WO2019235856A1

WO2019235856A1 - Antibody binding to tie2 and use thereof

Info

Publication number: WO2019235856A1
Application number: PCT/KR2019/006820
Authority: WO
Inventors: 고규영; 배점일; 김재령
Original assignee: 기초과학연구원; 한국과학기술원
Priority date: 2018-06-07
Filing date: 2019-06-05
Publication date: 2019-12-12

Abstract

The present invention relates to an antibody against Tie-2 or an antigen binding fragment thereof, a nucleic acid for coding same, a vector comprising said nucleic acid, a cell transformed with said vector, a method for producing said antibody or the antigen binding fragment thereof, and a composition for preventing or treating angiogenic diseases, comprising said antibody or the antigen binding fragment thereof.

Description

Antibodies that bind to TIE2 and uses thereof

The present invention provides an antibody against Tie-2 or an antigen-binding fragment thereof, a nucleic acid encoding the same, a vector comprising the nucleic acid, a cell transformed with the vector, a method for producing the antibody or an antigen-binding fragment thereof, and a blood vessel comprising the same. It relates to a composition for preventing or treating neoplastic disease.

Angiogenesis occurs dynamically by a variety of regulatory factors during the development, growth, preservation and maintenance of homeostasis. In this process, newly formed blood vessels serve as transport channels for various biomaterials such as nutrients, oxygen and hormones to surrounding cells. Functional and structurally abnormal blood vessels are a direct and indirect cause of the onset and progression of various diseases. Tumor blood vessels exacerbate hypoxia with functional and structural defects, leading to tumor progression and metastasis to other tissues, and to poor delivery of anticancer agents to the center of the tumor tissue. Defective blood vessels can be identified in various diseases or disorders, in addition to cancer. Examples include acute inflammatory reactions such as various eye diseases (eg diabetic macular edema, senile macular degeneration), viral infections and sepsis. Thus, if a therapeutic agent is available that can normalize pathological vessels, it can be applied to the treatment of various patients with vascular abnormalities.

The Angiopoietin family plays an important role in the formation and maintenance of blood vessels and includes four Angiopoietins (Ang1, Ang2, Ang3 and Ang4). Angiopoietin-1 (Ang1) binds to Tie2 receptors present on the surface of vascular endothelial cells to phosphorylate and activate Tie2 receptors, thereby stabilizing blood vessels. Angiopoietin-2 (Ang2), on the other hand, binds to the Tie2 receptor but acts as an antagonist that induces inactivation of the Tie2 receptor, leading to blood vessel destabilization and vascular leakage. Expression levels of Ang2 have been reported to be very increased in blood, eye disease, viral infections and bacterial infections and inflammatory diseases in cancer patients (Saharinen P et al., 2017, Nature Review Drug Discovery). However, Ang2 is known to act as an agent that induces activation of the Tie2 receptor in many processes, including lymphatic vessel formation and maintenance. Therefore, Ang2 seems to perform various functions depending on the environment and organization.

To date, various anti-Ang2 antibodies have been developed and clinical trials have been conducted by many biopharmaceuticals (eg, US Pat. No. 7,658,924 and US Pat. No. 8,987,420). These Ang2 antibodies have been shown to inhibit the binding of Ang2 to Tie2, and this Ang2 neutralizing effect ultimately appears to interfere with the formation of angiogenesis. Angiogenesis inhibition and anticancer activity of anti-Ang2 antibodies has been demonstrated in many preclinical models, and several types of anti-Ang2 antibodies have been clinically tested in various cancer patients. However, it has been demonstrated that inhibition of angiogenesis through Ang2 neutralization is not sufficient for anticancer treatment. For example, in a phase 3 clinical trial conducted by Amgen, the anticancer effect of Ang2 antibodies in ovarian cancer patients was not significant (Marth C et al., 2017, Eur. J. Cancer). In addition to cancer models, the efficacy of the Ang2 neutralizing antibody Nesvacumab was tested in ophthalmic patients, but the efficacy of Eylea (anti-VEGF) could not be improved in the Phase 2 combo-study.

In contrast to the approach of neutralizing Ang2, direct activation of Tie2 is considered a new approach to inhibit abnormal angiogenesis and reduce vascular permeability. Recombinant proteins that directly bind to the Tie2 receptor to induce Tie2 phosphorylation and activation have also been developed, and their therapeutic effects have been tested in many preclinical cancer and ocular models. COMP-Angl and Vasculotide are examples. These agents exhibited anti-angiogenic and anti-invasive activity, but have the disadvantage of having very short half-lives and unstable physicochemical properties. In addition, small molecule compounds (AKB-9778) have been developed as inhibitors of dephosphatase VE-PTP. VE-PTP serves to inactivate Tie2 by removing phosphate groups from phosphorylated Tie2. These compounds have the disadvantage of nonspecifically activating other receptors, but they also indirectly increase Tie2 activity by inhibiting VE-PTP. In addition, Tie2 activating antibodies have been developed (US6365154B1, US20170174789A1). These antibodies inhibited vascular leakage by increasing the survival rate of vascular endothelial cells. Interestingly, it has been claimed that one of the plant extracts induces Tie2 activity and can be used as a skin care cosmetic (eg JP2011102273A, JP2018043949A, JP2015168656A).

Under this technical background, the inventors of the present application sought to develop an antibody that specifically binds Tie2. As a result, the present inventors have developed a Tie2 antibody that binds Tie2 with high affinity, and confirms that the Tie2 antibody can play a role as a therapeutic agent for angiogenic diseases by inducing Tie2 phosphorylation and activation of the Tie2 receptor. The present invention has been completed.

발명의 요약Summary of the Invention

It is an object of the present invention to provide a novel antibody or antigen binding fragment thereof against Tie2.

Another object of the present invention is to provide a nucleic acid encoding the antibody or antigen-binding fragment thereof.

Another object of the present invention is to provide a vector comprising the nucleic acid, a cell transformed with the vector, and a method of manufacturing the same.

Still another object of the present invention is to provide a composition for preventing or treating angiogenic diseases comprising the antibody or antigen-binding fragment thereof.

Still another object of the present invention is to provide a composition for co-administration with another therapeutic agent for angiogenic diseases, including the antibody or antigen-binding fragment thereof.

In order to achieve the above object, the present invention provides an anti-Tie2 antibody or antigen binding fragment thereof that binds to a Tie2 Ig3-FNIII (1-3) domain comprising the sequence of SEQ ID NO: 2.

Specifically, the present invention also provides a heavy chain variable region comprising a heavy chain CDR comprising the amino acid sequence of SEQ ID NO: 3 to 5, a light chain variable region comprising a light chain CDR comprising the amino acid sequence of SEQ ID NO: 6 to 8; A heavy chain variable region comprising a heavy chain CDR comprising the amino acid sequence of SEQ ID NOs: 13 to 15, a light chain variable region comprising a light chain CDR comprising the amino acid sequence of SEQ ID NOs: 16 to 18; A heavy chain variable region comprising a heavy chain CDR comprising the amino acid sequence of SEQ ID NOs: 23 to 25, a light chain variable region comprising a light chain CDR comprising the amino acid sequence of SEQ ID NOs: 26 to 28; A heavy chain variable region comprising a heavy chain CDR comprising the amino acid sequence of SEQ ID NOs: 33 to 35, a light chain variable region comprising a light chain CDR comprising the amino acid sequence of SEQ ID NOs: 36 to 38; Or an antibody binding to Tie2 comprising a heavy chain variable region comprising a heavy chain CDR comprising the amino acid sequence of SEQ ID NOs: 43 to 45, a light chain variable region comprising a light chain CDR comprising the amino acid sequence of SEQ ID NO: 46 to 48, or Its antigen binding fragment is provided.

The present invention also provides a nucleic acid encoding the antibody or antigen-binding fragment thereof.

The present invention also provides a vector comprising the nucleic acid.

The present invention also provides a cell transformed with the vector.

The present invention also provides a method for producing the antibody or antigen-binding fragment thereof comprising the following steps: (a) culturing the cells; And (b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.

The present invention also provides a composition for preventing or treating angiogenesis-related diseases comprising the antibody or antigen-binding fragment thereof as an active ingredient.

The present invention also provides a composition for co-administration with another therapeutic agent for angiogenic diseases, comprising the antibody or antigen-binding fragment thereof.

Figure 1 shows the analysis results for Akt phosphorylation induced by anti-Tie2 antibody. HUVECs remained serum-starved for 6 hours and incubated with COMP-Ang1 (CA1, 0.5 μg / ml) or anti-Tie2 antibodies (11C4, 4A4, 3B2, 3E12 and 3H7) for 30 minutes. . Lysates of the cultured cells were subjected to SDS-PAGE / Western blotting and probed with p-Akt antibody (S473) or Akt antibody.

FIG. 2 shows the assay results for dose-dependent Tie2 phosphorylation (pTie2) induced by anti-Tie2 antibody 3H7. The Tie2 phosphorylation capacity of 3H7 antibody was analyzed through immunoprecipitation and western blotting analysis. Serum starved HUVECs were incubated with various concentrations of 3H7 for 30 minutes. As a control, HUVECs were incubated at the same time in Ang2 / Control antibody (Control Ab) mixture. After immunoprecipitation of the Tie2 protein in the cell lysate using the Tie2 antibody, the degree of phosphorylation of Tie2 was measured by SDS-PAGE / Western blot analysis. Tie2 phosphorylation was probed by 4G10, a tyrosine phosphate (pY) antibody derived from mice.

FIG. 3 shows the results of translocation of FOXO1 and endocytosis of Tie2 receptor due to anti-Tie2 antibody 3H7. HUVECs were placed in serum starvation for 6 hours and then incubated with negative control (Control), Ang2 / Control antibody mixture (A2 + Control Ab) or 3H7 for 30 minutes. After cell fixation, the cell nuclei were blue with DAPI staining, the Tie2 receptor was green with an anti-Tie2 antibody, and FOXO1 was stained red with an anti-FOXO1 antibody to examine the location of FOXO1 and clustered Tie2 receptors. Arrows in the figure labeled Tie2 receptors introduced into the cell.

Figure 4 is a result showing the inhibition of vascular permeability induced by VEGF or TNF-a through the Tie2 antibody 3H7. HUVECs were inoculated into a trans well chamber and incubated for 3 days. At 100% confluence, HUVECs were pretreated with Ang2 (A2, 1 μg / ml), Ang2 / control antibody mixture (A2 + Control Ab, 1 μg / ml) or 3H7 (1 μg / ml) for 30 minutes each. Treatment with VEGF (500 ng / ml) for 45 minutes in the upper chamber (A) or TNF-a (100 ng / ml) for 22 hours (B). The upper chamber was then treated with FITC-dextran for 20 minutes and the vascular permeability was analyzed by measuring the amount of FITC fluorescence transmitted through the lower chamber. The measured value is mean ± standard deviation. According to the one way ANOVA, * is p <0.05, ** is p <0.01, and *** is p <0.001.

FIG. 5 shows a heat map showing a significant difference region in deuterium uptake as a result of analyzing Tie2 alone or Tie2 / 3H7 complex through hydrogen / deuterium (H / D) exchange-mass spectrometry. H / D exchange-mass spectrometry data in the presence or absence of anti-Tie2 antibody 3H7 was used as heat maps for deuterium uptake per residue for hTie2 antigen at 0.333, 10, 60 and 240 minutes. The color scale represents the percentage of H / D exchange per residue between Tie2 alone and Tie2 / 3H7 mixture at each time. Red color indicates increased deuterium exchange and blue color was not absorbed.

6 is a schematic showing 3H7-binding epitope in Tie2. Binding-epitope (red) of anti-Tie2 antibody (3H7) in hTie2 antigen analyzed by H / D exchange-mass spectrometry, visualized with hTie2 FNIII (1-3) crystal structure (PDB: 5UTK) using PyMol software will be.

Figure 7 shows the results of phosphorylation (pAkt) of Akt induced by humanized anti-Tie2 antibodies. Serum starved HUVECs were incubated with humanized anti-Tie2 antibodies for 30 minutes. Cell lysates were then blotted with SDS-PAGE / Western and probed with p-Akt (s473) antibody or anti-Akt antibody.

FIG. 8 shows the increase in Schlemm's canal (SC) area and the decrease in IOP by humanized Tie2 antibody (3H7H12G4) in primary open-angle glaucoma mouse model. Tamoxifen was administered to 8-week-old A1: A2 ^{iΔ / Δ} mice for inducible deletion of Angioprotein-1 and Angioprotein-2. At 12 weeks of age, 3H7H12G4 (one eye, 1 mg injection of 5 mg / ml solution) and Fc (opposing eye, 1 μ injection of 5 mg / ml solution) were administered intraocularly. Intraocular pressure (IOP) was measured periodically at 12, 13, and 14 weeks of age. The immunostaining intensity of Prox1 and Tie2 at CD144 ⁺ SC area and CD144 ⁺ SC was measured 2 weeks after administration of 3H7H12G4. The reference scale is 100 μm, with n = 5 for each group. Values in this figure are mean ± standard deviation. According to the Kruskal-Wallis test followed by Tukey's HSD test with ranks, * is p <0.05.

9 shows the results of choroidal neovascularization (CNV) and vascular leakage inhibition by intravitreally injected of 3H7H12G4 in a laser induced choroidal neovascularization (CNV) model. Antibodies (5 mg / ml solution, 1 μl injection) were administered intravitreally on day 7 after laser photocoagulation. CD31 ⁺ CNV volume was measured, and on day 6 and / or 14 days after laser photocoagulation, the sum of the high-fluorescence regions measured in the FA image divided by the sum of the CNV regions measured in the ICGA image was around the CNV. The leaked area of was calculated. N = 11 for each group and the value is mean ± standard deviation. one-way ANOVA followed by Student-Newman-Keuls post-test, where * is p <0.05 and *** is p <0.001; According to the Paired Student's t-test, ## is p <0.01 and ### is p <0.001.

FIG. 10 is a fluorescence photograph showing co-localization of 3H7H12G4 and CD31 in endothelial cells in the choroidal neovascularization (CNV) region. One day after laser photocoagulation, 3H7H12G4 was subcutaneously administered. Co-localization of 3H7H12G4 and CD31 in endothelial cells around choroidal neovascularization (CNV) at 2, 4 and 8 days after laser photocoagulation were stained and analyzed using anti-human IgG secondary antibody.

Figure 11 shows inhibition of choroidal neovascularization (CNV) by subcutaneous injection of 3H7H12G4. One day after laser photocoagulation, 3H7H12G4 was subcutaneously administered. Eight days after laser photocoagulation, the amount of CD31 ⁺ CNV was measured. The reference scale is 100 μm, with n = 10 in each group. Values are mean ± standard deviation and *** is p <0.001 by unpaired Student's t-test.

발명의 상세한 설명 및 바람직한 구현예Detailed Description of the Invention and Preferred Embodiments

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The inventors of the present application have identified a Tie2 antibody as an agent that increases the activity of Tie2 by binding to a Tie2 Ig3-FNIII (1-3) domain comprising the sequence of SEQ ID NO: 2 of the human Tie2 full length sequence of SEQ ID NO: 1.

Tie2 is a receptor protein that promotes the differentiation and stabilization of blood vessels. It is highly expressed in blood vessels of diseases such as cancer, and activating the Tie2 receptor stabilizes cancer blood vessels and aggregates surrounding supporting cells. By the antibody or antigen-binding fragment according to the present invention, activating Tie2 of cancer vessels normalizes the cancer vessels, relieves the increase of hypoxia within the tumors, increases blood flow into the tumors, and provides sufficient oxygen supply. May increase the penetration of immune cells.

In this respect, the present invention relates to a Tie2 antibody or antigen binding fragment thereof that binds to a Tie2 Ig3-FNIII (1-3) domain comprising the sequence of SEQ ID NO: 2.

As used herein, the term "antibody" refers to an antibody that specifically binds to Tie2. The scope of the present invention includes not only complete antibody forms that specifically bind Tie2, but also antigen binding fragments of such antibody molecules.

A complete antibody is a structure having two full length light chains and two full length heavy chains, each of which is linked by heavy and disulfide bonds. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types and subclasses gamma 1 (γ1), gamma 2 (γ2), and gamma 3 (γ3). ), Gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant regions of the light chains have kappa (κ) and lambda (λ) types.

An antigen binding fragment or antibody fragment of an antibody means a fragment having an antigen binding function and includes Fab, F (ab '), F (ab') 2, Fv and the like. Fab in the antibody fragment has a structure having a variable region of the light and heavy chains, a constant region of the light chain and the first constant region (CH1) of the heavy chain has one antigen binding site. Fab ′ is a hinge region comprising one or more cysteine residues at the C-terminus of the heavy chain CH1 domain

It differs from Fab in that it has a region. F (ab ') 2 antibodies are produced by disulfide bonds of cysteine residues in the hinge region of Fab'. Fv is the minimum antibody fragment which has only a heavy chain light region and a light chain variable region. Double-chain Fv is a non-covalent bond in which a heavy chain variable region and a light chain variable region are linked, and a single chain Fv (single-chain Fv, scFv) is generally a variable region of the heavy chain and the light chain through a peptide linker. This covalent linkage or the C-terminus is directly linked to form a dimer-like structure such as a double-chain Fv. Such antibody fragments can be obtained using proteolytic enzymes (e.g., restriction digestion of the entire antibody with papain yields Fab and cleavage with pepsin yields F (ab ') 2 fragments). It can also be produced by recombinant technology.

In one embodiment, the antibody according to the invention is in Fv form (eg scFv) or is in the form of a complete antibody. In addition, the heavy chain constant region may be selected from any one isotype of gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε). For example, the constant region is gamma 1 (IgG1), gamma 3 (IgG3) or gamma 4 (IgG4). The light chain constant region may be of kappa or lambda type.

As used herein, the term “heavy chain” refers to a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and a full length heavy chain comprising three constant region domains CH1, CH2 and CH3 And fragments thereof. In addition, the term "light chain" as used herein refers to a full-length light chain and fragment thereof comprising a variable region domain VL and a constant region domain CL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to the antigen. All means.

Antibodies of the invention include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFV), single chain antibodies, Fab fragments, F (ab ') fragments, disulfide-binding Fvs (sdFV) And anti-idiotype (anti-Id) antibodies, or epitope-binding fragments of the antibodies, and the like.

Said monoclonal antibody refers to the same except for possible naturally occurring mutations in which antibodies obtained from substantially homogeneous antibody populations, ie, individual antibodies in the population, may be present in trace amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. In contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

"Epitope" refers to a protein determinant to which an antibody can specifically bind. Epitopes usually consist of a group of chemically active surface molecules, such as amino acids or sugar side chains, and generally have specific three dimensional structural characteristics as well as specific charge characteristics. Three-dimensional epitopes and non-stereo epitopes are distinguished in that the binding to the former is lost but not to the latter in the presence of a denatured solvent.

Tie2 antibody or antigen-binding fragment thereof according to the present invention, when identifying epitopes through hydrogen / deuterium exchange, TLSDILPPQPEN and / or amino acids 633 to 644 of Tie2 comprising the sequence of SEQ ID NO: 1 Binding to amino acids 713-726 of FAENNIGSSNPAFS.

The non-human (eg murine) antibody of the “humanized” form may comprise one or more amino acid sequences derived from one or more non-human antibodies (donor or source antibody) containing a minimal sequence derived from a non-human immunoglobulin (eg Eg, a CDR sequence). In most cases, humanized antibodies are non-human species (donor antibodies) that retain the desired specificity, affinity, and capacity for residues from the hypervariable region of the recipient, for example mice, rats, rabbits, or non-humans. Human immunoglobulins (receptor antibodies) replaced with residues from the hypervariable regions of primates. For humanization, residues in one or more framework domains (FR) of the variable region of the recipient human antibody may be replaced with corresponding residues from the non-human species donor antibody. This helps to maintain proper three-dimensional organization of the grafted CDR (s), thereby improving affinity and antibody stability. Humanized antibodies may include new residues that do not appear in additional recipient antibodies or donor antibodies, for example to further refine antibody performance.

The term “human antibody” refers to a molecule derived from human immunoglobulin, in which all amino acid sequences constituting the antibody including complementarity determining regions and structural regions are composed of human immunoglobulins.

While the heavy and / or light chain portions are the same or homologous to the corresponding sequences in an antibody derived from a particular species or belonging to a particular antibody class or subclass, the remaining chain (s) are derived from another species or another antibody class or Included are "chimeric" antibodies (immunoglobulins) that are identical or homologous to the corresponding sequences in antibodies belonging to the subclass, as well as fragments of such antibodies that exhibit the desired biological activity.

According to a specific example of the present application, 11C4, 4A4, 3B2, 3E12, and 3H7 were prepared using a mouse-derived Tie2 antibody, and 3H7H11G4, 3H7H12G4, 3H7H21G4 or 3H7H22G4 were CDR-grafted and humanized antibodies from a mouse-derived 3H7 antibody as a source antibody. Produced.

As used herein, “antibody variable domain” refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of complementarity determining regions (CDRs; ie CDR1, CDR2, and CDR3), and framework regions (FR). . VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

“Complementarity Determining Regions” (CDRs; ie CDR1, CDR2, and CDR3) refer to amino acid residues of antibody variable domains that are required for antigen binding, each variable domain typically identified as CDR1, CDR2 and CDR3. Three CDR regions.

In one embodiment, the Tie2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a heavy chain CDR comprising the amino acid sequence of SEQ ID NOs: 3 to 5, a light chain CDR comprising the amino acid sequence of SEQ ID NOs: 6 to 8 Light chain variable region;

A heavy chain variable region comprising a heavy chain CDR comprising the amino acid sequence of SEQ ID NOs: 13 to 15, a light chain variable region comprising a light chain CDR comprising the amino acid sequence of SEQ ID NOs: 16 to 18;

A heavy chain variable region comprising a heavy chain CDR comprising the amino acid sequence of SEQ ID NOs: 23 to 25, a light chain variable region comprising a light chain CDR comprising the amino acid sequence of SEQ ID NOs: 26 to 28;

A heavy chain variable region comprising a heavy chain CDR comprising the amino acid sequence of SEQ ID NOs: 33 to 35, a light chain variable region comprising a light chain CDR comprising the amino acid sequence of SEQ ID NOs: 36 to 38; or

A heavy chain variable region comprising a heavy chain CDR comprising the amino acid sequence of SEQ ID NO: 43 to 45, and a light chain variable region comprising a light chain CDR comprising the amino acid sequence of SEQ ID NO: 46 to 48.

"Framework regions" (FRs) are variable domain residues other than CDR residues. Each variable domain typically has four FRs identified as FR1, FR2, FR3 and FR4.

Tie2 antibodies are monovalent or bivalent and include single or double chains. Functionally, the binding affinity of Tie2 antibodies is in the range of 10-5M to 10-12M. For example, the binding affinity of a Tie2 antibody is 10 ⁻⁶ M to 10 ⁻¹² M, 10 ⁻⁷ M to 10 ⁻¹² M, 10 ⁻⁸ M to 10 ⁻¹² M, 10 ⁻⁹ M to 10 ⁻¹² M, 10 ^-5 M to 10 ^-11 M, 10 ^-6 M to 10 ^-11 M, 10 ^-7 M to 10 ^-11 M, 10 ^-8 M to 10 ^-11 M, 10 ^-9 M to 10 ^-11 M, 10 ^-10 M to 10 ^-11 M, 10 ^-5 M to 10 ^-10 M, 10 ^-6 M to 10 ^-10 M, 10 ^-7 M to 10 ^-10 M, 10 ^-8 M to 10 ^-10 M, 10 ^-9 M to 10 ^-10 M, 10 ^-5 M to 10 ^-9 M, 10 ^-6 M to 10 ^-9 M, 10 ^-7 M to 10 ^-9 M, 10 ^-8 M to 10 ^-9 M, 10 ^-5 M to 10 ^-8 M, 10 ^-6 M to 10 ^-8 M, 10 ^-7 M to 10 ^-8 M, 10 ^-5 M to 10 ^-7 M, 10 ^-6 M to 10 ^-7 M or 10 ^-5 M to 10 ^-6 M.

The antibody or antigen-binding fragment thereof that binds Tie2 may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 9 and a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 11;

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21;

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31;

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 41;

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 51;

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54;

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 58;

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 61 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 62; or

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 65 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 66.

Antibodies or antibody fragments of the present invention may include not only the sequences of the anti-Tie2 antibodies of the present invention described herein, but also biological equivalents thereof, as long as they can specifically recognize Tie2. For example, further changes can be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions and / or substitutions of amino acid sequence residues of the antibody. Such amino acid variations are made based on the relative similarity of amino acid side chain substituents such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have a similar shape. Thus, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; Phenylalanine, tryptophan and tyrosine are biologically equivalent functions.

Considering the above-described variations having biologically equivalent activity, the amino acid sequence possessed by the antibody of the present invention or the nucleic acid molecule encoding the same is interpreted to include a sequence showing substantial identity with the sequence described in SEQ ID NO. The above substantial identity is at least 90% when the sequences of the present invention are aligned as closely as possible with any other sequences, and the aligned sequences are analyzed using algorithms commonly used in the art. By a homology, most preferably at least 95% homology, at least 96%, at least 97%, at least 98%, at least 99% homology. Alignment methods for sequence comparison are known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI and the like and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn and tblastx on the Internet. BLSAT is accessible at www.ncbi.nlm.nih.gov/BLAST/. Sequence homology comparisons using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

Based on this, the antibody or antigen-binding fragment thereof of the present invention is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% compared to the specified sequence or all described in the specification. , 99%, or more homology. Such homology can be determined by sequence comparison and / or alignment by methods known in the art. For example, sequence comparison algorithms (ie, BLAST or BLAST 2.0), manual alignment, visual inspection can be used to determine the percent sequence homology of nucleic acids or proteins of the invention.

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

The nucleic acid may comprise the sequence of SEQ ID NO: 10, 12, 20, 22, 30, 32, 40, 42, 50, 52, 55, 56, 59, 60, 63, 64, 67 or 68.

The nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention can be isolated to recombinantly produce the antibody or antigen-binding fragment thereof. The nucleic acid is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or for further expression. Based on this, the present invention relates to a vector comprising the nucleic acid in another aspect.

"Nucleic acid" is meant to encompass DNA (gDNA and cDNA) and RNA molecules inclusively, and the nucleotides that are the basic building blocks of nucleic acids include natural nucleotides as well as analogs with modified sugar or base sites. . The sequences of nucleic acids encoding heavy and light chain variable regions of the invention can be modified. Such modifications include addition, deletion, or non-conservative or conservative substitutions of nucleotides.

The DNA encoding the antibody is readily isolated or synthesized using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to the DNA encoding the heavy and light chains of the antibody). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: signal sequence, origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences.

As used herein, the term "vector" refers to a plasmid vector as a means for expressing a gene of interest in a host cell; Cosmid vector; Viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors, and the like. The nucleic acid encoding the antibody in the vector is operably linked with a promoter.

“Operatively linked” means a functional binding between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences, or transcriptional regulator binding sites) and another nucleic acid sequence, whereby the regulatory sequence is the other nucleic acid. To control transcription and / or translation of the sequence.

In the case of a prokaryotic cell as a host, powerful promoters capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), ribosome binding sites for initiation of translation, and transcription / detox termination sequences. Also, for example, when the eukaryotic cell is a host, a promoter derived from the genome of the mammalian cell (e.g., a metallothionine promoter, a β-actin promoter, a human heroglobin promoter and a human muscle creatine promoter) or a mammal Promoters derived from animal viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse breast tumor virus (MMTV) promoter, LTR promoter of HIV) , Promoter of the Moroni virus Epsteinbar virus (EBV) and Loose Sacoma virus (RSV) promoter) can be used, and generally has a polyadenylation sequence as a transcription termination sequence.

In some cases, the vector may be fused with other sequences to facilitate purification of the antibody expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA).

Such vectors include antibiotic resistance genes commonly used in the art as selectable markers and include, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline. There is a resistance gene.

In another aspect, the present invention relates to a cell transformed with the above-mentioned vector. The cells used to produce the antibodies of the invention can be prokaryote, yeast or higher eukaryote cells, but are not limited thereto.

Bacillus strains such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g. Pseudomonas putida), Proteus Prokaryotic host cells such as Proteus mirabilis and Staphylococcus (eg, Staphylocus carnosus) can be used.

However, the greatest interest in animal cells, examples of useful host cell lines are COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0 , U20S, or HT1080, but is not limited thereto.

In another aspect, the present invention, (a) culturing the cells; And (b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.

The cells can be cultured in various media. It can be used as a culture medium without limitation among commercial media. All other necessary supplements known to those skilled in the art may be included at appropriate concentrations. Culture conditions, such as temperature, pH, and the like, are already in use with host cells selected for expression, which will be apparent to those skilled in the art.

The recovery of the antibody or antigen-binding fragment thereof can be removed by, for example, centrifugation or ultrafiltration, and the resultant can be purified using, for example, affinity chromatography or the like. Further other purification techniques such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography and the like can be used.

In another aspect, the present invention relates to a composition for preventing or treating angiogenic diseases comprising the antibody or antigen-binding fragment thereof as an active ingredient.

Angiogenesis refers to the formation or growth of new blood vessels from previously existing blood vessels, and “angiogenesis-related disease” refers to a disease associated with the development or progression of angiogenesis. If the antibody can be treated, the disease can be included within the scope of angiogenesis-related diseases without limitation. Examples of angiogenesis-related diseases include cancer, metastasis, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, macular degeneration, angiogenesis Neovascular glaucoma, erythrosis, proliferative retinopathy, psoriasis, hemophilic arthritis, capillary formation of atherosclerotic plaques, keloid , Wound granulation, vascular adhesion, rheumatoid arthritis, osteoarthritis, autoimmune diseases, Crohn's disease, restenosis , Atherosclerosis, intestinal adhesions, cat scratch disease, ulcers, liver cirrhosis, nephritis, diabetic kidney One comprising a ring (diabetic nephropathy), diabetes mellitus (diabetes mellitus), inflammatory diseases (inflammatory diseases) and neurodegenerative disorders (neurodegenerative diseases), but is not limited to such. In addition, the cancer is esophageal cancer (esophageal cancer), stomach cancer (stomach cancer), colon cancer (large intestine cancer), rectal cancer (rectal cancer), oral cancer (oral cancer), pharyngeal cancer (pharynx cancer), larynx cancer (larynx cancer), Lung cancer, colon cancer, breast cancer, breast cancer, uterine cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer testis cancer, bladder cancer, renal cancer, liver cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, Selected from the group consisting of brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma, and multiple myeloid blood cancer It is not.

As used herein, the term "prevention or prophylaxis" refers to any measure which results in inhibiting or delaying the onset of the disease of interest by administering the antibody or composition of the invention. The term “treatment or therapy” refers to any measure by which an antibody or composition of the invention is administered to improve or ameliorate the symptoms of a disease of interest.

Compositions comprising the antibodies of the invention are preferably pharmaceutical compositions and may further comprise suitable vehicles, excipients or diluents typically used in the art.

Pharmaceutical compositions comprising pharmaceutically acceptable vehicles include tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, lyophilizates and suppositories. It can be in various oral or parenteral dosage forms. In this regard, the pharmaceutical compositions of the present invention may be formulated in combination with diluents or excipients such as fillers, thickeners, binders, wetting agents, disintegrants, surfactants and the like. Solid preparations for oral administration may be in the form of tablets, pills, powders, granules, capsules and the like. With respect to these solids, the compounds of the present invention may be formulated in combination with one or more excipients such as starch, calcium carbonate, sucrose, lactose, or gelatin. Lubricants such as simple excipients, magnesium stearate, talc and the like can be further used. Liquid preparations for oral administration may be suspensions, internal solutions, emulsions, syrups and the like. Various excipients such as simple diluents such as water or liquid paraffin, wetting agents, sweeteners, aromatics, preservatives and the like can be included in the liquid formulation. In addition, the pharmaceutical compositions of the present invention may be in parenteral dosage forms such as sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilisates, suppositories, and the like. Injectable propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and esters such as ethyl oleate may be suitable for non-aqueous solvents and suspensions. Base materials for suppositories include Whitepsol, macrogol, Tween 61, cacao butter, laurin butter and glycerogelatin.

The composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” refers to the amount of a pharmaceutical composition for treating a disease sufficient to a reasonable benefit and risk ratio applicable to all medical treatments. Effective amounts include the severity of the disease to be treated, the age and sex of the patient, the type of disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration, the rate of secretion, the duration of treatment, the coadministration of the drug, and other known in the art. It depends on various factors including the parameters. The compositions of the present invention can be administered alone or in combination with other therapies. In such cases, they may be administered sequentially or simultaneously with conventional therapies. In addition, the composition may be administered in one or multiple doses. Given these factors completely, it is important to administer the minimum amount sufficient to achieve the maximum effect without side effects, which dose can be readily determined by one of ordinary skill in the art. The dosage of the pharmaceutical composition of the present invention is not particularly limited, but varies depending on various factors including the patient's health condition and weight, the severity of the disease, the type of drug, the route of administration, and the time of administration. The compositions are typically accepted routes into mammals, including rats, mice, livestock, humans, etc., once or in multiple doses per day, for example, orally, rectally, intravenously, subcutaneously. It may be administered intrauterinely or intratracerebrovascularly.

In another aspect, the present invention relates to a method for inhibiting angiogenesis, a method for preventing or treating angiogenesis-related diseases, comprising administering the antibody or the composition to a subject in need.

The methods of the present invention comprise administering a pharmaceutically effective amount of the pharmaceutical composition to a subject in need thereof. The subject may be a mammal such as a dog, cow, horse, rabbit, mouse, rat, chicken and human, but is not limited thereto. The pharmaceutical composition includes parenterally, subcutaneously, intraperitoneally, intrapulmonarily or intranasally, if necessary intratralesional administration for topical treatment. Can be administered by any suitable method. Preferred dosages of the pharmaceutical compositions of the present invention vary depending on various factors, including the health and weight of the individual, the severity of the disease, the type of medicament, the route of administration and the time of administration, and can be readily determined by one skilled in the art.

In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising the antibody, or a method for preventing or treating cancer, comprising administering the antibody or the composition to an individual in need thereof. The terms 'antibody', 'prophylaxis' and 'treatment' are as mentioned above.

If treatable with the antibodies of the invention, the cancer is not limited. In detail, the antibody of the present invention can prevent the development or progression of cancer by inhibiting angiogenesis. Examples of cancer include esophageal cancer, stomach cancer, large intestine cancer, rectal cancer, oral cancer, pharynx cancer, larynx cancer, lung cancer Lung cancer, colon cancer, breast cancer, breast cancer, uterine cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer testis cancer, bladder cancer, renal cancer, liver cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, Brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma and multiple myeloid blood cancer.

In addition, the antibodies of the invention can be used in combination with other antibodies or biologically active agents or materials for various purposes. The present invention relates to a composition for co-administration with other therapeutic agents for angiogenic diseases comprising the antibody or antigen-binding fragment thereof in this respect.

Such other angiogenic disease therapeutic agents include anti-angiogenic drugs, anti-inflammatory drugs and / or anticancer drugs. This can overcome the resistance of each other and improve the efficacy.

When administered in combination with another therapeutic agent for angiogenic diseases in the composition according to the present invention, the Tie2 antibody and the therapeutic agent for other angiogenic diseases may be administered sequentially or simultaneously. For example, after administering an anti-angiogenic drug, an anti-inflammatory drug, and / or an anti-cancer drug to a subject, a composition comprising an antibody against Tie2 or an antigen binding fragment thereof as an active ingredient is administered to the subject, or the composition is administered. After administration to a subject, anti-angiogenic drugs, anti-inflammatory drugs, and / or anticancer drugs are administered to the subject. If desired, the composition may be administered to the subject simultaneously with the anti-angiogenic drug, anti-inflammatory drug and / or anticancer drug.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Preparation of mouse-derived Tie2 monoclonal antibody

1.1. Mouse Immunization with Human Tie2

As an immunogen, the Ig3-FNIII (1-3) domain of human Tie2 (hTie2-Ig3-FNIII (1-3), SEQ ID NO: 2) was cloned into a vector containing a CMV promoter and transfected into a HEK293F cell line to express temporarily. . After incubation for 5 days, the expressed recombinant hTie2-Ig3-FNIII (1-3) protein was purified by an affinity column using Protein A. Twice a week for 6 weeks, 5 weeks old BALB / c mice were immunized with the injection of purified hTie2-Ig3-FNIII (1-3) mixed with adjuvant (100 μg / injection). Tie2 antibody titers in the sera of immunized mice were examined with a human Tie2 (hTie2) ELISA kit (R & D). When antibody titers (1: 5,000 dilution) were properly increased (OD> 1.0), spleens were extracted from immunized mice, B lymphocytes were isolated from the spleen, and myeloma cells (SP2 / 0) were cultured. Fused. The fused cells were cultured in HAT medium containing hypoxanthine, aminopterin and thymidine, and hybridoma cells containing only fusions of myeloma cells and B lymphocytes were selected and cultured. Surviving hybridoma cells were seeded in 96-well plates and culture supernatants were analyzed by hTie2 ELISA. Hybridoma pools displaying positive signals were selected for clonal selection through limiting dilution. Finally 37 single hybridoma cell lines were constructed. Several of the Tie2 binding antibodies showed a Tie2 activation effect. Candidate antibodies were selected based on Tie2 activation level and high affinity for human Tie2 and later processed for humanization.

1.2. Preparation and Purification of Mouse Anti-Tie2 Monoclonal Antibodies

To produce anti-Tie2 antibodies selected based on ELISA positive signals, hybridoma cells were cultured in 10% FBS containing-DMEM (Dulbesco Modified Eagle's Medium) in T75 (75 cm ² area) flasks. When the concentration of cells reached about 90%, the cells were washed with PBS, incubated in 50 ml of serum-free medium (SFM, Gibco), and then incubated at 37 ° C for 3 days. Thereafter, the culture solution containing the antibody secreted from each monoclonal hybridoma was collected, centrifuged to remove cells, and the culture supernatant was collected and filtered. Antibodies were purified from the culture supernatant using an AKTA purification apparatus (GE Healthcare.) Equipped with a Protein G protein affinity column (GE Healthcare.). Thereafter, the buffer of the antibody solution was replaced with PBS using a centrifugal filter unit (Amicon), and the purified antibody was concentrated.

1.3. Screening and Screening of Aieistic Tie2 Antibodies.

To investigate whether the mouse anti-Tie2 antibody induces a downstream signaling process of the Tie2 receptor in endothelial cells, after treatment with HUVEC (Lonza) with an anti-Tie2 antibody, the major downstream signal of the Tie2 receptor The transfer protein Akt phosphorylation level was analyzed by immunoblotting technique. As a positive control, COMP-Angl (CA1) was treated with the cells.

Specifically, HUVEC (1 × 10 ⁵ cells / ml) was incubated at 37 ° C. in EGM-2 medium (Lonza) in a 60 mm culture dish. Cells of 90% density were incubated for 6 hours in serum-free EBM-2 medium to bring serum starvation. Serum starved HUVECs were treated with anti-Tie2 antibody and further incubated for 30 minutes. Cells were washed with cold PBS, then treated with lysis buffer and lysed at 4 ° C. for 20 minutes. Subsequently, the precipitate was removed by centrifugation at 13000 rpm for 15 minutes to obtain cell lysate. 5x SDS sample buffer was added to the cell lysate, followed by protein electrophoresis (SDS-PAGE), followed by transfer to nitrocellulose membrane (NC membrane, GE).

To check for Akt phosphorylation, TBS-T containing 5% skim milk was blocked for 1 hour at room temperature (RT). Incubated with p-Akt antibody (S473) for 8 hours at 4 ° C. p-Akt signal was detected by ECL (Enhanced ChemiLuminescence). The membrane was then incubated for 15 minutes in a stripping buffer (thermo) for 15 minutes and then probed again with an anti-Akt antibody to confirm the total amount of Akt. As a result, several groups treated with anti-Tie2 antibodies such as 11C4, 4A4, 3B2, 3E12 and 3H7 strongly induced S473 phosphorylation of Akt (FIG. 1).

1.4. Determination of the affinity of Tie2 antibodies for hTie2 by Octet assay.

The affinity of the mouse monoclonal antibody for hTie2 was measured using an octet system (ForteBio) using Black 96-well plate (Greiner). The biosensors used for affinity measurements were hydrated for 10 minutes before being measured with an AR2G tip (ForteBio Octet). After hydration, hTie2 was diluted to 10 μg / ml concentration in 10 mM sodium acetate, pH6.0 buffer, fixed on AR2G biosensor and blocked with 1M ethanolamine. Mouse anti-Tie2 monoclonal antibodies were diluted to 50, 25, 12.5, 6.25, 3.125 and 0 nM with 1 × kinetic buffer, then bound for 300 seconds and dissociated for 900 seconds. For affinity measurements (KD), the association rate (K-on) and the dissociation rate (K-off) were analyzed globally and 1: 1 using Octet data analysis program (v.9.0.0.10). Fit to coupled model. Table 2 shows the affinity of mouse anti-Tie2 antibodies.

1.5. Tie2 phosphorylation induced by mouse Tie2 antibody (3H7).

Anti-Tie2 antibodies according to the present invention appear to bind to Tie2 and induce Tie2 clustering, ultimately leading to Tie2 activation. Experiments were performed to analyze the effect of anti-Tie2 antibodies on Tie2 phosphorylation using HUVECs.

Specifically, HUVEC was incubated in 37%, 5% CO ₂ concentration conditions using EGM-2 (Lonza) medium in a 100mm culture dish. When the cells reached a confluency of 80-90%, the medium was replaced with EBM-2 (Lonza) to bring serum starvation and the cells were further incubated for 6 hours. Various concentrations of anti-Tie2 antibody (0.02 μg / ml to 50 μg / ml) were treated to the cultured cells and further incubated for 30 minutes. Wash cells twice with cold PBS and add 1000μl of lysis buffer (10mM Tris-Cl pH 7.4, 150mM NaCl, 5mM EDTA, 10% glycerol, 1% Triton X-100, protease inhibitor, phosphatase inhibitor) Incubate for 60 minutes at. The cells were centrifuged at 12,000 rpm for 10 minutes to obtain cell lysates and supernatants were obtained. Protein concentration of the supernatant was quantified by BCA assay.

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

Membrane was blocked for 1 hour at room temperature using TBS-T mixed with 5% (v / v) BSA and incubated with anti-phosphotyrosine antibody (4G10, Millipore) at 4 ° C. for 8 hours before HRP conjugate The anti-mouse antibody (HRP conjugated anti-mouse antibody) was incubated and analyzed by Western blot. To determine the amount of immunoprecipitated Tie2, the membrane was reacted in stripping buffer (Thermo) for 15 minutes, then blocked again and probed again with anti-Tie2 antibodies (R & D systems, AF313). As shown in FIG. 2, the addition of anti-Tie2 antibody (3H7) to HUVEC cells induced a dose-dependently strong phosphorylation of Tie2. These data show that anti-Tie2 antibody (3H7) induces activation of Tie2 receptor directly in human vascular endothelial cells.

1.6 Endocytosis of Tie2 Clustered in HUVEC by Tie2 Antibody (3H7) and Translocation of FOXO1

Intracellular influx of Tie2 by 3H7 antibody in HUVEC and FOXO1 translocation from nucleus to cytoplasm were examined by immunofluorescence. Specifically, HUVECs were inoculated in an 8-well slide chamber (Lab-TekII) and maintained in EGM-2 medium for 2-3 days. At 100% confluence, cells were serum starved for 4 hours using EBM-2 medium and then treated with 1 μg / ml Tie2 antibody (3H7) for 30 minutes. Cells were then fixed with 4% formaldehyde / PBS solution for 10 minutes at room temperature (RT), permeabilized with 0.1% Triton X-100 / PBS solution, blocked with 1% BSA / PBS solution for 60 minutes at room temperature and , Incubated with primary antibody for 1 hour at RT. As primary antibodies, antibodies against hTie2 and FOXO1 were used. Thereafter, the cells were incubated with fluorescently labeled secondary antibody in dark conditions at room temperature for 1 hour, and mounted with a Vectashield mounting medium containing DAPI (Vector Labs) for nuclear staining. Cell images were then taken and analyzed with a laser scanning confocal microscope (LSM880, Carl Zeiss).

As shown in FIG. 3, the influx into Tie2 cells was strongly induced as in the control Ang2 antibody (Han et al., 2016, Science Translation Medicine), which is known to induce Tie2 clustering and activation upon 3H7 treatment. As reported earlier (Zhang et al, JBC 2002, 277, 45276-45284) that FOXO1 translocates from the nucleus to the cytoplasm when phosphorylated, FOXO1 of the negative control in serum starvation is present in the nucleus. In contrast (red), 3H7 treatment disappeared the signal of FOXO1 in the nucleus and was mostly detected in the cytoplasm.

1.7. Inhibition of vascular permeability induced by VEGF or TNF-a upon HUVEC treatment with anti-Tie2 antibody (3H7)

Vascular leak analysis in HUVEC was performed using the In Vitro Vascular Permeability Assay Kit (Millipore) according to the manufacturer's instructions. HUVECs were inoculated into transwell plate inserts and incubated for 3 days until 100% density. Ang2 (1 μg / ml) alone, a mixture of Ang2 (1 μg / ml) and control antibody (1 μg / ml) or 3H7 antibody (1 μg / ml) alone was preincubated in HUVECs for 30 minutes each, followed by VEGF (500 ng / ml). Or TNF-α (100 ng / ml) and cells were incubated separately at 37 ° C. for 45 minutes or 22 hours. FITC-dextran was then added to the upper chamber and incubated for 20 minutes. The amount of FITC-dextran that penetrated the HUVEC monolayer and accumulated in the lower chamber was measured with a fluorescence reader at excitation and emission wavelengths of 485 and 535 nm, respectively. As shown in FIG. 4, pretreatment of anti-Tie2 antibody (3H7) inhibited vascular leakage caused by VEGF or TNF-α, a vascular leak promoter.

Example 2. DNA sequencing of mouse-derived Tie2 antibody genes.

DNA sequences of the antibodies selected from Example 1.3 (derived from hybridoma cells) were analyzed. Specifically, hybridoma cells (2 × 10 ⁶ cells / ml) were incubated in DMEM containing 10% FBS and total RNA was obtained using RNeasy mini kit (Qiagen). Next, RNA concentration was measured and cDNA was synthesized through reverse transcription (RT) reaction. To amplify heavy and light chain variable region gene sequences, PCR was performed using a mouse Ig-primer set (Novagen) under the following conditions with cDNA as a template: 94 ° C. 5 min; [35 min at 94 ° C., 1 min at 50 ° C., 2 min at 72 ° C.] × 35 times; 6 minutes at 72 ° C .; Cooled to 4 ° C. PCR products obtained in each reaction were cloned into TA vectors to sequence DNA, and as a result, base sequences encoding CDRs, heavy chain variable regions, and light chain variable regions of each antibody were obtained (Tables 3 to 12).

Example 3. Epitope Mapping of Anti-Tie2 Antibodies Against hTie2

Epitopes of hTie2 recognized by mouse monoclonal antibody (3H7) were analyzed by HDX-MS (hydrogen / deuterium exchange-mass spectrometry) technique. HDX-MS analysis techniques are described in Houde D, Engen JR (2013) Methods Mol. Biol. 988: 269-89 and Houde et al. (2011) J. Pharm. Sci. 100 (6), 2071.

Binding-epitope of antibody (3H7) was analyzed using recombinant hTie2-Ig3-FNIII (1-3) protein. The hTie2-Ig3-FNIII (1-3) / antibody mixture was reacted for at least 3 hours prior to the deuterium labeling reaction to maintain the maximum binding state (100%) in 15-fold diluted deuterium labeling buffer (K _D = 25 nM). The prepared hTie2-Ig3-FNIII (1-3) / antibody complex was diluted 15-fold with deuterium labeling buffer, the labeling proceeded several times, and quenched using the same volume of quenching buffer. Labeling reaction times were 0 minutes (undeuterium), 0.33 minutes, 10 minutes, 60 minutes and 240 minutes, respectively. However, in deuterium conditions, deuterium labeling buffer was replaced with equilibrium buffer and the reaction was immediately stopped with quenching buffer. To perform mass spectrometry, deuterium-labeled hTie2-Ig3-FNIII (1-3) / antibody samples were loaded into a pepsin column and peptide digestion was performed. Mass spectrometry yielded 82.6% coverage data from a total of 50 peptides.

The differences in deuterium uptake between hTie2-Ig3-FNIII (1-3) and hTie2-Ig3-FNIII (1-3) / antibody complex conditions were analyzed. Corresponds to site or structurally altered site. The deuterium uptake difference between hTie2-Ig3-FNIII (1-3) and hTie2-Ig3-FNIII (1-3) / antibody complex is considered significant when 0.5-1 Da or more, and is shown in bold in Table 13 .

H / D exchange-mass spectrometry data (FIG. 5) in the presence or absence of anti-Tie2 antibody (3H7) was used to create a heatmap for deuterium uptake per residue at 0.333, 10, 60 and 240 minutes for hTie2 antigen. It was. The color scale represents the percentage of H / D exchange per residue between the antigen alone and the mixture of antigen / monoclonal antibodies at each individual time. Red color indicates increased deuterium exchange and blue color indicates no absorption. Heatmap analysis of the difference in deuterium uptake indicated that the epitope to which the antibody (3H7) binds were 633 to 644 residues (SEQ ID NO: 1, TLSDILPPQPEN) and 713 to 726 residues (SEQ ID NO: 2, FAENNIGSSNPAFS) of hTie2 (Fig. 13). Epitopes are shown in red in the hTie2-FNIII 3D structure generated using PyMol software (FIG. 6).

Example 4. Humanization and Full-length IgG Conversion of Mouse Anti-Tie2 Antibodies

The antibody was humanized in the following manner to remove the immunogenicity of the mouse anti-Tie2 antibody (3H7) when administered to humans.

4.1. Heavy chain humanization

The human antibody heavy chain variable gene IGHV1-46-01 showed 66% homology with the heavy chain sequence of antibody 3H7. Based on this analysis, three CDR regions of the 3H7 antibody were implanted into the human antibody heavy chain variable gene IGHV1-46-01. In this process, two humanized heavy chain antibody genes were designed (Table 14). Transplanted CDRs are underlined in the protein sequence. A back mutation to the mouse sequence was introduced into the heavy chain gene of humanized 3H7, shown in bold in the protein sequences of Table 14.

4.2. Light chain humanization

IGKV1-17-01, a human antibody light chain variable gene, showed 68% homology with the light chain sequence of antibody 3H7. Based on this analysis, three CDR regions of the 3H7 antibody were implanted into the human antibody light chain variable gene IGKV1-17-01. In this process, two humanized light chain antibody genes were designed (Table 14). Transplanted CDRs are underlined in the protein sequence. A back mutation into the mouse sequence was introduced into the light chain gene of humanized 3H7, shown in bold in the protein sequences of Table 14.

4.3. Synthesis of humanized genes and cloning into human full-length IgG antibodies

The humanized variable regions of the antibodies listed in Table 14 were cloned into the heavy and light chain vectors of the human IgG4 isotype backbone vector. DNA fragments of the humanized heavy chain variable region (VH) of the antibody were synthesized into the sequence 'EcoRI-signal sequence-VH-NheI-CH-XhoI' (Bioneer, Inc.). DNA fragments of the humanized light chain variable region (VL) of the antibody were also synthesized into the sequence 'EcoRI-signal sequence-VL-BsiWI-CL-XhoI'. The DNA fragments encoding the heavy and light chains were cloned into pOptiVEC ™ or pcDNA ™ 3.3 vectors, respectively.

4.4. Preparation and Purification of Humanized Anti-Tie2 Antibodies

To produce humanized Tie2 antibodies, Expi293F (Gibco) was used, which can produce highly efficient recombinant proteins. Expi293F cells (2x10 ⁶ cells / ml) were cultured in Erlenmeyer flasks and plasmids encoding heavy and light chains were co-transfected into Expi293F cells using the ExpiFectamine 293 transformation kit. Cells were then incubated for 5 days at 37 ° C., 8% CO ₂ conditions in a shaking incubator (orbital shaker, 125 rpm). The resulting cell culture medium was collected and centrifuged to remove the cells. Culture supernatants containing secreted antibodies were isolated and stored at 4 ° C. or immediately purified using an AKTA purification system (GE Healthcare) equipped with Protein A agarose column (GE Healthcare). Purified antibody was concentrated through a protein centrifuge filter (Amicon) to replace antibody buffer with PBS.

Example 5. Determination of the Affinity of Humanized Anti-Tie2 Antibodies for hTie2

The affinity of the humanized Tie2 antibody for hTie2 was measured using Octet system (ForteBio) using black 96-well plates (96 well F-type black plates, Greiner). Biosensors used for affinity measurements were hydrated for 10 minutes before being measured with an AR2G tip (ForteBio Octet). After hydration the humanized anti-Tie2 antibody was diluted to 10 μg / ml concentration in 10 mM sodium acetate, pH6.0 buffer, fixed in AR2G biosensor and blocked with 1 M ethanolamine. Recombinant hTie2 was diluted to 50, 25, 12.5, 6.25, 3.125 and 0 nM using 1 × kinetic buffer, bound for 300 seconds and dissociated for 900 seconds. Analyze K-on (association rate) and K-off (dissociation rate) globally for affinity measurements (KD) and use 1: 1 occlusion model using octet data analysis program (v9.0.0.10) To fit. KD values are shown in Table 15 below.

Example 6 In vitro Biological Characterization of Selected Humanized Anti-Tie2 Antibodies

6.1. Akt phosphorylation

To investigate whether the humanized anti-Tie2 antibody induces downstream signal transduction of the Tie2 receptor in vascular endothelial cells, HUVECs were treated with humanized Tie2 antibodies. Thereafter, the level of Akt phosphorylation, a major downstream signaling protein of the Tie2 receptor, was measured by immunoblotting. To compare the level of Akt activation, COMP-Ang1 (CA1) was used as a positive control. Specifically, HUVEC cells (1 × 10 ⁵ cells / ml) were incubated at 37 ° C. in EGM-2 medium (Lonza) in a 60 mm culture dish. 90% confluency cells were incubated with EBM-2 (Lonza) for 4 hours. Serum-starved HUVECs were treated with anti-Tie2 antibodies and further incubated for 30 minutes. The cells were washed with cold PBS, then treated with lysis buffer and lysed at 4 ° C. for 20 minutes. Cell lysates were then prepared by centrifugation at 13000 rpm for 15 minutes. 5 × SDS sample buffer was added to the cell lysate and the mixture was heated at 95 ° C. for 5 minutes. The mixture was then SDS-PAGE followed by Western blot.

To investigate the phosphorylation of Akt, the membrane was blocked for 1 hour at room temperature using TBST containing 5% skim milk and incubated with p-Akt antibody (S473) at 4 ° C. for about 8 hours. The amount of p-Akt was detected by ECL (Enhanced ChemiLuminescence). The membrane was then incubated for 15 minutes in stripping buffer (Thermo) and then probed again with anti-Akt antibody to determine the total amount of Akt.

As shown in FIG. 7, Akt phosphorylation was significantly increased by treatment of humanized 3H7 antibody. These data indicate that humanized anti-Tie2 antibodies can strongly induce the activation of Akt, a downstream signaling molecule of the Tie2 receptor in vascular endothelial cells.

Example 7 Effect of 3H7H12G4 in Primary Open-angle Glaucoma Mouse Model

To test whether Tie2 activation by 3H7H12G4 can restore the degraded Schlemm's Canal and lower the intraocular pressure (IoP), a primary open angle glaucoma mouse model was used. For inducible deletion of the Angiopoietin-1 / -2 double gene, a model was constructed by administering tamoxifen to 8-week-old Angiopoietin-1 / Angiopoietin-2 deficient ( A1: A2 ^{iΔ / Δ} ) mice (FIG. 8A). At 12 weeks of age, 3H7H12G4 (˜5 μg, one eye) and FC (˜5 μg, the other eye) were administered intraocularly (FIG. 8A). Each reagent containing 5 μg (˜1 μl, 5 mg / ml) was injected into the vitreous cavity using a Nanoliter 2000 micro-injector (World Precision Instruments) equipped with a glass capillary pipette. IoP was measured using a rebound tonometer (TonoLab) at 12, 13 and 14 weeks of age (FIG. 8A). IoP was measured by placing the tip of the pressure sensor about 1/8 inch from the center of the cornea immediately after anesthetizing the mouse. Digital readings of five consecutive intraocular pressure measurements were obtained on a tonometer. In wild-type mice, there was no intraocular pressure difference between eyes treated with 3H7H12G4 and eyes treated with Fc (FIG. 8C). On the other hand, A1: A2 ^{iΔ / Δ} mice showed a significant IoP reduction of 25.6% in eyes treated with 3H7H12G4 compared to eyes treated with Fc (FIG. 8D). Intensity of Prox1 and Tie2 immunostaining in the CD144 ⁺ SC region and CD144 ⁺ SC was measured 2 weeks after 3H7H12G4 administration. Anti-CD144 antibody (1: 200, BD Biosciences), anti-Prox1 antibody (1: 200, Relia Tech) and anti-Tie2 antibody (1: 200, R & D systems) were used. The CD144 ⁺ SC region of the entire cornea listed was calculated as a percentage of the CD144 ⁺ SC region divided by the control region. In the A1: A2 ^{iΔ / Δ} mice, the SC area of the eye treated with 3H7H12G4 was increased by 114.8% compared to the SC area of the eye treated with FC (FIGS. 8B and 8E). To quantify the relative expression of Prox1, the intensity in the nuclear region of CD144 ⁺ SC was measured. A1: A2 ^{iΔ / Δ} mice showed a significant increase of Prox1 intensity in eyes treated with 3H7H12G4 compared with Prox1 intensity in eyes treated with FC (88.4%) (FIGS. 8B, 8F). The intensity in the CD144 ⁺ SC region was measured to quantify the expression of Tie2. A1: A2 ^{iΔ / Δ} mice showed a significant increase of 63.0% in the Tie2 intensity of eyes treated with 3H7H12G4 compared to the Tie2 intensity of eyes treated with FC (FIGS. 8B, 8F). Overall, these results indicate that Tie2 activation by ^3H7H12G4 restores impaired SC in A1: A2 ^{iΔ / Δ} mice.

Example 8 Inhibition of CNV Degeneration and Vascular Leakage by Intravitreal 3H7H12G4 Injection in Laser-Induced Choroidal Neovascularization (CNV) Model

A laser induced CNV model was used to test the ability of 3H7H12G4 to suppress choroidal neovascularization (CNV), which is characteristic of wet age-related macular degeneration (AMD). Dilated pupils with 5mg / ml phenylephrine and 5mg / ml tropicamide eye drops (Santen pharmaceutical) and drop 0.5% proparacaine hydrochloride eye drops (Alcon) for local anesthesia, and slit lamp delivery The laser photocoagulator (Lumenis Inc.) and glass coverslips were used as contact lenses to visualize the retina. Sufficient laser energy (532nm wavelength, 250mW power, 100ms duration, 50μm spot size) was delivered to four areas of each eye (positions 3, 6, 9, and 12 o'clock of the posterior pole). The study only included bubbles that formed during laser photocoagulation, indicating that the Bruch's membrane was destroyed. Spots containing bleeding at the laser irradiation site were excluded from the analysis. In summary, 3H7H12G4 (5 μg) was administered into the vitreous cavity of mice 7 days after laser photocoagulation (FIG. 9A). As a control or for comparative analysis, Fc or VEGF-Trap (5 μg) was administered to mice in the same manner. To administer the reagents, a Nanoliter 2000 micro-injector (World Precision Instruments) equipped with a glass capillary pipette was used and each reagent containing 5 μg (~ 1 μl, 5 mg / ml) was added to the vitreous cavity. Injection. At 14 days after laser photocoagulation, CD31 ⁺ CNV volumes for the retinal pigment epithelium (RPE) -choroid-scleral planar mounts were calculated using the MATLAB image processing toolbox (MathWorks). CD31 antibody (1: 200, Millipore) was used to detect endothelial cells of CNV. VEGF-Trap effectively induced CNV degeneration by 64.4% compared to Fc, and 3H7H12G4 similarly induced CNV degeneration (65.7%) (FIG. 9B). Fluorescein angiography (FA) and indocyanine green angiography (ICGA) were combined to measure vascular leakage in neovascularization around the area of laser injury. Continuous-wave laser modules of 488nm and 755nm were used as stimulators of Fluorescein and ICG, respectively. The raster scanning pattern of the stimulus laser consists of a scanner system consisting of a rotating polygonal mirror (MC-5; Lincoln Laser) and a galvanometer-based scanning mirror (6230H; Cambridge technology), which is transmitted to the rear aperture of the imaging lens. It became. An objective lens (PlanApo λ, NA 0.75; Nikon) with a high numerical aperture (NA) was used as the imaging lens for wide-field fundus fluorescence images. Fluorescence signals detected with a photoamplifier tube (R9110; Hamamatsu Photonics) were digitized with a frame grabber and reconstructed into an image of 512x512 pixels per frame. In order to visualize late-phase FA and ICGA images using angiography, 10 mg of fluorescein sodium (Alcon) and 0.15 mg of Daiichi Pharmaceutical (ICG) were administered intraperitoneally and intravenously, respectively. . Imaging procedures were performed with general anesthesia and pupil dilation to improve the quality of the image. Java-based imaging software (ImajeJ; National Institutes of Health) was used to calculate the leakage area of CNV by dividing the total amount of hyperfluorescence measured in FA images by the CNV region measured in ICGA images. Compared to Fc, VEGF-Trap (37.0%) and 3H7H12G4 (24.6%) similarly inhibited vascular leakage (FIG. 9C). In particular, the Fc treated group showed no significant difference in vascular leakage between 6 and 14 days after laser photocoagulation, but VEGF-Trap and 3H7H12G4 significantly reduced vascular leakage (45.6% and 42.5%, respectively). Therefore, there was no quantitative difference in the magnitude of inhibition of CNV and vascular leakage between VEGF-Trap and 3H7H12G4 in a mouse model of laser induced CNV.

Example 9 Co-localization of 3H7H12G4 and CD31 in CNV Endothelial Cells.

To investigate whether subcutaneous injection of 3H7H12G4 could also have a therapeutic effect on CNV, co-localization of 3H7H12G4 and CD31 in CNV endothelial cells was evaluated. One day after laser photocoagulation, 3H7H12G4 (25 mg / kg) was subcutaneously administered. Fc (25 mg / kg) was administered to mice in the same manner as a control. Two, four and eight days after laser photocoagulation, co-localization of 3H7H12G4 and CD31 antibodies was directly detected in endothelial cells of CNV via anti-human IgG antibody (1: 1000, Jackson ImmunoResearch Laboratories) (FIG. 10A). The administered 3H7H12G4 was detectable to a high extent in CD31 ⁺ endothelial cell CNV (FIGS. 10B-10D).

Example 10. CNV inhibitory effect by 3H7H12G4 antibody subcutaneous injection.

Subcutaneous administration of 3H7H12G4 (25 mg / kg) was performed one day after laser photocoagulation to determine the effect of 3H7H12G4 subcutaneous injection on CNV inhibition. Fc (25 mg / kg) was administered to mice in the same manner as a control. Anti-CD31 antibody (1: 200, Millipore) was used for detection in CNV endothelial cells and CD31 ⁺ CNV volume for retinal pigment epithelium (RPE) -choroid-scleral plane mounts on day 8 laser photocoagulation Was calculated using the MATLAB image processing toolbox (MathWorks) (FIG. 11A). 3H7H12G4 effectively inhibited CNV production by 69.9% compared to Fc (FIGS. 11B, 11C), indicating that not only intravitreal injection but also subcutaneous injection of 3H7H12G4 has an inhibitory effect on CNV.

The antibody or antigen-binding fragment thereof that binds Tie2 according to the present invention binds to Tie2 with high affinity while maintaining cross-reactivity with humans and mice, and exhibits the desired antigen reactivity. In addition, the Tie2 antibody may be usefully used for the prevention or treatment of the desired angiogenic disease by inducing phosphorylation of Tie2 and activation of the Tie2 receptor.

As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Electronic file attached.

Claims

An antibody or antigen binding fragment thereof against Tie2 that binds to a Tie2 Ig3-FNIII (1-3) domain comprising the sequence of SEQ ID NO: 2.
The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody binds to amino acids 633 to 644 and / or 713 to 726 amino acids of Tie2 comprising the sequence of SEQ ID NO: 1.
According to claim 1, Heavy chain variable region comprising a heavy chain CDR comprising the amino acid sequence of SEQ ID NO: 3 to 5, Light chain variable region comprising a light chain CDR comprising the amino acid sequence of SEQ ID NO: 6 to 8;

A heavy chain variable region comprising a heavy chain CDR comprising the amino acid sequence of SEQ ID NOs: 13 to 15, a light chain variable region comprising a light chain CDR comprising the amino acid sequence of SEQ ID NOs: 16 to 18;

A heavy chain variable region comprising a heavy chain CDR comprising the amino acid sequence of SEQ ID NOs: 23 to 25, a light chain variable region comprising a light chain CDR comprising the amino acid sequence of SEQ ID NOs: 26 to 28;

A heavy chain variable region comprising a heavy chain CDR comprising the amino acid sequence of SEQ ID NOs: 33 to 35, a light chain variable region comprising a light chain CDR comprising the amino acid sequence of SEQ ID NOs: 36 to 38; or

An antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising a heavy chain CDR comprising the amino acid sequence of SEQ ID NOs: 43 to 45, and a light chain variable region comprising a light chain CDR comprising the amino acid sequence of SEQ ID NO: 46 to 48.
The method of claim 1,

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11;

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21;

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31;

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 41;

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 51;

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54;

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 58;

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 61 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 62; or

An antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 65 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 66.
A nucleic acid encoding the antibody of claim 1 or an antigen binding fragment thereof.
The method of claim 5, characterized in that it comprises the sequence of SEQ ID NO: 10, 12, 20, 22, 30, 32, 40, 42, 50, 52, 55, 56, 59, 60, 63, 64, 67 or 68 Nucleic acid.
An expression vector comprising the nucleic acid of claim 5.
A cell transformed with the expression vector of claim 7.
A method for preparing an antibody or antigen-binding fragment thereof that binds Tie2, comprising the following steps:

(a) culturing the cells of claim 8; And

(b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.
A composition for preventing or treating angiogenic diseases, comprising the antibody of claim 1 or an antigen-binding fragment thereof as an active ingredient.
11. The method of claim 10, wherein the angiogenic disease is cancer, metastasis, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, macular degeneration. , Neovascular glaucoma, neovascular glaucoma, erythrosis, proliferative retinopathy, psoriasis, hemophilic arthritis, capillary formation of atherosclerotic plaques, Keloid, wound granulation, vascular adhesion, rheumatoid arthritis, osteoarthritis, autoimmune diseases, Crohn's disease, lesteno Restenosis, atherosclerosis, intestinal adhesions, cat scratch disease, ulcers, liver cirrhosis, nephritis, diabetes Renal diseases (diabetic nephropathy), diabetes mellitus (diabetes mellitus), inflammatory diseases (inflammatory diseases) and neurodegenerative disorders (neurodegenerative diseases) a composition according to claim, selected from the group consisting of.
The method of claim 11, wherein the cancer is esophageal cancer, stomach cancer, large intestine cancer, rectal cancer, oral cancer, pharynx cancer, laryngeal cancer larynx cancer, lung cancer, colon cancer, breast cancer, breast cancer, uterine cervical cancer, endometrial cancer, ovarian cancer, prostate cancer ), Testis cancer, bladder cancer, renal cancer, liver cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer ( skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma and multiple myeloid blood cancer Characterized in that the composition.
A composition for co-administration with another therapeutic agent for angiogenic diseases, comprising the antibody of claim 1 or an antigen-binding fragment thereof.