Anti-GPC3 antibody or antigen-binding fragment and its use

Abstract

The present invention provides an anti-GPC-3 antibody and its antigen-binding fragment, and further provides a polynucleotide encoding the antibody, an expression vector and host cells for expressing the antibody, and a method for producing the antibody. Furthermore, the present invention further provides a pharmaceutical composition containing the antibody and the use of the antibody in the manufacture of a drug for treating cancer.

Description

[Technical Field]

[0001] This invention belongs to the field of biopharmaceutical technology, and more specifically, relates to an anti-GPC3 antibody or its antigen-binding fragment and its use. [Background technology]

[0002] GPC3 is a heparan sulfate proteoglycan expressed on the surface of various malignant cells, including hepatocellular carcinoma (HCC) cells. Glypican-3 is linked to the cell surface via a glycosylphosphatidylinositol anchor (GPI). GPC3 is highly expressed in over 70% of hepatocellular carcinoma biopsy tissues, but its expression is not observed in adjacent non-tumor tissues. The disease-free survival rate of GPC3-positive HCC patients is significantly lower than that of GPC3-negative HCC patients.

[0003] A certain antibody that binds to GPC3 has been found to have cell growth inhibitory activity via antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cell-mediated cytotoxicity (CDC) activity (International Patent Application WO 2003 / 000883). Furthermore, it has been shown that GPC3 is degraded in vivo and secreted into the bloodstream as a secreted form of GPC3, suggesting that antibodies capable of detecting secreted GPC3 could be used for tumor diagnosis (International Publication Nos. 2004 / 022739, 03 / 100429, and 2004 / 018667).

[0004] Antibody-drug conjugates (ADCs) are a novel targeted therapy that combines an antibody with a small molecule drug that has potent cytotoxic properties. Combining the potent killing power of small molecule drugs with the high targeting capabilities of monoclonal antibodies, ADCs have become a focus of research and development in targeted tumor therapy. ADCs typically consist of three components: an antibody or antibody ligand, a linker, and a small molecule drug, linked in a specific manner. The targeting of an ADC is due to its antibody component, while its toxicity is primarily due to the small molecule drug. The antibody component may also be toxic. After binding to an antigen on the surface of tumor cells, the antibody component is taken up into the cell. Subsequently, the ADC drug is broken down in lysosomes, releasing toxic substances—active chemicals—that damage DNA or inhibit tumor cell division, ultimately killing the tumor cells. Compared to other therapies, ADCs have the following characteristics: high therapeutic efficacy, high specificity to tumor cells, low false-positive rates, and a wide therapeutic safety window. They also exhibit low immunogenicity and are less likely to cause drug resistance. It has a longer circulating time in the serum (shorter than naked antibodies). It exhibits low cytotoxicity against non-target cells.

[0005] The following are some of the anti-GPC3 antibodies that have been reported to date:

[0006] Chinese Patent No. 1842540 discloses an anti-GPC3 antibody, e.g., GC33, which has higher ADCC and CDC activity compared to conventional antibodies. Its antibody epitope is located at the 544th to 553rd sequence (PKDNEISTFH) of the C-terminus of GPC3, although its binding ability to the GPC3 epitope is still relatively weak.

[0007] Chinese Patent No. 10452033 discloses a high-affinity monoclonal antibody against phosphatidylinositol proteoglycan-3 and its use. For example, YP7 is an antibody (DGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHS) produced by peptide immunotherapy consisting of 50 residues, and has high affinity for GPC3, although there is still room for improvement in its affinity for GPC3.

[0008] Chinese Patent Application Publication No. 115850492 also discloses an anti-phosphatidylinositol proteoglycan-3 monoclonal antibody, a polynucleotide, a method of production, and its use, claiming to solve the technical problem of low affinity between GPC3 monoclonal antibodies and target antigens in the prior art. However, there is still room for improvement in affinity with GPC3.

[0009] This indicates that previously reported anti-GPC3 antibodies have insufficient affinity (especially at the cellular level) or low toxicity. Therefore, there is an urgent need to develop anti-GPC3 antibodies or antigen-binding fragments that possess strong binding ability to GPC3 and excellent cytotoxic effects. [Overview of the project] [Problems that the invention aims to solve]

[0010] The present invention aims to provide an anti-GPC3 antibody or its antigen-binding fragment and its use in order to solve problems that exist in the prior art, such as low affinity and low cell-killing effect. [Means for solving the problem]

[0011] In one embodiment, the present invention provides an anti-GPC3 antibody or an antigen-binding fragment thereof, the anti-GPC3 antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), The VH includes HCDR1, HCDR2, and HCDR3 regions, and each of the HCDR1, HCDR2, and HCDR3 regions contains a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the CDR1, CDR2, and CDR3 regions of the amino acid sequence indicated by SEQ ID No: 1-2 or 6-9, or contains a sequence in which at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutation has occurred compared to the CDR1, CDR2, and CDR3 regions of the amino acid sequence indicated by SEQ ID No: 1-2 or 6-9, and the mutations may be selected from insertions, deletions, and / or substitutions, and the substitutions are preferably substitutions of conserved amino acids, and the amino acid sequence SEQ ID CDR1, CDR2, and CDR3 regions No. 1-2 and 6-9 are defined by the IMGT, Kabat, Chothia, AbM, or Contact methods. The VL includes LCDR1, LCDR2, and LCDR3 regions, each containing a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the CDR1, CDR2, and CDR3 regions of amino acid sequence SEQ ID NO:3, or each containing a sequence having at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutation compared to the CDR1, CDR2, and CDR3 regions of amino acid sequence SEQ ID NO:3, wherein the mutations may be selected from insertions, deletions, and / or substitutions, and the substitutions are preferably substitutions of conserved amino acids. The CDR1, CDR2, and CDR3 regions of No:3 are defined by the IMGT, Kabat, Chothia, AbM, or Contact methods.

[0012] In some embodiments, the position of the mutation is one or more selected from the 56th position (D56), 100th position (Q100), or 102nd position (S102) of the amino acid sequence represented by any of SEQ ID No: 1-2, 6-9. In some embodiments, the position of the mutation includes the 56th position (D56) and the 102nd position (S102) of the amino acid sequence represented by any of SEQ ID No: 1-2, 6-9. In some preferred embodiments, the mutation is one or more selected from D56A, D56K, Q100R, or S102R. In some preferred embodiments, the mutation is selected from Q100R, S102R, D56A, D56K+Q100R, D56A+S102R, or D56K+S102R.

[0013] In some embodiments, the HCDR1, HCDR2, and HCDR3 regions each contain the same sequence as the CDR1, CDR2, and CDR3 regions of the amino acid sequence represented by any of SEQ ID No: 1-2, 6-9, 23-28, and the LCDR1, LCDR2, and LCDR3 regions each contain a sequence that is the same as the CDR1, CDR2, and CDR3 regions of amino acid sequence SEQ ID No: 3.

[0014] In some embodiments, the VH contains a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of SEQ ID No: 1-2, 6-9, 23-28. In some embodiments, the VL contains a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID No: 3.

[0015] In some embodiments, the VH contains a sequence that is the same as any of the amino acid sequences SEQ ID No: 1-2, 6-9, 23-28, and / or the VL contains a sequence that is the same as the amino acid sequence SEQ ID NO: 3.

[0016] In some embodiments, the antibody or its antigen-binding fragment further comprises a heavy chain constant region (CH) and a light chain constant region (CL), wherein the heavy chain constant region (CH) may be the heavy chain constant region of human IgG1, and the light chain constant region (CL) may be the human κ light chain constant region. Specifically, the heavy chain constant region (CH) comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with amino acid sequence SEQ ID No: 4, 19, or 20, and the light chain constant region (CL) comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with amino acid sequence SEQ ID No: 5.

[0017] In some embodiments, the heavy chain constant region (CH) includes the same sequence as amino acid sequence SEQ ID No: 4, 19, or 20, and the light chain constant region (CL) includes the same sequence as amino acid sequence SEQ ID No: 5.

[0018] In some embodiments, the heavy chain (H) includes a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of the amino acid sequences SEQ ID No: 11-18 or 29-34, and / or the light chain (L) includes a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence SEQ ID No: 10.

[0019] As a preferred technical solution of the present invention, the heavy chain (H) contains the same sequence as any of the amino acid sequences SEQ ID No: 11-18 or 29-34, and the light chain (L) contains the same sequence as the amino acid sequence SEQ ID No: 10.

[0020] In some embodiments, the anti-GPC3 antibody or its antigen-binding fragment of the present invention is a humanized monoclonal antibody.

[0021] In some specific embodiments, the anti-GPC3 antibody or its antigen-binding fragment is (1) a chimeric antibody or its fragment, (2) a humanized antibody or its fragment, or (3) a fully human antibody or its fragment.

[0022] In some embodiments, the anti-GPC3 antibody or its antigen-binding fragment of the present invention has one or more of the following biological functions. (1) Specifically binds to the antigen represented by SEQ ID No: 37 or 38 and does not bind to the antigen represented by SEQ ID No: 36 (2) Specifically binds to human and monkey GPC3 proteins and does not bind to mouse GPC3 protein

[0023] In some embodiments, the specific binding of the anti-GPC3 antibody or its antigen-binding fragment to the antigen is represented by an equilibrium dissociation constant KD, for example, 10 -6 M or less (for example, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 M).

[0024] In one embodiment, the present invention provides an anti-GPC3 antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment specifically binds to an antigen indicated by SEQ ID No: 37 or 38, but does not bind to an antigen indicated by SEQ ID No: 36. In some embodiments, the antibody or its antigen-binding fragment is represented as VL indicated by SEQ ID No:39 and VH indicated by SEQ ID No:40, VL indicated by SEQ ID No:41 and VH indicated by SEQ ID No:42, VL indicated by SEQ ID No:43 and VH indicated by SEQ ID No:44, VL indicated by SEQ ID No:45 and VH indicated by SEQ ID No:46, VL indicated by SEQ ID No:47 and VH indicated by SEQ ID No:48, VL indicated by SEQ ID No:49 and VH indicated by SEQ ID No:50, VL indicated by SEQ ID No:51 and VH indicated by SEQ ID No:52, VL indicated by SEQ ID No:53 and VH indicated by SEQ ID No:54, VL indicated by SEQ ID No:55 and VH indicated by SEQ ID No:56, or VL indicated by SEQ ID No:57 and SEQ ID Includes VH as indicated by No:58.

[0025] In one embodiment, the present invention provides an anti-GPC3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment competitively binds to the same epitope of a reference antibody and the GPC3 protein, and the reference antibody comprises a heavy chain indicated by SEQ ID NO:11 and a light chain indicated by SEQ ID NO:10.

[0026] In some embodiments, the anti-GPC3 antibody or its antigen-binding fragment can block at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the binding of the reference antibody to the GPC3 protein. This competitive binding can be measured by a competitive binding assay. A competitive binding assay, well known to those skilled in the art, is an immunological assay that detects and quantifies an unknown substance by its ability to inhibit the binding of a labeled known antigen to its specific antibody; it is also called a competitive inhibition assay. For example, an antigen is pre-coated onto a microplate, serially diluted unlabeled test antibodies and specific concentrations of labeled known antibodies are added to the coated microplate and incubated. After washing, the amount of known antibody bound to the plate at different dilutions of the test antibody is measured. The stronger the ability of the test antibody to competitively bind to the known antibody and the antigen, the weaker the ability of the known antibody to bind to the antigen, and the less known antibody binds to the plate. The ability of the test antibody to block the labeled reference antibody can be measured using radioimmunoassay, enzyme immunoassays such as ELISA, or fluorescence immunoassays.

[0027] Another object of the present invention is to address the shortcomings of the prior art, which fail to meet the needs for developing anti-GPC3 antibodies, vaccines, and related diagnostic reagents, and to provide a novel GPC3 antigen epitope peptide to offer an effective tool for developing anti-GPC3 antibodies that have strong binding ability to GPC3 or relatively good cytotoxic effects.

[0028] In one embodiment, the present invention provides a GPC3 antigen epitope peptide, which is immunogenic and can induce an immune response in the body to produce antibodies against GPC3.

[0029] In one embodiment, the present invention provides a GPC3 antigen epitope peptide, the GPC3 antigen epitope peptide comprising at least seven consecutive amino acid residues from human GPC3 protein residues 485-496, the amino acid sequence of the human GPC3 protein being indicated by SEQ ID No:35, and the GPC3 antigen epitope peptide having one or more of the following biological functions. (1) Specifically binds to the anti-GPC3 antibody, (2) Induce an immune response to GPC3 in the subject's body (e.g., a humoral immune response), (3) Inducing the production of anti-GPC3 antibodies in the subject's body, (4) To prevent and / or treat GPC3-related diseases in subjects.

[0030] In some embodiments, the specific binding of the GPC3 antigen epitope peptide to the anti-GPC3 antibody can be measured by an ELISA method. In some embodiments, the ELISA method is as described in Example 12 and includes coating an ELISA plate with the GPC3 antigen epitope peptide, adding the anti-GPC3 antibody in a stepwise gradient dilution to the ELISA plate and incubating, and after incubation is complete, washing, color development, stopping, and measuring the OD value at 450 nm. In some embodiments, the specific binding of the GPC3 antigen epitope peptide to the anti-GPC3 antibody is measured by the OD of the antigen-antibody complex measured by ELISA. 450 This is reflected in the value. In some embodiments, when the concentration of the GPC3 antigen epitope peptide is 6 μg / mL, the OD when the anti-GPC3 antibody is saturated is 450 The value is 1.5±0.1 or greater, or 2±0.1 or greater, or 2.5±0.1 or greater, or 3±0.1 or greater. In some embodiments, the OD 450The values ​​are 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, or 3.9 or greater. In some embodiments, the anti-GPC3 antibody that specifically binds to the GPC3 antigen epitope peptide comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence indicated by SEQ ID No: 11 and the light chain comprises an amino acid sequence indicated by SEQ ID No: 10.

[0031] In some embodiments, the length of the GPC3 antigen epitope peptide is 7, 8, 9, 10, 11, or 12 amino acids. In some embodiments, the GPC3 antigen epitope peptide includes at least one of asparagine at position 487 and phenylalanine at position 493.

[0032] In some embodiments, the GPC3 antigen epitope peptide consists of consecutive amino acid residues at positions 487-493 of the human GPC3 protein residues. In some preferred embodiments, the GPC3 antigen epitope peptide consists of the amino acid sequence indicated by SEQ ID No: 38.

[0033] In one embodiment, the present invention provides a recombinant antigen comprising the GPC3 antigen epitope peptide and a carrier protein. The recombinant antigen can enhance the immunogenicity of the epitope peptide so that it is recognized by the body's immune system and an immune response is induced.

[0034] In some embodiments, the recombinant antigen has one or more of the following biological functions: (1) Specifically binds to the anti-GPC3 antibody, (2) Induce an immune response to GPC3 in the subject's body (e.g., a humoral immune response), (3) Inducing the production of anti-GPC3 antibodies in the subject's body, (4) To prevent and / or treat GPC3-related diseases in subjects.

[0035] In some embodiments, the anti-GPC3 antibody that specifically binds to the recombinant antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence indicated by SEQ ID No: 11 and the light chain comprises an amino acid sequence indicated by SEQ ID No: 10.

[0036] In some embodiments, the GPC3 antigen epitope peptide of the present invention is directly linked to a carrier protein or linked via a linker. In some embodiments, the linker may be a rigid or flexible linker, for example, a peptide linker, which comprises one or more serine and / or glycine.

[0037] In some embodiments, the GPC3 antigen epitope peptide of the present invention is ligated to the N-terminus and / or C-terminus of the carrier protein and / or inserted into the interior of the carrier protein. In some preferred embodiments, the GPC3 antigen epitope peptide of the present invention is ligated to the C-terminus of the carrier protein. In some preferred embodiments, the GPC3 antigen epitope of the present invention is ligated to the N-terminus of the carrier protein.

[0038] In some embodiments, the carrier protein includes, but is not limited to, keyhole hemocyanin (KLH), bovine serum albumin (BSA), thyroglobulin, fibrinogen, gelatin, a polyantigen peptide including diphtheria toxin DT, the transmembrane domain DTT of diphtheria toxin, rotavirus VP7, the heat shock protein of leishmania, flagellin of Campylobacter jejuni, the major outer membrane protein of Chlamydia trachomatis, ovalbumin (OVA), or the Fc domain of immunoglobulin, such as the Fc domain of IgG1, IgG2, IgG3, or IgG4. In some preferred embodiments, the carrier protein is selected from KLH and BSA.

[0039] In one embodiment, the present invention provides a chimeric antigen receptor comprising the anti-GPC3 antibody of the present invention or an antigen-binding fragment thereof.

[0040] In another embodiment, the present invention provides a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain comprises the anti-GPC3 antibody of the present invention or an antigen-binding fragment thereof.

[0041] In one embodiment, the present invention further provides a pharmaceutical composition comprising the anti-GPC3 antibody or its antigen-binding fragment or antibody conjugate (e.g., antibody-drug conjugate), or oncolytic virus, or chimeric antigen receptor, or bispecific or multispecific antibody molecule, or GPC3 antigen epitope peptide, or recombinant antigen, and one or more pharmaceutically acceptable carriers. If the composition comprises one or more antibodies (or their antigen-binding fragment or antibody conjugate, or oncolytic virus), the antibodies (or their antigen-binding fragment or antibody conjugate, or oncolytic virus) may be administered in multiple doses. The composition may optionally contain one or more other pharmacoactive ingredients, for example, another antibody or drug, for example, an antitumor agent.

[0042] The pharmaceutical composition of the present invention may be a vaccine, and the vaccine includes, but is not limited to, a protein vaccine or a nucleic acid vaccine.

[0043] Pharmaceutical compositions may contain any amount of excipients. Available excipients include vectors, surfactants, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, or combinations thereof. The use and selection of appropriate excipients are described in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20th edition (Lippincott Williams & Wilkins 2003), which is incorporated herein by reference.

[0044] In another embodiment, the present invention provides a kit comprising the anti-GPC3 antibody or its antigen-binding fragment, a GPC3 antigen epitope peptide, a recombinant antigen, an antibody-drug conjugate, or a bispecific or multispecific antibody molecule.

[0045] In some embodiments, the kit comprises the GPC3 antigen epitope peptide of the present invention and a tool for detecting the antibody.

[0046] In some embodiments, the kit is used to detect anti-GPC3 antibodies. In some embodiments, the kit is used to detect whether or not anti-GPC3 antibodies are present in a sample. In some embodiments, the kit is used to detect the level of anti-GPC3 antibodies in a sample. In some embodiments, the anti-GPC3 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence indicated by SEQ ID NO:11 and the light chain comprises an amino acid sequence indicated by SEQ ID NO:10.

[0047] In another embodiment, the present invention provides polynucleotides encoding the anti-GPC3 antibody or its antigen-binding fragment, antigen epitope peptide, or recombinant antigen. The polynucleotide of the present invention may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the polynucleotide is a cDNA molecule. The polynucleotide of the present invention can be produced or obtained by known methods, such as automated DNA synthesis and / or recombinant DNA technology, based on the amino acid sequence information of the present invention.

[0048] As is well known in the art, multiple codons can code for the same amino acid. Therefore, nucleic acids that code for protein sequences also include nucleic acids that exhibit codon degeneracy. The amino acid sequences described in this invention can be coded by various nucleic acids. The genetic code is universal and well known. Nucleic acids that code for any of the amino acid sequences described in this invention can be readily devised based on common sense in the art and optimized for production. Although the number of possible nucleic acid sequences that code for a particular amino acid is enormous, if a standard table of the genetic code is provided and with the help of a computer, a person skilled in the art can easily generate all possible combinations of nucleic acid sequences that code for a particular amino acid.

[0049] In another embodiment, the present invention provides an expression vector comprising the polynucleotide of the present invention. The expression vector includes a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vectors.

[0050] In another embodiment, the present invention provides a host cell comprising the polynucleotide of the present invention or the expression vector described above, the host cell including a prokaryotic cell, a yeast cell or a mammalian cell, such as a CHO cell, an NSO cell or other mammalian cell, but preferably a CHO cell.

[0051] In another embodiment, the present invention provides a bispecific or multispecific antibody molecule comprising the anti-GPC3 antibody or its antigen-binding fragment.

[0052] In another embodiment, the present invention provides an antibody-drug conjugate comprising the anti-GPC3 antibody or its antigen-binding fragment and a drug or toxin, wherein the drug or toxin is one or more selected from SN-38, MMAE, PBD dimer, DX-8951 (DXd), or DUBA.

[0053] The antibody and the drug can be bound via a linker to form an antibody-drug conjugate (ADC). Typically, an ADC comprises the anti-GPC3 antibody or antigen-binding fragment of the present invention linked to a drug or toxin via a linker. The linker may be degradable or non-degradable. Degradable linkers are typically readily degraded in the intracellular environment, thereby releasing the therapeutic agent from the antibody. Suitable degradable linkers include enzymatically degradable linkers such as peptide-containing linkers that can be degraded by intracellular lysosomal proteases, or sugar linkers such as glucuronide-containing linkers that can be degraded by glucuronidases. Peptide linkers may include dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include pH-sensitive linkers (e.g., hydrazone linkers that are hydrolyzed below pH 5.5) and linkers that are degraded under reducing conditions (e.g., disulfide linkage linkers). Non-degradable linkers typically release drugs under conditions where the antibody is hydrolyzed by a protease.

[0054] The linker has a reactive group that can react with specific amino acid residues before linking to the antibody, and linkage is achieved via this reactive group. Mercapto-specific reactive groups are preferred, such as maleimide compounds, haloamides, haloesters, halomethyl ketones, benzyl halides, vinyl sulfones, pyridyl disulfide, mercury derivatives, and polymethylene dimethyl sulfide thiosulfonates. The linker may include, for example, a maleimide linked to the antibody via thiosuccinimide.

[0055] Preferably, the drug or toxin linked via the linker is selected from CL2A-SN-38 (CAS No.: 1279680-68-0), mc-vc-PAB-MMAE (CAS No.: 646502-53-6), Tesirine (SG3249, CAS No.: 1595275-62-9), Deruxtecan (CAS No.: 1599440-13-7), and Vc-seco-DUBA (SYD985, CAS No.: 1345681-58-4). The molecular structure is shown in the following figure. [ka]

[0056] In this invention, the anti-GPC3 antibody or its antigen-binding fragment is bound to SN-38 via a CL2A linker.

[0057] In the present invention, the anti-GPC3 antibody or its antigen-binding fragment is bound to MMAE via an mc-VC-PAB linker.

[0058] In the present invention, the anti-GPC3 antibody or its antigen-binding fragment is bound to the PBD dimer via a maleimide-dPEG8-VA-PABA linker.

[0059] Preferably, the drug may be a cytotoxic agent, a cell growth inhibitor, or an immunosuppressant. In the embodiment, the linker links the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug may have an amino group, carboxyl group, mercapto group, hydroxyl group, or ketone group capable of forming a bond with the linker. When the drug is directly linked to the linker, the drug has a reactive functional group before linking to the antibody.

[0060] Preferably, the cytotoxic agent is selected from the group consisting of antitubulin agents, DNA supraclution junction reagents, DNA replication inhibitors, DNA alkylating reagents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.

[0061] Preferably, particularly useful examples of cytotoxic agents include, for example, DNA supraclution-binding reagents, DNA alkylation reagents, and tubulin inhibitors. Typical cytotoxic agents include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBD), indolinobenzodiazepines, oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

[0062] Preferably, the toxin is an auristatin derivative (e.g., auristatin E, auristatin F, MMAE, and MMAF), oleomycin, methanesol, lysine, lysine A-chain, combretastatin, docamycin, dorastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, or vincristine. The following are selected from the group consisting of vinblastine, colchicine, dihydroxyanthracine dione, actinomycin, diphtheria toxin, Pseudomonas aeruginosa exotoxin (PE) A, PE40, abrin, abrin A chain, modesin A chain, α-octupoccocci, geronin, mitogellin, restrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria officinalis inhibitors, glucocorticoids, or combinations thereof.

[0063] Preferably, the drug or toxin is one or more selected from SN-38 (NK012, CAS No.: 86639-52-3), MMAE (Monomethyl auristatin E, CAS No.: 474645-27-7), PBD dimer (SG3199, CAS No.: 1595275-71-0), DX-8951 (Exatecan, CAS No.: 171335-80-1), or DUBA (duocarmycin-hydroxybenzamide-azaindole).

[0064] In the present invention, the anti-GPC3 antibody or its antigen-binding fragment is conjugated to DX-8951(DXd) via a maleimide-GGFG linker.

[0065] In this invention, the anti-GPC3 antibody or its antigen-binding fragment is bound to DUBA via a Vc-seco linker.

[0066] In another aspect, the present invention further provides the use of the anti-GPC3 antibody or its antigen-binding fragment, or the pharmaceutical composition of the present invention, or the antibody-drug conjugate of the present invention in the manufacture of a drug for treating or preventing cancer, wherein the cancer is preferably liver cancer.

[0067] In another embodiment, the use of the GPC3 antigen epitope peptide, recombinant antigen, nucleic acid molecule, vector, or host cell of the present invention is provided in any of the following: (1) To manufacture an anti-GPC3 antibody or its antigen-binding fragment, (2) Manufacturing a product for treating and / or preventing and / or diagnosing a disease related to GPC3 in a subject, preferably the disease being a GPC3-positive cancer. (3) To manufacture a product for detecting an anti-GPC3 antibody or its antigen-binding fragment, wherein the anti-GPC3 antibody comprises a heavy chain indicated by SEQ ID NO:11 and a light chain indicated by SEQ ID NO:10. (4) Detection of an anti-GPC3 antibody or its antigen-binding fragment, wherein the anti-GPC3 antibody comprises a heavy chain indicated by SEQ ID NO:11 and a light chain indicated by SEQ ID NO:10. (5) Screening for anti-GPC3 antibodies or their antigen-binding fragments.

[0068] In some embodiments, the disease is a GPC3-positive cancer, such as liver cancer, hepatic cancer, colorectal cancer, or ovarian cancer.

[0069] In one embodiment, the present invention provides a method for producing an anti-GPC3 antibody or an antigen-binding fragment thereof, comprising the step of stimulating the animal immune system with the GPC3 antigen epitope peptide, recombinant antigen, nucleic acid molecule, vector, or host cell of the present invention to cause the animal to produce an antibody.

[0070] In some embodiments, the animal is selected from mammals such as humans, mice, rabbits, monkeys, cattle, sheep, and alpacas.

[0071] In one embodiment, the present invention provides a method for screening an anti-GPC3 antibody or its antigen-binding fragment, comprising contacting the GPC3 antigen epitope peptide of the present invention with an antibody or its antigen-binding fragment to be analyzed, and detecting the binding of the GPC3 antigen epitope peptide to the antibody or its antigen-binding fragment, wherein if the GPC3 antigen epitope peptide is bound to the antibody or its antigen-binding fragment, the antibody or its antigen-binding fragment becomes a candidate for an anti-GPC3 antibody or its antigen-binding fragment.

[0072] In some embodiments, the anti-GPC3 antibody or its antigen-binding fragment binds to the GPC3 antigen epitope peptide, and the binding is measured by an ELISA method. In some embodiments, the ELISA method is as described in Example 12, in which the GPC3 antigen epitope peptide is coated onto an ELISA plate, the anti-GPC3 antibody or its antigen-binding fragment is grade-diluted, added to the ELISA plate and incubated, and after incubation is complete, washing, color development, stopping, and the OD value at 450 nm can be measured. In some embodiments, the specific binding of the GPC3 antigen epitope peptide to the anti-GPC3 antibody or its antigen-binding fragment is measured by the ELISA for the OD of the antigen-antibody complex. 450 This is reflected in the value. In some embodiments, when the GPC3 antigen epitope peptide concentration is 6 μg / mL, the OD when the anti-GPC3 antibody or its antigen-binding fragment is saturated. 450 The value is 1.5±0.1 or greater, or 2±0.1 or greater, or 2.5±0.1 or greater, or 3±0.1 or greater. In some embodiments, the OD 450 The values ​​are 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, or 3.9 or higher. Furthermore, the present invention further provides a method for producing the anti-GPC3 antibody or its antigen-binding fragment.

[0073] The DNA molecular sequences of the anti-GPC3 antibody or its antigen-binding fragment described in the present invention can be obtained using conventional techniques such as hybridoma PCR amplification or phage display library screening. Furthermore, the coding sequences of the light chain and heavy chain can be fused to form a single-chain antibody (e.g., scFV).

[0074] Once the relevant sequence is obtained, it can be cloned into a vector, transformed into a host bacterium, and then the vector can be extracted from the host bacterium using conventional methods.

[0075] Furthermore, the relevant sequences can also be artificially synthesized, especially if the fragment length is short. Currently, the DNA sequences encoding the antibodies (or fragments thereof, or derivatives thereof) of the present invention can be obtained entirely by chemical synthesis. Moreover, mutations can also be introduced into the protein sequences of the present invention by chemical synthesis.

[0076] The present invention also relates to vectors comprising the above-mentioned suitable DNA sequence and suitable promoter or regulatory sequence. These vectors can be used to transform suitable host cells and express proteins therein.

[0077] The anti-GPC3 antibody or its antigen-binding fragment of the present invention can be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, or other properties. These methods are well known to those skilled in the art. Typically, transformed host cells are cultured under conditions suitable for the expression of the antibody of the present invention, and then purified using conventional immunoglobulin purification steps such as protein A-Sepharose affinity chromatography, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or other conventional separation and purification methods, or combinations thereof, to obtain the anti-GPC3 antibody or its antigen-binding fragment of the present invention.

[0078] A preferred embodiment of the method for producing the anti-GPC3 antibody or its antigen-binding fragment according to the present invention is a method for separating and purifying the anti-GPC3 antibody or its antigen-binding fragment, which is a protein A affinity chromatography method, a cation exchange method, or an anion exchange method.

[0079] The obtained monoclonal or bispecific antibodies can be identified using conventional methods. For example, antibody binding specificity can be measured by immunoprecipitation or in vitro binding assays such as enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). Antibody binding affinity can be measured, for example, by Scatchard analysis as described in Munson et al., Anal. Biochem., 107:220 (1980).

[0080] In the present invention, the antibody-drug conjugate is The present invention involves reducing the interchain disulfide bonds of the anti-GPC3 antibody or its antigen-binding fragment to generate 2n (e.g., 2, 4, 6, or 8) mercapto groups, The steps include crosslinking a drug-linker compound with the mercapto group of the reduced antibody to generate the corresponding antibody-drug conjugate, It is produced by a method comprising the step of further purifying by ultrafiltration and desalting to obtain the product.

[0081] For clarity, this specification defines common terms used to describe compounds.

[0082] Unless otherwise specified, the following terms and phrases used herein shall have the following meanings. Unless otherwise explicitly defined, any particular term or phrase should not be considered ambiguous or unclear, but should be understood in its ordinary sense. Where a trade name is mentioned herein, it refers to the corresponding product or its active ingredient. As used herein, “pharmaceutically acceptable” means a compound, material, composition, and / or dosage form suitable, in proportion to a reasonable benefit-to-risk ratio, for use in contact with human and animal tissues, within the bounds of reliable medical judgment, without causing excessive toxicity, irritation, allergic reactions, or other problems or complications.

[0083] GPC3 (also known as phosphatidylinositol proteoglycan 3) is a member of the heparin sulfate proteoglycan family and is anchored to the cell membrane surface via glycosyl-phosphatidylinositol (GPI). The human GPC3 gene is located on the X chromosome (Xp26) and codes for a 70 kDa protein containing 580 amino acids. This protein is cleaved between Arg358 and Ser359 by a furin-like convertase, producing a 40 kDa N-terminal subunit and a 30 kDa C-terminal subunit, the C-terminal subunit which also contains two heparan sulfate (HS) chains.

[0084] As used herein, the term “antibody” includes complete antibodies, their antigen-binding fragments (i.e., “antigen-binding moieties”), or single-chain antibodies. A complete antibody is a glycoprotein containing two heavy chains (H chains) and two light chains (L chains) linked by disulfide bonds. Each heavy chain consists of a variable heavy chain region (abbreviated herein as VH) and a constant heavy chain region. The constant heavy chain region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a variable light chain region (abbreviated herein as VL) and a constant light chain region. The constant light chain region consists of one domain, CL. The VH and VL regions are further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)) separated by more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding regions that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissue or factors. These host tissues or factors include various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0085] The term “antigen-binding fragment” (or simply “antibody portion”) refers to one or more fragments of an antibody that specifically bind to an antigen (e.g., the GPC3 protein). The antigen-binding function of an antibody has been demonstrated to be realized by fragments of a full-length antibody. Examples of binding fragments encompassed by the term “antigen-binding fragment” of an antibody include: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide bonds at a hinge region; (iii) Fd fragments consisting of a VH domain and a CH1 domain; (iv) Fv fragments consisting of the VL and VH domains of one arm of the antibody; (v) dAb fragments consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity-determining regions (CDRs); and (vii) nanobodies containing a heavy chain variable region with a single variable domain and two constant domains. Furthermore, although the two domains VL and VH of the FV fragment are encoded by separate genes, they can be linked via a linker using recombination, thereby obtaining a single protein chain (called single-chain Fv (scFv)) in which the VL and VH regions pair up to form a monovalent molecule. See, for example, Bird et al., (1988) Science 242:423-426, and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies also fall within the scope of the term "antigen-binding fragment" in the term antibody. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragment screening performed for use is the same as for complete antibodies.

[0086] As used herein, “isolated antibody” means an antibody that substantially does not contain other antibodies with different antigen specificities. For example, an isolated antibody that specifically binds to the GPC3 protein substantially does not contain antibodies that specifically bind to antigens other than GPC3. However, in some examples, an isolated antibody that specifically binds to the human GPC3 protein may cross-react with other antigens (e.g., other types of GPC3 proteins). Furthermore, an isolated antibody may substantially not contain other cellular material and / or chemical substances.

[0087] As used herein, the term "monoclonal antibody" refers to a formulation of an antibody molecule having a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0088] The term "chimeric antibody" refers to an antibody created by combining genetic material from a non-human organism with genetic material from a human organism. More generally, a chimeric antibody refers to an antibody that contains genetic material from one species and genetic material from another species.

[0089] The terms "bispecificity" or "multispecificity" refer to the ability of an antibody and / or antigen-binding molecule to specifically bind to two or more different antigenic determinants. Typically, a bispecific or multispecific antibody or antigen-binding molecule has two antigen-binding sites, each specific to a different antigenic determinant. In some embodiments, the bispecific or multispecific antibody or antigen-binding molecule can simultaneously bind to two or more antigenic determinants, particularly two or more antigenic determinants expressed on two or more different cells.

[0090] As used herein, the term "humanized antibody" refers to an antibody derived from a non-human species whose protein sequence has been modified to increase its similarity to naturally occurring human antibody variants.

[0091] The term "antibody-drug conjugate" refers to a device that utilizes the characteristic of antibodies to specifically recognize certain antigens on the surface of tumor cells to precisely deliver antitumor therapeutic agents (e.g., cytotoxins or cell inhibitors, radioisotopes, small molecule chemotherapeutic agents, etc.) to tumor target cells, accumulate within the cells, and then release them, thereby achieving the objective of precisely killing tumor cells. ADCs are considered one of the most promising antitumor drugs because they have an appropriate molecular weight, high stability, potent targeting, and low toxicity and side effects. In addition to monoclonal antibodies, bispecific antibodies can also be conjugated to therapeutic agents. In some embodiments, the portion that conjugates to the antibody or bispecific antibody of the present invention to form an antibody-drug conjugate is a cytotoxin. The cytotoxin is a substance that inhibits or blocks cellular function and / or causes cell damage, and includes small molecule cytotoxins. In some embodiments, the cytotoxin is selected from SN-38, MMAE, PBD dimer, DX-8951 (DXd), or DUBA.

[0092] As used herein, the terms “includes,” “incorporates,” and “possesses” are used interchangeably to indicate the comprehensiveness of a scheme, meaning that such scheme may include elements other than those described herein. Furthermore, as used herein, the terms “includes,” “incorporates,” and “possesses” should be understood to also include schemes consisting of…

[0093] The term "antibody" as used herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, complete antibodies, antigen-binding fragments, naked antibodies, conjugated antibodies, humanized antibodies, or fully human antibodies.

[0094] As used herein, the term “epitope” refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. “Epitope” is also called an “antigenic determinant” in the art. Epitopes, or antigenic determinants, are typically composed of chemically active surface groups of molecules such as amino acids, carbohydrates, or sugar side chains, and usually possess specific three-dimensional structural and charge characteristics. For example, an epitope typically consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial structure that may be “linear” or “conformal.” See, for example, EpitopeMapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996). In linear epitopes, all interaction points between a protein and an interacting molecule (e.g., an antibody) are linear along the primary amino acid sequence of the protein. On the other hand, in three-dimensional structural epitopes, interaction points exist across protein amino acid residues that are far apart from each other.

[0095] As used herein, the term “epitope peptide” refers to a peptide segment on an antigen that can function as an epitope. In some cases, an epitope peptide alone can be specifically recognized / bound to an antibody targeting the epitope. In other cases, it may be necessary to fuse the epitope peptide with a carrier protein for it to be recognized by a specific antibody.

[0096] As used herein, the term "carrier protein" refers to a protein capable of functioning as a carrier for an epitope peptide. That is, an epitope peptide can be inserted at a specific location (e.g., inside the protein, at the N-terminus, or at the C-terminus), resulting in the presentation of the epitope peptide and recognition by an antibody or immune system.

[0097] In this specification, the term "conserved amino acid" generally refers to amino acids belonging to the same class or possessing similar properties (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain structure, rigidity, etc.). Exemplarily, amino acids belonging to each of the following groups are conserved amino acid residues, and substitutions of amino acid residues within a group constitute substitutions of conserved amino acids.

[0098] 1) Alanine (A), Serine (S), Threonine (T) 2) Aspartic acid (D), glutamic acid (E) 3) Asparagine (N), Glutamine (Q) 4) Arginine (R), Lysine (K), Histidine (H) 5) Isoleucine (I), leucine (L), methionine (M), valine (V), and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W) The terms “identity” and “sequence…identity” as used herein are interchangeable and are calculated as follows: To determine the percentage of “identity” between two amino acid sequences or two nucleic acid sequences, these sequences are aligned for optimal comparison (for example, caps may be introduced on one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be excluded for comparison). Next, amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0099] The "+" symbol used herein indicates a combination of mutations. The following terms are used to indicate mutations: S102R indicates that the 102nd amino acid residue of the parent sequence, serine (S), is substituted with arginine (R).

[0100] The term "subject" includes all humans and non-human animals. The term "non-human animal" includes all vertebrates, including mammals and non-mammals. Examples include non-human primates, rodents, rabbits, pigs, dogs, cats, chickens, amphibians, and reptiles, but mammals such as non-human primates and rodents are preferred.

[0101] The term "therapeutic dose" refers to an amount of the anti-GPC3 antibody or its antigen-binding fragment of the present invention sufficient to prevent or improve symptoms associated with a disease or condition (e.g., cancer) and / or reduce the severity of the disease or condition. The therapeutic dose should be understood in relation to the condition being treated, and the actual effective dose can be readily determined by those skilled in the art. The antibody of the present invention is a monoclonal antibody structurally and chemically characterized as described below and in the following examples. The amino acid sequence ID numbers (SEQ ID No:) of the variable and constant regions of the heavy / light chains of the antibody are summarized in Tables 1 and 5.

[0102] The heavy chain variable region CDRs and light chain variable region CDRs listed in Tables 1 and 5 are defined by the Kabat, Chothia, IMGT, AbM, or Contact numbering system / method. Exemplary CDR region sequences of the anti-GPC3 antibody of the present invention are shown in Table 3.

[0103] [Table 1] TIFF2026521594000004.tif238164 TIFF2026521594000005.tif238164 TIFF2026521594000006.tif227164

[0104] [Table 2]

[0105] [Table 3]

[0106] Other features and advantages disclosed in this invention will become apparent from the following detailed description and examples, but should not be construed as limiting. All references, Genbank entries, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference. [Brief explanation of the drawing]

[0107] [Figure 1A] The phage levels of the positive monoclonal antibodies screened in Example 1 and the ELISA detection results for GPC3-B peptide are shown. [Figure 1B] The phage levels of the positive monoclonal antibodies screened in Example 1 and the ELISA detection results for GPC3-B peptide are shown. [Figure 2] This shows the affinity of GC90 and its mutant monoclonal antibodies for the GPC3 protein. [Figure 3] This shows the affinity of GC90 and its mutant monoclonal antibodies for the GPC3 protein. [Figure 4] This shows the binding of humanized monoclonal antibodies GC90 and hYP7HM to human liver cancer cells HepG2, Hep3B, and Huh-7 at different concentrations. [Figure 5A] This shows the binding of GC90 and its mutant monoclonal antibodies to human liver cancer cells. [Figure 5B] This shows the binding of GC90 and its mutant monoclonal antibodies to human liver cancer cells. [Figure 5C] This shows the binding of GC90 and its mutant monoclonal antibodies to human liver cancer cells. [Figure 5D] This shows the binding of GC90 and its mutant monoclonal antibodies to human liver cancer cells. [Figure 6] This shows the uptake of humanized monoclonal antibodies GC90 and hYP7HM at 1 nM concentrations into human liver cancer cells HepG2 or Hep3B. [Figure 7] This demonstrates the killing of human liver cancer cells by a drug conjugate of GC90 and its monoclonal antibody mutant. [Figure 8A] This invention demonstrates in vivo inhibition of tumors by the antibody-drug conjugate. [Figure 8B] This invention demonstrates in vivo inhibition of tumors by the antibody-drug conjugate. [Figure 9] The results of detecting the binding strength of GC90 and each duplicate peptide by ELISA are shown. The underlined amino acid sequences correspond to the specific sequences of each duplicate peptide. [Figure 10] This shows the amino acid sequence alignment analysis of human GPC3 protein and mouse GPC3 protein. [Figure 11] This shows the distribution of GC90 and other reported anti-GPC3 antibodies in the antigen recognition region of the GPC3 protein. [Modes for carrying out the invention]

[0108] Example 1 Acquisition of a fully human antibody-positive clone molecule targeting a novel GPC3 epitope Phage library eluates were obtained by panning using a naturally derived, fully human phage display library and GPC3-B peptide (a humanized recombinant antibody library was screened using amino acid residues Met at position 478 to Asp at position 531 of the extracellular domain of the GPC3 protein as antigens). The neutralized phage panning eluates were added to the prepared TG1 bacterial suspension and mixed uniformly. The mixture was then allowed to stand at 37°C to infect TG1 host bacteria for 45 minutes. After sufficient infection, the bacterial suspension was gradient diluted and spread onto agar plates containing the corresponding antibiotics. These plates were then incubated overnight at 37°C. The following day, a sterile 96-well deep-well plate was prepared with 0.5 mL of 2YT medium (containing 0.2% w / v glucose and 0.1 mg / mL ampicillin). Single colonies cultured in the plate were selected using a sterile pipette tip and transferred to the corresponding well plate. These plates were then cultured overnight (16-18 hours) with shaking at 37°C and 220 rpm. The following day, transfer 0.05-0.1 mL of the single-clonal bacterial suspension cultured overnight to a newly prepared sterile deep-well plate (containing 0.5 mL of 2YT medium with a final concentration of 0.1 mg / mL ampicillin), and then OD 600 The cells were cultured until the value reached approximately 0.6-0.8. Helper phages were added at a fixed rate, mixed uniformly by shaking, and then allowed to stand at 37°C for 45 minutes to infect the cells. After that, 0.25 mL of 2YT medium (0.1 mg / mL ampicillin and a final concentration of 0.05 mg / mL kanamycin after addition) was added, and the cells were cultured overnight (16-18 hours) at 30°C and 220 rpm with shaking. The bacterial suspension that had been expressed overnight was centrifuged at 4000 rpm for 10 minutes to obtain the phage display expression supernatant. Using this supernatant, ELISA binding detection against the GPC3-B peptide and FACS binding detection against the corresponding cells were performed to obtain a positive fully human antibody clone.

[0109] The binding ability of phage display antibodies to GPC3-B peptide in the supernatant was evaluated using an indirect ELISA method. ELISA plates were coated overnight at 4°C with 100 μL / well of CBS coating reagent containing 4 μg / mL GPC3-B peptide. The plates were washed with PBST (containing 0.05% Tween) and blocked at 37°C for 1 hour with 300 μL / well of PBS containing 3% skim milk. The blocking solution was then discarded, and 50 μL of serially diluted phage expression supernatant, 50 μL of 0.05% PBST, and a negative control (LP1: ipilimumab) were added to each plate, followed by incubation at room temperature for 2 hours. The plates were washed three times with 0.05% PBST, and 100 μL / well of horseradish peroxidase-conjugated goat anti-M13 phage antibody (Gikyo Co., Ltd.) was incubated at room temperature for 45 minutes. The plate was washed six times with 0.05% PBST, TMB chromogenic solution (GenScript) was added, and the plate was incubated at room temperature in the dark for 10 minutes. The reaction was stopped by adding 50 μl of 1 M HCl stop solution (Sigma). The plate was read at 450 nm using a microplate reader. The results of the binding activity detection are shown in Figures 1A-1B, demonstrating that multiple positive monoclonal antibodies had good binding activity to the GPC3-B peptide at the phage level.

[0110] The recognition of the GPC3-A peptide by the positive monoclonal antibodies obtained from screening was further investigated by ELISA. The specific procedure was as follows: GPC3-A peptide and GPC3-B peptide (6 μg / mL) were coated into 96-well plates at 100 μL / well and incubated overnight at 4°C. Next, the plates were blocked at 37°C for 2 hours with PBST containing 1% BSA (containing 0.05% Tween-20) (200 μL / well) and washed three times with PBST. The positive monoclonal antibodies GC008, GC010, GC011, GC025, GC035, GC037, GC053, GC067, GC139, GC147, GC90, and the control antibody hYP7HM were each grade-diluted with PBST containing 1% BSA and then sequentially added to 96-well plates (100 μL / well). The working concentrations were 30 nM and 15 nM. For concentrations below 15 nM, the solutions were diluted 4-fold in eight steps. The solutions were incubated at 37°C for 1 hour and washed three times with PBST. Next, 100 μL / well of anti-human IgG-FC-HRP (Sigma, 1 / 30000 dilution) was added, incubated at 37°C for 1 hour, washed three times with PBST, and then 50 μL of TMB (SURMOPICS) was added to allow the reaction to proceed. The reaction was stopped with 1 M H2SO4. The OD value at 450 nm was measured using a microplate reader. The results are shown in Table 4. The positive monoclonal antibodies screened in this example specifically bind only to the GPC3-B peptide, while the control antibody hYP7HM recognizes only the GPC3-A peptide (consistent with the patent document). This indicates that the positive monoclonal antibodies screened in this example have a different antigen recognition site than the control anti-GPC3 antibody hYP7HM.

[0111] [Table 4]

[0112] The positive clones screened in this example were sequenced, and the amino acid sequences of the heavy chain variable region and light chain variable region of the antibody are shown in Table 5.

[0113] [Table 5] TIFF2026521594000011.tif196164

[0114] Example 2 Production of humanized monoclonal antibodies 2.1 Vector Construction First, a nucleotide sequence encoding the human IgG1 heavy chain constant region (IgG1-CH) was ligated with a secretory signal peptide (SP) encoding sequence at the beginning and a stop codon (TAG) at the end to synthesize the gene. This gene was then cloned into a pcDNA3.1 vector (Shanghai Biotechnology Co., Ltd.) and inserted downstream of the CMV promoter in the pCHOGUN vector using in-fusion cloning. Specifically, primers with a particular insertion site and a high-fidelity PCR enzyme (HiFi PCR Premix, TAKARA) were designed, and the synthesized gene fragment and the pCHOGUN vector plasmid fragment were amplified, respectively. After gel recovery, the IgG1 heavy chain gene fragment and the linearized vector fragment were ligated (In-fusion Snap Assembly Master Mix, TAKARA) to obtain the heavy chain constant region vector pCHOGUN-IgG1. Following the above procedure, a signal peptide (SP) coding sequence was added to the leading end of the coding sequence of the human immunoglobulin κ light chain constant region (IgG-CK), and a stop codon (TAG) was added to the trailing end. After synthesizing the gene, it was inserted downstream of the CMV promoter in the pCHOGUN vector to obtain the light chain constant region vector pCHOGUN-CK.

[0115] For the construction of each human monoclonal antibody expression vector, the coding sequences of the heavy chain variable region (VH) and light chain variable region (VL) of the GC90 monoclonal antibody and its optimized mutant were synthesized. These sequences were then inserted between the SP and constant region of the pCHOGUN-IgG1 or pCHOGUN-CK vector according to the in-fusion cloning method described above to obtain the heavy chain and light chain expression vectors for the monoclonal antibodies. The specific antibody sequences are shown in Table 6.

[0116] [Table 6] TIFF2026521594000013.tif242164 TIFF2026521594000014.tif243164 TIFF2026521594000015.tif29164

[0117] 2.2 Cell transfection expression Transfection was performed according to the instructions for TransIT-PRO® Transfection Reagent (Mirus). As described above, ExpiCHO-S cells (Thermo) were cultured in complete medium (CHOgro® High Yield Expression System, containing 30 mL / L Poloxamer 188 solution 10% and 20 mL / L L-glutamine 200 mM, Mirus). Subculture was performed 24 hours before transfection, and the cell density the following day was 4 × 10⁶ 6 To achieve a cell / mL ratio, add 2 × 10 cells. 6The cells were diluted to 1 / mL. Light chain and heavy chain plasmids were added in a 1:1 ratio of 25 μg each to 12.5 mL of complete medium and mixed uniformly. Next, 50 μL of TransIT-PRO® Transfection Reagent (Mirus) was added, and after gentle inversion mixing, it was allowed to stand for 4 minutes. Then, while shaking, it was added dropwise to 50 mL of diluted cells, followed by the addition of 1 mL of CHOgro-titer Enhancer (Mirus). Immediately after the completion of transfection, the cells were placed in an incubator at 32°C and 5% CO2. 5% Efficient Feed C + AGT Supplement (Thermo) was added every other day, and the cells were cultured for a total of 7 days.

[0118] 2.3 Purification of Antibodies After culturing in a shaking flask for 7 days, the cell supernatant was collected, centrifuged at 4000 rpm for 20 minutes, and the supernatant was removed and filtered through a 0.22 μm filtration membrane (Milipore) for purification by protein A affinity chromatography. In short, a HiTrap Mabselect suRe prepacked column (Cytiva) was equilibrated with 5 to 10 times the column volume of 20 mM PB + 0.15 M NaCl buffer. Using an AKTA Avant 150 chromatography system (Cytiva), the filtered supernatant was loaded, and the purified column was then rinsed with 3 times the column volume of 20 mM PB + 0.15 M NaCl buffer, and then 1 time the column volume of 20 mM MPB + 1 M NaCl buffer, and finally washed with 20 mM PB until the baseline stabilized. Finally, the antibody was eluted with 20 mM citrate (adjusted to pH 3.0 with 20 mM sodium citrate), and the peak shape of 200 mAu-200 mAu was collected. The eluted antibody was immediately neutralized with neutralizing buffer (1 M Tris-HCl, pH 9.0), transferred to a 1.5 mL tube, and frozen at -80°C for later use.

[0119] Example 3 ELISA detection of affinity of humanized monoclonal antibodies The relative binding activity of each antibody to human GPC3 protein was measured by ELISA. The specific procedure was as follows: Recombinant human GPC3 protein (1 μg / mL) was coated onto 96-well plates at 100 μL / well and incubated overnight at 4°C. Subsequently, the plates were blocked at 37°C for 2 hours using PBST containing 1% BSA (containing 0.05% Tween-20) (200 μL / well). After washing three times with PBST, each humanized monoclonal antibody was grade-diluted with PBST containing 1% BSA and then sequentially added to 96-well plates (100 μL / well). The working concentrations of the humanized monoclonal antibodies GC90, GC90-4mu, GC90LH1, GC90LH2, GC90LH3, GC90LH4, and GC90LH5 were 15 nM and 3.75 nM. For concentrations below 3.75 nM, eight 4-fold dilutions were performed. The plates were incubated at 37°C for 1 hour and washed three times with PBST. Next, 100 μL / well of anti-human IgG-FC-HRP (Sigma, 1 / 30000 dilution) was added, incubated at 37°C for 1 hour, washed three times with PBST, then 50 μL of TMB (SURMOPICS) was added and the reaction was stopped with 1 M H2SO4, and the OD values ​​at 450 nm to 570 nm were measured using a microplate reader. The results in Figure 2 show that the GC90 monoclonal antibody and its mutants of the present invention have excellent binding affinity to the GPC3 protein.

[0120] Example 4 ELISA detection of affinity-matured monoclonal antibodies The relative binding activity of each antibody to human GPC3 protein was measured by ELISA. The specific procedure was as follows: Recombinant human GPC3 protein (1 μg / mL) was coated onto 96-well plates at 100 μL / well and incubated overnight at 4°C. Next, the plates were blocked at 37°C for 2 hours using PBST containing 1% BSA (containing 0.05% Tween-20) (200 μL / well). After washing three times with PBST, each humanized monoclonal antibody was grade-diluted with PBST containing 1% BSA and added sequentially to 96-well plates (100 μL / well). The working concentrations of the humanized monoclonal antibodies GC90-4mu, GC90LH6, GC90LH7, and GC90LH8 were 10000 ng / μL and 3333.33 ng / μL, respectively. For concentrations below 1111.11 ng / μL, 10 step-by-step dilutions were performed. The working concentrations of GC90LH8 and GC90LH10 were 10,000 ng / μL and 2,500 ng / μL, respectively. For concentrations below 625 ng / μL, eight 4-fold dilutions were performed. The solutions were incubated at 37°C for 1 hour and washed three times with PBST. Next, 100 μL / well of anti-human IgG-FC-HRP (Sigma, 1 / 30000 dilution) was added, incubated at 37°C for 1 hour, washed three times with PBST, then 50 μL of TMB (SURMOPICS) was added, and the reaction was stopped with 1 M H2SO4. The OD values ​​at 450 nm to 570 nm were measured using a microplate reader. The results in Figure 3 show that the GC90 monoclonal antibody and its mutants of the present invention have excellent binding affinity to the GPC3 protein.

[0121] Example 5 Flow cytometry detection of binding of humanized monoclonal antibodies to tumor cells The affinity of each anti-GPC3 antibody for HepG2, Hep3B, and Huh7 cells was measured using flow cytometry. The specific procedure was as follows: Each humanized monoclonal antibody was grade-diluted in FACS buffer (PBS + 5% FBS) and added sequentially to a 96-well U-shaped plate. The specific procedure was as follows: The working concentrations of each humanized monoclonal antibody were 100 nM and 25 nM. For concentrations below 6.25 nM, six 4-fold dilutions were performed. HepG2, Hep3B, and Huh7 cells were digested with trypsin, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were added to FACS-containing buffer in a 2 × 10⁶ ratio. 6 The antibody was resuspended at a concentration of cells / mL, and 50 μL was added to each well of a 96-well U-shaped plate. Then, 50 μL of the diluted antibody was added to each well, and the mixture was gently and uniformly mixed. The cells were incubated on ice for 90 minutes. After centrifugation at 4°C and 3500 rpm for 3 minutes, the supernatant was discarded. Next, 250 μL of pre-cooled FACS buffer was added to resuspend the cells, and centrifugation and washing were repeated twice. Diluted PE anti-human IgGFC fluorescent secondary antibody (BioLegend, 0.5 μL / 1 × 10⁶) was added. 5 100 μL of the cell-prepared antibody was added to each well and incubated on ice in the dark for 30 minutes. The supernatant was discarded and the cells were washed twice. Finally, the cells were resuspended in 200 μL of FACS buffer. MFI values ​​were detected using an Attune NxT flow cytometer (Thermo), and the data were processed using GraphPad Prism software. The results in Figure 4 show that the monoclonal antibody of the present invention has excellent affinity for HepG2, Hep3B, and Huh-7 cells.

[0122] Example 6 Flow cytometry detection of binding of humanized monoclonal antibodies to tumor cells The affinity of each anti-GPC3 antibody for HepG2 or Hep3B cells was measured by flow cytometry. The specific procedure was as follows: Each humanized monoclonal antibody was grade-diluted in FACS buffer (PBS + 5% FBS) and added sequentially to a 96-well U-shaped plate. The specific procedure was as follows: The working concentrations of GC90-4mU, GC90, GC90LH1, GC90LH2, GC90LH3, GC90LH4, and GC90LH5 were 100 nM and 25 nM. For concentrations below 6.25 nM, eight 4-fold dilutions were performed. HepG2 and Hep3B cells were digested with trypsin, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were added to FACS buffer in 2 × 10⁶ units. 6 The antibody was resuspended at a concentration of cells / mL, and 50 μL was added to each well of a 96-well U-shaped plate. Then, 50 μL of the diluted antibody was added to each well, and the mixture was gently and uniformly mixed. The cells were incubated on ice for 90 minutes. After centrifugation at 4°C and 3500 rpm for 3 minutes, the supernatant was discarded. Next, 250 μL of pre-cooled FACS buffer was added to resuspend the cells, and centrifugation and washing were repeated twice. Diluted PE anti-human IgGFC fluorescent secondary antibody (BioLegend, 0.5 μL / 1 × 10⁶) was added. 5 100 μL of the cell-prepared antibody was added to each well and incubated on ice in the dark for 30 minutes. The supernatant was discarded and the cells were washed twice. Finally, the cells were resuspended in 200 μL of FACS buffer. MFI values ​​were detected using an AttuneNxT flow cytometer (Thermo), and the data were processed using GraphPad Prism software. The results are shown in Figures 5A-5B. The monoclonal antibody of the present invention was shown to have very good affinity for both HepG2 cells and Hep3B cells.

[0123] Example 7 Flow cytometry detection of binding of affinity-matured monoclonal antibodies to tumor cells The affinity of each anti-GPC3 antibody for HepG2 cells was measured using flow cytometry. The specific procedure was as follows: Each humanized monoclonal antibody was grade-diluted in FACS buffer (PBS + 5% FBS) and added sequentially to a 96-well U-shaped plate. The working concentrations of IgG1, GC90-4mu, GC90LH6, GC90LH7, GC90LH8, GC90LH9, GC90LH10, ​​and GC90LH11 were 100 nM and 25 nM. For concentrations below 6.25 nM, 8-9 step dilutions were performed in 4-fold increments. HepG2 cells were digested with trypsin, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were added to FACS buffer in 2 × 10⁶ cells. 6 The antibody was resuspended at a concentration of cells / mL. 50 μL was added to each well of a 96-well U-shaped plate, then 50 μL of the diluted antibody was added to each well, gently mixed uniformly, and incubated on ice for 90 minutes. After centrifugation at 4°C and 3500 rpm for 3 minutes, the supernatant was discarded. Next, 250 μL of pre-cooled FACS buffer was added to resuspend the cells, and centrifugation and washing were repeated twice. Diluted PE anti-human IgGFC fluorescent secondary antibody (BioLegend, 0.5 μL / 1 × 10⁶) 5 100 μL of the prepared antibody (prepared in cells) was added to each well and incubated on ice in the dark for 30 minutes. The supernatant was discarded and the cells were washed twice. Finally, the cells were resuspended in 200 μL of FACS buffer. MFI values ​​were detected using an Attune NxT flow cytometer (Thermo), and the data was processed using GraphPad Prism software. The results are shown in Figures 5C-5D. The monoclonal antibody of the present invention was shown to have excellent affinity for HepG2 cells.

[0124] Example 8 Flow cytometry detection of the internalization efficiency of humanized monoclonal antibodies The internalization efficiency of each anti-GPC3 antibody was measured in HepG2 and Hep3B cells using flow cytometry. The specific procedure was as follows: GC90 and hYP7HM monoclonal antibodies were diluted to an experimental concentration of 1 nM in FACS buffer and added to 50 μL / well of a 96-well U-shaped plate. Antibody binding to cells was performed as in Example 5. After washing away unbound antibodies, the cells were transferred to 37°C and incubated for 0, 1, 2, and 4 hours, respectively. Immediately after incubation, 100 μL of pre-cooled FACS buffer was added to stop internalization. Then, the cells were centrifuged at 3500 rpm for 3 minutes, and the supernatant was discarded. Secondary antibodies were prepared as described in Example 5, and cells were resuspended in 100 μL of secondary antibody in each well and incubated on ice for 30 minutes. The cells were centrifuged twice, and finally resuspended in 200 μL of FACS buffer. The MFI value was detected using an instrument, and the data was processed using GraphPad Prism software. The intracellular internalization efficiency of the humanized monoclonal antibody was calculated using the formula (MFI value of the antibody at 1-37°C / MFI value of the antibody at 4°C) × 100%. The results are shown in Figure 6. The results in Figure 6 demonstrate that the humanized monoclonal antibody of the present invention has excellent internalization efficiency.

[0125] Example 9 Manufacturing of antibody-drug conjugates 9.1 Site-specific binding By mutating the residue at position 239 of the antibody heavy chain to cysteine ​​(S239C, EU code, corresponding to position 243 of the GC90-4mu heavy chain), site-specific binding to the drug was achieved via the linker through this cysteine. Specifically, the monoclonal antibody stock solution was replaced with 20 mM PBS buffer (pH=7.2) and the concentration was adjusted to approximately 5 mg / mL. A 250 mM EDTA solution was added in a volume ratio of 50:1 (antibody:EDTA) and thoroughly mixed. Next, depending on the combination of different monoclonal antibodies and different linker payloads (LPs), tris(2-carboxyethyl)phosphonate (TCEP) in an excess molar ratio of 1 to 12 times (relative to antibody) was added, thoroughly mixed, and the reduction reaction was carried out at room temperature (25°C) for 3 hours. An appropriate amount of DMSO was added to the above reaction solution, and then LP drug (5 mM / 10 mM pre-dissolved in DMSO) in an excess molar ratio of 6 to 12 times (relative to the antibody) was added, ensuring that the volume percentage of DMSO in the reaction system did not exceed 15%, and the mixture was thoroughly mixed. The reaction was then allowed to proceed at room temperature for 1.5 hours. After that, N-acetyl-L-cysteine ​​(NAC) solution was added, and the mixture was allowed to stand at room temperature for 10 minutes to stop the reaction.

[0126] The reaction mixture was desalted by ultrafiltration, transferred to a 10KD ultrafiltration tube (Millipore), supplemented with PBS buffer (pH 6.0), and concentrated to the desired volume by centrifugation at 3500g. This centrifugation and concentration process was repeated five times with additional PBS. The product was filtered through a 0.22μm filtration membrane (Millipore) and stored at -80°C.

[0127] Following the binding reaction, site-specific binding products of IgG1-PBD, GC90-4mu-PBD, GC90LH8-PBD, GC90LH9-PBD, GC90LH10-PBD, GC90LH11-PBD, IgG1-DUBA, GC90-4mu-DUBA, and hYP7HM-DUBA were obtained. The purity of the ADC products was analyzed by size exclusion chromatography (SEC), and the drug-antibody binding ratio (DAR) and the proportion of unbound antibodies were analyzed by hydrophobic interaction chromatography (HIC).

[0128] [Table 7]

[0129] 9.2 Random Joint The monoclonal antibody stock solution was replaced with 20 mM NaAc-HAc buffer (pH 5.5) and the concentration was adjusted to 5 ± 1 mg / mL. The required amount of tris(2-carboxyethyl)phosphine (TCEP) was added to the reaction system so that the molar ratio of tris(2-carboxyethyl)phosphine to antibody was 20:1, and after homogeneous mixing, the reduction reaction was carried out at 37°C for 3 hours. After the reduction reaction, the mixture was centrifuged in a 10 kD ultrafiltration centrifuge tube and replaced with 20 mM NaAc / Tris, 1 mM EDTA, pH 7.0 buffer (3500 × g, 4 times) (3500 × g, 4 times) to adjust the concentration to 5 ± 1 mg / mL. An appropriate amount of DMSO (approximately 10%) was added to the above reaction solution, and the required amount of LP solution (5-10 mM, pre-dissolved in DMSO) was added to the reaction system so that the molar ratio of LP drug to antibody was 10:1, and after homogeneous mixing, the binding reaction was carried out at 21-25°C for 1 hour. Next, N-acetylcysteine ​​(NAC) solution was added and mixed uniformly to achieve a molar ratio of 40:1 between N-acetylcysteine ​​(NAC) and antibody. The reaction was then stopped at 21-25°C for 1 hour. After the stopping reaction was complete, the solution was replaced with 20 mM His / His-HCl, pH 6.0 buffer (3500 × g, 4x) in a 10 kDa ultrafiltration centrifuge tube to adjust to the target concentration. After filtration through a 0.22 μm sterile filter, the solution was stored at -20°C or below.

[0130] After the binding reaction, randomly bound products of IgG1-Dxd and GC90-4mu-Dxd were obtained. The purity of the ADC products was analyzed by size exclusion chromatography (SEC), and the drug-antibody binding ratio (DAR) and the proportion of unbound antibodies were analyzed by hydrophobic interaction chromatography (HIC).

[0131] Example 10 Detection of antibody-drug conjugate killing of tumor cells The killing effects of various antibody-drug conjugates (ADCs) against HepG2, Hep3B, and Huh7 liver cancer cells were measured using Cell Counting Kit-8 (Dojindo). The specific procedure was as follows: HepG2, Hep3B, and Huh7 cells were cultured in 10% FBS (Gibco) + DMEM medium (Corning). When the degree of cell confluence reached 75% or higher, the cells were digested with trypsin (0.25% trypsin-EDTA) and the cell count was recorded. Cells were placed in 96-well plates in a 1.5 × 10⁶ format. 4 Cells / mL were plated at 160 μL / well (2400 cells / well) and incubated overnight at 37°C and 5% CO2. Next, various antibody-drug conjugates were diluted to 333.5 nM using 10% FBS + DMEM medium and added to 96-well plates containing 160 μL / well, resulting in a 5-fold dilution. Starting with an initial concentration of 66.7 nM, eight consecutive dilutions were performed, creating duplicate wells. Subsequently, the cells were incubated in a 37°C, 5% CO2 incubator. On day 4, tumor cell viability was detected using Cell Counting Kit-8. Specific cytotoxicity results are shown in Figure 7. The antibody-drug conjugates of the present invention exhibit good cytotoxicity against various types of liver cancer cells as the drug concentration increases.

[0132] Example 11 11.1 Antitumor Test of Test Substance 1 Creation of a tumor-bearing nude mouse model by subcutaneous xenografting of the human liver cancer cell line Hep3B. Hep3B cells in the exponential growth phase were harvested, and the cells were resuspended in a 1:1 mixture of PBS and matrix gel to achieve a cell density of 5 × 10⁶. 7 Adjusted to 5x10 6 Hep3B cells (0.1 mL / mice) were subcutaneously inoculated into the right anterior dorsal region of experimental mice. Tumor growth was observed periodically. The average volume was 100 mm³. 3When the tumors grew to a certain size, the mice were randomly divided into groups of six based on tumor size. The day of group division was designated as day 0. On the day of group division, IgG1-PBD or GC90-4mu-PBD was administered once via tail vein injection at a dose of 1.5 mg / kg. Tumor volume was measured twice a week. The formula for calculating volume is as follows: Tumor volume (mm 3 ) = 1 / 2 × (a × b 2 (a represents the major axis, b represents the minor axis)

[0133] Results: The data showed that GC90-4mu-PBD (1.5 mg / kg) had a significant antitumor effect (Figure 8A).

[0134] 11.2 Antitumor Test of Test Substance 2 Creation of a tumor-bearing nude mouse model by subcutaneous xenografting of the human liver cancer cell line Hep3B. Hep3B cells in the exponential growth phase were harvested, and the cells were resuspended in a 1:1 mixture of PBS and matrix gel to achieve a cell density of 5 × 10⁶. 7 Adjusted to 5x10 6 Hep3B cells (0.1 mL / mice) were subcutaneously inoculated into the right anterior dorsal region of experimental mice. Tumor growth was observed periodically. The average volume was 100 mm³. 3 When the tumors grew to a certain size, the mice were randomly divided into groups of six based on tumor size. The day of group division was designated as day 0. On the day of group division, IgG1-DUBA, hYP7HM-DUBA, or GC90-4mu-DUBA were administered once via tail vein injection at 5 or 10 mg / kg each. Tumor volume was measured twice a week. The volume calculation formula is as follows: Tumor volume (mm 3 ) = 1 / 2 × (a × b 2 (a represents the major axis, b represents the minor axis)

[0135] Results: The data showed that both GC90-4mu-DUBA (5 mg / kg) and hYP7HM-DUBA (10 mg / kg) had significant antitumor effects (Figure 8B).

[0136] 11.3 Antitumor Test of Test Substance 3 We created a tumor-bearing nude mouse model by subcutaneous xenografting of the human liver cancer cell line Hep3B.

[0137] Hep3B cells in the exponential growth phase were harvested, and the cells were resuspended in a 1:1 mixture of PBS and matrix gel to achieve a cell density of 5 × 10⁶. 7 Adjusted to 5x10 6 Hep3B cells (0.1 mL / mice) were subcutaneously inoculated into the right anterior dorsal region of experimental mice. Tumor growth was observed periodically. The average volume was 80-150 mm². 3 (Main experimental group) or 300-500 mm 3 When the tumors grew to the size of a satellite group, the mice were randomly divided into groups of six (one mouse in each of the IgG-PBD satellite group) based on tumor size and body weight. Day 0 was defined as the second day after group division. On day 0, the satellite group received a single tail vein injection of GC90-4mu-PBD and IgG1-PBD at a dose of 1.5 mg / kg. In the IgG-PBD satellite group, the maximum volume of blood was collected 24 hours after administration to collect serum, and the tumors were removed. In the GC90-4mu-PBD group, blood collection and tumor removal were performed alternately (serum was collected after 15 minutes, and serum and tumors were collected after 7 hours, on D1, D2, D3, D5, and D7). In the main experimental group, IgG1-PBD (0.6 mg / kg), GC90-4mu-PBD (0.1, 0.3, 0.6 mg / kg), or GC90-4mu-Dxd (3, 10 mg / kg) were administered once via tail vein injection on day 0. Mouse body weight and tumor size were measured twice a week. The formula for calculating tumor volume is as follows: Tumor volume (mm 3 ) = 1 / 2 × (a × b 2 (a represents the major axis, b represents the minor axis)

[0138] Example 12 Characterization of GC90 antibody function 12.1 Cross-reactions between species The binding of GC90 to different species of GPC3 proteins was measured by ELISA. The specific procedure was as follows: Each GPC3 protein (6 μg / mL) was coated into each well of a 96-well plate at a rate of 100 μL / well and incubated overnight at 4°C. Next, the plates were blocked at 37°C for 2 hours with PBST containing 1% BSA (containing 0.05% Tween-20) (200 μL / well). After washing three times with PBST, GC90 was grade-diluted with PBST containing 1% BSA and added sequentially to the 96-well plate (100 μL / well). The working concentrations were 30 nM and 15 nM. Below 15 nM, eight 4-fold dilutions were performed. The plates were incubated at 37°C for 1 hour and washed three times with PBST. Next, 100 μL / well of anti-human IgG-FC-HRP (Sigma, 1 / 30000 dilution) was added, incubated at 37°C for 1 hour, washed three times with PBST, then 50 μL of TMB (SURMOPICS) was added and the reaction was stopped with 1 M H2SO4. The OD value at 450 nm was measured using a microplate reader. The results are shown in Table 8.

[0139] [Table 8]

[0140] 12.2 Precise localization of the recognized area The amino acid sequence of the GPC3-B peptide was split into multiple duplicate peptides. The amino acid sequences of the duplicate peptides are shown in Table 9. The duplicate peptides were synthesized, and the binding properties to GC90 were measured for each peptide by ELISA to further accurately identify the antigen epitopes recognized by GC90. The specific processing was as follows: Each duplicate peptide (6 μg / mL) was coated into a 96-well plate at 100 μL / well and incubated overnight at 4°C. Next, the plates were blocked at 37°C for 2 hours using PBST containing 1% BSA (containing 0.05% Tween-20), and washed three times with PBST (200 μL / well). GC90 was grade-diluted with PBST containing 1% BSA (negative control: 1% BSA) and added sequentially to the 96-well plate (100 μL / well). The working concentrations were 30 nM and 15 nM. For concentrations below 15 nM, eight 4-fold dilutions were performed. The mixture was incubated at 37°C for 1 hour and washed three times with PBST. Next, 100 μL / well of anti-human IgG-FC-HRP (Sigma, 1 / 30000 dilution) was added, incubated at 37°C for 1 hour, washed three times with PBST, and then 50 μL of TMB (SURMOPICS) was added and the reaction was allowed to proceed. The reaction was stopped with 1 M H2SO4. The OD value at 450 nm was measured using a microplate reader. The results are shown in Figure 9. OD of the measured antigen-antibody complex. 450 The values ​​were used to indicate the binding strength between GC90 and each peptide. The results showed that the antibody exhibited strong binding activity only to GPC3-B3 or long fragments containing GPC3-B3. The GPC3 antigen epitope region was determined to be located in GPC3-B3, specifically at human GPC3 protein residues 485-496 (DKNLDEEGFESG).

[0141] [Table 9]

[0142] Furthermore, analysis of the homology between human GPC3 protein and mouse GPC3 protein based on amino acid sequences (Figure 10) revealed two amino acid residue differences between the two in the GPC3-B3 region (see GenbankID:XP_005594665.1 for monkey GPC3 protein, including GPC3-B3), namely the amino acid residues at position 487 (N) and position 493 (F) of the human GPC3 protein. Combining this with cross-detection results between species, it was found that mutations occurred in the mouse GPC3 protein at positions corresponding to asparagine at position 487 and phenylalanine at position 493 of the human GPC3 protein, preventing GC90 from binding to the mouse GPC3 protein. This suggests that positions 487 and / or 493 of the human GPC3 protein are one of the core binding sites of the antigen epitope region.

[0143] Figure 11 summarizes the antigen recognition regions of various anti-GPC3 antibodies on the GPC3 protein. The antigen epitope to which the antibody of the present invention binds is significantly different from the antigen-binding regions of other anti-GPC3 antibodies reported to date, revealing that it recognizes a novel GPC3 antigen epitope. The present invention identifies a novel GPC3 antigen epitope, and the antibody that binds to this epitope has high affinity and high internalization efficiency. The production of immunogens using this GPC3 antigen epitope opens up new research directions in the field of anti-GPC3 antibodies, reduces uncertainty in antibody production and screening, and is advantageous for the development of novel antibodies with improved function.

[0144] Although the present invention has been described by one or more embodiments, it should be understood that the present invention is not limited to these embodiments, and that the specification of the present invention is intended to encompass all alternatives, modifications, and changes that fall within the spirit and broad scope of the appended claims. All references cited in the present invention are incorporated into the present invention as a whole by reference.

Claims

1. An anti-GPC3 antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), The heavy chain variable region (VH) includes HCDR1, HCDR2, and HCDR3 regions, and each of the HCDR1, HCDR2, and HCDR3 regions includes a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the CDR1, CDR2, and CDR3 regions of the amino acid sequence indicated by SEQ ID No: 1-2 or 6-9. Alternatively, the sequence may contain at most three, two, or one mutation compared to the CDR1, CDR2, and CDR3 regions of the amino acid sequence indicated by SEQ ID No: 1-2 or 6-9, respectively. The light chain variable region (VL) includes the LCDR1, LCDR2, and LCDR3 regions, and each of the LCDR1, LCDR2, and LCDR3 regions includes a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the CDR1, CDR2, and LCDR3 regions of amino acid sequence SEQ ID No:

3. Alternatively, an anti-GPC3 antibody or its antigen-binding fragment is characterized by containing sequences in which at most three, two, or one mutation has occurred compared to the CDR1, CDR2, and CDR3 regions of amino acid sequence SEQ ID NO:3, respectively.

2. The antibody or antigen-binding fragment according to claim 1, characterized in that the antibody or antigen-binding fragment has a biological function of specifically binding to the antigen indicated by SEQ ID No: 37 or 38 and not binding to the antigen indicated by SEQ ID No:

36.

3. The location of the mutation is one or more selected from position 56 (D56), position 100 (Q100), or position 102 (S102) of the amino acid sequence indicated by SEQ ID No: 1-2 or 6-9. Preferably, the mutation is selected from Q100R, S102R, D56A, D56K+Q100R, D56A+S102R, or D56K+S102R, the antibody or antigen-binding fragment according to claim 1.

4. The antibody or antigen-binding fragment according to claim 3, characterized in that the HCDR1, HCDR2, and HCDR3 regions each contain the same sequences as the CDR1, CDR2, and CDR3 regions of the amino acid sequence indicated by any of SEQ ID No: 1-2, 6-9, or 23-28, and the LCDR1, LCDR2, and LCDR3 regions each contain the same sequences as the CDR1, CDR2, and CDR3 regions of the amino acid sequence SEQ ID No:

3.

5. The antibody or antigen-binding fragment according to any one of claims 1 to 4, characterized in that the CDR1, CDR2, and CDR3 regions are defined by the IMGT, Kabat, Chothia, AbM, or Contact method.

6. The VH includes a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence indicated by any of SEQ ID No: 1-2, 6-9, or 23-28, and / or The antibody or antigen-binding fragment according to claim 5, characterized in that the VL contains a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID No:

3.

7. The antibody or antigen-binding fragment according to claim 6, characterized in that VH contains the same sequence as any of amino acid sequences SEQ ID No: 1-2, 6-9, or 23-28, and VL contains the same sequence as amino acid sequence SEQ ID No:

3.

8. The antibody or antigen-binding fragment according to any one of claims 1 to 7, further comprising a heavy chain constant region (CH) and a light chain constant region (CL), preferably wherein the CH comprises the same sequence as amino acid sequence SEQ ID No: 4, 19, or 20, and the CL comprises the same sequence as amino acid sequence SEQ ID No:

5.

9. The antibody or antigen-binding fragment according to claim 8, characterized in that the heavy chain (H) comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of the amino acid sequences SEQ ID No: 11-18 or 29-34, and / or the light chain (L) comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence SEQ ID No:

10.

10. The antibody or antigen-binding fragment according to claim 9, characterized in that the heavy chain (H) contains the same sequence as any of the amino acid sequences SEQ ID No: 11-18 or 29-34, and the light chain (L) contains the same sequence as the amino acid sequence SEQ ID No:

10.

11. An anti-GPC3 antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment binds to the antigen indicated by SEQ ID No: 37 or 38, and does not bind to the antigen indicated by SEQ ID No:

36. Alternatively, the antibody or its antigen-binding fragment competitively binds to the same epitope as the reference antibody and the GPC3 protein, wherein the reference antibody comprises a heavy chain indicated by SEQ ID NO: 11 and a light chain indicated by SEQ ID NO:

10. Preferably, the anti-GPC3 antibody or its antigen-binding fragment is characterized in that it can block at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the binding of the reference antibody to the GPC3 protein.

12. A GPC3 antigen epitope peptide, wherein the GPC3 antigen epitope peptide consists of at least seven consecutive amino acid residues from human GPC3 protein residues 485 to 496, the amino acid sequence of the human GPC3 protein is indicated by SEQ ID NO: 35, and contains at least one of asparagine at position 487 and phenylalanine at position 493, and the GPC3 antigen epitope peptide has one or more of the following biological functions. (1) Specifically binds to the anti-GPC3 antibody, (2) Induce an immune response to GPC3 in the subject's body (e.g., a humoral immune response), (3) Inducing the production of anti-GPC3 antibodies in the subject's body, (4) To prevent and / or treat GPC3-related diseases in subjects.

13. The GPC3 antigen epitope peptide according to claim 12, characterized in that the GPC3 antigen epitope peptide consists of the amino acid sequence indicated by SEQ ID NO:

38.

14. A recombinant antigen, characterized in that the recombinant antigen comprises the GPC3 antigen epitope peptide described in claim 12 or 13 and a carrier protein.

15. A chimeric antigen receptor comprising an anti-GPC3 antibody or its antigen-binding fragment as described in any one of claims 1 to 11.

16. A polynucleotide encoding an anti-GPC3 antibody or its antigen-binding fragment according to any one of claims 1 to 11, a GPC3 antigen epitope peptide according to any one of claims 12 to 13, or a recombinant antigen according to claim 14.

17. An expression vector comprising the polynucleotide described in claim 16.

18. A host cell comprising the polynucleotide described in claim 16 or the expression vector described in claim 17.

19. An antibody-drug conjugate comprising an anti-GPC3 antibody or its antigen-binding fragment according to any one of claims 1 to 11, and a drug or toxin.

20. The antibody-drug conjugate according to claim 19, characterized in that the drug or toxin is one or more selected from SN-38, MMAE, PBD dimer, DX-8951 (DXd), or DUBA.

21. A bispecific or multispecific antibody molecule characterized by comprising an anti-GPC3 antibody or its antigen-binding fragment as described in any one of claims 1 to 11.

22. A pharmaceutical composition comprising an anti-GPC3 antibody or its antigen-binding fragment according to any one of claims 1 to 11, or a GPC3 antigen epitope peptide according to any one of claims 12 to 13, or a recombinant antigen according to claim 14, or a chimeric antigen receptor according to claim 15, or a polynucleotide according to claim 16, or an expression vector according to claim 17, or a host cell according to claim 18, or an antibody-drug conjugate according to any one of claims 19 to 20, or a bispecific or multispecific antibody molecule according to claim 21, and one or more pharmaceutically acceptable carriers.

23. A kit comprising an anti-GPC3 antibody or its antigen-binding fragment according to any one of claims 1 to 11, or a GPC3 antigen epitope peptide according to any one of claims 12 to 13, or a recombinant antigen according to claim 14, or an antibody-drug conjugate according to any one of claims 19 to 20, or a bispecific or multispecific antibody molecule according to claim 21.

24. Use of an anti-GPC3 antibody or antigen-binding fragment according to any one of claims 1 to 11, a GPC3 antigen epitope peptide according to any one of claims 12 to 13, a recombinant antigen according to claim 14, a chimeric antigen receptor according to claim 15, a polynucleotide according to claim 16, an expression vector according to claim 17, a host cell according to claim 18, an antibody-drug conjugate according to any one of claims 19 to 20, a bispecific or multispecific antibody molecule according to claim 21, or a kit according to claim 23, wherein the cancer is preferably liver cancer.

25. Use of the GPC3 antigen epitope peptide according to any one of claims 12 to 13, or the recombinant antigen according to claim 14, in any of the following. (1) To manufacture an anti-GPC3 antibody or its antigen-binding fragment, (2) Manufacturing products for treating and / or preventing and / or diagnosing GPC3-related diseases in subjects, (3) To manufacture a product for detecting anti-GPC3 antibodies or their antigen-binding fragments, (4) To detect an anti-GPC3 antibody or its antigen-binding fragment, (5) Screening for anti-GPC3 antibodies or antigen-binding fragments.

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