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

Abstract

The present invention relates to an anti-angiopoietin-2 antibody or antigen-binding fragment thereof, which specifically binds to angiopoietin 2 (Ang2) and induces activation of endothelial cell TEK tyrosine kinase, characterized by comprising: (a) a heavy chain complementarity determining region; and (b) a light chain complementarity determining region, the above-mentioned (a) heavy chain complementarity determining region comprising: CDRH1 of 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 CDRH3 of the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 20, or SEQ ID NO: 28, the above-mentioned (b) light chain complementarity determining region comprising: 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 CDRL3 of 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] This invention relates to an anti-angiogenic 2 antibody or a fragment thereof that specifically binds to angiopoietin-2 (Ang2), an inducing factor for angiogenesis, and binds together with angiopoietin-2 (Ang2) to the endothelial cell TEK tyrosine kinase (Tie2) receptor. Background Technology

[0002] The angiopoietin proteome consists of proteins that play an important role in the formation and maintenance of blood vessels, and there are four angiopoietins (angiopoietin-1 (Ang1), angiopoietin-2, angiopoietin-3 (Ang3), and angiopoietin-4 (Ang4)).

[0003] Angiopoietin-1 binds to the TEK tyrosine kinase receptor on endothelial cells and plays an important role in vascular maturation, attachment, migration, and survival.

[0004] Conversely, while angiopoietin-2 binds to the receptor endothelial cell TEK tyrosine kinase (TEK kinase) present on vascular endothelial cells, it acts as an antagonistic ligand. It inhibits TEK tyrosine kinase signaling by competing with angiopoietin-1 (Ang1), the agonist of TEK tyrosine kinase, for binding. Due to this mechanism, under conditions of VEGF overexpression or inflammation, vascular endothelial cells are activated, increasing vascular permeability. In this state, angiopoietin-1 induces endothelial cell stabilization and reduces vascular permeability. Conversely, the increased angiopoietin-2 in activated endothelial cells inhibits endothelial cell stabilization induced by angiopoietin-1 by competing with it. Therefore, in the presence of vascular endothelial growth factor, angiopoietin-2 inhibits the binding of angiopoietin-1-endothelial cell TEK tyrosine kinase (Ang1-Tie2), which maintains the stability of vascular endothelial cells, and its signal transduction, resulting in increased angiogenesis, leading to increased angiogenesis, vascular instability, and increased vascular permeability. On the other hand, in addition to its antagonist role in inactivating the endothelial cell TEK tyrosine kinase receptor, angiopoietin-2 has been reported to act as an agonist in inducing the activity of the endothelial cell TEK tyrosine kinase receptor under several specific conditions, including lymphangiogenesis and maintenance. Therefore, it is believed that it simultaneously possesses the functions of an antagonist and a weak agonist, and performs multiple functions depending on the situation.

[0005] Angiogenesis is essential for cancer growth; therefore, attempts have been made to inhibit angiogenesis and prevent further cancer growth by suppressing the function of angiopoietin-2, which is endothelial cell TEK tyrosine kinase-dependent, as described above. However, in most cases, this attempt only prevents angiopoietin-2 from acting as an antagonist by inhibiting its binding to endothelial cell TEK tyrosine kinase. In addition to antibodies that block the binding of angiopoietin-2 to endothelial cell TEK tyrosine kinase, recombinant proteins or antibodies that directly bind to the endothelial cell TEK tyrosine kinase receptor to induce phosphorylation and activation have also been reported.

[0006] Recently, antibodies that bind to angiopoietin-2 and together with endothelial cell TEK tyrosine kinase have been reported. These antibodies phosphorylate and activate endothelial cell TEK tyrosine kinase through endothelial cell TEK tyrosine kinase aggregation (Tie2 clustering). This not only inhibits the antagonistic effect of angiopoietin-2 but also acts as an agonist, suggesting the possibility of more effective vascular normalization.

[0007] Existing patent literature

[0008] Korean Patent Publication No. 10-2020-0144536 Summary of the Invention

[0009] Technical issues

[0010] The purpose of this invention is to provide an anti-angiogenic 2 antibody or its antigen-binding fragment that specifically binds to angiopoietin-2, which is an inducing factor for angiogenesis, and binds together with angiopoietin-2 to the endothelial cell TEK tyrosine kinase receptor to effectively induce the activation of the endothelial cell TEK tyrosine kinase receptor.

[0011] Another object of the present invention is to provide a pharmaceutical composition comprising the above-mentioned anti-angiogenic-2 antibody or its antigen-binding fragment as an active ingredient.

[0012] Another object of the present invention is to provide a pharmaceutical composition comprising the above-mentioned anti-angiogenic-2 antibody or its antigen-binding fragment as an active ingredient for treating diseases associated with angiogenesis, increased vascular permeability and / or reduced normal angiogenesis.

[0013] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer, comprising the above-mentioned anti-angiogenic-2 antibody or its antigen-binding fragment as an active ingredient.

[0014] Another object of the present invention is to provide a composition comprising the above-described anti-angiogenic-2 antibody or an antigen-binding fragment thereof for diagnosing diseases associated with angiopoietin-2 overexpression.

[0015] Another object of the present invention is to provide an antibody encoded thereon or an antigen-binding fragment thereof, a vector comprising the above-mentioned nucleic acid and a host cell, and a method for preparing an anti-angiogenic-2 antibody or an antigen-binding fragment thereof using the thereof.

[0016] Technical solution

[0017] To achieve the above objectives, the present invention provides an anti-angiogenic-2 antibody or its antigen-binding fragment thereof, which, as an antibody or its antigen-binding fragment that specifically binds to angiopoietin-2 and induces activation of endothelial cell TEK tyrosine kinase, is characterized by comprising:

[0018] (a) Heavy chain complementarity-determining region (CDR); and (b) Light chain complementarity-determining region. The aforementioned (a) heavy chain complementarity-determining region comprises: CDRH1 of the amino acid sequence of sequence 2, sequence 10, sequence 18, or sequence 26;

[0019] CDRH2 of amino acid sequences 3, 11, 19, or 27; and

[0020] CDRH3 of the amino acid sequences of sequence 4, sequence 12, sequence 20, or sequence 28. (The above...)

[0021] (b) The light chain complementarity-determining region contains: CDRL1 of the amino acid sequence of sequence 5, sequence 13, sequence 21 or sequence 29;

[0022] CDRL2 of the amino acid sequences of sequence 6, sequence 14, sequence 22, or sequence 30; and

[0023] CDRL3 of the amino acid sequences of sequence 7, sequence 15, sequence 23 or sequence 31.

[0024] In one embodiment of the present invention, preferably, the antibody or its antigen-binding fragment comprises:

[0025] (a) The heavy chain complementarity-determining region, comprising CDRH1 of amino acid sequence 2, CDRH2 of amino acid sequence 3, and CDRH3 of sequence 4; and

[0026] (b) Light chain complementarity-determining region, comprising CDRL1 of amino acid sequence 5, CDRL2 of amino acid sequence 6, and CDRL3 of amino acid sequence 7.

[0027] In yet another embodiment of the invention, preferably, the antibody or its antigen-binding fragment comprises:

[0028] (a) Heavy chain complementarity-determining region, comprising CDRH1 of amino acid sequence 10, CDRH2 of amino acid sequence 11, and CDRH3 of sequence 12; and

[0029] (b) Light chain complementarity-determining region, including, but not limited to, the CDRL1 of the amino acid sequence of sequence 13, the CDRL2 of the amino acid sequence of sequence 14, and the CDRL3 of the amino acid sequence of sequence 15.

[0030] In yet another embodiment of the invention, preferably, the antibody or its antigen-binding fragment comprises:

[0031] (a) Heavy chain complementarity-determining region, comprising CDRH1 of amino acid sequence 18, CDRH2 of amino acid sequence 19, and CDRH3 of sequence 20; and

[0032] (b) Light chain complementarity-determining region, including, but not limited to, the CDRL1 of the amino acid sequence of sequence 21, the CDRL2 of the amino acid sequence of sequence 22 and the CDRL3 of the amino acid sequence of sequence 23.

[0033] In another embodiment of the invention, preferably, the antibody or its antigen-binding fragment comprises:

[0034] (a) Heavy chain complementarity-determining region, comprising CDRH1 of amino acid sequence 26, CDRH2 of amino acid sequence 27, and CDRH3 of sequence 28; and

[0035] (b) CDRL1 of the amino acid sequence of sequence 29, CDRL2 of the amino acid sequence of sequence 30 and CDRL3 of the amino acid sequence of sequence 31, but not limited thereto.

[0036] In one embodiment of the present invention, preferably, the antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of sequence 8, sequence 16, sequence 24 and sequence 32; and a light chain variable region comprising an amino acid sequence selected from the group consisting of sequence 9, sequence 17, sequence 25 and sequence 33, but not limited thereto.

[0037] In one embodiment of the present invention, preferably, the antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, or a humanized antibody, but is not limited thereto.

[0038] The term "chimeric antibody" as used in this specification includes antibodies derived from other species, such as those with a variable region sequence derived from one species and a constant region sequence, or those with a variable region sequence derived from a mouse antibody and a constant region sequence derived from a human antibody.

[0039] As used in this specification, the term "humanized antibody" includes antibodies that graft a CDR sequence from germline cells of other mammalian species, such as mice, onto a human structure-forming region. Modifications to the added structure-forming region can occur not only within CDR sequences from germline cells of other mammalian species but also within human structure-forming sequences.

[0040] Furthermore, the present invention provides isolated nucleic acids encoding the anti-angiogenic-2 antibody or its antigen-binding fragment described above.

[0041] In one embodiment of the present invention, preferably, the antibody or its fragment specifically binds to angiopoietin-2 and binds together with angiopoietin-2 to the endothelial cell TEK tyrosine kinase receptor, but is not limited thereto.

[0042] In one embodiment of the present invention, preferably, the antigen-binding fragment is selected from the group consisting of scFv, (scFv)2, scFv-Fc, Fab, Fab' and F(ab')2, but is not limited thereto.

[0043] Furthermore, the present invention provides a pharmaceutical composition comprising the anti-angiogenic 2 antibody or its antigen-binding fragment of the present invention as an active ingredient for the prevention or treatment of diseases associated with angiopoietin-2 overexpression, angiogenesis, or increased vascular permeability.

[0044] In one embodiment of the present invention,

[0045] Preferably, the diseases associated with angiopoietin-2 overexpression, angiogenesis, or increased vascular permeability are cancer, cancer metastasis, inflammatory diseases, infections, cardiovascular diseases, kidney diseases, hereditary hemorrhagic telangiectasia, asthma, or edema, but are not limited to these.

[0046] In one embodiment of the present invention, the aforementioned cancers include carcinomas, lymphomas, blastomas, sarcomas, and leukemias, but are not limited thereto. 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 non-squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric or gastrointestinal cancer (including gastrointestinal cancer and gastrointestinal stromal carcinoma), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, hepatocellular carcinoma, bladder cancer, liver cancer, breast cancer, colorectal cancer, rectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer or renal cell carcinoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer and various forms of head and neck cancer, melanoma, superficial diffuse melanoma, malignant melanoma, acral lentigines-like melanoma, nodular melanoma, and also includes: B-cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma (NHL)); Small lymphocytic (SL) non-Hodgkin lymphoma; intermediate / follicular non-Hodgkin lymphoma; intermediate diffuse non-Hodgkin lymphoma; high-grade immunoblastic non-Hodgkin lymphoma; high-grade lymphoblastic non-Hodgkin lymphoma; high-grade small non-cracked cell non-Hodgkin lymphoma; large tumor non-Hodgkin lymphoma; mantle cell lymphoma; acquired immunodeficiency syndrome (AIDS)-associated lymphoma; Waldenström macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; post-transplant lymphoproliferative disorder (PTLD); and more, including but not limited to nevus, edema (related to brain cancer, etc.), and abnormal angiogenesis such as Megs syndrome.

[0047] Furthermore, the present invention provides a pharmaceutical composition comprising the angiopoietin-2 antibody or its antigen-binding fragment of the present invention as an active ingredient for the prevention or treatment of diseases associated with reduced normal angiogenesis.

[0048] In one embodiment of the present invention, preferably, the diseases associated with reduced normal angiogenesis are myocardial infarction, angina pectoris, cerebral infarction, stroke, Burglar disease, ischemic necrosis, foot ulcers, or erectile dysfunction, but are not limited thereto.

[0049] Furthermore, the present invention provides a pharmaceutical composition comprising the anti-angiogenic-2 antibody or its antigen-binding fragment as an active ingredient, the pharmaceutical composition being used to prevent or treat the following diseases: Alzheimer's disease; inflammatory autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, or psoriasis; ophthalmic diseases such as age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy (DR), or glaucoma; or coronavirus infections such as novel coronavirus infection.

[0050] Furthermore, the present invention provides a combination therapy composition for cancer treatment, wherein the cancer treatment comprises administering the anti-angiogenic-2 antibody or its antigen-binding fragment of the present invention simultaneously, alone or sequentially with radiation irradiation, chemotherapy agents, cytotoxic agents and / or immunotoxic agents.

[0051] In one embodiment of the present invention, preferably, the chemotherapeutic agent, cytotoxic agent and / or immunotoxic agent is selected from one or more of temozolomide, cisplatin, oxaliplatin, carboplatin, 5-FU, dacarbazine, procarbazine, vincristine, irinotecan, paclitaxel, paclitaxel, docetaxel, gemcitabine, glibenclamide, gefitinib, tarida, terbutaline, sorafenib, herceptin, bevacizumab, cetuximab, nimotuzumab, sorafenib, sunitinib and ZD6474 (ZACTIMATM), more preferably, is selected from one or more of temozolomide, cisplatin, oxaliplatin, vincristine, paclitaxel, gemcitabine, glibenclamide and gefitinib, but is not limited thereto.

[0052] The present invention will now be described.

[0053] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used in this specification is well-known and commonly used in the art to which this invention pertains.

[0054] This invention provides antibodies or their antigen-binding fragments that, while specifically binding to angiopoietin-2, do not inhibit the binding of angiopoietin-2 to the endothelial cell TEK tyrosine kinase receptor. These antibodies, forming a complex (antibody / angiopoietin-2 / endothelial cell TEK tyrosine kinase) with angiopoietin-2, bind to the endothelial cell TEK tyrosine kinase receptor to activate it like angiopoietin-1, thereby possessing a dual function of inducing endothelial cell TEK tyrosine kinase downstream signaling and inducing vascular endothelial cell stabilization.

[0055] Furthermore, the following anti-angiogenic-2 antibody and its medical uses are provided: in addition to binding with endothelial cell TEK tyrosine kinase receptors together with angiopoietin-2 to activate endothelial cell TEK tyrosine kinase receptors like angiopoietin-1, it also inhibits other proteins associated with cancer cell growth and / or cancer metastasis, such as integrin, to further enhance cancer cell growth inhibition and / or cancer metastasis inhibition effects by binding with angiopoietin-2, thereby also exerting the above effects in cells that do not express endothelial cell TEK tyrosine kinase.

[0056] In one embodiment of the present invention, a polypeptide molecule is provided, comprising: the heavy chain complementarity-determining region, the light chain complementarity-determining region, or a combination thereof of the aforementioned anti-angiogenic-2 antibody; or the heavy chain variable region, the light chain variable region, or a combination thereof. The aforementioned polypeptide molecule can function not only in the preparation of antibodies or their antigen-binding fragments, but also as a precursor or constituent of an angiopoietin-2 antagonist. For example, the aforementioned polypeptide molecule can act as an angiopoietin-2 antigen-binding site and may contain constituents including a protein scaffold (e.g., peptide antibody, nanobody) having a structure similar to that of an antibody, a bispecific antibody, a multispecific antibody, etc.

[0057] The term "antagonist" is defined as a concept that includes all molecules that partially or completely block, inhibit, or neutralize the biological activity of a target substance (such as angiopoietin-2).

[0058] The term "peptide-antibody" refers to a fusion protein in which all or part of the constant regions of a peptide and an antibody, such as the Fc region, are fused together. The peptide acts as an antigen-binding site (heavy chain and / or light chain CDR or variable region), and has a scaffold and function similar to that of an antibody.

[0059] The term "nanobody," also known as a single-domain antibody, refers to an antibody fragment containing a single variable domain in monomeric form. Similar to a complete antibody, it selectively binds to a specific antigen. Nanobodies typically have a molecular weight of approximately 12 kDa to 15 kDa, which is very small compared to the typical molecular weight (approximately 150 kDa to 160 kDa) of a complete antibody (containing two heavy chains and two light chains), and, depending on the case, even smaller than Fab fragments or scFv fragments.

[0060] The term "bispecific antibody" or "multispecific antibody" refers to an antibody that recognizes and / or binds to two or more different antigens (bispecific antibody) or multispecific antibody, or recognizes and / or binds to different sites of the same antigen, wherein one of the antigen-binding sites of the aforementioned bispecific antibody or multispecific antibody may contain the aforementioned polypeptide.

[0061] In a specific example, the aforementioned polypeptide molecule may comprise one or more of the following groups: polypeptides comprising amino acid sequences of sequence 2, sequence 10, sequence 18 or 26; polypeptides comprising amino acid sequences of sequence 3, sequence 11, sequence 19 or 27; and polypeptides comprising amino acid sequences of sequence 4, sequence 12, sequence 20 or 28.

[0062] The polypeptide is selected from one or more of the group consisting of a polypeptide containing an amino acid sequence of sequence 5, sequence 13, sequence 21 or sequence 29, a polypeptide containing an amino acid sequence of sequence 6, sequence 14, sequence 22 or sequence 30, and a polypeptide containing an amino acid sequence of sequence 7, sequence 15, sequence 23 or sequence 31; or a combination thereof.

[0063] In a specific example, the aforementioned polypeptide molecule may contain amino acid sequences of sequence 8, sequence 16, sequence 24 or sequence 32 and amino acid sequences of sequence 9, sequence 17, sequence 25 or sequence 33 or combinations thereof.

[0064] The terms “Kabat numbering,” “Kabat definition,” and “Kabat tag” are used interchangeably in this specification. These terms, recognized in the art to which this invention pertains, refer to a numbering system of amino acid residues that indicates a greater diversity (i.e., hypervariable) of the relevant sites of variable regions or antigen-binding sites of the heavy and light chains of an antibody than other amino acid residues (Kabat et al. (1971), Ann. NY Acad. Sci. 190: 382-391; Kabat, E.A. et al. (1991), Immunologically Significant Protein Sequences, 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).

[0065] As described above, the aforementioned bispecific or multispecific antibodies comprise antigen-binding sites for two or more distinct antigens, simultaneously recognizing and / or binding to two or more antigens. One of the antigen-binding sites may contain the polypeptide molecule described above. Specifically, the polypeptide molecule that functions as the angiopoietin-2 antigen-binding site can form a bispecific or multispecific antibody by forming a dimer or polymer with antigen-binding sites of other relevant antigens. Therefore, in one specific example, a bispecific or multispecific antibody comprising the aforementioned polypeptide molecule as an angiopoietin-2 antigen-binding site is provided.

[0066] In other examples, a protein scaffold is provided comprising one or more (e.g., 1 to 5 or 2 to 4) polypeptide molecules comprising one or more of the aforementioned polypeptide molecules or repeats of the aforementioned polypeptide molecules linked by a linker (hereinafter referred to as "first peptide") and a polypeptide that performs a structural function (hereinafter referred to as "second peptide"; for example, a constant region of the heavy or light chain of an antibody (IgG, IgA, IgE, IgD, IgM, etc.) or an Fc fragment of an antibody), wherein the one or more peptide complexes are bound in the second peptide (e.g., the Fc fragment) to have a multimeric structure.

[0067] In this invention, antibodies include animal-derived antibodies, chimeric antibodies, humanized antibodies, and human antibodies. Animal-derived antibodies, produced by immunizing an immunized animal with a desired antigen, often induce immune rejection when administered to humans for therapeutic purposes. Chimeric antibodies have been developed to suppress such immune rejection. A chimeric antibody is created by replacing the constant region of an animal-derived antibody, which would otherwise cause an anti-isotype reaction, with the constant region of a human antibody using genetic engineering. While chimeric antibodies offer a significant improvement in anti-isotype reactions compared to animal-derived antibodies, the variable region still contains animal-derived amino acids, thus carrying the potential side effect of anti-idiotypic reactions. Humanized antibodies have been developed to mitigate this side effect. This is achieved by transplanting the CDR (complementarity determining regions) region, which plays a crucial role in antigen binding, from the variable region of a chimeric antibody into a human antibody scaffold.

[0068] The most crucial aspect of CDR grafting technology for preparing humanized antibodies is selecting the optimal human antibody that best accepts the CDR site of the animal-derived antibody. This can be achieved using antibody databases, crystal structure analysis, and molecular modeling techniques. However, even when the CDR site of an animal-derived antibody is grafted into an optimized human antibody scaffold, some amino acids remaining in the animal-derived antibody scaffold still affect antigen binding, resulting in a significant portion of antigen-binding capacity not being preserved. Therefore, the application of additional antibody engineering techniques to restore antigen-binding capacity is essential.

[0069] According to a specific example, the above-mentioned antibodies can be mouse-derived antibodies, mouse-human chimeric antibodies, humanized antibodies, or human antibodies.

[0070] In this invention, "antibody" refers to a substance produced within the immune system in response to antigen stimulation, and its type is not particularly limited. Recently, antibodies have been widely used as therapeutic agents for diseases. Antibodies are stable both in vitro and in vivo, and have long half-lives, thus facilitating large-scale expression and production. Furthermore, antibodies inherently possess a dimer structure, resulting in very high affinity.

[0071] A complete antibody has a structure consisting of two full-length light chains and two full-length heavy chains, with each light chain linked to the heavy chain by a disulfide bond. The constant regions of an antibody are divided into heavy chain constant regions and light chain constant regions. Heavy chain constant regions include gamma (γ), muon (μ), alpha (α), delta (6), and epsilon (ε) types, with subtypes including gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). Light chain constant regions include kappa (κ) and lambda (λ) types.

[0072] The term "heavy chain" is defined as including a full-length heavy chain and fragments thereof, comprising a variable region domain (VH) with a sufficient amino acid sequence to confer specificity against the antigen, three constant region domains (CH1, CH2, and CH3), and a hinge. Similarly, the term "light chain" is defined as including a full-length light chain and fragments thereof, comprising a variable region domain (VL) with a sufficient amino acid sequence to confer specificity against the antigen, and a constant region domain (CL).

[0073] The term "complementarity determining region (CDR)" refers to the amino acid sequence of the hypervariable region of the heavy and light chains of an immunoglobulin. The heavy and light chains may each contain three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). These CDRs provide the major contact residues in the binding of antibodies to antigens or epitopes. On the other hand, in this specification, the terms "specific binding" or "specific recognition" have the same meaning as commonly known to those skilled in the art to which this invention pertains, referring to the elicitation of an immune response through the specific interaction of antigen and antibody.

[0074] The antigen-binding fragment of the antibody provided in this invention may be a fragment containing one or more of the above-mentioned complementarity-determining regions.

[0075] The term "antigen-binding fragment" refers to a portion of a polypeptide containing the part capable of binding to an antigen, as opposed to the entire structure of an immunoglobulin. Examples include, but are not limited to, scFv, (scFv)2, scFv-Fc, Fab, Fab', or F(ab')2.

[0076] In the aforementioned antigen-binding fragments, Fab is a structure with variable regions of both the light and heavy chains, a constant region of the light chain, and the first constant region (CH1) of the heavy chain, possessing 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 CH1 domain of the heavy chain. The F(ab')2 antibody is generated by forming disulfide bonds between cysteine ​​residues in the hinge region of Fab'. Fv is the smallest antibody fragment containing only the variable regions of the heavy and light chains. Recombinant techniques for generating Fv fragments are well-known in the art to which this invention pertains. Two-chain Fv connects the variable regions of the heavy and light chains via non-covalent bonds, while single-chain Fv typically connects the variable regions of the heavy and single chains via peptide linkers, either covalently or directly at the C-terminus, thus forming a dimer-like structure similar to two-chain Fv. The aforementioned linker can be a peptide linker formed from any number of amino acids, ranging from 1 to 100 or 2 to 50, and suitable sequences are well known in the art to which this invention pertains. The aforementioned antigen-binding fragment can be obtained using proteolytic enzymes (e.g., Fab can be obtained by limited cleavage of an intact antibody using papain, and F(ab')2 fragments can be obtained by cleavage using pepsin), or it can be prepared using gene recombination technology.

[0077] The term "hunge region" refers to the region containing the heavy chain of an antibody, located between the CH1 and CH2 regions. It describes the region that provides flexibility for the antigen-binding site within the antibody. For example, this hinge can be derived from human antibodies, specifically from IgA, IgE, or IgG, such as IgG1, IgG2, IgG3, or IgG4.

[0078] When animal-derived antibodies undergo chimerization, although the animal-derived IgG1 hinge is replaced with a human IgG1 hinge, the length of the animal-derived IgG1 hinge is shorter than that of the human IgG1 hinge, and the number of disulfide bonds between the two heavy chains is reduced from three to two, the rigidity of the hinge exhibits different effects. Therefore, modification of the hinge region can increase the antigen-binding effectiveness of humanized antibodies. Methods for deleting, adding, or substituting amino acids in the amino acid sequence used to modify the aforementioned hinge region are well known to those skilled in the art to which this invention pertains.

[0079] The portion of the anti-angiogenic-2 antibody other than the variable region can be a constant region derived from human antibodies, specifically, a constant region derived from IgA, IgE, or IgG, for example, a constant region derived from IgG1, IgG2, IgG3, or IgG4.

[0080] Anti-angiogenic factor-2 antibodies can be monoclonal antibodies. Monoclonal antibodies can be prepared using methods well-known in the art to which this invention pertains. For example, they can be prepared using phage display technology. Alternatively, anti-angiogenic factor-2 antibodies can be prepared from mouse monoclonal antibodies using the method described in the paper by 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).

[0081] On the other hand, individual monoclonal antibodies can be screened based on their binding affinity to angiopoietin-2 using typical enzyme-linked immunosorbent assays (ELISA). For the conjugates, inhibitory activity can be tested using functional analyses such as competitive ELISA (for examining molecular interactions) or cell-based assays. Then, for monoclonal antibody members selected based on strong inhibitory activity, their affinity for angiopoietin-2 (Kd values) is individually tested.

[0082] The final selected antibody, excluding the antigen-binding site, can be prepared not only as an antibody by human immunoglobulinization, but also as a humanized antibody. The method for preparing humanized antibodies is well-known in the technical field to which this invention pertains (Almagro, J.C. and Fransson, J., "Humanization of antibodies," Frontiers in Bioscience, 13 (2008), 1619-1633).

[0083] Other examples provide pharmaceutical compositions for inhibiting angiogenesis, comprising the aforementioned anti-angiogenic-2 antibody or its antigen-binding fragment as an active ingredient. Other examples provide methods for inhibiting angiogenesis, including the step of administering a pharmaceutically effective amount of the aforementioned anti-angiogenic-2 antibody or its antigen-binding fragment to a patient requiring inhibition of angiogenesis. The methods for inhibiting angiogenesis may further include, prior to the administration step, a step of identifying the patient requiring inhibition of angiogenesis.

[0084] Other examples provide pharmaceutical compositions for reducing vascular permeability that contain the aforementioned anti-angiogenic-2 antibody or its antigen-binding fragment as an active ingredient. Other examples provide methods for reducing vascular permeability, including the step of administering a pharmaceutically effective amount of the aforementioned anti-angiogenic-2 antibody or its antigen-binding fragment to a patient requiring reduced vascular permeability. The methods for reducing vascular permeability may further include, prior to the administration step, a step of identifying the patient requiring reduced vascular permeability.

[0085] Other examples provide pharmaceutical compositions for the prevention and / or treatment of diseases associated with angiopoietin-2 overexpression, angiogenesis, and / or increased vascular permeability, comprising the aforementioned anti-angiogenic-2 antibody or its antigen-binding fragment as an active ingredient. Other examples provide methods for the prevention and / or treatment of diseases associated with angiopoietin-2 overexpression, angiogenesis, and / or increased vascular permeability, comprising administering a pharmaceutically effective amount of the aforementioned anti-angiogenic-2 antibody or its antigen-binding fragment to a patient requiring prevention and / or treatment of diseases associated with angiopoietin-2 overexpression, angiogenesis, and / or increased vascular permeability. The aforementioned prevention and / or treatment methods may further include, prior to the administration step, a step of identifying a patient requiring prevention and / or treatment of diseases associated with angiopoietin-2 overexpression, angiogenesis, and / or increased vascular permeability.

[0086] Other examples provide pharmaceutical compositions for inducing normal angiogenesis that contain the aforementioned anti-angiogenic-2 antibody or its antigen-binding fragment as an active ingredient. Other examples provide methods for increasing normal angiogenesis, including the step of administering a pharmaceutically effective amount of the aforementioned anti-angiogenic-2 antibody or its antigen-binding fragment to a patient who requires the induction of normal angiogenesis. The methods for increasing normal angiogenesis may further include, prior to the administration step, a step of identifying a patient who requires the induction of normal angiogenesis.

[0087] Other examples provide pharmaceutical compositions for the prevention and / or treatment of diseases associated with reduced normal angiogenesis, comprising the aforementioned anti-angiogenic-2 antibody or its antigen-binding fragment as an active ingredient. Other examples provide methods for the prevention and / or treatment of diseases associated with reduced normal angiogenesis, including the step of administering a pharmaceutically effective amount of the aforementioned anti-angiogenic-2 antibody or its antigen-binding fragment to a patient requiring prevention and / or treatment of a disease associated with reduced normal angiogenesis. The aforementioned prevention and / or treatment methods may further include, prior to the administration step, a step of identifying a patient requiring prevention and / or treatment of a disease associated with reduced normal angiogenesis.

[0088] Other examples provide compositions comprising the antibodies described above or their antigen-binding fragments for the diagnosis of diseases associated with neovascularization and / or increased vascular permeability and / or decreased normal angiogenesis.

[0089] The above-mentioned pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, which is commonly used in the formulation of the drug and may be one or more selected from the group consisting of lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil, but is not limited thereto. Furthermore, the above-mentioned pharmaceutical composition may also comprise one or more selected from the group consisting of diluents, excipients, lubricants, humectants, sweeteners, flavorings, emulsifiers, suspending agents, and preservatives commonly used in the preparation of pharmaceutical compositions.

[0090] The pharmaceutically effective amount of the above-described pharmaceutical composition or the above-described antibody or its antigen-binding fragment can be administered orally or parenterally. In the case of parenteral administration, it can be administered via intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, lesion administration, intranasal administration, intrapulmonary administration, and rectal administration. For oral administration, since proteins or peptides are digested, the oral composition can be coated with the active pharmaceutical ingredient or formulated in a manner that protects it from degradation. Furthermore, the above-described composition can be administered via any device capable of enabling the active substance to move to target cells.

[0091] The content of the anti-angiogenic 2 antibody or its antigen-binding fragment in the above-mentioned pharmaceutical composition can be varied depending on factors such as the preparation method, administration route, patient's age, weight, sex, disease state, diet, administration time, administration interval, route of administration, metabolic rate, and response sensitivity. For example, the daily dose of the above-mentioned anti-angiogenic 2 antibody or its antigen-binding fragment can range from 0.001 mg / kg to 1000 mg / kg, specifically from 0.01 mg / kg to 100 mg / kg, and more specifically from 0.1 mg / kg to 50 mg / kg, but is not limited thereto. The above-mentioned daily dose can be prepared as a single-dose formulation, or in appropriate quantities, or in multi-dose containers. The above-mentioned "pharmaceuticalally effective amount" can refer to the content or dosage of the active ingredient that can exhibit the desired pharmacological effect, and can be determined in various ways depending on factors such as the preparation method, administration route, patient's age, sex, disease state, diet, administration time, administration interval, route of administration, metabolic rate, and response sensitivity.

[0092] The above-mentioned pharmaceutical composition can be formulated as a solution, suspension, syrup or emulsion in an oil or aqueous medium, or as an extract, powder, granule, tablet or capsule, etc., and may also contain dispersants or stabilizers for dosage form.

[0093] In particular, pharmaceutical compositions containing the aforementioned anti-angiogenic-2 antibody or its antigen-binding fragment contain an antibody or antigen-binding fragment, and therefore can be formulated into immunoliposomes. Liposomes containing antibodies can be prepared using methods well-known in the art to which this invention pertains. The aforementioned immunoliposomes, as lipid compositions containing phosphatidylcholine, cholesterol, and polyethylene glycol-derived phosphatidylethanolamine, can be prepared by reverse evaporation. For example, the Fab' fragment of the antibody can be linked to the liposome via a disulfide substitution reaction.

[0094] On the other hand, the aforementioned anti-angiogenic-2 antibody or its antigen-binding fragment specifically binds to angiopoietin-2, thus enabling the detection of angiopoietin-2 and the confirmation of whether angiopoietin-2 is overexpressed. Therefore, other embodiments of the present invention provide an angiopoietin-2 detection composition comprising the aforementioned anti-angiogenic-2 antibody or its antigen-binding fragment, and a diagnostic composition for diseases related to angiopoietin-2 overexpression.

[0095] The effects of the invention

[0096] This invention proposes an antibody that promotes downstream signaling by simultaneously inhibiting angiopoietin-2 and activating the endothelial cell Tek tyrosine kinase receptor, thereby revealing a novel method to inhibit angiogenesis induced by angiopoietin-2 and reduce vascular permeability. Furthermore, the antibody proposed in this invention holds promise for the diagnosis and treatment of abnormal angiogenesis-related diseases other than cancer and / or diseases induced by increased vascular permeability. This antibody can be used in combination therapies with chemotherapy drugs and other anticancer agents, and leveraging its specific recognition of angiopoietin-2, it holds promise for use in antibody fragments, bi- or multi-specific antibodies, protein scaffolds, etc. Attached Figure Description

[0097] Figure 1 To illustrate the results of Western blot analysis of AKT and ERK 42 / 44 phosphorylation of anti-angiogenic-2 antibody and angiopoietin-2 in HUVEC cells.

[0098] Figure 2 To illustrate the results of enzyme-linked immunosorbent assay (ELISA) showing the concentration-dependent induction of AKT phosphorylation in HUVEC cells by anti-angiogenic antibody and angiopoietin-2. The clones are listed sequentially from the curve with the highest value: 1D3, then 2C8, then 1A9, then 1H10, and the clone with the lowest value is clone 67, which served as the control group.

[0099] Figure 3 The binding affinity of anti-angiogenic-2 antibodies for human and mouse angiopoietin-2 was analyzed by enzyme-linked immunosorbent assay (ELISA).

[0100] Figure 4 This study aims to demonstrate the results of an enzyme-linked immunosorbent assay (ELISA) showing the formation of a complex by binding anti-angiogenic-2 antibody with angiopoietin-2 and endothelial cell Tek tyrosine kinase.

[0101] Figure 5 The results of enzyme-linked immunosorbent assay (ELISA) were used to demonstrate that anti-angiogenic-2 antibody 2C8 does not inhibit the binding of angiopoietin-2 to endothelial cell TEK tyrosine kinase.

[0102] Figure 6 The results show the effect of anti-angiogenic-2 antibody 2C8 on tumor growth inhibition in an LLC lung cancer cell tumor model (error bar: standard error of mean).

[0103] Figure 7 The combined administration of anti-angiogenic-2 antibody 2C8 and cisplatin showed significantly better tumor growth inhibition in an LLC lung cancer cell tumor model compared to administration of either antibody alone (error bars: standard error of the mean).

[0104] Figure 8 The combined administration of anti-angiogenic-2 antibody 2C8 and 5FU showed that in the MC38 colorectal cancer cell tumor model, the combined administration of these two antibodies had a significantly better tumor growth inhibition effect than the administration of either antibody alone (error bars: standard error of the mean). Detailed Implementation

[0105] The present invention will now be described in more detail by way of embodiments, but these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. It will be apparent to those skilled in the art that the embodiments described below can be modified without departing from the essential spirit of the invention.

[0106] Example 1: Preparation and screening of anti-angiogenic factor-2 antibodies

[0107] As an antigen, an antibody that specifically binds to human angiopoietin-2 (Sequence 1) was commissioned from Abclon (Korea). The preparation of the antibody of the present invention is based on Kohler and Milstein, Eur. J. Immunol. 6, 511 (1976).

[0108] In simple terms, human angiopoietin-2 was mixed with an adjuvant (Sigma) and injected twice into mice. The presence of antibodies was determined by enzyme-linked immunosorbent assay (ELISA). After the two immunizations, spleens were harvested from mice immunized with increased antibody titers (1:5000), and B lymphocytes were isolated and fused with sp2 / 0 cells. The fused cells were cultured in HAT medium supplemented with hypoxantine, aminopterine, and thymidine to selectively screen for B lymphocyte-sp2 / 0 cell fusion (hybridoma). The obtained hybridoma cells were then repeatedly separated into positive and negative cells using a serial dilution method (cloning) to prepare monoclonal cells for producing antibodies that react with the antigen.

[0109] After the obtained antibody-producing hybridoma cells were cultured in Dulbecco's Modified Eagle's Mediaum (DMEM) containing 10% (v / v) fetal bovine serum (FBS) at 37°C and 5% CO2, the antibody-producing cells were separated by centrifugation. The culture medium containing the isolated antibodies was then separated and the antibodies were purified using an affinity column (Protein A / G agarose column, Protein A / G (GenDEPOT)).

[0110] Example 2: Western blot analysis of activation of endothelial cell TEK tyrosine kinase signaling induced by anti-angiogenic factor-2 antibody.

[0111] Angiopoietin-2 acts as a weak agonist or antagonist by binding to the endothelial cell TEK tyrosine kinase receptor expressed in vascular endothelial cells. The anti-angiopoietin-2 antibody developed in this invention binds to angiopoietin-2 to form a complex with angiopoietin-2-endothelial cell TEK tyrosine kinase, thereby promoting downstream signal transduction by activating the endothelial cell TEK tyrosine kinase receptor. To analyze the effect of the anti-angiopoietin-2 antibody on downstream signal transduction of endothelial cell TEK tyrosine kinase using cell-based assays, ERK and AKT phosphorylation experiments were performed. To compare the degree of activation of downstream signal transduction of endothelial cell TEK tyrosine kinase, the same experiments were performed on groups treated with angiopoietin-2 (Sino Biological) and other angiopoietin-2 control antibodies (Korean Patent Publication No. 10-2015-0136031).

[0112] Specifically, HUVEC (Lonza) cells (2×10⁻⁶) were cultured in EGM-2 (Lonza) medium at 37°C. 5After the confluence reached 80%–90%, the medium was replaced with 0.5% fetal bovine serum basal medium (FBS basal media) (Lonza Biosciences) and cultured at 37°C for 16–24 hours. A mixture of 60 nM anti-angiogenic-2 antibody and 40 nM angiopoietin-2 protein (Sinochem Biosciences) was prepared and incubated for 30 minutes. The resulting cultured cells were then treated and cultured for another 10 minutes. For comparison, groups were prepared treated with 40 nM angiopoietin-2 and groups treated with 40 nM angiopoietin-2 plus 60 nM anti-angiogenic-2 control antibody. After washing the cells with phosphate-buffered saline (PBS), the cells were treated with lysis buffer (BIOSESANG Ripa buffer, 0.15 M sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl, pH 7.5, and 2 mM EDTA) and then centrifuged at 13,000 rpm for 15 minutes to recover the supernatant to obtain cell lysates.

[0113] 35 μg of cell lysate was added to sample buffer (BIOSESANG) containing a reducing agent and boiled at 95°C for 5 minutes. The mixture was then electrophoresed in a 10% Tris-Glycine gel and transferred to a nitrocellulose membrane (GVS). To confirm the phosphorylation of Akt and ERK42 / 44, which are downstream signaling pathways of the endothelial cell TEK tyrosine kinase receptor, the blot was blocked for 1 hour with phosphate-Tween buffer (PBST) containing 5% (v / v) skimmilk (Seoul milk). Anti-phosphorylated Akt antibody (Cell Signaling), anti-AKT antibody (Cell Signaling), anti-phosphorylated ERK42 / 44 antibody (Cell Signaling), and anti-ERK42 / 44 antibody (Santa Cruz) were then applied. The results obtained are as follows: Figure 1 As shown.

[0114] like Figure 1 As shown, compared with the negative control group, 1A9, 1D3, 2C8, and 1H10 clones exhibited AKT and ERK activation signals.

[0115] Example 3: Enzyme-linked immunosorbent assay (ELISA) for activation of downstream signaling of endothelial cell Tek tyrosine kinase induced by anti-angiogenic-2 antibody.

[0116] To quantitatively analyze the effect of anti-angiogenic-2 antibody on downstream signal transduction of TEK tyrosine kinase in endothelial cells, AKT phosphorylation level was determined by enzyme-linked immunosorbent assay (ELISA).

[0117] HUVEC (Lonza) cells (2×10⁻⁶) were cultured in EGM-2 (Lonza) medium at 37°C. 5 After reaching 80%–90% confluence, the culture medium was replaced with 0.5% fetal bovine serum (FBS) and cultured at 37°C for 16–24 hours. A mixture of 60 nM anti-angiogenic-2 antibody and 40 nM angiopoietin-2 protein (Sinochem) was added and incubated for 30 minutes. The cultured cells were then treated and cultured for another 10 minutes. For comparison, groups treated with 40 nM angiopoietin-2 and groups treated with 40 nM angiopoietin-2 + 60 nM anti-angiogenic-2 (h10D6-OPTI-67, US 10934350) were prepared. Cells were washed with phosphate-buffered saline (PBS), treated with lysis buffer (BIOSESANG), and centrifuged at 13000 rpm for 15 minutes to obtain cell lysates.

[0118] For the enzyme-linked immunosorbent assay (ELISA), the capture antibody of the PathScan Phospho-Akt1 (Ser473) sandwich ELISA antibody pair (Cell Signaling) was diluted 1:100 and 50 μL was added to a 96-well ps half-area ELISA plate (Greiner Bio-One) for coating. The plate was then washed four times with phosphate-Tween buffer (phosphate buffer containing 0.05% (v / v) Tween-20), and blocked at 37°C for 2 hours with phosphate-Tween buffer containing 1% (v / v) skim milk. Then, the ELISA plate was washed four times with phosphate-Tween buffer (PBST) containing 0.05% Tween 20, and cell lysates were added to allow phosphorylated AKT to bind with the capture antibody at 37°C for 2 hours. After washing the ELISA plate four times with phosphate-Tween buffer, secondary antibody diluted 1:1000 with 1% skim milk was added and allowed to bind for 1 hour at 37°C, followed by four washes with phosphate-Tween buffer. Finally, 50 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Kementec) was added to the ELISA plate to induce a colorimetric reaction for 10 minutes. Then, 50 μL of stop solution was added to terminate the reaction, and the OD450 value was measured on a plate reader (BioTek). The results obtained are as follows: Figure 2 As shown.

[0119] like Figure 2 As shown, it can be confirmed that, compared with existing known control antibodies, the monoclonal antibodies 1A9, 1D3, 2C8, and 1H10 of the present invention induce significantly stronger downstream signals of endothelial cell TEK tyrosine kinase.

[0120] Example 4: Gene Cloning of Mouse Anti-Angiopoietin-2 Antibody

[0121] RNA was isolated from the hybridomas obtained in Example 1 above using the AccuPrep Universal RNA extraction kit (Bioneer). Then, using this as a template, complementary deoxyribonucleic acid (cDNA) was synthesized and cloned according to known methods (Meyer L et al., Asimplified workflow for monoclonal antibody sequencing 2019, PLoS ONE). CDNA synthesis was performed using the SuperiorScript III cDNA Synthesis kit (Enzynomics). The synthesized cDNA was amplified by polymerase chain reaction (PCR) and cloned in a vector using the TOPcloner Blunt core kit (Enzynomics). DNA base sequence analysis was performed to obtain the complementarity-determining regions (CDRs), heavy chain variable regions, and light chain variable regions encoding each antibody, along with their base and amino acid sequences (Tables 1 to 8).

[0122] Table 1

[0123]

[0124]

[0125] Table 1 shows the CDR sequence of mouse anti-angiogenic-2 antibody 1A9.

[0126] Table 2

[0127]

[0128] Table 2 shows the variable region sequence of mouse anti-angiogenic-2 antibody 1A9.

[0129] Table 3

[0130]

[0131] Table 3 shows the CDR sequence of mouse anti-angiogenic-2 antibody 2C8.

[0132] Table 4

[0133]

[0134]

[0135] Table 4 shows the variable region sequence of mouse anti-angiogenic-2 antibody 2C8.

[0136] Table 5

[0137]

[0138] Table 5 shows the CDR sequence of mouse anti-angiogenic-2 antibody 1D3.

[0139] Table 6

[0140]

[0141] Table 6 shows the variable region sequence of mouse anti-angiogenic-2 antibody 1D3.

[0142] Table 7

[0143]

[0144] Table 7 shows the CDR sequence of mouse anti-angiogenic-2 antibody 1H10.

[0145] Table 8

[0146]

[0147] Table 8 shows the variable region sequence of mouse anti-angiogenic-2 antibody 1H10.

[0148] Example 5: Enzyme-linked immunosorbent assay (ELISA) of anti-angiogenic-2 antibody binding to human or mouse angiopoietin-2

[0149] To confirm the binding affinity of the anti-angiogenic-2 antibody to human or mouse angiopoietin-2, an enzyme-linked immunosorbent assay (ELISA) was performed. The assay was conducted in 96-well MaxiSorp... TM Flat bottom (96-well MaxiSorp) TMFlat-bottom ELISA plates were coated with 1 μg / mL of human angiopoietin-2 (Essential Biotech) or mouse angiopoietin-2 (Acrobiosystems). The plates were then washed five times with phosphate-Tween buffer (containing 0.05% (v / v) Tween 20), and blocked for 2 hours at room temperature with phosphate-Tween buffer containing 1% (v / v) skim milk. After washing five times with phosphate-Tween buffer containing 0.05% Tween 20, the plates were inoculated with 10 serially diluted (3-fold) concentrations of anti-angiogenic 2 starting at 300 nM and bound for 2 hours at room temperature. After washing five times with phosphate-Tween buffer containing 0.05% Tween 20, the plate was then bound with 1% skim milk containing a 1:3000 dilution of goat anti-mouse IgG / horseradish peroxidase (Goat anti-Mouse IgG / HRP) (Solarbio) for one hour, followed by six washes with phosphate-Tween buffer. Finally, 100 μL of 3,3',5,5'-tetramethylbenzidine substrate (Commentech) was added to the plate to induce a colorimetric reaction for 10 minutes. The reaction was then terminated by adding 100 μL of stop solution (2M sulfuric acid (H2SO4)), and the OD450 value was measured using a microplate reader (Berten). The results obtained are as follows: Figure 3 As shown.

[0150] Example 6: Enzyme-linked immunosorbent assay (ELISA) for confirming the formation of the antibody-angiopoietin-2-endothelial cell Tek tyrosine kinase complex for anti-angiopoietin-2 antibody

[0151] To confirm the formation of a complex between anti-angiogenic 2 antibody and angiopoietin-2 and the endothelial cell TEK tyrosine kinase receptor, an enzyme-linked immunosorbent assay (ELISA) was performed. The assay was performed in 96-well MaxiSorp... TMFlat-bottomed ELISA plates were coated with 2 μg / ml of endothelial cell TEK tyrosine kinase ECD (Sinochem Biotech). The plates were then washed five times with phosphate-Tween buffer (containing 0.05% (v / v) Tween 20 phosphate buffer), followed by blocking at room temperature for 2 hours with phosphate-Tween buffer containing 1% (v / v) skim milk. The plates were then washed five times with phosphate-Tween buffer containing 0.05% Tween 20, and a pre-prepared anti-angiogenic-2 antibody-angiogenic-2 complex was added and reacted at room temperature for 2 hours to bind to the endothelial cell TEK tyrosine kinase. The anti-angiogenic-2 antibody-angiogenic-2 complex was prepared as follows: Anti-angiogenic-2 antibody was added to 1% skim milk at a concentration of 600 nM, and then serially diluted 3-fold with 1% skim milk to prepare 10 concentrations. Angiopoietin-2 was mixed 1:1 with a sample diluted to 1 μg / mL in 1% skim milk. After washing the ELISA plate 5 times with phosphate-Tween buffer containing 0.05% Tween 20, goat anti-mouse IgG / horseradish peroxidase (Solepro) diluted 1:3000 in 1% skim milk was conjugated for 1 hour, followed by washing 6 times with phosphate-Tween buffer. Finally, 100 μL of 3,3',5,5'-tetramethylbenzidine substrate (Comtech) was added to the above ELISA plate to induce the colorimetric reaction for 10 minutes. Then, 100 μL of stop solution (2M sulfuric acid) was added to terminate the reaction, and the OD450 value was measured on an ELISA reader (Berten). The results obtained are as follows: Figure 4 As shown.

[0152] Example 7: Competitive enzyme-linked immunosorbent assay (ELISA) of anti-angiogenic-2 antibody binding to angiopoietin-2-endothelial cell TEK tyrosine kinase.

[0153] Angiopoietin-2 binding competition ELISA was performed using an anti-angiopoietin-2 antibody in endothelial cells to bind TEK tyrosine kinase. More specifically, in 96-well MaxiSorp... TMFlat-bottomed ELISA plates were coated with 4 μg / ml of endothelial cell TEK tyrosine kinase ECD (Sinochem Biotechnology Co., Ltd.). The plates were then washed five times with phosphate-Tween buffer (containing 0.05% (v / v) Tween 20 phosphate buffer solution), followed by blocking at room temperature for 2 hours with phosphate-Tween buffer containing 1% (v / v) bovine serum albumin (BSA). After washing five times with phosphate-Tween buffer containing 0.05% Tween 20, the plates were incubated with a biotin-labeled human angiopoietin-2 and anti-angiogenic-2 antibody complex or a mixture of biotin-labeled human angiopoietin-2 and unlabeled human angiopoietin-2 for 2 hours at room temperature to bind to the endothelial cell TEK tyrosine kinase. In this case, biotin-labeled human angiopoietin-2 is prepared as follows: 10 μl of biotin-labeled human angiopoietin-2 (>1 mg / ml) is mixed with 1 μl of a modifier reagent. The cap of the vial containing the biotin conjugation mix is ​​removed, and the angiopoietin-2-modifier solution is added. The mixture is reacted at room temperature in the dark for 15 minutes. Then, 1 μl of a quencher reagent is added, and the reaction is continued for 5 minutes. The complex of anti-angiogenic-2 antibody and biotin-labeled angiopoietin-2 was prepared as follows: Anti-angiogenic-2 antibody was added to 1% bovine serum albumin at a concentration of 1.2 μM, and then diluted 5-fold with 1% bovine serum albumin to prepare 10 concentrations. This was then mixed 1:1 with a sample containing biotin-labeled human angiopoietin-2 diluted to 1 μg / mL in 1% bovine serum albumin. The mixture of biotin-labeled human angiopoietin-2 and unlabeled human angiopoietin-2 was prepared as follows: Angiopoietin-2 was added to 1% bovine serum albumin at a concentration of 1.2 μM, and then serially diluted 5-fold with 1% bovine serum albumin to prepare 10 concentrations. This was then mixed 1:1 with a sample containing biotin-labeled human angiopoietin-2 diluted to 1 μg / mL in 1% bovine serum albumin. After washing the microplate five times with phosphate-Tween buffer containing 0.05% Tween 20, it was bound to streptavidin horseradish peroxidase (streptavidin HRP) (Abcam) diluted 1:10000 in 1% bovine serum albumin for 1 hour, and then washed six times with phosphate-Tween buffer.Finally, 100 μL of 3,3',5,5'-tetramethylbenzidine substrate (Comtech) was added to the above ELISA plate to induce the colorimetric reaction for 10 minutes. Then, 50 μL of stop solution (2M sulfuric acid) was added to terminate the reaction, and the OD450 value was measured on an ELISA reader (Berten). The results obtained are as follows. Figure 5 As shown.

[0154] Example 8: Tumor growth inhibition effect of anti-angiogenic-2 antibody in mouse tumor model

[0155] To confirm the tumor growth inhibitory effect of the anti-angiogenic-2 antibody, a mouse tumor model was used. The tumor size (V) was calculated using the following formula:

[0156] V = Tumor volume (mm) 3 = (length × width) 2 ) / 2(V=Tumor volume(mm 3 ) = (length × width) 2 ) / 2).

[0157] A. LLC lung cancer cell tumor model

[0158] To confirm the tumor growth inhibitory effect of anti-angiogenic factor-2, a syngeneic mouse model of lung cancer was used, utilizing the Lewis lung cancer (LLC, Lewis lung carcinoma, ATCC) cell line. LLC cells were cultured in DuPont modified Igor medium (DMEM) (Welgene) supplemented with 10% fetal bovine serum (Gibco). LLC cells (100 μL containing 1 × 10⁶ cells / mL) were cultured in this medium. 6 After subcutaneous inoculation of 100 μL of PBS + 100 μL of matrigel into C57BL / 6 mice (central experimental animals) of the same genotype, anti-angiogenic 2 antibody (2C8) was injected intraperitoneally on days 7, 9, 1, 14 and 16 from the date of cancer cell inoculation, and the size of the tumor was measured.

[0159] like Figure 6 As shown, the anti-angiogenic-2 antibody 2C8 was confirmed to inhibit tumor growth.

[0160] To confirm the efficacy of combined administration of anti-angiogenic-2 antibody with other existing anticancer agents, a combination therapy of anti-angiogenic-2 antibody 2C8 and cisplatin was conducted in an LLC lung cancer model. LLC cell lines were cultured in DuPont modified Igor medium (Weijian Company) supplemented with 10% fetal bovine serum (Gibco). LLC cells (100 μL containing 1 × 10⁶ cells / mL) were... 6 After subcutaneous inoculation of 100 μL of phosphate-buffered saline solution (100 cells + 100 μL of matrix) into C57BL / 6 mice (Orient) of the same genotype, when the tumor size reached 50 mm... 3 -100mm 3 During this period, angiopoietin-2 antibody was administered intraperitoneally at a dose of 5 mg / kg three times a week for two weeks. Cisplatin (Sigma-Aldrich) was administered once a week at a dose of 5 mg / kg. The experiment was conducted in the Ig (control group), 2C8 group, cisplatin group, and 2C8+cisplatin group.

[0161] like Figure 7 As shown, when anti-angiogenic-2 antibody 2C8 was administered in combination with cisplatin, tumor growth was inhibited by approximately 48% compared to the control group, demonstrating highly significant efficacy compared to the use of anti-angiogenic-2 antibody or 5FU alone. These results demonstrate that, compared to treatment with existing anticancer agents alone, the combination of anti-angiogenic-2 antibody with other anticancer agents can more significantly and potently inhibit cancer growth.

[0162] B. MC38 colon cancer cell growth model

[0163] To confirm the inhibitory effect of anti-angiogenic factor-2 antibody on tumor growth and its combined effect with 5-fluorouracil (5FU) in a colorectal cancer model, a colorectal cancer homologous mouse model using the MC38 cell line, a mouse colorectal cancer cell line, was used. The MC38 cell line was cultured in DuPont modified Igor medium (Weijian Company) supplemented with 10% fetal bovine serum (Weijian Company). MC38 cell line (100 μL containing 1×10⁶ cells / mL) was cultured in this medium. 6 After subcutaneous inoculation of phosphate-buffered saline (PPS) into C57BL / 6 mice (Orient) of the same genotype, when the tumor size reached 70 mm... 3 -100mm 3 Five doses of 5-FU were administered intraperitoneally every three days. Two doses of 20 mg / kg 5-FU were administered. The experiment was conducted in the Ig (control group), 2C8 group, 5FU (Sigma-Aldrich) group, and 2C8+5FU group.

[0164] like Figure 8 As shown, anti-angiogenic-2 antibody 2C8 and 5FU alone showed similar levels of tumor growth inhibition. In the combination of anti-angiogenic-2 antibody 2C8 and 5FU, tumor growth was inhibited by approximately 58% compared to the control group, demonstrating a highly significant efficacy compared to either anti-angiogenic-2 antibody or 5FU alone. These results demonstrate that, compared to treatment with existing anticancer agents alone, combination therapy with anti-angiogenic-2 antibodies can more significantly and potently inhibit cancer growth.

Claims

1. An anti-angiogenic-2 antibody or its antigen-binding fragment, which specifically binds to angiopoietin-2 and induces activation of endothelial cell TEK tyrosine kinase, characterized in that, Include: (a) Heavy chain complementarity determinant region; and (b) Light chain complementarity determinant region, The heavy chain complementarity determination region (a) above includes: CDRH1, amino acid sequence of sequence 18; The amino acid sequence of sequence 19 is CDRH2; and The amino acid sequence of sequence 20 is CDRH3. The complementarity determination region of the light chain in (b) above includes: CDRL1 of the amino acid sequence of sequence 21; CDRL2 of the amino acid sequence of sequence 22; and CDRL3 is the amino acid sequence of sequence 23.

2. The anti-angiopoietin-2 antibody or antigen-binding fragment thereof according to claim 1, characterized in that, The above-mentioned antibody or its antigen-binding fragment includes: The heavy chain variable region contains the amino acid sequence of sequence 24; and The light chain variable region contains the amino acid sequence of sequence 25.

3. The anti-angiopoietin-2 antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that, The aforementioned antibody or its fragments specifically bind to angiopoietin-2 and, together with angiopoietin-2, bind to the TEK tyrosine kinase receptor on endothelial cells.

4. An isolated nucleic acid, comprising, Encodes the anti-angiogenic-2 antibody of claim 1 or its antigen-binding fragment.

5. The anti-angiopoietin-2 antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, The antigen-binding fragments mentioned above are selected from the group consisting of scFv, (scFv)2, scFv-Fc, Fab, Fab' and F(ab')2.

6. The anti-angiopoietin-2 antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, The antibodies or their antigen-binding fragments mentioned above are mouse antibodies, chimeric antibodies, or humanized antibodies.

7. A pharmaceutical composition for preventing or treating lung cancer and colon cancer, characterized by comprising the compound of claim 1 or 2 as an active ingredient. It contains the anti-angiogenic-2 antibody as described in claim 1 or its antigen-binding fragment as an active ingredient.

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