Anti-GPC3 protein nano antibody and application thereof

Abstract

The invention relates to an anti-GPC3 protein nano antibody and application thereof. The amino acid sequence of the complementarity determining region CDR1 of the nano antibody comprises a sequence shown in SEQ ID NO.1, the amino acid sequence of the complementarity determining region CDR2 comprises a sequence shown in SEQ ID NO.2, and the amino acid sequence of the complementarity determining region CDR3 comprises a sequence shown in SEQ ID NO.3. The brand-new anti-GPC3 protein nano antibody is excavated, can specifically recognize and combine with GPC3 protein, has high affinity and small molecular weight, can be effectively applied to detection of GPC3 protein and diagnosis and treatment of GPC3 protein related diseases, and provides reference for development and development of new products for diagnosis and treatment of GPC3 protein related diseases.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an anti-GPC3 protein nano antibody and an application thereof. Background Art

[0002] Liver cancer is one of the most common malignant tumors in the world. The treatment of liver cancer mainly includes liver transplantation, tumor resection and non-resectable local therapy such as transcatheter arterial chemoembolization. Since liver cancer, especially primary liver cancer (HCC), is prone to metastasis in the early stage and relapse after treatment, it is of great significance to find an indicator that can accurately judge the prognosis and an effective target for liver cancer treatment. Glypican-3 (GPC3) is a heparan sulfate (HS) glycoprotein anchored on the cell membrane surface. It is highly expressed in liver cancer cells, but not expressed or low expressed in normal liver tissue; in addition, GPC3 is also expressed to a certain extent in some melanomas, lung cancers and ovarian cancers. Therefore, GPC3 can be used as a therapeutic target for liver cancer, melanoma, lung cancer and ovarian cancer.

[0003] At present, the clinical treatment methods using GPC3 mainly include: monoclonal antibodies targeting GPC3, GPC3-derived peptide / DNA vaccines, immunotoxins, etc., but the efficacy is limited in clinical trials and preclinical trials. In addition, the long development cycle of fully humanized traditional antibodies, high production costs, insufficient stability and many other factors limit their clinical application. It can be seen that although there are currently a variety of treatments for liver cancer, there are still certain limitations, especially in the efficient and low side effect treatment of specific tumor markers or targets. In this context, nano antibodies, as a new type of biological drug, are gradually showing their great potential in the treatment of liver cancer. Nano antibodies have the advantages of small size, high affinity, easy transformation and production, and can more accurately identify and attack liver cancer cells while reducing damage to normal cells. For example, CN108659129A discloses the use of prokaryotically expressed GPC3 fusion protein to immunize camels, extract RNA from camel spleen tissue, use it as a template to reverse into cDNA, construct it on the carrier of phage outer capsid protein, construct phage display library, and calculate the library capacity and titer. Then, using the antigen-antibody affinity principle, nanoantibodies that specifically bind to GPC3 were screened from the library through three rounds of panning.

[0004] In summary, the development of new nanoantibodies that can specifically bind to GPC3 is expected to provide new means and new ideas for the diagnosis and / or treatment of diseases such as liver cancer. Summary of the invention

[0005] In view of the deficiencies of the prior art and actual needs, the present invention provides an anti-GPC3 protein nanobody and its application.

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

[0007] In the first aspect, the present invention provides an anti-GPC3 protein nanobody, wherein the amino acid sequence of the complementary determining region CDR1 of the nanobody includes the sequence shown in SEQ ID NO.1, the amino acid sequence of the complementary determining region CDR2 includes the sequence shown in SEQ ID NO.2, and the amino acid sequence of the complementary determining region CDR3 includes the sequence shown in SEQ ID NO.3.

[0008] The present invention explores a new anti-GPC3 protein nanoantibody that can specifically recognize and bind to the GPC3 protein and has high affinity and small molecular weight. It can be effectively applied to the detection of the GPC3 protein and the diagnosis and treatment of GPC3 protein-related diseases, providing a reference for the development and expansion of new products for the diagnosis and treatment of GPC3 protein-related diseases.

[0009] SEQ ID NO.1: GFTLDTYA.

[0010] SEQ ID NO.2: ITSSNGHT.

[0011] SEQ ID NO. 3: AADRAMSTPQAMCVLWQA.

[0012] Preferably, the Nanobody further comprises framework regions FR1, FR2, FR3 and FR4.

[0013] Preferably, the amino acid sequence of the Nanobody comprises the sequence shown in SEQ ID NO.4.

[0014] SEQ ID NO.4:

[0015] QLQLVESGGGLVQPGGSLRLSCEASGFTLDTYAIGWFRQAPGKEREGVSCITSSNGHTY STDSVRGRFTISRDNAKNTVYLHMNSLEPEDTAVYYCAADRAMSTPQAMCVLWQAWGVS YWGQGTQVTVSS.

[0016] It is understood that in the art, when conservative substitutions are made with amino acids with similar or similar properties, the function of the protein is usually not changed, such as substitutions of amino acids with similar properties in CDR regions and / or FR regions, and amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues are or are not encoded by the genetic code, and therefore, nanobodies obtained by conservative substitutions with amino acids with similar or similar properties are also within the scope of protection of the present invention.

[0017] In a second aspect, the present invention provides a biomaterial, wherein the biomaterial comprises at least one of the following:

[0018] (1) a nucleic acid molecule encoding the anti-GPC3 protein nanobody described in the first aspect;

[0019] (2) a recombinant vector, wherein the recombinant vector contains the nucleic acid molecule described in (1);

[0020] (3) a host cell, wherein the host cell expresses the anti-GPC3 protein nanobody described in the first aspect;

[0021] (4) A polypeptide molecule, wherein the polypeptide molecule contains the anti-GPC3 protein nanobody described in the first aspect.

[0022] In a third aspect, the present invention provides the anti-GPC3 protein nanobody described in the first aspect or the biomaterial described in the second aspect having at least one of the following applications:

[0023] (1) Application in the detection of GPC3 protein for non-disease diagnosis purposes;

[0024] (2) Application in the preparation of products for detecting GPC3 protein;

[0025] (3) Application in the preparation of products for diagnosing diseases related to GPC3 protein;

[0026] (4) Use in the preparation of drugs for preventing, delaying or treating diseases related to GPC3 protein;

[0027] (5) Application in the preparation, evaluation or screening of drugs for treating GPC3 protein-related diseases.

[0028] In the present invention, due to the degeneracy of the genetic code, a large number of nucleic acid molecules that can be used to encode the Nanobodies of the present invention can be obtained. Therefore, in the case of a specific amino acid sequence that has been identified, a person skilled in the art can prepare any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the encoded protein, and can select a more preferred polynucleotide after codon optimization according to the preference of the host cell used in the actual preparation process. The nucleic acid molecules can be obtained by conventional methods, such as PCR amplification or artificial synthesis methods.

[0029] The recombinant cell of the present invention expresses the nanobody described in the first aspect, which may contain a nucleic acid molecule encoding the nanobody or a recombinant vector containing the nucleic acid molecule, and the host cell may be a prokaryotic cell, a lower eukaryotic cell or a higher eukaryotic cell, a prokaryotic cell such as a bacterial cell, a lower eukaryotic cell such as a yeast cell, and a higher eukaryotic cell such as a mammalian cell. Representative examples include Escherichia coli, yeast cells, etc.

[0030] Preferably, the product for detecting GPC3 protein and the product for diagnosing diseases associated with GPC3 protein each independently comprises any one of a reagent, a kit, a test strip, a detection device or a pharmaceutical composition.

[0031] Preferably, the reagent comprises at least one of an enzyme-linked immunosorbent assay reagent, an immunoblotting reagent or an immunohistochemistry assay reagent.

[0032] In the present invention, the ELISA reaction can be an indirect ELISA reaction or a sandwich ELISA reaction. For example, in an indirect ELISA reaction, the nanobody of the present invention can be used as a primary antibody, or in a sandwich ELISA reaction, the nanobody of the present invention can be coated on a well plate as a capture antibody.

[0033] In the present invention, the developed anti-GPC3 protein nanoantibody has high specificity and affinity, and can effectively mediate the ADCC killing effect of NK cells, and has the potential to be used for the development of related drugs.

[0034] Preferably, the GPC3 protein-related diseases include tumors expressing GPC3 protein, such as liver cancer.

[0035] In a fourth aspect, the present invention provides a GPC3 protein detection kit, wherein the kit comprises the anti-GPC3 protein nanobody described in the first aspect.

[0036] Preferably, the kit further comprises a reagent for binding or pairing detection with the anti-GPC3 protein nanobody.

[0037] In a fifth aspect, the present invention provides a pharmaceutical composition, comprising the anti-GPC3 protein nanobody described in the first aspect and / or the biomaterial described in the second aspect.

[0038] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0039] Preferably, the auxiliary material includes at least one of a carrier, a binder, a wetting agent, a disintegrant, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer.

[0040] In a sixth aspect, the present invention provides a chimeric antigen receptor, which comprises a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain and an intracellular signal transduction domain.

[0041] Preferably, the antigen binding domain comprises the anti-GPC3 protein nanobody described in the first aspect.

[0042] Preferably, the signal peptide comprises human CD8 signal peptide.

[0043] Preferably, the hinge region comprises a human IgG1 hinge region.

[0044] Preferably, the transmembrane domain comprises a human CD8 transmembrane domain.

[0045] Preferably, the intracellular co-stimulatory domain comprises a human 4-1BB intracellular co-stimulatory domain.

[0046] Preferably, the intracellular signaling domain comprises human CD3ζ.

[0047] In a seventh aspect, the present invention provides a chimeric antigen receptor cell, wherein the chimeric antigen receptor cell expresses the chimeric antigen receptor described in the sixth aspect.

[0048] Preferably, the starting cells of the chimeric antigen receptor cells include any one of T cells, TIL cells, B cells, NK cells or macrophages, or a combination of at least two of them.

[0049] Compared with the prior art, the present invention has at least the following beneficial effects:

[0050] The present invention develops a new nano-antibody targeting GPC3 protein, which has high affinity and small 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1A This is the result of the first round of PCR amplification of the VHH region of the alpaca antibody.

[0052] Figure 1B This is the result of the second round of PCR amplification of the VHH region of the alpaca antibody.

[0053] Figure 2 This is a graph showing the specificity results of recombinant single domain antibodies detected by flow cytometry.

[0054] Figure 3Schematic diagram of the molecular structure of CAR targeting GPC3.

[0055] Figure 4 This is a graph showing the expression results of BVHGC3-012 in T cells detected by flow cytometry.

[0056] Figure 5 This is a graph showing the results of flow cytometry detection of the binding efficiency of BN108 and BVHGC3-012 to GPC3 protein.

[0057] Figure 6 This is a diagram of the killing effect of CAR-T cells targeting GPC3 analyzed by IncuCyte real-time quantitative living cell imaging.

[0058] Figure 7 The figure shows the result of 194-G3-1-D02 antibody-mediated NK cell ADCC killing tumor cells. DETAILED DESCRIPTION

[0059] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0060] 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.

[0061] In the specific embodiment of the present invention, the sources of experimental materials are as follows:

[0062] CHO-S cells: Gibco, R80007;

[0063] SK-Hep-1 cells: ATCC, HTB-52;

[0064] Huh7 cells: Cell Bank of Chinese Academy of Sciences, SCSP-526;

[0065] FITC-labeled Rabbit anti-Llama IgG (H+L) antibody: Invitrogen, A16155;

[0066] Anti-M13-HRP antibody: SinoBiological, 11973-MM05T-H;

[0067] PE-labeled anti-human IgG antibody: Biolegend, 410708;

[0068] AF647-labeled Anti-human IgG antibody: Jackson ImmunoResearch, 109-606-170;

[0069] Anti-VHH antibody: GenScript, A02017;

[0070] AF647-labeled anti-human GPC3 antibody: R&D system, FAB2119A.

[0071] Example 1

[0072] In this example, alpaca immunization and antibody titer determination were performed.

[0073] (1) Construction of GPC3 overexpressing cells

[0074] Based on human GPC3 (UniProt Accession: P51654), a lentiviral expression vector was constructed and infected into CHO-S cells to obtain the GPC3-overexpressing cell line CHO-S-GPC3.

[0075] (2) Alpaca immunization and serum titer testing

[0076] The CHO-S-GPC3 cells constructed above were used to immunize camels (numbered 194#), once every three weeks, for a total of 5 times. 5 mL of peripheral blood was collected before each immunization, and the centrifuge tube containing the blood sample was placed in a centrifuge. After centrifugation, the supernatant was transferred to a new sterile centrifuge tube to collect the immune serum. The titer of alpaca serum was detected by ELISA. The results showed that the anti-GPC3 specific antibody content in the alpaca serum before immunization was extremely low. On the contrary, the binding titer of the serum to the GPC3 protein after the fourth immunization was significantly increased, indicating that the content of GPC3-specific antibodies in the alpaca serum was significantly increased after multiple immunizations, and the immunization was successful, which can be used for the construction of phage surface display libraries.

[0077] Example 2

[0078] This example constructs a nanobody phage surface display library.

[0079] (1) VHH antibody fragment cloning

[0080] After confirming that alpaca serum contained GPC3-specific antibodies, 100 mL of peripheral blood was collected, PBMCs were separated using lymphocyte separation solution, and RNA was extracted. TMII 1st Strand cDNA Synthesis Kit was used for reverse transcription to obtain cDNA. Using PBMC cDNA as a template, specific primers (upstream primer binds to the signal peptide of VHH antibody ORF, downstream primer binds to CH2 region) were used to PCR amplify the alpaca heavy chain antibody sequence. 1% agarose was used for electrophoresis analysis of PCR products, and the target fragment with a molecular weight of about 750 bp was recovered and separated ( Figure 1A ). The first round PCR product was used as a template and specific primers (the upstream primer binds to the antibody FR1 region, and the 5' end contains the SfiI restriction site GGCCCAGCCGGCC (SEQ ID NO.17); the downstream primer binds to the antibody hinge (Hinge) and FR4 region, and the 5' end contains the SfiI restriction site GGCCACGAAGGCC (SEQ ID NO.18)) 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 ).

[0081] (2) Electroporation of library vectors and detection of library capacity and diversity

[0082] 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 overnight at 16°C using T4 ligase. A mixture of 300 μL of the above-mentioned connection product and E. coli SS320 competent cells was added to a pre-cooled electroporation cup, and the connection product was transformed into E. coli by electroporation. 20 mL of SOC medium was added to resuspend the bacteria, and the shaking was revived and cultured at 37°C for 1 hour. 15 mL of bacterial solution was taken for subsequent phage production enrichment, and the remaining 5 mL of electroporation product was added with an equal volume of 50% glycerol, mixed evenly, and stored at -80°C. In addition, 20 μL of bacterial solution was taken and diluted with 2YT medium in a gradient manner and evenly coated on an LB plate containing ampicillin, cultured at 37°C overnight, and the number of clones that can be produced by each connection reaction was calculated to obtain the capacity of the nanoantibody phage surface display library. Twenty single clones on the plate were picked and Sanger sequencing was performed with M13R primers. The results showed that the phage library sequences were highly different, with no repeated sequences and good diversity.

[0083] (3) Enrichment of phage display libraries

[0084] Take 15 mL of the bacterial solution after electroporation and recovery culture, and dilute it with 2YT to adjust the OD 600 When the OD is about 0.25, add ampicillin with a final concentration of 100 μg / mL and place in a constant temperature shaker at 37°C and 225 rpm. 600When the value was 0.6, M13KO7 helper phage was added (the volume of M13KO7 helper phage added = 10 × bacterial solution volume × OD 600 ×5×10 8 / M13KO7 titer), shake well and let stand at 37℃ for 30min, then culture at 37℃ shaker at 225rpm for 1h. After the helper phage completes the infection of the target strain, centrifuge at 6000rpm for 10min, discard the supernatant, resuspend with 2YT-AK medium, and culture overnight at 25℃ shaker at 200rpm. Then centrifuge the bacterial solution at 10000rpm for 15min, transfer the supernatant containing phage particles to a new centrifuge tube (add 1 / 5 of the volume of bacterial solution PEG / NaCl to the tube), mix well and let stand at 4℃. After standing for 2h, centrifuge at 10000rpm for 30min at 4℃, collect the phage precipitate, and resuspend with PBS 1 / 50 of the original volume. Transfer the resuspended phage to a 1.5mL EP tube and centrifuge at 12000×g for 5min at 4℃ to remove insoluble impurities. The supernatant was then transferred to a new 1.5 mL EP tube, 250 μL of PEG / NaCl was added to each tube, mixed and allowed to stand at 4°C for 10 min, centrifuged at 12,000 × g for 10 min at 4°C, and the supernatant was discarded. After resuspending in 1 mL PBS, the supernatant was centrifuged at 12,000 × g for 5 min at 4°C, and the supernatant was transferred to a new 1.5 mL EP tube to obtain the original library displayed on the surface of the nanoantibody phage.

[0085] 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.

[0086] Example 3

[0087] In this example, a target antibody library was screened.

[0088] (1) First round of panning and product amplification

[0089] Seal the 1.5 mL centrifuge tube with 3% MPBS and incubate at 4°C overnight. Dilute the GPC3 antigen to 50 μg / mL with CBS solution and add to the 96-well solid phase plate. Coat at 4°C overnight. Take 1×10 7CHO-S-GPC3 cells were washed 3 times with PBS, resuspended in 3% PBSA, and blocked at 37°C for 1h on a 360° low-speed rotating mixer. At the same time, 150 μL of the precipitated product of the original library displayed on the surface of the nanoantibody phage was taken, 350 μL of 1% PBSA was added to the 360° low-speed rotating mixer, and blocked at 4°C for 1h as a premix. The blocked CHO-S cells were centrifuged at 500×g for 10min, the supernatant was removed, and the phage premix was added and incubated at 4°C for 1h on a 360° low-speed rotating mixer to remove phage clones that non-specifically bind to CHO-S. The GPC3 protein in the 96-well plate was discarded, and 200 μL of 3% MPBS buffer was added and allowed to stand at room temperature. After blocking for 1h, 3% MPBS buffer. CHO-S-GPC3 cells were centrifuged at 500×g for 10 min, the supernatant was taken and added to the GPC3 protein wells respectively, and incubated at room temperature for 1 h. The phage supernatant in the GPC3 protein wells was discarded, and 0.05% PBST was used to wash 6 times and PBS was washed 4 times. 100 μL of pH 2.2 Gly-HCl elution solution was added to each well, and incubated at 37°C for 8 min. The specifically bound phages were eluted twice, and the eluted products were stored in pre-sealed centrifuge tubes (washed twice with PBS after sealing) at 4°C.

[0090] Take 20 mL of 2YT medium, add tetracycline at a final concentration of 100 μg / mL and 20 μL of Escherichia coli ER2738 and culture in an incubator at 37°C and 225 rpm until the OD 600 The eluted phage product was added to the ER2738 bacterial solution, mixed and incubated at 37°C for 30 min, then 20 mL of 2YT medium was added and cultured at 37°C and 225 rpm for 30 min. 600 When the value was 0.5 again, M13KO7 helper phage was added (the added volume of M13KO7 = 10 × bacterial solution volume × OD 600 ×5×10 8 / M13KO7 titer), shake well and let stand at 37℃ for 30min. Add ampicillin at a final concentration of 100μg / mL to the bacterial solution, culture at 225rpm at 37℃ for 45min, centrifuge at 8000rpm for 20min, discard the supernatant and collect the bacteria. Resuspend with 40mL 2YT-AK medium and culture overnight at 210rpm at 30℃. Transfer the phage suspension infected and amplified overnight to a 50mL centrifuge tube, centrifuge at 8000rpm at 4℃ for 30min, and divide the supernatant into 40mL 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 at 8000rpm at 4℃ for 30min. Discard the supernatant and resuspend the phage with 1mL sterile PBS, centrifuge at 12000×g at 4℃ for 5min to remove insoluble impurities. Transfer 1 mL of phage suspension to a new 1.5 mL centrifuge tube, add 250 μL of PEG / NaCl, mix well, and let stand at 4°C for 10 min to precipitate phage. Centrifuge at 12,000 × g for 10 min at 4°C, and discard the supernatant. Resuspend the phage with 1 mL of PBS, centrifuge at 12,000 × g for 5 min at 4°C to remove insoluble impurities, and obtain the phage product obtained in the first round of panning amplification.

[0091] (2) Second to fourth rounds of panning and product amplification

[0092] 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.

[0093] (3) ELISA detection and sequencing of monoclonal phage

[0094] The ER2738 bacteria were infected with the nanoantibody phage surface display library after multiple rounds of panning, mixed and placed in a 37°C water bath for 10 minutes, then spread on the 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 overnight culture solution was added to each well, and cultured 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 225rpm for 45min. Centrifuged at 3900rpm for 10min, discarded the supernatant, resuspended in 500μL 2YT-AK medium per well, cultured at 30°C and 220rpm overnight. Centrifuged at 3900rpm for 10min, the supernatant obtained was the monoclonal phage particles.

[0095] While amplifying the phage monoclonal, coat the GPC3 antigen protein with CBS at pH 9.6 to the ELISA plate (2μg / mL, 100μL / well). After overnight coating at 4°C, discard the antigen, wash three times with PBST, add 250μL 3% MPBS to each well, and block overnight at 4°C. Discard the blocking solution, add 200μL 0.05% PBST to each well, wash four times, add 50μL 0.1% PBST, and then add 50μL of the above monoclonal phage supernatant one by one. After incubation at 4°C for 1h, wash five times with 0.05% PBST. Dilute the anti M13-HRP antibody (1:5000) with 0.05% PBST, add 100μL to each well, and incubate at 4°C for 45min. 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 the nanobody and named 194-G3-1-D02.

[0096] The CDR1 amino acid sequence of the nanobody 194-G3-1-D02 is shown in SEQ ID NO.1, the CDR2 amino acid sequence of the nanobody 194-G3-1-D02 is shown in SEQ ID NO.2, the CDR3 amino acid sequence of the nanobody 194-G3-1-D02 is shown in SEQ ID NO.3, and the amino acid sequence of the nanobody 194-G3-1-D02 is shown in SEQ ID NO.4.

[0097] Example 4

[0098] In this example, the recombinant antibody was purified and the half effective concentration (EC 50 ) determination.

[0099] The sequence of the nanobody 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. After transient transfection of the obtained expression plasmid into HEK293 cells, the harvested cell culture supernatant contained each recombinant nanobody. In the same way, the GPC3-specific antibody HYP7 was expressed as a positive control.

[0100] Human IgG1 Fc amino acid sequence (SEQ ID NO.5):

[0101] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0102] HYP7 light chain variable region amino acid sequence (SEQ ID NO.6):

[0103] DIVMSQSPSSLVVSIGEKVTMTCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASS RESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYNYPLTFGAGTKLELK.

[0104] HYP7 heavy chain variable region amino acid sequence (SEQ ID NO.7):

[0105] EVQLVETGGGMVQPEGSLKLSCAASGFTFNKNAMNWVRQAPGKGLEWVARIRNKTN NYATYYADSVKARFTISRDDSQSMLYLQMNNLKIEDTAMYYCVAGNSFAYWGQGTLVTVS A.

[0106] To determine the EC of 194-G3-1-D02 antibody 50, transiently transfect the relevant expression plasmid into 293F cells, and shake the flask to culture for antibody expression and purification. Since the target recombinant antibody contains human IgG fragments, Protein A magnetic beads can be used for affinity purification. Wash the Protein A magnetic beads twice with 30mL PBS buffer, 0.1M sodium hydroxide, and PBS buffer in sequence. Add the corresponding volume of Protein A magnetic beads (calculated as 20mg IgG / mL Protein A magnetic beads) to the 293F cell shake flask according to the required amount of the sample. Incubate at room temperature for 3h at 120rpm in a shaking incubator. Collect the Protein A magnetic beads with a magnetic separation rack and transfer them to a 50mL centrifuge tube. After washing twice with 30mL PBS buffer and 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 Protein A 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 23°C for 2 hours, then dialyzed at 5°C for 14 hours after changing the solution once. Finally, the protein transfer 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.

[0107] 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, and resuspend the cells with 500μL PBS for flow cytometry analysis.

[0108] The results are shown in Table 1. The EC of positive control antibody HYP7 against CHO-S-GPC3 50 The value is 0.09428 μg / mL, and the EC value of 194-G3-1-D02 for CHO-S-GPC3 is 50 The value is 0.5663μg / mL, which has a relatively high affinity.

[0109] Table 1

[0110]

[0111]

[0112] Example 5

[0113] This example detects the binding of recombinant single domain antibodies to tumor cells.

[0114] The binding specificity of each antibody was tested. The culture supernatant containing each antibody was mixed with 3×10 5 Huh7 (naturally expressing 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 and analyzed by flow cytometry. For the negative control group, only 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 and analyzed by flow cytometry.

[0115] The results are as follows Figure 2 As shown, the 194-G3-1-D02 antibody significantly bound to Huh7 cells but not to 293T cells.

[0116] Example 6

[0117] This example constructs CAR-T cells based on the target antibody.

[0118] (1) Design of CAR molecules

[0119] The target gene structure of the lentiviral vector involved in this example is as follows Figure 3 shown.

[0120] BVHGC3-012 is composed of the following structures in series: human CD8 signal peptide (SP for short), anti-human GPC3 nanoantibody 194-G3-1-D02 [VHH(194-G3-1-D02) 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).

[0121] 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).

[0122] Anti-human GPC3 single-chain antibody light chain variable region amino acid sequence (SEQ ID NO.8):

[0123] DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNR FSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKR.

[0124] Anti-human GPC3 single-chain antibody heavy chain variable region amino acid sequence (SEQ ID NO.9):

[0125] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKT GDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS.

[0126] Human CD8 signal peptide amino acid sequence (SEQ ID NO.10):

[0127] MALPVTALLLPLALLLHAARPS.

[0128] Human IgG4 hinge amino acid sequence (SEQ ID NO.11):

[0129] ESKYGPPCPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0130] Human CD8 transmembrane domain amino acid sequence (SEQ ID NO.12):

[0131] CDIYIWAPLAGTCGVLLLSLVITLYCNHRNR.

[0132] Human 4-1BB intracellular co-stimulatory domain amino acid sequence (SEQ ID NO.13):

[0133] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0134] Human CD3ζ intracellular signal transduction domain amino acid sequence (SEQ ID NO.14):

[0135] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0136] The amino acid sequence of the anti-human GPC3 chimeric antigen receptor (BVHGC3-012) (SEQ ID NO.15):

[0137] MALPVTALLLPLALLLHAARPSQLQLVESGGGLVQPGGSLRLSCEASGFTLDTYAIGWFRQAPGKEREGVSCITSSNGHTYSTDSVRGRFTISRDNAKNTVYLHMNSLEPEDTA VYYCAADRAMSTPQAMCVLWQAWGVSYWGQGTQVTVSSESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKCDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC ELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0138] The amino acid sequence of the anti-human GPC3 chimeric antigen receptor (BN108) (SEQ ID NO.16):

[0139] MALPVTALLLPLALLLHAARPSDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPP TFGQGTKLEIKRGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCT RFYSYTYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0140] (2) Lentivirus preparation

[0141] 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 with lentiviral packaging plasmids pRSV-Rev, pMDLg / pRRE and pMD2.G using the transfection reagent Lipofectamine 3000 according to the relevant instructions. 293T cells. The viral supernatant was collected 48 hours after transfection, centrifuged at 3000 rpm for 15 minutes at 4°C, filtered through a 0.45μm pore size filter membrane, and finally ultracentrifuged at 25,000 rpm for 3 hours at 4°C. The obtained virus concentrates were stored at -80°C and named BVHGC3-012 and BN108, respectively.

[0142] (3) CAR-T cell preparation

[0143] 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 / mL for transduction. At the same time, 25 ng / mL anti-CD3 antibody, 25 ng / mL anti-CD28 antibody and 300 IU / mL recombinant hIL-2 were supplemented and cultured in a cell culture incubator (culture temperature was 37 ° C, carbon dioxide concentration was 5%). After 24 h, the cell density was adjusted to 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 / 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.

[0144] (4) Detection of CAR molecule expression

[0145] The BVHGC3-012 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 BVHGC3-030 cells was detected by flow cytometry. The results are shown in Figure 4 The expression rates of BVHGC3-012 CAR were shown to be 51.9%.

[0146] (4) Detection of CAR molecule binding efficiency to GPC3

[0147] The BN108 and BVHGC3-012 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 BVHGC3-030 CAR-T cells binding to GPC3 was detected by flow cytometry. The results are shown in Figure 5 The binding efficiencies of BN108 and BVHGC3-012 CAR to GPC3 were 47.7% and 27.6%, respectively, indicating that both can effectively bind to the GPC3 protein.

[0148] Example 7

[0149] This example studies the function of CAR-T cells.

[0150] In vitro killing experiments were performed using BVHGC3-012 and BN108 CAR-T cells. 5 Huh7 cells labeled with mCherry were resuspended at a density of 100 μL / mL and inoculated in a 96-well plate at a volume of 100 μL per well. After being cultured overnight in the IncuCyte SX5 live cell imaging analysis system, CAR-T cells were added for co-culture at an effective target ratio of 1:2 to the effective number of cells, and the killing effect of CAR-T cells on tumor cells was recorded in real time. In addition, the target cell group and the Mock group (T cells without lentiviral transduction) were set as controls. After the co-culture, the changes in the mCherry fluorescence signals of the target cells in each group were calculated by the IncuCyte SX5 software. The lower the signal value, the fewer the number of cells in the group, and the better the killing effect of CAR-T cells.

[0151] The results are as follows Figure 6As shown, under the condition of 1:2 effect-target ratio, for GPC3-positive Huh7 cells, the killing effect of BN108 group (relative fluorescence area at the last time point was 1.68±0.012) was significantly improved compared with Mock group (relative fluorescence area at the last time point was 2.28±0.004), and the killing effect of BVHGC3-012 group (relative fluorescence area at the last time point was 1.36±0.010) was significantly better than that of BN108 group (P<0.05). The above results show that compared with existing antibodies, CAR-T cells constructed based on 194-G3-1-D02 antibody have stronger specific killing function against GPC3-positive tumor cells.

[0152] Example 8

[0153] Study on antibody-dependent cellular cytotoxicity (ADCC) mediated by 194-G3-1-D02.

[0154] 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 194-G3-1-D02 was added at a final concentration of 5 μg / mL.

[0155] 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 7 As shown, compared with the NK cell group without antibody, 194-G3-1-D02 antibody can significantly enhance the killing effect of NK cells on tumor cells (P<0.05), indicating that 194-G3-1-D02 antibody can effectively mediate the ADCC killing effect of NK cells.

[0156] In summary, the present invention develops a new GPC3 nanobody with good specificity and affinity, and has broad application prospects. It can be effectively used to prepare reagents targeting GPC3, including detection reagents and drugs, such as constructing chimeric antigen receptors and corresponding immune cells expressing chimeric antigen receptors, which can effectively and specifically kill tumor cells.

[0157] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention are within the protection scope and disclosure scope of the present invention.

Claims

1. An anti-GPC3 protein nanobody, characterized in that: The amino acid sequence of the complementary determining region CDR1 of the nanobody includes the sequence shown in SEQ ID NO.1, the amino acid sequence of the complementary determining region CDR2 includes the sequence shown in SEQ ID NO.2, and the amino acid sequence of the complementary determining region CDR3 includes the sequence shown in SEQ ID NO.

3.

2. The anti-GPC3 protein nanobody according to claim 1, characterized in that: The Nanobody also includes framework regions FR1, FR2, FR3 and FR4; The amino acid sequence of the nanobody includes the sequence shown in SEQ ID NO.

4.

3. A biomaterial, characterized in that: The biological material comprises at least one of the following: (1) a nucleic acid molecule encoding the anti-GPC3 protein nanobody according to claim 1 or 2; (2) a recombinant vector, wherein the recombinant vector contains the nucleic acid molecule described in (1); (3) a host cell, wherein the host cell expresses the anti-GPC3 protein nanobody according to claim 1 or 2; (4) A polypeptide molecule comprising the anti-GPC3 protein nanobody according to claim 1 or 2.

4. The anti-GPC3 protein nanobody according to claim 1 or 2 or the biomaterial according to claim 3 has at least one of the following applications: (1) Application in the detection of GPC3 protein for non-disease diagnosis purposes; (2) Application in the preparation of products for detecting GPC3 protein; (3) Application in the preparation of products for diagnosing diseases related to GPC3 protein; (4) Use in the preparation of drugs for preventing, delaying or treating diseases related to GPC3 protein; (5) Application in the preparation, evaluation or screening of drugs for treating GPC3 protein-related diseases.

5. The use according to claim 4, characterized in that: The product for detecting GPC3 protein and the product for diagnosing diseases related to GPC3 protein each independently include any one of a reagent, a kit, a test strip, a detection device or a pharmaceutical composition; The reagents include at least one of an enzyme-linked immunosorbent assay reagent, an immunoblotting reagent or an immunohistochemical assay reagent.

6. A GPC3 protein detection kit, characterized in that: The kit comprises the anti-GPC3 protein nanobody according to claim 1 or 2; The kit also includes a reagent for binding or pairing detection with the anti-GPC3 protein nanobody.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the anti-GPC3 protein nanobody according to claim 1 or 2 and / or the biomaterial according to claim 3.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable excipient; The auxiliary materials include at least one of a carrier, a binder, a wetting agent, a disintegrant, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer.

9. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain and an intracellular signal transduction domain; The antigen binding domain comprises the anti-GPC3 protein nanobody according to claim 1 or 2.

10. A chimeric antigen receptor cell, characterized in that The chimeric antigen receptor cell expresses the chimeric antigen receptor of claim 9; The starting cells of the chimeric antigen receptor cells include any one of T cells, TIL cells, B cells, NK cells or macrophages, or a combination of at least two of them.

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