KR20150014077A - Anti-Ang2 antibody inducing binding to Tie2 receptor - Google Patents

Abstract

Provided are an anti-Ang2 antibody or an antigen binding fraction thereof which specifically binds to Angiopoietin-2 (Ang2) which is an angiogenesis inducing factor, and binds to a Tie2 acceptor with Ang2; and uses thereof.

Description

Anti-Ang2 antibody inducing binding to Tie2 receptor < RTI ID = 0.0 >

An anti-Ang2 antibody or antigen-binding fragment thereof, which specifically binds to angiopoietin-2 (Ang2), a neovascularization-inducing factor and binds to Tie2 receptor together with Ang2, and uses thereof are provided .

Angiopoietin-2 (Ang2) is an antagonistic ligand of the receptor Tie2 present in vascular endothelial cells and is an agonist of Angiopoietin-1 ; Ang1) and Tie2 binding, thereby suppressing signal transduction by Tie2. Ang1, a ligand that activates the Tie2 receptor, acts as a key regulator to maintain blood vessel stabilization by maintaining the barrier function of vascular endothelial cells. Overexpression or inflammation of VEGF activates vascular endothelial cells and increases vascular permeability. At this time, Ang1 induces stabilization of vascular endothelial cells and promotes vascular permeability by promoting junctional integrity of vascular endothelial cells, while increased Ang2 in activated vascular endothelial cells compete with Ang1, It plays a role in inhibiting endothelial cell stabilization. Thus, Ang2 inhibits Ang1-Tie2 binding and signaling through it, which maintains the stability of vascular endothelial cells, and consequently promotes angiogenesis through dynamic rearrangement of blood vessels.

Since angiogenesis is an essential component of cancer growth, it has been known from studies of various preclinical models that inhibition of Tie2-dependent Ang2 function and inhibition of angiogenesis by inhibiting angiogenesis is possible, There have been various attempts to prevent the progression of cancer using anti-Ang2 antibodies.

Ang2 overexpression has been reported in a variety of solid tumors and blood cancers and the degree of Ang2 expression in serum may be a biomarker of poor prognosis. Overexpression of Ang2 has been reported in various diseases such as sepsis, bacterial infection, lung injury and kidney injury. In addition, there is a strong correlation between vascular leakage Much has been studied. Due to its association with these pathological conditions, Ang2 has been considered as an important target in therapeutic intervention. Many drugs are currently under clinical or preclinical development, including peptibodies, bispecific antibodies, and monoclonal antibodies.

However, most of these Ang2 target drugs have a mechanism of action that inhibits the activation of Tie2 receptor and downstream signaling by inhibiting Ang2 binding to the Tie2 receptor.

An example of the present invention relates to an angi2 antibody or antigen-binding fragment thereof that specifically binds to angiogenesis inducing factor Ang2 (angiopoietin-2) and binds to Tie2 receptor together with Ang2 to induce activation of Tie2 receptor to provide.

Another example provides a hybridoma cell line that produces a monoclonal antibody of said anti-Ang2 antibody.

Another example provides the anti-Ang2 antibody-Ang2 complex conjugated to Ang2 with the anti-Ang2 antibody or antigen-binding fragment thereof.

Another example provides a composition for activating Tie2 receptor comprising the anti-Ang2 antibody-Ang2 complex as an active ingredient.

Another example provides a pharmaceutical composition for inhibiting angiogenesis comprising the anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient.

Another example provides a pharmaceutical composition for reducing vascular permeability comprising the anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient.

Another example provides a pharmaceutical composition for inducing normal angiogenesis comprising the anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient.

Another example provides a pharmaceutical composition for the treatment of diseases associated with angiogenesis, increased vascular permeability, and / or reduced normal angiogenesis, comprising the anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient.

Another example provides a pharmaceutical composition for Ang2 inhibition and / or Tie2 receptor activation comprising said anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient.

Another example provides a composition for the diagnosis of diseases associated with Ang2 overexpression comprising said anti-Ang2 antibody or antigen-binding fragment thereof.

Other examples provide conjugates wherein said anti-Ang2 antibody or antigen-binding fragment thereof, Ang2, and Tie2 receptor are bound.

The present inventors have found that an antibody that specifically binds Ang2 but does not inhibit the binding of Ang2 to the Tie2 receptor and that forms a complex (antibody / Ang2 / Tie2) together with Ang2 and Tie2 receptors induces dimerization of the antibody Through which it is possible to induce activation of the Tie2 receptor and its downstream signaling by effectively clustering the complexed Tie2 receptor therewith. Antibodies with this mechanism of action bind to Ang2 to induce internalization and degradation, thereby inhibiting Ang2 function and lowering the level of circulating Ang2. In addition, Ang2 binds to Tie2 receptor and binds Tie2 receptor To induce Tie2 downstream signaling and to induce stabilization of vascular endothelial cells.

The present invention relates to a therapeutic antibody targeting angiogenesis inducing factor Ang2, which not only inhibits Ang2 function by specifically binding to Ang2 but also binds to Tie2 together with Ang2 to induce activation of Tie2 Anti-Ang2 < / RTI > The anti-Ang2 antibody binds to the Tei2 receptor together with Ang2 and activates the Tei2 receptor by downregulation of ang2 in diseases related to abnormal activation of blood vessels and dysfunction such as cancer, sepsis and eye diseases, To induce structural / functional normalization of the disease.

Specifically, one example of the present invention provides an anti-Ang2 antibody or antigen-binding fragment thereof that specifically binds (recognizes) Ang2 (angiopoietin-2), a neovascularization-inducing factor, and binds to Tie2 receptor together with Ang2 do. That is, the anti-Ang2 antibody or antigen-binding fragment thereof may specifically recognize Ang2 and bind to both Ang2 and Tie2 receptors. In addition, the anti-Ang2 antibody or antigen-binding fragment thereof may be one that induces activation of the Tie2 receptor. Activation of such Tie2 receptors may result in increased phosphorylation of the Tie2 receptor and / or at least one phosphorylation selected from the group consisting of proteins associated with the lower signal pathway, such as Akt (NM_005163), eNOS (NM_000603), 42/44 (NM_002745) Lt; / RTI > In addition, the anti-Ang2 antibody or antigen-binding fragment thereof may induce internalization of the Tie2 receptor into the cell. That is, the anti-Ang2 antibody or its antigen-binding fragment specifically binds to Ang2, but unlike the existing anti-Ang2 antibody, it does not inhibit the binding of Ang2 to the Tie2 receptor and binds to the Tie2 receptor together with Ang2, And induce the activation of the Tie2 receptor. The Ang2 protein, which acts as an antigen of the antibody provided in the present invention, is closely related to neovascularization and is a soluble ligand present in the blood, which is an angiogenesis ), Metastasis, and invasion of cancer cells. The Ang2 may be derived from a mammal such as a primate such as a human, a monkey, or a rodent such as a mouse or a rat. Examples of the Ang2 include Ang2 (for example, NCBI Accession No. O15123), Monkey Ang2 (for example, NCBI Accession No. Q8MIK6 Etc.), mouse Ang2 (e.g., NCBI Accession No. O35608 etc.), rat Ang2 (e.g. NCBI Accession No. O35462 etc.), and the like.

Tie tyrosine kinase is an angiopoietin-1 receptor and is expressed in endothelial cells of various mammals such as mouse (NM_013690; NP_038718), rat, human (NM_000459; NP_000450) It is involved in downstream signaling.

As described above, the anti-Ang2 antibody or its antigen-binding fragment provided by the present invention binds specifically to Ang2 but does not inhibit the binding of Ang2 to the Tie2 receptor and binds to the complex (antibody / Ang2 / Tie2) together with Ang2 and Tie2 receptors. Antibody has the property of inducing dimerization of the antibody and by effectively clustering the Tie2 receptor complexed therewith through this, it is possible to activate the Tie2 receptor and its downstream signaling Can be derived. Due to this mechanism, the antibody or its antigen-binding fragment of the present invention binds to Ang2 to induce internalization and degradation, thereby inhibiting Ang2 function and lowering the circulating Ang2 level, Tie2 receptor and activates Tie2 receptor like Ang1 to induce stabilization of vascular endothelial cells. As a result, not only the symptoms (diseases) caused by Ang2 overexpression but also the degradation of vascular endothelial cells such as vascular permeability (Disease) caused by an increase in the amount of the drug.

The anti-Ang2 antibody or antigen-binding fragment thereof may comprise all or part of the loop 1 (the region from position 417 to amino acid 434 of SEQ ID NO: 11) of human Ang2 (hAng2; SEQ ID NO: 11; Accession # O15123) Or one or more amino acid residues selected from the group consisting of consecutive or non-consecutive amino acid residues that are exposed to the outside of the loop 1 in SEQ ID NO: To 15, 2 to 10, or 2 to 5 amino acids, as an epitope, or specifically binds to the site.

Ang2 (SEQ ID NO: 11)

MWQIVFFTLS CDLVLAAAYN NFRKSMDSIG KKQYQVQHGS CSYTFLLPEM DNCRSSSSPY

VSNAVQRDAP LEYDDSVQRL QVLENIMENN TQWLMKLENY IQDNMKKEMV EIQQNAVQNQ

TAVMIEIGTN LLNQTAEQTR KLTDVEAQVL NQTTRLELQL LEHSLSTNKL EKQILDQTSE

INKLQDKNSF LEKKVLAMED KHIIQLQSIK EEKDQLQVLV SKQNSIIEEL EKKIVTATVN

NSVLQKQQHD LMETVNNLLT MMSTSNSAKD PTVAKEEQIS FRDCAEVFKS GHTTNGIYTL

TFPNSTEEIK AYCDMEAGGG GWTIIQRRED GSVDFQRTWK EYKVGFGNPS GEYWLGNEFV

SQLTNQQRYV LKIHLKDWEG NEAYSLYEHF YLSSEELNYR IHLKGLTGTA GKISSISQPG

NDFSTKDGDN DKCICKCSQM LTGGWWFDAC GPSNLNGMYY PQRQNTNKFN GIKWYYWKGS

GYSLKATTMM IRPADF

For example, the anti-Ang2 antibody may comprise a combination of Q418, P419, Q418 and P419 located in loop 1 of SEQ ID NO: 11, or a combination of Q418, P419, Q418 and P419 in SEQ ID NO: Or may specifically recognize an amino acid sequence region containing 2 to 20, 2 to 15, 2 to 10, or 2 to 5 amino acid residues as an epitope or specifically bind to this site. In one embodiment, the anti-Ang2 antibody may recognize an amino acid residue of Q418 and P419 of SEQ ID NO: 11 as an epitope, or specifically bind to this moiety.

Q418, P419, or an amino acid region comprising the specific binding site of the anti-Ang2 antibody is an exposed amino acid residue located in loop 1 of the three-dimensional structure of Ang2, and is directly involved in binding between Ang2 and Tie2 receptors , And it is considered to be a site to control this.

In the Q418, P419, or the amino acid region containing the specific anti-Ang2 antibody binding site, the "non-contiguous amino acid" is adjacent to each other on the secondary or tertiary structure of the protein, but is not continuous on the amino acid sequence It may mean an amino acid that does not exist. Thus, "contiguous or non-contiguous amino acid residues" in the present context may refer to contiguous amino acid residues in the primary, secondary or tertiary structure of the protein.

In addition to an anti-Ang2 antibody that recognizes and / or specifically binds to the site, an antibody or antigen-binding fragment thereof that competitively binds to the anti-Ang2 antibody also inhibits Ang2 and complexes with Ang2 and Tie2 receptors (I.e., antibody-Ang2 binds to the Tie2 receptor) to activate Tie2. Such competitively binding antibodies may be antibodies that recognize the previously described sites and adjacent sites on the three dimensional structure as epitopes and / or specific binding sites. The competitively binding antibody may have a binding affinity for Ang2 of 0.1 pM to 50 nM, specifically 1 pM to 30 nM, 2 pM to 20 nM, or 1 nM to 10 nM.

Accordingly, the anti-Ang2 antibody or antigen-binding fragment thereof of the present invention may be at least one selected from the group consisting of an antibody or an antigen-binding fragment which recognizes or specifically binds to the site described above as an epitope.

In an embodiment, the anti-Ang2 antibody or antigen-

A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (CDR-H1), a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (CDR-H2), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 A heavy chain complementarity determining region (CDR) comprising at least one member selected from the group consisting of a heavy chain variable region,

(CDR-L3) comprising the amino acid sequence of SEQ ID NO: 4, a polypeptide (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 5, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 ), Or a light chain variable region comprising the light chain complementarity determining region;

A combination of at least one heavy chain complementarity determining region and at least one light chain complementarity determining region; or

The combination of the heavy chain variable region and the light chain variable region

: ≪ / RTI >

More specifically, the anti-Ang2 antibody or antigen-binding fragment thereof

A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (CDR-H1), a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (CDR-H2), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 ), Or a heavy chain variable region comprising the heavy chain complementarity determining region: and

(CDR-L3) comprising the amino acid sequence of SEQ ID NO: 4, a polypeptide (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 5, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 ), Or a light chain variable region containing the light chain complementarity determining region

. ≪ / RTI >

Specifically, the heavy chain complementarity determining region of the anti-Ang2 antibody or antigen-binding fragment thereof may have, for example, the amino acid sequence shown in Table 1 below.

Heavy chain CDR amino acid sequence CDRH1-KABAT CDRH2-KABAT CDRH3-KABAT SDYAWN
(SEQ ID NO: 1) YINYSGNTDYNPSLKS
(SEQ ID NO: 2) GNFEGAMDY
(SEQ ID NO: 3)

The light chain complementarity determining region of the anti-Ang2 antibody or antigen-binding fragment thereof may be, for example, one having the amino acid sequence shown in Table 2 below.

Light chain CDR amino acid sequence CDRL1-KABAT CDRL2-KABAT CDRL3-KABAT KASQSVSNDVA
(SEQ ID NO: 4) YASNRYP
(SEQ ID NO: 5) QQDYSSPWT
(SEQ ID NO: 6)

In one embodiment, the heavy chain variable region of the antibody or antigen-binding fragment thereof may comprise the amino acid sequence of SEQ ID NO: 7.

VQLQESGPGLVKPSQSLSLTCTVTGYSIT SDYAWN WIRQFPGNKLEWMG YINYSGNTDYNPSLKS RSSITRDTSKNQFFLQLNSVTTGDTATYYCAR GNFEGAMDY WG (SEQ ID NO: 7)

(In SEQ ID NO: 7, the underlined bold letters are CDRH1, CDRH2, CDRH3 in order)

The light chain of the antibody according to one embodiment may comprise the amino acid sequence of SEQ ID NO: 9.

SIVMTQTPKFLLVSAGDRVTITC KASQSVSNDVA WYQQKPGQSPKLLIY YASNRYP GVPDRFTGSGYGTDFTFTISTVQAEDLAVYFC QQDYSSPWT FGGGTKLEIK (SEQ ID NO: 9)

(In SEQ ID NO: 9, underlined bold letters are CDRL1, CDRL2, CDRL3 in order)

Thus, the anti-Ang2 antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9, or a combination of the heavy chain variable region and the light chain variable region May include.

For example, the anti-Ang2 antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

In one example, the anti-Ang-2 antibody or antigen-binding fragment thereof may not consist of only one or more amino acid sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO:

In another example, the epitopes described above may be used to screen candidate agents, such as anti-Ang2 antibodies, for the diagnosis, prevention and / or treatment of diseases associated with Ang2 overexpression, angiogenesis, vascular permeability enhancement, and / A screening method is provided. In the screening method,

(a) contacting the candidate compound with an epitope of the tertiary structure of angiopoietin-2 as described above; And

(b) measuring the binding force between the epitope and the candidate compound

. ≪ / RTI >

The screening method may further comprise the step of (c) confirming the binding between Ang2 and Tie2 receptors. The step (c) may be performed to confirm whether the candidate compound inhibits the binding between Ang2 and Tie2 receptors, and may be performed before, after, or simultaneously with the step (b). Through the above step c), an anti-Ang2 antibody that does not inhibit the binding between Ang2 and Tie2 receptor, that is, a binding between Ang2 and Tie2 receptor, can be screened by binding to Ang2.

The screening method may further include the step of (d) confirming the Ang2 inhibition and / or the activation of the Tie2 receptor, and the sequence of the Ang2 inhibition is not limited. Through the above step d), an anti-Ang2 antibody which binds to Ang2 and inhibits its activity but binds to Ang2 to induce binding between Ang2 and Tie2 receptor and exhibits a Tie2 receptor activation effect such as Ang1 can be screened.

In the above screening method. The epitope and the candidate compound exhibit a binding force (kd) in the range of 10 nM or less, such as 0.1 pM to 10 nM, or 10 pM to 10 nM, or 100 pM to 10 nM, When the binding between Ang2 and Tie2 receptor is confirmed with the same binding force (i.e., when the candidate compound does not inhibit Ang2 and Tie2 receptor binding), the candidate compound can be used for the treatment of Ang2 overexpression, angiogenesis, vascular permeability enhancement, / RTI > and / or candidate substances for diagnosis, prevention, and / or treatment of diseases associated with decreased normal angiogenesis, such as anti-Ang2 antibody candidates.

The epitope may include all or some of the loop 1 (the region from position 417 to amino acid 434 of SEQ ID NO: 11) of human Ang-2 (hAng-2; SEQ ID NO: 11; Accession # O15123) Or two or more amino acid residues that are consecutive or non-consecutive, 2 to 15 amino acid residues that are exposed to the outside of loop 1 in SEQ ID NO: 11, , Or two to ten amino acids, for example, a combination of Q418, P419, Q418 and P419 located in loop 1 of SEQ ID NO: 11, or two or more contiguous or non-contiguous To 20, 2 to 15, 2 to 10, or 2 to 5 amino acid residues.

The candidate compound may be an artificially synthesized or naturally occurring compound, a polypeptide, an oligopeptide, a peptide or a protein structure (for example, an antibody, a peptibody, a nanobody, etc.), a polynucleotide, an oligonucleotide, an antisense- hairpin RNA), siRNA (small interference RNA), platamer, natural product extract, and the like.

The step of measuring the binding force between the epitope and the candidate compound can be carried out through various methods known in the art. For example, the binding force can be measured using a Biacore device. Generally, the range in which the binding force is recognized as a therapeutic agent can be defined as a binding constant, a KD value, of 10 nM or less. That is, for example, when the binding force between the epitope of angiopoietin-2 and the sample (for example, antibody) to be analyzed is measured by a surface plasmon resonance method such as a Biacore apparatus, (E. G., Antibody) to Ang2 overexpression, angiogenesis, vascular permeability enhancement, and / or normal angiogenesis < RTI ID = 0.0 > It can be judged as a candidate substance for diagnosis, prevention and / or treatment of a related disease.

The confirmation of the binding between Ang2 and Tie2 and the confirmation of Ang2 inhibition and activation of Tie2 receptor are described below.

In another example, the heavy chain complementarity determining region, the light chain complementarity determining region, or a combination thereof of the anti-Ang2 antibody described above; Or a polypeptide chain comprising a heavy chain variable region, a light chain variable region, or a combination thereof. The polypeptide molecule may function as a precursor or constituent of an antagonist to Ang2, as well as to produce an antibody or antigen-binding fragment thereof. For example, the polypeptide molecule may serve as an Ang2 antigen binding site and may be composed of a protein scaffold (e.g., a peptibody, a nanobody, etc.) having a structure similar to an antibody, a bispecific antibody, ≪ / RTI >

The term "antagonist" is interpreted to include all molecules that partially or completely block, inhibit or neutralize one or more of the biological activities of the target (e.g., Ang2).

The term "peptide + antibody" is a fusion protein in which all or part of a constant region, such as the Fc portion of a peptide and an antibody, is fused, wherein the peptide has an antigen binding site (heavy chain and / or light chain CDR or variable region) Quot; means a protein having a skeleton and function similar to an antibody.

The term "nanobody ", also referred to as a single-domain antibody, refers to an antibody fragment that contains a single variable domain of an antibody in the form of a monomer, . The molecular weight of the nanobodies is generally about 12 kDa to about 15 kDa and is very small compared to the typical molecular weight (about 150 kDa to about 160 kDa) of complete antibodies (including two heavy chains and two light chains) Is smaller than Fab fragment or scFv fragment.

The term "bispecific antibody" or "multispecific antibody" refers to an antibody that recognizes and / or binds different antigens of two (bispecific or multispecific) or different regions of the same antigen and / Refers to an antibody that binds to the antigen, wherein the antigen binding site of one of the bispecific antibody or the multispecific antibody comprises the polypeptide.

In an embodiment, the polypeptide molecule comprises

At least one selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, a polypeptide including the amino acid sequence of SEQ ID NO: 2, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 3;

At least one selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, a polypeptide including the amino acid sequence of SEQ ID NO: 6, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 6; or

Combinations thereof

. ≪ / RTI >

In an embodiment, the polypeptide molecule may comprise the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9, or a combination thereof.

In one example, the polypeptide molecule may not consist solely of one or more amino acid sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO:

As described above, the above-described bispecific antibody or multispecific antibody may comprise an antibody that simultaneously recognizes and / or binds two or more antigens, including respective antigen binding sites for two or more different antigens , And one of the antigen binding sites may include the polypeptide molecule as described above. Specifically, the polypeptide molecule serving as the Ang2 antigen binding site may form a duplex or multispecific antibody by forming a dimer or polymorphism with an antigen binding site for another antigen. Thus, in one embodiment, there is provided a bispecific or multispecific antibody comprising the polypeptide molecule as an Ang2 antigen binding site.

In another example, a polypeptide having at least one of the above-mentioned polypeptide molecules or a repeating structure (hereinafter, referred to as a 'first peptide') and a structural function (hereinafter, referred to as a 'second peptide' (E.g., 1 to 5, or 2 to 4, or 5 to 10) peptide complexes comprising a peptide complex comprising a heavy chain variable region (e.g., a heavy or light chain constant region of an antibody (e.g., IgG, IgA, IgE, IgD, IgM, ), And the at least one peptide complex is bound at a second peptide (e.g., an Fc fragment) to provide a protein skeleton having a multimer structure.

In the present invention, the antibody includes both an animal-derived antibody, a chimeric antibody, a humanized antibody, and a human antibody. An animal-derived antibody that produces a desired antigen by immunizing an immunized animal generally has immunomodulation upon administration to a human for therapeutic purposes, and a chimeric antibody has been developed in order to suppress such immune rejection. The chimeric antibody uses a genetic engineering method to replace the constant region of the animal-derived antibody, which is responsible for the anti-isotype reaction, with the constant region of the human antibody. Chimeric antibodies have been significantly improved in anti-isotype reactions compared to animal-derived antibodies, but still animal-derived amino acids are present in the variable region and have side effects on potential anti-idiotypic responses . Humanized antibodies have been developed to improve these side effects. It is constructed by grafting complementarity determining regions (CDRs), which play an important role in the binding of antigens in the variable regions of chimeric antibodies, to the human antibody framework.

The most important of the CDR grafting techniques for making humanized antibodies is to select an optimized human antibody that can best accommodate the CDR region of the animal-derived antibody. For this purpose, use of the antibody database, crystal structure structure analysis, and molecular modeling techniques. However, even if the CDR region of the animal-derived antibody is transplanted into the optimized human antibody backbone, the amino acid that affects the antigen binding may exist in the skeleton of the animal-derived antibody, The application of additional antibody engineering techniques to restore antigen binding is essential.

According to one embodiment, the antibody may be a mouse-derived antibody, a mouse-human chimeric antibody, a humanized antibody, or a human antibody.

The term "antibody" in the present invention means a substance produced by stimulation of an antigen in the immune system, and the kind thereof is not particularly limited. Antibodies have recently been widely used for the treatment of disease. Antibodies are very stable in vivo as well as in vivo and have a long half-life, which is advantageous for mass production and production. In addition, the antibody has an inherently dimer structure and thus has a very high avidity.

A complete antibody is a structure with two full length light chains and two full length heavy chains, each light chain linked by a heavy chain and a disulfide bond. The constant region of the antibody is divided into a heavy chain constant region and a light chain constant region and the heavy chain constant region has the type of gamma, mu, alpha, delta and epsilon, Gamma 1, gamma 2, gamma 3, gamma 4, alpha 1, and alpha 2, respectively. The constant region of the light chain has kappa (kappa) and lambda (lambda) types.

The term "heavy chain" refers to a variable region domain V _H comprising an amino acid sequence having a sufficient variable region sequence sufficient to confer antigen specificity, and three constant domain domains C _H1 , C _H2 and C _H3 and a hinge hinge < / RTI > and fragments thereof. The term "light chain" also encompasses both the full-length light chain comprising the variable region domain V _L and the constant region domain C _L comprising the amino acid sequence having a sufficient variable region sequence to confer specificity to the antigen and fragments thereof Is interpreted to mean inclusive.

The term "CDR (complementarity determining region)" refers to the amino acid sequence of the heavy chain of the immunoglobulin and the hypervariable region of the light chain. The heavy and light chains may each contain three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide the major contact residues for binding of the antibody to the antigen or epitope. In the present specification, the terms "specifically binding" or "specifically recognizing" are the same as those conventionally known to those skilled in the art, and the antigen and the antibody specifically interact to effect an immunological reaction .

The antigen-binding fragment of the antibody provided herein may be a fragment containing at least one of the complementarity determining sites.

The term "antigen binding fragment" refers to a fragment of a polypeptide comprising an immunoglobulin entire structure and a portion to which an antigen can bind. But are not limited to, for example, scFv, (scFv) ₂ , scFv-Fc, Fab, Fab 'or F (ab') ₂ .

Among the antigen-binding fragments, Fab has one antigen-binding site in a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (C _H1 ) of a heavy chain. Fab 'differs from Fab in that it has a hinge region that contains at least one cysteine residue at the C-terminus of the heavy chain C _H1 domain. The F (ab ') ₂ antibody is produced when the cysteine residue of the hinge region of the Fab' forms a disulfide bond. Recombinant techniques for producing Fv fragments with minimal antibody fragments having only heavy chain variable regions and light chain variable regions are well known in the art. The double-chain Fv is a non-covalent bond, and the variable region of the heavy chain and the light chain variable region are connected to each other. The single-chain Fv generally shares the variable region of the heavy chain and the variable region of the short chain through the peptide linker Or directly connected at the C-terminus to form a dimer-like structure like the double-stranded Fv. The linker may be a peptide linker consisting of 1 to 100 or 2 to 50 arbitrary amino acids, and sequences suitable for the art are known. The antigen-binding fragment can be obtained using a protein hydrolyzing enzyme (for example, when the whole antibody is restricted to papain, a Fab can be obtained and when cut with pepsin, an F (ab ') ₂ fragment can be obtained) Can be produced through recombinant DNA technology.

The term "hinge region" refers to a region that is included in the heavy chain of the antibody, which is present between the CH1 and CH2 regions and which functions to provide flexibility of the antigen binding site in the antibody. For example, the hinge may be derived from a human antibody, and specifically may be derived from IgA, IgE, or IgG, such as IgG1, IgG2, IgG3, or IgG4.

When the animal-derived antibody is subjected to a chimerization process, the animal-derived IgG1 hinge is replaced with a human IgG1 hinge. However, the animal-derived IgG1 hinge is shorter in length than the human IgG1 hinge, and the disulfide bond disulfide bond decreases from 3 to 2, and the rigidity of the hinge is different from each other. Thus, modification of the hinge region can increase the antigen binding efficiency of the humanized antibody. Methods of deletion, addition, or substitution of amino acids to modify the amino acid sequence of the hinge region are well known to those skilled in the art.

The portion of the anti-Ang2 antibody other than the variable region may be a constant region derived from a human antibody and specifically may be a constant region derived from IgA, IgE, or IgG, such as IgG1, IgG2, IgG3, or IgG4 .

The anti-Ang2 antibody may be a monoclonal antibody. Monoclonal antibodies can be prepared by methods well known in the art. For example, it can be produced using a phage display technique. Alternatively, the anti-Ang2 antibody can be prepared from a mouse monoclonal antibody by the method described in Schwaber et al. Using mouse monoclonal antibody (Schwaber, J and Cohen, EP, "Human x Mouse Somatic Cell Hybrid Clones Secreting Immunoglobulins of Both Parental Types, "Nature, 244 (1973), 444-447).

On the other hand, individual monoclonal antibodies can be screened based on their ability to bind Ang2 using a typical ELISA (Enzyme-Linked ImmunoSorbent Assay) format. Can be assayed for inhibitory activity through functional assays such as competitive ELISA (competitively ELISA) or cell-based assays to test molecular associations for conjugates. Each affinity for Ang2 (Kd values) is then tested for selected monoclonal antibody members based on strong inhibitory activity.

The finally selected antibodies can be used as humanized antibodies as well as human immunoglobulin antibodies except for the antigen binding portion. Methods of making humanized antibodies are well known in the art (Almagro, J.C. and Fransson, J., "Humanization of antibodies," Frontiers in Bioscience, 13 (2008), 1619-1633).

Another example provides a hybridoma cell line that produces a monoclonal antibody of said anti-Ang2 antibody. The hybridoma cell line may be a cell strain that is a Accession Number (KCLRF-BP-00295).

As described above, the anti-Ang2 antibody or its antigen-binding fragment proposed in the present invention is characterized in that Ang2 binds to Ang2 but does not inhibit Ang2 binding to Tie2 receptor and induces binding of Ang2 to Tie2 receptor. In addition, the thus conjugated anti-Ang2 antibody or its antigen-binding fragment and Ang2 conjugate acts like Angl to bind to the Tie2 receptor (Ang2 portion of the complex binds) and activates the Tie2 receptor Function.

Thus, another example provides the anti-Ang2 antibody-Ang2 complex conjugated to Ang2 with the anti-Ang2 antibody or antigen-binding fragment thereof. Another example provides a composition for inducing binding of Ang2 to a Tie2 receptor comprising the anti-Ang2 antibody-Ang2 complex as an active ingredient. Another example provides a method of inducing the binding of Ang2 to a Tie2 receptor, comprising administering the anti-Ang2 antibody-Ang2 complex to a patient in need of Ang2 and Tie2 receptor binding. The method of inducing the binding of Ang2 to the Tie2 receptor may further comprise identifying a patient in need of binding Ang2 and Tie2 receptors prior to the administering step. Another example provides a composition for activating Tie2 receptor comprising the above anti-Ang2 antibody-Ang2 complex as an active ingredient. Another example provides a method of activating Tie2 receptors comprising administering the anti-Ang2 antibody-Ang2 complex to a patient in need of activation of the Tie2 receptor. The Tie2 receptor activation method may further comprise identifying a patient in need of activation of the Tie2 receptor prior to the administering step. The patient may be a mammal including a primate including a human, a monkey and the like, a rodent including a mouse, a rat, etc., or a cell, a tissue, a body fluid or the like separated or artificially cultured therefrom. Another example provides for the use of said anti-Ang2 antibody-Ang2 complex for Tie2 receptor activation.

As described above, since the anti-Ang2 antibody or its antigen-binding fragment proposed in the present invention has a function of inhibiting abnormal angiogenesis by inhibiting the function of Ang2, the prevention of various diseases (for example, cancer) associated with abnormal angiogenesis , Alleviation, amelioration and / or treatment (see Example 13). In addition, the anti-Ang2 antibody or antigen-binding fragment thereof does not inhibit binding between Ang2 and Tie2 (see Example 3 and Figure 1), activating Tie2 (see Example 5 and Figure 2) (See Example 6 and FIG. 3) to promote vascular endothelial or lymphatic endothelial production and increase mobility (see Example 10), inhibiting increased vascular permeability (see Example 11), associated with vascular permeability It is also applicable to the prevention, alleviation, amelioration and / or treatment of various diseases (e.g., sepsis, eye diseases, etc.). In addition, as described above, the anti-Ang2 antibody or antigen-binding fragment thereof promotes the formation of vascular endothelium or lymphatic endothelium, thereby increasing the formation of healthy blood vessels and normalizing blood vessels. Thus, formation of healthy blood vessels such as wound healing or ischemic diseases And / or treatment of a variety of diseases or conditions requiring the treatment of cancer, and reduces the likelihood of cancer growth and metastasis by altering an abnormally formed cancer blood vessel into a structurally and functionally normal state. In addition, the anti-Ang2 antibody or antigen-binding fragment thereof has an effect of inhibiting an inflammatory reaction (see Example 12), and is applicable to prevention, alleviation, improvement, and / or treatment of various inflammatory diseases.

Another example provides a pharmaceutical composition for inhibiting angiogenesis comprising the anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient. Another example provides a method of inhibiting angiogenesis comprising administering a pharmaceutically effective amount of the anti-Ang2 antibody or antigen-binding fragment thereof to a patient in need of inhibition of angiogenesis. The method of inhibiting angiogenesis may further comprise identifying a patient in need of inhibition of angiogenesis prior to the administering step.

Another example provides a pharmaceutical composition for reducing vascular permeability comprising the anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient. Another example provides a method for reducing vascular permeability comprising administering a pharmaceutically effective amount of the anti-Ang2 antibody or antigen-binding fragment thereof to a patient in need of a reduction in vascular permeability. The method of reducing vascular permeability may further comprise identifying a patient in need of a reduction in vascular permeability prior to the administering step.

Another example provides a pharmaceutical composition for preventing and / or treating diseases associated with Ang2 overexpression, angiogenesis, and / or increased vascular permeability comprising the anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient. Another example comprises administering a therapeutically effective amount of the anti-Ang2 antibody or antigen-binding fragment thereof to a patient in need of prevention and / or treatment of a disease associated with Ang2 overexpression, angiogenesis, and / or increased vascular permeability A method for the prevention and / or treatment of diseases associated with Ang2 overexpression, angiogenesis, and / or increased vascular permeability. The method of preventing and / or treating may further comprise the step of identifying a patient in need of prevention and / or treatment of a disease associated with Ang2 overexpression, angiogenesis and / or vascular permeability increase prior to said administering step have.

Another example provides a pharmaceutical composition for inducing normal angiogenesis comprising the anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient. Another example provides a method for increasing normal angiogenesis comprising administering a pharmaceutically effective amount of the anti-Ang2 antibody or antigen-binding fragment thereof to a patient in need of induction of normal angiogenesis. The method of reducing vascular permeability may further comprise identifying a patient in need of induction of normal angiogenesis prior to the administering step.

Another example provides a pharmaceutical composition for the prevention and / or treatment of diseases associated with decreased normal angiogenesis, comprising the anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient. Another example is a method of treating a disease associated with decreased normal angiogenesis, comprising administering a therapeutically effective amount of the anti-Ang2 antibody or antigen-binding fragment thereof to a patient in need of prevention and / or treatment of a disease associated with decreased normal angiogenesis / RTI > and / or < / RTI > The method of preventing and / or treating may further include identifying a patient in need of prevention and / or treatment of a disease associated with a reduction in normal angiogenesis prior to the administering step.

Another example provides a pharmaceutical composition for tissue regeneration and / or wound healing comprising the anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient. Another example provides a method for tissue regeneration and / or wound healing comprising administering a therapeutically effective amount of the anti-Ang2 antibody or antigen-binding fragment thereof to a patient in need of tissue regeneration and / or wound healing. The method may further comprise identifying a patient in need of tissue regeneration and / or wound healing prior to the administering step. The subject to be administered with the active ingredient may be, for example, a patient suffering from damage to skin tissue or organ tissue, or having undergone skin transplantation.

Another example provides a pharmaceutical composition for Ang2 inhibition and / or Tie2 receptor activation comprising said anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient. Another example provides a method of activating Ang2 inhibition and / or Tie2 receptor comprising administering a therapeutically effective amount of said anti-Ang2 antibody or antigen-binding fragment thereof to a patient in need of Ang2 inhibition and / or Tie2 receptor activation. The Ang2 inhibition and / or Tie2 receptor activation method may further comprise identifying a patient in need of Ang2 inhibition and / or Tie2 receptor activation prior to the administering step. The anti-Ang2 antibody or antigen-binding fragment thereof may be in a state bound to the antigen Ang2.

Since the anti-Ang2 antibody or antigen-binding fragment thereof, which is an active ingredient of the pharmaceutical composition, is activated by binding to Ang2, the pharmaceutical composition may further include Ang2 in order to further enhance the function of the antibody or antigen- . The method may further comprise administering to the patient a pharmaceutically effective amount of Ang2.

Another example provides a composition for the diagnosis of diseases associated with increased angiogenesis and / or vascular permeability and / or decreased normal angiogenesis, comprising the antibody or antigen-binding fragment thereof.

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, which is usually used for the formulation of drugs, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate , Alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. , But the present invention is not limited thereto. The pharmaceutical composition may further comprise at least one member selected from the group consisting of a diluent, an excipient, a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent,

The pharmaceutical composition, or a pharmaceutically effective amount of the antibody or antigen-binding fragment thereof, can be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and intrathecal administration. In oral administration, the protein or peptide is extinguished, so that the oral composition can be formulated to coat the active agent or protect it from degradation from above. In addition, the composition may be administered by any device capable of transferring the active agent to the target cell.

The content of the anti-Ang2 antibody or antigen-binding fragment thereof in the pharmaceutical composition may be determined by various methods such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration interval, administration route, It can be prescribed variously by factors. For example, the daily dose of the anti-Ang2 antibody or antigen-binding fragment thereof may be in the range of 0.001 to 1000 mg / kg, specifically 0.01 to 100 mg / kg, more specifically 0.1 to 50 mg / kg, no. The daily dose may be formulated into a single dosage form in unit dose form, formulated in an appropriate amount, or intruded into a multi-dose container. The "pharmaceutically effective amount" may mean the amount or dose of the active ingredient that can exhibit the desired pharmacological effect, and may be determined by the formulation method, the manner of administration, the age, weight, sex, , Administration interval, route of administration, excretion rate, and responsiveness.

The pharmaceutical composition may be in the form of solutions, suspensions, syrups or emulsions in an oil or aqueous medium, or may be formulated in the form of excipients, powders, granules, tablets or capsules, May additionally include topics.

In particular, the pharmaceutical composition comprising the anti-Ang2 antibody or antigen-binding fragment thereof comprises an antibody or an antigen-binding fragment and can therefore be formulated as an immunoliposome. Liposomes containing antibodies can be prepared according to methods well known in the art. The immunoliposome is a lipid composition comprising phosphatidylcholine, cholesterol and polyethylene glycol-derivatized phosphatidylethanolamine and can be prepared by reverse phase evaporation. For example, a Fab 'fragment of an antibody can be conjugated to a liposome via a disulfide-replacement reaction.

On the other hand, the anti-Ang2 antibody or antigen-binding fragment thereof specifically binds to Ang2, so that Ang2 can be detected using the anti-Ang2 antibody, thereby confirming overexpression of Ang2. Thus, another embodiment of the present invention provides a composition for detecting Ang2 comprising said anti-Ang2 antibody or antigen-binding fragment thereof and a composition for diagnosing diseases associated with Ang2 overexpression.

In another example, treating the anti-Ang2 antibody or antigen-binding fragment thereof with a biological sample obtained from a patient (or isolated); And confirming whether or not an antigen-antibody reaction has occurred. Treating the biological sample obtained from the patient with the anti-Ang2 antibody or antigen-binding fragment thereof; And a method of providing information to diagnosis of a disease associated with Ang2 overexpression comprising the step of confirming whether the antigen-antibody reaction is present. When the antigen-antibody reaction is detected in the step of confirming whether the antigen-antibody reaction is detected, the method of providing information to the diagnosis may be to determine that the patient has Ang2 overexpression symptoms or Ang2 over-expression related disease . The biological sample may be selected from the group consisting of cells, tissues, body fluids, etc. obtained from the patient (or separated).

The step of confirming whether the antigen-antibody reaction is confirmed can be carried out through various methods known in the art. For example, it can be measured by a conventional enzyme reaction, fluorescence, luminescence and / or radiation detection, and specifically, immunochromatography, immunohistochemistry, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), Western blotting ), And the like, but the present invention is not limited thereto.

The subject to be administered or diagnosed with the pharmaceutical composition or antibody or antigen-binding fragment may be a mammal including a primate including a human, a monkey and the like, a rodent including a mouse, a rat, etc., or a cell isolated or artificially cultured therefrom, Tissue, body fluids, and the like.

Diseases associated with said angiogenesis and / or vascular permeability increase and / or Ang2 overexpression are cancer; Cancer; Ocular vascular diseases such as retinopathy of prematurity, macular degeneration (e.g., age-related macular degeneration), diabetic retinopathy, angiogenic glaucoma; Inflammatory diseases such as psoriasis, asthma, rheumatoid arthritis, pneumonia, chronic inflammation; Infectious disease (infection); Cardiovascular diseases such as hypertension and arteriosclerosis; Kidney disease; blood poisoning; asthma; edema; Hereditary hemorrhagic telangiectasia (HHT), and the like. The cancer overexpresses Ang2, which may be solid or hematocrit, including but not limited to squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, skin cancer, Endometrioid cancer, papillary cancer, soft tissue sarcoma, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatocellular carcinoma, gastric cancer, pancreatic cancer, , Ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, endometrial or uterine cancer, salivary cancer, renal cancer, prostate cancer, mucin cancer, thyroid cancer, head and neck cancer, brain cancer, osteosarcoma It can be more than a species.

The disease associated with decreased normal angiogenesis is a disease requiring the induction of normal angiogenesis, and includes diseases such as myocardial infarction, angina pectoris, ischemic diseases such as cerebral infarction and stroke (cerebral ischemia), Burger's disease (occlusive thrombosis), avascular necrosis avascular necrosis, foot ulcers (e.g., diabetic foot ulcers, etc.), erectile dysfunction, and the like.

Other examples provide conjugates wherein said anti-Ang2 antibody or antigen-binding fragment thereof, Ang2, and Tie2 receptor are bound. The complex may be present in vivo or in a cell isolated from a living body. The antibody in the complex may also form a dimer with an antibody in an adjacent complex to cluster two or more complexes to form a cluster comprising two or more complexes (see Example 8). This action is related to the Tie2 receptor activation function of the anti-Ang2 antibody. The complex may be used to monitor the action of the anti-Ang2 antibody, i.e., inhibition of Ang2 and / or Tie2 receptor, or may itself be used to inhibit Ang2 and / or activate Tie2 receptor.

Another example is a method for screening candidates for prevention and / or treatment of diseases associated with neovascularization and / or vascular permeability enhancement by ascertaining the complex formation and / or the activation of Ang2 inhibition and / or Tie2 receptor to provide.

In one embodiment, the screening method comprises:

Contacting a candidate compound with a sample comprising Ang2 and Tie2 receptor; And

Confirming the formation of a complex with the candidate compound, Ang2, and Tie2 receptor bound

. ≪ / RTI >

The step of confirming the formation of the complex with which the candidate compound, Ang2, and Tie2 receptor is bound can be confirmed by confirming the presence of a complex with the candidate compound, Ang2, and Tie2 receptor bound thereto, Lt; RTI ID = 0.0 > Tie2 < / RTI > The screening method may further comprise confirming the activation of the Ang2 inhibitor and / or the Tie2 receptor before, after, or simultaneously with the step of confirming the formation of the complex with the candidate compound, Ang2, and Tie2 receptor bound thereto have.

In the above screening method, when the formation of a complex in which the candidate compound, Ang2, and Tie2 receptor is bound is confirmed, that is, when the presence of the complex with the candidate compound, Ang2, and Tie2 receptor bound is confirmed, When the liver binding and the binding between Ang2 and Tie2 receptor are confirmed, the candidate compound is used as a candidate substance for the prevention and / or treatment of diseases related to Ang2 overexpression, angiogenesis, vascular permeability enhancement, and / or reduction of normal angiogenesis It can be judged.

In another example, the screening method comprises:

 Contacting a candidate compound with a sample comprising Ang2 and Tie2 receptor; And

Identifying Ang2 inhibition and / or activation of the Tie2 receptor

. ≪ / RTI > In the screening method, when the Ang2 inhibition and / or the activation of the Tie2 receptor is confirmed, the candidate compound may be used to prevent and / or prevent Ang2 overexpression, angiogenesis, vascular permeability enhancement, and / It can be judged as a candidate substance for treatment. The degree of inhibition (decomposition) of Ang2 can be determined by measuring the level of Ang2 before or after the treatment of the candidate compound with the same sample, or by dividing the same sample into the treatment group of the candidate compound and the untreated group, Before and after treatment, or with the levels of Ang2 in the treated and untreated groups. Or when the Ang2 level in the treated group or after the candidate compound treatment is reduced compared to the pre-treatment or untreated group of the candidate compound, it can be determined that Ang2 is inhibited. The activation of the Tie2 receptor may also be determined by comparing the degree of phosphorylation of one or more of the proteins involved in the degree of phosphorylation of the Tie2 receptor and / or the downstream signaling of the Tie2 receptor (e.g., Akt, eNOS, 42/44, Or before or after the treatment of the compound, or by dividing the same sample into the treatment group of the candidate compound and the untreated group, and comparing the degree of phosphorylation of the protein in the untreated group before and after the treatment with the candidate compound. At this time, if the degree of phosphorylation of the candidate compound or the treated group is increased compared to the pretreatment or non-treated group of the candidate compound, it can be judged that the Tie2 receptor is activated. Alternatively, activation of the Tie2 receptor can be determined by confirming the internalization of the Tie2 receptor.

In another example, the screening method comprises:

 Contacting a candidate compound with a sample comprising Ang2 and Tie2 receptor; And

Formation of a complex with candidate compound, Ang2, and Tie2 receptor bound, and activation of Ang2 inhibition and / or activation of Tie2 receptor

. ≪ / RTI > The formation of the complex, Ang2 inhibition, and activation of the Tie2 receptor are as described above.

Determination of the complex formation, measurement of Ang2 levels, measurement of the degree of phosphorylation of Tie2, measurement of the degree of phosphorylation of proteins involved in downstream signaling, and / or identification of internalization of the Tie2 receptor are well known in the art For example, by a conventional enzyme reaction, fluorescence, luminescence and / or detection of radiation. Specific examples thereof include immunochromatography, immunohistochemistry, immunohistochemistry, Enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (ELISA) LIA), Western blotting, and the like, but the present invention is not limited thereto The.

The candidate compound may be an artificially synthesized or naturally occurring compound, a polypeptide, an oligopeptide, a peptide structure or a protein structure (for example, an antibody, a peptibody, a nanobody, etc.), a polynucleotide, an oligonucleotide, an antisense- short hairpin RNA), siRNA (small interference RNA), platamer, natural product extract, and the like.

The sample comprising the Ang2 and Tie2 receptor is a cell or tissue isolated or artificially cultured in vivo and originally contains (expresses) an Ang2 and Tie2 receptor, or an Ang2 and / or Tie2 receptor is treated or Ang2 and / or Lt; RTI ID = 0.0 > Tie2 < / RTI >

Previously, there have been attempts to inhibit Ang2-induced angiogenesis by inhibiting the binding of Ang2 to its receptor Tie2, but it has been shown that Ang2 binds specifically to Ang2 and induces intracellular migration and degradation of Ang2, Antibodies that act to activate Tie2 receptors while maintaining binding potency and forming the antibody-Ang2-Tie2 receptor complex are not known to date. In this situation, the present invention suggests a novel method of inhibiting Ang2-induced angiogenesis and reducing vascular permeability by proposing an antibody that inhibits Ang2 while simultaneously activating Tie2 receptor and promoting downstream signal transduction. In addition, the antibody proposed in the present invention is expected to be applicable to diagnosis and treatment of abnormal angiogenesis-related diseases other than cancer and / or diseases caused by increased vascular permeability. The antibody can be used in combination with chemotherapeutic drugs and other anti-cancer therapies. It can be used for antibody fragments, bi- or multi-specific antibodies, and protein scaffolds using Ang2-specific cognitive functions.

FIG. 1 shows the Ang2-Tie2 competition ELISA showing the degree of inhibition of binding between Tie2 receptor and Ang2 according to the concentration of the anti-Ang2 antibody treatment according to one embodiment.
Figure 2 shows immunoblot results showing the level of Tie2 receptor and phosphorylated Tie2 receptor according to the treatment concentration of anti-Ang2 antibody according to one embodiment.
FIG. 3 is a result of immunoblotting showing the degree of protein phosphorylation involved in Downstream signaling of Tie2 receptor upon treatment with anti-Ang2 antibody according to one embodiment.
FIG. 4 shows the results of ELISA showing the degree to which the anti-Ang2 antibody according to one embodiment binds to Ang2 and Tie2 to form Complex.
FIG. 5 shows the results of immunoblotting showing the degree of phosphorylation of proteins involved in downstream signaling of Tie2 receptor and Tie2 receptor of a monomeric anti-Ang2 antibody according to one embodiment.
Figure 6 shows immunocyte staining results showing intracellular internalization of Tie2 receptors similar to Ang1 and anti Ang2 antibodies according to one embodiment.
FIG. 7 shows the effect of the anti-Ang2 antibody according to one embodiment on the proliferation of blood vessels and lymphatic endothelial cells.
FIG. 8 shows the effect of the anti-Ang2 antibody according to one embodiment on the migration of lymphatic endothelial cells.
Figure 9 shows the degree of inhibition of TNF or LPS-induced cell permeability in Angl or Ang2 antibody treatment according to Example 9.
10 shows results of mRNA expression of inflammation-related cell adhesion substances (ICAM-1, E-selectin) induced by LPS stimulation upon treatment with Ang1 or Ang2 antibody according to the 10th embodiment.
FIG. 11 is a graph showing relative intensities of fluorescence obtained from inflammatory cells in the treatment of anti-Ang2 antibody according to one embodiment, and shows the number of inflammatory cells attached to vascular endothelial cells.
Figure 12 shows the efficacy of an anti-Ang2 antibody according to one embodiment in a Colo205 tumor model.

Hereinafter, the present invention will be described in more detail with reference to the following examples, which should not be construed as limiting the scope of the present invention. It will be apparent to those skilled in the art that the embodiments described below may be modified without departing from the essential spirit of the invention.

Example  1. Section Ang2  Preparation of antibodies

The anti-Ang2 antibody was prepared by inducing an immune response by administering human Ang2 protein (R & D systems: 623-AN-025 / CF) with 5-week old BALB / c mice together with an adjuvant and then, by a known method described in Schwaber et al. (Schwaber, J and Cohen, EP, "Human x Mouse Somatic Cell Hybrid Clones Secreting Immunoglobulins of Both Parental Types," Nature, 244 (1973), 444-447).

More specifically, 100 ug of human Ang2 protein (R & D Systems) was added to 5 5-week old BALB / c mice (Japan SLC, Inc.) to obtain immunized mice necessary for development of hybridoma cell lines. (Freund ' s adjuvant) were mixed and injected intraperitoneally into 4-6 week old BALB / c mice (Japan SLC, Inc.). After 2 weeks, the antigen (half of the injected dose) was injected intraperitoneally in the same manner as above with the incomplete Freund's adjuvant mixed. Three days after the last boosting was performed, blood was collected from the tail of the mouse to obtain serum. After dilution with PBS at 1/1000, the antibody titer of Ang2 was increased by ELISA. As a result of the above, mice in which sufficient amounts of antibody were obtained were selected and cell fusion procedures were carried out.

Three days before the cell fusion experiment, a mixture of 50 ug of PBS and 100 ug of human Ang2 protein (R & D systems) was injected into the abdominal cavity of a BALB / c mouse (Japan SLC, Inc.), anesthetized with an immunized mouse, The spleen located on the left side of the spleen was removed. The extracted spleen was grinded to separate cells and mixed with a culture medium (DMEM, Hyclone) to prepare a spleen cell suspension. The suspension was centrifuged to recover the cell layer. 1x10 ⁸ spleen cells obtained above and 1x10 ⁷ myeloma cells (Sp2 / 0) were mixed and centrifuged to precipitate the cells. The centrifuged precipitate was slowly dispersed and treated with 1 ml of 45% polyethylene glycol (PEG 1500) in a culture medium (DMEM), maintained at 37 ° C for 1 min, and then 1 ml of culture medium (DMEM) Respectively. Then, 10 ml of the culture medium (DMEM) was added for 1 minute, allowed to stand in water at 37 ° C for 5 minutes, and then centrifuged again to 50 ml. The cell precipitate was resuspended in a separation medium (HAT medium) at a concentration of 1 to 2 × 10 ⁵ / ml, and 0.1 ml of each of the cells was dispensed into a 96-well plate and cultured in a 37 ° C. carbon dioxide incubator to prepare a hybridoma cell group Respectively.

Example  2. The anti- Ang2  Production of antibodies

2.1. term- Ang2  Production of antibody clones and antibody purification

Hybridomas producing 95 anti-Ang2 monoclonal antibodies in hybridomas differentiated from the parent hybridoma were selected based on their binding ability to Ang2 using the typical ELISA format from the antibody-producing hybridomas obtained above.

More specifically, to select hybridoma cells that specifically react only with Ang2 protein among the hybridoma cell groups prepared in Example 1, ELISA analysis using human Ang2 protein as an antigen was screened.

Human Ang2 protein was added to the microtiter plate in an amount of 100 ng / well, and the unreacted antigen was washed and removed. 50 쨉 l of the culture solution of the hybridoma cells obtained in Example 1 was added to each well and reacted for 1 hour, followed by thorough washing with phosphate buffer solution-Tween 20 (TBST) to remove unreacted culture medium. Mouse IgG-horseradish peroxidase (goat anti-mouse IgG-HRP) was added thereto, followed by reaction at room temperature for 1 hour, followed by washing with TBST solution. Then, the reaction was carried out by adding a substrate solution (OPD) of peroxidase, and the degree of the reaction was measured by absorbance at 450 nm with an ELISA reader The Breiddoma cell lines were repeatedly selected. Cloning of hybridoma cell lines producing a monoclonal antibody by limiting dilution of the hybridoma cell line obtained through repeated selection was finally obtained. The prepared hybridoma was deposited on May 13, 2013 with the deposit number KCLRF-BP-00295 in the Korean Cell Line Bank located in Yeongeon-dong, Jongno-gu, Seoul, Korea.

Each of the obtained hybridomas was cultured in DMEM (Dulbecco's Modified Eagle's Medium), and the culture solution was collected and the anti-Ang2 monoclonal antibody produced in each hybridoma was purified by protein G-affinity chromatography.

First, the hybridoma cells cultured in 50 ml of a culture medium (DMEM) medium containing 10% (v / v) FBS were centrifuged, and the cell precipitate was washed twice with 20 ml of PBS to remove FBS 50 ml of the culture medium (DMEM) medium was resuspended in the cell suspension and cultured in a 37 ° C carbon dioxide incubator for 3 days. Then, the cells producing the antibody were removed by centrifugation, and the culture medium in which the antibodies were secreted was separated and stored at 4 ° C or directly collected from the culture solution of 50 ml to 300 ml of the affinity column (Protein G agarose column, Pharmacia, USA) The purified antibody was purified by using the AKTA purification device (GE Healthcare), and the purified antibody was stored in PBS for protein aggregation filter (Amicon) to be used in the subsequent experiments. One of the antibodies obtained from each of the above hybridomas was designated 10D6.

The binding affinity of the antibody to human Ang-2 protein was measured by SPR method using BIAcore T100 (GE Healthcare). The SPR method is based on the principle that the refractive index of light passing through the chip changes according to the state of a substance coated on the sensor chip. When an antibody or an antigen is spilled on a chip with an antigen or an antibody coated thereon, And a Kd value is calculated from the measured value.

First, the anti-His antibody was immobilized on a CM5 sensor chip (GE Healthcare) to a level of 8,000 RU using a pH 5.0 acetate solution and an amine coupling kit (GE Healthcare). Here, a recombinant hAng-2 (C-His, R & D Systems) protein at a concentration of 6 ug / ml was flown and captured at 100-200 RU level. The antibody obtained in Example 2 was diluted twice from the concentration of 100 nM sequentially and then allowed to bind, on, off and dissolve 10 mM NaOH solution) and the antigen-antibody affinity was measured. hAng1 were tested in the same manner as described above, and the results are shown in Table 3 below.

Name of antibody hAng2 (Kd) SAIT-ANG2-AB-m10D6 8.0 nM

2.2. term Ang2  The gene of the antibody Cloning

Total RNA was obtained from the antibody-producing hybridoma (2x10 ⁶ cells) obtained in Example 2.1 using the RNeasy mini kit (Qiagen). Then, using the OneStep RT-PCR kit (Qiagen), Mouse Ig-Primer Set (Novagen) and Thermocycler (GeneAmp PCR System 9700, Applied Biosystem) as a template, Only the heavy and light chain variable region gene sequences of monoclonal antibodies produced in chopping boards were amplified: 94 ° C for 5 minutes; [30 min at 50 < 0 > C, 15 min at 95 [deg.] C), 35 cycles of 1 min at 94 [deg.] C, 1 min at 50 [ 72 ° C for 6 minutes; Cool to 4 ° C.

The PCR products obtained from each reaction were directly subjected to DNA sequencing to obtain CDRs, heavy chain variable regions and light chain variable regions of each antibody and nucleotide sequences encoding them. The obtained results are shown in Tables 4 7:

Name of antibody Heavy chain CDR sequence CDRH1-KABAT CDRH2-KABAT CDRH3-KABAT SAIT-ANG2-AB-m10D6 SDYAWN
(SEQ ID NO: 1) YINYSGNTDYNPSLKS
(SEQ ID NO: 2) GNFEGAMDY
(SEQ ID NO: 3)

Name of antibody Light chain CDR amino acid sequence CDRL1-KABAT CDRL2-KABAT CDRL3-KABAT SAIT-ANG2-AB-m10D6 KASQSVSNDVA
(SEQ ID NO: 4) YASNRYP
(SEQ ID NO: 5) QQDYSSPWT
(SEQ ID NO: 6)

Name of antibody Heavy chain variable region sequence SAIT-ANG2-AB-m10D6 VQLQESGPGLVKPSQSLSLTCTVTGYSIT SDYAWN WIRQFPGNKLEWMG YINYSGNTDYNPSLKS RSSITRDTSKNQFFLQLNSVTTGDTATYYCAR GNFEGAMDY WG (SEQ ID NO: 7) GATGTGCAGCTTCAGGAGTCGGGACCTGACCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTATGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGGCTACATAAACTACAGTGGTAACACTGACTACAACCCATCTCTCAAAAGTCGAAGCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGGGGACACAGCCACATATTACTGTGCAAGAGGTAACTTCGAAGGTGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 8)

Name of antibody Light chain variable region sequence SAIT-ANG2-AB-m10D6 SIVMTQTPKFLLVSAGDRVTITC KASQSVSNDVA WYQQKPGQSPKLLIY YASNRYP GVPDRFTGSGYGTDFTFTISTVQAEDLAVYFC QQDYSSPWT FGGGTKLEIK (SEQ ID NO: 9) agtattgtgatgacccagactcccaaattcctgcttgtatcagcaggagacagggttaccataacctgcaaggccagtcagagtgtgagtaatgatgtagcttggtaccaacagaagccagggcagtctcctaaactgctgatatactatgcatccaatcgctaccctggagtccctgatcgcttcactggcagtggatatgggacggatttcactttcaccatcagcactgtgcaggctgaagacctggcagtttatttctgtcagcaggattatagctctccgtggacgttcggtggaggcaccaagctggaaatcaaa (SEQ ID NO: 10)

(In Tables 6 and 7, the underlined bold letters are CDR1, CDR2, CDR3 in order)

Example  3. Ang2 - Tie2  10 for binding D6  Antibody competition ELISA assay

The Ang2-Tie2 binding competition ELISA was performed using the antibody binding to Ang-2 prepared in Example 2-1.

More specifically, a 96-well MaxiSorp ™ flat-bottom plate (Nunc) was coated with hTie2-Fc (R & D Systems), a protein bound with 4 μg / ml human IgG1 Fc. Then, the plate was washed five times with PBS (Phosphate Buffer Saline) containing 0.05% (v / v) Tween-20, and then washed with PBS containing 1% (v / v) BSA (Bovine serum albumin; Sigma) At room temperature for 2 hours.

For the Ang2-Tie2 competition ELISA, each anti-Ang2 antibody prepared in Example 2 was incubated with various concentrations of 1% (v / v) BSA and 400 ng / ml FLAG-tagged hAng- 2 was added to each well coated with the hTie-2 / Fc fusion protein. The plate was allowed to react at room temperature for 2 hours, and then the plate was washed five times with PBST. HRP-conjugated anti-FLAG antibody (Sigma) was diluted with PBS containing 1% (v / v) BSA at a ratio of 1: 5,000 (v / v) and added to each well in an amount of 100 μl. For 1 hour, and washed 5 times with PBST. Finally, 100 ul (microliter) of TMB substrate was added to each well of the plate to induce a color reaction for 3 minutes. After that, 100 ul of stop signal (Cell Signaling) was added to stop the reaction, OD450 values were measured on a plate reader (Molecular Devices).

For comparison, the same test as above was performed using 4H10, an anti-Ang2 antibody that inhibits Ang2-Tie2 binding. The 4H10 is an antibody having a heavy chain variable region and a light chain variable region as follows:

Heavy chain variable region (SEQ ID NO: 12):

EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYDMS WVRQAPGKGLEWVS LISPDSSSIYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYC AKDLISFWRGGFDY WGQGTLVTVSS

The light chain variable region (SEQ ID NO: 13)

QSVLTQPPSASGTPGQRVTISC SGSSSNIGSNYVN WYQQLPGTAPKLLIY ADSNRPS GVPDRFSGSKSGTSASLAISGLRSEDEADYYC GSWDYSLSG YVFGGGTKLTVLG

The degree (%) of inhibition of Ang-Tie2 binding is shown in Fig. As shown in Fig. 1, unlike 4H10, which is an anti-Ang2 antibody that inhibits Ang2-Tie2 binding, it was confirmed that 10D6 antibody does not inhibit Ang2-Tie2 receptor binding.

Example  4. Ang2  Antibody Antigen recognition site  ( epitope ) Confirm

In order to confirm the epitope (or specific binding site) of the anti-Ang2 antibody prepared in Example 2, the receptor binding domain (RBD) of the Ang2 protein tagged with Flag was transformed into an artificially mutated recombinant protein Were used for ELISA.

Each well of a 96-well MaxiSorp flat-bottom plate (Nunc) was coated with 50 ul of microliter of 1000 nM antibody. Then, the plate was washed 5 times with PBS (PBST) containing 0.05% (v / v) Tween-20, and then blocked with PBS containing 1% (v / v) BSA at room temperature for 2 hours. Mutation of the S417, Q418, P419, N421, I434, D448, A449, P452, Y460, N467, K468, or F469 residues of the Ang2 protein tagged with the N-terminal of the FLAG sequence (DYKDDDDK, Sigma) Each mutant protein (Mutant Ang2) was added to each well of the plate in an amount of 250 ng, and the plate was allowed to react at room temperature for 2 hours.

After washing 5 times with PBS containing 0.05% (v / v) Tween-20, HRP-conjugated anti-FLAG antibody (SIGMA) was diluted with PBS containing 1% (v / v) 5,000 (v / v), reacted at room temperature for 1 hour, and washed 5 times with PBS containing 0.1% (v / v) Tween-20.

Finally, 50 ul of TMB substrate was added to each well of the plate to induce a color development reaction at room temperature for 3 minutes. After that, 50 ul of Stop signal (Cell signaling) was added to stop the reaction , OD450 values were measured on a plate reader (Molecular Devices). The epitope for Ang2 antibodies was confirmed by comparing the binding of Ang2 with mutation to that of unmutated Ang2. The results of the measurement of the binding force (%) with Mutant Ang2 for the binding force with the obtained native Ang2 are shown in Table 8 below:

Loop 1 Loop 2 Loop 3 Loop4 417 418 419 421 434 448 449 452 460 467 468 469 10D6 101.17 38.94 41.08 109.09 109.49 104.55 97.86 102.15 103.15 106.84 110.27 108.79

Example  5. 10 D6  By antibody Tie2  Induction test of receptor phosphorylation

Since Ang2 binds to Tie2 receptor expressed in vascular endothelial cells and induces phosphorylation of receptor and induces the change of vascular endothelial cell, the effect of Ang2 on Tie2 phosphorylation of anti Ang2 antibody is analyzed using cell-based assay Experiments were performed.

For this, HUVEC (ATCC) cells (1 × 10 ⁶ cells) were cultured in EGM-2 (Lonza) medium at 37 ° C. in a 100 mm culture dish and changed to serum-free medium (Lonza) Lt; RTI ID = 0.0 > 37 C < / RTI > After washing once with PBS, the cells were changed to serum-free medium (Lonza) mixed with 1 nM sodium orthovanadate (Sigma) and further incubated for 10 minutes. After washing once with PBS, Ang2 antibody (10D6) at various concentrations (600 to 0.06 nM) was mixed with 40 nM of Ang2 protein (R & D systems) for 20 minutes, treated with the cultured cells and further incubated for 10 minutes . The cells were washed with PBS and treated with 400ul of lysis buffer (Roche). The cells were collected in a tube, and dissolved at 4 ° C for 30 minutes. The cells were centrifuged at 13,000 rpm for 15 minutes, .

 0.8 mg of Tie2 antibody (R & D system) was added to the cell lysate and reacted at 4 ° C overnight. Then, protein A bead (GE Healthcare) was added to immunoprecipitate. The obtained reaction product was centrifuged at 13,000 rpm for 15 minutes to obtain pellets. The pellet was washed with lysis buffer (Roche) 2 to 3 times, and a sample buffer (Invitrogen) mixed with a reducing agent was added. After boiling at 95 ° C for 5 minutes, NuPAGE Novex 4-12% Bis-Tris gel (Invitrogen) running and transferred to nitrocellulose membrane (Invitrogen).

The membranes were blocked with PBST mixed with 3% (v / v) skim milk (Sigma) for 30 min and confirmed with HRP-conjugated anti-phospho tyrosine antibody (Millipore) Respectively. For confirmation of Tie2, the blot was reacted in a stripping buffer (Thermo) for 15 minutes, then blocked again and confirmed with Tie2 antibody (Santa Cruz).

The results obtained are shown in Fig. As shown in FIG. 2, when 10D6 antibody was added together with Ang2 compared to Ang2 alone treatment group, it was confirmed that Tie2 phosphorylation was more strongly induced in all antibody concentration ranges tested.

Example  6: 10 D6  By antibody Tie2 signaling Activation induction test

In order to confirm whether the 10D6 antibody induces the activation of downstream signaling of the Tie2 receptor as well as the Tie2 receptor, the degree of phosphorylation of the proteins involved in downstream signaling upon treatment of Ang2 alone or with Ang2 and 10D6 antibodies in HUVEC (ATCC) . In order to compare the degree of downstream signaling, a group treated with Ang1 (R & D systems) and Ang2 (R & D systems) and Regeneron's anti Ang2 antibody (RG antibody; control antibody with MOA inhibiting Ang2-Tie2 binding) The same test was carried out.

Specifically, HUVEC (ATCC) cells (1 × 10 ⁵ cells) were cultured in EGM-2 (Lonza) medium at 37 ° C. in a 6-well culture dish and then cultured in serum-free medium (Lonza) And incubated at 37 [deg.] C for 6 to 16 hours. After washing once with PBS, 60 nM of Ang2 antibody (10D6) was mixed with 40 nM of Ang2 protein (R & D systems) for 20 minutes, treated with the cultured cells, and further cultured for 30 minutes. For comparison, groups were prepared in which 4 nM of Ang1 (R & D systems), 40 nM of Ang2 (R & D systems), and 40 nM of Ang2 (R & D systems) and 60 nM of anti-Ang2 antibody (Regeneron), respectively.

The cells were washed with PBS and treated with lysis buffer (Roche). The cells were collected in a tube, dissolved at 4 ° C for 30 minutes, and then centrifuged at 13,000 rpm for 15 minutes to quantify the supernatant. (Invitrogen) mixed with 25 μg of a reducing agent and boiled for 5 minutes at 95 ° C. The mixture was then run on NuPAGE Novex 4-12% Bis-Tris gel (Invitrogen) and transferred to nitrocellulose membrane .

To confirm the phosphorylation of Akt, eNOS, 42/44 involved in the downstream signaling of the Tie2 receptor, the above blot was blocked with PBST mixed with 3% (v / v) skim milk (Sigma) for 30 minutes, Anti-pAkt antibody, anti-p-eNOS antibody and anti-p-42/44 antibody (abnormal cell signaling). Blot was reacted in a stripping buffer (Thermo) for 15 minutes and then blocked again to detect Akt, eNOS, and 42/44 using anti-Akt antibody, anti-eNOS antibody, and 42/44 antibody (abnormal cell signaling).

The obtained results are shown in Fig. As shown in FIG. 3, downstream signaling was strongly induced in Ang1 alone treatment group and Ang2 alone treatment group and Ang2 and 10D6 antibody treatment group, and Ang2 and 10D6 antibody In the group treated with Angl alone was equal to or more than that in the group treated with Angl alone.

Example  7: 10 D6 - Ang2 - Tie2 complex  For confirmation of formation ELISA assay

Since the 10D6 anti-Ang2 antibody did not inhibit Ang2-Tie2 binding and activated Tie2 signaling, ELISA was performed to determine whether the complex formed between the antibody and the Ang2 Tie2 receptor.

Were coated with 4 ug / ml Tie2-Fc (R & D systems) or BSA (Sigma) in a 96-well MaxiSorp flat-bottom plate (Nunc). Then, the plate was washed five times with PBS (Phosphate Buffer Saline) containing 0.05% (v / v) Tween-20, and then washed with PBS containing 1% (v / v) BSA (Bovine serum albumin; Sigma) At room temperature for 2 hours. 0.25 ug / ml of Ang2 and 2 ug / ml of 10D6 antibody were added to each well. The plate was reacted at room temperature for 2 hours, and the plate was washed five times with PBST. HRP conjugated anti-mouse IgG antibody (Sigma) was diluted 1: 5,000 (v / v) with PBS containing 1% (v / v) BSA and added to each well in an amount of 100 μl After reacting at room temperature for 1 hour, it was washed 5 times with PBST. Finally, 100 ul (microliter) of TMB substrate was added to each well of the plate to induce a color reaction for 3 minutes. After that, 100 ul of stop signal (Cell Signaling) was added to stop the reaction, OD450 values were measured on a plate reader (Molecular Devices).

The results obtained are shown in Fig. As shown in FIG. 4, it was confirmed that 10D6 antibody binds to Ang2 bound to Tie2 to form a complex.

Example 8 . 10 D6  Antibody dimerization On by Tie2  Receptor clustering Induce activation through

To confirm the activation of Tie2 receptor by dimerization of 10D6 antibody, the Fab fragment of 10D6 antibody was purified and used for the experiment. Fab fragments were obtained from 10D6 antibody using Fab Preparation Kit (Pierce). PBS containing 20 mM EDTA and 20 mM L-cysteine was used as a digestion buffer. Immobilized papain was treated at room temperature for 2 hours to separate into Fab and Fc. The Fc fragment was removed from the reaction solution using a MabSelectSuRe column (GE Healthcare), and pure Fab fragments were separated and purified.

In a 100 mm culture dish, HUVEC (ATCC) cells (1 × 10 ⁶ ) were cultured at 37 ° C. using EGM-2 (Lonza) medium and then showed 80-90% confluency. (Lonza) and incubated at 37 [deg.] C for 6 to 16 hours. After washing once with PBS, the cells were changed to serum-free medium (Lonza) mixed with 1 nM sodium orthovanadate (Sigma) and further incubated for 10 minutes. After washing once with PBS, 60 nM of 10D6 Ang2 antibody or 10D6 Fab was mixed with 40 nM Ang2 protein (R & D systems) for 20 minutes, treated with the cultured cells, and further cultured for 10 minutes. The cells were washed with PBS and treated with 400ul of lysis buffer (Roche). The cells were collected in a tube, and dissolved at 4 ° C for 30 minutes. The cells were centrifuged at 13,000 rpm for 15 minutes, .

 0.8 mg of Tie2 antibody (R & D system) was added to the cell lysate and reacted at 4 ° C overnight. Then, protein A bead (GE Healthcare) was added to immunoprecipitate. The obtained reaction product was centrifuged at 13,000 rpm for 15 minutes to obtain pellets. The pellet was washed with lysis buffer (Roche) 2 to 3 times, and a sample buffer (Invitrogen) mixed with a reducing agent was added. After boiling at 95 ° C for 5 minutes, NuPAGE Novex 4-12% Bis-Tris gel (Invitrogen) running and transferred to nitrocellulose membrane (Invitrogen).

The membranes were blocked with PBST mixed with 3% (v / v) skim milk (Sigma) for 30 min and confirmed with HRP-conjugated anti-phospho tyrosine antibody (Millipore) Respectively. For confirmation of Tie2, the blot was reacted in a stripping buffer (Thermo) for 15 minutes, then blocked again and confirmed with Tie2 antibody (Santa Cruz).

To confirm the phosphorylation of Akt, eNOS, 42/44 involved in the downstream signaling of the Tie2 receptor, the above blot was blocked with PBST mixed with 3% (v / v) skim milk (Sigma) for 30 minutes, Anti-pAkt antibody, anti-p-eNOS antibody and anti-p-42/44 antibody (abnormal cell signaling). Blot was reacted in a stripping buffer (Thermo) for 15 minutes and then blocked again to detect Akt and 42/44 using anti-Akt antibody and anti-42/44 antibody (abnormal cell signaling).

The results obtained are shown in Fig. As shown in FIG. 5, activation of Tie2 and Akt, 42/44 was observed when 10D6 antibody was added together with Ang2, but activation was not observed when 10D6 Fab was treated. As a result, dimerization of 10D6 antibody resulted in Tie2 The receptor was clustered to confirm that the phosphorylation occurred strongly.

Example 9 .10 D6  By antibody Tie2  Receptor internalization  Confirm

HUVEC (ATCC) cells (1 × 10 ⁶ cells) were grown in 8-wells (# 80826, ibidi) on m-slide for 24 hours and serum starvation was performed in serum-free EBM-2 media (Lonza) for 3 hours. The cells were incubated at the time indicated in the figure after 200ng / ml of Ang1 (R & D systems) or 2ug / ml of Ang2 (R & D systems) and 10ug / ml of 10D6 antibody diluted in serum-free EBM-2 media. Cells fixed with 4% formaldehyde (Sigma) were first stained with anti-Tie2 antibody (R & D systems) and then stained with anti-goat Alexa 555 antibody (red, Life technology) and anti- mouse Alexa 488 antibody ). ≪ / RTI > The stained cells were treated with Vectashield mounting medium with DAPI (Vector labs) and observed with confocal laser scanning microscopy (Carl Zeiss).

The results are shown in Figs. 6A and 6B. As shown in FIG. 6 (a), the Ang2 and 10D6 antibodies were treated similarly to Tie2 endocytosis in Ang1 treatment, indicating that internalization of the Tie2 receptor started from 30 minutes (dots in the enlarged picture). And that the internalized Tie2 and 10D6 antibodies were located in the early endosome, which is the cell organelle, by the anti-EEA1 antibody (Cell Signaling) staining of FIG. 6b.

Example 10 . 10 D6  Identification of cell growth and mobility increase by antibody

The proliferation of blood vessels and lymphatic endothelial cells was assayed using the BrdU assay kit (Roche) Cell. P3 to P8 vascular endothelial cells (HUVEC, ATCC) or lymphatic endothelial cells (Lonza) were placed in a collagen coated 96 well plate (BD Bioscience) at 3000-5000 cells / well and then diluted with EGM-2 medium Time. 2 μg / ml Ang2 (R & D systems) and 10 μg / ml 10D6 antibody were mixed in 2% EBM medium, and the plate was placed in a 96-well plate washed with PBS and cultured for 3 days.

For comparison of efficacy, anti-Ang2 antibody from MedImmune Co. was used. For the BrdU assay, 10 μl of 1: 1,000 diluted BrdU solution was added to each well to label the cells. After incubation for 1 hour, the culture solution was removed, and the Fixation / Denaturation solution (Roche) was added to each well in an amount of 100 μl. The anti-BrdU antibody (Roche) conjugated with peroxidase was diluted to 1: 100 with dilution buffer (Roche), incubated for 1 hour in each well, and washed 4 times with washing buffer (Roche) . After adding 100 ul of TMB substrate, the absorbance was measured at 450 nm using a microplate reader (Perkin Elmer).

The results obtained are shown in Fig. As shown in FIG. 7, in both the endothelial cells of the vascular endothelial cells and the endothelial cells of the lymphatic vessels, the growth of the cells was observed in the group containing 10D6 antibody to Ang2, and in the case of MEDI antibody, the growth of Ang2 was decreased Respectively.

In addition, the mobility of lymphatic endothelial cells was measured using a xCelligence Real Time Cell Analyzer (RTCA) from GE Healthcare. RTCA is a non-invasive cell monitoring system that can detect cell changes by measuring impedance in real time. In order to perform cell migration assay, CIM-plate16 (GE Healthcare) composed of lower chamber and upper chamber is used, and microelectrodes for measuring impedance are arranged in the upper chamber. When cells seeded in chamber are moved through fine holes, It is attached to the electrode to confirm the degree of migration of the cell, which is indicated by the migration index. Lymphocyte endothelial cells (P5-7; Lonza) grown in EGM-2 medium (Lonza) were cultured in EBM medium supplemented with 1% FBS for 6 hours. 2 ug / ml Ang2 and 10D6 antibody were added to EBM medium supplemented with 2% FBS in each lower chamber of CIM-plate16 and then assembled with upper chamber coated with fibronectin (Sigma). For comparison of efficacy, anti-Ang2 antibody from MedImmune Co. was used. 30 μl of serum-free EBM medium was added to the upper chamber, and the CIM-plate was placed in the incubator for 1 hour to equilibrate between the plate and the culture medium and the background value was measured. Lymphocyte endothelial cells resuspensioned with serum-free media were seeded at 60,000 cells / well, allowed to settle down for 15 minutes, and mounted on a device to measure cell migration in real time. The degree of cell migration was expressed as Slope (1 / hr).

The results obtained are shown in Fig. As shown in FIG. 8, it was confirmed that migration of lymphocyte endothelial cells containing 10D6 antibody together with Ang2 was increased, but migration of Ang2 by the MEDI antibody was inhibited.

Example 11 . 10 D6  Antibody-mediated inhibition of cell permeability

In vitro vascular permeability assay kit (Millipore) was used to confirm the cytotoxicity of 10D6 antibody. Confluent monolayer was prepared by incubating HUVEC (ATCC) cells in collagen coated transwells at 5 × 10 ⁴ for 2 days. After treatment with 200 ng / ml of Ang2 or 200 ng / ml of Ang1 (R & D systems) alone or 200 ng / ml of Ang2 and 1 ug / ml of 10D6 antibody and preincubation for 30 min, LPS was added to induce permeability of the HUVEC monolayer ; Sigma) 100 ng / ml or TNF-a (R & D systems) 100 ng / ml. LPS or TNF-a weakens the binding between HUVEC cells to form a gap, thereby increasing the transport of material between the HUVEC monolayer. The LPS treatment group was treated with incuabtion for 6 hours and the TNF-a treatment group was treated with FITC-dextran (Millipore) in the upper chamber after incubation for 22 hours and incubated for 18 minutes. The fluorescence of FITC-Dextran in the lower chamber was measured at 485/535 nm (Ex / Em) setting using an Envision 2104 multilabel reader (Perkinelmer).

The results obtained are shown in Fig. FIG. 9 is a graph showing the intensity of the fluorescence measured in relative folds, which means vascular permeability induced by LPS or TNF-a. These results indicate that the increased vascular permeability by LPS or TNF-a is reduced to the contorl level in the Angl alone treatment group and the Ang2 and 10D6 antibody treatment groups.

Example 12 . 10 D6  Anti-inflammatory effects of antibodies

To examine the anti-inflammatory effect of 10D6 antibody, Ang1 alone or Ang2 and 10D6 antibodies were treated with 4X10 ⁵ of HUVEC (ATCC) cells and Ang1 and Ang2 (R & D systems) and Regeneron antibodies (RG antibody) Group treated together. HUVEC cells were treated with 0.2 ug / ml of Ang1, 0.1 ug / ml of Ang2 or 0.1 ug / ml of Ang2 and 0.5 g / ml of antibody, respectively. After preincubation for 30 min, LPS (Sigma) 100 ng / ml, and incubated for 5 hours.

The extent of inflammation was determined by measuring the mRNA expression of inflammatory adhesions (ICAM-1, E-selectin). The cells were washed with PBS, RNA was extracted using RNeasy Mini kit (Qiagen), and 2g RNA was synthesized by using Transcriptor First Strand cDNA synthesis kit (Roche). The pPCR reaction was performed using RT2 SYBR ^™ Green Mastermix (Qiagen) and LightCycler ^™ 480 Real-Time PCR System (Roche). HPRT1 is used as an internal control to correct the amount of RNA in the sample, and qPCR follows the procedure for all primers. Step 1: 95 ° C, 10 min; Step 2 (45 cycles): Step 2-1: 95 ° C, 15 sec; Step 2-2: 60 ° C, 1 min; Step 3: 65 ° C, 15 sec; Step 4: 95 ° C, continuous (every 20 ° C); Step 6: 40 ° C, 10 sec.

The primer sequences used in qPCR are summarized in Table 9 below:

The measured amounts of relative expression of ICAM-1 and E-selectin are shown in FIG. As shown in FIG. 10, it can be confirmed that the expression of ICAM-1 and E-selectin induced by LPS is significantly inhibited in Angl alone treatment group and Ang2 and 10D6 antibody treatment groups.

On the other hand, when the expression of inflammatory adherent substances (ICAM-1, E-selectin, etc.) of vascular endothelial cells is increased, inflammation is induced by promoting binding between inflammatory cells and vascular endothelial cells. In order to confirm this, experiments were carried out to measure the binding between HL-60 (ATCC) cells and HUVEC, an inflammatory cell.

HUVEC cells (4 × 10 ⁵ ) were cultured in a 24-multiwell plate and treated with 0.1 ug / ml of Ang2 alone or 0.1 ug / ml of Ang2 and 0.5 ug / ml of 10D6 antibody. As a control, Ang1 0.2 ug / ml and Ang2 0.1 ug / / ml and 0.5 ug / ml of Regeneron's antibody (RG antibody). As described above, HUVEC cells were treated with Angiopoietin and the antibody was preincubated for 30 min. After incubation for 2 hr 30 min, 1 ng / ml of TNF-a (eBioscience) was treated to induce the inflammatory reaction.

After HUVEC cells were washed with complete media (Lonza), HL-60 cells stained with Calcein-AM (BD bioscience) solution were added at 2.5 × 10 ⁵ per well and incubated for 1 hour. To remove HL-60 cells not bound to HUVEC cells, the cells were washed three times with PBS and then treated with lysis buffer (0.1% SDS, 50 mM Tris.HCl, pH 8.0). The lysate was transferred to a 96-well plate and the intensity of fluorescence from HL-60 cells was measured using an Envision 2104 multilabel reader (Perkin Elmer) at 485/535 nm (Ex / Em) setting.

The results obtained are shown in Fig. The graph of FIG. 11 shows the intensity of the fluorescence measured in relative folds, which is proportional to the number of HL-60 cells attached to HUVEC cells. These results indicate that the binding of HUVEC cells and HL-60 cells induced by TNF-a is significantly inhibited in Angl alone treatment group and Ang2 and 10D6 antibody treatment groups.

Example  Section 13 Ang -2 antibody Colo205  Tumor growth inhibitory effect

A colon cancer Xenograft model using Colo205 (ATCC), a human colon cancer cell line, was used to confirm the tumor growth inhibitory effect of Ang-2 antibody.

Colo205 cells were cultured in RPMI-1640 (Gibco) supplemented with 10% FBS (Gibco). ⁵ × 10 ⁵ Colo205 cell lines were resuspended in 100 μl of serum-free medium, and subcutaneously injected into 4-5 week old BALB / c nude mice (Shanghai SLAC Laboratory Animal Co. Ltd.) anesthetized with 1-2% isoflurane Respectively. When tumor size reached 100 to 200 mm ³ , the anti Ang-2 antibody (10D6) was intraperitoneally injected at a dose of 10 mg / kg twice a week, and the size of the tumor was measured.

The size of the tumor (V) was calculated using the following equation:

V = (length x width ² ) / 2

The obtained results are shown in Fig. The x-axis in FIG. 12 represents the day on which the antibody treatment was performed by Days after grouping. As shown in Fig. 12, it was confirmed that the antibody 10D6 inhibited the growth of colon cancer.

Korea Cell Line Research Foundation KCLRFBP00029 20130513

<110> SAMSUNG ELECTRONICS CO., LTD. <120> Anti-Ang2 antibody inducing binding to Tie2 receptor <130> DPP20132768KR <160> 13 <170> Kopatentin 1.71 <210> 1 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR-H1 of anti-Ang2 antibody <400> 1 Ser Asp Tyr Ala Trp Asn   1 5 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR-H2 of anti-Ang2 antibody <400> 2 Tyr Ile Asn Tyr Ser Gly Asn Thr Asp Tyr Asn Pro Ser Leu Lys Ser   1 5 10 15 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR-H3 of anti-Ang2 antibody <400> 3 Gly Asn Phe Glu Gly Ala Met Asp Tyr   1 5 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR-L1 of anti-Ang2 antibody <400> 4 Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala   1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR-L2 of anti-Ang2 antibody <400> 5 Tyr Ala Ser Asn Arg Tyr Pro   1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR-L3 of anti-Ang2 antibody <400> 6 Gln Gln Asp Tyr Ser Ser Pro Trp Thr   1 5 <210> 7 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region of an anti-Ang2 antibody <400> 7 Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Ser   1 5 10 15 Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp Tyr              20 25 30 Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met          35 40 45 Gly Tyr Ile Asn Tyr Ser Gly Asn Thr Asp Tyr Asn Pro Ser Leu Lys      50 55 60 Ser Arg Ser Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu  65 70 75 80 Gln Leu Asn Ser Val Thr Thr Gly Asp Thr Ala Thr Tyr Tyr Cys Ala                  85 90 95 Arg Gly Asn Phe Glu Gly Ala Met Asp Tyr Trp Gly             100 105 <210> 8 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> Coding DNA of heavy chain variable region of anti-Ang2 antibody <400> 8 gatgtgcagc ttcaggagtc gggacctgac ctggtgaaac cttctcagtc tctgtccctc 60 acctgcactg tcactggcta ctcaatcacc agtgattatg cctggaactg gatccggcag 120 tttccaggaa acaaactgga gtggatgggc tacataaact acagtggtaa cactgactac 180 aacccatctc tcaaaagtcg aagctctatc actcgagaca catccaagaa ccagttcttc 240 ctgcagttga attctgtgac tactggggac acagccacat attactgtgc aagaggtaac 300 ttcgaaggtg ctatggacta ctggggtcaa ggaacctcag tcaccgtctc ctca 354 <210> 9 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region of anti-Ang2 antibody <400> 9 Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp              20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile          35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Pro Gly Val Pro Asp Arg Phe Thr Gly      50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala  65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Trp                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 10 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Coding DNA of light chain variable region of anti-Ang2 antibody <400> 10 agtattgtga tgacccagac tcccaaattc ctgcttgtat cagcaggaga cagggttacc 60 ataacctgca aggccagtca gagtgtgagt aatgatgtag cttggtacca acagaagcca 120 gggcagtctc ctaaactgct gatatactat gcatccaatc gctaccctgg agtccctgat 180 cgcttcactg gcagtggata tgggacggat ttcactttca ccatcagcac tgtgcaggct 240 gaagacctgg cagtttattt ctgtcagcag gattatagct ctccgtggac gttcggtgga 300 ggcaccaagc tggaaatcaa a 321 <210> 11 <211> 496 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of Ang2 <400> 11 Met Trp Gln Ile Val Phe Phe Thr Leu Ser Cys Asp Leu Val Leu Ala   1 5 10 15 Ala Ala Tyr Asn Asn Phe Arg Lys Ser Met Asp Ser Ile Gly Lys Lys              20 25 30 Gln Tyr Gln Val Gln His Gly Ser Cys Ser Tyr Thr Phe Leu Leu Pro          35 40 45 Glu Met Asp Asn Cys Arg Ser Ser Ser Ser Pro Tyr Val Ser Asn Ala      50 55 60 Val Gln Arg Asp Ala Pro Leu Glu Tyr Asp Asp Ser Val Gln Arg Leu  65 70 75 80 Gln Val Leu Glu Asn Ile Met Glu Asn Asn Thr Gln Trp Leu Met Lys                  85 90 95 Leu Glu Asn Tyr Ile Gln Asp Asn Met Lys Lys Glu Met Val Glu Ile             100 105 110 Gln Gln Asn Ala Val Gln Asn Gln Thr Ala Val Met Ile Glu Ile Gly         115 120 125 Thr Asn Leu Leu Asn Gln Thr Ala Glu Gln Thr Arg Lys Leu Thr Asp     130 135 140 Val Glu Ala Gln Val Leu Asn Gln Thr Thr Arg Leu Glu Leu Gln Leu 145 150 155 160 Leu Glu His Ser Leu Ser Thr Asn Lys Leu Glu Lys Gln Ile Leu Asp                 165 170 175 Gln Thr Ser Glu Ile Asn Lys Leu Gln Asp Lys Asn Ser Phe Leu Glu             180 185 190 Lys Lys Val Leu Ala Met Glu Asp Lys His Ile Ile Gln Leu Gln Ser         195 200 205 Ile Lys Glu Glu Lys Asp Gln Leu Gln Val Leu Val Ser Lys Gln Asn     210 215 220 Ser Ile Ile Glu Glu Leu Glu Lys Lys Ile Val Thr Ala Thr Val Asn 225 230 235 240 Asn Ser Val Leu Gln Lys Gln Gln His Asp Leu Met Glu Thr Val Asn                 245 250 255 Asn Leu Leu Thr Met Met Ser Thr Ser Asn Ser Ala Lys Asp Pro Thr             260 265 270 Val Ala Lys Glu Glu Gln Ile Ser Phe Arg Asp Cys Ala Glu Val Phe         275 280 285 Lys Ser Gly His Thr Asn Gly Ile Tyr Thr Leu Thr Phe Pro Asn     290 295 300 Ser Thr Glu Glu Ile Lys Ala Tyr Cys Asp Met Glu Ala Gly Gly Gly 305 310 315 320 Gly Trp Thr Ile Ile Gln Arg Arg Glu Asp Gly Ser Val Asp Phe Gln                 325 330 335 Arg Thr Trp Lys Glu Tyr Lys Val Gly Phe Gly Asn Pro Ser Gly Glu             340 345 350 Tyr Trp Leu Gly Asn Glu Phe Val Ser Gln Leu Thr Asn Gln Gln Arg         355 360 365 Tyr Val Leu Lys Ile His Leu Lys Asp Trp Glu Gly Asn Glu Ala Tyr     370 375 380 Ser Leu Tyr Glu His Phe Tyr Leu Ser Ser Glu Glu Leu Asn Tyr Arg 385 390 395 400 Ile His Leu Lys Gly Leu Thr Gly Thr Ala Gly Lys Ile Ser Ser Ile                 405 410 415 Ser Gln Pro Gly Asn Asp Phe Ser Thr Lys Asp Gly Asp Asn Asp Lys             420 425 430 Cys Ile Cys Lys Cys Ser Gln Met Leu Thr Gly Gly Trp Trp Phe Asp         435 440 445 Ala Cys Gly Pro Ser Asn Leu Asn Gly Met Tyr Tyr Pro Gln Arg Gln     450 455 460 Asn Thr Asn Lys Phe Asn Gly Ile Lys Trp Tyr Tyr Trp Lys Gly Ser 465 470 475 480 Gly Tyr Ser Leu Lys Ala Thr Thr Met Met Ile Arg Pro Ala Asp Phe                 485 490 495 <210> 12 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> heavy chain variable region of 4H10 <400> 12 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr              20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Leu Ile Ser Pro Asp Ser Ser Ser Ile 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 Lys Asp Leu Ile Ser Phe Trp Arg Gly Gly Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 13 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region of 4H10 <400> 13 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln   1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn              20 25 30 Tyr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Ala Asp Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg  65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Tyr Ser Leu                  85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly             100 105 110

Claims (31)

Angiopoietin-2 (Ang2) and binds to the Tie2 receptor together with Ang2, or an antigen-binding fragment thereof. The method according to claim 1,
Lt; RTI ID = 0.0 &gt; Tie2 &lt; / RTI &gt; receptor,
An anti-Ang-2 antibody or antigen-binding fragment thereof. The method according to claim 1,
Increases activity by increasing phosphorylation of the Tie2 receptor. &Lt; RTI ID = 0.0 &gt;
An anti-Ang-2 antibody or antigen-binding fragment thereof. The method according to claim 1,
&Lt; / RTI &gt; Akt, eNOS, and 42/44.
An anti-Ang-2 antibody or antigen-binding fragment thereof. The method according to claim 1,
A combination of Q418, P419, Q418 and P419 of human Ang2 (SEQ ID NO: 11), or a combination of Q418, P419, or Q418 and P419 of SEQ ID NO: 11 ,
An anti-Ang-2 antibody or antigen-binding fragment thereof. The method according to claim 1,
A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (CDR-H1), a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (CDR-H2), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 A heavy chain complementarity determining region (CDR) comprising the at least one heavy chain complementarity determining region, or a heavy chain variable region comprising the at least one heavy chain complementarity determining region;
(CDR-L3) comprising the amino acid sequence of SEQ ID NO: 4, a polypeptide (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 5, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 ), Or a light chain variable region comprising the light chain complementarity determining region or the light chain variable region comprising the at least one light chain complementarity determining region;
A combination of the at least one heavy chain complementarity determining region and the at least one light chain complementarity determining region; or
The combination of the heavy chain variable region and the light chain variable region
Lt; RTI ID = 0.0 &gt; Ang-2 &lt; / RTI &gt; antibody or antigen-binding fragment thereof. The method according to claim 6,
A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (CDR-H1), a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (CDR-H2), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 ), Or a heavy chain variable region comprising said heavy chain complementarity determining region: and
(CDR-L3) comprising the amino acid sequence of SEQ ID NO: 4, a polypeptide (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 5, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 ), Or a light chain variable region comprising the light chain complementarity determining region
Lt; RTI ID = 0.0 &gt; Ang-2 &lt; / RTI &gt; antibody or antigen-binding fragment thereof. The method according to claim 6,
A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:
Anti-Ang2 antibody or antigen-binding fragment thereof. 9. The antigen-binding fragment of any one of claims 1 to 8, wherein the anti-Ang2 antibody is a monoclonal antibody. 10. An anti-Ang2 antibody or antigen-binding fragment thereof according to claim 9, wherein the anti-Ang2 antibody is obtained from a hybridoma of Accession No. KCLRF-BP-00295. 9. The antigen-binding fragment of any one of claims 1 to 8, wherein the anti-Ang2 antibody is a mouse-derived antibody, a mouse-human chimeric antibody, a humanized antibody or a human antibody. 9. The antibody fragment according to any one of claims 1 to 8, wherein said antigen binding fragment is selected from the group consisting of scFv, scFv2, scFv-Fc, Fab, Fab 'and F (ab') 2 of said anti- Lt; RTI ID = 0.0 &gt; Ang2 &lt; / RTI &gt; antibody or antigen-binding fragment thereof. Hybridoma cell line of Accession No. KCLRF-BP-00295 producing anti-Ang2 antibody. 9. A pharmaceutical composition for preventing or treating diseases associated with Ang2 overexpression, angiogenesis, or vascular permeability increase, comprising the anti-Ang2 antibody or antigen-binding fragment thereof of any one of claims 1 to 8 as an active ingredient. 15. The method of claim 14, wherein said Ang2 overexpression, angiogenesis, or vascular permeability related disease is selected from the group consisting of cancer, metastasis, inflammatory disease, infection, cardiovascular disease, renal disease, hereditary hemorrhagic capillary dilation, asthma, A pharmaceutical composition. 15. The pharmaceutical composition of claim 14, further comprising Ang2. 9. A pharmaceutical composition for the prevention or treatment of diseases associated with reduced normal angiogenesis, comprising the anti-Ang2 antibody or antigen-binding fragment thereof of any one of claims 1 to 8 as an active ingredient. 18. The pharmaceutical composition according to claim 17, wherein the disease associated with decreased normal angiogenesis is myocardial infarction, angina pectoris, cerebral infarction, stroke, Burger's disease, avascular necrosis, foot ulcer, or erectile dysfunction. 18. The pharmaceutical composition of claim 17, further comprising Ang2. 9. A pharmaceutical composition for tissue regeneration or wound healing, comprising the anti-Ang2 antibody of any one of claims 1 to 8 or an antigen-binding fragment thereof as an active ingredient. 21. The pharmaceutical composition of claim 20, further comprising Ang2. 9. A composition for the diagnosis of diseases associated with Ang2 overexpression, comprising the anti-Ang2 antibody of any one of claims 1 to 8 or an antigen-binding fragment thereof as an active ingredient. 23. The diagnostic composition of claim 22, wherein said Ang2 overexpression related disease is cancer, metastasis, infection, or cardiovascular disease. 9. A complex comprising an Ang2 antibody or an antigen-binding fragment thereof of any one of claims 1 to 8 bound to Ang2, and a Tie2 receptor. Contacting a candidate compound with a sample comprising Ang2 and Tie2 receptor; And
Confirming the formation of a complex with the candidate compound, Ang2, and Tie2 receptor bound, confirming Ang2 inhibition and activation of Tie2 receptor, or confirming the complex formation and confirming Ang2 inhibition and activation of Tie2 receptor Step All
/ RTI &gt;
A method for screening candidate substances for the prevention or treatment of Ang2 overexpression, neovascularization, vascular permeability enhancement, or a disease associated with decreased normal angiogenesis. 26. The method of claim 25,
The step of confirming the formation of a complex with which the candidate compound, Ang2, and Tie2 receptor is bound can be confirmed by confirming the presence of a complex with the candidate compound, Ang2, and Tie2 receptor, or the presence of a candidate compound with Ang2, Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;
A method for screening candidate substances for the prevention or treatment of Ang2 overexpression, neovascularization, vascular permeability enhancement, or a disease associated with decreased normal angiogenesis. 26. The method of claim 25, wherein the activation of the Tie2 receptor is confirmed by the degree of phosphorylation of the Tie2 receptor, the degree of phosphorylation of the protein involved in the downstream signal of the Tie2 receptor, intracellular migration of the Tie2 receptor,
A method for screening candidate substances for the prevention or treatment of Ang2 overexpression, neovascularization, vascular permeability enhancement, or a disease associated with decreased normal angiogenesis. At least one selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, a polypeptide including the amino acid sequence of SEQ ID NO: 2, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 3;
At least one selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, a polypeptide including the amino acid sequence of SEQ ID NO: 6, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 6; or
Combinations thereof
/ RTI &gt; 29. The polypeptide molecule of claim 28, comprising the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, or a combination thereof. Contacting the candidate compound with an epitope of Ang2 having the amino acid sequence of SEQ ID NO: 11; And
Measuring binding force between the epitope and the candidate compound; And
Step of confirming binding of Ang2 to Tie2 receptor
Lt; / RTI &gt;
Wherein the epitope of Ang2 is a combination of Q418, P419, Q418 and P419 of Ang2 of SEQ ID NO: 11 or a combination of Q418, P419, or Q418 and P419 of SEQ ID NO: 11,
Lt; RTI ID = 0.0 &gt; Ang2 &lt; / RTI &gt; antibody. 9. A conjugate of Ang2 antibody or antigen binding fragment thereof according to any one of claims 1 to 8 and Ang2.

Images