Development and application of a new tumor adapter therapeutic drug

Abstract

The present disclosure relates to the development and application of a novel tumor adapter therapeutic drug. The therapeutic drug comprises a bispecific antibody binding to GPC3 and CD3, which comprises a first binding domain binding to GPC3 on the surface of target cells and a second binding domain binding to CD3 on the surface of T cells. The bispecific antibody can effectively inhibit the growth of transplanted tumors in immune-reconstituted mice at an extremely low dose, i.e. can inhibit tumor growth; the toxicology study in cynomolgus monkeys also shows that the animals are well tolerated to the bispecific antibody, and the bispecific antibody is superior to the same antibody in terms of efficacy and safety.

Description

Technical Field

[0001] This disclosure relates to a bispecific antibody and its application, particularly the development and application of a novel tumor connector therapeutic agent. Background Technology

[0002] Glypican-3 (GPC3, also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS or SGBS1) is a phosphatidylinositol proteoglycan belonging to the heparan sulfate proteoglycans (HSPGs) family. It is anchored to the outer surface of the cell membrane via glycophosphatidyl inositol (GPI). The GPC3 protein core consists of 580 amino acids with a molecular weight of approximately 65 kDa. The protease Furin is cleaved between 358Arg and 359Ser (355R-356Q-357Y-358R-359S) to form two subunits. Two heparan sulfate polysaccharide chains are attached to the C-terminal subunit near the membrane. It is an important component of the extracellular matrix and may participate in the activation of cells through multiple signaling pathways such as Wnt, Hh (Hedgehog), and fibroblast growth factor (FGF) (Li Net al, Glypicans as Cancer Therapeutic Targets, Trends Cancer. 2018).GPC3 is expressed in various fetal tissues (liver, lung, kidney, and placenta). After birth, due to DNA methylation modification, the GPC3 gene ceases to be expressed. However, in various tumor tissues, such as 66-90% of hepatocellular carcinoma, 54-65% of squamous cell carcinoma of the lung, 16-64% of small cell lung cancer, 20-30% of gastrointestinal tumors (esophageal cancer, gastric cancer, cardia cancer), and some other types of cancer cells, GPC3 gene expression 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 squamouscell carcinoma and lung adenocarcinoma and its clinical significance, GenetMol Res. 2015 Aug). 28;14(3):10185-92;Ishiguro T et al,An anti-glypican 3 / CD3bispecific T cell-redirecting antibody for treatment of solid tumors,SciTransl Med.2017 Oct 4;9(410):eaal4291;Baumhoer D et al,Glypican 3 expressionin human nonneoplastic,preneoplastic,andneoplastic 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 low differentiation of HCC cells and disease prognosis.Recent reports indicate that GPC3 is a marker of tumor stem cells. GPC3-specific cytotoxic T cells (CTLs) can effectively kill tumor stem cells and inhibit tumor growth in mice (Okada Met al, Selective elimination of undifferentiated human pluripotent stem cells using pluripotent state-specific immunogenic antigen Glypican-3, Biochem Biophys ResCommun. 2019 Apr 9; 511(3):711-717).

[0003] T-cell bispecific antibodies (or T-cell connectors) are artificially constructed special antibody molecules that recognize target cell surface antigens (antigen arm) at one end and bind to the T-cell CD3 receptor (CD3 arm) at the other end. Using antibodies targeting the CD3ε chain, CD3 aggregates on T cells in a manner similar to TCR / peptide / HLA, thereby activating T cells and killing tumor cells. The BCMA×CD3 bispecific molecule is a T-cell bispecific (TCB) antibody that targets BCMA expressed on myeloma cells and the CD3ε chain (CD3e) present on T cells. The mechanism of action of the BCMA×CD3 bispecific molecule involves simultaneously binding to BCMA+ myeloma cells and CD3+ T cells, inducing T-cell activation and T-cell-mediated cell killing.

[0004] Codrituzumab is the first therapeutic monoclonal antibody targeting GPC3. It has shown significant antitumor activity in mouse tumor models. However, in the phase II clinical trial (NCT01507168), there was no significant difference in disease progression and overall survival between Codrituzumab and 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 bring clinical benefits to patients with high GPC3 expression in tumors or with CD16 high affinity mutations (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 a bispecific anti-GPC3 / CD3 antibody that can effectively kill various GPC3-positive tumor cells through T cell-mediated killing effects. Its activity is superior to that of monoclonal antibodies. However, due to its strong side effects, clinical research was suspended in 2018. In 2019, after the Phase I clinical trial was restarted, it was used in combination with the IL-6 antibody Tocilizumab to avoid cytokine release syndrome. Summary of the Invention

[0005] To address the problems existing in the prior art, this disclosure provides a novel GPC3 antibody with high affinity for the GPC3 protein. Furthermore, this disclosure also provides a novel GPC3-CD3κλ bispecific antibody formed by the GPC3 antibody and a CD3 antibody. The GPC3 antibody can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) against target cells, and can inhibit GPC3-induced endocytosis. The GPC3-CD3κλ bispecific antibody adopts a full-length IgG conformation. Through the introduction of charge and affinity optimization, the κλ bispecific antibody preferentially targets GPC3+ tumor tissue, recruiting and activating T cells at low concentrations to effectively kill target cells, while not activating T cells in the absence of target cells. Simultaneously, the κλ bispecific antibody does not bind to receptors such as FcγRI, FcγRIIA, and FcγRIIIA, reducing the risk of cytokine storm. Pharmacological studies have confirmed that this novel GPC3-CD3κλ bispecific antibody can inhibit tumor growth at extremely low doses and effectively suppress the growth of xenografts in immune-reconstituted mice. Toxicological studies in cynomolgus monkeys have also shown that the animals tolerate the novel GPC3-CD3κλ bispecific antibody well, and that the efficacy and safety of the novel GPC3-CD3κλ bispecific antibody are superior to those of similar antibodies.

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

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

[0008] In one respect, this disclosure provides nucleic acids encoding bispecific antibodies or their antigen-binding portions as described above.

[0009] In one respect, this disclosure provides a vector containing nucleic acids as described above.

[0010] In one respect, this disclosure provides cells containing nucleic acids or vectors as described above.

[0011] The antibody or its antigen-binding portion is based on any of the foregoing aspects, wherein the antibody or its antigen-binding portion is humanized.

[0012] In one aspect, this disclosure provides pharmaceutical compositions or kits comprising an antibody or its antigen-binding portion or its encoded nucleic acid as described above and a pharmaceutically acceptable carrier.

[0013] In one aspect, this disclosure provides antibody-drug conjugates comprising an antibody or its antigen-binding portion, a bispecific or multispecific molecule, covalently attached to a therapeutic portion of any of the foregoing aspects.

[0014] In one aspect, this disclosure provides a method for treating GPC3-related conditions, comprising the steps of administering to a mammal a therapeutically effective amount of an antibody or antigen-binding fragment thereof, nucleic acid, vector, cell, and / or pharmaceutical composition thereof.

[0015] In one aspect, this disclosure provides the use of antibodies or antigen-binding fragments thereof, nucleic acids, vectors, cells and / or pharmaceutical compositions of any of the foregoing aspects in the preparation of medicaments or kits for treating GPC3-related diseases in mammals.

[0016] The antibody disclosed herein can be used for a variety of applications, including the detection of GPC3 protein, diagnosis, treatment or prevention of GPC3-related diseases. Attached Figure Description

[0017] Figure 1 The results of non-reduced SDS-PAGE of the GPC3 recombinant protein are shown.

[0018] Figure 2 The flow cytometry results of GPC3 stable cells are shown.

[0019] Figure 3 The SDS-PAGE results of the CD3εγ-Fc recombinant protein are shown.

[0020] Figure 4 The flow cytometry results for GPC3 antibody and human GPC3 stable cells are shown.

[0021] Figure 5 The binding of antibody 49G8 to GPC3 antigens from different species is shown.

[0022] Figure 6 The antibody 49G8-mediated ADCC activity was demonstrated.

[0023] Figure 7 The binding of the humanized antibody to the GPC3 antigen was demonstrated.

[0024] Figure 8 The binding of the humanized antibody to GPC3 stable cells was demonstrated.

[0025] Figure 9 Octet affinity assays for humanized antibodies are shown.

[0026] Figure 10 The SPR affinity assay for the humanized antibody is shown.

[0027] Figure 11 The results of endocytosis of humanized antibodies are shown.

[0028] Figure 12The binding of the humanized CD3 antibody to the human CD3εγ antigen was demonstrated.

[0029] Figure 13 The binding of the CD3 humanized antibody to Jurkat cells was demonstrated.

[0030] Figure 14 The comparison of affinity between humanized CD3 antibodies and human and monkey CD3εγ antigens is shown.

[0031] Figure 15 The binding of the GPC3×CD3κλ bispecific antibody to GPC3 stable cells was demonstrated.

[0032] Figure 16 The binding of the GPC3×CD3κλ bispecific antibody to HepG2 cells was demonstrated.

[0033] Figure 17 The binding of the GPC3×CD3κλ bispecific antibody to Jurkat cells was demonstrated.

[0034] Figure 18 The binding of the GPC3×CD3κλ bispecific antibody to peripheral blood T cells was demonstrated.

[0035] Figure 19 illustrates the TDCC mediated by the GPC3×CD3κλ bispecific antibody. Figure 19A It showed killing effect on HepG2 cells. Figure 19B It shows activation of T cells.

[0036] Figure 20 This study demonstrates the effect of the GPC3×CD3κλ bispecific antibody on the NFAT signaling pathway in T cells.

[0037] Figure 21 The activation effect of the GPC3×CD3κλ bispecific antibody on PBMCs was demonstrated.

[0038] Figure 22 The binding of the GPC3×CD3κλ bispecific antibody to the FcγR activating receptor was demonstrated.

[0039] Figure 23 The inhibitory effect of the GPC3×CD3κλ bispecific antibody in an immune-reconstituted mouse HepG2 xenograft model was demonstrated.

[0040] Figure 24 The inhibitory effect of the GPC3×CD3κλ bispecific antibody on the CD3 humanized mouse Hepa1-6 / hGPC3 xenograft model was demonstrated. Detailed Implementation

[0041] I. Definition

[0042] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0043] As used herein, the term “GPC3” may refer to the concept of GPC3 itself, which is present in animals and preferably in humans, as well as any variants, isotypes and paralogs.

[0044] The term "human GPC3" refers to GPC3 derived from humans.

[0045] The terms "anti-GPC3 antibody" and "GPC3-binding antibody" refer to antibodies capable of binding to GPC3 with sufficient affinity, making the antibody useful as a diagnostic and / or therapeutic agent targeting GPC3. In one embodiment, the anti-GPC3 antibody binds to less than about 10% of its binding to unrelated non-GPC3 proteins, as measured by, for example, radioimmunoassay (RIA). In some embodiments, the GPC3-binding antibody has a concentration of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 μM). -8 M or lower, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (Kd) of M). In some embodiments, the anti-GPC3 antibody binds to conserved GPC3 epitopes from different species of GPC3.

[0046] “CD3” refers to any native CD3 from any vertebrate source, 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 as well as any form of CD3 derived from cell processing. The term also covers naturally occurring variants of CD3, such as splice variants or allelic variants. In one embodiment, CD3 is human CD3, specifically the epsilon 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 [Macacafascicularis] CD3ε is shown in NCBI GenBank no. BAB71849.1.

[0047] The term “cell surface” is used according to its normal meaning in this field, and therefore includes the cell exterior that can be accessed by binding to proteins and other molecules.

[0048] As used herein and unless otherwise stated, the terms “about” or “approximately” mean within 10% of a given value or range. Where an integer is required, the term means within 10% of a given value or range, rounded up or down to the nearest integer.

[0049] Regarding antibody chain polypeptide sequences, the phrase "substantially identical" can be understood as an antibody chain exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a reference polypeptide sequence. Regarding nucleic acid sequences, the term can be understood as a nucleotide sequence exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with a reference nucleic acid sequence.

[0050] The term "sequence identity" or "identity" has a generally accepted meaning in the art, and the percentage of sequence similarity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available techniques. Sequence identity can be measured along the full length of the polynucleotide or polypeptide or along a region of the molecule. Although many methods exist for measuring the identity between two polynucleotides or polypeptides, the term "identity" is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0051] A "substitution" variant is a variant in which at least one amino acid residue is removed from the natural sequence and replaced by a different amino acid at the same position. The substitution can be single, where only one amino acid is substituted in the molecule; or it can be multiple, where two or more amino acids are substituted in the same molecule. Multiple substitutions can be located at consecutive sites. Similarly, an amino acid can be substituted by multiple residues, and such variants include both substitution and insertion. An "insertion" variant is a variant in which one or more amino acids are inserted into an amino acid immediately adjacent to a specific position in the natural sequence. An adjacent amino acid is defined as one attached to the α-carboxyl or α-amino functional group of that amino acid. A "deletion" variant is a variant in which one or more amino acids are removed from the natural amino acid sequence. Typically, deletion variants have one or two amino acids missing from a specific region of their molecule.

[0052] Regarding the variable domains of antibodies, the term "variable" refers to certain portions of related molecules with extensive sequence differences between antibodies, used for the specific recognition and binding of a particular antibody to its specific target. However, variability is not uniformly distributed throughout the entire variable domain of an antibody. Variability is concentrated in three segments known as complementarity-determining regions (CDRs; namely CDR1, CDR2, and CDR3) or hypervariable regions, all located within the variable domains of the light and heavy chains. More conserved portions within the variable domain are called framework (FR) regions or framework sequences. Each variable domain of the natural heavy and light chains comprises four FR regions, primarily employing a β-sheet configuration, linked by three CDRs. These CDRs form loops that connect the β-sheet structure and, in some cases, partially form the β-sheet structure. The CDRs of each chain are typically linked in proximity by the FR regions, and the presence of CDRs from other chains contributes to the formation of antibody target binding sites (epitopes or determinants). As used herein, immunoglobulin amino acid residue numbering follows the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise stated. A CDR may have the ability to specifically bind associated epitopes.

[0053] As used herein, an "antibody fragment" or "antigen-binding fragment" refers to any portion of a full-length antibody that is less than the full length but contains at least a portion of the antibody's variable region (e.g., one or more CDRs and / or one or more antibody-binding sites) that binds to an antigen, and thus retains binding specificity and at least a portion of the full-length antibody's specific binding ability. Therefore, an antigen-binding fragment refers to an antibody fragment containing an antigen-binding portion that binds to the same antigen as an antibody fragment derived from the antibody fragment. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, as well as synthetically produced derivatives, such as recombinant derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, biantibodies, Fd and Fd' fragments, and other fragments, including modified fragments. The fragment may comprise multiple chains linked together, for example by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that acquires immune-specific binding (i.e., exhibits at least or at least about 10) when inserted into an antibody frame (e.g., by replacing the corresponding region). 7 -10 8 M -1 Antibodies against the Ka antigen. A “functional fragment” or “anti-GPC3 antibody analog” is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind ligands or initiate signal transduction. As used herein, a functional fragment generally has the same meaning as “antibody fragment”, and in the context of antibodies, it can refer to a fragment that prevents or substantially reduces the ability of the receptor to bind ligands or initiate signal transduction, such as Fv, Fab, F(ab')2, etc. An “Fv” fragment is a dimer (V) formed by non-covalent binding of a variable domain of a heavy chain and a variable domain of a light chain. H -V L (Dimer) composition. In this configuration, the three CDRs of each variable domain interact to determine V. H -V L The target binding sites on the surface of the dimer are the same as in the case of the intact antibody. The six CDRs collectively confer target binding specificity to the intact antibody. However, even a single variable domain (or half the Fv of only including three target-specific CDRs) can still have the ability to recognize and bind to the target.

[0054] As used herein, the term "bispecific antibody" (BsAb) refers to an antibody and / or antigen-binding molecule that specifically binds to two different antigenic determinants. Typically, a bispecific antibody and / or antigen-binding molecule contains two antigen-binding sites, each specific to a different antigenic determinant. In some embodiments, the bispecific antibody and / or antigen-binding molecule is capable of binding to two antigenic determinants simultaneously, particularly two antigenic determinants expressed on two different cells.

[0055] As used herein, “monoclonal antibody” refers to a population of identical antibodies, meaning that each individual antibody molecule in a population of monoclonal antibodies is identical to the others. This characteristic contrasts with that of a polyclonal population of antibodies, which contains antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by a number of well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared from immortalized B cells, for instance, by fusing with myeloma cells to generate hybridoma cell lines or by infecting B cells with a virus such as EBV. Recombinant techniques can also be used to prepare antibodies in vitro from a clonal population of host cells by transforming host cells with plasmids carrying artificial sequences of nucleotides encoding the antibody.

[0056] As used herein, the term "hybridoma" or "hybridoma cell" refers to a cell or cell line (typically myeloma or lymphoma cells) resulting from the fusion of antibody-producing lymphocytes and non-antibody-producing cancer cells. As is known to those skilled in the art, hybridomas can proliferate and continuously supply the production of specific monoclonal antibodies. Methods for generating hybridomas are known in the art. When referring to the term "hybridoma" or "hybridoma cell," it also includes subclones and progeny cells of the hybridoma.

[0057] 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 antibodies naturally produced by antibody-secreting B cells and synthetically produced antibodies with the same domains.

[0058] The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0059] "Humanized" antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) containing minimal sequences derived from non-human immunoglobulins. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues of the complementarity-determining region (CDR) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) with the desired specificity, affinity, and capability, such as mouse, rat, or rabbit.

[0060] Furthermore, in humanization, amino acid residues in the CDR1, CDR2, and / or CDR3 regions of VH and / or VL may be mutated to improve one or more binding properties (e.g., affinity) of the antibody. Mutations can be introduced, for example, through PCR-mediated mutations, and their effects on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conserved mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. Additionally, mutations within the CDRs typically do not exceed one or two. Therefore, the humanized antibodies described in this disclosure also cover antibodies containing one or two amino acid mutations within the CDRs.

[0061] As used herein, the term "CDR" refers to the complementarity-determining region, and each heavy and light chain of an antibody molecule is known to have three CDRs. CDRs, also known as hypervariable regions, are located in the variable regions of each heavy and light chain of the antibody and are highly variable sites in the primary structure of the CDR. In this specification, the CDRs of the heavy chain are represented by CDR1, CDR2, and CDR3 from the N-terminal sequence of the heavy chain, and the CDRs of the light chain are represented by CDR1, CDR2, and CDR3 from the N-terminal sequence of the light chain. These sites are adjacent to each other in the tertiary structure and determine the specificity of the antigen to which the antibody binds.

[0062] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which an antibody binds at its complementary site. Epitope determinants typically comprise chemically active surface subtypes of a molecule, such as amino acid or sugar side chains, and often possess specific three-dimensional structural features as well as specific charge characteristics.

[0063] As used herein, the terms “specific binding” and “immune-specific binding” for antibodies or their antigen-binding fragments are used interchangeably and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with the same antigen through a non-covalent interaction between the antibody and the antigen’s antibody-binding site. The antigen may be an isolated antigen or present in tumor cells. Typically, antibodies that immune-specifically bind (or specifically bind) antigens are present in quantities of approximately 1 × 10⁻⁶.7 M -1 Or 1×10 8 M -1 Or a larger affinity constant Ka (or 1×10) -7 M or 1×10 -8 The affinity constant (M or lower) binds to the antigen. The affinity constant can be determined by standard kinetic methods of antibody reactions, such as immunoassay, 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 assays known in the art; see also U.S. Patent No. 7,229,619, which describes exemplary SPR and ITC methods for calculating the binding affinity of antibodies. Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see Malmqvist (2000) Biochem. Soc. Trans. 27:335).

[0064] As used herein, the terms “polynucleotide” and “nucleic acid molecule” refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) typically linked together by phosphodiester bonds. As used herein, the term “nucleic acid molecule” is intended to include both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded and can be cDNA.

[0065] As used herein, isolated nucleic acid molecules are nucleic acid molecules isolated from other nucleic acid molecules present in natural sources of nucleic acid molecules. “Isolated” nucleic acid molecules, such as cDNA molecules, may be substantially free of other cellular material or culture medium when prepared by recombinant technology, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding provided antibody or antigen-binding fragments.

[0066] As used herein, “operably linked” in relation to a nucleic acid sequence, region, element, or domain indicates 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, thereby regulating or mediating the transcription of that nucleic acid.

[0067] Also provided are “conserved sequence modifications” of the sequences listed herein, i.e., nucleotide and amino acid sequence modifications that do not eliminate the binding of antibodies to antigens encoded by nucleotide sequences or containing amino acid sequences. These conserved sequence modifications include conserved nucleotide and amino acid substitutions, as well as nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the sequence listings herein using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conserved sequence modifications include conserved amino acid substitutions, wherein amino acid residues are replaced with amino acid residues having similar side chains. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar 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). Therefore, the predicted non-essential amino acid residues in anti-GPC3 antibodies are preferably replaced by another amino acid residue from the same side chain family. Methods for identifying nucleotides and conserved amino acid substitutions that do not eliminate antigen binding are well known in the art (e.g., see Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0068] As an alternative, in another embodiment, mutations can be randomly introduced along all or part of the coding sequence of the anti-GPC3 antibody, for example, through saturation mutagenesis, and the resulting modified anti-GPC3 antibody can be screened for improved binding activity.

[0069] As used herein, “expression” refers to the process by which a polypeptide is produced through the transcription and translation of polynucleotides. The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from host cells. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, Coomassie blue staining following gel electrophoresis, Lowry protein assays, and Bradford protein assays.

[0070] As used herein, a “host cell” is a cell used to receive, maintain, replicate, and amplify a vector. Host cells can also be used to express the polypeptide encoded by the vector. When a host cell divides, the nucleic acids contained in the vector replicate, thereby amplifying the nucleic acids. Host cells can be eukaryotic or prokaryotic cells. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells such as HEK 293 cells.

[0071] As used herein, a "vector" is a reproducible nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into a suitable host cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can typically be introduced via restriction enzyme digestion and ligation. Vectors also include those containing nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for amplification of nucleic acids or for expression / display of the polypeptide encoded by the nucleic acid. Vectors are typically kept free but can be designed to integrate genes or portions thereof into the chromosome of the genome. Vectors for artificial chromosomes, such as yeast artificial vectors and mammalian artificial chromosomes, are also considered. The selection and use of such vectors are well known to those skilled in the art.

[0072] As used in this article, vectors also include “viral vectors” or “vectors of viruses.” Viral vectors are engineered viruses that are operatively linked to a foreign gene to transfer (as a medium or shuttle) the foreign gene into cells.

[0073] As used herein, "expression vector" includes a vector capable of expressing DNA operatively linked to regulatory sequences, such as promoter regions, that influence the expression of such DNA fragments. These additional fragments may include promoter and terminator sequences and optionally include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector, which, when introduced into a suitable host cell, results in the expression of clonal DNA. Suitable expression vectors are well known to those skilled in the art and include reproducible expression vectors in eukaryotic and / or prokaryotic cells, as well as expression vectors that remain free or are integrated into the host cell genome.

[0074] As used herein, “treatment” for an individual suffering from a disease or disease condition means that the individual’s symptoms are partially or completely relieved, or remain unchanged after treatment. Therefore, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing underlying disease and / or preventing the worsening of symptoms or the development of disease. Treatment also includes any antibodies or antigen-binding fragments thereof provided, and any pharmaceutical use of the compositions provided herein.

[0075] As used in this article, “therapeutic effect” refers to the effect resulting from treatment of an individual, which alters, usually improves or enhances the symptoms of a disease or condition, or cures a disease or condition.

[0076] As used herein, "therapeutic effective amount" or "therapeutic effective dose" refers to an amount of substance, compound, material, or composition containing a compound that, when applied to a subject, is at least sufficient to produce a therapeutic effect. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or condition.

[0077] As used herein, "preventive effective dose" or "preventive effective amount" refers to the amount of a substance, compound, material, or composition containing a compound that, when applied to a subject, would have the intended preventive effect, such as preventing or delaying the onset or recurrence of a disease or symptom, or reducing the likelihood of the onset or recurrence of a disease or symptom. A fully preventive effective dose does not necessarily occur through the administration of a single dose and can occur only after a series of doses have been administered. Therefore, a preventive effective dose can be administered in one or more applications.

[0078] As used in this article, the term "patient" refers to mammals, such as humans.

[0079] II. Detailed Implementation Plan

[0080] In one aspect, this disclosure provides a bispecific antibody or its antigen-binding portion, comprising:

[0081] (a) A first antigen-binding moiety or antigen-binding fragment thereof of the GPC3 antigen bound to the surface of a target cell, the first antigen-binding moiety comprising a first heavy chain and a first light chain, the first antigen-binding moiety comprising a first binding domain binding to the first antigen, wherein the first binding domain comprises a heavy chain CDR selected from amino acid sequences 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 amino acid sequences 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

[0082] (b) A second antigen-binding moiety or an antigen-binding fragment thereof of a CD3 antigen bound to the surface of a T cell, the second antigen-binding moiety comprising a second heavy chain and a second light chain, the second antigen-binding moiety comprising a second binding domain that binds to the second antigen.

[0083] According to the antibody or its antigen-binding portion in the preceding aspect, the first binding domain comprises a heavy chain CDR1 selected from amino acid sequences SEQ ID NO:11, 32, 64, 79 or any variant thereof, a heavy chain CDR2 selected from amino acid sequences SEQ ID NO:12, 21, 33, 65, 72, 80, 101, 106 or any variant thereof, and a heavy chain CDR3 selected from amino acid sequences SEQ ID NO:13, 66, 73, 81 or any variant thereof; and / or the first binding domain comprises a light chain CDR1 selected from amino acid sequences SEQ ID NO:16, 38, 46, 60 or any variant thereof, a light chain CDR2 selected from amino acid sequences SEQ ID NO:17, 47 or any variant thereof, and a light chain CDR3 selected from amino acid sequences SEQ ID NO:18, 26, 29, 39, 48, 51, 54, 57, 61, 69, 76 or any variant thereof.

[0084] According to any of the foregoing aspects of the antibody or its antigen-binding portion, the heavy chain CDR1, CDR2, and CDR3 sequences of the first binding domain are selected from: the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 11, 12, 13; the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 11, 21, 13; the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 32, 33, 13; the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 64, 65, 66; the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 11, 72, 73; the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 79, 80, 81; the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 11, 101, 13; and the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 11, 101, 13. The first heavy chain CDR1, CDR2 and CDR3 sequences of NO:11, 106 and 13;The light chain CDR1, CDR2, and CDR3 sequences of the first binding domain are selected from: the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 16, 17, and 18; the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 16, 17, and 26; the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 16, 17, and 29; the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 38, 17, and 39; the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 46, 47, and 48; the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 16, 17, and 51; the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 16, 17, and 54; and the amino acid sequences SEQ ID NO: 16, 17, and 54. The first light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:16, 17, and 57; the first light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:60, 17, and 61; the first light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:16, 17, and 69; the first light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:16, 17, and 76; the first light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:92, 17, and 61; the first light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:95, 17, and 61; and the first light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:98, 17, and 61.

[0085] In some embodiments, the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:11, 12, and 13, and the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:16, 17, and 18.

[0086] In some embodiments, the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:11, 21, and 13, and the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:16, 17, and 18.

[0087] In some embodiments, the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:11, 12, and 13, and the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:16, 17, and 26.

[0088] In some embodiments, the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:11, 12, and 13, and the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:16, 17, and 29.

[0089] In some embodiments, the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:32, 33, and 13, and the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:16, 17, and 18.

[0090] In some embodiments, the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:11, 21, and 13, and the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:38, 17, and 39.

[0091] In some embodiments, the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:11, 21, and 13, and the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:16, 17, and 18.

[0092] In some embodiments, the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:11, 12, and 13, and the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:46, 47, and 48.

[0093] In some embodiments, the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:11, 12, and 13, and the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:16, 17, and 51.

[0094] In some embodiments, the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:11, 12, and 13, and the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:16, 17, and 54.

[0095] In some embodiments, the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:11, 12, and 13, and the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:16, 17, and 57.

[0096] In some embodiments, the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:11, 12, and 13, and the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:60, 17, and 61.

[0097] In some embodiments, the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:64, 65, and 66, and the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:16, 17, and 69.

[0098] In some embodiments, the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:11, 72, and 73, and the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:16, 17, and 76.

[0099] In some embodiments, the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:79, 80, and 81, and the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:16, 17, and 69.

[0100] In some embodiments, the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:11, 106, and 13, and the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:60, 17, and 61.

[0101] In some embodiments, the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:11, 106, and 13, and the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:92, 17, and 61.

[0102] In some embodiments, the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 11, 106, and 13, and the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 95, 17, and 61.

[0103] In some embodiments, the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:11, 106, and 13, and the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:98, 17, and 61.

[0104] According to any of the foregoing aspects, the antibody or its antigen-binding portion comprises a first heavy chain variable region selected from amino acid sequences SEQ ID NO:9, 19, 30, 40, 62, 70, 77, 99, 102, 104, 107, 109, 111 or any variant thereof, and / or comprises a first light chain variable region selected from amino acid sequences 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] According to any of the foregoing aspects of the antibody or its antigen-binding portion, the second binding domain is selected from heavy chain CDRs of amino acid sequences SEQ ID NO:134, 135, 136, 139, 142, 145, 148, 151, 154, 155 or any variant thereof; and / or contains light chain CDRs selected from amino acid sequences SEQ ID NO:115, 116, 117, 122, 123, 128, 131 or any variant thereof.

[0126] In some embodiments, the second binding domain comprises a second heavy chain CDR1 selected from amino acid sequences SEQ ID NO:134, 139, 154 or any variant thereof, a second heavy chain CDR2 selected from amino acid sequences SEQ ID NO:135, 155 or any variant thereof, a second heavy chain CDR3 selected from amino acid sequences SEQ ID NO:136, 142, 145, 148, 151 or any variant thereof; and / or a second light chain CDR1 selected from amino acid sequences SEQ ID NO:115, 122 or any variant thereof, a second light chain CDR2 selected from amino acid sequences SEQ ID NO:116, 123, 131 or any variant thereof, and a second light chain CDR3 selected from amino acid sequences SEQ ID NO:117, 128 or any variant thereof.

[0127] In some embodiments, the heavy chain CDR1, CDR2, and CDR3 sequences of the second binding domain are selected from: the second heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 134, 135, and 136; the second heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 139, 135, and 136; the second heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 139, 135, and 142; the second heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 139, 135, and 145; the second heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 139, 135, and 148; the second heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 139, 135, and 151; and the second heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 139, 135, and 151. The second heavy chain CDR1, CDR2, and CDR3 sequences of NO:154, 155, and 136; and the light chain CDR1, CDR2, and CDR3 sequences of the second binding domain are selected from: the second light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:115, 116, and 117; the second light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:122, 123, and 117; the second light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:115, 116, and 128; and the second light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:115, 131, and 128.

[0128] In some embodiments, the second binding domain comprises the second heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:134, 135, and 136, and the second light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:115, 116, and 128.

[0129] In some embodiments, the second binding domain comprises the second heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:134, 135, and 136, and the second light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:115, 116, and 117.

[0130] In some embodiments, the second binding domain comprises the second heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:134, 135, and 136, and the second light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO:122, 123, and 117.

[0131] In some embodiments, the second binding domain comprises a second heavy chain variable region selected from amino acid sequences SEQ ID NO:132, 137, 140, 143, 146, 149, 152, 156, 158, 160 or any variant thereof, and / or comprises a second light chain variable region selected from amino acid sequences SEQ ID NO:113, 118, 120, 124, 126, 129 or any variant thereof.

[0132] In some embodiments, the second binding domain comprises a second heavy chain variable region of amino acid sequence SEQ ID NO:132 or any variant thereof, and a second light chain variable region of amino acid sequence SEQ ID NO:126 or any variant thereof.

[0133] 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:113 or any variant thereof.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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:126 or any variant thereof.

[0139] Based on any of the aforementioned aspects of the antibody or its antigen-binding moiety, the first light chain of the first antigen-binding moiety is a κ-type light chain, and the second light chain of the second antigen-binding moiety is a λ-type light chain.

[0140] In some embodiments, the first light chain variable region of the first antigen-binding moiety has Gln 43 Lys mutation (Vκ) GPC3 :Gln 43 Lys). In some embodiments, the first heavy chain variable region of the first antigen-binding moiety has Gln. 39 Glu(VH GPC3 :Gln 39 Glu) mutation.

[0141] In some embodiments, the second light chain variable region of the second antigen-binding moiety has Gln 40 Glu mutation (Vλ) CD3 :Gln 40 Glu). In some embodiments, the second heavy chain variable region of the second antigen-binding moiety has Gln. 39 Lys mutation (VH) CD3 :Gln 39 Lys).

[0142] In some embodiments, the Fc portion of the first antigen-binding region and the second antigen-binding region of the bispecific antibody employs a knock-in-hole structure. In some preferred embodiments, a human IgG4 knock-in-hole structure is employed.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] In some preferred embodiments, the second heavy chain comprises a heavy chain of the amino acid sequence SEQ ID NO:168 or any variant thereof, and the second light chain comprises a light chain of the amino acid sequence SEQ ID NO:166 or any variant thereof.

[0147] In one specific embodiment, the first heavy chain of the bispecific antibody comprises a heavy chain of the amino acid sequence SEQ ID NO:164 or any variant thereof, and the first light chain of the bispecific antibody comprises a light chain of the amino acid sequence SEQ ID NO:162 or any variant thereof; the second heavy chain comprises a heavy chain of the amino acid sequence SEQ ID NO:168 or any variant thereof, and the second light chain comprises a light chain of the amino acid sequence SEQ ID NO:166 or any variant thereof.

[0148] In one specific embodiment, the first heavy chain comprises a heavy chain of the amino acid sequence SEQ ID NO:172 or any variant thereof, the first light chain of the bispecific antibody comprises a light chain of the amino acid sequence SEQ ID NO:170 or any variant thereof; the second heavy chain comprises a heavy chain of the amino acid sequence SEQ ID NO:168 or any variant thereof, and the second light chain comprises a light chain of the amino acid sequence SEQ ID NO:166 or any variant thereof.

[0149] In one aspect, this disclosure provides an antibody or antigen-binding portion thereof that binds to GPC3, wherein the antibody comprises the aforementioned first antigen-binding portion.

[0150] In some embodiments, the antibody binding to GPC3 or its antigen-binding portion has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with any of the aforementioned antibodies or their antigen-binding portions.

[0151] In one aspect, this disclosure provides nucleic acids encoding antibodies or their antigen-binding portions as described above, or nucleic acid molecules having sequence identity with them of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher.

[0152] In some embodiments, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding moiety is selected from nucleotide sequences SEQ ID NO:10, 20, 31, 41, 63, 71, 78, 100, 103, 105, 108, 110, 112 or any variant thereof; and / or the nucleic acid encoding the first light chain variable region of the first antigen-binding moiety is selected from nucleotide sequences 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.

[0153] In some preferred embodiments, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:10; and the nucleic acid encoding the first light chain variable region is a nucleotide sequence SEQ ID NO:15.

[0154] In some preferred embodiments, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:20; and the nucleic acid encoding the first light chain variable region is a nucleotide sequence SEQ ID NO:23.

[0155] In some preferred embodiments, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:10; and the nucleic acid encoding the first light chain variable region is a nucleotide sequence SEQ ID NO:25.

[0156] In some preferred embodiments, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:10; and the nucleic acid encoding the first light chain variable region is a nucleotide sequence SEQ ID NO:28.

[0157] In some preferred embodiments, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:31; and the nucleic acid encoding the first light chain variable region is a nucleotide sequence SEQ ID NO:35.

[0158] In some preferred embodiments, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:20; and the nucleic acid encoding the first light chain variable region is a nucleotide sequence SEQ ID NO:37.

[0159] In some preferred embodiments, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:41; and the nucleic acid encoding the first light chain variable region is a nucleotide sequence SEQ ID NO:43.

[0160] In some preferred embodiments, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:10; and the nucleic acid encoding the first light chain variable region is a nucleotide sequence SEQ ID NO:45.

[0161] In some preferred embodiments, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:10; and the nucleic acid encoding the first light chain variable region is a nucleotide sequence SEQ ID NO:50.

[0162] In some preferred embodiments, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:10; and the nucleic acid encoding the first light chain variable region is a nucleotide sequence SEQ ID NO:53.

[0163] In some preferred embodiments, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:10; and the nucleic acid encoding the first light chain variable region is a nucleotide sequence SEQ ID NO:56.

[0164] In some preferred embodiments, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:10; and the nucleic acid encoding the first light chain variable region is a nucleotide sequence SEQ ID NO:59.

[0165] In some preferred embodiments, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:63; and the nucleic acid encoding the first light chain variable region is a nucleotide sequence SEQ ID NO:68.

[0166] In some preferred embodiments, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:71; and the nucleic acid encoding the first light chain variable region is a nucleotide sequence SEQ ID NO:75.

[0167] In some preferred embodiments, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:78; and the nucleic acid encoding the first light chain variable region is a nucleotide sequence SEQ ID NO:83.

[0168] In some preferred embodiments, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:112; and the nucleic acid encoding the first light chain variable region is a nucleotide sequence SEQ ID NO:87.

[0169] In some preferred embodiments, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:112; and the nucleic acid encoding the first light chain variable region is a nucleotide sequence SEQ ID NO:89.

[0170] In some preferred embodiments, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:112; and the nucleic acid encoding the first light chain variable region is a nucleotide sequence SEQ ID NO:91.

[0171] In some preferred embodiments, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:112; and the nucleic acid encoding the first light chain variable region is a nucleotide sequence SEQ ID NO:94.

[0172] In some preferred embodiments, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:112; and the nucleic acid encoding the first light chain variable region is a nucleotide sequence SEQ ID NO:97.

[0173] Preferably, the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from nucleotide sequences SEQ ID NO:165, 173 or any variant thereof; and / or the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from nucleotide sequences SEQ ID NO:163, 171 or any variant thereof.

[0174] In some preferred embodiments, the nucleic acid encoding the first heavy chain of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:165, and the nucleic acid encoding the first light chain of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:163.

[0175] In some preferred embodiments, the nucleic acid encoding the first heavy chain of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:173, and the nucleic acid encoding the first light chain of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:171.

[0176] In some embodiments, the nucleic acid encoding the second heavy chain variable region of the second antigen-binding moiety is selected from nucleotide sequences SEQ ID NO:133, 138, 141, 144, 147, 150, 153, 157, 159, 161 or any variant thereof; and / or the nucleic acid encoding the second light chain variable region of the second antigen-binding moiety is selected from nucleotide sequences SEQ ID NO:114, 119, 121, 125, 127, 130 or any variant thereof.

[0177] In some preferred embodiments, the nucleic acid encoding the second heavy chain variable region of the second antigen-binding portion is a nucleotide sequence SEQ ID NO:133; and the nucleic acid encoding the second light chain variable region is a nucleotide sequence SEQ ID NO:127.

[0178] In some preferred embodiments, the nucleic acid encoding the second heavy chain variable region of the second antigen-binding portion is a nucleotide sequence SEQ ID NO:157; and the nucleic acid encoding the second light chain variable region is a nucleotide sequence SEQ ID NO:114.

[0179] In some preferred embodiments, the nucleic acid encoding the second heavy chain variable region of the second antigen-binding portion is a nucleotide sequence SEQ ID NO:157; and the nucleic acid encoding the second light chain variable region is a nucleotide sequence SEQ ID NO:127.

[0180] In some preferred embodiments, the nucleic acid encoding the second heavy chain variable region of the second antigen-binding portion is a nucleotide sequence SEQ ID NO:159; and the nucleic acid encoding the second light chain variable region is a nucleotide sequence SEQ ID NO:114.

[0181] In some preferred embodiments, the nucleic acid encoding the second heavy chain variable region of the second antigen-binding portion is a nucleotide sequence SEQ ID NO:159; and the nucleic acid encoding the second light chain variable region is a nucleotide sequence SEQ ID NO:119.

[0182] In some preferred embodiments, the nucleic acid encoding the second heavy chain variable region of the second antigen-binding portion is a nucleotide sequence SEQ ID NO:159; and the nucleic acid encoding the second light chain variable region is a nucleotide sequence SEQ ID NO:121.

[0183] In some preferred embodiments, the nucleic acid encoding the second heavy chain variable region of the second antigen-binding portion is a nucleotide sequence SEQ ID NO:159; and the nucleic acid encoding the second light chain variable region is a nucleotide sequence SEQ ID NO:127.

[0184] Preferably, the nucleic acid encoding the second heavy chain of the second antigen-binding portion is the nucleotide sequence SEQ ID NO:169 or any variant thereof; and / or the nucleic acid encoding the second light chain of the second antigen-binding portion is the nucleotide sequence SEQ ID NO:167 or any variant thereof.

[0185] In some preferred embodiments, the nucleic acid encoding the second heavy chain of the second antigen-binding portion is a nucleotide sequence SEQ ID NO:169, and the nucleic acid encoding the second light chain of the second antigen-binding portion is a nucleotide sequence SEQ ID NO:167.

[0186] In one specific embodiment, the nucleic acid encoding the first heavy chain of the first antigen-binding portion of the bispecific antibody is a nucleotide sequence SEQ ID NO:165, and the nucleic acid encoding the first light chain of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:163; the nucleic acid encoding the second heavy chain of the second antigen-binding portion is a nucleotide sequence SEQ ID NO:169, and the nucleic acid encoding the second light chain of the second antigen-binding portion is a nucleotide sequence SEQ ID NO:167.

[0187] In one specific embodiment, the nucleic acid encoding the first heavy chain of the first antigen-binding portion of the bispecific antibody is a nucleotide sequence SEQ ID NO:173, and the nucleic acid encoding the first light chain of the first antigen-binding portion is a nucleotide sequence SEQ ID NO:171; the nucleic acid encoding the second heavy chain of the second antigen-binding portion is a nucleotide sequence SEQ ID NO:169, and the nucleic acid encoding the second light chain of the second antigen-binding portion is a nucleotide sequence SEQ ID NO:167.

[0188] On the one hand, this disclosure provides a vector containing the aforementioned nucleic acid.

[0189] In one respect, this disclosure provides cells containing the aforementioned nucleic acids or vectors.

[0190] In one aspect, this disclosure provides compositions comprising the aforementioned bispecific antibody or its antigen-binding portion, nucleic acid, vector, and / or cells.

[0191] In one respect, this disclosure provides antibody-drug conjugates comprising the aforementioned bispecific antibody or its antigen-binding portion covalently attached to a therapeutic portion.

[0192] Preferably, the therapeutic component is selected from cytotoxic components, chemotherapeutic agents, cytokines, immunosuppressants, immunostimulants, cleavage peptides, or radioisotopes.

[0193] The antibodies disclosed herein can be used as therapeutic or diagnostic tools in various diseases in which GPC3 is poorly expressed or found.

[0194] In one embodiment of a GPC3-related disease, GPC3 expression in cells of a diseased tissue or organ is increased compared to its state in a healthy tissue or organ. An increase is defined as an increase of at least 10%, particularly at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000%, or even more. In one embodiment, expression is found only in the diseased tissue, while expression is suppressed in the corresponding healthy tissue. According to this disclosure, GPC3-related diseases include tumors.

[0195] In some implementations, the tumor is cancer. In some implementations, the cancer is GPC3-positive cancer, for example, it can be 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.

[0196] In some implementations, the therapeutic agent contains an antibody that specifically binds to an activated T-cell antigen.

[0197] In one embodiment, the therapeutic agent comprises an antibody that specifically binds to CD3, particularly CD3ε.

[0198] Methods for treating diseases and symptoms using the bispecific antibodies disclosed herein include the following steps: administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof, or nucleic acid molecule, or carrier, or cell, or pharmaceutical composition to a mammal.

[0199] In some embodiments, this disclosure provides a method of treating or preventing cancer, comprising administering an antibody capable of binding to GPC3 to a patient, wherein the antibody is administered to provide a serum level of at least 40 μg / ml. In various embodiments, the antibody is administered to provide a serum level of at least 50 μg / ml, at least 150 μg / ml, at least 300 μg / ml, at least 400 μg / ml, or at least 500 μg / ml. In various embodiments, the antibody is administered to provide a serum level not exceeding 800 μg / ml, 700 μg / ml, 600 μg / ml, 550 μg / ml, or 500 μg / ml. In one embodiment, the serum level provided is from 40 μg / ml to 700 μg / ml, preferably from 40 μg / ml to 600 μg / ml, preferably from 50 μg / ml to 500 μg / ml, such as from 150 μg / ml to 500 μg / ml or from 300 μg / ml to 500 μg / ml. As used herein, the term "serum level" refers to the concentration of the substance in serum. In one embodiment, serum levels are provided for at least 7 days or at least 14 days. In one embodiment, the method includes administering at least 300 mg / m². 2 Antibody dosage, such as at least 600 mg / m² 2 And preferably up to 1500 mg / m² 2 At most 1200 mg / m 2 Or up to 1000 mg / m 2 .

[0200] In some embodiments, this disclosure provides a method of treating or preventing cancer, comprising administering to a patient an antibody capable of binding to GPC3, wherein the antibody is at a concentration of at least 300 mg / m².2 such as at least 600mg / m 2 And preferably up to 1500 mg / m² 2 At most 1200 mg / m 2 Or up to 1000 mg / m 2 The antibody was administered at the prescribed dosage.

[0201] In some embodiments, this disclosure provides a method for treating or preventing cancer, comprising administering an antibody capable of binding to GPC3 to a patient, wherein at least 50%, preferably 60%, 70%, 80%, or 90% of the patient's cancer cells are GPC3 positive and / or at least 40%, preferably 50%, or 60% of the patient's cancer cells are GPC3 surface-expressing positive. In this respect, this disclosure also provides a method for treating or preventing cancer, the method comprising: a. identifying a patient showing at least 50%, preferably 60%, 70%, 80%, or 90% of GPC3-positive cancer cells and / or at least 40%, preferably 50%, or 60% of cancer cells that are GPC3 surface-expressing positive; and b. administering an antibody capable of binding to GPC3 to the patient. In one embodiment, at least 95% or at least 98% of the patient's cancer cells are GPC3 positive. In one embodiment, at least 70%, at least 80%, or at least 90% of the patient's cancer cells are GPC3 surface-expressing positive.

[0202] In one embodiment of the methods described herein, the treatment outcome for cancer is the achievement of disease stabilization. In one embodiment, disease stabilization is achieved for at least 2 months, at least 3 months, or at least 6 months.

[0203] In some embodiments, this disclosure provides a method for achieving disease stabilization in cancer patients, which includes administering an antibody capable of binding to GPC3 to the patient. In one embodiment, disease stabilization is achieved for at least 2 months, at least 3 months, or at least 6 months.

[0204] In one embodiment of the method described herein, the antibody is administered in a single dose or multiple doses.

[0205] In some embodiments, this disclosure provides methods for treating or preventing cancerous diseases, including administering an antibody capable of binding to GPC3 to a patient, wherein the antibody is administered in multiple doses.

[0206] If the antibody is administered in multiple doses according to this disclosure, it is preferred to administer the antibody in at least 3, 4, 5, 6, 7, 8, 9, or 10 doses, and more preferably in up to 30, 25, 20, 15, or 10 doses. It is preferred to administer the antibody at intervals of at least 7, 10, 14, or 20 days. It is also preferred to administer the antibody at intervals of 7 to 30 days, 10 to 20 days, and most preferably about 14 days.

[0207] In one embodiment, the antibody is administered to provide a serum level of at least 40 μg / ml. In various embodiments, the antibody is administered to provide a serum level of at least 50 μg / ml, at least 150 μg / ml, at least 300 μg / ml, at least 400 μg / ml, or at least 500 μg / ml. In various embodiments, the antibody is administered to provide a serum level not exceeding 800 μg / ml, 700 μg / ml, 600 μg / ml, 550 μg / ml, or 500 μg / ml. In one embodiment, the provided serum level is from 40 μg / ml to 700 μg / ml, preferably from 40 μg / ml to 600 μg / ml, preferably from 50 μg / ml to 500 μg / ml, such as from 150 μg / ml to 500 μg / ml or from 300 μg / ml to 500 μg / ml. In one embodiment, the serum level is provided for at least 7 days or at least 14 days. In one embodiment, the method includes administering at least 300 mg / ml. 2 such as at least 600mg / m 2 And preferably up to 1500 mg / m² 2 Up to 1200mg / m 2 Or up to 1000 mg / m 2 The dosage of antibodies.

[0208] The use of any of the foregoing antibodies or their antigen-binding fragments or nucleic acid molecules or carriers or cells or pharmaceutical compositions in the preparation of a medicament for the treatment of GPC3-related diseases in mammals.

[0209] Optionally, according to any of the foregoing aspects, the antibody may be conjugated to other drugs, such as labeled or cytotoxic conjugates.

[0210] In one aspect, this disclosure also includes kits, such as those containing antibodies, fragments thereof, homologs thereof, derivatives thereof, etc., such as labeled or cytotoxic conjugates, as well as instructions for use of antibodies, conjugates that kill specific cell types, etc. These instructions may include guidance on the use of antibodies, conjugates, etc., in vitro, in vivo, or ex vivo. Antibodies may be in liquid or solid form, typically lyophilized. The kit may contain other suitable reagents, such as buffers, reconstitution solutions, and other necessary components for the intended use. Consideration is given to pre-packaged reagent combinations with instructions for their intended use, such as for therapeutic purposes or for diagnostic assays. When the antibody is labeled, such as enzyme-labeled, the kit may include substrates and cofactors required for the enzyme (e.g., providing substrate precursors for detecting chromophores or fluorophores). Furthermore, other additives, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), may also be included. The relative amounts of various reagents can be varied to provide concentrated reagent solutions, which provides user flexibility, space savings, and reagent savings. These reagents may also be provided in dry powder form, typically lyophilized, and include excipients that, when dissolved, provide a reagent solution of appropriate concentration.

[0211] The use of any of the foregoing antibodies or their functional fragments or nucleic acid molecules or vectors or cells or pharmaceutical compositions or kits in the preparation of reagents for inhibiting GPC3 binding.

[0212] Furthermore, the antibodies disclosed herein can also be used in immunoassays, purification methods, and other methods that utilize immunoglobulins or fragments thereof. Such uses are well known in the art.

[0213] Accordingly, this disclosure also provides compositions comprising an antibody or fragment thereof against GPC3 disclosed herein, wherein the antibody is conveniently combined with a pharmaceutically acceptable carrier, diluent or excipient, as is common practice in the art.

[0214] As used in this disclosure, the term "pharmaceutical composition" refers to a formulation of a variety of preparations. Formulations containing a therapeutically effective amount of a multivalent antibody are in the form of a sterile liquid solution, liquid suspension, or lyophilized form, optionally containing a stabilizer or excipient.

[0215] The antibodies disclosed herein can be used as a single-use composition or in combination with other active agents.

[0216] In some embodiments, the humanized antibody of this disclosure is conjugated to a therapeutic portion (i.e., a drug). The therapeutic portion may be, for example, a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulant, a cleaved peptide, or a radioisotope. Such conjugates are referred to herein as “antibody-drug conjugates” or “ADCs”.

[0217] In some implementations, the antibody is conjugated to the cytotoxic portion. The cytotoxic portion may be selected, for example, from the following: paclitaxel; cytochalasin B; bacitracin D; ethidium bromide; emetine; mitomycin; etoposide; teniposide; vincristine; vinblastine; colchicine; doxorubicin; daunorubicin; dihydroxyanthradinone; microtubule inhibitors such as maytansin or their analogues or derivatives; antimitotic agents such as monomethylolpropionate E or F or their analogues or derivatives; salivarius toxin 10 or 15 or... Its analogues; irinotecan or its analogues; mitoxantrone; sclerosomycin; actinomycin D; 1-dehydrotestosterone; glucocorticoids; procaine; tetracaine; lidocaine; propranolol; puromycin; chachomycin or its analogues or derivatives; antimetabolites such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, sebazine, hydroxyurea, asparaginase, gemcitabine or cladribine; alkyl Antibiotics such as dichloromethyldiethylamine, thiopurine, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C; platinum derivatives such as cisplatin or carboplatin; docamycin A, docamycin SA, resveratrol (CC-1065) or their analogues or derivatives; antibiotics such as actinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, chloramphenicol, mitomycin, mitoxantrone, praziamic acid, benzomycin (AMC); pyrrolo[2,1-c][1,4]-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 toxin, cholera toxin, shiga-like toxins such as SLT. I, SLT II, ​​SLT III, LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alarin, saponins, saccharin, gellingin, absinthecin A chain, saccharin A chain, α-sarcin, Aleurites fordii protein, caryophyllin protein, American pokeweed proteins such as PAPI, PAPII and PAP-S, momordica charantia inhibitor, lacrimalin, croton toxin, sapaonaria officinalis inhibitor, white tree toxin, mitomycin, localized aspergillin, phenolmycin and enoxamycin toxin; ribonuclease (RNase); DNase I, staphylococcal endotoxin A; pokeweed antiviral protein; diphtheria toxin and Pseudomonas endotoxin.

[0218] In some embodiments, the antibody is conjugated to oligristatin or its peptide analogs, derivatives, or prodrugs. Oligstatin has been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division, and to possess anticancer and antifungal activities. For example, oligristatin E can react with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEB, respectively. Other typical oligristatin derivatives include AFP, MMAF (monomethyl oligristatin F), and MMAE (monomethyl oligristatin E). Suitable olistatin and its analogues, derivatives and prodrugs, as well as suitable adapters for conjugating olistatin to Ab, are described, for example, in U.S. Patent Nos. 5,635,483, 5,780,588 and 6,214,345 and International Patent Application Publications WO02088172, WO2004010957, WO2005081711, WO2005084390, WO2006132670, WO03026577, WO200700860, WO207011968 and WO205082023.

[0219] In some implementations, the antibody is conjugated to pyrrolo[2,1-c][1,4]-benzodiazepine (PDB) or its peptide analogs, derivatives, or prodrugs. Suitable PDBs and 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 PW et al., Bioorg Med Chem Lett 2009; 19:6463-6466; and Sagnou et al., Bioorg Med Chem Lett 2000; 10(18):2083-2086.

[0220] In some implementations, the antibody is conjugated to a cytotoxic moiety selected from the following: anthracycline antibiotics, maytansine, chachomycosis, docamycosis, resveratrol (CC-1065), salipodoxomil 10, salipodoxomil 15, irinotecan, monomethylolpropionate E, monomethylolpropionate F, PDB, or any analogues, derivatives or prodrugs thereof.

[0221] In some embodiments, the antibody is conjugated with anthracycline antibiotics or their analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with maytansine or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with chachiomycin or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with docalomycin or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with resveratrol (CC-1065) or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with salinomycin 10 or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with salinomycin 15 or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with monomethylolpropionate E or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with monomethylolpropionate F or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with pyrrolo[2,1-c][1,4]-benzodiazepine or its analogues, derivatives, or prodrugs. In some implementations, the antibody is conjugated with irinotecan or its analogues, derivatives, or prodrugs.

[0222] In some implementations, the antibody is conjugated to cytokines such as 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, ansistatin, and TNFα.

[0223] In some embodiments, the antibody is conjugated to a radioisotope or a chelate containing a radioisotope. For example, the antibody may be conjugated to a chelating agent linker (e.g., DOTA, DTPA, or thiacetam) that allows the antibody to complex with the radioisotope. The antibody may also, or optionally, contain 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, radioactive isotopes that emit beta or alpha particle radiation, such as... 131 I, 90 Y、 211 At、212 Bi、 67 Cu、 186 Re、 188 Re and 212 Pb.

[0224] The technique of conjugating molecules to antibodies is well known in the art. Typically, nucleic acid molecules are covalently linked to lysine or cysteine ​​residues on antibodies via N-hydroxysuccinimide or maleimide functional groups, respectively. It has been reported that conjugation methods using engineered cysteine ​​residues or integrating non-natural amino acids can improve the homogeneity of conjugates. In particular, those skilled in the art can also anticipate generating reactive, endogenous glutamine-engineered Fc-containing peptides using tags containing acyl donor glutamine (e.g., tags containing Gin peptides or Q-tags) or through peptide engineering (e.g., through amino acid deletion, insertion, substitution, or mutation on the peptide). Transglutaminase can then be covalently crosslinked with an amine donor agent (e.g., a small molecule containing or linked to a reactive amine) to form a stable and homogeneous pool of engineered Fc-containing peptide conjugates, wherein the amine donor agent specifically conjugates the Fc-containing peptide via an acyl donor glutamine tag or an accessible / exposed / reactive endogenous glutamine site (WO2012059882).

[0225] It should be understood that the therapeutic agent according to the above-described embodiments will be administered together with a suitable pharmaceutically acceptable carrier, excipient, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, etc. A large number of suitable formulations can be found in all pharmacopoeias known to medicinal chemists: Remington's Pharmaceutical Sciences (15th edition, Mack Publishing Company, Easton, Pa. (1975)), particularly Chapter 87 of Blaug and Seymour. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, and lipid-containing (cationic or anionic) carriers (e.g., Lipofectin). TM ( ), DNA conjugates, anhydrous absorbents, oil-in-water and water-in-oil emulsions, emulsion polyethylene glycol (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycol. Any of the foregoing mixtures may be used for treatments or therapies according to this disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable for the route of administration.

[0226] In one implementation, the antibody may be used as a therapeutic agent. Such agents will typically be used to treat, alleviate, and / or prevent diseases or pathologies in subjects associated with abnormal GPC3 expression, activity, and / or signaling. Treatment protocols can be implemented using standard methods by identifying subjects, such as those with (or at risk of developing) diseases or disorders associated with abnormal GPC3 expression, activity, and / or signaling, such as GPC3-related disorders. The antibody preparation, preferably one with high specificity and high affinity for its target antigen, is administered to the subject and will typically produce an effect due to its binding to the target. The administered antibody may eliminate or inhibit or impede the expression, activity, and / or signaling function of the target (e.g., GPC3). The administered antibody may eliminate or inhibit or impede the binding of the target (e.g., GPC3) to its naturally bound endogenous ligand. For example, the antibody binds to the target and modulates, blocks, inhibits, reduces, antagonizes, neutralizes, and / or otherwise impedes GPC3 expression, activity, and / or signaling. In some implementations, antibodies with heavy and light chain CDRs may be administered to subjects to treat diseases or disorders associated with abnormal GPC3 expression.

[0227] In another embodiment, antibodies against GPC3 can be used in methods known in the art related to the localization and / or quantification of GPC3 (e.g., for determining the level of GPC3 in appropriate physiological samples, for diagnostic methods, for protein imaging, etc.). In a given embodiment, an antibody that is specific to GPC3 or its derivatives, fragments, analogs, or homologues and comprises an antigen-binding domain derived from the antibody is used as a pharmaceutically active compound (hereinafter referred to as a "therapeutic agent").

[0228] In another embodiment, the GPC3 peptide can be isolated using antibodies specific to GPC3 via standard techniques such as immunoaffinity, chromatography, or immunoprecipitation. Antibodies (or fragments thereof) targeting the GPC3 protein can be used to detect the protein in a biological sample. In some embodiments, the detection of GPC3 in a biological sample is part of a clinical testing process, for example, to determine the efficacy of a given treatment. Conjugating (i.e., physically linking) an antibody to a detectable substance can facilitate detection. Examples of 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 cofactor complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazine luciferin, dansyl chloride, or phycoerythrin; one example of a luminescent material is luminol; examples of bioluminescent materials include luciferase, luciferin, and jellyfish protein; and examples of suitable radioactive materials include...125 I, 131 I, 35 S or 3 H.

[0229] In another embodiment, the antibody according to this disclosure can be used as a reagent to detect the presence of GPC3 or a protein fragment thereof in a sample. In some embodiments, the antibody contains a detectable label. The antibody is a polyclonal antibody, or more preferably a monoclonal antibody. A complete antibody or a fragment thereof (e.g., Fab, scFv, or F(ab')2) is used. The term "label" with respect to the antibody is intended to include both direct labeling of the antibody by conjugation (i.e., physical linking) to the antibody and indirect labeling of the antibody by reaction with another directly labeled reagent. Examples of indirect labeling include detecting a first antibody using a fluorescently labeled second antibody, and end-labeling an antibody with biotin to enable 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 within the subject. Thus, the term "biological sample" as used includes blood and fractions or components of blood, including serum, plasma, or lymph. In other words, the detection methods of the above embodiments can be used to detect analytes mRNA, protein, or genomic DNA in biological samples in vitro and in vivo. For example, in vitro detection techniques for analyte mRNA include Norhtern hybridization and in situ hybridization. In vitro detection techniques for analyte proteins 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, JRCrowther (ed.), Human Press, Totowa, NJ, 1995; "Immunoassay," E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and "Practice and Theory of Enzyme Immunoassays," P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo detection techniques for analyte proteins involve introducing labeled anti-analyte protein antibodies into the subject. For example, antibodies can be labeled with radioactive markers, and then the presence and location of the radiolabel in the subject's body can be detected using standard imaging techniques.

[0230] The antibodies described herein and their derivatives, fragments, analogs, and homologues may be incorporated into pharmaceutical compositions suitable for administration. The principles and considerations involved in preparing such compositions, as well as guidelines for selecting components, are well known in the art.

[0231] Such compositions typically contain an antibody and a pharmaceutically acceptable carrier. When using antibody fragments, the minimally inhibitory fragment that specifically binds to the target protein binding domain is preferred. For example, peptide molecules that retain the ability to bind to the target protein sequence can be designed based on the variable region sequence of the antibody. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology (see, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)).

[0232] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delayers compatible with drug administration. Suitable pharmaceutically acceptable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, the standard bibliography in the art, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, glucose solution, and 5% human serum albumin. Liposomes and non-aqueous carriers, such as immobilized oils, may also be used. The use of such media and reagents for pharmaceutically active substances is well known in the art. The use of any conventional media or reagent in the composition is contemplated, except that it may be incompatible with the antibody.

[0233] The pharmaceutical compositions of the above embodiments are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., local), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: sterile diluents for injection such as water, saline solutions, fixative oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates; and osmotic pressure adjusting agents such as sodium chloride or dextran. pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0234] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (in this case, water-soluble) or dispersions, as well as sterile powders for immediate preparation of sterile injections or dispersions. For intravenous administration, suitable pharmaceutically acceptable carriers include physiological saline, antibacterial water, Cremophor EL... TM (BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be fluid enough for easy injection. It must be stable under manufacturing and storage conditions and must be resistant to contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin to maintain the desired particle size in the dispersion case, and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents in the composition, such as sugars, polyols (such as mannitol, sorbitol), and sodium chloride. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0235] As needed, a sterile injectable solution can be prepared by incorporating the antibody in the desired amount into a suitable solvent having one or a combination of the components listed above (as required), followed by filtration sterilization. Generally, a dispersion is prepared by incorporating the antibody into a sterile carrier containing an alkaline dispersion medium and any other desired components listed above. For sterile powders used to prepare sterile injectable solutions, the preparation method involves obtaining a powder containing the active ingredient and any other desired components derived from a sterile filtrate solution of the aforementioned components through vacuum drying and freeze-drying.

[0236] For inhalation administration, the compound is delivered in the form of an aerosol spray from a pressurized container or dispenser or nebulizer containing a suitable propellant such as carbon dioxide.

[0237] Systemic administration can also be achieved via mucosal or transdermal routes. For mucosal or transdermal administration, a permeabilizing agent suitable for the permeability barrier is used in the formulation. Such permeabilizing agents are generally known in the art and include detergents, bile salts, and fusidic acid derivatives, such as those used for mucosal administration. Mucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, one or more antibodies can be formulated into ointments, ointments, gels, or creams as generally known in the art.

[0238] The compound can also be prepared in the form of suppositories (e.g., having a conventional suppository base, such as cocoa butter or other glycerides) or retention enemas for rectal delivery.

[0239] In one embodiment, the antibody can be prepared using a carrier that prevents it from being rapidly eliminated by the body, such as a sustained-release / controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene-vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art.

[0240] Particularly advantageous is the formulation of parenteral compositions in unit dosage form for ease of administration and dosage consistency. As used herein, unit dosage form refers to physically separable units suitable as unit doses for use in the subject to be treated; each unit contains a predetermined amount of one or more antibodies calculated to bind with the desired drug carrier to produce the desired therapeutic effect. The specifications of the unit dosage form in the above embodiments are indicated by and directly depend on the unique characteristics of the antibody and the specific therapeutic effect to be achieved, and the inherent limitations in the field of formulation of such antibodies for the individual to be treated.

[0241] The pharmaceutical composition may be placed in a container, package, or dispenser together with the instructions for use.

[0242] The formulations described herein may also contain more than one antibody, depending on the specific condition to be treated, preferably those with complementary activities that do not negatively affect each other. Alternatively or in addition, the composition may, for example, contain agents that enhance its function, such as cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitors. Such molecules are appropriately combined in amounts effective for the intended purpose. For example, they may be combined in a kit or in use.

[0243] In one embodiment, one or more antibodies may be administered in combination therapy, i.e., in combination with other agents, such as therapeutic agents (which can be used to treat pathological conditions or disorders, such as various forms of cancer, autoimmune disorders, and inflammatory diseases). The term "combination" herein refers to the administration of the agents substantially synchronously, simultaneously, or sequentially. If administered sequentially, the first of the two compounds is still preferably detected at an effective concentration at the therapeutic site when the second compound is initiated. In one case, "combination" may also mean that the reagent kit simultaneously contains the antibodies of this disclosure and other therapeutic agents.

[0244] For example, combination therapy may comprise one or more antibodies described herein formulated and / or co-administered 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 growth inhibitors, as detailed below). Such combination therapies can advantageously utilize lower doses of the administered therapeutic agent, thus avoiding the potential toxicities or complications associated with various monotherapy approaches.

[0245] In one implementation, the treatment regimen effectively reduced the release of cytokines associated with the administration of the T-cell activating therapeutic agent in the subject compared to a corresponding treatment regimen without the administration of the anti-GPC3 antibody.

[0246] For the purpose of clarity and concise description, the features are described herein as part of some identical or separate embodiments; however, it will be understood that the scope of this disclosure may include some embodiments having a combination of all or some of the features described.

[0247] Example

[0248] Example 1: Antigen Expression and Construction of Stable Transfected Cells

[0249] Using human GPC3 (Sino Biological, HG10088) as a template, the extracellular region (25-563) of human GPC3 was amplified, transfected into HEK293E cells, and expressed to obtain recombinant human GPC3 protein (SEQ ID NO.1). Alternatively, the near-membrane end sequence (aa 524-563) was inserted into the EcoRI site of the pGEX3X vector using the Gibson assembly method. The plasmid was transfected into E. coli BL21(DE3), and expression and purification were performed by IPTG to obtain GST-human GPC3stem recombinant protein (SEQ ID NO.2). GST-cynomolgus monkey GPC3stem recombinant protein (SEQ ID NO.3) and GST-mouse GPC3stem recombinant protein (SEQ ID NO.4) were expressed using the same method. Figure 1 The results of non-reduced SDS-PAGE of the GPC3 recombinant protein are shown.

[0250] HEK293T cells were co-transfected with the Lenti vector containing the full-length sequence of human and rhesus monkey GPC3 and the packaging plasmid (Genecoepia). Forty-eight hours after transfection, the culture supernatant containing pseudoviruses was collected, and 10 μL was used to infect 1×10⁻⁶ cells. 6 CHO cells were subjected to puromycin resistance selection, and finally stable transgenic cells CHO-hGPC3 (clone 2B10) and CHO-cynoGPC3 (clone 4D6) expressing high levels of human or monkey GPC3 were isolated. Figure 2 The flow cytometry results of GPC3 stable cells are shown.

[0251] Constructing human or monkey CD3εγ heterodimers: Human CD3γ (UniProt P09693, Gln23-Asn116) and CD3ε (UniProt P07766, Gln23-Asp126) extracellular nucleotide sequences were synthesized, and their C-termini were fused with human IgG Fc holes or Fcknob, respectively, to form human CD3εγ-Fc heterodimers (the amino acid sequence of human CD3γIgG Fc(hole) is shown in SEQ ID NO.5, and the amino acid sequence of human CD3εIgG 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 their C-termini were fused with cynomolgus monkey IgG Fc holes or Fc... Knob fusion was performed to express and form cynomolgus CD3εγ-Fc heterodimers (the amino acid sequence of cynomolgus CD3γIgGFc(hole) is shown in SEQ ID NO.7, and the amino acid sequence of cynomolgus CD3γIgG Fc(knob) is shown in SEQ ID NO.8). 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-293CD03DPM). After culturing at 37℃ and 120 rpm in 5% CO2 for 7 days, the supernatant was collected and purified by Protein A affinity chromatography to obtain human or cynomolgus CD3εγ-Fc recombinant protein. The SDS-PAGE purity was higher than 95%. Figure 3 ).

[0252] Example 2: Animal immunization and preparation of GPC3 antibodies

[0253] 1. GPC3 antibody screening

[0254] Female Balb / C mice aged 4-6 weeks were selected. A transient expression plasmid was constructed using the full-length or near-membrane end sequence of the human GPC3 extracellular region (aa 524-563). Mice were immunized with DNA using a gene gun, with 20 μg of plasmid per mouse immunized weekly. Serum titers were measured after 8 immunizations. The mice were then transfected with CHO-hGPC3 stable cells (5 × 10⁻⁶ cells). 6Mice were subjected to shock immunization (cells / mouse). Three days later, mice were euthanized by cervical dislocation, and their spleens and peripheral lymph nodes were collected. Total RNA was extracted after grinding and centrifugation, and reverse transcription was performed followed by PCR amplification using light and heavy chain-specific primers to obtain cDNA libraries of the antibody's light and heavy chain variable regions. A GPC3 immunoreceptor library based on filamentous phage M13 was constructed using phage display technology for subsequent phage panning.

[0255] Using a classic panning method, recombinant human GPC3 protein was coated onto immunotubes, and a 1mL LGPC3 phage immunoglobulin library was added for incubation and panning. Phages displaying high affinity Fab were eluted and used to infect TG1 bacteria. Phages were then prepared again for the next round of panning. After two rounds of panning and screening, single clones were inoculated into 96-well U-shaped plates for IPTG-induced expression. The culture supernatant was collected for ELISA detection, yielding 323 positive clones recognizing the piscisal end of the extracellular region of human GPC3.

[0256] Selected clones were generated by sequencing. The light 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. These vectors were co-transfected into HEK293E cells and cultured at 37℃, 120 rpm, and 5% CO2 for 5-6 days. The supernatant was collected and purified using a Protein A chromatography column to obtain the IgG anti-GPC3 chimeric antibody. The control antibody GC33 was synthesized according to the literature by Nakano K et al. (2009) and expressed as a human IgG1 chimeric antibody (GC33 for short).

[0257] The variable region sequence of the GPC3 antibody is shown below:

[0258] Table 1. Variable region sequence of GPC3 murine antibody

[0259]

[0260]

[0261] 5D6

[0262] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.9, and the nucleic acid it encodes is shown in SEQ ID NO.10. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.11, 12 and 13, respectively.

[0263]

[0264] Nucleic acid sequence

[0265]

[0266] The amino acid sequence of the light chain VK is shown in SEQ ID NO.14, and its encoded nucleic acid is shown in SEQ ID NO.15. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.16, 17 and 18, respectively.

[0267]

[0268] Nucleic acid sequence

[0269]

[0270] 29A10

[0271] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.19, and its encoded nucleic acid is shown in SEQ ID NO.20. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.11, 21 and 13, respectively.

[0272]

[0273] Nucleic acid sequence

[0274]

[0275] The amino acid sequence of the light chain VK is shown in SEQ ID NO.22, and its encoded nucleic acid is shown in SEQ ID NO.23. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.16, 17 and 18, respectively.

[0276]

[0277] Nucleic acid sequence

[0278]

[0279] 34H1

[0280] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.9, and the nucleic acid it encodes is shown in SEQ ID NO.10. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.11, 12 and 13, respectively.

[0281]

[0282] Nucleic acid sequence

[0283]

[0284] The amino acid sequence of the light chain VK is shown in SEQ ID NO.24, and its encoded nucleic acid is shown in SEQ ID NO.25. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.16, 17 and 26, respectively.

[0285]

[0286] Nucleic acid sequence

[0287]

[0288] 19F1

[0289] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.9, and the nucleic acid it encodes is shown in SEQ ID NO.10. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.11, 12 and 13, respectively.

[0290]

[0291] Nucleic acid sequence

[0292]

[0293] The amino acid sequence of the light chain VK is shown in SEQ ID NO.27, and its encoded nucleic acid is shown in SEQ ID NO.28. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.16, 17 and 29, respectively.

[0294]

[0295] Nucleic acid sequence

[0296]

[0297] 30B7

[0298] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.30, and its encoded nucleic acid is shown in SEQ ID NO.31. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.32, 33 and 13, respectively.

[0299]

[0300] Nucleic acid sequence

[0301]

[0302] The amino acid sequence of the light chain VK is shown in SEQ ID NO.34, and its encoded nucleic acid is shown in SEQ ID NO.35. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.16, 17 and 18, respectively.

[0303]

[0304] Nucleic acid sequence

[0305]

[0306] 28G4

[0307] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.19, and its encoded nucleic acid is shown in SEQ ID NO.20. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.11, 21 and 13, respectively.

[0308]

[0309] Nucleic acid sequence

[0310]

[0311] The amino acid sequence of the light chain VK is shown in SEQ ID NO.36, and its encoded nucleic acid is shown in SEQ ID NO.37. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.38, 17 and 39, respectively.

[0312]

[0313] Nucleic acid sequence

[0314]

[0315] 30A4

[0316] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.40, and the nucleic acid it encodes is shown in SEQ ID NO.41. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.11, 21 and 13, respectively.

[0317]

[0318] Nucleic acid sequence

[0319]

[0320] The amino acid sequence of the light chain VK is shown in SEQ ID NO.42, and its encoded nucleic acid is shown in SEQ ID NO.43. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.16, 17 and 18, respectively.

[0321]

[0322] Nucleic acid sequence

[0323]

[0324] 38D2

[0325] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.9, and the nucleic acid it encodes is shown in SEQ ID NO.10. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.11, 12 and 13, respectively.

[0326]

[0327]

[0328] Nucleic acid sequence

[0329]

[0330] The amino acid sequence of the light chain VK is shown in SEQ ID NO.44, and its encoded nucleic acid is shown in SEQ ID NO.45. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.46, 47 and 48, respectively.

[0331]

[0332] Nucleic acid sequence

[0333]

[0334] 39G4

[0335] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.9, and the nucleic acid it encodes is shown in SEQ ID NO.10. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.11, 12 and 13, respectively.

[0336]

[0337] Nucleic acid sequence

[0338]

[0339] The amino acid sequence of the light chain VK is shown in SEQ ID NO.49, and its encoded nucleic acid is shown in SEQ ID NO.50. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.16, 17 and 51, respectively.

[0340]

[0341] Nucleic acid sequence

[0342]

[0343] 41F1

[0344] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.9, and the nucleic acid it encodes is shown in SEQ ID NO.10. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.11, 12 and 13, respectively.

[0345]

[0346] Nucleic acid sequence

[0347]

[0348] The amino acid sequence of the light chain VK is shown in SEQ ID NO.52, and its encoded nucleic acid is shown in SEQ ID NO.53. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.16, 17 and 54, respectively.

[0349]

[0350] Nucleic acid sequence

[0351]

[0352] 41H1

[0353] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.9, and the nucleic acid it encodes is shown in SEQ ID NO.10. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.11, 12 and 13, respectively.

[0354]

[0355] Nucleic acid sequence

[0356]

[0357] The amino acid sequence of the light chain VK is shown in SEQ ID NO.55, and its encoded nucleic acid is shown in SEQ ID NO.56. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.16, 17 and 57, respectively.

[0358]

[0359] Nucleic acid sequence

[0360]

[0361] 49G8

[0362] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.9, and the nucleic acid it encodes is shown in SEQ ID NO.10. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.11, 12 and 13, respectively.

[0363]

[0364] Nucleic acid sequence

[0365]

[0366] The amino acid sequence of the light chain VK is shown in SEQ ID NO.58, and its encoded nucleic acid is shown in SEQ ID NO.59. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.60, 17 and 61, respectively.

[0367]

[0368] Nucleic acid sequence

[0369]

[0370] 46C3

[0371] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.62, and its encoded nucleic acid is shown in SEQ ID NO.63. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.64, 65 and 66, respectively.

[0372] Nucleic acid sequence

[0373]

[0374] The amino acid sequence of the light chain VK is shown in SEQ ID NO.67, and its encoded nucleic acid is shown in SEQ ID NO.68. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.16, 17 and 69, respectively.

[0375]

[0376] Nucleic acid sequence

[0377]

[0378] 42A1

[0379] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.70, and its encoded nucleic acid is shown in SEQ ID NO.71. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.11, 72 and 73, respectively.

[0380]

[0381] Nucleic acid sequence

[0382]

[0383] The amino acid sequence of the light chain VK is shown in SEQ ID NO.74, and its encoded nucleic acid is shown in SEQ ID NO.75. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.16, 17 and 76, respectively.

[0384]

[0385] Nucleic acid sequence

[0386]

[0387] 47B6

[0388] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.77, and its encoded nucleic acid is shown in SEQ ID NO.78. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.79, 80 and 81, respectively.

[0389]

[0390] Nucleic acid sequence

[0391]

[0392] The amino acid sequence of the light chain VK is shown in SEQ ID NO.82, and its encoded nucleic acid is shown in SEQ ID NO.83. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.16, 17 and 69, respectively.

[0393]

[0394] Nucleic acid sequence

[0395]

[0396] The binding of the above-mentioned antibody to human GPC3 stable cells was detected using FACS. CHO-human GPC3 stable cells prepared in Example 1 during the logarithmic growth phase were taken and adjusted to 5 × 10⁶ cells / year with 4% fetal bovine serum (Hyclone, SH30626.06). 5 Cells / ml: Add 100 μl / well of cell suspension to a 96-well U-shaped plate, centrifuge at 300g for 5 minutes, discard the supernatant, add 100 μL of serially diluted antibody to each well (starting concentration 200 nM, 3-fold dilution, 12 gradients), and incubate at 4°C for 45 minutes. Add 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution), incubate on ice for 45 minutes, and analyze by flow cytometry. Figure 4 The results of flow cytometry analysis of chimeric antibodies and human GPC3-CHO stable cells are shown.

[0397] Using the BLI biomembrane interference technique (Octet), the antibody to be tested was captured on the Protein A probe, and its binding ability with human GPC3 protein (starting concentration 10 μg / ml, 2-fold gradient, 5 concentration points in total) was detected. The affinity constant KD was obtained based on the binding and dissociation rates.

[0398] Based on the combined results of FACS and Octet assays, 15 high-affinity positive monoclonal antibodies were identified that recognize the GPC3 receptor on the cell membrane and simultaneously bind to recombinant GPC3 protein. Table 2 shows the affinity constants of the monoclonal antibodies for human GPC3.

[0399] Table 2. Binding activity of monoclonal antibodies to human GPC3

[0400]

[0401] 2. Humanization of GPC3 candidate antibodies

[0402] 2.1 Cross-species reaction of candidate antibodies

[0403] The high-affinity positive clone 49G8 was selected to detect its binding activity with the full-length recombinant GPC3 protein from humans, cynomolgus monkeys, and mice, as well as the recombinant protein from the juxtamembrane end of the extracellular region of human GPC3. 1 μg / ml of the above protein was coated overnight at 4°C. After blocking, 49G8 chimeric antibody (starting at 200 nM, serially diluted 3-fold for a total of 12 concentrations) was added, and the reaction was incubated at room temperature for 1 hour. Secondary antibody was added with HPR-labeled goat anti-human IgG Fc. After color development with TMB solution, the reaction was terminated with concentrated sulfuric acid, and the absorbance was read at 450 nm. ELISA results are as follows: Figure 5 As shown, the 49G8 antibody can bind to the full-length human and cynomolgus monkey GPC3 protein and the near-membrane end of the extracellular region of human GPC3, with a slightly higher affinity than the control antibody, but a weak affinity for mouse GPC3 protein (Table 3).

[0404] Table 3. ELISA results of 49G8 and full-length GPC3 protein from different species, and human GPC3 juxtamembrane protein.

[0405]

[0406] 2.2 Antibody-dependent cell-mediated cytotoxicity (ADCC)

[0407] Using HepG2 human liver cancer cells expressing GPC3 as target cells, the target cell density was adjusted to 5 × 10⁻⁶. 4 Cells / well, modulated effector cells NK92MI-CD16a1×10 5 Cells / wells were added with serially diluted antibody (starting concentration 66.7 nM, 10-fold increments, for a total of 6 dilutions), and incubated at 37°C in a 5% CO2 incubator for 4 hours. The supernatant was then used to detect lactate dehydrogenase (LDH) release. Results are as follows: Figure 6 As shown, the 49G8 chimeric antibody can mediate ADCC activity against HepG2 target cells.

[0408] 2.3 Humanization of the 49G8 antibody

[0409] The variable region sequence of the murine antibody 49G8 was humanized through CDR transplantation and point mutation. The light chain of the mAb49G8 murine antibody was mouse IMGT_mVK1-110, with human IMGT_hVK2-28, which has the highest homology with its framework region, selected for CDR transplantation. FM4 was selected from human IGKJ4*01, which has the highest homology. The heavy chain of the murine antibody was IGHV1-15, with human germline gene IMGT_hVH1-69 selected for CDR transplantation. FM4 was selected from human IGHJ4*01, which has the highest homology. Reversal mutations were designed, resulting in the synthesis of six humanized variants of the heavy chain variable region and six humanized variants of the light chain variable region. After pairing, these variants were transiently expressed in HEK293E cells. After culturing at 37℃, 120 rpm, and 5% CO2 for 5-6 days, the supernatant was collected and purified using a Protein A chromatography column to obtain the anti-GPC3 humanized antibody.

[0410] The binding activity of the humanized 49G8 antibody to the full-length human and cynomolgus monkey GPC3 protein was determined using ELISA; the binding of the humanized 49G8 antibody to human GPC3-CHO stable transfected cells was detected using flow cytometry. ELISA results are shown below. Figure 7 As shown, the 49G8 humanized antibody can bind to human and cynomolgus monkey GCP3 recombinant protein with high affinity. FACS results are as follows... Figure 8 As shown, the affinity of the 49G8 humanized antibody for stable human GPC3-CHO cells is comparable to that of the pre-humanized mouse antibody, and slightly higher than that of the control antibody. The heavy / light chain variable region sequence of the humanized antibody is as follows:

[0411] Table 4. Light chain variable region sequence of humanized GPC3 antibody

[0412]

[0413] h49G8VKv1

[0414] The amino acid sequence of the light chain VK is shown in SEQ ID NO.84, and its encoded nucleic acid is shown in SEQ ID NO.85. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.60, 17 and 61, respectively.

[0415]

[0416] Nucleic acid sequence

[0417]

[0418] h49G8VKv2

[0419] The amino acid sequence of the light chain VK is shown in SEQ ID NO.86, and its encoded nucleic acid is shown in SEQ ID NO.87. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.60, 17 and 61, respectively.

[0420]

[0421] Nucleic acid sequence

[0422]

[0423] h49G8VKv3

[0424] The amino acid sequence of the light chain VK is shown in SEQ ID NO.88, and its encoded nucleic acid is shown in SEQ ID NO.89. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.60, 17 and 61, respectively.

[0425]

[0426] Nucleic acid sequence

[0427]

[0428] h49G8VKv4

[0429] The amino acid sequence of the light chain VK is shown in SEQ ID NO.90, and its encoded nucleic acid is shown in SEQ ID NO.91. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.92, 17 and 61, respectively.

[0430]

[0431] Nucleic acid sequence

[0432]

[0433] h49G8VKv5

[0434] The amino acid sequence of the light chain VK is shown in SEQ ID NO.93, and its encoded nucleic acid is shown in SEQ ID NO.94. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.95, 17 and 61, respectively.

[0435]

[0436]

[0437] Nucleic acid sequence

[0438]

[0439] h49G8VKv6

[0440] The amino acid sequence of the light chain VK is shown in SEQ ID NO.96, and its encoded nucleic acid is shown in SEQ ID NO.97. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.98, 17 and 61, respectively.

[0441]

[0442] Nucleic acid sequence

[0443]

[0444] Table 5. Heavy chain variable region sequence of humanized GPC3 antibody

[0445]

[0446] h49G8VHv1

[0447] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.99, and its encoded nucleic acid is shown in SEQ ID NO.100. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.11, 101 and 13, respectively.

[0448]

[0449] Nucleic acid sequence

[0450]

[0451] h49G8VHv2

[0452] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.102, and its encoded nucleic acid is shown in SEQ ID NO.103. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.11, 101 and 13, respectively.

[0453]

[0454] Nucleic acid sequence

[0455]

[0456] h49G8VHv3

[0457] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.104, and its encoded nucleic acid is shown in SEQ ID NO.105. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.11, 106 and 13, respectively.

[0458]

[0459] Nucleic acid sequence

[0460]

[0461] h49G8VHv4

[0462] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.107, and its encoded nucleic acid is shown in SEQ ID NO.108. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.11, 101 and 13, respectively.

[0463]

[0464] Nucleic acid sequence

[0465]

[0466] h49G8VHv5

[0467] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.109, and its encoded nucleic acid is shown in SEQ ID NO.110. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.11, 101 and 13, respectively.

[0468]

[0469] Nucleic acid sequence

[0470]

[0471] h49G8VHv6

[0472] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.111, and its encoded nucleic acid is shown in SEQ ID NO.112. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.11, 106 and 13, respectively.

[0473]

[0474] Nucleic acid sequence

[0475]

[0476] 2.3 Affinity of humanized antibodies

[0477] 1) Biolayer Interferometry (BLI)

[0478] Using the BLI (Biomembrane Interference) technique (Octet), the antibody to be tested was captured on a Protein A probe. The binding affinity to human GPC3 protein (starting concentration 10 μg / ml, 2-fold gradient, 5 concentration points in total) was detected. The affinity constant KD was obtained based on the binding and dissociation rates. Results are as follows: Figure 9 As shown, the 49G8 humanized antibody binds to the GPC3 recombinant protein with high affinity. Affinity data are shown in Table 6.

[0479] Table 6. Affinity analysis results of BLI between 49G8 humanized antibody and full-length human GPC3 protein.

[0480]

[0481] 2) Surface Plasmon Resonance (SPR)

[0482] Humanized antibodies with high affinity were selected to compare their binding affinity to human and cynomolgus monkey GPC3. The humanized antibody h49G8VHv6VKv3 was captured using a ProteinA microarray (GE Healthcare). Serially diluted human and cynomolgus monkey GPC3 recombinant proteins (5 μg / ml as the starting concentration, 2-fold gradient, 5 concentration points in total) were flowed through the microarray 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 the Langmuir 1:1 kinetics model using Biacore T200 evaluation software. Results are as follows: Figure 10 As shown, the humanized antibody h49G8VHv6VKv3 binds to human and cynomolgus monkey GPC3 with high affinity. The affinity for human GPC3 is 1.39 nM, and the affinity for cynomolgus monkey GPC3 is 2.10 nM, which are comparable. Affinity data are shown in Table 7.

[0483] Table 7. Affinity results of SPR analysis between candidate antibodies and full-length GPC3 protein from humans and cynomolgus monkeys.

[0484]

[0485] 2.4 Endocytosis of humanized antibodies

[0486] Take HepG2 cells in the logarithmic growth phase, wash the cells once with pre-warmed medium, centrifuge at 1000 rpm for 3 minutes, discard the supernatant, and resuspend the cells in serum-free medium to 1×10⁻⁶. 6 Cells / mL; Pipettes 200 μL of cells into each EP tube, adds 500 μL of pre-chilled 0.5% BSA / PBS, centrifuges at 1000 rpm for 3 minutes, discards the supernatant, adds 10 μg / mL GPC3 antibody, and incubates at 4℃ or 37℃ for 60 min, 120 min, 240 min, 120 min, 180 min, and 240 min, respectively. After primary antibody incubation at each time point, washes twice with 500 μL of pre-chilled 0.5% BSA / PBS, then directly adds 1:300 diluted secondary antibody AF647-labeled goat anti-human IgG Fc and incubates for 45 minutes. After secondary antibody incubation, washes twice with 500 μL of 0.5% BSA / PBS, resuspends the cells in 200 μL for FACS detection. Flow cytometry results are as follows. Figure 11 As shown, no significant endocytosis was observed in the GPC3 humanized antibody under experimental conditions of 37°C and 4°C.

[0487] Example 3: Construction of GPC3×CD3 bispecific antibody

[0488] 1. Construction of CD3 humanized antibody

[0489] Murine hybridoma CD3 antibodies (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) recognize human and monkey CD3 receptors, and their sequences are as follows:

[0490] The light chain amino acid sequence of the anti-CD3 mouse monoclonal antibody is shown in SEQ ID NO.174:

[0491]

[0492] The heavy chain amino acid sequence of the anti-CD3 mouse monoclonal antibody is shown in SEQ ID NO.175:

[0493]

[0494] The anti-CD3 mouse monoclonal antibody was humanized. The human germline gene IMGT_hVL7-43, with the highest homology, was selected for light chain CDR transplantation, and human IGLJ3*02 was used for FM4. Human IMGT_hVH3-73 was selected for heavy chain CDR transplantation, and human IGHJ4*01 was used for FM4. Different heavy chain and light chain variants were obtained (Tables 8 and 9).

[0495] Table 8. Light chain variable region sequence of CD3 humanized antibody

[0496]

[0497] The amino acid sequence of hVL1 is shown in SEQ ID NO.113, the encoded nucleic acid is shown in SEQ ID NO.114, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.115, 116 and 117, respectively.

[0498]

[0499] Nucleic acid sequence

[0500]

[0501] The amino acid sequence of hVL2 is shown in SEQ ID NO.118, the encoded nucleic acid is shown in SEQ ID NO.119, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.115, 116 and 117, respectively.

[0502]

[0503] Nucleic acid sequence

[0504]

[0505] The amino acid sequence of hVL3 is shown in SEQ ID NO.120, the encoded nucleic acid is shown in SEQ ID NO.121, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.122, 123 and 117, respectively.

[0506]

[0507] Nucleic acid sequence

[0508]

[0509]

[0510] The amino acid sequence of hVL4 is shown in SEQ ID NO.124, its encoded nucleic acid is shown in SEQ ID NO.125, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.122, 123 and 117, respectively.

[0511]

[0512] Nucleic acid sequence

[0513]

[0514] The amino acid sequence of hVL5 is shown in SEQ ID NO.126, its encoded nucleic acid is shown in SEQ ID NO.127, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.115, 116 and 128, respectively.

[0515]

[0516] Nucleic acid sequence

[0517]

[0518] The amino acid sequence of hVL6 is shown in SEQ ID NO.129, its encoded nucleic acid is shown in SEQ ID NO.130, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.115, 131 and 128, respectively.

[0519]

[0520] Nucleic acid sequence

[0521]

[0522] Table 9. Heavy chain variable region sequence of CD3 humanized antibody

[0523]

[0524] The amino acid sequence of hVH1 is shown in SEQ ID NO.132, the encoded nucleic acid is shown in SEQ ID NO.133, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.134, 135 and 136, respectively.

[0525]

[0526] Nucleic acid sequence

[0527]

[0528] The amino acid sequence of hVH2 is shown in SEQ ID NO.137, the encoded nucleic acid is shown in SEQ ID NO.138, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.139, 135 and 136, respectively.

[0529]

[0530] Nucleic acid sequence

[0531]

[0532] The amino acid sequence of hVH3 is shown in SEQ ID NO.140, the encoded nucleic acid is shown in SEQ ID NO.141, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.139, 135 and 142, respectively.

[0533]

[0534] Nucleic acid sequence

[0535]

[0536] The amino acid sequence of hVH4 is shown in SEQ ID NO.143, and its encoded nucleic acid is shown in SEQ ID NO.144. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.139, 135 and 145, respectively.

[0537]

[0538] Nucleic acid sequence

[0539]

[0540] The amino acid sequence of hVH5 is shown in SEQ ID NO.146, the encoded nucleic acid is shown in SEQ ID NO.147, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.139, 135 and 148, respectively.

[0541]

[0542] Nucleic acid sequence

[0543]

[0544] The amino acid sequence of hVH6 is shown in SEQ ID NO.149, and its encoded nucleic acid is shown in SEQ ID NO.150. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.139, 135 and 151, respectively.

[0545]

[0546] Nucleic acid sequence

[0547]

[0548] The amino acid sequence of hVH7 is shown in SEQ ID NO.152, its encoded nucleic acid is shown in SEQ ID NO.153, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.154, 155 and 136, respectively.

[0549]

[0550] Nucleic acid sequence

[0551]

[0552] The amino acid sequence of hVH8 is shown in SEQ ID NO.156, its encoded nucleic acid is shown in SEQ ID NO.157, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.134, 135 and 136, respectively.

[0553]

[0554] Nucleic acid sequence

[0555]

[0556]

[0557] The amino acid sequence of hVH9 is shown in SEQ ID NO.158, its encoded nucleic acid is shown in SEQ ID NO.159, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.134, 135 and 136, respectively.

[0558]

[0559] Nucleic acid sequence

[0560]

[0561] The amino acid sequence of hVH10 is shown in SEQ ID NO.160, the encoded nucleic acid is shown in SEQ ID NO.161, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.134, 135 and 136, respectively.

[0562]

[0563] Nucleic acid sequence

[0564]

[0565] After synthesizing the full sequences of the humanized light and heavy chain variants, they were cloned into eukaryotic expression vectors containing the constant region of the antibody lambda light chain or the constant region CH1-CH3 of the human IgG4 heavy chain. These vectors were then co-transfected into HEK293E cells and cultured at 37°C, 120 rpm, and 5% CO2 for 5-6 days. The supernatant was then collected and purified using a Protein A chromatography column.

[0566] 2. Affinity of CD3 humanized antibodies

[0567] Human CD3εγ protein was coated and incubated overnight at 4°C. After blocking with 2% skim milk, CD3 antibody at different dilutions was added to each well and incubated for 1 hour. HPR-labeled goat anti-human IgG Fc was added to the secondary antibody, and after development with TMB solution, the reaction was terminated with concentrated sulfuric acid, and the absorbance was read at 450 nm (results are shown in the figure). Figure 12 (As shown in Table 8). Figure 12 The combination of anti-CD3 humanized antibodies (including aCD3-hVH8 / VL1, aCD3-hVH9 / VL1, aCD3-hVH9 / VL2, aCD3-hVH9 / VL3, aCD3-hVH1 / VL5, aCD3-hVH8 / VL5, and aCD3-hVH9 / VL5) shows binding to human CD3εγ protein. The CD3 humanized antibodies bind to the recombinant CD3εγ protein with high affinity.

[0568] 3. CD3 humanized antibody recognizes Jurkat T cells

[0569] Jurkat cells in the logarithmic growth phase were blocked with 3% BSA for 30 minutes, and then seeded at a density of 5 × 10⁶ cells per well. 4 Cells were added to 96-well U-plates, centrifuged, and the supernatant was discarded. 50 μL of serially diluted antibody (antibody concentration starting at 30 μg / mL, 5 three-fold dilutions) was added to each well, and the plates were incubated at 4°C for 1 hour. After washing away the primary antibody, secondary antibody was added to 1:300 diluted Alexa Fluro647-labeled goat anti-human IgG Fc (Jackson ImmunoResearch, 109-606-170), and the plates were incubated at 4°C for 45 minutes. After washing, each well was resuspended in 50 μL of PBS for FACS (iQue, Intellicyt) detection (results shown in the figure). Figure 13 (As shown in Table 10).

[0570] Table 10. Affinity of CD3 humanized antibodies

[0571] ELISA (CD3εγ) FACS Jurkat aCD3-hVH1 / VL5 0.60nM 10nM aCD3-hVH8 / VL1 0.65nM 15nM aCD3-hVH8 / VL5 0.74nM Weak aCD3-hVH9 / VL1 0.49nM 7nM aCD3-hVH9 / VL2 0.44nM 25nM aCD3-hVH9 / VL3 0.70nM weak aCD3-hVH9 / VL5 0.42nM 20nM OKT3-hIgG1 ND 0.27nM KLH-hIgG4 - -

[0572] ND: Not detected

[0573] -: Not combined

[0574] Figure 13 The combination of anti-CD3 humanized antibodies showed binding to Jurkat cells, with the CD3 humanized antibodies hVH9 / VL5 (aCD3-hVH9 / VL5) and hVH9 / VL2 (aCD3-hVH9 / VL2) both significantly weaker than the control antibody OKT3, binding to Jurkat cells with moderate affinity.

[0575] 4. Cross-recognition between humanized CD3 antibodies and human and monkey CD3εγ antigens

[0576] Human CD3εγ protein and monkey CD3εγ protein were coated separately and incubated overnight at 4°C. After blocking with 2% skim milk, CD3 antibody of different dilutions was added to each well and incubated for 1 hour. HPR-labeled goat anti-human IgG Fc was added as secondary antibody, and after color development with TMB solution, the reaction was terminated with concentrated sulfuric acid and the absorbance was read at 450 nm. Figure 14 The humanized CD3 antibodies hVH9 / VL5 (aCD3-hVH9 / VL5) and hVH9 / VL2 (aCD3-hVH9 / VL2) can both bind to human CD3εγ and monkey CD3εγ proteins simultaneously.

[0577] 5. Construction of GPC3×CD3κλ humanized bispecific antibody

[0578] A novel humanized bispecific antibody with a native IgG conformation, GPC3-CD3κλ, was constructed by combining humanized antibody 49G8VHv6VKv3 with humanized CD3 antibody hVH9 / VL5 containing the λ light chain (aCD3-hVH9 / VL5). Simultaneously, a charge variant (Vκλ) was introduced into both the GPC3 antigen arm and the CD3 arm. GPC3 :Gln 43 Lys;VH GPC3 :Gln 39 Glu; Vλ CD3 :Gln 40 Glu; VH CD3 :Gln 39 (Lys)(Table 11). The Fc region of the bispecific antibody adopts the human IgG4knob-into-hole structure to achieve heterodimer pairing (Atwell et al., 1997), and the hinge region is stabilized by mutations in Ser228Pro, Leu235Glu and Pro329Ala, while completely eliminating the interaction with the Fcγ receptor and C1q.

[0579] Plasmids encoding the corresponding antibody fragments were mixed at a ratio of κ light chain:λ light chain:heavy chain 1:heavy chain 2 = 2:2:1:1, and then mixed with 3 mg / mL PEI. This mixture was then co-transfected into CHO-S cells and cultured in 500 mL CD CHO AGT medium (Gibco#12490-001) at 37°C with 5% CO2 at 150 rpm. On days 2, 4, and 6 after transient transfection, 4% CHO Feed C+ (Gibco#A25031-05) was added. When cell viability dropped to approximately 85%, the fermentation broth was harvested, filtered, and preliminarily purified by Protein A affinity chromatography. SEC-HPLC showed a monomer content higher than 92%. Further purification using Butyl HP hydrophobic chromatography and Capto Q anion exchange chromatography further increased the monomer content to over 99.5% (Table 12).

[0580] Table 11. Humanized Bispecific Antibody for GPC3-CD3κλ

[0581] GPC3 Arm Light Chain GPC3 arm heavy chain CD3 Arm Light Chain CD3 Arm Heavy Chain GPC3×CD3κλ002 SEQ ID NO.162 SEQ ID NO.164 SEQ ID NO.166 SEQ ID NO.168 GPC3×CD3κλ003 SEQ ID NO.170 SEQ ID NO.172 SEQ ID NO.166 SEQ ID NO.168

[0582] GPC3×CD3κλ002:

[0583] GPC3 arm light chain SEQ ID NO.162

[0584]

[0585] Nucleotide sequence SEQ ID NO.163

[0586]

[0587] GPC3 arm heavy chain SEQ ID NO.164

[0588]

[0589] Nucleotide sequence SEQ ID NO.165

[0590]

[0591] CD3 arm light chain SEQ ID NO.166

[0592]

[0593] Nucleotide sequence SEQ ID NO.167

[0594]

[0595]

[0596] CD3 arm heavy chain SEQ ID NO.168

[0597]

[0598] Nucleotide sequence SEQ ID NO.169

[0599]

[0600] GPC3×CD3κλ003:

[0601] GPC3 arm light chain SEQ ID NO.170

[0602]

[0603] Nucleotide sequence SEQ ID NO.171

[0604]

[0605] GPC3 arm heavy chain SEQ ID NO.172

[0606]

[0607] Nucleotide sequence SEQ ID NO.173

[0608]

[0609]

[0610] CD3 arm light chain SEQ ID NO.166

[0611]

[0612] Nucleotide sequence SEQ ID NO.167

[0613]

[0614] CD3 arm heavy chain SEQ ID NO.168

[0615]

[0616] Nucleotide sequence SEQ ID NO.169

[0617]

[0618] Table 12. Purification of GPC3-CD3 humanized bispecific antibody

[0619]

[0620] Example 4: Binding activity of GPC3×CD3κλ bispecific antibody

[0621] The affinity of the bispecific antibody GPC3 antigen arm was determined by detecting its binding to recombinant GPC3 protein, overexpressing stable cells, or GPC3+ tumor cells, respectively. The affinity of the bispecific antibody CD3 arm was determined by detecting its binding to Jurkat cells.

[0622] 1. Affinity determination of GPC3×CD3κλ bispecific antibody with GPC3

[0623] Antibody at a concentration of 1 μg / ml was captured in a Protein A chip (GE Healthcare). Different concentrations of human or cynomolgus GPC3 recombinant protein (5 μg / ml as the starting concentration, 2-fold gradient, 6 concentration points in total) were used as analytes and flowed through the chip at a flow rate of 30 μL / min. The binding time was 120 seconds and the dissociation time was 400 seconds. By recording the changes in SPR signal during protein binding and dissociation, the kinetic constants were obtained by fitting a 1:1 binding model using Biacore T200 evaluation software.

[0624] 10 μg / mL of human or monkey CD3εγ recombinant antigen was conjugated to a CM5 chip (GE Healthcare) via amino-coupling, controlling the antigen binding amount to approximately 200 RU. After baseline stabilization, serially diluted antibodies (starting from 10 μg / mL, with 7 two-fold dilutions) were flowed through the chip at a flow rate of 30 μL / min, with a binding time of 350 seconds and a dissociation time of 600 seconds. Kinetic constants were obtained by fitting a 1:1 binding model using Biacore T200 evaluation software. Affinity assay results are shown in Table 13.

[0625] Table 13. Affinity of GPC3×CD3κλ bispecific antibody to GPC3 and CD3 recombinant antigens

[0626] KD(nM) Human GPC3 Monkey GPC3 Human CD3εγ Monkey CD3εγ GPC3×CD3κλ002 1.35 1.60 15.9 23.9 GPC3×CD3κλ003 2.11 1.91 12.1 17.8

[0627] 2. Binding of GPC3×CD3κλ bispecific antibody to GPC3 stable cells

[0628] Log-phase CHO-human GPC3 and CHO-cynomolgus monkey GPC3 cells were stably transfected and adjusted to 5 × 10⁶ cells / year with 4% fetal bovine serum (Hyclone, SH30626.06). 5Cells / ml: Add 100 μl / well of cell suspension to a 96-well U-shaped plate, centrifuge at 300g for 5 minutes, discard the supernatant, add 100 μL of serially diluted antibody to each well (starting concentration 1800 nM, 3-fold dilution, 10 gradients), and incubate at 4°C for 60 minutes. Add 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution), incubate on ice for 20 minutes, wash once, add 50 μL / well of propidium iodide solution (1:300), incubate for 5 minutes, and analyze by flow cytometry. Figure 15 As shown in Table 14, the GPC3×CD3κλ bispecific antibody binds to the GPC3 receptor in cells with high affinity, and the affinity on monkey GPC3-stable cells is comparable to that on human GPC3-stable cells.

[0629] Table 14. Binding of GPC3×CD3κλ bispecific antibody to stable cells

[0630] <![CDATA[EC 50 (nM)]]> human GPC3-CHO monkeyGPC3-CHO GPC3×CD3κλ002 2.8 3.0 GPC3×CD3κλ003 2.9 3.0 KLH×CD3 - -

[0631] 3. Binding of GPC3×CD3κλ bispecific antibody to GPC3+ tumor cells

[0632] HepG2 cells in logarithmic growth phase were taken, and 200 μg / mL mouse IgG (Jackson ImmunoResearch, 115-005-03) was added and the cells were blocked on ice for 30 minutes. The cell volume was then adjusted to 5 × 10⁶ cells / mL with 4% fetal bovine serum. 5 Cells / mL, 100 μL per well of a 96-well U-shaped plate, centrifuged at 300g for 5 minutes, supernatant discarded, 100 μL of serially diluted antibody (starting concentration 1800 nM, 3-fold dilution, 10 gradients) added to each well, incubated at 4°C for 60 minutes. After washing away the primary antibody, 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) was added, incubated on ice for 20 minutes, washed once, and then 50 μL / well of PI was added, incubated for 5 minutes, and analyzed by flow cytometry. Results are shown below. Figure 16 According to Table 15, the GPC3×CD3κλ bispecific antibody binds to GPC3+ tumor cells HepG2 with high affinity.

[0633] Table 15. Binding of GPC3×CD3κλ bispecific antibody to GPC3+ tumor cells

[0634] <![CDATA[EC 50 (nM)]]> HepG2 GPC3×CD3κλ002 2.1 GPC3×CD3κλ003 2.5 KLH×CD3 -

[0635] 4. Binding of GPC3×CD3κλ bispecific antibody to Jurkat cells

[0636] Jurkat cells in logarithmic growth phase were taken and 200 μg / mL mouse IgG (Jackson ImmunoResearch, 115-005-03) was added. The cells were then incubated on ice for 30 minutes. The cell volume was adjusted to 5 × 10⁶ cells / mL with 4% fetal bovine serum. 5 Cells / mL, 100 μL per well of a 96-well U-shaped plate, centrifuged at 300g to remove supernatant, 100 μL of serially diluted antibody (starting concentration 1800 nM, 3-fold dilution, 10 gradients) per well, incubated at 4°C for 60 min. Secondary antibody was added to 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution), incubated on ice for 20 min, washed once, and then 50 μL / well of PI was added, incubated for 5 min, and analyzed by flow cytometry (BD C6). Detection results are as follows. Figure 17 According to Table 16, the GPC3×CD3κλ bispecific antibody binds with intermediate affinity to the human leukemia T cell line Jurkat cells, EC... 50 Approximately 20-40 nM.

[0637] Table 16. Binding of GPC3×CD3κλ bispecific antibody to Jurkat cells

[0638] <![CDATA[EC 50 (nM)]]> Jurkat GPC3×CD3κλ002 36 GPC3×CD3κλ003 26 KLH×CD3 27

[0639] 5. Binding of GPC3×CD3κλ bispecific antibody to peripheral blood T cells

[0640] Fresh human peripheral blood was collected, and PBMCs were isolated using Ficoll-Paque Plus (GE, 17-1440-03). The PBMCs were adjusted to 5 × 10⁶ cells / mL with 4% fetal bovine serum (Hyclone, SH30626.06). 5 Cells / mL, 100 μL / well added to a 96-well U-shaped plate, centrifuged and supernatant discarded, 100 μL of serially diluted antibody (starting concentration 1800 nM, 3-fold dilution, 11 gradients) added to each well, incubated at 4°C for 60 min. Secondary antibody added 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution), incubated on ice for 20 min, washed once, then 50 μL / well of PI added, incubated for 5 min, and detected by flow cytometry (BD Celesta). Detection results are shown below. Figure 18 As shown, the GPC3×CD3κλ bispecific antibody binds to human peripheral blood T cells with low affinity.

[0641] Table 17. Binding of GPC3×CD3κλ bispecific antibody to peripheral blood T cells

[0642] <![CDATA[EC 50 (nM)]]> CD4+ T cells CD8+T cell GPC3×CD3κλ002 65 65 GPC3×CD3κλ003 56 69 KLH×CD3 77 84

[0643] Example 5: GPC3×CD3κλ bispecific antibody-mediated TDCC action

[0644] Freshly isolated PBMCs were mixed with HepG2 target cells in logarithmic growth phase at an effector / target cell ratio of 10:1. 50 μL of serially diluted antibody (starting from 66.7 nM, 10-fold dilution, 7 gradients) was added to each well, and the cells were incubated at 37°C with 5% CO2 for 24 hours. After incubation, 50 μL of supernatant was transferred to a new black ELISA plate, and 50 μL / well of LDH detection substrate was added. The reaction was stopped after 10 minutes, and LDH release was detected. The remaining cells in the wells were washed twice with 4% fetal bovine blood, incubated with 100 μg / mL human IgG for 10 minutes, and then T cell activation detection antibodies (CD25-BV421, CD4-FITC, CD69-BV605, and CD8-APC) were added. The cells were incubated on ice for 20 minutes. After washing and discarding the supernatant, 60 μL / well of PI was added, and the cells were incubated on ice for 5 minutes. Flow cytometry was then used for detection. Figure 19 shows that the maximum killing rate of the GPC3×CD3κλ bispecific antibody against HepG2 cells was slightly higher or equivalent to that of the control antibody in the in vitro activity study. Figure 19A However, the changes in CD69 (early activation) and CD25 (late activation) of T cells during TDCC both suggest that the GPC3×CD3κλ bispecific antibody is milder in activating T cells than the control antibody.

[0645] Example 6: Activation of T cell activation pathway by GPC3×CD3κλ bispecific antibody

[0646] Take target cells (CHO-human GPC3) in the logarithmic growth phase, centrifuge, discard the supernatant, and resuspend to 2×10⁻⁶. 5 Cells / ml. Seed 50 μL / well of target cells into 96-well plates and incubate overnight at 37°C with 5% CO2. Centrifuge Jurkat-NFAT-luc reporter cells in logarithmic growth phase at 300g for 5 minutes, discard the supernatant, and resuspend to 4×10⁻⁶ cells / ml. 6 Cells / ml, remove the supernatant from the 96-well plate, and seed 25 μL / well of Jurkat-NFAT-luc reporter cells into each well. Add 25 μL of serially diluted GPC3×CD3κλ bispecific antibody or control antibody KLH×CD3 (starting concentration 20 μg / ml, 3-fold dilution, 10 gradients) to each well. Incubate at 37°C for 6 hours with 5% CO2. After incubation, add 100 μL of detection reagent to each well according to the ONE-Glo Luciferase Assay System instructions and analyze using a microplate reader (MDSpectraMax i3x). Results are shown below. Figure 20When the GPC3×CD3κλ bispecific antibody targets CHO-hGPC3 cells, it can activate the NFAT signaling pathway in T cells, with a slightly lower activation level than the control antibody.

[0647] Example 7: Non-specific activation of PBMCs by GPC3×CD3κλ bispecific antibody

[0648] Freshly isolated PBMCs were incubated at 37°C (5% CO2) for 24 hours with 100 μL of antibody (10 μg / mL). Cells were washed twice with 4% fetal bovine blood, then incubated with 100 μg / mL human IgG for 10 minutes. T-cell activation detection antibodies (CD25-BV421, CD4-FITC, CD69-BV605, and CD8-APC) were added, and the cells were incubated on ice for 20 minutes. The supernatant was discarded after washing, and 60 μL of PI (pill-in-place) solution was added to each well. The cells were incubated on ice for 5 minutes and then analyzed by flow cytometry. Results are shown below. Figure 21 In the absence of target cells, the GPC3×CD3κλ bispecific antibody had no activating effect on peripheral blood T cells, which was comparable to the negative control KLH×CD3.

[0649] Example 8: Binding of GPC3×CD3κλ bispecific antibody to Fc receptor

[0650] A 50 μg / ml His-Tag antibody was amino-conjugated to a CM5 chip to capture His-tagged recombinant proteins FcγRI, FcγRIIAH131, and FcγRIIIAV158 (Sino Biological, #10256-H08H / 10374-H08H1 / 10389-H08H1) at a capture time of 40 seconds and a flow rate of 10 μL / min. After baseline stabilization, serially diluted antibody (initial concentration 37.5 μg / mL, 2-fold dilution) was flowed through the chip at a flow rate of 30 μL / min for a binding time of 120 seconds and a dissociation time of 200 seconds. Affinity constants were obtained using Biacore evaluation software. Figure 22 It can be seen that the GPC3×CD3κλ bispecific antibody did not bind to FcγRI, FcγRIIAH131 and FcγRIIIAV158; the wild-type IgG4 control antibody bound to FcγRI with strong affinity and had weak binding to FcγRIIAH131.

[0651] Example 9: Immunoreconstituted mouse subcutaneous HepG2 xenograft model

[0652] Female B-NGD mice (6-8 weeks old, Biocytogen Biosciences Co., Ltd.) were selected and subcutaneously inoculated with HepG2 cells (7×10⁶ cells per 10 ... 6 / each), until the tumor grows to 60-100mm3 Mice were randomly assigned to four groups: a treatment group (3.0 mg / kg), a treatment group (1.0 mg / kg), a treatment group (0.3 mg / kg), and a negative control group (KLH×CD3 3 mg / kg). Each mouse was injected via tail vein with 1×10 mg / kg of KLH×CD3. 7 Three days after the administration of PBMC cells, mice were given the first dose, followed by two doses every five days. Tumor volume and body weight were monitored. Mice were euthanized by cervical dislocation at the end of the experiment, and tumors were collected, weighed, and recorded. Results are shown below. Figure 23 The in vivo efficacy of the GPC3×CD3κλ bispecific antibody showed a dose-related relationship, with tumor inhibition rates (from low to high doses) of 76.7%, 81.3%, and 95.9%, respectively. Tumor-bearing mice tolerated the above doses well, with no adverse reactions such as weight loss.

[0653] Example 10: CD3 humanized mouse Hepa1-6 / hGPC3 xenograft model

[0654] Six-week-old female C57 / BL6-hCD3 mice (Biocytok Biotechnology Co., Ltd.) were selected and Hepa1-6 / hGPC3 (6×10⁻⁶) were introduced. 6 (Each mouse) was subcutaneously inoculated until the tumor volume reached 60-100 mm². 3 Mice were randomly assigned to groups: a treatment group (10 mg / kg), a treatment group (3 mg / kg), a treatment group (1 mg / kg), and a negative control group (KLH×CD3 10 mg / kg). Dosing was administered every 3 days for a total of 3 doses. Tumor volume and body weight were monitored. Mice were euthanized by cervical dislocation at the end of the experiment, and tumors were collected, weighed, and recorded. Results are shown below. Figure 24 In a CD3 humanized mouse model, the GPC3×CD3κλ bispecific antibody significantly inhibited the growth of Hepa1-6 / hGPC3 tumors and reduced tumor volume. sequence list <110> Connoya Biomedical Technology (Chengdu) Co., Ltd. <120> Development and application of a novel tumor connector therapy drug <130> MTP20181 <160> 175 <170> PatentIn version 3.5 <210> 1 <211> 552 <212> PRT <213> Homo sapiens <400> 1 Gln Pro Pro Pro Pro Pro Pro Asp Ala Thr Cys His Gln Val Arg Ser 1 5 10 15 Phe Phe Gln Arg Leu Gln Pro Gly Leu Lys Trp Val Pro Glu Thr Pro 20 25 30 Val Pro Gly Ser Asp Leu Gln Val Cys Leu Pro Lys Gly Pro Thr Cys 35 40 45 Cys Ser Arg Lys Met Glu Glu Lys Tyr Gln Leu Thr Ala Arg Leu Asn 50 55 60 Met Glu Gln Leu Leu Gln Ser Ala Ser Met Glu Leu Lys Phe Leu Ile 65 70 75 80 Ile Gln Asn Ala Ala Val Phe Gln Glu Ala Phe Glu Ile Val Val Arg 85 90 95 His Ala Lys Asn Tyr Thr Asn Ala Met Phe Lys Asn Asn Tyr Pro Ser 100 105 110 Leu Thr Pro Gln Ala Phe Glu Phe Val Gly Glu Phe Phe Thr Asp Val 115 120 125 Ser Leu Tyr Ile Leu Gly Ser Asp Ile Asn Val Asp Asp Met Val Asn 130 135 140 Glu Leu Phe Asp Ser Leu Phe Pro Val Ile Tyr Thr Gln Leu Met Asn 145 150 155 160 Pro Gly Leu Pro Asp Ser Ala Leu Asp Ile Asn Glu Cys Leu Arg Gly 165 170 175 Ala Arg Arg Asp Leu Lys Val Phe Gly Asn Phe Pro Lys Leu Ile Met 180 185 190 Thr Gln Val Ser Lys Ser Leu Gln Val Thr Arg Ile Phe Leu Gln Ala 195 200 205 Leu Asn Leu Gly Ile Glu Val Ile Asn Thr Thr Asp His Leu Lys Phe 210 215 220 Ser Lys Asp Cys Gly Arg Met Leu Thr Arg Met Trp Tyr Cys Ser Tyr 225 230 235 240 Cys Gln Gly Leu Met Met Val Lys Pro Cys Gly Gly Tyr Cys Asn Val 245 250 255 Val Met Gln Gly Cys Met Ala Gly Val Val Glu Ile Asp Lys Tyr Trp 260 265 270 Arg Glu Tyr Ile Leu Ser Leu Glu Glu Leu Val Asn Gly Met Tyr Arg 275 280 285 Ile Tyr Asp Met Glu Asn Val Leu Leu Gly Leu Phe Ser Thr Ile His 290 295 300 Asp Ser Ile Gln Tyr Val Gln Lys Asn Ala Gly Lys Leu Thr Thr Thr 305 310 315 320 Ile Gly Lys Leu Cys Ala His Ser Gln Gln Arg Gln Tyr Arg Ser Ala 325 330 335 Tyr Tyr Pro Glu Asp Leu Phe Ile Asp Lys Lys Val Leu Lys Val Ala 340 345 350 His Val Glu His Glu Glu Thr Leu Ser Ser Arg Arg Arg Glu Leu Ile 355 360 365 Gln Lys Leu Lys Ser Phe Ile Ser Phe Tyr Ser Ala Leu Pro Gly Tyr 370 375 380 Ile Cys Ser His Ser Pro Val Ala Glu Asn Asp Thr Leu Cys Trp Asn 385 390 395 400 Gly Gln Glu Leu Val Glu Arg Tyr Ser Gln Lys Ala Ala Arg Asn Gly 405 410 415 Met Lys Asn Gln Phe Asn Leu His Glu Leu Lys Met Lys Gly Pro Glu 420 425 430 Pro Val Val Ser Gln Ile Ile Asp Lys Leu Lys His Ile Asn Gln Leu 435 440 445 Leu Arg Thr Met Ser Met Pro Lys Gly Arg Val Leu Asp Lys Asn Leu 450 455 460 Asp Glu Glu Gly Phe Glu Ser Gly Asp Cys Gly Asp Asp Glu Asp Glu 465 470 475 480 Cys Ile Gly Gly Ser Gly Asp Gly Met Ile Lys Val Lys Asn Gln Leu 485 490 495 Arg Phe Leu Ala Glu Leu Ala Tyr Asp Leu Asp Val Asp Asp Ala Pro 500 505 510 Gly Asn Ser Gln Gln Ala Thr Pro Lys Asp Asn Glu Ile Ser Thr Phe 515 520 525 His Asn Leu Gly Asn Val His Ser Pro Leu Lys Ala Ser Gly Gly Gly 530 535 540 Gly Ser His His His His His His 545 550 <210> 2 <211> 218 <212> PRT <213> Homo sapiens <400> 2 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140 Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys 210 215 <210> 3 <211> 282 <212> PRT <213> Macaca fascicularis <400> 3 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg Asp Gly Gly Asp Lys Trp Arg Asn Lys Phe Glu Leu 35 40 45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60 Leu Three Gln Ser Met With Arg Tyr With Asp Lys His Asn 65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95 Gly Val Leu Asp With Arg Tyr Gly Val Ser Arg With Tyr Ser 100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125 Met Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140 Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Ile Glu Gly 210 215 220 Arg Gly Ile Pro Gly Ala Glu Leu Ala Tyr Asp Leu Asp Val Asp Asp 225 230 235 240 Val Pro Gly Asn Asn Gln Gln Ala Thr Pro Lys Asp Asn Glu Ile Ser 245 250 255 Thr Phe His Asn Leu Gly Asn Val His Ser Pro Leu Lys Ala Ser Gly 260 265 270 Gly Gly Gly Ser His His His His His His 275 280 <210> 4 <211> 282 <212> PRT <213> Mus musculus <400> 4 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140 Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Ile Glu Gly 210 215 220 Arg Gly Ile Pro Gly Ala Glu Leu Ala Tyr Asp Leu Asp Val Asp Asp 225 230 235 240 Ala Pro Gly Asn Lys Gln His Gly Asn Gln Lys Asp Asn Glu Ile Thr 245 250 255 Thr Ser His Ser Val Gly Asn Met Pro Ser Pro Leu Lys Ala Ser Gly 260 265 270 Gly Gly Gly Ser His His His His His His 275 280 <210> 5 <211> 306 <212> PRT <213> Homo sapiens <400> 5 Gln Ser Ile Lys Gly Asn His Leu Val Lys Val Tyr Asp Tyr Gln Glu 1 5 10 15 Asp Gly Ser Val Leu Leu Thr Cys Asp Ala Glu Ala Lys Asn Ile Thr 20 25 30 Trp Phe Lys Asp Gly Lys Met Ile Gly Phe Leu Thr Glu Asp Lys Lys 35 40 45 Lys Trp Asn Leu Gly Ser Asn Ala Lys Asp Pro Arg Gly Met Tyr Gln 50 55 60 Cys Lys Gly Ser Gln Asn Lys Ser Lys Pro Leu Gln Val Tyr Tyr Arg 65 70 75 80 Met Cys Gln Asn Cys Ile Glu Leu Asn Ala Pro Glu Ala Ala Gly Gly 85 90 95 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 100 105 110 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 115 120 125 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 130 135 140 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 145 150 155 160 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 165 170 175 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu 180 185 190 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys 195 200 205 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 210 215 220 Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 225 230 235 240 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 245 250 255 Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp 260 265 270 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 275 280 285 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 290 295 300 Gly Lys 305 <210> 6 <211> 339 <212> PRT <213> Homo sapiens <400> 6 Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys 1 5 10 15 Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro 20 25 30 Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp 35 40 45 Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys 50 55 60 Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg 65 70 75 80 Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg 85 90 95 Val Cys Glu Asn Cys Met Glu Met Asp Ala Pro Glu Ala Ala Gly Gly 100 105 110 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 115 120 125 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 130 135 140 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 145 150 155 160 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 165 170 175 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 180 185 190 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu 195 200 205 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 210 215 220 Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 225 230 235 240 Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 245 250 255 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 260 265 270 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 275 280 285 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 290 295 300 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 305 310 315 320 Gly Lys Gly Ser Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu 325 330 335 Trp His Glu <210> 7 <211> 305 <212> PRT <213> Macaca fascicularis <400> 7 Gln Ser Phe Glu Glu Asn Arg Lys Leu Asn Val Tyr Asn Gln Glu Asp 1 5 10 15 Gly Ser Val Leu Leu Thr Cys His Val Lys Asn Thr Asn Ile Thr Trp 20 25 30 Phe Lys Glu Gly Lys Met Ile Asp Ile Leu Thr Ala His Lys Asn Lys 35 40 45 Trp Asn Leu Gly Ser Asn Thr Lys Asp Pro Arg Gly Val Tyr Gln Cys 50 55 60 Lys Gly Ser Lys Asp Lys Ser Lys Thr Leu Gln Val Tyr Tyr Arg Met 65 70 75 80 Cys Gln Asn Cys Ile Glu Leu Asn Ala Pro Glu Ala Ala Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser 210 215 220 Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 8 <211> 330 <212> PRT <213> Macaca fascicularis <400> 8 Gln Asp Gly Asn Glu Glu Met Gly Ser Ile Thr Gln Thr Pro Tyr Gln 1 5 10 15 Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Ser Gln His Leu 20 25 30 Gly Ser Glu Ala Gln Trp Gln His Asn Gly Lys Asn Lys Glu Asp Ser 35 40 45 Gly Asp Arg Leu Phe Leu Pro Glu Phe Ser Glu Met Glu Gln Ser Gly 50 55 60 Tyr Tyr Val Cys Tyr Pro Arg Gly Ser Asn Pro Glu Asp Ala Ser His 65 70 75 80 His Leu Tyr Leu Lys Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp 85 90 95 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 100 105 110 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 115 120 125 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 130 135 140 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 145 150 155 160 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 165 170 175 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 180 185 190 Ala Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 195 200 205 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu 210 215 220 Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro 225 230 235 240 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 245 250 255 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 260 265 270 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 275 280 285 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 290 295 300 Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Ser Gly Leu Asn Asp Ile 305 310 315 320 Phe Glu Ala Gln Lys Ile Glu Trp His Glu 325 330 <210> 9 <211> 115 <212> PRT <213> Mus musculus <400> 9 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Leu Gly Tyr Thr Phe Ala Asp Tyr 20 25 30 Glu Ile His Trp Val Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile His Pro Gly Ser Gly Gly Thr Ala Tyr Asn Gln Thr Phe 50 55 60 Glu Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Tyr Tyr Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ala 115 <210> 10 <211> 345 <212> DNA <213> Mus musculus <400> 10 caggtccagc tgcagcagtc tggggctgag ctggtgaggc ctggggcttc agtgaagctg 60 tcctgcaagg ctttggggcta cacatttgct gactatgaaa tacactgggt gaagcagaca 120 cctgtgcatg gcctggagtg gattggagct attcatccag gaagtggtgg tactgcctac 180 aatcagacgt tcgagggcaa ggccacactg actgcagaca aatcctccac cacagcctac 240 atggagctca gcagcctgac atctgaggac tctgctgtct attactgtac aagatactat 300 tcctttgctt actggggcca agggactctg gtcactgtct ctgca 345 <210> 11 <211> 6 <212> PRT <213> Mus musculus <400> 11 Ala Asp Tyr Glu Ile His 1 5 <210> 12 <211> 17 <212> PRT <213> Mus musculus <400> 12 Ala Ile His Pro Gly Ser Gly Gly Thr Ala Tyr Asn Gln Thr Phe Glu 1 5 10 15 Gly <210> 13 <211> 6 <212> PRT <213> Mus musculus <400> 13 Tyr Tyr Ser Phe Ala Tyr 1 5 <210> 14 <211> 112 <212> PRT <213> Mus musculus <400> 14 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 15 <211> 336 <212> DNA <213> Mus musculus <400> 15 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120 tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg 300 ctcacgttcg gtgctgggac caagctggag ctgaaa 336 <210> 16 <211> 16 <212> PRT <213> Mus musculus <400> 16 Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1 5 10 15 <210> 17 <211> 7 <212> PRT <213> Mus musculus <400> 17 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 18 <211> 9 <212> PRT <213> Mus musculus <400> 18 Ser Gln Ser Thr His Val Pro Leu Thr 1 5 <210> 19 <211> 115 <212> PRT <213> Mus musculus <400> 19 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Leu Gly Tyr Thr Phe Ala Asp Tyr 20 25 30 Glu Ile His Trp Val Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile His Pro Gly Ser Gly Gly Thr Ala Tyr Asn Gln Thr Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Tyr Tyr Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ala 115 <210> 20 <211> 345 <212> DNA <213> Mus musculus <400> 20 gaggttcaac tgcagcagtc tggggctgag ctggtgaggc ctggggcttc agtgaagctg 60 tcctgcaagg ctttggggcta cacatttgct gactatgaaa tacactgggt gaagcagaca 120 cctgtgcatg gcctggagtg gattggagct attcatccag gaagtggtgg tactgcctac 180 aatcagacgt tcaagggcaa ggccacactg actgcagaca aatcctccac cacagcctac 240 atggagctca gcagcctgac atctgaggac tctgctgtct attactgtac aagatactat 300 tcctttgctt actggggcca agggactctg gtcactgtct ctgca 345 <210> 21 <211> 17 <212> PRT <213> Mus musculus <400> 21 Ala Ile His Pro Gly Ser Gly Gly Thr Ala Tyr Asn Gln Thr Phe Lys 1 5 10 15 Gly <210> 22 <211> 112 <212> PRT <213> Mus musculus <400> 22 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 23 <211> 336 <212> DNA <213> Mus musculus <400> 23 gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120 tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg 300 ctcacgttcg gtgctgggac caagctggaa ataaaa 336 <210> 24 <211> 112 <212> PRT <213> Mus musculus <400> 24 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Ile His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 25 <211> 336 <212> DNA <213> Muscles <400> 25 gatgttgtga tgacccaac tccactcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagccttgta cacagtaatg gaacaccta tttacattgg 120 tacctgcaga agccaggcca gtctccaag ctcctgatct acaaagtttc siaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcagatc 240 agcagagtgg aggctgagga tctgggagtt tattctctgct ctcaagtat acatgttccg 300 ctcacgttcg gtgctgggac caagctggag ctgaaa 336 <210> 26 <211> 9 <212> PRT <213> Mus musculus <400> 26 Ser Gln Ser Ile His Val Pro Leu Thr 1 5 <210> 27 <211> 111 <212> PRT <213> Mus musculus <400> 27 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Ile Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 28 <211> 333 <212> DNA <213> Mus musculus <400> 28 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120 tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatattccc 300 acgtcggtg gaggcaccaa gctggagctg aaa 333 <210> 29 <211> 8 <212> PRT <213> Mus musculus <400> 29 Ser Gln Ser Thr His Ile Pro Thr 1 5 <210> 30 <211> 115 <212> PRT <213> Mus musculus <400> 30 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Arg Leu Ser Cys Lys Ala Leu Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile His Pro Gly Ser Gly Thr Ala Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Tyr Tyr Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Wing 115 <210> 31 <211> 345 <212> DNA <213> Muscles <400> 31 caggttcagc tgcagcagtc tgggctgag ctggtgaggc ctggggctc atgaggctg 60 tcctgcaagg ctttgggcta cacatttact gactatgaa tgcactggt gagcagaca 120 cctgtgcatg gcctggaatg gattggagct attcatccag gaagtggtgg tactgcctac 180 aatcagaagt tcaagggcaa ggccactg actgcagaca aatcctccag cacagcctac 240 atggagctca gcagcctgac atctgaggac tctgctgtct attactgtac aagatactat 300 tccttgctt actggggcca agggactctg gtcactgtct ctgca 345 <210> 32 <211> 6 <212> PRT <213> Mus musculus <400> 32 Thr Asp Tyr Glu Met His 1 5 <210> 33 <211> 17 <212> PRT <213> Mus musculus <400> 33 Ala Ile His Pro Gly Ser Gly Gly Thr Ala Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 34 <211> 112 <212> PRT <213> Mus musculus <400> 34 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 35 <211> 336 <212> DNA <213> Muscles <400> 35 gatgttttga tgacccaac tccactcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagccttgta cacagtaatg gaacaccta tttacattgg 120 tacctgcaga agccaggcca gtctccaag ctcctgatct acaaagtttc siaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcagatc 240 agcagagtgg aggctgagga tctgggagtt tattctctgct ctcaagtac acatgttccg 300 ctcacgttcg gtgctgggac caagctggaa atcaa 336 <210> 36 <211> 112 <212> PRT <213> Mus musculus <400> 36 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 37 <211> 336 <212> DNA <213> Mus musculus <400> 37 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagcattgta catagtaatg gaaacaccta tttacaatgg 120 tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttccg 300 tacacgtcg gaggggggac caagctggaa ataaaa 336 <210> 38 <211> 16 <212> PRT <213> Mus musculus <400> 38 Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Gln 1 5 10 15 <210> 39 <211> 9 <212> PRT <213> Mus musculus <400> 39 Phe Gln Gly Ser His Val Pro Tyr Thr 1 5 <210> 40 <211> 115 <212> PRT <213> Mus musculus <400> 40 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ser Cys Lys Ala Leu Gly Tyr Ala Phe Ala Asp Tyr 20 25 30 Glu Ile His Trp Val Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile His Pro Gly Ser Gly Gly Thr Ala Tyr Asn Gln Thr Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Tyr Tyr Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Wing 115 <210> 41 <211> 345 <212> DNA <213> Muscles <400> 41 caggtccagc tgcagcagtc tgggctgag ctggtgaggc ctggggctc atgaagctg 60 tcctgcaagg ctttgggcta cgcatttgct gactatgaaa tacactgggt gaagcagaca 120 cctgtgcatg gcctggagtg gattggagct attcatccag gaagtggtgg tactgcctac 180 aatcagacgt tcaagggcaa ggccacactg actgcagaca aatcctccac cacagcctac 240 atggagctca gcagcctgac atctgaggac tctgctgtct attactgtac aagatactat 300 tccttgctt actggggcca agggactctg gtcactgtct ctgca 345 <210> 42 <211> 112 <212> PRT <213> Mus musculus <400> 42 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 43 <211> 336 <212> DNA <213> Mus musculus <400> 43 gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120 tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg 300 ctcacgtcg gtgctgggac caagctggag ctgaaa 336 <210> 44 <211> 112 <212> PRT <213> Mus musculus <400> 44 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu With Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Leu 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Leu Gln Val 85 90 95 Thr His Val Pro Tyr Thr Phe Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 45 <211> 336 <212> DNA <213> Muscles <400> 45 gatgttgtga tgacccaac tccactcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gtctagtca gagccttga aacagtaatg gaacaccta ttgaactgg 120 tacctccaga aaccaggcca gtctccacag ctcctgatct acaggtttc siaccgattt 180 tctggggtcc tagacaggtt cagtggtagt ggatcaggga cagatttcac actgaaaatc 240 agcagagtgg aggctgagga ttgggaggtt tattctctgcc tccaagttac acatgtcccg 300 tacacgttcg gaggggggac caagctggaa aaaa 336 <210> 46 <211> 16 <212> PRT <213> Mus musculus <400> 46 Arg Ser Ser Gln Ser Leu Glu Asn Ser Asn Gly Asn Thr Tyr Leu Asn 1 5 10 15 <210> 47 <211> 7 <212> PRT <213> Mus musculus <400> 47 Arg Val Ser Asn Arg Phe Ser 1 5 <210> 48 <211> 9 <212> PRT <213> Mus musculus <400> 48 Leu Gln Val Thr His Val Pro Tyr Thr 1 5 <210> 49 <211> 111 <212> PRT <213> Mus musculus <400> 49 Asp Val Val Val Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 50 <211> 333 <212> DNA <213> Muscles <400> 50 gatgttgtgg tgactcaac tccactcc ctgctgtca gtcttggaga tcaggcctcc 60 atctcttgca gatctagtca gagccttgta cacagtaatg gaacaccta tttacattgg 120 tacctgcaga agccaggcca gtctccaag ctcctgatct acaaagtttc siaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcagatc 240 agcagagtgg aggctgagga tctgggagtt tattctctgct ctcaagtac acatgttccg 300 acgttcggtg gaggcaccaa gctggaaatc aaa 333 <210> 51 <211> 8 <212> PRT <213> Mus musculus <400> 51 Ser Gln Ser Thr His Val Pro Thr 1 5 <210> 52 <211> 112 <212> PRT <213> Mus musculus <400> 52 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Asn 85 90 95 Thr His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 53 <211> 336 <212> DNA <213> Mus musculus <400> 53 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120 tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaaatac acatgttccg 300 ctcacgtcg gtgctgggac caagctggag ctgaaa 336 <210> 54 <211> 9 <212> PRT <213> Mus musculus <400> 54 Ser Gln Asn Thr His Val Pro Leu Thr 1 5 <210> 55 <211> 112 <212> PRT <213> Mus musculus <400> 55 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Leu His Trp Leu Tyr Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr Arg Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 56 <211> 336 <212> DNA <213> Muscles <400> 56 gatgttgtga tgacccagac tccactcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagccttgta cacagtaatg gaacaccta tttacattgg 120 tacctgcaga agccaggcca gtctccaag ctcctgatct acaaagtttc siaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaggatc 240 agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acgtgttccg 300 ctcacgttcg gtgctgggac caagctggag ctgaaa 336 <210> 57 <211> 9 <212> PRT <213> Mus musculus <400> 57 Ser Gln Ser Thr Arg Val Pro Leu Thr 1 5 <210> 58 <211> 112 <212> PRT <213> Mus musculus <400> 58 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Leu Gln Val 85 90 95 Thr His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 59 <211> 336 <212> DNA <213> Mus musculus <400> 59 gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagcattgta catagtaatg gaaacaccta tttagaatgg 120 tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tatttctgcc tccaagttac acatgtcccg 300 ctcacgtcg gtgctgggac caagctggag ctgaaa 336 <210> 60 <211> 16 <212> PRT <213> Mus musculus <400> 60 Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 <210> 61 <211> 9 <212> PRT <213> Mus musculus <400> 61 Leu Gln Val Thr His Val Pro Leu Thr 1 5 <210> 62 <211> 111 <212> PRT <213> Mus musculus <400> 62 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Asn Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 110 <210> 63 <211> 333 <212> DNA <213> Mus musculus <400> 63 caggtccagt tgcaacgtc tggacctgag ctggtgaagc ctggggcttc agtgaagata 60 tcctgcaagg cttctggcta caccttcact gactactata taaactgggt gaagcagaag 120 cctggacagg gacttgagtg gattggatgg attatcctg gaagcggtaa tactaagtac 180 aatgagaagt tcaagggcaa ggccacattg actgtagaca catcctccag cacagcctac 240 atgcagctca gcagcctgac atccgaggac actgctgtct atttctgtgc aagggggcgc 300 tggggccaag ggactctggt cactgtctct gca 333 <210> 64 <211> 6 <212> PRT <213> Mus musculus <400> 64 Thr Asp Tyr Tyr Ile Asn 1 5 <210> 65 <211> 17 <212> PRT <213> Mus musculus <400> 65 Trp Ile Tyr Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 66 <211> 2 <212> PRT <213> Mus musculus <400> 66 Gly Arg 1 <210> 67 <211> 112 <212> PRT <213> Mus musculus <400> 67 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Arg 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 68 <211> 336 <212> DNA <213> Mus musculus <400> 68 gatgttgtga tgacccagac tccactctcc ctgcctgtca gtcttcgaga tcaagcctcc 60 atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120 tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttcct 300 cggacgtcg gtggaggcac caagctggag ctgaaa 336 <210> 69 <211> 9 <212> PRT <213> Mus musculus <400> 69 Ser Gln Ser Thr His Val Pro Arg Thr 1 5 <210> 70 <211> 115 <212> PRT <213> Mus musculus <400> 70 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Leu Gly Tyr Thr Phe Ala Asp Tyr 20 25 30 Glu Ile His Trp Val Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile His Pro Gly Ser Gly Thr Ala Tyr Ser Gln Thr Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Tyr Phe Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Wing 115 <210> 71 <211> 345 <212> DNA <213> Muscles <400> 71 caggttcagc tgcagcagtc tgggctgag ctggtgaggc ctggggctc atgaagctg 60 tcctgcaagg ctttgggcta cacatttgct gactatgaaa tacactgggt gaagcagaca 120 cctgtgcatg gcctggaatg gattggagct attcatccag gaagtggtgg tactgcctac 180 agtcagacgt tcaagggcaa ggccactg actgcagaca aatcctccac cacagcctac 240 atggagctca gcagcctgac atctgaggac tctgctgtct attactgtac aagatacttt 300 tccttgctt actggggcca agggactctg gtcactgtct ctgca 345 <210> 72 <211> 17 <212> PRT <213> Mus musculus <400> 72 Ala Ile His Pro Gly Ser Gly Gly Thr Ala Tyr Ser Gln Thr Phe Lys 1 5 10 15 Gly <210> 73 <211> 6 <212> PRT <213> Mus musculus <400> 73 Tyr Phe Ser Phe Ala Tyr 1 5 <210> 74 <211> 112 <212> PRT <213> Mus musculus <400> 74 Asp Val Val Val Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Ala Pro Tyr Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys 100 105 110 <210> 75 <211> 336 <212> DNA <213> Muscles <400> 75 gatgttgtgg tgactcaac tccactcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagccttgta cacagtaatg gaacaccta tttacattgg 120 tacctgcaga agccaggcca gtctccaag ctcctgatct acaaagtttc siaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcagatc 240 agcagagtgg aggctgagga tctgggatt tattctctgct ctcaagtac acatgctccg 300 tacacgttcg gaggggggac cagactggaa aaaa 336 <210> 76 <211> 9 <212> PRT <213> Mus musculus <400> 76 Ser Gln Ser Thr His Ala Pro Tyr Thr 1 5 <210> 77 <211> 111 <212> PRT <213> Mus musculus <400> 77 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr 20 25 30 Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 110 <210> 78 <211> 333 <212> DNA <213> Mus musculus <400> 78 caggttcagc tgcaacgtc tggagctgag ctgatgaagc ctggggcctc agtgaagata 60 tcctgcaagg ctactggcta cacattcagt agctactgga tagagtgggt aaagcagagg 120 cctggacatg gccttgagtg gattggagag atttacctg gaagtggtag tactaactac 180 aatgagaagt tcaagggcaa ggccacattc actgcagata catcctccaa cacagcctac 240 atgcaactca gcagcctgac atctgaggac tctgccgtct attactgtgc gaggggttac 300 tggggccaag ggactctggt cactgtctct gca 333 <210> 79 <211> 6 <212> PRT <213> Mus musculus <400> 79 Dear Dear Tyr Trp Ile Glu 1 5 <210> 80 <211> 17 <212> PRT <213> Mus musculus <400> 80 Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 81 <211> 2 <212> PRT <213> Mus musculus <400> 81 Gly Tyr 1 <210> 82 <211> 112 <212> PRT <213> Mus musculus <400> 82 Asp Ile Val Met Thr Gln Ala Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 83 <211> 336 <212> DNA <213> Mus musculus <400> 83 gatattgtga tgacgcaggc tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120 tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttcct 300 cggacgttcg gtggaggcac caagctggaa atcaaa 336 <210> 84 <211> 112 <212> PRT <213> Homo sapiens <400> 84 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Tyr Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Leu Gln Val 85 90 95 Thr His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 85 <211> 336 <212> DNA <213> Homo sapiens <400> 85 gacgtggtca tgacacagag ccctctgagc ctgcctgtga cactgggaca gcctgccagc 60 atcagctgta gaagcagcca gagcatcgtg cacagcaact acaacacata cctggaatgg 120 tatcagcaga ggcctggcca gtctcctaga ctgctgatct acaaggtgtc caacagattc 180 agcggcgtgc ccgacagatt ctctggctct ggatctggca ccgacttcac cctgaagatc 240 tccagagtgg aagccgagga cgtgggcgtg tacttctgtc tccaggtcac acacgtgccc 300 ctgacatttg gccagggcac caagctggaa atcaag 336 <210> 86 <211> 112 <212> PRT <213> Homo sapiens <400> 86 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Tyr Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Leu Gln Val 85 90 95 Thr His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 87 <211> 336 <212> DNA <213> Homo sapiens <400> 87 gacgtggtca tgacacagag ccctctgagc ctgcctgtga cacctggcga acctgccagc 60 atcagctgta gaagcagcca gagcatcgtg cacagcaact acaacacata cctggagtgg 120 tatctgcaga agcccggcca gtctcctcag ctgctgatct acaaggtgtc caacagattc 180 agcggcgtgc ccgacagatt ctctggctct ggatctggca ccgacttcac cctgaagatc 240 tccagagtgg aagccgagga cgtgggcgtg tacttctgtc tccaggtcac acacgtgccc 300 ctgacatttg gccagggcac caagctggaa atcaag 336 <210> 88 <211> 112 <212> PRT <213> Homo sapiens <400> 88 Asp Val Val Put Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Leu Gln Val 85 90 95 Thr His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 89 <211> 336 <212> DNA <213> Homo sapiens <400> 89 gacgtggtca tgacacagag ccctctgagc ctgcctgtga cacctggcga acctgccagc 60 atcagctgta gaagcagcca gagcatcgtg cacagcaacg gcaacacata cctggagtgg 120 tatctgcaga agcccggcca gtctcctcag ctgctgatct acaaggtgtc caacagattc 180 agcggcgtgc ccgacagatt ctctggctct ggatctggca ccgacttcac cctgaagatc 240 tccagagtgg aagccgagga cgtgggcgtg tacttctgtc tccaggtcac acacgtgccc 300 ctgacatttg gccagggcac caagctggaa atcaag 336 <210> 90 <211> 112 <212> PRT <213> Homo sapiens <400> 90 Asp Val Val Put Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Ser Asn Thr Leu Glu Glu Trp Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Leu Gln Val 85 90 95 Thr His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 91 <211> 336 <212> DNA <213> Homo sapiens <400> 91 gacgtgtca tgacacagag cccctgagc ctgcctgtga cacctggcga acctgccagc 60 atcagctgta gaagcagcca gagcatcgtg cacagcaca gcacacata cctggagtgg 120 tatctgcaga agcccggcca gtctcctcag ctgctgatct acaagtgtc caacagattc 180 agcggcgtgc ccgacagatt ctctggctct ggatctggca ccgactcac cctgaagattc 240 tccagagtgg aagccgagga cgtgggcgtg tacttctgtc tccaggtcac acacgtgccc 300 ctgacatttg gccagggcac caagctggaa atcaag 336 <210> 92 <211> 16 <212> PRT <213> Homo sapiens <400> 92 Arg Ser Ser Gln Ser Ile Val His Ser Asn Ser Asn Thr Tyr Leu Glu 1 5 10 15 <210> 93 <211> 112 <212> PRT <213> Homo sapiens <400> 93 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Thr Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Leu Gln Val 85 90 95 Thr His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 94 <211> 336 <212> DNA <213> Homo sapiens <400> 94 gacgtggtca tgacacagag ccctctgagc ctgcctgtga cacctggcga acctgccagc 60 atcagctgta gaagcagcca gagcatcgtg cacagcaaca caaacacata cctggagtgg 120 tatctgcaga agcccggcca gtctcctcag ctgctgatct acaaggtgtc caacagattc 180 agcggcgtgc ccgacagatt ctctggctct ggatctggca ccgacttcac cctgaagatc 240 tccagagtgg aagccgagga cgtgggcgtg tacttctgtc tccaggtcac acacgtgccc 300 ctgacatttg gccagggcac caagctggaa atcaag 336 <210> 95 <211> 16 <212> PRT <213> Homo sapiens <400> 95 Arg Ser Ser Gln Ser Ile Val His Ser Asn Thr Asn Thr Tyr Leu Glu 1 5 10 15 <210> 96 <211> 112 <212> PRT <213> Homo sapiens <400> 96 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Ala Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Leu Gln Val 85 90 95 Thr His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 97 <211> 336 <212> DNA <213> Homo sapiens <400> 97 gacgtggtca tgacacagag ccctctgagc ctgcctgtga cacctggcga acctgccagc 60 atcagctgta gaagcagcca gagcatcgtg cacagcaacg ccacacata cctggagtgg 120 tatctgcaga agcccggcca gtctcctcag ctgctgatct acaagtgtc caacagattc 180 agcggcgtgc ccgacagatt ctctggctct ggatctggca ccgactcac cctgaagattc 240 tccagagtgg aagccgagga cgtgggcgtg tactctgtc tccaggtcac acacgtgccc 300 ctgacatttg gccaggggcac caagctggaa atcaag 336 <210> 98 <211> 16 <212> PRT <213> Homo sapiens <400> 98 Arg Ser Ser Gln Ser Ile Val His Ser Asn Ala Asn Thr Tyr Leu Glu 1 5 10 15 <210> 99 <211> 115 <212> PRT <213> Homo sapiens <400> 99 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asp Tyr 20 25 30 Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Trp Glu Met 35 40 45 Gly Ala Ile His Pro Gly Ser Gly Thr Ala Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Tyr Tyr Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 100 <211> 345 <212> DNA <213> Homo sapiens <400> 100 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcagg ctagcggcta caccttcgcc gactacgaga tccactgggt ccgacaggct 120 ccaggacagg gacttgaatg gatggggcgct atccatcctg gctctggcgg cacagcttac 180 aaccagaaat tccagggcag agtgaccctg accgccgaca agtctagcac caccgcctac 240 atggaactga gcagcctgag aagcgaggac accgccgtgt actactgcac ccggtactac 300 agcttcgcct actggggaca gggaaccctg gtcacagtca gctct 345 <210> 101 <211> 17 <212> PRT <213> Homo sapiens <400> 101 Ala Ile His Pro Gly Ser Gly Gly Thr Ala Tyr Asn Gln Lys Phe Gln 1 5 10 15 Gly <210> 102 <211> 115 <212> PRT <213> Homo sapiens <400> 102 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asp Tyr 20 25 30 Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Ile His Pro Gly Ser Gly Gly Thr Ala Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Tyr Tyr Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Dear Dear 115 <210> 103 <211> 345 <212> DNA <213> Homo sapiens <400> 103 caggttcagc tggttcagtc tggcgccgaa ctgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcaagg ctagcggcta caccttcgcc gactacgaga tccactgggt ccgacaggct 120 ccaggacagg gacttgaatg gatgggcgct atccatcctg gctctggcgg cacagcttac 180 aaccagaaat tccagggcag agtgaccctg accgccgaca agtctagcac caccgcctac 240 atggaactga gcagcctgag aagcgaggac accgccgtgt actactgcac ccggtactac 300 agcttcgcct actggggaca gggaaccctg gtcacagtca gctct 345 <210> 104 <211> 115 <212> PRT <213> Homo sapiens <400> 104 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asp Tyr 20 25 30 Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Trp Glu Met 35 40 45 Gly Ala Ile His Pro Gly Ser Gly Thr Ala Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Tyr Tyr Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 105 <211> 345 <212> DNA <213> Homo sapiens <400> 105 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcagg ctagcggcta caccttcgcc gactacgaga tccactgggt ccgacaggct 120 ccaggacagg gacttgaatg gatggggcgct atccatcctg gctctggcgg cacagcttac 180 gcccagaaat tccagggcag agtgaccctg accgccgaca agtctagcac caccgcctac 240 atggaactga gcagcctgag aagcgaggac accgccgtgt actactgcac ccggtactac 300 agcttcgcct actggggaca gggaaccctg gtcacagtca gctct 345 <210> 106 <211> 17 <212> PRT <213> Homo sapiens <400> 106 Ala Ile His Pro Gly Ser Gly Gly Thr Ala Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 107 <211> 115 <212> PRT <213> Homo sapiens <400> 107 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asp Tyr 20 25 30 Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Ile His Pro Gly Ser Gly Gly Thr Ala Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Tyr Tyr Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 108 <211> 345 <212> DNA <213> Homo sapiens <400> 108 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcaagg ctagcggcta caccttcgcc gactacgaga tccactgggt ccgacaggct 120 ccaggacagg gacttgaatg gatgggcgct atccatcctg gctctggcgg cacagcttac 180 aaccagaaat tccagggcag agtgaccctg accgccgacg agtctagcac caccgcctac 240 atggaactga gcagcctgag aagcgaggac accgccgtgt actactgcac ccggtactac 300 agcttcgcct actggggaca gggaaccctg gtcacagtca gctct 345 <210> 109 <211> 115 <212> PRT <213> Homo sapiens <400> 109 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asp Tyr 20 25 30 Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Ile His Pro Gly Ser Gly Gly Thr Ala Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Tyr Tyr Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 110 <211> 345 <212> DNA <213> Homo sapiens <400> 110 caggttcagc tggttcagtc tggcgccgaa ctgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcagg ctagcggcta caccttcgcc gactacgaga tccactgggt ccgacaggct 120 ccaggacagg gacttgaatg gatggggcgct atccatcctg gctctggcgg cacagcttac 180 aaccagaaat tccagggcag agtgaccctg accgccgacg agtctagcac caccgcctac 240 atggaactga gcagcctgag aagcgaggac accgccgtgt actactgcac ccggtactac 300 agcttcgcct actggggaca gggaaccctg gtcacagtca gctct 345 <210> 111 <211> 115 <212> PRT <213> Homo sapiens <400> 111 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asp Tyr 20 25 30 Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Trp Glu Met 35 40 45 Gly Ala Ile His Pro Gly Ser Gly Thr Ala Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Tyr Tyr Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Dear Dear 115 <210> 112 <211> 345 <212> DNA <213> Homo sapiens <400> 112 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcaagg ctagcggcta caccttcgcc gactacgaga tccactgggt ccgacaggct 120 ccaggacagg gacttgaatg gatgggcgct atccatcctg gctctggcgg cacagcttac 180 gcccagaaat tccagggcag agtgaccctg accgccgacg agtctagcac caccgcctac 240 atggaactga gcagcctgag aagcgaggac accgccgtgt actactgcac ccggtactac 300 agcttcgcct actggggaca gggaaccctg gtcacagtca gctct 345 <210> 113 <211> 109 <212> PRT <213> Homo sapiens <400> 113 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 114 <211> 327 <212> DNA <213> Homo sapiens <400> 114 caggctgtgg tcacacaaga gcctagcctg acagtgtctc ctggcggcac agtgaccctg 60 acctgtagat cttctacagg cgccgtgacc accagcaact acgctaattg ggtgcagcag 120 aagcccggcc aggctcctag aggactgatc ggcggaacaa acaagagagc cccttggaca 180 cccgccagat tctctggatc tctgctcggc ggaaaggccg ctctgacact ttctggtgct 240 cagcctgagg acgaggccga gtactattgt gccctgtggt acagcaacct gtgggtgttc 300 ggcggaggca ccaaactgac agttctg 327 <210> 115 <211> 14 <212> PRT <213> Homo sapiens <400> 115 Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 116 <211> 8 <212> PRT <213> Homo sapiens <400> 116 Gly Gly Thr Asn Lys Arg Ala Pro 1 5 <210> 117 <211> 9 <212> PRT <213> Homo sapiens <400> 117 Ala Leu Trp Tyr Ser Asn Leu Trp Val 1 5 <210> 118 <211> 109 <212> PRT <213> Homo sapiens <400> 118 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 119 <211> 327 <212> DNA <213> Homo sapiens <400> 119 caggctgtgg tcacacaaga gcctagcctg acagtgtctc ctggcggcac agtgaccctg 60 acctgtagat cttctacagg cgccgtgacc accagcaact acgctaattg ggtgcaggag 120 aagcccggcc aggctcctag aggactgatc ggcggaacaa acaagagagc cccttggaca 180 cccgccagat tctctggatc tctgctcggc ggaaaggccg ctctgacact ttctggtgct 240 cagcctgagg acgaggccga gtactattgt gccctgtggt acagcaacct gtgggtgttc 300 ggcggaggca ccaaactgac agttctg 327 <210> 120 <211> 109 <212> PRT <213> Homo sapiens <400> 120 Glu Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Glu Ser Ser Asp Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Glu Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 121 <211> 327 <212> DNA <213> Homo sapiens <400> 121 gaggctgtgg tcacacaaga gcctagcctg acagtgtctc ctggcggcac agtgaccctg 60 acctgtgagt cttctgacgg cgccgtgacc accagcaact acgctaattg ggtgcaggag 120 aagcccggcc aggctcctag aggactgatc ggcggaacaa acaaggaggc cccttggaca 180 cccgccagat tctctggatc tctgctcggc ggaaaggccg ctctgacact ttctggtgct 240 cagcctgagg acgaggccga gtactattgt gccctgtggt acagcaacct gtgggtgttc 300 ggcggaggca ccaaactgac agttctg 327 <210> 122 <211> 14 <212> PRT <213> Homo sapiens <400> 122 Glu Ser Ser Asp Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 123 <211> 8 <212> PRT <213> Homo sapiens <400> 123 Gly Gly Thr Asn Lys Glu Ala Pro 1 5 <210> 124 <211> 109 <212> PRT <213> Homo sapiens <400> 124 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Glu Ser Ser Asp Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Glu Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 125 <211> 327 <212> DNA <213> Homo sapiens <400> 125 caggctgtgg tcacacaaga gcctagcctg acagtgtctc ctggcggcac agtgaccctg 60 acctgtgagt cttctgacgg cgccgtgacc accagcaact acgctaattg ggtgcaggag 120 aagcccggcc aggctcctag aggactgatc ggcggaacaa acaaggaggc cccttggaca 180 cccgccagat tctctggatc tctgctcggc ggaaaggccg ctctgacact ttctggtgct 240 cagcctgagg acgaggccga gtactattgt gccctgtggt acagcaacct gtgggtgttc 300 ggcggaggca ccaaactgac agttctg 327 <210> 126 <211> 109 <212> PRT <213> Homo sapiens <400> 126 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 127 <211> 327 <212> DNA <213> Homo sapiens <400> 127 caggctgtgg tcacacaaga gcctagcctg acagtgtctc ctggcggcac agtgaccctg 60 acctgtagat cttctacagg cgccgtgacc accagcaact acgctaattg ggtgcaggag 120 aagcccggcc aggctcctag aggactgatc ggcggaacaa acaagagagc cccttggaca 180 cccgccagat tctctggatc tctgctcggc ggaaaggccg ctctgacaat cactggtgct 240 caggctgagg acgaggccga gtactattgt gtgctgtggt acagcaacct gtgggtgttc 300 ggcggaggca ccaaactgac agttctg 327 <210> 128 <211> 9 <212> PRT <213> Homo sapiens <400> 128 Val Leu Trp Tyr Ser Asn Leu Trp Val 1 5 <210> 129 <211> 109 <212> PRT <213> Homo sapiens <400> 129 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Phe Gln Glu Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Tyr Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 130 <211> 327 <212> DNA <213> Homo sapiens <400> 130 caggctgtgg tcacacaaga gcctagcctg acagtgtctc ctggcggcac agtgaccctg 60 acctgtagat cttctacagg cgccgtgacc accagcaact acgctaattg gttccaggag 120 aagcccggcc aggctcctag aggactgatc tacggaacaa acaagagagc cccttggaca 180 cccgccagat tctctggatc tctgctcggc ggaaaggccg ctctgacact ttctggtgct 240 caggctgagg acgaggccga gtactattgt gtcctgtggt acagcaacct gtgggtgttc 300 ggcggaggca ccaaactgac agttctg 327 <210> 131 <211> 8 <212> PRT <213> Homo sapiens <400> 131 Tyr Gly Thr Asn Lys Arg Ala Pro 1 5 <210> 132 <211> 125 <212> PRT <213> Homo sapiens <400> 132 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Only Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Tyr and Gln Met Asn Served With Arg and Glu Asp Thr and Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Only Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 133 <211> 375 <212> DNA <213> Homo sapiens <400> 133 gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60 tcttgtgccg ccagcggctt caccttcaac acctacgcta tgaacgggt ccgacaggcc 120 cctggcaaag gactggaatg gtgtccaga atcaggtcca agtacaacaa ctacgccacc 180 tactacgccg acagcgtga ggacagattc accatcagca gggacgacag caagacacc ctgtacctgc agatgaacag cctgagagcc gaggacaccg ccgtgtacta ctgtgtcaga cacggcaact tcggcaacag ctatgtgtct tggtttgcct actggggcca gggcacactg 360 gtcacagtta gctct 375 <210> 134 <211> 6 <212> PRT <213> Homo sapiens <400> 134 Asn Thr Tyr Ala With Asn 1 5 <210> 135 <211> 18 <212> PRT <213> Homo sapiens <400> 135 Ile Arg Ser Light Tire Asn Asn Tire Ala Thr Tyr Tyr Ala Asp Ser Val 1 5 10 15 Light Asp <210> 136 <211> 14 <212> PRT <213> Homo sapiens <400> 136 His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 137 <211> 125 <212> PRT <213> Homo sapiens <400> 137 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 138 <211> 375 <212> DNA <213> Homo sapiens <400> 138 gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60 tcttgtgccg ccagcggctt caccttctcc acctacgcta tgaactgggt ccgaaaggcc 120 cctggcaaag gactggaatg ggtgtccaga atcaggtcca agtacaacaa ctacgccacc tactacgccg acagcgtga ggacagattc accatcagca gggacgacag caagacacc ctgtacctgc agatgaacag cctgagagcc gaggacaccg ccgtgtacta ctgtgtcaga cacggcaact tcggcaacag ctatgtgtct tggtttgcct actggggcca gggcacactg 360 gtcacagtta gctct 375 <210> 139 <211> 6 <212> PRT <213> Homo sapiens <400> 139 Ser Thr Tyr Ala With Asn 1 5 <210> 140 <211> 125 <212> PRT <213> Homo sapiens <400> 140 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Glu Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 141 <211> 375 <212> DNA <213> Homo sapiens <400> 141 gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60 tcttgtgccg ccagcggctt caccttctcc acctacgcta tgaactgggt ccgaaaggcc 120 cctggcaaag gactggaatg ggtgtccaga atcaggtcca agtacaacaa ctacgccacc 180 tactacgccg acagcgtgaa ggacagattc accatcagca gggacgacag caagaacacc 240 ctgtacctgc agatgaacag cctgagagcc gaggacaccg ccgtgtacta ctgtgtcaga 300 cacggcaact tcggcgagag ctatgtgtct tggtttgcct actggggcca gggcacactg 360 gtcacagtta gctct 375 <210> 142 <211> 14 <212> PRT <213> Homo sapiens <400> 142 His Gly Asn Phe Gly Glu Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 143 <211> 125 <212> PRT <213> Homo sapiens <400> 143 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Free Mp3 Download 85 90 95 Tyrant Cys Val Arg His Gly Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Free Mp3 Download 115 120 125 <210> 144 <211> 375 <212> DNA <213> Homo sapiens <400> 144 60. gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg tcttgtgccg ccagcggctt caccttctcc acctacgcta tgaactgggt ccgaaaggcc 120 cctggcaaag gactggaatg ggtgtccaga atcaggtcca agtacaacaa ctacgccacc tactacgccg acagcgtga ggacagattc accatcagca gggacgacag caagacacc ctgtacctgc agatgaacag cctgagagcc gaggacaccg ccgtgtacta ctgtgtcaga cacggcaact tcggccagag ctatgtgtct tggtttgcct actggggcca gggcacactg 360 gtcacagtta gctct 375 <210> 145 <211> 14 <212> PRT <213> Homo sapiens <400> 145 His Gly Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 146 <211> 125 <212> PRT <213> Homo sapiens <400> 146 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asp Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Free Mp3 Download 115 120 125 <210> 147 <211> 375 <212> DNA <213> Homo sapiens <400> 147 60. gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg tcttgtgccg ccagcggctt caccttctcc acctacgcta tgaactgggt ccgaaaggcc 120 cctggcaaag gactggaatg ggtgtccaga atcaggtcca agtacaacaa ctacgccacc tactacgccg acagcgtga ggacagattc accatcagca gggacgacag caagacacc ctgtacctgc agatgaacag cctgagagcc gaggacaccg ccgtgtacta ctgtgtcaga 360. cacggcaact tcggcgacag ctatgtgtct tggtttgcct actggggcca gggcacactg gtcacagtta gctct 375 <210> 148 <211> 14 <212> PRT <213> Homo sapiens <400> 148 His Gly Asn Phe Gly Asp Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 149 <211> 125 <212> PRT <213> Homo sapiens <400> 149 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Thr Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 150 <211> 375 <212> DNA <213> Homo sapiens <400> 150 60. gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg tcttgtgccg ccagcggctt caccttctcc acctacgcta tgaactgggt ccgaaaggcc 120 cctggcaaag gactggaatg ggtgtccaga atcaggtcca agtacaacaa ctacgccacc tactacgccg acagcgtga ggacagattc accatcagca gggacgacag caagacacc ctgtacctgc agatgaacag cctgagagcc gaggacaccg ccgtgtacta ctgtgtcaga 360. cacggcaact tcggcaccag ctatgtgtct tggtttgcct actggggcca gggcacactg gtcacagtta gctct 375 <210> 151 <211> 14 <212> PRT <213> Homo sapiens <400> 151 His Gly Asn Phe Gly Thr Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 152 <211> 125 <212> PRT <213> Homo sapiens <400> 152 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Ala Met Asn Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Glu Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Tyr and Gln Met Asn Served With Arg and Glu Asp Thr and Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Only Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 153 <211> 375 <212> DNA <213> Homo sapiens <400> 153 gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60 tcttgtgccg ccagcggctt caccttctcc gactacgcta tgaacggt ccgaaggcc 120 cctggcaaag gactggaatg ggtgtccaga atcaggtcca agtacaacaa ctacgccacc tactacgccg acagcgtgga ggacagattc accatcagca gggacgacag caagaacacc ctgtacctgc agatgaacag cctgagagcc gaggacaccg ccgtgtacta ctgtgtcaga cacggcaact tcggcaacag ctatgtgtct tggtttgcct actggggcca gggcacactg 360 gtcacagtta gctct 375 <210> 154 <211> 6 <212> PRT <213> Homo sapiens <400> 154 Ser Asp Tyr Wing With Ass 1 5 <210> 155 <211> 18 <212> PRT <213> Homo sapiens <400> 155 Ile Arg Ser Light Tire Asn Asn Tire Ala Thr Tyr Tyr Ala Asp Ser Val 1 5 10 15 Glu Asp <210> 156 <211> 125 <212> PRT <213> Homo sapiens <400> 156 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg With Arg Ser Tyr Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Tyr Leu Gln Met Asn Served Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Only Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 157 <211> 375 <212> DNA <213> Homo sapiens <400> 157 gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60 tcttgtgccg ccagcggctt caccttcaac acctacgcta tgaacggt ccgaaggcc 120 cctggcaaag gactggaatg ggtgggaaga atcaggtcca agtacaacaa ctacgccacc tactacgccg acagcgtgaa ggacagattc accatcagca gggacgacag caagaacagc ctgtacctgc agatgaacag cctgaaaacc gaggacaccg ccgtgtacta ctgtgccaga cacggcaact tcggcaacag ctatgtgtct tggtttgcct actggggcca gggcacactg 360 gtcacagtta gctct 375 <210> 158 <211> 125 <212> PRT <213> Homo sapiens <400> 158 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Light Tire Asphalt Asphalt Tyre Ala Thr Tyre Tyre Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Light Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyrant Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Free Mp3 Download 115 120 125 <210> 159 <211> 375 <212> DNA <213> Homo sapiens <400> 159 60. gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg tcttgtgccg ccagcggctt caccttcaac acctacgcta tgaactgggt ccgaaaggcc 120 cctggcaaag gactggaatg ggtgggaaga atcaggtcca agtacaacaa ctacgccacc tactacgccg acagcgtgaa ggacagattc accatcagca gggacgacag caagaacagc ctgtacctgc agatgaacag cctgaaaacc gaggacaccg ccgtgtacta ctgtgtcaga cacggcaact tcggcaacag ctatgtgtct tggtttgcct actggggcca gggcacactg 360 gtcacagtta gctct 375 <210> 160 <211> 125 <212> PRT <213> Homo sapiens <400> 160 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 161 <211> 375 <212> DNA <213> Homo sapiens <400> 161 gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60 tcttgtgccg ccagcggctt caccttcaac acctacgcta tgaactgggt ccgaaaggcc 120 cctggcaaag gactggaatg ggtggccaga atcaggtcca agtacaacaa ctacgccacc 180 tactacgccg acagcgtgaa ggacagattc accatcagca gggacgacag caagaacagc 240 ctgtacctgc agatgaacag cctgaaaacc gaggacaccg ccgtgtacta ctgtgtcaga 300 cacggcaact tcggcaacag ctatgtgtct tggtttgcct actggggcca gggcacactg 360 gtcacagtta gctct 375 <210> 162 <211> 219 <212> PRT <213> Homo sapiens <400> 162 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Lys Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Leu Gln Val 85 90 95 Thr His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 163 <211> 657 <212> DNA <213> Homo sapiens <400> 163 gacgtggtca tgacacagag ccctctgagc ctgcctgtga cacctggcga acctgccagc 60 atcagctgta gaagcagcca gagcatcgtg cacagcaacg gcaacacata cctggagtgg 120 tatctgaaga agcccggcca gtctcctcag ctgctgatct acaaggtgtc caacagattc 180 agcggcgtgc ccgacagatt ctctggctct ggatctggca ccgacttcac cctgaagatc 240 tccagagtgg aagccgagga cgtgggcgtg tacttctgtc tccaggtcac acacgtgccc 300 ctgacatttg gccagggcac caagctggaa atcaagcgaa ctgtggctgc accatctgtc 360 ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 420 ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 480 tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc 540 agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa 600 gtcacccatc agggcctgag ctcgcccgtc acaaagagct tcaacagggg agagtgt 657 <210> 164 <211> 442 <212> PRT <213> Homo sapiens <400> 164 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asp Tyr 20 25 30 Glu Ile His Trp Val Arg Glu Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Ile His Pro Gly Ser Gly Gly Thr Ala Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Tyr Tyr Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315 320 Lys Gly Leu Ala Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu Glu 340 345 350 Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn 405 410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 165 <211> 1326 <212> DNA <213> Homo sapiens <400> 165 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcaagg ctagcggcta caccttcgcc gactacgaga tccactgggt ccgagaggct 120 ccaggacagg gacttgaatg gatgggcgct atccatcctg gctctggcgg cacagcttac 180 gcccagaaat tccagggcag agtgaccctg accgccgacg agtctagcac caccgcctac 240 atggaactga gcagcctgag aagcgaggac accgccgtgt actactgcac ccggtactac 300 agcttcgcct actggggaca gggaaccctg gtcacagtca gctctgctag caccaagggc 360 cccagcgtgt tccccctggc cccttgcagc agaagcacca gcgagagcac agccgccctg 420 ggctgcctgg tgaaggacta cttccccgag cccgtgaccg tgtcctggaa cagcggcgct 480 ctgaccagcg gcgtgcatac cttccccgcc gtgctccaga gcagcggact gtactccctg 540 agcagcgtgg tgaccgtgcc ttccagcagc ctgggcacca agacctacac ctgcaacgtg 600 gaccacaagc ccagcaacac caaggtggac aagagagtgg agagcaagta cggccctccc 660 tgcccccctt gccctgcccc cgagttcgag ggcggaccta gcgtgttcct gttccccccc 720 aagcccaagg acaccctgat gatcagcaga acccccgagg tgacctgcgt ggtggtggac 780 gtgtcccagg aggaccccga ggtccagttt aattggtacg tggacggcgt ggaagtgcat 840 aacgccaaga ccaagcccag agaggagcag ttcaacagca cctacagagt ggtgtccgtg 900 ctgaccgtgc tgcaccagga ctggctgaac ggcaaggaat acaagtgcaa ggtctccaac 960 aagggcctgg ccagcagcat cgagaagacc atcagcaagg ccaagggcca gccacggggag 1020 ccccaggtct gcaccctgcc acctagccaa gaggagatga ccaagaacca ggtgtccctg 1080 agctgtgccg tgaaaggctt ctatcccagc gatatcgccg tggagtggga gagcaacggc 1140 cagcccgaga acaactacaa gaccaccccc cctgtgctgg acagcgacgg cagcttcttc 1200 ctggtttcca agctgaccgt ggacaagtcc agatggcagg agggcaacgt cttcagctgc 1260 tccgtgatgc acgaggccct gcacaaccac tacacccaga agtccctgag cctgagcctg 1320 ggcaag 1326 <210> 166 <211> 215 <212> PRT <213> Homo sapiens <400> 166 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100 105 110 Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 115 120 125 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 130 135 140 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala 145 150 155 160 Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 165 170 175 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 180 185 190 Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 195 200 205 Val Ala Pro Thr Glu Cys Ser 210 215 <210> 167 <211> 645 <212> DNA <213> Homo sapiens <400> 167 caggctgtgg tcacacaaga gcctagcctg acagtgtctc ctggcggcac agtgaccctg 60 acctgtagat cttctacagg cgccgtgacc accagcaact acgctaattg ggtgcaggag 120 aagcccggcc aggctcctag aggactgatc ggcggaacaa acaagagagc cccttggaca 180 cccgccagat tctctggatc tctgctcggc ggaaaggccg ctctgacaat cactggtgct 240 caggctgagg acgaggccga gtactattgt gtgctgtggt acagcaacct gtgggtgttc 300 ggcggaggca ccaactgac agttctggtt cagcccaagg cggcgccctc ggtcactctg 360 ttcccgccct cctctgagga gcttcaagcc aaaaggcca cactgtgtg tctcataagt 420 gacttctatc cgggagccgt gatagtggcc tggaggcag atagcagccc cgtcaggcg 480 ggagtggaga caccacacc ctccaaaaa agcacaaca agtacgcggc cagcagctac 540 ctgagcctga cgcctgagca gtggaagtcc cacagaagct acagctgcca ggtcacgcat 600 gaagggagca ccgtggagaa gaagtggcc cctacagaat gttca 645 <210> 168 <211> 452 <212> PRT <213> Homo sapiens <400> 168 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg With Arg Ser Tyr Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 210 215 220 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Ala Ser Ser 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Cys Gln Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Leu Gly Lys 450 <210> 169 <211> 1356 <212> DNA <213> Homo sapiens <400> 169 gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60 tcttgtgccg ccagcggctt caccttcaac acctacgcta tgaactgggt ccgaaaggcc 120 cctggcaaag gactggaatg ggtgggaaga atcaggtcca agtacaacaa ctacgccacc 180 tactacgccg acagcgtgaa ggacagattc accatcagca gggacgacag caagaacagc 240 ctgtacctgc agatgaacag cctgaaaacc gaggacaccg ccgtgtacta ctgtgtcaga 300 cacggcaact tcggcacag ctatgtgtct tggtttgcct actggggcca gggcacactg 360 gtcacagtta gctctgctag caccaagggc cccagcgt tccccctggc cccttgcagc 420 agaagcacca gcgagagcac agccgccctg ggctgcctgg tgaaggacta cttccccgag 480 cccgtgaccg tgtcctggaa cagcggcgct ctgaccagcg gcgtgcatac cttccccgcc 540 gtgctccaga gcagcggact gtactccctg agcagcgtgg tgaccgtgcc ttccagcagc 600 ctgggcacca agacctacac ctgcaacgtg gaccacaagc ccaggcaccac caggtggac 660 aagagagtgg agcaagta cggccctccc tgccccctt gccctgcccc cgagttcgag 720 ggcggaccta gcgtgttcct gttcccccc aagcccagg acacctgat gatcagcaga 780 acccccgagg tgacctgcgt ggtgtggac gtgtcccagg aggaccccga ggtccagttt 840 aattggtacg tggacgggt ggaagtgcat aacgccaga ccaagcccag agaggagcag 900 ttcacagca cctacagagt gtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 960 ggcaggaat acagtgcaa ggtctccaac aagggcctgg ccagcagcat cgagaagacc 1020 atcagcaagg ccaagggcca gccacggggag ccccaggtct acaccctgcc accttgtcaa 1080 gaggagatga ccaagaacca ggtgtccctg tggtgtctgg tgaaaggctt ctatcccagc 1140 gatatcgccg tggagtggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 1200 cctgtgctgg acagcgacgg cagcttcttc ctgtactcca agctgaccgt ggacaagtcc 1260 agatggcagg agggcaacgt cttcagctgc tccgtgatgc acgaggccct gcacaaccac 1320 taacacccaga agtccctgag cctgagcctg ggcaag 1356 <210> 170 <211> 219 <212> PRT <213> Homo sapiens <400> 170 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Lys Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Leu Gln Val 85 90 95 Thr His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Lys 115 120 125 Lys Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 171 <211> 657 <212> DNA <213> Homo sapiens <400> 171 gacgtggtca tgacacagag ccctctgagc ctgcctgtga cacctggcga acctgccagc 60 atcagctgta gaagcagcca gagcatcgtg cacagcaacg gcaacata cctggagtgg 120 180 agcggcgtgc ccgacagatt ctctggctct ggatctggca ccgacttcac cctgaagaatc 240 tccagagtgg aagccgagga cgtgggcgtg tacttctgtc tccaggtcac acacgtgccc 300 ctgacatttg gccagggcac caagctgggaa atcaagcgaa ctgtggctgc accatctgtc 360 ttcatcttcc cgccatctga taagaaattg aaatctggaa ctgcctctgt tgtgtgcctg 420 ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 480 tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc 540 agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa 600 gtcacccatc agggcctgag ctcgcccgtc aaaagagct tcaacagggg agagtgt 657 <210> 172 <211> 442 <212> PRT <213> Homo sapiens <400> 172 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asp Tyr 20 25 30 Glu Ile His Trp Val Arg Glu Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Ile His Pro Gly Ser Gly Gly Thr Ala Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Tyr Tyr Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Glu Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315 320 Lys Gly Leu Ala Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu Glu 340 345 350 Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn 405 410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 173 <211> 1326 <212> DNA <213> Homo sapiens <400> 173 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcaagg ctagcggcta caccttcgcc gactacgaga tccactgggt ccgagaggct 120 ccaggacagg gacttgaatg gatgggcgct atccatcctg gctctggcgg cacagcttac 180 gcccagaaat tccagggcag agtgaccctg accgccgacg agtctagcac caccgcctac 240 atggaactga gcagcctgag aagcgaggac accgccgtgt actactgcac ccggtactac 300 agcttcgcct actggggaca gggaaccctg gtcacagtca gctctgctag caccaagggc 360 cccagcgtgt tccccctggc cccttgcagc agaagcacca gcgagagcac agccgccctg 420 ggctgcctgg tggaggacta cttccccgag cccgtgaccg tgtcctggaa cagcggcgct 480 ctgaccagcg gcgtgcatac cttccccgcc gtgctccaga gcagcggact gtactccctg 540 agcagcgtgg tgaccgtgcc ttccagcagc ctgggcacca agacctacac ctgcaacgtg 600 gaccacaagc ccagcaacac caaggtggac gagagagtgg agagcaagta cggccctccc 660 tgcccccctt gccctgcccc cgagttcgaa ggcggaccta gcgtgttcct gttccccccc 720 aagcccaagg acaccctgat gatcagcaga acccccgagg tgacctgcgt ggtggtggac 780 gtgtcccagg aggaccccga ggtccagttt aattggtacg tggacggcgt ggaagtgcat 840 aacgccaaga ccaagcccag agaggagcag ttcaacagca cctacagagt ggtgtccgtg 900 ctgaccgtgc tgcaccagga ctggctgaac ggcaaggaat acaagtgcaa ggtctccaac 960 aagggcctgg ccagcagcat cgagaagacc atcagcaagg ccaagggcca gccacggggag 1020 ccccaggtct gcaccctgcc acctagccaa gaggagatga ccaagaacca ggtgtccctg 1080 agctgtgccg tgaaaggctt ctatcccagc gatatcgccg tggagtggga gagcaacggc 1140 cagcccgaga acaactacaa gaccaccccc cctgtgctgg acagcgacgg cagcttcttc 1200 ctggtttcca agctgaccgt ggacaagtcc agatggcagg agggcaacgt cttcagctgc 1260 tccgtgatgc acgaggccct gcacaaccac tacacccaga agtccctgag cctgagcctg 1320 ggcaag 1326 <210> 174 <211> 109 <212> PRT <213> Mus musculus <400> 174 Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Asp His Leu Phe Thr Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 175 <211> 125 <212> PRT <213> Mus musculus <400> 175 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125

Claims

1. A bispecific antibody or its antigen-binding portion, comprising: (a) A first antigen-binding moiety of a GPC3 antigen bound to the surface of a target cell, the first antigen-binding moiety comprising a first heavy chain and a first light chain, the first antigen-binding moiety comprising a first binding domain for binding to a first antigen, wherein, The first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 11, 106, and 13, and the first light chain CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 60, 17, and 61; and (b) A second antigen-binding portion of the CD3 antigen bound to 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 for binding to the second antigen, wherein the second binding domain comprises the second heavy chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 134, 135 and 136, and the second light chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 115, 116 and 128.

2. The bispecific antibody or its antigen-binding moiety according to claim 1, wherein, The first light chain of the first antigen-binding portion is a κ-type light chain; the first binding domain includes a first heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 111, and a first light chain variable region with an amino acid sequence as shown in SEQ ID NO: 88; the first light chain variable region has Gln 43 Lys mutation (Vκ) GPC3 Gln 43 Lys); the first heavy chain variable region has Gln 39 Glu (VH) GPC3 :Gln 39 Glu mutation.

3. The bispecific antibody or its antigen-binding portion according to claim 1 or 2, wherein, The second light chain of the second antigen-binding portion is a λ-type light chain; the second binding domain includes a second heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 158, and a second light chain variable region with an amino acid sequence as shown in SEQ ID NO: 126; the second light chain variable region has Gln 40 Glu mutation (Vλ) CD3 Gln 40 Glu); the second heavy chain variable region has Gln 39 Lys mutation (VH) CD3 Gln 39 Lys).

4. The bispecific antibody or its antigen-binding portion according to claim 3, wherein the Fc portion of the first antigen-binding portion and the second antigen-binding portion of the bispecific antibody adopts a knock-in-hole structure.

5. The bispecific antibody or its antigen-binding portion according to claim 4, wherein the Fc portion of the first antigen-binding portion and the second antigen-binding portion of the bispecific antibody adopts a human IgG4 knob-into-hole structure.

6. The bispecific antibody or its antigen-binding portion according to claim 5, wherein, The first heavy chain comprises a heavy chain selected from the heavy chain shown in SEQ ID NO: 164, and the first light chain comprises a light chain selected from the light chain shown in SEQ ID NO:

162.

7. The bispecific antibody or its antigen-binding portion according to claim 5, wherein, The first heavy chain comprises a heavy chain selected from the heavy chain shown in SEQ ID NO: 172, and the first light chain comprises a light chain selected from the light chain shown in SEQ ID NO:

170.

8. The bispecific antibody or its antigen-binding portion according to claim 6 or 7, wherein the second heavy chain comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 168, and the second light chain comprises a light chain with an amino acid sequence as shown in SEQ ID NO:

166.

9. The bispecific antibody or its antigen-binding portion according to claim 8, wherein, The first heavy chain of the bispecific antibody comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 164, and the first light chain of the bispecific antibody comprises a light chain with an amino acid sequence as shown in SEQ ID NO: 162; the second heavy chain comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 168, and the second light chain comprises a light chain with an amino acid sequence as shown in SEQ ID NO:

166.

10. The bispecific antibody or its antigen-binding portion according to claim 8, wherein, The first heavy chain of the bispecific antibody comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 172, and the first light chain of the bispecific antibody comprises a light chain with an amino acid sequence as shown in SEQ ID NO: 170; the second heavy chain comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 168, and the second light chain comprises a light chain with an amino acid sequence as shown in SEQ ID NO:

166.

11. An antibody or antigen-binding portion thereof that binds to GPC3, said antibody comprising the first antigen-binding portion of any one of claims 1-10.

12. Nucleic acid encoding the antibody or antigen-binding moiety thereof that binds to GPC3 according to claim 11.

13. The nucleic acid according to claim 12, wherein, The nucleotide sequence encoding the nucleic acid of the first heavy chain variable region of the first antigen-binding region is shown in SEQ ID NO: 112; and the nucleotide sequence encoding the nucleic acid of the first light chain variable region is shown in SEQ ID NO:

89.

14. The nucleic acid according to claim 12, wherein, The nucleotide sequence encoding nucleic acid of the first heavy chain of the first antigen-binding portion is shown in SEQ ID NO: 173, and the nucleotide sequence encoding nucleic acid of the first light chain of the first antigen-binding portion is shown in SEQ ID NO:

171.

15. The nucleic acid according to claim 12, wherein, The nucleotide sequence encoding nucleic acid of the first heavy chain of the first antigen-binding portion is shown in SEQ ID NO: 165, and the nucleotide sequence encoding nucleic acid of the first light chain of the first antigen-binding portion is shown in SEQ ID NO:

163.

16. Nucleic acid encoding the bispecific antibody or its antigen-binding portion according to any one of claims 1-10.

17. The nucleic acid according to claim 16, wherein, The nucleotide sequence encoding the nucleic acid of the first heavy chain variable region of the first antigen-binding portion of the bispecific antibody is shown in SEQ ID NO: 112; and the nucleotide sequence encoding the nucleic acid of the first light chain variable region is shown in SEQ ID NO:

89.

18. The nucleic acid according to claim 16, wherein, The nucleotide sequence encoding nucleic acid of the first heavy chain of the first antigen-binding portion of the bispecific antibody is shown in SEQ ID NO: 173, and the nucleotide sequence encoding nucleic acid of the first light chain of the first antigen-binding portion is shown in SEQ ID NO:

171.

19. The nucleic acid according to claim 16, wherein, The nucleotide sequence encoding nucleic acid of the first heavy chain of the first antigen-binding portion of the bispecific antibody is shown in SEQ ID NO: 165, and the nucleotide sequence encoding nucleic acid of the first light chain of the first antigen-binding portion is shown in SEQ ID NO:

163.

20. The nucleic acid according to any one of claims 16-19, wherein, The nucleotide sequence encoding the nucleic acid of the second heavy chain variable region of the second antigen-binding portion is shown in SEQ ID NO: 159; and the nucleotide sequence encoding the nucleic acid of the second light chain variable region is shown in SEQ ID NO:

127.

21. The nucleic acid according to any one of claims 16-19, wherein, The nucleotide sequence encoding nucleic acid of the second heavy chain of the second antigen-binding portion is shown in SEQ ID NO: 169, and the nucleotide sequence encoding nucleic acid of the second light chain of the second antigen-binding portion is shown in SEQ ID NO:

167.

22. The nucleic acid according to claim 16, wherein, The nucleotide sequence encoding nucleic acid of the first heavy chain of the first antigen-binding portion of the bispecific antibody is shown in SEQ ID NO: 165, the nucleotide sequence encoding nucleic acid of the first light chain of the first antigen-binding portion is shown in SEQ ID NO: 163, the nucleotide sequence encoding nucleic acid of the second heavy chain of the second antigen-binding portion is shown in SEQ ID NO: 169, and the nucleotide sequence encoding nucleic acid of the second light chain of the second antigen-binding portion is shown in SEQ ID NO:

167.

23. The nucleic acid according to claim 16, wherein, The nucleotide sequence encoding nucleic acid of the first heavy chain of the first antigen-binding portion of the bispecific antibody is shown in SEQ ID NO: 173, the nucleotide sequence encoding nucleic acid of the first light chain of the first antigen-binding portion is shown in SEQ ID NO: 171, the nucleotide sequence encoding nucleic acid of the second heavy chain of the second antigen-binding portion is shown in SEQ ID NO: 169, and the nucleotide sequence encoding nucleic acid of the second light chain of the second antigen-binding portion is shown in SEQ ID NO:

167.

24. A vector containing the nucleic acid according to any one of claims 12-23.

25. A cell containing the nucleic acid of any one of claims 12-23 or the vector of claim 24.

26. A pharmaceutical composition comprising the bispecific antibody or its antigen-binding portion as described in any one of claims 1-10, the antibody or its antigen-binding portion that binds to GPC3 as described in claim 11, the nucleic acid as described in any one of claims 12-23, the carrier as described in claim 24, and / or the cell as described in claim 25.

27. A kit comprising the bispecific antibody or its antigen-binding portion according to any one of claims 1-10, the antibody or its antigen-binding portion that binds to GPC3 according to claim 11, the nucleic acid according to any one of claims 12-23, the vector according to claim 24, the cell according to claim 25, and / or the composition according to claim 26.

28. The use of the bispecific antibody or its antigen-binding portion according to any one of claims 1-10 in the preparation of a medicament or kit for the diagnosis, treatment, or prevention of GPC3-positive tumors; wherein, The GPC3-positive tumor is a GPC3-positive cancer; the GPC3-positive cancer is a GPC3-positive liver cancer.

29. The use according to claim 28, wherein, The GPC3-positive liver cancer is GPC3-positive hepatocellular carcinoma.

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