Novel Anti-ang-2 antibody and uses thereof

Abstract

The present invention relates to: a novel single domain antibody (sdAb) or antigen-binding fragment thereof that specifically binds to angiopoietin-2 (Ang-2); a multispecific fusion protein comprising same; a polynucleotide encoding the antibody or fusion protein; an expression vector carrying the polynucleotide; a host cell containing the expression vector; a method for producing the antibody, including culturing the host cell; a pharmaceutical composition comprising the antibody for the prevention or treatment of diseases associated with angiopoietin-2 activation or overproduction; and a method for preventing or treating diseases associated with angiopoietin-2 activation or overproduction using same. The novel anti-Ang-2 antibody of the present invention has high affinity and specificity for Ang-2, and thus can be effectively used for the prevention, treatment, or diagnosis of diseases associated with angiopoietin-2 activation or overproduction.

Description

Novel anti-ANG-2 antibodies and uses thereof

[0001] The present invention relates to a novel single domain antibody (sdAb) that specifically binds to angiopoietin-2 (Ang-2) or an antigen-binding fragment thereof; a multispecific fusion protein comprising the same; a polynucleotide encoding the antibody or fusion protein; an expression vector comprising the polynucleotide; a host cell comprising the expression vector; a method for producing the antibody comprising a step of culturing the cell; a pharmaceutical composition for preventing or treating a disease associated with angiopoietin-2 activation or overproduction comprising the antibody; and a method for preventing or treating a disease associated with angiopoietin-2 activation or overproduction using the same.

[0002]

[0003] Angiogenesis, the process by which new blood vessels extend from pre-existing vessels, plays a crucial role in a wide range of physiological and pathological conditions. Numerous anti-angiogenic and pro-angiogenic factors are involved in blood vessel formation and maintenance.

[0004] Representative angiogenic factors include vascular endothelial cell growth factors (VEGF) A, B, C, and D, basic fibroblast growth factor (bFGF), placental growth factor (PIGF), and angiopoietin (Ang). Antiangiogenic factors include thrombospondins (TSP), angiostatin, endostatin, pigment epithelium-derived factor (PEDF), and soluble decoy receptors consisting of the extracellular domains of receptors for angiogenic factors. Various VEGF families are activated in the form of homodimers or heterodimers, and among them, VEGF-A exists as six isoforms, VEGF121, VEGF145, VEGF165, VEGF183, VEGF189, and VEGF206, through alternative splicing during the expression process. Various VEGF families regulate downstream cell signaling through homodimers or heterodimers of three receptors consisting of vascular endothelial growth factor receptor 1 (VEGFR1, Flt-1), vascular endothelial growth factor receptor 2 (VEGFR2, KDR), and vascular endothelial growth factor receptor 3 (VEGFR3, Flt-4). In addition to the VEGF family, angiopoietins are involved in angiogenesis during embryonic growth and adult development.Representative examples include angiopoietin-1 (Ang-1), which acts as an agonist of the TEK receptor tyrosine kinase (TEK / Tie2) pathway, and angiopoietin-2 (Ang-2), which acts as an agonist or antagonist depending on the surrounding conditions.

[0005] These various angiogenic factors, each with its own role, stimulate surrounding vascular endothelial cells and bind to various receptors, activating cells through downstream signaling pathways to promote migration and proliferation. Conversely, angiogenic inhibitors suppress excessive blood vessel formation from the end of development until adulthood, or help existing immature blood vessels maintain a normal structure. However, if the balance between these numerous factors, each with its own opposing roles, is disrupted, vascular diseases can occur or existing pathological conditions can worsen. Abnormal angiogenesis is known to be a cause of various diseases, including retinal vascular ophthalmic diseases, rheumatoid arthritis, and psoriasis.

[0006]

[0007] Meanwhile, it is known that the inactivation of the TEK / Tie2 receptor, a membrane kinase present in vascular endothelial cells, among diseases related to abnormal angiogenesis, is very deeply related to the progression of retinal vascular disease. TEK / Tie2 is a tyrosine kinase receptor present in vascular endothelial cells and perivascular cells, and is mainly expressed in vascular endothelial cells, playing a role in regulating the stabilization and restructuring function of neovascularization (Dumont DJ, et al. Oncogene 8: 1293-1301, 1993). To date, four ligands for the TEK / Tie2 receptor have been identified: Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), Angiopoietin-3 (Ang-3), and Angiopoietin-4 (Ang-4) (Yancopoulos GD et al. Nature 407:242-248, 2000). Among them, the roles of Ang-1 and Ang-2 have been most actively studied. Ang-1 acts as a potent agonist for TEK / Tie2 receptor signaling, and Ang-2 acts as an antagonist or partial agonist depending on the status and morphology of cells expressing the TEK / Tie2 receptor. It has been confirmed that the concentration of Ang-2 in the vitreous of patients with retinal vascular diseases, including wAMD, DMR, DR, and RVO, is elevated compared to normal individuals. (EMBO Molecular Medicine 2016 8: 1265-1288). Overexpression of Ang-2 causes retinal perivascular cells (pericytes) to detach from endothelial cells, increases endothelial cell sensitivity to VEGF and proinflammatory factors, and induces TEK / Tie2 inactivation. Consequently, Ang-2 inhibits TEK / Tie2, resulting in loss of vascular stabilization function and exacerbation of vascular leakage.On the other hand, Ang-1 contributes to vascular stabilization and regeneration by recruiting perivascular cells including pericytes and maintaining endothelial cell dormancy through activation of the TEK / Tie2 pathway.

[0008] Although various vascular disease-related drugs are being developed (Korean registered patent KR 10-1913940 B1), there is still a lack of new development of anti-Ang-2 antibodies that have improved binding affinity and affinity for Ang-2 protein and excellent activity in treating related diseases.

[0009]

[0010] The problem to be solved by the present invention is to provide a single domain antibody (sdAb) or an antigen-binding fragment thereof that specifically binds to angiopoietin-2 (Ang-2); a multispecific fusion protein comprising the same; a polynucleotide encoding the antibody or fusion protein; an expression vector comprising the polynucleotide; a host cell comprising the expression vector; a method for producing the antibody comprising a step of culturing the cell; a pharmaceutical composition for preventing or treating a disease associated with angiopoietin-2 activation or overproduction comprising the antibody; and a method for preventing or treating a disease associated with angiopoietin-2 activation or overproduction using the same.

[0011]

[0012] One object of the present invention is to provide a single domain antibody (sdAb) or an antigen-binding fragment thereof that specifically binds to angiopoietin-2 (Ang-2), wherein the single domain antibody comprises: (i) a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 35, or SEQ ID NO: 38; (ii) a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, or SEQ ID NO: 39; and (iii) a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, or SEQ ID NO: 40.

[0013] Another object of the present invention is to provide a multispecific fusion protein comprising a single domain antibody (sdAb) or an antigen-binding fragment thereof that specifically binds to angiopoietin-2 (Ang-2); and a second antigen-binding substance.

[0014] Another object of the present invention is to provide a bispecific antibody that simultaneously binds to Ang-2 and VEGF, comprising a single domain antibody (sdAb) that specifically binds to angiopoietin-2 (Ang-2) or an antigen-binding fragment thereof; and an anti-VEGF antibody that specifically binds to VEGF or an antigen-binding fragment thereof.

[0015] Another object of the present invention is to provide a single domain antibody or antigen-binding fragment thereof that specifically binds to angiopoietin-2 (Ang-2), or a polynucleotide encoding the fusion protein.

[0016] Another object of the present invention is to provide an expression vector comprising the polynucleotide.

[0017] Another object of the present invention is to provide a host cell comprising the expression vector.

[0018] Another object of the present invention is to provide a method for producing an antibody comprising a step of culturing the host cell.

[0019] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease associated with angiopoietin-2 activation or overproduction, comprising a single domain antibody specifically binding to angiopoietin-2 (Ang-2) or an antigen-binding fragment thereof or a fusion protein thereof as an active ingredient.

[0020] Another object of the present invention is to provide a method for preventing or treating a disease associated with angiopoietin-2 activation or overproduction, comprising administering the pharmaceutical composition to a subject.

[0021]

[0022] The novel anti-Ang-2 antibody of the present invention has high affinity and specificity for Ang-2. Accordingly, the antibody of the present invention can effectively inhibit the binding of Ang-2 to TEK / Tie2, thereby activating the TEK / Tie2 pathway. Furthermore, the anti-Ang-2 antibody of the present invention has high binding affinity for Ang-2 while simultaneously having no or minimal affinity for Ang-1, thereby effectively inhibiting the action of Ang-2 without interfering with the activation of TEK / Tie2 by Ang-1. Accordingly, the anti-Ang-2 antibody of the present invention can be effectively utilized for the prevention, treatment, or diagnosis of diseases associated with angiopoietin-2 activation or overproduction.

[0023] Based on the excellent activity described above, the anti-Ang-2 antibody of the present invention can be provided in the form of a fusion protein with a substance that binds to another target molecule, and for example, can be provided in the form of a fusion protein with a VEGF binding substance to exhibit a synergistic effect in the treatment of vascular diseases.

[0024]

[0025] Figure 1 is a diagram showing the SDS electrophoresis results of purified anti-Ang-2 single domain antibody.

[0026] Figure 2 is a diagram showing the results of a comparison of the specific binding affinity (EC50) of an anti-Ang-2 single domain antibody (GB1 fusion sdAb) to Ang-1 and Ang-2 antigens by ELISA titration.

[0027] Figure 3 is a diagram showing the results of a comparison of the specific binding affinity (EC50) of an anti-Ang-2 single domain antibody (Fc fusion sdAb) to Ang-1 and Ang-2 antigens by ELISA titration.

[0028] Figure 4 is a diagram showing the results of measuring the inhibitory activity of anti-Ang-2 single domain antibody on TEK / Tie2 receptor signaling in HUVEC cells.

[0029] Figure 5 is a diagram measuring the angiogenesis inhibitory activity of an anti-Ang-2 antibody in a CNV animal model. The experimental group for the anti-Ang-2 antibody of the present invention is designated as Anti-Ang2.

[0030] Figure 6 is a diagram showing the results of expression and purification of a multispecific fusion protein containing an anti-Ang-2 antibody.

[0031] Figure 7 shows the results of expression, purification, and purity (SE-HPLC analysis) of three different forms of multivalent and multivalent fusion proteins of the ALTS54B clone among anti-Ang-2 antibodies.

[0032] Figure 8 is a diagram showing the concentration and SDS-PAGE results of a multispecific fusion protein containing an anti-Ang-2 antibody.

[0033] Figure 9 is a diagram showing the direct binding (EC50) of a multispecific fusion protein containing an anti-Ang-2 antibody to VEGF-A165.

[0034] Figure 10 is a diagram showing the change in VEGF-A165 binding absorbance according to the concentration change of a multispecific fusion protein containing an anti-Ang-2 antibody.

[0035] Figure 11 is a diagram showing the binding inhibition (IC50) between VEGFR1 and VEGF-A165 of a multispecific fusion protein containing an anti-Ang-2 antibody.

[0036] Figure 12 is a diagram showing the absorbance change of competitive binding inhibition of biotinylated VEGF-A165 and VEGF receptor 1 (VEGFR1) by a multispecific fusion protein containing an anti-Ang-2 antibody.

[0037] Figure 13 is a diagram showing the direct binding affinity (EC50) of a multispecific fusion protein containing an anti-Ang-2 antibody to Ang-1 and Ang-2.

[0038] Figure 14 is a diagram showing the binding absorbance for Ang-2 or Ang-1 according to changes in the concentration of a multispecific fusion protein containing an anti-Ang-2 antibody.

[0039] Figure 15 is a diagram showing the competitive binding inhibition (IC50) of Ang-1 and Ang-2 to TEK / Tie2 receptors by a multispecific fusion protein containing an anti-Ang-2 antibody.

[0040] Figure 16 is a diagram showing the change in absorbance of competitive binding inhibition between biotinylated Ang-2 and TEK / Tie2 receptor according to the concentration change of multispecific fusion protein containing anti-Ang-2 antibody.

[0041] Figure 17 is a diagram showing the inhibitory activity on TEK / Tie2 receptor signaling in HUVEC cells induced by angiopoietin-2 (Ang-2) when treated with a multispecific fusion protein containing an anti-Ang-2 antibody.

[0042] Figure 18 is a diagram showing the inhibitory activity of angiopoietin-2 (Ang-2)-induced TEK / Tie2 receptor signaling in HUVEC cells upon treatment with a multispecific fusion protein containing an anti-Ang-2 antibody.

[0043] Figure 19 is a diagram showing the recovery activity of Akt phosphorylation in HUVEC cells according to the inhibition of Ang-2 and VEGF by a multispecific fusion protein containing an anti-Ang-2 antibody. V indicates VEGF, A1 indicates Ang-1, A2 indicates Ang-2, and + indicates combined treatment.

[0044] Figure 20 is a diagram showing the VEGF-induced HUVEC cell growth inhibitory activity of multispecific fusion proteins containing anti-Ang-2 antibodies.

[0045] Figure 21 is a diagram showing the inhibitory activity of a multispecific fusion protein containing an anti-Ang-2 antibody on HUVEC monolayer permeability induced by single treatment with VEGF.

[0046] Figure 22 is a diagram showing the inhibitory activity of a multispecific fusion protein containing an anti-Ang-2 antibody on HUVEC monolayer permeability induced by single treatment with VEGF.

[0047] Figure 23 is a diagram showing the inhibitory activity of multispecific fusion proteins containing anti-Ang-2 antibodies on HUVEC monolayer permeability induced by complex treatment with VEGF, Ang1, and Ang-2.

[0048] Figure 24 is a diagram measuring the angiogenesis inhibitory activity of a multispecific fusion protein containing an anti-Ang-2 antibody in a CNV animal model.

[0049]

[0050] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below. In addition, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated into this specification in their entirety by reference to more clearly explain the level of the technical field to which the present invention belongs and the contents of the present invention.

[0051] Furthermore, those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Furthermore, such equivalents are intended to be encompassed by the present invention.

[0052]

[0053] Throughout this specification, conventional one-letter and three-letter codes for naturally occurring amino acids are used. Amino acids referred to herein by abbreviation are described according to the IUPAC-IUB nomenclature.

[0054] Alanine Ala, A Arginine Arg, R

[0055] Asparagine Asn, N Aspartic acid Asp, D

[0056] Cysteine ​​Cys, C Glutamic acid Glu, E

[0057] Glutamine Gln, Q Glycine Gly, G

[0058] Histidine His, H Isoleucine Ile, I

[0059] Leucine Leu, L Lysine Lys, K

[0060] Methionine Met, M Phenylalanine Phe, F

[0061] Proline Pro, P Serine Ser, S

[0062] Threonine Thr, T Tryptophan Trp, W

[0063] Tyrosine Tyr, Y Valine Val, V

[0064]

[0065] anti-Ang-2 antibody

[0066] One aspect of the present invention provides an antibody that specifically binds to angiopoietin-2 (Ang-2). The antibody of the present invention that specifically binds to angiopoietin-2 (Ang-2) may also be referred to as an "anti-Ang-2 antibody" hereinafter.

[0067] Specifically, the anti-Ang-2 antibody of the present invention may be a single domain antibody (sdAb) or an antigen-binding fragment thereof that specifically binds to angiopoietin-2 (Ang-2). In the present specification, the single domain antibody that specifically binds to angiopoietin-2 (Ang-2) may also be referred to as "anti-Ang-2 sdAb" or "anti-Ang-2 single domain antibody" hereinafter.

[0068]

[0069] As used herein, the term "antibody" refers to a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that immunologically has reactivity with a specific antigen, and includes polyclonal antibodies, monoclonal antibodies, whole antibodies, and antibody fragments. For the purposes of the present invention, the antibody may be an antibody that specifically binds to Ang-2 protein. The term also includes chimeric antibodies, humanized antibodies, human antibodies, and bivalent or bispecific molecules (e.g., bispecific antibodies), diabodies, triabodies, and tetrabodies. The term further includes single-chain antibodies, scabs, derivatives of antibody constant regions, and artificial antibodies based on protein scaffolds that have binding function to FcRn. A whole antibody has a structure having two full-length light chains and two full-length heavy chains, each light chain being linked to a heavy chain by a disulfide bond. The above whole antibodies include IgA, IgD, IgE, IgM, and IgG, and IgG includes subtypes such as IgG1, IgG2, IgG3, and IgG4. The above antibody fragments refer to fragments having an antigen-binding function, and include Fd, Fab, Fab', F(ab')2, and Fv. The Fd refers to the heavy chain portion included in the Fab fragment. The Fab has one antigen-binding site in a structure having variable regions of the light and heavy chains, a constant region of the light chain, and the first constant region (CH1 domain) of the heavy chain. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. The F(ab')2 antibody is generated when the cysteine ​​residues in the hinge region of Fab' form a disulfide bond. Fv (variable fragment) refers to the smallest antibody fragment having only the heavy chain variable region and the light chain variable region.Double disulfide Fv (dsFv) has a heavy chain variable region and a light chain variable region linked by a disulfide bond, and single-chain Fv (scFv) has a heavy chain variable region and a light chain variable region covalently linked, typically via a peptide linker. These antibody fragments can be obtained using proteolytic enzymes (for example, restriction digestion of a whole antibody with papain can yield a Fab fragment, and digestion with pepsin can yield an F(ab')2 fragment), and specifically, can be produced using genetic recombination technology. Specifically, the antibody can be a single-domain antibody, for example, a heavy chain-only antibody.

[0070] In the present invention, the term "single domain antibody (sdAb)" refers to a single antigen-binding polypeptide having three complementarity determining regions (CDRs). sdAb refers to the minimum unit of human heavy chain variable domain (VH) or human light chain variable domain (VL) that provides specific binding affinity in a complete antibody, and the single variable domain of single heavy chain antibody (V) such as shark, llama, camel, and alpaca. H H) is an antibody containing only Ang-2. For the purpose of the present invention, the single-domain antibody may refer to an sdAb having an epitope that is a protein-determining site for Ang-2 protein as a single-domain antibody (sdAb) that specifically binds to Ang-2.

[0071] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains are, respectively, "V H " and "V L" may be referred to as these domains. These domains are generally the most variable part of the antibody (compared to other antibodies of the same class) and contain the antigen binding site. For example, V H has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and where CDR1 to CDR3 refer to complementarity determining regions 1 to 3.

[0072] An "isolated" antibody is one that has been identified, separated, and / or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide is free from all other components from its production environment. Contaminant components of the production environment, such as those arising from recombinant transfected cells, are materials that typically interfere with research, diagnostic, or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the polypeptide will be purified to homogeneity by (1) being greater than 95% by weight of the antibody, and in some embodiments, greater than 99% by weight, as determined, for example, by the Lowry method; (2) using a rotating cup arrayer to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence; or (3) by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. An isolated antibody comprises the antibody in situ within a recombinant cell, since at least one component of the antibody's natural environment will be absent. However, typically, an isolated polypeptide or antibody will be prepared by at least one purification step.

[0073]

[0074] In the present invention, the term "Angiopoietin-2 (Ang-2)" includes variants and isoforms of Ang-2 naturally expressed by the ANGPT2 gene. Angiopoietin-2 loosens the bond between cells or induces apoptosis, which ultimately causes blood vessel atrophy. Angiopoietin-2 activates or inhibits the TEK / Tie2 pathway depending on the distribution environment of surrounding angiogenesis regulators according to normal and abnormal conditions. That is, angiopoietin-2 acts as an antagonist or partial agonist for the TEK / Tie2 receptor, which is a receptor on the cell surface, depending on the surrounding environment, and interacts with other vascular endothelial cell growth factors including VEGF to maintain homeostasis related to angiogenesis in the normal state. At this time, angiopoietin-2 (Ang-2) It competitively binds to angiopoietin-1 (Ang-1) and TEK / Tie2 receptors. In a normal state where homeostasis regarding angiogenesis and inhibition is maintained, the expression levels of Ang-1 and Ang-2 are balanced. However, in angiogenesis-related diseases including cancer, diabetic retinopathy, and age-related macular degeneration, the level of Ang-2 is relatively high, resulting in an imbalance.

[0075] Accordingly, antibodies that specifically recognize Ang-2 can be used for the diagnosis, prevention, or treatment of diseases associated with Ang-2 activation or overproduction, and the present inventors have developed antibodies that bind with high affinity to the Ang-2 protein. The anti-Ang-2 sdAb of the present invention binds with high affinity to Ang-2 antibodies while exhibiting no reactivity toward Ang-1, and thus can be usefully utilized in the field of treating related diseases.

[0076] The angiopoietin-2 (Ang-2) protein of the present invention may be obtained recombinantly by a conventional method or may be purchased from a conventional commercial source. The Ang-2 of the present invention may be in the form of an isolated protein or in the form of a combination with another protein or polysaccharide, and specifically, may be in the form of a combination with a bacterial exotoxin, but is not limited thereto. In one embodiment, the Ang-2 may be human Ang-2, and in one example, the Ang-2 protein may comprise the amino acid sequence represented by SEQ ID NO: 42.

[0077]

[0078] The anti-Ang-2 sdAb of the present invention comprises (i) a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 35, or SEQ ID NO: 38;

[0079] (ii) a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, or SEQ ID NO: 39; and

[0080] (iii) may comprise a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, or SEQ ID NO: 40.

[0081]

[0082] In one embodiment, the anti-Ang-2 sdAb of the present invention

[0083] (a) CDR 1 of SEQ ID NO: 11, CDR2 of SEQ ID NO: 12, and CDR3 of SEQ ID NO: 13; (b) CDR 1 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16; (c) CDR 1 of SEQ ID NO: 17, CDR2 of SEQ ID NO: 18, and CDR3 of SEQ ID NO: 19; (d) CDR 1 of SEQ ID NO: 20, CDR2 of SEQ ID NO: 21, and CDR3 of SEQ ID NO: 22; (e) CDR 1 of SEQ ID NO: 23, CDR2 of SEQ ID NO: 24, and CDR3 of SEQ ID NO: 25; (f) CDR 1 of SEQ ID NO: 26, CDR2 of SEQ ID NO: 27, and CDR3 of SEQ ID NO: 28; (g) CDR 1 of SEQ ID NO: 29, CDR2 of SEQ ID NO: 30, and CDR3 of SEQ ID NO: 31; (h) CDR 1 of SEQ ID NO: 32, CDR2 of SEQ ID NO: 33, and CDR3 of SEQ ID NO: 34; (i) CDR 1 of SEQ ID NO: 35, CDR2 of SEQ ID NO: 36, and CDR3 of SEQ ID NO: 37; or (j) CDR 1 of SEQ ID NO: 38, CDR2 of SEQ ID NO: 39, and CDR3 of SEQ ID NO: 40.

[0084] In one example, the anti-Ang-2 sdAb of the present invention may comprise CDR 1 of SEQ ID NO: 17, CDR2 of SEQ ID NO: 18, and CDR3 of SEQ ID NO: 19; or CDR 1 of SEQ ID NO: 29, CDR2 of SEQ ID NO: 30, and CDR3 of SEQ ID NO: 31.

[0085] In one embodiment of the present invention, clones highly reactive with Ang-2 and low reactivity with Ang-1 were screened to derive 10 novel antibodies and their respective CDRs having high antigen-binding activity selective for Ang-2. This suggests that the anti-Ang-2 sdAb of the present invention can be useful in fields requiring recognition of the Ang-2 protein, such as the diagnosis or treatment of diseases in which the Ang-2 protein is overexpressed.

[0086] In one embodiment, the anti-Ang-2 sdAb of the present invention may comprise a sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the CDR sequence.

[0087] In one example, since the CDR sequence may include a mutation for exclusion of an N-glycosylation site, the sequence prior to the mutation for exclusion of N-glycosylation may also be included within the scope of the present invention as a biological equivalent.

[0088]

[0089] In another embodiment, the anti-Ang-2 sdAb of the present invention comprises a heavy chain variable region, wherein the heavy chain variable region can comprise a sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 10.

[0090] In one example, an anti-Ang-2 sdAb of the invention comprises a heavy chain variable region, wherein the heavy chain variable region can comprise an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 7, or a sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 7.

[0091]

[0092] In certain embodiments, amino acid sequence variants of the anti-Ang-2 sdAb of the present invention are contemplated. For example, they may improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecule or by peptide synthesis. Such modifications include, for example, deletions of residues from the amino acid sequence of the antibody, and / or insertions of residues into such amino acid sequence, and / or substitutions of residues within such amino acid sequence. Any combination of various alterations, including deletions, insertions, and substitutions, can be performed to arrive at the final construct, provided that the final construct retains the desired properties, e.g., antigen-binding properties. Target sites for substitutional mutagenesis can include the heavy chain variable region and the framework region.

[0093] In one embodiment, the variant may comprise a biological equivalent of the anti-Ang-2 sdAb of the present invention, as long as the antigen-binding properties are maintained. For example, arginine, lysine, and histidine are all positively charged residues, aspartate and glutamate are negatively charged residues, serine, threonine, asparagine, and glutamine are polar uncharged residues, alanine, valine, isoleucine, leucine, and methionine are hydrophobic residues, and phenylalanine, tyrosine, and tryptophan are hydrophobic residues with an aromatic ring structure. Therefore, a variant comprising a mutation based on the relative similarity of these amino acid residues may be considered a biologically functional equivalent.

[0094] In one embodiment, the biological equivalent may comprise conservative substitutions. The term "conservative substitution" herein is defined as follows, and conservative substitutable candidate amino acids are shown in parentheses: Ala (Gly, Ser); Arg (Gly, Gln); Asn (Gln; His); Asp (Glu); Cys (Ser); Gln (Asn, Lys); Glu (Asp); Gly (Ala, Pro); His (Asn; Gln); Ile (Leu; Val); Leu (Ile; Val); Lys (Arg; Gln); Met (Leu, Ile); Phe (Met, Leu, Tyr); Ser (Thr; Gly); Thr (Ser; Val); Trp (Tyr); Tyr (Trp; Phe); Val (Ile; Leu).

[0095]

[0096] In one embodiment, the anti-Ang-2 antibody of the present invention may be provided in a multivalent form in which two or more anti-Ang-2 sdAbs are linked. Specifically, the anti-Ang-2 antibody of the present invention may be provided in the form of a single monovalent sdAb, or in a bivalent, tetravalent, or higher multivalent form, thereby enhancing binding affinity and activity to a target molecule. In this case, the sdAbs may be directly linked to each other or linked via a linker.

[0097] In the present invention, the linker may be, but is not limited to, a non-peptide linker or a peptide linker. More specifically, the peptide linker may include one or more amino acids, for example, from 1 to 1000 amino acids, and includes any peptide linker known in the art, such as a [GS]x linker, a [GGGS]x linker, and a [GGGGS]x linker, wherein x may be a natural number of 1 or more (for example, 1, 2, 3, 4, 5, or more), but is not limited thereto. In one example, the linker may use a peptide linker comprising a sequence of 7 to 20 amino acids, and in a more specific example, the [GGGGS]3 linker of SEQ ID NO: 67 or the GGGGSGGGS linker of SEQ ID NO: 68 may be used, but is not limited thereto.

[0098]

[0099] The anti-Ang-2 sdAb of the present invention has the property of binding with high affinity to angiopoietin-2.

[0100] In one embodiment, the anti-Ang-2 sdAb can specifically bind to the fibrinogen-like domain (FLD) of the Ang-2 protein. The FLD of Ang-2 may be the fibrinogen-like domain of the human Ang-2 protein. In one example, the fibrinogen-like domain of Ang-2 may comprise the amino acid sequence represented by SEQ ID NO: 43.

[0101] In one embodiment of the present invention, it was confirmed that the anti-Ang-2 sdAb of the present invention has high affinity and specificity for Ang-2 and can effectively induce activation of the TEK / Tie2 pathway by preventing Ang-2 from binding to the TEG / Tie2 receptor.

[0102] In one example, the binding capacity of the anti-Ang-2 antibody of the present invention to the Ang-2 antigen is 10 based on the half-maximal reaction concentration (EC50). -6 M to 10 -12 M may be, and more specifically, the EC50 of Ang-2 antibody is 10 -7 M to 10 -12 , 10 -8 M to 10 -12 , 10 -9 M to 10 -12 , 10 -10 M to 10 -12 M, 10 -6 M to 10 -11 , 10 -7 M to 10 -11 , 10 -8 M to 10 -11 , 10 -9 M to 10 -11 M, 10 -10 M to 10 -11 M, 10 -6 M to 10 -10 , 10 -7 M to 10 -10 , 10 -8M to 10 -10 , 10 -9 M to 10 -10 It may be M. At this time, the EC50 may be measured by an ELISA titration method of an antibody to the antigen.

[0103]

[0104] The anti-Ang-2 sdAb of the present invention is a single domain antibody that has the characteristic of not binding to angiopoietin-1 (ANG-1) or having low reactivity.

[0105] In one embodiment of the present invention, the results of comparing the competitive binding inhibition of Ang-1 and Ang-2 to the TEK / Tie2 receptor confirmed that the median binding concentration of the anti-Ang-2 sdAb to Ang-2 was 1000 times lower than that of Ang-1 based on the EC50% (Examples 2 and 3), confirming that the antibody of the present invention can effectively inhibit the action of Ang-2 without interfering with the activation of TEK / Tie2 by Ang-1. In one example, Ang-1 may include an amino acid sequence represented by SEQ ID NO: 44, and the FLD of Ang-1 may include an amino acid sequence represented by SEQ ID NO: 45.

[0106] The anti-Ang-2 sdAb of the present invention can activate the TEK / Tie2 pathway by inhibiting the binding of angiopoietin-2 to the TEK / Tie2 receptor. The TEK / Tie2 pathway is known to play a role in helping to structurally stabilize blood vessels during the development of angiogenesis. In one embodiment of the present invention, it was confirmed that the anti-Ang-2 sdAb of the present invention effectively inhibits Ang-2 binding to the TEK / Tie2 receptor (Example 3), and as a result, exhibits high TEK / Tie2 receptor signaling inhibition activity (Example 4).

[0107]

[0108] In one embodiment, the anti-Ang-2 antibody of the present invention may be in a form fused with a biocompatible substance or carrier capable of increasing its stability or duration of activity. Specifically, the anti-Ang-2 antibody of the present invention may be in a form linked to the biocompatible substance or carrier via a linker or covalent bond.

[0109] In the present invention, the terms "biocompatible material" and "carrier" refer to a material that can extend the stability or activity duration of the anti-Ang-2 antibody of the present invention when directly or indirectly linked to the anti-Ang-2 antibody of the present invention by covalent or non-covalent bonds to form a complex. The types of biocompatible materials or carriers that can be used in the present invention are diverse, and can be selected from the group consisting of, for example, polyethylene glycol, fatty acids, cholesterol, albumin and fragments thereof, albumin binding materials, polymers of repeating units of a specific amino acid sequence, FcRn binding materials, in vivo connective tissues, nucleotides, fibronectin, transferrin, saccharides, high molecular weight polymers, and combinations thereof. Such biocompatible materials or carriers include biocompatible materials that extend the in vivo half-life by covalent or non-covalent bonding.

[0110]

[0111] In one embodiment, the biocompatible material may be an immunoglobulin Fc region or a GB1 domain.

[0112] Specifically, the immunoglobulin Fc region may be selected from the group consisting of (a) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; (b) a CH1 domain and a CH2 domain; (c) a CH1 domain and a CH3 domain; (d) a CH2 domain and a CH3 domain; (e) a combination of one or more of the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain with an immunoglobulin hinge region or a portion of a hinge region; and (f) a dimer of each domain of a heavy chain constant region and a light chain constant region. In the present invention, the immunoglobulin Fc region may also be used interchangeably as "Fc", "Fc region", and "Fc portion".

[0113] In one example, the immunoglobulin Fc region is characterized in that a modification selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof has occurred in at least one amino acid in the native immunoglobulin Fc region.

[0114] In one example, the immunoglobulin Fc region may have a site capable of forming a disulfide bond removed, some amino acids at the N-terminus of a native Fc removed, a methionine residue added to the N-terminus of a native Fc, a complement binding site removed, or an ADCC (antibody dependent cell mediated cytotoxicity) site removed.

[0115] In one example, the immunoglobulin Fc region may be aglycosylated.

[0116] In one example, the immunoglobulin Fc region may be derived from IgG, IgA, IgD, IgE, or IgM. In one example, the immunoglobulin Fc region may be a rabbit immunoglobulin IgG region or a human immunoglobulin IgG region.

[0117] In one example, the immunoglobulin Fc region may be a hybrid of domains of different origins derived from immunoglobulins selected from the group consisting of IgG, IgA, IgD, IgE, and IgM.

[0118] In another example, the immunoglobulin Fc may comprise, but is not limited to, the amino acid sequence of SEQ ID NO: 47 or SEQ ID NO: 69, or a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 47 or SEQ ID NO: 69.

[0119]

[0120] Specifically, the GB1 domain refers to the B1 domain of the immunoglobulin-binding protein “protein G.” In the present invention, the GB1 domain may be used interchangeably as “GB1” or “protein G B1 domain.”

[0121] In one example, the GB1 domain may be a Streptococcal Protein G B1 domain of protein G derived from Streptococcus, or may be a GB1 domain of protein G derived from another organism or microorganism.

[0122] In one example, the GB1 domain is characterized in that a modification selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof has occurred in at least one amino acid in the native GB1 domain.

[0123] In another example, the GB1 domain may include, but is not limited to, the amino acid sequence of SEQ ID NO: 46 or a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 46.

[0124]

[0125] multispecific fusion proteins

[0126] Another aspect of the present invention provides a multispecific fusion protein comprising a single domain antibody (sdAb) or an antigen-binding fragment thereof that specifically binds to angiopoietin-2 (Ang-2); and a second antigen-binding substance.

[0127] The definitions of the above “angiopoietin-2”, “antibody”, “sdAb”, and “single domain antibody that specifically binds to angiopoietin-2” are the same as described above.

[0128]

[0129] In the present invention, the term multi-specific fusion protein is a binding molecule having two or more binding specificities. The term "multi-specific" specifically includes "bispecific" and "trispecific", as well as higher-order specific binding affinities. The term "multi-specific fusion protein" includes "multi-specific antibody", "multi-specific heavy chain-only antibody", "multi-specific heavy chain antibody", "multi-specific UniAb". TM ", and "multispecific binding compound" are used herein in the broadest sense, including all binding molecules having more than one binding specificity.

[0130] The anti-Ang-2 antibody of the present invention, based on its high Ang-2 specificity, can be applied in a manner that reduces mutual resistance by fusing with other therapeutic proteins, angiogenesis inhibitors, or antibodies, thereby exhibiting a synergistic effect in the treatment of vascular-related diseases and enhancing efficacy. From this perspective, the anti-Ang-2 antibody of the present invention can be provided in the form of a fusion protein conjugated to various substances.

[0131]

[0132] In the present invention, the term "second antigen binding substance" may include, without limitation, any substance that specifically binds to a target molecule other than angiopoietin-2, i.e., a second antigen, and examples thereof may include antibodies, antigen-binding fragments, peptides, aptamers, compounds, non-antibody protein structures, recombinant proteins, or nucleic acids. As a more specific example, it may be a receptor or antibody that binds to a vascular or inflammation regulation-related factor other than angiopoietin-2. The second antigen binding substance may be used without limitation as long as it is a substance that can be combined with the anti-Ang-2 antibody of the present invention to enhance efficacy.

[0133] The multispecific fusion protein of the present invention may additionally include, in addition to the second antigen-binding substance, a third antigen-binding substance, a fourth antigen-binding substance, ..., an n-th antigen-binding substance without limitation. In this case, n is an integer greater than or equal to 3. The n-th antigen-binding substance may be, for non-limiting examples, an antibody, an antigen-binding fragment, a peptide, an aptamer, a compound, a non-antibody protein structure, a recombinant protein, or a nucleic acid.

[0134]

[0135] For example, the multispecific fusion protein of the present invention may modulate two or more pathways selected from the group consisting of the VEGF receptor 1 pathway, the VEGF receptor 2 pathway, the VEGF receptor 3 pathway, the VEGF receptor 4 pathway, the Tie1 or TEK / Tie2 receptor pathway, the Wnt / β-Catenin axis pathway, the Norrin pathway, the complement system pathway, the integrin pathway, the VE-PTP pathway, the Notch1, 2, 3, 4 / DLL1, 3, 4 axis pathway, and the tumor necrosis factor (TNF-α) receptor pathway, or may multiply modulate two or more pathways.

[0136] For example, the second antigen binding agent may be an anti-VEGF antibody or a soluble VEGF receptor.

[0137] The multispecific fusion protein of the present invention comprises a single domain antibody (sdAb) or an antigen-binding fragment thereof that specifically binds to angiopoietin-2 (Ang-2); and a second antigen-binding agent, which may be directly linked or linked via a linker, or may additionally include another protein moiety, but is not limited thereto. In this case, the "linker" is defined in the same manner as described above. The method and position of linkage of the multispecific fusion protein of the present invention can be used without limitation by any method or position practiced in the art, as long as it does not change the structure or activity of the linked protein.

[0138]

[0139] In one embodiment, the second antigen-binding agent may be, for example, an antibody. In this case, when the second antigen-binding agent in the present invention is an antibody, it may be referred to as a "second antibody."

[0140] In one embodiment, the multispecific fusion protein of the present invention may comprise the anti-Ang-2 antibody or antigen-binding fragment thereof; and a second antibody or antigen-binding fragment thereof.

[0141] The term "second antibody" in the present invention refers to an antibody whose target molecule is different from that of the anti-Ang-2 antibody, and which specifically binds to a target molecule other than angiopoietin-2. The second antibody is not limited thereto as long as it has specificity for a target molecule different from that of the anti-Ang-2 sdAb of the present invention, thereby providing a synergistic effect or additional activity.

[0142] Meanwhile, when the multispecific fusion protein of the present invention is an n-th specific antibody having a triple specificity or higher, the multispecific fusion protein may additionally include a second antibody, a third antibody, ..., n-th antibody, each having a target molecule different from Ang-2. In this case, n is an integer of 3 or greater.

[0143] In one embodiment, the anti-Ang-2 antibody and the second antibody may each be provided independently in a monovalent form, or may be provided in a bivalent, tetravalent, or higher multivalent form, wherein each molecule may be provided in a form in which they are directly bound to each other or linked via a linker.

[0144] In one embodiment, the multispecific fusion protein of the present invention may have an anti-Ang-2 antibody and a second antibody sequentially linked from the N-terminus, or may have a second antibody and an anti-Ang-2 antibody sequentially linked from the N-terminus.

[0145]

[0146] In one embodiment, the anti-Ang-2 antibody and the second antibody may each independently be in a form in which the biocompatible material or carrier described above is fused, and in one example, the biocompatible material or carrier may be an immunoglobulin Fc region or a GB1 domain.

[0147] In one example, the multispecific fusion protein of the present invention may be a fusion protein in which an anti-Ang-2 antibody, a biocompatible material or carrier, and a second antibody are sequentially linked from the N-terminus. In another example, the multispecific antibody of the present invention may be a fusion protein in which a second antibody, a biocompatible material or carrier, and an anti-Ang-2 antibody are sequentially linked from the N-terminus. Independently, the second antibody and the biocompatible material or carrier may be directly linked to each other or linked through a linker, and the biocompatible material or carrier and the anti-Ang-2 antibody may be directly linked to each other or linked through a linker.

[0148]

[0149] In one embodiment, the second antibody is not limited as long as it targets a molecule different from Ang-2, but may be, for example, an anti-VEGF antibody, an anti-C3b antibody, an anti-C5 antibody, an anti-ACC2 antibody, an anti-RAMP2 antibody, an anti-Tie2 antibody, an anti-EGFR antibody, an anti-αvβ5 antibody, an anti-RANKL antibody, an anti-influenza antibody, an anti-malarial antibody, an anti-HCV antibody, an anti-HIV antibody, an anti-HBV antibody, an anti-CK-MB antibody, an anti-troponin I antibody, an anti-myoglobin antibody, an anti-PSA antibody, an anti-AFP antibody, an anti-CEA antibody, an anti-TSH antibody, an anti-CSH antibody, an anti-hcG antibody, an anti-cortisol antibody, an anti-progesterone antibody, an anti-testosterone antibody, an anti-GAD antibody, an anti-insulin antibody, an anti-IGF antibody, an anti-B cell antibody, or an anti-BlyS antibody.

[0150]

[0151] In one embodiment, the second antibody may be an anti-VEGF antibody that specifically binds to VEGF. In this case, the multispecific fusion protein of the present invention may be a bispecific antibody that simultaneously binds to Ang-2 and VEGF, comprising a single domain antibody (sdAb) that specifically binds to angiopoietin-2 (Ang-2) or an antigen-binding fragment thereof; and an anti-VEGF antibody or an antigen-binding fragment thereof that specifically binds to VEGF.

[0152] In the present invention, the term "vascular endothelial growth factor" or "VEGF" refers to a type of signaling protein that plays an important role in vasculogenesis and angiogenesis. Various series of VEGF are activated in the form of homodimers or heterodimers, and among them, VEGF-A exists as six isoforms of VEGF121, VEGF145, VEGF165, VEGF183, VEGF189, and VEGF206 through alternative splicing during the expression process. The various VEGF families regulate downstream cell signaling through homodimers or heterodimers of three receptors: vascular endothelial growth factor receptor 1 (VEGFR1, Flt-1), vascular endothelial growth factor receptor 2 (VEGFR2, KDR), and vascular endothelial growth factor receptor 3 (VEGFR3, Flt-4). VEGF functions as part of a system that stores and supplies oxygen to tissues when blood flow is inadequate, and its general function is to create new blood vessels during embryonic development, muscle after injury or exercise, and formation of blood vessels to bypass infarcted blood vessels. However, increased amounts of VEGF can cause abnormal angiogenesis.

[0153] In the present invention, as long as VEGF exhibits effects of promoting regeneration of damaged skin, regeneration and growth of hair through inducing angiogenesis, proliferation of epidermal cells, promotion of cell migration, and increase in microvascular receptivity, its amino acid sequence is not particularly limited. The entire amino acid sequence of VEGF may be used, a mutated amino acid sequence thereof may be used, or a partial fragment thereof may be used. The specific amino acid sequence of VEGF or the base sequence information of the gene encoding it can be obtained from known databases such as NCBI's GenBank.

[0154]

[0155] VEGF is one of the most important angiogenic factors in retinal vascular diseases, and anti-VEGF therapy, a strategy that blocks the function of this factor, has been established as the most effective treatment to date. However, this single-factor inhibition treatment cannot solve the problem of ischemia in the blood vessels around the choroid due to excessive angiogenesis inhibition, and can even cause side effects that worsen symptoms. Therefore, anti-VEGF therapy alone can raise serious concerns about side effects in elderly or diabetic patients who require new blood vessel formation in the ischemic brain, heart, and limbs. Furthermore, anti-VEGF therapy non-responsiveness in up to 30% of patients with wAMD and up to 50% of patients with diabetic macular edema (DME) remains a significant area of ​​improvement. Moreover, anti-VEGF monotherapy still has significant unmet medical needs, as there is still much room for improvement in the drug response rate related to visual acuity improvement and drying effect, and the drug's sustained effect related to the interval between intravitreal injections (IVT).

[0156] According to one embodiment of the present invention, a multispecific fusion protein that simultaneously binds to Ang-2 and VEGF can solve the problems of the conventional anti-VEGF treatment described above, and can induce stabilization and remodeling of new blood vessels through regulation of the TEK / Tie2 pathway, while effectively inhibiting the proliferation of new blood vessels through regulation of the VEGF pathway. Accordingly, the dual specific antibody of the present invention that simultaneously binds to Ang-2 and VEGF can exhibit a synergistic effect on inhibiting new blood vessel formation by simultaneously regulating the VEGF pathway and the TEK / Tie2 pathway.

[0157] In one embodiment, the inventors of the present invention prepared a fusion protein combining the anti-Ang-2 antibody and the anti-VEGF antibody, and verified that the fusion protein of the present invention effectively neutralizes Ang-2, thereby inhibiting phosphorylation signaling of the TEK / Tie2 pathway, and at the same time possesses high binding properties to VEGF, thereby exhibiting HUVEC growth inhibitory activity. Furthermore, they confirmed remarkable angiogenesis inhibitory activity in a mouse choroidal neovascularization animal model. In other words, it was confirmed that while the activity of the existing anti-Ang-2 antibody was maintained or enhanced, the intrinsic activity of the anti-VEGF antibody itself, which is the second antigen-binding substance to be bound, was also maintained, ultimately exhibiting a synergistic effect on angiogenesis.

[0158]

[0159] In one example, the anti-VEGF antibody may be aflibercept, bevacizumab, brolucizumab, Conbercept, or ranibizumab, but is not limited thereto as long as it is a VEGF-specific antibody. In a more specific example, the anti-VEGF antibody may be aflibercept, and the aflibercept may be comprised of a VEGFR1 domain, a VEGFR2 domain, and an IgG Fc.

[0160] In one example, the multispecific fusion protein of the present invention may be in a form in which an anti-Ang-2 antibody is linked to the N-terminus or C-terminus of aflibercept. In this case, the anti-Ang-2 antibody may include a tetravalent or bivalent anti-Ang-2 sdAb. For example, when the anti-Ang-2 antibody includes a tetravalent anti-Ang-2 sdAb, two sdAbs may be fused in series to one of the two heavy chains of the IgG1 Fc domain of aflibercept that are linked by a disulfide bond, and the remaining two sdAbs may be fused in series to the remaining heavy chain of the two IgG1 Fc domain heavy chains. For example, when the anti-Ang-2 antibody comprises two anti-Ang-2 sdAbs, one sdAb may be fused to one of the two heavy chains of the IgG1 Fc domain of aflibercept linked by a disulfide bond, and the other sdAb may be fused to the other of the two IgG1 Fc domain heavy chains. The structural diagrams of the multispecific fusion protein of the present invention according to one embodiment of the invention in each fusion form are shown in FIGS. 6 and 7.

[0161] In one embodiment of the present invention, the multispecific fusion protein of the present invention exhibited excellent activity in various forms, and was also confirmed to be smoothly expressed and produced. This indicates that the high heat resistance and protein folding stability of the antibody of the present invention facilitate the formation of fusion proteins in various forms.

[0162]

[0163] In one example, the multispecific fusion protein of the present invention can comprise a sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to any one sequence selected from the group consisting of SEQ ID NOs: 48 to 65.

[0164]

[0165] Polynucleotides, vectors, host cells and methods of production

[0166] The anti-Ang-2 antibody of the present invention and the multispecific fusion protein comprising the same can be produced by any antibody production technique and fusion protein production technique known in the art.

[0167]

[0168] Another aspect of the present invention provides a polynucleotide encoding a single domain antibody (sdAb) that specifically binds to angiopoietin-2 (Ang-2) or an antigen-binding fragment thereof, or a multispecific fusion protein comprising the same.

[0169] The definitions of “angiopoietin-2”, “antibody”, “single-domain antibody”, “single-domain antibody that specifically binds to angiopoietin-2”, and “multispecific fusion protein” are the same as those described above.

[0170]

[0171] Such polynucleotide molecules may encode the heavy chain variable region or heavy chain CDR region of the anti-Ang-2 antibody, or the amino acid sequence of a multispecific fusion protein. In one example, the sequence of the polynucleotide molecule encoding the heavy chain variable region and CDR region of the anti-Ang-2 antibody according to the present invention may be the polynucleotide sequence of SEQ ID NO: 70 to SEQ ID NO: 79, as described in Table 4. In one example, the sequence of the polynucleotide molecule encoding the fusion protein according to the present invention may be the polynucleotide sequence of SEQ ID NO: 80 to SEQ ID NO: 97.

[0172] The above "polynucleotide" has a comprehensive meaning that includes DNA (gDNA and cDNA) and RNA molecules, and can also be used interchangeably with "nucleic acid molecule." Nucleotides, which are the basic structural units of polynucleotides, include not only natural nucleotides but also analogs with modified sugar or base moieties. The sequence of the polynucleotide encoding the anti-Ang-2 antibody of the present invention and the fusion protein comprising the same may be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides. The polynucleotide of the present invention is also interpreted to include a nucleotide sequence that exhibits substantial identity to the above-described nucleotide sequence. The above substantial identity means a nucleotide sequence that exhibits 80% or more homology, in one specific example 90% or more homology, in another specific example 95% or more homology, and in yet another specific example 98% or more homology, when the nucleotide sequence of the present invention and any other sequence are aligned to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0173]

[0174] According to another aspect of the present invention, one or more expression vectors comprising the polynucleotide are provided. The definition of the "polynucleotide" is as described above.

[0175] The term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid linked to it. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted. Another type of vector is a viral vector, in which virally derived DNA or RNA sequences are present in the vector for packaging into the virus. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genome. Furthermore, certain vectors can drive the expression of a gene to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Typical expression vectors useful in recombinant DNA techniques often exist in the form of plasmids. In this specification, the terms "plasmid" and "vector" may be used interchangeably, as the plasmid is the most commonly used form of vector. However, the present invention is intended to encompass other forms of expression vectors that serve equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0176]

[0177] According to another aspect of the present invention, a host cell comprising the expression vector is provided. In this case, the definition of the "expression vector" is as described above.

[0178] The host cell may be a cell transformed with the recombinant vector of the present invention. Any host cell known in the art that allows stable and continuous cloning and expression of the recombinant vector may be used. For example, the host cell may be a prokaryotic cell, yeast, or eukaryotic cell. For example, suitable prokaryotic host cells include strains of Bacillus species such as Escherichia coli, Bacillus subtilis, and B. thuringensis, Enterobacteriaceae, and strains such as Salmonella typhymurium, Serratia marcescens, and various Pseudomonas species. Suitable eukaryotic host cells to be transformed include yeast, such as Saccharomyces cerevisiae, insect cells, plant cells, and animal cells, such as Sp2 / 0, Chinese hamster ovary (CHO) K1, CHO DXB-11, CHO DG44, PER.C6, CHO / -DHFR, HEK293, HEK293F, VERO, HELA, BRL 3A, SP2 / 0, NS-0, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, and MDCK cell lines. The term "host cell" is also used to refer to a transformed cell or a cell that is capable of expressing a selected gene of interest following transformation with a nucleic acid sequence. The term includes progeny of the parent cell, so long as the selected gene is present, regardless of whether the progeny are identical in morphology or genetic makeup to the original parent.

[0179]

[0180] Another aspect of the present invention provides a method for producing an antibody or fusion protein, comprising the step of culturing the host cell.

[0181] Host cell culture for the production of the above antibodies, antigen-binding fragments thereof, and multispecific fusion proteins comprising them can be performed using appropriate media and culture conditions known in the art. These culture processes can be easily adjusted and used by those skilled in the art depending on the host cell selected. The culture processes are divided into suspension culture and adherent culture depending on the cell growth type, and batch, fed-batch, and continuous culture depending on the culture type. Various culture processes are disclosed, for example, in the literature ["Biochemical Engineering" by James M. Lee, Prentice-Hall International Editions, pp. 138-176].

[0182] For example, the above culture can be performed using CHO cells as a culture medium without limitation among commercially available media. The temperature, pH, feeding frequency, time, etc. of the culture can be appropriately adjusted together with the host cell selected for expression, and the protein expression and cell culture steps can be performed using a transient expression or stable expression system with specialized culture conditions, which will be apparent to those skilled in the art. The antibody and multispecific fusion protein of the present invention have the characteristic of being capable of high-efficiency expression of 300 mg or more per liter of culture medium even under the transient expression conditions of the examples due to their stable sequence and structure.

[0183]

[0184] In one embodiment, the manufacturing method may further comprise a step of recovering the antibody or fusion protein from the cultured cells.

[0185] Any method known in the art for purifying immunoglobulins may be used to recover the antibodies, for example, chromatography (ion exchange, affinity (e.g., protein A), hydrophobic, size exclusion, etc.), centrifugation, differential solubility, or other standard techniques for protein purification.

[0186] For example, the recovery may be performed by removing cells, for example, by filtration, obtaining a cell-free recovery fluid (Harvest Cell Culture Fluid, HCCF), and performing a one-step purification of the resultant through Protein A affinity chromatography. In one embodiment, it was confirmed that the antibody of the present invention and the multispecific fusion protein comprising the same exhibited an average purity of about 97% or more by SE-HPLC analysis when subjected to only one purification step of Protein A affinity chromatography due to their stable sequence and structure. That is, the antibody of the present invention or the fusion protein comprising the same can recover an average of 300 mg or more of the fusion protein per liter of culture medium with a purity of 98% or more with only one step of Protein A affinity chromatography, and this means that the antibody or fusion protein of the present invention can easily recover a high amount of protein by performing a conventional purification step in the art once due to its excellent physical properties, so that the production of a highly concentrated preparation can be facilitated.

[0187]

[0188] It was confirmed that the anti-Ang-2 antibody of the present invention or the fusion protein comprising it can be recovered with high yield and high purity only through a Protein A affinity chromatography step from the culture supernatant using only a transient expression system in CHO cells due to the stability of its properties. For example, the anti-Ang-2 antibody of the present invention has high thermostability and can have a high aggregation temperature at the level of a general antibody, thereby exhibiting excellent properties. Considering the common technical knowledge in the art that fusion proteins are generally difficult to express easily and have a high possibility of forming protein aggregates and low molecular weight fragments, the antibody of the present invention or the fusion protein comprising it is significant in that it can produce a protein in a highly concentrated and highly pure preparation.

[0189]

[0190] pharmaceutical composition

[0191] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating a disease associated with angiopoietin-2 activation or overproduction, comprising as an active ingredient a single domain antibody (sdAb) specifically binding to angiopoietin-2 (Ang-2) of the present invention, an antigen-binding fragment thereof, or a multispecific fusion protein of the present invention.

[0192] The definitions of “angiopoietin-2”, “antibody”, “single-domain antibody”, “single-domain antibody that specifically binds to angiopoietin-2”, and “multispecific fusion protein” are the same as those described above.

[0193]

[0194] In the present invention, the term "disease associated with angiopoietin-2 activation or overproduction" is interpreted to comprehensively refer to diseases caused by abnormal activation or excessive production of the angiopoietin-2 protein. Normally, Ang-2 and Ang-1 are expressed at balanced levels, but in various vascular diseases, Ang-2 levels become relatively high, creating an imbalance.

[0195] Specifically, the disease associated with the above angiopoietin-2 activation or overproduction may be a vascular disease, and more specifically may include a disease associated with angiogenesis, vascular endothelial cell stabilization, and / or vascular permeability.

[0196] For example, diseases related to the above angiopoietin-2 activation or overproduction include age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy (DR), retinal vein occlusion (RVO), retinopathy of prematurity (ROP), neovascular glaucoma, proliferative retinopathy, vascular adhesion, Clarkson's disease / ISCLS, osteoarthritis, rheumatoid arthritis, psoriasis, Crohn's disease, ulcers, diabetic kidney disease These may include, but are not limited to, Nephropathy, Nephritis, Liver Cirrhosis, Neurodegenerative Diseases, or Atherosclerosis.

[0197]

[0198] The anti-Ang-2 antibody of the present invention can bind to Ang-2 with high affinity and induce inhibition and / or neutralization of the activity of Ang-2 protein, and thus has high utility in the prevention or treatment of diseases in which Ang-2 protein is activated or overexpressed. In one embodiment, the anti-Ang-2 antibody of the present invention confirmed excellent Ang-2 binding affinity and inhibitory ability of TEK / Tie2 pathway phosphorylation signal transduction, and accordingly, it was confirmed that it has a significant inhibitory ability on angiogenesis in an actual animal model, and thus can be usefully used in the treatment of diseases related to Ang-2 activation or overproduction.

[0199] In the present invention, the term "prevention" may mean any action that inhibits or delays the onset of a disease associated with angiopoietin-2 activation or overproduction by administering the composition, and the term "treatment" may mean any action that improves or beneficially changes the symptoms of the disease by administering the composition.

[0200] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.

[0201] In the present invention, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate a living organism and does not inhibit the biological activity and properties of the administered compound. In a composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

[0202] The above pharmaceutical composition may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations are prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0203] The above pharmaceutical composition may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.

[0204] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.

[0205] As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined according to factors including the type and severity of the individual, age, sex, type of disease, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered singly or in multiple doses. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects by taking all of the above factors into consideration, and it can be easily determined by those skilled in the art.

[0206] The pH of the above pharmaceutical composition may be 4-8, and more specifically, it may exhibit high stability when it is pH 4.8-5.7, pH 4.5-5.5, pH 5-5.7, pH 5-5.5, pH 7-8, pH 7-7.8, or pH 7.2-7.5.

[0207] As described above, the anti-Ang-2 antibody or fusion protein of the present invention can be easily produced as a highly concentrated and highly purified preparation due to its excellent physical properties, thereby providing an advantageous advantage in providing a pharmaceutical composition for intravenous (IVT) injection, for example, where volume is limited. For example, the pharmaceutical composition of the present invention can provide a preparation containing a highly concentrated active ingredient of 100 mg / ml or more.

[0208]

[0209] In one embodiment, the pharmaceutical composition may comprise a fusion protein comprising an anti-Ang-2 antibody and an anti-VEGF antibody as an active ingredient, in which case the pharmaceutical composition may exhibit superior efficacy in treating vascular diseases by inhibiting Ang-2 and VEGF. Anti-VEGF treatment alone may cause ischemia of blood vessels around the choroid due to excessive inhibition of angiogenesis, and may have serious side effects, for example, in elderly or diabetic patients who require new blood vessel formation in the ischemic brain, heart, or limbs. Therefore, the pharmaceutical composition according to one embodiment of the present invention may exhibit excellent efficacy in stabilizing and normalizing neovascularization without the side effects of anti-VEGF treatment by simultaneously inhibiting Ang-2 and VEGF. In this case, the ease of high-concentration and high-purity formulation of the fusion protein of the present invention can increase the amount of the active ingredient contained in the same volume of composition, which can provide a great technical advantage in use as an anti-VEGF therapeutic agent mainly manufactured for intravitreal injection (IVT).

[0210]

[0211] Treatment methods

[0212] Another aspect of the present invention provides a method for preventing or treating a disease associated with angiopoietin-2 activation or overproduction, comprising administering the pharmaceutical composition to a subject.

[0213] The above “pharmaceutical composition”, “angiopoietin-2”, “disease associated with angiopoietin-2 activation or overproduction”, “treatment”, or “prevention” are defined as above.

[0214] The method for treating a disease associated with the above angiopoietin-2 activation or overproduction may be a method comprising a step of administering to a subject suffering from or suspected of suffering from a disease associated with angiopoietin-2 activation or overproduction a pharmaceutical composition further comprising an antibody and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is the same as described above. The method for treating a disease associated with angiopoietin-2 activation or overproduction may specifically be a method comprising a step of administering to a subject suffering from a disease associated with angiopoietin-2 activation or overproduction a pharmaceutical composition comprising the anti-Ang-2 antibody of the present invention.

[0215] The above-mentioned subject includes mammals, birds, etc., including cows, pigs, sheep, chickens, dogs, humans, etc., and the subject whose disease is treated by administration of the composition of the present invention is included without limitation.

[0216] At this time, the composition can be administered singly or multiple times in a pharmaceutically effective amount. At this time, the composition can be administered in the form of a liquid, powder, aerosol, capsule, enteric-coated tablet, or capsule or suppository. Routes of administration include, but are not limited to, intravitreal administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration. However, since peptides are digested when administered orally, oral compositions must be formulated to coat the active agent or protect it from degradation in the stomach. Furthermore, the pharmaceutical composition can be administered by any device that allows the active agent to travel to target cells.

[0217]

[0218] therapeutic purposes

[0219] Another aspect of the present invention provides a use of the pharmaceutical composition for the treatment of a disease associated with angiopoietin-2 activation or overproduction.

[0220] The above “pharmaceutical composition”, “angiopoietin-2”, “disease associated with angiopoietin-2 activation or overproduction”, and “treatment” are defined as above.

[0221]

[0222] Diagnostic methods

[0223] Another aspect of the present invention provides a method for providing information for diagnosing vascular disease, comprising the step of detecting Ang-2 protein in a separated biological sample from a subject suspected of having vascular disease through an antigen-antibody reaction using the anti-Ang-2 antibody. Furthermore, this method may be a method for diagnosing vascular disease.

[0224] The above “anti-Ang-2 antibody”, “Ang-2”, and “vascular disease” are defined as above.

[0225]

[0226] The method for providing the above information or the diagnostic method can detect Ang-2 protein by reacting an antibody specific to Ang-2 of the present invention with a separated biological sample of an individual suspected of having a vascular disease or an individual suffering from a vascular disease and detecting the formation of an antigen-antibody complex, thereby providing information for diagnosing or predicting the prognosis of a vascular disease.

[0227] Specifically, the method for providing the above information or the diagnostic method may be a method including: (a) a step of treating a separated biological sample of a subject suspected of having a vascular disease with the antibody to detect Ang-2 protein through an antigen-antibody reaction; and (b) a step of comparing the level of Ang-2 protein detected in (a) with a control group, and determining that the subject is a patient with a vascular disease if the level of Ang-2 protein is higher than that of the control group.

[0228] The term "biological sample" as used herein includes, but is not limited to, tissues, cells, whole blood, serum, plasma, autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatants, ruptured eukaryotic cells, and bacterial expression systems. These biological samples can be reacted with the antibodies of the present invention, with or without manipulation, to determine the presence of Ang-2 protein or vascular disease.

[0229] In the present invention, the term "antigen-antibody complex" means a combination of Ang-2 protein antigen in a sample and an anti-Ang-2 antibody according to the present invention that recognizes the same, and the formation of such an antigen-antibody complex can be detected by any method selected from the group consisting of a colorimetric method, an electrochemical method, a fluorimetric method, a luminometry method, a particle counting method, a visual assessment method, and a scintillation counting method. However, it is not necessarily limited to these and various applications and applications are possible.

[0230] In the present invention, various labels can be used to detect antigen-antibody complexes. Specific examples include, but are not limited to, enzymes, fluorescent substances, ligands, luminescent substances, microparticles, and radioactive isotopes.

[0231] Specifically, antigen-antibody complexes can be detected using an enzyme-linked immunosorbent assay (ELISA). ELISA includes various ELISA methods, including a direct ELISA that uses a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA that uses a labeled secondary antibody that recognizes a capture antibody in a complex of antibodies that recognize the antigen attached to the solid support, a direct sandwich ELISA that uses another labeled antibody that recognizes an antigen in a complex of antibodies and antigens attached to a solid support, an indirect sandwich ELISA that uses a labeled secondary antibody that recognizes an antibody after reacting with another antibody that recognizes an antigen in a complex of antibodies and antigens attached to a solid support, and a competitive ELISA that competes between different antigens having the same antibody binding site.

[0232]

[0233] Example 1: Screening of anti-angiopoietin-2 (Ang-2) antibodies from a phage display synthetic single-domain antibody library.

[0234] Example 1-1. Biopanning of phage libraries for Ang-2-specific antibody screening

[0235] For phage library panning, human Ang-2 protein was diluted in PBS to a concentration of 10 μg / ml, and 100 μl was added to a 96-well microplate (Cat. No. 32296, SPL, South Korea) and coated at 4°C for 16 h. The coated plate was washed with PBS (Phosphate Buffer and Saline), and 100 μl of PBS solution containing 2% skim milk was added, blocked at 4°C for 12 h, and then washed three times with PBS.

[0236] The first panning was performed with a phage solution (4 x 10 1270 μl of phage / ml, 2% BSA / 0.1% Tween-20) was added to the Ang-2-coated microplate and incubated at 37°C for 1 hour. The incubated plate was washed 15 times with PBST (0.1% Tween 20 / PBS 200 μl) and then washed 5 more times with PBS. From the second panning, the number of PBST washes was increased according to the number of pannings, and 1 μg of Ang-1 protein was added to the wells to remove antibodies binding to Ang-1. After washing, 5 μg HRV 3C protease in 50 μl PBS was added to the microplate wells and incubated at room temperature for 3 hours to elute the bound phage. The eluted phage was infected into 100 μl of XL1-blue bacteria cultured in 2xTY medium containing 10 μg / ml tetracycline to an optical density (OD600nm) of 0.5. After 1 h of incubation at 37°C, the medium was replaced with LB medium containing 100 μg / ml ampicillin and incubated at 37°C for more than 16 h.

[0237] The following day, phage production was performed by culturing E. coli in LB medium containing 100 μg / ml ampicillin and 50 μg / ml kanamycin after infection with VCSM13 helper phage. 1 ml of the culture was centrifuged at 3,500 rpm for 10 min, and the supernatant was transferred to a new tube. 250 μl of 20% PEG / 2.5 M NaCl solution was added, mixed, and incubated on ice for 1 h. The phage particles were then precipitated by centrifugation at 12,000 rpm for 15 min at 4°C. The phage pellet was resuspended in 70 μl of 2% milk / 0.1% Tween-20 (PBST) for the subsequent panning process. The above process was performed identically for two synthetic single domain antibody phage libraries A and B.

[0238]

[0239] Example 1-2. Obtaining individual clones using enzyme-linked immunosorbent assay (ELISA)

[0240] After centrifuging 200 μl of the cells infected with the eluted phages in the final panning step, the pellet obtained was suspended in 100 μl of PBS, lysed by sonication, and centrifuged to collect only the supernatant. 4 μl of the supernatant was reacted with anti-mouse IgG HRP (Horseradish Peroxidase) secondary antibody (Cat. No. A9169, Sigma) diluted 1:5,000 in 100 μl of PBS / 0.1% Tween-20 for 1 h at room temperature on an Ang-2-coated microplate. In addition, to confirm the specificity for Ang-2, the reaction was performed on a microplate coated with Ang-1 protein, a homologous protein with high sequence homology. After washing five times with PBST (PBS / 0.1% Tween-20), the cells were reacted at room temperature for 10 minutes using the chromogenic substrate 3,3',5,5' tetramethylbenzidine (TMB) and the color development was confirmed. To select specific individual clones, cells from the final panning round were diluted and plated on LB agar medium containing ampicillin. Single colonies of the final amplification population (192 colonies each from two phage libraries A and B), a total of 384 colonies, were collected and inoculated into 1 ml of LB medium, respectively, and cultured at 37 °C for 18 hours with shaking at 200 rpm. The pellet obtained by centrifuging each 1 ml of culture was suspended in 100 μl of PBS and analyzed by ELISA as above. After color development, TMB reaction stop solution was added, and absorbance was measured at a wavelength of 450 nm using a TECAN spectrometer to obtain individual clones that were highly reactive with Ang-2 and lowly reactive with Ang-1. The number of clones obtained through the above process is shown in Table 1.

[0241] [Table 1]

[0242]

[0243] The selected clones were individually sequenced by PCR, and the amino acid sequences of Ang-2-specific single-domain antibodies (sdAbs) were obtained as shown in Table 2 below. In addition, the complementarity determining region (CDR) sequences of the obtained single-domain individual antibodies are shown in Table 3 based on Kabat numbering. In addition, the nucleic acid sequences of the single-domain antibodies are shown in Table 4.

[0244] [Table 2]

[0245]

[0246] Clone type CDR1 CDR2 CDR3 ALTS2 5 A*sdAbFSFADYNMA (SEQ ID NO: 11) STITSSGSGTS (SEQ ID NO: 12) ATGGSIDN (SEQ ID NO: 13) ALTS3 5 AsdAbFSFDEYSMS (SEQ ID NO: 14) STIASNGADKT (SEQ ID NO: 15) ATLGQIDN (SEQ ID NO: 16) ALTS4 9 AsdAbFSFGTSDMA (SEQ ID NO: 17) SSITYDGSDKT (SEQ ID NO: 18) ATTNSPMDN (SEQ ID NO: 19) ALTS5 3 AsdAbFSFSTTDMS (SEQ ID NO: 20) STIGSRGGTIY (SEQ ID NO: 21) ATGGELDN (SEQ ID NO: 22) ALTS8 3 AsdAbFSFADYVMT (SEQ ID NO: 23) SSIASDGGNKA (SEQ ID NO: 24)ATDGPLDN(SEQ ID NO: 25)ALTS3BsdAbFILSDPYIA(SEQ ID NO: 26)AGIYSKRNWAN(SEQ ID NO: 27)AAQSLQHIEHGPSMIY(SEQ ID NO: 28)ALTS54BsdAbFIFSDVQIS(SEQ ID NO: 29)AEIHSDRRTY(SEQ ID NO: 30)DTPDMPLPEHQKMEY(SEQ ID NO: 31)ALTS59BsdAbIISSWSNIA(SEQ ID NO: 32)AEINSASQTN(SEQ ID NO: 33)DTWNPALSAMRVDY(SEQ ID NO: 34)ALTS67BsdAbITFSTIPIA(SEQ ID NO: 35)SEINSKGQTY(SEQ ID NO: 36)DTTAATADPDY(SEQ ID NO: 37)ALTS95BsdAbSISSDHTMS(SEQ ID NO: 38)ATIYSGGSTY(SEQ ID NO: 39)NAVDPSPLWFRTY(SEQ ID NO: 40)

[0247]

[0248]

[0249] Example 1-3. Gene cloning of Ang-2-specific single-domain antibody

[0250] The Ang-2-specific antibody gene was isolated from individual clone cultures by polymerase chain reaction (PCR) with forward and reverse primers using HF PCR premix. The amplified gene was confirmed as a DNA band of the expected size on a 1% agarose gel and isolated using a gel extraction kit. The isolated gene was inserted into a bacterial expression vector expressing the Streptococcal Protein G B1 domain (GB1) of SEQ ID NO: 46, which has 18 bp homology at both ends, or the rabbit IgG fragment crystallizable region of SEQ ID NO: 47, using an overlapping PCR method. The ligation reaction product was transformed into XL1-Blue bacteria, which were then plated on LB agar medium containing ampicillin and cultured at 37°C for more than 12 hours. A single colony was cultured to isolate the plasmid, which was then transformed into the antibody-expressing strain.

[0251]

[0252] Example 1-4. Production of Ang-2-specific single-domain antibodies

[0253] Each antibody clone obtained from the phage library was purified using the immobilized metal affinity chromatography (IMAC) method to produce and purify it. After transforming the above expression vector into the BL21 strain (LysS), each colony was inoculated into a 1 L LB culture medium containing ampicillin and cultured. 1 ml of the culture medium was obtained, centrifuged, suspended using 2X sample buffer, and then subjected to electrophoresis (SDS-PAGE) to confirm the expression of the target protein. The entire culture medium containing the confirmed target protein was centrifuged to obtain a cell pellet, which was then suspended in binding buffer (20 mM Tris-HCl pH 7.9, 500 mM NaCl, 40 mM imidazole). The cells were disrupted using an ultrasonicator, centrifuged at 12,000 rpm, and the supernatant was collected to obtain a whole cell extract. The obtained whole cell extract was bound to a resin linked with Ni-iminodiacetic acid. After this, the resin was washed three or more times with washing buffer (20 mM Tris-HCl pH 7.9, 500 mM NaCl, 40 mM imidazole). After washing was completed, the target protein was eluted with elution buffer. The composition of the elution buffer used for elution was 20 mM Tris-HCl pH 7.5, 100 mM NaCl, 60 mM Imidazole, and 60, 100, 250, and 500 mM imidazole were used according to the imidazole concentration to obtain the eluted fractions, respectively. Each fraction was subjected to protein quantification using the Bradford method. Fractions in which protein was detected were subjected to protein electrophoresis (SDS-PAGE), and the eluate containing only the target protein was collected. The buffer was exchanged with PBS buffer using an Amicon stirred cell system, 10K MWCO, (Cat. No. UFSC05001, Merk, USA), and then stored in 50% glycerol PBS to a concentration of 1 mg / ml.Protein electrophoresis was performed under reducing and non-reducing conditions to verify the final concentration and condition, and stored at -20°C. The results are shown in Figure 1.

[0254] Through the above process, 10 types of anti-Ang-2 single domain antibodies were developed, and finally, anti-Ang-2 single domain antibodies in the form of fusion with GB1 and Fc for stability of expression and production were obtained.

[0255]

[0256] Example 2: Direct binding analysis of anti-Ang-2 single-domain antibodies to Ang1 and Ang-2

[0257] To analyze the direct binding of the single domain antibody obtained in Example 1 to Ang1 and Ang-2, a direct binding ELISA was performed.

[0258] A 96-well Maxibinding plate (SPL) was coated with 2 μg (microgram) / ml of human Ang-2 antigen and human Ang-1 antigen (R&D Systems). The plate was then washed five times with PBST (PBS containing 0.1% Tween-20) and blocked with PBS containing 2% skim milk at room temperature. The prepared sdAb-GB1 and sdAb-Fc were added to each well of the antigen-coated plate at various concentrations ranging from 1 μg / mL to 0.01 μg / mL, and the plate was incubated at room temperature for 1 hour. The plate was then washed five times with 0.1% Tween-20 PBST, and HRP-conjugated anti-mouse IgG antibody (Sigma) was added to each well and incubated at room temperature. Finally, 100 μL of TMB substrate (SeraCare) was added to each well of the plate, and the color reaction was induced for 30 minutes, after which the reaction was stopped using 1 M H2SO4 solution. The OD (450 nm) value was measured using a microplate absorbance meter (Molecular Devices).

[0259] As a result, the specific binding (EC50) of the GB1 fusion anti-Ang-2 single domain antibody (sdAb-GB1) to Ang-1 and Ang-2 antigens by ELISA titration is shown in Fig. 2, and the specific binding (EC50) of the Fc fusion anti-Ang-2 single domain antibody (Fc-GB1) to Ang-1 and Ang-2 antigens by ELISA titration is shown in Fig. 3.

[0260]

[0261] Example 3: TEK / Tie2 competitive binding assay of anti-Ang-2 single-domain antibodies to Ang1 and Ang-2

[0262] For the single domain antibodies obtained in Example 1, in order to analyze the inhibition of TEK / Tie2 receptor binding by Ang-2 binding of the anti-Ang-2 domain and the selectivity for Ang-1, a competition ELISA was performed to determine the competitive binding (IC50) of the antibody of the present invention to the TEK / Tie2 receptor with biotinylated antigen proteins (biotinylated Ang-1, biotinylated Ang-2).

[0263] Specifically, 96-well MaxiSorp TMhTie2-Fc (R&D Systems) was coated on a flat-bottom plate (Nunc) at a concentration of 100 ng / well. Next, the plate was washed three times with PBST (Phosphate Buffer Saline, 0.05% Tween-20) containing 0.05% (v / v) Tween-20, and blocked with TBS (Tris-Buffered Saline) containing 1% (v / v) BSA (Bovine serum albumin; Sigma) for 2 h at room temperature. The prepared anti-Ang-2 sdAb-GB1 antibody and anti-Ang-2 sdAb-Fc antibody were reacted with biotinylated Ang-1 or biotinylated Ang-2 at various concentrations ranging from 1 μM to 0.01 pM for 2 h at room temperature to induce binding. After adding the sdAb and biotinylated antigen protein (Ang-1 or Ang-2) reaction solution to each well coated with hTie2-Fc fusion protein, the plate was incubated at room temperature for 2 hours, and then washed three times with PBST. Streptavidin-conjugated HRP (Horseradish Peroxidase) was added to the well, incubated at room temperature for 1 hour, and then washed three times with PBST. Finally, 100 μL of TMB (3,3',5,5'-Tetramethylbenzidine) substrate (SeraCare) was added to each well of the plate, and the color reaction was induced for 30 minutes, and the reaction was stopped using 1 M H2SO4 solution. The OD (450 nm) value was measured using a microplate absorbance meter (Molecular devices).

[0264] As a result, the competitive binding potency (IC50) of anti-Ang-2 sdAb-GB1 antibody to Ang-1 and Ang-2 is shown in Table 5, and the competitive binding potency (IC50) of anti-Ang-2 sdAb-Fc antibody to Ang-1 and Ang-2 is shown in Table 6. It was confirmed that all anti-Ang-2 sdAb-GB1 antibodies did not inhibit the binding of Tie2 to Ang-1, and the binding inhibition ability of Tie2 to Ang-2 showed different binding inhibition abilities (IC50) depending on the anti-Ang2 single-domain sequence, confirming that the discovered single-domain antibody sequence had excellent Ang-2-specific binding despite the high sequence homology between Ang-1 and Ang-2 antigens. In particular, sdAb-GB1 fused with the ALTS95B domain showed the highest inhibitory potency with an IC50 of 0.424 nM (Table 5). Anti-Ang-2 sdAb-Fc antibody also did not inhibit the binding of Tie2 to Ang-1, similar to sdAb-GB1. In particular, sdAb-Fc fused to the ALTS59B domain showed the highest inhibitory effect at 0.268 nM (Table 6).

[0265] [Table 5]

[0266]

[0267] [Table 6]

[0268]

[0269] Example 4: Analysis of TEK / Tie2 receptor signaling inhibitory activity of anti-Ang-2 single-domain antibodies.

[0270] Next, we aimed to analyze the TEK / Tie2 receptor signaling inhibitory activity of the anti-Ang-2 single-domain antibody (sdAb-Fc). The anti-Ang-2 sdAb-Fc antibody obtained in Example 1 was used.

[0271] Human umbilical vein endothelial cells (HUVEC, Cell Systems) were suspended in endothelial cell medium (ECM) containing growth factors and seeded at 2 x 10 per well in a 6-well culture plate. 5Cells were seeded so that they would become . After culturing for 1 day, the existing medium was replaced with ECM containing 0.5% FBS (fetal bovine serum) and cultured for 6 hours. 500 ng / ml of angiopoietin-2 (angiopoietin2, Ang-2) and each anti-Ang-2 Fc fusion single-domain antibody (hereinafter, “antibody”) at various concentrations were pre-mixed with the ECM to induce the formation of Ang-2-antibody complexes for 30 minutes. The cells cultured in 6 wells were washed once with HBSS (Hanks balanced salt solution), and 1 ml of the Ang-2-antibody complexes that had completed the reaction were treated to each well and cultured in a CO2 incubator for 30 minutes. Afterwards, each well was washed once with HBSS and lysed with a cell lysis buffer containing protease inhibitors and phosphatase inhibitors. Protein concentration was quantified using the BCA quantitation method (Pierce), and all samples were immunoprecipitated overnight at refrigerated temperature using anti-Tie2 antibody (R&D Systems) to ensure that the total protein content was the same. Anti-TEK / Tie2 antibody was recovered from the lysate using Dynabeads (Invitrogen), washed three times with PBS, and subjected to 6% SDS-PAGE. The lysate from which anti-TEK / Tie2 had been removed was subjected to 8% SDS-PAGE to separate proteins by size. Proteins were transferred from the gel to the membrane using a nitrocellulose membrane (Millipore), blocked with blocking buffer (5% nonfat milk or 4% BSA in TBST) at room temperature, and stored overnight in the refrigerator with membrane-specific anti-phospho-tyrosine antibody (Millipore) or anti-phospho-Akt (Ptotein Kinase B and rac) antibody (Cell Signal Technology). The secondary antibody used for each membrane was anti-IgG-HRP, which was treated for 1 hour at room temperature according to the species of the primary antibody produced.The membrane was washed three times with TBST at each step. The membrane was visualized with a Chemidoc instrument (ATTO) using an ECL kit (BioRad) and quantified using the CS4 analyzer program. After color development, the membrane was treated with stripping buffer at 60°C for 60 minutes to remove the antibody, and treated with blocking buffer at room temperature for 1 hour. After treatment with anti-TEK / Tie2 antibody (R&D systems), anti-Akt antibody (Cell Signal Technology) for 1 hour at room temperature, and secondary antibody for 1 hour at room temperature, the membrane was visualized using the same method. Using the signal values ​​calculated by the CS4 analyzer, the ratio of phosphorylated Tie2 to total Tie2 (pTie2 / Tie2) and the ratio of phosphorylated Akt to total Akt (pAkt / Akt) were calculated to indicate the degree of phosphorylation.

[0272] As a result, the analysis of the TEK / Tie2 receptor signaling inhibition activity of the anti-Ang-2 single-domain antibody is shown in Fig. 4. To evaluate Tie2 and AKT phosphorylation induced by Ang-2, the solvent-treated group was set as the negative control (NC) and the Ang-2 alone-treated group was set as the positive control (PC) in each phosphorylation assay. When sdAb-Fc was simultaneously treated with the target protein Ang-2 at a 2-fold molar concentration, pTie2 / Tie2 was suppressed to below the NC level in all protein-treated groups except for the ALTS3B sdAb-Fc protein. In addition, pAkt / Akt was suppressed to a level close to the NC in all protein-treated groups except for the ALTS54B-sdAb-Fc.

[0273]

[0274] Example 5: Validation of activity in an animal model of laser-induced choroidal neovascularization

[0275] The angiogenesis inhibitory ability of the anti-Ang-2 single-domain antibody prepared above was evaluated in a mouse model of laser-induced choroidal neovascularization (CNV). Using the sdAb of sequence number 3, an anti-Ang-2 antibody was expressed in a structure in which one corresponding domain antibody was fused to the Fc C-terminus and assembled into a homodimer to form two antigen-binding sites.

[0276] To this end, four laser spots were irradiated on each eye of 8-week-old C57BL / 6J male mice to induce damage to the Bruch's membrane. Immediately after laser irradiation, the test substance was administered intravitreously to both eyes at a single dose of 1 μL / eye.

[0277] On the 13th day after CNV induction, both eyes were enucleated and fixed in a 4% paraformaldehyde solution for 30 minutes. The choroid was then isolated and reacted with a blocking solution containing 5% BSA, 5% Normal goat serum, and 0.5% Tx-100 for 1 hour, and then stained overnight with fluorescently labeled Isolectin GS-IB4 (Invitrogen). After washing with PBS, the choroid was flattened on a slide and images were acquired using a fluorescence microscope (Olympus). The neovascular area of ​​each spot in the acquired images was quantitatively analyzed using ImageJ software (NIH). The graph sets the vehicle group to 100%, and the experimental groups are expressed as relative percentage values ​​based on this value. As a representative example, the results comparing the vehicle group with the anti-Ang-2 antibody (SEQ ID NO: 3) are shown in Figure 5.

[0278] As can be seen in Fig. 5, the anti-Ang-2 antibody of the present invention was confirmed to exhibit a remarkable angiogenesis inhibitory effect of close to 50% compared to the vehicle group.

[0279]

[0280] Example 6: Production of anti-Ang-2 antibody fusion multispecific proteins

[0281] Example 6-1. Sequence synthesis

[0282] DNA fragments of the desired sequence according to the present invention were produced according to the specifications of GeneScript (Seoul, Republic of Korea).

[0283] Basic methods for DNA manipulation were performed according to the instructions in the material below.

[0284] Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). The use of the following molecular biological reagents followed the manufacturer's instructions.

[0285]

[0286] Example 6-2. Expression vector production and culture

[0287] The DNA sequence encoding the multispecific fusion protein that simultaneously binds to VEGF and Ang-2 was synthesized in a cassette format by adding restriction enzymes (XhoI and NotI) and a leader peptide sequence to the base sequence encoding amino acids 48 to 65 of the sequence listing, which includes the anti-Ang-2 single-domain antibody sequence according to the present invention and the sequence of aflibercept. The synthesized DNA was introduced into pcDNA3.4 (Invitrogen) using XhoI and NotI restriction enzymes to produce a fusion protein expression vector. The pcDNA3.4 vector containing the DNA of the multispecific fusion protein that simultaneously binds VEGF and Ang-2 was introduced into a CHO-S cell line and transiently expressed. CHO-S host cell gene ExpiCHO TM Expression System Kit (Gibco) and OptiPRO TM Genes were introduced using SFM (Gibco), CHO-S cell line and ExpiFectamine TM The expression system guide of the CHO Transfection Kit was followed. Specifically, ExpiCHO TM Using Expression Medium (Gibco), survival rate was over 90%, and viable cell concentration was 6x10 6 CHO-S cells (cells / mL) were cultured in a CO2 incubator at 37°C, humidity ≥80%, and CO2 8%. 0.8 μg of fusion protein DNA expression vector was introduced per 1 ml of culture volume into the prepared cells using Expifectamine CHO reagent and OptiPRO TM Cells were transiently transfected using SFM. After transfection, ExpiCHO Feed was added within 18–22 hours according to the guide, and expression was maintained for up to 8 days in a CO2 incubator at 32°C, humidity >80%, and CO25%. After complete expression, the culture medium was centrifuged to separate the culture medium and cells.

[0288] Example 6-3. Purification of fusion protein

[0289] The expression solution separated by centrifugation was purified by affinity chromatography using a Protein A column (HiScreen Fibro™ PrismA, Cytiva). To purify the fusion protein, the column was equilibrated in 20 mM sodium phosphate, 150 mM NaCl (pH 7.0) buffer before purification. After binding the fusion protein to the column, impurities were removed by flowing more than five times the column volume (5 CV) of 20 mM Tris, 500 mM NaCl (pH 7.0) buffer and 20 mM sodium citrate (pH 6.0) buffer. After completing this step, the affinity-bound protein was eluted using 20 mM sodium citrate (pH 3.5) buffer, and the eluted fusion protein was immediately neutralized to pH 7.0 to pH 7.4 using 1 M Tris-HCl solution.

[0290] As a result, the expression and purification results of the anti-Ang-2 antibody fusion multispecific protein are disclosed in FIGS. 6 and 7.

[0291] As shown in Fig. 6, the anti-Ang-2 antibody according to the present invention exhibited very advantageous properties in terms of commercialization and mass production of the material in terms of expression, purification, and stability of the fusion protein in all cases when introduced in the form of a module at the N-terminus, C-terminus, or in the form of a monovalent or multivalent fusion protein. Fig. 7 shows the results of expression, purification, and purity (SE-HPLC analysis) of three different forms of multivalent and multivalent fusion proteins of the ALTS54B clone (SEQ ID NO: 7) among the anti-Ang-2 single domain antibody sequences. It was confirmed that the anti-Ang-2 fusion protein according to the present invention has excellent thermostability and expression level regardless of the fusion position or number of the anti-Ang-2 single domain antibody, and has the characteristics of maintaining stable properties that enable production with high purity and high concentration.

[0292]

[0293] Example 6-4. Concentration and formulation

[0294] Among the anti-Ang-2 fusion proteins manufactured above, the fusion protein purified and neutralized by Protein A affinity chromatography for ALTS49AafC4 (SEQ ID NO: 50) was concentrated using a membrane filter (Centricon-100, Amicon) with a molecular weight cut-off of 100 KDa. Simultaneously, the PBS buffer, pH 7.4 (Phosphate Buffered Saline), or PBS buffer, pH 7.0 (Phosphate Buffered Saline), or histidine buffer, pH 5.0 (10 mM Histidine-HCl, 7% sucrose, 0.03% Tween20), or histidine buffer, pH 5.5 (10 mM Histidine-HCl, 7% sucrose, 0.03% Tween20), or histidine buffer, pH 6.0 (10 mM Histidine-HCl, 7% sucrose, 0.03% Tween20), or histidine buffer, pH 6.5 (10 mM Histidine-HCl, 7% sucrose, 0.03% Tween20) was replaced. Specifically, the protein was concentrated by centrifugation at 4000 rpm and 4°C using a centrifugal filter unit (Amicon® Millipore), and the concentrated protein was divided into PBS and histidine buffer, and the buffer was replaced using a centrifugal filter unit. The results of replacing the buffer after concentrating the purified product are shown in Table 7 and Figure 8.

[0295] As a result, the ALTS49AafC4 fusion protein was characterized as being stably maintained under minimal excipient conditions at concentrations of about 20 mg / ml to 30 mg / ml at pH 5.0 to pH 5.5 and pH 7.0 to pH 7.4. After concentration and buffer exchange of ALTS49AafC4, protein electrophoresis (SDS-PGAE) and SE-HPLC were performed on each fusion protein under reduced or non-reduced conditions, and the results showed that the concentration and formulation were stable with a purity of over 99% under pH 5.0, pH 5.5, and pH 7.4 conditions.

[0296]

[0297] Buffer compositionFusion protein concentration (mg / ml)Recovery rate after buffer replacement (%)Visually observed particlesSEC-HPLCHistidine buffer, pH 5.0 (10 mM Histidine-HCl, 7% Sucrose, 0.03% Tween20)22.879None99.7Histidine buffer, pH 5.5 (10 mM Histidine-HCl, 7% Sucrose, 0.03% Tween20)22.875None99.6Histidine buffer, pH 6.0 (10 mM Histidine-HCl, 7% Sucrose, 0.03% Tween20)2.8647Aggregate-histidine buffer, pH 6.5 (10 mM Histidine-HCl, 7% Sucrose, 0.03% Tween20)0.5345Aggregate-histidine buffer, pH 7.0 (10 mM Histidine-HCl, 7% Sucrose, 0.03% Tween20) 18.850 None - PBS, pH 7.4 (10 mM Na2HPO4, 1.8 mM KH2PO4, NaCl 137 mM, KCL 2.7 mM) 31.676 None 99.9

[0298]

[0299] Example 7: Direct Binding Characterization of Anti-Ang-2 Antibody Fusion Multispecific Proteins to VEGF-A165

[0300] The binding properties of the anti-Ang-2 antibody fusion multispecific protein prepared in Example 6 to recombinant human VEGF-A165 were analyzed using a direct binding ELISA. Specifically, a 96-well MaxiSorp TM Recombinant human VEGF-A165 (R&D System) was coated on a flat-bottom plate (Nunc). The coated plate was washed three times with phosphate buffer saline (PBS) containing 0.05% (v / v) Tween-20, and then blocked with PBS containing 1% (v / v) bovine serum albumin (BSA) and sucrose for 2 hours at room temperature. Anti-Ang-2 antibody fusion multispecific protein was reacted at various concentrations ranging from 33 nM to 1 pM for 2 hours at room temperature to induce binding. After binding was completed, the plate was washed three times with PBST, and HRP-conjugated anti-human Fc secondary antibody (Invitrogen) was added to each well and reacted for 1 hour at room temperature. After the reaction was completed, the plate was washed three times with PBST, and 100 μL of TMB substrate (SeraCare) was added to each well of the plate to induce a color reaction for 30 minutes. Finally, the reaction was stopped using a 1 M H2SO4 solution, and the absorbance (OD450 nm) value was measured using a plate reader (Molecular devices). The absorbance EC50 (half-maximal reaction concentration) value according to the concentration of the fusion protein was analyzed using SoftMax (Molecular devices).

[0301] As a result, the direct binding affinity (EC50) values ​​of the anti-Ang-2 fusion multispecific proteins according to SEQ ID NOs: 48 to 65 of the present invention to recombinant human VEGF-A165 are shown in FIG. 9, and the change in VEGF-A165 binding absorbance according to the change in the concentration of the anti-Ang-2 antibody fusion multispecific protein is shown in FIG. 10. As a result of the analysis, it was confirmed that ALTS53AafC4 (SEQ ID NO: 51) and ALTS53AafC4 (SEQ ID NO: 52) had EC50s of 109 pM and 112 pM for VEGF-A165, and all of the remaining fusion proteins had EC50s of several tens of pM. Through this, it was confirmed that all proteins fused with the anti-Ang-2 domain had high binding affinity for VEGF-A165.

[0302]

[0303] Example 8: Analysis of the competitive binding properties of anti-Ang-2 antibody fusion multispecific proteins between VEGF and VEGF receptor 1 (VEGFR1)

[0304] Next, the binding inhibition properties of the anti-Ang-2 antibody fusion multispecific protein prepared in Example 6 above between VEGF-A165 and the VEGF receptor were analyzed using a competitive binding method ELISA.

[0305] Specifically, 96-well MaxiSorp TMA flat-bottom plate (Nunc) was coated with hVEGFR1-Fc fusion protein (R&D Systems). The plate was then washed three times with phosphate buffer saline (PBS) containing 0.05% (v / v) Tween-20 and blocked with blocking buffer containing 1% casein for 2 hours at room temperature. Anti-Ang-2 antibody fusion multispecific protein was reacted with biotinylated recombinant human VEGF-A165 (R&D System) at various concentrations for 2 hours at room temperature to induce binding. After adding the anti-Ang-2 antibody fusion multispecific protein and biotinylated antigen protein reaction solution to each well coated with recombinant human VEGFR1-Fc fusion protein, the plate was reacted for 2 hours at room temperature and then washed three times with PBST. Streptavidin-conjugated HRP was added to the wells and reacted at room temperature for 1 hour, then washed three times with PBST. Finally, 100 μL of TMB substrate (SeraCare) was added to each well of the plate to induce color development for 30 minutes, and the reaction was stopped using 1 M H2SO4 solution. The OD450 nm value was measured using a plate reader (Molecular Devices). The absorbance IC50 (half-maximal inhibitory concentration) value according to the concentration of the fusion protein was analyzed using SoftMax (Molecular device).

[0306] As a result, the IC50 values ​​for competitively inhibiting the binding between recombinant human VEGF receptor 1 (VEGFR1) and biotinylated VEGF-A165 of the anti-Ang-2 fusion multispecific protein according to SEQ ID NOs: 48 to 65 of the present invention are shown in FIG. 11, and the absorbance changes for the competitive binding of biotinylated VEGF-A165 of the anti-Ang-2 antibody fusion multispecific protein and VEGF receptor 1 (VEGFR1) are shown in FIG. The binding inhibition ability (IC50) of VEGF-A165 to VEGF receptor 1 (VEGFR1) was shown to be strong competitive binding inhibition (IC50) at the nM level for all anti-Ang-2 antibody fusion multispecific proteins.

[0307]

[0308] Example 9: Direct Binding Characterization of Anti-Ang-2 Antibody Fusion Multispecific Proteins to Ang-1 / Ang-2

[0309] Next, the binding properties of the anti-Ang2 antibody fusion multispecific protein to Ang-1 and Ang-2 were analyzed using a direct binding (direct) ELISA.

[0310] Specifically, recombinant human Ang-1 (R&D System) and recombinant human Ang-2 (R&D System) were coated on 96-well MaxiSorpTM flat-bottom plates (Nunc). The coated plates were washed three times with phosphate buffer saline (PBS) containing 0.05% (v / v) Tween-20, and then blocked with PBS containing 1% (v / v) BSA (Bovine serum albumin) and sucrose for 2 hours at room temperature. Anti-Ang-2 antibody fusion multispecific proteins were diluted to various concentrations starting from 42 nM and reacted for 2 hours at room temperature to induce binding. After binding was completed, the plates were washed three times with PBST, and HRP-conjugated anti-human Fc secondary antibody (Invitrogen) was added to each well and reacted for 1 hour at room temperature. After the reaction was completed, the plate was washed three times with PBST, and 100 μL of TMB substrate (SeraCare) was added to each well of the plate to induce color development for 30 minutes. Finally, the reaction was stopped using 1 M H2SO4 solution, and the OD450 nm value was measured using a plate reader (Molecular Devices). The absorbance EC50 (half-maximal reaction concentration) value according to the concentration of the fusion protein was analyzed using SoftMax (Molecular device).

[0311] As a result, the binding of the anti-Ang-2 fusion multi-specific protein according to SEQ ID NO: 48 to 65 of the present invention to recombinant human Ang-2 or Ang-1 was expressed as an EC50 value (Fig. 13), and the binding absorbance to Ang-2 or Ang-1 according to the concentration change of the anti-Ang-2 antibody fusion multi-specific protein is shown in Fig. 14. The binding (EC50) value to Ang-2 showed strong binding at the level of several hundred pM at most. Nonspecific binding to Ang-1 was very minimal for all fusion proteins, confirming that the selective binding to the Ang-2 antigen was very high.

[0312]

[0313] Example 10: Analysis of the competitive binding properties of anti-Ang-2 antibody fusion multispecific proteins between Ang-2 and TEK / Tie2 receptors

[0314] The inhibitory properties of anti-Ang-2 antibody fusion multispecific proteins competing with TEK / Tie2 proteins for binding to Ang-2 or Ang-1 were analyzed using a competition-type ELISA.

[0315] Specifically, 96-well MaxiSorpTM flat-bottom plates (Nunc) were coated with recombinant human Tie2-Fc (R&D Systems). The plates were then washed three times with phosphate buffer saline (PBS) containing 0.05% (v / v) Tween-20 and blocked with blocking buffer containing 1% casein for 2 hours at room temperature. Anti-Ang-2 antibody fusion multispecific proteins were reacted with biotinylated recombinant human Ang-1 or biotinylated recombinant human Ang-2 (R&D System) at various concentrations for 2 hours at room temperature to induce binding. After adding the anti-Ang-2 antibody fusion multispecific proteins and biotinylated antigen protein reaction solution to each well coated with TEK / Tie2-Fc fusion protein, the plates were reacted for 2 hours at room temperature, and then washed three times with PBST. Streptavidin-conjugated HRP was added to the wells and reacted at room temperature for 1 hour, then washed three times with PBST. Finally, 100 μL of TMB substrate (SeraCare) was added to each well of the plate to induce color development for 30 minutes, and the reaction was stopped using 1 M H2SO4 solution. The OD450 nm value was measured using a plate reader (Molecular devices). The absorbance IC50 (half-maximal inhibitory concentration) value according to the concentration of the fusion protein was analyzed using SoftMax (Molecular Device).

[0316] As a result, the IC50 values ​​for competitively inhibiting the binding between recombinant human TEK / Tie2-Fc protein and biotinylated Ang-2 of the anti-Ang-2 fusion multi-specific protein according to SEQ ID NOs: 48 to 65 of the present invention are shown in FIG. 15, and the change in absorbance for competitive binding inhibition between biotinylated Ang-2 and TEK / Tie2 receptor according to the change in the concentration of the anti-Ang-2 antibody fusion multi-specific protein is shown in FIG. SEQ ID NOs: 48, 49, 50, 52, 53, and 54 showed very strong competitive binding inhibition (IC50) between Tie2 / Ang-2 at the level of tens of pM, and the remaining fusion protein sequences showed IC50 values ​​at the level of nM. In general, it was confirmed that the stronger the competitive binding due to the avidity effect for the antigen, the more equivalent sdAb was fused, and it was confirmed that the competitive binding inhibition activity was maintained regardless of the fusion position at the N-terminus or C-terminus.

[0317]

[0318] Example 11: Analysis of the inhibitory activity of anti-Ang-2 antibody fusion multispecific protein on TEK / Tie2 receptor signaling in HUVEC cells.

[0319] We aimed to measure the inhibitory activity of anti-Ang-2 antibody fusion multispecific protein on TEK / Tie2 receptor signaling in HUVEC cells.

[0320] Human umbilical vein endothelial cells (HUVEC, Cell Systems) were suspended in endothelial cell medium (ECM) containing growth factors and seeded at 2 x 10 per well in a 6-well culture plate. 5Cells were seeded so that they would become . After culturing for 1 day, the existing medium was replaced with ECM containing 0.5% FBS (fetal bovine serum) and cultured for 6 hours. 500 ng / ml angiogenic factor 2 (angiopoietin 2, Ang-2) and the fusion proteins of SEQ ID NO: 54, SEQ ID NO: 60, SEQ ID NO: 63, and SEQ ID NO: 64 were pre-mixed with the ECM at each concentration to induce the formation of Ang-2-antibody complexes for 30 minutes. The cells cultured in 6 wells were washed once with HBSS (Hanks balanced salt solution), and 1 ml of the Ang-2-antibody complexes that had completed the reaction were treated to each well and cultured in a CO2 incubator for 30 minutes. Afterwards, each well was washed once with HBSS and lysed with a cell lysis buffer containing protease inhibitors and phosphatase inhibitors. Protein concentration was quantified using the BCA assay (Pierce), and all samples were adjusted to have the same total protein amount, and immunoprecipitated overnight at room temperature using anti-TEK / Tie2 antibody (R&D Systems). Anti-TEK / Tie2 antibody was recovered from the lysate using Dynabeads (Invitrogen), washed three times with PBS, and subjected to 6% SDS-PAGE to separate TEK / Tie2 protein. The cell lysate from which Dynabeads had been removed was subjected to 8% SDS-PAGE to separate Akt protein. Proteins were transferred from the gel to a nitrocellulose membrane (Millipore), blocked at room temperature with blocking buffer (5% nonfat milk or 4% BSA in TBST), and immunoblotted overnight at room temperature with anti-phospho-tyrosine antibody (Millipore) and anti-phospho-Akt antibody (Cell signal technology) for each membrane. The secondary antibody used on each membrane was anti-IgG-HRP treated at room temperature for 1 hour according to the production species of the primary antibody.The membrane was washed three times with TBST at each step. The membrane was visualized on a Chemidoc instrument (ATTO) using an ECL kit (BioRad) and quantified using the CS4 analyzer program. After color development, the membrane was treated with riblot buffer at 60°C for 20 minutes to remove the antibody, and then treated with blocking buffer at room temperature for 1 hour. After treatment with anti-TEK / Tie2 antibody (R&D systems), anti-Akt antibody (Cell Signal Technology) for 1 hour at room temperature, and secondary antibody for 1 hour at room temperature, the membrane was visualized using the same method. The signal values ​​calculated with the CS4 analyzer were quantified.

[0321] As a result, the inhibitory activity of the anti-Ang-2 antibody fusion multispecific protein on TEK / Tie2 receptor signaling in HUVEC cells is shown in Figures 17 and 18.

[0322] When target protein Ang-2 and the fusion proteins of SEQ ID NO: 54, SEQ ID NO: 60, and SEQ ID NO: 64 of the present invention were treated to HUVEC cells at a molar concentration of 1:1, TEK / Tie2 receptor phosphorylation (PC of the left graph) and its downstream signal, Akt phosphorylation (PC of the right graph), by Ang-2 were suppressed to the level of the solvent-treated group (NC), as shown in Figure 17.

[0323] In addition, the minimum effective concentrations of the fusion proteins of SEQ ID NO: 54, SEQ ID NO: 60, SEQ ID NO: 63, and SEQ ID NO: 64 that inhibit TEK / Tie2 and Akt phosphorylation by Ang-2 to the NC level were confirmed. Figure 18 shows that when the fusion proteins of SEQ ID NO: 54, SEQ ID NO: 60, SEQ ID NO: 63, and SEQ ID NO: 64 were treated at 0.28, 0.50, 0.28, and 0.25 times molar concentrations, respectively, compared to Ang-2, the phosphorylation of both TEK / Tie2 and Akt was inhibited to the NC level.

[0324] From the above results, the excellent TEK / Tie2 receptor phosphorylation inhibition and Akt phosphorylation inhibition effects of the fusion protein of the present invention were confirmed.

[0325]

[0326] Example 12: Analysis of Ang-1-mediated HUVEC Akt phosphorylation amplification activity according to Ang-2 and VEGF inhibition by anti-Ang-2 / VEGF antibody fusion multispecific protein

[0327] Human umbilical vein endothelial cells (HUVEC, Cell Systems) were suspended in endothelial cell medium (ECM) containing growth factors and seeded at 3 x 10 per well in a 6-well culture plate. 5Cells were seeded so that they would become spherical. After culturing for 1 day, the existing medium was replaced with ECM containing 0.5% FBS (fetal bovine serum) and cultured for 6 hours. 100 ng / ml Ang-1, 500 ng / ml Ang-2, 10 ng / ml VEGF, and each anti-Ang-2 and anti-VEGF antibody fusion multispecific protein at different concentrations were pre-mixed with the ECM and induced to form VEGF-antibody-Ang-2 complexes for 30 minutes. The cells cultured in 6-well plates were washed once with HBSS (Hanks balanced salt solution), and 1 ml of the Ang-2-antibody complexes that had completed the reaction were treated to each well and cultured in a CO2 incubator for 30 minutes. Afterwards, each well was washed once with HBSS and lysed with cell lysis buffer containing protease inhibitors and phosphatase inhibitors. Protein concentration was quantified using the BCA assay (Pierce), and the cell lysates, adjusted to the same total protein amount for all samples, were subjected to 8% SDS-PAGE to isolate Akt protein. Proteins were transferred from the gel to the membrane using a nitrocellulose membrane (Millipore), blocked at room temperature with blocking buffer (5% nonfat milk in TBST), and immunoblotted overnight at room temperature with membrane-specific anti-phospho-Akt antibodies (Cell signal technology). Secondary antibodies were treated with anti-rabbit IgG-HRP for 1 hour at room temperature according to the species of the primary antibody. The membrane was washed three times with TBST after each step. The ECL kit (BioRad) was used for visualization on a Chemidoc instrument (ATTO), and the data were quantified using the CS4 analyzer program. After the color development was completed, the membrane was treated with a riblot buffer at 60°C for 20 minutes to remove the antibody, and then treated with a blocking buffer at room temperature for 1 hour.After treatment with anti-Akt antibody (Cell Signal Technology) for 1 hour at room temperature and secondary antibody for 1 hour at room temperature, the cells were visualized using the same method. The signal values ​​calculated using the CS4 analyzer were quantified.

[0328] The activity of the anti-Ang-2 / VEGF antibody fusion multispecific protein on the amplification of Akt phosphorylation mediated by Ang-1 in HUVEC cells following Ang-2 and VEGF inhibition is shown in Figure 19. To confirm Ang-1-mediated Akt phosphorylation, the Ang-1 alone treatment group was set to 100% Akt phosphorylation. When Ang-2 was simultaneously treated with Ang-1 at 20-fold and VEGF at 0.3-fold the molar concentration of Ang-1, which is the condition for confirming the decrease in Akt phosphorylation activity by Ang-2 and VEGF on the phosphorylated Akt by Ang-1, Akt phosphorylation was reduced to 87%. In addition, when the fusion proteins of SEQ ID NO: 63 and SEQ ID NO: 64 were additionally treated at 2-fold molar concentration compared to Ang-2, the reduced Akt phosphorylation due to Ang-2 and VEGF was amplified to 180% and 156%, respectively. This is a result confirming that the fusion antibody of the present invention can exert a synergistic effect by smoothly restoring the suppressed Tie2 pathway by simultaneously suppressing VEGF and Ang-2.

[0329]

[0330] Example 13: Analysis of the growth inhibitory activity of anti-Ang-2 antibody fusion multispecific protein on human umbilical vein endothelial cells (HUVECs)

[0331] The growth inhibitory activity of anti-Ang-2 antibody fusion multispecific protein on HUVEC cells was evaluated.

[0332] Human umbilical vein endothelial cells (HUVEC, Cell systems) were suspended in assay medium (2% FBS, ECM) and seeded at 2 x 10 per well in a 96-well culture plate. 3Cells were seeded so that they became VEGF-A165 (R&D Systems). VEGF-A165 and anti-Ang-2 antibody fusion multispecific protein or aflibercept were mixed in the assay medium so that the concentration of recombinant human VEGF-A165 (R&D Systems) was 20 ng / ml, and complex formation was induced at 37°C for 3 hours. The complex medium was added to the 96-well plate to which cells were attached so that the volume of the assay medium and the volume of the assay medium containing the complex were 1:1, and the 96-well plate treated with the complex medium was cultured in a 37°C incubator for 66 hours. Afterwards, to measure the degree of cell growth, 96-well plates were treated with CellTiter-Blue (Promega) and cultured in a 37℃ incubator for 6 hours. Relative fluorescence units (RFU) were measured at 560 / 590 nm wavelength using a microplate reader (SpectraMax M5, Molecular device). The RFU IC50 (Half-maximal inhibitory concentration) values ​​according to the concentration of the fusion protein were analyzed using SoftMax (Molecular device).

[0333] As a result, the human umbilical vein endothelial cell (HUVEC) growth inhibitory activity of the anti-Ang-2 antibody fusion multi-specific proteins of the present invention, SEQ ID NO: 54, SEQ ID NO: 60, SEQ ID NO: 63, and SEQ ID NO: 64, is shown in FIG. 20. It was confirmed that the SEQ ID NO: 54, SEQ ID NO: 60, SEQ ID NO: 63, and SEQ ID NO: 64 fusion proteins of the present invention had IC50 values ​​of 56 pM, 52 pM, 87 pM, and 57 pM, respectively, for the target protein VEGF-A165. This confirmed that all anti-Ang-2 antibody fusion multi-specific proteins had similar VEGF-A165-induced HUVEC cell growth inhibitory abilities.

[0334]

[0335] Example 14: Inhibitory activity of anti-Ang-2 antibody fusion multispecific protein on human umbilical vein endothelial cell (HUVEC) monolayer permeability

[0336] The effect of anti-Ang-2 antibody fusion multispecific protein on vascular permeability was determined using an in vitro vascular permeability assay (Sigma).

[0337] Human umbilical vein endothelial cells were cultured on a permeable insert made of a permeable membrane to form a single endothelial layer, and after inducing changes in vascular permeability, vascular permeability was confirmed by measuring the degree of passage of substances through the HUVEC cell layer of the permeable insert. Human umbilical vein endothelial cells (HUVEC, Cell systems) were suspended in endothelial cell growth medium (EGM) containing growth factors and seeded on the permeable insert at a density of 1 x 10 per insert. 5 Cells were seeded. After culturing for 1 day, the existing medium was replaced with EGM (analytical medium) containing 0.5% FBS (fetal bovine serum) and cultured for 24 hours. Anti-Ang-2 antibody fusion multispecific protein was mixed with the analytical medium so that the concentration of recombinant human VEGF-A165 (R&D systems), a vascular permeability inducer, was 10 ng / ml, and the existing medium was replaced and cultured for 24 hours.

[0338] Human umbilical vein endothelial cells (HUVEC, Cell systems) were suspended in endothelial cell growth medium (EGM) containing growth factors, and 1 x 10 5Cells were seeded into each well of the permeable insert and cultured for 1 day. Afterwards, the existing medium was washed with HBSS (Welgene), and EGM (analytical medium) containing 0.5% FBS (fetal bovine serum) was added and cultured for 24 hours. The concentrations of recombinant human VEGF-A165 (R&D systems), which are vascular permeability inducers, and Ang-2 (R&D systems), and Ang-1 (R&D systems), which are vascular stabilizing factors, were adjusted to 10 ng / ml, 2000 ng / ml, and 100 ng / ml, respectively. After mixing anti-Ang-2 antibody fusion multispecific protein into the analytical medium, the existing medium was replaced and cultured for an additional 24 hours.

[0339] Afterwards, FITC-dextran (Sigma) was treated to a concentration of 1.5 mg / ml in the permeable insert, and the monolayer permeability was measured every 1 to 2 hours. The degree of permeability was determined by mixing a portion of the assay medium contained in the receiver well with DPBS, transferring it to a 96-well black plate, and confirming it as relative fluorescence units (RFU) at a wavelength of 485 / 535 nm using a microplate reader (SpectraMax M5, Molecular device).

[0340] The results of measuring the HUVEC monolayer permeability inhibition activity of the anti-Ang-2 antibody fusion multispecific protein are shown in Figures 21, 22, and 23.

[0341] First, to confirm the inhibitory activity of VEGF-A165 on HUVEC monolayer permeability, the VEGF-only treatment group was set as a positive control group (PC), and the anti-Ang-2 antibody fusion multi-specific protein was additionally treated. As a result, as can be seen in Fig. 21, when the fusion proteins of SEQ ID NO: 54 and SEQ ID NO: 64 of the present invention were treated to HUVEC at a molar concentration of 1:1 with respect to the target protein VEGF-A165, the monolayer permeability increased by VEGF-A165 was inhibited by 84% and 92%, respectively, compared to the positive control group (PC). Also, as can be seen in Fig. 22, when the fusion proteins of SEQ ID NO: 63 and SEQ ID NO: 64 of the present invention were treated at a molar concentration of 1:1 with respect to the target protein VEGF-A165, the HUVEC monolayer permeability induced by VEGF-A165 was recovered by 105% and 81%, respectively, compared to the positive control group (PC).

[0342] In order to confirm the change in HUVEC monolayer permeability when both VEGF-A165 and Ang-2 were inhibited, the experimental group treated with both VEGF, Ang-2, and Ang-1 was set as a positive control group (PC), and treated with an anti-Ang-2 antibody fusion multi-specific protein. As a result, as can be seen in Fig. 23, when the fusion proteins of SEQ ID NO: 54 and SEQ ID NO: 64 of the present invention were treated with VEGF-A165 at a molar concentration of 1:1 for the target proteins VEGF-A165 and Ang-2, the monolayer permeability increased by VEGF-A165 and Ang-2 was inhibited by 95% and 98%, respectively, compared to the positive control group (PC).

[0343] From these results, the excellent HUVEC monolayer permeability inhibition activity of the fusion protein of the present invention was confirmed.

[0344]

[0345] Example 15: Activity of an Anti-Ang-2 Antibody Fusion Multispecific Protein in an Animal Model of Laser-Induced Choroidal Neovascularization

[0346] The inhibitory effects of anti-Ang-2 antibody-fused multispecific proteins on vascular leakage and angiogenesis were evaluated in a mouse model of laser-induced choroidal neovascularization (CNV). To this end, four laser spots were irradiated to each eye of 8-week-old C57BL / 6J male mice to induce damage to the Bruch's membrane. Immediately after laser irradiation, the test substance was administered intravitreally into the vitreous of both eyes at a single dose of 1 μL / eye.

[0347] On day 11 after CNV induction, general anesthesia and mydriasis were induced in mice using ketamine injection and eye drops, followed by intraperitoneal injection of fluorescent contrast agent. After general anesthesia was induced, local anesthesia was induced using eye drops, and the mice were placed on a stage, and FFA images were captured using an imaging camera (Phoenix). Retinal images were acquired within 10 minutes of fluorescent contrast agent administration. The leakage area of ​​each spot in the acquired images was quantitatively analyzed using ImageJ software (NIH). The graphs set the vehicle group as 100%, and the remaining experimental groups are expressed as relative percentage values ​​based on this value.

[0348] On the 13th day after CNV induction, both eyes were enucleated and fixed in a 4% paraformaldehyde solution for 30 minutes. The choroid was isolated and reacted for 1 hour in a blocking solution containing 5% BSA, 5% Normal goat serum, and 0.5% Tx-100, and then stained overnight with fluorescently labeled Isolectin GS-IB4 (Invitrogen). After washing with PBS, the choroid was flattened on a slide and images were acquired using a fluorescence microscope (Olympus). The neovascular area of ​​each spot in the acquired images was quantitatively analyzed using ImageJ software (NIH). The graphs set the vehicle group as 100%, and the remaining experimental groups are expressed as relative percentage values ​​based on this value.

[0349] As a result, the vascular leakage inhibition and angiogenesis inhibition activities of the anti-Ang-2 antibody fusion multi-specific protein were confirmed in an animal model, and are shown in Fig. 24. Anti-Ang-2 antibody and anti-VEGF antibody were set as controls, and a protein fused to the Fc C-terminus of sequence number 3 according to the present invention and aflibercept were used, respectively. The sequence number 63 fusion protein according to the present invention was administered at low doses (19.2 μg / eye, molar equivalent to 15 μg of aflibercept) and high doses (25.6 μg / eye, molar equivalent to 20 μg of aflibercept), and showed dose-dependent results. In addition, the high-dose group administered at the same molar equivalent to the control group showed a superior effect compared to the control group. These results demonstrated that the anti-Ang-2 antibody fusion multispecific protein had a synergistic effect on angiogenesis inhibition and vascular stabilization by simultaneously inhibiting VEGF and Ang-2 (Fig. 24).

[0350]

[0351] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A single domain antibody (sdAb) or an antigen-binding fragment thereof that specifically binds to angiopoietin-2 (Ang-2), The above single domain antibody (i) a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 35, or SEQ ID NO: 38; (ii) a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, or SEQ ID NO: 39; and (iii) An antibody or antigen-binding fragment thereof comprising a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, or SEQ ID NO:

40.

2. In the first paragraph, the single domain antibody (a) CDR 1 of SEQ ID NO: 11, CDR2 of SEQ ID NO: 12, and CDR3 of SEQ ID NO: 13; (b) CDR 1 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16; (c) CDR 1 of SEQ ID NO: 17, CDR2 of SEQ ID NO: 18, and CDR3 of SEQ ID NO: 19; (d) CDR 1 of SEQ ID NO: 20, CDR2 of SEQ ID NO: 21, and CDR3 of SEQ ID NO: 22; (e) CDR 1 of SEQ ID NO: 23, CDR2 of SEQ ID NO: 24, and CDR3 of SEQ ID NO: 25; (f) CDR 1 of SEQ ID NO: 26, CDR2 of SEQ ID NO: 27, and CDR3 of SEQ ID NO: 28; (g) CDR 1 of SEQ ID NO: 29, CDR2 of SEQ ID NO: 30, and CDR3 of SEQ ID NO: 31; (h) CDR 1 of SEQ ID NO: 32, CDR2 of SEQ ID NO: 33, and CDR3 of SEQ ID NO: 34; (i) CDR 1 of SEQ ID NO: 35, CDR2 of SEQ ID NO: 36, and CDR3 of SEQ ID NO: 37; or (j) An antibody or antigen-binding fragment thereof comprising CDR 1 of SEQ ID NO: 38, CDR2 of SEQ ID NO: 39, and CDR3 of SEQ ID NO:

40.

3. In the first paragraph, the single domain antibody comprises a heavy chain variable region, An antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 10 or a sequence having at least 90% homology with any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 10.

4. In the first paragraph, the single domain antibody is an antibody or antigen-binding fragment thereof that does not bind to angiopoietin-1 (Ang-1) or has low reactivity with it.

5. In the first paragraph, the single domain antibody is an antibody or antigen-binding fragment thereof that activates the TEK / Tie2 pathway by inhibiting the binding of angiopoietin-2 to the TEK / Tie2 receptor.

6. In the first paragraph, the single domain antibody is an antibody or antigen-binding fragment thereof in a form in which a biocompatible material is fused.

7. In paragraph 6, the biocompatible material is an antibody or an antigen-binding fragment thereof selected from the group consisting of polyethylene glycol (PEG), cholesterol, albumin and fragments thereof, albumin binding material, polymers of repeating units of a specific amino acid sequence, FcRn binding material, in vivo connective tissue or derivatives thereof, nucleotides, fibronectin, transferrin, saccharides, polymers, and combinations thereof.

8. A multispecific fusion protein comprising a single domain antibody (sdAb) or an antigen-binding fragment thereof that specifically binds to angiopoietin-2 (Ang-2) of paragraph 1; and a second antigen-binding substance.

9. A multispecific fusion protein according to claim 8, wherein the single domain antibody (sdAb) or antigen-binding fragment thereof that specifically binds to angiopoietin-2 (Ang-2) is directly linked to the second antigen-binding substance through a peptide bond or indirectly through a linker.

10. A multispecific fusion protein according to claim 8, wherein the second antigen-binding substance is an anti-VEGF antibody or an antigen-binding fragment thereof that specifically binds to VEGF.

11. A multispecific fusion protein according to claim 10, wherein the anti-VEGF antibody is aflibercept, bevacizumab, brolucizumab, or ranibizumab.

12. In claim 10, the fusion protein is a multispecific fusion protein comprising a sequence having at least 90% homology with any one sequence selected from the group consisting of SEQ ID NOs: 48 to 65 or any one sequence selected from the group consisting of SEQ ID NOs: 48 to 65.

13. A dual specific antibody that simultaneously binds to Ang-2 and VEGF, comprising a single domain antibody (sdAb) or an antigen-binding fragment thereof that specifically binds to angiopoietin-2 (Ang-2) of paragraph 1; and an anti-VEGF antibody or an antigen-binding fragment thereof that specifically binds to VEGF.

14. A polynucleotide encoding a single domain antibody or antigen-binding fragment thereof that specifically binds to angiopoietin-2 (Ang-2) according to any one of claims 1 to 7, a fusion protein according to any one of claims 8 to 12, or a dual specific antibody according to claim 13.

15. An expression vector comprising the polynucleotide of Article 14.

16. A host cell comprising the expression vector of item 14.

17. A method for producing an antibody, comprising a step of culturing the cell of claim 16.

18. A pharmaceutical composition for preventing or treating a disease associated with angiopoietin-2 activation or overproduction, comprising a single domain antibody or an antigen-binding fragment thereof that specifically binds to angiopoietin-2 (Ang-2) according to any one of claims 1 to 7 as an active ingredient.

19. A pharmaceutical composition according to claim 18, wherein the disease associated with angiopoietin-2 activation or overproduction is a vascular disease.

20. In claim 19, the vascular disease is Age-related Macular Degeneration (AMD), Diabetic Macular Edema (DME), Diabetic Retinopathy (DR), Retinal Vein Occlusion (RVO), Retinopathy of Prematurity (ROP), Neovascular Glaucoma, Proliferative Diabetic Retinopathy (pDR), Vascular Adhesion, Clarkson's Disease / ISCLS, Osteoarthritis, Rheumatoid Arthritis, Psoriasis, Crohn's Disease, Ulcer, Diabetic Nephropathy, Nephritis A pharmaceutical composition selected from the group consisting of (Nephritis), liver cirrhosis, neurodegenerative diseases, and atherosclerosis.

21. A method for preventing or treating a disease associated with angiopoietin-2 activation or overproduction, comprising administering to a subject the pharmaceutical composition of claim 18.

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