WO2024085606A1 - Bispecific antibody including first antigen-binding site that specifically binds to human angiopoietin-2, and use thereof - Google Patents

Abstract

The present invention relates to a bispecific antibody including a first antigen-binding site that specifically binds to human angiopoietin-2 (ANG-2), and a second antigen-binding site that specifically binds to at least one antigen selected from the group consisting of human vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-beta, T-cell immunoglobulin and mucin domain-containing protein (TIM)-3 ligand, and pigment epithelium-derived factor receptor (PEDF-R), wherein the first antigen-binding site that specifically binds to the ANG-2 includes, in the heavy chain variable domain, a CDRH1 region of SEQ ID NO: 1 or 7 or 13, a CDRH2 region of SEQ ID NO: 2 or 8 or 14, and a CDRH3 region of SEQ ID NO: 3 or 9 or 15, and includes, in the light chain variable domain, a CDRL1 region of SEQ ID NO: 4 or 10 or 16, a CDRL2 region of SEQ ID NO: 5 or 11 or 17, and a CDRL3 region of SEQ ID NO: 6 or 12 or 18.

Description

Bispecific antibody comprising a first antigen binding site that specifically binds to human angiopoietin-2 and applications thereof

The present invention relates to a bispecific antibody comprising a first antigen binding site that specifically binds to human angiopoietin-2 and its applications.

Angiopoietin-2 (hereinafter also referred to as 'ANG-2') binds to the receptor Tie2 present on vascular endothelial cells, but acts as an antagonistic ligand, producing angiophore, an agonist of Tie2. It acts to inhibit signal transduction by Tie2 by competing with Angiopoietin-1 (Ang1) for binding to Tie2. Due to this mechanism of action, when vascular endothelial growth factor (Vascular Epidermal Growth Factor, hereinafter referred to as 'VEGF') is overexpressed or in a state of inflammation, vascular endothelial cells are activated and vascular permeability increases. In this case, Ang1 While it induces stabilization of endothelial cells and reduces vascular permeability, increased Ang2 in activated vascular endothelial cells competes with Ang1, thereby inhibiting stabilization of vascular endothelial cells by Ang1. Therefore, Ang2 inhibits Ang1-Tie2 binding and signaling through Ang1-Tie2, which maintains the stability of vascular endothelial cells in the presence of VEGF, and ultimately promotes angiogenesis in blood vessels, resulting in increased angiogenesis, destabilization of blood vessels, and vascular permeability. causes an increase Meanwhile, in addition to its role as an antagonist that induces the inactivation of the Tie2 receptor, Ang2 has been reported to have agonist properties that induce the activity of the Tie2 receptor in some specific situations, including the formation and maintenance of lymphatic vessels. Therefore, it functions as an antagonist and a weak agonist. It is believed to have all of the following functions and perform various functions depending on the situation.

Since the process of neovascularization is an essential factor in the growth of cancer, there have been attempts to prevent further growth of cancer by inhibiting neovascularization by inhibiting the Tie2-dependent function of Ang2 as described above. However, in most cases, these are It is known to have the function of inhibiting binding and preventing its role as an antagonist. In addition to antibodies that interfere with the binding of Ang2 to Tie2, recombinant proteins or antibodies that directly bind to the Tie2 receptor and induce phosphorylation and activation have been reported.

Recently, an antibody that binds to Ang2 and binds to Tie2 to phosphorylate and activate Tie2 through Tie2 clustering has been reported. This does not simply block Ang2's role as an antagonist, but rather acts as an agonist, leading to more effective vascular normalization. It suggests a possibility.

Meanwhile, Ranibizumab (brand name Lucentis®) is a monoclonal antibody fragment derived from the same parent murine antibody as bevacizumab (Avastin). However, this antibody has been affinity matured to provide stronger binding to VEGF-A (International Patent Application Publication No. WO 98/45331). As VEGF-A blockade may be associated with some systemic toxicity, it is known that ranibizumab is losing its Fc portion to reduce serum half-life and consequently systemic toxicity.

International Patent Application Publication Nos. WO 2010/040508 and WO 2011/117329 relate to bispecific anti-VEGF/anti-ANG-2 antibodies.

The object of the present invention is a bispecific antibody comprising a site that specifically binds to human ANG-2 and a site that specifically binds to human VEGF, TGF-beta, TIM-3 ligand or PEDF-R (PNPLA2, ATGL). It provides antibodies.

Another object of the present invention is to provide a pharmaceutical composition containing the bispecific antibody as an active ingredient.

In order to achieve the above object, the present invention provides a first antigen binding site that specifically binds to human angiopoietin (ANG)-2, human vascular endothelial growth factor (VEGF), transforming growth factor, Hereinafter referred to as 'TGF')-beta, the ligand of T-cell immunoglobulin and mucin-domain containing protein (T-cell immunoglobulin and mucin-domain containing, hereinafter referred to as 'TIM')-3, and pigment epithelium-derived factor receptor. A bispecific antibody comprising a second antigen binding site that specifically binds to one antigen selected from the group consisting of (pigment epithelium-derived factor receptor, hereinafter referred to as 'PEDF-R'),

i) The first antigen binding site that specifically binds to the ANG-2 is the CDRH1 region of SEQ ID NO: 1 or 7 or 13, the CDRH2 region of SEQ ID NO: 2 or 8 or 14, and the CDRH3 of SEQ ID NO: 3 or 9 or 15 comprising a region within a heavy chain variable domain, a CDRL1 region of SEQ ID NO: 4 or 10 or 16, a CDRL2 region of SEQ ID NO: 5 or 11 or 17 and a CDRL3 region of SEQ ID NO: 6 or 12 or 18 within a light chain variable domain;

ii) the second antigen binding site specifically binding to VEGF comprises a VEGFR1 D2 domain, and the second antigen binding site specifically binding to TGF-beta comprises TGF-beta RII ECD, The second antigen binding site that specifically binds to the ligand of TIM-3 includes TIM-3 ECD, and the second antigen binding site that specifically binds to the PEDF-R includes a 44-mer. Contains a PEDF of

iii) The antibody provides a bispecific antibody that induces Tie2 activation.

In one embodiment of the present invention, the VEGFR1 D2 domain is the VEGFR1 D2 domain of SEQ ID NO: 21, the TGF-beta RII ECD is the TGF-beta RII ECD of SEQ ID NO: 24, and the TIM-3 ECD is the TIM of SEQ ID NO: 27 -3 ECD and the 44-mer PEDF preferably include the amino acid sequence of SEQ ID NO: 30, but are not limited thereto.

In another embodiment of the present invention, the VEGFR1 D2 domain, the TGF-beta RII ECD, the TIM-3 ECD, and the 44-mer PEDF are preferably connected to an immunoglobulin by a linker, and the linker It is more preferable that the linker is SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, or SEQ ID NO: 29, respectively, but is not limited thereto.

Additionally, the present invention provides a pharmaceutical composition for the treatment of cancer containing the antibody according to the present invention.

Additionally, the present invention provides a pharmaceutical composition for the treatment of vascular diseases containing the antibody according to the present invention.

The present invention also provides a nucleic acid encoding the bispecific antibody according to the present invention.

The present invention also provides a method for producing a bispecific antibody according to the present invention, comprising expressing a nucleic acid encoding the bispecific antibody in a prokaryotic or eukaryotic host cell and recovering the bispecific antibody from the cell or cell culture supernatant. Provides a method for producing a bispecific antibody.

A “bispecific antibody” according to the invention is an antibody with two different antigen-binding specificities. If an antibody has more than one specificity, the epitope recognized may be associated with a single antigen or more than one antigen. The antibodies of the invention are specific for two different antigens, ANG-2 as the first antigen and VEGF, TGF-beta, the ligand of TIM-3 or PEDF-R as the second antigen.

Bispecific antibodies of the invention include, for example, multivalent single chain antibodies, diabodies and triabodies, as well as additional antigen binding sites (e.g., single chain Fv, It includes antibodies having the constant domain structure of a full-length antibody to which a VH domain and/or a VL domain, Fab, or (Fab)2) are linked. Antibodies can be full length from a single species, chimeric or humanized. For antibodies with two or more antigen binding sites, some of the binding sites may be identical, as long as the protein has binding sites for two different antigens. That is, the first binding site is specific for ANG-2, while the second binding site is specific for VEGF, TGF-beta, TIM-3 ligand, and PEDF-R, and vice versa.

In certain embodiments, the antibodies of the invention further comprise immunoglobulin constant regions of one or more immunoglobulin classes. Immunoglobulin classes include IgG, IgM, IgA, IgD and IgE isotypes and, in the case of IgG and IgA, their subtypes. In a preferred embodiment, the antibodies of the invention have the constant domain structure of an IgG type antibody, but have four antigen binding sites.

The term “chimeric antibody” refers to an antibody comprising a variable region, i.e., a binding region, from one source or species and at least a portion of a constant region from a different source or species, typically produced by recombinant DNA technology. Chimeric antibodies comprising murine variable regions and human constant regions are preferred. Other preferred forms of “chimeric antibodies” encompassed by the present invention are those wherein the constant portions are altered or varied from the constant portions of the original antibody, thereby producing properties according to the present invention, particularly with regard to C1q binding and/or Fc receptor (FcR) binding. will be. Such chimeric antibodies are also referred to as “class-switched antibodies”. Chimeric antibodies are the product of expressed immunoglobulin genes comprising a DNA segment encoding an immunoglobulin variable region and a DNA segment encoding an immunoglobulin constant region. Methods for producing chimeric antibodies include conventional recombinant DNA and gene transfection techniques well known in the art. For example, Morrison, S.L., et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; See US 5,202,238 and US 5,204,244.

The term “humanized antibody” refers to an antibody whose framework or “complementarity determining regions” (CDRs) have been altered to include the CDRs of an immunoglobulin of different specificity compared to the parent immunoglobulin. In a preferred embodiment, murine CDRs are grafted into the framework region of a human antibody to produce a “humanized antibody.” For example, Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M.S., et al., Nature 314 (1985) 268-270. Particularly preferred CDRs correspond to those representing sequences recognizing the above-mentioned antigens for chimeric antibodies.

As used herein, the term “human antibody” is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the art (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that can produce the full repertoire or selection of human antibodies upon immunization in the absence of endogenous immunoglobulin production. Transfer of a human germline immunoglobulin gene sequence into such germline mutant mice will result in the production of human antibodies upon challenge (e.g. Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, M., et al., Year Immunol. Human antibodies can also be produced from phage display libraries (Hoogenboom, H.R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J.D., et al., J. Mol. Biol 222 (1991) 581-597). For the production of monoclonal antibodies, Cole, A., et al. and Boerner, P., et al.'s techniques are also available (Cole, A., et al., Monoclonal Antibodies and Cancer Therapy, Liss, A.L., p. 77 (1985); and Boerner, P., et al. al., J. Immunol. 147 (1991) 86-95).

As used herein, the term “recombinant human antibody” refers to any human antibody that is manufactured, expressed, produced or isolated by recombinant methods, such as from a host cell such as NS0 or CHO cells or from an animal transgenic for human immunoglobulin genes (e.g. It is intended to include antibodies isolated from (e.g., mouse), or antibodies expressed using recombinant expression vectors transfected into host cells. The recombinant human antibody has variable and constant regions in rearranged form.

As used herein, the term “complementarity determining region (CDR)” refers to the amino acid sequence of the hypervariable region of the heavy and light chains of immunoglobulins. The heavy and light chains may each contain three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDR may provide key contact residues for the antibody to bind to the antigen or epitope. Meanwhile, in the present specification, the term "specifically binds" or "specifically recognizes" has the same meaning commonly known to those skilled in the art, and refers to a condition in which an antigen and an antibody specifically interact to produce an immunological reaction. means that

As used herein, “variable domain” (variable domain of the light chain (VL), variable region of the heavy chain (VH)) refers to each of the pairs of light and heavy chains that are directly involved in antibody binding to antigen. The domains of the variable human light and heavy chains have the same general structure, with each domain consisting of four framework (FR) regions of broadly conserved sequence, linked by three "hypervariable regions" (or complementarity determining regions, CDRs). Includes. The framework region has a β-sheet conformation, and the CDRs can form loops connecting the β-sheet structures. The CDRs within each chain maintain their three-dimensional structure by framework regions and, together with CDRs from other chains, form an antigen-binding site.

As used herein, the term “hypervariable region” or “antigen-binding site of an antibody” refers to the amino acid residues of an antibody that are responsible for antigen-binding. Hypervariable regions include amino acid residues from the “complementarity determining region” or “CDR”.

The “framework” or “FR” region is the variable domain region excluding the hypervariable region residues as defined herein. Therefore, the light and heavy chains of the antibody comprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The CDRs on each chain are separated by the framework amino acids. In particular, CDR3 of the heavy chain is the region that most contributes to antigen binding. CDR and FR regions are determined according to the standard definitions of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

Bispecific antibodies according to the invention also include said antibodies with “conservative sequence alterations” (this means “variants” of bispecific antibodies). This means nucleotide and amino acid sequence changes that do not affect or alter the above-mentioned properties of the antibody according to the invention. Changes can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions involve the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. This family includes amino acids with basic side chains (e.g. lysine, arginine, histidine), amino acids with acidic side chains (e.g. aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g. threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Accordingly, a predicted non-essential amino acid residue in a bispecific antibody may be replaced with another amino acid residue, preferably from the same side chain family.

Therefore, as used herein, a "variant" bispecific antibody is one whose amino acid sequence differs from the "parent" bispecific antibody amino acid sequence by at most 10, preferably about 2 to about 5, additions, deletions, and deletions within one or more variable or constant regions of the parent antibody. /or refer to a different molecule by substitution. Amino acid substitutions are as described in Riechmann, L., et al., Nature 332 (1988) 323-327 and Queen, C, et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033 may be performed by mutagenesis based on molecular modeling. “Variant” bispecific antibodies according to the invention also include bispecific antibody forms in which the linker (if present) therein is modified or replaced by another linker.

As used herein, the term “binding” or “specific binding” refers to the binding of an antibody to an epitope of an antigen (human VEGF, ANG-2, TGF-beta, TIM-3 ligand or PEDF-R) in an in vitro assay. Mention.

The term “epitope” includes any polypeptide determinant capable of specifically binding to an antibody. In certain embodiments, epitope determinants include amino acids, sugar side chains, chemically active surface groups of molecules such as phosphoryl or sulfonyl, and, in certain embodiments, may have specific three-dimensional structural properties and/or specific charge properties. there is. An epitope is a region of an antigen that is bound by an antibody.

As used herein, the term “linker” refers to a peptide having an amino acid sequence, preferably of synthetic origin. Typically, the peptides link a) VH-CH1 to VL-CL, b) VL-CL to VH-CH1, c) VH-CL to VL-CH1, or d) VL-CH1 to VH-CL. The linker according to the present invention is used to connect human immunoglobulin G4 and human VEGFR1 domain 2, TGF-beta RII ECD, TIM-3 ECD, and the PEDF sequence of the 44-mer.

As used herein, “pharmaceutical carrier” includes any and all physiologically compatible solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion).

The composition of the present invention can be administered by various methods known in the art. As will be appreciated by those skilled in the art, the route and/or mode of administration will vary depending on the desired results. To administer an antibody of the invention by a particular route of administration, it may be necessary to coat the antibody with or co-administer the antibody with a material that will prevent its inactivation. For example, the antibody can be administered to a subject in a suitable carrier, such as liposomes or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffered solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media or agents for pharmaceutically active ingredients is known in the art.

As used herein, the phrases “parenteral administration” and “administered parenterally” refer to any mode of administration other than enteral and topical administration, typically by injection, including intravenous, intramuscular, intraarterial, intrathecal, and intraarticular. Including, without limitation, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, intraepithelial, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injections and infusions.

As used herein, the term cancer refers to proliferative diseases such as lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin and intraocular melanoma, and uterine cancer. , ovarian cancer, rectal cancer, anal area cancer, stomach cancer, gastrointestinal cancer, colon cancer, breast cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, Thyroid cancer, parathyroid cancer, adrenal cancer, soft sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or urethral cancer, renal cell cancer, renal pelvis cancer, mesothelioma, hepatocellular cancer, biliary tract cancer, and tumors of the central nervous system (CNS) , spine tumor, brainstem tumor, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma and Ewing's sarcoma, and a refractory version of any of the above cancers or one of the above cancers. Includes combinations of the above.

Another aspect of the invention is the bispecific antibody or said pharmaceutical composition according to the invention as an anti-angiogenic agent. The anti-angiogenic substances can be used for the treatment of cancer, especially solid tumors, and other vascular diseases.

One embodiment of the invention is a bispecific antibody according to the invention for the treatment of vascular diseases.

Another aspect of the present invention is the pharmaceutical composition for treating vascular diseases.

Another aspect of the invention is the use of the antibody according to the invention for the manufacture of a medicament for the treatment of vascular diseases.

Another aspect of the invention is a method of treating a patient suffering from vascular disease by administering an antibody according to the invention to the patient in need of treatment.

The term “vascular disease” includes cancer, inflammatory disease, atherosclerosis, ischemia, trauma, sepsis, COPD, asthma, diabetes, AMD, retinopathy, stroke, adiposity, acute lung injury, hemorrhage, e.g. cytokine-induced Vascular effusion, allergy, Graves' disease, Hashimoto's autoimmune thyroiditis, idiopathic thrombocytopenic purpura, giant cell arteritis, rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, Crohn's disease, multiple sclerosis, ulcerative colitis, especially solid tumors. For, intraocular neovascular syndromes such as proliferative retinopathy or age-related macular degeneration (AMD), rheumatoid arthritis and psoriasis (Folkman, J., et al., J. Biol. Chem. 267 (1992) 10931-10934; Klagsbrun, M., et al., Rev. Physiol. 53 (1991) 217-239; and Garner, A., Pathobiology of ocular disease, A dynamic approach. ., and Klintworth, G.K., (eds.), 2nd edition, Marcel Dekker, New York (1994), pp 1625-1710).

The composition may also include adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured by the above sterilization procedures and by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic substances such as sugars, sodium chloride, etc. into the composition. Additionally, sustained absorption of injectable pharmaceutical forms can be obtained by the inclusion of substances that delay absorption, such as aluminum monostearate and gelatin.

Regardless of the route of administration chosen, the antibodies of the invention, which can be used in suitable hydrated forms and/or pharmaceutical compositions of the invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

The actual dosage levels of the active ingredient in the pharmaceutical compositions of the invention may vary to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, while not being toxic to the patient. The dosage level selected will depend on the activity of the particular composition of the invention employed, the route of administration, the time of administration, the rate of excretion of the particular antibody to be employed, the duration of treatment, the other drugs, compounds and/or substances to be combined with the particular composition to be employed, the treatment. It will vary depending on various pharmacokinetic factors, including factors well known in the medical field, such as the patient's age, gender, weight, condition, general health, and past medical history.

The compositions of the present invention must be fluid and sterile to the extent that the compositions can be delivered by syringe. In addition to water, the carrier is preferably isotonic buffered saline. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintenance of the required particle size in case of dispersion, and by the use of surfactants. In many cases, it is desirable to include isotonic substances such as sugars, polyhydric alcohols such as mannitol or sorbitol, and sodium chloride in the composition.

The present invention proposes an antibody that promotes downstream signaling by inhibiting Ang2, VEGF, TGF-beta, and TIM-3 ligands or activating PEDF-R while simultaneously activating the Tie2 receptor, thereby inhibiting neovascularization and improving vascular permeability. Suggests a new way to reduce it. In addition, the antibody proposed in the present invention is expected to have applicability in the diagnosis and treatment of cancer, diseases related to abnormal angiogenesis, and/or diseases caused by increased vascular permeability.

Figure 1 is a schematic diagram of a bispecific antibody comprising a first antigen binding site that specifically binds to human angiopoietin-2 developed in the present invention.

Figure 2 shows the SDS-PAGE results of purified anti-Ang2/VEGF antibody.

Figures 3 and 4 show the results of SDS-PAGE purified anti-Ang2/TGF-β antibody (Figure 3) and the results of Western blot confirmation of the TGF-β binding region, the second antigen binding site (Figure 4), respectively.

Figures 5 and 6 show ELISA results analyzing the binding affinity of anti-Ang2/VEGF antibodies (Figure 5: 2C8-D2, Figure 6: 1D3-D2) to human Ang2, respectively.

Figures 7 and 8 are ELISA results showing the binding ability of anti-Ang2/VEGF antibody (Figure 7, 2C8-D2, Figure 8, 1D3-D2) to human VEGF or mouse VEGF, respectively.

Figure 9 shows ELISA results showing that anti-Ang2/VEGF antibody can bind to VEGF and Ang2 simultaneously.

Figure 10 is a Western blot result showing that anti-Ang2/VEGF antibody and anti-Ang2/TGF-β antibody phosphorylate AKT and ERK 42/44 in HUVEC along with Ang2.

Figure 11 is a result showing that anti-Ang2/VEGF antibody binds to VEGF competitively with soluble VEGFR2.

Figure 12 shows the results of anti-Ang2/VEGF antibody inhibiting HUVEC proliferation caused by VEGF.

Figures 13 to 15 show the inhibition of neovascularization by anti-Ang2/VEGF antibodies in the mouse laser-induced CNV (wet AMD) model (Figure 13. representative FFA images & CTF, Figure 14. representative OCT images & CNV lesion) and improvement in vision, respectively. This is a result showing the effect (Figure 15. ERG).

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

Example 1: Construction of a bispecific antibody comprising a first antigen binding site that specifically binds to human angiopoietin-2

An anti-Ang2 antibody known to activate the Tie2 receptor using Ang2 to create a bispecific antibody containing a first antigen binding site that specifically binds to human angiopoietin-2 (Korean Patent Application No. 10- 2022-0028751) and D2, the VEGF binding domain of human VEGFR1 (SEQ ID NO: 19, Accession: P17948) or TGF-βextracellular domain (SEQ ID NO: 24) or human TIM-3 extracellular domain (SEQ ID NO: 27) or human PEDF 44-mer Peptide (SEQ ID NO: 30) was used.

The heavy chain of the anti-Ang2/VEGF antibody is a heavy chain CDR sequence of 1A9 or 2C8 or 1D3 (SEQ ID NOs: 1 to 3 or 7 to 9 or 13 to 15) and C- The human immunoglobulin G4 heavy chain sequence and linker sequence (SEQ ID NO: 20) and VEGFR1 D2 sequence (SEQ ID NO: 21), in which terminal lysine was replaced with alanine, were composed into one and synthesized in GenScript. The light chain of the anti-Ang2/VEGF antibody used was the light chain CDR sequence (SEQ ID NO: 4 to 6 or 10 to 12 or 16 to 18) of 1A9 or 2C8 or 1D3 described in Korean Patent Application No. 10-2022-0028751. .

The heavy chain of the anti-Ang2/TGF-β antibody is the heavy chain CDR sequence of 1A9 or 2C8 or 1D3 (SEQ ID NOs: 1 to 3 or 7 to 9 or 13 to 15) described in Korean Patent Application No. 10-2022-0028751. The human immunoglobulin G4 heavy chain sequence and linker sequence (SEQ ID NO: 23), in which lysine at the C-terminal was replaced with alanine, and the TGF-βextracellular domain (SEQ ID NO: 24) were constructed as one, and then synthesized in GenScript. The light chain of the anti-Ang2/TGF-β antibody has the light chain CDR sequence (SEQ ID NO: 4 to 6 or 10 to 12 or 16 to 18) of 1A9 or 2C8 or 1D3 described in Korean Patent Application No. 10-2022-0028751. used.

The heavy chain of the anti-Ang2/TIM-3 ligand antibody is the heavy chain CDR sequence of 1A9 or 2C8 or 1D3 (SEQ ID NOs: 1 to 3 or 7 to 9 or 13 to 15) described in Korean Patent Application No. 10-2022-0028751. And the human immunoglobulin G4 heavy chain sequence and linker sequence (SEQ ID NO: 26), in which lysine at the C-terminal was replaced with alanine, and the human TIM-3 extracellular domain (SEQ ID NO: 27) were composed into one, and then synthesized in GenScript. The light chain of the anti-Ang2/TIM-3 ligand antibody is the light chain CDR sequence of 1A9 or 2C8 or 1D3 (SEQ ID NOs: 4 to 6 or 10 to 12 or 16 to 18) described in Korean Patent Application No. 10-2022-0028751. was used.

The heavy chain of the anti-Ang2/PEDF-R antibody is the heavy chain CDR sequence of 1A9 or 2C8 or 1D3 (SEQ ID NOs: 1 to 3 or 7 to 9 or 13 to 15) described in Korean Patent Application No. 10-2022-0028751, and The human immunoglobulin G4 heavy chain sequence and linker sequence (SEQ ID NO: 29), in which lysine at the C-terminal was replaced with alanine, and the human PEDF 44-mer peptide (SEQ ID NO: 30) were combined into one, and then synthesized in GenScript. The light chain of the anti-Ang2/PEDF-R antibody has the light chain CDR sequence (SEQ ID NO: 4 to 6 or 10 to 12 or 16 to 18) of 1A9 or 2C8 or 1D3 described in Korean Patent Application No. 10-2022-0028751. used.

Domain	CDR	CDR sequence	sequence number
Heavy chain	CDRH1-Kabat	DYWIG	SEQ ID NO: 1
	CDRH2-Kabat	DIYPGGGYTNCNEKFKG	SEQ ID NO: 2
	CDRH3-Kabat	SDYRNDEGFAD	SEQ ID NO: 3
Light chain	CDRL1-Kabat	SASSSVSSSYLH	SEQ ID NO: 4
	CDRL2-Kabat	RTSNLAS	SEQ ID NO: 5
	CDRL3-Kabat	QQWSGYPYT	SEQ ID NO: 6

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

Protein	CDR	CDR sequence	sequence number
Heavy chain	CDRH1-Kabat	NYWIG	SEQ ID NO: 7
	CDRH2-Kabat	IYPGGGYTNYNEKFK	SEQ ID NO: 8
	CDRH3-Kabat	SDYRDDEGFAY	SEQ ID NO: 9
Light chain	CDRL1-Kabat	SASSSVSSSYLH	SEQ ID NO: 10
	CDRL2-Kabat	RTSNLAS	SEQ ID NO: 11
	CDRL3-Kabat	QQWSGYPYT	SEQ ID NO: 12

Table 2 shows the CDR sequence of anti-Ang2 antibody 2C8

Protein	CDR	CDR sequence	sequence number
Heavy chain	CDRH1-Kabat	DYGWN	SEQ ID NO: 13
	CDRH2-Kabat	ISYSGTTSYNPSLK	SEQ ID NO: 14
	CDRH3-Kabat	SEGTGFYAMDY	SEQ ID NO: 15
Light chain	CDRL1-Kabat	KASQSVSNDVA	SEQ ID NO: 16
	CDRL2-Kabat	YASNRYT	SEQ ID NO: 17
	CDRL3-Kabat	QQDYSSPT	SEQ ID NO: 18

Table 3 shows the CDR sequence of anti-Ang2 antibody 1D3

Protein	Amino acid sequence	sequence number
Human VEGFR1	DLAARNILLSENNVVKICDFGLARDIYKNPDYVRKGDTRLPLKWMAPESIFDKIYSTKSDVWSYGVLLWEIFSLGGSPYPGVQMDEDFCSRLREGMRAPEYSTPEIYQIMLDCWHRDPKERPRFAELVEKLGDLLQANVQQDGKDYIPINAILTGNSGFTYSTPAFSEDFFKESISAPKFNSGSSDDVRYVNAFKFMSLERIKT FEELLPNATSMFDDYQGDSSTLLASPMLKRFTWTDSKPKASLKIDLRVTSKSKESGLSDVSRPSFCHSSCGHVSEGKRRFTYDHAELERKIACCSPPPDYNSVVLYSTPPI	SEQ ID NO: 19
Linker connecting Human IgG4 and VEGFR1 D2	GGGGSGGGG	SEQ ID NO: 20
(129-229) sequence including Human VEGFR1 D2	SDTGRPFVEMYSEKPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTI	SEQ ID NO: 21

Table 4 shows the linker connecting human immunoglobulin G4 and the (129-229) sequence containing human VEGFR1 domain 2, and the (129-229) sequence containing human VEGFR1 domain 2,

Protein	Amino acid sequence	sequence number
Human TGF-β RII isoform B	MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEF SEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYMAP EVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK	SEQ ID NO: 22
Linker connecting human IgG4 and human TGF-β RII extracellular domain	GGGGSGGGGSGGGGSGGGGSG	SEQ ID NO: 23
Human TGF-β RII isoform B extracellular domain (23-159) sequence	TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD	SEQ ID NO: 24

Table 5 shows the linker connecting human immunoglobulin G4 and human TGF-βRII extracellular domain sequence and human TGF-βRII extracellular domain sequence,

Protein	Amino acid sequence	sequence number
Human TIM-3	MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIGIYIGAGICAGL ALALIFGALIFKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQPSQPLGCRFAMP	SEQ ID NO: 25
Linker connecting human IgG4 and human TIM-3 extracellular domain	GGGGSGGGGSGGGGSGGGG	SEQ ID NO: 26
Human TIM-3 extracellular domain (22-200) sequence	SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIR	SEQ ID NO: 27

Table 6 shows the linker connecting human immunoglobulin G4 and human TIM-3 extracellular domain sequence and human TIM-3 extracellular domain sequence,

Protein	Amino acid sequence	sequence number
Human PEDF	MQALVLLLCIGALLGHSSCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARSTKEIPDEISILL LGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGALLFIGKILDPRGP	SEQ ID NO: 28
Linker connecting human IgG4 and human PEDF 44-mer peptide	GGGGSGGGGS	SEQ ID NO: 29
Human PEDF 44-mer peptide (78-121) sequence	VLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGT	SEQ ID NO: 30

Table 7 shows the linker connecting human immunoglobulin G4 and human PEDF 44-mer peptide sequence and human PEDF 44-mer peptide sequence,

Example 2: Antibody expression and purification

The ExpiCHO expression system (Thermo Fisher) was used to produce a bispecific antibody containing a first antigen binding site that specifically binds to human angiopoietin-2. ExpiCHO was cultured in a 125 mL flask (Corning) using 30 mL of ExpiCHO expression medium (Thermo Fisher) until the cell number reached ⁶ A total of 25 μg of heavy chain and light chain DNA prepared in Example 1 was prepared at a 1:1.5 ratio (w/w) using 1 mL of OptiPRO SFM (Thermo Fisher). 80 μL of ExpiFectamine CHO reagent (Thermo Fisher) was prepared by adding it to 920 μL of OptiPRO SFM. After mixing the prepared DNA and ExpiFectamine CHO reagent, transfection was performed by adding it to 25 mL of the ExpiCHO culture medium prepared earlier within 5 minutes, and cultured at 37°C, 8% CO ₂ , and 125 rpm. On the first day after transfection, 150 μL of ExpiFectamine CHO Enhancer (Thermo Fisher) and 4 mL of ExpiCHO Feed (Thermo Fisher) were added to the culture medium, and the conditions were changed to 32°C, 5% CO ₂ , and 125 rpm. On the fifth day after transfection, an additional 4 mL of ExpiCHO Feed was added. On the 7th day after transfection, the culture was transferred to a conical tube (SPL) and centrifuged at 3,500 g for 30 minutes at 4°C. The supernatant was collected and filtered using a 0.22 μm CA syringe filter (Sartorius), and -70 Stored in a deep freezer at ℃.

Purification was performed using the culture medium prepared previously. A Hitrap Mabselect Sure (Cytiva) 5mL column was mounted on a Semi-Prep HPLC system (Youngin Chromas). Equilibrium was achieved by flowing over 5 CV of PBS at a flow rate of 2.5 mL/min. The harvested culture was filtered through a 0.2 μm filter and loaded into an equilibrated column at a flow rate of 2.5 mL/min. After loading was completed, PBS was again flowed until the UV value reached equilibrium, and elution buffer (0.1 M glycine, pH 2.7) was flowed to elute the anti-Ang2/VEGF antibody. The pH of the eluted solution was adjusted to 7.0 using 1 M Tris buffer.

Amicon 30 kDa (Millipore) was used to change the buffer of the purified bispecific antibody to PBS. 5 mL of PBS was added to Amicon, centrifuged at 4°C, 5,000 rpm, and cleaned. After centrifugation, all of the solution was discarded, 10 mL of the sample eluted earlier was added, and then centrifuged at 4°C and 5,000 rpm to concentrate to about 3 mL. After adding 12 mL of PBS to the concentrated 3 mL, it was centrifuged again and concentrated to 3 mL. This process was repeated four additional times and the buffer was exchanged for PBS. Bispecific antibodies were prepared by performing sterile filtration on the sample after buffer exchange was completed using a 0.22 μm syringe filter (Millipore).

Example 3: SDS-PAGE analysis of purified anti-Ang2/VEGF antibody samples

To confirm the purity and integrity of the anti-Ang2/VEGF antibody purified in Example 2, it was analyzed by SDS-PAGE. To 15 μL of a sample of appropriate protein concentration, add SDS-Sample Buffer 4X reducing (Gendepot) or Native SDS-Sample Buffer 4X non reducing (Gendepot) to 1X each, and heat the reducing sample at 98°C for 10 minutes using a heat block. Samples were prepared by heat treatment. NuPAGE 4-12%, Bis-Tris mini protein gel (Invitrogen) was installed in the XCell SureLock Mini-Cell (Invitrogen), and NuPage 1X MOPS SDS running buffer (Invitrogen) was added to prepare for electrophoresis. TOPview Prestained Protein Ladder Marker (Enzynomics) and the sample prepared above were loaded into the prepared gel well, and then electrophoresed at 200V for 42 minutes using PowerEase Touch 600W Power Supply (Invitrogen) equipment. The gel after electrophoresis was placed in sun gel staining solution (LPS solution) for staining. The gel for which staining was completed was placed in distilled water for destaining.

As shown in Figure 2, a band of the desired size was confirmed under reducing (R) and non-reducing (NR) conditions, and it was confirmed that there was almost no impurity.

Example 4: SDS-PAGE and Western blot analysis of purified anti-Ang2/TGF-β antibody samples

To confirm the purity and integrity of the anti-Ang2/TGF-β antibody purified in Example 2 in the same manner as in Example 3, it was analyzed by SDS-PAGE. As shown in Figure 3, a band of the desired size was confirmed under reducing (R) and non-reducing (NR) conditions, and it was confirmed that there was almost no impurity. To confirm the TGF-β binding region of the anti-Ang2/TGF-β antibody, Western blot analysis was performed using anti-hTGF-β RII antibody. 60 ng and 600 ng of purified anti-Ang2/TGF-β antibody were subjected to SDS-PAGE electrophoresis in the same manner as in Example 3. After electrophoresis, the gel was mounted on the After the transfer, the membrane was placed in 0.05% TBST (Tris Buffered Saline containing 0.05% (v/v) Tween-20) containing 5% (w/v) skim milk and blocked for 1 hour. After washing the blocked membrane three times with 0.1% TBST, it was added to primary antibody buffer (2 μg/mL Goat anti-hTGF-β RII antibody (R&D systems), 5% (w/v) skim milk, 0.05% TBST). A membrane was added and allowed to bind for 2 hours. After washing the membrane with complete primary antibody binding three times with 0.1% TBST, secondary antibody buffer (1:5000 Rabbit anti-Goat IgG(H+L)-HRP (Gendepot), 5% (w/v) skim milk, 0.05 % TBST) and allowed to bind for 1 hour. After washing the membrane with secondary antibody binding three times with 0.1% TBST, a 1:1 mixture of Clarity Western ECL Substrate peroxide solution (Bio-rad) and Clarity Western ECL Substrate Luminol/enhancer (Bio-rad) was applied to the membrane. After spraying, the band was confirmed using ImageQuant LAS 500 (Cytiva) equipment.

As shown in Figure 4, Western blot analysis was performed using an anti-TGFβRII antibody under reducing (R) and non-reducing (NR) conditions, and the integrity of the material was confirmed by confirming a band of heavy chain size.

Example 5: Binding properties of anti-Ang2/VEGF antibody

ELISA was performed to confirm the binding ability of the anti-Ang2/VEGF antibody with human Ang2, human VEGF, or mouse VEGF. A 96-well MaxiSorp™ flat-bottom plate was coated with 1 μg/mL of human Ang2 (Sino Biological), human VEGF 165 (Sino Biological), or mouse VEGF 164 (Sino Biological). Then, the plate was washed five times with PBST (Phosphate Buffer Saline containing 0.05% (v/v) Tween-20) and then washed with PBST containing 1% (v/v) skim milk at room temperature for 2 hours. Blocked. Afterwards, the plate was washed five times with PBST containing 0.05% Tween-20, and 11 different concentrations of anti-Ang2/VEGF antibodies serially diluted 3 times from 300 nM were added and allowed to bind at room temperature for 2 hours. After washing the plate 5 times with PBST, Goat anti-Human Kappa/HRP (Novus) diluted 1:10,000 in PBST was added for 1 hour, and then washed 6 times with PBST. Finally, 100 μL of TMB substrate (Kementec) was added to the plate to induce a color reaction for 10 minutes, then 50 μL of Stop solution (2 MH ₂ SO ₄ ) was added to stop the reaction, and the OD450 value was measured using a plate reader (BioTek). ) was measured. As shown in Figures 5 to 8, it was confirmed that the anti-Ang2/VEGF antibodies (2C8-D2 and 1D3-D2) developed in the present invention bind to Ang2 and VEGF.

ELISA was performed to confirm the ability of the anti-Ang2/VEGF antibody to simultaneously bind Ang2 and VEGF. 1 μg/mL human VEGF 165 (Sino Biological) was coated on a 96-well MaxiSorp™ flat-bottom plate. The coated plate was washed five times with PBST and then blocked with PBST containing 1% (v/v) skim milk at room temperature for 2 hours. 60 nM of human Ang2 (Sino Biological) was mixed in advance with 11 different concentrations of anti-Ang2/VEGF antibody, serially diluted 3 times from 300 nM, and left at room temperature for 30 minutes. After washing the plate on which blocking was completed 5 times with PBST, previously prepared samples were added and allowed to bind at room temperature for 2 hours. After washing the plate 5 times with PBST, Mouse anti-His tag/HRP (SouthernBiotech) diluted 1:20,000 in PBST was bound to the plate for 1 hour, and then washed 6 times with PBST. Finally, 100 μL of TMB substrate (Kementec) was added to the plate to induce a color reaction for 10 minutes, then 100 μL of Stop solution (2 MH ₂ SO ₄ ) was added to stop the reaction, and the OD450 value was measured using a plate reader (BioTek). ) was measured.

As shown in Figure 9, it was confirmed that the anti-Ang2/VEGF antibody developed in the present invention can bind to Ang2 and VEGF at the same time.

Example 6: Binding properties of anti-Ang2/TGF-β antibody

Bio-layer interferometry (BLI) analysis was performed to confirm the binding ability of the anti-Ang2/TGF-β antibody to Ang2 or TGF-β. Octet Amine Reactive Second-Generation (AR2G) Biosensor (Sartorius) was placed in 1X kinetics buffer included in the AR2G kit and hydrated for 10 minutes. After completing hydration, the biosensor was placed in a 0.5 mL LightSafe micro centrifuge tube (Sigma) containing 1X kinetics buffer, and the initial baseline was measured for 60 seconds. The biosensor was placed in a 0.5 mL LightSafe micro centrifuge tube containing 225 μL of distilled water and 12.5 μL each of 400 mM EDC and 200 mM S-NHS and activated for 300 seconds. After loading 4 μL of 10 mM acetate buffer (pH 5.0) containing 20 μg/mL TGF-β (Acrobiosystems) into the drop holder, the activated biosensor was added and immobilized for 300 seconds. The immobilized biosensor was placed in a 0.5ml LightSafe micro centrifuge tube containing 250 μL of 1 M ethanolamine (pH 8.5) and quenched for 300 seconds. After quenching was completed, the biosensor was placed back in 1 proceeded. After the association was completed, the biosensor was placed in 1X kinetics buffer and dissociation was measured for 600 seconds. After completing the analysis, ka, kd, and KD were calculated by global fitting.

	ks [M-1s-1]	kd [s-1]	K.D. [nM]
hAng2 RBD	5.31 x 10 4	6.64 x 10 -4	12.5
hTGF-β1	4.82 x 10 4	9.65 x 10 -4	20.0

As shown in Table 8, it was confirmed that the anti-Ang2/TGF-β antibody developed in the present invention can bind to Ang2 and TGF-β.

Example 7: Western blot analysis to confirm activation of Tie2 signaling by anti-Ang2/VEGF antibody or anti-Ang2/TGF-β antibody

Ang2 binds to the Tie2 receptor expressed on vascular endothelial cells and acts as a weak agonist or antagonist. The anti-Ang2/VEGF antibody or anti-Ang2/TGF-β antibody developed in the present invention can bind to Ang2 and form a complex with Ang2-Tie2, thereby activating the Tie2 receptor and promoting downstream signaling. To analyze the effect of anti-Ang2/VEGF antibody or anti-Ang2/TGF-β antibody on Tie2 downstream signaling, ERK and AKT phosphorylation experiments were performed in HUVEC. In order to compare the degree of activation of Tie2 downstream signaling, the same test was performed on the group treated only with Ang2 (Sino Biological).

Specifically, HUVEC (Lonza) cells ( ² Cultured at 37°C for 16 to 24 hours. 60 nM of anti-Ang2/VEGF antibody or anti-Ang2/TGF-β antibody was mixed with 40 nM of Ang2 protein (Sino Biological) and left for 30 minutes. Then, the cultured cells were treated and incubated for another 10 minutes. For comparison, a group treated with only 40 nM Ang2 without antibody was also prepared. After washing the cells using PBS, lysis buffer (Ripa buffer (Biosesang), 0.15M Sodium chloride, 1% Triton , and 2 mM EDTA), centrifuged at 13,000 rpm for 15 minutes, and the supernatant was recovered to obtain a cell lysate.

Add sample buffer (GenDEPOT) mixed with reducing agent to 20-30 μg of cell lysate, boil at 95°C for 5 minutes, and then electrophoresed on 10% Tris-Glycine gel or 4-12% Bis-Tris gel (Invitrogen). , transferred onto NC or PVDF membranes (Millipore). To check the phosphorylation of Akt and ERK 42/44, which are involved in downstream signaling of the Tie2 receptor, the above blot was blocked for 1 hour with PBST mixed with 5% (v/v) skim milk (Seoul Milk). , 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).

As shown in Figure 10, anti-Ang2/VEGF antibody and anti-Ang2/TGF-β antibody showed AKT and ERK activation signals compared to the negative control group.

Example 8: Competitive VEGF binding of anti-Ang2/VEGF antibody to VEGFR2

VEGF is a representative pro-angiogenic cytokine that promotes blood vessel formation. VEGF is known to regulate angiogenesis by binding to VEGFR2 and activating downstream signals. The anti-Ang2/VEGF antibody designed in the present invention can bind to VEGF competitively with VEGFR2 and inhibit VEGF from activating the VEGFR2 downstream signal. ELISA analysis was performed to confirm competitive VEGF binding of anti-Ang2/VEGF antibody and VEGFR2.

Greiner Bio-One 96-well Half Area ELISA Microplates were coated with 2 μg/mL human VEGFR2 ECD (Sino Biological). The plate was washed five times with PBST (Phosphate Buffer Saline containing 0.05% (v/v) Tween-20) and then blocked with PBST containing 1% (w/v) skim milk at room temperature for 2 hours. After washing the plate 5 times with PBST, pre-incubate 150 μg/mL of human VEGF (Sino Biological) with AVI tag and 11 different concentrations of anti-Ang2/VEGF antibody serially diluted 3 times from 1,500 nM for 10 minutes at room temperature. The samples were placed in the plate prepared above and combined for 2 hours at room temperature. After washing the plate 5 times with PBST, Anti-Streptavidin-HRP (Abcam) diluted 1:15,000 in PBST was added to the plate, allowed to bind for 1 hour, and washed again 6 times with PBST. 50 μL of TMB substrate (Kementec) was added to the plate to induce a color reaction for 10 minutes, then 25 μL of Stop solution (2 MH ₂ SO ₄ ) was added to stop the reaction, and the OD450 value was recorded using a plate reader (BioTek). Measured. As shown in Figure 11, it was confirmed that the amount of VEGF bound to VEGFR2 decreased as the concentration of the anti-Ang2/VEGF antibody increased, thereby confirming that the anti-Ang2/VEGF antibody binds to VEGF competitively with VEGFR2.

Example 9: Inhibition of HUVEC proliferation by anti-Ang2/VEGF antibody

VEGF is a representative pro-angiogenic cytokine that promotes blood vessel formation, and VEGF is known to promote HUVEC proliferation. Anti-Ang2/VEGF antibodies can inhibit HUVEC proliferation by binding to VEGF in the culture medium and inhibiting VEGF from binding to VEGFR2 of HUVEC. To confirm this, an experiment was performed to check the proliferation of HUVEC by adding VEGF or VEGF and anti-Ang2/VEGF antibody to HUVEC.

Specifically, using EBM-2 media (Lonza) containing 0.5% FBS, HUVEC (Lonza) were placed in a 96 well cell culture plate (SPL) coated with 0.1% gelatin at 50,000 - 0 cells/well. , cultured in an incubator (37°C, 5% CO ₂ ) for 16 hours. EBM-2 media containing 0.5% FBS was prepared 30 minutes before treating the cells with VEGF 30 ng/mL, VEGF 30 ng/mL + anti-Ang2/VEGF antibody 2.5 nM. After removing the supernatant of the plate after 16 hours of incubation, 100 μL of the previously prepared sample was added to each well and cultured in an incubator (37°C, 5% CO ₂ ) for 72 hours. After 72 hours, 20 μL of MTS (Promega) was added to each well of the plate and reacted again in the incubator for 4 hours. After completion of the reaction, OD was measured at 490 nm using a plate reader (BioTek). As shown in the results in Figure 12, when VEGF was added, the OD value was higher at the same cell number than when VEGF was not added, confirming HUVEC proliferation caused by VEGF. When VEGF and anti-Ang2/VEGF antibody were added together, the OD value was lower for the same number of cells than when VEGF alone was added, suggesting that anti-Ang2/VEGF antibody binds to VEGF in the medium and inhibits HUVEC proliferation caused by VEGF. It was confirmed that it was possible.

Example 10: Inhibition of neovascularization and improvement of visual acuity of anti-Ang2/VEGF antibody in Mouse Laser-induced CNV model

A study was conducted to confirm the therapeutic potential of anti-Ang2/VEGF antibodies in a wet AMD model caused by abnormal neovascularization with severe leakage. Using the mouse Laser-induced CNV (choroidal neovascularization) model, which is widely used as a wet macular degeneration model, the efficacy of anti-Ang2/VEGF antibody in suppressing choroidal neovascularization and restoring retinal function was compared with aflibercept, a positive control drug.

After inducing laser CNV in C57BL/6 mice, a single IVT (intravitreal) injection of CNV control group and positive control group, IgG and aflibercept, was administered at 5 μg/μL/eye (about 35 μM) and 20 μg/μL/eye (about 177 μM), respectively. injection) was administered. The test group, anti-Ang2/VEGF antibody, was administered as a single IVT at a dose of 5.8 μg/μL/eye (about 35 μM). For retinal imaging evaluation, fundus fluorescein angiography (FFA) and optical coherence tomography (OCT) were performed 11 days after CNV induction to measure the size and degree of leakage of the CNV lesion, and the lesion volume, respectively. Electroretinogram evaluation was performed on the scotopic ERG (electroretinogram) 12 days after CNV induction. Electroretinogram changes in response to mono-flash (0.9 log cds/m2) stimulation were evaluated using B-wave amplitude.

When anti-Ang2/VEGF antibody is administered as a single IVT immediately after CNV induction at a dose of 5.8 μg/μL/eye, the size and leakage of CNV lesions are reduced (Figures 13 and 14), and the B-wave amplitude on scotopic ERG is reduced. It was confirmed that the was significantly improved (FIG. 15).

Reference Example 1: Preparation and screening of anti-Ang2 antibody

An antibody that specifically binds to human Ang2 as an antigen was ordered from AbClone (Korea), and the antibody of the present invention was manufactured by Kohler and Milstein, Eur. J Immunol. 6, 511 (1976). In summary, human Ang2 mixed with an adjuvant (Sigma) as an antigen was injected twice into mice, and antibody production was confirmed by ELISA. After two immunizations, the antibody titer (1:5000) increased, so the spleens were removed from the immunized mice, and B lymphocytes were isolated and fused with sp2/0 cells. The fused cells were cultured in a medium (HAT mediaum) supplemented with hypoxantin, aminopterine, and thymidine, and cells (hybridoma) in which B lymphocytes and sp2/0 cells were fused were selectively selected. The obtained hybridoma cells were separated into positive and negative cells using a serial dilution method, and the cloning process was repeated to prepare monoclonal cells that produce antibodies that react to the antigen. The obtained antibody-producing hybridoma cells were cultured in DMEM (Dulbeco's Modified Eagle's Mediaum) containing 10% (v/v) FBS at 37°C and 5% CO ₂ conditions, and then centrifuged to obtain antibody-producing cells. The culture medium in which the antibodies were secreted was separated, and the antibodies were purified using an affinity column (Protein A/G agarose column, Protein A/G (GenDEPOT)).

Claims (8)

1. A first antigen binding site that specifically binds to human angiopoietin (ANG)-2, and

   Human vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-beta, T-cell immunoglobulin and mucin domain-containing protein (TIM)-3 ligand, and pigment epithelium-derived factor receptor (PEDF). -R), a bispecific antibody comprising a second antigen binding site that specifically binds to one antigen selected from the group consisting of,

   i) The first antigen binding site that specifically binds to the ANG-2 is the CDRH1 region of SEQ ID NO: 1 or 7 or 13, the CDRH2 region of SEQ ID NO: 2 or 8 or 14, and the CDRH3 of SEQ ID NO: 3 or 9 or 15 comprising a region within a heavy chain variable domain, a CDRL1 region of SEQ ID NO: 4 or 10 or 16, a CDRL2 region of SEQ ID NO: 5 or 11 or 17 and a CDRL3 region of SEQ ID NO: 6 or 12 or 18 within a light chain variable domain;

   ii) the second antigen binding site that specifically binds to VEGF comprises a VEGFR1 D2 domain,

   The second antigen binding site that specifically binds to TGF-beta includes TGF-beta RII ECD,

   The second antigen binding site that specifically binds to the ligand of TIM-3 includes the TIM-3 ECD,

   The second antigen binding site that specifically binds to the PEDF-R includes 44-mer PEDF,

   iii) the antibody is a bispecific antibody that induces Tie2 activation.
2. The method of claim 1, wherein the VEGFR1 D2 domain is the VEGFR1 D2 domain of SEQ ID NO: 21,

   The TGF-beta RII ECD is the TGF-beta RII ECD of SEQ ID NO: 24,

   The TIM-3 ECD is TIM-3 ECD of SEQ ID NO: 27,

   The 44-mer PEDF is a bispecific antibody characterized in that it contains the amino acid sequence of SEQ ID NO: 30.
3. The method of claim 1 or 2, wherein the VEGFR1 D2 domain, the TGF-beta RII ECD, the TIM-3 ECD, and the 44-mer PEDF are linked to an immunoglobulin by a linker. Specific antibodies.
4. The bispecific antibody according to claim 3, wherein the linker is each of SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, or SEQ ID NO: 29.
5. A pharmaceutical composition for the treatment of cancer comprising the antibody according to any one of claims 1 to 4.
6. A pharmaceutical composition for the treatment of vascular diseases comprising the antibody according to any one of claims 1 to 4.
7. A nucleic acid encoding a bispecific antibody according to any one of claims 1 to 4.
8. A method for producing a bispecific antibody according to any one of claims 1 to 4, comprising expressing a nucleic acid encoding said bispecific antibody in a prokaryotic or eukaryotic host cell and producing the bispecific antibody from the cell or cell culture supernatant. A method for producing a bispecific antibody, characterized in that recovery.

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