Antibody for recognizing multiple different epitopes of glypican 3 and application thereof

Abstract

The present invention provides a plurality of monoclonal antibodies capable of recognizing glypican 3 (GPC3) with high affinity. The monoclonal antibodies not only have high affinity, but also cover a plurality of epitopes of GPC3, and can be used for detecting a tumor marker GPC3 by a sandwich method. The monoclonal antibody provided by the invention not only has good thermal stability, but also has excellent cytotoxic activity after being conjugated with pseudomonas exotoxin PE24, is obviously superior to the existing immunotoxin HN3-PE24, and can be used for developing immunotoxin or antibody-drug conjugate (ADC) drugs with stronger activity and better stability.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to high-affinity monoclonal antibodies that recognize multiple different antigenic epitopes of glypican-3 and their applications. Background Art

[0002] Cancer has become the second leading cause of human death after cardiovascular diseases, and the global incidence and mortality rates of liver cancer rank sixth and fourth, respectively. Although surgical operation is the standard treatment for liver cancer, due to the insidious onset and high malignancy of liver cancer patients, most of them are diagnosed at the middle or advanced stage once diagnosed, so only 5-10% of liver cancer patients can undergo surgical treatment. Middle and advanced 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, phosphatidylinositol proteoglycan 3) is a member of the heparan sulfate proteoglycan family and is anchored to the cell membrane surface by glycosyl-phosphatidylinositol (GPI). The human GPC3 gene is located on the X chromosome (Xp26) and encodes a 70 kDa protein that contains 580 amino acids and is endoproteolytically cleaved by a furin-like convertase between Arg358 and Ser359, generating a 40 kDa N-terminal subunit and a 30 kDa C-terminal subunit, and there are also two heparan sulfate (HS) chains on the C-terminal subunit. Studies have shown that the expression level of GPC3 is significantly upregulated in approximately 72% of hepatocellular carcinoma (HCC) compared to normal hepatocytes, cholangiocarcinoma, and liver metastatic carcinoma, but it is not expressed in normal adult liver tissues. In addition, high expression of GPC3 in HCC patients is often associated with a poor prognosis, all of which indicate the potential role of GPC3 as a biomarker in HCC. Currently, GPC3 has been proposed 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) immunotherapies.

[0004] Antibody-drug conjugates (ADCs) are highly effective tumor-targeted therapeutic agents. Antibody fragments can also be fused with proteinaceous toxin fragments to generate chimeric structures called immunotoxins. The immunotoxin Lumoxiti 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 the inhibition of protein synthesis and ultimately lead to cell death. Immunotoxins are considered to trigger tumor regression through two mechanisms: antibody-induced inactivation of cell signaling and toxin-induced inhibition of protein synthesis. Therefore, the cytotoxicity of antibodies conjugated with the same toxin may vary greatly depending on their different mechanisms of action. According to the research results of Wei Gao et al., although the affinity of the antibody HN3 against GPC3 is much weaker than that of YP7, the cytotoxicity and in vivo tumor suppression activity of the immunotoxin HN3-PE24 targeting GPC3 are higher than those of YP7-PE24 (Wei Gao et al. Nat Commun. 2015 Mar 11; 6:6536.). Therefore, focusing on the research and development of antibodies and finding better antibodies are important strategies for synthesizing more active ADCs or antibody-toxin conjugates. SUMMARY OF THE INVENTION

[0005] The object of the present invention is to provide monoclonal antibodies that highly affinity recognize multiple epitopes of phosphatidylinositol proteoglycan 3. The provided antibodies include immunoconjugates of antibody fragments (such as single-chain variable region fragments (scFv)) and effector molecules (such as toxin proteins). Compositions including antibodies that specifically bind to GPC3, nucleic acid molecules encoding these antibodies, expression vectors containing the nucleic acid molecules, and isolated host cells expressing the nucleic acid molecules are also provided.

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

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

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

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

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

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

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

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

[0014] Alternatively, the heavy chain variable region of the antibody of (8) has complementarity-determining regions shown by amino acid residues at positions 26-35, 50-66, and 97-117 of SEQ ID NO:15; the light chain variable region of the antibody has complementarity-determining regions shown by amino acid residues at positions 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 set forth 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 set forth 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; and 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] Human GPC3 has four known subtypes (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 of the present invention can bind to one or more of the four human GPC3 subtypes, or conservative variants thereof.

[0022] Specifically, the antibody of phosphatidylinositol proteoglycan 3 of the present invention is:

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

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

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

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

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

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

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

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

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

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

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

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

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

[0036] Alternatively, the amino acid sequence of the heavy chain variable region of the antibody described in (14) is as shown in SEQ ID NO: 27, and the amino acid sequence of the light chain variable region 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 antibody of the present invention can be of any isotype. It can be, for example, an IgM or IgG antibody, such as IgG1 or IgG2. The types of antibodies that can specifically bind to GPC3 can be converted into each other according to known methods (for example, IgG can be converted into IgM). Isotype switching can also be used to convert one IgG subclass into another subclass, for example, from IgG1 to IgG2.

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

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

[0041] (2) Fab', an antibody molecule fragment obtained by treating a complete antibody with pepsin and then reducing it to produce a complete light chain and a part of the heavy chain; two Fab' fragments are obtained for each antibody molecule;

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

[0043] (4) Fv, a genetically engineered fragment containing the variable regions of the light and heavy chains expressed as two chains;

[0044] (5) Single-chain antibody (e.g., scFv), a genetically engineered molecule containing the variable regions of the light and heavy chains and linked by a suitable polypeptide linker into a genetically fused single-chain molecule;

[0045] (6) Dimer of single-chain antibody (scFv2), defined as a dimer of scFv (also referred to as a "minibody");

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

[0047] Those skilled in the art will understand that conservative variants of antibodies can be prepared. Amino acid substitutions can be made in the VH and / or VL regions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions), and the resulting VH and VL still retain the ability to bind GPC3 or have a stronger binding ability to GPC3. Conservative substitutions of functionally similar amino acids are well known to those of ordinary skill in the art. The following six groups are examples of amino acids 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, which 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. Effector molecules include but are not limited to toxins, drugs, or detectable markers.

[0056] The drugs described in the present invention are substances with cytotoxic or anti-tumor activities, such as Monomethylauristatin E (MMAE), Monomethylauristatin F (MMAF), Pyrrolobenzodiazepine (PBD) dimer, N2'-deacetyl-N2'-(3-Mercapto-1-oxopropyl)-Maytansine (DM1), vinblastine, daunomycin, etc., and also such as radioactive reagents 125 I 32 P 14 C 3 H and 35 S, etc.

[0057] The toxins described in the present invention are toxic proteins with cytotoxic or anti-tumor activities, 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 A-F, as well as truncated mutants and point mutants of these toxins. These toxins can be obtained commercially (for example, Sigma Chemical Company, St. Louis, MO). The toxins also include variants of the above toxins (for example, 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 natural sequence, the cytotoxic fragment of the natural sequence, and conservatively modified variants of the natural sequence or its cytotoxic fragment. These modifications include but are not limited to removing multiple amino acid deletions, single or multiple amino acid substitutions in domains Ia, Ib, II, and III, and adding one or more sequences at the carboxyl terminus (for example, see Siegall et al., J. Biol. Chem. 264: 14256-14261, 1989). The cytotoxic fragments of Pseudomonas exotoxin include PE24, PE40, PE38, and PE35, etc.

[0058] The detectable markers described in the present invention are substances that can be detected by isotope analyzers, enzyme-labeled instruments, bioluminescence detectors, chemiluminescence detectors, electrochemiluminescence detectors, fluorescence analyzers, or visualized with the naked eye. 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 In125 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 ruthenium trisbipyridine), colloidal gold.

[0059] The effector molecule can be linked to the antibody of the present invention in any manner known to those skilled in the art. For example, the antibody can be functionally linked (by chemical conjugation, gene fusion, non-covalent association, or others) to one or more other molecular entities. Depending on the chemical structure of the effector molecule, the method of linking the effector molecule and the antibody is different. Polypeptides generally contain multiple functional groups; for example, carboxylic acid (COOH), free amino group (-NH2) or thiol 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 link additional reactive functional groups. The derivatization can include linking any of a variety of known linker molecules. The linker can be any molecule used to join the antibody and the effector molecule. The linker is capable of forming a covalent bond with the antibody and the effector molecule. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight-chain or branched-chain 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 constituent amino acids or to the α-carbon amino and carboxyl groups of the terminal amino acids through its side groups (such as through the disulfide bond of cysteine). Generally, the antibody or a part thereof is derivatized such that the 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 necessary to release the effector molecule from the antibody. Therefore, in these cases, the immunoconjugate will contain a cleavable bond near the target site. Cleavage of the linker to release the effector molecule from the antibody can be caused by enzymatic activity or by the conditions in which the immunoconjugate is located inside 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. The vectors include: bacterial plasmids, phages, 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 antibody, recombinant protein, immunoconjugate, polynucleotide, vector or genetically engineered host cell of the present invention. The pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The antibody, recombinant protein, immunoconjugate, polynucleotide, vector or genetically engineered host cell is soluble in an aqueous carrier such as buffered saline. It may also contain pharmaceutically excipients required for near 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 antibody, recombinant protein, immunoconjugate, polynucleotide, vector or genetically engineered host cell of the present invention in the preparation of therapeutic drugs or diagnostic reagents for autoimmune diseases, viral infections or cancers.

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

[0066] The monoclonal antibody disclosed by the present invention can also be used to prepare a chimeric antigen receptor (CAR; also known as chimeric T cell receptor, artificial T cell receptor or chimeric immune receptor) or a bispecific antibody.

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

[0068] The antibodies and compositions provided by the present invention can be used for various purposes, such as for the molecular diagnosis of tumors, to confirm the expression of GPC3 in samples from patients with liver cancer and other tumors. The samples can be any samples, including but not limited to tissues from biopsies, autopsies, and pathological specimens. Biological samples also include tissue sections, 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: 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 combinations provided by the present invention cover 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 (the Kd values are all in the nM and pM levels, or even higher), but also have good thermal stability. The monoclonal antibodies provided by the present invention have superior cytotoxic activity after conjugation with Pseudomonas exotoxin PE24, significantly superior to 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. [[ID=ll]]Brief Description of the Drawings

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

[0073] Figure 2 The monoclonal antibodies described in the present invention are used for detecting GPC3 by the sandwich ELISA method. The first vertical column is the coated antibody. After adding GPC3 protein and co-incubating, the monoclonal antibody in the first horizontal row is used as the detection antibody for detecting GPC3. The light gray squares indicate that the antigenic epitopes of the coated antibody and the detection antibody do not overlap, which is suitable for detecting GPC3 in biological samples by the double antibody sandwich method. The black squares indicate that the antigenic epitopes of the coated 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 epitope recognized by the antibody of the present invention. The overlapping intersections indicate that there is partial overlap of the antigenic epitopes of these monoclonal antibodies.

[0075] Figure 4 ELISA method was used to detect the affinity of the monoclonal antibody of the present invention for binding to GPC3 protein. Figure 4 a is human GPC3 protein, Figure 4 b is murine GPC3 protein.

[0076] Figure 5 FACS method was used to detect the binding activity of the monoclonal antibody of the present invention to GPC3-negative / positive cell lines. Figure 5 a is GPC3-negative A431 cell line, Figure 5 b is GPC3-positive cell line G1, Figure 5 c is GPC3-positive hepatocellular carcinoma cell line HepG2, Figure 5 d is GPC3-positive hepatocellular carcinoma cell line Hep3B, Figure 5 e is GPC3-positive hepatocellular carcinoma cell line Huh7.

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

[0078] Figure 7 In vivo antitumor activity of immunotoxin J80A. Hep3B cells were inoculated subcutaneously into NSG mice. After tumor formation, they were treated with different doses of immunotoxin J80A-PE24. The treatment method was intravenous injection via the tail vein, once every 2 days. Detailed implementation manners

[0079] The present invention discloses and describes the preparation and identification of monoclonal antibodies that bind to GPC3. Specific implementation examples 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) of the present invention can strongly kill GPC3-positive tumor cells in vitro, providing experimental evidence for drug development.

[0080] The present invention will be further described in detail below with reference to specific examples and data. It should be understood that these examples are only for illustrative purposes of the present invention and do not limit the scope of the present invention in any way. The terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art unless otherwise specified. In the following examples, 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 ELISA enzyme-linked immunosorbent assay HS heparin sulfate

[0084] FACS fluorescence activated cell sorting Ig immunoglobulin

[0085] GPC3 Glypican 3 mAb monoclonal antibody

[0086] Pfu colony-forming unit PE Pseudomonas exotoxin SPR surface plasmon resonance APC allophycocyanin

[0087] Example 1: Preparation of Monoclonal Antibodies Against Glypican 3 of the Present Invention

[0088] This example describes the generation of high-affinity mAbs against tumor-associated GPC3.

[0089] Four-month-old chickens were immunized with GPC3-hFc protein (Sino Biological, Cat: 10088-H02H2) at a dose of 300 μg each time, with an interval of 2 weeks between each immunization. The spleens of the chickens were collected after 4 immunizations, and the total RNA in the spleens was extracted and reverse 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 (antibody sequences are shown in Tables 1 and 2).

[0090] 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:

[0091] Table 1. CDR amino acid sequences of 14 monoclonal antibodies (according to Kabat and IMGT)

[0092]

[0093]

[0094] Table 2 Amino acid sequences of variable regions of 14 monoclonal antibodies

[0095]

[0096]

[0097] Example 2 Detection of GPC3 protein using the monoclonal antibodies of the present invention

[0098] The scFvs of the above 14 monoclonal antibodies were fused with hFc to construct an expression vector pPBSPS - scFv - hFc, which was expressed in 293F cells. The expression product was purified using a protein A chromatography column (GE healthcare). The purified antibody and the sandwich ELISA method were used to detect GPC3 protein.

[0099] First, 14 monoclonal antibodies were used as coating antibodies and were respectively coated on the bottom of the ELISA plate ( Figure 2 , the first vertical column), and then GPC3 protein standard or biological samples containing GPC3 (such as serum from liver cancer patients) were added. Then, these 14 biotin - labeled monoclonal antibodies were respectively used as detection antibodies ( Figure 2 , the first horizontal column) and incubated with them. The binding of the detection antibody to GPC3 was detected using HRP - labeled streptavidin. The grey squares indicate that the antibody combinations at these positions can detect GPC3 protein with high sensitivity, and there is no repulsion between the coating antibody and the detection antibody, which can be used for the detection and content analysis of GPC3 in biological samples. The black squares indicate that there is partial repulsion when the antibody combinations at these positions bind to GPC3 protein, which also shows that there is partial overlap in the epitopes of these antibodies. According to this result, the epitopes of the 14 monoclonal antibodies (J58, J80B, A5, H49, F5, G15, A43, A18, F67, C46, I34, J80A, I82, I88) can be further reduced to 12 (since the antigenic epitopes of J58 and J80B overlap more with A5, so A5 is used as a representative) ( Figure 3 ).

[0100] Example 3 In vitro characterization of antibodies

[0101] 1. Human - mouse cross - reaction of the antibody prepared in Example 1 with GPC3 protein

[0102] The affinity of the antibodies prepared in Example 1 for human and murine GPC3 proteins (SinoBiological, Cat: 50989-M08B) was determined by ELISA. The human and murine GPC3 proteins were respectively coated on ELISA plates, and incubated with antibodies diluted at gradient concentrations. Anti-hFc tag antibodies were used to detect the binding ability of the antibodies to human and murine GPC3. The ELISA results showed that all 14 antibodies could bind very strongly to human GPC3 protein. Except for H49 and I82 which could not bind murine GPC3, other antibodies could bind murine GPC3, but the affinity of J58 for murine GPC3 was very weak ( Figure 4 a and Figure 4 b). Antibodies A5, A18, A43, C46, F5, F67, G15, I34, I88, J80A, J80B that could bind both human and murine GPC3 could be used for the detection of murine GPC3 protein and also for the therapeutic research of tumors with positive murine GPC3 expression.

[0103] 2. SPR determination of the binding kinetics and affinity of the antibodies prepared in Example 1 for GPC3

[0104] 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 ProteOn software was used to fit the binding and dissociation curves to the 1:1 Langmiur binding model. The measured affinities of the monoclonal antibodies are shown in Table 3.

[0105] Table 3 Affinity constant Kd values of monoclonal antibodies determined by Biacore

[0106]

[0107] The results showed that the antibodies prepared in Example 1 had high affinities for GPC3. The affinity of A18 reached 0.0214 pM, and the affinity of A43 reached 1.52 pM. The affinity of the control antibody HN3 was only 1.95 nM.

[0108] 3. Detection of antibody stability

[0109] 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 drug-likeness evaluation. A higher Tm value means the protein conformation is more stable. The stability of the antibody prepared in Example 1 was measured using the Prometheus NT.48 from Nano Temper: The antibody concentration was diluted to 50 μg / ml, then loaded, and the temperature was raised from 25 °C to 95 °C at a rate of 1.5 °C / min within 40 minutes. Finally, the Tm value of the antibody was obtained. The results are shown in Table 4.

[0110] Table 4 Tm values of the monoclonal antibodies of the present invention

[0111]

[0112] The results show that most of the antibodies have relatively high thermal stability. Among them, the Tm values of A18 and F5 can reach above 70 °C and 80 °C, showing significant advantages in terms of drug-likeness.

[0113] Example 4 FACS detection of the binding of the antibodies of the present invention to GPC3-positive cell lines

[0114] A431, G1 (A431 cell line overexpressing GPC3) (Phung Yet al. MAbs 2012; 4:592-599), and hepatoma cell lines HepG2, Hep3B, and HuH-7 were cultured in adherent mode. The medium used was DMEM 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 respectively combined with A431, G1, HepG2, Hep3B, and Huh7 cells, and APC-labeled goat anti-human secondary antibody was added to detect the antibodies bound to the cell surface. The results are as Figure 5 shown. The antibodies prepared in Example 1 can strongly bind to G1 cells and HepG2, Hep3B, and Huh7 cells, but do not bind to GPC3-negative A431 cells, indicating that the antibodies prepared in Example 1 can specifically recognize and bind to the GPC3 protein on the cell surface.

[0115] Example 5 Cytotoxicity of the antibodies of the present invention fused with toxins to GPC3-positive cells

[0116] 1. Construction of cell lines

[0117] Package lentiviral particles expressing luciferase and GFP genes for infecting A431, G1, HepG2, Hep3B, and Huh7 cells. After 3 days of infection, screen with puromycin, and then use FACS to screen GFP-positive cells. The obtained positive cells can co-express luciferase and are used for subsequent detection of cell viability. The positive cells are renamed A431LG, G1LG, HepG2LG, Hep3BLG, and Huh7LG.

[0118] 2. Preparation of immunotoxin

[0119] Fuse the monoclonal antibody (scFv) provided by the present invention with Pseudomonas exotoxin PE24 to construct an immunotoxin, and additionally add 6 histidines (6x His) to the N-terminus of the immunotoxin to facilitate the purification of the immunotoxin. The expression of the immunotoxin uses Escherichia coli HB2151 strain. Inoculate the strain into 2 L of 2YT medium and culture at 37 °C for 4 - 5 h until the OD 600 reaches 0.9, add 1 mM IPTG for induction, and express for 10 h at 30 °C. Collect the bacterial cells, break the bacterial cells by high pressure and then centrifuge, filter and process the supernatant, and then purify through a nickel column.

[0120] 3. Cytotoxicity of immunotoxin

[0121] Start with 1000 ng / ml of the immunotoxin, perform a 1:10 serial dilution, and then co-incubate with A431LG, G1LG, HepG2LG, Hep3BLG, and Huh7LG cells for 72 hours, and then detect its killing activity against the cells. Considering that HN3-PE24 is currently the immunotoxin with the best killing effect targeting GPC3, use HN3-PE24 as a reference for parallel comparison.

[0122] The results are as Figure 6As shown. The 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 selectivity for GPC3-positive tumor cells. The activity intensity of the immunotoxin is represented by the IC50 value, and the measured IC50 values are shown in Table 5. The activities of the vast majority of the immunotoxins provided by the present invention are stronger than those of HN3-PE24, such as J80A-PE24, A43-PE24, C46-PE24, J80B-PE24, A5-PE24, G15-PE24, I82-PE24, A18-PE24, H49-PE24. Among them, J80A-PE24 has the strongest killing activity, and the IC50 value for Hep3BLG reaches 7.974 ng / ml, while the IC50 of HN3-PE24 is as high as 109 ng / ml, indicating that the antitumor activity of J80A-PE24 is much stronger than that of HN3-PE24.

[0123] Table 5. IC50 values of monoclonal antibody-based immunotoxins against GPC3-positive tumor cells

[0124]

[0125] Example 6 In vivo antitumor activity of immunotoxin J80A

[0126] In this example, taking immunotoxin J80A as an example, the in vivo antitumor activity of J80A was studied. 5×10 6 Hep3B cells were inoculated subcutaneously into NSG mice. When the volume of the formed tumor reached 200 mm 3 , the tumor-bearing mice were treated. The treatment groups were injected intravenously through the tail vein with different doses of immunotoxin (2.5, 5, 10 mg / kg body weight), and the control group was injected with PBS buffer. The treatment was performed once every 2 days. The results showed that all three dose groups could significantly inhibit the growth of tumors ( Figure 7 ), especially the 10 mg / kg and 5 mg / kg dose groups, which could significantly reduce the tumor size, and the tumors of some mice could completely disappear after treatment.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that recognizes different epitopes of glypican-3, including a heavy-chain variable region and a light-chain variable region, characterized in that: (1) The heavy-chain variable region of the antibody has a complementarity-determining region HCDR1 shown by the amino acid residues at positions 26-35 of SEQ ID NO:11, a complementarity-determining region HCDR2 shown by the amino acid residues at positions 50-66, and a complementarity-determining region HCDR3 shown by the amino acid residues at positions 97-114; the light-chain variable region of the antibody has a complementarity-determining region LCDR1 shown by the amino acid residues at positions 23-30 of SEQ ID NO:12, a complementarity-determining region LCDR2 shown by the amino acid residues at positions 47-53, and a complementarity-determining region LCDR3 shown by the amino acid residues at positions 86-95. Or, (2) The heavy-chain variable region of the antibody has a complementarity-determining region HCDR1 shown by the amino acid residues at positions 26-35 of SEQ ID NO:25, a complementarity-determining region HCDR2 shown by the amino acid residues at positions 50-66, and a complementarity-determining region HCDR3 shown by the amino acid residues at positions 97-114; the light-chain variable region of the antibody has a complementarity-determining region LCDR1 shown by the amino acid residues at positions 23-30 of SEQ ID NO:26, a complementarity-determining region LCDR2 shown by the amino acid residues at positions 47-53, and a complementarity-determining region LCDR3 shown by the amino acid residues at positions 86-95.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: (1) The amino acid sequence of the heavy-chain variable region of the antibody is as shown in SEQ ID NO:11, and the amino acid sequence of the light-chain variable region is as shown in SEQ ID NO:12; Or, (2) The amino acid sequence of the heavy-chain variable region of the antibody is as shown in SEQ ID NO:25, and the amino acid sequence of the light-chain variable region is as shown in SEQ ID NO:

26.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein 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, wherein The antibody is a Fab fragment, a Fab' fragment, an F(ab)'2 fragment, a single-chain variable fragment scFv or a disulfide-stabilized variable fragment dsFv.

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

6. An immunoconjugate, characterized in that Comprising the monoclonal antibody according to any one of claims 1-4 and an effector molecule, and the effector molecule is Pseudomonas exotoxin.

7. A polynucleotide, characterized in that It encodes the antibody according to any one of claims 1-4, 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 Containing 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 Including one or several of the antibody according to any one of claims 1-4, 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 - 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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