CN114685668B

CN114685668B - Human GPC3 monoclonal antibody and conjugate thereof

Info

Publication number: CN114685668B
Application number: CN202011589109.5A
Authority: CN
Inventors: 孙召朋; 姚兵; 唐昭娜; 王欣; 袁灿; 王艳翠; 彭秀娥; 王翠; 刘伯宁; 惠希武
Original assignee: Jushi Biopharmaceutical Co ltd
Current assignee: Jushi Biopharmaceutical Co ltd
Priority date: 2020-12-28
Filing date: 2020-12-28
Publication date: 2023-10-13
Anticipated expiration: 2040-12-28
Also published as: CN114685668A

Abstract

The present application relates to an antibody to GPC3 and related conjugates that can be effectively used for the treatment and/or prevention of GPC 3-positive tumors, including, but not limited to, cancers typified by liver cancer.

Description

Human GPC3 monoclonal antibody and conjugate thereof

Technical Field

The application relates to the field of biological medicine, in particular to an antibody drug conjugate/conjugate for resisting Gypican3 and application thereof in the aspect of treating tumors.

Background

Hepatocellular carcinoma (hepatocellular carcinoma, HCC) is abbreviated as liver cancer. Liver cancer is one of major diseases threatening human health, and the incidence rate is 6 th and the death rate is 2 nd of malignant tumors in the world. About 81 ten thousand liver cancer patients die each year worldwide, and the number of deaths in China is more than 50%. At present, liver cancer has become a serious problem for the health of the whole population.

Since early clinical symptoms of liver cancer are not obvious, most patients are found to be middle and late stages, and the optimal treatment time is missed, which is also a main cause of poor prognosis and cancer recurrence. The liver cancer treatment modes are numerous due to different tumor stages, including hepatectomy, liver transplantation, ablation, TACE, targeted drug treatment and the like.

For patients with advanced liver cancer, systemic treatment methods comprise systemic chemotherapy, molecular targeted therapy, immunotherapy and the like, and the life time of the patients with middle and advanced liver cancer can be prolonged and the life quality of the patients can be improved through the therapy.

(1) Systemic chemotherapy

Traditional cytotoxic drugs have too great damage to the liver, may exacerbate cirrhosis and hepatitis, and may not produce significant therapeutic benefit to patients.

Arsenic trioxide has a certain treatment effect on middle and late stage liver cancer, and can improve the survival quality of patients.

(2) Molecular targeted therapy

Sorafenib and regorafenib are molecular targeted drugs approved for the treatment of advanced liver cancer. Two large international multicenter phase III clinical trials fully prove that sorafenib has certain survival benefits for advanced liver cancer in different national regions and different liver disease backgrounds. The most common adverse effects are diarrhea, weight loss, hand-foot syndrome, rash, myocardial ischemia, hypertension, etc.

(3) Immunotherapy

In recent years, the rising immunotherapy provides a new method for treating the hepatocellular carcinoma, and the research on a new strategy for treating the liver cancer more effectively is a current research hotspot at home and abroad.

Currently, PD-1 mab is recommended by the CSCO guidelines as a second line treatment regimen for advanced liver cancer. The PD-1 has better treatment effect by combining the treatment methods of bevacizumab, lenvatinib, apatinib and the like. In addition, cellular immunotherapy and methods of treatment with immunomodulators (including thymosin alpha 1, interferon alpha, etc.) also have certain antitumor effects.

Currently, the common curative treatment modes of liver cancer are surgical excision, liver transplantation and local ablation, however, most liver cancer patients have advanced stage when they find the liver cancer, and the possibility of curative treatment is lost.

Based on the above circumstances, there is an urgent need for more technical means and therapeutic drugs that can effectively treat liver cancer and have acceptable toxic and side effects.

Since 1996 Glypican (GPC) was discovered, its mechanism of action in liver cancer has attracted widespread attention to scholars both at home and abroad. Several studies in recent years have shown that phosphatidylinositol protein 3 (Glypican-3, GPC3, hereinafter referred to as GPC 3) is a currently accepted early marker for liver cancer diagnosis and a target for immunotherapy.

GPC3 protein is a member of the heparan sulfate glycoprotein (Heparan sulfate proteoglycan, HSPG) family, a class of proteoglycans distributed on the cell membrane surface that are anchored to the cell outer membrane by glycosyl phosphatidylinositol (GlycosyIphosphatidyIinositoI, GPI). GPC3 is not expressed or expressed in adult tissues, is highly expressed in embryonic hepatocytes, but is not expressed in hepatocytes 3 weeks after birth. GPC3 is specifically highly expressed in liver cancer tissues, whereas cholangiocellular carcinoma and normal liver tissues are not. GPC3 can reduce the adhesion between collagen type I fibronectin of HCC cells under pathological conditions, and enhance migration and invasion ability of cells.

GPC3 has obvious sensitivity and specificity to liver cancer diagnosis, and can be used as a new target for liver cancer treatment. The antibody medicine has high specificity, low side effect and good treatment effect, and becomes a new pet for each big medicine enterprise, so that the development of the antibody medicine targeting GPC3 has important significance. At present, at least 14 items for targeting GPC3 at home and abroad exist, and most items are monoclonal antibodies, double antibodies, vaccines and CAR-T for targeting GPC3. The fastest development progress in all projects is Codrituzumab (GC-33) which is jointly developed by Chinese and foreign pharmacy and Roche, and GC33 is the first antibody targeting GPC3, is also the GPC3 antibody with the largest clinical trial registered at present, is in the clinical second stage at present, and the preliminary results of clinical study show that: codrituzumab showed no clinical benefit in this previously treated HCC population, thus it was seen that the monoclonal antibody drug GC33 (Roche) phase II targeting GPC3 had poor clinical efficacy, and there was no significant difference in the overall survival and progression-free survival of the drug and control groups.

The first clinical trial results of CAR-GPC3-T clinical trials for GPC3 targets for the treatment of refractory recurrent hepatocellular carcinoma using CAR-T cells were published by keji biology company (CARsgen Therapeutics) at the American Society of Clinical Oncology (ASCO) 2017, at day 6 and 5, worldwide, showing primary efficacy and good safety. However, this approach to CAR-T treatment has inherent drawbacks such as: CAR-T is difficult to use effectively for solid tumor treatment, mainly because: firstly, the specific target of the solid tumor is difficult to select, the CAR-T adverse reaction is related to the selection of the target, if the target is improperly selected, the off-target effect can occur, and more adverse reactions are generated; secondly, CAR-T is an activated T cell, which needs to act inside the tumor tissue, however, only 1% -2% of T cells infused intravenously can reach the deep part of the tumor tissue, which is insufficient to exert an effective antitumor effect. Finally, after the CAR-T enters the deep part of tumor tissues, multiple barriers in the tumor immune microenvironment need to be overcome, so that the anti-tumor effect can be continuously exerted.

Based on the above shortcomings of the prior art, the present application has conducted the following studies:

1. obtaining high affinity anti-GPC 3 monoclonal antibody which can be used as candidate antibody for preparing subsequent antibody drug conjugate/conjugate;

2. based on GPC3 monoclonal antibody, 16C8-8E6 ADC is prepared, and experimental data show that the preparation method has remarkable in vitro growth inhibition effect on liver cancer cells HepG2 and Hep3 b;

3. the heterologous scFv gene is fused with the curtailed toxin gene using DNA recombination techniques and cloned into a suitable vector, translating the single chain chimeric protein in e.coli, thus also referred to as a genetically engineered immunotoxin (one of the conjugates of the application). The immunotoxin is cloned into a proper expression vector, so that the immunotoxin is efficiently transcribed and translated in escherichia coli, and the immunotoxin has the characteristics of short expression period, simplicity and convenience in operation, low cost, easiness in control and mass production. The monoclonal antibody with high GPC3 affinity or the functional fragment thereof obtained by the application carries toxin to tumor tissues with specific antigen, binds with antigen on target cells, internalizes the toxin, deactivates ribosome, inhibits protein synthesis, kills cells, and researches show that: the immunotoxins prepared by the present application also have unexpected effects in killing tumor cells.

Disclosure of Invention

In one aspect, the application provides antibodies or functional fragments thereof capable of specifically binding GPC3. In particular, the antibodies comprise a heavy chain and a light chain, wherein

(i) The heavy chain comprises three CDR regions, wherein at least one of the CDR regions has an amino acid sequence as set forth in SEQ ID NO: 1.2 or 3 or a sequence having at least 80% (preferably 85%, 90%, 95%, 98% or 99%) sequence identity thereto; and is also provided with

(ii) The light chain comprises three CDR regions, wherein at least one of the CDR regions has an amino acid sequence as set forth in SEQ ID NO: 4. 5 or 6 or a sequence having at least 80% (preferably 85%, 90%, 95%, 98% or 99%) sequence identity thereto.

In some particular embodiments, the antibody comprises a heavy chain and a light chain, wherein

(i) The heavy chain comprises three CDR regions having the amino acid sequence set forth in SEQ ID NO: 1.2 and 3; and/or

(ii) The light chain comprises three CDR regions having the amino acid sequence set forth in SEQ ID NO: 4. 5 and 6.

In certain embodiments, the antibodies or functional fragments thereof of the application are isolated.

In certain embodiments, the antibodies or functional fragments thereof of the application are monoclonal antibodies.

In certain embodiments, the antibodies or functional fragments thereof of the application may be chimeric antibodies, as well as humanized antibodies, including semi-humanized antibodies and fully-humanized antibodies.

In certain embodiments, the antibodies or functional fragments thereof of the application have ADCC activity.

In certain embodiments, the antibodies or functional fragments thereof of the present application have CDC activity.

In certain embodiments, the antibodies or functional fragments thereof of the application comprise: diabodies, fab fragments, fc fragments, fab 'fragments, F (ab)' ₂ scFv, dsFv, and single domain antibodies; the scFv protein is a fusion protein of a light chain variable region and a heavy chain variable region of immunoglobulin combined through a joint; in the dsFv, the chain is mutated to introduce disulfide bonds to stabilize the linkage of the chain;

in certain embodiments, the antibodies or functional fragments thereof of the application are IgM, igD, igG, igA and IgE, preferably IgG1 antibodies.

In certain embodiments, the antibodies or functional fragments thereof of the application are useful in the treatment or prevention of cancer, wherein the cancer overexpresses GPC3.

In another aspect, the application provides an isolated polynucleotide encoding an antibody of the application.

In yet another aspect, the application provides a combination of isolated polynucleotides comprising a polynucleotide encoding the light chain of an antibody or functional fragment thereof of the application and a polynucleotide encoding the heavy chain of an antibody or functional fragment thereof of the application.

In another aspect, the application provides an expression vector comprising a polynucleotide of the application or a combination of polynucleotides of the application operably linked to regulatory sequences that allow expression of the polypeptide encoded thereby in a host cell or cell-free expression system.

In some embodiments of the application, the host cell may be a prokaryotic host cell, a eukaryotic host cell, or a phage. The prokaryotic host cell can be escherichia coli, bacillus subtilis, streptomycete, proteus mirabilis or the like. The eukaryotic host cell can be fungi such as Pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces, trichoderma, etc., insect cells such as myxoplasma pratensis, etc., plant cells such as tobacco, etc., and mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc. In some embodiments, the host cell of the application is preferably a mammalian cell, more preferably a BHK cell, CHO cell, NSO cell or COS cell.

In another aspect, the application provides an antibody-drug conjugate comprising an antibody of the application or a functional fragment thereof coupled to one or more drugs, preferably the drug is a cytotoxic drug (e.g. an antimetabolite, an antitumor antibiotic, an alkaloid), a toxin, an immunopotentiator or a radioisotope, more preferably the drug is selected from the group consisting of an auristatin derivative, a maytansinoid derivative (e.g. Ansamitocin) or maytansine (Mertansine)), a dolastatin peptide (dolastatin) and derivatives thereof), a camptothecin analogue, a DNA topoisomerase I inhibitor and derivatives thereof; most preferably the drug is selected from MMAE (Monomethyl auristatin E) and MMAF (Monomethyl auristatin F).

In some embodiments, an antibody having the ability to bind GPC3 is covalently linked to the drug moiety through a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker may be cleaved under intracellular conditions. In one embodiment, the linker is hydrolyzable at a pH of less than 5.5. In some embodiments, the linker may be cleaved by an intracellular protease. In one embodiment, the linker is a cathepsin cleavable linker. In some embodiments, the linker comprises a dipeptide. In some embodiments, the dipeptide is valine (Val) -citrulline (Cit); in some embodiments, the antibody is linked to the linker through a cysteine thiol of the antibody. In one embodiment, the antibody is linked to the linker through an amino group of the antibody (in particular an amino group of a glutamine residue).

In certain embodiments, the antibody-drug conjugates of the application have the general formula Ab- (L-U) _n Wherein Ab represents a monoclonal antibody targeting GPC3, L is a linker selected from NH2- (PEG) _m Val-Cit, mc-Val-Cit-pAB (p-amino-benzyl), mc-Val-Cit-pABC (p-amino-benzoyloxy) or Val-Cit, U is a drug selected from MMAE, MMAF, DXD and SN38, and n represents a drug antibody ratio DAR, an integer of 1 to 8, preferably 2, 4, 6, 8, more preferably 2.m is an integer from 1 to 8, preferably 3, 4, 5, 6, 7, 8.

In certain embodiments, the toxins described herein are preferably Pseudomonas exotoxins (Pseudomonas) or variants thereof, most preferably Pseudomonas exotoxin (PE 38).

In certain specific embodiments, PE38 of the present application comprises SEQ ID NO:44, and a polypeptide comprising the amino acid sequence of 44.

In certain specific embodiments, the antibody-drug conjugates of the application comprise a single chain antibody scFv that targets human GPC3, and an immunotoxin PE38 linked to the single chain antibody scFv.

In certain specific embodiments, the single chain antibody scFv of the present application has the structure of a heavy chain variable region-connecting peptide-light chain variable region, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:7, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:8, and a polypeptide sequence shown in the figure; the immunotoxin is PE38KDEL, and has the sequence shown in SEQ ID NO:44, and a polypeptide having the amino acid sequence shown in seq id no.

In certain specific embodiments, the single chain antibody scFv of the application is linked to an immunotoxin by a linker.

In certain specific embodiments, the linker peptides of the application comprise: GGGGSGGGGSGGGGS, GGGGS, GGGGSGGGGS.

In certain embodiments, the linker of the application comprises: ASGG, GGGGS;

in another aspect, the application also relates to a Chimeric Antigen Receptor (CAR) comprising the monoclonal antibody described above.

In another aspect, the application provides a pharmaceutical formulation comprising an antibody-drug conjugate of the application, and a pharmaceutically acceptable diluent, carrier or excipient.

In another aspect, the application provides a medical article (medical preparation) comprising an antibody-drug conjugate of the application. In some embodiments, the medical article is in the form of a kit comprising a container containing the antibody-drug conjugate. In one embodiment, the medical article further comprises printed instructions (printed instructions) for using the article in a method of treating or preventing cancer, particularly GPC 3-expressing cancer.

In another aspect, the present application provides antibody-drug conjugates for the effective treatment and/or prevention of cancers associated with GPC 3-expressing cells, such as gastric cancer, esophageal cancer, pancreatic cancer, non-small cell lung cancer (non small cell lung cancer, NSCLC), ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gall bladder cancer, and metastases thereof, particularly gastric cancer metastasis, peritoneal metastasis, and lymph node metastasis. Particularly preferred such cancer diseases are adenocarcinomas of the stomach, esophagus, pancreatic duct, bile duct, lung and ovary; most preferred is liver cancer.

In another aspect, the application provides the use of the aforementioned conjugate with an antiproliferative agent in the manufacture of a medicament for the treatment of a tumor.

In another aspect, the application provides a pharmaceutical composition comprising the conjugate as described above and an antiproliferative agent.

In certain embodiments, the antiproliferative agent comprises paclitaxel, doxorubicin, docetaxel, cisplatin, carboplatin, iproplatin. In certain embodiments, the anti-proliferative agent may also be an antibody, an antibody drug conjugate, a fusion protein.

Drawings

FIG. 1GPC3 structural diagram

FIG. 2 is a schematic diagram of 16C8-8E6 ADC architecture

FIG. 3 shows the in vitro growth inhibition effect of 16C8-8E6 ADC on hepatoma cells HepG2 and Hep3 b;

fig. 3A: hepG2; fig. 3B: hep3b

FIG. 4 SDS-PAGE detection of purified 8E6-PE38

Annotation:

1.8E6-PE38-28a inclusion body becomes renatured and then passes through Histrap column to be FT

2.8E6-PE38-32a inclusion body becomes renatured and then passes through Histrap column to be FT

3.Marker

FIG. 5 inhibition of in vitro growth of liver cancer cell Hep3b by immunotoxins

FIG. 6 inhibition of in vitro growth of hepatoma cell HepG2 by immunotoxins

FIG. 7 in vivo efficacy test of immunotoxins on Hep3b tumor-bearing mice

FIG. 8 in vivo efficacy test of immunotoxins on HepG2 tumor-bearing mice

Detailed Description

Definition:

unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For definitions and terms in the art, the expert may refer specifically to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

Notwithstanding that the numerical ranges and approximations of the parameters set forth in the broad scope of the application, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. In addition, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a range recited as "1 to 10" should be considered to include any and all subranges between (inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g., 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10. In addition, any reference referred to as being "incorporated herein" should be understood as being incorporated in its entirety.

The terms "pharmaceutical composition," "combination drug" and "pharmaceutical combination" as used herein are used interchangeably to refer to a combination of at least one drug and optionally a pharmaceutically acceptable carrier or adjuvant, combined together to achieve a particular purpose. In certain embodiments, the pharmaceutical compositions comprise combinations that are separated in time and/or space, so long as they are capable of co-acting to achieve the objects of the present application. For example, the components contained in the pharmaceutical composition (e.g., antibodies, nucleic acid molecules, nucleic acid molecule combinations and/or conjugates according to the application) may be administered to a subject in whole or separately. When the components contained in the pharmaceutical composition are separately administered to a subject, the components may be administered to the subject simultaneously or sequentially. Preferably, the pharmaceutically acceptable carrier is water, buffered aqueous solution, isotonic saline solution such as PBS (phosphate buffered saline), dextrose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol or polyalkylene glycols such as polypropylene glycol, triglycerides and the like. The type of pharmaceutically acceptable carrier used depends inter alia on whether the composition according to the application is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The composition according to the application may comprise as additives wetting agents, emulsifiers or buffer substances.

The pharmaceutical composition, vaccine or pharmaceutical formulation according to the application may be administered by any suitable route, for example, orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously.

As used herein, "therapeutically effective amount" or "effective amount" refers to a dosage sufficient to demonstrate the benefit of the subject to which it is administered. The actual amount administered, as well as the rate and time course of administration, will depend on the subject's own condition and severity. The prescription of treatment (e.g., decision on dosage, etc.) is ultimately the responsibility of and depends on the general practitioner and other physician, typically considering the disease being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to the physician.

The term "subject" as used herein refers to a mammal, such as a human, but may also be other animals, such as wild animals (e.g., aigrette, geranium, crane, etc.), domestic animals (e.g., duck, goose, etc.), or laboratory animals (e.g., gorilla, monkey, rat, mouse, rabbit, guinea pig, woodchuck, ground squirrel, etc.).

The term "antibody" refers to an intact antibody and any antigen-binding fragment ("antigen-binding portion") or single chain thereof. "full length antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated VH) and a heavy chain constant region. The heavy chain constant region comprises three domains (domains), CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated VL) and a light chain constant region. The light chain constant region comprises a domain, CL. The VH and VL regions can also be subdivided into regions of high variability, known as Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, known as Framework Regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of an antibody may mediate the binding of an immunoglobulin to a host's tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). Chimeric or humanized antibodies are also encompassed by the antibodies according to the application.

The term "humanized antibody" refers to an antibody that can comprise CDR regions derived from a human antibody, and the remainder of the antibody molecule is derived from a human antibody (or antibodies). Furthermore, to preserve binding affinity, some residues of the backbone (referred to as FR) segment may be modified; the humanized antibody or a fragment thereof according to the present application may be prepared by techniques known to those skilled in the art;

the term "chimeric antibody" refers to antibodies in which the variable region sequences are from one species and the constant region sequences are from another species, e.g., antibodies in which the variable region sequences are from a mouse antibody and the constant region sequences are from a human antibody. The chimeric antibody or a fragment thereof according to the present application can be prepared by using a genetic recombination technique. For example, the chimeric antibodies may be produced by cloning recombinant DNA comprising a promoter and sequences encoding the variable regions of a non-human, particularly murine, monoclonal antibody according to the application, as well as sequences encoding the constant regions of a human antibody. The chimeric antibody of the application encoded by such a recombinant gene will be, for example, a murine-human chimeric, the specificity of which is determined by the variable region derived from murine DNA and the isotype of which is determined by the constant region derived from human DNA. For methods of preparing chimeric antibodies, reference may be made, for example, to verhoeyen et al (BioEssays, 8:74, 1988).

The term "monoclonal antibody" refers to a preparation of antibody molecules having a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The term "functional fragment" as used herein refers in particular to an antibody fragment such as Fv, scFv (sc refers to single chain), fab, F (ab ') 2, fab', scFv-Fc fragment or diabody (diabody), or any fragment that should be able to increase half-life by chemical modification, e.g. addition of a poly (alkylene) glycol such as polyethylene glycol ("pegylation, pegylation") (known as Fv-PEG, scFv-PEG, fab-PEG, F (ab ') 2-PEG or pegylated fragment of Fab' -PEG) ("PEG" is polyethylene glycol), which has GPC3 binding activity). Preferably, the functional fragment will consist of or comprise a partial sequence of the heavy or light chain variable chain of the antibody from which it is derived, which partial sequence is sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, such a functional fragment will comprise a minimum of 5 amino acids, preferably 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence from which it is derived.

In general, for the preparation of monoclonal Antibodies or functional fragments thereof, in particular of murine origin, reference can be made to the techniques described in particular in the handbook "Antibodies" (Harlow and Lane, antibodies: A Laboratory Manual, cold Spring Harbor Laboratory, cold Spring Harbor NY, pp.726, 1988) or to the techniques described by Kohler and Milstein for preparation from hybridoma cells (Nature, 256:495-497, 1975).

In the present application, the anti-GPC 3 monoclonal antibody can be produced in cells of CHO-K1 (ATCC Number: CCL-61, lot No.: 59965043) using methods known in the art.

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

Conservative variants: "conservative" amino acid substitutions are those substitutions that do not substantially affect or reduce the affinity of the protein, such as the affinity of the antibody for GPC3. For example, a human antibody that specifically binds GPC3 can include up to about 1, up to about 2, up to about 5, up to about 10, or up to about 15 conservative substitutions, and specifically binds a GPC3 polypeptide. The term conservative variation also includes the use of a substituted amino acid instead of the unsubstituted parent amino acid, provided that the antibody specifically binds GPC3. Non-conservative substitutions are those that reduce activity or bind to GPC3.

Separating: an "isolated" biological component (e.g., a nucleic acid, protein (including antibodies), or organelle) has been substantially separated or purified from other biological components (i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles) in the environment in which the component naturally occurs (e.g., a cell). Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also includes nucleic acids and proteins prepared by recombinant expression in a host cell and chemically synthesized nucleic acids.

Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma and other sarcomas, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma sebaceous gland carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer and central nervous system tumors (e.g., glioma, astrocytoma, medulloblastoma, cranial glioma (crantiomonoma), ependymoma, pineal tumor, angioblastoma, neuroma, oligodendroglioma, meningioma (mengioma), melanoma, and retinoblastoma).

In some embodiments, the tumor is a liver cancer, such as HCC or hepatoblastoma, melanoma, squamous cell carcinoma, such as lung squamous cell carcinoma, clear cell carcinoma, such as ovarian clear cell carcinoma, thyroid carcinoma, wilms' tumor, neuroblastoma, or testicular germ cell tumor. The hepatocellular carcinoma (HCC) is a primary liver malignancy, typically occurring in patients with inflammatory liver caused by viral hepatitis, hepatotoxin, or cirrhosis (often caused by alcoholism). HCC is also known as malignant hepatoma.

EXAMPLE 1GPC3 monoclonal antibody screening and preparation

1.1 antigen preparation

Full length GPC3 was cloned into pcDNA3.1 (done by Nanjing Style Biotechnology Co., ltd.) and transformed into TOP10 competent cells according to the mRNA and amino acid sequences of GPC3 called in NCBI database (SEQ ID NO: NM-004484.4 and NP-004475.1).

The strain was cultured, and the cells were collected, and plasmid extraction was performed using a plasmid miniprep kit (Tiangen Biochemical technology (Beijing), inc., DP 103). HEK293 and CHO K1 cells are respectively transfected electrically, G418 is subjected to pressure screening, stable transgenic lines of high expression GPC3 are obtained, namely, the monoclonal HEK293-GPC3 is used for immunization, and CHO K1-GPC3 (4E 1 clone) is used for screening detection.

Three 6-8 week old Balb/c mice were selected. Take 1x10 ⁷ Each HEK293-GPC3 cell was emulsified in 200ul of adjuvant (An Bi Qi organism, cat# CDN-A001) and 100ul was injected subcutaneously and 100ul was injected intraperitoneally at four points per mouse. Specific immunization protocols are shown in table 1:

1.2 preparation and screening of hybridoma cells

Lymphocytes were isolated and fused by electrofusion to mouse myeloma cell SP2/0-Ag14 (Nanjac Bai Biotechnology Co., ltd.; cat# CBP 60824), and the resulting hybridomas were used to screen as anti-GPC 3-specific antibodies.

Electrofusion, standing for 10min, transferring into HAT screening culture medium, incubating in 37 deg.C incubator for 1 hr, and spreading 96-well plate (approbamateey 2x 10) ⁴ cells/well), 7 days of liquid exchange Clonacell-HY ^TM Medium E (Stemcell, cat# 03805), 2 days after changing.

Clones screened by using a Cystation cell imaging multifunctional microplate detection system and FACS (flow cytometry fluorescence sorting technology) were named 4-22E10-G7, 1-15C8-2H6, 66B6-1A6, 1E7-1D4, and 16C8-8E6, respectively, and sequenced (by Nanjing Jinsrey biosciences Co., ltd.).

Chimeric antibodies were constructed from the 5 selected cloned variable regions and human IgG1 constant region (trastuzum constant region sequence), HEK293F transiently expressed, protein A was purified in one step, and ELISA and FACS assays were performed as shown in Table 2:

TABLE 2 affinity assay results

Luo Shiyuan grinding

As can be seen from the above table, 16C8-8E6 (hereinafter referred to as 8E 6) has higher affinity than Luo Shiyuan ground antibody GC33, and can be used as a candidate antibody for the preparation of subsequent antibody drug conjugates.

TABLE 3 related sequences of antibodies to which the present application relates

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Example 2 16C8-8E6 ADC preparation and in vitro efficacy

2.1 16C8-8E6 ADC preparation

A volume of LND1002 (purchased by the organism of the genus Linning, structure of which is shown in FIG. 2) was dissolved in DMSO, 10 Xreaction buffer, 16C8-8E6 antibody, mTGase (transglutaminase), H, respectively ₂ O is transferred into an elastic ethylene-vinyl acetate disposable reaction bag by a peristaltic pump according to a certain sequence, and the reaction bag is sealed and mixed uniformly and then is placed at 30-37 ℃ for 48-168 hours. The coupling rate was checked by liquid phase analysis every 24h samples during the reaction, and when the coupling rate was 90%, the reaction was terminated and immediately purified. The detection result shows that the modification rate is more than 95%, and the average dar=2. The amino acid sequence of the transglutaminase in practical use is as follows:

(sEQ ID No：41)

2.2 16C8-8E6 ADC in vitro drug efficacy

Performing in-vitro drug effect detection by taking liver cancer cells Hep3b and HepG2 as target cells, collecting target cells to be resuspended as single cell suspension, and identifying cell viability and cell count by trypan blue staining method; cell density was adjusted to 1X10 ⁵ cells/ml; 100 μl per well was added to a 96-well black flat bottom cell culture plate; adding 20 mu.l of diluted test sample to the 96-well black flat bottom cell culture plate inoculated with cells; placing in a cell incubator (37 ℃,5% CO) ₂ ) Incubating for 66+ -3 hr; add sodium resazurin solution (0.03%), 20 μl per well; the mixture is acted for 3 to 4 hours at 37 ℃, the fluorescence value is read by an enzyme labelling instrument at 550nm/610nm, magellan6 or similar mapping software is used for mapping, and the semi-inhibition concentration IC of the reference standard and the sample is fitted ₅₀ 。

The experimental results are shown in fig. 3, and show that the 16C8-8E6 ADC has a certain inhibition effect on the growth of HepG2 and Hep3b cancer cells in vitro.

EXAMPLE 3 immunotoxin preparation

According to the sequencing result of 16C8-8E6, the scFv-PE38 recombinant immunotoxin 8E6-PE38 is constructed, and the recombinant plasmid is named 8E6-PE38/pET22b. Wherein the linker of VH and VL is GGGGSGGGGSGGGGS (SEQ ID NO: 42), and the linker of VL and PE38 is ASGG (SEQ ID NO: 43). Genes were synthesized by the company Kirschner Biotech Co. The immunotoxin PE38 was named: PE38KDEL has the amino acid sequence:

(SEQ ID NO：44)

3.1 construction of recombinant plasmids

1. Primer synthesis

A set of primers (T7 and T7 Ter) was synthesized from the multiple cloning sites of the 8E6-PE38/pET22b gene sequences, pET28a and pET32a vectors (pET 28a: novagen TB074; pET32a: novagen TB 122), and were synthesized by the company of Kirschner Biotech Co.

Primer P1 (T7): 5'-TAATACGACTCACTATAGGG-3'; (SEQ ID NO: 45)

Primer P2 (T7 Ter): 5'-GCTAGTTATTGCTCAGCGG-3' (SEQ ID NO: 46)

PCR primer amplification of 8E6-PE38 target Gene

The 8E6-PE38 gene was amplified using 8E6-PE38/pET22b as template and P1 and P2 as primers, wherein KAPA HiFi HotStart ReadyMix (2X) for PCR was purchased from Kapabiosystems.

The PCR reaction system is shown in Table 3:

TABLE 4 PCR reaction System

Composition of the components	Volume of
		KAPA HiFi HotStart ReadyMix(2×)	50μl
Primer P1	3μl
		Primer P2	3μl
Template	2.5μl
		Water and its preparation method	Up to 100. Mu.l

After the PCR reaction, the PCR product was detected by 0.8% agarose gel electrophoresis, and the target band was subjected to gel cutting recovery, purification and concentration measurement.

3. Ligation and identification of recombinant plasmids

The amplified gene, pET28a and pET32a were digested with NcoI and XhoI, respectively, and the expression vector was ligated with the digested product using T4 DNA ligase, and transformed into E.coli Top10 competent cells. 8E6-PE38/pET28a positive clones were selected on LB plate medium containing kanamycin (50. Mu.g/mL) and 8E6-PE38/pET32a positive clones were selected on ampicillin-containing (100. Mu.g/mL) LB plate medium.

The P1 and P2 are used as primers to verify by a colony PCR method and a method for enzyme digestion of recombinant plasmids, and the result shows that the construction is successful, thus obtaining recombinant expression vectors 8E6-PE38/pET28a and 8E6-PE38/pET32a.

Example 4: expression and purification of immunotoxins

1. Solution preparation

Buffer A：20mM Tris，200mM NaCl，pH 7.0

Buffer B:20mM Tris,200mM NaCl,1M Midazole, pH 7.0

Buffer C:20mM Tris,200mM NaCl,8M Urea, pH 7.0

Buffer D：20mM Tris，200mM NaCl，0.2mM GSSG，1mM GSH，pH 7.0

Buffer E:20mM Tris,200mM NaCl,0.2mM GSSG,1mM GSH,8M Urea, pH 7.0

2. Inducible expression of recombinant proteins

The 8E6-PE38/pET28a and 8E6-PE38/pET32a recombinant plasmids are transformed into BL21 (DE 3) competent cells, and monoclonal is selected for PCR identification. Inoculating the PCR positive monoclonal of BL21 (DE 3)/8E 6-PE38/pET28a to LB culture medium (Kan+), inoculating the PCR positive monoclonal of BL21 (DE 3)/8E 6-PE38/pET32a to LB culture medium (amp+), and shake culturing at 220rpm for 5-7 h at 37 ℃ until OD600 is between 0.6 and 0.8. Shaking culture is carried out at the temperature of 16 ℃ by a shaking table 220r/min, IPTG (final concentration of 1 mM) induction is carried out, and induction is carried out overnight for 16-20 h.

3. Thallus collection and crushing

The fermentation broth was centrifuged at 6000rpm at 4℃for 10min, and the cells were collected. The cells were resuspended at a rate of 20mL buffer A (20mM Tris,200mM NaCl,pH 7.0) per gram of wet cell weight. Ultrasonic crushing is carried out in ice bath, the power is 22W, the working time is 3s, the interval time is 5s, and the ultrasonic crushing is carried out for 15min. Centrifuge at 12000rpm for 30min at 4deg.C, collect supernatant and precipitate.

SDS-PAGE analysis of protein expression

SDS-PAGE detection results show that the protein is expressed in the form of inclusion bodies, so that further inclusion body renaturation experiments are needed.

4. Renaturation of target protein

(1) The inclusion bodies were resuspended in a ratio of 1mL of denaturing solution (Buffer C+2% Titon-100) per 40mg of inclusion bodies, and the mixture was allowed to denature overnight at 4 ℃.

(2) Centrifugal at 10000rpm for 15min at 4deg.C, collecting supernatant.

(3) The supernatant was added to a 25kDa dialysis bag and placed tightly on both ends in 6M urea buffer (6M urea, 20mM Tris,200mM NaCl,0.2mM GSSG,1mM GSH,pH 7.0) and shaken on ice for 2h.

(4) Sequentially adding into 4M urea buffer solution, 2M urea buffer solution and 0M urea buffer solution (4M/2M/0M urea, 20mM Tris,200mM NaCl,0.2mM GSSG,1mM GSH,pH 7.0), and respectively oscillating on ice for 2h.

5. Affinity layer purification of target protein

(1) The supernatant was centrifuged at 12000rpm at 4℃for 30min and filtered through a 0.45 μm filter.

(2) Proteins were purified using AKTA purification system and nickel ion affinity chromatography column (GE, 5 mL). The column 10CV was equilibrated with BufferA.

(3) The renaturation solution is loaded at a flow rate of 1mL/min, balanced by Buffer A after loading, and the flow-through solution is collected.

(4) Elution was performed with 2%, 10%, 30%, 50% and 100% buffer B, respectively.

(5) SDS-PAGE detects the flow through and elutes the sample, and the result shows that the protein is not combined with the affinity chromatography column and the protein is in the flow through. SDS-PAGE detection results are shown in FIG. 4. It follows that the purification of the protein of interest meets the requirements of further experiments.

EXAMPLE 4 in vitro efficacy of immunotoxins

Performing in-vitro drug effect detection by taking Hep3b and HepG2 as target cells, collecting the target cells to be resuspended into single cell suspension, and identifying the cell viability and cell count by trypan blue staining; cell density was adjusted to 1X10 ⁵ cells/ml; 100 μl per well was added to a 96-well black flat bottom cell culture plate; adding 20 mu.l of diluted test sample to the 96-well black flat bottom cell culture plate inoculated with cells; incubating in a cell incubator (37deg.C, 5% CO 2) for 66+ -3 hr; add sodium resazurin solution (0.03%), 20 μl per well; the mixture is acted for 3 to 4 hours at 37 ℃, the fluorescence value is read by an enzyme labelling instrument at 550nm/610nm, magellan6 or similar mapping software is used for mapping, and the semi-inhibition concentration IC50 of the reference standard and the sample is fitted.

The results show that the immunotoxins have better growth inhibition effect on liver cancer cells Hep3b (figure 5) and HepG2 (figure 6). Based on the in vitro results, an immunotoxin was selected for in vivo efficacy validation (hereinafter referred to as 8E6-PE 38) using 8E6-PE38 (pET 32a expression).

EXAMPLE 5 alternative immunotoxin in vivo efficacy

1. In vivo efficacy test of Hep3b tumor-bearing mice

(1) Seed tumor: establishing a mouse model of subcutaneous liver cancer of human, dividing 20 BALB/c nu/nu mice into 4 groups, 5 mice/group, and inoculating 5×10 mice each ⁶ A group of mice was randomly taken as control for liver cancer Hep3b cells.

(2) Administration: at a tumor volume of 100mm ³ The test drugs are intravenously administered to tumor-bearing mice at doses of 0.5mg/kg, 1.0mg/kg and 10.0mg/kg. Intravenous administration was performed once every other day, eight times. Tumor growth changes were dynamically observed by measuring tumor diameters at various times after dosing (two weeks after dosing was stopped, and observation was continued).

(3) Results: the results are shown in FIG. 7, wherein the 10.0mg/kg group had the best effect, and the tumor growth was better inhibited, compared with the 1.0mg/kg and 0.5mg/kg groups, which showed slightly poorer tumor inhibition effect, and overall, the therapeutic effect showed a certain concentration-dependent (dose-dependent manger) trend.

2. In vivo efficacy test of HepG2 tumor-bearing mice

(1) Seed tumor: establishing a mouse model of subcutaneous liver cancer of human, dividing 20 BALB/c nu/nu mice into 4 groups, 5 mice/group, each vaccinated with 5×10 ⁶ A group of mice was randomly taken as controls for liver cancer HepG2 cells.

(3) Results: the results are shown in FIG. 8, wherein the 10.0mg/kg group had the best effect, and the tumor growth was better inhibited, compared with the 1.0mg/kg and 0.5mg/kg groups, which showed slightly poorer tumor inhibition effect, and overall, the therapeutic effect showed a certain concentration-dependent (dose-dependent manger) trend.

In conclusion, the antibody drug conjugate and immunotoxin obtained by the application with GPC3 as a target point have good technical effects of inhibiting tumor growth in-vivo and in-vitro experiments on tumor cells represented by liver cancer cells.

The above description is of preferred embodiments only, which are exemplary only and not limiting of the combination of features necessary to practice the application. The headings provided are not meant to limit the various embodiments of the application. Terms such as "comprising," "including," and "comprising" are not intended to be limiting. Furthermore, unless otherwise indicated, the absence of a numerical modification includes the plural form, and "or", "or" means "and/or". Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

All publications and patents mentioned in this specification are herein incorporated by reference. Various modifications and variations of the described methods and compositions of the application will be apparent to those skilled in the art without departing from the scope and spirit of the application. Although the application has been described in terms of specific preferred embodiments, it should be understood that the application as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the application which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Sequence listing

<110> stone pharmaceutical group giant stone biopharmaceutical Co., ltd

<120> a human GPC3 monoclonal antibody and conjugate thereof

<130> 1

<160> 46

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8

<212> PRT

<213> Artificial sequence (unknown)

<400> 1

Gly Tyr Ile Phe Thr Asn Tyr Gly

1 5

<210> 2

<211> 8

<212> PRT

<213> Artificial sequence (unknown)

<400> 2

Ile Asn Thr Tyr Thr Gly Glu Pro

1 5

<210> 3

<211> 14

<212> PRT

<213> Artificial sequence (unknown)

<400> 3

Ala Arg Lys Gly Thr Val Arg Ala Ser Tyr Ala Met Asp Tyr

1 5 10

<210> 4

<211> 6

<212> PRT

<213> Artificial sequence (unknown)

<400> 4

Glu Asn Ile Tyr Asn Tyr

1 5

<210> 5

<211> 3

<212> PRT

<213> Artificial sequence (unknown)

<400> 5

Leu Ala Lys

1

<210> 6

<211> 9

<212> PRT

<213> Artificial sequence (unknown)

<400> 6

Gln His His Tyr Gly Ser Pro Tyr Thr

1 5

<210> 7

<211> 121

<212> PRT

<213> Artificial sequence (unknown)

<400> 7

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

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

20 25 30

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

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Asn Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Asn Leu Lys Asn Glu Asp Met Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Lys Gly Thr Val Arg Ala Ser Tyr Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 8

<211> 107

<212> PRT

<213> Artificial sequence (unknown)

<400> 8

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Ile Tyr Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Leu Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Lys Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr His Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His His Tyr Gly Ser Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 9

<211> 8

<212> PRT

<213> Artificial sequence (unknown)

<400> 9

Gly Tyr Thr Phe Thr Asn Tyr Gly

1 5

<210> 10

<211> 8

<212> PRT

<213> Artificial sequence (unknown)

<400> 10

Ile Thr Thr Tyr Thr Gly Glu Ser

1 5

<210> 11

<211> 14

<212> PRT

<213> Artificial sequence (unknown)

<400> 11

Ala Arg Lys Gly Thr Val Arg Ala Ser Tyr Ala Met Asp Tyr

1 5 10

<210> 12

<211> 6

<212> PRT

<213> Artificial sequence (unknown)

<400> 12

Glu Asn Ile Tyr Ser Tyr

1 5

<210> 13

<211> 3

<212> PRT

<213> Artificial sequence (unknown)

<400> 13

Asn Ala Lys

1

<210> 14

<211> 9

<212> PRT

<213> Artificial sequence (unknown)

<400> 14

Gln His His Tyr Gly Val Pro Tyr Thr

1 5

<210> 15

<211> 121

<212> PRT

<213> Artificial sequence (unknown)

<400> 15

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Gly Asp Met Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Lys Gly Thr Val Arg Ala Ser Tyr Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 16

<211> 107

<212> PRT

<213> Artificial sequence (unknown)

<400> 16

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

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Gly Val Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 17

<211> 9

<212> PRT

<213> Artificial sequence (unknown)

<400> 17

Gly Tyr Ser Ile Thr Tyr Asp Tyr Ser

1 5

<210> 18

<211> 7

<212> PRT

<213> Artificial sequence (unknown)

<400> 18

Ile His Tyr Arg Gly Tyr Thr

1 5

<210> 19

<211> 12

<212> PRT

<213> Artificial sequence (unknown)

<400> 19

Ala Ser Pro Tyr Gly Asn Tyr Ala Trp Phe Ala Tyr

1 5 10

<210> 20

<211> 6

<212> PRT

<213> Artificial sequence (unknown)

<400> 20

Gln Ser Ile Ser Asn Asn

1 5

<210> 21

<211> 3

<212> PRT

<213> Artificial sequence (unknown)

<400> 21

Tyr Val Ser

1

<210> 22

<211> 9

<212> PRT

<213> Artificial sequence (unknown)

<400> 22

Gln Gln Ser Asn Ser Trp Pro Phe Thr

1 5

<210> 23

<211> 119

<212> PRT

<213> Artificial sequence (unknown)

<400> 23

Asp Val Gln Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln

1 5 10 15

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

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile His Tyr Arg Gly Tyr Thr Tyr Tyr Ser Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Ser Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Ile Ser Val Thr Glu Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Ser Pro Tyr Gly Asn Tyr Ala Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Ala Leu Val Ser Val Ser Ala

115

<210> 24

<211> 107

<212> PRT

<213> Artificial sequence (unknown)

<400> 24

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

1 5 10 15

Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Val Ser Gln Ser Ile Ser Gly Asn Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Thr

65 70 75 80

Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 25

<211> 8

<212> PRT

<213> Artificial sequence (unknown)

<400> 25

Ala Tyr Thr Phe Thr Asp Tyr Ser

1 5

<210> 26

<211> 8

<212> PRT

<213> Artificial sequence (unknown)

<400> 26

Ile Asn Thr Glu Thr Gly Glu Pro

1 5

<210> 27

<211> 4

<212> PRT

<213> Artificial sequence (unknown)

<400> 27

Ala Ser Ile Arg

1

<210> 28

<211> 11

<212> PRT

<213> Artificial sequence (unknown)

<400> 28

Gln Ser Leu Leu Asn Ser Asp Gly Lys Thr Tyr

1 5 10

<210> 29

<211> 3

<212> PRT

<213> Artificial sequence (unknown)

<400> 29

Leu Val Ser

1

<210> 30

<211> 9

<212> PRT

<213> Artificial sequence (unknown)

<400> 30

Cys Gln Gly Thr His Phe Pro Arg Thr

1 5

<210> 31

<211> 111

<212> PRT

<213> Artificial sequence (unknown)

<400> 31

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

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

20 25 30

Ser Ile His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

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

100 105 110

<210> 32

<211> 112

<212> PRT

<213> Artificial sequence (unknown)

<400> 32

Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Phe Tyr Tyr Cys Cys Gln Gly

85 90 95

Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 33

<211> 8

<212> PRT

<213> Artificial sequence (unknown)

<400> 33

Gly Phe Thr Phe Asn Thr Asn Ala

1 5

<210> 34

<211> 10

<212> PRT

<213> Artificial sequence (unknown)

<400> 34

Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr

1 5 10

<210> 35

<211> 11

<212> PRT

<213> Artificial sequence (unknown)

<400> 35

Val Arg Glu Gly Val Leu Gly Ser Leu Ala Tyr

1 5 10

<210> 36

<211> 5

<212> PRT

<213> Artificial sequence (unknown)

<400> 36

Ser Ser Val Ser Tyr

1 5

<210> 37

<211> 3

<212> PRT

<213> Artificial sequence (unknown)

<400> 37

Ser Thr Ser

1

<210> 38

<211> 9

<212> PRT

<213> Artificial sequence (unknown)

<400> 38

Gln Gln Arg Ser Ser Tyr Pro Phe Thr

1 5

<210> 39

<211> 120

<212> PRT

<213> Artificial sequence (unknown)

<400> 39

Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Lys Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met

65 70 75 80

Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Val Arg Glu Gly Val Leu Gly Ser Leu Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 40

<211> 106

<212> PRT

<213> Artificial sequence (unknown)

<400> 40

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Phe Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 41

<211> 331

<212> PRT

<213> Artificial sequence (unknown)

<400> 41

Asp Ser Asp Glu Arg Val Thr Pro Pro Ala Glu Pro Leu Asp Arg Met

1 5 10 15

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

20 25 30

Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly Arg

35 40 45

Lys Gln Gln Met Thr Glu Glu Gln Arg Glu Trp Leu Ser Tyr Gly Cys

50 55 60

Val Gly Val Thr Trp Val Asn Ser Gly Gln Tyr Pro Thr Asn Arg Leu

65 70 75 80

Ala Phe Ala Phe Phe Asp Glu Asp Lys Tyr Lys Asn Glu Leu Lys Asn

85 90 95

Gly Arg Pro Arg Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg Val

100 105 110

Ala Lys Asp Ser Phe Asp Glu Ala Lys Gly Phe Gln Arg Ala Arg Asp

115 120 125

Val Ala Ser Val Met Asn Lys Ala Leu Glu Asn Ala His Asp Glu Gly

130 135 140

Ala Tyr Leu Asp Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn Asp Ala

145 150 155 160

Leu Arg Asn Glu Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg Asn

165 170 175

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

180 185 190

Met Lys Ala Val Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp Arg

195 200 205

Ser Gly Ser Ser Asp Lys Arg Lys Tyr Gly Asp Pro Glu Ala Phe Arg

210 215 220

Pro Asp Arg Gly Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn Ile

225 230 235 240

Pro Arg Ser Pro Thr Ser Pro Gly Glu Ser Phe Val Asn Phe Asp Tyr

245 250 255

Gly Trp Phe Gly Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr Val Trp

260 265 270

Thr His Gly Asn His Tyr His Ala Pro Asn Gly Ser Leu Gly Ala Met

275 280 285

His Val Tyr Glu Ser Lys Phe Arg Asn Trp Ser Asp Gly Tyr Ser Asp

290 295 300

Phe Asp Arg Gly Ala Tyr Val Val Thr Phe Val Pro Lys Ser Trp Asn

305 310 315 320

Thr Ala Pro Asp Lys Val Thr Gln Gly Trp Pro

325 330

<210> 42

<211> 15

<212> PRT

<213> Artificial sequence (unknown)

<400> 42

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 43

<211> 4

<212> PRT

<213> Artificial sequence (unknown)

<400> 43

Ala Ser Gly Gly

1

<210> 44

<211> 235

<212> PRT

<213> Artificial sequence (unknown)

<400> 44

Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu Pro Thr Gly Ala Glu

1 5 10 15

Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln

20 25 30

Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu

35 40 45

Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala

50 55 60

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

65 70 75 80

Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr

85 90 95

Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn

100 105 110

Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe

115 120 125

Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val

130 135 140

Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr

145 150 155 160

Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro

165 170 175

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

180 185 190

Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu

195 200 205

Gln Ala Thr Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro

210 215 220

Pro His His His His His His Lys Asp Glu Leu

225 230 235

<210> 45

<211> 20

<212> DNA

<213> Artificial sequence (unknown)

<400> 45

taatacgact cactataggg 20

<210> 46

<211> 19

<212> DNA

<213> Artificial sequence (unknown)

<400> 46

gctagttatt gctcagcgg 19

Claims

1. An antibody capable of specifically binding GPC3, the antibody comprising a heavy chain variable region and a light chain variable region, wherein (i) the heavy chain variable region comprises three CDR regions, CDR1, CDR2, and CDR3, respectively; the amino acid sequence of CDR1 is shown in SEQ ID NO:1, the amino acid sequence of CDR2 is shown in SEQ ID NO:2, the amino acid sequence of CDR3 is shown in SEQ ID NO:3 is shown in the figure; and (ii) the light chain variable region comprises three CDR regions, CDR1, CDR2 and CDR3, respectively; the amino acid sequence of CDR1 is shown in SEQ ID NO:4, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 6.

2. The antibody of claim 1, which is selected from the group consisting of monoclonal antibodies, diabodies, fab fragments, fab 'fragments, F (ab)' ₂ Single chain antibodies (scfvs), disulfide stabilized single chain antibodies (dsfvs).

3. A conjugate comprising the antibody of claim 1 capable of specifically binding GPC3, which is a single chain antibody scFv, and an immunotoxin PE38 linked to the single chain antibody scFv; the single chain antibody scFv has the structure of a heavy chain variable region-a connecting peptide-a light chain variable region, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:7, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:8, and a polypeptide sequence shown in the figure; the immunotoxin is PE38, having the sequence of SEQ ID NO:44, and a polypeptide comprising the amino acid sequence shown in seq id no; the single-chain antibody scFv is connected with the immunotoxin through a connector; the connecting peptide is selected from the group consisting of: GGGGSGGGGSGGGGS, GGGGS, GGGGSGGGGS; the linker is selected from: ASGG, GGGGS.

4. A pharmaceutical composition comprising the antibody of any one of claims 1 to 2 or the conjugate of claim 3.

5. Use of the antibody of any one of claims 1 to 2, the conjugate of claim 3 or the pharmaceutical composition of claim 4 in the manufacture of a medicament for the treatment of liver cancer.