Development and Use of Novel Tumor Engagement Therapeutic Agents

Abstract

This disclosure relates to the development and use of novel tumor-engaging therapeutic agents. The therapeutic agents include bispecific antibodies that bind to GPC3 and CD3, with a first binding domain that binds to GPC3 on the surface of target cells and a second binding domain that binds to CD3 on the surface of T cells. The bispecific antibodies inhibit tumor growth at very low doses and can effectively inhibit the growth of tumors xenografted in immune-reconstituted mice. Toxicological studies conducted in cynomolgus monkeys indicate that the bispecific antibodies are well tolerated by the animals. The efficacy and safety of the bispecific antibodies are superior to those of similar antibodies.

Description

[Technical field]

[0001] The present disclosure relates to bispecific antibodies and uses thereof, in particular the development and use of novel tumor engager therapeutics. [Background technology]

[0002] Glypican-3 (also called GPC3, DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS, or SGBS1) is a phosphatidylinositol proteoglycan that belongs to the heparan sulfate proteoglycans (HSPGs) family and is anchored to the outer surface of the cell membrane by glycosyl phosphatidyl inositol (GPI). The core of the GPC3 protein consists of 580 amino acids and has a molecular weight of approximately 65 kDa. It is cleaved by the proteinase Furin between 358Arg-359Ser (355R-356Q-357Y-358R-359S) to form two subunits. The two heparan sulfate polysaccharide chains are connected to the C-terminal subunit of the proximal membrane, which is an important part of the extracellular matrix and may contribute to the activation of cells by multiple signaling pathways such as Wnt, Hh (Hedgehog), and fibroblast growth factor (FGF) (Li N et al, Glypicans as Cancer Therapeutic Targets, Trends Cancer. 2018).GPC3 is expressed in various fetal tissues (liver, lung, kidney and placenta), and after birth, due to DNA methylation, the GPC3 gene is no longer expressed. However, in various tumor tissues, such as 66-90% of hepatocellular carcinomas, 54-65% of lung squamous cell carcinomas, 16-64% of small cell lung cancers, 20-30% of gastrointestinal tumors (esophageal cancer, gastric cancer, cardia cancer), and some other types of cancer cells, the expression of the GPC3 gene is restored (Kawaida M et al,Diffuse and canalicular patterns of glypican-3 expression reflect malignancy of hepatocellular carcinoma,Pathol Int.2019 Mar;69(3):125-134;Yu X et al,Differential expression of glypican-3 (GPC3) in lung squamous cell carcinoma and lung adenocarcinoma and its clinical significance,Genet Mol Res.2015 Aug 28;14(3):10185-92;Ishiguro T et al,An anti-glypican3 / CD3 bispecific T cell-redirecting antibody for treatment of solid tumors,Sci Transl Med.2017 Oct 4;9(410):eaal4291;Baumhoer D et al,Glypican3 expression in human nonneoplastic,preneoplastic,and neoplastic tissues: a tissue microarray analysis of 4,387 tissue samples,Am J Clin Pathol.2008 Jun;129(6):899-906). In pathological sections, diffuse expression of GPC3 is associated with the degree of poor differentiation of HCC cells and disease prognosis.Recently, it has been reported that GPC3 is a tumor stem cell marker, and that GPC3-specific cytotoxic T lymphocytes (CTLs) can effectively kill tumor stem cells and suppress tumor growth in mice (Okada Metal, Selective elimination of undifferentiated human pluripotent stem cells using pluripotent state-specific immunogenic antigen Glypican-3, Biochem Biophys Res Commun. 2019 Apr 9; 511(3): 711-717).

[0003] T cell bispecific antibodies (also called T cell adaptors) are special antibody molecules that recognize the surface antigens (antigen arms) of target cells through one end and bind to the CD3 receptors (CD3 arms) of T cells through the other end, activating T cells and killing tumors by agglutinating CD3 of T cells in a manner similar to TCR / peptide / HLA. BCMA×CD3 bispecific molecules are T cell bispecific (TCB) antibodies that target BCMA expressed on myeloma cells and the CD3ε chain (CD3e) present on T cells. The mechanism of action of BCMA×CD3 bispecific molecules is to simultaneously bind BCMA + Myeloma cells and CD3 + This includes binding to T cells, leading to T cell activation and T cell-mediated cell killing.

[0004] Codrituzumab is the first therapeutic monoclonal antibody targeting GPC3, and shows significant tumor-suppressing activity in mouse tumor models, but in a phase II clinical trial (NCT01507168), the codrituzumab group showed no significant difference in disease progression-free time and overall survival compared to the control group (Abou-Alfa GK et al, Randomized phase II placebo controlled study of codrituzumab in previously treated patients with advanced hepatocellular carcinoma, J Hepatol. 2016 Aug; 65(2): 289-95). Studies have shown that codrituzumab may provide clinical benefit to patients with tumors with high GPC3 expression and high affinity mutations of CD16 (Chen G et al,Combining expression of GPC3 in tumors and CD16 on NK cells from peripheral blood to identify patients responding to codrituzumab,Oncotarget.2018 Jan 2;9(12):10436-10444). ERY974 is an anti-GPC3 / CD3 bispecific antibody that can effectively kill various GPC3-positive tumor cells by T cell killing effectors and is superior to monoclonal antibodies in activity, but due to relatively strong side effects, clinical studies were suspended in 2018, and after phase I clinical trials were resumed in 2019, it was combined with IL-6 antibody tocilizumab to avoid cytokine release syndrome. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present disclosure provides a novel GPC3 antibody having high affinity to GPC3 protein. The present disclosure also provides a novel GPC3-CD3 κλ bispecific antibody formed of GPC3 antibody and CD3 antibody. The GPC3 antibody can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) against target cells, and the GPC3 antibody can suppress GPC3-induced endocytosis. The GPC3-CD3 κλ bispecific antibody adopts a full-length IgG configuration, introduces charges, and optimizes affinity, so that the κλ bispecific antibody can preferentially localize to GPC3+ tumor tissues, recruit activated T cells at low concentrations, and cause effective killing of target cells, and in the absence of target cells, it does not activate T cells, and the κλ bispecific antibody does not bind to receptors such as FcγRI, FcγRIIA, and FcγRIIIA, thereby reducing the risk of cytokine storm. In efficacy tests, the novel GPC3-CD3κλ bispecific antibody inhibits tumor growth at a very low dose and can effectively inhibit the growth of tumors transplanted into immune reconstituted mice. Toxicological studies conducted in cynomolgus monkeys also show that the animals have good tolerance to the novel GPC3-CD3κλ bispecific antibody, and the efficacy and safety of the novel GPC3-CD3κλ bispecific antibody are both superior to those of similar antibodies.

[0006] In one aspect, the disclosure provides an antibody, or antigen-binding portion thereof, that binds to GPC3.

[0007] In one aspect, the disclosure provides a bispecific antibody, or antigen-binding portion thereof.

[0008] In one aspect, the present disclosure provides a nucleic acid encoding a bispecific antibody or antigen-binding portion thereof according to the above aspect.

[0009] In one aspect, the present disclosure provides a vector comprising a nucleic acid according to the above aspect.

[0010] In one aspect, the present disclosure provides a cell comprising a nucleic acid or vector according to the previous aspect.

[0011] According to the antibody or antigen-binding portion thereof of any of the above aspects, the antibody or antigen-binding portion thereof is humanized.

[0012] In one aspect, the present disclosure provides a pharmaceutical composition or kit comprising an antibody or antigen-binding portion thereof according to any of the preceding aspects, or an encoding nucleic acid thereof, and a pharma- ceutically acceptable carrier.

[0013] In one aspect, the disclosure provides an antibody-drug conjugate comprising the antibody, or antigen-binding portion thereof, bispecific or multispecific molecule according to any of the preceding aspects covalently linked to a therapeutic moiety.

[0014] In one aspect, the disclosure provides a method of treating a GPC3-associated disease, comprising administering to a mammal a therapeutically effective amount of an antibody or antigen-binding fragment thereof, nucleic acid, vector, cell and / or pharmaceutical composition according to any preceding aspect.

[0015] In one aspect, the present disclosure provides the use of an antibody or antigen-binding fragment thereof, nucleic acid, vector, cell, and / or pharmaceutical composition according to any of the preceding aspects in the manufacture of a medicament or kit for treating a GPC3-related disease in a mammal.

[0016] The antibodies according to the present disclosure are used in a variety of applications, including detection of GPC3 protein, diagnosis, treatment, or prevention of GPC3-related diseases, and the like. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows the results of non-reducing SDS-PAGE of recombinant GPC3 protein. [Diagram 2] FIG. 1 shows the results of flow cytometry detection of GPC3 stably transfected cells. [Diagram 3]FIG. 1 shows the results of SDS-PAGE of CD3εγ-Fc recombinant protein. [Figure 4] FIG. 1 shows the results of flow cytometry of GPC3 antibody and human GPC3 stably transfected cells. [Diagram 5] FIG. 1 shows the binding of antibody 49G8 to different species GPC3 antigens. [Figure 6] FIG. 1 shows the ADCC effect of antibody 49G8. [Figure 7] FIG. 1 shows binding of humanized antibodies to the GPC3 antigen. [Figure 8] FIG. 1 shows binding of humanized antibodies to GPC3 stably transfected cells. [Figure 9] FIG. 1 shows Octet affinity measurements of humanized antibodies. [Figure 10] FIG. 1 shows SPR affinity measurements of humanized antibodies. [Figure 11] FIG. 1 shows the cell endocytosis results of humanized antibodies. [Figure 12] FIG. 1 shows binding of CD3 humanized antibodies to human CD3 εγ antigen. [Figure 13] FIG. 1 shows binding of CD3 humanized antibodies to Jurkat cells. [Figure 14] FIG. 1 shows a comparison of the affinity of humanized CD3 antibodies to human and monkey CD3εγ antigens. [Figure 15] FIG. 1 shows binding of GPC3×CD3κλ bispecific antibody to GPC3 stably transfected cells. [Figure 16] FIG. 1 shows binding of GPC3×CD3κλ bispecific antibody to HepG2 cells. [Figure 17] FIG. 1 shows binding of GPC3×CD3κλ bispecific antibody to Jurkat cells. [Figure 18] FIG. 1 shows binding of GPC3×CD3κλ bispecific antibody to peripheral blood T cells. [Figure 19]FIG. 19 shows the TDCC effect of the GPC3×CD3κλ bispecific antibody, with FIG. 19A showing the killing of HepG2 cells and FIG. 19B showing the activation of T cells. [Figure 20] FIG. 1 shows the effect of a GPC3×CD3κλ bispecific antibody on the T cell NFAT signal pathway. [Figure 21] FIG. 1 shows the activating effect of GPC3×CD3κλ bispecific antibody on PBMCs. [Figure 22] FIG. 1 shows binding of GPC3×CD3κλ bispecific antibody to FcγR activating receptors. [Figure 23] FIG. 13 shows the suppressive effect of the GPC3×CD3κλ bispecific antibody in an immune reconstituted mouse HepG2 tumor xenograft model. [Figure 24] FIG. 13 shows the suppressive effect of GPC3×CD3κλ bispecific antibody in a CD3-humanized mouse Hepa1-6 / hGPC3 transplanted tumor model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] I. Definition In this disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology used herein are terms and routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0019] The term "GPC3" as used herein can refer to a concept that collectively refers to GPC3 itself present in animals, preferably humans, and any mutants, isoforms, and paralogs thereof.

[0020] The term "human GPC3" refers to GPC3 derived from humans.

[0021] The terms "anti-GPC3 antibody" and "antibody that binds to GPC3" refer to an antibody that can bind to GPC3 with sufficient affinity such that the antibody is useful for targeting GPC3 as a diagnostic and / or therapeutic agent. In one embodiment, the anti-GPC3 antibody has a binding level to unrelated non-GPC3 proteins that is less than about 10% of the binding level of the antibody to GPC3, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the antibody that binds to GPC3 has a binding level of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 Dissociation constant (K d In some embodiments, the anti-GPC3 antibody binds to a conserved GPC3 epitope among GPC3s from different species.

[0022] "CD3" refers to any native CD3 from any vertebrate, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The term covers "full-length", unprocessed CD3, and any form of CD3 processed intracellularly. The term further covers naturally occurring variants of CD3, such as splice variants or allelic variants. In one embodiment, the CD3 is human CD3, and in particular the ε subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is shown in UniProt (www.uniprot.org) accession number P07766 (version 144), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. The amino acid sequence of cynomolgus monkey [Macaca fascicularis] CD3ε is shown in NCBI GenBank no. BAB71849.1.

[0023] The term "cell surface" is used according to its ordinary meaning in the art, and includes the exterior of the cell that is accessible for binding to proteins and other molecules.

[0024] As used herein, the term "about" or "approximately" means, unless otherwise specified, within ±10% of a given value or range. When required to be an integer, the term shall be rounded up or down to the nearest integer within ±10% of the given value or range.

[0025] With respect to antibody chain polypeptide sequences, the term "substantially identical" may be understood as antibody chains exhibiting 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more sequence identity to a reference polypeptide sequence. With respect to nucleic acid sequences, the term may be understood as a nucleotide sequence exhibiting at least 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or more sequence identity to a reference nucleic acid sequence.

[0026] Sequence "homology" or "identity" has a meaning well known in the art, and the disclosed techniques can be used to calculate the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide, or along a region of the molecule. There are several ways to measure homology between two polynucleotides or polypeptides, but the term "homology" is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0027] "Substitutional" variants are those in which at least one amino acid residue in a native sequence has been removed and a different amino acid has been inserted in the same position. The substitutions may be single, where only one amino acid has been replaced in the molecule, or multiple, where two or more amino acids have been replaced in the same molecule. Multiple substitutions are present at contiguous sites. Similarly, an amino acid may be replaced by multiple residues, and such variants include both substitutions and insertions. "Insertional" variants are those in which one or more amino acids have been inserted immediately adjacent to an amino acid at a particular position in a native sequence. Immediately adjacent to an amino acid means bonded to the α-carboxyl or α-amino functionality of that amino acid. "Deletional" variants are those in which one or more amino acids in a native amino acid sequence have been removed. Typically, deletional variants are those in which one or two amino acids have been deleted in a particular region of the molecule.

[0028] With respect to the variable domain of an antibody, the term "variable" refers to the specific portions of the related molecule that differ greatly in sequence among antibodies and are used for the specific recognition and binding of a particular antibody to a particular target. However, variability is not evenly distributed throughout the variable domain of an antibody. The variability is concentrated in three segments called complementarity determining regions (CDRs, i.e., CDR1, CDR2, and CDR3) or hypervariable regions, which are present in all light and heavy chain variable domains. The more highly conserved parts of the variable domains are called framework (FR) regions or framework sequences. Each naturally occurring heavy and light chain variable domain, respectively, contains four FR regions that are primarily in a β-sheet configuration, connected by three CDRs, which form loops that connect to, and in some cases form part of, the β-sheet structure. The CDRs of each chain are usually adjacently connected by FR regions and contribute to the formation of the target binding site (epitope or determinant) of the antibody with the CDRs from the other chain. As used herein, immunoglobulin amino acid residue numbering is according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise specified. A CDR may have the ability to specifically bind to an associated epitope.

[0029] 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 contains at least a portion of the variable region of said antibody that binds to an antigen (e.g., one or more CDRs and / or one or more antibody binding sites), thereby retaining at least a portion of the binding specificity and specific binding ability of said full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment is derived. Antibody fragments include antibody derivatives produced by enzyme-catalyzed treatment of a full-length antibody, as well as synthetically produced derivatives, e.g., recombinantly produced derivatives. Antibodies include antibody fragments. Illustrative examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single chain Fv (scFv), Fv, dsFv, diabody, Fd, and Fd' fragments, as well as other fragments, including modified fragments. The fragments may, for example, contain multiple chains linked by disulfide bonds and / or peptide linkers. Antibody fragments usually contain at least or about 50 amino acids, and typically contain at least or about 200 amino acids. Antigen-binding fragments are those that immunospecifically bind (i.e., bind at least 10 amino acids) when inserted into an antibody framework (e.g., by replacing the corresponding region). 7 ~10 8 M -1 or at least about 10 7 ~10 8 M -1 The term "functional fragment" or "anti-GPC3 antibody analog" includes any antibody fragment that provides an antibody that binds to an antigen (indicating a Ka of 0.01 to 0.1). A "functional fragment" or "anti-GPC3 antibody analog" is a fragment or analog that inhibits or substantially reduces the ability of the receptor to bind to a ligand or initiate signal transduction. As used herein, a functional fragment generally has the same meaning as an "antibody fragment" and, in the context of an antibody, may refer to a fragment that inhibits or substantially reduces the ability of the receptor to bind to a ligand or initiate signal transduction, such as Fv, Fab, F(ab')2, etc. An "Fv" fragment is a dimer (V) formed by the non-covalent association of one heavy chain variable domain and one light chain variable domain.H -V L In this configuration, the three CDRs of each variable domain interact to form a V H -V L The six CDRs define a target binding site on the surface of the dimer. The six CDRs confer target binding specificity to the intact antibody. However, a single variable domain (or half of an Fv containing only three CDRs specific for a target) can also function to recognize and bind to a target.

[0030] The term "bispecific antibody" (BsAb) as used herein refers to an antibody and / or antigen-binding molecule capable of specifically binding to two different antigenic determinants, and typically a bispecific antibody and / or antigen-binding molecule comprises two antigen-binding sites, each having specificity for a different antigenic determinant. In some embodiments, the bispecific antibody and / or antigen-binding molecule can simultaneously bind to two antigenic determinants, in particular two antigenic determinants expressed on two different cells.

[0031] As used herein, "monoclonal antibody" refers to a population of identical antibodies, meaning that each individual antibody molecule in a monoclonal antibody population is identical to the other antibody molecules. This property is the opposite of that of a polyclonal population of antibodies, which contains antibodies with multiple distinct sequences. Monoclonal antibodies can be prepared in many well-known ways (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 fusing with myeloma cells to produce hybridoma cell lines, or by infection of B cells with a virus such as EBV. Recombinant techniques can also be used to prepare antibodies from a clonal population of host cells in vitro by transforming the host cells with a plasmid carrying an artificial sequence of nucleotides encoding the antibody.

[0032] The term "hybridoma" or "hybridoma cell" as used herein refers to a cell or cell line produced by the fusion of an antibody-producing lymphocyte with a non-antibody-producing cancer cell (usually a myeloma or lymphoma cell). As known to those skilled in the art, a hybridoma can grow and produce and continuously supply a specific monoclonal antibody. Methods for producing hybridomas are known in the art. When the term "hybridoma" or "hybridoma cell" is referred to, it also includes subclones and progeny cells of the hybridoma.

[0033] As used herein, a full length antibody is an antibody having two full length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full length light chains (VL-CL) and a hinge region, such as an antibody produced naturally by an antibody-secreting B cell, or an antibody having the same domains produced synthetically.

[0034] 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, e.g., the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0035] "Humanized" antibodies refer to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Preferably, humanized antibodies are human immunoglobulins (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.

[0036] In humanization, it is also possible to improve one or more binding properties (e.g., affinity) of the antibody by mutating amino acid residues in the CDR1, CDR2 and / or CDR3 regions of VH and / or VL. For example, mutations can be introduced by PCR-mediated mutagenesis, and the effect on antibody binding or other functional properties can be evaluated by in vitro or in vivo testing as described herein. Typically, conservative mutations are introduced. Such mutations may be amino acid substitutions, additions, or deletions. Also, mutations in the CDRs usually do not exceed one or two. Thus, the humanized antibody of the present disclosure further includes antibodies containing one or two amino acid mutations in the CDRs.

[0037] The term "CDR" as used herein refers to a complementarity-determining region, and each heavy and light chain of an antibody molecule is known to have three CDRs. CDRs, also called hypervariable regions, are present in the variable regions of each heavy and light chain of an antibody, and are highly variable sites in the primary structure of the CDRs. Herein, the CDRs of the heavy chain are designated CDR1, CDR2, and CDR3 at the amino terminus of the amino terminal sequence derived from the heavy chain, and the CDRs of the light chain are designated CDR1, CDR2, and CDR3 at the amino terminus of the amino terminal sequence derived from the light chain. These sites are adjacent to each other in the tertiary structure and determine the specificity of the antigen that binds to the antibody.

[0038] The term "epitope" as used herein refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

[0039] As used herein, with respect to an antibody or antigen-binding fragment thereof, the terms "specifically binds" or "immunospecifically binds" are used interchangeably herein and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with a cognate antigen by non-covalent interactions between the antibody and the antibody-binding site of the antigen. The antigen may be an isolated antigen or may be present on tumor cells. Typically, an antibody that immunospecifically binds (or specifically binds) to an antigen is present at approximately 1×10 7 M -1 Or about 1 x 10 8 M -1 or higher affinity constant Ka (1×10 -7 Or 1×10 -8 The antibody binds to the antigen with a dissociation constant (Kd) of M or less. The affinity constant can be measured by standard kinetic methods of antibody reactions, such as immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC) or other kinetic interaction measurements well known in the art, see U.S. Pat. No. 7,229,619, which describes exemplary SPR and ITC methods for calculating the binding affinity of antibodies. Devices and methods for real-time detection and monitoring of binding kinetics are known and commercially available (see BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335).

[0040] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer containing two or more linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), typically linked by phosphodiester bonds. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules, RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, and may be cDNA.

[0041] As used herein, an isolated nucleic acid molecule is a nucleic acid molecule that is isolated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. For example, an "isolated" nucleic acid molecule of a cDNA molecule is substantially free of other cellular material or culture medium when prepared by recombinant techniques, or is substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules that encode the provided antibodies or antigen-binding fragments.

[0042] As used herein, "operably linked" with respect to a nucleic acid sequence, region, element, or domain means that the nucleic acid regions are functionally related to each other. For example, a promoter can be operably linked to a nucleic acid encoding a polypeptide such that it can regulate or mediate transcription of the nucleic acid.

[0043] "Conservative sequence modifications" of the sequences in the sequence listing herein, i.e., nucleotide and amino acid sequence modifications that do not eliminate the binding of the antibody encoded by the nucleotide sequence or containing the amino acid sequence to the antigen, are also provided. These conservative sequence modifications include conservative nucleotide and amino acid substitutions, and nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the sequence listing herein by standard techniques well known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative sequence modifications include conservative amino acid substitutions, in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine and histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, predicted non-essential amino acid residues in anti-GPC3 antibodies are preferably replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art (see, e.g., Brummell et al, Biochem. 32:1180-1187 (1993); Kobayashi et al, Protein Eng. 12(10):879-884 (1999); Burkset et al, Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0044] Alternatively, in another embodiment, mutations can be introduced randomly along all or part of the sequence encoding the anti-GPC3 antibody, for example by saturation mutagenesis, and the resulting modified anti-GPC3 antibodies can be screened for improved binding activity.

[0045] "Expression" as used herein 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, a method for determining the amount of a polypeptide produced from a host cell. Such methods can include, but are not limited to, quantification of a polypeptide in a cell lysate by ELISA, gel electrophoresis followed by Coomassie blue staining, Lowry protein assay, and Bradford protein assay.

[0046] As used herein, a "host cell" is a cell used to receive, maintain, replicate, and amplify a vector. A host cell can also be used to express a polypeptide encoded by a vector. When a host cell divides, the nucleic acid contained in the vector is replicated and the nucleic acid is amplified. A host cell can be a eukaryotic or prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells, such as HEK293 cells.

[0047] A "vector" as used herein is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into a suitable host cell. Vectors include vectors that can introduce a nucleic acid encoding a polypeptide or a fragment thereof, typically by restriction digestion and ligation. Vectors further include vectors that contain a nucleic acid encoding a polypeptide. Vectors are used to introduce a nucleic acid encoding a polypeptide into a host cell for amplification of the nucleic acid or for expression / display of the polypeptide encoded by the nucleic acid. Vectors usually remain episomal, but may also be engineered into chromosomes where a gene or a part thereof is integrated into the genome. Also contemplated are vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. The selection and use of such vehicles is well known to those skilled in the art.

[0048] Vector as used herein further includes "viral vectors" or "viral vectors." Viral vectors are engineered viruses that are operably linked to exogenous genes to transfer the exogenous genes into cells (as a vehicle or shuttle).

[0049] As used herein, an "expression vector" includes a vector capable of expressing DNA, said DNA being operably linked to a regulatory sequence, e.g., a promoter region, that affects the expression of the DNA fragment. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression carriers can generally be derived from plasmid or viral DNA, or contain elements of both. Thus, an expression carrier refers to a recombinant DNA or RNA construct, e.g., a plasmid, phage, recombinant virus, or other vector, that, when introduced into a suitable host cell, results in the expression of the 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, and expression vectors that remain episomal or that integrate into the host cell genome.

[0050] As used herein, "treating" an individual with a disease or a symptom of a disease means that the individual's symptoms are partially or totally ameliorated or remain unchanged following treatment. Thus, treatment includes prevention, therapy and / or cure. Prevention refers to the prevention of underlying disease and / or the prevention of worsening of symptoms or progression of disease. Treatment further includes any pharmaceutical use of any of the antibodies or antigen-binding fragments thereof provided and compositions provided herein.

[0051] As used herein, "therapeutic benefit" means the benefit obtained by treating an individual, which is an alteration in the symptoms of a disease or disease state, typically an improvement or amelioration of the disease or disease state, or a cure of the disease or disease state.

[0052] As used herein, a "therapeutically effective amount" or a "therapeutically effective dose" refers to an amount of a drug, compound, substance, or composition containing a compound that is at least sufficient to achieve a therapeutic effect following administration to a subject, and thus is the amount necessary to prevent, cure, ameliorate, suppress or partially suppress the symptoms of a disease or condition.

[0053] As used herein, a "prophylactically effective amount" or a "prophylactically effective dose" refers to the amount of a substance, compound, material, or composition containing a compound that, upon administration to a subject, has a desired prophylactic effect, such as, for example, preventing or delaying the onset or recurrence of a disease or condition, or reducing the likelihood of the onset or recurrence of a disease or condition. A complete prophylactically effective dose need not be achieved by administering a single dose, but may be achieved only when a series of doses are administered. Thus, a prophylactically effective amount may be administered in one or more administrations.

[0054] As used herein, the term "patient" refers to a mammal, such as a human.

[0055] II. MODE FOR CARRYING OUT THE DISCLOSURE

[0056] In one aspect, the disclosure provides a method for the preparation of a polypeptide comprising: (a) a first antigen-binding portion or antigen-binding fragment thereof that binds to a GPC3 antigen on a target cell surface, the first antigen-binding portion comprising a first heavy chain and a first light chain, the first antigen-binding portion comprising a first binding domain that binds to the first antigen, the first binding domain comprising a heavy chain CDR selected from the amino acid sequence SEQ ID NO: 11, 12, 13, 21, 32, 33, 64, 65, 66, 72, 73, 79, 80, 81, 101, 106, or any variant thereof, and / or a light chain CDR selected from the amino acid sequence SEQ ID NO: 16, 17, 18, 26, 29, 38, 39, 46, 47, 48, 51, 54, 57, 60, 61, 69, 92, 95, 98, or any variant thereof; and (b) a second antigen-binding portion or antigen-binding fragment thereof that binds to a CD3 antigen on the surface of a T cell, the second antigen-binding portion comprising a second heavy chain and a second light chain, the second antigen-binding portion comprising a second binding domain that binds to a second antigen. According to the antibody or antigen-binding portion thereof, the first binding domain comprises a heavy chain CDR1 selected from the amino acid sequence of SEQ ID NO:11, 32, 64, 79, or any variant thereof, a heavy chain CDR2 selected from the amino acid sequence of SEQ ID NO:12, 21, 33, 65, 72, 80, 101, 106, or any variant thereof, and a heavy chain CDR3 selected from the amino acid sequence of SEQ ID NO:13, 66, 73, 81, or any variant thereof, and / or the first binding domain comprises a light chain CDR1 selected from the amino acid sequence of SEQ ID NO:16, 38, 46, 60, or any variant thereof, a light chain CDR2 selected from the amino acid sequence of SEQ ID NO:17, 47, or any variant thereof, and a light chain CDR3 selected from the amino acid sequence of SEQ ID NO:18, 26, 29, 39, 48, 51, 54, 57, 61, 69, 76, or any variant thereof.

[0057] In the antibody or antigen-binding portion thereof according to any of the above embodiments, the heavy chain CDR1, CDR2 and CDR3 sequences of the first binding domain are selected from the group consisting of the first heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NOs: 11, 12, 13, the first heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NOs: 11, 21, 13, the first heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NOs: 32, 33, 13, the first heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NOs: 64, 65, 66, the first heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NOs: 11, 72, 73, the first heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NOs: 79, 80, 81, the first heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NOs: 82, 83, 84, the first heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NOs: 85, 86, the first heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NOs: 87, 88, 89, the first heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NOs: 89, 90, 91, the first heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NOs: 92, 93, the first heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NOs: 94, 95, 96, the first heavy chain CDR1, CDR2 and CDR3 sequences of amino acid and the light chain CDR1, CDR2 and CDR3 sequence of the first binding domain is selected from the first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO: 16, 17, 18, the first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO: 16, 17, 26, the first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO: 16, 17, 29, the first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO: 38, 17, 39, the first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO: 39, 40, 41, 42, 43, and the light chain CDR1, CDR2 and CDR3 sequence of the first binding domain is selected from the first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO: 16, 17, 18, the first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO: 16, 17, 26, the first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO: 16, 17, 29, the first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO: 38, 17, 39, the first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO: 39, 42, 43, and the light chain CDR1, CDR2 and CDR3 sequence of the first binding domain is selected from the first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO: 16, 17, 18, the first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO the first light chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NOs: 16, 17, 51; the first light chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NOs: 16, 17, 54; the first light chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NOs: 16, 17, 57; the first light chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NOs: 60, 17, 61; the first light chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NOs: 16, 17, 69;the first light chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NO: 92, 17, 61; the first light chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NO: 95, 17, 61; and the first light chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NO: 98, 17, 61.

[0058] In some embodiments, the first binding domain comprises a first heavy chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:11, 12, 13, and a first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:16, 17, 18.

[0059] In some embodiments, the first binding domain comprises a first heavy chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:11, 21, 13, and a first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:16, 17, 18.

[0060] In some embodiments, the first binding domain comprises a first heavy chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:11, 12, 13, and a first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:16, 17, 26.

[0061] In some embodiments, the first binding domain comprises a first heavy chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:11, 12, 13, and a first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:16, 17, 29.

[0062] In some embodiments, the first binding domain comprises a first heavy chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:32, 33, 13, and a first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:16, 17, 18.

[0063] In some embodiments, the first binding domain comprises a first heavy chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:11, 21, 13, and a first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:38, 17, 39.

[0064] In some embodiments, the first binding domain comprises a first heavy chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:11, 21, 13, and a first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:16, 17, 18.

[0065] In some embodiments, the first binding domain comprises a first heavy chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:11, 12, 13, and a first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:46, 47, 48.

[0066] In some embodiments, the first binding domain comprises a first heavy chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:11, 12, 13, and a first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:16, 17, 51.

[0067] In some embodiments, the first binding domain comprises a first heavy chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:11, 12, 13, and a first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:16, 17, 54.

[0068] In some embodiments, the first binding domain comprises a first heavy chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:11, 12, 13, and a first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:16, 17, 57.

[0069] In some embodiments, the first binding domain comprises a first heavy chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:11, 12, 13, and a first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:60, 17, 61.

[0070] In some embodiments, the first binding domain comprises a first heavy chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:64, 65, 66, and a first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:16, 17, 69.

[0071] In some embodiments, the first binding domain comprises a first heavy chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:11, 72, 73, and a first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:16, 17, 76.

[0072] In some embodiments, the first binding domain comprises a first heavy chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:79, 80, 81, and a first light chain CDR1, CDR2 and CDR3 sequence of amino acid sequence SEQ ID NO:16, 17, 69.

[0073] In some embodiments, the first binding domain comprises a first heavy chain CDR1, CDR2 and CDR3 sequence of the amino acid sequence SEQ ID NO:11, 106, 13, and a first light chain CDR1, CDR2 and CDR3 sequence of the amino acid sequence SEQ ID NO:60, 17, 61.

[0074] In some embodiments, the first binding domain comprises a first heavy chain CDR1, CDR2 and CDR3 sequence of the amino acid sequence SEQ ID NO:11, 106, 13, and a first light chain CDR1, CDR2 and CDR3 sequence of the amino acid sequence SEQ ID NO:92, 17, 61.

[0075] In some embodiments, the first binding domain comprises a first heavy chain CDR1, CDR2 and CDR3 sequence of the amino acid sequence SEQ ID NO:11, 106, 13, and a first light chain CDR1, CDR2 and CDR3 sequence of the amino acid sequence SEQ ID NO:95, 17, 61.

[0076] In some embodiments, the first binding domain comprises a first heavy chain CDR1, CDR2 and CDR3 sequence of the amino acid sequence SEQ ID NO:11, 106, 13, and a first light chain CDR1, CDR2 and CDR3 sequence of the amino acid sequence SEQ ID NO:98, 17, 61.

[0077] In the antibody or antigen-binding portion thereof according to any of the above embodiments, the first binding domain comprises a first heavy chain variable region selected from the amino acid sequence of SEQ ID NO: 9, 19, 30, 40, 62, 70, 77, 99, 102, 104, 107, 109, 111, or any variant thereof, and / or a first light chain variable region selected from the amino acid sequence of SEQ ID NO: 14, 22, 24, 27, 34, 36, 42, 44, 49, 52, 55, 58, 67, 74, 82, 84, 86, 88, 90, 93, 96, or any variant thereof.

[0078] In some embodiments, the first binding domain comprises a first heavy chain variable region of amino acid sequence SEQ ID NO:9 or any variant thereof, and a first light chain variable region of amino acid sequence SEQ ID NO:14 or any variant thereof.

[0079] In some embodiments, the first binding domain comprises a first heavy chain variable region of amino acid sequence SEQ ID NO:19, or any variant thereof, and a first light chain variable region of amino acid sequence SEQ ID NO:22, or any variant thereof.

[0080] In some embodiments, the first binding domain comprises a first heavy chain variable region of amino acid sequence SEQ ID NO:9 or any variant thereof, and a first light chain variable region of amino acid sequence SEQ ID NO:24 or any variant thereof.

[0081] In some embodiments, the first binding domain comprises a first heavy chain variable region of amino acid sequence SEQ ID NO:9 or any variant thereof, and a first light chain variable region of amino acid sequence SEQ ID NO:27 or any variant thereof.

[0082] In some embodiments, the first binding domain comprises a first heavy chain variable region of amino acid sequence SEQ ID NO:30, or any variant thereof, and a first light chain variable region of amino acid sequence SEQ ID NO:34, or any variant thereof.

[0083] In some embodiments, the first binding domain comprises a first heavy chain variable region of amino acid sequence SEQ ID NO:19 or any variant thereof, and a first light chain variable region of amino acid sequence SEQ ID NO:36 or any variant thereof.

[0084] In some embodiments, the first binding domain comprises a first heavy chain variable region of amino acid sequence SEQ ID NO:40, or any variant thereof, and a first light chain variable region of amino acid sequence SEQ ID NO:42, or any variant thereof.

[0085] In some embodiments, the first binding domain comprises a first heavy chain variable region of amino acid sequence SEQ ID NO:9 or any variant thereof, and a first light chain variable region of amino acid sequence SEQ ID NO:44 or any variant thereof.

[0086] In some embodiments, the first binding domain comprises a first heavy chain variable region of amino acid sequence SEQ ID NO:9 or any variant thereof, and a first light chain variable region of amino acid sequence SEQ ID NO:49 or any variant thereof.

[0087] In some embodiments, the first binding domain comprises a first heavy chain variable region of amino acid sequence SEQ ID NO:9 or any variant thereof, and a first light chain variable region of amino acid sequence SEQ ID NO:52 or any variant thereof.

[0088] In some embodiments, the first binding domain comprises a first heavy chain variable region of amino acid sequence SEQ ID NO:9 or any variant thereof, and a first light chain variable region of amino acid sequence SEQ ID NO:55 or any variant thereof.

[0089] In some embodiments, the first binding domain comprises a first heavy chain variable region of amino acid sequence SEQ ID NO:9 or any variant thereof, and a first light chain variable region of amino acid sequence SEQ ID NO:58 or any variant thereof.

[0090] In some embodiments, the first binding domain comprises a first heavy chain variable region of amino acid sequence SEQ ID NO:62 or any variant thereof, and a first light chain variable region of amino acid sequence SEQ ID NO:67 or any variant thereof.

[0091] In some embodiments, the first binding domain comprises a first heavy chain variable region of amino acid sequence SEQ ID NO:70, or any variant thereof, and a first light chain variable region of amino acid sequence SEQ ID NO:74, or any variant thereof.

[0092] In some embodiments, the first binding domain comprises a first heavy chain variable region of amino acid sequence SEQ ID NO:77, or any variant thereof, and a first light chain variable region of amino acid sequence SEQ ID NO:82, or any variant thereof.

[0093] In some embodiments, the first binding domain comprises a first heavy chain variable region of amino acid sequence SEQ ID NO:111, or any variant thereof, and a first light chain variable region of amino acid sequence SEQ ID NO:86, or any variant thereof.

[0094] In some embodiments, the first binding domain comprises a first heavy chain variable region of amino acid sequence SEQ ID NO:111, or any variant thereof, and a first light chain variable region of amino acid sequence SEQ ID NO:88, or any variant thereof.

[0095] In some embodiments, the first binding domain comprises a first heavy chain variable region of amino acid sequence SEQ ID NO:111, or any variant thereof, and a first light chain variable region of amino acid sequence SEQ ID NO:90, or any variant thereof.

[0096] In some embodiments, the first binding domain comprises a first heavy chain variable region of amino acid sequence SEQ ID NO:111, or any variant thereof, and a first light chain variable region of amino acid sequence SEQ ID NO:93, or any variant thereof.

[0097] In some embodiments, the first binding domain comprises a first heavy chain variable region of amino acid sequence SEQ ID NO:111, or any variant thereof, and a first light chain variable region of amino acid sequence SEQ ID NO:96, or any variant thereof.

[0098] In the antibody or antigen-binding portion thereof of any of the above embodiments, the second binding domain comprises a heavy chain CDR selected from the amino acid sequence of SEQ ID NO: 134, 135, 136, 139, 142, 145, 148, 151, 154, 155, or any variant thereof, and / or a light chain CDR selected from the amino acid sequence of SEQ ID NO: 115, 116, 117, 122, 123, 128, 131, or any variant thereof.

[0099] In some embodiments, the second binding domain comprises a second heavy chain CDR1 selected from the amino acid sequence SEQ ID NO: 134, 139, 154, or any variant thereof, a second heavy chain CDR2 selected from the amino acid sequence SEQ ID NO: 135, 155, or any variant thereof, a second heavy chain CDR3 selected from the amino acid sequence SEQ ID NO: 136, 142, 145, 148, 151, or any variant thereof, and / or a second light chain CDR1 selected from the amino acid sequence SEQ ID NO: 115, 122, or any variant thereof, a second light chain CDR2 selected from the amino acid sequence SEQ ID NO: 116, 123, 131, or any variant thereof, and a second light chain CDR3 selected from the amino acid sequence SEQ ID NO: 117, 128, or any variant thereof.

[0100] In some embodiments, the heavy chain CDR1, CDR2 and CDR3 sequences of the second binding domain are selected from the group consisting of the second heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NO: 134, 135, 136, the second heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NO: 139, 135, 136, the second heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NO: 139, 135, 142, the second heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NO: 139, 135, 145, the second heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NO: 139, 135, 148, the second heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NO: 139, 135, 151, the second heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NO: 139, 135, 152, the second heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NO: 139, 135, 153, the second heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NO: 139, 135, 154, the second heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NO: 139, 135, 155, the second heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NO: 139, 135, 156, the second heavy chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NO: 139, 135, 157, and the second binding domain-like light chain CDR1, CDR2 and CDR3 sequence is selected from the second light chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NOs: 115, 116, 117, the second light chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NOs: 122, 123, 117, the second light chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NOs: 115, 116, 128, the second light chain CDR1, CDR2 and CDR3 sequences of amino acid sequences SEQ ID NOs: 115, 131, 128.

[0101] In some embodiments, the second binding domain comprises a second heavy chain CDR1, CDR2 and CDR3 sequence of the amino acid sequence SEQ ID NO:134, 135, 136, and a second light chain CDR1, CDR2 and CDR3 sequence of the amino acid sequence SEQ ID NO:115, 116, 128. In some embodiments, the second binding domain comprises a second heavy chain CDR1, CDR2 and CDR3 sequence of the amino acid sequence SEQ ID NO:134, 135, 136, and a second light chain CDR1, CDR2 and CDR3 sequence of the amino acid sequence SEQ ID NO:115, 116, 117.

[0102] In some embodiments, the second binding domain comprises a second heavy chain CDR1, CDR2 and CDR3 sequence of the amino acid sequence SEQ ID NO:134, 135, 136, and a second light chain CDR1, CDR2 and CDR3 sequence of the amino acid sequence SEQ ID NO:122, 123, 117.

[0103] In some embodiments, the second binding domain comprises a second heavy chain variable region selected from the amino acid sequence of SEQ ID NO:132, 137, 140, 143, 146, 149, 152, 156, 158, 160, or any variant thereof, and / or a second light chain variable region selected from the amino acid sequence of SEQ ID NO:113, 118, 120, 124, 126, 129, or any variant thereof.

[0104] In some embodiments, the second binding domain comprises a second heavy chain variable region having the amino acid sequence SEQ ID NO:132, or any variant thereof, and a second light chain variable region having the amino acid sequence SEQ ID NO:126, or any variant thereof.

[0105] In some embodiments, the second binding domain comprises a second heavy chain variable region having the amino acid sequence SEQ ID NO:156, or any variant thereof, and a second light chain variable region having the amino acid sequence SEQ ID NO:113, or any variant thereof.

[0106] In some embodiments, the second binding domain comprises a second heavy chain variable region of amino acid sequence SEQ ID NO:156, or any variant thereof, and a second light chain variable region of amino acid sequence SEQ ID NO:126, or any variant thereof.

[0107] In some embodiments, the second binding domain comprises a second heavy chain variable region of amino acid sequence SEQ ID NO:158, or any variant thereof, and a second light chain variable region of amino acid sequence SEQ ID NO:113, or any variant thereof.

[0108] In some embodiments, the second binding domain comprises a second heavy chain variable region of amino acid sequence SEQ ID NO:158, or any variant thereof, and a second light chain variable region of amino acid sequence SEQ ID NO:118, or any variant thereof.

[0109] In some embodiments, the second binding domain comprises a second heavy chain variable region of amino acid sequence SEQ ID NO:158, or any variant thereof, and a second light chain variable region of amino acid sequence SEQ ID NO:120, or any variant thereof.

[0110] In some embodiments, the second binding domain comprises a second heavy chain variable region having the amino acid sequence SEQ ID NO:158 or any variant thereof, and a second light chain variable region having the amino acid sequence SEQ ID NO:126 or any variant thereof.

[0111] In the antibody or antigen-binding portion thereof according to any of the preceding aspects, the first light chain of the first antigen-binding portion is a κ light chain and the second light chain of the second antigen-binding portion is a λ light chain; In some embodiments, the first light chain variable region of the first antigen binding portion comprises the amino acid sequence of Gln 43 Lys mutation (Vκ GPC3 : Gln 43 In some embodiments, the first heavy chain variable region of the first antigen binding portion has a Gln 39 Glu(VH GPC3 :Gln 39 Glu) mutation.

[0112] In some embodiments, the second light chain variable region of the second antigen binding portion comprises the amino acid sequence of Gln 40 Glu mutation (Vλ CD3 : Gln 40 In some embodiments, the second heavy chain variable region of the second antigen binding portion has Gln 39 Lys mutation (VH CD3 :Gln 39 Lys).

[0113] In some embodiments, the Fc portions of the first and second antigen-binding portions of the bispecific antibody adopt a knob-into-hole conformation, hi some preferred embodiments, a human IgG4 knob-into-hole conformation.

[0114] In some embodiments, the first heavy chain comprises a heavy chain selected from SEQ ID NO: 164, 172, or any variant thereof, and the first light chain comprises a light chain selected from SEQ ID NO: 162, 170, or any variant thereof.

[0115] In some preferred embodiments, the first heavy chain comprises a heavy chain selected from SEQ ID NO: 164 or any variant thereof, and the first light chain comprises a light chain selected from SEQ ID NO: 162 or any variant thereof.

[0116] In some preferred embodiments, the first heavy chain comprises a heavy chain selected from SEQ ID NO:172 or any variant thereof, and the first light chain comprises a light chain selected from SEQ ID NO:170 or any variant thereof.

[0117] In some preferred embodiments, the second heavy chain comprises a heavy chain of amino acid sequence SEQ ID NO:168 or any variant thereof, and the second light chain comprises a light chain of amino acid sequence SEQ ID NO:166 or any variant thereof.

[0118] In one specific embodiment, the first heavy chain of the bispecific antibody comprises the heavy chain of the amino acid sequence SEQ ID NO:164 or any variant thereof, the first light chain of the bispecific antibody comprises the light chain of the amino acid sequence SEQ ID NO:162 or any variant thereof, the second heavy chain comprises the heavy chain of the amino acid sequence SEQ ID NO:168 or any variant thereof, and the second light chain comprises the light chain of the amino acid sequence SEQ ID NO:166 or any variant thereof.

[0119] In one specific embodiment, the first heavy chain comprises the heavy chain of the amino acid sequence SEQ ID NO:172 or any variant thereof, the first light chain of the bispecific antibody comprises the light chain of the amino acid sequence SEQ ID NO:170 or any variant thereof, the second heavy chain comprises the heavy chain of the amino acid sequence SEQ ID NO:168 or any variant thereof, and the second light chain comprises the light chain of the amino acid sequence SEQ ID NO:166 or any variant thereof.

[0120] In one aspect, the present disclosure provides an antibody or antigen-binding portion thereof that binds to GPC3, comprising the first antigen-binding portion described above. In some embodiments, an antibody or antigen-binding portion thereof that binds to GPC3 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to an antibody or antigen-binding portion thereof of any of the previous aspects.

[0121] In one aspect, the disclosure provides a nucleic acid encoding an antibody or antigen-binding portion thereof according to any of the previous aspects, or a nucleic acid molecule having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity thereto.

[0122] In some embodiments, the encoding nucleic acid of the first heavy chain variable region of the first antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:10, 20, 31, 41, 63, 71, 78, 100, 103, 105, 108, 110, 112, or any variant thereof, and / or the encoding nucleic acid of the first light chain variable region of the first antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:15, 23, 25, 28, 35, 37, 43, 45, 50, 53, 56, 59, 68, 75, 83, 85, 87, 89, 91, 94, 97, or any variant thereof.

[0123] In some preferred embodiments, the encoding nucleic acid for the first heavy chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:10, and the encoding nucleic acid for the first light chain variable region is the nucleotide sequence SEQ ID NO:15.

[0124] In some preferred embodiments, the encoding nucleic acid for the first heavy chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:20, and the encoding nucleic acid for the first light chain variable region is the nucleotide sequence SEQ ID NO:23.

[0125] In some preferred embodiments, the encoding nucleic acid of the first heavy chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:10, and the encoding nucleic acid of the first light chain variable region is the nucleotide sequence SEQ ID NO:25.

[0126] In some preferred embodiments, the encoding nucleic acid for the first heavy chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:10, and the encoding nucleic acid for the first light chain variable region is the nucleotide sequence SEQ ID NO:28.

[0127] In some preferred embodiments, the encoding nucleic acid of the first heavy chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:31, and the encoding nucleic acid of the first light chain variable region is the nucleotide sequence SEQ ID NO:35.

[0128] In some preferred embodiments, the encoding nucleic acid for the first heavy chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:20, and the encoding nucleic acid for the first light chain variable region is the nucleotide sequence SEQ ID NO:37.

[0129] In some preferred embodiments, the encoding nucleic acid for the first heavy chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:41, and the encoding nucleic acid for the first light chain variable region is the nucleotide sequence SEQ ID NO:43.

[0130] In some preferred embodiments, the encoding nucleic acid of the first heavy chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:10, and the encoding nucleic acid of the first light chain variable region is the nucleotide sequence SEQ ID NO:45.

[0131] In some preferred embodiments, the encoding nucleic acid of the first heavy chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:10, and the encoding nucleic acid of the first light chain variable region is the nucleotide sequence SEQ ID NO:50.

[0132] In some preferred embodiments, the encoding nucleic acid for the first heavy chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:10, and the encoding nucleic acid for the first light chain variable region is the nucleotide sequence SEQ ID NO:53.

[0133] In some preferred embodiments, the encoding nucleic acid of the first heavy chain variable region of the first antigen-binding portion has the nucleotide sequence SEQ ID NO:10, and the encoding nucleic acid of the first light chain variable region has the nucleotide sequence SEQ ID NO:56.

[0134] In some preferred embodiments, the encoding nucleic acid for the first heavy chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:10, and the encoding nucleic acid for the first light chain variable region is the nucleotide sequence SEQ ID NO:59.

[0135] In some preferred embodiments, the encoding nucleic acid for the first heavy chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:63, and the encoding nucleic acid for the first light chain variable region is the nucleotide sequence SEQ ID NO:68.

[0136] In some preferred embodiments, the encoding nucleic acid for the first heavy chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:71, and the encoding nucleic acid for the first light chain variable region is the nucleotide sequence SEQ ID NO:75.

[0137] In some preferred embodiments, the encoding nucleic acid for the first heavy chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:78, and the encoding nucleic acid for the first light chain variable region is the nucleotide sequence SEQ ID NO:83.

[0138] In some preferred embodiments, the encoding nucleic acid for the first heavy chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:112, and the encoding nucleic acid for the first light chain variable region is the nucleotide sequence SEQ ID NO:87.

[0139] In some preferred embodiments, the encoding nucleic acid of the first heavy chain variable region of the first antigen-binding portion has the nucleotide sequence SEQ ID NO:112, and the encoding nucleic acid of the first light chain variable region has the nucleotide sequence SEQ ID NO:89.

[0140] In some preferred embodiments, the encoding nucleic acid for the first heavy chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:112, and the encoding nucleic acid for the first light chain variable region is the nucleotide sequence SEQ ID NO:91.

[0141] In some preferred embodiments, the encoding nucleic acid of the first heavy chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:112, and the encoding nucleic acid of the first light chain variable region is the nucleotide sequence SEQ ID NO:94.

[0142] In some preferred embodiments, the encoding nucleic acid for the first heavy chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:112, and the encoding nucleic acid for the first light chain variable region is the nucleotide sequence SEQ ID NO:97.

[0143] Preferably, the coding nucleic acid of the first heavy chain of the first antigen-binding moiety is selected from the nucleotide sequence SEQ ID NO: 165, 173 or any variant thereof, and / or the coding nucleic acid of the first light chain of the first antigen-binding moiety is selected from the nucleotide sequence SEQ ID NO: 163, 171 or any variant thereof.

[0144] In some preferred embodiments, the encoding nucleic acid of the first heavy chain of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:165, and the encoding nucleic acid of the first light chain of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:163.

[0145] In some preferred embodiments, the encoding nucleic acid of the first heavy chain of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:173, and the encoding nucleic acid of the first light chain of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:171.

[0146] In some embodiments, the encoding nucleic acid of the second heavy chain variable region of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO: 133, 138, 141, 144, 147, 150, 153, 157, 159, 161, or any variant thereof, and / or the encoding nucleic acid of the second light chain variable region of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO: 114, 119, 121, 125, 127, 130, or any variant thereof.

[0147] In some preferred embodiments, the encoding nucleic acid for the second heavy chain variable region of the second antigen-binding portion is the nucleotide sequence SEQ ID NO:133, and the encoding nucleic acid for the second light chain variable region is the nucleotide sequence SEQ ID NO:127.

[0148] In some preferred embodiments, the coding nucleic acid for the second heavy chain variable region of the second antigen-binding portion is the nucleotide sequence SEQ ID NO:157, and the coding nucleic acid for the second light chain variable region is the nucleotide sequence SEQ ID NO:114.

[0149] In some preferred embodiments, the encoding nucleic acid for the second heavy chain variable region of the second antigen-binding portion is the nucleotide sequence SEQ ID NO:157, and the encoding nucleic acid for the second light chain variable region is the nucleotide sequence SEQ ID NO:127.

[0150] In some preferred embodiments, the encoding nucleic acid for the second heavy chain variable region of the second antigen-binding portion is the nucleotide sequence SEQ ID NO:159, and the encoding nucleic acid for the second light chain variable region is the nucleotide sequence SEQ ID NO:114.

[0151] In some preferred embodiments, the coding nucleic acid for the second heavy chain variable region of the second antigen-binding portion is the nucleotide sequence SEQ ID NO:159, and the coding nucleic acid for the second light chain variable region is the nucleotide sequence SEQ ID NO:119.

[0152] In some preferred embodiments, the coding nucleic acid for the second heavy chain variable region of the second antigen-binding portion is the nucleotide sequence SEQ ID NO:159, and the coding nucleic acid for the second light chain variable region is the nucleotide sequence SEQ ID NO:121.

[0153] In some preferred embodiments, the coding nucleic acid for the second heavy chain variable region of the second antigen-binding portion is the nucleotide sequence SEQ ID NO:159, and the coding nucleic acid for the second light chain variable region is the nucleotide sequence SEQ ID NO:127.

[0154] Preferably, the coding nucleic acid for the second heavy chain of the second antigen-binding moiety has the nucleotide sequence SEQ ID NO:169 or any variant thereof, and / or the coding nucleic acid for the second light chain of the second antigen-binding moiety has the nucleotide sequence SEQ ID NO:167 or any variant thereof.

[0155] In some preferred embodiments, the encoding nucleic acid for the second heavy chain of the second antigen binding moiety is the nucleotide sequence SEQ ID NO:169, and the encoding nucleic acid for the second light chain of the second antigen binding moiety is the nucleotide sequence SEQ ID NO:167.

[0156] In one specific embodiment, the coding nucleic acid of the first heavy chain of the first antigen-binding moiety of the bispecific antibody has the nucleotide sequence SEQ ID NO:165, and the coding nucleic acid of the first light chain of the first antigen-binding moiety has the nucleotide sequence SEQ ID NO:163, the coding nucleic acid of the second heavy chain of the second antigen-binding moiety has the nucleotide sequence SEQ ID NO:169, and the coding nucleic acid of the second light chain of the second antigen-binding moiety has the nucleotide sequence SEQ ID NO:167.

[0157] In one specific embodiment, the coding nucleic acid of the first heavy chain of the first antigen-binding moiety of the bispecific antibody has the nucleotide sequence SEQ ID NO:173, and the coding nucleic acid of the first light chain of the first antigen-binding moiety has the nucleotide sequence SEQ ID NO:171, the coding nucleic acid of the second heavy chain of the second antigen-binding moiety has the nucleotide sequence SEQ ID NO:169, and the coding nucleic acid of the second light chain of the second antigen-binding moiety has the nucleotide sequence SEQ ID NO:167.

[0158] In one aspect, the present disclosure provides a vector comprising the nucleic acid.

[0159] In one aspect, the disclosure provides a cell comprising the nucleic acid or vector.

[0160] In one aspect, the disclosure provides a composition comprising the bispecific antibody or antigen-binding portion thereof, nucleic acid, vector, and / or cell.

[0161] In one aspect, the disclosure provides an antibody-drug conjugate comprising said bispecific antibody, or an antigen-binding portion thereof, covalently linked to a therapeutic moiety.

[0162] Preferably, the therapeutic moiety is selected from a cytotoxic moiety, a chemotherapeutic agent, a cellular factor, an immunosuppressant, an immunostimulant, a degradative peptide, or a radioisotope.

[0163] The antibodies of the present disclosure are useful as therapeutic or diagnostic tools for various disorders in which GPC3 is adversely expressed or expressed. In one embodiment of the GPC3-related disease, GPC3 expression is increased in cells of diseased tissues or organs compared to the state in healthy tissues or organs. Increased is 10% or more, particularly 20% or more, 50% or more, 100% or more, 200% or more, 500% or more, 1000% or more, 10000% or more or more. In one embodiment, expression is found only in diseased tissues, and expression in corresponding healthy tissues is inhibited. According to the present disclosure, the GPC3-related disease includes tumors.

[0164] In some embodiments, the tumor is cancer. In some embodiments, the cancer is GPC3-positive cancer, such as GPC3-positive liver cancer, GPC3-positive hepatocellular carcinoma, GPC-positive pancreatic cancer, GPC-positive lung cancer, GPC-positive colon cancer, GPC-positive breast cancer, GPC-positive prostate cancer, GPC-positive leukemia or GPC-positive lymphoma.

[0165] In some embodiments, the therapeutic agent comprises an antibody that specifically binds to an activating T cell antigen.

[0166] In one embodiment, the therapeutic agent comprises an antibody that specifically binds to CD3, in particular CD3ε.

[0167] A method of treating diseases and conditions with a bispecific antibody according to the present disclosure comprises administering to a mammal a therapeutically effective amount of an antibody or antigen-binding fragment thereof, or a nucleic acid molecule, or a vector, or a cell, or a pharmaceutical composition according to any of the preceding aspects.

[0168] In some embodiments, the disclosure provides a method of treating or preventing a cancer disease, comprising administering to a patient an antibody capable of binding to GPC3 to a serum level of 40 μg / ml or more. In different embodiments, the antibody is administered to a serum level of 50 μg / ml or more, 150 μg / ml or more, 300 μg / ml or more, 400 μg / ml or more, or 500 μg / ml or more. In different embodiments, the antibody is administered to a serum level of 800 μg / ml or less, 700 μg / ml or less, 600 μg / ml or less, 550 μg / ml or less, or 500 μg / ml or less. In one embodiment, the serum level provided is between 40μg / ml and 700μg / ml, preferably between 40μg / ml and 600μg / ml, preferably between 50μg / ml and 500μg / ml, for example between 150μg / ml and 500μg / ml, or between 300μg / ml and 500μg / ml. The term "serum level" as used herein means the concentration in the serum of the substance under consideration. In one embodiment, serum levels are provided for 7 days or more or 14 days or more. In one embodiment, the method provides a serum level of 300mg / ml or more. 2 More than 600 mg / m 2 or more, and preferably 1500 mg / m 2 Below 1200mg / m 2 Less than or equal to 1000 mg / m 2 This involves administering the following doses of antibody:

[0169] In some embodiments, the present disclosure provides a method for administering a dose of 300 mg / m 2 More than 600 mg / m 2 or more, and preferably 1500 mg / m 2 Below 1200mg / m 2 Less than or equal to 1000 mg / m 2 The present invention provides a method for treating or preventing a cancer disease, comprising administering an antibody capable of binding to GPC3 at the following doses:

[0170] In some embodiments, the present disclosure provides a method for treating or preventing a cancer disease, comprising administering to a patient an antibody capable of binding to GPC3, wherein 50% or more, preferably 60% or more, 70% or more, 80% or more, or 90% or more of the patient's cancer cells are GPC3 positive, and / or 40% or more, preferably 50% or more, or 60% or more of the patient's cancer cells are positive for surface expression of GPC3. In this regard, the present disclosure further provides a method for treating or preventing a cancer disease, comprising: a. identifying a patient suffering from 50% or more, preferably 60% or more, 70% or more, 80% or more, or 90% or more of GPC3 positive cancer cells, and / or 40% or more, preferably 50% or more, or 60% or more of GPC3 positive cancer cells, and b. administering to the patient an antibody capable of binding to GPC3. In one embodiment, 95% or more, or 98% or more of the patient's cancer cells are positive for GPC3. In one embodiment, 70% or more, 80% or more, or 90% or more of the cancer cells in a patient are positive for surface expression of GPC3.

[0171] In one embodiment of the method according to any of the aspects of the present invention, the outcome of treating the cancer disease is achieving stabilization of the disease, hi one embodiment, the stabilization of the disease lasts for 2 months or more, 3 months or more, or 6 months or more.

[0172] In some embodiments, the present disclosure provides a method for stabilizing a disease state in a cancer patient, comprising administering to the patient an antibody capable of binding to GPC3. In one embodiment, the stabilization of the disease state lasts for 2 months or more, 3 months or more, or 6 months or more.

[0173] In one embodiment of the method according to any aspect described herein, the antibody is administered in a single dose or in multiple doses.

[0174] In some embodiments, the present disclosure provides a method of treating or preventing a cancer disease comprising administering to a patient multiple doses of an antibody capable of binding to GPC3.

[0175] According to the present disclosure, when the antibody is administered in multiple doses, preferably, the antibody is administered in 3 or more doses, 4 or more doses, 5 or more doses, 6 or more doses, 7 or more doses, 8 or more doses, 9 or more doses, or 10 or more doses, and preferably 30 or less doses, 25 or less doses, 20 or less doses, 15 or less doses, or 10 or less doses. Preferably, the antibody doses are administered at intervals of 7 days or more, 10 or more days, 14 or more days, or 20 or more days. Preferably, the antibody doses are administered at intervals of 7 to 30 days, 10 to 20 days, and preferably about 14 days.

[0176] In one embodiment, the antibody is administered to provide a serum level of 40 μg / ml or more. In a different embodiment, the antibody is administered to provide a serum level of 50 μg / ml or more, 150 μg / ml or more, 300 μg / ml or more, 400 μg / ml or more, or 500 μg / ml or more. In a different embodiment, the antibody is administered to provide a serum level of 800 μg / ml or less, 700 μg / ml or less, 600 μg / ml or less, 550 μg / ml or less, or 500 μg / ml or less. In one embodiment, the serum level provided is 40 μg / ml to 700 μg / ml, preferably 40 μg / ml to 600 μg / ml, preferably 50 μg / ml to 500 μg / ml, for example, 150 μg / ml to 500 μg / ml or 300 μg / ml to 500 μg / ml. In one embodiment, the serum level is provided for 7 days or more, or 14 days or more. In one embodiment, the method comprises administering a 300 mg / m 2 More than 600 mg / m 2 or more, and preferably 1500 mg / m 2 Below 1200mg / m 2 Less than or equal to 1000 mg / m 2 This includes administering the following doses of antibody:

[0177] There is provided use of an antibody or antigen-binding fragment thereof, or a nucleic acid molecule, or a vector, or a cell, or a pharmaceutical composition according to any of the preceding aspects in the preparation of a medicament for treating a GPC3-related disease in a mammal.

[0178] According to any of the above aspects, optionally the antibody is conjugated to another drug, for example a labeled or cytotoxic conjugate.

[0179] In one aspect, the present disclosure further includes kits including, for example, the antibodies, fragments, homologs, derivatives, etc., of the present disclosure, such as labeled or cytotoxic conjugates, and antibody instructions, conjugates that kill specific types of cells, etc. The instructions may include guides for in vitro, in vivo, or ex vivo use of the antibodies, conjugates, etc. The antibodies may be in liquid or solid form, and are typically lyophilized. The kits may further include other suitable reagents, such as buffers, reconstitution solutions, and other components as required depending on the intended use. Combinations of packaged reagents in predetermined amounts with instructions for their use, such as therapeutic or diagnostic assay use, are also contemplated. If the antibody is labeled, for example, with an enzyme, the kit may include substrates and cofactors required for the enzyme (e.g., substrate precursors that provide a detectable chromophore or fluorophore). In addition, other additives may be included, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers). The relative amounts of the various reagents can be varied to provide concentrates of the reagent solutions, thereby providing flexibility for the user, space savings, reagent savings, etc. These reagents may also be provided in the form of a dry powder, typically provided lyophilized, and containing excipients that upon dissolution provide a solution of the appropriate concentration of the reagent.

[0180] The present invention provides use of the antibody or functional fragment thereof, or the nucleic acid molecule, or the vector, or the cell, or the pharmaceutical composition, or the kit according to any one of the above aspects in preparing a reagent for inhibiting binding of GPC3.

[0181] The antibodies of the present disclosure may also be used in immunoassays, purification methods, and other methods that employ immunoglobulins or fragments thereof, such uses being well known to those of skill in the art.

[0182] Correspondingly, the present disclosure further provides a composition comprising an anti-GPC3 antibody or a fragment thereof according to the present disclosure, which can be conveniently combined with a pharma- ceutically acceptable carrier, diluent or excipient, which is a means known in the art.

[0183] The term "pharmaceutical composition" as used in this disclosure refers to a formulation of various preparations. The formulations containing a therapeutically effective amount of a multivalent antibody may be in a sterile liquid solution, liquid suspension, or lyophilized form, and may optionally contain stabilizers or excipients.

[0184] The antibodies of the present disclosure may be used as compositions to be administered alone or in combination with other active agents. In some embodiments, the humanized antibodies of the present disclosure are conjugated to a therapeutic moiety (i.e., a drug). The therapeutic moiety may be, for example, a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulant, a degradative peptide, or a radioisotope. Such conjugates are also referred to herein as "antibody-drug conjugates" or "ADCs."

[0185] In some embodiments, the antibody is conjugated to a cytotoxic moiety, which may be selected from the group consisting of taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine (cephaeline), mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, tubulin inhibitors such as maytansine or an analogue or derivative thereof, mitotic inhibitors such as monomethyl auristatin E or F or an analogue or derivative thereof, dolastatin 10 or 15 or an analogue thereof, irinotecan or an analogue thereof, mitoxantrone, mithramycin, actinomycin D, D), 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, calicheamicin or its analogs or derivatives, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, antimetabolites such as gemcitabine or cladribine, e.g., mechlorethamine, thiopurine, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclosporine, ... alkylating agents such as rofosfamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, and mitomycin C; platinum derivatives such as cisplatin or carboplatin; duocarmycin A, duocarmycin SA, rachelmycin (CC-1065) or analogs or derivatives thereof; antibiotics such as actinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, primycin, and anthramycin (AMC);4]-benzodiazepines (PDB), diphtheria toxin and related molecules such as diphtheria A chain and its active fragments and hybrid molecules, ricin such as ricin A or deglycosylated ricin A chain toxins, cholera toxin, shiga-like toxins such as SLT I, SLT II, ​​SLT IIV, LT toxin, C3 toxin, shiga toxin, pertussis toxin, tetanus toxin, Bowman-Birk soybean protease inhibitor, Pseudomonas exotoxins, allorin, saporin, modeccin, geranin, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins such as PAPI, PAPII and PAP-S, momordica charantia inhibitors, curcin, crotin, saponaria officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin and enomycin toxins, ribonuclease (RNase), DNase I, Staphylococcus aureus endotoxin A, pokeweed antiviral protein, diphtheria toxin, and Pseudomonas endotoxin.

[0186] In some embodiments, the antibody is conjugated to an auristatin or its peptide analog, derivative, or prodrug. Auristatins affect microtubule dynamics, GTP hydrolysis, and nuclear and cell division, and have been shown to have anticancer and antifungal activity. For example, auristatin E can react with p-acetylbenzoic acid or benzoylvaleric acid to generate AEB and AEVB, respectively. Other exemplary auristatin derivatives include AFP, MMAF (monomethyl auristatin F), and MMAE (monomethyl auristatin E). Suitable auristatins, analogs, derivatives and prodrugs of auristatins, as well as linkers suitable for conjugating auristatins with Abs, are described, for example, in U.S. Pat. Nos. 5,635,483, 5,780,588, 6,214,345 and International Patent Publications WO02088172, WO2004010957, WO2005081711, WO2005084390, WO2006132670, WO03026577, WO200700860, WO207011968 and WO205082023.

[0187] In some embodiments, the antibody is conjugated to pyrrolo[2,1-c][1,4]-benzodiazepine (PDB), its peptide analogs, derivatives, or prodrugs. Suitable PDBs, PDB derivatives, and related techniques are described, for example, in Hartley JA et al., Cancer Res 2010, 70(17):6849-6858, Antonow D. et al., Cancer J 2008, 14(3):154-169, Howard PWet al., Bioorg Med Chem Lett 2009;19:6463-6466, and Sagnou et al., Bioorg Med Chem Lett 2000;10(18):2083-2086.

[0188] In some embodiments, the antibody is conjugated to a cytotoxic moiety selected from an anthracycline antibiotic, mertansine, a calicheamicin, a duocarmycin, rachelmycin (CC-1065), dolastatin 10, dolastatin 15, irinotecan, monomethyl auristatin E, monomethyl auristatin F, PDB, or an analog, derivative, or prodrug of any of these.

[0189] In some embodiments, the antibody is conjugated to an anthracycline antibiotic or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to mertansine or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to calicheamicin or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to duocarmycin or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to rachelmycin (CC-1065) or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to dolastatin 10 or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to dolastatin 15 or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to monomethyl auristatin E or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to monomethylauristatin F or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to pyrrolo[2,1-c][1,4]-benzodiazepine or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to irinotecan or an analog, derivative, or prodrug thereof.

[0190] In some embodiments, the antibody is conjugated to a cytokine (e.g., IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNα, IFNβ, IFNγ, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFα).

[0191] In some embodiments, the antibody is conjugated to a radioisotope or a chelate containing a radioisotope. For example, the antibody can be conjugated to a chelating linker (e.g., DOTA, DTPA, or tiuxetan). The antibody can also, or alternatively, include or be conjugated to one or more radiolabeled amino acids or other radiolabeled molecules. Non-limiting examples of radioisotopes include: 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 125 I, 131 I, 186 Re, 213 Bi, 225 Ac and 227 For therapeutic purposes, radioisotopes that emit beta or alpha particle radiation, e.g. 131 I, 90 Y, 211 At, 212 Bi, 67 Cu, 186 Re, 188 Re and 212 Pb may also be used.

[0192] Techniques for conjugating molecules to antibodies are well known in the art. Usually, nucleic acid molecules are covalently linked to lysines or cysteines in antibodies via N-hydroxysuccinimide esters or maleimide functional groups, respectively. It has been reported that conjugation methods using engineered cysteines or incorporating unnatural amino acids can improve the identity of the conjugates. Those skilled in the art will particularly consider Fc-containing polypeptides engineered with acyl donor glutamine-containing tags (e.g., Gin-containing peptide tags or Q-tags) or endogenous glutamines made reactive by polypeptide engineering (e.g., by amino acid deletion, insertion, substitution, or mutation of the polypeptide). Transglutaminase can then covalently cross-link amine donating agents (e.g., small molecules that contain or are coupled to reactive amines) to form a stable and homogenous population of engineered Fc-containing polypeptide conjugates in which the amine donating agents are site-specifically conjugated to Fc-containing polypeptides via acyl donor glutamine-containing tags or accessible / exposed / reactive endogenous glutamines (WO2012059882).

[0193] It will be understood that the therapeutic agents of the embodiments are administered with suitable pharma- ceutically acceptable carriers, excipients, and other agents that are incorporated into the formulation to provide improved transfer, delivery, tolerance, and the like. Many suitable formulations are described in pharmacopoeias known to medicinal chemists, such as Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, Pa. (1975)), particularly Chapter 87 of Blaug, Seymour therein. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid (cationic or anionic)-containing carriers (e.g., Lipofectin, TM), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, emulsion polyethylene glycols (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycol. Any of the above mixtures may be used in the treatment or method of treatment of the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation, is physiologically compatible, and is tolerable to the route of administration.

[0194] In one embodiment, the antibody can be used as a therapeutic agent. Such agents are typically used to treat, ameliorate, and / or prevent a disease or condition associated with abnormal GPC3 expression, activity, and / or signaling in a subject. The treatment regimen can be performed by standard methods by identifying a subject, for example a human patient suffering from (or at risk of or developing) a disease or disorder associated with abnormal GPC3 expression, activity, and / or signaling, for example a GPC3-associated disorder. An antibody preparation, preferably an antibody preparation with high specificity and high affinity for a target antigen, is administered to the subject and generally exerts an effect by binding to the target. The administered antibody can eliminate, inhibit, or interfere with the expression, activity, and / or signaling function of the target (e.g., GPC3). The administered antibody can eliminate, inhibit, or interfere with the binding of the target (e.g., GPC3) to an endogenous ligand that naturally binds to it. For example, the antibody binds to a target and modulates, inhibits, suppresses, reduces, antagonizes, neutralizes, and / or otherwise interferes with the expression, activity, and / or signaling of GPC3. In some embodiments, an antibody having heavy and light chain CDRs can be administered to a subject to treat a disease or disorder associated with abnormal GPC3 expression.

[0195] In another embodiment, antibodies against GPC3 are used in methods related to localization and / or quantification of GPC3 known in the art (e.g., for determining the level of GPC3 and / or GPC3 in an appropriate biological sample, diagnostic methods, protein imaging, etc.). In a particular embodiment, antibodies specific for GPC3 or derivatives, fragments, analogs or homologs thereof, comprising an antigen-binding domain derived from the antibody, are used as pharma- ceutical active compounds (hereinafter referred to as "therapeutic agents").

[0196] In another embodiment, antibodies specific for GPC3 can be used to isolate GPC3 polypeptides by standard techniques such as immunoaffinity, chromatography, or immunoprecipitation. Antibodies (or fragments thereof) against GPC3 protein are used to detect the protein in biological samples. In some embodiments, GPC3 is detected in biological samples as part of a clinical trial procedure, for example to determine the effectiveness of a particular treatment regimen. Conjugating (i.e., physically linking) the antibody to a detectable substance is advantageous for detection. Detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminofluorescein, dansylamide chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, fluorescein and aequorin; examples of suitable radioactive materials include 125 I, 131 I, 35 S, or 3 Contains H.

[0197] In another embodiment, the antibody of the present disclosure can be used as a reagent for detecting the presence of GPC3 or a protein fragment thereof in a sample. In some embodiments, the antibody comprises a detectable label. The antibody is a polyclonal antibody or, more preferably, a monoclonal antibody. An intact antibody or a fragment thereof (e.g., Fab, scFv, or F(ab')2) is used. The term "labeling" with respect to an antibody includes direct labeling of the antibody by conjugating (i.e., physically linking) a detectable substance to the antibody, and indirect labeling of the antibody by reaction with another directly labeled reagent. Examples of indirect labeling include detection of a primary antibody with a fluorescently labeled secondary antibody, and end-labeling of an antibody with biotin to allow detection with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present in the subject's body. Thus, the term "biological sample" includes blood and fractions or components of blood, including serum, plasma, or lymph. In other words, the detection method according to the embodiment is used for detecting analyte mRNA, protein, or genomic DNA in a biological sample in vitro and in vivo. For example, in vitro detection techniques for analyte mRNA include Northern hybridization and in situ hybridization. In vitro detection techniques for analyte protein include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. In vitro detection techniques for analyte genomic DNA include Southern hybridization.Procedures for performing immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, edited by J.R.Crowther, Human Press, Totowa, NJ, 1995, "Immunoassay", E. Diamandis & T. Christopoulus, Academic Press, Inc., San Diego, Calif., 1996, and "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. In vivo detection techniques for analyte proteins also include introducing into a subject a labeled anti-analyte protein antibody. For example, the antibody is labeled with a radioactive label, and the presence and location of the radioactive label in the subject in vivo is detected by standard imaging techniques.

[0198] The antibodies and derivatives, fragments, analogs and homologs thereof described herein can be incorporated into pharmaceutical compositions suitable for administration. The principles, considerations involved in the preparation of such compositions, and guidelines for the selection of the components thereof, are well known in the art.

[0199] Such compositions usually include an antibody and a pharma- ceutically acceptable carrier. When an antibody fragment is used, it is preferably a minimal inhibitory fragment that specifically binds to the binding domain of the target protein. For example, based on the variable region sequence of an antibody, a peptide molecule can be designed that retains the ability to bind to the target protein sequence. Such peptides can be produced by chemical synthesis and / or recombinant DNA technology (see, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)).

[0200] The term "pharmaceutical acceptable carrier" as used herein is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, acceptable for pharmaceutical administration. Suitable pharmaceutical acceptable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is a standard reference text in the field and is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vectors such as liposomes and fixed oils can also be used. The use of such media and agents for pharmaceutical active substances is well known in the art. Except for conventional media or agents that are incompatible with the antibody, their use in the composition is contemplated.

[0201] The pharmaceutical compositions of the embodiments are prepared to suit their intended route of administration. Examples of routes of administration include, for example, parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration include a sterile injectable diluent, e.g., water, saline, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents, antibacterial agents, e.g., benzyl alcohol or methyl p-hydroxybenzoate, antioxidants, e.g., ascorbic acid or sodium bisulfite, chelating agents, e.g., ethylenediaminetetraacetic acid (EDTA), buffers, e.g., acetates, citrates, or phosphates, and osmolality regulators, e.g., sodium chloride or dextrose. The pH can be adjusted with acids or bases, e.g., hydrochloric acid or sodium hydroxide. Parenteral formulations can be packaged in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0202] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Pharmaceutically acceptable carriers suitable for intravenous administration include saline, sterile water, Cremophor EL, and the like. TM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid for easy injection. It must also be stable under the conditions of preparation and storage and must be capable of preventing the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. A coating, for example, lecithin, can be used to maintain a desired particle size in the case of dispersions, and surfactants can be used to maintain proper fluidity. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, such as sugars, polyols (for example, mannitol, sorbitol), sodium chloride, and the like, in the composition. Prolonged absorption of the injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0203] Sterile injectable solutions are prepared by incorporating the antibody in the required amount in a suitable solvent with one or a combination of the above listed ingredients (as needed), as required, and then filtered and sterilized. Usually, dispersions are prepared by incorporating the antibody in a sterile carrier containing a basic dispersion medium and other required ingredients from those listed above. For sterile powders for preparing sterile injectable solutions, the preparation method includes vacuum drying and freeze-drying of the powder containing the active ingredient and any additional desired ingredients from a sterile-filtered solution of the above ingredients.

[0204] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressured container or dispenser or nebulizer that is suitably supplied with a propellant, e.g., a gas such as carbon dioxide.

[0205] Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for permeating the barrier is used in the formulation. Such penetrants are well known in the art and include, for example, detergents for transmucosal administration, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved by using nasal drops or suppositories. For transdermal administration, one or more antibodies can be formulated into pastes, ointments, gels, or creams well known in the art.

[0206] The compounds can also be prepared for rectal delivery in the form of suppositories (eg, with conventional suppository bases such as cocoa butter or other glycerides) or retention enemas.

[0207] In one embodiment, the antibody can be prepared with a carrier that will protect it from rapid elimination from the body, such as a sustained / controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations are readily apparent to those skilled in the art.

[0208] It is particularly advantageous to prepare parenteral compositions in unit dosage form for ease of administration and uniformity of dosage.As used herein, unit dosage form refers to a physically discrete unit suitable for single administration to a subject to be treated, each unit containing a specific amount of one or more of the above antibodies calculated to produce a desired therapeutic effect in combination with the required pharmaceutical carrier.The specifications of the unit dosage form of the above embodiment are determined and directly depend on the inherent characteristics of the antibody and the specific therapeutic effect to be achieved, as well as the limitations inherent in the technology of preparing such antibodies to treat individuals.

[0209] The pharmaceutical compositions may be included in a container, pack, or dispenser together with instructions for administration.

[0210] The formulations herein may further comprise more than one of the above antibodies depending on the specific condition to be treated, preferably antibodies with complementary activities but not adversely affecting each other. Additionally or alternatively, the composition may comprise an agent that enhances the function of the composition, such as a cytotoxin, cytokine, chemotherapeutic agent, or growth inhibitor. Such molecules may be present in an appropriate combination in an effective amount for the intended purpose. For example, they may be present in a kit or in a combination at the time of use.

[0211] In one embodiment, one or more of the antibodies are used in combination therapy, i.e., in combination with other agents, e.g., therapeutic agents (for treating pathological conditions or disorders, e.g., various forms of cancer, autoimmune disorders, and inflammatory diseases). As used herein, the term "combination" refers to the administration of agents substantially simultaneously, simultaneously, or sequentially. When administered sequentially, preferably, the first compound of the two compounds can still be detected at effective concentrations at the treatment site when administration of the second compound begins. In some cases, "combination" may include the antibody of the present disclosure and another therapeutic agent simultaneously in a kit.

[0212] For example, combination therapy includes the co-preparation and / or co-administration of one or more antibodies according to the present disclosure with one or more additional therapeutic agents (e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxins or cell proliferation inhibitors, as described in more detail below.) Such combination therapy may utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with various monotherapies.

[0213] In one embodiment, administration of an anti-GPC3 antibody and administration of a therapeutic agent can reduce the number of B cells in the subject.

[0214] In one embodiment, the therapeutic regimen can effectively reduce cytokine release associated with administration of the T cell activation therapeutic agent to the subject, compared to a therapeutic regimen in which the corresponding anti-GPC3 antibody is not administered.

[0215] Although technical features are described herein as part of the same or separate embodiments for clarity and conciseness of description, it will be understood that the scope of the invention may include embodiments consisting of combinations of all or some of the described features. Working Example

[0216] Example 1: Antigen expression and construction of stably transfected cells Using human GPC3 (Sino biological, HG10088) as a template, human GPC3 extracellular domain (25-563) was amplified and transfected into HEK293E cells and expressed to obtain human GPC3 recombinant protein (SEQ ID NO.1); or the membrane proximal domain sequence (aa524-563) was inserted into the EcoRI site of pGEX3X vector by Gibson assembly method, and the plasmid was transfected into E. coli BL21 (DE3), expression was induced by IPTG, and purified to obtain GST-human GPC3 stem recombinant protein (SEQ ID NO.2). GST-cynomolgus monkey GPC3 stem recombinant protein (SEQ ID NO.3) and GST-mouse GPC3 stem recombinant protein (SEQ ID NO.4) were expressed in a similar manner. Figure 1 shows the results of non-reducing SDS-PAGE of GPC3 recombinant protein.

[0217] The Lenti vector containing the full-length GPC3 sequence of human and rhesus monkey and the packaging plasmid (Genecoepia) were co-transfected into HEK293T cells. 48 hours after transfection, the culture supernatant containing the pseudovirus was collected, 10 μL was taken, and 1 × 10 6CHO cells were infected, puromycin was added, and an antibody screening was performed, and finally stable transfected cells CHO-hGPC3 (clone 2B10) and CHO-cynoGPC3 (clone 4D6) that expressed human or monkey GPC3 at high levels were isolated. Figure 2 shows the flow cytometry detection results of GPC3 stable transfected cells.

[0218] Construction of human or monkey CD3εγ heterodimer: Human CD3γ (UniProt P09693, Gln23-Asn116) and CD3ε (UniProt P07766, Gln23-Asp126) extracellular domain nucleotide sequences were synthesized, and the C-terminus was fused to human IgG Fc hole or Fc knob, respectively, and expressed to form human CD3εγ-Fc heterodimers (the amino acid sequence of human CD3γ IgG Fc(hole) is shown in SEQ ID NO.5, and the amino acid sequence of human CD3ε IgG Fc(knob) is shown in SEQ ID NO.6). Similarly, cynomolgus monkey CD3γ (UniProt Q95LI7, Gln23-Asn110) and CD3ε (UniProt Q95LI5, Gln22-Asp117) were synthesized, and the C-terminus was fused to cynomolgus monkey IgG Fc hole or Fc knob, respectively. The amino acid sequence of the cynomolgus CD3εγ-Fc heterodimer formed by fusion with knob (cynomolgus CD3γ IgG Fc(hole) is shown in SEQ ID NO.7. The amino acid sequence of cynomolgus CD3γ IgG Fc(knob) is shown in SEQ ID NO.8. The recombinant plasmids expressing CD3γ-Fc and CD3ε-Fc were mixed with 3 mg / mL PEI (Polysciences, #24765-2) and co-transfected into HEK293E cells (medium OPM-293 CD03 DPM). After culturing at 37°C, 120 rpm, 5% CO2 for 7 days, the culture supernatant was collected and purified by Protein A affinity chromatography to obtain human or cynomolgus CD3εγ-Fc recombinant protein, with SDS-PAGE purity exceeding 95% (Figure 3).

[0219] Example 2: Animal immunization and production of GPC3 antibody 1. GPC3 antibody screening Balb / C female mice aged 4 to 6 weeks were selected, and a transient expression plasmid was constructed using the full length or membrane proximal region sequence (aa524-563) of human GPC3. The mice were then immunized with DNA using a gene gun. The mice were immunized with 20 μg of plasmid / mouse each time, once a week for a total of 8 immunizations. Serum titers were then detected, and CHO-hGPC3 stable transfected cells (5 × 10 6 Booster immunization was performed using 1000 cells / mouse. After 3 days, the mice were sacrificed by cervical dislocation, and the mouse spleens and peripheral lymph nodes were collected, polished, centrifuged, and collected. Total RNA was extracted and reverse transcribed, and then PCR amplification was performed using specific primers for light and heavy chains, respectively, to obtain antibody light and heavy chain variable region cDNA libraries. A GPC3 immune library based on the filamentous phage M13 in the phage antibody library display technology was constructed for subsequent phage panning.

[0220] A classical panning method was adopted, human GPC3 recombinant protein was coated on an immunotube, 1 mL of GPC3 phage immune library was added, and the Fab phages showing high affinity were eluted by incubation and panning, and then infected with TG1 bacteria to prepare the phages again for the next panning. After repeating the panning screening twice, the single clones were inoculated into a U-type 96-well plate, IPTG-induced expression was performed, the culture supernatant was taken, and ELISA detection was performed, and a total of 323 positive clones that recognized the membrane proximal region of human GPC3 extracellular domain were obtained.

[0221] A part of the clones was selected by sequencing, and the light chain and heavy chain genes were cloned into eukaryotic expression vectors containing the antibody light chain constant region or the human IgG1 heavy chain constant region CH1-CH3, respectively, and co-transfected into HEK293E cells. After culturing for 5-6 days at 37°C, 120 rpm, and 5% CO2, the culture supernatant was collected and purified by Protein A chromatography column to obtain an IgG anti-GPC3 chimeric antibody. Referring to the literature Nakano K et al. (2009), a control antibody GC33 was synthesized and expressed into a human IgG1 chimeric antibody (abbreviated as GC33).

[0222] The GPC3 antibody variable region sequences were as follows: [Table 1]

[0223] 5D6 The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.9, its encoding nucleic acid is shown in SEQ ID NO.10, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.11, 12 and 13, respectively. [ka] Nucleic acid sequence [ka]

[0224] The amino acid sequence of the light chain VK is shown in SEQ ID NO.14, its encoding nucleic acid is shown in SEQ ID NO.15, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.16, 17 and 18, respectively. [ka] Nucleic acid sequence [ka]

[0225] 29A10 The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.19, its encoding nucleic acid is shown in SEQ ID NO.20, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.11, 21 and 13, respectively. [ka] Nucleic acid sequence [ka]

[0226] The amino acid sequence of the light chain VK is shown in SEQ ID NO.22, its encoding nucleic acid is shown in SEQ ID NO.23, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.16, 17 and 18, respectively. [ka] Nucleic acid sequence [ka]

[0227] 34H1 The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.9, its encoding nucleic acid is shown in SEQ ID NO.10, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.11, 12 and 13, respectively. [ka] Nucleic acid sequence [ka]

[0228] The amino acid sequence of the light chain VK is shown in SEQ ID NO.24, its encoding nucleic acid is shown in SEQ ID NO.25, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.16, 17 and 26, respectively. [ka] Nucleic acid sequence [ka]

[0229] 19F1 The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.9, its encoding nucleic acid is shown in SEQ ID NO.10, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.11, 12 and 13, respectively. [ka] Nucleic acid sequence [ka]

[0230] The amino acid sequence of the light chain VK is shown in SEQ ID NO.27, its encoding nucleic acid is shown in SEQ ID NO.28, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.16, 17 and 29, respectively. [ka] Nucleic acid sequence [ka]

[0231] 30B7 The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.30, its encoding nucleic acid is shown in SEQ ID NO.31, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.32, 33 and 13, respectively. [ka] Nucleic acid sequence [ka]

[0232] The amino acid sequence of the light chain VK is shown in SEQ ID NO.34, its encoding nucleic acid is shown in SEQ ID NO.35, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.16, 17 and 18, respectively. [ka] Nucleic acid sequence [ka]

[0233] 28G4 The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.19, its encoding nucleic acid is shown in SEQ ID NO.20, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.11, 21 and 13, respectively. [ka] Nucleic acid sequence [ka]

[0234] The amino acid sequence of the light chain VK is shown in SEQ ID NO.36, its encoding nucleic acid is shown in SEQ ID NO.37, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.38, 17 and 39, respectively. [ka] Nucleic acid sequence [ka]

[0235] 30A4 The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.40, its encoding nucleic acid is shown in SEQ ID NO.41, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.11, 21 and 13, respectively. [ka] Nucleic acid sequence [ka]

[0236] The amino acid sequence of the light chain VK is shown in SEQ ID NO.42, its encoding nucleic acid is shown in SEQ ID NO.43, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.16, 17 and 18, respectively. [ka] Nucleic acid sequence [ka]

[0237] 38D2 The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.9, its encoding nucleic acid is shown in SEQ ID NO.10, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.11, 12 and 13, respectively. [ka] Nucleic acid sequence [ka]

[0238] The amino acid sequence of the light chain VK is shown in SEQ ID NO. 44, its encoding nucleic acid is shown in SEQ ID NO. 45, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs. 46, 47 and 48, respectively. [ka] Nucleic acid sequence [ka]

[0239] 39G4 The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.9, its encoding nucleic acid is shown in SEQ ID NO.10, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.11, 12 and 13, respectively. [ka] Nucleic acid sequence [ka]

[0240] The amino acid sequence of the light chain VK is shown in SEQ ID NO.49, its encoding nucleic acid is shown in SEQ ID NO.50, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.16, 17 and 51, respectively. [ka] Nucleic acid sequence [ka]

[0241] 41F1 The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.9, its encoding nucleic acid is shown in SEQ ID NO.10, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.11, 12 and 13, respectively. [ka] Nucleic acid sequence [ka]

[0242] The amino acid sequence of the light chain VK is shown in SEQ ID NO.52, its encoding nucleic acid is shown in SEQ ID NO.53, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.16, 17 and 54, respectively. [ka] Nucleic acid sequence [ka]

[0243] 41H1 The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.9, its encoding nucleic acid is shown in SEQ ID NO.10, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.11, 12 and 13, respectively. [ka] Nucleic acid sequence [ka]

[0244] The amino acid sequence of the light chain VK is shown in SEQ ID NO.55, its encoding nucleic acid is shown in SEQ ID NO.56, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.16, 17 and 57, respectively. [ka] Nucleic acid sequence [ka]

[0245] 49G8 The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.9, its encoding nucleic acid is shown in SEQ ID NO.10, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.11, 12 and 13, respectively. [ka] Nucleic acid sequence [ka]

[0246] The amino acid sequence of the light chain VK is shown in SEQ ID NO.58, its encoding nucleic acid is shown in SEQ ID NO.59, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.60, 17 and 61, respectively. [ka] Nucleic acid sequence [ka]

[0247] 46C3 The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.62, its encoding nucleic acid is shown in SEQ ID NO.63, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.64, 65 and 66, respectively. [ka] Nucleic acid sequence [ka]

[0248] The amino acid sequence of the light chain VK is shown in SEQ ID NO.67, its encoding nucleic acid is shown in SEQ ID NO.68, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.16, 17 and 69, respectively. [ka] Nucleic acid sequence [ka]

[0249] 42A1 The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.70, its encoding nucleic acid is shown in SEQ ID NO.71, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.11, 72 and 73, respectively. [ka] Nucleic acid sequence [ka]

[0250] The amino acid sequence of the light chain VK is shown in SEQ ID NO.74, its encoding nucleic acid is shown in SEQ ID NO.75, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.16, 17 and 76, respectively. [ka] Nucleic acid sequence [ka]

[0251] 47B6 The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.77, its encoding nucleic acid is shown in SEQ ID NO.78, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.79, 80 and 81, respectively. [ka] Nucleic acid sequence [ka]

[0252] The amino acid sequence of the light chain VK is shown in SEQ ID NO.82, its encoding nucleic acid is shown in SEQ ID NO.83, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.16, 17 and 69, respectively. [ka] Nucleic acid sequence [ka]

[0253] The binding of the antibody to human GPC3 stable transfected cells was detected by FACS. CHO-human GPC3 stable transfected cells produced in Example 1 in the logarithmic growth phase were taken, and the cells were diluted to 5 × 10 5 The cells / ml were adjusted, and 100 μl / well of the cell suspension was added to a U-shaped 96-well plate, centrifuged at 300 g for 5 minutes, the supernatant was discarded, and 100 μL of gradient diluted antibody (initial concentration 200 nM, 3-fold dilution, 12 gradients) was added to each well and incubated at 4 ° C for 45 minutes. Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) was added as a secondary antibody, incubated on ice for 45 minutes, and detected by flow cytometry. Figure 4 shows the results of detecting chimeric antibodies and human GPC3-CHO stable transfected cells by flow cytometry.

[0254] The test antibodies were captured by a Protein A probe using BLI biofilm interference technology (Octet) to detect their binding ability to human GPC3 protein (initial concentration 10 μg / ml, 2-fold gradient, total of 5 concentration points), and the affinity constant KD was calculated based on the binding rate and dissociation rate.

[0255] Combining the results of FACS and Octet detection, a total of 15 positive monoclonal antibodies were obtained that recognized the GPC3 receptor on the cell membrane with high affinity and bound to the GPC3 recombinant protein. Table 2 shows the affinity constants of the monoclonal antibodies against human GPC3. [Table 2]

[0256] 2. Humanization of GPC3 candidate antibodies

[0257] 2.1 Species cross-reactivity of candidate antibodies A positive clone 49G8 with high affinity was selected, and its binding activity to human, cynomolgus monkey, and mouse GPC3 full-length recombinant protein and the membrane proximal region recombinant protein of human GPC3 extracellular domain was detected. After coating and blocking overnight at 4°C with 1 μg / ml of the protein, 49G8 chimeric antibody (initial concentration 200 nM, 3-fold gradient dilution, total of 12 concentrations) was added and reacted at room temperature for 1 hour, HPR-labeled goat anti-human IgG Fc was added as a secondary antibody, the solution was colored with TMB, the reaction was terminated with concentrated sulfuric acid, and the absorbance at 450 nm was read. The ELISA results are shown in Figure 5, and the 49G8 antibody can bind to human and cynomolgus monkey GPC3 full-length protein and the membrane proximal region of human GPC3 extracellular domain, with a slightly higher affinity than the control antibody, and a weaker affinity to mouse GPC3 protein (Table 3). [Table 3]

[0258] 2.2 Antibody-dependent cell-mediated cytotoxicity (ADCC) The target cells were human liver cancer cells HepG2 expressing GPC3, and the target cell density was 5 × 10 4 Adjust effector cells NK92MI-CD16a to 1 x 10 cells / well. 5 The cells / well were adjusted and gradient-diluted antibodies (initial concentration 66.7 nM, 10-fold gradient, total of 6 dilutions) were added, and the cells were incubated at 37°C, 5% CO2 incubator for 4 hours, and the supernatant was taken to detect the release of lactate dehydrogenase (LDH). The results are shown in Figure 6, and the 49G8 chimeric antibody mediated the ADCC effect of the target cell HepG2.

[0259] 2.3 Humanization of 49G8 antibody Humanization was performed on the variable region sequence of mouse-derived antibody 49G8 by CDR grafting and point mutation. The mAb49G8 mouse-derived antibody light chain was mouse IMGT_mVK1-110, and human IMGT_hVK2-28, which has the highest identity with this framework region, was selected to perform CDR grafting, and human IGKJ4*01, which has the highest identity as FM4, was selected. The mouse-derived antibody heavy chain was IGHV1-15, and human germline gene IMGT_hVH1-69 was selected to perform CDR grafting, and human IGHJ4*01, which has the highest identity as FM4, was selected. Reverse mutations were designed, and a total of six humanized variants of heavy chain variable regions and six humanized variants of light chain variable regions were synthesized, combined and paired, and then transiently expressed in HEK293E cells. After culturing for 5 to 6 days at 37°C, 120 rpm, and 5% CO2, the supernatant of the culture medium was collected and purified by Protein A chromatography column to obtain anti-GPC3 humanized antibody.

[0260] The binding activity of the 49G8 humanized antibody to human and cynomolgus monkey GPC3 full-length proteins was measured by ELISA, and the binding of the 49G8 humanized antibody to human GPC3-CHO stable transfected cells was detected by flow cytometry. The ELISA results are shown in Figure 7, and the 49G8 humanized antibody bound both human and cynomolgus monkey GCP3 recombinant proteins with high affinity. The FACS results are shown in Figure 8, and the affinity of the 49G8 humanized antibody to human GPC3-CHO stable transfected cells was equivalent to the binding ability of the mouse antibody before humanization and slightly higher than that of the control antibody. The humanized antibody heavy chain / light chain variable region sequences were as follows. [Table 4]

[0261] h49G8VKv1 The amino acid sequence of the light chain VK is shown in SEQ ID NO.84, its encoding nucleic acid is shown in SEQ ID NO.85, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.60, 17 and 61, respectively. [ka] Nucleic acid sequence [ka]

[0262] h49G8VKv2 The amino acid sequence of the light chain VK is shown in SEQ ID NO.86, its encoding nucleic acid is shown in SEQ ID NO.87, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.60, 17 and 61, respectively. [ka] Nucleic acid sequence [ka]

[0263] h49G8VKv3 The amino acid sequence of the light chain VK is shown in SEQ ID NO.88, its encoding nucleic acid is shown in SEQ ID NO.89, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.60, 17 and 61, respectively. [ka] Nucleic acid sequence [ka]

[0264] h49G8VKv4 The amino acid sequence of the light chain VK is shown in SEQ ID NO.90, its encoding nucleic acid is shown in SEQ ID NO.91, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.92, 17 and 61, respectively. [ka] Nucleic acid sequence [ka]

[0265] h49G8VKv5 The amino acid sequence of the light chain VK is shown in SEQ ID NO.93, its encoding nucleic acid is shown in SEQ ID NO.94, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.95, 17 and 61, respectively. [ka] Nucleic acid sequence [ka]

[0266] h49G8VKv6 The amino acid sequence of the light chain VK is shown in SEQ ID NO.96, its encoding nucleic acid is shown in SEQ ID NO.97, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.98, 17 and 61, respectively. [ka] Nucleic acid sequence [ka] [Table 5]

[0267] h49G8VHv1 The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.99, its encoding nucleic acid is shown in SEQ ID NO.100, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.11, 101 and 13, respectively. [ka] Nucleic acid sequence [ka]

[0268] h49G8VHv2 The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.102, its encoding nucleic acid is shown in SEQ ID NO.103, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.11, 101 and 13, respectively. [ka] Nucleic acid sequence [ka]

[0269] h49G8VHv3 The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.104, its encoding nucleic acid is shown in SEQ ID NO.105, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.11, 106 and 13, respectively. [ka] Nucleic acid sequence [ka]

[0270] h49G8VHv4 The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.107, its encoding nucleic acid is shown in SEQ ID NO.108, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.11, 101 and 13, respectively. [ka] Nucleic acid sequence [ka]

[0271] h49G8VHv5 The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.109, its encoding nucleic acid is shown in SEQ ID NO.110, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.11, 101 and 13, respectively. [ka] Nucleic acid sequence [ka]

[0272] h49G8VHv6 The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.111, its encoding nucleic acid is shown in SEQ ID NO.112, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.11, 106 and 13, respectively. [ka] Nucleic acid sequence [ka]

[0273] 2.3 Affinity of humanized antibodies 1) Biolayer Interferometry (BLI) The antibody to be detected was captured by Protein A probe using BLI biofilm interference technology (Octet), and its binding ability to human GPC3 protein (initial concentration 10μg / ml, 2-fold gradient, total of 5 concentration points) was detected, and the affinity constant KD was obtained based on the binding rate and dissociation rate. The results are shown in Figure 9, and the 49G8 humanized antibody binds to GPC3 recombinant protein with high affinity, and the affinity data are shown in Table 6. [Table 6]

[0274] 2) Surface Plasmon Resonance (SPR) High-affinity humanized antibodies were selected and their binding ability to human and cynomolgus monkey GPC3 was compared. Humanized antibody h49G8VHv6VKv3 was captured using a Protein A chip (GE healthcare), and gradient-diluted human and cynomolgus monkey GPC3 recombinant proteins (initial concentration 5 μg / ml, 2-fold gradient, total of 5 concentration points) were run through the chip at a flow rate of 30 μL / min. The binding time was 150 seconds, and the dissociation time was 600 seconds. Kinetic constants were obtained by fitting using the Langmuir 1:1 kinetics model in the Biacore T200 evaluation software. The results are shown in Figure 10. The humanized antibody h49G8VHv6VKv3 bound to human and cynomolgus monkey GPC3 with high affinity, with an affinity of 1.39 nM for human GPC3 and 2.10 nM for cynomolgus monkey GPC3, which corresponded to the affinity data. [Table 7]

[0275] 2.4 Endocytosis of humanized antibodies HepG2 cells in logarithmic growth phase were taken, the cells were washed once with pre-warmed medium, centrifuged at 1000 rpm for 3 min, the supernatant was discarded, serum-free medium was added, and the cells were diluted to 1 × 10 6The cells were resuspended in 1000μL / mL, and 200μL / tube of cells were aspirated into EP tubes, 500μL of pre-chilled 0.5% BSA / PBS was added, centrifuged at 1000rpm for 3 minutes, the supernatant was discarded, 10μg / mL of GPC3 test antibody was added, and incubated at 4℃ or 37℃ for 60, 120, 240, 120, 180, and 240 minutes, respectively. After the incubation of the primary antibody was completed at each time point, the cells were washed twice with 500μL of pre-chilled 0.5% SA / PBS, and then AF647-labeled goat anti-human IgG Fc diluted at 1:300 was directly added as the secondary antibody and incubated for 45 minutes. After the incubation of the secondary antibody was completed, the cells were washed twice with 500μL of 0.5% BSA / PBS, and the cells were resuspended in 200μL and subjected to FACS detection. The results of flow cytometry detection are shown in FIG. 11. The humanized GPC3 antibody did not show significant endocytosis under either the test conditions of 37° C. or 4° C.

[0276] Example 3: Construction of GPC3xCD3 bispecific antibody 1. Construction of CD3 humanized antibody The mouse-derived hybridoma CD3 antibody (EMBO J. 1985.4(2):337-344; J. Immunol. 1986,137(4):1097-100; J. Exp. Med. 1991,174: 319-326; J. Immunol. 1991,147(9):3047-52) recognized the human and monkey CD3 receptors, and its sequence was as follows:

[0277] The anti-CD3 mouse monoclonal antibody light chain amino acid sequence is shown in SEQ ID NO.174. [ka]

[0278] The anti-CD3 mouse monoclonal antibody heavy chain amino acid sequence is shown in SEQ ID NO.175. [ka]

[0279] Anti-CD3 mouse monoclonal antibody was humanized, the most identical human germline gene IMGT_hVL7-43 was selected, light chain CDR grafting was performed, human IGLJ3*02 was used as FM4, and human IMGT_hVH3-73 was selected, heavy chain CDR grafting was performed, human IGHJ4*01 was used as FM4. Different heavy and light chain variants were designed and obtained (Tables 8 and 9). [Table 8]

[0280] The amino acid sequence of hVL1 is shown in SEQ ID NO.113, its encoding nucleic acid is shown in SEQ ID NO.114, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.115, 116 and 117, respectively. [ka] Nucleic acid sequence [ka]

[0281] The amino acid sequence of hVL2 is shown in SEQ ID NO.118, its encoding nucleic acid is shown in SEQ ID NO.119, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.115, 116 and 117, respectively. [ka] Nucleic acid sequence [ka]

[0282] The amino acid sequence of hVL3 is shown in SEQ ID NO.120, its encoding nucleic acid is shown in SEQ ID NO.121, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.122, 123 and 117, respectively. [ka] Nucleic acid sequence [ka]

[0283] The amino acid sequence of hVL4 is shown in SEQ ID NO.124, its encoding nucleic acid is shown in SEQ ID NO.125, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.122, 123 and 117, respectively. [ka] Nucleic acid sequence [ka]

[0284] The amino acid sequence of hVL5 is shown in SEQ ID NO.126, its encoding nucleic acid is shown in SEQ ID NO.127, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.115, 116 and 128, respectively. [ka] Nucleic acid sequence [ka]

[0285] The amino acid sequence of hVL6 is shown in SEQ ID NO.129, its encoding nucleic acid is shown in SEQ ID NO.130, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.115, 131 and 128, respectively. [ka] Nucleic acid sequence [ka] [Table 9]

[0286] The amino acid sequence of hVH1 is shown in SEQ ID NO.132, its encoding nucleic acid is shown in SEQ ID NO.133, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.134, 135 and 136, respectively. [ka] Nucleic acid sequence [ka]

[0287] The amino acid sequence of hVH2 is shown in SEQ ID NO.137, its encoding nucleic acid is shown in SEQ ID NO.138, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.139, 135 and 136, respectively. [ka] Nucleic acid sequence [ka]

[0288] The amino acid sequence of hVH3 is shown in SEQ ID NO.140, its encoding nucleic acid is shown in SEQ ID NO.141, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.139, 135 and 142, respectively. [ka] Nucleic acid sequence [ka]

[0289] The amino acid sequence of hVH4 is shown in SEQ ID NO.143, its encoding nucleic acid is shown in SEQ ID NO.144, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.139, 135 and 145, respectively. [ka] Nucleic acid sequence [ka]

[0290] The amino acid sequence of hVH5 is shown in SEQ ID NO.146, its encoding nucleic acid is shown in SEQ ID NO.147, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.139, 135 and 148, respectively. [ka] Nucleic acid sequence [ka]

[0291] The amino acid sequence of hVH6 is shown in SEQ ID NO.149, its encoding nucleic acid is shown in SEQ ID NO.150, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.139, 135 and 151, respectively. [ka] Nucleic acid sequence [ka]

[0292] The amino acid sequence of hVH7 is shown in SEQ ID NO.152, its encoding nucleic acid is shown in SEQ ID NO.153, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.154, 155 and 136, respectively. [ka] Nucleic acid sequence [ka]

[0293] The amino acid sequence of hVH8 is shown in SEQ ID NO.156, its encoding nucleic acid is shown in SEQ ID NO.157, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.134, 135 and 136, respectively. [ka] Nucleic acid sequence [ka]

[0294] The amino acid sequence of hVH9 is shown in SEQ ID NO.158, its encoding nucleic acid is shown in SEQ ID NO.159, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.134, 135 and 136, respectively. [ka] Nucleic acid sequence [ka]

[0295] The amino acid sequence of hVH10 is shown in SEQ ID NO.160, its encoding nucleic acid is shown in SEQ ID NO.161, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.134, 135 and 136, respectively. [ka] Nucleic acid sequence [ka]

[0296] The entire sequences of the humanized light and heavy chain mutants were synthesized, and then cloned into eukaryotic expression vectors containing the antibody λ light chain constant region or the human IgG4 heavy chain constant region CH1-CH3, and co-transfected into HEK293E cells. After culturing for 5-6 days at 37°C, 120 rpm, and 5% CO2, the culture supernatant was collected and purified using a Protein A chromatography column.

[0297] 2. Affinity of CD3 humanized antibodies Human CD3εγ protein was coated overnight at 4℃. After blocking with 2% skim milk, various dilutions of CD3 antibody were added to each well, incubated for 1 hour, HPR-labeled goat anti-human IgG Fc was added as secondary antibody, color developed with TMB solution, and the reaction was terminated with concentrated sulfuric acid, and the absorbance at 450 nm was read (results shown in Figure 12, Table 8). Figure 12 shows the binding combinations of anti-CD3 humanized antibodies to human CD3εγ protein (including aCD3-hVH8 / VL1, aCD3-hVH9 / VL1, aCD3-hVH9 / VL2, aCD3-hVH9 / VL3, aCD3-hVH1 / VL5, aCD3-hVH8 / VL5, aCD3-hVH9 / VL5), and CD3 humanized antibodies bound to CD3εγ recombinant protein with high affinity.

[0298] 3. Recognition of Jurkat T cells by CD3 humanized antibodies Jurkat cells in the logarithmic growth phase were blocked with 3% BSA for 30 min and plated in a U-shaped 96-well plate at 5 × 10 4 Cells / well were added, centrifuged, and the supernatant was discarded. 50 μL of gradient-diluted antibody (from an antibody concentration of 30 μg / ml, diluted 3-fold in a gradient of 5) was added to each well and incubated for 1 hour at 4° C. After washing to remove the primary antibody, Alexa Fluro647-labeled goat anti-human IgG Fc (Jackson ImmunoResearch, 109-606-170) diluted 1:300 was added as the secondary antibody and incubated for 45 minutes at 4° C. After washing, each well was resuspended in 50 μL of PBS and subjected to FACS (iQue, Intellicyt) detection (results shown in FIG. 13, Table 10). [Table 10]

[0299] FIG. 13 shows combinations of anti-CD3 humanized antibodies that bound to Jurkat cells, with the CD3 humanized antibodies hVH9 / VL5 (aCD3-hVH9 / VL5) and hVH9 / VL2 (aCD3-hVH9 / VL2) binding to Jurkat cells with intermediate affinity, significantly weaker than the control antibody OKT3.

[0300] 4. Cross-recognition of humanized CD3 antibodies to human and monkey CD3εγ antigens Human CD3εγ protein and monkey CD3εγ protein were coated overnight at 4℃. After blocking with 2% skim milk, CD3 antibody was added to each well at different dilutions and incubated for 1 hour. HPR-labeled goat anti-human IgG Fc was added as secondary antibody, color developed with TMB solution, and the reaction was terminated with concentrated sulfuric acid, and the absorbance at 450 nm was read. Figure 14 shows that both CD3 humanized antibodies hVH9 / VL5 (aCD3-hVH9 / VL5) and hVH9 / VL2 (aCD3-hVH9 / VL2) simultaneously bound human CD3εγ and monkey CD3εγ proteins.

[0301] 5. Construction of GPC3×CD3κλ humanized bispecific antibody A novel GPC3-CD3κλ humanized bispecific antibody was constructed with the humanized antibody 49G8VHv6VKv3 and the humanized CD3 antibody hVH9 / VL5 (aCD3-hVH9 / VL5) containing the λ light chain, and also with charge variants (Vκ) in the GPC3 antigen arm and CD3 arm. GPC3 :Gln 43 Lys, V.H. GPC3 :Gln 39 Glu, Vλ CD3 :Gln 40 Glu, VH CD3 :Gln 39To achieve heterodimeric pairing, the Fc portion of the bispecific antibody adopted the human IgG4 knob-into-hole structure (Atwell et al., 1997) with the mutations Ser228Pro, Leu235Glu and Pro329Ala to maintain stability of the hinge region and completely abolish interactions with Fcγ receptors and C1q (Table 11).

[0302] Plasmids encoding the corresponding antibody fragments were mixed in a ratio of κ light chain:λ light chain:heavy chain 1:heavy chain 2 = 2:2:1:1, mixed with 3 mg / mL PEI, and co-transfected into CHO-S cells. The cells were cultured in 500 mL of CD CHO AGT medium (Gibco #12490-001) at 37 °C, 5% CO2, and 150 rpm, and 4% CHO Feed C + feed (Gibco #A25031-05) was added on the 2nd, 4th, and 6th days after each transient transfection. When the cell activity decreased to about 85%, the fermentation liquid was harvested, filtered, and pre-purified by Protein A affinity chromatography. The SEC-HPLC monomer content was shown to be more than 92%, and the monomer content was further improved to more than 99.5% through Butyl HP hydrophobic chromatography and Capto Q anion chromatography (Table 12). [Table 11]

[0303] GPC3×CD3κλ002: The GPC3 arm light chain is shown in SEQ ID NO.162. [ka]

[0304] The nucleotide sequence is shown in SEQ ID NO.163. [ka]

[0305] The GPC3 arm heavy chain is shown in SEQ ID NO.164. [ka]

[0306] The nucleotide sequence is shown in SEQ ID NO.165. [ka]

[0307] The CD3 arm light chain is shown in SEQ ID NO.166. [ka]

[0308] The nucleotide sequence is shown in SEQ ID NO.167. [ka]

[0309] The CD3 arm heavy chain is shown in SEQ ID NO.168. [ka]

[0310] The nucleotide sequence is shown in SEQ ID NO.169. [ka]

[0311] GPC3×CD3κλ003: The GPC3 arm light chain is shown in SEQ ID NO.170. [ka]

[0312] The nucleotide sequence is shown in SEQ ID NO.171. [ka]

[0313] The GPC3 arm heavy chain is shown in SEQ ID NO.172. [ka]

[0314] The nucleotide sequence is shown in SEQ ID NO.173. [ka]

[0315] The CD3 arm light chain is shown in SEQ ID NO.166. [ka]

[0316] The nucleotide sequence is shown in SEQ ID NO.167. [ka]

[0317] The CD3 arm heavy chain is shown in SEQ ID NO.168. [ka]

[0318] The nucleotide sequence is shown in SEQ ID NO.169. [ka] [Table 12]

[0319] Example 4, Binding activity of GPC3×CD3κλ bispecific antibody The affinity of the bispecific antibody GPC3 antigen arm was determined by detecting binding to GPC3 recombinant protein, overexpressing stably transfected cells, or GPC3+ tumor cells, respectively, and the affinity of the bispecific antibody CD3 arm was determined by detecting binding to Jurkat cells.

[0320] 1. Determination of affinity of GPC3×CD3κλ bispecific antibody to GPC3 Antibodies at a concentration of 1 μg / ml were captured on a Protein A chip (GE healthcare), and different concentrations of human or cynomolgus monkey GPC3 recombinant protein (initial concentration: 5 μg / ml, 2-fold gradient, total of 6 concentration points) were flowed through the chip at a flow rate of 30 μL / min as samples. The binding time was 120 seconds, and the dissociation time was 400 seconds. The changes in SPR signal during the binding and dissociation processes to the protein were recorded, and the affinity constants were obtained by fitting with a 1:1 binding model using Biacore T200 evaluation software.

[0321] Human or monkey CD3εγ recombinant antigen 10μg / mL was coupled to a CM5 chip (GE healthcare) via the amino group, and the antigen binding amount was controlled to about 200RU. After the baseline was stabilized, gradient diluted antibody (diluted 2-fold with 7 gradients from 10μg / mL) was run through the chip at a flow rate of 30μL / min, the binding time was 350 seconds, and the dissociation time was 600 seconds. The kinetic constants were obtained by fitting with a 1:1 binding model using Biacore T200 evaluation software. The affinity measurement results are shown in Table 13. [Table 13]

[0322] 2. Binding of GPC3 × CD3κλ bispecific antibodies to GPC3 stably transfected cells CHO-human GPC3 and CHO-cynomolgus monkey GPC3 stably transfected cells in the logarithmic growth phase were cultured at 5 × 105 The concentration was adjusted to 100 cells / ml, and 100 μl / well of the cell suspension was added to a U-shaped 96-well plate, centrifuged at 300 g for 5 minutes, the supernatant was discarded, and 100 μL of gradient-diluted antibody (initial concentration 1800 nM, 3-fold dilution, 10 gradient) was added to each well and incubated at 4° C. for 60 minutes. Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) was added as a secondary antibody at 50 μL / well, incubated on ice for 20 minutes, washed once, and then propidium iodide solution (1:300) was added at 50 μL / well, incubated for 5 minutes, and detected by flow cytometry. As shown in FIG. 15 and Table 14, the GPC3×CD3κλ bispecific antibody bound to the cellular GPC3 receptor with high affinity, and the affinity to monkey GPC3 stably transfected cells was equivalent to the affinity to human GPC3 stably transfected cells. [Table 14]

[0323] 3. Binding of GPC3×CD3κλ bispecific antibodies to GPC3+ tumor cells HepG2 cells in the logarithmic growth phase were taken, and 200 μg / mL of mouse IgG (Jackson ImmunoResearch, 115-005-03) was added, blocked in an ice bath for 30 min, and the cells were diluted with 4% bovine serum at 5 × 10 5 The cells / mL were adjusted, 100 μL / well was added to a U-shaped 96-well plate, centrifuged at 300 g for 5 minutes, the supernatant was discarded, and 100 μL of gradient diluted antibody (initial concentration 1800 nM, 3-fold dilution, 10 gradient) was added to each well and incubated at 4 ° C for 60 minutes. The primary antibody was removed by washing, and Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) 50 μL / well was added, incubated on ice for 20 minutes, washed once, and then PI 50 μL / well was added, incubated for 5 minutes, and detected by flow cytometry. The detection results are shown in Figure 16 and Table 15, and the GPC3 × CD3 κ λ bispecific antibody bound to GPC3 + ​​tumor cells HepG2 with high affinity. [Table 15]

[0324] 4. Binding of GPC3×CD3κλ bispecific antibody to Jurkat cells Jurkat cells in the logarithmic growth phase were taken, and 200 μg / mL of mouse IgG (Jackson ImmunoResearch, 115-005-03) was added and incubated in an ice bath for 30 minutes. The cells were diluted to 5 × 10 5 The cells / mL were adjusted, 100 μL / well was added to a U-shaped 96-well plate, centrifuged at 300 g, the supernatant was discarded, and 100 μL of gradient diluted antibody (initial concentration 1800 nM, 3-fold dilution, 10 gradient) was added to each well and incubated at 4 ° C for 60 minutes. Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) 50 μL / well was added as a secondary antibody, incubated on ice for 20 minutes, washed once, PI 50 μL / well was added, incubated for 5 minutes, and detected by flow cytometry (BD C6). The detection results are shown in Figure 17 and Table 16. The GPC3 × CD3 κ λ bispecific antibody bound to human leukemia T cell line Jurkat cells with medium affinity and inhibited EC 50 was approximately 20-40 nM. [Table 16]

[0325] 5. Binding of GPC3×CD3κλ bispecific antibody to peripheral blood T cells Fresh human peripheral blood was drawn and PBMCs were isolated using Ficoll.Paque Plus (GE, 17-1440-03). PBMCs were cultured at 5 × 10 cells / mL in 4% bovine serum (Hyclone, SH30626.06). 5The concentration was adjusted to 100 cells / mL, 100 μL / well was added to a U-shaped 96-well plate, centrifuged, the supernatant was discarded, and gradient diluted antibody (initial concentration 1800 nM, 3-fold dilution, 11 gradient) 100 μL / well was added and incubated at 4 ° C for 60 minutes. Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) 50 μL / well was added as a secondary antibody, and the mixture was incubated in an ice bath for 20 minutes. After washing once, PI 50 μL / well was added, incubated for 5 minutes, and detected by flow cytometry (BD Celesta). The detection results are shown in Figure 18, and the GPC3 × CD3 κ λ bispecific antibody bound to human peripheral blood T cells with low affinity.

[0326] [Table 17]

[0327] Example 5: TDCC effect of GPC3×CD3κλ bispecific antibody Freshly isolated PBMCs were taken and mixed with target cells HepG2 cells in the logarithmic growth phase, with effector cells / target cells = 10:1, gradient diluted antibodies (antibody concentration was diluted 10-fold with 7 gradients from 66.7 nM) were added 50 μL / well, and the mixture was cultured at 5% CO2 and 37 °C for 24 hours. After the culture was completed, 50 μL of the supernatant was transferred to a new black immunoplate, and 50 μL / well of LDH detection substrate was added, and the reaction was terminated after 10 minutes to detect LDH release. The remaining cells in the wells were washed twice with 4% bovine serum, and then human IgG 100 μg / mL was added and incubated for 10 minutes, and T cell activation detection antibodies (CD25-BV421, CD4-FITC, CD69-BV605 and CD8-APC) were further added and incubated on ice for 20 minutes. After washing, the supernatant was discarded, PI (60 μL / well) was added, incubated on ice for 5 min, and detected by flow cytometry. As shown in Figure 19, in the in vitro activity test, the GPC3 × CD3 κλ bispecific antibody had a slightly higher or equivalent maximum killing rate against HepG2 cells compared to the control antibody (Figure 19A), but the changes in T cell CD69 (early activation) and CD25 (late activation) during the TDCC process both suggested that the activation of T cells by the GPC3 × CD3 κλ bispecific antibody was milder than that by the control antibody.

[0328] Example 6: Activation of T cell activation pathways by GPC3 x CD3 κλ bispecific antibodies Target cells CHO-human GPC3 in the logarithmic growth phase were taken and centrifuged, the supernatant was discarded, and 2 × 10 5 The target cells were inoculated into a 96-well plate at 50 μl / well and cultured overnight at 37°C with 5% CO2. Jurkat-NFAT-luc reporter cells in logarithmic growth phase were harvested and centrifuged at 300 g for 5 minutes, the supernatant was discarded, and 4 × 10 6The cells were resuspended at 100 μL / ml, the 96-well plate was removed, the supernatant was discarded, and 25 μL / well of Jurkat-NFAT-luc reporter cells were inoculated into the 96-well plate, and 25 μL of gradient-diluted GPC3×CD3κλ bispecific antibody or control antibody KLH×CD3 (initial concentration 20 μg / ml, 3-fold dilution, 10 gradient) was added to each well, and the cells were cultured at 5% CO2 and 37°C for 6 hours. After the culture was completed, 100 μL of detection reagent was added to each well according to the ONE-Glo Luciferase Assay System instruction manual, and detection was performed using a microplate reader (MD SpectraMax i3x). The detection results are shown in Figure 20. When CHO-hGPC3 was used as the target cell, the GPC3×CD3κλ bispecific antibody was able to activate the NFAT signal pathway of T cells, but the activation level was slightly lower than that of the control antibody.

[0329] Example 7. Non-specific activation of PBMCs by GPC3×CD3κλ bispecific antibody Freshly isolated PBMCs were taken, 100 μL of antibody (10 μg / mL) was added, and the cells were cultured at 5% CO2 and 37°C for 24 hours. After washing the cells in the wells twice with 4% bovine serum, 100 μg / mL of human IgG was added, incubated for 10 minutes, and T cell activation detection antibodies (CD25-BV421, CD4-FITC, CD69-BV605, and CD8-APC) were further added and incubated on ice for 20 minutes. After washing, the supernatant was discarded, 60 μL / well of PI was added, incubated on ice for 5 minutes, and detected by flow cytometry. The detection results are shown in Figure 21. In the absence of target cells, the GPC3×CD3κλ bispecific antibody had no activating effect on peripheral blood T cells, equivalent to the negative control KLH×CD3.

[0330] Example 8: Binding of GPC3×CD3κλ bispecific antibodies to Fc receptors 50μg / ml of His-Tag antibody was coupled to a CM5 chip via amino groups, and His-tagged FcγRI, FcγRIIAH131 and FcγRIIIAV158 recombinant proteins (Sino Biological, #10256-H08H / 10374-H08H1 / 10389-H08H1) were captured, the capture time was 40 seconds, the flow rate was 10μL / min, and after the baseline was stabilized, gradient diluted antibodies (initial concentration 37.5μg / mL, 2-fold dilution) were run through the chip at a flow rate of 30μL / min, the binding time was 120 seconds, and the dissociation time was 200 seconds. The affinity constants were obtained by fitting with Biacore evaluation software. As can be seen from FIG. 22, the GPC3×CD3κλ bispecific antibody was able to bind FcγRI, FcγRIIA H131 and FcγRIIIA V158 The wild-type IgG4 control antibody bound FcγRI with relatively high affinity and FcγRIIA H131 It bound slightly weakly to

[0331] Example 9: Immune reconstitution mouse subcutaneous HepG2 tumor transplant model Six to eight-week-old B-NGD female mice (Biocytogen Pharmaceuticals Co., Ltd.) were selected and subcutaneously injected with HepG2 cells (7 × 10 6 / animal) and tumors were 60-100 mm 3 When the mice reached the target concentration, they were randomly divided into groups: 3.0 mg / kg, 1.0 mg / kg, 0.3 mg / kg, and the negative control group, KLH×CD3 3 mg / kg. Each mouse was administered 1×10 PBMC cells. 7 The mice were injected with 100 mg of the drug into the tail vein, and the first administration was started 3 days later, with an administration interval of once every 5 days, for a total of two administrations. The tumor volume and body weight of the mice were monitored, and after the experiment was completed, the mice were sacrificed by cervical dislocation, and the tumors were weighed and recorded. The results are shown in Figure 23, and the GPC3xCD3κλ bispecific antibody showed a dose-dependent in vivo efficacy, with tumor inhibition rates of 76.7%, 81.3%, and 95.9%, respectively. The tumor-bearing mice tolerated the above doses well and did not show any adverse effects such as weight loss.

[0332] Example 10: CD3 humanized mouse Hepa1-6 / hGPC3 transplanted tumor model Six-week-old female C57 / BL6-hCD3 mice (Biocytogen Pharmaceuticals Co., Ltd.) were selected and cultured with Hepa1-6 / hGPC3 (6 × 10 6 / animal) was subcutaneously inoculated into mice, and the tumor volume was 60-100 mm 3 When the total weight of the mice reached 100 mg / kg, the mice were randomly divided into groups. The mice were divided into groups of 10 mg / kg, 3 mg / kg, 1 mg / kg, and 10 mg / kg KLH×CD3, respectively. The administration interval was once every 3 days, and the mice were administered three times in total. The tumor volume and body weight of the mice were monitored, and after the experiment, the mice were sacrificed by slaughter, and the tumors were weighed and recorded. The results are shown in Figure 24. In the CD3 humanized mouse model, the GPC3×CD3κλ bispecific antibody was able to significantly inhibit the growth of Hepa1-6 / hGPC3 tumors and reduce tumor volume.

Claims

1. (a) a first antigen-binding portion or antigen-binding fragment thereof that specifically binds to a GPC3 antigen on the surface of a target cell, the first antigen-binding portion comprises a first heavy chain and a first light chain, the first antigen-binding portion comprising a first binding domain that specifically binds to a first antigen; The first binding domain comprises a first antigen-binding portion or antigen-binding fragment thereof, the first antigen-binding portion or fragment thereof comprising a combination of heavy and light chain complementarity determining regions (CDRs); (b) a second antigen-binding portion or antigen-binding fragment thereof that specifically binds to a CD3 antigen on the surface of a T cell, the second antigen-binding portion comprising a second heavy chain and a second light chain, the second antigen-binding portion comprising a second binding domain that binds to a second antigen; a first heavy chain CDR1, CDR2 and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 11, 106, 13, respectively, and a first light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 60, 17, 61, respectively, a bispecific antibody or an antigen-binding portion thereof.

2. 2. The bispecific antibody or antigen-binding portion thereof of claim 1 , wherein the first binding domain comprises a first heavy chain variable region of amino acid sequence SEQ ID NO: 111 and a first light chain variable region of amino acid sequence SEQ ID NO:

88.

3. 2. The bispecific antibody or antigen-binding portion thereof of claim 1 , wherein the second binding domain comprises a second heavy chain CDR1, CDR2 and CDR3 sequence comprising the amino acid sequences set forth in SEQ ID NOs: 134, 135, 136, respectively, and a second light chain CDR1, CDR2 and CDR3 sequence comprising the amino acid sequences set forth in SEQ ID NOs: 115, 116, 128, respectively.

4. 4. The bispecific antibody or antigen-binding portion thereof of claim 3, wherein the second binding domain comprises a second heavy chain variable region of amino acid sequence SEQ ID NO: 158 and a second light chain variable region of amino acid sequence SEQ ID NO:

126.

5. The first light chain variable region of the first antigen-binding portion may have a Gln43Lys mutation (Vκ ＧＰＣ３ : Gln43Lys), and / or The first heavy chain variable region of the first antigen-binding portion may be Gln39Glu(VH ＧＰＣ３ 3. The bispecific antibody or antigen-binding portion thereof of claim 2, having a Gln39Glu) mutation.

6. The second light chain variable region of the second antigen-binding portion may have a Gln40Glu mutation (Vλ ＣＤ３ and the second heavy chain variable region of the second antigen-binding portion has a Gln39Lys mutation (VH:Gln40Glu), provided that the 39th amino acid in the amino acid sequence was originally Gln. ＣＤ３ 5. The bispecific antibody or antigen-binding portion thereof of claim 4, having the following amino acid sequence:

7. The bispecific antibody or antigen-binding portion thereof of claim 1, wherein the Fc portions of the first and second antigen-binding portions of the bispecific antibody adopt a knob-into-hole structure, including a human IgG4 knob-into-hole structure.

8. the first heavy chain comprises a heavy chain selected from the amino acid sequence of SEQ ID NO: 164, and the first light chain comprises a light chain selected from the amino acid sequence of SEQ ID NO: 162; or the first heavy chain comprises a heavy chain selected from the amino acid sequence SEQ ID NO: 172 and the first light chain comprises a light chain selected from the amino acid sequence SEQ ID NO: 170; and / or 2. The bispecific antibody or antigen-binding portion thereof of claim 1 , wherein the second heavy chain comprises a heavy chain of amino acid sequence SEQ ID NO: 168 and the second light chain comprises a light chain of amino acid sequence SEQ ID NO:

166.

9. An antibody or antigen-binding portion thereof that binds to GPC3, comprising a first antigen-binding portion as described in claim 1.

10. A nucleic acid encoding a bispecific antibody or an antigen-binding portion thereof according to claim 1, or an antibody or an antigen-binding portion thereof that binds to GPC3 according to claim 9.

11. 11. The nucleic acid of claim 10, wherein the coding nucleic acid of the first heavy chain variable region of the first antigen-binding portion has the nucleotide sequence SEQ ID NO: 112 and the coding nucleic acid of the first light chain variable region has the nucleotide sequence SEQ ID NO:

89.

12. the coding nucleic acid of the first heavy chain of the first antigen-binding portion of the bispecific antibody has the nucleotide sequence SEQ ID NO: 165, and the coding nucleic acid of the first light chain of the first antigen-binding portion has the nucleotide sequence SEQ ID NO: 163, the coding nucleic acid of the second heavy chain of the second antigen-binding portion has the nucleotide sequence SEQ ID NO: 169, and the coding nucleic acid of the second light chain of the second antigen-binding portion has the nucleotide sequence SEQ ID NO: 167, or the coding nucleic acid of the first heavy chain of the first antigen-binding portion of the bispecific antibody has the nucleotide sequence SEQ ID NO: 173, and the coding nucleic acid of the first light chain of the first antigen-binding portion has the nucleotide sequence SEQ ID NO: 171, the coding nucleic acid of the second heavy chain of the second antigen-binding portion has the nucleotide sequence SEQ ID NO: 169, and the coding nucleic acid of the second light chain of the second antigen-binding portion has the nucleotide sequence SEQ ID NO: 167; A nucleic acid according to claim 10 or 11.

13. A vector comprising the nucleic acid of claim 10.

14. A cell comprising the nucleic acid of claim 10 or the vector of claim 12.

15. A composition comprising a bispecific antibody or antigen-binding portion thereof described in claim 1, or an antibody or antigen-binding portion thereof that binds to GPC3 described in claim 9, or a nucleic acid described in claim 10, or a vector described in claim 13, or a cell described in claim 14.

16. 10. An antibody-drug conjugate comprising the bispecific antibody or antigen-binding portion thereof of claim 1 covalently linked to a therapeutic moiety, or an antibody or antigen-binding portion thereof that binds to GPC3 of claim 9.

17. The antibody-drug conjugate of claim 16, wherein the therapeutic moiety is selected from a cytotoxic moiety, a chemotherapeutic agent, a cellular factor, an immune inhibitor, an immune stimulant, a degradative peptide, or a radioisotope.

18. The cytotoxic moiety may be selected from the group consisting of tataxol, cytochalasin B, gramicidin D, ethidium bromide, emetine (cephaeline), mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, tubulin inhibitors, mitosis inhibitors, dolastatin 10 or 15, irinotecan, mitoxantrone, mithramycin, actinomycin D D), 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, calicheamicin, antimetabolites, alkylating agents, cisplatin or carboplatin, duocarmycin A, duocarmycin SA, rachelmycin (CC-1065), antibiotics, pyrrolo[2,1-c][1,4]-benzodiazepines (PDB), diphtheria toxin and related molecules, ricin, cholera toxin, shiga-like toxin, pertussis toxin, tetanus toxin, Bowman-Birk soybean protease inhibitor, Pseudomonas exotoxin, allorin, saporin, modeccin, geranin, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein, momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin and enomycin toxins, ribonuclease (RNase), DNase I, Staphylococcus aureus endotoxin A, pokeweed antiviral protein, diphtheria toxin, Pseudomonas endotoxin; the cellular factor is selected from IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNα, IFNβ, IFNγ, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFα; The radioisotope is ３ H. １４ C. １５ N. ３５ S. ６７ Cu, ９０ Y. ９９ Tc, １２５ I, １３１ I, １８６ Re, １８８ Re, ２１１ At, ２１２ Bi, ２１２ Pb, ２１３ Bi, ２２５ Ac and ２２７ Th, The antibody-drug conjugate of claim 17.

19. the tubulin inhibitor is selected from maytansine; the mitotic inhibitor is selected from monomethyl auristatin E or F; the antimetabolite is selected from methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine; the alkylating agent is selected from mechlorethamine, thiopurine, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine; The antibiotic is selected from bleomycin, idarubicin, primycin, and anthramycin (AMC); The diphtheria toxin and related molecules are selected from the diphtheria A chain and active fragments and hybrid molecules thereof; the ricin is selected from ricin A or a deglycosylated ricin A chain toxin; The shiga-like toxin is selected from SLT I, SLT II, ​​SLT IIV, LT toxin, C3 toxin, and shiga toxin; The antibody-drug conjugate of claim 18, wherein the pokeweed protein is selected from PAPI, PAPII and PAP-S.

20. A kit comprising a specific antibody or an antigen-binding portion thereof according to claim 1, or an antibody or an antigen-binding portion thereof that binds to GPC3 according to claim 9, or a nucleic acid molecule according to claim 10, or a vector according to claim 13, or a cell according to claim 14, or a composition according to claim 15, or an antibody-drug conjugate according to claim 16.

21. A specific antibody or antigen-binding portion thereof according to claim 1, an antibody or antigen-binding portion thereof that binds to GPC3 according to claim 9, a nucleic acid molecule according to claim 10, a vector according to claim 12, a cell according to claim 14, a composition according to claim 15 and / or an antibody-drug conjugate according to claim 16 for use in the diagnosis, treatment or prevention of a GPC3-related disease, The GPC3-related disease includes a tumor, the tumor is a cancer, the cancer is a GPC3-positive cancer, and the GPC3-positive cancer is selected from GPC3-positive liver cancer, GPC3-positive hepatocellular carcinoma, GPC-positive pancreatic cancer, GPC-positive lung cancer, GPC-positive colorectal cancer, GPC-positive breast cancer, GPC-positive prostate cancer, and GPC-positive lymphoma; Or, the GPC3-related disease is GPC-positive leukemia. The specific antibody or antigen-binding portion thereof according to claim 1, the antibody or antigen-binding portion thereof that binds to GPC3 according to claim 9, the nucleic acid molecule according to claim 10, the vector according to claim 13, the cell according to claim 14, the composition according to claim 15 and / or the antibody-drug conjugate according to claim 16.

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