An anti-GPC3 protein nanobody and its application

Abstract

The present invention relates to an anti-GPC3 protein nanobody and its application. The amino acid sequence of the complementary determining region (CDR1) of the nanobody comprises the sequence shown in SEQ ID NO. 1, the amino acid sequence of the complementary determining region (CDR2) comprises the sequence shown in SEQ ID NO. 2, and the amino acid sequence of the complementary determining region (CDR3) comprises the sequence shown in SEQ ID NO. 3. The present invention discovers a novel anti-GPC3 protein nanobody that can specifically recognize and bind to the GPC3 protein, possesses high affinity and a small molecular weight, and 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.

Description

Technical Field

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

[0002] Liver cancer is one of the most common malignant tumors worldwide. Treatment options primarily include liver transplantation, tumor resection, and non-resective local therapies such as transcatheter arterial chemoembolization. Because liver cancer, especially primary hepatocellular carcinoma (HCC), is prone to metastasis and recurrence after treatment in the early stages, identifying an accurate prognostic marker and an effective therapeutic target for HCC is crucial. Glypican-3 (GPC3) is a heparan sulfate (HS) glycoprotein anchored to the cell membrane. It is highly expressed in liver cancer cells but absent or at low levels in normal liver tissue. Furthermore, GPC3 is also expressed to a limited extent in some melanomas, lung cancers, and ovarian cancers. Therefore, GPC3 may serve as a therapeutic target for HCC, melanomas, lung cancer, and ovarian cancers.

[0003] Currently, clinical treatments targeting GPC3 primarily include monoclonal antibodies, GPC3-derived peptide / DNA vaccines, and immunotoxins. However, these have demonstrated limited efficacy in both clinical and preclinical trials. Furthermore, the long development cycle, high production costs, and limited stability of fully humanized conventional antibodies limit their clinical application. Consequently, despite the emergence of a variety of treatments for liver cancer, certain limitations remain, particularly in the area of ​​effective, low-side effect treatments targeting specific tumor markers or targets. Against this backdrop, nanobodies, as a new type of biopharmaceutical, are increasingly demonstrating their significant potential in liver cancer treatment. Nanobodies offer the advantages of small size, high affinity, and ease of modification and production. They can more accurately identify and attack liver cancer cells while minimizing damage to normal cells. For example, CN108659129A discloses the use of prokaryotically expressed GPC3 fusion protein to immunize camels. RNA was extracted from camel spleen tissue and converted into cDNA using this template. This cDNA was then constructed onto a phage outer capsid protein vector to construct a phage display library, and the library capacity and titer were calculated. 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 response to 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 has discovered 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 used in the detection of 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, conservative substitutions with amino acids having similar or similar properties generally do not alter the function of the protein, such as substitutions of amino acids with similar properties in CDR and / or FR regions. Amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. Therefore, Nanobodies obtained by conservative substitution with amino acids having 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 comprising 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 containing 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 GPC3 protein-related diseases;

[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 having identified a specific amino acid sequence, those 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. The more preferred polynucleotide can be selected through codon optimization according to the preferences 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 cells of the present invention express the Nanobodies described in the first aspect, which may comprise a nucleic acid molecule encoding a Nanobody or a recombinant vector containing said nucleic acid molecule. The host cells may be prokaryotic cells, lower eukaryotic cells, or higher eukaryotic cells. Prokaryotic cells are such as bacterial cells, lower eukaryotic cells are such as yeast cells, and higher eukaryotic cells are such as mammalian cells. Representative examples include Escherichia coli and yeast cells.

[0030] Preferably, the product for detecting GPC3 protein and the product for diagnosing GPC3 protein-related diseases each independently comprise 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 can be an indirect ELISA or a sandwich ELISA. For example, in an indirect ELISA, the Nanobody of the present invention can be used as a primary antibody, or in a sandwich ELISA, the Nanobody of the present invention can be used as a capture antibody coated on a well plate.

[0033] In the present invention, the anti-GPC3 protein nanoantibody developed 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, comprising 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 excipients include at least one of a carrier, a binder, a wetting agent, a disintegrant, an emulsifier, a solubilizer, 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 costimulatory domain comprises a human 4-1BB intracellular costimulatory 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 the 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 modification difficulty; 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 The figure shows 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 the result of flow cytometry detection of BVHGC3-012 expression in T cells.

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

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

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

[0059] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. However, the following examples are merely 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 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.

[0061] In the specific embodiments 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 the 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] This example involves immunization of alpacas and determination of antibody titers.

[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 alpacas (numbered 194#), and immunized 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 the alpaca serum was tested by ELISA. The results showed that the content of anti-GPC3 specific antibodies 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 medium, and RNA was extracted. TMReverse transcription was performed using the II 1st Strand cDNA Synthesis Kit to obtain cDNA. Using PBMC cDNA as a template, specific primers (upstream primer binds to the signal peptide of the VHH antibody ORF, downstream primer binds to the CH2 region) were used to PCR amplify the alpaca heavy chain antibody sequence. PCR products were analyzed by electrophoresis using 1% agarose gel, and the target fragment with a molecular weight of approximately 750 bp 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 (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 products were analyzed by electrophoresis using 1% agarose gel, 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 VHH fragment library were ligated overnight at 16°C using T4 ligase. 300 μL of the above ligation product and E. coli SS320 competent cells were added to a pre-chilled electroporation cuvette and transformed into E. coli by electroporation. 20 mL of SOC medium was added to resuspend the cells and shaken at 37°C for 1 hour. 15 mL of the bacterial solution was used for subsequent phage production and enrichment. The remaining 5 mL of the electroporated product was added to an equal volume of 50% glycerol, mixed evenly, and stored at -80°C. Separately, 20 μL of the bacterial solution was diluted with 2YT medium and evenly spread on LB plates containing ampicillin. The cells were incubated at 37°C overnight. The number of clones produced by each ligation reaction was calculated to obtain the capacity of the nanobody phage surface display library. Twenty single clones were picked from the plate and subjected to Sanger sequencing using the M13R primer. The results showed that the phage library sequences were highly different, with no repeated sequences and good diversity.

[0083] (3) Enrichment of phage surface 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 value 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 OD value is 0.6, add M13KO7 helper phage (the volume of M13KO7 helper phage added = 10 × the volume of bacterial solution × OD 600 ×5×10 8 / M13KO7 titer), shake well and let it stand at 37℃ for 30 minutes, then culture on a shaker at 37℃ at 225rpm for 1 hour. After the helper phage completes the infection of the target strain, centrifuge at 6000rpm for 10 minutes, discard the supernatant, resuspend with 2YT-AK medium, and culture overnight on a shaker at 25℃ at 200rpm. Then centrifuge the bacterial solution at 10000rpm for 15 minutes, transfer the supernatant containing phage particles to a new centrifuge tube (add 1 / 5 of the bacterial solution volume of PEG / NaCl to the tube), mix well and let it stand at 4℃. After standing for 2 hours, centrifuge at 10000rpm for 30 minutes at 4℃, collect the phage precipitate, and resuspend it with PBS with 1 / 50 of the original volume. Transfer the resuspended phage to a 1.5mL EP tube and centrifuge at 12000×g for 5 minutes 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. After mixing, the tube was allowed to stand at 4°C for 10 minutes. The tube was then centrifuged at 12,000 × g for 10 minutes at 4°C and the supernatant was discarded. After resuspending in 1 mL of PBS, the tube was centrifuged at 12,000 × g for 5 minutes at 4°C. The supernatant was transferred to a new 1.5 mL EP tube to obtain the original nanobody phage display library.

[0085] 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.

[0086] Example 3

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

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

[0089] Seal a 1.5 mL centrifuge tube with 3% MPBS and incubate at 4°C overnight. Dilute GPC3 antigen to 50 μg / mL with CBS solution and add to a 96-well solid phase plate. Coat at 4°C overnight. 7CHO-S-GPC3 cells were washed three times with PBS, resuspended in 3% PBSA, and blocked at 37°C for 1 hour on a 360° low-speed vortexer. Simultaneously, 150 μL of the precipitate from the original nanobody phage display library was added to 350 μL of 1% PBSA and blocked at 4°C for 1 hour on a 360° low-speed vortexer to prepare a premix. The blocked CHO-S cells were centrifuged at 500 × g for 10 minutes, the supernatant removed, and the phage premix added. The cells were incubated at 4°C for 1 hour on a 360° low-speed vortexer to remove phage clones that nonspecifically bound 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 1 hour, 3% MPBS buffer was added. CHO-S-GPC3 cells were centrifuged at 500 × g for 10 minutes. The supernatant was removed and added to the GPC3 protein wells and incubated at room temperature with shaking for 1 hour. The phage supernatant in the GPC3 protein wells was discarded and washed six times with 0.05% PBST and four times with PBS. 100 μL of pH 2.2 Gly-HCl elution buffer was added to each well and incubated at 37°C with shaking for 8 minutes. Specifically bound phage were eluted twice. The eluted product was stored in a pre-sealed centrifuge tube (washed twice with PBS after blocking) 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 well, and incubated at 37°C for 30 minutes. Then 20 mL of 2YT medium was added and the culture was continued at 37°C and 225 rpm for 30 minutes. 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 it stand at 37℃ for 30 minutes. Add ampicillin to the bacterial solution at a final concentration of 100μg / mL, culture at 37℃ and 225rpm for 45 minutes, centrifuge at 8000rpm for 20 minutes, discard the supernatant and collect the bacteria. Resuspend with 40mL 2YT-AK medium and culture overnight at 30℃ and 210rpm. Transfer the phage suspension that has been infected and amplified overnight to a 50mL centrifuge tube, centrifuge at 8000rpm at 4℃ for 30 minutes, and then divide the supernatant into 40mL centrifuge tubes. Add 10mL PEG / NaCl to each tube, mix well and place on ice, let it stand for 1 hour to precipitate the phage, and centrifuge at 8000rpm at 4℃ for 30 minutes. Discard the supernatant and resuspend the phage with 1mL sterile PBS, centrifuge at 12000×g at 4℃ for 5 minutes 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 thoroughly, and incubate at 4°C for 10 minutes to precipitate the phage. Centrifuge at 12,000 × g for 10 minutes at 4°C, and discard the supernatant. Resuspend the phage in 1 mL of PBS and centrifuge at 12,000 × g for 5 minutes at 4°C to remove insoluble impurities. This will yield the phage product from the first round of panning.

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

[0092] 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.

[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. After mixing, the cells were placed in a 37°C water bath for 10 minutes, and then spread on LB plates 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 cells were 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 8After 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.

[0095] While amplifying the phage monoclonals, coat the GPC3 antigen protein onto an ELISA plate (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. Discard the blocking buffer, wash four times with 200 μL of 0.05% PBST per well, then add 50 μL of 0.1% PBST, and then add 50 μL of the monoclonal phage supernatant to each well. Incubate at 4°C for 1 hour, then 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 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] Nanobody sequences were amplified by PCR and cloned into the eukaryotic expression vector pcDNA3.4, where they were expressed at the C-terminus fused to the human IgG1 Fc fragment. The resulting expression plasmids were transiently transfected into HEK293 cells, and the harvested cell culture supernatant contained each recombinant Nanobody. A GPC3-specific antibody, HYP7, was expressed in the same manner 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 50293F 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 3 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 23°C for 2 h, then the solution was changed once and then dialyzed at 5°C for 14 h. Finally, the protein content was determined and the sample was filtered through a 0.22 μm sterile filter membrane, aliquoted, and stored at -80°C until use.

[0107] 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.

[0108] The results are shown in Table 1. The EC of positive control antibody HYP7 against CHO-S-GPC3 50 The value was 0.09428 μg / mL, and the EC value of 194-G3-1-D02 on CHO-S-GPC3 was 50 The value is 0.5663μg / mL, which has a 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 three times with PBS. 100μL of 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 three times with PBS. The cells were resuspended in 500μL of PBS and analyzed by flow cytometry. For the negative control group, only 100μL of 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 three times with PBS. The cells were resuspended in 500μL of 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 embodiment is as follows Figure 3 shown.

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

[0121] 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).

[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 costimulatory 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 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 BVHGC3-012 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 transduced with lentivirus at an MOI of 5 TU / mL. 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 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 / 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.

[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). FITC-labeled anti-VHH antibodies were 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 BVHGC3-030 cells was detected by flow cytometry. The results are shown in Figure 2. Figure 4 The expression rates of BVHGC3-012 CAR were 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, 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. 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 2. 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 into 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 effect-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, a target cell group only and a mock group (T cells not transduced with lentivirus) were set as controls. After the co-culture, the changes in the mCherry fluorescence signals of the target cells in each group were calculated using the IncuCyte SX5 software. The lower the signal value, the fewer cells in the group and the better the killing effect of CAR-T cells.

[0151] The results are as follows Figure 6As shown, under the condition of an effector-target ratio of 1:2, the killing effect of the BN108 group (relative fluorescence area at the last time point was 1.68±0.012) on GPC3-positive Huh7 cells was significantly improved compared with the mock group (relative fluorescence area at the last time point was 2.28±0.004), and the killing effect of the BVHGC3-012 group (relative fluorescence area at the last time point was 1.36±0.010) was significantly better than that of the BN108 group (P<0.05). These results show that compared with existing antibodies, CAR-T cells constructed based on the 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 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 a 96-well plate at a density of 1:1 / well and cultured overnight. NK cells were added at a 2:1 effector-to-target ratio, along with the target antibody 194-G3-1-D02 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 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 7 As shown in the results, compared with the NK cell group without antibody addition, 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 embodiment of the present invention, but the scope of protection 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 fall within the scope of protection and disclosure 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 is shown in SEQ ID NO.1, the amino acid sequence of the complementary determining region CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the complementary determining region CDR3 is 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 is shown in SEQ ID NO.

4.

3. A biomaterial, characterized in that The biological material is 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 containing the nucleic acid molecule described in (1); (3) A host cell expressing the anti-GPC3 protein nanobody according to claim 1 or 2.

4. Use of the anti-GPC3 protein nanobody according to claim 1 or 2 or the biomaterial according to claim 3 in at least one of the following: (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 liver cancer; (4) Use in the preparation of drugs for preventing, delaying or treating liver cancer.

5. The use according to claim 4, characterized in that The product for detecting GPC3 protein and the product for diagnosing liver cancer 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 immunohistochemistry assay reagent.

6. A detection kit for GPC3 protein, 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 pharmaceutically acceptable excipients; 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 costimulatory domain and an intracellular signal transduction domain; The antigen binding domain is composed of 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 are T cells and / or NK cells.

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