CN114478780A

CN114478780A - Antibody for recognizing multiple different epitopes of glypican 3 and application thereof

Info

Publication number: CN114478780A
Application number: CN202011541805.9A
Authority: CN
Inventors: 丰明乾; 李静文; 陈鑫
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2020-10-23
Filing date: 2020-12-23
Publication date: 2022-05-13

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) medicines with stronger activity and better stability.

Description

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

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a high-affinity monoclonal antibody for recognizing multiple different epitopes of glypican 3 and application thereof.

Background

Cancer has become the second leading cause of human death after cardiovascular disease, with the global incidence and mortality of liver cancer being the sixth and fourth sites, respectively. Although surgery is the standard treatment of liver cancer, only 5-10% of liver cancer patients can be treated by surgery because liver cancer patients have latent disease and high malignancy, and once diagnosis is mostly in the middle and late stages. Patients in the middle and late stages do not respond well to most chemotherapeutic drugs because of this type of cancer. Therefore, the development of new drugs with different mechanisms of action is urgently required. Immunotherapy represents a new approach, but it remains a challenge, mainly because there are no good tumor-specific targets.

Glypican-3(GPC3, Glypican 3) is a member of heparin sulfate proteoglycans, and is anchored to the surface of cell membranes by glycosyl-phosphatidylinositol (GPI). The human GPC3 gene is located on the X chromosome (Xp26) and encodes a 70kDa protein, containing 580 amino acids, which is cleaved endonucleases between Arg358 and Ser359 with a furin-like convertase to yield a 40kDa N-terminal subunit and a 30kDa C-terminal subunit, with two Heparan Sulfate (HS) chains on the C-terminal subunit. Studies have shown that GPC3 is expressed at a significantly up-regulated level in approximately 72% hepatocellular carcinoma (HCC) compared to normal hepatocytes, cholangiocarcinomas and liver metastases, but not in liver tissue of normal adults. In addition, high expression of GPC3 in liver cancer patients is often accompanied by poor prognosis, all suggesting a potential role for GPC3 as a biomarker in HCC. GPC3 has been proposed as targets for antibody (Ishiguro et al, Cancer Res 68: 9832-.

Antibody-drug conjugates (ADCs) are very effective tumor-targeted therapeutic drugs. Antibody fragments may also be fused to protein toxoid fragments to produce chimeric structures known as immunotoxins. The CD 22-targeted immunotoxin Lumoxiti has been FDA approved for the treatment of relapsed or refractory Hairy Cell Leukemia (HCL). Pseudomonas Exotoxin (PE) a is a commonly used toxin fragment in immunotoxins that can induce a block in protein synthesis and ultimately cell death. Immunotoxins are thought to trigger tumor regression by two mechanisms: antibody-induced inactivation of cell signals and toxin-induced inhibition of protein synthesis, and therefore the cytotoxicity resulting from conjugation of antibodies of different mechanisms of action to the same toxin can vary greatly. According to the results of the study by Wei Gao et al, although the affinity of antibody HN3 of GPC3 is much weaker than that of YP7, the cytotoxicity and in vivo tumor suppression activity of immunotoxin HN3-PE24 against GPC3 are higher than those of YP7-PE24(Wei Gao et al. nat Commun.2015Mar 11; 6: 6536.). Therefore, focusing on antibody development, searching for better antibodies, and synthesizing better ADC or antibody-toxin conjugates are important strategies for developing ADC or immunotoxin drugs.

Disclosure of Invention

The purpose of the present invention is to provide a monoclonal antibody that recognizes multiple epitopes of glypican 3 with high affinity. Antibodies provided include immunoconjugates of antibody fragments (e.g., single chain variable fragments (scfvs)) and effector molecules (e.g., toxin proteins). Also provided are compositions comprising antibodies that specifically bind GPC3, nucleic acid molecules encoding these antibodies, expression vectors comprising the nucleic acid molecules, and isolated host cells that express the nucleic acid molecules.

The specific technical scheme of the invention is as follows:

a monoclonal antibody of 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 complementarity determining regions represented by amino acid residues at positions 26-35, 50-66 and 97-115 of SEQ ID NO: 1; the variable region of the light chain of the antibody has complementarity determining regions shown by amino acid residues 23-29, 46-52 and 85-97 of SEQ ID NO 2;

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

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

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

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

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

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

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

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

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

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

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

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

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

There are four known subtypes (subtypes 1-4) of human GPC 3. The nucleic acid and amino acid sequences of 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 may bind to one or more of the four human GPC3 subtypes, or conservative variants thereof.

Specifically, the antibody of glypican 3 of the invention is:

(1) the heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 1, and the light chain variable amino acid sequence is shown as SEQ ID NO. 2;

or (2) the heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 3, and the light chain variable amino acid sequence is shown as SEQ ID NO. 4;

or (3) the heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO.5, and the light chain variable amino acid sequence is shown as SEQ ID NO. 6;

or (4) the heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 7, and the light chain variable amino acid sequence is shown as SEQ ID NO. 8;

or (5) the heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 9, and the light chain variable amino acid sequence is shown as SEQ ID NO. 10;

or (6) the heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 11, and the light chain variable amino acid sequence is shown as SEQ ID NO. 12;

or (7) the heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 13, and the light chain variable amino acid sequence is shown as SEQ ID NO. 14;

or (8) the heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 15, and the light chain variable amino acid sequence is shown as SEQ ID NO. 16;

or (9) the heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 17, and the light chain variable region amino acid sequence is shown as SEQ ID NO. 18;

or (10) the heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 19, and the light chain variable amino acid sequence is shown as SEQ ID NO. 20;

or (11) the heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 21, and the light chain variable amino acid sequence is shown as SEQ ID NO. 22;

or (12) the heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 23, and the light chain variable amino acid sequence is shown as SEQ ID NO. 24;

or (13) the heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 25, and the light chain variable amino acid sequence is shown as SEQ ID NO. 26;

or (14) the antibody has the heavy chain variable amino acid sequence shown as SEQ ID NO. 27 and the light chain variable amino acid sequence shown as SEQ ID NO. 28.

The antibody of the invention is a single chain antibody, a double chain antibody, a monoclonal antibody or a chimeric antibody.

The monoclonal antibodies of the invention can be of any isotype. May be, for example, an IgM or IgG antibody, such as IgG1 or IgG 2. The classes of antibodies that can specifically bind GPC3 can be converted from one another according to known methods (e.g., IgG can be converted to IgM). Class switching can also be used to switch one IgG subclass to another, for example from IgG1 to IgG 2.

The antibody of the invention may be:

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

(2) fab' antibody molecule fragments obtainable by treatment of an intact antibody with pepsin followed by reduction to produce a portion of the intact light and heavy chains; two Fab' fragments were obtained per antibody molecule;

(3) (Fab')2, an antibody fragment obtainable by treating an intact antibody with pepsin, but not subsequently reducing it; f (ab ')2 is a dimer of two Fab' fragments joined together by two disulfide bonds;

(4) fv, a gene-engineered fragment containing the variable regions of the light and heavy chains expressed as 2 chains;

(5) single chain antibodies (e.g., scFv), genetically engineered molecules that contain a light chain variable region and a heavy chain variable region and are linked by a suitable polypeptide linker into genetically fused single chain molecules;

(6) dimers of single chain antibodies (scFv2), defined as dimers of scFv (also known as "minibodies");

(7) a VH single domain antibody, an antibody fragment consisting of the variable region of the heavy chain.

One skilled in the art will appreciate that conservative variants of the antibody may be made. Amino acid substitutions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions) may be made in the VH and/or VL regions, with the substituted VH and VL still retaining the ability to bind GPC3, or being more capable of binding GPC 3. 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 that are considered conservative substitutions for one another:

1) alanine (a), serine (S), threonine (T);

2) aspartic acid (D), glutamic acid (E);

3) asparagine (N), glutamine (Q);

4) arginine (R), lysine (K);

5) isoleucine (I), leucine (L), methionine (M), valine (V);

6) phenylalanine (F), tyrosine (Y), tryptophan (W).

It is another object of the present invention to provide a recombinant protein comprising the antibody of the present invention and a tag sequence to facilitate expression and/or purification. The tag sequence includes but is not limited to a6 × His tag.

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 labels.

The drug of the present invention is a substance having cytotoxic or antitumor activity, such as Monomethylauristatin E (MMAE), Monomethylauristatin F (MMAF), Pyrolobe diazepine (PBD) dimer, N2'-deacetyl-N2' - (3-Mercapto-1-oxopropyl) -Maytansine (Maytansine DM1), vinblastine, daunomycin and the like, and radioactive agents¹²⁵I、³²P、¹⁴C、³H and³⁵s and the like.

The toxin is a toxic protein with cytotoxicity or antitumor activity, and can be conjugated with the antibody to form immunotoxin, including but not limited to pseudomonas exotoxin, ricin, abrin, diphtheria toxin and subunits thereof, and botulinum toxin A-F, and truncated mutants and point mutants of the toxins. These toxins are commercially available (e.g., Sigma Chemical Company, st. louis, MO). The toxins also include variants of the above toxins (see, e.g., U.S. patent nos. 5,079,163 and 4,689,401). In one embodiment, the toxin is Pseudomonas Exotoxin (PE) (U.S. Pat. No.5,602,095). The "pseudomonas exotoxin" includes its native sequence, a cytotoxic fragment of the native sequence, and conservatively modified variants of the native sequence or a cytotoxic fragment thereof. These modifications include, but are not limited to, the removal of multiple amino acid deletions in domains Ia, Ib, II and III, single or multiple amino acid substitutions and the addition of one or more sequences at the carboxy terminus (see, e.g., Siegel et al, J.biol. chem.264: 14256-14261, 1989). Cytotoxic fragments of pseudomonas exotoxin include PE24, PE40, PE38, PE35, and the like.

The detectable label of the present invention is a substance that can be detected by an isotope analyzer, a microplate reader, a bioluminescent detector, a chemiluminescent detector, an electrochemiluminescent detector, a fluorescent analyzer, or visualized by the naked eye, including, but not limited to, radioisotopes (e.g., radioisotopes)³H、¹⁴C、¹⁵N、³⁵S、⁹⁰Y、、⁹⁹Tc、¹¹¹In、¹²⁵I、¹³¹I) Enzymes (such as horseradish peroxidase, beta-galactosidase, alkaline phosphatase, glucose oxidase and the like), 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 and the like) which can be used for detectionProteins, fluorescent dyes Cy3, Cy5, rare earth phosphors, quantum dots, etc.), biotin, magnetic reagents (e.g., gadolinium), electrochemiluminescent reagents (e.g., ruthenium terpyridyl), colloidal gold.

Effector molecules can be attached to the antibodies of the invention using any means known to those skilled in the art. For example, the antibody may be functionally linked (by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities. The method of linking the effector molecule and the antibody varies depending on the chemical structure of the effector molecule. Polypeptides generally contain multiple functional groups; for example carboxylic acids (COOH), free amino groups (-NH)₂) Or a sulfhydryl group (-SH) which may be used to react with a suitable functional group on the antibody to bind the effector molecule. Alternatively, the antibody may be derivatized to expose or attach additional reactive functional groups. The derivatization may include attaching any of a variety of known linker molecules. The linker may be any molecule used to join the antibody to an effector molecule. The linker is capable of forming a covalent bond with the antibody and effector molecule. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight or branched chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and effector molecule are polypeptides, the linker may be conjugated to the alpha carbon amino and carboxyl groups of the constituent amino acids or to the terminal amino acids through its side groups (e.g., through disulfide bonds 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.

In certain cases, it is desirable to release the effector molecule from the antibody when the immunoconjugate has reached its target site. Thus, in these cases, the immunoconjugate will comprise a bond cleavable in the vicinity of 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 under which the immunoconjugate is located within the target cell or in the vicinity of the target site.

It is another object of the present invention to provide a polynucleotide encoding the antibody, recombinant protein or immunoconjugate of the present invention.

Another objective of the invention is to provide a vector containing the polynucleotide of the invention. The carrier comprises: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

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 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 may be soluble in an aqueous carrier, such as buffered saline or the like. May also contain pharmaceutically acceptable adjuvants required to approximate physiological conditions, such as pH regulator and buffer, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.

Another objective of the invention is to provide an application of the antibody, the recombinant protein, the immunoconjugate, the polynucleotide, the vector or the genetically engineered host cell in preparing a therapeutic drug or a diagnostic reagent for autoimmune diseases, viral infections or cancers.

The cancer is liver cancer, stomach cancer, colorectal cancer, lung cancer or ovarian cancer, or any other type of cancer expressing GPC 3.

The monoclonal antibodies disclosed herein can also be used to make chimeric antigen receptors (CAR; also known as chimeric T cell receptors, artificial T cell receptors, or chimeric immunoreceptors) or bispecific antibodies.

The specific examples of the present invention disclose antibodies a5, a18, a43, C46, F5, F67, G15, H49, I34, I82, I88, J58, J80A, J80B, which have high affinity and recognize different epitopes. The invention also discloses the effect of the immunotoxin of the antibody fusion pseudomonas exotoxin A (PE) on treating liver cancer. These antibodies can also be constructed as bispecific antibodies, antibody-drug conjugates (ADCs), and the like, for antibody-targeted therapy, or as CAR-T, CAR-NK, and the like, for cell therapy. The antibodies and compositions can be used to diagnose tumors that are positive for GPC3 expression.

The antibodies and compositions provided by the present invention can be used for a variety of purposes, such as molecular diagnostics of tumors, confirming the expression of GPC3 in liver cancer and other tumor patient samples. The sample may be any sample, including but not limited to tissue from biopsies, autopsies, and pathological specimens. Biological samples also include sections of tissue, such as frozen sections taken for histological purposes. Biological samples also include bodily fluids such as blood, serum, plasma, sputum, spinal fluid, or urine. Biological samples are typically obtained from mammals, including humans, non-human primates, mice, and the like.

The invention also provides a method of treating a subject having a cancer, such as liver cancer: selecting a subject having a cancer that expresses GPC3, administering to the subject a therapeutically effective amount of a monoclonal antibody to GPC3, or an immunoconjugate comprising the antibody.

The invention has the advantages that:

the combination of the monoclonal antibodies provided by the invention covers a plurality of different epitopes in a GPC3 molecule, so that different monoclonal antibodies can be combined and paired to prepare a detection reagent or a kit for GPC 3. The monoclonal antibody provided by the invention not only has high affinity (Kd values are both in nM and pM grades, even higher), but also has good thermal stability. The monoclonal antibody provided by the invention has excellent cytotoxic activity after being conjugated with pseudomonas exotoxin PE24, and is obviously superior to the existing immunotoxin HN3-PE 24. The antibody of the invention can be used for developing antibody-drug conjugates or immunotoxin drugs with better activity.

Drawings

FIG. 123 epitope clustering analysis of monoclonal antibodies. The 23 monoclonal antibodies were initially divided into 14 epitopes by monoclonal phage competition ELISA (represented by J58, J80B, a5, H49, F5, G15, a43, a18, F67, C46, I34, J80A, I82, I88, respectively).

FIG. 2 the monoclonal antibody of the present invention was used for detection of GPC3 by a sandwich ELISA method. The first column is the coating antibody, after adding the GPC3 protein and co-incubating, the detection of GPC3 was performed using the monoclonal antibody of the first row as the detection antibody. The light grey squares indicate that the epitopes of the coating antibody and the detection antibody do not overlap and are suitable for the detection of GPC3 in biological samples by the double antibody sandwich method. The black squares indicate that the coating antibody overlaps the epitope of the detection antibody and is not suitable for pairing by the double antibody sandwich method.

FIG. 3 is a simplified diagram of the epitopes recognized by the antibodies of the present invention. The overlapping intersection indicates that there is partial overlap of the epitopes of these monoclonal antibodies.

FIG. 4 ELISA method for detecting the affinity of the monoclonal antibody of the present invention for binding to GPC3 protein. FIG. 4a shows human GPC3 protein, and FIG. 4b shows murine GPC3 protein.

FIG. 5 FACS method the binding activity of the monoclonal antibody of the invention to GPC3 negative/positive cell line was examined. FIG. 5a is a GPC3 negative A431 cell line, FIG. 5b is a GPC3 positive cell line G1, FIG. 5c is a GPC3 positive liver cancer cell line HepG2, FIG. 5d is a GPC3 positive liver cancer cell line Hep3B, and FIG. 5e is a GPC3 positive liver cancer cell line Huh 7.

FIG. 6 killing activity of immunotoxins of the invention against GPC3 negative cell line A431LG and GPC3 positive cell lines G1LG, Hep3BLG, HepG2LG and Huh7 LG.

Figure 7 in vivo tumor suppressive activity of immunotoxin J80A. Hep3B cells were inoculated subcutaneously into NSG mice and, after nodulation, treated with various doses of immunotoxin J80A-PE 24. The treatment mode is tail vein administration, and the administration is performed once every 2 days.

Detailed Description

The present disclosure describes the preparation and identification of monoclonal antibodies that bind GPC 3. Particular embodiments disclose the isolation and characterization of monoclonal antibodies targeting GPC 3. The specific data disclosed herein demonstrate that these antibodies bind cell surface associated GPC3 with high affinity, and the relationship between different antibodies binding different epitopes of GPC 3. The antibody fusion toxin (immunotoxin) can strongly kill GPC3 positive tumor cells in vitro, and provides experimental evidence for drug development.

The invention is described in further detail below with reference to specific examples and data, it being understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way. Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified. In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

Abbreviations

CDR complementarity determining region HCC hepatocellular carcinoma

CTL cytotoxic T lymphocyte hFc human Fc

ELISA enzyme-linked immunosorbent assay HS heparin sulfate

FACS fluorescence activated cell sorting Ig immunoglobulins

GPC3 glypican 3 mAb monoclonal antibody

Pfu colony Forming Unit Pseudomonas PE (Pseudomonas) exotoxin

SPR surface plasmon resonance APC allophycocyanin

Example 1: preparation of monoclonal antibody of glypican 3 of the invention

This example describes the generation of high affinity mabs against tumor-associated GPC 3.

Immunizing 4-month-old chicken with GPC3-hFc protein (Sino Biological, Cat:10088-H02H2), immunizing 300 μ g each time, 2 weeks apart each time, collecting spleen of chicken after immunizing 4 times, extracting total RNA in spleen, reverse transcribing into cDNA, using the cDNA as template for establishing phage antibody library, and constructing chicken antibody phage library (library capacity 2.7 × 10)⁹). After 4 panning of the phage library using GPC3 protein as antigen, 300 clones were randomly picked and sequenced, resulting in 23 highly enriched representative clones, named A5, a20, a31, a41, a64, B81, a61, F5, G41, I68, a43, C15, a18, F67, C46, I34, J80A, I82, I88, J58, J80B, H49, G15, respectively. Phage competition ELISA experiments showed that 23 representative clones could be initially classified as 14 epitopes (figure 2),represented by J58, J80B, a5, H49, F5, G15, a43, a18, F67, C46, I34, J80A, I82, I88, respectively (see tables 1 and 2 for antibody sequences).

The amino acid sequences of the complementarity determining regions (CDR regions) and the variable regions of the above-mentioned antibodies are shown in tables 1 and 2:

TABLE 1.14 CDR amino acid sequences of monoclonal antibodies (according to Kabat and IMGT)

TABLE 2.14 variable region amino acid sequences of monoclonal antibodies

Example 2 detection of GPC3 protein Using the monoclonal antibody of the present invention

The scFv of the 14 monoclonal antibodies was fused with hFc to construct an expression vector pPBSPS-scFv-hFc, which was expressed in 293F cells. The expression product was purified by protein A chromatography (GE healthcare). GPC3 protein was detected using purified antibodies and sandwich ELISA.

First, 14 monoclonal antibodies are used as coating antibodies to be coated on the bottom of an ELISA plate (figure 2, first column), then GPC3 protein standard or biological samples containing GPC3 (such as serum of liver cancer patients) are added, then the 14 biotin-labeled monoclonal antibodies are used as detection antibodies (figure 2, first column) to be incubated with the detection antibodies, and the binding of the detection antibodies and GPC3 is detected by using HRP-labeled streptavidin. The gray squares show that the combination of the antibodies at the position can carry out high-sensitivity detection on the GPC3 protein, and the coated antibodies and the detection antibodies have no repulsion, so that the combination can be used for detecting and analyzing the content of GPC3 in a biological sample. The black squares indicate that the combination of antibodies at this position is partially repulsive when bound to the GPC3 protein, also indicating that the epitopes of these antibodies partially overlap. From this result, the epitopes of 14 monoclonal antibodies (J58, J80B, a5, H49, F5, G15, a43, a18, F67, C46, I34, J80A, I82, I88) were further reduced to 12 (represented by a5 since the epitopes of J58 and J80B overlap a5 more) (fig. 3).

Example 3 in vitro characterization of antibodies

1. Human murine Cross-reactivity of the antibodies prepared in example 1 with the GPC3 protein

The affinity of the antibody prepared in example 1 to human-derived and murine GPC3 protein (Sino Biological, Cat:50989-M08B) was determined by ELISA. Human and mouse GPC3 proteins were coated on ELISA plates, respectively, incubated with a gradient of diluted antibody, and the binding ability of the antibody to human and mouse GPC3, respectively, was detected using anti-hFc-tagged antibodies. The ELISA results showed that all 14 antibodies bound very strongly to human GPC3 protein, except H49, I82 did not bind murine GPC3, and all others bound murine GPC3, but that J58 bound murine GPC3 with very weak affinity (fig. 4a and 4 b). Antibodies a5, a18, a43, C46, F5, F67, G15, I34, I88, J80A, J80B capable of binding human and mouse GPC3 can be used for detection of mouse GPC3 protein and therapeutic study of tumors positive for expression of mouse GPC 3.

SPR measurement of binding kinetics and affinity of the antibody prepared in example 1 to GPC3

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 analyte and running buffer were injected into the chip at a flow rate of 30 μ L/min with a sample binding time of 180 seconds followed by a dissociation time of 600 seconds. Binding and dissociation curves were fitted to a 1:1Langmiur binding model using ProteOn software. As a result, the affinities of the monoclonal antibodies were measured as shown in Table 3.

TABLE 3 affinity constant Kd values of monoclonal antibodies determined by Biacore

The results show that the affinity of the antibody prepared in example 1 and GPC3 is high, the affinity of A18 reaches 0.0214pM, and the affinity of A43 reaches 1.52 pM. The affinity of the control antibody HN3 was only 1.95nM.

3. Detection of antibody stability

The Tm value is a parameter for quantitatively describing the thermal stability of a protein, and is one of the most commonly used indicators in pharmaceutical evaluation. A higher Tm value means a more stable protein conformation. The stability of the antibody prepared in example 1 was determined using Prometheus NT.48 from Nano tester: the antibody concentration was diluted to 50ug/ml and then loaded, the temperature rose from 25 ℃ to 95 ℃ within 40min at a rate of 1.5 ℃/min. And finally obtaining the Tm value of the antibody. The results are shown in Table 4.

TABLE 4 Tm values of the monoclonal antibodies of the invention

The result shows that most antibodies have high thermal stability, wherein Tm of A18 and F5 can reach more than 70 ℃ and 80 ℃, and the antibody has remarkable advantages in the aspect of pharmacy.

Example 4 FACS detection of binding of the antibodies of the invention to a GPC3 positive cell line

A431, G1 (A431 cell line overexpressing GPC3) (Phung Yet. MAbs 2012; 4: 592. 599) and liver cancer cell lines HepG2, Hep3B, HuH-7 were cultured adherently in DMEM medium (Invitrogen, CarlsbadCA) 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, respectively, and APC-labeled goat anti-human secondary antibodies were added to detect the antibodies bound to the cell surface. As shown in FIG. 5, the antibody prepared in example 1 strongly bound to G1 cells and HepG2, Hep3B and Huh7 cells, but did not bind to GPC 3-negative A431 cells, indicating that the antibody prepared in example 1 specifically recognizes and binds to the GPC3 protein on the cell surface.

Example 5 cytotoxicity of the antibodies of the invention after fusion of the toxin to GPC 3-positive cells

1. Construction of cell lines

Lentiviral particles expressing luciferase and GFP genes were packaged for infection with a431, G1, HepG2, Hep3B, Huh7 cells, and 3 days after infection were screened for puromycin followed by FACS screening for GFP positive cells. The obtained positive cells can co-express luciferase and are used for detecting the survival rate of the cells subsequently. The positive cells were renamed A431LG, G1LG, HepG2LG, Hep3BLG, Huh7 LG.

2. Preparation of immunotoxins

The monoclonal antibody (scFv) provided by the invention is fused with pseudomonas exotoxin PE24 to construct an immunotoxin, and 6 histidines (6 XHis) are additionally added at the N-terminal of the immunotoxin to facilitate purification of the immunotoxin. Expression of immunotoxin was carried out using E.coli strain HB 2151. Inoculating the strain into 2L 2YT medium, culturing at 37 deg.C for 4-5 hr, and culturing to obtain OD₆₀₀Reaching 0.9, adding 1mM IPTG for induction, and expressing for 10h at 30 ℃. Collecting thallus, crushing thallus under high pressure, centrifuging, filtering to obtain supernatant, and purifying with nickel column.

3. Cytotoxicity of immunotoxins

Starting with 1000ng/ml of immunotoxin, a 1: 10 gradient dilution, then with A431LG, G1LG, HepG2LG, Hep3BLG, Huh7LG cells were incubated for 72 hours, then its killing activity on cells was examined. Considering that HN3-PE24 is the immunotoxin having the best killing effect targeting GPC3 at present, HN3-PE24 was used as a reference for parallel comparison.

The results are shown in FIG. 6. The immunotoxin has strong cytotoxic activity on GPC3 positive G1LG, Hep3BLG, HepG2LG and Huh7LG cells, but does not kill GPC3 negative A431LG, and shows that the immunotoxin has high selectivity on GPC3 positive tumor cells. The intensity of the activity of the immunotoxin was expressed as IC50 value, and the measured IC50 value was shown in Table 5. The activity of most of immunotoxins provided by the invention is stronger than that of HN3-PE24, such as J80A-PE24, A43-PE24, C46-PE24, J80B-PE24, A5-PE24, G15-PE24, I82-PE24, A18-PE24 and H49-PE24, wherein the killing activity of J80A-PE24 is strongest, the IC50 value of Hep3BLG reaches 7.974ng/ml, and the IC50 of HN3-PE24 is as high as 109ng/ml, which shows that the tumor inhibition activity of J80A-PE24 is stronger than that of HN3-PE 24.

TABLE 5 killing activity of monoclonal antibody based immunotoxins on GPC3 positive tumor cells IC50 values

Example 6 in vivo tumor inhibiting Activity of immunotoxin J80A

In this example, immunotoxin J80A was used as an example to study the in vivo tumor suppressive activity of J80A. Will be 5X 10⁶Hep3B cells were inoculated subcutaneously into NSG mice to form tumors of 200mm³Tumor-bearing mice were treated at this time. The treatment groups were injected with different doses of immunotoxin (2.5, 5, 10mg/kg body weight) via tail vein and the control group was injected with PBS buffer. The treatment is performed every 2 days. The results show that all three dose groups can significantly inhibit the growth of tumors (FIG. 7), especially the 10mg/kg and 5mg/kg dose groups can significantly reduce the size of the tumor body, and the tumors can completely disappear after part of mice are treated.

Sequence listing

<110> university of agriculture in Huazhong

<120> antibody recognizing multiple different epitopes of glypican 3 and use thereof

<160> 28

<170> SIPOSequenceListing 1.0

<210> 1

<211> 126

<212> PRT

<213> Artificial Sequence (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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. The monoclonal antibody of glypican 3 comprises a heavy chain variable region and a light chain variable region, and is characterized in that:

(1) the heavy chain variable region of the antibody has complementarity determining regions shown by amino acid residues 26-35, 50-66 and 97-115 of SEQ ID NO 1; the variable region of the light chain of the antibody has complementarity determining regions shown by amino acid residues 23-29, 46-52 and 85-97 of SEQ ID NO 2;

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

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

2. The monoclonal antibody of claim 1, characterized in that:

or (9) the heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 17, and the light chain variable amino acid sequence is shown as SEQ ID NO. 18;

3. The monoclonal antibody 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 of claim 1, characterized in that the antibody is a VH single domain antibody, a Fab fragment, a Fab 'fragment, a F (ab)'2 fragment, a single chain variable fragment (scFv) or a disulfide stabilized variable fragment (dsFv).

5. A recombinant protein comprising the monoclonal antibody of any one of claims 1-4 and a tag sequence to facilitate expression and/or purification.

6. An immunoconjugate characterized by comprising the monoclonal antibody of any one of claims 1-4 and an effector molecule.

7. The immunoconjugate according to claim 6, characterized in that the effector molecule is a toxin, a drug or a detectable label.

8. The immunoconjugate according to claim 7, characterized in that the toxin is a protein with cytotoxic or anti-tumor activity, including but not limited to abrin or a variant thereof, ricin or a variant thereof, pseudomonas exotoxin or a variant thereof, diphtheria toxin or a variant thereof, botulinum toxin or a variant thereof.

9. The immunoconjugate according to claim 7, characterized in that the drug is a compound with cytotoxic or anti-tumor activity, including but not limited to vinblastine, daunomycin, monomethyyl auristatin E, monomethyyl auristatin F, Pyrrolobizodiazepine dimer, N2'-deacetyl-N2' - (3-Mercapto-1-oxopropyl) -Maytansine.

10. The immunoconjugate of claim 7, characterized in that the detectable label is a substance that can be detected by an isotope analyzer, an enzyme reader, a bioluminescent detector, a chemiluminescent detector, an electrochemiluminescent detector, a fluorescent analyzer, or visualized by the naked eye, including but not limited to radioisotopes (e.g., 125I, 131I, 32P, 14C, 3H, and 35S), enzymes useful for detection (e.g., horseradish peroxidase, beta-galactosidase, alkaline phosphatase, glucose oxidase, etc.), fluorescent proteins (e.g., Green Fluorescent Protein (GFP), Yellow Fluorescent Protein (YFP), allophycocyanin APC, phycoerythrin PE), bioluminescent markers (e.g., luciferase), fluorescent compounds (e.g., fluorescein isothiocyanate, rhodamine, 5-dimethylamino-1-naphthalenesulfonyl chloride, Cytosyl chloride, Cytosine, and the like, Phycoerythrin, fluorescent dyes Cy3, Cy5, rare earth phosphors, quantum dots, etc.), biotin, a magnetic reagent (e.g., gadolinium), an electrochemiluminescent reagent (e.g., ruthenium terpyridyl), and colloidal gold.

11. A polynucleotide characterised in that it encodes an antibody according to any one of claims 1 to 4, or a recombinant protein according to claim 5, or an immunoconjugate according to any one of claims 6 to 10.

12. A vector comprising the polynucleotide of claim 11.

13. A genetically engineered host cell comprising the vector of claim 12, or having the polynucleotide of claim 11 integrated into its genome.

14. A pharmaceutical composition comprising one or more of the antibody of any one of claims 1 to 4, the recombinant protein of claim 5, the immunoconjugate of any one of claims 6 to 10, the polynucleotide of claim 11, the vector of claim 12 or the genetically engineered host cell of claim 13.

15. Use of the antibody of any one of claims 1 to 4, the recombinant protein of claim 5, the immunoconjugate of any one of claims 6 to 10, the polynucleotide of claim 11, the vector of claim 12 or the genetically engineered host cell of claim 13 for the manufacture of a medicament or diagnostic agent for the treatment of an autoimmune disease, a viral infection or cancer.