KR20230097598A - Antibody specifically binding to angiopoietin-2, or its fragment - Google Patents

Abstract

The present invention relates to an anti-Ang2 antibody or antigen-binding fragment thereof which is an antibody specifically binding to angiopoietin 2 (Ang2) and inducing Tie2 activation or antigen-binding fragment thereof, and comprises: (a) a heavy chain complementarity determining region (CDR) including CDRH1 of an amino acid sequence of sequence number 18 or sequence number 26, CDRH2 of an amino acid sequence of sequence number 19 or sequence number 27, and CDRH3 of an amino acid sequence of sequence number 20 or sequence number 28; and (b) a light chain CDR including CDRL1 of an amino acid sequence of sequence number 21 or sequence number 29, CDRL2 of an amino acid sequence of sequence number 22 or sequence number 30, and CDRL3 of an amino acid sequence of sequence number 23 or sequence number 31. Accordingly, Ang2-induced neovascularization may be inhibited and vascular permeability may be reduced.

Description

Antibody specifically binding to angiopoietin-2, or its fragment}

The present invention relates to an anti-Ang2 antibody or fragment thereof that specifically binds to angiogenesis inducer, Angiopoietin-2 (Ang2), and binds to a Tie2 receptor together with Ang2.

The angiopoietin protein group is proteins that play an important role in the generation and maintenance of blood vessels, and there are four types of angiopoietin (Ang1, Ang2, Ang3, Ang4).

Angiopoietin-1 binds to the Tie2 receptor and plays an important role in vascular maturation, adhesion, migration and survival.

On the other hand, Angiopoietin-2 binds to the receptor Tie2 present in vascular endothelial cells, but acts as an antagonistic ligand, and is an agonist of Tie2, Angiopoietin-1 (Ang1). ) and Tie2 binding, thereby inhibiting signal transduction by Tie2. Due to this mechanism of action, in the state of overexpression or inflammation of VEGF, vascular endothelial cells are activated and vascular permeability is increased. At this time, Ang1 induces stabilization of vascular endothelial cells and reduces vascular permeability, whereas activated Increased Ang2 in vascular endothelial cells competes with Ang1 to inhibit stabilization of vascular endothelial cells by Ang1. Therefore, Ang2 inhibits Ang1-Tie2 binding and signaling through it, which maintains the stability of vascular endothelial cells when VEGF is present, and consequently promotes angiogenesis of blood vessels, resulting in increased angiogenesis, destabilization of blood vessels, and vascular permeability. cause an increase On the other hand, in addition to its role as an antagonist inducing inactivation of the Tie2 receptor, Ang2 has been reported to have properties as an agonist that induces the activity of the Tie2 receptor in some specific situations, including lymphatic vessel formation and maintenance. It is considered to have all the functions of and perform various functions depending on the situation.

Since the angiogenesis process is an essential factor for cancer growth, attempts have been made to prevent additional growth of cancer by inhibiting angiogenesis by inhibiting the function of Tie2-dependent Ang2 as described above. It is known to have a function of inhibiting binding and preventing its role as an antagonist. In addition to antibodies that interfere with the binding of Ang2 to Tie2, recombinant proteins or antibodies that directly bind to the Tie2 receptor to induce phosphorylation and activation have also been reported.

Recently, antibodies that phosphorylate and activate Tie2 through Tie2 clustering by binding to Tie2 by binding to Ang2 have also been reported. It presents possibilities.

[Prior Patent Literature]

Republic of Korea Patent Publication No. 10-2020-0144536

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

Another object of the present invention is to provide a pharmaceutical composition containing the anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the treatment of diseases associated with angiogenesis, increased vascular permeability, and/or decreased normal blood vessel formation, comprising the anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising the anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient.

Another object of the present invention is to provide a composition for diagnosing a disease related to Ang2 overexpression comprising the anti-Ang2 antibody or antigen-binding fragment thereof.

Another object of the present invention is to provide a nucleic acid encoding the antibody or antigen-binding fragment thereof, a vector and host cell containing the nucleic acid, and a method for preparing an anti-Ang2 antibody or antigen-binding fragment thereof using the same.

In order to achieve the above object, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to angiopoietin 2 (Ang2) and induces Tie2 activation,

(a) CDRH1 of the amino acid sequence of SEQ ID NO: 18, or SEQ ID NO: 26;

CDRH2 of the amino acid sequence of SEQ ID NO: 19, or SEQ ID NO: 27, and

a heavy chain complementarity determining region (CDR) comprising CDRH3 of the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 28; and

(b) CDRL1 of the amino acid sequence of SEQ ID NO: 21, or SEQ ID NO: 29;

CDRL2 of the amino acid sequence of SEQ ID NO: 22, or SEQ ID NO: 30, and

It provides an anti-Ang2 antibody or antigen-binding fragment thereof comprising a light chain CDR comprising CDRL3 of SEQ ID NO: 23 or SEQ ID NO: 31 amino acid sequence.

In one embodiment of the present invention, the antibody or antigen-binding fragment thereof

(a) a heavy chain complementarity determining region (CDR) comprising CDRH1 of the amino acid sequence of SEQ ID NO: 18, CDRH2 of the amino acid sequence of SEQ ID NO: 19, and CDRH3 of SEQ ID NO: 20; and

(b) a light chain CDR comprising CDRL1 of the amino acid sequence of SEQ ID NO: 21, CDRL2 of the amino acid sequence of SEQ ID NO: 22, and CDRL3 of the amino acid sequence of SEQ ID NO: 23;

In another embodiment of the present invention, the antibody or antigen-binding fragment thereof

(a) a heavy chain complementarity determining region (CDR) comprising CDRH1 of the amino acid sequence of SEQ ID NO: 26, CDRH2 of the amino acid sequence of SEQ ID NO: 27, and CDRH3 of SEQ ID NO: 28; and

(b) a light chain CDR comprising CDRL1 of the amino acid sequence of SEQ ID NO: 29, CDRL2 of the amino acid sequence of SEQ ID NO: 30, and CDRL3 of the amino acid sequence of SEQ ID NO: 31;

In another embodiment of the present invention, the antibody or antigen-binding fragment thereof is a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 24 and SEQ ID NO: 32, and the group consisting of SEQ ID NO: 25 and SEQ ID NO: 33 It is preferred, but not limited to, that it includes a selected light chain variable region.

In one embodiment of the present invention, the antibody or fragment thereof specifically binds to angiopoietin-2 and preferably binds to the Tie2 receptor together with Ang2, but is not limited thereto.

In one embodiment of the present invention, the antigen-binding fragment is preferably selected from the group consisting of scFv, (scFv)2, scFv-Fc, Fab, Fab' and F(ab')2, but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for preventing or treating diseases associated with Ang2 overexpression, angiogenesis, or increased vascular permeability, comprising the anti-Ang2 antibody or antigen-binding fragment thereof of the present invention as an active ingredient.

In one embodiment of the present invention,

The disease associated with Ang2 overexpression, angiogenesis, or increased vascular permeability is preferably cancer, cancer metastasis, inflammatory disease, infection, cardiovascular disease, kidney disease, hereditary hemorrhagic telangiectasia, asthma, or edema, but is not limited thereto. .

In addition, the present invention provides a pharmaceutical composition for preventing or treating a disease associated with reduced normal angiogenesis, comprising the Ang2 antibody or antigen-binding fragment thereof of the present invention as an active ingredient.

In one embodiment of the present invention, the disease associated with reduced normal angiogenesis is preferably myocardial infarction, angina pectoris, cerebral infarction, stroke, Buerger's disease, avascular necrosis, foot ulcer, or erectile dysfunction, but is not limited thereto.

The present invention will be described below

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.

The present invention specifically binds to Ang2 but does not inhibit the binding between Ang2 and Tie2 receptors, and an antibody forming a complex (antibody/Ang2/Tie2) with Ang2 and Tie2 receptors binds to Tie2 receptors together with Ang2 and binds to Tie2 receptors like Ang1. Provided is an antibody or antigen-binding fragment thereof having a dual function of inducing Tie2 downstream signaling by activating a receptor and inducing stabilization of vascular endothelial cells.

In addition, in addition to the effect of binding to the Tie2 receptor together with Ang2 and activating the Tie2 receptor like Ang1, it inhibits the binding of other proteins involved in cancer cell growth and/or cancer metastasis, such as integrin and Ang2, thereby enhancing cancer cell growth. An anti-Ang2 antibody having inhibitory and/or cancer metastasis inhibitory effects and capable of exhibiting such effects even in cells that do not express Tie2 and pharmaceutical uses thereof are provided.

In one embodiment of the present invention, the heavy chain complementarity determining region of the anti-Ang2 antibody described above, the light chain complementarity determining region, or a combination thereof; or a polypeptide molecule comprising a heavy chain variable region, a light chain variable region, or a combination thereof. The polypeptide molecule can function as a precursor or component of an antagonist to Ang2 as well as the construction of an antibody or antigen-binding fragment thereof. For example, the polypeptide molecule may serve as an Ang2 antigen binding site, and may be composed of a protein scaffold having a structure similar to an antibody (e.g., peptibody, nanobody, etc.), bispecific antibody, multispecific antibody, etc. components may be included.

The term “antagonist” is intended to include any molecule that partially or completely blocks, inhibits or neutralizes 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 a peptide and an Fc portion of an antibody is fused, and the peptide serves as an antigen binding site (heavy chain and / or light chain CDR or variable region) It refers to a protein having a skeleton and function similar to that of an antibody.

The term "nanobody", also called a single-domain antibody, refers to an antibody fragment comprising a single variable domain of an antibody in the form of a monomer, and, similar to a fully structured antibody, selectively binds to a specific antigen. has the characteristic of The molecular weight of nanobodies is usually about 12 kDa to about 15 kDa, which is very small compared to the typical molecular weight of a complete antibody (containing two heavy chains and two light chains) (about 150 kDa to about 160 kDa), in some cases Therefore, they are smaller than Fab fragments or scFv fragments.

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

In an embodiment, the polypeptide molecule comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 18 or 26, a polypeptide comprising the amino acid sequence of SEQ ID NO: 19 or 27, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 20 or 28 At least one selected from the group consisting of peptides;

At least one selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 21 or 29, a polypeptide comprising the amino acid sequence of SEQ ID NO: 22 or 30, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 23 or 31; or a combination thereof.

In embodiments, the polypeptide molecule may include the amino acid sequence of SEQ ID NO: 24 or 32, the amino acid sequence of SEQ ID NO: 25 or 33, or a combination thereof.

As described above, the bispecific antibody or multispecific antibody is an antibody that simultaneously recognizes and/or binds two or more antigens, including antigen binding sites for two or more different antigens. As such, 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 dimer 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 above polypeptide molecule as an Ang2 antigen binding site is provided.

In another example, one or more of the above-described polypeptide molecules or a repeat of the polypeptide molecule repeatedly linked by a linker (hereinafter referred to as 'first peptide') and a polypeptide having a structural function (hereinafter referred to as 'second peptide') ; For example, one or more (eg, 1 to 5, or 2 to 4) peptide complexes including the constant region of the heavy or light chain of an antibody (IgG, IgA, IgE, IgD, IgM, etc.), or the Fc fragment of an antibody ), and the one or more peptide complexes are linked in a second peptide (eg, Fc fragment) to provide a protein scaffold having a multimeric structure.

Antibodies in the present invention include all of animal-derived antibodies, chimeric antibodies, humanized antibodies, and human antibodies. Animal-derived antibodies produced by immunizing an animal to be immunized with a desired antigen may generally cause immune rejection when administered to humans for therapeutic purposes, and chimeric antibodies have been developed to suppress such immune rejection. A chimeric antibody is one in which the constant region of an animal-derived antibody, which causes an anti-isotype reaction, is substituted with the constant region of a human antibody using a genetic engineering method. Compared to animal-derived antibodies, chimeric antibodies have improved significantly in anti-isotype response, but animal-derived amino acids are still present in the variable region, resulting in side effects for potential anti-idiotypic responses. are doing What was developed to improve these side effects is a humanized antibody. It is produced by grafting complementarity determining regions (CDRs), which play an important role in antigen binding, in the variable region of a chimeric antibody to a human antibody framework.

The most important thing in the CDR grafting technology for producing humanized antibodies is to select an optimized human antibody that can best accept the CDR region of an animal-derived antibody. structure analysis, molecular modeling techniques, etc. are utilized. However, even when the CDR region of an animal-derived antibody is grafted onto an optimized human antibody backbone, there are cases in which amino acids that affect antigen binding are present while being located in the backbone of an animal-derived antibody, so antigen-binding ability is not preserved in many cases. Since it exists, it can be said that the application of additional antibody engineering techniques for restoring 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, "antibody" refers to a substance produced by stimulation of an antigen in the immune system, and the type is not particularly limited. Antibodies have recently been widely used for the treatment of diseases. Antibodies are advantageous for mass expression and production because they are very stable in vitro as well as in vivo and have a long half-life. In addition, since antibodies essentially have a dimer structure, 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 to the heavy chain by a disulfide bond. The antibody constant region 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, subclasses It has 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 is of the kappa (κ) and lambda (λ) type.

The term "heavy chain" includes a variable region domain VH comprising an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen and a hinge with three constant region domains CH1, CH2 and CH3. It is interpreted as meaning including both full-length heavy chains and fragments thereof. In addition, the term "light chain" refers to a full-length light chain comprising a variable region domain VL and a constant region domain CL comprising an amino acid sequence having sufficient variable region sequence to impart specificity to an antigen, and fragments thereof. interpreted as meaning

The term "complementarity determining region (CDR)" refers to the amino acid sequence of the hypervariable region of the heavy and light chains of immunoglobulin. Heavy and light chains may each contain three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide key contact residues for antibody binding to an antigen or epitope. On the other hand, in the present specification, the term "specifically binding" or "specifically recognizing" has the same meaning as commonly known to those skilled in the art, and an immunological reaction by specifically interacting with an antigen and an antibody. means that

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

The term "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure, and refers to a portion of a polypeptide including a portion capable of binding to an antigen. 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 light and heavy chain variable regions, a light chain constant region, and a first constant region (CH1) 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 CH1 domain. An F(ab')2 antibody is produced by forming a disulfide bond between cysteine residues in the hinge region of Fab'. Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region, and a recombination technique for generating an Fv fragment is widely known in the art. In two-chain Fv, the heavy chain variable region and the light chain variable region are connected by a non-covalent bond, and in single-chain Fv, the heavy chain variable region and the short chain variable region are generally shared through a peptide linker. They are linked by bonds or directly linked at the C-terminus, so that they can form a dimer-like structure like double-chain Fv. The linker may be a peptide linker consisting of 1 to 100 or 2 to 50 amino acids, and appropriate sequences are known in the art. The antigen-binding fragment can be obtained using a proteolytic enzyme (eg, Fab can be obtained by restriction digestion of whole antibody with papain, and F(ab') ₂ fragment can be obtained by digestion with pepsin), It can be produced through genetic recombination technology.

The term "hinge region" is a region included in the heavy chain of an antibody, which is present between the CH1 and CH2 regions, and refers to a region that functions to provide flexibility of the antigen binding site in the 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 an 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 than the human IgG1 hinge, and the disulfide bond between the two heavy chains ( disulfide bonds are reduced from 3 to 2, resulting in different effects on the rigidity of the hinge. Thus, modification of the hinge region can increase the antigen binding efficiency of a humanized antibody. Methods for deleting, adding or substituting amino acids to modify the amino acid sequence of the hinge region are well known to those skilled in the art.

The portion of the anti-Ang2 antibody except for the variable region may be a constant region derived from a human antibody, and specifically, may be a constant region derived from IgA, IgE, or IgG, such as IgG1, IgG2, IgG 3, or IgG4. .

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

Meanwhile, individual monoclonal antibodies may be screened based on their binding ability to Ang2 using a typical enzyme-linked immunosorbent assay (ELISA) format. Inhibitory activity can be assayed through functional assays such as competitive ELISA for assaying molecular interactions with the conjugates or functional assays such as cell-based assays. Then, each affinity (Kd values) for Ang2 is assayed for the monoclonal antibody members selected based on the strong inhibitory activity.

The final selected antibodies may be prepared and used as humanized antibodies as well as antibodies in which the remaining parts except for the antigen-binding portion are made into human immunoglobulin antibodies. Methods for preparing 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 embodiment provides a pharmaceutical composition for inhibiting angiogenesis comprising the anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient. Another embodiment 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 of angiogenesis inhibition. The angiogenesis inhibition method may further include identifying a patient in need of angiogenesis inhibition prior to the administering.

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

Another embodiment provides a pharmaceutical composition for preventing and/or treating diseases associated with Ang2 overexpression, angiogenesis, and/or increased vascular permeability, comprising the anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient. Another example is to administer 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 Ang2 overexpression, angiogenesis and/or increased vascular permeability. Provided are methods for preventing and/or treating diseases associated with Ang2 overexpression, angiogenesis and/or increased vascular permeability. The prevention and/or treatment method may further include identifying a patient in need of prevention and/or treatment of a disease associated with Ang2 overexpression, angiogenesis, and/or increased vascular permeability prior to the administering step. there is.

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

Another embodiment provides a pharmaceutical composition for preventing and/or treating diseases associated with reduced normal angiogenesis, comprising the anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient. Another example is a treatment of a disease associated with reduced normal angiogenesis 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 a disease associated with reduced normal angiogenesis. Prophylactic and/or therapeutic methods are provided. The prevention and/or treatment method may further include identifying a patient in need of prevention and/or treatment of a disease associated with reduced normal angiogenesis prior to the administering step.

Another embodiment provides a composition for diagnosing a disease associated with angiogenesis and/or increased vascular permeability and/or decreased normal blood vessel formation, comprising the antibody or antigen-binding fragment thereof.

The pharmaceutical composition may further include a pharmaceutically acceptable carrier, which is commonly used in drug formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, and 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 at least one selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, which are commonly used in the preparation of pharmaceutical compositions.

A pharmaceutically effective amount of the pharmaceutical composition or 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, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc. may be administered. When administered orally, since the protein or peptide is digested, the oral composition may be formulated to coat the active agent or protect it from degradation in the stomach. In addition, the composition may be administered by any device capable of transporting an active substance to a target cell.

The content of the anti-Ang2 antibody or antigen-binding fragment thereof in the pharmaceutical composition may vary depending on the formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, administration interval, administration route, excretion rate and response sensitivity. It can be prescribed in various ways by factors. For example, the daily dose of the anti-Ang2 antibody or antigen-binding fragment thereof may be in the range of 0.001 to 1000 mg/kg, specifically 0.01 to 100 mg/kg, and more specifically 0.1 to 50 mg/kg, but is not limited thereto no. The daily dose may be formulated as one formulation in unit dose form, formulated in appropriate portions, or prepared by placing it in a multi-dose container. The "pharmaceutically effective amount" may refer to the amount or dosage of an active ingredient capable of exhibiting a desired pharmacological effect, and may include formulation method, administration method, patient's age, weight, sex, medical condition, food, and administration time. , it can be variously determined by factors such as administration interval, route of administration, rate of excretion, and response sensitivity.

The pharmaceutical composition may be formulated in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or in the form of extracts, powders, powders, granules, tablets or capsules. Additional topics may be included.

In particular, since the pharmaceutical composition containing the anti-Ang2 antibody or antigen-binding fragment thereof includes the antibody or antigen-binding fragment, it can be formulated as an immunoliposome. Liposomes containing antibodies can be prepared according to methods well known in the art. The immune liposome is a lipid composition containing phosphatidylcholine, cholesterol, and polyethylene glycol-derivatized phosphatidyl ethanolamine and can be prepared by reverse phase evaporation. For example, a Fab' fragment of an antibody can be conjugated to a liposome via a disulfide-exchange 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 the anti-Ang2 antibody, and through this, it is possible to determine whether Ang2 is overexpressed. Accordingly, another embodiment of the present invention provides a composition for detecting Ang2 and a composition for diagnosing diseases associated with overexpression of Ang2, including the anti-Ang2 antibody or antigen-binding fragment thereof.

The present invention proposes an antibody that inhibits Ang2 and simultaneously activates the Tie2 receptor to promote downstream signal transduction, thereby providing a new method for inhibiting Ang2-induced angiogenesis and reducing vascular permeability. In addition, the antibody proposed in the present invention is expected to be applicable to the diagnosis and treatment of abnormal angiogenesis-related diseases other than cancer and/or diseases caused by increased vascular permeability. The antibody can be used for combination therapy with chemical drugs and other anticancer drugs, and is expected to be used for antibody fragments, bi- or multi-specific antibodies, protein scaffolds, etc. by using Ang2-specific recognition.

Figure 1 is a western blot showing that anti-Ang2 antibody phosphorylate AKT and ERK 42/44 in HUVEC cells together with Ang2.
2 is an ELISA result showing that the anti-Ang2 antibody induces AKT phosphorylation in HUVEC cells together with Ang2 in an antibody concentration-dependent manner.
Figure 3 analyzed the binding affinity of the anti-Ang2 antibody to human Ang2 and mouse Ang2 through ELISA.
4 is an ELISA result showing that the anti-Ang2 antibody binds to Ang2 and Tie2 to form a complex.
5 is an ELISA result showing that the anti-Ang2 antibody 2C8 does not interfere with the binding between Ang2 and Tie2.

Hereinafter, the present invention will be described in more detail with examples, but this is only illustrative and is not intended to limit the scope of the present invention. It is apparent to those skilled in the art that the embodiments described below may be modified within a range that does not deviate from the essential gist of the invention.

Example 1: Preparation and screening of anti-Ang2 antibodies

An antibody that specifically binds to human Ang2 (SEQ ID NO: 1) as an antigen was ordered from Abclon (Korea), and the antibody production of the present invention was performed by Kohler and Milstein, Eur. J. Immunol. 6, 511 (1976).

In summary, human Ang2 as an antigen was mixed with an adjuvant (Sigma) and injected twice into mice, and then antibody production was confirmed by ELISA. After two rounds of immunization, the antibody titer (1:5000) increased, so the spleen was removed from the immunized mice, and B lymphocytes were isolated and fused with sp2/0 cells. The fused cells were cultured in a medium (HAT mediaum) supplemented with hypoxantin, aminopterine, and thymidine, and cells in which B lymphocytes and sp2/0 cells were fused (hybridoma) were selectively selected. Monoclonal cells producing antigen-reactive antibodies were prepared by repeating the process of separating positive and negative cells from the obtained hybridoma cells using the serial dilution method (cloning).

The antibody-producing hybridoma cells obtained above were cultured in DMEM (Dulbeco's Modified Eagle's Mediaum) containing 10% (v/v) FBS at 37°C and 5% CO ₂ conditions, followed by centrifugation to produce antibody cells. was removed, and the antibody-secreted culture medium was separated, and the antibody was pure purified using an affinity column (Protein A/G agarose column, Protein A/G (GenDEPOT)).

Example 2: Induction of activation of Tie2 signaling by anti-Ang2 antibody Western blot

Ang2 acts as a weak agonist or antagonist by binding to the Tie2 receptor expressed on vascular endothelial cells. Since the anti-Ang2 antibody developed in the present invention binds to Ang2 and forms a complex with Ang2-Tie2, thereby activating the Tie2 receptor and promoting downstream signaling, anti-Ang2 antibody downstream of Tie2 signaling using a cell-based assay ERK and AKT phosphorylation experiments were performed to analyze the effect on . In order to compare the degree of activation of Tie2 lower signaling, the same test was performed on the group treated with Ang2 (Sino Biological) and other Ang2 control antibodies (Korean Patent Publication No. 10-2015-0136031).

Specifically, after culturing HUVEC (Lonza) cells (2X10 ⁵ pieces) at 37 ° C using EGM-2 (Lonza) medium, and showing 80 to 90% confluency, change to 0.5% FBS Basal media (Lonza) for 16 to 10 days. Incubated at 37°C for 24 hours. After mixing 60 nM of anti-Ang2 antibody with 40 nM of Ang2 protein (Sino Biological) and incubating for 30 minutes, the cultured cells were treated and further cultured for 10 minutes. For comparison, groups treated with Ang2 40 nM and Ang2 40 nM + anti-Ang2 control antibody 60 nM, respectively, were prepared. After washing the cells with PBS, lysis buffer (Ripa buffer, 0.15M Sodium chloride, 1% Triton X-100, 1% Sodium deoxycholate, 0.1% SDS, 50mM Tris-HCl, pH 7.5, and 2 mM EDTA), centrifuged at 13,000 rpm for 15 minutes, and the supernatant was recovered to obtain a cell lysate.

35 ug of the cell lysate was added with a sample buffer (Biosesang) mixed with a reducing agent, boiled at 95 ° C for 5 minutes, electrophoresed on a 10% Tris-Glycine gel, and transferred to a nitrocellulose membrane (GVS). In order to confirm the phosphorylation of Akt and ERK 42/44 involved in downstream signaling of Tie2 receptor, the above blot was blocked with PBST mixed with 5% (v/v) skim milk (Seoul Milk) for 1 hour. , anti-phosphorylated Akt antibody (Cell signaling), anti-AKT antibody (Cell signaling), anti-phosphorylated ERK 42/44 antibody (Cell signaling), and anti-ERK 42/44 antibody (Santa cruz). The obtained results are shown in FIG. 1 .

As shown in Figure 1, the 1A9, 1D3, 2C8, and 1H10 clones showed AKT and ERK activation signals compared to the negative control group.

Example 3: Induction of activation of Tie2 lower signaling by anti-Ang2 antibody ELISA

In order to quantitatively analyze the effect of the anti-Ang2 antibody on Tie2 downstream signaling, the level of AKT phosphorylation was measured by ELISA.

After culturing HUVEC (Lonza) cells (2X10 ⁵ ) at 37°C using EGM-2 (Lonza) medium, and showing 80-90% confluency, change to 0.5% FBS Basal media (Lonza) for 16 to 24 hours. Incubated at 37°C. After mixing 60 nM of anti-Ang2 antibody with 40 nM of Ang2 protein (Sino Biological) and incubating for 30 minutes, the cultured cells were treated and further cultured for 10 minutes. For comparison, groups treated with 40 nM of Ang2 and 60 nM of Ang2 40 nM + anti-Ang2 control antibody (h10D6-OPTI-67, US 10934350), respectively, were prepared. After washing the cells with PBS, treating them with lysis buffer (Biosesang), centrifuging at 13,000 rpm for 15 minutes, and recovering the supernatant to obtain a cell lysate.

For ELISA, 50 μL of PathScan®Phospho-Akt1 (Ser473) Sandwich ELISA Antibody Pair (Cell Signaling) capture antibody diluted 1:100 was added to a 96 well ps half area plate (Greiner cio-one) and coated. Then, after washing the plate 4 times with PBST (Phosphate Buffer Saline containing 0.05% (v/v) Tween-20), the plate was washed 2 times at 37°C with PBST containing 1% (v/v) skim milk. blocked for an hour. After that, the plate was washed 4 times with PBST containing 0.05% Tween-20, and then the cell lysate was added and the phosphorylated AKT was bound to the capture antibody for 2 hours at 37 ° C. After washing the plate 4 times with PBST, the secondary antibody diluted 1:1000 in 1% skim milk was bound at 37°C for 1 hour, and washed 4 times with PBST. Finally, 50 μL of TMB substrate (Kementec) was added to the plate to induce a color reaction for 10 minutes, then the reaction was stopped by adding 50 μL of Stop solution, and the OD450 value was measured on a plate reader (BioTek) . The obtained results are shown in FIG. 2 .

As shown in FIG. 2, it was confirmed that the monoclonal antibodies 1A9, 1D3, 2C8, and 1H10 of the present invention showed significantly stronger Tie2 lower signal induction compared to previously known control antibodies.

Example 4: Gene cloning of mouse anti-Ang2 antibody

RNA was isolated from the hybridoma obtained in Example 1 using the AccuPrep Universal RNA extraction kit (Bioneer). And using this as a template, cDNA synthesis and cloning were performed according to a previously known method (Meyer L et al., “A simplified workflow for monoclonal antibody sequencing” 2019, PLoS ONE). For cDNA synthesis, the SuperiorScript III cDNA Synthesis kit (Enzynomics) was used, and the synthesized cDNA was amplified by PCR and cloned into a vector using the TOPcloner Blunt core kit (Enzynomics). Base sequences encoding the regions and light chain variable regions were obtained (Tables 1 to 8).

Domain CDR CDR sequences sequence number heavy chain CDRH1-Kabat DYWIG SEQ ID NO: 2 CDRH2-Kabat DIYPGGGYTNCNEKFKG SEQ ID NO: 3 CDRH3-Kabat SDYRNDEGFAD SEQ ID NO: 4 light chain CDRL1-Kabat SASSSVSSSYLH SEQ ID NO: 5 CDRL2-Kabat RTSNLAS SEQ ID NO: 6 CDRL3-Kabat QQWSGYPYT SEQ ID NO: 7

Table 1 shows the CDR sequence of the mouse anti-Ang2 antibody 1A9.

Domain Variable region sequence sequence number heavy chain QVQLQQSGAELVRPGTSVKMSCKAAGYTFTDYWIGWVKQRPGHGLEWIGDIYPGGGYTNCNEKFKGKA
TLTADTSSSTAYMQLSSLTSEDSAIYYCSRSDYRNDEGFADWGQGTLVTVSAAK SEQ ID NO: 8 light chain ENVLTQSPAIMTASLGQKVTMTCSASSSVSSSYLHWYQQKSGASPKPLIHRTSNLASGVPARFSGSGSGT
SYSLTISSVEAEDDATYYCQQWSGYPYTFGGGTKLEIKRADAAPTVS SEQ ID NO: 9

Table 2 shows the sequence of the variable region of the mouse anti-Ang2 antibody 1A9.

Domain CDR CDR sequences sequence number heavy chain CDRH1-Kabat NYWIG SEQ ID NO: 10 CDRH2-Kabat DIYPGGGYTNYNEKFKG SEQ ID NO: 11 CDRH3-Kabat SDYRDDEGFAY SEQ ID NO: 12 light chain CDRL1-Kabat SASSSVSSSYLH SEQ ID NO: 13 CDRL2-Kabat RTSNLAS SEQ ID NO: 14 CDRL3-Kabat QQWSGYPYT SEQ ID NO: 15

Table 3 shows the CDR sequences of the mouse anti-Ang2 antibody 2C8.

Domain Variable region sequence sequence number heavy chain QVHLQQSGAELVRPGTSVKMSCKAAGYTFTNYWIGWVKQRPGHGLEWIGDIYPGGGYTNYNEKFKGKA
TLTADTSSSTAYMQLSSLTSEDSAIYYCSRSDYRDDEGFAYWGQGTLVTVSAAKTTPPKLYPLA SEQ ID NO: 16 light chain ENVLTQSPAIMAASLGQKVTMTCSASSSVSSSYLHWYQQKSGASPKPLIHRTSNLASGVPARFSGSGSGS
GTSYSLTISTVEAEDAATYYCQQWSGYPYTFGGGTKLEIKRADAAPTVSAAASLS SEQ ID NO: 17

Table 4 shows the sequence of the variable region of the mouse anti-Ang2 antibody 2C8.

Domain CDR CDR sequences sequence number heavy chain CDRH1-Kabat SDYGWN SEQ ID NO: 18 CDRH2-Kabat YISYSGTTSYNPSLKS SEQ ID NO: 19 CDRH3-Kabat SEGTGFYAMDY SEQ ID NO: 20 light chain CDRL1-Kabat KASQSVSNDVA SEQ ID NO: 21 CDRL2-Kabat YASNRYT SEQ ID NO: 22 CDRL3-Kabat QQDYSSPT SEQ ID NO: 23

Table 5 shows the CDR sequences of the mouse anti-Ang2 antibody 1D3.

Domain Variable region sequence sequence number heavy chain DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYGWNWIRQFPGNKLEWMGYISYSGTTSYN
PSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARSEGTGFYAMDYWGQGTSVTVSSA
KTTPPKLY SEQ ID NO: 24 light chain SIVMTQTPKFLLVSAGDRVTIICKASQSVSNDVAWYQQKPGQSPKLLIYYASNRYTGVPDRFT
GSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPTFGGGTKLEIKRA SEQ ID NO: 25

Table 6 shows variable region sequences of mouse anti-Ang2 antibody 1D3

Domain CDR CDR sequences sequence number heavy chain CDRH1-Kabat NYWIG SEQ ID NO: 26 CDRH2-Kabat IYPGGGYTNYNEKFK SEQ ID NO: 27 CDRH3-Kabat SDYRFDEGFAY SEQ ID NO: 28 light chain CDRL1-Kabat SASSSVSSSYLH SEQ ID NO: 29 CDRL2-Kabat RTSNLAS SEQ ID NO: 30 CDRL3-Kabat QQWSGYPYT SEQ ID NO: 31

Table 7 shows the CDR sequences of the mouse anti-Ang2 antibody 1H10.

Domain Variable region sequence sequence number heavy chain QVQLQQSGAELVRPGTSVKMSCKATGYTFTNYWIGWVKQRPGHGLEWIGDIY
PGGGYTNYNEKFKGKATLTADTSSSTACMQLSSLTSEDSAIYYCARSDYRFDEG
FAYWGQGTLVTIS SEQ ID NO: 32 light chain ENVLTQSPAIMAASLGQKVTMTCSASSSVSSSYLHWYQQKSGASPKPLIHRT
SNLASGVPARFSGSGSGTSYSLTISSVEAEDDATYYCQQWSGYPYTF
GGGTKLEIKRA SEQ ID NO: 33

Table 8 shows variable region sequences of mouse anti-Ang2 antibody 1H10.

Example 5: ELISA of human Ang2 or mouse Ang2 binding of anti-Ang2 antibodies

ELISA was performed to confirm the binding ability of the anti-Ang2 antibody to human Ang2 or mouse Ang2. A 96-well MaxiSorp™ flat-bottom plate was coated with 1 ug/mL of human Ang2 (Sino Biological) or mouse Ang2 (Acrobiosystemsl). Then, after washing the plate 5 times with PBST (Phosphate Buffer Saline containing 0.05% (v/v) Tween-20), the plate was washed with PBST containing 1% (v/v) skim milk for 2 hours at room temperature. Blocked. Thereafter, the plate was washed 5 times with PBST containing 0.05% Tween-20, and then anti-Ang2 was added at 10 different concentrations, starting from 300 nM in a 3-fold serial dilution, and allowed to bind at room temperature for 2 hours. After washing the plate 5 times with PBST of 0.05% Tween-20, Goat anti-Mouse IgG/HRP (Solarbio) diluted 1:3000 in 1% skim milk was combined for 1 hour and washed 6 times with PBST. . Finally, 100 μL (microliter) of TMB substrate (Kementec) was added to the plate to induce a color reaction for 10 minutes. Then, the reaction was stopped by adding 100 μL of Stop solution (2 MH ₂ SO ₄ ), and the OD450 Values were measured on a plate reader (BioTek). The obtained results are shown in FIG. 3 .

Example 6: ELISA for confirming formation of antibody-Ang2-Tie2 complex of anti-Ang2 antibody

ELISA was performed to determine whether a complex was formed between the anti-Ang2 antibody and the Ang2 Tie2 receptor. A 96-well MaxiSorp™ flat-bottom plate was coated with 2 ug/ml Tie2 ECD (Sino Biological). Then, after washing the plate 5 times with PBST (Phosphate Buffer Saline containing 0.05% (v/v) Tween-20), the plate was washed with PBST containing 1% (v/v) skim milk for 2 hours at room temperature. Blocked. Then, after washing the plate 5 times with PBST containing 0.05% Tween-20, anti-Ang2 antibody and Ang2 complex prepared in advance were added to bind to Tie2 at room temperature for 2 hours. Anti-Ang2 antibody and Ang2 complex were prepared by adding anti-Ang2 antibody to 1% skim milk at a concentration of 600 nM, then serially diluting 3-fold using 1% skim milk to prepare 10 concentrations, and Ang2 to 1% skim milk. It was prepared by mixing 1 ug/mL diluted sample and 1:1. After washing the plate 5 times with PBST of 0.05% Tween-20, Goat anti-Mouse IgG/HRP (Solarbio) diluted 1:3000 in 1% skim milk was combined for 1 hour and washed 6 times with PBST. . Finally, 100 μL (microliter) of TMB substrate (Kementec) was added to the plate to induce a color reaction for 10 minutes. Then, the reaction was stopped by adding 50 μL of Stop solution (2 MH ₂ SO ₄ ), and the OD450 Values were measured on a plate reader (BioTek). The obtained results are shown in FIG. 4 .

Example 7: Competition ELISA of anti-Ang2 antibodies for Ang2-Tie2 binding

Ang2-Tie2 binding competition ELISA was performed using an anti-Ang2 antibody. More specifically, 96-well MaxiSorp™ flat-bottom plates were coated with 4 ug/ml Tie2 ECD (Sino Biological). Then, after washing the plate 5 times with PBST (Phosphate Buffer Saline containing 0.05% (v/v) Tween-20), blocking for 2 hours at room temperature with PBST containing 1% (v/v) BSA made it Then, after washing the plate 5 times with PBST containing 0.05% Tween-20, a biotin-labeled human Ang2 and anti-Ang2 antibody complex or a mixture of biotin-labeled human Ang2 and unlabeled human Ang2 was added at room temperature for 2 hours. It was allowed to bind to Tie2. At this time, for biotin-labeled human Ang2, mix 1 ul of modifier reagent with 10 ul of human Ang2 (>1mg/ml), remove the vial cap containing Biotin conjugation mix, add Ang2-modifier solution, mix, block light at room temperature, and After reacting for 15 minutes or longer, 1 ul Quencher reagent was added and reacted for 5 minutes. The anti-Ang2 antibody and biotin-labeled Ang2 complex were prepared by adding the anti-Ang2 antibody to a concentration of 1.2 μM in 1% BSA and then serially diluting 5-fold using 1% BSA to prepare 10 concentrations, and biotin-labeled human Ang2 was then added to 1 It was prepared by mixing 1:1 with a sample diluted to 1 μg/mL in % BSA. A mixture of biotin-labeled human Ang2 and unlabeled human Ang2 was prepared by adding Ang2 to 1% BSA at a concentration of 1.2 μM in 1% BSA. , 10 concentrations were prepared by serial dilution 5-fold using 1% BSA, and biotin-labeled human Ang2 was mixed with a sample diluted to 1 μg/mL in 1% BSA at a ratio of 1:1. After washing the plate 5 times with PBST of 0.05% Tween-20, streptavidin HRP (Abcam) diluted 1:10000 in 1% BSA was combined for 1 hour and washed 6 times with PBST. Finally, 100 μL (microliter) of TMB substrate (Kementec) was added to the plate to induce a color reaction for 10 minutes. Then, the reaction was stopped by adding 50 μL of Stop solution (2 MH ₂ SO ₄ ), and the OD450 Values were measured on a plate reader (BioTek). The obtained results are shown in FIG. 5 .

<110> NEORTESBIO Inc. <120> Antibody specifically binding to angiopoietin-2, or its fragment <130> P21-0050HS <160> 33 <170> KoPatentIn 3.0 <210> 1 <211> 496 <212> PRT <213> Homo sapiens <400> 1 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> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 <400> 2 Asp Tyr Trp Ile Gly 1 5 <210> 3 <211> 17 <212> PRT <213> Artificial Sequence <220 > <223> CDRH2 <400> 3 Asp Ile Tyr Pro Gly Gly Gly Tyr Thr Asn Cys Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 <400> 4 Ser Asp Tyr Arg Asn Asp Glu Gly Phe Ala Asp 1 5 10 <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 <400> 5 Ser Ala Ser Ser Ser Val Ser Ser Ser Ser Tyr Leu His 1 5 10 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDRL2 <400> 6 Arg Thr Ser Asn Leu Ala Ser 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 <400> 7 Gln Gln Trp Ser Gly Tyr Pro Tyr Thr 1 5 <210> 8 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 8 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ala Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Trp Ile Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Gly Gly Gly Tyr Thr Asn Cys Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ser Arg Ser Asp Tyr Arg Asn Asp Glu Gly Phe Ala Asp Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala Ala Lys 115 120 <210> 9 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 9 Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Thr Ala Ser Leu Gly 1 5 10 15 Gln Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Pro Leu 35 40 45 Ile His Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu 65 70 75 80 Ala Glu Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro 85 90 95 Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala 100 105 110 Ala Pro Thr Val Ser 115 <210> 10 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 <400> 10 Asn Tyr Trp Ile Gly 1 5 <210> 11 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDRH2 <400> 11 Asp Ile Tyr Pro Gly Gly Gly Tyr Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 12 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 <400> 12 Ser Asp Tyr Arg Asp Asp Glu Gly Phe Ala Tyr 1 5 10 <210> 13 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 <400> 13 Ser Ala Ser Ser Ser Val Ser Ser Ser Ser Tyr Leu His 1 5 10 <210> 14 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDRL2 <400> 14 Arg Thr Ser Asn Leu Ala Ser 1 5 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 <400> 15 Gln Gln Trp Ser Gly Tyr Pro Tyr Thr 1 5 <210> 16 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 16 Gln Val His Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ala Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Gly Gly Gly Tyr Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ser Arg Ser Asp Tyr Arg Asp Asp Glu Gly Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Lys Leu 115 120 125 Tyr Pro Leu Ala 130 <210 > 17 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 17 Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ala Ala Ser Leu Gly 1 5 10 15 Gln Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Pro Leu 35 40 45 Ile His Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Thr Val Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro 85 90 95 Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala 100 105 110 Ala Pro Thr Val Ser Ala Ala Ala Ser Leu Ser 115 120 <210> 18 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 <400 > 18 Ser Asp Tyr Gly Trp Asn 1 5 <210> 19 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDRH2 <400> 19 Tyr Ile Ser Tyr Ser Gly Thr Thr Ser Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 20 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 <400> 20 Ser Glu Gly Thr Gly Phe Tyr Ala Met Asp Tyr 1 5 10 <210> 21 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 <400> 21 Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala 1 5 10 <210> 22 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDRL2 <400> 22 Tyr Ala Ser Asn Arg Tyr Thr 1 5 <210> 23 <211> 8 <212> PRT <213> Artificial Sequence <220> < 223> CDRL3 <400> 23 Gln Gln Asp Tyr Ser Ser Pro Thr 1 5 <210> 24 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 24 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 Gly Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Ser Gly Thr Thr Ser Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Ser Glu Gly Thr Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Lys Leu 115 120 125 Tyr <210> 25 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 25 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 Ile 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 Thr 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 Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala 100 105 <210> 26 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 <400> 26 Asn Tyr Trp Ile Gly 1 5 <210> 27 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> CDRH2 <400> 27 Ile Tyr Pro Gly Gly Gly Tyr Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 <210> 28 <211> 11 <212 > PRT <213> Artificial Sequence <220> <223> CDRH3 <400> 28 Ser Asp Tyr Arg Phe Asp Glu Gly Phe Ala Tyr 1 5 10 <210> 29 <211> 12 <212> PRT <213> Artificial Sequence < 220> <223> CDRL1 <400> 29 Ser Ala Ser Ser Ser Val Ser Ser Ser Ser Tyr Leu His 1 5 10 <210> 30 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDRL2 < 400> 30 Arg Thr Ser Asn Leu Ala Ser 1 5 <210> 31 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 <400> 31 Gln Gln Trp Ser Gly Tyr Pro Tyr Thr 1 5 <210> 32 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 32 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Thr Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Gly Gly Gly Tyr Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Cys 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Tyr Arg Phe Asp Glu Gly Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Ile Ser 115 <210> 33 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 33 Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ala Ala Ser Leu Gly 1 5 10 15 Gln Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Pro Leu 35 40 45 Ile His Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu 65 70 75 80 Ala Glu Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro 85 90 95 Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala 100 105 110

Claims (10)

An antibody or antigen-binding fragment thereof that specifically binds to angiopoietin 2 (Ang2) and induces Tie2 activation,
(a) CDRH1 of the amino acid sequence of SEQ ID NO: 18, or SEQ ID NO: 26;
CDRH2 of the amino acid sequence of SEQ ID NO: 19, or SEQ ID NO: 27, and
a heavy chain complementarity determining region (CDR) comprising CDRH3 of the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 28; and
(b) CDRL1 of the amino acid sequence of SEQ ID NO: 21, or SEQ ID NO: 29;
CDRL2 of the amino acid sequence of SEQ ID NO: 22, or SEQ ID NO: 30, and
An anti-Ang2 antibody or antigen-binding fragment thereof comprising a; light chain CDR comprising CDRL3 of the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 31. The method of claim 1, wherein the antibody or antigen-binding fragment thereof
(a) a heavy chain complementarity determining region (CDR) comprising CDRH1 of the amino acid sequence of SEQ ID NO: 18, CDRH2 of the amino acid sequence of SEQ ID NO: 19, and CDRH3 of SEQ ID NO: 20; and
(b) a light chain CDR comprising CDRL1 of the amino acid sequence of SEQ ID NO: 21, CDRL2 of the amino acid sequence of SEQ ID NO: 22, and CDRL3 of the amino acid sequence of SEQ ID NO: 23; anti-Ang2 antibody or antigen binding thereof, characterized in that it comprises snippet. The method of claim 1, wherein the antibody or antigen-binding fragment thereof
(a) a heavy chain complementarity determining region (CDR) comprising CDRH1 of the amino acid sequence of SEQ ID NO: 26, CDRH2 of the amino acid sequence of SEQ ID NO: 27, and CDRH3 of SEQ ID NO: 28; and
(b) a light chain CDR comprising CDRL1 of the amino acid sequence of SEQ ID NO: 29, CDRL2 of the amino acid sequence of SEQ ID NO: 30, and CDRL3 of the amino acid sequence of SEQ ID NO: 31; anti-Ang2 antibody or antigen binding thereof, characterized in that it comprises snippet. The method of claim 1, wherein the antibody or antigen-binding fragment thereof is a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 24 and SEQ ID NO: 32, and a light chain variable selected from the group consisting of SEQ ID NO: 25 and SEQ ID NO: 33 An anti-Ang2 antibody or antigen-binding fragment thereof comprising a site. The anti-Ang2 antibody or fragment thereof according to claim 1, wherein the antibody or fragment thereof specifically binds to angiopoietin-2 and binds to the Tie2 receptor together with Ang2. The method according to any one of claims 1 to 5, wherein the antigen-binding fragment is selected from the group consisting of scFv, (scFv)2, scFv-Fc, Fab, Fab' and F(ab')2, An anti-Ang2 antibody or antigen-binding fragment thereof. A pharmaceutical composition for the prevention or treatment of diseases associated with Ang2 overexpression, angiogenesis, or increased vascular permeability, comprising the anti-Ang2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 as an active ingredient. According to claim 7,
The disease associated with Ang2 overexpression, angiogenesis, or increased vascular permeability is cancer, cancer metastasis, inflammatory disease, infection, cardiovascular disease, kidney disease, hereditary hemorrhagic telangiectasia, asthma, or edema Pharmaceutical composition. Claims 1 to 5, wherein any one of the anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient, a pharmaceutical composition for the prevention or treatment of diseases associated with reduced normal angiogenesis. The pharmaceutical composition according to claim 9, wherein the disease associated with reduced normal blood vessel production is myocardial infarction, angina pectoris, cerebral infarction, stroke, Buerger's disease, avascular necrosis, foot ulcer, or erectile dysfunction.

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