Antibodies recognizing multiple different epitopes of glypican 3 and their applications

Abstract

The present invention provides a plurality of monoclonal antibodies that can recognize glycan 3 (GPC3) with high affinity. These monoclonal antibodies not only have high affinity, but also cover multiple antigenic epitopes of GPC3, and can be used to detect tumor marker GPC3 by sandwich method. The monoclonal antibodies provided by the present invention not only have good thermal stability, but also have excellent cytotoxic activity after being conjugated with Pseudomonas exotoxin PE24, which is significantly better than the existing immunotoxin HN3-PE24, and can be used to develop immunotoxins or antibody-drug conjugates (ADC) with stronger activity and better stability.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a high-affinity monoclonal antibody capable of recognizing a plurality of different antigenic epitopes of phosphatidylinositol proteoglycan 3 and an application thereof. Background Art

[0002] Cancer has become the second leading cause of death after cardiovascular disease, among which the global incidence and mortality of liver cancer rank sixth and fourth respectively. Although surgery is the standard treatment for liver cancer, due to the hidden nature of liver cancer and its high malignancy, most patients are diagnosed in the middle or late stages, so only 5-10% of liver cancer patients can undergo surgical treatment. Middle and late stage patients do not respond well to most chemotherapy drugs because of this type of cancer. Therefore, there is an urgent need to develop new drugs with different mechanisms of action. Immunotherapy represents a new approach, but it remains a challenge, mainly because there are no good tumor-specific targets.

[0003] Glypican-3 (GPC3) is a member of the heparan sulfate proteoglycans and is anchored to the cell membrane surface by glycosyl-phosphatidylinositol (GPI). The human GPC3 gene is located on chromosome X (Xp26) and encodes a 70kDa protein containing 580 amino acids. The protein is endocleaved by furin-like convertase between Arg358 and Ser359 to produce a 40kDa N-terminal subunit and a 30kDa C-terminal subunit. The C-terminal subunit also has two heparan sulfate (HS) chains. Studies have shown that the expression level of GPC3 in about 72% of hepatocellular carcinoma (HCC) is significantly upregulated compared with normal hepatocellular carcinoma, cholangiocarcinoma and liver metastasis, but it is not expressed in normal adult liver tissue. In addition, high expression of GPC3 in patients with liver cancer is often associated with poor prognosis, which indicates the potential role of GPC3 as a biomarker in HCC. GPC3 has been suggested as a target for antibody (Ishiguro et al., Cancer Res 68:9832-9838, 2008; Nakano et al., Biochem Biophys Res Commun 378:279-284, 2009; Nakano et al., Anticancer Drugs 21:907-916, 2010) and cell-based (Nakatsura et al., Clin Cancer Res 10:8630-8640, 2004; Komori et al., Clin Cancer Res 12:2689-2697, 2006) immunotherapy.

[0004] Antibody-drug conjugates (ADCs) are very effective tumor-targeted therapeutics. Antibody fragments can also be fused with protein toxin fragments to produce chimeric structures called immunotoxins. Lumoxiti, an immunotoxin targeting CD22, has been approved by the FDA for the treatment of relapsed or refractory hairy cell leukemia (HCL). Pseudomonas exotoxin (PE) A is a commonly used toxin fragment in immunotoxins, which can induce protein synthesis blockage and ultimately lead to cell death. Immunotoxins are believed to trigger tumor regression through two mechanisms: antibody-induced cell signal inactivation and toxin-induced protein synthesis inhibition, so antibodies with different mechanisms of action conjugated to the same toxin may have very different cytotoxicity. According to the results of Wei Gao et al., although the affinity of the antibody HN3 for GPC3 is much weaker than that of YP7, the cytotoxicity and in vivo tumor inhibition activity of the immunotoxin HN3-PE24 for GPC3 are higher than those of YP7-PE24 (Wei Gao et al. Nat Commun. 2015 Mar 11; 6: 6536.). Therefore, focusing on antibody research and development and finding better antibodies can synthesize ADCs or antibody-toxin conjugates with better activity. This is an important strategy for developing ADCs or immunotoxin drugs. Summary of the invention

[0005] The present invention aims to provide monoclonal antibodies that recognize multiple antigenic epitopes of GPC3 with high affinity. The antibodies provided include immunoconjugates of antibody fragments (e.g., single-chain variable region fragments (scFv)) and effector molecules (e.g., toxin proteins). Also provided are compositions comprising antibodies that specifically bind to GPC3, nucleic acid molecules encoding these antibodies, expression vectors comprising the nucleic acid molecules, and isolated host cells expressing the nucleic acid molecules.

[0006] The specific technical solutions of the present invention are as follows:

[0007] A monoclonal antibody against glypican 3, comprising a heavy chain variable region and a light chain variable region, wherein (1) the heavy chain variable region of the antibody has the complementary determining regions shown by amino acid residues 26-35, 50-66 and 97-115 of SEQ ID NO:1; the light chain variable region of the antibody has the complementary determining regions shown by amino acid residues 23-29, 46-52 and 85-97 of SEQ ID NO:2;

[0008] Alternatively, (2) the heavy chain variable region of the antibody has the complementary determining regions shown by amino acid residues 26-35, 50-66 and 97-116 of SEQ ID NO:3; the light chain variable region of the antibody has the complementary determining regions shown by amino acid residues 23-30, 47-53 and 86-95 of SEQ ID NO:4;

[0009] Alternatively, (3) the heavy chain variable region of the antibody has the complementary determining regions shown by amino acid residues 26-35, 50-66 and 97-117 of SEQ ID NO:5; the light chain variable region of the antibody has the complementary determining regions shown by amino acid residues 23-30, 47-53 and 86-97 of SEQ ID NO:6;

[0010] Alternatively, (4) the heavy chain variable region of the antibody has the complementary determining regions shown by amino acid residues 26-35, 50-66 and 97-111 of SEQ ID NO:7; the light chain variable region of the antibody has the complementary determining regions shown by amino acid residues 23-31, 48-54 and 87-95 of SEQ ID NO:8;

[0011] Alternatively, (5) the heavy chain variable region of the antibody has the complementary determining regions shown by amino acid residues 26-35, 50-66 and 97-115 of SEQ ID NO:9; the light chain variable region of the antibody has the complementary determining regions shown by amino acid residues 23-30, 47-53 and 86-97 of SEQ ID NO:10;

[0012] Alternatively, (6) the heavy chain variable region of the antibody has the complementary determining regions shown by amino acid residues 26-35, 50-66 and 97-114 of SEQ ID NO:11; the light chain variable region of the antibody has the complementary determining regions shown by amino acid residues 23-30, 47-53 and 86-95 of SEQ ID NO:12;

[0013] Alternatively, (7) the heavy chain variable region of the antibody has the complementary determining regions shown by amino acid residues 26-35, 50-66 and 97-117 of SEQ ID NO:13; the light chain variable region of the antibody has the complementary determining regions shown by amino acid residues 23-30, 47-53 and 86-94 of SEQ ID NO:14;

[0014] Alternatively, (8) the heavy chain variable region of the antibody has the complementary determining regions shown by amino acid residues 26-35, 50-66 and 97-117 of SEQ ID NO:15; the light chain variable region of the antibody has the complementary determining regions shown by amino acid residues 23-30, 47-53 and 86-98 of SEQ ID NO:16;

[0015] Alternatively, (9) the heavy chain variable region of the antibody has the complementary determining regions shown by amino acid residues 26-35, 50-66 and 97-108 of SEQ ID NO:17; the light chain variable region of the antibody has the complementary determining regions shown by amino acid residues 23-35, 52-58 and 91-104 of SEQ ID NO:18;

[0016] Alternatively, (10) the heavy chain variable region of the antibody has the complementary determining regions shown by amino acid residues 26-35, 50-65 and 96-115 of SEQ ID NO:19; the light chain variable region of the antibody has the complementary determining regions shown by amino acid residues 23-30, 47-53 and 86-95 of SEQ ID NO:20;

[0017] Alternatively, (11) the heavy chain variable region of the antibody has the complementary determining regions shown by amino acid residues 26-35, 50-66 and 97-117 of SEQ ID NO:21; the light chain variable region of the antibody has the complementary determining regions shown by amino acid residues 23-32, 49-55 and 88-97 of SEQ ID NO:22;

[0018] Alternatively, (12) the heavy chain variable region of the antibody has the complementary determining regions shown by amino acid residues 26-35, 50-66 and 97-115 of SEQ ID NO:23; the light chain variable region of the antibody has the complementary determining regions shown by amino acid residues 23-29, 46-52 and 85-96 of SEQ ID NO:24;

[0019] Alternatively, (13) the heavy chain variable region of the antibody has the complementary determining regions shown by amino acid residues 26-35, 50-66 and 97-114 of SEQ ID NO:25; the light chain variable region of the antibody has the complementary determining regions shown by amino acid residues 23-30, 47-53 and 86-95 of SEQ ID NO:26;

[0020] Alternatively, (14) the heavy chain variable region of the antibody has the complementary determining regions shown by amino acid residues 26-35, 50-66 and 97-115 of SEQ ID NO:27; the light chain variable region of the antibody has the complementary determining regions shown by amino acid residues 23-30, 47-53 and 86-96 of SEQ ID NO:28.

[0021] There are four known subtypes of human GPC3 (subtypes 1-4). The nucleic acid and amino acid sequences of the four subtypes of GPC3 are known, including GenBank accession numbers: NM_001164617 and NP_001158089 (subtype 1); NM_004484 and NP_004475 (subtype 2); NM_001164618 and NP_001158090 (subtype 3); and NM_001164619 and NP_001158091 (subtype 4). The antibodies described in the present invention may bind to one or more of the four human GPC3 subtypes, or conservative variants thereof.

[0022] Specifically, the antibody against glypican 3 of the present invention is:

[0023] (1) The heavy chain variable region amino acid sequence of the antibody is shown in SEQ ID NO: 1, and the light chain variable amino acid sequence is shown in SEQ ID NO: 2;

[0024] Alternatively, (2) the heavy chain variable region amino acid sequence of the antibody is as shown in SEQ ID NO:3, and the light chain variable amino acid sequence is as shown in SEQ ID NO:4;

[0025] Alternatively, (3) the heavy chain variable region amino acid sequence of the antibody is as shown in SEQ ID NO:5, and the light chain variable amino acid sequence is as shown in SEQ ID NO:6;

[0026] Alternatively, (4) the heavy chain variable region amino acid sequence of the antibody is as shown in SEQ ID NO:7, and the light chain variable amino acid sequence is as shown in SEQ ID NO:8;

[0027] Alternatively, (5) the heavy chain variable region amino acid sequence of the antibody is as shown in SEQ ID NO:9, and the light chain variable amino acid sequence is as shown in SEQ ID NO:10;

[0028] Alternatively, (6) the heavy chain variable region amino acid sequence of the antibody is as shown in SEQ ID NO: 11, and the light chain variable amino acid sequence is as shown in SEQ ID NO: 12;

[0029] Alternatively, (7) the heavy chain variable region amino acid sequence of the antibody is as shown in SEQ ID NO: 13, and the light chain variable amino acid sequence is as shown in SEQ ID NO: 14;

[0030] Alternatively, (8) the heavy chain variable region amino acid sequence of the antibody is as shown in SEQ ID NO: 15, and the light chain variable amino acid sequence is as shown in SEQ ID NO: 16;

[0031] Alternatively, (9) the heavy chain variable region amino acid sequence of the antibody is as shown in SEQ ID NO: 17, and the light chain variable amino acid sequence is as shown in SEQ ID NO: 18;

[0032] Alternatively, (10) the heavy chain variable region amino acid sequence of the antibody is as shown in SEQ ID NO: 19, and the light chain variable amino acid sequence is as shown in SEQ ID NO: 20;

[0033] Alternatively, (11) the heavy chain variable region amino acid sequence of the antibody is as shown in SEQ ID NO: 21, and the light chain variable amino acid sequence is as shown in SEQ ID NO: 22;

[0034] Alternatively, (12) the heavy chain variable region amino acid sequence of the antibody is as shown in SEQ ID NO: 23, and the light chain variable amino acid sequence is as shown in SEQ ID NO: 24;

[0035] Alternatively, (13) the heavy chain variable region amino acid sequence of the antibody is as shown in SEQ ID NO: 25, and the light chain variable amino acid sequence is as shown in SEQ ID NO: 26;

[0036] Alternatively, the heavy chain variable region amino acid sequence of the antibody (14) is as shown in SEQ ID NO:27, and the light chain variable amino acid sequence is as shown in SEQ ID NO:28.

[0037] The antibody of the present invention is a single-chain antibody, a double-chain antibody, a monoclonal antibody or a chimeric antibody.

[0038] The monoclonal antibodies of the present invention can be of any isotype. For example, they can be IgM or IgG antibodies, such as IgG1 or IgG2. The types of antibodies that can specifically bind to GPC3 can be converted to each other according to known methods (for example, IgG can be converted to IgM). Type conversion can also be used to convert one IgG subclass to another, such as from IgG1 to IgG2.

[0039] The antibody of the present invention may be:

[0040] (1) Fab, a fragment comprising a monovalent antigen-binding fragment of an antibody molecule, which can be produced by digesting an intact antibody with papain to produce a complete light chain and a portion of one heavy chain;

[0041] (2) Fab', an antibody molecule fragment that can be obtained by treating an intact antibody with pepsin followed by reduction to produce an intact light chain and a portion of a heavy chain; two Fab' fragments are obtained per antibody molecule;

[0042] (3) (Fab')2, an antibody fragment obtained by treating an intact antibody with pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments linked together by two disulfide bonds;

[0043] (4) Fv, a gene fragment containing the light chain variable region and the heavy chain variable region expressed as two chains;

[0044] (5) Single-chain antibodies (e.g., scFv), genetically engineered molecules containing a light chain variable region and a heavy chain variable region connected by a suitable polypeptide linker into a genetically fused single-chain molecule;

[0045] (6) dimers of single-chain antibodies (scFv2), defined as dimers of scFv (also known as “minibodies”);

[0046] (7) VH single-domain antibody, an antibody fragment consisting of the heavy chain variable region.

[0047] Those skilled in the art will appreciate that conservative variants of antibodies can be prepared. Amino acid substitutions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions) can be made in the VH and / or VL regions, and the substituted VH and VL still retain the ability to bind to GPC3, or have a stronger binding ability to GPC3. Conservative substitutions of amino acids with similar functions are well known to those of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for each other:

[0048] 1) Alanine (A), serine (S), threonine (T);

[0049] 2) Aspartic acid (D), glutamic acid (E);

[0050] 3) Asparagine (N), glutamine (Q);

[0051] 4) Arginine (R), Lysine (K);

[0052] 5) Isoleucine (I), leucine (L), methionine (M), valine (V);

[0053] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0054] Another object of the present invention is to provide a recombinant protein, wherein the recombinant protein comprises the antibody of the present invention and a tag sequence for assisting expression and / or purification. The tag sequence includes but is not limited to a 6x His tag.

[0055] The GPC3 antibody of the present invention can be conjugated to an effector molecule, which includes but is not limited to a toxin, a drug or a detectable marker.

[0056] The drug of the present invention is a substance having cytotoxic or anti-tumor activity, such as Monomethylauristatin E (MMAE), Monomethylauristatin F (MMAF), Pyrrolobenzodiazepine (PBD) dimer, N2'-deacetyl-N2'-(3-Mercapto-1-oxopropyl)-Maytansine (Maytansine DM1), vinblastine, daunomycin, etc., and also radioactive reagents 125 I. 32 P. 14 C. 3 H and 35 S et al.

[0057] The toxins described in the present invention are toxic proteins with cytotoxic or anti-tumor activity, which can be conjugated with the antibodies described in the present invention to form immunotoxins, including but not limited to Pseudomonas exotoxin, ricin, abrin, diphtheria toxin and its subunits and botulinum toxin AF, as well as truncated mutants and point mutants of these toxins. These toxins can be purchased commercially (e.g., Sigma Chemical Company, St. Louis, MO). The toxins also include variants of the above toxins (e.g., see U.S. Patent Nos. 5,079,163 and 4,689,401). In one embodiment, the toxin is Pseudomonas exotoxin (PE) (U.S. Patent No. 5,602,095). The "Pseudomonas exotoxin" includes its native sequence, the cytotoxic fragment of the native sequence, and conservatively modified variants of the native sequence or its cytotoxic fragment. These modifications include, but are not limited to, removal of domains Ia, Ib, multiple amino acid deletions in II and III, single or multiple amino acid substitutions, and addition of one or more sequences at the carboxyl terminus (e.g., see Siegall et al., J. Biol. Chem. 264: 14256-14261, 1989). Cytotoxic fragments of Pseudomonas exotoxin include PE24, PE40, PE38, PE35, and the like.

[0058] The detectable markers of the present invention are substances that can be detected by an isotope analyzer, an enzyme marker, a bioluminescence detector, a chemiluminescence detector, an electrochemiluminescence detector, a fluorescence analyzer, or can be visualized by the naked eye, and these substances include but are not limited to radioactive isotopes (such as 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, 111 In,125 I. 131 I), enzymes that can be used for detection (such as horseradish peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, etc.), fluorescent proteins (such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), allophycocyanin APC, phycoerythrin PE), bioluminescent markers (such as luciferase), fluorescent compounds (such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamino-1-naphthalenesulfonyl chloride, phycoerythrin, fluorescent dyes Cy3, Cy5, rare earth inorganic luminescent materials, quantum dots, etc.), biotin, magnetic reagents (such as gadolinium), electrochemiluminescent reagents (such as terpyridine ruthenium), and colloidal gold.

[0059] The effector molecule can be connected to the antibody of the present invention using any means known to those skilled in the art. For example, the antibody can be functionally connected (by chemical coupling, genetic fusion, non-covalent association or other) to one or more other molecular entities. Depending on the chemical structure of the effector molecule, the method of connecting the effector molecule and the antibody is different. Polypeptides generally contain multiple functional groups; for example, carboxylic acid (COOH), free amino group (-NH2) or sulfhydryl group (-SH), which can be used to react with suitable functional groups on the antibody to bind to the effector molecule. Alternatively, the antibody can be derivatized to expose or connect additional reactive functional groups. The derivatization may include connecting any of a variety of known linker molecules. The linker can be any molecule used to join the antibody to the effector molecule. The linker can form a covalent bond with the antibody and the effector molecule. Suitable linkers are well known to those skilled in the art, including but not limited to linear or branched carbon linkers, heterocyclic carbon linkers or peptide linkers. In the case where the antibody and the effector molecule are polypeptides, the linker can be joined to the α-carbon amino group and carboxyl group of the constituent amino acids or to the terminal amino acid through its side groups (e.g., through the disulfide bond of cysteine). Typically, the antibody or portion thereof is derivatized such that binding to the target antigen is not adversely affected by the derivatization or labeling.

[0060] In some cases, when the immunoconjugate has reached its target site, it is desirable to release the effector molecule from the antibody. Therefore, in these cases, the immunoconjugate will contain a bond that is cleavable near the target site. Cleavage of the linker to release the effector molecule from the antibody may be caused by enzymatic activity, or by the conditions to which the immunoconjugate is exposed within the target cell or near the target site.

[0061] Another object of the present invention is to provide a polynucleotide encoding the antibody, recombinant protein or immunoconjugate of the present invention.

[0062] Another object of the present invention is to provide a vector containing the polynucleotide of the present invention, including bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0063] Another object of the present invention is to provide a pharmaceutical composition, comprising one or more of the antibodies, recombinant proteins, immunoconjugates, polynucleotides, vectors or genetically engineered host cells described in the present invention. The pharmaceutical composition also includes a pharmaceutically acceptable carrier. The antibodies, recombinant proteins, immunoconjugates, polynucleotides, vectors or genetically engineered host cells are soluble in aqueous carriers, such as buffered saline. It may also contain pharmaceutically acceptable excipients required to approach physiological conditions, such as pH regulators and buffering agents, sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate.

[0064] Another object of the present invention is to provide the use of the antibodies, recombinant proteins, immunoconjugates, polynucleotides, vectors or genetically engineered host cells of the present invention in the preparation of therapeutic drugs or diagnostic reagents for autoimmune diseases, viral infections or cancers.

[0065] The cancer is liver cancer, gastric cancer, colorectal cancer, lung cancer or ovarian cancer, or any other type of cancer expressing GPC3.

[0066] The monoclonal antibodies disclosed in the present invention can also be used to prepare chimeric antigen receptors (CAR; also known as chimeric T cell receptors, artificial T cell receptors or chimeric immunoreceptors) or bispecific antibodies.

[0067] The specific embodiments of the present invention disclose antibodies A5, A18, A43, C46, ​​F5, F67, G15, H49, I34, I82, I88, J58, J80A, and J80B with high affinity and recognition of different epitopes. The present invention also discloses the effect of these antibodies fused with Pseudomonas exotoxin A (PE) immunotoxins for treating liver cancer. These antibodies can also be constructed into bispecific antibodies, antibody-drug conjugates (ADCs), etc. for antibody targeted therapy, or constructed into CAR-T, CAR-NK, etc. for cell therapy. The antibodies and compositions can be used to diagnose tumors that are positive for GPC3 expression.

[0068] The antibodies and compositions provided by the present invention can be used for a variety of purposes, such as for molecular diagnosis of tumors, to confirm whether GPC3 is expressed in samples from patients with liver cancer and other tumors. The sample can be any sample, including but not limited to tissues from biopsies, autopsies, and pathological specimens. Biological samples also include sections of tissues, such as frozen sections obtained for histological purposes. Biological samples also include body fluids, such as blood, serum, plasma, sputum, cerebrospinal fluid, or urine. Biological samples are generally obtained from mammals, including humans, non-human primates, mice, etc.

[0069] The present invention also provides a method for treating a subject suffering from cancer, such as liver cancer, by selecting a subject suffering from cancer expressing GPC3, and administering to the subject a therapeutically effective amount of a monoclonal antibody against GPC3, or an immunoconjugate comprising the antibody.

[0070] Advantages of the present invention:

[0071] The monoclonal antibody combination provided by the present invention covers multiple different antigenic epitopes in the GPC3 molecule. Therefore, different monoclonal antibodies can be combined and paired to prepare detection reagents or kits for GPC3. The monoclonal antibodies provided by the present invention not only have high affinity (Kd values ​​are all at nM and pM levels, or even higher), but also have good thermal stability. The monoclonal antibodies provided by the present invention have excellent cytotoxic activity after being conjugated with Pseudomonas exotoxin PE24, which is significantly better than the existing immunotoxin HN3-PE24. The antibodies described in the present invention can be used to develop antibody-drug conjugates or immunotoxin drugs with better activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 Epitope clustering analysis of 23 monoclonal antibodies. The 23 monoclonal antibodies were initially divided into 14 epitopes (represented by J58, J80B, A5, H49, F5, G15, A43, A18, F67, C46, ​​I34, J80A, I82, and I88) using monoclonal phage competitive ELISA.

[0073] Figure 2 The monoclonal antibody of the present invention is used for detecting GPC3 by sandwich ELISA. The first vertical column is the coating antibody. After adding GPC3 protein and co-incubating, the monoclonal antibody in the first horizontal column is used as the detection antibody to detect GPC3. The light gray square indicates that the antigenic epitopes of the coating antibody and the detection antibody do not overlap, which is suitable for the double antibody sandwich method to detect GPC3 in biological samples. The black square indicates that the antigenic epitopes of the coating antibody and the detection antibody overlap, which is not suitable for pairing by the double antibody sandwich method.

[0074] Figure 3Simplified diagram of the antigenic epitopes recognized by the antibodies of the present invention. The overlapping intersections indicate that the antigenic epitopes of these monoclonal antibodies partially overlap.

[0075] Figure 4 The affinity of the monoclonal antibody of the present invention for binding to the GPC3 protein was detected by ELISA. Figure 4 A is human GPC3 protein, Figure 4 B is mouse GPC3 protein.

[0076] Figure 5 The binding activity of the monoclonal antibody of the present invention to the GPC3 negative / positive cell line was detected by FACS. Figure 5 a is the GPC3 negative A431 cell line, Figure 5 b is the GPC3 positive cell line G1, Figure 5 c is the GPC3-positive liver cancer cell line HepG2, Figure 5 d is the GPC3-positive liver cancer cell line Hep3B, Figure 5 e is the GPC3-positive liver cancer cell line Huh7.

[0077] Figure 6 The immunotoxin of the present invention has killing activity against the GPC3-negative cell line A431LG and the GPC3-positive cell lines G1LG, Hep3BLG, HepG2LG and Huh7LG.

[0078] Figure 7 In vivo tumor inhibition activity of immunotoxin J80A. Hep3B cells were inoculated subcutaneously into NSG mice, and after tumor formation, different doses of immunotoxin J80A-PE24 were used for treatment. The treatment was administered via tail vein once every 2 days. DETAILED DESCRIPTION

[0079] The present invention discloses the preparation and identification of monoclonal antibodies that bind to GPC3. Specific embodiments disclose the isolation and characterization of monoclonal antibodies targeting GPC3. The specific data disclosed in the present invention demonstrate that these antibodies bind to cell surface-associated GPC3 with high affinity, as well as the relationship between different antibodies binding to different epitopes of GPC3. The antibody fusion toxin (immunotoxin) described in the present invention can strongly kill GPC3-positive tumor cells in vitro, providing experimental evidence for drug development.

[0080] The present invention is further described in detail below in conjunction with specific embodiment and with reference to data, it should be understood that these embodiments are only for illustrating the present invention, but not to limit the scope of the present invention in any way. The terms used in the present invention, unless otherwise indicated, generally have the meaning commonly understood by those of ordinary skill in the art. In the following examples, the various processes and methods not described in detail are conventional methods well known in the art.

[0081] abbreviation

[0082] CDR Complementarity determining region HCC Hepatocellular carcinoma

[0083] CTL cytotoxic T lymphocyte hFc human Fc

[0084] ELISA enzyme-linked immunosorbent assay HS heparan sulfate

[0085] FACS fluorescence activated cell sorting Ig immunoglobulin

[0086] GPC3 Glypican 3 mAb Monoclonal Antibody

[0087] Pfu colony forming unit PE Pseudomonas exotoxin

[0088] SPR Surface plasmon resonance APC Allophycocyanin

[0089] Example 1: Preparation of monoclonal antibodies against glypican 3 of the present invention

[0090] This example describes the generation of high affinity mAbs against tumor-associated GPC3.

[0091] Four-month-old chickens were immunized with GPC3-hFc protein (Sino Biological, Cat: 10088-H02H2), 300 μg each time, 2 weeks apart each time. After 4 immunizations, the spleens of the chickens were collected, and the total RNA in the spleens was extracted and reversely transcribed into cDNA as a template for establishing a phage antibody library. A chicken antibody phage library (library capacity 2.7×10 9 ). Using GPC3 protein as an antigen, the phage library was panned four times, and 300 clones were randomly selected for sequencing. As a result, 23 highly enriched representative clones were obtained, which were named A5, A20, A31, A41, A64, B81, A61, F5, G41, I68, A43, C15, A18, F67, C46, ​​I34, J80A, I82, I88, J58, J80B, H49, and G15. Phage competition ELISA experiments showed that the 23 representative clones could be preliminarily classified into 14 antigenic epitopes ( Figure 2 ), represented by J58, J80B, A5, H49, F5, G15, A43, A18, F67, C46, ​​I34, J80A, I82, and I88, respectively (the antibody sequences are shown in Tables 1 and 2).

[0092] The amino acid sequences of the complementary determining regions (CDR regions) and variable regions of the above antibodies are shown in Tables 1 and 2:

[0093] Table 1. CDR amino acid sequences of 14 monoclonal antibodies (according to Kabat and IMGT)

[0094]

[0095]

[0096] Table 2. Amino acid sequences of the variable regions of 14 monoclonal antibodies

[0097]

[0098]

[0099] Example 2 Detection of GPC3 protein using the monoclonal antibody of the present invention

[0100] The scFv of the above 14 monoclonal antibodies was fused with hFc to construct the expression vector pPBSPS-scFv-hFc, and expressed in 293F cells. The expression product was purified using a protein A chromatography column (GE healthcare). The GPC3 protein was detected using the purified antibody and sandwich ELISA method.

[0101] First, 14 monoclonal antibodies were used as coating antibodies and coated on the bottom of the ELISA plate ( Figure 2 , first column), then add GPC3 protein standard or biological sample containing GPC3 (such as serum of liver cancer patients), and then use the 14 biotinylated monoclonal antibodies as detection antibodies ( Figure 2 , first horizontal row), incubated with it, and the binding of the detection antibody to GPC3 was detected with HRP-labeled streptavidin. The gray squares show that the antibody combination at this position can detect the GPC3 protein with high sensitivity, and there is no rejection between the coated antibody and the detection antibody, which can be used for the detection and content analysis of GPC3 in biological samples. The black squares show that the antibody combination at this position has a partial rejection effect when binding to the GPC3 protein, which also shows that the epitopes of these antibodies are partially overlapped. According to this result, the epitopes of 14 monoclonal antibodies (J58, J80B, A5, H49, F5, G15, A43, A18, F67, C46, ​​I34, J80A, I82, I88) can be further reduced to 12 (because the antigenic epitopes of J58 and J80B overlap more with A5, so A5 is represented) ( Figure 3 ).

[0102] Example 3 In vitro characterization of antibodies

[0103] 1. Human-mouse cross-reaction of the antibody prepared in Example 1 with the GPC3 protein

[0104] The affinity of the antibodies prepared in Example 1 to human and mouse GPC3 proteins (SinoBiological, Cat: 50989-M08B) was determined by ELISA. The human and mouse GPC3 proteins were coated on ELISA plates, respectively, and gradient concentrations of diluted antibodies were added for incubation. The binding ability of the antibodies to human and mouse GPC3 was detected with anti-hFc tag antibodies. The ELISA results showed that these 14 antibodies could bind to human GPC3 protein very strongly. Except for H49 and I82, which could not bind to mouse GPC3, the other antibodies could bind to mouse GPC3, but the affinity of J58 to mouse GPC3 was very weak ( Figure 4 A and Figure 4 B). Antibodies A5, A18, A43, C46, ​​F5, F67, G15, I34, I88, J80A, and J80B that can bind to human and mouse GPC3 can be used for the detection of mouse GPC3 protein and can also be used for therapeutic research on mouse GPC3-positive tumors.

[0105] 2. SPR determination of the binding kinetics and affinity between the antibody prepared in Example 1 and GPC3

[0106] The GPC3-his protein was immobilized on a carboxymethyl sensor chip (S series sensor chip CM5) by standard amine coupling. The chip was washed to obtain a stable baseline, and then different concentrations of antibody analytes and running buffer were injected into the chip at a flow rate of 30 μL / min. The sample binding time was 180 seconds and the subsequent dissociation time was 600 seconds. The binding and dissociation curves were fitted to a 1:1 Langmiur binding model using ProteOn software. The affinities of the monoclonal antibodies measured are shown in Table 3.

[0107] Table 3 Affinity constants Kd values ​​of monoclonal antibodies determined by Biacore

[0108]

[0109] The results showed that the antibodies prepared in Example 1 had high affinity for GPC3, with A18 having an affinity of 0.0214 pM and A43 having an affinity of 1.52 pM. The affinity of the control antibody HN3 was only 1.95 nM.

[0110] 3. Antibody stability testing

[0111] The Tm value is a parameter that quantitatively describes the thermal stability of a protein and is one of the most commonly used indicators in drugability evaluation. The higher the Tm value, the more stable the protein conformation. The stability of the antibody prepared in Example 1 was determined using Nano Temper's Prometheus NT.48: the antibody concentration was diluted to 50ug / ml, then loaded, and the temperature rose from 25°C to 95°C within 40min at a rate of 1.5°C / min. Finally, the Tm value of the antibody was obtained. The results are shown in Table 4.

[0112] Table 4 Tm values ​​of the monoclonal antibodies of the present invention

[0113]

[0114] The results showed that most antibodies had relatively high thermal stability, among which A18 and F5 had Tm values ​​of over 70°C and 80°C, respectively, showing significant advantages in terms of drugability.

[0115] Example 4 FACS detection of the binding of the antibody of the present invention to the GPC3 positive cell line

[0116] A431, G1 (A431 cell line overexpressing GPC3) (Phung Yet al. MAbs 2012; 4: 592-599) and liver cancer cell lines HepG2, Hep3B, HuH-7 were cultured in an adherent manner. The culture medium used was DMEM culture medium (Invitrogen, Carlsbad CA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 1% L-glutamine and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA). After harvesting the cells, the antibodies prepared in Example 1 were bound to A431, G1, HepG2, Hep3B, Huh7 cells, and APC-labeled goat anti-human secondary antibodies were added to detect the antibodies bound to the cell surface. The results are shown in FIG. Figure 5 As shown, the antibody prepared in Example 1 can strongly bind to G1 cells and HepG2, Hep3B, Huh7 cells, but not to GPC3-negative A431 cells, indicating that the antibody prepared in Example 1 can specifically recognize and bind to the GPC3 protein on the cell surface.

[0117] Example 5 Cytotoxicity of the Antibody of the Present Invention after Fusion with Toxin to GPC3-Positive Cells

[0118] 1. Construction of cell lines

[0119] Lentiviral particles expressing luciferase and GFP genes were packaged and used to infect A431, G1, HepG2, Hep3B, and Huh7 cells. After 3 days of infection, puromycin was used for selection, and then GFP-positive cells were screened by FACS. The obtained positive cells can co-express luciferase and are used for subsequent detection of cell viability. The positive cells were renamed A431LG, G1LG, HepG2LG, Hep3BLG, and Huh7LG.

[0120] 2. Preparation of Immunotoxin

[0121] The monoclonal antibody (scFv) provided by the present invention was fused with Pseudomonas exotoxin PE24 to construct an immunotoxin, and 6 histidines (6x His) were additionally added to the N-terminus of the immunotoxin to facilitate the purification of the immunotoxin. The immunotoxin was expressed using the Escherichia coli HB2151 strain. The strain was inoculated into 2L of 2YT medium and cultured at 37°C for 4-5h. The OD 600 When the p-value reached 0.9, 1 mM IPTG was added for induction and the expression was carried out at 30°C for 10 h. The bacterial cells were collected, crushed under high pressure and centrifuged, the supernatant was filtered and purified by nickel column.

[0122] 3. Cytotoxicity of Immunotoxins

[0123] The immunotoxin was diluted 1:10 starting from 1000ng / ml, and then incubated with A431LG, G1LG, HepG2LG, Hep3BLG, and Huh7LG cells for 72 hours, and then its cell killing activity was detected. Considering that HN3-PE24 is the best immunotoxin targeting GPC3, HN3-PE24 was used as a reference for parallel comparison.

[0124] The results are as follows Figure 6The immunotoxin has strong cytotoxic activity against GPC3-positive G1LG, Hep3BLG, HepG2LG, and Huh7LG cells, but has no killing effect on GPC3-negative A431LG, indicating that the immunotoxin has a high degree of selectivity for GPC3-positive tumor cells. The activity intensity of the immunotoxin is expressed by the IC50 value, and the measured IC50 values ​​are shown in Table 5. The activities of most of the immunotoxins provided by the present invention are stronger than HN3-PE24, such as J80A-PE24, A43-PE24, C46-PE24, J80B-PE24, A5-PE24, G15-PE24, I82-PE24, A18-PE24, and H49-PE24. Among them, J80A-PE24 has the strongest killing activity, with an IC50 value of 7.974 ng / ml for Hep3BLG, while the IC50 value of HN3-PE24 is as high as 109 ng / ml, which indicates that the anti-tumor activity of J80A-PE24 is much stronger than that of HN3-PE24.

[0125] Table 5. IC50 values ​​of monoclonal antibody-based immunotoxins against GPC3-positive tumor cells

[0126]

[0127] Example 6 In vivo anti-tumor activity of immunotoxin J80A

[0128] This example uses the immunotoxin J80A as an example to study the in vivo tumor inhibition activity of J80A. 6 Hep3B cells were inoculated subcutaneously into NSG mice until the tumor volume reached 200 mm 3 The tumor-bearing mice were treated at 2.5, 5, and 10 mg / kg body weight in the treatment group and PBS buffer in the control group. The treatment was performed once every 2 days. The results showed that all three dose groups could significantly inhibit tumor growth ( Figure 7 ), especially the 10mg / kg and 5mg / kg dosage groups, can significantly reduce the size of tumors, and the tumors of some mice can completely disappear after treatment. Sequence Listing <110> Huazhong Agricultural University <120> Antibodies recognizing multiple different epitopes of glypican 3 and their applications <160> 28 <170> SIPOSequenceListing 1.0 <210> 1 <211> 126 <212> PRT <213> Artificial Sequence <400> 1 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Asn Arg Tyr 20 25 30 Cys Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Asp Ser Asp Ser Gly Gly Thr Asp Tyr Gly Ala Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Lys Ser Ala Tyr Gly Gly Trp Cys Gly Ser Arg Val Ala Pro Trp 100 105 110 Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser 115 120 125 <210> 2 <211> 107 <212> PRT <213> Artificial Sequence <400> 2 Gln Ala Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Gly Trp Phe Gln 20 25 30 Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp Asn Asp 35 40 45 Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Leu Ser Gly 50 55 60 Ser Thr Gly Thr Leu Thr Ile Thr Gly Val Arg Ala Glu Asp Glu Ala 65 70 75 80 Val Tyr Tyr Cys Gly Ser Ser Glu Asn Ser Tyr Val Gly His Val Ala 85 90 95 Ile Phe Gly Ala Gly Thr Thr Leu Thr Val Leu 100 105 <210> 3 <211> 127 <212> PRT <213> Artificial Sequence <400> 3 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Arg Thr Asn 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Asn Ser Ala Gly Gly Trp Thr Gly Tyr Gly Pro Ala Val 50 55 60 Lys Gly His Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg 65 70 75 80 Leu Gln Leu Thr Asn Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Lys Ser Ala Gly Gly Trp Cys Asp Ser Gly Asp Tyr Gly Ala Gly 100 105 110 Cys Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser 115 120 125 <210> 4 <211> 105 <212> PRT <213> Artificial Sequence <400> 4 Gln Ala Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Leu Gly Gly 1 5 10 15 Thr Val Lys Ile Thr Cys Ser Gly Gly Ser Gly Ser Tyr Gly Trp Phe 20 25 30 Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp Asn 35 40 45 Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Thr Ser 50 55 60 Gly Ser Thr Gly Thr Leu Thr Ile Thr Gly Val Gln Ala Asp Asp Glu 65 70 75 80 Ala Val Tyr Phe Cys Gly Ser Thr Asp Ser Ser Tyr Val Gly Ile Phe 85 90 95 Gly Ala Gly Thr Thr Leu Thr Val Leu 100 105 <210> 5 <211> 128 <212> PRT <213> Artificial Sequence <400> 5 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Phe Gly Gly Ser His Thr Gly Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Thr Arg Asp Asn Gly Gln Ser Thr Met Arg 65 70 75 80 Leu Gln Leu Ser Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 Thr Arg Gly Gly Gly Tyr Phe Cys Thr Tyr Gly Trp Cys Pro Gly Gly 100 105 110 Gly Glu Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser 115 120 125 <210> 6 <211> 107 <212> PRT <213> Artificial Sequence <400> 6 Gln Ala Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Leu Gly Gly 1 5 10 15 Thr Val Lys Ile Thr Cys Ser Gly Gly Ser Gly Ser Tyr Gly Trp Phe 20 25 30 Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp Asn 35 40 45 Asp Gln Arg Pro Ser Asn Ile Pro Ser Arg Phe Ser Gly Ser Lys Ser 50 55 60 Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Val Asp Asp Glu 65 70 75 80 Ala Val Tyr Tyr Cys Gly Ser Gly Asp Ser Ser Ser Gly Asp Ser Gly 85 90 95 Val Phe Gly Ala Gly Thr Thr Leu Thr Val Leu 100 105 <210> 7 <211> 122 <212> PRT <213> Artificial Sequence <400> 7 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Gly Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Lys Asn Asp Gly Ser Phe Ala Leu Tyr Gly Ala Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Ser Gly Gln Ser Thr Val Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Phe Cys 85 90 95 Ala Lys Ser Ala Gly Val Val Gly Gly Pro Asp Asp Ile Asp Ala Trp 100 105 110 Gly His Gly Thr Glu Val Ile Val Ser Ser 115 120 <210> 8 <211> 105 <212> PRT <213> Artificial Sequence <400> 8 Gln Ala Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu 1 5 10 15 Thr Val Arg Ile Thr Cys Ser Gly Ser Ser Tyr Ser Tyr Tyr Gly Trp 20 25 30 Tyr Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp 35 40 45 Asn Asp Asn Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Thr 50 55 60 Ser Gly Ser Thr Gly Thr Leu Thr Ile Thr Gly Val Arg Ala Glu Asp 65 70 75 80 Glu Ala Val Tyr Tyr Cys Gly Asn Tyr Gly Ser Ser Ala Gly Ile Phe 85 90 95 Gly Ala Gly Thr Thr Leu Thr Val Leu 100 105 <210> 9 <211> 126 <212> PRT <213> Artificial Sequence <400> 9 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Gly Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Asp Phe Ser Ser Tyr 20 25 30 Gly Met Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Asp Gly Ala Gly Ser Asn Thr Tyr Tyr Ala Thr Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Lys Glu Ser Cys Ile Gly Ser Gly Cys Gly Phe Leu Val Gly Arg 100 105 110 Ile Asp Ala Trp Gly Gln Gly Thr Glu Val Ile Val Ser Ser 115 120 125 <210> 10 <211> 107 <212> PRT <213> Artificial Sequence <400> 10 Gln Ala Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Leu Gly Gly 1 5 10 15 Thr Val Lys Ile Thr Cys Ser Gly Ser Ser Gly Ser Tyr Gly Trp Tyr 20 25 30 Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Tyr Asn 35 40 45 Asp Lys Arg Pro Ser Asn Ile Pro Ser Arg Phe Ser Gly Ser Thr Ser 50 55 60 Gly Ser Thr Gly Thr Leu Thr Ile Thr Gly Val Gln Ala Glu Asp Glu 65 70 75 80 Ala Val Tyr Phe Cys Gly Ser Tyr Asp Ser Ser Ala Gly Tyr Val Gly 85 90 95 Ile Phe Gly Ala Gly Thr Thr Leu Thr Val Leu 100 105 <210> 11 <211> 125 <212> PRT <213> Artificial Sequence <400> 11 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Tyr Asn Asp Gly Ser Thr Pro Asn Tyr Gly Ser Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Lys Thr Thr Gly Gly Phe Ser Tyr Tyr Tyr Asp Ala Gly Asp Ile 100 105 110 Asp Thr Trp Gly His Gly Thr Glu Val Ile Val Ser Ser 115 120 125 <210> 12 <211> 105 <212> PRT <213> Artificial Sequence <400> 12 Gln Ala Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Thr Cys Ser Gly Gly Ser Tyr Ser Tyr Gly Trp Phe 20 25 30 Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp Asn 35 40 45 Thr Asn Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Lys Ser 50 55 60 Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Glu Asp Glu 65 70 75 80 Ala Val Tyr Phe Cys Gly Ser Ala Asp Ser Ser Tyr Ala Gly Ile Phe 85 90 95 Gly Ala Gly Thr Thr Leu Thr Val Leu 100 105 <210> 13 <211> 128 <212> PRT <213> Artificial Sequence <400> 13 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ala Thr Ile Ser Phe Gly Gly Ser His Thr Gly Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 Thr Arg Gly Gly Gly Tyr Phe Cys Ser Tyr Gly Trp Cys Pro Gly Gly 100 105 110 Gly Glu Ile Asp Thr Trp Gly His Gly Thr Glu Val Ile Val Ser Ser 115 120 125 <210> 14 <211> 104 <212> PRT <213> Artificial Sequence <400> 14 Gln Ala Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Thr Cys Ser Gly Ser Ser Gly Ser Tyr Gly Trp Tyr 20 25 30 Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp Asn 35 40 45 Asp Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Glu Ser 50 55 60 Gly Ser Met Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Lys Asp Glu 65 70 75 80 Ala Val Tyr Phe Cys Gly Ser Arg Asp Asn Ser Ala Ala Ile Phe Gly 85 90 95 Ala Gly Thr Thr Leu Thr Val Leu 100 <210> 15 <211> 128 <212> PRT <213> Artificial Sequence <400> 15 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Ser Phe Ser Ser Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Gly Thr Asp Ser Ser Phe Pro Asn Tyr Gly Ala Ala Val 50 55 60 Lys Gly Arg Ala Ile Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Lys Ser Val Tyr Gly Gly Trp Cys Thr Ser Gly Ser Cys Ser Gly 100 105 110 Arg Arg Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser 115 120 125 <210> 16 <211> 108 <212> PRT <213> Artificial Sequence <400> 16 Gln Ala Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Leu Gly Glu 1 5 10 15 Thr Val Lys Ile Thr Cys Ser Gly Ser Ser Gly Ser Tyr Gly Trp Tyr 20 25 30 Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp Asn 35 40 45 Thr Asp Arg Pro Ser Asn Ile Pro Ser Arg Phe Ser Gly Ser Lys Ser 50 55 60 Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Glu Asp Glu 65 70 75 80 Ala Val Tyr Phe Cys Gly Ser Arg Asp Ser Ser Gly Thr Gly Tyr Val 85 90 95 Gly Ile Phe Gly Ala Gly Thr Thr Leu Thr Val Leu 100 105 <210> 17 <211> 119 <212> PRT <213> Artificial Sequence <400> 17 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Gly Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Asp Phe Ser Asp Tyr 20 25 30 Gly Met Ser Trp Met Arg Gln Ala Pro Asp Lys Gly Leu Glu Tyr Val 35 40 45 Ala Ala Ile Ser Asn Asp Gly Ser Ser Thr Leu Tyr Gly Ala Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg 65 70 75 80 Leu His Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Phe Cys 85 90 95 Ala Arg Ser Gly Gly Gly Ala Gly Asp Ile Asp Ala Trp Gly Arg Gly 100 105 110 Thr Glu Val Ile Val Ser Ser 115 <210> 18 <211> 114 <212> PRT <213> Artificial Sequence <400> 18 Gln Ala Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Thr Cys Ser Gly Gly Gly Ser Ser Ser Tyr Gly Tyr 20 25 30 Asn Tyr Gly Trp Tyr Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Thr 35 40 45 Val Ile Tyr Ser Asn Asp Asn Arg Pro Ser Asn Ile Pro Ser Arg Phe 50 55 60 Ser Gly Ser Ala Ser Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val 65 70 75 80 Gln Val Glu Asp Glu Ala Val Tyr Tyr Cys Gly Ser Tyr Glu Gly Ser 85 90 95 Ile Asp Gly Arg Tyr Val Gly Val Phe Glu Ala Gly Thr Thr Leu Thr 100 105 110 Val Leu <210> 19 <211> 126 <212> PRT <213> Artificial Sequence <400> 19 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser His 20 25 30 Gly Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Thr Thr Gly Ser Tyr Thr Gly Tyr Gly Ser Ala Val Asp 50 55 60 Gly Arg Ala Thr Ile Ser Arg Asp Asp Gly Gln Ser Thr Val Arg Leu 65 70 75 80 Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Phe Cys Ala 85 90 95 Lys Ser Thr Asp Gly Gly Cys Ile Ser Tyr Gly Ile Cys Pro Asp Glu 100 105 110 Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser 115 120 125 <210> 20 <211> 105 <212> PRT <213> Artificial Sequence <400> 20 Gln Ala Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Thr Cys Ser Gly Gly Gly Asn Trp Tyr Gly Trp Tyr 20 25 30 Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp Asn 35 40 45 Thr Asn Arg Pro Ser Asn Ile Pro Ser Arg Phe Ser Gly Ser Thr Ser 50 55 60 Gly Ser Thr Ser Thr Leu Thr Ile Thr Gly Val Gln Ala Asp Asp Glu 65 70 75 80 Ala Ile Tyr Tyr Cys Gly Ser Tyr Asp Ser Thr Tyr Asp Asp Ile Phe 85 90 95 Gly Ala Gly Thr Thr Leu Thr Val Leu 100 105 <210> 21 <211> 128 <212> PRT <213> Artificial Sequence <400> 21 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Gly Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Ser Val 35 40 45 Ala Gln Ile Thr Ser Thr Gly Ser Ser Thr Tyr Tyr Gly Ser Ala Val 50 55 60 Asp Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 Ala Lys Thr Thr Gly Ser Gly Ser Cys Thr Tyr Gly Trp Asn Cys Ala 100 105 110 Gly Asn Ile Asp Ser Trp Gly His Gly Thr Glu Val Ile Val Ser Ser 115 120 125 <210> 22 <211> 107 <212> PRT <213> Artificial Sequence <400> 22 Gln Ala Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Leu Gly Gly 1 5 10 15 Thr Val Glu Ile Thr Cys Ser Gly Asp Asp Asn Tyr Asp Tyr Tyr Gly 20 25 30 Trp Phe Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr 35 40 45 Glu Ser Asn Lys Arg Pro Ser Asn Ile Pro Leu Arg Phe Ser Gly Ser 50 55 60 Thr Ser Gly Ser Thr Asn Thr Leu Thr Ile Thr Gly Val Gln Ala Asp 65 70 75 80 Asp Glu Ala Val Tyr Phe Cys Gly Thr Ser Asp Asn Gly Gly Val Gly 85 90 95 Thr Phe Gly Ala Gly Thr Thr Leu Thr Val Leu 100 105 <210> 23 <211> 126 <212> PRT <213> Artificial Sequence <400> 23 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Cys Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Asp Ser Asp Ser Gly Gly Thr Asp Tyr Gly Ala Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asp Gly Gln Ser Thr Val Arg 65 70 75 80 Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Lys Ser Gly Tyr Gly Gly Trp Cys Gly Gly Arg Asp Val Ala Trp 100 105 110 Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser 115 120 125 <210> 24 <211> 106 <212> PRT <213> Artificial Sequence <400> 24 Gln Ala Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Gly Trp Tyr Gln 20 25 30 Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp Asn Thr 35 40 45 Asn Arg Pro Ser Asn Ile Pro Ser Arg Phe Ser Gly Ser Leu Ser Gly 50 55 60 Ser Thr Ser Thr Leu Thr Ile Thr Gly Val Gln Ala Asp Asp Glu Ala 65 70 75 80 Val Tyr Phe Cys Gly Ser Tyr Asp Ser Ser Ala Asp Tyr Val Gly Ile 85 90 95 Phe Gly Ala Gly Thr Thr Leu Thr Val Leu 100 105 <210> 25 <211> 125 <212> PRT <213> Artificial Sequence <400> 25 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Gly Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ala Asp Ile Ser Ser Thr Gly Ser Ser Thr Tyr Tyr Ala Leu Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Phe Cys 85 90 95 Ala Lys Ser Gly Tyr Val Gly Ser Trp Tyr Ile Asp Ser Pro Trp Ile 100 105 110 Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser 115 120 125 <210> 26 <211> 105 <212> PRT <213> Artificial Sequence <400> 26 Gln Ala Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Thr Cys Ser Gly Ser Ser Ser Tyr Ser Gly Trp Tyr 20 25 30 Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp Asn 35 40 45 Thr Lys Arg Pro Ser Asn Ile Pro Ser Arg Phe Ser Gly Ser Lys Ser 50 55 60 Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Ile Gln Ala Asp Asp Glu 65 70 75 80 Ala Val Tyr Tyr Cys Gly Ser Thr Asp Asn Asn Tyr Asp Gly Ile Phe 85 90 95 Gly Ala Gly Thr Thr Leu Thr Val Leu 100 105 <210> 27 <211> 126 <212> PRT <213> Artificial Sequence <400> 27 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Asn Arg Asn 20 25 30 Cys Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Asp Ser Asp Ser Gly Gly Thr Glu Tyr Gly Ala Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asp Gly Gln Ser Thr Leu Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 Ala Lys Ser Gly Tyr Gly Gly Trp Cys Gly Ser Arg Ser Ala Ala Trp 100 105 110 Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser 115 120 125 <210> 28 <211> 106 <212> PRT <213> Artificial Sequence <400> 28 Gln Ala Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Leu Gly Glu 1 5 10 15 Thr Val Lys Ile Thr Cys Ser Gly Ser Ser Gly Asn Tyr Gly Trp Tyr 20 25 30 Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Ser Ser 35 40 45 Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ala Leu Ser 50 55 60 Gly Ser Thr Ala Thr Leu Ser Ile Thr Gly Val Arg Ala Asp Asp Glu 65 70 75 80 Ala Val Tyr Phe Cys Gly Gly Tyr Asp Gly Ile Asn Arg Ala Ala Ile 85 90 95 Phe Gly Ala Gly Thr Thr Leu Thr Val Leu 100 105

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that recognizes different antigenic epitopes of glypican 3, comprising a heavy chain variable region and a light chain variable region, characterized in that: (1) The heavy chain variable region of the antibody comprises a complementary determining region HCDR1 as represented by amino acid residues 26-35, a complementary determining region HCDR2 as represented by amino acid residues 50-66, and a complementary determining region HCDR3 as represented by amino acid residues 97-115 of SEQ ID NO: 1; the light chain variable region of the antibody comprises a complementary determining region LCDR1 as represented by amino acid residues 23-29, a complementary determining region LCDR2 as represented by amino acid residues 46-52, and a complementary determining region LCDR3 as represented by amino acid residues 85-97 of SEQ ID NO: 2; Alternatively, (2) the heavy chain variable region of the antibody comprises a complementarity determining region HCDR1 as shown by amino acid residues 26-35, a complementarity determining region HCDR2 as shown by amino acid residues 50-66, and a complementarity determining region HCDR3 as shown by amino acid residues 97-116 of SEQ ID NO: 3; and the light chain variable region of the antibody comprises a complementarity determining region LCDR1 as shown by amino acid residues 23-30, a complementarity determining region LCDR2 as shown by amino acid residues 47-53, and a complementarity determining region LCDR3 as shown by amino acid residues 86-95 of SEQ ID NO:

4.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: (1) The heavy chain variable region amino acid sequence of the antibody is shown in SEQ ID NO: 1, and the light chain variable amino acid sequence is shown in SEQ ID NO: 2; Alternatively, (2) the heavy chain variable region amino acid sequence of the antibody is as shown in SEQ ID NO:3, and the light chain variable amino acid sequence is as shown in SEQ ID NO:

4.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that The antibody is a single-chain antibody, a double-chain antibody or a chimeric antibody.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that The antibody is a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a single chain variable fragment scFv or a disulfide bond stabilized variable fragment dsFv.

5. A recombinant protein, characterized in that The recombinant protein is the monoclonal antibody according to any one of claims 1 to 4 and a tag sequence that assists expression and / or purification.

6. An immunoconjugate, characterized in that The method comprises the monoclonal antibody according to any one of claims 1 to 4 and an effector molecule, wherein the effector molecule is a Pseudomonas exotoxin.

7. A polynucleotide characterized in that It encodes the antibody according to any one of claims 1 to 4, or the recombinant protein according to claim 5.

8. A carrier, characterized in that Containing the polynucleotide according to claim 7.

9. A genetically engineered host cell, characterized in that Contains the vector according to claim 8, or the polynucleotide according to claim 8 is integrated into the genome.

10. A pharmaceutical composition, characterized in that The method comprises one or more of the antibody according to any one of claims 1 to 4, the recombinant protein according to claim 5, the immunoconjugate according to claim 6, the polynucleotide according to claim 7, the vector according to claim 8 or the genetically engineered host cell according to claim 9.

11. Use of the antibody according to any one of claims 1 to 4, the recombinant protein according to claim 5, the immunoconjugate according to claim 6, the polynucleotide according to claim 7, the vector according to claim 8 or the genetically engineered host cell according to claim 9 in the preparation of a therapeutic drug or diagnostic reagent for liver cancer.

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