KR102118691B1 - Composition for blocking vascular leakage containing an Anti-Ang2 antibody - Google Patents

Abstract

An angi2 antibody or an antigen-binding fragment thereof, which specifically binds to angiopoietin-2 (Ang2), an angiogenesis inducer, and binds to the Tie2 receptor with Ang2, and vascular leakage and/or Or use is provided for the prevention and/or treatment of diseases with vascular inflammation.

Description

Composition for blocking vascular leakage containing an Anti-Ang2 antibody}

An angi2 antibody or an antigen-binding fragment thereof, which specifically binds to angiopoietin-2 (Ang2), an angiogenesis inducer, and binds to the Tie2 receptor with Ang2, and vascular leakage and/or Or use is provided for the prevention and/or treatment of diseases with vascular inflammation.

Angiopoietin-2 (Angiopoietin-2; Ang2) is an antagonistic ligand of the receptor Tie2 present in vascular endothelial cells. Angiopoietin-1, an agonist of Tie2 ; Ang1) and Tie2 binding by competing for Tie2 signaling. Ang1, a ligand that activates the Tie2 receptor, acts as a key regulator that maintains the stabilization of blood vessels by maintaining the barrier function of vascular endothelial cells. In the overexpression or inflammation state of VEGF, vascular endothelial cells are activated, and vascular permeability is increased. At this time, Ang1 promotes junctional integrity of vascular endothelial cells, thereby inducing stabilization of vascular endothelial cells and reducing vascular permeability, whereas increased Ang2 in activated vascular endothelial cells competes with Ang1 to cause vascularization caused by Ang1 It serves to inhibit endothelial cell stabilization. Thus, Ang2 inhibits Ang1-Tie2 binding, which maintains the stability of vascular endothelial cells, and signaling through it, and consequently promotes angiogenesis through dynamic rearrangement of blood vessels.

Since the angiogenesis process is an essential element for the growth of cancer, it has been known by studies of several preclinical models that it can prevent the further growth of cancer by inhibiting the function of Tie2-dependent Ang2 as described above to inhibit angiogenesis. Various attempts have been made to prevent the progression of cancer using anti- Ang2 antibodies.

Overexpression of Ang2 has been reported in various solid and hematologic cancers, and it is also a biomarker of poor prognosis of Ang2 in serum. Overexpression of Ang2 has been reported in various diseases such as sepsis, bacterial infection, lung injury, and kidney injury, as well as cancer, and among them, the association with vascular leakage It has been studied a lot. Due to its association with the pathological condition, Ang2 has been considered an important target in therapeutic intervention. A large number of drugs are currently being developed in clinical or preclinical stages, and their forms are diverse, such as peptibody, bispecific antibody, and monoclonal antibody.

However, most of these Ang2 target drugs have an action mechanism that inhibits the activation of Tie2 receptor and downstream signaling by inhibiting Ang2 from binding to Tie2 receptor, and the effect of vascular normalization by Tie2 receptor and downstream signaling cannot be expected.

An example of the present invention specifically binds to Ang2 (Angiopoietin-2), an angiogenesis inducer, and binds to the Tie2 receptor together with Ang2 to induce activation of the Tie2 receptor, an anti Ang2 antibody or antigen-binding fragment thereof to provide.

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

Another example provides an anti-Ang2 antibody-Ang2 complex in which Ang2 is bound to the anti-Ang2 antibody or antigen-binding fragment thereof.

Another example provides a composition for activating a 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 blocking blood vessel leakage 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 prevention and/or 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 the prevention and/or treatment of diseases accompanying vascular leakage, which includes the anti- Ang2 antibody or antigen-binding fragment thereof as an active ingredient.

Another example provides a pharmaceutical composition for preventing and/or treating diseases accompanying vascular inflammation, including the anti- Ang2 antibody or antigen-binding fragment thereof as an active ingredient.

Another example provides a pharmaceutical composition for preventing and/or treating sepsis 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 the anti- Ang2 antibody or antigen-binding fragment thereof as an active ingredient.

Another example provides a composition for diagnosing a disease related to Ang2 overexpression comprising the anti- Ang2 antibody or antigen-binding fragment thereof.

Another example provides a complex bound to the anti-Ang2 antibody or antigen-binding fragment thereof, Ang2, and Tie2 receptor.

The present inventors specifically bind to Ang2, but do not inhibit the binding of Ang2 and Tie2 receptors, and the antibody that forms a complex (antibody/Ang2/Tie2) with Ang2 and Tie2 receptors induces dimerization of the antibody It has been confirmed that by effectively clustering the Tie2 receptor complexed thereto, activation of the Tie2 receptor and its downstream signaling can be induced. Antibodies having this mechanism of action bind to Ang2 and induce intracellularization (internalization) and decomposition, thereby inhibiting Ang2 function and lowering circulating Ang2 level, and at the same time, binding to Tie2 receptor with Ang2 and Tie2 receptor like Ang1 It activates and induces Tie2 downstream signaling and has a dual function to induce stabilization of vascular endothelial cells.

The present invention relates to a therapeutic antibody targeting Ang2, an angiogenesis inducer, specifically inhibiting Ang2 function by specifically binding to Ang2 to inhibit angiogenesis and reduce blood vessel density in cancer tissue. , It relates to an anti-Ang2 antibody that binds to Tie2 together with Ang2 and induces activation of Tie2 to normalize blood vessels structurally and/or functionally. The anti- Ang2 antibody binds to the Tie2 receptor together with Ang2 and the Tie2 receptor with downregulation of Ang2 in diseases related to abnormal activation and dysfunction of blood vessels, such as cancer, sepsis, and ocular disease. It has the effect of treating diseases by inducing structural/functional normalization of blood vessels by activating.

Specifically, an example of the present invention provides an anti-Ang2 antibody or antigen-binding fragment thereof that specifically binds (recognizes) Ang2 (Angiopoietin-2), an angiogenesis inducer, and binds to the Tie2 receptor 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 to induce activation of the Tie2 receptor. Activation of the Tie2 receptor may include one or more phosphorylation selected from the group consisting of proteins related to increased phosphorylation of the Tie2 receptor and/or its lower signaling pathway, such as Akt (NM_005163), eNOS (NM_000603), 42/44 (NM_002745), etc. Can be induced by. In addition, the anti- Ang2 antibody or antigen-binding fragment thereof may be one that induces internalization of the Tie2 receptor into cells. That is, while the anti-Ang2 antibody or antigen-binding fragment thereof specifically binds Ang2, unlike the conventional anti-Ang2 antibody, it does not inhibit the binding of Ang2 to the Tie2 receptor, thereby binding to the Tie2 receptor together with Ang2 complex And may induce 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 the formation of angiogenesis and is a soluble ligand present in the blood.It is widely used for angiogenesis, metastasis, and cancer cell invasion. Works. The Ang2 may be derived from mammals such as primates such as humans and monkeys, rodents such as mice, rats, etc., for example, human Ang2 (eg, NCBI Accession No. O15123, etc.), monkey Ang2 (eg, NCBI Accession No. Q8MIK6) Etc.), mouse Ang2 (eg, NCBI Accession No. O35608, etc.), rat Ang2 (eg, NCBI Accession No. O35462, etc.), and the like, but are not limited thereto.

The Tie2 receptor (TEK tyrosine kinase) is an angiopoietin-1 receptor, expressed in endothelial cells of various mammals such as mice (NM_013690; NP_038718), rats, and humans (NM_000459; NP_000450). It is involved in downstream signaling.

As described above, the anti-Ang2 antibody or antigen-binding fragment provided in the present invention specifically binds Ang2, but does not inhibit the binding of Ang2 and Tie2 receptors and complexes with Ang2 and Tie2 receptors (antibody/Ang2/Tie2) The antibody constituting, has the property of inducing dimerization of the antibody, and thereby effectively clustering the Tie2 receptor complexed therein, thereby activating the Tie2 receptor and its downstream signaling. Characterized in that it can induce. Due to this mechanism of action, the antibody or antigen-binding fragment thereof of the present invention binds to Ang2 and induces intracellular migration (internalization) and degradation, thereby inhibiting Ang2 function and lowering circulating Ang2 level, along with Ang2. By binding to the Tie2 receptor and having a dual function that activates the Tie2 receptor like Ang1 to induce the stabilization of vascular endothelial cells, not only the symptoms (disease) caused by overexpression of Ang2, but also the stabilization of vascular endothelial cells, that is, vascular permeability It can also be useful in the treatment of symptoms (diseases) due to the increase.

The anti- Ang2 antibody or antigen-binding fragment thereof is all or part of the loop 1 (sites 417 to 434 amino acids of SEQ ID NO: 11) of human Ang2 (hAng2; SEQ ID NO: 11; Accession # O15123) (e.g., loop) Of 2 to 20 or 2 consecutive or non-contiguous one or more amino acid residues exposed to the outside of loop 1 in SEQ ID NO: 11) It may be an amino acid sequence region containing 15 to 2, 10 to 10, or 2 to 5 amino acids as an epitope, or may specifically bind to this region.

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 be contiguous or contiguous comprising amino acid residues 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: 11. 2 to 20, 2 to 15, 2 to 10, or 2 to 5 amino acid sequence regions comprising 2 to 5 amino acids may be recognized as epitopes or may specifically bind to this site. In one example, the anti-Ang2 antibody may recognize amino acid residues of Q418 and P419 of SEQ ID NO: 11 as epitopes or specifically bind to this portion.

The anti-Ang2 antibody specifically binding site, Q418, P419, or an amino acid site comprising the same is an exposed amino acid residue located in loop 1 of AngD's three-dimensional structure, directly involved in binding between Ang2 and Tie2 receptors, or , It is believed to be a site that regulates this.

In the anti-Ang2 antibody specifically binding site Q418, P419, or an amino acid site containing the same, "non-contiguous amino acids" are adjacent to each other on the secondary or tertiary structure of the protein, but not on the amino acid sequence. Does not mean amino acids. Thus, as used herein, "consecutive or non-contiguous amino acid residues" may refer to contiguous amino acid residues on a protein's primary, secondary or tertiary structure.

In addition, not only the anti-Ang2 antibody that recognizes and/or specifically binds the site, but also the antibody or antigen-binding fragment thereof that binds to the anti-Ang2 antibody competitively inhibits Ang2 and at the same time forms a complex with Ang2 and Tie2 receptors. (Ie, antibody-Ang2 binds to the Tie2 receptor), which can activate Tie2. The competitively binding antibody may be an antibody that recognizes the site described above and an adjacent site in a three-dimensional structure as an epitope and/or a specific binding site. The competitively binding antibody may have a binding affinity with 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 one or more selected from the group consisting of antibodies or antigen-binding fragments that specifically recognize or bind to the site described above as an epitope.

In an embodiment, 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 (CDR-H3) Heavy chain variable region comprising one or more selected from the group consisting of) (complementarity determining region, CDR), or heavy chain variable region comprising the heavy chain complementarity determining region:

A polypeptide comprising the amino acid sequence of SEQ ID NO: 4 (CDR-L1), a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 (CDR-L2), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 (CDR-L3) ), a light chain variable region comprising at least one selected from the group consisting of, or a light chain variable region comprising the light chain complementarity determining region;

A combination of said one or more heavy chain complementarity determining regions and one or more light chain complementarity determining regions; or

Combination of the heavy chain variable region and the light chain variable region

It may include:

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 (CDR-H3) ) A heavy chain complementarity determining region, or a heavy chain variable region comprising the heavy chain complementarity determining region: and

A polypeptide comprising the amino acid sequence of SEQ ID NO: 4 (CDR-L1), a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 (CDR-L2), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 (CDR-L3) ) A light chain complementarity determining region, or a light chain variable region comprising the light chain complementarity determining region

It may be to include.

Specifically, the heavy chain complementarity determining region of the anti- Ang2 antibody or antigen-binding fragment thereof may be, for example, having an amino acid sequence of 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)

In addition, the light chain complementarity determining region of the anti- Ang2 antibody or antigen-binding fragment thereof may have, for example, an 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 be one comprising the amino acid sequence of SEQ ID NO:7:

DVQLQESGPGLVKPSQSLSLTCTVTGYSIT SDYAWN WIRQFPGNKLEWMG YINYSGNTDYNPSLKS RSSITRDTSKNQFFLQLNSVTTGDTATYYCAR GNFEGAMDY WGQGTSVTVSS (SEQ ID NO: 7)

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

The light chain of the antibody according to one embodiment may include 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, the underlined bold letters are CDRL1, CDRL2, and CDRL3 in order)

Accordingly, 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. It may be included.

For example, the anti-Ang2 antibody or antigen-binding fragment thereof may include 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: 9.

In one example, the anti-ang-2 antibody or antigen-binding fragment thereof may not be composed 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: 5.

In another example, the epitopes described above are used to identify candidate substances for the diagnosis, prevention and/or treatment of diseases associated with Ang2 overexpression, angiogenesis, enhancement of vascular permeability, and/or reduction of normal angiogenesis, such as anti Ang2 antibodies. Methods of screening are provided. The screening method,

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

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

It may include.

The screening method may further include (c) confirming the binding between the Ang2 and Tie2 receptors. The step (c) is for confirming whether the candidate compound inhibits the binding between Ang2 and the Tie2 receptor, and can be performed before, after, or simultaneously with the step (b). Through step c), while binding to Ang2, the binding between the Ang2 and Tie2 receptors is not inhibited, that is, an anti-Ang2 antibody having a property of inducing binding between the Ang2 and Tie2 receptors can be screened.

In addition, the screening method may further include (d) confirming Ang2 inhibition and/or activation of the Tie2 receptor, and the order in which it is included is not limited. Through step d), it is possible to screen anti-Antibody antibodies that bind to Ang2 and inhibit its activity, but bind to Ang2 to induce binding between Ang2 and Tie2 receptors, and exhibit Tie2 receptor activation effects such as Ang1.

In the screening method. When the epitope and the candidate compound exhibit a binding force (kd) of 10 nM or less, for example, in the range of 0.1 pM to 10 nM, or in the range of 10 pM to 10 nM, or 100 pM to 10 nM, or the epitope and the candidate compound are When the binding between Ang2 and the Tie2 receptor is confirmed while showing the same binding force (that is, when the candidate compound does not inhibit the binding between the Ang2 and the Tie2 receptor), the candidate compound is over-expressed in Ang2, angiogenesis, enhancement of vascular permeability, and And/or a candidate substance for diagnosis, prevention, and/or treatment of a disease associated with a decrease in normal angiogenesis, such as an anti- Ang2 antibody candidate substance.

The epitope is all, part (eg, outside of loop 1) of loop 1 (sites from amino acids 417 to 434 of SEQ ID NO: 11) of human Ang-2 (hAng-2; SEQ ID NO: 11; Accession # O15123) At least one selected from the group consisting of amino acid residue sites exposed to), or from 2 to 20, 2 to 15 consecutive or non-contiguous sequences comprising at least one amino acid residue exposed outside of loop 1 in SEQ ID NO: 11 , Or an amino acid sequence region comprising 2 to 10 amino acids, e.g., Q418, P419, combination of Q418 and P419 located in loop 1 of SEQ ID NO: 11, or contiguous or non-contiguous two comprising them To 20, 2 to 15, 2 to 10, or 2 to 5 amino acids, and may be one or more selected from the group consisting of amino acid sequence regions.

The candidate compound may be artificially synthesized or natural various compounds, polypeptides, oligopeptides, peptides or protein structures (eg, antibodies, peptibody, nanobody, etc.), polynucleotides, oligonucleotides, antisense-RNA, shRNA (short hairpin RNA), siRNA (small interference RNA), aptamers, natural product extracts, and the like.

The step of measuring the binding force between the epitope and the candidate compound may be performed through various methods known in the art. For example, the binding force can be measured using a Biacore device. In general, the range in which the binding force is recognized as a therapeutic drug may be defined as a binding constant KD value of 10 nM or less. That is, for example, the binding force between the above-mentioned epitope of angiopoietin-2 and the sample to be analyzed (for example, an antibody) was measured by a surface plasmon resonance method such as a Biacore device, 0.1 pM to 50 nM, specifically 0.5 pM to 35 nM, more specifically 1 pM to 10 nM, the sample (e.g., antibody) Ang2 overexpression, neovascularization, vascular permeability enhancement, and/or reduction in normal angiogenesis It can be judged as a candidate substance for diagnosis, prevention and/or treatment of a related disease.

The method for confirming the binding between Ang2 and Tie2 and for inhibiting Ang2 and activating the Tie2 receptor are as described below.

In another example, the heavy chain complementarity determining region, light chain complementarity determining region, or combinations thereof of the anti- Ang2 antibody described above; Or a polypeptide molecule comprising a heavy chain variable region, a light chain variable region, or a combination thereof is provided. The polypeptide molecule may function as a precursor or component of an antagonist to Ang2 as well as the production of an antibody or antigen-binding fragment thereof. For example, the polypeptide molecule may be an Ang2 antigen binding site, and a protein scaffold (eg, a peptide body, a nanobody, etc.) having a structure similar to that of an antibody, a bispecific antibody, a multispecific antibody, etc. It can be included as an ingredient.

The term "antagonist" is interpreted as a concept encompassing all molecules that partially, completely block, inhibit, or neutralize one or more of the biological activities of a target (eg, Ang2).

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

The term "nanobody" is also referred to as a single-domain antibody, and refers to an antibody fragment comprising a single variable domain of an antibody in a monomeric form, and selectively binds to a specific antigen similar to a fully structured antibody. It has the characteristic. The molecular weight of the nanobody is generally about 12 kDa to about 15 kDa, very small compared to the general molecular weight of the complete antibody (including two heavy chains and two light chains) (about 150 kDa to about 160 kDa), in some cases Is smaller than the Fab fragment or scFv fragment.

The term “bispecific antibody” or “multispecific antibody” recognizes and/or binds two (bispecific antibodies) or more (multispecific antibodies) different antigens, or recognizes different sites of the same antigen and/or It means an antibody that binds, and the antigen-binding site of one of the bispecific antibodies or multispecific antibodies may include the polypeptide.

In an embodiment, the polypeptide molecule is

At least one selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, a polypeptide comprising 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 comprising the amino acid sequence of SEQ ID NO: 6, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 6; or

Combination of these

It may be to include.

In an embodiment, the polypeptide molecule may include 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 be composed 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: 5.

As described above, the bispecific antibody or multispecific antibody includes two or more different antigen-binding sites, and recognizes and/or binds two or more antigens simultaneously. As, one of the antigen-binding sites may include the polypeptide molecule described above. Specifically, the polypeptide molecule serving as the Ang2 antigen binding site may form a dimeric or multimer with an antigen binding site for another antigen to construct a bispecific antibody or a multispecific antibody. Thus, in one embodiment, a bispecific antibody or multispecific antibody comprising the polypeptide molecule as an Ang2 antigen binding site is provided.

In another example, one or more polypeptide molecules described above or a polypeptide having a structural function with a repeater (hereinafter referred to as a'first peptide') repeatedly linked by a linker (hereinafter referred to as a'second peptide') ; For example, one or more peptide complexes comprising an antibody (IgG, IgA, IgE, IgD, IgM, etc.) heavy chain or light chain constant region, or an antibody Fc fragment) (e.g. 1-5, or 2-4) ), wherein the one or more peptide complexes are combined in a second peptide (such as an Fc fragment) to provide a protein framework having a multimeric structure.

Antibodies in the present invention include animal-derived antibodies, chimeric antibodies, humanized antibodies, and human antibodies. Animal-derived antibodies produced by immunizing an immunized animal with a desired antigen may generally have an immune rejection reaction when administered to a human for therapeutic purposes, and chimeric antibodies have been developed to suppress such immune rejection. Chimeric antibodies are those that replace the constant regions of animal-derived antibodies that cause anti-isotype reactions with the constant regions of human antibodies using genetic engineering methods. Chimeric antibodies have been significantly improved in anti-isotype reactions compared to animal-derived antibodies, but still contain animal-derived amino acids in the variable region, thus implicating side effects on potential anti-idiotypic responses. Doing. Humanized antibodies have been developed to improve these side effects. It is produced by grafting a complementary determining region (CDR) region that plays an important role in antigen binding among variable regions of a chimeric antibody into a human antibody framework.

The most important thing in the CDR grafting technology for the production of humanized antibodies is to select an optimized human antibody that can best accept the CDR regions of animal-derived antibodies. To this end, use of an antibody database and crystal structure structure), and molecular modeling technology. However, even when the CDR regions of animal-derived antibodies are grafted onto the optimized human antibody skeleton, there are cases where amino acids that affect the antigen binding while being located in the skeleton of the animal-derived antibody are often not preserved. Since it exists, it can be said that the application of additional antibody engineering techniques to restore antigen binding ability 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.

In the present invention, the term "antibody" refers to a substance produced by stimulation of an antigen in the immune system, and its type is not particularly limited. Antibodies have been recently used for the treatment of disease. Antibodies are very stable both in vitro and in vivo, and have a long half-life, which is advantageous for mass expression and production. In addition, since the antibody has a dimer structure in nature, the avidity is very high.

A complete antibody has a structure having two full length light chains and two full length heavy chains, and each light chain is 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 gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, and subclasses. Gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.

The term "heavy chain" refers to the variable region domain V _H and the three constant region domains C _H1 , C _H2 and C _H3 and the hinge ( It is interpreted as including both the full-length heavy chain and the fragments thereof. In addition, the term “light chain” refers to both the full-length light chain comprising the variable region domain V _L and the constant region domain C _L and a fragment thereof comprising an amino acid sequence having sufficient variable region sequences to confer specificity to the antigen. It is interpreted as including meaning.

The term "complementarity determining region" (CDR) refers to the amino acid sequence of the hypervariable regions of the heavy and light chains of immunoglobulins. The heavy chain and light chain may each include three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs can provide a major contact residue for the antibody to bind to an antigen or epitope. On the other hand, in the present specification, the term, "specifically binding" or "specifically recognized" has the same meaning as commonly known to those skilled in the art, and the antigen and antibody specifically interact to perform an immunological reaction. Means

The antigen-binding fragment of the antibody provided in the present invention may be a fragment containing one or more of the complementarity determining regions.

The term, “antigen-binding fragment” is a fragment of the entire structure of an immunoglobulin, meaning a portion of a polypeptide that includes a portion to which an antigen can bind. For example, it may be scFv, (scFv) ₂ , scFv-Fc, Fab, Fab' or F(ab') ₂ , but is not limited thereto.

Among the antigen-binding fragments, Fab has 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, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain C _H1 domain. The F(ab') ₂ antibody is produced by cysteine residues in the hinge region of Fab' forming disulfide bonds. Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region, and recombinant techniques for generating Fv fragments are well known in the art. The double-chain Fv (two-chain Fv) is a non-covalently linked heavy chain variable region and a light chain variable region, and the single-chain Fv (single-chain Fv) generally shares the variable region of the heavy chain and the variable region of the single chain through a peptide linker. It can be linked by a bond or directly linked at the C-terminus to form a dimer-like structure such as a double chain Fv. The linker may be a peptide linker consisting of 1 to 100 or 2 to 50 arbitrary amino acids, and sequences suitable in the art are known. The antigen-binding fragments can be obtained using proteolytic enzymes (e.g., a restriction digestion of the whole antibody with papain yields Fab, and digestion with pepsin yields F(ab') ₂ fragment), It can be produced through genetic recombination technology.

The term “hunge region” refers to a region included in the heavy chain of an antibody, which exists between CH1 and CH2 regions, and functions to provide flexibility of an antigen binding site in an antibody. For example, the hinge may be derived from a human antibody, specifically, IgA, IgE, or IgG, such as IgG1, IgG2, IgG 3, or IgG4.

When the animal-derived antibody undergoes a chimerization process, the animal-derived IgG1 hinge is replaced with a human IgG1 hinge, but the animal-derived IgG1 hinge is shorter in length than the human IgG1 hinge and disulfide bond between the two heavy chains ( The disulfide bond is reduced from 3 to 2, and the rigidity of the hinge shows different effects. 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 excluding the variable region of the anti Ang2′ antibody may be a constant region derived from a human antibody, specifically, IgA, IgE, or IgG, such as a constant region derived from IgG1, IgG2, IgG 3, or IgG4. .

The anti Ang2′ antibody can be a monoclonal antibody. Monoclonal antibodies can be prepared by methods well known in the art. For example, it can be manufactured using a phage display technique. Alternatively, the anti-Ang2 antibody can be produced from a mouse-derived monoclonal antibody by a method described in a paper such as Schwaber using a 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).

Meanwhile, individual monoclonal antibodies can be screened based on the ability to bind Ang2 using a typical Enzyme-Linked ImmunoSorbent Assay (ELISA) format. Inhibitory activity can be assayed through functional assays such as competitive ELISA (Competitive ELISA) or cell-based assays to assay molecular interactions for conjugates. Each affinity for Ang2 (Kd values) is then assayed for selected monoclonal antibody members based on strong inhibitory activity.

The final selected antibodies can be prepared and used as humanized antibodies, as well as humanized 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 the anti Ang2 antibody. The hybridoma cell line may be a cell line having accession number (KCLRF-BP-00295).

As described above, the anti-Ang2 antibody or antigen-binding fragment proposed in the present invention is characterized by inducing the binding of Ang2 to the Tie2 receptor without inhibiting Ang2 from binding to the Tie2 receptor while binding to Ang2. In addition, the conjugate of the anti- Ang2 antibody or antigen-binding fragment thereof and Ang2 thus bound, acts like Ang1, binds to the Tie2 receptor (the Ang2 portion of the complex binds), and activates the Tie2 receptor. It is characterized by having a function.

Accordingly, another example provides an anti-Ang2 antibody-Ang2 complex in which Ang2 is bound to the anti-Ang2 antibody or an antigen-binding fragment thereof. Another example provides a composition for activating a Tie2 receptor comprising the anti- Ang2 antibody-Ang2 complex as an active ingredient. Another example provides a method of activating a Tie2 receptor, 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 include identifying a patient in need of activation of the Tie2 receptor prior to the administering step. The patient may be a human, a primate including a monkey, a mammal, a rodent containing a mouse, a rat, or the like, or a cell, tissue, body fluid, or the like, artificially or cultured therefrom. Another example is provided for the use of the anti Ang2 antibody-Ang2 complex for Tie2 receptor activation.

As described above, the anti- Ang2 antibody or antigen-binding fragment proposed in the present invention has a function of inhibiting abnormal angiogenesis by inhibiting the function of Ang2, thereby preventing various diseases (eg, cancer) related to abnormal angiogenesis , Alleviation, improvement and/or treatment (see Example 13). In addition, the anti-Ang2 antibody or antigen-binding fragment thereof does not inhibit the binding between Ang2 and Tie2 (see Examples 3 and 1), thereby activating Tie2 (see Examples 5 and 2), thereby signaling Tie2 signaling. By activating (see Example 6 and Figure 3), promoting the production of vascular endothelial or lymphatic endothelium and increasing mobility (see Example 10), and inhibiting the increase in vascular permeability (see Example 11), It is also applicable to prevention, alleviation, improvement and/or treatment of various diseases (eg, sepsis, ocular disease, etc.). In addition, as described above, the anti- Ang2 antibody or antigen-binding fragment thereof promotes the production of vascular endothelial or lymphatic endothelium, thereby increasing the formation of healthy blood vessels and normalizing the blood vessels, thereby forming healthy blood vessels such as wound healing or ischemic disease. It is applicable to the prevention, alleviation, improvement, and/or treatment of various diseases or symptoms that require the disease, and reduces the likelihood of cancer growth and metastasis by changing abnormally formed cancer vessels to structural and functional normal conditions. In addition, the anti- Ang2 antibody or antigen-binding fragment thereof has an effect of inhibiting an inflammatory response (see Example 12), and thus 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 for inhibiting angiogenesis, comprising administering a pharmaceutically effective amount of the anti- Ang2 antibody or antigen-binding fragment thereof to a patient in need thereof. The method for inhibiting angiogenesis may further include identifying a patient in need of inhibition of angiogenesis prior to the administration 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 of reducing vascular permeability comprising administering a pharmaceutically effective amount of the anti- Ang2 antibody or antigen-binding fragment thereof to a patient in need thereof. The method for reducing vascular permeability may further include 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 related to Ang2 overexpression, angiogenesis, and/or increased vascular permeability, including the anti- Ang2 antibody or antigen-binding fragment thereof as an active ingredient. Another example comprises the step of administering a pharmaceutically effective amount of the anti- Ang2 antibody or antigen-binding fragment thereof to a patient in need of prevention and/or treatment of diseases associated with Ang2 overexpression, angiogenesis and/or increased vascular permeability. It provides a method for preventing and/or treating diseases related to Ang2 overexpression, angiogenesis, and/or increased vascular permeability. The method of prevention and/or treatment may further include identifying a patient in need of prevention and/or treatment of diseases associated with Ang2 overexpression, angiogenesis and/or increased vascular permeability prior to the 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 of 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 inducing normal angiogenesis. The method for increasing normal angiogenesis may further include identifying a patient in need of inducing normal angiogenesis prior to the administration.

Another example provides a pharmaceutical composition for the prevention and/or treatment of diseases associated with a decrease in normal angiogenesis, including the anti- Ang2 antibody or antigen-binding fragment thereof as an active ingredient. Another example comprises administering a pharmaceutically 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 a decrease in normal angiogenesis, Prophylactic and/or therapeutic methods are provided. The method of prevention and/or treatment may further include identifying a patient in need of prevention and/or treatment of a disease associated with a decrease 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 of tissue regeneration and/or wound healing comprising administering a pharmaceutically 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 include identifying a patient in need of tissue regeneration and/or wound healing prior to the administering step. The patient to be administered the active ingredient may be, for example, a patient with skin tissue or organ tissue damage, or a skin transplant.

Another example provides a pharmaceutical composition for Ang2 inhibition and/or Tie2 receptor activation comprising the anti- Ang2 antibody or antigen-binding fragment thereof as an active ingredient. Another example provides a method of Ang2 inhibition and/or Tie2 receptor activation comprising administering a pharmaceutically effective amount of the 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 include 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 as an active ingredient of the pharmaceutical composition is activated by binding to Ang2, in order to further enhance the function of the antibody or antigen-binding fragment, the pharmaceutical composition further includes Ang2 It may be. In addition, the method may further include administering a pharmaceutically effective amount of Ang2 to the patient.

As described above, the anti-Ang2 antibody or antigen-binding fragment thereof may be useful for the prevention and/or treatment of diseases accompanying vascular leakage by inducing blood vessel normalization and inhibiting an increase in vascular permeability.

Accordingly, another example of the present invention provides a pharmaceutical composition for blocking blood vessel leakage, including the anti- Ang2 antibody or antigen-binding fragment thereof as an active ingredient. Another example provides a method of blocking vascular leakage, comprising administering a pharmaceutically effective amount of the anti- Ang2 antibody or antigen-binding fragment thereof to a patient in need thereof. The vascular leak blocking method may further include a step of identifying a patient in need of vascular leak blocking prior to the administration step. Another example provides the use of the anti- Ang2 antibody or antigen-binding fragment thereof for blocking vascular leakage, or for use in the manufacture of vascular leakage blocking agents. 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 as an active ingredient of the pharmaceutical composition is activated by binding to Ang2, in order to further enhance the function of the antibody or antigen-binding fragment, the pharmaceutical composition further includes Ang2 It may be. In addition, the method may further include administering a pharmaceutically effective amount of Ang2 to the patient.

Another example provides a pharmaceutical composition for the prevention and/or treatment of diseases accompanying vascular leakage, comprising the anti- Ang2 antibody or antigen-binding fragment thereof as an active ingredient. Another example is the prevention of diseases associated with vascular leakage, comprising administering a pharmaceutically effective amount of the anti- Ang2 antibody or antigen-binding fragment thereof to a patient in need of prevention and/or treatment of diseases associated with vascular leakage, and And/or provide treatment. The method for preventing and/or treating diseases associated with vascular leakage may further include identifying a patient in need of prevention and/or treatment of diseases accompanying vascular leakage prior to the administration step. Another example is the use of the anti- Ang2 antibody or antigen-binding fragment thereof for use in the prevention and/or treatment of diseases with vascular leakage, or pharmaceutical compositions for the prevention and/or treatment of diseases with vascular leakage. Provides use for use in. 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 as an active ingredient of the pharmaceutical composition is activated by binding to Ang2, in order to further enhance the function of the antibody or antigen-binding fragment, the pharmaceutical composition further includes Ang2 It may be. In addition, the method may further include administering a pharmaceutically effective amount of Ang2 to the patient.

Another example provides a pharmaceutical composition for preventing and/or treating diseases accompanying vascular inflammation, including the anti- Ang2 antibody or antigen-binding fragment thereof as an active ingredient. Another example is the prevention of diseases associated with vascular inflammation, comprising administering a pharmaceutically effective amount of the anti- Ang2 antibody or antigen-binding fragment thereof to a patient in need of prevention and/or treatment of diseases associated with vascular inflammation, and And/or provide treatment. The method for preventing and/or treating diseases associated with vascular inflammation may further include identifying a patient in need of prevention and/or treatment of diseases accompanying vascular inflammation prior to the administration step. Another example is the use of the anti- Ang2 antibody or antigen-binding fragment thereof for use in the prevention and/or treatment of diseases with vascular inflammation, or pharmaceutical compositions for the prevention and/or treatment of diseases with vascular inflammation. Provides use for use in. 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 as an active ingredient of the pharmaceutical composition is activated by binding to Ang2, in order to further enhance the function of the antibody or antigen-binding fragment, the pharmaceutical composition further includes Ang2 It may be. In addition, the method may further include administering a pharmaceutically effective amount of Ang2 to the patient.

Another example provides a pharmaceutical composition for preventing and/or treating sepsis comprising the anti- Ang2 antibody or antigen-binding fragment thereof as an active ingredient. Another example provides a method of preventing and/or treating sepsis, comprising administering a pharmaceutically effective amount of the anti- Ang2 antibody or antigen-binding fragment thereof to a patient in need thereof. The method for preventing and/or treating sepsis may further include identifying a patient in need of prevention and/or treatment of sepsis prior to the administering step. Another example provides the use of the anti- Ang2 antibody or antigen-binding fragment thereof for use in the prevention and/or treatment of sepsis, or the use of pharmaceutical compositions for the prevention and/or treatment of sepsis for use in manufacturing. The anti- Ang2 antibody or antigen-binding fragment thereof may be in a state bound to the antigen, Ang2. Since the function of the anti- Ang2 antibody or antigen-binding fragment thereof as an active ingredient of the pharmaceutical composition is activated by binding with Ang2, in order to further enhance the function of the antibody or antigen-binding fragment, the pharmaceutical composition further includes Ang2 It may be. In addition, the method may further include administering a pharmaceutically effective amount of Ang2 to the patient.

Another example provides a composition for the diagnosis of diseases related to angiogenesis and/or increased vascular permeability and/or reduced normal angiogenesis and/or vascular leakage and/or vascular inflammation, comprising the antibody or antigen-binding fragment thereof.

The pharmaceutical composition may further include a pharmaceutically acceptable carrier, which is commonly used for the formulation of drugs, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate , Alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It may be one or more selected from the group consisting of, but is not limited thereto. The pharmaceutical composition may further include one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., which are commonly used in the manufacture of pharmaceutical compositions.

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

The content of the anti-Ang2 antibody or antigen-binding fragment thereof in the pharmaceutical composition may include formulation methods, administration methods, patient age, weight, sex, morbidity, food, administration time, administration interval, administration route, excretion rate, and response sensitivity. It can be variously prescribed by factors. For example, the daily dose of the anti- Ang2 antibody or antigen-binding fragment thereof may range from 0.001 to 1000 mg/kg, specifically 0.01 to 100 mg/kg, more specifically 0.1 to 50 mg/kg, but is not limited thereto. no. The daily dosage may be formulated as a single dosage form in unit dose form, or may be formulated in appropriate portions, or may be prepared by incorporating into a multi-dose container. The "pharmaceutical effective amount" may mean a content or dosage of an active ingredient capable of exhibiting a desired pharmacological effect, and a formulation method, a administration method, a patient's age, weight, sex, pathological condition, food, and administration time , Administration interval, route of administration, rate of excretion, and response responsiveness.

The pharmaceutical composition may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be formulated in the form of ex-agent, powder, granule, tablet or capsule, and dispersant or stable for formulation Topics may be further included.

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

On the other hand, since the anti-Ang2 antibody or antigen-binding fragment thereof specifically binds to Ang2, it is possible to detect Ang2 using it, thereby confirming whether Ang2 is overexpressed. Accordingly, another example of the present invention provides a composition for detecting Ang2 comprising the anti- Ang2 antibody or an antigen-binding fragment thereof, and a composition for diagnosing a disease related to Ang2 overexpression.

In another example, treating a biological sample obtained (or isolated) from a patient with the anti- Ang2 antibody or antigen-binding fragment thereof; And it provides an Ang2 detection method comprising the step of determining whether the antigen-antibody reaction. In addition, the step of treating the anti-Ang2 antibody or antigen-binding fragment thereof to a biological sample obtained from a patient; And it provides a method for providing information in the diagnosis of diseases associated with Ang2 overexpression comprising the step of determining whether the antigen-antibody reaction. A method of providing information to the diagnosis may be to determine that the patient has an Ang2 overexpression symptom or has an Ang2 overexpression-related disease when an antigen-antibody reaction is detected in the step of determining whether the antigen-antibody reaction is present. . The biological sample may be selected from the group consisting of (or isolated) cells, tissues, and body fluids obtained from a patient.

The step of determining whether the antigen-antibody reaction may be performed may be performed through various methods known in the art. For example, it can be measured through conventional enzymatic reaction, fluorescence, luminescence and/or radiation detection, specifically, immunochromatography, immunohistochemistry, enzyme linked immunosorbent assay: ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), Western blotting (Western blotting) ) May be measured by a method selected from the group consisting of, but is not limited thereto.

Patients subject to administration or diagnosis of the pharmaceutical composition or antibody or antigen-binding fragment are mammals including primates including humans, monkeys, etc., rodents including mice, rats, etc., or cells isolated or artificially cultured therefrom, Tissue, body fluids, and the like.

Diseases associated with angiogenesis and/or increased vascular permeability and/or overexpression of Ang2 include cancer; Cancer metastasis; Ocular vascular diseases such as retinopathy of prematurity, macular degeneration (eg, age-related macular degeneration), diabetic retinopathy, and angiogenic glaucoma; Inflammatory diseases such as psoriasis, asthma, rheumatoid arthritis, pneumonia, and chronic inflammation; Infectious diseases (infection); Cardiovascular diseases such as hypertension and arteriosclerosis; Kidney disease; blood poisoning; asthma; edema; Hereditary hemorrhagic telangiectasia (HHT), and the like. The cancer is an overexpression of Ang2, which may be solid cancer or blood cancer, but is not limited to squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, skin cancer, skin or eyeball My melanoma, rectal cancer, rectal cancer, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocyte lymphoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer , Ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, colon cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulva cancer, thyroid cancer, head and neck cancer, brain cancer, osteosarcoma, etc. 1 It may be more than a species.

The disease associated with the reduction of normal angiogenesis is a disease requiring induction of normal angiogenesis, ischemic diseases such as myocardial infarction, angina, cerebral infarction, stroke (cerebral ischemia), Berger's disease (occlusive thrombo vasculitis), avascular necrosis ( avascular necrosis), foot ulcers (eg, diabetic foot ulcers, etc.), erectile dysfunction, and the like.

The diseases associated with vascular leakage include sepsis, vascular leak syndrome, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), diabetic vascular disease complications).

The disease accompanying vascular inflammation may be selected from the group consisting of sepsis, vascular infection or infectious disease.

In particular, sepsis is a disease accompanied by a systemic inflammatory reaction and vascular leakage, and the anti-Ang2 antibody or antigen-binding fragment thereof having an excellent inflammation-reducing effect and vascular permeability-reducing effect can be particularly useful.

In the treatment of diseases associated with angiogenesis and/or increased vascular permeability and/or Ang2 overexpression, diseases with vascular leakage, diseases with vascular inflammation, such as sepsis, the anti Ang2 antibody or antigen binding fragment thereof Increased therapeutic effects can be expected in combination with existing treatments for diseases associated with increased angiogenesis and/or increased vascular permeability and/or Ang2 overexpression, diseases associated with vascular leakage, vascular inflammation, such as sepsis. have. Therefore, the anti- Ang2 antibody or antigen-binding fragment thereof is also useful as a pharmaceutical composition for combined administration.

In this aspect, the pharmaceutical composition for the prophylaxis and/or treatment of diseases associated with angiogenesis and/or increased vascular permeability and/or Ang2 overexpression, diseases with vascular leakage, diseases with vascular inflammation, such as sepsis It may further include diseases related to angiogenesis and/or increased vascular permeability and/or Ang2 overexpression, diseases with vascular leakage, diseases with vascular inflammation, or conventional therapeutic agents for sepsis. In another aspect, another example of the present invention is accompanied by diseases related to the anti- Ang2 antibody or antigen-binding fragment thereof and angiogenesis and/or increased vascular permeability and/or overexpression of Ang2, diseases with vascular leakage, and vascular inflammation It provides a composition for a combination administration comprising at least one selected from the group consisting of a conventional treatment for a disease or sepsis. The conventional therapeutic agents include prophylaxis, treatment, relief of symptoms, such as diseases associated with angiogenesis and/or increased vascular permeability and/or Ang2 overexpression, diseases with vascular leakage, diseases with vascular inflammation, such as sepsis, and / Or any compound, antibody, gene (e.g., antisense oligonucleotide, siRNA, shRNA, microRNA, etc.), aptamer, cell therapy, radiation therapy, etc. For example, it may be one or more selected from the group consisting of antibiotics, drotrecogin alpha, corticosteroid, and vasopressin, but is not limited thereto.

Another example provides a complex bound to the anti-Ang2 antibody or antigen-binding fragment thereof, Ang2, and Tie2 receptor. The complex may be present in a living body or may be present in cells isolated from the living body. In addition, the antibody in the complex may form a dimer with an antibody in an adjacent complex, and cluster two or more complexes to form a cluster comprising two or more complexes (see Example 8). This action is related to the function of activating the Tie2 receptor of the anti-Ang2 antibody. The complex may be used for monitoring the action of the anti-Ang2 antibody, that is, inhibiting Ang2 and/or activating the Tie2 receptor, or inhibiting Ang2 by itself and/or activating the Tie2 receptor.

Another example is a method for screening candidate substances for the prevention and/or treatment of diseases associated with angiogenesis and/or enhancement of vascular permeability by determining whether the complex formation and/or Ang2 inhibition and/or activation of the Tie2 receptor is activated. to provide.

In one embodiment, the screening method,

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

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

It may include.

The step of confirming the formation of the complex to which the candidate compound, Ang2, and Tie2 receptor is bound, confirms the existence of the complex to which the candidate compound, Ang2, and Tie2 receptor is bound, or whether the candidate compound is bound to Ang2 and Ang2 And Tie2 receptors. The screening method may further include the step of confirming Ang2 inhibition and/or activation of the Tie2 receptor before, after, or simultaneously with the step of confirming formation of the complex to which the candidate compound, Ang2, and Tie2 receptor are bound. have.

In the above screening method, when the formation of the complex to which the candidate compound, Ang2, and Tie2 receptor is bound is confirmed, that is, the presence of the complex to which the candidate compound, Ang2, and Tie2 receptor is bound is confirmed, or the candidate compound and Ang2 If liver binding and binding between Ang2 and Tie2 receptors are identified, the candidate compound is a candidate substance for the prevention and/or treatment of diseases associated with Ang2 overexpression, angiogenesis, increased vascular permeability, and/or reduced normal angiogenesis. I can judge.

In another example, the screening method,

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

Confirming Ang2 inhibition and/or activation of the Tie2 receptor

It may include. In the above screening method, when Ang2 inhibition and/or activation of the Tie2 receptor is confirmed, the candidate compound is prevented and/or prevented from diseases related to Ang2 overexpression, angiogenesis, increased vascular permeability, and/or reduced normal angiogenesis. It can be judged as a candidate substance for treatment. Inhibition (degradation) of the Ang2 measures the level of Ang2 before and after treatment of the candidate compound with respect to the same sample, or by dividing the same sample into the treated group and the untreated group to measure the level of Ang2, the candidate compound It can be confirmed before and after treatment or by comparison with the level of Ang2 in the treated and untreated groups. Alternatively, at this time, it may be determined that Ang2 is inhibited when the candidate compound is treated or when the level of Ang2 in the treated group is decreased compared to before or after the treatment of the candidate compound. In addition, the activation of the Tie2 receptor is a candidate for the same sample for the same degree of phosphorylation of one or more of the phosphorylation degree of the Tie2 receptor and/or the protein (eg, Akt, eNOS, 42/44, etc.) involved in downstream signaling of the Tie2 receptor. It can be determined before and after the treatment of the compound, or by dividing the same sample into the treatment group and the untreated group of the candidate compound, and comparing and comparing the phosphorylation degree of the protein in the treatment group and the untreated group before and after the treatment with the candidate compound. At this time, it can be determined that the Tie2 receptor is activated after the treatment of the candidate compound or when the degree of phosphorylation of the treatment group is increased compared to the treatment with or without the treatment of the candidate compound. Alternatively, the activation of the Tie2 receptor can be determined by confirming whether the Tie2 receptor is internalized.

In another example, the screening method,

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

Confirmation of the formation of a complex to which the candidate compound, Ang2, and Tie2 receptor is bound, and/or activation of Ang2 inhibition and/or Tie2 receptor

It may include. Details of formation of the complex, inhibition of Ang2, and activation of the Tie2 receptor are as described above.

Confirmation of the complex formation, measurement of Ang2 level, measurement of the degree of phosphorylation of Tie2, measurement of the degree of phosphorylation of proteins involved in downstream signaling, and/or confirmation of intracellularization of Tie2 receptors are known in the art. It can be carried out through various methods, for example, it can be measured through conventional enzymatic reaction, fluorescence, luminescence and/or radiation detection, specifically, immunochromatography, immunohistochemistry, Enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay: LIA), Western blotting (Western blotting) may be measured by a method selected from the group consisting of, but is not limited thereto.

The candidate compound may be artificially synthesized or natural various compounds, polypeptides, oligopeptides, peptide structures or protein structures (eg, antibodies, peptibody, nanobody, etc.), polynucleotides, oligonucleotides, antisense-RNA, shRNA ( It may be one or more selected from the group consisting of short hairpin RNA (siRNA), small interference RNA (siRNA), aptamer, natural product extract, and the like.

The sample containing the Ang2 and Tie2 receptors is a cell or tissue cultured or artificially isolated from a living body, and essentially contains (expresses) the Ang2 and Tie2 receptors, or the Ang2 and/or Tie2 receptors are treated or Ang2 and/or It may be engineered to express the Tie2 receptor.

Previously, attempts have been made to inhibit the formation of angiogenesis by Ang2 by inhibiting the binding of Ang2 to its receptor, Tie2, but specifically binds to Ang2 and induces intracellular migration and decomposition while Ang2 and Tie2 receptors Antibodies that act to activate the Tie2 receptor while maintaining the binding ability to form the antibody-Ang2-Tie2 receptor complex are not known to date. In this situation, the present invention proposes a new method capable of inhibiting angiogenesis by Ang2 and reducing vascular permeability by suggesting an antibody that inhibits Ang2 while simultaneously activating the Tie2 receptor to promote downstream signal transduction. In addition, the antibody proposed in the present invention is expected to be applied to diagnosis and treatment of abnormal vascular formation-related diseases other than cancer and/or diseases caused by increased vascular permeability. The antibody can be used in combination therapy with chemical drugs and other anti-cancer drugs, and is expected to be used for antibody fragments, bi- or multi-specific antibodies, protein scaffolds, etc. by using Ang2 specific cognitive action.

Figure 1 is an Ang2-Tie2 competition ELISA results showing the degree of inhibiting the binding between the Tie2 receptor and Ang2 according to the anti Ang2 antibody treatment concentration according to an embodiment.
Figure 2a is a result of immunoblotting showing the change in the level of the Tie2 receptor and phosphorylated Tie2 receptor according to the concentration of the anti- Ang2 antibody according to an embodiment.
Figure 2b is a result of immunoblotting showing a change in the level of Tie2 receptor and phosphorylated Tie2 receptor by an anti-Ang2 antibody according to an embodiment compared to a control antibody (RG), Figure 2c shows the ratio of phosphorylated Tyr in Tie2 It is a graph.
3A is an immunoblotting result showing the degree of phosphorylation of a protein involved in downstream signaling of the Tie2 receptor during anti-Ang2 antibody treatment according to an embodiment.
Figure 3b is a result of immunoblotting showing the degree of phosphorylation of a protein (Akt) involved in Tie2 receptor phosphorylation and downstream signaling when anti-Ang2 antibody treatment according to an embodiment in an animal model, Figure 3c is the result of Figure 3b It is a graph showing the numerical value.
FIG. 4 is an ELISA result showing the degree of formation of a complex by antiang2 antibody and Angie2, and Tie2 according to an embodiment.
5 is a result of immunoblotting showing the degree of phosphorylation of a protein involved in downstream signaling of the Tie2 receptor and the Tie2 receptor of the monomeric anti-Ang2 antibody according to an embodiment.
6 is an immunocytostaining result showing intracellular internalization of Tie2 receptors having similar Ang1 and anti Ang2 antibodies according to an embodiment.
7 is a result showing the effect of the anti- Ang2 antibody according to an embodiment on the proliferation of vascular and lymphatic endothelial cells.
8 is a result showing the effect of the anti- Ang2 antibody on the movement of lymphatic endothelial cells according to an embodiment.
9 is a result showing the degree of cell permeability inhibition induced by TNF or LPS when treating Ang1 or Ang2 antibody according to an embodiment.
10 is a result showing the mRNA expression level of the inflammation-associated cell adhesion material (ICAM-1, E-selectin) induced by LPS stimulation when Ang1 or Ang2 antibody treatment according to an embodiment.
11 is a graph showing the intensity of fluorescence obtained from inflammatory cells when treated with anti- Ang2 antibody according to an embodiment in a relative fold, and shows the number of inflammatory cells attached to vascular endothelial cells.
12 is a result showing the efficacy of the anti- Ang2 antibody in one example in the Colo205 tumor model.
13 is a result showing the efficacy of increasing survival rate in a sepsis model induced by LPS injection of an anti-ang2 antibody according to an embodiment.
14 is a result showing the efficacy of augmentation of survival in a sepsis model induced by CLP of an anti Ang2 antibody according to an embodiment.
15 is a result of confirming the anti-vascular leakage efficacy of the anti-ang2 antibody according to an embodiment in the lung tissue of the sepsis model induced by CLP.
16 is a result of confirming the anti-inflammatory efficacy of the anti-ang2 antibody according to an embodiment in lung tissue of a sepsis model induced by CLP.

Hereinafter, the present invention will be described in more detail with reference to examples, but these are merely illustrative and are not intended to limit the scope of the present invention. It is apparent to those skilled in the art that the embodiments described below can be modified without departing from the essential gist of the invention.

Example 1. Section Ang2 Preparation of antibodies

The anti-Ang2 antibody was administered by adjuvant with human Ang2 protein (R&D systems; 623-AN-025/CF) to a 5-week-old BALB/c mouse to induce an immune response, followed by a known method described in a paper by Schwaber et al. Hybridomas producing individual antibodies were prepared (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, in order to obtain an immunized mouse required for the development of a hybridoma cell line, 100 ug (microgram) of human Ang2 protein per 100 animals per 5 5 week old BALB/c mice (Japan SLC, Inc.) ( R&D Systems) and the same amount of Freund's adjuvant were mixed and injected intraperitoneally into 4-6 week old BALB/c mice (Japan SLC, Inc.). After 2 weeks, the antigen (first half of the injected amount) was mixed with incomplete Freund's adjuvant and injected into the abdominal cavity of the mouse in the same manner as above. After a week, the final boosting was performed, and after 3 days, blood was collected from the tail of the mouse to obtain serum, and it was confirmed that the titer of the antibody that recognizes Ang2 is increased by ELISA by diluting it in PBS with 1/1000. As a result of the above, a mouse in which a sufficient amount of antibody was obtained was selected and cell fusion was performed.

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.), and the body was anesthetized after the immunized mouse was anesthetized. The spleen located on the left side of the was removed. The extracted spleen was ground with a mesh to separate cells, and mixed with a culture medium (DMEM, Hyclon) to make a spleen cell suspension. The suspension was centrifuged to recover the cell layer. The obtained spleen cells 1x10 ⁸ and myeloma cells (Sp2/0) 1x10 ⁷ were mixed and then centrifuged to precipitate the cells. The centrifuged precipitate was slowly dispersed, treated with 1 ml of 45% polyethylene glycol (PEG 1500) in culture medium (DMEM), maintained at 37° C. for 1 minute, and then 1 ml of culture medium (DMEM) was added. Did. Thereafter, 10 ml of culture medium (DMEM) was added for 1 minute, left in water at 37°C for 5 minutes, and then adjusted to 50 ml and centrifuged again. Cell sediment was resuspended in 1~2×10 ⁵ /ml in a separation medium (HAT medium), dispensed 0.1 ml in 96-well plates, and incubated in a 37°C carbon dioxide incubator to prepare a hybridoma cell population. Did.

Example 2. Anti- Ang2 Production of antibodies

2.1. term- Ang2 Production of antibodies clone selection and antibody purification

Hybridomas producing 95 anti-Ang2 monoclonal antibodies were selected from hybridomas differentiated from murine hybridomas based on the ability to bind to Ang2 using a typical ELISA format from the obtained antibody-producing hybridomas.

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

100 ng of human Ang2 protein was added to each microtiter plate and attached to the plate surface, and unreacted antigen was washed and removed. 50 µl of each of the hybridoma cells obtained in Example 1 was added to each well and reacted for 1 hour, followed by washing with phosphate buffer solution-Tween 20 (TBST) solution to remove unreacted culture. Goat anti-mouse IgG-horseradish peroxidase (goat anti-mouse IgG-HRP) was added thereto, reacted at room temperature for 1 hour, and then thoroughly washed with TBST solution. Subsequently, a substrate solution (OPD) of peroxidase is added to react, and the degree of the reaction is measured by absorbance at 450 nm with an ELISA Reader to secrete antibodies with high binding specificity only to human Ang2 protein. Bridoma cell lines were selected repeatedly. By limiting dilution of the hybridoma cell line obtained through repeated screening, 58 clones of hybridoma cell lines producing monoclonal antibodies were finally obtained. The produced hybridomas were deposited with the Korea Cell Line Bank located in Yeongeon-dong, Jongno-gu, Seoul, Korea on April 23, 2013, and received accession number KCLRF-BP-00295.

After each of the obtained hybridomas was cultured in DMEM (Dulbeco's Modified Eagle's Medium), the culture medium was collected and purified by protein G-affinity chromatography to anti-Angle2 monoclonal antibodies produced in each hybridoma.

First, the hybridoma cells cultured in 50 ml of culture medium (DMEM) medium containing 10% (v/v) FBS were centrifuged to wash cell sediment twice or more with 20 ml PBS to remove FBS. 50 ml of the culture medium (DMEM) medium was resuspended in the cell precipitate, and then cultured in a 37°C carbon dioxide incubator for 3 days. Then, the cells producing the antibody are removed by centrifugation, and the culture medium from which the antibodies are secreted is isolated and stored at 4°C or collected immediately to obtain an affinity column (Protein G agarose column; Pharmacia, USA) from 50 mL to 300 mL of the culture medium. After purification of the antibody using an attached AKTA purification device (GE Healthcare), the purified antibody was stored by substituting the supernatant with PBS using a protein aggregation filter (Amicon), and used in subsequent experiments. One of the antibodies obtained from each hybridoma 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 uses the principle that the refractive index of light passing through the chip changes depending on the state of the material coated on the sensor chip.If the antibody or antigen is spilled while the antigen or antibody is coated on the chip, the refractive index changes due to the binding between them Occurs, and Kd value is calculated from the measured value.

First, an anti-His antibody was immobilized to 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 flowed and captured at a level of 100 to 200 RU. Here, the antibody obtained in Example 2 is diluted 2 times sequentially from a concentration of 100 nM, and then bound (on), separated (off), and dissociated (regeneration, 10 mM) with the antigen captured on the sensor chip by flowing each method. NaOH solution) was used to measure antigen-antibody affinity. hAng1 was tested in the same manner as above, and the results are shown in Table 3 below.

Antibody name hAng2 (Kd) SAIT-ANG2-AB-m10D6 8.0 nM

2.2. term Ang2 Gene of antibody Cloning

Total RNA was obtained from the antibody-producing hybridoma (2x10 ⁶ cells) obtained in Example 2.1 using an RNeasy mini kit (Qiagen). Then, using this as a template, using the OneStep RT-PCR kit (Qiagen) and Mouse Ig-Primer Set (Novagen) and a thermocycler (GeneAmp PCR System 9700, Applied Biosystem), each hybrid was subjected to the following conditions: Only the variable region gene sequences of the heavy and light chains of the monoclonal antibody produced by chopping board were amplified: 5 minutes at 94°C; [30 min at 50°C, 15 min at 95°C], [1 min at 94°C, 1 min at 50°C, 2 min at 72°C] x 35 cycles; 6 minutes at 72°C; Cool to 4°C.

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

Antibody name Heavy chain CDR sequences 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)

Antibody name 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)

Antibody name Heavy chain variable region sequence SAIT-ANG2-AB-m10D6 DVQLQESGPGLVKPSQSLSLTCTVTGYSIT SDYAWN WIRQFPGNKLEWMG YINYSGNTDYNPSLKS RSSITRDTSKNQFFLQLNSVTTGDTATYYCAR GNFEGAMDY WGQGTSVTVSS (SEQ ID NO: 7) GATGTGCAGCTTCAGGAGTCGGGACCTGACCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTATGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGGCTACATAAACTACAGTGGTAACACTGACTACAACCCATCTCTCAAAAGTCGAAGCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGGGGACACAGCCACATATTACTGTGCAAGAGGTAACTTCGAAGGTGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 8)

Antibody name 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 above, the underlined bold letters are CDR1, CDR2, and CDR3 in order)

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

An 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 binding to Fc of 4 ug (microgram)/ml of human IgG1. Then, after washing the plate 5 times with PBS (Phosphate Buffer Saline) containing 0.05% (v/v) Tween-20, PBS containing 1% (v/v) BSA (Bovine serum albumin; Sigma) It was blocked at room temperature for 2 hours.

For the Ang2-Tie2 competition ELISA, each anti Ang2 antibody prepared in Example 2 above was mixed with 1% (v/v) BSA at 400 nM to 0.001 nM in various concentrations and 400 ng/ml FLAG-Tagging hAng- Together with 2, after putting the hTie-2/Fc fusion protein into each well coated, the plate was reacted for 2 hours at room temperature, and then the plate was washed 5 times with PBST. HRP-conjugated anti-FLAG antibody (Sigma) was diluted with PBS containing 1% (v/v) of BSA at a ratio of 1:5,000 (v/v), and then added to each well in an amount of 100 ul (microliter). After putting and reacting for 1 hour at room temperature, it was washed 5 times with PBST. Finally, 100 ul (microliter) TMB substrate (Cell Signaling) was added to each well of the plate to induce a color reaction for 3 minutes, and then 100 ul of Stop solution (Cell Signaling) was added to stop the reaction, OD450 values were measured on a plate reader (Molecular Devices).

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

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

EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYDMS WVRQAPGKGLEWVS LISPDSSSIYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYC AKDLISFWRGGFDY WGQGTLVTVSS

Light chain variable region (SEQ ID NO: 13)

QSVLTQPPSASGTPGQRVTISC SGSSSNIGSNYVN WYQQLPGTAPKLLIY ADSNRPS GVPDRFSGSKSGTSASLAISGLRSEDEADYYC GSWDYSLSG YVFGGGTKLTVLG

The degree (%) of inhibiting Ang-Tie2 binding is shown in FIG. 1. As shown in Figure 1, unlike the anti-Ang2 antibody 4H10, which inhibits Ang2-Tie2 binding, it was confirmed that the 10D6 antibody does not inhibit the binding between Ang2-Tie2 receptors.

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

Recombinant protein artificially mutated the receptor binding domain (RBD) of the Ang2 protein in the form of flagging in order to confirm the epitope (or specific binding site) of the anti- Ang2 antibody prepared in Example 2 ELISA was performed using.

Each well of a 96-well MaxiSorp ^™ flat-bottom plate (Nunc) was coated with 50 ul (microliter) of antibody at 1000 nM. Thereafter, 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. SAG, Q418, P419, N421, I434, D448, A449, P452, Y460, N467, K468, or F469 residues of Ang2 protein tagged with N-terminal FLAG sequence (DYKDDDDK, Sigma) with alanine mutation Each of the obtained mutant proteins (Mutant Ang2) was added to each well of the plate by 250 ng, and the plate was reacted 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 added to PBS containing 1% (v/v) BSA 1: After diluting at a 5,000 (v/v) ratio and reacting for 1 hour at room temperature, it was washed 5 times with PBS containing 0.1% (v/v) Tween-20.

Lastly, 50 ul of TMB substrate (Cell signaling) was added to each well of the plate to induce a color reaction for 3 minutes at room temperature. Then, 50 ul of Stop solution (Cell signaling) was added to stop the reaction. , OD450 values were measured on a plate reader (Molecular Devices). Each epitope for Ang2 antibodies was identified by comparing the binding capacity of Ang2 with Mutation to Ang2 without mutation. Table 8 shows the results of measuring the binding force (%) of Mutant Ang2 with respect to the binding force of the obtained native Ang2:

Example 5. 10 D6 By antibody Tie2 Receptor phosphorylation induction test

Ang2 acts to induce changes in vascular endothelial cells by inducing and activating phosphorylation of receptors by binding to Tie2 receptors expressed on vascular endothelial cells, so cell-based assays are used to analyze the effects of anti-Ang2 antibodies on Tie2 phosphorylation The following experiment was performed.

To this end, HUVEC (ATCC) cells (1×10 ⁶ cells) in a 100 mm culture dish were cultured at 37° C. using EGM-2 (Lonza) medium, and showed 80 to 90% confluency. (Lonza) and cultured at 37°C for 6 to 16 hours. After washing once with PBS, the culture medium was further cultured for 10 minutes by changing to a serum-free medium (Lonza) mixed with 1 nM of sodium orthovanadate (Sigma). After washing once with PBS, various concentrations (600-0.06 nM) of Ang2 antibody (10D6) were mixed with 40 nM of Ang2 protein (R&D systems) for 20 minutes, treated with the cultured cells, and further cultured for 10 minutes. . After washing the cells with PBS, after treating 400ul of lysis buffer (Roche), the cells are collected in a tube, lysed at 4℃ for 30 minutes, and centrifuged at 13,000 rpm for 15 minutes to nanodrop the supernatant. It was quantified using.

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

To confirm Tie2 phosphorylation, the membrane was blocked with PBST mixed with 3% (v/v) skim milk (Sigma) for 30 minutes, and then confirmed using HRP-conjugated anti-phospho tyrosine antibody (Millipore). Did. To confirm Tie2, the blot was reacted in a stripping buffer (Thermo) for 15 minutes, and then blocked again to confirm the Tie2 antibody (Santa Cruz).

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

In addition, referring to the method described above, the phosphorylation degree of Tie2 in the treatment of 60 nM with 10D6 antibody and Ang2 is a control antibody (Anti Ang2 antibody from Regeneron; control antibody with MOA inhibiting Ang2-Tie2 binding; expressed as RG antibody) The results of immunoblotting compared to are shown in Figure 2b. After antibody treatment, the band density of the blots of pTyr and Ty2 was measured using ImageJ software, and the pTyr/Tie2 ratio was calculated and the results are shown in FIG. 2C.

2B and 2C show the Tie2 phosphoric acid and the result in the group not treated with antibody and Ang2 in NC. 2B and 2C, when the 10D6 antibody was treated, the phosphorylation degree of the Tie2 receptor was increased by about 180% compared to the case where only Ang2 was treated without antibody treatment, whereas when treated with the control antibody, RG antibody, without antibody treatment Compared with only Ang2 treatment, it was reduced by about 67%, and the Tie2 phosphorylation effect of 10D6 antibody was about 8.6 times higher than that of the control antibody.

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

To confirm whether the 10D6 antibody induces the activation of downstream signaling of Tie2 receptor as well as Tie2, immunoblotting of the degree of phosphorylation of proteins participating in downstream signaling when treating Ang2 alone or Ang2 and 10D6 antibodies to HUVEC (ATCC) cells Were tested. To compare the degree of downstream signaling activation, 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) were treated together. The same test was performed for the same.

Specifically, HUVEC (ATCC) cells (1X105) in a 6 well culture dish were cultured at 37°C using EGM-2 (Lonza) medium, and when 80-90% confluency was shown, the serum-free medium (Lonza) was changed. Incubated at 37°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), left for 20 minutes, treated with the cultured cells, and further cultured for 30 minutes. For comparison, groups treated with Angn (R&D systems) 4nM, Ang2 (R&D systems) 40nM, and Ang2 (R&D systems) 40nM + anti Ang2 antibody (Regeneron) 60nM were prepared.

After washing the cells with PBS, after treating the lysis buffer (Roche), the cells were collected in a tube, lysed at 4°C for 30 minutes, and centrifuged at 13,000 rpm for 15 minutes to quantify the supernatant. Add sample buffer (Invitrogen) containing reducing agent to 25 ug of cell lysate, boil at 95°C for 5 minutes, run NuPAGE Novex 4-12% Bis-Tris gel (Invitrogen), and move to nitrocellulose membrane (Invitrogen). Ordered.

To confirm the phosphorylation of Akt, eNOS, and 42/44 involved in downstream signaling of the Tie2 receptor, the above blot was blocked for 30 minutes with PBST mixed with 3% (v/v) skim milk (Sigma), Anti-pAkt antibody, anti-p-eNOS antibody, and anti-p-42/44 antibody (abnormal cell signaling) were treated. After reacting the blot with stripping buffer (Thermo) for 15 minutes, blocking again to confirm 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. 3A. As shown in Figure 3a, compared to the group treated with Ang2 alone and Ang2 and RG antibodies, downstream signaling is strongly induced in the group treated with Ang1 alone and Ang2 and 10D6 antibodies, and Ang2 and 10D6 antibodies It can be confirmed that the effect in the group treated with is equal to or higher than the Ang1 alone treatment group.

In addition, the animal model ( in the degree of phosphorylation of the protein (Akt) involved in Tie2 receptor phosphorylation and downstream signaling during anti- Ang2 antibody treatment ( in in vivo ). Specifically, 5 mg/kg of antibody was injected into the C57BL6 mice of 7-8 weeks of age alone or with 20 ug of Ang2 through the tail vein, and after 1 hour, lung tissue was obtained from the mice. The degree of activity of Tie2 and Akt in tissue lysate obtained by homogenization lysis of the prepared lung tissue using lysis buffer (Roche) and FastPrep kit (MP biomedicals) was confirmed by the above method.

The obtained results are shown in FIGS. 3B and 3C. In FIGS. 3B and 3C, REGN or RG is the result of the control antibody ((Anti Ang2 antibody from Regeneron). As can be seen in FIGS. 3B and 3C, the 10D6 antibody showed significant Tie2 and compared to the control antibody even in the in vivo test. It showed the phosphorylation effect of protein (Akt) involved in its downstream signaling.

Example 7 . 10 D6 - Ang2 - Tie2 complex To confirm formation ELISA assay

Since it was confirmed that the 10D6 anti-Ang2 antibody did not inhibit Ang2-Tie2 binding and activate Tie2 signaling, ELISA was performed to confirm whether a complex was formed between the antibody and the Ang2 Tie2 receptor.

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

The obtained results are shown in FIG. 4. As shown in Figure 4, it was confirmed that the 10D6 antibody forms a complex by binding to Ang2 bound to Tie2.

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

The Fab fragment of the 10D6 antibody was purified and used in the experiment to confirm whether the activation of the Tie2 receptor was an effect by dimerization of the 10D6 antibody. 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 the digestion buffer, and when immobilized papain was treated for 2 hours at room temperature, it was separated into Fab and Fc. The Fc fragment was removed from the reaction solution using MabSelectSuRe column (GE Healthcare), and only pure Fab fragments were isolated and purified.

HUVEC (ATCC) cells (1×10 ⁶ cells) in a 100 mm culture dish were cultured at 37° C. using EGM-2 (Lonza) medium, and when 80 to 90% confluency was shown, the serum-free medium (Lonza) was changed to 6 to 16. Incubated at 37°C for an hour. After washing once with PBS, the culture medium was further cultured for 10 minutes by changing to a serum-free medium (Lonza) mixed with 1 nM of sodium orthovanadate (Sigma). 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. After washing the cells with PBS, after treating 400ul of lysis buffer (Roche), the cells are collected in a tube, lysed at 4℃ for 30 minutes, and centrifuged at 13,000 rpm for 15 minutes to nanodrop the supernatant. It was quantified using.

1 mg of Tie2 antibody (R&D system) was added to the 0.8 mg cell lysate and reacted overnight at 4°C, followed by immunoprecipitation by adding protein A bead (GE Healthcare). After the obtained reactant was centrifuged at 13,000 rpm for 15 minutes to obtain pellets, washed 2 to 3 times with lysis buffer (Roche), put sample buffer (Invitrogen) with reducing agent in it, boiled at 95° C. for 5 minutes, NuPAGE Novex 4-12% Bis-Tris gel (Invitrogen) was run and transferred to a nitrocellulose membrane (Invitrogen).

To confirm Tie2 phosphorylation, the membrane was blocked with PBST mixed with 3% (v/v) skim milk (Sigma) for 30 minutes, and then confirmed using HRP-conjugated anti-phospho tyrosine antibody (Millipore). Did. To confirm Tie2, the blot was reacted in a stripping buffer (Thermo) for 15 minutes, and then blocked again to confirm the Tie2 antibody (Santa Cruz).

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

The obtained results are shown in FIG. 5. As shown in FIG. 5, activation of Tie2 and Akt, 42/44 occurs when 10D6 antibody is added together with Ang2, whereas activation is not observed when treated with 10D6 Fab, Tie2 by dimerization of 10D6 antibody It was confirmed that the phosphorylation was strongly caused by the receptor clustering.

Example 9 .10 D6 By antibody Tie2 Receptor internalization Confirm

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

The results are shown in FIG. 6A (red: Tie2, blue: nucleus, green; 10D6) and 6b. As shown in FIG. 6A, it was found that the internalization of the Tie2 receptor starts from 30 minutes when the Ang2 and 10D6 antibodies are treated as in the case of Tie2 endocytosis during Ang1 treatment (dots in enlarged picture). And it was confirmed by the anti-EEA1 antibody (Cell Signaling) staining of Figure 6b that the internalized Tie2 and 10D6 antibodies were located in the early endosome, an organelle in cells.

Example 10 . 10 D6 Confirm the effect of increasing cell growth and mobility by antibodies

Measurement of proliferation of vascular and lymphatic endothelial cells was performed using BrdU assay kit (Roche) Cell. Put P3-P8 vascular endothelial cells (HUVEC, ATCC) or lymphatic endothelial cells (Lonza) into Collagen coated 96 well plates (BD Bioscience) at 3000-5000 cells/well, and then use EGM-2 medium (Lonza) 16-24 Incubated for hours. After preparing and mixing 2ug/ml Ang2 (R&D systems) and 10ug/ml 10D6 antibody in 2% EBM medium, put them into a 96-well plate washed with PBS and incubate for 3 days.

For efficacy comparison, anti-Ang2 antibody from MedImmune was used. For BrdU assay analysis, 10 μl of BrdU solution diluted 1;1,000 was added to each well to label the cells. After incubation for 1 hour, after removal of the culture, Fixation/Denaturation solution (Roche) was added to each well in 100 ul and waited for 30 minutes to remove. Peroxidase-conjugated Anti-BrdU antibody (Roche) was diluted to 1;100 using dilution buffer (Roche), put into each well, reacted for 1 hour, and then washed 4 times using washing buffer (Roche). . After adding 100ul of TMB substrate and reacting, absorbance was measured at 450nm using a Microplate reader (Perkin Elmer).

The obtained results are shown in FIG. 7. As shown in FIG. 7, it was confirmed that the growth of cells was increased in the group in which 10D6 antibody was added to Ang2 in both vascular endothelial cells and lymphatic endothelial cells, and in the case of MEDI antibodies, the cell growth by Ang2 was decreased. Confirmed.

In addition, the mobility of lymphatic endothelial cells was measured using GE Healthcare's xCelligence RTCA (Realtime cell analyzer). RTCA is a non-invasive cell monitoring system that can check cell changes by measuring impedance in real time. To perform the cell migration assay, CIM-plate16 (GE Healthcare) composed of lower chamber and upper chamber is used, and microelectrodes measuring impedance in the upper chamber are arranged. It can be attached to the electrode to check the degree of cell migration, which is indicated by the migration index. Lymphatic endothelial cells (P5-7; Lonza) grown in EGM-2 medium (Lonza) were cultured in EBM medium with 1% FBS for 6 hours. Lower chamber of CIM-plate16 2 ug/ml Ang2 and 10D6 antibody were added to EBM medium containing 2% FBS in each well, and then assembled with fibronectin (Sigma) coated upper chamber. For efficacy comparison, anti-Ang2 antibody from MedImmune was used. After adding 30 ul of serum-free EBM medium to the upper chamber, the equilibrium between the plate and the medium was placed in an incubator for 1 hour, the CIM-plate was mounted to a device station in the incubator, and the background value was measured. After the lymphatic endothelial cells resuspensioned with serum-free media were seeded at an amount of 60,000 cells/well, left to settle down for 15 minutes, mounted on a device, and the cell movement was measured in real time. The degree of cell migration was expressed as Slope (1/hr).

8 shows the obtained results. As shown in FIG. 8, the movement of lymphatic endothelial cells containing 10D6 antibody with Ang2 increased, but in the case of MEDI antibody, it was confirmed that the movement of cells by Ang2 was inhibited.

Example 11 . 10 D6 Confirmation of the effect of suppressing cell permeability by antibodies

In vitro vascular permeability assay kit (Millipore) was used to confirm the effect of inhibiting cell permeability of 10D6 antibody. 5x10 ⁴ HUVEC (ATCC) cells were added to the collagen coated transwell and cultured for 2 days to make a confluent monolayer. Angps 200 ng/ml or Ang1 (R&D systems) 200 ng/ml alone, or treated with 200 ng/ml of Ang2 and 1 ug/ml of 10D6 antibody, and after 30 minutes of pre-incubation, LPS (Lipopolysaccharide) to induce permeability of 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 movement of substances between HUVEC monolayers. The LPS treatment group was incubated for 6 hours, the TNF-a treatment group was treated with FITC-dextran (Millipore) in the upper chamber after 22 hours incubation, and incubated for 18 minutes. The fluorescence of FITC-Dextran moved to the lower chamber was measured at 485/535 nm (Ex /Em) setting using Envision 2104 multilabel Reader (Perkinelmer).

The obtained results are shown in FIG. 9. 9 is a graph showing the measured fluorescence intensity as a relative fold, and indicates vascular permeability induced by LPS or TNF-a. Through the above results, it can be confirmed that vascular permeability increased by LPS or TNF-a decreased to contorl level in the Ang1-only group and the group treated with Ang2 and 10D6 antibodies together.

Example 12 . 10 D6 Anti-inflammatory effect confirmation by antibody

In order to confirm the anti-inflammatory effect by the 10D6 antibody, HUVEC (ATCC) cells 4X10 ⁵ were treated with Ang2 alone or with Ang2 and 10D6 antibodies together, and Ang1 and Ang2 (R&D systems) and Regeneron's antibodies (RG antibodies) were used as controls. Groups treated together were included. 0.2 ug/ml Ang1, 0.1 ug/ml Ang2, or 0.1 ug/ml Ang2 and 0.5 ug/ml antibody were each treated to HUVEC cells, and after 30 minutes pre-incubation, LPS (Sigma) 100 to induce an inflammatory reaction. ng/ml was treated and incubated for 5 hours.

The extent of the inflammatory response was confirmed by measuring the mRNA expression level of the inflammatory attachments (ICAM-1, E-selectin). After washing the cells with PBS, RNA was extracted using an RNeasy Mini kit (Qiagen), and 2 ug of the RNA was synthesized into cDNA using the Transcriptor First Strand cDNA synthesis kit (Roche). The pPCR reaction was performed using RT2 SYBR ^TM Green Mastermix (Qiagen) and LightCycler ^TM 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 following procedure for all primers. Step1: 95°C, 10 min; Step2 (45 cycles): Step 2-1: 95°C, 15 sec; Step 2-2: 60° C., 1 min; Step3: 65° C., 15 sec; Step4: 95°C, continuous (every 20°C); Step6: 40°C, 10 sec.

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

The relative expression levels of the measured ICAM-1 and E-selectin are shown in FIG. 10. As shown in Figure 10, it can be seen that the expression of ICAM-1 and E-selectin induced by LPS is significantly inhibited in the group treated with Ang1 alone and the group treated with Ang2 and 10D6 antibody together.

On the other hand, when the expression of inflammatory endothelial substances (ICAM-1, E-selectin, etc.) of vascular endothelial cells increases, the association between inflammatory cells and vascular endothelial cells is promoted, leading to an inflammatory response. To confirm this, an experiment was conducted to measure the binding between HL-60 (ATCC) cells, which are inflammatory cells, and HUVEC.

HUVEC cells (4X10 ⁵ ) were cultured in 24-multiwell plates to treat 0.1 ug/ml of Ang2 alone or 0.1 ug/ml of Ang2 and 0.5 ug/ml of 10D6 antibody together, 0.2 ug/ml of Ang1, and 0.1 ug of Ang2 as a control. The group treated with /ml and 0.5 ug/ml of Regeneron antibody (RG antibody) was included. Angiopoietin and antibody were treated with HUVEC cells as described above, and after 30-minute pre-incubation, 1 ng/ml of TNF-a (eBioscience) was incubated for 2 hours and 30 minutes to induce an inflammatory reaction.

After washing the HUVEC cells with complete media (Lonza), 2.5 x 10 ⁵ HL-60 cells stained with Calcein-AM (BD bioscience) solution were added to each well and incubated for 1 hour. After washing three times with PBS to remove HL-60 cells not bound to HUVEC cells, lysis buffer (0.1% SDS, 50mM Tris.HCl, pH8.0) was treated. The obtained lysate was transferred to a 96 well plate, and the intensity of fluorescence derived from HL-60 cells was measured in an 485/535 nm (Ex /Em) setting using an Envision 2104 multilabel Reader (Perkin Elmer).

Fig. 11 shows the obtained results. The graph of FIG. 11 shows the measured fluorescence intensity as a relative fold, and is proportional to the number of HL-60 cells attached to HUVEC cells. Through the above results, it can be confirmed that the binding of HUVEC cells and HL-60 cells induced by TNF-a was significantly inhibited in the group treated with Ang1 alone and the group treated with Ang2 and 10D6 antibodies together.

Example 13. Section Ang -2 of antibodies Colo205 Tumor growth inhibitory effect

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

The Colo205 cell line was cultured using RPMI-1640 (Gibco) medium to which 10% FBS (Gibco) was added. After resuspension of 5X10 ⁵ Colo205 cell lines in 100 ul of serum-free medium, subcutaneously in ^4-5 week old BALB/c nude mice (Shanghai SLAC Laboratory Animal Co. Ltd.) anesthetized with 1~2% isoflurane. Was injected. When the tumor size reached 100-200 mm ³ , the size of the tumor was measured after intraperitoneal injection of anti-ang-2 antibody (10D6) twice a week at a concentration of 10 mg/kg.

Tumor size (V) was calculated using the following equation:

V=(length x width ² )/2

The obtained results are shown in Fig. 12. The x-axis in FIG. 12 refers to the day of antibody treatment by Days after grouping. As shown in Figure 12, it was confirmed that 10D6 antibody inhibits the growth of colon cancer.

Example 14. LPS Induced sepsis animal model 10 D6 Confirmation of the effect of improving survival rate by antibody

10D6 antibody was observed by injecting LPS (Lipopolysaccharide; Sigma) into the abdominal cavity of a mouse and increasing the survival rate by 10D6 antibody in an animal model inducing septicemia accompanied by an increase in blood Ang2 concentration and systemic inflammation and vascular leakage. We have confirmed its potential as a treatment for sepsis.

Adult C57BL/6 mice (Jackson Laboratory, 6-8 weeks, male) were injected intraperitoneally with 5 mg/kg of 10D6 antibody. For comparison, animals infused into the abdominal cavity with PBS in an amount of 5 mg/kg each as Regeneron antibody (RE GN antibody), Enbrel (TNF-a blocker; Amgen), Avastin (VEGF-A blocker; Genentech), and control A model was used. After 1 hour of injection, LPS (Sigma) was injected into the abdominal cavity in an amount of 15 mg/kg to induce sepsis, and survival rates for each group were recorded hourly for 5 days to confirm the prevention and/or treatment efficacy of sepsis by antibodies.

The obtained results are shown in FIG. 13. 13 is a graph showing the survival rate of mice in %. As can be seen in Figure 13, it can be seen that the group treated with 10D6 antibody had a significantly increased survival rate compared to all control groups including the control group. In particular, the survival rate of the control group was 13.3% based on 120 hours, and the survival rate of the control group was 40% or less, whereas the survival rate of the 10D6 antibody treatment group was about 60%. These results show excellent anti-septic treatment effect of anti-ang2 according to the present invention.

Example 15. CLP ( Cecal ligation puncture ) Sepsis in animal models 10 D6 Antibiotic improvement effect and anti-vascular leakage and anti-inflammatory effect confirmed by antibody

In order to confirm the efficacy of the 10D6 antibody in other sepsis animal models other than LPS, the 10D6 antibody affects survival rate, vascular leakage, and inflammatory response in the CLP model in which intestinal bacteria are ejected into the abdominal cavity by binding the appendix and punctured with a needle. I checked the impact.

For the experiment, adult C57BL/6 mice (Jackson Laboratory, 6-8 weeks, male) were used. For comparison of efficacy with 10D6 antibody 10mg/kg, Regeneron antibody (REGN antibody), COMP-Ang1 (Cartilage oligomeric matrix protein-angiopoietin 1; Tie2 receptor activator; Genexel) and 0.5% BSA as a control, respectively 10mg/kg It was injected into the abdominal cavity by volume. After 30 minutes of injection, the mice are anesthetized, 75% of the appendix is threaded and punctured with a 21-gauge neendle to induce sepsis, and the survival rate for each group is recorded hourly for 80 hours to prevent and/or treat sepsis caused by antibodies Efficacy was confirmed.

14 shows the obtained results. 14 is a graph showing the survival rate of mice in %. As can be seen in Figure 14, it can be seen that the group treated with 10D6 antibody had a significantly increased survival rate compared to all control groups including the control group.

In order to confirm the anti-vascular leakage and anti-inflammatory efficacy of the 10D6 antibody in the sepsis animal model, lung tissue of each group was obtained and analyzed using H&E staining (Hematoxilin and Eosin, DAKO) 16 hours after induction of sepsis in the animal model.

Fig. 15 shows the obtained results. As can be seen in FIG. 15, swelling of the lung tissue of the mice in which sepsis was induced was observed with edema of the alveoli due to vascular leakage and an increase in the number of inflammatory cells. Unlike the control group including the control group, in the group treated with the 10D6 antibody, it was confirmed that the lung tissue was maintained similar to the normal tissue.

In addition, immunostaining was performed as follows to measure the degree of inflammation in the lungs. The frozen section of lung tissue was blocked for 1 hour using a 0.3% TBST (TBS with 0.3% Triton X-100) buffer containing 5% goat serum (Jackson Immunoresearch). After adding anti-ICAM antibody (rabbit, BD bioscience) and DAPI (Molecular Probe) to the blocking buffer, it was added to the frozen section and reacted for 3 hours. After washing three times with TBS, FITC-conjugated anti-rabbit IgG antibody (Jackson Immunoresearch) was added to the blocking buffer and added to the frozen section for reaction for 2 hours. After washing with TBS three times, the coverslip (Marenfield) was covered with Fluorescence mounting medium (DAKO). The frozen section of the stained tumor was observed with an LSM5 (Zeiss) confocal microscope to obtain a fluorescence image.

The obtained results are shown in Fig. 16. As can be seen in FIG. 16, it was observed that the ICAM-1 (inflammatory attachment substance) expression of the lung tissue increased by the induction of sepsis decreased to the normal lung tissue level only in the group treated with the 10D6 antibody.

Korea Cell Line Research Foundation KCLRFBP00295 20130423

<110> SAMSUNG ELECTRONICS CO., LTD. <120> Composition for blocking vascular leakage containing an Anti-Ang2 antibody <130> DPP20133384KR <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> 118 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region of an anti-Ang2 antibody <400> 7 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Asn Tyr Ser Gly Asn Thr Asp Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ser Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Gly Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gly Asn Phe Glu Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <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 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 (15)

Specifically binds to angiopoietin-2 (Ang2), binds to the Tie2 receptor along with Ang2,
Includes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 (CDR-H1), a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 (CDR-H2), and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 (CDR-H3). A heavy chain complementarity determining region, or a heavy chain variable region comprising the heavy chain complementarity determining region; And
Includes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4 (CDR-L1), a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5 (CDR-L2), and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6 (CDR-L3). Light chain complementarity determining region, or light chain variable region comprising the light chain complementarity determining region
As a pharmaceutical composition for the prevention or treatment of diseases associated with vascular leakage comprising an anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient,
The disease associated with vascular leakage is in the group consisting of vascular leak syndrome, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), and diabetic vascular complications. The pharmaceutical composition selected. delete Specifically binds to angiopoietin-2 (Ang2), binds to the Tie2 receptor along with Ang2,
Includes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 (CDR-H1), a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 (CDR-H2), and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 (CDR-H3). A heavy chain complementarity determining region, or a heavy chain variable region comprising the heavy chain complementarity determining region; And
Includes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4 (CDR-L1), a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5 (CDR-L2), and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6 (CDR-L3). Light chain complementarity determining region, or light chain variable region comprising the light chain complementarity determining region
Containing, pharmaceutical composition for the prevention or treatment of diseases associated with vascular inflammation comprising an anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient. The pharmaceutical composition of claim 3, wherein the disease accompanying vascular inflammation is vascular infection. Specifically binds to angiopoietin-2 (Ang2), binds to the Tie2 receptor along with Ang2,
Includes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 (CDR-H1), a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 (CDR-H2), and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 (CDR-H3). A heavy chain complementarity determining region, or a heavy chain variable region comprising the heavy chain complementarity determining region; And
Includes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4 (CDR-L1), a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5 (CDR-L2), and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6 (CDR-L3). Light chain complementarity determining region, or light chain variable region comprising the light chain complementarity determining region
A pharmaceutical composition for the prevention or treatment of sepsis, comprising an anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient. The method according to any one of claims 1 and 3 to 5,
The anti-ang-2 antibody or antigen-binding fragment thereof increases activity by increasing phosphorylation of the Tie2 receptor,
Pharmaceutical composition. The method according to any one of claims 1 and 3 to 5,
The anti-ang-2 antibody or antigen-binding fragment thereof increases phosphorylation of at least one selected from the group consisting of Akt, eNOS, and 42/44,
Pharmaceutical composition. The method according to any one of claims 1 and 3 to 5,
The anti-ang-2 antibody or antigen-binding fragment thereof comprises a combination of Q418, P419, Q418 and P419 of human Ang2 (SEQ ID NO: 11), or the combination of Q418, P419, or Q418 and P419 in SEQ ID NO: 11 It specifically binds to 20 to 20 amino acid residues,
Pharmaceutical composition. delete delete The anti-ang-2 antibody or antigen-binding fragment thereof according to any one of claims 1 and 3 to 5,
Comprising 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: 9,
Pharmaceutical composition. The pharmaceutical composition according to any one of claims 1 and 3 to 5, wherein the anti- Ang2 antibody or antigen-binding fragment thereof is obtained from a hybridoma of accession number KCLRF-BP-00295. The pharmaceutical composition according to any one of claims 1 and 3 to 5, wherein the anti- Ang2 antibody is a mouse-derived antibody, a mouse-human chimeric antibody, a humanized antibody or a human antibody. The antigen-binding fragment according to any one of claims 1 and 3 to 5, wherein the antigen-binding fragment is scFv, (scFv)2, scFv-Fc, Fab, Fab' and F(ab') of the anti-Ang2 antibody. The pharmaceutical composition is selected from the group consisting of 2. The pharmaceutical composition according to any one of claims 1 and 3 to 5, further comprising Ang2.

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