Bispecific antibody specifically binding to tie2 and VEGF, and use thereof

Abstract

The present invention provides a novel anti-Tie2 antibody or fragment thereof, and a bispecific antibody comprising the antibody and a VEGF-specific binding site. The anti-Tie2 antibody according to the present invention induced phosphorylation by specifically binding to the Tie2 protein. At this time, the antigen specificity and affinity of the anti-Tie2 antibody were excellent compared to existing ligands (Ang-1, Ang-2). In addition, the bispecific antibody comprising the anti-Tie2 antibody and the VEGF binding site inhibited VEGF while activating the Tie2 protein. In particular, the bispecific antibody effectively inhibited side effects that can be caused by an inhibitor of VEGF. In addition, the anti-Tie2 antibody or bispecific antibody exhibited a therapeutic effect on diseases in a mouse model of vascular abnormality-related diseases. Thus, the anti-Tie2 antibody and the bispecific antibody comprising the anti-Tie2 antibody and the VEGF-specific binding site according to the present invention can be used as a therapeutic agent for vascular abnormality-related diseases.

Description

Bispecific antibodies specifically binding to TIE2 and VEGF and uses thereof

[0001] The present invention relates to a novel anti-Tie2 antibody or binding fragment thereof and a bispecific antibody comprising a VEGF binding site, and to pharmaceutical uses thereof.

[0002] Tie2 (Tyrosine kinase receptor 2) protein is a receptor-type tyrosine kinase (TK) specifically expressed in vascular endothelial cells. It is activated through phosphorylation by its specific ligand, Ang-1 (Agiopoietin-1). Tie2 protein is known to play a very important role in angiogenesis or structural or functional normalization and stabilization of blood vessels in various physiological or pathological situations (Korean Patent Publication No. 10-2195957). Therefore, Tie2 protein is attracting attention as a treatment for ischemic diseases. In addition, activation of Tie2 protein is known to suppress vascular leakage, vascular inflammation, and vascular abnormality induced by vascular endothelial growth factor (VEGF). Recently, a method has been proposed to suppress abnormal angiogenesis and induce normalization and stabilization of blood vessels by inhibiting VEGF activation and activating Tie2 protein by Ang-1 protein as a treatment strategy for diabetic retinopathy, sepsis, and tumors.

[0003] However, the physiologically activated form of Ang-1, the Ang-1 tetramer, is very difficult to produce as a recombinant protein, and it has a short half-life in vivo, making it difficult to utilize as a therapeutic agent. To solve the problems of Ang-1, research is being conducted on various Tie2-specific activators that can replace Ang-1 protein, but problems such as low productivity and concerns about inducing an immune response limit their use as therapeutic agents. In addition, VEGF inhibitors are known to exhibit various side effects.

[0004] Therefore, there is a need for research and development of methods to treat various vascular abnormalities related to angiogenesis, endothelial signaling, inflammation, and / or vascular leakage regulation through more effective induction of Tie2 protein activity and inhibition of VEGF activity.

[0005] Accordingly, the inventors of the present invention completed the present invention by conducting research to develop a treatment agent for vascular abnormality-related diseases and confirming that a novel anti-Tie2 antibody and a bispecific antibody comprising the antibody and a VEGF binding site improve vascular abnormality-related diseases in a vascular disease mouse model.

[0006] In order to achieve the above object, one aspect of the present invention provides an antibody or fragment thereof that specifically binds to Tie2, comprising (a) a heavy chain variable region (VH) comprising i) a heavy chain complementary determining region 1 (HCDR1) represented by the amino acid sequence of SEQ ID NO: 84, ii) a heavy chain complementary determining region 2 (HCDR2) represented by the amino acid sequence of SEQ ID NO: 85, and iii) a heavy chain complementary determining region 3 (HCDR3) represented by any one amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 15, and SEQ ID NO: 86; and (b) a light chain variable region (VL) comprising iv) a light chain complementary determining region 1 (LCDR1) represented by the amino acid sequence of SEQ ID NO: 87, v) a light chain complementary determining region 2 (LCDR2) represented by the amino acid sequence of SEQ ID NO: 88, and vi) a light chain complementary determining region 3 (LCDR3) represented by the amino acid sequence of SEQ ID NO: 89. In the amino acid sequence X1X2X3FX4X5X6X7 of SEQ ID NO: 84, X1 is G or S, X2 is F or D, X3 is T or S, X4 is N, T or A, X5 is S or G, X6 is Y, N or Q, and X7 is G, W or A; X8X9X of SEQ ID NO: 85 10 DX 11 X 12 X 13 X 14 X8 of the amino acid sequence is T or I, X9 is S or Y, and X 10 is N, P or W, and X 11 is G, D or S, and X 12 is S or G, and X 13 is T, D or G, and X 14 is T or I; ARKVVRGYX of sequence number 86 15 X 16 X of HDAFDI amino acid sequence 15 is S, H or L, and X 16 is Y or P; QX of sequence number 87 17 X18 X 19 SX 20 X in the amino acid sequence 17 is S, G or D, and X 18 is V or I, and X 19 is S or D, and X 20 is Y or N; X of sequence number 88 21 X 22 X of S amino acid sequence 21 is G or A, and X 22 is A or S; QQX of sequence number 89 23 X 24 X 25 X 26 PX 27 X of T amino acid sequence 23 is Y, A or G, and X 24 is G, N or Y, and X 25 is T, S or D, and X 26 is T or F, and X 27 is Y, L or W.

[0007] Another aspect of the present invention provides a polynucleotide encoding the antibody or a fragment thereof, an expression vector comprising the polynucleotide, and a cell transformed with the vector.

[0008] Another aspect of the present invention provides a method for producing an antibody or a fragment thereof, comprising the steps of culturing the transformed cell and obtaining the antibody or a fragment thereof from the culture medium.

[0009] Another aspect of the present invention provides a bispecific antibody comprising the anti-Tie2 antibody or a fragment thereof; and a VEGF-specific binding site.

[0010] Another aspect of the present invention provides a polynucleotide encoding the bispecific antibody, a vector comprising the polynucleotide, and a cell transformed with the vector.

[0011] Another aspect of the present invention provides a method for producing a bispecific antibody, comprising the steps of culturing the transformed cell and obtaining a bispecific antibody from the culture solution.

[0012] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating a vascular abnormality-related disease, comprising as an active ingredient the anti-Tie2 antibody or fragment thereof; or the anti-Tie2 antibody or fragment thereof and a bispecific antibody comprising a VEGF-specific binding site.

[0013] Another aspect of the present invention provides a use of the anti-Tie2 antibody or fragment thereof; or a bispecific antibody comprising the anti-Tie2 antibody or fragment thereof and a VEGF-specific binding site, for the prevention or treatment of a vascular abnormality-related disease.

[0014] Another aspect of the present invention provides a method for preventing or treating a vascular abnormality-related disease, comprising administering to a subject the anti-Tie2 antibody or fragment thereof; or a bispecific antibody comprising the anti-Tie2 antibody or fragment thereof and a VEGF-specific binding site.

[0015] The anti-Tie2 antibody of the present invention specifically bound to the Tie2 protein and induced phosphorylation. At this time, the antigen specificity and affinity of the anti-Tie2 antibody were superior to those of existing ligands (Ang-1, Ang-2). In addition, the bispecific antibody comprising the anti-Tie2 antibody and a VEGF binding site activated the Tie2 protein while simultaneously inhibiting VEGF. In particular, the bispecific antibody effectively suppressed side effects that may occur due to VEGF inhibitors. In addition, the anti-Tie2 antibody or bispecific antibody showed a therapeutic effect on diseases related to vascular abnormalities in a mouse model of a disease. Therefore, the anti-Tie2 antibody according to the present invention and the bispecific antibody comprising the anti-Tie2 antibody and a VEGF-specific binding site can be used as therapeutic agents for diseases related to vascular abnormalities.

[0016] Figures 1a to 1c show the results of Western blot analysis of the phosphorylation level of Tie2 and its downstream signaling factors in HEK293 cells, 3T3-J2 cells, or HUVEC (Human umbilical vein endothelial cells), which are cells expressing human or mouse Tie2, using an anti-Tie2 antibody (MT-100).

[0017] Figures 2a to 2d show the results of Western blot analysis of the phosphorylation level of Tie2 and its downstream signal transduction factors by treatment with anti-Tie2 antibodies (MT-101, MT-110, or MT-111) in HUVEC.

[0018] Figure 3 shows the results of Western blot analysis of the phosphorylation levels of Tie2 and its downstream signaling factors by treatment with an IgG1-type antibody (MT-101 (IgG1)), an antibody with the constant region of isotype IgG4 (MT-101 (IgG4)), and an antibody with the L234A / L235A amino acid substitution mutation in the IgG1-type CH2 region (MT-101 (IgG1-LALA)) in HUVEC.

[0019] Figure 4 is a diagram showing the results of screening for induction of phosphorylation of 49 types of human tyrosine receptor proteins (Receptor tyrosine kinases, RTKs) after treatment with anti-Tie2 antibody (MT-100) in HUVEC.

[0020] Figures 5a to 5d are graphs showing the results of confirming the cross-reactivity of anti-Tie2 antibodies (MT-100, MT-101 (IgG1), MT-101 (IgG1-LALA), or MT-101 (IgG4)).

[0021] Figure 6 shows the results confirming the vascular network stabilization inducing effect of anti-Tie2 antibody (MT-100).

[0022] Figures 7a and 7b are graphs showing the results of confirming apoptosis after treatment with anti-Tie2 antibody (MT-101 (IgG1), MT-101 (IgG1-LALA), or MT-101 (IgG4)) in HUVEC cultured under serum-free and hypoxic conditions.

[0023] Figures 8a and 8b are graphs and diagrams showing the results of examining vascular permeability (vascular leakage) in HUVECs treated with VEGF (Vascular endothelial growth factor) and anti-Tie2 antibody (MT-100 or MT-101). Figure 8c shows the results of examining the expression of ICAM-1 (Intercellular adhesion molecule-1) and VCAM-1 (Vascular cell adhesion molecule-1), which are inflammatory response mediators, by Western blot after treating HUVECs with VEGF and anti-Tie2 antibody (MT-100 or MT-101).

[0024] Figures 9a and 9b are graphs showing the results of confirming the effect of anti-Tie2 antibody (MT-101) on the interaction between Tie2 protein and Ang-2 (Angiopoietin-2) protein using bio-layer interferometry (BLI).

[0025] Figure 10 is a diagram showing the results of Western blot analysis of the phosphorylation level of Tie2 and its downstream signaling factors after pretreatment or co-treatment with an anti-Tie2 antibody (MT-101) of Ang-2 protein in HUVEC.

[0026] Figures 11a to 11c show the results of confirming the efficacy of inhibiting acute kidney injury after administration of anti-Tie2 antibody (MT-101 (MT-101 (IgG1), MT-101 (IgG4), or MT-101 (IgG1-LALA)) in an acute kidney injury (AKI) mouse model induced by ischemia reperfusion (I / R). Figure 11a is a diagram showing the experimental schedule, and Figure 11b shows the results of serum creatinine analysis. Figure 11c shows the results of blood urea nitrogen (BUN) analysis.

[0027] Figures 12a and 12b show the results of confirming tubular-interstitial injury through H&E (Hematoxylin & eosin) staining after administration of anti-Tie2 antibody (MT-101 (IgG1), MT-101 (IgG4), or MT-101 (IgG1-LALA)) in an I / R-induced AKI mouse model. Figure 12a is a drawing showing the staining results, and Figure 12b is a graph showing the results in numerical form.

[0028] Figure 13a shows the results of immunostaining for phosphorylation of Tie2 protein (P-Tie2) in renal blood vessels after administration of anti-Tie2 antibody (MT-101 (IgG1-LALA)) in an I / R-induced AKI mouse model. Figure 13b shows the results of immunostaining for vascular endothelial cell marker protein (CD31) and vascular endothelial pericyte marker protein (PDGFRβ).

[0029] Figure 14a is a diagram showing the results of immunostaining for the expression of inflammatory mediators (VCAM-1) in renal blood vessels (CD31) after administration of anti-Tie2 antibody (MT-101 (IgG1-LALA)) in an I / R-induced AKI mouse model. Figure 14b is a diagram showing the results of immunostaining for Gr-1 protein for the degree of neutrophil cell adhesion or influx. Figure 14c is a graph showing the results of polymerase chain reaction (Real-time PCR) analysis for the expression of cytokines (CCL2, IL-6, or IL-1β) that induce immune responses in renal tissue.

[0030] Figures 15a to 15d show the results of confirming the efficacy of anti-Tie2 antibody (MT-101 (IgG1-LALA)) after intravenous administration to mice in an acute kidney injury mouse model (LPS-induced AKI) induced by lipopolysaccharide (LPS). Figure 15a is a diagram showing the experimental schedule. Figures 15b and 15c are graphs showing the results of measuring serum creatinine and blood urea nitrogen (BUN). Figure 15d is a diagram showing the results of confirming the degree of kidney damage and renal vascular (CD31) damage through immunostaining for KIM-1 (Kidney injury molecule-1) protein and CD31 protein, respectively.

[0031] Figures 16a to 16c show the results of confirming the efficacy of anti-Tie2 antibody (MT-101 (IgG1-LALA)) administered to a mouse model of chronic kidney disease (CKD) induced by ischemia and reperfusion. Figure 16a is a diagram showing the experimental schedule. Figure 16b is a diagram showing the results of confirming renal tubular damage in kidney tissue using H&E staining. Figure 16c is a diagram showing the results of confirming kidney fibrosis using Masson's trichrome staining, Sirius red staining, and immunostaining for fibrosis markers (Collagen IV, α-SMA).

[0032] Figures 17a to 17d show the results of confirming the efficacy of anti-Tie2 antibody (MT-101 (IgG1-LALA)) administered to a CKD mouse model induced by adenine. Figure 17a is a diagram showing the experimental schedule. Figure 17b shows the results of measuring serum creatinine, and Figure 17c shows the results of confirming the degree of kidney damage through the gene expression of KIM-1 and NGAL (Neutrophil gelatinase-associated lipocalin). Figures 17d and 17e are diagrams and graphs showing the results of H&E and PAS (Periodic acid-Schiff) staining.

[0033] Figures 18a to 18d show the results of confirming renal fibrosis in mice after administration of anti-Tie2 antibody (MT-101 (IgG1-LALA)) at various concentrations (2 mg / kg, 5 mg / kg, 10 mg / kg) in an adenine-induced CKD mouse model. Figures 18a to 18c show the results of Sirius red staining and immunostaining for fibrosis markers (Collagen IV or α-SMA), respectively. Figure 18d shows the results of confirming Col1a1, Col3a1, ACTA2, or TGF-β gene expression through polymerase chain reaction.

[0034] Figures 19a to 19c show the results of confirming damage to renal tubules (Lotus tetragonolobus lectin, LTL) and vascular endothelial cells after administration of anti-Tie2 antibody (MT-101 (IgG1-LALA)) at various concentrations (2 mg / kg, 5 mg / kg, 10 mg / kg) in an adenine-induced CKD mouse model. Figure 19a shows the results of immunostaining, and Figure 19b shows the results of confirming gene expression of immune response inducers (MCP-1, TNF-α, or IL-1β) and immune cell marker proteins (CD4, CD11c, F4 / 80, or CD206) by polymerase chain reaction.

[0035] Figures 20a to 20c show the results of confirming the efficacy of anti-Tie2 antibody (MT-101 (IgG1-LALA)) in a CKD mouse model induced by unilateral ureteral obstruction (UUO). Figure 20a is a diagram showing the experimental schedule. Figures 20b to 20e show the results of confirming the degree of tubular-interstitial injury and fibrosis through H&E staining, Masson's trichrome staining, and immunostaining for fibrosis marker proteins (Collagen IV or α-SMA).

[0036] Figures 21a and 21b show the results of confirming the efficacy of anti-Tie2 antibody (MT-101 (IgG1-LALA)) administered to a mouse model of sepsis induced by lipopolysaccharide (LPS). Figure 21a is a diagram showing the experimental schedule. Figure 21b is a graph showing the results of confirming the survival rate (percent survival (%)) of the mice.

[0037] Figures 22a to 22d show the results of confirming the efficacy of anti-Tie2 antibodies (MT-100 and MT-101) administered at different concentrations (1 mg / kg, 5 mg / kg) in a hindlimb ischemic mouse model. Figure 22a shows the results of observing blood flow and tissue necrosis. Figure 22b shows the results of analyzing blood flow (LDPI ratio), and Figure 22c shows the results of analyzing tissue necrosis. Figure 22d shows the results of analyzing angiogenesis (α-SMA). + CD31 + This is the result of analyzing the vessels.

[0038] Figures 23a to 23c show the results of measuring the intracavernosal pressure (ICP; indicating the degree of erectile power) of the corpus cavernosum penis in response to penile nerve electrical stimulation after administering anti-Tie2 antibodies (MT-100 or MT-101) at different concentrations (1 μg, 10 μg) in a diabetic erectile dysfunction mouse model. Figure 23a shows the results of measuring the intracavernosal pressure, Figure 23b shows the results of analyzing the maximum intracavernosal pressure, and Figure 23c shows the results of analyzing the total intracavernosal pressure.

[0039] Figures 24a to 24c show the results of confirming angiogenesis through immunostaining for vascular endothelial cell marker protein (CD31) and vascular endothelial cell marker protein (NG2) in corpus cavernosum tissue after administration of anti-Tie2 antibodies (MT-100 and MT-101) in a diabetic erectile dysfunction mouse model. Figure 24a shows the results of immunostaining, Figure 24b shows the results of analyzing the expression area of ​​vascular endothelial cell marker protein (CD31), and Figure 24c shows the results of analyzing the expression area of ​​vascular endothelial cell marker protein (NG2).

[0040] Figures 25a and 25b are graphs showing the results of confirming the degree of capillary-like tube formation by anti-Tie2 antibody (MT-100 or MT-101) treatment in HUVEC treated with high glucose (HG) and analyzing the vascular network formation value.

[0041] Figures 26a to 26c show the results of intravitreal administration of anti-Tie2 antibodies (MT-100 or MT-101) or a control drug (EYLEA) in a mouse model of laser-induced choroidal neovascularization (CNV), and the results confirming the efficacy. Figure 26a is a diagram showing the experimental schedule. Figure 26b shows the results of observing choroidal neovascularization, and Figure 26c shows the results of measuring choroidal vascular leakage values ​​in comparison to choroidal neovascularization.

[0042] Figures 27a to 27d show the results of analyzing the expression of endomucin, the mass, area, and volume of CNV after intravitreal administration of anti-Tie2 antibody (MT-100 or MT-101) in a mouse model of laser-induced choroidal neovascularization (CNV).

[0043] Figures 28a to 28c show the results of observing avascular areas and neovascular tuft areas in the retina after intravitreal administration of anti-Tie2 (MT-101) or a control drug (EYLEA) in an oxygen-induced retinopathy (OIR) mouse model. Figure 28a is a diagram showing the experimental schedule. Figures 28b and 28c show the results of observing avascular areas and neovascular tuft areas in the retina, respectively.

[0044] Figures 29a and 29b show the results of examining the degree of proliferation and leakage of retinal vascular endothelial cells induced by ischemia after intravitreal administration of an anti-Tie2 antibody (MT-101) in an oxygen-induced retinopathy mouse model (OIR).

[0045] FIG. 30 is a drawing showing the structure of an anti-Tie2 antibody (MT-101), which is one specific example of the present invention, and a bispecific antibody (MT-103-1, MT-103-2, MT-103-3, MT-103-4, and MT-103-5) containing a VEGF (Vascular endothelial growth factor) binding site.

[0046] Figures 31a to 31e are drawings showing the results of Western blot analysis of phosphorylation of VEGFR2 and its downstream signaling factors after treatment of HUVEC with VEGF and a bispecific antibody (MT-103-1, MT-103-2, MT-103-3, MT-103-4, or MT-103-5) or a control drug (EYLEA, Avastin, or Beovu).

[0047] Figures 32a and 32b are drawings showing the results of Western blot analysis of phosphorylation of Tie2 and its downstream signaling factors by treatment with anti-Tie2 antibody (MT-101) or bispecific antibody (MT-103-1 or MT-103-4) in HUVEC.

[0048] Figure 33a is a graph showing the results of confirming cell death after treating HUVEC with anti-Tie2 antibody (MT-101) or bispecific antibody (MT-103-1 or MT-103-4) at various concentrations (1 nM, 10 nM). Figures 33b and 33c are drawings and graphs showing the results of confirming the increase in vascular endothelial cell permeability induced by VEGF through immunostaining of adherens junction protein (VE-cadherin) after treating HREC cells (Human retinal endothelial cells) with VEGF and bispecific antibody (MT-103-1 or MT-103-4) at various concentrations (0.125 nM, 0.5 nM, 2 nM, 8 nM, 32 nM). Figure 33d is a graph showing the increase in vascular endothelial cell permeability induced by VEGF and Ang-2, expressed as the degree of permeation of fluorescently labeled dextran, after treating HREC cells with VEGF and Ang-2 and a bispecific antibody (MT-103-1 or MT-103-4) at different concentrations (0.05 nM, 0.5 nM, 5 nM).

[0049] Figure 34 is a diagram showing the results of confirming the binding of Ang-2 and Tie2 according to the binding of a dual specific antibody (MT-103-1) and VEGF using a biolayer interferometer.

[0050] Figure 35a is a diagram showing experimental conditions for confirming activation of VEGFR and Tie2 downstream signaling factors by treatment with Ang-1, Ang-2, and a dual-specific antibody (MT-103-1 or MT-103-4) in HUVEC, and Figure 35b is a diagram showing the results of confirming phosphorylation of VEGFR2, Tie2, and their downstream signaling factors under each condition by Western blot.

[0051] Figure 36a shows the results of Western blot analysis of phosphorylation of VEGFR2 and Tie2 downstream signaling factors in HUVECs treated with VEGF, and a bispecific antibody (MT-103-1 or MT-103-4) or a control drug (Vabysmo). Figure 36b shows the results of comparing the degree of apoptosis in HUVECs treated with a bispecific antibody (MT-103-1 or MT-103-4) or a control drug (Vabysmo, EYLEA, or Beovu) and cultured under serum-free and hypoxic conditions.

[0052] Figures 37a to 37c show the results of confirming the angiogenesis inhibitory efficacy after intravitreal administration of an anti-Tie2 antibody (MT-101) or a bispecific antibody (MT-103-1 or MT-103-4) in a mouse model of choroidal neovascularization (CNV) induced by laser irradiation. Figure 37a is a diagram showing the experimental schedule. Figure 37b shows the results of confirming choroidal vascular leakage, and Figure 37c shows the results of analyzing the CNV vascular area.

[0053] Figures 38a to 38c show the results of intravitreal administration of a bispecific antibody (MT-103-1 or MT-103-4) or a control drug (EYLEA) in a mouse model of laser-induced choroidal neovascularization (CNV). Figure 38a is a diagram illustrating the experimental schedule. Figure 38b shows the results of confirming choroidal vascular leakage, and Figure 38c shows the results of analyzing the CNV vascular area.

[0054] Figures 39a to 39c show the results of intravitreal administration of a bispecific antibody (MT-103-1 or MT-103-4) or a control drug (EYELA) in a mouse model of oxygen-induced retinopathy (OIR), confirming the efficacy. Figure 39a is a diagram illustrating the experimental schedule. Figures 39b and 39c show the results of observing the avascular area and neovascular tuft area in the mouse retina, respectively.

[0055] Definition of Terms

[0056] The term "antibody" as used herein refers to immunoglobulin molecules and multimers thereof, each having a structure in which two heavy chains are linked to each other by a disulfide bond and one light chain is linked by a disulfide bond. The light chain (LC) contains two regions, namely, one variable region (Variable region of light chain, VL) and one constant region (Constant region of light chain, CL). In addition, the heavy chain (HC) contains four regions, namely, one variable region (Variable region of heavy chain, VH) and three constant regions (Constant region of heavy chain, CH; CH1, CH2 and CH3). The constant regions of the light and heavy chains play a role in conferring biological properties such as binding between light and / or heavy chains, secretion, complement binding, and binding to Fc receptors (FcR), while the variable regions of the light and heavy chains determine antigen recognition and binding specificity. The antibody can act as an agonist or an antagonist.

[0057] As used herein, the term "antigen-binding fragment" means any polypeptide or glycoprotein comprising a portion of an intact antibody, particularly an antigen-binding site or variable region of an intact antibody. Such antigen-binding fragments may be produced by recombinant DNA techniques or by enzymatic or chemical digestion of an intact antibody, and examples of such antigen-binding fragments include, but are not limited to, Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, Diabodies, and the like, as well as bispecific and multispecific antibodies formed therefrom.

[0058] The term “single-chain Fv” or “scFv” as used herein refers to a protein in which the VH and VL domains of an antibody are connected by a linker consisting of a peptide chain of about 15 amino acids. The sequence of light chain variable region (VL)-linker-heavy chain variable region (VH) or heavy chain variable region (VH)-linker-light chain variable region (VL) is possible, and it has the same or similar antigen specificity as the original antibody.

[0059] As used herein, the term "heavy chain" refers to a full-length heavy chain and fragments thereof, including a variable region (VH) and three constant regions, CH1, CH2, and CH3. Heavy chain constant regions (CH) exhibit different amino acid compositions and sequences, and thus possess different types of antigenicity. Therefore, immunoglobulins can be classified into five categories and referred to as immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE. The corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain, respectively. In addition, depending on the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds, the same type of immunoglobulin can be classified into different subtypes. For example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4.

[0060] As used herein, the term "light chain" refers to two types, λ and κ, consisting of approximately 211 to 217 amino acids. Each human antibody has exactly one such chain. The light chain is composed of a continuous variable region (VL) and a constant region (CL). The light chain may include both full-length light chains and fragments thereof. Any CL and CH1 regions of these antibody classes may be used in the present disclosure.

[0061] The above "variable region" refers to the region of an antibody to which an antigen binds. The variable region includes three hypervariable regions called complementarity determining regions (CDRs) and four framework regions (FRs).

[0062] As used herein, the term "complementarity determining region (CDR)" refers to a region among the variable regions of an antibody that confers antigen binding specificity. The CDR primarily plays a role in binding to an epitope of the antigen. The heavy chain and the light chain each contain three complementarity determining regions. The CDRs of each chain are typically called CDR1, CDR2, and CDR3, sequentially starting from the N-terminus, and are identified by the chain on which the particular CDR is located. The FRs of each chain are typically called FR1, FR2, FR3, or FR4, sequentially starting from the N-terminus, and are identified by the chain on which the particular FR is located.

[0063] As used herein, the term "Fc region" refers to the Fc domain of an immunoglobulin. The Fc region refers to a protein that includes the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of an immunoglobulin, but does not include the variable regions of the heavy and light chains of the immunoglobulin, the heavy chain constant region 1 (CH1), and the light chain constant region (CL).

[0064] The Fc region may be a wild-type Fc domain or an Fc domain fragment. For example, the Fc region fragment may have lysine (K) deleted from the C-terminus. Alternatively, the Fc region fragment may contain only CH3. The immunoglobulin may be IgG, IgA, IgE, IgD, or IgM. Additionally, it may be IgG1, IgG2, IgG3, or IgG4, which are subclasses of IgG, or IgA1 or IgA2, which are subclasses of IgA.

[0065] As used herein, the term "Fc region variant" refers to a variant that has a different glycosylation pattern than the wild-type Fc region, or may have increased glycosylation compared to the wild-type Fc region, decreased glycosylation compared to the wild-type Fc region, or a deglycosylated form. An aglycosylated Fc region is also included. The Fc region or variant may have a modified number of sialic acid, fucosylation, or glycosylation through culture conditions or genetic manipulation of the host.

[0066] Additionally, the sugar chains of the Fc region of the immunoglobulin can be modified by conventional methods, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. In addition, the Fc domain variant may be a mixed form of the Fc regions of the immunoglobulin IgG, IgA, IgE, IgD, or IgM. In addition, the Fc region variant may be a form in which some amino acids of the Fc region are substituted.

[0067] The “amino acid” introduced by the above substitution and / or addition may be any one selected from the group consisting of lysine (K), alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).

[0068] Specifically, the Fc region variant may have at least one amino acid substitution, for example, about 1 to about 10 amino acid substitutions, or about 1 to about 5 amino acid substitutions, compared to a native sequence Fc region. In addition, the variant Fc region may have at least about 80% homology, at least about 90% homology, or at least about 95% homology with a native sequence Fc region. In one embodiment of the present invention, the Fc region or variant thereof may comprise the amino acid sequence of SEQ ID NO: 54, SEQ ID NO: 63, or SEQ ID NO: 66.

[0069] As used herein, the term "antigen" refers to a structure capable of selectively binding to an antibody. A target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. Specifically, an antigen may be a polypeptide or a protein present on the cell surface or within a cell.

[0070] As used herein, the term "vector" refers to a material for carrying or expressing a nucleic acid sequence comprising a nucleic acid sequence encoding a multispecific fusion protein (e.g., an antibody) as described herein. Specifically, vectors include expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes.

[0071] As used herein, the term "polynucleotide," also referred to as "nucleic acid," refers to a polymer of nucleotides of any length. Specifically, the polynucleotide may be DNA or RNA.

[0072] As used herein, the term "bispecific antibody" refers to a substance that binds to at least one target or antigen. In the present invention, the bispecific antibody may include an antigen-binding site that specifically binds to Tie2 and a binding site that specifically binds to the VEGF protein.

[0073] Hereinafter, the present invention will be described in detail.

[0074] <Anti-Tie2 antibody or fragment thereof>

[0075] An antibody or fragment thereof that specifically binds to Tie2

[0076] The present invention provides an anti-Tie2 antibody or fragment thereof that specifically binds to Tie2.

[0077] The term "Tie2" as used herein refers to a receptor-type tyrosine kinase specifically expressed in vascular endothelial cells. It is also known as TEK tyrosine kinase, Antiopoietin-1 receptor, or CD202B (Cluster of differentiation 202B), and is a protein encoded by the TEK gene in humans. The Tie2 protein is activated by phosphorylation, typically by Ang-1, a Tie2 ligand. The Tie2 protein is composed of an extracellular domain (ECD) consisting of Ig-like domain 1, Ig-like domain 2, three EGF-like domains, Ig-like domain 3, and three fibronectin type-III domains; a transmembrane domain; and an intracellular tyrosine kinase domain (ITK). Tie2 protein has been reported to play a very important role in angiogenesis, structural / functional normalization and stabilization of blood vessels in various physiological / pathological situations.

[0078] In the present invention, the Tie2 protein may be included without limitation as long as it is derived from mammals, including primates such as humans and monkeys, and rodents such as rats and mice.

[0079] Specifically, the Tie2 protein may be the extracellular domain of the Tie protein. It generally refers to a polypeptide comprising the amino acid sequence of the Tie2 protein. The amino acid sequence and polynucleotide sequence for the Tie2 protein can be obtained from known databases such as the GenBank of the National Institutes of Health (NCBI) in the United States. In one specific example, it may comprise the amino acid sequence of SEQ ID NO: 91.

[0080] Additionally, the Tie protein may comprise a fragment thereof. The term "fragment" refers to a protein in which a portion of the N-terminus and / or C-terminus of the protein is deleted. Accordingly, the Tie protein fragment may be a Tie2 protein in which a portion of the N-terminus and / or C-terminus is deleted. Specifically, the Tie2 protein may be a Tie protein in which the N-terminus and / or C-terminus of the extracellular domain is deleted.

[0081] Additionally, the protein may be composed of a sequence in which one or more amino acids of the protein are added, deleted, or substituted, as long as it has the same activity as the protein or has the same gene location encoding the Tie2 on the chromosome. Specifically, the Tie2 protein may comprise or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity, or 100% identity, with the amino acid sequence of SEQ ID NO: 91.

[0082] In the present invention, the antibody or fragment thereof may include (a) a heavy chain variable region (VH) comprising i) a heavy chain complementary determining region 1 (HCDR1) represented by the amino acid sequence of SEQ ID NO: 84, ii) a heavy chain complementary determining region 2 (HCDR2) represented by the amino acid sequence of SEQ ID NO: 85, and iii) a heavy chain complementary determining region 3 (HCDR3) represented by any one amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 15, and SEQ ID NO: 86; and (b) a light chain variable region (VL) comprising iv) a light chain complementary determining region 1 (LCDR1) represented by the amino acid sequence of SEQ ID NO: 87, v) a light chain complementary determining region 2 (LCDR2) represented by the amino acid sequence of SEQ ID NO: 88, and vi) a light chain complementary determining region 3 (LCDR3) represented by the amino acid sequence of SEQ ID NO: 89. In the amino acid sequence X1X2X3FX4X5X6X7 of SEQ ID NO: 84, X1 is G or S, X2 is F or D, X3 is T or S, X4 is N, T or A, X5 is S or G, X6 is Y, N or Q, and X7 is G, W or A; X8X9X of SEQ ID NO: 85 10 DX 11 X 12 X 13 X 14 X8 of the amino acid sequence is T or I, X9 is S or Y, and X 10 is N, P or W, and X 11 is G, D or S, and X 12 is S or G, and X 13 is T, D or G, and X 14 is T or I; ARKVVRGYX of sequence number 86 15 X 16 X of HDAFDI amino acid sequence 15 is S, H or L, and X 16 is Y or P; QX of sequence number 87 17 X 18 X 19 SX 20X in the amino acid sequence 17 is S, G or D, and X 18 is V or I, and X 19 is S or D, and X 20 is Y or N; X of sequence number 88 21 X 22 X of S amino acid sequence 21 is G or A, and X 22 is A or S; QQX of sequence number 89 23 X 24 X 25 X 26 PX 27 X of T amino acid sequence 23 is Y, A or G, and X 24 is G, N or Y, and X 25 is T, S or D, and X 26 is T or F, and X 27 is Y, L or W.

[0083] Specifically, the antibody or fragment thereof may include a heavy chain variable region comprising an HCDR1 comprising an amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising an amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising an amino acid sequence of SEQ ID NO: 86, and a light chain variable region comprising an LCDR1 comprising an amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising an amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising an amino acid sequence of SEQ ID NO: 6.

[0084] In addition, specifically, the antibody or fragment thereof may include a heavy chain variable region comprising HCDR1 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 13, HCDR2 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 14, and HCDR3 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, and SEQ ID NO: 20; and a light chain variable region comprising LCDR1 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 10, and SEQ ID NO: 16, LCDR2 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 11, and SEQ ID NO: 17, and LCDR3 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 12, and SEQ ID NO: 18.

[0085] In one specific embodiment, the antibody may comprise a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0086] In one specific embodiment, the heavy chain variable region may include an HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 9; and a light chain variable region including an LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 12.

[0087] In one specific embodiment, the heavy chain variable region may include an HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 15; and a light chain variable region including an LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 18.

[0088] In one embodiment, the heavy chain variable region may include a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 19; and a light chain variable region may include a LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6. In one embodiment, the heavy chain variable region may include a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 20; and a light chain variable region may include a LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0089] At this time, the antibody may include a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 21 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 22; a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 24; a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 26; a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 22; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 28 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 22.

[0090] In the present invention, the anti-Tie2 antibody or fragment thereof can bind to the Tie2 protein and activate Tie2. The binding of the anti-Tie2 antibody or fragment thereof to the Tie2 protein can be confirmed by measuring binding affinity.

[0091] As used herein, the term “binding affinity” refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule and its binding partner. The affinity of a molecule X for its partner Y is generally expressed as the dissociation constant (K D ) can be presented by the dissociation and association rate constants (K respectively). off and K on ) is the ratio.

[0092] The binding affinity of the anti-Tie2 antibody refers to the ability of the anti-Tie2 antibody or fragment thereof to bind to the Tie2 protein, and the binding can be measured by a method known to those skilled in the art. In one embodiment of the present invention, the anti-Tie antibody or fragment thereof may exhibit a dissociation constant of about 1.0E-11 M to about 1.0E-8 M with respect to the human-derived Tie2 protein. In one embodiment, the anti-Tie antibody or fragment thereof may exhibit a dissociation constant of about 4.75E-10 M to about 3.42E-09 M.

[0093] In addition, the anti-Tie2 antibody or fragment thereof of the present invention can bind to Tie2 proteins derived from mammals other than humans, such as monkeys, mice, rats, dogs, and pigs. In one specific embodiment, the anti-Tie2 antibody or fragment thereof of the present invention can exhibit a dissociation constant of about 1.0E-10 M to about 5.0E-7 M with respect to the Tie2 protein derived from mice. In one embodiment, it can exhibit a dissociation constant of about 6.66E-9 M to about 1.44E-8 M.

[0094] Polynucleotide encoding an antibody or fragment thereof

[0095] Another aspect of the present invention provides a polynucleotide encoding an anti-Tie2 antibody or fragment thereof. The anti-Tie2 antibody and fragment thereof are as described above.

[0096] Specifically, the polynucleotide encoding the heavy chain variable region of the anti-Tie2 antibody may include a base sequence encoding an amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, or SEQ ID NO: 28. The polynucleotide encoding the light chain variable region of the anti-Tie2 antibody may include a base sequence encoding an amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26.

[0097] Additionally, if the polynucleotide encodes the same polypeptide, one or more bases may be mutated by substitution, deletion, insertion, or a combination thereof. When producing a polynucleotide sequence by chemical synthesis, synthetic methods widely known in the art can be used, such as the method described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988), and examples thereof include triester, phosphite, phosphoramidite, and H-phosphate methods, PCR and other autoprimer methods, and oligonucleotide synthesis methods on solid supports.

[0098] In one specific example, the polypeptide encoding the heavy chain variable region can comprise a base sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to a polynucleotide encoding the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, or SEQ ID NO: 28.

[0099] In one specific example, the polypeptide encoding the light chain variable region can comprise a base sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to a polynucleotide encoding the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26.

[0100] The polynucleotide may additionally include a signal sequence or a leader sequence. The term "signal sequence" as used herein refers to a nucleic acid encoding a signal peptide that directs the secretion of a target protein. The signal peptide is cleaved after translation in the host cell. Specifically, the signal sequence of the present invention is a polynucleotide encoding an amino acid sequence that initiates the movement of a protein across the ER (Endoplasmic Reticulum) membrane.

[0101] Signal sequences are well-characterized in the art and typically contain 16 to 30 amino acid residues, although they may contain more or fewer. A typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region contains 4 to 12 hydrophobic residues that anchor the signal sequence through the membrane lipid bilayer during movement of the immature polypeptide.

[0102] After initiation, the signal sequence is cleaved within the lumen of the ER by cellular enzymes commonly known as signal peptidases. At this time, the signal sequence may be a secretion signal sequence of tPa (tissue Plasminogen Activation), HSV gDs (Signal sequence of herpes simplex virus glycoprotein D), or growth hormone. Preferably, a secretion signal sequence used in higher eukaryotic cells including mammals can be used. In addition, it can be used by substituting a codon with a high expression frequency in the host cell. In one specific example, the signal sequence may include the amino acid sequence of SEQ ID NO: 51.

[0103] vector loaded with polynucleotides

[0104] Another aspect of the present invention provides a vector loaded with a polynucleotide encoding the anti-Tie2 antibody or a fragment thereof. Specifically, the vector may include a polynucleotide encoding a heavy chain of the anti-Tie2 antibody or a fragment thereof. In this case, the polynucleotide may include a base sequence encoding a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, or SEQ ID NO: 28. The vector may include a polynucleotide encoding a light chain of the anti-Tie2 antibody. In this case, the polynucleotide may include a base sequence encoding a light chain variable region comprising an amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26. The polynucleotide encoding the heavy chain region and the polynucleotide including the light chain region may be loaded into separate vectors or into a single vector.

[0105] The vector can be introduced into a host cell and recombined and integrated into the host cell genome. Alternatively, the vector is understood to be a nucleic acid vehicle containing a polynucleotide sequence capable of autonomously replicating as an episome. The vector includes linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

[0106] Specifically, the vector may be a plasmid DNA, phage DNA, etc., and may be a commercially developed plasmid (e.g., pUC18, pBAD, pIDTSAMRT-AMP, etc.), an E. coli-derived plasmid (e.g., pYG601BR322, pBR325, pUC118, pUC119, etc.), a Bacillus subtilis-derived plasmid (e.g., pUB110, pTP5, etc.), a yeast-derived plasmid (e.g., Yep13, Yep24, Ycp50, etc.), a phage DNA (e.g., Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), an animal virus vector (e.g., retrovirus, adenovirus, vaccinia virus, etc.), an insect virus vector (e.g., baculovirus, etc.). There is. Since the protein expression amount and formula of the above vector differ depending on the host cell, it is desirable to select and use the host cell most suitable for the purpose.

[0107] As used herein, the terms "gene expression" or "expression" of a protein of interest are understood to mean transcription of a DNA sequence, translation of an mRNA transcript, and secretion of an antibody product or fragment thereof. A useful expression vector may be RcCMV (Invitrogen, Carlsbad) or a variant thereof. The expression vector may include a human CMV (Cytomegalovirus) promoter to promote continuous transcription of the gene of interest in mammalian cells, and a polyadenylation signal sequence to increase the steady-state level of RNA after transcription.

[0108] Transformed cells

[0109] Another aspect of the present invention provides a transformed cell into which an expression vector comprising a polynucleotide encoding the anti-Tie2 antibody or a fragment thereof has been introduced.

[0110] As used herein, the term "transformed cell" refers to prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. Such transformed cells can be produced by introducing the vector into a host cell and transforming it. Furthermore, the polynucleotide contained in the vector can be expressed to produce an antibody according to the present invention.

[0111] The above transformation can be performed by various methods. As long as the antibody of the present invention can be produced, it is not particularly limited thereto. Specifically, the transformation method may be a CaCl2 precipitation method, a Hanahan method that increases efficiency by using a reducing substance called DMSO (Dimethyl sulfoxide) in the CaCl2 precipitation method, electroporation, calcium phosphate precipitation, protoplast fusion, a stirring method using silicon carbide fibers, an Agrobacterium-mediated transformation method, a transformation method using PEG, dextran sulfate, lipofectamine, and a drying / inhibition-mediated transformation method. In addition, a target object can be delivered into a cell using a virus particle by infection. In addition, a vector can be introduced into a host cell by gene bombardment, etc.

[0112] In addition, the host cell used for producing the transformed cell is not particularly limited as long as it can produce the antibody of the present invention or a fragment thereof. Specifically, the host cell may include, but is not limited to, a prokaryotic cell, a eukaryotic cell, a mammal, a plant, an insect, a fungus, or a cell of cellular origin. An example of the prokaryotic cell may be Escherichia coli. In addition, an example of the eukaryotic cell may be yeast. In addition, the mammalian cell may be CHO cells, F2N cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, SP2 / 0 cells, human lymphoblastoid, NSO cells, HT-1080 cells, PERC.6 cells, HEK293 cells, or HEK293T cells, but is not limited thereto, and any cell that can be used as a mammalian host cell known to those skilled in the art may be used.

[0113] In addition, in order to optimize the properties of the multi-specific fusion protein according to the present invention as a therapeutic agent or for other purposes, the glycosylation-related genes of the host cell can be manipulated using a method known to those skilled in the art to adjust the sugar chain pattern of the antibody (e.g., sialic acid, fucosylation, glycosylation).

[0114] Method for producing an antibody or fragment thereof

[0115] Another aspect of the present invention provides a method for producing the anti-Tie2 antibody or fragment thereof.

[0116] The method for producing the above antibody may include the steps of i) culturing the transformed cell; and ii) obtaining the antibody or a fragment thereof from the culture solution. In this case, the anti-Tie2 antibody, the fragment thereof, and the transformed cell are the same as described above.

[0117] The term “cultivation” as used herein refers to a method of growing microorganisms (e.g., the transformed cells) under appropriately artificially controlled environmental conditions.

[0118] The above-mentioned method of culturing the transformed cells can be performed using methods widely known in the art. Specifically, the culturing is not particularly limited as long as it can express and produce the protein of the present invention. Specifically, the culturing can be performed continuously in a batch process, fed batch process, or repeated fed batch process.

[0119] In addition, the step of obtaining the protein from the culture can be performed by a method known in the art. Specifically, the obtaining method is not particularly limited as long as it can obtain the produced protein of the present invention. Preferably, the obtaining method can be a method such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion), etc.

[0120] Dual-specific antibodies

[0121] Another aspect of the present invention provides a bispecific antibody comprising the anti-Tie 2 antibody or fragment thereof and a VEGF-specific binding site. The anti-Tie 2 antibody and fragment thereof are the same as described above.

[0122] At this time, at least one VEGF-specific binding site may be bound to the anti-Tie 2 antibody or fragment thereof. Preferably, two VEGF-specific binding sites may be bound to the anti-Tie 2 antibody or fragment thereof. In addition, the VEGF-specific binding site may be in a form bound to the C-terminus of the heavy chain constant region of the anti-Tie 2 antibody, but is not limited thereto.

[0123] VEGF binding site

[0124] As used herein, the term "VEGF" refers to vascular endothelial growth factor, which is produced by cells that stimulate angiogenesis. VEGF is an important signaling protein involved in angiogenesis by inducing the proliferation and division of vascular endothelial cells and increasing vascular permeability. VEGF binds to VEGF receptors (VEGFRs) to induce angiogenesis and lymphangiogenesis. VEGF may include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and / or PlGF (placental growth factor).

[0125] In the present invention, the VEGF protein may be included without limitation as long as it is derived from mammals, including primates such as humans and monkeys, and rodents such as rats and mice. In addition, the VEGF protein may be included without limitation as long as it binds to the VEGF receptor (VEGFR) and exhibits activity equivalent to or similar to VEGF. Here, "activity" may mean, for example, specifically binding to the VEGF receptor, and this specific binding can be measured using methods known to those skilled in the art.

[0126] In addition, the VEGF protein may include native VEGF and fragments thereof. The native VEGF protein refers to a natural VEGF protein. The amino acid sequence and polynucleotide sequence for the native VEGF protein can be obtained from known databases such as the GenBank of the National Institutes of Health (NCBI) of the United States. In one specific embodiment of the present invention, the VEGF protein may include the amino acid sequence of SEQ ID NO: 79, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, or SEQ ID NO: 95.

[0127] In addition, as long as it has the same activity as the VEGF protein or has the same gene location encoding the VEGF protein on the chromosome, it may be composed of a sequence in which one or more amino acids of the protein are added, deleted, or substituted. Specifically, the VEGF may comprise or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 79, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, or SEQ ID NO: 95.

[0128] In the present invention, the VEGF-specific binding site may be selected from the group consisting of receptors, ligands, antibodies, and fragments thereof that bind to VEGF, but is not limited thereto as long as it specifically binds to VEGF and inhibits the activity of VEGF. Specifically, the VEGF-specific binding site may be a receptor. In this case, the receptor may be a VEGF receptor (VEGFR).

[0129] The above “VEGF receptor (VEGFR)” refers to a transmembrane protein that binds to vascular endothelial growth factor (VEGF) and regulates angiogenesis. VEGFR is a tyrosine kinase receptor and exists in three types: VEGFR1, VEGFR2, and VEGFR3. VEGF binds to the extracellular domain of each VEGFR and activates the receptor. VEGF-A binds to both VEGFR1 and VEGFR2, and both receptors are expressed on vascular endothelial cells. VEGF-B and PlGF bind only to VEGFR1. On the other hand, VEGF-C and VEGF-D are known to bind to VEGFR2 and VEGFR3, and VEGFR3 is mainly expressed on lymphatic endothelial cells. The VEGFR comprises seven extracellular domains, a transmembrane domain, and an intracellular tyrosine kinase domain.

[0130] In the present invention, VEGFR may be an extracellular domain of VEGFR.

[0131] As used herein, the term "extracellular domain of a VEGF receptor" refers to the domain of a VEGF receptor that binds to VEGF. Specifically, it refers to a portion excluding the transmembrane domain and the intracellular tyrosine kinase domain of the VEGF receptor. Specifically, it may be the extracellular domain of VEGFR1 (SEQ ID NO: 96) or VEGFR2 (SEQ ID NO: 80). In one specific example, the VEGFR1 extracellular domain and the VEGFR2 extracellular domain may comprise the amino acid sequences of SEQ ID NO: 97 and SEQ ID NO: 98, respectively.

[0132] In the present invention, the receptor may include a fragment thereof. The “fragment” is the same as described above. Specifically, the receptor fragment may be one in which a portion of the N-terminus and / or C-terminus of VEGFR is deleted. More specifically, the receptor fragment may be one in which a portion of the N-terminus and / or C-terminus of the extracellular domain of VEGFR is deleted. The “fragment of the extracellular domain of VEGFR” may include domains D1, D2, D3 of VEGFR, which are regions to which VEGF binds, or a combination thereof. Preferably, the fragment of VEGFR may include D2 and / or D3.

[0133] In addition, VEGFR may be a fusion form of extracellular domain fragments of VEGFR subtypes. In one specific example, it may be a fusion form of an extracellular domain fragment of VEGFR1 and an extracellular domain fragment of VEGFR2. In one specific example, it may be a fusion form of an extracellular domain fragment of VEGFR1 and an extracellular domain fragment of VEGFR3. In one specific example, it may be a fusion form of an extracellular domain fragment of VEGFR2 and an extracellular domain fragment of VEGFR3. In this case, the extracellular domains may be directly linked or linked via a linker. The linker is a peptide linker, and the peptide linker is described below. Preferably, the VEGFR may be a fusion form of the D2 domain of VEGFR1 and the D3 domain of VEGFR2. In one embodiment of the present invention, the receptor may comprise the amino acid sequence of SEQ ID NO: 83.

[0134] Specifically, the VEGF-specific binding site may be an antibody or a fragment thereof. The antibody and fragment thereof are the same as described above.

[0135] More specifically, the antibody or fragment thereof that specifically binds to VEGF may include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 69 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 71, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 72 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 73, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 74 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 76, or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 77 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 78. In this case, the antibody or fragment thereof that specifically binds to VEGF may be linked via a peptide linker. Here, the peptide linker is described below. In one specific example, the peptide linker may include the amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 70, or SEQ ID NO: 75.

[0136] In the present invention, the antibody or fragment thereof that specifically binds to VEGF can specifically bind to VEGF and inhibit the activity of VEGF.

[0137] Bispecific antibody structure

[0138] The bispecific antibody of the present invention may comprise an anti-Tie2 antibody and a VEGF-specific binding site. The VEGF-specific binding site may be bound to the Fc region of the anti-Tie2 antibody or a variant thereof. Furthermore, the binding site may be linked via a linker.

[0139] More specifically, the bispecific antibody may be a fusion protein dimer comprising the following structural formulas (I) and (II):

[0140] N'-X'-[L1]p-Fc region or a variant thereof-[L2]qYC' (I); and

[0141] N'-X''-C' (II)

[0142] At this time, in the structural formulas (I) and (II),

[0143] The above N' is the N-terminus,

[0144] The above C' is the C-terminal,

[0145] The above - means combination,

[0146] X' represents the heavy chain variable region (VH) and heavy chain constant region (CH1) of the anti-Tie2 antibody,

[0147] X'' represents the light chain variable region (VL) and light chain constant region (CL) of the anti-Tie2 antibody,

[0148] X' and X'' combine to form a binding site (X) that specifically binds to Tie2,

[0149] The above Y is a VEGF-specific binding site,

[0150] The above L1 and L2 are peptide linkers,

[0151] The above p and q are each independently O or 1.

[0152] The anti-Tie2 antibody, VEGF specific binding site, Fc region or variant thereof, heavy chain variable region (VH), heavy chain constant region (CH), light chain variable region (VL) and light chain constant region (CL) are the same as described above.

[0153] As used herein, the term "peptide linker" refers to a peptide composed of one or more amino acids. Typically, it may be composed of 1 to 100 consecutive amino acids, 5 to 80 consecutive amino acids, 7 to 70 consecutive amino acids, 10 to 60 consecutive amino acids, or 12 to 50 amino acids. Peptide linkers are known in the art or are described herein. In one embodiment, the peptide linker L1 or L2 may be composed of 30 amino acids. Specifically, the linker L1 may comprise at least one cysteine. Specifically, it may comprise one, two, or three cysteines. Furthermore, the peptide linker L1 may be derived from the hinge of an immunoglobulin. In one embodiment, the linker L1 may comprise the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO: 65. The linker L2 may comprise (G4S)n and may further comprise (G)n or 1 to 10 amino acids. In addition, the linker L2 may be (G)n or (GS3)n. In this case, n may each independently be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one specific example, the linker L2 may comprise the amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 70, or SEQ ID NO: 75.

[0154] At this time, the VEGF-specific binding site is the same as described above.

[0155] Specific examples of the above bispecific antibodies are as follows.

[0156] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22, and the VEGF-specific binding site may comprise the amino acid sequence of SEQ ID NO: 83.

[0157] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 21 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 22, and the VEGF-specific binding site may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 69 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 71.

[0158] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 21 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 22, and the VEGF-specific binding site may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 72 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 73.

[0159] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 21 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 22, and the VEGF-specific binding site may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 74 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 76.

[0160] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 21 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 22, and the VEGF-specific binding site may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 77 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 78.

[0161] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24, and the VEGF-specific binding site may comprise the amino acid sequence of SEQ ID NO: 83.

[0162] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 24, and the VEGF-specific binding site may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 69 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 71.

[0163] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24, and the VEGF-specific binding site may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 72 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 73.

[0164] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 24, and the VEGF-specific binding site may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 74 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 76.

[0165] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24, and the VEGF-specific binding site may include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 77 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 78.

[0166] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 26, and the VEGF-specific binding site may comprise the amino acid sequence of SEQ ID NO: 83.

[0167] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may include a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 26, and the VEGF-specific binding site may include a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 69 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 71.

[0168] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 26, and the VEGF-specific binding site may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 72 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 73.

[0169] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 26, and the VEGF-specific binding site may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 74 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 76.

[0170] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may include a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 26, and the VEGF-specific binding site may include a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 77 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 78.

[0171] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22, and the VEGF-specific binding site may comprise the amino acid sequence of SEQ ID NO: 83.

[0172] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 22, and the VEGF-specific binding site may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 69 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 71.

[0173] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 22, and the VEGF-specific binding site may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 72 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 73.

[0174] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 22, and the VEGF-specific binding site may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 74 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 76.

[0175] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 22, and the VEGF-specific binding site may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 77 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 78.

[0176] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 28 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22, and the VEGF-specific binding site may comprise the amino acid sequence of SEQ ID NO: 83.

[0177] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 28 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 22, and the VEGF-specific binding site may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 69 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 71.

[0178] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 28 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 22, and the VEGF-specific binding site may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 72 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 73.

[0179] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 28 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 22, and the VEGF-specific binding site may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 74 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 76.

[0180] In one specific example, the anti-Tie2 antibody or fragment thereof of the bispecific antibody may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 28 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 22, and the VEGF-specific binding site may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 77 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 78.

[0181] Polynucleotide encoding a bispecific antibody

[0182] Another aspect of the present invention provides a polynucleotide encoding the bispecific antibody. The bispecific antibody is the same as described above.

[0183] Specifically, the polynucleotide may include a base sequence encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46. Additionally, the polynucleotide may include a base sequence encoding a light chain comprising the amino acid sequence of SEQ ID NO: 47.

[0184] Additionally, if the polynucleotide encodes the same polypeptide, one or more bases may be mutated by substitution, deletion, insertion, or a combination thereof. When producing a polynucleotide sequence by chemical synthesis, synthetic methods widely known in the art can be used, such as the method described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988), and examples thereof include triester, phosphite, phosphoramidite, and H-phosphate methods, PCR and other autoprimer methods, and oligonucleotide synthesis methods on solid supports.

[0185] In one specific example, the polynucleotide encoding the heavy chain can comprise a base sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to a polynucleotide encoding the amino acid sequence of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46.

[0186] In one specific example, the polynucleotide encoding the light chain can comprise a base sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to a polynucleotide encoding the amino acid sequence of SEQ ID NO: 47.

[0187] The above polynucleotide may additionally include a signal sequence or a leader sequence. The signal sequence is the same as described above.

[0188] vector loaded with polynucleotides

[0189] Another aspect of the present invention provides a vector loaded with a polynucleotide encoding the bispecific antibody. The polynucleotide is the same as described above. Specifically, the vector may include a polynucleotide encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46. The vector may include a polynucleotide encoding a light chain comprising the amino acid sequence of SEQ ID NO: 47.

[0190] At this time, each polynucleotide can be loaded into each vector, or can be loaded into the same vector.

[0191] Transformed cells

[0192] Another aspect of the present invention provides a cell transformed with a vector loaded with a polynucleotide encoding the bispecific antibody. The bispecific antibody is the same as described above.

[0193] Method for producing bispecific antibodies

[0194] Another aspect of the present invention provides a method for producing the bispecific antibody.

[0195] The method for producing the above bispecific antibody may include the steps of i) culturing the transformed cell; and ii) obtaining the bispecific antibody from the culture solution.

[0196] <Pharmaceutical Composition>

[0197] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating a vascular abnormality-related disease, comprising as an active ingredient the anti-Tie2 antibody or fragment thereof; or the anti-Tie2 antibody or fragment thereof and a bispecific antibody comprising a VEGF-specific binding site. The anti-Tie2 antibody or fragment thereof and the bispecific antibody are the same as those described above.

[0198] As used herein, the term “vascular abnormality-related disease” refers to a disease associated with angiogenesis abnormalities or structural and / or functional alterations of blood vessels.

[0199] The above vascular abnormality-related disease may be a disease related to angiogenesis abnormality, or a structural and / or functional variation of blood vessels. The above "angiogenesis" refers to the formation or growth of new blood vessels from existing blood vessels, and the above "angiogenesis-related disease" refers to a disease related to the occurrence or progression of angiogenesis. Specifically, the above "angiogenesis abnormality" may be caused by excessive increase, decrease, or deficiency. The above "vascular structural and / or functional variation-related disease" may be caused by any one or more selected from the group consisting of structural abnormality of blood vessels, dysfunction of blood vessels, and damage to blood vessels.

[0200] The above vascular abnormality-related diseases may be specifically caused by, but are not limited to, vascular leakage, inflammation, and local ischemia. For example, it may be any one selected from the group consisting of acute renal injury, chronic renal failure, macular degeneration, diabetic retinopathy, diabetic macular edema, retinal vessel occlusion, proliferative retinopathy, retinopathy of prematurity, corneal graft rejection, glaucoma, critical limb ischemia, diabetic erectile dysfunction, sepsis, acute respiratory distress syndrome, vasculitis, Alzheimer's disease, Parkinson's disease, multiple sclerosis, stroke, thrombosis, occlusion, cancer, systemic erythropoiesis, psoriasis, hemophilic arthritis, associated sclerosis, capillary formation of atherosclerotic plaques, keloids, wound granulation, vascular adhesions, rheumatoid arthritis, osteoarthritis, autoimmune diseases, Crohn's disease, restenosis, atherosclerosis, intestinal adhesions, cat scratch disease, ulcers, cirrhosis, nephritis, diabetic nephropathy, diabetes, inflammatory diseases, and neurodegenerative diseases, but is not limited thereto.

[0201] The term "prevention" as used herein refers to any measure intended to suppress or delay the onset of a disease. Furthermore, the term "treatment" as used herein refers to any measure intended to improve or alleviate the symptoms of an existing disease. Desirable effects of such treatment include preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of the disease, improving or alleviating the condition, and achieving remission or improved prognosis.

[0202] The preferred dosage of the above pharmaceutical composition varies depending on the patient's condition and weight, the severity of the disease, the drug form, the route and duration of administration, but can be appropriately selected by those skilled in the art. In the pharmaceutical composition for treating or preventing a disease of the present invention, the active ingredient may be included in any amount (effective amount) depending on the intended use, formulation, mixing purpose, etc., as long as it can exhibit therapeutic activity against the disease. Here, "effective amount" refers to the amount of the active ingredient that can induce an improvement in the condition of the disease or a therapeutic effect. Such an effective amount can be experimentally determined within the normal ability of those skilled in the art.

[0203] Pharmacokinetic parameters, such as bioavailability, and underlying parameters, such as clearance rate, can also influence efficacy. Therefore, "enhanced efficacy" (e.g., improved efficacy) can be attributed to improved pharmacokinetic parameters and improved efficacy, and can be measured by comparing parameters, such as clearance rate and disease treatment or improvement, in test animals or human subjects.

[0204] In addition to the active ingredient, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier or additive. The term "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not exhibit toxicity exceeding the adaptability of the subject of application (prescription). The carrier refers to a compound that facilitates the addition of the antibody of the present invention or an antigen-binding fragment thereof into cells or tissues, and the additive refers to any component necessary for preparing the pharmaceutical composition into a required dosage form, such as an excipient, a disintegrant, a binder, a flavoring agent, a preservative, a lubricant, a stabilizer, a viscosifier, etc.

[0205] When the above pharmaceutical composition is prepared as a parenteral dosage form, it can be formulated in the form of injections, transdermal administration, nasal inhalation, and suppositories using a suitable carrier according to a method known in the art. When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, and preferably, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, and isotonic solutions such as 5% dextrose can be used. Methods for formulating pharmaceutical compositions are known in the art, and specific references may be made to literature [Remington's Pharmaceutical Sciences (19th ed, 1995)], etc. The above literature is considered to be a part of the present specification.

[0206] The above-described active ingredient of the present invention or a pharmaceutical composition containing the above-described active ingredient can be administered to a subject in a pharmaceutically effective amount. The term "pharmaceutically effective amount" refers to an amount that sufficiently exhibits a preventive or therapeutic effect with a reasonable benefit / risk ratio applicable to all medical treatments. In addition, the "pharmaceutically effective amount" varies depending on various factors including the severity of the disease to be treated, the patient's age and sex, 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 treatment period, and other parameters known in the art.

[0207] The above-mentioned subject may be a mammal, preferably a human. In addition, the above-mentioned "subject" refers to a subject suffering from, or likely to suffer from, a vascular abnormality-related disease.

[0208] The term "administration" as used herein refers to introducing a given substance into an individual through an appropriate method, and the route of administration of the composition may be administered through any common route as long as it can reach the target tissue. Specifically, it may be parenteral administration. The parenteral administration may include, but is not limited to, intravenous administration, subcutaneous administration, intraperitoneal administration, intrauterine administration, intracerebrovascular administration, etc. In addition, it may be administered directly locally to the diseased area.

[0209] The preferred dosage of the above pharmaceutical composition may range from 0.01 μg / kg to 10 g / kg per day, or from 0.01 mg / kg to 1 g / kg, depending on the patient's condition, weight, sex, age, severity of the condition, and route of administration. Administration may be administered once a day or divided into several times. Such dosage should not be construed as limiting the scope of the present invention in any way.

[0210] The pharmaceutical composition may be administered alone or in combination with other treatments. In such cases, it may be administered sequentially or simultaneously with conventional treatments. Furthermore, the composition may be administered in a single dose or in multiple divided doses. Taking these factors into full consideration, it is important to administer the minimum amount sufficient to achieve maximum effect without adverse effects, and this dosage can be readily determined by a specialist in the field. The dosage of the pharmaceutical composition is not particularly limited but may vary depending on various factors, including the patient's health and weight, the severity of the disease, the type of drug, the route of administration, and the time of administration.

[0211] In addition, the active ingredient of the present invention or the pharmaceutical composition containing the active ingredient may be administered in combination with other vascular abnormality-related disease treatment agents, anticancer agents, etc. In this case, the active ingredient of the present invention or the pharmaceutical composition may be administered simultaneously or sequentially with other treatment agents, such as vascular abnormality-related disease treatment agents, anti-inflammatory drugs, anticancer agents, etc. For example, other treatment agents, such as vascular abnormality-related disease treatment agents, anti-inflammatory drugs, anticancer agents, etc., may be first administered to a subject, and then the antibody of the present invention or an antigen-binding fragment thereof, or the pharmaceutical composition may be additionally administered to the subject, or the antibody of the present invention or an antigen-binding fragment thereof, or the pharmaceutical composition may be first administered to a subject, and then other treatment agents, such as vascular abnormality-related disease treatment agents, anti-inflammatory drugs, anticancer agents, etc., may be additionally administered to the subject. In addition, depending on the case, the antibody of the present invention or an antigen-binding fragment thereof, or the pharmaceutical composition and other treatment agents, such as vascular abnormality-related disease treatment agents, anti-inflammatory drugs, anticancer agents, etc., may be administered to a subject simultaneously.

[0212] Another aspect of the present invention provides a use of the anti-Tie2 antibody or fragment thereof; or a bispecific antibody comprising the anti-Tie2 antibody or fragment thereof and a VEGF-specific binding site for preventing or treating a vascular abnormality-related disease. Another aspect of the present invention provides a method for preventing or treating a vascular abnormality-related disease, comprising administering to a subject the anti-Tie2 antibody or fragment thereof; or a bispecific antibody comprising the anti-Tie2 antibody or fragment thereof and a VEGF-specific binding site.

[0213] The above anti-Tie2 antibodies, fragments, VEGF, bispecific antibodies, entities, prevention, treatment and vascular abnormality related diseases are the same as described above.

[0214] Hereinafter, the present invention will be described in more detail through the following examples. However, the following examples are intended to exemplify the present invention, and the scope of the present invention is not limited to these examples.

[0215] I. Antibodies that specifically bind to human Tie2 protein

[0216] Example 1. Production of an antibody that specifically binds to human Tie2 protein

[0217] Example 1.1. Screening of antibodies that specifically bind to human Tie2 protein

[0218] For the identification of effective antibodies included in the present invention, a human antibody library (diversity: 2.5x10 11 ) and the Fc region (mFc) of mouse IgG were combined with recombinant human Tie2 extracellular domain (hTie2-ECD-mFc) protein (SEQ ID NO: 99) to perform phage display panning.

[0219] Specifically, hTie2-ECD-mFc, used as an antigen, was fixed to an immunotube at a concentration of 10 μg / mL, and phage molecules expressed from an antibody library were bound for 1 hour, and then washed seven times with PBST (PBS / 0.05% Tween 20) buffer. The phage molecules bound to the antigen were recovered by treating with a 100 mM Triethylamine solution for 10 minutes, and the TG1 E. coli host was infected, and the TG1 host was obtained by spreading on a solid medium containing 2x YT / ampicillin / 2% glucose.

[0220] After that, phage molecules were expressed again from the TG1 host obtained using M13KO7 helper phage and used in the subsequent panning. The method described above was carried out up to a total of 3 panning rounds, and the washing process was performed 15 times in the 2nd round and 20 times in the 3rd round to recover antibody-expressing phages with high binding affinity. After that, the single clones recovered in the 3rd round were inoculated into 2x YT / ampicillin / 2% glucose liquid medium dispensed at 150 μL / well in a 96-well plate, and cultured with shaking at 37℃ for 16 hours. After adding 50 μL of 2x YT / 50% glycerol solution to the culture medium to prepare a master plate, 10 μL was taken from the master plate and inoculated into 2x YT / ampicillin / 2% glucose liquid medium dispensed at 150 μL / well again, and cultured with shaking at 37℃ for approximately 3 hours. After incubation, the plate was centrifuged at 4,000 rpm for 15 min to remove the supernatant, and the E. coli pellet was resuspended in 150 μL of 2x YT / ampicillin / 1 mM IPTG liquid medium and cultured at 30°C for 16 h.

[0221] After centrifugation of the culture solution cultivated by the above method, the E. coli pellet was treated with 150 μL of periplasmic extraction solution (30 mM Tris-HCl, 20% sucrose, 1 mM EDTA, pH 8.0) and left on ice for 30 minutes. Subsequently, periplasmic proteins were recovered from the supernatant by centrifugation at 4,000 rpm for 10 minutes. Periplasmic proteins were treated on a 96-well ELISA plate coated with 100 ng / well of hTie2-ECD-mFc or mouse Tie2 extracellular domain (mTie2-ECD-mFc) to screen for periplasmic scFv antibodies.

[0222] Subsequently, scFv antibodies that cross-bind to hTie2-ECD (SEQ ID NO: 91) and mTie2-ECD (SEQ ID NO: 100) were screened through screening, and through sequence analysis of antibodies that showed binding affinity, five different antibodies (named 'MT-100', 'MT-110', 'MT-120', 'MT-101', and 'MT-111', respectively) having complementary binding regions (CDRs) in the heavy chain and heavy chain variable region were discovered (Table 1).

[0223] The heavy chain variable region (VH) and light chain variable region (VL) sequences of each antibody are shown in Tables 2 to 6. The antibodies were cloned into pcDNA3.4-based heavy and light chain expression vectors (Invitrogen) each introducing an IgG1 heavy chain constant region (CH1-hinge-CH2-CH3) and a light chain constant region (CL) for transient expression in the form of IgG, and used for transient expression and purification. The following antibodies can be used interchangeably as MT-100 or MT-100 antibody, MT-110 or MT-110 antibody, MT-120 or MT-120 antibody, MT-101 or MT-101 antibody, and MT-111 or MT-111 antibody.

[0224] Antibody name CDR amino acid sequence sequence number MT-100 MT-100-HCDR1GFTFNSYG1 MT-100-HCDR2TSNDGSTT2 MT-100-HCDR3ARKVVRGYSYHDAFDI3 MT-100-LCDR1QSVSSY4 MT-100-LCDR2GAS5 MT-100-LCDR3QQYGTTPYT6 MT-110 MT-110-HCDR1SDSFTSNW7MT -110-HCDR2IYPDDSDT8MT-110-HCDR3ARQIDTGYFDY9MT-110-LCDR1QGISSN10MT-110-LCDR2AAS11MT-11 0-LCDR3QQANSFPLT12MT-120MT-120-HCDR1GFTFAGQA13MT-120-HCDR2ISWDSGGI14MT-120-HCDR3ARGST AFPRYFEY15MT-120-LCDR1QDIDSY16MT-120-LCDR2ASS17MT-120-LCDR3QQGYDFPWT18MT-101MT-101-HC DR1GFTFNSYG1MT-101-HCDR2TSNDGSTT2MT-101-HCDR3ARKVVRGYHYHDAFDI19MT-101-LCDR1QSVSSY4MT- 101-LCDR2GAS5MT-101-LCDR3QQYGTTPYT6MT-111MT-111-HCDR1GFTFNSYG1MT-111-HCDR2TSNDGSTT2MT -111-HCDR3ARKVVRGYLPHDAFDI20MT-111-LCDR1QSVSSY4MT-111-LCDR2GAS5MT-111-LCDR3QQYGTTPYT6

[0225] MT-100 Amino Acid Sequence Sequence Number: MT-100 HC signal peptide: MGWSCIILFLVATATGVHS51VHEVQLVESGGGVVQPGRSLRLSCAASGFTFNSYGMQWVRQAPGKGLEWVAVTSNDGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARKVVRGYSYHDAFDIWGQGTMVTVSS21 CH1: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV52 First Linker: EPKSCDKTHTCPPCP53 Fc (CH2 + CH3): APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG54 MT-100 LC signal peptide: MGWSCIILFLVATATGVHS51VLDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYRQKPGQAPRLLIYGASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVFYCQQYGTTPYTFGQGTKVEIK22 CLR: TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC82

[0226] MT-110 Amino Acid SequenceSEQ ID NO: MT-110 HCsignal peptideMGWSCIILFLVATATGVHS51VHQMQLVQSGAEVKKPGESLKISCKGSSDSFTSNWIAWVRQKPGKGLEWMGIIYPDDSDTRYSPSFQGQVTMSADKSTSTAYLQWSSLKASDTAMYYCARQIDTGYFDYWGQGTLITVSS23CH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV52First LinkerEPKSCDKTHTCPPCP53Fc (CH2 + CH3)APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG54MT-110 LCsignal peptideMGWSCIILFLVATATGVHS51VLDIQMTQYPSSLAASTGDRVTITCRASQGISSNLAWYQQKPGNSPKLLIYAASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGPGTKVEIK24CLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC82

[0227] MT-120아미노산 서열서열번호MT-120 HCsignal peptideMGWSCIILFLVATATGVHS51VHQLQLVQSGGDLVQPGRSLRLSCAASGFTFAGQAMHWVRQAPGKGLEWVSSISWDSGGIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGSTAFPRYFEYWGQGTLVTVSS25CH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV52제1 링커EPKSCDKTHTCPPCP53Fc(CH2+CH3)APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG54MT-120 LCsignal peptideMGWSCIILFLVATATGVHS51VLDIQMTQSPSSVSASIGDKVTITCRSSQDIDSYLAWYQQRPGQTPNLLIYASSTLQDGVPSRFSGSGSGTHFTLTITNLQPEDSATYHCQQGYDFPWTFGQGTKVEIK26CLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC82

[0228] MT-101 Amino Acid SequenceSequence Number MT-101 HCsignal peptideMGWSCIILFLVATATGVHS51VHEVQLVESGGGVVQPGRSLRLSCAASGFTFNSYGMQWVRQAPGKGLEWVAVTSNDGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARKVVRGYHYHDAFDIWGQGTMVTVSS27CH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV52First LinkerEPKSCDKTHTCPPCP53Fc (CH2 + CH3)APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG54MT-101 LCsignal peptideMGWSCIILFLVATATGVHS51VLDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYRQKPGQAPRLLIYGASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVFYCQQYGTTPYTFGQGTKVEIK22CLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC82

[0229] MT-111 amino acid sequence SEQ ID NO. MT-111 HC signal peptide MGWSCIILFLVATATGVHS51VHEVQLVESGGGVVQPGRSLRLSCAASGFTFNSYGMQWVRQAPGKGLEWVAVTSNDGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARKVVRGYLPHDAFDIWGQGTMVTVSS28CH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV521 LinkerEPKSCDKTHTCPPCP53Fc(CH2+CH3)APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG54MT-111 LCsignal peptideMGWSCIILFLVATATGVHS51VLDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYRQKPGQAPRLLIYGASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVFYCQQYGTTP YTFGQGTKVEIK22CLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC82

[0230] Example 1.2. Design of IgG1-LALA and IgG4 mutant antibodies based on MT-101.

[0231] Since IgG1 antibodies can bind to the Fc-gamma receptor (FcγR) group and induce non-specific immune cell activation side effects, two mutant antibodies with reduced Fc reactive function were generated and evaluated from an IgG1 Tie2-binding antibody (MT-101). Specifically, a mutant antibody was generated in which amino acids 234 and 235 of the CH2 region of IgG1, Leucine, were substituted with Alanine. Specifically, primers capable of inducing DNA sequence mutations such that Leucine at positions 234 and 235 in the Fc region of MT-101 (IgG1) was generated to substitute Alanine, thereby inducing point mutations (site-directed mutagenesis) in a polynucleotide encoding the heavy chain of MT-101. As a result, an expression vector loaded with a polynucleotide encoding the MT-101 (IgG1-LALA) heavy chain (Table 9, SEQ ID NO: 29) was constructed. Additionally, in order to produce an isotypic MT-101 (IgG4) mutant antibody with low binding affinity to FcγR in the natural state, the heavy chain variable region of MT-101 was cloned into a pcDNA3.4 vector into which a human IgG4 constant region was introduced, thereby constructing an MT-101 (IgG4) heavy chain (Table 9, SEQ ID NO: 30) expression vector. The two types of mutant antibodies were transiently expressed and purified using each heavy chain expression vector and the MT-101 (IgG1) light chain expression vector.

[0232] Amino acid sequence of MT-101 (IgG1-LALA) Sequence number: MT-101 (IgG1-LALA) HC Signal peptide: MGWSCIILFLVATATGVHS 51 VHEVQLVESGGGVVQPGRSLRLSCAASGFTFNSYGMQWVRQAPGKGLEWVAVTSNDGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARKVVRGYHYHDAFDIWGQGTMVTVSS 27 CH1: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV 52 First linker: EPKSCDKTHTCPPC 53 Fc (CH2 + CH3): APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 63 MT-101 (IgG1-LALA) LC Signal peptide: MGWSCIILFLVATATGVHS 51 VLDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYRQKPGQAPRLLIYGASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVFYCQQYGTTPYTFGQGTKVEIK 22 CLR: TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 82

[0233] Amino acid sequence of MT-101(IgG4) Sequence number: MT-101(IgG4) HC Signal peptide: MGWSCIILFLVATATGVHS 51: VHEVQLVESGGGVVQPGRSLRLSCAASGFTFNSYGMQWVRQAPGKGLEWVAVTSNDGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARKVVRGYHYHDAFDIWGQGTMVTVSS 27: CH1: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV 90: First linker: ESKYGPPCPPCP 65: Fc(CH2+CH3): APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 66: MT-101(IgG4) LC Signal peptide: MGWSCIILFLVATATGVHS 51: VLDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYRQKPGQAPRLLIYGASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVFYCQQYGTTPYTFGQGTKVEIK 22: CL: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 82

[0234] 서열 정보서열서열번호MT-101(IgG1-LALA)-HCEVQLVESGGGVVQPGRSLRLSCAASGFTFNSYGMQWVRQAPGKGLEWVAVTSNDGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARKVVRGYHYHDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG29MT-101(IgG4)-HCEVQLVESGGGVVQPGRSLRLSCAASGFTFNSYGMQWVRQAPGKGLEWVAVTSNDGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARKVVRGYHYHDAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK30

[0235] 실시예 1.3. 대조군 항체 설계

[0236] In order to compare the MT-101 of the present invention with the previously applied Tie2 activating antibodies, a total of five control Tie2 activating antibodies were designed and produced. Specifically, the 2F2 antibody and the 4E2 antibody included in the international patent publication WO 2021-029746 A2, the TIE-1-Igγ1 antibody included in the U.S. registered patent US 9902782 B2, the IGT-427 antibody included in the U.S. patent publication US 2023-0287109 A1, and the #3 antibody included in the international patent publication WO 2021-102173 A1 were set as controls. After reverse translation with reference to the heavy and light chain variable region amino acid sequences of each antibody, the genes were synthesized by performing a codon optimization process for production in Expi293F cells. Each synthesized heavy and light chain variable region gene was cloned into the pcDNA3.4 vector containing the IgG1 heavy and light chain constant regions, respectively, to construct heavy and light chain expression vectors for a total of five antibodies. The amino acid sequences of the heavy and light chains of the control antibody are shown in Table 10, and were used in the experiments after transient expression and purification.

[0237]

[0238] Example 1.4. Production of an antibody that specifically binds to human Tie2 protein.

[0239] The pcDNA3.4 vector (Invitrogen) loaded with polynucleotides encoding the heavy and light chains of the antibodies produced by the methods of Examples 1.1 to 1.3 was introduced into Expi293F cells or ExpiCHO cells to perform transient expression and purification of the antibodies. Specifically, for transient expression of antibodies in Expi293 cells, 50 μg each of the heavy and light chain expression vectors (total 100 μg) was diluted in Opti-MEM medium (6 mL), and ExpiFectamine TM 293 Transfection Kit reagent (ExpiFectamine TM 293 Reagent) 320 μL was diluted in Opti-MEM medium (6 mL) and allowed to stand for 5 minutes each. The medium with the vector added and the medium with the reagent added were mixed and allowed to stand for an additional 10 minutes, and then 100 mL (2.0 x 10) of Expi293F cells were added. 6 cells / mL) and cultured in suspension for 24 hours.

[0240] ExpiFectamine was added to cells cultured using the method described above. TM After adding 293 Transfection Enhancer and further culturing for 4 days, the supernatant was collected by centrifugation at 10,000xg for 30 min and filtered through a 0.2 μm filter. To express ExpiCHO-based antibodies, 40 μg each of heavy and light chain expression vectors (total 80 μg) were added to OptiPRO TM Dilute in SFM medium (4 mL), ExpiFectamine TM Reagents of CHO Transfection Kit (ExpiFectamine TM CHO Reagent) 320 μL OptiPRO TMDiluted in 3.7 mL of SFM medium, each was allowed to stand for 4 minutes. The medium with the vector added and the medium with the reagent added were mixed and allowed to stand for another 5 minutes, and then 100 mL (3.0 x 10) of ExpiCHO cells were added. 6 cells / mL) and cultured in suspension for 22 hours.

[0241] ExpiFectamine was added to the cells cultured as described above. TM CHO Transfection Enhancer 1 600 μL with ExpiCHO TM Treat 16 mL of feed solution and ExpiCHO 5 days later TM The cells were cultured for 12 days after additional treatment with 16 mL of feed solution. Afterwards, the culture solution was centrifuged at 10,000 × g for 40 minutes and filtered through a 0.2 μm filter to recover the supernatant. The supernatant recovered from Expi293F or ExpiCHO was bound to an affinity column (MabSelect PrismA, Cytiva) using an AKTA Avant 150 instrument (Cytiva) and eluted with an acidic eluent (100 mM glycine buffer, pH 3.5). The eluted antibody was finally purified into PBS buffer using a desalting column (Hipprep 26 / 10). The concentration of the purified antibody was measured by absorbance or BCA assay, and then SDS-PAGE analysis was performed under non-reducing and reducing conditions. As a result, an antibody with a total molecular weight of approximately 150 kDa, including a heavy chain (approximately 50 kDa) and a light chain (approximately 25 kDa), was identified.

[0242] Example 2. Confirmation of binding affinity of anti-Tie2 antibody

[0243] The binding affinity of the antibody prepared through the above Example 1.4 to human Tie2 protein was confirmed.

[0244] The affinity of antibodies was measured using an Octet K2 (ForteBio) device based on the BLI (Biolayer interferometry) technique. Specifically, the AR2G biosensor tip was hydrated with distilled water for 10 minutes and then activated using a buffer containing 20 mM EDC and 10 mM Sulfo-NHS. Then, 60 nM hTie2-ECD-6xHis protein (SEQ ID NO: 101) or mTie2-ECD-mFc protein as an antigen was diluted in an immobilization buffer (10 mM sodium acetate; pH 6.0) and reacted with the activated AR2G tip for 10 minutes to immobilize the antigen on the AR2G tip.

[0245] After that, the antigen immobilization reaction was terminated by treating with 1 M ethanolamine (pH 8.5) solution, and the AR2G tip was reacted in a PBS buffer solution for 60 seconds to obtain a baseline of antigen-antibody binding. Then, MT-100 (IgG1), MT-111 (IgG1), MT-101 (IgG1), MT-101 (IgG4), or MT-101 (IgG1-LALA) was diluted in three sections at a concentration ranging from 5 nM to 100 nM using a PBS buffer solution, and was associated with the AR2G tip on which the antigen was immobilized for 800 seconds, and then dissociated for 800 seconds. Based on the reaction curve confirmed through the analysis of the association and dissociation reactions, the K on , K off and K D The affinity of each antibody was measured by calculating the value.

[0246] Antigen-antibody K D (M)K on (1 / Ms)K off(1 / s) Human Tie2 MT-100 3.42E-09 3.38E-05 1.15E-03 MT-11 16.41E-101.04E-05 6.67E-05 MT-101 (IgG1) 4.71E-101.93E-05 9.12E-05 MT-101 (IgG4) 7.21E-107.40E-05 5.34E-04 MT-101 (IgG1-LALA) 4.75E-107.87E-05 3.74E-04 Mouse Tie2MT-1001.44E-085.84E058.44E-03MT-1111.95E-092.43E054.75E-04MT-101(IgG1)6.66E-092.73E051.82E-03

[0247] As a result, as shown in Table 11 above, it was confirmed that among MT-100, MT-111, and MT-101, MT-101 (IgG1) had the highest affinity for human Tie2 protein. In addition, it was confirmed that the MT-101 (IgG4) and MT-101 (IgG1-LALA) variants having the same variable region as MT-101 (IgG1) also had affinities for human Tie2 protein of 0.47 nM to 0.72 nM, similar to MT-101 (IgG1).

[0248] Example 3. Confirmation of Tie2 activation by anti-Tie2 antibody

[0249] In order to confirm the Tie2 activating ability of antibodies (MT-100, MT-101, MT-110, and MT-111) backboneed with the IgG1-type Fc prepared in Example 1.4 above, and the homologous antibody of MT-101 (MT-101 (IgG4)) and the mutant antibody (MT-101 (IgG1-LALA)), the phosphorylation of Tie2 protein (human or mouse) and the downstream signal transduction factors (AKT, ERK, eNOS, or FOXO1) of the Tie2 protein (P-Tie2, P-AKT, P-ERK, P-eNOS, or P-FOXO1) of the Tie2 protein was confirmed through Western blot.

[0250] Specifically, HEK293 cells (1x106 cells) or 3T3-J2 cells (5x10 ) expressing mouse Tie2 5 Cells) were cultured in a 60 mm culture dish using DMEM medium at 37°C. The cells cultured as described above were maintained in serum-free DMEM medium for 6 hours to create a serum-starved state, and then treated with the antibody (MT-100) at a concentration range of 0.1 μg / mL to 20 μg / mL for 15 minutes, and then the cells were lysed with a lysis buffer to obtain a cell lysate.

[0251] SDS-PAGE was performed on the cell lysate obtained as above, and then transferred to a PVDF membrane. The membrane was treated with 0.1% Tween20 TBST buffer solution diluted with 3% BSA and reacted at room temperature for 2 hours. Then, the membrane was treated with primary antibodies diluted 1:1,000 and reacted at room temperature for 2 hours. At this time, the primary antibodies used were anti-phospho-tyrosine antibody (Sigma, 05-321), anti-Tie2 antibody (R&D systems, AF313), anti-phospho-AKT antibody (Cell Signaling, 9271), anti-AKT antibody (Cell Signaling, 9272), anti-phospho-ERK antibody (Cell Signaling, 9106), anti-ERK antibody (Cell Signaling, 9102), anti-phospho-eNOS antibody (Cell Signaling, 9571), anti-eNOS antibody (Cell Signaling, 5880), anti-phsopho-FOXO1 antibody (Cell Signaling, 9461), and anti-FOXO1 antibody (Cell Signaling, 2880). After the primary antibody reaction, the sections were treated with a peroxidase-conjugated secondary antibody diluted 1:5,000 and incubated at room temperature for 1 hour. The secondary antibodies used were anti-rabbit IgG antibody (Invitrogen, 31460) or anti-mouse IgG antibody (Abcam, ab97023). After the secondary antibody reaction was completed, protein expression was confirmed by treatment with ECL solution.

[0252] As a result, it was confirmed that MT-100 induced phosphorylation of Tie2, AKT, or ERK proteins in humans or mice (Fig. 1a and Fig. 1b).

[0253] In addition, human umbilical vein endothelial cells (HUVEC) were used instead of HEK293 cells, and EGM-2 medium and serum-free M199 medium were used as media, and the cells were lysed to confirm the phosphorylation of Tie2. Afterwards, the phosphorylation of Tie2 protein and the phosphorylation of the signal transduction factor were confirmed in HUVEC expressing human Tie2 using the same method as above, except that immunoprecipitation was used.

[0254] As a result, it was confirmed that the antibody (MT-100, MT-101, MT-110, MT-111, MT-101(IgG4) or MT-101(IgG1-LALA)) induced phosphorylation of Tie2, AKT, ERK, eNOS or FOXO1 proteins in HUVECs, similar to that in HEK293 cells (Figs. 1c to 3).

[0255] Example 4. Confirmation of the specificity of anti-Tie2 antibodies for the Tie2 antigen protein.

[0256] It was confirmed whether the anti-Tie2 antibody obtained in Example 1.4 above specifically phosphorylates only the Tie2 protein in vascular endothelial cells or phosphorylates other receptor proteins in addition to the Tie2 protein.

[0257] Specifically, HUVECs (3x10 6Cells) were cultured in a 100 mm culture dish using EGM-2 medium at 37°C. HUVECs cultured as described above were maintained in M199 medium containing 1% serum for 6 hours to create a serum-starved state, and then treated with MT-100 at a concentration of 5 μg / mL for 30 minutes. The cells were treated with lysis buffer, and the cells were lysed to obtain a cell lysate. The cell lysates obtained by the above method were quantified using the BCA protein quantification method, and then 49 human tyrosine receptor proteins (Receptor tyrosine kinases (RTKs); ALK / CD246, Axl, DDR1, DDR2, Dtk, EGF R, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, ErbB2, ErbB3, ErbB4, FGF R1, FGF R2 alpha, FGF R3, FGF R4, Flt-3 / Flk-2, HGF R / c-MET, IGF-I R, Insulin R / CD220, M-CSF R, Mer, MSP R / Ron, MuSK, PDGF R alpha, PDGF R beta, The membrane was treated with antibodies against c-Ret, ROR1, ROR2, Ryk, SCF R / c-kit, Tie-1, Tie-2, TrkA, TrkB, TrkC, VEGF R1 / Flt-1, VEGF R2 / KDR, and VEGF R3 / Flt-4) and reacted at 4°C for 12 hours. The membrane was treated with anti-tyrosine phosphorylation antibody (Anti-phospho-tyrosine-HRP) and reacted at room temperature for 1 hour, and then treated with ECL solution to confirm the phosphorylation of 49 RTKs.

[0258] As a result, it was confirmed that MT-100 selectively phosphorylates only Tie2 protein among 49 RTKs in HUVEC (Fig. 4).

[0259] Example 5. Confirmation of cross-species binding ability of anti-Tie2 antibodies to Tie2 antigen protein.

[0260] We confirmed whether anti-Tie2 antibodies (MT-100, MT-101(IgG1), MT-101(IgG4), and MT-101(IgG1-LALA)) that bind to interspecies Tie2 proteins can also cross-bind to Tie2 proteins from other species.

[0261] Here, the Tie2 gene of monkey, mouse, rat, dog, or pig was synthesized and cloned into pcDNA3.4 vector to produce Tie2 expression vectors for each species (SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 106). 10 μg of each Tie2 expression vector and 20 μL of Lipofectamine 2,000 (Invitrogen) were diluted in 1 mL of Opti-MEM medium and transfected into HEK293T cells (2.0 x 10 6 The cells were treated and cultured for 2 days. The culture solution was centrifuged (300xg, 5 min) to separate and remove the supernatant, and the cells were washed with FACS buffer.

[0262] As described above, the cells expressing each type of Tie2 were treated with anti-Tie2 antibodies (MT-100, MT-101 (IgG1), MT-101 (IgG4), or MT-101 (IgG1-LALA)) at a concentration of 1 μg / mL, reacted at room temperature for 1 hour, and then washed twice with FACS buffer. Then, 100 μL of goat anti-human IgG-FITC (FITC-conjugated goat anti-human IgG, Ivitrogen) diluted 1:200 was added, reacted at room temperature for 30 minutes, and washed twice again with FACS buffer. After that, antibody binding was confirmed using a NovoCyte Flow Cytometer (Agilent).

[0263] As a result, it was confirmed that anti-Tie2 antibodies (MT-100, MT-101 (IgG1), MT-101 (IgG4), MT-101 (IgG1-LALA)) bind to Tie2 proteins derived not only from humans but also from monkeys, mice, rats, dogs, or pigs (Figs. 5a to 5d).

[0264] Example 6. Confirmation of the vascular endothelial cell activation effect of anti-Tie2 antibody.

[0265] Example 6.1. Analysis of the effects of anti-Tie2 antibodies on angiogenesis and network structure stabilization.

[0266] We analyzed whether anti-Tie2 antibodies induce tube formation and network stabilization of vascular endothelial cells on Matrigel.

[0267] Specifically, HUVECs (1x10) were serum-starved by culturing for 6 h in M199 medium containing 1% serum. 5 Cells) were seeded onto a 24-well culture dish coated with GFR (Growth factor reduced)-Matrigel. The HUVECs were treated with MT-100 at a concentration of 10 μg / mL and the control substance, Ang-1 (R&D systems, 923-AN), at a concentration of 500 ng / mL, and cultured for 18 hours to determine the degree of capillary-like tube formation.

[0268] As a result, it was confirmed that the vascular network in the MT-100 treatment group was increased and maintained for a long period of time, similar to the positive control group, Ang-1 treatment group (Fig. 6).

[0269] Example 6.2. Analysis of the inhibitory effect of anti-Tie2 antibodies on vascular endothelial cell damage.

[0270] The inhibitory effect of anti-Tie2 antibody on vascular endothelial cell damage was confirmed through analysis of apoptosis of vascular endothelial cells induced when cultured under serum-free and hypoxic conditions.

[0271] Specifically, HUVECs (5x10 4 Cells) were seeded in 24-well plates and cultured at 37°C for 48 hours using EGM-2 medium. The next day, HUVECs were treated with anti-Tie2 antibody (MT-101 (IgG1)) in serum-free M199 medium at the concentrations shown in Figure 7a (0.01 nM, 0.1 nM, 1 nM, 10 nM, or 100 nM) and cultured for 40 hours under hypoxic (less than 1% oxygen) culture conditions at 37°C. The cells cultured using the above method were fixed and stained with Crystal violet reagent for 10 minutes. After washing four times with distilled water, the stained reagent was eluted with distilled water containing 1% SDS, and the absorbance value (OD 570 nm) was measured.

[0272] In addition, anti-Tie2 antibodies (MT-101 (IgG1), MT-101 (IgG4), and MT-101 (IgG1-LALA)) were treated at the concentrations (0.5 nM or 5 nM) shown in Figure 7b in the same manner as above, and the absorbance value (OD 570 nm) was measured.

[0273] As a result, it was confirmed that the MT-101 (IgG1) treatment group inhibited apoptosis in a concentration-dependent manner compared to the control group (Fig. 7a). In addition, it was confirmed that MT-101 (IgG4) and MT-101 (IgG1-LALA) also had the same apoptosis inhibition efficacy as MT-101 (IgG1) (Fig. 7b).

[0274] Example 6.3. Analysis of the effects of anti-Tie2 antibodies on vascular endothelial permeability (leakage) and inflammation inhibition.

[0275] The inhibitory effect on increased vascular endothelial permeability (so-called leakage phenomenon) due to abnormality of blood vessels was confirmed using cell immunochemical staining.

[0276] Specifically, HUVECs (1x10) were seeded in the upper chamber of a 12-well Transwell plate (0.4 μm pore). 5 After seeding with HUVECs (cells), the cells were cultured for 3 days and then maintained in a serum-starved state for 6 hours. Afterwards, VEGF (R&D systems, 293-VE) was treated at 50 ng / mL to artificially increase vascular permeability, and MT-100 was treated at a concentration of 20 μg / mL for 5 hours. Afterwards, fluorescently labeled dextran (FITC-Dextran, 70 kDa) was added to the culture medium, and dextran permeated between HUVECs was compared and analyzed after 30 minutes using a fluorometer (SpectraMAx i3x).

[0277] As a result, it was confirmed that vascular permeability increased in cells treated with VEGF alone, whereas vascular permeability was completely inhibited in cells treated with VEGF and MT-100 (Fig. 8a).

[0278] Additionally, HUVECs (1x10 5Cells) were seeded on 35 mm confocal dishes (SPL) and cultured for 2 days, and then maintained in a serum-starved state for 6 hours. Then, VEGF (50 ng / mL) or VEGF (50 ng / mL) and MT-100 (10 μg / mL) were treated and reacted for 3 hours. After that, the cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized for 10 minutes using a phosphate-buffered saline solution containing 0.2% Triton X-100, and blocked with a phosphate-buffered saline solution containing 1% BSA for 1 hour at room temperature. Anti-VE-cadherin antibody (Santacruz, sc-9989) was diluted 1:50 as the primary antibody and reacted for 2 hours at room temperature. Then, the cells were treated with a fluorescently labeled secondary antibody (Alexa Fluor 488 donkey anti-goat, Invitrogen) diluted 1:200 and incubated at room temperature for 1 hour. Finally, the cells were treated with DAPI diluted 1:1,000, and the cell nuclei were stained for 3 minutes and observed under a fluorescence microscope.

[0279] As a result, it was confirmed that in cells treated with VEGF alone, cell-cell junctions were formed significantly weaker, but in cells treated with MT-100, cell-cell junctions were well maintained and the boundary surface was strongly developed (Fig. 8b).

[0280] In addition, the anti-Tie2 antibody's inhibitory effect on vascular endothelial inflammation was analyzed through the expression of proteins (Intercellular adhesion molecule-1 (ICAM-1), Vascular cell adhesion molecule-1 (VCAM-1)) that mediate the adhesion of vascular endothelial cells and inflammatory cells (leukocytes).

[0281] Specifically, HUVECs (5x10 5Cells) were seeded in a 60 mm culture dish and cultured for 24 hours, and then maintained in serum-free M199 medium for 6 hours to create a serum-starved state. Thereafter, VEGF alone at 20 ng / mL and / or MT-100 at a concentration of 10 μg / mL were treated and reacted for 6 hours. The cells were then lysed to obtain a cell lysate.

[0282] Western blot was performed using the cell lysate obtained as described above as a sample in the same manner as in Example 3. At this time, anti-ICAM-1 antibody (Santacruz, sc-8439) or anti-VCAM-1 antibody (Santacruz, sc-13160) was used, and anti-mouse IgG antibody (Abcam, ab97023) was used as a secondary antibody to confirm the expression of ICAM-1 and VCAM-1.

[0283] As a result, it was confirmed that the expression of ICAM-1 and VCAM-1 induced by VEGF treatment was reduced in the MT-100 treatment group (Fig. 8c).

[0284] Example 7. Confirmation of the inhibitory effect of anti-Tie2 antibody on Ang-2 binding to Tie2.

[0285] The inhibitory effect of anti-Tie2 antibody on the mutual binding of Tie2 and Ang-2 in pathological situations where Ang-2 is increased was confirmed in the same manner as in Example 2.

[0286] Specifically, the AR2G biosensor tip was hydrated with distilled water for 10 minutes and then activated using a buffer containing 20 mM EDC and 10 mM Sulfo-NHS. Then, 100 nM hTie2-ECD-6x His protein as an antigen was diluted in an immobilization buffer (10 mM sodium acetate, pH 6.0) and reacted with the activated AR2G tip for 10 minutes to immobilize the antigen on the tip.

[0287] Afterwards, the antigen immobilization reaction was terminated by treatment with 1 M ethanolamine (pH 8.5) solution. First, to measure the baseline of antigen-antibody binding, the AR2G tip was reacted in a phosphate-buffered saline (PBS) buffer solution for 60 seconds. Then, 200 nM MT-101 (IgG1) or PBS buffer solution was associated with the AR2G tip with the antigen immobilized thereon for 600 seconds, and then 40 nM recombinant human Ang-2 protein was associated and dissociated for 600 seconds, respectively. The association and dissociation reaction values ​​were analyzed by global fitting with the Data Analysis HT 12.0 program.

[0288] As a result, it was confirmed that Ang-2 did not bind to Tie2 when MT-101 (IgG1) was bound to Tie2 (Fig. 9a and Fig. 9b).

[0289] Example 8. Confirmation of the Tie2 activation-inducing effect of anti-Tie2 antibodies in the presence of Ang-2.

[0290] We analyzed whether anti-Tie2 antibodies could induce phosphorylation of Tie2 expressed in vascular endothelial cells in the presence of Ang-2.

[0291] Specifically, HUVECs (3x10 6 Cells) were seeded in 100 mm culture dishes and cultured for 24 hours, and then maintained in serum-free M199 medium for 6 hours to create a serum-starved state. Afterwards, Ang-2 (5 nM) was pretreated 20 minutes before treatment with anti-Tie2 antibody (MT-101 (1 nM)), and Ang-2 (5 nM) and MT-101 (1 nM) were treated alone or in combination for 20 minutes. After the reaction was completed, the cells were lysed with a lysis buffer to obtain a cell lysate.

[0292] Anti-Tie2 antibody (R&D systems, AF313) was added at a concentration of 1 μg / mL to the cell lysate obtained as above, and the reaction was performed at 4°C for 12 hours. 30 μL of protein G beads (Millipore, 16-266) were added to perform immunoprecipitation to obtain a sample. At this time, total protein (Whole cell lysate, WCL) was also obtained and used as a sample.

[0293] Western blot was performed using the sample prepared as described above in the same manner as in Example 3. At this time, the primary antibodies used were anti-phospho-tyrosine antibody (Sigma, 05-321), anti-Tie2 antibody, anti-phospho-AKT antibody, anti-AKT antibody, anti-phospho-ERK antibody, or anti-ERK antibody.

[0294] As a result, it was confirmed that MT-101 strongly induced phosphorylation of Tie2, AKT, or ERK proteins even in the presence of Ang-2 (Fig. 10).

[0295] Example 9. Confirmation of the inhibitory effect of anti-Tie2 antibodies on acute renal injury.

[0296] Example 9.1. Analysis of the effect of anti-Tie2 antibodies in an animal model of acute renal injury induced by ischemia and reperfusion.

[0297] The acute renal injury inhibitory efficacy of the anti-Tie2 antibody prepared in Example 1.4 above was confirmed using an acute renal injury animal model induced by ischemia and reperfusion.

[0298] Specifically, 100 μL of Control IgG (10 mg / kg) or anti-Tie2 antibodies (MT-101 (IgG1), MT-101 (IgG4), and MT-101 (IgG1-LALA)) were administered into the tail vein of 8-week-old C57BL / 6J mice in the normal group (Sham) or ischemia reperfusion (I / R) group at the concentrations (5 mg / kg or 10 mg / kg) shown in Fig. 11b and Fig. 11c. After 30 minutes of injection, a solution mixed with general anesthetics for animals, Ketamin (60 mg / kg) and Xylazine (6 mg / kg), was injected intraperitoneally (0.2 μL / g) to anesthetize the mice, and then an approximately 1.5 cm incision was made on the skin of the mouse abdomen. Through the incision site, a thin cotton swab soaked in PBS buffer and a microscopy were used to remove the surrounding kidneys. The right kidney was secured, avoiding the fat, and the artery and vein connected to the kidney were secured using microclamps. Ischemia was induced in the left kidney using the same method (Fig. 11a).

[0299] Additionally, the body temperature of the mice was maintained at 35°C to 36°C using a heating pad and a mouse rectal thermometer, and the microclamp was removed after 30 minutes to allow blood to be reperfused to the kidneys. The incised abdomen was then closed with sutures, and the external wound was closed with metal staples. The mice were observed and allowed to recover at a temperature of 22°C to 25°C. Twenty-four hours after surgery, blood was collected from the inferior vena cava of the anesthetized mice and centrifuged at 4°C to obtain serum. Serum creatinine (s-creatinine) and blood urea nitrogen (BUN) were analyzed using an automatic chemical analyzer (Hitachi 7180).

[0300] As a result, it was confirmed that in the Control IgG group, the concentrations of serum creatinine and BUN increased due to I / R, whereas in the anti-Tie2 antibody (MT-101 (IgG1), MT-101 (IgG4), or MT-101 (IgG1-LALA)) administration group, they significantly decreased (Fig. 11b and Fig. 11c).

[0301] Example 9.2. Analysis of the inhibitory effect of anti-Tie2 antibody on tubular damage

[0302] To confirm the inhibitory effect of anti-Tie2 antibody on acute renal injury, the degree of renal tubular damage was analyzed through histological analysis.

[0303] Specifically, the kidneys were removed from the experimental mice of Example 9.1, fixed in 10% formalin for 24 hours, and embedded in paraffin to produce blocks. The paraffin blocks were sectioned into 4 μm thick sections, attached and fixed on slides, to produce tissue section slides. Afterwards, the section slides were treated with xylene to remove paraffin, and stained with hematoxylin-eosin (Figure 12a) reagent to observe damage to the renal tubules, which were evaluated as follows using a damage score: 0: Normal, 1: 25% or less damage, 2: 25% to 50% damage, 3: 50% to 75% damage, 4: 75% or more damage.

[0304] As a result, it was confirmed that tubular damage was more evident in the acute renal injury-induced group (Control IgG) than in the normal group (Sham), whereas tubular damage was inhibited in a concentration-dependent manner in the anti-Tie2 antibody (MT-101 (IgG1), MT-101 (IgG4), or MT-101 (IgG1-LALA)) administration group (Fig. 12a and Fig. 12b).

[0305] Example 9.3. Analysis of the effects of anti-Tie2 antibody on inducing Tie2 activation and inhibiting vascular endothelial cell damage.

[0306] To confirm whether the acute renal injury inhibitory effect of the anti-Tie2 antibody confirmed in Example 9.1 above was induced by inhibition of renal vascular endothelial cell damage through Tie2 activation, Tie2 phosphorylation and immunostaining of renal vascular endothelial cells and perivascular endothelial cells were performed.

[0307] Specifically, the mouse kidney of Example 9.1 was extracted, fixed in 4% paraformaldehyde for 2 hours, dehydrated in a PBS buffer solution containing 15% sucrose at 4°C for 2 hours, and then in a PBS buffer solution containing 30% sucrose at 4°C for 24 hours. Then, a frozen block was prepared using an optical crystallization transducer (OCT). The frozen block was sectioned into 7 μm thick sections, fixed on slides with 4% paraformaldehyde for about 5 minutes, washed three times with a washing buffer (2% FBS + 0.1% Sodium Azide in PBS), and then blocked with a non-specific protein blocking buffer (1% BSA in PBS) for 1 hour.

[0308] The membranes were treated with primary antibodies (anti-CD31 antibody (Sigma, MAB1398Z, 1:200), anti-p-Tie2 antibody (R&D system, AF2720, 1:100), or anti-PDGFRβ (Invitrogen, 14-1402-82, 1:100)) and incubated at 4°C for 16 hours. After completion of the reaction, the membranes were washed three times with washing buffer, and then treated with secondary antibodies labeled with FITC or Alexa flour 594 (FITC-labeled anti-rabbit IgG, Invitrogen, A21206; Alexa flour 594-labeled anti-hamster IgG, Invitrogen, A21113; FITC-labeled anti-hamster IgG, Invitrogen, A21110; Alexa flour 594-labeled anti-rat IgG, Invitrogen, A21209, each 1:1,000). The cells were incubated at room temperature for 2 hours. After washing twice with washing buffer, the cells were treated with DAPI (4',6-diamidino-2-phenylindole; 1 μg / mL) and incubated at room temperature for 1 minute to stain the cell nuclei. After the reaction was completed, the cells were washed twice with washing buffer and the tissue sections were observed using a fluorescence microscope.

[0309] As a result, it was confirmed that phosphorylation of Tie2 protein was not induced in the acute renal injury-induced group (Control IgG), whereas phosphorylation of Tie2 protein was increased in the renal blood vessels in the MT-101 (IgG1-LALA)-administered group (Fig. 13a). In addition, it was confirmed that vascular endothelial cells and perivascular cells in the MT-101 (IgG1-LALA)-administered group were maintained similarly to the normal group (Sham) (Fig. 13b).

[0310] Example 9.4. Analysis of the anti-inflammatory efficacy of anti-Tie2 antibodies.

[0311] A major factor in acute renal injury caused by ischemia and reperfusion is an excessive inflammatory response. When ischemia and reperfusion increase reactive oxygen species (ROS), the expression of proteins (e.g., VCAM-1, vascular cell adhesion molecule-1) that mediate the adhesion of inflammatory cells, such as neutrophils, increases on the surface of renal vascular endothelial cells. This increases neutrophil adhesion to the vessel wall and influx into the tubular interstitium, triggering a persistent inflammatory response and exacerbating tubular damage.

[0312] In order to analyze whether the acute renal injury inhibitory effect of the anti-Tie2 antibody in Example 9.1 above was induced through inflammation inhibition, the expression of CD31, a vascular endothelial cell marker protein, VCAM-1, an inflammatory cell adhesion mediator protein, or Gr-1, a neutrophil marker, was analyzed through immunostaining.

[0313] Specifically, the kidney was extracted from the mouse of Example 9.1, and tissue staining was performed in the same manner as in Example 9.3. At this time, the primary antibody (anti-CD31 antibody (Sigma, MAB1398Z, 1:200), anti-VCAM-1 antibody (Santacruz, sc-13160, 1:100), or anti-Gr-1 antibody (Abcam, ab25377, 1:100)) was used, and the secondary antibody labeled with FITC or Alexa flour 594 (FITC-labeled anti-mouse IgG, Invitrogen, A21202; FITC-labeled anti-rat IgG, Invitrogen, A21208; Alexa flour 594-labeled anti-hamster IgG, Invitrogen, A21113, each 1:1,000) was used.

[0314] As a result, it was confirmed that the expression of VCAM-1 in renal vascular endothelial cells increased in the acute renal injury-induced group (Control IgG) compared to the normal group (Sham). On the other hand, in the MT-101 (IgG1-LALA) administration group, it was decreased similar to the normal group (Fig. 14a). At this time, it was confirmed that the influx of neutrophils into the tubulointerstitium was also reduced (Fig. 14b).

[0315] Example 9.5. Analysis of the inhibitory effect of anti-Tie2 antibodies on the expression of inflammatory response-inducing factors.

[0316] Changes in the expression of cytokines (CCL2, IL-6, or IL-1β) that induce an inflammatory response in mouse kidney tissue treated with anti-Tie2 antibody (MT-101 (IgG1-LALA)) were confirmed through real-time polymerase chain reaction (PCR) analysis.

[0317] Specifically, the mouse kidney of Example 9.1 was extracted, homogenized in 1 mL of TRIzol (Invitrogen) solution using a homogenizer, and left for 5 minutes to completely dissociate the nucleoprotein complex. 200 μL of chloroform was added, shaken for 15 seconds, and reacted at room temperature. The mixture was centrifuged at 14,000 rpm at 4°C for 15 minutes to separate the supernatant, and approximately 400 μL was transferred to a new tube, after which an equal volume of isopropanol was added to precipitate the RNA. The precipitate was centrifuged at 14,000 rpm at 4°C for 10 minutes, washed with 1 mL of 75% ethanol, and centrifuged for 5 minutes under the same conditions to collect the RNA pellet. After drying the pellet, 50 μL to 100 μL of RNase-free water was added and dissolved at 55°C for 10 minutes to prepare RNA.

[0318] The RNA prepared for cDNA synthesis was diluted to a concentration of 1 to 1.5 μg / μL, and then RNA (2 μg), oligo dT primer (1 μL), and RNase-free water were mixed to make a total volume of 10 μL, incubated at 70°C for 10 minutes, and cooled on ice for 5 minutes. Afterwards, the cDNA synthesis mixture was prepared, incubated at 42°C for 1 hour, and then treated at 70°C for 10 minutes to inactivate RTase. qPCR was performed using Solg 2X Real-Time PCR Smart mix (solgent) on a Bio-rad instrument (96-well PCR plate). The PCR reaction was performed at 95°C for 15 minutes for enzyme activation, followed by 35 cycles (95°C, 20 s; 58°C, 40 s; 72°C, 30 s), and finally, a PCR melting curve step was performed.

[0319] As a result, it was confirmed that the expression of inflammatory factors, CCL2 (CC motif chemokine ligand2), IL-6, and IL-1β, was inhibited in the MT-101 (IgG1-LALA) administration group compared to the Control IgG treatment group (Figure 14c).

[0320] Example 10. Confirmation of the inhibitory effect of anti-Tie2 antibodies on sepsis-induced acute kidney injury.

[0321] Example 10.1. Analysis of the effect of anti-Tie2 antibodies in an animal model of acute renal injury induced by lipopolysaccharide-induced sepsis.

[0322] The acute renal injury inhibitory efficacy of the anti-Tie2 antibody prepared in Example 1.4 above was also confirmed in a mouse model of acute renal injury induced by sepsis induced by lipopolysaccharide (LPS).

[0323] Specifically, 8-week-old C57BL / 6J mice were randomly divided into groups and given a single intraperitoneal injection of LPS (sigma, L3024) at a dose of 10 mg / kg. Control IgG or MT-101 (IgG1-LALA) was injected intravenously into the tail vein at a concentration of 10 mg / kg, 100 μL each, 1 hour before or 1 hour after LPS administration. After 24 hours, blood was collected from the inferior vena cava of the anesthetized mice and centrifuged at 4°C to obtain serum. Serum creatinine (s-creatinine) and blood urea nitrogen (BUN) were analyzed using an automatic chemical analyzer (Hitachi 7180) (Fig. 15a).

[0324] As a result, it was confirmed that the concentrations of s-creatinine and BUN increased in the control IgG administration group, but were significantly reduced in the group administered 1 hour before or after MT-101 (IgG1-LALA) (Fig. 15b and Fig. 15c).

[0325] Example 10.2. Analysis of the inhibitory effect of anti-Tie2 antibodies on renal vascular endothelial cell and tubular damage in an animal model of sepsis-induced acute renal injury.

[0326] In Example 10.1 above, the acute renal injury inhibitory efficacy of the anti-Ti2 antibody was analyzed through immunostaining of KIM-1, a tubular damage marker protein, and CD31, a vascular endothelial cell marker protein, in renal tissue.

[0327] Specifically, the mouse kidney of Example 10.1 was extracted and tissue staining was performed in the same manner as in Example 9.3. At this time, the primary antibody (anti-CD31 antibody (Sigma, MAB1398Z, 1:200) or anti-KIM-1 antibody (R&D system, MAB18171, 1:200)) was used, and the secondary antibody labeled with FITC or Alexa flour 594 (FITC-labeled anti-rabbit IgG (Invitrogen, A21206), Alexa flour 594-labeled anti-hamster IgG (Invitrogen, A21113), each 1:1,000) was used.

[0328] As a result, it was confirmed that damage to renal tubules and renal blood vessels was significantly reduced in the group administered 1 hour before or after MT-101 (IgG1-LALA) (Fig. 15d).

[0329] Example 11. Confirmation of the therapeutic effect of anti-Tie2 antibodies on chronic renal failure.

[0330] Example 11.1. Analysis of the effect of anti-Tie2 antibodies in an animal model of chronic renal failure induced by ischemia and reperfusion.

[0331] The therapeutic efficacy of the anti-Tie2 antibody prepared in Example 1.4 above for chronic renal failure was analyzed in an animal model of chronic renal failure induced by ischemia and reperfusion.

[0332] Specifically, 8-week-old C57BL / 6J mice were randomly divided, and a solution containing a mixture of ketamine (60 mg / kg) and xylazine (6 mg / kg), which are general anesthetics for animals, was injected intraperitoneally (0.2 μL / g) to anesthetize the mice. Then, an incision of approximately 1.5 cm was made on the skin of the abdomen of the mice. Through the incision site, the right kidney was secured using a thin cotton swab soaked in PBS buffer and a microscopist, avoiding the fat around the kidney, and the artery and vein connected to the kidney were fixed using a micro clamp. Ischemia was induced in the left kidney using the same method (Fig. 16a).

[0333] In addition, the body temperature of the mice was maintained at 35°C to 36°C using a heating pad and a mouse rectal thermometer, and after 27 minutes, the microclamp was removed to reperfuse blood to the kidney. Then, the incised abdomen was closed with suture, and the external wound was closed with a metal staple, and the mice were observed and recovered at a temperature of 22°C to 25°C. Thereafter, on days 2, 4, and 6, 100 μL of Control IgG or anti-Tie2 antibody (MT-101 (IgG-LALA)) was injected into the tail vein at a concentration of 5 mg / kg, and blood was collected from the inferior vena cava on days 3, 7, or 14 and centrifuged at 4°C to obtain serum. And on the 14th day, the mouse kidney was extracted, and tissue staining was performed using hematoxylin-eosin (H&E) reagent in the same manner as in Example 9.2 to observe damage to the renal tubules, and the damage score was evaluated as follows: 0: Normal, 1: 25% or less damage, 2: 25% to 50% damage, 3: 50% to 75% damage, 4: 75% or more damage.

[0334] As a result, it was confirmed that renal tubular damage was significantly observed in the chronic renal failure-induced group (Control IgG) compared to the normal group (Sham), whereas renal tubular damage was significantly suppressed in the MT-101 (IgG1-LALA) administration group (Fig. 16b).

[0335] Example 11.2. Analysis of the anti-fibrotic effect of anti-Tie2 antibodies in an animal model of chronic renal failure induced by ischemia and reperfusion.

[0336] The anti-renal fibrosis efficacy of MT-101 was confirmed through Masson's trichrome staining, sirus red staining, and immunostaining of collagen IV or α-SMA, which are fibrosis marker proteins.

[0337] Specifically, kidneys were removed from the 14-day mice in Example 11.1, and paraffin blocks were prepared using the same method as in Example 9.2. To determine the degree of fibrosis using Masson's trichrome method, kidney tissue embedded in paraffin was sectioned into 4 μm thick sections, attached to slides, and then deparaffinized and hydrated with distilled water. Bouin's solution was preheated to 60°C in a hood, and the slides were immersed in the solution for 60 minutes and cooled for 10 minutes. The slides were rinsed with tap water to completely clear the tissue, and then washed once with distilled water.

[0338] Weigert's Iron Hematoxylin Solution A and B were mixed in equal proportions and stained for 5 minutes, and the slides were washed in running water for 2 minutes. Afterwards, Biebrich Scarlet / Acid Fuchsin solution was applied to the slides, stained for 15 minutes, and washed with distilled water. After differentiation treatment with Phosphomolybdic / Phosphotungstic acid solution for 15 minutes or until collagen no longer appeared red, the slides were immersed in Aniline Blue solution for 5 to 10 minutes without washing, and dehydrated very quickly in two sequential 95% alcohol cycles. Finally, the slides were cleared with xylene and embedded in synthetic resin.

[0339] In addition, for confirmation of fibrosis using Sirius red, paraffin-embedded kidney tissue was sectioned into 4 μm thick sections and attached to slides. The sections were deparaffinized by immersing them in xylene three times for 3 minutes each, rehydrated three times in 100% ethanol for 2 minutes each, 95% to 50% ethanol for 2 minutes each, and deionized water for 5 minutes each. The slides were stained by immersing them in Sirius Red solution for 60 minutes, and then washed twice with an acetic acid solution. Afterwards, the slides were dehydrated by tapping them five times in 100% ethanol, cleared three times in xylene for 3 minutes each, and mounted using Permount.

[0340] In addition, to identify fibrosis marker proteins, collagen IV (abcam, ab6586) and α-SMA (abcam, ab7817), paraffin-embedded kidney tissues were sectioned into 4 μm thick sections and attached to slides. The sections were deparaffinized by immersing them in xylene twice for 5 minutes each, and rehydrated twice in 100% ethanol for 5 minutes each, 95% to 50% ethanol for 2 minutes, and deionized water for 5 minutes each. Antigen retrieval was performed at 95°C for 20 minutes in sodium citrate buffer (pH 6.0), followed by washing with 1x TBST buffer for 5 minutes. After 20 minutes of treatment in 0.1 M TBS buffer containing 0.2% Triton X-100 for cell membrane permeabilization, the sections were blocked for 20 minutes with a solution containing 0.1 M TBST, 2% BSA, 2% serum, and 10% Avidin D, and washed with 0.1 M TBST buffer. The primary antibody was diluted in 0.1 M TBST buffer containing 2% BSA, 2% serum, and 10% Biotin, and incubated overnight at 4°C in a wet chamber, followed by two washes with 1x TBS buffer for 10 minutes each. Afterwards, the sections were treated for 15 minutes in a 0.1 M TBS and 0.3% H2O2 solution, and washed twice.

[0341] Secondary antibodies were diluted in 0.1 M PBS buffer and incubated for 1 hour at room temperature. After the reaction, the sections were washed twice with 0.1 M TBS buffer. Vectastain Elite ABC reagent (Vector laboratories) was diluted and incubated for 1 hour at room temperature, washed twice with 0.1 M TBS buffer for 5 minutes each, and then treated with DAB solution and incubated for 5 minutes. Finally, the sections were washed twice with distilled water, counterstained with hematoxylin, and washed with distilled water for 10 minutes. Dehydration was performed in 70% ethanol for 1 minute, 95% ethanol for 2 minutes three times, and 100% ethanol for 2 minutes three times, and then treated with xylene for 3 minutes three times. Finally, the slides were confirmed using the diaminobenzidine (DAB) method using Permount.

[0342] As a result, the chronic renal failure-induced group (Control IgG) showed a marked progression of fibrosis compared to the normal group (Sham). In contrast, the MT-101 (IgG1-LALA) administration group showed a significant reduction in fibrosis progression and marker protein expression compared to the chronic renal failure-induced group (Fig. 16c).

[0343] Example 11.3. Analysis of the Effect of Anti-Tie2 Antibodies in an Animal Model of Chronic Renal Failure Induced by Adenine

[0344] The therapeutic efficacy of the anti-Tie2 antibody prepared in Example 1.4 above for chronic renal failure was also analyzed in an animal model of chronic renal failure induced by adenine intake.

[0345] Specifically, 8-week-old C57BL / 6J mice were randomly divided into 5 groups, and each group was orally administered adenine (Sigma, A5665) at a dose of 50 mg / kg daily for 15 days. At this time, 100 μL of Control IgG (10 mg / kg) or MT-101 (2 mg / kg, 5 mg / kg, or 10 mg / kg) was administered intravenously into the tail vein on days 3, 8, 13, and 18, respectively. On day 21 from the first adenine administration, the mice were sacrificed, and their serum was obtained in the same manner as in Example 9.1, and serum creatinine was measured. In addition, to confirm the inhibition of the expression of KIM-1 and NGAL (Neutrophil gelatinase-associated lipocalin), which are tubular damage factors, real-time polymerase reaction was performed in the same manner as in Example 9.5 (Fig. 17a).

[0346] As a result, the concentration of serum creatinine decreased in the MT-101 (IgG1-LALA) administration group (Fig. 17b), and it was confirmed that tubular damage was suppressed through gene expression analysis of KIM-1 or NGAL (Fig. 17c).

[0347] Additionally, the efficacy in suppressing renal and tubular damage was analyzed through histological analysis.

[0348] Specifically, after the kidneys were removed from the mice sacrificed on the 21st day, section slides were prepared using the same method as in Example 9.2, and damage to the renal tubules was observed through hematoxylin-eosin staining (Fig. 17d).

[0349] Basement membrane damage of glomeruli and tubules was confirmed using the following method.

[0350] The above kidney tissue section slides were deparaffinized, hydrated in distilled water, and immersed in periodic acid solution for 10 minutes. They were then washed four times in distilled water and immersed in Schiff's solution for 30 minutes. The section slides were then washed twice in distilled water, stained with hematoxylin for 1 minute, dehydrated in ethanol, and mounted in xylene (Fig. 17e). Vacuolization of the renal cortex, leukocyte infiltration in the tubules / proximal tubules, and proximal tubule simplification were evaluated as a tubular damage score using the following scoring system: 0: Normal, 1: 25% or less damage, 2: 25% to 50% damage, 3: 50% to 75% damage, and 4: 75% or more damage.

[0351] As a result, in the control IgG administration group, renal damage caused by adenine was clearly observed, but in the MT-101 (IgG1-LALA) administration group, it was confirmed that damage to the renal cortex and tubules was suppressed in a concentration-dependent manner (Fig. 17d and Fig. 17e).

[0352] Example 11.4. Analysis of the anti-fibrotic efficacy of anti-Tie2 antibodies in a model of chronic renal failure induced by adenine.

[0353] The anti-Tie2 antibody's inhibitory effect on renal fibrosis was confirmed through Sirus red staining and immunostaining for fibrosis marker proteins (Collagen IV, α-SMA). Specifically, kidneys from the experimental mice of Example 11.3 were removed, and Sirus red staining (Figure 18a) and immunostaining for fibrosis marker proteins, collagen IV (Figure 18b) or α-SMA (Figure 18c), were performed in the same manner as in Example 11.2.

[0354] As a result, it was confirmed that in the Control IgG administration group in which chronic renal failure was induced by adenine, fibrosis increased in the renal glomeruli and tubules compared to the normal group (sham), whereas fibrosis was reduced in the MT-101 (IgG1-LALA) administration group (Figs. 18a to 18c).

[0355] Additionally, the antifibrotic efficacy of MT-101 (IgG1-LALA) was analyzed using real-time polymerase chain reaction.

[0356] Specifically, kidneys were extracted from the experimental mice of Example 11.3, and the gene expression levels of Col1a1, Col3a1, ACTA2, and TGF-β within each group were measured through polymerase chain reaction in the same manner as in Example 9.5.

[0357] As a result, it was confirmed that the expression of fibrosis marker proteins was reduced in a concentration-dependent manner in the MT-101 (IgG1-LALA) administration group, similar to the results shown in the above immunostaining (Figs. 18a to 18c) (Fig. 18d).

[0358] Example 11.5. Analysis of the Inhibitory and Stabilizing Effects of Anti-Tie2 Antibodies on Vascular Damage in a Model of Chronic Renal Failure Induced by Adenine

[0359] To confirm the efficacy of anti-Tie2 antibody in suppressing chronic renal failure, immunostaining of LTL, a tubular marker protein, and CD31, a vascular endothelial cell marker protein, was performed in renal tissue.

[0360] Specifically, the kidney was removed from the experimental mouse of Example 11.3 and fixed in 4% paraformaldehyde for 2 hours. Then, it was immersed in a PBS buffer solution containing 15% sucrose and reacted at 4°C for 2 hours, then immersed in a PBS buffer solution containing 30% sucrose and immersed at 4°C for 24 hours to dehydrate. Then, a frozen block was prepared using an optically transient chromatography (OCT) embedding agent. The frozen block was sectioned into 7 μm thick sections, fixed on slides with 4% paraformaldehyde for about 5 minutes, washed three times with a washing buffer (2% FBS + 0.1% sodium azide in PBS), and then blocked with a non-specific protein blocking buffer (1% BSA in PBS) for 1 hour.

[0361] Afterwards, the sections were treated with primary antibodies (anti-LTL antibody (Verot laboratories, RL-1321-2, 1:400), anti-CD31 antibody (Sigma, MAB1398Z, 1:200)) and reacted at 4°C for 16 hours. After the reaction was completed, the sections were washed three times with washing buffer, and then treated with anti-LTL antibody (1:1,000) or Alexa Flour594-labeled secondary antibodies (Alexa Flour594-labeled anti-hamster IgG (Invitrogen, A21113), 1:1,000) and reacted at room temperature for 2 hours. Afterwards, the sections were washed twice again with washing buffer, and then treated with DAPI (1 μg / mL) and reacted for 1 minute at room temperature to stain cell nuclei. After the reaction was completed, the tissue sections were observed using a fluorescence microscope after washing twice with washing buffer.

[0362] As a result, it was confirmed that damage to renal tubules and renal blood vessels was suppressed in the MT-101 (IgG1-LALA) administration group (Fig. 19a).

[0363] Example 11.6. Analysis of the anti-inflammatory efficacy of anti-Tie2 antibodies.

[0364] Chronic renal failure and fibrosis are aggravated by persistent inflammatory responses within the kidney. Therefore, real-time polymerase chain reaction (PCR) was performed to determine whether the expression of inflammatory factors was suppressed in the MT-101 (IgG1-LALA) administration group.

[0365] Specifically, the kidney was extracted from the experimental mouse of Example 11.3, and the gene expression levels of inflammatory inducers MCP-1 (Monocyte chemoattractant protein-1), TNF-α (Tumor necrosis factor-α), and IL-1β; and immune cell markers CD4, CD11c, F4 / 80, and CD206 were measured through polymerase chain reaction in the same manner as Example 9.5.

[0366] As a result, compared to the control IgG administration group, it was confirmed that the expression of inflammatory factors and immune cell markers was significantly reduced in the MT-101 (IgG1-LALA) administration group (Figure 19b).

[0367] Example 11.7. Analysis of the therapeutic efficacy of anti-Tie2 antibodies in an animal model of renal injury induced by unilateral ureteral obstruction.

[0368] The anti-Tie2 antibody prepared in Example 1.4 above was additionally confirmed to have an inhibitory effect on renal damage in a unilateral ureteral obstruction (UUO) mouse model.

[0369] Specifically, 8-week-old C57BL / 6J mice were randomly divided, and the mice were anesthetized by intraperitoneal injection (0.2 μL / g) of a solution containing ketamine (60 mg / kg) and xylazine (6 mg / kg), which are general anesthetics for animals. Under the anesthesia, the skin of the mouse abdomen was incised approximately 1.5 cm, and the left ureter was exposed through a lateral incision using a thin cotton swab and microscopy soaked in PBS buffer, and ligated using two sutures at the level of the inferior pole of the kidney. On days 2, 4, and 6 after surgery, 100 μL of Control IgG (5 mg / kg) or MT-101 (5 mg / kg) was intravenously injected. On day 7 after the surgery, the mice were sacrificed, and blood and urine were collected, and kidney tissues were removed (Fig. 20a).

[0370] The extracted kidney tissue was stained with hematoxylin-eosin reagent in the same manner as in Example 9.2, and then the damage to the renal tubules was observed and the damage score was evaluated (0: normal group, 1: damage of 25% or less, 2: damage of 25% to 50%, 3: damage of 50% to 75%, 4: damage of 75% or more).

[0371] As a result, it was confirmed that tubular damage was more evident in the Control IgG administration group than in the normal group (Sham), whereas tubular damage was reduced in the MT-101 (IgG1-LALA) administration group (Fig. 20b).

[0372] Example 11.8. Analysis of the anti-fibrotic efficacy of anti-Tie2 antibodies in an animal model of renal injury induced by unilateral ureteral obstruction.

[0373] The inhibitory efficacy of MT-101 on renal fibrosis was confirmed in an animal model of renal injury induced by unilateral ureteral obstruction (UUO) through immunohistochemistry. Specifically, Masson's trichrome staining (Fig. 20c) and immunohistochemistry for collagen IV (Fig. 20d) or α-SMA (Fig. 20e) were performed on renal tissues extracted from the experimental mice of Example 11.7 using the same method as in Example 11.2.

[0374] As a result, it was confirmed that in the Control IgG administration group, fibrosis increased compared to the normal group (sham), and the expression of collagen IV and α-SMA increased, whereas in the MT-101 (IgG1-LALA) administration group, it decreased compared to the Control IgG administration group (Figures 20c to 20e).

[0375] Example 12. Confirmation of the sepsis treatment effect of anti-Tie2 antibodies.

[0376] The sepsis treatment efficacy of the anti-Tie2 antibody prepared in Example 1.4 above was confirmed in a lipopolysaccharide (LPS)-induced sepsis mouse model.

[0377] Specifically, 8-week-old C57BL / 6J mice were randomly divided and given a single intraperitoneal injection of LPS (sigma, L3024) at a dose of 10 mg / kg. 100 μL of Control IgG (10 mg / kg) or MT-101 (IgG1-LALA) (5 mg / kg or 10 mg / kg) was injected into the tail vein 1 hour before or 1 hour after LPS administration, and the survival of the mice was analyzed for up to 168 hours (7 days) (Fig. 21a).

[0378] As a result, in the group administered Control IgG 1 hour after LPS administration, the mortality rate increased over time, and the mouse survival rate decreased to 43.75% on the 7th day (168 hours) after inducing sepsis with LPS. On the other hand, in the group administered MT-101 (IgG1-LALA) (10 mg / kg) 1 hour before LPS injection, all mice survived. In addition, in the groups administered 5 mg / kg or 10 mg / kg of MT-101 (IgG1-LALA) 1 hour after LPS injection, the mouse survival rate increased to 93.75% or 100%, respectively (Fig. 21b).

[0379] Example 13. Confirmation of the therapeutic effect of anti-Tie2 antibodies on critical limb ischemia.

[0380] Example 13.1. Analysis of therapeutic effects in an animal model of lower extremity ischemia

[0381] The therapeutic efficacy of the anti-Tie2 antibody prepared in Example 1.4 above was confirmed using an animal model of ischemic stroke.

[0382] Specifically, 6-week-old male BALB / CA-nu / nu mice (average weight: 20 g to 24 g) were anesthetized using an inhalation anesthesia system, and the legs of the mice were fixed with tape. Using forceps and scissors, the skin was incised approximately 1 cm from the knee to the mid-thigh, and a retractor was used to secure the surgical site. Through the surgical site, the proximal and distal parts between the femoral and popliteal arteries were each tied with two sutures under a dissecting microscope, occluded, and then resected to block blood flow to the lower extremities.

[0383] Afterwards, HBSS solution, Control IgG (5 mg / kg), or anti-Tie2 antibody (MT-100 or MT-101) (1 mg / kg or 5 mg / kg) were injected into the peri-ischemic tissue of the lower limbs, respectively, and then the blood flow of the lower limbs of the mice was measured using Laser Doppler perfusion imaging (LDPI, Moor Instruments Ltd). Blood flow was measured using Laser Doppler images on the day of surgery (day 0), days 7, 14, 21, and 28. Perfusion of the ischemic and non-ischemic lower limbs was calculated based on the pixel types generated in the color histogram, where red and blue represent high and low perfusion levels, respectively. Blood perfusion is expressed as the LDPI index, which represents the ratio of blood flow in the ischemic and non-ischemic limbs, where a preoperative ratio of 100 indicates equal blood perfusion in both paws. The extent of ischemic hindlimb necrosis was measured 28 days after surgery, and the necrosis score was evaluated as follows: 0, limb recall; 1, toe amputation; 2, foot amputation; 3, limb amputation.

[0384] As a result, it was confirmed that blood flow was significantly improved in the lower extremities of mice administered MT-100 or MT-101 compared to the lower extremities of the HBSS or Control IgG (5 mg / kg) administration groups (Figs. 22a and 22b). In addition, it was confirmed that necrosis was suppressed in the MT-101 treatment group (Fig. 22c).

[0385] Example 13.2. Confirmation of functional angiogenesis induction in the lower extremity ischemic area.

[0386] In order to confirm whether the effect of improving lower extremity ischemia in Example 13.1 above was achieved by inducing functional angiogenesis in the area where lower extremity ischemia occurred, the expression of CD31, a vascular endothelial cell marker protein, and α-SMA, a perivascular cell marker protein, was confirmed through immunostaining.

[0387] Specifically, after ischemia-induced lower extremity muscles were excised from the experimental mice of Example 13.1, section slides were prepared in the same manner as in Example 9.3. At this time, the section slides were manufactured to a thickness of 7 μm. The section slides were fixed by treating them with 4% paraformaldehyde and reacting them for about 5 minutes. After that, they were washed three times with washing buffer (2% FBS + 0.1% sodium azide in PBS), and then blocked with non-specific protein blocking buffer (5% BSA in PBS) for 1 hour. The tissue section slides prepared as above were subjected to immunostaining in the same manner as in Example 9.3.

[0388] At this time, the primary antibody used was anti-CD31 antibody (Sigma, MAB1398Z, 1:200) or anti-α-SMA antibody (Abcam, ab5694, 1:200). After treating the section slides with the primary antibody, they were reacted at 4℃ for 16 hours, washed three times with washing buffer, and then treated with the secondary antibody diluted 1:1,000 and reacted at room temperature for 2 hours. At this time, the secondary antibody used was Cy3-labeled anti-rabbit IgG antibody (Jackson ImmunoResearch, 128-165-160) or TRITC-labeled anti-hamster IgG antibody (Jackson ImmunoResearch, 101-025-165). Capillary (CD31) density and α-SMA cell counts were assessed for each mouse using three randomly selected microscopic fields from three consecutive sections of each tissue block.

[0389] As a result, it was confirmed that angiogenesis increased in the lower extremities of mice administered MT-100 or MT-101 compared to the lower extremities of the HBSS solution or Control IgG (5 mg / kg) administration groups. At this time, it was confirmed that the newly formed blood vessels were well surrounded by cells surrounding the blood vessels (Fig. 22d).

[0390] Example 14. Confirmation of the therapeutic effect of anti-Tie2 antibodies on erectile dysfunction.

[0391] Example 14.1. Analysis of therapeutic effects in an animal model of diabetic erectile dysfunction.

[0392] The erectile dysfunction treatment efficacy of the anti-Tie2 antibody prepared in Example 1.4 above was confirmed using a streptozotocin (STZ)-induced diabetic erectile dysfunction animal model.

[0393] Specifically, 8-week-old male C57BL / 6J mice (average weight: 20–25 g) were intraperitoneally injected with STZ (0.1 M sodium citrate buffer, pH 4.5, Sigma-Aldrich) for 5 days. After 8 weeks of administration, fasting and postprandial blood glucose levels were measured using an Accu-Chek blood glucose meter (Roche Diagnostics), and mean systolic blood pressure (MSBP) was measured using a noninvasive tail cuff system (Visitech system). To confirm the erectile function improvement effect of anti-Tie2 antibodies (MT-100, MT-101), 20 μL of PBS, Control IgG (10 μg), MT-100 (1 μg or 10 μg), or MT-101 (1 μg or 10 μg) was each administered to the intracavernous corpus callosum of STZ-induced diabetic mice using a 30-gauge insulin syringe.

[0394] Two weeks later, to measure erectile function, the left superficial part of the lower abdomen of each mouse was opened, and the penile cavernosal nerve (penile nerve) located on the posterolateral side of the prostate was prepared so that it could be clearly seen. For electrical stimulation of the penile nerve, a platinum electrode was placed on the penile nerve, and electrical stimulation was applied for about 1 minute at an intensity of 5 V and 12 Hz to induce penile erection. At this time, the intracavernosal pressure (ICP) during erection was measured using a pressure transmitter (Biospec system) connected to a computer through a catheter inserted into the corpora cavernosa.

[0395] The ICP on the vertical axis in Fig. 23a represents the pressure inside the penis during erection, and is generally an indicator of erectile power. The horizontal axis represents the time after electrical stimulation, and the 1-minute electrical stimulation period is indicated by a black bar on the horizontal axis. In addition, Fig. 23b shows the value obtained by dividing the maximum intracavernosal pressure (Maximal ICP) by the mean systolic blood pressure (MSBP). Fig. 23c shows the value obtained by dividing the area of ​​the intracavernosal pressure curve (Total ICP (area under the curve)) by the mean systolic blood pressure (MSBP). At this time, blood pressure is a value checked to measure erectile power because it can affect the intracavernosal pressure.

[0396] As a result, the group administered MT-100 (10 μg) or MT-101 (1 μg or 10 μg) showed a higher erectile function improvement effect compared to the PBS or Control IgG treatment group, and it was confirmed that the erectile function was restored to about 90% or more of the level of the non-diabetic group (Normal). In particular, the peak intracavernosal pressure and the area of ​​the penile pressure curve related to penile erection were improved to normal levels in the anti-Tie2 antibody (MT-100 and MT-101) administration group (Figs. 23a to 23c).

[0397] Example 14.2. Analysis of the efficacy of inducing functional angiogenesis and stabilization in the corpora cavernosa.

[0398] The penis is composed of spongy tissue, which is responsible for erectile function, and small blood vessels that supply nutrients to it. However, under pathological conditions such as diabetes, hyperlipidemia, or nerve damage, these blood vessels can lose their function and become unstable, leading to erectile dysfunction.

[0399] In order to confirm whether the erectile improvement effect of the anti-Tie2 antibody of Example 14.1 above was induced through functional angiogenesis and stabilization in the corpus cavernosum, the degree of blood vessels in the corpus cavernosum was confirmed through immunostaining for the expression of CD31, a vascular endothelial cell marker protein, and NG2, a perivascular cell marker protein.

[0400] Specifically, the dorsal penile nerve and corpus cavernosum were isolated from the penile tissue of each mouse, and then each tissue was fixed in 4% paraformaldehyde at 4°C for 24 hours. The fixed tissue was fixed using a cryoembedding agent, and then sectioned into 7 μm thick sections using a cryostat to prepare tissue section slides. Next, the prepared tissue sections were fixed on slides with 4% paraformaldehyde for approximately 5 minutes, and then washed three times with washing buffer (2% FBS + 0.1% sodium azide in PBS). The tissue section slides were treated with nonspecific protein blocking buffer (5% BSA in PBS) and blocked for 1 hour.

[0401] Afterwards, the primary antibody was treated with anti-CD31 antibody (Sigma, MAB1398Z, 1:200) or NG2 antibody (Sigma, ZRB5320, 1:100) and reacted at 4℃ for 16 hours. After the reaction was completed, the cells were washed three times with washing buffer, and then treated with FITC- or TRITC-labeled secondary antibodies (FITC-labeled anti-hamster IgG (Invitrogen, A21110), Alexa Fluor 594-labeled anti-rabbit IgG (Invitrogen, A21207), each 1:1,000) and reacted at room temperature for 2 hours. Afterwards, the cells were washed twice again with washing buffer, and then treated with DAPI (4',6-diamidino-2-phenylindole; 2 μg / mL) and reacted for 5 minutes at room temperature to stain the cell nuclei. After the reaction was completed, the tissue sections were washed twice with washing buffer and then observed using a fluorescence microscope.

[0402] As a result, among the group of mice induced with diabetes (DM) by streptozotocin (STZ), it was confirmed that the blood vessel distribution and coverage of perivascular cells in the corpus cavernosum of the group administered PBS or Control IgG (10 μg) were reduced compared to the normal group, whereas in the group administered MT-100 (10 μg) or MT-101 (1 μg or 10 μg), the blood vessel distribution and coverage of perivascular cells were restored to similar levels as in the normal group (Figs. 24a to 24c).

[0403] Example 14.3. Analysis of the efficacy of inducing angiogenesis and stabilizing network structure under hyperglycemic conditions.

[0404] Angiogenesis is induced by the proliferation, migration, and differentiation of endothelial cells into tubular structures, ultimately maintaining a network. However, exposure to various conditions (such as hypoxia or hyperglycemia) can lead to endothelial cell abnormalities, inhibiting their differentiation into lumen and disrupting cell-cell junctions, which are crucial for network formation.

[0405] To verify the functional angiogenesis-inducing effect of the anti-Tie2 antibody confirmed in Example 14.2 above, vascular endothelial cells were exposed to high blood glucose, and then the tube formation-inducing and stabilizing effects of MT-100 or MT-101 were analyzed.

[0406] Specifically, HUVECs were treated with 5 mM glucose, which is similar to normal blood glucose levels, and 30 mM glucose, which corresponds to high blood glucose levels, and cultured for 48 hours. At this time, PBS, Control IgG (5 μg / mL), or anti-Tie2 antibody (MT-100 (1 μg / mL, 10 μg / mL) or MT-101 (0.1 μg / mL, 1 μg / mL, 5 μg / mL)) were treated together with the glucose. After that, each HUVEC (2x10 5 Cells) were inoculated and cultured in a 37°C, 5% CO2 incubator for 16 hours, while observing under a microscope.

[0407] As a result, the degree of blood vessel formation was significantly reduced in the high-concentration glucose (HG) treatment group (HG-PBS or HG-Control IgG) compared to the normal-concentration glucose (NG) treatment group. On the other hand, the high-concentration glucose (HG) and MT-100 (10 μg / mL) or MT-101 (1 μg / mL or 5 μg / mL) co-treatment group showed a significant increase in blood vessel formation compared to the normal-concentration glucose (NG) treatment group, and it was confirmed that the network was well maintained (Fig. 25a and Fig. 25b).

[0408] Example 15. Confirmation of the therapeutic effect of anti-Tie2 antibodies on retinal diseases.

[0409] Example 15.1. Analysis of therapeutic effects in an animal model of choroidal neovascularization induced by laser irradiation.

[0410] The therapeutic efficacy of the anti-Tie2 antibody prepared in Example 1.4 for age-related macular degeneration (AMD) was confirmed using a laser-induced neovascularization (CNV) mouse model.

[0411] Specifically, 7-week-old C57BL6 mice were locally anesthetized with 0.5% proparacaine hydrochloride (Alcon). The pupils were dilated with 1% tropicamide (Santen pharmaceutical), and Bruch's membrane was ruptured in four regions (3, 6, 9, and 12 o'clock positions of the posterior pole) of each eye using laser photocoagulation (Wavelength 532 nm, Diameter 50 μm, Duration 80 mS, Power level 200 mW) using a laser photocoagulator. When the laser is irradiated to the retinal surface, retinal tissue burns occur, resulting in the formation of bubbles on the retinal surface. Mice in which no bubbles were observed were classified and excluded.

[0412] On the third day after laser photocoagulation, 1 μL each of Control IgG (10 μg), MT-100 (10 μg), MT-101 (10 μg), or the control drug EYLEA (10 μg) was injected intravitreally into the mouse. On the seventh day after laser photocoagulation, indocyanine green angiography (ICGA) was performed to observe abnormal choroidal neovascularization. At the same time, fluorescein angiography (FA) was also performed to analyze the leakage of choroidal neovascularization. On the eighth day after laser photocoagulation, each mouse eye was enucleated, fixed in 4% paraformaldehyde at room temperature for 1 hour, and then washed three times with phosphate-buffered saline (PBS) buffer (Fig. 26a).

[0413] Afterwards, a radial incision was made through the cornea using a surgical scalpel under a dissecting microscope, and the sclera was carefully peeled toward the optic nerve starting from the incised area using forceps, and the lens was finally removed. After cutting the retina into a four-leaf clover shape, tissue slides were prepared by permeabilization in PBST (0.5% Triton X-100 in PBS) buffer at 4°C for 16 hours, and immunohistochemical staining was performed.

[0414] The permeabilized retinas were washed three times with PBS buffer and then blocked in PBST-BSA (0.5% Triton X-100 + 3% BSA in PBS) solution at 4°C for 16 hours. Anti-endomucin antibody (Santacruz, SC-65495, 1:200) was used as the primary antibody and reacted at 4°C for 16 hours. Afterwards, the retinas were washed four times with PBST buffer at 20-minute intervals, and the secondary antibody (Cy3-labeled anti-antibody 1:100) was used and reacted at room temperature for 1 hour. The tissue slides were washed four times with PBST buffer at 20-minute intervals, treated with hydrosoluble mounting medium, and observed using a confocal microscope.

[0415] The yellow dotted line area in Figure 27a represents the mass of CNV, and the white dotted line area represents the area of ​​CNV. The volume of CNV can be obtained using the following [Mathematical Formula 1].

[0416] [Mathematical Formula 1]

[0417] CNV volume = CNV mass size x z-Stack height (z-Scan 1 μm) x 2 / 3 (Hemisphere coefficient)

[0418] As a result, the CNV area and choroidal blood vessel leakage were significantly reduced in the MT-100 or MT-101 administration group. In particular, it was confirmed that the reduction effect was similar to the effect in the control drug EYLEA administration group (Figs. 26b and 26c). In addition, the results of endomucin expression in the tissues confirmed that the MT-100 or MT-101 administration group reduced the area, mass, or volume of CNV (Figs. 27a to 27d).

[0419] Example 15.2. Analysis of therapeutic effects in an animal model of retinopathy induced by oxygen conditions.

[0420] The therapeutic efficacy of the anti-Tie2 antibody prepared in Example 1.4 was verified in ischemic retinal diseases including retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO) using an oxygen-induced retinopathy (OIR) mouse model.

[0421] Specifically, C57BL / 6J mice on the 7th day after birth were exposed to a hyperbaric oxygen chamber maintained at 75% oxygen concentration for 5 days. On the 12th day, the mice were removed from the hyperbaric oxygen chamber, and 1 μL of Control IgG (10 μg), MT-101 (10 μg), or the control drug EYLEA (10 μg) was injected into the vitreous cavity of the mice, and the mice were maintained under normal atmospheric conditions (21% oxygen concentration) for 5 days (Fig. 28a). On the 17th day, the eyes were removed from the mice, fixed in 4% paraformaldehyde at room temperature for 1 hour, and then washed three times with PBS buffer. The retinas were removed from the ocular tissues in the same manner as in Example 15.1, and tissue slides were prepared and immunohistochemical staining was performed. At this time, anti-CD31 antibody was used as the primary antibody. The tissue stained using the above method was observed using a confocal microscope.

[0422] As a result, it was confirmed that the avascular area (Fig. 28b) and neovascular tuft area (Fig. 28c) in the retina were significantly reduced in the MT-101 (10 μg) administration group compared to the control drug EYLEA administration group.

[0423] Example 15.3. Analysis of the efficacy of inhibiting retinal vascular leakage.

[0424] In retinal diseases associated with angiogenesis, abnormalities in retinal blood vessels increase their permeability, leading to leakage into surrounding tissues. Therefore, the inhibitory effect of the anti-Tie2 antibody according to the present invention on retinal blood vessel leakage was confirmed using Evans Blue staining in the mouse model of Example 15.2.

[0425] Specifically, Control IgG or MT-101 antibody was injected intraocularly into mice using the same method as in Example 15.2. On the 17th day, Evans Blue dye was administered into the femoral vein under a microscope, and the eyes were extracted 2 hours later and fixed in 4% paraformaldehyde for 1 hour. The fixed tissue was treated with a cryo-embedding agent (OCT) to produce a frozen block. The frozen block was sectioned to a thickness of 10 μm to produce tissue section slides, and the tissue section slides were fixed by treating the tissue with 4% paraformaldehyde for approximately 5 minutes. The sensitivity of Evans Blue dye leaking around the retinal blood vessels was analyzed by observing the tissue section slides using a laser scanning confocal microscope.

[0426] As a result, it was confirmed that leakage around the retinal blood vessels was significantly reduced in the MT-101 administration group compared to the Control IgG administration group (Fig. 29a and Fig. 29b).

[0427] II. Bispecific antibodies

[0428] Example 16. Production of a bispecific antibody for Tie2 activation and VEGF signaling pathway inhibition.

[0429] A bispecific antibody with a triple function of activating vascular endothelial cell-specific Tie2 and its downstream signaling factors, while inhibiting the binding of Ang-2 to Tie2 and the signaling pathway of VEGF or VEGFR2 was created.

[0430] Example 16.1. Structure design of a bispecific antibody

[0431] To inhibit Tie2 activation, Ang-2 binding, and VEGF signaling pathway, a fusion protein or bispecific antibody construct was designed using the MT-101 antibody as a backbone and introducing five VEGF signaling pathway inhibitors (SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46) into the C-terminal portion of the Fc region (Fig. 30).

[0432] Specifically, MT-103-1 fusion protein (SEQ ID NO: 42) in which Aflibercept VEGFR1-D2 and VEGFR2-D3 domains were introduced, MT-103-2 bispecific antibody (SEQ ID NO: 43) in which Bevacizumab heavy chain variable region and light chain variable region were introduced in scFv form, MT-103-3 bispecific antibody (SEQ ID NO: 44) in which Ranibizumab heavy chain variable region and light chain variable region were introduced in scFv form, MT-103-4 bispecific antibody (SEQ ID NO: 45) in which Brolucizumab light chain variable region and heavy chain variable region were introduced in scFv form, and MT-103-5 (SEQ ID NO: 46) in which Ramucirumab heavy chain variable region and light chain variable region were introduced in scFv form were designed.

[0433] At this time, the amino acid sequences of each of the five VEGF signal pathway inhibitors were reverse translated and then codon optimized for expression in Expi293F cells, and then the coding genes were synthesized. The synthesized five VEGF signal pathway inhibitor genes were cloned into the C-terminal region of the pcDNA3.4 (Invitrogen)-based MT-101 heavy chain expression vector and introduced in a form connected to MT-101 IgG with a linker containing the amino acid sequence of SEQ ID NO: 49 or SEQ ID NO: 50. As a control bispecific antibody, the heavy chain variable region (SEQ ID NO: 40) of p2.3 was cloned into the heavy chain variable region (SEQ ID NO: 27) of MT-101-1 to construct a heavy chain expression vector.

[0434] Table 12 shows the CDR amino acid sequences of the bispecific antibodies, and Tables 13 to 18 show the amino acid sequences of the heavy chains of MT-103-1, MT-103-2, MT-103-3, MT-103-4, and MT-103-5, the amino acid sequence of the common MT-103 light chain, and the amino acid sequence of the heavy chain of the control p2.3-1. The light chain of the control p2.3-1 fusion protein is described in SEQ ID NO: 41. Transient expression of the fusion protein or bispecific antibody was performed using the constructed heavy and light chain expression vectors, respectively.

[0435] 항체서열 정보서열서열번호MT-103-1MT-103-1-HCDR1GFTFNSYG1MT-103-1-HCDR2TSNDGSTT2MT-103-1-HCDR3ARKVVRGYHYHDAFDI19MT-103-1-LCDR1QSVSSY4MT-103-1-LCDR2GAS5MT-103-1-LCDR3QQYGTTPYT6MT-103-2MT-103-2-HCDR1GFTFNSYG1MT-103-2-HCDR2TSNDGSTT2MT-103-2-HCDR3ARKVVRGYHYHDAFDI19MT-103-2-LCDR1QSVSSY4MT-103-2-LCDR2GAS5MT-103-2-LCDR3QQYGTTPYT6MT-103-3MT-103-3-HCDR1GFTFNSYG1MT-103-3-HCDR2TSNDGSTT2MT-103-3-HCDR3ARKVVRGYHYHDAFDI19MT-103-3-LCDR1QSVSSY4MT-103-3-LCDR2GAS5MT-103-3-LCDR3QQYGTTPYT6MT-103-4MT-103-4-HCDR1GFTFNSYG1MT-103-4-HCDR2TSNDGSTT2MT-103-4-HCDR3ARKVVRGYHYHDAFDI19MT-103-4-LCDR1QSVSSY4MT-103-4-LCDR2GAS5MT-103-4-LCDR3QQYGTTPYT6MT-103-5MT-103-5-HCDR1GFTFNSYG1MT-103-5-HCDR2TSNDGSTT2MT-103-5-HCDR3ARKVVRGYHYHDAFDI19MT-103-5-LCDR1QSVSSY4MT-103-5-LCDR2GAS5MT-103-5-LCDR3QQYGTTPYT6

[0436] MT-103-1 Amino acid sequence SEQ ID NO. MT-103-1 HCMT-101 HC signal peptide MGWSCIILFLVATATGVHS51VH1EVQLVESGGGVVQPGRSLRLSCAASGFTFNSYGMQWVRQAPGKGLEWVAVTSNDGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARKVVRGYHYHDAFDIWGQGTMVTVSS27CH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV521 LinkerEPKSCDKTHTCPPCP53Fc(CH2+CH3)APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG63VEGF Specific binding receptor fragment second linkerGGGGSGGGGSGGGGS49D2 DomainSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIID67D3 DomainVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK68MT-103-1 LCsignalpeptideMGWSCIILFLVATATGVHS51VL1DIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYRQKPGQAPRLLIYGASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVFYCQQYGTTPYTFGQGTKVEIK22CLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC82

[0437] MT-103-2 Amino acid sequence SEQ ID NO. MT-103-2 HCMT-101 HC signal peptide MGWSCIILFLVATATGVHS51VH1EVQLVESGGGVVQPGRSLRLSCAASGFTFNSYGMQWVRQAPGKGLEWVAVTSNDGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARKVVRGYHYHDAFDIWGQGTMVTVSS27CH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV521 LinkerEPKSCDKTHTCPPCP53Fc(CH2+CH3)APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG63VEGF Specific binding antibody fragment 2 LinkerGGGGSGGGGSGGGGSKL50VH2EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSS69Part 3 LinkerSGGGGSGGGGSGGGGS70VL2DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK71MT-103-2 LCsignalpeptideMGWSCIILFLVATATGVHS51VL1DIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYRQKPGQAPRLLIYGASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVFYCQQYGTTPYTFGQGTKVEIK22CLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC82

[0438] MT-103-3 amino acid sequence SEQ ID NO. MT-103-3 HCMT-101 HC signal peptide MGWSCIILFLVATATGVHS51VH1EVQLVESGGGVVQPGRSLRLSCAASGFTFNSYGMQWVRQAPGKGLEWVAVTSNDGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARKVVRGYHYHDAFDIWGQGTMVTVSS27CH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV521 LinkerEPKSCDKTHTCPPCP53Fc(CH2+CH3)APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG63VEGF Specific binding antibody fragment 2 LinkerGGGGSGGGGSGGGGSKL50VH2EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSS72Part 3 LinkerGGGGSGGGGSGGGGS49VL2DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK73MT-103-3 LCsignalpeptideMGWSCIILFLVATATGVHS51VL1DIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYRQKPGQAPRLLIYGASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVFYCQQYGTTPYTFGQGTKVEIK22CLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC82

[0439] MT-103-4 amino acid sequence SEQ ID NO. MT-103-4 HCMT-101 HC signal peptide MGWSCIILFLVATATGVHS51VH1EVQLVESGGGVVQPGRSLRLSCAASGFTFNSYGMQWVRQAPGKGLEWVAVTSNDGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARKVVRGYHYHDAFDIWGQGTMVTVSS27CH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV521 LinkerEPKSCDKTHTCPPCP53Fc(CH2+CH3)APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG63VEGF Specific binding antibody fragment 2 LinkerGGGGSGGGGSGGGGS49VH2EVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGDHNSGWGLDIWGQGTLVTVSS74Part 3 LinkerGGGGGSGGGGSGGGGSGGGGS75VL2EIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFLTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTVL76MT-103-4 LCsignalpeptideMGWSCIILFLVATATGVHS51VL1DIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYRQKPGQAPRLLIYGASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVFYCQQYGTTPYTFGQGTKVEIK22CLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC82

[0440] MT-103-5 amino acid sequence SEQ ID NO. MT-103-5 HCMT-101 HC signal peptide MGWSCIILFLVATATGVHS51VH1EVQLVESGGGVVQPGRSLRLSCAASGFTFNSYGMQWVRQAPGKGLEWVAVTSNDGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARKVVRGYHYHDAFDIWGQGTMVTVSS27CH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV521 LinkerEPKSCDKTHTCPPCP53Fc(CH2+CH3)APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG63VEGF Specific binding antibody fragment 2 LinkerGGGGSGGGGSGGGGSKL50VH2EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDIWGQGTMVTVSS77Part 3 LinkerGGGGSGGGGSGGGGS49VL2DIQMTQSPSSVSASIGDRVTITCRASQGIDNWLGWYQQKPGKAPKLLIYDASNLDTGVPSRFSGSGSGTYFTLTISSLQAEDFAVYFCQQAKAFPPTFGGGTKVDIK78MT-103-5 LCsignalpeptideMGWSCIILFLVATATGVHS51VL1DIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYRQKPGQAPRLLIYGASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVFYCQQYGTTPYTFGQGTKVEIK22CLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC82

[0441]

[0442] Hereinafter, a bispecific fusion protein containing a VEGF receptor is also described as a bispecific antibody.

[0443] 16.2. Expression and purification of bispecific antibodies

[0444] The constructed MT-103-1, MT-103-2, MT-103-3, MT-103-4, or MT-103-5 heavy chain expression vectors were introduced into Expi293F or ExpiCHO cells in the same manner as in Example 1.4 to transiently express and purify the antibodies. The concentration of the finally purified bispecific antibodies in PBS buffer was measured by absorbance or BCA analysis, and then analyzed by SDS-PAGE analysis under non-reducing and reducing conditions.

[0445] As a result, it was confirmed that MT-103-1 had a heavy chain of approximately 80 kDa, a light chain of approximately 25 kDa, and a total molecular weight of approximately 210 kDa. In addition, it was confirmed that MT-103-2, MT-103-3, MT-103-4, or MT-103-5 had a heavy chain of approximately 75 kDa, a light chain of approximately 25 kDa, and a total molecular weight of approximately 200 kDa, respectively.

[0446] Example 16.3. Confirmation of target affinity of a bispecific antibody

[0447] The target affinity of the dual specific antibody (MT-103-1 or MT-103-4) was analyzed using the BLI technique in the same manner as in Example 2.

[0448] Specifically, for affinity measurement for Tie2, the AR2G biosensor tip was hydrated with distilled water for 10 minutes and then activated using a buffer containing 20 mM EDC and 10 mM Sulfo-NHS. Then, 100 nM hTie2-ECD-6xHis protein was diluted in immobilization buffer (10 mM sodium acetate, pH 6.0) and reacted with the activated AR2G tip for 10 minutes to immobilize the antigen on the AR2G tip. Afterwards, the antigen immobilization reaction was terminated by treatment with 1 M ethanolamine (pH 8.5) solution, and the AR2G tip was reacted in a PBS buffer solution for 60 seconds to obtain a baseline of antigen-antibody binding.

[0449] After that, 60 nM MT-103-1 or MT-103-4 was bound and dissociated from the AR2G tip immobilized with hTie2-ECD-6xHis antigen for 800 s, respectively. For affinity measurement for VEGF, 200 nM MT-103-1 or MT-103-4 was immobilized on the AR2G tip as described above, and then 60 nM recombinant human VEGF (hVEGF) protein was bound and dissociated from the tip immobilized with bispecific antibodies for 800 s, respectively. Based on the response curve confirmed through the analysis of binding and dissociation reactions, the K on , K off , and K D The values ​​were calculated and the affinity for each antigen of MT-103-1 or MT-103-4 was measured, and the results are shown in Table 1.

[0450] As a result, it was confirmed that MT-103-1 and MT-103-4 antibodies showed high affinity for hTie2 and hVEGF (Table 19).

[0451] Antigen-antibody K D (M)K on (1 / Ms)Koff (1 / s) Human Tie2 MT-103-17.92E-111.92E051.52E-05 MT-103-45.58E-101.49E058.32E-05 Human VEGF MT-103-1<1.00E-121.78E05<1.00E-07 MT-103-4<1.00E-121.02E05<1.00E-07

[0452] Example 17. Confirmation of activation of bispecific antibodies

[0453] The activity of the bispecific antibody was confirmed through analysis of phosphorylation-inducing activity of Tie2 and its downstream signaling factors in vascular endothelial cells, apoptosis and vascular endothelial permeability inhibition activity, VEGF-VEGFR2 signaling pathway inhibition activity, and Ang-2 binding inhibition activity to Tie2.

[0454] Example 17.1. Confirmation of the VEGF signaling pathway inhibition efficacy of a bispecific antibody.

[0455] The VEGF signal transduction pathway inhibitory efficacy of the bispecific antibody prepared by the method of Example 16.2 above was analyzed by comparing the degree of phosphorylation of VEGFR2 and its downstream signal transduction factors by VEGF in vascular endothelial cells with that of the control drug (Aflibercept: EYLEA, Bevacizumab: Avastin or Brolucizumab: Beovu).

[0456] Specifically, HUVECs (5x10 5Cells) were cultured in 60 mm culture dishes at 37°C using EGM-2 medium. HUVECs cultured as described above were cultured in M199 medium containing 1% serum for 6 hours to achieve serum starvation. Subsequently, bispecific antibodies (MT-103-1, MT-103-2, MT-103-3, MT-103-4) or control drugs (EYLEA, Avastin, or Beovu) and VEGF (at different concentrations as shown in Figures 31a to 31e) were mixed and reacted for 20 minutes. The mixture was then treated to HUVECs and reacted for 15 minutes.

[0457] Afterwards, cells were lysed using a lysis buffer to extract proteins, and SDS-PAGE was performed on the proteins. Then, the cells were transferred to a PVDF membrane, and the membrane was blocked with a 0.1% Tween-20 TBST buffer solution diluted with 3% BSA for 2 hours at room temperature. Afterwards, the membrane was treated with the primary antibody diluted 1:1,000 and reacted for 2 hours at room temperature. At this time, the primary antibodies used were anti-phospho-VEGFR2 antibody (Cell signaling, 3770), anti-phospho-AKT antibody, and anti-phospho-ERK antibody. Afterwards, the secondary antibody (peroxidase-conjugated) was diluted 1:3,000 and treated with the membrane, and reacted for 1 hour at room temperature. After the reaction was completed, the membrane was treated with ECL solution to confirm the degree of phosphorylation of the VEGF signaling pathway factor.

[0458] As a result, MT-103-1 or MT-103-4 significantly reduced the phosphorylation of VEGFR2 to a level comparable to that of the respective control drugs, EYLEA or Beovu (Figs. 31a and 31d). Phosphorylation of VEGFR2 was also reduced in a concentration-dependent manner in the MT-103-2, MT-103-3, or MT-103-5 treatment groups (Figs. 31b, 31c, and 31e). In addition, phosphorylation of AKT or ERK was significantly increased in the bispecific antibody treatment group, which was confirmed to be increased by the bispecific antibody activating the Tie2 signaling pathway (Figs. 31a to 31e).

[0459] Example 17.2. Confirmation of Tie2 signaling pathway activation by bispecific antibodies

[0460] A comparative analysis was conducted to determine whether the bispecific antibody prepared by the method of Example 16.2 above induces phosphorylation of Tie2 and its downstream signaling factors in vascular endothelial cells. The experiment was performed in the same manner as in Example 3 above.

[0461] Specifically, HUVECs (3x10 6 Cells) were cultured in a 100 mm culture dish using EGM-2 medium at 37°C. The HUVECs cultured as described above were cultured in M199 medium containing 1% serum for 6 hours to achieve serum starvation. Subsequently, anti-Tie2 antibody (MT-101) or bispecific antibody (MT-103-1 or MT-103-4) was treated at the concentrations (0.5 nM, 1 nM, 5 nM, or 10 nM) shown in Figure 32a and reacted for 20 minutes. Thereafter, the cells were lysed with a lysis buffer to obtain a cell lysate. Using the cell lysate as a sample, immunoprecipitation and Western blotting were performed in the same manner as in Example 3.

[0462] As a result, the bispecific antibody MT-103-1 induced phosphorylation of Tie2, AKT, or ERK proteins in the same manner as the single antibody MT-101 (Fig. 32a). In addition, MT-103-4 exhibited phosphorylation-inducing activity at a level similar to that of MT-103-1 (Fig. 32b).

[0463] Example 17.3. Confirmation of the efficacy of a dual-specific antibody in inhibiting vascular endothelial cell damage and permeability.

[0464] The vascular endothelial cell activation efficacy of the bispecific antibody prepared in Example 16.2 above was confirmed through analysis of vascular endothelial cell apoptosis induced when cultured under serum-free and hypoxic conditions and vascular endothelial cell permeability induced by VEGF.

[0465] Specifically, HUVECs (5x10 4 Cells) were cultured in 24-well plates using EGM-2 medium at 37°C for 48 hours. The following day, HUVECs were treated with anti-Tie2 antibody (MT-101) and bispecific antibody (MT-103-1 or MT-103-4) in serum-free M199 medium at the concentrations (1 nM or 10 nM) shown in Figure 24a, and cultured for 40 hours under hypoxic (less than 1% oxygen) culture conditions at 37°C. The surviving cells were then fixed and stained with Crystal violet reagent for 10 minutes. The cells were washed approximately four times with distilled water, and the stained reagent was eluted with distilled water containing 1% SDS, and the absorbance value was measured.

[0466] As a result, it was confirmed that the MT-103-1 or MT-103-4 treatment group inhibited cell death to an equal or greater extent than the MT-101 treatment group (Fig. 33a).

[0467] HREC cells (1x10 5Cells) were seeded on 35 mm Confocal dishes (SPL) and cultured for 2 days, and then maintained in a serum-starved state for 4 hours. Then, VEGF (1 nM), Control IgG (2 nM), or bispecific antibodies (MT-103-1 or MT-103-4) were treated together at various concentrations (0.125 nM, 0.5 nM, 2 nM, 8 nM, 32 nM) and reacted for 30 minutes. Afterwards, the cells were fixed with 4% paraformaldehyde for 10 minutes, and the cell membrane was permeabilized for 30 minutes using a PBS solution containing 0.2% Triton X-100. After that, the cells were blocked for 1 hour at room temperature using a PBS solution containing 3% BSA. After treatment with anti-VE-cadherin antibody (R&D, 1:100) and incubation for 2 hours at room temperature, the cells were treated with a fluorescently labeled secondary antibody (Alexa Fluor 488 donkey anti-goat, Invitrogen) diluted 1:200 and incubated for 1 hour at room temperature. The cells were treated with DAPI diluted 1:1,000 to stain the cell nuclei for 3 minutes and then observed under a fluorescence microscope.

[0468] As a result, it was confirmed that the groups treated with VEGF and Control IgG formed significantly weaker cell-to-cell junctions. On the other hand, in the groups treated with MT-103-1 or MT-103-4 together with VEGF, it was confirmed that cell-to-cell junctions were well maintained and the interfaces were strongly developed (Fig. 33b). Furthermore, it was confirmed that this aspect increased as the treatment concentration increased for both types of dual-specific antibodies (Fig. 33c).

[0469] Additionally, HREC cells (1x10 5Cells) were seeded in 12-transwells and cultured at 37°C for 72 hours using EGM-2 medium, and then cultured at 37°C for 48 hours using EGM-2 medium without VEGF. The cells were treated with a 40 kDa FITC-dextran solution at a concentration of 200 ng / mL and reacted at 37°C for 30 minutes, and the initial fluorescence leakage was measured. VEGF and Ang-2, and control IgG (5 nM) or bispecific antibodies (MT-103-1 or MT-103-4) were treated at various concentrations (0.05 nM, 0.5 nM, 5 nM). At the completion of the treatment, the cells were treated with a 40 kDa FITC-dextran solution at a concentration of 200 ng / mL and reacted at 37°C for 30 minutes, and the fluorescence leakage after 24 hours was measured, and the change compared to the initial measurement value was calculated.

[0470] As a result, it was confirmed that the relative fluorescence leakage was reduced in all groups treated with MT-103-1 or MT-103-4 compared to the VEGF and Ang-2, and Control IgG treatment groups. At this time, the group treated with 5 nM of the dual-specific antibodies VEGF and Ang-2 all showed the highest inhibitory efficacy. In particular, it was confirmed that the inhibitory efficacy of MT-103-4 was superior to that of MT-103-1 (Fig. 33d).

[0471] Example 18. Confirmation of the triple efficacy of a bispecific antibody.

[0472] In pathological situations where both VEGF and Ang-2 are increased, the triple efficacy (Tie2 binding, inhibition of Ang-2 binding to Tie2, and VEGF binding) of MT-103-1 or MT-103-4 was confirmed based on the BLI technique described in Example 2 above.

[0473] Specifically, the AR2G biosensor tip was hydrated with distilled water for 10 min, activated using a buffer containing 20 mM EDC and 10 mM Sulfo-NHS, and then immobilized on the tip by reacting with 200 nM MT-103-1, MT-103-4, or p2.3-1 control bispecific antibodies for 10 min. After antibody immobilization, 100 nM hTie2-ECD-6xHis protein was bound and dissociated for 600 s to analyze Tie2 binding ability, and 40 nM recombinant Ang-2 protein was again bound and dissociated for 600 s to analyze Ang-2 binding inhibition ability. In the final step, to evaluate VEGF binding ability, the tip was treated with 60 nM hVEGF protein and bound and dissociated for 600 s, respectively. The above binding and dissociation reactions were analyzed by global fitting in the Data Analysis HT 12.0 program.

[0474] As a result, MT-103-1 and MT-103-4 bound to Tie2 and inhibited the binding of Ang-2 to Tie2. They also bound to hVEGF. The control p2.3-1 double antibody bound to Tie2 and hVEGF but did not inhibit the binding of Ang-2 to Tie2 (Fig. 34).

[0475] Example 19. Confirmation of the efficacy of a bispecific antibody in activating the Tie2 signaling pathway and inhibiting the VEGF signaling pathway.

[0476] We confirmed that the bispecific antibody activates Tie2 expressed in vascular endothelial cells while inhibiting VEGF under conditions where Ang-1, Ang-2, or VEGF is expressed.

[0477] Specifically, HUVECs (3x10 6Cells) were seeded in 100 mm culture dishes and cultured for 24 hours, and then maintained in serum-free M199 medium for 6 hours. Then, MT-103-1 (1 nM or 5 nM) or MT-103-4 (1 nM or 5 nM) was added to the medium containing Ang-2 (5 nM) and VEGF (1 nM) or Ang-1 (5 nM), Ang-2 (5 nM) and VEGF (1 nM) and reacted for 10 minutes. After treating the cells for 15 minutes each under the same conditions as in Fig. 35a, the cells were lysed with a lysis buffer to obtain a cell lysate.

[0478] The cell lysate obtained as described above was treated with anti-Tie2 antibody (R&D) at a concentration of 1 μg / mL and reacted at 4°C for 12 hours, and then 30 μL of protein G beads were added to perform immunoprecipitation. Western blotting was performed using the sample prepared as described above in the same manner as in Example 3. At this time, the primary antibody was used by diluting anti-phospho-tyrosine antibody, anti-phospho-VEGFR2 antibody, anti-phospho-AKT antibody, or anti-phospho-ERK antibody at a ratio of 1:1,000.

[0479] As a result, it was confirmed that MT-103-1 or MT-103-4 strongly induced phosphorylation of Tie2, AKT, or ERK proteins in the presence of Ang-1, Ang-2, and VEGF, while inhibiting phosphorylation of VEGFR2 (Fig. 35b).

[0480] Example 20. Comparison of Tie2 activation and VEGF inhibition efficacy of bispecific antibodies and control drugs.

[0481] The efficacy of the bispecific antibody prepared in Example 16.2 above and the bispecific antibody (Faricimab: Vabysmo) that simultaneously inhibits Ang-2 and VEGF, which is currently used as a therapeutic agent, was compared.

[0482] Specifically, HUVECs (3x10 6Cells) were seeded in a 60 mm culture dish and cultured for 24 hours, and then maintained in serum-free M199 medium for 6 hours. Then, the cells were treated with MT-103-1, MT-103-4, or Vabysmo at various concentrations (0.5 nM, 1 nM, 2 nM, 5 nM, or 10 nM) for 15 minutes, and then the cells were lysed with lysis buffer and Western blotting was performed in the same manner as in Example 17.1.

[0483] As a result, the control drug Vabysmo did not induce phosphorylation of Tie2 downstream signaling pathway factors (AKT and ERK). In contrast, MT-103-1 or MT-103-4 induced phosphorylation of Tie2 downstream signaling pathway factors (AKT and ERK). In addition, it was confirmed that MT-103-1 showed an approximately 8-fold higher inhibitory effect on VEGFR2 phosphorylation compared to Vabysmo, and MT-103-4 showed an approximately 20-fold lower inhibitory effect (Fig. 36a).

[0484] In addition, as a result of analyzing the apoptosis inhibition efficacy using the same method as in Example 17.3, it was confirmed that MT-103-1 or MT-103-4 effectively reduced apoptosis of HUVECs induced when cultured in serum-free and hypoxic conditions. In contrast, no apoptotic effect was observed by Vabysmo (Fig. 36b).

[0485] Example 21. Confirmation of the therapeutic effect of a dual-specific antibody on retinal disease.

[0486] Example 21.1. Analysis of the inhibitory effect of choroidal neovascularization in an animal model of choroidal neovascularization induced by laser irradiation.

[0487] The therapeutic efficacy for age-related macular degeneration (AMD) was confirmed using a laser-induced neovascularization (CNV) mouse model. In this mouse model, VE-cad-creERT, which expresses tdTomato fluorescent markers in vascular endothelial cells by tamoxifen, was used.2 -tdTomato mice (Max Plank Institute and Jackson Laboratories) were used to quantitatively analyze neovascularization.

[0488] Specifically, 7-week-old VE-cad-creERT 2 -tdTomato After local anesthesia with 0.5% proparacaine hydrochloride (Alcon), the pupils were dilated with 1% tropicamide (Santen pharmaceutical), and Bruch's membrane was ruptured in four regions (3, 6, 9, and 12 o'clock positions of the posterior pole) of each eye using a laser photocoagulator (Wavelength 532 nm, Diameter 50 μm, Duration 80 mS, Power level 200 mW). When the laser is irradiated to the retinal surface, retinal tissue is burned, resulting in the formation of bubbles on the retinal surface. Mice in which no bubbles were observed were classified and excluded.

[0489] On the third day after laser photocoagulation, 1 μL each of Control IgG (20 μg), MT-101 (20 μg), MT-103-1 (20 μg), MT-103-4 (20 μg), or the control drug EYLEA (10 or 20 μg) was injected into the vitreous cavity of mice. On the seventh day after laser photocoagulation, indocyanine green angiography (ICGA) was performed to observe abnormal choroidal neovascularization. At the same time, fluorescein angiography (FA) was performed to analyze the leakage of choroidal neovascularization (Fig. 37a).

[0490] Eight days after laser photocoagulation, each mouse eye was enucleated, fixed in 4% paraformaldehyde at room temperature for 1 hour, and then washed three times with PBS buffer. Then, a radial incision was made through the cornea using a surgical scalpel under a dissecting microscope. Starting from the incision site, the sclera was carefully peeled toward the optic nerve with forceps, and the lens was finally removed. After cutting the retina into a four-leaf clover shape, some tissue was collected for Western blot analysis. The remaining tissue was permeabilized in PBST (0.5% Triton X-100 in PBS) at 4°C for 16 hours to prepare tissue slides. The permeabilized retina was washed three times with PBS buffer, treated with a hydrosoluble mounting medium, and observed using a confocal microscope.

[0491] As a result, the leakage of choroidal blood vessels was significantly reduced in the anti-Tie2 antibody (MT-101) or dual-specific antibody (MT-103-1 or MT-103-4) administration groups. In particular, the reduction effect was confirmed to be superior to that in the control drug EYLEA two-concentration administration group (Fig. 37b). In addition, it was confirmed that the CNV vascular area expressing the dtTomato fluorescent label in the tissue was significantly reduced in the dual-specific antibody administration group (MT-103-1 or MT-103-4) compared to the control drug EYELA administration group (Fig. 37c).

[0492] Example 21.2. Analysis of the inhibitory effect on pre-formed choroidal neovascularization in an animal model of choroidal neovascularization induced by laser irradiation.

[0493] The inhibitory effect on pre-formed choroidal neovascularization was confirmed using a laser-induced neovascularization (CNV) mouse model using the same method as in Example 21.1. In this mouse model, VE-cad-creERT, which expresses tdTomato fluorescent markers in vascular endothelial cells by tamoxifen, was also confirmed. 2- New blood vessels were quantitatively analyzed using tdTomato mice.

[0494] In this experiment, indocyanine green angiography (ICGA) was performed 7 days after laser photocoagulation to observe abnormal choroidal neovascularization. Simultaneously, fluorescein angiography (FA) was performed to analyze the leakage of choroidal neovascularization. Then, 1 μL each of control IgG (20 μg), MT-103-1 antibody (20 μg), MT-103-4 antibody (20 μg), or the control drug EYLEA (10 or 20 μg) was injected into the vitreous cavity of mice. ICGA and FA were performed 14 days after laser photocoagulation, and the degree of inhibition of leakage caused by unstable neovascularization after laser photocoagulation and the efficacy in reducing the area of ​​pre-formed choroidal neovascularization were analyzed by comparing the data obtained 7 days after photocoagulation.

[0495] As a result, choroidal vascular leakage and choroidal neovascularization were significantly reduced in the dual-specific antibody (MT-103-1 or MT-103-4) administration group. In particular, the reduction effect was confirmed to be equivalent to or greater than that of the control drug EYLEA (two concentrations) administration group (Fig. 38b). In addition, it was confirmed that the CNV vascular area expressing the dtTomato fluorescent marker in the tissue was significantly reduced in the dual-specific antibody administration group (MT-103-1 or MT-103-4) compared to the control drug EYELA administration group (Fig. 38c).

[0496] Example 21.3. Analysis of therapeutic effects in an animal model of retinopathy induced by oxygen conditions.

[0497] The therapeutic efficacy of bispecific antibodies (MT-103-1 or MT-103-4) in ischemic retinal diseases including retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO) was analyzed using a hyperoxia-induced retinopathy (OIR) mouse model.

[0498] Specifically, C57BL / 6J mice on the 7th day after birth were exposed to hyperoxia by breeding them in a hyperbaric oxygen chamber where the oxygen concentration was maintained at 75% for 5 days. On the 12th day, the mice were removed from the hyperbaric oxygen chamber, and 1 μL each of Control IgG (20 μg), MT-103-1 antibody (20 μg), MT-103-4 antibody (20 μg), or the control drug EYLEA (10 μg or 20 μg) was injected into the vitreous cavity of the mice, and the mice were maintained under normal atmospheric conditions (21% oxygen concentration) for 5 days. On the 17th day, the eyes were extracted from the mice, fixed in 4% paraformaldehyde at room temperature for 1 hour, and then washed three times with PBS buffer. The retinas were extracted from the ocular tissues in the same manner as in Example 15.1, and tissue slides were prepared and immunostaining was performed. At this time, the primary antibody used was an anti-CD31 antibody. The tissue stained using the above method was observed using a confocal microscope (Figure 39a).

[0499] As a result, it was analyzed that the area of ​​intraretinal ischemic neovascularization was significantly reduced in the MT-103-1 or MT-103-4 administration group compared to the control drug EYELA administration group. In addition, it was confirmed that the area of ​​pathological neovascular plexus was also significantly reduced in the MT-103-1 administration group compared to the EYLEA administration group (Figures 39b and 39c).

Claims

1. (a) a heavy chain variable region (VH) comprising i) a heavy chain complementarity determining region 1 (HCDR1) represented by the amino acid sequence of SEQ ID NO: 84; ii) a heavy chain complementarity determining region 2 (HCDR2) represented by the amino acid sequence of SEQ ID NO: 85; and iii) a heavy chain complementarity determining region 3 (HCDR3) represented by any one amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 15, and SEQ ID NO: 86; and (b) an antibody or fragment thereof that specifically binds to Tie2, comprising a light chain variable region (VL) comprising: (b) a light chain complementary determining region 1 (LCDR1) represented by the amino acid sequence of SEQ ID NO: 87; (v) a light chain complementary determining region 2 (LCDR2) represented by the amino acid sequence of SEQ ID NO: 88; and (vi) a light chain complementary determining region 3 (LCDR3) represented by the amino acid sequence of SEQ ID NO: 89; X1X2X3FX4X5X6X7 (SEQ ID NO: 84) where X1 is G or S, X2 is F or D, X3 is T or S, X4 is N, T or A, X5 is S or G, X6 is Y, N or Q, and X7 is G, W or A; X8X9X 10 DX 11 X 12 X 13 X 14 (Sequence number 85) X8 is T or I, X9 is S or Y, and X 10 is N, P or W, and X 11 is G, D or S, and X 12 is S or G, and X 13 is T, D or G, and X 14 is T or I; ARKVVRGYX 15 X 16 HDAFDI (SEQ ID NO: 86) X 15 is S, H or L, and X 16 is Y or P; QX 17 X 18 X 19 SX 20 (Sequence number 87) X 17 is S, G or D, and X 18 is V or I, and X 19 is S or D, and X 20 is Y or N; X 21 X 22 S (SEQ ID NO: 88) X 21 is G or A, and X 22 is A or S; QQX 23 X 24 X 25 X 26 PX 27 T (SEQ ID NO: 89) X 23 is Y, A or G, and X 24 is G, N or Y, and X 25 is T, S or D, and X 26 is T or F, and X 27 is Y, L or W.

2. In paragraph 1, The antibody or fragment thereof comprises a heavy chain variable region comprising an HCDR1 comprising an amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising an amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising an amino acid sequence of SEQ ID NO: 86; and An antibody or fragment thereof that specifically binds to Tie2, comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6: ARKVVRGYX 15 X 16 HDAFDI (SEQ ID NO: 86) Here, X 15 is S, H or L, and X 16 is Y or P.

3. In paragraph 1, The antibody comprises a heavy chain variable region comprising an HCDR1 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 13, an HCDR2 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 14, and an HCDR3 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, and SEQ ID NO: 20; and An antibody or fragment thereof that specifically binds to Tie2, comprising a light chain variable region comprising LCDR1 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 10, and SEQ ID NO: 16, LCDR2 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 11, and SEQ ID NO: 17, and LCDR3 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 12, and SEQ ID NO:

18.

4. In paragraph 1, The antibody comprises a heavy chain variable region comprising an HCDR1 comprising an amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising an amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising an LCDR1 comprising an amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising an amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising an amino acid sequence of SEQ ID NO: 6; A heavy chain variable region comprising an HCDR1 comprising an amino acid sequence of SEQ ID NO: 7, an HCDR2 comprising an amino acid sequence of SEQ ID NO: 8, and an HCDR3 comprising an amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising an LCDR1 comprising an amino acid sequence of SEQ ID NO: 10, an LCDR2 comprising an amino acid sequence of SEQ ID NO: 11, and an LCDR3 comprising an amino acid sequence of SEQ ID NO: 12; A heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 15, and a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 18; A heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 19, and a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; or An antibody or fragment thereof that specifically binds to Tie2, comprising a heavy chain variable region comprising an HCDR1 comprising an amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising an amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising an amino acid sequence of SEQ ID NO: 20, and a light chain variable region comprising an LCDR1 comprising an amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising an amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising an amino acid sequence of SEQ ID NO:

6.

5. In paragraph 4, The antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 21 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 22; A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24; A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 26; A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22; or An antibody or fragment thereof that specifically binds to Tie2, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 28 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

22.

6. The anti-Tie2 antibody or fragment thereof of paragraph 1; and a bispecific antibody comprising a VEGF-specific binding site.

7. In paragraph 6, A bispecific antibody, wherein the VEGF-specific binding site is any one selected from the group consisting of a receptor, a ligand, an antibody and a fragment thereof.

8. In paragraph 7, A bispecific antibody wherein the VEGF-specific binding site is a receptor.

9. In paragraph 8, A bispecific antibody, wherein the receptor comprises an amino acid sequence of SEQ ID NO:

83.

10. In paragraph 7 A bispecific antibody, wherein the VEGF-specific binding site is an antibody or a fragment thereof.

11. In paragraph 10, The antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 69 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 71; A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 72 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 73; A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 74 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 76; or A bispecific antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 77 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

78.

12. In paragraph 6, A bispecific antibody wherein the anti-Tie2 antibody or fragment thereof and the VEGF-specific binding site are linked via a linker.

13. In paragraph 6, The above bispecific antibody is a fusion protein dimer comprising the following structural formulas (I) and (II): N'-X'-[L1]p-Fc region or a variant thereof-[L2]qYC' (I); and N'-X''-C' (II) At this time, in the structural formulas (I) and (II), The above N' is the N-terminus, The above C' is the C-terminal, The above - means combination, X' is the heavy chain variable region (VH) and heavy chain constant region (CH1) of the anti-Tie2 antibody of the first clause, X'' represents the light chain variable region (VL) and light chain constant region (CL) of the anti-Tie2 antibody of the first clause, X` and X`` combine to form a binding site (X) that specifically binds to Tie2, The above Y is a VEGF-specific binding site, The above L1 and L2 are peptide linkers, The above p and q are each independently O or 1.

14. A polynucleotide encoding the antibody of paragraph 1 or a fragment thereof; or the bispecific antibody of paragraph 6.

15. An expression vector comprising the polynucleotide of clause 14.

16. Cells transformed with the vector of Article 15. 17.i) A step of culturing the transformed cell of clause 16; and ii) A method for producing an anti-Tie2 antibody or a fragment thereof; or a bispecific antibody, comprising a step of obtaining an anti-Tie2 antibody or a fragment thereof; or a bispecific antibody from the cell culture medium; 18. A pharmaceutical composition for preventing or treating a disease related to vascular abnormalities, comprising the anti-Tie2 antibody or fragment thereof of paragraph 1 or the bispecific antibody of paragraph 6 as an active ingredient.

19. In paragraph 18, A pharmaceutical composition, wherein the above vascular abnormality-related disease is a disease related to angiogenesis abnormality or structural and / or functional variation of blood vessels.

20. In paragraph 19, The above-mentioned angiogenesis-related disease is caused by excessive increase, decrease, or deficiency. Pharmaceutical composition.

21. In paragraph 19, A pharmaceutical composition, wherein the disease related to structural and / or functional mutations of the blood vessels is caused by at least one selected from the group consisting of structural abnormality of blood vessels, dysfunction of blood vessels, and damage to blood vessels.

22. In paragraph 18, The above vascular abnormality-related disease is any one selected from the group consisting of acute renal injury, chronic renal failure, macular degeneration, diabetic retinopathy, diabetic macular edema, retinal vessel occlusion, proliferative retinopathy, retinopathy of prematurity, corneal graft rejection, glaucoma, critical limb ischemia, diabetic erectile dysfunction, sepsis, acute respiratory distress syndrome, vasculitis, Alzheimer's disease, Parkinson's disease, multiple sclerosis, stroke, thrombosis, occlusion, cancer, systemic erythema, psoriasis, hemophilic arthritis, associated sclerosis, capillary formation of atherosclerotic plaques, keloid, wound granulation, vascular adhesion, rheumatoid arthritis, osteoarthritis, autoimmune disease, Crohn's disease, restenosis, atherosclerosis, intestinal adhesions, cat scratch disease, ulcer, cirrhosis, nephritis, diabetic nephropathy, diabetes, inflammatory disease, and neurodegenerative disease.

23. Use of the anti-Tie2 antibody or fragment thereof of paragraph 1; or the bispecific antibody of paragraph 6 for the prevention or treatment of a disease related to vascular abnormalities.

24. A method for preventing or treating a vascular abnormality-related disease, comprising administering to a subject the anti-Tie2 antibody or fragment thereof of paragraph 1; or the bispecific antibody of paragraph 6.

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