at 150 μL per well, and 20 μL of the overnight culture solution was added to each well, and incubated at 37°C and 225 rpm until OD 600 Add M13KO7 helper phage, mix well and let stand at 37℃ for 15min (M13KO7 volume = 10 × bacterial solution volume × OD 600 ×5×10 8 / M13KO7 titer), cultured at 37°C and 225 rpm for 45 min. Centrifuged at 3900 rpm for 10 min, discarded the supernatant, resuspended in 500 μL 2YT-AK medium per well, cultured at 30°C and 220 rpm overnight. Centrifuged at 3900 rpm for 10 min, the supernatant obtained was the monoclonal phage particles.

[0117] While amplifying the phage monoclonal, coat the GPC3 antigen protein with CBS at pH 9.6 to the ELISA plate (2 μg / mL, 100 μL / well). After overnight coating at 4°C, discard the antigen, wash three times with PBST, add 250 μL 3% MPBS to each well, and block overnight at 4°C. After the blocking solution is mixed, add 200 μL 0.05% PBST to each well, wash four times, add 50 μL 0.1% PBST, and then add 50 μL of the above monoclonal phage supernatant one by one. After incubation at 4°C for 1 hour, wash five times with 0.05% PBST. Dilute the anti M13-HRP antibody (1:5000) with 0.05% PBST, add 100 μL to each well, and incubate at 4°C for 45 minutes. After washing five times with 0.05% PBST, 100 μL TMB was added to develop color at room temperature for 10 min, and then 50 μL 0.2 M hydrochloric acid was added to terminate the color development. 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 74-G3-1-G11.

[0118] Example 4

[0119] Expression of recombinant single domain antibodies and detection of binding to target proteins.

[0120] The sequence of 74-G3-1-G11 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 74-G3-1-G11 antibody. In the same manner, the GPC3-specific antibody hYP7 was expressed as a positive control (YP7 VL amino acid sequence (SEQ ID NO. 13): DIVMSQSPSSLVVSIGEKVTMTCKSSQSLLYSSNQKNYLAW YQQKPGQSPKLLIYWASSRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYNYPL TFGAGTKLELK. YP7 VH amino acid sequence (SEQ ID NO. 14): EVQLVETGGGMVQPEGSLKLSCAASGFTFNKNAMNWVRQAPGKGLEWVARIRNKTNNYATYYADSVKARFTISRDDSQSML YLQMNNLKIEDTAMYYCVAGNSFAYWGQGTLVTVSA).

[0121] The binding specificity of 74-G3-1-G11 was tested. The culture supernatant containing 74-G3-1-G11 antibody was mixed with 3×10 5Huh7 (naturally expressing GPC3), SK-Hep-1 overexpressing GPC3, and 293T (GPC3-negative cell line) cells were incubated at room temperature for 1 hour. After centrifugation 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. After centrifugation at 800×g for 5 minutes at room temperature, the supernatant was discarded and the cells were washed 3 times with PBS. The cells were resuspended in 500μL PBS for flow cytometry analysis.

[0122] The results are as follows Figure 2 As shown in the figure, the expression supernatant of the negative control antibody group had no significant binding to Huh7 and 293T; the 74-G3-1-G11 recombinant single domain antibody had significant binding to the Huh7 cell line. At the same time, the binding level of the 74-G3-1-G11 recombinant single domain antibody to 293T was significantly lower than that of the negative control antibody, indicating that the 74-G3-1-G11 recombinant single domain antibody can bind to GPC3 more specifically.

[0123] Example 5

[0124] Purification and half effective concentration (EC 50 ) determination.

[0125] To determine the EC of 74-G3-1-G11 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, ProteinA magnetic beads can be used for affinity purification. Wash the ProteinA magnetic beads twice with 30mL PBS buffer, 0.1M sodium hydroxide, and PBS buffer in sequence. Add the corresponding volume of ProteinA magnetic beads (calculated as 20mg IgG / mL ProteinA magnetic beads) to the 293F cell shake flask according to the required amount of the sample. Incubate at room temperature for 4h at 120rpm in a shaking incubator. Collect the ProteinA magnetic beads with a magnetic separation rack and transfer them to a 50mL centrifuge tube. After washing twice with 30mL PBS buffer and deionized water, resuspend them with 1mL 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 ProteinA magnetic beads twice, combine the eluates and add neutralization buffer to adjust the pH of the solution. The eluted sample was dialyzed with PBS at least 100 times the volume of the sample, first dialyzed at 25°C for 2 hours, then dialyzed at 8°C for 16 hours after changing the solution once. Finally, the protein concentration was determined and the sample was filtered with a 0.22 μm sterile filter membrane and packaged, and stored in a -80°C refrigerator for use.

[0126] Serial dilutions of the target antibody were added to 3×10 5 CHO-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 for 5 minutes at room temperature, discard the supernatant containing antibodies, wash the cells 3 times with PBS, resuspend the cells with 500μL PBS, and perform flow cytometry analysis.

[0127] The results are as follows Figure 3 As shown in Table 1, Table 1 is the EC of recombinant single domain antibodies detected by flow cytometry 50 Results. EC of positive control antibody HYP7 against CHO-S-GPC3 50 was 12.93 μg / ml, while the EC of 74-G3-1-G11 for CHO-S-GPC3 50 It is 0.2731 μg / ml, indicating that 74-G3-1-G11 has a very strong binding ability to GPC3, which is significantly increased by about two orders of magnitude compared with the positive control.

[0128] Example 6

[0129] Construction of CAR-T cells based on target antibodies.

[0130] (1) Design of CAR molecules

[0131] The target gene structure of the lentiviral vector involved in this example is as follows Figure 4A shown.

[0132] BVHGC3-015 is composed of the following structures in series: human CD8 signal peptide (SP for short), anti-human GPC3 single domain antibody 74-G3-1-G11 [VHH(74-G3-1-G11) 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).

[0133] 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).

[0134] GC33 VL amino acid sequence (SEQ ID NO.15):

[0135] DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSN RFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKR.

[0136] GC33 VH amino acid sequence (SEQ ID NO.16):

[0137] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPK TGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS.

[0138] (2) Lentivirus preparation

[0139] 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 above lentiviral vector expression plasmids were co-transfected into 293T cells with the lentiviral packaging plasmids pRSV-Rev, pMDLg / pRRE and pMD2.G according to the relevant instructions using the transfection reagent Lipofectamine 3000. The viral supernatant was collected 48 hours after transfection, centrifuged at 3000rpm for 15 minutes at 4°C, filtered through a 0.45μm pore size filter membrane, and finally ultracentrifuged at 25,000rpm for 3 hours at 4°C. The obtained virus concentrate was stored at -80°C and named BVHGC3-015 and BN108 respectively. Finally, Jurkat cells were used as materials to detect the activity titer of the above lentivirus.

[0140] (3) CAR-T cell preparation

[0141] 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 for 24 h (culture temperature was 37 ° C, carbon dioxide concentration was 5%). 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 (culture temperature was 37 ° C, carbon dioxide concentration was 5%). After 24 h, the cell density was adjusted to 1.5×10 6 ~2×10 6 / mL, and supplemented with 300IU / mL of hIL-2. On the 4th day after transduction, the cells were washed to remove the residual lentiviral particles in the supernatant, and then cultured in a cell culture incubator for 5 days (culture temperature was 37°C, carbon dioxide concentration was 5%), during which the cell density was maintained at 1×10 6 ~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 Ctrl T.

[0142] (4) Detection of CAR molecule expression

[0143] The BVHGC3-015 CAR-T cells to be tested 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, the FITC-labeled anti-VHH antibody was incubated with the CAR-T cells for 1 hour. The supernatant was then removed by centrifugation, and the cells were washed twice with FACS buffer and resuspended. Using Ctrl T cells as negative control, the CAR molecule expression rate of BVHGC3-015 cells was detected by flow cytometry. The results are shown in Figure 4B The expression rates of BVHGC3-015CAR were shown to be 65.8%.

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

[0145] The BN108 and BVHGC3-015 CAR-T cells to be tested were washed twice with PBS and resuspended with FACS buffer (PBS containing 0.1% sodium azide and 0.4% BSA). According to the antibody instructions, the GPC-3-His tag protein was incubated with the CAR-T cells for 1 hour, then the supernatant was removed by centrifugation, and the cells were washed twice with FACS buffer and resuspended. The CAR-T cells were then incubated with PE-labeled anti-G4SLinker antibody for 1 hour. The supernatant was then removed by centrifugation, and the cells were washed twice with FACS buffer and resuspended. Using Ctrl T cells as negative controls, the efficiency of BN108 and BVHGC3-015 CAR-T cells binding to GPC3 was detected by flow cytometry. The results are shown in Figure 4C The binding efficiencies of BN108 and BVHGC3-015 CAR to GPC3 were shown to be 56.9% and 77.1%, respectively.

[0146] Example 7

[0147] Study on the functions of CAR-T cells in vivo and in vitro.

[0148] In vitro killing experiment of BVHGC3-015 and BN108 CAR-T cells

[0149] Use culture medium at 1×10 5 Resuspend SK-Hep-1-GPC3 at a density of 100 g / mL OE -mCherry, Huh7-Luc-mCherry, and Hep3B-mCherry cells were 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 1:4, 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 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. The results are shown in Figure 5A-5C As shown, under the condition of effector-target ratio of 1:4, for GPC3 positive SK-Hep-1-GPC3 OE-Luc-mCherry 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 BVHGC3-015 group was also significantly improved (P<0.05), and its effect was significantly better than that of the BN108 group. In addition, consistent results can be obtained in the Huh7 and Hep3B liver cancer cell line models. The above results show that compared with existing antibodies, CAR-T cells constructed based on 74-G3-1-G11 antibodies have significant specific killing function against GPC3-positive tumor cells.

[0150] Example 8

[0151] Study on antibody-dependent cellular cytotoxicity (ADCC) mediated by 74-G3-1-G11.

[0152] HuH7 hepatoma cells to be labeled with mCherry fluorescence were selected to detect the ADCC function of the target antibody. After washing the target cells three times with OptiVitro NK cell expansion medium, 1×10 4 The target cells were seeded in a 96-well plate at a density of 1:1 / well and cultured overnight. NK cells were added at a 2:1 effector-target ratio, and the target antibody 74-G3-1-G11 was added at a final concentration of 5 μg / mL.

[0153] The real-time quantitative live cell imaging and analysis platform IncuCyte was used for detection. The changes in the fluorescence signals of 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 NK cell killing effect. Figure 6 As shown, compared with the NK cell group without antibody, 74-G3-1-G11 antibody can significantly enhance the killing effect of NK cells on tumor cells (P<0.05), indicating that 74-G3-1-G11 antibody can effectively mediate the ADCC killing effect of NK cells.

[0154] In summary, the tumor-specific glycoprotein antibody G11 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.

[0155] 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 G11, characterized in that: The amino acid sequence of CDR1 of the tumor-specific glycoprotein antibody G11 includes the sequence shown in SEQ ID NO.1; The amino acid sequence of CDR2 of the tumor-specific glycoprotein antibody G11 includes the sequence shown in SEQ ID NO.2; The amino acid sequence of CDR3 of the tumor-specific glycoprotein antibody G11 includes the sequence shown in SEQ ID NO.

3.

2. The tumor-specific glycoprotein antibody G11 according to claim 1, characterized in that: The amino acid sequence of the tumor-specific glycoprotein antibody G11 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 G11 according to claim 1 or 2 and the full-length or partial amino acid sequence of the crystallizable segment Ig Fc of human immunoglobulin; Preferably, the Ig Fc comprises the full-length or partial sequence of the Fc segment of IgG1, IgG2, IgG3 or IgG4, or a combination thereof; Preferably, the Fc amino acid sequence of IgG1 includes the sequence shown in SEQ ID NO.5; Preferably, 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 D8 according to claim 1 or 2 or the single domain 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; Preferably, the antigen binding domain comprises the tumor-specific glycoprotein antibody G11 according to claim 1 or 2; 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; Preferably, the hinge region comprises any one or a combination of at least two of IgG1, IgG4, TCRα, TCRβ, CD8, CD28 or CD137; Preferably, the transmembrane region includes any one of TCRα, TCRβ, CD40, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, CD314, CD28, CD3ε, CD3ζ, CD3γ or CD3δ, or a combination of at least two thereof; Preferably, the signal transduction domain includes a co-stimulatory domain and a human CD3 intracellular signal transduction domain; Preferably, the costimulatory domain comprises any one or a combination of at least two of 4-1BB, PD1, Dap10, CDS, ICAM-1, ICOS, NKG2D, GITR, OX40L, CD3ε, CD3γ, CD3δ, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70 or CD134; Preferably, the human CD3 intracellular signal transduction domain comprises any one or a combination of at least two of CD3ζ, CD3ε, CD3γ or CD3δ; Preferably, the signal peptide comprises a human CD8 signal peptide having an amino acid sequence as shown in SEQ ID NO.7; Preferably, the hinge region comprises the human IgG4 hinge region as shown in SEQ ID NO.8; Preferably, the transmembrane region comprises the human CD8 transmembrane region as shown in SEQ ID NO.9; 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; Preferably, the chimeric antigen receptor comprises the human CD8 signal peptide shown in SEQ ID NO.7, the tumor-specific glycoprotein antibody G11 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; Preferably, the amino acid sequence of the chimeric antigen receptor comprises 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 G11 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; Preferably, the expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated viral 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; Preferably, the chimeric antigen receptor immune cell comprises the expression vector of claim 7; 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.

9. Use of the tumor-specific glycoprotein antibody G11 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; Preferably, the tumor comprises a GPC3-positive solid tumor; and the cancer comprises any one of liver cancer, ovarian cancer, lung cancer or melanoma.

10. Use of the tumor-specific glycoprotein antibody G11 according to claim 1 or 2 or the chimeric antigen receptor according to claim 5 in the preparation of a preparation for killing NK cells, macrophages or neutrophils.

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