Anti-Glypican-3 fully humanized single-domain antibody and application thereof

Abstract

The invention relates to a fully humanized single-domain antibody for resisting Glypican-3 and an application of the fully humanized single-domain antibody. The antibody comprises three heavy chain complementary determining regions (CDRs), and the amino acid sequence of the CDRs is the 31st-35th amino acid sequence of SEQ ID NO: 1; the amino acid sequence of the CDR2 is the 50th-66th amino acid sequence of SEQ ID NO: 1; the amino acid sequence of the CDR3 is the 99-107th amino acid sequence of SEQ ID NO: 1. According to the invention, a bacteriophage display technology is utilized, antibody screening is carried out through targeting human GPC3 protein, and one anti-human GPC3 specific fully-humanized monoclonal single-domain antibody is obtained; the antibody can be specifically combined with human GPC3, can effectively inhibit activation of a Wnt signal, provides an effective alternative antibody drug for HCC treatment with GPC3 as a target, and has a potential clinical application prospect.

Description

Technical Field

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

[0002] Among primary liver cancers, 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 has been increasing year by year, especially in Asia (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 low, which makes it particularly important to develop targeted and efficient HCC treatment options.

[0003] Glypican-3 (GPC3) is a member of the heparan sulfate proteoglycan (HSPG) family and is anchored on the cell surface through glycosylphosphatidylinositol (GPI). To date, many studies have shown that GPC3 plays a major role in regulating signal transduction on the cell membrane surface (Filmus J, Capurro M, Rast J. Glypicans [J]. Genome Biol, 2008, 9 (5): 224), and can act as a Wnt co-receptor to activate the canonical Wnt / β-catenin signaling pathway and regulate cell behavior, such as promoting cell proliferation. Under physiological conditions, GPC3 is highly expressed in the liver and intestine during the embryonic period, and its expression is turned off after the individual development is complete (Filmus J. Glypicans in growth control and cancer [J]. Glycobiology, 2001, 11 (3): 19R-23R). In HCC patients, 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), so GPC3 has become a diagnostic marker and 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). In addition to naked antibody therapy, antibodies targeting GPC3 can also be used as antibody-drug conjugates and derived chimeric antigen receptor T cell therapy (i.e., CAR-T cell therapy) as a means of treating hepatocellular carcinoma, with direct clinical translation potential.At present, targeted immunotherapy against GPC3 has made great progress, such as immunotoxins, bispecific antibodies and CAR-T cell therapy (Fu Y, Urban DJ, 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 a very small molecular weight of only 12-15 kDa and contain only a single heavy chain variable region (V H), has similar antigen binding affinity to traditional antibodies, but combines the advantages of small molecules and monoclonal antibodies, has 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. The above characteristics make single-domain antibodies widely used in basic research, diagnosis and treatment. Currently, more than 30 single-domain antibody compounds are under clinical development (Kunz S, Durandy M, Seguin L, Feral CC. NANOBODY ((R)) Molecule, a Giga Medical Tool in Nanodimensions [J]. Int J Mol Sci, 2023, 24 (17)), and studies have also successfully developed sdAb-derived GPC3-targeted ImmunoPET imaging strategies, which have shown 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] In view of the shortcomings of the 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 a fully human monoclonal single-domain antibody specific for human GPC3. The obtained single-domain antibody can specifically bind to human GPC3 and can effectively inhibit the activation of Wnt signaling, providing an effective alternative antibody drug for the treatment of HCC targeting GPC3, and has potential clinical application prospects.

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

[0007] In a first aspect, the present invention protects a fully humanized single domain antibody against Glypican-3, which comprises three heavy chain complementary determining regions CDR, wherein:

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

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

[0010] The amino acid sequence of CDR3 is the amino acid sequence at positions 99 to 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; and 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) has 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 to the amino acid sequence of 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 to the amino acid sequence shown in SEQ ID NO:1.

[0017] In a second aspect, the present invention also protects the nucleic acid molecule of the fully humanized single-domain antibody against Glypican-3 described above, the sequence of which is 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, including but not limited to: plasmids, bacteriophages (such as lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), 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 herpes viruses (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 can be a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant or a lubricant.

[0025] In specific embodiments, 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.

[0026] In a seventh aspect, the present invention protects the use of any of the above-mentioned fully humanized single-domain antibodies against Glypican-3 for preparing a drug for treating hepatocellular carcinoma.

[0027] In a specific embodiment, the treatment comprises: administering to the subject a pharmaceutical composition comprising an effective amount of any of the foregoing fully humanized single-domain antibodies against Glypican-3 and an acceptable carrier.

[0028] Beneficial Effects

[0029] The present invention uses phage display technology to screen antibodies by targeting human GPC3 protein, and obtains a fully human monoclonal single-domain antibody specific to human GPC3. The obtained single-domain antibody can specifically bind to human GPC3 and effectively inhibit the activation of Wnt signaling, providing an effective alternative antibody drug for the treatment of HCC targeting GPC3, and has potential clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[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 lines (FACS);

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

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

[0039] Fig.10 .Analysis of the effect of M2B6 antibody on Wnt signaling pathway. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below with reference to the examples. The reagents or instruments used without 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). The cells were cultured in a 5% CO2, 37°C incubator using DMEM complete medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 0.1 mg / ml streptomycin. TMGPC3 cDNA was transfected into A431 cells using Invitrogen 2000 (Invitrogen, Carlsbad, CA), and the GPC3 overexpressing cell line A431-GPC3 was obtained by Zeocin screening. GPC3-his fusion protein (Q25-S550) was purchased from ACRO Biosystems.

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

[0044] 1. Phage Display

[0045] Construction of single domain antibody phage library. Purified human GPC3-his protein (Q25-S550) was used as the screening antigen in the single domain antibody phage library (phage library size * 10 12 pFu / ml). 50μg / ml purified GPC3-his protein was used to coat the ELISA plate overnight at 4℃; 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 1:1 and incubated at room temperature for 1 hour, added to the blocked ELISA plate (100μl / well), and incubated at room temperature for 1 hour; the ELISA plate was washed 20 times with PBST solution; 100μl, 100mM Triethylamine elution buffer was used for elution at room temperature in the dark for 30 minutes; the eluted phages were infected with TG1 cells in the logarithmic growth phase, and amplified and recovered for the next round of screening. After three rounds of screening, the enrichment of positive phages was analyzed by ELISA.

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

[0047] 2. Phage FACS Sorting

[0048] Take 2Mil of Hep3B-GPC3 KO-ΔHS (liver cancer cell line Hep3B, GPC3 knocked out using CRISPR-Cas9 technology and then overexpressed GPC3 with heparan sulfate chain (HS chain) deletion, i.e. Hep3B-GPC3 KO-ΔHS) cells and filter through a 70μm cell filter. Block the cells with 3% PBS-milk solution at room temperature for 1h. Block the phage library obtained from the third round of screening with 6% PBS-milk solution at room temperature for 1h in a 1:1 ratio. Mix the phage and cell suspension in a 1:1 ratio and incubate at room temperature for 1h. After washing with PBS, add Anti-M13 Antibody (PE) and Mouse Monoclonal (SinoBiological) at a ratio of 5μl / test and incubate on ice for 1 hour. After washing with PBS, resuspend the cells for sorting. Sorting 20% ​​of strongly positive cells that bind to the single domain antibody library phage. The sorted cells were eluted with 100 mM Triethylamine elution buffer to remove phages, and then infected with TG1 and amplified to obtain the sorted phage library ( Figure 2 ).

[0049] 3. Phage ELISA

[0050] 3.1 Polyclonal phage ELISA

[0051] 5 μg / ml GPC3-his protein and BSA were used to coat the ELISA plate overnight at 4°C; the ELISA plate was blocked with 5% PBST-milk solution at room temperature for 1 hour; each round of enriched phage was amplified and recovered, incubated with 10% PBS-milk solution at room temperature for 1 hour at a ratio of 1:1, added to the blocked ELISA plate (100 μl / well), and incubated at room temperature for 1 hour; the ELISA plate was washed 5 times with PBST solution; HRP / Anti-M13 Monoclonal conjugate was mixed with 3% The PBS-milk solution was mixed, added to the washed ELISA plate (50 μl / well), and incubated at room temperature for 1 hour; the ELISA plate was washed 3 times with PBST solution; TMB color development solution was added to the ELISA plate (100 μl / well), and after color development at room temperature for 3 minutes, 0.5 M sulfuric acid was added to stop the color development (100 μl / well); the absorbance value was detected at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) and the affinity of the phage after each round of amplification was analyzed.

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

[0053] 3.2 Monoclonal phage ELISA

[0054] The TG1 cells were infected with the enriched phage library after sorting, and 188 monoclones were randomly selected from them, amplified and recovered phages. 5μg / ml GPC3-his protein and BSA were used to coat the ELISA plate at 4℃ overnight; the ELISA plate was blocked with 3% PBST-milk solution at room temperature for 1 hour; the 188 amplified monoclonal phages were incubated with 6% PBS-milk at room temperature for 1 hour at a ratio of 1:1, and added to the ELISA plate coated with GPC3-his protein and negative control his protein and blocked (50μl / well), and incubated at room temperature for 1 hour; the ELISA plate was washed 5 times with PBST solution; HRP / Anti-M13 Monoclonal conjugate was mixed with 3% The PBS-milk solution was mixed, added to the washed ELISA plate (50 μl / well), and incubated at room temperature for 1 hour; the ELISA plate was washed 5 times with PBST solution; TMB color development solution was added to the ELISA plate (100 μl / well), and after color development at room temperature for 3 minutes, 0.5 M sulfuric acid was added to stop the color development (100 μl / well); the absorbance value was detected at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) and the binding ability of the monoclonal phage to the GPC3 protein was analyzed.

[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 clone by PCR, and treat the DNA with restriction endonucleases to make it present sticky ends; connect the pFUSE-hFc plasmid vector backbone and DNA fragment at 16°C overnight with T4 ligase; transform the ligation product into DH5α competent medium, culture and pick the monoclonal clone, shake the bacteria at 37°C, 250rpm overnight; extract the plasmid and sequence it. The monoclonal sequence was named M2B6, single domain antibody format ( Figure 4 ). The single domain antibody is composed of a framework region FR1, a complementary determining region CDR1, a framework region FR2, a complementary determining region CDR2, a framework region FR3, a complementary determining region CDR3 and a framework region FR4 in sequence, and the specific information is as follows: its amino acid sequence is shown in SEQ ID No.1. Positions 1-30 of SEQ ID No.1 are framework region FR1, positions 31-35 are complementary determining region CDR1, positions 36-49 are framework region FR2, positions 50-66 are complementary determining region CDR2, positions 67-98 are framework region FR3, positions 99-107 are complementary determining region CDR3, and positions 108-118 are framework region FR4. The nucleic acid molecule encoding the single domain antibody is shown in SEQID NO:2.

[0058] 4.2 Protein expression and purification

[0059] 5Mil HEK293T cells were seeded in a cell culture dish with DMEM complete medium supplemented with 10% fetal bovine serum, 100U / ml penicillin, and 0.1mg / ml streptomycin, and cultured in a 5% CO2, 37°C incubator. When the cell density reached 60-80%, 10μg pFUSE-M2B6-VHH plasmid was added to 0.5ml DMEM medium, and 30μg PEI was added to 0.5ml DMEM medium, and the mixture was allowed to stand for 5 minutes; the PEI and DMEM mixture was added to the plasmid and DMEM mixture, and the mixture was allowed to stand for 20 minutes. During the standing period, the DMEM complete medium was replaced for HEK293T cells. The mixed DMEM medium of plasmid and PEI was added to the HEK293T cell culture dish; the medium was replaced 24h after transfection, and the supernatant was recovered every 24 hours, and the DMEM complete medium was replaced to continue the culture. The recovered supernatant was filtered through a 0.45 μm microporous filter membrane to remove debris; the supernatant was separated and purified by a Protein A-Agarose (GE Healthcare, Piscataway, NJ) affinity column to obtain the M2B6-VHH recombinant protein.

[0060] 4.3SDS-PAGE

[0061] The concentration of M2B6-VHH recombinant protein was determined by BCA protein quantification method, and 5 μg of M2B6-VHH protein was subjected to polyacrylamide gel electrophoresis to obtain the bands of M2B6-VHH recombinant protein under denaturing reduction and denaturing non-reducing environment, and the purity of M2B6-VHH recombinant protein was identified ( Figure 5 ).

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

[0063] 5.1 Binding specificity

[0064] 5 μg / ml GPC3-his (Q25-S550) protein and BSA were used to coat the ELISA plate at 4°C overnight; the ELISA plate was blocked with 3% PBST-milk solution at room temperature for 1 hour; the M2B6-VHH recombinant protein was diluted to 5 μg / ml with 3% PBS-milk solution, 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 γHRP (Jackson ImmunoResearch) was mixed with 3% PBS-milk solution at a ratio of 1:2000, added to the washed ELISA plate (50 μl / well), and incubated at room temperature for 1 hour; the ELISA plate was washed 3 times with PBST solution; TMB color development solution was added to the ELISA plate (100 μl / well), and after color development at room temperature for 3 minutes, 0.5 M sulfuric acid was added to stop the color development (100 μl / well); the absorbance value was detected at a wavelength of 450 nm using an enzyme-linked immunosorbent assay and the specificity of the M2B6-VHH recombinant protein to the GPC3 protein was analyzed.

[0065] The results are as follows Figure 6 As shown, the M2B6 antibody has no specific recognition effect on the negative control protein (BSA), but specifically recognizes GPC3, which indicates that the M2B6 antibody has recognition specificity for GPC3 at the protein level.

[0066] 5.2 Binding affinity

[0067] 5 μg / ml GPC3-his protein was used to coat the ELISA plate at 4°C overnight; the ELISA plate was blocked with 3% PBST-milk solution at room temperature for 1 hour; 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), 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 ELISA plate (50 μl / well), and incubated at room temperature for 1 hour; the ELISA plate was washed 3 times with PBST solution; TMB color development solution was added to the ELISA plate (100 μl / well), and after color development at room temperature for 3 minutes, 0.5 M sulfuric acid was added to stop color development (100 μl / well); the absorbance value was detected at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) and the affinity of the M2B6-VHH recombinant protein was analyzed by fitting the affinity curve.

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

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

[0070] 6.1 Binding Specificity

[0071] GPC3 overexpressing cell lines (A431-GPC3, A431 as negative control) or HCC cell lines (Hep3B, Huh-7) (0.5Mil cells / sample) were cultured, centrifuged at 2000rpm, 4℃ for 5 minutes, the supernatant was discarded, the cells were resuspended with PBS, centrifuged again and the supernatant was discarded, and the cells were resuspended with PBS solution containing 1.5μg / ml purified M2B6-VHH recombinant protein and 5% BSA, and incubated on ice for 1 hour; centrifuged at 2000rpm, 4℃ for 5 minutes, the supernatant was discarded, and the cells were resuspended with PBS, centrifuged again and the supernatant was discarded, Goatanti-Human IgG Fc Secondary Antibody, PE (invitrogen) was mixed with PBS solution containing 5% BSA at a ratio of 1:200 and the cells were resuspended, incubated on ice for 1 hour, centrifuged at 2000rpm, 4℃ for 5 minutes, the supernatant was discarded, and the cells were resuspended with PBS, centrifuged again, the supernatant was discarded, and 0.3ml The cells were resuspended in PBS, and the PE fluorescent labeling signal was detected by flow cytometry to analyze the specificity of M2B6-VHH recombinant protein to GPC3-positive cells.

[0072] The results are as follows Figure 7 As shown in the figure, the M2B6 antibody can specifically recognize GPC3 on the surface of A431-GPC3 cells, but has no specific recognition effect on A431 cells, which indicates that the M2B6 antibody has specific recognition of GPC3 at the cellular level. The M2B6 antibody can also recognize GPC3 expressed on the surface of Hep3B cells and Huh-7 cells, which indicates that the M2B6 antibody also has recognition effect on HCC cell lines expressing GPC3 ( Figure 8 ).

[0073] 6.2 Binding affinity

[0074] GPC3 overexpressing cell lines (A431-GPC3, A431 as negative control) or HCC cell lines (Hep3B, Huh-7) (0.5Mil cells / sample) were cultured, centrifuged at 2000rpm, 4°C for 5 minutes, the supernatant was discarded, resuspended with PBS, centrifuged again and the supernatant was discarded, and the cells were resuspended with PBS solution containing 40, 13.3333, 4.4444, 1.4815, 0.4938, 0.1646, 0.0549, 0.0183, 0.0061μg / ml purified M2B6-VHH recombinant protein and 5% BSA, and incubated on ice for 1 hour; centrifuged at 2000rpm, 4°C for 5 minutes, the supernatant was discarded, and the cells were resuspended with PBS, centrifuged again and the supernatant was discarded, and Goat anti-Human IgG FcSecondary Antibody, PE (invitrogen) was mixed with 5% The cells were resuspended in PBS solution of BSA and incubated on ice for 1 hour, then centrifuged at 2000 rpm and 4°C for 5 minutes, the supernatant was discarded, and the cells were resuspended in PBS. The cells were centrifuged again, the supernatant was discarded, and the cells were resuspended in 0.3 ml PBS. The PE fluorescent labeling signal was detected by flow cytometry, and the affinity curve of the M2B6-VHH recombinant protein was fitted and the affinity was calculated.

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

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

[0077] HEK293-Topflash cells were seeded in 48-well plates (0.075Mil / well). After 36 hours, when the cell density reached 60-80%, Lipofectamine TM2000 cells were transfected with 250ng pcDNA3.1-GPC3 plasmid and 10ng PTK-Renilla-luc plasmid per well. After 24 hours, serum-free DMEM medium was replaced and cells were starved for 12 hours. 100μl M2B6-VHH / hIgG antibody (0.69μM) was added to each well for pretreatment for 1 hour. After incubation, 100μl Wnt-3a / L cell supernatant was added to the Wnt stimulation group and the control group, respectively. After 12 hours of stimulation, the cells were washed once with PBS, 50μl PLB lysis buffer was added to each well, and the cells were placed in a -80℃ refrigerator for overnight lysis. After lysis, the cells were thawed on ice, transferred to a 1.5ml EP tube, centrifuged at 10000g for 1 minute, and 20μl supernatant was taken from each tube for detection. RLU is the value of Luciferase / Renilla in each group. The results showed that M2B6-VHH antibody can inhibit the activation of the Wnt signaling pathway in cells ( Fig.10 ).

[0078] SEQ ID NO:1

[0079] QVQLVQSGGGLVQPGGSLRLSCAASDFAFSYEMSWVRQAPGKGLEWVASINQDGSEKYYVDSVKGRFTISR

[0080] DNSKNTLYLQMNTLRAEDTATYYCASSTVTLLFDYWGQGTLVTVSS

[0081] SEQ ID NO:2

[0082] CAGGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGAC

[0083] TCTCCTGTGCAGCCTCTGATTTCGCTTTCTCTTATTATGAAATGAGCTGGGTCCGCCAGG

[0084] CTCCAGGGAAGGGTCTAGAGTGGGTGGCCAGCATAAACCAAGATGGAAGTGAGAAATA

[0085] CTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGC

[0086] TGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCACATATTACTGTGCGAGT

[0087] TCTACAGTAACTCTCCTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0088] The protection content 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 thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

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 of claim 3 or a recombinant vector containing the expression cassette of claim 4.

6. A recombinant microorganism containing the nucleic acid molecule according to claim 3, or a recombinant microorganism containing the expression cassette according to claim 4, or a recombinant microorganism containing the recombinant vector according to claim 5.

7. 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.

8. The pharmaceutical composition according to claim 7, 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.

9. 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.

10. The use according to claim 9, characterized in that: The treatment comprises: administering to the subject a pharmaceutical composition comprising an effective amount of any of the above-mentioned fully humanized single-domain antibodies against Glypican-3 and an acceptable carrier.

Images