Antibody trans-binding to tie2 of different cells, and use thereof

Abstract

The present invention relates to an antibody trans-binding to a TEK receptor tyrosine kinase 2 (Tie2) of different cells, and a use thereof, and, more specifically, to: an antibody and use thereof, the antibody trans-binding to Tie2 of different cells so as to improve integrity between endothelial cells, perivascular cells, or endothelial cells and perivascular cells, thereby exhibiting preventive, ameliorative, or therapeutic effects on diseases associated with angiogenesis and increased vascular permeability, or diseases associated with decreased normal angiogenesis. The antibody trans-binding to an extracellular domain of the Tie2 protein, of the present invention, has excellent in vivo vascular protective activity and angiogenesis inhibitory activity, and the cluster of the antibody exhibits the activity of promoting angiogenesis, and thus can be very effectively used in the development of therapeutic agents for preventing, alleviating, or treating diseases associated with angiogenesis, increased vascular permeability, or decreased normal angiogenesis.

Description

Antibodies that trans-bind to Tie2 in different cells and uses thereof

[0001] This application claims the benefit of Republic of Korea Patent Application No. 10-2024-0037888, filed March 19, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to an interferon beta dry powder formulation and a method for producing the same, and more particularly, to an interferon dry powder formulation of spherical particles with high morphological perfection produced by spray drying a liquid composition containing interferon beta, and a method for producing the same.

[0003]

[0004] Angiopoietin (Ang)-Tie2 signaling plays a crucial role in modulating various dysregulated leaky vascular diseases. Tie2 is a receptor tyrosine kinase primarily expressed on the surface of endothelial cells (ECs) and plays a crucial role in regulating vascular function. While Ang1 is known to be an agonist for Tie2, its antagonistic effects are conditionally dependent. Ang1-induced Tie2 phosphorylation (i) stabilizes vascular endothelial cadherin (VE-cadherin) and EC adhesion molecules via the VE protein tyrosine phosphatase (VE-PTP) pathway, (ii) induces AKT phosphorylation via phosphoinositide 3-kinase, and (iii) promotes pericyte recruitment for further stabilization of the vasculature. However, Ang1 has also been reported to promote vascular remodeling and expansion, and to synergize the pro-angiogenic response of VEGF in glioblastoma.

[0005]

[0006] The detailed mechanisms of Tie2-mediated vascular stabilization remain unclear, as dissecting the intercellular mode of action of Tie2, which depends on the innate multimeric structure of Ang1, is difficult.

[0007]

[0008] Based on the multimeric nature of Ang1, COMP-Ang1 and the polyethylene glycol (PEG)-clustered Tie2-agonistic peptide vasculotide have been developed to treat vascular diseases by promoting Tie2 clustering. Recently, Ang2-binding and Tie2-activating antibodies (ABTAAs) have been developed to overcome the short in vivo half-life of COMP-Ang1. More recently, drugs that directly target Tie2 have also been developed, such as PEGylated hexavalent Fab conjugates and engineered tetravalent anti-human Tie2 antibodies. Importantly, although other anti-Tie2 antibodies have been reported to induce Tie2 cis-clustering and signaling, no in vivo angiogenic activity has been reported for any Tie2-targeting bivalent antibody form. Furthermore, no therapeutic directly targeting Tie2 has entered clinical trials to date.

[0009]

[0010] The present inventors conducted a comparative study of an antibody that showed excellent efficacy in a laser-induced choroidal neovascularization monkey model, an animal model of age-related macular degeneration, and another antibody that showed strong Tie2 signaling activity but did not show an in vivo vascular stabilizing effect, thereby clearly elucidating the mechanism for achieving an in vivo vascular stabilizing effect using an antibody targeting Tie2.

[0011]

[0012] Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating diseases related to angiogenesis, increased vascular permeability, or decreased normal blood vessel formation, which comprises as an active ingredient an antibody that trans-binds to the extracellular domain of Tie2 (TEK receptor tyrosine kinase 2) protein, an antigen-binding fragment thereof, or a cluster thereof.

[0013]

[0014] Another object of the present invention is to provide an antibody or an antigen-binding fragment thereof that trans-binds to the extracellular domain of the Tie2 (TEK receptor tyrosine kinase 2) protein.

[0015]

[0016] Another object of the present invention is to provide a use of an antibody, an antigen-binding fragment thereof, or a cluster thereof that trans-binds to the extracellular domain of the Tie2 (TEK receptor tyrosine kinase 2) protein for preparing a pharmaceutical composition for treating a disease associated with angiogenesis, increased vascular permeability, or a disease associated with decreased normal angiogenesis.

[0017]

[0018] Another object of the present invention is to provide a method for treating a disease associated with angiogenesis, increased vascular permeability, or a disease associated with decreased normal angiogenesis, which comprises administering to a subject in need thereof an effective amount of a composition comprising, as an active ingredient, an antibody that trans-binds to the extracellular domain of the Tie2 (TEK receptor tyrosine kinase 2) protein, an antigen-binding fragment thereof, or a cluster thereof.

[0019]

[0020] In order to achieve the above-described object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating diseases related to angiogenesis, increased vascular permeability, or decreased normal blood vessel formation, comprising as an active ingredient an antibody that trans-binds to the extracellular domain of Tie2 (TEK receptor tyrosine kinase 2) protein, an antigen-binding fragment thereof, or a cluster thereof.

[0021]

[0022] In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating diseases related to angiogenesis, increased vascular permeability, or decreased normal blood vessel formation, comprising an antibody that trans-binds to the extracellular domain of Tie2 (TEK receptor tyrosine kinase 2) protein, an antigen-binding fragment thereof, or a cluster thereof.

[0023]

[0024] In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating diseases related to angiogenesis, increased vascular permeability, or decreased normal blood vessel formation, which is essentially composed of an antibody that trans-binds to the extracellular domain of Tie2 (TEK receptor tyrosine kinase 2) protein, an antigen-binding fragment thereof, or a cluster thereof.

[0025]

[0026] In order to achieve another object of the present invention, the present invention provides an antibody or an antigen-binding fragment thereof that trans-binds to the extracellular domain of Tie2 (TEK receptor tyrosine kinase 2) protein.

[0027]

[0028] In order to achieve another object of the present invention, the present invention provides the use of an antibody, an antigen-binding fragment thereof, or a cluster thereof that trans-binds to the extracellular domain of the Tie2 (TEK receptor tyrosine kinase 2) protein for preparing a pharmaceutical composition for treating a disease associated with angiogenesis, increased vascular permeability, or a disease associated with decreased normal angiogenesis.

[0029]

[0030] In order to achieve another object of the present invention, the present invention provides a method for treating a disease associated with angiogenesis, increased vascular permeability, or a disease associated with decreased normal angiogenesis, which comprises administering to a subject in need thereof an effective amount of a composition comprising, as an active ingredient, an antibody that trans-binds to the extracellular domain of a Tie2 (TEK receptor tyrosine kinase 2) protein, an antigen-binding fragment thereof, or a cluster thereof.

[0031]

[0032] Hereinafter, the present invention will be described in detail.

[0033]

[0034] The present invention provides a pharmaceutical composition for preventing or treating diseases related to angiogenesis, increased vascular permeability, or decreased normal blood vessel formation, comprising as an active ingredient an antibody that trans-binds to the extracellular domain of Tie2 (TEK receptor tyrosine kinase 2) protein, an antigen-binding fragment thereof, or a cluster thereof.

[0035]

[0036] In this specification, the term "comprising" is used with the same meaning as "including" or "characterized by", and does not exclude additional components, etc. that are not specifically mentioned in the composition according to the present invention. In addition, the term "consisting of" means excluding additional elements, etc. that are not separately described. The term "essentially consisting of" means that, within the scope of the composition, it may include materials that do not substantially affect the basic characteristics thereof in addition to the materials described.

[0037]

[0038] In the present invention, the Tie2 is an angiopoietin receptor, also known as CD202B. Tie2 is known to be expressed almost exclusively in mouse, rat, and human endothelial cells. This receptor possesses a unique extracellular domain comprising two immunoglobulin-like loops separated by three epidermal growth factor-like repeats linked to three fibronectin type III-like repeats. The ligand for Tie2 is angiopoietin-1. Defects in Tie2 are associated with hereditary venous malformations. In cancer patients, Tie2 is expressed in a subset of monocytes located in tumors, where it is essential for new blood vessel formation. The gene encoding Tie2 is known as Gene ID: 7010 in humans and Gene ID: 21687 in mice. The mRNA reference sequences of human Tie2 are known as NM_000459, NM_001290077, NM_001290078, NM_001375475, NM_001375476, etc., and the protein reference sequences are known as NP_000450, NP_001277006, NP_001277007, NP_001362404, NP_001362405, etc. The mRNA reference sequences of mouse Tie2 are known as NM_001290549, NM_001290551, NM_013690, etc., and the protein reference sequences are known as NP_001277478, NP_001277480, NP_038718, etc.As described above, the Tie2 protein is composed of Ig like 1 - Ig like 2 - EGF like 1, 2, 3 - Ig like 3 - Fibronectin 1, 2, 3, - Transmembrane - protein kinase in that order, and the region including Ig like 1 to Ig like 3 can be divided into a ligand binding domain, and the region including Ig like 1 to Fibronectin 1, 2, 3 can be divided into an extracellular domain.

[0039]

[0040] In a preferred embodiment of the present invention, the Tie2 protein may be composed of an amino acid sequence of SEQ ID NO: 1, wherein the ligand binding domain may be composed of an amino acid sequence of SEQ ID NO: 2, which is amino acid sequences 13 to 443, and the extracellular domain may be composed of an amino acid sequence of SEQ ID NO: 3, which is amino acid sequences 13 to 735.

[0041]

[0042] [Sequence number 1]

[0043] WSYGVLLWEIVSLGGTPYCGMTCAELYEKLPQGYRLEKPLNCDDEVYDLMRQCWREKPYERPSFAQILVSLNRMLEERKTYVNTTLYEKFTYAGIDCSAEEAA

[0044]

[0045] In the present invention, 'antibody' is also called immunoglobulin (Ig), and is a general term for proteins that selectively act on antigens and participate in biological immunity. In the present invention, antibody is used in the broadest sense, and specifically includes monoclonal antibodies (including monoclonal antibodies and full-length monoclonal antibodies), polyclonal antibodies (polyclonal antibodies), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., variable regions and other parts of antibodies that exhibit the desired biological activity (e.g., binding to c-Met)). Whole antibodies found in nature are generally composed of two pairs of light chains (LC) and heavy chains (HC), which are polypeptides composed of multiple domains, or have as their basic units the structures of two pairs of HC / LC. There are five types of heavy chains that make up mammalian antibodies, represented by the Greek letters α, δ, ε, γ, and μ, and depending on the type of heavy chain, they make up different types of antibodies, such as IgA, IgD, IgE, IgG, and IgM, respectively. There are two types of light chains that make up mammalian antibodies, represented by λ and κ. The heavy and light chains of antibodies are structurally divided into variable and constant regions according to the variability of the amino acid sequence. The constant region of the heavy chain is composed of three or four heavy chain constant regions, such as CH1, CH2, and CH3 (IgA, IgD, and IgG antibodies) and CH4 (IgE and IgM antibodies), depending on the type of antibody, and the light chain is composed of one constant region, CL. The variable regions of the heavy and light chains each consist of one domain, the heavy chain variable region (VH) or the light chain variable region (VL). The light and heavy chains are linked by a single covalent disulfide bond with their variable and constant regions aligned side by side, and the heavy chains of the two molecules bound to the light chain are linked by two covalent disulfide bonds to form the entire antibody.Whole antibodies specifically bind to antigens through the variable regions of the heavy and light chains, and since whole antibodies are composed of two pairs of heavy and light chains (HC / LC), one molecule of whole antibodies has bivalent monospecificity that binds to the same two antigens through the two variable regions. The variable region, which includes the site where the antibody binds to the antigen, is divided into a framework region (FR) with little sequence variability and a complementary determining region (CDR), which is a hypervariable region with high sequence variability. VH and VL each have three CDRs and four FRs arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus. The CDR with the highest sequence variability within the variable region of the antibody is the site that directly binds to the antigen and is most important for the antigen specificity of the antibody.

[0046]

[0047] In the present invention, the antibody includes both monoclonal and polyclonal antibodies, and is an antibody that contains a specific amino acid sequence in the light and heavy chain CDRs so as to be able to selectively bind to Tie2, and is preferably a monoclonal antibody. In addition, the antibody of the present invention includes all of chimeric antibodies, humanized antibodies, and human antibodies, and is preferably a human antibody.

[0048]

[0049] In the present invention, the monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies bind very specifically to a single antigen epitope.

[0050]

[0051] The term "monoclonal" in the present invention indicates that the antibody is obtained from a substantially homologous population and that the antibody has the characteristics of an antibody, but does not necessarily mean that the antibody must be produced by a specific method. For example, the monoclonal antibody of the present invention may be produced by the hybridoma method first described in the literature (Kohler et al. (1975) Nature 256: 495)) or may be produced by the recombinant DNA method (see U.S. Patent No. 4,816,567). In addition, for example, the monoclonal antibody may be isolated from a phage antibody library using the technique described in the literature (see Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597 and Presta (2005) J. Allergy Clin. Immunol. 116: 731).

[0052]

[0053] The antibodies of the present invention specifically include chimeric antibodies, in which a portion of the heavy and / or light chain may be from a particular species or be identical with or homologous to corresponding sequences in a particular antibody, while the remaining portion may be from another species or be identical with or homologous to corresponding sequences in another antibody, as long as the antibody of the present invention exhibits the desired biological activity (e.g., selective binding to an NRS) (U.S. Patent No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855).

[0054]

[0055] A humanized antibody is an antibody that contains sequences from both human and non-human (e.g., murine, rat) antibodies, typically human antibodies, except for the epitope-binding region (CDR), which may contain sequences of non-human origin. A fully human antibody contains only human immunoglobulin protein sequences, and may be produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell, or by phage display.

[0056]

[0057] Natural antibodies produced in vivo are typically heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by a single covalent disulfide bond, although the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end; the constant domain of the light chain aligns with the first constant domain of the heavy chain, and the light chain variable domain aligns with the variable domain of the heavy chain. Special amino acid residues are believed to form the interface between the light-chain variable domain and the heavy-chain variable domain. The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the antibody's heavy or light chain. The variable region of the heavy chain is designated "VH," and the variable region of the light chain is designated "VL." These domains are generally the most variable portions of the antibody and contain the antigen-binding site.

[0058]

[0059] In the present invention, 'hypervariable' refers to the fact that several sequences within the variable region differ extensively in sequence between antibodies and include residues directly involved in the binding and specificity of each particular antibody for its specific antigenic determinants. In both the light and heavy chain variable regions, hypervariability is concentrated in three segments known as complementarity determining regions (CDRs) or hypervariable loops (HVLs). CDRs are defined by sequence comparisons in the literature (Kabat et al., 1991, In: Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD.), whereas HVLs are structurally defined by the three-dimensional structure of the variable region, as disclosed in the literature (Chothia and Lena, 1987, J. Mol. Biol. 196:901-917).

[0060]

[0061] The three CDRs within each of the heavy and light chains are separated by framework regions (FRs), which contain sequences that tend to be less variable. From the amino terminus to the carboxy terminus of the heavy and light chain variable regions, the FRs and CDRs are arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The large β-sheet arrangement of the FRs brings the CDRs within each chain close to each other as well as to the CDRs from the other chain. The resulting conformation contributes to the antigen-binding site (see Kabat et al., 1991, NIH Publ. No. 91-3242, Vol. I, pages 647-669), but not all CDR residues need be directly involved in antigen binding.

[0062]

[0063] In the present invention, the antigen binding fragment may be characterized as being a fragment selected from the group consisting of diabody, minibody, F(ab)2, and F(ab')2.

[0064]

[0065] In the present invention, an antibody fragment means an antibody fragment that maintains the antigen-specific binding affinity of the entire antibody, and preferably, the fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the human-derived Tie2 protein affinity of the parent antibody.

[0066]

[0067] Fab (fragment antigen-binding) is an antigen-binding fragment of an antibody, consisting of one variable domain and one constant domain of each heavy chain and light chain. F(ab')2 is a fragment produced by hydrolyzing an antibody with pepsin, and has a structure in which two Fabs are linked by a disulfide bond at the heavy chain hinge. F(ab') is a monomeric antibody fragment in which a heavy chain hinge is added to Fab, which is obtained by reducing the disulfide bond of the F(ab')2 fragment. Fv (variable fragment) is an antibody fragment composed only of the variable domains of each heavy chain and light chain. scFv (single chain variable fragment) is a recombinant antibody fragment in which the heavy chain variable region (VH) and the light chain variable region (VL) are linked by a flexible peptide linker. A diabody is a fragment in which the VH and VL of an scFv are connected by a very short linker, so that they cannot bind to each other, but instead bind to the VL and VH of another scFv of the same type, forming a dimer. A minibody is a fragment in which two scFvs are bound to the CH3 that constitutes the Fc region of an antibody.

[0068]

[0069] Additionally, the antibody or fragment thereof of the present invention described above may be conjugated to, but is not limited to, enzymes, fluorescent substances, radioactive substances, proteins, and the like. Furthermore, methods for conjugating such substances to antibodies are well known in the art.

[0070]

[0071] The antibody of the present invention may be derived from any animal, including mammals, birds, and the like, including humans. Preferably, the antibody may be an antibody of a human, mouse, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken, and most preferably, a human or mouse.

[0072]

[0073] A human antibody is an antibody having the amino acid sequence of a human immunoglobulin, including antibodies isolated from a human immunoglobulin library or antibodies isolated from an animal that has been transgenic for one or more human immunoglobulins and does not express endogenous immunoglobulins.

[0074]

[0075] According to one embodiment of the present invention, it was confirmed that an antibody that trans-binds to the extracellular domain of the Tie2 protein contributes to the stabilization of blood vessels by trans-binding to Tie2 expressed in endothelial cells, pericytes, or endothelial cells and pericytes.

[0076]

[0077] In the present invention, the term "trans-binding" can be interpreted as having the same meaning as "trans-mode" or "trans-acting," and refers to the binding of two variable regions constituting an antibody to antigens expressed on different cells. Conversely, "cis-binding" refers to the binding of two variable regions constituting an antibody to antigens expressed on the same cell.

[0078]

[0079] In the present invention, the antibody may be characterized as an antibody that trans-binds to the extracellular domain of the Tie2 protein, and preferably, may be characterized as trans-binding to the ligand binding domain of the Tie2 protein.

[0080]

[0081] In the present invention, the antibody may be characterized by specifically binding to an epitope present in the extracellular domain of the Tie2 protein, preferably within the ligand binding domain.

[0082]

[0083] In the present invention, the epitope refers to a protein determinant capable of specifically binding to an antibody. An epitope is generally composed of a molecular surface group such as an amino acid or sugar side chain, and generally has specific three-dimensional structural characteristics and specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to a conformational epitope is lost in the presence of a denaturing solvent, but not to a non-conformational epitope. An epitope may include amino acid residues directly involved in binding (also referred to as the immunogenic component of the epitope) and other amino acid residues not directly involved in binding, such as amino acid residues that are effectively blocked by a specific antigen-binding peptide (i.e., the amino acid residues are present within the footprint of the specific antigen-binding peptide).

[0084]

[0085] In the present invention, the antigenic determinant to which the antibody binds is not particularly limited in its specific sequence as long as it is a region including a continuous amino acid sequence existing in the extracellular domain of the Tie2 protein, preferably a ligand binding domain, and is 5 to 50 consecutive amino acids existing in the ligand binding domain of SEQ ID NO: 2 or the extracellular domain of SEQ ID NO: 3, specifically, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, It may be a region comprising a sequence of 46, 47, 48, 49 or 50 amino acids. The epitope to which the antibody binds may comprise two or more regions comprising consecutive amino acids within the extracellular domain of the Tie2 protein, preferably the ligand binding domain.

[0086]

[0087] In a preferred embodiment of the present invention, the antibody can bind to the Ig-like 1 and 2 domains of the Tie2 protein, and specifically, the antigenic determinant to which the antibody binds can be characterized in that it exists in the Ig-like 1 and 2 domains of the Tie2 protein. The Ig-like 1 and 2 domains of the Tie2 protein can be composed of the amino acid sequence of SEQ ID NO: 4. The above antigenic determinant is not particularly limited in its specific sequence as long as it is a region including a continuous amino acid sequence present in the Ig-like 1 and 2 domains of the Tie2 protein, and is 5 to 50 consecutive amino acids present in the Ig-like 1 and 2 domains of SEQ ID NO: 4, specifically, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, It may be a region comprising a sequence of 49 or 50 amino acids. The antigenic determinant to which the antibody binds may comprise two or more regions comprising consecutive amino acids within the Ig-like 1 and 2 domains of the Tie2 protein.

[0088]

[0089] In another aspect of the present invention, the distance between the C-terminal ends of each Tie2 protein extracellular domain trans-bound to the Tie2 protein expressed on the surface of different cells may be 300 to 450 angstroms (Å). The C-terminal end of the Tie2 protein extracellular domain may be residue 745 in the amino acid sequence of SEQ ID NO: 1.

[0090]

[0091] In one aspect of the present invention, the antibody is preferably a monospecific antibody specific for Tie2, and may be characterized by being bivalent and trans-binding to Tie2 proteins expressed in different cells.

[0092]

[0093] In one embodiment of the present invention, when the antibody trans-binds to the Tie2 protein expressed in different cells in a bivalent manner, each variable region constituting the antibody may bind to the same region of the Tie2 protein, i.e., the same antigenic determinant, or may bind to different antigenic determinants, but preferably, they may bind to the same antigenic determinant.

[0094]

[0095] In one aspect of the present invention, the antibody may be characterized by trans-binding to Tie2 of an endothelial cell, a pericyte, or an endothelial cell and a pericyte.

[0096]

[0097] In one embodiment of the present invention, the antibody may be characterized in that the ratio of trans binding to the Tie2 protein is higher than the ratio of cis binding. For example, the ratio of trans binding to cis binding to the Tie2 protein of the antibody may be 51:49 to 100:0, and specifically, may be 51:49, 60:40, 70:30, 80:20, 90:10, or 100:0, but is not limited thereto.

[0098]

[0099] According to one embodiment of the present invention, it was confirmed that antibodies that trans-bind to the Tie2 protein exhibit excellent in vivo vascular protection, vascular stabilization, and angiogenesis inhibition activities, whereas antibodies that cis-bind to the Tie2 protein exhibit strong Tie2 signaling activity but fail to exhibit vascular protection, vascular stabilization, or angiogenesis inhibition activities. Therefore, antibodies that trans-bind to the Tie2 protein may exhibit preventive, ameliorating, or therapeutic effects on diseases associated with angiogenesis and increased vascular permeability.

[0100] The 'treatment' of the present invention comprehensively refers to improving symptoms caused by a disease related to the formation of new blood vessels and increased vascular permeability, which may include curing, substantially preventing, or improving the condition of the disease, and includes, but is not limited to, alleviating, curing, or preventing one or most of the symptoms resulting from the disease.

[0101]

[0102] According to another embodiment of the present invention, a cluster of antibodies that trans-bind to Tie2 protein has been identified as a potent activator of Tie2 signaling and exhibits potential angiogenic activity, and thus, the cluster of antibodies may exhibit preventive, ameliorating, or therapeutic effects on diseases associated with decreased normal angiogenesis.

[0103]

[0104] Accordingly, the present invention provides a pharmaceutical composition for preventing or treating diseases associated with angiogenesis, increased vascular permeability, or decreased normal angiogenesis, comprising as an active ingredient the antibody, an antigen-binding fragment thereof, or a cluster thereof that trans-binds to the extracellular domain of the Tie2 protein.

[0105]

[0106] In the present invention, the disease related to the formation of new blood vessels and increased vascular permeability may be selected from the group consisting of cancer, Clarkson's disease, macular degeneration, diabetic macular edema, diabetic retinopathy, glaucoma, corneal neovascularization, retinal neovascularization, choroidal neovascularization, critical limb ischemia, hereditary hemorrhagic telangiectasia, diabetic nephropathy, postrenal renal failure, prerenal azotemia, and renal failure, but is not limited thereto.

[0107]

[0108] In the present invention, the disease associated with the decrease in normal blood vessel formation may be selected from the group consisting of myocardial infarction, angina pectoris, cerebral infarction, stroke, Buerger's disease, avascular necrosis, foot ulcer, and erectile dysfunction, but is not limited thereto.

[0109]

[0110] In the present invention, the antibody cluster refers to a group formed by at least two antibodies or fragments thereof binding to each other. Preferably, the antibody cluster may be characterized as being formed by the binding of two or more antibodies and an anti-Fc antibody.

[0111]

[0112] The composition of the present invention may further comprise a pharmaceutically acceptable additive, and pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, maltose, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight with respect to the composition, but is not limited thereto.

[0113]

[0114] In addition, the composition of the present invention can be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, it can be prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0115]

[0116] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations can be prepared by mixing the compound of chemical formula 1 with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives can be included.

[0117]

[0118] Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cocoa butter, laurin butter, and glycerogelatin.

[0119]

[0120] Meanwhile, the injection may contain conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.

[0121]

[0122] In addition, the therapeutic compositions of the present invention may further comprise any physiologically acceptable carrier, excipient or stabilizer (Remington: The Science and Practice of Pharmacy, 19th Edition, Alfonso, R., ed, Mack Publishing Co. (Easton, PA: 1995)). Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphoric acid, citric acid and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or nonionic surfactants, such as Tween, Pluronics, or polyethylene glycol (PEG).

[0123]

[0124] In the present invention, the content of the composition is not particularly limited depending on the purpose or aspect of use, and may be, for example, 0.01 to 99 wt%, preferably 0.5 to 50 wt%, and more preferably 1 to 30 wt%, based on the total weight of the composition. The pharmaceutical composition of the present invention may contain 0.1 to 99.9 wt% of an antibody, an antigen-binding fragment thereof, or a cluster thereof prepared by the method of the present invention, and 99.9 to 0.1 wt% of a carrier.

[0125]

[0126] The dosage of the pharmaceutical composition of the present invention for the human body may vary depending on the patient's age, body weight, sex, dosage form, health condition, and disease severity, and is generally 0.01 to 100 mg / kg / day, preferably 0.1 to 20 mg / kg / day, and more preferably 5 to 10 mg / kg / day. In addition, it may be administered in divided doses at regular intervals according to the judgment of a doctor or pharmacist.

[0127]

[0128] The present invention also provides a food composition for preventing or improving diseases related to angiogenesis, increased vascular permeability, or decreased normal blood vessel formation, comprising as an active ingredient the antibody, antigen-binding fragment thereof, or cluster thereof that trans-binds to the extracellular domain of the Tie2 protein.

[0129]

[0130] The term "food" used in the present invention includes meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and includes all foods in the conventional sense.

[0131]

[0132] The food composition of the present invention may include a health functional food. The term "health functional food" as used herein refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients having functional properties useful to the human body. Here, "functionality" means obtaining a beneficial effect for health purposes, such as regulating nutrients for the structure and function of the human body or physiological effects. The health functional food of the present invention can be manufactured by a method commonly used in the art, and during the manufacturing process, it can be manufactured by adding raw materials and ingredients commonly added in the art.

[0133]

[0134] In addition, the formulation of the above health functional food can be manufactured without limitation as long as it is a formulation recognized as a health functional food. The food composition of the present invention can be manufactured in various forms of formulation, and unlike general drugs, it has the advantage of not having side effects that may occur with long-term use of drugs as it uses food as a raw material, and it is highly portable, so the health functional food of the present invention can be consumed as a supplement to enhance the effects of anti-inflammatory or anti-allergic agents.

[0135]

[0136] In addition, the food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, it may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in mixtures. The ratio of these additives is not particularly important, but is generally selected from the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the food composition of the present invention, but is not limited thereto.

[0137]

[0138] In addition, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional beverages. The carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, a natural sweetener such as thaumatin or stevia extract, or a synthetic sweetener such as saccharin or aspartame may be used. The proportion of the natural carbohydrate may generally be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the composition of the present invention, but is not limited thereto.

[0139]

[0140] In order to achieve another object of the present invention, the present invention provides the use of an antibody, an antigen-binding fragment thereof, or a cluster thereof that trans-binds to the extracellular domain of the Tie2 (TEK receptor tyrosine kinase 2) protein for preparing a pharmaceutical composition for treating a disease associated with angiogenesis, increased vascular permeability, or a disease associated with decreased normal angiogenesis.

[0141]

[0142] In order to achieve another object of the present invention, the present invention provides a method for treating a disease associated with angiogenesis, increased vascular permeability, or a disease associated with decreased normal angiogenesis, which comprises administering to a subject in need thereof an effective amount of a composition comprising, as an active ingredient, an antibody that trans-binds to the extracellular domain of a Tie2 (TEK receptor tyrosine kinase 2) protein, an antigen-binding fragment thereof, or a cluster thereof.

[0143]

[0144] The 'effective amount' of the present invention refers to an amount that, when administered to a subject, improves, treats, detects, diagnoses, or inhibits or reduces a disease related to angiogenesis, increased vascular permeability, or a disease related to decreased normal blood vessel production. The 'subject' may be an animal, preferably a mammal, particularly an animal including a human, and may also be a cell, tissue, organ, etc. derived from an animal. The subject may be a patient in need of the above effect.

[0145]

[0146] The antibody of the present invention, which trans-binds to the extracellular domain of the Tie2 protein, has excellent in vivo vascular protection activity and angiogenesis inhibition activity, and since the cluster of the antibody exhibits angiogenesis-promoting activity, it can be very usefully utilized in the development of agents for the prevention, improvement, or treatment of diseases related to angiogenesis, increased vascular permeability, or diseases related to decreased normal angiogenesis.

[0147]

[0148] Figures 1a to 1h show the results confirming that the 2F2 antibody activates the Tie2 signal more strongly than the 4E2 antibody (Figures 1a and 1b. 2F2, 2F2 in HUVEC cells Fab, 4E2 and 4E2 Fab The results are shown in the immunofluorescence images of the expression of cell adhesion protein VE-Cadherin after each treatment. Fig. 1c. The results of quantifying the leakage of FITC-dextran in HUVEC cells treated with each antibody and VEGF. Fig. 1d and Fig. 1e. The results of detecting and quantifying p-VEGFR2 in HUVEC cells treated with each antibody. Fig. 1f to Fig. 1h. The results of detecting and quantifying p-TIE2 and p-AKT in HUVEC cells treated with each antibody.)

[0149] Figures 2a to 2d show the results confirming that the 4E2 antibody reduces retinal thickness and leakage in a CNV monkey model.

[0150] Figure 3 is a diagram showing the amino acid sequence of the Tie2 protein.

[0151] Figures 4a and 4b are the structural results of the trans-binding mode of 4E2 antibody and the cis-binding mode of 2F2 antibody on the cell surface (Figure 4a. Red-labeled 4E2 ( FL 4E2) and 2F2 ( FL 2F2) This is the result confirmed by fluorescence microscopy after treating HUVEC with antibody. Fig. 4b. This is the result of staining HUVEC and HBVPs (human brain vascular pericytes) with Cytolight Rapid Dye (red) and Vybrant DiO Cell-Labeling Solution (green), respectively.).

[0152] Figure 5 is 2F2:Tie2 452 and 4E2:Tie2 452 This shows the SEC elution profile of the protein.

[0153] Figures 6a to 6e show 2F2:Tie2 through SAXS-MD calculations. ECD and 4E2:Tie2 ECDThe results of evaluating the conformational space of (Fig. 6a. Two attached Tie2 ECD To estimate the structural space occupied by the digital terminal of 2F2:Tie2 452 and 4E2:Tie2 344 MD simulations were performed using SAXS data of IgG:Tie2 complex. SAXS experiments of IgG:Tie2 complex showed that Tie2 was dimerized to avoid Fn3 domain-mediated dimerization of Tie2. ECD Instead, Tie2 452 was performed using. Six linker regions (a and a', b and b', c and c') connecting five rigid bodies (R1, R2 and R2', R3 and R3') were allowed to move. The lowest SAXS-χ 2 2F2:Tie2 representing the value 452 and 4E2:Tie2 344 Each of the seven structures was selected for further analysis. Fig. 6b. MD simulations using two conformers yielded better χ 2 Because the values ​​could not be generated, SAXS fitting was more efficient than fitting using a single conformer, 2F2:Tie2. 452 and 4E2:Tie2 452 MD fitting was performed using a single conformer. Fig. 6c. 2F2:Tie2 452 The calculated structure (left) and 4E2Tie2 452 (Right) were confirmed to fit well with the experimental SAXS curves of 2F2:Tie2 at various concentrations (4.50, 2.25, and 1.13 mg / ml from top to bottom). 452 and 4E2:Tie2 452 Guinier plot (qRg, less than 1.3) of 2F2:Tie2 452 and 4E2:Tie2 452 4.5 mg / ml sample of 4E2:Tie2 452There were no detectable insertions. The estimated Rg values ​​(Å) from Guinier plot analysis are shown in Figs. 6d and 6e. Tie2 ECD The two coordinates are 2F2:Tie2 respectively 452 and 4E2:Tie2 452 2F2:Tie2 according to the SAXS-MD structure of ECD and 4E2:Tie2 ECD A structural model was generated. (Left) SAXS-χ 2 Two binding Tie2 for the seven SAXS-MD structures with the lowest values ECD The end-to-end distance was calculated. (Right) 2F2:Tie2 ECD and 4E2:Tie2 ECD Four representative structural models are shown, where the Fc region is excluded and the Fab domain is shown in gray to simplify the figure. Tie2 on one side ECD Molecules overlap (black) and two attached Tie2 ECD The distance (Å) between the digital ends is displayed.).

[0154] Figures 7a to 7c show the results confirming that 4E2 induces trans-binding of Tie2, while 2F2 induces cis-binding. (Figure 7a. Phase-contrast microscopic images of cell aggregation and quantitative analysis of aggregated cells after treating Tie2-CHO-K1 cells with IgG1, 2F2, or 4E2 (each 30 μg / ml). Figure 7b. His6-tagged IgG-Fc (IgG-Fc His ) or Tie2 His Quantitative analysis of attached cells after treatment with IgG1, 2F2, or 4E2 (each 30 μg / ml) on Tie2-CHO-K1 cells attached to Ni-NTA wells coated with Tie2. Figure 7c. Tie2 cells were treated with IgG1, 2F2, or 4E2 (each 30 μg / ml). His- Results of immunoblotting analysis of Tie2 protein attached to Tie2-CHO-K1 cells after treatment with coated magnetic beads and quantitative analysis graph.

[0155] Figures 8a and 8b show the results confirming that 4E2 induces trans-binding of Tie2, whereas 2F2 induces cis-binding (Figure 8a. Images analyzing the distribution of Tie2 using a confocal fluorescence microscope after treating HUVECs with 4E2 or 2F2. Tie2 (red) was stained with an anti-Tie2 antibody, EC junctions (green) with an FITC-conjugated anti-VE-cadherin antibody, and nuclei (blue) with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI). Tie2 was mainly distributed at the cell-cell contacts when treated with 4E2, but was not observed in the 2F2 treatment group. Figure 8b. HUVECs and human brain vascular pericytes (HBVPs) were stained with Incucyte Cytolight Rapid Dye (red) and Vybrant DiO, respectively. (Results of quantitative analysis of the degree of mobilization of surrounding cells in EC-pericyte co-formed vascular structures after staining with Cell-Labeling Solution (green).)

[0156] Figures 9a and 9b show the results of measuring the change in tumor volume (Figure 9a) and the final tumor weight (Figure 9b) according to treatment with mouse PD-1 antibody, 4E2 antibody, or a combination thereof in the CT-26 tumor model.

[0157] Figures 10a and 10b show the results of immunohistochemistry (IHC) (Red: CD8+ T cells, Brown: Foxp3+ Treg cells) and quantitative analysis of changes in tumor infiltrating lymphocytes (TILs) in tumor tissues after treatment with mouse PD-1 antibody, 4E2 antibody, or a combination thereof in the CT-26 tumor model.

[0158] Figures 11a to 11d show the results of flow cytometry analysis of changes in tumor infiltrating lymphocytes (TILs) in tumor tissues after treatment with mouse PD-1 antibody, 4E2 antibody, or a combination thereof in the CT-26 tumor model.

[0159] Figures 12a and 12b show the results of measuring the antitumor efficacy of combined radiation therapy and 4E2 treatment in the CT-26 tumor model by measuring the change in tumor volume (Figure 12a) and the final tumor weight (Figure 12b).

[0160] Figure 13 shows immunofluorescence staining images (Green: pimonidazole (hypoxyprobe), Blue: DAPI) and quantitative analysis results for changes in hypoxic areas (after administration) due to combined radiation therapy and 4E2 therapy in the CT-26 tumor model.

[0161] Figure 14 shows the results of observing the survival period of animals after administering each experimental substance to an animal model in which sepsis was induced by LPS.

[0162] Figures 15a and 15b show the results of evaluating the ability to inhibit vascular leakage through FFA measurement after administering each experimental substance to a mouse model of laser-induced choroidal neovascular disease.

[0163] Figures 16a and 16b show the results of evaluating the efficacy of reducing retinal edema through OCT measurement after administering each experimental substance to a mouse model of laser-induced choroidal neovascular disease.

[0164] Figure 17 shows the results of evaluating the optic nerve recovery efficacy through ERG measurement after administering each experimental substance to a mouse model of laser-induced choroidal neovascular disease.

[0165] Figure 18 is a diagram showing the experimental design in a naturally occurring NPDR / DME monkey model.

[0166] Figure 19 shows the results of changes in the central retinal thickness compared to the baseline after administration of an experimental substance in a naturally occurring NPDR / DME monkey model.

[0167] Figure 20 shows the results of observing changes in macular thickness in nine ETDRS subfields compared to baseline after administration of an experimental substance in a naturally occurring NPDR / DME monkey model.

[0168] Figure 21 is a diagram showing an experimental design for evaluating therapeutic activity after administering a test substance to a dexamethasone (DEX)-induced ocular hypertension animal model.

[0169] Figures 22a and 22b show the results of evaluating individual intraocular pressure (IOP) for each eye between groups at each evaluation point for 4 weeks after administering an experimental substance to a dexamethasone (DEX)-induced ocular hypertension animal model.

[0170] Figures 23a to 23g show the results of analyzing the avascular area by taking a retinal flat-mounted image after administering an experimental substance to an OIR (Oxygen Induced Retinopathy) model and evaluating the degree of retinopathy using the OIR score.

[0171] Figures 24a and 24b show the results of quantitative evaluation of the number of extraretinal budding by taking H&E retinal images after administering an experimental substance to an OIR (Oxygen Induced Retinopathy) model (black arrows indicate new blood vessel masses growing toward the vitreous body (extraretinal budding).).

[0172] Figures 25a to 25g are drawings evaluating the tumor vessel expansion and leakage improvement activity of 4E2 using a GBM (glioblastoma multiforme) mouse model ((Figure 25a) Vessel dilation observed using PECAM stain in tumor blood vessels located in the GBM center. (Figure 25b) Improved blood flow observed using Lectin perfusion. (Figure 25c) Vascular leakage observed using Evans blue perfusion.)

[0173] Figure 26 is a diagram showing the experimental design for evaluating the glomerular damage recovery efficacy of 4E2 using an STZ-diabetic nephropathy mouse model.

[0174] Figures 27a to 27c show the results of measuring kidney weight, hematological values, and urine albumin after administering each experimental substance to an STZ-diabetic nephropathy mouse model.

[0175] Figures 28a to 28c show the results of measuring diabetic glomerular damage based on MT stain analysis after administering each experimental substance to an STZ-diabetic nephropathy mouse model.

[0176] Figures 29a to 29c show the results of measuring diabetic renal interstitial damage based on H&E staining after administering each experimental substance to an STZ-diabetic nephropathy mouse model.

[0177] Figures 30a to 30e show the results of measuring diabetic glomerular expansion based on PAS stain analysis after administering each experimental substance to an STZ-diabetic nephropathy mouse model.

[0178] Figures 31a and 32b show the results of measuring the survival rate of GBM mouse models following combined administration of 4E2 and anti-PD-1 antibodies. Antibody administration schedule for survival analysis (Figure 31a). Survival curves of GBM mice administered IgG, 4E2 anti-PD-1 antibodies, or a combination of 4E2 and anti-PD-1 antibodies (Figure 31b).

[0179]

[0180] Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and the present invention is not limited thereto.

[0181]

[0182] Screening and production of Tie2-active antibodies

[0183] An in-house library of scFvs was used to screen Tie2-targeting antibodies. Clones 2F2 and 4E2 were selected based on their ability to mimic Ang1. Their IgG1 forms (2F2 and 4E2, see Korean Patent Publication No. 10-2021-0020839) were expressed in stable CHO cell lines. The harvested cell culture fluid (HCCF) was purified through three column chromatography steps. (i) 2F2 and 4E2 were first purified by protein-A affinity chromatography (PAC) using MabSelect PrismA™ and MabSelect SuRe™ LX columns (Cytiva #17549803 and #17547403, respectively). HCCF was loaded onto a PAC column pre-equilibrated with buffer (pH 7.0, 50 mM sodium citrate) and washed with buffer (pH 6.0, 50 mM sodium citrate, 1 M NaCl). Elution was performed with buffer (pH 3.4, 50 mM sodium citrate). (ii) The PAC eluate was purified by cation exchange chromatography using a Capto S ImpAct column (Cytiva #17371702). Protein binding was performed in buffer (pH 5.0, 20 mM sodium citrate), and the column was washed with buffer (pH 5.5, 20 mM sodium citrate). Elution was performed using buffer (pH 6.0, 20 mM sodium citrate, 50 mM NaCl). (iii) Finally, the pH value and salt concentration of the eluent of Capto S ImpAct were adjusted for subsequent column chromatography with 4E2 (pH 5.6, 20 mM sodium citrate, 100 mM NaCl) and 2F2 (pH 6.0, 20 mM sodium citrate, 50 mM NaCl). The flow-through of the Capto adhesive multimode column (Cytiva #17544403) was then collected for further study.

[0184]

[0185] 2F2 is a more powerful Tie2 activator than 4E2.

[0186] 4E2 is human (Tie2), monkey ( Mnk Tie2) and mouse( Mos Tie2) While Tie2 protein has broad cross-species reactivity, 2F2 has Tie2 and Mnk It bound only to Tie2 (Table 1).

[0187]

[0188] [Table 1]

[0189]

[0190]

[0191] Their 1-valent fragment antigen-binding domain (4E2 Fab and 2F2 Fab ) did not show any activity (Fig. 1a and 1b). Both 4E2 and 2F2 were effective in preventing VEGF-induced downregulation of VE-cadherin on the surface of HUVECs (Fig. 1a and 1b). 4E2 and 2F2 showed similar activity in preventing dextran leakage (Fig. 1c) and inhibiting VEGF-induced VEGFR2 phosphorylation (Fig. 1d and 1e).

[0192] In a previous study, the present inventors confirmed that 4E2 can inhibit VEGFR2 phosphorylation through VE-PTP, a phosphatase that regulates the phosphorylation of receptor tyrosine kinases in endothelial cells (ECs) (Exp Mol Med 55, 470-484 (2023)). 2F2 clearly increased the phosphorylation of Tie2 and AKT, whereas 4E2 only slightly increased Tie2 phosphorylation, and its phosphorylation activity of AKT was unclear (Figs. 1f-1h). Therefore, 2F2 was determined to be a more potent activator than 4E2 in Tie2-mediated signaling in HUVECs.

[0193]

[0194] In a monkey model of laser-induced choroidal neovascularization, only 4E2, not 2F2, exhibited vasoprotective activity.

[0195] Based on the above cell-based in vitro characterization, the inventors hypothesized that 2F2 would have a potent vascular protective function in vivo. 2F2 Mos Because it did not bind to Tie2, we investigated its vascular protective activity using a laser-induced choroidal neovascularization (CNV) monkey model (Table 2). After inducing CNV in the eyes of rhesus monkeys, 4E2 and 2F2 were injected intravitreally. Human IgG1 and aflibercept (Eylea) were also administered as negative and positive controls, respectively. Aflibercept binds to circulating VEGF and acts as a VEGF trap. In the aflibercept and 4E2 treatment groups, ocular thickness and retinal leakage area were significantly reduced. Surprisingly, the 2F2 treatment group showed no significant change in retinal thickness, similar to the IgG1 negative control group (Figures 2a and 2b). Moreover, the retinal leakage area was slightly increased in both the 2F2 treatment group and the negative control group (Figures 2c and 2d).

[0196]

[0197] Whole retinas from each monkey were isolated and the expression levels of nine genes were assessed by qPCR analysis (Table 2).

[0198]

[0199] [Table 2]

[0200]

[0201]

[0202] No significant differences were observed in the expression of genes related to inflammation across all four groups. Interestingly, 4E2 exhibited clear antiangiogenic activity similar to that of aflibercept. The 4E2 and aflibercept-treated groups exhibited similar expression levels of genes involved in vascularization and angiogenesis. Specifically, (i) levels of PDGFR-beta (a marker of pericytes) and CD31 (a marker of ECs) were significantly reduced, indicating that ECs were stabilized, and (ii) levels of VEGF and Ang2 were also reduced compared to the human IgG1 group. However, the antiangiogenic activity of 2F2 was not clearly evident. Although the expression level of Ang2 was clearly reduced in the 2F2-treated group, the levels of other genes were similar to those in the human IgG1 group. To elucidate why 2F2, a more potent Tie2 activator than 4E2, did not exhibit any vascular protective activity in vivo, additional studies were conducted.

[0203]

[0204] Analysis of the different Tie2 binding modes of 4E2 and 2F2

[0205] Based on the previous experimental results, we determined that Ang1-Tie2 signaling, such as phosphorylation of Tie2 and AKT, was not correlated with the in vivo anti-angiogenic activity of 4E2.

[0206]

[0207] To investigate the molecular mechanisms underlying the diverse in vivo activities of 4E2 and 2F2 targeting Tie2, various Tie2 proteins (Tie2 745 , residues 23-745; Tie2 452 , residues 23-452, Tie2 344 , residues 23-344) using 4E2 Fab and 2F2 Fab We attempted to determine the complex crystal structure of Tie2. 745 Wow Tie2 452Each contains an entire extracellular domain (ECD) and a ligand binding domain (LBD) (Fig. 3). Finally, 4E2 Fab :Tie2 344 and 2F2 Fab :Tie2 452 The crystal structures were determined at resolutions of 3.9 and 3.0 Å, respectively (Table 3 and Fig. 4a).

[0208] [Table 3]

[0209]

[0210]

[0211] 4E2 Fab Tie2 is composed of Ig-like 1-2 domains LBD It was confirmed that the 4E2 antibody recognizes the tip region of the Tie2 protein consisting of the amino acid sequence of sequence number 1 as an epitope. Specifically, it was confirmed that the 4E2 antibody recognizes the region below as an epitope in the Tie2 protein consisting of the amino acid sequence of sequence number 1.

[0212]

[0213] 4E2 epitope sequence (Ig like 1-2 domain): ILINSLPLVSD (SEQ ID NO: 5, 27-37 of SEQ ID NO: 1), AKKVVWKREKASKINGAY (SEQ ID NO: 6, 83-100 of SEQ ID NO: 1), RIRTMKMRQQASFL (SEQ ID NO: 7, 112-125 of SEQ ID NO: 1), KEEDAV (SEQ ID NO: 8, 149-154 of SEQ ID NO: 1), SARYIGGNLF (SEQ ID NO: 9, 190-199 of SEQ ID NO: 1)

[0214]

[0215] On the other hand, 2F2 Fab Tie2, which is composed of EGF-like 1 / 2 / 3 and Ig-like 3 LBD It was confirmed to bind to the lateral region of 2F2 and 4E2 (Fig. 4b). Since the biological activity of 2F2 and 4E2 was confirmed only in IgG1 cotext, not Fab (Fig. 1a and Fig. 1b), the two Tie2ECD -The binding IgG1 model is 2F2 Fab :Tie2 452 and 4E2 Fab :Tie2 344 It was constructed using the crystal structure of . Interestingly, 2F2 Fab and 4E2 Fab The different Tie2 bonding modes are each composed of two bonded Tie2 ECD The cis and trans configurations of the molecule can be induced (Fig. 4b). 4E2:Tie2 ECD The model has an extended shape that favors recognition of two Tie2 molecules on adjacent cell surfaces. However, 2F2:Tie2 ECD The model has a staggered U shape, suggesting that 2F2 likely binds to two Tie2 molecules on the same cell surface.

[0216]

[0217] Therefore, the inventors of the present invention have found two Tie2 attached to 2F2 and 4E2, respectively, through SAXS (small-angle X-ray scattering) experiments. ECD We estimated the structural space covered by the digital terminal of Tie2. ECD The Fn3 domain of Tie2 mediates dimerization in SAXS experiments. 452 IgG1:Tie2 (1:2) complexes of 2F2 and 4E2 were prepared using (Fig. 5).

[0218] 2F2:Tie2 with the lowest SAXS-χ2 value using molecular dynamics (MD) simulations 452 and 4E2:Tie2 452 We calculated various structures of Tie2. ECD Tie2 452 Molecular fitting to IgG1:Tie2 ECD (1:2) The model was reconstructed (Fig. 6a). In fact, 2F2:Tie2 ECD Model's Tie2 ECDThe molecule exhibited a staggered conformation in the cis mode, but 4E2:Tie2 ECD The model molecules were found to prefer an extended conformation in the trans mode (Figs. 6a to 6e). Therefore, 2F2 is likely to bind to two Tie2 molecules on the same cell surface (intracellular cis-action), whereas 4E2 promotes recognition of two Tie2 molecules on adjacent cell surfaces (intercellular trans-action). Referring to the results in Figs. 6d and 6e, when the 4E2 antibody bound to Tie2 proteins expressed on the surfaces of different cells in the trans mode, the distance between the two Tie2 protein ECD C-terminal residues (specifically, residue 745 of the Tie2 protein amino acid sequence of SEQ ID NO: 1) was found to be approximately 300 to 450 angstroms (Å).

[0219]

[0220] 4E2 is located primarily at the EC-EC junction, while 2F2 is located at the EC surface.

[0221] The proposed Tie2 binding mode of 2F2 and 4E2, as determined through structural studies, was verified at the cellular level. First, to confirm trans-binding of Tie2, Tie2-CHO-K1 cells were cultured in suspension and treated with IgG1, 2F2, or 4E2, and the degree of cell aggregation was assessed. As a result, aggregation of more than 10 cells was observed only when treated with 4E2, while such aggregation was not observed in the IgG1 and 2F2 treatment groups (Fig. 7a).

[0222] For further validation, His6-tagged recombinant Tie2 (Tie2 His ) and Tie2-CHO-K1 cells were analyzed for adhesion. Tie2 was detected only when treated with 4E2. HisBinding between Tie2-CHO-K1 cells was confirmed, and no significant adhesion was observed in the IgG1 and 2F2 treatment groups (Fig. 7b).

[0223] Additionally, Ni-NTA magnetic beads were used in Tie2 His After coating with Tie2, the binding to Tie2-CHO-K1 cells was evaluated by treating with IgG, 2F2, and 4E2, respectively. As a result, consistent with the previous experiment, Tie2 binding was observed only in the 4E2-treated group. His -Coated beads adhered to Tie2-CHO-K1 cells, while no adhesion was observed in the IgG1 and 2F2-treated groups (Fig. 7c). These results support the possibility that 4E2 induces trans-binding of Tie2 between endothelial cells, whereas 2F2 mediates cis-binding of Tie2 on the EC surface.

[0224] Furthermore, to analyze the binding pattern of Tie2, HUVECs were treated with 4E2 or 2F2, stained with anti-Tie2 antibody, and the distribution of Tie2 was observed using a confocal fluorescence microscope. As a result, 2F2 was uniformly distributed across the cell surface, whereas 4E2 was primarily observed at intercellular junctions (Fig. 8a). These observations suggest that 2F2 likely induces cis-binding of Tie2, whereas 4E2 likely mediates trans-binding of Tie2 between cells.

[0225]

[0226] Pericytes are spaced at regular intervals along the capillary wall and are crucial for the formation and maintenance of the endothelial barrier. Therefore, the extent of pericytes is a crucial factor in vascular maturation. Pericytes also express Tie2, and Ang-Tie2 signaling can regulate the association of ECs with pericytes. VEGF significantly reduced the number of pericytes, while Ang2 enhanced their detachment from ECs. We confirmed that 4E2 inhibited VEGF- and Ang2-induced pericyte detachment, whereas 2F2 did not (Fig. 7E). The intercellular trans-association of Tie2 by 4E2 stabilizes attached pericytes to ECs and maintains endothelial barrier integrity.

[0227]

[0228] The diverse roles of cis- and trans-binding of Tie2 are well known. Cis-clustering of Tie2 in the EC matrix contributes to endothelial migration and proliferation, whereas trans-binding of Tie2 at the EC-EC junction is involved in vascular protection.

[0229] Although Tie2 signaling, such as phosphorylation of Tie2 and AKT, has been used to screen Tie2 agonist molecules, comparative studies of 4E2 and 2F2 in the present invention have confirmed that these markers have little relevance to actual angiogenic activity.

[0230] In summary, although 4E2 was less effective in activating Tie2 signaling, it induced intercellular association of Tie2, and inducing such intercellular trans-association was judged to be an essential characteristic that antibody therapeutics should exhibit for angiogenesis-prevention of vascular diseases.

[0231]

[0232] Evaluation of the efficacy of combined immunotherapy in a CT26 colon cancer animal model

[0233] Using a CT26 colon cancer cell transplantation tumor model, we aimed to evaluate the synergistic anti-tumor effect and observe the anti-tumor effect through combined administration of PD-1 antibody and 4E2 antibody due to increased lymphocyte influx into the tumor. Seven days after CT26 cell transplantation into BALB / c mice, 4E2 (30 mg / kg) was administered intravenously once a week and mouse PD-1 antibody (10 mg / kg) was administered intraperitoneally three times a week, and the mice were sacrificed on the 10th day.

[0234] Looking at the results on the final day (day 10), the mPD-1 10 mg / kg, 4E2 30 mg / kg single administration group and the mPD-mPD-1 10 mg / kg+4E2 30 mg / kg combined administration group showed statistically significant tumor growth inhibition effects of 24.0% (p<0.05), 35.6% (p<0.05), and 58.9% (p<0.05), respectively, compared to the isotype control (hlgG1) group (Fig. 9a).

[0235] On the 10th day after the start of drug administration, the CT26 tumor was resected and its weight was measured. As a result, compared to the isotype control (hlgG1) group, there was a statistically significant decrease in tumor weight of 26.8% (p<0.05), 41.8% (p<0.05), and 64.0% (p<0.05) in the mPD-1 10 mg / kg, 4E2 30 mg / kg single administration group, and mPD-1 10 mg / kg + 4E2 30 mg / kg combined administration group, respectively (Fig. 9b).

[0236] For TIL analysis, tumor tissues from three mice per group were obtained and subjected to IHC analysis. After fixing the tumor tissues in 4% PFA and embedding them into paraffin blocks, they were stained for CD8+ (red) and Foxp3+ Treg (brown) cells using immunohistochemical staining, and quantified. As a result, the mouse PD-1 antibody, 4E2, and combination treatment groups showed increased expression of immune cells compared to isotypes (Figs. 10a and 10b).

[0237] Tumor tissues were obtained from three mice per group and flow cytometric analysis was performed. After isolating immune cells from tumor tissues, CD4, CD8, Gr-1, and CD11b antibodies were conjugated to the immune cells, respectively, and flow cytometric analysis was performed. CD4+ T cells were not measured in any group, and CD8+ T cells were significantly measured only in the 4E2 and 4E2 combination groups. MDSC (gr-1+, CD11b+) were observed to increase in all mouse PD-1, 4E2, and 4E2 combination groups (Figures 11a to 11d).

[0238] From the above results, when mPD-1 (10 mg / kg) and 4E2 (30 mg / kg) were administered alone or in combination via the intraperitoneal or intravenous route to CT26 transplanted BALB / c mice, statistically significant tumor growth inhibition activity was observed in all groups, and in particular, it was confirmed that the combination administration group had a high anticancer activity of 58.9%.

[0239]

[0240] Evaluation of the efficacy of combined radiation therapy in a CT-26 tumor animal model

[0241] In this study, 6- to 8-week-old BALB / c mice were injected with 5X10 CT-26 cell lines into the right thigh. 6After cell injection, treatment was administered when the tumor diameter reached 1 cm. The experimental groups were composed of: (1) Isotype antibody treatment group (mouse IgG2a, 10 mg / Kg, twice a week, for 2 weeks), (2) radiation therapy group (10 Gy once, total 10 Gy, Co-60 gamma-irradiator, GammaBeam100-80, dose rate: 108 cGy / min, 3Х30 cm^2 collimator), (3) radiation therapy and isotype antibody combination treatment group (10 Gy once, total 10 Gy + Isotype, 10 mg / Kg, twice a week, for 2 weeks), (4) radiation therapy and 4E2 antibody combination treatment group (10 Gy once, total 10 Gy + 4E2, 10 mg / Kg, twice a week, for 2 weeks). Tumor volume and body weight were measured three times a week with n=7~8 per group. Tumor volume was measured by measuring the long axis X short axis. 2 / 2(mm 3 ) was calculated.

[0242] Additionally, to confirm the hypoxia-improving effect, mice (n=3 per group) were injected into the tail vein with pimonidazole 90 minutes before euthanasia on the 7th day after treatment. Afterwards, tumors were excised, frozen sections were prepared, and immunofluorescence staining using anti-pimonidazole antibody-FITC and anti-CD31 antibodies was performed to evaluate the hypoxic area (%). Statistical analysis was performed using GraphPad Prism 5.0 software using a two-way ANOVA with Bonferroni post-tests, and a difference of P<0.05 was considered statistically significant.

[0243]

[0244] As a result of the experiment, a significant tumor growth inhibition effect was observed in the radiation therapy group, RT+Isotype combination therapy group, and RT+4E2 combination therapy group compared to the Isotype antibody therapy group and 4E2 antibody therapy group (Fig. 12a).

[0245] Twenty-three days after treatment, the tumors were removed, photographed, and weighed. There was no significant difference in tumor weight between the radiation therapy group and the radiation therapy + isotype antibody combination treatment group. However, the radiation therapy + 4E2 antibody combination treatment group showed a significant decrease in tumor weight compared to the radiation therapy group and the radiation therapy + isotype antibody combination treatment group (P<0.05, Fig. 12b).

[0246] To assess the hypoxia-improving effect of pimonidazole, we analyzed the hypoxic area within the tumor. The hypoxic area (%) significantly decreased in the radiation therapy + 4E2 antibody combination treatment group compared to the radiation therapy and radiation therapy + isotype antibody combination treatment groups (Fig. 13). These results suggest that the 4E2 antibody, when combined with radiation therapy, may induce vascular normalization, improving the hypoxic tumor microenvironment and enhancing anticancer efficacy.

[0247]

[0248] Evaluation of therapeutic activity in Clarkson disease (inflammatory vascular leakage-related disease, LPS-induced sepsis model, E-cigarette lung) models

[0249] We aimed to evaluate the therapeutic activity of the 4E2 antibody against inflammatory vascular leak-related diseases in an animal model of LPS-induced sepsis. C57BL / 6 mice were pre-administered a single dose of isotype control (mlgG2a) and 4E2 via the intravenous and oral routes, respectively, 48 hours before LPS administration, and 2 hours before LPS administration, respectively. The mice were then observed for general symptoms and survival over time.

[0250] As a result of the experiment, in the isotype control (mlgG2a) + LPS group, crouching posture, decreased spontaneous movement, hypothermia, and tremors were observed from 22 hours, and the first death was confirmed within 22 hours, and the last mouse died at 30 hours. In the 4E2 + LPS group, similar symptoms appeared from 22 hours, and the first death was confirmed within 22 hours, and the last mouse died at 48 hours. In the Dexa + LPS group, the same symptoms appeared from 22 hours, and the first death was confirmed at 30 hours, and the last mouse died at 96 hours. In the 4E2 + Dexa + LPS group, similar symptoms appeared from 22 hours, and the first death was confirmed at 30 hours, but one mouse survived until the final observation date.

[0251] In the 4E2 + Dexa + LPS group, deaths were observed over a period of 22 to 96 hours, and the median survival time up to the final 96 hours was analyzed to be 25 hours in the isotype control (mlgG2a) + LPS group. In contrast, the survival time was extended to 38 hours in the 4E2 + LPS group, 46 hours in the Dexa + LPS group, and more than 48 hours in the 4E2 + Dexa + LPS group (Fig. 14).

[0252]

[0253] Evaluation of therapeutic activity in a mouse model of macular degeneration

[0254] Ten days after laser irradiation to induce choroidal CNV in C57BL / 6 mouse eyes, contrast agent was injected intraperitoneally into the mice, and then FFA (Fluorescein Angiography) of the retinal blood vessels was taken. Based on the images obtained, the size of the CNV lesion was measured by corrected total fluorescence (CTF). Based on this, an experiment was conducted to compare and evaluate the effect on the size of the CNV lesion and the electroretinogram (ERG) between the CNV control group (G2) and the Aflibercept administration group (G3) or the 4E2 antibody administration group (G4 and G5).

[0255] In the group administered Eylea® 20 μg / μL / eye intravitreally (IVT), both the size of CNV lesions on FFA and the volume of CNV lesions on OCT were statistically significantly reduced compared to the CNV control group administered hIgG1 (P<0.01 and P<0.0001, respectively). Therefore, the efficacy verification process through this trial was deemed valid.

[0256] Meanwhile, in the 4E2 antibody administration group, when comparing the size of CNV lesions on FFA with the isotype control group, it was confirmed that the size of CNV lesions was reduced more significantly than in the test group administered Eylea® by IVT (P<0.01) (Figures 15a and 15b).

[0257] Similarly, when comparing the lesion volume on OCT (Optical Coherence Tomography) with the excipient group, in the case of G4 administered 4E2 20 μg / μL / eye, the lesion volume was confirmed to be statistically significantly reduced to a level similar to that of the Eylea® administered group (P<0.0001). In the case of G5 administered 4E2 40 μg / μL / eye, the lesion volume was also confirmed to be statistically significantly reduced compared to the CNV control group (Figs. 16a and 16b, P<0.001).

[0258] In the results of another evaluation item, the electroretinogram (ERG), it was confirmed that the electroretinogram in the test group administered the test substance 4E2 was significantly increased compared to the CNV control group at a level similar to that in the Eylea® administered group (Fig. 17, P<0.0001, P<0.0001, respectively).

[0259] In summary of the results of this study, when the test substance 4E2 was administered intravitreally at a dose of 20 μg / μL / eye or 40 μg / μL / eye in a mouse CNV model, it was observed that the size of the CNV lesion was reduced on FFA and OCT, and the electroretinogram (ERG) increased, which was reduced by the lesion.

[0260]

[0261] Evaluation of therapeutic activity in an animal model of diabetic macular edema (retinopathy)

[0262] To evaluate the efficacy and safety of the 4E2 antibody in improving retinal thickness and other characteristics in a monkey model of spontaneous nonproliferative diabetic retinopathy (NPDR) / diabetic macular edema (DME). Sixteen spontaneous NPDR / DME monkeys were divided into four groups, and four monkeys in each group received two intravitreal injections of vehicle, aflibercept 2 mg / eye, or 4E2 2 mg / eye or 4 mg / eye at a dose of 0.05 mL / eye, once a month. Fundus photography (FP) and optical coherence tomography (OCT) were performed before administration (baseline) and on days 28, 56, and 84 after administration, and fluorescein angiography (FFA) was performed before administration, on days 56 and 84. Body weight and blood biochemistry tests were performed every two weeks (Fig. 18).

[0263] (1) Fundus photography (FP) and fluorescein angiography (FFA):

[0264] Vehicle group: All four animals showed NPDR-related changes at baseline, but there were no significant changes after administration.

[0265] Eylea 2 mg / eye group: All four animals showed NPDR-related changes at baseline. Some improvement was observed on day 56 after administration, but some remained similar to baseline or worsened on day 84. Compared to the vehicle group, NPDR-related changes improved in two of the four animals.

[0266] 4E2 2mg / eye group: All four animals showed NPDR-related changes at baseline, and some improvement was observed 56 days after administration. Compared to the vehicle group, NPDR-related changes were significantly improved in one of the four animals.

[0267] 4E2 4mg / eye group: All four animals showed NPDR-related changes at baseline, and some improvement was observed 56 days after administration, with significant improvement in NPDR-related changes in two of the four animals (results not shown).

[0268]

[0269] (2) Optical coherence tomography (OCT):

[0270] Foveal thickness: The vehicle group showed no significant change after administration. The Eylea group showed no significant change due to large individual differences. The 4E2 2mg / eye group showed a decreasing trend, and the 4mg / eye group showed a significant decrease compared to baseline on days 28 and 84 after administration, and a significant decrease at all time points compared to the vehicle group (Fig. 19).

[0271] Mean macular thickness across nine ETDRS zones: The vehicle group showed no significant change after administration. The Eylea group also showed no significant change. The 4E2 2 mg / eye group showed a decreasing trend, and the 4 mg / eye group showed a decreasing trend at all time points after administration (Fig. 20).

[0272] This study evaluated the efficacy and safety of 4E2 in improving retinal thickness in a naturally occurring NPDR / DME monkey model. Results of FP / FFA showed that NPDR-related changes were improved more frequently in the 4E2-treated group compared to the vehicle-treated group. OCT results showed that foveal thickness and mean macular thickness were significantly reduced in the 4E2 4 mg / eye group compared to the vehicle-treated group. Therefore, 4E2 showed potential for the treatment of NPDR / DME.

[0273]

[0274] Evaluation of therapeutic activity in a glaucoma mouse model

[0275] This study was conducted to evaluate the intraocular pressure (IOP) control and optic nerve protection efficacy of 4E2 using a dexamethasone (DEX)-induced ocular hypertension (OH) model in mice. After checking the basal IOP using a rebound tonometer, 12-week-old mice were treated with dexamethasone ophthalmic solution (DEX), one drop (5 μL) in each eye (OD & OS) three times daily for 4 weeks. IOP was measured again at 4 weeks after induction, and animals with a 30% or greater increase compared to the basal IOP were selected and divided into groups. IOP was measured in both eyes at the same time after fixing the mice in a correction frame without local anesthesia, and a total of 5 measurements were taken per eye for each measurement, and the average value was taken (Fig. 21).

[0276]

[0277] As a result of clinical symptom observation, no significant symptoms or changes related to test substance administration were observed during the entire test period.

[0278] Intraocular pressure was measured at one-week intervals from the time of group separation until the fourth week after test substance administration, and the intraocular pressure measured in the evening was higher than the intraocular pressure measured in the morning throughout the entire evaluation period.

[0279] Based on the right eye (OD) administered with the test substance, in the test group that received a single IVT administration of the formulation buffer after the start of administration, the intraocular pressure was maintained high at 14 mmHg or higher throughout the entire test period, whereas in the test group that received repeated eye drops of the control drug Xalatan, the intraocular pressure decreased by about 10% more than the time of group separation in the evaluation one week after administration, and then showed a tendency for the intraocular pressure to steadily decrease by about 4 to 8% at each evaluation time point. On the other hand, in the test group that received a single IVT administration of the test substance 4E2, the intraocular pressure showed a large decrease of about 15% compared to the time of group separation in the evaluation one week after administration, and then the intraocular pressure value decreased in the first week was maintained consistently without further decrease in intraocular pressure for the four weeks during the evaluation (Figs. 22a and 22b).

[0280] In summary, the test results suggest that a single intraocular pressure lowering effect of 80 ug / eye of test substance 4E2 via intraocular injection in a dexamethasone-induced ocular hypertension mouse model is similar to that of daily eye drops of the control drug Xalatan for 4 weeks.

[0281]

[0282] Evaluation of therapeutic activity in the OIR (Oxygen Induced Retinopathy) model

[0283] The aim of this study was to evaluate the therapeutic activity of the test substance 4E2 in corneal neovascularization, retinal neovascularization, and choroidal neovascularization in the OIR mouse model. This study was conducted to compare and evaluate the efficacy of the test substance 4E2 in improving abnormal retinal neovascularization in the mouse OIR (Oxygen Induced Retinopathy) model, a disease model for retinopathy of prematurity (ROP).

[0284] On the 7th day after birth, newborn mice were placed in a 75% oxygen chamber with their mothers and raised for 5 days. After the oxygen chamber was terminated, both mothers and their pups were transferred to a normoxia room air environment, and the pups were administered the test substance and control drug by intravitreal injection (IVT), and raised until the end of the test (post-natal day (PN) 17). On postnatal day 17, the right eye (OD) of the newborn mouse was extracted and prepared as a retinal flat-mount, and then stained with isolectin-IB4 for retinopathy scoring. The left eye (OS) was extracted and prepared as a paraffin slide, and then H&E staining was performed to count extra-retinal budding, which is an abnormal vascular mass that grows from the retina toward the vitreous body (G1: vehicle (PBS), intravenous administration (IVT), 1 dose. G2: OIR control, formulation buffer, IVT, 1 dose. G3: Aflibercept (40 μg / μL / eye), IVT, 1 dose. G4: OIR-4E2 (40 μg / μL / eye), IVT, 1 dose).

[0285]

[0286] The test results are as follows. In the retinopathy score evaluated on flat-mount slides, in the test group that received a single IVT administration of the test substance 4E2 at a dose of 40 μg / eye, a statistically significant decrease was confirmed in the avascular area, vessel tuft, and total retinopathy score among the detailed retinopathy score evaluation items compared to the OIR control group (Avascular area: P<0.05, Vessel tuft: P<0.001, Total retinopathy score: P<0.01).

[0287] In the total retinopathy score, when the test substance 4E2 was administered at a dose of 40 μg / eye, it was confirmed that the therapeutic activity was equivalent to that of the control group that received a single IVT administration of the reference drug aflibercept at a dose of 40 μg / eye (P=0.2226) (Figs. 23a to 23g).

[0288] Extra-retinal budding, which is an abnormal vascular mass that grows from the retina toward the vitreous, was counted in H&E-stained eyes, and it was confirmed that when a single IVT administration of each of the test substances 4E2 at a dose of 40 μg / eye was performed, the extra-retinal budding lesions induced by OIR were reduced to a level similar to that when the control drug aflibercept was administered at a dose of 40 μg / eye (P<0.01, P<0.05) (Fig. 24a and Fig. 24b).

[0289] In summary, the test results showed that a single intravenous (IV) administration of test substance 4E2 at a dose of 40 μg / eye significantly reduced the total retinopathy score for lesions induced by OIR. This result was consistently confirmed by the significant reduction in extra-retinal budding in the same test group on H&E-stained histopathology slides.

[0290]

[0291] Evaluation of therapeutic activity in hereditary hemorrhagic telangiectasia (GBM) vessels

[0292] To confirm the vascular normalization function of the 4E2 antibody on dilated diseased blood vessels, experiments were conducted using an EGFRviii-overexpressing GBM (glioblastoma multiforme) mouse model. In this model, vessel abnormalities were observed depending on the location of the cancer caused by EGFRviii overexpression in the brain, and increased VEGF expression, angiogenesis, and vessel leakage were confirmed centered on this. Using this model, 4E2 antibody (= 4E2) (40 mg / kg, IP injection) and the anti-VEGFR2 antibody DC101 (40 mg / kg, IP injection) as a control were administered twice a week for 2 weeks.

[0293]

[0294] As a result of the test, in the negative control group administered human IgG1, GBM vessels showed abnormal vessel dilation and reduced branching, and lectin perfusion, which observes blood flow, decreased and Evans blue leakage, which observes vascular leakage, increased.

[0295] In contrast, as a result of 4E2 antibody treatment, vessel dilation within the GBM was reduced (Fig. 25a), vessel branching was increased (Fig. 25a), lectin perfusion was increased (Fig. 25b), and Evans blue leakage was clearly reduced (Fig. 25c), and all of the corresponding results showed significant improvement.

[0296] The 4E2 antibody can be judged to have the efficacy to normalize diseased blood vessels that have expanded and leaked through TIE2 activation.

[0297]

[0298] Evaluation of therapeutic activity in renal disease using a STZ-induced glomerulus injury mouse model

[0299] To evaluate the therapeutic activity of the 4E2 antibody against renal diseases caused by angiogenesis, such as diabetic nephropathy, postrenal insufficiency, and nephrolithiasis, a STZ-induced glomerulus injury mouse model was created by administering STZ intraperitoneally twice to mice, excluding the normal control group. Details of the experimental design are shown in Figure 26.

[0300]

[0301] As a result of kidney organ weight analysis, the absolute and relative kidney weights were relatively lower in the 4E2 antibody alone group and the 4E2 antibody + Dapagliflozin combination group compared to the vehicle administration group. As a result of serum biochemical tests, BUN and creatinine were decreased in the 4E2 antibody alone group, the Dapagliflozin alone group, and the 4E2 antibody + Dapagliflozin combination group compared to the vehicle administration group. As a result of urinary albumin analysis using ELISA, the amount of urinary albumin was decreased in the 4E2 antibody alone group, the Dapagliflozin alone group, and the 4E2 antibody + Dapagliflozin combination group compared to the vehicle administration group (Figures 27a to 27c).

[0302]

[0303] Renal lesions induced by diabetic nephropathy were evaluated by dividing them into tubular, interstitial, and glomerular lesions. Renal tubules were evaluated semiquantitatively for tubular degeneration and tubular necrosis according to severity. Renal interstitium was evaluated for interstitial fibrosis and inflammatory cell infiltration. Renal glomeruli were evaluated for mesangial proliferation, mesangiolysis, and fibrosis.

[0304] Histopathological examination of the kidneys revealed that renal lesions were alleviated in the 4E2 antibody-only, Dapagliflozin-only, and 4E2 antibody+Dapagliflozin combination groups compared to the vehicle group (Figs. 28a to 29c). In addition, the diameter and area of ​​renal glomeruli were measured to be significantly reduced in the 4E2 antibody-only and 4E2 antibody+Dapagliflozin combination groups compared to the vehicle group (Figs. 30a to 30e).

[0305]

[0306] In conclusion, test substance 4E2 was judged to have an effect in alleviating renal disease caused by angiogenesis based on serum biochemical and histopathological findings.

[0307]

[0308] Evaluation of the efficacy of combined immunotherapy in an animal model of glioblastoma

[0309] Glioblastoma (GBM) is a highly invasive and treatment-resistant malignant brain tumor, with current treatments showing only limited efficacy. Abnormal vascular structures in the tumor microenvironment impede immune cell infiltration, thereby reducing the effectiveness of immunotherapy. Therefore, in this study, we aimed to determine whether 4E2, which induces vascular normalization, enhances the therapeutic efficacy of immune checkpoint inhibitors (anti-PD-1 antibodies). To establish a glioblastoma animal model, tumor cells were transplanted into the brains of mice. The mice were then divided into four groups, with nine mice per group. IgG was administered as a control, while the experimental groups included 4E2 alone (40 mg / kg, twice weekly), anti-PD-1 alone (8 mg / kg, twice weekly), and 4E2 plus anti-PD-1 combination. Survival rates were measured for 50 days after two weeks of drug administration (Figure 31a). The median survival time in the control group (IgG administration) was 19 days, 21 days in the 4E2 monotherapy group, and 30 days in the anti-PD-1 antibody monotherapy group. In contrast, the median survival time in the 4E2 and anti-PD-1 antibody combination group was 35 days. Furthermore, in the combination treatment group, three out of nine mice survived for more than 40 days, confirming an increased long-term survival rate (Figure 31b). This study confirmed that the 4E2 protein induces vascular normalization and, when administered in combination with an immune checkpoint inhibitor, can suppress tumor growth and increase survival. This suggests that vascular normalization can be a strategy to maximize the effect of immunotherapy. Therefore, the combination of vascular normalization and immunotherapy is expected to be an important approach in the future treatment of glioblastoma.

[0310]

[0311] The antibody of the present invention, which trans-binds to the extracellular domain of the Tie2 protein, has excellent in vivo vascular protection activity and angiogenesis inhibition activity, and since the cluster of the antibody exhibits angiogenesis-promoting activity, it can be very usefully utilized in the development of a treatment for diseases related to angiogenesis, increased vascular permeability, or decreased normal blood vessel formation, and thus has high industrial applicability.

Claims

1. A pharmaceutical composition for preventing or treating diseases related to angiogenesis, increased vascular permeability, or decreased normal blood vessel formation, comprising as an active ingredient an antibody that trans-binds to the extracellular domain of the Tie2 (TEK receptor tyrosine kinase 2) protein, an antigen-binding fragment thereof, or a cluster thereof.

2. A pharmaceutical composition according to claim 1, wherein the antibody binds to a ligand binding domain of the Tie2 protein.

3. A pharmaceutical composition according to claim 2, wherein the ligand binding domain is composed of an amino acid sequence of sequence number 2.

4. A pharmaceutical composition according to claim 1, characterized in that the antibody binds to Ig-like 1 and 2 domains of the Tie2 protein.

5. A pharmaceutical composition according to claim 3, wherein the antibody comprises Ig-like 1 and 2 domains of the Tie2 protein having an amino acid sequence of SEQ ID NO:

4.

6. A pharmaceutical composition according to claim 1, characterized in that the distance between the C-terminal ends of the extracellular domains of the Tie2 protein to which the antibody is trans-bound is 300 to 450 angstroms (Å).

7. A pharmaceutical composition according to claim 6, characterized in that the C-terminus of the extracellular domain of the Tie2 protein is residue 745 in the amino acid sequence of SEQ ID NO:

1.

8. A pharmaceutical composition according to claim 1, characterized in that the antibody is selected from the group consisting of IgG, IgA, IgM, IgE, and IgD.

9. A pharmaceutical composition according to claim 1, wherein the fragment is selected from the group consisting of diabodies, minibodies, F(ab)2, and F(ab')2.

10. A pharmaceutical composition according to claim 1, wherein the antibody is characterized in that it trans-binds to Tie2 of an endothelial cell, a pericyte, or an endothelial cell and a pericyte.

11. A pharmaceutical composition according to claim 1, characterized in that the antibody is bivalent.

12. A pharmaceutical composition according to claim 1, characterized in that the disease associated with neovascularization and increased vascular permeability is selected from the group consisting of cancer, Clarkson's disease, macular degeneration, diabetic macular edema, diabetic retinopathy, glaucoma, corneal neovascularization, retinal neovascularization, choroidal neovascularization, critical limb ischemia, hereditary hemorrhagic telangiectasia, diabetic nephropathy, postrenal renal failure, prerenal azotemia, and renal failure.

13. A pharmaceutical composition according to claim 1, characterized in that the disease associated with the decrease in normal blood vessel formation is selected from the group consisting of myocardial infarction, angina pectoris, cerebral infarction, stroke, Buerger's disease, avascular necrosis, foot ulcer, and erectile dysfunction.

14. A pharmaceutical composition according to claim 1, wherein the cluster of the antibody is formed by binding of the antibody and an anti-Fc antibody. 15.An antibody or antigen-binding fragment thereof that trans-binds to the extracellular domain of the Tie2 (TEK receptor tyrosine kinase 2) protein.

16. Use of an antibody, an antigen-binding fragment thereof, or a cluster thereof that trans-binds to the extracellular domain of the Tie2 (TEK receptor tyrosine kinase 2) protein for preparing a pharmaceutical composition for the treatment of a disease associated with angiogenesis, increased vascular permeability, or a disease associated with decreased normal angiogenesis.

17. A method for treating a disease associated with angiogenesis, increased vascular permeability, or decreased normal blood vessel formation, comprising administering to a subject in need thereof an effective amount of a composition comprising, as an active ingredient, an antibody that trans-binds to the extracellular domain of the Tie2 (TEK receptor tyrosine kinase 2) protein, an antigen-binding fragment thereof, or a cluster thereof.

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