CN118922443A - Fusion proteins binding VEGF and Tie2 and uses thereof - Google Patents

Abstract

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

Description

Fusion protein combining VEGF and Tie2 and its use

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/299,177, filed on January 13, 2022, U.S. Provisional Application No. 63/310,359, filed on February 15, 2022, and U.S. Provisional Application No. 63/335,805, filed on April 28, 2022, under 35 U.S.C. §119(e). The entire disclosures of these prior applications are incorporated herein by reference in their entirety.

Sequence Listing

This application contains a sequence listing submitted electronically in XML format. The 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.

Technical Field

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

Introduction and Overview

During the development, growth, maintenance and homeostasis of an organism, angiogenesis occurs dynamically through a variety of regulatory factors. The newly formed blood vessels in this process serve as transport channels for various biomaterials (such as nutrients, oxygen and hormones) in surrounding cells. Functional and structurally abnormal blood vessels are direct or indirect causes of the occurrence and progression of various diseases. Tumor blood vessels increase hypoxia due to their functional and structural defects, causing tumor progression and metastasis to other tissues, resulting in poor delivery of anticancer drugs to the core of tumor masses. In addition to cancer, defective blood vessels have also been found in various other diseases and conditions. Examples include various eye diseases (such as diabetic macular edema, wet age-related macular degeneration, diabetic retinopathy), viral infections, and acute inflammatory reactions, such as sepsis. Thus, if a therapeutic agent capable of normalizing pathological blood vessels is available, it can be applied to treat various patients with vascular abnormalities.

The angiopoietin family plays an important role in the formation and maintenance of blood vessels, and includes four angiopoietins (Ang1, Ang2, Ang3 and Ang4). Angiopoietin-1 (Ang1) binds to the Tie2 receptor present on the surface of vascular endothelial cells to phosphorylate and activate the Tie2 receptor, resulting in the stabilization of blood vessels and the inhibition of vascular leakage. On the other hand, angiopoietin-2 (Ang2) binds to the Tie2 receptor, but acts as an antagonist to induce the inactivation of the Tie2 receptor and inhibit the binding of Ang1, resulting in the instability of blood vessels and vascular leakage, thus tending to promote the growth of new blood vessels. It is reported that the expression level of Ang2 is greatly improved 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, it is also known that Ang2 acts as an agonist to induce the activation of Tie2 receptors in several processes (including lymphangiogenesis and maintenance), and it is believed that Ang2 performs various functions according to the context.

To date, the development and clinical testing of various anti-Ang2 antibodies have been the focus of many biopharmaceutical companies (e.g., U.S. Patent Nos. 7,658,924 and 8,987,420). These Ang2 antibodies are reported to inhibit the binding of Ang2 to Tie2, and the Ang2 neutralization is reported to hinder the formation of new blood vessels. The anti-angiogenic and anti-cancer activities of these anti-Ang2 antibodies have been confirmed in many preclinical models, and different anti-Ang2 antibodies are being clinically tested in various cancer patients. However, their anti-cancer efficacy has been shown to be insufficient. For example, a phase 3 clinical trial conducted by Amgen showed that the anti-cancer efficacy of Ang2 antibodies in ovarian cancer patients was not significant (Marth C et al., 2017, Eur. J. Cancer, 70: 111-121). In addition to cancer models, the Ang2 neutralizing antibody Nesvacumab was also tested in ophthalmic patients, but it failed to improve in a phase 2 clinical study. (anti-VEGF) efficacy. See, for example, “Regeneron provides update on (aflibercept)injection and nesvacumab (ang2antibody)combination program” news release, which is available at: investor.regeneron.com/news-releases/news-release-details/regeneron-provides-update-eylear-aflibercept-injection-and.

In contrast to the above-mentioned Ang2 neutralization method, direct Tie2 activation is also considered to be an alternative method for inhibiting angiogenesis and inhibiting vascular permeability. Recombinant proteins that directly bind to Tie2 receptors and induce phosphorylation and activation of Tie2 have also been developed and tested in many preclinical cancer and ophthalmology models. Examples include cartilage oligomeric matrix protein (COMP)-Angl (Cho et al., 2004, PNAS101 (15): 5547-5552) and vascular peptide (Vasculotide) (David S et al., 2011, Am J Physiol Lung Cell Mol Physiol 300 (6): L851-L862). Although these agents show anti-angiogenesis and anti-permeability activities, they tend to have very short half-lives and unstable physicochemical properties. In addition, a small molecule compound (AKB-9778) has been developed as an inhibitor of the phosphatase VE-PTP, which inactivates Tie2 by removing the phosphate group from phosphorylated Tie2 (Goel S et al., 2013, J Natl Cancer Inst 105(16):1188-1201). Although this compound has the disadvantage of also activating other receptors, it indirectly increases Tie2 activity by inhibiting VE-PTP. 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. In addition, agonistic Tie2 antibodies have been developed. See, e.g., U.S. Patent No. 6365154B1; and U.S. Patent Publication No. 20170174789A1. These antibodies improve endothelial cell survival and inhibit vascular leakage. Interestingly, herbal extracts show Tie2 activation activity and are claimed to be used as skin care cosmetics. See, e.g., Japanese Patent Application Nos. JP2011102273A, JP2018043949A, JP2015168656A.

Tie2 is a receptor protein that promotes the differentiation and stabilization of blood vessels and is highly expressed in blood vessels. If activated, the Tie2 receptor stabilizes the blood vessels and can recruit surrounding support cells. For example, activated Tie2 in cancer vessels normalizes cancer vessels and reduces vascular leakage, which eliminates increased hypoxia within the tumor, supplies sufficient oxygen by increasing blood flow into the tumor, and increases the delivery of other anti-cancer drugs and the penetration of immune cells.

Vascular endothelial growth factor (VEGF) inhibitors are the standard of care for first-line treatment of diabetic macular edema (DME) and wet age-related macular degeneration (wAMD), although moderately frequent intraocular administration and a significantly insufficient responder population limit the effectiveness of these treatments.

Therefore, there is a need for improved methods for modulating angiogenesis, endothelial signaling, inflammation and/or vascular leakage, and/or 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, persistence, and/or reduced frequency of treatment (to reduce patient burden and compliance issues). The present disclosure is intended to meet one or more of these needs, provide other benefits, or at least provide the public with a useful choice.

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

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

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 to VEGF and thereby preventing VEGF from exerting its activity.

In one embodiment, the VEGF binding domain comprises the extracellular domains of two VEGF receptor subtypes, such as the extracellular domains of both VEGF receptor 1 and VEGF receptor 2 (eg, the Ig2 domain of VEGF receptor 1 and the Ig3 domain of VEGF receptor 2).

In one embodiment, the VEGF binding domain comprises an anti-VEGF antibody or antibody binding fragment thereof. In another embodiment, the anti-VEGF antibody or fragment thereof comprises the variable binding domain of bevacizumab, ranibizumab, or HuMab G6-31 (US2007/0141065).

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

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 to Tie2 and provides, preserves or enhances Tie2 or Tie2 activity at the cell surface membrane.

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.

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

In one embodiment, the anti-Tie2 antibody is of the IgG1 isotype.

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

In one embodiment, the VEGF binding domain comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical 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.

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

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

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

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

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

In one embodiment, the fusion protein comprises a CH domain comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical 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 one embodiment, the fusion protein comprises one or more mutations in the constant region domain of the heavy chain to reduce or eliminate the effective interaction with the Fc receptors on other immune cells in the body. In one embodiment, the one or more mutations include L234, L235, G236 and G237 (EU numbering) are changed to LAGA mutations, FEGG mutations, AAGG mutations, AAGA mutations, LALA mutations or combinations thereof. In one embodiment, the one or more mutations include LALA mutations and mutations at K322 (eg, K322A) and P331 (eg, P331S) (EU numbering).

In one embodiment, the fusion protein is included in 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 is included in one or more mutations at L234A, L235A and/or H310A. In one embodiment, the fusion protein is included in 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).

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

In one embodiment, the fusion protein comprises a linker between the VEGF binding domain and the anti-Tie2 antibody or its antibody fragment from the C-terminus to one or more heavy chain constant domains of the anti-Tie2 antibody or its antigen-binding fragment and in order from the N-terminus to the 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.

In one embodiment, the fusion protein comprises a CH domain comprising the amino acid sequence of SEQ ID NO: 17, 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 its antibody fragment, and a VEGF binding domain comprising the amino acid sequence of SEQ ID NO: 15.

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.

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

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

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.

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.

In one embodiment, the fusion protein binds to the Tie2 Ig3-FNIII(1-3) domain comprising SEQ ID NO: 2, 3 or 4 with an affinity _KD (M) of less than 3E ^-9M .

In one embodiment, the fusion protein is pegylated. PEGylation (or pegylation) refers to the covalent attachment or incorporation of chains (e.g., PEG polymers) comprising multiple polyethylene glycol (PEG, also known as macrogol) units. In one embodiment, PEG has a molecular weight of about 40 kDa or about 20 kDa.

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.

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.

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 chemicals, biopolymers or peptides that increase the half-life of the fusion protein.

In one embodiment, a polypeptide comprising a chain monomer of a fusion protein of the invention is provided.

In one embodiment, the polypeptide comprises a heavy chain monomer of a fusion protein of the present invention. In another 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 with the sequence of SEQ ID NO: 11 or 26. In one embodiment, the polypeptide consists of the sequence of SEQ ID NO: 26.

In one embodiment, the polypeptide comprises a light chain monomer of a fusion protein of the present invention. In another 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, with SEQ ID NO:12. In one embodiment, the polypeptide consists of the sequence of SEQ ID NO:12.

As used herein, "biopolymer" refers to a polymer produced by a natural source, which is chemically synthesized by biomaterials or completely biosynthesized by living organisms or microorganisms. In some embodiments, the biopolymer includes but is 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 alginate), natural rubber (polymers of isoprene), subarin, lignin (complex polyphenol polymers), cutin and glue film (complex polymers of long-chain fatty acids), melanin, metabolites and other structural molecules.

In one embodiment, the one or more half-life extension modulators comprise: a biopolymer containing PEG (polyethylene glycol), hyaluronic acid (HA), or phosphorylcholine; albumin; an albumin binding peptide; and/or an HA binding protein fragment.

In another embodiment, the present disclosure provides a nucleic acid encoding the fusion protein.

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

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 with SEQ ID NO: 27 or 29. In one embodiment, the nucleic acid molecule consists of SEQ ID NO: 27 or 29.

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 with SEQ ID NO: 28. In one embodiment, the nucleic acid molecule consists of SEQ ID NO: 28.

In one embodiment, the disclosure provides a collection of one or more polynucleotides, wherein each polynucleotide encodes at least one of the monomeric chains of a fusion protein of the invention, such that both chains (ie, a light chain and a heavy chain) of the fusion protein are encoded.

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

In one embodiment, a collection of one or more vectors is provided, which collectively comprise a collection of one or more polynucleotides of the present invention, such that both chains (ie, light chain and heavy chain) of the fusion protein are encoded in the collection of vectors.

In one embodiment, the vector is an animal virus, such as a virus selected from the group consisting of retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses, varicella virus, baculoviruses, papillomaviruses, anelloviruses, and papovaviruses.

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

In another embodiment, the present disclosure provides cells transformed with the expression vector.

In another embodiment, the present disclosure provides a method for producing a fusion protein that binds Tie2 and VEGF, comprising the steps of: culturing cells transformed with the expression vector; and recovering the fusion protein from the cultured cells. In another embodiment, the present disclosure provides a method for producing a fusion protein of the present invention by expression from a vector or a collection of vectors.

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

In another embodiment, the present disclosure provides use of the fusion protein for manufacturing a medicament for treating angiogenic diseases in a subject in need thereof.

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

In some embodiments, the angiogenic disease or vascular disease is cancer, metastasis, diabetic retinopathy, retinopathy of prematurity, diabetic macular edema, corneal transplant rejection, macular degeneration, glaucoma such as neovascular glaucoma, erythema cutanea generalis, proliferative retinopathy, psoriasis, hemophilic arthritis, combined sclerosis, capillarization 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.

In some embodiments, the cancer is esophageal cancer, stomach cancer, colorectal 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 lymphoma, lymphoma, or multiple myeloid leukemia.

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

In some embodiments, the inflammation is from sepsis, acute respiratory distress syndrome, and/or a viral infectious disease.

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 domestic pet.

In some embodiments, the present disclosure provides a pharmaceutical composition comprising the fusion protein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.

Additional objects and advantages will be set forth in the following parts of the description, and in part will be understood from the description, or may be learned through practice. These objects and advantages will be realized and attained by the elements and combinations particularly pointed out in the appended claims.

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 claims. Section headings are provided only for the convenience of the reader and do not limit the disclosure.

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

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1C provide sensorgrams of IGT-427 binding to human (Figure 1A), rabbit (Figure 1B), and mouse (Figure 1C) Tie2-Fc fusion proteins.

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 hours and incubated with IGT-427 (Figure 2A) or aflibercept (Figure 2B) at the indicated concentrations for 30 minutes and then treated with recombinant human VEGF for 2 minutes. Cell lysates were subjected to SDS-PAGE/protein blotting and blots were probed with anti-phosphorylated VEGFR2 (Tyr1175), anti-VEGFR2, anti-phosphorylated Akt (S473) or total Akt antibodies. VEGF reporter assays showed comparable inhibitory effects of IGT-427, faricimab, and aflibercept (Figure 2C).

Figure 3 shows stronger and more sustained levels of phosphorylated Tie2 signaling by IGT-427 when compared to Angiopoietin-1. Chinese hamster ovary (CHO) cells overexpressing human Tie2 were serum starved for 4 hours and then treated with 10 nM of IGT-427 or Angiopoietin-1 for the indicated durations. Lysates were subjected to phosphorylated Tie2 and total Tie2 ELISA assays.

FIG. 4 shows the dose-dependent activation of Akt signaling by IGT-427 in HUVECs.

Figure 5 shows that IGT-427 overcomes and bypasses the effects of angiopoietin-2 on Tie2 signaling in HUVECs, leading to further activation of Akt signaling in the presence of pre-treated Ang2.

Figure 6 shows that IGT-427 binds simultaneously to Tie2 and VEGF in complex with Ang2. Ang2 was captured in a CM5 chip and human Tie2, IGT-427 and human VEGF were subsequently injected in a surface plasmon resonance (SPR) (Biocore ^™ ) analysis.

Figure 7 shows the inhibition of TNF-α-induced apoptosis by IGT-427. Pre-treated for 1 hour and then treated with TNF (50ng/ml) for 24 hours. Apoptotic cells were stained by APO-BrdU ^TM TUNEL Assay Kit (Thermo Fisher, A23210) and assayed by Attune (Thermo Fisher). Values are mean ± SE. By one-way ANOVA, ***p<0.001, ****p<0.0001.

FIG8 shows the cell surface levels of Tie2 on Chinese hamster ovary cells overexpressing human Tie2 (CHO-Tie2) treated with various agents at different time points.

Figure 9 shows that IGT-427 blocks Tie2 cleavage by MMP14. Recombinant human MMP14, human Tie2-ECD-human IgG Fc fusion protein or IGT-427 were mixed and incubated as indicated.

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 shows that IGT-427 restores impaired endothelial barrier integrity caused by VEGF treatment.TEER (transendothelial electrical resistance) was assessed in HUVECs.

Figure 12 shows the effect of intravitreal injection of IGT-427 or Inhibition of CNV (choroidal neovascularization). Antibodies (50 μl injection volume/eye, (800 μg), IGT-427 (885 μg), control IgG (716 μg)) were administered intravitreally. The molar ratio of 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 14 days after laser photocoagulation. For each group, 4-7 rabbits were tested, with 6 laser-induced CNV lesions tested per rabbit. CNV lesions (n=24-42) in each group were imaged. Values are mean ± SEM. By one-way ANOVA, *p<0.05, **p<0.001.

A schematic diagram of IGT-427 variants used for PEGylation is shown in Figure 13. All constructs have a common light chain and five different heavy chains that generate two labile cysteines per antibody.

Figure 14 shows the SDS-PAGE data of the optimized PEGylation conditions for five IGT-427 variants. PRO593, PRO594 and PRO595 experienced complete conversion with no unmodified antibody in the reaction mixture. The reaction mixtures of PRO592 and PRO596 had some unmodified antibody at the end of the reaction.

Figure 15 shows SPR binding of IGT-427 and PEGylated variants to Tie2 and VEGF surfaces. There were slight differences in the binding signals to either antigen, but the PEGylated variants bound both antigens similarly relative to unmodified IGT-427.

SEC-HPLC chromatograms of five ocular PK study test articles are shown in Figure 16. With the exception of the 20 kDa PEGylated species which contained 15% of high molecular weight species, all constructs were greater than 90% pure.

Figure 17 shows non-reducing SDS-PAGE data for ocular PK study test articles. Lane 1: Lane 2: faricitumab; Lane 3: IGT-427; Lane 4: (2×20 kDa linear PEG)-IGT-427; Lane 5: (2×40 kDa branched PEG)-IGT-427.

Figures 18A-18C show SPR binding data for ocular PK test articles to rabbit VEGF (Figure 18A), Tie2 (Figure 18B), and Ang2 (Figure 18C). A summary of the measured binding constants is tabulated.

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 repeated measurements from a single rabbit eye. The exponential fit for each data set is shown in red, and the fit parameters and extracted eye half-life are shown below each curve. For the PEGylated IGT-427 variant, 14 day measurements are included in the figure but excluded from the fit and extracted half-life analysis. A summary of the observed half-life for each test article is shown in the lower right.

Sequence Description

Table 1 provides a listing of certain sequences mentioned herein.

Implementation Description

definition

Unless otherwise indicated, the following terms and phrases as used herein are intended to have the following meanings:

As used herein, the term "anti-Tie2 antibody" refers to an antibody that specifically binds to Tie2, and in addition to a complete antibody that specifically binds to Tie2, it also includes an antigen-binding fragment of an antibody molecule. In some embodiments, the complete antibody has a structure of two full-length light chains and two full-length heavy chains, and each light chain is connected to the 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 the following subclasses: 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.

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

Fab' is different 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 forming disulfide bonds between cysteine residues in the hinge region of Fab'. Fv is the smallest antibody fragment, which has only the variable region of the heavy chain and the variable region of the light chain. Double-chain Fv (Fv of two chains) is formed by non-covalent bonds between the variable region of the heavy chain and the variable region of the light chain, and single-chain Fv (scFv) is usually formed covalently by a 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 similar to double-chain Fv. The fragment can be obtained by proteolytic enzymes (for example, Fab can be obtained by restrictive digestion of the whole antibody with papain, and F(ab')2 fragments can be obtained by cutting with pepsin), or it can be made by genetic manipulation technology.

The antibody may be, for example, in the form of an Fv (e.g., scFv) or a complete antibody. In addition, the constant region of the heavy chain may be selected from any isotype of gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε). For example, the constant region is γ1 (IgG1), γ3 (IgG3), or γ4 (IgG4). The light chain constant region may be of κ or λ type.

Antibodies include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fv (scFV), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-bonded Fv (sdFV) and anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of the above antibodies, etc., but are not limited thereto.

The term "heavy chain" or "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, which has an amino acid sequence containing sufficient variable regions to provide antigen specificity. In addition, as used herein, the term "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, which has an amino acid sequence containing sufficient variable regions to provide antigen specificity. The heavy chain constant region domain (e.g., the heavy chain constant region domain of human IgG1) may contain one or more modifications to reduce or eliminate effective interactions with Fc receptors on immune cells in vivo. Such mutations can eliminate its binding to Fc receptors and reduce or eliminate antibody-directed cytotoxicity. Such modifications may include changing L ₂₃₄ L ₂₃₅ G ₂₃₆ G ₂₃₇ residues (EU numbering) to LAGA mutations, FEGG mutations, AAGG mutations, AAGA mutations, LALA mutations, or a combination thereof. In some embodiments, the IgG constant region comprises mutations at K322 and P331 (EU numbering) to reduce or eliminate immune cell-mediated cytotoxicity (e.g., in combination with any of the aforementioned mutations). In some embodiments, the IgG constant region comprises a LALA mutation and mutations at K322 and P331 (e.g., K322A and P331S) (EU numbering) to reduce or eliminate immune cell-mediated cytotoxicity.

"Monoclonal antibody" is an antibody produced by a single clone of a cell or cell line and is composed of identical antibody molecules. Monoclonal antibodies can have monovalent affinity and only bind to the same epitope. In contrast, polyclonal antibodies bind to multiple epitopes and are usually prepared by several different plasma cell lineages that secrete antibodies. Bispecific monoclonal antibodies can also be engineered by increasing the target of one monoclonal antibody to two epitopes.

"Epitope" refers to a protein determinant to which an antibody can specifically bind (e.g., the portion of an antigen recognized by an antibody). An epitope can be a group of chemically active surface molecules, such as amino acids or sugar side chains, and generally has specific charge characteristics as well as specific three-dimensional structural characteristics.

The "humanized" form of non-human (e.g., mouse) antibodies is a chimeric antibody comprising one or more amino acid sequences (e.g., one or more CDR sequences, such as 6 CDR sequences) from a non-human antibody (donor antibody or source antibody) and other minimal sequences derived from non-human immunoglobulin. In some embodiments, humanized antibodies are human immunoglobulins (receptor antibodies), whose hypervariable regions are replaced by residues from non-human primates, mice, rats, rabbits, or non-human primates hypervariable regions (receptor antibodies), and the human immunoglobulins have the required specificity, affinity, and ability of the residues from the receptor hypervariable regions. For humanization, one or more residues in the framework domain (FR) can be replaced by the corresponding residues of non-human donor antibodies. This can contribute to maintaining the appropriate three-dimensional configuration of the transplanted CDR, thereby improving affinity and antibody stability. Humanized antibodies, for example, can alternatively or additionally include new residues that do not appear in the original receptor antibody or donor antibody to further improve the other performance of the antibody.

Included are any "chimeric" antibodies (immunoglobulins) that exhibit the desired biological activity, as well as fragments of the above-mentioned antibodies, in which a portion of the heavy chain and/or light chain is derived from a particular species, or is identical or homologous to the corresponding sequence in antibodies belonging to that subclass, while the remaining chain is derived from another species, or belongs to another antibody class or is identical to the corresponding sequence in antibodies belonging to that subclass.

As used herein, "antibody variable domain" refers to a domain comprising a complementarity determining region (CDR; e.g., CDR1, CDR2, and CDR3) and a framework region (FR). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. "Framework region" (FR) is the variable domain segment outside the CDR. Each variable domain typically has four FRs identified as FR1, FR2, FR3, and FR4.

"Complementarity determining regions" (CDRs; ie, CDR1, CDR2, and CDR3) refer to the amino acid residues of the variable domains of antibodies that are essential for antigen binding. Each variable domain typically contains three CDR regions identified as CDR1, CDR2, and CDR3.

As used herein, "half-life extension regulator" refers to a chemical, biopolymer, peptide, polypeptide or protein fragment that can be added to an antibody or antibody fragment to increase its half-life. The half-life extension regulator may include: a biopolymer containing PEG (polyethylene glycol), hyaluronic acid (HA) or phosphorylcholine; albumin; an albumin binding peptide; or an HA binding protein fragment.

For example, the half-life can also be reduced by eliminating the interaction of the recycling of antibody or antibody fragment and neonatal Fc receptor (FcRn) mediation. For example, it is known that the combined mutation at multiple sites affects the half-life of the antibody. In some embodiments, mutation includes 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, mutation includes L235A, L236A, and H310A (EU numbering). A reduction in 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 desirable due to established on-target toxicity issues with VEGF inhibition (bleeding, hypertension, etc.).

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

As used herein, "fusion protein" encompasses any polypeptide comprising sequences from multiple sources. Fusion proteins can be produced, for example, by genetic fusion (eg, polynucleotides encoding the sequences of the fusion protein) or by chemically linking separately produced or synthesized polypeptides.

"Angiogenic diseases", also referred to as "angiogenesis-related diseases", are diseases in which angiogenesis occurs or is associated with the progression of angiogenesis.

As used herein, a "subject" can be a human or an animal. For example, the subject can be a human. In some embodiments, the subject can be a mammal. In some embodiments, the subject can include a companion animal (also referred to as a "pet"). The term "companion animal" refers to a domesticated animal that can be kept as a pet or otherwise used for companionship purposes, and includes but is not limited to dogs, cats, rabbits, ferrets, horses, donkeys, mules, and hamsters. In some embodiments, the companion animal is a mammalian companion animal.

As used herein, the term "prevention" refers to any action that inhibits the target disease, delays its onset, or reduces the likelihood of its occurrence by administering a fusion protein or composition. The term "treatment" or "therapy" refers to any action that reduces, delays, or alleviates the symptoms of the target disease, or cures the target disease, reduces its severity, or delays its progression.

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

The "sequence identity" between two nucleic acid sequences refers to the percentage of identical nucleotides between the sequences. The "sequence identity" between two amino acid sequences refers to the percentage of identical amino acids between the sequences.

The terms "% identical", "% identity" or similar terms are particularly intended to refer to the percentage of identical nucleotides or amino acids in the best alignment between the sequences to be compared. The 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. The comparison of two sequences is usually performed by comparing the sequences after the best alignment in terms of segments or "comparison windows" to identify local regions of corresponding sequences. The best alignment for comparison can be performed manually, or with the help of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, with the help of the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the help of the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the help of computer programs that use such algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

The percent identity is obtained by determining the number of corresponding identical positions in the sequences being compared, dividing that number by the number of positions for comparison (eg, the number of positions in the reference sequence), and multiplying the result by 100.

In some embodiments, the degree of identity is given for 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 full length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, which are contiguous nucleotides in some embodiments. In some embodiments, the degree of identity is given for the full length of the reference sequence.

A nucleic acid sequence or amino acid sequence having a specific 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, and in some cases, is functionally equivalent to the given sequence. An important property includes the ability to act as a cytokine, particularly when administered to a subject. In some embodiments, a nucleic acid sequence or amino acid sequence having a specific degree of identity to a given nucleic acid sequence or amino acid sequence is functionally equivalent to the given sequence.

As used herein, the term "about" means a degree of variation that does not substantially affect the properties of the subject matter described, for example, within 10%, 5%, 2% or 1%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not attempting 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.

Overview

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

Fusion Protein

The present disclosure relates to fusion proteins 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.

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: 5-7, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 8-10.

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

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

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

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.

In one embodiment, the fusion protein binds to amino acids 633-644 (SEQ ID NO: 20) and/or amino acids 713-726 (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 to amino acids 633-644 (SEQ ID NO: 19) and/or amino acids 713-726 (SEQ ID NO: 20) of Tie2 having the amino acid sequence of SEQ ID NO: 1.

In one embodiment, the anti-Tie2 antibody is of the IgG1 isotype.

In one embodiment, the VEGF binding domain comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical 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.

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 chain comprises a Tie2 binding domain at the N-terminal end that binds to a Tie2 Ig3-FNIII (1-3) domain comprising SEQ ID NO: 2, 3 or 4, and a VEGF binding domain at the C-terminal end. Suitably, the Tie2 binding domain is an anti-Tie2 antibody, wherein the heavy chain pair of the fusion protein comprises the heavy chain of an antibody, and the light chain pair of the fusion protein comprises the light chain of an antibody.

In one embodiment, the fusion protein comprises:

a. A dimer of a first and a second heavy chain monomer, wherein each heavy chain monomer comprises a single-chain polypeptide comprising, from N-terminus to C-terminus:

i. A heavy chain variable domain (VH) that binds to Tie2 together with a light chain variable domain, which is linked to

ii. A constant heavy chain domain (CH) comprising a CH1 domain and an Fc region or a fragment thereof, linked to

iii. a VEGF binding domain comprising an extracellular domain of a VEGF receptor, such as the VEGF-A binding region of VEGF receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2); and

b. a first and a second light chain monomer, each light chain monomer comprising, from N-terminus to C-terminus, a light chain variable domain (VL) that binds to Tie2 together with VH, which is connected to a constant light chain domain (CL1);

wherein the first and second monomers dimerize via their Fc regions or fragments thereof;

And wherein the Tie2 binding domain is formed by pairing each heavy chain monomer with one of the light chain monomers such that the VH1 and CH1 of each heavy chain monomer pair are paired with the VL and CL1 domains of the light chain monomer.

In one embodiment, the fusion protein comprises a linker between the VEGF receptor and the anti-Tie2 antibody or antibody fragment thereof. In another embodiment, the linker comprises between 5 and 50 residues, such as between 10 and 40 residues, such as between 15 and 30 residues, such as 20 residues.

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

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

In one embodiment, the fusion protein comprises a linker between the VEGF binding domain and the anti-Tie2 antibody or its antibody fragment from the C-terminus to one or more heavy chain constant domains of the anti-Tie2 antibody or its antigen-binding fragment and in order from the N-terminus to the C-terminus, such as wherein the linker comprises a sequence having between 5 and 50 amino acid residues, such as between 10 and 40 residues, such as between 15 and 30 residues, such as 20 residues.

In one embodiment, the fusion protein comprises a linker between the VEGF binding domain and the anti-Tie2 antibody or its antibody fragment from the C-terminus to one or more heavy chain constant domains of the anti-Tie2 antibody or its antigen-binding fragment and in order from the N-terminus to the 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.

In one embodiment, the fusion protein comprises a CH domain comprising the amino acid sequence of SEQ ID NO: 17, 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 its antibody fragment, and a VEGF binding domain comprising the amino acid sequence of SEQ ID NO: 15.

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.

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

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

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.

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 of the heavy chain to reduce or eliminate the effective interaction with Fc receptors on other immune cells in the body. In some embodiments, the one or more modifications or mutations include changing L ₂₃₄ L ₂₃₅ G ₂₃₆ G ₂₃₇ (EU numbering) to LAGA mutation, FEGG mutation, AAGG mutation, AAGA mutation, LALA mutation or a combination thereof. In some embodiments, the IgG constant region comprises any of the aforementioned mutations (e.g., LALA mutation) and mutations at K322 and P331 (EU numbering) to reduce and eliminate immune cell-mediated cytotoxicity.

Tie2 antibodies are monovalent or bivalent and contain single or double chains. Functionally, the binding affinity or dissociation constant ( _KD ) is the molar (M) concentration of the ligand when half of the ligand binding sites on the protein are occupied in the equilibrium of the system. It is calculated by dividing the dissociation rate ( _Koff ) value by the association rate ( _Kon ) value. In the context of the dissociation constant, lower values are consistent with stronger binding. The _KD of Tie2 antibodies ranges from ^10-5M to ^10-12M . For example, the Tie2 antibody has a binding affinity ( _KD ) of ^10-6 M to ^10-12 M, ^10-7 M to ^10-12 M, ^10-8 M to ^10-12 M, ^{10-9 M} to ^10-12 M, 10-5 M to ^10-11 M, ^10-6 M to ^10-11 M, ^10-7 M to ^10-11 M, ^10-8 M to ^10-11 M, ^10-9 M to ^10-11 M, ^10-10 M to ^10-11 M, ^10-5 M to ^10-10 M, ^10-6 M to ^10-10 M, ^10-7 M to ^10-10 M, ^10-8 M to ^10-10 M, ^10-9 M to ^10-10 M, ^10-5 M to ^10-9 M, ^10-6 M to ^10-9 M ^{, 10-7 M} to 10 ^-9 M, 10 ^-8 M to 10 ^-9 M, 10 ^-5 M to 10 ^-8 M, 10 ^-6 M to 10 ^-8 M, 10 ^-7 M to 10 ^-8 M, 10 ^-5 M to 10 ^-7 M, 10 ^-6 M to 10 ^-7 M, or 10 ^-5 M to 10 ^-6 M.

IGT-427 is an exemplary fusion protein described herein that simultaneously inhibits VEGF signaling and activates the Tie2 signaling pathway. IGT-427 binds human Tie-2 with a _KD <1 nM in a bivalent interaction similar to a cell surface interaction, and binds human VEGF with a _KD <10 pM. With VEGF on the surface, IGT-427 binds with an apparent affinity of <500 pM. With IGT-427 on the surface, the _KD is <10 pM. IGT-427 exhibits a comparable high affinity to the rabbit ortholog.

In one embodiment, the fusion protein binds to the Tie2 Ig3-FNIII (1-3) domain comprising SEQ ID NO: 2, 3 or 4 with an affinity _KD (M) of less than 3E ^-9 M (where E ^-X represents the number of negative exponents and zero, e.g., 3E ^-9 is the same as .0000000003), less than 3.5E ^{-9 M} , less than 4E-10 M, or less. A _KD value of less than a given amount means that the _KD is numerically less than the given amount, which indicates stronger binding.

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

The Tie2 antibody or antibody fragment in the fusion protein may include its bioequivalent within the scope of specifically recognizing Tie2. For example, it may include changes in the amino acid sequence to further improve the binding affinity and/or other biological properties of the antibody. Such modifications may include, for example, deletions, insertions and/or substitutions of residues in the amino acid sequence of the antibody. These amino acid variations may be based on the relative similarity of the amino acid substituents, such as the hydrophobicity, hydrophilicity, charge, size, etc. of the amino acid side chains, thereby providing conservative substitutions. By analyzing the size, shape and type of the amino acid side chain substituents, it is known that arginine, lysine and histidine are all positively charged residues; alanine, glycine and serine have similar sizes; phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, we can say that arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine are conservative substitutions.

Taking into account the above variations with bioequivalent activity, in some embodiments, the amino acid sequence of the fusion protein includes the sequences 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, such as at least 85% identity, 90% identity, 95% identity, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity.

Based on this, the fusion protein or its components can have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity when compared to a specified sequence or all sequences described in the specification.

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

In one embodiment, the fusion protein further comprises the sequence of SEQ ID NO:22, and site-specific modification (e.g., pegylation) is performed on the cysteine residues of the sequence of SEQ ID NO:22 with any half-life extension regulator 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.

In one embodiment, PEG has a molecular weight of about 40 kDa.

In one embodiment, a polypeptide comprising a chain monomer of a fusion protein of the invention is provided.

In one embodiment, the polypeptide comprises a heavy chain monomer of a fusion protein of the present invention. In another 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 with the sequence of SEQ ID NO: 11 or 26. In one embodiment, the polypeptide consists of the sequence of SEQ ID NO: 26.

In one embodiment, the polypeptide comprises a light chain monomer of a fusion protein of the present invention. In another 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, with SEQ ID NO:12. In one embodiment, the polypeptide consists of the sequence of SEQ ID NO:12.

Polynucleotides; Production methods; Vectors and host cells

In another aspect, the present disclosure relates to a nucleic acid encoding the fusion protein.

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

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 with SEQ ID NO: 27 or 29. In one embodiment, the nucleic acid molecule consists of SEQ ID NO: 27 or 29.

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 with SEQ ID NO: 28. In one embodiment, the nucleic acid molecule consists of SEQ ID NO: 28.

In one embodiment, the disclosure provides a collection of one or more polynucleotides, wherein each polynucleotide encodes at least one of the monomeric chains of a fusion protein of the invention, such that both chains (ie, a light chain and a heavy chain) of the fusion protein are encoded.

The fusion protein can be recombinantly produced by constructing or isolating a nucleic acid encoding the fusion protein. Further cloning (DNA amplification) by isolating the nucleic acid and inserting it into a replicable vector can be performed, or further expression can be performed. Based on this, the present disclosure relates to a vector containing the nucleic acid on the other hand.

In some embodiments, the disclosure relates to nucleic acids comprising the fusion protein. "Nucleic acid" means to include DNA (gDNA and cDNA) and RNA molecules, and nucleotides (the basic structural unit of nucleic acids) include nucleotides in nature, and analogs with modified sugar or base parts. The sequence of nucleic acids encoding heavy and light chain variable regions can be modified. Modifications include addition, deletion, or non-conservative or conservative substitutions of nucleotides.

The DNA encoding the fusion protein is easily isolated or synthesized using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to the DNA encoding the heavy and light chains). Many vectors are available. Vector components typically 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.

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

In one embodiment, a collection of one or more vectors is provided, which collectively comprise a collection of one or more polynucleotides of the present invention, such that both chains (ie, light chain and heavy chain) of the fusion protein are encoded in the collection of vectors.

In one embodiment, the vector is an animal virus, such as a virus selected from the group consisting of retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses, varicella virus, baculoviruses, papillomaviruses, and papovaviruses.

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

As used herein, the term "vector" as a means for expressing a target gene in a host cell includes plasmid vectors, cosmid vectors, phage vectors, adenoviral vectors, retroviral vectors and viral vectors, such as adeno-associated viral vectors. The nucleic acid encoding the antibody in the vector is operably linked to a promoter.

As used herein, the term "operably connected" or "connected" refers to the functional connection between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or a transcription regulator binding site array) and different nucleic acid sequences. Thus, the control sequence controls the transcription and/or translation of other nucleic acid sequences.

In some embodiments, provided herein are host cells comprising polynucleotides encoding the fusion proteins described herein. In some embodiments, provided herein are host cells comprising vectors comprising polynucleotides encoding the fusion proteins described herein. The host cell may be prokaryotic or eukaryotic. The host cell may be isolated, such as cultured or not a 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.

When prokaryotic cells are used as hosts, a strong promoter capable of 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 and T7 promoter, etc.), a ribosome binding site for translation initiation and a transcription/translation termination sequence are generally included. In addition, for example, in the case of eukaryotic cells as hosts, promoters derived from mammalian cell genomes (examples: metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (examples: adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, 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.

In some cases, the vector can be fused to other sequences to facilitate purification of the expressed antibody. The sequences to be fused are, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6×His (hexahistidine; Qiagen, USA).

The vector contains an antibiotic resistance gene commonly used as a selection marker in the art, for example, resistance genes to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

In another aspect, the present disclosure relates to cells transformed with the above-mentioned expression vectors. Cells used to produce antibodies can be prokaryotes, yeasts and higher eukaryotic cells, but are not limited thereto.

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

Exemplary animal host cell lines that can be used are 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, but are not limited thereto.

In one embodiment, the present disclosure provides a method for producing a fusion protein that binds Tie2 and VEGF, comprising the steps of: culturing cells containing a nucleic acid (such as an expression vector) encoding the fusion protein (e.g., transforming with an expression vector containing a nucleic acid encoding the fusion protein); and recovering the fusion protein from the cultured cells.

Cells can be cultured in a variety of culture media. Any commercial culture media 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, etc., and selected host cells are known to those skilled in the art.

Recovery of the antibody or antigen-binding fragment thereof can be carried out by, for example, removing impurities using centrifugation or ultrafiltration and, for example, using affinity chromatography, etc. Additional purification techniques such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, etc. can be used.

Uses of fusion proteins; Therapy

In another aspect, the present disclosure relates to methods and compositions for preventing or treating angiogenic diseases or vascular diseases by administering an effective amount of fusion proteins. In other aspects, the present embodiments relate to methods and compositions for regulating angiogenesis, endothelial signaling, inflammation and/or vascular leakage by administering fusion proteins to subjects in need thereof. In some embodiments, the fusion proteins described herein are used in therapy.

Angiogenesis refers to the formation or growth of new blood vessels from pre-existing blood vessels. Examples of angiogenesis diseases or angiogenesis-related diseases include, but are not limited to, cancer, metastasis, diabetic retinopathy, diabetic macular edema, retinopathy of prematurity, corneal transplant rejection, macular degeneration, glaucoma (such as neovascular glaucoma), systemic erythema, proliferative retinopathy, psoriasis, hemophilic arthritis, capillary formation in atherosclerotic plaques, keloids, wound granulation, vascular adhesions, rheumatoid arthritis, degenerative osteoarthritis, autoimmune diseases, Crohn's disease, restenosis, atherosclerosis, intestinal adhesions, cat scratch disease, ulcers, cirrhosis, nephritis, diabetic nephropathy, diabetes, inflammatory diseases, and neurodegenerative diseases. In addition, exemplary cancers include, but are not limited to, esophageal cancer, gastric cancer, colorectal 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 lymphoma, lymphoma and multiple myeloid leukemia.

Fusion proteins can be administered to regulate angiogenesis, endothelial signaling, inflammation, and/or vascular leakage. For example, fusion proteins can be administered to improve vascular endothelial cell membrane integrity and/or reduce vascular leakage. Vascular leakage is known to promote visual impairment in several common eye diseases, including but not limited to diabetic macular edema (DME), diabetic retinopathy (DR), and age-related macular degeneration (AMD). In some embodiments, inflammation is from sepsis, respiratory distress syndrome, and/or viral infectious diseases.

The fusion protein and its composition can be applied to mammals, including rats, mice, livestock, companion animals, people, etc. In some embodiments, the subject is a person. In some embodiments, the subject is a companion animal, such as a mammal companion animal. In some embodiments, the companion animal is a dog, cat, rabbit, ferret, horse, mule, donkey or hamster. The fusion protein or its composition can be applied every day. The fusion protein or its composition can be applied once every 1, 2, 3, 4, 6 or 12 months. In one embodiment, the fusion protein is applied once every six months (i.e. twice a year). It is applied via a commonly accepted route, such as oral, rectal, intravitreal, intravenous, subcutaneous, intrauterine or intracerebrovascular administration. In some embodiments, it is applied by intravitreal injection. In one embodiment, a fusion protein between 1mg and 10mg is applied by intravitreal injection every day, every month or every six months. In one embodiment, a fusion protein less than 10mg is applied by intravitreal injection approximately once every six months (e.g. twice a year).

Pharmaceutical composition; administration

In some embodiments, the composition comprising the fusion protein is a pharmaceutical composition.In some embodiments, the composition includes a suitable vehicle, excipient or diluent commonly used in the art.

Suitable vehicles, excipients, diluents are described in Remington: The Science and Practice of Pharmacy (19th Edition) ed. A. R. Gennaro, Mack Publishing Company, Easton, Pa. 1995. Suitable pharmaceutically acceptable vehicles, excipients or diluents include, for example, one or more of water, saline, phosphate buffered saline, dextrose, histidine, glycerol, sucrose, polysorbate, ethanol, etc., and combinations thereof. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 6 to about 7. It will be apparent to those skilled in the art that certain carriers may be more preferred depending on, for example, the route of administration and concentration.

The pharmaceutical composition with pharmaceutically acceptable vehicle can be 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, lyophilized products and suppositories. The pharmaceutical composition can include diluents or excipients (which can be combined), such as fillers, thickeners, adhesives, wetting agents, disintegrants, surfactants, etc. The solid preparations for oral administration can be in the form of tablets, pills, powders, granules, capsules, etc. About agglomeration, compounds can be prepared by combining one or more excipients, such as starch, calcium carbonate, sucrose, lactose or gelatin. In addition, simple excipients and lubricants can be used, such as magnesium stearate, talcum, etc.

Liquid preparations for oral administration can be suspensions, oral solutions, emulsions, syrups, etc. Excipients (such as water) or simple diluents (such as wet paraffin), various wetting agents, sweeteners, aromatics, preservatives, etc. can be included in the liquid preparations. In addition, the pharmaceutical composition can be a parenteral dosage form, such as sterile aqueous solution, non-aqueous solvent, suspension, emulsion, lyophilized material, suppository, etc. Injectable propylene glycol, polyethylene glycol, vegetable oil, such as olive oil and ester, such as ethyl oleate can be applicable to insoluble solvents and suspensions. The basic material of suppository includes Witepsol, polyethylene glycol, Tween61, cocoa butter, laurel fat and glycerin gelatin.

The composition is applied in a pharmaceutically effective amount. Terms used herein, such as "pharmaceutically effective amount" refer to the amount of the pharmaceutical composition sufficient to treat the disease with an appropriate benefit/risk ratio applicable to all medical treatments. The effective amount can be different according to various factors, including parameters such as the severity of the disease, the age and gender of the patient, the type of disease, drug activity, drug sensitivity, administration time, administration route, secretion rate, treatment duration and other factors. In addition, the above-mentioned composition can be applied in a single dose or divided into multiple doses. When these factors are fully considered, it is important to apply the minimum amount sufficient to obtain the maximum effect without side effects. The dosage of the pharmaceutical composition is not particularly limited, but it varies according to various factors, including the patient's health status and body weight, the severity of the disease, drug type, administration route and administration time.

The composition can be administered to mammals, including rats, mice, livestock, companion animals, people, etc., once or multiple times a day via commonly accepted routes (e.g., oral, rectal, intravenous, subcutaneous, intrauterine, intravitreal, or intracerebrovascular). In some embodiments, the composition is 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 domestic pet.

The present disclosure further relates to a method for preventing or treating angiogenic diseases or vascular diseases, and an anti-angiogenic method, which comprises the step of administering the antibody or the above composition to an individual in need thereof.

The fusion protein can be provided in a pharmaceutical composition.

The method disclosed herein includes a procedure for administering a pharmaceutically effective dose of a pharmaceutical composition to an individual who needs to inhibit angiogenesis. The individual can be a mammal, such as a dog, a cat, a ferret, a cow, a horse, a rabbit, a mouse, a rat or a human, but is not limited thereto. For example, in one embodiment, the individual can be a chicken, a turkey or other non-mammal. The pharmaceutical composition can be administered via a suitable manner, including parenteral, subcutaneous, intraperitoneal, intrapulmonary or intranasal, and, if necessary, for local treatment of the lesion. In some embodiments, the dosage of the pharmaceutical composition changes according to various factors, including but not limited to the individual's health status and body weight, the severity of the disease, the type of drug, the route of administration and time, and the dosage of the pharmaceutical composition can be easily determined by those skilled in the art.

In other aspects, the present disclosure relates to a method for cancer prevention or treatment, which comprises a procedure for administering the composition or antibody to an individual in need of the antibody and the composition or a pharmaceutical composition for cancer prevention or treatment comprising the antibody.

Cancer is not limited as long as it can be treated with the antibodies disclosed herein. Specifically, antibodies can prevent the occurrence or progression of cancer by inhibiting angiogenesis. Examples of cancer include esophageal cancer, gastric cancer, colorectal 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 leukemia, but are not limited thereto.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

Example

The following examples are intended only to illustrate the present invention and are not intended to limit the present invention.

Example 1. Preparation of fusion protein

A. Construct Design

A fusion protein IGT-427 was designed in which the VEGF-A binding regions of VEGF receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2) were fused to the C-terminus of the heavy chain (HC) of the anti-Tie2 antibody. The construct was made into a protein expression vector containing a 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.

B. Expression and purification of IGT-427

According to the manufacturer's instructions (Gibco), the plasmid DNA encoding IGT-427 is transiently cotransfected into ExpiCHO-S cells. Use equal amounts of heavy and light chain DNA. Subsequently, the transfected cells are incubated under vibration at 32 ° C and 5% _CO in air in a humidified atmosphere to obtain a maximum titer scheme. After incubation for 12 days, the culture medium containing the secreted fusion protein is collected, centrifuged to remove cells, and the culture supernatant is collected and filtered. Using the AKTAPure purification system (Cytiva) with the affinity column of HiTrap MabSelect SureProtein A (Cytiva), the fusion protein is purified in binding/washing buffer (20mM sodium phosphate, pH 7.2 and 150mM NaCl) and elution buffer (25mM sodium acetate, pH 3.3), thus 100 μl of 1M Tris-HCl, pH 8.8 are added by every milliliter of fraction to neutralize immediately. The combined fractions were then buffer exchanged into PBS, pH 7.4 using a centrifugal filter unit (Amicon). The final sample was then stored until further use. In parallel, the fusion protein was subjected to quality control studies such as SDS-PAGE (Invitrogen ^™ Novex ^™ WedgeWell), SEC-HPLC (Agilent Infinity 1260 and pore size columns) and endotoxin measurement (Charles River Cartridge <0.05EU/ml sensitivity) and surface plasmon resonance (SPRBiacore).

Example 2. Preparation of orthologous Tie2 protein

A. Construct Design

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

B. Expression and purification of Tie2 orthologs

To produce Tie2 orthologs, Expi293F (Gibco) cells capable of efficiently producing recombinant proteins were used. Orthologous Tie2 proteins were transiently expressed in the Expi293F cell line by referring to the manufacturer's instructions (Gibco). After transfection and incubation for 4 days at 37 ° C and 8% CO ₂ in the air under shaking, the resulting culture medium was collected and centrifuged to remove cells. The culture supernatant containing secreted antibodies was separated and 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 binding and washing buffer systems (20mM sodium phosphate, pH 7.2 and 150mM NaCl) and elution buffer (25mM sodium acetate, pH 3.3). Tie2 protein was immediately neutralized by adding 100 μl of 1M Tris-HCl, pH 8.8 per milliliter fraction. The fractions were pooled and subsequently buffer exchanged into PBS, pH 7.4, through a protein centrifugal filter (Amicon) and stored at -80°C. As a follow-up, the proteins were analyzed for quality control studies such as SDS-PAGE (Invitrogen ^™ Novex ^TMWedgeWell ), SEC-HPLC (Agilent Infinity 1260 and pore size columns) and surface plasmon resonance (SPR Biacore).

Example 3. Affinity measurement of fusion protein (IGT-427) for Tie2 orthologs

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

Table 2. Affinity of fusion protein IGT-427 for the antigen Tie2-Ig3-FNIII(1-3) orthologs

antigen K _on (M ^-1 s ^-1 ) K _off (1/s) K _D (M) Human Tie2 dimer 1.06E+05 4.70E-05 4.42E-10 Rabbit dimer Tie2 1.28E+05 6.82E-05 5.32E-10 Mouse dimer Tie2 8.87E+04 3.07E-04 3.46E-09 Human monomer Tie2 1.59E+05 4.00E-05 2.50E-10 Rabbit monomer Tie2 1.65E+05 6.49E-05 3.93E-10 Mouse monomeric Tie2 1.19E+04 4.33E-04 3.62E-09

Example 4. Inhibition of VEGFR2 phosphorylation by IGT-427

HUVECs (1×10 ⁵ cells/ml) were cultured in EGM-2 medium (Lonza) at 37°C in 60 mm culture dishes. Cells at 90% confluence were incubated with EBM-2 medium without supplements for 4 hours for starvation. Starved HUVECs were treated with IGT-427 or Pretreatment for 30 minutes, and then treated with recombinant human VEGF for 2 minutes. The cells were washed with cold PBS, treated with lysis buffer (10mM Tris-Cl pH 7.4, 150mM NaCl, 5mM EDTA, 10% glycerol, 1% Triton X-100, protease inhibitors, phosphatase inhibitors), and lysed at 4°C for 20 minutes. Subsequently, cell lysates were prepared by centrifugation at 13000rpm for 15 minutes. The protein concentration in the supernatant was quantitatively determined by BCA. Cell lysates were prepared and SDS PAGE was performed on the cell lysates by adding 4×SDS sample buffer, and the proteins were transferred to nitrocellulose membranes (GE).

To study VEGFR2 and Akt phosphorylation, blots were blocked with TBS-T containing 5% skim milk for 1 h at room temperature (RT) and incubated with anti-phospho-VEGFR2 antibody (Tyr1175) or anti-phospho-Akt (S473) antibody for approximately 16 h at 4°C. The signal of phosphorylated VEGFR2 (Tyr1175) or pAkt (S473) was visualized by enhanced chemiluminescence (ECL). Subsequently, the membrane was incubated in stripping buffer (Thermo) for 15 min and then reprobed with anti-VEGFR2 or anti-Akt antibodies to determine the amount of total VEGFR2 and total Akt. IGT-427 inhibited VEGF-induced VEGFR2 phosphorylation at Tyr1175 in a concentration-dependent manner, and this inhibition was In addition, IGT-427 induced the phosphorylation of Akt in a dose-dependent manner.

VEGF reporter assays were performed using a 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, faricitumab, or aflibercept was added at a concentration of 3× concentration of 50 nM and three-fold serial dilutions in 25 μl of assay buffer, and recombinant human VEGF at a 3× concentration of 40 ng/ml in 25 μl of assay buffer was added to the plate. After incubation for 6 hours at 37°C, 75 μl of BIO-GLO ^™ reagent provided in the kit was added and the assay buffer was used. Discovery System (PROMEGA ^™ ) measured luminescence. The IC 50 values of IGT-427, faricimab, or aflibercept were approximately 0.48 nM, 0.32 nM, or 0.75 nM, respectively ( FIG. 2C ), indicating that VEGF inhibition by IGT-427 was comparable to that by faricimab or aflibercept.

Example 5. Stronger and more sustained activation of Tie2 by IGT-427

CHO cells overexpressing full-length human Tie2 (CHO-hTie2, 1×10 ⁵ cells/ml) were cultured in DMDM medium (Thermo Fisher) at 37°C in 60 mm culture dishes. Cells at 90% confluence were incubated with DMEM medium without supplements for 4 hours for starvation. Starved CHO cells were treated with IGT-427 or recombinant human angiopoietin-1 (R&D ) for different durations (30 min, 1 h, 2 h, 6 h and 24 h). The 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 at 4 ° C for 20 min. Subsequently, cell lysates were prepared by centrifugation at 13000 rpm for 15 min. The lysates were then used for the pTie2 and total Tie2 ELISA assay kits (R&D ) The levels of phosphorylated Tie2 and total Tie2 signals were detected. The phosphorylated Tie2 signal was normalized by the total Tie2 signal. Compared with angiopoietin-1, IGT-427 showed a stronger and more persistent pTie2 signal (Figure 3).

Example 6. Dose-dependent activation of Akt signaling by IGT-427

HUVEC (1×10 ⁵ cells/ml) were cultured in EGM-2 medium (Lonza) at 37°C in 60mm culture dishes. Cells at 90% confluence were incubated with EBM-2 medium without supplements for 4 hours for starvation. Starved HUVEC were treated with various concentrations of IGT-427 (0.3, 1, 3, 10, 30, 100 nM) for 30 minutes. The 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 at 4°C for 20 minutes. Subsequently, cell lysates were prepared by centrifugation at 13000 rpm for 15 minutes. Protein concentration was quantified by BCA assay. Cell lysates were prepared and cell lysates were subjected to SDS PAGE by adding 4×SDS sample buffer, and the proteins were transferred to nitrocellulose membranes (GE). To study Akt phosphorylation, the blot was blocked with TBS-T containing 5% skim milk at room temperature (RT) for 1 hour and incubated with anti-phospho-Akt (S473) antibody at 4°C for about 16 hours. The signal of pAkt (S473) was visualized by enhanced chemiluminescence (ECL). Subsequently, the membrane was incubated in stripping buffer (Thermo) for 15 minutes and then re-probed with anti-Akt antibody to determine the amount of total Akt. As shown in Figure 4, IGT-427 had a dose-dependent gradual increase in phosphorylated Akt signaling.

Example 7. Bypassing Ang2 signaling by IGT-427

HUVEC (1×10 ⁵ cells/ml) were cultured in EGM-2 medium (Lonza) at 37°C in 60 mm culture dishes. Cells at 90% confluence were incubated with EBM-2 medium without supplements for 4 hours for starvation. Starved HUVEC were treated with recombinant human angiopoietin-2 (R&D ) pretreatment for 30 minutes, and then treated with 2×20kDa PEGylated IGT-427 at a concentration of 30nM for 60 minutes. The cells were washed with cold PBS, treated with lysis buffer (10mM Tris-Cl pH 7.4, 150mM NaCl, 5mM EDTA, 10% glycerol, 1% Triton X-100, protease inhibitors, phosphatase inhibitors), and lysed at 4°C for 20 minutes. Subsequently, cell lysates were prepared by centrifugation at 13000rpm for 15 minutes. Protein concentration was quantitatively determined by BCA. Cell lysates were prepared and SDS PAGE was performed on the cell lysates by adding 4×SDS sample buffer, and the proteins were transferred to nitrocellulose membranes (GE). In order to study Akt phosphorylation, the blot was blocked at room temperature (RT) for 1 hour with TBS-T containing 5% skim milk, and incubated for about 16 hours at 4°C with anti-phosphorylated Akt (S473) antibodies. The signal of pAkt (S473) was visualized by enhanced chemiluminescence (ECL). Subsequently, the membrane was incubated in stripping buffer (Thermo) for 15 minutes and then re-probed 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, indicating that IGT-427 overcomes Ang2 signaling (Figure 5).

Example 8. Dual Binding Capacity of IGT-427

The dual binding properties of IGT-427 to its antigens Tie2 and VEGF were examined by surface plasmon resonance (SPR) system (BIACORE ^TM 3000). ) were captured on a CM5 SPR chip (Cytiva) and injected first with the human dimer form of Tie2 (100 nM), followed by sequential injections of IGT-427 (150 nM) and human VEGF (20 nM) (R&D ). The sensorgrams showed a sequential increase in signals, indicating that IGT-427 was able to bind to both the Ang2-Tie2 complex and VEGF simultaneously ( FIG. 6 ).

Example 9. Inhibition of TNF-α-induced apoptosis by IGT-427

HUVEC (1×10 ⁵ cells/ml) were cultured in EGM-2 medium (Lonza) at 37°C in 60 mm culture dishes. Cells at 90% confluence were treated with IGT-427 or Pretreatment for 60 minutes, and then treated with recombinant human TNF-α (50ng/ml) for 24 hours. Apoptotic cells are stained by APO-BrdU ^TM TUNEL assay kit (Thermo Fisher, A23210) and measured by Attune (Thermo Fisher). In short, cells are suspended in 0.5mL of PBS, and 1% (w/v) paraformaldehyde in 5mL of PBS is added to the cell suspension, and then the cell suspension is placed on ice for 15 minutes. Cells are centrifuged at 300 × g for 5 minutes and washed twice in 5mL of PBS. Cells are resuspended in 0.5mL of PBS, and 5mL of ice-cold 70% (v/v) ethanol is added to the cell suspension, and the cell suspension is placed in a -20°C refrigerator for a minimum of 30 minutes. In order to remove 70% (v/v) ethanol, the cell suspension is centrifuged at 300 × g for 5 minutes, and then the cell pellet is resuspended with the washing buffer provided in the 1mL test 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 dH ₂ O) at 37°C for 60 minutes. At the end of the incubation time, 1.0 mL of rinsing buffer was added to the cells, and the cells were centrifuged at 300×g for 5 minutes. After washing again with rinsing buffer, the cells were incubated in 100 μL of antibody staining solution (5.0 μL of Alexa Fluor ^TM 488 dye-labeled anti-BrdU antibody and 95 μL of rinsing buffer) at room temperature for 30 minutes. Apoptotic cells were analyzed by Attune flow cytometry (Thermo Fisher). TNF-α induced apoptosis in HUVEC cells, and IGT-427 significantly inhibited TNF-α-induced apoptosis, while Not so (Figure 7).

Example 10. Measuring the 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 minutes, and resuspended at a density of 0.05×10 ⁶ cells/mL. To inoculate the assay plate, 1 mL of the cell suspension was aliquoted into each well of a 24-well tissue culture plate and the plate was incubated overnight at 370°C. The next day, the culture medium was removed from the tissue culture plate and replaced with 0.5 mL of DMEM alone for the unstimulated group or DMEM containing 10 nM angiopoietin 1 (R&D #923-AN), 10 nM IGT-301 (ITP-006), or 10 nM IGT-427 (ITP-016) in DMEM. The assay plates were returned to the 370°C tissue culture incubator for a predetermined length of time (0.5 h, 1 h, 2 h, 4 h, 6 h, or 24 h).

After incubation, the tissue culture plates were placed on ice, washed with cold DPBS, and lysed by adding 0.5 mL of non-enzymatic cell dissociation buffer ( #CPD-125) separation. The cells were incubated on ice for 5 minutes and collected by adding 1 mL of FACS buffer (0.5% BSA in DPBS) and gently pipetting to separate the cells still adhering 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 precipitate the cells, and the supernatant was aspirated. The cells were washed once with 1 mL of FACS buffer, and the supernatant was aspirated, leaving ~100 μL of residual FACS buffer in the tube. Subsequently, 100 μL of 99.5 μL FACS buffer and 0.5 μL of PE-Tie2 ( The cells were stained with a staining mixture consisting of 50 μl of 1% parafilm and incubated at 40°C in the dark for 30 minutes.

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

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 global 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 considered to be the major Tie2 cleavage, and Tie2 is cleaved by matrix metalloproteinase-14 at multiple sites within the fibronectin type 3 domain (Idowu, TO et al., eLife, August 24, 2020, 9: e59520).

Recombinant human MMP-14 (R&D Cat.#, 918-MP-010, 0.5 μg) was mixed with dimeric human Tie2 extracellular domain (ECD)-human IgG fusion protein (hTie2-ECD, 5 μg). 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 in intact hTie2-ECD, with the appearance of shorter, cleaved fragments. However, IGT-427 significantly blocked the cleavage and reduction of intact human Tie2-ECD protein by MMP-14 (Figure 9).

Without wishing to be bound by theory, and based on this evidence, it is believed that a bispecific antibody comprising an anti-Tie2 antibody or antigen-binding fragment that binds to a Tie2Ig3-FNIII(1-3) domain containing 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 will reduce the shedding of Tie2 by combining the effects of blocking the binding of MMP-14 to human Tie2 with the effects of inhibiting the cleavage of human Tie2-ECD protein caused by VEGF. This provides a particularly beneficial result of retaining Tie2 at the cell surface membrane.

Example 12. Measurement of soluble Tie2 (sTie2)

HUVEC (1×10 ⁵ cells/ml) were cultured in EGM-2 medium (Lonza) at 37°C in 60 mm culture dishes. Cells at 90% confluence were treated with IGT-427, faricitumab or recombinant human angiopoietin-1 (R&D ) for 60 minutes and then treated with recombinant human TNF-α (50 ng/ml) for 24 hours. The cell supernatant was collected and centrifuged at 13000 rpm for 10 minutes. The total Tie2 ELISA kit (R&D ) Soluble Tie2 in cell supernatants was measured. IGT-427 inhibited soluble Tie2 levels both under basal and TNF-α-induced conditions, while the other agents did not ( FIG. 10 ).

Example 13. TEER (transendothelial electrical resistance) measurement

HUVEC (2×10 ⁵ cells) were cultured in cell culture inserts in 24-well plates (Corning) at 37°C in EGM-2 medium (Lonza). After two days of incubation, the medium was replaced with EGM-2 medium containing 0.5% FBS. After the next two days of incubation, the cell culture inserts were placed in (NanoAnalytics), and TEER (transendothelial electrical resistance) was continuously measured at 37°C in a _CO2 incubator. After confirming the formation of the cell barrier, recombinant human VEGF (R&D) at a concentration of 10 ng/ml was added to the lower compartment. Various agents such as IGT-427, faricizumab, aflibercept or recombinant human angiopoietin-1 (R&D) at a concentration of 10 nM were added to the lower compartment, and TEER was monitored over the next 48 hours after VEGF treatment. VEGF destroys endothelial barrier integrity, and IGT-427 is superior to faricizumab and aflibercept and Ang1 in restoring endothelial barrier integrity from VEGF-induced damage (Figure 11).

Example 14. Inhibition of CNV (choroidal neovascularization) by intravitreal injection of IGT-427

Chinchilla rabbits (male, 2.0-2.5 kg) were anesthetized by applying eye drops (Mydriacyl ophthalmic solution, 1%) to the right eyeball and illuminated by a 532 nm laser (Elite, The right eyeball was irradiated with a 40 μg/ml irradiation kit (USA) to produce six spots around the optic nerve. On the day of choroidal neovascularization (CNV) induction, IGT-427, or control IgG (50 μL injection volume/eye, (800 μg), IGT-427 (885 μg), control IgG (716 μg)). The molar ratio of IGT-427 and control IgG was 1:0.65:0.68. Due to some restrictions on concentration and volume, different molar numbers were used. On days 0, 7, and 14, animals were anesthetized by applying eye drops (Mydriacyl ophthalmic solution, 1%) to the right eyeball, and 1 mL of fluorescein sodium salt solution (2%) was injected intravenously to take fundus images using a fundus camera (TRC-50IX, TOPCON, Japan) within 2 minutes. Retinal CNV area and efficacy were assessed using retinal fluorescein fundus photography, and image analysis was performed using ImageJ software (NIH, Bethesda, MD) to verify the fluorescence intensity of CNV lesions. Compared with control IgG, IGT-427 inhibited retinal leakage by 30%. In contrast, It was able to inhibit retinal leakage by 20% ( FIG. 12 ).

Example 15. IGT-427 variants for PEGylation

In order to extend the ocular half-life of IGT-427, the heavy and light chain plasmids of IGT-427 were modified to prepare five different antibody constructs for PEGylation (summarized in Figure 13). All five variants have a light chain containing a mutation C214S (EU numbering), which, in combination with the C218S mutation (EU numbering) on each heavy chain, removes the interchain disulfide bond between the heavy chain and the light chain. In addition, one or two heavy chain cysteines in the hinge region of IGT-427 are replaced by serine, leaving one or zero interchain disulfide bonds between the heavy chains. In the case of PRO592 and PRO596, a single disulfide bond is retained for reduction and PEGylation. 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 on the C-terminal of the heavy chain, while PRO594 and PRO595 each have a cysteine introduced on the linker domain between the Fc domain and the VEGF trap domain. Together, these variants represent a collection of constructs with different PEGylation sites, which are tested for PEGylation efficiency, antigen binding, and biological activity. Each IGT-427 variant is expressed using the Expi293 transient expression system. Clarified cell culture supernatants were purified using HiTrap mAbselect PrismA columns (Cytiva). The eluted antibody is dialyzed into 1 × PBS and concentrated to 10 mg/mL for PEGylation studies.

Example 16. PEGylation reaction screening

Each IGT-427 variant at 10 mg/mL 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. The reaction mixture was analyzed by non-reducing SDS-PAGE, which revealed that the variants showed up to 50% conversion to the di-PEGylated species.

In order to further optimize the PEGylation efficiency, PRO593 (which is a variant that shows the best conversion under mild reducing conditions) is used to screen the reducing conditions. The PRO593 of 10mg/mL is reduced for 1 hour with 10 or 100mM cysteamine, reduced for 15 minutes with 1 or 10mM DTT, or reduced for 15 minutes with 2 or 20 times of molar excess of TCEP (tris (2-carboxylethyl) phosphine). After each reduction, PRO593 buffer is exchanged into PEGylation buffer, and PEGylated for 1.5 hours with 10 times of molar excess of 20kDa linear PEG-maleimide. From this reducing condition group, it is observed that 20 times of molar excess of TCEP causes PRO593 to be almost completely converted into the species with two PEG additions. Then all five IGT-427 variants are reduced at room temperature for 15 minutes with 20 times of molar excess of TCEP, followed by buffer exchange into PEGylation buffer and PEGylated with 10 times of molar excess of 20kDa linear PEG-maleimide. The result of the optimized reaction conditions is shown in Figure 14. The antibody with two PEG additions was purified from the reaction mixture using a HiTrap SP-HP cation exchange chromatography column (Cytiva) and gradient salt elution. The PEGylated antibody was dialyzed into 1× PBS and concentrated to 10 mg/mL, formulated with 0.01% polysorbate-20, and filtered through a 0.2 μm filter.

Example 17. Characterization of PEGylated IGT-427 variants

Binding to Tie2 or VEGF was characterized by SPR (Figure 15). A 100 nM solution of each antibody was injected at 30 μL/min for 300 seconds over a surface containing immobilized Tie2 or VEGF. The relative binding signal after 300 seconds was used to determine differences in binding.

Example 18. Ocular PK Study

To evaluate the effects of PEGylation and PEG molecular weight on the ocular pharmacokinetics of IGT-427, male New Zealand white rabbits were administered 500 μg intravitreal injection of faricitumab, IGT-427, (2×20 kDa PEG)-IGT-427 and (2×40 kDa PEG)-IGT-427. For each test article group, three or four animals were dosed on day 0. Blood was collected from three or four animals on days 1, 3, 7 and 14 after dosing, and then they were euthanized by intravenous overdose of barbiturates, and then both eyes were harvested, quickly frozen in liquid nitrogen and dissected to collect aqueous humor, vitreous humor, retina and choroid. The vitreous humor samples were diluted 1:5 in 1×PBST without homogenization and stored at -70°C until further analysis.

Purchased from commercial sources, Dialyze against formulation buffer (10 mM sodium phosphate, 40 mM NaCl, 0.03% polysorbate-20, 5% sucrose) and dilute to 10 mg/mL in the same buffer.

Farasimab was expressed using the Expi293 transient expression system (ThermoFisher). Clarified cell culture supernatants were purified using HiTrapMabSelect PrismA columns (Cytiva), washed with 2% ethanol to remove endotoxins, followed by pH gradient elution to remove incompletely assembled antibodies. The 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.

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

PEGylated IGT-427. PRO593 was expressed using the Expi293 transient expression system (ThermoFisher). The clarified cell culture supernatant was purified using a HiTrapMabSelect PrismA column (Cytiva) and washed with 2% ethanol to remove endotoxins. 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 20 kDa linear or 40 kDa branched PEG maleimide at room temperature for 1.5 hours. The PEGylation reaction mixture was purified using a HiTrap SP-HP column (Cytiva) at pH 4.6 with a NaCl gradient. The PEGylated antibody in the eluate was dialyzed into PBS, concentrated to 10 mg/mL, formulated with 0.01% polysorbate-20, and filtered through a 0.2 μm filter.

Example 19. Characterization of samples 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 minutes without heating, followed by staining with SafeStain (ThermoFisher). 5 μg of each test article was loaded onto a 4-12% bis-tris gel at 150 V for 60 minutes in 100 mM arginine, 1× PBS, pH 6.7 at 0.5 mL/min. SEC-HPLC analysis was performed on a size exclusion column (SEC)-300. Binding to rabbit Tie2 and VEGF was characterized by SPR.

SPR was used to evaluate the binding of PEGylated IGT-427 variants to Tie2 or VEGF. Figure 3 shows the resulting sensorgrams from 100 nM injections of the variants on the surface of human Tie2 or VEGF. Relative to unmodified IGT-427, the binding signal is dramatically reduced due to the presence of PEG. However, the relative binding signal of each variant to the rest of the variants can be used as a measure of the relative affinity for the antigen. Although there are slight differences in the binding signals of each variant, overall, the binding to Tie2 or VEGF is largely independent of the PEGylation site.

All test articles were formulated at 10 mg/mL and were greater than 90% pure by SEC-HPLC (FIG. 16) and non-reducing SDS-PAGE (FIG. 17), except for 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 corresponding antigens with expected affinity (FIG. 18A-18C).

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 the PEGylated form of IGT-427 were captured onto mouse anti-human IgG coated plates and detected with Tie2-HRP conjugate. The ELISA was performed by capturing the antibody onto a mouse anti-human IgG coated plate and detecting it with a polyclonal goat anti-human IgG-HRP conjugate. Finally, faricimab was captured onto a VEGF coated plate and detected with a polyclonal goat anti-human IgG-HRP conjugate. For all ELISA analyses, samples were assayed in duplicate between dilutions of 1:1250 and 1:62500 and quantified using Molecular Devices. M5 plate reader reads the luminescence of the assay plate. For all ELISA analyses, the sample luminescence values were within the linear range of the standard curve. The entire standard curve was fitted using a 4-parameter equation, and the sample luminescence values were converted to concentrations via a 4-parameter fit.

Anti-human IgG capture/Tie2 detection ELISA. 1 μg/mL anti-human IgG in carbonate buffer (pH 9.5) was used to coat high binding plates overnight at 4°C. Wash with wash buffer (1× PBST with 150 mM NaCl (containing The coated plates were washed with 5% BSA in PBST (20% phosphate buffered saline) and blocked at 37°C for 1 hour with 5% BSA in PBST (shaking at 420 rpm). The blocked plates were washed and the samples, standard curves and blanks were incubated on the plates at room temperature for 1.5 hours (shaking at 420 rpm). After the sample incubation, the plates were washed and incubated with 1 μg/mL biotinylated human Tie2 at room temperature for 1 hour (covered, shaking at 420 rpm). The plates were washed again and incubated with streptavidin-HRP in the dark for 1 hour. The plates were washed a final time and then developed with chemiluminescent HRP substrate. All luminescent wavelengths were read in a plate reader after 30 seconds.

Anti-human IgG capture/anti-huFc detection ELISA. 1 μg/mL anti-human IgG 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 dried at 4°C after the last wash. Samples, standard curves, and blanks were incubated on the plates at 4°C overnight. After sample incubation, the plates were washed and incubated with polyclonal goat anti-human IgG-HRP conjugate at room temperature for 1 hour (covered, shaken at 420 rpm). The plates were washed a final time and then developed with chemiluminescent HRP substrate. All luminescent wavelengths were read in a plate reader after 30 seconds.

VEGF capture/anti-huFc detection ELISA. 1 μg/mL VEGF 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 dried at 4°C after the last wash. Samples, standard curves, and blanks were incubated on the plates at 4°C overnight. After sample incubation, the plates were washed and incubated with polyclonal goat anti-human IgG-HRP conjugate at room temperature for 1 hour (covered, shaken at 420 rpm). The plates were washed a final time and then developed with chemiluminescent HRP substrate. All luminescent wavelengths were read in a plate reader after 30 seconds.

Example 21. Measurement of ocular PK

Total drug levels for each test article were measured in the vitreous humor at 1, 3, 7, and 14 days after dosing. Eyes from three or four animals were harvested at each time point, yielding six or eight vitreous humor samples for each test article at each time point. Total drug levels in animals dosed with IGT-427 or PEGylated IGT-427 were quantified by anti-human IgG capture and Tie2 detection ELISA. Finally, total drug levels in animals dosed with faricimab were quantified by VEGF capture and anti-human Fc detection ELISA. The results of each of these ELISAs are shown in Figure 19. Its Cmax value is 433.8 μg/mL, and the half-life is 4.2 days. Faresimab was measured in the vitreous, and its Cmax was 154.2 μg/mL, and it decayed with a half-life of 4.4 days. IGT-427 was measured in the vitreous, and its Cmax value was 217.3 μg/mL, and the half-life was 3.8 days. IGT-427 modified with two 20kDa linear PEG molecules was measured in the vitreous, and its Cmax was 406.6 μg/mL, and it decayed with a half-life of 8.3 days. IGT-427 modified with two 40kDa branched PEG molecules was measured in the vitreous, and its Cmax value was 268.8 μg/mL, and the half-life was 8.0 days.

Industrial Applicability

The fusion protein that binds Tie2 and VEGF can bind Tie2 and VEGF with high affinity, maintain cross-reactivity with humans, mice and rabbits, and show desired antigen reactivity. In addition, it can be used to prevent or treat target angiogenic diseases or vascular diseases by inducing Tie2 phosphorylation, activation of Tie2 receptors and inhibition of VEGF.

Equivalent

The foregoing written description is considered sufficient to enable one skilled in the art to practice the embodiment. The foregoing description and examples detail certain embodiments and describe the best mode contemplated by the inventor. However, it should be understood that no matter how detailed the foregoing may appear in text, the embodiments may be practiced in many ways and should be interpreted in accordance with the appended claims and any equivalents thereof.

When a term such as at least and about precedes a list of values or ranges, the term modifies all values or ranges provided in the list. In some cases, the term about can include values rounded to the nearest significant figure. Any stated range includes endpoints where there is no explicit language to the contrary; for example, "between 5 and 50" encompasses the values 5 and 50.

Claims (83)

1. A fusion protein comprising an anti-Tie2 antibody or an antigen-binding fragment thereof and a vascular endothelial growth factor (VEGF) binding domain, wherein the fusion protein binds to the Tie2 Ig3-FNIII (1-3) domain comprising SEQ ID NO: 2, 3 or 4 and binds to VEGF. 2. The fusion protein of claim 1, wherein the VEGF binding domain comprises a VEGF receptor extracellular domain. 3. A fusion protein comprising: a vascular endothelial growth factor (VEGF) binding domain, wherein the VEGF binding domain comprises the vascular endothelial growth factor (VEGF)-A binding region of VEGF receptor 1 (VEGFR1) of SEQ ID NO: 13 and the vascular endothelial growth factor (VEGF)-A binding region of VEGF receptor 2 (VEGFR2) of SEQ ID NO: 14; and an anti-Tie2 antibody or an antibody binding fragment thereof, wherein the fusion protein binds to the Tie2 Ig3-FNIII (1-3) domain comprising SEQ ID NO: 2, 3 or 4 and VEGF. 4. The fusion protein of any one of the preceding claims, wherein 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. 5. The fusion protein according to any one of the preceding claims, wherein the fusion protein binds to the amino acid sequence of SEQ ID NO: 20 of Tie2 and/or the amino acid sequence of SEQ ID NO: 21 of Tie2. 6. The fusion protein of any of the preceding claims, wherein the anti-Tie2 antibody is of the IgG1 isotype. 7. The fusion protein of any of the preceding claims, wherein the anti-Tie2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR comprising an amino acid sequence of SEQ ID NOs: 5-7, and the light chain variable region comprises a light chain CDR comprising an amino acid sequence of SEQ ID NOs: 8-10. 8. The fusion protein of any of the preceding claims, wherein the VEGF binding domain comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:15. 9. The fusion protein of any one of claims 1 to 7, wherein the VEGF binding domain comprises the amino acid sequence of SEQ ID NO: 15. 10. The fusion protein of any of the preceding claims, wherein the fusion protein comprises a linker between the VEGF binding domain and the anti-Tie2 antibody or antibody fragment thereof, optionally wherein the linker comprises a sequence having between 5 and 50 amino acid residues, between 10 and 40 residues, between 15 and 30 residues, or 20 residues. 11. The fusion protein of any of the preceding claims, wherein the fusion protein comprises a linker between the VEGF binding domain and the anti-Tie2 antibody or antibody fragment thereof, and wherein the linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:16. 12. The fusion protein of claim 11, wherein the fusion protein comprises a linker between the VEGF binding domain and the anti-Tie2 antibody or antibody fragment thereof, and wherein the linker comprises the amino acid sequence of SEQ ID NO: 16. 13. The fusion protein of any one of claims 1 to 10, wherein the fusion protein comprises a linker between the VEGF binding domain and the anti-Tie2 antibody or antibody fragment thereof, and 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, optionally wherein the VEGF binding domain is a VEGF receptor. 14. The fusion protein of claim 13, wherein the fusion protein comprises a linker between the VEGF binding domain and the anti-Tie2 antibody or antibody fragment thereof, and wherein the linker comprises the amino acid sequence of SEQ ID NO: 25. 15. The fusion protein of any of the preceding claims, wherein the fusion protein comprises a CH domain comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 17. The fusion protein of claim 15 , wherein the fusion protein comprises a CH domain comprising the amino acid sequence of SEQ ID NO: 17. 17. The fusion protein of any of the preceding claims, wherein the fusion protein comprises one or more mutations in the heavy chain constant region that reduce or eliminate interaction with an Fc receptor. 18. The fusion protein of claim 17, wherein 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. 19. The fusion protein of claim 18, wherein the one or more mutations comprise a LALA mutation and mutations at K322 and P331 (EU numbering), optionally K322A and P331S (EU numbering). 20. The fusion protein of any one of claims 1 to 18, wherein the fusion protein comprises one or more mutations at L234, L235, H310, M252, I253, S254, T256, H433, N434 and/or H435 (EU numbering), optionally wherein the mutations are: a) L234A, L235A and H310A; or b) M252, I253, S254, T256, H433, N434 and/or H435, optionally M252Y; I253A, I253M or I253V; S254T; T256D; H433K; N434F; and/or H435A, H435Q or H435R. 21. The fusion protein of any of the preceding claims, wherein the fusion protein comprises a linker between the VEGF binding domain and the anti-Tie2 antibody or antibody fragment thereof from the C-terminus to one or more heavy chain constant domains of the anti-Tie2 antibody or antigen-binding fragment thereof and in order from the N-terminus to the C-terminus. 22. The fusion protein of any one of the preceding claims, 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. 23. The fusion protein of any of the preceding claims, wherein the fusion protein comprises a CH domain comprising the amino acid sequence of SEQ ID NO: 17, 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, and a VEGF binding domain comprising the amino acid sequence of SEQ ID NO: 15. 24. The fusion protein of any one of the preceding claims, wherein 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. 25. The fusion protein of any of the preceding claims, wherein the fusion protein comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 11 or 26. 26. The fusion protein of claim 25, wherein the fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 or 26. 27. The fusion protein of any of the preceding claims, wherein 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. 28. The fusion protein of claim 27, wherein the fusion protein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 12. 29. The fusion protein of any one of the preceding claims, wherein the fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 or 26, and a light chain comprising the amino acid sequence of SEQ ID NO: 12. 30. The fusion protein of any one of the preceding claims, wherein the fusion protein binds to the Tie2 Ig3-FNIII(1-3) domain comprising SEQ ID NO: 2, 3 or 4 with an affinity _KD (M) of less than 3E- ^9M . 31. The fusion protein of any one of the preceding claims, wherein the fusion protein is pegylated. 32. The fusion protein of claim 31, wherein the fusion protein is site-specifically PEGylated. 33. The fusion protein of claim 32, wherein the fusion protein is site-specifically PEGylated at a cysteine residue. 34. The fusion protein of claim 31, wherein the fusion protein further comprises the sequence of SEQ ID NO: 22 and is site-specifically PEGylated at the cysteine residue of the sequence of SEQ ID NO: 22. 35. The fusion protein of claim 34, wherein the sequence of SEQ ID NO:22 is present at the C-terminus of the heavy chain. 36. The fusion protein of claim 34, wherein the heavy chain comprises the sequence of SEQ ID NO:23. 37. The fusion protein of claim 34, wherein the heavy chain comprises the sequence of SEQ ID NO:24. 38. The fusion protein of any one of claims 31-37, wherein the PEG has a molecular weight of about 40 kDa. 39. The fusion protein of any one of the preceding claims, wherein the fusion protein further comprises one or more half-life extension regulators. 40. The fusion protein of claim 39, wherein the one or more half-life extension modulators comprise a chemical, biopolymer, or peptide that increases the half-life of the fusion protein. 41. The fusion protein of claim 39 or claim 40, wherein the one or more half-life extension regulators comprise: a biopolymer containing PEG (polyethylene glycol), hyaluronic acid (HA) or phosphorylcholine; albumin; an albumin binding peptide, and/or an HA binding protein fragment. 42. A nucleic acid encoding the fusion protein of any one of claims 1 to 41. 43. An expression vector comprising the nucleic acid of claim 42. 44. The expression vector of claim 43, further comprising a human cytomegalovirus IE1 (CMV-IE1) promoter/enhancer. 45. A cell transformed with the expression vector of claim 43 or claim 44. 46. A method for producing a fusion protein that binds Tie2 and VEGF, the method comprising the steps of: Cultivating the cell of claim 45; and The fusion protein is recovered from the cultured cells. 47. A method for preventing or treating angiogenic diseases or vascular diseases, the method comprising administering an effective amount of the fusion protein of any one of claims 1 to 41 to a subject in need thereof. 48. Use of the fusion protein of any one of claims 1 to 41 for the manufacture of a medicament for treating angiogenic diseases or vascular diseases in a subject in need thereof. 49. The fusion protein of any one of claims 1 to 41 for use in treating angiogenic diseases or vascular diseases in a subject in need thereof. 50. The method, use, or fusion protein for use of any one of claims 47 to 49, wherein the angiogenic disease or vascular disease is cancer, metastasis, diabetic retinopathy, retinopathy of prematurity, diabetic macular edema, corneal transplant rejection, macular degeneration, glaucoma, optionally wherein the glaucoma is neovascular glaucoma, erythropoiesis systemica, proliferative retinopathy, psoriasis, hemophilic arthritis, combined sclerosis, capillarization 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. 51. The method, use, or fusion protein for use of claim 50, wherein the cancer is esophageal cancer, gastric cancer, colorectal 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 lymphoma, lymphoma, or multiple myeloid leukemia. 52. A method for modulating angiogenesis, endothelial signaling, inflammation, hypoxia and/or vascular leakage, the method comprising administering to a subject in need thereof an effective amount of the fusion protein of any one of claims 1 to 41. 53. The method of claim 52, wherein the inflammation is from sepsis, acute respiratory distress syndrome, and/or a viral infectious disease. 54. The method, use, or fusion protein for use of any one of claims 47 to 53, wherein the subject is a human. 55. The method, use, or fusion protein for use of any one of claims 47 to 53, wherein the subject is a companion animal, optionally a mammalian companion animal. 56. The method, use, or fusion protein for use of claim 55, wherein the companion animal is a dog, cat, rabbit, ferret, horse, mule, donkey, or hamster. 57. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 41. 58. A polypeptide comprising chain monomers of the fusion protein of any one of claims 1 to 41. 59. The polypeptide of claim 58, wherein the polypeptide comprises a heavy chain monomer of the fusion protein of any one of claims 1 to 41. 60. The polypeptide of claim 59, wherein the polypeptide comprises a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the sequence of SEQ ID NO: 11 or 26. 61. The polypeptide of claim 60, wherein the polypeptide comprises SEQ ID NO: 11 or 26. 62. The polypeptide of claim 61, wherein the polypeptide consists of SEQ ID NO: 11 or 26. 63. The polypeptide of claim 58, wherein the polypeptide comprises a light chain monomer of the fusion protein of any one of claims 1 to 41. 64. The polypeptide of claim 63, wherein the polypeptide comprises a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 12. 65. The polypeptide of claim 64, wherein the polypeptide comprises SEQ ID NO:12. 66. The polypeptide of claim 65, wherein the polypeptide consists of SEQ ID NO: 12. 67. A nucleic acid molecule encoding the polypeptide of any one of claims 58 to 66. 68. The nucleic acid molecule of claim 67, wherein the nucleic acid molecule encodes the polypeptide of any one of claims 59 to 62. 69. The nucleic acid molecule of claim 68, wherein the nucleic acid molecule comprises a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 27 or 29. 70. The nucleic acid molecule of claim 69, wherein the nucleic acid molecule comprises SEQ ID NO: 27 or 29. 71. The nucleic acid molecule of claim 70, wherein the nucleic acid molecule consists of SEQ ID NO: 27 or 29. 72. The nucleic acid molecule of claim 67, wherein the nucleic acid molecule encodes the polypeptide of any one of claims 63 to 66. 73. The nucleic acid molecule of claim 72, wherein the nucleic acid molecule comprises a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:28. 74. The nucleic acid molecule of claim 73, wherein the nucleic acid molecule comprises SEQ ID NO:28. 75. The nucleic acid molecule of claim 74, wherein the nucleic acid molecule consists of SEQ ID NO:28. 76. A collection of one or more polynucleotides, each of which encodes at least one of the monomeric chains of a fusion protein according to any one of claims 1 to 41, such that both chains (i.e., a light chain and a heavy chain) of the fusion protein are encoded. 77. A vector comprising the nucleic acid of any one of claims 67 to 75. 78. The vector of claim 77, wherein the vector comprises a nucleic acid encoding a heavy chain sequence and a light chain sequence of the fusion protein. 79. A collection of one or more vectors, which collectively comprise a collection of one or more polynucleotides of claim 76, such that both chains (i.e., the light chain and the heavy chain) of the fusion protein are encoded in the collection of vectors. 80. The vector or vector collection of any one of claims 77 to 79, wherein the vector is an animal virus, such as a virus selected from retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses, varicella virus, baculoviruses, papillomaviruses, anelloviruses and papovaviruses. 81. A host cell expressing the vector of any one of claims 77 to 80. 82. A pharmaceutical composition comprising a nucleic acid molecule according to any one of claims 67 to 75, a collection of one or more polynucleotides according to claim 76, or a vector or collection of vectors according to any one of claims 77 to 79, and a pharmaceutically acceptable carrier, diluent or excipient. 83. A method of producing a fusion protein according to any one of claims 1 to 41 by expression from a vector or a collection of vectors.