WO2022059800A1

WO2022059800A1 - Bispecific binding molecules against vegf and ang2

Info

Publication number: WO2022059800A1
Application number: PCT/JP2021/035211
Authority: WO
Inventors: Chiungkuang CHEN; Aishwarya KUNDU; Sreenivasu Mudumba; Masaaki Kageyama; Akira Nonoyama; Atsushi Yoshida; Yasuo YANAGI; Chui Ming Gemmy Cheung; Tokiyoshi Matsushita; Chie SHINTANI; Merry GUNAWAN; Erika KIMURA; Ming Chuan Richard YU
Original assignee: Santen Pharmaceutical Co., Ltd.; Singapore Health Services Pte Ltd
Priority date: 2020-09-15
Filing date: 2021-09-14
Publication date: 2022-03-24
Also published as: TW202227509A

Abstract

The present disclosure relates to bispecific binding molecules against human vascular endothelial growth factor (VEGF/VEGF-A) and against human angiopoietin-2 (ANG-2), including protein sequences, methods for their production, pharmaceutical compositions containing the bispecific binding molecules, and therapeutic and diagnostic uses thereof.

Description

DESCRIPTION

TITLE OF INVENTION

BISPECIFIC BINDING MOLECULES AGAINST VEGF AND ANG2 TECHNICAL FIELD

[0001] The present disclosure relates to bispecific binding molecules against human vascular endothelial growth factor (VEGF/VEGF-A) and against human angiopoietin-2 (ANG-2), including protein sequences, methods for their production, pharmaceutical compositions containing the bispecific binding molecules, and therapeutic and diagnostic uses thereof.

BACKGROUND ART

[0002] Angiogenesis is implicated in the pathogenesis of a variety of disorders including solid tumors, intraocular neovascular syndromes such as proliferative retinopathies or age-related macular degeneration (AMD), rheumatoid arthritis, and psoriasis (Folkman, J., et al., J. Biol. Chem.267 (1992) 10931-10934; Klagsbrun, M., et al, Annu. Rev. Physiol.53 (1991) 217- 239; and Gamer, A., Vascular Diseases, in: Pathobiology of Ocular Disease, A Dynamic Approach, Gamer, A., and Klintworth, G. K. (eds.), 2nd edition, Marcel Dekker, New York (1994), pp. 1625- 1710).

[0003] Human vascular endothelial growth factor (VEGF/VEGF-A) is described in, e.g., Leung, D.W., et al, Science 246 (1989) 1306-9; Keck, P.J., et al, Science 246 (1989) 1309- 12 and Connolly, D.T., et al, J. Biol. Chem. 264 (1989) 20017-24. The expression of VEGF is potentiated in response to hypoxia, by activated oncogenes, and by a variety of cytokines. VEGF is involved in the regulation of normal and abnormal angiogenesis and neovascularization associated with tumors and intraocular disorders (Ferrara, N., et al, Endocr. Rev. 18 (1997) 4- 25; Berkman, R.A., et al, J. Clin. Invest.91 (1993) 153-159; Brown, L.F., et al, Human Pathol. 26 (1995) 86- 91; Brown, L.F., et al, Cancer Res. 53 (1993) 4727-4735; Mattern, J., et al, Brit. J. Cancer.73 (1996) 931-934; and Dvorak, H.F., et al, Am. J. Pathol.146 (1995) 1029-1039).

[0004] Ocular vascular diseases such as age-related macular degeneration (AMD) and diabetic retinopathy (DR) are caused by abnormal choroidal and retinal neovascularization, respectively, and are the leading causes of visual loss. Since the retina consists of well-defined layers of neuronal, glial, and vascular elements, relatively small disturbances of vascular proliferation or edema can lead to significant loss of visual function. Inherited retinal degenerations, such as Retinitis Pigmentosa (RP), are also associated with vascular abnormalities, such as arteriolar narrowing and vascular atrophy. They affect as many as 1 in 3500 individuals and are characterized by progressive night blindness, visual field loss, optic nerve atrophy, arteriolar attenuation, and central vision loss to complete blindness.

[0005] In retinopathies, partial or general ischemia of the retina is accompanied by overexpression of VEGF and hyperproliferation of blood vessels, blindness can result (Aiello, L. P et al., 1994. N. Engl. J. Med.331, 1480-1487; Adamis, A. P., et al., , Am. J. Ophthalmol. 118, 445-450). Inhibition of VEGF expression in such ophthalmic disease states may be able to treat or prevent resulting blindness.

[0006] Human angiopoietin-2 (ANG-2 or Ang-2 or Ang2) (alternatively referred to as ANGPT2 or ANG2) is described in Maisonpierre, et al., Science 277 (1997) 55-60 and Cheung, A. H., et al, Genomics 48 (1998) 389-91. Ang2 plays an important role in angiogenesis and its expression levels have been correlated with cancer and eye diseases (D. Gerald, et al., Cancer Res. 2013, 73(6)4649-57; Watanabe, et al., Am. J. Ophthalmol. 2005, 139(3):476-81).

[0007] For ocular diseases via intravitreal application, smaller antibody fragments like

Fab or F(ab’)2 are often used as they have lower systemic toxicities due to their shorter serum half- life. In addition, these smaller fragments typically have also shorter intravitreal half-lives (e.g., due to faster diffusion into the serum) and typically are dosed more frequently.

SUMMARY OF INVENTION

[0008] The present disclosure provides bispecific binding molecules capable of inhibiting VEGF and/or Ang2. In some embodiments, the bispecific binding molecules may have a longer half-life based on a larger molecule size compared to a monospecific inhibitor. In some embodiment, the bispecific binding molecules are designed to inhibit angiogenesis and/or treating ocular diseases. Methods for producing the bispecific binding molecules, including the processes involving nucleic acids, vectors, expression vectors, and host vector system, are also disclosed. The present disclosure includes the following embodiments.

[1] A bispecific binding molecule comprising a polypeptide comprising an anti-angiopoetin2 (ANG2)-binding domain and a vascular endothelial growth factor (VEGF)-binding domain, wherein the anti-ANG2-b inding domain is an ANG2 antibody or an antigen binding fragment thereof, wherein the VEGF-binding domain comprises a fusion protein which binds to VEGF polypeptide comprising an extracellular domain of VEGF receptor operatively linked to Fc domain, wherein the anti-ANG2-binding domain is a Fab fragment or a scFv fragment.

[2] The bispecific binding molecule of [1], the anti-ANG2 -binding domain comprises an amino acid sequence of SEQ ID NO: 3 and/or an amino acid sequence of SEQ ID NO: 4, wherein the VEGF-binding domain comprises an amino acid sequence of SEQ ID NO: 18, wherein the Fc domain is IgGl.

[3] The bispecific binding molecule of [2], the anti-ANG2 -binding domain comprises an amino acid sequence of SEQ ID NO: 1 and/or an amino acid sequence of SEQ ID NO: 2, wherein the IgGl is mutated.

[4] The bispecific binding molecule of [2], wherein the Fc domain has an amino acid sequence with substitutions, deletions, insertions, and/or additions at, at least one site selected from 235th and 309th position of the IgGl in accordance with EU/Kabat numbering scheme.

[5] The bispecific binding molecule of [4], wherein the Fc domain has an amino acid sequence with substitutions at 309th position of the IgGl in accordance with EU/Kabat numbering scheme.

[6] The bispecific binding molecule of [5], wherein the Fc domain has an amino acid sequence with substitutions into K. [7] The bispecific binding molecule of [4], wherein the Fc domain has an amino acid sequence with substitutions at 235th position of the IgGl in accordance with EU/Kabat numbering scheme.

[8] The bispecific binding molecule of [7], wherein the Fc domain has an amino acid sequence with substitutions into K.

[9] The bispecific binding molecule of any one of [1 ]-[6], wherein the VEGF-binding domain linked with the N-terminus of the ANG2 -binding domain.

[10] The bispecific binding molecule of any one of [l]-[6], wherein the ANG2-binding domain linked with the N-terminus of the VEGF-binding domain.

[11] The bispecific binding molecule of any one of [1 ]-[ 10], wherein the anti-ANG2 -binding domain is a Fab fragment.

[12] The bispecific binding molecule of any one of [1 ]-[l 0], wherein the anti-ANG2-binding domain is a scFv fragment.

[13] The bispecific binding molecule of any one of [1]-[12], wherein the VEGF-binding domain linked with the ANG2-binding domain via a linker.

[14] The bispecific binding molecule of any one of [1]-[13], wherein the binding molecule comprises an amino acid sequence of SEQ ID NO: 10.

[15] The bispecific binding molecule of [14], wherein the binding molecule comprises an amino acid sequence of SEQ ID NO: 11.

[16] The bispecific binding molecule of [14], wherein the binding molecule comprises an amino acid sequence of SEQ ID NO: 12.

[17] The bispecific binding molecule of any one of [1 ]-[ 13], wherein the binding molecule comprises an amino acid sequence of SEQ ID NO: 2.

[18] The bispecific binding molecule of [17], wherein the binding molecule comprises an amino acid sequence of SEQ ID NO: 11.

[19] The bispecific binding molecule of [17], wherein the binding molecule comprises an amino acid sequence of SEQ ID NO: 12.

[20] The bispecific binding molecule of any one of [1]-[13], wherein the binding molecule comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

[21] The bispecific binding molecule of [1], wherein the anti-ANG2 -binding domain is a Fab fragment comprise an anti-ANG2 binding Fab heavy and light chains, wherein the anti-ANG2 binding Fab heavy chain is fused to N-terminus of the VEGF-binding domain optionally through a peptide linker, wherein the anti-ANG2 binding Fab heavy chain comprises an amino acid sequence of SEQ. ID NO: 3 and the anti-ANG2 binding Fab light chain comprises an amino acid sequence of SEQ. ID NO: 4 and the VEGF-binding domain comprises an amino acid sequence of SEQ. ID NO: 9.

[22] The bispecific binding molecule of [21], wherein the anti -ANG2 -bin ding domain is a Fab fragment comprise an anti-ANG2 binding Fab heavy and light chains, wherein the anti-ANG2 binding Fab heavy chain is fused to N-terminus of the VEGF-binding domain optionally through a peptide linker, wherein the anti-ANG2 binding Fab heavy chain comprises an amino acid sequence of SEQ. ID NO: 1 and the anti-ANG2 binding Fab light chain comprises an amino acid sequence of SEQ. ID NO: 2 and the VEGF-binding domain comprises an amino acid sequence of SEQ. ID NO: 9.

[23] The bispecific binding molecule of [21], wherein the anti -AN G2 -binding domain is a Fab fragment comprise an anti-ANG2 binding Fab heavy and light chains, wherein the anti-ANG2 binding Fab heavy chain is fused to N-terminus of the VEGF-binding domain optionally through a peptide linker, wherein the anti-ANG2 binding Fab heavy chain comprises an amino acid sequence of SEQ. ID NO: 1 and the anti-ANG2 binding Fab light chain comprises an amino acid sequence of SEQ. ID NO: 10 and the VEGF-binding domain comprises an amino acid sequence of SEQ. ID NO: 9.

[24] The bispecific binding molecule of [21 ]-[23], the peptide linker comprises an amino acid sequence of SEQ. ID NO: 7

[25] The bispecific binding molecule of [21 ]-[23], the peptide linker comprises an amino acid sequence of SEQ. ID NO: 8

[26] The bispecific binding molecule of [1], wherein the anti -AN G2 -binding domain is a Fab fragment comprise an anti-ANG2 binding Fab heavy and light chains, wherein the anti-ANG2 binding Fab heavy chain is fused to C-terminus of the VEGF-binding domain optionally through a peptide linker, wherein the anti-ANG2 binding Fab heavy chain comprises an amino acid sequence of SEQ. ID NO: 3 and the anti-ANG2 binding Fab light chain comprises an amino acid sequence of SEQ. ID NO: 4 and the VEGF-binding domain comprises an amino acid sequence of SEQ. ID NO: 9.

[27] The bispecific binding molecule of [1], wherein the anti-ANG2 -binding domain is a Fab fragment comprise an anti-ANG2 binding Fab heavy and light chains, wherein the anti-ANG2 binding Fab heavy chain is fused to C-terminus of the VEGF-binding domain optionally through a peptide linker, wherein the anti-ANG2 binding Fab heavy chain comprises an amino acid sequence of SEQ. ID NO: 1 and the anti-ANG2 binding Fab light chain comprises an amino acid sequence of SEQ. ID NO: 2 and the VEGF-binding domain comprises an amino acid sequence of SEQ. ID NO: 9.

[28] The bispecific binding molecule of [1], wherein the anti-ANG2 -binding domain is a Fab fragment comprise an anti-ANG2 binding Fab heavy and light chains, wherein the anti-ANG2 binding Fab heavy chain is fused to C-terminus of the VEGF-binding domain optionally through a peptide linker, wherein the anti-ANG2 binding Fab heavy chain comprises an amino acid sequence of SEQ. ID NO: 1 and the anti-ANG2 binding Fab light chain comprises an amino acid sequence of SEQ. ID NO: 10 and the VEGF-b inding domain comprises an amino acid sequence of SEQ. ID NO: 9.

[29] The bispecific binding molecule of [26]-[28], the peptide linker comprises an amino acid sequence of SEQ. ID NO: 7

[30] The bispecific binding molecule of [26]-[28], the peptide linker comprises an amino acid sequence of SEQ. ID NO:8

[31] The bispecific binding molecule of [1 ], wherein the anti-ANG2 -binding domain is a scFv fragment, wherein the scFv fragment is fused to N-terminus of the VEGF -binding domain optionally through a first peptide linker, wherein the scFv fragment comprises VH and VL through a second peptide linker, the VH and VL comprise amino acid sequence of SEQ. ID NO: 3 and 4 respectively and the VEGF-binding domain comprises an amino acid sequence of SEQ. ID NO: 9.

[32] The bispecific binding molecule of [1], wherein the anti-ANG2 -binding domain is a scFv fragment, wherein the scFv fragment is fused to C-terminus of the VEGF-binding domain optionally through a first peptide linker, wherein the scFv fragment comprises VH and VL through a second peptide linker, the VH and VL comprise amino acid sequence of SEQ. ID NO: 3 and 4 respectively and the VEGF-binding domain comprises an amino acid sequence of SEQ. ID NO: 9.

[33] The bispecific binding molecule of [31 ]-[32], the second peptide linker comprises an amino acid sequence of SEQ. ID NO: 17

[34] The bispecific binding molecule of [31 ]-[33], the first peptide linker comprises an amino acid sequence of SEQ. ID NO: 7

[35] The bispecific binding molecule of [31 ]-[33], the first peptide linker comprises an amino acid sequence of SEQ. ID NO:8

[36] The bispecific binding molecule of [1], wherein the anti-ANG2-binding domain is a scFv fragment, wherein the scFv fragment is fused to N-terminus of the VEGF-binding domain optionally through a peptide linker, wherein the scFv comprises an amino acid sequence of SEQ. ID NO: 5 and the VEGF-binding domain comprises an amino acid sequence of SEQ. ID NO: 9.

[37] The bispecific binding molecule of [1], wherein the anti-ANG2-binding domain is a scFv fragment, wherein the scFv fragment is fused to C-terminus of the VEGF-binding domain optionally through a peptide linker, wherein the scFv comprises an amino acid sequence of SEQ. ID NO: 5 and the VEGF-binding domain comprises an amino acid sequence of SEQ. ID NO: 9.

[38] The bispecific binding molecule of [1 ], wherein the anti -ANG2 -binding domain is a scFv fragment, wherein the scFv fragment is fused to N-terminus of the VEGF-binding domain optionally through a peptide linker, wherein the scFv comprises an amino acid sequence of SEQ. ID NO: 23 and the VEGF-binding domain comprises an amino acid sequence of SEQ. ID NO: 9. [39] The bispecific binding molecule of [1], wherein the anti-ANG2 -binding domain is a scFv fragment, wherein the scFv fragment is fused to C-terminus of the VEGF-binding domain optionally through a peptide linker, wherein the scFv comprises an amino acid sequence of SEQ. ID NO: 23 and the VEGF-binding domain comprises an amino acid sequence of SEQ. ID NO: 9.

[40] The bispecific binding molecule of [36]-[39], the peptide linker comprises an amino acid sequence of SEQ. ID NO: 7

[41] The bispecific binding molecule of [36]-[39], the peptide linker comprises an amino acid sequence of SEQ. ID NO:8

[42] The bispecific binding molecule of claim 1, wherein the anti-ANG2 -binding domain is a Fab fragment, wherein the Fab fragment is fused to N-terminus of the VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence that is SEQ. ID NO: 11, wherein the bispecific binding molecule comprises a light chain amino acid sequence that is SEQ. ID NO: 10.

[43] The bispecific binding molecule of claim 1, wherein the anti-ANG2 -binding domain is a Fab fragment, wherein the Fab fragment is fused to N-terminus of the VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence that is SEQ. ID NO: 11, wherein the bispecific binding molecule comprises a light chain amino acid sequence that is SEQ. ID NO: 2.

[44] The bispecific binding molecule of any one of [1 ]-[43], wherein the binding molecule is a dimer of the polypeptide thereof.

[45] A pharmaceutical composition comprises the bispecific binding molecule of any one of [l]-[44] and pharmaceutically acceptable excipient.

[46] A method of inhibiting angiogenesis using the bispecific binding molecule of any one of [l]-[44],

[47] A method of improve and/or treat clinical conditions selected from angiogenesis, vascular permeability, edema, and inflammation using the bispecific binding molecule of any one of [l]-[44],

[48] A method of treating ocular disease administering the bispecific binding molecule of any one of [l]-[44],

[49] The method of [48], wherein the ocular disease is selected from the group consisting of AMD, macular edema, CNV, DME, pathological myopia, vascular glaucoma, GA, retinal vein occlusion and ROP and corneal neovascularization.

[50] A composition comprising the bispecific binding molecule of any one of [l]-[44], for inhibiting angiogenesis or treating ocular disease.

[51] The bispecific binding molecule of any one of [l]-[44] for use in inhibiting angiogenesis or treating ocular disease. [52] Use of the binding molecule of any one of [ 1 ]-[44] in the manufacture of a medicament for inhibiting angiogenesis or treating ocular disease.

[53] A nucleic acid encoding the bispecific binding molecule of any one of [l]-[44],

[54] A vector comprises the nucleic acid of [53],

[55] An expression vector comprising a nucleic acid of [54], wherein the nucleic acid is operatively linked to an expression control sequence.

[56] A host vector system for production of a fusion polypeptide which comprises the expression vector of [55], in a suitable host cell.

[57] The host vector system of [56], wherein the suitable host cell is a bacterial cell, yeast cell, insect cell, or mammalian cell.

[58] The host vector system of [56], wherein the suitable host cell is a CHO cell.

[59] A method of producing the bispecific binding molecule, comprising growing cells of the host-vector system of any one of [56]-[58], under conditions permitting production of the fusion polypeptide and recovering the fusion polypeptide so produced.

BREIIF DESCRIPTION OF DRAWINGS

[0009] Figure 1: Figure 1(a) shows a schematic diagram of VEGF Binding Domain - linker - Ang-2 Binding Domain (scFv). Figure 1 (b) shows a schematic diagram of VEGF Binding Domain - linker - Ang-2 Binding Domain(Fab). Figure 1(c) shows a schematic diagram of Ang- 2 Binding Domain (Fab) - linker - VEGF Binding Domain. Figure 1 (d) shows a schematic diagram of ANG-2 Binding Domain (scFv) - linker - VEGF Binding Domain. Figure 1 (e) shows a schematic diagram of First monomer: ANG-2 Binding Domain (Fab) - linker - VEGF Binding Domain and Second monomer: VEGF Binding Domain - linker - ANG-2 Binding Domain (Fab). Figure 1 (f) shows a schematic diagram of First monomer: ANG-2 Binding Domain (scFv) - linker - VEGF Binding Domain and Second monomer: VEGF Binding Domain - linker - ANG-2 Binding Domain (scFv). Figure 1 (g) shows a schematic diagram of First monomer: ANG-2 Binding Domain (Fab) - linker - VEGF Binding Domain and Second monomer: VEGF Binding Domain - linker - ANG-2 Binding Domain (scFv). Figure 1 (h) shows a schematic diagram of First monomer: ANG-2 Binding Domain (scFv) - linker - VEGF Binding Domain and Second monomer: VEGF Binding Domain - linker - ANG-2 Binding Domain (Fab).

[0010] Figure 2: Schematic diagram of ELISA setting

[0011] Figure 3 : (a) result of ELISA-ANG2 for BSP2 and (b) result of ELISA-VEGF for BSP2.

[0012] Figure 4: Result of Biacore-VEGF and ANG2 for BSP2.

[0013] Figure 5: Result of Biacore-VEGF and ANG2 for BSP1.

[0014] Figure 6: Representative concentration response curves that deduced IC50 (A) of BSP2 and (B) Control 2. HUVEC endothelial cells were treated with 5ng/ml of recombinant human VEGF- 165 followed by different concentrations of BSP2 or Control 2 for 3 days and end point cell viability was measured by using redox dye WST-8. Total 9 independent experiments with minimum 3 replicates in each experiment were performed

[0015] Figure 7: Concentration-response curve that deduced tube formation treated with BSP2 and Control 1 (averaged response of three independent experiments were shown).

[0016] Figure 8: Effect of BSP2 on angiopoietin-2-induced phosphorylation of Tie-2 in HUVEC.

[0017] Figure 9: Effect of BSP2 on angipoietin-2-induced Akt phosphorylation in HUVEC cells. HUVEC was treated with angiopoietin-2 with the indicated concentration of the bispecific antibody for 15 min. Then, cells were harvested and lysed. After that, ELISA for Akt phosphorylation was performed on cell lysate. (N=2)

[0018] Figure 10: Concentration response curve for BSP2 (Sample 1) on angiopoietin

2-induced Akt phosphorylation in HUVEC. [0019] Figure 11 : Concentration response curve for BSP2 (Sample 2) on angiopoietin

2-induced Akt phosphorylation in HUVEC.

[0020] Figure 12: Concentration response curve for Control 1 on angiopoietin-2 induced Akt Phosphorylation in HUVEC.

[0021] Figure 13: Animal model compound treatment timepoints plot against vascular leakage percentage area calculation based on FA images. Vascular leakage area of each treatment group was normalized based on original baseline obtained at beginning of the study. No animals were lost during design 6 months study period due to safety or other AE. (N=4 Control 2 group; N=6, BSP2 l.Omg and N=5, 3.0mg).

[0022] Figures 14-28: FA images per each group at week 0, 1, 12 and 24 for compound treated vascular leakage evaluation (OD) and compound treated non-DLAAA induced eye as control (OS). Figure 14: Control 2 0.5mg baseline; Figure 15: Control 2 0.5mg, week 1; Figure 16: Control 2 0.5mg, weekl2; Figure 17: Control 20.5mg, week24; Figure 18: BSP2 l.Omg, baseline; Figure 19: BSP2 l.Omg, week 1; Figure 20: BSP2 l.Omg, week 12; Figure 21: BSP2 l .Omg, week 24; Figure 22: BSP2 3.0mg, baseline; Figure 23. BSP2 3.0mg, week 1; Figure 24: BSP2 3.0mg, week 12; Figure 25: BSP2 3.0mg, week 24; Figure 26: BSP2 3.0mg, baseline, noninduced eye; Figure 27. BSP23.0mg, week 12, non-induced; and Figure 28. BSP2 3.0mg, week24, non-induced eye.

DESCRIPTION OF EMBODIMENTS

[0023] In one embodiment, bispecific binding molecules of the present disclosure include, for example, novel bispecific binding molecules capable of binding to human VEGF and human ANG-2 comprising a first antigen-binding domain that specifically binds to human VEGF and a second antigen-binding domain that specifically binds to human ANG-2, characterized in that i) said VEGF binding domain is derived from aflibercept (VEGF receptor-Fc fusion protein which “traps” VEGF (herein, referred to as a “VEGF trap”) and competes with the naturally occurring VEGF cellular receptor to inhibit VEGF), which has an amino acid sequence as shown in SEQ ID NO:6; and ii) said ANG-2 binding domain comprises in the heavy chain variable domain as shown in SEQ ID NO: 3 and in the light chain variable domain as shown in SEQ ID No 4.

[0024] Various aspects and embodiments are described herein. These aspects and embodiments may, however, be embodied in many different forms and should not be construed as being limited to the specific embodiments described herein; rather, the specific embodiments disclosed herein are exemplary and are provided to inform a person of ordinary skill in the art how to make and use the bispecific binding molecules, compositions, devices, methods of treatment, kits and methods of manufacture of pharmaceutical products described herein. All books, publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

[0025] As may be appreciated from the foregoing and following description, each and every feature described herein, and each and every combination of two or more of such features, is included within the scope of the present disclosure provided that the features included in such a combination are not mutually inconsistent. For example, any embodiment whose use is consistent with any other embodiment is contemplated and thus included in this description. Other aspects and embodiments are set forth in the following description and claims, and also when considered in conjunction with the accompanying examples and drawings.

[0026] As used in this specification and the appended claims, the singular forms “a,”

“an,” and “the” include plural references unless the context clearly dictates otherwise. For example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure.

A. Definitions

[0027] In order that the present disclosure may be more readily understood, certain terms will be defined as follows. Additional definitions are set forth throughout the detailed description.

[0028] As used herein, “nucleic acid” includes both DNA and RNA, including DNA and RNA containing non-standard nucleotides. A “nucleic acid” contains at least one polynucleotide (a “nucleic acid strand”). A “nucleic acid” may be single-stranded or doublestranded.

[0029] As used herein, "vector" refers to a nucleic acid molecule capable of introducing or transporting another nucleic acid molecule. The introduced nucleic acid is generally linked to a vector nucleic acid molecule, e.g., inserted therein. The vector may contain sequences which induce autonomous replication in the cell, or may contain sufficient sequences to allow its integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication defective retroviruses and lentiviruses.

[0030] As used herein, "binding molecule" refers to a binding protein that comprises antigen-binding site. The terms "binding site," "antigen-binding site," or “binding domain” as used herein denotes the region(s) of a binding molecule to which a ligand actually binds. The term "binding site," "antigen-binding site," or "binding domain" includes heavy chain variable domain (VH) and/or light chain variable domain (VL), or pairs of VH/VL, and can be derived from whole antibody or antibody fragment such as single chain Fv, a VH domain and/or a VL domain, Fab, or (Fab)2. In one embodiment, each of the antigen-binding sites comprises a heavy chain variable domain (VH) and/or a light chain variable domain (VL), and preferably may be formed by a pair consisting of a light chain variable domain (VL) and a heavy chain variable domain (VH). Binding molecules can be fusion proteins or binding agents.

[0031] The binding domain, and particularly heavy chain variable domains (VH) and/or light chain variable domains (VL), that specifically bind to human vascular endothelial growth factor (VEGF) may be derived from (i) anti-VEGF antibodies or (ii) anti-VEGF binding molecules, including aflibercept obtained by de novo immunization methods using inter alia either human VEGF protein or nucleic acid or fragments thereof or by phage display.

[0032] The binding domain, and particularly heavy chain variable domains (VH) and/or light chain variable domains (VL), that specifically bind to human angiopoietin-2 (ANG- 2) may be derived from (i) anti- ANG-2 antibody molecules disclosed in US7521053 and W02003/030833A2; or (ii) from anti-ANG-2 antibody molecules obtained for example by de novo immunization methods using inter alia either the human ANG-2 protein or nucleic acid or fragments thereof or by phage display. Anti- ANG-2 antibody molecules disclosed in US7521053 include Ab536, comprising a VH comprising SEQ ID NO: 3 and VL comprising SEQ ID NO: 4 of the present application. Specificity of the binding molecule refers to selective recognition for a particular epitope of an antigen. Natural antibodies, for example, are monospecific.

[0033] As used herein, the term "binding" or "specifically binding" refers to the binding of the molecule to an epitope of the antigen (including either human VEGF or human ANG-2 or any other VEGF or ANG-2) in an in vitro assay, preferably in a plasmon resonance assay (BIAcore, GE- Healthcare Uppsala, Sweden) with purified wild-type antigen. The affinity of the binding is defined by the terms k_a (rate constant for the association of the binding molecule from the binding molecule/antigen complex), kjo (dissociation constant), and KD (kfj/ka). Binding or specifically binding means a binding affinity (KD) of 10'⁸ mol/1 or less, preferably 10’⁹ M to 10‘¹³ mol/1, and more preferably I O'¹⁰ M to 10’¹³ mol/1.

[0034] "Bispecific binding molecules" according to the disclosure also abbreviated as "Bispecific binding antibodies", that are binding molecules which have two different antigenbinding specificities. Where a bispecific binding molecule has more than one specificity, the recognized epitopes may be associated with a single antigen or more than one antigen. Bispecific binding molecules of the present disclosure may be specific for two different antigens, for example, VEGF as a first antigen and ANG-2 as second antigen.

[0035] The term "monospecific" binding molecule as used herein denotes a molecule that comprises one or more binding sites each of which bind to the same epitope of the same antigen.

[0036] The term "valent" as used herein denotes the presence of a specified number of binding sites in a binding molecule. As such, the terms "bivalent", "tetravalent", and "hexavalent" denote the presence of two binding site, four binding sites, and six binding sites, respectively, in a binding molecule. The bispecific binding molecules according to the present disclosure are at least "bivalent" and may be multivalent, for example, trivalent, tetravalent, pentavalent, or hexavalent. Preferably the bispecific binding molecule according to the present disclosure is bivalent, trivalent or tetravalent. In one embodiment, the bispecific binding molecule is bivalent. In another embodiment, the bispecific binding molecule is trivalent. In yet another embodiment, the bispecific binding molecule is tetravalent.

[0037] The term “Fc fragment”, "immunoglobulin Fc region," or “ Fc domain” as used herein equivalently and interchangeably, refers to a protein that contains at least the heavy-chain constant region 2 (CH2) and the heavy-chain constant region 3 (CH3) of an immunoglobulin. In one embodiment, the Fc region excludes the variable regions of the heavy and light chains, the heavy-chain constant region 1 (CHI) and the light-chain constant region 1 (CL1) of the immunoglobulin. The Fc region may further include a hinge region at the heavy-chain constant region. Also, the immunoglobulin Fc region disclosed herein may contain a part or all of the Fc region including the heavy-chain constant region 1 (CHI) and/or the light-chain constant region 1 (CL1), except for the variable regions of the heavy and light chains, as long as it has a physiological function substantially similar to or better than the native protein. Also, the immunoglobulin Fc region may be a fragment having a deletion in a relatively long portion of the amino acid sequence of CH2 and/or CH3.

[0038] The immunoglobulin Fc region disclosed herein may include a native amino acid sequence or a sequence analogue thereof. An amino acid sequence analogue is a sequence that is different from the native amino acid sequence due to a deletion, an insertion, a nonconservative or conservative substitution or combinations thereof of one or more amino acid residues.

[0039] The terms "antagonist" or "inhibitor" as used herein equivalently and interchangeably and include a binding molecule that is capable of inhibiting and/or neutralizing the biological signaling activity of a protein, for example by blocking binding or substantially reducing binding of a protein to its ligand and thus inhibiting or reducing the signalization pathway triggered by the protein and/or inhibiting or reducing a protein-mediated cell response like angiogenesis.

[0040] The terms "monoclonal antibody" as used herein refer to a preparation of antibody molecules of a single amino acid composition. The term "chimeric antibody" refers to an antibody comprising a variable region, i.e., binding region, from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques.

[0041] The "variable domain" or "variable domain of a light chain (VL)" or "variable region of a heavy chain (VH)" as used herein denotes each of the pair of light and heavy chains which is involved directly in binding the binding molecule to the antigen. The domains of variable human light and heavy chains have the same general structure and each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a p-sheet conformation and the CDRs may form loops connecting the P-sheet structure. The CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain the antigen binding site. The heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the binding molecule according to the invention and therefore provide a further object of the invention.

[0042] The terms "hypervariable region" or "antigen-binding portion" when used herein refer to the amino acid residues of a binding molecule which are responsible for antigenbinding. The hypervariable region comprises amino acid residues from the "complementarity determining regions" or "CDRs". "Framework" or "FR" regions are those variable domain regions other than the hypervariable region residues as herein defined. Therefore, the light and heavy chains of a binding molecule comprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRs on each chain are separated by such framework amino acids. Especially, CDR3 of the heavy chain is the region which contributes most to antigen binding. CDR and FR regions are determined according to the standard definition of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health,

Bethesda, MD (1991).

[0043] The term "epitope" includes any polypeptide determinant capable of specific binding to a binding molecule. In certain embodiments, epitope determinant includes chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and or specific charge characteristics. An epitope is a region of an antigen that is bound by a binding molecule. In certain embodiments, a binding molecule is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.

[0044] “Fab” is antibody fragment that comprises one constant domain (CHI, CL) and one variable domain (VH, VL) formed by dimerization of the heavy chain-derived sequence and the light chain-derived sequence, respectively, wherein the variable domains of VH and VL constitute the antigen binding sites. The two Fab’ fragments are bound to the Fc portion via the hinge region as an F(ab’)2 fragment at the N-terminus.

[0045] “Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of the sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994).

[0046] Furthermore said single chain Fv may be preferably disulfide-stabilized. Such further disulfide-stabilization of single chain antibodies is achieved by the introduction of a disulfide bond between the variable domains of the single chain antibodies and is described e.g in WO 94/029350, Rajagopal, V., et al., Prot. Engin. 10 (12) (1997) 1453-59; Kobayashi, H., et al., Nuclear Medicine & Biology 25 (1998) 387-393; or Schmidt, M, et al., Oncogene 18 (1999) 171 1-1721.

[0047] “VEGF inhibitor” used herein refers to any compound that reduces or inhibits the expression or biological activity of VEGF.

[0048] The term "peptide-linker" as used within the invention denotes a peptide with amino acid sequences, which is preferably of synthetic origin. These peptide- linkers according to invention are used to link the different antigen-binding sites and/or antibody fragments eventually comprising the different antigen-binding sites (e.g. single chain Fv, full length antibodies, a VH domain and/or a VL domain, Fab, (Fab)2, Fc part) together to form a bispecific antibody according to the invention.

[0049] Anti-Ang 2 inhibitors are described in W02003/030833A2 and U.S. Patent No. 7,521,053, each of which is incorporated herein by reference in its entirety, including an anti-Ang2 inhibitor’s structure comprising a 536HC heavy chain and a 536kappa light chain, properties, methods for making and using, and other related antibodies. Anti-ang2 inhibitors and related antibodies, etc. are also described in W02004/092215A2, which is incorporated herein by reference in its entirety, in particular with respect to the anti-ang2 inhibitor and related peptides and proteins, their structure and properties, and methods for making and using them.

[0050] Aflibercept, one of the VEGF inhibitors, is a recombinant fusion protein consisting of VEGF-binding portions from the extracellular domains of human VEGF receptors 1 and 2 that are fused to the Fc portion of the human IgGl immunoglobulin. Aflibercept has been approved in the United States and Europe for the treatment of wet macular degeneration under the trade name Eylea™.

B. Description of Bispecific Binding Molecules

[0051] Binding Moieties

[0052] The bispecific binding molecules of the present disclosure may comprise an anti-Ang-2 binding domain, which binds to Angiopoietin 2 (Ang-2) and inhibits the binding of Ang-2 to its receptor. In one embodiment, the anti-Ang-2 binding domain may comprise the Fab region derived from VH, VL, CHI and CL of an Ang-2 inhibitor, as described in W02003/030833A2 and US Patent No. 7,521,053. In one embodiment, the anti-Ang-2 binding Fab heavy and light chains are derived from VH and VL of an Ang-2 inhibitor as shown in SEQ ID NO: 3 and 4.

[0053] In another embodiment, the anti-Ang-2 binding domain may comprise a singlechain Fv region derived from VH and VL of an anti-Ang-2 inhibitor having an amino acid sequence that is from at least about 70% to 100% identical to SEQ. ID NO: 3 and/or SEQ ID NO: 4 respectively. In another embodiment, the anti-Ang-2 binding domain may comprise a VH having an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% to SEQ. ID NO: 3, and/or a VL having an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% to SEQ. ID NO: 4. In another embodiment, the VL may be fused to the N-terminus of the VH via a linker. In another embodiment, the VH may be fused to the N-terminus of the VL via a linker.

[0054] In one embodiment, the anti-Ang-2 binding domain may comprise any or all of HCDR1 of SEQ ID NO: 24, HCDR2 of SEQ ID NO: 25, HCDR3 of SEQ ID NO: 26, LCDR1 of SEQ ID NO: 27, LCDR2 of SEQ ID NO: 28, and LCDR3 of SEQ ID NO: 29. [0055] In one embodiment, the anti-Ang-2 binding domain comprises SEQ. ID NO: 5. In another embodiment, the Ang-2 binding domain comprises a sequence identity of at least about 75% to SEQ. ID NO: 5. In another embodiment, the Ang-2 binding domain comprises a sequence identity of at least about 80% to SEQ. ID NO: 5. In another embodiment, the Ang-2 binding domain comprises a sequence identity of at least about 85% to SEQ. ID NO: 5. In another embodiment, the Ang-2 binding domain comprises a sequence identity of at least about 90% to SEQ. ID NO: 5. In another embodiment, the Ang-2 binding domain comprises a sequence identity of at least about 95% to SEQ. ID NO: 5. In another embodiment, the Ang-2 binding domain comprises a sequence identity of about 100% to SEQ. ID NO: 5.

[0056] In one embodiment, the anti-Ang-2 binding domain comprises SEQ. ID NO: 23. In another embodiment, the Ang-2 binding domain comprises a sequence identity of at least about 75% to SEQ. ID NO: 23. In another embodiment, the Ang-2 binding domain comprises a sequence identity of at least about 80% to SEQ. ID NO: 23. In another embodiment, the Ang-2 binding domain comprises a sequence identity of at least about 85% to SEQ. ID NO: 23. In another embodiment, the Ang-2 binding domain comprises a sequence identity of at least about 90%to SEQ. ID NO: 23. In another embodiment, the Ang-2 binding domain comprises a sequence identity of at least about 95% to SEQ. ID NO: 5. In another embodiment, the Ang-2 binding domain comprises a sequence identity of about 100% to SEQ. ID NO: 23.

[0057] The bispecific binding molecules of the present disclosure may comprise a VEGF binding domain, which may “trap” VEGF (herein, referred to as a “VEGF trap”) and competes with the naturally occurring VEGF cellular receptor to inhibit VEGF. In one embodiment, the VEGF binding domain is aflibercept, which has an amino acid sequence as shown in SEQ ID NO: 6. In some embodiments, the VEGF binding domain may comprise an amino acid sequence having about 70% to 100% identity to SEQ ID NO: 6 . In one embodiment, the VEGF binding domain may comprise SEQ. ID NO: 6 . In another embodiment, the VEGF binding domain comprises a sequence identity of at least about 75% to SEQ. ID NO: 6 . In another embodiment, the VEGF binding domain comprises a sequence identity of at least about 80% to SEQ. ID NO: 6. In another embodiment, the VEGF binding domain comprises a sequence identity of at least about 85% to SEQ. ID NO: 6. In another embodiment, the VEGF binding domain comprises a sequence identity of at least about 90% to SEQ. ID NO: 6. In another embodiment, the VEGF binding domain comprises a sequence identity of at least about 95% to SEQ. ID NO: 6. In another embodiment, the VEGF binding domain comprises a sequence identity of about 100% to SEQ. ID NO: 6.

[0058] In some embodiments, the VEGF binding domain comprises Aflibercept variant with substitution, deletion, insertion and/or addition in the Fc domain to improve aggregation. In some embodiments, the VEGF binding domain comprises the Fc domain having an amino acid sequence with substitutions, deletions, insertions, and/or additions at, at least one site selected from 235^th and 309^th positions of the IgGl in accordance with EU/Kabat numbering scheme. In some embodiments, substitution with lysin (K) is preferred at 235^th and 309^th positions of the IgGl in accordance with EU/Kabat numbering scheme. In some embodiments, the VEGF binding domain comprises the Fc domain having an amino acid sequence with substitutions, deletions, insertions, and/or additions at 235^th position of the IgGl in accordance with EU/Kabat numbering scheme. In a preferred embodiment, the VEGF binding domain comprises the Fc domain having an amino acid sequence with substitutions, deletions, insertions, and/or additions at 309^th position of the IgGl in accordance with EU/Kabat numbering scheme. [0059] In one embodiment, the VEGF binding domain comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the VEGF binding domain may comprise an amino acid sequence having about 70% to 100% identity to SEQ ID NO: 9. In one embodiment, the VEGF binding domain comprises a sequence identity of at least about 70% to SEQ. ID NO: 9. In another embodiment, the VEGF binding domain comprises a sequence identity of at least about 75% to SEQ. ID NO: 9. In another embodiment, the VEGF binding domain comprises a sequence identity of at least about 80% to SEQ. ID NO: 9. In another embodiment, the VEGF binding domain comprises a sequence identity of at least about 85% to SEQ. ID NO: 9. In another embodiment, the VEGF binding domain comprises a sequence identity of at least about 90% to SEQ. ID NO: 9. In another embodiment, the VEGF binding domain comprises a sequence identity of at least about 95% to SEQ. ID NO: 9. In another embodiment, the Ang-2 antagonist binding domain comprises a sequence identity of about 100% to SEQ. ID NO: 9.

[0060] The bispecific binding molecules of the present disclosure may comprise a VEGF binding domain, which may “VEGF trap” and competes with the naturally occurring VEGF cellular receptor to inhibit VEGF. In one embodiment, the VEGF binding domain is aflibercept, which has an amino acid sequence as shown in SEQ ID NO: 20. In some embodiments, the VEGF binding domain may comprise an amino acid sequence having about 70% to 100% identity to SEQ ID NO: 20. In one embodiment, the VEGF binding domain may comprise SEQ. ID NO: 20. In another embodiment, the VEGF binding domain comprises a sequence identity of at least about 75% to SEQ. ID NO: 20. In another embodiment, the VEGF binding domain comprises a sequence identity of at least about 80% to SEQ. ID NO: 6 or 20. In another embodiment, the VEGF binding domain comprises a sequence identity of at least about 85% to SEQ. ID NO: 20. In another embodiment, the VEGF binding domain comprises a sequence identity of at least about 90% to SEQ. ID NO: 20. In another embodiment, the VEGF binding domain comprises a sequence identity of at least about 95% to SEQ. ID NO: 20. In another embodiment, the VEGF binding domain comprises a sequence identity of about 100% to SEQ. ID NO: 20.

[0061] The bispecific binding molecules disclosed herein may comprise an anti- ANG2 binding domain fused to a VEGF binding domain. In one embodiment, the anti-ANG2 binding domain may be fused to the C-terminus of the VEGF binding domain. In another embodiment, the anti-ANG2 binding domain may be fused to the N-terminus of the VEGF binding domain.

[0062] In one embodiment, the bispecific binding molecules disclosed herein may comprise an anti-ANG2 binding domain fused to the C-terminus of a VEGF binding domain, wherein the anti-ANG2 binding domain is a single chain Fv and has an amino acid sequence identity of at least about 70% to 100% of SEQ ID NO: 5. In one embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 70% of SEQ. ID NO: 5. In another embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 75% of SEQ. ID NO: 5. In another embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 80% of SEQ. ID NO: 5. In another embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 85% of SEQ. ID NO: 5. In another embodiment, the anti- ANG2 binding domain has a sequence identity of at least about 90% of SEQ. ID NO: 5. In another embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 95% of SEQ. ID NO: 5. In still another embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 100% of SEQ. ID NO: 5. In another embodiment, the anti-ANG2 binding domain comprises SEQ. ID NO: 5. [0063] In another embodiment, the bispecific binding molecules disclosed herein may comprise an anti-ANG2 binding domain fused to the N-terminus of a VEGF binding domain, wherein the anti-ANG2 binding domain is a single chain Fv and has an amino acid sequence identity of at least about 70% to 100% of SEQ ID NO: 5. In one embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 70% of SEQ. ID NO: 5. In another embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 75% of SEQ. ID NO: 5. In another embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 80% of SEQ. ID NO: 5. In another embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 85% of SEQ. ID NO: 5. In another embodiment, the anti- ANG2 binding domain has a sequence identity of at least about 90% of SEQ. ID NO: 5. In another embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 95% of SEQ. ID NO: 5. In still another embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 100% of SEQ. ID NO: 5. In another embodiment, the anti-ANG2 binding domain comprises SEQ. ID NO: 5.

[0064] In one embodiment, the bispecific binding molecules disclosed herein may comprise an anti-ANG2 binding domain fused to the C-terminus of a VEGF binding domain, wherein the anti-ANG2 binding domain is a single chain Fv and has an amino acid sequence identity of at least about 70% to 100% of SEQ ID NO: 23. In one embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 70% of SEQ. ID NO: 23. In another embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 75% of SEQ. ID NO: 23. In another embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 80% of SEQ. ID NO: 23. In another embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 85% of SEQ. ID NO: 23. In another embodiment, the anti- ANG2 binding domain has a sequence identity of at least about 90% of SEQ. ID NO: 23. In another embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 95% of SEQ. ID NO: 23. In still another embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 100% of SEQ. ID NO: 5. In another embodiment, the anti-ANG2 binding domain comprises SEQ. ID NO: 23.

[0065] In another embodiment, the bispecific binding molecules disclosed herein may comprise an anti-ANG2 binding domain fused to the N-terminus of a VEGF binding domain, wherein the anti-ANG2 binding domain is a single chain Fv and has an amino acid sequence identity of at least about 70% to 100% of SEQ ID NO: 23. In one embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 70% of SEQ. ID NO: 23. In another embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 75% of SEQ. ID NO: 23. In another embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 80% of SEQ. ID NO: 23. In another embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 85% of SEQ. ID NO: 23. In another embodiment, the anti- ANG2 binding domain has a sequence identity of at least about 90% of SEQ. ID NO: 23. In another embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 95% of SEQ. ID NO: 23. In still another embodiment, the anti-ANG2 binding domain has a sequence identity of at least about 100% of SEQ. ID NO: 23. In another embodiment, the anti-ANG2 binding domain comprises SEQ. ID NO: 23.

[0066] In one embodiment, the bispecific binding molecules disclosed herein may comprise an anti-ANG2 binding domain fused to the C-terminus or N-terminus of a VEGF binding domain, wherein the anti-ANG2 binding domain is a Fab fragment comprising a heavy chain amino acid sequence having a sequence identity of at least about 70% to 100% identical to SEQ ID NO: 1 or 21 and a light chain amino acid sequence having a sequence identity of at least about 70% to 100% identical to SEQ ID NO: 2 or 22 or 10.

[0067] In one embodiment, the anti-ANG2 Fab fragment heavy chain amino acid sequence comprises SEQ. ID NO: 1 . In another embodiment, the anti-ANG2 Fab fragment heavy chain amino acid sequence has a sequence identity of at least about 70% of SEQ. ID NO: 1 . In another embodiment, the anti-ANG2 Fab fragment heavy chain amino acid sequence has a sequence identity of at least about 75% of SEQ. ID NO: 1. In another embodiment, the anti-ANG2 Fab fragment heavy chain amino acid sequence has a sequence identity of at least about 80% of SEQ. ID NO: 1. In another embodiment, the anti-ANG2 Fab fragment heavy chain amino acid sequence has a sequence identity of at least about 85% of SEQ. ID NO: 1. In another embodiment, the anti-ANG2 Fab fragment heavy chain amino acid sequence has a sequence identity of at least about 90% of SEQ. ID NO: 1. In another embodiment, the anti-ANG2 Fab fragment heavy chain amino acid sequence has a sequence identity of at least about 95% of SEQ. ID NO: 1 . In another embodiment, the anti-ANG2 Fab fragment heavy chain amino acid sequence has a sequence identity of at least about 100% of SEQ. ID NO: 1.

[0068] In one embodiment, the anti-ANG2 Fab fragment heavy chain amino acid sequence comprises SEQ. ID NO: 21. In another embodiment, the anti-ANG2 Fab fragment heavy chain amino acid sequence has a sequence identity of at least about 70% of SEQ. ID NO: 21. In another embodiment, the anti-ANG2 Fab fragment heavy chain amino acid sequence has a sequence identity of at least about 75% of SEQ. ID NO: 21. In another embodiment, the anti- ANG2 Fab fragment heavy chain amino acid sequence has a sequence identity of at least about 80% of SEQ. ID NO: 21 . In another embodiment, the anti-ANG2 Fab fragment heavy chain amino acid sequence has a sequence identity of at least about 85% of SEQ. ID NO: 21. In another embodiment, the anti-ANG2 Fab fragment heavy chain amino acid sequence has a sequence identity of at least about 90% of SEQ. ID NO: 21. In another embodiment, the anti-ANG2 Fab fragment heavy chain amino acid sequence has a sequence identity of at least about 95% of SEQ. ID NO: 21. In another embodiment, the anti-ANG2 Fab fragment heavy chain amino acid sequence has a sequence identity of at least about 100% of SEQ. ID NO: 21.

[0069] In one embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence comprises SEQ. ID NO: 2. In another embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence has a sequence identity of at least about 70% of SEQ. ID NO: 2. In another embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence has a sequence identity of at least about 75% of SEQ. ID NO: 2. In another embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence has a sequence identity of at least about 80% of SEQ. ID NO: 2. In another embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence has a sequence identity of at least about 85% of SEQ. ID NO: 2. In another embodiment, the anti- ANG2 Fab fragment light chain amino acid sequence has a sequence identity of at least about 90% of SEQ. ID NO: 2. In another embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence has a sequence identity of at least about 95% of SEQ. ID NO: 2. In another embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence has a sequence identity of about 100% of SEQ. ID NO: 2.

[0070] In one embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence comprises SEQ. ID NO: 10. In another embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence has a sequence identity of at least about 70% of SEQ. ID NO: 10. In another embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence has a sequence identity of at least about 75% of SEQ. ID NO: 10. In another embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence has a sequence identity of at least about 80% of SEQ. ID NO: 10. In another embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence has a sequence identity of at least about 85% of SEQ. ID NO: 10. In another embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence has a sequence identity of at least about 90% of SEQ. ID NO: 10. In another embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence has a sequence identity of at least about 95% of SEQ. ID NO: 10. In another embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence has a sequence identity of about 100% of SEQ. ID NO: 10.

[0071] In one embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence comprises SEQ. ID NO: 22. In another embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence has a sequence identity of at least about 70% of SEQ. ID NO: 22. In another embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence has a sequence identity of at least about 75% of SEQ. ID NO: 22. In another embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence has a sequence identity of at least about 80% of SEQ. ID NO: 22. In another embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence has a sequence identity of at least about 85% of SEQ. ID NO: 22. In another embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence has a sequence identity of at least about 90% of SEQ. ID NO: 22. In another embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence has a sequence identity of at least about 95% of SEQ. ID NO: 22. In another embodiment, the anti-ANG2 Fab fragment light chain amino acid sequence has a sequence identity of at least about 100% of SEQ. ID NO: 22.

[0072] In one embodiment, the bispecific binding molecules disclosed herein may comprise an anti-ANG2 binding domain fused to the C-terminus orN-terminus of a VEGF binding domain, wherein the VEGF binding domain comprises an amino acid sequence having a sequence identity of at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% and 100% identical to SEQ. ID NO: 6.

[0073] In one embodiment, the bispecific binding molecules disclosed herein may comprise an anti-ANG2 binding domain fused to the C-terminus or N-terminus of a VEGF binding domain, wherein the VEGF binding domain comprises an amino acid sequence having a sequence identity of at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% and 100% identical to SEQ. ID NO: 9.

[0074] In one embodiment, the bispecific binding molecules disclosed herein may comprise an anti-ANG2 binding domain fused to the C-terminus or N-terminus of a VEGF binding domain, wherein the VEGF binding domain comprises an amino acid sequence having a sequence identity of at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% and 100% identical to SEQ. ID NO: 20.

[0075] Peptide Linker Sequences

[0076] In the present disclosure, the anti-ANG2 binding domain and VEGF binding domain may be fused by a linker. In some embodiments, the anti-Ang-2 binding domain may comprise a single-chain, and Fv region may be derived from VH and VL fused by a linker. In one embodiment, the linker comprises a peptide comprising the amino acid sequence (GGGGS)_n, where n may be from 1 to 15. In one embodiment, the linker comprises the amino acid sequence (GGGGS)_n, where n may be from 2-10. In another embodiment, the linker comprises the amino acid sequence (GGGGS)_n, where n may be from 3-7. In yet another embodiment, the linker comprises the amino acid sequence (GGGGS)«, where n may be from 4-6. In still another embodiment, the linker comprises the amino acid sequence (GGGGS)„, where n is 1. In one another embodiment, the linker comprises the amino acid sequence (GGGGS)„, where n is 2. In another embodiment, the linker comprises the amino acid sequence (GGGGS)_n, where n is 3. In yet another embodiment, the linker comprises the amino acid sequence (GGGGS)„, where n is 4. In still another embodiment, the linker comprises the amino acid sequence (GGGGS)„, where n is 5. In still another embodiment, the linker comprises the amino acid sequence (GGGGS)_n, where n is 6. In still another embodiment, the linker comprises the amino acid sequence (GGGGS)„, where n is 7. In still another embodiment, the linker comprises the amino acid sequence (GGGGS)_n, where n is 8. In still another embodiment, the linker comprises the amino acid sequence (GGGGS)„, where n is 9. In another embodiment, the linker comprises the amino acid sequence (GGGGS)„, where n is 10. In another embodiment, the linker comprises SEQ ID NO: 7. In another embodiment, the linker comprises SEQ ID NO: 8. In another embodiment, the linker comprises SEQ ID NO: 17.

[0077] Linker considerations include the effect on physical or pharmacokinetic properties of the resulting compound, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable as well as planned degradation), viscosity, rigidity, flexibility, immunogenicity, modulation of antibody binding, the ability to be incorporated into a micelle or liposome, and the like.

[0078] Bispecific Binding Molecule Embodiments

[0079] The present disclosure relates to novel bispecific binding molecules comprising an anti-ANG2 binding domain to both VEGF and Ang-2. In some embodiments, the bispecific binding molecules disclosed herein may comprise one or two anti-ANG2 binding domains and one or two VEGF-binding domains, wherein: a) the anti-ANG2 binding domain is a peptide, Fab or scFv; and wherein said peptide, Fab, or scFv comprises light chain CDRs as derived from a light chain with an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 95%, at least about 99% or at least about 100% identical to SEQ ID NO: 2, 10, or 22, and heavy chain CDRs as derived from an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 95%, at least about 99% or at least about 100% identical to SEQ ID NO. 1 or 21, or CDRs derived from a scFv with an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 95%, at least about 99% or at least about 100% identical to SEQ ID NO. 5 or 23; b) the VEGF-binding domain is a VEGF-binding molecule, wherein said VEGF-binding molecule comprises a binding domain with an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 95%, at least about 99% or at least about 100% identical to SEQ ID NO: 6, 9, or 20.

[0080] In one embodiment, the bispecific binding molecule may comprise an anti- ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence that is SEQ. ID NO: 11. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 70% sequence identity to SEQ. ID NO: 11. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 75% sequence identity to SEQ. ID NO:11. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 80% sequence identity to SEQ. ID NO:11. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 85% sequence identity to SEQ. ID NO: 11. In yet another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 90% sequence identity to SEQ. ID NO:11. In another embodiment, the bispecific binding molecule may comprise an anti- ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 95% sequence identity to SEQ. ID NO:11. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 99% sequence identity to SEQ. ID NO:11.

[0081] In one embodiment, the bispecific binding molecule comprises a heavy chain amino acid sequence that is SEQ. ID NO:11 and a light chain amino acid sequence that is SEQ. ID NO:2. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 70% sequence identity to SEQ. ID NO: 11 and a light chain amino acid sequence having about 70% sequence identity to SEQ. ID NO:2. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 75% sequence identity to SEQ. ID NO: 11 and a light chain amino acid sequence having about 75% sequence identity to SEQ. ID NO:2. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 80% sequence identity to SEQ. ID NO:11 and a light chain amino acid sequence having about 80% sequence identity to SEQ. ID NO:2. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 85% sequence identity to SEQ. ID NO: 11 and a light chain amino acid sequence having about 85% sequence identity to SEQ. ID NO:2. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 90% sequence identity to SEQ. ID NO: 11 and a light chain amino acid sequence having about 90% sequence identity to SEQ. ID NO:2. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 95% sequence identity to SEQ. ID NO: 11 and a light chain amino acid sequence having about 95% sequence identity to SEQ. ID NO:2. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 100% sequence identity to SEQ. ID NO:11 and a light chain amino acid sequence having about 100% sequence identity to SEQ. ID NO:2.

[0082] In one embodiment, the bispecific binding molecule comprises a heavy chain amino acid sequence that is SEQ. ID NO:11 and a light chain amino acid sequence that is SEQ. ID NO: 10. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 70% sequence identity to SEQ. ID NO: 11 and a light chain amino acid sequence having about 70% sequence identity to SEQ. ID NO: 10. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 75% sequence identity to SEQ. ID NO: 11 and a light chain amino acid sequence having about 75% sequence identity to SEQ. ID NO: 10. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 80% sequence identity to SEQ. ID NO: 11 and a light chain amino acid sequence having about 80% sequence identity to SEQ. ID NO: 10. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 85% sequence identity to SEQ. ID NO: 11 and a light chain amino acid sequence having about 85% sequence identity to SEQ. ID NO: 10. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 90% sequence identity to SEQ. ID NO: 11 and a light chain amino acid sequence having about 90% sequence identity to SEQ. ID NO: 10. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 95% sequence identity to SEQ. ID NO: 11 and a light chain amino acid sequence having about 95% sequence identity to SEQ. ID NO: 10. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 100% sequence identity to SEQ. ID NO: 11 and a light chain amino acid sequence having about 100% sequence identity to SEQ. ID NO: 10.

[0083] In one embodiment, the bispecific binding molecule may comprise an anti-

ANG2 binding domain that is fused to one or both of the C-terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence that is SEQ. ID NO: 12. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the C- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 70% sequence identity to SEQ. ID NO: 12. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the C-terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 75% sequence identity to SEQ. ID NO: 12. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the C-terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 80% sequence identity to SEQ. ID NO: 12. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the C-terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 85% sequence identity to SEQ. ID NO: 12. In yet another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the C-terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 90% sequence identity to SEQ. ID NO: 12. In another embodiment, the bispecific binding molecule may comprise an anti- ANG2 binding domain that is fused to one or both of the C-terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 95% sequence identity to SEQ. ID NO: 12. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the C-terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 99% sequence identity to SEQ. ID NO: 12.

[0084] In one embodiment, the bispecific binding molecule comprises a heavy chain amino acid sequence that is SEQ. ID NO: 12 and a light chain amino acid sequence that is SEQ. ID NO:2. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 70% sequence identity to SEQ- ID NO: 12 and a light chain amino acid sequence having about 70% sequence identity to SEQ. ID NO:2. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 75% sequence identity to SEQ. ID NO: 12 and a light chain amino acid sequence having about 75% sequence identity to SEQ. ID NO:2. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 80% sequence identity to SEQ. ID NO: 12 and a light chain amino acid sequence having about 80% sequence identity to SEQ. ID NO:2. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 85% sequence identity to SEQ. ID NO: 12 and a light chain amino acid sequence having about 85% sequence identity to SEQ. ID NO:2. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF -binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 90% sequence identity to SEQ. ID NO: 12 and a light chain amino acid sequence having about 90% sequence identity to SEQ. ID NO:2. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 95% sequence identity to SEQ. ID NO: 12 and a light chain amino acid sequence having about 95% sequence identity to SEQ. ID NO:2. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 100% sequence identity to SEQ. ID NO:12 and a light chain amino acid sequence having about 100% sequence identity to SEQ. ID NO:2.

[0085] In one embodiment, the bispecific binding molecule comprises a heavy chain amino acid sequence that is SEQ. ID NO: 12 and a light chain amino acid sequence that is SEQ. ID NO: 10. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 70% sequence identity to SEQ. ID NO: 12 and a light chain amino acid sequence having about 70% sequence identity to SEQ. ID NO: 10. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF -binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 75% sequence identity to SEQ. ID NO: 12 and a light chain amino acid sequence having about 75% sequence identity to SEQ. ID NO: 10. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 80% sequence identity to SEQ. ID NO: 12 and a light chain amino acid sequence having about 80% sequence identity to SEQ. ID NO: 10. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 85% sequence identity to SEQ. ID NO: 12 and a light chain amino acid sequence having about 85% sequence identity to SEQ. ID NO: 10. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 90% sequence identity to SEQ. ID NO: 12 and a light chain amino acid sequence having about 90% sequence identity to SEQ. ID NO: 10. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 95% sequence identity to SEQ. ID NO: 12 and a light chain amino acid sequence having about 95% sequence identity to SEQ. ID NO: 10. In one embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is fused to one or both of the N- terminus of a VEGF-binding domain optionally through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence having about 100% sequence identity to SEQ. ID NO: 12 and a light chain amino acid sequence having about 100% sequence identity to SEQ. ID NO: 10.

[0086] In one embodiment, the bispecific binding molecule may comprise an anti- ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having SEQ. ID NO: 13. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 70% of SEQ. ID NO: 1 . In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 75% of SEQ. ID NO: 13. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N- terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 80% of SEQ. ID NO: 13. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 85% of SEQ. ID NO: 13. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF -binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 90% of SEQ. ID NO: 13. In another embodiment, the bispecific binding molecule may comprise an anti- ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 95% of SEQ. ID NO: 13. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 99% of SEQ. ID NO: 13.

[0087] In one embodiment, the bispecific binding molecule may comprise an anti- ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having SEQ. ID NO: 14. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 70% of SEQ. ID NO: 14. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 75% of SEQ. ID NO: 14. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N- terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 80% of SEQ. ID NO: 14. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 85% of SEQ. ID NO: 14. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 90% of SEQ. ID NO: 14. In another embodiment, the bispecific binding molecule may comprise an anti- ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 95% of SEQ. ID NO: 14. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 99% of SEQ. ID

NO: 14. [0088] In one embodiment, the bispecific binding molecule may comprise an anti- ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having SEQ. ID NO: 15. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 70% of SEQ. ID NO: 15. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 75% of SEQ. ID NO: 15. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N- terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 80% of SEQ. ID NO: 15. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 85% of SEQ. ID NO: 15. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 90% of SEQ. ID NO: 15. In another embodiment, the bispecific binding molecule may comprise an anti- ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 95% of SEQ. ID NO: 15. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 99% of SEQ. ID NO: 15.

[0089] In one embodiment, the bispecific binding molecule may comprise an anti- ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having SEQ. ID NO: 16. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 70% of SEQ. ID NO: 16. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 75% of SEQ. ID NO: 16. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N- terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 80% of SEQ. ID NO: 16. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 85% of SEQ. ID NO: 16. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 90% of SEQ. ID NO: 16. In another embodiment, the bispecific binding molecule may comprise an anti- ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 95% of SEQ. ID NO: 16. In another embodiment, the bispecific binding molecule may comprise an anti-ANG2 binding domain that is a single-chain Fv, which is fused to the N-terminus or the C-terminus of a VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises an amino acid sequence having a sequence identity of at least about 99% of SEQ. ID NO: 16.

[0090] The bispecific binding molecules of the present disclosure comprising binding domains to both VEGF and Ang-2 have, without limitation, the exemplary bispecific binding molecule configurations in N-terminal to C-terminus direction connected by a linker in Figure 1.

[0091] The term "N- terminus" denotes the last amino acid of the N-terminus. The term "C-terminus" denotes the last amino acid of the C-terminus. [0092] In some embodiments, said bispecific binding molecule may have an Fc region that is derived from IgG, IgA, IgD, IgE and IgM, or that is made by combinations thereof or hybrids thereof. In a preferred embodiment, the Fc region is derived from IgG. In another preferred embodiment, the Fc region is derived from IgGl .

[0093] In one embodiment, the bispecific binding molecule of the present disclosure blocks the interaction between Ang-2 and its receptor Tie-2 with an IC50 at 100 pM or less, 80 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 28 pM or less, 26 pM or less, 24pM or less, as determined by a method described in Example 1 of the present disclosure. In one embodiment, the bispecific binding molecule of the present disclosure binds to VEGF with an EC50 at 500 pM or less, 400 pM or less, 300 pM or less, 200 pM or less, 150 pM or less, 130 pM or less, 120 pM or less, 110 pM or less, 108 pM or less, 105 pM or less, 102 pM or less, as determined by a method described in Example 1 of the present disclosure.

[0094] In one embodiment, the bispecific binding molecule of the present disclosure binds to VEGF with Kd of 1000 pM or less, 500 pM or less, 400 pM or less, 300 pM or less, 250 pM or less, 220 pM or less, 200 pM or less, 190 pM or less, 160 pM or less, 130 pM or less, 100 pM or less, 90 pM or less, 80 pM or less, 70 pM or less, 60 pM or less, 58 pM or less, 56 pM or less, as determined by BIACORE described in Example 2 of the present disclosure. In one embodiment, the bispecific binding molecule of the present disclosure binds to Ang2 with Kd of 1000 pM or less, 800 pM or less, 500 pM or less, 450 pM or less, 400 pM or less, 350 pM or less, 300 pM or less, 250 pM or less, 200 pM or less, or 180 pM or less, or 160 pM or less , or 150 pM or less, 140 pM or less, as determined by BIACORE described in Example 2 of the present disclosure. [0095] In one embodiment, the bispecific binding molecule of the present disclosure binds to VEGF with Kd of 1000 pM or less, 500 pM or less, 400 pM or less, 300 pM or less, 250 pM or less, 220 pM or less, 200 pM or less, 195 pM or less, 190 pM or less, 188 pM or less, as determined by BIACORE described in Example 2 of the present disclosure. In one embodiment, the bispecific binding molecule of the present disclosure binds to Ang2 with Kd of 1000 pM or less, 800 pM or less, 500 pM or less, 450 pM or less, 400 pM or less, 350 pM or less, 300 pM or less, 280 pM or less, 260 pM or less, 255 pM or less, as determined by BIACORE described in Example 2 of the present disclosure.

[0096] In one embodiment, the bispecific binding molecule of the present disclosure inhibits proliferation of UUVEC cells at IC50 of 1000 pM or less, 700 pM or less, 500 pM or less, 400 pM or less, 350 pM or less, 345 pM or less, 340 pM or less, 337 pM or less, as determined by anti-VEGF cell proliferation functional assay described in Example 5-A of the present disclosure. In one embodiment, the bispecific binding molecule of the present disclosure inhibits tube formation of HUVEC cells at IC50 of 50 nM or less, 20 nM or less, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, as determined by Ang2 -mediated tube formation assay described in Example 5-B of the present disclosure. In one embodiment, the bispecific binding molecule of the present disclosure inhibits Akt phosphorylation at IC 50 of 20nM or less, lOnM or less, 5nM or less, 4nM or less, 3nM or less, 2.5nM or less, as determined by phospho-Akt ELISA assay described in Example 5-D of the present disclosure.

[0097] In one embodiment, the bispecific binding molecules disclosed herein may have superior effect (such as vascular leaking inhibition capability, leaky vessels regression and longer duration) in comparison with aflibercept and/or ANG2 antibody such as Ab536.

[0098] Description of the Production/Manufacturing [0099] The bispecific binding molecules disclosed herein are produced by techniques used for producing multispecific antibodies or recombinant proteins, which include, but are not limited to, plasmid preparation, cell culture, transient transfection, purification and analysis.

[0100] 1. Plasmid Preparation

[0101] Target DNA sequence was designed, optimized and synthesized (Genwiz). The synthesized sequence was sub-cloned into pcDNA3.4 vector. Then, transfection grade plasmids were maxi-prepared for Expi HEK 293F (Thermo fisher Scientific) cell expression.

[0102] 2. Cell Culture and Transient Transfection

[0103] Expi HEK 293F cells were grown in serum-free HD 293F™ Expression

Medium (Thermo Fisher Scientific) and the grown cells were maintained in Erlenmeyer Flasks (Coming Inc.) at 37°C with 8% CO2 on an orbital shaker(VWR Scientific). Then, one day before transfection, the cells were seeded at an appropriate density in Coming Erlenmeyer Flasks. On the day of transfection, DNA and transfection reagent were mixed at an optimal ratio and added into the flask with cells ready for transfection. The recombinant plasmids encoding target antibody were transiently co-transfected into a suspension of HD293F cell cultures. The cell culture supernatants collected on day 6 from the' transfection were used for purification.

[0104] 3. Purification and Analysis

[0105] Cell culture broth was centrifuged and filtered. Filtered cell culture supernatant was loaded onto the MabSelect SuRe™ LX (GE Healthcare Life Sciences) at an appropriate flowrate. After washing and elution with appropriate buffers, the eluted fractions were pooled and buffer exchanged to PBS 7.2. To improve the purity, the desalting sample was pooled, concentrated and loaded onto HiLoadl 6/600 Superdex200pg 320ml column. After SEC purification, the target fractions were pooled and loaded onto ion-exchange chromatography column at an appropriate flow rate to reduce endotoxin level and subsequently concentrated. The purified protein was analyzed by SDS-PAGE, Western blot, HPLC analysis to determine the molecular weight and purity. The concentration was determined by A280 method.

[0106] The binding specificity and affinity of the bispecific binding molecule of the present invention can be determined by assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA) as well as Biacore (GE Healthcare, Chicago, IL) and mass spectroscopy (MS, CovalX).

[0107] The present disclosure also includes DNAs encoding bispecific binding molecules, which can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. The inserted protein-coding sequence may be operatively linked to an expression control sequence. A variety of host-vector systems may be utilized to express the protein-coding sequence in host cells. For example, mammalian cells (e.g., CHO cell), insect cells, yeast cells, and bacterial cells can be used as host cells. These host-vector systems include mammalian cell systems infected with a virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with a virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA. Depending on the host-vector system utilized, any one of suitable transcription and translation elements may be used.

[0108] The present disclosure also includes bispecific binding molecules obtained by growing cells of the host-vector system under conditions suitable for the production of the fusion polypeptide and recovering the fusion polypeptide so produced.

[0109] The present disclosure also provides a pharmaceutical composition comprising the bispecific binding molecules disclosed herein for administration to a subject. The pharmaceutical compositions disclosed herein may further include a pharmaceutically acceptable carrier, excipient, or diluent. As used herein, the term "pharmaceutically acceptable" means that the composition is sufficient to achieve the therapeutic effects without deleterious side effects, and may be readily determined depending on the type of the diseases, the patient's age, body weight, health conditions, gender, and drug sensitivity, administration route, administration mode, administration frequency, duration of treatment, drugs used in combination or coincident with the composition disclosed herein, and other factors known in medicine.

[0110] The pharmaceutical compositions comprising the bispecific binding molecules disclosed herein may be administered locally, regionally or systemically, such as, for example, administration by subcutaneous, subcutaneous, intravitreal, intradermal, intravenous, intra-arterial, intraperitoneal or intramuscular injection. In the case of intraocular injection, the pharmaceutical compositions comprising the bispecific binding molecules disclosed herein may be administered intravitreally or subconjunctivally.

[0111] The pharmaceutical compositions may comprise from about 0.01 mg/mL to about 500 mg/mL of the bispecific binding molecules disclosed herein.

[0112] The pharmaceutical compositions comprising the bispecific binding molecules disclosed herein may further include a pharmaceutically acceptable carrier. For injectable preparations, the carrier may include a buffering agent, a preserving agent, an analgesic, a solubilizer, an isotonic agent, a diluent, and a stabilizer. For preparations for topical administration, the carrier may include a base, an excipient, a lubricant, a stabilizer and a preserving agent.

[0113] Further, the pharmaceutical composition comprising the bispecific binding molecules disclosed herein may be formulated into a single dosage form suitable for the patient's body, and preferably is formulated into a preparation useful for protein drugs according to the typical method.

[0114] The pharmaceutical composition comprising the bispecific antibodies disclosed herein is expected to have in-vivo longer duration of efficacy and therapeutic level of the bispecific antibody titer, thereby remarkably reducing the number and frequency of administration thereof

[0115] The administration dose and frequency of the pharmaceutical composition comprising the bispecific binding molecules disclosed herein are determined by the type of active ingredient, together with various factors such as the disease to be treated, administration route, patient's age, gender, and body weight, and disease severity.

[0116] Methods of treatment

[0117] The bispecific binding molecules disclosed herein may be used to inhibit angiogenesis. The bispecific binding molecules disclosed herein may be used to improve and/or treat clinical conditions selected from angiogenesis, vascular permeability, edema, and inflammation. The bispecific binding molecules disclosed herein may be used to treat any angiogenesis-mediated disorders or diseases, including, without limitation, chronic wounds, peripheral arterial disease, ischemic heart disease, chronic inflammation such as rheumatoid arthritis, malignant tumors, diabetic retinopathy, and atherosclerosis.

[0118] The bispecific binding molecules disclosed herein may be used to treat any angiogenesis-mediated ocular disorders or diseases, including, without limitation, cancer, proliferative retinopathies, proliferative diabetic retinopathies, age-related macular degeneration (AMD), macular edema, choroidal neovascularization (CNV), vascular glaucoma, geographic atrophy (GA), retinal vein occlusion, retinopathy of prematuriy (ROP), corneal neovascularization. The bispecific binding molecules disclosed herein also may be used to treat any angiogenesis-mediated ocular disorders or diseases, including, macular edema following retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), neovascular (Wet) age-related macular degeneration, dry AMD, diabetic macular edema (DME), diabetic retinopathy (DR) in patients with DME, and neovascular age-related macular degeneration, AMD with subfoveal choroidal neovascularization, myopic choroidal neovascularization, and neovascular glaucoma.

C. Bispecific Binding Molecules Assays

[0119] Molecular assays were developed to assess the bispecific binding molecules’ affinity and specificity to Ang-2 and VEGF, and the effect on the Ang-2:Tie2 interaction and VEGF:VEGFR interaction. Functional assays were developed to assess the cells viability treated by the bispecific binding molecule and the effect of the bispecific binding molecule on angiopoietin-2 induced Tie-2 phosphorylation and the effect of the bispecific binding molecule on angiopoietin-2 mediated tube formation and the effect of the bispecific binding molecule by IVT injection in the persistent retinal neovascularization rabbit model.

[0120] The test samples used in the following Examples are shown below unless otherwise noted.

[0121] Table 1

[0122] BSP1 or BSP2 was synthesized and expressed in mammalian HEK 293F cell through pay per service contract research organization (GenScript Biotech). Once a target antigen was determined, codons for the bispecific binding molecule were optimized based on the target antigen sequence using computer programing. After the correct protein sequence was confirmed, the optimized gene was cloned into mammalian expression vector pcDNA3.4. Thereafter, a supercoiled, low endotoxin transfection grade plasmid was prepared, and the bispecific binding molecule was expressed in the HEK 293F cell. The expressed bispecific binding molecule was purified via one step protein A purification, western blot (WB), high performance liquid chromatography (HPLC), sodium dodecylsulfate-polyacrylamide gel electrophoretic system (SDS-PAGE), and size exclusion chromatography (SEC), and endotoxin was removed. Control 1 and Control 2 were synthesized as below.

[0123] Target DNA sequence of Control 1 (SEQ ID No.l and SEQ ID No.2) and Control 2 (SEQ ID No.6) were synthesized by Genwitz with reference to US75721053B and W02000/075319. The synthesized sequence was sub-cloned into pcDNA3.4 vector. Then, transfection grade plasmids were maxi-prepared for Expi HEK 293F (Thermo fisher Scientific) cell expression. Expi HEK 293F cells were grown in serum-free HD293™ Expression Medium (Thermo Fisher Scientific) and the grown cells were maintained in Erlenmeyer Flasks (Coming Inc.) at 37°C with 8% CO2 on an orbital shaker (VWR Scientific). Then, one day before transfection, the cells were seeded at an appropriate density in Coming Erlenmeyer Flasks. On the day of transfection, DNA and transfection reagent were mixed at an optimal ratio and added into the flask with cells ready for transfection. The recombinant plasmids encoding target antibody were transiently co-transfected into a suspension of HD293F cell cultures. The cell culture supernatants collected on day 6 from the transfection were used for purification. Cell culture broth was centrifuged and filtered. Filtered culture supernatant was loaded onto the MabSeloct SuRe™LX (GE Health Life Sciences) at an appropriate flowrate. After washing and elution with appropriate buffers, the eluted fractions were pooled and buffer exchanged to PBS 7.2. To improve the purity, the desalting sample was pooled, concentrated and loaded onto HiLoad 16/600 Superdex200pg 320ml column. After SEC purification, the target fractions were pooled and loaded onto ion-exchange chromatography column at an appropriate flow rate to reduce endotoxin level and subsequently concentrated. The purified protein was analyzed by SDS-PAGE, Western blot, HPLC analysis to determine the molecular weight and purity. The concentration was determined by A280 method.

[0124] EXAMPLE 1

[0125] Binding Specificity

[0126] The binding specificity of the present invention can be determined by in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).

[0127] Methods

[0128] ELISA setting is shown in Figure 2.

[0129] VEGF Coating

[0130] For the VEGF binding assay, a carbonate buffer was first created by dissolving a carbonate-bicarbonate buffer capsule (Sigma Aldrich (Lot # SLBS9151)) in lOOmL of milli-Q water. rhVEGF (100 pg/mL; Peptro Tech) was diluted with the carbonate buffer (50mM, pH 9.6) to make a 1 pg/mL solution. MaxiSorp plate (96-well plate; Thermo Scientific (Lot # 155467)) was coated with 100 μL of 1 pg/mL rhVEGF165, sealed with a plate sealer, and incubated at 4°C overnight (16-24 hrs).

[0131] Tie2-Fc coating (for Ang2) [0132] For the Ang2 binding assay, Tie2-Fc (200 pg/mL, 55 μL/tube; Fitzgerald

Industries) was diluted with PBS (Wako Lot # (KCP7032)) to achieve a concentration of 1 pg/mL, and the MaxiSorp plate (96-well plate; Thermo Scientific (Lot # 155467)) was coated 100 μL of 1 pg/mL Tie2-Fc, sealed with a plate sealer, and incubated at 4°C for overnight (16-24 hrs).

[0133] Buffer Preparation [0134] The wash buffer PBS-T was prepared by dissolving 2 tablets of PBS-T (Takara

Bio (Lot # SLBS9251)) in 2000 mL milli-Q water. The blocking solution was prepared by diluting 10% BSA (Secare/PBL (Lot# 10385708)) 10-fold with the PBS-T, sterilized by filtration through a 0.22 pm membrane, and stored at 4°C. The diluent was prepared by mixing the blocking solution 10-fold with the PBS-T

[0135] Analyte preparation

[0136] Serial dilutions for each sample were prepared in Protein LoBind plate

(Eppendorf) as outlined below.

[0137] Table 2

[0138] Blocking

[0139] The plates were washed three times with 300 μL/well PBS-T, and blocked with

200 μL of BSA/PBS-T. They were sealed with a plate sealer, and incubated at room temperature for 1 hr.

[0140] Ang2+Analyte

[0141] The FLAG-tagged ligands (Ang2-FLAG, 30 pg/mL, 30 μL/tube; BPS

Bioscience) were diluted with diluents as shown in table6. After 6-8 minutes of blocking, 200 μL of Ang2-FLAG (20 ng/mL) was diluted 2-fold with 200 μL of analyte in Protein LoBind plate (Eppendorf), and incubated at room temperature for 1 hr on a plate shaker (200 rpm).

[0142] Table 3

[0143] Analyte application

[0144] After the plates were washed three times with Wash Buffer (300 μL/well PBS-

T), 100 μL of standards and samples was added to the plates, sealed with a plate sealer, and incubated at room temperature on a plate shaker (500 rpm) for Ihr.

[0145] For VEGF detection antibody, anti-human IgG Fc-HRP stock (0.8 mg/mL;

Jackson Immuno Research) was prepared as outlined below to be used within Ihr of preparation.

[0146] Table 4

[0147] For anti-Ang2 detection antibody, anti-FLAG Tag stock (Mouse-Mono(M2),

HRP, 50 pg/mL, 20x; SIGMA) was prepared as outlined below to be used within Ihr of preparation.

[0148] Table 5

[0149] 1-Step Ultra TMB ELISA (Thermo Scientific) was removed from storage and equilibrated to room temperature in the dark.

[0150] After the plates were washed three times with Wash Buffer (300 yL/well (PBS- T)), 100 μL of the above prepared detection antibody was added to each well, and the plate was covered with a plate sealer and incubated on a plate shaker (500 rpm, Ihr, RT).

[0151] The plate was again washed with Wash Buffer (300 μL/well x 5), and 100 μL of 1-Step Ultra TMB ELISA was added to each well. Then, the plate was covered with a plate sealer and incubated in the dark (RT) for 5-15 min. After the incubation, 100 μL of stop solution (IM phosphoric acid; Signal Aldrich) was added to each well. Finally, within 5 minutes of adding the stop solution, Benchmark Plus (BioRad) was used to analyze the plate according to the below specification.

[0152] Table 6

[0153] Results [0154] The results are shown in Table 7 and Figures 3(a) and 3(b).

[0155] As shown in Table 7, BSP2 blocked the interaction between Ang-2 and its receptor Tie-2 with an IC50 at 23.8 pM, whereas Control 1, an ANG2 mAb, blocked the interaction between Ang-2 and its receptor Tie-2 with an IC50 at 40.3 pM. BSP2 bound to VEGF with an EC50 at 101.1 pM, whereas Control 2, a VEGF inhibitor, bound to VEGF with an EC50 at 81.0 pM.

[0156] Table 7

[0157] EXAMPLE 2

[0158] Binding Affinity Assay

[0159] The binding affinity of the present invention can be determined by Biacore (GE Healthcare, Chicago, IL).

[0160] Method

[0161] The assay was performed at 25°C and the running buffer was HBS-EP (10 mM HEPES, 150 mM NaCI, 3 mM EDTA, 0.05% Tween 20, pH 7.4; GE Healthcare (Lot. No. BCBV7062)). Diluted antibodies were injected over the surface as capture phase, diluted antigen were injected over the surface as association phase, followed by injecting running buffer as dissociation phase.

[0162] An affinity analysis of bispecific binding molecule (BSP1) was performed on a Biacore 8K, 29215379-2177839 (GE Healthcare, Chicago, IL) with PBS and 0.005 percent P20 surfactant (GE Healthcare, Chicago, IL.) as running buffer according to the manufacture’s recommended protocol. Binding assays were carried out by first attaching around 100 Ru of BSP1 to the Series S Sensor Chip coated with Protein A, then various concentration of human recomb inant- VEGF protein (R&D systems, 0-1 OnM) or human recombinant Ang2 protein (R&D systems, 0-1 OOnM) will inject over the BSP bound surface at flow rate of 30 (Ang2) or 100 (VEGF) ul/min for 120 seconds. Antibody binding kinetics including ka (association rate constant), kd (dissociation rate constant) and KD (dissociation equilibrium constant) were determined by using Biacore 8K evaluation software version 1.1 (Chicago, IL.). lOmM Glycine-HCl PH 1.5, (Lot. No. 20181221) was used to dissociate bound analyte from the sensor chip after sample injection.

[0163] Results [0164] The binding affinity of BSP 1 against ANG2 was measured twice at different time points, and BSP1 had a Kd of 139 pM and 230 pM, respectively (See Figure 5). BSP1 had a Kd of 55pM against VEGF.

[0165] BSP2 and Control 1 (anti-Ang2 mAb) had Kd of 254 and 316pM against Ang2, respectively. BSP2 and Control 2 (anti-VEGF) had Kd of 187 and 187pM, respectively against VEGF (See Figure 4, 5). Table 8 shows the results of the binding affinity against ANG2 and VEGF of the present invention.

[0166] Table 8

[0167] EXAMPLE 3 [0168] Assays for binding affinity between Ang2/Tie2 and Ang2/BSP2 blocking Ang2/Tie2 complex formation

[0169] The binding affinity of Tie2 to Ang2 after being blocked by the present invention can be determined by Biacore.

[0170] Method

[0171] This study was performed using Biacore 8K (GE Healthcare, Chicago, IL) to measure the binding affinity of Tie2 to Ang2 after being blocked by an antibody. The immobilization of human recombinant Ang2 protein (R&D Systems) was performed under 25 degrees Celsius while HBS-EP was used as the running buffer. The sensor chip surface of flow cells 1 and 2 were activated by freshly mixed 50 mmol/L N-Hydroxysuccinimide (NHS) and 200 mmol/L l-ethyl-3 -(3 -dimethylaminopropyl) carbodiimide hydrochloride (EDC) for 200s (10 μL/min). Afterwards, Ang2 diluted in 30 mmol/L NaAC (pH 4.5) was injected into the flow cell 2 to achieve conjugation of appropriate Response Unit respectively, while one of the flow cell 1 was set as blank. After the amine coupling reaction, the remaining active coupling sites on chip surface were blocked with 200s injection of 1 mol/L ethanolamine hydrochloride.

[0172] The assay was performed at 25°C and the running buffer was HBS-EP. lOOnM BSP1, Control 1 were injected over the surface of the flow cell 2 for 60s. Then, diluted recombinant Tie2-Fc fusion protein (R&D systems) was injected over the surface of the flow cells 1 and 2 as association phase, followed by injecting running buffer as dissociation phase. Single-cycle kinetics model was used, where analyte concentrations are injected sequentially in a single cycle, with no regeneration between injections. Single cycle model was used to determine binding affinity and interaction between Ang2 /Tie2-Fc and (Ang2+BSP2)/Tie2-Fc.

[0173] Results [0174] As shown in Table 9, Ang2 had a Kd of 4.26 nM for Tie2-Fc. Once Ang2 bound to BSP2, it was no longer be able to bind to Tie2-Fc to form the Ang2-Tie2 complex. Control 1 also produced the same result as BSP2.

[0175] Table 9

[0176] EXAMPLE 4

[0177] Simultaneous Binding of VEGF and ANG2

[0178] Materials and Methods

[0179] Instrumentation

[0180] For the integrity/aggregation test, the measurements were performed using an Autoflex II MALDI ToF ToF mass spectrometer (Bruker) equipped with CovalX’s HM4 interaction module. CovalX’s interaction module contains a special detecting system designed to optimize detection up to 2MDa with nano-molar sensitivity.

[0181] 1 p.1 of each dilution obtained was mixed with 1 pl of a matrix composed of a re-crystallized sinapinic acid matrix (10 mg/ml) in acetonitrile/water (1 :1, v/v), TFA 0.1% (K200 MALDI Kit). After mixing, 1 pl of each sample was spotted on the MALDI plate (SCOUT 384).

After crystallization at room temperature, the plate was introduced in the MALDI mass spectrometer and analysed immediately in High -Mass MALDI mode. The analysis has been repeated in triplicate.

[000182] Cross-link Experiments [0183] The cross-linking experiments allow the direct analysis of non-covalent interaction by High-Mass MALDI mass spectrometry. By mixing a protein sample containing non- covalent interactions with a specially developed cross-linking mixture (Bich, C et al. Anal. Chem., 2010, 82 (1), pp 172-179), it is possible to specifically detect non-covalent complex with high- sensitivity. The covalent binding generated allows the interacting species to survive the sample preparation process and the MALDI ionization. A special High-Mass detection system allows characterizing the interaction in the High-Mass range.

[0184] Each mixture prepared for the control experiment (9 pl left) was submitted to cross-linking using CovalX’s K200 MALDI MS analysis kit. The protein solutions (9 pl left) (from 1 to 1/128) were mixed with 1 pl of K200 Stabilizer reagent (2 mg/ml) and incubated at room temperature. After the incubation time (180 minutes) the samples were prepared for MALDI analysis as for Control experiments. The samples were analysed by High-Mass MALDI MS immediately after crystallization.

[0185] Target proteins, rhVGEF and rhAng2 were purchased from Sino Biological (PA, US) and PeproTech (NJ, US), respectively.

[0186] Table 10

[0187] Control Experiments

[0188] For this experiment, rhVEGF and BSP1 were detected with MH+= 39.338 kDa and MH+= 209.620 kDa. [0189] Table 11

[0190] Cross-Link Experiments

[0191] The cross-linking experiment was completed after 180 minutes incubation time with the cross-linking reagent. After cross-linking, one additional peak was detected with MH+= 249.593 kDa,.

[0192] Table 12

[0193] Control Experiments

[0194] For this experiment, rhAng2 and BSP2 were detected with MH+= 65.241 kDa and MH+= 210.432 kDa. [0195] Table 13

[0196] Cross-Link Experiments

[0197] The cross-linking experiment was completed after 180 minutes incubation time with the cross-linking reagent. After cross-linking, two additional peaks were detected with MH+= 287.904 kDa and MH+=352.067 kDa. [0198] Table 14

[0199] Control Experiments

[0200] For this experiment, rhVEGF, rhAng2 and BSP were detected with MH+=

39.801 kDa, MH+=64.451 kDa and MH+= 220.889 kDa.

[0201] Table 15

[0202] Cross-Link Experiments

[0203] The cross-linking experiment was completed after 180 minutes incubation time with the cross-linking reagent. After cross-linking, two additional peaks were detected with MH+= 254.016 kDa and MH+= 388.535 kDa.

[0204] Table 16

[0205] Results [0206] As shown in Tables 7, 9, and 11-16, which list the results obtained from the above described experiments, BSP2 as designed was capable of binding to one VEGF and two Ang2 molecules simultaneously. In other words, upon binding to one target (e.g., VEGF or ANG2), BSP1 was additionally capable of simultaneously binding to another target, confirming its bispecific binding.

[0207] Example 5

[0208] A. cellular functional assay for anti- VEGF

[0209] The binding affinity of the present invention can be determined by anti-VEGF cell proliferation functional assay.

[0210] Anti-VEGF growth inhibitory assay:

[0211] HUVEC cells (C2519A, Lonza, Basel, Switzerland) were cultured and maintained in T75 NUNC easy flaks (Cat. No. 156499, Thermo Scientific, MA, USA) with endothelial growth medium (Cat. No. cAP-02, Angio-proteomie, MA, USA) until 80% confluency at 37 °C in a humidified incubator (NUAIRE, NU-5831E, MN, USA) containing 5% CO2 and 95% air. HUVEC cells passage from P2 to P7 were used for the assays (P2 and P7 refers to Passage numbers 2 and 7, respectively). Cells were lifted by using Accutase (Cat 12679-54, Naclai tesque, Kyoto, Japan) and seeded 5 x 10³ cells per well of a flat bottom 96-well plate (Cat 3596, Coming, NY, USA) in 70 pl of the medium which was made of 1:3 mixture of endothelial cells growth medium (Cat. No. cAP-02, Angioproteomie, MA, USA) and endothelial cell serum-free defined medium without phenol red (113PR-500, Sigma-Aldrich, USA). Cells were incubated for overnight.

[0212] Materials and method: A medium containing following components was prepared freshly each time for 4 units of 96-well plates. 40 mL of medium 113PR-500 (Sigma Aldrich), 400 μL of FBS (Cat: F4135, Sigma Aldrich), 400 μL of ECGS (Cat: 356006, Coming), 400 μL of Heparin stock 2mg/ml (Cat: 07980, Stemcells), FBS (Cat: F4153, Sigma Aldrich), 200 μL of Hydrocortisone stock 96ug/ml (Cat: 07925, Stemcells), 500 μL of L-Ascorbic (Cat: A7631, Sigma). The medium in the 96-well plate incubated overnight was aspirated gently and replaced with 70 uL per well of the freshly prepared the medium above. The cells in each well were treated by topping up with 15uL each of the recombinant human VEGF-165 (Stemcell Technologies, Cat#78073) at a final concentration of 5 ng/ml in all the wells except the control wells. The antibodies (BSP2 and Control 2) were prepared in serial dilutions accordingly as stocks required (15 μL per well x n) in order to reach desired final concentrations later in wells with total medium volume 100 μL. BSP2 and Control 2 treated wells with HUVEC cells in 85 μL of medium plus VEGF were topped up 15 μL each of respective serially diluted antibody solution. VEGF only treated wells with HUVEC cells in 85 μL of medium were topped up 15 μL each of medium alone. Untreated control wells with HUVEC cells in 70 μL of medium were topped up 30 μL each medium alone and incubated the 96-well plate for 3 days at 37 °C in a humidified incubator containing 5% CO2 and 95% air. Each well was added 10 uL each of WST 8 (CCK-8, Dojindo, Japan) per well was added and incubated for 4 hrs followed by measuring the absorbance at 450 nm wavelength by using infinite M200 (Tecan, Switzerland). IC50 doses together with concentration response curves were determined by GraphPad prism software (San Diego, CA, USA).

[0213] Results: The results are shown in Table 17 and Figure 6.

[0214] Table 17: Comparison of endpoint average IC50 of BSP2 and Control 2 from nine independent experiments each was shown.

[0215] B. cellular functional assay for anti-Ang2

[0216] The study aims to evaluate the efficacy of compounds against Ang2-mediated tube formation in Human Umbilical Vein Endothelial Cells (HUVEC).

[0217] Materials and Method

[0218] Tube formation assay can be used to model the reorganization stage of angiogenesis. The assay measures the plated sub-confluent endothelial cells to form tube like structures with the extracellular matrix support. It is an assay used to evaluate the ability of various compounds to promote or inhibit tube formation. Compounds that are able to inhibit tube formation could be useful in various diseases studies. The principal of tube formation is upon endothelial cells growth, the cells attach and generate mechanical forces on the surrounding extracellular support matrix. These forces can create tracks to facilitate cell migrating along, in turn these cells will form tube structure.

[0219] Materials: EGM™-2 Endothelial Cell Growth Medium-2 BulletKit™ (Cat: CC-3162, Lonza), Human Umbilical Vein Endothelial Cells (Cat: C2519A, Lonza), Recombinant Human Angiopoietin-2 (rhAng2) (Cat: 623-AN/CF, R&D systems) and Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Cat: A1413202, Thermo Fisher Scientific) were used.

[0220] Method: HUVEC were resuspended in EGM-2 medium without fetal bovine serum (FBS) and VEGF. 10,000 cells/well were seeded in 96-well plate on 50pl of Basement Membrane Extract (BME; GeltrexTM, Thermo Fisher Scientific) in the presence of rhAng2 and increasing dose of either BSP2 or Control 1. (Sample concentration used during individual experiments were shown in Table 22). Phase contrast images were acquired 6 hrs after treatment. [0221] Table 22.

[0222] Results: Total tube length was measured using an image analysis software (Image!; Angiogenesis Analyzer plugin). Each measurement was normalized against the tube length difference induced by rhAng2 in the presence of highest and lowest dose of BSP2. IC50 was analyzed based on the normalized total length for each experiment using a scientific graphing and biostatistics software (GraphPad Prism). In this study, IC50 was analyzed to measure the inhibitory efficacy of BSP2 and control 1 against rhAng2-mediated tube formation relative to each other. The concentration-response curve for BSP2 and Control 1 based on the averaged response of three independent experiments were shown in Figure 7. Average IC50 value were shown in Table 23.

[0223] Table 23.

[0224] C. cellular functional assay for anti-Ang2 function of bispecific antibody by assessing Tie-2 phosphorylation [0225] The study aims to investigate the selective inhibitory effect of the bispecific antibody, on Angiopoietin-2 signaling by determining phosphorylation status of Tie-2, an Angiopoietin-2 receptor in HUVEC cells after Angiopoietin-2 stimulation. Analysis of phosphorylation is done by western blot. Besides the bispecific antibody, Control 1 (Anti-Ang2 Ab536) is used as a positive control and Control 2 (Aflibercept) is used as a negative control.

[0226] Materials and Methods

[0227] Under normal physiological conditions, in absence of Angiopoietin-1, Angiopoietin-2 acts as an agonist of the Tie-2 receptor. The effect of the bispecific antibody on the phosphorylation status of Tie-2 was captured by changes in its protein levels by western blots.

[0228] Drug substance

[0229] Sample and reagent preparation

[0230] Reagents

[0231] Instruments

[0232] Cells

Human Umbilical Vein Endothelial Cells

Name: (HUVEC)

Source: Kurabo Industries LTD

The number of passage

upon receipt:

The number of passage at

the study start:

[0233] Culture condition

[0234] Temperature and CO2 concentration are as follows: - 37 °C, 5% CO2/95% air. [0235] Composition and preparation of the growth media - 5 mL of Penicillinstreptomycin (10,000 U/mL) was added to CS-C complete medium. Immediately before use, 1/1000 volume of 10 pg/mL FDF1 were added to prepare complete media.

[0236] Method

[0237] Experimental design

[0238] 1. Schedule

[0239] 2. Study group

[0240] The test substances were grouped and measured according to the table below. For BSP2, the same sample was measured at two points and labeled as Sample 1 and Sample 2, respectively.

[0241] 3. Rationale for the concentration of the test substances [0242] The dose range for BSP2 titration was set based on the reported IC50 values of Ab536. As a negative control, 1 pM, an excess concentration of Control 2, was set for this assay to negate any influence of anti-VEGF activity on the assay. As a positive control in this assay, the titration of Control 1 was set based on the reported IC50 value of anti-ANG2 antibody known for Ab536.

[0243] Experimental Procedure

[0244] Cryorecovery

[0245] Cryopreserved HUVEC cells were thawed in water bath at 37 °C. The cell suspension and 5.5 mL of growth media supplemented with FGF1 were mixed and the cells were seeded in Collagen I-coated 100 mm dish. The cells were cultured for 1 day in CO2 incubator. On the next day, the media was changed to fresh growth media supplemented with FGF1.

[0246] Cell passage

[0247] Upon reaching sub-confluent, the media was discarded and the cells were rinsed with PBS (-) twice. 0.05% Trypsin-EDTA was added and the culture dish was placed in CO2 incubator until the cells were detached. The detached cells were suspended in 3.5 mL of growth media. The cells were centrifuged at 230xg, RT, for 5 min. Supernatant was discarded and re-suspended in 1 mL of growth media. 10 μL of cell suspension and 10 μL of 2 x trypan blue solution were mixed and the number of the cell with Countess II automated Cell Counter was measured. Cell suspension was diluted toward 2.5-5.0 x 10³ cell/cm² with growth media based on the result of cell count. 10 mL of the prepared cell suspension supplemented with FGF1 was added into Collagen I-coated 100 mm dish. The cells were cultured in CO2 incubator.

[0248] Cell seeding for the assay [0249] The cell suspension was obtained and cell density of the cell suspension was adjusted to 5 x 10⁵ cell/mL with growth media. 1 mL of the prepared cell suspension was added into 6-well plate. 1 mL of growth media supplemented with FGF1 (5xl0⁵ cells/well) was added.

The cells were cultured in CO2 incubator for overnight.

[0250] Drug instillation

[0251] Antibodies and angiopoietin-2 were mixed and incubated at 37 °C for 15 min.

Old media was aspirated and replaced with the Ab/Angiopoietin-2 solution. The solution was incubated at 37 °C for 15 min.

[0252] Cell harvest and cell lysate preparation

[0253] Lysis buffer with sample diluent Concentrate 2 (2x), distilled water and protease inhibitor cocktail were prepared. 1 mM sodium orthovanadate I PBS (+) with sodium orthovanadate, D-PBS (+), and PBS (-) were prepared. Antibody/Angiopoietin-2 solution was aspirated after the incubation and the cells were rinsed with 1 mM Sodium Orthovanadate / PBS (+). The cells were removed from the vessel by scraping and the cells were harvested in 1.5 mL tube. The cells were centrifuged at 15,000 rpm, for 1 min, at 4 °C. Supernatant was discarded. 100 μL of lysis buffer was added and the tube was kept on ice for 15 min, vortexing occasionally. The buffer was centrifuged at 15,000 rpm for 1 min at 4 °C. Supernatant (cell lysate) was transferred to new tube and it was stored at -30 °C

[0254] Sample preparation for SDS-PAGE

[0255] 1/10 vol. of 2-Mercaptoethanol was added to 4 x Leammli Sample Buffer 4. 3 vol. of 4 x Leammli Sample Buffer was added to each 1 vol. of the samples. Heated them at 95 °C for 5 min. The samples were kept on ice. SDS-PAGE was performed. 10 x Tris I Glycine / SDS Buffer was diluted with ddWater to prepare 1 x Tris / Glycine / SDS Buffer. Mini-PROTEAN TGX Gel (7.5%) was set to Mini-PROTEAN Tetra System. Electrophoresis tank was filled with 1 x Tris / Glycine / SDS Buffer. The samples and precision plus western standard were applied. Electrophoresis was performed at 150 V.

[0256] Western Blot

[0257] 10 x Tris / Glycine Buffer, methanol, and ddWater (1:2:7) were mixed to prepare Towbin Buffer. Immobilon-P membrane was hydrophilized with methanol. The fiber pad, the gel, the hydrophilized-membrane, and the filter paper were soaked in Towbin Buffer. The fiber pad, filter paper, gel, the membrane were placed on the cassette. The cassette was set to the electrophoresis tank. Electrophoresis was performed at 100 V for 45 min. The membrane was blocked with PVDF Blocking Reagent for Can Get Signal for Ihr. The membrane was washed with TBS-T three-time. 1st antibody reaction solution with Can get signal immunoreaction enhancer solution 1 was prepared. The membrane was soaked in 1^st antibody reaction solution at 4°C for overnight. The membrane was washed with TBS-T three-time. 2^nd antibody reaction solution with Can get signal immunoreaction enhancer solution 2 was prepared. The membrane was soaked in 2^nd antibody reaction solution at R.T. for 1 hr. The membrane was washed with TBS-T three-time. The membrane was soaked in Chemi-Lumi One Super prepared immediately before the detection. ECL was detected with ChemiDoc™ XRS. Stripping & Western Blot. The membrane was rinsed with TBS-T. The membrane was soaked into warmed-westem blot stripping buffer at 37 °C for 10-15 min. The membrane was rinsed with TBS-T. Western blot was performed for another antibody according to aforementioned Western Blot protocol.

[0258] Antibodies used in the study

[0259] Data analysis

[0260] Analysis items - Membrane images obtained with molecular imager

(ChemiDoc™ XRS+)

[0261] Analysis method - Contrast adjustment was performed with Molecular Imager.

[0262] Result: Result is shown in Figure 8.

[0263] Conclusion

[0264] In this study, western blot analysis was performed to investigate the selective inhibitory effect of the novel bispecific antibody on angiopoietin-2-induced Tie-2 phosphorylation in HUVEC.

[0265] As shown in Figure 8, upregulation of Tie-2 phosphorylation by Angiopoietin-

2 stimuli in HUVEC was confirmed and Tie-2 phosphorylation was inhibited by the addition of the novel bispecific antibody. In conclusion, the study shows that the novel bispecific antibody can suppress the phosphorylation of Tie-2 by inhibiting the interaction between Angiopoietin-2 and Tie-2.

[0266] D. cellular functional assay for anti-Ang2 function of bispecific antibody by assessing Akt phosphorylation

[0267] Purpose [0268] In this study, effect of the bispecific antibody on angiopoietin-2 induced Akt phosphorylation in HUVEC was examined with phospho-Akt ELISA assay.

[0269] Materials and Method

[0270] Drug Substance

[0271] Sample and reagent preparation

[0272] Reagents

[0273] Instruments

[0274] Cells

Name: Human Umbilical Vein Endothelial Cells (HUVEC)

Source: Kurabo Industries LTD

The number of passage upon receipt: 2

The number of passage at the study start: 6

[0275] Culture condition

[0276] Temperature and CO2 concentration are as follows: 37 °C, 5% CO2/95% air [0277] Composition and preparation of the growth media: 5 mL of Penicillinstreptomycin (10,000 U/mL) was added to CS-C complete medium. Immediately before use, 1/1000 volume of 10 pg/mL FDF1 was added to prepare complete media

[0278] Method

[0279] Experimental design

[0280] 1. Schedule

[0281] 2. Study group

[0282] The test substances were grouped and measured according to the table below.

For BSP2, the same sample was measured at two points and labeled as Sample 1 and Sample 2, respectively.

[0283] 3. Rationale for the concentration of the test substances

The dose range for BSP2 titration was set based on the reported IC50 values of Ab536. As a negative control, 1 pM, an excess concentration of Control 2, was set for this assay to deny any influence of anti-VEGF activity on the assay. As a positive control, the titration of Control 1 was set based on the reported IC50 value of anti-ANG2 antibody known for Ab536.

[0284] Experimental Procedure

[0285] Cryorecovery and Cell passage

[0286] HUVEC cells were cryopreserved and prepared using the same procedure as Example5 -C

[0287] Cell seeding for the assay

[0288] For Akt ELISA, collected the prepared HUVEC cells and cell density of the cell suspension was adjusted toward 5 x 10⁵ cell/mL with growth media and 1 mL of the prepared cell suspension was added into 6-well plate. Then, 1 mL of growth media supplemented with FGF1 (5xl0⁵ cells/well) was added to the well. The cells were cultured in CO2 incubator for overnight

[0289] Drug instillation

[0290] Antibodies and angiopoietin-2 were mixed and incubated at 37 °C for 15 min.

Old media was aspirated and replaced with the Ab/Angiopoietin-2 solution. The solution was incubated at 37 °C for 15 min. [0291] Cell harvest and cell lysate preparation

[0292] 1 x Cell extraction buffer with 5 x Cell extraction buffer, 50 x Cell extraction buffer and protease cocktail were prepared. The cells were rinsed with PBS (-).150 μL of lx Cell Extraction Buffer was added in each well of 6-well plate. The cell suspension was harvested to 1.5 mL tube and sonicated on ice in ultrasonic bath sonicator for 15 min. The suspension was centrifuge at 15,000 rpm for 1 min at 4 °C. Supernatant (cell lysate) was transferred to new tube and it was stored at -30 °C

[0293] ELISA

[0294] 50 μL of cell lysate was added to FastScan ELISA Microwell Plate. Akt (Pan)

Rabbit Capture Antibody and Phospho-Akt (Ser473) Rabbit HRP-Linked were mixed to prepare antibody cocktail. Antibody cocktail was added to each well. The plate was shaken at 400 rpm for 1 hr at R. T. The plate was washed with lx wash buffer three time. TMB substrate was added and the plate was shaken at 400 rpm for 15 min. at R. T. Stop solution was added to each well. Absorbance at 450 and 540 nm was measured.

[0295] Data analysis

[0296] Analysis method

[0297] The absorbance value of each group was represented by the bar graph and sigmoid curve. The sigmoid curve was created using "Quest Graph ™ Four Parameter Logistic (4PL) Curve Calculator," AAT Bioquest, Inc from the calculated values of each test concentration sample obtained, and the IC50 of each test substance was calculated. IC50 was calculated from the formula of the fitting curve drawn by Sample 1, Sample 2, and Control 1 titration.

[0298] Results

[0299] The results are shown in Tables 24 and Figure 9-12. [0300] Table 24: IC50 value (nM)

[0301] Conclusion

[0302] The aim of this study was to determine the inhibitory effect of the bispecific antibody on Akt phosphorylation. For that purpose, ELISA for phospho-Akt was performed to investigate the selective inhibitory effect of the bispecific antibody on Angiopoietin-2-induced Akt phosphorylation in HUVEC. Akt phosphorylation was dose-dependently inhibited by the addition of each two different batches of the novel bispecific antibody with IC50 value of 2.5 nM and 2.4 nM, respectively.

[0303] EXAMPLE6 Animal model for persistent retinal neovascularization

[0304] The purpose of this study is to evaluate the efficacy of BSP2 in comparison with aflibercept by IVT injection in the persistent retinal neovascularization (PRNV/DLAAA) rabbit model. The PRNV/DLAAA model simulates angiogenic retinal diseases in order to identify indications that may benefit from the drug or novel therapies of the diseases in human. Model induction, study protocol and vascular leakage evaluation were designed based on published article (A novel model of persistent retinal neovascularization for the development of sustained anti-VEGF therapies, Li, et al Exp Eye Res Res. 2018 Sep;174:98-106. doi: 10.1016/j.exer.2018.05.027.)

[0305] Materials and Methods:

[0306] Be brief, Dutch Belted rabbits were used in this study. For each animal, only one eye was used to establish the retinal injury model for characterization and the fellow eye was used as a control. For further drug testing, retinal injury was done on one eye. However, the compound was administered to both eyes to investigate the drug toxicity/effect with and without disease. A unique component of this protocol is the evaluation of ophthalmologic endpoints on conscious rather than on anesthetized animals. This removes potential interferences from anesthetic agents during the examinations, and moreover, increases the likelihood that animals will survive for what could amount to years of follow-up.

[0307] A, Slit lamp examination:

[0308] Slit-lamp examination is to reveal an important inflammatory reaction in anterior chamber of the rabbit’s eyes that received the treatment of the test articles.

[0309] The observations was performed by an ophthalmologist for all eyes immediately after the injections and follow up at the day 1 and day 3. If there are signs of inflammation, the follow up was continued based on the drug effects and durations.

[0310] The standardization of Uveitis Nomenclature (SUN) were applied in all our studies. Cells and flare in the anterior chamber were observed using a slit lamp beam of 1 X 1 mm in height and width, when thrown at an angle of 45-60°. Based on the finding, the inflammation can be categorized from 0 to 4+ grade.

[0311] B, Efficacy measurement/evaluation in Persistent Retinal Neovascularization (PRNV) Rabbit Model Instruments:

[0312] Fundus photography (FP): Fundus pictures taken serially are useful to monitor treatment response, inflammatory changes and the drug distribution. Fluorescein Angiography (FA): FA imaging location is same as color images. FA images are taken according to the following schedule: Optical coherence tomography (OCT): OCT is an established medical imaging technique that uses light to capture with micrometre-resolution three-dimensional images of the retinal and choroid structure, map and measure their thickness.

[0313] C, Standards of PRNV model for pharmacology study [0314] All animals enrolled in this study had persistent retinal neovascularization (PRNV) with the FA Leakage over 2 months prior to dosing. Neovascular angiographic leak area and intensity were defined by NaF angiography (0.5 mis, 10%, OCT7FA in3 fields, 55°). About 60- 70% of animals enrolled in this study had stable angiographic leak area > 2 disc diameters and leak severity of > score 2 (may difference with study purpose). Efficacy of the treatment and vascular regression quantification analysis was evaluated by the quantitative measurement of FA photography.

[0315] D, Safety: the observation and scoring for anterior segment inflammation after the dosing was recorded using slit lamp. Retinal toxic effects such as vitreous clouding and retinal cloudy was identified by fundus photography, OCT as well as FA during the study period in any of the treatment eyes.

[0316] E, Drug deposition and distribution: Drug deposition and distribution was referenced in vitreous. The formulation effects on the deposition and clearance of the injected drug was observed by photography of the color fundus camera for the all-time points post the dosing.

[0317] Results: Animal model compound treatment timepoints plot against vascular leakage percentage area (%) calculation based on FA images were shown Figure 13. Figures 14-28 are FA images per each group at week 0, 1, 12 and 24 for compound treated vascular leakage evaluation (OD) and compound treated non-DLAAA induced eye as control (OS).

[0318] As shown in animal study figures 14 to 28, BSP2 has demonstrated superior vascular leaking inhibition capability, leaky vessels regression and longer duration in comparison with Control 1 known VEGF inhibitor for aflibercept.

[0319] Sequences described herein are briefly explained in the table below. BRIEF DESCRIPTION OF SEQUENCES IN SEQUENCE LISTING

* : 2 substitutions from leucine to lysine at 235th and 309th positions of the IgGl in accordance with EU/Kabat SEQUENCES

SEQ ID NO: 1

EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIY YADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

SEQ ID NO: 2

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRAS

GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKLEIKRTVAAPSV

FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS

LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 3

EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIY

YADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGYWGQGT LVTVSS

SEQ ID NO: 4

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRAS

GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKLEIK

SEQ ID NO: 5

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRAS

GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKLEIK

GGGGSGGGGS

EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIY

YADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGYWGQGT LVTVSS

SEQ ID NO: 6

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKG

FIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNC

TARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLY

TCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE

VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL

NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP

SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

HNHYTQKSLSLSPG(K) SEQ ID NO: 7

(GGGGS)n,n=l-10

SEQ ID NO: 8

(GGGGS)n,n=4

SEQ ID NO: 9

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKG

FIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNC

TARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLY

TCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELKGGPSVFLFPPKPKDTLMISRTPE

VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVKHQDWL

NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP

SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG

SEQ ID NO: 10

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRAS

GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKLEIKRRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 11

EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIY

YADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGYWGQGT

LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

GGGGSGGGGSGGGGSGGGGS

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKG

FIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNC

TARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLY

TCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELKGGPSVFLFPPKPKDTLMISRTPE

VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVKHQDWL

NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP

SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

HNHYTQKSLSLSPG

SEQ ID NO: 12 SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKG FIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNC TARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLY TCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELKGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVKHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG

GGGGSGGGGSGGGGSGGGGS

EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIY YADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

SEQ ID NO: 13

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLlYLGSNRAS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKLEIK GGGGSGGGGS

EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIY YADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGYWGQGT LVTVSS

GGGGSGGGGSGGGGSGGGGS

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKG FIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNC

TARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLY TCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELKGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVKHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG

SEQ ID NO: 14

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKG FIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNC TARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLY TCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELKGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVKHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG

GGGGSGGGGSGGGGSGGGGS

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRAS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKLEIK GGGGSGGGGS EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIY

YADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGYWGQGT LVTVSS

SEQ ID NO: 15

EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIY

YADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGYWGQGT LVTVSS

GGGGSGGGGS

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRAS

GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKLEIK

GGGGSGGGGSGGGGSGGGGS

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKG

FIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNC

TARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLY

TCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELKGGPSVFLFPPKPKDTLMISRTPE

VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVKHQDWL

NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG

SEQ ID NO: 16

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKG

FIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNC

TARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLY

TCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELKGGPSVFLFPPKPKDTLMISRTPE

VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVKHQDWL

NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP

SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

HNHYTQKSLSLSPG

GGGGSGGGGSGGGGSGGGGS

EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIY

YADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGYWGQGT LVTVSS

GGGGSGGGGS

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRAS

GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKLEIK

SEQ ID NO: 17

GGGGSGGGGS SEQ ID NO: 18

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIW DSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSV GEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLT IDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHE

SEQ ID NO: 19

KDKTHTCPPCPAPELLGGPSVF

LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID NO: 20

TARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLY TCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE

VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG

SEQ ID NO: 21

AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID NO: 22

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRA

SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKLEIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 23

EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIY

YADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGYWGQGT LVTVSS

GGGGSGGGGS

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRAS

GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKLEIK

SEQ ID NO: 24

GFTFSSYGMHW

SEQ ID NO: 25

YISSSGSTIYYADSVKG

SEQ ID NO: 26

CARDLLDYDILTGYGYW

SEQ ID NO: 27

CRSSQSLLHSNGYNYLDW

SEQ ID NO: 28

YLGSNRASG

SEQ ID NO: 29

CMQGTHWPPT

Claims

CLAIMS A bispecific binding molecule comprising a polypeptide comprising an anti-angiopoetin2 (ANG2)-binding domain and a vascular endothelial growth factor (VEGF)-binding domain, wherein the anti-ANG2-binding domain is an ANG2 antibody or an antigen binding fragment thereof, wherein the VEGF -binding domain comprises a fusion protein which binds to VEGF polypeptide comprising an extracellular domain of VEGF receptor operatively linked to Fc domain, wherein the anti-ANG2-binding domain is a Fab fragment or a scFv fragment. The bispecific binding molecule of claim 1, the anti-ANG2-binding domain comprises an amino acid sequence of SEQ ID NO: 3 and/or an amino acid sequence of SEQ ID NO: 4, wherein the VEGF-binding domain comprises an amino acid sequence of SEQ ID NO: 18, wherein the Fc domain is IgGl . The bispecific binding molecule of claim 2, the anti -ANG2 -binding domain comprises an amino acid sequence of SEQ ID NO: 1 and/or an amino acid sequence of SEQ ID NO: 2, wherein the IgGl is mutated. The bispecific binding molecule of claim 2, wherein the Fc domain has an amino acid sequence with substitutions, deletions, insertions, and/or additions at, at least one site selected from 235th and 309th position of the IgGl in accordance with EU/Kabat numbering scheme. The bispecific binding molecule of claim 4, wherein the Fc domain has an amino acid sequence with substitutions at 309th position of the IgGl in accordance with EU/Kabat numbering scheme. The bispecific binding molecule of claim 5, wherein the Fc domain has an amino acid sequence with substitutions into K. The bispecific binding molecule of claim 4, wherein the Fc domain has an amino acid sequence with substitutions at 235th position of the IgGl in accordance with EU/Kabat numbering scheme. The bispecific binding molecule of claim 7, wherein the Fc domain has an amino acid sequence with substitutions into K. The bispecific binding molecule of claim 1 , wherein the VEGF-binding domain linked with the N-terminus of the ANG2 -binding domain. The bispecific binding molecule of claim 1 , wherein the ANG2-binding domain linked with the N-terminus of the VEGF-binding domain. The bispecific binding molecule of claim 2, wherein the anti-ANG2 -binding domain is a Fab fragment. The bispecific binding molecule of claim 2, wherein the anti -ANG2 -binding domain is a scFv fragment. The bispecific binding molecule of claim 1, wherein the VEGF-binding domain linked with the ANG2 -binding domain via a linker. The bispecific binding molecule of claim 1, wherein the binding molecule comprises an amino acid sequence of SEQ ID NO: 10. The bispecific binding molecule of claim 14, wherein the binding molecule comprises an amino acid sequence of SEQ ID NO: 11. The bispecific binding molecule of claim 14, wherein the binding molecule comprises an amino acid sequence of SEQ ID NO: 12. The bispecific binding molecule of claim 1, wherein the binding molecule comprises an amino acid sequence of SEQ ID NO: 2. The bispecific binding molecule of claim 17, wherein the binding molecule comprises an amino acid sequence of SEQ ID NO: 11. The bispecific binding molecule of claim 17, wherein the binding molecule comprises an amino acid sequence of SEQ ID NO: 12. The bispecific binding molecule of claim 1, wherein the binding molecule comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, , SEQ ID NO: 15 and SEQ ID NO: 16. The bispecific binding molecule of claim 1 , wherein the anti -ANG2 -binding domain is a Fab fragment, wherein the Fab fragment is fused to N-terminus of the VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence that is SEQ. ID NO: 11, wherein the bispecific binding molecule comprises a light chain amino acid sequence that is SEQ. ID NO: 10. The bispecific binding molecule of claim 1, wherein the anti-ANG2 -binding domain is a Fab fragment, wherein the Fab fragment is fused to N-terminus of the VEGF-binding domain through a peptide linker, wherein the bispecific binding molecule comprises a heavy chain amino acid sequence that is SEQ. ID NO: 11, wherein the bispecific binding molecule comprises a light chain amino acid sequence that is SEQ. ID NO: 2. The bispecific binding molecule of claim 1, wherein the binding molecule is a dimer of the polypeptide thereof. A pharmaceutical composition comprises the bispecific binding molecule of claim 1 and pharmaceutically acceptable excipient. A method of inhibiting angiogenesis using the bispecific binding molecule of claim 1. A method of improve and/or treat clinical conditions selected from angiogenesis, vascular permeability, edema, and inflammation using the bispecific binding molecule of claim 1. 7. A method of treating ocular disease administering the bispecific binding molecule of claim 1. 8. The method of claim 27, wherein the ocular disease is selected from the group consisting of AMD, macular edema, CNV, DME, pathological myopia, vascular glaucoma, GA, retinal vein occlusion, ROP and corneal neovascularization. 9. A composition comprising the bispecific binding molecule of claim 1, for inhibiting angiogenesis or treating ocular disease. 0. The bispecific binding molecule of claim 1 for use in inhibiting angiogenesis or treating ocular disease. 1. Use of the binding molecule of claim 1 in the manufacture of a medicament for inhibiting angiogenesis or treating ocular disease. 2. A nucleic acid encoding the bispecific binding molecule of claim 1. 3. A vector comprises the nucleic acid of claim 33. 4. An expression vector comprising a nucleic acid of claim 32, wherein the nucleic acid is operatively linked to an expression control sequence. 5. A host vector system for production of a fusion polypeptide which comprises the expression vector of claim 34, in a suitable host cell. 6. The host vector system of claim 35, wherein the suitable host cell is a bacterial cell, yeast cell, insect cell, or mammalian cell. 7. The host vector system of claim 35, wherein the suitable host cell is a CHO cell. 8. A method of producing the bispecific binding molecule, comprising a growing cells of the host-vector system of claim 34, under conditions permitting production of the fusion polypeptide and recovering the fusion polypeptide so produced.