CN119013305A

CN119013305A - Monoclonal antibodies targeting GPC3 and antibody drug conjugates comprising such antibodies

Info

Publication number: CN119013305A
Application number: CN202380033927.2A
Authority: CN
Inventors: 黄超兰; 黎玮; 韩佩; 马翔; 徐强; 卢芷惠
Original assignee: Yuyan Biotechnology Shanghai Co ltd; Yuyan Technology Beijing Co ltd
Current assignee: Yuyan Biotechnology Shanghai Co ltd; Yuyan Technology Beijing Co ltd
Priority date: 2022-04-24
Filing date: 2023-03-28
Publication date: 2024-11-22
Also published as: WO2023207475A1

Abstract

本发明提供了一种靶向GPC3的单克隆抗体或其抗原结合片段、由此单克隆抗体制备的抗体药物缀合物、包含本发明的抗体药物缀合物的药物组合物或诊断/预后组合物。本发明的抗体可以优异地结合GPC3蛋白，且本发明的抗体药物缀合物对于表达GPC3蛋白的肿瘤细胞具有很好的杀伤效果。The present invention provides a monoclonal antibody or an antigen-binding fragment thereof targeting GPC3, an antibody-drug conjugate prepared from the monoclonal antibody, and a pharmaceutical composition or a diagnostic/prognostic composition comprising the antibody-drug conjugate of the present invention. The antibody of the present invention can bind to the GPC3 protein excellently, and the antibody-drug conjugate of the present invention has a good killing effect on tumor cells expressing the GPC3 protein.

Description

Monoclonal antibodies targeting GPC3 and antibody drug conjugates comprising such antibodies

The present application claims priority from chinese patent application 202210435793.4 entitled "monoclonal antibody targeting GPC3 and antibody drug conjugate comprising such antibody" filed 24 at 2022, 04, the entire contents of which priority text is incorporated herein by reference.

Technical Field

The invention relates to the field of biological medicine. In particular, the invention relates to a monoclonal antibody or antigen binding fragment thereof targeting GPC3, an antibody drug conjugate comprising the antibody, and the use of the antibody and/or antibody drug conjugate in the treatment of related diseases.

Background

Malignant tumors (cancers) have become the "first-hand" killer that threatens the lives and health of people worldwide. The number of tumor patients in each year is over 1400 ten thousand worldwide, and the number of tumor patients newly increased in China is over 300 ten thousand annually.

The root cause of high mortality in cancer is the spread of cancer cells, metastasis and susceptibility to relapse and drug resistance in most patients after treatment. Clinically, the existing treatment means, such as surgery, radiotherapy and chemotherapy, have very little curative effect on cancer cell metastasis, recurrence and drug resistance, or only have recent curative effect, and can not change the long-term survival condition of patients. Currently, surgical resection works well for about 10-20% of early patients, but is almost ineffective for patients who have developed diffuse metastases. Radiotherapy can only treat local lesions, often as an adjuvant treatment before and after surgery and as a radical treatment for a few kinds of cancers. Chemotherapy can be used for patients who have developed diffusion metastasis, but because of the large toxic and side effects, recent or long-term drug resistance is easy to generate, so that the chemotherapy has obvious recent curative effect only for about 20-30% of patients. Even if comprehensive treatment measures of combined application of surgery, radiotherapy and chemotherapy are adopted, the long-term curative effect of 5 years of survival is wandering for 20-30% for many years, and about 70-80% of patients die within 5 years after treatment due to metastasis, recurrence and drug resistance. Even early cancer patients who have no metastasis at the time of treatment still have a part of the patients who die after treatment after metastasis and recurrence.

GPC3 is a tumor-associated antigen that is specifically overexpressed by tumor cells, particularly in cancer patients such as liver cancer, small cell lung cancer, and the like. Aiming at cancer species such as liver cancer, small cell lung cancer and the like, the existing effective treatment means are relatively lacking. The applicant has found that many antibody drugs targeting GPC3 alone do not have a significant inhibitory effect on the growth of tumor cells per se, and thus it is desirable to achieve treatment by developing a pharmaceutical form of an antibody-drug conjugate targeting GPC 3-positive tumor patients.

Disclosure of Invention

In one aspect, the invention provides a monoclonal antibody or antigen-binding fragment thereof that targets GPC3, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,

The light chain variable region comprises:

VL CDR1 comprising the amino acid sequence shown in SEQ ID NO. 12 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO. 12,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO. 13 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO. 13, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO. 14 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO. 14;

And/or the number of the groups of groups,

The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO. 32 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO. 32,

A VH CDR2 comprising the amino acid sequence shown in SEQ ID NO. 33 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO. and

A VH CDR3 comprising the amino acid sequence shown in SEQ ID No. 34 or comprising an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID No. 34.

In another aspect, the invention provides an antibody drug conjugate of the formula:

Ab-L-D, wherein:

the symbol "Ab" represents at least one of the monoclonal antibodies of the invention described previously or an antigen-binding fragment portion thereof;

The symbol "L" represents a linker linking the antibody moiety and the partner molecule; and

The symbol "D" represents a partner molecule.

In another aspect, the invention provides an antibody drug conjugate, ab- (L-D) p, of the formula wherein:

The symbol "L" represents a linker linking the antibody moiety and the partner molecule;

the symbol "D" represents a partner molecule; and

The symbol "p" represents the number of linker/partner molecules (preferably drug carriers) conjugated to the aforementioned monoclonal antibodies of the invention or antigen binding fragment portions thereof.

In yet another aspect, the invention provides a pharmaceutical composition comprising a monoclonal antibody of the invention or an antigen-binding fragment thereof or an antibody drug conjugate of the invention, and optionally a pharmaceutically acceptable carrier.

In yet another aspect, the invention provides a diagnostic/prognostic composition comprising a monoclonal antibody of the invention or an antigen-binding fragment thereof or an antibody drug conjugate of the invention, and optionally a pharmaceutically acceptable carrier.

In yet another aspect, the invention provides an isolated nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment thereof of the invention.

In yet another aspect, the invention provides an expression vector comprising a nucleic acid molecule of the invention.

In yet another aspect, the invention provides a host cell comprising a nucleic acid molecule of the invention or an expression vector of the invention.

In yet another aspect, the invention provides a hybridoma cell expressing a monoclonal antibody or antigen-binding fragment portion thereof of the invention.

In a further aspect, the invention provides the use of a monoclonal antibody of the invention or an antigen-binding fragment thereof or an antibody drug conjugate of the invention in the preparation of a pharmaceutical composition; optionally, the pharmaceutical composition is used for treating malignancy, preventing and/or treating metastasis or recurrence of malignancy in a patient.

In yet another aspect, the invention provides a method of treating malignancy, preventing and/or treating metastasis or recurrence of malignancy in a patient, the method comprising administering to the patient an effective amount of a monoclonal antibody or antigen-binding fragment portion thereof of the invention, an antibody-drug conjugate of the invention, or a pharmaceutical composition of the invention.

In a further aspect, the invention provides the use of a monoclonal antibody of the invention or an antigen-binding fragment thereof or an antibody drug conjugate of the invention in the preparation of a diagnostic/prognostic composition; optionally, the diagnostic/prognostic composition is used to detect the presence of a malignancy in a patient; alternatively, the diagnostic/prognostic composition is used to prognose malignant tumor recurrence or progression in a patient.

In yet another aspect, the invention provides a method for prognosis of recurrence or progression of a malignancy in a patient, the method comprising:

(a) Isolating a biological sample comprising tumor cells from the patient;

(b) Contacting the biological sample comprising tumor cells with a monoclonal antibody or antigen-binding fragment thereof of the invention or an antibody drug conjugate of the invention; and

(C) Identifying the presence of tumor cells that bind to the monoclonal antibody or antigen binding fragment thereof,

Thereby prognosing the recurrence or progression of the malignancy in the patient.

In yet another aspect, the present invention provides a method for detecting/diagnosing the presence of a malignancy in a patient, comprising:

a) Contacting a biological sample obtained from the patient with a monoclonal antibody or antigen-binding fragment thereof of the invention or an antibody drug conjugate of the invention;

b) Detecting binding of the monoclonal antibody or antigen binding fragment thereof to a target antigen in the biological sample, wherein detection of the binding is indicative of the presence of malignancy in the patient.

In yet another aspect, the invention provides a method for detecting the presence of tumor cells in a biological sample, comprising:

a) Contacting the biological sample with a monoclonal antibody or antigen-binding fragment thereof of the invention or an antibody drug conjugate of the invention;

b) Detecting binding of said monoclonal antibody or antigen binding fragment thereof or said antibody drug conjugate to a target antigen in said biological sample,

Wherein detection of said binding is indicative of the presence of a tumor cell in said biological sample.

In yet another aspect, the invention provides a method for isolating tumor cells, the method comprising:

(a) Providing a population of cells suspected of comprising tumor cells;

(b) Identifying a subpopulation of said cells that binds to a monoclonal antibody or antigen-binding fragment thereof of the invention or said antibody drug conjugate; and

(C) Isolating the subpopulations.

In yet another aspect, the invention provides a method of producing a monoclonal antibody or antigen-binding fragment thereof that targets human tumor cells expressing GPC3, comprising:

(i) Culturing a host cell of the invention under conditions suitable for expression of the nucleic acid molecule or expression vector, and

(Ii) Isolating and purifying the antibody or antigen binding fragment thereof expressed by the nucleic acid molecule or expression vector.

The antibody of the present invention has excellent binding to GPC3 protein, and can bind to tumor cells expressing GPC3, especially liver cancer cells and small cell lung cancer cells. The antibody drug conjugate of the invention has excellent killing effect on tumor cells expressing GPC3, especially liver cancer cells and small cell lung cancer cells.

Drawings

FIG. 1 detection of EC50 of GPC3 antibody binding to HepG2 cells by flow cytometry;

FIG. 2 median fluorescence intensity (max MFI) at the maximum antibody concentration of GPC3 antibody binding to HepG2 cells was measured by flow cytometry.

Sequence description:

SEQ ID NO. 1: chimeric murine antibody light chain coding sequence (FS 001-mVL-LC (kappa))

SEQ ID NO. 2: humanized antibody light chain coding sequence (FS 001-VL1-LC (kappa))

SEQ ID NO. 3: humanized antibody light chain coding sequence (FS 001-VL2-LC (kappa))

SEQ ID NO. 4: humanized antibody light chain coding sequence (FS 001-VL3-LC (kappa))

SEQ ID NO. 5: humanized antibody light chain coding sequence (FS 001-VL4-LC (kappa))

SEQ ID NO. 6: chimeric murine antibody heavy chain coding sequence (FS 001-mVH-HC (IgG 1))

SEQ ID NO. 7: humanized antibody heavy chain coding sequence (FS 001-VH1-HC (IgG 1))

SEQ ID NO. 8: humanized antibody heavy chain coding sequence (FS 001-VH2-HC (IgG 1))

SEQ ID NO. 9: humanized antibody heavy chain coding sequence (FS 001-VH3-HC (IgG 1))

SEQ ID NO. 10: humanized antibody heavy chain coding sequence (FS 001-VH4-HC (IgG 1))

SEQ ID NO. 11: murine antibody light chain variable region (FS 001-mVL)

SEQ ID NO. 12: CDR1 of FS001-mVL (mVL-CDR 1)

SEQ ID NO. 13: CDR2 of FS001-mVL (mVL-CDR 2)

SEQ ID NO. 14: CDR3 of FS001-mVL (mVL-CDR 3)

SEQ ID NO. 15: humanized light chain variable region (FS 001-VL 1)

SEQ ID NO. 16: CDR1 of FS001-VL1 (VL 1-CDR 1)

SEQ ID NO. 17: CDR2 of FS001-VL1 (VL 1-CDR 2)

SEQ ID NO. 18: CDR3 of FS001-VL1 (VL 1-CDR 3)

SEQ ID NO. 19: humanized light chain variable region (FS 001-VL 2)

SEQ ID NO. 20: CDR1 of FS001-VL2 (VL 2-CDR 1)

SEQ ID NO. 21: CDR2 of FS001-VL2 (VL 2-CDR 2)

SEQ ID NO. 22: CDR3 of FS001-VL2 (VL 2-CDR 3)

SEQ ID NO. 23: humanized light chain variable region (FS 001-VL 3)

SEQ ID NO. 24: CDR1 of FS001-VL3 (VL 3-CDR 1)

SEQ ID NO. 25: CDR2 of FS001-VL3 (VL 3-CDR 2)

SEQ ID NO. 26: CDR3 of FS001-VL3 (VL 3-CDR 3)

SEQ ID NO. 27: humanized light chain variable region (FS 001-VL 4)

SEQ ID NO. 28: CDR1 of FS001-VL4 (VL 4-CDR 1)

SEQ ID NO. 29: CDR2 of FS001-VL4 (VL 4-CDR 2)

SEQ ID NO. 30: CDR3 of FS001-VL4 (VL 4-CDR 3)

SEQ ID NO. 31: murine antibody heavy chain variable region (FS 001-mVH)

SEQ ID NO. 32: CDR1 of FS001-mVH (mVH-CDR 1)

SEQ ID NO. 33: CDR2 of FS001-mVH (mVH-CDR 2)

SEQ ID NO. 34: CDR3 of FS001-mVH (mVH-CDR 3)

SEQ ID NO. 35: humanized heavy chain variable region (FS 001-VH 1)

SEQ ID NO. 36: CDR1 of FS001-VH1 (VH 1-CDR 1)

SEQ ID NO. 37: CDR2 of FS001-VH1 (VH 1-CDR 2)

SEQ ID NO. 38: CDR3 of FS001-VH1 (VH 1-CDR 3)

SEQ ID NO. 39: humanized heavy chain variable region (FS 001-VH 2)

SEQ ID NO. 40: CDR1 of FS001-VH2 (VH 2-CDR 1)

SEQ ID NO. 41: CDR2 of FS001-VH2 (VH 2-CDR 2)

SEQ ID NO. 42: CDR3 of FS001-VH2 (VH 2-CDR 3)

SEQ ID NO. 43: humanized heavy chain variable region (FS 001-VH 3)

SEQ ID NO. 44: CDR1 of FS001-VH3 (VH 3-CDR 1)

SEQ ID NO. 45: CDR2 of FS001-VH3 (VH 3-CDR 2)

SEQ ID NO. 46: CDR3 of FS001-VH3 (VH 3-CDR 3)

SEQ ID NO. 47: humanized heavy chain variable region (FS 001-VH 4)

SEQ ID NO. 48: CDR1 of FS001-VH4 (VH 4-CDR 1)

SEQ ID NO. 49: CDR2 of FS001-VH4 (VH 4-CDR 2)

SEQ ID NO. 50: CDR3 of FS001-VH4 (VH 4-CDR 3)

SEQ ID NO. 51: human light chain constant region

SEQ ID NO. 52: human heavy chain constant region

SEQ ID NO. 53: murine antibody light chain variable region+human light chain constant region (FS 001-mVL-LC (kappa))

SEQ ID NO. 54: humanized light chain variable region + human light chain constant region (FS 001-VL1-LC (kappa))

SEQ ID NO. 55: humanized light chain variable region + human light chain constant region (FS 001-VL2-LC (kappa))

SEQ ID NO. 56: humanized light chain variable region + human light chain constant region (FS 001-VL3-LC (kappa))

SEQ ID NO. 57: humanized light chain variable region + human light chain constant region (FS 001-VL4-LC (kappa))

SEQ ID NO. 58: murine antibody heavy chain variable region+human heavy chain constant region (FS 001-mVH-HC (IgG 1))

SEQ ID NO 59: humanized heavy chain variable region + human heavy chain constant region (FS 001-VH1-HC (IgG 1))

SEQ ID NO. 60: humanized heavy chain variable region + human heavy chain constant region (FS 001-VH2-HC (IgG 1))

SEQ ID NO. 61: humanized heavy chain variable region + human heavy chain constant region (FS 001-VH3-HC (IgG 1))

SEQ ID NO. 62: humanized heavy chain variable region + human heavy chain constant region (FS 001-VH4-HC (IgG 1))

SEQ ID NO. 63: CDR1 of a murine antibody light chain variable region (or humanized light chain variable region)

SEQ ID NO. 64: CDR2 of a murine antibody heavy chain variable region (or humanized heavy chain variable region)

Detailed Description

(One), general definition

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether naturally or partially or fully synthetically (e.g., recombinantly) produced, including any fragment thereof comprising at least a portion of the variable region of an immunoglobulin molecule that retains the binding specificity of a full-length immunoglobulin. Thus, antibodies include any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen binding domain (antibody binding site). Antibodies include antibody fragments, such as anti-tumor stem cell antibody fragments. As used herein, the term antibody thus includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as, but not limited to, fab fragments, fab ' fragments, F (ab ') ₂ fragments, fv fragments, disulfide-linked Fv (dsFv), fd fragments, fd ' fragments, single chain Fv (scFv), single chain Fab (scFab), diabodies, anti-idiotype (anti-Id) antibodies, or antigen-binding fragments of any of the above. Antibodies provided herein include members of any immunoglobulin class (e.g., igG, igM, igD, igE, igA and IgY), any class (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2), or subclass (e.g., igG2a and IgG2 b).

As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full length, but that comprises at least a portion of the variable region (e.g., one or more CDRs and/or one or more antibody binding sites) of the antibody that binds an antigen, and thus retains binding specificity as well as at least a portion of the specific binding capacity of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that comprises an antigen-binding portion that binds the same antigen as an antibody from which the antibody fragment was derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, fab ', F (ab ') ₂, single chain Fv (scFv), fv, dsFv, diabodies, fd, and Fd ' fragments, and others, including modified fragments (see, e.g., ,Methods in Molecular Biology,Vol 207:Recombinant Antibodies for Cancer Therapy Methods and Protocols(2003);Chapter 1;p 3-25,Kipriyanov)., which may include multiple chains linked together, e.g., by disulfide bonds and/or by peptide linkers, antibody fragments generally comprise at least or about 50 amino acids, and typically at least or about 200 amino acids, antigen binding fragments include any antibody fragment that upon insertion into an antibody framework (e.g., by replacement of the corresponding region) attains immunospecific binding (i.e., exhibits a Ka antigen of at least or at least about 10 ⁷-10⁸M^-1).

As used herein, "monoclonal antibody" refers to a population of identical antibodies, meaning that each individual antibody molecule in the monoclonal antibody population is identical to the other antibody molecules. This characteristic is in contrast to the characteristic of a polyclonal population of antibodies comprising antibodies having a plurality of different sequences. Monoclonal antibodies can be prepared by a number of well known methods (Smith et al (2004) J.Clin. Pathol.57,912-917; and Nelson et al, J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared by immortalizing B cells, e.g., by fusion with myeloma cells to produce hybridoma cell lines or by infecting B cells with a virus such as EBV. Recombinant techniques can also be used to produce antibodies from clonal populations of host cells in vitro by transforming the host cells with plasmids carrying artificial sequences of nucleotides encoding the antibodies.

As used herein, the term "hybridoma" or "hybridoma cell" refers to a cell or cell line (typically myeloma or lymphoma cells) produced by fusion of an antibody-producing lymphocyte and a non-antibody-producing cancer cell. As known to those of ordinary skill in the art, hybridomas can proliferate and continue to supply for the production of specific monoclonal antibodies. Methods for producing hybridomas are known in the art (see, e.g., harlow & Lane, 1988). When referring to the term "hybridoma" or "hybridoma cell," it also includes subclones and progeny cells of a hybridoma.

As used herein, "conventional antibody" refers to an antibody comprising two heavy chains (which may be labeled H and H ') and two light chains (which may be labeled L and L') and two antigen binding sites, wherein each heavy chain may be a full-length immunoglobulin heavy chain or any functional region thereof that retains antigen binding capacity (e.g., heavy chains including but not limited to V _H, V _H-C_H 1, and V _H-C_H1-C_H2-C_H 3 chains), and each light chain may be a full-length light chain or any functional region (e.g., light chains including but not limited to V _L and V _L-C_L chains). Each heavy chain (H and H ') is paired with a light chain (L and L', respectively).

As used herein, a full length antibody is an antibody having two full length heavy chains (e.g., V _H-C_H1-C_H2-C_H or V _H-C_H1-C_H2-C_H3-C_H) and two full length light chains (V _L-C_L) and a hinge region, e.g., an antibody naturally produced by B cells by antibody secretion and an antibody synthetically produced with the same domains.

As used herein, dsFv refers to Fv with an engineered intermolecular disulfide bond that stabilizes the V _H-V_L pair.

As used herein, fab fragments are antibody fragments obtained by digestion of full length immunoglobulins with papain, or fragments of the same structure synthetically produced, e.g., by recombinant methods. The Fab fragment comprises a light chain (comprising V _L and C _L) and another chain comprising the variable domain of the heavy chain (V _H) and one constant region domain of the heavy chain (C _H).

As used herein, a F (ab') ₂ fragment is an antibody fragment resulting from digestion of an immunoglobulin with pepsin at a pH of 4.0-4.5, or a fragment having the same structure, e.g., synthetically produced by recombinant methods. The F (ab') ₂ fragment essentially comprises two Fab fragments, wherein each heavy chain portion comprises an additional few amino acids, including cysteines forming disulfide bonds linking the two fragments.

As used herein, a Fab 'fragment is a fragment comprising half (one heavy and one light chain) of the F (ab') ₂ fragment.

As used herein, an scFv fragment refers to an antibody fragment comprising a variable light chain (V _L) and a variable heavy chain (V _H) covalently linked in any order by a polypeptide linker. The linker length is such that the two variable domains bridge substantially undisturbed. Exemplary linkers are (Gly-Ser) _n residues interspersed with some Glu or Lys residues to increase solubility.

The term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

By "humanized" antibody is meant a form of non-human (e.g., mouse) antibody that is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (e.g., fv, fab, fab ', F (ab') ₂ or other antigen-binding subsequence of the antibody) that contains minimal sequence derived from a non-human immunoglobulin. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the Complementarity Determining Regions (CDRs) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.

Furthermore, in humanization, it is also possible to mutate amino acid residues within the CDR1, CDR2 and/or CDR3 regions of VH and/or VL, thereby improving one or more binding properties (e.g., affinity) of the antibody. Mutations, such as PCR-mediated mutations, can be introduced, and their effect on antibody binding or other functional properties can be assessed using in vitro or in vivo assays described herein. Typically, conservative mutations are introduced. Such mutations may be amino acid substitutions, additions or deletions. In addition, mutations within the CDRs typically do not exceed one or two. Thus, the humanized antibodies of the present invention also encompass antibodies comprising 1 or 2 amino acid mutations within the CDRs.

As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitope determinants generally comprise chemically active surface groupings of molecules such as amino acids or sugar side chains, and generally have specific three dimensional structural characteristics as well as specific charge characteristics.

As used herein, a variable domain or variable region is a particular Ig domain of an antibody heavy or light chain that comprises amino acid sequences that vary between different antibodies. Each light chain and each heavy chain has one variable region domain V _L and V _H, respectively. The variable domains provide antigen specificity and are therefore responsible for antigen recognition. Each variable region comprises a CDR, which is part of an antigen binding site domain, and a Framework Region (FR).

As used herein, "antigen binding domain" and "antigen binding site" are synonymously used to refer to a domain within an antibody that recognizes and physically interacts with the cognate (cognate) antigen. The natural conventional full length antibody molecule has two conventional antigen binding sites, each comprising a heavy chain variable region portion and a light chain variable region portion. Conventional antigen binding sites comprise loops linking antiparallel β chains within the variable region domain. The antigen binding site may comprise other portions of the variable region domain. Each conventional antigen binding site comprises 3 hypervariable regions from the heavy chain and 3 hypervariable regions from the light chain. The hypervariable regions are also known as Complementarity Determining Regions (CDRs).

As used herein, "hypervariable region," "HV," "complementarity determining region," and "CDR," and "antibody CDR" are used interchangeably to refer to one of a plurality of portions within each variable region that together form an antigen binding site of an antibody. Each variable region domain contains 3 CDRs, designated CDR1, CDR2, and CDR3. For example, the light chain variable region domain comprises 3 CDRs, designated VL CDR1, VL CDR2, and VL CDR3; the heavy chain variable region domain comprises 3 CDRs, designated VH CDR1, VH CDR2 and VH CDR3. The 3 CDRs in the variable region are discontinuous along the linear amino acid sequence but are contiguous in the folded polypeptide. The CDRs are located within loops connecting parallel chains of the β -sheet of the variable domain. As described herein, CDRs are known to those of skill in the art and can be identified based on Kabat or Chothia numbering (see, e.g., ,Kabat,E.A.et al.(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242, and Chothia, c.et al (1987) j.mol.biol.196:901-917).

As used herein, a Framework Region (FR) is a domain located within the variable region domain of an antibody within the β -sheet; the FR region is relatively more conserved than the hypervariable region in terms of amino acid sequence.

As used herein, a "constant region" domain is a domain in an antibody heavy or light chain that comprises an amino acid sequence that is relatively more conserved than the amino acid sequence of a variable region domain. In conventional full length antibody molecules, each light chain has a single light chain constant region (C _L) domain, while each heavy chain comprises one or more heavy chain constant region (C _H) domains, including C _H1、C_H2、C_H and C _H 4. Full length IgA, igD and IgG isotypes comprise C _H1、C_H2、C_H and hinge regions, while IgE and IgM comprise Fab arms of C _H1、C_H2、C_H and C _H4.C_H 1 and C _L domain extended antibody molecules, thus facilitating interaction with antigen and turning of antibody arms. The antibody constant regions may serve effector functions such as, but not limited to, clearing antigens, pathogens, and toxins that the antibody specifically binds, such as by interacting with various cells, biomolecules, and tissues.

As used herein, a functional region of an antibody is an antibody portion comprising at least V _H、V_L、C_H (e.g., C _H1、C_H 2 or C _H3)、C_L or hinge region domains or at least functional regions thereof) of the antibody.

As used herein, a functional region of a V _H domain is at least a portion of a complete V _H domain that retains at least part of the binding specificity of the complete V _H domain (e.g., by retaining one or more CDRs of the complete V _H domain), such that the functional region of the V _H domain binds antigen alone or in combination with another antibody domain (e.g., V _L domain) or region thereof. The functional region of an exemplary V _H domain is a region comprising CDR1, CDR2, and/or CDR3 of the V _H domain.

As used herein, a functional region of a V _L domain is at least a portion of a complete V _L domain that retains at least part of the binding specificity of the complete V _L domain (e.g., by retaining one or more CDRs of the complete V _L domain), such that the functional region of the V _L domain binds antigen alone or in combination with another antibody domain (e.g., V _H domain) or region thereof. The functional region of an exemplary V _L domain is a region comprising CDR1, CDR2, and/or CDR3 of the V _L domain.

As used herein, "specific binding" or "immunospecifically binding" with respect to an antibody or antigen-binding fragment thereof is used interchangeably herein and refers to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with an alloantigen through non-covalent interactions between the antibody and the antibody binding site of the antigen. The antigen may be an isolated antigen or present in a tumor cell.

The term "kasloc" or "Ka" as used herein is intended to denote the rate of binding of a particular antibody-antigen interaction, while the term "Kdis" or "Kd" as used herein is intended to denote the rate of dissociation of a particular antibody-antigen interaction. The term "KD" as used herein is intended to mean the dissociation constant, which is derived from the ratio of KD to Ka (i.e. KD/Ka) and is expressed as the molar concentration (M). The KD value of an antibody can be determined using methods known in the art. Methods of determining the KD of an antibody may be, for example, surface Plasmon Resonance (SPR) (Rich and Myszka (2000) curr. Opin. Biotechnol11:54; englebiene (1998) analysis.123:1599), isothermal Titration Calorimetry (ITC) or other kinetic interaction assays known in the art (see, e.g., paul, ed., fundamental Immunology,2nd ed., RAVEN PRESS, new York, pages 332-336 (1989); see also U.S. Pat. No. 7,229,619) which describes exemplary SPR and ITC methods for calculating binding affinity of an antibody. Instruments and methods for detecting and monitoring binding rates in real time are known and commercially available (see ,BiaCore 2000,Biacore AB,Upsala,Sweden and GE Healthcare Life Sciences;Malmqvist(2000)Biochem.Soc.Trans.27:335).

The term "conservative sequence modification" or "conservative amino acid substitution" as used herein refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications may be introduced into the antibodies of the invention by standard techniques well known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A "conservative amino acid modification" or "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having similar properties and/or structure. Families of amino acid residues with similar side chains have been defined in the art.

There are 20 common amino acids and many classifications. For example, amino acids are classified according to their binding groups: amino acids can be classified into aliphatic amino acids, aromatic amino acids, heterocyclic amino acids, sulfur-containing amino acids, iodine-containing amino acids. Aliphatic amino acids include alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, lysine, arginine, glycine, serine, threonine, cysteine, asparagine, and glutamine. The aromatic amino acids include: phenylalanine and tyrosine. Heterocyclic amino acids include: histidine and tryptophan. Heterocyclic imino acids include: proline. Amino acid classification based on chemical properties: amino acids can be classified into acidic amino acids, basic amino acids, and neutral amino acids. Neutral amino acids include glycine, alanine, leucine, isoleucine, valine, cystine, cysteine, methionine, threonine, serine, phenylalanine, tyrosine, tryptophan, proline, methionine and hydroxyproline. Acidic amino acids include glutamic acid and aspartic acid. Basic amino acids include lysine, arginine, and histidine.

Furthermore, amino acids can also be classified as follows: amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

As used herein, "polypeptide" refers to two or more amino acids that are covalently linked. The terms "polypeptide" and "protein" are used interchangeably herein.

An "isolated protein," "isolated polypeptide," or "isolated antibody" means that the protein, polypeptide, or antibody (1) is not associated with components that accompany it in its natural state, (2) is free of other proteins from the same species, (3) is expressed by cells from a different species, or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cell system that differs from the naturally derived cell of the polypeptide will be "isolated" from its naturally associated components. The protein may also be isolated such that it is substantially free of naturally associated components, i.e., using protein purification techniques well known in the art.

"Substitution", "deletion" or "addition" of an amino acid residue refers to an amino acid modification in a peptide or protein that is conservative, does not alter the function and/or biological activity of the peptide or protein. For example, substitutions of amino acid residues may be conservative amino acid substitutions, suitable conservative amino acid substitutions being known to those skilled in the art, and generally may be made without altering the biological activity of the resulting molecule. In general, one skilled in the art recognizes that single amino acid substitutions in the non-essential region of a polypeptide do not substantially alter biological activity (see, e.g., watson et al Molecular Biology of the Gene,4th Edition,1987,The Benjamin/Cummings pub. Co., p.224).

As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) that are typically linked together by phosphodiester bonds.

As used herein, an isolated nucleic acid molecule is a nucleic acid molecule that is isolated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium when prepared by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibodies or antigen binding fragments.

Sequence "identity" has art-recognized meanings and the percent sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using the disclosed techniques. Sequence identity may be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule. (see, e.g., ：Computational Molecular Biology,Lesk,A.M.,ed.,Oxford University Press,New York,1988;Biocomputing:Informatics and Genome Projects,Smith,D.W.,ed.,Academic Press,New York,1993;Computer Analysis of Sequence Data,Part I,Griffin,A.M.,and Griffin,H.G.,eds.,Humana Press,New Jersey,1994;Sequence Analysis in Molecular Biology,von Heinje,G.,Academic Press,1987;and Sequence Analysis Primer,Gribskov,M.and Devereux,J.,eds.,M Stockton Press,New York,1991). although there are many methods of measuring identity between two polynucleotides or polypeptides, the term "identity" is well known to the skilled artisan (carrello, H. & Lipman, d., SIAM J APPLIED MATH 48:1073 (1988)).

As used herein, "operably linked" with respect to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other. For example, a promoter may be operably linked to a nucleic acid encoding a polypeptide such that the promoter regulates or mediates transcription of the nucleic acid.

As used herein, "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of a polypeptide can be assessed using any method known in the art, including, for example, methods of determining the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, coomassie blue staining after gel electrophoresis, lowry protein assay, and Bradford protein assay.

As used herein, a "host cell" is a cell that is used to receive, hold, replicate, and amplify a vector. Host cells may also be used to express the polypeptides encoded by the vectors. When the host cell is divided, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The host cell may be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, heLa cells, HEK cells such as HEK 293 cells.

As used herein, a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction digestion and ligation. Vectors also include those comprising nucleic acids encoding polypeptides. Vectors are used to introduce a nucleic acid encoding a polypeptide into a host cell, for amplifying the nucleic acid or for expressing/displaying the polypeptide encoded by the nucleic acid. Vectors typically remain episomal, but may be designed to integrate a gene or portion thereof into the chromosome of the genome. Vectors for artificial chromosomes are also contemplated, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles is well known to those skilled in the art.

As used herein, an "expression vector" includes vectors capable of expressing DNA operably linked to regulatory sequences, such as promoter regions, capable of affecting the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both. Thus, expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, that when introduced into an appropriate host cell results in expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic and/or prokaryotic cells as well as expression vectors that remain episomal or are integrated into the genome of a host cell.

As used herein, "treating" an individual with a disease or condition means that the symptoms of the individual are partially or fully alleviated, or remain unchanged after treatment. Thus, treatment includes prophylaxis, treatment and/or cure. Prevention refers to preventing an underlying disease and/or preventing worsening of symptoms or disease progression. Treatment also includes any antibody or antigen-binding fragment thereof provided, and any pharmaceutical use of the compositions provided herein.

As used herein, "therapeutic effect" refers to the effect resulting from treatment of an individual that alters, generally improves or ameliorates symptoms of, or cures a disease or condition.

As used herein, a "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, block or partially block the symptoms of a disease or disorder.

As used herein, a "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, e.g., prevent or delay the onset or recurrence of a disease or symptom, reducing the likelihood of the onset or recurrence of a disease or symptom. The fully prophylactically effective dose need not occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

As used herein, the term "patient" refers to a mammal, such as a human.

(II) antibodies targeting GPC3 protein

The light chain variable region comprises:

And/or the number of the groups of groups,

The heavy chain variable region comprises:

GPC3 is known as Glypican-3 (Glypican-3), wherein the human GPC3 protein is numbered P51654 in the Uniprot database, see https:// www.uniprot.org/Uniprot/P51654.GPC3 is a tumor-associated antigen that is specifically overexpressed by tumor cells, particularly in cancer patients such as liver cancer, small cell lung cancer, and the like.

For this potential target of GPC3 protein, the inventors designed chimeric antibodies using murine light/heavy chain variable regions and human light/heavy chain constant regions, as well as variants of a variety of different humanized antibodies. Experiments in fig. 1 and 2 demonstrate that the EC50 or max MFI binding capacity of the multiple monoclonal antibodies of the invention on HepG2 cells is better than that of the positive control antibody Codrituzumab. With the excellent binding capacity of the monoclonal antibodies of the invention, partner molecules such as cytotoxins can be efficiently delivered to tumor cells.

In some embodiments, the VL CDR1 comprises the amino acid sequence shown in SEQ ID NO. 12 or has 1 or 2 amino acid residue substitutions relative to SEQ ID NO. 12. In some embodiments, the VL CDR2 comprises the amino acid sequence shown in SEQ ID NO. 13 or has 1 or 2 amino acid residue substitutions relative to SEQ ID NO. 13. In some embodiments, the VL CDR3 comprises the amino acid sequence of SEQ ID NO. 14 or has 1 or 2 amino acid residue substitutions relative to SEQ ID NO. 14. In some embodiments, the amino acid substitution is a conservative amino acid substitution that does not alter the function and/or activity of the amino acid sequence.

In some embodiments, the VH CDR1 comprises the amino acid sequence shown in SEQ ID NO. 32 or has 1 or 2 amino acid residue substitutions relative to SEQ ID NO. 32. In some embodiments, the VH CDR2 comprises the amino acid sequence shown in SEQ ID NO. 33 or has 1 or 2 amino acid residue substitutions relative to SEQ ID NO. 33. In some embodiments, the VH CDR3 comprises the amino acid sequence shown as SEQ ID NO:34 or comprises 1 or 2 amino acid residue substitutions relative to SEQ ID NO: 34. In some embodiments, the amino acid substitution is a conservative amino acid substitution that does not alter the function and/or activity of the amino acid sequence.

In some embodiments, a monoclonal antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region comprising VL CDR1, VL CDR2, and VL CDR3.

In some embodiments, the VL CDR1 comprises or consists of the amino acid sequence of X ₁ SSQSLVHSNGNTYLQ (SEQ ID NO: 63) or of the amino acid sequence of X ₁ SSQSLVHSNGNTYLQ (SEQ ID NO: 63), wherein the symbol X ₁ represents:

-an aliphatic amino acid;

-an amino acid selected from any one of the following: alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, lysine, arginine, glycine, serine, threonine, cysteine, asparagine and glutamine; optionally: alanine or asparagine;

-an amino acid selected from any one of the following: lysine, arginine, and histidine; or alternatively

Basic amino acids, such as lysine (K) or arginine (R).

In some embodiments, the VL CDR1 comprises or consists of an amino acid sequence selected from the group consisting of: RSSQSLVHSNGNTYLQ and KSSQSLVHSNGNTYLQ.

In some embodiments, the VL CDR2 comprises or consists of an amino acid sequence shown by KVSNRFS.

In some embodiments, the VL CDR3 comprises or consists of the amino acid sequence shown at CSQSIHVPY or CSQSIHVPY.

In some embodiments, a monoclonal antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region comprising VH CDR1, VH CDR2, and VH CDR3.

In some embodiments, the VH CDR1 comprises or consists of the amino acid sequence shown at DYEMH or DYEMH.

In some embodiments, the VH CDR2 comprises or consists of the amino acid sequence shown as GIHPGSGGTAYX ₂QKFX₃ G (SEQ ID NO: 64),

Wherein the symbol X ₂ represents:

-an aliphatic amino acid;

-an amino acid selected from any one of the following: alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, lysine, arginine, glycine, serine, threonine, cysteine, asparagine and glutamine; or alternatively

Alanine (A) or asparagine (N), and/or

Wherein the symbol X ₃ represents:

-an aliphatic amino acid;

Lysine (K) or glutamine (Q).

In some embodiments, the VH CDR3 comprises or consists of the amino acid sequence shown at YYSFAYWGQG or YYSFAYWGQG.

In some embodiments, the monoclonal antibodies or antigen binding fragments thereof of the invention comprise a light chain variable region and a heavy chain variable region,

The light chain variable region comprises:

VL CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of: RSSQSLVHSNGNTYLQ and KSSQSLVHSNGNTYLQ, respectively,

VL CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of: KVSNRFS, and

VL CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of: CSQSIHVPY;

And/or the number of the groups of groups,

The heavy chain variable region comprises:

A VH CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of: DYEMH the process of the preparation of the pharmaceutical composition,

A VH CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of: GIHPGSGGTAYNQKFKG, GIHPGSGGTAYNQKFQG and GIHPGSGGTAYAQKFQG, and

A VH CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of: YYSFAYWGQG.

The light chain variable region comprises:

VL CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of: RSSQSLVHSNGNTYLQ the process of the preparation of the pharmaceutical composition,

And

The heavy chain variable region comprises:

A VH CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of: GIHPGSGGTAYNQKFKG, and

The light chain variable region comprises:

VL CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of: KSSQSLVHSNGNTYLQ the process of the preparation of the pharmaceutical composition,

And

The heavy chain variable region comprises:

A VH CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of: GIHPGSGGTAYAQKFQG, and

The light chain variable region comprises:

And

The heavy chain variable region comprises:

A VH CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of: GIHPGSGGTAYNQKFQG, and

In some embodiments, the invention provides a monoclonal antibody, or antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region, wherein the VL CDR1, VL CDR2, and VL CDR3, and VH CDR1, VH CDR2, and VH CDR3 in the light chain variable region and heavy chain variable region comprise or consist of the amino acid sequences shown in table 1 below:

TABLE 1

In some embodiments, a monoclonal antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 11 and a heavy chain variable region. In some embodiments, a monoclonal antibody or antigen binding fragment thereof of the invention comprises a light chain variable region comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID No. 11 and which has the same or similar biological function and/or activity as SEQ ID No. 11.

In some embodiments, a monoclonal antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 31. In some embodiments, a monoclonal antibody or antigen binding fragment thereof of the invention comprises a light chain variable region comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID No. 31 and said amino acid sequence has the same or similar biological function and/or activity as SEQ ID No. 31.

In some embodiments, the monoclonal antibodies of the invention, or antigen-binding fragments thereof, are capable of binding GPC3 protein in vitro with an EC50 of less than 1 nM. In some embodiments, the monoclonal antibodies of the invention, or antigen-binding fragments thereof, are capable of binding GPC3 protein on the surface of HepG2 cells with an EC50 of less than 5 nM. In some embodiments, the monoclonal antibodies or antigen binding fragments thereof of the invention can be effective in killing tumor cells (e.g., hepG 2) after conjugation to a cytotoxin (e.g., vcMMAE).

In some embodiments, a monoclonal antibody or antigen binding fragment thereof of the invention comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises or consists of an amino acid sequence selected from any one of: SEQ ID NOs 11, 15, 19, 23 and 27.

In some embodiments, a monoclonal antibody or antigen binding fragment thereof of the invention comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises or consists of an amino acid sequence selected from any one of: SEQ ID NOs 31, 35, 39, 43 and 47.

In some embodiments, a monoclonal antibody or antigen binding fragment thereof of the invention comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises or consists of an amino acid sequence selected from any one of: 11, 15, 19, 23 and 27; and wherein the heavy chain variable region comprises or consists of an amino acid sequence selected from any one of the following: SEQ ID NOs 31, 35, 39, 43 and 47.

In some embodiments, the invention provides monoclonal antibodies or antigen-binding fragments thereof comprising or consisting of the amino acid sequences shown below in table 2:

TABLE 2

In some embodiments, the monoclonal antibodies of the invention, or antigen-binding fragments thereof, further comprise a heavy chain constant region. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence: SEQ ID NO. 51.

In some embodiments, the monoclonal antibodies of the invention, or antigen binding fragments thereof, further comprise a light chain constant region. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence: SEQ ID NO. 52.

In some embodiments, the monoclonal antibodies of the invention, or antigen binding fragments thereof, further comprise a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region comprises or consists of the amino acid sequences: SEQ ID NO. 51; and the light chain constant region comprises or consists of the amino acid sequence: SEQ ID NO. 52.

In some embodiments, a monoclonal antibody or antigen binding fragment thereof of the invention comprises a heavy chain comprising a heavy chain variable region comprising or consisting of an amino acid sequence selected from any one of the following: 31, 35, 39, 43 and 47, and said heavy chain constant region comprises or consists of the amino acid sequence: SEQ ID NO. 51.

In some embodiments, a monoclonal antibody or antigen binding fragment thereof of the invention comprises a heavy chain and a light chain comprising a light chain variable region comprising or consisting of an amino acid sequence selected from any one of the following: 11, 15, 19, 23 and 27, and the light chain constant region comprises or consists of the amino acid sequence: SEQ ID NO. 52.

In some embodiments, a monoclonal antibody or antigen binding fragment thereof of the invention comprises a heavy chain comprising a heavy chain variable region comprising or consisting of an amino acid sequence selected from any one of the following: 31, 35, 39, 43 and 47, and said heavy chain constant region comprises or consists of the amino acid sequence: SEQ ID NO. 51; and the light chain comprises a light chain variable region comprising or consisting of an amino acid sequence selected from any one of the following: 11, 15, 19, 23 and 27, and the light chain constant region comprises or consists of the amino acid sequence: SEQ ID NO. 52.

In some embodiments, the invention provides monoclonal antibodies or antigen-binding fragments thereof comprising or consisting of the amino acid sequences shown below in table 3:

TABLE 3 Table 3

In some embodiments, the invention provides monoclonal antibodies or antigen-binding fragments thereof comprising or consisting of the amino acid sequences shown below in table 4:

TABLE 4 Table 4

(III), nucleic acid, vector, host cell, hybridoma and antibody production method

In one aspect, the invention provides an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the invention as described above. In some embodiments, the nucleic acid molecules of the invention encode the heavy and/or light chain variable regions, the individual CDRs in the heavy and/or light chain variable regions, and the heavy and/or light chain constant regions of the antibodies of the invention described previously. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon optimized for the host cell used for expression. In some embodiments, the nucleic acid molecules of the invention are operably linked to expression control sequences.

In some embodiments, a nucleic acid molecule of the invention comprises or consists of a nucleotide sequence selected from any one of the following: 1,2, 3, 4 and 5, and/or said nucleic acid molecule comprises or consists of a nucleotide sequence selected from any one of the following: SEQ ID NOs 6, 7, 8, 9 and 10. In some embodiments, the nucleic acid molecules of the invention comprise or consist of a nucleotide sequence selected from any one of the following combinations: SEQ ID NO. 1 and 6, SEQ ID NO. 7 and 2, SEQ ID NO. 8 and 2, SEQ ID NO. 9 and 2, SEQ ID NO. 10 and 2, SEQ ID NO. 8 and 3, SEQ ID NO. 9 and 3, SEQ ID NO. 10 and 3, SEQ ID NO. 7 and 4, SEQ ID NO. 8 and 4, SEQ ID NO. 9 and 4, SEQ ID NO. 10 and 4, SEQ ID NO. 8 and 5, SEQ ID NO. 9 and 5, and SEQ ID NO. 10 and 5.

The invention also provides an expression vector comprising at least one of the nucleic acid molecules of the invention as described above.

The invention also provides host cells transformed with at least one of the nucleic acid molecules or expression vectors of the invention described above.

The invention also provides hybridoma cells that express at least one of the monoclonal antibodies of the invention described above or antigen binding fragment portions thereof.

The invention also provides a method of producing an antibody or antigen-binding fragment thereof of the invention comprising:

(Ii) Isolating and purifying the antibody or antigen binding fragment thereof expressed by the host cell.

In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon optimized for the host cell used for expression. In some embodiments, the nucleic acid molecule is operably linked to an expression control sequence. In some embodiments, the nucleic acid molecule comprises or consists of a nucleotide sequence selected from any one of the following: 1,2, 3, 4 and 5, and/or said nucleic acid molecule comprises or consists of a nucleotide sequence selected from any one of the following: SEQ ID NOs 6, 7, 8, 9 and 10. In some embodiments, the nucleic acid molecule comprises or consists of a nucleotide sequence selected from any one of the following combinations: SEQ ID NO. 1 and 6, SEQ ID NO. 7 and 2, SEQ ID NO. 8 and 2, SEQ ID NO. 9 and 2, SEQ ID NO. 10 and 2, SEQ ID NO. 8 and 3, SEQ ID NO. 9 and 3, SEQ ID NO. 10 and 3, SEQ ID NO. 7 and 4, SEQ ID NO. 8 and 4, SEQ ID NO. 9 and 4, SEQ ID NO. 10 and 4, SEQ ID NO. 8 and 5, SEQ ID NO. 9 and 5, and SEQ ID NO. 10 and 5.

(i) Preparing a hybridoma cell capable of expressing a monoclonal antibody or antigen-binding fragment portion thereof of the invention, and

(Ii) Isolating and purifying the antibody or antigen binding fragment thereof expressed by the hybridoma cells.

(IV), antibody drug conjugates

In one aspect, the invention provides an Antibody Drug Conjugate (ADC) prepared from an Antibody or antigen binding fragment thereof of the invention.

Specifically, the invention provides an antibody drug conjugate of the formula Ab-L-D, wherein:

The symbol "D" represents a partner molecule.

As used herein, an ADC is prepared by linking a partner molecule to an antibody or antigen binding fragment thereof via a linker. In the ADC of the invention, the antibody performs a targeting function: the antibody directs the conjugate to a target tissue or cell in which its antigen is found by binding to the target tissue or cell. Where the linker is, for example, cleaved, releasing the partner molecule to perform its desired biological function.

The partner molecule may be a drug cargo or a marker molecule. The drug cargo may be a therapeutic agent, for example, the therapeutic agent may be, for example, a cytotoxic agent, a chemotherapeutic agent, a cytostatic agent, an immunomodulatory agent, and the like. In some embodiments, the therapeutic agent is a cytotoxic agent, i.e., a cytotoxin. As used herein, the terms "cytotoxic agent" and "cytotoxin" are used interchangeably.

The labeling molecule may be any label that produces a detectable signal, such as a radiolabel, a fluorescent label, or an enzyme that catalyzes a detectable modification of a substrate.

As used herein, the terms "drug", "payload" and "drug payload" are used interchangeably. As used herein, antibody-drug conjugates (ADCs) encompass conjugates formed by the attachment of an Antibody or antigen binding fragment thereof of the invention to a drug carrier, as well as conjugates formed by the attachment of an Antibody or antigen binding fragment thereof of the invention to a labeling molecule.

The proportion of the partner molecule that is linked to the antibody may vary depending on factors such as the amount of partner molecule used during the conjugation reaction and experimental conditions. The ratio of partner molecule to antibody may be 1 to 6,1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 to 1.5.

Accordingly, the present invention also provides an antibody drug conjugate, ab- (L-D) p, of the formula wherein:

the symbol "D" represents a partner molecule; and

In some embodiments, p is selected from the following values ：1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、 and 8.0.

In some embodiments, the linker of the invention is selected from one of the following: vc, mc-GGFG, acLysvc, mc, malPeg, m (H20) c, and m (H20) cvc; and/or the linker is cleavable. In some embodiments, the linker of the invention is vc.

In some embodiments, the partner molecule of the invention is a drug cargo that is a therapeutic molecule; optionally, the drug cargo is selected from one of: cytotoxic agents, cytostatic agents, immunomodulatory agents, and chemotherapeutic agents; optionally, the drug cargo is selected from one of: MMAE, MMAF, DM1, DM4, dxd, SN-38, topotecan and derivatives thereof, exatecan and derivatives thereof, belotecan and derivatives thereof, camptothecin, calicheamicin and PBD.

In some embodiments, the drug cargo is MMAE. "MMAE" refers to monomethyl auristatin E, as shown in the following figure, wherein the wavy line indicates covalent attachment to the linker (L) of the antibody drug conjugate:

In some embodiments, the drug cargo is MMAF. MMAF refers to monomethyl auristatin F, where the wavy line indicates covalent attachment to the linker (L) of the antibody drug conjugate (US 2005/023849):

In some embodiments, the linker-partner molecule is vcMMAE. The vcMMAE drug linker moiety and conjugation method are disclosed in WO2004010957, US7659241, US7829531, US7851437 and US 11/833,028 (SEATTLEGENETICS, inc.) which are incorporated herein by reference. The vcMMAE drug linker moiety is conjugated to the antibodies of the invention using methods similar to those disclosed in the above-mentioned documents.

For further discussion of the types of cytotoxins, linkers and methods for conjugating therapeutic agents to antibodies, see further Saito, g.et al (2003) adv. Drug deliv. Rev.55:199-215; trail, p.a. et al (2003)Cancer.Immunol.Immunother.52：328-337;Payne,G.(2003)Cancer Cell 3：207-212;Allen,T.M.(2002)Nat.Rev.Cancer 2：750-763;Pastan,I. and Kreitman, r.j. (2002) curr.opin.investig.drugs 3:1089-1091; senter, p.d. and Springer, c.j. (2001) adv. Drug deliv. Rev.53:247-264, which are incorporated herein by reference.

In some embodiments, the drug cargo may further be a radioisotope, thereby producing a cytotoxic radiopharmaceutical (also referred to as an antibody radioisotope conjugate). Examples of radioisotopes that have killing on tumor cells include, but are not limited to, iodine 131, indium 111, yttrium 90, and lutetium 177. Methods of preparing antibody radioisotope conjugates are known in the art.

In some embodiments, the partner molecule of the invention is a marker molecule; optionally, the marker molecule is selected from one of the following: radiolabels, fluorescent labels, and detectably modified enzymes. In some embodiments, the radiolabel is a radioisotope, such as iodine 131, indium 111, yttrium 90, and lutetium 177.

Experiments prove that the GPC3 protein can be used as an effective target of antibody drug conjugates. The antibody drug conjugate obtained by coupling the antibody with the small molecular toxin can generate excellent killing effect on tumors expressing GPC3 protein.

Fifth, pharmaceutical compositions, therapeutic uses and methods

The present invention provides pharmaceutical compositions comprising a therapeutically effective amount of at least one monoclonal antibody of the invention as described above or an antigen binding fragment portion thereof or at least one antibody drug conjugate of the invention as described above, therapeutic uses of such pharmaceutical compositions and methods of treatment. In the antibody drug conjugates of the invention, the antibodies of the invention are conjugated to a drug carrier.

In one aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one monoclonal antibody or antigen binding fragment portion thereof of the invention described above or at least one antibody drug conjugate of the invention described above. In some embodiments, the pharmaceutical compositions of the present invention may further comprise a pharmaceutically acceptable carrier.

A "therapeutically effective amount" of at least one of the foregoing monoclonal antibodies of the invention or antigen binding fragment portions thereof or at least one of the foregoing antibody drug conjugates of the invention preferably results in a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease asymptomatic periods, or prevention of injury or disability due to disease distress. For example, for the treatment of a tumor, a "therapeutically effective amount" of at least one of the foregoing monoclonal antibodies of the invention, or antigen-binding fragment portions thereof, or at least one of the foregoing antibody drug conjugates of the invention, preferably inhibits cell growth or tumor growth by at least about 10%, preferably by at least about 20%, more preferably by at least about 30%, more preferably by at least about 40%, more preferably by at least about 50%, more preferably by at least about 60%, more preferably by at least about 70%, more preferably by at least about 80%, relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in an animal model system that predicts efficacy against human tumors. Alternatively, it may be assessed by examining the ability to inhibit cell growth, which inhibition may be determined in vitro by assays well known to those of skill in the art. An effective amount of an antibody or antigen-binding fragment thereof of the invention is capable of reducing tumor size or otherwise alleviating a symptom in a subject, such as preventing and/or treating metastasis or recurrence. Such amounts may be determined by one skilled in the art based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.

In another aspect, the invention provides the use of at least one monoclonal antibody or antigen binding fragment portion thereof of the invention as described above or at least one antibody drug conjugate of the invention as described above in the preparation of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is used to treat malignancy, prevent and/or treat metastasis or recurrence of malignancy in a patient.

In another aspect, the present invention provides a pharmaceutical composition comprising at least one monoclonal antibody or antigen binding fragment portion thereof of the invention as described above or at least one antibody drug conjugate of the invention as described above for use in a method of treating malignancy, preventing and/or treating metastasis or recurrence of malignancy in a patient.

In another aspect, the present invention provides a method of treating malignancy, preventing and/or treating metastasis or recurrence of malignancy in a patient, the method comprising administering to the patient an effective amount of at least one monoclonal antibody or antigen-binding fragment portion thereof of the invention described above, at least one antibody drug conjugate of the invention described above, and/or at least one pharmaceutical composition of the invention described above.

In some embodiments, the malignancy is a tumor that expresses GPC3 protein. In some embodiments, the malignancy is selected from breast cancer, colorectal cancer, pancreatic cancer, prostate cancer, liver cancer, lung cancer (small cell lung cancer), and gastric cancer. In some embodiments, the malignancy is liver cancer or lung cancer (small cell lung cancer).

Sixth, diagnostic/prognostic compositions, diagnostic/prognostic uses and methods

As described herein, GPC3 is a tumor-associated antigen that is specifically overexpressed by tumor cells, particularly in cancer patients such as liver cancer, small cell lung cancer, and the like, and is expressed on the surface of tumor cells.

The present invention provides diagnostic/prognostic compositions comprising at least one monoclonal antibody or antigen-binding fragment portion thereof of the invention as described above or at least one antibody drug conjugate of the invention as described above, diagnostic/prognostic uses of such compositions and diagnostic/prognostic methods. In the diagnostic/prognostic drug conjugates of the present invention, the antibodies of the present invention are conjugated to a labeling molecule. For diagnostic/prognostic compositions, diagnostic/prognostic uses and diagnostic/prognostic methods, the antibody drug conjugates of the present invention described above refer to conjugates formed by the linking of an antibody or antigen-binding fragment thereof of the present invention to a marker molecule.

In one aspect, the invention provides the use of at least one monoclonal antibody or antigen binding fragment portion thereof of the invention as described above or at least one antibody drug conjugate of the invention as described above in the preparation of a diagnostic/prognostic composition. In some embodiments, the diagnostic composition is used to detect the presence of a malignancy in a patient. In some embodiments, the prognostic composition is used to prognose the recurrence or progression of a malignancy in a patient.

In another aspect, the present invention provides a method for detecting/diagnosing the presence of a malignancy in a patient, the method comprising administering to the patient an effective amount of at least one monoclonal antibody or antigen-binding fragment portion thereof of the invention described above, at least one antibody drug conjugate of the invention described above, and/or at least one diagnostic/prognostic composition of the invention described above.

In some embodiments, the invention provides a method of detecting/diagnosing the presence of a malignancy in a patient, comprising:

a) Contacting a biological sample obtained from said patient with at least one monoclonal antibody or antigen binding fragment portion thereof of the invention described above, at least one antibody drug conjugate of the invention described above, and/or at least one diagnostic/prognostic composition of the invention described above;

b) Detecting binding of at least one of the foregoing monoclonal antibodies of the invention or antigen-binding fragment portions thereof, at least one of the foregoing antibody drug conjugates of the invention, and/or at least one of the foregoing diagnostic/prognostic compositions of the invention to a target antigen in the biological sample, wherein detection of binding of the antibody to the target antigen is indicative of the presence of malignancy in the patient.

In another aspect, the invention provides a method for prognosis of recurrence or progression of a malignancy in a patient, the method comprising administering to the patient an effective amount of at least one monoclonal antibody or antigen-binding fragment portion thereof of the invention described above, at least one antibody drug conjugate of the invention described above, and/or at least one diagnostic/prognostic composition of the invention described above.

In some embodiments, the invention provides a method for prognosis of malignant tumor recurrence or progression in a patient, the method comprising:

(a) Isolating a biological sample comprising tumor cells from the patient;

(b) Contacting the biological sample comprising tumor cells with at least one of the foregoing monoclonal antibodies of the invention or antigen-binding fragment portions thereof, at least one of the foregoing antibody drug conjugates of the invention, and/or at least one of the foregoing diagnostic/prognostic compositions of the invention; and

(C) Identifying binding of the antibody to the tumor cell is indicative of the presence of the tumor cell,

In some embodiments, the progression of the malignancy comprises metastasis of the malignancy in a patient.

In some embodiments, the identification of the presence of tumor cells that bind to at least one of the foregoing monoclonal antibodies of the invention or antigen binding fragment portions thereof, at least one of the foregoing antibody drug conjugates of the invention, and/or at least one of the foregoing diagnostic/prognostic compositions of the invention is indicative of a high risk of recurrence or progression of a malignancy in the patient.

In some embodiments, the partner molecule of the invention is a marker molecule; optionally, the marker molecule is selected from one of the following: radiolabels (e.g., isotopes), fluorescent labels (e.g., fluorochromes), chemicals, and enzymes and tags that can detect modifications.

In some embodiments, the biological sample is selected from one of the following: blood samples, tissue samples, and lymph samples.

Methods for detecting antibody-antigen binding are known in the art, such as ELISA and the like.

(Seventh), kit

Also included within the scope of the invention is a kit for use in the methods of the invention, the kit comprising at least one monoclonal antibody of the invention described above or antigen binding fragment thereof, at least one antibody drug conjugate of the invention described above, at least one pharmaceutical composition of the invention described above, or one diagnostic/prognostic composition of the invention described above, and instructions for use.

In some embodiments, the kit may further comprise at least one additional detection reagent for detecting the presence of at least one of the aforementioned monoclonal antibodies of the invention or antigen binding fragments thereof, at least one of the aforementioned antibody drug conjugates of the invention, at least one of the aforementioned pharmaceutical compositions of the invention, or one of the aforementioned diagnostic/prognostic compositions of the invention. Kits generally include a label that indicates the intended use and/or method of use of the kit contents. The term label includes any written or recorded material provided on or with or otherwise with the kit.

Examples

A further understanding of the present application may be obtained by reference to the specific examples which are set forth to illustrate, but are not intended to limit the scope of the present application. It will be apparent that various modifications and variations can be made to the present application without departing from the spirit of the application, and therefore, such modifications and variations are also within the scope of the application as claimed.

EXAMPLE 1 preparation of GPC3 antibody

1. Vector construction

Design, vector construction, expression, purification, identification, and the like of the GPC3 antibody sequence of the present invention were completed by Santa biological medicine (Shanghai) Limited. The construction process mainly comprises the steps of constructing the sequence of the GPC3 antibody on a pcDNA3.4 vector, and obtaining plasmids after PCR, enzyme digestion, connection, transformation, identification, sequencing, alignment and extraction. Vectors comprising the sequence of the GPC3 antibody of the present invention are as follows (light chain 5 plasmids: 5 Kappa types; heavy chain 5 plasmids: 5 IgG1 types):

light chain coding sequence (shaded portion corresponds to constant region sequence):

Chimeric murine antibody light chain coding sequence: FS001-mVL-LC (kappa) (SEQ ID NO: 1) for expressing murine light chain variable region+human light chain constant region

Humanized antibody light chain coding sequence: FS001-VL1-LC (kappa) (SEQ ID NO: 2) for expressing a humanized light chain variable region+a human light chain constant region

Humanized antibody light chain coding sequence: FS001-VL2-LC (kappa) (SEQ ID NO: 3) for expressing a humanized light chain variable region+a human light chain constant region

Humanized antibody light chain coding sequence: FS001-VL3-LC (kappa) (SEQ ID NO: 4) for expressing a humanized light chain variable region+a human light chain constant region

Humanized antibody light chain coding sequence: FS001-VL4-LC (kappa) (SEQ ID NO: 5) for expressing a humanized light chain variable region+human light chain constant region

Heavy chain coding sequence (shaded portion corresponds to constant region sequence):

Chimeric murine antibody heavy chain coding sequence: FS001-mVH-HC (IgG 1) (SEQ ID NO: 6) for expressing murine heavy chain variable region+human heavy chain constant region

Humanized antibody heavy chain coding sequence: FS001-VH1-HC (IgG 1) (SEQ ID NO: 7) for expression of humanized heavy chain variable region+human heavy chain constant region

Humanized antibody heavy chain coding sequence: FS001-VH2-HC (IgG 1) (SEQ ID NO: 8) for expression of humanized heavy chain variable region+human heavy chain constant region

Humanized antibody heavy chain coding sequence: FS001-VH3-HC (IgG 1) (SEQ ID NO: 9) for expression of humanized heavy chain variable region+human heavy chain constant region

Humanized antibody heavy chain coding sequence: FS001-VH4-HC (IgG 1) (SEQ ID NO: 10) for expression of humanized heavy chain variable region+human heavy chain constant region

The amino acid sequences of the light chain variable region and the heavy chain variable region in the antibody expressed by the above vector are as follows:

light chain variable region (amino acid sequence):

( 1) Murine antibody light chain variable region (FS 001-mVL: SEQ ID NO. 11 )

The underlined italics are respectively labeled in turn mVL-CDR1(SEQ ID NO:12：RSSQSLVHSNGNTYLQ)、mVL-CDR2(SEQ ID NO:13：KVSNRFS)、mVL-CDR3(SEQ ID NO:14：CSQSIHVPY).

( 2) Humanized light chain variable region (FS 001-VL1: SEQ ID NO. 15 )

The underlined italics are respectively labeled in turn VL1-CDR1(SEQ ID NO:16：RSSQSLVHSNGNTYLQ)、VL1-CDR2(SEQ ID NO:17：KVSNRFS)、VL1-CDR3(SEQ ID NO:18：CSQSIHVPY).

( 3) Humanized light chain variable region (FS 001-VL2: SEQ ID NO. 19 )

The underlined italics are respectively labeled in turn VL2-CDR1(SEQ ID NO:20：RSSQSLVHSNGNTYLQ)、VL2-CDR2(SEQ ID NO:21：KVSNRFS)、VL2-CDR3(SEQ ID NO:22：CSQSIHVPY).

( 4) Humanized light chain variable region (FS 001-VL3: SEQ ID NO. 23 )

The underlined italics are respectively labeled in turn VL3-CDR1(SEQ ID NO:24：RSSQSLVHSNGNTYLQ)、VL3-CDR2(SEQ ID NO:25：KVSNRFS)、VL3-CDR3(SEQ ID NO:26：CSQSIHVPY).

( 5) Humanized light chain variable region (FS 001-VL4: SEQ ID NO. 27 )

The underlined italics are respectively labeled in turn VL4-CDR1(SEQ ID NO:28：KSSQSLVHSNGNTYLQ)、VL4-CDR2(SEQ ID NO:29：KVSNRFS)、VL4-CDR3(SEQ ID NO:30：CSQSIHVPY).

Heavy chain variable region (amino acid sequence):

( 1) Murine antibody heavy chain variable region (FS 001-mVH: SEQ ID NO. 31 )

The underlined italics indicate mVH-CDR1 (SEQ ID NO:32:)、mVH-CDR2(SEQ ID NO:33：)、mVH-CDR3(SEQ ID NO:34：)。

( 2) Humanized heavy chain variable region (FS 001-VH1: SEQ ID NO. 35 )

The underlined italics indicate VH1-CDR1 (SEQ ID NO:36:)、VH1-CDR2(SEQ ID NO:37：)、VH1-CDR3(SEQ ID NO:38：)。

( 3) Humanized heavy chain variable region (FS 001-VH2: SEQ ID NO. 39 )

The underlined italics indicate VH2-CDR1 (SEQ ID NO:40:)、VH2-CDR2(SEQ ID NO:41：)、VH2-CDR3(SEQ ID NO:42：)。

( 4) Humanized heavy chain variable region (FS 001-VH3: SEQ ID NO. 43 )

The underlined italics indicate VH3-CDR1 (SEQ ID NO:44:)、VH3-CDR2(SEQ ID NO:45：)、VH3-CDR3(SEQ ID NO:46：)。

( 5) Humanized heavy chain variable region (FS 001-VH4: SEQ ID NO. 47 )

The underlined italics indicate VH4-CDR1 (SEQ ID NO:48:)、VH4-CDR2(SEQ ID NO:49：)、VH4-CDR3(SEQ ID NO:50：)。

the heavy chain variable region is directly linked to the amino acid sequence of the human heavy chain constant region below, and the light chain variable region is directly linked to the amino acid sequence of the human light chain constant region below:

human light chain constant region (SEQ ID NO: 51):

Human heavy chain constant region (SEQ ID NO: 52):

the amino acid sequences of the full length heavy chain and full length light chain of the antibodies prepared in this example are as follows:

Light chain (amino acid sequence) (shaded portion corresponds to constant region sequence):

( 1) Murine antibody light chain variable region+human light chain constant region (FS 001-mVL-LC (kappa): SEQ ID NO. 53 )

( 2) Humanized light chain variable region + human light chain constant region (FS 001-VL1-LC (kappa): SEQ ID NO. 54 )

( 3) Humanized light chain variable region + human light chain constant region (FS 001-VL2-LC (kappa): SEQ ID NO. 55 )

( 4) Humanized light chain variable region + human light chain constant region (FS 001-VL3-LC (kappa): SEQ ID NO. 56 )

( 5) Humanized light chain variable region + human light chain constant region (FS 001-VL4-LC (kappa): SEQ ID NO. 57 )

Heavy chain (amino acid sequence) (shaded portion corresponds to constant region sequence):

( 1) Murine heavy chain variable region+human heavy chain constant region (FS 001-mVH-HC (IgG 1): SEQ ID NO. 58 )

( 2) Humanized heavy chain variable region + human heavy chain constant region (FS 001-VH1-HC (IgG 1): SEQ ID NO. 59 )

( 3) Humanized heavy chain variable region + human heavy chain constant region (FS 001-VH2-HC (IgG 1): SEQ ID NO. 60 )

( 4) Humanized heavy chain variable region + human heavy chain constant region (FS 001-VH3-HC (IgG 1): SEQ ID NO. 61 )

( 5) Humanized heavy chain variable region + human heavy chain constant region (FS 001-VH4-HC (IgG 1): SEQ ID NO. 62 )

The antibodies constructed in this example were as follows:

TABLE 1

2. Expression and purification of antibodies, and SDS-PAGE identification

The plasmids were expressed in CHO cells as required for 7 days using ExpiCHO-s expression system and purified using Protein A affinity chromatography on day 6 to give antibodies.

Subsequently, purity of the resulting antibody was checked by SDS-PAGE. In SDS-PAGE, reducing band purity or reducing heavy chain plus light chain sum purity is calculated by imageJ according to a peak area normalization method; the molecular weight of the non-reducing band of the reference IPI is about 150kDa, and the purity is more than 90 percent; the molecular weight of the reduced heavy chain is about 55kDa, the molecular weight of the light chain is about 25kDa, and the purity of the heavy chain and the light chain is more than 90 percent.

3. Affinity kinetic detection of antibodies

And opening GATOR the instrument and related software, and selecting a kinetic experimental mode. The analysis procedure was performed as follows:

TABLE 2

The results of the Global fit pattern affinity kinetics measurements were as follows:

TABLE 3 Table 3

Experimental results show that the correlation coefficient R ² of all antibodies in the Global fitting mode is larger than 0.95, meets the requirement of system adaptability, and has reliable results.

Example 2-determination of affinity of GPC3 antibodies to GPC3 at the protein level by ELISA

The affinity of the aforementioned GPC3 antibodies to GPC3 at the protein level was determined using the following method, including: ELISA plates were coated with GPC3-His (2. Mu.g/mL, 1X PBS) and incubated overnight at 4℃in 30. Mu.L/well; plates were washed 3 times with PBST and blocked with 5% pbs-milk for 2 hours at room temperature; the plates were washed and diluted antibodies (1% milk dilution) were added to the ELISA plates at 30 μl per well and incubated for 60 minutes at room temperature; plates were washed 3 times with PBST, then anti-human Fc-HRP (abcam; ab97225;1% milk dilution) was added at 1:4000 and incubated for 60 minutes at room temperature; plates were washed 3 times with PBST; adding tetramethyl benzidine, 30 mu L per well; the reaction was stopped with 2M stop solution and the OD450 reading was taken.

The results of the affinity assay are shown in table 4. As shown in the table, all 15 antibodies bind well to GPC3 protein with EC50 less than 1nM.

TABLE 4 Table 4

Example 3-determination of affinity of GPC3 antibodies to GPC3 at the cytological level by flow cytometry

Human hepatoma cells HepG2 were plated into 96-well plates, 50000 cells per well, and then washed twice with PBS repeatedly, and the PBS was removed. GPC3 antibody to be detected was used as primary antibody, and working concentration was 30nM prepared with 1% FBS/PBS solution, and diluted 3-fold in sequence, for a total of 5 specific concentration points. The prepared antibody dilutions were added to corresponding 96-well plates and incubated at 4℃for 30 min. The washing was then repeated twice with 1% FBS/PBS solution, and the antibody supernatant was aspirated after centrifugation with PBS. PBS solution with 10% fbs content was used according to 1:300 goat anti-human secondary IgG (h+l) was formulated to remove cross recognition capacity of other species and added to 96-well plates and incubated at 4 ℃ for 30 min. The affinity of the antibodies to GPC3 targets was assessed comprehensively by EC50 and median fluorescence intensity (Medium Fluorescence Intensity, MFI) at the highest concentration point, collected and analyzed using a flow cytometer. Antibody Condrituzumab (purchased from the Baiying organism) was used as a positive control antibody for binding to GPC 3.

The results of the EC50 determination are shown in figure 1. The results are shown in fig. 1: each of these 15 GPC3 antibodies was able to bind efficiently to HepG2 cells. Of these 15 antibodies, 11 antibodies had EC50 of less than 5nM. Specifically, the ability of FS001-huVH + huVL1 to bind to HepG2 cells was 1.43 times Codrituzumab, FS001-huVH + huVL1 was 1.66 times, FS001-huVH2+ huVL was 1.45 times, FS001-huVH + huVL2 was 1.82 times, FS001-huVH4+ huVL2 was 2.76 times, FS001-huVH4+ huVL3 was 1.89 times, and FS001-huVH4+ huVL4 was 1.45 times. The other 4 antibodies, including FS001-VH4-VL1, have slightly weaker binding capacities than the positive control antibody, but also bind GPC3 well.

The results of the MFI determination are shown in fig. 2. As shown in fig. 2, all of the 15 GPC3 antibodies could also bind to HepG2 cells.

Example 4 preparation of antibody-drug conjugate (ADC)

Antibody-drug conjugates were prepared using the following method

Materials and methods:

Coupling buffer: 25mM Na ₂B₄O₇, 25mM NaCl, 1mM DPTA, pH 7.4

Dialysis buffer: 1 XPBS, pH 7.4

Concentration of antibody reduction: 5mg/mL

Tris (2-chloroethyl) phosphate (TCEP): 5mM, binding buffer

TCEP: antibody = 2.3:1

Payload: 10mM linker-payload, dimethyl sulfoxide (DMSO)

DMSO in the final reaction: 10 percent of

Payload, antibody=10:1

Reduction temperature and time: 25 ℃ and 2 hours

Coupling temperature and time: 25 ℃ and 4 hours

And (3) a coupling step:

thawing and dialyzing the antibodies at 4 ℃ using a coupling buffer for >4 hours;

concentrating the antibody solution to 2-5mg/ml if necessary;

adding TCEP working solution to reduce antibody, and incubating in a water bath at 25 ℃;

preparing a linker-cargo solution in DMSO (10 mM);

Calculating the volume of each conjugated effective linker-cargo solution as a function of molar concentration;

mixing the antibody with the linker-payload in the desired molar ratio and incubating in a 25 ℃ water bath;

Dialyzing the conjugate at 4 ℃ using ADC storage buffer for more than 4 hours, replacing the buffer at least every 4 hours; and

The ADC was extracted and filtered using a 0.22 μm filter and the samples were submitted for UV, SEC-HPLC and HIC-HPLC analysis.

By the above method, antibody-drug conjugates of FS001-VH4-VL1, FS001-VH3-VL2, FS001-VH4-VL2 and FS001-VH4-VL3 were prepared in this example using vc-MMAE (mc-vc-PAB-MMAE, C68H105N11O15, supplied by Mitsubiquity chemistry) as linker-loads.

TABLE 5

By the above method, also using Dxd (MC-GGFG-DXD, HY13MY2801, supplied by Mitsui chemistry) as linker-payload, antibody-drug conjugates of FS001-VH3-VL2 and FS001-VH4-VL3 were prepared in this example.

TABLE 6

Example 5 biological Activity of antibody-drug conjugates (ADCs) Using toxin MMAE against cancer cell killing

This example tests the biological activity of the antibody conjugated toxins of the present invention against cancer cell killing after they were made into ADCs.

Human hepatoma cells HepG2 from ATCC, human hepatoma cells Huh7 from thomson and chinese hamster ovary cells CHO from negative control ATCC were plated in 96-well plates, 2500 per well. Incubate in a 5% CO ₂ incubator overnight at 37 ℃. The next day half of the medium of the cell plate was removed. A total medium was used to prepare a FS001-VH4-VL1、FS001-VH4-VL1-vc-MMAE、FS001-VH3-VL2-vc-MMAE、FS001-VH4-VL2-vc-MMAE、FS001-VH4-VL3-vc-MMAE、Codrituzumab-vc-MMAE(GPC3 positive control ADC at 2x, supplied by Miniture Chemie), hIgG1-vcmMAE (negative control ADC, supplied by Miniture Chemie), followed by 3-fold gradient dilutions of 11 spots of specific concentration, serial dilutions of FS001-VH4-VL1、FS001-VH4-VL1-vc-MMAE、FS001-VH3-VL2-vc-MMAE、FS001-VH4-VL2-vc-MMAE、FS001-VH4-VL3-vc-MMAE、Codrituzumab-vc-MMAE and hIgG1-vc-MMAE were added to appropriate wells and incubated in a 5% CO ₂ incubator at 37℃for 5 days. After the incubation, 100uL of Cell-Titer-Glo 2.0 reagent was added to the 96-well plate, mixed well on a plate shaker for 2 minutes, and finally equilibrated at room temperature for 10 minutes. The reading was performed using a microplate reader. The biological activity of the ADC on each cell line was finally compared by IC 50.

The results are shown in Table 7. The results are shown in table 7: FS001-VH4-VL1-vc-MMAE, FS001-VH3-VL2-vc-MMAE, FS001-VH4-VL2-vc-MMAE, and FS001-VH4-VL3-vc-MMAE had good killing effect on both human hepatoma cell lines HepG2 and HuH7, but no killing effect on negative control cells CHO. Neither the GPC3 antibody unconjugated vc-MMAE (FS 001-VH4-VL 1) nor the negative control hIgG1-vc-MMAE effectively killed HepG2 and HuH7 cells.

The above results suggest that GPC3 antibodies alone cannot be used to kill tumor cells in vitro, but surprisingly, GPC3 proteins were found to be effective targets for antibody drug conjugates. The antibody drug conjugate obtained by coupling the antibody with the small molecular toxin can generate excellent killing effect on tumors expressing GPC3 protein.

TABLE 7

Example 6 biological Activity of antibody-drug conjugate (ADC) Using toxin Dxd against cancer cells

Human hepatoma cells HepG2 from ATCC, human hepatoma cells Huh7 from thomson, human hepatoma cells JHH7 from thomson, and chinese hamster ovary cells CHO from negative control ATCC were plated in 96-well plates, 2500/well. Incubate in a 5% CO ₂ incubator overnight at 37 ℃. The next day half of the medium of the cell plate was removed. 2X sequential dilutions of FS001-VH3-VL2, FS001-VH4-VL3, FS001-VH3-VL2-Dxd, FS001-VH4-VL3-Dxd and hIgG1 (negative control antibody, supplied by Baiying organism) were prepared with complete medium followed by 3-fold gradient dilutions, 11 specific concentration points total, and FS001-VH3-VL2, FS001-VH4-VL3, FS001-VH3-VL2-Dxd, FS001-VH4-VL3-Dxd and hIgG1 were added to appropriate wells and incubated for 5 days in a 5% CO ₂ incubator at 37 ℃. After the incubation, 100uL of Cell-Titer-Glo 2.0 reagent was added to the 96-well plate, mixed well on a plate shaker for 2 minutes, and finally equilibrated at room temperature for 10 minutes. The reading was performed using a microplate reader. The biological activity of the ADC on each cell line was finally compared by IC 50.

The results are shown in table 8.

TABLE 8

The results are shown in the table: FS001-VH3-VL2-Dxd and FS001-VH4-VL3-Dxd have better killing effect on human liver cancer cell lines HepG2, huH7 and JHH7, but have no killing effect on negative control CHO cells, and the killing IC50 value is similar to the killing effect of the ADC drugs of vc-MMAE version in table 7, which indicates that the GPC3 antibody of the invention can realize excellent anti-tumor activity when being coupled with different types of small molecular toxins. Neither the GPC3 antibody unconjugated Dxd (FS 001-VH3-VL2, FS001-VH4-VL 3) nor the negative control antibody hIgG1 effectively killed HepG2, huH7 and JHH7 cells. The above results also suggest that GPC3 antibodies alone cannot be used to kill tumor cells in vitro, but GPC3 protein can be a target for effective antibody drug conjugates. The antibody drug conjugate obtained by coupling different types of small molecular toxins to the antibody can generate excellent killing effect on tumors expressing GPC3 protein.

The technical scheme is as follows:

1. A monoclonal antibody or antigen-binding fragment thereof that targets GPC3, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,

The light chain variable region comprises:

And/or the number of the groups of groups,

The heavy chain variable region comprises:

2. The isolated monoclonal antibody, or antigen-binding fragment thereof, of claim 1, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,

The light chain variable region comprises:

VL CDR1 comprising or consisting of the amino acid sequence shown in X ₁ SSQSLVHSNGNTYLQ,

Wherein the symbol X ₁ represents:

-an aliphatic amino acid;

-Lysine (K) or arginine (R);

VL CDR2 comprising or consisting of the amino acid sequence shown by KVSNRFS, and

VL CDR3 comprising or consisting of the amino acid sequence shown in CSQSIHVPY;

And/or

The heavy chain variable region comprises:

VH CDR1 comprising or consisting of the amino acid sequence shown in DYEMH,

VH CDR2 comprising or consisting of the amino acid sequence shown in GIHPGSGGTAYX ₂QKFX₃ G,

Wherein the symbol X ₂ represents:

-an aliphatic amino acid;

Alanine (A) or asparagine (N),

Wherein the symbol X ₃ represents:

-an aliphatic amino acid;

-Lysine (K) or glutamine (Q), and

VH CDR3 comprising or consisting of the amino acid sequence shown in YYSFAYWGQG.

3. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,

The light chain variable region comprises:

And/or the number of the groups of groups,

The heavy chain variable region comprises:

4. The isolated monoclonal antibody, or antigen-binding fragment thereof, of any one of claims 1-3, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,

The light chain variable region comprises:

And

The heavy chain variable region comprises:

5. The isolated monoclonal antibody, or antigen-binding fragment thereof, of any one of claims 1-3, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,

The light chain variable region comprises:

And

The heavy chain variable region comprises:

6. The isolated monoclonal antibody, or antigen-binding fragment thereof, of any one of claims 1-3, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,

The light chain variable region comprises:

And

The heavy chain variable region comprises:

7. The isolated monoclonal antibody, or antigen-binding fragment thereof, of any one of claims 1-6, wherein the monoclonal antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 11 or an amino acid sequence having at least 80%, 85%, at least 90%, at least 95% or more sequence identity to SEQ ID No. 11;

And/or

The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 31 or an amino acid sequence having at least 80%, 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 31.

8. The isolated monoclonal antibody, or antigen-binding fragment thereof, of claim 7, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,

The light chain variable region comprises or consists of an amino acid sequence selected from any one of the following: 11, 15, 19, 23 and 27;

And/or

The heavy chain variable region comprises or consists of an amino acid sequence selected from any one of the following: SEQ ID NOs 31, 35, 39, 43 and 47.

9. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 7 or 8, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,

Wherein the light chain variable region and heavy chain variable region each comprise or consist of an amino acid sequence selected from the group consisting of: SEQ ID NO. 11 and SEQ ID NO. 31, SEQ ID NO. 15 and SEQ ID NO. 35, SEQ ID NO. 15 and SEQ ID NO. 39, SEQ ID NO. 15 and SEQ ID NO. 43, SEQ ID NO. 15 and SEQ ID NO. 47, SEQ ID NO. 19 and SEQ ID NO. 39, SEQ ID NO. 19 and SEQ ID NO. 43, SEQ ID NO. 19 and SEQ ID NO. 47, SEQ ID NO. 23 and SEQ ID NO. 35, SEQ ID NO. 23 and SEQ ID NO. 39, SEQ ID NO. 23 and SEQ ID NO. 43, SEQ ID NO. 23 and SEQ ID NO. 47, SEQ ID NO. 27 and SEQ ID NO. 39, SEQ ID NO. 27 and SEQ ID NO. 43, and SEQ ID NO. 27 and SEQ ID NO. 47.

10. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-9,

The monoclonal antibody comprises a heavy chain constant region; alternatively, the monoclonal antibody comprises a heavy chain constant region comprising or consisting of the amino acid sequence: SEQ ID NO. 51;

And/or

The monoclonal antibody comprises a light chain constant region; alternatively, the monoclonal antibody comprises a light chain constant region comprising or consisting of the amino acid sequence: SEQ ID NO. 52.

11. The isolated monoclonal antibody, or antigen-binding fragment thereof, of any one of claims 1-10, comprising a heavy chain and a light chain,

The heavy chain comprises a heavy chain variable region comprising or consisting of an amino acid sequence selected from any one of the following: 31, 35, 39, 43 and 47, and said heavy chain constant region comprises or consists of the amino acid sequence: SEQ ID NO. 51; and/or

The light chain comprises a light chain variable region comprising or consisting of an amino acid sequence selected from any one of the following: 11, 15, 19, 23 and 27, and the light chain constant region comprises or consists of the amino acid sequence: SEQ ID NO. 52.

12. The isolated monoclonal antibody, or antigen-binding fragment thereof, of any one of claims 1-11, comprising a heavy chain and a light chain,

The heavy chain comprises or consists of an amino acid sequence selected from any one of the following: SEQ ID NOs 58, 59, 60, 61 and 62; and

The light chain comprises or consists of an amino acid sequence selected from any one of the following: SEQ ID NOs 53, 54, 55, 56 and 57.

13. The isolated monoclonal antibody, or antigen-binding fragment thereof, of any one of claims 1-12, comprising a heavy chain and a light chain, the heavy chain and the light chain each comprising an amino acid sequence selected from the group consisting of: SEQ ID NO 58 and SEQ ID NO 53, SEQ ID NO 59 and SEQ ID NO 54, SEQ ID NO 60 and SEQ ID NO 54, SEQ ID NO 61 and SEQ ID NO 54, SEQ ID NO 62 and SEQ ID NO 54, SEQ ID NO 60 and SEQ ID NO 55, SEQ ID NO 61 and SEQ ID NO 55, SEQ ID NO 62 and SEQ ID NO 55, SEQ ID NO 59 and SEQ ID NO 56, SEQ ID NO 60 and SEQ ID NO 56, SEQ ID NO 61 and SEQ ID NO 56, SEQ ID NO 62 and SEQ ID NO 56, SEQ ID NO 60 and SEQ ID NO 57, SEQ ID NO 61 and SEQ ID NO 57, and SEQ ID NO 62 and SEQ ID NO 57.

14. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-13,

Wherein all monoclonal antibodies are capable of binding GPC3 protein in vitro with an EC50 of less than 1 nM; and/or

Wherein the monoclonal antibody is capable of binding GPC3 protein on the surface of HepG2 cells with an EC50 of less than 5 nM.

15. An isolated nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-14; or alternatively

The nucleic acid molecule comprises or consists of a nucleotide sequence selected from any one of the following: 1, 2, 3, 4 and 5, and/or said nucleic acid molecule comprises or consists of a nucleotide sequence selected from any one of the following: SEQ ID NOs 6, 7, 8, 9 and 10; or alternatively

The nucleic acid molecule comprises or consists of a nucleotide sequence selected from any one of the following combinations: SEQ ID NO. 1 and 6, SEQ ID NO. 7 and 2, SEQ ID NO. 8 and 2, SEQ ID NO. 9 and 2, SEQ ID NO. 10 and 2, SEQ ID NO. 8 and 3, SEQ ID NO. 9 and 3, SEQ ID NO. 10 and 3, SEQ ID NO. 7 and 4, SEQ ID NO. 8 and 4, SEQ ID NO. 9 and 4, SEQ ID NO. 10 and 4, SEQ ID NO. 8 and 5, SEQ ID NO. 9 and 5, and SEQ ID NO. 10 and 5.

16. The isolated nucleic acid molecule of claim 15, said nucleic acid molecule being operably linked to an expression control sequence.

17. An expression vector comprising the nucleic acid molecule of claim 15 or 16.

18. A host cell comprising the nucleic acid molecule of claim 15 or 16 or the expression vector of claim 17.

19. A hybridoma cell expressing the monoclonal antibody or antigen binding fragment portion thereof of any one of claims 1-14.

20. An antibody drug conjugate of the formula:

Ab-L-D, wherein:

The symbol "D" represents a partner molecule;

Or alternatively

Ab- (L-D) p, wherein:

the symbol "D" represents a partner molecule; and

The symbol "p" represents the number of linker/partner molecules (preferably drug carriers) conjugated to the aforementioned monoclonal antibodies of the invention or antigen binding fragment portions thereof; alternatively, p is selected from the following values ：1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、 and 8.0.

21. The antibody drug conjugate of claim 20, wherein the linker is selected from one of: vc, mc-GGFG, acLysvc, mc, malPeg, m (H20) c, and m (H20) cvc; and/or the linker is cleavable.

22. The antibody drug conjugate of claim 20 or 21 wherein

The partner molecule is a drug cargo, which is a therapeutic molecule; optionally, the drug cargo is selected from one of: cytotoxic agents, cytostatic agents, immunomodulatory agents, and chemotherapeutic agents; optionally, the drug cargo is selected from one of: MMAE, MMAF, DM1, DM4, dxd, SN-38, topotecan and derivatives thereof, exatecan and derivatives thereof, belotecan and derivatives thereof, camptothecin, calicheamicin and PBD; or alternatively

The partner molecule is a labeling molecule; optionally, the marker molecule is selected from one of the following: radiolabels (e.g., isotopes), fluorescent labels (e.g., fluorochromes), chemicals, and enzymes and tags that can detect modifications.

23. The antibody drug conjugate of any of claims 20-22, wherein the monoclonal antibody or antigen-binding fragment portion thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region and heavy chain variable region each comprise or consist of an amino acid sequence set forth in any combination of: SEQ ID NO. 23 and SEQ ID NO. 47;

preferably, the monoclonal antibody comprises a heavy chain and a light chain, said heavy chain and said light chain comprising or consisting of the amino acid sequences shown in the following combinations, respectively: SEQ ID NO. 62 and SEQ ID NO. 56;

Wherein the linker is mc-GGFG; and is also provided with

The drug load is Dxd and p is 3.5 to 8.0.

24. A pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-14 or the antibody drug conjugate of any one of claims 20-23, and optionally a pharmaceutically acceptable carrier.

25. Use of the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-14 or the antibody drug conjugate of any one of claims 20-23 in the preparation of a pharmaceutical composition; optionally, the pharmaceutical composition is for treating malignancy, preventing and/or treating metastasis or recurrence of malignancy in a patient; optionally, the pharmaceutical composition is for detecting the presence of a malignancy in a patient; alternatively, the pharmaceutical composition is for prognosis of malignant tumor recurrence or progression in a patient.

26. A method of treating malignancy, preventing and/or treating metastasis or recurrence of malignancy in a patient, the method comprising administering to the patient an effective amount of the monoclonal antibody or antigen-binding fragment portion thereof of any one of claims 1-14, the antibody drug conjugate of any one of claims 20-23, or the pharmaceutical composition of claim 24.

27. The method of claim 26, wherein the malignancy is selected from the group consisting of breast cancer, colorectal cancer, pancreatic cancer, prostate cancer, liver cancer, lung cancer (small cell lung cancer), and gastric cancer.

28. The method of claim 26 or 27, further comprising administering to the patient other anti-tumor therapeutic means, such as administration of a chemotherapeutic agent, an antibody targeting other tumor-specific antigens, or radiation therapy.

29. A method of detecting the presence of tumor cells in a biological sample, comprising:

a) Contacting the biological sample with the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-14;

b) Detecting binding of said monoclonal antibody or antigen binding fragment thereof to a target antigen in said biological sample,

30. A method of detecting the presence of a malignancy in a patient, comprising:

a) Contacting a biological sample obtained from the patient with the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-14;

31. A method for prognosis of malignant tumor recurrence or progression in a patient, the method comprising:

(a) Isolating a biological sample comprising tumor cells from the patient;

(b) Contacting the biological sample comprising tumor cells with the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-14; and

32. The method of claim 31, wherein the progression of the malignancy comprises metastasis of the malignancy in a patient.

33. The method of claim 31 or 32, wherein the biological sample is selected from one of the following: blood samples, tissue samples, and lymph samples.

34. The method of any one of claims 31-33, wherein the malignancy is selected from the group consisting of breast cancer, colorectal cancer, pancreatic cancer, prostate cancer, liver cancer, lung cancer (small cell lung cancer), and gastric cancer.

35. A method of producing a monoclonal antibody or antigen-binding fragment thereof that targets human tumor cells expressing GPC3, comprising:

(i) Culturing the host cell of claim 18 with expression of said nucleic acid molecule or expression vector, and

Claims

A monoclonal antibody or antigen-binding fragment thereof that targets GPC3, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,

The light chain variable region comprises:

VL CDR1 comprising the amino acid sequence shown in SEQ ID NO. 12 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO. 12,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO. 13 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO. 13, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO. 14 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO. 14;

And/or the number of the groups of groups,

The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO. 32 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO. 32,

A VH CDR2 comprising the amino acid sequence shown in SEQ ID NO. 33 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO. and

A VH CDR3 comprising the amino acid sequence shown in SEQ ID No. 34 or comprising an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID No. 34.
The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,

The light chain variable region comprises:

VL CDR1 comprising or consisting of the amino acid sequence shown in X ₁ SSQSLVHSNGNTYLQ,

Wherein the symbol X ₁ represents:

-an aliphatic amino acid;

-an amino acid selected from any one of the following: alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, lysine, arginine, glycine, serine, threonine, cysteine, asparagine and glutamine; optionally: alanine or asparagine;

-an amino acid selected from any one of the following: lysine, arginine, and histidine; or alternatively

-Lysine (K) or arginine (R);

VL CDR2 comprising or consisting of the amino acid sequence shown by KVSNRFS, and

VL CDR3 comprising or consisting of the amino acid sequence shown in CSQSIHVPY;

And/or

The heavy chain variable region comprises:

VH CDR1 comprising or consisting of the amino acid sequence shown in DYEMH,

VH CDR2 comprising or consisting of the amino acid sequence shown in GIHPGSGGTAYX ₂QKFX₃ G,

Wherein the symbol X ₂ represents:

-an aliphatic amino acid;

-an amino acid selected from any one of the following: alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, lysine, arginine, glycine, serine, threonine, cysteine, asparagine and glutamine; or alternatively

Alanine (A) or asparagine (N),

Wherein the symbol X ₃ represents:

-an aliphatic amino acid;

-an amino acid selected from any one of the following: alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, lysine, arginine, glycine, serine, threonine, cysteine, asparagine and glutamine; or alternatively

-Lysine (K) or glutamine (Q), and

VH CDR3 comprising or consisting of the amino acid sequence shown in YYSFAYWGQG.
The isolated monoclonal antibody or antigen-binding fragment thereof of claim 2, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,

The light chain variable region comprises:

VL CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of: RSSQSLVHSNGNTYLQ and KSSQSLVHSNGNTYLQ, respectively,

VL CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of: KVSNRFS, and

VL CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of: CSQSIHVPY;

And/or the number of the groups of groups,

The heavy chain variable region comprises:

A VH CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of: DYEMH the process of the preparation of the pharmaceutical composition,

A VH CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of: GIHPGSGGTAYNQKFKG, GIHPGSGGTAYNQKFQG and GIHPGSGGTAYAQKFQG, and

A VH CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of: YYSFAYWGQG.
The isolated monoclonal antibody or antigen-binding fragment thereof of claim 3, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,

The light chain variable region comprises:

VL CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of: RSSQSLVHSNGNTYLQ the process of the preparation of the pharmaceutical composition,

VL CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of: KVSNRFS, and

VL CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of: CSQSIHVPY;

And

The heavy chain variable region comprises:

A VH CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of: DYEMH the process of the preparation of the pharmaceutical composition,

A VH CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of: GIHPGSGGTAYNQKFKG, and

A VH CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of: YYSFAYWGQG.
The isolated monoclonal antibody or antigen-binding fragment thereof of claim 4, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,

The light chain variable region comprises:

VL CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of: KSSQSLVHSNGNTYLQ the process of the preparation of the pharmaceutical composition,

VL CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of: KVSNRFS, and

VL CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of: CSQSIHVPY;

And

The heavy chain variable region comprises:

A VH CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of: DYEMH the process of the preparation of the pharmaceutical composition,

A VH CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of: GIHPGSGGTAYAQKFQG, and

A VH CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of: YYSFAYWGQG.
The isolated monoclonal antibody or antigen-binding fragment thereof of claim 5, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,

The light chain variable region comprises:

VL CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of: RSSQSLVHSNGNTYLQ the process of the preparation of the pharmaceutical composition,

VL CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of: KVSNRFS, and

VL CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of: CSQSIHVPY;

And

The heavy chain variable region comprises:

A VH CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of: DYEMH the process of the preparation of the pharmaceutical composition,

A VH CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of: GIHPGSGGTAYNQKFQG, and

A VH CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of: YYSFAYWGQG.
The isolated monoclonal antibody, or antigen-binding fragment thereof, of claim 1, wherein the monoclonal antibody comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID No. 11 or an amino acid sequence having at least 80%, 85%, at least 90%, at least 95% or more sequence identity to SEQ ID No. 11;

And/or

The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 31 or an amino acid sequence having at least 80%, 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 31.
The isolated monoclonal antibody or antigen-binding fragment thereof of claim 7, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,

The light chain variable region comprises or consists of an amino acid sequence selected from any one of the following: 11, 15, 19, 23 and 27;

And/or

The heavy chain variable region comprises or consists of an amino acid sequence selected from any one of the following: SEQ ID NOs 31, 35, 39, 43 and 47.
The isolated monoclonal antibody or antigen-binding fragment thereof of claim 7 or 8, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,

Wherein the light chain variable region and heavy chain variable region each comprise or consist of an amino acid sequence selected from the group consisting of: SEQ ID NO. 11 and SEQ ID NO. 31, SEQ ID NO. 15 and SEQ ID NO. 35, SEQ ID NO. 15 and SEQ ID NO. 39, SEQ ID NO. 15 and SEQ ID NO. 43, SEQ ID NO. 15 and SEQ ID NO. 47, SEQ ID NO. 19 and SEQ ID NO. 39, SEQ ID NO. 19 and SEQ ID NO. 43, SEQ ID NO. 19 and SEQ ID NO. 47, SEQ ID NO. 23 and SEQ ID NO. 35, SEQ ID NO. 23 and SEQ ID NO. 39, SEQ ID NO. 23 and SEQ ID NO. 43, SEQ ID NO. 23 and SEQ ID NO. 47, SEQ ID NO. 27 and SEQ ID NO. 39, SEQ ID NO. 27 and SEQ ID NO. 43, and SEQ ID NO. 27 and SEQ ID NO. 47.
The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1 or 9,

The monoclonal antibody comprises a heavy chain constant region; alternatively, the monoclonal antibody comprises a heavy chain constant region comprising or consisting of the amino acid sequence: SEQ ID NO. 51;

And/or

The monoclonal antibody comprises a light chain constant region; alternatively, the monoclonal antibody comprises a light chain constant region comprising or consisting of the amino acid sequence: SEQ ID NO. 52.
The isolated monoclonal antibody or antigen-binding fragment thereof of claim 10, comprising a heavy chain and a light chain,

The heavy chain comprises a heavy chain variable region comprising or consisting of an amino acid sequence selected from any one of the following: 31, 35, 39, 43 and 47, and said heavy chain constant region comprises or consists of the amino acid sequence: SEQ ID NO. 51; and/or

The light chain comprises a light chain variable region comprising or consisting of an amino acid sequence selected from any one of the following: 11, 15, 19, 23 and 27, and the light chain constant region comprises or consists of the amino acid sequence: SEQ ID NO. 52.
The isolated monoclonal antibody or antigen-binding fragment thereof of claim 11, comprising a heavy chain and a light chain,

The heavy chain comprises or consists of an amino acid sequence selected from any one of the following: SEQ ID NOs 58, 59, 60, 61 and 62; and

The light chain comprises or consists of an amino acid sequence selected from any one of the following: SEQ ID NOs 53, 54, 55, 56 and 57.
The isolated monoclonal antibody or antigen binding fragment thereof of claim 12, comprising a heavy chain and a light chain, the heavy chain and the light chain comprising or consisting of an amino acid sequence selected from any one of the following combinations: SEQ ID NO 58 and SEQ ID NO 53, SEQ ID NO 59 and SEQ ID NO 54, SEQ ID NO 60 and SEQ ID NO 54, SEQ ID NO 61 and SEQ ID NO 54, SEQ ID NO 62 and SEQ ID NO 54, SEQ ID NO 60 and SEQ ID NO 55, SEQ ID NO 61 and SEQ ID NO 55, SEQ ID NO 62 and SEQ ID NO 55, SEQ ID NO 59 and SEQ ID NO 56, SEQ ID NO 60 and SEQ ID NO 56, SEQ ID NO 61 and SEQ ID NO 56, SEQ ID NO 62 and SEQ ID NO 56, SEQ ID NO 60 and SEQ ID NO 57, SEQ ID NO 61 and SEQ ID NO 57, and SEQ ID NO 62 and SEQ ID NO 57.

Preferably, said heavy chain and said light chain comprise or consist of the amino acid sequences shown in the following combinations, respectively: SEQ ID NO. 62 and SEQ ID NO. 57.
The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1 or 7,

Wherein the monoclonal antibody is capable of binding GPC3 protein in vitro with an EC50 of less than 1 nM; and/or

Wherein the monoclonal antibody is capable of binding GPC3 protein on the surface of HepG2 cells with an EC50 of less than 5 nM.
An antibody drug conjugate of the formula:

Ab- (L-D) p, wherein:

the symbol "Ab" represents at least one monoclonal antibody or antigen binding fragment portion thereof according to any one of claims 1-14;

The symbol "L" represents a linker linking the antibody moiety and the partner molecule;

the symbol "D" represents a partner molecule; and

The symbol "p" represents the amount of drug cargo conjugated to at least one monoclonal antibody or antigen binding fragment portion thereof according to any one of claims 1-14, p being selected from the following values ：1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、 and 8.0.
The antibody drug conjugate of claim 20, wherein the linker is selected from one of: vc, mc-GGFG, acLysvc, mc, malPeg, m (H20) c, and m (H20) cvc; and/or the linker is cleavable.
The antibody drug conjugate of claim 15 or 16, wherein

The drug cargo is selected from one of the following: cytotoxic agents, cytostatic agents, immunomodulatory agents, and chemotherapeutic agents; preferably, the drug load is selected from one of the following: MMAE, MMAF, DM1, DM4, dxd, SN-38, topotecan and derivatives thereof, exatecan and derivatives thereof, belotecan and derivatives thereof, camptothecin, calicheamicin and PBD.
The antibody drug conjugate of any of claims 15-17, wherein the monoclonal antibody or antigen-binding fragment portion thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region and heavy chain variable region each comprise or consist of an amino acid sequence set forth in any combination of: SEQ ID NO. 23 and SEQ ID NO. 47;

preferably, the monoclonal antibody comprises a heavy chain and a light chain, said heavy chain and said light chain comprising or consisting of the amino acid sequences shown in the following combinations, respectively: SEQ ID NO. 62 and SEQ ID NO. 56;

Wherein the linker is mc-GGFG; and is also provided with

The drug load is Dxd and p is 3.5 to 8.0.
A pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-14 or the antibody drug conjugate of any one of claims 15-18, and optionally a pharmaceutically acceptable carrier.
Use of the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-14 or the antibody drug conjugate of any one of claims 15-18 in the preparation of a pharmaceutical composition; optionally, the pharmaceutical composition is for treating malignancy, preventing and/or treating metastasis or recurrence of malignancy in a patient; optionally, the pharmaceutical composition is for detecting the presence of a malignancy in a patient; alternatively, the pharmaceutical composition is for prognosis of malignant tumor recurrence or progression in a patient.
A method of treating malignancy, preventing and/or treating metastasis or recurrence of malignancy in a patient, the method comprising administering to the patient an effective amount of the monoclonal antibody or antigen-binding fragment portion thereof according to any one of claims 1-14, the antibody drug conjugate according to any one of claims 15-18, or the pharmaceutical composition of claim 19.
The method of claim 21, wherein the malignancy is selected from the group consisting of breast cancer, colorectal cancer, pancreatic cancer, prostate cancer, liver cancer, lung cancer (small cell lung cancer), and gastric cancer.