Anti-Tie2 antibody and its use

Abstract

To provide an antibody against Tie-2 or an antigen binding fragment thereof, a nucleic acid for coding the same, a vector comprising the nucleic acid, a cell transformed with the vector, a method for producing the antibody or the antigen binding fragment thereof, and a composition for preventing or treating angiogenic diseases, comprising the antibody or the antigen binding fragment thereof.SOLUTION: The present invention provides an anti-Tie2 antibody or an antigen binding fragment thereof that binds to the Tie2 Ig3-FNIII (1-3) domain comprising a specific amino acid sequence.SELECTED DRAWING: None

Description

Technical Field

[0001] [1] The present invention relates to an antibody against Tie-2 or an antigen-binding fragment thereof, a nucleic acid encoding the same, a vector containing the nucleic acid, a cell transformed with the vector, a method for producing the antibody or the antigen-binding fragment thereof, and a pharmaceutical composition for preventing or treating an angiogenesis disease. [2]

Background Art

[0002] [3] Angiogenesis occurs dynamically by various regulatory factors during the development, growth, maintenance, and homeostasis of organisms. The newly formed blood vessels in this process function as transport channels for various biological materials such as nutrients, oxygen, and hormones in the surrounding cells. Functionally and structurally abnormal blood vessels are the direct or indirect cause of the onset and progression of various diseases. Tumor blood vessels exacerbate hypoxia due to their functional and structural defects, resulting in tumor progression and metastasis to other tissues, and also insufficient delivery of anticancer drugs to the center of the tumor mass. Defects in blood vessels are also seen in various other diseases and conditions in addition to cancer. Examples include various eye diseases (e.g., diabetic macular edema, wet age-related macular degeneration), acute inflammatory reactions such as viral infections and sepsis. Therefore, if a therapeutic agent capable of normalizing pathological blood vessels is available, it can be applied to the treatment of various patients with vascular abnormalities.

[0003] [4] The angiopoietin family plays an important role in the formation and maintenance of blood vessels and consists of four angiopoietins (Ang1, Ang2, Ang3, and Ang4). Angiopoietin-1 (Ang1) binds to the Tie2 receptor present on the surface of vascular endothelial cells, phosphorylates and activates the Tie2 receptor, and consequently leads to vascular stabilization. On the other hand, angiopoietin-2 (Ang2) binds to the Tie2 receptor but acts as an antagonist to induce inactivation of the Tie2 receptor, resulting in vascular destabilization and vascular leakage. The expression level of Ang2 has been reported to be highly increased in the blood of cancer patients, eye diseases, viral and bacterial infections, and inflammatory diseases (Saharinen P et al., 2017, Nature Review Drug Discovery). However, Ang2 is also known to act as an agonist that induces activation of the Tie2 receptor in some processes including the formation and maintenance of lymphatic vessels and is thought to perform various functions depending on the situation.

[0004] [5] So far, the development and clinical trials of various anti-Ang2 antibodies have been intensively carried out by many biopharmaceutical companies (e.g., U.S. Patent Nos. 7,658,924 and 8,987,420). These Ang2 antibodies inhibit the binding of Ang2 to Tie2, and their Ang2 neutralizing effect has finally been shown to prevent the formation of new blood vessels. The anti-angiogenic and anti-cancer activities of these anti-Ang2 antibodies have been demonstrated in many preclinical models, and various anti-Ang2 antibodies are being clinically tested in various cancer patients. However, their anti-cancer effects have been proven to be insufficient. For example, in a Phase 3 clinical trial conducted by Amgen, the anti-cancer effect of the Ang2 antibody in ovarian cancer patients was shown to be only slight (Marth C et al., 2017, Eur. J. Cancer). In addition to cancer models, nesvacumab, an Ang2 neutralizing antibody, has been tested in eye patients, but its efficacy of Eylea (anti-VEGF) could not be improved in its Phase 2 clinical combination trial.

[0005] [6] In contrast to the Ang2 neutralization approach described above, direct Tie2 activation has also been considered as an alternative approach for inhibiting angiogenesis and suppressing vascular permeability. Recombinant proteins that directly bind to the Tie2 receptor and induce phosphorylation and activation of Tie2 have also been developed and tested in many preclinical cancer and eye models. Examples thereof include COMP-Angl (Cho et al., 2004, PNAS) and Vasculotide (David S et al., 2011, Am J Physiol Lung Cell Mol Physiol). These agents showed anti-angiogenic and anti-permeability activities but had very short half-lives and unstable physicochemical properties. Furthermore, a small molecule compound (AKB-9778) was developed as an inhibitor of phosphatase VE-PTP that inactivates Tie2 by removing phosphate groups from phosphorylated Tie2 (Goel S, 2013, J Natl Cancer Inst). This compound indirectly increases Tie2 activity by inhibiting VE-PTP but has the drawback of also activating other receptors (Frye M, 2015, J Exp.Med, Hayashi M, 2013, Nature Communication, Mellberg S et al., 2009, FASEB J.). Furthermore, agonist Tie2 antibodies have been developed (U.S. Patent No. 6365154, U.S. Patent Application Publication No. 20170174789). These antibodies increased endothelial cell survival and suppressed vascular leakage. Interestingly, herb extracts have been shown to activate Tie2 activity and their use in skin care cosmetics has been claimed (e.g., Japanese Patent Application Publication No. 2011102273, Japanese Patent Application Publication No. 2018043949, Japanese Patent Application Publication No. 2015168656).

[0006] [7] Based on this technical background, the inventors of the present application made efforts to develop antibodies that specifically bind to Tie2. As a result, the inventors developed Tie2 antibodies that bind with affinity and confirmed that these Tie2 antibodies can serve as therapeutic agents for angiogenesis diseases by inducing phosphorylation and activation of the Tie2 receptor, thereby completing the present invention.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Summary of the Invention

Problems to be Solved by the Invention

[0009] [9] (Summary of the Invention)

[10] An object of the present invention is to provide a novel anti-Tie2 antibody or an antigen-binding fragment thereof.

[0010]

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

[0011]

[12] Another object of the present invention is to provide a vector containing the nucleic acid, a cell transformed with the vector, and a method for producing the same.

[0012]

[13] Another object of the present invention is to provide a composition for preventing or treating an angiogenesis disease, which contains the antibody or an antigen-binding fragment thereof.

[0013]

[14] Another object of the present invention is to provide a composition containing the antibody or an antigen-binding fragment thereof, and a composition for co-administration with other therapeutic agents for angiogenesis diseases.

Means for Solving the Problems

[0014]

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

[0015]

[16] Specifically, the present invention provides a heavy chain variable region containing a heavy chain CDR having the amino acid sequences of SEQ ID NOs: 3 to 5, a light chain variable region containing a light chain CDR having the amino acid sequences of SEQ ID NOs: 6 to 8; a heavy chain variable region containing a heavy chain CDR having the amino acid sequences of SEQ ID NOs: 13 to 15, a light chain variable region containing a light chain CDR having the amino acid sequences of SEQ ID NOs: 16 to 18; a heavy chain variable region containing a heavy chain CDR having the amino acid sequences of SEQ ID NOs: 23 to 25, a light chain variable region containing a light chain CDR having the amino acid sequences of SEQ ID NOs: 26 to 28; a heavy chain variable region containing a heavy chain CDR having the amino acid sequences of SEQ ID NOs: 33 to 35, a light chain variable region containing a light chain CDR having the amino acid sequences of SEQ ID NOs: 36 to 38; or a heavy chain variable region containing a heavy chain CDR having the amino acid sequences of SEQ ID NOs: 43 to 45, a light chain variable region containing a light chain CDR having the amino acid sequences of SEQ ID NOs: 46 to 48, and also provides an anti-Tie2 antibody or an antigen-binding fragment thereof.

[0016]

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

[0017]

[18] The present invention also provides a vector encoding the nucleic acid.

[0018]

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

[0019]

[20] The present invention also provides a method for producing the antibody or an antigen-binding fragment thereof, comprising the following steps: (a) a step for culturing the cell; and (b) a step for recovering the antibody or an antigen-binding fragment thereof from the cell.

[0020]

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

[0021]

[22] The present invention also provides a composition for co-administration with other angiogenesis disease therapeutics, comprising the antibody or an antigen-binding fragment thereof.

[23]

Brief Description of the Drawings

[0022]

Figure 1

[24] Figure 1 shows the analysis results regarding Akt phosphorylation induced by anti-Tie2 antibodies. HUVECs were 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. Cell lysates were subjected to SDS-PAGE / Western blotting, and the blots were examined using anti-phospho-Akt (S473) or anti-Akt antibodies.

[0023]

Figure 2

[25] Figure 2 shows the analysis results regarding dose-dependent Tie2 phosphorylation (pTie2) induced by anti-Tie2 antibody 3H7. The ability of 3H7 antibody to induce Tie2 phosphorylation was examined by immunoprecipitation and Western blotting analysis. Serum-starved HUVECs were incubated with various concentrations of 3H7 antibody for 30 minutes. As a control, HUVECs were incubated with Ang2 / control Ab mixture. After immunoprecipitating the cell lysates with anti-Tie2 antibody, SDS-PAGE / Western blotting analysis was performed. Tie2 phosphorylation was examined using mouse anti-phosphotyrosine (pY) antibody 4G10.

[0024]

Figure 3

[26] Figure 3 shows the results regarding Tie2 endocytosis and FOXO1 translocation induced by anti-Tie2 antibody 3H7. HUVECs were serum-starved for 6 hours and incubated with 3H7 or Ang2 (A2) and control anti-Ang2 antibody (control Ab, 1 μg / ml) for 30 minutes. After fixation, HUVECs were stained with DAPI (blue), anti-Tie2 antibody (green), and anti-FOXO1 antibody (red), and the localization of clustered Tie2 receptor and FOXO1 was examined. Arrowheads indicate Tie2 receptors endocytosed by 3H7.

[0025]

Figure 4

[27] Figure 4 shows the results regarding the inhibition of VEGF- or TNF-α-induced vascular permeability by 3H7. HUVECs were seeded in transwell chambers and grown for 3 days. HUVECs at 100% confluence were pretreated for 30 minutes with Ang2 (A2, 1 μg / ml), Ang2 and control Ab (A2 + control Ab, 1 μg / ml) or 3H7 (1 μg / ml), and then treated in the upper chamber with VEGF (500 ng / ml) for 45 minutes (A) or TNF-α (100 ng / ml) for 22 hours (B). Vascular permeability was evaluated by measuring the FITC fluorescence in the lower chamber after adding FITC-dextran to the upper chamber for 20 minutes. Values are mean ± SD. By one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001.

[0026]

Figure 5

[28] Figure 5 shows the results regarding a heat map indicating regions of significant difference in deuterium incorporation when Tie2 alone or the Tie2 / 3H7 complex was tested by hydrogen / deuterium (H / D) exchange mass spectrometry. Using the data from H / D exchange mass spectrometry, heat maps of deuterium incorporation per residue for the hTie2 antigen at 0.333 minutes, 10 minutes, 60 minutes, and 240 minutes in the absence or presence of the anti-Tie2 antibody 3H7 were created. The color scale indicates the percentage of H / D exchange per residue between Tie2 alone and the Tie2 / 3H7 mixture at each individual time. Red indicates regions where deuterium exchange increased, and blue indicates no incorporation.

[0027]

Figure 6

[29] Figure 6 is a schematic diagram showing the 3H7 binding epitope on Tie2. The anti-Tie2 antibody 3H7 binding epitope (red) on the hTie2 antigen analyzed by H / D exchange mass spectrometry was visualized on an image of the hTie2 FNIII(1-3) crystal structure (PDB: 5UTK) using PyMol software.

[0028]

Figure 7

[30] Figure 7 shows the results regarding the phosphorylation of Akt (pAkt) induced by the humanized anti-Tie2 antibody. Serum-starved HUVECs were incubated with the humanized anti-Tie2 antibody for 30 minutes. Thereafter, the cell lysates were subjected to SDS-PAGE / Western blotting, and the blots were probed with anti-phospho-Akt (S473) or anti-Akt antibody.

[0029]

Figure 8

[31] Figure 8 shows the results regarding the increase in SC area and the decrease in IOP by the humanized Tie2 antibody 3H7H12G4 in a mouse model of primary open-angle glaucoma. Tamoxifen administration for the inducible deletion of both angiopoietin-1 and -2 was performed in 8-week-old A1:A2iΔ / Δ mice. Intravitreal administration of 3H7H12G4 (one eye, 1 μl injection of a 5 mg / ml solution) and Fc (the contralateral eye, 1 μl injection of a 5 mg / ml solution) was performed at 12 weeks of age. Regular measurements of intraocular pressure (IOP) were performed at 12, 13, and 14 weeks of age. The CD144+ SC area and the intensity of Prox1 and Tie2 immunostaining within CD144+ SC were measured 2 weeks after 3H7H12G4 administration. Scale bar, 100 μm. n = 5 per group. Values are mean ± SD. *p < 0.05 by Kruskal-Wallis test followed by Tukey's ranked HSD test.

[0030]

Figure 9

[32] Figure 9 shows the results regarding the suppression of CNV (Choroidal Neo-Vascularization) and vascular leakage by 3H7H12G4 injected intravitreally in the laser-induced CNV model. Intravitreal administration of the antibody (1 μl injection of a 5 mg / ml solution) was performed 7 days after laser photocoagulation. The CD31+ CNV volume was measured, and the leakage area around CNV was calculated by dividing the total area of strong fluorescence measured in the FA images 6 days and / or 14 days after laser photocoagulation by the total CNV area measured in the ICGA image. n = 11 per group. Values are mean ± SD. By one-way ANOVA followed by Student-Newman-Keuls post hoc test, *p < 0.05, ***p < 0.001. By paired Student's t-test, ##p < 0.01, p < 0.001.

[0031]

Figure 10

[33] Figure 10 shows the results regarding the co-localization of 3H7H12G4 and CD31 in endothelial cells within the CNV region. Subcutaneous administration of 3H7H12G4 was performed 1 day after laser photocoagulation. The co-localization of 3H7H12G4 and CD31 in the endothelial cells of CNV was directly detected with an anti-human IgG secondary antibody 2 days, 4 days, and 8 days after laser photocoagulation.

[0032]

Figure 11

[34] Figure 11 shows the results regarding the suppression by subcutaneously injected 3H7H12G4. Subcutaneous administration of 3H7H12G4 was performed on the first day after laser photocoagulation. The CD31+ CNV volume was measured 8 days after laser photocoagulation. Scale bar, 100 μm. n = 10 per group. Values are mean ± SD. By unpaired Student's t-test, ***p < 0.001.

Mode for Carrying Out the Invention

[0033]

[35]

[36] Detailed Description and Preferred Embodiments of the Present Invention

[37] Unless otherwise defined, 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 disclosure pertains.

[0034]

[38] The inventors of the present application have confirmed that the Tie2 antibody is an agent that increases Tie2 activity by binding to the Tie2 Ig3-FNIII(1-3) domain containing the sequence of SEQ ID NO: 2.

[0035]

[39] Tie2 is a receptor protein that promotes vascular differentiation and stabilization and is highly expressed in blood vessels. When the Tie2 receptor is activated, it stabilizes cancer blood vessels, which enables it to gather surrounding supporting cells. The antibody or antigen-binding fragment thereof according to the present invention normalizes the active Tie2 in cancer blood vessels, eliminates increased hypoxia in the tumor, increases blood flow to the tumor to supply sufficient oxygen, and increases the delivery of other anticancer agents and the penetration of immune cells.

[0036]

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

[0037]

[41] As used herein, the term "antibody" means an antibody that specifically binds to Tie2. Within the scope of the present invention, in addition to a complete antibody that specifically binds to Tie2, antigen-binding fragments of the antibody molecule are also included.

[0038]

[42] A complete antibody has a structure of two full-length light chains and two full-length heavy chains, and each light chain is linked to the heavy chain by a disulfide bond. The constant regions of the heavy chains include gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and the subclasses include gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The constant regions of the light chains include kappa (κ) type and lambda (λ) type.

[0039]

[43] An antigen-binding fragment of an antibody or a fragment of an antibody means a fragment capable of binding to an antigen, and includes Fab, F(ab’), F(ab’)2 and Fv. Among antibody fragments, Fab has one antigen-binding site and includes a structure having the variable regions of the light and heavy chains, the constant region of the light chain, and the first CH1 of the heavy chain.

[0040]

[44] Fab’ differs from Fab in having a hinge region containing one or more cysteine residues at the C-terminus of the CH1 domain. An F(ab’)2 antibody is produced by the formation of a disulfide bond between the cysteine residues of the hinge region of Fab’. Fv is the smallest antibody fragment having only the variable regions of the heavy and light chains. A double-stranded Fv (dsFv) is formed by a non-covalent bond between the heavy-chain variable region and the light-chain variable region, and a single-chain Fv (scFv) is generally formed through a covalent peptide linker between the heavy-chain variable region and the light-chain variable region or is linked by directly forming a dimer-like structure such as dsFv at its C-terminus. These fragments can be obtained by proteolytic enzymes (for example, Fab can be obtained by limited digestion of a complete antibody with papain, and F(ab’)2 can be obtained by cleavage with pepsin), and can also be produced by genetic engineering techniques.

[0041]

[45] In one embodiment, the antibody according to the present invention is in the form of Fv (for example, scFv) or in the form of a complete antibody. Further, the constant region of the heavy chain may be selected from any 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 the kappa or lambda type.

[0042]

[46] As used herein, the term "heavy chain" means a full-length heavy chain or a fragment thereof, which comprises a variable region domain VH and three constant region domains CH1, CH2 and CH3, and has an amino acid sequence with a variable region sufficient to provide antigen specificity. Further, the term "light chain" as used herein means a full-length light chain or a fragment thereof, which comprises a variable region domain VL and a constant region domain CL, and has an amino acid sequence with a variable region sufficient to provide antigen specificity.

[0043]

[47] The antibodies of the present invention include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fv (scFv), single-chain antibodies, Fab fragments, F(ab’) fragments, disulfide-bonded Fv (sdFV) and anti-idiotype (anti-Id) antibodies, or epitope-binding fragments of the above antibodies, and analogs thereof.

[0044]

[48] A monoclonal antibody is an antibody obtained from a substantially homogeneous population of antibodies, i.e., it means the same particular antibody in a population that may be present in trace amounts except for possible natural mutations. Monoclonal antibodies are highly specific and are induced against a single antigenic site. In contrast to conventional (polyclonal) antibodies, which typically include different antibodies directed by different epitopes, monoclonal antibodies are each directed by a single determinant.

[0045]

[49] "Epitope" means a protein determinant to which an antibody can specifically bind. An epitope is usually a group of chemically active surface molecules, such as amino acid or sugar side chains, and generally has specific charge characteristics as well as specific three-dimensional structural characteristics. Conformational epitopes and non-conformational epitopes lose their binding to the former in the presence of a denaturing solvent, but do not lose their binding to the latter.

[0046]

[50] When the epitope is identified through hydrogen / deuterium exchange, the Tie2 antibody or antigen-binding fragment thereof according to the present invention binds to the amino acids TLSDILPPQPEN at positions 633-644 and / or the amino acids FAENNIGSSNPAFS at positions 713-726 of Tie2 that constitute the sequence of SEQ ID NO: 1.

[0047]

[51] The "humanized" form of a non-human (e.g., mouse) antibody is a chimeric antibody having a minimal sequence derived from a non-human immunoglobulin that includes one or more amino acid sequences (e.g., CDR sequences) from the non-human antibody (e.g., donor or source antibody). In most cases, the humanized antibody is a human immunoglobulin (recipient antibody) whose hypervariable regions are replaced with residues from the hypervariable regions of a non-human primate, mouse, rat, rabbit, or non-human primate (recipient antibody), and that has the desired specificity, affinity, and ability of the residues from the recipient's hypervariable regions. For humanization, one or more of the variable regions within the residues of the framework domain (FR) of the recipient human antibody can be replaced with the corresponding residues from a donor antibody of a non-human species. This maintains the appropriate three-dimensional conformation of the grafted CDRs, thereby potentially improving affinity and antibody stability. The humanized antibody can further refine additional properties of the antibody, including, for example, new residues not present in the antibody or donor antibody.

[0048]

[52] A "humanized antibody" as a molecule derived from a human immunoglobulin means that the entire amino acid sequence constituting the antibody, including the complementarity-determining regions and structural regions, is composed of a human immunoglobulin.

[0049]

[53] Includes any "chimeric" antibody (immunoglobulin) that exhibits the desired biological activity and fragments of said antibody, where the chimeric antibody has a part of the heavy chain and / or light chain that is derived from a particular species or is identical or homologous to the corresponding sequence in an antibody belonging to its subclass, while the remaining chain is derived from another species or belongs to another antibody class or is identical to the corresponding sequence in an antibody belonging to its subclass.

[0050]

[54] For certain examples of this application, mouse-derived 11C4, 4A4, 3B2, 3E12, and 3H7 antibodies were produced, the CDRs of the 3H7 antibody as a donor antibody were grafted, and humanized 3H7H11G4, 3H7H12G4, 3H7H21G4, or 3H7H22G4 antibodies were produced.

[0051]

[55] As used herein, "antibody variable domain" refers to a part of the light and heavy chains of an antibody that includes the amino acid sequences of the complementarity-determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and the framework regions (FR). VH represents the variable domain of the heavy chain. VL represents the variable domain of the light chain.

[0052]

[56] "Complementarity-determining region" (CDR; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of the variable domain of an antibody necessary for antigen binding. Each variable domain typically includes three CDR regions, usually identified as CDR1, CDR2, and CDR3.

[0053]

[57] In one embodiment, the Tie2 antibody or an antigen-binding fragment thereof may include a heavy chain variable region comprising a heavy chain CDR having the amino acid sequence of SEQ ID NO: 3-5, and a light chain variable region comprising a light chain CDR having the amino acid sequence of SEQ ID NO: 6-8;

[0054]

[58] a heavy chain variable region comprising a heavy chain CDR having the amino acid sequence of SEQ ID NO: 13-15, and a light chain variable region comprising a light chain CDR having the amino acid sequence of SEQ ID NO: 16-18;

[0055]

[59] a heavy chain variable region comprising a heavy chain CDR having the amino acid sequence of SEQ ID NO: 23-25, and a light chain variable region comprising a light chain CDR having the amino acid sequence of SEQ ID NO: 26-28;

[0056]

[60] a heavy chain variable region comprising a heavy chain CDR having the amino acid sequence of SEQ ID NO: 33-35, and a light chain variable region comprising a light chain CDR having the amino acid sequence of SEQ ID NO: 36-38;

[0057]

[61] A heavy chain variable region containing a heavy chain CDR having the amino acid sequences of SEQ ID NOs: 43 to 45, and a light chain variable region containing a light chain CDR having the amino acid sequences of SEQ ID NOs: 46 to 48.

[0058]

[62] The "framework region" (FR) is the variable domain residue other than the CDR residue. Each variable domain usually has four FRs identified as FR1, FR2, FR3, and FR4.

[0059]

[63] The Tie2 antibody is monovalent or divalent and includes single-stranded or double-stranded. Functionally, the binding affinity of the Tie2 antibody is 10 -5 M to 10 -12 M. For example, the binding affinity of the 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 is.

[0060]

[64] The Tie2 antibody or its antigen-binding fragment may contain a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 9 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 11;

[0061]

[65] A heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 19 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 21;

[0062]

[66] A heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 29 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 31;

[0063]

[67] A heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 39 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 41;

[0064]

[68] A heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 49 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 51;

[0065]

[69] A heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 53 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 54;

[0066]

[70] A heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 57 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 58;

[0067]

[71] A heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 61 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 62;

[0068]

[72] A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 19 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 21.

[0069]

[73] The antibody or antibody fragment of the present invention may include a biologically equivalent thereof as long as it can specifically recognize Tie2. For example, in order to further improve the binding affinity and / or other biological properties of the antibody, changes can be introduced into the amino acid sequence. Such modifications may include, for example, deletions, insertions and / or substitutions of amino acid sequence residues of the antibody. These amino acid variations are made based on the relative similarity of amino acid substituents such as the hydrophobicity, hydrophilicity, charge, size of the amino acid side chain. By analysis of the size, shape and type of amino acid side chain substituents, it is known that arginine, lysine and histidine are all positively charged residues; alanine, glycine and serine have similar sizes; and phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; phenylalanine, tryptophan and tyrosine can be said to be biologically functionally equivalent.

[0070]

[74] Considering the above mutations having biologically equivalent activities, the amino acid sequence of the antibody of the present invention or the nucleic acid molecule encoding the same antibody is construed to include the sequence of the sequence number and any sequence showing substantial identity. Substantial identity means at least 90% homology, most preferably at least 95% homology, 96% or more, 97% or more, 98% or more, 99% or more sequence homology when the sequences described in the present invention and any other sequence are aligned as much as possible and analyzed by algorithms commonly used in the art. Alignment methods for sequence comparison are known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) can be accessed from NBCI etc. and can be used in combination with sequence analysis programs such as blastp, blasm, blastx, tblastn and tblastx on the Internet. BLSAT can be accessed at www.ncbi.nlm.nih.gov / BLAST / . The sequence homology comparison method using this program can be found at www.ncbi.nlm.nih.gov / BLAST / blast_help.html.

[0071]

[75] Based on this, the antibody or antigen-binding fragment thereof of the present invention may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology when compared to the sequences specified herein or all sequences. This homology can be determined by sequence comparison and / or alignment by methods known in the art. For example, the percent sequence homology of the nucleic acid or protein of the present invention can be determined using a sequence comparison algorithm (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection.

[0072]

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

[0073]

[77] 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.

[0074]

[78] The antibody or its antigen-binding fragment can be recombinantly produced by isolating the nucleic acid encoding the antibody or its antigen-binding fragment of the present invention. Further cloning (DNA amplification) can be performed by isolating the nucleic acid and inserting it into a replicable vector, or further expression can be performed. Based on this, the present invention relates to a vector containing nucleic acid in another aspect.

[0075]

[79] "Nucleic acid" means to comprehensively include DNA (gDNA and cDNA) and RNA molecules, and the nucleotide, which is the basic structural unit of nucleic acid, essentially includes nucleotides and analogs having modified sugar or base moieties. The sequence of the nucleic acid encoding the heavy and light chain variable regions of the present invention can be modified. The modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0076]

[80] The DNA encoding the antibody can be easily isolated or synthesized using conventional processes (for example, by using oligonucleotide probes that can specifically bind to the DNA encoding the heavy and light chains). Many vectors can be utilized. The 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 element, promoter, and transcription termination sequence.

[0077]

[81] As used herein, the term "vector" includes viral vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors as a means for expressing the gene of interest in a host cell. The nucleic acid encoding the antibody in the vector is operably linked to a promoter.

[0078]

[82] "Operably linked" means a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, signal sequence, or sequence of a transcriptional regulatory factor binding site) and a different nucleic acid sequence, whereby said control sequence controls the transcription and / or translation of said other nucleic acid sequence.

[0079]

[83] When the host is a prokaryotic cell, strong promoters capable of processing 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 translation initiation, and transcription / translation termination sequences are generally included. In addition, for example, when the host is a eukaryotic cell, promoters derived from the genomes of mammalian cells (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used, and generally, a polyadenylation sequence can be included as a transcription termination sequence.

[0080]

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

[0081]

[85] The vector contains antibiotic resistance genes commonly used in the art as selection markers, such as resistance genes to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0082]

[86] In another aspect, the present invention relates to a cell transformed with the above vector. The cells used to produce the antibodies of the present invention may be, but are not limited to, prokaryotes, yeasts, and higher eukaryotic cells.

[0083]

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

[0084]

[88] However, the greatest interest lies in animal cells, and examples of useful host cell lines 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, HepG2, SK-Hep, MMT, TRI, MRC5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0085]

[89] In another aspect, the present invention relates to a method for producing an antibody and an antigen-binding fragment thereof, comprising (a) steps for culturing cells; and (b) steps for recovering the antibody or its antigen-binding fragment from the cultured cells.

[0086]

[90] Cells can be cultured in various media. Any commercially available medium can be used without limitation. All other essential supplements known to those skilled in the art can be included at appropriate concentrations. Culture conditions such as temperature, pH, and the selected host cell have already been used and will be apparent to those skilled in the art.

[0087]

[91] The recovery of the antibody or its antigen-binding fragment can be carried out by removing impurities using, for example, centrifugation or ultrafiltration, and by using, for example, affinity chromatography and the like. Further additional purification techniques, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography and similar techniques can be used.

[0088]

[92] In another aspect, the present invention relates to a composition for preventing or treating an angiogenesis disease comprising an active ingredient of an antibody or its antigen-binding fragment as an active ingredient.

[0089]

[93] Angiogenesis means the formation or growth of new blood vessels from existing blood vessels. "Angiogenesis-related disease" means a disease related to the occurrence or progression of angiogenesis. If a disease can be treated with an antibody, the disease can be included in the scope of angiogenesis-related diseases without limitation. Examples of angiogenesis-related diseases include, but are not limited to, cancer, metastasis, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, macular degeneration, neovascular glaucoma, exfoliative dermatitis, proliferative retinopathy, psoriasis, hemophilic arthritis, capillary formation in atherosclerotic plaques, keloid, wound granulation, vascular adhesion, rheumatoid arthritis, osteoarthritis, autoimmune diseases, Crohn's disease, restenosis, atherosclerosis, rheumatoid arthritis, cat scratch wounds, ulcers, cirrhosis, nephritis, diabetic nephropathy, diabetes, inflammatory diseases and neurodegenerative diseases. In addition, cancer includes, but is not limited to, esophageal cancer, gastric cancer, colorectal cancer, rectal cancer, oral cancer, pharynx cancer, larynx cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin lymphoma, lymphoma and multiple myeloma blood cancer selected from the group consisting of, but not limited to, these.

[0090]

[94] As used herein, the term "prevention" means any action that prevents or delays the onset of a disease of interest by administering the antibody or composition of the present invention. The term "treatment or therapy" indicates any action that improves or ameliorates the symptoms of a disease of interest by administering the antibody or composition of the present invention.

[0091]

[95] The composition containing the antibody of the present invention is preferably a pharmaceutical composition and may contain a suitable vehicle, excipient or diluent typically used in the art.

[0092]

[96] A pharmaceutical composition having a pharmaceutically acceptable vehicle may be in various oral or parenteral dosage forms, such as tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterile aqueous liquids, non-aqueous liquids, suspensions, lyophilized agents and suppositories. Regarding the pharmaceutical composition of the present invention, it may be a diluent or excipient that can be formulated with a combination of, for example, 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. In connection with binding, the compounds of the present invention can be formulated by combining one or more excipients, such as starch, calcium carbonate, sucrose, lactose or gelatin. Simple excipients and lubricants, such as magnesium stearate, talc and the like can be further used. Liquid preparations for oral administration may be suspensions, oral solutions, emulsions, syrups or the like. Excipients, such as simple diluents like water or wet paraffin, various wetting agents, sweeteners, fragrances, preservatives and the like can be included in the liquid preparation. In addition, the pharmaceutical composition of the present invention may be in parenteral dosage forms, such as sterile aqueous liquids, non-aqueous solvents, suspensions, emulsions, lyophilized agents, 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 insoluble solvents and suspensions. The basic substances of suppositories include witepsol, macrogol, tween 61, cocoa butter, laureth butter and glycerogelatin.

[0093]

[97] The composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount of a pharmaceutical composition sufficient for treating a disease, with an appropriate benefit / risk ratio applicable to all medical treatments. The effective amount may vary depending on various factors including the severity of the disease, the age and gender of the patient, the type of disease, drug activity, drug sensitivity, administration time, administration route, secretion rate, treatment period, co-administration of drugs, and other factors known in the art, and other factors known in the art. The composition of the present invention can be administered alone or in combination with other treatments. In this case, the composition can be administered sequentially or simultaneously with conventional therapies. Also, the above composition can be administered as a single dose or divided into multiple doses. Considering these factors sufficiently, it is important to administer the minimum amount sufficient to obtain the maximum effect without side effects, and the dosage can be easily set by an expert. The dosage of the pharmaceutical composition of the present invention is not particularly limited, but it varies depending on various factors such as the health status and weight of the patient, the severity of the disease, the type of drug, the administration route, and the administration time. The composition may be administered once or multiple times a day to mammals including rats, mice, livestock, humans, etc. via typically acceptable routes, such as orally, rectally, intravenously, subcutaneously, intrauterinely, or intracerebrovascularly.

[0094]

[98] From another aspect, the present invention relates to a method for preventing or treating an angiogenesis disease, which comprises the step of administering the antibody or the composition to an individual in need thereof.

[0095]

[99] The method of the present invention includes a procedure for administering a pharmaceutically effective dosage of a pharmaceutical composition to an individual in need of inhibiting angiogenesis. The subject is a mammal, such as a dog, cow, horse, rabbit, mouse, rat, chicken, and human, but is not limited thereto. The pharmaceutical composition can be administered by appropriate methods including parenterally, subcutaneously, intraperitoneally, intratracheally, or intranasally, and, if necessary, into the wound for local treatment. The preferred dosage of the pharmaceutical composition of the present invention varies depending on various factors including the health status and weight of the individual, the severity of the disease, the type of drug, the route and time of administration, and it can be easily determined by those skilled in the art.

[0096]

[0100] In other aspects, the present invention relates to a method for preventing or treating cancer, comprising administering to an individual in need of an antibody and a composition the composition or the procedure of the antibody, and a composition or a pharmaceutical composition for preventing or treating cancer, comprising the antibody.

[0097]

[0101] Cancer is not limited as long as it is treatable with the antibody of the present invention. Specifically, the antibody of the present invention can prevent the occurrence or progression of cancer by inhibiting angiogenesis. Examples of cancer include, but are not limited to, esophageal cancer, gastric cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin lymphoma, lymphoma and multiple myeloma, blood cancer.

[0098]

[0102] In addition, the antibody of the present invention can be used in combination with other antibodies or biologically active agents or materials for various purposes. In this regard, the present invention relates to a composition for co-administration with other therapeutic agents for angiogenesis diseases, comprising an antibody or an antigen-binding fragment thereof.

[0099]

[0103] Other therapeutic agents for angiogenesis diseases include anti-angiogenic agents, anti-inflammatory agents and / or anti-cancer agents. Thereby, mutual resistance can be overcome and efficacy can be improved.

[0100]

[0104] In the composition according to the present invention, when administered in combination with other therapeutic agents for angiogenesis diseases, the Tie2 antibody and other therapeutic agents for angiogenesis diseases may be administered sequentially or simultaneously. For example, after administering an anti-angiogenic agent, an anti-inflammatory agent and / or an anti-cancer agent to a target individual, a composition having an antibody against Tie2 or an antigen-binding fragment thereof as an active ingredient can be administered, or after administering the composition, an anti-angiogenic agent, an anti-inflammatory agent and / or an anti-cancer agent can be administered. Optionally, the composition and the anti-angiogenic agent, the anti-inflammatory agent and / or the anti-cancer agent can be administered to the target individual simultaneously.

[0101]

[0105]

[0106] Example

[0107] Hereinafter, the present invention will be described in detail by way of examples. The following examples are merely intended to illustrate the present invention and do not limit the present invention.

[0102]

[0108]

[0109] Example 1. Preparation of mouse monoclonal anti-Tie2 antibody

[0110] 1.1 Immunization of mice with human Tie2

[0111] The Ig3-FNIII(1-3) domain of human Tie2 (hTie2-Ig3-FNIII(1-3), SEQ ID NO: 2) was cloned into a vector containing the CMV promoter using it as an immunogen and transiently expressed by transfection into the HEK293F cell line. After 5 days of incubation, the expressed recombinant hTie2-Ig3-FNIII(1-3) protein was purified using an affinity column with Protein A. Five-week-old BALB / c mice were immunized twice a week for 6 weeks using purified hTie2-Ig3-FNIII(1-3) (100 μg / injection) mixed with an adjuvant. The titer of anti-Tie2 antibody in the serum of the immunized mice was examined using a human Tie2 (hTie2) ELISA kit (R&D). When the antibody titer (1:5,000 dilution) increased appropriately (OD > 1.0), the spleen was removed from the immunized mice, B lymphocytes were isolated from it, 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 fusions of myeloma cells and B lymphocytes were selected and cultured. The surviving hybridoma cells were seeded in 96-well plates, and their culture supernatants were tested by hTie2 ELISA. For clone selection, a hybridoma pool showing a positive signal was selected by limiting dilution. Finally, 37 monoclonal hybridoma lines were established. Among them, some Tie2-binding antibodies showed Tie2 activation effects. The candidate antibodies were selected based on the Tie2 activation level and high affinity for human Tie2 and were later humanized.

[0103]

[0112]

[0113] Table 1. Full-length human Tie2 (hTie2) and Ig3-FNIII(1-3) sequences

Table 1-1

[0104]

[0114]

Table 1-2

[0105]

[0115]

[0116] 1.2 Production and Purification of Mouse Monoclonal Tie2 Antibody To produce anti-Tie2 antibodies selected based on ELISA positive signals, hybridoma cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS in a T75 (75 cm 2 area) flask. When the cell confluence reached approximately 90%, the cells were washed with PBS, incubated with 50 ml of serum-free medium (SFM, Gibco), and cultured at 37°C for 3 days. Then, the medium in which antibodies were secreted from each monoclonal hybridoma was collected, centrifuged to remove cells, and the culture supernatant was collected and filtered. Next, the antibody was purified using an AKTA purification device (GE Healthcare) equipped with a Protein G affinity column (GE Healthcare). The purified antibody was concentrated by replacing the supernatant with PBS using a centrifugal filter unit (Amicon).

[0106]

[0118]

[0119] 1.3 Identification and Screening of Antagonist Tie2 Antibody

[0120] To investigate whether mouse anti-Tie2 antibody induces downstream signaling of Tie2 receptor in endothelial cells, HUVEC (Lonza) was treated with anti-Tie2 antibody, and then the level of Akt phosphorylation, a major downstream signaling protein, and Tie2 receptor were analyzed by immunoblotting. As a positive control, COMP-Ang1 (CA1) was treated with the cells.

[0107]

[0121] Specifically, HUVEC (1×10 5Cells / ml) were cultured in EGM-2 medium (Lonza) at 37 °C in a 60 mm culture dish. Cells at 90% confluence were incubated for 6 hours in serum-free EBM-2 medium for serum starvation. Serum-starved HUVECs were treated with anti-Tie2 antibody and further incubated for 30 minutes. The cells were washed with cold PBS, treated with lysis buffer, and lysed at 4 °C for 20 minutes. Subsequently, cell lysates were prepared by centrifugation at 13,000 rpm for 15 minutes. Cell lysates were prepared by adding 5×SDS sample buffer, and the cell lysates were subjected to SDS PAGE and the proteins were transferred to a nitrocellulose membrane (GE).

[0108]

[0122] To examine Akt phosphorylation, the blot was blocked with TBS-T containing 5% skim milk at room temperature (RT) for 1 hour and incubated with anti-phospho-Akt antibody (S473) at 4 °C for approximately 8 hours. The signal of phospho-Akt was visualized by enhanced chemiluminescence (ECL). Subsequently, the membrane was incubated with stripping buffer (Thermo) for 15 minutes and reprobed with anti-Akt antibody to measure the total amount of Akt. Akt phosphorylation at S473 was strongly induced in several groups treated with anti-Tie2 antibodies such as 11C4, 4A4, 3B2, 3E12, and 3H7 (Figure 1).

[0109]

[0123]

[0124] 1.4. Measurement of the affinity of anti-Tie2 antibodies for hTie2 by octet analysis

[0125] The affinity of the mouse monoclonal antibody against hTie2 was measured using the Octet system (ForteBio) with a black 96-well plate (96-well F-type black plate, Greiner). The biosensor used for affinity measurement was hydrated for 10 minutes before measurement using an AR2G chip (ForteBio Octet). After hydration, hTie2 was diluted to a concentration of 10 μg / ml with 10 mM sodium acetate, pH 6.0 buffer, immobilized on the AR2G biosensor, and blocked with 1 M ethanolamine. The mouse monoclonal anti-Tie2 antibody was diluted to 50, 25, 12.5, 6.25, 3.125, and 0 nM with 1× kinetic buffer, and subjected to 300 seconds of association and 900 seconds of dissociation. For the affinity measurement (KD), the association rate (K-on) and dissociation rate (K-off) were analyzed by the binding curve (global) and fitted to a 1:1 binding model using the Octet data analysis v9.0.0.10 program. The affinity of the mouse anti-Tie2 antibody is shown in Table 2.

[0110]

[0126]

[0127] Table 2. Affinity of mouse anti-Tie2 antibody for hTie2

Table 2

[0111]

[0128]

[0129] 1.5. Tie2 phosphorylation induced by mouse Tie2 antibody 3H7 The anti-Tie2 antibody developed in the present invention is shown to bind to and induce Tie2 clustering, ultimately inducing Tie2 activation. Experiments were conducted to analyze the effect of the anti-Tie2 antibody on Tie2 phosphorylation using HUVEC.

[0112]

[0131] Specifically, HUVEC (Lonza) was cultured in EGM-2 (Lonza) in a 100 mm culture dish at 37 °C and 5% CO2 concentration. At 80-90% confluence, the cells were exchanged to EBM-2 (Lonza) medium for 6 hours for serum starvation. The cultured cells were treated with anti-Tie2 antibodies at various concentrations (0.02 μg / ml to 50 μg / ml) and further incubated for 30 minutes. The cells were washed twice with cold PBS and lysed with 1000 μl of lysis buffer (10 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, protease inhibitor, phosphatase inhibitor), and then incubated at 4 °C for 60 minutes. Cell extracts were prepared and centrifuged at 12,000 rpm for 10 minutes. The protein concentration in the supernatant was quantified by BCA assay.

[0113]

[0132] For Tie2 immunoprecipitation, 1 μg of Tie2 antibody (R&D Systems, AF313) was added to 0.5 mg of lysate and incubated overnight at 4 °C with shaking. Then, Dynabeads® ProteinG (Life Technologies) was added and reacted for 2 hours. The beads were fixed to one side of the tube using a magnet, washed 3 times with lysis buffer, and then incubated at 70 °C for 10 minutes with 2×SDS sample buffer containing a reducing agent. The beads were removed from the sample, electrophoresed on a 4-15% SDS protein gel (Bio-Rad), and then transferred to a 0.45 μm PVDF membrane.

[0114]

[0133] The membrane was blocked with TBS-T mixed with 5% (v / v) BSA at room temperature for 1 hour, incubated with anti-phosphotyrosine antibody (4G10, Millipore) at 4°C for 8 hours, followed by incubation with HRP-labeled anti-mouse antibody and then Western blotting analysis. To measure the amount of immunoprecipitated Tie2, the membrane was reacted in stripping buffer (Thermo) for 15 minutes, then blocked again and reprobed with anti-Tie2 antibody (R&D Systems, AF313). As shown in Figure 2, when anti-Tie2 antibody 3H7 was added to HUVEC cells, phosphorylation of Tie2 was strongly induced in a dose-dependent manner. These data indicate that anti-Tie2 antibody 3H7 directly induces activation of the Tie2 receptor in human endothelial cells.

[0115]

[0134]

[0135] 1.6. Clathrin-mediated Tie2 endocytosis and FOXO1 translocation in HUVEC by Tie2 antibody (3H7)

[0136] The effects of 3H7 on Tie2 localization and FOXO1 translocation from the nucleus to the cytoplasmic solute were examined by immunofluorescence in HUVEC. Specifically, HUVEC were seeded in 8-well slide chambers (Lab-TekII) and maintained in EGM-2 medium for 2 - 3 days. At 100% confluence, the cells were serum-starved in EBM-2 medium for 4 hours and treated with 1 μg / ml anti-Tie2 antibody 3H7 for 30 minutes. Then, the cells were fixed with 4% formaldehyde in PBS at room temperature (RT) for 10 minutes, permeabilized with 0.1% Triton X-100 in PBS, blocked with 1% BSA in PBS at RT for 60 minutes, and incubated with the primary antibody at RT for 1 hour. Primary antibodies for hTie2 and FOXO1 were used. Next, the cells were incubated with the secondary antibody (Invitrogen) at room temperature in the dark for 1 hour and mounted with Vectashield mounting medium containing DAPI (VectorLabs). Images were taken using a laser scanning confocal microscope (LSM880, Carl Zeiss).

[0116]

[0137] As shown in Fig. 3, 3H7 treatment significantly induced Tie2 endocytosis, similar to the control Ang2 antibody (Han et al., 2016, Science Translation Medicine) known to induce Tie2 clustering and activation. Consistent with previous reports showing that FOXO1 localizes to the cytoplasm after phosphorylation (Zhang et al., JBC 2002, 277, 45276 - 45284), FOXO1 was localized in the nucleus under basal serum starvation conditions, but disappeared from the nucleus after 3H7 treatment, as compared to the serum starvation control (red).

[0117]

[0138]

[0139] 1.7. Inhibition of angiogenesis induced by VEGF or TNF-α upon treatment of HUVEC with anti-Tie2 antibody 3H7.

[0140] The vascular leakage assay was performed on HUVEC using an in vitro angiogenesis permeability assay kit (Millipore) according to the manufacturer's instructions. HUVEC were seeded onto inserts of transwell plates and cultured for 3 days until 100% confluence. HUVEC were pre-incubated with Ang2 (1 μg / ml), Ang2 (1 μg / ml) together with a control antibody (1 μg / ml), or 3H7 antibody (1 μg / ml) alone for 30 minutes, then VEGF (500 ng / ml) or TNF-α (100 ng / ml) was added, and the cells were incubated at 37°C for 45 minutes or 22 hours, respectively. FITC-dextran was added to the upper chamber and incubated for 20 minutes. 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. As shown in Fig. 4, pretreatment with anti-Tie2 antibody 3H7 significantly inhibited vascular leakage induced by the angiogenesis promoters VEGF or TNF-α.

[0118] 「141]

[0142] Example 2. DNA Gene Sequence Analysis of Mouse Anti-Tie2 Antibody

[0143] The DNA nucleotide sequences of the antibodies (derived from hybridoma cells) selected in Example 1.3 were analyzed. Specifically, hybridoma cells (2×10 6 cells / ml) were cultured in DMEM containing 10% FBS, and then total RNA was obtained using an RNeasy Mini Kit (Qiagen). Next, the RNA concentration was measured, and cDNA was synthesized by reverse transcription (RT) reaction. To amplify the gene sequences of the heavy and light chain variable regions, PCR was performed under the following conditions using the above cDNA as a template and a mouse Ig primer set (Novagen): 5 minutes at 94°C; [1 minute at 94°C, 1 minute at 50°C, 2 minutes at 72°C] × 35 cycles; 6 minutes at 72°C; cooled to 4°C. The PCR products obtained in each reaction were cloned into a TA vector and subjected to DNA sequencing, thereby obtaining the nucleotide sequences encoding the CDRs, heavy chain variable regions, and light chain variable regions of each antibody (Tables 3 to 12).

[0119]

[0144]

[0145] Table 3. CDR Sequences of Mouse Anti-Tie2 Antibody 3B2

Table 3

[0120]

[0146]

[0147] Table 4. Variable Region Sequences of Mouse Anti-Tie2 Antibody 3B2

Table 4

[0121]

[0148]

[0149] Table 5. CDR Sequences of Mouse Anti-Tie2 Antibody 3E12

Table 5

[0122]

[0150]

[0151] Table 6. Variable Region Sequences of Mouse Anti-Tie2 Antibody 3E12 [Table 6]

[0123]

[0152]

[0153] Table 7. CDR Sequences of Mouse Anti-Tie2 Antibody 3H7 [Table 7]

[0124]

[0154]

[0155] Table 8. Variable Region Sequences of Mouse Anti-Tie2 Antibody 3H7 [Table 8]

[0125]

[0156]

[0157] Table 9. CDR Sequences of Mouse Anti-Tie2 Antibody 4A4 [Table 9]

[0126]

[0158]

[0159] Table 10. Variable Region Sequences of Mouse Anti-Tie2 Antibody 4A4 [Table 10]

[0127]

[0160]

[0161] Table 11. CDR Sequences of Mouse Anti-Tie2 Antibody 11C4 [Table 11]

[0128]

[0162]

[0163] Table 12. Variable Region Sequences of Mouse Anti-Tie2 Antibody 11C4 [Table 12]

[0129]

[0164]

[0165] Example 3. Epitope Mapping of Mouse Anti-Tie2 Antibodies against hTie2

[0166] The antigenic determinant (epitope) of hTie2 recognized by the mouse monoclonal antibody 3H7 was analyzed by HDX-MS (hydrogen / deuterium exchange mass spectrometry) technology. The HDX-MS analysis method is described in the following literature: Houde D, Engen JR (2013) Methods Mol. Biol. 988:269-89 and Houde et al., (2011) J. Pharm. Sci. 100(6), 2071.

[0130]

[0167] The binding epitope of antibody 3H7 was analyzed using recombinant hTie2-Ig3-FNIII(1-3) protein. Prior to the deuterium labeling reaction, the hTie2-Ig3-FNIII(1-3) / antibody mixture was incubated for more than 3 hours and maintained at maximum binding (100%) under 15-fold diluted deuterium labeling buffer (KD = 25 nM). The prepared hTie2-Ig3-FNIII(1-3) / antibody complex was diluted 15-fold with deuterium labeling buffer, labeled at various times, and then quenched with an equal volume of quenching buffer. The labeling reaction times were 0 minutes (non-deuterium), 0.33 minutes, 10 minutes, 60 minutes, and 240 minutes. However, in the non-deuterium state, the deuterium labeling buffer was exchanged with the equilibration buffer, and the reaction was immediately stopped using the quenching buffer. In mass spectrometry, the deuterium-labeled hTie2-Ig3-FNIII(1-3) / antibody sample was loaded onto a pepsin column for peptide digestion. Mass spectrometry showed that 82.6% coverage data were obtained from a total of 50 digestible peptides.

[0131]

[0168] The difference in deuterium incorporation between hTie2-Ig3-FNIII(1-3) alone and the hTie2-Ig3-FNIII(1-3) / antibody complex condition was compared and analyzed. Regions showing a clear decrease in deuterium incorporation are either the peptides to which the antibody directly binds or regions with structural changes. When the difference in deuterium incorporation between hTie2-Ig3-FNIII(1-3) alone and the hTie2-Ig3-FNIII(1-3) / antibody complex is 0.5 - 1 Da or more, it is regarded as significant and is shown in bold in Table 13.

[0132]

[0169] Using the data of H / D exchange mass spectrometry (Figure 5), heat maps of deuterium incorporation per residue of the hTie2 antigen at 0.333, 10, 60, and 240 minutes were created in the absence or presence of the anti-Tie2 antibody 3H7. The color scale indicates the percentage of H / D exchange per residue between the antigen alone and the antigen / mAb mixture at each time. Red indicates regions where deuterium exchange has increased, and blue indicates no incorporation. Heat map analysis of the deuterium incorporation difference showed that the epitopes to which the antibody 3H7 binds are residues 633 - 644 (SEQ ID NO: 1, TLSDILPPQPEN) and residues 713 - 726 (SEQ ID NO: 1, FAENNIGSSNPAFS) of hTie2 (Table 13). The epitopes were shown in red on the 3D structure of hTie2-FNIII generated using PyMol software (Figure 6).

[0133]

[0170]

[0171] Table 13. Epitope mapping analysis of 3H7 binding to hTie2 by HDX-MS

Table 13-1

[0134]

[0172]

Table 13-2

[0135]

[0173]

[0174] Example 4. Humanization and full-length IgG conversion of mouse anti-Tie2 antibody

[0175] To eliminate the immunogenicity of mouse anti-Tie2 antibody 3H7 when administered to humans, the antibody was humanized as follows.

[0136]

[0176] 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 these analyses, the three CDR regions of the 3H7 antibody were grafted onto the human antibody heavy chain variable gene IGHV1-46-01. In this process, two humanized heavy chain antibody genes were designed (Table 14). The grafted CDRs are underlined in the protein sequence. Reverse mutations to the mouse sequence were introduced into the heavy chain gene of humanized 3H7 and are shown in bold in the protein sequence of Table 14.

[0137]

[0178]

[0179] 4.2 Light chain humanization

[0180] The human antibody light chain variable gene IGKV1-17-01 showed 68% homology with the light chain sequence of antibody 3H7. Based on these analyses, the three CDR regions of the 3H7 antibody were grafted onto the human antibody light chain variable gene IGKV1-17-01. In this process, two humanized light chain antibody genes were designed (Table 14). The grafted CDRs are underlined in the protein sequence. Reverse mutations to the mouse sequence were introduced into the light chain gene of humanized 3H7 and are shown in bold in the protein sequence of Table 14.

[0138]

[0181]

[0182] 4.3. Humanized gene synthesis and cloning into human full-length IgG antibody

[0183] The humanized variable regions of the antibodies in Table 14 were cloned into the heavy and light chain vectors of a human IgG4 isotype backbone vector. The DNA fragment of the humanized heavy chain variable region (VH) of the antibody was synthesized as the sequence of "EcoRI - signal sequence - VH - NheI - CH - XhoI" (Bioneer). The DNA fragment of the humanized light chain variable region (VL) of the antibody was also synthesized as the sequence of "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.

[0139]

[0184] Table 14. Humanized anti - Tie2 antibody derived from mouse 3H7 antibody

[0185]

Table 14 - 1

[0140]

[0186]

Table 14 - 2

[0141]

[0187]

Table 14 - 3

[0142]

[0188]

[0189] 4.4. Production and purification of humanized anti - Tie2 antibody

[0190] To produce the humanized anti - Tie2 antibody, Expi293F (Gibco) cells, which can produce recombinant proteins with high efficiency, were used. Expi293F cells (2×10 6Cells / ml) were cultured in a Erlenmeyer flask, and plasmids encoding the heavy and light chains were co-transfected into Expi293F cells using the ExpiFectamine 293 transfection kit. The cells were cultured for 5 days at 37 °C in a shaking incubator (orbital shaker, 125 rpm) under 8% CO2. The resulting medium was collected and centrifuged to remove the cells. The culture supernatant containing the secreted antibody was isolated 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 it through a protein centrifugal filter (Amicon), and the solution was exchanged with PBS.

[0143]

[0191]

[0192] Example 5. Measurement of the Affinity of a Humanized Anti-Tie2 Antibody for hTie2

[0193] The affinity of the humanized anti-Tie2 antibody for hTie2 was measured using an Octet system (ForteBio) with a black 96-well plate (96-well F-type black plate, Greiner). The biosensor used for the affinity measurement was hydrated for 10 minutes before measurement using an AR2G chip (ForteBio Octet). After hydration, the humanized anti-Tie2 antibody was diluted to a concentration of 10 μg / ml in 10 mM sodium acetate, pH 6.0 buffer, immobilized on the 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 and subjected to 300 seconds of association and 900 seconds of dissociation. For the affinity measurement (KD), the association rate (K-on) and dissociation rate (K-off) were analyzed by a binding curve (global) and fitted to a 1:1 binding model using the Octet data analysis v9.0.0.10 program. The KD values are shown in Table 15 below.

[0144]

[0194]

[0195] Table 15. Affinity of Humanized 3H7 Antibody for hTie2-Ig3-FNIII(1-3) [Table 15]

[0145]

[0196]

[0197] Example 6. Analysis of In Vitro Biological Characteristics of Selected Humanized Anti-Tie2 Antibodies

[0198] 6.1. Akt Phosphorylation

[0199] To examine whether the humanized anti-Tie2 antibody induces downstream signaling of the Tie2 receptor in endothelial cells, HUVEC (Lonza) was treated with the humanized anti-Tie2 antibody. Next, the level of Akt phosphorylation, which is a major downstream signaling protein of the Tie2 receptor, was measured by immunoblotting. To compare the degree of Akt activation, in the experiment, cells were treated with COMP-Ang1 (CA1) as a positive control. Specifically, HUVEC cells (1×10 5 cells / ml) were cultured at 37°C in EGM-2 (Lonza) in a 60 mm culture dish. Cells at 90% confluence were incubated with EBM-2 (Lonza) for 4 hours. Serum-starved HUVEC was treated with the anti-Tie2 antibody and further incubated for 30 minutes. The cells were washed with cold PBS, treated with lysis buffer, and lysed at 4°C for 20 minutes. Next, cell lysates were prepared by centrifugation at 13,000 rpm for 15 minutes. 5×SDS sample buffer was added to the cell lysates, and the mixture was boiled at 95°C for 5 minutes. Next, the mixture was subjected to SDS-PAGE and subsequently to Western blotting.

[0146]

[0200] To examine Akt phosphorylation, the membrane was blocked with TBST containing 5% skim milk at RT for 1 hour and incubated with anti-phospho-Akt antibody (S473) at 4°C for approximately 8 hours. The amount of phosphorylated Akt was visualized by enhanced chemiluminescence (ECL). Next, the membrane was incubated in stripping buffer (Thermo) for 15 minutes and then reprobed with anti-Akt antibody to measure the total amount of Akt.

[0147]

[0201] As shown in Figure 7, Akt phosphorylation was significantly increased by treatment with the humanized 3H7 antibody. These data indicate that the humanized anti-Tie2 antibody can potently induce the activation of Akt, a major downstream signaling molecule of the Tie2 receptor in endothelial cells.

[0148]

[0202]

[0203] Example 7. Effect of 3H7H12G4 in a primary open-angle glaucoma mouse model.

[0204] To test whether activation of Tie2 by 3H7H12G4 can rescue regressed SC (Schlemm's canal) and lower IOP (intraocular pressure), a primary open-angle glaucoma mouse model was used. This model was generated by inducible deletion of both the angiopoietin-1 and -2 genes, resulting in tamoxifen treatment of 8-week-old double angiopoietin-1 / angiopoietin-2 deficient (A1:A2 iΔ / Δ)Produced by administering to mice (Figure 8A). Intravitreal administration of 3H7H12G4 (~5 μg, one eye) and Fc (~5 μg, the opposite eye) was performed at 12 weeks of age (Figure 8A). To intravitreally administer the indicated reagents, ~1 μl (5 mg / ml) containing 5 μg of each reagent was injected into the vitreous cavity using a Nanoliter 2000 microinjector (World Precision Instruments) equipped with a glass capillary pipette. IOP measurements were performed using a rebound tonometer (TonoLab) at 12, 13, and 14 weeks (Figure 8A). IOP was measured by placing the tip of the pressure sensor approximately 1 / 8 inch from the center of the cornea immediately after anesthetizing the mice. Digital readout information of five consecutive IOP measurements was obtained from the tonometer. In wild-type mice, there was no difference in IOP between the eyes treated with 3H7H12G4 and the eyes treated with Fc (Figure 8C). On the other hand, in A1:A2 iΔ / Δ In mice, it was shown that IOP was significantly decreased by 25.6% in the eyes treated with 3H7H12G4 compared to the eyes treated with Fc (Figure 8D). CD144 + The SC area and CD144 + The intensity of Prox1 and Tie2 immunostaining in the SC were measured 2 weeks after administration of 3H7H12G4. Anti-CD144 antibody (1:200, BD Biosciences), anti-Prox1 antibody (1:200, ReliaTech), and anti-Tie2 antibody (1:200, R&D Systems) were used. CD144 of the entire mounted cornea + The SC area was calculated as the percentage of the CD144 + area divided by the area of its control. A1:A2 iΔ / Δ In mice, it was shown that the SC region was significantly increased by 114.8% in the 3H7H12G4-treated eyes compared to the Fc-treated eyes (Figure 8B, E). To quantify the relative expression of Prox1, CD144 + The intensity in the nuclear region of the SC was measured. A1:A2 iΔ / ΔIn mice, it was shown that Prox1 intensity was significantly increased by 88.4% in the 3H7H12G4-treated eyes compared to the Fc-treated eyes (Figure 8B, F). To quantify the expression of Tie2, the intensity was measured in the CD144 + SC region. A1:A2 iΔ / Δ In mice, it was shown that Tie2 intensity was significantly increased by 63.0% in the 3H7H12G4-treated eyes compared to the Fc-treated eyes (Figure 8B, F). Overall, these findings indicate that the activation of Tie2 by 3H7H12G4 rescues the impaired SC in A1:A2 iΔ / Δ mice.

[0149]

[0205]

[0206] Example 8. CNV regression and suppression of vascular leakage by intravitreally injected 3H7H12G4 in a laser-induced CNV model.

[0207] 3H7H12G4 was tested for its ability to inhibit choroidal neovascularization (CNV), a feature of age-related macular degeneration (AMD), using a laser-induced choroidal neovascularization (CNV) model. The pupils were dilated using 5 mg / ml phenylephrine and 5 mg / ml tropicamide eye drops (Santen Pharmaceutical), and after instilling 0.5% proparacaine hydrochloride eye drops (Alcon) for topical anesthesia, a laser photocoagulation device (Lumenis) equipped with a slit lamp delivery system was used with a glass coverslip as a contact lens to visualize the retina. Sufficient laser energy (wavelength of 532 nm, output of 250 mW, duration of 100 ms, spot size of 50 μm) was delivered to four locations (3 o'clock, 6 o'clock, 9 o'clock, and 12 o'clock positions in the posterior pole) in each eye. Bubbles were generated during laser photocoagulation, and only burns indicating rupture of Bruch's membrane were included in this study. Spots containing bleeding at the laser sites were excluded from the analysis. To summarize the clinical situation, 3H7H12G4 (5 μg) was intravitreally administered to mice 7 days after laser photocoagulation (Figure 9A). As a control or comparison, Fc or VEGF-Trap (5 μg each) was administered to mice in the same manner. To intravitreally administer the predetermined reagents, approximately 1 μl (5 mg / ml) containing 5 μg of each reagent was injected into the vitreal cavity using a Nanoject 2000 microinjector (World Precision Instruments) equipped with a glass capillary pipette. CD31 of retinal pigment epithelium (RPE)-choroid-sclera flat mounts +The CNV volume was calculated 14 days after laser photocoagulation using the MATLAB Image Processing Toolbox (MathWorks). Anti-CD31 antibody (1:200, Millipore) was used for the detection of endothelial cells of CNV. VEGF-Trap effectively induced CNV regression up to 64.4% compared to Fc, and 3H7H12G4 also induced CNV regression (65.7%) (Figure 9B). By combining fluorescein angiography (FA) and indocyanine green angiography (ICGA), it became possible to measure the vascular leakage of neovascularization around the laser injury site. Continuous wave laser modules of 488 nm and 785 nm were used as excitation sources for fluorescein and ICG, respectively. The raster scanning pattern of the excitation laser was executed by a scanner system composed of a rotating polygon mirror (MC-5; Lincoln Laser) and a galvanometer-based scan mirror (6230H; Cambridge technology), and was sent to the back aperture of the imaging lens. An objective lens with a high numerical aperture (NA) (PlanApoλ, NA 0.75; Nikon) was used as the imaging lens to provide a wide-field fundus fluorescence image. 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 pixel size of 512×512 per frame. To visualize the late-phase (6 minutes) FA and ICGA images using the angiography system, 10 mg of sodium fluorescein (Alcon) and 0.15 mg of ICG (Daiichi Pharmaceutical) were administered intraperitoneally and intravenously, respectively. The imaging procedure was performed under general anesthesia and pupil dilation to improve the image quality. The leakage area from CNV was calculated by dividing the total high-fluorescence area measured in the FA image by the total area of CNV measured in the ICGA image using Java-based imaging software (ImageJ; National Institutes of Health). Both VEGF-Trap (37.0%) and 3H7H12G4 (24.6%) similarly suppressed vascular leakage compared to Fc (Figure 9C).Notably, there was no significant difference in vascular leakage between the Fc treatment group from 6 to 14 days after laser photocoagulation, but vascular leakage was significantly reduced with VEGF-Trap and 3H7H12G4 (45.6% and 42.5%, respectively) (Figure 9C). Thus, the magnitude of CNV and vascular leakage suppression could not be quantitatively distinguished between VEGF-Trap and 3H7H12G4 in a mouse model of laser-induced CNV.

[0150]

[0208]

[0209] Example 9. Co-localization of 3H7H12G4 and CD31 in endothelial cells of CNV.

[0210] To investigate whether subcutaneously injected 3H7H12G4 can also exert a therapeutic effect on CNV, we first evaluated the co-localization of 3H7H12G4 and CD31 in endothelial cells of CNV. Subcutaneous administration of 3H7H12G4 (25 mg / kg) was performed 1 day after laser photocoagulation. As a control, Fc (25 mg / kg) was administered to mice in the same manner. Co-localization of 3H7H12G4 and anti-CD31 antibody (1:200, Millipore) in endothelial cells of CNV was directly detected with an anti-human IgG antibody (1:1000, Jackson ImmunoResearch Laboratories) 2, 4, and 8 days after laser photocoagulation (Figure 10A). The administered 3H7H12G4 was highly detectable in endothelial cell CNV (Figure 10B-D). + Endothelial cell CNV was highly detectable (Figure 10B-D).

[0151]

[0211]

[0212] Example 10. CNV inhibitory effect of subcutaneously injected 3H7H12G4 antibody.

[0213] To determine the effect of subcutaneously injected 3H7H12G4 in CNV inhibition, subcutaneous administration of 3H7H12G4 (25 mg / kg) was performed one day after laser photocoagulation. As a control, Fc (25 mg / kg) was administered to mice in the same manner. For the detection of endothelial cells of CNV, anti-CD31 antibody (1:200, Millipore) was used, and CD31 of RPE-choroid-sclera flat mount was used with the MATLAB image processing toolbox (MathWorks) 8 days after laser photocoagulation. + CNV volume was calculated (Figure 11A). 3H7H12G4 effectively inhibited CNV formation up to 69.9% compared to Fc (Figure 11B, C), indicating that 3H7H12G4 has an inhibitory effect on CNV not only by intravitreal injection but also by subcutaneous injection.

[0152]

[0214] Industrial Applicability

[0215] The antibody or antigen-binding fragment thereof that binds to Tie2 according to the present invention binds to Tie2 with high affinity, maintains cross-reactivity with respect to humans and mice, and exhibits a desired antigen reactivity. In addition, by inducing phosphorylation of Tie2 and activation of the Tie2 receptor, it can be usefully used for preventing or treating a target angiogenesis disease.

[0153]

[0216]

[0217] So far, specific parts of the content of the present invention have been described in detail, but it will be clear to those skilled in the art that these specific techniques are merely preferred embodiments. Therefore, the scope of the present invention is not limited to these embodiments.

[0154]

[0218]

[0219] The present disclosure relates to, for example, the following. <1> An anti-Tie2 antibody or an antigen-binding fragment thereof that binds to the Tie2 Ig3-FNIII(1-3) domain containing SEQ ID NO: 2. <2> The method according to <1>, wherein the antibody or an antigen-binding fragment thereof binds to amino acids 633-644 and / or amino acids 713-726 of Tie2 constituting the sequence of SEQ ID NO: 1. <3> The antibody or an antigen-binding fragment thereof A heavy chain variable region containing a heavy chain CDR containing the amino acid sequences of SEQ ID NOs: 3-5 and a light chain variable region containing a light chain CDR containing the amino acid sequences of SEQ ID NOs: 6-8; A heavy chain variable region containing a heavy chain CDR containing the amino acid sequences of SEQ ID NOs: 13-15 and a light chain variable region containing a light chain CDR containing the amino acid sequences of SEQ ID NOs: 16-18; A heavy chain variable region containing a heavy chain CDR containing the amino acid sequences of SEQ ID NOs: 23-25 and a light chain variable region containing a light chain CDR containing the amino acid sequences of SEQ ID NOs: 26-28; A heavy chain variable region containing a heavy chain CDR containing the amino acid sequences of SEQ ID NOs: 33-35 and a light chain variable region containing a light chain CDR containing the amino acid sequences of SEQ ID NOs: 36-38; or A heavy chain variable region containing a heavy chain CDR containing the amino acid sequences of SEQ ID NOs: 43-45 and a light chain variable region containing a light chain CDR containing the amino acid sequences of SEQ ID NOs: 46-48 The method according to <1>, comprising. <4> The antibody or an antigen-binding fragment thereof A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 9 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 11; A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 19 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 21; A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 29 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 31; A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 39 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 41; A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 49 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 51; A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 53 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 54; A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 57 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 58; A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 61 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 62; A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 65 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 66 The method according to <1> above, comprising the same. <5> A nucleic acid encoding the antibody or an antigen-binding fragment thereof according to any one of <1> to <4> above. <6> The method according to <5> above, wherein the nucleic acid comprises SEQ ID NO: 10, 12, 20, 22, 30, 32, 40, 42, 50, 52, 55, 56, 59, 60, 63, 64, 67 or 68. <7> An expression vector containing the nucleic acid of <5> above. <8> A cell transformed with the expression vector of <7> above. <9> A method for producing an antibody or an antigen-binding fragment thereof that binds to Tie2, comprising: (a) A culturing process of the cell of <8> above; and (b) A process for recovering an antibody or an antigen-binding fragment thereof from the cultured cells. <10> A composition for preventing or treating an angiogenesis disease, comprising the antibody or an antigen-binding fragment thereof according to any one of <1> to <4> above as an active ingredient. <11> The method according to <10> above, wherein the composition is characterized by the following: The angiogenesis disease is selected from the group consisting of cancer, metastasis, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, macular degeneration, neovascular glaucoma, generalized exfoliative dermatitis, proliferative retinopathy, psoriasis, hemophilic arthritis, related sclerosis, capillary formation of atherosclerotic plaque, keloid, wound granulation, vascular adhesion, rheumatoid arthritis, osteoarthritis, autoimmune disease, Crohn's disease, restenosis, atherosclerosis, reactive arthritis, cat scratch, ulcer, cirrhosis, nephritis, diabetic nephropathy, diabetes, inflammatory disease, and neurodegenerative disease. <12> The method according to <11> above, wherein the composition is characterized by the following: The cancer is selected from the group consisting of esophageal cancer, gastric cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin lymphoma, lymphoma, and multiple myeloma blood cancer. <13> A composition for combination therapy with other therapeutic agents for angiogenesis diseases, comprising the antibody or an antigen-binding fragment thereof according to any one of <1> to <4> above.

Claims

**Claim 1** An anti-Tie2 antibody or an antigen-binding fragment thereof that binds to the Tie2 Ig3-FNIII(1-3) domain having the sequence of SEQ ID NO: 2, wherein the antibody or antigen-binding fragment thereof (a) a complementarity-determining region-H1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5, a heavy chain variable region comprising, and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 6, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, a light chain variable region comprising, An antibody or antigen-binding fragment thereof comprising. **Claim 2** The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof binds to amino acids 633-644 and / or amino acids 713-726 of Tie2 constituting the sequence of SEQ ID NO:

1. **Claim 3** The antibody or antigen-binding fragment thereof A heavy chain variable region comprising an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 9 and a light chain variable region comprising an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 11 The antibody or antigen-binding fragment thereof according to claim 1, comprising. **Claim 4** The antibody or antigen-binding fragment thereof A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11 The antibody or antigen-binding fragment thereof according to claim 3, comprising. **Claim 5** The affinity (K Ｄ ) of the anti-Tie2 antibody is less than 1.0E －12 M, the antibody or antigen-binding fragment thereof according to claim 1. **Claim 6** A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5. **Claim 7** The nucleic acid according to claim 6, wherein the nucleic acid comprises SEQ ID NO: 10 and / or 12. **Claim 8** An expression vector comprising the nucleic acid according to claim 6 or 7. **Claim 9** A cell transformed with the expression vector according to claim 8. **Claim 10** A method for producing an antibody or antigen-binding fragment that binds to Tie2, comprising (a) culturing the cell according to claim 9; and (b) recovering the antibody or antigen-binding fragment thereof from the cultured cell. **Claim 11** A pharmaceutical composition for preventing or treating an angiogenesis disease, comprising an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, and a pharmaceutically acceptable vehicle, excipient or diluent.

12. The pharmaceutical composition according to claim 11, further comprising another antibody or a biologically active agent.

13. The pharmaceutical composition according to claim 11 or 12, wherein the angiogenesis disease is selected from cancer, metastasis, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, macular degeneration, neovascular glaucoma, generalized exfoliative dermatitis, proliferative retinopathy, psoriasis, hemophilic arthritis, associated sclerosis, capillary formation in atherosclerotic plaques, keloid, wound granulation, vascular adhesion, rheumatoid arthritis, osteoarthritis, autoimmune diseases, Crohn's disease, restenosis, atherosclerosis, reactive arthritis, cat scratch disease, ulcer, cirrhosis, nephritis, diabetic nephropathy, diabetes, inflammatory diseases, and neurodegenerative diseases.

14. The pharmaceutical composition according to claim 13, wherein the cancer is selected from esophageal cancer, gastric cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma, and multiple myeloma blood cancer.

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