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

Abstract

Provided in the present invention are an anti-GPC-3 antibody and an antigen-binding fragment thereof, and also provided are a polynucleotide encoding the antibody, an expression vector and a host cell for expressing the antibody, and a method for preparing the antibody. In addition, further provided in the present invention are a pharmaceutical composition comprising the antibody and the use of the antibody in the preparation of a drug for treating cancers.

Description

An anti-GPC3 antibody or antigen-binding fragment and its use Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an anti-GPC3 antibody or an antigen-binding fragment thereof and uses thereof. Background Art

[0002] GPC3 is a heparan sulfate proteoglycan expressed on the surface of various malignant cells, such as hepatocellular carcinoma (HCC) cells. Glypican-3 is linked to the cell surface via a glycosylphosphatidylinositol (GPI) anchor. GPC3 has been shown to be highly expressed in over 70% of HCC biopsies but absent in adjacent non-tumor tissue. Patients with GPC3-positive HCC have significantly lower disease-free survival than those with GPC3-negative HCC.

[0003] It has been discovered that certain types of antibodies that bind to GPC3 have cytostatic activity through antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities (International Patent Application WO 2003 / 000883). Furthermore, it has been shown that GPC3 is cleaved in vivo and secreted into the blood as a secreted form of GPC3, and that tumor diagnosis can be performed using antibodies capable of detecting the secreted form of GPC3 (International Patent Applications WO 2004 / 022739, WO 03 / 100429, and WO 2004 / 018667).

[0004] Antibody-drug conjugates (ADCs) are a novel targeted drug therapy, combining antibodies with highly cytotoxic small-molecule drugs. They combine the potent lethality of small-molecule drugs with the high targeting properties of monoclonal antibodies, making them a hot topic in the research and development of targeted cancer therapy. ADCs generally consist of three components, linked in a specific manner: an antibody or antibody-like ligand, a linker, and a small-molecule drug. The targeting properties of ADCs come from the antibody component, while the toxicity primarily comes from the small-molecule drug, although the antibody component can also be toxic. After binding to tumor cell surface antigens, the antibody component is internalized and then degraded in the lysosome, releasing active chemical toxins that damage DNA or inhibit tumor cell division, ultimately killing the cells. Compared to other therapeutic modalities, ADCs offer the following advantages: strong therapeutic efficacy; high tumor cell specificity, low false negative rates, and a wider therapeutic safety window; low immunogenicity, making them less susceptible to drug resistance; long circulation time in serum (shorter than naked antibodies); and low toxicity to non-target cells.

[0005] Currently reported anti-GPC3 antibodies include:

[0006] Patent CN1842540B discloses an anti-GPC3 antibody, such as GC33, which has higher ADCC and CDC activities than traditional antibodies. The antibody epitope is located within the sequence of positions 544 to 553 (PKDNEISTFH) at the C-terminus of GPC3. However, the binding ability to the GPC3 epitope is still weak.

[0007] Patent CN10452033B discloses a high-affinity monoclonal antibody for glypican 3 and its uses, such as YP7, which is an antibody obtained by immunization with a 50-residue peptide (DGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHS) and has a high affinity for GPC3, but its affinity for GPC3 still needs to be further improved;

[0008] Patent CN115850492A also discloses a monoclonal antibody and polynucleotide against GPC-3, as well as preparation methods and applications thereof, and claims to have solved the technical problem of low affinity between GPC3 monoclonal antibodies and target antigens in the prior art. However, its affinity to GPC3 still needs to be further improved.

[0009] It can be seen that the anti-GPC3 antibodies reported in the prior art still have problems such as insufficient affinity (especially affinity at the cellular level) or poor killing ability. Therefore, there is an urgent need to develop an anti-GPC3 antibody or antigen-binding fragment that has strong binding ability to GPC3 and good cell killing effect.

[0010] Summary of the Invention

[0011] The object of the present invention is to provide an anti-GPC3 antibody or an antigen-binding fragment thereof and uses thereof, so as to solve the problems of low affinity and poor cell killing effect existing in the prior art.

[0012] In one aspect, the present invention provides an anti-GPC3 antibody or an antigen-binding fragment thereof, wherein the anti-GPC3 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL);

[0013] The VH comprises HCDR1, HCDR2 and HCDR3 regions, and the HCDR1, HCDR2 and HCDR3 regions respectively comprise sequences that are at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CDR1, CDR2 and CDR3 regions of any one of the amino acid sequences shown in SEQ ID Nos: 1-2, 6-9; or respectively comprise sequences that have at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations compared to the CDR1, CDR2 and CDR3 regions of any one of the amino acid sequences shown in SEQ ID Nos: 1-2, 6-9; the mutations may be selected from insertions, deletions and / or substitutions, and the substitutions are preferably substitutions of conservative amino acids; the amino acid sequence SEQ ID Nos: 1-2, 6-9 CDR1, CDR2, and CDR3 regions were defined according to IMGT, Kabat, Chothia, AbM, or Contact;

[0014] The VL comprises LCDR1, LCDR2 and LCDR3 regions, wherein the LCDR1, LCDR2 and LCDR3 regions respectively comprise sequences that are 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 of SEQ ID NO: 3; or respectively comprise sequences that have at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations compared to the CDR1, CDR2 and CDR3 regions of the amino acid sequence of SEQ ID NO: 3; the mutations may be selected from insertions, deletions and / or substitutions, and the substitutions are preferably conservative amino acid substitutions; the CDR1, CDR2 and CDR3 regions of the amino acid sequence of SEQ ID No: 3 are defined according to IMGT, Kabat, Chothia, AbM or Contact.

[0015] In some embodiments, the position of the mutation is selected from one or more of position 56 (D56), position 100 (Q100), or position 102 (S102) of the amino acid sequence shown in any one of SEQ ID Nos: 1-2, 6-9. In some embodiments, the position of the mutation includes position 56 (D56) and position 102 (S102) of the amino acid sequence shown in any one of SEQ ID Nos: 1-2, 6-9. In some preferred embodiments, the mutation is selected from one or more of D56A, D56K, Q100R, or S102R. In some preferred embodiments, the mutation is selected from Q100R, S102R, D56A, D56K+Q100R, D56A+S102R, or D56K+S102R.

[0016] In some embodiments, the HCDR1, HCDR2 and HCDR3 regions have the same sequences as the CDR1, CDR2 and CDR3 regions of any one of the amino acid sequences shown in SEQ ID Nos: 1-2, 6-9, 23-28, respectively; and the LCDR1, LCDR2 and LCDR3 regions respectively comprise the same sequences as the CDR1, CDR2 and CDR3 regions of the amino acid sequence of SEQ ID No: 3.

[0017] In some embodiments, the VH comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID Nos: 1-2, 6-9, 23-28. In some embodiments, the VL comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No: 3.

[0018] In some embodiments, the VH comprises a sequence identical to any one of the amino acid sequences of SEQ ID Nos: 1-2, 6-9, 23-28; and / or the VL comprises a sequence identical to the amino acid sequence of SEQ ID NO: 3.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region (CH) and a light chain constant region (CL); the heavy chain constant region (CH) can be a heavy chain constant region of human IgG1; the light chain constant region (CL) can be a human κ light chain constant region; specifically, the heavy chain constant region (CH) comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID Nos: 4, 19 or 20; the light chain constant region (CL) comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No: 5.

[0020] In some embodiments, the heavy chain constant region (CH) comprises a sequence identical to the amino acid sequence of SEQ ID Nos: 4, 19, or 20; and the light chain constant region (CL) comprises a sequence identical to the amino acid sequence of SEQ ID No: 5.

[0021] In some embodiments, the heavy chain (H) comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of any one of SEQ ID Nos: 11-18, 29-34; and / or the light chain (L) comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No: 10.

[0022] As a preferred technical solution of the present invention, the heavy chain (H) comprises a sequence identical to any one of the amino acid sequences SEQ ID Nos: 11-18, 29-34; the light chain (L) comprises a sequence identical to the amino acid sequence SEQ ID No: 10.

[0023] In some embodiments, the anti-GPC3 antibody or antigen-binding fragment thereof described herein is a humanized monoclonal antibody.

[0024] In some specific embodiments, the anti-GPC3 antibody or antigen-binding fragment thereof is: (1) a chimeric antibody or a fragment thereof; (2) a humanized antibody or a fragment thereof; or (3) a fully human antibody or a fragment thereof.

[0025] In some embodiments, the anti-GPC3 antibodies or antigen-binding fragments thereof described herein have one or more of the following biological functions:

[0026] ) specifically binds to the antigen shown in SEQ ID Nos: 37 or 38, and does not bind to the antigen shown in SEQ ID No: 36;

[0027] ) specifically binds to human and monkey GPC3 proteins, but does not bind to mouse GPC3 protein.

[0028] In some embodiments, the specific binding of the anti-GPC3 antibody or antigen-binding fragment thereof to the antigen is confirmed by an equilibrium dissociation constant KD of, for example, 10 -4 M or smaller (e.g. 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) to express.

[0029] In one aspect, the present invention provides an anti-GPC3 antibody or an antigen-binding fragment thereof, which specifically binds to the antigen shown in SEQ ID Nos: 37 or 38 and does not bind to the antigen shown in SEQ ID No: 36. In some embodiments, the antibody or antigen-binding fragment thereof comprises: the VL shown in SEQ ID No: 39 and the VH shown in SEQ ID No: 40; the VL shown in SEQ ID No: 41 and the VH shown in SEQ ID No: 42; the VL shown in SEQ ID No: 43 and the VH shown in SEQ ID No: 44; the VL shown in SEQ ID No: 45 and the VH shown in SEQ ID No: 46; the VL shown in SEQ ID No: 47 and the VH shown in SEQ ID No: 48; the VL shown in SEQ ID No: 49 and the VH shown in SEQ ID No: 50; the VL shown in SEQ ID No: 51 and the VH shown in SEQ ID No: 52; the VL shown in SEQ ID No: 53 and the VH shown in SEQ ID No: 54; the VL shown in SEQ ID No: 55 and the VH shown in SEQ ID No: 56; or, the VL shown in SEQ ID No: 57 and the VH shown in SEQ ID No: 58.

[0030] In one aspect, the present invention provides an anti-GPC3 antibody or an antigen-binding fragment thereof, which competes with a reference antibody for binding to the same epitope of the GPC3 protein, wherein the reference antibody comprises a heavy chain as set forth in SEQ ID NO: 11 and a light chain as set forth in SEQ ID NO: 10.

[0031] In some embodiments, the anti-GPC3 antibody or antigen-binding fragment thereof is capable of blocking the binding of a reference antibody to the GPC3 protein by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%. Competitive binding can be determined by a competitive binding assay. Competitive binding assays, well known to those skilled in the art, are immunological assays that detect and quantify unknown substances by their ability to inhibit the binding of a labeled, known antigen to its specific antibody, also known as competitive inhibition assays. For example, an antigen is pre-coated on a microplate, and then a serial dilution of an unlabeled test antibody and a labeled, known antibody at a specific concentration are added to the pre-coated microplate. The plate is then incubated, and after washing, the amount of known antibody bound to the plate at different dilutions of the test antibody is measured. The greater the ability of the test antibody to compete with the known antibody for antigen binding, the weaker the ability of the known antibody to bind to the antigen, and the less known antibody is bound to the plate. The ability of the test antibody to block the labeled reference antibody can be determined using radioimmunoassays, enzyme immunoassays such as ELISA, or fluorescent immunoassays.

[0032] Another object of the present invention is to provide a novel GPC3 antigen epitope peptide to address the unmet development needs of anti-GPC3 antibodies, vaccines, and related diagnostic reagents in the prior art, and to provide an effective tool for developing anti-GPC3 antibodies with strong binding ability to GPC3 or good cell-killing effects.

[0033] In one aspect, the present invention provides a GPC3 antigen epitope peptide, which is immunogenic and can induce an immune response in an organism to produce antibodies against GPC3.

[0034] In one aspect, the present invention provides a GPC3 antigen epitope peptide, wherein the GPC3 antigen epitope peptide consists of at least 7 consecutive amino acid residues within residues 485-496 of the human GPC3 protein, the amino acid sequence of the human GPC3 protein is shown in SEQ ID No: 35, and the GPC3 antigen epitope peptide has one or more of the following biological functions:

[0035] (1) Specific binding to anti-GPC3 antibodies;

[0036] (2) inducing an immune response (e.g., a humoral immune response) against GPC3 in a subject;

[0037] (3) inducing the production of anti-GPC3 antibodies in the subject;

[0038] (4) Preventing and / or treating GPC3-related diseases in a subject.

[0039] In some embodiments, the specific binding of the GPC3 antigen epitope peptide to the anti-GPC3 antibody is determined by ELISA. In some embodiments, the ELISA method is described in Example 12, comprising coating the GPC3 antigen epitope peptide on an ELISA plate, serially diluting the anti-GPC3 antibody, adding the ELISA plate to the plate for incubation, washing and developing the color after the incubation is complete, and measuring the OD value at 450 nm after termination. In some embodiments, the specific binding of the GPC3 antigen epitope peptide to the anti-GPC3 antibody is determined by ELISA, and the OD value of the antigen-antibody complex is determined by ELISA. 450 In some embodiments, when the GPC3 epitope peptide concentration is 6 μg / mL, the OD value at which the anti-GPC3 antibody is saturated is 450 The value is not less than 1.5±0.1, or not less than 2±0.1, or not less than 2.5±0.1, or not less than 3±0.1. In some embodiments, the OD 450The value is not less than 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. In some embodiments, the anti-GPC3 antibody that specifically binds to the GPC3 antigen epitope peptide comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID No: 11, and the light chain comprising the amino acid sequence of SEQ ID No: 10.

[0040] In some embodiments, the GPC3 antigen epitope peptide is 7, 8, 9, 10, 11, or 12 amino acids in length. In some embodiments, the GPC3 antigen epitope peptide comprises at least one of asparagine at position 487 and phenylalanine at position 493.

[0041] In some embodiments, the GPC3 antigen epitope peptide consists of consecutive amino acid residues from residues 487 to 493 of the human GPC3 protein. In some preferred embodiments, the GPC3 antigen epitope peptide consists of the amino acid sequence shown in SEQ ID No: 38.

[0042] In one aspect, the present invention provides a recombinant antigen comprising the GPC3 antigen epitope peptide of the present invention and a carrier protein. The recombinant antigen can enhance the immunogenicity of the epitope peptide, allowing it to be recognized by the body's immune system and induce an immune response.

[0043] In some embodiments, the recombinant antigen has one or more of the following biological functions:

[0044] (1) Specific binding to anti-GPC3 antibodies;

[0045] (2) inducing an immune response (e.g., a humoral immune response) against GPC3 in a subject;

[0046] (3) inducing the production of anti-GPC3 antibodies in the subject;

[0047] (4) Preventing and / or treating GPC3-related diseases in a subject.

[0048] 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 the amino acid sequence shown in SEQ ID No: 11, and the light chain comprises the amino acid sequence shown in SEQ ID No: 10.

[0049] In some embodiments, the GPC3 antigen epitope peptide of the present invention is directly linked to a carrier protein or is linked via a linker. In some embodiments, the linker can be a rigid or flexible linker, such as a peptide linker comprising one or more serines and / or glycines.

[0050] In some embodiments, the GPC3 antigen epitope peptide of the present invention is linked to the N-terminus and / or C-terminus of the carrier protein, and / or is inserted into the interior of the carrier protein. In some preferred embodiments, the GPC3 antigen epitope peptide of the present invention is linked to the C-terminus of the carrier protein. In some preferred embodiments, the GPC3 antigen epitope of the present invention is linked to the N-terminus of the carrier protein.

[0051] In some embodiments, the carrier protein includes but is not limited to keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), thyroglobulin, fibrinogen, gelatin, multimeric antigenic peptides, including diphtheria toxin DT, diphtheria toxin transmembrane domain DTT, rotavirus VP7, Leishmania heat shock protein, Campylobacter jejuni flagellin, Chlamydia trachomatis major outer membrane protein, chicken ovalbumin (OVA) or immunoglobulin Fc domain, such as IgG1, IgG2, IgG3 or IgG4 Fc domain. In some preferred embodiments, the carrier protein is selected from KLH and BSA.

[0052] In one aspect, the present invention provides a chimeric antigen receptor comprising an anti-GPC3 antibody or antigen-binding fragment thereof of the present invention.

[0053] In another aspect, 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 or antigen-binding fragment thereof of the present invention.

[0054] In one aspect, the present invention also provides a pharmaceutical composition comprising an anti-GPC3 antibody or antigen-binding fragment thereof, or an antibody conjugate (e.g., an antibody-drug conjugate), or an oncolytic virus, or a chimeric antigen receptor, or a bispecific or multispecific antibody molecule, or a GPC3 antigen epitope peptide, or a recombinant antigen, and one or more pharmaceutically acceptable carriers. When the composition comprises more than one antibody (or its antigen-binding fragment, or antibody conjugate, or oncolytic virus), the antibody (or its antigen-binding fragment, or antibody conjugate, or oncolytic virus) can be administered in batches. The composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug, such as an anti-tumor drug.

[0055] The pharmaceutical composition of the present invention may be a vaccine, including but not limited to a protein vaccine or a nucleic acid vaccine.

[0056] The pharmaceutical composition can include any number of excipients. Operable excipients include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, coloring agents, flavorings, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, or combinations thereof. Selection and use of suitable excipients are taught in the following, Gennaro writes, Remington: The Science and Practice of Pharmacy, 20th edition (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0057] In another aspect, the present invention provides a kit comprising the anti-GPC3 antibody or antigen-binding fragment thereof, GPC3 antigen epitope peptide, recombinant antigen, antibody-drug conjugate, or bispecific or multispecific antibody molecule of the present invention.

[0058] In some embodiments, the kit comprises the GPC3 antigen epitope peptide of the present invention, and a means for detecting antibodies.

[0059] In some embodiments, the kit is used to detect anti-GPC3 antibodies. In some embodiments, the kit is used to detect the presence of anti-GPC3 antibodies 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 the amino acid sequence set forth in SEQ ID NO: 11 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0060] In another aspect, the present invention provides a polynucleotide encoding an anti-GPC3 antibody, or antigen-binding fragment thereof, antigenic epitope peptide, or recombinant antigen of the present invention. The polynucleotide of the present invention can be, for example, DNA or RNA, and may or may not contain intronic sequences. In a preferred embodiment, the polynucleotide is a cDNA molecule. The polynucleotide of the present invention can be prepared or obtained by known means based on the amino acid sequence information of the present invention, such as by automated DNA synthesis and / or recombinant DNA technology.

[0061] As is well known in the art, multiple codons can encode the same amino acid. Therefore, nucleic acids encoding protein sequences include nucleic acids with codon degeneracy. The amino acid sequence of the present invention can be encoded by a variety of nucleic acids. The genetic code is universal and well known. The nucleic acid encoding any amino acid sequence of the present invention can be easily conceived based on the common knowledge in the art, and can be optimized for production. Although the possible number of nucleic acid sequences encoding a given amino acid is very large, given the standard table of the genetic code, and with the assistance of a calculator, those skilled in the art can easily produce every possible combination of nucleic acid sequences encoding a given amino acid.

[0062] In another aspect, the present invention provides an expression vector comprising the polynucleotide of the present invention, wherein 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.

[0063] In another aspect, the present invention provides a host cell comprising the polynucleotide of the present invention or the aforementioned expression vector; the host cell comprises a prokaryotic cell, yeast or mammalian cell, such as a CHO cell, NS0 cell or other mammalian cell, preferably a CHO cell.

[0064] In another aspect, the present invention provides a bispecific or multispecific antibody molecule comprising the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention.

[0065] In another aspect, the present invention provides an antibody-drug conjugate comprising the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention and a drug or toxin; the drug or toxin is selected from one or more of SN-38, MMAE, PBD dimer, DX-8951 (DXd) or DUBA.

[0066] The antibody and the drug can be coupled via a linker to form an antibody-drug conjugate (ADC). Typically, the ADC comprises an anti-GPC3 antibody or antigen-binding fragment of the present invention, connected to a drug or toxin via a linker. The linker can be a degradable or non-degradable linker. Degradable linkers typically readily degrade in the intracellular environment, thereby releasing the therapeutic agent from the antibody. Suitable degradable linkers include enzymatically degradable linkers, such as linkers containing peptidyl groups that can be degraded by intracellular lysosomal proteases, or sugar linkers, such as linkers containing glucuronides that can be degraded by glucuronidases. Peptide linkers can include dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include pH-sensitive linkers (e.g., hydrazone linkers that hydrolyze at a pH below 5.5) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug when the antibody is hydrolyzed by proteases.

[0067] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved via the active reactive group. Preferred are sulfhydryl-specific active reactive groups, such as maleimides, halogenated amides, halogenated esters, halogenated methyl ketones, benzyl halides, vinyl sulfones, pyridyl disulfides, mercury derivatives, and polymethylene dimethyl sulfide thiosulfonates. The linker can include, for example, a maleimide attached to the antibody via thiosuccinimide.

[0068] Preferably, the linker-connected drug or toxin is selected from the group consisting of: 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 figure below:

[0069] In the present invention, the anti-GPC3 antibody or antigen-binding fragment thereof is coupled to SN-38 via a CL2A linker.

[0070] In the present invention, the anti-GPC3 antibody or antigen-binding fragment thereof is coupled to MMAE via a mc-VC-PAB linker.

[0071] In the present invention, the anti-GPC3 antibody or antigen-binding fragment thereof is coupled to the PBD dimer via a maleimide-dPEG8-VA-PABA linker.

[0072] Preferably, the drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In one embodiment, a linker connects the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group capable of forming a bond with the linker. In the case where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0073] Preferably, the cytotoxic drug is selected from the group consisting of anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, DNA alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or a combination thereof.

[0074] Preferably, examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, and typical cytotoxic drugs 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 (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines) and vinca alkaloids, or a combination thereof.

[0075] Preferably, the toxin is selected from the group consisting of auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), chlortetracycline, maytansinoids, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, adriamycin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxybenzoate, daptomycin, acetaminophen ... anthracnose dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, a Sapaonaria officinalis inhibitor, a glucocorticoid, or a combination thereof.

[0076] Preferably, the drug or toxin is selected from one or more of 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).

[0077] In the present invention, the anti-GPC3 antibody or antigen-binding fragment thereof is coupled to DX-8951 (DXd) via a maleimide-GGFG linker.

[0078] In the present invention, the anti-GPC3 antibody or antigen-binding fragment thereof is coupled to DUBA via a Vc-seco linker.

[0079] In another aspect, the present invention also provides a use of the anti-GPC3 antibody or antigen-binding fragment thereof, the pharmaceutical composition, or the antibody-drug conjugate of the present invention in the preparation of a medicament for treating or preventing cancer, preferably liver cancer.

[0080] In another aspect, the present invention provides a use of the GPC3 antigen epitope peptide, recombinant antigen, nucleic acid molecule, vector or host cell in any of the following:

[0081] (1) preparing an anti-GPC3 antibody or an antigen-binding fragment thereof;

[0082] (2) preparing a product for treating and / or preventing and / or diagnosing a GPC3-related disease in a subject; preferably, the disease is a GPC3-positive cancer;

[0083] (3) preparing a product for detecting an anti-GPC3 antibody or an antigen-binding fragment thereof; preferably, the anti-GPC3 antibody comprises a heavy chain as set forth in SEQ ID NO: 11 and a light chain as set forth in SEQ ID NO: 10;

[0084] (4) detecting an anti-GPC3 antibody or an antigen-binding fragment thereof; preferably, the anti-GPC3 antibody comprises a heavy chain as set forth in SEQ ID NO: 11 and a light chain as set forth in SEQ ID NO: 10;

[0085] (5) Screening for anti-GPC3 antibodies or antigen-binding fragments thereof.

[0086] In some embodiments, the disease is a GPC3-positive cancer, such as liver cancer, colorectal cancer, ovarian cancer, etc.

[0087] In one aspect, the present invention provides a method for preparing an anti-GPC3 antibody or an antigen-binding fragment thereof, comprising the step of stimulating an animal's 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 antibodies.

[0088] In some embodiments, the animal is selected from mammals such as humans, mice, rabbits, monkeys, cows, sheep, or alpacas.

[0089] In one aspect, the present invention provides a method for screening anti-GPC3 antibodies or antigen-binding fragments thereof, comprising contacting a GPC3 antigen epitope peptide of the present invention with an antibody or antigen-binding fragment thereof to be analyzed, and detecting binding of the GPC3 antigen epitope peptide to the antibody or antigen-binding fragment thereof. If the GPC3 antigen epitope peptide and the antibody or antigen-binding fragment thereof exhibit binding, the antibody or antigen-binding fragment thereof is a candidate anti-GPC3 antibody or antigen-binding fragment thereof.

[0090] In some embodiments, the anti-GPC3 antibody or antigen-binding fragment thereof binds to the GPC3 antigen epitope peptide, and the binding is determined by an ELISA method. In some embodiments, the ELISA method is described in Example 12, comprising coating the GPC3 antigen epitope peptide on an ELISA plate, serially diluting the anti-GPC3 antibody or antigen-binding fragment thereof, adding the ELISA plate to the plate for incubation, washing and developing the color after the incubation is complete, and measuring the OD value at 450 nm after termination. In some embodiments, the specific binding of the GPC3 antigen epitope peptide to the anti-GPC3 antibody or antigen-binding fragment thereof is determined by ELISA, and the OD value of the antigen-antibody complex is determined by ELISA. 450In some embodiments, when the concentration of the GPC3 antigen epitope peptide is 6 μg / mL, the OD value of the anti-GPC3 antibody or its antigen-binding fragment at saturation is 450 The value is not less than 1.5±0.1, or not less than 2±0.1, or not less than 2.5±0.1, or not less than 3±0.1. In some embodiments, the OD 450 The value is not less than 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. Furthermore, the present invention also provides a method for preparing the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention:

[0091] The DNA sequence of the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention can be obtained using conventional techniques, such as hybridoma PCR amplification or phage display library screening. In addition, the coding sequences of the light chain and heavy chain can be fused together to form a single-chain antibody (e.g., scFV).

[0092] Once the relevant sequence is obtained, it can be cloned into a vector, then transferred into host bacteria, and then the relevant vector can be extracted from the host bacteria by conventional methods.

[0093] In addition, artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is relatively short. Currently, DNA sequences encoding the antibodies of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely through chemical synthesis. In addition, mutations can also be introduced into the protein sequences of the present invention through chemical synthesis.

[0094] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0095] The anti-GPC3 antibodies or antigen-binding fragments thereof of the present invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be isolated and purified using various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention, and then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose affinity chromatography, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, and other conventional separation and purification methods, or a combination of these methods, to obtain the anti-GPC3 antibodies or antigen-binding fragments thereof of the present invention.

[0096] As a preferred embodiment of the method for preparing the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention, the method for separating and purifying the anti-GPC3 antibody or antigen-binding fragment thereof is protein A affinity chromatography, cation exchange method or anion exchange method.

[0097] The resulting monoclonal antibodies or bispecific antibodies can be characterized by conventional means. For example, the binding specificity of the antibody can be determined by immunoprecipitation or in vitro binding assays such as enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). The binding affinity of the antibody can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0098] In the present invention, the antibody-drug conjugate is prepared according to a method comprising the following steps:

[0099] The interchain disulfide bonds of the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention are reduced to generate 2n (e.g., 2, 4, 6, 8) thiol groups;

[0100] The drug-linker compound cross-links with the reduced antibody sulfhydryl group to generate the corresponding antibody-drug conjugate;

[0101] The product was further purified by ultrafiltration and desalting.

[0102] For the sake of clarity, general terms used in the description of the compounds are defined herein.

[0103] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered as undefined or unclear in the absence of a specific definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient. The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of sound medical judgment without excessive toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0104] The term "GPC3" (also known as glypican 3) refers to a member of the heparan sulfate proteoglycan family, anchored to the cell membrane by glycosylphosphatidylinositol (GPI). The human GPC3 gene is located on chromosome X (Xp26) and encodes a 70 kDa protein containing 580 amino acids. This protein is cleaved endonucleolytically by a furin-like convertase between Arg358 and Ser359 to produce a 40 kDa N-terminal subunit and a 30 kDa C-terminal subunit, which also contains two heparan sulfate (HS) chains.

[0105] The term "antibody" as used herein includes intact antibodies and any antigen-binding fragments thereof (i.e., "antigen-binding portions") or single chains thereof. An intact antibody is a glycoprotein comprising two heavy (H) chains and two light (L) chains connected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be 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 from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (CIq) of the classical complement system.

[0106] The term "antigen-binding fragment" (or simply "antibody portion") refers to one or more fragments of an antibody that specifically binds to an antigen (e.g., a GPC3 protein). It has been shown that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); (vi) an isolated complementarity-determining region (CDR); and (vii) a nanobody, a heavy chain variable region comprising a single variable domain and two constant domains. In addition, although the two domains VL and VH of the FV fragment are encoded by separate genes, they can be connected by a linker using recombinant methods to form a single protein chain, wherein the VL region and the VH region are paired to form a monovalent molecule (called single-chain Fv (scFv); 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 are also included in the term "antigen-binding fragment" of an antibody. These antibody fragments can be obtained by conventional techniques known to those skilled in the art, and the fragment screening for use is the same as that for intact antibodies.

[0107] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities. For example, an isolated antibody that specifically binds to a GPC3 protein is substantially free of antibodies that specifically bind to antigens other than GPC3. However, for example, in some embodiments, an isolated antibody that specifically binds to a human GPC3 protein may have cross-reactivity with other antigens (e.g., GPC3 proteins from other species). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemical substances.

[0108] As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0109] The term "chimeric antibody" refers to an antibody made by combining genetic material from a non-human source with genetic material from a human. Or more generally, a chimeric antibody is an antibody that has genetic material from one species and genetic material from another species.

[0110] The term "bispecific" or "multispecific" refers to an antibody and / or antigen-binding molecule that is capable of specifically binding to two or more different antigenic determinants. Typically, a bispecific or multispecific antibody or antigen-binding molecule comprises two antigen-binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, the bispecific or multispecific antibody or antigen-binding molecule is capable of simultaneously binding to two or more antigenic determinants, particularly two or more antigenic determinants expressed on two or more different cells.

[0111] As used herein, the term "humanized antibody" refers to an antibody from a non-human species whose protein sequence has been modified to increase its similarity to naturally occurring antibody variants in humans.

[0112] The term "antibody-drug conjugate" refers to the use of antibodies to specifically recognize specific antigens on the surface of tumor cells, thereby achieving accurate delivery of anti-tumor therapeutic agents (such as cytotoxins or cytostatics, radioactive isotopes, small molecule chemotherapy drugs, etc.) to tumor target cells, resulting in intracellular accumulation and release, to achieve the purpose of accurately killing tumors. ADC is also considered to be the most promising anti-tumor drug because of its suitable molecular weight, high stability, strong targeting, and low toxic side effects. In addition to monoclonal antibodies, bispecific antibodies can also be coupled to therapeutic agents. In some embodiments, the part coupled to the antibody or bispecific antibody of the present invention to form an antibody conjugate is a cytotoxin, which refers to a substance that inhibits or prevents cell function and / or causes cell destruction, and includes small molecule cytotoxins. In some embodiments, the cytotoxin is selected from SN-38, MMAE, PBD dimer, DX-8951 (DXd) or DUBA.

[0113] As used herein, the terms "include," "comprising," and "having" are used interchangeably and are intended to indicate the inclusiveness of a solution, meaning that other elements may be present in addition to the listed elements. It should also be understood that the use of "include," "comprising," and "having" in this document also provides a "consisting of" solution.

[0114] The term "antibody" herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, whole antibodies, antigen-binding fragments, naked antibodies, conjugated antibodies, humanized antibodies, or fully human antibodies.

[0115] As used herein, the term "epitope" refers to the site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope" is also referred to as an "antigenic determinant" in the art. An epitope or antigenic determinant is typically composed of chemically active surface groups of a molecule, such as amino acids or carbohydrates or sugar side chains, and typically has specific three-dimensional structural characteristics and specific charge characteristics. For example, an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 continuous or non-continuous amino acids in a unique spatial conformation, which can be "linear" or "conformational". See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996). In a linear epitope, all interacting points between a protein and an interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interacting points exist across separate protein amino acid residues.

[0116] As used herein, the term "epitope peptide" refers to a peptide segment on an antigen that can serve as an epitope. In some cases, an epitope peptide alone can be specifically recognized / bound by an antibody directed against the epitope. In other cases, it may be necessary to fuse the epitope peptide to a carrier protein in order for the epitope peptide to be recognized by a specific antibody.

[0117] As used herein, the term "carrier protein" refers to a protein that can act as a carrier of an epitope peptide, i.e., it can insert an epitope peptide at a specific position (e.g., inside the protein, at the N-terminus or C-terminus) so that the epitope peptide can be presented, thereby enabling the epitope peptide to be recognized by antibodies or the immune system.

[0118] The term "conservative amino acid" herein generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). For example, the amino acids within each of the following groups are conservative amino acid residues of each other, and substitutions of amino acid residues within the group are substitutions of conservative amino acids:

[0119] 1) Alanine (A), serine (S), threonine (T);

[0120] 2) Aspartic acid (D), glutamic acid (E);

[0121] 3) Asparagine (N), glutamine (Q);

[0122] 4) Arginine (R), Lysine (K), Histidine (H);

[0123] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0124] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0125] The terms "identity" and "sequence ... identity" are used interchangeably herein and are calculated as follows: To determine the percent "identity" of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When 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.

[0126] As used herein, the symbol "+" indicates a combination of mutations. Herein, the following terms are used to designate mutations: S102R indicates that the amino acid residue serine (S) at position 102 of the parent sequence is substituted with arginine (R).

[0127] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, for example, non-human primates, rodents, rabbits, pigs, dogs, cats, chickens, amphibians and reptiles, although mammals such as non-human primates and rodents are preferred.

[0128] The term "therapeutically effective amount" refers to an amount of an anti-GPC3 antibody or antigen-binding fragment thereof of the present invention sufficient to prevent or ameliorate symptoms associated with a disease or condition (e.g., cancer) and / or lessen the severity of the disease or condition. A therapeutically effective amount should be understood in the context of the condition being treated, wherein one skilled in the art can readily identify an actual effective amount. The antibodies of the present invention are structurally and chemically characterized monoclonal antibodies as described below and in the following examples. The amino acid sequence ID numbers (SEQ ID Nos.) of the heavy and light chain variable and constant regions of the antibodies are summarized in Tables 1 and 5.

[0129] The heavy chain variable region CDRs and light chain variable region CDRs in Tables 1 and 5 are defined by the Kabat, Chothia, IMGT, AbM, or Contact numbering systems / methods. The CDR region sequences of exemplary anti-GPC3 antibodies of the present invention are detailed in Table 3.

[0130] Table 1 Amino acid sequences of heavy chain / light chain variable and constant regions of anti-GPC3 antibodies

[0131] Table 2 Amino acid sequence of GPC3 antigen

[0132] Table 3 Numbering system / method definition of CDRs of heavy and light chain variable regions of anti-GPC3 antibodies

[0133] By the following detailed description and examples, other features and advantages disclosed by the present invention will become apparent, which should not be construed as limiting. The contents of all references, Genbank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0134] FIG1A and FIG1B show the ELISA test results of the positive monoclonal antibodies screened in Example 1 at the phage level and the GPC3-B peptide.

[0135] Figures 2 and 3 show the affinity of GC90 and its mutant monoclonal antibodies to GPC3 protein.

[0136] FIG4 shows the binding of humanized monoclonal antibodies GC90 and hYP7HM to human liver cancer cells HepG2, Hep3B and Huh-7 at different concentrations.

[0137] Figures 5A to 5D show the binding of GC90 and its mutant monoclonal antibodies to human liver cancer cells.

[0138] FIG6 shows the internalization of humanized mAbs GC90 and hYP7HM in human hepatoma cells HepG2 or Hep3B at a concentration of 1 nM.

[0139] FIG7 shows the killing effect of GC90 and its mutant monoclonal antibody drug conjugates on human liver cancer cells.

[0140] Figures 8A and 8B show the inhibition of tumors in vivo by the antibody-drug conjugates of the present invention.

[0141] FIG9 shows the binding strength results between GC90 and overlapping peptides detected by ELISA, wherein the underlined amino acid sequences correspond to the specific sequences of the overlapping peptides.

[0142] FIG10 shows an amino acid sequence alignment analysis between human GPC3 protein and mouse GPC3 protein.

[0143] Figure 11 shows the distribution of antigen recognition regions of GC90 and other reported anti-GPC3 antibodies on the GPC3 protein.

[0144] Example 1

[0145] Obtaining a fully human antibody positive clone targeting a new epitope of GPC3

[0146] Three rounds of panning were performed using a natural fully human phage display library and the GPC3-B peptide (amino acid residue Met at position 478 to amino acid residue Asp at position 531 of the extracellular domain of the GPC3 protein, used as an antigen to screen the humanized recombinant antibody library) to obtain a phage library eluate. The neutralized phage panning eluate was added to the prepared TG1 bacterial solution, mixed well, and incubated at 37°C for 45 minutes to infect the TG1 host bacteria. After sufficient infection, the bacterial solution was gradiently diluted and spread on agar plates with relevant resistance, and inverted to culture overnight at 37°C. The next day, a 96-well sterile deep-well plate containing 0.5mL of 2YT medium (containing 0.2% w / v glucose and 0.1mg / mL ampicillin) was prepared. Monoclonal colonies cultured on the plate were picked with a sterile pipette tip and placed in the corresponding well plate. The plates were shaken at 37°C and cultured overnight (16-18 hours). The next day, 0.05-0.1 mL of overnight cultured monoclonal bacterial solution was transferred to a newly prepared sterile deep-well plate (containing 0.5 mL of 2YT medium containing 0.1 mg / mL ampicillin antibiotic at a final concentration) and cultured until OD 600 The value is approximately 0.6-0.8. After adding a certain proportion of helper phage and shaking to mix, the cells were allowed to infect at 37°C for 45 minutes. 0.25 mL of 2YT medium (containing 0.1 mg / mL ampicillin and kanamycin to a final concentration of 0.05 mg / mL) was added and cultured overnight at 30°C and 220 rpm for 16-18 hours. The overnight expression culture was centrifuged at 4000 rpm for 10 minutes to obtain the phage display expression supernatant for ELISA binding detection against the GPC3-B peptide and FACS binding detection against the corresponding cells to obtain positive fully human antibody clones.

[0147] Indirect ELISA was used to assess the binding capacity of phage-displayed antibodies in the supernatant for the GPC3-B peptide. ELISA plates were coated with 100 μl / well of 4 μg / mL of GPC3-B peptide in CBS coating reagent at 4°C overnight. The plates were washed with PBST (containing 0.05% Tween) and blocked with 300 μl / well of PBS containing 3% skim milk at 37°C for 1 hour. The blocking solution was then discarded, and 50 μl of serially diluted phage expression supernatant and 50 μl of 0.05% PBST, as well as a negative control (IP1: ipilimumab), were added to each plate and incubated at room temperature for 2 hours. The plates were washed three times with 0.05% PBST and incubated with 100 μl / well of horseradish peroxidase-conjugated goat anti-M13 phage antibody (Sino-Bio) at room temperature for 45 minutes. The plate was washed six times with 0.05% PBST, and then TMB colorimetric solution (GenScript) was added and incubated at room temperature in the dark for 10 minutes. The reaction was terminated by adding 50 μl of 1 M HCl stop solution (Sigma). The plate was read at 450 nm using a microplate reader. The binding capacity test results are shown in Figures 1A-1B, showing that multiple positive monoclonal antibodies have good binding capacity with the GPC3-B peptide at the phage level.

[0148] ELISA was further used to identify whether the positive monoclonal antibodies obtained by screening recognized GPC3-A peptide. The specific operation was as follows: GPC3-A peptide and GPC3-B peptide (6 μg / mL) were coated in a 96-well plate at 100 μL / well and incubated at 4°C overnight. The plates were then blocked with 1% BSA in PBST (containing 0.05% Tween-20) for 2 hours at 37°C (200 μL / well), washed three times with PBST, and serially diluted with 1% BSA in PBST (100 μL / well) to a 96-well plate. The working concentrations of the positive monoclonal antibodies GC008, GC010, GC011, GC025, GC035, GC037, GC053, GC067, GC139, GC147, and GC90, as well as the control antibody hYP7HM, were added to the plate (100 μL / well) at a working concentration of 30 nM, 15 nM, and 15 nM, followed by eight dilutions in 4 steps. The plates were incubated at 37°C for 1 hour, and washed three times with PBST. Then, 100 μL / well of Anti-Human IgG-FC-HRP (Sigma, 1 / 30,000 dilution) was added, incubated at 37°C for 1 hour, washed three times with PBST, and then 50 μL of TMB (SURMOPICS) was added for reaction. The reaction was terminated with 1 M H2SO4, and the OD value was measured at 450 nm on 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 only recognizes the GPC3-A peptide (consistent with the patent literature), indicating that the positive monoclonal antibodies screened in this example have different antigen recognition sites compared to the control anti-GPC3 antibody hYP7HM.

[0149] Table 4 Binding of anti-GPC3 antibodies to GPC3-A peptide and GPC3-B peptide

[0150] Note: “-” indicates no binding; “++++” indicates ELISA detection OD 450 The value is greater than 1.5.

[0151] 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.

[0152] Table 5 Light chain and heavy chain variable region sequences of anti-GPC3 antibodies

[0153] Example 2

[0154] Preparation of humanized monoclonal antibodies

[0155] 2.1 Vector construction

[0156] First, the nucleotide sequence encoding the human IgG1 heavy chain constant region (IgG1-CH) was synthesized and cloned into the pcDNA3.1 vector (Shanghai Sangon Biotech Co., Ltd.) with a secretion signal peptide (SP) coding sequence at the front and a termination codon (TAG) at the rear. The gene was then inserted downstream of the CMV promoter in the pCHOGUN vector via in-fusion cloning. Specifically, the synthesized gene fragment and the pCHOGUN vector plasmid fragment were amplified using primers designed for the specific insertion site and a high-fidelity PCR enzyme (HiFi PCR Premix, TAKARA). After gel recovery, the IgG1 heavy chain gene fragment and the linearized vector fragment were ligated (In-fusion Snap Assembly Master Mix, TAKARA) to generate the heavy chain constant region vector pCHOGUN-IgG1. According to the above implementation method, the coding sequence of the human immunoglobulin kappa light chain constant region (IgG-CK) was synthesized, with the signal peptide (SP) coding sequence at the front and the termination codon TAG at the rear. After gene synthesis, it was inserted downstream of the CMV promoter of the pCHOGUN vector to obtain the light chain constant region vector pCHOGUN-CK.

[0157] To construct expression vectors for each humanized monoclonal antibody, the coding sequences for the heavy chain variable region (VH) and light chain variable region (VL) of the GC90 monoclonal antibody and its optimized mutants were synthesized and inserted between the SP and constant regions of the pCHOGUN-IgG1 or pCHOGUN-CK vectors using the in-fusion cloning method described above to generate the monoclonal antibody heavy and light chain expression vectors. Specific antibody sequences are shown in Table 6.

[0158] Table 6 Light chain and heavy chain sequences of humanized mAbs

[0159] 2.2 Cell transfection and expression

[0160] according to Transfection was performed according to the instructions of Transfection Reagent (Mirus). ExpiCHO-S cells (Thermo) were cultured in complete medium ( High Yield Expression System (containing 30 mL / L of Poloxamer 188 solution 10% and 20 mL / L of L-glutamine 200 mM, Mirus) was used for subculture 24 hours before transfection. The cells were diluted to 2 × 10 6cells / mL to ensure that the cell density is 4x10 6 cells / mL. Add 25 μg of light and heavy chain plasmids at a ratio of 1:1 to 12.5 mL of complete culture medium and mix well. Then add 50 μL of transfection reagent. Gently invert the Transfection Reagent (Mirus) to mix thoroughly, let it sit for 4 minutes, then add it dropwise to the diluted cells (50 mL) while shaking. Add 1 mL of CHOgro-titer Enhancer (Mirus) dropwise. Immediately after transfection, place the cells in a 32°C, 5% CO2 incubator. Add 5% Efficient Feed C + AGT Supplement (Thermo) every other day for a total of 7 days.

[0161] 2.3 Antibody Purification

[0162] After 7 days of culture in a shake flask, the cell supernatant was collected, centrifuged at 4000 rpm for 20 minutes, and the supernatant was taken and filtered with a 0.22 μm filter (Milipore). The antibody was purified by protein A affinity chromatography. Briefly, a HiTrap Mabselect suRe prepacked column (Cytiva) was equilibrated with 20 mM PB + 0.15 M NaCl buffer for 5 to 10 column volumes, and the filtered supernatant was loaded using an AKTA Avant 150 chromatography system (Cytiva). The purification column was then washed with 3 column volumes of 20 mM PB + 0.15 M NaCl buffer, 1 column volume of 20 mM PB + 1 M NaCl buffer, and then washed with 20 mM PB until the baseline was stable. Finally, the antibody was eluted with 20 mM citric acid (adjusted to pH 3.0 with 20 mM sodium citrate), and the peak 200 mAu-200 mAu was collected. The eluted antibody was immediately neutralized with neutralization buffer (1 M Tris-HCl, pH 9.0), placed in a 1.5 mL tube, and frozen at -80 for later use.

[0163] Example 3

[0164] ELISA affinity detection of humanized monoclonal antibodies

[0165] The relative binding activity of each antibody against human GPC3 protein was determined by ELISA. The following procedures were performed: 100 μL / well of recombinant human GPC3 protein (1 μg / mL) was coated onto a 96-well plate and incubated overnight at 4°C. The plates were then blocked with 1% BSA in PBST (containing 0.05% Tween-20) for 2 hours at 37°C (200 μL / well). The plates were washed three times with PBST. Each humanized monoclonal antibody was serially diluted in PBST containing 1% BSA and added to the 96-well plate (100 μL / well). The working concentrations of humanized monoclonal antibodies GC90, GC90-4mu, GC90LH1, GC90LH2, GC90LH3, GC90LH4, and GC90LH5 were 15 nM, 3.75 nM, and then diluted 4 times from 3.75 nM to 8 dilutions. The cells were incubated at 37°C for 1 hour and washed three times with PBST. Anti-Human IgG-FC-HRP (Sigma, 1 / 30,000 dilution) was then added at 100 μL / well, incubated at 37°C for 1 hour, washed three times with PBST, and then 50 μL of TMB (SURMOPICS) was added for reaction. The reaction was terminated with 1 M H2SO4, and the OD value was measured on a microplate reader at 450 nm-570 nm. As shown in Figure 2, the GC90 monoclonal antibody and its mutants of the present invention all have very good binding affinity to the GPC3 protein.

[0166] Example 4

[0167] ELISA detection of affinity matured monoclonal antibody molecules

[0168] The relative binding activity of each antibody to human GPC3 protein was determined by ELISA. The specific operation was as follows: recombinant human GPC3 protein (1 μg / mL) was coated in a 96-well plate at 100 μL / well and incubated at 4°C overnight. The plates were then blocked with 1% BSA in PBST (containing 0.05% Tween-20) for 2 hours at 37°C (200 μL / well), washed three times with PBST, and serially diluted with 1% BSA in PBST before being added to a 96-well plate (100 μL / well). The working concentrations of humanized mAbs GC90-4mu, GC90LH6, GC90LH7, and GC90LH8 were 10,000 ng / ul, 3,333.33 ng / ul, and 1,111.11 ng / ul, followed by 3-fold dilutions for a total of 10 points. The working concentrations of GC90LH8 and GC90LH10 were 10,000 ng / ul, 2,500 ng / ul, and 625 ng / ul, followed by 4-fold dilutions for a total of 8 points. The plates were incubated at 37°C for 1 hour and washed three times with PBST. Then, 100 μL / well of Anti-Human IgG-FC-HRP (Sigma, 1 / 30,000 dilution) was added, incubated at 37°C for 1 hour, washed three times with PBST, and then 50 μL of TMB (SURMOPICS) was added for reaction. The reaction was terminated with 1 M H₂SO₄, and the OD value was measured on a microplate reader at 450 nm-570 nm. As shown in Figure 3, the GC90 monoclonal antibody and its mutants of the present invention all have very good binding affinity to the GPC3 protein.

[0169] Example 5

[0170] Flow cytometry detection of humanized monoclonal antibody binding to tumor cells

[0171] Flow cytometry was used to determine the affinity of each anti-GPC3 antibody to HepG2, Hep3B, and Huh7 cells. The specific operation was as follows: each humanized monoclonal antibody was serially diluted with FACS Buffer (PBS + 5% FBS) and added to a 96-well U-shaped plate in sequence. The specific operation was as follows: the working concentration of each humanized monoclonal antibody was: 100nM, 25nM, and 6.25nM, and then diluted in multiples of 4 for 6 points. HepG2, Hep3B, and Huh7 cells were digested with trypsin, centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in FACS Buffer at a concentration of 2×10 6Cells were centrifuged at 4°C for 3 minutes at 3500 rpm, and the supernatant was discarded. The cells were then resuspended in 250 μL of pre-cooled FACS Buffer and centrifuged twice. 100 μL of diluted PE anti-human IgG FC fluorescent secondary antibody (BioLegend, 0.5 μL / 1×10 5 The cells were incubated on ice for 30 min in the dark. The supernatant was discarded and washed twice, and the cells were resuspended in 200 μL FACS buffer. MFI values ​​were measured using an Attune NxT flow cytometer (Thermo), and data were processed using GraphPad Prism software. The results in Figure 4 demonstrate that the monoclonal antibodies of the present invention have good affinity for HepG2 cells, Hep3B cells, and Huh-7 cells.

[0172] Example 6

[0173] Flow cytometry detection of humanized monoclonal antibody binding to tumor cells

[0174] Flow cytometry was used to determine the affinity of each anti-GPC3 antibody to HepG2 or Hep3B cells. The specific operation was as follows: each humanized monoclonal antibody was serially diluted with FACS Buffer (PBS + 5% FBS) and added to a 96-well U-shaped plate in sequence. The specific operation was as follows: GC90-4mu, GC90, GC90LH1, GC90LH2, GC90LH3, GC90LH4 and GC90LH5, the working concentrations were: 100nM, 25nM, 6.25nM and so on, diluted in multiples of 4 for a total of 8 points. HepG2 and Hep3B cells were digested with trypsin, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cells were resuspended in FACS Buffer at a concentration of 2×10 6 / mL, 50μL was added to each well of a 96-well U-bottom plate, and then 50μL of the diluted antibody was added to each well, gently mixed, and incubated on ice for 90min. After centrifugation at 4°C 3500rpm for 3min, the supernatant was discarded, and then 250μL of pre-cooled FACS Buffer was added to resuspend the cells, and the centrifugation and washing were repeated twice. 100μL of diluted PE anti-human IgG FC fluorescent secondary antibody (BioLegend, 0.5μL / 1×105 cells) was added to each well and incubated on ice in the dark for 30min. The supernatant was discarded and washed twice, and finally the cells were resuspended with 200μL FACS Buffer. The MFI value was detected using an Attune NxT flow cytometer (Thermo), and the data were processed using GraphPad Prism software. The results are shown in Figures 5A-5B. It can be seen that the monoclonal antibody of the present invention has a good affinity for HepG2 cells or Hep3B cells.

[0175] Example 7

[0176] Flow cytometry detection of affinity-matured monoclonal antibody binding to tumor cells

[0177] Flow cytometry was used to determine the affinity of each anti-GPC3 antibody to HepG2 cells. The specific operation was as follows: each humanized monoclonal antibody was serially diluted with FACS Buffer (PBS + 5% FBS) and added to a 96-well U-shaped plate in sequence. The specific operation was as follows: IgG1, GC90-4mu, GC90LH6, GC90LH7, GC90LH8, GC90LH9, GC90LH10 and GC90LH11, the working concentrations were: 100nM, 25nM, 6.25nM and then diluted in multiples of 4 for a total of 8-9 points. HepG2 cells were digested with trypsin, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cells were resuspended in FACS Buffer at a concentration of 2×10 6 Cells were centrifuged at 4°C for 3 minutes at 3500 rpm, and the supernatant was discarded. The cells were then resuspended in 250 μL of pre-cooled FACS Buffer and centrifuged twice. 100 μL of diluted PE anti-human IgG FC fluorescent secondary antibody (BioLegend, 0.5 μL / 1×10 5The cells were incubated on ice for 30 minutes in the dark. The supernatant was discarded, and the cells were washed twice, then resuspended in 200 μL FACS buffer. MFI values ​​were measured using an Attune NxT flow cytometer (Thermo), and data were processed using GraphPad Prism software. The results are shown in Figures 5C-5D, showing that the monoclonal antibodies of the present invention have good affinity for HepG2 cells.

[0178] Example 8

[0179] Flow cytometry detection of internalization efficiency of humanized monoclonal antibodies

[0180] In HepG2 and Hep3B cells, flow cytometry was used to determine the internalization efficiency of each anti-GPC3 antibody. The specific operation was as follows: the GC90 and hYP7HM monoclonal antibodies were diluted to an experimental concentration of 1 nM with FACS Buffer, and 50 μL / well was added to a 96-well U-shaped plate. The antibodies and cells were bound as in Example 5. After washing away the unbound antibodies, the cells were transferred to 37°C and incubated for 0, 1, 2, and 4 hours, respectively. After the incubation was completed, 100 μL of pre-cooled FACS buffer was immediately added to terminate internalization, followed by centrifugation at 3500 rpm for 3 minutes and the supernatant was discarded. The secondary antibody was configured as in Example 5, and the cells were resuspended with 100 μL of secondary antibody per well and incubated on ice for 30 minutes. The cells were washed twice by centrifugation and finally resuspended in 200 μL FACS Buffer. The MFI value was detected by FACS. The data were processed using GraphPad Prism software. The cellular internalization efficiency of the humanized monoclonal antibody was calculated as follows: (1-antibody MFI value at 37°C / antibody MFI value at 4°C) × 100%. The results are shown in Figure 6. According to the results in Figure 6, it can be seen that the humanized monoclonal antibody of the present invention has a good internalization efficiency.

[0181] Example 9

[0182] Preparation of Antibody-Drug Conjugates

[0183] 9.1 Site-directed coupling

[0184] The 239th residue of the antibody heavy chain was mutated to cysteine ​​(S239C, EU numbering, corresponding to the 243rd position of the GC90-4mu heavy chain), thereby site-specifically coupling the drug through the linker through the cysteine. Specifically, the single antigen solution was replaced with 20mM PBS buffer (pH = 7.2) and the concentration was adjusted to about 5mg / mL, and 250mM EDTA solution was added at a volume ratio of 50:1 (antibody: EDTA) and mixed thoroughly. Then, according to the different monoclonal antibodies and different linker-payload (LP) combinations, tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was added at a 1-12 times excess molar ratio (relative to the antibody), mixed thoroughly, and placed at room temperature (25°C) for reduction reaction for 3 hours. Add an appropriate amount of DMSO to the reaction solution, followed by a 6-12-fold molar excess (relative to the antibody) of the LP drug (pre-dissolved in DMSO at 5mM / 10mM), ensuring that the DMSO volume in the reaction system does not exceed 15%. Mix thoroughly and react at room temperature for 1.5 hours. Then, add N-acetyl-L-cysteine ​​(NAC) solution and allow to stand at room temperature for 10 minutes to terminate the reaction.

[0185] Ultrafiltration was used for desalting. The reaction solution was transferred to a 10KD ultrafiltration tube (Millipore), supplemented with PBS buffer (pH 6.0), and concentrated to the desired volume by centrifugation at 3500 g. PBS was added and the centrifugation was repeated five times. The product was filtered through a 0.22 μm filter membrane (Millipore) and stored at -80°C.

[0186] After the coupling reaction, IgG1-PBD, GC90-4mu-PBD, GC90LH8-PBD, GC90LH9-PBD, GC90LH10-PBD, GC90LH11-PBD and IgG1-DUBA, GC90-4mu-DUBA, and hYP7HM-DUBA site-specific coupling products were obtained. The purity of the ADC products was analyzed by size exclusion chromatography (SEC), and the drug-antibody coupling ratio (DAR) and naked antibody ratio were analyzed by hydrophobic interaction chromatography (HIC).

[0187] Table 7 ADC product detection analysis

[0188] 9.2 Random Coupling

[0189] The single antigen solution was replaced with 20 mM NaAc-HAc buffer (pH 5.5) and adjusted to a concentration of 5±1 mg / mL. The desired amount of tris(2-carboxyethyl)phosphine (TCEP) was added to the reaction system at a molar ratio of 20:1 to the antibody, mixed thoroughly, and then reduced at 37°C for 3 hours. After the reduction reaction, the solution was centrifuged in a 10 kD ultrafiltration tube and replaced with 20 mM NaAc / Tris, 1 mM EDTA, pH 7.0 buffer (3500 × g, four times) to adjust the concentration to 5±1 mg / mL. An appropriate amount of DMSO (~10%) was added to the reaction solution. The desired amount of LP solution (5-10 mM, pre-dissolved in DMSO) was added to the reaction system at a molar ratio of LP drug to antibody of 10:1, mixed thoroughly, and allowed to react at 21-25°C for 1 hour. Then, add N-acetylcysteine ​​(NAC) solution at a molar ratio of 40:1 to the antibody, mix thoroughly, and terminate the reaction at 21-25°C for 1 hour. After termination, replace the buffer with 20 mM His / His-HCl, pH 6.0 buffer (3500 × g, 4 times) using a 10 kD ultrafiltration centrifuge tube and adjust to the target concentration. After filtering through a 0.22 μm sterilizing filter, store at ≤-20°C.

[0190] After the coupling reaction, IgG1-Dxd and GC90-4mu-Dxd random coupling products were obtained. The purity of the ADC products was analyzed by size exclusion chromatography (SEC), and the drug-antibody coupling ratio (DAR) and naked antibody ratio were analyzed by hydrophobic interaction chromatography (HIC).

[0191] Example 10

[0192] Antibody-drug conjugates for tumor cell killing assay

[0193] The cell cytotoxicity of each antibody-drug conjugate against HepG2, Hep3B, and Huh7 liver cancer cells was determined using the Cell Counting Kit-8 (Dojindo). Specifically, HepG2, Hep3B, and Huh7 cells were cultured in 10% FBS (Gibco) + DMEM medium (Corning). When the cell confluence reached 75% or higher, the cells were digested with trypsin (0.25% Trypsin-EDTA) and counted. 1.5 × 10 4Cells / mL, 160 μL / well (2400 cells / well) were plated onto 96-well plates and cultured overnight at 37°C and 5% CO2. Each antibody-drug conjugate was then diluted to 333.5 nM using 10% FBS+DMEM medium and added to a 96-well plate containing 160 ul / well at a rate of 40 μL / well for a 5-fold dilution, with an initial concentration of 66.7 nM. Eight concentration points were serially diluted, and replicates were made; the cells were then cultured in a 37°C, 5% CO2 constant temperature incubator. On the fourth day, the viability of the tumor cells was detected using a Cell Counting Kit-8. The specific killing results are shown in Figure 7. As the administration concentration increased, the antibody-drug conjugate of the present invention showed a good cell killing effect on various types of liver cancer cells.

[0194] Example 11

[0195] 11.1 Antitumor Test of Test Substance 1

[0196] A subcutaneous xenograft tumor-bearing nude mouse model of human hepatocellular carcinoma Hep3B cell line was established.

[0197] Hep3B cells in the exponential growth phase were collected and resuspended in 1:1 PBS and Matrigel to adjust the cell density to 5×10 7 The experimental mice were subcutaneously inoculated with 5×10 6 Hep3B cells (0.1 mL / mouse) were added and the tumor growth was observed regularly. When the tumor grew to an average volume greater than 100 mm 3 The mice were randomly divided into groups of 6 based on tumor size. The day of grouping was defined as day 0. On the day of grouping, 1.5 mg / kg of IgG1-PBD or GC90-4mu-PBD was administered via a single tail vein injection. Tumor volume was measured twice weekly. The volume was calculated using the following formula:

[0198] Tumor volume (mm 3 )=1 / 2×(a×b 2 )(where a represents the major diameter and b represents the minor diameter)

[0199] Results: The data showed that GC90-4mu-PBD (1.5 mg / kg) had a significant anti-tumor effect ( FIG8A ).

[0200] 11.2 Antitumor Test of Test Substance 2

[0201] A subcutaneous xenograft tumor-bearing nude mouse model of human hepatocellular carcinoma Hep3B cell line was established.

[0202] Hep3B cells in the exponential growth phase were collected and resuspended in 1:1 PBS and Matrigel to adjust the cell density to 5×10 7The experimental mice were subcutaneously inoculated with 5×10 6 Hep3B cells (0.1 mL / mouse) were added and the tumor growth was observed regularly. When the tumor grew to an average volume greater than 100 mm 3 Rats were randomly divided into groups of 6 based on tumor size. The day of grouping was defined as day 0. On the day of grouping, 5 or 10 mg / kg of IgG1-DUBA, hYP7HM-DUBA, or GC90-4mu-DUBA were administered via a single tail vein injection. Tumor volume was measured twice weekly. The volume was calculated using the following formula:

[0203] Tumor volume (mm 3 )=1 / 2×(a×b 2 )(where a represents the major diameter and b represents the minor diameter)

[0204] Results: The data showed that both GC90-4mu-DUBA (5 mg / kg) and hYP7HM-DUBA (10 mg / kg) had significant anti-tumor effects (Figure 8B).

[0205] 11.3 Antitumor Tests of Test Substances 3

[0206] Establishment of a subcutaneous xenograft tumor-bearing nude mouse model of human hepatocellular carcinoma Hep3B cell line

[0207] Hep3B cells in the exponential growth phase were collected and resuspended in 1:1 PBS and Matrigel to adjust the cell density to 5×10 7 The experimental mice were subcutaneously inoculated with 5×10 6 Hep3B cells (0.1 mL / cell) were added, and tumor growth was observed regularly. When the tumor grew to an average volume of 80-150 mm 3 (Main experimental group) or 300-500mm 3(Satellite group) Mice were randomly divided and dosed according to tumor size and body weight, with 6 mice per group (1 mouse in the satellite IgG-PBD group). The second day of grouping was defined as Day 0: On Day 0, the satellite group received a single tail vein injection of 1.5 mg / kg GC90-4mu-PBD and IgG1-PBD. Blood was collected and serum was collected 24 hours after administration in the satellite IgG-PBD group, and tumors were harvested. Blood and tumors were collected alternately in the GC90-4mu-PBD group (serum was collected 15 minutes later, and serum and tumors were collected 7 hours, D1, D2, D3, D5, and D7). On Day 0, the main experimental group received a single tail vein injection of IgG1-PBD (0.6 mg / kg), GC90-4mu-PBD (0.1, 0.3, and 0.6 mg / kg), and GC90-4mu-Dxd (3 and 10 mg / kg). Mouse body weight and tumor size were measured twice weekly. The volume calculation formula is as follows:

[0208] Tumor volume (mm 3 )=1 / 2×(a×b 2 )(where a represents the major diameter and b represents the minor diameter)

[0209] Example 12

[0210] Functional characterization of GC90 antibody

[0211] 12.1 Species Cross-Reactivity

[0212] GC90 binding to GPC3 proteins from different species was determined by ELISA. Specifically, 100 μL / well of each GPC3 protein (6 μg / mL) was coated onto a 96-well plate and incubated overnight at 4°C. The plates were then blocked with 1% BSA in PBST (containing 0.05% Tween-20) for 2 hours at 37°C (200 μL / well), washed three times with PBST, and serially diluted GC90 in PBST containing 1% BSA. The plates were then added to the 96-well plate (100 μL / well) at a working concentration of 30 nM, 15 nM, and 15 nM, followed by eight dilutions in 4 steps. The plates were incubated at 37°C for 1 hour, and washed three times with PBST. Then, 100 μL / well of Anti-Human IgG-FC-HRP (Sigma, 1 / 30000 dilution) was added, incubated at 37°C for 1 h, washed three times with PBST, and then 50 μL of TMB (SURMOPICS) was added to react. The reaction was terminated with 1 M H2SO4, and the OD value was measured at 450 nm by a microplate reader. The results are shown in Table 8.

[0213] Table 8 Binding of GC90 to GPC3 proteins from different species

[0214] Note: √ represents affinity recognition, and × represents no recognition.

[0215] 12.2 Fine Mapping of Recognition Epitopes

[0216] The amino acid sequence of the GPC3-B peptide was divided into multiple overlapping peptides, the amino acid sequences of which are shown in Table 9. Overlapping peptides were synthesized and the binding properties of each peptide to GC90 were measured using ELISA to further accurately identify the epitope recognized by GC90. Specifically, each overlapping peptide (6 μg / mL) was coated in a 96-well plate at 100 μL / well and incubated overnight at 4°C. The plates were then blocked with 1% BSA in PBST (containing 0.05% Tween-20) for 2 hours at 37°C (200 μL / well), washed three times with PBST, and serially diluted GC90 in PBST with 1% BSA (negative control: 1% BSA) and added to the 96-well plate (100 μL / well) at a working concentration of 30 nM, 15 nM, and 15 nM, followed by 8 dilutions in 4 steps. The plates were incubated at 37°C for 1 hour and washed three times with PBST. Then, 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 react. The reaction was terminated with 1M H2SO4 and the OD value was measured at 450 nm using a microplate reader. The results are shown in Figure 9. 450 The values ​​show the binding strength between GC90 and each peptide. It can be seen that the antibody has strong binding activity only with GPC3-B3 or long fragments containing GPC3-B3. It can be determined that the GPC3 antigen epitope region is located in GPC3-B3, namely, residues 485-496 of the human GPC3 protein (DKNLDEEGFESG).

[0217] Table 9 Amino acid sequences of overlapping peptides

[0218] Further, amino acid sequence analysis of the homology between human and mouse GPC3 proteins ( FIG10 ) revealed two amino acid residue differences within the GPC3-B3 region (referenced for monkey GPC3 protein, Genbank ID: XP_005594665.1, also including GPC3-B3), namely, amino acid residue 487 (N) and amino acid residue 493 (F) in the human GPC3 protein. Combined with the results of cross-species detection, it was determined that GC90 was unable to bind to the mouse GPC3 protein due to mutations in the mouse GPC3 protein at positions corresponding to asparagine 487 and phenylalanine 493 in the human GPC3 protein. This indicates that positions 487 and / or 493 of the human GPC3 protein are one of the core binding sites in the antigen epitope region.

[0219] Figure 11 summarizes the antigen recognition regions of various anti-GPC3 antibodies on the GPC3 protein. It can be seen that the antigen epitope bound by the antibodies of the present invention is distinct from the antigen binding regions of other reported anti-GPC3 antibodies, recognizing a novel GPC3 epitope. The present invention identifies a novel GPC3 epitope, and antibodies that bind to it exhibit high affinity and internalization efficiency. Using this GPC3 epitope to prepare immunogens opens up a new research and development direction in the field of anti-GPC3 antibodies, reducing the uncertainty of antibody preparation and screening and facilitating the development of new antibodies with improved functionality.

[0220] 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 the present description is intended to cover all substitutions, modifications and variations 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 by reference in their entirety.

Claims

1. An anti-GPC3 antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL); The heavy chain variable region (VH) comprises HCDR1, HCDR2 and HCDR3 regions, and the HCDR1, HCDR2 and HCDR3 regions respectively comprise sequences that are at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CDR1, CDR2 and CDR3 regions of any of the amino acid sequences shown in SEQ ID Nos: 1-2, 6-9; or a sequence comprising at most 3, 2 or 1 mutations in the CDR1, CDR2 and CDR3 regions of the amino acid sequence shown in any one of SEQ ID Nos: 1-2, 6-9, respectively; The light chain variable region (VL) comprises LCDR1, LCDR2 and LCDR3 regions, and the LCDR1, LCDR2 and LCDR3 regions respectively comprise sequences that are 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 SEQ ID No: 3; Or a sequence comprising at most 3, 2 or 1 mutations compared to the CDR1, CDR2 and CDR3 regions of the amino acid sequence of SEQ ID NO: 3, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof has the following biological function: specifically binding to the antigen shown in SEQ ID Nos: 37 or 38, and not binding to the antigen shown in SEQ ID No:

36.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the position of the mutation is selected from one or more of position 56 (D56), position 100 (Q100) or position 102 (S102) of the amino acid sequence shown in any one of SEQ ID Nos: 1-2, 6-9; Preferably, the mutation is selected from Q100R, S102R, D56A, D56K+Q100R, D56A+S102R or D56K+S102R.

4. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The HCDR1, HCDR2 and HCDR3 regions respectively have the same sequences as the CDR1, CDR2 and CDR3 regions of the amino acid sequence shown in any one of SEQ ID Nos: 1-2, 6-9, 23-28; and the LCDR1, LCDR2 and LCDR3 regions respectively 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 thereof according to any one of claims 1 to 4, characterized in that: The CDR1, CDR2 and CDR3 regions are defined according to IMGT, Kabat, Chothia, AbM or Contact.

6. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that: The VH comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in any one of SEQ ID Nos: 1-2, 6-9, 23-28; and / or The VL comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No:

3.

7. The antibody or antigen-binding fragment thereof according to claim 6, characterized in that: The VH comprises a sequence identical to any one of the amino acid sequences SEQ ID Nos: 1-2, 6-9, 23-28; and the VL comprises a sequence identical to the amino acid sequence SEQ ID NO:

3.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, characterized in that: It also comprises a heavy chain constant region (CH) and a light chain constant region (CL); preferably, the CH comprises a sequence identical to the amino acid sequence of SEQ ID Nos: 4, 19 or 20; and the CL comprises a sequence identical to the amino acid sequence of SEQ ID No:

5.

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

10.

10. The antibody or antigen-binding fragment thereof according to claim 9, characterized in that: The heavy chain (H) comprises a sequence identical to any one of the amino acid sequences SEQ ID Nos: 11-18, 29-34; the light chain (L) comprises a sequence identical to the amino acid sequence SEQ ID No:

10.

11. An anti-GPC3 antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof binds to the antigen set forth in SEQ ID Nos: 37 or 38, and does not bind to the antigen set forth in SEQ ID No: 36; Alternatively, the antibody or antigen-binding fragment thereof competes with a reference antibody for binding to the same epitope of the GPC3 protein, wherein the reference antibody comprises a heavy chain as set forth in SEQ ID NO: 11 and a light chain as set forth in SEQ ID NO: 10; Preferably, the anti-GPC3 antibody or antigen-binding fragment thereof is capable of blocking 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, characterized in that: The GPC3 antigen epitope peptide consists of at least 7 consecutive amino acid residues within residues 485-496 of the human GPC3 protein, the amino acid sequence of which is shown in 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) Specific binding to anti-GPC3 antibodies; (2) inducing an immune response (e.g., a humoral immune response) against GPC3 in a subject; (3) inducing the production of anti-GPC3 antibodies in a subject; (4) Preventing and / or treating a GPC3-related disease in a subject.

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

38.

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

15. A chimeric antigen receptor comprising the anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11. 16 . A polynucleotide encoding the anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 , the GPC3 antigen epitope peptide according to any one of claims 12 to 13 , or the recombinant antigen according to claim 14 . An expression vector comprising the polynucleotide of claim 16 .

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

19. An antibody-drug conjugate, characterized in that: The method comprises the anti-GPC3 antibody or antigen-binding fragment thereof 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 selected from one or more of SN-38, MMAE, PBD dimer, DX-8951 (DXd) or DUBA.

21. A bispecific or multispecific antibody molecule, characterized in that: The invention comprises the anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

22. A pharmaceutical composition, characterized in that It comprises the anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, or the GPC3 antigen epitope peptide according to any one of claims 12 to 13, or the recombinant antigen according to claim 14, or the chimeric antigen receptor according to claim 15, or the nucleic acid molecule according to claim 16, or the vector according to claim 17, or the host cell according to claim 18, or the antibody-drug conjugate according to any one of claims 19 to 20, or the bispecific or multispecific antibody molecule according to claim 21; and one or more pharmaceutically acceptable carriers.

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

24. Use of the anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the GPC3 antigen epitope peptide according to any one of claims 12 to 13, the recombinant antigen according to claim 14, the chimeric antigen receptor according to claim 15, the nucleic acid molecule according to claim 16, the vector according to claim 17, the host cell according to claim 18, the antibody-drug conjugate according to any one of claims 19 to 20, the bispecific or multispecific antibody molecule according to claim 21, or the kit according to claim 23 in the preparation of a medicament for treating or preventing cancer, 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) preparing an anti-GPC3 antibody or an antigen-binding fragment thereof; (2) preparing a product for treating and / or preventing and / or diagnosing a GPC3-related disease in a subject; (3) preparing products for detecting anti-GPC3 antibodies or antigen-binding fragments thereof; (4) detecting anti-GPC3 antibodies or antigen-binding fragments thereof; (5) Screening anti-GPC3 antibodies or antigen-binding fragments thereof.