Fusion proteins binding VEGF and TIE2 and uses thereof - Patents.com

Abstract

The present disclosure refers to a fusion protein having an antibody or antigen-binding fragment thereof against Tie-2 and a vascular endothelial growth factor (VEGF) binding domain, a method for making the fusion protein, and pharmaceutical compositions and methods for preventing or treating angiogenic diseases or modulating angiogenesis, endothelial signaling, inflammation and / or vascular leakage comprising the fusion protein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 299,177, filed January 13, 2022, U.S. Provisional Patent Application No. 63 / 310,359, filed February 15, 2022, and U.S. Provisional Patent Application No. 63 / 335,805, filed April 28, 2022. The entire disclosures of these earlier applications are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a sequence listing that has been submitted electronically in XML format. Said XML copy, created on January 11, 2023, is named "2023-01-12_01262-0004-00PCT_ST26.xml" and is 48,489 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

[0003] Field The present disclosure relates to a fusion protein comprising an anti-Tie-2 antibody or antigen-binding fragment thereof and a vascular endothelial growth factor (VEGF) binding domain, the fusion protein simultaneously binding to Tie2 and VEGF, a nucleic acid encoding the fusion protein, a vector containing the nucleic acid, a cell transformed with the vector, a method for preparing the fusion protein, and methods and compositions for modulating angiogenesis, inflammation and vascular leakage. In some embodiments, the disclosure relates to methods and pharmaceutical compositions for preventing or treating angiogenic and vascular diseases. [Background technology]

[0004] Introduction and Overview Angiogenesis occurs dynamically during the development, growth, maintenance and homeostasis of an organism through various regulatory factors. In this process, newly formed blood vessels function as transport channels for various biomaterials, such as nutrients, oxygen and hormones in the surrounding cells. Functionally and structurally abnormal blood vessels are the direct or indirect cause of the initiation and progression of various diseases. Tumor blood vessels, due to their defective functions and structures, aggravate hypoxia, leading to tumor progression and metastasis to other tissues, and poor delivery of anticancer drugs to the center of the tumor mass. Defective blood vessels are also found in various other diseases and conditions besides cancer. Examples include various eye diseases (e.g., diabetic macular edema, wet age-related macular degeneration, diabetic retinopathy), viral infections and acute inflammatory responses such as sepsis. Therefore, if a therapeutic agent capable of normalizing pathological blood vessels were available, it could be applied to the treatment of various patients / animals with vascular abnormalities.

[0005] The angiopoietin family plays an important role in the formation and maintenance of blood vessels and is composed of four angiopoietins (Ang1, Ang2, Ang3, Ang4). Angiopoietin-1 (Ang1) binds to the Tie2 receptor present on the surface of vascular endothelial cells, phosphorylating and activating the Tie2 receptor, resulting in stabilization of blood vessels and suppression of vascular leakage. On the other hand, angiopoietin-2 (Ang2) binds to the Tie2 receptor but acts as an antagonist to induce inactivation of the Tie2 receptor, inhibiting binding by Ang1, resulting in vascular destabilization and vascular leakage, thereby tending to promote the growth of new blood vessels. It has been reported that the expression level of Ang2 is highly increased in the blood of cancer patients, eye diseases, viral and bacterial infections, and inflammatory diseases (Saharinen P et al., 2017, Nature Review Drug Discovery 16:635-661). However, Ang2 is also known to act as an agonist inducing activation of the Tie2 receptor in several processes, including lymphatic vessel formation and maintenance, and Ang2 is thought to fulfill various functions depending on the situation.

[0006] To date, the development and clinical trials of various anti-Ang2 antibodies have attracted the interest of many biopharmaceutical companies (e.g., U.S. Patent Nos. 7,658,924 and 8,987,420). These Ang2 antibodies have been reported to inhibit the binding of Ang2 to Tie2, and the Ang2 neutralizing effect has been reported to prevent the formation of new blood vessels. The anti-angiogenic and anti-cancer activities of these anti-Ang2 antibodies have been demonstrated in many preclinical models, and various anti-Ang2 antibodies have been clinically tested in various cancer patients. However, their anti-cancer effects have been demonstrated to be insufficient. For example, a phase 3 clinical trial conducted by Amgen showed that the anti-cancer effect of Ang2 antibodies in ovarian cancer patients was slight (Marth C et al, 2017, Eur. J. Cancer, 70:111-121). In addition to cancer models, nesvacumab, an Ang2 neutralizing antibody, was tested in ocular patients, but failed to improve the efficacy of EYLEA® (anti-VEGF) in a phase 2 clinical trial. See, for example, the "Regeneron provides update on EYLEA(R) (aflibercept) injection and nesvacumab (ang2 antibody) combination program" press release, available at investor.regeneron.com / news-releases / news-release-details / regeneron-provides-update-eylear-aflibercept-injection-and

[0007] In contrast to the Ang2 neutralization approach mentioned above, direct Tie2 activation has also been considered as an alternative approach to inhibit angiogenesis and suppress vascular permeability. Recombinant proteins that directly bind to the Tie2 receptor and induce Tie2 phosphorylation and activation have also been developed and tested in many preclinical cancer and eye models. Examples include cartilage oligomeric matrix protein (COMP)-Angl (Cho et al., 2004, PNAS 101(15):5547-5552) and vasculotide (David S et al., 2011, Am J Physiol Lung Cell Mol Physiol 300(6):L851-L862). Although these agents have shown antiangiogenic and antipermeability activity, they tend to have very short half-lives and unstable physicochemical properties. Furthermore, a small molecule compound (AKB-9778) has been developed as an inhibitor of VE-PTP, a phosphatase that inactivates Tie2 by removing phosphate groups from phosphorylated Tie2 (Goel S et al., 2013, J Natl Cancer Inst 105(16):1188-1201). This compound indirectly increases Tie2 activity by inhibiting VE-PTP, but has the drawback of also activating other receptors. See, for example, Frye M.et al., 2015, J Exp.Med., 212(13):2267-2287; Hayashi M,et al., 2013, Nature Communication, 4:1672; and Mellberg S.et al., 2009, FASEB J., 23(5):1490-1502). Furthermore, Tie2 antibodies with agonistic activity have been developed. See, for example, U.S. Patent No. 6,365,154 and U.S. Patent Application Publication No. 20170174789. These antibodies increased endothelial cell survival and inhibited vascular leakage. Interestingly, herbal extracts have been shown to activate Tie2 activity and claimed for use as skin care cosmetics. See, for example, JP2011102273, JP2018043949, JP2015168656.

[0008] Tie2 is a receptor protein that promotes the differentiation and stabilization of blood vessels and is highly expressed in blood vessels. When activated, Tie2 receptors stabilize blood vessels and allow surrounding support cells to gather. For example, activated Tie2 in cancer blood vessels normalizes cancer blood vessels, reduces vascular leakage, eliminates increased hypoxia in tumors, and provides sufficient oxygen by increasing blood flow into tumors, increasing the delivery of other anticancer drugs and the penetration of immune cells.

[0009] Vascular endothelial growth factor (VEGF) inhibitors are the standard first-line treatment for diabetic macular edema (DME) and wet age-related macular degeneration (wAMD), but the efficacy of these treatments is limited by the significant frequency of intraocular administration and a significant subpopulation of poor responders. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Patent No. 7,658,924 [Patent Document 2] U.S. Patent No. 8,987,420 [Patent Document 3] U.S. Patent No. 6,365,154 [Patent Document 4] US Patent Application Publication No. 2017 / 0174789 [Patent Document 5] Patent No. 2011102273 [Patent Document 6] Patent No. 2018043949 [Patent Document 7] Patent No. 2015168656 [Non-patent literature]

[0011] [Non-Patent Document 1] Saharinen P et al.,2017,Nature Review Drug Discovery 16:635-661 [Non-Patent Document 2] Marth C et al,2017,Eur.J.Cancer,70:111-121 [Non-Patent Document 3] Cho et al., 2004, PNAS 101(15):5547-5552 [Non-Patent Document 4] David S et al.,2011,Am J Physiol Lung Cell Mol Physiol 300(6):L851-L862 [Non-Patent Document 5] Goel S et al.,2013,J Natl Cancer Inst 105(16):1188-1201 [Non-Patent Document 6] Frye M. et al.,2015,J Exp.Med.,212(13):2267-2287 [Non-Patent Document 7] Hayashi M,et al.,2013,Nature Communication,4:1672 [Non-Patent Document 8] Mellberg S. et al.,2009,FASEB J.,23(5):1490-1502 Summary of the Invention [Problem to be solved by the invention]

[0012] Therefore, there is a need for improved methods for modulating angiogenesis, endothelial signaling, inflammation and / or vascular leakage, and / or for treating angiogenic diseases. For example, there is a need for treatments for angiogenic diseases, such as cancer, with improved efficacy, affinity, half-life, stability, pharmacodynamics, durability, and / or reduced frequency of treatment to reduce patient burden and compliance issues. The present disclosure aims to meet one or more of these needs, provide other benefits, or at least provide the public with a useful option. [Means for solving the problem]

[0013] The present disclosure provides a fusion protein comprising an anti-Tie2 antibody or antigen-binding fragment thereof and a vascular endothelial growth factor (VEGF) binding domain. In some embodiments, the fusion protein binds to a Tie2 Ig3-FNIII(1-3) domain comprising SEQ ID NO:2, 3, or 4 and VEGF.

[0014] In one embodiment, the VEGF binding domain comprises a VEGF receptor extracellular domain.

[0015] In one embodiment, the VEGF binding domain is an antagonist VEGF binding domain. Thus, in one embodiment, the VEGF binding domain antagonizes (i.e., inhibits) the activity of VEGF in a cell. The VEGF binding domain achieves its antagonist effect by binding VEGF and thereby preventing VEGF from exerting its activity.

[0016] In one embodiment, the VEGF binding domain comprises the extracellular domains of two VEGF receptor isoforms, for example the extracellular domains of both VEGF receptor 1 and VEGF receptor 2 (e.g., the Ig2 domain of VEGF receptor 1 and the Ig3 domain of VEGF receptor 2).

[0017] In one embodiment, the VEGF binding domain comprises an anti-VEGF antibody or antibody binding fragment thereof. In a further embodiment, the anti-VEGF antibody or fragment thereof comprises the variable binding domain of bevacizumab, ranibizumab or HuMab G6-31 (US Patent Application Publication No. 2007 / 0141065).

[0018] In some embodiments, the present disclosure provides a vascular endothelial growth factor (VEGF) binding domain comprising a vascular endothelial growth factor (VEGF)-A binding region of VEGF receptor 1 (VEGFR1) of SEQ ID NO: 13 and a vascular endothelial growth factor (VEGF)-A binding region of VEGF receptor 2 (VEGFR2) of SEQ ID NO: 14; an anti-Tie2 antibody or an antibody-binding fragment thereof; A fusion protein comprising: Here, the fusion protein provides a fusion protein that binds to the Tie2 Ig3-FNIII(1-3) domain comprising SEQ ID NO: 2, 3 or 4 and VEGF.

[0019] In one embodiment, the anti-Tie2 antibody or antigen-binding fragment thereof is an agonist anti-Tie2 antibody or antigen-binding fragment thereof. Thus, in one embodiment, the anti-Tie2 antibody or antigen-binding fragment thereof binds Tie2 and provides, preserves, or enhances Tie2 or Tie2 activity at the cell surface membrane.

[0020] In one embodiment, the VEGF binding domain is linked to the C-terminus of the heavy chain (HC) of the anti-Tie2 antibody or antigen-binding fragment thereof.

[0021] In one embodiment, the fusion protein binds amino acids 633 to 644 (SEQ ID NO: 19) and / or amino acids 713 to 726 (SEQ ID NO: 20) of Tie2 of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the anti-Tie2 antibody, or antibody-binding fragment thereof, binds amino acids 633 to 644 (SEQ ID NO: 19) and / or amino acids 713 to 726 (SEQ ID NO: 20) of Tie2 of the amino acid sequence of SEQ ID NO: 1.

[0022] In one embodiment, the anti-Tie2 antibody has an IgG1 isotype.

[0023] In one embodiment, the anti-Tie2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a heavy chain CDR comprising the amino acid sequence of any of SEQ ID NOs: 5 to 7, and a light chain variable region comprising a light chain CDR comprising the amino acid sequence of any of SEQ ID NOs: 8 to 10.

[0024] In one embodiment, the VEGF binding domain comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:15.

[0025] In one embodiment, the VEGF binding domain comprises the amino acid sequence of SEQ ID NO:15.

[0026] In one embodiment, the fusion protein comprises a linker between the VEGF binding domain (e.g., a VEGF receptor) and the anti-Tie2 antibody or antibody fragment thereof, hi a further embodiment, the linker comprises 5 to 50 amino acid residues, such as 10 to 40 residues, such as 15 to 30 residues, for example 20 residues.

[0027] In one embodiment, the fusion protein comprises a linker between the VEGF binding domain (e.g., a VEGF receptor) and the anti-Tie2 antibody or antibody fragment thereof, wherein the linker comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:16.

[0028] In one embodiment, the fusion protein comprises a linker between the VEGF binding domain (eg, a VEGF receptor) and the anti-Tie2 antibody or antibody fragment thereof, wherein the linker comprises the amino acid sequence of SEQ ID NO:16.

[0029] In one embodiment, the fusion protein comprises a linker between the VEGF binding domain (e.g., a VEGF receptor) and the anti-Tie2 antibody or antibody fragment thereof, wherein the linker comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:25.

[0030] In one embodiment, the fusion protein comprises a linker between the VEGF binding domain (eg, a VEGF receptor) and the anti-Tie2 antibody or antibody fragment thereof, wherein the linker comprises the amino acid sequence of SEQ ID NO:25.

[0031] In one embodiment, the fusion protein comprises a CH domain comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:17.

[0032] In one embodiment, the fusion protein comprises a CH domain comprising the amino acid sequence of SEQ ID NO:17.

[0033] In one embodiment, the fusion protein comprises one or more mutations in the constant region domain of the heavy chain to reduce or eliminate efficient interaction with Fc receptors on other immune cells in the body. In one embodiment, the one or more mutations comprise changing L234, L235, G236 and G237 (EU numbering) to a LAGA mutation, a FEGG mutation, an AAGG mutation, an AAGA mutation, a LALA mutation or a combination thereof. In one embodiment, the one or more mutations comprise a LALA mutation and a mutation at K322 (e.g., K322A) and a mutation at P331 (e.g., P331S) (EU numbering).

[0034] In one embodiment, the fusion protein comprises one or more mutations at L234, L235, H310, M252, I253, S254, T256, H433, N434 and / or H435 (EU numbering). In one embodiment, the fusion protein comprises one or more mutations at L234A, L235A and / or H310A. In one embodiment, the fusion protein comprises one or more mutations at M252 (e.g., M252Y), I253 (e.g., I253A, I253M or I253V), S254 (e.g., S254T), T256 (e.g., T256D), H433 (e.g., H433K), N434 (e.g., N434F) and / or H435 (e.g., H435A, H435Q or H435R).

[0035] In one embodiment, the fusion protein comprises a linker between the VEGF-binding domain and the anti-Tie2 antibody or antibody fragment thereof, on the C-terminal side of one or more heavy chain constant domains of the anti-Tie2 antibody or antigen-binding fragment thereof, and in order from N-terminus to C-terminus, for example the linker comprises a sequence having 5 to 50 residues, for example 10 to 40 residues, for example 15 to 30 residues, for example 20 residues, and the VEGF-binding domain comprises the amino acid sequence of SEQ ID NO: 15.

[0036] In one embodiment, the fusion protein comprises a linker between the VEGF binding domain and the anti-Tie2 antibody or antibody fragment thereof, on the C-terminal side of one or more heavy chain constant domains of the anti-Tie2 antibody or antigen-binding fragment thereof and in order from N-terminus to C-terminus, wherein the linker comprises the amino acid sequence of SEQ ID NO: 16 or 25, and the VEGF binding domain comprises the amino acid sequence of SEQ ID NO: 15.

[0037] In one embodiment, the fusion protein comprises a CH domain comprising the amino acid sequence of SEQ ID NO: 17 and a linker comprising the amino acid sequence of SEQ ID NO: 16 or 25 between the VEGF binding domain and the anti-Tie2 antibody or antibody fragment thereof, wherein the VEGF binding domain comprises the amino acid sequence of SEQ ID NO: 15.

[0038] In one embodiment, the fusion protein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0039] In one embodiment, the fusion protein comprises a heavy chain that comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:11.

[0040] In one embodiment, the fusion protein comprises a heavy chain that comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:26.

[0041] In one embodiment, the fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:11.

[0042] In one embodiment, the fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:26.

[0043] In one embodiment, the fusion protein comprises a light chain that comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:12.

[0044] In one embodiment, the fusion protein comprises a light chain comprising the amino acid sequence of SEQ ID NO:12.

[0045] In one embodiment, the fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:11 and a light chain comprising the amino acid sequence of SEQ ID NO:12.

[0046] In one embodiment, the fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:26 and a light chain comprising the amino acid sequence of SEQ ID NO:12.

[0047] In one embodiment, the fusion protein is -9 Affinity K less than M D (M) binds to the Tie2 Ig3-FNIII(1-3) domain comprising SEQ ID NO: 2, 3 or 4.

[0048] In one embodiment, the fusion protein is pegylated. Pegylation (or pegylation) refers to the covalent attachment or fusion of a chain comprising multiple polyethylene glycol (PEG, also called macrogol) units (e.g., PEG polymers). In one embodiment, the PEG has a molecular weight of about 40 kDa or about 20 kDa.

[0049] In one embodiment, the fusion protein is site-specifically PEGylated. In one embodiment, the fusion protein is site-specifically PEGylated on a cysteine ​​residue. In one embodiment, the fusion protein further comprises the sequence of SEQ ID NO: 22 and is site-specifically PEGylated on a cysteine ​​residue of the sequence of SEQ ID NO: 22.

[0050] In one embodiment, the sequence of SEQ ID NO: 22 is present at the C-terminus of the heavy chain. In one embodiment, the heavy chain comprises the sequence of SEQ ID NO: 23. In one embodiment, the heavy chain comprises the sequence of SEQ ID NO: 24.

[0051] In one embodiment, the fusion protein further comprises one or more half-life extension modulators. In one embodiment, the one or more half-life extension modulators comprise a chemical, biopolymer, or peptide that increases the half-life of the fusion protein.

[0052] In one embodiment, a polypeptide is provided that comprises a chain of monomers of a fusion protein of the invention.

[0053] In one embodiment, the polypeptide comprises a heavy chain monomer of the fusion protein of the invention. In a further embodiment, the polypeptide comprises a sequence having at least 70% identity, such as at least 75% identity, such as at least 80% identity, such as at least 85% identity, such as at least 90% identity, such as at least 91% identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as 100% identity to the sequence of SEQ ID NO: 11 or 26. In one embodiment, the polypeptide consists of the sequence of SEQ ID NO: 11. In one embodiment, the polypeptide consists of the sequence of SEQ ID NO: 26.

[0054] In one embodiment, the polypeptide comprises a light chain monomer of the fusion protein of the present invention. In a further embodiment, the polypeptide comprises a sequence having at least 70% identity to SEQ ID NO: 12, such as at least 75% identity, such as at least 80% identity, such as at least 85% identity, such as at least 90% identity, such as at least 91% identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as 100% identity. In one embodiment, the polypeptide consists of the sequence of SEQ ID NO: 12.

[0055] As used herein, "biopolymer" refers to a polymer produced from a natural source, either chemically synthesized from biological materials or entirely biosynthesized by an organism or microorganism. In some embodiments, biopolymers include, but are not limited to, polypeptides or proteins (i.e., polymers of amino acids, such as collagen, actin, and fibrin), polynucleotides (i.e., polymers of nucleic acids, such as DNA or RNA), polysaccharides (i.e., carbohydrates and glycosylated molecules, such as cellulose, starch, or alginates), natural rubber (polymers of isoprene), suberin, lignin (complex polyphenol polymers), cutin and cutan (complex polymers of long chain fatty acids), melanin, metabolites, and other structural molecules.

[0056] In one embodiment, the one or more half-life extension modulators comprise a biopolymer containing PEG (polyethylene glycol), hyaluronic acid (HA) or phosphorylcholine; albumin; albumin binding peptides and / or HA binding protein fragments.

[0057] In another embodiment, the disclosure provides a nucleic acid encoding the fusion protein.

[0058] In another embodiment, the disclosure provides a nucleic acid encoding a polypeptide comprising a linear monomer of the fusion protein of the invention. In one embodiment, the disclosure provides a nucleic acid encoding a heavy chain of the fusion protein. In another embodiment, the disclosure provides a nucleic acid encoding a light chain of the fusion protein.

[0059] In one embodiment the nucleic acid molecule comprises a sequence having at least 70% identity, such as at least 75% identity, such as at least 80% identity, such as at least 85% identity, such as at least 90% identity, such as at least 91% identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as 100% identity to SEQ ID NO: 27 or 29. In one embodiment the nucleic acid molecule consists of SEQ ID NO: 27 or 29.

[0060] In one embodiment, the nucleic acid molecule comprises a sequence having at least 70% identity, such as at least 75% identity, such as at least 80% identity, such as at least 85% identity, such as at least 90% identity, such as at least 91% identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as 100% identity to SEQ ID NO:28. In one embodiment, the nucleic acid molecule consists of SEQ ID NO:28.

[0061] In one embodiment, the disclosure provides a set of one or more polynucleotides, each polynucleotide encoding at least one of the monomer chains of a fusion protein of the invention, wherein both such chains (i.e., a light chain and a heavy chain) of the fusion protein are encoded.

[0062] In another embodiment, the disclosure provides an expression vector comprising the nucleic acid. In another embodiment, a vector is provided comprising the nucleic acid encoding one heavy chain sequence and one light chain sequence of the fusion protein.

[0063] In one embodiment, a set of one or more vectors is provided that collectively comprise a set of one or more polynucleotides of the invention, such that both chains of a fusion protein (i.e., a light chain and a heavy chain) are encoded in said set of vectors.

[0064] In one embodiment, the vector is a virus selected from an animal virus, such as a reverse transcriptase virus (including a lentivirus), an adenovirus, an adeno-associated virus, a herpes virus, a varicella virus, a baculovirus, a papilloma virus, anellovirus, and a papovavirus.

[0065] In one embodiment, the expression vector further comprises the human cytomegalovirus IE1 (CMV-IE1) promoter / enhancer.

[0066] In another embodiment, the disclosure provides a cell transformed with the expression vector.

[0067] In another embodiment, the disclosure provides a method for producing a fusion protein that binds Tie2 and VEGF, comprising culturing cells transformed with an expression vector and recovering the fusion protein from the cultured cells. In another embodiment, the disclosure provides a method for the production of the fusion proteins of the invention by expression from a vector or set of vectors.

[0068] In another embodiment, the present disclosure provides a method for preventing or treating an angiogenic disease, comprising administering to a subject in need thereof an effective amount of a fusion protein.

[0069] In another embodiment, the disclosure provides for the use of the fusion protein for the manufacture of a medicament for treating an angiogenic disease in a subject in need thereof.

[0070] In another embodiment, the present disclosure provides a fusion protein for use in treating an angiogenic or vascular disease in a subject in need thereof.

[0071] In some embodiments, the angiogenic or vascular disease is cancer, metastasis, diabetic retinopathy, retinopathy of prematurity, diabetic macular edema, corneal graft rejection, macular degeneration, glaucoma such as neovascular glaucoma, systemic erythroderma, proliferative retinopathy, psoriasis, hemophilic arthropathy, analogous sclerosis, capillary formation of atherosclerotic plaques, keloids, wound granulation, vascular adhesions, rheumatoid arthritis, osteoarthritis, autoimmune diseases, Crohn's disease, restenosis, atherosclerosis, intestinal adhesions, cat scratch disease, ulcers, cirrhosis, nephritis, diabetic nephropathy, diabetes, inflammatory diseases, or neurodegenerative diseases.

[0072] In some embodiments, the cancer is esophageal cancer, gastric cancer, colon cancer, rectal cancer, oral cavity cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma, or multiple myeloid blood cancer.

[0073] In some embodiments, the present disclosure provides a method for modulating angiogenesis, endothelial signaling, inflammation and / or vascular leakage, comprising administering an effective amount of a fusion protein to a subject in need of modulating angiogenesis, endothelial signaling, inflammation and / or vascular leakage.

[0074] In some embodiments, the inflammation results from sepsis, acute respiratory distress syndrome, and / or viral infectious disease.

[0075] In some embodiments, the subject is a human. In some embodiments, the subject is a companion animal, such as a mammalian companion animal. In some embodiments, the mammalian companion animal is a dog, cat, rabbit, ferret, horse, mule, donkey, or hamster, or other domesticated pet.

[0076] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the fusion protein. In some embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier, diluent, or excipient.

[0077] Additional objects and advantages will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0078] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the scope of the claims. Section headings are provided merely for the convenience of the reader and are not intended to limit the scope of the disclosure.

[0079] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate specific embodiments and, together with the description, serve to explain the principles described herein. [Brief description of the drawings]

[0080] [Figure 1A] 1A-1C provide sensorgrams for the binding of IGT-427 to human (FIG. 1A), rabbit (FIG. 1B) and mouse (FIG. 1C) Tie2-Fc fusion proteins. [Figure 1B] 1A-1C provide sensorgrams for the binding of IGT-427 to human (FIG. 1A), rabbit (FIG. 1B) and mouse (FIG. 1C) Tie2-Fc fusion proteins. [Figure 1C] 1A-1C provide sensorgrams for the binding of IGT-427 to human (FIG. 1A), rabbit (FIG. 1B) and mouse (FIG. 1C) Tie2-Fc fusion proteins. [Figure 2A]Figures 2A-2C show inhibition of VEGFR2 phosphorylation and AKT activation by IGT-427. Human umbilical vein endothelial cells (HUVEC) were serum-starved for 4 h, incubated with the indicated concentrations of IGT-427 (Figure 2A) or aflibercept (Figure 2B) for 30 min, and then treated with recombinant human VEGF for 2 min. Cell lysates were subjected to SDS-PAGE / Western blotting, and blots were probed with anti-phospho-VEGFR2 (Tyr1175), anti-VEGFR2, anti-phospho-Akt (S473) or total Akt antibodies. VEGF reporter assays show comparable inhibitory effects of IGT-427, faricimab, and aflibercept (Figure 2C). [Figure 2B] Figures 2A-2C show inhibition of VEGFR2 phosphorylation and AKT activation by IGT-427. Human umbilical vein endothelial cells (HUVEC) were serum-starved for 4 h, incubated with the indicated concentrations of IGT-427 (Figure 2A) or aflibercept (Figure 2B) for 30 min, and then treated with recombinant human VEGF for 2 min. Cell lysates were subjected to SDS-PAGE / Western blotting, and blots were probed with anti-phospho-VEGFR2 (Tyr1175), anti-VEGFR2, anti-phospho-Akt (S473) or total Akt antibodies. VEGF reporter assays show comparable inhibitory effects of IGT-427, faricimab, and aflibercept (Figure 2C). [Figure 2C] Figures 2A-2C show inhibition of VEGFR2 phosphorylation and AKT activation by IGT-427. Human umbilical vein endothelial cells (HUVEC) were serum-starved for 4 h, incubated with the indicated concentrations of IGT-427 (Figure 2A) or aflibercept (Figure 2B) for 30 min, and then treated with recombinant human VEGF for 2 min. Cell lysates were subjected to SDS-PAGE / Western blotting, and blots were probed with anti-phospho-VEGFR2 (Tyr1175), anti-VEGFR2, anti-phospho-Akt (S473) or total Akt antibodies. VEGF reporter assays show comparable inhibitory effects of IGT-427, faricimab, and aflibercept (Figure 2C). [Diagram 3]Figure 3 shows stronger and more sustained levels of phospho-Tie2 signaling by IGT-427 compared to Angiopoietin-1. Chinese Hamster Ovary (CHO) cells overexpressing human Tie2 were serum starved for 4 hours and then treated with 10 nM IGT-427 or Angiopoietin-1 for the indicated periods. Lysates were subjected to phospho-Tie2 and total Tie2 ELISA assays. [Figure 4] FIG. 4 shows dose-dependent activation of Akt signaling by IGT-427 in HUVEC. [Diagram 5] FIG. 5 shows that IGT-427 overcomes and circumvents the effect of angiopoietin-2 on Tie2 signaling in HUVECs, leading to further activation of Akt signaling in the presence of pre-treated Ang2. [Figure 6] Figure 6 shows that IGT-427 binds simultaneously to Tie2 and VEGF complexed with Ang2. Ang2 was captured on a CM5 chip and subsequently human Tie2, IGT-427 and human VEGF were injected in a surface plasmon resonance (SPR) (Biocore™) analysis. [Figure 7] Figure 7 shows the inhibition of TNF-α-induced apoptosis by IGT-427. HUVECs were pretreated with IGT-427 or Eylea® for 1 h, then treated with TNF (50 ng / ml) for 24 h. Apoptotic cells were stained with APO-BrdU™ TUNEL Assay Kit (Thermo Fisher, A23210) and determined by Attune (Thermo Fisher). Values ​​are mean ± SEM. ***p<0.001, ****p<0.0001 by one-way ANOVA. [Figure 8] FIG. 8 shows cell surface levels of Tie2 on Chinese hamster ovary cells overexpressing human Tie2 (CHO-Tie2) treated with various agents at various time points. [Figure 9]Figure 9 shows that IGT-427 blocks Tie2 cleavage by MMP 14. Recombinant human MMP 14, human Tie2-ECD-human IgG Fc fusion protein or IGT-427 were mixed and incubated as indicated. [Figure 10] Figure 10 shows that IGT-427 inhibits sTie2 production in basal or TNF-α-treated HUVECs. sTie2 levels were measured by Tie2 ELISA assay. [Figure 11] Figure 11 shows that IGT-427 restores impaired endothelial barrier integrity due to VEGF treatment.TEER (transendothelial electrical resistance) was assessed in HUVECs. [Figure 12] FIG. 12 shows the inhibition of CNV (choroidal neovascularization) by intravitreally injected IGT-427 or Eylea® in a laser-induced CNV model. Intravitreous administration of antibodies (50 μl injection volume / eye, Eylea® (800 μg), IGT-427 (885 μg), control IgG (716 μg)) was performed on day 0 after laser photocoagulation. The molar ratio of Eylea®, IGT-427 and control IgG was 1:0.65:0.68. Fluorescence intensity in the leakage area around CNV was measured in FA images taken on day 14 after laser photocoagulation. For each group, 4 to 7 rabbits were tested, and 6 laser-induced CNV lesions were tested for each rabbit. CNV lesions (n=24-42) were imaged for each group. Values ​​are mean ± SEM. *p<0.05, **p<0.001 by one-way ANOVA. [Figure 13] Figure 13 shows a schematic of IGT-427 variants for PEGylation. All constructs have a common light chain with five different heavy chains generating two labile cysteines per antibody. [Figure 14]Figure 14 shows the SDS-PAGE data of the optimized PEGylation conditions for five IGT-427 variants. PRO593, PRO594 and PRO595 undergo complete conversion with no unmodified antibody in the reaction mixture. The reaction mixtures of PRO592 and PRO596 have some unmodified antibody at the end of the reaction. [Figure 15] Figure 15 shows the SPR binding of IGT-427 and PEGylated variants to Tie2 and VEGF surfaces. Although there are slight differences in the binding signals to either antigen, the PEGylated variants bind both antigens similarly compared to unmodified IGT-427. [Figure 16] Figure 16 shows the SEC-HPLC chromatograms of the five ocular PK study test articles. All constructs were greater than 90% pure, except for the 20 kDa PEGylated species, which contained 15% high molecular weight species. [Figure 17] Figure 17 shows non-reduced SDS-PAGE data of ocular PK study test articles: Lane 1: Eylea®; Lane 2: Faricimab; Lane 3: IGT-427; Lane 4: (2 x 20 kDa linear PEG)-IGT-427; Lane 5: (2 x 40 kDa branched PEG)-IGT-427. [Figure 18A] 18A-18C show SPR binding data for ocular PK test articles to rabbit VEGF (FIG. 18A), Tie2 (FIG. 18B), and Ang2 (FIG. 18C). A summary of the measured binding constants is provided in the table. [Figure 18B] 18A-18C show SPR binding data for ocular PK test articles to rabbit VEGF (FIG. 18A), Tie2 (FIG. 18B), and Ang2 (FIG. 18C). A summary of the measured binding constants is provided in the table. [Figure 18C] 18A-18C show SPR binding data for ocular PK test articles to rabbit VEGF (FIG. 18A), Tie2 (FIG. 18B), and Ang2 (FIG. 18C). A summary of the measured binding constants is provided in the table. [Figure 19-1]Figure 19 shows a summary of total drug measurements from New Zealand White rabbit vitreous. Measured drug levels from ELISA quantification are plotted over time. Each data point represents duplicate measurements from a single rabbit eye. Exponential fits to each data set are shown in red with fitted parameters, and extracted ocular half-lives are shown below each curve. For PEGylated IGT-427 variants, 14-day measurements are included in the plot but excluded from the fitting and extracted half-life analysis. A summary of observed half-lives for each test article is shown at the bottom right. [Figure 19-2] Figure 19 shows a summary of total drug measurements from New Zealand White rabbit vitreous. Measured drug levels from ELISA quantification are plotted over time. Each data point represents duplicate measurements from a single rabbit eye. Exponential fits to each data set are shown in red with fitted parameters, and extracted ocular half-lives are shown below each curve. For PEGylated IGT-427 variants, 14-day measurements are included in the plot but excluded from the fitting and extracted half-life analysis. A summary of observed half-lives for each test article is shown at the bottom right. [Figure 19-3] Figure 19 shows a summary of total drug measurements from New Zealand White rabbit vitreous. Measured drug levels from ELISA quantification are plotted over time. Each data point represents duplicate measurements from a single rabbit eye. Exponential fits to each data set are shown in red with fitted parameters, and extracted ocular half-lives are shown below each curve. For PEGylated IGT-427 variants, 14-day measurements are included in the plot but excluded from the fitting and extracted half-life analysis. A summary of observed half-lives for each test article is shown at the bottom right. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0081] Array Description Table 1 provides a list of the specific sequences referenced herein. [Table 1]

[0082] TIFF2025503704000003.tif232151

[0083] TIFF2025503704000004.tif233151

[0084] TIFF2025503704000005.tif232149

[0085] TIFF2025503704000006.tif232151

[0086] TIFF2025503704000007.tif233148

[0087] Description of the embodiments definition Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings:

[0088] As used herein, the term "anti-Tie2 antibody" refers to an antibody that specifically binds to Tie2, including antigen-binding fragments of antibody molecules, as well as intact antibodies that specifically bind to Tie2. In some embodiments, intact antibodies have a structure of two full-length light chains and two full-length heavy chains, with each light chain connected to a heavy chain by a disulfide bond. The constant region of the heavy chain has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, with subclasses of gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.

[0089] "Antigen-binding fragment" or "antibody fragment of an antibody" refers to a fragment capable of binding to an antigen, and includes Fab, F(ab'), F(ab')2, and Fv. Among the antibody fragments, Fab has one antigen-binding site having the structure of the light chain and heavy chain variable regions, the light chain constant region, and the first CH1 of the heavy chain.

[0090] Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the CH1 domain. F(ab')2 antibodies are produced by disulfide bond formation between cysteine ​​residues in the hinge region of Fab'. Fv is the smallest antibody fragment that has only the variable region of the heavy chain and the variable region of the light chain. A double-chain Fv (two-chain Fv) is formed by non-covalent bonding between the variable region of the heavy chain and the variable region of the light chain, and a single-chain Fv (scFv) is generally formed via a covalently bonded peptide linker between the variable region of the heavy chain and the variable region of the light chain, or directly connected at the C-terminus by forming a dimer-like structure like a double-chain Fv. This fragment can be obtained by proteolytic enzymes (for example, Fab can be obtained by limited digestion of the whole antibody using papain, and F(ab')2 fragments can be obtained by cleavage with pepsin), and can also be produced by genetic engineering techniques.

[0091] The antibody may be, for example, in Fv format (e.g., scFv) or in full antibody format. Furthermore, the heavy chain constant region may be selected from any isotype: gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε). For example, the constant region is gamma 1 (IgG1), gamma 3 (IgG3) or gamma 4 (IgG4). The light chain constant region may be of kappa or lambda type.

[0092] Antibodies include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFVs), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFVs) and anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of the above antibodies.

[0093] The term "heavy chain" of "HC" refers to a full-length heavy chain or a fragment thereof comprising a variable region domain VH and three constant region domains CH1, CH2 and CH3, with an amino acid sequence having sufficient variable regions to provide antigen specificity. Additionally, as used herein, "light chain" or "LC" refers to a full-length light chain or a fragment thereof comprising a variable region domain VL and a constant region domain CL, with an amino acid sequence having sufficient variable regions to provide antigen specificity. The heavy chain constant region domain (e.g., of human IgG1) can contain one or more modifications to reduce or eliminate efficient interaction with Fc receptors on immune cells in the body. Such mutation(s) can inhibit its binding to Fc receptors and reduce or eliminate antibody-induced cytotoxicity. Such modifications can be used to modify the L 234 L 235 G 236 G 237 The IgG constant region may include altering residues (EU numbering) to LAGA, FEGG, AAGG, AAGA, LALA, or combinations thereof. In some embodiments, the IgG constant region includes mutations at K322 and P331 (EU numbering) (e.g., in combination with any of the preceding mutations) to reduce or eliminate immune cell-mediated cytotoxicity. In some embodiments, the IgG constant region includes a LALA mutation and mutations at K322 and P331 (e.g., K322A and P331S) (EU numbering) to reduce or eliminate immune cell-mediated cytotoxicity.

[0094] A "monoclonal antibody" is an antibody produced by a single clone of a cell or cell line and consisting of identical antibody molecules. Monoclonal antibodies can have a monovalent affinity and bind only to the same epitope. In contrast, polyclonal antibodies bind to multiple epitopes and are usually produced by several different antibody-secreting plasma cell lines. Bispecific monoclonal antibodies can also be designed by increasing the target of one monoclonal antibody to two epitopes.

[0095] "Epitope" means a protein determinant to which an antibody can specifically bind (e.g., that part of an antigen recognized by an antibody). An epitope can be a chemically active surface molecule, such as a group of amino acids or sugar side chains, and generally has specific charge characteristics and specific three-dimensional structural characteristics.

[0096] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain one or more amino acid sequences (e.g., one or more CDR sequences, such as six CDR sequences) from a non-human antibody (donor or source antibody) and other minimal sequences from a non-human immunoglobulin. In some embodiments, a humanized antibody is a human immunoglobulin (recipient antibody) whose hypervariable regions have been replaced by residues from a hypervariable region of a non-human primate, mouse, rat, rabbit or non-human primate (recipient antibody) that have the desired specificity, affinity and capacity of the residues from the recipient's hypervariable region. For humanization, one or more residues in the framework domain (FR) may be replaced by the corresponding residues of the non-human donor antibody. This may help to maintain the proper three-dimensional configuration of the grafted CDR(s), thereby improving affinity and antibody stability. A humanized antibody may, for example, alternatively or additionally, include new residues that do not appear in the original recipient or donor antibody to further refine the performance of the antibody.

[0097] Included are any "chimeric" antibodies (immunoglobulins) and fragments of such antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a subclass, while the remaining chain(s) are identical to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, exhibiting the desired biological activity.

[0098] As used herein, an "antibody variable domain" refers to a domain comprising complementarity determining regions (CDRs; e.g., CDR1, CDR2 and CDR3) and framework regions (FRs). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. "Framework regions" (FRs) are the variable domain segments outside the CDRs. Each variable domain typically has four FRs identified as FR1, FR2, FR3 and FR4.

[0099] "Complementarity determining region" (CDR; i.e., CDR1, CDR2 and CDR3) refers to the amino acid residues of the variable domain of an antibody that are necessary for antigen binding. Each variable domain typically contains three CDR regions identified as CDR1, CDR2 and CDR3.

[0100] As used herein, a "half-life extension modulator" refers to a chemical, biopolymer, peptide, polypeptide, or protein fragment that can be added to an antibody or antibody fragment to increase the half-life of the antibody or antibody fragment. Half-life extension modulators can include biopolymers containing PEG (polyethylene glycol), hyaluronic acid (HA), or phosphorylcholine; albumin; albumin-binding peptides; or HA-binding protein fragments.

[0101] Half-life can also be decreased, for example, by abolishing the interaction of the antibody or antibody fragment with neonatal Fc receptor (FcRn)-mediated recycling. For example, combinatorial mutations at multiple sites are known to affect antibody half-life. In some embodiments, the mutations include M252 (e.g., M252Y), I253 (e.g., I253A, I253M, or I253V), S254 (e.g., S254T), T256 (e.g., T256D), H310 (e.g., H310A), H433 (e.g., H433K), N434 (e.g., N434F), and / or H435 (e.g., H435A, H435Q, or H435R) (EU numbering). In some embodiments, the mutations include L235A, L236A, and H310A (EU numbering). A reduced systemic half-life may be advantageous for non-systemic disease treatments, such as ocular disease treatments, where minimal systemic exposure and reduced systemic half-life are desired due to established on-target toxicity issues of VEGF inhibition (bleeding, hypertension, etc.).

[0102] As used herein, "or" has an inclusive meaning (ie, equivalent to and / or) unless the context clearly dictates otherwise.

[0103] As used herein, a "fusion protein" encompasses any polypeptide that contains sequences from more than one source. Fusion proteins can be produced, for example, from a genetic fusion (e.g., a polynucleotide encoding the sequences of the fusion protein) or by chemically linking separately produced or synthesized polypeptides.

[0104] "Angiogenic disease", also called "angiogenesis-associated disease", refers to a disease associated with the development of angiogenesis or the progression of angiogenesis.

[0105] As used herein, a "subject" may be a human or an animal. For example, a subject may be a human. In some embodiments, a subject may be a mammal. In some embodiments, a subject may include a companion animal (also called a "pet"). The term "companion animal" refers to domesticated animals that may be kept as pets or for other entertainment purposes, including, but not limited to, dogs, cats, rabbits, ferrets, horses, donkeys, mules, and hamsters. In some embodiments, a companion animal is a mammalian companion animal.

[0106] The term "prevention" as used herein refers to any action of inhibiting, delaying the onset, or reducing the likelihood of occurrence of the disease of interest by administering a fusion protein or composition. The term "treatment" or "therapy" refers to any action of reducing, delaying, or alleviating the symptoms of the disease of interest, or curing the disease of interest, reducing the severity of the disease of interest, or slowing the progression of the disease of interest.

[0107] The present disclosure describes nucleic acid and amino acid sequences that have a degree of identity to a given nucleic acid or amino acid sequence, respectively (a reference sequence).

[0108] "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences.

[0109] The terms "% identical", "% identity" or similar terms are intended to refer in particular to the percentage of nucleotides or amino acids that are identical in optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may, but are not necessarily, randomly distributed over the entire length of the sequences to be compared. Comparison of two sequences is usually performed by comparing said sequences over segments or "windows of comparison" after optimal alignment in order to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or with the aid of the local affinities algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local affinities algorithm by Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin).

[0110] Percentage identity is obtained by determining the number of identical positions where the sequences to be compared match, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100.

[0111] In some embodiments, the degree of identity is given over a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given over at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides in consecutive nucleotides in some embodiments. In some embodiments, the degree of identity is given over the entire length of the reference sequence.

[0112] A nucleic acid sequence or amino acid sequence that has a certain degree of identity to a given nucleic acid sequence or amino acid sequence, respectively, can have at least one functional property of the given sequence, for example, in some cases, is functionally equivalent to the given sequence. One important property includes the ability to act as a cytokine, especially when administered to a subject. In some embodiments, a nucleic acid sequence or amino acid sequence that has a certain degree of identity to a given nucleic acid sequence or amino acid sequence is functionally equivalent to the given sequence.

[0113] As used herein, the term "about" indicates a degree of variation, for example, within 10%, 5%, 2% or 1%, that does not substantially affect the properties of the described subject matter. Thus, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending on the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0114] overview Fusion proteins that specifically bind to both Tie2 and VEGF are provided herein. The fusion proteins are designed to address both the limitations of current Tie2 and VEGF therapies as well as the limitations of other currently available therapies for angiogenic or vascular diseases. As a result, fusion proteins are provided that include an anti-Tie2 antibody or antigen-binding fragment thereof and a vascular endothelial growth factor (VEGF) binding domain. These fusion proteins can serve as beneficial therapeutic agents for angiogenic or vascular diseases due to their dual functions of VEGF inhibition and Tie2 activation.

[0115] Fusion proteins The present disclosure relates to a fusion protein comprising a Tie2 antibody or antigen-binding fragment thereof and a VEGF binding domain. In some embodiments, the Tie-2 antibody or antigen-binding fragment thereof binds to a Tie2 Ig3-FNIII(1-3) domain comprising the sequence of SEQ ID NO:2.

[0116] In some embodiments, the Tie2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 5 to 7, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 8 to 10.

[0117] In some embodiments, the fusion protein binds to a Tie2 Ig3-FNIII(1-3) domain comprising SEQ ID NO:2, 3 or 4 and VEGF.

[0118] In some embodiments, the fusion protein comprises a vascular endothelial growth factor (VEGF) binding domain comprising a vascular endothelial growth factor (VEGF)-A binding region of VEGF receptor 1 (VEGFR1) of SEQ ID NO: 13 and a vascular endothelial growth factor (VEGF)-A binding region of VEGF receptor 2 (VEGFR2) of SEQ ID NO: 14, and an anti-Tie2 antibody or antibody binding fragment thereof, wherein the fusion protein binds to a Tie2 Ig3-FNIII(1-3) domain comprising SEQ ID NO: 2, 3 or 4 and VEGF.

[0119] In some embodiments, the VEGF binding domain comprises an anti-VEGF antibody or antibody binding fragment thereof. In further embodiments, the anti-VEGF antibody or fragment thereof comprises the variable binding domain of bevacizumab, ranibizumab, or HuMab G6-31 (U.S. Patent Application Publication No. 2007 / 0141065).

[0120] In one embodiment, the VEGF binding domain is linked to the C-terminus of the heavy chain (HC) of the anti-Tie2 antibody or antigen-binding fragment thereof.

[0121] In one embodiment, the fusion protein binds amino acids 633 to 644 (SEQ ID NO: 20) and / or amino acids 713 to 726 (SEQ ID NO: 21) of Tie2 having the amino acid sequence of SEQ ID NO: 1. In one embodiment, the anti-Tie2 antibody, or antibody-binding fragment thereof, binds amino acids 633 to 644 (SEQ ID NO: 19) and / or amino acids 713 to 726 (SEQ ID NO: 20) of Tie2 of the amino acid sequence of SEQ ID NO: 1.

[0122] In one embodiment, the anti-Tie2 antibody has an IgG1 isotype.

[0123] In one embodiment, the VEGF binding domain comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 15. In one embodiment, the VEGF binding domain comprises the amino acid sequence of SEQ ID NO:15.

[0124] In one embodiment the fusion protein comprises four polypeptide chains, two of which are a pair of heavy chains and two of which are a pair of light chains. Suitably the heavy chains comprise a Tie2 binding domain at the N-terminus which binds to the Tie2 Ig3-FNIII(1-3) domain comprising SEQ ID NO: 2, 3 or 4, and a VEGF binding domain at the C-terminus. Suitably the Tie2 binding domain is an anti-Tie2 antibody, and the pair of heavy chains of the fusion protein comprises an antibody heavy chain and the pair of light chains of the fusion protein comprises an antibody light chain.

[0125] In one embodiment, the fusion protein comprises: a. a dimer of a first and a second heavy chain monomer, each heavy chain monomer comprising, from N-terminus to C-terminus: i. A heavy chain variable domain (VH) domain that binds Tie2 together with a light chain variable domain linked to the constant heavy chain domain (CH) of ii.; ii. a constant heavy domain (CH) comprising a CH1 domain and an Fc region or a fragment thereof linked to the VEGF binding domain of iii.; iii. A VEGF-binding domain including a VEGF receptor extracellular domain (such as the VEGF-A binding region of VEGF receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2)); a dimer of the first and second heavy chain monomers comprising a single polypeptide chain comprising: b. first and second light chain monomers, each light chain monomer comprising, from N-terminus to C-terminus, a light chain variable domain (VL) that, together with the VH, binds Tie2, linked to a constant light chain domain (CL1); Including, the first and second monomers dimerize via their Fc regions or fragments thereof; The Tie2 binding domain is formed by pairing each heavy chain monomer with one of the light chain monomers, such that the VH and CH1 of each heavy chain monomer pairs with the VL and CL1 domains of the light chain monomer.

[0126] In one embodiment, the fusion protein comprises a linker between the VEGF receptor and the anti-Tie2 antibody or antibody fragment thereof, hi a further embodiment, the linker comprises 5 to 50 residues, such as 10 to 40 residues, such as 15 to 30 residues, for example 20 residues.

[0127] In some embodiments, the fusion protein comprises a linker between the VEGF binding domain and the anti-Tie2 antibody or antibody fragment thereof, wherein the linker comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the linker comprises the sequence of SEQ ID NO:16.

[0128] In some embodiments, the fusion protein comprises a linker between the VEGF receptor and the anti-Tie2 antibody or antibody fragment thereof, wherein the linker comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the linker comprises the sequence of SEQ ID NO: 25.

[0129] In one embodiment, the fusion protein comprises a linker between the VEGF-binding domain and the anti-Tie2 antibody or antibody fragment thereof, on the C-terminal side of one or more heavy chain constant domains of the anti-Tie2 antibody or antigen-binding fragment thereof, and in N-terminal to C-terminal order, for example the linker comprises a sequence having 5 to 50 amino acid residues, for example 10 to 40 residues, for example 15 to 30 residues, for example 20 residues.

[0130] In one embodiment, the fusion protein comprises a linker between the VEGF binding domain and the anti-Tie2 antibody or antibody fragment thereof, on the C-terminal side of one or more heavy chain constant domains of the anti-Tie2 antibody or antigen-binding fragment thereof and in order from N-terminus to C-terminus, wherein the linker comprises the amino acid sequence of SEQ ID NO: 16 or 25, and the VEGF binding domain comprises the amino acid sequence of SEQ ID NO: 15.

[0131] In one embodiment, the fusion protein comprises a CH domain comprising the amino acid sequence of SEQ ID NO: 17 and a linker comprising the amino acid sequence of SEQ ID NO: 16 or 25 between the VEGF binding domain and the anti-Tie2 antibody or antibody fragment thereof, wherein the VEGF binding domain comprises the amino acid sequence of SEQ ID NO: 15.

[0132] In one embodiment, the fusion protein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0133] In one embodiment, the fusion protein comprises a heavy chain that comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 11. In one embodiment, the fusion protein comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO:11.

[0134] In one embodiment, the fusion protein comprises a heavy chain that comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 26. In one embodiment, the fusion protein comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO:26.

[0135] In one embodiment, the fusion protein comprises a light chain comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 12. In one embodiment, the fusion protein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 12. In one embodiment, the fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 12.

[0136] In one embodiment, the fusion protein comprises a CH domain comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 17. In one embodiment, the fusion protein comprises a CH domain comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the fusion protein comprises one or more modifications or mutations in the constant region domain in the heavy chain to reduce or eliminate efficient interaction with Fc receptors on other immune cells in the heavy chain constant region domain. In some embodiments, the one or more modifications or mutations are in the L 234 L 235 G 236 G 237 (EU numbering) to a LAGA mutation, a FEGG mutation, an AAGG mutation, an AAGA mutation, a LALA mutation, or a combination thereof. In some embodiments, the IgG constant region comprises any of the foregoing mutations (e.g., a LALA mutation) and mutations at K322 and P331 (EU numbering) to reduce and eliminate immune cell-mediated cytotoxicity.

[0137] Tie2 antibodies can be monovalent or bivalent and contain single or two chains. Functionally, the binding affinity or dissociation constant (K D ) is the molar (M) concentration of ligand at which half of the ligand binding sites on the protein are occupied at system equilibrium. D ) is the dissociation rate (K off ) value is the association rate (K on In the context of dissociation constants, a lower value corresponds to stronger binding. The K D is 10 -5 M~10 -12 For example, the binding affinity (K D ) is 10 -6 M~10 -12 M, 10 -7 M~10 -12 M, 10 -8 M~10 -12 M, 10 -9 M~10 -12 M, 10-5 M~10 -11 M, 10 -6 M~10 -11 M, 10 -7 M~10 -11 M, 10 -8 M~10 -11 M, 10 -9 M~10 -11 M, 10 -10 M~10 -11 M, 10 -5 M~10 -10 M, 10 -6 M~10 -10 M, 10 -7 M~10 -10 M, 10 -8 M~10 -10 M, 10 -9 M~10 -10 M, 10 -5 M~10 -9 M, 10 -6 M~10 -9 M, 10 -7 M~10 -9 M, 10 -8 M~10 -9 M, 10 -5 M~10 -8 M, 10 -6 M~10 -8 M, 10 -7 M~10 -8 M, 10 -5 M~10 -7 M, 10 -6 M~10 -7 M or 10 -5 M~10 -6 It's M.

[0138] IGT-427, an exemplary fusion protein described herein, simultaneously inhibits VEGF signaling and activates the Tie2 signaling pathway. IGT-427 has a K of less than 1 nM in bivalent interactions as well as cell surface interactions. D Binds human Tie-2 with a K of less than 10 pM D IGT-427 binds human VEGF with an apparent affinity of less than 500 pM to surface VEGF. IGT-427 binds human VEGF with an apparent affinity of less than 500 pM to surface VEGF. Dis less than 10 pM. IGT-427 shows a similar high affinity to the rabbit orthologue.

[0139] In one embodiment, the fusion protein is -9 M (where E -X represents the negative exponent and the number of zeros, e.g., 3E -9 is the same as .0000000003.) Less than 3.5E -9 Less than M, 4E -10 Affinity K less than or equal to M D (M) binds to the Tie2 Ig3-FNIII(1-3) domain comprising SEQ ID NO: 2, 3 or 4. D The value is K D is numerically smaller than a given amount, indicating stronger binding.

[0140] IGT-427 has the potential to provide superior efficacy compared to agents that inhibit VEGF alone and / or have more potent Tie-2 agonist activity compared to Ang2 inhibitors.

[0141] The Tie2 antibody or antibody fragment in the fusion protein may include its biological equivalent within the range of being able to specifically recognize Tie2. For example, the Tie2 antibody or antibody fragment in the fusion protein may include changes to the amino acid sequence to further improve the binding affinity and / or other biological properties of the antibody. Such modifications may include, for example, deletion, insertion and / or substitution of amino acid sequence residues of the antibody. These amino acid variations may be made based on the relative similarity of the amino acid substitutes, such as the hydrophobicity, hydrophilicity, charge, size of the amino acid side chain, to provide conservative substitutions. By analyzing the size, shape and type of amino acid side chain substitutes, it is known that arginine, lysine and histidine are all positively charged residues, alanine, glycine and serine have similar sizes, and phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, it can be said that arginine, lysine and histidine alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine are conservative substitutions.

[0142] Taking into account the above variations with biologically equivalent activity, in some embodiments, the amino acid sequence of the fusion protein comprises the sequence of the six CDRs in SEQ ID NOs: 5-10, and / or the heavy and light chains of SEQ ID NOs: 11 and 12, or any sequence showing substantial identity. Substantial identity means at least 80% identity, e.g., at least 85% identity, 90% identity, 95% identity, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity.

[0143] On this basis, a fusion protein or a component thereof may have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity when compared to the specified or all sequences described herein.

[0144] In one embodiment, the fusion protein is pegylated. In one embodiment, the fusion protein is modified with a half-life extension modulator, such as PEG, hyaluronic acid, or phosphorylcholine. In one embodiment, the fusion protein is site-specifically pegylated. In one embodiment, the fusion protein is site-specifically pegylated on a cysteine ​​residue. In one embodiment, the fusion protein is site-specifically (e.g., on a cysteine ​​residue) modified with a half-life extension modulator, which may include PEG, hyaluronic acid, or phosphorylcholine. Alternatively, the half-life extension modulator may include albumin, an albumin binding peptide, and / or an HA binding protein fragment.

[0145] In one embodiment, the fusion protein further comprises the sequence of SEQ ID NO:22, which is site-specifically modified (e.g., pegylated) on a cysteine ​​residue of the sequence of SEQ ID NO:22 with any of the half-life extension modulators described herein. In one embodiment, the sequence of SEQ ID NO:22 is present at the C-terminus of the heavy chain. In one embodiment, the heavy chain comprises the sequence of SEQ ID NO:23. In one embodiment, the heavy chain comprises the sequence of SEQ ID NO:24.

[0146] In one embodiment, the PEG has a molecular weight of about 40 kDa.

[0147] In one embodiment, a polypeptide is provided that comprises a chain of monomers of a fusion protein of the invention.

[0148] In one embodiment, the polypeptide comprises a heavy chain monomer of the fusion protein of the invention. In a further embodiment, the polypeptide comprises a sequence having at least 70% identity, such as at least 75% identity, such as at least 80% identity, such as at least 85% identity, such as at least 90% identity, such as at least 91% identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as 100% identity to the sequence of SEQ ID NO: 11 or 26. In one embodiment, the polypeptide consists of the sequence of SEQ ID NO: 11. In one embodiment, the polypeptide consists of the sequence of SEQ ID NO: 26.

[0149] In one embodiment, the polypeptide comprises a light chain monomer of the fusion protein of the present invention. In a further embodiment, the polypeptide comprises a sequence having at least 70% identity to SEQ ID NO: 12, such as at least 75% identity, such as at least 80% identity, such as at least 85% identity, such as at least 90% identity, such as at least 91% identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as 100% identity. In one embodiment, the polypeptide consists of the sequence of SEQ ID NO: 12.

[0150] Polynucleotides; Production methods; Vectors and host cells In another aspect, the disclosure relates to a nucleic acid encoding the fusion protein.

[0151] In another embodiment, the disclosure provides a nucleic acid encoding a polypeptide comprising a linear monomer of the fusion protein of the invention. In one embodiment, the disclosure provides a nucleic acid encoding a heavy chain of the fusion protein. In another embodiment, the disclosure provides a nucleic acid encoding a light chain of the fusion protein.

[0152] In one embodiment the nucleic acid molecule comprises a sequence having at least 70% identity, such as at least 75% identity, such as at least 80% identity, such as at least 85% identity, such as at least 90% identity, such as at least 91% identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as 100% identity to SEQ ID NO: 27 or 29. In one embodiment the nucleic acid molecule consists of SEQ ID NO: 27 or 29.

[0153] In one embodiment, the nucleic acid molecule comprises a sequence having at least 70% identity, such as at least 75% identity, such as at least 80% identity, such as at least 85% identity, such as at least 90% identity, such as at least 91% identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as 100% identity to SEQ ID NO:28. In one embodiment, the nucleic acid molecule consists of SEQ ID NO:28.

[0154] In one embodiment, the disclosure provides a set of one or more polynucleotides, each polynucleotide encoding at least one of the monomer chains of a fusion protein of the invention, wherein both such chains (i.e., a light chain and a heavy chain) of the fusion protein are encoded.

[0155] Fusion proteins can be produced recombinantly by constructing or isolating a nucleic acid that encodes the fusion protein. The nucleic acid can be isolated and further cloned (DNA amplification) by inserting it into a replicable vector or further expressed. Based on this, the present disclosure relates in another aspect to a vector containing the nucleic acid.

[0156] In some embodiments, the present disclosure relates to nucleic acids, including fusion proteins. "Nucleic acid" is intended to comprehensively encompass DNA (gDNA and cDNA) and RNA molecules, and the basic structural unit of nucleic acid, nucleotide, includes natural nucleotides and analogs having modified sugar or base moieties. The sequences of nucleic acids encoding heavy chain variable regions and light chain variable regions may be modified. Modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0157] DNA encoding the fusion protein is readily isolated or synthesized using conventional processes (e.g., by using oligonucleotide probes capable of specifically binding to DNA encoding the heavy and light chains). Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0158] In some embodiments, the disclosure provides an expression vector comprising a nucleic acid encoding a fusion protein, hi another embodiment, a vector is provided comprising a nucleic acid encoding one heavy chain sequence and one light chain sequence of the fusion protein.

[0159] In one embodiment, a set of one or more vectors is provided that collectively comprise a set of one or more polynucleotides of the invention, such that both chains of a fusion protein (i.e., a light chain and a heavy chain) are encoded in said set of vectors.

[0160] In one embodiment, the vector is a virus selected from an animal virus, such as a reverse transcriptase virus (including a lentivirus), an adenovirus, an adeno-associated virus, a herpes virus, a varicella virus, a baculovirus, a papilloma virus, and a papovavirus.

[0161] In some embodiments, the expression vector further comprises a promoter / enhancer, such as the human cytomegalovirus IE1 (CMV-IE1) promoter / enhancer.

[0162] As used herein, the term "vector" as a means for expressing a gene of interest in a host cell includes plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, and viral vectors such as adeno-associated virus vectors. The nucleic acid encoding the antibody in the vector is operably linked to a promoter.

[0163] As used herein, the term "operably linked" or "linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0164] In some embodiments, provided herein is a host cell comprising a polynucleotide encoding a fusion protein described herein. In some embodiments, provided herein is a host cell comprising a vector comprising a polynucleotide encoding a fusion protein described herein. The host cell may be a prokaryotic or eukaryotic cell. The host cell may be isolated, e.g., cultured, or not part of a multicellular organism. In some embodiments, the host cell is a member of a cell line. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is an immortalized mammalian cell.

[0165] In the case of prokaryotic cells as hosts, a strong promoter capable of processing transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for initiation of translation, and a transcription / translation termination sequence are generally included. Furthermore, for example, in the case of eukaryotic cells as hosts, promoters derived from the genome of mammalian cells (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used, and generally, a polyadenylation sequence can be included as a transcription termination sequence.

[0166] In some cases, the vector may be fused to other sequences to facilitate purification of the expressed antibody, such as glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6xHis (hexahistidine; Qiagen, USA).

[0167] The vectors contain antibiotic resistance genes commonly used in the art as selectable markers, such as genes for resistance to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

[0168] In another aspect, the present disclosure relates to a cell transformed with the above expression vector. The cells used to produce the antibodies can be, but are not limited to, prokaryotic, yeast and higher eukaryotic cells.

[0169] Prokaryotic host cells such as Escherichia coli, strains of Bacillus, such as Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus (e.g., Staphylococcus carnosus) can be used.

[0170] Exemplary useful animal host cell lines include, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0171] In one embodiment, the disclosure provides a method for producing a fusion protein that binds Tie2 and VEGF, the method comprising culturing cells that contain a nucleic acid, such as an expression vector, encoding the fusion protein (e.g., transformed with an expression vector that contains a nucleic acid encoding the fusion protein), and recovering the fusion protein from the cultured cells.

[0172] Cells can be cultured in various media. Any commercially available culture medium can be used without limitation. All other necessary supplements known to those skilled in the art can be included at appropriate concentrations. Culture conditions such as temperature, pH, and the host cell selected are known to those skilled in the art.

[0173] The antibody or antigen-binding fragment thereof can be recovered, for example, by centrifugation or ultrafiltration, and by removing impurities, for example, by affinity chromatography. Further additional purification techniques, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, etc., can be used.

[0174] Uses of Fusion Proteins; Therapeutics In another aspect, the disclosure relates to methods and compositions for preventing or treating angiogenic or vascular diseases by administering an effective amount of the fusion protein. In other aspects, the present embodiments relate to methods and compositions for modulating angiogenesis, endothelial signaling, inflammation and / or vascular leakage by administering the fusion protein to a subject in need thereof. In some embodiments, the fusion proteins described herein are for use in therapy.

[0175] Angiogenesis refers to the formation or growth of new blood vessels from pre-existing vessels. Examples of angiogenic or angiogenic-related diseases include, but are not limited to, cancer, metastasis, diabetic retinopathy, diabetic macular edema, retinopathy of prematurity, corneal graft rejection, macular degeneration, glaucoma such as neovascular glaucoma, generalized erythroderma, proliferative retinopathy, psoriasis, hemophilic arthropathy, capillary formation in atherosclerotic plaques, keloids, wound granulation, vascular adhesion, rheumatoid arthritis, osteoarthritis, autoimmune diseases, Crohn's disease, restenosis, atherosclerosis, intestinal adhesions, cat scratch disease, ulcers, liver cirrhosis, nephritis, diabetic nephropathy, diabetes, inflammatory diseases, and neurodegenerative diseases. Further exemplary cancers include, but are not limited to, esophageal cancer, gastric cancer, colon cancer, rectal cancer, oral cavity cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma, and multiple myeloid blood cancer.

[0176] The fusion protein may be administered to regulate angiogenesis, endothelial signaling, inflammation and / or vascular leakage. For example, the fusion protein may be administered to increase vascular endothelial cell membrane integrity and / or reduce vascular leakage. Vascular leakage is known to contribute to visual impairment in several common eye disorders, including but not limited to diabetic macular edema (DME), diabetic retinopathy (DR) and age-related macular degeneration (AMD). In some embodiments, the inflammation is due to sepsis, respiratory distress syndrome and / or viral infectious disease.

[0177] The fusion proteins and compositions thereof may be administered to mammals, including rats, mice, livestock, pets, humans, and the like. In some embodiments, the subject is a human. In some embodiments, the subject is a pet, such as a mammalian pet. In some embodiments, the pet is a dog, cat, rabbit, ferret, horse, mule, donkey, or hamster. The fusion protein or compositions thereof may be administered daily. The fusion protein or compositions thereof may be administered once every 1, 2, 3, 4, 6, or 12 months. In one embodiment, the fusion protein is administered once every 6 months (i.e., twice a year). Administration is by a typically accepted route, such as oral, rectal, intravitreal, intravenous, subcutaneous, intrauterine, or intracerebrovascular administration. In some embodiments, administration is by intravitreal injection. In one embodiment, 1 mg to 10 mg of the fusion protein is administered by intravitreal injection daily, monthly, or every 6 months. In one embodiment, less than 10 mg of the fusion protein is administered by intravitreal injection about once every six months (eg, twice a year).

[0178] Pharmaceutical Compositions; Administration In some embodiments, the composition comprising the fusion protein is a pharmaceutical composition. In some embodiments, the composition comprises a suitable vehicle, excipient or diluent typically used in the art.

[0179] Suitable vehicles, excipients, and diluents are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. AR Gennaro, Mack Publishing Company, Easton, Pa. 1995. Suitable pharma- ceutically acceptable vehicles, excipients, or diluents include, for example, one or more of water, saline, phosphate buffered saline, dextrose, histidine, glycerol, sucrose, polysorbate, ethanol, and the like, and combinations thereof. The pH of the solution is preferably about 5 to about 8, more preferably about 6 to about 7. It will be apparent to one of skill in the art that certain carriers may be more preferable, for example, depending on the route of administration and concentration.

[0180] The pharmaceutical composition with a pharmaceutically acceptable vehicle may be in various oral or parenteral dosage forms, such as tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, lyophilisates and suppositories. The pharmaceutical composition may contain diluents or excipients that may be formulated in combination, such as fillers, thickeners, binders, wetting agents, disintegrants, surfactants, etc. The solid preparation for oral administration may be in the form of tablets, pills, powders, granules, capsules, etc. In connection with binding, the compound may be formulated by combining one or more excipients, such as starch, calcium carbonate, sucrose, lactose or gelatin. Simple excipients and lubricants, such as magnesium stearate, talc, etc. may be further used.

[0181] Liquid preparations for oral administration may be suspensions, oral solutions, emulsions, syrups, etc. Excipients, such as simple diluents such as water or wet paraffin, various wetting agents, sweeteners, flavorings, preservatives, etc., may be included in liquid preparations. In addition, pharmaceutical compositions may be parenteral dosage forms such as sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilisates, suppositories, etc. Injectable propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and esters such as ethyl oleate may be suitable for insoluble solvents and suspensions. The basic substances of suppositories include witepsol, macrogol, Tween 61, cocoa butter, lauric butter, and glycerogelatin.

[0182] The composition is administered in a pharmacologic effective amount. Terms used herein, such as "pharmacologic effective amount," refer to a sufficient amount of a pharmaceutical composition for disease treatment with a reasonable benefit / risk ratio that may be applicable to all medical treatments. The effective amount may vary depending on various factors, including parameters such as the severity of the disease, the age and sex of the patient, the type of disease, drug activity, drug sensitivity, administration time, administration route, excretion rate, duration of treatment, and other factors. Also, the composition may be administered in a single dose or divided into multiple doses. Taking these factors into full consideration, it is important to administer the minimum amount sufficient to obtain maximum effect without side effects. The dosage of the pharmaceutical composition varies depending on various factors, including, but not limited to, the health and weight of the patient, the severity of the disease, the type of drug, the administration route, and the administration time.

[0183] The compositions may be administered to mammals, including rats, mice, livestock, pets, humans, etc., via typically accepted routes, such as orally, rectally, intravenously, subcutaneously, intrauterinely, intravitreally, or intracerebrovascularly, once or multiple times daily. In some embodiments, the compositions are administered by intravitreal injection. In some embodiments, the subject is a human. In some embodiments, the subject is a companion animal, such as a mammalian companion animal. In some embodiments, the companion animal is a dog, cat, rabbit, ferret, horse, mule, donkey, hamster, or other domesticated pet.

[0184] In another aspect, the disclosure refers to a method of prevention or treatment for angiogenic or vascular diseases, and anti-angiogenic methods, comprising administering an antibody or composition as described above to an individual in need thereof.

[0185] The fusion protein may be provided in a pharmaceutical composition.

[0186] The method of the present disclosure includes a procedure for administering a pharma- ceutically effective dose of a pharmaceutical composition to an individual in need of angiogenesis inhibition. The individual may be a mammal, such as, but not limited to, a dog, cat, ferret, cow, horse, rabbit, mouse, rat, or human. For example, in one embodiment, the individual may be a chicken, turkey, or other non-mammalian. The pharmaceutical composition may be administered in an appropriate manner, including parenterally, subcutaneously, intraperitoneally, intrapulmonary or intranasally, and, optionally, intralesionally for localized treatment. In some embodiments, the dosage of the pharmaceutical composition varies depending on various factors, including, but not limited to, the individual's health and weight, the severity of the disease, the type of drug, the route of administration, and the time of administration, and may be easily determined by one of ordinary skill in the art.

[0187] In another aspect, the disclosure refers to a method of preventing or treating cancer comprising administering the antibody or the composition comprising the antibody to an individual in need thereof.

[0188] The cancer is not limited as long as it can be treated with the antibody of the present disclosure. Specifically, the antibody can prevent the occurrence or progression of cancer by inhibiting angiogenesis. Examples of cancer include, but are not limited to, esophageal cancer, gastric cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma, and multiple myeloid cancer blood cancer.

[0189] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0190] [Example] The following examples are intended merely to illustrate the invention and are not to be construed as limiting thereof.

[0191] [Example 1] Preparation of fusion proteins A. Design of the structure A fusion protein, IGT-427, was designed in which the VEGF-A binding domains of VEGF receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2) were fused to the C-terminus of the heavy chain (HC) of an anti-Tie2 antibody. The construct was in a protein expression vector containing the human cytomegalovirus IE1 (CMV-IE1) promoter / enhancer. The amino acid sequences of the heavy chain (HC) and light chain (LC) are shown in Table 1 above.

[0192] B. Expression and Purification of IGT-427 Plasmid DNA encoding IGT-427 was transiently co-transfected into ExpiCHO-S cells according to the manufacturer's instructions (Gibco). Equal amounts of heavy and light chain DNA were used. The transfected cells were then incubated at 32°C in a humidified atmosphere of 5% CO2 in air with shaking for the Max titer protocol. After 12 days of incubation, the culture medium containing the secreted fusion protein was harvested, centrifuged to remove the cells, and the culture supernatant was collected and filtered. The fusion protein was purified using the AKTAPure purification system (Cytiva) with an affinity column of HiTrap MabSelect Sure Protein A (Cytiva) in binding / washing buffer (20 mM sodium phosphate, pH 7.2 and 150 mM NaCl) and elution buffer (25 mM sodium acetate, pH 3.3), and therefore immediately neutralized by adding 100 μl of 1 M Tris-HCl, pH 8.8 per milliliter fraction. The pooled fractions were then buffer exchanged into PBS, pH 7.4 using centrifugal filter units (Amicon). The resulting final samples were stored until further use. Concurrently, the fusion proteins were subjected to quality control studies such as SDS-PAGE (Invitrogen™ Novex™ WedgeWell), SEC-HPLC (Agilent Infinity 1260 and 300Å pore size column) and endotoxin determination (Charles River Cartridge<0.05EU / ml sensitivity) and surface plasmon resonance (SPR Biacore).

[0193] [Example 2] Preparation of orthologous Tie2 proteins A. Design of the structure The Ig3 and FNIII (1-3) domains of three orthologous proteins of Tie2 (human, rabbit and mouse) were selected as binding proteins to IGT-427. The domains of each Tie2 ortholog were cloned into the pFuse-mouse IgG1 Fc2 vector, which consists of the elongation factor 1-alpha (EF-1α) promoter and IL2 signal peptide, with consecutive C-terminal hexa-histidine and thrombin protease cleavage sites (LVPRGS) between the Tie2 and IgG1 Fc sequences. The sequences of the human (SEQ ID NO:2), rabbit (SEQ ID NO:3) and mouse (SEQ ID NO:4) Tie2 orthologs are shown in Table 1 above.

[0194] B. Expression and purification of Tie2 orthologs To produce Tie2 orthologues, Expi293F (Gibco) cells, which can produce recombinant proteins with high efficiency, were used. Orthologous Tie2 proteins were transiently expressed in Expi293F cell line by referring to the manufacturer's instructions (Gibco). After 4 days of transfection and incubation at 37°C in a humidified atmosphere of 8% CO2 in air with shaking, the resulting culture medium was collected and centrifuged to remove cells. Culture supernatants containing secreted antibodies were isolated and either stored at 4°C or immediately purified using an AKTAPure purification device (Cytiva) equipped with an affinity column (HiTrap MabSelect Sure Protein A, Cytiva) in two different buffer systems: binding and washing (20 mM sodium phosphate, pH 7.2 and 150 mM NaCl) and elution buffer (25 mM sodium acetate, pH 3.3). Tie2 protein was immediately neutralized by adding 100 μl of 1 M Tris-HCl, pH 8.8 per milliliter fraction. Fractions were pooled and then buffer exchanged into PBS, pH 7.4 through a protein centrifuge filter (Amicon) and stored at −80° C. The protein was then analyzed for quality control studies such as SDS-PAGE (Invitrogen™ Novex™ WedgeWell), SEC-HPLC (Agilent Infinity 1260 and 300 Å pore size columns) and surface plasmon resonance (SPR).

[0195] [Example 3] Affinity measurement of the fusion protein (IGT-427) for Tie2 orthologues The affinity of the fusion proteins to the orthologous Tie2 protein was measured by an SPR system (Biacore 3000). CM5 SPR chips (Cytiva) were modified with either the monomeric or dimeric forms of human, rabbit or mouse Tie2 using EDC / NHS chemistry (where the dimeric form was an Fc fusion). After activating the sensor chip with EDC (0.2 M) and NHS (0.05 M) for 7 min, 2.5 μg mL in 10 mM sodium acetate, pH 5 was added to reach an antigen surface density of approximately 200 RU. -1 The Tie2 constructs were then injected at 100 nM, 33.3 nM, 11.1 nM, 3.7 nM and 1.2 nM. The surfaces were then deactivated with a 7 min injection of 1 M ethanolamine·HCl pH 8.5. Activation and deactivation reagents were purchased from Cytiva. After immobilization of the Tie2 constructs, the surfaces were conditioned with two 5 s pulses of 300 mM phosphoric acid. Fusion proteins were injected over these immobilized Tie2 surfaces at 100 nM, 33.3 nM, 11.1 nM, 3.7 nM and 1.2 nM in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 2 mg / mL BSA and 0.05% polysorbate-20) for 5 min at 30 μL / min and dissociation was monitored for 20 min. Surface regeneration was performed with a 10 s injection of 300 mM phosphoric acid. The sensorgrams were fitted to a 1:1 Langmuir binding model using Scrubber software (BioLogic). K D The values ​​and SPR sensorgrams are shown in Table 2 below and Figures 1A to 1C, respectively. [Table 2]

[0196] [Example 4] Inhibition of VEGFR2 phosphorylation by IGT-427 HUVECs (1 × 10 5HUVECs were cultured at 1000 x 1000 cells / ml. To starve, 90% confluent cells were incubated with EBM-2 medium without supplements for 4 hours. Starved HUVECs were pretreated with IGT-427 or Eylea® at the indicated concentrations for 30 minutes, and then treated with recombinant human VEGF for 2 minutes. Cells were washed with cold PBS, treated with lysis buffer (10 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, protease inhibitors, phosphatase inhibitors) and lysed for 20 minutes at 4°C. Cell lysates were then prepared by centrifugation at 13000 rpm for 15 minutes. Protein concentrations in the supernatants were quantified by BCA assay. Cell lysates were prepared by adding 4x SDS sample buffer, cell lysates were subjected to SDS PAGE, and proteins were transferred to nitrocellulose membranes (GE).

[0197] To examine phosphorylation of VEGFR2 and Akt, blots were blocked with TBS-T containing 5% skim milk for 1 hour at room temperature (RT) and incubated with anti-phospho-VEGFR2 (Tyr1175) or anti-phospho-Akt (S473) antibodies for approximately 16 hours at 4°C. Phospho-VEGFR2 (Tyr1175) or pAkt (S473) signals were visualized by enhanced chemiluminescence (ECL). Membranes were then incubated for 15 minutes in stripping buffer (Thermo) and then reprobed with anti-VEGFR2 or anti-Akt antibodies to determine the amount of total VEGFR2 and total Akt. VEGF-induced VEGFR2 phosphorylation at Tyr1175 was inhibited by IGT-427 in a concentration-dependent manner, and the inhibition was comparable to that by Eylea (Figures 2A and 2B). Furthermore, IGT-427 induced phosphorylation of Akt in a dose-dependent manner.

[0198] VEGF reporter assays were performed using the VEGF Reporter Bioassay Kit (PROMEGA™). Briefly, 0.4 ml of KDR / NFAT-RE HEK293 cells were added to 4.6 ml of DMEM medium containing 10% FBS (assay buffer), and 25 μl of the cell suspension was plated in a white flat-bottom 96-well assay plate (Corning). IGT-427, faricimab or aflibercept was added at a 3-fold higher concentration of 50 nM and 3-fold serial dilutions in 25 μl of assay buffer, and recombinant human VEGF at a 3-fold higher concentration of 40 ng / ml in 25 μl of assay buffer was added to the plate. After 6 hours of incubation at 37° C., 75 μl of BIO-GLO™ reagent provided in the kit was added, and luminescence was measured using the GloMax® Discovery System (PROMEGA™). The IC50 values ​​of IGT-427, faricimab or aflibercept were approximately 0.48 nM, 0.32 nM or 0.75 nM, respectively (Figure 2C), suggesting that VEGF inhibition by IGT-427 was comparable to that by either faricimab or aflibercept.

[0199] [Example 5] Stronger and more sustained activation of Tie2 by IGT-427 CHO cells overexpressing full-length human Tie2 (CHO-hTie2, 1x105 cells / ml) were cultured in DMDM ​​medium (Thermo Fisher) at 37°C in 60 mm culture dishes. To starve, 90% confluent cells were incubated with DMEM medium without supplements for 4 hours. Starved CHO cells were treated with IGT-427 or recombinant human angiopoietin-1 (R&D Systems®) at a concentration of 10 nM for various periods (30 min, 1 h, 2 h, 6 h, and 24 h). Cells were washed with cold PBS and lysed with lysis buffer (10 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, protease inhibitors, phosphatase inhibitors) for 20 min at 4°C. Cell lysates were then prepared by centrifugation at 13000 rpm for 15 min. The lysates were then used to detect the levels of phospho-Tie2 and total Tie2 signals using pTie2 and total Tie2 ELISA assay kits (R&D Systems®). Phospho-Tie2 signals were normalized by total Tie2 signals. IGT-427 showed stronger and more sustained pTie2 signals compared to angiopoietin-1 (Figure 3).

[0200] [Example 6] Dose-dependent activation of Akt signaling by IGT-427 HUVECs (1x105 cells / ml) were cultured in EGM-2 medium (Lonza) at 37°C in 60mm culture dishes. To starve, 90% confluent cells were incubated with EBM-2 medium without supplements for 4 hours. Starved HUVECs were treated with various concentrations of IGT-427 (0.3, 1, 3, 10, 30, 100 nM) for 30 minutes. Cells were washed with cold PBS and treated with lysis buffer (10 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, protease inhibitors, phosphatase inhibitors) and lysed for 20 minutes at 4°C. Cell lysates were then prepared by centrifugation at 13000 rpm for 15 minutes. Protein concentrations were quantified by BCA assay. Cell lysates were prepared by adding 4xSDS sample buffer, the cell lysates were subjected to SDS PAGE, and proteins were transferred to nitrocellulose membranes (GE). To examine phosphorylation of Akt, blots were blocked with 5% skim milk in TBS-T for 1 hour at room temperature (RT) and incubated with anti-phospho-Akt (S473) antibody for approximately 16 hours at 4°C. pAkt (S473) signal was visualized by enhanced chemiluminescence (ECL). Membranes were then incubated for 15 minutes in stripping buffer (Thermo) and then reprobed with anti-Akt antibody to determine the amount of total Akt. As shown in Figure 4, there was a dose-dependent gradual increase in phospho-Akt signaling by IGT-427.

[0201] [Example 7] Bypassing Ang2 signaling by IGT-427 HUVECs (1x105 cells / ml) were cultured in EGM-2 medium (Lonza) at 37°C in 60mm culture dishes. To starve, 90% confluent cells were incubated with EBM-2 medium without supplements for 4 hours. Starved HUVECs were pretreated with recombinant human angiopoietin-2 (R&D Systems®) at concentrations of 0.01, 0.1, 1 nM for 30 minutes, and then treated with 2x20 kDa pegylated IGT-427 at a concentration of 30 nM for 60 minutes. Cells were washed with cold PBS and lysed with lysis buffer (10 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, protease inhibitors, phosphatase inhibitors) for 20 minutes at 4°C. Cell lysates were then prepared by centrifugation at 13000 rpm for 15 min. Protein concentration was quantified by BCA assay. Cell lysates were prepared by adding 4xSDS sample buffer, the cell lysates were subjected to SDS PAGE, and proteins were transferred to nitrocellulose membranes (GE). To examine phosphorylation of Akt, blots were blocked with TBS-T containing 5% skim milk for 1 h at room temperature (RT) and incubated with anti-phospho-Akt (S473) antibody for approximately 16 h at 4°C. pAkt (S473) signals were visualized by enhanced chemiluminescence (ECL). Membranes were then incubated for 15 min in stripping buffer (Thermo) and then reprobed with anti-Akt antibody to determine the amount of total Akt. Recombinant angiopoietin-2 (Ang2) induced weak phosphorylation of Akt in a concentration-dependent manner. However, IGT-427 further increased Akt phosphorylation in the presence of recombinant angiopoietin-2, demonstrating that IGT-427 overcomes Ang2 signaling (Figure 5).

[0202] [Example 8] Double binding capacity of IGT-427 The dual binding properties of IGT-427 to its antigens, Tie2 and VEGF, were investigated by a surface plasmon resonance (SPR) system (BIACORE™ 3000). Recombinant human full-length Ang2 (R&D Systems®) was captured on a CM5 SPR chip (Cytiva), and human dimeric Tie2 (100 nM) was first injected, followed by IGT-427 (150 nM) and human VEGF (20 nM) (R&D Systems®). The sensorgrams showed a progressive increase in signal, demonstrating that IGT-427 can bind to Ang2-Tie2 complex and VEGF simultaneously ( FIG. 6 ).

[0203] [Example 9] Inhibition of TNF-α-induced apoptosis by IGT-427 HUVECs (1x105 cells / ml) were cultured in EGM-2 medium (Lonza) at 37°C in 60mm culture dishes. 90% confluent cells were pretreated with IGT-427 or Eylea® for 60 min, then treated with recombinant human TNF-α (50ng / ml) for 24 h. Apoptotic cells were stained by APO-BrdU™ TUNEL Assay Kit (Thermo Fisher, A23210) and determined by Attune (Thermo Fisher). Briefly, cells were suspended in 0.5mL PBS, 5mL 1% (w / v) paraformaldehyde in PBS was added to the cell suspension, and then the cell suspension was placed on ice for 15 min. Cells were centrifuged at 300xg for 5 min and washed twice with 5mL PBS. The cells were resuspended in 0.5 mL of PBS, 5 mL of ice-cold 70% (v / v) ethanol was added to the cell suspension, and the cell suspension was placed in a -20°C freezer for a minimum of 30 minutes. To remove the 70% (v / v) ethanol, the cell suspension was centrifuged at 300 x g for 5 minutes, and the cell pellet was then resuspended in 1 mL of wash buffer provided in the kit. The cell pellet was incubated in 50 μL of DNA labeling solution (10 μL of reaction buffer, 0.75 μL of TdT enzyme, 8.0 μL of BrdUTP, and 31.25 μL of dH2O) for 60 minutes at 37°C. At the end of the incubation period, 1.0 mL of rinse buffer was added to the cells, and the cells were centrifuged at 300 x g for 5 minutes. After further washing with rinse buffer, cells were incubated in 100 μL of antibody staining solution (5.0 μL of Alexa Fluor™ 488 dye-conjugated anti-BrdU antibody and 95 μL of rinse buffer) for 30 min at room temperature. Apoptotic cells were analyzed by Attune flow cytometry (Thermo Fisher). TNF-α induced apoptosis in HUVEC cells, and IGT-427, but not Eylea, significantly inhibited apoptosis induced by TNF-α (Figure 7).

[0204] [Example 10] Measurement of surface Tie2 level In preparation for the assay, CHO-hTie2 cells were grown to confluence on 10 cm tissue culture dishes. CHO-hTie2 cells were washed with Dulbecco's Phosphate Buffered Saline (DPBS), trypsinized to detach the cells, counted, centrifuged at 200 g for 5 min, and resuspended at a density of 0.05×106 cells / mL. To seed the assay plates, 1 mL of cell suspension was dispensed into each well of a 24-well tissue culture plate, and the plate was incubated overnight at 370°C. The next day, medium was removed from the tissue culture plates and replaced with 0.5 mL of either DMEM alone or DMEM containing 10 nM Angiopoietin 1 (R&D Systems®#923-AN), 10 nM IGT-301 (ITP-006), or 10 nM IGT-427 (ITP-016) for the unstimulated group. The assay plates were returned to a 370° C. tissue culture incubator for the indicated times (0.5 h, 1 h, 2 h, 4 h, 6 h or 24 h).

[0205] After incubation, the tissue culture plates were placed on ice, washed with cold DPBS, and detached by adding 0.5 mL of non-enzymatic cell dissociation buffer (PeproTech® #CPD-125). Cells were incubated on ice for 5 minutes, added 1 mL of FACS buffer (0.5% BSA in DPBS), and collected by gentle pipetting to detach cells still attached to the culture surface. The resulting cell suspension was collected in a 5 mL Falcon FACS tube, centrifuged at 300 g for 5 minutes to pellet the cells, and the supernatant was aspirated. Cells were washed once with 1 mL of FACS buffer, and the supernatant was aspirated, leaving approximately 100 uL of residual FACS buffer in the tube. Cells were then stained by adding 100 uL of staining cocktail consisting of 99.5 uL of FACS buffer and 0.5 uL of PE-Tie2 (BioLegend® #334205). The samples were covered with parafilm and incubated in the dark at 40° C. for 30 minutes.

[0206] After incubation, cells were washed twice with 1 mL of FACS buffer as above, then resuspended in a final volume of 400 uL of FACS buffer and either analyzed immediately or fixed for analysis the next day. For fixation, cells were washed twice as above, resuspended in 200 uL of 4% paraformaldehyde for 30 minutes at 40°C in the dark, washed once with 1 mL of FACS buffer, resuspended in a final volume of 400 uL of FACS buffer, and stored covered overnight at 40°C. Samples were acquired using an Attune NXT Acoustic flow cytometer (Thermo Fisher) and files were analyzed using FlowJo™ (Tree Star, Inc.). IGT-427 treatment resulted in a slower disappearance and greater presence of surface Tie2 compared to the endogenous Tie2 agonist angiopoietin-1 (Ang1) (Figure 8).

[0207] [Example 11] Blocking Tie2 cleavage Tie2 is known to be cleaved and downregulated in humans, mice and endothelial cells in various inflammatory conditions, resulting in increased levels of soluble Tie2 (sTie2) (Thamm et al., Critical Care Medicine, 2018.46:e928-e936). Blocking a broad range of matrix metalloproteinases (MMPs) is known to be sufficient to prevent Tie2 cleavage and vascular leakage (Thamm et al., Critical Care Medicine, 2018.46:e928-e936; Sung et al., 2011. The Journal of Clinical Endocrinology & Metabolism 96:E1148-E1152; Findley et al., 2007, Arteriosclerosis, Thrombosis, and Vascular Biology 27:2619-2626). MMP-14 is believed to be the major Tie2 cleavage, and Tie2 is cleaved at multiple sites within the fibronectin type 3 domain by matrix metalloproteinase-14 (Idowu, TO et al., eLife, August 24, 2020, 9: e59520).

[0208] Recombinant human MMP-14 (R&D Systems®, Cat# 918-MP-010, 0.5ug) was mixed with dimeric human Tie2 extracellular domain (ECD)-human IgG fusion protein (hTie2-ECD, 5ug) in the absence or presence of IGT-427 (same molar concentration of hTie2-ECD). The mixture was incubated at 30°C for 16 hours and subjected to SDS-PAGE (reducing conditions). Consistent with previous reports, co-incubation of MMP-14 and human Tie2 resulted in a significant reduction of intact hTie2-ECD and the appearance of shorter cleaved fragments. However, IGT-427 significantly blocked the cleavage and degradation of intact human Tie2-ECD protein by MMP-14 (Figure 9).

[0209] Without wishing to be bound by theory, based on this evidence, it is believed that a bispecific comprising an anti-Tie2 antibody or antigen-binding fragment that binds to the Tie2 Ig3-FNIII(1-3) domain comprising SEQ ID NO:2, 3 or 4, or more specifically the amino acid sequence of SEQ ID NO:20 and / or the amino acid sequence of SEQ ID NO:21, and a VEGF-binding domain, reduces Tie2 shedding by combining the effects of blocking MMP-14 binding to human Tie2 and inhibiting VEGF-induced cleavage of the human Tie2-ECD protein, providing the particularly beneficial result of retaining Tie2 at the cell surface membrane.

[0210] [Example 12] Soluble Tie2 (sTie2) measurement HUVECs (1x105 cells / ml) were cultured in EGM-2 medium (Lonza) at 37°C in 60mm culture dishes. 90% confluent cells were pretreated with IGT-427, faricimab or recombinant human angiopoietin-1 (R&D Systems®) at a concentration of 10nM for 60 minutes, and then treated with recombinant human TNF-α (50ng / ml) for 24 hours. Cell supernatants were collected and centrifuged at 13000rpm for 10 minutes. Soluble Tie2 in cell supernatants was measured by a total Tie2 ELISA assay kit (R&D Systems®). IGT-427, but not other drugs, suppressed soluble Tie2 levels in both basal and TNF-α-induced conditions (Figure 10).

[0211] [Example 13] TEER (Transendothelial Electrical Resistance) Assay HUVECs (2x105 cells) were cultured in cell culture inserts in 24-well plates (Corning) in EGM-2 medium (Lonza) at 37°C. After 2 days of incubation, the medium was changed to EGM-2 medium containing 0.5% FBS. After a further 2 days of incubation, the cell culture inserts were placed in a CellZscope® (NanoAnalytics) and TEER (transendothelial electrical resistance) was measured continuously in a CO2 incubator at 37°C. After confirming cell barrier formation, recombinant human VEGF (R&D) at a concentration of 10ng / ml was added to the lower compartment. Various drugs, such as IGT-427, faricimab, aflibercept or recombinant human angiopoietin-1 (R&D) at a concentration of 10nM, were added to the lower compartment and TEER was monitored for 48 hours after VEGF treatment. VEGF disrupts endothelial barrier integrity, and IGT-427 was superior to faricimab and aflibercept, as well as Ang1, in restoring endothelial barrier integrity from VEGF-induced damage (Figure 11).

[0212] [Example 14] Inhibition of CNV (choroidal neovascularization) by intravitreally injected IGT-427 Chinchilla rabbits (male, 2.0-2.5 kg) were anesthetized by applying eye drops (Mydriacyl eye drops, 1%) to the right eyeball, and the right eyeball was irradiated with a laser (Elite, Lumenis®, USA) at 532 nm, power output of 150 mW, duration of 0.1 s to generate six spots around the optic nerve. IGT-427, Eylea® or control IgG (50 μl injection volume / eye, Eylea® (800 μg), IGT-427 (885 μg), control IgG (716 μg)) was administered intravitreally to the right eye of anesthetized animals on the day of choroidal neovascularization (CNV) induction using a syringe with a 3-gauge needle. The molar ratio of Eylea®, IGT-427 and control IgG is 1:0.65:0.68. Due to some limitations on concentration and volume, different molarities were used. On days 0, 7, and 14, animals were anesthetized by applying eye drops (Mydriacyl eye drops, 1%) to the right eyeball, and 1 mL of fluorescein sodium salt solution (2%) was intravenously injected to take fundus images using a fundus camera (TRC-50IX, TOPCON, Japan) within 2 minutes. Retinal fluorescein fundus photographs were used to evaluate retinal CNV area and efficacy, and image analysis was performed using ImageJ software (NIH, Bethesda, MD) to verify the fluorescence intensity of the CNV lesion site. IGT-427 inhibited retinal leakage by 30% compared to the control IgG. Meanwhile, Eylea® was able to inhibit retinal leakage by 20% (Figure 12).

[0213] [Example 15] IGT-427 variants for PEGylation To extend the ocular half-life of IGT-427, the heavy and light chain plasmids of IGT-427 were modified to generate five different antibody constructs for PEGylation (outlined in FIG. 13). All five variants have a light chain with the mutation C214S (EU numbering) that removes the interchain disulfide bond between the heavy and light chains in combination with a C218S mutation (EU numbering) on ​​each heavy chain. In addition, one or both heavy chain cysteines in the hinge region of IGT-427 were replaced with serine, leaving one or none of the interchain disulfide bonds between the heavy chains. In the case of PRO592 and PRO596, a single disulfide bond remains reduced and PEGylated. In the case of PRO593, PRO594, and PRO595, all interchain disulfide bonds are removed and new cysteine ​​residues are introduced for PEGylation. PRO593 has a cysteine ​​introduced at the C-terminus of the heavy chain, whereas PRO594 and PRO595 each have a cysteine ​​introduced in the linker domain between the Fc domain and the VEGF trap domain. Together, these variants represent a set of constructs with diverse PEGylation sites that were tested for PEGylation efficiency, antigen binding, and bioactivity. Each IGT-427 variant was expressed using the Expi293 transient expression system. Clarified cell culture supernatants were purified on a HiTrap mAbselect PrismA column (Cytiva). The eluted antibody was dialyzed into 1x PBS and concentrated to 10mg / mL for PEGylation studies.

[0214] [Example 16] PEGylation reaction screening 10 mg / mL of each of the IGT-427 variants was reduced with 10 or 20 mM cysteamine for 1 hour at room temperature and then buffer exchanged into PEGylation buffer (50 mM sodium phosphate, 150 mM NaCl, 2.5 mM EDTA, pH 7.2). 10 molar equivalents of 20 kDa linear PEG-maleimide were added to the buffer exchanged antibody and the PEGylation reaction was allowed to proceed for 1.5 hours at room temperature. Analysis of the reaction mixtures by non-reducing SDS-PAGE revealed that the variants showed up to about 50% conversion to the doubly PEGylated species.

[0215] To further optimize PEGylation efficiency, reduction conditions were screened using PRO593, the variant that showed the best conversion under mild reduction conditions. 10 mg / mL PRO593 was reduced with 10 or 100 mM cysteamine for 1 hour, 1 or 10 mM DTT for 15 minutes, or 2 or 20-fold molar excess of TCEP (tris(2-carboxyethyl)phosphine) for 15 minutes. After each reduction, PRO593 was buffer exchanged into PEGylation buffer and PEGylated with a 10-fold molar excess of 20 kDa linear PEG-maleimide for 1.5 hours. From this set of reduction conditions, it was observed that a 20-fold molar excess of TCEP resulted in nearly complete conversion of PRO593 to species with two PEG attachments. All five IGT-427 variants were then reduced with a 20-fold molar excess of TCEP for 15 min at room temperature, followed by buffer exchange into PEGylation buffer and PEGylation with a 10-fold molar excess of 20 kDa linear PEG-maleimide. The results of the optimized reaction conditions are shown in Figure 14. Antibodies with two PEG attachments were purified from the reaction mixture using a HiTrap SP-HP cation exchange chromatography column (Cytiva) and gradient salt elution. The PEGylated antibodies were dialyzed into 1x PBS, concentrated to 10 mg / mL, formulated with 0.01% polysorbate-20, and filtered through a 0.2 μm filter.

[0216] [Example 17] Characterization of PEGylated IGT-427 variants Binding of Tie2 or VEGF was characterized by SPR (FIG. 15). A 100 nM solution of each antibody was injected over the surface containing immobilized Tie2 or VEGF at 30 μL / min for 300 s. The relative binding signal after 300 s was used to determine the difference in binding.

[0217] [Example 18] Ocular PK studies To evaluate the effect of PEGylation and PEG molecular weight on the ocular pharmacokinetics of IGT-427, 500 μg intravitreal injections of Eylea®, faricimab, IGT-427, (2×20 kDa PEG)-IGT-427, and (2×40 kDa PEG)-IGT-427 were administered to male New Zealand White rabbits. For each test article group, three or four animals were dosed on day 0. Blood was collected from either three or four animals on days 1, 3, 7, and 14 after dosing, and then both eyes were harvested, flash frozen in liquid nitrogen, and dissected for collection of aqueous humor, vitreous humor, retina, and choroid after euthanasia by intravenous barbiturate overdose. Vitreous humor samples were diluted 1:5 in 1×PBST without homogenization and stored at −70° C. until further analysis.

[0218] Zaltrap® was purchased from a commercial source, dialyzed against EYLEA® formulation buffer (10 mM sodium phosphate, 40 mM NaCl, 0.03% polysorbate-20, 5% sucrose) and diluted to 10 mg / mL in the same buffer.

[0219] Faricimab was expressed using the Expi293 transient expression system (ThermoFisher). Clarified cell culture supernatants were purified on HiTrap MabSelect PrismA columns (Cytiva) with a 2% ethanol wash to remove endotoxin, followed by pH gradient elution to remove incompletely assembled antibody. Eluted antibody was dialyzed into 1x PBS, concentrated to 10 mg / mL, formulated with 0.01% polysorbate-20, and filtered through a 0.2 μm filter.

[0220] IGT-427 was expressed using the Expi293 transient expression system. Clarified cell culture supernatants were purified on HiTrap MabSelect PrismA columns (Cytiva) with a 2% ethanol wash to remove endotoxin. Eluted antibodies were dialyzed into 1× PBS, concentrated to 10 mg / mL, formulated with 0.01% polysorbate-20, and filtered through a 0.2 μm filter.

[0221] PEGylated IGT-427. PRO593 was expressed using the Expi293 transient expression system (ThermoFisher). Clarified cell culture supernatant was purified on a HiTrap MabSelect PrismA column (Cytiva) with a 2% ethanol wash to remove endotoxin. The eluted antibody was dialyzed into 1× PBS and concentrated to 10 mg / mL for PEGylation. The concentrated antibody was reduced with a 20-fold molar excess of TCEP, then desalted into PEGylation buffer and incubated with a 10-fold molar excess of either a 20 kDa linear or 40 kDa branched PEG maleimide for 1.5 h at room temperature. The PEGylation reaction mixture was purified using a HiTrap SP-HP column (Cytiva) at pH 4.6 with a NaCl gradient. The eluting PEGylated antibody was dialyzed into PBS, concentrated to 10 mg / mL, formulated with 0.01% polysorbate-20, and filtered through a 0.2 μm filter.

[0222] [Example 19] Sample characterization for PK studies Non-reducing SDS-PAGE analysis of each test article was performed by mixing 5 μg of protein with 4× LDS (lithium dodecyl sulfate) and running the samples on a 4-12% Bis-Tris gel at 150 V for 60 min without heating, followed by staining with SafeStain (ThermoFisher). SEC-HPLC analysis was performed by loading 5 μg of each test article onto a Zenix® size-exclusion column (SEC)-300 at 0.5 mL / min in 100 mM arginine, 1× PBS, pH 6.7. Binding to rabbit Tie2 and VEGF was characterized by SPR.

[0223] SPR was used to evaluate the binding of PEGylated IGT-427 variants to either Tie2 or VEGF. Figure 3 shows the sensorgrams obtained from 100 nM injection of variants onto human Tie2 or VEGF surfaces. Compared to unmodified IGT-427, there is a dramatic reduction in binding signal due to the presence of PEG. However, the relative binding signal of each variant to the remaining variants can be used as a measure of relative affinity for the antigen. Although there are slight differences in the binding signal of each variant, overall, binding to either Tie2 or VEGF is largely independent of the PEGylation site.

[0224] All test articles were formulated at 10 mg / mL and were >90% pure by SEC-HPLC (Figure 16) and non-reducing SDS-PAGE (Figure 17), except for the 20 kDa PEGylated IGT-427, which contained 15% high molecular weight species. Endotoxin levels were all below 0.1 EU / mg, and all test articles bound their respective antigens with the expected affinity (Figures 18A-18C).

[0225] [Example 20] Total Drug ELISA for Vitreous Humor Measurements Total drug levels in the vitreous humor were quantified by three different ELISAs depending on the drug administered. IGT-427 and a PEGylated version of IGT-427 were captured on mouse anti-human IgG-coated plates and detected with a Tie2-HRP conjugate. Eylea® was captured on mouse anti-human IgG-coated plates and detected with a polyclonal goat anti-human IgG-HRP conjugate. Finally, faricimab was captured on a VEGF-coated plate and detected with a polyclonal goat anti-human IgG-HRP conjugate. For all ELISA analyses, samples were assayed in duplicate at dilutions between 1:1250 and 1:62500, and the luminescence of the assay plates was read using a Molecular Devices SpectraMax® M5 plate reader. For all ELISA analyses, sample luminescence values ​​were within the linear range of the standard curve. The entire standard curve was fitted using a four-parameter equation, and sample luminescence values ​​were converted to concentrations by a four-parameter fit.

[0226] Anti-human IgG capture / Tie2 detection ELISA. High binding plates were coated overnight at 4° C. with 1 μg / mL anti-human IgG in carbonate buffer (pH 9.5). The coated plates were washed with wash buffer (1×PBST (Phosphate Buffered Saline with TWEEN®20) containing 150 mM NaCl) and blocked with 5% BSA in PBST for 1 hour at 37° C. (shaking at 420 RPM). The blocked plates were washed and samples, standard curve and blanks were incubated on the plates for 1.5 hours at room temperature (shaking at 420 RPM). After sample incubation, the plates were washed and incubated with 1 μg / mL biotinylated human Tie2 for 1 hour at room temperature (covered and shaking at 420 RPM). The plates were washed again and incubated with streptavidin-HRP for 1 hour, protected from light. The plates were given a final wash and then developed with chemiluminescent HRP substrate. After 30 seconds, all emission wavelengths were read on a plate reader.

[0227] Anti-human IgG capture / anti-huFc detection ELISA. High binding plates were coated overnight at 4° C. with 1 μg / mL anti-human IgG in carbonate buffer (pH 9.5). The coated plates were washed with wash buffer and blocked with 1% BSA in PBST for 4 hours at 4° C. The blocked plates were washed and after the final wash the plates were dried at 4° C. Samples, standard curve and blanks were incubated on the plates overnight at 4° C. After sample incubation the plates were washed and incubated with polyclonal goat anti-human IgG-HRP conjugate for 1 hour at room temperature (capped and shaking at 420 RPM). The plates were given a final wash and then developed with chemiluminescent HRP substrate. All emission wavelengths were read on a plate reader after 30 seconds.

[0228] VEGF capture / anti-huFc detection ELISA. VEGF at 1 μg / mL in carbonate buffer (pH 9.5) was used to coat high binding plates overnight at 4° C. The coated plates were washed with wash buffer and blocked with 1% BSA in PBST for 4 hours at 4° C. The blocked plates were washed and after the final wash the plates were dried at 4° C. Samples, standard curve and blanks were incubated on the plates overnight at 4° C. After sample incubation the plates were washed and incubated with polyclonal goat anti-human IgG-HRP conjugate for 1 hour at room temperature (capped and shaking at 420 RPM). The plates were given a final wash and then developed with chemiluminescent HRP substrate. All emission wavelengths were read on a plate reader after 30 seconds.

[0229] [Example 21] Measurement of ocular PK Total drug levels of each test article were measured in the vitreous humor on days 1, 3, 7, and 14 after dosing. Eyes were harvested from three or four animals at each time point, resulting in either six or eight vitreous humor samples per test article per time point. Total drug levels were quantified from animals dosed with IGT-427 or PEGylated IGT-427 by anti-human IgG capture and Tie2 detection ELISA. Total drug levels were quantified from animals dosed with EYLEA® by anti-human IgG capture and anti-human Fc detection ELISA. Finally, total drug levels were quantified from animals dosed with faricimab by VEGF capture and anti-human Fc detection ELISA. The results of each of these ELISAs are shown in FIG. 19. EYLEA® was measured in the vitreous with a Cmax value of 433.8 μg / mL and a half-life of 4.2 days. Faricimab was measured intravitreally with a Cmax of 154.2 μg / mL and a half-life of 4.4 days. IGT-427 was measured intravitreally with a Cmax of 217.3 μg / mL and a half-life of 3.8 days. IGT-427 modified with two 20 kDa linear PEG molecules was measured intravitreally with a Cmax of 406.6 μg / mL and a half-life of 8.3 days. IGT-427 modified with two 40 kDa branched PEG molecules was measured intravitreally with a Cmax of 268.8 μg / mL and a half-life of 8.0 days. [Industrial Applicability]

[0230] The fusion protein that binds to Tie2 and VEGF can bind to Tie2 and VEGF with high affinity, maintain cross-reactivity to humans, mice and rabbits, and exhibit desired antigen reactivity. Furthermore, by inducing Tie2 phosphorylation, activation of Tie2 receptor and inhibition of VEGF, the fusion protein that binds to Tie2 and VEGF can be used to prevent or treat angiogenic or vascular diseases of interest.

[0231] Equivalent The foregoing specification is believed to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and examples detail certain embodiments and set forth the best mode contemplated by the inventors. However, no matter how detailed the foregoing may be in writing, it will be understood that the embodiments can be practiced in many ways and should be construed in accordance with the appended claims and their equivalents.

[0232] When terms such as at least and about are present before a list of numerical values ​​or ranges, those terms modify all of the values ​​or ranges provided in the list. In some cases, the term about may include numerical values ​​that are rounded to the nearest significant figure. Any recited range includes the endpoints unless expressly stated to the contrary; for example, "5 to 50" includes the values ​​5 and 50.

Claims

1. (a) a vascular endothelial growth factor (VEGF) binding domain comprising a VEGF receptor extracellular domain; and (b) an anti-Tie2 antibody, (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO:5, a heavy chain complementarity determining region 2 (HCDR2) having the amino acid sequence of SEQ ID NO:6, and a heavy chain complementarity determining region 3 (HCDR3) having the amino acid sequence of SEQ ID NO:7; wherein the VH comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 18; and (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 8, a light chain complementarity determining region 2 (LCDR2) having the amino acid sequence of SEQ ID NO: 9, and a light chain complementarity determining region 3 (LCDR3) having the amino acid sequence of SEQ ID NO: 10, wherein the VL comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 19; Fusion proteins.

2. The fusion protein of claim 1, wherein the fusion protein comprises a VH comprising the amino acid sequence of SEQ ID NO: 18 and a VL comprising the amino acid sequence of SEQ ID NO:

19.

3. The fusion protein is (a) a heavy chain (HC) comprising at least 95% identity to the amino acid sequence of SEQ ID NO:26; and (b) a light chain (LC) comprising at least 95% identity to the amino acid sequence of SEQ ID NO:

12.

4. The fusion protein of claim 3, wherein the fusion protein comprises a HC comprising the amino acid sequence of SEQ ID NO:26 and a LC comprising the amino acid sequence of SEQ ID NO:

12.

5. The fusion protein is (a) a HC that comprises at least 95% identity to the amino acid sequence of SEQ ID NO:11; and (b) a LC comprising at least 95% identity to the amino acid sequence of SEQ ID NO:

12.

6. The fusion protein of claim 5 , wherein the fusion protein comprises a HC comprising the amino acid sequence of SEQ ID NO:11 and a LC comprising the amino acid sequence of SEQ ID NO:

12.

7. The fusion protein of claim 1 , wherein the VEGF receptor extracellular domain comprises the amino acid sequence of SEQ ID NO: 13 and the amino acid sequence of SEQ ID NO:

14.

8. The fusion protein of claim 1 , wherein the anti-Tie2 antibody has an IgG1 isotype constant region including a constant heavy chain domain (CH).

9. The fusion protein of claim 8, wherein the CH comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

17.

10. The fusion protein of claim 8, wherein the CH comprises the amino acid sequence of SEQ ID NO:

17.

11. the IgG1 isotype constant region comprises one or more mutations that reduce or eliminate interaction with an Fc receptor, 9. The fusion protein of claim 8, wherein the one or more mutations are selected from the group consisting of K322, P331, L234, L235, H310, M252, I253, S254, T256, H433, N434 and H436, wherein the numbering of amino acid positions is according to EU numbering.

12. The fusion protein of claim 8 , wherein the VEGF-binding domain is linked to the C-terminus of the CH of the anti-Tie2 antibody by a linker.

13. The linker is (a) an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 25; or (b) an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 16; The fusion protein of claim 12, comprising:

14. The linker is (a) the amino acid sequence of SEQ ID NO: 25, or (b) the amino acid sequence of SEQ ID NO: 16; The fusion protein of claim 13 .

15. (a) a HC comprising, in order from N-terminus to C-terminus: (i) a VH comprising the amino acid sequence of SEQ ID NO: 18; (ii) CH comprising the amino acid sequence of SEQ ID NO: 17; (iii) a linker comprising the amino acid sequence of SEQ ID NO: 25, and (vi) a VEGF receptor extracellular domain comprising the amino acid sequence of SEQ ID NO: 13 and SEQ ID NO: 14, and (b) a LC comprising, in order from N-terminus to C-terminus: (i) a VL comprising the amino acid sequence of SEQ ID NO: 19; and (ii) a constant light domain (CL) comprising the amino acid sequence of SEQ ID NO: 30; A fusion protein comprising:

16. (a) a HC comprising, in order from N-terminus to C-terminus: (i) a VH comprising the amino acid sequence of SEQ ID NO: 18; (ii) CH comprising the amino acid sequence of SEQ ID NO: 17; (iii) a linker comprising the amino acid sequence of SEQ ID NO: 16, and (iv) a VEGF receptor extracellular domain comprising the amino acid sequence of SEQ ID NO: 13 and SEQ ID NO: 14, and (b) a LC comprising, in order from N-terminus to C-terminus: (i) a VL comprising the amino acid sequence of SEQ ID NO: 19; and (ii) CL comprising the amino acid sequence of SEQ ID NO: 30; A fusion protein comprising:

17. A nucleic acid encoding a fusion protein according to any one of claims 1 to 16.

18. 18. One or more expression vectors comprising the nucleic acid of claim 17.

19. A cell comprising one or more expression vectors according to claim 18.

20. (a) a first nucleic acid encoding a HC of a fusion protein, said HC comprising, in order from N-terminus to C-terminus: (i) a VH comprising the amino acid sequence of SEQ ID NO: 18; (ii) CH comprising the amino acid sequence of SEQ ID NO: 17; (iii) a linker comprising the amino acid sequence of SEQ ID NO: 25, and (iv) a VEGF receptor extracellular domain comprising the amino acid sequence of SEQ ID NO: 13 and SEQ ID NO: 14; and (b) a second nucleic acid encoding a LC of the fusion protein, said LC comprising, in order from N-terminus to C-terminus: (i) a VL comprising the amino acid sequence of SEQ ID NO: 19; and (ii) CL comprising the amino acid sequence of SEQ ID NO: 30; A nucleic acid composition comprising:

21. (a) a first nucleic acid encoding a HC comprising the nucleic acid sequence of SEQ ID NO:29; and (b) a second nucleic acid encoding an LC comprising the nucleic acid sequence of SEQ ID NO: 28; 21. The nucleic acid composition of claim 20.

22. 21. The nucleic acid composition of claim 20, wherein the HC comprises the amino acid sequence of SEQ ID NO:26 and the LC comprises the amino acid sequence of SEQ ID NO:

12.

23. 21. An expression vector system comprising the nucleic acid composition of claim 20.

24. 24. A cell comprising the expression vector system of claim 23, wherein the expression vector system encodes both the HC and LC of the fusion protein.

25. (a) a HC of the fusion protein, said HC comprising, in order from N-terminus to C-terminus: (i) a VH comprising the amino acid sequence of SEQ ID NO: 18; (ii) CH comprising the amino acid sequence of SEQ ID NO: 17; (iii) a linker comprising the amino acid sequence of SEQ ID NO: 25, and (iv) a VEGF receptor extracellular domain comprising the amino acid sequence of SEQ ID NO: 13 and SEQ ID NO: 14, and (b) a LC of the fusion protein, said LC comprising, in order from N-terminus to C-terminus: (i) a VL comprising the amino acid sequence of SEQ ID NO: 19; and (ii) CL comprising the amino acid sequence of SEQ ID NO: 30; An isolated nucleic acid encoding

26. 26. The isolated nucleic acid of claim 25, comprising the nucleic acid sequence of SEQ ID NO:29 and the nucleic acid sequence of SEQ ID NO:

28.

27. 26. The isolated nucleic acid of claim 25, wherein the HC comprises the amino acid sequence of SEQ ID NO:26 and the LC comprises the amino acid sequence of SEQ ID NO:

12.

28. 26. One or more expression vectors comprising the isolated nucleic acid of claim 25.

29. 30. A cell comprising one or more expression vectors according to claim 28, wherein the one or more expression vectors encode both the HC and the LC.

30. (a) a first nucleic acid encoding a HC of a fusion protein, said HC comprising, in order from N-terminus to C-terminus: (i) a VH comprising the amino acid sequence of SEQ ID NO: 18; (ii) CH comprising the amino acid sequence of SEQ ID NO: 17; (iii) a linker comprising the amino acid sequence of SEQ ID NO: 16, and (iv) a VEGF receptor extracellular domain comprising the amino acid sequence of SEQ ID NO: 13 and SEQ ID NO: 14, and (b) a second nucleic acid encoding a LC of the fusion protein, said LC comprising, in order from N-terminus to C-terminus: (i) a VL comprising the amino acid sequence of SEQ ID NO: 19; and (ii) CL comprising the amino acid sequence of SEQ ID NO: 30; A nucleic acid composition comprising:

31. (a) a first nucleic acid encoding a HC comprising the nucleic acid sequence of SEQ ID NO:27; and (b) a second nucleic acid encoding an LC comprising the nucleic acid sequence of SEQ ID NO: 28; The nucleic acid composition of claim 30.

32. 31. The nucleic acid composition of claim 30, wherein the HC comprises the amino acid sequence of SEQ ID NO:11 and the LC comprises the amino acid sequence of SEQ ID NO:

12.

33. 31. An expression vector system comprising the nucleic acid composition of claim 30.

34. 34. A cell comprising the expression vector system of claim 33, wherein the expression vector system encodes both the HC and LC of the fusion protein.

35. (a) a HC of the fusion protein, said HC comprising, in order from N-terminus to C-terminus: (i) a VH comprising the amino acid sequence of SEQ ID NO: 18; (ii) CH comprising the amino acid sequence of SEQ ID NO: 17; (iii) a linker comprising the amino acid sequence of SEQ ID NO: 16, and (iv) a VEGF receptor extracellular domain comprising the amino acid sequence of SEQ ID NO: 13 and SEQ ID NO: 14; and (b) a LC of the fusion protein, said LC comprising, in order from N-terminus to C-terminus: (i) a VL comprising the amino acid sequence of SEQ ID NO: 19; and (ii) CL comprising the amino acid sequence of SEQ ID NO: 30; An isolated nucleic acid encoding

36. 36. The isolated nucleic acid of claim 35, comprising the nucleic acid sequence of SEQ ID NO:27 and the nucleic acid sequence of SEQ ID NO:

28.

37. 36. The isolated nucleic acid of claim 35, wherein the HC comprises the amino acid sequence of SEQ ID NO:11 and the LC comprises the amino acid sequence of SEQ ID NO:

12.

38. 36. One or more expression vectors comprising the isolated nucleic acid of claim 35.

39. 40. A cell comprising one or more expression vectors according to claim 38, wherein the one or more expression vectors encode both the HC and the LC.

40. 1. A method for producing a fusion protein that binds Tie2 and VEGF, comprising: Culturing the cell of claim 19 to produce a fusion protein; recovering the fusion protein from the cultured cells; A method comprising:

41. 1. A method for producing a fusion protein that binds Tie2 and VEGF, comprising: Culturing the cell of claim 24, 29, 34 or 39 to produce a fusion protein; recovering the fusion protein from the cultured cells; A method comprising:

42. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 16 and a pharma- ceutically acceptable carrier, diluent or excipient.

43. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 16 for use in the treatment of diabetic macular edema in a patient in need thereof.

44. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 16 for use in the treatment of macular degeneration in a patient in need thereof.

45. 45. The pharmaceutical composition of claim 44, wherein the macular degeneration is age-related macular degeneration.

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