A tumor-specific glycoprotein antibody G11 and its application

Abstract

The present invention discloses a tumor-specific glycoprotein antibody G11 and its application. 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. The tumor-specific glycoprotein antibody G11 of the present invention has affinity for GPC3 and a smaller molecular weight. The engineered immune cells developed based on the tumor-specific glycoprotein antibody G11 have higher exogenous gene expression levels, better effectiveness, better drug efficacy, and lower modification difficulty.

Description

Technical Field

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

[0002] GPC3 (Glypican-3) is a carcinoembryonic antigen involved in cell proliferation, differentiation, migration, and apoptosis. It is a biomarker for a variety of malignant tumors, particularly expressed in up to 70-80% of hepatocellular carcinomas, while it is almost not expressed in normal tissues. Due to its tumor specificity, GPC3 is considered a "golden" target that can accurately attack liver cancer cells. 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] Existing technologies primarily develop therapeutic monoclonal antibodies, bispecific antibodies, and chimeric antigen receptor T cells. The GPC3 protein is easily cleaved into an N-terminal soluble protein and a C-terminal membrane protein, which are then released from cancer cells into the microenvironment. Therefore, antibody screening requires specific antibodies that bind to the 30 kDa C-terminal membrane protein. Steric hindrance and other factors can also affect the binding efficiency of ScFv antibodies to GPC3. Furthermore, existing technologies primarily utilize mouse / humanized antibodies, which are subject to molecular weight limitations and hinder high penetration.

[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 response to the deficiencies of existing technologies 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 modification difficulty; the detection reagents developed based on it have higher sensitivity and lower false positive rates.

[0012] Heavy-chain antibodies, a special type of antibody lacking light chains, are found in the blood of camelids and elasmobranchs. Unlike conventional antibodies, these antibodies contain only a single heavy-chain variable region (VHH) and two conventional CH2 and CH3 domains. Cloning these variable regions can yield single-domain antibodies (also known as nanobodies) composed solely of the heavy-chain variable region. These antibodies possess advantages such as 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 (PBMCs) 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. The phage display library is then enriched and selected for 3-4 rounds using virus-like particles (VLPs) containing GPC3. Monoclones are picked from the enriched products, and the specific binding of the candidate antibodies to GPC3-VLPs is tested by ELISA. After the positive antibodies are expressed and purified, their affinity to CHO-S cells expressing GPC3 is tested respectively. Antibody clones with strong affinity for GPC3 were selected, and the GPC3 single-domain antibody sequence was cloned into a CAR expression vector. CAR-T cells using the GPC3 single-domain antibody as the antigen-binding domain were constructed, and their ability to kill GPC3-positive Huh7 cells in vivo and in vitro was tested. The results showed that compared with existing technologies, the GPC3 single-domain antibody obtained in this invention has a specific killing effect on cell lines expressing GPC3.

[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 region of human immunoglobulin Ig Fc.

[0018] 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.

[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 or a combination of at least two of CD8, CD28, CD137, IgG1, IgG4, TCRα or TCRβ.

[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 includes a costimulatory domain and a human CD3 intracellular signal transduction domain.

[0031] Preferably, the costimulatory domain includes 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 or a combination of at least two of CD3ζ, CD3ε, CD3γ or CD3δ.

[0033] Preferably, the signal peptide comprises the 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 includes 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 comprising 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 cells include 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 use of the tumor-specific glycoprotein antibody G11 described in the first aspect or the chimeric antigen receptor described in the fourth aspect in preparing 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 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 Figure 2 shows the specificity of recombinant single domain antibodies detected by flow cytometry.

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

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

[0068] Figure 4B This is the result of flow cytometry detection of BVHGC3-015 expression in T cells;

[0069] Figure 4C This is the result 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 results 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 the 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 with reference to the embodiments and drawings. It should be understood that the specific embodiments 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 experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0086] Example 1

[0087] Alpaca immunization and serum titer testing.

[0088] (1) Construction of GPC3 overexpressing cells

[0089] Based on 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] Three alpacas (73#, 74# and 194#) were immunized using the CHO-S-GPC3 cells constructed above, 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, third and fourth immunizations, and the centrifuge tubes containing the blood samples were placed in a centrifuge and centrifuged at 800 × g for 10 minutes to collect the upper serum. GPC3-His recombinant protein was diluted with sterile PBS to a final concentration of 1 μg / mL. A new 96-well enzyme-labeled plate was taken and 100 μL / well of 1 μg / mL of GPC3-His recombinant protein was added and coated at 4°C overnight; then the antigen coating solution was shaken off and washed 5 times with PBST (containing 0.05% Tween 20); 200 μL / well of blocking solution was added and blocked at 37°C for 2 hours; after shaking off the blocking buffer, the plate was washed 5 times with PBST; the serum collected above was gradiently diluted with PBS, and 100 μL of the gradiently diluted serum was added to the 96-well enzyme-labeled plate and incubated at room temperature for 1 hour. The control well was PBS; the liquid in the well was shaken off and washed 5 times with PBST; 100 μL of HRP was added Anti-Llama IgG (H+L) antibody (1:50,000 dilution) was incubated at room temperature for 1 hour; after shaking off the liquid in the wells, the wells were washed 5 times with PBST; 100 μL / well TMB colorimetric solution was added; incubated at room temperature in the dark for 10 minutes; 50 μL / well stop solution was added; and the OD450 value in the wells was read using a microplate reader. The results are shown in Tables 1, 2, and 3. The binding titers of the sera of the three alpacas before immunization to the GPC3 protein were all lower than 1:2000 (the antibody titer is expressed in the form of 1:X, where X is the maximum dilution factor at which the antibody can be detected; the higher the dilution factor, the higher the antibody titer), indicating that the content of anti-GPC3 specific antibodies contained therein was extremely low. On the contrary, the binding titer of the serum of 73# and 74# alpacas after the fourth immunization to GPC3 protein was more than 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 serum 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 a 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). PCR products were analyzed by electrophoresis using 1% agarose gel, and the target fragment with a molecular weight of approximately 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 regions, and the 5' end contains the SfiI restriction site GGCCACGAAGGCC) were used to amplify the heavy chain antibody VHH fragment. 1% agarose gel was used for electrophoresis analysis of the PCR products, 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 could 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 storage capacity of 1.32×10 920 monoclonal clones were picked from the plate. The single domain antibody phage display library obtained from 194# alpaca PBMC had a storage capacity of 1.15×10 9 20 single clones were picked from the plate and Sanger sequencing was performed using the M13R primer. The results showed that the phage library sequences were highly different, with no repeated sequences and good diversity.

[0105] (3) Enrichment of phage surface display libraries

[0106] Take the above electroporation and recovery culture solution and culture it to OD 600 When the pH reached 0.6, add M13KO7 helper phage, shake well, and incubate at 37°C for 30 minutes, then incubate on a shaker for 1 hour. After the helper phage has successfully infected the target strain, centrifuge and discard the supernatant, resuspend and culture overnight. The bacterial solution is centrifuged, and the supernatant containing phage particles is incubated at 4°C for 2 hours. The phage pellet is then collected by centrifugation and resuspended to remove insoluble impurities, thereby obtaining the original single-domain antibody phage surface display library.

[0107] Take 10 μL of precipitate and make 10-fold serial dilutions, add 200 μL of OD 600 The titer of the phage display library was determined by counting plaques and quantifying the phage display library.

[0108] Example 3

[0109] Target antibody library selection.

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

[0111] Dilute GPC3 antigen and add it to 96-well solid phase plate, and coat it at 4℃ overnight. 7 CHO-S-GPC3 cells were washed three times with PBS, resuspended in PBSA, and blocked for 1 hour. Simultaneously, the precipitate from the original library displayed on the surface of single-domain antibodies (SDOs) was collected and blocked at 4°C for 1 hour to prepare the premix. The blocked CHO-S cells were centrifuged at 500 × g and the supernatant removed. Phage clones that non-specifically bound to CHO-S were incubated at 4°C to remove them. The GPC3 protein in the 96-well plate was discarded, and buffer was added and allowed to stand at room temperature. After blocking, the CHO-S-GPC3 cells were centrifuged, the supernatant removed, and added to the GPC3 protein wells, incubating at room temperature with shaking for 1 hour. The phage supernatant in the GPC3 protein wells was discarded, washed, and Gly-HCl elution buffer was added to each well. The cells were incubated with shaking. Specifically bound phage were eluted twice, and the eluted products were stored in pre-blocked centrifuge tubes at 4°C.

[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 value of the bacterial solution was 600 When the pH is 0.5, add M13KO7 helper phage, shake well, let stand, centrifuge, discard the supernatant, collect the phage, resuspend, and culture overnight. Centrifuge the phage suspension that has been infected and amplified overnight, and aliquot the supernatant into centrifuge tubes. Add 10 mL of PEG / NaCl to each tube, mix well, place on ice, let stand for 1 hour to precipitate the phage, and centrifuge for 30 minutes. Discard the supernatant and resuspend the phage in 1 mL of sterile PBS. Centrifuge to remove insoluble impurities. This is the phage product obtained from the first round of panning amplification.

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

[0114] GPC3 antigen was diluted to 10 μg / mL in CBS solution, added to a 96-well solid phase plate, and coated overnight at 4°C. Phage products amplified from the first round of panning were used for the second to fourth rounds of panning, following the same steps 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 display library after multiple rounds of panning was used to infect ER2738 Escherichia coli, mixed well, placed in a 37°C water bath, let stand for 10 minutes, and 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 cultured 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 above overnight culture solution was added to each well. The culture was cultured at 37°C and 225 rpm until the OD 600 Add M13KO7 helper phage, mix well and let stand at 37℃ for 15min (M13KO7 volume = 10×bacteria solution volume×OD 600 ×5×10 8 After titer ( / M13KO7), incubate at 37°C, 225 rpm, and 45 min. Centrifuge at 3900 rpm for 10 min, discard the supernatant, and resuspend each well in 500 μL of 2YT-AK medium. Incubate overnight at 30°C, 220 rpm, and centrifuge at 3900 rpm for 10 min. The supernatant obtained is the monoclonal phage particle.

[0117] While amplifying the phage monoclonals, coat the GPC3 antigen protein onto ELISA plates (2 μg / mL, 100 μL / well) using CBS (pH 9.6). After overnight coating at 4°C, discard the antigen, wash three times with PBST, add 250 μL of 3% MPBS to each well, and block overnight at 4°C. After the blocking solution is fully diluted, add 200 μL of 0.05% PBST to each well, wash four times, add 50 μL of 0.1% PBST, and then add 50 μL of the monoclonal phage supernatant to each well. After incubation at 4°C for 1 hour, wash five times with 0.05% PBST. Dilute anti-M13-HRP antibody (1:5000) in 0.05% PBST, add 100 μL to each well, and incubate at 4°C for 45 minutes. After washing 5 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 value was read on the enzyme plate. 450 The sample / negative control ratio 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 74-G3-1-G11 sequence was amplified by PCR and cloned into the eukaryotic expression vector pcDNA3.4, where it was expressed at its C-terminus in fusion with the human IgG1 Fc fragment. The resulting expression plasmid was transiently transfected into HEK293 cells, and 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 detected. The culture supernatant containing 74-G3-1-G11 antibody was mixed with 3×10 5Incubate Huh7 (natively expressing GPC3), SK-Hep-1 overexpressing GPC3, and 293T (GPC3-negative cell line) cells at room temperature for 1 hour. Centrifuge at 800 × g for 5 minutes at room temperature, discard the supernatant, and wash the cells three times with PBS. Add 100 μL of PE-conjugated anti-human IgG antibody (1:500 dilution) and incubate at room temperature for 45 minutes in the dark. Centrifuge at 800 × g for 5 minutes at room temperature, discard the supernatant, and wash the cells three times with PBS. Resuspend the cells in 500 μL of PBS for flow cytometry analysis.

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

[0123] Example 5

[0124] Purification of recombinant antibodies and half-maximal effective concentration (EC 50 ) determination.

[0125] To determine the EC of 74-G3-1-G11 antibody 50 293F cells were transiently transfected with the relevant expression plasmids and cultured in shake flasks for antibody expression and purification. Since the target recombinant antibody contains human IgG fragments, Protein A magnetic beads were used for affinity purification. The Protein A magnetic beads were washed twice with 30 mL of PBS buffer, 0.1 M sodium hydroxide, and PBS buffer, respectively. Based on the required sample volume, the appropriate volume of Protein A magnetic beads (calculated at 20 mg IgG / mL Protein A magnetic beads) was added to the 293F cell shake flask. The cells were incubated in a shaking incubator at 120 rpm for 4 hours at room temperature. The Protein A magnetic beads were collected using a magnetic separation rack and transferred to a 50 mL centrifuge tube. After washing twice with 30 mL of PBS buffer and deionized water, the beads were resuspended in 1 mL of elution buffer. After incubation at room temperature for 5 minutes, the beads were collected using a magnetic separation rack, and the supernatant containing the target antibody was transferred to a 15 mL centrifuge tube. The Protein A magnetic beads were eluted twice, the eluates were combined, and the pH of the solution was adjusted by adding neutralization buffer. The eluted sample was dialyzed against PBS (at least 100 times the volume of the sample) at 25°C for 2 h, then the solution was changed once and then dialyzed at 8°C for 16 h. Finally, the protein concentration was determined and the sample was filtered through a 0.22 μm sterile filter membrane and aliquoted and stored at -80°C until use.

[0126] Serial dilutions of the target antibody were added to 3×10 5 Incubate CHO-S-GPC3 and CHO-S cells at room temperature for 1 hour. Centrifuge at 800 × g for 5 minutes at room temperature, discard the supernatant containing the antibody, and wash the cells three times with PBS. Add 100 μL of PE-conjugated anti-human IgG antibody (1:500 dilution), mix thoroughly, and incubate at room temperature in the dark for 45 minutes. Centrifuge at 800 × g for 5 minutes at room temperature, discard the supernatant containing the antibody, wash the cells three times with PBS, and resuspend the cells in 500 μL of PBS for flow cytometry analysis.

[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 on CHO-S-GPC3 was 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 embodiment is as follows Figure 4A shown.

[0132] BVHGC3-015 is composed of the following structures in series: human CD8 signal peptide (abbreviated as SP), anti-human GPC3 single-domain antibody 74-G3-1-G11 [abbreviated as VHH(74-G3-1-G11)], human IgG4 hinge region (abbreviated as IgG4 hinge), human CD8 transmembrane domain (abbreviated as CD8 TM), human 4-1BB intracellular co-stimulatory domain (abbreviated as 4-1BB ICD), and human CD3ζ intracellular signal transduction domain (abbreviated as CD3ζICD).

[0133] BN108 is composed of the following structures in series: human CD8 signal peptide (SP), anti-human GPC3 single-chain antibody [scFv (GC33)], human CD8 hinge region (CD8 hinge), human CD8 transmembrane domain (CD8 TM), human 4-1BB intracellular co-stimulatory domain (4-1BB ICD), and human CD3ζ intracellular signal transduction domain (CD3ζICD).

[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 CAR expression sequences were fully synthesized and ligated into the lentiviral vector pCDH-EF1α-MCS via molecular cloning, enabling expression under the control of the human EF-1α promoter and Kozak sequence. Using the transfection reagent Lipofectamine 3000, the lentiviral expression plasmids were co-transfected into 293T cells along with the lentiviral packaging plasmids pRSV-Rev, pMDLg / pRRE, and pMD2.G, respectively, according to the manufacturer's instructions. Viral supernatants were collected 48 hours after transfection, centrifuged at 3000 rpm for 15 minutes at 4°C, filtered through a 0.45 μm pore size filter, and finally ultracentrifuged at 25,000 rpm for 3 hours at 4°C. The resulting viral concentrates were stored at -80°C and designated BVHGC3-015 and BN108, respectively. Finally, the lentiviral activity titers were assayed using Jurkat cells.

[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 transduced with lentivirus at an MOI of 5TU / cell. 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 hIL-2. On the 4th day after transduction, the cells were washed to remove the residual lentiviral particles in the supernatant and continued to be cultured in a cell culture incubator for 5 days (culture temperature 37°C, carbon dioxide concentration 5%), during which the cell density was maintained at 1×10 6 ~2×10 6 / mL. Cells were harvested 10 days after transduction and frozen in liquid nitrogen until use. The resulting CAR-T cells were named after the corresponding CAR molecule; T cells not transduced with lentivirus were designated 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). FITC-labeled anti-VHH antibody was incubated with CAR-T cells for 1 hour according to the antibody instructions. The supernatant was then removed by centrifugation, washed twice with FACS buffer, and resuspended. Ctrl T cells were used as negative controls, and the CAR molecule expression rate of BVHGC3-015 cells was detected by flow cytometry. The results are shown in Figure 2. 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, and 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. Ctrl T cells were used as negative controls, and the efficiency of BN108 and BVHGC3-015 CAR-T cells binding to GPC3 was detected by flow cytometry. The results are shown in Figure 2. Figure 4C The binding efficiencies of BN108 and BVHGC3-015 CARs 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 assay 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 μl / mL OE -mCherry, Huh7-Luc-mCherry, and Hep3B-mCherry cells were seeded in 96-well plates 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 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 2. Figures 5A-5C As shown, under the condition of effector-target ratio of 1:4, for GPC3 positive SK-Hep-1-GPC3 OEIn the 74-G3-1-G11 antibody-based CAR-T cells, the killing effect of the BN108 group was significantly improved compared to the mock group (P<0.05). 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 were obtained in the Huh7 and Hep3B liver cancer cell line models. These results show that compared with existing antibodies, CAR-T cells constructed based on the 74-G3-1-G11 antibody have significantly higher specificity 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 test the ADCC function of the target antibody. After washing the target cells three times with OptiVitro NK cell expansion medium, 1×10 4 Target cells were seeded into 96-well plates at a density of 1:1 / well and cultured overnight. NK cells were added at an effector-to-target ratio of 2:1, 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 signal of target cells in each group were calculated using 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 in the results, compared with the NK cell group without antibody addition, 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. Antibody drugs developed based on it have better effectiveness, stronger tissue penetration, better in vivo stability, and better efficacy. Engineered immune cells developed based on it have higher exogenous gene expression levels, better effectiveness, better efficacy, and lower modification difficulty. Detection reagents developed based on it have higher sensitivity and lower false positive rates.

[0155] The applicant states that the present invention is intended to illustrate the detailed methods of the present invention through the above-described embodiments, but the present invention is not limited to the above-described detailed methods, that is, it does not mean that the present invention must rely on the above-described detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure 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 is the sequence shown in SEQ ID NO.1; The amino acid sequence of CDR2 of the tumor-specific glycoprotein antibody G11 is the sequence shown in SEQ ID NO.2; The amino acid sequence of CDR3 of the tumor-specific glycoprotein antibody G11 is 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 region of human immunoglobulin Ig Fc.

4. The single domain antibody according to claim 3, characterized in that The Ig Fc comprises the full-length or partial sequence of the Fc segment of IgG1, IgG2, IgG3 or IgG4, or a combination thereof.

5. The single domain antibody according to claim 4, characterized in that The Fc amino acid sequence of the IgG1 includes the sequence shown in SEQ ID NO.

5.

6. The single domain antibody according to claim 3, characterized in that The amino acid sequence of the single-domain antibody includes the sequence shown in SEQ ID NO.

6.

7. A composition for detecting tumor-specific glycoprotein, characterized in that: The composition comprises the tumor-specific glycoprotein antibody G11 according to claim 1 or 2 or the single-domain antibody according to any one of claims 3-6.

8. 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 is the tumor-specific glycoprotein antibody G11 according to claim 1 or 2.

9. The chimeric antigen receptor according to claim 8, characterized in that The signal peptide includes any one or a combination of at least two of CD8, GM-CSF, CD4, CD28, CD137, IgG, IgE, TCRα or TCRβ.

10. The chimeric antigen receptor according to claim 8, characterized in that The hinge region includes any one or a combination of at least two of IgG1, IgG4, TCRα, TCRβ, CD8, CD28 or CD137.

11. The chimeric antigen receptor according to claim 8, characterized in that 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 of them.

12. The chimeric antigen receptor according to claim 8, characterized in that The signal transduction domain includes a costimulatory domain and a human CD3 intracellular signal transduction domain.

13. The chimeric antigen receptor according to claim 12, characterized in that The costimulatory domain includes 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.

14. The chimeric antigen receptor according to claim 12, characterized in that 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.

15. The chimeric antigen receptor according to claim 9, characterized in that The signal peptide includes a human CD8 signal peptide with an amino acid sequence as shown in SEQ ID NO.

7.

16. The chimeric antigen receptor according to claim 10, wherein The hinge region includes the human IgG4 hinge region shown in SEQ ID NO.

8.

17. The chimeric antigen receptor according to claim 11, wherein The transmembrane region includes the human CD8 transmembrane region shown in SEQ ID NO.

9.

18. The chimeric antigen receptor according to claim 12, wherein The signal transduction domain includes the human 4-1BB intracellular region shown in SEQ ID NO.10 and / or the human CD3ζ intracellular region shown in SEQ ID NO.

11.

19. The chimeric antigen receptor according to claim 8, characterized in that The chimeric antigen receptor includes 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.

20. The chimeric antigen receptor according to claim 8, wherein The amino acid sequence of the chimeric antigen receptor includes the sequence shown in SEQ ID NO.

12.

21. 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 any one of claims 3 to 6, or the chimeric antigen receptor according to any one of claims 8 to 20.

22. An expression vector, characterized in that The expression vector comprises the nucleic acid molecule of claim 21.

23. The expression vector according to claim 22, characterized in that The expression vector is any one of a retroviral vector and an adeno-associated viral vector containing the gene encoding the chimeric antigen receptor according to any one of claims 8 to 20.

24. A chimeric antigen receptor immune cell, characterized in that The chimeric antigen receptor immune cell expresses the chimeric antigen receptor according to any one of claims 8 to 20.

25. The chimeric antigen receptor immune cell according to claim 24, characterized in that The chimeric antigen receptor immune cell comprises the expression vector of claim 22 or 23.

26. The chimeric antigen receptor immune cell according to claim 24, characterized in that 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.

27. Use of the tumor-specific glycoprotein antibody G11 according to claim 1 or 2, the chimeric antigen receptor according to any one of claims 8 to 20, the expression vector according to claim 22 or 23, or the chimeric antigen receptor immune cell according to any one of claims 24 to 26 in the preparation of a product for preventing and / or treating GPC3-positive liver cancer or GPC3-positive liver cancer tumor cells.

28. Use of the tumor-specific glycoprotein antibody G11 according to claim 1 or 2 or the chimeric antigen receptor according to any one of claims 8 to 20 in the preparation of a preparation for killing NK cells, macrophages or neutrophils.

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