Anti-gpc3 fully humanized single-domain antibody and application thereof

Abstract

The application relates to the technical field of biological medicine, and provides an anti-GPC3 full humanization single-domain antibody and application thereof. The application uses phage display technology to obtain one anti-human GPC3 specificity full humanization monoclonal single-domain antibody through antibody screening by targeting human GPC3 protein. The obtained single-domain antibody can be specifically combined with human GPC3, and can effectively inhibit the nuclear translocation of YAP, provides an effective alternative antibody drug for GPC3-targeted HCC treatment, and has a potential clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a fully humanized single-domain antibody against Glypican-3 and its applications. Background Technology

[0002] Liver cancer is one of the leading causes of cancer-related deaths worldwide, ranking among the highest mortality rates. Hepatocellular carcinoma (HCC) is the most common pathological type of primary liver cancer, and its incidence is on the rise globally, particularly in Asia where the disease burden is significant. HCC patients have a poor prognosis and low 5-year survival rate; therefore, developing highly effective therapeutic strategies targeting specific molecular targets is of significant clinical importance.

[0003] Glypican-3 (GPC3) is a member of the heparan sulfate proteoglycan (HSPG) family, anchored to the cell surface via glycosylphosphatidylinositol (GPI). Under physiological conditions, GPC3 expression is confined to the liver and placental tissues during embryonic development, and its expression is significantly downregulated in adulthood. However, GPC3 is often abnormally overexpressed in hepatocellular carcinoma (HCC), and its expression level is closely related to tumor malignancy and poor patient prognosis, making it widely recognized as an HCC-specific biomarker and a potential therapeutic target. Studies have shown that GPC3 can promote HCC cell proliferation, inhibit apoptosis, and enhance invasion and metastasis by interacting with multiple signaling pathways (such as Wnt and Hippo-YAP). Among these signaling pathways, YAP (Yes-associated protein) is a transcriptional coactivator and a key downstream effector of the Hippo signaling pathway. Currently, various targeted therapies targeting GPC3 have been developed, including monoclonal antibodies, antibody-drug conjugates (ADCs), and chimeric antigen receptor T-cell (CAR-T) therapy, some of which have entered clinical trials and shown potential therapeutic value. However, existing strategies mainly utilize GPC3 membrane surface expression for targeted delivery or immune recognition, rather than directly intervening in its mediated endogenous oncogenic signaling, which may be an important reason for the limited response of some current treatments. Studies have reported that anti-GPC3 monoclonal antibodies can significantly inhibit the activity of the Wnt / β-catenin signaling pathway in hepatocellular carcinoma cell lines and suppress tumor growth. However, no specific blocking antibodies against GPC3 and YAP signaling have been reported.

[0004] Single-domain antibodies (sdAbs), also known as heavy-chain single-domain antibodies (VHHs), are proteins composed of the variable domain of the antibody heavy chain. They are small antibody fragments with a molecular weight of approximately 12-15 kDa, consisting of only a single heavy-chain variable domain (VH). Despite their simplified structure, sdAbs exhibit antigen-binding affinity comparable to traditional antibodies, while combining the advantages of small molecule drugs and monoclonal antibodies, exhibiting higher solubility, thermal stability, and specificity. Due to their small molecular size and unique structural features, sdAbs show significant advantages in recognizing occult or spatially restricted antigenic epitopes. These properties have led to widespread interest in sdAbs in basic research, diagnostic reagents, and therapeutic formulation development. According to recent research, more than 30 compounds based on sdAbs are currently in clinical development, and the ImmunoPET probe, an sdAb derivative targeting GPC3, has been successfully developed, demonstrating excellent diagnostic accuracy in preclinical models of hepatocellular carcinoma (HCC). In summary, single-domain antibodies, due to their advantages such as small molecular weight, strong penetration, low immunogenicity, and ease of engineering modification, have become an important direction for the development of novel diagnostic and therapeutic drugs, and have broad clinical application value and commercialization potential. Summary of the Invention

[0005] This invention provides a fully humanized single-domain antibody against Glypican-3, which can specifically bind to human GPC3 and effectively inhibit the nuclear translocation of YAP, providing an effective alternative antibody drug for the treatment of HCC targeting GPC3.

[0006] In a first aspect, the present invention provides an anti-GPC3 single-domain antibody comprising complementarity-determining regions CDR1, CDR2, and CDR3, as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0007] Specifically as follows:

[0008] SEQ ID NO: 1: DYEMS

[0009] SEQ ID NO: 2: RINSDGTVVNYADSVKG

[0010] SEQ ID NO: 3: LTGGWWDLY

[0011] As an alternative, the complete amino acid sequence of the anti-GPC3 single-domain antibody is shown in SEQ ID NO: 4.

[0012] The single-domain antibody of the present invention can be expressed in prokaryotic or eukaryotic expression systems, such as Escherichia coli, yeast, insect cells or mammalian cells, through genetic engineering technology to obtain biologically active single-domain antibody proteins.

[0013] In a second aspect, the present invention provides a nucleic acid encoding the above-described anti-GPC3 single-domain antibody.

[0014] Alternatively, the nucleic acid may contain the complete DNA nucleotide sequence of an anti-GPC3 single-domain antibody, as shown in SEQ ID NO: 5.

[0015] A third aspect of the present invention provides an expression vector comprising the aforementioned nucleic acid. In this invention, the expression vector may be a plasmid, bacteriophage, granule, or viral vector, etc.

[0016] In a fourth aspect, the present invention provides a host cell comprising the aforementioned nucleic acid or expression vector. The host cell may be a transgenic cell line or recombinant bacteria, and may also include eukaryotic or prokaryotic cells, such as Escherichia coli, yeast, or animal cells and cell lines.

[0017] A fifth aspect of the present invention provides an immunoconjugate comprising an antibody portion and a coupling portion coupled to said antibody portion.

[0018] The antibody portion comprises the anti-GPC3 single-domain antibody described above in this invention.

[0019] The conjugation portion is selected from drugs, especially small molecule drugs, thereby forming antibody-drug conjugates.

[0020] The coupling portion can also be selected from fluorescent markers, chemiluminescent markers, chromogenic markers, or enzymes. This allows for the development of detection products for GPC3, such as detection reagents, kits, and chips. The detection method includes: binding an anti-GPC3 antibody to the GPC3 antigen in the sample to form an antibody-antigen or antibody fragment-antigen complex; subsequently, detecting this sample complex to determine the presence of the target antigen in the sample. During the detection process, the presence or quantity of the target antigen is indicated by the aforementioned markers.

[0021] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the anti-GPC3 single-domain antibody described above.

[0022] The pharmaceutical compositions of the present invention may also include a pharmaceutically acceptable carrier.

[0023] The pharmaceutical compositions of the present invention may also include other drugs or preparations capable of killing tumor cells.

[0024] The pharmaceutical compositions of the present invention may further include immune cells. These immune cells include T cells, NK cells, macrophages, or dendritic cells (DCs), etc.

[0025] Alternatively, the immune cells may express a chimeric antigen receptor on their surface, and the antigen recognition domain of the chimeric antigen receptor may contain the sequence of the anti-GPC3 single-domain antibody. Therefore, the anti-GPC3 single-domain antibody of the present invention can also be applied to chimeric antigen receptor T-cell therapy, etc.

[0026] The anti-GPC3 single-domain antibody M2C5 of this invention is a fully humanized single-domain antibody that specifically binds to human GPC3. It exhibits high specificity and affinity, and effectively inhibits the nuclear translocation of YAP. Besides its application in naked antibody therapy, it can also be used in antibody-drug conjugates and derived chimeric antigen receptor T-cell therapy (CAR-T cell therapy) as a treatment for hepatocellular carcinoma, providing an effective alternative antibody drug for the treatment of GPC3-targeted HCC, and has potential clinical application prospects. Attached Figure Description

[0027] Figure 1 To detect the binding of enriched bacteriophages to antigen proteins using ELISA.

[0028] Figure 2 The image shows the pFUSE-M2C5-hFc plasmid.

[0029] Figure 3 The purified M2C5-hFc protein was detected by SDS-PAGE.

[0030] Figure 4 This assay is used to detect the specific binding of the M2C5 antibody to the GPC3 protein using an ELISA.

[0031] Figure 5 This study aims to detect the specific binding of the M2C5 antibody to GPC3-overexpressing cell lines using fine-grained chromatograms (FACS).

[0032] Figure 6 For the detection of M2C5 antibody binding to HCC cell lines (FACS).

[0033] Figure 7 Affinity analysis of M2C5 antibody.

[0034] Figure 8 Functional analysis of the M2C5 antibody. Detailed Implementation

[0035] Example 1: Screening of fully humanized antibody M2C5 targeting human GPC3 using phage display technology

[0036] 1.1 Cell lines and antigens

[0037] A431 (human epithelial carcinoma 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 incubator at 37°C. Lipofectamine was used to... TM GPC3 cDNA was transfected into A431 cells at Invitrogen (Carlsbad, CA) in 2000, and the GPC3 overexpressing cell line A431-GPC3 was obtained by Zeocin selection. The GPC3-his fusion protein (Q25-S550) was purchased from ACRO Biosystems.

[0038] 1.2 Phage Display

[0039] Construct a 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 Screening was performed using pFu / ml. 50 μg / ml purified GPC3-his protein was used to coat the ELISA plate overnight at 4°C; the plate was blocked for 1 hour at room temperature with PBST-milk solution containing 5% skim milk powder and 0.1% Tween-20; a single-domain antibody phage library was mixed with 10% PBS-milk at a 1:1 ratio and incubated at room temperature for 1 hour, then added to the blocked ELISA plate (100 μl / well) and incubated for 1 hour at room temperature; the plate was washed 20 times with PBST solution; elution was performed for 30 minutes at room temperature in the dark with 100 μl of 100 mM Triethylamine elution buffer; the eluted phages were used to infect logarithmic growth phase TG1 cells, amplified, and recovered for the next round of screening. After three rounds of screening, ELISA analysis showed that the enrichment of positive phages significantly increased the affinity of the enriched phage population for the GPC3 antigen. Figure 1 BSA was used as a negative control.

[0040] 1.3 Monoclonal phage ELISA

[0041] TG1 cells were infected with a sorted and enriched phage library, and 188 single clones were randomly selected, amplified, and recovered. Coat the microplate with 5 μg / ml GPC3-his protein and BSA overnight at 4°C; block the microplate with 3% PBST-milk solution at room temperature for 1 hour; incubate 188 amplified monoclonal phages at a 1:1 ratio with 6% PBS-milk at room temperature for 1 hour, then add 50 μl / well to the microplates coated with GPC3-his protein and the negative control his protein, respectively, and incubate at room temperature for 1 hour; wash the microplate 5 times with PBST solution; mix HRP / Anti-M13 Monoclonal conjugate with 3% PBS-milk solution at a 1:4000 ratio, add 50 μl / well to the washed microplates, and incubate at room temperature for 1 hour; wash the microplate 5 times with PBST solution; add TMB chromogenic solution to the microplate (100 μl / well), and after 3 minutes of color development at room temperature, add 0.5... Color development was stopped with sulfuric acid (100 μl / well); absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the binding ability of monoclonal phage to GPC3 protein was analyzed.

[0042] By detecting monoclonal phages with high antigen affinity using ELISA, monoclonal phages were obtained. Further analysis of the sequences of the antigen-positive monoclonal phages was conducted, and the enriched monoclonal sequences were named M2C5, in the form of a single-domain antibody.

[0043] Its DNA nucleotide sequence (SEQ ID NO: 5) is as follows:

[0044] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGATTTCGATTTCTATGATTATGAAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGATCTCACGTATCAACTCTGATGGGACTGTTGTCAAC TACGCGGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCACGTATTACTGTGTGGGTCTTACCGGTGGCTGGTGGGACTTATACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA

[0045] Its protein amino acid sequence (SEQ ID NO: 4) is as follows:

[0046] QVQLVQSGGGLVQPGGSLRLSCAASDFDFY DYEMS WVRQAPGKGLEWIS RINSDGTVVNYADSVKG RFTISRDNSKNTLYLQMNTLRAEDTATYYCVG LTGGWWDLY WGQGTLVTVSS

[0047] The CDR regions of the antibody are the underlined portions of the aforementioned amino acid sequences, as follows:

[0048] CDR1: amino acid sequence positions 31-35 (SEQ ID NO: 1); CDR2: amino acid sequence positions 50-66 (SEQ ID NO: 2); CDR3: amino acid sequence positions 99-107 (SEQ ID NO: 3).

[0049] Example 2: Expression and purification of M2C5 antibody

[0050] 2.1 Molecular Cloning

[0051] Forward primers (primer-F) and reverse primers (primer-R) were designed to amplify the DNA fragment from a single phage clone using PCR. The DNA was then treated with restriction endonucleases to produce sticky ends. The pFUSE-hFc plasmid vector backbone and the DNA fragment were ligated overnight at 16°C using T4 ligase. The ligation product was transformed into DH5α competent cells, cultured, and single clones were picked and incubated overnight at 37°C and 250 rpm. The plasmid was extracted and sequenced for verification. The M2C5-hFc protein expression plasmid using pFUSE as a vector was constructed. Figure 2 Human Fc tags are beneficial for antibody purification using Protein A adsorption columns.

[0052] 2.2 M2C5-VHH protein expression and purification

[0053] Inoculate 5 × 10⁶ cells into cell culture dishes using DMEM complete medium (supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 0.1 mg / ml streptomycin). 6HEK293T cells were cultured in a 5% CO2 incubator at 37°C. When the cell density reached 60-80%, 10 μg of pFUSE-M2C5-VHH plasmid was added to 0.5 ml of DMEM medium, and 30 μg of PEI was added to 0.5 ml of DMEM medium. The mixture was allowed to stand for 5 minutes. Then, a mixture of PEI and DMEM was added to the plasmid and DMEM mixture, and the mixture was allowed to stand for 20 minutes. During this period, the DMEM medium was replaced for HEK293T cells. The plasmid and PEI mixture in DMEM medium was added to HEK293T cell culture dishes. The medium was replaced 24 hours after transfection, and the supernatant was collected every 24 hours and replaced with DMEM medium for further culture. The collected supernatant was filtered through a 0.45 μm microporous membrane to remove debris. The supernatant was then purified by separating and purifying the M2C5-VHH recombinant protein using a Protein A-Agarose (GE Healthcare, Piscataway, NJ) affinity column.

[0054] 2.3 SDS-PAGE

[0055] The concentration of M2C5-VHH recombinant protein was determined by BCA protein quantification. 5 μg of M2C5-VHH protein was subjected to polyacrylamide gel electrophoresis to obtain bands of M2C5-VHH recombinant protein under denaturing and non-denaturing conditions, thus identifying the purity of the M2C5-VHH recombinant protein. Figure 3 The results showed that a single, clear main band was visible at the expected molecular weight (approximately 70 kDa), with sharp edges and no obvious tailing or extraneous protein bands. Under denaturing-reducing conditions, the protein completely dissociated into monomers; under denaturing-non-reducing conditions, no high-molecular-weight polymer bands appeared, indicating that the M2C5-VHH recombinant protein had high purity and met the requirements for subsequent experiments.

[0056] Example 3: ELISA detection of the specificity and affinity of M2C5 binding to antigen protein

[0057] 3.1 Binding Specificity

[0058] Coat the microplate with 5 μg / ml GPC3-his(Q25-S550) protein and BSA overnight at 4°C; block the microplate with 3% PBST-milk solution at room temperature for 1 hour; dilute the M2C5-VHH recombinant protein to 5 μg / ml with 3% PBS-milk solution and add it to the blocked microplate (50 μl / well), incubate at room temperature for 1 hour; wash the microplate three 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 and added to the washed ELISA plate (50 μl / well). The plate was incubated at room temperature for 1 hour. The plate was washed three times with PBST solution. TMB chromogenic buffer was added to the plate (100 μl / well), and after incubation at room temperature for 3 minutes, 0.5 M sulfuric acid (100 μl / well) was added to stop the incubation. The absorbance was measured at 450 nm using an ELISA reader, and the specificity of the M2C5-VHH recombinant protein for GPC3 protein was analyzed. The results showed that the M2C5 antibody did not specifically recognize the negative control protein (BSA), but it could specifically recognize GPC3. This indicates that the M2C5 antibody possesses GPC3 recognition specificity at the protein level. Figure 4 ).

[0059] 3.2 Combining Affinity

[0060] Coat the microplate with 5 μg / ml GPC3-his protein overnight at 4°C; block the microplate with 3% PBST-milk solution at room temperature for 1 hour; dilute M2C5-VHH protein 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, and 0.0002 μg / ml (3-fold dilution), add to the blocked microplate (50 μl / well), and incubate at room temperature for 1 hour; wash the microplate three times with PBST solution (340 μl / well); add Goat anti-Human Fc γ HRP was mixed with 3% PBS-milk solution at a ratio of 1:2000 and added to the washed ELISA plate (50 μl / well), and incubated at room temperature for 1 hour. The ELISA plate was washed three times with PBST solution. TMB chromogenic solution was added to the ELISA plate (100 μl / well), and after incubation at room temperature for 3 minutes, 0.5M sulfuric acid (100 μl / well) was added to stop the incubation. The absorbance was measured at 450 nm using an ELISA reader, and an affinity curve was fitted to analyze the affinity of the M2C5-VHH recombinant protein. The results showed that the affinity of the M2C5 antibody for GPC3 protein was 13.84 nM (…). Figure 7 (A in the middle).

[0061] Example 4: FACS detection of M2C5-VHH cell binding specificity and affinity

[0062] 4.1 Binding Specificity

[0063] Culture GPC3-overexpressing cell lines (A431-GPC3, A431 as a negative control) or HCC cell lines (Hep3B, Huh-7) (0.5 Mil cells / sample). Centrifuge at 2000 rpm, 4°C for 5 minutes, discard the supernatant, resuspend in PBS, centrifuge again, discard the supernatant, resuspend the cells in PBS containing 1.5 μg / ml purified M2C5-VHH recombinant protein and 5% BSA, and incubate on ice for 1 hour. Centrifuge at 2000 rpm, 4°C for 5 minutes, discard the supernatant, resuspend in PBS, centrifuge again, discard the supernatant, mix Goatanti-Human IgG Fc Secondary Antibody, PE (invitrogen) at a ratio of 1:200 with PBS containing 5% BSA and resuspend the cells. Incubate on ice for 1 hour, centrifuge at 2000 rpm, 4°C for 5 minutes, discard the supernatant, resuspend in PBS, centrifuge again, discard the supernatant, and resuspend in 0.3 ml of PBS. Cells were resuspended in PBS, and PE fluorescent labeling signals were detected by flow cytometry to analyze the specificity of the M2C5-VHH recombinant protein for GPC3-positive cells. Flow cytometry was used to detect the recognition of the M2C5 antibody on the GPC3-overexpressing cell line (A431-GPC3). The results showed that the M2C5 antibody could specifically recognize GPC3 on the surface of A431-GPC3 cells, but did not have specific recognition activity on A431 cells. This indicates that the M2C5 antibody has GPC3 recognition specificity at the cellular level. Figure 5 ).

[0064] Flow cytometry was used to detect the recognition of GPC3-positive HCC cell lines (Hep3B and Huh-7) by M2C5 antibody. The results showed that M2C5 antibody could also recognize GPC3 expressed on the cell membrane surface of Hep3B and Huh-7 cells, indicating that M2C5 antibody also has a recognition function for HCC cell lines expressing GPC3. Figure 6 ).

[0065] 4.2 Combining Affinity

[0066] GPC3 overexpressing cell lines (A431-GPC3, A431 as a negative control) or HCC cell lines (Hep3B, Huh-7) (0.5 Mil cells / sample) were cultured, centrifuged at 2000 rpm, 4°C for 5 minutes, and the supernatant was discarded. The cells were resuspended in PBS, centrifuged again, and the supernatant was discarded. The cells were resuspended in PBS containing 40, 13.3333, 4.4444, 1.4815, 0.4938, 0.1646, 0.0549, 0.0183, and 0.0061 μg / ml purified M2C5-VHH recombinant protein and 5% BSA, and incubated on ice for 1 hour. After centrifugation at 2000 rpm, 4°C for 5 minutes, the supernatant was discarded, and the cells were resuspended in PBS, centrifuged again, and the supernatant was discarded. Goat anti-Human IgGFc Secondary Antibody, PE (invitrogen), was diluted 1:200 with PBS containing 5% BSA. The cells were resuspended in PBS after mixing with BSA and incubated on ice for 1 hour. Then, the cells were centrifuged at 2000 rpm for 5 minutes at 4°C, the supernatant was discarded, and the cells were resuspended in PBS. After another centrifugation and discarding the supernatant, the cells were resuspended in 0.3 ml of PBS. The PE fluorescent labeling signal was detected by flow cytometry. An affinity curve for the M2C5-VHH recombinant protein was fitted, and the affinity was calculated. The results showed that the affinity of the M2C5 antibody for A431-GPC3 cells was 23.31 nM (…). Figure 7 (B in the middle).

[0067] Example 5: Functional analysis of M2C5 antibody

[0068] 5.1 Western blot analysis to detect the effect of M2C5 on phosphorylated YAP

[0069] Hep3B cells were seeded in 6-well plates (0.6 Mil / well). After 36 hours, when the cell density reached 80%, 100 μl of M2C5-VHH / hIgG antibody (11 mg / mL) was added to each well for pretreatment for 24 hours. Cells were washed three times with PBS, and 100 μl of RIPA lysis buffer was added to each well. After lysis, the cells were transferred to 1.5 ml EP tubes, centrifuged at 10000 g for 10 minutes, and 80 μl of supernatant lysis buffer was collected. 20 μl of loading buffer was added to each tube, and the cells were incubated at 100°C for 5 minutes. After cooling to room temperature, SDS-PAGE gel chromatography was performed to detect the effect of M2C5 on phosphorylated YAP and YAP protein. The results showed that M2C5-VHH antibody could increase YAP phosphorylation (…). Figure 8 (A in the middle).

[0070] 5.2 Nucleocytoplasmic separation experiment to detect the effect of M2C5 on YAP

[0071] Hep3B cells were seeded in 6-well plates (0.6 Mil / well). After 36 h, when the cell density reached 80%, 100 μl of M2C5-VHH / h IgG antibody (11 mg / mL) was added to each well for pretreatment for 24 h. Cells were washed three times with pre-cooled PBS, centrifuged at 500g for 10 min, resuspended in 40 μl of CE1, centrifuged at 1000g for 10 min, and 40 μl of supernatant was collected to obtain cytoplasmic proteins. The cell pellet was resuspended in 40 μl of CE2, centrifuged at 6000g for 10 min, and the supernatant was discarded. Nuclease was added and 40 μl of CE3 was added to resuspend the pellet, centrifuged at 6800g for 10 min, and 40 μl of supernatant was collected to obtain nuclear proteins. 10 μl of loading buffer was added to each tube, and the cells were incubated at 100℃ for 5 min. After cooling to room temperature, SDS-PAGE gel electrophoresis was performed to detect the expression of YAP in the cytoplasm and nucleus. The results showed that M2C5-VHH antibody could inhibit the nuclear translocation of YAP in cells. Figure 8 (B in the middle).

[0072] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the present invention, and all such modifications and improvements fall within the scope of the present invention.

Claims

1. An anti-GPC3 fully humanized single-domain antibody, characterized in that, complementarity determining regions CDR1, CDR2 and CDR3 comprising the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively. 2.The anti-GPC3 fully humanized single-domain antibody of claim 1, characterized in that, The amino acid sequence of the anti-GPC3 fully humanized single-domain antibody is set forth in SEQ ID NO:

4.

3. A nucleic acid encoding the anti-GPC3 fully humanized single-domain antibody of claim 1 or 2.

4. The nucleic acid of claim 3, wherein, comprises a nucleotide sequence set forth in SEQ ID NO:

5.

5. An expression vector comprising the nucleic acid of claim 3 or 4.

6. A host cell comprising the nucleic acid of claim 3 or 4 or the expression vector of claim 5.

7. An immunoconjugate comprising an antibody moiety selected from the anti-GPC3 fully humanized single-domain antibody of claim 1 or 2 and a conjugating moiety coupled to the antibody moiety, the conjugating moiety being selected from a fluorescent label, a chemiluminescent label or a chromogenic label.

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