CN113444179B - anti-GPC 3antibody and pharmaceutical composition containing same - Google Patents

Abstract

The invention belongs to the technical field of anti-cancer drugs, and particularly relates to an anti-GPC 3antibody and a pharmaceutical composition comprising the same. The light chain variable region and the heavy chain variable region of the anti-GPC 3antibody respectively contain amino acid sequences shown in SEQ ID NO 3 and SEQ ID NO 4; or sequences having at least 90% identity with the amino acid sequences shown in SEQ ID NO 3 and SEQ ID NO 4, respectively. The novel anti-human GPC 3antibody provided by the invention enhances the ADCC effect of the anti-GPC 3antibody by enhancing the affinity of the antibody and Fc gamma RIIIa (CD16A), thereby greatly improving the killing effect of the antibody on tumors, particularly liver cell tumors.

Description

anti-GPC 3antibody and pharmaceutical composition containing same

Technical Field

The invention belongs to the technical field of anti-cancer drugs, and particularly relates to an anti-GPC 3antibody and a pharmaceutical composition comprising the same.

Background

Glypican 3 (GPC 3) (SEQ ID NO 1) core protein consisted of an N-terminal protein with a relative molecular mass of 40kD and a membrane-bound C-terminal protein of 30 kD. The C-terminal is anchored to the cell surface via a glycosyl-phosphatidylinositol anchor protein (GPI), which binds to heparin-binding proteins, such as growth factors, and is an extracellular matrix component that regulates cell growth, proliferation, differentiation, adhesion, migration, and may also be involved in inhibiting or regulating the growth of most mesodermal tissues and organs. The GPC3 gene is expressed in different tissues in a large difference, and is highly expressed in hepatocellular carcinoma tissues, but is low or even not expressed in non-tumor liver tissues, hepatocellular adenoma, bile duct cancer, intrahepatic cholangiocellular carcinoma, gallbladder cancer and metastatic tumors of the liver. Monoclonal antibodies recognizing the carboxy-terminus of the GPC3 protein produce antibody-dependent cellular cytotoxicity (ADCC). anti-GPC 3antibody or protein fragment binding to GPC3 is known from patents such as US9790267B2, US20100248359A1, WO2004022739, US9409994B2, CN104140974B and CN109988240A, and these antibodies or binding fragments are used for the treatment of cancer, particularly liver cancer, by recombinant expression of humanized antibodies or chimeric antigen receptor T-cells (CAR-T) or the like. CAR-T cells constructed by taking GPC3 as a target are an effective cell therapy means, but CAR-T cell therapy technology has the toxicity problem of clinical therapy. In the current clinical trial process, after CAR-T is infused back into a patient, a series of reactions such as fever, chills, leukopenia, tumor lysis syndrome, cytokine storm, graft-versus-host reaction and the like often occur, and the death of the patient may be directly caused. In addition, CAR-T technology is also quite expensive. The development of therapeutic antibodies against GPC3 is a safer and more effective form. As a result of research, the research on the antibody GPC3 has been mainly focused abroad, and as in the patent documents published in China, patent CN200880103166.9 of Mideless corporation in the United states discloses a monoclonal antibody against glypican-3. The U.S. government (represented by the ministry of health and human services) developed monoclonal antibody patents (human monoclonal antibody specific to glypican 3 and its use in CN 201280029201.3), and high affinity monoclonal antibody for glypican 3 and its use in CN201380039993.7, with different sequences from different viewpoints of specificity and affinity, respectively.

However, no drug is marketed at home and abroad with a therapeutic antibody against GPC3 until the date of application. GC33 was the first humanized antibody to enter clinical studies (Ishiguro T, equivalent, anti-tibial 3antibody as a potential antibody agent for human liver cancer. cancer Res.2008; 68 (23): 9832-. GC33 is an antibody obtained by performing humanized transformation on a murine maternal antibody, GC33 recognizes a polypeptide epitope at the carboxyl terminal (542-563) of GPC3, and mainly exerts an anti-Tumor effect through antibody-dependent ADCC effect and recruitment of Tumor Infiltrating Lymphocytes (TIL). However, the GC33 antibody did not reach the pre-set focus in the secondary clinical study, which failed.

The GC33 antibody uses a wild-type Fc fragment of human IgG1, which has a certain ADCC and CDC effects, and after the antibody binds to GPC3 protein on the surface of tumor cells, the antibody exerts the ADCC and CDC effects of the antibody to kill the tumor. The Fc fragment of the antibody mainly interacts with fcyriiia (CD16A) in the human Fc receptor to exert ADCC effect, but wild-type IgG1 antibody does not exert sufficient ADCC effect, and particularly in patients with low affinity receptors (containing subtype F158), ADCC of the antibody exerts antitumor effect and is further reduced. Approximately 80% to 90% of patients carry Fc low affinity receptor mutations. The insufficient ADCC effect of the antibody is an important cause of clinical failure of GC 33.

Disclosure of Invention

In order to solve the above problems in the prior art, it is an object of the present invention to provide a novel anti-GPC 3antibody that kills cell tumors by further enhancing ADCC effect.

Another object of the present invention is to provide a pharmaceutical composition comprising the anti-GPC 3antibody, which is a combination of specific binding of tumor cells and blocking of tumor angiogenesis, thereby achieving better anti-tumor activity.

In order to achieve the above object, the present invention provides the following technical solutions:

an anti-GPC 3antibody, wherein the anti-GPC 3antibody is an anti-human GPC 3antibody, and a light chain variable region and a heavy chain variable region of the anti-GPC 3antibody respectively comprise amino acid sequences shown in SEQ ID NO 3 and SEQ ID NO 4; or sequences having at least 90% identity to the amino acid sequences shown in SEQ ID NO 3 and SEQ ID NO 4, respectively.

Preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%; and more preferably 100% identical.

Preferably, the heavy chain constant region of said anti-human GPC 3antibody comprises the amino acid sequence set forth in SEQ ID NO 5, or a sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO 5.

Preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%; and more preferably 100% identical.

The anti-human GPC 3antibody further enhances the affinity of the antibody with Fc gamma RIIIa (CD16A) and enhances the ADCC effect of the anti-GPC 3antibody by mutating and modifying wild Fc, thereby greatly improving the killing effect of the antibody on tumors, particularly liver cell tumors.

The invention also provides pharmaceutical compositions comprising an anti-GPC 3antibody, the compositions comprising an effective amount of an anti-human GPC 3antibody and at least one VEGF/VEGFR inhibitor.

Preferably, the VEGF/VEGFR inhibitor is an antibody or fusion protein; further preferably, the VEGF/VEGFR inhibitor is selected from Bevacizumab, Ranibizumab, Brolucizumab, Olinvicimab, Gentuximab, Alacizumab pegol, Aflibercept or Conbercept.

Preferably, the VEGF/VEGFR inhibitor is a small molecule compound; further preferably, the VEGF/VEGFR inhibitor is selected from Apatinib, Axitinib, Linifinib, Sorafenib, Vandertinib, Cabozantinib, Regorafenib, Pazopanib, Sunitinib, Lenvatinib, Tivozanib, Brivanib, Nintedanib, Motesanib, Cediranib, or Fruquintinib.

Preferably, the content ratio of the anti-human GPC 3antibody to the VEGF/VEGFR inhibitor in the composition is: when the VEGF/VEGFR inhibitor in the composition is an antibody or fusion protein, the composition comprises 60-1200mg of anti-human GPC 3antibody and 60-1200mg of VEGF/VEGFR inhibitor; when the VEGF/VEGFR inhibitor in the composition is a small molecule, the composition comprises 60-1200mg of anti-human GPC 3antibody and 100-1000mg of the VEGF/VEGFR inhibitor.

Preferably, the pharmaceutical composition is a single compound preparation.

Preferably, the pharmaceutical composition is a combination of two separate formulations.

Preferably, the two separate formulations are administered sequentially.

Preferably, the two separate formulations are administered simultaneously.

The pharmaceutical composition can be in a single compound preparation form, and can also be a combination of two separate preparations; when two separate preparations are combined, the administration may be simultaneous or sequential,

for example, the subject received at least one, at least two, at least three, or at least four doses of the VEGF/VEGFR inhibitor prior to use of the anti-human GPC3 antibody.

The subject may also receive at least one, at least two, at least three, or at least four doses of the human GPC 3antibody prior to administration of the VEGF/VEGFR inhibitor.

Preferably, the anti-human GPC 3antibody is administered at a dose of 0.1-15 mg/kg; for example, the anti-human GPC 3antibody is administered at a dose of 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15 mg/kg.

Preferably, the VEGF/VEGFR inhibitor is an antibody or fusion protein administered at a dose of 0.1-15 mg/kg. For example, the VEGF/VEGFR inhibitor is administered at a dose of 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15 mg/kg.

Preferably, the VEGF/VEGFR inhibitor small molecule compound is administered at a dose of 0.1-100 mg/kg.

For example, the VEGF/VEGFR small molecule inhibitor is administered at a dose of 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 mg/kg.

Preferably, the anti-human GPC 3antibody and VEGF/VEGFR antibody or fusion protein is administered once every one, two, three, four or five weeks, such as once a week, or once every two weeks, or once every three weeks.

Preferably, the VEGF/VEGFR small molecule inhibitor is administered once daily.

Preferably, the cancer is liver cancer, lung cancer, colon cancer, stomach cancer, prostate cancer, pancreatic cancer, lymphoma and metastatic cancer thereof.

Further preferably liver cancer, wherein the liver cancer is recurrent or progressive after hepatectomy, liver cancer radio frequency ablation, hepatic artery interventional therapy, liver transplantation, chemotherapy, radiotherapy-proton therapy or immunotherapy.

The invention also discloses application of the pharmaceutical composition in preparing a medicament for treating cancer.

The treatment may be a combination therapy or chemotherapy.

Compared with the prior art, the invention has the following beneficial effects:

(1) the novel anti-human GPC 3antibody provided by the invention enhances the affinity of the antibody and Fc gamma RIIIa (CD16A), enhances the ADCC effect of the anti-GPC 3antibody, and further greatly improves the killing effect of the antibody on tumors, particularly liver cell tumors.

(2) The anticancer combined drug composition combines the specific combination of tumor cells and the blocking of tumor angiogenesis, has better antitumor activity than a single drug, and provides a new strategy and thought for the treatment of cancer.

Drawings

FIG. 1 shows the SEC-HPLC detection result of Bevacizumab;

FIG. 2 shows SEC-HPLC detection results of Anti-GPC3 antibody;

FIG. 3 is a graph showing the pharmacodynamic relationship of the test sample in a CDX model established by a HepG2 cell line;

FIG. 4 is a vector map of pTT5 plasmid.

Detailed Description

The method of the present invention is described below with reference to specific examples to make it easier to understand and understand the technical solution of the present invention, but the present invention is not limited thereto. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

Interpretation of terms

In the present invention, unless defined otherwise, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

Exemplary techniques for use in conjunction with recombinant DNA, oligonucleotide synthesis, tissue culture and transformation (e.g., electroporation, lipofection), enzymatic reactions, and purification techniques are known in the art. Many such techniques and procedures are described, for example, in Sambrook et al, Molecular Cloning: a Laboratory Manual (2 nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), and many others. In addition, exemplary techniques for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and patient treatment are also known in the art.

In this application, the use of "or" means "and/or" unless stated otherwise. In the case of multiple dependent claims, the use of "or" in the alternative merely refers to more than one of the foregoing independent or dependent claims. Also, terms such as "element" or "component" encompass elements and components comprising one unit as well as elements and components comprising more than one subunit, unless specifically stated otherwise.

As described herein, any concentration range, percentage range, ratio range, or integer range is to be understood as including the value of any integer within the range, and where appropriate including fractions thereof (such as tenths and hundredths of integers), unless otherwise indicated.

Units, prefixes, and symbols are denoted in their international system of units (SI) recognized form. Numerical ranges include the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure which can be had by reference to the specification as a whole.

Unless otherwise indicated, as used in accordance with this disclosure, the following terms are to be understood to have the following meanings:

by "administering" is meant physically introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Routes of administration of the anti-GPC3 and/or anti-VEGF/VEGFR antibodies include intravenous, intramuscular, subcutaneous, intraperitoneal, or other parenteral routes of administration, e.g., by injection or infusion. The phrase "parenteral administration" as used herein refers to modes of administration, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, typically by injection rather than enteral and topical administration. Non-parenteral routes include topical, epidermal or mucosal routes of administration. Administration can also be performed, for example, once, multiple times, and/or over one or more extended periods.

An "antibody" refers to a molecule comprising at least the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain and at least the CDR1, CDR2 and CDR3 of the light chain, wherein the molecule is capable of binding to an antigen. The term antibody includes, but is not limited to, fragments capable of binding antigen, such as Fv, single chain Fv (scFv), Fab ', and (Fab') 2. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species, such as mouse, human, cynomolgus monkey, and the like.

In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region. In some embodiments, the antibody comprises at least one heavy chain comprising a heavy chain variable region and at least a portion of a heavy chain constant region, and at least one light chain comprising a light chain variable region and at least a portion of a light chain constant region. In some embodiments, the antibody comprises two heavy chains and two light chains, wherein each heavy chain comprises a heavy chain variable region and at least a portion of a heavy chain constant region, and wherein each light chain comprises a light chain variable region and at least a portion of a light chain constant region. As used herein, a single chain fv (scfv), or any other antibody comprising a single polypeptide chain comprising, for example, all six CDRs (three heavy chain CDRs and three light chain CDRs), is considered to have one heavy chain and one light chain. In some such embodiments, the heavy chain is an antibody region comprising three heavy chain CDRs and the light chain is an antibody region comprising three light chain CDRs.

"heavy chain variable region" refers to the region comprising heavy chain CDR1, Framework (FR)2, CDR2, FR3, and CDR 3. In some embodiments, the heavy chain variable region further comprises at least a portion of FR1 and/or at least a portion of FR 4. In some embodiments, the heavy chain CDR1 corresponds to Kabat residues 26 to 35; heavy chain CDR2 corresponds to Kabat residues 50 to 65; and heavy chain CDR3 corresponds to Kabat residues 95 to 102(Johnson, G., Kabat, E.A., & Wu, T.T. (1996). Kabat database of sequences of proteins of immunological interest. in L.A.Herzenberg, W.M.Weir, & C.Blackwell (Eds.), Weir's Handbook of Experimental Immunology I.immunology and Molecular Immunology (pp.6.1-6.21.) Cambridge, MA: Blackwell Science Inc..).

By "heavy chain constant region" is meant a region comprising at least three heavy chain constant domains, CH1, CH2, and CH 3. Non-limiting exemplary heavy chain constant regions include γ, δ, and α. Non-limiting exemplary heavy chain constant regions also include epsilon and mu. Each heavy chain constant region corresponds to one antibody isotype. For example, an antibody comprising a gamma constant region is an IgG antibody, an antibody comprising a delta constant region is an IgD antibody, and an antibody comprising an alpha constant region is an IgA antibody. Furthermore, the antibody comprising a mu constant region is an IgM antibody, and the antibody comprising an epsilon constant region is an IgE antibody. Certain isoforms may be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (comprising a γ 1 constant region), IgG2 (comprising a γ 2 constant region), IgG3 (comprising a γ 3 constant region), and IgG4 (comprising a γ 4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (comprising an α 1 constant region) and IgA2 (comprising an α 2 constant region) antibodies; IgM includes, but is not limited to, IgM1 and IgM 2. In some embodiments, the heavy chain constant region comprises one or more mutations (or substitutions), additions, or deletions that confer the desired characteristics to the antibody.

"heavy chain" (abbreviated HC) refers to a polypeptide comprising at least a heavy chain variable region with or without a leader sequence. In some embodiments, the heavy chain comprises at least a portion of a heavy chain constant region. The term "full-length heavy chain" as used herein refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

"light chain variable region" refers to the region comprising light chain CDR1, Framework (FR)2, CDR2, FR3, and CDR 3. In some embodiments, the light chain variable region further comprises FR1 and/or FR 4. In some embodiments, the light chain CDR1 corresponds to Kabat residues 24 to 34; light chain CDR2 corresponds to Kabat residues 50 to 56; and light chain CDR3 corresponds to Kabat residues 89 to 97(Johnson, G., Kabat, E.A., & Wu, T.T. (1996). Kabat database of sequences of proteins of immunological interest. in L.A.Herzenberg, W.M.Weir, & C.Blackwell (Eds.), Weir's Handbook of Experimental Immunology I.immunology and Molecular Immunology (pp.6.1-6.21.) Cambridge, MA: Blackwell Science Inc..).

"light chain" (abbreviated LC) refers to a polypeptide comprising at least a light chain variable region with or without a leader sequence. In some embodiments, the light chain comprises at least a portion of a light chain constant region. The term "full-length light chain" as used herein refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

"host cell" refers to a cell that may be or has been the recipient of a vector or isolated polynucleotide. The host cell may be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; a plant cell; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, and 293 and CHO cells and derivatives thereof, such as 293-6E and DG44 cells, respectively.

"Fc domain" or "Fc fragment" refers to the "fragment crystallizable" region of an immunoglobulin heavy chain. In general, an Fc domain is capable of interacting with another Fc domain to form a dimeric complex. The Fc domain may bind to a cell surface receptor (Fc receptor) and/or a protein of the complement system, or may be modified to impair or enhance such binding activity. The Fc domain may be derived from IgG, IgA, IgD, IgM, or IgE antibodies and produce immune functions such as opsonization, cell lysis, mast cell degranulation, among other Fc receptor dependent processes. The original immunoglobulin source of the native Fc is preferably an immunoglobulin of human origin, preferably IgG1 and IgG 2. In the present invention, the Fc domain is preferably composed of CH2 and CH3 of human IgG 1.

In certain embodiments, an "Fc variant" refers to a molecule or sequence that is modified by a native Fc and yet can still bind an Fc receptor. "Fc variants" include molecules or sequences humanized from non-human native Fc "Fc domains" include native Fc as described above and Fc variant molecules or sequences, which comprise molecules in monomeric or multimeric form, obtained from intact antibody breakdown, or by gene recombination expression or by other means.

An "Fc Receptor (FcR)" is an immunoglobulin expressed on the surface of a particular immune cell for recognizing an antibody Fc region that mediates an immune response. After the Fab region of the antibody recognizes an infection source antigen, the Fc region of the antibody is combined with an Fc receptor on immune cells to start the response function of the immune cells, such as phagocytosis, cytotoxic injury and the like, and destroy and remove the infection source. Fc receptors are mainly classified into three types, Fc γ R, Fc α R and Fc ∈ R, according to the type of antibody recognized by the Fc receptor and the difference of cells expressed by the Fc receptor, and Fc γ R is further classified into four subtypes, Fc γ RI (CD64), Fc γ RII (CD32), Fc γ RIII (CD16), and fcrn (neonatal fcrcepter). Fc γ RIII (CD16) has two highly homologous subtypes Fc γ RIIIa and Fc γ RIIIb in different cell types, in which Fc γ RIIIa has medium/low affinity for the Fc region of lgG antibody and is involved in immune response processes such as phagocytosis, inflammation mediation, antibody-mediated cell killing, and mast cell degranulation and elimination. In the Fc gamma RIIIa population, a Single Nucleotide Polymorphism (SNP) site causes two subtypes of high affinity (176Val/V158) and low affinity (176 Phe/F158). By modifying the amino acid sequence or glycosylation of the Fc region, the affinity of the antibody for Fc receptors can be increased or decreased, thereby improving antibody efficacy and half-life.

"ADCC" is antibody-dependent cell-mediated cytotoxicity (ADCC), and refers to binding of Fab fragment of antibody to epitope of virus-infected cell or tumor cell, binding of Fc fragment to FcR on surface of killer cell (NK cell, macrophage, etc.) and direct killing of target cell by killer cell. NK cells, mononuclear macrophages and neutrophils can kill tumor cells in an ADCC mode under the mediation of specific IgG. The NK cells have broad-spectrum anti-swelling and pain functions, and can kill and dissolve tumor cells coated by specific IgG through an ADCC mode, and can directly kill the tumor cells through close contact with the tumor cells. ADCC effects can be enhanced or reduced by engineering the native Fc region (Wang X, Mathieu M, Brezski R J, et al. IgG Fc engineering to modular antibody effects factors [ J ]. Protein & Cell,2018,9(1): 63-73.).

CDC refers to Complement Dependent Cytotoxicity (CDC), i.e., a classical pathway of complement is activated by a complex formed by binding of a specific antibody to a corresponding antigen on the surface of a cell membrane, and the formed tapping complex exerts a lytic effect on a target cell.

The angiogenesis inhibitor is used for inhibiting the growth and metastasis of tumors by blocking angiogenesis promoting factors and receptors thereof which play a key role in the formation of new blood vessels in the tumors. The VEGF (SEQ ID NO 2) and VEGFR signaling pathways dominate in playing an angiogenic role. The VEGF target medicine can be combined with a specific endothelial cell receptor to generate an inhibition effect on the growth of tumor cells, thereby achieving the purpose of treatment. Anti-angiogenic agents include those substances that bind to and block the angiogenic activity of angiogenic factors or their receptors. For example, the anti-angiogenic agent is an antibody or other antagonist of an angiogenic agent, such as an antibody to VEGF-a (e.g., Bevacizumab) or VEGF-a receptor (e.g., KDR receptor or Flt-1 receptor), an anti-PDGFR inhibitor such as imatinib mesylate, a small molecule that blocks VEGF receptor signaling (e.g., PTK787/ZK2284, SU6668,/SU 11248 (sunitinib malate), AMG706, or those small molecules described in, e.g., international patent application WO 2004/113304). Anti-angiogenic agents also include natural angiogenesis inhibitors such as angiostatin, endostatin, and the like.

"cancer" is customarily used to refer broadly to all malignant tumors. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific non-limiting examples of such cancers include squamous cell cancer, small-cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous cell non-small cell lung cancer), adenocarcinoma of the lung, squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer (liver cancer), bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland carcinoma, kidney cancer, renal cell carcinoma, liver cancer, prostate cancer, vulval cancer, thyroid cancer, liver cancer (hepatoma), brain cancer, endometrial cancer, testicular cancer, cholangiocarcinoma, gallbladder cancer, gastric cancer, melanoma, and various types of head and neck cancer (including head and neck squamous cell carcinoma).

"chemotherapy/chemotherapy" is a compound used to treat cancer. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents, such as thiotepa and cyclophosphamide; nitrogen mustards, such as chlorambucil, estramustine, ifosfamide, mechlorethamine, melphalan, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorouramicin, fotemustine, lomustine, nimustine and ranimustine; antibiotics, such as enediyne antibiotics e.g., calicheamicin; daptomycin, including daptomycin a; bisphosphonates, such as clodronate; aclacinomycin, actinomycin, azaserine, bleomycin, actinomycin C, carubicin, carminomycin, actinomycin D, daunorubicin, doxorubicin, epirubicin, esorubicin, idarubicin, sisomicin, mitomycins such as mitomycin C, mycophenolic acid, noramycin, olivomycin, pelomycin, pofiomycin, puromycin, doxorubicin, roxithromycin, streptonigrin, ubenimex, setastatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin; purine analogs such as fludarabine, 6-mercaptopurine, thioimidine, thioguanine; pyrimidine analogs such as ancitabine, 6-azauridine, cytarabine, enocitabine, floxuridine; androgens such as carpoterone, drotandrosterone propionate, epithioandrostanol, meindrotane, testolactone; anti-adrenaline, such as aminoglutethimide, mitotane, trostane; folic acid replenisher such as folinic acid; acetic acid glucurolactone; an aldphosphoramide glycoside; (ii) aminolevulinic acid; eniluracil; amoxicillin; edatrexae; desphosphamide; dimecorsine; diazaquinone; eflornithine; saddle of vinegar; an epothilone; etoglut; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanol; nisridine; pentostatin; melphalan; pirarubicin; losoxanone; 2-ethyl hydrazide; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; vincristine; vinorelbine; (ii) teniposide; daunomycin; aminopterin; (ii) Hirodad; ibandronate; irinotecan (CPT-11) (therapeutic regimens including irinotecan with 5-FU and leucovorin); capecitabine; an inhibitor of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib), and VEGF-A that reduces cell proliferation, and a pharmaceutically acceptable salt, acid, or derivative of any of the foregoing.

"treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathological condition or disorder. In certain embodiments, the term "treatment" encompasses any administration or use of a disease therapeutic agent in a mammal (including a human), and includes inhibiting or slowing disease or disease progression; partial or complete response to disease, e.g., by causing regression, or recovery or repair of lost, lost or defective function; a stimulus-ineffective process; or to smooth the disease to a reduced severity. The term "treating" also includes reducing the severity of any phenotypic feature and/or reducing the incidence, extent or likelihood of that feature. The subject in need of treatment includes those already having the disorder as well as those predisposed to having the disorder, or those in which the disorder is to be prevented.

"combination therapy" refers to a pharmaceutical therapeutic use that may be administered alone or in combination with other therapeutic modalities. These antibodies can be provided prior to, substantially simultaneously with, or subsequent to other treatment modalities, such as surgery, chemotherapy, radiation therapy, or administration of a biological agent (e.g., another therapeutic antibody). In some embodiments, the cancer has relapsed or progressed following a treatment selected from surgery, chemotherapy, radiation therapy, or a combination thereof. To treat cancer, the antibodies can be administered with one or more additional anti-cancer agents, e.g., chemotherapeutic agents, growth inhibitory agents, anti-angiogenic agents, and/or anti-tumor compositions, as discussed herein. Non-limiting examples of chemotherapeutic agents, growth inhibitory agents, anti-angiogenic agents, anti-cancer agents, and anti-tumor compositions that can be used in combination with the antibodies of the invention are provided below the "definitions" herein.

Example 1 molecular construction of anti-GPC 3antibody and anti-VEGF antibody

The light and heavy chain amino acid sequences of the antibodies in table 1 were codon optimized according to human host cells and the genes were synthesized conventionally, with the addition of 5'(EcoRI) and 5' UTR ([ SEQ ID NO 11]) and 3'UTR ([ tgtgatga ]) and 3' (HindIII), and the genes were cloned into the vector pTT5 (vector map shown in fig. 4) by 5'EcoRI and 3' HindIII, the pTT5 vector sequence being shown as SEQ ID NO 12. Selecting clone for sequencing, selecting correctly sequenced thallus for conservation and enlarged culture, and using the enlarged thallus for extraction of plasmid.

TABLE 1 sequence combinations of anti-GPC 3antibody and anti-VEGF antibody

Antibodies	Light chain sequence	Heavy chain sequence
			Anti-GPC3	SEQ ID NO 6	SEQ ID NO 7
Bevacizumab	SEQ ID NO 8	SEQ ID NO 9
			Anti-GPC3(WT)	SEQ ID NO 6	SEQ ID NO 10

EXAMPLES expression and purification of anti-GPC 3antibody and anti-VEGF antibody

HEK293 cell transfection and protein separation and purification of the extracted plasmid were performed as follows

1. The cell density, determined to be greater than 95% viable, was adjusted to 3X 106 cells/mL using HEK293 cell culture medium (already pre-warmed), gently shaken and aliquoted (transfection system 90%) into cells with a volume not exceeding 1/3 of the shake flask specification in the shake flask and placed on a shaker until use.

2. The volume of the transfection buffer opti-MEM was calculated from the volume of transfected cells and was 1/10 for the transfection system; calculating the amount of a transfection reagent polyethylene, wherein the proportion of the transfection reagent polyethylene is 3 mu L/mL of transfected cells; the total amount of transfected DNA was calculated at a ratio of 1. mu.g/mL transfected cells.

The specific transfection procedure was as follows:

adding 10% MEM into 1 50ml centrifuge tube, adding plasmid, mixing, filtering, standing for 5min, adding polyethylene imine into DNA suspension, mixing (mixing by gently inverting for 2-3 times), and standing for 15-20 min. Then, the compound is gently added into the subpackaged cells, and the shake flask is gently shaken while the compound is added; the transfected cells were cultured in a shaker at 37 ℃.

Day 1 feed

After 20h, the corresponding amount of enhancer was added at 1:1000 transfected cell volume and Feed at 2% transfected cell volume.

Determination of expression amount of transfected cells on day 4

Taking 200uL of a sample to be detected, detecting the protein expression quantity by using a label-free analyzer, using 293 culture medium as a negative control, and using a solution with known protein concentration as a positive control during analysis of the analyzer.

Collecting samples on days 5-6

Detecting the cell viability (65-75% or higher), centrifuging the sample at 3000-. The SEC-HPLC detection results after purification of the expressed protein after construction of the above clone are shown in FIGS. 1 and 2, wherein the peak characteristics in the graph are shown in the following tables 2 and 3:

TABLE 2 Bevacizumab SEC-HPLC DETECTION RESULT PEAK FEATURE

TABLE 3 SEC-HPLC DETECTION OF ANTI-GPC 3ANTIBODY WITH PEAK FEATURES

Example three Anti-GPC3 and Anti-GPC3(WT) affinity assays for human CD16A

By using Gator ^TM The Anti-GPC3 and Anti-GPC3(WT) expressed as described above were subjected to affinity detection with human CD16A Protein (available from Novoprotein, cat # CS11), respectively, using a Protein A biosensor to capture an antibody sample, and then the captured antibody sample was subjected to kinetic detection of binding and dissociation with human CD16A Protein. Kinetics were performed using 1:1 the fitting analysis is performed in conjunction with the model. The brief steps are shown in table 4 below:

TABLE 4

Step (ii) of	Time
		Protein loading	200s
Bonding of	180s
		Dissociation	300s
Regeneration	30s

Using Gator ^TM The instrumental affinity data are shown in table 5 below:

TABLE 5

The affinity of the Anti-GPC 3antibody after modification is improved by about 10.8 times compared with that of the Anti-GPC3(WT) antibody before modification to the human CD16A protein.

Example four HepG2 cell line establishment of mouse subcutaneous CDX model and drug efficacy test

4.1 cell culture

HepG2 cells were cultured in MEM +0.01mM NEAA medium containing 10% fetal bovine serum. The exponentially growing HepG2 cells were collected and PBS resuspended to appropriate concentrations for subcutaneous tumor inoculation in nude mice.

4.2 animal modeling

Experimental mice were inoculated subcutaneously on the right back with 1X 10 ⁷ HepG2 cells, resuspended in 1:1 (0.1 ml/piece) in PBS and matrigel, and observing the growth of tumor until the tumor grows to 80-120mm of average volume ³ (about 100 mm) ³ ) The administration was randomized and divided into groups according to the tumor size and the mouse body weight. The day of tumor cell inoculation was defined as day 0.

The detailed administration method, administration dose and administration route were conducted as shown in table 6 below.

TABLE 6 administration route, dosage and regimen in subcutaneous xenograft model of human hepatoma HepG2 cell line

Note: 1. the administration volume was 10. mu.l/g; 2. the order and time intervals for administration of the fourth two test agents were: bevacizumab was administered 30 minutes before Anti-GPC 3.

4.3 random grouping

Before the start of dosing, all animals were weighed and tumor volume was measured with a vernier caliper. Since tumor volume affects the effectiveness of the treatment, mice were grouped according to their tumor volume using a random grouping design to ensure similar tumor volumes between groups. The grouping is done using StudyDirectorTM (version number 3.1.399.19, vendor studio System, inc., s.san Francisco, CA, USA).

4.4 Experimental Observation and data Collection

After tumor cell inoculation, routine monitoring includes the effects of tumor growth and treatment on the normal behavior of the animal, including the activity of the experimental animal, food intake and water intake, weight gain or loss, eyes, hair follicles and other abnormal conditions. Clinical symptoms observed during the trial were recorded in the raw data. The second dose was administered on day 7 (i.e., day 27 of tumor inoculation) after the start of dosing on the day after the cohort (i.e., day 20 of tumor inoculation). Body weight and tumor size of mice were measured 2-3 times per week. Tumor volume calculation formula: tumor volume (mm3) 1/2 × (a × b) ² ) (wherein a represents a long diameter and b represents a short diameter). StudyDirector (TM) was used in the experiment(version number 3.1.399.19, supplier studio System, Inc.) software data were collected including measurements of the long and short diameter of the tumor and weighing of the animal body weight. The following results of the in vivo efficacy test in animals were obtained based on weekly measurements. As shown in FIG. 3, the statistical results show that the combination of Bevacizumab and anti-GPC3 inhibited tumor growth better than either Bevacizumab or anti-GPC3 alone.

Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, and that the simple modifications or equivalent substitutions of the technical solutions of the present invention by those of ordinary skill in the art can be made without departing from the spirit and scope of the technical solutions of the present invention.

Sequence listing

<110> Suzhou pulekang pharmaceutical science and technology Co., Ltd

<120> an anti-GPC 3antibody and pharmaceutical composition comprising the same

<130> 2020.03.02

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 556

<212> PRT

<213> GPC3

<400> 1

Gln Pro Pro Pro Pro Pro Pro Asp Ala Thr Cys His Gln Val Arg Ser

1 5 10 15

Phe Phe Gln Arg Leu Gln Pro Gly Leu Lys Trp Val Pro Glu Thr Pro

20 25 30

Val Pro Gly Ser Asp Leu Gln Val Cys Leu Pro Lys Gly Pro Thr Cys

35 40 45

Cys Ser Arg Lys Met Glu Glu Lys Tyr Gln Leu Thr Ala Arg Leu Asn

50 55 60

Met Glu Gln Leu Leu Gln Ser Ala Ser Met Glu Leu Lys Phe Leu Ile

65 70 75 80

Ile Gln Asn Ala Ala Val Phe Gln Glu Ala Phe Glu Ile Val Val Arg

85 90 95

His Ala Lys Asn Tyr Thr Asn Ala Met Phe Lys Asn Asn Tyr Pro Ser

100 105 110

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

115 120 125

Ser Leu Tyr Ile Leu Gly Ser Asp Ile Asn Val Asp Asp Met Val Asn

130 135 140

Glu Leu Phe Asp Ser Leu Phe Pro Val Ile Tyr Thr Gln Leu Met Asn

145 150 155 160

Pro Gly Leu Pro Asp Ser Ala Leu Asp Ile Asn Glu Cys Leu Arg Gly

165 170 175

Ala Arg Arg Asp Leu Lys Val Phe Gly Asn Phe Pro Lys Leu Ile Met

180 185 190

Thr Gln Val Ser Lys Ser Leu Gln Val Thr Arg Ile Phe Leu Gln Ala

195 200 205

Leu Asn Leu Gly Ile Glu Val Ile Asn Thr Thr Asp His Leu Lys Phe

210 215 220

Ser Lys Asp Cys Gly Arg Met Leu Thr Arg Met Trp Tyr Cys Ser Tyr

225 230 235 240

Cys Gln Gly Leu Met Met Val Lys Pro Cys Gly Gly Tyr Cys Asn Val

245 250 255

Val Met Gln Gly Cys Met Ala Gly Val Val Glu Ile Asp Lys Tyr Trp

260 265 270

Arg Glu Tyr Ile Leu Ser Leu Glu Glu Leu Val Asn Gly Met Tyr Arg

275 280 285

Ile Tyr Asp Met Glu Asn Val Leu Leu Gly Leu Phe Ser Thr Ile His

290 295 300

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

305 310 315 320

Ile Gly Lys Leu Cys Ala His Ser Gln Gln Arg Gln Tyr Arg Ser Ala

325 330 335

Tyr Tyr Pro Glu Asp Leu Phe Ile Asp Lys Lys Val Leu Lys Val Ala

340 345 350

His Val Glu His Glu Glu Thr Leu Ser Ser Arg Arg Arg Glu Leu Ile

355 360 365

Gln Lys Leu Lys Ser Phe Ile Ser Phe Tyr Ser Ala Leu Pro Gly Tyr

370 375 380

Ile Cys Ser His Ser Pro Val Ala Glu Asn Asp Thr Leu Cys Trp Asn

385 390 395 400

Gly Gln Glu Leu Val Glu Arg Tyr Ser Gln Lys Ala Ala Arg Asn Gly

405 410 415

Met Lys Asn Gln Phe Asn Leu His Glu Leu Lys Met Lys Gly Pro Glu

420 425 430

Pro Val Val Ser Gln Ile Ile Asp Lys Leu Lys His Ile Asn Gln Leu

435 440 445

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

450 455 460

Asp Glu Glu Gly Phe Glu Ser Gly Asp Cys Gly Asp Asp Glu Asp Glu

465 470 475 480

Cys Ile Gly Gly Ser Gly Asp Gly Met Ile Lys Val Lys Asn Gln Leu

485 490 495

Arg Phe Leu Ala Glu Leu Ala Tyr Asp Leu Asp Val Asp Asp Ala Pro

500 505 510

Gly Asn Ser Gln Gln Ala Thr Pro Lys Asp Asn Glu Ile Ser Thr Phe

515 520 525

His Asn Leu Gly Asn Val His Ser Pro Leu Lys Leu Leu Thr Ser Met

530 535 540

Ala Ile Ser Val Val Cys Phe Phe Phe Leu Val His

545 550 555

<210> 2

<211> 206

<212> PRT

<213> VEGF

<400> 2

Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys

1 5 10 15

Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu

20 25 30

Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys

35 40 45

Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu

50 55 60

Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile

65 70 75 80

Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe

85 90 95

Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg

100 105 110

Gln Glu Lys Lys Ser Val Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys

115 120 125

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

130 135 140

Cys Cys Leu Met Pro Trp Ser Leu Pro Gly Pro His Pro Cys Gly Pro

145 150 155 160

Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr Cys

165 170 175

Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu

180 185 190

Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg

195 200 205

<210> 3

<211> 112

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 3

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 4

<211> 115

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 4

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 5

<211> 330

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 5

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

1 5 10 15

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

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

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

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

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

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 6

<211> 219

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 6

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

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

115 120 125

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

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 7

<211> 445

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 7

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys

325 330 335

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

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 8

<211> 214

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 8

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

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

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 9

<211> 453

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 9

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

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

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

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

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

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

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

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

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 10

<211> 445

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 10

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

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

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 11

<211> 66

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

gccaccatgg agacagatac cctgctgctg tgggtgctgc tgctgtgggt ccctggcagc 60

accgga 66

<210> 12

<211> 4401

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

gtacatttat attggctcat gtccaatatg accgccatgt tgacattgat tattgactag 60

ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt 120

tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac 180

gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg 240

ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag 300

tccgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat 360

gaccttacgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat 420

ggtgatgcgg ttttggcagt acaccaatgg gcgtggatag cggtttgact cacggggatt 480

tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga 540

ctttccaaaa tgtcgtaata accccgcccc gttgacgcaa atgggcggta ggcgtgtacg 600

gtgggaggtc tatataagca gagctcgttt agtgaaccgt cagatcctca ctctcttccg 660

catcgctgtc tgcgagggcc agctgttggg ctcgcggttg aggacaaact cttcgcggtc 720

tttccagtac tcttggatcg gaaacccgtc ggcctccgaa cggtactccg ccaccgaggg 780

acctgagcga gtccgcatcg accggatcgg aaaacctctc gagaaaggcg tctaaccagt 840

cacagtcgca aggtaggctg agcaccgtgg cgggcggcag cgggtggcgg tcggggttgt 900

ttctggcgga ggtgctgctg atgatgtaat taaagtaggc ggtcttgaga cggcggatgg 960

tcgaggtgag gtgtggcagg cttgagatcc agctgttggg gtgagtactc cctctcaaaa 1020

gcgggcatta cttctgcgct aagattgtca gtttccaaaa acgaggagga tttgatattc 1080

acctggcccg atctggccat acacttgagt gacaatgaca tccactttgc ctttctctcc 1140

acaggtgtcc actcccaggt ccaagtttaa acggatctct agcgaattcc ctctagaggg 1200

cccgtttctg ctagcaagct tgctagcggc cgctcgaggc cggcaaggcc ggatcccccg 1260

acctcgacct ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt 1320

ttgtgtctct cactcggaag gacatatggg agggcaaatc atttggtcga gatccctcgg 1380

agatctctag ctagaggatc gatccccgcc ccggacgaac taaacctgac tacgacatct 1440

ctgccccttc ttcgcggggc agtgcatgta atcccttcag ttggttggta caacttgcca 1500

actgaaccct aaacgggtag catatgcttc ccgggtagta gtatatacta tccagactaa 1560

ccctaattca atagcatatg ttacccaacg ggaagcatat gctatcgaat tagggttagt 1620

aaaagggtcc taaggaacag cgatgtaggt gggcgggcca agataggggc gcgattgctg 1680

cgatctggag gacaaattac acacacttgc gcctgagcgc caagcacagg gttgttggtc 1740

ctcatattca cgaggtcgct gagagcacgg tgggctaatg ttgccatggg tagcatatac 1800

tacccaaata tctggatagc atatgctatc ctaatctata tctgggtagc ataggctatc 1860

ctaatctata tctgggtagc atatgctatc ctaatctata tctgggtagt atatgctatc 1920

ctaatttata tctgggtagc ataggctatc ctaatctata tctgggtagc atatgctatc 1980

ctaatctata tctgggtagt atatgctatc ctaatctgta tccgggtagc atatgctatc 2040

ctaatagaga ttagggtagt atatgctatc ctaatttata tctgggtagc atatactacc 2100

caaatatctg gatagcatat gctatcctaa tctatatctg ggtagcatat gctatcctaa 2160

tctatatctg ggtagcatag gctatcctaa tctatatctg ggtagcatat gctatcctaa 2220

tctatatctg ggtagtatat gctatcctaa tttatatctg ggtagcatag gctatcctaa 2280

tctatatctg ggtagcatat gctatcctaa tctatatctg ggtagtatat gctatcctaa 2340

tctgtatccg ggtagcatat gctatcctca tgataagctg tcaaacatga gaattaattc 2400

ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat 2460

ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 2520

atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 2580

tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 2640

cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 2700

agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 2760

taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 2820

tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg 2880

catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac 2940

ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 3000

ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa 3060

catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc 3120

aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt 3180

aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga 3240

taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 3300

atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa 3360

gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa 3420

tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt 3480

ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt 3540

gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 3600

agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt 3660

aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 3720

agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac 3780

tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac 3840

atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 3900

taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg 3960

gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca 4020

gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt 4080

aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta 4140

tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc 4200

gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc 4260

cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa 4320

ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag 4380

cgagtcagtg agcgaggaag c 4401

Claims (6)

1. A pharmaceutical composition comprising an effective amount of an anti-GPC 3antibody and a VEGF/VEGFR inhibitor;

the variable regions of the light chain and the heavy chain of the anti-GPC 3antibody are respectively amino acid sequences shown in SEQ ID NO 3 and SEQ ID NO 4;

the heavy chain constant region of the anti-GPC 3antibody is the amino acid sequence shown in SEQ ID NO 5,

the VEGF/VEGFR inhibitor is Bevacizumab.

2. The pharmaceutical composition of claim 1, wherein said anti-GPC 3antibody is

The content of the VEGF/VEGFR inhibitor is 60-1200 mg.

3. The pharmaceutical composition of claim 1, wherein said anti-GPC 3antibody is administered at a dose of 0.1-15 mg/kg.

4. The pharmaceutical composition of claim 1, wherein the VEGF/VEGFR inhibitor is administered at a dose of 0.1-15 mg/kg.

5. Use of a pharmaceutical composition according to claim 1 in the manufacture of a medicament for the treatment of cancer, wherein the cancer is liver cancer.

6. The use of claim 5, wherein the liver cancer is recurrent or progressive after hepatectomy, radiofrequency ablation of liver cancer, hepatic artery intervention, liver transplantation, chemotherapy, radiotherapy-proton therapy or immunotherapy.

Images