KR20220059888A - A Novel anti-Tie2 antibody or its antigen-binding fragments, and the use thereof - Google Patents

Abstract

The present invention relates to a novel anti-Tie2 antibody or antigen-binding fragment thereof, and use thereof. The antibody or antigen-binding fragment thereof includes: a heavy chain variable region including CDR1-H having the amino acid sequence of X1-X2-X3-X4-X5-X6-Y-X7-M-H-W-X8; and a light chain variable region including CDR1-L having the amino acid sequence of X17-X18-X19-X20-X21-X22-X23-N-X24-X25-X26-Y. The antibody or antigen-binding fragment thereof is applied as an active ingredient of a human pharmaceutical composition without concern for a rejection reaction.

Description

Novel anti-Tie2 antibody or antigen-binding fragments thereof, and uses thereof

The present invention relates to novel anti-Tie2 antibodies or antigen-binding fragments thereof and uses thereof.

Tie2 protein is a receptor-type tyrosine kinase specifically expressed in vascular endothelial cells, and is known to be phosphorylated and activated by a specific ligand, typically a multimeric glycoprotein Ang1 (Angiopoietin-1). It is known to play a very important role in angiogenesis, structural and functional normalization and stabilization of blood vessels.

Activation of the Tie2 protein by Ang1 can induce structurally and functionally mature blood vessels by itself, and in particular is spotlighted as a therapeutic agent for ischemic diseases. In addition, it has the ability to suppress vascular leakage, vascular inflammation, and blood vessel abnormality induced by VEGF, and recently, as a treatment strategy for diabetic retinopathy, sepsis and tumors, abnormal angiogenesis through VEGF inhibition is inhibited Tie2 activation by Ang1 is receiving more attention as a strategy for inducing normalization and stabilization of pre-generated abnormal blood vessels through activation of the Tie2 receptor is suggested.

However, the Ang1 is synthesized in the form of a tetramer in vivo and exhibits physiological activity. Since the Ang1 tetramer is a recombinant protein, it is very difficult to produce, and it is difficult to use as a therapeutic agent because it has a short half-life in vivo. . In order to solve the problem of Ang1, research on various Tie2-specific activators that can replace Ang1 is being conducted, but there are problems such as low productivity and concerns about inducing an immune response, so there is a limit to its use as a therapeutic agent. Accordingly, there is still a need for research and development on a Tie2-specific activator that can replace Ang1 as described above.

One object of the present invention is to provide a novel antibody or antigen-binding fragment thereof that specifically binds to the Tie2 protein.

Another object of the present invention is to provide a gene encoding the antibody or antigen-binding fragment thereof, and an expression vector and transformant comprising the gene.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating angiogenesis-related diseases comprising the antibody or antigen-binding fragment thereof.

In order to achieve the above object, one aspect of the present invention is an amino acid sequence of X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -YX ₇ -MHWX ₈ (in the amino acid sequence, X ₁ to X ₈ is each independently any amino acid), a heavy chain variable region comprising a CDR1-H, and X ₁₇ -X ₁₈ -X ₁₉ -X ₂₀ -X ₂₁ -X ₂₂ -X ₂₃ -NX ₂₄ -X ₂₅ - The amino acid sequence of X ₂₆ -Y (in the amino acid sequence, X ₁₇ to X ₂₀ and X ₂₂ to X ₂₆ are each independently any amino acid, and X ₂₁ is any one selected from the group consisting of tyrosine, phenylalanine and tryptophan ), an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising CDR1-L.

The antibody or antigen-binding fragment thereof may specifically bind to human or monkey Tie2 protein.

The antibody or antigen fragment thereof may specifically phosphorylate human or monkey Tie2 protein.

In order to achieve the above object, another aspect of the present invention provides a gene encoding the antibody or antigen-binding fragment thereof, and an expression vector and transformant comprising the gene.

In order to achieve the above object, another aspect of the present invention provides a pharmaceutical composition for preventing or treating angiogenesis-related diseases comprising the antibody or antigen-binding fragment thereof.

The novel antibody or antigen-binding fragment of the present invention binds to human or monkey Tie2 protein and phosphorylates it, while in terms of specificity or affinity for the antigen, it is not compatible with the existing ligands (Ang1, Ang2) or antibodies. shows a much better effect than

In addition, since the novel antibody or antigen-binding fragment thereof of the present invention is a fully human antibody, it can be applied as an active ingredient of a pharmaceutical composition for humans without fear of rejection, so that angiogenesis is insufficient or abnormal blood vessels are excessive. There is an advantage that can be effectively used for the treatment or prevention of induced angiogenesis-related diseases.

However, the effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

1 shows the results of analyzing the major amino acids through alanine scanning technique for the CDRs of the heavy chain variable region and the light chain variable region of the primary antibody (Ang1mab1).
2 shows the results of confirming the phosphorylation activity of the first and second primary antibodies (Ang1mab1, Ang1mab4 and Ang1mab2-1) on Tie2, AKT, and ERK proteins in HEK293 cells expressing human Tie2 protein.
3 shows the results of confirming the phosphorylation activity of the mutant antibodies on the Tie2 protein in HEK293 cells expressing the human Tie2 protein.
4 shows the results of confirming the phosphorylation activity of the first and second primary antibodies (Ang1mab1, Ang1mab4, and Ang1mab2-1) and mutant antibodies on Tie2, AKT, and ERK proteins in HUVEC.
5 shows the results of confirming that the primary antibody (Ang1mab4) specifically phosphorylates only the Tie2 protein in HUVEC and HEK293 cells expressing human Tie2 protein.
6 to 8 show the results of confirming cross-species cross-linking of the first and second primary antibodies (Ang1mab1, Ang1mab4, and Ang1mab2-1) and the variant antibodies.
9 schematically shows a variant designed by removing major domains from human Tie2 protein one by one in order to identify antigenic determinants of the antibody of the present invention.
10 shows the results of confirming which domain of the human Tie2 protein the primary antibody (Ang1mab4) binds.
11 shows the results of confirming whether the first and second primary antibodies (Ang1mab1, Ang1mab4, and Ang1mab2-1) and mutant antibodies promote the angiogenesis of HUVECs.
12 shows the results of confirming whether the primary antibody (Ang1mab4) inhibits HUVEC cell death (apoptosis).
13 shows the results of confirming whether the human Tie2 protein is properly expressed in the blood vessels of the transgenic mouse prepared in a specific example of the present invention.
14 shows the results of ex vivo confirmation of whether primary antibodies (Ang1mab1 and Ang1mab4) promote vascular sprouting by activating human Tie2 protein expressed in transgenic mice.
15 and 16 show whether the primary antibody (Ang1mab4) promotes wound healing through the promotion of angiogenesis by activating the human Tie2 protein expressed in the transgenic mouse, and furthermore, the blood vessels by well surrounding the pericyte. It shows the results of confirming in vivo whether or not to stabilize.
17 shows the results of confirming in vivo whether the primary antibody (Ang1mab4) can improve lower extremity ischemia through the promotion of angiogenesis by activating human Tie2 protein expressed in transgenic mice.
18 shows the results of confirming whether the primary antibody (Ang1mab4) can effectively inhibit the increase in vascular permeability caused by vascular endothelial growth factor (VEGF) in HUVECs, the so-called leakage phenomenon.
19 shows the results of confirming in vivo whether the primary antibody (Ang1mab4) can effectively inhibit sepsis by activating human Tie2 protein expressed in transgenic mice to inhibit vascular leakage.
20 and 21 show the results of confirming in vivo whether the primary antibody (Ang1mab1) can effectively inhibit tumor growth caused by excessive abnormal angiogenesis by activating human Tie2 protein expressed in transgenic mice. will be.
22 shows whether the primary antibody (Ang1mab1) can increase the influx of anti-tumor immune cells that inhibit tumor growth into tumors by activating human Tie2 protein expressed in transgenic mice to normalize abnormal tumor blood vessels. Shows the results confirmed in Vivo.
23 shows whether or not tumor growth can be more effectively inhibited when the primary antibody (Ang1mab1) that activates human Tie2 protein and the immune checkpoint inhibitory antibody (Anti-PD-1) that inhibits immune evasion are administered in combination. Shows the results confirmed in vivo.

First, the terms used in the specification of the present invention will be described.

The term 'antibody' as used herein refers to immunoglobulin molecules and multimers thereof having a structure in which one light chain is connected to each other by a disulfide bond and one light chain to each of two heavy chains connected to each other by a disulfide bond. The light chain contains two regions, one variable region (light chain variable region, V _L ) and one constant region (light chain constant region, _CL ), and the heavy chain contains four regions, namely one variable region (heavy chain variable region). region, V _H ) and three constant regions (heavy chain constant region, C _H ; C _H 1 , C _H 2 and C _H 3 ). The constant regions of the light and heavy chains serve to confer biological properties such as binding between the light and/or heavy chains, secretion, complement binding, binding to Fc receptors (FcR), etc., and the variable regions of the light and heavy chains are antigen recognition and binding specificity. In particular, the binding specificity of the antibody is due to the structural complementarity between the antibody binding site and the antigenic determinant (epitope). Such an antibody binding site is mainly a complementarity determining region contained in the variable regions of the heavy and light chains. Since it consists of residues derived from three hypervariable regions called (complementarity determining region, CDR), such a complementarity determining region is referred to as amino acid sequences defining the binding specificity of an antibody. These three CDRs are arranged between four relatively well-conserved regions called framework regions (FR), which are FR1, CDR1, FR2, CDR2, They are arranged in the order of FR3, CDR3 and FR4 and are linked. In this case, the three CDRs included in the heavy chain variable region are referred to as CDR1-H, CDR2-H and CDR3-H, respectively, and the three CDRs included in the light chain variable region are respectively CDR1-L, CDR2-L and CDR3-H. It is called L.

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

A 'monoclonal antibody' referred to in the present invention refers to an antibody molecule of a single amino acid sequence, also referred to as a 'mAb', which targets a specific antigen, and is not construed as requiring antibody production by any special method. For example, monoclonal antibodies may be produced by a monoclonal or hybridoma of B cells, but may also be recombinant.

A 'chimeric antibody' as used herein refers to an antibody produced by genetic engineering, which, in its broadest sense, contains one or more sites from one antibody and one or more sites from one or more other antibodies. For example, a chimeric antibody comprises the heavy and light chain variable regions of an antibody derived from a non-human animal such as mouse, rat, hamster, and rabbit together with the heavy and light chain constant regions of a human antibody. A chimeric antibody may also refer to a multispecific antibody that exhibits specificity for at least two different antigens.

A 'humanized antibody' as referred to in the present invention is derived wholly or in part from a non-human animal, and to avoid or minimize an immune response in humans, for example, to replace certain amino acids in the framework regions of the variable regions of heavy and light chains. Refers to a modified antibody. The constant regions of humanized antibodies are in most cases the constant regions of human heavy and light chains.

As used herein, the term 'human antibody' is meant to include antibodies having variable and constant regions derived from human immunoglobulin sequences. Human antibodies of the invention are encoded by human immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, in particular in CDR3. It may contain non-amino acid residues. However, the 'human antibody' referred to in the present invention does not include an antibody in which CDR sequences derived from germlines derived from other non-human animals such as mice are grafted onto human framework sequences.

As used herein, the term 'epitope' refers to a site of an antigen that can specifically bind to an antigen binding site of an antibody known as a paratope. A single antigen may have more than one antigenic determinant, and even if they bind to the same antigen, when the specific site of the antigen to which the antibodies bind is different, these antibodies have different antigenic determinants. The antigenic determinant is usually composed of a group of chemically active surface molecules, such as amino acids or sugar side chains, and generally has specific three-dimensional structural characteristics as well as specific charge characteristics. In addition, the antigenic determinant may have a three-dimensional or non-stereoscopic structure, and the three-dimensional antigenic determinant and the non-steric antigenic determinant lose binding to the stereogenic antigenic determinant in the presence of a denaturing solvent, but the binding to the non-steric antigenic determinant is not lost. distinguished in that they do not

As used herein, "conservative substitution" means that an amino acid residue is substituted with an amino acid residue having a similar side chain. For example, substitution among amino acid residues having basic side chains such as lysine, arginine, and histidine corresponds to a conservative substitution. In addition, amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; amino acid residues having β-branched side chains such as threonine, valine, isoleucine; Substitutions among amino acid residues with aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine are also conservative substitutions.

Hereinafter, the present invention will be described in detail.

One. Novel antibody or antigen-binding fragment thereof

One aspect of the present invention provides a novel antibody or antigen-binding fragment thereof.

The antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region.

The heavy chain variable region included in the antibody or antigen-binding fragment thereof of the present invention has an amino acid sequence of X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -YX ₇ -MHWX ₈ (SEQ ID NO: 4) Including CDR1-H, in the amino acid sequence of SEQ ID NO: 4, X ₁ to X ₈ may each independently be any amino acid.

In the amino acid sequence of SEQ ID NO: 4, X ₁ may be any one selected from the group consisting of glycine, alanine, valine, isoleucine and leucine, or any amino acid that can be conservatively substituted therewith, and X ₁ is glycine , may be any one selected from the group consisting of alanine and valine, wherein X ₁ may be any one selected from the group consisting of glycine and alanine.

In the amino acid sequence of SEQ ID NO: 4, X ₂ is any one selected from the group consisting of glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine and tryptophan, or any amino acid that can be conservatively substituted therewith , wherein X ₂ may be any one selected from the group consisting of alanine, valine, isoleucine, leucine, tyrosine and phenylalanine, wherein X ₂ is selected from the group consisting of alanine, valine, isoleucine, leucine, tyrosine and phenylalanine may be any one, wherein X ₂ may be any one selected from the group consisting of alanine, isoleucine, leucine, tyrosine and phenylalanine, and X ₂ is any one selected from the group consisting of alanine, leucine and phenylalanine can be one

In the amino acid sequence of SEQ ID NO: 4, X ₃ is any one selected from the group consisting of serine, threonine, asparagine, glutamine, glycine, alanine, valine, isoleucine and leucine, or any amino acid that can be conservatively substituted and X ₃ may be any one selected from the group consisting of serine, threonine, alanine, valine, isoleucine and leucine, and X ₃ is any one selected from the group consisting of serine, threonine, alanine and valine may be one, and X ₃ may be any one selected from the group consisting of threonine and alanine.

In the amino acid sequence of SEQ ID NO: 4, X ₄ is any one selected from the group consisting of glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine and tryptophan, or any amino acid that can be conservatively substituted therewith , wherein X ₄ may be any one selected from the group consisting of glycine, alanine, valine, isoleucine, leucine, tyrosine and phenylalanine, and X ₄ is selected from the group consisting of glycine, alanine, valine, tyrosine and phenylalanine and X ₄ may be any one selected from the group consisting of glycine, alanine, tyrosine and phenylalanine, and X ₄ may be any one selected from the group consisting of alanine and phenylalanine .

In the amino acid sequence of SEQ ID NO: 4, X ₅ is any one selected from the group consisting of serine, threonine, asparagine, glutamine, glycine, alanine, valine, isoleucine and leucine, or any amino acid that can be conservatively substituted and X ₅ may be any one selected from the group consisting of serine, threonine, alanine, valine, isoleucine and leucine, and X ₅ is any one selected from the group consisting of serine, threonine, alanine and valine may be one, and X ₅ may be any one selected from the group consisting of serine and alanine.

In the amino acid sequence of SEQ ID NO: 4, X ₆ is any one selected from the group consisting of serine, threonine, asparagine, glutamine, glycine, alanine, valine, isoleucine and leucine, or any amino acid that can be conservatively substituted and X ₆ may be any one selected from the group consisting of serine, threonine, asparagine, glutamine, glycine, alanine and valine, and X ₆ is serine, threonine, asparagine, glutamine, alanine and valine It may be any one selected from the group consisting of, wherein X ₆ may be any one selected from the group consisting of serine, asparagine and alanine.

In the amino acid sequence of SEQ ID NO: 4, X ₇ may be any one selected from the group consisting of glycine, alanine, valine, isoleucine and leucine, or any amino acid that can be conservatively substituted therewith, and X ₇ is glycine , and may be any one selected from the group consisting of alanine and valine, wherein X ₇ may be any one selected from the group consisting of glycine and alanine, and X ₇ may be alanine.

In the amino acid sequence of SEQ ID NO: 4, X ₈ may be any one selected from the group consisting of glycine, alanine, valine, isoleucine and leucine, or any amino acid that can be conservatively substituted therewith, and X ₈ is glycine , may be any one selected from the group consisting of alanine and valine, wherein X ₈ may be any one selected from the group consisting of alanine and valine.

The amino acid sequence of SEQ ID NO: 4 is GLTFSNYAMHWV (SEQ ID NO: 10), ALTFSNYAMHWV (SEQ ID NO: 21), GATFSNYAMHWV (SEQ ID NO: 22), GLTFANYAMHWV (SEQ ID NO: 23), GLTFSNYAMHWA (SEQ ID NO: 24) and GFTFSSYAMHWV (SEQ ID NO: 16) It may be any one amino acid sequence selected from the group consisting of, or an amino acid sequence having 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more homology with the amino acid sequence.

The heavy chain variable region included in the antibody or antigen-binding fragment thereof of the present invention may further include CDR2-H comprising the amino acid sequence of IX ₉ -YX ₁₀ -X ₁₁ -X ₁₂ -NK (SEQ ID NO: 5). And, in the amino acid sequence of SEQ ID NO: 5, X ₉ to X ₁₂ may each independently be any amino acid.

In the amino acid sequence of SEQ ID NO: 5, X ₉ may be any one selected from the group consisting of serine, threonine, asparagine, glutamine, glycine, alanine, valine, isoleucine and leucine, or any amino acid that can be conservatively substituted therewith and X ₉ may be any one selected from the group consisting of serine, threonine, alanine, valine, isoleucine and leucine, and X ₉ is any one selected from the group consisting of serine, threonine, alanine and valine and X ₉ may be any one selected from the group consisting of serine and alanine.

In the amino acid sequence of SEQ ID NO: 5, X ₁₀ is any one selected from the group consisting of aspartic acid, glutamic acid, glycine, alanine, valine, isoleucine and leucine, or any amino acid that can be conservatively substituted therewith, X ₁₀ may be any one selected from the group consisting of aspartic acid, glutamic acid, glycine, alanine and valine, and X ₁₀ may be any one selected from the group consisting of aspartic acid, glutamic acid, alanine and valine. and X ₁₀ may be any one selected from the group consisting of aspartic acid and alanine.

In the amino acid sequence of SEQ ID NO: 5, X ₁₁ may be any one selected from the group consisting of glycine, alanine, valine, isoleucine and leucine, or any amino acid that can be conservatively substituted therewith, and X ₁₁ is glycine , may be any one selected from the group consisting of alanine and valine, wherein X ₁₁ may be any one selected from the group consisting of glycine and alanine.

In the amino acid sequence of SEQ ID NO: 5, X ₁₂ is any one selected from the group consisting of aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, glycine, alanine, valine, isoleucine and leucine, or a conservative substitution thereof It may be any possible amino acid, wherein X ₁₂ may be any one selected from the group consisting of aspartic acid, glutamic acid, serine, threonine, glycine, alanine, valine, isoleucine and leucine, wherein X ₁₂ is aspartic acid, It may be any one selected from the group consisting of glutamic acid, serine, threonine, glycine, alanine and valine, and X ₁₂ may be any one selected from the group consisting of glutamic acid, serine and alanine.

The amino acid sequence of SEQ ID NO: 5 is any one amino acid sequence selected from the group consisting of ISYDGENK (SEQ ID NO: 11) and ISYDGSNK (SEQ ID NO: 17), or 70% or more, 80% or more, 90% of the amino acid sequence It may be an amino acid sequence having at least 95% homology, or at least 99% homology.

In addition, the heavy chain variable region included in the antibody or antigen-binding fragment thereof of the present invention may further include CDR3-H.

The CDR3-H may include the amino acid sequence of X ₁₃ -TPKGX ₁₄ -X ₁₅ -LEX ₁₆ (SEQ ID NO: 6) or the amino acid sequence of ARQYSGVFEY (SEQ ID NO: 18), wherein X ₁₃ in the amino acid sequence of SEQ ID NO: 6 to X ₁₆ may each independently be any amino acid.

In the amino acid sequence of SEQ ID NO: 6, X ₁₃ may be any one selected from the group consisting of glycine, alanine, valine, isoleucine and leucine, or any amino acid that can be conservatively substituted therewith, and X ₁₃ is glycine , and may be any one selected from the group consisting of alanine and valine, wherein X ₁₃ may be any one selected from the group consisting of glycine and alanine, and X ₁₃ may be alanine.

In the amino acid sequence of SEQ ID NO: 6, X ₁₄ may be any one selected from the group consisting of glycine, alanine, valine, isoleucine and leucine, or any amino acid that can be conservatively substituted therewith, and X ₁₄ is glycine , may be any one selected from the group consisting of alanine and valine, wherein X ₁₄ may be any one selected from the group consisting of glycine and alanine.

In the amino acid sequence of SEQ ID NO: 6, X ₁₅ may be any one selected from the group consisting of glycine, alanine, valine, isoleucine and leucine, or any amino acid that can be conservatively substituted therewith, and X ₁₅ is glycine , and may be any one selected from the group consisting of alanine and valine, wherein X ₁₅ may be any one selected from the group consisting of glycine and alanine, and X ₁₅ may be alanine.

In the amino acid sequence of SEQ ID NO: 6, X ₁₆ may be any one selected from the group consisting of glycine, alanine, valine, isoleucine and leucine, or any amino acid that can be conservatively substituted therewith, and X ₁₆ is alanine , may be any one selected from the group consisting of valine, isoleucine and leucine, wherein X ₁₆ may be any one selected from the group consisting of alanine and isoleucine.

The amino acid sequence of SEQ ID NO: 6 is the amino acid sequence of ATPKGGALEI (SEQ ID NO: 12), or an amino acid sequence having 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more homology to the amino acid sequence can

On the other hand, the CDR3-H is the amino acid sequence of ARQYSGVFEY (SEQ ID NO: 18) or the amino acid sequence of SEQ ID NO: 18 and 70% or more, 80% or more, 90% or more, 95% or more, or an amino acid sequence having 99% or more homology may include

In addition, the heavy chain variable region included in the antibody or antigen-binding fragment thereof of the present invention is FR1-H, RQAPGKGLEWVAV (SEQ ID NO: 30) having an amino acid sequence of QVQLVQSGGGVVQPGRSLRLSCAAS (SEQ ID NO: 29) or an amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAAS (SEQ ID NO: 37) ), FR2-H having the amino acid sequence of YYADSVKGRFTISRDNSKNMLQLQMNRLRSEDTAVYYC (SEQ ID NO: 31) or FR3-H having the amino acid sequence of YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 38), and WGQGTLITVVITVSS (SEQ ID NO: 32) 39) may include at least one framework selected from the group consisting of FR4-H having the amino acid sequence. For example, the heavy chain variable region included in the antibody or antigen-binding fragment thereof of the present invention may have the amino acid sequence of SEQ ID NO: 43 or the amino acid sequence of SEQ ID NO: 44.

In addition, the light chain variable region included in the antibody or antigen-binding fragment thereof of the present invention is X ₁₇ -X ₁₈ -X ₁₉ -X ₂₀ -X ₂₁ -X ₂₂ -X ₂₃ -NX ₂₄ -X ₂₅ -X ₂₆ - a CDR1-L having the amino acid sequence of Y (SEQ ID NO: 7), wherein in the amino acid sequence of SEQ ID NO: 7, X ₁₇ to X ₂₀ and X ₂₂ to X ₂₆ are each independently any amino acid, and X ₂₁ is tyrosine , may be any one selected from the group consisting of phenylalanine and tryptophan, or any amino acid that can be conservatively substituted therewith.

In the amino acid sequence of SEQ ID NO: 7, X ₁₇ is any one selected from the group consisting of serine, threonine, asparagine, glutamine, glycine, alanine, valine, isoleucine and leucine, or any amino acid that can be conservatively substituted and X ₁₇ may be any one selected from the group consisting of asparagine, glutamine, glycine, alanine, valine, isoleucine and leucine, and X ₁₇ is selected from the group consisting of asparagine, glutamine, alanine and valine. and X ₁₇ may be any one selected from the group consisting of glutamine and alanine.

In the amino acid sequence of SEQ ID NO: 7, X ₁₈ is any one selected from the group consisting of serine, threonine, asparagine, glutamine, glycine, alanine, valine, isoleucine, and leucine, or any amino acid that can be conservatively substituted and X ₁₈ may be any one selected from the group consisting of serine, threonine, glycine, alanine, valine, isoleucine and leucine, and X ₁₈ is selected from the group consisting of serine, threonine, alanine and valine and X ₁₈ may be any one selected from the group consisting of serine and alanine.

In the amino acid sequence of SEQ ID NO: 7, X ₁₉ may be any one selected from the group consisting of glycine, alanine, valine, isoleucine and leucine or any amino acid that can be conservatively substituted therewith, and X ₁₉ is glycine , may be any one selected from the group consisting of alanine and valine, wherein X ₁₉ may be any one selected from the group consisting of alanine and valine.

In the amino acid sequence of SEQ ID NO: 7, X ₂₀ may be any one selected from the group consisting of glycine, alanine, valine, isoleucine and leucine or any amino acid that can be conservatively substituted therewith, and X ₂₀ is alanine , may be any one selected from the group consisting of valine, isoleucine and leucine, wherein X ₂₀ may be any one selected from the group consisting of alanine and leucine.

In the amino acid sequence of SEQ ID NO: 7, X ₂₁ may be any one selected from the group consisting of tyrosine, phenylalanine and tryptophan or any amino acid that can be conservatively substituted therewith, and X ₂₁ is tyrosine and phenylalanine It may be any one selected from the group being

In the amino acid sequence of SEQ ID NO: 7, X ₂₂ is any one selected from the group consisting of arginine, histidine, lysine, serine, threonine, asparagine, glutamine, glycine, alanine, valine, isoleucine and leucine, or a conservative substitution thereof may be any possible amino acid, wherein X ₂₂ may be any one selected from the group consisting of arginine, lysine, serine, threonine, glycine, alanine, valine, isoleucine and leucine, and X ₂₂ is arginine, lysine , may be any one selected from the group consisting of serine, threonine, glycine, alanine and valine, wherein X ₂₂ may be any one selected from the group consisting of arginine, lysine, serine, threonine, alanine and valine, , wherein X ₂₂ may be any one selected from the group consisting of arginine, serine and alanine.

In the amino acid sequence of SEQ ID NO: 7, X ₂₃ is any one selected from the group consisting of serine, threonine, asparagine, glutamine, glycine, alanine, valine, isoleucine and leucine, or any amino acid that can be conservatively substituted and X ₂₃ may be any one selected from the group consisting of serine, threonine, glycine, alanine, valine, isoleucine and leucine, and X ₂₃ is selected from the group consisting of serine, threonine, alanine and valine and X ₂₃ may be any one selected from the group consisting of serine and alanine.

In the amino acid sequence of SEQ ID NO: 7, X ₂₄ is any one selected from the group consisting of serine, threonine, asparagine, glutamine, glycine, alanine, valine, isoleucine and leucine, or any amino acid that can be conservatively substituted and X ₂₄ may be any one selected from the group consisting of asparagine, glutamine, glycine, alanine, valine, isoleucine and leucine, and X ₂₄ is asparagine, glutamine, glycine, alanine, and valine. may be any one selected from, and X ₂₄ may be any one selected from the group consisting of asparagine and alanine.

In the amino acid sequence of SEQ ID NO: 7, X ₂₅ is any one selected from the group consisting of arginine, histidine, lysine, glycine, alanine, valine, isoleucine and leucine, or any amino acid that can be conservatively substituted therewith , wherein X ₂₅ may be any one selected from the group consisting of arginine, lysine, glycine, alanine, valine, isoleucine and leucine, and X ₂₅ is selected from the group consisting of arginine, lysine, glycine, alanine and valine and X ₂₅ may be any one selected from the group consisting of arginine, lysine, alanine and valine, and X ₂₅ is any one selected from the group consisting of arginine, lysine and alanine can

In the amino acid sequence of SEQ ID NO: 7, X ₂₆ is any one selected from the group consisting of serine, threonine, asparagine, glutamine, glycine, alanine, valine, isoleucine and leucine, or any amino acid that can be conservatively substituted and X ₂₆ may be any one selected from the group consisting of asparagine, glutamine, glycine, alanine, valine, isoleucine and leucine, and X ₂₆ is asparagine, glutamine, glycine, alanine, and valine. may be any one selected from, and X ₂₆ may be any one selected from the group consisting of asparagine and alanine.

The amino acid sequence of SEQ ID NO: 7 is QSVLYSSNNKNY (SEQ ID NO: 13), ASVLYSSNNKNY (SEQ ID NO: 25), QSALYSSNNKNY (SEQ ID NO: 26), QSVLYSANNKNY (SEQ ID NO: 27), QSVLFSSNNKNY (SEQ ID NO: 28) and QSVLYRSNNRNY (SEQ ID NO: 19) It may be any one amino acid sequence selected from the group consisting of, or an amino acid sequence having 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more homology with the amino acid sequence.

The light chain variable region included in the antibody or antigen-binding fragment thereof of the present invention may further include a CDR-L2 comprising the amino acid sequence of WX ₂₇ -X ₂₈ (SEQ ID NO: 8), and the amino acid of SEQ ID NO: 8 In the sequence, X ₂₇ and X ₂₈ may each independently be any amino acid.

In the amino acid sequence of SEQ ID NO: 8, X ₂₇ may be any one selected from the group consisting of glycine, alanine, valine, isoleucine and leucine, or any amino acid that can be conservatively substituted therewith, and X ₂₇ is glycine , may be any one selected from the group consisting of alanine and valine, the X ₂₇ may be any one selected from the group consisting of glycine and alanine, the X ₂₇ may be alanine.

In the amino acid sequence of SEQ ID NO: 8, X ₂₈ is any one selected from the group consisting of serine, threonine, asparagine, glutamine, glycine, alanine, valine, isoleucine and leucine, or any amino acid that can be conservatively substituted and X ₂₈ may be any one selected from the group consisting of serine, threonine, glycine, alanine, valine, isoleucine and leucine, and X ₂₈ is selected from the group consisting of serine, threonine, alanine and valine and X ₂₈ may be any one selected from the group consisting of serine and alanine.

The amino acid sequence of SEQ ID NO: 8 is the amino acid sequence of WAS (SEQ ID NO: 14), or 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more or 90% of the amino acid sequence It may be an amino acid sequence having more than one homology.

In addition, the light chain variable region included in the antibody or antigen-binding fragment thereof of the present invention may further include a CDR3-L comprising the amino acids of QQYX ₂₉ -SX ₃₀ -PX ₃₁ -X ₃₂ (SEQ ID NO: 9), , In the amino acid of SEQ ID NO: 9, X ₃₀ and X ₃₂ may each independently be any amino acid, and X ₂₉ is any one selected from the group consisting of tyrosine, phenylalanine and tryptophan, or any one in which conservative substitution is possible It may be an amino acid, and X ₃₁ may be any one selected from the group consisting of isoleucine and tyrosine, or any amino acid that can be conservatively substituted therewith.

In the amino acid sequence of SEQ ID NO: 9, X ₂₉ may be any one selected from the group consisting of tyrosine, phenylalanine and tryptophan, or any amino acid that can be conservatively substituted therewith, and X ₂₉ is composed of tyrosine and phenylalanine It may be any one selected from the group being

In the amino acid sequence of SEQ ID NO: 9, X ₃₀ may be any one selected from the group consisting of glycine, alanine, valine, isoleucine and leucine or any amino acid that can be conservatively substituted therewith, and X ₃₀ is alanine , may be any one selected from the group consisting of valine, isoleucine and leucine, wherein X ₂₀ may be any one selected from the group consisting of alanine and isoleucine.

In the amino acid sequence of SEQ ID NO: 9, X ₃₁ may be any one selected from the group consisting of isoleucine and tyrosine, or any amino acid capable of conservative substitution therewith.

In the amino acid sequence of SEQ ID NO: 9, X ₃₂ is any one selected from the group consisting of serine, threonine, asparagine, glutamine, glycine, alanine, valine, isoleucine and leucine, or any amino acid that can be conservatively substituted and X ₃₂ may be any one selected from the group consisting of serine, threonine, alanine, valine, isoleucine and leucine, and X ₃₂ is any one selected from the group consisting of serine, threonine, alanine and valine may be one, and X ₃₂ may be any one selected from the group consisting of threonine and alanine.

The amino acid sequence of SEQ ID NO: 9 is any one amino acid sequence selected from the group consisting of QQYYSAPIT (SEQ ID NO: 15) and QQYFSIPYT (SEQ ID NO: 20), or 70% or more, 80% or more, 90% of the amino acid sequence It may be an amino acid sequence having at least 95% homology, or at least 99% homology.

In addition, the light chain variable region included in the antibody or antigen-binding fragment thereof of the present invention is FR1-L, LAWYQQKPGQPPNLLMY (SEQ ID NO: 34) having the amino acid sequence of DIQMTQSPDSLAVSLGERATINCKSS (SEQ ID NO: 33), or LAWYQQKLGQPPQLLIY (SEQ ID NO: 40) ), FR2-L having the amino acid sequence of TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 35) or FR3-L having the amino acid sequence of TRESGVPDRFSGSGSGADFTLTISSLQAEDVAVYYC (SEQ ID NO: 41), and FGQGTRLEIK (SEQ ID NO: 36) 42) may include at least one framework selected from the group consisting of FR4-L having the amino acid sequence. For example, the light chain variable region included in the antibody or antigen-binding fragment thereof of the present invention may have the amino acid sequence of SEQ ID NO: 45 or the amino acid sequence of SEQ ID NO: 46.

The antibody or antigen-binding fragment thereof of the present invention may be a humanized antibody or human antibody or antigen-binding fragment thereof, and in particular, a human antibody or antigen-binding fragment thereof. In particular, the antibody or antigen-binding fragment thereof of the present invention is a heavy chain constant region comprising any one amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 47 and the amino acid sequence of SEQ ID NO: 49 and/or SEQ ID NO: 51 It may include a light chain constant region including an amino acid sequence.

The antibody or antigen-binding fragment thereof of the present invention may specifically bind to the amino acid sequence of LPATLTMTVDK (SEQ ID NO: 3). In other words, the amino acid sequence of SEQ ID NO: 3 is an antigenic determinant of the antibody or antigen-binding fragment thereof of the present invention. Therefore, it can also specifically bind to a polypeptide or protein comprising the amino acid sequence of SEQ ID NO: 3. As a polypeptide or protein comprising the amino acid sequence of SEQ ID NO: 3, a typical Tie2 protein or an Ig2 domain thereof may be mentioned. The Tie2 protein may be derived from mammals, such as humans, monkeys, rats, mice, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and the like, and among them, may be derived from humans, monkeys, etc. . In the present invention, unless it is specified as a non-human species, the Tie2 protein means that it is derived from a human. In particular, the human-derived Tie2 protein may have the amino acid sequence of SEQ ID NO: 1, and its Ig2 domain is the amino acid of SEQ ID NO: 2 It may have a sequence. In this case, the amino acid sequence of SEQ ID NO: 3 is the region of 125th to 135th among the amino acid sequence of SEQ ID NO: 1. Accordingly, the antibody or antigen-binding fragment thereof of the present invention may specifically bind to a Tie2 protein or an Ig2 domain thereof, particularly a human Tie2 protein or an Ig2 domain thereof.

In addition, the antibody or antigen-binding fragment thereof of the present invention may have a dissociation constant (K _D ) value of 10 nM or less, 7 nM or less, 5 nM or less, 3 nM or less, 2 nM or less, 1.7 nM or less, or 1.5 nM or less. For example, the antibody or antigen-binding fragment thereof of the present invention is 1.4nM or less, 1.3nM or less, 1.2nM or less, 1.1nM or less, 1.0nM or less, 0.97nM or less, 0.95nM or less, 0.93nM or less, 0.90nM or less, 0.87 or less nM or less, 0.85 nM or less, 0.83 nM or less, 0.80 nM or less, 0.77 nM or less, 0.75 nM or less, 0.73 nM or less, 0.70 nM or less, 0.67 nM or less, 0.65 nM or less, 0.63 nM or less, 0.60 nM or less, 0.57 nM or less , 0.55 nM or less, 0.53 nM or less, 0.50 nM or less, 0.47 nM or less, 0.45 nM or less, 0.43 nM or less, 0.40 nM or less, 0.37 nM or less, 0.35 nM or less, 0.33 nM or less, 0.30 nM or less, 0.27 nM or less, 0.25 nM or less, 0.23 nM or less, 0.20 nM or less, 0.17 nM or less, 0.15 nM or less, 0.13 nM or less, 0.10 nM or less, 0.07 nM or less, 0.05 nM or less, 0.03 nM or less, 0.02 nM or less, or 0.01 nM or less ( K _D ) can bind to the amino acid sequence of SEQ ID NO: 3 above.

In addition, the antibody or antigen-binding fragment thereof of the present invention is 1×10 ⁴ M ^-1 s ^-1 or more, 2×10 ⁴ M ^-1 s ^-1 or more, 3×10 ⁴ M ^-1 s ^-1 or more, 4 A binding rate constant of at least ×10 ⁴ M ^-1 s ^-1 , at least 5×10 ⁴ M ^-1 s ^-1 , at least 6×10 ⁴ M ^-1 s ^-1 , or at least 7×10 ⁴ M ^-1 s ^-1 ( K _on ) may have a value. For example, the antibody or antigen-binding fragment thereof of the present invention is 7.3×10 ⁴ M ^-1 s ^-1 or more, 7.5×10 ⁴ M ^-1 s ^-1 or more, 7.7×10 ⁴ M ^-1 s ^-1 or more, 8.0 ×10 ⁴ M ^-1 s ^-1 or more, 8.3×10 ⁴ M ^-1 s ^-1 or more, 8.5×10 ⁴ M ^-1 s ^-1 or more, 8.7×10 ⁴ M ^-1 s ^-1 or more, 8.8×10 ⁴ M ^-1 s ^-1 or more, 8.9×10 ⁴ M ^-1 s ^-1 or more, 9.0×10 ⁴ M ^-1 s ^-1 or more, 9.1×10 ⁴ M ^-1 s ^-1 or more, 9.2×10 ⁴ M ^-1 s ^-1 or more, 9.3×10 ⁴ M ^-1 s ^-1 or more, 9.4×10 ⁴ M ^-1 s ^-1 or more, 9.5×10 ⁴ M ^-1 s ^-1 or more, 9.6×10 ⁴ M ^-1 s ^-1 or more, 9.7×10 ⁴ M ^-1 s ^-1 or more, 9.8×10 ⁴ M ^-1 s ^-1 or more, 9.9×10 ⁴ M ^-1 s ^-1 or more, 1.0×10 ⁵ M ^-1 s ^{- 1} or more, 1.05×10 ⁵ M ^-1 s ^-1 or more, 1.1×10 ⁵ M ^-1 s ^-1 or more, 1.15×10 ⁵ M ^-1 s ^-1 or more, 1.2×10 ⁵ M ^-1 s ^-1 or more , 1.25×10 ⁵ M ^-1 s ^-1 or more, 1.3×10 ⁵ M ^-1 s ^-1 or more, 1.35×10 ⁵ M ^-1 s ^-1 or more, 1.4×10 ⁵ M ^-1 s ^-1 or more, 1.45 It may bind to the amino acid sequence of SEQ ID NO: 3 with a binding rate constant (K _on ) value of at least ×10 ⁵ M ⁻¹ s ⁻¹ or greater than or equal to 1.5×10 ⁵ M ⁻¹ s ⁻¹ .

In addition, the antibody or antigen-binding fragment thereof of the present invention is 1×10 ^-3 s ^-1 or less, 7×10 ^-4 s ^-1 or less, 5×10 ^-4 s ^-1 or less, 3×10 ^-4 s ^- It may be one having a dissociation rate constant (K _off ) value of ¹ or less or 2×10 ^-4 s ^-1 or less. For example, the antibody or antigen-binding fragment thereof of the present invention is 1.7×10 ^-4 s ^-1 or less, 1.5×10 ^-4 s ^-1 or less, 1.0×10 ^-4 s ^-1 or less, 9.7×10 ^-5 s ^{- 1} or less, 9.5×10 ^-5 s ^-1 or less, 9.3×10 ^-5 s ^-1 or less, 9.0×10 ^-5 s ^-1 or less, 8.7×10 ^-5 s ^-1 or less, 8.5×10 ^-5 s ^{- 1} or less, 8.3×10 ^-5 s ^-1 or less, 8.0×10 ^-5 s ^-1 or less, 7.7×10 ^-5 s ^-1 or less, 7.5×10 ^-5 s ^-1 or less ^, 7.3×10 ^-5 s -1 or less ¹ or less, 7.0×10 ^-5 s ^-1 or less, 6.7×10 ^-5 s ^-1 or less, 6.5×10 ^-5 s ^-1 or less, 6.3×10 ^-5 s ^-1 or less ^, 6.0×10 ^-5 s -1 or less ¹ or less, 5.7×10 ^-5 s ^-1 or less, 5.5×10 ^-5 s ^-1 or less, 5.3×10 ^-5 s ^-1 or less, 5.0×10 ^-5 s ^-1 or less ^, 4.7×10 ^-5 s -1 or less ¹ or less, 4.5×10 ^-5 s ^-1 or less, 4.3×10 ^-5 s ^-1 or less, 4.0×10 ^-5 s ^-1 or less, 3.7×10 ^-5 s ^-1 or less ^, 3.5×10 ^-5 s -1 or less ¹ or less, 3.3×10 ^-5 s ^-1 or less, 3.0×10 ^-5 s ^-1 or less, 2.7×10 ^-5 s ^-1 or less, 2.5×10 ^-5 s ^-1 or less, 2.3×10 ^-5 s ^{- 1} or less, 2.0×10 ^-5 s ^-1 or less, 1.7×10 ^-5 s ^-1 or less, 1.5×10 ^-5 s ^-1 or less, 1.3×10 ^-5 s ^-1 or less, or 1.0×10 ^-5 s ^- It can bind to the amino acid sequence of SEQ ID NO: 3 with a dissociation rate constant (K _off ) value of ¹ or less.

In addition, the antibody or antigen-binding fragment thereof of the present invention may have a melting temperature (T _m ) of 70°C or higher, 73°C or higher, 75°C or higher, 77°C or higher, or 80°C or higher. For example, the antibody or antigen-binding fragment thereof of the present invention is 81 ° C. or higher, 82 ° C. or higher, 83 ° C. or higher, 84 ° C. or higher, 85 ° C. or higher, 86 ° C. or higher, 87 ° C. or higher, 88 ° C. or higher, 89 ° C. or higher, 90 It may have a melting temperature (T _m ) of ℃ or higher, 91 °C or higher, 92 °C or higher, 93 °C or higher, 94 °C or higher, or 95 °C or higher.

On the other hand, when the antibody or antigen-binding fragment thereof of the present invention binds to the Tie2 protein comprising the amino acid sequence of SEQ ID NO: 3, the Tie2 protein is self-phosphorylated regardless of binding to its ligands Ang1, Ang2, etc. can be, thereby activating a signal transduction pathway in the cell. Accordingly, the antibody or binding fragment thereof of the present invention may serve to activate the Tie2 protein by replacing the ligand of the Tie2 protein.

2. Related genes of novel antibodies or antigen-binding fragments thereof, and vectors and transformants containing the same

Another aspect of the present invention provides a gene encoding all or a part of the antibody or antigen-binding fragment thereof of the present invention.

In one embodiment of the present invention, a gene encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention and a gene encoding the light chain variable region of the antibody or antigen-binding fragment thereof of the present invention may be provided, respectively. . The gene encoding the heavy chain variable region may, for example, encode the amino acid sequence of SEQ ID NO: 43 or the amino acid sequence of SEQ ID NO: 44, and the gene encoding the light chain variable region may include, for example, the amino acid sequence of SEQ ID NO: 45 or SEQ ID NO: 46 It may be one that encodes the amino acid sequence of In the gene encoding the variable region of the heavy or light chain, various modifications may be made to the coding region within a range that does not change the amino acid sequence of the variable regions expressed from the coding region, and the expression of the gene is also performed in parts other than the coding region. Various mutations can be made within the range that does not affect the Consequently, as long as the gene of the variable regions encodes a protein having an equivalent activity, one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, and such mutated genes are also included within the scope of the present invention. do.

In another embodiment of the present invention, there is provided a gene encoding each of the CDRs included in the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention. Specifically, a gene encoding CDR1-H, a gene encoding CDR2-H, and a gene encoding CDR3-H are each provided individually. In particular, the gene encoding the CDR1-H may encode the amino acid sequence of SEQ ID NO: 4, for example, the gene encoding the CDR1-H is the amino acid sequence of SEQ ID NO: 10, the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: It may encode any one amino acid sequence selected from the group consisting of the amino acid sequence of 22, the amino acid sequence of SEQ ID NO: 23, the amino acid sequence of SEQ ID NO: 24 and SEQ ID NO: 16. In addition, the gene encoding the CDR2-H may encode the amino acid sequence of SEQ ID NO: 5, for example, the amino acid sequence of SEQ ID NO: 11 or the amino acid sequence of SEQ ID NO: 17. In addition, the gene encoding the CDR3-H may encode the amino acid sequence of SEQ ID NO: 6, particularly, the amino acid sequence of SEQ ID NO: 12, or encode the amino acid sequence of SEQ ID NO: 18.

In another embodiment of the present invention, there is provided a gene encoding each of the CDRs included in the light chain variable region of the antibody or antigen-binding fragment thereof of the present invention. Specifically, a gene encoding CDR1-L, a gene encoding CDR2-L and a gene encoding CDR3-L are each provided individually. In particular, the gene encoding the CDR1-L may encode the amino acid sequence of SEQ ID NO: 7, for example, the gene encoding the CDR1-L includes the amino acid sequence of SEQ ID NO: 13, the amino acid sequence of SEQ ID NO: 25, and SEQ ID NO: It may encode any one amino acid sequence selected from the group consisting of the amino acid sequence of 26, the amino acid sequence of SEQ ID NO: 27, the amino acid sequence of SEQ ID NO: 28, and the amino acid sequence of SEQ ID NO: 19. In addition, the gene encoding the CDR2-L may be one encoding the amino acid sequence of SEQ ID NO: 8, particularly the amino acid sequence of SEQ ID NO: 14, and the gene encoding the CDR3-L is the amino acid sequence of SEQ ID NO: 9, particularly the sequence It may be one encoding the amino acid sequence of No. 15 or the amino acid sequence of SEQ ID NO: 20.

In the gene encoding the CDRs, various modifications can be made to the coding region within a range that does not change the amino acid sequence of the CDRs expressed from the coding region, and within a range that does not affect the expression of the gene in parts other than the coding region. Various mutations can be made in As a result, one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof as long as the gene of the CDRs encodes a protein having an equivalent activity, and such a mutated gene is also included in the scope of the present invention. .

In addition, another aspect of the present invention provides a recombinant expression vector containing a gene encoding all or part of the antibody or antigen-binding fragment thereof of the present invention, and a transformant into which the expression vector is introduced.

The recombinant expression vector of the present invention includes a gene encoding all or part of the antibody or antigen-binding fragment thereof of the present invention, and the gene may be operably linked to a promoter.

The expression vector includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector.

The recombinant expression vector of the present invention is a promoter, terminator, enhancer, etc. according to the type of host cell in which all or part of the antibody or antigen-binding fragment thereof of the present invention is to be expressed or produced. The same expression control sequence, or a sequence for secretion, etc. can be appropriately combined depending on the purpose.

The expression vector may further include a selection marker for selecting a host cell into which the vector is introduced, and in the case of an expression vector capable of replication, it may include an origin of replication.

In addition, the recombinant expression vector may include a sequence for facilitating purification of the expression protein, specifically, a tag for separation and purification so as to be operable on a gene encoding all or part of the antibody or antigen-binding fragment thereof of the present invention Genes encoding for can be linked. In this case, as the tag for separation and purification, GST, poly-Arg, FLAG, histidine-tag (His-tag), c-myc, etc. may be used alone or two or more of them may be sequentially connected. The gene encoding all or part of the antibody or antigen-binding fragment thereof of the present invention may be cloned through a restriction enzyme cleavage site, and when a gene encoding a protease recognition site is used in the vector, the present invention It is linked in frame with the gene encoding all or part of the antibody or antigen-binding fragment thereof of the present invention, and after obtaining all or part of the antibody or antigen-binding fragment thereof of the present invention, cleavage with a proteolytic enzyme In this case, all or part of the antibody or antigen-binding fragment thereof of the present invention in its original form can be produced.

In addition, a recombinant expression vector containing a gene encoding all or part of the antibody or antigen-binding fragment thereof of the present invention is introduced into the transformant of the present invention.

Transformants can be prepared by transforming the recombinant expression vector according to the present invention into any suitable host cell selected from the group consisting of bacteria, yeast, E. coli, fungi, plant cells and animal cells depending on the purpose of expression. . For example, the host cell may be E. coli (E. coli BL21(DE3), DH5α, etc.) or yeast cells (Saccharomyces genus, Pichia genus, etc.). In this case, an appropriate culture method and medium conditions according to the type of host cell can be easily selected by those skilled in the art from known techniques in the art.

As a method for introducing a recombinant expression vector for producing a transformant of the present invention, a known technique, that is, a heat shock method, an electric shock method, or the like may be used.

Since the protein expressed from the transformant is the antibody or antigen-binding fragment thereof of the present invention, mass production of the antibody or antigen-binding fragment thereof of the present invention is facilitated by mass culturing the transformant to express the gene. can do.

3. Use of novel antibodies or antigen-binding fragments thereof

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

The angiogenesis-related disease refers to the formation or growth of new blood vessels from existing blood vessels, and the angiogenesis-related disease refers to a disease related to the occurrence or progression of angiogenesis. As long as it can be prevented or treated by the antibody or antigen-binding fragment thereof of the present invention, the disease may be included in the scope of the angiogenesis-related disease without limitation.

The antibody or antigen-binding fragment thereof of the present invention can bind to the Tie2 protein comprising the amino acid sequence of SEQ ID NO: 3 to self-phosphorylate the Tie2 protein, thereby transducing signals involved in angiogenesis in cells path can be activated. As described above, when angiogenesis-related signal transduction pathways are activated due to phosphorylation of Tie2 protein, necessary angiogenesis is promoted (Fukuhara et al. , Histol. Histopathol. , 25 , 387-396 (2010)), abnormally The blood vessels that have been generated are matured and stabilized (Koh, Trends Mol. Med. , 19 (1), 31-39 (2013); Anisimov et al. , Circulation, 127 , 424-434 (2013); Park et al. , Cancer Cell, 30 , 953-967 (2016)., Lee et al. , Science Translational Medicine, 5 (203), 203ra127 (2013); Cahoon et al. , Diabetes, 64 (12), 4247-4259 (2015) ; Han et al. , Science Translational Medicine, 8 (335), 335ra55 (2016)). Therefore, the antibody or antigen-binding fragment thereof of the present invention can be used as an active ingredient in the prevention or treatment of diseases caused by insufficient angiogenesis or diseases caused by abnormal blood vessels.

In one embodiment of the present invention, the angiogenesis-related disease may be a disease caused by insufficient angiogenesis. As described above, diseases caused by insufficient angiogenesis include, for example, ischemia occurring in the brain, heart, and peripheral tissues, cerebral infarction, myocardial infarction, lower extremity ischemia, wound delay, foot ulcer, retinopathy of prematurity, and erectile dysfunction. , organ transplantation, etc., but is not limited thereto.

Also, in another embodiment of the present invention, the angiogenesis-related disease may be a disease caused by abnormal blood vessels or excessive abnormal angiogenesis. As described above, diseases caused by abnormal blood vessels or excessive abnormal angiogenesis may be caused by leakage of blood vessels, inflammation, or local ischemia, for example, cancer, metastatic cancer, diabetic retinopathy, macular degeneration, sepsis, Angiogenic glaucoma, psoriasis, rheumatoid arthritis, diabetic nephropathy, diabetic foot ulcers, vascular adhesion, atherosclerosis, It may be capillary formation of atherosclerotic plaques, acute liver failure, acute renal failure, acute lung failure, systemic inflammatory reaction syndrome, and the like, but is not limited thereto.

The prophylaxis refers to any measure that suppresses or delays the onset of a disease. In addition, the treatment refers to all measures to improve or ameliorate the symptoms of the expressed disease, and desirable effects of the treatment include prevention of the occurrence or recurrence of the disease, alleviation of symptoms, any direct or indirect pathological pathology of the disease. reducing outcome, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and remission or improved prognosis.

The pharmaceutical composition may further include a pharmaceutically acceptable carrier or additive in addition to the antibody or antigen-binding fragment thereof of the present invention.

The meaning of 'pharmaceutically acceptable' means that it does not inhibit the activity of the active ingredient and does not have more toxicity than the application (prescription) target can adapt to. The carrier means a compound that facilitates the addition of the antibody or antigen-binding fragment thereof of the present invention into a cell or tissue, and the additive is any component necessary to prepare a pharmaceutical composition into the required dosage form, such as an excipient , means a disintegrant, a binder, a flavoring agent, an antiseptic, a lubricant, a stabilizer, a viscous agent, and the like.

In addition, the pharmaceutical composition may be administered in combination with therapeutic agents for each disease. In this case, the pharmaceutical composition may further include at least one therapeutic agent for each disease in addition to the antibody or antigen-binding fragment thereof of the present invention. The other treatment for each disease may include an anti-angiogenic drug, an anti-inflammatory drug, and/or an anti-cancer drug.

In addition, another aspect of the present invention provides a method for preventing or treating angiogenesis-related diseases, comprising administering the antibody or antigen-binding fragment thereof of the present invention, or the pharmaceutical composition to a subject in need thereof.

The subject may be a mammal, for example a human, and in particular a patient. In addition, the 'individual in need' refers to an individual suffering from, or likely to suffer from, an angiogenesis-related disease.

The antibody or antigen-binding fragment thereof of the present invention, or the pharmaceutical composition may be administered in a pharmaceutically effective amount. The 'pharmaceutically effective amount' means an amount sufficiently exhibiting the effect of prevention or treatment with a reasonable benefit/risk ratio applicable to all medical treatments. In addition, the 'pharmaceutically effective amount' refers to the severity of the disease to be treated, the age and sex of the patient, the type of disease, the activity of the drug, the sensitivity to the drug, the administration time, the administration route, the secretion rate, the treatment period, the drug depends on a variety of factors, including co-administration of the drug and other parameters known in the art. The pharmaceutical composition may be administered alone or in combination with other therapies. In such cases, they may be administered sequentially or simultaneously with conventional therapy. In addition, the composition may be administered in a single dose or divided into multiple doses. When these factors are fully considered, it is important to administer a minimum amount sufficient to obtain the maximum effect without side effects, and this dosage can be readily determined by a person skilled in the art. The dosage of the pharmaceutical composition is not particularly limited, but varies according to various factors including the patient's health condition and weight, the severity of the disease, the type of drug, the administration route, and the administration time. In particular, the pharmaceutical composition is administered in a single or multiple daily doses into mammals, including humans, by routes typically accepted by routes such as orally, rectally, intravenously, subcutaneously, intraperitoneally. It may be administered (intraperitoneally), intrauterinely (intrauterinely) or intracerebrovascularly (intracerebrovascularly).

In addition, the antibody or antigen-binding fragment thereof of the present invention, or the pharmaceutical composition may be administered in combination with other therapeutic agents for angiogenesis-related diseases. In this case, the antibody or antigen-binding fragment thereof of the present invention, or the pharmaceutical composition may be administered simultaneously or sequentially with other therapeutic agents such as anti-angiogenic drugs, anti-inflammatory drugs and/or anti-cancer drugs. For example, after administration of another therapeutic agent such as an anti-angiogenic drug, an anti-inflammatory drug and/or an anticancer drug to a subject first, the antibody or antigen-binding fragment thereof, or the pharmaceutical composition of the present invention is added to the subject Or, after administering the antibody or antigen-binding fragment thereof of the present invention, or the pharmaceutical composition to the subject first, other therapeutic agents such as anti-angiogenic drugs, anti-inflammatory drugs and/or anti-cancer drugs are additionally administered to the subject can be administered. In addition, depending on the case, the antibody or antigen-binding fragment thereof of the present invention, or the pharmaceutical composition and other therapeutic agents such as anti-angiogenic drugs, anti-inflammatory drugs and / or anti-cancer drugs may be administered to the subject at the same time.

Hereinafter, the present invention will be described in detail by way of Examples.

However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

Design of Antibodies that Specific Binding to Human Tie2 Protein

[1-1] Design and fabrication of antibodies

[1-1-1] Design of the first primary antibody

The amino acid sequences of CDR1-H, CDR2-H and CDR3-H are designed as shown in Table 1 below, and each of them is FR1-H having the amino acid sequence of SEQ ID NO: 29, FR2-H having the amino acid sequence of SEQ ID NO: 30, The first primary antibody, Ang1mab1, is interposed in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 between FR3-H having the amino acid sequence of SEQ ID NO: 31 and FR4-H having the amino acid sequence of SEQ ID NO: 32 and the heavy chain variable region of Ang1mab4 were designed.

CDR1-H CDR2-H CDR3-H GLTFSNYAMHWV ISYDGENK ATPKGGALEI SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 12

Similarly, the amino acid sequences of CDR1-L, CDR2-L and CDR3-L are designed as shown in Table 2 below, and each of them is FR1-L having the amino acid sequence of SEQ ID NO: 33, FR2- having the amino acid sequence of SEQ ID NO: 34 L, FR3-L having the amino acid sequence of SEQ ID NO: 35 and FR4-L having the amino acid sequence of SEQ ID NO: 36 Interposed in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the first primary antibody The light chain variable regions of Ang1mab1 and Ang1mab4 were designed.

CDR1-L CDR2-L CDR3-L QSVLYSSNNKNY WAS QQYYSAPIT SEQ ID NO: 13 SEQ ID NO: 14 SEQ ID NO: 15

The heavy chain variable region designed as described above, the N-terminus of the heavy chain constant region having the amino acid sequence of SEQ ID NO: 47, in which the three constant regions are arranged in the order of C _H 1-hinge-C _H 2 -C _H 3 The heavy chain of the primary antibody Ang1mab1, and the heavy chain variable region designed as described above, were placed at the N-terminus of the heavy chain constant region having the amino acid sequence of SEQ ID NO: 49, which is a subclass of IgG4 heavy chain constant region, Each of the heavy chains of the primary antibody Ang1mab4 was designed. And, the light chain variable region designed as described above was placed at the N-terminus of the light chain constant region (CL) having the amino acid sequence of SEQ ID NO: 51 to design the light chains of the first primary antibodies _Ang1mab1 and Ang1mab4.

[1-1-2] Design of the second primary antibody Ang1mab2-1

The amino acid sequences of CDR1-H, CDR2-H and CDR3-H are designed as shown in Table 3 below, and each of them is FR1-H having the amino acid sequence of SEQ ID NO: 37, FR2-H having the amino acid sequence of SEQ ID NO: 30; The heavy chain of the Ang1mab2-1 antibody by interposing in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 between FR3-H having the amino acid sequence of SEQ ID NO: 38 and FR4-H having the amino acid sequence of SEQ ID NO: 39 Variable regions were constructed.

CDR1-H CDR2-H CDR3-H GFTFSSYAMHWV ISYDGSNK ARQYSGVFEY SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18

Similarly, the amino acid sequences of CDR1-L, CDR2-L and CDR3-L are designed as shown in Table 4 below, and each of them is FR1-L having the amino acid sequence of SEQ ID NO: 33, FR2- having the amino acid sequence of SEQ ID NO: 40 A second primary antibody interposed in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 between L, FR3-L having the amino acid sequence of SEQ ID NO: 41 and FR4-L having the amino acid sequence of SEQ ID NO: 42 The light chain variable region of Ang1mab2-1 was constructed.

CDR1-L CDR2-L CDR3-L QSVLYRSNNRNY WAS QQYFSIPYT SEQ ID NO: 19 SEQ ID NO: 14 SEQ ID NO: 20

The heavy chain variable region designed as described above, the N-terminus of the heavy chain constant region having the amino acid sequence of SEQ ID NO: 47, in which the three constant regions are arranged in the order of C _H 1-hinge-C _H 2 -C _H 3 to design the heavy chain of the second primary antibody Ang1mab2-1. Then, the light chain variable region designed as described above was arranged at the N-terminus of the light chain constant region (CL) having the amino acid sequence of SEQ ID NO: 51 to design the light chain of the second basic antibody Ang1mab2-1.

[1-1-3] Production of antibodies

Base sequences encoding the heavy and light chains of the three types of antibodies (Ang1mab1, Ang1mab4 and Ang1mab2-1) designed in Examples [1-1-1] and [1-1-2] were introduced into pcDNA3.4 vector Thus, five types of expression vectors were prepared.

Then, a total of 100 ug of each 50 ug each of the heavy and light chain expression vectors was diluted in 6 ml of Opti-MEM medium, and 320 ul of the ExpiFectamine ^TM 293 Transfection Kit reagent (ExpiFectamine ^TM 293 Reagent) was diluted in 6 ml of Opti-MEM medium and allowed to stand for 5 minutes each. . The vector-added medium and the reagent-added medium were mixed and left still for 10 minutes, then added to 100 ml of Expi293F ^TM cells (2.0x10 ⁶ cells/ml) and suspended in culture for 24 hours. ExpiFectamine ^TM 293 Transfection Enhancer was added to the cultured cells as described above and further cultured for 4 days, then centrifuged at 10,000xg for 30 minutes and filtered through a 0.2um filter to recover the supernatant. Then, the protein expressed in the supernatant was bound to an affinity column (MabSelect prismA) using an AKTA purifier100 instrument, and then eluted with an acidic eluate (50 mM acetic acid buffer, pH 4.0). The protein eluted as described above was finally purified with PBS buffer using a desalting column (Hiprep 26/10). The purified protein as described above is an antibody having a total molecular weight of about 150 kDa having a heavy chain (about 50 kDa) and a light chain (about 25 kDa) through SDS-PAGE analysis under non-reducing and reducing conditions. It was confirmed that

[1-2] Identification of key residues in CDRs

As a representative technique for confirming the contribution of a specific residue to the stability and function of a protein, there is an alanine scanning technique. Alanine is not large in size, is chemically inactive, and has a methyl functional group that mimics the secondary structure preference of other amino acids. It is a technique to confirm the contribution of a specific amino acid by checking how the function changes. For example, if there is no significant change in the stability or function of a protein despite the substitution of a specific amino acid with alanine, the specific amino acid is highly likely to be an amino acid that does not affect the stability or function of the protein. If there is a significant change in the stability or function of the protein, it can be determined that the specific amino acid is highly likely to be an amino acid that affects the stability or function of the protein (Morrison et al. , Curr. Opin. Chem. Biol. , 5 (3):302-307 (2001)). In the present invention, according to the alanine scanning technique as described above, it was confirmed what the major amino acids were in the CDRs designed in Example [1-1].

Specifically, each amino acid in the CDRs of Tables 1 and 2 constituting the heavy chain variable region and light chain variable region of Ang1mab1, which is one of the first basic antibodies actually implemented in Example [1-1-3], is as follows. As shown in Table 5, preliminary mutant antibodies substituted with alanine were designed. And, by point mutagenesis, the DNA codon encoding the amino acid at each position was converted into the DNA codon encoding the alanine to prepare expression vectors of the heavy and light chains of the preliminary mutated antibodies. The preliminary mutant antibodies prepared as described above were expressed and separated/purified in the same manner as in Example [1-1-3].

CDRs of heavy chain variable regions CDRs of light chain variable regions One CDR1-H HC1-G01A One CDR1-L LC1-Q01A 2 CDR1-H HC1-L02A 2 CDR1-L LC1-S02A 3 CDR1-H HC1-T03A 3 CDR1-L LC1-V03A 4 CDR1-H HC1-F04A 4 CDR1-L LC1-L04A 5 CDR1-H HC1-S05A 5 CDR1-L LC1-Y05A 6 CDR1-H HC1-N06A 6 CDR1-L LC1-S06A 7 CDR1-H HC1-Y07A 7 CDR1-L LC1-S07A 8 CDR1-H HC1-M09A 8 CDR1-L LC1-N08A 9 CDR1-H HC1-H10A 9 CDR1-L LC1-N09A 10 CDR1-H HC1-W11A 10 CDR1-L LC1-K10A 11 CDR1-H HC1-V12A 11 CDR1-L LC1-N11A 12 CDR2-H HC2-I01A 12 CDR1-L LC1-Y12A 13 CDR2-H HC2-S02A 13 CDR2-L LC2-W01A 14 CDR2-H HC2-Y03A 14 CDR2-L LC2-S03A 15 CDR2-H HC2-D04A 15 CDR3-L LC3-Q01A 16 CDR2-H HC2-G05A 16 CDR3-L LC3-Q02A 17 CDR2-H HC2-E06A 17 CDR3-L LC3-Y03A 18 CDR2-H HC2-N07A 18 CDR3-L LC3-Y04A 19 CDR2-H HC2-K08A 19 CDR3-L LC3-S05A 20 CDR3-H HC3-T02A 20 CDR3-L LC3-P07A 21 CDR3-H HC3-P03A 21 CDR3-L LC3-I08A 22 CDR3-H HC3-K04A 22 CDR3-L LC3-T09A 23 CDR3-H HC3-G05A 24 CDR3-H HC3-G06A 25 CDR3-H HC3-L08A 26 CDR3-H HC3-E09A 27 CDR3-H HC3-I10A

For the 49 preliminary mutant antibodies obtained as described above, binding ability to human Tie2 protein was confirmed through ELISA analysis.

Specifically, 100 μl of human Tie2-Fc recombinant protein (antigen) at a concentration of 1 ug/ml was dispensed in a 96-well ELISA plate (Maxisorp) and left at 4 °C for 16 hours to immobilize the antigen on the surface of each well. Then, a blocking buffer containing 3% skim milk was treated and reacted at room temperature for 2 hours, and then 49 preliminary mutant antibodies obtained as above were added to each well at 0.5ug/ml 100ul each was treated with a concentration of , and the reaction was further carried out for 1 hour. Each well was washed twice with 0.1% TBS-Tween20 (TBST), and then treated with goat anti-human IgG-HRP (HRP-conjugated goat anti-human IgG) and reacted for 1 hour. Again, each well was washed 3 times with TBST, and then treated with 50ul each of o-phenylenediamine, a substrate, and reacted for 5 minutes. And after the reaction was terminated by treating each well with 50ul of a reaction stop solution (2.5MH ₂ SO ₄ ), the absorbance was measured at 450 nm using an ELISA reader to compare the antigen binding power of the preliminary mutant antibodies.

As a result, as shown in FIG. 1 , in the case of the 1st to 6th and 12th amino acids of CDR1-H, the 2nd and 4th to 6th amino acids of CDR2-H, and the 6th amino acid of CDR3-H And in the case of the 10th amino acid, binding to human Tie2 protein was not reduced despite the substitution with alanine, and in the case of the 1st to 4th, 6th, 7th and 9th to 11th amino acids of CDR1-L, In the case of the 3rd amino acid of CDR2-L and the 9th amino acid of CDR3-L, it was confirmed that the binding affinity to human Tie2 protein did not decrease to less than 75% compared to the primary antibody despite the substitution with alanine.

From the above results, among the 12 amino acids constituting the CDR1-H of the heavy chain variable region, 7 amino acids (the 1st to 6th and 12th amino acids) identified above and the original 8th amino acid, which is alanine, are excluded from a total of 4 A total of 4 amino acids excluding the 4 amino acids (2nd and 4th to 6th amino acids) identified above among 8 amino acids constituting CDR2-H of the heavy chain variable region, and CDR3 of the heavy chain variable region Of the 10 amino acids constituting -H, a total of 6 amino acids excluding the 2 amino acids (6th and 10th amino acids) identified above and the 1st and 7th amino acids which are originally alanine, the basic antibody Ang1mab1 is a human Tie2 protein It was found to be an essential amino acid involved in binding to In addition, a total of 3 amino acids excluding the 9 amino acids (1st to 4th, 6th, 7th and 9th to 11th amino acids) identified above among the 12 amino acids constituting CDR1-L of the light chain variable region Among the three amino acids constituting CDR2-L of the light chain variable region, one amino acid (third amino acid) identified above and a total of one amino acid excluding the second amino acid, which is originally alanine, and CDR3 of the light chain variable region Of the 9 amino acids constituting -L, a total of 7 amino acids except for 1 amino acid (9th amino acid) identified above and the 6th amino acid, which is the original alline, maintains the binding ability of the basic antibody Ang1mab1 to human Tie2 protein It was found to be an essential amino acid that is involved in

[1-3] Design and manufacture of mutant antibodies

Based on the results of Example [1-2] above, a mutant antibody in which the 1st, 2nd, 5th and 12th amino acids of the CDR1 of the heavy chain variable region of the basic antibody Ang1mab4 are substituted with alanine, and the CDR1 of the light chain variable region Mutant antibodies in which the 1st, 3rd and 7th amino acids were substituted with alanine were produced and separated/purified in the same manner as in Example [1-1-3], and additionally, CDR1 of the light chain variable region of the primary antibody Ang1mab4 A mutant antibody in which the 5th amino acid was substituted with phenylalanine was additionally designed and produced and separated/purified in the same manner as in Example [1-1-3], a total of 8 mutant antibodies having CDRs as shown in Table 6 below was prepared.

mutant antibody CDR1-H CDR2-H CDR3-H CDR1-L CDR2-L CDR3-L H1
G01A A LTFSNYAMHWV
(SEQ ID NO: 21) ISYDGENK
(SEQ ID NO: 11) ATPKGGALEI
(SEQ ID NO: 12) QSVLYSSNNKNY
(SEQ ID NO: 13) WAS
(SEQ ID NO: 14) QQYYSAPIT
(SEQ ID NO: 15) H1
L02A G A TFSNYAMHWV
(SEQ ID NO: 22) same as above same as above same as above same as above same as above H1
S05A GLTF A NYAMHWV
(SEQ ID NO: 23) same as above same as above same as above same as above same as above H1
V12A GLTFSNYAMHW A
(SEQ ID NO: 24) same as above same as above same as above same as above same as above L1
Q01A GLTFSNYAMHWV
(SEQ ID NO: 10) same as above same as above A SVLYSSNNKNY
(SEQ ID NO: 25) same as above same as above L1
V03A same as above same as above same as above QS A LYSSNNKNY
(SEQ ID NO: 26) same as above same as above L1
S07A same as above same as above same as above QSVLYS A NNKNY
(SEQ ID NO: 27) same as above same as above L1
Y05F same as above same as above same as above QSVL F SSNNKNY
(SEQ ID NO: 28) same as above same as above

[1-4] Design and Construction of Control Antibodies

In the first basic antibodies Ang1mab1 and Ang1mab4, the CDR3-H of the heavy chain variable region was designed by changing the amino acid sequence of SEQ ID NO: 53, and produced and isolated / in the same manner as in Example [1-1-3] After purification, two control antibodies (IgG1 control and IgG4 control) that do not bind to human Tie2 protein were prepared, respectively.

Confirmation of Affinity for Antigens of Primary and Variant Antibodies

For the three first and second primary antibodies (Ang1mab1, Ang1mab4 and Ang1mab2-1) prepared in Example [1-1] and the 8 mutant antibodies prepared in Example [1-3], antigens Binding ability to human Tie2 protein was confirmed.

The affinity of the antibodies was measured with an Octet K2 (ForteBio) device based on the BLI (Biolayer Interferometry) technique. Specifically, the AR2G biosensor tip was hydrated with tertiary distilled water for 10 minutes and then 20 mM EDC, 10 mM Sulfo-NHS It was activated using a buffer containing Then, 45 nM of human Tie2-Fc protein (antigen) was diluted in immobilization buffer (10 mM sodium acetate; pH 6.0) and reacted with the activated AR2G tip for 10 minutes to immobilize the antigen to the AR2G tip. . Thereafter, the antigen immobilization reaction was terminated by treatment with a 1M ethanolamine (ethanolamine, pH 8.5) solution, and the AR2G tip was reacted in PBS buffer for 60 seconds to obtain a baseline of antigen-antibody binding. Then, the first and second primary antibodies or the eight variant antibodies were diluted in PBS to concentrations of 30, 15, 7.5, 3.75 and 1.63 nM, and the antigen was bound to the immobilized AR2G tip for 1,000 seconds. After (association), it was dissociated for 1,800 seconds. Based on the reaction curve confirmed through the association and dissociation reaction analysis, the affinity of each antibody was measured by calculating K _on , K _off , and K _D values through global fitting analysis of the Data Analysis HT 12.0 program.

antibody K _on (1/Ms) K _off (1/s) K _D (M) Ang1mab1 9.70×10 ⁴ 4.62×10 ^-5 4.76×10 ^-10 Ang1mab4 8.70×10 ⁴ 4.60×10 ^-5 5.29×10 ^-10 Ang1mab4 (H1G01A) 9.38×10 ⁴ 7.12×10 ^-5 7.61×10 ^-10 Ang1mab4 (H1L02A) 9.71×10 ⁴ 4.56×10 ^-5 4.71×10 ^-10 Ang1mab4 (H1S05A) 8.89×10 ⁴ 8.31×10 ^-5 9.34×10 ^-10 Ang1mab4 (H1V12A) 7.67×10 ⁴ 7.62×10 ^-5 9.97×10 ^-10 Ang1mab4 (L1Q01A) 1.09×10 ⁵ 1.48×10 ^-4 1.36×10 ^-9 Ang1mab4 (L1V03A) 8.77×10 ⁴ 4.72×10 ^-5 5.38×10 ^-10 Ang1mab4 (L1S07A) 1.11×10 ⁵ 7.54×10 ^-5 6.82×10 ^-10 Ang1mab4 (L1Y05F) 1.01×10 ⁵ 4.80×10 ^-5 4.74×10 ^-10 Ang1mab2-1 8.42×10 ⁵ 1.14×10 ^-5 1.35×10 ^-10

As a result, as shown in Table 7, both the first and second primary antibodies and the variant antibodies had a K _D value of 1.4 nM or less, mostly at a level of several hundreds of pM, indicating excellent affinity for the antigen human Tie2 protein. .

Confirmation of antigen activation by primary and mutant antibodies

For the three primary antibodies (Ang1mab1, Ang1mab4 and Ang1mab2-1) prepared in Example [1-1] and the 8 mutant antibodies prepared in Example [1-3], these antibodies were human Tie2 protein and It was confirmed whether phosphorylation (P-Tie2, P-AKT, P-ERK) of lower signaling factors (AKT, ERK) on the signal transduction pathway involving the Tie2 protein was induced.

Specifically, HEK293 cells (1x10 ⁶ cells) expressing human Tie2 protein were cultured in a 60 mm culture dish at a temperature of 37 ^o C using DMEM medium. The cells cultured as described above were maintained in serum-free DMEM medium for 6 hours to achieve serum starvation, and then the first and second primary antibodies (Ang1mab1, Ang1mab4 and Ang1mab2-1) and mutant antibodies (10-g) /ml) and the positive control Ang1 (300ng/ml) were treated hourly or for 30 minutes, and then the cells were lysed with a lysis buffer to obtain a cell lysate. After performing SDS-PAGE on the cell lysate obtained as described above, it was transferred to a PVDF membrane and reacted with 0.1% Tween20 TBST buffer diluted with 3% BSA at room temperature for 2 hours. Then, the primary antibody (P-Tie2, Tie2, P-AKT, AKT, P-ERK or ERK) diluted at 1:1000 was treated and reacted at room temperature for 2 hours, and peroxidase-conjugated at 1:5000 diluted. (peroxidase-conjugated) secondary antibody was treated and reacted at room temperature for 1 hour, and then treated with ECL solution to reduce human Tie2 protein and lower signal transduction factors (AKT, ERK) on the signal transduction pathway involving the Tie2 protein. It was confirmed whether phosphorylation was induced.

As a result, as shown in FIGS. 2 and 3, it was confirmed that the first and second primary antibodies (Ang1mab1, Ang1mab4 and Ang1mab2-1) and mutant antibodies induce phosphorylation of Tie2, AKT, and ERK proteins. , It was confirmed that all of the first and second primary antibodies (Ang1mab1, Ang1mab4 and Ang1mab2-1) exhibited an effect equal to or greater than that of the positive control, Ang1 (FIG. 2). In addition, it was confirmed that the mutant antibodies also induce phosphorylation of human Tie2 protein similarly to the first primary antibodies (Ang1mab1 and Ang1mab4) ( FIG. 3 ).

Furthermore, in the same manner as above, except that human umbilical vein endothelial cells (HUVEC) and M199 as a medium were used instead of HEK293 cells, the first and second primary antibodies (Ang1mab1, Ang1mab4 and Ang1mab2) -1) and phosphorylation (P-Tie2, P-AKT, P-ERK) was checked.

As a result, as shown in FIG. 4 , as in HEK293 cells, phosphorylation of Tie2, AKT, and ERK proteins in the first and second primary antibodies (Ang1mab1, Ang1mab4, and Ang1mab2-1) and mutant antibodies in HUVEC. was confirmed to induce (Fig. 4).

Confirmation of specificity of primary antibody for antigen receptor

Whether Ang1mab4, one of the first primary antibodies obtained in Example [1-1], specifically phosphorylates only the Tie2 protein receptor in HEK293 cells expressing HUVEC and human Tie2 protein, or other protein receptors other than the Tie2 protein It was also confirmed that phosphorylation was carried out.

Specifically, HUVECs (3x10 ⁶ cells) and HEK293 cells (1x10 ⁷ cells) were cultured in a 100 mm culture dish at a temperature of 37 ^o C using M199 and DMEM medium, respectively. HUVEC and HEK293 cells cultured as described above were maintained in M199 and DMEM medium containing 1% serum for 6 hours, respectively, to make them into a serum starvation state, treated with Ang1mab4 hourly (10 minutes or 30 minutes), and then lysed Cells were lysed with a buffer solution to obtain a cell lysate. The cell lysate obtained as described above was quantified by the BCA protein quantification method, and then reacted with a membrane containing specific phosphorylated antibodies to 49 human protein receptors. Then, a protein receptor induced by phosphorylation by Ang1mab4 was identified through western blotting.

As a result, as shown in FIG. 5 , it was confirmed that the primary antibody Ang1mab4 selectively phosphorylates only the Tie2 protein receptor in HUVECs (A) and HEK293 cells expressing human Tie2 protein (B).

Identification of cross-linking of primary and variant antibodies to cross-species Tie2 protein

Whether the first and second primary antibodies (Ang1mab1, Ang1mab4 and Ang1mab2-1) and mutant antibodies that bind to human Tie2 protein can cross-link to Tie2 protein from other species (cross-species binding) Confirmed. To this end, Tie2 genes of mice, rats, dogs, pigs, and monkeys were synthesized and cloned into pcDNA3.4 vector to prepare Tie2 expression vectors for each species. 10 ug each of the Tie2 expression vector and 20 ul of Lipofectamine 2000 (invitrogen) were diluted in 1 ml of Opti-MEM medium, treated with HEK293T cells (2.0x10 ⁶ cells), and cultured for 2 days. Then, the culture solution was centrifuged (300xg, 5 minutes) to separate and remove the supernatant, and the cells were washed with FACS buffer. The first and second primary antibodies (Ang1mab1, Ang1mab4 and Ang1mab2-1) and mutant antibodies were treated with the Tie2 expression cells obtained as described above at a concentration of 5-g/ml, and after reaction at room temperature for 1 hour, FACS buffer was used. Washed twice. Then, 100-l of goat anti-human IgG-FITC (FITC-conjugated goat anti-human IgG; invitrogen) diluted 1:200 was added at a time and reacted at room temperature for 30 minutes, followed by washing twice again with FACS buffer. After that, antibody binding was checked using a FACS calibur device (BD Bioscience).

As a result, as shown in FIGS. 6 to 8 , the first and second primary antibodies (Ang1mab1, Ang1mab4 and Ang1mab2-1) and mutant antibodies were Tie2 protein derived from human ( FIG. 6 ) and monkey ( FIG. 7 ). It was confirmed that, although it did not bind to Tie2 protein derived from another species (FIG. 8).

Identification of epitope of primary antibody

To which part of the human Tie2 protein the primary antibody binds, that is, the antigenic determinant of the antibody was analyzed.

To this end, as shown in FIG. 9, mutants (Tie2ΔIg1; Tie2ΔIg1-2; Tie2ΔIg1-2,EGF; Tie2Δ) in which the full-length or extracellula domain of the human Tie2 protein has been partially removed as shown in FIG. Binding of the primary antibody to Ig1-2, EGF, Ig3) was confirmed. Specifically, the portion encoding the Ig1, Ig2, EGF or IgG3 domain was removed from the nucleotide sequence encoding the human Tie2 protein by recombinant-PCR, and then cloned into a pcDNA3 vector. The vector 10-g and Lipofectamine 2000 (invitrogen) 20-l into which the nucleotide sequence encoding the full length or part of the human Tie2 protein is inserted were diluted in 1 ml of Opti-MEM medium, and this was added to HEK293T cells (2.0x10 ⁶ cells). treated and cultured for 2 days. The culture was centrifuged (300xg, 5 minutes) to separate and remove the supernatant, and the cells were washed with FACS buffer. The cells expressing the entire length or part of the human Tie2 protein obtained as described above were treated with the basic antibody Ang1mab4 at a concentration of 10-g/ml, reacted at room temperature for 1 hour, and then washed twice with FACS buffer. Then, 100 μl of goat anti-human IgG-FITC (FITC-conjugated goat anti-human IgG; invitrogen) diluted 1:200 was added, reacted at room temperature for 30 minutes, and washed twice again with FACS buffer. Antibody binding was confirmed using Image FACS, FlowSight (Amnis).

As a result, as shown in FIG. 10 , the Ang1mab4 primary antibody bound to Tie2 Full and the Ig1-removed mutant Tie2ΔIg1, but the Ig2 removed Tie2 mutants (Tie2ΔIg1-2; Tie2ΔIg1-2). , EGF; Tie2ΔIg1-2, EGF, Ig3) was confirmed not to bind to all (FIG. 10).

From the above results, it can be seen that the basic and mutant antibodies bind to the Ig2 domain of human Tie2 protein, and thus the Ig2 domain of human Tie2 protein is an antigenic determinant of the basic and mutant antibodies.

Confirmation of effects of basic and mutant antibodies according to human Tie2 protein activation - Effects related to promoting angiogenesis

[7-1] In Vitro ( in vitro ) - angiogenesis promoting effect

It was confirmed whether the basic and mutant antibodies promote the angiogenesis of HUVECs.

Specifically, HUVECs (1x10 ⁵ cells), which became serum starved by maintaining for 6 hours in M199 medium containing 1% serum, were dispensed in a GFR-Matrigel-coated 24-well culture dish, and the HEVECs in the above example The degree of angiogenesis was checked while incubating for 18 hours by treating the basic and mutant antibodies (20-g/ml) and control antibody (IgG4 control; 20-g/ml) prepared in 1 above.

As a result, as shown in FIG. 11 , both of the first and second primary antibodies (Ang1mab1, Ang1mab4 and Ang1mab2-1) and mutant antibodies increase blood vessel formation and maintain for a long time compared to the control antibody. was confirmed (FIG. 11).

[7-2] In Vitro ( in vitro ) - Inhibitory effect of apoptosis of vascular cells

It was confirmed whether the primary antibody inhibits apoptosis of HUVECs. Specifically, HUVECs (3x10 ⁶ cells) were cultured in a 100 mm culture dish using M199 medium at a temperature of 37 ^o C for 24 hours. The next day, in serum-free M199 medium, HUVECs were treated with the primary antibody Ang1mab4 (20-g/ml) and the positive control, Ang1 (300ng/ml), and cultured for 36 hours. Cells were lysed to obtain a cell lysate. The cell lysate obtained as described above was quantified by BCA protein quantitation, followed by SDS-PAGE, and transferred to a PVDF membrane and reacted with 0.1% Tween20 TBST buffer diluted with 3% BSA at room temperature for 2 hours. Then, the primary antibodies (caspase-3, caspase-8 or actin) diluted at 1:1000 were treated and reacted at room temperature for 2 hours, and peroxidase-conjugated (peroxidase-conjugated) diluted at 1:5000 was treated. ) After the secondary antibody was treated and reacted at room temperature for 1 hour, the expression of the caspase-3, caspase-8 and actin proteins was checked by treating the ECL solution.

As a result, as shown in FIG. 12 , it was confirmed that the primary antibody suppressed the cell death of HUVECs induced in the starvation state at a level similar to that of the positive control Ang1 ( FIG. 12 (A) ). And, this inhibitory effect on cell death could be verified by confirming that the activities of cleaved Cas-3 and Cas-8, which are proteins inducing cell death, were inhibited (FIG. 12 (B)).

[7-3] in vivo ( in vivo ) Construction of transgenic mice to confirm the effect

In order to confirm in vivo the effect achieved by activating only human Tie2 protein by the basic and mutant antibodies, a transgenic mouse (EC-hTie2 Tg) in which human Tie2 protein is specifically expressed in mouse vascular endothelial cells was constructed did

Specifically, a linear nucleotide sequence consisting of a mouse Tie2 promoter gene, a human Tie2 gene, a poly A gene, and a mouse Tie2 enhancer gene in order was prepared.

Superovulation was induced by injecting pregnant mare serum gonadotropin and human chorionic gonadotropin into C57BL/6N female mice. As described above, superovulation-induced female C57BL/6N mice were crossed with C57BL/6N male mice, and fertilized eggs were obtained from C57BL/6N female mice whose pregnancy was confirmed. In the male pronucleus of zygote among the fertilized eggs obtained as described above, a linear nucleotide sequence consisting of a mouse Tie2 promoter gene, a human Tie2 gene, a poly A gene, and a mouse Tie2 enhancer gene was prepared in this order. injected. After the fertilized egg injected with the linear nucleotide sequence as described above was again transplanted into the ICR surrogate mother, a mouse having the human Tie2 gene (SEQ ID NO: 1) was obtained through genotyping. Then, in order to check whether the human Tie2 protein is normally expressed in the blood vessels of the mouse, the brain tissue was separated from the mouse obtained as above, fixed with formalin (Sigma) for 1 day, and then 30% sucrose was added. After processing the PBS for 3 days, it was made into a frozen block (frozen block). Then, a tissue slide was prepared by cryosectioning the frozen block, and an antibody of CD31 (Millipore, 1:200), which is a blood vessel-specific marker, and a Tie2 antibody that specifically binds to human Tie2 protein (Biolegend 1:200) The mouse brain blood vessels were immunostained using Each antibody was treated at 4-C for 16 hours, and then further treated with a secondary antibody attached to Cy3 or Alexa Fluor 488 blood vessels at room temperature for 2 hours, and then the tissue slides were used using Mounting Medium With DAPI (Vector). After immunostaining, it was observed through a fluorescence microscope.

As a result, as shown in FIG. 13 , it was confirmed that the human Tie2 protein was specifically expressed only in blood vessels of transgenic mice (EC-hTie2 Tg) ( FIG. 13 ).

[7-4] X Vivo ( ex vivo ) - Promotes blood vessel sprouting

Using the transgenic mouse prepared in Example [7-3], the primary antibodies (Ang1mab1 and Ang1mab4) prepared in Example [1-1] induce vascular endothelial cell sprouting in arterial tissue. It was analyzed in ex vivo whether or not it exhibits the efficacy.

Specifically, the aortic blood vessel was separated from the mouse prepared in Example [7-3], cut to a predetermined size (1 mm), and seeded on a matrigel-coated plate. Then, the primary antibodies Ang1mab1 (50 μg/ml) and Ang1mab4 (10, 50 μg/ml) and control antibodies (IgG4 control; 50 μg/ml), and positive controls VEGF (100 ng/ml) and Ang1 (200 ng/ml) ) was added and cultured for 6 days at an interval of 2 days, respectively, and the degree of germination of vascular endothelial cells from the arterial tissue was confirmed.

As a result, as shown in FIG. 14 , both of the primary antibodies (Ang1mab1, Ang1mab4) were confirmed to significantly increase the germination of arteriovascular endothelial cells compared to the control antibody, and also compared to the positive control (VEGF, Ang1). It was confirmed to significantly increase the germination of arteriovascular endothelial cells (FIG. 14).

[7-5] in vivo ( in vivo ) - promote wound healing

In vivo analysis of how much the primary antibody prepared in Example [1-1] can promote wound healing through induction of angiogenesis using the transgenic mouse prepared in Example [7-3] did

Specifically, after subcutaneous hair removal of the transgenic mouse prepared in Example [7-3], a circular wound with a diameter of 4 mm was induced, and Ang1mab4, a basic antibody, and a control antibody (IgG4 control) were added to the wound site at 7.5 mg. After intraperitoneal injection 3 times at 2 day intervals at a dose of /kg, the degree of wound healing was comparatively analyzed for 6 days.

As a result, as shown in FIG. 15 , in the group treated with the primary antibody Ang1mab4, it was confirmed that the wound healed much faster than the group treated with the control antibody ( FIG. 15 ).

Furthermore, in order to verify the efficacy of promoting wound healing as described above, a tissue slide was prepared and immunostained in the same manner as in Example [7-3] after separating the recovered wound tissue from the mouse. On the tissue slides, an antibody to CD31, a blood vessel-specific marker (Millipore, 1:200) and an antibody to αSMC, a marker of perivascular cells that maintain blood vessel stabilization (Abcam, 1:100), were added at 4-C for 16 hours. After treatment with secondary antibodies, Cy3 anti-Hamster (Thermo, 1:500) and Alexa Fluor 488 goat anti-rabbit (Invitrogen, 1:500) at room temperature for 2 hours, Mounting Medium With DAPI (Vector) ) was used to confirm whether the formation and stabilization of blood vessels was improved through a fluorescence microscope after immunostaining the tissue slides.

As a result, as shown in FIG. 16 , it was confirmed that angiogenesis increased in the Ang1mab4-treated tissue, and that the blood vessel was well surrounded by cells around the blood vessel ( FIG. 16 ).

[7-6] in vivo ( in vivo ) - Improvement of lower extremity ischemia

In vivo analysis of how much the primary antibody prepared in Example [1-1] can improve lower extremity ischemia by promoting angiogenesis using the transgenic mouse prepared in Example [7-3] did

Specifically, the transgenic mice prepared in Example [7-3] were anesthetized using a respiratory anesthesia system, and then the mouse legs were fixed with tape. After removing the hair on the surgical site with a hair removal cream, disinfect it with an alcohol swab, make an incision in the skin about 1 cm from the knee to the middle of the thigh using forceps and scissors, secure the view of the surgical site with a retractor, and then wash with PBS under a dissecting microscope. The subcutaneous fat around the femoral muscle was removed with a thin cotton swab, microscopic scissors, and fine scissors. Then, the femoral artery and vein in the groin area were separated from the nerve, and two 7-0 silks were passed through the lower part of the proximal end of the femoral artery/vein, and the femoral artery/vein was tied and occluded at regular intervals. Also, two 7-0 silks were passed under the distal part of the femoral artery/vein, tied at a certain distance and occluded. The tractor was removed and the mice were recovered on a heated mat by sutures with 5-0 vicryl sutures. The primary antibody, Ang1mab4, was injected into the tissue surrounding the lower extremity ischemia of a mouse in which lower extremity ischemia was induced by cutting the arteries and veins in the lower extremity and blocking the flow of blood. Then, in order to compare blood vessel formation and blood flow, the blood flow in the lower extremities of the mouse was measured using a laser Doppler imaging system. Laser Doppler images were measured for mice before surgery, on days 1, 3, and 7 after surgery. After lower extremity ischemia surgery, the mice were divided into two groups of 7 mice each, and a dose of 10 mg/kg was administered on the 1st and 3rd days after the lower extremity ischemia surgery. The primary antibody, Ang1mab4, and the control antibody (IgG4 control) were divided and injected into the tissues surrounding the surgery of each group of mice. And by taking Doppler images and comparing the blood flow of the non-operated leg and the operated leg, the effect of improving lower extremity ischemia was confirmed.

As a result, as shown in FIG. 17 , in the group treated with the Ang1mab4, it was confirmed that angiogenesis in the lower extremity tissue was increased compared to the group treated with the control antibody (FIG. 17(A)), blood flow It was confirmed that the flow was also significantly improved (FIG. 17 (B)).

Effect of primary and mutant antibodies according to human Tie2 protein activation

[8-1] In Vitro ( in vitro ) - Check the effect of inhibiting vascular permeation (inhibiting leakage)

It was analyzed how effectively the basic antibodies could inhibit the increase in vascular permeability caused by the abnormality of blood vessels, the so-called leakage phenomenon.

Specifically, HUVECs (1X10 ⁵ cells) were seeded in the upper chamber of a 12-well Transwell plate (0.4 μm pore) and cultured for 3 days, and then maintained in a serum starvation state for 6 hours. Then, in order to artificially increase vascular permeability, 50 ng/ml of VEGF was treated in all of the negative control, positive control, and experimental groups, while 100 ng/ml of Ang1 was administered to the positive control group, and Ang1mab4, the primary antibody, was administered to the experimental group by 10 each. , 25 and 50 μg/ml were treated for 5 hours. Then, fluorescence-labeled dextran (FITC-Dextran, 70 kDa) was added to the culture medium, and 30 minutes later, dextran transmitted through HUVECs was compared and analyzed with a fluorescence analyzer (SpectraMAx i3x).

As a result, as shown in (A) of FIG. 18, while the vascular permeability was increased in the cells of the negative control group treated with VEGF alone, the cells of the test group treated with the primary antibody Ang1mab4 were positive with Ang1 treatment. It was confirmed that the vascular permeation phenomenon was completely suppressed similarly to the cells of the control group (FIG. 18 (A)).

In addition, HUVECs (1X10 ⁵ cells) were seeded in 35 mm confocal dish (SPL) and cultured for 2 days, and then maintained in a serum starvation state for 6 hours. Then, while the negative control was treated with 50ng/ml of VEGF, the positive control was treated with 100ng/ml of Ang1, and the experimental group was treated with the primary antibody Ang1mab4 at a concentration of 25μg/ml for 5 hours. Then, it was fixed with 4% paraformaldehyde for 10 minutes, and then permeated through the cell membrane for 10 minutes using PBS containing 0.2% Triton X-100, and then with PBS containing 1% BSA for 1 hour. Blocked at room temperature. After diluting the VE-cadherin antibody (Santacruz) 1:50 at room temperature for 2 hours, dilute the fluorescence-labeled secondary antibody (Alexa Fluor 488 donkey anti-goat, Invitrogen) 1:200 at room temperature. It was treated for 1 hour, stained with DAPI diluted 1:1000 for 3 minutes, and observed with a fluorescence microscope.

As a result of confirming the change in the position of VE-cadherin involved in junction formation between HUVECs through cytoimmunochemical staining analysis, as shown in FIG. 18(B) , in the negative control treated with VEGF alone, cells Although junctions are formed remarkably weakly, in the cells of the test group treated with the primary antibody Ang1mab4, cell junctions between cells are well formed, and the interface between cells is strongly developed, equal to or greater than in the cells of the positive control group treated with Ang1. was confirmed (FIG. 18 (B)).

[8-2] in vivo ( in vivo ) - the effect of improving sepsis

Using the transgenic mouse prepared in Example [7-3], the primary antibody (Ang1mab4) prepared in Example [1-1] inhibits vascular leakage and enhances vascular endothelial integrity, resulting in sepsis was analyzed in vivo to see how much it can be inhibited.

Specifically, the transgenic mouse prepared in Example [7-3] was anesthetized using a respiratory anesthesia system, the abdominal hairs were removed and wiped with a sterile alcohol swab, and the integument was applied to the central part of the abdomen in a size of 1 cm. incised. Then, the endothelium was further incised and the cecum of the mouse was removed, and the 75% position of the cecum was tied with 4-0 black silk suture. Then, using a 21-gauge needle, a perforation was induced in the lower area near the site tied with the black silk suture (a total of two perforations in the front and back of the cecum), and the cecum was pressed so that the contents in the cecum could escape, and then the cecum was inserted into the abdominal cavity A sepsis model was prepared by cecal ligation and puncture (CLP) by putting the incision in the ventilator and suturing the incision and then recovering the mouse on a heating mat. Afterwards, the primary antibody Ang1mab4 and the control antibody (IgG4 control) at a dose of 10 mg/kg were injected into the tail vein of the mouse, and the survival of the mice was investigated for 7 days.

As a result, as shown in FIG. 19 , in the group treated with the control antibody, the mortality rate increased over time, and the survival rate was 0% at 3.5 days (84 hours) after CLP induction, whereas the primary antibody Ang1mab4 was treated In one group, all mice survived (100%) up to 3.5 days (84 hours) after CLP induction, and it was confirmed that the survival rate of 40% was maintained until the 7th day (FIG. 19).

[8-3] in vivo ( in vivo ) - tumor growth inhibitory effect

Using the transgenic mouse prepared in Example [7-3], it was examined how effectively the primary antibody prepared in Example [1-1] can inhibit tumor growth caused by abnormal angiogenesis. Analyzed in vivo.

Specifically, GL261 cells (5x10 ³ cells/μL), which are mouse glioblastomas, were injected subcutaneously into the transgenic mice prepared in Example [7-3]. Changes in tumor size were measured at intervals of 2 days after tumor injection, using vernier calipers for the long axis and short axis of the tumor. By calculating the size of the measured tumor as (long axis x short axis x short axis)/2, when the size grew to an average of 30 mm ³ , a control antibody (IgG1 control; 7.5 mg/kg) was used in the control group, and the primary antibody Ang1mab1 in the experimental group. (7.5 mg/kg) was administered to the tail vein 5 times at 3-day intervals. On the 23rd day of tumor injection, all subjects were killed using CO ² gas, and then laparotomy was performed to visually check for organ abnormalities and measure the weight of the tumor.

As a result, as shown in FIG. 20, it was confirmed that the tumor size in mice treated with the primary antibody, Ang1mab1, was inhibited by about 50% or more compared to the control group (IgG1 control) (FIG. 20 (A)), It was confirmed that the weight of the tumor was also reduced by 70% or more (FIG. 20 (B)).

Furthermore, in order to confirm whether the tumor growth inhibitory effect as described above was induced through normalization of abnormal tumor blood vessels, after separating the tumor tissue from the mouse, a tissue slide was performed in the same manner as in Example [7-3]. was prepared and immunostained. In the tumor tissue, the antibody of CD31 (Millipore, 1:200), which is a blood vessel-specific marker, and the antibody of αSMC, a marker of perivascular cells that maintain blood vessel stabilization (Abcam, 1:100) were mixed at 4-C for 16 hours. After treatment, secondary antibodies Cy3 anti-Hamster (Thermo, 1:500) and Alexa Fluor 488 goat anti-rabbit (Invitrogen, 1:500) were treated at room temperature for 2 hours, and Mounting Medium With DAPI (Vector) After immunostaining the tissue slides with a fluorescence microscope, the improvement of stable blood vessel development was observed.

As a result, as shown in FIG. 21 , it was confirmed that the blood vessels in the tumor tissue treated with the primary antibody Ang1mab1 were well surrounded by perivascular cells and stabilized ( FIG. 21 ).

[8-4] in vivo ( in vivo ) - Effect of increasing the influx of anti-tumor immune cells into the tumor

It was confirmed whether the primary antibody identified in Example [8-3] suppressed tumor growth by increasing the influx of anti-tumor immune cells into the tumor due to the normalization and stabilization-inducing effect of abnormal tumor blood vessels.

Example [8-3] After the tumor was excised and cut into small pieces, it was placed in 3 ml of RPMI1640 culture medium containing collagenase (2 μg/mL) and maintained at 37° C. for 1 hour. Then, the tissue was put into a 70 μm cell strainer and finely pulverized, and then washed twice with 3 ml of PBS. For observation of changes in the influx of immune cells into tumors using flow cytometry, hematopoietic cell markers CD45, NK cell markers NK1.1, T cell markers CD3, CD4, CD8, and macrophage markers CD11b, F4/80 Antibodies were stained for 30 minutes at a concentration of 1 μg/ml and washed twice with 1x PBS. And in order to accurately check the number of infiltrated immune cells, 50 μl of CountBright Absolute count beads (Invitrogen) were added to perform the analysis.

As a result, as shown in FIG. 22, NK cells (FIG. 22(A)), CD8, CD4 T cells (FIG. 22(B)), macrophages (FIG. 22(C)) It was confirmed that the influx into the tumor was increased, and through this, it was confirmed that the primary antibody Ang1mab1 inhibits tumor growth by increasing the influx of anti-tumor immune cells through normalization and stabilization of abnormal tumor blood vessels.

[8-5] in vivo ( in vivo ) - Effect of concurrent administration with primary antibody and other anticancer drugs

When using the transgenic mouse prepared in Example [7-3], the primary antibody prepared in Example [1-1] and the immune checkpoint inhibitory antibody (Anti-PD-1), which is one of the other anticancer agents, were concurrently administered , it was confirmed whether the tumor growth could be more effectively inhibited than when each was treated alone.

Specifically, LLC cells (5x10 ³ cells/μl), which are mouse lung tumor cells, were injected subcutaneously into the transgenic mice prepared in Example [7-3]. Changes in tumor size were measured at intervals of 2 days after tumor injection, using vernier calipers for the long axis and short axis of the tumor. When the size of the measured tumor was calculated as (long axis x short axis x short axis)/2 and the average size grew to about 30 mm ³ , the control antibody (IgG1 control; 7.5 mg / kg), the primary antibody Ang1mab1 (7.5 mg / kg) And Anti-PD-1 antibody (20mg/kg) was treated alone or the primary antibody, Ang1mab1, and Anti-PD-1 antibody were co-administered. Each antibody was administered to the tail vein a total of 5 times at 3-day intervals, and on the 23rd day of tumor injection, all subjects were killed using CO ² gas, and then laparotomy was performed to visually check for organ abnormalities and the weight of the tumor. was measured.

As a result, as shown in FIG. 23, it was confirmed that the size of the tumor was inhibited by about 85% in the mouse group administered with the primary antibody Ang1mab1 and the Anti-PD1 antibody (FIG. 23 (A)), and the tumor It was confirmed that the weight was also reduced by more than 87% (FIG. 23 (B)).

In the above, preferred embodiments of the present invention have been exemplarily described, but the scope of the present invention is not limited to the specific embodiments as described above, and those of ordinary skill in the art will It will be possible to change it appropriately.

<110> Korea Research Institute of Bioscience & Biotechnology <120> A Novel anti-Tie2 antibody or its antigen-binding fragments, and the use thereof <130> 2021DPA4143 <150> KR 10-2020-0144961 <151> 2020-11- 03 <160> 53 <170> KoPatentIn 3.0 <210> 1 <211> 1124 <212> PRT <213> Homo sapiens <400> 1 Met Asp Ser Leu Ala Ser Leu Val Leu Cys Gly Val Ser Leu Leu Leu 1 5 10 15 Ser Gly Thr Val Glu Gly Ala Met Asp Leu Ile Leu Ile Asn Ser Leu 20 25 30 Pro Leu Val Ser Asp Ala Glu Thr Ser Leu Thr Cys Ile Ala Ser Gly 35 40 45 Trp Arg Pro His Glu Pro Ile Thr Ile Gly Arg Asp Phe Glu Ala Leu 50 55 60 Met Asn Gln His Gln Asp Pro Leu Glu Val Thr Gln Asp Val Thr Arg 65 70 75 80 Glu Trp Ala Lys Lys Val Val Trp Lys Arg Glu Lys Ala Ser Lys Ile 85 90 95 Asn Gly Ala Tyr Phe Cys Glu Gly Arg Val Arg Gly Glu Ala Ile Arg 100 105 110 Ile Arg Thr Met Lys Met Arg Gln Gln Ala Ser Phe Leu Pro Ala Thr 115 120 125 Leu Thr Met Thr Val Asp Lys Gly As p Asn Val Asn Ile Ser Phe Lys 130 135 140 Lys Val Leu Ile Lys Glu Glu Asp Ala Val Ile Tyr Lys Asn Gly Ser 145 150 155 160 Phe Ile His Ser Val Pro Arg His Glu Val Pro Asp Ile Leu Glu Val 165 170 175 His Leu Pro His Ala Gln Pro Gln Asp Ala Gly Val Tyr Ser Ala Arg 180 185 190 Tyr Ile Gly Gly Asn Leu Phe Thr Ser Ala Phe Thr Arg Leu Ile Val 195 200 205 Arg Arg Cys Glu Ala Gln Lys Trp Gly Pro Glu Cys Asn His Leu Cys 210 215 220 Thr Ala Cys Met Asn Asn Gly Val Cys His Glu Asp Thr Gly Glu Cys 225 230 235 240 Ile Cys Pro Pro Gly Phe Met Gly Arg Thr Cys Glu Lys Ala Cys Glu 245 250 255 Leu His Thr Phe Gly Arg Thr Cys Lys Glu Arg Cys Ser Gly Gln Glu 260 265 270 Gly Cys Lys Ser Tyr Va l Phe Cys Leu Pro Asp Pro Tyr Gly Cys Ser 275 280 285 Cys Ala Thr Gly Trp Lys Gly Leu Gln Cys Asn Glu Ala Cys His Pro 290 295 300 Gly Phe Tyr Gly Pro Asp Cys Lys Leu Arg Cys Ser Cys Asn Asn Gly 305 310 315 320 Glu Met Cys Asp Arg Phe Gln Gly Cys Leu Cys Ser Pro Gly Trp Gln 325 330 335 Gly Leu Gln Cys Glu Arg Glu Gly Ile Pro Arg Met Thr Pro Lys Ile 340 345 350 Val Asp Leu Pro Asp His Ile Glu Val Asn Ser Gly Lys Phe Asn Pro 355 360 365 Ile Cys Lys Ala Ser Gly Trp Pro Leu Pro Thr Asn Glu Glu Met Thr 370 375 380 Leu Val Lys Pro Asp Gly Thr Val Leu His Pro Lys Asp Phe Asn His 385 390 395 400 Thr Asp His Phe Ser Val Ala Ile Phe Thr Ile His Arg Ile Leu Pro 405 410 415 Pro Asp Se r Gly Val Trp Val Cys Ser Val Asn Thr Val Ala Gly Met 420 425 430 Val Glu Lys Pro Phe Asn Ile Ser Val Lys Val Leu Pro Lys Pro Leu 435 440 445 Asn Ala Pro Asn Val Ile Asp Thr Gly His Asn Phe Ala Val Ile Asn 450 455 460 Ile Ser Ser Glu Pro Tyr Phe Gly Asp Gly Pro Ile Lys Ser Lys Lys 465 470 475 480 Leu Leu Tyr Lys Pro Val Asn His Tyr Glu Ala Trp Gln His Ile Gln 485 490 495 Val Thr Asn Glu Ile Val Thr Leu Asn Tyr Leu Glu Pro Arg Thr Glu 500 505 510 Tyr Glu Leu Cys Val Gln Leu Val Arg Arg Gly Glu Gly Gly Glu Gly 515 520 525 His Pro Gly Pro Val Arg Arg Phe Thr Thr Ala Ser Ile Gly Leu Pro 530 535 540 Pro Pro Arg Gly Leu Asn Leu Leu Pro Lys Ser Gln Thr Thr Leu Asn 545 550 555 56 0 Leu Thr Trp Gln Pro Ile Phe Pro Ser Ser Glu Asp Asp Phe Tyr Val 565 570 575 Glu Val Glu Arg Arg Ser Val Gln Lys Ser Asp Gln Gln Asn Ile Lys 580 585 590 Val Pro Gly Asn Leu Thr Ser Val Leu Leu Asn Asn Leu His Pro Arg 595 600 605 Glu Gln Tyr Val Val Arg Ala Arg Val Asn Thr Lys Ala Gln Gly Glu 610 615 620 Trp Ser Glu Asp Leu Thr Ala Trp Thr Leu Ser Asp Ile Leu Pro Pro 625 630 635 640 Gln Pro Glu Asn Ile Lys Ile Ser Asn Ile Thr His Ser Ser Ala Val 645 650 655 Ile Ser Trp Thr Ile Leu Asp Gly Tyr Ser Ile Ser Ser Ile Thr Ile 660 665 670 Arg Tyr Lys Val Gln Gly Lys Asn Glu Asp Gln His Val Asp Val Lys 675 680 685 Ile Lys Asn Ala Thr Ile Thr Gln Tyr Gln Leu Lys Gly Leu Glu Pro 690 695 700 Glu Th r Ala Tyr Gln Val Asp Ile Phe Ala Glu Asn Asn Ile Gly Ser 705 710 715 720 Ser Asn Pro Ala Phe Ser His Glu Leu Val Thr Leu Pro Glu Ser Gln 725 730 735 Ala Pro Ala Asp Leu Gly Gly Gly Lys Met Leu Leu Ile Ala Ile Leu 740 745 750 Gly Ser Ala Gly Met Thr Cys Leu Thr Val Leu Leu Ala Phe Leu Ile 755 760 765 Ile Leu Gln Leu Lys Arg Ala Asn Val Gln Arg Arg Met Ala Gln Ala 770 775 780 Phe Gln Asn Val Arg Glu Glu Pro Ala Val Gln Phe Asn Ser Gly Thr 785 790 795 800 Leu Ala Leu Asn Arg Lys Val Lys Asn Asn Pro Asp Pro Thr Ile Tyr 805 810 815 Pro Val Leu Asp Trp Asn Asp Ile Lys Phe Gln Asp Val Ile Gly Glu 820 825 830 Gly Asn Phe Gly Gln Val Leu Lys Ala Arg Ile Lys Lys Asp Gly Leu 835 840 845 Arg Met Asp Ala Ala Ile Lys Arg Met Lys Glu Tyr Ala Ser Lys Asp 850 855 860 Asp His Arg Asp Phe Ala Gly Glu Leu Glu Val Leu Cys Lys Leu Gly 865 870 875 880 His His Pro Asn Ile Ile Asn Leu Leu Gly Ala Cys Glu His Arg Gly 885 890 895 Tyr Leu Tyr Leu Ala Ile Glu Tyr Ala Pro His Gly Asn Leu Leu Asp 900 905 910 Phe Leu Arg Lys Ser Arg Val Leu Glu Thr Asp Pro Ala Phe Ala Ile 915 920 925 Ala Asn Ser Thr Ala Ser Thr Leu Ser Ser Gln Gln Leu Leu His Phe 930 935 940 Ala Ala Asp Val Ala Arg Gly Met Asp Tyr Leu Ser Gln Lys Gln Phe 945 950 955 960 Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Gly Glu Asn Tyr 965 970 975 Val Ala Lys Ile Ala Asp Phe Gly Leu Ser Arg Gly Gln Glu Val Tyr 980 985 990 Val Lys Lys Thr Met Gly Arg Leu Pro Val Arg Trp Met Ala Ile Glu 995 1000 1005 Ser Leu Asn Tyr Ser Val Tyr Thr Thr Asn Ser Asp Val Trp Ser Tyr 1010 1015 1020 Gly Val Leu Leu Trp Glu Ile Val Ser Leu Gly Gly Thr Pro Tyr Cys 1025 1030 1035 1040 Gly Met Thr Cys Ala Glu Leu Tyr Glu Lys Leu Pro Gln Gly Tyr Arg 1045 1050 1055 Leu Glu Lys Pro Leu Asn Cys Asp Asp Glu Val Tyr Asp Leu Met Arg 1060 1065 1070 Gln Cys Trp Arg Glu Lys Pro Tyr Glu Arg Pro Ser Phe Ala Gln Ile 1075 1080 1085 Leu Val Ser Leu Asn Arg Met Leu Glu Glu Arg Lys Thr Tyr Val Asn 1090 1095 1100 Thr Thr Leu Tyr Glu Lys Phe Thr Tyr Ala Gly Ile Asp Cys Ser Ala 1105 1110 1115 1120 Glu Glu Ala Ala <210> 2 <211> 94 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (1)..(94) <223> Ig2 domain of Tie2 Protein <400> 2 Gln Gln Ala Ser Phe Leu Pro Ala Thr Leu Thr Met Thr Val Asp Lys 1 5 10 15 Gly Asp Asn Val Asn Ile Ser Phe Lys Lys Val Leu Ile Lys Glu Glu 20 25 30 Asp Ala Val Ile Tyr Lys Asn Gly Ser Phe Ile His Ser Val Pro Arg 35 40 45 His Glu Val Pro Asp Ile Leu Glu Val His Leu Pro His Ala Gln Pro 50 55 60 Gln Asp Ala Gly Val Tyr Ser Ala Arg Tyr Ile Gly Gly Asn Leu Phe 65 70 75 80 Thr Ser Ala Phe Thr Arg Leu Ile Val Arg Arg Cys Glu Ala 85 90 <210> 3 <211> 11 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (1)..(11) <223> Epitope for the anti-hTie2 antibody <400> 3 Leu Pro Ala Thr Leu Thr Met Thr Val Asp Lys 1 5 10 <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223 > General amino acid sequence of CDR1-H in the 1st and 2nd basic anti-hTie2 antibodies <220> <221> MISC_FEATURE <222> (1) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222 > (2) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (3) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (4) <223 > Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (5) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (6) <223> Xaa can be any amino aci d <220> <221> MISC_FEATURE <222> (8) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (12) <223> Xaa can be any amino acid <400> 4 Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Met His Trp Xaa 1 5 10 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> General amino acid sequence of CDR2-H in the 1st and 2nd basic anti-hTie2 antibodies <220> <221> MISC_FEATURE <222> (2) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (4) <223> Xaa can be any amino acid < 220> <221> MISC_FEATURE <222> (5) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (6) <223> Xaa can be any amino acid <400> 5 Ile Xaa Tyr Xaa Xaa Xaa Asn Lys 1 5 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> General amino acid sequence of CDR3-H in the 1st basic anti-hTie2 antibody <220> < 221> MISC_FEATURE <222> (1) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (6) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (7) <2 23> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (10) <223> Xaa can be any amino acid <400> 6 Xaa Thr Pro Lys Gly Xaa Xaa Leu Glu Xaa 1 5 10 <210> 7 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> General amino acid sequence of CDR1-L in the 1st and 2nd basic anti-hTie2 antibodies <220> <221> MISC_FEATURE <222> (1 ) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (2) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (3) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (4) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (5) <223> Xaa can be Tyr, Phe or Trp <220> <221> MISC_FEATURE <222> (6) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (7) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (9) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (10) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (11 ) <223> Xaa can be any amino acid <400> 7 Xa a Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Tyr 1 5 10 <210> 8 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> General amino acid sequence of CDR2-L in the 1st and 2nd basic anti-hTie2 antibodies <220> <221> MISC_FEATURE <222> (2) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (3) <223> Xaa can be any amino acid <400> 8 Trp Xaa Xaa 1 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> General amino acid sequence of CDR3-L in the 1st and 2nd basic anti-hTie2 antibodies < 220> <221> MISC_FEATURE <222> (4) <223> Xaa can be Tyr, Phe or Trp <220> <221> MISC_FEATURE <222> (6) <223> Xaa can be any amino acid <220> <221 > MISC_FEATURE <222> (8) <223> Xaa can be Ile or Tyr <220> <221> MISC_FEATURE <222> (9) <223> Xaa can be any amino acid <400> 9 Gln Gln Tyr Xaa Ser Xaa Pro Xaa Xaa 1 5 <210> 10 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR1-H in the 1st basic anti-hTie2 antibody <400> 10 Gly Leu Th r Phe Ser Asn Tyr Ala Met His Trp Val 1 5 10 <210> 11 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR2-H in the 1st basic anti-hTie2 antibody <400> 11 Ile Ser Tyr Asp Gly Glu Asn Lys 1 5 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR3-H in the 1st basic anti -hTie2 antibody <400> 12 Ala Thr Pro Lys Gly Gly Ala Leu Glu Ile 1 5 10 <210> 13 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR1-L in 1st basic anti-hTie2 antibody <400> 13 Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr 1 5 10 <210> 14 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR2-L in the 1st basic anti-hTie2 antibody <400> 14 Trp Ala Ser 1 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR3 -L in the 1st basic anti-hTie2 antibody <400> 15 Gln Gln Tyr Tyr Ser Ala Pro Ile Thr 1 5 <210> 16 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR1-H in the 2nd basic anti-hTie2 antibody <400> 16 Gly Phe Thr Phe Ser Ser Tyr Ala Met His Trp Val 1 5 10 <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR2-H in the 2nd basic anti-hTie2 antibody <400> 17 Ile Ser Tyr Asp Gly Ser Asn Lys 1 5 <210> 18 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR3-H in the 2nd basic anti-hTie2 antibody <400 > 18 Ala Arg Gln Tyr Ser Gly Val Phe Glu Tyr 1 5 10 <210> 19 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR1-L in the 2nd basic anti -hTie2 antibody <400> 19 Gln Ser Val Leu Tyr Arg Ser Asn Asn Arg Asn Tyr 1 5 10 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR3 -L in the 2nd basic anti-hTie2 antibody <400> 20 Gln Gln Tyr Phe Ser Ile Pro Tyr T hr 1 5 <210> 21 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR1-H in the mutated anti-hTie2 antibody H1G01A <400> 21 Ala Leu Thr Phe Ser Asn Tyr Ala Met His Trp Val 1 5 10 <210> 22 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR1-H in the mutated anti-hTie2 antibody H1L02A <400 > 22 Gly Ala Thr Phe Ser Asn Tyr Ala Met His Trp Val 1 5 10 <210> 23 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR1-H in the mutated anti-hTie2 antibody H1S05A <400> 23 Gly Leu Thr Phe Ala Asn Tyr Ala Met His Trp Val 1 5 10 <210> 24 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR1-H in the mutated anti-hTie2 antibody H1V12A <400> 24 Gly Leu Thr Phe Ser Asn Tyr Ala Met His Trp Ala 1 5 10 <210> 25 <211> 12 <212> PRT <213> Artificial Sequence <220 > <223> amino acid sequence of CDR1-L in the mutated anti -hTie2 antibody L1Q01A <400> 25 Ala Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr 1 5 10 <210> 26 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR1-L in the mutated anti-hTie2 antibody L1V03A <400> 26 Gln Ser Ala Leu Tyr Ser Ser Asn Asn Lys Asn Tyr 1 5 10 <210> 27 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR1-L in the mutated anti-hTie2 antibody L1S07A <400> 27 Gln Ser Val Leu Tyr Ser Ala Asn Asn Lys Asn Tyr 1 5 10 <210> 28 <211> 12 <212> PRT < 213> Artificial Sequence <220> <223> Amino acid sequence of CDR1-L in the mutated anti-hTie2 antibody L1Y05F <400> 28 Gln Ser Val Leu Phe Ser Ser Asn Asn Lys Asn Tyr 1 5 10 <210> 29 <211 > 25 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of FR1 in the heavy chain variable region of the 1st basic anti-hTie2 antibody <400> 29 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 30 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of FR2 in the heavy chain variable region of the 1st basic anti-hTie2 antibody <400> 30 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val 1 5 10 <210> 31 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of FR3 in the heavy chain variable region of the 1st basic anti-hTie2 antibody <400> 31 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ser Lys Asn Met Leu Gln Leu Gln Met Asn Arg Leu Arg Ser Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 32 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of FR4 in the heavy chain variable region of the 1st basic anti-hTie2 antibody <400> 32 Trp Gly Gln Gly Thr Val Ile Thr Val Ser Ser 1 5 10 <210> 33 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of FR1 in the light chain variable region of the 1st basic anti-hTie2 antibody <400> 33 Asp Ile Gln Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser 20 25 <210> 34 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of FR 2 in the light chain variable region of the 1st basic anti-hTie2 antibody <400> 34 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Asn Leu Leu Met 1 5 10 15 Tyr <210> 35 <211> 36 <212 > PRT <213> Artificial Sequence <220> <223> Amino acid sequence of FR3 in the light chain variable region of the 1st basic anti-hTie2 antibody <400> 35 Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala 20 25 30 Val Tyr Tyr Cys 35 <210> 36 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of FR4 in the light chain variable region of the 1st basic anti-hTie2 antibody <400> 36 Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 1 5 10 <210> 37 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of FR1 in the heavy chain variable region of the 2nd basic anti-hTie2 antibody <400> 37 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Ar g 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 38 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of FR3 in the heavy chain variable region of the 2nd basic anti-hTie2 antibody <400> 38 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 39 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of FR4 in the heavy chain variable region of the 2nd basic anti-hTie2 antibody <400> 39 Trp Gly Gln Gly Thr Leu Ile Thr Val Ser Ser 1 5 10 <210> 40 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of FR2 in the light chain variable region of the 2nd basic anti-hTie2 antibody <400> 40 Leu Ala Trp Tyr Gln Gln Lys Leu Gly Gln Pro Pro Gln Leu Leu Ile 1 5 10 15 Tyr <210> 41 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of FR3 in the light chain variable region of the 2nd basic anti-hTie2 antibody <400> 41 Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Ala Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala 20 25 30 Val Tyr Tyr Cys 35 <210> 42 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of FR4 in the light chain variable region of the 2nd basic anti-hTie2 antibody <400> 42 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 43 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> General amino acid sequence of the heavy chain variable region in the 1st basic anti-hTie2 antibody <220> <221> MISC_FEATURE <222> (26) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> ( 27) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (28) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (29) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (30) < 223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (31) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (33) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (37) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (52) <223> Xaa can be any amino acid <220> < 221> MISC_FEATURE <222> (54) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (55) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (56) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (97) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (102) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (103) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (106) <223> Xaa can be any amino acid <400> 43 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Xaa Xaa Xaa Xaa Xaa Xaa Tyr 20 25 30 Xaa Met His Trp Xaa Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Xaa Tyr Xaa Xaa Xaa Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Met Leu Gln 65 70 75 80 Leu Gln Met Asn Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Xaa Thr Pro Lys Gly Xaa Xaa Leu Glu Xaa Trp Gly Gln Gly Thr Val 100 105 110 Ile Thr Val Ser Ser 115 <210> 44 <211> 117 <212 > PRT <213> Artificial Sequence <220> <223> General amino acid sequence of the heavy chain variable region in the 2nd basic anti-hTie2 antibody <220> <221> MISC_FEATURE <222> (26) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (27) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (28) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (29) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (30) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222 > (31) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (33) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (37) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (52) <223> Xaa can be any amino acid < 220> <221> MISC_FEATURE <222> (54) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (55) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (56) <223> Xaa can be any amino acid <400> 44 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Xaa Xaa Xaa Xaa Xaa Xaa Tyr 20 25 30 Xaa Met His Trp Xaa Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Xaa Tyr Xaa Xaa Xaa Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Tyr Ser Gly Val Phe Glu Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Ile Thr Val Ser Ser 115 <210> 45 <211> 113 <212> PRT <213> Artifi cial Sequence <220> <223> General amino acid sequence of the light chain variable region in the 1st basic anti-hTie2 antibody <220> <221> MISC_FEATURE <222> (27) <223> Xaa can be any amino acid <220 > <221> MISC_FEATURE <222> (28) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (29) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE < 222> (30) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (31) <223> Xaa can be Tyr, Phe or Trp <220> <221> MISC_FEATURE <222> (32 ) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (33) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (35) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (36) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (37) <223> Xaa can be any amino acid <220 > <221> MISC_FEATURE <222> (57) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (58) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE < 222> (98) <223> Xaa can be Tyr, Phe or Trp <220> <221> MISC_FEATURE <222> (100) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (102) <223> Xaa can be Ile or Tyr <220> <221> MISC_FEATURE <222> (103) <223> Xaa can be any amino acid <400> 45 Asp Ile Gln Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Asn Xaa Xaa Xaa Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Asn Leu Leu Met Tyr Trp Xaa Xaa Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Xaa Ser Xaa Pro Xaa Xaa Phe Gly Gln Gly Thr Arg Leu Glu Ile 100 105 110 Lys <210> 46 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> General amino acid sequence of the light chain variable region in the 2nd basic anti-hTie2 antibody <220> <221> MISC_FEATURE <222> (27) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (28) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (29) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (30) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (31) <223> Xaa can be Tyr, Phe or Trp <220> < 221> MISC_FEATURE <222> (32) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (33) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (35) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (36) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (37) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (57) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (58) <223> Xaa can be any amino acid <220> <221> MISC_FEATURE <222> (98) <223> Xaa can be Tyr, Phe or Trp <220> <221> MISC_FEATURE <222> (100) <223> Xaa can be any amino acid <220> < 221> MISC_FEATURE <222> (102) <223> Xaa can be Ile or Tyr <220> <221> MISC_FEAT URE <222> (103) <223> Xaa can be any amino acid <400> 46 Asp Ile Gln Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Asn Xaa Xaa Xaa Tyr Leu Ala Trp Tyr Gln Gln Lys Leu Gly Gln 35 40 45 Pro Gln Leu Leu Ile Tyr Trp Xaa Xaa Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Ala Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Xaa Ser Xaa Pro Xaa Xaa Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 47 <211> 329 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the heavy chain constant region in the Ang1mab1 <400> 47 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 48 <211> 987 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of the heavy chain constant region in the Ang1mab1 <400> 48 ccctccacca ag ggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctac a gcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960 cagaagagcc tctccctgtc cccgggt 987 <210> 49 <211> 327 <212> PRT <213> Artificial Sequence of the Amino acid sequence <2201> 327 <212> PRT <213> Artificial Sequence <2201> <400> 49 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu V al Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp S er Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 50 <211> 981 <212> DNA <213> Artificial Sequence <220> <223 > Nucleotide sequence of the heavy chain constant region in the Ang1mab4 <400> 50 gccagcacca agggccccag cgtgttcccc ctggccccct gcagccggag caccagcgag 60 agcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgagc 120 tggaacagcg gcgccctgac cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc 180 ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcagcctggg caccaagacc 240 tacacctgca acgtggacca caagcccagc aacaccaagg tggacaagcg ggtggagagc 300 aagtacggcc ccccctgccc cccctgcccc gcccccgagt tcctgggcgg ccccagcgtg 360 ttcctgttcc cccccaagcc caaggacacc ctgatgatca gccggacccc cgaggtgacc 420 tgcgtggtgg tggacgtgag ccaggaggac cccgaggtgc agttcaactg gtacgtggac 480 ggcgtggagg tgcacaacgc caagaccaag ccccgggagg agcagttcaa cagcacctac 540 cgggtggtga gcgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaa g 600 tgcaaggtga gcaacaaggg cctgcccagc agcatcgaga agaccatcag caaggccaag 660 ggccagcccc gggagcccca ggtgtacacc ctgcccccca gccaggagga gatgaccaag 720 aaccaggtga gcctgacctg cctggtgaag ggcttctacc ccagcgacat cgccgtggag 780 tgggagagca acggccagcc cgagaacaac tacaagacca ccccccccgt gctggacagc 840 gacggcagct tcttcctgta cagccggctg accgtggaca agagccggtg gcaggagggc 900 aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagagc 960 ctgagcctga gcctgggcaa g 981 <210 > 51 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the light chain constant region in the Ang1mab1 and Ang1mab4 <400> 51 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 52 <211> 321 <212> DNA <213> Artificial Sequence <220 > <223> Nucleotide sequence of the light chain constant region in the Ang1mab1 and Ang1mab4 <400> 52 ggtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagttcgcc cgtcacaaag 300 agcttcaaca ggggagagtg t 321 <210> 53 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR3-H in the control antibodies<400> 53 Ala Thr Gln Lys Gly Gly Ala Leu Glu Ile 1 5 10

Claims (17)

The amino acid sequence of X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -YX ₇ -MHWX ₈ (SEQ ID NO: 4) (in the amino acid sequence of SEQ ID NO: 4, X ₁ to X ₈ are each independently a heavy chain variable region comprising CDR1-H having any amino acid; and
The amino acid sequence of X ₁₇ -X ₁₈ -X ₁₉ -X ₂₀ -X ₂₁ -X ₂₂ -X ₂₃ -NX ₂₄ -X ₂₅ -X ₂₆ -Y (SEQ ID NO: 7) (in the amino acid sequence of SEQ ID NO: 7, X ₁₇ to X ₂₀ and X ₂₂ to X ₂₆ are each independently any amino acid, and X ₂₁ is any one selected from the group consisting of tyrosine, phenylalanine and tryptophan) a light chain variable region comprising a CDR1-L;
comprising, an antibody or antigen-binding fragment thereof. The method according to claim 1,
Wherein X ₁ , X ₇ , X ₈ , X ₁₉ and X ₂₀ are each independently any one selected from the group consisting of glycine, alanine, valine, isoleucine and leucine;
wherein X ₂ and X ₄ are each independently any one selected from the group consisting of glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine and tryptophan;
Wherein X ₃ , X ₅ , X ₆ , X ₁₇ , X ₁₈ , X ₂₃ , X ₂₄ and X ₂₆ are each independently from the group consisting of serine, threonine, asparagine, glutamine, glycine, alanine, valine, isoleucine and leucine any one selected;
wherein X ₂₁ is any one selected from the group consisting of tyrosine and phenylalanine;
wherein X ₂₂ is any one selected from the group consisting of serine, threonine, asparagine, glutamine, arginine, histidine, lysine, glycine, alanine, valine, isoleucine and leucine; or
wherein X ₂₅ is any one selected from the group consisting of arginine, histidine, lysine, glycine, alanine, valine, isoleucine and leucine;
Which will, an antibody or antigen-binding fragment thereof. 3. The method according to any one of claims 1 and 2,
The heavy chain variable region is CDR2-H comprising the amino acid sequence of IX ₉ -YX ₁₀ -X ₁₁ -X ₁₂ -NK (SEQ ID NO: 5), and X ₁₃ -TPKGX ₁₄ -X ₁₅ -LEX ₁₆ (SEQ ID NO: 6) or at least one selected from the group consisting of CDR3-H comprising the amino acid sequence of ARQYSGVFEY (SEQ ID NO: 18); or
The light chain variable region comprises a CDR2-L comprising the amino acid sequence of WX ₂₇ -X ₂₈ (SEQ ID NO: 8) and a CDR3-L comprising the amino acid sequence of QQYX ₂₉ -SX ₃₀ -PX ₃₁ -X ₃₂ (SEQ ID NO: 9). The antibody or antigen-binding fragment thereof further comprising at least one selected from the group consisting of.
(In the amino acid sequences, X ₉ to X ₁₆ , X ₂₇ , X ₂₈ , X ₃₀ and X ₃₂ are each independently any amino acid, and X ₂₉ is any one selected from the group consisting of tyrosine, phenylalanine and tryptophan and X ₃₁ is any one selected from the group consisting of isoleucine and tyrosine) 4. The method according to claim 3,
Wherein X ₉ , X ₂₈ and X ₃₂ are each independently any one selected from the group consisting of serine, threonine, asparagine, glutamine, glycine, alanine, valine, isoleucine and leucine;
wherein X ₁₀ is any one selected from the group consisting of aspartic acid, glutamic acid, glycine, alanine, valine, isoleucine and leucine;
Wherein X ₁₁ , X ₁₃ to X ₁₆ , X ₂₇ and X ₃₀ are each independently any one selected from the group consisting of glycine, alanine, valine, isoleucine and leucine;
wherein X ₁₂ is any one selected from the group consisting of aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, glycine, alanine, valine, isoleucine and leucine;
X ₂₉ is any one selected from the group consisting of tyrosine and phenylalanine; or
X ₃₁ is any one selected from the group consisting of isoleucine and tyrosine;
Which will, an antibody or antigen-binding fragment thereof. The method according to claim 1
The antibody is a fully human antibody, antibody or antigen-binding fragment thereof. The method according to claim 1,
The antibody or antigen-binding fragment thereof specifically binds to human or monkey Tie2 protein, the antibody or antigen-binding fragment thereof. The method according to claim 1,
The antigen or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to the Ig2 domain of the human or monkey Tie2 protein. The method according to claim 1,
Wherein the antibody or antigen-binding fragment thereof binds to human or monkey Tie2 protein with a K _D value of 1.4 nM or less. 9. The method according to any one of claims 6 to 8,
Wherein the antibody or antigen-binding fragment thereof specifically phosphorylates human or monkey Tie2 protein, the antibody or antigen-binding fragment thereof. A gene encoding the antibody of claim 1 or an antigen-binding fragment thereof. A recombinant expression vector comprising the gene of claim 10 . A transformant in which the recombinant expression vector of claim 11 is introduced into a host cell. A method for producing an antigen or antigen-binding fragment thereof that specifically binds to a human or monkey Tie2 protein, comprising the step of culturing the transformant of claim 12 . A pharmaceutical composition for preventing or treating angiogenesis-related diseases comprising the antibody of claim 1 or a binding fragment thereof. 15. The method of claim 14,
The angiogenesis-related disease is a pharmaceutical composition for the prevention or treatment of angiogenesis-related diseases, which is caused by a decrease or deficiency of angiogenesis. 15. The method of claim 14,
The angiogenesis-related disease is a pharmaceutical composition for preventing or treating angiogenesis-related diseases, which is caused by abnormal blood vessels, or excessive abnormal angiogenesis. at least one disease treatment; and
The antibody of claim 1 or an antigen-binding fragment thereof;
A pharmaceutical composition for co-administration for the prevention or treatment of angiogenesis-related diseases comprising a.

Images