Anti-angiopoietin-2 antibodies and uses thereof

Abstract

The present invention relates to mouse antibodies that bind to angiopoietin-2 (Ang2), humanized anti-Ang2 antibodies derived therefrom, and their uses. The anti-Ang2 antibodies have the dual functions of activating the Tie2 receptor and neutralizing Ang2. The anti-Ang2 antibodies show the property of normalizing abnormal and pathological blood vessels, and thus exhibit therapeutic efficacy for various diseases and disorders associated with abnormal blood vessels. The present invention provides angiogenesis inhibitors and compositions comprising the antibodies as active ingredients for preventing and treating diseases associated with abnormal Ang2 expression and Tie2 dysregulation, as well as compositions comprising the antibodies for diagnosing diseases associated with Ang2 inhibition and Tie2 activation.

Description

Technical Field

[0001] The present invention includes anti-Ang2 antibodies or antigen-binding fragments thereof, which specifically bind to angiopoietin-2 (Ang2), a ligand known as the one that controls blood vessel formation and maintenance; pharmaceutical compositions containing the antibodies or antigen-binding fragments thereof; nucleic acids encoding the antibodies or antigen-binding fragments thereof; vectors containing the nucleic acids; host cells transformed with the vectors; and methods for producing the antibodies or antigen-binding fragments thereof. Background Art

[0002] In the development, growth, maintenance and homeostasis of an organism, angiogenesis occurs dynamically through a variety of regulatory factors. The newly formed blood vessels in this process serve as transport channels for various biomaterials (such as nutrients, oxygen and hormones in surrounding cells). Functional and structurally abnormal blood vessels are direct or indirect causes of the occurrence and development of various diseases. Tumor blood vessels aggravate hypoxia due to their defective functions and structures, thereby causing tumor progression and metastasis to other tissues, and also causing poor delivery of anticancer drugs to the core of the tumor mass. In addition to cancer, defective blood vessels have also been found in various other diseases and conditions. Examples thereof include various eye diseases (e.g., diabetic macular edema, wet age-related macular degeneration), viral infections and acute inflammatory reactions (such as sepsis). Therefore, if a therapeutic agent capable of normalizing pathological blood vessels is available, it can be applied to treat various patients with vascular abnormalities.

[0003] The angiopoietin family plays an important role in the formation and maintenance of blood vessels, and is composed of four angiopoietins (Ang1, Ang2, Ang3 and Ang4). Angiopoietin-1 (Ang1) binds to the Tie2 receptor present on the surface of vascular endothelial cells to phosphorylate and activate the Tie2 receptor, thereby causing vascular stability. On the other hand, angiopoietin-2 (Ang2) binds to the Tie2 receptor, but acts as an antagonist to induce the inactivation of the Tie2 receptor, thereby causing vascular instability and vascular leakage. It is reported that in the blood of cancer patients, eye diseases, viral and bacterial infections, and inflammatory diseases, the expression level of Ang2 is greatly improved (Saharinen P et al., 2017, Nature Review Drug Discovery). However, it is also known that Ang2 acts as an agonist to induce the activation of Tie2 receptors in several processes (including lymphangiogenesis and maintenance), and it is therefore believed that Ang2 performs a variety of functions according to the context.

[0004] Ang2 binding antibodies have been reported in several documents (e.g., US 7,658,924 and US 8,987,420). It is known that most of the Ang2 antibodies reported so far inhibit the binding of Ang2 to Tie2, and thus inhibit the formation of new blood vessels through this Ang2 neutralization effect. Currently, a variety of Ang2 antibodies are being clinically tested in various cancer patients, but their anticancer efficacy is considered insufficient. For example, a phase 3 clinical trial conducted by Amgen showed that the anticancer efficacy of the Ang2 antibody in ovarian cancer patients was not significant (Marth C et al., 2017, Eur. J. Cancer).

[0005] In addition to antibodies, recombinant proteins that directly bind to Tie2 receptors to induce Tie2 phosphorylation and activation have also been reported. Examples include COMP-Angl (Cho et al., 2004, PNAS) and Vasculotide (David S et al., 2011, Am J Physiol Lung Cell Mol Physiol) peptides composed of five angiopoietin-1 protein fragments. However, it is believed that these proteins have very short half-lives and unstable physicochemical properties. In addition, there is a phosphatase called VE-PTP, which removes the phosphate group from phosphorylated Tie2 to inactivate Tie2, and a low molecular weight compound (AKB-9778) has also been developed, which indirectly maintains Tie2 activity by inhibiting the activity of the enzyme VE-PTP (Goel S, 2013, J Natl Cancer Inst). However, this compound has the disadvantage of activating other receptors in addition to Tie2 (Frye M, 2015, J Exp. Med, Hayashi M, 2013, Nature Communication, Mellberg S et al., 2009, FASEB J.). Summary of the invention

[0006] The present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to human angiopoietin-2 and induces Tie2 activation, wherein the antibody or antigen-binding fragment thereof binds to amino acids 289-299 of SEQ ID NO: 1, amino acids 316-322 of SEQ ID NO: 1, or amino acids 336-353 of SEQ ID NO: 1, as determined by a hydrogen / deuterium exchange method.

[0007] The antibody or antigen-binding fragment thereof may bind to human and mouse Ang2. The antibody may be polyclonal or monoclonal. The antigen-binding fragment may be scFv or Fab. The antibody or fragment thereof may be humanized.

[0008] In another aspect, the invention relates to an antibody or antigen-binding fragment comprising:

[0009] (a) a complementarity determining region (CDR) of a heavy chain variable region having a HCDR1 amino acid sequence of SEQ ID NO: 3, a HCDR2 amino acid sequence of SEQ ID NO: 4, and a HCDR3 amino acid sequence of SEQ ID NO: 5; and

[0010] (b) CDRs of a light chain variable region comprising the LCDR1 amino acid sequence of SEQ ID NO:6, the LCDR2 amino acid sequence of SEQ ID NO:7, and the LCDR3 amino acid sequence of SEQ ID NO:8.

[0011] In another aspect, the invention relates to an antibody or antigen-binding fragment comprising:

[0012] (a) a complementarity determining region (CDR) of a heavy chain variable region having a HCDR1 amino acid sequence of SEQ ID NO: 13, a HCDR2 amino acid sequence of SEQ ID NO: 14, and a HCDR3 amino acid sequence of SEQ ID NO: 15; and

[0013] (b) CDRs of a light chain variable region comprising the LCDR1 amino acid sequence of SEQ ID NO:16, the LCDR2 amino acid sequence of SEQ ID NO:17, and the LCDR3 amino acid sequence of SEQ ID NO:18.

[0014] In one aspect, the present invention relates to an antibody or an antigen-binding fragment thereof comprising the complementarity determining regions (CDRs) of an antibody produced by a cell line deposited with accession number KCLRF-BP-00417 or KCLRF-BP-00418.

[0015] In yet another aspect, the present invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of the above-mentioned antibody or antigen-binding fragment thereof associated with a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise a small molecule inhibitor or vascular endothelial growth factor (VEGF) antagonist used in chemotherapy. In one aspect, the VEGF antagonist may be an anti-VEGF antibody, a VEGF inhibitory fusion protein, or a small molecule kinase inhibitor.

[0016] In yet another aspect, the present invention relates to a method for inhibiting tumor growth in a patient, comprising administering to the patient a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment. The method may further comprise administering simultaneously or stepwise with the administration of the antibody or fragment thereof of the present invention a small molecule inhibitor or vascular endothelial growth factor (VEGF) antagonist used in chemotherapy. The VEGF antagonist may be an anti-VEGF antibody, a VEGF inhibitory fusion protein or a small molecule kinase inhibitor.

[0017] In yet another aspect, the present invention relates to a method for inhibiting choroidal neovascularization, inhibiting ocular vascular leakage, or simultaneously triggering choroidal capillary regeneration in an eye disease patient, the method comprising administering the above-mentioned pharmaceutical composition to the patient. The method may further comprise administering a small molecule inhibitor or vascular endothelial growth factor (VEGF) antagonist used in chemotherapy simultaneously or stepwise with the administration of the antibody or fragment thereof of the present invention. The VEGF antagonist may be an anti-VEGF antibody, a VEGF inhibitory fusion protein, or a small molecule kinase inhibitor. The eye disease is wet age-related macular degeneration (wAMD), diabetic macular edema (DME), or diabetic retinopathy (DR).

[0018] These and other objects of the present invention will be more fully understood from the following description of the invention, the accompanying drawings and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be more fully understood from the detailed description given hereinafter and the accompanying drawings, which are given by way of illustration only and therefore do not limit the present invention, and in which;

[0020] Figure 1 Akt phosphorylation induced by anti-Ang2 antibodies. HUVECs were serum starved for 6 hr and incubated with COMP-Ang1 (CA1, 0.5 μg / ml) or anti-Ang2 antibodies (control, 2C8, 4B9, 2F10 and 4E2, respectively) for 30 min in the absence or presence of human Ang2 (1 μg / ml). Cell lysates were subjected to SDS-PAGE / Western blotting and blots were probed with anti-phospho-Akt (S473) or anti-Akt antibodies.

[0021] Figure 2.Schematic diagram showing the epitopes of anti-Ang2 antibodies, which have been analyzed by hydrogen / deuterium exchange mass spectrometry. Recombinant hAng2-RBD alone or hAng2-RBD / Ang2-antibody complex was labeled with deuterium. The labeled proteins were digested in a pepsin column and analyzed by mass spectrometry. Deuterium uptake of hAng2-RBD alone and hAng2-RBD / Ang-2 antibody complex was analyzed and the difference in deuterium uptake was compared. Peptides with a mass difference of more than 0.5-1 Da in deuterium uptake were identified as specific epitopes that mediate binding to anti-Ang2 antibodies. The 2C8 epitope (red) and the 4B9 epitope (green) are visualized in an image of the Ang2-RBD crystal structure (PDB: 2GY7) generated using PyMol software.

[0022] Figure 3 Dose-dependent Akt phosphorylation (pAkt) by humanized anti-Ang2 antibodies 4B9H11 and 2C8H11 Serum-starved HUVECs were incubated with human Ang2, anti-Ang2 antibodies, or human Ang2 and various concentrations of anti-Ang2 antibodies for 30 min. Cell lysates were subjected to SDS-PAGE / Western blotting.

[0023] Figure 4 Dose-dependent phosphorylation of Tie2 (pTie2) by humanized anti-Ang2 antibodies 4B9H11 and 2C8H11. Immunoprecipitation and Western blot analysis were performed to investigate the phosphorylation of 4B9H11 ( Figure 4 A) and 2C8H11( Figure 4 B) Ability of antibodies to induce Tie2 phosphorylation. Serum-starved HUVECs were incubated with human Ang2, anti-Ang2 antibody alone, or human Ang2 and various concentrations of anti-Ang2 antibody for 30 min. Cell lysates were immunoprecipitated with anti-Tie2 antibody and subsequently analyzed by SDS-PAGE / Western blot.

[0024] Figure 5 Tie2 receptor aggregation and FOXO1 translocation caused by humanized Ang2 antibody. HUVECs were serum starved for 6 hr and incubated with COMP-Ang1 (CA1), Ang2 (A2) or Ang2 and anti-Ang2 antibodies (control Ab, 2C8H11 or 4B9H11) for 30 min. After fixation, HUVECs were stained with DAPI (blue), anti-Tie2 antibody (green), anti-FOXO1 antibody (red) and anti-human Fc (cyan) to study Tie2 aggregation at the cell surface, translocation of FOXO1 from the nucleus, and the presence of humanized Ang2 antibodies in the intercellular junction area. Arrows indicate Tie2 aggregated at intercellular contacts and co-localized Ang2 antibodies.

[0025] Figure 6 .Time course of Tie2 receptor aggregation, FOXO1 translocation and Ang2 antibody localization in cell-to-cell junctions in HUVECs. Serum-starved HUVECs were incubated with anti-Ang2 antibodies (control Ab or 2C8H11) for different time points (from 10 min to 240 min). After cell fixation, Tie2 receptors aggregated at the cell surface and endocytosed Tie2 receptors were studied by staining with anti-Tie2 antibodies. Humanized anti-Ang2 antibodies at the cell surface and cytosol were probed with anti-human Fc antibodies. Arrows indicate Tie2 aggregated at cell-to-cell contacts and co-localized Ang2 antibodies.

[0026] Figure 7 . Inhibition of vascular permeability by humanized anti-Ang2 antibody. HUVECs were seeded on transwell chambers and grown for 3 days. At 100% confluence, HUVECs were pretreated with COMP-Ang1 (CA1, 0.5 μg / ml), Ang2 (A2, 1 μg / ml), Ang2 and control Ab (A2+control Ab, 1 μg / ml), 2C8H11 (A2+2C8H11, 1 μg / ml) or 4B9H11 (A2+4B9H11, 1 μg / ml) for 30 min and treated with TNF-a (100 ng / ml) for 22 hr into the upper chamber. Vascular permeability was assessed by measuring FITC fluorescence in the lower chamber 20 min after adding FITC-dextran in the upper chamber. Values ​​are mean ± SD. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA.

[0027] Figure 8 . The EC of mouse Ang2 by anti-Ang2 antibody obtained by ELISA 50 EC values ​​obtained by ELISA 50 To measure the binding affinity of humanized anti-Ang2 antibodies to mouse Ang2 (mAng2). Recombinant mAng2 was coated and incubated with serially diluted anti-Ang2 antibodies 4B9H11 and 2C8H11. Next, the plate was reacted with anti-human IgG (Fab)-HRP secondary antibody. The plate was treated with TMB solution and the absorbance was measured at 450 nm. EC was analyzed using the WorkOut 2.5 program from PerkinElmer. 50 value.

[0028] Fig. 9Humanized 2C8H11 antibody and cisplatin (Cpt) inhibit tumor growth in LLC tumor model. LLC tumor growth was compared in mice treated as indicated starting 7 days after tumor implantation. Black arrows indicate injections of antibody, while red arrows indicate single injections of Cpt. For each group, n=7-9. Values ​​are mean ± SD. *p<0.05 relative to Fc; #p<0.05 relative to Fc+Cpt.

[0029] Fig.10 Normalization of tumor vasculature by humanized 2C8H11 antibody. Comparison of PDGFRβ on tumors in LLC subcutaneous tumor model + Pericyte coverage and CD31 in intratumoral regions + BV. Scale bar, 100 μm. For each group, n=5. Values ​​are mean±SD. *p<0.05 vs. Fc; #p<0.05 vs. Fc+Cpt.

[0030] Fig.11 Hypoxia-induced perfusion reduction in tumor vessels by humanized 2C8H11 antibody. Tumor vascular perfusion was analyzed and compared with lectin and hypoxia probes in LLC tumors. + Hypoxia area. Hypoxia probe + Area is expressed as a percentage of the total cross-sectional area. Scale bar, 100 μm. For each group, n=5. Values ​​are mean±SD. *p<0.05 vs. Fc; #p<0.05 vs. Fc+Cpt.

[0031] Fig.12 .Enhanced delivery of Cpt drug to the tumor core by humanized 2C8H11 antibody. Using anti-Cpt-modified DNA antibody, Cpt was delivered to the tumor core in the tumor harvested on day 21. + Area imaging. + Area was measured as a percentage of total cross-sectional area. Scale bar, 100 μm. For each group, n=5. Values ​​are mean ± SD. #p<0.05 relative to Fc+Cpt.

[0032] Fig.13 Inhibition of CNV regression and vascular leakage by intravitreal injection of 2C8H11 antibody in a laser-induced CNV model Intravitreal administration of the antibody was performed 7 days after laser photocoagulation Measurement of CD31 +CNV volume, and leakage area around CNV was calculated as the total hyperfluorescent area measured in FA images divided by the total CNV area measured in ICGA images 6 days and / or 14 days after laser photocoagulation. Scale bar, 100 μm. For each group, n = 11. Values ​​are mean ± SD. By one-way ANOVA followed by Student-Newman-Keuls post hoc test, ***p < 0.001; by paired Student's t-test, ### p<0.001.

[0033] Fig.14 . Regression of CNV and choriocapillaris regeneration caused by intravitreal injection of 2C8H11 antibody. Intravitreal administration of the antibody was performed 7 days after laser photocoagulation. The CNV volume (area delineated by a white dashed box) and the avascular space surrounding the CNV (area delineated by a yellow dashed box) were measured by OCTA imaging of the eyes at 6, 14, 21, and 35 days after laser photocoagulation. For each group, n=11. Values ​​are mean ± SD. *p<0.05, **p<0.005 relative to Fc by one-way ANOVA followed by Student-Newman-Keuls post hoc test.

[0034] Fig.15 .Colocalization of 2C8H11 antibody and CD31 in CNV endothelial cells. 2C8H11 antibody was administered subcutaneously 1 day after laser photocoagulation. Colocalization of 2C8H11 antibody and CD31 in CNV endothelial cells was directly detected by anti-human IgG antibody 2, 4, and 8 days after laser photocoagulation.

[0035] Fig.16 CNV inhibition by subcutaneously injected 2C8H11 antibody. Subcutaneous administration of 2C8H11 antibody was performed 1 day after laser photocoagulation. CD31 was measured 8 days after laser photocoagulation. + CNV volume. Scale bar, 100 μm. For each group, n=10. Values ​​are mean±SD. ***p<0.001 by unpaired Student's t-test. DETAILED DESCRIPTION

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the terms used herein are well known in the art and are used in an ordinary manner.

[0037] In this application, "a" and "an" are used to refer to both singular and plural objects.

[0038] In one aspect, the present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to human angiopoietin-2 and induces Tie2 activation, wherein the antibody or the antigen-binding fragment thereof binds to the amino acid of SEQ ID NO:115, the amino acid of SEQ ID NO:116, or the amino acid of SEQ ID NO:117.

[0039] The amino acids of SEQ ID NO:115 correspond to amino acids 336-353 of SEQ ID NO:1, the amino acids of SEQ ID NO:116 correspond to amino acids 289-299 of SEQ ID NO:1, and the amino acids of SEQ ID NO:117 correspond to amino acids 316-322 of SEQ ID NO:1.

[0040] As used herein, the term "antibody that specifically binds to Ang2" refers to an antibody that binds to Ang2, thereby resulting in inhibition of the biological activity of Ang2, and is used interchangeably with "anti-Ang2 antibody" and "Ang2-binding antibody".

[0041] "Antibody" as used herein is an immunoglobulin molecule that reacts immunologically with a specific antigen, and means a protein molecule that acts as a receptor for specific antigen recognition, and may include all of polyclonal antibodies, monoclonal antibodies (single clone antibodies), whole antibodies, and antibody fragments. In addition, the antibody may include chimeric antibodies (e.g., humanized mouse antibodies) and bivalent or bispecific molecules (e.g., bispecific antibodies), double antibodies, three antibodies, and four antibodies.

[0042] A full antibody has a structure comprising two full-length light chains and two full-length heavy chains, and each light chain may be linked to a heavy chain by a disulfide bond. Full antibodies include IgA, IgD, IgE, IgM, and IgG, and IgG is a subtype and includes IgG1, IgG2, IgG3, and IgG4.

[0043] In the present disclosure, the antibody or antigen-binding fragment thereof can bind to human and mouse Ang2.

[0044] The antibody fragment means a fragment that retains the antigen binding function, and includes Fab, Fab', F(ab')2, scFv, Fv and the like.

[0045] Fab has a structure of the variable regions of the light chain and the heavy chain and the constant region of the light chain and the first constant region (CH1 domain) of the heavy chain, and has one antigen binding site. Fab' is different from Fab in that Fab' has a hinge region including one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are produced by achieving disulfide bonds of cysteine ​​residues in the hinge region of Fab'.

[0046] Fv (variable fragment) refers to the smallest antibody fragment with only heavy chain variable region and light chain variable region. In double-chain Fv (dsFv), heavy chain variable region and light chain variable region are connected by disulfide bonds. In single-chain Fv (scFv), heavy chain variable region and light chain variable region are usually connected by covalent bonds using peptide linkers. These antibody fragments can be obtained by using proteolytic enzymes (for example, Fab can be obtained by limiting the whole antibody with papain, and F (ab') 2 fragments can be obtained by cutting with pepsin), and can be constructed by recombinant DNA technology (for example, by using DNA encoding the heavy chain or its variable region of antibody and DNA encoding the light chain or its variable region as templates and using the PCR (polymerase chain reaction) method of primer pairs to amplify, and using the combination of DNA encoding the peptide linker of the primer pairs to amplify, so that the two ends of the peptide linker are respectively connected to the heavy chain or its variable region and the light chain or its variable region).

[0047] In the present disclosure, the antibody or antigen-binding fragment thereof may be humanized. Preferably, the anti-Ang2 antibody according to the present invention may be a fully human antibody selected from a human antibody library, but is not limited thereto.

[0048] The antibody or antigen-binding fragment thereof according to the present invention is characterized in that it contains a heavy chain variable region, which includes a heavy chain CDR1 having an amino acid sequence of SEQ ID NO:3, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO:4, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO:5; and a light chain variable region, which includes a light chain CDR1 having an amino acid sequence of SEQ ID NO:6, a light chain CDR2 having an amino acid sequence of SEQ ID NO:7, and a light chain CDR3 having an amino acid sequence of SEQ ID NO:8.

[0049] The antibody or antigen-binding fragment thereof according to the present invention is characterized in that it contains a heavy chain variable region, which includes a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 13, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 14, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 15; and a light chain variable region, which includes a light chain CDR1 having an amino acid sequence of SEQ ID NO: 16, a light chain CDR2 having an amino acid sequence of SEQ ID NO: 17, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 18.

[0050] In the present invention, the antibody or its antigen-binding fragment is characterized in that it contains a heavy chain variable region, which comprises the amino acid sequence of SEQ ID NO: 9, 19, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107 or 111; and a light chain variable region, which comprises the amino acid sequence of SEQ ID NO: 11, 21, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108 or 112, but is not limited thereto.

[0051] The amino acid sequence of the antibody can be substituted by conservative substitution. The "conservative substitution" refers to the modification of a polypeptide, which includes at least one amino acid being substituted by an amino acid having a biochemical property similar to that of the corresponding polypeptide, without causing loss of biological or biochemical function. "Conservative amino acid substitution" refers to such a substitution, in which an amino acid residue is substituted by an amino acid residue with a similar side chain. The art defines the types of amino acid residues with similar side chains. These types 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 uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), 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). It is expected that the antibodies of the invention can have conservative amino acid substitutions while still retaining activity.

[0052] In the present invention, the antibody or antigen-binding fragment thereof is characterized by containing the complementarity determining region (CDR) of the antibody produced by the cell line deposited with the deposit number KCLRF-BP-00417 or KCLRF-BP-00418.

[0053] The anti-Ang2 antibody sequences of the present invention may differ from the sequences provided in this application. For example, the amino acid sequences may differ from those listed above in that: (a) the variable region may be separated from the constant domain of the light chain, (b) the amino acids may differ from those listed above without thereby significantly affecting the chemical properties of the residues (so-called conservative substitutions), (c) the amino acids may differ from those listed above by a given percentage, for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology. Alternatively, the nucleic acid encoding the antibody may (a) be separated from the constant domain of the light chain, (b) differ from those listed above without thereby altering the encoded residues, or (c) may differ from those listed above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

[0054] When making conservative changes to the amino acid sequence, the hydropathic index of the amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biological function to a protein is generally understood in the art. It is generally recognized that the relative hydropathic character of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.).

[0055] It is also understood in the art that similar amino acid substitutions can be effectively performed based on hydrophilicity. For example, the maximum local average hydrophilicity of a protein, such as that governed by the hydrophilicity of its neighboring amino acids, is related to the biological properties of the protein. It should be understood that an amino acid can be substituted by another amino acid with similar hydrophilicity and produce a biologically or immunologically modified protein. In such changes, substitutions of amino acids with hydrophilicity values ​​within + / -2 are preferred, those within + / -1 are particularly preferred, and those within + / -0.5 are even more particularly preferred.

[0056] As described above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions taking into account various of the foregoing characteristics are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0057] In another aspect, the present invention relates to a pharmaceutical composition containing the antibody or antigen-binding fragment thereof as an active ingredient.

[0058] The pharmaceutical composition is characterized by containing a pharmaceutically effective amount of the antibody or antigen-binding fragment thereof according to the present invention and a pharmaceutically acceptable carrier.

[0059] The pharmaceutical composition may further comprise a small molecule inhibitor or vascular endothelial growth factor (VEGF) antagonist used in chemotherapy. The VEGF antagonist may be an anti-VEGF antibody, a VEGF inhibitory fusion protein or a small molecule kinase inhibitor.

[0060] In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating an eye disease, comprising the antibody or antigen-binding fragment thereof as an active ingredient.

[0061] In another aspect, the present invention relates to a method for inhibiting choroidal neovascularization, inhibiting ocular vascular leakage, or simultaneously triggering choriocapillaris regeneration in an ocular patient, the method comprising administering to the patient the above-mentioned pharmaceutical composition.

[0062] The pharmaceutical composition for preventing or treating ocular diseases may further comprise a small molecule inhibitor or vascular endothelial growth factor (VEGF) antagonist used in chemotherapy. The VEGF antagonist may be an anti-VEGF antibody, a VEGF inhibitory fusion protein or a small molecule kinase inhibitor.

[0063] The method may further comprise administering a small molecule inhibitor or vascular endothelial growth factor (VEGF) antagonist used in chemotherapy simultaneously or stepwise with the administration of the antibody or fragment thereof of the present invention. The VEGF antagonist may be an anti-VEGF antibody, a VEGF inhibitory fusion protein or a small molecule kinase inhibitor.

[0064] The anti-Ang2 antibody or the antigen-binding fragment thereof has a function of inhibiting abnormal angiogenesis by inhibiting the function of Ang2, and thus has an effect of preventing or treating eye diseases accompanied by abnormal blood vessels.

[0065] As used herein, the term "prevention" refers to any effect of inhibiting or slowing the progression of an eye disease by administering the composition according to the present invention, and the term "treatment" refers to inhibiting, alleviating or eliminating the development of an eye disease.

[0066] In the present invention, the eye disease is wet age-related macular degeneration (wAMD), diabetic macular edema (DME) or diabetic retinopathy (DR), but is not limited thereto.

[0067] As used herein, the term "macular degeneration" refers to a disease in which new blood vessels form abnormal growth, thereby causing damage to the macula and affecting vision. Macular degeneration mainly occurs in people over 50 years old and is divided into non-exudative (dry) or exudative (wet). In the case of wet AMD, blindness may result, in particular. The cause of AMD has not yet been elucidated, but the known risk factors are age; and environmental factors, including smoking, hypertension, obesity, genetic susceptibility, excessive UV exposure, low serum antioxidant concentrations, etc.

[0068] As used herein, the term "macular edema" refers to swelling of the macula of the retina, and the swelling occurs due to fluid leakage of retinal blood vessels. Blood leaks from weak blood vessel walls into a localized area of ​​the macula of the retina, which is a color-sensitive nerve ending and in which retinal cones are abundant. The image then fades to the right of the center or to the center of the central area. Vision gradually decreases over a few months. As used herein, the term "diabetic retinopathy" refers to a complication of the eye in which vision is reduced due to retinal microcirculation disorders caused by peripheral circulatory disorders caused by diabetes. Initially, it may cause mild problems with vision, but may eventually lead to blindness. Diabetic retinopathy can occur in anyone with type 1 diabetes or type 2 diabetes.

[0069] The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an anti-Ang2 antibody and a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" is a material that can be added to an active ingredient to aid in the formulation or stabilization of the preparation and that does not cause significant adverse toxicological effects on patients.

[0070] The carrier refers to a carrier or diluent that does not inhibit the biological activity and properties of the administered compound and does not irritate the patient. The pharmaceutically acceptable carrier in the composition to be formulated as a liquid solution is sterilized and suitable for living organisms. Saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol can be used as a carrier, or at least one component thereof can be mixed for use, and other conventional additives such as antioxidants, buffers, antibacterial agents, etc. can be added as needed. In addition, by further adding diluents, dispersants, surfactants, binders and lubricants thereto, the composition can be prepared into an injection (such as an aqueous solution, suspension, emulsion, etc.), a pill, a capsule, a granule or a tablet. Other carriers are described, for example, in [Remington's Pharmaceutical Sciences (EWMartin)]. The composition may contain a therapeutically effective amount of at least one anti-Ang2 antibody.

[0071] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for preparing temporary sterile injectable solutions or dispersions. The use of such media and medicaments for pharmaceutically active materials is known in the art. Preferably, the composition is formulated for parenteral injection. The composition can be formulated into solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. In some cases, the composition can include isotonic agents (e.g., sugars), polyols (such as mannitol, sorbitol) or sodium chloride. A sterile injection solution can be prepared by incorporating the required amount of active compound with one of the above-mentioned components or a combination thereof into an appropriate solvent, followed by aseptic microfiltration as required. Typically, dispersions are prepared by incorporating active compounds into sterile vehicles containing basic dispersion media and other required components from the above-mentioned components. Sterile powders for the preparation of sterile injectable solutions are obtained by vacuum drying or freeze drying (lyophilization) of a powder of the active ingredient and any additional desired ingredient from a previously sterile filtered solution.

[0072] The pharmaceutical composition can be administered orally or parenterally, and its dosage and frequency can be changed depending on the severity of the patient. The composition can be administered to the patient by push injection or by continuous infusion as needed. For example, the push injection of the antibody of the present invention presented as a Fab fragment can have an amount of 0.0025 to 100 mg / kg body weight, 0.025 to 0.25 mg / kg, 0.010 to 0.10 mg / kg or 0.10 to 0.50 mg / kg. For continuous infusion, the antibody of the present invention presented as a Fab fragment can be given 1 to 24 hours, 1 to 12 hours, 2 to 12 hours, 6 to 12 hours, 2 to 8 hours or 1 to 2 hours with 0.001 to 100 mg / kg minute, 0.0125 to 1.25 mg / kg / minute, 0.010 to 0.75 mg / kg / minute, 0.010 to 1.0 mg / kg / minute or 0.10 to 0.50 mg / kg / minute. When administering an antibody of the invention presented as a full-length antibody (with intact constant regions), the administration amount may be about 1 to 10 mg / kg body weight, 2 to 8 mg / kg, or 5 to 6 mg / kg. The full-length antibody is usually administered by injection lasting 30 to 35 minutes. The frequency of administration depends on the severity of the disease. The frequency may be 3 times per week to once a week or once every two weeks.

[0073] In addition, the composition can be administered to the patient by subcutaneous injection. For example, the anti-Ang2 antibody having an administration amount of 10 to 100 mg can be administered to the patient by subcutaneous injection weekly, biweekly or monthly.

[0074] As used herein, "therapeutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, as well as an amount of a combination of anti-Ang2 antibodies. The exact amount may vary depending on many factors including the components and physical characteristics of the therapeutic composition, the intended patient population, individual patient considerations, etc., but is not limited thereto, and can be easily determined by a person skilled in the art. When these factors are fully considered, it is important to give the minimum amount sufficient to obtain the maximum effect without side effects, and this dosage can be easily determined by an expert in the field.

[0075] The dosage of the pharmaceutical composition of the present invention is not particularly limited, but varies according to various factors, including the patient's health and body weight, the severity of the disease, and the type of drug, the route of administration, and the time of administration. The composition can be administered in a single dose or multiple doses per day by a route (e.g., oral, rectal, intravenous, subcutaneous, intrauterine, or intracerebrovascular) generally permitted in mammals (including rats, mice, cattle, humans, etc.).

[0076] In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising the antibody or antigen-binding fragment thereof as an active ingredient.

[0077] In another aspect, the present invention relates to a method for inhibiting tumor growth and treating cancer in a patient, comprising administering to the patient a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment. The method may further comprise administering simultaneously or stepwise with the administration of the antibody or fragment thereof of the present invention a small molecule inhibitor or vascular endothelial growth factor (VEGF) antagonist used in chemotherapy. The VEGF antagonist may be an anti-VEGF antibody, a VEGF inhibitory fusion protein or a small molecule kinase inhibitor.

[0078] As used herein, the terms "cancer" or "tumor" generally refer to or describe the physiological condition in mammals that is characterized by uncontrolled cell growth / proliferation.

[0079] The cancer that can be treated with the composition of the present invention is not particularly limited, and includes both solid cancer and hematological cancer. Examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, skin cancer, skin or intraocular melanoma, rectal cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumors, breast cancer, colon cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, osteosarcoma, etc., but are not limited thereto.

[0080] The composition for preventing or treating cancer comprises an anti-Ang2 antibody, and its constitution is the same as that comprised in the composition for preventing or treating ocular diseases, and thus the description of each constitution is equally applicable to the composition for preventing or treating cancer.

[0081] The present application also contemplates the use of the anti-Ang2 antibodies described herein in combination with chemotherapeutic or radiotherapeutic interventions or other treatments. Specifically, combining anti-Ang2 antibodies with other therapies targeting different aspects of Ang2 function may also prove effective.

[0082] In another embodiment, the antibodies of the present invention can be linked to at least one agent to form an antibody conjugate to increase the efficacy of the antibody molecule as a diagnostic or therapeutic agent.

[0083] In another aspect of the present invention, the present invention relates to a nucleic acid encoding the antibody or antigen-binding fragment thereof.

[0084] Nucleic acids used herein may be present in cells, cell lysates, or may also be present in partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially pure" when purified from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) by standard techniques including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. Nucleic acids of the present invention may be, for example, DNA or RNA, and may or may not contain intron sequences.

[0085] In still another aspect of the present invention, the present invention relates to a recombinant expression vector comprising the nucleic acid.

[0086] To express the antibodies or fragments thereof, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma expressing the target antibody), and the DNA can be "operably associated" with transcription and translation control sequences to be inserted into an expression vector.

[0087] The term "operably combined" as used herein can indicate that the antibody gene is connected to a vector so that the transcription and translation control sequences in the vector have the expected function of controlling the transcription and translation of the antibody gene. Expression vectors and expression control sequences are selected to have compatibility with the host cell to be used for expression. The light chain gene of the antibody and the heavy chain gene of the antibody are inserted into a separate vector, or the two genes are inserted into the same expression vector. The antibody is inserted into an expression vector by standard methods (for example, the antibody gene fragment is connected to the complementary restriction enzyme site on the vector, or a flat end connection is performed when there is no restriction enzyme site at all). In some cases, the recombinant expression vector can encode a signal peptide that promotes host cell secretion of the antibody chain. The antibody chain gene can be cloned into a vector so that the signal peptide is combined with the amino terminus of the antibody chain gene according to the framework. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (that is, a signal peptide derived from a protein other than an immunoglobulin). In addition, the recombinant expression vector has a regulatory sequence that controls the expression of the antibody chain gene in the host cell. "Regulatory sequences" may include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody chain genes. Those skilled in the art will recognize that the design of the expression vector may be altered by changing the regulatory sequences, depending on factors such as the choice of host cells to be transformed, the expression level of the protein, etc.

[0088] In still another aspect, the invention relates to a cell transformed with a recombinant expression vector.

[0089] The cells used to produce the antibodies of the present disclosure may be prokaryotes, yeast or higher eukaryotic cells, but are not limited thereto.

[0090] In particular, strains of Bacillus (e.g., Escherichia coli, Bacillus subtilis, and Bacillus tuligensis), Streptomyces, Pseudomonas (e.g., Pseudomonas putida), and prokaryotic host cells such as Proteus mirabilis and Staphylococcus (e.g., Staphylococcus carnosus) can be used.

[0091] Examples of animal cell lines of greatest interest and use include, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0092] The nucleic acid or vector is transfected into a host cell. For "transfection", various commonly used techniques (such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, lipofection, etc.) can be used to introduce exogenous nucleic acid (DNA or RNA) into a prokaryotic host cell or a eukaryotic host cell. Considering that it is suitable for use in mammalian cells, the antibodies according to the present invention can be expressed in eukaryotic cells, preferably in mammalian host cells. Mammalian host cells suitable for expressing the antibody can include Chinese hamster ovary (CHO) cells (e.g., including dhfr-CHO cells used with a DHFR selection marker), NSO myeloma cells, COS cells or SP2 cells, etc. as examples.

[0093] In another aspect, the present invention relates to a method for producing an anti-Ang2 antibody or an antigen-binding fragment thereof, comprising culturing a host cell and expressing the antibody or the antigen-binding fragment thereof.

[0094] When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody can be produced by culturing the host cell for a sufficient period of time to allow the antibody to be expressed in the host cell, or more preferably, for a sufficient period of time to allow the antibody to be secreted into the medium in which the host cells are cultured.

[0095] In some cases, the expressed antibody can be separated from the host cell and purified to obtain uniformity. The separation or purification of the antibody can be carried out by separation methods, purification methods (e.g., chromatography) commonly used for proteins. The chromatography can include, for example, affinity chromatography, ion exchange chromatography, or hydrophobic chromatography including protein A columns and protein G columns. In addition to chromatography, the antibody can also be separated and purified by further combination with filtration, ultrafiltration, salting out, dialysis, etc.

[0096] Example

[0097] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only used to illustrate the present invention, and it is clear to those skilled in the art that the scope of the present invention should not be interpreted as limiting these examples.

[0098] Example 1: Preparation of mouse monoclonal anti-Ang2 antibody

[0099] 1-1: Immunization of mice with human Ang2

[0100] For use as an antigen, the receptor binding domain (RBD) of human Ang2 (hAng2, SEQ ID NO: 2) was cloned into a vector containing a CMV promoter and transiently expressed by transfection into a HEK293F cell line. After incubation for 5 days, the expressed recombinant human Ang2-RBD was purified by an affinity column. Five-week-old BALB / c mice were immunized twice a week with purified human Ang2-RBD (100 μg / injection) mixed with adjuvant for 6 weeks. The anti-Ang2 antibody titer in the serum of immunized mice was examined by hAng2 ELISA. When the antibody titer (1:5,000 dilution) increased appropriately (OD>1.0), the spleen was extracted from the immunized mouse, and B lymphocytes were isolated therefrom and fused with cultured myeloma cells (SP2 / 0). The fused cells were cultured in HAT medium containing hypoxanthine, aminopterin and thymidine, and hybridoma cells consisting only of the fusion of myeloma cells and B lymphocytes were selected and cultured. Surviving hybridoma cells were seeded in 96-well plates and the culture supernatants were tested by hAng2 ELISA. Hybridoma pools showing positive signals were selected by limiting dilution for clonal selection. Ultimately, about 50 monoclonal hybridoma cell lines were established. Among them, several Ang2-binding antibodies showed Tie2 activation activity. Candidate antibodies were selected based on Tie2 activation levels and high affinity for human Ang2 and then processed for humanization.

[0101] [Table 1]

[0102] Human angiopoietin-2 full length (hAng2) and receptor binding domain (RBD) sequences

[0103]

[0104] 1-2: Production and purification of mouse monoclonal anti-Ang2 antibodies

[0105] To generate anti-Ang2 antibodies selected based on ELISA positive reactions, the cells were plated at T75 (75 cm 2Hybridoma cells were cultured in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS in a flask (10% area). When the cell confluence reached about 90%, the cells were washed with PBS, incubated with 50ml serum-free medium (SFM, Gibco) and cultured at 37°C for 3 days. Then, the culture medium in which the antibody was secreted from each monoclonal hybridoma was collected, centrifuged to remove the cells, and the culture supernatant was collected and filtered. The antibodies were then purified using an AKTA purification device (GE Healthcare) equipped with a Protein G affinity column (GE Healthcare). The purified antibodies were concentrated using a centrifugal filter unit (Amicon) by replacing the supernatant with PBS.

[0106] 1-3: Identification and screening of anti-Ang2 antibodies that activate Tie2 receptors

[0107] To investigate whether mouse anti-Ang2 antibodies induce downstream signaling of Tie2 receptors in endothelial cells, HUVECs (Lonza) were treated with a combination of hAng2 protein and anti-Ang2 antibodies, and then the phosphorylation level of Akt, a major downstream signaling protein of Tie2 receptors, was analyzed by immunoblotting. As a negative control group, full-length hAng2 alone (R&D systems) was treated into the cells.

[0108] Specifically, HUVEC (1×10 5 Cells / ml) were cultured in EGM-2 medium (Lonza) at 37°C in 60mm culture dishes. Cells (90% confluence) were incubated with serum-free EBM-2 medium for 4 hours for serum starvation. Serum-starved HUVECs were treated with a mixture of anti-Ang2 antibody and hAng2 protein (1 μg / ml, R&D system) and further incubated for 30 min. The cells were washed with cold PBS, treated with lysis buffer, and lysed at 4°C for 20 min. Cell lysates were then prepared by centrifugation at 13000 rpm for 15 min. 5x SDS sample buffer was added to the cell lysate, and the cell lysate was boiled at 95°C for 5 minutes. The cell lysate was then subjected to SDS PAGE, and the protein was transferred to a nitrocellulose membrane (GE).

[0109] To study Akt phosphorylation, the blot was blocked with 5% skim milk in TBS-T at room temperature (RT) for 1 hr and incubated with anti-phospho-Akt antibody (S473) at 4°C for about 8 hr. The amount of phospho-Akt was visualized by enhanced chemiluminescence (ECL). The membrane was then incubated in stripping buffer (Thermo) for 15 min and then probed again with anti-Akt antibody to determine the amount of total Akt.

[0110] In several groups treated with a combination of hAng2 and anti-Ang2 antibodies (such as 2C8, 4B9, 2F10, and 4E2), Akt phosphorylation at S473 was strongly induced ( Figure 1 ).

[0111] 1-4: Affinity measurement of anti-Ang2 antibodies against hAng2 by Octet analysis

[0112] The affinity of mouse monoclonal antibodies against hAng2 was measured using the Octet system (ForteBio). Specifically, a black 96-well plate (96-well F-type black plate, Greiner) was used to measure the buffer and sample in a total of 200 μl / well. The biosensor for affinity measurement was hydrated for 10 min and then measured using an AR2G tip (ForteBio Octet). After hydration, hAng2 was diluted in 10 mM sodium acetate buffer (pH 6.0) at a concentration of 10 μg / ml, immobilized on the AR2G biosensor, and blocked with 1 M ethanolamine. Mouse monoclonal anti-Ang2 antibodies were diluted to 50, 25, 12.5, 6.25, 3.125, and 0 nM with 1× kinetic buffer, and association was performed for 300 seconds and dissociation for 900 seconds. For affinity measurements (K D ), the association rate (K-on) and dissociation rate (K-off) were analyzed by binding curve (global) and fitted to a 1:1 binding model using Octet data analysis v9.0.0.10 program. K D The values ​​are shown in Table 2 below. The affinity of mouse anti-Ang2 antibodies to hAng2 is shown in Table 2.

[0113] [Table 2]

[0114] Affinity of mouse anti-Ang2 antibodies for hAng2

[0115] Antibody Kon(1 / Ms) Koff(1 / s) <![CDATA[K D (M)]]> 2C8 7.78E+04 3.54E-06 4.55E-11 2F10 1.24E+05 1.71E-05 1.38E-10 4B9 1.37E+05 5.04E-07 3.68E-12 4E2 2.83E+04 1.34E-04 4.74E-09

[0116] Example 2: DNA gene sequence analysis of mouse anti-Ang2 antibody

[0117] The DNA nucleotide sequences of the antibodies (derived from hybridoma cells) selected in Examples 1-3 were analyzed. Specifically, hybridoma cells (2×10 6Cells / ml), and then total RNA was obtained using the RNeasy mini kit (Qiagen). Next, the RNA concentration was measured, and cDNA was synthesized by reverse transcription (RT) reaction. In order to amplify the heavy chain and light chain variable region gene sequences of the monoclonal antibodies produced in each hybridoma cell, PCR was performed using the above cDNA as a template using a mouse Ig-primer set (Novagen) under the following conditions: 94°C for 5 minutes; [94°C for 1min, 50°C for 1min, 72°C for 2min] × 35 cycles; at 72°C for 6min; cooled to 4°C. The PCR products obtained from each reaction were cloned into a TA vector and subjected to DNA sequencing to obtain the nucleotide sequences encoding the CDR, heavy chain variable region, and light chain variable region of each antibody (Tables 3 to 10).

[0118] [Table 3]

[0119] CDR sequences of mouse anti-Ang2 antibody 4B9

[0120]

[0121]

[0122] [Table 4]

[0123] Variable region sequence of mouse anti-Ang2 antibody 4B9

[0124] [Table 5]

[0125] CDR sequences of mouse anti-Ang2 antibody 2C8

[0126]

[0127] [Table 6]

[0128] Variable region sequence of mouse anti-Ang2 antibody 2C8

[0129] [Table 7]

[0130] CDR sequences of mouse anti-Ang2 antibody 2F10

[0131]

[0132] [Table 8]

[0133] Variable region sequence of mouse anti-Ang2 antibody 2F10

[0134] [Table 9]

[0135] CDR sequences of mouse anti-Ang2 antibody 4E2

[0136]

[0137] [Table 10]

[0138] Variable region sequence of mouse anti-Ang2 antibody 4E2

[0139]

[0140] Example 3: Epitope mapping of mouse anti-Ang2 antibodies against hAng2

[0141] The antigenic determinants (epitopes) of hAng2 recognized by mouse monoclonal antibodies 2C8 and 4B9 were analyzed by HDX-MS (hydrogen / deuterium exchange mass spectrometry) technology. The HDX-MS analysis method is described in the following articles: Houde D, Engen JR (2013) Methods Mol. Biol. 988: 269-89 and Houde et al. (2011) J. Pharm. Sci. 100 (6), 2071.

[0142] The recombinant hAng2-RBD protein was used to analyze the epitopes of antibodies 2C8 and 4B9. Before the deuterium labeling reaction, the hAng2-RBD / antibody mixture was incubated for more than 3 hrs in a 15× diluted deuterium labeling buffer (K D =25nM) to maintain maximum binding (100%). The prepared hAng2-RBD / antibody complex was diluted 15 times with deuterium labeling buffer labeled at different times and then quenched with the same volume of quenching buffer. The labeling reaction time was 0min (non-deuterium), 0.33min, 10min, 60min and 240min. However, under non-deuterium conditions, the deuterium labeling buffer was replaced with the equilibrium buffer and the reaction was immediately terminated with the quenching buffer. For mass spectrometry analysis, the deuterium-labeled hAng2-RBD / antibody sample was loaded onto a pepsin column and peptide digestion was performed. Mass spectrometry analysis showed that the 13 peptides at the N-terminus of hAng2-RBD and the peptic peptides corresponding to 25-40 amino acids were not detected, and 83.7% coverage data was obtained from a total of 45 peptic peptides.

[0143] The difference in deuterium uptake between hAng2-RBD alone and hAng2-RBD-antibody complex conditions was analyzed comparatively, and regions showing a significant decrease in deuterium uptake were peptides directly bound by the antibody or regions of structural alteration. The difference in deuterium uptake between hAng2-RBD alone and hAng2-RBD-antibody complex was considered significant when it was 0.5-1 Da or greater.

[0144] Analysis of the differences in deuterium uptake indicated that the epitope bound by antibody 2C8 was residues 61 to 78 of SEQ ID NO: 2 of hAng2-RBD -QRTWKEYKVGFGNPSGEY (SEQ ID NO: 115) (Table 11), and the epitope bound by antibody 4B9 was residues 14 to 24 of SEQ ID NO: 2 -KSGHTTNGIYT (SEQ ID NO: 116) and residues 41 to 47 of SEQ ID NO: 2 -EAGGGGW (SEQ ID NO: 117) (Table 12). In the case of antibody 4B9, it cannot be excluded that an undetermined region (residues 25 to 40 of SEQ ID NO: 2) may be included within the scope of the epitope. The results of the epitope analysis for each antibody are shown in different colors on the 3D structure of hAng2-RBD generated using the PyMol software ( Figure 2 ).

[0145] [Table 11]

[0146] Epitope mapping analysis of 2C8 binding to hAng2 by HDX-MS

[0147]

[0148]

[0149] [Table 12]

[0150] Epitope mapping analysis of 4B9 binding to hAng2 by HDX-MS

[0151]

[0152] Example 4: Humanization of mouse anti-Ang2 antibody and conversion to full-length IgG

[0153] To eliminate the immunogenicity of mouse anti-Ang2 antibodies 2C8 and 4B9 when administered to humans, the antibodies were humanized as follows.

[0154] 4-1: Heavy chain humanization

[0155] The human antibody heavy chain variable gene showing 64% homology with the heavy chain sequence of antibody 2C8 is IGHV1-46-01. Based on these analyses, the CDR region of 2C8 antibody was transplanted into the human antibody heavy chain variable gene IGHV1-46-01. In this process, 5 humanized heavy chain antibody genes were designed (Table 13). In the heavy chain gene of humanized 2C8, a reverse mutation of the mouse sequence was introduced, which is represented in bold in the protein sequence of Table 13.

[0156] The human antibody heavy chain variable gene showing 80% homology with the heavy chain sequence of antibody 4B9 is IGHV3-11-01. Based on the analysis, the CDR region of 4B9 antibody is transplanted into the human antibody heavy chain variable gene IGHV3-11-01. As a result, 3 humanized heavy chain antibody genes (Table 13) were designed in this process. In the heavy chain gene of humanized 4B9, the reverse mutation of mouse sequence was introduced, which is represented in bold in the protein sequence of Table 13.

[0157] 4-2: Light chain humanization

[0158] The human antibody light chain variable gene showing 67% homology with the light chain sequence of antibody 2C8 is IGKV1-9-01. Based on these analyses, the CDR region of the 2C8 antibody was transplanted into the human antibody light chain variable gene IGKV1-9-01. In this process, 3 humanized light chain antibody genes were designed (Table 13). In the light chain gene of humanized 2C8, a reverse mutation of the mouse sequence was introduced, which is represented in bold in the protein sequence of Table 13.

[0159] The human antibody light chain variable gene showing 70% homology with the light chain sequence of antibody 4B9 is IGKV1-39-01. Based on these analyses, the CDR region of the 4B9 antibody was transplanted into the human antibody light chain variable gene IGKV1-39-01. In this process, a humanized light chain antibody gene was designed (Table 13).

[0160] 4-3: Humanized gene synthesis and cloning of full-length human IgG antibodies

[0161] The humanized variable regions of the antibodies in Table 15 were incorporated into the heavy chain and light chain vectors of human IgG1 antibodies. The coding nucleotides corresponding to the humanized heavy chain variable region (VH) of the antibody were synthesized by Bioneer, Inc., thereby constituting "EcoRI-signal sequence-VH-NheI-CH-XhoI". The coding nucleotides corresponding to the humanized light chain variable region (VL) of the antibody were synthesized by Bioneer, Inc., thereby constituting "EcoRI-signal sequence-VL-BsiWI-CL-XhoI". EcoRI and XhoI were used to establish a vector expressing a full-length human IgG antibody, and the polynucleotides encoding the heavy chain were cloned into OptiCHOTM pOptiVEC included in the Antibody Expression Kit (Invitrogen) TM -TOPO TA cloning kit, and clone the polynucleotide encoding the light chain into pcDNA TM 3.3-TOPO TA cloning kit (Invitrogen) vector. For the construction of human IgG4 class antibodies of 2C8H11 and 4B9H11, respectively designated as 2C8H11G4 and 4B9H11G4, the constant regions (CH1-hinge-CH2-CH3) of the 2C8H11 heavy chain gene and the 4B9 heavy chain gene were replaced with polynucleotides encoding IgG4 class heavy chain constant regions.

[0162] [Table 13]

[0163] Humanized anti-Ang2 antibody derived from mouse 4B9 and 2C8 antibodies

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174] 4-4: Production and purification of humanized anti-Ang2 antibodies

[0175] To produce humanized anti-Ang2 antibodies, Expi293F (Gibco) cells were used, which are capable of producing recombinant proteins with high efficiency. Expi293F cells (2 × 10 6Cells / ml), and the plasmids encoding heavy and light chains were co-transfected into Expi293F cells using the ExpiFectamine293 transfection kit. The cells were cultured for 5 days at 37°C, 8% CO2 in an oscillating incubator (orbital oscillator, 125rpm). The resulting culture medium was collected and centrifuged to remove the cells. The culture supernatant containing the secreted antibody was separated and stored at 4°C, or immediately purified using an AKTA purification system (GE Healthcare) equipped with an affinity column (protein A agarose column, GE Healthcare). The purified antibody was concentrated by passing through a 0.2 μm protein centrifugal filter (Amicon), while replacing the solution with PBS.

[0176] Example 5: Affinity measurement of humanized anti-Ang2 antibodies against hAng2

[0177] The affinity of humanized anti-Ang2 antibodies for hAng2 was measured using the Octet system (ForteBio). Specifically, a black 96-well plate (96-well F-type black plate, Greiner) was used to measure the buffer and sample in a total of 200 μl / well. The biosensor for affinity measurement was hydrated for 10 min and then measured using an AR2G tip (ForteBio Octet). After hydration, the humanized anti-Ang2 antibody was diluted at a concentration of 10 μg / ml in 10 mM sodium acetate buffer (pH 6.0), immobilized on the AR2G biosensor, and blocked with 1 M ethanolamine. Recombinant hAng2 was diluted to 50, 25, 12.5, 6.25, 3.125, and 0 nM with 1× kinetic buffer, and association was performed for 300 seconds and dissociation for 900 seconds. For affinity measurements (K D ), the association rate (K-on) and dissociation rate (K-off) were analyzed by binding curve (global) and fitted to a 1:1 binding model using Octet data analysis v9.0.0.10 program. K D The values ​​are shown in Tables 14-15 below.

[0178] The affinity of the humanized 4B9 antibody to hAng2 is summarized in Table 14. The affinity of the humanized 2C8 antibody to hAng2 is in Table 15. In addition, the IgG4 class 2C8H11G4 and 4B9H11G4 antibodies also showed sub-nanomolar high affinity to the hAng2 antigen (Table 16).

[0179] [Table 14]

[0180] Affinity of humanized 4B9 antibody for hAng2

[0181] Antibody Kon(1 / Ms) Kdis(1 / s) <![CDATA[K D (M)]]> 4B9H11 9.29E+04 1.58E-06 1.71E-11 4B9H21 7.37E+04 8.94E-06 1.21E-10 4B9H31 9.39E+04 1.56E-05 1.67E-10

[0182] [Table 15]

[0183] Affinity of humanized 2C8 antibody for hAng2

[0184]

[0185]

[0186] [Table 16]

[0187] Affinity of IgG4-class 2C8H11G4 and 4B9H11G4 antibodies for hAng2

[0188] Antibody Kon(1 / Ms) Kdis(1 / s) <![CDATA[K D (M)]]> 2C8H11G4 4.15E+05 4.35E-06 1.05E-11 4B9H11G4 4.32E+05 3.46E-05 8.00E-11

[0189] Example 6: In vitro biological properties analysis of selected humanized anti-Ang2 antibodies

[0190] 6-1: Akt phosphorylation

[0191] To investigate whether humanized anti-Ang2 antibodies induce downstream signaling of Tie2 receptors in endothelial cells, HUVEC (Lonza) were treated with human Ang2 protein and humanized anti-Ang2 antibodies. Then, phosphorylation levels of Akt, a major downstream signaling protein of Tie2 receptors, were measured by immunoblotting. To compare the extent of Akt activation, cells were treated with full-length hAng2 (R&D systems) alone or with antibodies alone in the experiment.

[0192] Specifically, HUVEC cells (1×10 5 Cells / ml) were cultured in EGM-2 (Lonza) at 37°C in 60 mm culture dishes. Cells at 90% confluence were incubated with EBM-2 (Lonza) for 4 hr. Serum-starved HUVECs were treated with a mixture of anti-Ang2 antibody and hAng2 protein (1 μg / ml, R&D system) and further incubated for 30 min. The cells were washed with cold PBS, treated with lysis buffer, and lysed at 4°C for 20 min. Cell lysates were then prepared by centrifugation at 13000 rpm for 15 min. 5x SDS sample buffer was added to the cell lysate, and the mixture was boiled at 95°C for 5 min. The mixture was then subjected to SDS-PAGE and subsequent protein blotting.

[0193] To study the phosphorylation of Akt, the membrane was blocked with 5% skim milk in TBST at RT for 1 hr and incubated with anti-phospho-Akt antibody (S473) at 4°C for about 8 hr. The amount of phospho-Akt was visualized by enhanced chemiluminescence (ECL). The membrane was then incubated in stripping buffer (Thermo) for 15 min and then probed again with anti-Akt antibody to determine the amount of total Akt.

[0194] like Figure 3 As shown, Akt phosphorylation was significantly increased by treatment with 0.5 μg / ml anti-Ang2 antibody in the presence of hAng2, and it was maintained up to an antibody concentration of 50 μg / ml in both the 4B9H11-treated group and the 2C8H11-treated group. These data indicate that humanized anti-Ang2 antibodies can strongly induce activation of Akt, a major downstream signaling molecule of the Tie2 receptor, in endothelial cells. Similar patterns were observed when humanized 4B9H11- or 2C8H11-IgG4 antibodies were tested.

[0195] 6-2: Tie2 phosphorylation induced by humanized anti-Ang2 antibody

[0196] Ang2 binds to the Tie2 receptor and acts as a weak agonist or antagonist. The anti-Ang2 antibody developed in the present invention binds to Ang2 to induce an Ang2-antibody complex, thereby further causing the aggregation of the Tie2 receptor and thus enhancing the activation of the Tie2 receptor. An experiment was conducted to analyze the effect of the anti-Ang2 antibody on Tie2 phosphorylation using HUVEC.

[0197] Specifically, HUVEC (Lonza) was cultured in EGM-2 (Lonza) at 37°C and 5% CO2 concentration in a 100 mm culture dish. At 80%-90% confluence, the cells were replaced with EBM-2 (Lonza) medium for 2 to 6 hrs for serum starvation. Various concentrations (0.02 μg / ml to 50 μg / ml) of humanized anti-Ang2 antibodies were mixed with hAng2 protein (1 μg / ml, R&D systems) for 30 min. Then, the mixture was treated with cultured cells and further incubated for 30 min. The cells were washed twice with cold PBS and then lysed in 1000 μl lysis buffer (10 mM Tris-Cl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, protease inhibitors, phosphatase inhibitors), and then lysed at 4°C for 60 min. Cell extracts were prepared and centrifuged at 12,000 rpm for 10 min. The supernatant was quantified by BCA assay.

[0198] 1 μg of Tie2 antibody (R&D systems, AF313) was added to 0.5 mg of cell lysate and incubated overnight at 4°C. Then, Dynabeads TM Protein G (Life technologies) was reacted for 2 hr and immunoprecipitated. The beads were fixed on one side of the test tube using a magnet, washed three times with lysis buffer, and then incubated at 70°C with 2x SDS sample buffer containing a reducing agent for 10 min. The beads were removed from the sample and electrophoresed on a 4%-15% SDS protein gel (Bio-Rad) and then transferred to a 0.45 μm PVDF membrane.

[0199] The membrane was blocked with TBS-T mixed with 5% (v / v) BSA for 1 hr at room temperature and incubated with anti-phosphotyrosine antibody (4G10, Millipore) for 8 hr at 4°C, followed by incubation with HRP-conjugated anti-mouse antibody and subsequent Western blotting. To measure the amount of immunoprecipitated Tie2, the membrane was reacted in stripping buffer (Thermo) for 15 min, then blocked again and probed again with anti-Tie2 antibody (R&D systems, AF313). Figure 4 As shown in Figure 5, when anti-Ang2 antibody was added to HUVEC cells together with Ang2, the phosphorylation of Tie2 was strongly induced in a dose-dependent manner, which was consistent with Figure 3 Similar patterns were observed when humanized 4B9H11- or 2C8H11-IgG4 antibodies were tested. These data indicate that humanized anti-Ang2 antibodies 2C8H11 and 4B9H11 directly induce activation of Tie2 receptors in human endothelial cells.

[0200] 6-3: Tie2 aggregation and FOXO1 translocation in HUVECs

[0201] Tie2 aggregation at the intercellular junction area caused by anti-Ang2 antibodies and translocation of FOXO1 from the nucleus to the cytosol were detected in HUVEC by immunofluorescence. Specifically, HUVECs were seeded on 8-well slide chambers (Lab-TekII) and maintained in EGM-2 medium for 2 to 3 days. At 100% confluence, the cells were serum starved for 4 hr with EBM-2 medium and then treated with 1 μg / ml anti-Ang2 antibodies and 1 μg / ml hAng2 for 30 min. Thereafter, the cells were fixed with 4% formaldehyde in PBS for 10 min at room temperature (RT), permeabilized with 0.1% Triton X-100 in PBS, blocked with 1% BSA in PBS for 60 min at RT, and incubated with the primary antibody for 1 hr at RT. Primary antibodies for hTie2, FOXO1, and human Fc were used. The cells were then incubated with secondary antibodies (Invitrogen) for 1 hr at RT in the dark and mounted with Vectashield mounting medium with DAPI (Vector Labs). Images were captured using a laser scanning confocal microscope (LSM880, Carl Zeiss).

[0202] like Figure 5 As shown, treatment with 2C8H11 or 4B9H11 together with hAng2 induced Tie2 translocation / aggregation to cell-cell contacts, just like Comp-Ang1 (CA1) or control Ang2 antibodies known to induce Tie2 aggregation and activation (Han et al., 2016, Science Translation Medicine). Consistent with previous reports showing that FOXO1 is localized in the cytoplasm after phosphorylation (Zhang et al., JBC 2002, 277, 45276–45284), when FOXO1 is localized in the nucleus under basal, serum-starved conditions, FOXO1 is significantly absent from the nucleus when treated with 2C8H11+hAng2 or 4B9H11+hAng2 compared to serum-starved controls (red). At the same time, Ang2 treatment negligibly induced FOXO1 translocation from the nucleus to the cytosol. Interestingly, humanized antibodies 2C8H11, 4B9H11 (cyan) were found to colocalize with aggregated Tie2 receptors at cell-cell contacts and that the antibodies internalized Tie2 receptors in the cytosol ( Figure 5 ), indicating that the anti-Ang2 antibody forms a ternary complex with the Tie2 receptor by binding to Ang2.

[0203] In a time course study (from 10 min to 240 min), 2C8H11-induced Tie2 aggregation and FOXO1 translocation were examined. Figure 6As shown, in the presence of hAng2, the control Ang2 Ab induced Tie2 aggregation at cell-cell contacts (green) within 10 min and triggered endocytosis of aggregated Tie2 receptors. After 30 min of treatment with the control Ang2 Ab+hAng2, the Tie2 receptors at cell-cell contacts were significantly reduced, and most of the Tie2 receptors disappeared within 120 min and 240 min. When the control Ang2 antibody was stained with an anti-human Fc antibody (cyan), it showed a similar pattern as that of Tie2 receptors. In contrast, in the case of 2C8H11 and hAng2, Tie2 aggregation at cell-cell contacts was maintained even after 240 min of treatment. Consistently, the 2C8H11 antibody co-localized with Tie2 at cell-cell contacts was also maintained until 240 min ( Figure 6 ).

[0204] 6-4: Inhibition of vascular permeability by humanized anti-Ang2 antibody

[0205] Vascular leakage assays were performed in HUVEC using an in vitro vascular permeability assay kit (Millipore) according to the manufacturer's instructions. HUVECs were seeded into inserts of penetration plates and cultured for 3 days to reach 100% confluence. HUVECs were pre-incubated with Ang2 (1 μg / ml), Ang2 (1 μg / ml) and control, 2C8H11 or 4B9H11 antibodies (1 μg / ml) for 30 min, then TNF-a (100 ng / ml) was added and the cells were incubated at 37°C for 22 hr. FITC-dextran was added to the upper chamber and incubated for 20 min. The passage of FITC-dextran across the HUVEC monolayer was measured by a fluorescence reader at excitation and emission wavelengths of 485 and 535 nm, respectively. Figure 7 As shown, pretreatment with anti-Ang2 antibody and Ang2 significantly inhibited vascular leakage induced by vascular leakage promoting factor TNF-a.

[0206] Example 7: Affinity measurement of humanized anti-Ang2 antibodies against mAng2

[0207] The affinity of humanized antibodies to mouse Ang2 (mAng2) was analyzed by ELISA. Specifically, mAng2 was diluted in 30 μl coating buffer (0.1M sodium carbonate buffer) at 20 ng per well in a half 96-well plate (Corning 3690) and incubated overnight at 4 ° C. After washing 3 times with TBS-T solution, the well plate was blocked with 3% skim milk for 1 hr at room temperature and then washed again. 2C8H11 and 4B9H11 were continuously diluted from 3 mg / ml to 300 ng / ml. After 30 μl of diluted anti-Ang2 antibody was loaded into the well, the well plate was incubated at room temperature for 2 hr. Next, 30 μl of anti-human IgG (Fab)-HRP (Jackson) secondary antibody 1: 3000 dilution was added to each well and incubated at room temperature for 1 hr. After all reactions were completed, the plate was washed again with TBS-T, and each well was treated with 30 μl of TMB solution. After 5 min of development, the plate was treated with 1 N sulfuric acid to stop the reaction, and the absorbance was measured at 450 nm. Based on the measured OD values, EC was analyzed using the WorkOut 2.5 program from PerkinElmer. 50 EC values ​​of 4B9H11 and 2C8H11 for mAng2 binding 50 105μg / ml and 97μg / ml respectively ( Figure 8 ).

[0208] Example 8: Evaluation of tumor growth inhibition in LLC subcutaneous model.

[0209] The ability of 2C8H11 anti-Ang2 antibody to inhibit tumor growth was tested in the LLC (Lewis Lung Carcinoma) cell line tumor model. Specifically, LLC cell line (ATCC) was cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco). LLC cells (1×10 6 ) were injected subcutaneously into 6- to 8-week-old C57BL / 6 mice (Jackson Laboratory) anesthetized with a mixture of ketamine and xylazine. 3 10 mg / kg of 2C8H11 antibody was intraperitoneally administered to mice every 2 to 3 days. In both the monotherapy and combination therapy groups, cisplatin (Cpt) was injected intraperitoneally once at a dose of 3 mg / kg. Changes in tumor volume were tracked over the next few days. Tumor volume (V) was measured using the following formula:

[0210] V=(width 2 × length) / 2

[0211] The experiment was conducted in 4 groups: Fc (control group), Fc+Cpt group, 2C8H11 group, and 2C8H11+Cpt group. Fig. 9 As shown, the 2C8H11 antibody inhibited tumor growth by 29% compared to Fc, which is similar to the tumor growth inhibition obtained by Fc+Cpt injection. At the same time, treatment with 2C8H11 and Cpt combined delayed tumor growth by 47% compared to Fc. Therefore, these results demonstrate that treatment with 2C8H11 combined with Cpt most effectively inhibits tumor growth.

[0212] Example 9: Tumor vascular normalization effect of 2C8H11 antibody.

[0213] In order to study the changes in tumor blood vessels caused by 2C811 antibodies, we obtained frozen sections of tumor samples and performed immunofluorescence analysis by staining with vascular-specific marker CD31 and pericyte-specific marker PDGFRβ. Specifically, tumor samples were harvested from mice from the experiment described in Example 8, fixed in 4% paraformaldehyde (PFA, Merck), dehydrated in 30% sucrose (Junsei), embedded in OCT compound (Leica), and sliced ​​using a cryostat (Leica). The resulting frozen sections were blocked for 1 hr using protein blocking buffer (DAKO). The sections were then stained with hamster anti-CD31 antibodies (1:200, Millipore) and rat anti-PDGFRβ antibodies (1:200, eBioscience) in PBS for 8 hr at 4 °C. After washing 3 times with PBS, the sections were stained with Alexa488-conjugated anti-hamster IgG antibody and Alexa594-conjugated anti-rat IgG antibody (1:1000, Jackson Immunoresearch) in PBS for 1 hr at room temperature. After washing 3 more times with PBS, the sections were mounted in fluorescent mounting medium (DAKO) using coverslips (Marienfeld). The stained sections were imaged using a LSM880 confocal microscope (Zeiss).

[0214] The results are Fig.10 Shown in the figure; red signal indicates CD31 + region, and the green signal indicates PDGFR-β + Compared with tumors treated with Fc or Fc+Cpt, tumor blood vessel (BV) density was reduced by 56% in tumors treated with 2C8H11 or 2C8H11+Cpt, and the morphology of these vessels normalized to resemble normal vessels. In addition, tumors treated with 2C8H11 or 2C8H11+Cpt had increased PDGFRβ +The pericyte coverage was increased (increased 2.4-fold), and blood vessels and pericytes were more closely associated with each other. These results show that the 2C8H11 antibody can reduce the density of blood vessels within the tumor mass and normalize its morphology.

[0215] Example 10: Increasing tumor vascular functionality by 2C8H11 antibody.

[0216] In order to analyze the functionality of tumor blood vessels after treatment with 2C8H11, vascular perfusability and hypoxia were evaluated. Before harvesting tumor masses, mice were intravenously injected with 100 μl DyLight488-lectin (Vector laboratory) and intraperitoneally injected with 60 mg / kg pimonidazole hydrochloride (Hypoxyprobe) dissolved in PBS for 30 min, and then killed. Mice were fixed by perfusion with 4% PFA. We obtained frozen sections from tumor masses and stained them with hamster anti-CD31 antibodies (1:200, Millipore) and 4.3.11.3 mouse Pacific blue-Mab (1:50, Hypoxyprobe) in PBS. The sections were imaged using a LSM880 confocal microscope (Zeiss), and the images obtained were analyzed using ImageJ software (http: / / rsb.info.nih.gov / ij) to quantify lectins. + Area / CD31 + Area and Hypoxia Probes + area.

[0217] The results are Fig.11 Compared to tumors treated with Fc or Fc+Cpt, tumors treated with 2C8H11 or 2C8H11+Cpt showed normalized tumor vasculature with enhanced perfusion, as evidenced by increased lectin + Area (green) / CD31 + In addition, when compared to tumors treated with Fc or Fc+Cpt, the hypoxia probe in tumors treated with 2C8H11 or 2C8H11+Cpt was significantly higher than that in tumors treated with Fc or Fc+Cpt. + Hypoxia, indicated by the area (green), was reduced by 72%. These results indicate that the 2C8H11 antibody not only normalizes the morphology of tumor vessels, but also enhances their functionality by increasing vascular perfusion, which subsequently leads to reduced hypoxia.

[0218] Example 11: Increasing the delivery of anticancer drugs into tumor masses by 2C8H11 antibody.

[0219] The drug Cpt inhibits tumor growth by inhibiting DNA synthesis and has been widely used in human cancer patients. To evaluate whether the 2C8 antibody can increase the delivery of this drug to tumors by normalizing tumor blood vessels, frozen sections of tumors were stained with anti-cisplatin-modified DNA antibodies (1:100, Abcam) and hamster anti-CD31 antibodies (1:200, Millipore). Fig.12 As shown, the level of Cpt-modified DNA (green) in the 2C8H11 / Cpt-treated group was significantly increased by 2.1-fold compared to the Fc+Cpt-treated group. This result shows that the delivery of Cpt to the tumor mass is enhanced due to the normalization of tumor blood vessels by the 2C8H11 antibody, which subsequently effectively inhibits tumor growth.

[0220] Example 12: CNV regression and vascular leakage inhibition effects of 2C8H11 antibody in laser-induced CNV model.

[0221] The ability of 2C8H11 antibody to inhibit choroidal neovascularization (CNV) was tested using a laser-induced CNV model, which is a hallmark of wet age-related macular degeneration (AMD). After dilating the pupil with 5 mg / ml phenylephrine and 5 mg / ml tropicamide eye drops (Santen Pharmaceutical), and instilling 0.5% proparacaine hydrochloride eye drops (Alcon) for local anesthesia, a glass cover glass was used as a contact lens, and a laser photocoagulator (Lumenis Inc.) with a slit lamp delivery system was used to visualize the retina. Sufficient laser energy (532 nm wavelength, 250 mW power, 100 ms duration, 50 μm spot size) was delivered at 4 positions (3, 6, 9, and 12 o'clock positions at the posterior pole) for each eye. This study only includes burns that produce bubbles (indicating rupture of Bruch's membrane) during laser photocoagulation. Spots containing hemorrhage at the laser site were excluded from analysis. To recapitulate the clinical situation, mice were intravitreally administered 2C8H11 (5 μg) 7 days after laser photocoagulation ( Fig.13 A). As a control or for comparison, mice were given Fc or VEGF-Trap (5 μg each) in the same manner. For intravitreal administration of the indicated agents, approximately 1 μl (5 mg / ml) containing 5 μg of each agent was injected into the vitreous cavity using a Nanoliter 2000 microinjector (World Precision Instruments) equipped with a glass capillary pipette. CD31 expression in retinal pigment epithelium (RPE)-choroid-sclera flat mounts was calculated 14 days after laser photocoagulation using the MATLAB Image Processing Toolbox (MathWorks). +CNV volume. Anti-CD31 antibody (1:200, Millipore) was used to detect endothelial cells with CNV. VEGF-Trap effectively induced CNV regression by 64.4% compared to Fc, and 2C8H11 similarly induced CNV regression (65.3%) ( Fig.13 B. Fig.13 C). Combined fluorescein angiography (FA) and indocyanine green angiography (ICGA) enabled us to measure vascular leakage at the neovascularization around the laser-injured site. Continuous-wave laser modules at 488 nm and 785 nm were used as excitation sources for fluorescein and ICG, respectively. The raster-scan pattern of the excitation laser was obtained by a scanner system consisting of a rotating polygon mirror (MC-5; Lincoln Laser) and a galvanometer-based scanning mirror (6230H; Cambridge technology) and delivered to the back aperture of the imaging lens. A high numerical aperture (NA) objective lens (PlanApoλ, NA 0.75; Nikon) was used as an imaging lens to provide wide-angle fundus fluorescence images. The fluorescence signal detected by a photomultiplier tube (R9110; Hamamatsu Photonics) was digitized by a frame grabber and reconstructed in real time into an image with a size of 512 × 512 pixels per frame. To visualize late (6 min) FA and ICGA images using the angiography system, 10 mg sodium fluorescein (Alcon) and 0.15 mg ICG (Daiichi Pharmaceutical) were administered intraperitoneally and intravenously, respectively. Imaging procedures were performed under general anesthesia and pupil dilation to improve image quality. The leakage area of ​​CNV was calculated as the total measured hyperfluorescent area in the FA image divided by the total measured CNV area in the ICGA image using Java-based imaging software (ImajeJ; National Institutes of Health). Both VEGF-Trap (37.0%) and 2C8H11 (38.3%) similarly inhibited vascular leakage compared to Fc ( Fig.13 B. Fig.13 D). Notably, the Fc-treated group showed no significant difference in vascular leakage between 6 and 14 days after laser photocoagulation, whereas VEGF-Trap and 2C8H11 significantly reduced vascular leakage (45.6% and 50.0%, respectively) ( Fig.13 B. Fig.13 D) Thus, in the laser-induced CNV mouse model, the extent of inhibition of CNV and vascular leakage was quantitatively indistinguishable between VEGF-Trap and 2C8H11.

[0222] Example 13: CNV regression and choriocapillaris regeneration effects of 2C8H11 antibody.

[0223] To determine the role of 2C8H11 in CNV regression and choriocapillaris regeneration after CNV establishment, Fc, VEGF-Trap, control antibody, or 2C8H11 (5 μg each) were administered intravitreally to mice via a Nanoliter 2000 microinjector (World Precision Instruments) 7 days after laser photocoagulation. In vivo optical coherence tomography angiography (OCTA) was performed 6, 14, 21, and 35 days after laser photocoagulation ( Fig.14 A). The retinal choroidal layer was imaged using a prototype high-speed swept-source optical coherence tomography (OCT) system using a custom ring cavity wavelength scanning laser centered at 1048 nm at an A scan rate of 230 kHz. OCT images were collected in a 1.7 mm x 1.7 mm field of view within the retinal choroidal layer to monitor the regeneration of the choroidal vasculature at the laser photocoagulation site after intravitreal injection of agents. In order to obtain cross-sectional OCT angiograms (which allow selective visualization of blood vessels in the absence of retinal and choroidal parenchyma), we compared the repeatedly recorded B-scan images and detected the pixel-by-pixel intensity decorrelation of those images, which was mainly caused by the movement of red blood cells within the blood vessels. Then, by using an automatic layer flattening and segmentation algorithm, the cross-sectional OCT angiogram was flattened to the RPE, and a frontal OCT angiogram was generated by individually projecting each flattened cross-sectional OCT angiogram within three depth ranges: the inner retina, the outer retina, and the choroidal layer. The outer plexiform layer and Bruch's membrane are defined as the boundaries separating the inner retina, the outer retina, and the choroidal layer. The density of retinal and choroidal vessels was automatically calculated as the proportion of the measured area occupied by flowing vessels, which were defined as pixels with decorrelation values ​​above a threshold level. Avascular pixels were detected from en face OCT angiograms representing the choroidal layer with the help of the Image Processing Toolbox of MATLAB (MathWorks). The total volume of the avascular space surrounding the laser injury site was then calculated by multiplying the number of avascular pixels by the volume of one pixel. To analyze the changes in the volume of the avascular space, the serial measurements for each eye were converted into percentage changes relative to the baseline values. The CNV volume in the outer retinas treated with Fc was slightly reduced, but those treated with VEGF-Trap and 2C8H11 showed a significant reduction in CNV volume ( Fig.14 B. Fig.14 C). Meanwhile, a slight reduction in avascular space was observed in the choroids treated with Fc. Interestingly, at 14, 21, and 35 days after laser photocoagulation, the choroids in the eyes treated with 2C8H11 showed a continuous and significant reduction in avascular space of 30.1%, 36.4%, and 37.0%, respectively ( Fig.14 B. Fig.14 D). Similarly, the choroids in control Ab-treated eyes showed a 21.7%, 30.2%, and 38.0% reduction in avascular space at 14, 21, and 35 days after laser photocoagulation, respectively ( Fig.14 B. Fig.14 D). However, at D14, D21, and D35, the choroids in VEGF-Trap treated eyes showed an increase in avascular space by 11.4%, 16.0%, and 18.1%, respectively ( Fig.14 B. Fig.14 D). Overall, these findings indicate that in the laser-induced CNV model, both 2C8H11 and control Ab promoted regeneration of the choriocapillaris layer, whereas VEGF-Trap caused choriocapillaris regression.

[0224] Example 14: Co-localization of 2C8H11 antibody and CD31 in CNV endothelial cells.

[0225] To investigate whether subcutaneously injected 2C8H11 can also exert a therapeutic effect on CNV, we first evaluated the colocalization of 2C8H11 antibody and CD31 in CNV endothelial cells. 2C8H11 antibody (25 mg / kg) was administered subcutaneously 1 day after laser photocoagulation. As a control, Fc (25 mg / kg) was administered to mice in the same manner. The colocalization of 2C8H11 antibody and anti-CD31 antibody (1:200, Millipore) in CNV endothelial cells was directly detected 2, 4, and 8 days after laser photocoagulation. Fig.15 A) CD31 in the CNV region of administered 2C8H11 + Highly detectable in endothelial cells ( Fig.15 B- Fig.15 D).

[0226] Example 15: CNV inhibition effect of subcutaneously injected 2C8H11 antibody.

[0227] To determine the effect of subcutaneously injected 2C8H11 antibody on CNV inhibition, 2C8H11 antibody (25 mg / kg) was administered subcutaneously 1 day after laser photocoagulation. As a control, Fc (25 mg / kg) was administered to mice in the same manner. Anti-CD31 antibody (1:200, Millipore) was used to detect endothelial cells in CNV, and CD31 was calculated in RPE-choroid-sclera paving using MATLAB image processing toolbox (MathWorks) 8 days after laser photocoagulation. + CNV volume ( Fig.16A). Compared with Fc, 2C8H11 effectively inhibited CNV formation by 69.9% ( Fig.16 B. Fig.16 C), indicating that both intravitreal and subcutaneous injections of 2C8H11 have an inhibitory effect on CNV.

[0228] The microorganism of the present invention is named 2C8 and was deposited in the Korean Cell Line Bank (KCLB) of the Cancer Research Institute of Seoul National University College of Medicine, 28 Yangon-dong, Jongno-gu, Seoul, South Korea, at 110-744 on January 30, 2018 (Accession No.: KCLRF-BP-00417).

[0229] The microorganism of the present invention is named 4B9 and was deposited in the Korean Cell Line Bank (KCLB) of the Cancer Research Institute of Seoul National University College of Medicine, 28 Yangon-dong, Jongno-gu, Seoul, South Korea, at 110-744 on January 30, 2018 (Accession No.: KCLRF-BP-00418).

[0230] Industrial Applicability

[0231] The present invention relates to antibodies that inhibit Ang2 and simultaneously activate Tie2 receptors, thereby promoting downstream signal transduction. It provides a method for inhibiting Ang2-induced angiogenesis and reducing vascular permeability. In addition, the antibodies according to the present invention can be used to diagnose and treat abnormal angiogenesis-related diseases, such as eye diseases or cancers and / or diseases caused by increased vascular permeability.

[0232] The present invention has been described in detail based on its specific features, and it is obvious to those skilled in the art that these specific techniques are only preferred embodiments, and therefore the scope of the present invention is not limited to the embodiments. Therefore, the true scope of the present invention will be defined by the appended claims and their equivalents.

[0233] Sequence Listing Free Text

[0234] Attached to the electronic file. <110> Institute of Basic Sciences KAIST <120> Anti-angiopoietin-2 antibodies and uses thereof <130> PP-B2174 <150> US 62 / 633,038 <151> 2018-02-20 <150> KR 10-2019-0018769 <151> 2019-02-18 <160> 117 <170> KoPatentIn 3.0 <210> 1 <211> 496 <212> PRT <213> Artificial Sequence <220> <223> Human Angiopoietin-2 Full Length <400> 1 Met Trp Gln Ile Val Phe Phe Thr Leu Ser Cys Asp Leu Val Leu Ala 1 5 10 15 Ala Ala Tyr Asn Asn Phe Arg Lys Ser Met Asp Ser Ile Gly Lys Lys 20 25 30 Gln Tyr Gln Val Gln His Gly Ser Cys Ser Tyr Thr Phe Leu Leu Pro 35 40 45 Glu Met Asp Asn Cys Arg Ser Ser Ser Ser Pro Tyr Val Ser Asn Ala 50 55 60 Val Gln Arg Asp Ala Pro Leu Glu Tyr Asp Asp Ser Val Gln Arg Leu 65 70 75 80 Gln Val Leu Glu Asn Ile Met Glu Asn Asn Thr Gln Trp Leu Met Lys 85 90 95 Leu Glu Asn Tyr Ile Gln Asp Asn Met Lys Lys Glu Met Val Glu Ile 100 105 110 Gln Gln Asn Ala Val Gln Asn Gln Thr Ala Val Met Ile Glu Ile Gly 115 120 125 Thr Asn Leu Leu Asn Gln Thr Ala Glu Gln Thr Arg Lys Leu Thr Asp 130 135 140 Val Glu Ala Gln Val Leu Asn Gln Thr Thr Arg Leu Glu Leu Gln Leu 145 150 155 160 Leu Glu His Ser Leu Ser Thr Asn Lys Leu Glu Lys Gln Ile Leu Asp 165 170 175 Gln Thr Ser Glu Ile Asn Lys Leu Gln Asp Lys Asn Ser Phe Leu Glu 180 185 190 Lys Lys Val Leu Ala Met Glu Asp Lys His Ile Ile Gln Leu Gln Ser 195 200 205 Ile Lys Glu Glu Lys Asp Gln Leu Gln Val Leu Val Ser Lys Gln Asn 210 215 220 Ser Ile Ile Glu Glu Leu Glu Lys Lys Ile Val Thr Ala Thr Val Asn 225 230 235 240 Asn Ser Val Leu Gln Lys Gln Gln His Asp Leu Met Glu Thr Val Asn 245 250 255 Asn Leu Leu Thr Met Met Ser Thr Ser Asn Ser Ala Lys Asp Pro Thr 260 265 270 Val Ala Lys Glu Glu Gln Ile Ser Phe Arg Asp Cys Ala Glu Val Phe 275 280 285 Lys Ser Gly His Thr Thr Asn Gly Ile Tyr Thr Leu Thr Phe Pro Asn 290 295 300 Ser Thr Glu Glu Ile Lys Ala Tyr Cys Asp Met Glu Ala Gly Gly Gly 305 310 315 320 Gly Trp Thr Ile Ile Gln Arg Arg Glu Asp Gly Ser Val Asp Phe Gln 325 330 335 Arg Thr Trp Lys Glu Tyr Lys Val Gly Phe Gly Asn Pro Ser Gly Glu 340 345 350 Tyr Trp Leu Gly Asn Glu Phe Val Ser Gln Leu Thr Asn Gln Gln Arg 355 360 365 Tyr Val Leu Lys Ile His Leu Lys Asp Trp Glu Gly Asn Glu Ala Tyr 370 375 380 Ser Leu Tyr Glu His Phe Tyr Leu Ser Ser Glu Glu Leu Asn Tyr Arg 385 390 395 400 Ile His Leu Lys Gly Leu Thr Gly Thr Ala Gly Lys Ile Ser Ser Ile 405 410 415 Ser Gln Pro Gly Asn Asp Phe Ser Thr Lys Asp Gly Asp Asn Asp Lys 420 425 430 Cys Ile Cys Lys Cys Ser Gln Met Leu Thr Gly Gly Trp Trp Phe Asp 435 440 445 Ala Cys Gly Pro Ser Asn Leu Asn Gly Met Tyr Tyr Pro Gln Arg Gln 450 455 460 Asn Thr Asn Lys Phe Asn Gly Ile Lys Trp Tyr Tyr Trp Lys Gly Ser 465 470 475 480 Gly Tyr Ser Leu Lys Ala Thr Thr Met Met Ile Arg Pro Ala Asp Phe 485 490 495 <210> 2 <211> 221 <212> PRT <213> Artificial sequence <220> <223> Human angiopoietin-2 receptor binding domain (RBD) <400> 2 Glu Glu Gln Ile Ser Phe Arg Asp Cys Ala Glu Val Phe Lys Ser Gly 1 5 10 15 His Thr Thr Asn Gly Ile Tyr Thr Leu Thr Phe Pro Asn Ser Thr Glu 20 25 30 Glu Ile Lys Ala Tyr Cys Asp Met Glu Ala Gly Gly Gly Gly Trp Thr 35 40 45 Ile Ile Gln Arg Arg Glu Asp Gly Ser Val Asp Phe Gln Arg Thr Trp 50 55 60 Lys Glu Tyr Lys Val Gly Phe Gly Asn Pro Ser Gly Glu Tyr Trp Leu 65 70 75 80 Gly Asn Glu Phe Val Ser Gln Leu Thr Asn Gln Gln Arg Tyr Val Leu 85 90 95 Lys Ile His Leu Lys Asp Trp Glu Gly Asn Glu Ala Tyr Ser Leu Tyr 100 105 110 Glu His Phe Tyr Leu Ser Ser Glu Glu Leu Asn Tyr Arg Ile His Leu 115 120 125 Lys Gly Leu Thr Gly Thr Ala Gly Lys Ile Ser Ser Ile Ser Gln Pro 130 135 140 Gly Asn Asp Phe Ser Thr Lys Asp Gly Asp Asn Asp Lys Cys Ile Cys 145 150 155 160 Lys Cys Ser Gln Met Leu Thr Gly Gly Trp Trp Phe Asp Ala Cys Gly 165 170 175 Pro Ser Asn Leu Asn Gly Met Tyr Tyr Pro Gln Arg Gln Asn Thr Asn 180 185 190 Lys Phe Asn Gly Ile Lys Trp Tyr Tyr Trp Lys Gly Ser Gly Tyr Ser 195 200 205 Leu Lys Ala Thr Thr Met Met Ile Arg Pro Ala Asp Phe 210 215 220 <210> 3 <211> 5 <212> PRT <213> Artificial sequence <220> <223> CDRH1 <400> 3 Asp Tyr Tyr Met Tyr 1 5 <210> 4 <211> 17 <212> PRT <213> Artificial sequence <220> <223> CDRH2 <400> 4 Thr Ile Ser Val Gly Gly Ser Phe Thr Tyr Tyr Pro Asp Ser Val Lys 1 5 10 15 Gly <210> 5 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDRH3 <400> 5 Asp Trp Gly Leu Arg Pro Trp Phe Val Tyr 1 5 10 <210> 6 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CDRL1 <400> 6 Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala 1 5 10 <210> 7 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CDRL2 <400> 7 Trp Ala Ser Thr Arg His Thr 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDRL3 <400> 8 Gln Gln His Tyr Ser Thr Pro Pro Thr 1 5 <210> 9 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Val Gly Gly Ser Phe Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Asp Trp Gly Leu Arg Pro Trp Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115 <210> 10 <211> 357 <212> DNA <213> Artificial sequence <220> <223> Heavy chain variable region <400> 10 gaagtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt gactattaca tgtattgggt tcgccagact 120 ccggaaaaga ggctggagtg ggtcgcaacc attagtgttg gtggtagttt cacctactat 180 ccagacagtg tgaaggggcg attcaccatc tccagagaca atgccaagaa caacctgtac 240 ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attactgtgc aagagactgg 300 ggattacgac cctggtttgt ttactggggc caagggactc tggtcactgt ctctgca 357 <210> 11 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Light chain variable region <400> 11 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Pro 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 12 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Light chain variable region <400> 12 gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc 60 atcacctgca aggccagtca ggatgtgagt actgctgtag cctggtatca acaaaaacca 120 gggcaatctc ctaaactact gatttactgg gcatccaccc ggcacactgg agtccctgat 180 cgcttcacag gcagtggatc tgggacagat tatactctca ccatcagcag tgtgcaggct 240 gaagacctgg cactttatta ctgtcagcaa cattatagca ctcctcccac gttcggctcg 300 gggacaaagt tggaaataaa a 321 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 <400> 13 Ser Tyr Trp Met His 1 5 <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDRH2 <400> 14 Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 15 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 <400> 15 Arg Phe Tyr Tyr Gly Ser Asp Trp Tyr Phe Asp Val 1 5 10 <210> 16 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CDRL1 <400> 16 Lys Ala Ser Gln Asp Val Gly Thr Ala Val Ala 1 5 10 <210> 17 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CDRL2 <400> 17 Trp Ala Ser Thr Arg His Thr 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDRL3 <400> 18 Gln Gln Tyr Ser Ser Tyr Pro Leu Thr 1 5 <210> 19 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <400> 19 Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Ser Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Gly Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Phe Tyr Tyr Gly Ser Asp Trp Tyr Phe Asp Val Trp Gly 100 105 110 Ala Gly Ser Thr Val Thr Val Ser Ser 115 120 <210> 20 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Heavy Chain Variable Region <400> 20 caggtgcaac tgcagcagtc tgggcctcag ctggttaggc ctggggcttc agtgaagata 60 tcctgcaagg cttctggtta ctcattcacc agctactgga tgcactgggt gaagcagagg 120 cctggacaag gtcttgagtg gattggcatg attgatcctt ccgatagtga aactaggtta 180 aatcagaagt tcaaggacaa ggcctcattg actgtagaca aatcctccag cacagcctac 240 atgcaactca gcagcccgac atctggggac tctgcggtct attactgtgc aagacgtttt 300 tactacgggt cggactggta cttcgatgtc tggggcgcag ggtccacggt caccgtctcc 360 tca 363 <210> 21 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light Chain Variable Region <400> 21 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 22 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Light chain variable region <400> 22 gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc 60 atcacctgca aggccagtca ggatgtgggt actgctgtag cctggtatca acagaaacca 120 ggtcaatctc ctaaactact gatttactgg gcatccaccc ggcacactgg agtccctgat 180 cgcttcacag gcagtggatc tgggacagat ttcactctca ccattagcaa tgtgcagtct 240 gaagacttgg cagattattt ctgtcagcaa tatagcagct atcctctcac gttcggctcg 300 gggacaaagt tggaaataaa a 321 <210> 23 <211> 5 <212> PRT <213> Artificial sequence <220> <223> CDRH1 <400> 23 Asp Tyr Tyr Met Tyr 1 5 <210> 24 <211> 17 <212> PRT <213> Artificial sequence <220> <223> CDRH2 <400> twenty four Thr Ile Asn Asp Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys 1 5 10 15 Gly <210> 25 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDRH3 <400> 25 Asp Trp Gly Leu Arg Pro Trp Phe Val Tyr 1 5 10 <210> 26 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CDRL1 <400> 26 Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala 1 5 10 <210> 27 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CDRL2 <400> 27 Trp Ala Ser Thr Arg His Thr 1 5 <210> 28 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDRL3 <400> 28 Gln Gln His Tyr Thr Thr Pro Pro Thr 1 5 <210> 29 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain Variable Region <400> 29 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Tyr Trp Ile Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Asp Trp Gly Leu Arg Pro Trp Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115 <210> 30 <211> 357 <212> DNA <213> Artificial sequence <220> <223> Heavy chain variable region <400> 30 gaagtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt gactattaca tgtattggat tcgccagact 120 ccggaaaaga ggctggagtg ggtcgcaacc attaatgatg gtggtagtta cacctactat 180 ccagacagtg tgaaggggcg attcaccatc tccagagaca atgccaagaa caacctgtac 240 ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attactgtgc aagagactgg 300 ggattacgac cctggtttgt ttactggggc caagggactc tggtcactgt ctctgca 357 <210> 31 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Light chain variable region <400> 31 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 32 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Light chain variable region <400> 32 gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc 60 atcacctgca aggccagtca ggatgtgagt actgctgtag cctggtatca acaaaaacca 120 gggcaatctc ctaaactact gatttactgg gcatccaccc ggcacactgg agtccctgat 180 cgcttcacag gcagtggatc tgggacagat tatactctca ccatcagcag tgtgcaggct 240 gaagacctgg cactttatta ctgtcagcaa cattatacca ctcctcccac gttcggctcg 300 gggacaaagt tggaaataaa a 321 <210> 33 <211> 5 <212> PRT <213> Artificial sequence <220> <223> CDRH1 <400> 33 Gly Tyr Asn Met Asn 1 5 <210> 34 <211> 17 <212> PRT <213> Artificial sequence <220> <223> CDRH2 <400> 34 Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 35 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CDRH3 <400> 35 Tyr Gly Asn Tyr Val Asp Tyr 1 5 <210> 36 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CDRL1 <400> 36 Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala 1 5 10 <210> 37 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CDRL2 <400> 37 Trp Ala Ser Thr Arg His Thr 1 5 <210> 38 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDRL3 <400> 38 Gln Gln His Tyr Asn Thr Pro Pro Thr 1 5 <210> 39 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <400> 39 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Asn Met Asn Trp Val Lys Gln Ser Asn Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Val Arg Tyr Gly Asn Tyr Val Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115 <210> 40 <211> 342 <212> DNA <213> Artificial sequence <220> <223> Heavy chain variable region <400> 40 cagctgcagc agtctggacc tgagctggag aagcctggcg cttcagtgaa gatatcctgc 60 aaggcttctg gttactcatt cactggctac aacatgaact gggtgaagca gagcaatgga 120 aagagccttg agtggattgg aaatattgat ccttactatg gtggtactag ctacaaccag 180 aagttcaagg gcaaggccac attgactgta gacaaatcct ccagcacagc ctacatgcag 240 ctcaagagcc tgacatctga ggactctgca gtctattact gtgtaaggta tggtaactac 300 gtggactact ggggccaagg caccactctc acagtctcct ca 342 <210> 41 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Light chain variable region <400> 41 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Asn Thr Pro Pro 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 42 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Light chain variable region <400> 42 gacattgtga tgacccagtc ccacaaattc atgtccacat cagtaggaga cagggtcagc 60 atcacctgca aggccagtca ggatgtgagt actgctgtag cctggtatca acaaaaacca 120 gggcaatctc ctaaactact gatttactgg gcatccaccc ggcacactgg agtccctgat 180 cgcttcacag gcagtggatc tgggacagat tatactctca ccatcagcag tgtgcaggct 240 gaagacctgg cactttatta ctgtcagcaa cattataaca ctcctcccac gttcggctcg 300 gggacaaagt tggaaataaa a 321 <210> 43 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 43 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Tyr Met Tyr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Val Gly Gly Ser Phe Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Trp Gly Leu Arg Pro Trp Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 44 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 44 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 45 <211> 357 <212> DNA <213> Artificial sequence <220> <223> VH <400> 45 caggtacagc tcgtggagtc tggtggaggc ttggtgaaac ctggagggtc cctgagactt 60 agctgtgcag cttccggctt cacattttca gactattata tgtattggat cagacaggct 120 cccgggaagg gcttggagtg ggtttcaacc attagtgttg gcggatcttt tacttactac 180 ccagacagtg tgaaggggag attcacaatc tccagggata acgcgaaaaa cagcctgtat 240 ctccaaatga atagcctgag agccgaagat accgccgtgt actactgcgc cagagactgg 300 ggattacggc cctggttcgt gtactggggc cagggaaccc tggtcaccgt ctcctca 357 <210> 46 <211> 321 <212> DNA <213> Artificial sequence <220> <223> VL <400> 46 gacatccaga tgacacagtc cccaagctcc ctgtctgcat ctgtgggaga ccgggtgacc 60 atcacttgta aggcctcaca ggatgtttct actgctgtcg catggtacca gcaaaagccg 120 ggtaaagctc ccaagctttt gatatactgg gccagcacca ggcacacagg cgtgccatca 180 agattcagtg ggtccggatc cggcacggat tttacactca ctattagctc actgcaacct 240 gaagactttg ccacctatta ctgccagcag cattatagca cccctcccac cttcggtcag 300 ggcactaaag tagaaatcaa a 321 <210> 47 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 47 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Val Gly Gly Ser Phe Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Trp Gly Leu Arg Pro Trp Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 48 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 48 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 49 <211> 357 <212> DNA <213> Artificial sequence <220> <223> VH <400> 49 caggtccagc tggtggaatc cggcggaggc ttggtgaagc ctggaggcag cctaagactc 60 tcctgtgcag cctctggctt caccttctct gactattaca tgtattgggt ccgccaggct 120 ccagggaaag ggctcgagtg ggtttcaaca attagtgtag gtggaagctt cacctactat 180 cctgactccg tgaaaggaag atttacgatc tctagggata atgccaagaa ctcactgtac 240 cttcagatga acagcctgag agcggaggac acagccgtgt actactgcgc tagagattgg 300 ggattaagac cctggtttgt ttattggggc cagggaaccc tggtcaccgt ctcctca 357 <210> 50 <211> 321 <212> DNA <213> Artificial sequence <220> <223> VL <400> 50 gacatccaga tgacacagtc cccaagctcc ctgtctgcat ctgtgggaga ccgggtgacc 60 atcacttgta aggcctcaca ggatgtttct actgctgtcg catggtacca gcaaaagccg 120 ggtaaagctc ccaagctttt gatatactgg gccagcacca ggcacacagg cgtgccatca 180 agattcagtg ggtccggatc cggcacggat tttacactca ctattagctc actgcaacct 240 gaagactttg ccacctatta ctgccagcag cattatagca cccctcccac cttcggtcag 300 ggcactaaag tagaaatcaa a 321 <210> 51 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 51 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Val Gly Gly Ser Phe Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Trp Gly Leu Arg Pro Trp Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 52 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 52 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 53 <211> 357 <212> DNA <213> Artificial sequence <220> <223> VH <400> 53 caggtgcagc tggtcgaatc tggaggaggc ttggtgaaac ctggggggtc cctgagactc 60 tcttgtgcag cctccggctt taccttttct gactactaca tgtattgggt tcgccaggct 120 cccggtaagg ggttagagtg ggtggctacc attagtgttg gcggttcatt tacttattac 180 ccagatagtg tgaaaggacg gttcaccatc agcagggaca atgcaaagaa ctcactctat 240 ctacaaatga atagcctgag agccgaggat acagcggtgt attactgcgc cagagattgg 300 ggacttcgac catggttcgt ctactggggc cagggaaccc tggtcaccgt ctcctca 357 <210> 54 <211> 321 <212> DNA <213> Artificial sequence <220> <223> VL <400> 54 gacatccaga tgacacagtc cccaagctcc ctgtctgcat ctgtgggaga ccgggtgacc 60 atcacttgta aggcctcaca ggatgtttct actgctgtcg catggtacca gcaaaagccg 120 ggtaaagctc ccaagctttt gatatactgg gccagcacca ggcacacagg cgtgccatca 180 agattcagtg ggtccggatc cggcacggat tttacactca ctattagctc actgcaacct 240 gaagactttg ccacctatta ctgccagcag cattatagca cccctcccac cttcggtcag 300 ggcactaaag tagaaatcaa a 321 <210> 55 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 55 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Phe Tyr Tyr Gly Ser Asp Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 56 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 56 Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 57 <211> 363 <212> DNA <213> Artificial sequence <220> <223> VH <400> 57 caggtgcagc tggtgcagag tggagctgag gtaaaaaagc ccggcgccag tgtgaaggtt 60 agttgcaagg cctctggata caccttcaca agctattgga tgcactgggt gcgacaagct 120 cctgggcagg ggcttgagtg gatgggaatg atcgacccat ccgattcaga aactaggctc 180 aaccagaaat tcaaagatag agtgactatg accagggaca cctccacgag cacagtctac 240 atggaattgt caagcctgcg ctctgaggac acagccgtgt actattgtgc aagacggttt 300 tactatggta gcgattggta ctttgatgtt tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 58 <211> 321 <212> DNA <213> Artificial sequence <220> <223> VL <400> 58 gacatacagt tgacccagtc tccttccttc ctgtccgcct ccgtgggcga tagagttacc 60 attacttgca aagctagtca ggacgtgggt accgcagtgg cctggtatca gcagaaacca 120 ggtaaagccc ctaagctcct gatctactgg gcatcaacac ggcacacagg ggtcccaagc 180 aggttttctg gcagcggatc aggaaccgaa tttacactga cgatctcgtc tctgcagccc 240 gaggatttcg ctacttacta ctgtcaacaa tatagtagct atcccctcac tttcggtcag 300 ggcactaaag tagaaatcaa a 321 <210> 59 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 59 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Methionine, Glutamic acid, Leucine, Serine, Serine, Leucine, Arginine, Serine, Glutamic acid, Aspartic acid, Threonine, Alanine, Valine, Tyrosine, Tyrosine, Cysteine 85 90 95 Alanine, Arginine, Arginine, Phenylalanine, Tyrosine, Tyrosine, Glycine, Serine, Aspartic acid, Tryptophan, Tyrosine, Phenylalanine, Aspartic acid, Valine, Tryptophan, Glycine 100 105 110 Glutamine, Glycine, Threonine, Leucine, Valine, Threonine, Valine, Serine, Serine 115 120 <210> 60 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 60 Aspartic acid, Isoleucine, Glutamine, Leucine, Threonine, Glutamine, Serine, Proline, Serine, Phenylalanine, Leucine, Serine, Alanine, Serine, Valine, Glycine 1 5 10 15 Aspartic acid, Arginine, Valine, Threonine, Isoleucine, Threonine, Cysteine, Lysine, Alanine, Serine, Glutamine, Aspartic acid, Valine, Glycine, Threonine, Alanine 20 25 30 Valine, Alanine, Tryptophan, Tyrosine, Glutamine, Glutamine, Lysine, Proline, Glycine, Lysine, Alanine, Proline, Lysine, Leucine, Leucine, Isoleucine 35 40 45 Tyrosine, Tryptophan, Alanine, Serine, Threonine, Arginine, Histidine, Threonine, Glycine, Valine, Proline, Serine, Arginine, Phenylalanine, Serine, Glycine 50 55 60 Serine, Glycine, Serine, Glycine, Threonine, Glutamic acid, Phenylalanine, Threonine, Leucine, Threonine, Isoleucine, Serine, Serine, Leucine, Glutamine, Proline 65 70 75 80 Glutamic acid, Aspartic acid, Phenylalanine, Alanine, Threonine, Tyrosine, Tyrosine, Cysteine, Glutamine, Glutamine, Tyrosine, Serine, Serine, Tyrosine, Proline, Leucine 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 61 <211> 363 <212> DNA <213> Artificial sequence <220> <223> VH <400> 61 caggtgcaac tcgtgcagtc tggagctgaa gtgaagaaac ccggggcctc agtgaaggtg 60 agttgcaaag catctgggta ctcatttacc agctattgga tgcactgggt gcggcaggcc 120 ccaggacaag gcctggagtg gattggcatg atcgaccctt ccgatagtga aacgaggctg 180 aaccagaagt ttaaagatcg cgtcaccatg accagggaca caagtacttc tacagtctac 240 atggagttga gcagcctgag atcagaggac acagccgttt actactgtgc tagacgattc 300 tattatggca gcgactggta tttcgatgta tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 62 <211> 321 <212> DNA <213> Artificial sequence <220> <223> VL <400> 62 gacatacagt tgacccagtc tccttccttc ctgtccgcct ccgtgggcga tagagttacc 60 attacttgca aagctagtca ggacgtgggt accgcagtgg cctggtatca gcagaaacca 120 ggtaaagccc ctaagctcct gatctactgg gcatcaacac ggcacacagg ggtcccaagc 180 aggttttctg gcagcggatc aggaaccgaa tttacactga cgatctcgtc tctgcagccc 240 gaggatttcg ctacttacta ctgtcaacaa tatagtagct atcccctcac tttcggtcag 300 ggcactaaag tagaaatcaa a 321 <210> 63 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 63 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Ser Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Phe Tyr Tyr Gly Ser Asp Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 64 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 64 Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 65 <211> 363 <212> DNA <213> Artificial sequence <220> <223> VH <400> 65 caggtgcaac tggtgcagtc tggtgctgag gtgaagaaac caggcgcttc agtcaaggta 60 agctgcaaag caagtggata ctccttcacc tcttattgga tgcactgggt tagacaggcc 120 cctggtcaag gcctcgagtg gattggcatg atcgacccct ctgacagcga aactaggctg 180 aatcagaaat ttaaggacaa ggcctccatg acacgggata catccacaag caccgtttac 240 atggaactga gctcgctgag aagtgaggac actgccgtgt attactgtgc gagacgcttt 300 tattacgggt cagattggta cttcgatgtg tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 66 <211> 321 <212> DNA <213> Artificial sequence <220> <223> VL <400> 66 gacatacagt tgacccagtc tccttccttc ctgtccgcct ccgtgggcga tagagttacc 60 attacttgca aagctagtca ggacgtgggt accgcagtgg cctggtatca gcagaaacca 120 ggtaaagccc ctaagctcct gatctactgg gcatcaacac ggcacacagg ggtcccaagc 180 aggttttctg gcagcggatc aggaaccgaa tttacactga cgatctcgtc tctgcagccc 240 gaggatttcg ctacttacta ctgtcaacaa tatagtagct atcccctcac tttcggtcag 300 ggcactaaag tagaaatcaa a 321 <210> 67 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 67 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Ser Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Methionine Glutamic acid Leucine Serine Serine Leucine Arginine Serine Glutamic acid Aspartic acid Threonine Alanine Valine Tyrosine Tyrosine Cysteine 85 90 95 Alanine Arginine Arginine Phenylalanine Tyrosine Tyrosine Glycine Serine Aspartic acid Tryptophan Tyrosine Phenylalanine Aspartic acid Valine Tryptophan Glycine 100 105 110 Glutamine Glycine Threonine Leucine Valine Threonine Valine Serine Serine 115 120 <210> 68 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 68 Aspartic acid Isoleucine Glutamine Leucine Threonine Glutamine Serine Proline Serine Phenylalanine Leucine Serine Alanine Serine Valine Glycine 1 5 10 15 Aspartic acid Arginine Valine Threonine Isoleucine Threonine Cysteine Lysine Alanine Serine Glutamine Aspartic acid Valine Glycine Threonine Alanine 20 25 30 Valine Alanine Tryptophan Tyrosine Glutamine Glutamine Lysine Proline Glycine Lysine Alanine Proline Lysine Leucine Leucine Isoleucine 35 40 45 Tyrosine Tryptophan Alanine Serine Threonine Arginine Histidine Threonine Glycine Valine Proline Serine Arginine Phenylalanine Serine Glycine 50 55 60 Serine Glycine Serine Glycine Threonine Glutamic acid Phenylalanine Threonine Leucine Threonine Isoleucine Serine Serine Leucine Glutamine Proline 65 70 75 80 Glutamic acid Aspartic acid Phenylalanine Alanine Threonine Tyrosine Tyrosine Cysteine Glutamine Glutamine Tyrosine Serine Serine Tyrosine Proline Leucine 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 69 <211> 363 <212> DNA <213> Artificial sequence <220> <223> VH <400> 69 caggtgcagc tggtgcagtc tggggctgag gtgaaaaagc caggcgcttc cgtcaaagtt 60 tcctgcaagg catctggtta ctcttttaca agctattgga tgcactgggt gaagcaggcc 120 cccggacaag ggctcgagtg gattggcatg atcgatcctt ccgatagtga aacacgcttg 180 aatcagaaat tcaaggacaa ggccagtatg accagggata ctagcacaag cactgtatat 240 atggagctta gctcactgag atcagaagac acggccgtgt actactgtgc gagacggttt 300 tactatggct ccgactggta tttcgacgtc tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 70 <211> 321 <212> DNA <213> Artificial sequence <220> <223> VL <400> 70 gacatacagt tgacccagtc tccttccttc ctgtccgcct ccgtgggcga tagagttacc 60 attacttgca aagctagtca ggacgtgggt accgcagtgg cctggtatca gcagaaacca 120 ggtaaagccc ctaagctcct gatctactgg gcatcaacac ggcacacagg ggtcccaagc 180 aggttttctg gcagcggatc aggaaccgaa tttacactga cgatctcgtc tctgcagccc 240 gaggatttcg ctacttacta ctgtcaacaa tatagtagct atcccctcac tttcggtcag 300 ggcactaaag tagaaatcaa a 321 <210> 71 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 71 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Ser Leu Thr Val Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Phe Tyr Tyr Gly Ser Asp Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 72 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 72 Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 73 <211> 363 <212> DNA <213> Artificial sequence <220> <223> VH <400> 73 caggtgcagc tggtgcagtc tggcgctgag gtgaagaaac ctggggcctc agtgaaggtt 60 tcctgtaaag caagtggata ctctttcacc agctactgga tgcactgggt gaaacaggcc 120 cccggccaag ggcttgagtg gattggtatg atcgatccat ccgacagcga aactaggctc 180 aaccagaagt tcaaggataa agcgtccttg acagtagata catccacgag cacagtttat 240 atggagctgt ctagtctgcg gtctgaagac accgccgtgt attattgcgc tagacgcttt 300 tattacggct cggactggta ctttgacgtc tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 74 <211> 321 <212> DNA <213> Artificial sequence <220> <223> VL <400> 74 gacatacagt tgacccagtc tccttccttc ctgtccgcct ccgtgggcga tagagttacc 60 attacttgca aagctagtca ggacgtgggt accgcagtgg cctggtatca gcagaaacca 120 ggtaaagccc ctaagctcct gatctactgg gcatcaacac ggcacacagg ggtcccaagc 180 aggttttctg gcagcggatc aggaaccgaa tttacactga cgatctcgtc tctgcagccc 240 gaggatttcg ctacttacta ctgtcaacaa tatagtagct atcccctcac tttcggtcag 300 ggcactaaag tagaaatcaa a 321 <210> 75 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 75 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Phe Tyr Tyr Gly Ser Asp Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 76 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 76 Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 77 <211> 363 <212> DNA <213> Artificial sequence <220> <223> VH <400> 77 caggtgcagc tggtgcagag tggagctgag gtaaaaaagc ccggcgccag tgtgaaggtt 60 agttgcaagg cctctggata caccttcaca agctattgga tgcactgggt gcgacaagct 120 cctgggcagg ggcttgagtg gatgggaatg atcgacccat ccgattcaga aactaggctc 180 aaccagaaat tcaaagatag agtgactatg accagggaca cctccacgag cacagtctac 240 atggaattgt caagcctgcg ctctgaggac acagccgtgt actattgtgc aagacggttt 300 tactatggta gcgattggta ctttgatgtt tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 78 <211> 321 <212> DNA <213> Artificial sequence <220> <223> VL <400> 78 gatattcaac tcacccagag tccatccttc ctgtctgcct cagtgggcga cagagtgtca 60 atcacatgca aggcaagcca ggatgttggc actgctgtgg cttggtatca gcaaaaacca 120 ggtaaggccc ccaaactgct tatttactgg gcatcaaccc ggcacacggg tgtccccgac 180 aggttcagcg gcagtggatc tgggacagag tttaccctga ctatcagctc cctgcagcct 240 gaagactttg ccacttatta ctgtcagcag tactctagct atcctctcac cttcggtcag 300 ggcactaaag tagaaatcaa a 321 <210> 79 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 79 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Phe Tyr Tyr Gly Ser Asp Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 80 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 80 Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 81 <211> 363 <212> DNA <213> Artificial sequence <220> <223> VH <400> 81 caggtgcaac tcgtgcagtc tggagctgaa gtgaagaaac ccggggcctc agtgaaggtg 60 agttgcaaag catctgggta ctcatttacc agctattgga tgcactgggt gcggcaggcc 120 ccaggacaag gcctggagtg gattggcatg atcgaccctt ccgatagtga aacgaggctg 180 aaccagaagt ttaaagatcg cgtcaccatg accagggaca caagtacttc tacagtctac 240 atggagttga gcagcctgag atcagaggac acagccgttt actactgtgc tagacgattc 300 tattatggca gcgactggta tttcgatgta tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 82 <211> 321 <212> DNA <213> Artificial sequence <220> <223> VL <400> 82 gatattcaac tcacccagag tccatccttc ctgtctgcct cagtgggcga cagagtgtca 60 atcacatgca aggcaagcca ggatgttggc actgctgtgg cttggtatca gcaaaaacca 120 ggtaaggccc ccaaactgct tatttactgg gcatcaaccc ggcacacggg tgtccccgac 180 aggttcagcg gcagtggatc tgggacagag tttaccctga ctatcagctc cctgcagcct 240 gaagactttg ccacttatta ctgtcagcag tactctagct atcctctcac cttcggtcag 300 ggcactaaag tagaaatcaa a 321 <210> 83 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 83 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Ser Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Phe Tyr Tyr Gly Ser Asp Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 84 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 84 Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 85 <211> 363 <212> DNA <213> Artificial sequence <220> <223> VH <400> 85 caggtgcaac tggtgcagtc tggtgctgag gtgaagaaac caggcgcttc agtcaaggta 60 agctgcaaag caagtggata ctccttcacc tcttattgga tgcactgggt tagacaggcc 120 cctggtcaag gcctcgagtg gattggcatg atcgacccct ctgacagcga aactaggctg 180 aatcagaaat ttaaggacaa ggcctccatg acacgggata catccacaag caccgtttac 240 atggaactga gctcgctgag aagtgaggac actgccgtgt attactgtgc gagacgcttt 300 tattacgggt cagattggta cttcgatgtg tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 86 <211> 321 <212> DNA <213> Artificial sequence <220> <223> VL <400> 86 gatattcaac tcacccagag tccatccttc ctgtctgcct cagtgggcga cagagtgtca 60 atcacatgca aggcaagcca ggatgttggc actgctgtgg cttggtatca gcaaaaacca 120 ggtaaggccc ccaaactgct tatttactgg gcatcaaccc ggcacacggg tgtccccgac 180 aggttcagcg gcagtggatc tgggacagag tttaccctga ctatcagctc cctgcagcct 240 gaagactttg ccacttatta ctgtcagcag tactctagct atcctctcac cttcggtcag 300 ggcactaaag tagaaatcaa a 321 <210> 87 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 87 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Ser Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Phe Tyr Tyr Gly Ser Asp Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 88 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 88 Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 89 <211> 363 <212> DNA <213> Artificial sequence <220> <223> VH <400> 89 caggtgcagc tggtgcagtc tggggctgag gtgaaaaagc caggcgcttc cgtcaaagtt 60 tcctgcaagg catctggtta ctcttttaca agctattgga tgcactgggt gaagcaggcc 120 cccggacaag ggctcgagtg gattggcatg atcgatcctt ccgatagtga aacacgcttg 180 aatcagaaat tcaaggacaa ggccagtatg accagggata ctagcacaag cactgtatat 240 atggagctta gctcactgag atcagaagac acggccgtgt actactgtgc gagacggttt 300 tactatggct ccgactggta tttcgacgtc tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 90 <211> 321 <212> DNA <213> Artificial sequence <220> <223> VL <400> 90 gatattcaac tcacccagag tccatccttc ctgtctgcct cagtgggcga cagagtgtca 60 atcacatgca aggcaagcca ggatgttggc actgctgtgg cttggtatca gcaaaaacca 120 ggtaaggccc ccaaactgct tatttactgg gcatcaaccc ggcacacggg tgtccccgac 180 aggttcagcg gcagtggatc tgggacagag tttaccctga ctatcagctc cctgcagcct 240 gaagactttg ccacttatta ctgtcagcag tactctagct atcctctcac cttcggtcag 300 ggcactaaag tagaaatcaa a 321 <210> 91 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 91 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Ser Leu Thr Val Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Phe Tyr Tyr Gly Ser Asp Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 92 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 92 Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 93 <211> 363 <212> DNA <213> Artificial sequence <220> <223> VH <400> 93 caggtgcagc tggtgcagtc tggcgctgag gtgaagaaac ctggggcctc agtgaaggtt 60 tcctgtaaag caagtggata ctctttcacc agctactgga tgcactgggt gaaacaggcc 120 cccggccaag ggcttgagtg gattggtatg atcgatccat ccgacagcga aactaggctc 180 aaccagaagt tcaaggataa agcgtccttg acagtagata catccacgag cacagtttat 240 atggagctgt ctagtctgcg gtctgaagac accgccgtgt attattgcgc tagacgcttt 300 tattacggct cggactggta ctttgacgtc tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 94 <211> 321 <212> DNA <213> Artificial sequence <220> <223> VL <400> 94 gatattcaac tcacccagag tccatccttc ctgtctgcct cagtgggcga cagagtgtca 60 atcacatgca aggcaagcca ggatgttggc actgctgtgg cttggtatca gcaaaaacca 120 ggtaaggccc ccaaactgct tatttactgg gcatcaaccc ggcacacggg tgtccccgac 180 aggttcagcg gcagtggatc tgggacagag tttaccctga ctatcagctc cctgcagcct 240 gaagactttg ccacttatta ctgtcagcag tactctagct atcctctcac cttcggtcag 300 ggcactaaag tagaaatcaa a 321 <210> 95 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 95 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Methionine Glutamic acid Leucine Serine Serine Leucine Arginine Serine Glutamic acid Aspartic acid Threonine Alanine Valine Tyrosine Tyrosine Cysteine 85 90 95 Alanine Arginine Arginine Phenylalanine Tyrosine Tyrosine Glycine Serine Aspartic acid Tryptophan Tyrosine Phenylalanine Aspartic acid Valine Tryptophan Glycine 100 105 110 Glutamine Glycine Threonine Leucine Valine Threonine Valine Serine Serine 115 120 <210> 96 <211> 107 <212> PRT <213> Artificial sequence <220> <223> VL <400> 96 Aspartic acid Isoleucine Glutamine Leucine Threonine Glutamine Serine Proline Serine Phenylalanine Leucine Serine Alanine Serine Valine Glycine 1 5 10 15 Aspartic acid Arginine Valine Serine Isoleucine Threonine Cysteine Lysine Alanine Serine Glutamine Aspartic acid Valine Glycine Threonine Alanine 20 25 30 Valine Alanine Tryptophan Tyrosine Glutamine Glutamine Lysine Proline Glycine Lysine Alanine Proline Lysine Leucine Leucine Isoleucine 35 40 45 Tyrosine Tryptophan Alanine Serine Threonine Arginine Histidine Threonine Glycine Valine Proline Aspartic acid Arginine Phenylalanine Serine Glycine 50 55 60 Serine Glycine Serine Glycine Threonine Glutamic acid Phenylalanine Threonine Leucine Threonine Isoleucine Serine Serine Leucine Glutamine Proline 65 70 75 80 Glutamic acid Aspartic acid Phenylalanine Alanine Aspartic acid Tyrosine Phenylalanine Cysteine Glutamine Glutamine Tyrosine Serine Serine Tyrosine Proline Leucine 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 97 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> VH <400> 97 caggtgcagc tggtgcagag tggagctgag gtaaaaaagc ccggcgccag tgtgaaggtt 60 agttgcaagg cctctggata caccttcaca agctattgga tgcactgggt gcgacaagct 120 cctgggcagg ggcttgagtg gatgggaatg atcgacccat ccgattcaga aactaggctc 180 aaccagaaat tcaaagatag agtgactatg accagggaca cctccacgag cacagtctac 240 atggaattgt caagcctgcg ctctgaggac acagccgtgt actattgtgc aagacggttt 300 tactatggta gcgattggta ctttgatgtt tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 98 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> VL <400> 98 gacatccagt tgacccaatc accatccttt ctgtctgcct ctgtgggaga tagagtctcc 60 attacttgca aggccagtca ggatgtgggg accgctgttg cctggtacca gcaaaaaccc 120 ggaaaggcac ctaaactcct tatctactgg gcatccaccc ggcacacagg agtgccagac 180 aggtttagcg ggtcaggctc tggtacagag ttcactctga caatttctag cctgcagcct 240 gaagacttcg ctgattattt ctgtcagcag tatagcagtt accccctcac gttcggtcag 300 ggcactaaag tagaaatcaa a 321 <210> 99 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 99 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Methionine Glutamic acid Leucine Serine Serine Leucine Arginine Serine Glutamic acid Aspartic acid Threonine Alanine Valine Tyrosine Tyrosine Cysteine 85 90 95 Alanine Arginine Arginine Phenylalanine Tyrosine Tyrosine Glycine Serine Aspartic acid Tryptophan Tyrosine Phenylalanine Aspartic acid Valine Tryptophan Glycine 100 105 110 Glutamine Glycine Threonine Leucine Valine Threonine Valine Serine Serine 115 120 <210> 100 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 100 Aspartic acid Isoleucine Glutamine Leucine Threonine Glutamine Serine Proline Serine Phenylalanine Leucine Serine Alanine Serine Valine Glycine 1 5 10 15 Aspartic acid Arginine Valine Serine Isoleucine Threonine Cysteine Lysine Alanine Serine Glutamine Aspartic acid Valine Glycine Threonine Alanine 20 25 30 Valine Alanine Tryptophan Tyrosine Glutamine Glutamine Lysine Proline Glycine Lysine Alanine Proline Lysine Leucine Leucine Isoleucine 35 40 45 Tyrosine Tryptophan Alanine Serine Threonine Arginine Histidine Threonine Glycine Valine Proline Aspartic acid Arginine Phenylalanine Serine Glycine 50 55 60 Serine Glycine Serine Glycine Threonine Glutamic acid Phenylalanine Threonine Leucine Threonine Isoleucine Serine Serine Leucine Glutamine Proline 65 70 75 80 Glutamic acid Aspartic acid Phenylalanine Alanine Aspartic acid Tyrosine Phenylalanine Cysteine Glutamine Glutamine Tyrosine Serine Serine Tyrosine Proline Leucine 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 101 <211> 363 <212> DNA <213> Artificial sequence <220> <223> VH <400> 101 caggtgcaac tcgtgcagtc tggagctgaa gtgaagaaac ccggggcctc agtgaaggtg 60 agttgcaaag catctgggta ctcatttacc agctattgga tgcactgggt gcggcaggcc 120 ccaggacaag gcctggagtg gattggcatg atcgaccctt ccgatagtga aacgaggctg 180 aaccagaagt ttaaagatcg cgtcaccatg accagggaca caagtacttc tacagtctac 240 atggagttga gcagcctgag atcagaggac acagccgttt actactgtgc tagacgattc 300 tattatggca gcgactggta tttcgatgta tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 102 <211> 321 <212> DNA <213> Artificial sequence <220> <223> VL <400> 102 gacatccagt tgacccaatc accatccttt ctgtctgcct ctgtgggaga tagagtctcc 60 attacttgca aggccagtca ggatgtgggg accgctgttg cctggtacca gcaaaaaccc 120 ggaaaggcac ctaaactcct tatctactgg gcatccaccc ggcacacagg agtgccagac 180 aggtttagcg ggtcaggctc tggtacagag ttcactctga caatttctag cctgcagcct 240 gaagacttcg ctgattattt ctgtcagcag tatagcagtt accccctcac gttcggtcag 300 ggcactaaag tagaaatcaa a 321 <210> 103 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 103 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Ser Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Phe Tyr Tyr Gly Ser Asp Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 104 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 104 Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 105 <211> 363 <212> DNA <213> Artificial sequence <220> <223> VH <400> 105 caggtgcaac tggtgcagtc tggtgctgag gtgaagaaac caggcgcttc agtcaaggta 60 agctgcaaag caagtggata ctccttcacc tcttattgga tgcactgggt tagacaggcc 120 cctggtcaag gcctcgagtg gattggcatg atcgacccct ctgacagcga aactaggctg 180 aatcagaaat ttaaggacaa ggcctccatg acacgggata catccacaag caccgtttac 240 atggaactga gctcgctgag aagtgaggac actgccgtgt attactgtgc gagacgcttt 300 tattacgggt cagattggta cttcgatgtg tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 106 <211> 321 <212> DNA <213> Artificial sequence <220> <223> VL <400> 106 gacatccagt tgacccaatc accatccttt ctgtctgcct ctgtgggaga tagagtctcc 60 attacttgca aggccagtca ggatgtgggg accgctgttg cctggtacca gcaaaaaccc 120 ggaaaggcac ctaaactcct tatctactgg gcatccaccc ggcacacagg agtgccagac 180 aggtttagcg ggtcaggctc tggtacagag ttcactctga caatttctag cctgcagcct 240 gaagacttcg ctgattattt ctgtcagcag tatagcagtt accccctcac gttcggtcag 300 ggcactaaag tagaaatcaa a 321 <210> 107 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 107 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Ser Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Phe Tyr Tyr Gly Ser Asp Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 108 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 108 Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 109 <211> 363 <212> DNA <213> Artificial sequence <220> <223> VH <400> 109 caggtgcagc tggtgcagtc tggggctgag gtgaaaaagc caggcgcttc cgtcaaagtt 60 tcctgcaagg catctggtta ctcttttaca agctattgga tgcactgggt gaagcaggcc 120 cccggacaag ggctcgagtg gattggcatg atcgatcctt ccgatagtga aacacgcttg 180 aatcagaaat tcaaggacaa ggccagtatg accagggata ctagcacaag cactgtatat 240 atggagctta gctcactgag atcagaagac acggccgtgt actactgtgc gagacggttt 300 tactatggct ccgactggta tttcgacgtc tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 110 <211> 321 <212> DNA <213> Artificial sequence <220> <223> VL <400> 110 gacatccagt tgacccaatc accatccttt ctgtctgcct ctgtgggaga tagagtctcc 60 attacttgca aggccagtca ggatgtgggg accgctgttg cctggtacca gcaaaaaccc 120 ggaaaggcac ctaaactcct tatctactgg gcatccaccc ggcacacagg agtgccagac 180 aggtttagcg ggtcaggctc tggtacagag ttcactctga caatttctag cctgcagcct 240 gaagacttcg ctgattattt ctgtcagcag tatagcagtt accccctcac gttcggtcag 300 ggcactaaag tagaaatcaa a 321 <210> 111 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 111 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Ser Leu Thr Val Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Phe Tyr Tyr Gly Ser Asp Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 112 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 112 Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 113 <211> 363 <212> DNA <213> Artificial sequence <220> <223> VH <400> 113 caggtgcagc tggtgcagtc tggcgctgag gtgaagaaac ctggggcctc agtgaaggtt 60 tcctgtaaag caagtggata ctctttcacc agctactgga tgcactgggt gaaacaggcc 120 cccggccaag ggcttgagtg gattggtatg atcgatccat ccgacagcga aactaggctc 180 aaccagaagt tcaaggataa agcgtccttg acagtagata catccacgag cacagtttat 240 atggagctgt ctagtctgcg gtctgaagac accgccgtgt attattgcgc tagacgcttt 300 tattacggct cggactggta ctttgacgtc tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 114 <211> 321 <212> DNA <213> Artificial sequence <220> <223> VL <400> 114 gacatccagt tgacccaatc accatccttt ctgtctgcct ctgtgggaga tagagtctcc 60 attacttgca aggccagtca ggatgtgggg accgctgttg cctggtacca gcaaaaaccc 120 ggaaaggcac ctaaactcct tatctactgg gcatccaccc ggcacacagg agtgccagac 180 aggtttagcg ggtcaggctc tggtacagag ttcactctga caatttctag cctgcagcct 240 gaagacttcg ctgattattt ctgtcagcag tatagcagtt accccctcac gttcggtcag 300 ggcactaaag tagaaatcaa a 321 <210> 115 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Epitope <400> 115 Gln Arg Thr Trp Lys Glu Tyr Lys Val Gly Phe Gly Asn Pro Ser Gly 1 5 10 15 Glu Tyr <210> 116 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Epitope <400> 116 Lys Ser Gly His Thr Thr Asn Gly Ile Tyr Thr 1 5 10 <210> 117 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Epitope <400> 117 Glu Ala Gly Gly Gly Gly Trp 1 5

Claims

1. An antibody or an antigen-binding fragment thereof that specifically binds to human angiopoietin-2 and induces Tie2 activation, comprising: (a) a complementarity determining region (CDR) of a heavy chain variable region and a CDR of a light chain variable region, wherein the CDR of the heavy chain variable region consists of a complementarity determining region 1 (HCDR1) represented by the amino acid sequence of SEQ ID NO: 3, a HCDR2 represented by the amino acid sequence of SEQ ID NO: 4, and a HCDR3 represented by the amino acid sequence of SEQ ID NO: 5, and the CDR of the light chain variable region consists of: LCDR1 represented by the amino acid sequence of SEQ ID NO: 6, LCDR2 represented by the amino acid sequence of SEQ ID NO: 7, and LCDR3 represented by the amino acid sequence of SEQ ID NO: 8; or (b) a CDR of a heavy chain variable region and a CDR of a light chain variable region, wherein the CDR of the heavy chain variable region consists of: LCDR1 shown by the amino acid sequence of SEQ ID NO: 13, LCDR2 shown by the amino acid sequence of SEQ ID NO: 14, and LCDR3 shown by the amino acid sequence of SEQ ID NO: 15, the CDR of the light chain variable region consists of: LCDR1 shown by the amino acid sequence of SEQ ID NO: 16, LCDR2 shown by the amino acid sequence of SEQ ID NO: 17, and LCDR3 shown by the amino acid sequence of SEQ ID NO:

18.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises the CDR in (a) of claim 1 and is bound to the amino acid of SEQ ID NO: 116 and / or the amino acid of SEQ ID NO:

117.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises the CDR in (b) of claim 1 and is bound to the amino acid of SEQ ID NO:

115.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof binds to human and mouse Ang2.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody is polyclonal or monoclonal.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antigen-binding fragment is scFv or Fab.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody is humanized.

8. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising: (a) a CDR of a heavy chain variable region consisting of: HCDR1 shown by the amino acid sequence of SEQ ID NO: 3, HCDR2 shown by the amino acid sequence of SEQ ID NO: 4, and HCDR3 shown by the amino acid sequence of SEQ ID NO: 5; and (b) The CDRs of the light chain variable region consist of: LCDR1 represented by the amino acid sequence of SEQ ID NO: 6, LCDR2 represented by the amino acid sequence of SEQ ID NO: 7, and LCDR3 represented by the amino acid sequence of SEQ ID NO:

8.

9. The antibody or antigen-binding fragment thereof according to claim 1 or 3, comprising: (a) a CDR of a heavy chain variable region consisting of: a HCDR1 shown by the amino acid sequence of SEQ ID NO: 13, a HCDR2 shown by the amino acid sequence of SEQ ID NO: 14, and a HCDR3 shown by the amino acid sequence of SEQ ID NO: 15; and (b) The CDRs of the light chain variable region consist of: LCDR1 shown by the amino acid sequence of SEQ ID NO: 16, LCDR2 shown by the amino acid sequence of SEQ ID NO: 17, and LCDR3 shown by the amino acid sequence of SEQ ID NO:

18.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising: (a) the CDRs of claim 1 (a) and a heavy chain variable region selected from SEQ ID NO: 9, 43, 47 or 51; and a light chain variable region selected from SEQ ID NO: 11, 44, 48 or 52; or (b) the CDRs of claim 1 (b) and a heavy chain variable region selected from SEQ ID NO: 19, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107 or 111; and a light chain variable region selected from SEQ ID NO: 21, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108 or 112.

11. A pharmaceutical composition comprising a pharmaceutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

12. The pharmaceutical composition according to claim 11, further comprising a small molecule inhibitor used in chemotherapy.

13. The pharmaceutical composition according to claim 11, further comprising a vascular endothelial growth factor (VEGF) antagonist.

14. The pharmaceutical composition of claim 13, wherein the VEGF antagonist is an anti-VEGF antibody, a VEGF inhibitory fusion protein, or a small molecule kinase inhibitor.

15. Use of a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 11 to 14 in the preparation of a medicament for inhibiting tumor growth in a cancer patient, wherein the cancer is selected from squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, rectal cancer, adrenal cancer, hepatocellular carcinoma, gastrointestinal cancer, cervical cancer, ovarian cancer, breast cancer, colon cancer, endometrial cancer or uterine cancer, kidney cancer, prostate cancer, thyroid cancer, head and neck cancer and brain cancer.

16. The use according to claim 15, wherein the cancer is squamous cell carcinoma of the lung.

17. Use of the pharmaceutical composition according to any one of claims 11-14 in the preparation of a medicament for treating an eye disease in a patient, wherein the eye disease is wet age-related macular degeneration (wAMD), diabetic macular edema (DME) or diabetic retinopathy (DR).

18. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

19. An expression vector comprising the nucleic acid according to claim 18.

20. A host cell transformed with the expression vector according to claim 19.

21. A method for producing an anti-Ang2 antibody or an antigenic fragment thereof, the method comprising culturing the host cell according to claim 20.

Images