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

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

Antibodies Recognizing Multiple Different Epitopes of Glypican 3 and Their Applications

technical field

The invention belongs to the field of biotechnology, and in particular relates to a high-affinity monoclonal antibody that recognizes three different antigenic epitopes of glypican and its application.

Background technique

Cancer has become the second leading cause of human death after cardiovascular disease, and liver cancer ranks sixth and fourth in global incidence and mortality, respectively. Although surgery is the standard treatment for liver cancer, only 5-10% of liver cancer patients can be treated with surgery due to the insidious onset and high degree of malignancy of liver cancer patients. Patients with advanced disease 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.

Glypican-3 (GPC3, Glypican 3) is a member of heparin sulfate proteoglycans, which is anchored to the cell membrane surface through glycosyl-phosphatidylinositol (GPI). The human GPC3 gene is located on the X chromosome (Xp26) and encodes a 70kDa protein containing 580 amino acids that is endo-cleaved by a furin-like convertase between Arg358 and Ser359, resulting in a 40kDa N-terminal subunit and a 30kDa C-terminal subunit There are two heparan sulfate (HS) chains on the C-terminal subunit. Studies have shown that the expression level of GPC3 is significantly up-regulated in about 72% of hepatocellular carcinoma (HCC) compared with normal hepatocytes, cholangiocarcinoma and liver metastases, but it is not expressed in normal adult liver tissue. In addition, high expression of GPC3 in HCC patients is often accompanied by poor prognosis, which all suggest the potential role of GPC3 as a biomarker in HCC. Currently GPC3 has been suggested as an 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) targets for immunotherapy.

Antibody-drug conjugates (ADCs) are very effective tumor-targeted therapeutic drugs. Antibody fragments can also be fused to protein toxoid fragments to create chimeric structures called immunotoxins. The CD22-targeting immunotoxin, Lumoxiti, has been approved by the FDA for the treatment of relapsed or refractory hairy cell leukemia (HCL). Pseudomonas exotoxin (PE) A, a commonly used toxin fragment in immunotoxins, can induce blockade of protein synthesis and ultimately cell death. Immunotoxins are thought to trigger tumor regression through two mechanisms: antibody-induced inactivation of cell signaling and toxin-induced inhibition of protein synthesis, so the cytotoxicity of antibodies conjugated to the same toxin with different mechanisms of action may vary widely. According to the research results of Wei Gao et al., although the affinity of the GPC3 antibody HN3 is much weaker than that of YP7, the cytotoxicity and in vivo antitumor activity of the GPC3 immunotoxin HN3-PE24 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 can synthesize ADCs or antibody-toxin conjugates with better activity, which is an important strategy for developing ADCs or immunotoxin drugs.

SUMMARY OF THE INVENTION

An object of the present invention is to provide monoclonal antibodies that recognize multiple epitopes of Glypican with high affinity. Provided antibodies include immunoconjugates of antibody fragments (eg, single-chain variable fragment (scFv)) and effector molecules (eg, 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 expressing the nucleic acid molecules.

The specific technical scheme of the present invention is as follows:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Human GPC3 has four known isoforms (isoforms 1-4). The nucleic acid and amino acid sequences of the four isoforms of GPC3 are known, including GenBank accession numbers: NM_001164617 and NP_001158089 (isoform 1); NM_004484 and NP_004475 (isoform 2); NM_001164618 and NP_001158090 (isoform 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 isoforms, or conservative variants thereof.

Specifically, the antibody of Glypican 3 of the present invention is:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The antibodies of the present invention are single chain antibodies, diabodies, monoclonal antibodies or chimeric antibodies.

The monoclonal antibodies of the present invention can be of any isotype. It can be eg an IgM or IgG antibody, eg IgGl or IgG2. The classes of antibodies that can specifically bind to GPC3 can be converted to each other according to known methods (eg, IgG can be converted to IgM). Class switching can also be used to switch from one IgG subclass to another, eg, from IgG1 to IgG2.

The antibody of the present invention can be:

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

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

(3) (Fab')2, an antibody fragment that can be obtained by treating an intact antibody with pepsin without subsequent reduction; F(ab')2 is two Fab' fragments linked by two disulfide bonds dimers together;

(4) Fv, containing the gene process fragment of the variable region of the light chain and the variable region of the heavy chain expressed as two chains;

(5) Single-chain antibodies (such as scFv), genetically engineered molecules that contain a light chain variable region and a heavy chain variable region and are linked into a genetically fused single-chain molecule through a suitable polypeptide linker;

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

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

Those skilled in the art will appreciate that conservative variants of antibodies can be prepared. Amino acid substitutions (eg, 1, 2, 3, 4, or 5 amino acid substitutions) can be made in the VH and/or VL regions, and the substituted VH and VL still retain the ability to bind to GPC3, or to GPC3. Stronger binding ability. 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 each other:

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).

Another object of the present invention is to provide a recombinant protein comprising the antibody of the present invention and a tag sequence for assisting in expression and/or purification. The tag sequence includes, but is not limited to, a 6xHis tag.

The GPC3 antibodies described herein can be conjugated to effector molecules. Effector molecules include, but are not limited to, toxins, drugs, or detectable labels.

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

The toxins of the present invention are toxic proteins with cytotoxic or anti-tumor activity, which can be conjugated with the antibodies of the present invention to form immunotoxins, including but not limited to Pseudomonas exotoxin, ricin, and acacia proteins, diphtheria toxin and its subunits, and botulinum toxins A-F, and truncated and point mutants of these toxins. These toxins are commercially available (eg, Sigma Chemical Company, St. Louis, MO). The toxins also include variants of the above toxins (see, eg, US Pat. Nos. 5,079,163 and 4,689,401). In one embodiment, the toxin is Pseudomonas exotoxin (PE) (US Patent No. 5,602,095). The "Pseudomonas exotoxin" includes its native sequence, cytotoxic fragments of said native sequence, and conservatively modified variants of the native sequence or cytotoxic fragments thereof. These modifications include, but are not limited to, removal of multiple amino acid deletions in Domain Ia, Domain Ib, II, and III, single or multiple amino acid substitutions, and addition of one or more sequences at the carboxy terminus (see, eg, Siegall et al. , J. Biol. Chem. 264: 14256-14261, 1989). Cytotoxic fragments of Pseudomonas exotoxin include PE24, PE40, PE38, and PE35, among others.

The detectable label of the present invention is a substance that can be detected by an isotope analyzer, a microplate reader, a bioluminescence detector, a chemiluminescence detector, an electrochemiluminescence detector, a fluorescence analyzer, or visualized with the naked eye, and these substances include But not limited to radioisotopes (such as ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I), enzymes that can be used for detection (such as horseradish peroxidase) , β-galactosidase, alkaline phosphatase, glucose oxidase, etc.), fluorescent proteins (such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), allophycocyanin APC, phycoerythrin PE), biological Luminescent labels (such as luciferase), fluorescent compounds (such as luciferin, fluorescein isothiocyanate, rhodamine, 5-dimethylamino-1-naphthalenesulfonyl chloride, phycoerythrin, fluorescent dyes Cy3, Cy5 , rare earth inorganic light-emitting materials, quantum dots, etc.), biotin, magnetic reagents (such as gadolinium), electrochemiluminescence reagents (such as ruthenium terpyridine), colloidal gold.

Effector molecules can be linked to the antibodies of the invention using any means known to those of skill in the art. For example, the antibody can be functionally linked (by chemical conjugation, genetic fusion, non-covalent association, or otherwise) 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 varies. Polypeptides typically contain multiple functional groups; eg, carboxylic acid (COOH), free amino groups ( _-NH2 ), or sulfhydryl groups (-SH), which can be used to react with appropriate functional groups on the antibody to bind to the effector molecule. Alternatively, the antibody can be derivatized to expose or attach additional reactive functional groups. The derivatization can include attachment of any of a variety of known linker molecules. The linker can be any molecule used to join the antibody and 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, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and effector molecule is a polypeptide, the linker may be attached via its side groups (eg, via a cysteine disulfide bond) to the constituent amino acids or to the alpha carbon amino and carboxyl groups of terminal amino acids. Typically, the antibody or portion thereof is derivatized so that binding to the target antigen is not adversely affected by the derivatization or labeling.

In certain instances, the effector molecule needs to be released from the antibody when the immunoconjugate has reached its target site. Thus, 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 may result from enzymatic activity, or from the conditions in which the immunoconjugate is placed within the target cell or near the target site.

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

Another object of the present invention is to provide a vector containing the polynucleotide of the present invention. The vectors include: 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 also includes 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 and the like. It can also contain pharmaceutically acceptable excipients required by close to physiological conditions, such as pH adjusting agents and buffering agents, etc., sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate, etc.

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

The cancer is liver, stomach, colorectal, lung or ovarian cancer, or any other type of cancer that expresses GPC3.

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

Specific examples of the present invention disclose antibodies A5, A18, A43, C46, F5, F67, G15, H49, I34, I82, I88, J58, J80A, J80B with high affinity and recognizing different epitopes. The invention also discloses the effect of these antibodies fused with the immunotoxin of Pseudomonas exotoxin A (PE) in treating liver cancer. These antibodies can also be constructed into bispecific antibodies, antibody-drug conjugates (ADC), etc. for antibody-targeted therapy, or CAR-T, CAR-NK, etc. for cell therapy. The antibodies and compositions can be used to diagnose GPC3-positive tumors.

The antibodies and compositions provided by the present invention can be used for various purposes, such as molecular diagnosis of tumors, and confirmation of GPC3 expression in liver cancer and other tumor patient samples. The sample can be any sample including, but not limited to, tissue from biopsy, autopsy, and pathology specimens. Biological samples also include sections of tissue, such as frozen sections obtained for histological purposes. Biological samples also include bodily fluids, such as blood, serum, plasma, sputum, spinal fluid, or urine. Biological samples are generally obtained from mammals, including humans, non-human primates, mice, and the like.

The present invention also provides a method of treating a subject with cancer, such as liver cancer: selecting a subject with a GPC3-expressing cancer, administering to the subject a therapeutically effective amount of a monoclonal antibody to GPC3, or An immunoconjugate comprising said antibody.

Advantages of the present invention:

The monoclonal antibody combination provided by the present invention covers a plurality of different antigenic epitopes in the GPC3 molecule. Therefore, different monoclonal antibodies can be combined and paired to prepare a detection reagent or kit for GPC3. The monoclonal antibody provided by the present invention not only has high affinity (Kd values are both at nM and pM levels, or even higher), but also has good thermal stability. The monoclonal antibody provided by the present invention has superior cytotoxic activity after being conjugated with Pseudomonas exotoxin PE24, which is significantly better than the existing immunotoxin HN3-PE24. The antibodies of the present invention can be used to develop antibody-drug conjugates or immunotoxin drugs with better activity.

Description of drawings

Figure 1. Epitope clustering analysis of 23 monoclonal antibodies. The 23 monoclonal antibodies were preliminarily divided into 14 epitopes (J58, J80B, A5, H49, F5, G15, A43, A18, F67, C46, I34, J80A, I82, I88 to represent).

Figure 2. The monoclonal antibody of the present invention is used to detect GPC3 by sandwich ELISA. The first column is the coating antibody. After adding GPC3 protein and co-incubating, the monoclonal antibody in the first column is used as the detection antibody to detect GPC3. The light gray squares indicate that the epitopes of the coating antibody and the detection antibody do not overlap, which is suitable for the double-antibody sandwich method to detect GPC3 in biological samples. Black squares indicate that the epitope of the coating antibody and the detection antibody overlap, which is not suitable for double-antibody sandwich method.

Figure 3 is a simplified diagram of the antigenic epitopes recognized by the antibodies of the invention. Overlapping intersections indicate that the epitopes of these monoclonal antibodies partially overlap.

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

Figure 5 FACS method to detect the binding activity of the monoclonal antibodies of the present invention to GPC3 negative/positive cell lines. Figure 5a shows the GPC3-negative A431 cell line, Figure 5b shows the GPC3-positive cell line G1, Figure 5c shows the GPC3-positive liver cancer cell line HepG2, Figure 5d shows the GPC3-positive liver cancer cell line Hep3B, and Figure 5e shows the GPC3-positive liver cancer cell line Huh7 .

Figure 6 Killing activity of the immunotoxin of the present invention on GPC3-negative cell line A431LG and GPC3-positive cell line G1LG, Hep3BLG, HepG2LG and Huh7LG.

Figure 7 In vivo tumor suppressor activity of immunotoxin J80A. Hep3B cells were inoculated subcutaneously into NSG mice, and after tumor formation, they were treated with different doses of immunotoxin J80A-PE24. The mode of treatment is tail vein administration, once every 2 days.

Detailed ways

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

The present invention will be described in further detail below in conjunction with specific embodiments and with reference to data. It should be understood that these embodiments are only for illustrating the present invention, rather than limiting the scope of the present invention in any way. 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 procedures and methods not described in detail are conventional methods well known in the art.

abbreviation

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 Immunoglobulin

GPC3 Glypican 3 mAb Monoclonal Antibody

Pfu Colony Forming Unit PE Pseudomonas Exotoxin

SPR Surface Plasmon Resonance APC Allophycocyanin

Example 1: Preparation of monoclonal antibody to Glypican 3 of the present invention

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

4-month-old chickens were immunized with GPC3-hFc protein (Sino Biological, Cat: 10088-H02H2), 300 μg each time, 2 weeks apart each time. After 4 times of immunization, the spleen of the chicken was collected, and the total RNA in the spleen was extracted and reverse transcribed. cDNA was used as a template for building a phage antibody library, and a chicken antibody phage library was constructed (library capacity 2.7×10 ⁹ ). Using GPC3 protein as the antigen, after 4 times of panning of the phage library, 300 clones were randomly selected and sequenced. As a result, 23 highly enriched representative clones were obtained, named A5, A20, A31, A41, A64, B81, A61, F5, G41, I68, A43, C15, A18, F67, C46, I34, J80A, I82, I88, J58, J80B, H49, G15. Phage competition ELISA experiments showed that 23 representative clones could be preliminarily classified into 14 epitopes (Fig. J80A, I82, and I88 are represented (see Tables 1 and 2 for antibody sequences).

The above-mentioned antibody complementarity determining regions (CDR regions) and variable region amino acid sequences are shown in Tables 1 and 2:

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

Table 2. The variable region amino acid sequences of 14 monoclonal antibodies

Example 2 Using the monoclonal antibody of the present invention to detect GPC3 protein

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

First, 14 monoclonal antibodies were used as coating antibodies, which were respectively coated on the bottom of the ELISA plate (Figure 2, the first column), and then GPC3 protein standard or biological samples containing GPC3 (such as serum from patients with liver cancer) were added, and then The 14 biotin-labeled monoclonal antibodies were used as detection antibodies (Fig. 2, the first row) and incubated with them, and the binding of the detection antibodies to GPC3 was detected with HRP-labeled streptavidin. The gray square shows that the antibody combination at this position can detect GPC3 protein with high sensitivity, and there is no rejection 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 square shows that the antibody combination at this position has a partial repulsion effect when it binds to GPC3 protein, which also indicates that the epitopes of these antibodies partially overlap. According to this result, the epitopes of 14 monoclonal antibodies (J58, J80B, A5, H49, F5, G15, A43, A18, F67, C46, I34, J80A, I82, I88) can be further reduced to 12 ( Since the antigenic epitopes of J58 and J80B overlap more with A5, A5 is used as the representative) (Fig. 3).

Example 3 In vitro characterization of antibodies

1. Human-mouse cross-reaction between the antibody prepared in Example 1 and GPC3 protein

The affinities of the antibodies prepared in Example 1 with human and murine GPC3 proteins (SinoBiological, Cat: 50989-M08B) were determined by ELISA. Human and murine GPC3 proteins were coated on ELISA plates respectively, and incubated with antibodies diluted in gradient concentrations. Anti-hFc tag antibodies were used to detect the binding ability of the antibodies to human and murine GPC3, respectively. ELISA results show that these 14 antibodies can bind very strongly to human GPC3 protein. Except H49 and I82, which cannot bind to mouse GPC3, other antibodies can bind to mouse GPC3, but J58 has a very weak affinity for binding mouse GPC3. (Fig. 4a and Fig. 4b). Antibodies A5, A18, A43, C46, F5, F67, G15, I34, I88, J80A, J80B that can bind to human and mouse GPC3 can be used for the detection of mouse GPC3 protein, and can also be used for mouse GPC3 positive expression Therapeutic research in tumors.

2. SPR assay for the 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 μL/min with a sample binding time of 180 s, followed by a dissociation time of 600 s. Binding and dissociation curves were fitted to a 1:1 Langmiur binding model using ProteOn software. The affinities of the measured monoclonal antibodies are shown in Table 3.

Table 3 Affinity constant Kd value of monoclonal antibody determined by Biacore

The results showed that the antibodies prepared in Example 1 had high affinity to GPC3, the affinity of A18 reached 0.0214pM, and the affinity of A43 reached 1.52pM. The affinity of the control antibody HN3 is only 1.95nM.

3. Detection of antibody stability

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 the evaluation of druggability. A higher Tm value means a more stable protein conformation. Use Prometheus NT.48 of Nano Temper to measure the stability of the antibody prepared in Example 1: Dilute the antibody concentration to 50ug/ml, then load the sample, at a speed of 1.5°C/min, the temperature rises from 25°C to 25°C within 40 minutes 95°C. Finally, the Tm value of the antibody was obtained. The results are shown in Table 4.

Table 4 Tm value of the monoclonal antibody of the present invention

The results show that most of the antibodies have relatively high thermal stability, and the Tm of A18 and F5 can reach above 70℃ and 80℃, which have significant advantages in druggability.

Example 4 FACS detection of the binding of the antibodies of the present invention to GPC3 positive cell lines

A431, G1 (A431 cell line overexpressing GPC3) (Phung Yet al. MAbs 2012; 4:592-599) and hepatoma cell lines HepG2, Hep3B, HuH-7 were cultured in an adherent manner, and the medium used was DMEM culture base (Invitrogen, Carlsbad CA) and supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 1% L-glutamine and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA). After the cells were harvested, the antibodies prepared in Example 1 were combined with A431, G1, HepG2, Hep3B, and Huh7 cells respectively, and APC-labeled goat anti-human secondary antibody was added to detect the antibodies bound to the cell surface. The results are shown in Figure 5. 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 It recognizes and binds to the GPC3 protein on the cell surface.

Example 5 Cytotoxicity to GPC3-positive cells after the antibody fusion toxin of the present invention

1. Construction of Cell Lines

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

2. Preparation of Immunotoxin

The monoclonal antibody (scFv) provided by the present invention is fused with Pseudomonas exotoxin PE24 to construct an immunotoxin, and an additional 6 histidines (6x His) are added to the N-terminal of the immunotoxin to facilitate the immunotoxin purification. Escherichia coli HB2151 strain was used for the expression of immunotoxin. The strains were inoculated into 2L of 2YT medium, and the OD ₆₀₀ reached 0.9 after culturing at 37°C for 4-5h, induced by adding 1mM IPTG, and expressed at 30°C for 10h. The cells were collected, crushed by high pressure, centrifuged, and the supernatant was filtered and purified by a nickel column.

3. Cytotoxicity of immunotoxins

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

The results are shown in Figure 6. The immunotoxin has strong cytotoxic activity on GPC3-positive G1LG, Hep3BLG, HepG2LG, and Huh7LG cells, but does not kill GPC3-negative A431LG, indicating that the immunotoxin has high selectivity for GPC3-positive tumor cells. The activity intensity of immunotoxin is expressed by IC50 value, and the measured IC50 value is shown in Table 5. Most of the immunotoxins provided by the present invention have stronger activity than HN3-PE24, such as J80A-PE24, A43-PE24, C46-PE24, J80B-PE24, A5-PE24, G15-PE24, I82-PE24, A18 -PE24, H49-PE24, among which J80A-PE24 has the strongest killing activity, and the IC50 value of Hep3BLG reaches 7.974ng/ml, while the IC50 of HN3-PE24 is as high as 109ng/ml, which shows that J80A-PE24 has far more antitumor activity Stronger than HN3-PE24.

Table 5. IC50 values of killing activity of monoclonal antibody-based immunotoxins on GPC3-positive tumor cells

Example 6 In vivo tumor inhibitory activity of immunotoxin J80A

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

sequence listing

<110> Huazhong Agricultural University

<120> Antibodies Recognizing Multiple Different Epitopes of Glypican 3 and Their Applications

<160> 28

<170> SIPOSequenceListing 1.0

<210> 1

<211> 126

<212> PRT

<213> Artificial Sequence

<400> 1

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Asn Arg Tyr

20 25 30

Cys Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Asp Ser Asp Ser Gly Gly Thr Asp Tyr Gly Ala Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg

65 70 75 80

Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Lys Ser Ala Tyr Gly Gly Trp Cys Gly Ser Arg Val Ala Pro Trp

100 105 110

Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser

115 120 125

<210> 2

<211> 107

<212> PRT

<213> Artificial Sequence

<400> 2

Gln Ala Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Gly Trp Phe Gln

20 25 30

Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp Asn Asp

35 40 45

Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Leu Ser Gly

50 55 60

Ser Thr Gly Thr Leu Thr Ile Thr Gly Val Arg Ala Glu Asp Glu Ala

65 70 75 80

Val Tyr Tyr Cys Gly Ser Ser Glu Asn Ser Tyr Val Gly His Val Ala

85 90 95

Ile Phe Gly Ala Gly Thr Thr Leu Thr Val Leu

100 105

<210> 3

<211> 127

<212> PRT

<213> Artificial Sequence

<400> 3

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Arg Thr Asn

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Asn Ser Ala Gly Gly Trp Thr Gly Tyr Gly Pro Ala Val

50 55 60

Lys Gly His Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg

65 70 75 80

Leu Gln Leu Thr Asn Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Lys Ser Ala Gly Gly Trp Cys Asp Ser Gly Asp Tyr Gly Ala Gly

100 105 110

Cys Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser

115 120 125

<210> 4

<211> 105

<212> PRT

<213> Artificial Sequence

<400> 4

Gln Ala Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Leu Gly Gly

1 5 10 15

Thr Val Lys Ile Thr Cys Ser Gly Gly Ser Gly Ser Tyr Gly Trp Phe

20 25 30

Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp Asn

35 40 45

Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Thr Ser

50 55 60

Gly Ser Thr Gly Thr Leu Thr Ile Thr Gly Val Gln Ala Asp Asp Glu

65 70 75 80

Ala Val Tyr Phe Cys Gly Ser Thr Asp Ser Ser Tyr Val Gly Ile Phe

85 90 95

Gly Ala Gly Thr Thr Leu Thr Val Leu

100 105

<210> 5

<211> 128

<212> PRT

<213> Artificial Sequence

<400> 5

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Phe Gly Gly Ser His Thr Gly Tyr Ala Pro Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Thr Arg Asp Asn Gly Gln Ser Thr Met Arg

65 70 75 80

Leu Gln Leu Ser Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys

85 90 95

Thr Arg Gly Gly Gly Tyr Phe Cys Thr Tyr Gly Trp Cys Pro Gly Gly

100 105 110

Gly Glu Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser

115 120 125

<210> 6

<211> 107

<212> PRT

<213> Artificial Sequence

<400> 6

Gln Ala Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Leu Gly Gly

1 5 10 15

Thr Val Lys Ile Thr Cys Ser Gly Gly Ser Gly Ser Tyr Gly Trp Phe

20 25 30

Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp Asn

35 40 45

Asp Gln Arg Pro Ser Asn Ile Pro Ser Arg Phe Ser Gly Ser Lys Ser

50 55 60

Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Val Asp Asp Glu

65 70 75 80

Ala Val Tyr Tyr Cys Gly Ser Gly Asp Ser Ser Ser Gly Asp Ser Gly

85 90 95

Val Phe Gly Ala Gly Thr Thr Leu Thr Val Leu

100 105

<210> 7

<211> 122

<212> PRT

<213> Artificial Sequence

<400> 7

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Gly Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Lys Asn Asp Gly Ser Phe Ala Leu Tyr Gly Ala Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Ser Gly Gln Ser Thr Val Arg

65 70 75 80

Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Phe Cys

85 90 95

Ala Lys Ser Ala Gly Val Val Gly Gly Pro Asp Asp Ile Asp Ala Trp

100 105 110

Gly His Gly Thr Glu Val Ile Val Ser Ser

115 120

<210> 8

<211> 105

<212> PRT

<213> Artificial Sequence

<400> 8

Gln Ala Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu

1 5 10 15

Thr Val Arg Ile Thr Cys Ser Gly Ser Ser Tyr Ser Tyr Tyr Gly Trp

20 25 30

Tyr Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp

35 40 45

Asn Asp Asn Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Thr

50 55 60

Ser Gly Ser Thr Gly Thr Leu Thr Ile Thr Gly Val Arg Ala Glu Asp

65 70 75 80

Glu Ala Val Tyr Tyr Cys Gly Asn Tyr Gly Ser Ser Ala Gly Ile Phe

85 90 95

Gly Ala Gly Thr Thr Leu Thr Val Leu

100 105

<210> 9

<211> 126

<212> PRT

<213> Artificial Sequence

<400> 9

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Gly Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Asp Phe Ser Ser Tyr

20 25 30

Gly Met Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Asp Gly Ala Gly Ser Asn Thr Tyr Tyr Ala Thr Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg

65 70 75 80

Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Lys Glu Ser Cys Ile Gly Ser Gly Cys Gly Phe Leu Val Gly Arg

100 105 110

Ile Asp Ala Trp Gly Gln Gly Thr Glu Val Ile Val Ser Ser

115 120 125

<210> 10

<211> 107

<212> PRT

<213> Artificial Sequence

<400> 10

Gln Ala Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Leu Gly Gly

1 5 10 15

Thr Val Lys Ile Thr Cys Ser Gly Ser Ser Gly Ser Tyr Gly Trp Tyr

20 25 30

Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Tyr Asn

35 40 45

Asp Lys Arg Pro Ser Asn Ile Pro Ser Arg Phe Ser Gly Ser Thr Ser

50 55 60

Gly Ser Thr Gly Thr Leu Thr Ile Thr Gly Val Gln Ala Glu Asp Glu

65 70 75 80

Ala Val Tyr Phe Cys Gly Ser Tyr Asp Ser Ser Ala Gly Tyr Val Gly

85 90 95

Ile Phe Gly Ala Gly Thr Thr Leu Thr Val Leu

100 105

<210> 11

<211> 125

<212> PRT

<213> Artificial Sequence

<400> 11

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Tyr Asn Asp Gly Ser Thr Pro Asn Tyr Gly Ser Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg

65 70 75 80

Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Lys Thr Thr Gly Gly Phe Ser Tyr Tyr Tyr Asp Ala Gly Asp Ile

100 105 110

Asp Thr Trp Gly His Gly Thr Glu Val Ile Val Ser Ser

115 120 125

<210> 12

<211> 105

<212> PRT

<213> Artificial Sequence

<400> 12

Gln Ala Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Thr Cys Ser Gly Gly Ser Tyr Ser Tyr Gly Trp Phe

20 25 30

Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp Asn

35 40 45

Thr Asn Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Lys Ser

50 55 60

Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Glu Asp Glu

65 70 75 80

Ala Val Tyr Phe Cys Gly Ser Ala Asp Ser Ser Tyr Ala Gly Ile Phe

85 90 95

Gly Ala Gly Thr Thr Leu Thr Val Leu

100 105

<210> 13

<211> 128

<212> PRT

<213> Artificial Sequence

<400> 13

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val

35 40 45

Ala Thr Ile Ser Phe Gly Gly Ser His Thr Gly Tyr Ala Pro Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg

65 70 75 80

Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys

85 90 95

Thr Arg Gly Gly Gly Tyr Phe Cys Ser Tyr Gly Trp Cys Pro Gly Gly

100 105 110

Gly Glu Ile Asp Thr Trp Gly His Gly Thr Glu Val Ile Val Ser Ser

115 120 125

<210> 14

<211> 104

<212> PRT

<213> Artificial Sequence

<400> 14

Gln Ala Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Thr Cys Ser Gly Ser Ser Gly Ser Tyr Gly Trp Tyr

20 25 30

Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp Asn

35 40 45

Asp Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Glu Ser

50 55 60

Gly Ser Met Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Lys Asp Glu

65 70 75 80

Ala Val Tyr Phe Cys Gly Ser Arg Asp Asn Ser Ala Ala Ile Phe Gly

85 90 95

Ala Gly Thr Thr Leu Thr Val Leu

100

<210> 15

<211> 128

<212> PRT

<213> Artificial Sequence

<400> 15

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Ser Phe Ser Ser Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Gly Thr Asp Ser Ser Phe Pro Asn Tyr Gly Ala Ala Val

50 55 60

Lys Gly Arg Ala Ile Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg

65 70 75 80

Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Lys Ser Val Tyr Gly Gly Trp Cys Thr Ser Gly Ser Cys Ser Gly

100 105 110

Arg Arg Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser

115 120 125

<210> 16

<211> 108

<212> PRT

<213> Artificial Sequence

<400> 16

Gln Ala Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Leu Gly Glu

1 5 10 15

Thr Val Lys Ile Thr Cys Ser Gly Ser Ser Gly Ser Tyr Gly Trp Tyr

20 25 30

Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp Asn

35 40 45

Thr Asp Arg Pro Ser Asn Ile Pro Ser Arg Phe Ser Gly Ser Lys Ser

50 55 60

Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Glu Asp Glu

65 70 75 80

Ala Val Tyr Phe Cys Gly Ser Arg Asp Ser Ser Gly Thr Gly Tyr Val

85 90 95

Gly Ile Phe Gly Ala Gly Thr Thr Leu Thr Val Leu

100 105

<210> 17

<211> 119

<212> PRT

<213> Artificial Sequence

<400> 17

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Gly Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Asp Phe Ser Asp Tyr

20 25 30

Gly Met Ser Trp Met Arg Gln Ala Pro Asp Lys Gly Leu Glu Tyr Val

35 40 45

Ala Ala Ile Ser Asn Asp Gly Ser Ser Thr Leu Tyr Gly Ala Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg

65 70 75 80

Leu His Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Phe Cys

85 90 95

Ala Arg Ser Gly Gly Gly Ala Gly Asp Ile Asp Ala Trp Gly Arg Gly

100 105 110

Thr Glu Val Ile Val Ser Ser

115

<210> 18

<211> 114

<212> PRT

<213> Artificial Sequence

<400> 18

Gln Ala Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Thr Cys Ser Gly Gly Gly Ser Ser Ser Tyr Gly Tyr

20 25 30

Asn Tyr Gly Trp Tyr Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Thr

35 40 45

Val Ile Tyr Ser Asn Asp Asn Arg Pro Ser Asn Ile Pro Ser Arg Phe

50 55 60

Ser Gly Ser Ala Ser Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val

65 70 75 80

Gln Val Glu Asp Glu Ala Val Tyr Tyr Cys Gly Ser Tyr Glu Gly Ser

85 90 95

Ile Asp Gly Arg Tyr Val Gly Val Phe Glu Ala Gly Thr Thr Leu Thr

100 105 110

Val Leu

<210> 19

<211> 126

<212> PRT

<213> Artificial Sequence

<400> 19

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser His

20 25 30

Gly Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Thr Thr Gly Ser Tyr Thr Gly Tyr Gly Ser Ala Val Asp

50 55 60

Gly Arg Ala Thr Ile Ser Arg Asp Asp Gly Gln Ser Thr Val Arg Leu

65 70 75 80

Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Phe Cys Ala

85 90 95

Lys Ser Thr Asp Gly Gly Cys Ile Ser Tyr Gly Ile Cys Pro Asp Glu

100 105 110

Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser

115 120 125

<210> 20

<211> 105

<212> PRT

<213> Artificial Sequence

<400> 20

Gln Ala Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Thr Cys Ser Gly Gly Gly Asn Trp Tyr Gly Trp Tyr

20 25 30

Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp Asn

35 40 45

Thr Asn Arg Pro Ser Asn Ile Pro Ser Arg Phe Ser Gly Ser Thr Ser

50 55 60

Gly Ser Thr Ser Thr Leu Thr Ile Thr Gly Val Gln Ala Asp Asp Glu

65 70 75 80

Ala Ile Tyr Tyr Cys Gly Ser Tyr Asp Ser Thr Tyr Asp Asp Ile Phe

85 90 95

Gly Ala Gly Thr Thr Leu Thr Val Leu

100 105

<210> 21

<211> 128

<212> PRT

<213> Artificial Sequence

<400> 21

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Ala Leu Ser Leu Val Cys Lys Gly Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Ser Val

35 40 45

Ala Gln Ile Thr Ser Thr Gly Ser Ser Thr Tyr Tyr Gly Ser Ala Val

50 55 60

Asp Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg

65 70 75 80

Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys

85 90 95

Ala Lys Thr Thr Gly Ser Gly Ser Cys Thr Tyr Gly Trp Asn Cys Ala

100 105 110

Gly Asn Ile Asp Ser Trp Gly His Gly Thr Glu Val Ile Val Ser Ser

115 120 125

<210> 22

<211> 107

<212> PRT

<213> Artificial Sequence

<400> 22

Gln Ala Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Leu Gly Gly

1 5 10 15

Thr Val Glu Ile Thr Cys Ser Gly Asp Asp Asn Tyr Asp Tyr Tyr Gly

20 25 30

Trp Phe Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr

35 40 45

Glu Ser Asn Lys Arg Pro Ser Asn Ile Pro Leu Arg Phe Ser Gly Ser

50 55 60

Thr Ser Gly Ser Thr Asn Thr Leu Thr Ile Thr Gly Val Gln Ala Asp

65 70 75 80

Asp Glu Ala Val Tyr Phe Cys Gly Thr Ser Asp Asn Gly Gly Val Gly

85 90 95

Thr Phe Gly Ala Gly Thr Thr Leu Thr Val Leu

100 105

<210> 23

<211> 126

<212> PRT

<213> Artificial Sequence

<400> 23

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Asn Thr Tyr

20 25 30

Cys Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Asp Ser Asp Ser Gly Gly Thr Asp Tyr Gly Ala Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asp Gly Gln Ser Thr Val Arg

65 70 75 80

Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Lys Ser Gly Tyr Gly Gly Trp Cys Gly Gly Arg Asp Val Ala Trp

100 105 110

Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser

115 120 125

<210> 24

<211> 106

<212> PRT

<213> Artificial Sequence

<400> 24

Gln Ala Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Gly Trp Tyr Gln

20 25 30

Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp Asn Thr

35 40 45

Asn Arg Pro Ser Asn Ile Pro Ser Arg Phe Ser Gly Ser Leu Ser Gly

50 55 60

Ser Thr Ser Thr Leu Thr Ile Thr Gly Val Gln Ala Asp Asp Glu Ala

65 70 75 80

Val Tyr Phe Cys Gly Ser Tyr Asp Ser Ser Ala Asp Tyr Val Gly Ile

85 90 95

Phe Gly Ala Gly Thr Thr Leu Thr Val Leu

100 105

<210> 25

<211> 125

<212> PRT

<213> Artificial Sequence

<400> 25

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Ala Leu Ser Leu Val Cys Lys Gly Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val

35 40 45

Ala Asp Ile Ser Ser Thr Gly Ser Ser Thr Tyr Tyr Ala Leu Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg

65 70 75 80

Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Phe Cys

85 90 95

Ala Lys Ser Gly Tyr Val Gly Ser Trp Tyr Ile Asp Ser Pro Trp Ile

100 105 110

Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser

115 120 125

<210> 26

<211> 105

<212> PRT

<213> Artificial Sequence

<400> 26

Gln Ala Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Thr Cys Ser Gly Ser Ser Ser Tyr Ser Gly Trp Tyr

20 25 30

Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp Asn

35 40 45

Thr Lys Arg Pro Ser Asn Ile Pro Ser Arg Phe Ser Gly Ser Lys Ser

50 55 60

Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Ile Gln Ala Asp Asp Glu

65 70 75 80

Ala Val Tyr Tyr Cys Gly Ser Thr Asp Asn Asn Tyr Asp Gly Ile Phe

85 90 95

Gly Ala Gly Thr Thr Leu Thr Val Leu

100 105

<210> 27

<211> 126

<212> PRT

<213> Artificial Sequence

<400> 27

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Asn Arg Asn

20 25 30

Cys Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Asp Ser Asp Ser Gly Gly Thr Glu Tyr Gly Ala Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asp Gly Gln Ser Thr Leu Arg

65 70 75 80

Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys

85 90 95

Ala Lys Ser Gly Tyr Gly Gly Trp Cys Gly Ser Arg Ser Ala Ala Trp

100 105 110

Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser

115 120 125

<210> 28

<211> 106

<212> PRT

<213> Artificial Sequence

<400> 28

Gln Ala Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Leu Gly Glu

1 5 10 15

Thr Val Lys Ile Thr Cys Ser Gly Ser Ser Gly Asn Tyr Gly Trp Tyr

20 25 30

Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Ser Ser

35 40 45

Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ala Leu Ser

50 55 60

Gly Ser Thr Ala Thr Leu Ser Ile Thr Gly Val Arg Ala Asp Asp Glu

65 70 75 80

Ala Val Tyr Phe Cys Gly Gly Tyr Asp Gly Ile Asn Arg Ala Ala Ile

85 90 95

Phe Gly Ala Gly Thr Thr Leu Thr Val Leu

100 105

Claims (15)

1. The monoclonal antibody of Glypican 3, comprising a heavy chain variable region and a light chain variable region, characterized in that: (1) The heavy chain variable region of the antibody has the complementarity determining regions shown in amino acid residues 26-35, 50-66 and 97-115 of SEQ ID NO: 1; the light chain of the antibody can be The variable region has the complementarity determining regions shown in amino acid residues 23-29, 46-52 and 85-97 of SEQ ID NO:2; Or, (2) the variable region of the heavy chain of the antibody has the complementarity determining regions shown in amino acid residues 26-35, 50-66 and 97-116 of SEQ ID NO: 3; The chain variable region has the complementarity determining regions shown in amino acid residues 23-30, 47-53 and 86-95 of SEQ ID NO:4; Or, (3) the variable region of the heavy chain of the antibody has the complementarity determining regions shown in amino acid residues 26-35, 50-66 and 97-117 of SEQ ID NO: 5; The chain variable region has the complementarity determining regions shown in amino acid residues 23-30, 47-53 and 86-97 of SEQ ID NO:6; Alternatively, (4) the variable region of the heavy chain of the antibody has the complementarity determining regions shown in amino acid residues 26-35, 50-66 and 97-111 of SEQ ID NO: 7; The chain variable region has the complementarity determining regions shown in amino acid residues 23-31, 48-54 and 87-95 of SEQ ID NO:8; Or, (5) the variable region of the heavy chain of the antibody has the complementarity determining regions shown in amino acid residues 26-35, 50-66 and 97-115 of SEQ ID NO: 9; The chain variable region has the complementarity determining regions shown in amino acid residues 23-30, 47-53 and 86-97 of SEQ ID NO: 10; Alternatively, (6) the variable region of the heavy chain of the antibody has the complementarity determining regions shown in amino acid residues 26-35, 50-66 and 97-114 of SEQ ID NO: 11; The chain variable region has the complementarity determining regions shown in amino acid residues 23-30, 47-53 and 86-95 of SEQ ID NO: 12; Alternatively, (7) the variable region of the heavy chain of the antibody has the complementarity determining regions shown in amino acid residues 26-35, 50-66 and 97-117 of SEQ ID NO: 13; The chain variable region has the complementarity determining regions shown in amino acid residues 23-30, 47-53 and 86-94 of SEQ ID NO: 14; Alternatively, (8) the variable region of the heavy chain of the antibody has the complementarity determining regions shown in amino acid residues 26-35, 50-66 and 97-117 of SEQ ID NO: 15; The chain variable region has the complementarity determining regions shown in amino acid residues 23-30, 47-53 and 86-98 of SEQ ID NO: 16; Or, (9) the variable region of the heavy chain of the antibody has the complementarity determining regions shown in amino acid residues 26-35, 50-66 and 97-108 of SEQ ID NO: 17; The chain variable region has the complementarity determining regions shown in amino acid residues 23-35, 52-58 and 91-104 of SEQ ID NO: 18; Alternatively, (10) the variable region of the heavy chain of the antibody has the complementarity determining regions shown in amino acid residues 26-35, 50-65 and 96-115 of SEQ ID NO: 19; The chain variable region has the complementarity determining regions shown in amino acid residues 23-30, 47-53 and 86-95 of SEQ ID NO: 20; Alternatively, (11) the variable region of the heavy chain of the antibody has the complementarity determining regions shown in amino acid residues 26-35, 50-66 and 97-117 of SEQ ID NO: 21; The chain variable region has the complementarity determining regions shown in amino acid residues 23-32, 49-55 and 88-97 of SEQ ID NO: 22; Or, (12) the variable region of the heavy chain of the antibody has the complementarity determining regions shown in amino acid residues 26-35, 50-66 and 97-115 of SEQ ID NO: 23; The chain variable region has the complementarity determining regions shown in amino acid residues 23-29, 46-52 and 85-96 of SEQ ID NO: 24; Alternatively, (13) the variable region of the heavy chain of the antibody has the complementarity determining regions shown in amino acid residues 26-35, 50-66 and 97-114 of SEQ ID NO: 25; The chain variable region has the complementarity determining regions shown in amino acid residues 23-30, 47-53 and 86-95 of SEQ ID NO: 26; Alternatively, (14) the variable region of the heavy chain of the antibody has the complementarity determining regions shown in amino acid residues 26-35, 50-66 and 97-115 of SEQ ID NO: 27; The chain variable region has the complementarity determining regions shown in amino acid residues 23-30, 47-53 and 86-96 of SEQ ID NO:28. 2. monoclonal antibody according to claim 1, is characterized in that: (1) The heavy chain variable region amino acid sequence of the antibody is shown in SEQ ID NO: 1, and the light chain variable amino acid sequence is shown in SEQ ID NO: 2; Or, (2) the heavy chain variable region amino acid sequence of the antibody is shown in SEQ ID NO:3, and the light chain variable amino acid sequence is shown in SEQ ID NO:4; Or, (3) the heavy chain variable region amino acid sequence of the antibody is shown in SEQ ID NO:5, and the light chain variable amino acid sequence is shown in SEQ ID NO:6; Or, (4) the heavy chain variable region amino acid sequence of the antibody is shown in SEQ ID NO:7, and the light chain variable amino acid sequence is shown in SEQ ID NO:8; Or, (5) the heavy chain variable region amino acid sequence of the antibody is shown in SEQ ID NO:9, and the light chain variable amino acid sequence is shown in SEQ ID NO:10; Or, (6) the heavy chain variable region amino acid sequence of the antibody is shown in SEQ ID NO: 11, and the light chain variable amino acid sequence is shown in SEQ ID NO: 12; Or, (7) the heavy chain variable region amino acid sequence of the antibody is shown in SEQ ID NO: 13, and the light chain variable amino acid sequence is shown in SEQ ID NO: 14; Or, (8) the heavy chain variable region amino acid sequence of the antibody is shown in SEQ ID NO: 15, and the light chain variable amino acid sequence is shown in SEQ ID NO: 16; Or, (9) the heavy chain variable region amino acid sequence of the antibody is shown in SEQ ID NO: 17, and the light chain variable amino acid sequence is shown in SEQ ID NO: 18; Alternatively, (10) the heavy chain variable region amino acid sequence of the antibody is shown in SEQ ID NO: 19, and the light chain variable amino acid sequence is shown in SEQ ID NO: 20; Alternatively, (11) the heavy chain variable region amino acid sequence of the antibody is shown in SEQ ID NO: 21, and the light chain variable amino acid sequence is shown in SEQ ID NO: 22; Or, (12) the heavy chain variable region amino acid sequence of the antibody is shown in SEQ ID NO: 23, and the light chain variable amino acid sequence is shown in SEQ ID NO: 24; Alternatively, (13) the heavy chain variable region amino acid sequence of the antibody is shown in SEQ ID NO: 25, and the light chain variable amino acid sequence is shown in SEQ ID NO: 26; Alternatively, the heavy chain variable region amino acid sequence of the antibody described in (14) is shown in SEQ ID NO: 27, and the light chain variable amino acid sequence is shown in SEQ ID NO: 28. 3. The monoclonal antibody according to claim 1, characterized in that the antibody is a single-chain antibody, a diabody or a chimeric antibody. 4. The monoclonal antibody of claim 1, wherein the antibody is a VH single domain antibody, a Fab fragment, a Fab' fragment, an F(ab)'2 fragment, a single chain variable fragment (scFv) or Disulfide stabilized variable fragments (dsFv). 5. A recombinant protein, characterized in that, the recombinant protein comprises the monoclonal antibody of any one of claims 1-4 and a tag sequence that assists in 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 of claim 6, wherein the effector molecule is a toxin, a drug or a detectable label. 8. The immunoconjugate according to claim 7, wherein the toxin is a protein with cytotoxic or antitumor activity, including but not limited to acacia or its variant, ricin or its A variant, 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, wherein the drug is a compound with cytotoxicity or antitumor activity, including but not limited to vinblastine, daunomycin, Monomethyl auristatin E, Monomethyl auristatin F, Pyrrolobenzodiazepine dimer, N2'-deacetyl-N2'-(3-Mercapto-1-oxopropyl)-Maytansine. 10. The immunoconjugate according to claim 7, wherein the detectable label is capable of being detected by an isotope analyzer, a microplate reader, a bioluminescence detector, a chemiluminescence detector, and an electrochemiluminescence detector , Fluorescence analysis instrument detection, or naked-eye visualization of substances, these substances include but are not limited to radioisotopes (such as 125I, 131I, 32P, 14C, 3H and 35S), 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), bioluminescence Labels (such as luciferase), fluorescent compounds (such as luciferin, fluorescein isothiocyanate, rhodamine, 5-dimethylamino-1-naphthalenesulfonyl chloride, phycoerythrin, fluorescent dyes Cy3, Cy5, Rare earth inorganic light-emitting materials, quantum dots, etc.), biotin, magnetic reagents (eg gadolinium), electrochemiluminescence reagents (eg ruthenium terpyridine), colloidal gold. 11. 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, or the immunization according to any one of claims 6-10 conjugate. 12. A vector comprising the polynucleotide of claim 11. 13. A genetically engineered host cell, characterized by comprising the vector of claim 12, or the polynucleotide of claim 11 integrated into the genome. 14. A pharmaceutical composition, characterized in that it comprises the antibody according to any one of claims 1-4, the recombinant protein according to claim 5, and the immunoconjugate according to any one of claims 6-10 , one or more of the polynucleotide of claim 11 , the vector of claim 12 or the genetically engineered host cell of claim 13 . 15. The antibody of any one of claims 1-4, the recombinant protein of claim 5, the immunoconjugate of any one of claims 6-10, the polynucleotide of claim 11 , the application of the vector of claim 12 or the genetically engineered host cell of claim 13 in the preparation of therapeutic drugs or diagnostic reagents for autoimmune diseases, viral infections or cancer.

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