Monoclonal antibodies targeting different epitopes of GPC3 and application of monoclonal antibodies in immunotherapy of liver cancer

Abstract

The invention discloses a group of monoclonal antibodies targeting different epitopes of GPC3 and application of the monoclonal antibodies in immunotherapy of liver cancer. A group of high-affinity and high-specificity murine anti-GPC3 monoclonal antibodies are obtained through screening, and key epitopes at the near end of a GPC3 membrane are recognized. The antibody and derivative molecules thereof have excellent specificity and binding strength, can be applied to various scenes of liver cancer detection, molecular diagnosis, targeted therapy and the like, and have remarkable clinical and industrialization values.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a group of monoclonal antibodies targeting different epitopes of GPC3 and their application in the immunotherapy of liver cancer. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common and deadliest malignant tumors worldwide. While current treatments such as surgical resection, local ablation, targeted therapies, and immune checkpoint inhibitors have made some progress, most patients are diagnosed at an advanced stage, resulting in limited efficacy and a high recurrence rate. Identifying molecular targets that specifically recognize HCC cells but are expressed at low levels in normal tissues is a crucial direction for achieving precision medicine. Glycoemulsin-3 (GPC3) is a membrane-bound heparin sulfate proteoglycan that is highly expressed during embryonic development, almost absent in normal adult livers, but significantly upregulated in approximately 70%-80% of HCC tissues. Aberrant GPC3 expression can promote tumor cell growth and immune escape by activating signaling pathways such as Wnt / β-catenin, thus it is considered a specific molecular marker and ideal target for HCC.

[0003] GPC3 possesses a unique molecular structure: its precursor protein is cleaved into N-terminal and C-terminal parts by furinase, linked by disulfide bonds. The C-terminus contains a GPI anchoring signal and is fixed to the cell membrane surface. However, GPC3 may undergo further "unanchoring" or release into a soluble form in vivo, exposing or eliminating some antibody epitopes, leading to unstable antibody recognition targeting the upstream region. In contrast, the C-terminal fragment of GPC3 (segment 511-560) is located proximal to the membrane, representing the most stable region closest to the membrane surface, and typically remains on the cell surface even after protein processing or cleavage. Antibodies targeting this proximal region can maintain their recognition ability in different forms of GPC3, are less affected by protein cleavage or conformational changes, and can more accurately and consistently reflect the true expression of GPC3 on the tumor cell surface.

[0004] Therefore, developing specific antibodies targeting the GPC3 (511-560) segment is of great significance. These antibodies can not only serve as highly specific biomarkers in pathological diagnosis or immunoassay, but also as binding units for drug carriers or molecular imaging probes, significantly improving targeting accuracy and signal stability. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a group of monoclonal antibodies targeting different epitopes of GPC3 and their application in the immunotherapy of liver cancer. This invention obtains a group of high-affinity, high-specificity murine anti-GPC3 monoclonal antibodies through screening, recognizing key epitopes proximal to the GPC3 membrane. These antibodies and their derivative molecules possess excellent specificity and binding strength, and can be applied to various scenarios such as liver cancer detection, molecular diagnosis, and targeted therapy.

[0006] The present invention achieves the above-mentioned objectives by adopting the following technical solution: A first aspect of the present invention provides an antibody or antigen-binding fragment thereof that targets GPC3.

[0007] Furthermore, the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-3 and the light chain variable region comprises LCDR1-3; The HCDR1-3 and LCDR1-3 are selected from any one of the following groups: (1) HS01 antibody: HCDR1-3 in the heavy chain variable region as shown in SEQ ID NO:1, and LCDR1-3 in the light chain variable region as shown in SEQ ID NO:2; (2) HSO2 antibody: HCDR1-3 in the heavy chain variable region as shown in SEQ ID NO:3, and LCDR1-3 in the light chain variable region as shown in SEQ ID NO:4; (3) HSO3 antibody: HCDR1-3 in the heavy chain variable region as shown in SEQ ID NO:5, and LCDR1-3 in the light chain variable region as shown in SEQ ID NO:6.

[0008] Furthermore, the HCDR1-3 and LCDR1-3 are determined using any one or more of the numbering systems Kabat, IMGT, Chothia, AbM, Contact, Martin (enhanced Chothia), and Aho. Optionally, the HCDR1-3 and LCDR1-3 are determined using the Kabat numbering system; Optionally, the HCDR1-3 and LCDR1-3 are selected from any one of the following groups: (1) HS01 antibody: The amino acid sequences are HCDR1-3 as shown in SEQ ID NO:7-9 and LCDR1-3 as shown in SEQ ID NO:10-12 respectively; (2) HSO2 antibody: The amino acid sequences are HCDR1-3 as shown in SEQ ID NO:13-15 and LCDR1-3 as shown in SEQ ID NO:16-18, respectively; (3) HSO3 antibody: HCDR1-3 with amino acid sequences as shown in SEQ ID NO:19-21, and LCDR1-3 with amino acid sequences as shown in SEQ ID NO:22, SEQ ID NO:11, and SEQ ID NO:12.

[0009] In this invention, HCDR1-3 refers to the three CDRs contained in the heavy chain variable region (VH) of the antibody, namely HCDR1 (heavy chain first complementarity-determining region), HCDR2 (heavy chain second complementarity-determining region), and HCDR3 (heavy chain third complementarity-determining region). LCDR1-3 refers to the three CDRs contained in the light chain variable region (VL) of the antibody, namely LCDR1 (light chain first complementarity-determining region), LCDR2 (light chain second complementarity-determining region), and LCDR3 (light chain third complementarity-determining region).

[0010] It should be noted that the HCDR1-3 in the heavy chain variable region and the LCDR1-3 in the light chain variable region of the antibody described in this invention are not limited to the specific amino acid sequences as described above. The sequences corresponding to HCDR1-3 and LCDR1-3 obtained by defining the CDR1-3 in the heavy chain variable region and the light chain variable region of the antibody described in this invention using any CDR numbering scheme will fall within the protection scope of this invention.

[0011] In some implementations, the CDR numbering scheme includes, but is not limited to, any one or any combination of two or more of the following: Chothia numbering scheme, Kabat numbering scheme, IMGT numbering scheme, Contact numbering scheme, AbM numbering scheme, Martin (enhanced Chothia) numbering scheme, and Aho numbering scheme.

[0012] In some implementations, the HCDR1-3 of the HS01 antibody, determined using the IMGT numbering system, are GYTFGDYN, INPNSGTT, and AIWGIYYGNRPYYAMNY, respectively, and the LCDR1-3 are KSVSSSGYSY, LAS, and QHSRELPLT, respectively. The HCDR1-3 of the HS02 antibody, determined using the IMGT numbering system, are AYTFTDYS, INTETGEP, and SSLY, respectively, and the LCDR1-3 are QSLLYSDGKTY, LVS, and CQGTHFPRT, respectively. The HCDR1-3 of the HS03 antibody, determined using the IMGT numbering system, are GYTFTDYN, INPNNGGT, and AIWGIYYGSGPYYAMNY, respectively, and the LCDR1-3 are KSVSTSGYSY, LAS, and QHSRELPLT, respectively.

[0013] In some implementations, the HCDR1-3 of the HS01 antibody, determined using the Chothia numbering system, are GYTFGDY, NPNSGT, and WGIYYGNRPYYAMNY, while the LCDR1-3 are RASKSVSSSGYSYMH, LASNLES, and QHSRELPLT. The HCDR1-3 of the HS02 antibody, determined using the Chothia numbering system, are AYTFTDY, NTETGE, and LY, while the LCDR1-3 are KSSQSLLYSDGKTYLN, LVSKLDS, and CQGTHFPRT. The HCDR1-3 of the HS03 antibody, determined using the Chothia numbering system, are GYTFTDY, NPNNGG, and WGIYYGSGPYYAMNY, while the LCDR1-3 are RASKSVSTSGYSYMY, LASNLES, and QHSRELPLT.

[0014] In some embodiments, antibody sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to the amino acid sequences corresponding to the heavy chain variable regions and light chain variable regions of the antibodies provided by the present invention will fall within the protection scope of the present invention.

[0015] In this invention, homology refers to the degree of similarity between two sequences (amino acid sequences or nucleotide sequences), which is usually calculated through sequence alignment. Specifically, at the level of nucleic acid (DNA / RNA) or protein sequences, homology is analyzed through sequence alignment. When two sequences have a high proportion of identical or similar residues (nucleotides or amino acids), and this similarity cannot be explained by random events, it can be inferred that they are homologous.

[0016] Furthermore, the heavy chain variable region and light chain variable region of the antibody or its antigen-binding fragment are selected from any one of the following: (1) HS01 antibody: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:1 or has at least 80% homology with SEQ ID NO:1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:2 or has at least 80% homology with SEQ ID NO:2; (2) HSO2 antibody: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:3 or has at least 80% homology with SEQ ID NO:3, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:4 or has at least 80% homology with SEQ ID NO:4; (3) HSO3 antibody: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:5 or has at least 80% homology with SEQ ID NO:5, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:6 or has at least 80% homology with SEQ ID NO:6; Optionally, the GPC3 is the proximal end of a human GPC3; Optionally, the proximal end of the human GPC3 is amino acid 511-560 of the C-terminal region of the human GPC3, and its amino acid sequence is shown in SEQ ID NO:36; Optionally, the HS01 antibody and the HS03 antibody can specifically recognize and bind to amino acids 521-540 of the C-terminal region of GPC3, and their amino acid sequences are shown in SEQ ID NO:38. Optionally, the HSO2 antibody can specifically recognize and bind to amino acids 541-560 of the C-terminal region of GPC3, the amino acid sequence of which is shown in SEQ ID NO:40.

[0017] Furthermore, the antibody or its antigen-binding fragment is a humanized variant of the HS01 antibody; The humanized variant of the HS01 antibody comprises any combination of the following heavy chain variable regions and light chain variable regions: The heavy chain variable region is selected from: the heavy chain variable region HS01-hVH1 with the amino acid sequence as shown in SEQ ID NO:27, the heavy chain variable region HS01-hVH2 with the amino acid sequence as shown in SEQ ID NO:28, the heavy chain variable region HS01-hVH3 with the amino acid sequence as shown in SEQ ID NO:29, or the heavy chain variable region HS01-hVH4 with the amino acid sequence as shown in SEQ ID NO:30, or a humanized variant with at least 80% homology; The light chain variable region is selected from: the light chain variable region HS01-hVL1 with an amino acid sequence as shown in SEQ ID NO:23, the light chain variable region HS01-hVL2 with an amino acid sequence as shown in SEQ ID NO:24, the light chain variable region HS01-hVL3 with an amino acid sequence as shown in SEQ ID NO:25, or the light chain variable region HS01-hVL4 with an amino acid sequence as shown in SEQ ID NO:26, or a humanized variant with at least 80% homology; Optionally, the heavy chain variable region and the light chain variable region can be arbitrarily paired to form an antibody or a single-chain variable fragment (scFv). Optionally, the humanized variant of the HS01 antibody includes heavy chain variable regions and light chain variable regions selected from any one of the following groups: (1) HS01-h1: The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:27, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:24; (2) HS01-h2: The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:27, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:25; (3) HS01-h3: heavy chain variable region with amino acid sequence as shown in SEQ ID NO:28, and light chain variable region with amino acid sequence as shown in SEQ ID NO:25; (4) HS01-h4: heavy chain variable region with amino acid sequence as shown in SEQ ID NO:29, and light chain variable region with amino acid sequence as shown in SEQ ID NO:25; (5) HS01-h5: heavy chain variable region with amino acid sequence as shown in SEQ ID NO:29, and light chain variable region with amino acid sequence as shown in SEQ ID NO:26; Optionally, the amino acid sequences of the scFv corresponding to HS01-h1, HS01-h2, HS01-h3, HS01-h4, and HS01-h5 are as shown in SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, respectively, or are humanized variants with at least 80% homology.

[0018] A second aspect of the present invention provides an antigen-binding molecule that targets GPC3.

[0019] Furthermore, the antigen-binding molecule comprises a heavy chain variable region and a light chain variable region of an antibody or its antigen-binding fragment as described above, and the antigen-binding molecule is a full-length antibody and / or its antigen-binding fragment and / or fusion. Optionally, the antigen-binding molecule is full-length IgG, Fab, F(ab′)2, single-domain antibody, scFv, scFv-Fc, bivalent or multivalent antibody, bispecific or multispecific antibody or polymer thereof; Optionally, when the molecule is scFv or a fusion containing scFv, the heavy chain variable region and the light chain variable region are connected by a linking group; Optionally, the linking group includes peptide linkers or non-peptide linkers; Optionally, the linking group is a peptide linker; Optionally, the length of the peptide linker is 5-30 amino acids; Optionally, the peptide linker is a flexible linker rich in glycine / serine; Optionally, the peptide linker is (G4S). n , where n = 1-4.

[0020] A third aspect of the present invention provides a dual-site tandem antigen-binding molecule.

[0021] Furthermore, the dual-site tandem antigen-binding molecule contains two different antigen-binding domains, both of which are derived from the antigen-binding fragments of the antibody as described above. Optionally, the two antigen-binding domains are connected in tandem, enabling them to simultaneously bind two non-overlapping or substantially non-overlapping epitopes of GPC3, thereby enhancing epivalent multivalency. Optionally, the two antigen-binding domains are connected by a linker group; Optionally, the linker group includes a peptide linker or a non-peptide linker arm.

[0022] A fourth aspect of the present invention provides a chimeric antigen receptor (CAR).

[0023] Furthermore, the recognition domain of the chimeric antigen receptor is selected from: (1) scFv as described above; or (2) Antigen-binding molecules with two sites in tandem as described above; Optionally, the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, a signal peptide (SP), the recognition domain, a hinge / spacer region, a transmembrane region, a co-stimulatory signaling domain, and an intracellular signal transduction region. Optionally, the signal peptide is selected from the signal peptides of the following molecules: CD3, CD4, CD8, and GM-CSF; Optionally, the hinge / spacer region is selected from the hinge / spacer regions of the following molecules: CD8, 41BB, IgG1, IgG4, PD1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT, and variants thereof; Optionally, the transmembrane region is selected from the transmembrane regions of the following molecules: CD8, 41BB, IgG1, IgG4, PD1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT, and variants thereof; Optionally, the co-stimulatory signaling domain is selected from the co-stimulatory signaling domains of the following molecules: 4-1BB, HVEM, CD27, CD19, CD28, ICOS, CD4, CD8α, CD8β, CD40, OX40, DR3, CD2, GITR, CD30, TIM1, CD226, CD278 and variants thereof; Optionally, the intracellular signal transduction region is selected from the intracellular signal transduction regions of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, CD278, CD21, CD22, FcεRI, FcRγ, FcRβ, CD4, CD5, CD8, CD79a, CD79b, DAP10, DAP12, CD66d, ZAP70 and their variants; Optionally, the signal peptide is a CD8 signal peptide; Optionally, the hinge / spacer area is a CD8 hinge / spacer area; Optionally, the transmembrane region is a CD8 transmembrane region; Optionally, the co-stimulation signal structure domain is a 4-1BB co-stimulation signal structure domain; Optionally, the intracellular signal transduction region is the CD3ζ intracellular signal transduction region; Optionally, the amino acid sequence of the CD8 signal peptide is shown in SEQ ID NO:42; Optionally, the amino acid sequence of the CD8 hinge / spacer region is as shown in SEQ ID NO:43; Optionally, the amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO:44; Optionally, the amino acid sequence of the 4-1BB co-stimulatory signaling domain is shown in SEQ ID NO:45; Optionally, the amino acid sequence of the CD3ζ intracellular signal transduction region is shown in SEQ ID NO:46.

[0024] The fifth aspect of the invention provides any of the following products: (1) A nucleic acid molecule that encodes an antibody or an antigen-binding fragment thereof as described above, an antigen-binding molecule as described above, a two-site tandem antigen-binding molecule as described above, or a chimeric antigen receptor as described above. Optionally, the nucleotide sequence encoding the nucleic acid molecule of the chimeric antigen receptor as described above is shown in any one of SEQ ID NO:47-54; In some embodiments, the nucleic acid molecule may be chemically or biochemically modified, or may contain non-natural or derived nucleotide bases, as readily understood by those skilled in the art. Such modifications include, for example, labeling, methylation, internucleotide modifications such as non-electrolyte linkages (e.g., methylphosphonates, triphosphates, aminophosphates, carbamates, etc.), substitution of one or more naturally occurring nucleotides with analogs, side-linked portions (e.g., peptides), intercalating agents (e.g., acridine, psoralen, etc.), chelating agents, electrolyte linkages (e.g., thiophosphates, dithiophosphates, etc.), alkylating agents, and modified bonds (e.g., α-anomeric nucleic acids, etc.).

[0025] (2) An expression vector comprising the nucleic acid molecule; Optionally, the expression vector is a DNA vector or an RNA vector; Optionally, the expression vector is a virus-derived vector; Optionally, the vector from which the virus originates is a lentiviral vector, a retroviral vector, adenovirus vector, adeno-associated virus vector, poxvirus vector, or herpesvirus vector. Optionally, the nucleic acid molecule is operatively linked to a promoter in the expression vector; Optionally, the promoter is EF1α, PGK, SFFV, CMV, CAG, or SV40; Optionally, the expression vector comprises at least one sequence of papain site, packaging signal, and transposon element.

[0026] (3) A recombinant host cell, said recombinant host cell comprising the expression vector or expressing the chimeric antigen receptor as described above; Optionally, the host cell is a eukaryotic cell; Optionally, the eukaryotic cell is a mammalian cell, a plant cell, or a yeast cell; Optionally, the host cell is a T cell, NK cell, macrophage, dendritic cell, CTL cell, NKT cell, mast cell, or γδT cell; In some embodiments, the recombinant host cells are CAR-T cells or CAR-NK cells that have been transduced and expanded in vitro. Optionally, they further express cytokine support modules (such as IL-15, IL-7, IL-2, IL-12, IL-18, IL-21, etc.). IL-15 and IL-21 are used to prolong the in vivo half-life of CAR cells, IL-12 is used to enhance cytotoxic activity and resist immunosuppression, and IL-12 is used to activate local immunity and recruit other immune cells.

[0027] (4) An antibody derivative comprising an antibody or an antigen-binding fragment thereof as described above, an antigen-binding molecule as described above, or an antigen-binding molecule with two sites in tandem as described above; In some embodiments, the antibody derivative further comprises a detectable marker directly or indirectly conjugated to the antibody or its antigen-binding fragment; In some implementations, direct coupling refers to the direct connection between the antibody and the detectable marker via covalent bonds (such as amide bonds, disulfide bonds, or thioether bonds), while indirect coupling refers to the indirect binding of the antibody and the detectable marker via linkers (such as PEG-type linkers, amino acid linkers, or biotin-avidin / streptavidin-mediated linkers).

[0028] In some embodiments, the detectable markers include, but are not limited to: fluorescent markers (fluorescein-based (FITC, FAM, TAMRA), rhodamine-based (TRITC, Texas Red), near-infrared fluorescent dyes (Cy5, Cy7, Alexa Fluor 647), quantum dots), enzyme markers (horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, glucose oxidase), and radioactive markers (…). 125 I, 3 H, 14 C 99m Tc), chemiluminescent markers (acridinium ester, luminol and its derivatives, isoluminol and its derivatives), bioaffinity markers (biotin, avidin, streptavidin, neutral avidin), and nanoparticle markers (gold nanoparticles, silver nanoparticles, magnetic nanoparticles (Fe3O4)). The antibody-conjugates obtained after conjugation retain their specific binding activity to GPC3.

[0029] (5) A detection reagent comprising an antibody or an antigen-binding fragment thereof as described above, an antigen-binding molecule as described above, a two-site tandem antigen-binding molecule as described above, or an antibody derivative thereof; (6) A testing product comprising the testing reagent; In some implementations, the detection products include ELISA kits, Western blot (WB) kits, immunohistochemistry (IHC) kits, immunofluorescence (IF / ICC) kits, and flow cytometry (FCM) kits.

[0030] (7) A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described above, an antigen-binding molecule as described above, a two-site tandem antigen-binding molecule as described above, or the recombinant host cell; In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, specifically including but not limited to: buffer solutions, osmotic pressure regulators, stabilizers, fillers, preservatives, cell preservation solutions / suspensions, and cryoprotectants.

[0031] The buffer solutions include, but are not limited to, phosphate-buffered saline (PBS), citrate buffer, histidine buffer, Tris-HCl buffer, etc. Their function is to maintain the pH of the drug preparation within the physiologically appropriate range (usually 6.0-7.4), avoid protein denaturation and aggregation, and ensure the antigen-binding activity of the antibody.

[0032] The osmotic pressure regulators include, but are not limited to, sodium chloride, potassium chloride, mannitol, sorbitol, glucose, etc., and their function is to make the osmotic pressure of the preparation consistent with that of human blood / tissue fluid (isotonic), reduce local irritation of the body after injection (such as pain, redness and swelling), and prevent cell rupture (if it contains cellular components).

[0033] The stabilizers include, but are not limited to: protein protectants (sucrose, trehalose, lactose, maltose), surfactants (polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), poloxamer 188, and amino acids (glycine, arginine, lysine).

[0034] The fillers include, but are not limited to, mannitol, glycine, lactose, and dextran 40, which are used to maintain the volume and shape of the formulation during the freeze-drying process, prevent the drug from disintegrating after freeze-drying, and facilitate reconstitution.

[0035] The preservatives include, but are not limited to, benzyl alcohol, chlorobutanol, and thimerosal, which are used to inhibit microbial contamination in multi-dose formulations and extend the shelf life of the formulation after opening (such as antibody drugs that are injected multiple times).

[0036] The cell preservation solution / suspension includes, but is not limited to: physiological saline, PBS, RPMI 1640 medium, X-VIVO medium (serum-free / low-serum formulation), serum substitutes (such as albumin, human serum albumin HSA), cytokines (such as IL-2, IL-7, IL-15), anticoagulants (such as heparin sodium), and antibiotics (such as penicillin / streptomycin).

[0037] The cryoprotectants include, but are not limited to, dimethyl sulfoxide (DMSO, final concentration 5%-10%), glycerol, and propylene glycol, which can be used in combination with serum substitutes or albumin to reduce ice crystal damage during cell cryopreservation / thawing and maintain the activity and killing function of CAR immune cells (such as liquid nitrogen cryopreservation preparations for clinical CAR-T cells).

[0038] (8) A pharmaceutical preparation comprising the pharmaceutical composition.

[0039] In some embodiments, the present invention does not particularly limit the specific dosage form of the pharmaceutical preparation. Those skilled in the art can make conventional selections according to actual needs. For example, the dosage form of the pharmaceutical preparation includes, but is not limited to: injection solution, lyophilized powder for injection, sustained-release microspheres for injection, topical preparations (such as gels and creams), fresh cell suspension preparations, and cryopreserved cell preparations.

[0040] The sixth aspect of the present invention provides any of the following methods: (1) A method for preparing recombinant host cells as described above, the method comprising: introducing the expression vector as described above into host cells to obtain the recombinant host cells; (2) A method for detecting GPC3 protein in a test sample for non-diagnostic and non-therapeutic purposes, the method comprising: contacting the test sample with an antibody or its antigen-binding fragment as described above, an antigen-binding molecule as described above, a two-site tandem antigen-binding molecule as described above, or a test reagent as described above, to detect the formation of antigen-antibody immune complexes.

[0041] In some embodiments, the expression vector of the present invention as described above can be introduced into the desired host cell using any method, including but not limited to: DEAE-dextran-mediated transfection, calcium phosphate transfection, cationic lipid-mediated transfection, electroporation, etc. In the present invention, any suitable method known to those skilled in the art can be used to transiently or stably transfect the target nucleic acid into the desired host cell; those skilled in the art can make conventional selections according to actual needs.

[0042] Furthermore, the present invention also provides a method for preventing and / or treating diseases related to abnormal GPC3 expression, the method comprising: administering to a subject in need a preventive and / or therapeutically effective amount of the recombinant host cell, pharmaceutical composition, or pharmaceutical preparation of the present invention as described above.

[0043] In some embodiments, the present invention does not particularly limit the specific types of diseases related to the abnormal expression of GPC3, including but not limited to: hepatocellular carcinoma, hepatoblastoma, clear cell carcinoma of the ovary, serous cystadenocarcinoma of the ovary, gastric cancer, colorectal cancer, lung adenocarcinoma, nephroblastoma, neuroblastoma, melanoma, pancreatic cancer, esophageal cancer, or gallbladder cancer.

[0044] In some implementations, the subjects include various animal groups, including both human individuals and non-human animals. The specific types of non-human animals can be divided into vertebrates and non-vertebrates: Vertebrates include mammals and non-mammals. Mammals include non-human primates (especially higher primates closely related to humans), livestock such as sheep, pigs, and cattle, pets such as dogs and cats, and rodents commonly used in experimental research (such as mice and rats), guinea pigs, rabbits, and other mammals such as goats; non-mammals include birds such as chickens, amphibians (such as frogs and toads), reptiles (such as lizards and snakes), etc. In a specific implementation of this invention, the subjects are preferably humans.

[0045] In some implementations, the effective dose refers to the lowest drug dose (or cell count) that, when administered to a subject in need (preferably a human), can prevent the occurrence of diseases associated with abnormal GPC3 expression, inhibit disease progression (such as tumor growth / metastasis), alleviate disease symptoms (such as pain, tumor-related complications), improve subject prognosis (such as prolonged survival, improved quality of life), or cure the disease. This dose must simultaneously meet the dual requirements of clinical benefit and controllable safety, avoiding both ineffectiveness due to insufficient dosage and unacceptable toxic side effects (such as cytokine storms, immune rejection, etc.) due to excessive dosage.

[0046] The seventh aspect of the present invention provides for any of the following applications: (1) The use of the antibody or its antigen-binding fragment as described above, the antigen-binding molecule as described above, or the antigen-binding molecule with two sites in tandem as described above in the preparation of a detection reagent for detecting GPC3 protein; (2) The use of the antibodies or antigen-binding fragments thereof as described above, the antigen-binding molecules as described above, the antigen-binding molecules with two sites in tandem as described above, the antibody derivatives or detection reagents as described above in the preparation of detection products for detecting GPC3 protein; (3) The use of the antibodies or antigen-binding fragments thereof as described above, the antigen-binding molecules as described above, the antigen-binding molecules with two sites in tandem as described above, the antibody derivatives or detection reagents as described above in the preparation of diagnostic products for the diagnosis and / or auxiliary diagnosis of diseases related to abnormal GPC3 expression; (4) The use of the antibodies or antigen-binding fragments thereof as described above, the antigen-binding molecules as described above, the antigen-binding molecules with two sites in tandem as described above, the recombinant host cells or antibody derivatives as described above in the preparation of medicaments for the prevention and / or treatment of diseases related to abnormal GPC3 expression; (5) The use of the antibody or its antigen-binding fragment as described above, the antigen-binding molecule as described above, the antigen-binding molecule with two sites in tandem as described above, the recombinant host cell as described above, the antibody derivative or pharmaceutical composition as described above in the preparation of a pharmaceutical formulation for the prevention and / or treatment of diseases related to abnormal GPC3 expression; Optionally, the diseases associated with abnormal GPC3 expression include hepatocellular carcinoma, hepatoblastoma, clear cell ovarian carcinoma, serous cystadenocarcinoma of the ovary, gastric cancer, colorectal cancer, lung adenocarcinoma, nephroblastoma, neuroblastoma, melanoma, pancreatic cancer, esophageal cancer, or gallbladder cancer.

[0047] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: This invention obtains a novel group of high-affinity and high-specificity murine anti-GPC3 monoclonal antibodies through screening. These antibodies recognize key epitopes on the proximal end of the GPC3 membrane. The antibodies and their derivative molecules have excellent specificity and binding strength, and can be applied to various scenarios such as detection, molecular diagnosis and targeted therapy of GPC3-abnormal expression-related tumors such as hepatocellular carcinoma and hepatoblastoma. They have significant clinical and industrial value. Attached Figure Description

[0048] Figure 1 : Graph showing the results of GPC3 binding activity assay of anti-GPC3 antibodies HS01, HS02, and HS03; Figure 2 : HS01-03 CAR-T in vitro cell killing results; Figure 3 : A graph showing the changes in tumor volume and the final tumor weight and size in mice; Figure 4 : HS01 light and heavy chain sequence humanized binding detection results; Figure 5 : Affinity test results of humanized HS01 light and heavy chain sequences; Figure 6 The result graph of dynamic killing detection performed by the Real-Time Cell Impedance Analysis (RTCA) system. Detailed Implementation

[0049] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. The experimental consumables, reagents, and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following embodiments are for illustrative purposes only and should not limit the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0050] Example 1: Obtaining GPC3-terminal recognition antibodies by immunization of mice Recombinant GPC3 antigen proteins pGEX-4T-1-GST-GPC3(511-560)-His and pcDNA3.4-GPC3-His(S359-F) were emulsified with Freund's complete adjuvant and incomplete adjuvant and then subcutaneously injected into 10 mice to gradually enhance the immune response (5 times). Serum titers were collected after each immunization and detected by ELISA using the coated peptides Biotin-GPC3(511-530) / Biotin-GPC3(521-540) / Biotin-GPC3(531-550) / Biotin-GPC3(541-560) [corresponding to SEQ ID NO:37-40] (1 μg / mL, 100 μL / well). In the detection system, the primary antibody consisted of purified murine antibody and a positive control antibody (10 μg / mL, 100 μL / well), and the secondary antibodies were HRP-labeled anti-mouse IgG and anti-human IgG (1:10000, 100 μL / well), respectively. Two mice with the highest titers were selected based on peptide binding signal intensity for hybridoma preparation. After fusion, positive hybridomas underwent primary and secondary screening. Clones with high affinity for the peptide were selected for secondary subcloning to ensure the monoclonal characteristics and stability of the antibodies. Finally, the positive clones obtained from the secondary screening were sequenced and identified. After comparison with known databases and removal of repetitive sequences, three monoclonal antibody clones with unique sequences and high binding strength to the target peptides were obtained: HS01-03. Among them, HS01 and HS03 can be combined with GPC3 (521-540) segment, HS02 can be combined with GPC3 (541-560) segment, the heavy chain variable region and light chain variable region sequences of HS01 are shown as SEQ ID NO:1-2, the heavy chain variable region and light chain variable region sequences of HS02 are shown as SEQ ID NO:3-4, the heavy chain variable region and light chain variable region sequences of HS03 are shown as SEQ ID NO:5-6, and the sequence information of HS01-03 is shown in Table 1 below.

[0051] Table 1 Sequence Information of HS01-03

[0052] The absorbance changes detected by ELISA are shown in Table 2 below. The results show that in the detection group corresponding to the Biotin-GPC3 (521-540) fragment, the absorbance values ​​of HS01 and HS03 reached 4.3305 and 4.3479 respectively (far higher than other fragments), indicating that HS01 and HS03 can bind to the GPC3 (521-540) fragment with strong binding signal and high affinity. In the detection group corresponding to the Biotin-GPC3 (541-560) fragment, the absorbance value of HS02 reached 3.7895 (significantly higher than other fragments), indicating that HS02 can bind to the GPC3 (541-560) fragment with strong binding signal and high affinity. The absorbance values ​​corresponding to the other fragments (such as 511-530 and 531-550) were all low (mostly between 0.1 and 1.0), indicating that these three antibodies only specifically bind to the target peptide fragments and there is no obvious cross-binding. The above results indicate that the monoclonal antibody HS01-03 screened in this embodiment has a strong specific binding ability to different polypeptide fragments at the GPC3 terminal.

[0053] Table 2 Results of ELISA detection of absorbance changes

[0054] The designed HS01~3 antibody combinations were synthesized and subcloned into the pcDNA3.4-IgG1Fc expression vector. After the vector was verified by sequencing, endotoxin-free plasmids were prepared and humanized antibodies were expressed in 293F cells. Different concentrations of candidate antibodies (20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, 0.000256 μg / mL, 100 μL / well) were used; the secondary antibody was PE-Goat anti-Human IgG Fc (Invitrogen, Cat#: 12-4998-82) (1:1000 dilution), mixed with 293T-GPC3 (2×10⁻⁶). 5 After incubation with cells / wells, the flow cytometry fluorescence intensity was measured, and the affinity was calculated. Results are as follows: Figure 1 As shown in the figure, the results indicate that all three antibodies have the activity of binding to GPC3-positive cells, meaning that they can all achieve binding to GPC3.

[0055] Based on antibody sequencing results, HS01-03 CAR sequences were designed and corresponding pCDH-EF1α expression vectors were constructed, as shown in SEQ ID NO:47-49. The resulting plasmids were co-transfected into logarithmically growing adherent 293T cells using a four-plasmid lentiviral system (pCDH-EF1α expression vector, PsPAX2, pMD2.G, and helper plasmid pRev, mixed in a mass ratio of 4:3:2:1). Cell supernatants were collected after 48-72 hours, concentrated, filtered, and lentivirus was obtained and stored at -80℃ for later use. Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood, and T cells were isolated using the EasySep™ Human T Cell Isolation Kit (STEMCELL, #17951). T cells were normally activated 2-3 days after X-vivo (lonza) medium containing 10 ng / mL IL-7 (nearshore protein, GMP-C086), 5 ng / mL IL-15 (nearshore protein, GMP-C016), and ImmunoCult™ Human CD3 / CD28 / CD2 T Cell Activator (STEMCELL, #10970) antibody. The medium was then replaced with a medium without the activation antibody, and the cells were expanded to day 14. CAR-T cell positivity was detected using G4S antibody (Anti-(G4S)n(B02H1)mAb(PE), Hycells, #GS-ARPE100). Killing experiments were performed for 16 hours using a mixture of CAR-T positive cells and target cells (Huh-7) (effect-to-target ratio 1:1 and 2.5:1). GC33 served as a control (scFv SEQ ID NO:57). Results are as follows: Figure 2 As shown, the killing rate results indicate that the constructed HS01-H03 cells all have significant killing ability. It can be seen that HS01-03 CAR-T cells have efficient and specific killing ability against GPC3 positive target cells, and the killing activity increases with the increase of the effector-to-target ratio.

[0056] Tumorigenesis in mice: Healthy mice were selected, and the subcutaneous injection sites (approximately 1 cm below the right axilla) were shaved and marked to ensure accurate injection. During tumorigenesis preparation, the target hepatocellular carcinoma cells Huh-7 were injected at a rate of 5 × 10⁻⁶ cells per mouse. 6The number of cells was determined, and the cells were resuspended in 50 μL of PBS and gently mixed with an equal volume of matrix gel, avoiding air bubbles. The mixture was then placed on ice. Using a 1 mL syringe, 100 μL of the cell-matrix gel mixture was injected subcutaneously into mice after removing air, forming a palpable small tumor. The injection speed and force must be controlled during the injection process to avoid leakage or accidental entry into other tissue layers. Mice were housed in a constant-temperature environment after implantation, and tumor formation was monitored regularly. When the tumor stabilized and grew to a suitable volume (e.g., 100-200 mm), the tumor was considered complete. 3 Afterwards, mice were randomly divided into experimental and control groups, ensuring consistency in sex, weight, and tumor size among the groups. Subsequently, GPC3-specific CAR-T cells were intravenously injected into the experimental groups, while the control group received non-specific T cells or an equal volume of saline. Following administration, the long diameter (a) and short diameter (b) of the tumor were measured using calipers 2-3 times per week, and the result was calculated using the formula V = (a × b). 2 The tumor volume was calculated by 1 / 2, and the tumor growth curve was recorded and plotted. The in vivo antitumor activity and therapeutic effect of CAR-T cells were evaluated by dynamically observing changes in tumor volume and differences in the control group. The results are as follows: Figure 3 As shown, the results indicate that CAR-T cells constructed using the HS01-03 sequence exhibit significant anti-tumor efficacy in mouse models.

[0057] Example 2: Humanization and Detection of HS01 Antibody Sequence To further reduce the murine origin of the antibody, the present invention transplanted the CDRs (CDR1, CDR2, and CDR3) of the murine antibody screened in Example 1 into the backbone region (FR) of the human antibody to prepare a humanized antibody. Specifically, the heavy and light chains of the murine antibody HS01 sequence that specifically recognizes GPC3 were humanized. The degree of humanization was evaluated and screened by the T20 score, and four light chain VL sequences (HS01-hVL1, HS01-hVL2, HS01-hVL3, and HS01-hVL4, whose amino acid sequences are shown in SEQ ID NO:23-26) and four heavy chain VH sequences (HS01-hVH1, HS01-hVH2, HS01-hVH3, and HS01-hVH4, whose amino acid sequences are shown in SEQ ID NO:27-30) were constructed. These sequences were codon-optimized and then constructed in pairs in the pcDNA3.4-IgG1Fc expression vector. 100 μL of the supernatant from the expression of the humanized GPC3 antibody corresponding to the 293F expression plasmid was co-incubated with 293T cells overexpressing GPC3. A negative control was set up using ordinary 293T cells, and a positive control was set up using 9F2 cells (VH SEQ ID NO:58, VL SEQ ID NO:59). The secondary antibody used was 488-anti-Fc (1 μg / mL). The binding affinity between different combinations of humanized antibodies and 293T-GPC3 was detected. The binding detection results after humanization of the HS01 light and heavy chain sequences are shown below. Figure 4 As shown in the results, the MFI values ​​of the combinations VH1VL2, VH1VL3, VH2VL3, VH3VL3, and VH3VL4 were significantly higher than those of other combinations, indicating that these combinations possess strong binding activity while retaining GPC3 binding specificity. Based on the flow cytometry mean fluorescence intensity (MFI) results, the combinations VH1VL2, VH1VL3, VH2VL3, VH3VL3, and VH3VL4 were selected as the preferred humanized combinations.

[0058] The five selected combinations were expressed with 293F and then diluted to different concentrations: 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg / mL candidate antibodies (100 μL / well); the secondary antibody was PE-Goat anti-Human IgGFc (Invitrogen, Cat#: 12-4998-82) (1:1000 dilution), and 293T-GPC3 (2×10⁻⁶). 5 After incubation with cells / wells, the flow cytometry fluorescence intensity was measured, and the affinity was calculated. The results are as follows: Figure 5 As shown in the figure, the results indicate that the humanized antibodies can all achieve high affinity binding to GPC3, with 9F2 serving as a positive control.

[0059] The humanized sequences were converted into CAR sequences (the amino acid sequences of scFv(HS01-hscFv1, HS01-hscFv2, HS01-hscFv3, HS01-hscFv4, HS01-hscFv5) are shown in SEQ ID NO:31-35, respectively; the nucleic acid sequences of CAR(HS01-h1-CAR, HS01-h2-CAR, HS01-h3-CAR, HS01-h4-CAR, HS01-h5-CAR) are shown in SEQ ID NO:50-54, respectively) and constructed in the pCDH-EF1α expression vector. CAR-T cells were then prepared according to the above protocol. Using the human hepatocellular carcinoma cell line Huh7 as target cells, CAR-T cells were co-incubated at an effector-to-target ratio of 1:5, and dynamic killing was detected using a real-time cell impedance analysis (RTCA) system. The results are as follows: Figure 6 As shown, the results indicate that all humanized HS01CAR-T cells exhibited strong killing function, with HS01 serving as the non-humanized control and 9F2 as the positive control.

Claims

1. An antibody or antigen-binding fragment thereof targeting GPC3, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-3 and the light chain variable region comprises LCDR1-3. The HCDR1-3 and LCDR1-3 are selected from any one of the following groups: (1) HS01 antibody: HCDR1-3 in the heavy chain variable region as shown in SEQ ID NO:1, and LCDR1-3 in the light chain variable region as shown in SEQ ID NO:2; (2) HSO2 antibody: HCDR1-3 in the heavy chain variable region as shown in SEQ ID NO:3, and LCDR1-3 in the light chain variable region as shown in SEQ ID NO:4; (3) HSO3 antibody: HCDR1-3 in the heavy chain variable region as shown in SEQ ID NO:5, and LCDR1-3 in the light chain variable region as shown in SEQ ID NO:

6.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The HCDR1-3 and LCDR1-3 are determined using one or more of the following numbering systems: Kabat, IMGT, Chothia, AbM, Contact, Martin (enhanced Chothia), and Aho. Optionally, the HCDR1-3 and LCDR1-3 are determined using the Kabat numbering system; Optionally, the HCDR1-3 and LCDR1-3 are selected from any one of the following groups: (1) HS01 antibody: The amino acid sequences are HCDR1-3 as shown in SEQ ID NO:7-9 and LCDR1-3 as shown in SEQ ID NO:10-12 respectively; (2) HSO2 antibody: The amino acid sequences are HCDR1-3 as shown in SEQ ID NO:13-15 and LCDR1-3 as shown in SEQ ID NO:16-18, respectively; (3) HSO3 antibody: HCDR1-3 with amino acid sequences as shown in SEQ ID NO:19-21, and LCDR1-3 with amino acid sequences as shown in SEQ ID NO:22, SEQ ID NO:11, and SEQ ID NO:

12.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region and light chain variable region of the antibody or its antigen-binding fragment are selected from any one of the following: (1) HS01 antibody: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:1 or has at least 80% homology with SEQ ID NO:1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:2 or has at least 80% homology with SEQ ID NO:2; (2) HSO2 antibody: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:3 or has at least 80% homology with SEQ ID NO:3, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:4 or has at least 80% homology with SEQ ID NO:4; (3) HSO3 antibody: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:5 or has at least 80% homology with SEQ ID NO:5, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:6 or has at least 80% homology with SEQ ID NO:6; Optionally, the GPC3 is the proximal end of a human GPC3; Optionally, the proximal end of the human GPC3 is amino acid 511-560 of the C-terminal region of the human GPC3, and its amino acid sequence is shown in SEQ ID NO:

36. Optionally, the HS01 antibody and the HS03 antibody can specifically recognize and bind to amino acids 521-540 of the C-terminal region of GPC3, and their amino acid sequences are shown in SEQ ID NO:

38. Optionally, the HSO2 antibody can specifically recognize and bind to amino acids 541-560 of the C-terminal region of GPC3, the amino acid sequence of which is shown in SEQ ID NO:

40.

4. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment is a humanized variant of the HS01 antibody; The humanized variant of the HS01 antibody comprises any combination of the following heavy chain variable regions and light chain variable regions: The heavy chain variable region is selected from: the heavy chain variable region HS01-hVH1 with the amino acid sequence as shown in SEQ ID NO:27, the heavy chain variable region HS01-hVH2 with the amino acid sequence as shown in SEQ ID NO:28, the heavy chain variable region HS01-hVH3 with the amino acid sequence as shown in SEQ ID NO:29, or the heavy chain variable region HS01-hVH4 with the amino acid sequence as shown in SEQ ID NO:30, or a humanized variant with at least 80% homology; The light chain variable region is selected from: the light chain variable region HS01-hVL1 with an amino acid sequence as shown in SEQ ID NO:23, the light chain variable region HS01-hVL2 with an amino acid sequence as shown in SEQ ID NO:24, the light chain variable region HS01-hVL3 with an amino acid sequence as shown in SEQ ID NO:25, or the light chain variable region HS01-hVL4 with an amino acid sequence as shown in SEQ ID NO:26, or a humanized variant with at least 80% homology; Optionally, the heavy chain variable region and the light chain variable region can be arbitrarily paired to form an antibody or a single-chain variable fragment (scFv). Optionally, the humanized variant of the HS01 antibody includes heavy chain variable regions and light chain variable regions selected from any one of the following groups: (1) HS01-h1: The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:27, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:24; (2) HS01-h2: heavy chain variable region with amino acid sequence as shown in SEQ ID NO:27, and light chain variable region with amino acid sequence as shown in SEQ ID NO:25; (3) HS01-h3: The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:28, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:25; (4) HS01-h4: heavy chain variable region with amino acid sequence as shown in SEQ ID NO:29, and light chain variable region with amino acid sequence as shown in SEQ ID NO:25; (5) HS01-h5: The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:29, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:26; Optionally, the amino acid sequences of the scFv corresponding to HS01-h1, HS01-h2, HS01-h3, HS01-h4, and HS01-h5 are as shown in SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, respectively, or are humanized variants with at least 80% homology.

5. An antigen-binding molecule targeting GPC3, characterized in that, The antigen-binding molecule comprises the heavy chain variable region and the light chain variable region of the antibody or its antigen-binding fragment as described in any one of claims 1-4, and the antigen-binding molecule is a full-length antibody and / or its antigen-binding fragment and / or fusion complex. Optionally, the antigen-binding molecule is full-length IgG, Fab, F(ab′)2, single-domain antibody, scFv, scFv-Fc, bivalent or multivalent antibody, bispecific or multispecific antibody or polymer thereof; Optionally, when the molecule is scFv or a fusion containing scFv, the heavy chain variable region and the light chain variable region are connected by a linking group; Optionally, the linking group includes peptide linkers or non-peptide linkers; Optionally, the linking group is a peptide linker; Optionally, the length of the peptide linker is 5-30 amino acids; Optionally, the peptide linker is a flexible linker rich in glycine / serine; Optionally, the peptide linker is (G4S). n , where n = 1-4.

6. A dual-site tandem antigen-binding molecule, characterized in that, The dual-site tandem antigen-binding molecule comprises two different antigen-binding domains, both of which are derived from the antigen-binding fragment of the antibody according to any one of claims 1-4. Optionally, the two antigen-binding domains are connected in tandem, enabling them to simultaneously bind two non-overlapping or substantially non-overlapping epitopes of GPC3, thereby enhancing epivalent multivalency. Optionally, the two antigen-binding domains are connected by a linker group; Optionally, the linker group includes a peptide linker or a non-peptide linker arm.

7. A chimeric antigen receptor (CAR), characterized in that, The recognition domain of the chimeric antigen receptor is selected from: (1) The scFv as described in claim 5; or (2) The dual-site tandem antigen-binding molecule as described in claim 6; Optionally, the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, the following components in sequence: a signal peptide, the recognition domain, a hinge / spacer region, a transmembrane region, a co-stimulatory signaling domain, and an intracellular signal transduction region. Optionally, the signal peptide is selected from the signal peptides of the following molecules: CD3, CD4, CD8, and GM-CSF; Optionally, the hinge / spacer region is selected from the hinge / spacer regions of the following molecules: CD8, 41BB, IgG1, IgG4, PD1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT, and variants thereof; Optionally, the transmembrane region is selected from the transmembrane regions of the following molecules: CD8, 41BB, IgG1, IgG4, PD1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT, and variants thereof; Optionally, the co-stimulatory signaling domain is selected from the co-stimulatory signaling domains of the following molecules: 4-1BB, HVEM, CD27, CD19, CD28, ICOS, CD4, CD8α, CD8β, CD40, OX40, DR3, CD2, GITR, CD30, TIM1, CD226, CD278 and variants thereof; Optionally, the intracellular signal transduction region is selected from the intracellular signal transduction regions of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, CD278, CD21, CD22, FcεRI, FcRγ, FcRβ, CD4, CD5, CD8, CD79a, CD79b, DAP10, DAP12, CD66d, ZAP70, and their variants; Optionally, the signal peptide is a CD8 signal peptide; Optionally, the hinge / spacer area is a CD8 hinge / spacer area; Optionally, the transmembrane region is a CD8 transmembrane region; Optionally, the co-stimulation signal structure domain is a 4-1BB co-stimulation signal structure domain; Optionally, the intracellular signal transduction region is the CD3ζ intracellular signal transduction region; Optionally, the amino acid sequence of the CD8 signal peptide is shown in SEQ ID NO:42; Optionally, the amino acid sequence of the CD8 hinge / spacer region is as shown in SEQ ID NO:43; Optionally, the amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO:44; Optionally, the amino acid sequence of the 4-1BB co-stimulatory signaling domain is shown in SEQ ID NO:45; Optionally, the amino acid sequence of the CD3ζ intracellular signal transduction region is shown in SEQ ID NO:

46.

8. Any of the following products: (1) A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the antigen-binding molecule as described in claim 5, the dual-site tandem antigen-binding molecule as described in claim 6, or the chimeric antigen receptor as described in claim 7; Optionally, the nucleotide sequence encoding the chimeric antigen receptor of claim 7 is shown in any one of SEQ ID NO:47-54; (2) An expression vector, characterized in that the expression vector comprises the nucleic acid molecule; Optionally, the expression vector is a DNA vector or an RNA vector; Optionally, the expression vector is a virus-derived vector; Optionally, the vector from which the virus originates is a lentiviral vector, a retroviral vector, adenovirus vector, adeno-associated virus vector, poxvirus vector, or herpesvirus vector. Optionally, the nucleic acid molecule is operatively linked to a promoter in the expression vector; Optionally, the promoter is EF1α, PGK, SFFV, CMV, CAG, or SV40; Optionally, the expression vector comprises at least one sequence of papain site, packaging signal, and transposon element; (3) A recombinant host cell, characterized in that the recombinant host cell comprises the expression vector or expresses the chimeric antigen receptor of claim 7; Optionally, the host cell is a eukaryotic cell; Optionally, the eukaryotic cell is a mammalian cell, a plant cell, or a yeast cell; Optionally, the host cell is a T cell, NK cell, macrophage, dendritic cell, CTL cell, NKT cell, mast cell, or γδT cell; (4) An antibody derivative, characterized in that the antibody derivative comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the antigen-binding molecule as described in claim 5, or the dual-site tandem antigen-binding molecule as described in claim 6; (5) A detection reagent, characterized in that the detection reagent comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the antigen-binding molecule as described in claim 5, the dual-site tandem antigen-binding molecule as described in claim 6, or the antibody derivative thereof; (6) A testing product, characterized in that the testing product comprises the testing reagent; (7) A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the antibody or antigen-binding fragment thereof of any one of claims 1-4, the antigen-binding molecule of claim 5, the dual-site tandem antigen-binding molecule of claim 6, or the recombinant host cell; (8) A pharmaceutical preparation, characterized in that the pharmaceutical preparation comprises the pharmaceutical composition.

9. Any of the following methods: (1) A method for preparing the recombinant host cell of claim 8, characterized in that, The method includes: introducing the expression vector of claim 8 into a host cell to obtain the recombinant host cell; (2) A method for detecting GPC3 protein in a test sample for non-diagnostic and non-therapeutic purposes, characterized in that the method comprises: contacting the test sample with an antibody or antigen-binding fragment thereof as described in any one of claims 1-4, an antigen-binding molecule as described in claim 5, a dual-site tandem antigen-binding molecule as described in claim 6, or a detection reagent as described in claim 8, to detect the formation of antigen-antibody immune complexes.

10. Applied to any of the following aspects: (1) The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the antigen-binding molecule as described in claim 5, or the dual-site tandem antigen-binding molecule as described in claim 6 in the preparation of a detection reagent for detecting GPC3 protein; (2) The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the antigen-binding molecule as described in claim 5, the dual-site tandem antigen-binding molecule as described in claim 6, the antibody derivative or detection reagent as described in claim 8 in the preparation of a detection product for detecting GPC3 protein; (3) The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the antigen-binding molecule as described in claim 5, the dual-site tandem antigen-binding molecule as described in claim 6, the antibody derivative or detection reagent as described in claim 8 in the preparation of diagnostic products for the diagnosis and / or auxiliary diagnosis of diseases related to abnormal GPC3 expression; (4) The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the antigen-binding molecule as described in claim 5, the dual-site tandem antigen-binding molecule as described in claim 6, and the recombinant host cell or antibody derivative as described in claim 8 in the preparation of a medicament for the prevention and / or treatment of diseases related to abnormal GPC3 expression; (5) The use of any antibody or antigen-binding fragment thereof according to any one of claims 1-4, the antigen-binding molecule according to claim 5, the dual-site tandem antigen-binding molecule according to claim 6, the recombinant host cell, antibody derivative or pharmaceutical composition according to claim 8 in the preparation of a pharmaceutical formulation for the prevention and / or treatment of diseases related to abnormal GPC3 expression; Optionally, the diseases associated with abnormal GPC3 expression include hepatocellular carcinoma, hepatoblastoma, clear cell ovarian carcinoma, serous cystadenocarcinoma of the ovary, gastric cancer, colorectal cancer, lung adenocarcinoma, nephroblastoma, neuroblastoma, melanoma, pancreatic cancer, esophageal cancer, or gallbladder cancer.

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