An antibody or fragment thereof that specifically binds to angiopoietin-2

Abstract

The present invention relates to an anti-Ang2 antibody or antigen-binding fragment thereof that specifically binds to angiopoietin 2 (Ang2) and induces Tie2 activation, the antibody or antigen-binding fragment thereof comprising: (a) a heavy chain complementarity determining region (CDR) comprising a CDRH1 having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:18, or SEQ ID NO:26, a CDRH2 having the amino acid sequence of SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:19, or SEQ ID NO:27, and a CDRH3 having the amino acid sequence of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:20, or SEQ ID NO:28; and (b) a light chain CDR comprising a CDRL1 having the amino acid sequence of SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:21, or SEQ ID NO:29, a CDRL2 having the amino acid sequence of SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:22, or SEQ ID NO:30, and a CDRL3 having the amino acid sequence of SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:23, or SEQ ID NO:31.

Description

[Technical Field]

[0001] The present invention relates to an anti-Ang2 antibody or a fragment thereof that specifically binds to angiopoietin-2 (Ang2), a neovascularization-inducing factor, and binds to the Tie2 receptor together with Ang2. [Background technology]

[0002] The angiopoietin protein group is a group of proteins that play an important role in the generation and maintenance of blood vessels, and there are four types of angiopoietin (Ang1, Ang2, Ang3, and Ang4).

[0003] Angiopoietin-1 binds to the Tie2 receptor and plays an important role in vascular maturation, adhesion, migration and survival.

[0004] Angiopoietin-2 binds to the receptor Tie2 present on vascular endothelial cells and acts as an antagonistic ligand, competing with the Tie2 agonist angiopoietin-1 (Ang1) for Tie2 binding, thereby inhibiting Tie2-mediated signaling. Through this mechanism, overexpression of VEGF or inflammation activates vascular endothelial cells, increasing vascular permeability. Ang1 then induces endothelial cell stabilization and reduces vascular permeability. Meanwhile, increased Ang2 in activated endothelial cells competes with Ang1, thereby inhibiting Ang1-mediated stabilization of vascular endothelial cells. Therefore, Ang2 inhibits Ang1-Tie2 binding, which maintains endothelial cell stability in the presence of VEGF, and signaling through this binding, ultimately promoting angiogenesis, resulting in increased angiogenesis, vascular destabilization, and increased vascular permeability. On the other hand, in addition to its role as an antagonist that induces inactivity of the Tie2 receptor, Ang2 has also been reported to have agonist properties that induce activity of the Tie2 receptor in some specific situations, including the formation and maintenance of lymphatic vessels. Therefore, Ang2 is considered to have both antagonist and weak agonist functions and to perform various functions depending on the situation.

[0005] Because the process of angiogenesis is essential for cancer growth, attempts have been made to prevent further cancer growth by inhibiting the Tie2-dependent function of Ang2 to suppress angiogenesis, but most of these efforts are known to function by inhibiting the binding of Ang2 to Tie2, thereby preventing its role as an antagonist.In addition to antibodies that block the binding of Ang2 to Tie2, recombinant proteins and antibodies that directly bind to the Tie2 receptor to induce phosphorylation and activation have also been reported.

[0006] Recently, an antibody has been reported that binds to Ang2 and Tie2, thereby phosphorylating and activating Tie2 through Tie2 clustering. This antibody does not simply block the antagonistic role of Ang2, but rather acts as an agonist, suggesting the possibility of more effective vascular normalization. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Republic of Korea Patent Publication No. 10-2020-0144536 Summary of the Invention [Problem to be solved by the invention]

[0008] The 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), a neovascularization-inducing factor, and binds to the Tie2 receptor together with Ang2 to effectively induce activation of the Tie2 receptor.

[0009] Another object of the present invention is to provide a pharmaceutical composition containing the anti-Ang2 antibody or its antigen-binding fragment as an active ingredient.

[0010] Yet another object of the present invention is to provide a pharmaceutical composition for treating diseases associated with neovascularization, increased vascular permeability, and / or decreased normal angiogenesis, comprising the anti-Ang2 antibody or its antigen-binding fragment as an active ingredient.

[0011] A further object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which contains the anti-Ang2 antibody or its antigen-binding fragment as an active ingredient.

[0012] It is yet another object of the present invention to provide a composition for diagnosing diseases associated with overexpression of Ang2, comprising the anti-Ang2 antibody or its antigen-binding fragment.

[0013] A further object of the present invention is to provide nucleic acids encoding the antibodies or antigen-binding fragments thereof, vectors and host cells containing the nucleic acids, and methods for producing anti-Ang2 antibodies or antigen-binding fragments thereof using the same. [Means for solving the problem]

[0014] In order to achieve the above object, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to angiopoietin 2 (Ang2) and induces Tie2 activation, (a) a CDRH1 having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 18, or SEQ ID NO: 26; CDRH2 of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 19, or SEQ ID NO: 27, and a heavy chain complementarity determining region (CDR) comprising a CDRH3 of the amino acid sequence of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:20, or SEQ ID NO:28; (b) CDRL1 of the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 21, or SEQ ID NO: 29; CDRL2 of the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 22, or SEQ ID NO: 30, and and a light chain CDR comprising a CDRL3 having the amino acid sequence of SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:23, or SEQ ID NO:31; and an anti-Ang2 antibody or antigen-binding fragment thereof.

[0015] In one embodiment of the present invention, the antibody or antigen-binding fragment thereof is (a) a heavy chain complementarity determining region (CDR) comprising a CDRH1 having the amino acid sequence of SEQ ID NO: 2, a CDRH2 having the amino acid sequence of SEQ ID NO: 3, and a CDRH3 having the amino acid sequence of SEQ ID NO: 4; (b) a light chain CDR comprising a CDRL1 having the amino acid sequence of SEQ ID NO: 5, a CDRL2 having the amino acid sequence of SEQ ID NO: 6, and a CDRL3 having the amino acid sequence of SEQ ID NO: 7; In another embodiment of the present invention, the antibody or antigen-binding fragment thereof is (a) heavy chain complementarity-determining regions (CDRs) comprising a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 11, and a CDRH3 having the amino acid sequence of SEQ ID NO: 12; (b) a light chain CDR comprising CDRL1 having the amino acid sequence of SEQ ID NO: 13, CDRL2 having the amino acid sequence of SEQ ID NO: 14, and CDRL3 having the amino acid sequence of SEQ ID NO: 15;

[0016] In another embodiment of the present invention, the antibody or antigen-binding fragment thereof is (a) heavy chain complementarity determining regions (CDRs) comprising a CDRH1 having the amino acid sequence of SEQ ID NO: 18, a CDRH2 having the amino acid sequence of SEQ ID NO: 19, and a CDRH3 having the amino acid sequence of SEQ ID NO: 20; (b) a light chain CDR comprising CDRL1 having the amino acid sequence of SEQ ID NO: 21, CDRL2 having the amino acid sequence of SEQ ID NO: 22, and CDRL3 having the amino acid sequence of SEQ ID NO: 23;

[0017] In yet another embodiment of the present invention, the antibody or antigen-binding fragment thereof is (a) heavy chain complementarity determining regions (CDRs) comprising a CDRH1 having the amino acid sequence of SEQ ID NO: 26, a CDRH2 having the amino acid sequence of SEQ ID NO: 27, and a CDRH3 having the amino acid sequence of SEQ ID NO: 28; (b) a light chain CDR comprising CDRL1 having the amino acid sequence of SEQ ID NO: 29, CDRL2 having the amino acid sequence of SEQ ID NO: 30, and CDRL3 having the amino acid sequence of SEQ ID NO: 31;

[0018] In one embodiment of the present invention, the antibody or antigen-binding fragment thereof preferably comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24, and SEQ ID NO:32, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:25, and SEQ ID NO:33, but is not limited thereto.

[0019] In one embodiment of the present invention, the antibody or antigen-binding fragment thereof is preferably, but not limited to, a murine antibody, a chimeric antibody, or a humanized antibody.

[0020] As used herein, "chimeric antibody" includes antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as antibodies in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0021] As used herein, the term "humanized antibody" includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human structural regions. Further structural region modifications can be made within the human structural sequences as well as within the CDR sequences derived from the germline of yet another mammalian species.

[0022] The present invention also provides an isolated nucleic acid encoding the anti-Ang2 antibody or antigen-binding fragment thereof of the present invention.

[0023] In one embodiment of the present invention, the antibody or antigen-binding fragment thereof preferably specifically binds to angiopoietin-2 and binds to the Tie2 receptor together with Ang2, but is not limited thereto.

[0024] 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.

[0025] The present invention also provides a pharmaceutical composition for preventing or treating diseases associated with overexpression of Ang2, neovascularization, or increased vascular permeability, comprising the anti-Ang2 antibody or its antigen-binding fragment of the present invention as an active ingredient.

[0026] In one embodiment of the present invention, The diseases associated with overexpression of Ang2, neovascularization, or increased vascular permeability are preferably, but not limited to, cancer, cancer metastasis, inflammatory diseases, infection, cardiovascular diseases, kidney diseases, hereditary hemorrhagic telangiectasia, asthma, or edema.

[0027] In one embodiment of the present invention, the cancer includes, but is not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric or gastrointestinal cancer (including gastrointestinal carcinoma and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, hepatocellular carcinoma, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, endometrial and uterine cancer, salivary gland carcinoma, kidney and renal cell carcinoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, and various forms of head and neck cancer, melanoma, superficial diffuse melanoma, malignant melanoma, acral lentiginous melanoma, and nodular melanoma, as well as B-cell lymphoma (low-grade / follicular non-Hodgkin's lymphoma (NHL)). ; small lymphocytic (SL) NHL; intermediate / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-dividing cell NHL; bulky mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; including Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; post-transplant lymphoproliferative disorder (PTLD), as well as cancers including, but not limited to, nevus syndrome, edema (such as that associated with brain tumors), and abnormal blood vessel proliferation such as Meige syndrome.

[0028] The present invention also provides a pharmaceutical composition for preventing or treating diseases associated with a decrease in normal angiogenesis, comprising the anti-Ang2 antibody or antigen-binding fragment thereof of the present invention as an active ingredient.

[0029] In one embodiment of the present invention, the disease associated with a decrease in normal angiogenesis is preferably, but not limited to, myocardial infarction, angina pectoris, cerebral infarction, stroke, Buerger's disease, avascular necrosis, foot ulcer, or erectile dysfunction.

[0030] The present invention also provides a pharmaceutical composition for the prevention or treatment of Alzheimer's disease; inflammatory autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, or psoriasis; ophthalmological diseases such as age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy (DR), or glaucoma; or coronavirus infections such as coronavirus 19, comprising the anti-Ang2 antibody or antigen-binding fragment thereof of the present invention as an active ingredient.

[0031] The present invention also provides a combination therapeutic composition for use in cancer treatment, which comprises administering the anti-Ang2 antibody or antigen-binding fragment thereof of the present invention simultaneously, separately, or sequentially with radiation, a chemotherapeutic agent, a cytotoxic agent, and / or an immunotoxic agent.

[0032] In one embodiment of the present invention, the chemotherapeutic agent, cytotoxic agent and / or immunotoxic agent is preferably one or more selected from temozolomide, cisplatin, oxaliplatin, carboplatin, 5-FU, dacarbazine, procarbazine, vinblastine, vincristine, irinotecan, taxol, paclitaxel, docetaxel, gemcitabine, Gleevec, Iressa, Tarceva and Nexavar, Herceptin, bevacizumab, cetuximab, nimotuzumab, sorafenib, sunitinib and ZD6474 (ZACTIMATM), more preferably, but not limited to, temozolomide, cisplatin, oxaliplatin, vinblastine, taxol, gemcitabine, Gleevec and Iressa.

[0033] The present invention will be described below.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein is well known and commonly used in the art.

[0035] The present invention provides a dual-function antibody or antigen-binding fragment thereof that specifically binds to Ang2 but does not inhibit the binding of Ang2 to the Tie2 receptor, and that forms a complex (antibody / Ang2 / Tie2) with Ang2 and the Tie2 receptor, which binds to the Tie2 receptor together with Ang2, activates the Tie2 receptor like Ang1, induces Tie2 downstream signaling, and induces stabilization of vascular endothelial cells.

[0036] In addition, the present invention provides an anti-Ang2 antibody and its pharmaceutical uses, which, in addition to binding to the Tie2 receptor together with Ang2 and activating the Tie2 receptor like Ang1, inhibits the binding of Ang2 to other proteins involved in cancer cell growth and / or cancer metastasis, such as integrins, thereby having enhanced effects of inhibiting cancer cell growth and / or suppressing cancer metastasis, and which can exert such effects even in cells that do not express Tie2.

[0037] In one embodiment of the present invention, a polypeptide molecule is provided that includes a heavy chain complementarity determining region, a light chain complementarity determining region, or a combination thereof; or a heavy chain variable region, a light chain variable region, or a combination thereof, of the above-described anti-Ang2 antibody. The polypeptide molecule can function not only as an antibody or its antigen-binding fragment, but also as a precursor or component of an Ang2 antagonist. For example, the polypeptide molecule may function as an Ang2 antigen-binding site or as a component of a protein scaffold (e.g., peptibody, nanobody, etc.) with a structure similar to that of an antibody, a bispecific antibody, a multispecific antibody, etc.

[0038] The term "antagonist" is intended to encompass all molecules that partially or completely block, inhibit or neutralize one or more of the biological activities of a target (eg, Ang2).

[0039] The term "peptibody (peptide + antibody)" refers to a fusion protein in which a peptide is fused to all or part of an antibody invariant region such as the Fc region, and the peptide acts as an antigen-binding site (heavy chain and / or light chain CDR or variable region), and the protein has a framework and function similar to that of an antibody.

[0040] The term "nanobody," also known as a single-domain antibody, refers to an antibody fragment containing a single variable domain of an antibody in a monomeric form, and has the property of selectively binding to a specific antigen similar to that of an intact antibody. The molecular weight of a nanobody is generally about 12 kDa to about 15 kDa, which is much smaller than the typical molecular weight (about 150 kDa to about 160 kDa) of an intact antibody (containing two heavy chains and two light chains), and in some cases is smaller than a Fab fragment or an scFv fragment.

[0041] The term "bispecific antibody" or "multispecific antibody" refers to an antibody that recognizes and / or binds to two (bispecific antibody) or more (multispecific antibody) different antigens or recognizes and / or binds to different sites on the same antigen, and one antigen-binding site of the bispecific or multispecific antibody may comprise the polypeptide.

[0042] In particular embodiments, the polypeptide molecule is one or more selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:18, or SEQ ID NO:26, a polypeptide comprising the amino acid sequence of SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:19, or SEQ ID NO:27, and a polypeptide comprising the amino acid sequence of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:20, or SEQ ID NO:28; The polypeptide may comprise one or more selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:21 or SEQ ID NO:29, a polypeptide comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:22 or SEQ ID NO:30, and a polypeptide comprising the amino acid sequence of SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:23 or SEQ ID NO:31; or a combination thereof.

[0043] In specific examples, the polypeptide molecule may comprise the amino acid sequence of SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24 or SEQ ID NO:32, the amino acid sequence of SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:25 or SEQ ID NO:33, or a combination thereof.

[0044] The terms "Kabat number," "Kabat definition," and "Kabat labeling" are used interchangeably herein. These terms, recognized in the art, refer to a numbering system for amino acid residues that are more diverse (i.e., highly variable) than other amino acid residues in the variable regions or antigen-binding sites of antibody heavy and light chains (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391; Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, National Institutes of Health Publication No. 91-3242).

[0045] As described above, the bispecific or multispecific antibody is an antibody that simultaneously recognizes and / or binds to two or more antigens by containing antigen-binding sites for each of the two or more different antigens, and one of the antigen-binding sites may comprise the above-described polypeptide molecule. Specifically, the polypeptide molecule that serves as the Ang2 antigen-binding site may form a dimer or multimer with an antigen-binding site for another antigen to constitute the bispecific or multispecific antibody. Thus, in one embodiment, a bispecific or multispecific antibody is provided that contains the above-described polypeptide molecule as an Ang2 antigen-binding site.

[0046] In yet another example, a protein scaffold having a multimeric structure is provided, which comprises one or more (e.g., 1 to 5, or 2 to 4) peptide complexes each comprising one or more of the above-mentioned polypeptide molecules or a repeat of the polypeptide molecules linked together by linkers (hereinafter referred to as a "first peptide") and a polypeptide having a structural function (hereinafter referred to as a "second peptide"; for example, an invariant region of the heavy or light chain of an antibody (e.g., IgG, IgA, IgE, IgD, IgM, etc.), or an Fc fragment of an antibody), and the one or more peptide complexes are linked by the second peptide (e.g., the Fc fragment) to form a protein scaffold.

[0047] In the present invention, antibodies include animal-derived antibodies, chimeric antibodies, humanized antibodies, and human antibodies. Animal-derived antibodies, which are produced by immunizing a target animal with a desired antigen, generally cause immune rejection when administered to humans for therapeutic purposes. To prevent such immune rejection, chimeric antibodies have been developed. Chimeric antibodies are produced by replacing the constant region of an animal-derived antibody, which causes anti-isotype reactions, with the constant region of a human antibody using genetic engineering techniques. Although chimeric antibodies have significantly improved anti-isotype reactions compared to animal-derived antibodies, they still contain animal-derived amino acids in the variable region, which can lead to potential side effects such as anti-idiotypic reactions. To address these side effects, humanized antibodies have been developed. These antibodies are produced by grafting the complementarity determining regions (CDRs), which play an important role in antigen binding, from the variable region of a chimeric antibody onto a human antibody framework.

[0048] The most important aspect of CDR grafting technology for creating humanized antibodies is the selection of an optimized human antibody that can best accommodate the CDR region of an animal-derived antibody, and for this purpose, antibody databases, crystal structure analysis, molecular modeling technology, etc. are utilized. However, even when the CDR region of an animal-derived antibody is grafted onto an optimized human antibody framework, there are many cases in which the antigen-binding ability is not preserved due to the presence of amino acids located in the framework of the animal-derived antibody that affect antigen binding. Therefore, the application of additional antibody engineering techniques to restore the antigen-binding ability is essential.

[0049] In one embodiment, the antibody may be a murine-derived antibody, a mouse-human chimeric antibody, a humanized antibody, or a human antibody.

[0050] In the present invention, the term "antibody" refers to a substance produced in the immune system in response to antigen stimulation, and the type of antibody is not particularly limited. Antibodies have recently been widely used as therapeutic agents for diseases. Antibodies are highly stable in vivo as well as in vitro, and have a long half-life, which makes them advantageous for mass expression and production. Furthermore, antibodies inherently have a dimeric structure, which gives them very high avidity.

[0051] A complete antibody has two full-length light chains and two full-length heavy chains, each connected to a heavy chain by a disulfide bond. The constant regions of antibodies are divided into heavy-chain and light-chain constant regions. Heavy-chain constant regions are of gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, with subclasses of gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). Light-chain constant regions are of kappa (κ) and lambda (λ) types.

[0052] The term "heavy chain" is intended to encompass a full-length heavy chain and fragments thereof, including a variable region domain VH containing an amino acid sequence with sufficient variable region sequence to confer antigen specificity, three constant region domains CH1, CH2, and CH3, and a hinge. The term "light chain" is intended to encompass a full-length light chain and fragments thereof, including a variable region domain VL containing an amino acid sequence with sufficient variable region sequence to confer antigen specificity, and a constant region domain CL.

[0053] The term "CDR (complementarity determining region)" refers to the amino acid sequences of the hypervariable regions of the heavy and light chains of immunoglobulins. Each heavy and light chain can contain three CDRs (CDRH1, CDRH2, CDRH3, and CDRL1, CDRL2, CDRL3). The CDRs can provide key contact residues for antibody binding to an antigen or epitope. Meanwhile, the terms "specifically bind" and "specifically recognize" as used herein have the same meaning as commonly known to those skilled in the art, meaning that an antigen and an antibody specifically interact to cause an immunological reaction.

[0054] The antigen-binding fragment of the antibody provided by the present invention may be a fragment comprising one or more of the above-mentioned complementarity-determining sites.

[0055] The term "antigen-binding fragment" refers to a portion of a polypeptide that is a fragment of the entire immunoglobulin structure and contains the portion capable of binding to an antigen, such as, but not limited to, scFv, (scFv)2, scFv-Fc, Fab, Fab', or F(ab')2.

[0056] Among the antigen-binding fragments, Fab has a structure comprising light and heavy chain variable regions, a light chain constant region, and the first heavy chain constant region (CH1), 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. F(ab')2 antibodies are produced by disulfide bonding between cysteine ​​residues in the hinge region of Fab'. Fv is the minimum antibody fragment comprising only the heavy chain variable region and the light chain variable region, and recombinant techniques for producing Fv fragments are widely known in the art. In a two-chain Fv, the heavy-chain variable region and the light-chain variable region are linked non-covalently, while in a single-chain Fv, the heavy-chain variable region and the single-chain variable region are generally linked covalently via a peptide linker or directly at the C-terminus, thereby forming a dimer-like structure similar to that of a two-chain Fv. The linker may be a peptide linker consisting of 1 to 100 or 2 to 50 amino acids, and suitable sequences are known in the art. The antigen-binding fragment can be obtained using protease (for example, Fab can be obtained by limited digestion of a whole antibody with papain, or F(ab')2 fragments can be obtained by digestion with pepsin), or can be produced through genetic recombination techniques.

[0057] The term "hinge region" refers to a region contained in an antibody heavy chain, located between the CH1 and CH2 regions, and having the function of providing flexibility to the antigen-binding site within the antibody. For example, the hinge may be derived from a human antibody, specifically, from IgA, IgE, or IgG, such as IgG1, IgG2, IgG3, or IgG4.

[0058] When an animal-derived antibody undergoes chimerization, the animal-derived IgG1 hinge is replaced with a human IgG1 hinge. However, the animal-derived IgG1 hinge is shorter than the human IgG1 hinge, and the number of disulfide bonds between the two heavy chains is reduced from three to two, resulting in different hinge rigidity effects. Therefore, 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.

[0059] The portion of the anti-Ang2 antibody excluding the variable region may be a constant region derived from a human antibody, specifically, a constant region derived from IgA, IgE, or IgG, for example, IgG1, IgG2, IgG3, or IgG4.

[0060] The anti-Ang2 antibody may be a monoclonal antibody. Monoclonal antibodies can be produced according to methods well known in the art. For example, they may be produced using phage display technology. Alternatively, the anti-Ang2 antibody can be produced as a mouse-derived monoclonal antibody by the method described in Schwaber et al. (Schwaber, J and Cohen, EP, "Human x Mouse Somatic Cell Hybrid Clones Secreting Immunoglobulins of Both Parental Types," Nature, 244 (1973), 444-447).

[0061] Alternatively, individual monoclonal antibodies can be screened based on their binding ability to Ang2 using a typical enzyme-linked immunosorbent assay (ELISA) format. The binding partners can be assayed for inhibitory activity through functional assays such as competitive ELISA to test for molecular interactions, or cell-based assays. Subsequently, the affinities (Kd values) of the monoclonal antibodies selected based on their potent inhibitory activity for Ang2 are assayed.

[0062] The finally selected antibody can be produced and used as a humanized antibody, or an antibody in which the remaining portion, excluding the antigen-binding domain, is humanized to a human immunoglobulin. Methods for producing humanized antibodies are well known in the art (Almagro, J.C. and Fransson, J., "Humanization of antibodies," Frontiers in Bioscience, 13 (2008), 1619-1633).

[0063] Another example provides a pharmaceutical composition for inhibiting neovascularization, comprising the anti-Ang2 antibody or its antigen-binding fragment as an active ingredient. Another example provides a method for inhibiting neovascularization, comprising administering a pharmaceutically effective amount of the anti-Ang2 antibody or its antigen-binding fragment to a patient in need of such inhibition. The method for inhibiting neovascularization may further comprise identifying the patient in need of neovascularization inhibition prior to the administering step.

[0064] Another example provides a pharmaceutical composition for reducing vascular permeability, comprising the anti-Ang2 antibody or its antigen-binding fragment as an active ingredient. Another example provides a method for reducing vascular permeability, comprising administering a pharmaceutically effective amount of the anti-Ang2 antibody or its antigen-binding fragment to a patient in need of such reduction. The method may further comprise identifying the patient in need of such reduction prior to the administering step.

[0065] Another example provides a pharmaceutical composition for preventing and / or treating diseases associated with Ang2 overexpression, neovascularization, and / or increased vascular permeability, comprising the anti-Ang2 antibody or antigen-binding fragment thereof as an active ingredient. Another example provides a method for preventing and / or treating diseases associated with Ang2 overexpression, neovascularization, and / or increased vascular permeability, comprising administering a pharmaceutically effective amount of the anti-Ang2 antibody or antigen-binding fragment thereof to a patient in need of such prevention and / or treatment. The method for preventing and / or treating may further include identifying a patient in need of such prevention and / or treatment prior to the administering step.

[0066] Another example provides a pharmaceutical composition for inducing normal angiogenesis, comprising the anti-Ang2 antibody or its antigen-binding fragment as an active ingredient. Another example provides a method for increasing normal angiogenesis, comprising administering a pharmaceutically effective amount of the anti-Ang2 antibody or its antigen-binding fragment to a patient in need of such induction. The method may further include identifying a patient in need of such induction prior to the administering step.

[0067] Another example provides a pharmaceutical composition for preventing and / or treating a disease associated with decreased normal angiogenesis, comprising the anti-Ang2 antibody or its antigen-binding fragment as an active ingredient. Another example provides a method for preventing and / or treating a disease associated with decreased normal angiogenesis, comprising administering a pharmaceutically effective amount of the anti-Ang2 antibody or its antigen-binding fragment to a patient in need of such prevention and / or treatment. The method for preventing and / or treating may further include identifying a patient in need of such prevention and / or treatment prior to the administering step.

[0068] Another example provides a composition for diagnosing a disease associated with neovascularization and / or increased vascular permeability and / or decreased normal angiogenesis, comprising the antibody or antigen-binding fragment thereof.

[0069] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, which is one or more commonly used in drug formulations, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. The pharmaceutical composition may also further comprise one or more commonly used in the manufacture of pharmaceutical compositions, including diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0070] The pharmaceutical composition or a pharmaceutically effective amount of the antibody or antigen-binding fragment thereof can be administered orally or parenterally. Parenteral administration can be by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, or intrarectal administration. Since proteins or peptides are digested during oral administration, oral compositions may be formulated to coat the active agent or protect it from digestion in the stomach. Furthermore, the compositions may be administered by any device that can deliver the active agent to target cells.

[0071] The content of the anti-Ang2 antibody or its antigen-binding fragment in the pharmaceutical composition can be variously formulated depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, diet, administration time, administration interval, administration route, excretion rate, and reaction sensitivity. For example, the daily dose of the anti-Ang2 antibody or its antigen-binding fragment 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. The daily dose may be formulated in the form of a single unit dose, appropriately divided into individual doses, or packaged in a multi-dose container. The term "pharmaceutically effective amount" refers to the content or dosage of an active ingredient that can exhibit a desired pharmacological effect and can be variously determined depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, diet, administration time, administration interval, administration route, excretion rate, and reaction sensitivity.

[0072] The pharmaceutical composition may be in the form of a solution, suspension, syrup or emulsion in an oily or aqueous medium, or may be formulated into the form of an extract, powder, granule, tablet or capsule, etc., and may further contain a dispersing agent or stabilizer for formulation.

[0073] In particular, pharmaceutical compositions containing the anti-Ang2 antibody or its antigen-binding fragment can be formulated into immunoliposomes because they contain the antibody or antigen-binding fragment. Antibody-containing liposomes can be prepared by methods well known in the art. The immunoliposomes are lipid compositions containing phosphatidylcholine, cholesterol, and polyethylene glycol-derivatized phosphatidylethanolamine, and can be prepared by reverse phase evaporation. For example, the Fab' fragment of an antibody can be conjugated to liposomes via a disulfide exchange reaction.

[0074] Meanwhile, since the anti-Ang2 antibody or its antigen-binding fragment specifically binds to Ang2, it can be used to detect Ang2, thereby confirming the presence or absence of Ang2 overexpression. Thus, another embodiment of the present invention provides a composition for detecting Ang2, comprising the anti-Ang2 antibody or its antigen-binding fragment, and a composition for diagnosing diseases associated with Ang2 overexpression. [Effects of the Invention]

[0075] The present invention proposes a novel method for inhibiting Ang2-induced angiogenesis and reducing vascular permeability by providing an antibody that simultaneously inhibits Ang2 and activates the Tie2 receptor to promote downstream signaling. Furthermore, the antibody proposed in the present invention is expected to be applicable to the diagnosis and treatment of diseases other than cancer associated with abnormal angiogenesis and / or diseases caused by increased vascular permeability. The antibody can be used in combination with chemical drugs and other anti-cancer therapeutic agents, and is expected to be used in antibody fragments, bi- or multi-specific antibodies, protein scaffolds, etc., by utilizing its Ang2-specific recognition activity. [Brief explanation of the drawings]

[0076] [Figure 1] Western blot results showing that anti-Ang2 antibody, together with Ang2, phosphorylates AKT and ERK 42 / 44 in HUVEC cells. [Figure 2] 1 shows the results of ELISA demonstrating that anti-Ang2 antibodies, together with Ang2, induce AKT phosphorylation in HUVEC cells in an antibody concentration-dependent manner. The clones listed in descending order of the curve with the highest value are 1D3, 2C8, 1A9, and 1H10, and the clone with the lowest value is the control clone 67. [Figure 3]The binding affinity of the anti-Ang2 antibodies to human Ang2 and mouse Ang2 was analyzed by ELISA. [Figure 4] 1 shows the results of ELISA demonstrating that anti-Ang2 antibodies bind to Ang2 and Tie2 to form a complex. [Figure 5] 1 shows the results of ELISA demonstrating that the anti-Ang2 antibody 2C8 does not interfere with the binding of Ang2 to Tie2. [Figure 6] 1 shows that anti-Ang2 antibody 2C8 inhibits tumor growth in an LLC lung cancer cell tumor model (error bar: standard error of the mean). [Figure 7] This shows that combined administration of anti-Ang2 antibody 2C8 and cisplatin has significantly superior tumor growth inhibitory efficacy compared to either agent administered alone in an LLC lung cancer cell tumor model (error bar: standard error of mean). [Figure 8] This shows that combined administration of anti-Ang2 antibody 2C8 and 5FU has significantly superior tumor growth inhibitory efficacy compared to either agent administered alone in an MC38 colon cancer cell tumor model (error bar: standard error of mean). BEST MODE FOR CARRYING OUT THE INVENTION

[0077] The present invention will be described in more detail below with reference to examples, but these are merely illustrative and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that the examples described below can be modified without departing from the essential gist of the invention.

[0078] Example 1: Production and screening of anti-Ang2 antibodies An antibody that specifically binds to human Ang2 (SEQ ID NO: 1) was ordered from AbClon (Korea), and the antibody of the present invention was produced with reference to Kohler and Milstein, Eur. J. Immunol. 6, 511 (1976).

[0079] Briefly, mice were injected twice with human Ang2 as an antigen mixed with an adjuvant (Sigma), and antibody production was confirmed by ELISA. After the second immunization, antibody titers (1:5000) increased. Spleens were removed from the immunized mice, B lymphocytes were isolated, and fused with sp2 / 0 cells. The fused cells were cultured in HAT medium containing hypoxanthine, aminopterin, and thymidine, and hybridomas formed by fusion of B lymphocytes with sp2 / 0 cells were selectively selected. The resulting hybridoma cells were then cloned repeatedly using the serial dilution method to separate positive and negative cells, producing single clones producing antibodies that reacted with the antigen.

[0080] The obtained antibody-producing hybridoma cells were cultured in DMEM (Dulbeco's Modified Eagle's Medium) containing 10% (v / v) FBS at 37°C and 5% CO2, and then the antibody-producing cells were removed by centrifugation. The culture medium containing the antibody was separated and purified using an affinity column (Protein A / G agarose column, Protein A / G (GenDEPOT)).

[0081] Example 2: Western blot of anti-Ang2 antibody-induced activation of Tie2 signaling Ang2 binds to the Tie2 receptor expressed on vascular endothelial cells and acts as a weak agonist or antagonist. The anti-Ang2 antibody developed in the present invention binds to Ang2 to form an Ang2-Tie2 complex, activating the Tie2 receptor and promoting downstream signaling. Therefore, to analyze the effect of the anti-Ang2 antibody on Tie2 downstream signaling using a cell-based assay, we performed ERK and AKT phosphorylation experiments. To compare the level of activation of Tie2 downstream signaling, we also performed the same experiment on groups treated with Ang2 (Sino Biological) and another Ang2 control antibody (Korean Patent Publication No. 10-2015-0136031).

[0082] Specifically, HUVEC (Lonza) cells (2 × 10 5 Cells were cultured in EGM-2 (Lonza) medium at 37°C. When the cells reached 80-90% confluency, they were replaced with 0.5% FBS basal medium (Lonza) and cultured at 37°C for 16-24 hours. After 30 minutes, 60 nM of anti-Ang2 antibody was mixed with 40 nM of Ang2 protein (Sino Biological) and incubated. The cells were then treated with the anti-Ang2 antibody and further cultured for 10 minutes. For comparison, groups were treated with 40 nM Ang2 and 40 nM Ang2 plus 60 nM of anti-Ang2 control antibody. The cells were washed with PBS and then treated with a lysis buffer (Biosesang (Ripa buffer), 0.15 M sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 7.5, and 2 mM EDTA), followed by centrifugation at 13,000 rpm for 15 minutes to collect the supernatant and obtain a cell lysate.

[0083] 35 μg of cell lysate was added to a sample buffer (Biosesang) containing a reducing agent and boiled at 95°C for 5 minutes. The gel was then electrophoresed on a 10% Tris-glycine gel and transferred to a nitrocellulose membrane (GVS). To confirm the presence or absence of phosphorylation of Akt and ERK 42 / 44, which are involved in downstream signaling of the Tie2 receptor, the blot was blocked for 1 hour with PBST containing 5% (v / v) skim milk (Seoul Milk) and then treated with 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 results are shown in Figure 1.

[0084] As shown in FIG. 1, the 1A9, 1D3, 2C8, and 1H10 clones showed AKT and ERK activation signals compared to the negative control group.

[0085] Example 3: ELISA of anti-Ang2 antibody-induced activation of Tie2 subunit signaling To quantitatively analyze the effect of anti-Ang2 antibody on Tie2-dependent signal transduction, the phosphorylation level of AKT was measured by ELISA.

[0086] HUVEC (Lonza) cells (2 × 10 5Cells were cultured in EGM-2 (Lonza) medium at 37°C. When the cells reached 80-90% confluency, they were replaced with 0.5% FBS basal medium (Lonza) and cultured at 37°C for 16-24 hours. Anti-Ang2 antibody (60 nM) was mixed with Ang2 protein (40 nM) (Sino Biological) for 30 minutes, then treated with the cultured cells and further cultured for 10 minutes. For comparison, treatments with Ang2 (40 nM) and Ang2 (40 nM) plus anti-Ang2 control antibody (h10D6-OPTI-67, US10934350) (60 nM) were also prepared. The cells were washed with PBS, treated with lysis buffer (Biosesang), and centrifuged at 13,000 rpm for 15 minutes to collect the supernatant and obtain cell lysates.

[0087] For ELISA, 50 μL of the capture antibody from the PathScan RPhospho-Akt1 (Ser473) Sandwich ELISA Antibody Pair (Cell signaling) diluted 1:100 was coated onto a 96-well ps half-area plate (Greiner Bio-One). The plate was then washed four times with PBST (phosphate buffered saline containing 0.05% (v / v) Tween-20) and blocked for 2 hours at 37°C with PBST containing 1% (v / v) skim milk. The plate was then washed four times with PBST containing 0.05% Tween-20, and the cell lysate was added. The plate was then incubated at 37°C for 2 hours to allow phosphorylated AKT to bind to the capture antibody. After washing the plate four times with PBST, secondary antibodies diluted 1:1000 in 1% skim milk were incubated at 37°C for 1 hour and then washed four times with PBST. Finally, 50 μL of TMB substrate (Kementec) was added to the plate to induce color development for 10 minutes. The reaction was then terminated by adding 50 μL of Stop solution, and the OD450 value was measured using a plate reader (BioTek). The results are shown in Figure 2.

[0088] As shown in FIG. 2, the monoclonal antibodies 1A9, 1D3, 2C8, and 1H10 of the present invention were confirmed to induce significantly stronger Tie2 signaling than the known control antibodies.

[0089] Example 4: Gene cloning of mouse anti-Ang2 antibody RNA was isolated from the hybridomas obtained in Example 1 using an AccuPrep Universal RNA extraction kit (Bioneer). Using this as a template, cDNA was synthesized and cloned according to a known method (Meyer L et al., "A simplified workflow for monoclonal antibody sequencing," 2019, PLoS ONE). cDNA was synthesized using a SuperiorScript III cDNA Synthesis kit (Enzynomics), and the synthesized cDNA was amplified by PCR and cloned into a vector using a TOPcloner Blunt core kit (Enzynomics). DNA sequencing was performed to obtain the nucleotide and amino acid sequences encoding the CDRs, heavy chain variable region, and light chain variable region of each antibody (Tables 1 to 8).

[0090] JPEG0007749696000001.jpg42170Table 1 shows the CDR sequences of the mouse anti-Ang2 antibody 1A9.

[0091] JPEG0007749696000002.jpg41170Table 2 shows the variable region sequences of the mouse anti-Ang2 antibody 1A9.

[0092] JPEG0007749696000003.jpg42170Table 3 shows the CDR sequences of the mouse anti-Ang2 antibody 2C8.

[0093] JPEG0007749696000004.jpg41170Table 4 shows the variable region sequences of the mouse anti-Ang2 antibody 2C8.

[0094] JPEG0007749696000005.jpg42170 Table 5 shows the CDR sequences of the mouse anti-Ang2 antibody 1D3.

[0095] JPEG0007749696000006.jpg42170Table 6 shows the variable region sequences of the mouse anti-Ang2 antibody 1D3.

[0096] JPEG0007749696000007.jpg42170Table 7 shows the CDR sequences of the mouse anti-Ang2 antibody 1H10.

[0097] JPEG0007749696000008.jpg42170Table 8 shows the variable region sequences of the mouse anti-Ang2 antibody 1H10.

[0098] Example 5: ELISA for binding of anti-Ang2 antibodies to human Ang2 or mouse Ang2 ELISA was performed to confirm the binding ability of the anti-Ang2 antibody to human Ang2 or mouse Ang2. TMFlat-bottom plates were coated with 1 μg / mL human Ang2 (Sino Biological) or mouse Ang2 (Acrobiosystems). The plates were then washed five times with PBST (phosphate buffered saline containing 0.05% (v / v) Tween-20) and blocked for 2 hours at room temperature with PBST containing 1% (v / v) skim milk. The plates were then washed five times with PBST containing 0.05% Tween-20. Ten concentrations of anti-Ang2 antibodies, serially diluted 3-fold starting from 300 nM, were added and allowed to bind for 2 hours at room temperature. The plates were washed five times with PBST containing 0.05% Tween-20 and then bound with goat anti-mouse IgG / HRP (Solarbio) diluted 1:3000 in 1% skim milk for 1 hour, followed by six washes with PBST. Finally, 100 μL (microliter) of TMB substrate (Kementec) was added to the plate to induce the color reaction for 10 minutes, and then 100 μL of Stop solution (2 M HSO) was added to stop the reaction, and the OD450 value was measured on a plate reader (BioTek). The results are shown in Figure 3.

[0099] Example 6: ELISA for confirming the formation of antibody-Ang2-Tie2 complexes of anti-Ang2 antibodies ELISA was performed to confirm whether or not a complex between the anti-Ang2 antibody and the Ang2 Tie2 receptor was formed. TMA flat-bottom plate was coated with 2 μg / ml Tie2 ECD (Sino Biological). The plate was then washed five times with PBST (phosphate buffered saline containing 0.05% (v / v) Tween-20) and blocked for 2 hours at room temperature with PBST containing 1% (v / v) skim milk. After washing five times with PBST containing 0.05% Tween-20, the plate was then loaded with a pre-prepared anti-Ang2 antibody / Ang2 complex and allowed to bind to Tie2 for 2 hours at room temperature. The anti-Ang2 antibody / Ang2 complex was prepared by adding anti-Ang2 antibody at a concentration of 600 nM to 1% skim milk and serially diluting it three-fold with 1% skim milk to prepare 10 concentrations. The anti-Ang2 antibody / Ang2 complex was then mixed 1:1 with a 1 μg / mL Ang2 sample diluted in 1% skim milk. After washing the plate five times with 0.05% Tween-20 PBST, goat anti-mouse IgG / HRP (Solarbio) diluted 1:3000 in 1% skim milk was added for 1 hour and then washed six times with PBST. Finally, 100 μL (microliter) of TMB substrate (Kementec) was added to the plate to induce the color reaction for 10 minutes. The reaction was then stopped by adding 50 μL of Stop solution (2 M H2SO4), and the OD450 value was measured on a plate reader (BioTek). The results are shown in Figure 4.

[0100] Example 7: Competition ELISA of anti-Ang2 antibodies for Ang2-Tie2 binding Ang2-Tie2 binding competition ELISA was performed using anti-Ang2 antibody. More specifically, a 96-well MaxiSorp TMA flat-bottom plate was coated with 4 μg / ml Tie2 ECD (Sino Biological). The plate was then washed five times with PBST (phosphate buffered saline containing 0.05% (v / v) Tween-20) and blocked with PBST containing 1% (v / v) BSA at room temperature for 2 hours. The plate was then washed five times with PBST containing 0.05% Tween-20. A conjugate of biotin-labeled human Ang2 and an anti-Ang2 antibody, or a mixture of biotin-labeled human Ang2 and unlabeled human Ang2, was added and allowed to bind to Tie2 at room temperature for 2 hours. Biotin-labeled human Ang2 was prepared by mixing 10 μl of human Ang2 (>1 mg / ml) with 1 μl of Modifier reagent, removing the cap from the vial containing the Biotin conjugation mix, adding the Ang2-modifier solution, mixing, and incubating at room temperature in the dark for 15 minutes or more. After that, 1 μl of Quencher reagent was added and incubated for 5 minutes. Conjugates of anti-Ang2 antibody and biotin-labeled Ang2 were prepared by adding anti-Ang2 antibody to 1% BSA at a concentration of 1.2 μM, serially diluting the antibody five-fold with 1% BSA to produce 10 concentrations, and then mixing 1:1 with a sample of biotin-labeled human Ang2 diluted to 1 μg / mL in 1% BSA. Mixtures of biotin-labeled human Ang2 and unlabeled human Ang2 were prepared by adding Ang2 to 1% BSA at a concentration of 1.2 μM, serially diluting the antibody five-fold with 1% BSA to produce 10 concentrations, and then mixing 1:1 with a sample of biotin-labeled human Ang2 diluted to 1 μg / mL in 1% BSA. Plates were washed five times with 0.05% Tween-20 in PBST, then bound with streptavidin-HRP (Abcam) diluted 1:10,000 in 1% BSA for 1 hour, followed by six washes with PBST.Finally, 100 μL (microliter) of TMB substrate (Kementec) was added to the plate to induce the color reaction for 10 minutes, and then 50 μL of Stop solution (2 M H2SO4) was added to stop the reaction, and the OD450 value was measured on a plate reader (BioTek). The results are shown in Figure 5.

[0101] Example 8: Tumor growth suppression effect of anti-Ang2 antibody in mouse tumor model A mouse tumor model was used to confirm the tumor growth inhibitory effect of anti-Ang2 antibody. The tumor size (V) was calculated using the following formula: V = Tumor volume (mm 3 )=(length×width 2 ) / 2

[0102] A. LLC lung cancer cell tumor model To confirm the tumor growth inhibitory effect of anti-Ang2 antibody, a syngeneic mouse lung cancer model was used using the mouse lung cancer cell line, LLC (Lewis lung carcinoma, ATCC). LLC cells were cultured in DMEM (Welgene) supplemented with 10% FBS (Gibco). LLC cells (1 × 10 6 The tumor cells were subcutaneously inoculated into C57BL / 6 mice of the same genotype (Central Experimental Animals) with 100 μL of PBS + 100 μL of matrix. On days 7, 9, 11, 14, and 16 after cancer cell inoculation, anti-Ang2 antibody (2C8) was intraperitoneally injected at a dose of 5 mg / kg, and tumor size was measured.

[0103] As shown in Figure 6, it was confirmed that the anti-Ang2 antibody 2C8 inhibited tumor growth.

[0104] To confirm the efficacy of combined administration of anti-Ang2 antibodies with other existing anticancer drugs, we performed a combined administration test of the Ang2 antibody 2C8 and cisplatin in an LLC lung cancer model. LLC cell lines were cultured in DMEM (Welgene) supplemented with 10% FBS (Gibco). LLC cells (1 × 10 6in 100 μL of PBS) was subcutaneously inoculated into C57BL / 6 mice (Orient) of the same genotype, and tumors with a volume of 50–100 mm were observed. 3 When the tumor size reached 100 mg / kg, Ang2 antibody was administered intraperitoneally at a dose of 5 mg / kg three times a week for two weeks. Cisplatin (Sigma) was administered once at a dose of 5 mg / kg. Experiments were conducted on Ig (control group), 2C8 group, cisplatin group, and 2C8 + cisplatin group.

[0105] As shown in Figure 7, when anti-Ang2 antibody 2C8 was administered in combination with cisplatin, tumor growth was suppressed by approximately 48% compared to the control group, demonstrating significantly greater efficacy than the administration of anti-Ang2 antibody or 5FU alone. These results indicate that anti-Ang2 antibody, when administered in combination with other anticancer drugs, can suppress cancer growth much more potently than existing anticancer drug monotherapy.

[0106] B. MC38 colon cancer cell growth model To confirm the tumor growth inhibitory effect of anti-Ang2 antibodies in a colon cancer model and the effect of coadministration with 5-fluorouracil (5FU), a syngeneic mouse model of colon cancer using the MC38 cell line, a murine colon cancer cell line, was used. The MC38 cell line was cultured in DMEM (Welgene) supplemented with 10% FBS (Welgene). MC38 cells (1 × 10 6 in 100 μL of PBS) was subcutaneously inoculated into C57BL / 6 mice (Orient) of the same genotype, and tumors with a volume of 70–100 mm 3 When the mice reached 100% CI, Ang2 antibody (2C8) was intraperitoneally administered at a dose of 5 mg / kg for a total of five times at 3-day intervals. 5FU was administered twice at a dose of 20 mg / kg. The experiment was conducted on Ig (control group), 2C8 group, 5FU (Sigma) group, and 2C8 + 5FU group.

[0107] As shown in Figure 8, the anti-Ang2 antibody 2C8 and 5FU alone showed similar levels of tumor growth inhibitory effect, and in addition, when the anti-Ang2 antibody 2C8 and 5FU were administered in combination, tumor growth was inhibited by approximately 58% compared to the control group, demonstrating significantly greater efficacy than the administration of the anti-Ang2 antibody or 5FU alone. These results indicate that the anti-Ang2 antibody, when administered in combination with other anticancer drugs, can inhibit cancer growth much more potently than existing anticancer drug monotherapy.

Claims

1. An antibody or an antigen-binding fragment thereof that specifically binds to angiopoietin 2 (Ang2) and induces Tie2 activation, The antibody or antigen-binding fragment thereof (a) heavy chain complementarity determining regions (CDRs) comprising a CDRH1 having the amino acid sequence of SEQ ID NO: 2, a CDRH2 having the amino acid sequence of SEQ ID NO: 3, and a CDRH3 having the amino acid sequence of SEQ ID NO: 4; (b) a light chain CDR comprising a CDRL1 having the amino acid sequence of SEQ ID NO: 5, a CDRL2 having the amino acid sequence of SEQ ID NO: 6, and a CDRL3 having the amino acid sequence of SEQ ID NO: 7; and an anti-Ang2 antibody or antigen-binding fragment thereof.

2. An antibody or an antigen-binding fragment thereof that specifically binds to angiopoietin 2 (Ang2) and induces Tie2 activation, The antibody or antigen-binding fragment thereof (a) heavy chain complementarity determining regions (CDRs) comprising a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 11, and a CDRH3 having the amino acid sequence of SEQ ID NO: 12; (b) a light chain CDR comprising a CDRL1 having the amino acid sequence of SEQ ID NO: 13, a CDRL2 having the amino acid sequence of SEQ ID NO: 14, and a CDRL3 having the amino acid sequence of SEQ ID NO: 15; and an anti-Ang2 antibody or antigen-binding fragment thereof.

3. An antibody or an antigen-binding fragment thereof that specifically binds to angiopoietin 2 (Ang2) and induces Tie2 activation, The antibody or antigen-binding fragment thereof (a) heavy chain complementarity determining regions (CDRs) comprising a CDRH1 having the amino acid sequence of SEQ ID NO: 18, a CDRH2 having the amino acid sequence of SEQ ID NO: 19, and a CDRH3 having the amino acid sequence of SEQ ID NO: 20; (b) a light chain CDR comprising a CDRL1 having the amino acid sequence of SEQ ID NO: 21, a CDRL2 having the amino acid sequence of SEQ ID NO: 22, and a CDRL3 having the amino acid sequence of SEQ ID NO: 23; and an anti-Ang2 antibody or antigen-binding fragment thereof.

4. An antibody or an antigen-binding fragment thereof that specifically binds to angiopoietin 2 (Ang2) and induces Tie2 activation, The antibody or antigen-binding fragment thereof (a) heavy chain complementarity determining regions (CDRs) comprising a CDRH1 having the amino acid sequence of SEQ ID NO: 26, a CDRH2 having the amino acid sequence of SEQ ID NO: 27, and a CDRH3 having the amino acid sequence of SEQ ID NO: 28; (b) a light chain CDR comprising a CDRL1 having the amino acid sequence of SEQ ID NO: 29, a CDRL2 having the amino acid sequence of SEQ ID NO: 30, and a CDRL3 having the amino acid sequence of SEQ ID NO: 31; and an anti-Ang2 antibody or antigen-binding fragment thereof.

5. The antibody or antigen-binding fragment thereof The anti-Ang2 antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

9.

6. The antibody or antigen-binding fragment thereof, The anti-Ang2 antibody or antigen-binding fragment thereof according to claim 2, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

17.

7. The antibody or antigen-binding fragment thereof, The anti-Ang2 antibody or antigen-binding fragment thereof according to claim 3, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

25.

8. The antibody or antigen-binding fragment thereof, The anti-Ang2 antibody or antigen-binding fragment thereof according to claim 4, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 32 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

33.

9. The anti-Ang2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof specifically binds to angiopoietin-2 and binds to the Tie2 receptor together with Ang2.

10. An isolated nucleic acid encoding the anti-Ang2 antibody or antigen-binding fragment thereof of any one of claims 1 to 8.

11. The antigen-binding fragment may be an scFv, (scFv) ２ , scFv-Fc, Fab, Fab' and F(ab') ２ The anti-Ang2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which is selected from the group consisting of:

12. The anti-Ang2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, or a humanized antibody.

13. A pharmaceutical composition for preventing or treating cancer or an ophthalmic disease selected from the group consisting of age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy (DR), or glaucoma, comprising the anti-Ang2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 as an active ingredient.

14. The pharmaceutical composition according to claim 13, wherein the pharmaceutical composition is administered in combination with radiation and / or chemotherapy.

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