A fully humanized single-domain antibody against Glypican-3 and its application

Abstract

The present invention relates to a fully humanized single-domain antibody against Glypican-3 and its application; the antibody comprises 3 heavy-chain complementarity-determining regions (CDRs), wherein the amino acid sequence of CDR1 is the amino acid sequence at positions 31-35 of SEQ ID NO:1; the amino acid sequence of CDR2 is the amino acid sequence at positions 50-66 of SEQ ID NO:1; and the amino acid sequence of CDR3 is the amino acid sequence at positions 99-107 of SEQ ID NO:1. The present invention utilizes phage display technology to screen for antibodies by targeting human GPC3 protein, and obtains 1 specific fully human monoclonal single-domain antibody against human GPC3; the antibody can specifically bind to human GPC3 and can effectively inhibit the activation of Wnt signal, providing an effective alternative antibody drug for the treatment of HCC targeting GPC3, and having potential clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a fully humanized single-domain antibody against Glypican-3 and its application. Background Art

[0002] In primary liver cancer, hepatocellular carcinoma (HCC) is the most common pathological type, accounting for about 75%-85% (Xu W, Jian D, Yang H, et al. Aggregation-induced emission: Application in diagnosis and therapy of hepatocellular carcinoma[J]. Biosensors and Bioelectronics, 2024, 266). Globally, the incidence of HCC shows an increasing trend year by year, especially in Asian regions (Wang Y, Deng B. Hepatocellular carcinoma: molecular mechanism, targeted therapy, and biomarkers[J]. Cancer Metastasis Rev, 2023, 42(3): 629-52), and the overall survival rate of HCC patients (especially advanced patients) is relatively low, which also makes it particularly important to develop efficient HCC treatment regimens targeting specific targets.

[0003] Glypican-3 (GPC3) is one of the members of the heparan sulfate proteoglycan (HSPG) family and is anchored to the cell surface via a glycosylphosphatidylinositol (GPI) anchor. To date, multiple studies have shown that GPC3 mainly plays a role in regulating signal transduction on the cell membrane surface (Filmus J, Capurro M, Rast J. Glypicans[J]. Genome Biol, 2008, 9(5):224), can act as a Wnt co-receptor to activate the canonical Wnt / β-catenin signaling pathway and regulate cell behaviors such as promoting cell proliferation and so on. Under physiological conditions, GPC3 is highly expressed in the liver and intestine during embryonic development and its expression is turned off after complete individual development (Filmus J. Glypicans in growth control and cancer[J]. Glycobiology, 2001, 11(3):19R-23R). In patients with HCC, most of them have specific high expression of GPC3, which is highly correlated with poor prognosis (An S, Zhang D, Zhang Y, et al. GPC3-targeted immunoPET imaging of hepatocellular carcinomas[J]. Eur J Nucl Med Mol Imaging, 2022, 49(8):2682-92). Therefore, GPC3 has become a diagnostic marker and an important therapeutic target for HCC (Zhou F, Shang W, Yu X, Tian J. Glypican-3: A promising biomarker for hepatocellular carcinoma diagnosis and treatment[J]. Med Res Rev, 2018, 38(2):741-67). Antibodies targeting GPC3, in addition to being used in naked antibody therapy, antibody-drug conjugates and the derived chimeric antigen receptor T cell therapy (i.e., CAR-T cell therapy) can all be used as means to treat hepatocellular carcinoma and have direct clinical translation potential.Considerable progress has been made in current GPC3-targeted immunotherapies, such as immunotoxins, bispecific antibodies, and CAR-T cell therapies (Fu Y, Urban D J, Nani RR, et al. Glypican-3-Specific Antibody Drug Conjugates Targeting Hepatocellular Carcinoma[J]. Hepatology, 2019, 70(2):563-76).

[0004] Single-domain antibodies (sdAbs) have an extremely small molecular weight of only 12-15 kDa and contain only a single variable heavy chain region (V H) It has an antigen-binding affinity similar to that of traditional antibodies, but combines the advantages of small molecules and monoclonal antibodies, with higher solubility, stability, and specificity (Mohseni A, Molakarimi M, Taghdir M, et al. Exploring single-domain antibody thermostability by molecular dynamics simulation[J]. J Biomol Struct Dyn, 2019, 37(14):3686-96; Tang H, Gao Y, Han J. Application Progress of the Single Domain Antibody in Medicine[J]. Int J Mol Sci, 2023, 24(4)), and has unique advantages in recognizing antigen fragments. These characteristics enable single-domain antibodies to be widely used in basic research, diagnosis, and treatment. Currently, more than 30 single-domain antibody compounds are in clinical development (Kunz S, Durandy M, Seguin L, Feral C C. NANOBODY((R)) Molecule, a Giga Medical Tool in Nanodimensions[J]. Int J Mol Sci, 2023, 24(17)), and a GPC3-targeted ImmunoPET imaging strategy derived from sdAb has also been successfully developed, showing excellent diagnostic accuracy in preclinical HCC models (An S, Zhang D, Zhang Y, et al. GPC3-targeted immunoPET imaging of hepatocellular carcinomas[J]. Eur J Nucl Med Mol Imaging, 2022, 49(8):2682-92). Summary of the Invention

[0005] Aiming at the deficiencies of existing problems, the purpose of the present invention is to provide a fully humanized single-domain antibody against Glypican-3 and its application. The present invention uses phage display technology to screen antibodies by targeting human GPC3 protein, and obtains 1 specific fully human monoclonal single-domain antibody against human GPC3. The obtained single-domain antibody can specifically bind to human GPC3 and effectively inhibit the activation of Wnt signal, providing an effective alternative antibody drug for HCC treatment targeting GPC3, and having potential clinical application prospects.

[0006] The technical solution adopted by the present invention to solve its technical problems is:

[0007] In a first aspect, the present invention protects a fully humanized single-domain antibody against Glypican-3, which comprises 3 heavy-chain complementarity-determining regions (CDRs). Among them,

[0008] The amino acid sequence of CDR1 is the amino acid sequence at positions 31-35 of SEQ ID NO:1;

[0009] The amino acid sequence of CDR2 is the amino acid sequence at positions 50-66 of SEQ ID NO:1;

[0010] The amino acid sequence of CDR3 is the amino acid sequence at positions 99-107 of SEQ ID NO:1.

[0011] In a specific embodiment, the single-domain antibody further comprises a framework region.

[0012] In a more specific embodiment, the framework region FR1 of the single-domain antibody is the amino acid sequence at positions 1-30 of SEQ ID NO:1; the framework region FR2 of the single-domain antibody is the amino acid sequence at positions 36-49 of SEQ ID NO:1; the framework region FR3 of the single-domain antibody is the amino acid sequence at positions 67-98 of SEQ ID NO:1; the framework region FR4 of the single-domain antibody is the amino acid sequence at positions 108-118 of SEQ ID NO:1.

[0013] In a specific embodiment, the antibody is selected from any one of the following (A1)-(A3):

[0014] (A1) having the amino acid sequence shown in SEQ ID NO:1;

[0015] (A2) a sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more identity with the amino acid sequence shown in SEQ ID NO:1;

[0016] (A3) an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the amino acid sequence shown in SEQ ID NO:1.

[0017] In a second aspect, the present invention also protects a nucleic acid molecule of the fully humanized single-domain antibody against Glypican-3 described above, and its sequence is as shown in SEQ ID NO:2.

[0018] In a third aspect, the present invention also protects an expression cassette containing the nucleic acid molecule described above.

[0019] In a fourth aspect, the present invention also protects a recombinant vector containing the nucleic acid molecule described above or a recombinant vector containing the expression cassette shown above.

[0020] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, phages (such as λ phage or M13 filamentous phage, etc.), cosmids (i.e., cosmid plasmids), artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), P1 artificial chromosomes (PAC) or Ti plasmid artificial chromosomes (TAC), etc.), viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses or herpesviruses (such as herpes simplex virus), etc.).

[0021] In a more specific embodiment, the recombinant vector is pFUSE.

[0022] In a fifth aspect, the present invention protects a recombinant microorganism containing the nucleic acid molecule described above, or a recombinant microorganism containing the expression cassette described above, or a recombinant microorganism containing the recombinant vector described above.

[0023] In a sixth aspect, the present invention protects a pharmaceutical composition comprising a therapeutically or diagnostically effective amount of a fully humanized single-domain antibody against Glypican-3, and a pharmaceutically acceptable carrier.

[0024] In a specific embodiment, the pharmaceutically acceptable carrier may be a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption promoter, adsorption carrier, surfactant or lubricant.

[0025] In a specific embodiment, the pharmaceutical composition further comprises a therapeutically or diagnostically effective amount of at least one agent selected from therapeutic agents, diagnostic agents and theranostic agents.

[0026] In a seventh aspect, the present invention protects the use of the fully humanized single-domain antibody against Glypican-3 described in any one of the above in the preparation of a drug for treating hepatocellular carcinoma.

[0027] In a specific embodiment, the treatment includes: administering to the subject a pharmaceutical composition comprising an effective amount of the fully humanized single-domain antibody against Glypican-3 described in any one of the above and an acceptable carrier.

[0028] Beneficial effects

[0029] The present invention utilizes phage display technology to screen for antibodies by targeting the human GPC3 protein, and obtains 1 fully human monoclonal single-domain antibody specific to human GPC3. The obtained single-domain antibody can specifically bind to human GPC3 and can effectively inhibit the activation of the Wnt signal, providing an effective alternative antibody drug for the treatment of HCC targeting GPC3, and having potential clinical application prospects. Brief description of the drawings

[0030] Figure 1 . ELISA detection of the binding of enriched phages to antigen proteins;

[0031] Figure 2 . Flow sorting of cells with strong binding of phages to cell surface antigens;

[0032] Figure 3 . ELISA detection of the binding of enriched phages to antigen proteins after sorting;

[0033] Figure 4 . Plasmid map of pFUSE-M2B6-hFc;

[0034] Figure 5 . SDS-PAGE detection of purified M2B6-hFc protein;

[0035] Figure 6 . Specific binding detection of M2B6 antibody to GPC3 protein (ELISA);

[0036] Figure 7 . Specific binding detection of M2B6 antibody to GPC3 overexpressing cell line (FACS);

[0037] Figure 8 . Binding detection of M2B6 antibody to HCC cell line (FACS);

[0038] Figure 9 . Affinity analysis of M2B6 antibody;

[0039] Figure 10 . Analysis of the effect of M2B6 antibody on the Wnt signaling pathway. Detailed implementation manners

[0040] The following further describes the present invention in detail with reference to embodiments. Reagents or instrument and equipment not indicating the manufacturer are regarded as conventional products that can be purchased on the market.

[0041] Cell lines and antigens

[0042] A431 (human epithelial cancer cell line) was purchased from ATCC (American Type Culture Collection). It was cultured in DMEM complete medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 0.1 mg / ml streptomycin in a 5% CO2, 37 °C incubator. Using Lipofectamine TMThe GPC3 cDNA was transfected into A431 cells by 2000 (Invitrogen, Carlsbad, CA), and the GPC3-overexpressing cell line A431-GPC3 was obtained by screening with Zeocin. The GPC3-his fusion protein (Q25-S550) was purchased from ACRO Biosystems.

[0043] Example 1 Screening and Preparation of a Fully Humanized Single-Domain Antibody Against Glypican-3

[0044] 1. Phage Display

[0045] A single-domain antibody phage library was constructed. The purified human GPC3-his protein (Q25-S550) was used as the antigen for screening in the single-domain antibody phage library (phage library size * 10 12 pFu / ml). The ELISA plate was coated with 50 μg / ml of the purified GPC3-his protein at 4 °C overnight; the ELISA plate was blocked with PBST-milk solution containing 5% skim milk powder and 0.1% Tween-20 at room temperature for 1 hour; the single-domain antibody phage library was mixed with 10% PBS-milk at a ratio of 1:1 and incubated at room temperature for 1 hour, then added to the blocked ELISA plate (100 μl / well) and incubated at room temperature for 1 hour; the ELISA plate was washed 20 times with PBST solution; 100 μl of 100 mM Triethylamine elution buffer was used to elute the phage at room temperature in the dark for 30 minutes; the eluted phage was used to infect TG1 cells in the logarithmic growth phase, and after amplification and recovery, it was used for the next round of screening. After three rounds of screening, ELISA was used to analyze the enrichment of positive phage.

[0046] The results are as Figure 1 shown, the affinity of the enriched phage population for the GPC3 antigen increased ( Figure 1 ), while BSA as a negative control did not bind to the phage population.

[0047] 2. Phage FACS Sorting

[0048] Take 2 million Hep3B-GPC3 KO-ΔHS (Hep3B, a hepatocellular carcinoma cell line, overexpresses GPC3 lacking heparan sulfate chain (HS chain) after knocking out GPC3 using CRISPR-Cas9 technology, i.e., Hep3B-GPC3 KO-ΔHS) cells and filter them through a 70-μm cell strainer. Block the cells with 3% PBS-milk solution at room temperature for 1 h. Block the phage library obtained from the third round of screening with 6% PBS-milk solution at a ratio of 1:1 at room temperature for 1 h. Mix the phages with the cell suspension at a ratio of 1:1 and incubate at room temperature for 1 h. After washing with PBS, add Anti-M13 Antibody (PE), Mouse Monoclonal (SinoBiological) at a ratio of 5 μl / test and incubate on ice for 1 hour. After washing with PBS, resuspend the cells for sorting. Sort 20% of the strongly positive cells that bind to the phages of the single-domain antibody library. Elute the phages from the sorted cells with 100 mM Triethylamine elution buffer, infect TG1, and amplify and recover to obtain the sorted phage library ( Figure 2 ).

[0049] 3. Phage ELISA

[0050] 3.1 Polyclonal phage ELISA

[0051] Coat the ELISA plate overnight at 4 °C with 5 μg / ml GPC3-his protein and BSA; block the ELISA plate with 5% PBST-milk solution at room temperature for 1 h; amplify and recover the phages enriched in each round, incubate with 10% PBS-milk solution at a ratio of 1:1 at room temperature for 1 h, and add them to the blocked ELISA plate (100 μl / well), and incubate at room temperature for 1 h; wash the ELISA plate 5 times with PBST solution; mix HRP / Anti-M13 Monoclonal conjugate with 3% PBS-milk solution at a ratio of 1:4000, add it to the washed ELISA plate (50 μl / well), and incubate at room temperature for 1 h; wash the ELISA plate 3 times with PBST solution; add TMB chromogenic solution to the ELISA plate (100 μl / well), develop color at room temperature for 3 minutes, and then add 0.5 M sulfuric acid to stop the color development (100 μl / well); detect the absorbance at a wavelength of 450 nm with an enzyme-linked immunosorbent detector and analyze the affinity of the phages after each round of amplification.

[0052] The results are as Figure 3 shown. The affinity of the phage population enriched after sorting for the GPC3 antigen increased, while BSA, used as a negative control, did not bind to the phage population.

[0053] 3.2 Monoclonal phage ELISA

[0054] Infect TG1 cells with the enriched phage library after sorting, randomly pick 188 monoclonal colonies from them, amplify and recover the phages. Coat an ELISA plate overnight at 4°C with 5 μg / ml GPC3-his protein and BSA; block the ELISA plate with 3% PBST-milk solution at room temperature for 1 hour; incubate the 188 amplified monoclonal phages with 6% PBS-milk at a ratio of 1:1 at room temperature for 1 hour, and add them respectively to the ELISA plates coated with GPC3-his protein and negative control his protein and blocked well (50 μl / well), and incubate at room temperature for 1 hour; wash the ELISA plate 5 times with PBST solution; mix HRP / Anti-M13 Monoclonal conjugate with 3% PBS-milk solution at a ratio of 1:4000, add it to the washed ELISA plate (50 μl / well), and incubate at room temperature for 1 hour; wash the ELISA plate 5 times with PBST solution; add TMB chromogenic solution to the ELISA plate (100 μl / well), after chromogenic reaction at room temperature for 3 minutes, add 0.5 M sulfuric acid to stop chromogenic reaction (100 μl / well); detect the absorbance value with an ELISA reader at a wavelength of 450 nm and analyze the binding ability of monoclonal phages to GPC3 protein.

[0055] 4. Preparation of M2B6-VHH protein

[0056] 4.1 Molecular cloning

[0057] Design forward primer primer-F and reverse primer primer-R to amplify the DNA fragment in the phage monoclonal by PCR, and treat the DNA with restriction enzymes to make it have sticky ends; ligate the pFUSE-hFc plasmid vector backbone and the DNA fragment overnight at 16°C with T4 ligase; transform the ligation product into DH5α competent cells, culture and pick monoclonal colonies, shake the bacteria overnight at 37°C and 250 rpm in a shaker; extract the plasmid and perform sequencing verification. Name the monoclonal sequence M2B6, in the form of single-domain antibody ( Figure 4 ). The single-domain antibody is composed of framework region FR1, complementary determining region CDR1, framework region FR2, complementary determining region CDR2, framework region FR3, complementary determining region CDR3 and framework region FR4 in sequence. The specific information is as follows: Its amino acid sequence is shown in SEQ ID No.1. The 1st to 30th positions of SEQ ID No.1 are framework region FR1, the 31st to 35th positions are complementary determining region CDR1, the 36th to 49th positions are framework region FR2, the 50th to 66th positions are complementary determining region CDR2, the 67th to 98th positions are framework region FR3, the 99th to 107th positions are complementary determining region CDR3, and the 108th to 118th positions are framework region FR4. The nucleic acid molecule encoding the single-domain antibody is shown in SEQ ID NO:2.

[0058] 4.2 Protein expression and purification

[0059] Seed 5 million HEK293T cells in a cell culture dish with DMEM complete medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 0.1 mg / ml streptomycin, and place it in a 5% CO2, 37°C incubator for culture. When the cell density reaches 60 - 80%, add 10 μg of pFUSE-M2B6-VHH plasmid to 0.5 ml of DMEM medium, add 30 μg of PEI to 0.5 ml of DMEM medium, and let it stand for 5 minutes; add the PEI and DMEM mixture to the plasmid and DMEM mixture, and let it stand for 20 minutes. During the standing period, replace the DMEM complete medium for HEK293T cells. Add the mixed DMEM medium of plasmid and PEI to the HEK293T cell culture dish; replace the medium 24 h after transfection, and collect the supernatant every 24 hours, and replace it with DMEM complete medium to continue the culture. Filter the collected supernatant through a 0.45 μm microporous filter membrane to remove debris; purify the M2B6-VHH recombinant protein by separating it through a ProteinA-Agarose (GE Healthcare, Piscataway, NJ) affinity column.

[0060] 4.3 SDS-PAGE

[0061] Determine the concentration of M2B6-VHH recombinant protein by the BCA protein quantification method, and perform polyacrylamide gel electrophoresis on 5 μg of M2B6-VHH protein to obtain the bands of M2B6-VHH recombinant protein in a denatured reducing and denatured non-reducing environment, and identify the purity of M2B6-VHH recombinant protein ( Figure 5 )

[0062] 5. ELISA to detect the binding specificity and affinity of M2B6 to antigen protein

[0063] 5.1 Binding specificity

[0064] Use 5 μg / ml GPC3-his (Q25 - S550) protein and BSA to coat the ELISA plate overnight at 4°C; block the ELISA plate with 3% PBST-milk solution at room temperature for 1 hour; dilute the M2B6-VHH recombinant protein to 5 μg / ml with 3% PBS-milk solution, add it to the blocked ELISA plate (50 μl / well), and incubate at room temperature for 1 hour; wash the ELISA plate 3 times with PBST solution (340 μl / well); add Goat anti-Human Fc γHRP (Jackson ImmunoResearch) was mixed with 3% PBS-milk solution at a ratio of 1:2000, added to the washed enzyme-linked immunosorbent assay (ELISA) plate (50 μl / well), and incubated at room temperature for 1 hour; the ELISA plate was washed 3 times with PBST solution; TMB chromogenic solution was added to the ELISA plate (100 μl / well), and after chromogenic reaction at room temperature for 3 minutes, 0.5 M sulfuric acid was added to stop the chromogenic reaction (100 μl / well); the absorbance was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay detector, and the specificity of the M2B6-VHH recombinant protein to GPC3 protein was analyzed.

[0065] The results are as Figure 6 shown. The M2B6 antibody did not specifically recognize the negative control protein (BSA), but specifically recognized GPC3, indicating that the M2B6 antibody has recognition specificity for GPC3 at the protein level.

[0066] 5.2 Binding affinity

[0067] The ELISA plate was coated with 5 μg / ml GPC3-his protein at 4 °C overnight; the ELISA plate was blocked with 3% PBST-milk solution at room temperature for 1 hour; the M2B6-VHH protein was diluted with 3% PBS-milk solution to 40, 13.3333, 4.4444, 1.4814, 0.4938, 0.1646, 0.0549, 0.0183, 0.0061, 0.0020, 0.0007, 0.0002 μg / ml (3-fold dilution) respectively, added to the blocked ELISA plate (50 μl / well), and incubated at room temperature for 1 hour; the ELISA plate was washed 3 times with PBST solution (340 μl / well); Goat anti-Human Fc γ HRP was mixed with 3% PBS-milk solution at a ratio of 1:2000, added to the washed enzyme-linked immunosorbent assay (ELISA) plate (50 μl / well), and incubated at room temperature for 1 hour; the ELISA plate was washed 3 times with PBST solution; TMB chromogenic solution was added to the ELISA plate (100 μl / well), and after chromogenic reaction at room temperature for 3 minutes, 0.5 M sulfuric acid was added to stop the chromogenic reaction (100 μl / well); the absorbance was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay detector, and the affinity curve was fitted to analyze the affinity of the M2B6-VHH recombinant protein.

[0068] The results showed that the affinity of the M2B6 antibody to GPC3 protein was 5.06 nM ( Figure 9 ).

[0069] 6. FACS detection of the cell-binding specificity and affinity of M2B6-VHH

[0070] 6.1 Binding specificity

[0071] Culture GPC3 overexpressing cell lines (A431-GPC3, with A431 as the negative control) or HCC cell lines (Hep3B, Huh-7) (0.5 Mil cells / sample). After centrifugation at 2000 rpm for 5 minutes at 4°C, discard the supernatant, resuspend with PBS, centrifuge again and discard the supernatant. Resuspend the cells with PBS solution containing 1.5 μg / ml purified M2B6-VHH recombinant protein and 5% BSA, and incubate on ice for 1 hour. After centrifugation at 2000 rpm for 5 minutes at 4°C, discard the supernatant, and resuspend with PBS. Centrifuge again and discard the supernatant. Mix Goat anti-Human IgG Fc Secondary Antibody, PE (invitrogen) with PBS solution containing 5% BSA at a ratio of 1:200 and resuspend the cells. After incubating on ice for 1 hour, centrifuge at 2000 rpm for 5 minutes at 4°C, discard the supernatant, resuspend with PBS, centrifuge again, discard the supernatant, resuspend the cells with 0.3 ml PBS, and detect the PE fluorescence labeling signal with a flow cytometer to analyze the specificity of M2B6-VHH recombinant protein for GPC3-positive cells.

[0072] The results are as Figure 7 shown. The M2B6 antibody can specifically recognize GPC3 on the cell membrane surface of A431-GPC3, but does not specifically recognize A431 cells, indicating that the M2B6 antibody has recognition specificity for GPC3 at the cellular level. The M2B6 antibody can also recognize GPC3 expressed on the cell membrane surface of Hep3B cells and Huh-7 cells, indicating that the M2B6 antibody also has recognition effects on HCC cell lines expressing GPC3 ( Figure 8 ).

[0073] 6.2 Binding Affinity

[0074] Culture GPC3 overexpressing cell lines (A431-GPC3, with A431 as the negative control) or HCC cell lines (Hep3B, Huh-7) (0.5 Mil cells / sample). After centrifugation at 2000 rpm for 5 minutes at 4°C, discard the supernatant, resuspend with PBS, centrifuge again and discard the supernatant. Resuspend the cells with PBS solution containing purified M2B6-VHH recombinant protein at concentrations of 40, 13.3333, 4.4444, 1.4815, 0.4938, 0.1646, 0.0549, 0.0183, 0.0061 μg / ml and 5% BSA, and incubate on ice for 1 hour. After centrifugation at 2000 rpm for 5 minutes at 4°C, discard the supernatant, resuspend with PBS, centrifuge again and discard the supernatant. Mix Goat anti-Human IgG Fc Secondary Antibody, PE (invitrogen) with PBS solution containing 5% BSA at a ratio of 1:200 and resuspend the cells. After incubating on ice for 1 hour, centrifuge at 2000 rpm for 5 minutes at 4°C, discard the supernatant, resuspend with PBS, centrifuge again, discard the supernatant, resuspend the cells with 0.3 ml PBS, and detect the PE fluorescence labeling signal using a flow cytometer. Fit the affinity curve of M2B6-VHH recombinant protein and calculate the affinity.

[0075] The results showed that the affinity of M2B6 antibody for A431-GPC3 cells was 14 nM ( Figure 9 ).

[0076] 7. Detect the effect of M2B6 on the Wnt signaling pathway by Luciferase reporter assay

[0077] Seed HEK293-Topflash cells into 48-well plates (0.075 Mil / well). After 36 h, when the cell density reaches 60 - 80%, use Lipofectamine TMTransfect 250 ng of pcDNA3.1-GPC3 plasmid and 10 ng of PTK-Renilla-luc plasmid per well. After 24 hours, replace the serum-free DMEM medium to starve the cells for 12 hours, and add 100 μl of M2B6-VHH / hIgG antibody (0.69 μM) to each well for pretreatment for 1 hour; after incubation, add 100 μl of Wnt-3a / L cell supernatant to the Wnt stimulation group and the control group respectively. After stimulation for 12 h, wash the cells once with PBS, add 50 μl of PLB lysis buffer to each well, and place them in an -80 °C refrigerator for overnight lysis; after lysis is completed, thaw on ice, transfer to a 1.5 ml EP tube, centrifuge at 10000 g for 1 minute, and take 20 μl of the supernatant from each tube for detection. RLU is the value of Luciferase / Renilla in each group. The results show that the M2B6-VHH antibody can inhibit the activation of the Wnt signaling pathway in cells( Figure 10 ).

[0078] SEQ ID NO:1

[0079] QVQLVQSGGGLVQPGGSLRLSCAASDFAFSYYEMSWVRQAPGKGLEWVASINQDGSEKYYVDSVKGRFTISR

[0080] DNSKNTLYLQMNTLRAEDTATYYCASSTVTLLFDYWGQGTLVTVSS

[0081] SEQ ID NO:2

[0082] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGAC

[0083] TCTCCTGTGCAGCCTCTGATTTCGCTTTCTCTTATTATGAAATGAGCTGGGTCCGCCAGG

[0084] CTCCAGGGAAGGGTCTAGAGTGGGTGGCCAGCATAAACCAAGATGGAAGTGAGAAATA

[0085] CTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGC

[0086] TGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCACATATTACTGTGCGAGT

[0087] TCTACAGTAACTCTCCTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0088] The protection scope of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the appended claims are taken as the protection scope.

Claims

1. A fully humanized single-domain antibody against Glypican-3, comprising three heavy chain complementary determining regions (CDRs), wherein: The amino acid sequence of CDR1 is the amino acid sequence at positions 31 to 35 of SEQ ID NO: 1; The amino acid sequence of CDR2 is the amino acid sequence at positions 50 to 66 of SEQ ID NO: 1; The amino acid sequence of CDR3 is the amino acid sequence at positions 99 to 107 of SEQ ID NO:

1.

2. The fully humanized single-domain antibody against Glypican-3 according to claim 1, characterized in that: The antibody is selected from any one of the following (A1)-(A3): (A1) has the amino acid sequence shown in SEQ ID NO: 1; (A2) a sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more identity to the amino acid sequence of SEQ ID NO: 1; (A3) an amino acid sequence having one or more conservative amino acid mutations compared to the amino acid sequence shown in SEQ ID NO:

1.

3. The nucleic acid molecule of the fully humanized single-domain antibody against Glypican-3 according to claim 1 or 2, whose sequence is shown in SEQ ID NO:

2.

4. An expression cassette containing the nucleic acid molecule according to claim 3.

5. A recombinant vector containing the nucleic acid molecule according to claim 3.

6. A recombinant vector containing the expression cassette of claim 4.

7. A recombinant microorganism containing the nucleic acid molecule according to claim 3.

8. A recombinant microorganism containing the expression cassette of claim 4.

9. A recombinant microorganism containing the recombinant vector according to claim 5.

10. A pharmaceutical composition comprising a therapeutically or diagnostically effective amount of the fully humanized single-domain antibody against Glypican-3 according to claim 1 or 2, and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition according to claim 10, characterized in that The pharmaceutical composition further comprises a therapeutically or diagnostically effective amount of at least one agent selected from the group consisting of a therapeutic agent, a diagnostic agent, and a theranostic agent.

12. Use of the fully humanized single-domain antibody against Glypican-3 according to claim 1 or 2 in the preparation of a drug for treating hepatocellular carcinoma.

13. The use according to claim 12, characterized in that: The treatment comprises: administering to the subject a pharmaceutical composition comprising an effective amount of the fully humanized single-domain antibody against Glypican-3 according to any one of claims 1 or 2 and an acceptable carrier.

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