CA3034574C - Anti-vegf-a and anti-ang2 antibodies and uses thereof - Google Patents

Abstract

The present invention relates to bispecific antibodies having activity against a vascular endothelial growth factor (VEGF) and an angiopoietin (ANG), and methods of making and using such bispecific antibodies.

Description

ANTI-VEGF-A AND ANTI-ANG2 ANTIBODIES AND USES THEREOF Field of the Invention The invention relates to bispecific antibodies having activity against a vascular endothelial growth factor (VEGF) and an angiopoietin (ANG), and uses of such antibodies.

Background to the Invention Angiogenesis, the formation of new blood vessels from existing vasculature, is a complex biological process required for the formation and physiological functions of virtually all the organs. It is an essential element of embryogenesis, normal physiological growth, repair and pathological processes such as tumour expansion. Normally, angiogenesis is tightly regulated by the local balance of angiogenic and angiostatic factors in a multi-step process involving vessel sprouting, branching and tubule formation by endothelial cells (involving processes such as activation of endothelial cells (ECs), vessel destabilisation, synthesis and release of dcgradativc enzymes, EC migration, EC proliferation, EC organization and differentiation and vessel maturation).

In the adult, physiological angiogenesis is largely confined to wound healing and several components of female reproductive function and embryonic development. In disease-related angiogenesis which includes any abnormal, undesirable or pathological angiogenesis, the local balance between angiogenic and angiostatic factors is dysregulated leading to inappropriate and/or structurally abnormal blood vessel formation. Pathological angiogenesis has been associated with disease states including diabetic retinopathy, psoriasis, cancer, rheumatoid arthritis, atheroma, Kaposi's sarcoma and haemangioma (Fan et al, 1995, Trends Pharmacology. Science. 16: 57-66; Folkman, 1995, Nature Medicine 1: 27-31). In cancer, growth of primary and secondary tumours beyond 1-2 mm3 requires angiogenesis (Folkman, J. New England Journal of Medicine 1995; 33, 1757-1763).

VEGF is a potent and ubiquitous vascular growth factor. Prior to identification of the role of VEGF as a secreted mitogen for endothelial cells, it was identified as a vascular permeability factor, highlighting VEGF's ability to control many distinct aspects of endothelial cell behaviour, including proliferation, migration, specialization and survival (Ruhrberg, 2003 BioEssays 25:1052-1060). VEGF family members include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF- 1 E, placental growth factor (PIGF) and endocrine gland-derived VEGF (EG-VEGF). Active forms of VEGF are synthesised either as homodimers or heterodimers with other VEGF family members.

VEGF-A exists in six isoforms generated by alternative splicing: VEGF121, VEGF145, VEGF165, VEGF183, VEGF189 and VEGF206. These isofom1s differ primarily in their bioavailability, with VEGF165 being the predominant isoform (Podar, et al. 2005 Blood 105( 4): 1383-1395). The regulation of splicing during embryo genesis to produce stage- and tissuespecific ratios of the various isoforms creates rich potential for distinct and context dependent behaviour of endothelial cells in response to VEGF.

VEGF is believed to be an important stimulator of both normal and disease-related angiogenesis (Jakeman, et al. 1993 Endocrinology: 133,848-859; Kolch, et al. 1995 Breast Cancer Research and Treatment: 36, 139-155) and vascular permeability (Connolly, et al. 1989 J. Biol.

Chem: 264,20017-20024). Antagonism ofVEGF action by sequestration of VEGF with antibodies can result in a reduction in tumor growth (Kim, et al. 1993 Nature: 362,841-844). Heterozygous disruption of the VEGF gene resulted in fatal deficiencies in vascularisation (Carmeliet, et al. 1996 Nature 380:435-439; Ferrara, et al. 1996 Nature 380:439-442).

In addition to the VEGF family, the angiopoietins are thought to be involved in vascular development and postnatal angiogenesis. The angiopoietins include a naturally occurring agonist, angiopoietin-1 (ANG-I), as well as a naturally occurring antagonist, angiopoietin-2 (ANG-2). The role of ANG-I is thought to be conserved in the adult, where it is expressed widely and constitutively (Hanahan, Science, 277:48-50 (1997); Zagzag, et al., Exp Neurology, 159:391-400 (1999)). In contrast, ANG-2 expression is primarily limited to sites of vascular remodeling where it is thought to block the constitutive stabilizing or maturing function of ANG-I, allowing vessels to revert to, and remain in, a plastic state which may be more responsive to sprouting signals (Hanahan, 1997; Holash et al., Oncogene 18:5356-62 (1999); Maisonpierre, 1997). Studies of ANG-2 expression in disease-related angiogenesis have found many tumor types to show vascular ANG-2 expression (Maisonpierre et al., Science 277:55-60 (1997)). Functional studies suggest ANG-2 is involved in tumor angiogenesis and associate ANG-2 overexpression with increased tumor growth in a mouse xenograft model (Ahmad, et al., Cancer Res., 61:1255-1259 (2001)).

Other studies have associated ANG-2 overexpression with tumor hypervascularity (Etoh, et al., Cancer Res. 61:2145-53 (2001); Tanaka et al., Cancer Res. 62:7124-29 (2002)). 2 Using homology-based cloning approaches, Valenzuela et al. (Proc Natl Acad Sci US A. 1999 Mar 2;96(5):1904-9) identified 2 novel angiopoietins: angiopoietin-3 (ANG-3) in mouse, and angiopoietin-4 (ANG-4) in human. Although ANG-3 and ANG-4 are more structurally diverged from each other than are the mouse and human versions of ANG-1 and ANG-2, they appear to represent the mouse and human counterparts of the same gene locus. Very little is known about the biology of these members of the angiopoietin family. For example, ANG-4 is expressed at high levels only in the lung (Tsigkos, et al., Expert Opin. Investig. Drugs 12(6): 933-941 (2003); Valenzuela, ct al., Proc. Natl. Acad. Sci. 96:1904-1909 (1999)). ANG-4 expression levels arc known to increase in response to hypoxia, and endothelial cell growth factors lead to increasing levels of ANG-4 expression in a glioblastoma ce11 line and endothelial ce11s. However, the mechanism of expression regulation, and the resulting effect on physiological and disease-related angiogenesis are unknown (Lee, et al., FASEB J. 18: 1200-1208 (2004).

The angiopoietins were first discovered as ligands for the Tie receptor tyrosine kinase family that is selectively expressed within the vascular endothelium (Yancopoulos et al., Nature 407:242-48 (2000). ANG-1, ANG-2, ANG-3 and ANG-4 bind primarily to the Tie-2 receptor and so are also known as Tie-2 ligands. Binding of ANG-1 to Tie-2 induces tyrosine phosphorylation of the receptor via autophosphorylation and subsequently activation of its signalling pathways via signal transduction (Maisonpien-e, P. et al. 1997 Science: 277, 55-60). ANG-2 is a naturally occurring antagonist for ANG-1 acting through competitive inhibition of ANG-I-induced kinase activation of the Tie-2 receptor (Hanahan, 1997; Davis et al., Cell 87:1161-69 (1996); Maisonpierre et al., Science 277:55-60 (1997)).

Knock-out mouse studies of Tic-2 and ANG-I show similar phenotypes and suggest that ANG-1 stimulated Tie-2 phosphorylation mediates remodeling and stabilization of developing vessel, promoting blood vessel maturation during angiogenesis and maintenance of endothelial cell-support cell adhesion (Dumont et al., Genes & Development, 8: 1897-1909 (1994); Sato, Nature, 376:70-74 (1995); (Thurston, G. et al., 2000 Nature Medicine: 6, 460-463)).

In recent years ANG-1, ANG-2 and/or Tie-2 have been proposed as possible anti-cancer therapeutic targets (see, for example, US Patent Nos. 6,166,185, 5,650,490 and 5,814,464 each disclose anti-Tie-2 ligand and receptor antibodies). Studies using soluble Tie-2 have been reported to decrease the number and size of tumors in rodents. Also, some groups have reported the use of antibodies that bind to ANG-2 (see, for example, U.S. Patent No. 6,166,185 and U.S. Patent 3 Application Publication No. 2003/0124129) and antibodies that bind to VEGF-A (see, for example, US Patent No. 8,216,571). Additionally, there are examples of targeting VEGF-A and ANG-2 (see, for example, WO200197850, WO2007089445, and US Patent No. 8,268,314).

However, there is an unmet need is the medical arts for a bispecific antibody targeting VEGF-A and ANG-2 that is more tolerable or effective. More particularly, there is an unmet need related to improving the safety at least as it relates to toxicity associated with targeting VEGF-A (e.g., thromboembolic events, renal toxicity, etc.). To this end, the bispecific antibodies targeting VEGF-A and ANG-2 disclosed herein arc effective at reducing vascular dysrcgulation and tumor growth with a decrease in toxicity related to, for example, thromboembolic events and/or renal toxicity.

Summary of the Invention The invention relates to bispecific antibodies that bind to VEGF and ANG. The invention further relates to bispecific antibodies that bind to VEGF and ANG, and reduce the activity of at least one biological activity of VEGF and ANG. The invention even further relates to providing bispecific antibodies to a subject in need thereof that bind to VEGF and ANG, and reduce tumor growth and/or reduce tumor volume.

Brief Description of the Figures Figure 1. Depicts a schematic of the general structural format of five different bispecific antibody (BiS) backbones, BiSl, BiS2, BiS3, BiS4, and BiS5. The scFv is depicted in dark grey and the IgG Fv is depicted in light grey.

Figure 2. Depicts a schematic representation of the bispecific antibody BiS3Ab-VEGF Hl RK-ANG-2.

Figure 3. Depicts the DNA and protein sequences for the light chain of the bispecific antibody BiS3Ab-VEGF HlRK-ANG-2.

Figure 4. Depicts the DNA sequence of the heavy chain of the bispecific antibody BiS3AbVEGF HlRK-ANG-2. 4 Figure 5. Depicts the protein sequence of the heavy chain of the bispecific antibody BiS3Ab-VEGF HlRK-ANG-2.

Figure 6. Representative data for an elution profile for the bispecific antibody BiSAbVEGF HlRK-ANG-2.

Figure 7. Representative data for purification profiles for the bispecific antibody BiSAbVEGF HlRK-ANG-2.

Figure 8. Representative SDS-PAGE gel for the bispecific antibody BiS3Ab-VEGF H1RK-ANG-2. BsAb - Intact BiS3Ab-VEGF H1RK-ANG-2; Ab - Anti-VEGF mAb; H-BsAb - HeavychainofBiS3Ab-VEGFHlRK-ANG-2; H-Ab- Heavy chain of anti-VEGF mAb; L- Light chian of BiS3Ab-VEGF HlRK-ANG-2 and anti-VEGF.

Figure 9. Representative data after focusing for the bispecific antibody BiS3Ab-VEGF HlRK-ANG-2.

Figure 10. Representative data for transition temperatures for the bispecific antibody BiS3Ab-VEGF HlRK-ANG-2.

Figure 11. Representative data for concurrent binding of the bispecific antibody BiSAbVEGF HlRK-ANG-2 to VEGF-165 and ANG-2.

Figure 12. A. Representative data for concurrent binding of the bispecific antibody BiSAbVEGF H1RK-ANG-2 to VEGF-165 and ANG-2 using an ELISA based assay.

Figure 12. B. Representative data for concurrent binding of the bispecific antibody BiSAbVEGF HlRK-ANG-2 to VEGF-165 and ANG-2 using an ELISA based assay.

Figure 13. Representative data showing lack of binding to VEGF121 by the bispecific antibody BiSAb-VEGF HlRK-ANG-2.

Figure 14. Representative data showing lack of binding to VEGF189 by the bispecific antibody BiSAb-VEGF HlRK-ANG-2.

Figure 15. Representative data showing reduction in tumor volume in the presence of the bispecific antibody BiSAb-VEGF HlRK-ANG-2 in a 786-0 renal cell carcinoma model.

Figure 16. Representative data showing reduction in tumor volume in the presence of the bispecific antibody BiSAb-VEGF HlRK-ANG-2 in a BxPC3 pancreatic carcinoma model.

Figure 17. A. Representative data showing vasculogenesis without the presence of the bi specific antibody BiSAb-VEGF Hl RK-ANG-2.

Figure 17. B. Representative data showing vasculogenesis in the presence of the bispecific antibody BiSAb-VEGF HlRK-ANG-2.

Figure 18. Representative data showing reduction of the vessel migration (arrow) towards the periphery of the retina (dashed line) in the presence of the bispecific antibody BiSAb-VEGF HlRK-ANG-2. 4X magnification.

Figure 19. Representative data showing reduction of the vessel branching in the presence ofBiSAb-VEGF HlRK-ANG-2. 20X magnification.

Figure 20. A. Representative data showing renal pathology without the presence of the anti-VEGF antibody and the bispecific antibody BiSAb-VEGF HlRK-ANG-2.

Figure 20. B. Representative data showing renal pathology data in the presence of the antiVEGF antibody.

Figure 20. C. Representative data showing reduction in renal pathology in the presence of the bispecific BiSAb-VEGF HlRK-ANG-2 present. 6 WO 2018/037000 Detailed Description Definitions PCT/EP2017/071104 Before describing the present invention in detail, it is to be understood that this invention is not limited to specific compositions or process steps, as such can vary. As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" ( or "an"), as well as the terms "one or more," and "at least one" can be used interchangeably herein. Further it is understood that wherever aspects arc described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of' and/or "consisting essentially of' are also provided.

Complementarity determining regions (CDRs) are responsible for antibody binding to its antigen. CD Rs are determined by a number of methods in the art (inc1uding Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); Chothia (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); IMGT (ImMunoGeneTics) (Lefranc, M.P. et al., Dev. Comp. Immunol. 27: 55-77 (2003)); and other methods). Although specific CDR sequences are mentioned and claimed herein, the invention also encompasses CDR sequences defined by any method known in the art.

As use herein, the term "subject" refers to any member of the subphylum cordata, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaccous birds, ducks, geese, and the like arc also non-limiting examples.

Bispecific Antibodies Suitable bi specific antibodies of the invention can be or are derived from any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), sub-isotype (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or allotype (e.g., Gm, e.g., Glm(f, z, a or x), G2m(n), G3m(g, b, or c), Am, Em, and Km(l, 2 or 3)).

Such antibodies can include light chains classified as either lambda chains or kappa chains based on the amino acid sequence of the light chain constant region. Figure 1 shows a schematic of the orientation of five different bispecific backbones (BiS) (see, for example, PCT Patent Application Nos. PCT/US2016/035026 and PCT/US2015/025232). Specific linkers within the scFv and 7 linkers linking the scFv to a specified po1tion of bispecific antibodies of the invention ( e.g., GGGGSGGGGSGGGGSGGGGS) are described. However, any suitable linker within the scFv or linking the scFv to any specified portion of bispecific antibodies of the invention may be used (see, for example, PCT Patent Application Nos. PCT/US2016/035026 and PCT/US2015/025232).

Production of Binding Molecules Recombinant DNA methods for producing and screenmg for bispecific antibodies described herein arc known in the art (e.g. U.S. Patent No. 4,816,567). DNA encoding the bispecific antibodies, for example, DNA encoding a VH domain, a VL domain, a single chain variable fragment (scFv), or combinations thereof can be inserted into a suitable expression vector, which can then be transfected into a suitable host cell, such as E. coli cells, simian COS ce11s, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce an antibody, to obtain the bispecific antibodies of the invention.

Suitable expression vectors are known in the art. An expression vector can contain a polynucleotide that encodes a bispecific antibody linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., U.S.

Patent Nos. 5,981,216; 5,591,639; 5,658,759 and 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy chain (including the scFv portion), the entire light chain, or both the entire heavy and light chains. The expression vector can be transferred to a host cell by conventional techniques and the transfected cells can be cultured by conventional techniques to produce the bispecific antibodies.

Mammalian cell lines suitable as hosts for expression of recombinant antibodies arc known in the art and include many immortalized cell lines available from the American Type Culture Collection, including but not limit to CHO cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney 293 cells, and a number of other cell lines. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the bispecific antibodies. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include CHO, 8 VERY, BHK, Hela, COS, MOCK, 293, 3T3, Wl38, BT483, Hs578T, HTB2, BT2O and T47D, NSO (a murine myeloma cell line that does not endogenously produce any functional immunoglobulin chains), SP20, CRL7O3O and HsS78Bst cells. Human cell lines developed by immortalizing human lymphocytes can be used to recombinantly produce monoclonal antibodies.

The human cell line PER.C6® (Crucell, Netherlands) can be used to recombinantly produce monoclonal antibodies. Additional cell lines which may be used as hosts for expression of recombinant antibodies include insect cells (e.g. Sf21/Sf9, Trichoplusia ni Bti-Tn5bl-4), or yeast cells ( e.g. S. ccrcvisiac, Pichia, OS7326681; etc.), plants cells (US20080066200), or chicken cells (WO2008142124).

Bispecific antibodies can be stably expressed in a cell line using methods known in the art.

Stable expression can be used for long-tenn, high-yield production of recombinant proteins. For stable expression, host cells can be transformed with an appropriately engineered vector that includes expression control elements (e.g., promoter, enhancer, transcription terminators, polyadenylation sites, etc.), and a selectable marker gene. Following the introduction of the foreign DNA, cells are allowed to grow for 1-2 days in an enriched media, and are then switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells that have stably integrated the plasmid into their chromosomes to grow and form foci which in tum can be cloned and expanded into cell lines. Methods for producing stable cell lines with a high yield are known in the art and reagents are generally available commercially. Transient expression can also be carried out by using methods known in the art.

Transient transfection is a process in which the nucleic acid introduced into a cell does not integrate into the genome or chromosomal DNA of that cell and is maintained as an extra-chromosomal clement in the cell (e.g., as an cpisome).

A cell line expressing a bispecitic antibody, either stable or transiently transfected, can be maintained in cell culture medium and conditions known in the art resulting in the expression and production of the bispecific antibodies. Cell culture media can be based on commercially available media formulations, including, for example, DMEM or Ham's F12. In addition, the cell culture media can be modified to support increases in both cell growth and biologic protein expression.

As used herein, the terms "cell culture medium," "culture medium," and "medium formulation" refer to a nutritive solution for the maintenance, growth, propagation, or expansion of cells in an artificial in vitro environment outside of a multicellular organism or tissue. Cell culture medium 9 may be optimized for a specific cell culture use, including cell culture growth medium which is formulated to promote cellular growth or cell culture production medium which is formulated to promote recombinant protein production. The terms nutrient, ingredient, and component are used interchangeably herein to refer to the constituents that make up a cell culture medium. Cell lines can be maintained using a fed batch method. As used herein, "fed batch method," refers to a method by which a cell culture is supplied with additional nutrients after first being incubated with a basal medium. For example, a fed batch method may include adding supplemental media according to a determined feeding schedule within a given time period. Thus, a "fed batch cell culture" refers to a cell culture wherein the cells, typically mammalian, and culture medium are supplied to the culturing vessel initially and additional culture nutrients are fed, continuously or in discrete increments, to the culture during culturing, with or without periodic cell and/or product harvest before tem1ination of culture.

Cell culture media and the nutrients contained therein are known in the art. Cell culture medium may include a basal medium and at least one hydrolysate, e.g., soy-based hydrolysate, a yeast-based hydrolysate, or a combination of the two types of hydrolysates resulting in a modified basal medium. The additional nutrients may include only a basal medium, such as a concentrated basal medium, or may include only hydrolysates, or concentrated hydrolysates. Suitable basal media include Dulbecco's Modified Eagle's Medium (DMEM), DME/Fl2, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, a-Minimal Essential Medium (a-MEM), Glasgow's Minimal Essential Medium (G-MEM), PF CHO (see, e.g., CHO protein free medium (Sigma) or EX-CELL TM 325 PF CHO Serum-Free Medium for CHO Cells Protein-Free (SAFC Bioscience), and Iscovc's Modified Dulbecco's Medium. Other examples of basal media which may be used include BME Basal Medium (Gibco-Invitrogcn; sec also Eagle, H (1965) Proc. Soc. Exp. Biol. Med. 89, 36); Dulbecco's Modified Eagle Medium (DMEM, powder) (Gibco-Tnvitrogen (# 31600); see also Dulbecco and Freeman (1959) Virology. 8:396; Smith et al. (1960) Virology. 12:185. Tissue Culture Standards Committee, In Vitro 6:2, 93); CMRL 1066 Medium (Gibco-Invitrogen (#11530); see also Parker et al. (1957) Special Publications, N.Y. Academy of Sciences, 5:303).

The basal medium may be serum-free, meaning that the medium contains no serum (e.g., fetal bovine serum (FBS), horse serum, goat serum, or any other animal-derived serum known to one skilled in the art) or animal protein free media or chemically defined media.

The basal medium may be modified in order to remove certain non-nutritional components found in standard basal medium, such as various inorganic and organic buffers, surfactant(s), and sodium chloride. Removing such components from basal cell medium allows an increased concentration of the remaining nutritional components, and may improve overall cell growth and protein expression. In addition, omitted components may be added back into the cell culture medium containing the modified basal cell medium according to the requirements of the cell culture conditions. The cell culture medium may contain a modified basal cell medium, and at least one of the following nutrients, an iron source, a recombinant growth factor; a buffer; a surfactant; an osmolarity regulator; an energy source; and non-animal hydrolysates. In addition, the modified basal cell medium may optionally contain amino acids, vitamins, or a combination of both amino acids and vitamins. A modified basal medium may further contain glutamine, e.g, L-glutamine, and/or methotrexate.

Purification and Isolation Once a bispecific antibody has been produced, it may be purified by methods known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigens Protein A or Protein G, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the bispecific antibodies of the invention may be fused to heterologous polypeptide sequences (referred to herein as "tags") to facilitate purification.

Bispecific antibodies of the invention can be used in a number of ways. For example, bispecific antibodies of the invention can be used to bind to VEGF, ANG, or any combination of these proteins and thereby reduce at least one biological activity of VEGF, ANG, or any combination of these activities. More particularly, the bispecific antibodies of the invention can be used to bind to VEGF-165, ANG-2, or any combination of these proteins and thereby reduce at least one biological activity of VEGF-165, ANG-2, or any combination of these activities, which may include a reduction in activation or phosphorylation of their respective receptors and/or a reduction in angiogenesis in connection with cellular dysregulation. 11 Exemplary Embodiments An embodiment of the invention relates to a bispecific antibody comprising a first binding domain comprising heavy chain complementarity determining regions 1 - 3 (i.e., HCDRl, HCDR2, and HCDR3) and light chain complementarity determining regions 1 3 (i.e., LCDRl, LCDR2, and LCDR3) of a bispecific antibody described herein, and a second binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDR 1, LCDR2, and LCDR3 of a bispecific antibody described herein, wherein the first binding domain binds to VEGF-A and the second binding domain binds to ANG-2. In a further embodiment the bispecific antibody is BiS3AbVEGF HlRK-ANG-2.

Another embodiment relates to a bispecific antibody comprising a first binding domain comprising an HCDR 1, HCDR2, and HCDR3 and an LCDR 1, LCDR2, and LCDR3 of a bispecific antibody described herein, and a second binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3 of a bispecific antibody described herein, wherein the first binding domain binds to VEGF-A and the second binding domain binds to ANG-2 and wherein the bispecific antibody binds VEGF165. In a further embodiment the bispecific antibody is BiS3Ab-VEGF HlRK-ANG-2.

Another embodiment relates to a bispecific antibody comprising a first binding domain comprising anHCDRl, HCDR2, andHCDR3 andanLCDRl, LCDR2, andLCDR3 ofa bispecific antibody described herein and a second binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3 of a bispecific antibody described herein, wherein the first binding domain binds to VEGF-A and the second binding domain binds to ANG-2 and wherein the bispecific antibody binds VEGF165 with greater affinity compared to VEGF121. In a further embodiment the bispccific antibody is BiS3Ab-VEGF HlRK-ANG-2.

Another embodiment relates to a bispecific antibody comprising a first binding domain comprising an HCDR 1, HCDR2, and HCDR3 and an LCDR 1, LCDR2, and LCDR3 of a bi specific antibody described herein and a second binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3 of a bispecific antibody described herein, wherein the first binding domain binds to VEGF-A and the second binding domain binds to ANG-2 and wherein the bispecific antibody binds VEGF165 with greater affinity compared to VEGF189. In a further embodiment the bispecific antibody is BiS3Ab-VEGF HlRK-ANG-2. 12 Another embodiment relates to a bispecific antibody comprising a first binding domain comprising anHCDRl, HCDR2, andHCDR3 andanLCDRl, LCDR2, andLCDR3 ofa bispecific antibody described herein and a second binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3 of a bispecific antibody described herein, wherein the first binding domain binds to VEGF-A and the second binding domain binds to ANG-2 and wherein the bispecific antibody binds VEGF165 with greater affinity compared to VEGF121 and VEGF189. In a further embodiment the bispecific antibody is BiS3Ab-VEGF HlRK-ANG-2.

Another embodiment relates to a bispecific antibody comprising a first binding domain comprising anHCDRl, HCDR2, andHCDR3 andanLCDRl, LCDR2, andLCDR3 ofa bispecific antibody described herein and a second binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDR 1, LCDR2, and LCDR3 of a bi specific antibody described herein, wherein the first binding domain binds to VEGF-A and the second binding domain binds to ANG-2 and wherein the bispecific antibody reduces human VEGFR2 phosphorylation, murine VEGFR2 phosphorylation, or both human and murine VEGFR2 phosphorylation. In a further embodiment the bispecific antibody is BiS3Ab-VEGF HlRK-ANG-2.

Another embodiment relates to a bispecific antibody comprising a first binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3 ofa bispecific antibody described herein and a second binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3 of a bispecific antibody described herein, wherein the first binding domain binds to VEGF-A and the second binding domain binds to ANG-2 and wherein the bispecific antibody reduces human Tie2 receptor phosphorylation. In a further embodiment the bispecific antibody is BiS3Ab-VEGF HlRK-ANG-2.

Another embodiment relates to a bispecific antibody comprising a first binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3 of a bispecific antibody described herein and a second binding domain comprising an HCDRt, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3 of a bispecific antibody described herein, wherein the first binding domain binds to VEGF-A and the second binding domain binds to ANG-2 and wherein the bispecific antibody reduces angiogenesis.

Another embodiment relates to a bispecific antibody comprising a first binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3 of a bispecific antibody described herein and a second binding domain comprising an HCDRl, HCDR2, and 13 HCDR3 and an LCDRl, LCDR2, and LCDR3 of a bispecific antibody described herein, wherein the first binding domain binds to VEGF-A and the second binding domain binds to ANG-2 and wherein the bispecific antibody reduces tumor growth, reduces tumor volume, or reduces tumor growth and reduces tumor volume as a result of being provided to a subject having a tumor. In a further embodiment the bispecific antibody is BiS3Ab-VEGF HlRK-ANG-2.

Another embodiment relates to a bispecific antibody comprising a first binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3 of a bispecific antibody described herein and a second binding domain comprising an HCDRI, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3 of a bispecific antibody described herein, wherein the first binding domain binds to VEGF-A and the second binding domain binds to ANG-2 and wherein the bispecific antibody binds to ANG-2 with greater affinity than the parental ANG-2 antibody used to make the second binding domain. In a more particular embodiment, the binding affinity of the second binding domain to ANG-2 is increased by about I-fold to about 20-fold. In a further more particular embodiment, the binding affinity of the second binding domain to ANG- 2 is increased by about I-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about I2-fold, about 13- fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, or about 20-fold. In a further embodiment the bispecific antibody is BiS3Ab-VEGF HlRK-ANG-2.

Another embodiment relates to a bispecific antibody comprising a first binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3 of a bispecific antibody described herein and a second binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRI, LCDR2, and LCDR3 of a bispecific antibody described herein, wherein the first binding domain binds to VEGF-A and the second binding domain binds to ANG-2 and wherein the bispecific antibody has one or more or any combination of the characteristics described herein, including binding to VEGFl 65, binding to VEGFl 65 with greater affinity compared to VEGF121, binding to VEGF165 with greater affinity compared to VEGF189, binding to VEGF165 with greater affinity compared to VEGF121 and VEGF189, reducing human VEGFR2 phosphorylation, reducing murine VEGFR2 phosphorylation, reducing human and murine VEGFR2 phosphorylation, reducing human Tie2 receptor phosphorylation, reducing angiogenesis, reducing tumor growth, reducing tumor volume, reducing tumor growth and reducing tumor volume, and increasing affinity to ANG-2 through the second binding domain 14 compared to the parental ANG-2 antibody used to make the second binding domain. In a further embodiment the bispecific antibody is BiS3Ab-VEGF HlRK-ANG-2.

Another embodiment relates to a bispecific antibody comprising an antibody heavy chain having the formula VH-CH1-H-CH2-CH3, wherein VH is a heavy chain variable domain, CHI is a heavy chain constant region domain 1, His a hinge region, CH2 is a heavy chain constant region domain 2, and CH3 is a heavy chain constant region domain 3. In another further embodiment, the bispecific antibody includes an antibody light chain having the f01mula VL-CL, wherein VL is a variable light chain domain and CL is a light chain constant domain. In another even further embodiment, the bispecific antibody has the formula VH-CH1-H-CH2-CH3 and VL-CL. In a further embodiment the bispecific antibody is BiS3Ab-VEGF HIRK-ANG-2.

Another embodiment relates to a bispecific antibody comprising the formula VH-CH1-HCH2- CH3 and VL-CL wherein one or more scFv molecules are covalently attached to one or more N-terminal po1iions of the antibody heavy chain or antibody light chain. In another further embodiment the one or more scFv molecules are covalently attached to the N-terrninal domain of one or more VL of the bispecific antibody. In a more particular embodiment, the bispecific antibody includes the formula VH-CH1-H-CH2-CH3 and scFv-Ll-VL-CL, wherein Ll is a linker and the other various parts are previously described. In another more particular embodiment, the bispecific antibody includes the formula scFv-Ll-VH-CH1-CH2-CH3 and VL-CL.

Another embodiment relates to a bispecific antibody comprising the formula VH-CH1-HCH2- CH3 and VL-CL wherein one or more scFv molecules are covalently attached to one or more C-terminal portions of the antibody heavy chain. In a more particular embodiment, the bispecific antibody comprises the formula VH-CHI-CH2-CH3-Ll-scFv and VL-CL. In another more particular embodiment, the bispecific antibody comprises the formula VH-CH1-CH2-CH3-LlscFv- L2 and VL-CL, wherein L2 is a linker and is independent of LI and wherein Ll and L2 are covalently bound to CH3, with the other various parts being previously described. In another further more paiiicular embodiment, the bispecific antibody comprises the formula VH-CHl-LlscFv- L2-CH2-CH3 and VL-CL, wherein LI and L2 are independent linkers and wherein the heavy chain can contain a hinge region or be hingeless. In a furtl1er embodiment the bispecific antibody is BiS3Ab-VEGF HlRK-ANG-2.

In a specific embodiment, there is a bispecific antibody comprising a first binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3, wherein the first binding domain HCDRl, HCDR2, and HCDR3 and LCDRl, LCDR2, and LCDR3 comprise SEQ ID NOs: 17 -22, respectively; and a second binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3, wherein the second binding domain HCDRl, HCDR2, and HCDR3 and LCDRl, LCDR2, and LCDR3 comprise SEQ ID NOs: 23 28, respectively.

In another specific embodiment, there is a bispecific antibody first binding domain comprising a heavy chain and a light chain comprising SEQ ID NOs: 3 and 9, respectively, and a second binding domain comprising a heavy chain and a light chain comprising SEQ ID NOs: 5 and 11, respectively.

In another specific embodiment, there is a bispecific antibody comprising a heavy chain amino acid sequence comprising SEQ TD NO: I and a light chain amino acid sequence comprising SEQ ID NO: 7.

In another specific embodiment, there is a bispecific antibody comprising a fonnulahaving the parts VH-CH1-H-CH2-CH3, VL-CL, and one or more scFv, Ll, or optionally L2, wherein the formula can be: a. VH-CH1-CH2-CH3 and scFv-Ll-VL-CL; b. scFv-Ll-VH-CH1-CH2-CH3 and VL-CL; c. VH-CH1-CH2-CH3-Ll-scFv and VL-CL; d. VH-CH1-CH2-CH3-Ll-scFv-L2 and VL-CL, wherein Ll and L2 are covalently bound to CH3; e. VH-CH1-Ll-scFv-L2-CH2-CH3 and VL-CL, the heavy chain can contain a hinge region or be hingeless. ln another specific embodiment, there is a bispecific antibody with the formula VH-CH1-CH2- CH3-Ll-scFv and VL-CL.

In another specific embodiment, there is a bispecific antibody comprising a scFv comprising the amino acid sequence of SEQ ID NO: 13.

In another specific embodiment, there is a nucleic acid sequence comprising polynucleotides encoding a bispecific antibody comprising a first binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3, wherein the first binding domain HCDRl, HCDR2, and HCDR3 and LCDRl, LCDR2, and LCDR3 comprise SEQ ID NOs: 17 - 22, respectively; and a second binding domain comprising an HCDRl, HCDR2, and HCDR3 16 and an LCDRl, LCDR2, and LCDR3, wherein the second binding domain HCDRl, HCDR2, and HCDR3 and LCDRl, LCDR2, and LCDR3 comprise SEQ ID NOs: 23 - 28, respectively.

In another specific embodiment, there is a vector comprising polynucleotides encoding a bispecific antibody comprising a first binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3, wherein the first binding domain HCDRl, HCDR2, and HCDR3 and LCDR 1, LCDR2, and LCDR3 comprise SEQ ID NOs: 17 - 22, respectively; and a second binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3, wherein the second binding domain HCDRl, HCDR2, and HCDR3 and LCDRl, LCDR2, and LCDR3 comprise SEQ ID NOs: 23 - 28, respectively.

In another specific embodiment, there is a cell comprising a vector comprising polynucleotides encoding a bispecific antibody comprising a first binding domain comprising an HCDR 1, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3, wherein the first binding domain HCDRl, HCDR2, and HCDR3 and LCDRl, LCDR2, and LCDR3 comprise SEQ ID NOs: 17 - 22, respectively; and a second binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3, wherein the second binding domain HCDRl, HCDR2, and HCDR3 and LCDRl, LCDR2, and LCDR3 comprise SEQ ID NOs: 23 -28, respectively.

In another specific embodiment, there is a method of making a bispecific antibody comprising culturing a cell comprising a vector comprising polynucleotides encoding a bispecific antibody comprising a first binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3, wherein the first binding domain HCDRl, HCDR2, and HCDR3 and LCDRl, LCDR2, and LCDR3 comprise SEQ ID NOs: 17 - 22, respectively; and a second binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3, wherein the second binding domain HCDRl, HCDR2, and HCDR3 and LCDRI, LCDR2, and LCDR3 comprise SEQ ID NOs: 23 - 28, respectively.

In another specific embodiment, there is a method of reducing angiogenesis comprising providing a bispecific antibody to a subject wherein the bispecific antibody comprises a first binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3, wherein the first binding domain HCDRl, HCDR2, and HCDR3 and LCDRl, LCDR2, and LCDR3 comprise SEQ ID NOs: 17 - 22, respectively; and a second binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3, wherein the 17 second binding domain HCDRl, HCDR2, and HCDR3 and LCDRl, LCDR2, and LCDR3 comprise SEQ ID NOs: 23 - 28, respectively.

Sequences SEQ ID SEQUENCE Description NO 1 EVQLLESGGGL VQPGGSLRLSCAASGFTFSWYEMYWVRQA Amino acid sequence of the heavy PGKGLEWVSSISPSGGWTMYADSVKGRFTISRDNSKNTLYL chain of BiS3Ab-VEGF HlRKQMNSLRAEDTA VYYCATPL YSSDGLSAGDIWGQGTMVTVS ANG-2 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFP A VLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCL VKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLST ,SP GKGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSIT GSYLA WYQQKPGQAPRLLITGASSW ATGIPDRFSGSGSGTD FTL TISRLEPEDFAVYYCQQYSSSPITFGCGTRLEIKGGGGSG GGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASG FTFTNYGMHWVRQAPGKCLEWV A VISHDGNNKYYVDSVK GRFTISRDNSKNTLYLQMNSLRAEDTA VYYCAREGIDFWSG LNWFDPWGQGTL VTVSS 2 GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGC Nucleotide sequence of the heavy CTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCGGATTC chain of HiS3Ab-VEGF HlRKACTTTCTCTTGGTACGAGATGTATTGGGTTCGCCAAGCTC ANG-2 CTGGTAAAGGTTTGGAGTGGGTTTCTTCTATCTCTCCTTCT GGTGGCTGGACTATGTATGCTGACTCCGTTAAAGGTCGCT TCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTT GCAGATGAACAGC1TAAGGGCTGAGGACACGGCCGTGTA TTACTGTGCGACCCCCTTGTATAGCAGTGACGGGCTTTCG GCGGGGGATATCTGGGGCCAAGGGACAATGGTCACCGTC TCAAGCGCGTCGACCAAGGGCCCATCCGTCTTCCCCCTGG CACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCC TGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGA CGGTGTCCTGGAACTCAGGCGCTCTGACCAGCGGCGTGC ACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTC CCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGG CACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAG CAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTG TGACAAAAC1CACACATGCCCACCGTGCCCAGCACCTGA ACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAA CCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTC ACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAG CACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAG GACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTCTACACC 18 SEQ ID SEQUENCE Description NO 3 4 6 CTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTC AGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACA TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGC TCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCA GGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGC ATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT CCCTGTCTCCGGGTAAAGGCGGAGGGGGATCCGGCGGAG GGGGCTCTGAGATCGTGCTGACCCAGAGCCCCGGCACCC TGAGCCTGAGCCCTGGCGAGAGAGCCACCCTGAGCTGCC GGGCCAGCCAGTCCATCACCGGCAGCTACCTGGCTTGGT ATCAGCAGAAGCCCGGACAGGCCCCCAGACTGCTGATCA CCGGCGCTTCCAGCTGGGCCACCGGCATCCCCGACAGAT TCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCA TCAGCAGACTGGAGCCCGAGGACTTCGCCGTGTACTACT GCCAGCAGTACAGCAGCAGCCCCATCACCTTCGGAtgcGG CACCAGGCTGGAGATCAAGGGCGGAGGGGGCTCTGGGG GAGGGGGCAGCGGCGGCGGAGGATCTGGGGGAGGGGGC AGCCAGGTGCAGCTGGTCGAGTCTGGCGGCGGAGTGGTG CAGCCCGGCAGAAGCCTGAGACTGAGCTGCGCCGCCAGC GGCTTCACCTTCACCAACTACGGCATGCACTGGGTCCGCC AGGCCCCTGGCAAGtGCCTGGAGTGGGTGGCCGTGATCAG CCACGACGGCAACAACAAGTACTACGTGGACAGCGTGAA GGGCAGATTCACCATCAGCAGGGACAACAGCAAGAACAC CCTGTACCTCCAGATGAACAGCCTGAGAGCCGAGGACAC CGCCGTGTACTACTGCGCCAGAGAGGGCATCGACTTTTG GAGCGGCCTGAATTGGTTCGACCCCTGGGGCCAGGGCAC CCTGGTGACCGTGTCCAGC EVQLLESGGGL VQPGGSLRLSCAASGfTPSWYEM YWVRQA PGKGLEWVSSISPSGGWTMYADSVKGRFTISRDNSKNTL YL QMNSLRAEDT A VYYCATPL YSSDGLSAGDIWGQGTMVTVS s GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGC CTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCGGATTC ACTTTCTCTTGGTACGAGATGTATTGGGTTCGCCAAGCTC CTGGTAAAGGTTTGGAGTGGGTTTCTTCTATCTCTCCTTCT GGTGGCTGGACTATGTATGCTGACTCCGTTAAAGGTCGCT TCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTT GCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTA TTACTGTGCGACCCCCTTGTATAGCAGTGACGGGCTTTCG GCGGGGGATATCTGGGGCCAAGGGACAATGGTCACCGTC TCAAGC QVQL VESGGGVVQPGRSLRLSCAASGFTFTNYGMHWVRQA PGKCJ ,EWV AVJSHDGNNKYYVDSVKGRFTTSRDNSKNTI ,YJ, QMNSLRAEDT A VYYCAREGIDFWSGLNWFDPWGQGTLVT vss Amino acid sequence of the first binding domain heavy chain variable domain of BiS3Ab-VEGF HlRKANG- 2 Nucleotide sequence of the first binding domain heavy chain variable domain of BiS3Ab-VEGJ:-i HIRKANG- 2 Amino acid sequence of the second binding domain heavy chain of BiS3Ab-VEGF HlRK-ANG-2 CAGGTGCAGCTGGTCGAGTCTGGCGGCGGAGTGGTGCAG Nucleotide sequence of the second CCCGGCAGAAGCCTGAGACTGAGCTGCGCCGCCAGCGGC binding domain of the heavy chain of TTCACCTTCACCAACT ACGGCATGCACTGGGTCCGCCAGG BiS3Ab-VEGF HlRK-ANG-2 CCCCTGGCAAGTGCCTGGAGTGGGTGGCCGTGATCAGCC ACGACGGCAACAACAAGTACTACGTGGACAGCGTGAAGG GCAGATTCACCATCAGCAGGGACAACAGCAAGAACACCC TGTACCTCCAGATGAACAGCCTGAGAGCCGAGGACACCG 19 WO 2018/037000 SEQ ID SEQUENCE NO 7 8 9 11 12 CCGTGTACTACTGCGCCAGAGAGGGCATCGACTTTTGGA GCGGCCTGAATTGGTTCGACCCCTGGGGCCAGGGCACCC TGGTGACCGTGTCCAGC EIVLTQSP ATLSLSPGERATLSCRASQSVHSSYLA WYQQKPG QAPRJ J ,IYGASSRATGIPDRFSGSGSGTDFTJ ,TJSRT ,EPEDFA VYYCQQSYRTPSFGQGTRLEIKRTV AAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSF NRGEC GAGATCGTGCTGACCCAGTCTCCAGCCACCCTCTCTTTGT CTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTC AGAGTGTTCACAGCAGCTACTTAGCCTGGTACCAGCAGA AACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATC CAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAG TGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTG GAGCCTGAAGATTTTGCAGTTTACTACTGTCAACAGAGTT ACCGCACCCCTTCCTTCGGCCAAGGGACACGACTGGAGA TTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCC GCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTT GTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAA GTACAGTGGAAGGTGGATAACGCCCiCCAATCGGGTAAC TCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAG CACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGC AGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCAC CCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAA CAGGGGAGAGTGT EIVLTQSPATLSLSPGERATLSCRASQSVHSSYLA WYQQKPG QAPRLLIYGASSRATGTPDRFSGSGSGTDFTJ ,TJSRT ,EPEDFA VYYCQQSYRTPSFGQGTRLEIK GAGATCGTGCTGACCCAGTCTCCAGCCACCCTCTCTTTGT CTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTC AGAGTGTTCACAGCAGCTACTTAGCCTGGTACCAGCAGA AACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATC CAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAG TGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTG GAGCCTGAAGATTTTGCAGTTTACTACTGTCAACAGAGTT ACCGCACCCCTTCCTTCGGCCAAGGGACACGACTGGAGA TTAAA EIVT ,TQSPGTLST ,SPGERATLSCRASQSJTGSYJ ,A WYQQKPG QAPRLLITGASSW ATGIPDRFSGSGSGTDFTLTISRLEPEDFA VYYCQQYSSSPITFGCGTRLEIK GAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGCCTG AGCCCTGGCGAGAGAGCCACCCTGAGCTGCCGGGCCAGC CAGTCCATCACCGGCAGCTACCTGGCTTGGTATCAGCAG AAGCCCGGACAGGCCCCCAGACTGCTGATCACCGGCGCT TCCAGCTGGGCCACCGGCATCCCCGACAGATTCAGCGGC AGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGA CTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAG TACAGCAGCAGCCCCATCACCTTCGGAlgcGGCACCAGGC TGGAGATCAAG PCT/EP2017/071104 Description Amino acid sequence of the light chain of BiS3Ab-VEGF HlRKANG- 2 Nucleotide sequence of the light chain of BiS3Ab-VEGF H1 RKANG- 2 Amino acid sequence of the first binding domain light chain variable domain of BiS3Ab-VEGF HlRKANG- 2 Nucleotide sequence of the first binding domain light chain variable domain of BiS3Ab-VEGF HlRKANG- 2 Amino acid sequeuce of the secoud binding domain light chain domain of BiS3Ab-VEGF HlRK-ANG-2 Nucleotide sequence of the second binding domain light chain domain ofBiS3Ab-VEGF H1 RK-ANG-2 SEQ ID SEQUENCE Description NO 13 EIVLTQSPGTLSLSPGERATLSCRASQSITGSYLA WYQQKPG Amino acid sequence of the scFv of QAPRLLITGASSW ATGIPDRFSGSGSGTDFTLTISRLEPEDFA BiS3Ab-VEGF HlRK-ANG-2 VYYCQQYSSSPITFGCGTRLEIKGGGGSGGGGSGGGGSGGG GSQVQLVESGGGVVQPGRSLRLSCAASGFTfTNYGMHWVR QAPGKCLEWV A VISHDGNNKYYVDSVKGRFTISRDNSKNT LYLQMNSLRAEDTAVYYCAREGIDFWSGLNWFDPWGQGT J,VTVSS 14 GAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGCCTG Nucleotide sequence of the scFv of AGCCCTGGCGAGAGAGCCACCCTGAGCTGCCGGGCCAGC Bi83Ab-VEGF HlRK-ANG-2 CAGTCCATCACCGGCAGCTACCTGGCTTGGTATCAGCAG AAGCCCGGACAGGCCCCCAGACTGCTGATCACCGGCGCT TCCAGCTGGGCCACCGGCATCCCCGACAGATTCAGCGGC AGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGA CTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAG TACAGCAGCAGCCCCATCACCTTCGGAtgcGGCACCAGGC TGGAGATCAAGGGCGGAGGGGGCTCTGGGGGAGGGGGC AGCGGCGGCGGAGGATCTGGGGGAGGGGGCAGCCAGGT GCAGCTGGTCGAGTCTGGCGGCGGAGTGGTGCAGCCCGG CAGAAGCCTGAGACTGAGCTGCGCCGCCAGCGGCTTCAC CTTCACCAACTACGGCATGCACTGGGTCCGCCAGGCCCCT GGCAAGtGCCTGGAGTGGGTGGCCGTGATCAGCCACGAC GGCAACAACAAGTACTACGTGGACAGCGTGAAGGGCAG ATTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTA CCTCCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGT GTACTACTGCGCCAGAGAGGGCATCGACTTTTGGAGCGG CCTGAATTGGTTCGACCCCTGGGGCCAGGGCACCCTGGT GACCGTGTCCAGC 15 GGGGSGGGGSGGGGSGGGGS Amino acid sequence of the linker within the scFV 16 GGGGSGGGGS Amino acid sequence of the linker between the CH3 domain and the scFv 17 WYEMY HCDRl amino acid sequence of the first binding domain of BiS3Ab- VEGF HlRK-ANG-2 18 SISPSGGWTMYADSVKG HCDR2 amino acid sequence of the first binding domain of BiS3Ab- VEGF HlRK-ANG-2 19 PLYSSDGLSAGDI HCDR3 amino acid sequence of the first binding domain of BiS3Ab- VEGF HlRK-ANG-2 20 RASQSVHSSYLA LCDRl amino acid sequence of the first binding domain of BiS3Ab- VEGF HlRK-ANG-2 21 GASSRAT LCDR2 amino acid sequence of the first binding domain of BiS3Ab- VEGF HlRK-ANG-2 21 SEQ ID SEQUENCE Description NO 22 QQSYRTPS LCDR3 amino acid sequence of the first binding domain of BiS3Ab- VEGF HlRK-ANG-2 23 GFTFTNYGMH HCDRl amino acid sequence of the second binding domain of BiS3Ah- VEGF HlRK-ANG-2 24 VISHDGNNKYYVDSVKG HCDR2 amino acid sequence of the second binding domain of BiS3Ab- VEGF HlRK-ANG-2 25 EGIDFWSGLNWFDP HCDR3 amino acid sequence of the second binding domain of BiS3Ab- VEGF HlRK-ANG-2 26 RASQSITGSYLA LCDRl amino acid sequence of the second binding domain of BiS3Ab- VEGF HlRK-ANG-2 27 GASSWAT LCDR2 amino acid sequence of the second binding domain of BiS3Ab- VEGF HlRK-ANG-2 28 QQYSSSPIT LCDR3 amino acid sequence of the second binding domain of BiS3Ab- VEGF HlRK-ANG-2 Examples For the experiments described herein various antibodies were used, including MEDI3617 (hit J Oncol. 2012 May;40(5): 1321-30), Avastin® (Ferrara, Net al. Biochem Biophys Res Comm, 333:328-335, 2005), 06-31 (Liang, WC et al. J Biol Chem, 281: 951-961, 2006), B20-4.1 (Liang, WC et al. J Biol Chem, 281: 951-961, 2006), and an isotype control, designated R347, as a monospecific or a bispecific antibody as needed. An anti-VEOF IgO 1 antibody capable of binding all VEOF isoforms that is not cross-reactive with mouse can used as a positive control for some binding and functional studies. Where cross reactivity to mouse VEOF is needed the antibodies 06-31 and B20-4.1 can be used as a positive control.

EXAMPLE 1- FORMAT AND SEQUENCE OF BS3AB-VEOF H1RK-AN02.

BiS3Ab-VEOF HlRK-AN0-2 was designed to concurrently reduce one or more biological activities of VEOF-A and AN0-2 by reducing binding to their receptors, VEOFR and Tie2 respectively. Figure 2 is a schematic diagram of BiS3Ab-VEOF HlRK-AN0-2. The bispecific bivalent antibody is comprised of a full-length IgO molecule with a scFv linked to the C-terminus of each heavy chain as previously described by Dimasi et. al. (J Mol Biol. 2009). The binding specificity of the Fab region is anti-VEOF-A (first binding domain) and the scFv is anti-AN0-2 22 (second binding domain). The entire nucleotide sequence encoding the light chain of the first binding domain is shown in Figure 3. The translated amino acid sequence and the light chain variable region amino acid sequence is also shown in Figure 3. The anti-VEGF light chain was germline corrected at position 107 by mutating a threonine to lysine. The germline corrected antiVEGF sequence is referred to as HlRK. The complete nucleotide sequence of the heavy chain is shown in Figure 4 and the corresponding amino acid sequence is shown in Figure 5. The amino acid sequence of the heavy chain sequence can be further divided into the heavy chain variable region of the first binding domain, the heavy chain constant region including the CHl, CH2 and CH3 domain, the connecting glycine serine linker, the variable light chain of the second binding domain, the scFv glycine serine linker and the variable heavy region of the second binding domain.

EXAMPLE 2 - TRANSIENT TRANSFECTION Transient transfection of BiS3Ab-VEGF HlRK-ANG-2 and the parental antibodies were carried out in HEK 293F suspension cells cultured in FreeStyle ™ serum-free media (In vitro gen) at 120 rpm, 37° C and 8% CO2. The cells were split to 0.7 x 106 one day prior transfection. 300 μL of 293fectin TM transfection reagent (Invitrogen) and 200 μg of the DNA was separately diluted into 5mL of Opti-MEM® I Reduced Serum Medium (Invitrogen) and incubated for five minutes at room temperature. The DNA and 293fectin TM mixture was combined and incubated for an additional 30 minutes and then added to 300 mL of 1X106 HEK 293F cells per mL. The volume of the transfected culture was doubled every third day with FreeStyle ™ serum-free media. The culture was harvested on the eleventh day by centrifugation for 10 minutes 1500 X g and 0.2 mM filtered (Eppendorf).

Expression of BiSAb-VEGF HlRK-ANG-2 and parental antibodies were monitored using a protein A binding method. An aliquot of the cultured media was 0.2 μm filter (Eppendor±) and loaded onto a protein A column (POROS® A 20 μm Column, 4.6 x 50 mm, 0.8 mL ) using a HPLC system (Agilent 1100 Capillary LC). The column was washed with lX PBS pH 7.2, and antibodies were eluted with 0.1 % phosphoric acid (pH 1.8). The area under the eluted peak, determine by integrating the UV signal at A280 nm, was measured and used to calculate the expression level by compared to a known IgG standard. Table 1 shows the expression level of the parental antibodies and BiSAb-VEGF HlRK-ANG-2. 23 Table 1 Anti-VEGF mAb Anti-ANG-2 mAb BiSAb-VEGF HlRK-ANG-2 Transient expression 195 165 174 (after day 10 in 293 F (mg/L) EXAMPLE 3 - PROTEIN PURIFICATION AND CONCENTRATION DETERMINATION Antibodies were purified by standard protein A affinity chromatography methods. One liter of conditioned media was centrifuged at 1500 x g for 10 minutes and 0.2 μM vacuum filtered (Nalgene ). The filtered supernatant was loaded onto a mAbselect TM protein A columns (GE) using an Akta Explorer (GE). The protein A column was equilibrated with 20 column volumes of lX PBS, pH 7.2 and the filtered culture media was loaded using a flow rate of 5 mUmin. Unbound material was removed by using 20 column volumes of lX PBS, pH 7.2. Antibody elution was carried out using 10 column volumes of 0.1M glycine, 150 mM sodium chloride pH 3.2. The elution was monitored using absorbance of 280 nm. The protein A eluted antibodies were immediately neutralized by using 1/10 of volume per fraction of 1 M Tris-HCI pH 7.0. The antibodies were then filtered using a 0.22 μM syringe filter (Nalgene). The concentration of the purified antibodies was determined by reading the absorbance at 280 nm using a NanoDrop (NanoDrop) and an extinction coefficient of 1.4 M-1cm- 1.

Aggregate generated during the expression of the BiSAb-VEGF HlRK-ANG-2 can be efficiently removed by Ceramic Hydroxyapatite type II (GE) purification. The CHT column was pre-conditioned with five column volumes of lM sodium hydroxide and neutralize to pH 7.2 with lX PBS pH 7.2 at 5 mL/min. 20 column volumes of buffer A (20% lX PBS, pH 7.2 in sterile water) was used to equilibrate the column prior to use. BiSAb-VEGF HlRK-ANG-2 protein A eluant was directly loaded on the CHT column and washed with 20 column volumes of buffer A.

The monomer fraction was eluted with 15 % buffer A and 85% buffer B (5X PBS, pH 7.2) for 15 column volumes. The aggregate was eluted using 100% buffer B. A representative elution profile is shown in Figure 6. The monomer fraction was dialyzed overnight in lX PBS, pH 7.2.

Monomeric content of the BiS3Ab-VEGF HlRK-ANG-2 was measured after the protein A purification to determine the aggregate level and if a polishing step is needed. Analytical sizeexclusion chromatography (SEC-HPLC) was carried out using an Agilent 1100 HPLC (Agilent) 24 with a TSK GEL G3000SWXL column (Tosoh Bioscience). 250 μg ofbispecific antibodies were used for the analysis. The mobile phase used was 0.1 M sodium sulfate, 0.1 M sodium phosphate pH 6.8, and antibodies were monitored using an absorbable of 280 nm. Chemstation software (Agilent) was used for the analysis and the figures were prepared using Prism5 software (GraphPad). A representative monomeric content after protein purification and after ceramic hydroxyapatite purification is shown in Figure 7. At least 12% of aggregates in BiS3Ab-VEGF HlRK-ANG-2 can efficiently be removed by using ceramic hydroxyapatite chromatography.

EXAMPLE 4-ANALYTICAL CHARACTERIZATION OF BISAB-VEGF HlRK-ANG-2 BiS3Ab-VEGF H1RK-ANG-2 was analyzed by reducing and non-reducing SDS-PAGE. 2 μ.g of protein, anti-VEGF or BiS3Ab-VEGF HlRK-ANG-2, in 15 μL of l X PBS pH 7.2 and mixed with 5 μL of LDS-PAGE loading buffer, with m1d without IX NuPAGE reducing agent (lnvitrogen). 10 μL of the Novex Sharp Pre-Stained Protein Standard (lnvitrogen) was used as a protein ladder. The samples were heated at 70° C for 10 minutes, spun down at 13,500 rpm using a benchtop centrifuge and loaded onto 4 - 12% Nupage gel (Invitrogen). Electrophoresis was carried out in MOPS buffer at 200 volts for one hour. The SDS-PAGE gels were stained with SimplyBlue TM SafeStain (lnvitrogen) and de-stained in water overnight. A representative SDSPAGE gel is shown in Figure 8.

Imaged capillary isoelectric focusing of BiS3Ab-VEGF HlRK-ANG-2 was performed using an iCE2 analyzer (ProteinSimple). The pharmalytes pH 3 10 and 8 10.5 was obtained from Sigma. The FC cartridge Chemical Testing Kit for the performance evaluation of the iCE3 Analyzer, including anolyte (80 mM phosphoric acid in 0.1 % methyl cellulose), catholyte (100 mM sodium hydroxide in 0.1 %% methyl cellulose), 0.5% methylcellulose, hemoglobin and ampholytes and pl markers in 0.35<¾, methyl cellulose were purchased from ProteinSimple. 5.85 and 9.46 pl markers were obtained from ProteinSimple. The FC cartridge separation used was purchased from ProteinSimple BiS3Ab-VEGF HlRK-ANG-2 was prepared at 1 mg/mL in deionized water. 50 μl of l mg/ml Bs3Ab-VEGF-Ang2 solution, 2 1,tl of 5.85 pl marker, 2 μl of 9.46 pl marker, 140 1,tl of 0.5% methylcellulose, 2 1-tl of phannalytes 3-10 and 6 μl of 8-10.5 pharmalytes were combined; vortex for 45 sec and centrifuged at 10,000 rpm for 3 minutes.

Sample was introduced to the capillary using an autosampler (ProteinSimple). Sample separation was performed by pre-focus at 1000 kV for 1 minute/s followed by 3000 kV for 7 minute/s.

Detection was carried out with a deuterium lamp detector at 280 nm. Data were analyzed and figures were prepared using the iCE280 analyzer software. Representative focusing of BiS3AbVEGF HlRK-ANG-2 is shown in Figure 9; the pl of the protein is indicated.

BiS3Ab-VEGF HlRK-ANG-2 was dialyzed three times overnight in 25 mM Histidine pH 6.0 prior to differential scanning calorimetry analysis using a VP-DSC (Microcal). The final dialysis buffer was used for reference scans to obtain a stable base line for reference subtraction.

The reagents were degassed for a minimum of two minutes and proteins were diluted to 1 mg/mL in reference buffer and scanned at 1 °C/min from 20 °C to 110 °C using a 16 seconds filter period.

Representative transition temperatures for BiS3Ab-VEGF HlRK-ANG-2 are shown in Figure 10.

EXAMPLE 5 - BINDING AFFINITY OF BIS3AB-VEGF H1RK-ANG-2 TO ANG-2 BiS3Ab-VEGF HlRK-ANG-2 binding affinity to ANG-2 was determined. Equilibrium binding constants (KD) were obtained from measurements made on KinExA 3000 and 3200 instruments (Sapidyne Instruments, Boise, ID). Human ANG-2 (huAng2) protein was coated onto UltraLink® Biosupport beads (PIERCE, Rockford, IL) at concentrations of 5 mg/mL and 30 mg/mL in coating buffer (50 mM sodium carbonate buffer, pH 9). Coated beads were then separated (gentle pulse spin) from unreacted huAng2 protein solution, and blocked with lM Tris, pH 8, containing BSA at 10 mg/mL) for approximately 15 minutes at room temperature. After this, the bead slurry was spun to remove the blocking solution, and then the block step was repeated for approximately 2 hours using fresh block buffer, and stored at 4 ° C until used. Prior to use, the huAng2-coated beads were transferred to a bead vial, resuspended in approximately 27 mLs of instrument buffer (HBS-P buffer, pH 7.4; contains lOmM HEPES, 0.15M NaCl, 0.005% P20+0.02% NaN3), and affixed to the KinExA instrument. Briefly, solutions of BiS3Ab-VEGF HlRK-ANG-2 were prepared at 4 pM, 40 pM and 400 pM in instrument buffer (HBS-P buffer), then dispensed into three separate series of 13 tubes. These concentrations of bi specific antibody were chosen to allow measurements to be made under both receptor- and KD -controlled conditions, which would allow for more rigorous estimations of reagent activity and affinity, respectively. Two-fold serial dilutions ofhuAng2 protein were then titrated across nine of the tubes containing the bispecific solutions, followed by IO-fold-dilutions across two more tubes, leaving one tube as the bispecific-only, "zero" control. In so doing, this yielded concentration series' of huAng2protein that ranged from 39 fM-2 nM (4 pM bispecific experiment), 156 pM - 8 nM (40 26 pM and 400 pM bispecific experiments). Based on theory curve simulations available through the vendor software (Sapidyne Instruments, Boise, Idaho), the mixtures were incubated 1 - 3 days at room temperature to allow binding to reach equilibrium. At the end of this time, signal-testing experiments were conducted to determine the appropriate run conditions for each set of measurements. Detection of free antibody was made possible using a species-specific, secondary antibody reagent (Goat Anti-Human IgG (H+L)-DyLight649, Part #109-495-088, Jackson ImmunoResearch Laboratories), employed at 0.75 mg/mL or 1.0 mg/mL in instrument buffer containing BSA at 1 mg/mL. Data obtained from all sets of measurements was then simultaneously fitted to a one-site binding model using the software's' n-Curve analysis feature to obtain the equilibrium binding constant (KD) as reported in Table 2.

Table 2 JS-u, nM (95% CI) Binding *Ko.:..l!M Fit (Alternate Ligand (Std. Aff. Site Error model - model - ref Aetivitr [Ligand]) ref [lgG]) BiSAb- VEGF 24.0 (17.3- huVEGJ<' 3.06% 80% 30.1 HlRK- 34.2) ANG-2 BiSAb- VEGF 23.3 (11.2- huAng2 3.67% 536% 4.35 HlRK- 41.7) ANG-2 BiSAb-VEGF HlRK-ANG-2 binding affinity to VEGF was determined. As with the anti-hu-Ang2 measurements, equilibrium binding constants (KO) measurements were performed on KinExA 3000 and 3200 instruments (Sapidyne Instruments, Boise, ID). Human VEGF (huVEGF) protein was coated onto UltraLink® Biosupport beads (PIERCE, Rockford, IL) at concentrations of 3 mg/mL, 30 mg/mL and 50 mg/mL in coating buffer (50 mM sodium carbonate buffer, pH 9).

Coated beads were then separated (gentle pulse spin) from unreacted hu VEGF protein solution, 27 and blocked with IM Tris, pH8, containing BSA at 10 mg/mL) for approximately 15 minutes at room temperature. After this, the bead slurry was spun to remove the blocking solution, and then the block step was repeated for approximately 2 hours using fresh block buffer, and stored at 4 ° C until used. Prior to use, the huAng2-coated beads were transferred to a bead vial, resuspended in approximately 27 mLs of instrument buffer (lOmM HEPES+300mM NaC1+5mM CaC12+0.05% P20+0.02% NaN3, pH8), and affixed to the KinExA instrument. Briefly, solutions BiSAb-VEGF HlRK-ANG-2 were prepared at 10 pM, 100 pM and 2.5 nM in instrument buffer, then dispensed into three separate series of 13 tubes. These concentrations of bispecific were chosen to allow measurements to be made under both receptor- and KD -controlled conditions, which would allow for more rigorous estimations of reagent activity and affinity, respectively. Two-fold serial dilutions of huVEGF protein were then titrated across nine of the tubes containing the bi specific solutions, followed by IO-fold-dilutions across two more tubes, leaving one tube as the bispecificonly, "zero" control. In so doing, this yielded concentration series' of hu VEGF protein that ranged from 78 JM - 4 nM (10 pM bispecific experiment), 488 JM - 25 nM (100 pM bispecific experiment), and 3.91 pM - 200 nM (2.5 nM bispecific experiment). Based on theory curve simulations available through the vendor software (Sapidyne Instruments, Boise, Idaho), the mixtures were incubated 1 - 4 days at room temperature to allow binding to reach equilibrium. At the end of this time, signal-testing experiments were conducted to determine the appropriate run conditions for each set of measurements. Detection of free antibody was made possible using a species-specific, secondary antibody reagent (Goat Anti-Human IgG (H+L)-DyLight649, Part #109-495-088, Jackson ImmunoResearch Laboratories), employed at 0.75 mg/mL, 1.0 mg/mL or 2 mg/mL in instrument buffer containing BSA at 1 mg/mL. Data obtained from all sets of measurements was then simultaneously fitted to a one-site binding model using the software's' nCurve analysis feature to obtain the equilibrium binding constant (KD) as reported above in Table 2.

EXAMPLE 6-CONCURRENT BINDING BY BIS3AB-VEGF HlRK-ANG-2 TO ANG-2 AND VEGF165 Concurrent binding experiments were performed on a Biacore 3000 (GE Healthcare) at 25° C using lOnM of VEGF165, 100 nM of Ang2 and 10 nM of Bs3Ab-VEGF-Ang2 in 10 mM Acetate, pH 5 and immobilized to on CMS sensorchip surfaces, using standard amine coupling 28 protocols provided by the manufacturer (GE Healthcare). Using the solutions BiSAb-VEGF HlRK-ANG-2 immobilized chip, 100 nM of VEGF and a mixture of lO0nM of VEGF and 500nM of ANG-2 were prepared in HBS buffer (GE Healthcare). The VEGF solution was injected at a flow rate of 30 mUmin for 500 seconds. An additional injection of VEGF or the VEGF/ ANG-2 mixture was injected for 250 seconds after the first injection. A similar experiment was done by first injecting 500nM of ANG-2 followed by another ANG-2 injection of the VEGF/ANG-2 mixture. To further confirm concurrent binding, the VEGF and ANG-2 coated chips were used.

For the VEGF165 smface, 50nM of BiSAb-VEGF HlRK-ANG-2 was flowed at 30 mL/min for 600 seconds followed by a second injection of 50 nM BiSAb-VEGF HlRK-ANG-2 and 500nM of ANG-2. The ANG-2 surface was used for a simi1ar experiment. 50nM of BiSAb-VEGF Hl RKANG- 2 was used for the initial injection for 500 seconds at 30 mL/min. The second injection was done using either 50nM of BiSAb-VEGF HlRK-ANG-2 of a mixture of BiSAb-VEGF HlRKANG- 2 and lOOnM of VEGF165. The data were analyzed using BIAevaluation (GE healthcare) and the figure was prepared using Prism 5 (Graph Pad) and representative results are shown in Figure 11.

BiSAb-VEGF HlRK-ANG-2 antibodies were also screened for concurrent binding to VEGF and ANG-2 in a dual binding ELISA. Maxisorp plates (Nunc, Cat #439454) were coated with 100 μl of 1.0 μg/mL human or mouse VEGF (Peprotech) diluted in PBS without Ca++ or Mg++ and refrigerated overnight. Plates were decanted, then blocked for 1.5 hours with 200 μl of Blocking Buffer containing 3% BSA (Sigma, Cat #A-3059) and 0.1 % Tween-20 in lX PBS on a plate shaker. Plates were washed 3 times with l X PBS containing 0.1 % Tween-20. 50 μ1 of 60 nM and serial dilutions of BiSAb-VEGF HlRK-ANG-2 bispecific antibodies, Ang-2 antibody, or bispccific with r347 isotypc control arm (BS3Ab-r347-Ang2) in blocking buffer were added in duplicate and incubated for 1 hour on a plate shaker. Plates were washed 3 times with wash buffer, then 50 μ1 of 1 μg/ml human or mouse Ang2-biotin (R&D Systems) in blocking buffer was added to each well and incubated at room temperature for 1 hour on a plate shaker. Plates were washed, then 50 μl of 1:15,000 streptavidin HRP (Pierce) was added for 1 hour at room temperature on a plate shaker. Plates were washed, then developed by adding 50 μl ofTMB solution (KPL) to each well, then stopping the reaction with 50 μl of IM phosphoric acid. Plates were read at 450nm using a microplate reader. EC50 values were determined using non-linear regression analysis (log dose response, 4-parameter fit curves) in GraphPad Prism, version 5.01 (San Diego, CA). 29 Representative results are shown in Figure 12A (human) and Figure 12B (mouse). Strong concmTent binding to human and mouse VEGF and ANG-2 was exhibited by BiSAb-VEGF HlRK-ANG-2 (EC50 10.8 pM and 103.8 pM, respectively), compared to the Ang2 antibody (MEDI3617) alone and BS3Ab-r347-Ang2 which showed weak binding in this assay, denoting failure to bind VEGF and ANG-2 at the same time.

EXAMPLE 7 - SCREENING OF BISAB-VEGF HlRK-ANG-2 FOR REDUCED VEGF121 BINDING Antibodies were screened for VEGF121 binding in an ELISA format. 96-well half well maxisorp plates were coated with 25 μ1 of 2 μg/mL human VEGF (Peprotech) diluted in PBS without Ca++ or Mg++ and refrigerated overnight. Plates were decanted, then blocked for 1.5 hours at 37° C with 180 μI of Blocking Buffer containing 3% BSA (Sigma, Cat #A-3059) and 0.1% Tween-20 in lX PBS. Plates were washed 3 times with 1 X PBS containing 0.1% Tween- 20. 50 μ1 serial dilutions of anti-VEGF antibodies, Avastin® (positive control; anti-VEGF antibody) and r347 (negative control) in blocking buffer were added in duplicate and incubated at 37° C for 1 hour. Plates were washed 3 times with wash buffer, then 50 μ1 of 1 :5000 goat antihuman HRP IgG H+L (Jackson Immunoresearch) was added to each well and incubated at room temperature for 1 hour. Plates were developed by adding 50 μ1 of TMB solution (KPL) to each well, then stopping the reaction with 50 μ1 of lM phosphoric acid. Plates were read at 450nm using a m.icroplate reader. Representative results are shown in Figure 13. BiSAb-VEGF HlRK-ANG-2 lacked VEGF121 binding, in contrast to the positive control B20-4.1.

EXAMPLE 8 - SCREENING OF BISAB-VEGF HlRK-ANG-2 FOR REDUCED VEGF189 BINDING BiSAb-VEGF Hl RK-ANG-2 was screened for binding to VEG Fl 89 in an ELISA format. 96-well half well maxisorp plates were coated with 25 μI of 2 μg/mL human VEGF189 (R&D Systems) diluted in PBS without Ca++ or Mg++ and refrigerated overnight. Plates were decanted, then blocked for 1.5 hours at 37° C with 180 μI of Blocking Buffer containing 3% BSA (Sigma, Cat #A-3059) and 0.1% Tween-20 in lX PBS. Plates were washed 3 times with 1 X PBS containing 0.1 % Tween-20. 50 μ1 of 6.7 nM and serial dilutions of BiSAb-VEGF HlRK-ANG-2, 06-31 (positive control) and BS3Ab-r347-Ang2 (negative control) in blocking buffer were added in duplicate and incubated at 37° C for 1 hour. Plates were washed 3 times with wash buffer, then 50 μl of 1:5000 goat anti-human HRP IgG H+L (Jackson Immunoresearch) was added to each well and incubated at room temperature for 1 hour. Plates were developed by adding 50 μl of TMB solution (KPL) to each well, then stopping the reaction with 50 μl of lM phosphoric acid. Plates were read at 450nm using a microplate reader. Figure 14 shows representative results for BiSAbVEGF HlRK-ANG-2. BiSAb-VEGF HlRK-ANG-2 showed 5 fold lower binding to VEGF 189 compared to the positive control G6-31(EC500.057 nM vs 0.0096 nM).

EXAMPLE 9 - FUNCTIONAL ASSAYS TO DETERMINE POTENCY OF VEGF-ANG2 BISPECIFJC ANTIBODIES BiSAb-VEGF HJ RK-ANG-2 were screened in functional bioassays to detennine ability to reduce p VEGFR2 and pTie2 in cell lines with humm1, mouse and cyno receptors. Ad293- Hu VEGFR2 (Cl. E2), Hek293-Tie2, Ad293-muVEGFR2-muAng2 cells (Cl. DlO), Ad293- cynoVEGFR2-cynoAng2 cells (Cl. SB5) and Ad293-cynoTie2 cells (Cl. D12) were generated from stable transfections. Cells were seeded at subconfluency in 96-well poly-D-Lysine tissue culture plates (Costar, Tewksbury, MA) with 100 μl DMEM + 10% FBS (Life Technologies, Carlsbad, CA) and incubated overnight at 37° C and 5% CO2. The next day, media was aspirated and replaced with 50 μl starvation media (DMEM + 0.2% FBS + 0.1 % BSA) and cells were returned to the incubator overnight. At 24 hours, media was aspirated and 2660 nM (2X concentration) antibodies, BiSAb-VEGF HlRK-ANG-2 and BS3Ab-HPV-r347 negative control were serially diluted in serum free DMEM + 0.1 % BSA and added in duplicate to the plate for 30 minutes at 3 7° C. Then, 50 μI of 12 μg/ml human, mouse (R&D Systems) or cyno Ang2 (in-house preparation)+ 20nM of human, mouse (Peprotech, Rocky Hill, NJ), or cyno (in-house preparation) VEGF ( 4X) mixed 1: 1 was then added to the wells and incubated at 4 ° C for 30 minutes. Plates were then incubated at 37° C for an additional 7 minutes. Plates were decanted and we11s lysed with 55 μlice cold RIPA lysis buffer (Boston BioProducts, Boston, MA) containing protease and phosphatase inhibitors (Life Technologies, Carlsbad, CA). Human, cyno and murine pVEGFR2 were detected using pVEGFR2 whole cell lysate kits (Meso Scale Diagnostics, Rockville, MD).

Human and cyno pTie2 was determined using a protocol developed using the Meso Scale Diagnostics (MSD) platform. MSD high bind plates were coated overnight with 2 μg/ml of Tie2 antibody clone 16 (Abeam, Cambridge, MA). The next day, plates were washed with tris buffered 31 saline (TBS) only and blocked with 3% MSD Blocker A+ 0.05% Tween 20 (Sigma, St Louis, MO) in TBS for 1 hour at room temperature with rotary shaking. Plates were washed with TBS + 0.05% Tween 20 and lysates were added to plate, and then incubated for 1 hour at room temperature with rotary shaking. Plates were washed and 1 μg/ml of anti-human Tie2 antibody (AF2720, R&D Systems, Minneapolis, MN) was added for 1 hour at room temperature with rotary shaking. Plates were washed, then 1 μg/ml sulfo-tag goat anti-rabbit secondary antibody (MSD, Rockville, MD) was added to the plates for 1 hour at room temperature with rotary shaking. Plates were washed, Read Buffer T (MSD, Rockville, MD) was added, then plates read immediately using a Sector lmager 6000 (MSD, Rockville, MD).

Murine pTie2 was determined using a protocol developed using the Meso Scale Diagnostics (MSD) platfonn. MSD streptavidin plates were blocked with 3% MSD Blocker A + 0.05% Tween 20 (Sigma, St Louis, MO) in TBS for 1 hour at room temperature with rotary shaking. Plates were washed with TBS + 0.05% Tween 20 and then 25 μI/well of 2 μg/ml Biotin anti-mouse Tie2 antibody (Biolegend# 124006) in blocking buffer was incubated for 1 hour at room temperature with rotary shaking. Plates were decanted and washed 3 times. Then, 25 μI/well of lysate was added per well in duplicate and incubated at room temperature for 2 hours on a plate shaker. Plates were washed, then 25 μ1 of sulfo-tag PY20 (MSD) was added per well and incubated for 1 hour at room temperature on a plate shaker. Plates were washed, then 150 μl of 2X MSD read buffer Twas added and plates were read immediately using a Sector Imager 6000 (MSD, Rockville, MD).

Percent phosphorylation for pTie2 and pVEGFR2 was calculated by the formula: [average RLU (test samplc)/avcragc RLU (no antibody)]* 100. Representative results arc shown in Table 3. BiSAb-VEGF HlRK-ANG-2 potently reduced human, mouse and cyno pVEGFR2 and pTie2 showing that both arms are functional in the bispecific format. The Anti-ANG-2 activity of BiSAb-VEGF HlRK-ANG-2 showed remarkably greater activity when compared to the ANG-2 antibody (MEDI3617) used to the make the scFV anti-ANG-2 ofBiSAb-VEGF HlRK-ANG-2. 32 Table 3 Molecule Hu pVEGFR2 EC50 (nM) BS3Ab-VEGF H1RK-An 2 0.087 2.29 5.95 12.16 0.131 3.47 H1RK 0.071 not tested not tested not tested 0.099 not tested Controls B20-4.1 not tested not tested 26.25 not tested 4.25 not tested An 2 antibod not tested 2.65 nottested 137 not tested 33.17 BS3Ab-HPV-r347 (·) control NIA N/A NIA NIA NIA NIA EXAMPLE 10- IN VIVO ACTIVITY OF BISAB-VEGF HlRK-ANG-2 BiSAb-VEGF H lRK-ANG-2 was tested in vivo for efficacy in a 786-0 renal cell carcinoma and a BxPC3 pancreatic carcinoma model which included casting of the BxPC3 tumors to illustrate anti-angiogenesis within the tumor compartment. In addition, retinal vasculogenesis models were performed to further demonstrate the activity of BiSAb-VEGF HlRK-ANG-2. Even more, a model of thrombocytopenia was performed in mice to detennine if less toxicity occurred with BiSAb-VEGF HlRK-ANG-2 compared to an anti-VEGF positive control antibody (G6-31) that binds to all isoforms of VEGF. Finally, renal pathology was evaluated.

For the 786-0 renal cell carcinoma model, tumor fragments from a human renal cancer cell line, 786-0, were implanted subcutaneously into the right flank of nude mice. After tumor volume reached approximately 200 mm3 , dosing was initiated. Mice were treated twice per week for a total of 6 doses (triangles on axis). Doses were normalized based on molecular weight. BiSAbVEGF HlRK-ANG-2 was more effective at reducing tumor growth compared to either the ANG- 2 antibody (MEDI3617) or the VEGF antibody (Avastin®) alone. P-value = 0.03 as determined by one-way ANOVA analysis Graphpad Prism version 5.01 (San Diego California).

Representative data arc shown in Figure 15.

For the BxPC3 pancreatic carcinoma model, BxPC3 tumor fragments were implanted subcutaneously into the right flank of female SCID mice. After tumor volume reached approximately 200 mm3, dosing was initiated. Mice were dosed twice per week for a total of 6 doses (triangles on axis). Doses were normalized based on molecular weight. BiSAb-VEGF HlRK-ANG-2 was more effective at reducing tumor growth compared to either the ANG-2 33 antibody (MEDI3617) or the VEGF antibody (Avastin®) alone. P-value=0.02, as determined by one-way ANOV A analysis Graphpad Prism version 5.01 (San Diego California). Representative data are shown in Figure 16.

In addition to tumor volume, tumor vasculature was evaluated using tumors from BxPC3 pancreatic carcinoma model work. Mice were dosed with heparin to prevent blood clotting 15 minutes prior to euthanasia. A solution of O. lmM sodium nitroprusside was perfused at a rate of approximately 6 mUmin. Microfil MV-122 was prepared by mixing 8 mL of latex, 10 mL of diluent and 900 uL of cure. After the mixture settled (approximately 1 minute) it was perfused at a rate of 2mL /min until a total volume of 17 mL was administered. After 60-90 minutes the tumor was dissected and immersed in 10% NBF for 24 hours. The sample was then transferred through an ethanol gradient (25% ETOH/PBS, 50% ETOH/PBS, 75% ETOH/PBS, 95% ETOH, and then 100 % ETOH) for 24 hours each gradient level. After the final incubation the sample was immersed in methyl salicylate to clear the dehydrated tumor sample before imaging by light microscopy. Tumor vasculature was reduced in mice with BiSAb-VEGF HlRK-ANG-2.

Representative data are shown in Figure 17.

In addition to the models described above, BiSAb-VEGF HlRK-ANG-2 was evaluated in a retinal angiogenesis model. Using this model CDl mice were intraparatoneally dosed at birth, days 1, 3, and 5. At day 8 the mice were anesthetized and were infused with fluorescein-labeled dextran. Eyes were removed and fixed with 10% formalin before preparation of flat mounts. Flat mounts were examined by fluorescence microscopy.

Neonatal retinal angiogenesis is comprised of two processes, namely, vessel migration from the optic nerve (Figure 18 dot-arrow) to the edge of the retina and branching. BiSAb-VEGF HlRK-ANG-2 demonstrated reduced vessel migration compared to the extent of migration without BiSAb-VEGF HlRK-ANG-2 present. Representative results are shown in Figure 18.

BiSAb-VEGF H1 RK-ANG-2 demonstrated reduced vessel branching compare to the extent of branching without BiSAb-VEGF HlRK-ANG-2 present. Representative data are shown in Figure 19.

For the thrombocytopenia model, a method was adopted from Meyer et al. (J Thromb Haemost 7:171-81, 2009). Briefly PC gamma receptor 2A transgenic mice, 8-16 weeks old were injected with premixed VEGF165, 0.6 units heparin, and antibody into the lateral tail vein. Mice were then observed for behavioural signs of distress and scored as: (-) stopped and moved 34 constantly from comer to comer, breathing normal, ( +) signs of lethargy, stopped and moved in longer duration, breathing shallow, (++) very lethargic, stopped moving, staying in mostly one side of the box, breathing deeply, ( +++) sever thrombotic event-twitching and twirling, ( ++++) death. BiSAb-VEGF HlRK-ANG-2 had reduced thrombocytopenia as compared to the antiVEGF control (G6-31). Representative data are shown in Table 4.

Table 4 Observations Score Anti-VEGF* + VEGF165 + 0.6 units Heparin Labored breathing, twitching and +++ twirling BiSAb-VEGF HIRK-ANG-2 + 0.6 units Stopped and moved with glimpses of Heparin slowing down but recovers quickly, -/+ brealhes norrnally. * Anti VEGF binds all isoforms ofVEGF Kidneys from four animals per group were examined by staining via Periodic acid-Schiff (PAS). The PAS staining was used to examine kidney pathology after 14 doses of the treatments.

There was increased mesangial matrix and thickened capillary loops (arrows) in the anti-VEGF (G6-31) treated animals compared to the BiSAb-VEGF HlRK-ANG-2. Representative are shown in Table 5 and Figures 20A - C.

Table 5 Pathology Untreated Anti-VEGF BiSAb-VEGF HlRKANG- 2 Increased mesangial matrix 0 2.75 0 Thickened capillary loops 0 2 0 Grade O == absent, Grade 1 == minimal, Grade 2 = Mild, Grade 3 = Moderate, Grade 4 == Severe, Grade 5 = Very severe 85107200 Equivalents The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiments may be practiced in many ways and the claims include any equivalents thereof.

Sequence Listing in Electronic Form In accordance with Section 111(1) of the Patent Rules, this description contains a sequence listing in electronic form in ASCII text format (file: 85107200 Seq 06-MAY-19 vl.txt).

A copy of the sequence listing in electronic form is available from the Canadian Intellectual Property Office. 36 Date Rei;ue/Date Received 2023-12-04

Claims (12)

1. 85107200 CLAIMS: 1. A bispecific antibody comprising a first binding domain comprising heavy chain complementarity determining regions 1 - 3 (HCDRl, HCDR2, and HCDR3) and light chain complementarity determining regions 1 - 3 (LCDRl, LCDR2, and LCDR3), wherein the first binding domain HCDRl, HCDR2, and HCDR3 and LCDRl, LCDR2, and LCDR3 comprise SEQ ID NOs: 17 - 22, respectively, and a second binding domain comprising an HCDRl, HCDR2, and HCDR3 and an LCDRl, LCDR2, and LCDR3, wherein the second binding domain HCDRl, HCDR2, and HCDR3 and LCDRl, LCDR2, and LCDR3 comprise SEQ ID NOs: 23 - 28, respectively.
2. 2. The bispecific antibody of claim 1, wherein the first binding domain comprises a heavy chain and a light chain comprising SEQ ID NOs: 3 and 9, respectively, and wherein the second binding domain comprises a heavy chain and a light chain comprising SEQ ID NOs: 5 and 11, respectively.
3. 3. The bispecific antibody of claim 1, wherein the bispecific antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 7.
4. 4. The bispecific antibody of claim 1, wherein the bispecific antibody comprises a formula having the parts VH-CH1-H-CH2-CH3, VL-CL, and one or more scFv, LI and/or L2, wherein the individual part are VH = a heavy chain variable domain; CHI = a heavy chain constant region domain 1; H = a hinge region; CH2 = a heavy chain constant region domain 2; CH3 = a heavy chain constant region domain 3; VL = a variable light chain domain; CL= a light chain constant domain; LI= a linker; and L2 = a linker independent of LI, wherein the formula can be: a. VH-CH1-CH2-CH3 and scFv-LI-VL-CL; b. scFv-Ll-VH-CHl-CH2-CH3 and VL-CL; c. VH-CHl-CH2-CH3-LI-scFv and VL-CL; d. VH-CHl-CH2-CH3-LI-scFv-L2 and VL-CL, wherein LI and L2 are covalently bound to CH3; e. VH-CHl-LI-scFv-L2-CH2-CH3 and VL-CL, the heavy chain can contain a hinge region or be hingeless.
5. 5. The bispecific antibody of claim 4 comprising the formula VH-CHl-CH2-CH3-LI-scFv and VLCL. 37 Date Re<;ue/Date Received 2023-12-04 85107200
6. 6. The bispecific antibody of claim 5 wherein the scFv comprises the amino acid sequence of SEQ IDNO: 13.
7. 7. A nucleic acid sequence comprising polynucleotides encoding the bispecific antibody of claim 1.
8. 8. A vector comprising the nucleotide sequence of claim 7.
9. 9. A cell comprising the vector of claim 8.
10. 10. A method of making the bispecific antibody of claim 1 comprising culturing a cell comprising the vector of claim 9.
11. 11. Use of the bispecific antibody of claim 1 to reduce angiogenesis in a subject in need thereof, wherein the subject exhibits aberrant or unwanted angiogenesis.
12. 12. Use of the bispecific antibody of claim 1 in the manufacture of a medicament to reduce angiogenesis in a subject in need thereof, wherein the subject exhibits aberrant or unwanted angiogenesis. 38 Date Re<;ue/Date Received 2023-12-04