TW202144433A - Antibody or antigen-binding fragment, preparation method and pharmaceutical use thereof - Google Patents

Abstract

The invention relates to antibody or antigen-binding fragment, preparation method and pharmaceutical use thereof. Concretely, the invention relates to chimeric antibody and humanized antibody comprising CDR region of anti-GPC3 antibody, pharmaceutical composition comprising the anti-GPC3 antibody or the antigen-binding fragment thereof, and use as an anti-cancer medicament thereof. Specifically, this invention relates to humanized anti-GPC3 antibody, the preparation of medicament for treating diseases or conditions mediated by GPC3 thereof, and used as cell growth inhibitors or used for tumor detection and diagnosis thereof.

Description

Antibody or antigen-binding fragment thereof, preparation method and medical use thereof

The present invention relates to an anti-GPC3 antibody specifically immunoreactive to human GPC3 receptor, or an antigen-binding fragment thereof, a chimeric antibody comprising the CDR region of the anti-GPC3 antibody, a humanized antibody, and a human anti-GPC3 antibody Pharmaceutical compositions of antibodies and antigen-binding fragments thereof, as well as their use as cytostatic and anticancer drugs and use in detecting or diagnosing tumors.

Glypican 3 (Glypican3, GPC3) is a 70 kDa membrane protein belonging to the Glypicans family that functions as an extracellular matrix in organogenesis and plays a role in cell adhesion or as a receptor for cell growth factors. After GPC3 expression, it is cleaved by furin enzyme to generate the N-terminal 40kd soluble part and the 30kd part anchored to the C-terminus of the cell membrane by GPI molecules.

GPC3 is expressed in embryonic tissues, especially liver and kidney, and is an extracellular matrix protein involved in organ formation. In adult tissues, the expression of GPC3 is not observed outside the placenta, but it is observed in various cancer tissues such as hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, and lung squamous cell carcinoma. Seen, of GPC3 and AFP (α -fetoprotein; AFP), carcinoembryonic antigen (Carcinoembryonic antigen; CEA) and other proteins are similarly expressed in embryonic tissue protein, and therefore is classified as embryonal carcinoma antigen. That is, GPC3 exhibits characteristics that are not expressed in normal tissue cells but specifically expressed in cancer cells, and thus serves as a target molecule and tumor marker for cancer therapy. In addition, genomics and functional studies have shown that GPC3 plays an important role in maintaining the Wnt pathway and the initiation of the Hedgehogs pathway. For example, GPC3-coupled heparan sulfate molecules can enhance the binding of Wnts to their receptors and thus play an important role in maintaining the Wnt pathway. . GPC3 is expressed in the brain, digestive tract, bladder, gonad and skin and is highly expressed on the surface of hepatocellular carcinoma; Wnt pathway plays an important role in hepatocarcinogenesis, such as mutations in β- Catenin pathway and Frizzled-7 receptor in 20% of hepatocellular carcinomas Overexpression, so GPC3 may play a role in promoting the development of some hepatocellular carcinomas.

According to some embodiments of the present invention, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof comprising an antibody heavy chain variable region and an antibody light chain variable region, wherein the antibody heavy chain variable region comprises at least one selected from the group consisting of HCDR shown in the following sequences: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 13 ID NO: 14 or SEQ ID NO: 15,

The antibody light chain variable region comprises at least one LCDR selected from the following sequences: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26.

In some embodiments, the heavy chain variable region of an anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: HCDR1 set forth in SEQ ID NO:7, HCDR2 set forth in SEQ ID NO:10, and SEQ ID NO: HCDR3 shown in 13.

In some embodiments, the heavy chain variable region of an anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: HCDR1 set forth in SEQ ID NO:8, HCDR2 set forth in SEQ ID NO:11, and SEQ ID NO: HCDR3 shown in 14.

In some embodiments, the heavy chain variable region of an anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: HCDR1 set forth in SEQ ID NO:9, HCDR2 set forth in SEQ ID NO:12, and SEQ ID NO: HCDR3 shown in 15.

In some embodiments, the light chain variable region of an anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: LCDR1 as set forth in SEQ ID NO: 16, LCDR2 as set forth in SEQ ID NO: 21, and SEQ ID NO: 24 shown in LCDR3.

In some embodiments, the light chain variable region of an anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: LCDR1 set forth in SEQ ID NO: 17, LCDR2 set forth in SEQ ID NO: 22, and SEQ ID NO: LCDR3 shown in 25.

In some embodiments, the light chain variable region of an anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: LCDR1 set forth in SEQ ID NO: 18, LCDR2 set forth in SEQ ID NO: 23, and SEQ ID NO: LCDR3 shown in 26.

In some embodiments, the light chain variable region of an anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: LCDR1 as set forth in SEQ ID NO: 19, LCDR2 as set forth in SEQ ID NO: 21, and SEQ ID NO: 24 shown in LCDR3.

In some embodiments, the light chain variable region of an anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: LCDR1 set forth in SEQ ID NO:20, LCDR2 set forth in SEQ ID NO:23, and SEQ ID NO: LCDR3 shown in 26.

In some embodiments, the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: HCDR1 set forth in SEQ ID NO:7, HCDR2 set forth in SEQ ID NO:10, and HCDR3 set forth in SEQ ID NO:13; And the variable region of the light chain of the antibody comprises: LCDR1 shown in SEQ ID NO: 16, LCDR2 shown in SEQ ID NO: 21 and LCDR3 shown in SEQ ID NO: 24.

In some embodiments, the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: HCDR1 set forth in SEQ ID NO:8, HCDR2 set forth in SEQ ID NO:11, and HCDR3 set forth in SEQ ID NO:14; And the variable region of the light chain of the antibody comprises: LCDR1 shown in SEQ ID NO: 17, LCDR2 shown in SEQ ID NO: 22 and LCDR3 shown in SEQ ID NO: 25.

In some embodiments, the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: HCDR1 set forth in SEQ ID NO:9, HCDR2 set forth in SEQ ID NO:12, and HCDR3 set forth in SEQ ID NO:15; And the variable region of the light chain of the antibody comprises: LCDR1 shown in SEQ ID NO: 18, LCDR2 shown in SEQ ID NO: 23 and LCDR3 shown in SEQ ID NO: 26.

In some embodiments, the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: the antibody heavy chain variable region comprises: HCDR1 set forth in SEQ ID NO:7, HCDR2 set forth in SEQ ID NO:10, and SEQ ID NO:10 HCDR3 shown in ID NO: 13; and the antibody light chain variable region comprises: LCDR1 shown in SEQ ID NO: 19, LCDR2 shown in SEQ ID NO: 21, and LCDR3 shown in SEQ ID NO: 24.

In some embodiments, the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: the antibody heavy chain variable region comprises: HCDR1 set forth in SEQ ID NO:9, HCDR2 set forth in SEQ ID NO:12, and SEQ ID NO:12 HCDR3 shown in ID NO: 15; and the antibody light chain variable region comprises: LCDR1 shown in SEQ ID NO: 20, LCDR2 shown in SEQ ID NO: 23, and LCDR3 shown in SEQ ID NO: 26.

In some embodiments, the anti-GPC3 antibody or antigen-binding fragment thereof according to the invention is selected from the group consisting of: murine antibody or antigen-binding fragment thereof, chimeric antibody or antigen-binding fragment thereof, human antibody or antigen-binding fragment thereof, humanized Antibodies or antigen-binding fragments thereof.

In some embodiments, according to the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgGl, IgG2, IgG3 or IgG4 or a variant thereof ;

Preferably, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, IgG2 or IgG4;

More preferably, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as shown in SEQ ID NO:52.

In some embodiments, according to the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises an IgGl heavy chain constant region that is amino acid mutated to have enhanced ADCC toxicity.

In some embodiments, according to the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from human kappa chain, lambda chain, or a variant thereof;

Preferably, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from human kappa chain;

More preferably, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a light chain constant region as shown in SEQ ID NO:53.

In some embodiments, the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention, wherein the anti-GPC3 antibody or antigen-binding fragment thereof comprises a variable heavy chain selected from the sequence shown below region, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared to: SEQ ID NO: 27, SEQ ID NO: 29 , SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:37.

In some embodiments, the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention, wherein the anti-GPC3 antibody or antigen-binding fragment thereof comprises a light chain variable region selected from, or has a Light chain variable regions of at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity: SEQ ID NO:28, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:38.

In a preferred embodiment, the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention, wherein the anti-GPC3 antibody or antigen-binding fragment thereof comprises:

The heavy chain variable region shown in SEQ ID NO:27 and the light chain variable region shown in SEQ ID NO:28;

The heavy chain variable region shown in SEQ ID NO:29 and the light chain variable region shown in SEQ ID NO:28;

The heavy chain variable region shown in SEQ ID NO:30 and the light chain variable region shown in SEQ ID NO:28;

The heavy chain variable region shown in SEQ ID NO:31 and the light chain variable region shown in SEQ ID NO:28;

The heavy chain variable region shown in SEQ ID NO:32 and the light chain variable region shown in SEQ ID NO:33;

The heavy chain variable region shown in SEQ ID NO:32 and the light chain variable region shown in SEQ ID NO:34;

The heavy chain variable region shown in SEQ ID NO:32 and the light chain variable region shown in SEQ ID NO:35;

The heavy chain variable region set forth in SEQ ID NO:32 and the light chain variable region set forth in SEQ ID NO:36; or

The heavy chain variable region shown in SEQ ID NO:37 and the light chain variable region shown in SEQ ID NO:38.

In some embodiments, the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention, wherein the anti-GPC3 antibody or antigen-binding fragment thereof contains a heavy chain selected from the sequence shown below, or with Heavy chains having at least 80%, 85%, 90%, 95%, or 99% identity compared to the following sequences: SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 or SEQ ID NO:49.

In some embodiments, the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention, wherein the anti-GPC3 antibody or antigen-binding fragment thereof contains a light chain selected from the group consisting of the following sequences, or has at least 80% compared to the following sequences , 85%, 90%, 95% or 99% identity light chain: SEQ ID NO:40, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO : 50.

In a specific embodiment, the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention, wherein the anti-GPC3 antibody comprises:

The heavy chain set forth in SEQ ID NO:39 and the light chain set forth in SEQ ID NO:40;

The heavy chain set forth in SEQ ID NO:42 and the light chain set forth in SEQ ID NO:40;

The heavy chain set forth in SEQ ID NO:43 and the light chain set forth in SEQ ID NO:40;

The heavy chain set forth in SEQ ID NO:44 and the light chain set forth in SEQ ID NO:45;

The heavy chain set forth in SEQ ID NO:44 and the light chain set forth in SEQ ID NO:46;

The heavy chain set forth in SEQ ID NO:44 and the light chain set forth in SEQ ID NO:47;

The heavy chain set forth in SEQ ID NO:44 and the light chain set forth in SEQ ID NO:48; or,

The heavy chain shown in SEQ ID NO:49 and the light chain shown in SEQ ID NO:50.

According to some embodiments of the present invention, there is provided a polynucleotide encoding an anti-GPC3 antibody or antigen-binding fragment thereof of the present invention.

According to some embodiments of the present invention, there is provided an expression vector comprising a polynucleotide of the present invention.

According to some embodiments of the present invention, there is provided a host cell which introduces or contains the expression vector of the present invention. In a specific embodiment, the host cells are bacteria, preferably Escherichia coli.

In another specific embodiment, the host cell is a yeast, preferably Pichia.

In another specific embodiment, the host cells are mammalian cells, preferably CHO cells or HEK293 cells.

According to some embodiments of the present invention, there is provided a method of producing an anti-GPC3 antibody, comprising the steps of culturing a host cell of the present invention, isolating the antibody from the culture, and purifying the antibody.

According to some embodiments of the present invention, there is provided a pharmaceutical composition comprising the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable excipient, diluent or carrier.

According to some embodiments of the present invention, there is provided a detection or diagnostic kit comprising an anti-GPC3 antibody or antigen-binding fragment thereof of the present invention and an excipient, diluent or carrier useful for detection or diagnosis.

According to some embodiments of the present invention, there is provided a detection or diagnostic kit comprising an anti-GPC3 antibody or antigen-binding fragment thereof of the present invention, and optionally, one or more detection of the anti-GPC3 antibody or antigen-binding fragment thereof Reagents that bind to GPC3 or its epitope.

According to some embodiments of the present invention, there is provided an anti-GPC3 antibody or an antigen-binding fragment thereof of the present invention, or the use of the above-mentioned pharmaceutical composition in the preparation of a medicament for treating or preventing a GPC3-mediated disease or condition.

According to some embodiments of the present invention, there is provided the use of an anti-GPC3 antibody or antigen-binding fragment thereof, or the above-mentioned pharmaceutical composition according to the present invention, in the preparation of a kit, wherein the kit is used for detecting or diagnosing GPC3-mediated disease or condition.

In some embodiments, according to the above-described use of the invention, the disease or disorder is cancer.

In preferred embodiments, the disease or disorder is cancer expressing GPC3.

In a more preferred embodiment, the cancer is selected from breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma cell tumor and melanoma.

In specific embodiments, the disease or disorder is selected from the group consisting of: hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, lung squamous cell carcinoma.

According to some embodiments of the present invention, there is provided a method of treating or preventing a GPC3-mediated disease, comprising the step of providing a therapeutically effective amount or a prophylactically effective amount of an anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention to a subject .

According to some embodiments of the present invention, there is provided a method of treating or preventing a GPC3-mediated disease comprising the step of providing a subject with a therapeutically effective amount or a prophylactically effective amount of a pharmaceutical composition according to the present invention.

In some embodiments, the subject is suspected of having, has had, and is susceptible to a GPC3-mediated disease selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, Lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophagus cancer, cervical cancer, gallbladder cancer, glioblastoma and melanoma, preferably hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, lung squamous cell carcinoma.

The anti-GPC3 antibody or antigen-binding fragment of the present invention can specifically bind to the GPC3 antigen (or its epitope) and cells expressing GPC3, and has significant CDC activity and ADCC activity, and has a significant killing effect on tumors.

In addition, the anti-GPC3 antibody or the antigen-binding fragment thereof of the present invention has good endocytosis, and is suitable for conjugation with drugs to construct ADC. While maintaining high specificity and killing activity, the anti-GPC3 antibody or antigen-binding fragment of the present invention has lower immunogenicity and higher stability, and is significantly better than Codrituzumab (anti-Glypican 3 monoclonal antibody). ). The anti-GPC3 antibody or antigen-binding fragment of the present invention has better potential as an anti-cancer drug and ensures drug safety.

Detailed description of the invention

1. Terminology

For easier understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere herein, all other technical and scientific terms used herein have the meaning as commonly understood by one of ordinary skill in the art.

The three-letter and one-letter codes for amino acids used in the present invention are as described in J. Biol. Chem, 243, p3558 (1968).

The term "antibody" in the present invention refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement sequence of the constant region of immunoglobulin heavy chain are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ, δ, γ, alpha and epsilon chains. The same class of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of disulfide bonds in the heavy chain. For example, IgG can be divided into different subclasses. IgG1, IgG2, IgG3, IgG4. Light chains are classified into kappa chains or lambda chains by differences in constant regions. Each of the five classes of Ig can have a kappa chain or a lambda chain.

In the present invention, the antibody light chain variable region of the present invention may further comprise a light chain constant region comprising human or murine κ, λ chains or variants thereof.

In the present invention, the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region comprising human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.

The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions. The order from the amino end to the carboxyl end is: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3. The number and position of CDR amino acid residues in the VL and VH regions of the antibodies or antigen-binding fragments of the present invention conform to the known Kabat numbering rules and Kabat or ABM definition rules (http://bioinf.org.uk /abs/).

The term "antigen presenting cell" or "APC" is a cell that displays a foreign antigen complexed with MHC on its surface. T cells recognize this complex using the T cell receptor (TCR). Examples of APCs include, but are not limited to, dendritic cells (DCs), topical blood mononuclear cells (PBMCs), monocytes, B lymphoblasts, and monocyte-derived dendritic cells.

The term "antigen presentation" refers to the process by which APCs capture antigens and enable their recognition by T cells, eg, as a component of MHC-I/MHC-II conjugates.

The term "GPC3" includes any variant or isoform of GPC3 that is naturally expressed by a cell. Antibodies of the invention can cross-react with GPC3 derived from non-human species. Alternatively, the antibody may also be specific for human GPC3 and may not exhibit cross-reactivity with other species. GPC3, or any variant or isoform thereof, can be isolated from the cells or tissues in which they are naturally expressed, or produced by recombinant techniques using those techniques commonly used in the art and described herein. Preferably, the anti-GPC3 antibody targets human GPC3 with a normal glycosylation pattern.

The term "recombinant human antibody" includes human antibodies prepared, expressed, created or isolated by recombinant methods, the techniques and methods involved are well known in the art, such as:

1. Antibodies isolated from transgenic human immunoglobulin genes, transchromosomic animals (eg mice) or fusion tumors prepared therefrom;

2. Antibodies isolated from host cells transformed to express the antibodies, such as transfectomas;

3. An antibody isolated from a recombinant combinatorial human antibody library; and

4. Antibodies prepared, expressed, created or isolated by methods such as splicing of human immunoglobulin gene sequences to other DNA sequences.

Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as those that occur during antibody maturation.

The term "murine antibody" in the present invention is a monoclonal antibody to human GPC3 prepared according to the knowledge and skills in the art. In preparation, test articles are injected with GPC3 antigen, and fusionomas expressing antibodies with the desired sequence or functional properties are isolated. In a preferred embodiment of the present invention, the murine GPC3 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine κ, λ chain or a variant thereof, or further comprise murine IgG1, IgG2, Heavy chain constant regions of IgG3 or IgG4 or variants thereof.

The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. Human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human backbone sequences (ie, "humanized antibodies").

The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by grafting mouse CDR sequences into the framework of human antibody variable regions. Humanized antibodies can overcome the disadvantage of strong immune responses induced by chimeric antibodies because they carry a large number of mouse protein components. To avoid a decrease in activity while decreasing immunogenicity, the variable regions of the human antibody can be subjected to minimal reverse mutations to maintain activity.

The term "chimeric antibody" is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To build a chimeric antibody, select a fusion tumor that secretes a mouse-specific monoclonal antibody, then select the variable region gene from the mouse fusion tumor cells, and then select the constant region gene of the human antibody as needed. The variable region gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in the eukaryotic industrial system or the prokaryotic industrial system. The constant region of the human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or its variants, preferably comprising the heavy chain constant region of human IgG1, IgG2 or IgG4, or using amino acid mutation An IgG1 heavy chain constant region that enhances ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity.

The term "antigen-binding fragment" refers to antigen-binding fragments and antibody analogs of antibodies, which generally include at least a portion of the antigen-binding or variable regions (eg, one or more CDRs) of a parental antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody. Usually, when When the activity is expressed on a molar basis, the antibody fragment retains at least 10% of its parent binding activity. Preferably the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody for the target. Examples of antigen-binding fragments include, but are not limited to: Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson, 2005, Nat. Biotechnol. 23: 1126-1136.

A "Fab fragment" consists of the CH1 and variable regions of one light chain and one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

The "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domains.

A "Fab' fragment" contains a light chain and a portion of a heavy chain comprising the VH and CH1 domains and the region between the CH1 and CH2 domains, thus allowing between the two heavy chains of two Fab' fragments Interchain disulfide bonds are formed to form F(ab')2 molecules.

An "F(ab')2 fragment" contains two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

"Fv regions" comprise variable regions from both heavy and light chains, but lack constant regions.

The term "multispecific antibody" is used in its broadest sense to encompass antibodies with specificity for multiple epitopes. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH-VL unit has polyepitope specificity; has two or more VL and VH Region antibodies, each VH-VL unit binds to a different target or a different epitope of the same target; antibodies with two or more single variable regions, each single variable region to a different target point or the same target Binding to different epitopes; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, antibody fragments that have been covalently or non-covalently linked together, etc.

The term "single-chain antibody" is a single-chain recombinant protein composed of the heavy chain variable region VH and light chain variable region VL of an antibody linked by a linking peptide, which is the smallest antibody fragment with a complete antigen-binding site.

The term "domain antibody fragment" is an immunologically functional immunoglobulin fragment containing only the variable region of the heavy chain or the variable region of the light chain. In certain instances, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody fragment. The two VH regions of a bivalent domain antibody fragment can target the same or different antigens.

The term "binding to GPC3" in the present invention refers to the ability to interact with human GPC3.

The term "antigen-binding site" of the present invention refers to a three-dimensional site recognized by an antibody or antigen-binding fragment of the present invention.

The term "epitope" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from adjacent amino acids, or non-adjacent amino acids that are juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically include at least 3-15 amino acids in unique spatial conformations. Methods for determining what epitopes are bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation assays, among others. Methods for determining the spatial conformation of epitopes include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.

The terms "specifically binds" and "selectively binds" as used in the present invention refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, when used as an analyte-human GPC3 antibody, using as a ligand, measured by surface plasmon resonance technology (SPR) in the instrument, the antibody is lower than about 10 ^{- 7} M solution equilibrium or even smaller dissociation constant ( K _D ) binds to a predetermined antigen with at least twice the affinity for binding to the predetermined antigen or non-specific antigens other than the predetermined antigen or closely related antigens (eg, BSA, etc.). The term "antibody that recognizes an antigen" is used interchangeably herein with the term "antibody that specifically binds".

The term "cross-reactivity" refers to the ability of an antibody of the invention to bind to GPC3 from different species. For example, an antibody of the invention that binds to human GPC3 can also bind to GPC3 of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays (eg SPR and ELISA), or binding or functional interaction with cells that physiologically express GPC3. Methods to determine cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.

The terms "inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of the ligand preferably reduces or alters the normal level or type of activity that occurs when ligand binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable reduction in ligand binding affinity when contacted with an anti-GPC3 antibody compared to a ligand not contacted with an anti-GPC3 antibody.

The term "inhibiting growth" (eg, in relation to a cell) is intended to include any measurable reduction in cell growth.

The terms "inducing an immune response" and "enhancing an immune response" are used interchangeably and refer to the stimulation (ie, passive or adaptive) of an immune response to a particular antigen. The term "induction" with respect to inducing CDC or ADCC refers to stimulating a specific mechanism of direct cell killing.

The "ADCC" in the present invention, namely antibody-dependent cell-mediated cytotoxicity, refers to that cells expressing Fc receptors are The Fc segment that recognizes the antibody directly kills the target cell coated by the antibody. The ADCC effector function of the antibody can be reduced or eliminated by enhancing or reducing the modification of the Fc region of IgG. The modification refers to making mutations in the constant region of the heavy chain of the antibody.

Methods of producing and purifying antibodies and antigen-binding fragments are well known and can be found in the art, eg, Cold Spring Harbor's Technical Guide to Antibody Assays, Chapters 5-8 and 15. For example, mice can be immunized with human GPC3 or fragments thereof, and the resulting antibodies can be coated, purified, and amino acid sequenced using conventional methods. Antigen-binding fragments can likewise be prepared by conventional methods. The antibody or antigen-binding fragment of the present invention uses genetic engineering to add one or more human FR regions to the non-human CDR regions. Human FR germline sequences can be obtained from the website of ImMunoGeneTics (IMGT) at http://imgt.cines.fr, or from J. Immunoglobulins, 2001 ISBN012441351.

The engineered antibodies or antigen-binding fragments of the invention can be prepared and purified using general methods. The cDNA sequences of the corresponding antibodies can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more preferred prior art, mammalian-like expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminus of the FC region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones were expanded in serum-free medium in bioreactors for antibody production. The antibody-secreted culture medium can be purified and collected by conventional techniques. Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by general methods, such as molecular sieves, ion exchange. The obtained product should be frozen immediately, eg -70°C, or lyophilized.

Antibodies of the present invention refer to monoclonal antibodies. The monoclonal antibody (mAb) in the present invention refers to an antibody obtained from a single pure cell strain, and the cell strain is not limited to eukaryotic, prokaryotic or phage pure cell strains. Monoclonal antibodies or antigen-binding fragments can be obtained by recombinant techniques such as fusion tumor technology, recombinant technology, phage display technology, synthetic technology (eg, CDR-grafting), or other existing techniques.

"Administering," "administering," and "treating," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to exogenous drugs, therapeutic agents, diagnostic agents, or compositions that interact with the animal. , contact of humans, subjects, cells, tissues, organs or biological fluids. "Administering," "administering," and "treating" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, wherein the fluids are in contact with cells. "Administering," "administering," and "treating" also mean the in vitro and ex vivo treatment of, for example, cells by an agent, diagnostic, binding composition, or by another cell. "Treatment" when applied to human, veterinary or research subjects refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.

"Treatment" means administering an internal or external therapeutic agent, such as comprising any of the antibodies of the invention, to a patient having one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in a patient or population being treated, either by inducing regression of such symptoms or inhibiting the progression of such symptoms to any clinically measured extent. The amount of a therapeutic agent effective to relieve symptoms of any particular disease (also referred to as a "therapeutically effective amount") can vary depending on factors such as the patient's disease state, age and weight, and the ability of the drug to produce the desired effect in the patient. Whether symptoms of a disease have been alleviated can be assessed by any clinical test commonly used by physicians or other health care professionals to assess the severity or progression of the symptoms. Although embodiments of the present invention (eg, methods of treatment or articles of manufacture) may not be effective in alleviating symptoms of the target disease that each patient has, it can be determined according to any statistical test known in the art such as Student's t-test, chi-square test, based on Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra checked sum Wilcoxon test determined that it should reduce target disease symptoms in a statistically significant number of patients.

The term "consisting essentially of," or variations thereof, as used throughout the specification and scope of claims, means that all such elements or groups of elements are included, and optionally other elements of a similar or different nature to the elements, which other elements are not significant Change the basic or new properties of a given dosing regimen, method or composition.

The term "naturally occurring" as applied to an item as used herein refers to the fact that the item can be found in nature. For example, a polypeptide sequence or a polynucleotide sequence that exists in an organism (including a virus) that can be isolated from natural sources and has not been intentionally modified artificially in the laboratory is naturally occurring.

An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or conditions of the condition being treated. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the general health of the patient, the method, route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

"Exogenous" refers to a substance that is to be produced outside of an organism, cell or human in context.

"Endogenous" refers to a substance produced in a cell, organism or human body depending on the context.

"Homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example if every position in two DNA molecules is occupied by an adenine, then the molecules are homologous at that position of. The percent homology between the two sequences is a function of the number of matches or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, when sequences are optimally aligned, two sequences are 60% homologous if 6 of 10 positions in the sequences are matched or homologous. In general, comparisons are made when the two sequences are aligned for the greatest percent homology.

As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably and all such designations include progeny thereof. Thus, the words "transformants" and "transformed cells" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that, due to deliberate or unintentional mutations, all progeny may not be exactly the same in terms of DNA content. Mutant progeny that have the same function or biological activity as screened in the original transformed cell are included. Where a different name is meant, it is clear from the context.

"Optional" or "optionally" means that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs or instances where it does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that an antibody heavy chain variable region of a particular sequence may, but need not, be present.

"Pharmaceutical composition" means containing one or more of the antibodies or antigen-binding fragments thereof described herein, together with other components such as physiological/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the administration to the organism, facilitate the absorption of the active ingredient and then exert the biological activity.

The following examples are used to further describe the present invention, but these examples do not limit the scope of the present invention. The experimental methods that do not specify specific conditions in the examples of the present invention generally follow general conditions, such as the Antibody Technology Experiment Manual of Cold Spring Harbor, the Molecular Colonization Manual; or the conditions suggested by the manufacturers of raw materials or commodities. The reagents that do not indicate the specific source are general reagents purchased in the market.

Example 1: Antigen Preparation

The protein antigen is human GPC3 recombinant protein (UniProt #P51654, Gln 25-His 559) with a His tag at the C-terminus, namely human GPC3 His protein, purchased from AcroBiosystems (Cat #GP3-H52H4).

Table 1. Human GPC3 (Gln 25-His 559) amino acid sequence

Example 2: Acquisition of murine fusion tumors and antibody sequences

Animals were immunized with human GPC3 recombinant protein. A total of 5 C57bl/6 and 5 SJL mice, female, 10 weeks old, were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. Using Sigma Complete Freund's Adjuvant (CFA) and Sigma Incomplete Freund's Adjuvant (IFA), the immunogen and immune adjuvant are thoroughly mixed and emulsified in a 1:1 ratio to make a stable "water-in-oil" liquid; injection dose 25μg/200μL/mouse.

Immunization program

The immunized mouse serum was evaluated by indirect ELISA method as described in Example 3(1) to evaluate the serum titer and the ability to bind to cell surface antigens, and the control of the titer detection (more than 100,000-fold dilution) was used to determine the initiation of cell fusion. The immunized mice with strong serum titers were selected for a final immunization and then sacrificed. The spleen cells were fused with SP2/0 myeloma cells and then plated to obtain fusion tumors. The target fusion tumors were screened by indirect ELISA. The French strain is a single cell line. The obtained positive antibody strains were further screened by indirect ELISA to select fusion tumors that bind the recombinant protein. Log phase fusion tumor cells were harvested, RNA was extracted with Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript ^™ Reverse Transcriptase, Takara #2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and sequenced, and finally the sequences of mouse antibodies M1, M2 and M3 were obtained.

Table 2. Heavy and light chain variable region sequences of murine mAbs

Note: The bottom line represents the mouse mAb CDR sequence.

Table 3. Murine antibody CDR region sequences

Example 3: In vitro binding activity detection method of antibodies

(1) In vitro indirect ELISA binding experiment:

GPC3 His protein (AcroBiosystems, Cat # GP3-H52H4) was diluted to a concentration of 0.5 μg/ml with PBS pH 7.4, added to a 96-well high-affinity ELISA plate at a volume of 100 μL/well, and incubated at 4°C overnight ( 16-20 hours). After washing the plate three times with PBST (PBS containing 0.05% Tween-20 at pH 7.4), 200 μL/well of 1% bovine serum albumin (BSA) blocking solution diluted with PBST was added, and incubated at 37°C for 0.5 hours for blocking. After blocking, the blocking solution was discarded, and the plate was washed once with PBST buffer.

The antibody to be tested was diluted with PBST containing 1% BSA, starting at 100 nM, 5-fold serial dilution, 11 doses, added to the ELISA plate at 100 μL/well, and incubated at 37 °C for 1 hour. After the incubation, the plate was washed three times with PBST, and 200 μL/well HRP-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, cat#115-035-071) or HRP-conjugated goat anti-human secondary antibody diluted with PBST containing 1% BSA was added. Anti-(Rockland, cat#609-103-123), incubated at 37°C for 0.5 hours. After washing the plate 5 times with PBST, add 100 μL/well of TMB as a chromogenic substrate (Suzhou Yake Chemical Reagent Co., Ltd., cat# S0025), incubate at 25°C for 8-15 minutes in the dark, and add 50 μL/well of 1M HCl to stop After the reaction, the absorbance was read at 450 nm with a microplate reader (Thermo, Ascent), and the data were analyzed.

Do concentration signal value curve analysis results, as shown in the following table:

Table 4. Affinity of murine antibodies to human GPC3 antigen (EC ₅₀ values)

The results showed that the mouse antibody had a good affinity for human GPC3 antigen.

(2) In vitro FACS binding experiment:

GPC3 high expression tumor cells HepG2 (ATCC deposit number: HB-8065) were digested with trypsin, collected by centrifugation, adjusted for cell density with FACS buffer (1×PBS containing 2% FBS), and plated in 96-well U bottom plate , 1 x 10 ⁵ to 2 x 10 ⁵ cells per well. Centrifuge: 1200g for 5 minutes, discard the supernatant, add 100 μL of antibody solution that has been gradient diluted with FACS buffer, and incubate at 4°C for 1 hour. Centrifugation: 1200g, 5 minutes, discard the supernatant, wash the cells twice with PBS, add the fluorescently labeled secondary antibody working solution PE anti-human IgG Fc Antibody (Biolegend, Cat# 409304) or FITC anti-mouse prepared in FACS buffer IgG Antibody (Biolegend, Cat# 406001), 100 μL per well resuspended cells and incubated at 4°C for 1 hour. Centrifugation: 1200g, 5 minutes, discard the supernatant. After washing the cells twice with PBS, the cells were resuspended in PBS, and the fluorescence signal was detected by flow cytometer DxFlex, and the EC ₅₀ concentration of antibody-bound cells was analyzed by curve.

Table 5. Affinity of murine antibodies to human GPC3 high expressing cells (EC ₅₀ values)

The results showed that the mouse antibody had a good affinity for human GPC3 expressing cells.

Example 4: Mouse Antibody Chimerization Experiment

The mouse antibody obtained by screening is chimerized, and the variable region of the mouse antibody is cloned in the constant region of a human antibody to obtain a chimeric antibody. The constant region of an exemplary chimeric antibody can be a human IgG1 heavy chain constant region and kappa light. chain constant region.

(1) In vitro protein binding experiments:

The affinity of the chimeric antibody to the human GPC3 antigen was tested according to the in vitro indirect ELISA binding experiment and the Biacore method of Example 3(1).

The Biacore test method is as follows:

a) Wafer Preparation:

The mouse anti-human IgG (Fc) antibody was diluted to 25μg/mL with immobilization reagent (10mM sodium acetate, pH 5.0), about 100μL of mouse anti-human IgG (Fc) antibody was used for each channel of the wafer, and about 190μL was used to fix two channels. Reagents Add 10 μL of mouse anti-human IgG (Fc) antibody. First, the surface of the CM5 wafer was activated with 400 mM EDC and 100 mM NHS at a flow rate of 10 μL/min for 420 s. Next, 25 μg/mL of mouse anti-human IgG (Fc) antibody was injected into the experimental channel (FC4) at a flow rate of 10 μL/min About 420s, the fixed amount is about 9000 to 14000RU. Finally, the wafers were blocked with 1 M ethanolamine at 10 μL/min for 420 s. The reference channel (FC3) performs the same operation as the test channel (FC4).

b) Capture ligand:

The antibody stock solution was diluted to 4 μg/mL with running reagent, and injected into the experimental channel (FC4) at a flow rate of 10 μL/min to capture about 200 RU. The reference channel (FC3) does not require ligand capture.

c) Analyte multi-loop analysis:

The human GPC 3 protein was diluted 2-fold with the running reagent, and the diluted samples were sequentially injected into the experimental channel and the reference channel at a flow rate of 30 μL/min according to the corresponding binding time and dissociation time. After each concentration analysis, the wafers were regenerated with 3M magnesium chloride at a flow rate of 20 μL/min for 30 s to wash away ligands as well as undissociated analytes. For the next concentration analysis, the experimental channel needs to recapture the same amount of ligand.

d) Data analysis:

KD values for each antibody were calculated using Biacore T200 analysis software. The reference channel (FC3) is used for background subtraction.

Table 6. Affinity of chimeric antibodies to human GPC3 antigen

The ELISA and Biacore results are shown in Table 6, showing that the chimeric antibody has a good affinity for human GPC3 antigen.

(2) In vitro cell binding experiments:

GPC3 high expressing cells (CHO-K1 cells overexpressing human or monkey GPC3, and human hepatoma cells JHH-7 expressing GPC3) were trypsinized, collected by centrifugation, and washed with FACS buffer (1 ×PBS), after adjusting the cell density, it was plated in a 96-well U-bottom plate, with 1×10 ⁵ to 2×10 ⁵ cells per well. Centrifugation: 1200g, 5 minutes, discard the supernatant, added 100 μ L of serially diluted antibody solution was with FACS buffer and incubated for 4 ℃ 1 hour. Centrifugation: 1200g, 5 minutes, discard the supernatant, wash the cells twice with PBS, add the fluorescently labeled secondary antibody working solution PE anti-human IgG Fc Antibody (Biolegend, Cat# 409304) or FITC anti- mouse IgG Antibody (Biolegend, Cat # 406001), 100 μ L per well of cells were resuspended, incubated for 4 ℃ 1 hour. Centrifugation: 1200g, 5 minutes, discard the supernatant. After washing the cells twice with PBS, the cells were resuspended in PBS, and the fluorescence signal was detected by a flow cytometer DxFlex, and the EC ₅₀ concentration of the antibody-bound cells was analyzed by curve.

Table 7. GPC3 chimeric antibody expression Cells affinity (EC ₅₀ value)

The results showed that the chimeric antibodies had high affinity to cells expressing GPC3.

Example 5: Mouse Antibody Humanization Experiment

The humanization of the murine anti-human GPC3 monoclonal antibody was carried out as disclosed in many literatures in the art. Briefly, human constant domains are used to replace the constant domains of the parent (murine antibody), and human antibody sequences are selected based on the homology of the murine and human antibodies. Humanize.

Based on the obtained VH/VL CDR typical structures of murine antibodies, the variable region sequences of the heavy and light chains were compared with the human antibody germline database to obtain a human germline model with high homology.

The CDR regions of the murine antibody are transplanted to the corresponding humanized template selected. Then, based on the three-dimensional structure of the murine antibody, backmutation was performed on the embedded residues, the residues that directly interact with the CDR regions, and the residues that have important effects on the conformation of VL and VH, and the CDR regions were backmutated. The chemically unstable amino acid residues were optimized, and the antibody composed of the humanized heavy chain variable region HCVR and light chain variable region LCVR sequences was selected after performance testing and comparison of the number of back mutations. The sequences are as follows:

Table 8. Antibody heavy and light chain variable region CDR sequences

Table 9. Humanized Antibody CDR Regions

Table 10. Heavy and light chain variable region sequences of humanized antibodies

Note: The bottom line represents the monoclonal antibody CDR sequence.

The designed heavy and light chain variable region sequences are linked to the IgG1 heavy chain constant region and light chain constant region sequences. Exemplarily, the antibody heavy chain constant regions are selected from the human IgG1 native constant regions shown in SEQ ID NO: 52 , the light chain constant region is selected from the constant region of the human kappa chain whose sequence is shown in SEQ ID NO: 53, and the heavy chain and light chain sequences are obtained as follows:

Table 11. Heavy and light chain sequences, and heavy and light chain constant region sequences of humanized antibodies

Table 12. Sequence numbering of antibodies and their heavy, light, and variable regions

Example 6: Expression and purification of humanized antibodies

cDNA fragments were synthesized based on the amino acid sequences of the light and heavy chains of each of the humanized antibody above, and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20). The expression vector and transfection reagent PEI (Polysciences, Inc.Cat.No.23966) ratio of 1: 2 transfected HEK293 cells (Life Technologies Cat.No.11625019), and placed in incubator incubated ₂ CO.'S 4-5 sky. The cell culture medium was collected, centrifuged and filtered, and the sample was loaded onto the antibody purification affinity column. The column was washed with phosphate buffer, eluted with glycine hydrochloride buffer (pH 2.7, 0.1M Gly-HCl), and 1M Tris hydrochloric acid, pH 9.0. Neutralization and phosphate buffer dialysis were performed to obtain the humanized antibody protein of the present invention, and its concentration and purity were as shown in the following table.

Table 13. Concentration and Purity of Humanized Antibodies

Example 7: In vitro binding affinity experiments of humanized antibodies:

(1) In vitro indirect ELISA binding experiment:

_{The affinity (EC 50} ) of each humanized antibody to human GPC3 antigen was determined using the in vitro indirect ELISA binding experiment in Example 3(1). The results are shown in Table 14 below:

Table 14. The humanized antibody of human GPC3 antigen affinity (EC ₅₀₎

The results show that: the humanized antibody of the present invention has high affinity with human GPC3 antigen.

(2) In vitro FACS binding experiment:

Using the in vitro FACS binding experiment of Example 4 (2), the affinity (EC ₅₀ ) of each humanized antibody to GPC3 antigen-expressing cells was determined. The results are shown in Table 15 below:

Table 15. The humanized antibody of human GPC3 cells expressing high affinity (EC ₅₀ value)

The results show that the humanized antibody has high affinity with stable cell lines overexpressing human or monkey GPC3 antigen, and can bind to human hepatoma cell JHH-7.

Example 8: Endocytosis of Humanized Antibodies

To detect whether the antibody of the present invention can be endocytosed with human GPC3 after binding to GPC3, stable transfection cell line CHO-K1-human GPC3 and human hepatoma cell JHH-7 (CBP60204, purchased from Nanjing Kebai Biotechnology Co., Ltd.) were used. and HepG2 (SCSP-510, purchased from the Chinese Academy of Sciences Cell Bank) for evaluation.

The cells were trypsinized, collected and resuspended in pre-chilled FACS buffer to adjust the cell concentration to 1×10 ⁶ /mL. Take the EP tube, add 1 mL of cell suspension, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, add 1 mL of the prepared antibody to be tested to resuspend the cells, the final concentration of the antibody is 20 μg/mL, and incubate at 4°C for 1 hour on a shaker , the supernatant was discarded by centrifugation (4°C, 1500 rpm × 5 minutes), washed twice with FACS buffer, and the supernatant was discarded. Add 100 μL of fluorescently labeled secondary antibody working solution PE anti-human IgG Fc Antibody (Biolegend, Cat# 409304) or FITC anti-mouse IgG Antibody (Biolegend, Cat# 406001) to each tube, resuspend the cells, and incubate at 4°C for 30 minutes minutes, the supernatant was discarded by centrifugation (4°C, 1500 rpm × 5 minutes), washed twice with FACS buffer, and the supernatant was discarded. Add 1 mL of pre-warmed cell culture medium to each tube to resuspend the cells and mix well. Divide into 4 tubes of 200 μL each, which are divided into 0-minute group, blank group, 30-minute group and 2-hour group. On ice, the rest were placed in a 37 °C incubator for 30 minutes and 2 hours, respectively, and the EP tubes were taken out at the corresponding time points and placed on ice to pre-cool for 5 minutes. × 5 min), washed once with FACS buffer, and discarded the supernatant. Add 250 μL of strip buffer to the EP tubes of all treatment groups except the 0-minute group, incubate at room temperature for 8 minutes, centrifuge and discard the supernatant (4°C, 1500 rpm × 5 minutes), wash twice with FACS buffer, and discard the supernatant. All treatment groups were resuspended in 100 μL of PBS and detected by flow cytometry DxFlex.

Antibody endocytosis percentage = (fluorescence intensity value at each time point - average fluorescence intensity value of Blank group)/(average fluorescence intensity value at zero point - average fluorescence intensity value of Blank group)

Table 16. Endocytosis of humanized antibodies

The results show that: the humanized antibody of the present invention has good endocytosis in CHO-K1-human GPC3, JHH-7 and HepG2 cells.

Example 9: CDC experiments of humanized antibodies

The target cells in this experiment were human GPC3 overexpressing stable cell line CHO-K1-hGPC3. The experimental medium was FBS-free cell culture medium F12K (Gibco ^™ , Cat# 21127022) for cell and antibody resuspension or dilution. CHO-K1-hGPC3 cells were trypsinized, collected by centrifugation, resuspended in experimental medium to 1×10 ⁵ cells/ml, 50 μL were plated on a white 96-well plate (Corning, 3610), and 25 μL of 4× working concentration of antibody ( The initial working concentration of the antibody was 20nM, 5-fold dilution, 10 concentration points, and 0nM points were established), incubate at 37°C for 30 minutes, add 25μL of 80% human serum (GemCell ^TM US, Cat#100-512), and incubate at 37°C incubator for 24 hours. Add 50 μL of CellTiter-Glo (Promega, Cat#G7573), mix well at room temperature for 10 minutes in the dark, and read with a multi-plate reader (Thermofisher, Lux).

Antibody killing percentage (%)=(E-S)/(E-M)×100

E is the value of the well without antibody, that is, cells + culture medium + human serum;

S is the value of the sample well, that is, cells + antibodies + human serum;

M is the medium + human serum well value.

Antibody concentration as the abscissa, the ordinate is the percentage destruction, using GraphPad Prism 8.3.0 in the log (inhibitor) vs.response - Variable slope curve fitting, the concentration IC ₅₀ Analysis of antibody obtained killer cells, the results shown below surface.

Table 17. CDC effects of humanized antibodies

The results showed that the humanized antibodies H1-3, H2-3 and H3-1 all had significant CDC effects.

Example 10: Charge Heterogeneous Stability Experiment of Humanized Antibodies

In this experiment, the method of panoramic isoelectric focusing (iCIEF) was used to detect and compare the purity of the charged heterogeneity of the humanized antibody H3-1 at the initial 0 point and at 25°C and 40°C for one month, respectively. This examines the stability of the antibody.

A mixture of samples with amphoteric groups, ampholytes, buffers and auxiliary additives is injected into the capillary. When a DC voltage is applied to both ends of the capillary, the carrier ampholyte can form a pH gradient within a certain range. The sample set According to its charged properties, it migrates to the cathode or the anode. When the pH value in the capillary is the same as the isoelectric point (pI) of the component, the net charge of the solute molecule is zero, and the component will aggregate at this point macroscopically and no further Migration to achieve the purpose of separating components in complex samples. After collecting the spectrum, according to the linear relationship between the marker pI value and the migration time of the chromatographic peak, the pI value and peak ratio (main peak, acid peak, base peak) of the test sample can be obtained. The main steps are as follows:

1) System suitability sample preparation: Take out the suitability sample tube in the Maurice cIEF System Suitability Kit (Protein Simple, Cat#046-044), add 40μl deionized water and 160μl System Suitability Test Mix, mix well and transfer to 1.5 1 ml centrifuge tube, vortexed and centrifuged, and 160ul of the supernatant was transferred to a 96-well sample plate for use.

2) Prepare cIEF Master Mix solution: containing 37 μl ultrapure water, 35 μl 1% MC (Protein Simple, Cat#101876), 4 μl Pharmalyte pH 3-10 (Protein Simple, Cat#17-0456-01), 2 μl 500mM arginine (Protein Simple, Cat#042-691), and the corresponding two pI Marker 6.14 (protein simple, cat: 046-031) and 9.99 (protein simple, cat: 046-034)) 1 μl each, with a total volume of 80 μl .

3) Preparation of the test product: The sample is set to 3 conditions: the initial point is 0; The test chamber (Memmer, model HPP 1060) was placed for 1 month at a humidity of 65% and a temperature of 40°C. Take 20 μl of the corresponding sample, add it to the EP tube containing 80 μl of cIEF Master Mix solution in step 2), vortex and mix well, then centrifuge, take 80 μl of the supernatant and transfer it to a 96-well sample plate, and centrifuge for use.

4) On-board detection: turn on the capillary electrophoresis instrument (Protein Simple, maurice) and software, follow the instrument operation steps, perform instrument self-test, install the capillary cartridge (Protein Simple, Cat# PS-MC02-C), and place the 96-well sample. The plate is placed in the corresponding position of the instrument for cIEF analysis.

Table 18. iCIEF detection of humanized antibody H3-1

The results showed that compared with the initial 0 point, the proportion of the main peak of H3-1 did not change much after being placed at 25 °C for one month, and the stability was good. When placed under the forced degradation condition of 40 °C for one month, the antibody showed the expected degradation phenomenon. It is reflected in the reduction of the main peak ratio to 22.3%.

Example 11: Molecular variant stability experiments of humanized antibodies

In this experiment, by capillary electrophoresis (CE-SDS) method, the purity of the humanized antibody H3-1 at the initial 0 point and the molecular variants stored at 25°C and 40°C for one month were detected and compared. CE-SDS is based on the migration of protein samples in gel electrophoresis under denaturing conditions, and the separation is completed according to the difference in the migration time of proteins with different molecular weights, so as to obtain the detection results of the sample purity after non-reduction and reduction treatment.

The main steps are as follows:

1) Sample preparation: The sample was sampled at the initial 0 point; placed in a stability test chamber (Memmer, model HPP 1060) under the conditions of a humidity of 65% and a temperature of 25°C for 1 month; , Model HPP 1060) for 1 month at a humidity of 65% and a temperature of 40°C.

2) Non-reduced CE sample treatment: add samples to the EP tube, the amount of protein in each sample is 50 μg, add 1 μL of 10kD internal standard (Protein Simple, Cat#046-144), add 2.5 μL of 250 mM IAM (Sigma, Cat# I1149-5G) was added with 1× sample buffer (Protein Simple, Cat#046-567) to a final volume of 50 μL.

3) Reduction CE sample processing: add samples to the EP tube, the amount of protein in each sample is 50 μg, add 1 μL of 10kD internal standard (Protein Simple, Cat#046-144), and add 2.5 μL of β-mercaptoethanol ( Sigma, Cat# M3148-25ML), 1× sample buffer (Protein Simple, Cat#046-567) was added to a final volume of 50 μL.

4) After shaking and mixing, heat and incubate at 70°C for 10 min in a dry thermostat (Hangzhou Aosheng Instrument Co., Ltd., model MK20001), then take it out, incubate on ice for 5 min, and centrifuge at 12000 rpm for 5 min after cooling. After centrifugation, 35 μL of the supernatant was transferred to a 96-well matched with an instrument, and then centrifuged at 1000 rpm for 5 min.

5) Sample detection: Put the 96-well sample plate into the capillary electrophoresis instrument (Protein Simple, Maurice), open the instrument and software, follow the instrument operation steps, perform instrument self-check, and install the capillary cartridge (Protein Simple, Cat#090- 157), prepare the corresponding reagent and put it into the corresponding position of the instrument. According to the operation steps of the instrument, set the corresponding parameters to perform reduction or non-reduction CE analysis.

6) Data processing: After the sample detection is completed, set the corresponding integration parameters, and perform calculation and analysis with the software of the instrument to obtain the sample purity.

Table 19. CE-SDS detection of humanized antibody H3-1

The results of non-reduced CE showed that the proportion of the main peak of the humanized antibody H3-1 at 25°C for one month was similar to that at 0 point, and the main peak decreased at 40°C for one month; in reducing CE, compared with 0 point, no matter Whether it was placed at 25°C or 40°C for a month, the sum of the ratios of heavy and light chains of the antibody did not change much. In conclusion, the stability of the humanized antibody H3-1 is good.

<110> Mainland businessman Shanghai Hansoh Biomedical Technology Co., Ltd. (SHANGHAI HANSOH BIOMEDICAL CO.,LTD) Mainland businessman Jiangsu Hansoh Pharmaceutical Group Co., Ltd. (JIANGSU HANSOH PHARMACEUTICAL GROUP CO.,LTD.)

<120> Antibody or antigen-binding fragment thereof, preparation method and medical use thereof

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<150> 202010314338.X

<151> 2020-04-20

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<151> 2020-09-08

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<212> PRT

<213> Mouse (Mus musculus)

<220>

<221> Domain

<223> Mouse CDRs

<400> 10

<210> 11

<211> 19

<212> PRT

<213> Mouse (Mus musculus)

<220>

<221> Domain

<223> Mouse CDRs

<400> 11

<210> 12

<211> 17

<212> PRT

<213> Mouse (Mus musculus)

<220>

<221> Domain

<223> Mouse CDRs

<400> 12

<210> 13

<211> 9

<212> PRT

<213> Mouse (Mus musculus)

<220>

<221> Domain

<223> Mouse CDRs

<400> 13

<210> 14

<211> 9

<212> PRT

<213> Mouse (Mus musculus)

<220>

<221> Domain

<223> Mouse CDRs

<400> 14

<210> 15

<211> 2

<212> PRT

<213> Mouse (Mus musculus)

<220>

<221> Domain

<223> Mouse CDRs

<400> 15

<210> 16

<211> 16

<212> PRT

<213> Mouse (Mus musculus)

<220>

<221> Domain

<223> Mouse CDRs

<400> 16

<210> 17

<211> 11

<212> PRT

<213> Mouse (Mus musculus)

<220>

<221> Domain

<223> Mouse CDRs

<400> 17

<210> 18

<211> 16

<212> PRT

<213> Mouse (Mus musculus)

<220>

<221> Domain

<223> Mouse CDRs

<400> 18

<210> 19

<211> 16

<212> PRT

<213> Mouse (Mus musculus)

<220>

<221> Domain

<223> Mouse CDRs

<400> 19

<210> 20

<211> 16

<212> PRT

<213> Mouse (Mus musculus)

<220>

<221> Domain

<223> Mouse CDRs

<400> 20

<210> 21

<211> 7

<212> PRT

<213> Mouse (Mus musculus)

<220>

<221> Domain

<223> Mouse CDRs

<400> 21

<210> 22

<211> 7

<212> PRT

<213> Mouse (Mus musculus)

<220>

<221> Domain

<223> Mouse CDRs

<400> 22

<210> 23

<211> 7

<212> PRT

<213> Mouse (Mus musculus)

<220>

<221> Domain

<223> Mouse CDRs

<400> 23

<210> 24

<211> 9

<212> PRT

<213> Mouse (Mus musculus)

<220>

<221> Domain

<223> Mouse CDRs

<400> 24

<210> 25

<211> 9

<212> PRT

<213> Mouse (Mus musculus)

<220>

<221> Domain

<223> Mouse CDRs

<400> 25

<210> 26

<211> 9

<212> PRT

<213> Mouse (Mus musculus)

<220>

<221> Domain

<223> Mouse CDRs

<400> 26

<210> 27

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<221> Domain

<223> Humanized heavy chain variable region

<400> 27

<210> 28

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<221> Domain

<223> Humanized light chain variable region

<400> 28

<210> 29

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<221> Domain

<223> Humanized heavy chain variable region

<400> 29

<210> 30

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<221> Domain

<223> Humanized heavy chain variable region

<400> 30

<210> 31

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<221> Domain

<223> Humanized heavy chain variable region

<400> 31

<210> 32

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<221> Domain

<223> Humanized heavy chain variable region

<400> 32

<210> 33

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> Domain

<223> Humanized light chain variable region

<400> 33

<210> 34

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> Domain

<223> Humanized light chain variable region

<400> 34

<210> 35

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> Domain

<223> Humanized light chain variable region

<400> 35

<210> 36

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> Domain

<223> Humanized light chain variable region

<400> 36

<210> 37

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<221> Domain

<223> Humanized heavy chain variable region

<400> 37

<210> 38

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<221> Domain

<223> Humanized light chain variable region

<400> 38

<210> 39

<211> 448

<212> PRT

<213> Artificial Sequence

<220>

<221> Chain

<223> Humanized antibody heavy chain

<400> 39

<210> 40

<211> 219

<212> PRT

<213> Artificial Sequence

<220>

<221> Chain

<223> Humanized Antibody Light Chain

<400> 40

<210> 41

<211> 448

<212> PRT

<213> Artificial Sequence

<220>

<221> Chain

<223> Humanized antibody heavy chain

<400> 41

<210> 42

<211> 448

<212> PRT

<213> Artificial Sequence

<220>

<221> Chain

<223> Humanized antibody heavy chain

<400> 42

<210> 43

<211> 448

<212> PRT

<213> Artificial Sequence

<220>

<221> Chain

<223> Humanized antibody heavy chain

<400> 43

<210> 44

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<221> Chain

<223> Humanized antibody heavy chain

<400> 44

<210> 45

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> Chain

<223> Humanized Antibody Light Chain

<400> 45

<210> 46

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> Chain

<223> Humanized Antibody Light Chain

<400> 46

<210> 47

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> Chain

<223> Humanized Antibody Light Chain

<400> 47

<210> 48

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> Chain

<223> Humanized Antibody Light Chain

<400> 48

<210> 49

<211> 441

<212> PRT

<213> Artificial Sequence

<220>

<221> Chain

<223> Humanized antibody heavy chain

<400> 49

<210> 50

<211> 219

<212> PRT

<213> Artificial Sequence

<220>

<221> Chain

<223> Humanized Antibody Light Chain

<400> 50

<210> 51

<211> 535

<212> PRT

<213> Artificial Sequence

<220>

<221> variants

<223> Human GPC3 (Gln 25-His 559) antigen

<400> 51

<210> 52

<211> 330

<212> PRT

<213> Homo sapiens

<400> 52

<210> 53

<211> 107

<212> PRT

<213> Homo sapiens

<400> 53

Claims (22)

An anti-GPC3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: HCDR1 set forth in SEQ ID NO:7, HCDR2 set forth in SEQ ID NO:10, and HCDR3 set forth in SEQ ID NO:13, or, HCDR1 set forth in SEQ ID NO: 8, HCDR2 set forth in SEQ ID NO: 11, and HCDR3 set forth in SEQ ID NO: 14, or, HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 12, and HCDR3 shown in SEQ ID NO: 15; The light chain variable region comprises: LCDR1 of SEQ ID NO: 16, LCDR2 of SEQ ID NO: 21 and LCDR3 of SEQ ID NO: 24, or, LCDR1 shown in SEQ ID NO: 17, LCDR2 shown in SEQ ID NO: 22, and LCDR3 shown in SEQ ID NO: 25, or, LCDR1 of SEQ ID NO: 18, LCDR2 of SEQ ID NO: 23 and LCDR3 of SEQ ID NO: 26, or, LCDR1 of SEQ ID NO: 19, LCDR2 of SEQ ID NO: 21 and LCDR3 of SEQ ID NO: 24, or, LCDR1 shown in SEQ ID NO:20, LCDR2 shown in SEQ ID NO:23, and LCDR3 shown in SEQ ID NO:26. The anti-GPC3 antibody or antigen-binding fragment thereof according to claim 1, wherein, The heavy chain variable region comprises HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:10 and HCDR3 shown in SEQ ID NO:13; the light chain variable region comprises SEQ ID NO:16 LCDR1 shown in SEQ ID NO: 21, LCDR2 shown in SEQ ID NO: 21, and LCDR3 shown in SEQ ID NO: 24; or, The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 8, HCDR2 shown in SEQ ID NO: 11 and HCDR3 shown in SEQ ID NO: 14; the light chain variable region comprises the HCDR1 shown in SEQ ID NO: 17 LCDR1 shown in SEQ ID NO: 22, LCDR2 shown in SEQ ID NO: 22, and LCDR3 shown in SEQ ID NO: 25; or, The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 12 and HCDR3 shown in SEQ ID NO: 15; the light chain variable region comprises the HCDR1 shown in SEQ ID NO: 18 LCDR1 shown in SEQ ID NO: 23, LCDR2 shown in SEQ ID NO: 26, and LCDR3 shown in SEQ ID NO: 26, or, The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 7, HCDR2 shown in SEQ ID NO: 10 and HCDR3 shown in SEQ ID NO: 13; the light chain variable region comprises the HCDR1 shown in SEQ ID NO: 19 LCDR1 shown in SEQ ID NO: 21, LCDR2 shown in SEQ ID NO: 21, and LCDR3 shown in SEQ ID NO: 24; or, The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 12 and HCDR3 shown in SEQ ID NO: 15; the light chain variable region comprises the HCDR1 shown in SEQ ID NO: 20 LCDR1 shown in SEQ ID NO: 23, LCDR2 shown in SEQ ID NO: 23, and LCDR3 shown in SEQ ID NO: 26. The anti-GPC3 antibody or antigen-binding fragment thereof according to claim 1 or 2, which is selected from murine antibodies or antigen-binding fragments thereof, chimeric antibodies or antigen-binding fragments thereof, and human antibodies or antigen-binding fragments thereof , or a humanized antibody or an antigen-binding fragment thereof. The anti-GPC3 antibody or antigen-binding fragment thereof of claim 3, wherein the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, IgG2, IgG3 or IgG4 or a variant thereof; Preferably, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, IgG2 or IgG4; More preferably, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as shown in SEQ ID NO:52. The anti-GPC3 antibody or antigen-binding fragment thereof according to claim 3, wherein the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from human kappa chain, lambda chain or a variant thereof; Preferably, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from human kappa chain; More preferably, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a light chain constant region as shown in SEQ ID NO:53. The anti-GPC3 antibody or antigen-binding fragment thereof as claimed in claim 3, wherein the anti-GPC3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the following sequence, or has at least 70% compared with the following sequence , 75%, 80%, 85%, 90%, 95% or 99% identical heavy chain variable regions: SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 , SEQ ID NO: 32 or SEQ ID NO: 37; And/or, selected from the light chain variable region shown in the following sequence, or a light chain with at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared to the following sequence Variable region: SEQ ID NO: 28, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:38. The anti-GPC3 antibody or antigen-binding fragment thereof according to claim 6, wherein the anti-GPC3 antibody or antigen-binding fragment thereof comprises: The heavy chain variable region shown in SEQ ID NO:27 and the light chain variable region shown in SEQ ID NO:28; The heavy chain variable region shown in SEQ ID NO:29 and the light chain variable region shown in SEQ ID NO:28; The heavy chain variable region shown in SEQ ID NO:30 and the light chain variable region shown in SEQ ID NO:28; The heavy chain variable region shown in SEQ ID NO:31 and the light chain variable region shown in SEQ ID NO:28; The heavy chain variable region shown in SEQ ID NO:32 and the light chain variable region shown in SEQ ID NO:33; The heavy chain variable region shown in SEQ ID NO:32 and the light chain variable region shown in SEQ ID NO:34; The heavy chain variable region shown in SEQ ID NO:32 and the light chain variable region shown in SEQ ID NO:35; The heavy chain variable region set forth in SEQ ID NO:32 and the light chain variable region set forth in SEQ ID NO:36; or The heavy chain variable region shown in SEQ ID NO:37 and the light chain variable region shown in SEQ ID NO:38. The anti-GPC3 antibody or antigen-binding fragment thereof according to claim 6, wherein the anti-GPC3 antibody or antigen-binding fragment thereof contains a heavy chain selected from the following sequence, or has at least 80%, 85% compared with the following sequence , 90%, 95% or 99% identical heavy chain: SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 or SEQ ID NO:49 ; And/or, selected from the light chain shown in the following sequence, or a light chain with at least 80%, 85%, 90%, 95% or 99% identity compared to the following sequence: SEQ ID NO: 40, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:50. The anti-GPC3 antibody or antigen-binding fragment thereof of claim 8, wherein the anti-GPC3 antibody comprises: The heavy chain set forth in SEQ ID NO:39 and the light chain set forth in SEQ ID NO:40; The heavy chain set forth in SEQ ID NO:42 and the light chain set forth in SEQ ID NO:40; The heavy chain set forth in SEQ ID NO:43 and the light chain set forth in SEQ ID NO:40; The heavy chain set forth in SEQ ID NO:44 and the light chain set forth in SEQ ID NO:45; The heavy chain set forth in SEQ ID NO:44 and the light chain set forth in SEQ ID NO:46; The heavy chain set forth in SEQ ID NO:44 and the light chain set forth in SEQ ID NO:47; The heavy chain set forth in SEQ ID NO:44 and the light chain set forth in SEQ ID NO:48; or, The heavy chain shown in SEQ ID NO:49 and the light chain shown in SEQ ID NO:50. A polynucleotide encoding the anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9. An expression vector comprising the polynucleotide of claim 10. A host cell which introduces or contains the expression vector as claimed in claim 11. The host cell according to claim 12, wherein the host cell is selected from bacteria, yeast or mammalian cells; wherein, the bacteria are preferably Escherichia coli; the yeast is preferably Pichia; the mammalian cells are CHO cells or HEK293 cells are preferred. A method for producing an anti-GPC3 antibody, comprising the steps of: 1) culturing the host cell as described in claim 12 or 13; 2) isolating the antibody from the culture; and 3) Purify the antibody. A pharmaceutical composition comprising the anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, and a pharmaceutically acceptable excipient, diluent or carrier. A detection or diagnostic kit comprising the anti-GPC3 antibody or antigen-binding fragment thereof as described in any one of claims 1 to 9, optionally, also comprising one or more detection of the anti-GPC3 antibody or antigen-binding fragment thereof Reagents that bind to GPC3 or its epitope. An anti-GPC3 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 9 or a pharmaceutical composition as described in claim 15 in the preparation of a medicament for the treatment or prevention of GPC3-mediated diseases or conditions the use of. A use of the anti-GPC3 antibody or its antigen-binding fragment as described in any one of claims 1 to 9 or the pharmaceutical composition as described in claim 15 in the preparation of a kit, wherein the kit is used for detecting, Diagnosis, prognosis of GPC3 mediated diseases or disorders. Use as claimed in claim 17 or 18, wherein, the disease or condition is cancer; Preferably the disease or disorder is a cancer expressing GPC3; More preferably, the cancer is selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma and melanoma . The anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the pharmaceutical composition according to claim 15, for the treatment or prevention of GPC3-mediated diseases; The disease is selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophagus cancer, cervical cancer, gallbladder cancer, glioblastoma and melanoma. The anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the pharmaceutical composition according to claim 15, for use in the detection, diagnosis, and prognosis of GPC3-mediated diseases; The disease is selected from breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma and melanoma, preferably For liver cancer, melanoma, ovarian cancer, non-small cell lung cancer, prostate cancer. A method of treating or preventing a GPC3-mediated disease, comprising the steps of: providing the subject with a therapeutically effective amount or a prophylactically effective amount of the anti-GPC3 antibody or antigen-binding fragment thereof of any one of claims 1 to 9; or Providing a subject with a therapeutically effective amount or a prophylactically effective amount of the pharmaceutical composition of claim 15; wherein the GPC3-mediated disease is selected from breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma and Melanoma.