Treating neovascularization

Abstract

This document provides methods and materials for treating neovascularization (e.g., ocular neovascularization). In some cases, this document provides polypeptides (e.g., recombinant polypeptides) that include (a) one or more VEGFR-1 immunoglobulin (Ig) 2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more fragment crystallizable region (Fc) domains. In some cases, a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains can bind (e.g., bind and inhibit) two or more VEGF ligands and can be used as a VEGF antagonist. This document also provides methods for using the polypeptides provided herein to treat a mammal (e.g., a human) having, or at risk of developing, neovascularization (e.g., ocular neovascularization) and / or macular edema.

Description

[0001] Atorney Docket No. 44807-0507W01 / Pl 8657-02

[0002] TREATING NEOVASCULARIZATION

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Patent Application Serial No. 63 / 752,081, filed on January 31, 2025. The disclosure of the prior application is considered part of, and is incorporated by reference in, the disclosure of this application.

[0005] STATEMENT REGARDING FEDERAL FUNDING

[0006] This invention was made with Government support under grant number NNX16AO69A, awarded by the National Aeronautics and Space Administration, as well as under grant nos. CA240339, EY028996, EY031097, and EY033924, awarded by the National Institutes of Health, and under contract number HT942523C0005, awarded by the Defense Health Agency, Medical Research and Development Branch. The Government has certain rights in the invention.

[0007] TECHNICAL FIELD

[0008] This document relates to methods and materials for treating neovascularization (e.g., ocular neovascularization). In some cases, this document provides polypeptides (e g., recombinant polypeptides) that include (a) one or more vascular endothelial growth factor receptor (VEGFR)-1 immunoglobulin (Ig) 2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more fragment crystallizable region (Fc) domains. In some cases, a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains can bind (e.g., bind and inhibit) two or more VEGF ligands and can be used as a VEGF antagonist. This document also provides methods for using the polypeptides provided herein to treat a mammal (e.g., a human) having, or at risk of developing, neovascularization (e.g., ocular neovascularization) and / or macular edema.Atorney Docket No. 44807-0507W01 / Pl 8657-02

[0009] BACKGROUND

[0010] Vision loss is a global public health challenge (Saaddine, Ophthalmology 110:253— 254 (2003)) that is associated with poor health outcomes and significantly damages quality of life in affected patients (National Academies of Sciences, The Impact of Vision Loss . In Making Eye Health a Population Health Imperative: Vision for Tomorrow (National Academies Press (US) (2016)). Retinal and choroidal neovascular diseases constitute the most common causes of moderate-to-severe vision loss worldwide (Campochiaro, J Mol Med (Berl) 91 :311-321 (2013)). These diseases, which include neovascular age-related macular degeneration (NVAMD) and diabetic retinopathy (DR), are characterized by the development of abnormal blood vessels that leak fluid into the vitreous or grow into avascular areas within the retina. Ultimately, these irregular and leaky vessels interfere with retinal function and can lead to detachment, resulting in vision loss.

[0011] SUMMARY

[0012] This document provides methods and materials for treating neovascularization (e.g., ocular neovascularization such as vascular endothelial growth factor (VEGF)-induced ocular neovascularization) and / or macular edema. In some cases, this document provides polypeptides (e.g., recombinant polypeptides) that include (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains. Such polypeptides can bind (e.g., bind and inhibit) two or more VEGF ligands and can be used as VEGF antagonists (also referred to herein as VEGF antagonist polypeptides and VEGF receptor decoy polypeptides). This document also provides methods for using one or more VEGF antagonists provided herein (e.g., one or more polypeptides (e.g., one or more chimeric polypeptides) that each include (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) to treat a mammal (e.g., a human) having, or at risk of developing, neovascularization. This document also provides methods for using polypeptides provided herein to treat a mammal (e.g., a human) having, or at risk of developing, neovascularization (e.g., ocular neovascularization) and / or macular edema.Atorney Docket No. 44807-0507W01 / Pl 8657-02

[0013] As demonstrated herein, polypeptides that include (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains can simultaneously bind multiple VEGFR-1 and VEGFR-2 ligands. For example, polypeptides provided herein can simultaneously bind (e.g., bind and sequester) at least two ligands (e.g., can simultaneously bind both VEGF-A and VEGF-C). Also as demonstrated herein, polypeptides that include (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains can be used to block neovascularization (e.g., ocular neovascularization).

[0014] Having the ability to simultaneously target both VEGFR-1 and VEGFR-2 ligands (e.g., by administering one or more VEGF antagonist polypeptides provided herein that each include (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) provides a unique and unrealized opportunity to inhibit neovascularization (e.g., ocular neovascularization such as pathogenic ocular neovascularization). In some cases, the ability to inhibit neovascularization using the polypeptides provided herein can allow treatment of several debilitating eye diseases, including NVAMD and DR.

[0015] In general, one aspect of this document features VEGF antagonist polypeptides. The VEGF antagonist polypeptides can include, or consist essentially of, (a) a VEGFR-1 Ig2 domain, (b) a VEGFR-2 Ig2 domain, (c) a VEGFR-2 Ig3 domain, and (d) a Fc domain. The VEGF antagonist polypeptide can simultaneously bind VEGF-A and VEGF-C. The VEGFR-1 Ig2 domain can comprise, consist of, or consist essentially of SEQ ID NO: 1. The VEGFR-2 Ig2 domain can comprise, consist of, or consist essentially of SEQ ID NO:2. The VEGFR-2 lg3 domain can comprise, consist of, or consist essentially of SEQ ID NO:3. The Fc domain can comprise, consist of, or consist essentially of SEQ ID NO:4. The VEGF antagonist polypeptide can include (a), followed by (b), followed by (c), followed by (d). The VEGF antagonist polypeptide can comprise, consist of, or consist essentially of SEQ ID NO:5. The VEGF antagonist polypeptide include (b), followed by (c), followed by (a), followed by (d). The VEGF antagonist polypeptide can comprise, consist of, or consist essentially of SEQ IDAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0016] NO:6. This document also features compositions including such VEGF antagonist polypeptides. The composition can include a pharmaceutically acceptable carrier.

[0017] In another aspect, this document features nucleic acid encoding VEGF antagonist polypeptides that include, or consist essentially of, (a) a VEGFR-1 Ig2 domain, (b) a VEGFR-2 Ig2 domain, (c) a VEGFR-2 Ig3 domain, and (d) a Fc domain. The VEGF antagonist polypeptide can simultaneously bind VEGF-A and VEGF-C. The VEGFR-1 Ig2 domain can comprise, consist of, or consist essentially of SEQ ID NO: 1. The VEGFR-2 Ig2 domain can comprise, consist of, or consist essentially of SEQ ID NO:2. The VEGFR-2 Ig3 domain can comprise, consist of, or consist essentially of SEQ ID NO:3. The Fc domain can comprise, consist of, or consist essentially of SEQ ID NO:4. The VEGF antagonist polypeptide can include (a), followed by (b), followed by (c), followed by (d). The VEGF antagonist polypeptide can comprise, consist of, or consist essentially of SEQ ID NO:5. The VEGF antagonist polypeptide include (b), followed by (c), followed by (a), followed by (d). The VEGF antagonist polypeptide can comprise, consist of, or consist essentially of SEQ ID NO:6. This document also features compositions including nucleic acids encoding such VEGF antagonist polypeptides. The composition can include a pharmaceutically acceptable carrier.

[0018] In another aspect, this document features methods for treating a mammal having ocular neovascularization. The methods can include, or consist essentially of, administering, to the mammal, a VEGF antagonist polypeptide that includes, or consists essentially of, (a) a VEGFR-1 Ig2 domain, (b) a VEGFR-2 Ig2 domain, (c) a VEGFR-2 Ig3 domain, and (d) a Fc domain and / or a nucleic acid encoding such a VEGF antagonist polypeptide. The VEGF antagonist polypeptide can simultaneously bind VEGF-A and VEGF-C. The VEGFR-1 Ig2 domain can comprise, consist of, or consist essentially of SEQ ID NO: 1. The VEGFR-2 lg2 domain can comprise, consist of, or consist essentially of SEQ ID NO:2. The VEGFR-2 Ig3 domain can comprise, consist of, or consist essentially of SEQ ID NO:3. The Fc domain can comprise, consist of, or consist essentially of SEQ ID NO:4. The VEGF antagonist polypeptide can include (a), followed by (b), followed by (c), followed by (d). The VEGF antagonist polypeptide can comprise, consist of, or consist essentially of SEQ ID NO:5. The VEGF antagonist polypeptide include (b), followed by (c), followed by (a), followed by (d).Atorney Docket No. 44807-0507W01 / Pl 8657-02

[0019] The VEGF antagonist polypeptide can comprise, consist of, or consist essentially of SEQ ID NO:6. The mammal can be a human. The mammal can have been previously treated with an anti-VEGF therapy (e.g., and can be resistant to the previously administered anti-VEGF therapy).

[0020] In another aspect, this document features methods for treating a mammal having a disease or disorder associated with ocular neovascularization. The methods can include, or consist essentially of, administering to the mammal, a VEGF antagonist polypeptide that includes, or consists essentially of, (a) a VEGFR-1 Ig2 domain, (b) a VEGFR-2 Ig2 domain, (c) a VEGFR-2 Ig3 domain, and (d) a Fc domain and / or a nucleic acid encoding such a VEGF antagonist polypeptide. The VEGF antagonist polypeptide can simultaneously bind VEGF -A and VEGF-C. The VEGFR-1 Ig2 domain can comprise, consist of, or consist essentially of SEQ ID NO: 1. The VEGFR-2 Ig2 domain can comprise, consist of, or consist essentially of SEQ ID NO:2. The VEGFR-2 Ig3 domain can comprise, consist of, or consist essentially of SEQ ID NO:3. The Fc domain can comprise, consist of, or consist essentially of SEQ ID NO:4. The VEGF antagonist polypeptide can include (a), followed by (b), followed by (c), followed by (d). The VEGF antagonist polypeptide can comprise, consist of, or consist essentially of SEQ ID NO:5. The VEGF antagonist polypeptide include (b), followed by (c), followed by (a), followed by (d). The VEGF antagonist polypeptide can comprise, consist of, or consist essentially of SEQ ID NO:6. The disease or disorder associated with ocular neovascularization is neovascular age-related macular degeneration (NVAMD), polypoidal choroidal vasculopathy, diabetic retinopathy (DR), ocular histoplasmosis, myopic degeneration, angioid streaks, choroidal neovascularization, retinal neovascularization, or retinopathy of prematurity (ROP). The mammal can be a human. The mammal can have been previously treated with an anti-VEGF therapy (e.g., and can be resistant to the previously administered anti-VEGF therapy).

[0021] In another aspect, this document features methods for treating a mammal having macular edema. The methods can include, or consist essentially of, administering to the mammal, a VEGF antagonist polypeptide that includes, or consists essentially of, (a) a VEGFR-1 Ig2 domain, (b) a VEGFR-2 Ig2 domain, (c) a VEGFR-2 Ig3 domain, and (d) a Fc domain and / or a nucleic acid encoding such a VEGF antagonist polypeptide. The VEGFAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0022] antagonist polypeptide can simultaneously bind VEGF-A and VEGF-C. The VEGFR-1 Ig2 domain can comprise, consist of, or consist essentially of SEQ ID NO: 1. The VEGFR-2 Ig2 domain can comprise, consist of, or consist essentially of SEQ ID NO:2. The VEGFR-2 Ig3 domain can comprise, consist of, or consist essentially of SEQ ID NO:3. The Fc domain can comprise, consist of, or consist essentially of SEQ ID NO:4. The VEGF antagonist polypeptide can include (a), followed by (b), followed by (c), followed by (d). The VEGF antagonist polypeptide can comprise, consist of, or consist essentially of SEQ ID NO:5. The VEGF antagonist polypeptide include (b), followed by (c), followed by (a), followed by (d). The VEGF antagonist polypeptide can comprise, consist of, or consist essentially of SEQ ID NO:6. The macular edema can be diabetic macular edema or macular edema due to retinal vein occlusion. The mammal can be a human. The mammal can have been previously treated with an anti-VEGF therapy (e.g., and can be resistant to the previously administered anti-VEGF therapy).

[0023] Unless otherwise defined, 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 invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0024] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figures 1A-1E. Design and production of bispecific VEGF receptor-based receptor decoy polypeptides. Figure 1A) Diagram of VEGF family members and their respective receptors, with downstream receptor functions indicated. Monomers of VEGF-A and P1GF expressed by the same cell can result in VEGF / P1GF heterodimers, causingAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0027] heterodimerization of VEGFR-1 and VEGFR-2 receptors. Figure IB) Schematic of the domain layouts of VEGFR-1 and VEGFR-2 receptor tyrosine kinases. Figure 1C) Schematics of VEGF receptor decoy polypeptides. The engineered molecules 12-Fc and 21-Fc fuse the minimal ligand binding domains of VEGFR-1 (immunoglobulin (Ig)2) and VEGFR-2 (Ig2-3) as N-terminal fusions to a human IgGl Fc for high-affinity binding to both VEGFR-1 and VEGFR-2 ligands. The clinically approved antagonist protein aflibercept fuses VEGFR-1 Ig2 and VEGFR-2 Ig3 to a human IgGl Fc for affinity binding of VEGFR-1 ligands. Figure ID) Overlay of the size-exclusion chromatography (SEC) traces of recombinantly expressed 12-Fc, 21-Fc, and aflibercept. Figure IE) SDS-PAGE analysis of recombinantly expressed VEGF antagonist polypeptides under non-reducing (left) and reducing (right) conditions. See also Figure 7.

[0028] Figures 2A-2H. Bispecific receptor decoy polypeptides show binding to all VEGFR-1 and VEGFR-2 ligands. Figure 2A) Schematic of biolayer interferometry -based binding assay layout, with immobilized antagonist protein and soluble VEGF ligand. Figures 2B-2H) Equilibrium binding titrations of VEGF-A165 (Figure 2B), VEGF-A121 (Figure 2C), VEGF-B (Figure 2D), P1GF-1 (Figure 2E), P1GF-2 (Figure 2F), VEGF-C (Figure 2G), and VEGF-D (Figure 2H) ligands against engineered bispecific receptor decoy polypeptides and aflibercept. See also Figure 8 and Table 2.

[0029] Figures 3A-3D. Bispecific receptor decoy polypeptides bind two ligands simultaneously and block ligand interaction with VEGF receptors. Figure 3A) Engineered bispecific receptor decoy polypeptides (100 nM) were incubated with serial dilutions of VEGF-C to pre-block binding to the VEGFR-2 binding domains prior to measuring interaction with immobilized VEGF-A via biolayer interferometry. Binding signal was normalized to that of the engineered bispecific receptor decoy polypeptides without ligand preincubation. Figure 3B) Engineered bispecific receptor decoy polypeptides (100 nM) were incubated with serial dilutions of Pl GF- 1 to pre-block binding to the VEGFR-1 binding domain prior to measuring interaction with immobilized VEGF-C via biolayer interferometry. Binding signal was normalized to that of the engineered bispecific receptor decoy polypeptides without ligand preincubation. Figures 3C-3D) A saturating concentration (100 nM) of either VEGF-A (Figure 3C) or VEGF-C (Figure 3D) was preincubated with theAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0030] indicated antagonist polypeptides prior to measuring binding to immobilized VEGFR-2 via biolayer interferometry. Total signal was normalized to ligand binding in the absence of antagonist. See also Figure 10 and Tables 4 and 5.

[0031] Figures 4A-4C. Bispecific receptor decoy polypeptides attenuate VEGF ligand-induced angiogenic signaling. Figures 4A-4C) Immunoblot analyses of cell lysates from HMECs treated with 1.1 nM (50 ng / mL) VEGF-A (Figure 4A), 3.8 nM (100 ng / mL) VEGF-C (Figure 4B), or 0.22 nM (10 ng / mL) VEGF-A and 1.18 nM (31 ng / mL) VEGF-C (Figure 4C) in the absence or presence of the indicated antagonist polypeptides (100 nM). Blots were probed with anti-p VEGFR-2, anti-VEGFR-2, anti-pAkt, anti-pan Akt, anti-pERKl / 2, and anti-ERKl / 2. Blots were also probed with an anti- glyceraldehyde-3-phospahte dehydrogenase (GAPDH) antibody to confirm equivalent loading.

[0032] Figures 5A-5E. Bispecific receptor decoy polypeptides inhibit proliferation, migration, and survival of endothelial cells. Figures 5A-5B) HMEC proliferation (Figure 5A) and transwell cell migration (Figure 5B) were quantified via real-time cell analysis using an xCELLigence instrument. Saturating amounts of VEGF-A (1.1 nM; 50 ng / mL) (left) or VEGF-C (3.8 nM; 100 ng / mL) for proliferation studies and (1.9 nM; 50 ng / mL) for migration studies (right) incubated with titrations of the indicated antagonist polypeptides were added to HMEC cells. Cell index after 48 hours (proliferation) or 5 hours (migration), normalized to that of cells that received ligand only, is presented. Data represent mean±SD (n=4). Figure 5C) HMEC survival was measured after 48 hours in starvation media containing saturating amounts of VEGF-A (1.1 nM; 50 ng / mL) or VEGF-C (3.8 nM; 100 ng / mL) incubated with titrations of the indicated antagonist polypeptides. Cell viability was measured using the water-soluble tetrazolium salt WST-1. Signal was normalized to that of cells that received ligand only. Data represent mean±SD (n=3). Figure 5D) VEGF-A (0.22 nM; 10 ng / mL) and VEGF-C (1.18 nM; 31 ng / mL) incubated with titrations of the indicated antagonist polypeptides were added to HMEC cells. Cell index after 48 hours (proliferation), normalized to that of cells that received ligand only, is presented. Data represent mean±SD (n=4). Figure 5E) HMEC survival was measured after 48 hours in starvation media containing 0.22 nM (10 ng / mL) VEGF-A and 1.18 nM (31 ng / mL) VEGF-C incubated with titrations of the indicated antagonist polypeptides. Cell viability was measured using WST-1.Atorney Docket No. 44807-0507W01 / Pl 8657-02

[0033] Signal was normalized to that of cells that received ligand only. Data represent mean±SD (n=3). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to aflibercept cohort by 2-tailed Student’s t test. See also Figures 13-15, Table 6, and Table 7.

[0034] Figures 6A-6J. Engineered bispecific receptor decoy polypeptides lead to greater suppression of neovascularization relative to clinical antagonist polypeptides in a mouse models of ischemic retinopathy and choroidal neovascularization. Figures 6A-6C) Seven-day-old mouse pups were exposed to 75% oxygen in an airtight chamber for 5 days to induce retinopathy and then returned to room air. Pups were then given an intravitreal injection of 1 pg VEGF antagonist protein in one eye and saline in the fellow eye as a control. Five days later, mice were euthanized and retinas were isolated and incubated with fluorescent lectin to visualize area of neovascularization via microscopy. Representative images are shown of saline-treated fellow eye controls (Figures 6A, 6B, and 6C, top) adjacent to the corresponding eyes treated with aflibercept (Figure 6A, bottom), 12-Fc (Figure 6B, bottom), and 21-Fc (Figure 6C, bottom). Note that the hyaloid vessels (indicated by arrowheads) do not represent neovascularization; instead, the staining in the periphery represents buds of neovascularization. Neovascularization was quantified as percent of retinal area by image analysis and is shown in Figure 6D and Figure 6E. Figures 6F-6J) Choroidal neovascularization was induced in 6 to 8-week old mice via laser injury of Bruch’s membrane, and mice were administered intraocular injections of receptor decoy polypeptides in one eye and saline in the fellow eye. For the PBS control cohort, PBS was injected in one eye and no injection was administered to the fellow eye. Seven days later, mice were euthanized and eyes were fixed and incubated with fluorescent lectin to visualize area of neovascularization via microscopy. Representative images are shown for fellow eyes (Figures 6F-61, top) and treated eyes (Figures 6F-61, bottom). Quantification of choroidal neovascularization area by image analysis is presented in (Figure 6 J). Data represent mean±SEM. **p<0.01; ***p<0.001; ****p<0.0001 by Wilcoxon matched-pairs signed-rank test for (Figures 6D-6E) and by paired Student’s t-test for (Figure 6J). See also Figures 18, 19 and Table 7.

[0035] Figure 7. Structural model of bispecific receptor decoy polypeptides. The crystallographic structures of the VEGFR-2 D23 / VEGF-C complex (Protein Data BankAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0036] (PDB) ID: 3V2A), the VEGFR-1 / VEGF -A complex (PDB ID: 5T89), and a human IgGl antibody (PDB ID: 1HZH) were manually grafted using ChimeraX software to visualize the hypothetical layouts of engineered bispecific receptor decoy polypeptides 12-Fc and 21-Fc.

[0037] Figures 8A-8C. Interaction of VEGF receptor decoy polypeptides with VEGF family ligands. Kinetic traces from biolayer interferometry analysis of VEGF-A165, VEGF-A121, P1GF-1, P1GF-2, VEGF-B, VEGF-C, and VEGF-D binding to immobilized 12-Fc (Figure 8A), 21-Fc (Figure 8B), or aflibercept (Figure 8C).

[0038] Figures 9A-9I: VEGFR-1 and VEGFR-2 binding to VEGF family ligands. (A)-(G) Equilibrium binding titrations of VEGF-A165 (Figure 9A), VEGF-A121 (Figure 9B), VEGF-B (Figure 9C), P1GF-1 (Figure 9D), P1GF-2 (Figure 9E), VEGF-C (Figure 9F), and VEGF-D (Figure 9G) ligands against immobilized VEGF receptor binding domains (VEGFR-1 D23 Fc and VEGFR-2 D23 Fc). Figures 9H-9I) Kinetic traces from biolayer interferometry analysis of each VEGF ligand binding to VEGFR-1 D23 Fc (Figure 9H) and VEGFR-2 D23 Fc (Figure 91). See also Table 3.

[0039] Figures 10A-10B. Bispecific receptor decoy polypeptides compete with VEGF receptors for ligand binding. A saturating concentration (100 nM) of either VEGF-A (Figure 10A) or VEGF-C (Figure 10B) was preincubated with the indicated receptor decoy polypeptides prior to measuring binding to immobilized VEGFR-1 (Figure 10A) or VEGFR-3 (Figure 10B) via biolayer interferometry. Total signal was normalized to ligand binding in the absence of receptor decoy.

[0040] Figures 11A-1 IB. HMECs show greater magnitude of response to VEGF-C in survival assays compared to HUVECs. HUVECs (Figure 11 A) and HMECs (Figure 1 IB) were stimulated with titrations of VEGF-A or VEGF-C in starvation media. Cell survival was quantified after 48 hours of treatment using the water-soluble tetrazolium salt WST-1. Signal is presented as absorbance at 440 nm (A440), with subtraction of background from a well containing cells in assay media only. Data represent mean±SD (n=3).

[0041] Figures 12A-12D: Selection of saturating VEGF-A and VEGF-C doses for real-time cell analysis proliferation and migration studies. Figures 12A-12B) HMEC cells were titrated to determine saturating doses VEGF-A (Figure 12A) and VEGF-C (Figure 12B) for induction of proliferation using an xCELLigence instrument. Cell index was measured everyAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0042] 15 minutes for 72 hours. Figures 12C-12D) HMEC cells were titrated to determine saturating doses VEGF-A (Figure 12C) and VEGF-C (Figure 12D) for induction of proliferation using an xCELLigence instrument. For Figure 12C, 20,000 initial cells were added to the upper well, and cell index was measured every 5 minutes. For Figure 12D, 30,000 initial cells were added to the upper well, and cell index was measured every 15 minutes. All conditions were plated in duplicate. Data represent mean±SD (n=2). “Media only” indicates negative control wells that contained cells in assay media only.

[0043] Figures 13A-13C. Representative VEGF-A or VEGF-C-induced proliferation profiles measured via real-time cell analysis. Figures 13A-13C) HMEC cells were treated with VEGF-A (left, 1.1 nM) or VEGF-C (right, 3.8 nM) in the presence of titrated amounts of 12-Fc (Figure 13A), 21-Fc (Figure 13B), or aflibercept (Figure 13C). Cell index was measured every 15 minutes for 72 hours via real-time cell analysis using an xCELLigence instrument. Data represent mean±SD (n=2).

[0044] Figures 14A-14C. Representative VEGF-A or VEGF-C-induced transwell migration profiles measured via real-time cell analysis. HMECs were seeded in transwell plates and treated with VEGF-A (left, 1.1 nM) or VEGF-C (right, 1.9 nM) in the presence of titrated amounts of 12-Fc (Figure 14A), 21-Fc (Figure 14B), or aflibercept (Figure 14C). Cell index was measured every 5 minutes for 24 hours via real-time cell analysis using an xCELLigence instrument. Data represent mean±SD (n=2).

[0045] Figures 15A-15C. Representative xCELLigence VEGF-A and VEGF-C-induced -proliferation profiles . HMECs were treated with VEGF-A (0.22 nM) or VEGF-C (1.18 nM) in the presence of titrated amounts of 12-Fc (Figure 15A), 21-Fc (Figure 15B), or aflibercept (Figure 15C). Cell index was measured every 15 minutes for 72 hours via real-time cell analysis using an xCELLigence instrument. Data represent mean±SD (n=2).

[0046] Figures 16A-16B. Novel receptor decoys show similar isoelectric point to aflibercept and exhibit minimal binding to extracellular matrix. Figure 16A) Isoelectric focusing gel of the engineered receptor decoy polypeptides 12-Fc and 21-Fc, aflibercept, and Fc-fused VEGFR-1 domains 1-3 (VEGFR-1 D123 Fc, a high-pl control). Figure 16B) Schematic of the modified enzyme-linked immunosorbent assay (ELISA) used to determine receptor decoy protein binding to extracellular matrix (ECM) (top). Receptor decoy polypeptides wereAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0047] incubated in wells coated with a thin layer of Matrigel to simulate ECM, and binding was quantified using a fluorescent secondary antibody. ECM binding was measured for 12-Fc, 21-Fc, and aflibercept, as well as VEGFR-1 D123 Fc and Fc-fused VEGFR-2 domains 2 and 3 (VEGFR-2 D23 Fc) as controls (bottom). Data represent mean±SD (n=2).

[0048] Figures 17A-17E: Engineered receptor decoy polypeptides show high affinity binding to murine VEGF-A and VEGF-C. Figures 17A-17B) Equilibrium binding titrations of murine VEGF-Aies (Figure 17A) and murine VEGF-C (Figure 17B) ligands against immobilized engineered receptor decoy polypeptides and aflibercept. Figures 17C-17E) Kinetic traces from biolayer interferometry analysis of the interaction between murine VEGF-Aies and VEGF-C against immobilized for 12-Fc (Figure 17C), 21-Fc (Figure 17D), and aflibercept (Figure 17E). See also Table 2.

[0049] Figure 18. Aflibercept controls neovascularization in oxygen-induced retinopathy model down to dose of 2 pg . Seven-day-old mouse pups were exposed to 75% oxygen in a hyperbaric chamber for 5 days and then returned to room air to allow for the development of the oxygen-induced retinopathy phenotype. Pups then received intravitreal injection of aflibercept at the indicated dose in one eye and HEPES-buffered saline (FIBS) in the fellow eye as a control. Five days later, mice were sacrificed and the retinas were isolated, incubated with fluorescent lectin, and flat-mounted to visualize area of neovascularization via microscopy. Quantification of neovascularization area by image analysis is shown. Data represent mean±SEM. p<0.05 by Wilcoxon matched-pairs signed-rank test.

[0050] Figures 19A-19B. Engineered bispecific receptor decoy polypeptides lead to greater suppression of neovascularization relative to clinical receptor decoy polypeptides in a mouse model of ischemic retinopathy when quantified as total neovascular area. Seven-day-old mouse pups were exposed to 75% oxygen in an airtight chamber for 5 days to induce retinopathy and then returned to room air. Pups were then given an intravitreal injection of 1 pg VEGF receptor decoy protein in one eye and saline in the fellow eye as a control. Five days later, mice were euthanized and retinas were isolated and incubated with fluorescent lectin to visualize area of neovascularization via microscopy. Quantification of neovascularization area by image analysis is shown for 12-Fc (Figure 19A) and 21-FcAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0051] (Figure 19B) compared to aflibercept. Data represent mean±SEM. **p<0.01; ***p<0.001 by Wilcoxon matched pairs signed rank test.

[0052] DET ILED DESCRIPTION

[0053] This document provides methods and materials for treating neovascularization (e.g., ocular neovascularization) and / or macular edema. In some cases, this document provides polypeptides (e.g., recombinant polypeptides) that bind (e.g., bind and inhibit) two or more VEGF ligands. For example, this document provides polypeptides (e.g., VEGF antagonist polypeptides) that include (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains. Also provided are methods for using one or more VEGF antagonist polypeptides provided herein (e.g., one or more polypeptides (e.g., one or more chimeric polypeptides) that each include (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains). In some cases, this document provides methods and materials for using one or more VEGF antagonist polypeptides to treat a mammal (e.g., a human) having, or at risk of developing, neovascularization (e.g., ocular neovascularization). For example, one or more VEGF antagonist polypeptides can be administered to a mammal (e g., a human) having, or at risk of developing, neovascularization to treat the mammal. In some cases, this document provides methods and materials for using one or more VEGF antagonist polypeptides to treat a mammal (e.g., a human) having a disease or disorder associated with ocular vascularization. For example, one or more VEGF antagonist polypeptides can be administered to a mammal (e.g., a human) having a disease or disorder associated with ocular vascularization to treat the mammal. In some cases, this document provides methods and materials for using one or more VEGF antagonist polypeptides to treat a mammal (e.g., a human) having, or at risk of developing, macular edema. For example, one or more VEGF antagonist polypeptides can be administered to a mammal (e g., a human) having, or at risk of developing, macular edema to treat the mammal.

[0054] A VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or moreAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0055] VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can bind any appropriate VEGF ligand. In some cases, a VEGF ligand that can be bound (e.g., bound and sequestered) by a VEGF antagonist polypeptide provided herein can be in a dimeric form (e.g., a homodimeric form or a heterodimeric form). Examples of VEGF ligands that can be bound (e.g., bound and sequestered) by a VEGF antagonist polypeptide provided herein include, without limitation, VEGF-A (e.g., VEGF-A165 and VEGF-A121 isoforms), VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor 1 (P1GF-1), and P1GF-2. In some cases, a VEGF antagonist polypeptide can be simultaneously bind (e.g., bind and sequester) two or more VEGF ligands. In some cases, a VEGF antagonist polypeptide provided herein can be a bispecific VEGF antagonist polypeptide (e.g., can bind 2 VEGF ligands). In some cases, a VEGF antagonist polypeptide provided herein can bind (e.g., can simultaneously bind) VEGF-A and VEGF-C.

[0056] A VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR- 1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can include any appropriate one or more VEGFR- 1 Ig2 domains. A VEGFR- 1 Ig2 domain that can be included in a VEGF antagonist polypeptide provided herein can be any appropriate length (e.g., can include any number of amino acids). In some cases, a VEGFR-1 Ig2 domain can be from about 94 amino acids in length to about 102 amino acids in length (e.g., can be from about 94 to about 100, can be from about 94 to about 98, can be from about 94 to about 96, can be from about 96 to about 102, can be from about 98 to about 102, can be from about 100 to about 102, can be from about 96 to about 100, can be from about 96 to about 98, can be from about 98 to about 100, or can be about 94 amino acids in length).

[0057] A VEGFR-1 lg2 domain that can be included in a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can have any appropriate amino acid sequence. In some cases, a VEGFR-1 Ig2 domain can comprise, consist essentially of, or consist of an amino acid sequence set forth in SEQ IDNO:!.Atorney Docket No. 44807-0507W01 / Pl 8657-02

[0058] GRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRII WDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQ (SEQ ID NO:1)

[0059] A VEGFR-1 Ig2 domain that consists essentially of an amino acid sequence set forth in SEQ ID NO: 1 can include the amino acid sequence set forth in SEQ ID NO: 1 with zero, one, or two amino acid substitutions within the articulated sequence of the sequence identifier (e.g., SEQ ID NO: 1), with zero, one, two, three, four, or five amino acid residues preceding the articulated sequence of the sequence identifier (e g., SEQ ID NO:1), and / or with zero, one, two, three, four, or five amino acid residues following the articulated sequence of the sequence identifier (e.g., SEQ ID NO: 1), provided that the natriuretic peptide retains at least some VEGFR-1 Ig2 domain activity (e.g., the ability to bind VEGF-A).

[0060] In some cases, a VEGFR-1 Ig2 domain that can be included in a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can have an amino acid sequence set forth in the National Center for Biotechnology Information (NCBI) databases at, for example, accession no.

[0061] P17948 (version P17498.2).

[0062] In some cases, a VEGFR-1 Ig2 domain that can be included in a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can be as described elsewhere (see, e.g., U.S. Patent No. 7,087,411; and U.S. Patent No. 10,259,860).

[0063] A VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 lg2 domains, (b) one or more VEGFR-2 lg2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can include any appropriate one or more VEGFR-2 Ig2 domains. A VEGFR-2 Ig2 domain that can be included in a VEGF antagonist polypeptide provided herein can be any appropriate length (e.g., can include any number of amino acids). In some cases, a VEGFR-2 Ig2 domain can be from about 102 amino acids in length to about 109 amino acids in length (e.g., can be from about 102 to about 107, can be from about 102 to about 105, can be from about 103 to about 109, can beAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0064] from about 105 to about 109, can be from about 105 to about 107, can be from about 103 to about 105, can be from about 105 to about 107, or can be about 106 amino acids in length).

[0065] A VEGFR-2 Ig2 domain that can be included in a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can have any appropriate amino acid sequence. In some cases, a VEGFR-2 Ig2 domain can comprise, consist essentially of, or consist of an amino acid sequence set forth in SEQ TDNO:2.

[0066] DYRSPFIASVSDQHGVVYITENKNKTVVIPCLGSISNLNVSLCARYPEKRF VPDGNRISWDSKKGFTIPSYMISYAGMVFCEAKINDESYQSIMYIVVVVG YRIYD (SEQ ID NO:2)

[0067] A VEGFR-2 Ig2 domain that consists essentially of an amino acid sequence set forth in SEQ ID NO:2 can include the amino acid sequence set forth in SEQ ID NO:2 with zero, one, or two amino acid substitutions within the articulated sequence of the sequence identifier (e.g., SEQ ID NO:2), with zero, one, two, three, four, or five amino acid residues preceding the articulated sequence of the sequence identifier (e.g., SEQ ID NO:2), and / or with zero, one, two, three, four, or five amino acid residues following the articulated sequence of the sequence identifier (e.g., SEQ ID NO:2), provided that the natriuretic peptide retains at least some VEGFR-2 Ig2 domain activity (e.g., the ability to bind VEGF-C (e.g., the ability to bind VEGF-C when paired with a VEGFR-2 Ig3 domain)).

[0068] In some cases, a VEGFR-2 Ig2 domain that can be included in a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 lg2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can have an amino acid sequence set forth in the NCBI databases at, for example, accession no. 35968 (version 35968.2).

[0069] A VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can include any appropriate one orAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0070] more VEGFR-2 Ig3 domains. A VEGFR-2 Ig3 domain that can be included in a VEGF antagonist polypeptide provided herein can be any appropriate length (e.g., can include any number of amino acids). In some cases, a VEGFR-2 Ig3 domain can be from about 94 amino acids in length to about 104 amino acids in length (e.g., can be from about 94 to about 102, can be from about 94 to about 100, can be from about 94 to about 98, can be from about 94 to about 96, can be from about 96 to about 104, can be from about 98 to about 104, can be from about 100 to about 104, can be from about 102 to about 104, can be from about 96 to about 102, can be from about 98 to about 100, can be from about 96 to about 98, can be from about 98 to about 100, can be from about 100 to about 102, or can be about 102 amino acids in length).

[0071] A VEGFR-2 Ig3 domain that can be included in a VEGF antagonist polypeptide provided herein (e g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can have any appropriate amino acid sequence. In some cases, a VEGFR-2 Ig3 domain can comprise, consist essentially of, or consist of an amino acid sequence set forth in SEQ IDNO:3.

[0072] VVL SP SHGIEL S VGEKLVLNCTARTELNVGIDFNWEYP S SKHQHKKLVNR DLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVH EK (SEQ ID NO:3)

[0073] A VEGFR-2 Ig3 domain that consists essentially of an amino acid sequence set forth in SEQ ID NO:3 can include the amino acid sequence set forth in SEQ ID NO:3 with zero, one, or two amino acid substitutions within the articulated sequence of the sequence identifier (e.g., SEQ ID NO:3), with zero, one, two, three, four, or five amino acid residues preceding the articulated sequence of the sequence identifier (e.g., SEQ ID NO:3), and / or with zero, one, two, three, four, or five amino acid residues following the articulated sequence of the sequence identifier (e.g., SEQ ID NO:3), provided that the natriuretic peptide retains at least some VEGFR-2 Ig3 domain activity (e.g., the ability to bind VEGF-C).Atorney Docket No. 44807-0507W01 / Pl 8657-02

[0074] In some cases, a VEGFR-2 Ig3 domain that can be included in a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can have an amino acid sequence set forth in the NCBI databases at, for example, accession no. 35968 (version 35968.2).

[0075] In some cases, a VEGFR-2 Ig3 domain that can be included in a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can be as described elsewhere (see, e.g., U.S. Patent No. 7,087,411).

[0076] A VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR- 1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can include any appropriate one or more VEGFR-1 Ig2 domains. In some cases, a Fc domain can include an IgGl hinge, CH2, and CH3. For example, a Fc domain can include a human IgGl hinge, CH2, and CH3.

[0077] A Fc domain that can be included in a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can be any appropriate length (e.g., can include any number of amino acids). In some cases, a Fc domain can be from about 226 amino acids in length to about 279 amino acids in length (e.g., can be from about 226 to about 270, can be from about 226 to about 260, can be from about 226 to about 250, can be from about 226 to about 240, can be from about 226 to about 230, can be from about 230 to about 279, can be from about 240 to about 279, can be from about 250 to about 279, can be from about 260 to about 279, can be from about 270 to about 279, can be from about 230 to about 270, can be from about 240 to about 260, can be from about 230 to about 250, can be from about 240 to about 260, can be from about 250 to about 270, or can be about 226 amino acids in length).

[0078] AFc domain that can be included in a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more FcAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0079] domains) can have any appropriate amino acid sequence. In some cases, a Fc domain can comprise, consist essentially of, or consist of an amino acid sequence set forth in SEQ ID NO:4.

[0080] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:4)

[0081] A Fc domain that consists essentially of an amino acid sequence set forth in SEQ ID NO:4 can include the amino acid sequence set forth in SEQ ID NO:4 with zero, one, or two amino acid substitutions within the articulated sequence of the sequence identifier (e.g., SEQ ID NO:4), with zero, one, two, three, four, or five amino acid residues preceding the articulated sequence of the sequence identifier (e.g., SEQ ID NO:4), and / or with zero, one, two, three, four, or five amino acid residues following the articulated sequence of the sequence identifier (e.g., SEQ ID NO:4), provided that the natriuretic peptide retains at least some Fc domain activity (e.g., the ability to enhance ligand binding of the VEGF antagonist polypeptide and / or extended serum half-life of the VEGF antagonist polypeptide). In some cases, a Fc domain that consists essentially of an amino acid sequence set forth in SEQ ID NO:4 can include an amino acid substitution at residue 14 (as numbered in SEQ ID NO:4). In some cases, a Fc domain that consists essentially of an amino acid sequence set forth in SEQ ID NO:4 can include an amino acid substitution at residue 15 (as numbered in SEQ ID NO:4). In some cases, a Fc domain that consists essentially of an amino acid sequence set forth in SEQ ID NO:4 can include an amino acid substitution at residue 109 (as numbered in SEQ ID NO :4).

[0082] In some cases, a Fc domain that can be included in a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can have an amino acid set sequence forth in the NCBI databases at, forAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0083] example, accession no. P01857 (version P01857.2), accession no. P01871 (version P01871.5), accession no. P01876 (version P01876.3).

[0084] In some cases, a Fc domain that can be included in a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can be as described elsewhere (see, e.g., U.S. Patent No. 7,087,411).

[0085] In some cases, two or more VEGFR Ig domains present in a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can be present in the form of a dimer. In some cases where two or more VEGFR Ig domains present in a VEGF antagonist polypeptide provided herein are present in the form of a dimer, the two or more VEGFR Ig domains can be multimerized using a multimerizing domain (e.g., a dimerizing domain). An example of a multimerizing domain that can be used to multimerize two or more VEGFR Ig domains is a leucine zipper domain, may be used to multimerize VEGFR Ig domains.

[0086] The components of a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can be presented in any appropriate order. In some cases, a VEGF antagonist polypeptide provided herein can include, from N-terminal to C-terminal: one or more VEGFR-1 Ig2 domains, followed by one or more VEGFR-2 Ig2 domains, followed by one or more VEGFR-2 Ig3 domains followed by one or more Fc domains. In some cases, a VEGF antagonist polypeptide provided herein can include, from N-terminal to C-terminal: one or more VEGFR-2 lg2 domains, followed by one or more VEGFR-2 lg3 domains, followed by one or more VEGFR-1 Ig2 domains, followed by one or more Fc domains.

[0087] In some cases, two consecutive components of a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can be directedly connected.Atorney Docket No. 44807-0507W01 / Pl 8657-02

[0088] In some cases, two consecutive components of a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can be connected via a linker. In some cases, a linker can provide the VEGF antagonist polypeptide provided herein with flexibility (e.g., to allow for better binding sterics for the target VEGF ligands). Examples of linkers that can be used to connect two consecutive components of a VEGF antagonist polypeptide provided herein include, without limitation, GGS linkers, glycine linkers, and VEGFR inter-domain linkers (e.g., naturally occurring VEGFR inter-domain linkers).

[0089] In some cases, a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can be a 12-Fc polypeptide (see, e.g., Table 1). For example, a VEGF antagonist polypeptide provided herein can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ IDNO:5.

[0090] In some cases, a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can be a 21-Fc polypeptide (see, e.g., Table 1). For example, a VEGF antagonist polypeptide provided herein can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ IDNO:6.

[0091] In some cases, a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 lg3 domains, and (d) one or more Fc domains) can inhibit (e.g., reduce or eliminate) VEGF activity. For example, a VEGF antagonist polypeptide provided herein can inhibit proliferation, migration, and / or survival of an endothelial cell (e.g., a human endothelial cell).

[0092] Any appropriate method can be used to obtain a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one orAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0093] more Fc domains). For example, a VEGF antagonist polypeptide provided herein can be obtained by synthesizing the polypeptide of interest using appropriate polypeptide synthesizing techniques.

[0094] This document also provides nucleic acid molecules that can encode a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains). For example, this document provides nucleic acid molecules encoding a VEGF antagonist polypeptide that comprises, consists essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:5. For example, this document provides nucleic acid molecules encoding a VEGF antagonist polypeptide that comprises, consists essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:6

[0095] In some cases, a nucleic acid that can encode a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can be in the form of a vector (e.g., a viral vector or a non-viral vector).

[0096] When a vector including a nucleic acid sequence that can encode a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) is a viral vector, any appropriate viral vector can be used. Examples viral vectors include, without limitation, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and lentiviral vectors.

[0097] When a vector that can encode a VEGF antagonist polypeptide provided herein (e g., a polypeptide that includes (a) one or more VEGFR-1 lg2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) is a non-viral vector, any appropriate non-viral vector can be used. In some cases, a non-viral vector can be an expression plasmid (e.g., a cDNA expression vector). In some cases, a non-viral vector can be a polymeric nanoparticle vector.

[0098] In addition to including a nucleic acid sequence that can encode a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2Atorney Docket No. 44807-0507W01 / Pl 8657-02

[0099] domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains), a vector (e.g., a viral vector or a non-viral vector) can contain one or more regulatory elements operably linked to the nucleic acid sequence that can encode a VEGF antagonist polypeptide provided herein. Such regulatory elements can include promoter sequences, enhancer sequences, response elements, signal peptides, internal ribosome entry sequences, polyadenylation signals, terminators, and inducible elements that modulate expression (e.g., transcription or translation) of a nucleic acid sequence encoding a VEGF antagonist polypeptide. The choice of regulatory element(s) that can be included in a vector depends on several factors, including, without limitation, inducibility, targeting, and the level of expression desired. For example, a promoter can be included in a vector to facilitate transcription of a nucleic acid sequence encoding a nucleoporin polypeptide. A promoter can be a naturally occurring promoter or a recombinant promoter. A promoter can be ubiquitous or inducible (e.g., in the presence of tetracycline), and can affect the expression of a nucleic acid sequence encoding a polypeptide in a general or tissue-specific manner. Examples of promoters that can be used to drive expression of a VEGF antagonist polypeptide provided herein in cells include, without limitation, CMV promoters, SV40 promoters (e.g., an SV40 early promoter region), and CMV immediate enhancer / p-actin (CAG) promoters. As used herein, “operably linked” refers to positioning of a regulatory element relative to a nucleic acid sequence encoding a polypeptide in such a way as to permit or facilitate expression of the encoded polypeptide. For example, a vector can contain a promoter and a nucleic acid sequence that can encode a VEGF antagonist polypeptide provided herein. In this case, the promoter is operably linked to a nucleic acid sequence that can encode a VEGF antagonist polypeptide provided herein such that it drives expression of the VEGF antagonist polypeptide in cells.

[0100] Nucleic acid sequences that can encode a VEGF antagonist polypeptide provided herein (e.g., a polypeptide that includes (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can be produced by techniques including, without limitation, common molecular cloning, polymerase chain reaction (PCR), chemical nucleic acid synthesis techniques, and combinations of such techniques. For example, PCR or RT-PCR can be usedAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0101] with oligonucleotide primers designed to amplify nucleic acid (e.g., genomic DNA or RNA) that can encode a VEGF antagonist polypeptide provided herein.

[0102] One or more VEGF antagonist polypeptides provided herein (e.g., one or more polypeptides (e.g., one or more chimeric polypeptides) that each include (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) and / or nucleic acid sequences designed to express one or more VEGF antagonist polypeptides provided herein can be formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal having, or at risk of developing, neovascularization (e.g., ocular neovascularization) and / or macular edema. For example, a therapeutically effective amount of one or more VEGF antagonist polypeptides provided herein (and / or nucleic acid sequences designed to express one or more VEGF antagonist polypeptides provided herein) can be formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. A pharmaceutical composition can be formulated for administration in solid or liquid form including, without limitation, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules.

[0103] A composition (e.g., a pharmaceutically acceptable composition) including one or more VEGF antagonist polypeptides provided herein (e.g., one or more polypeptides (e.g., one or more chimeric polypeptides) that each include (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) and / or nucleic acid sequences designed to express one or more VEGF antagonist polypeptides provided herein can be administered locally or systemically. A composition containing one or more VEGF antagonist polypeptides provided herein (and / or nucleic acid sequences designed to express one or more VEGF antagonist polypeptides provided herein) can be designed for, parenteral (including intravitreal, subretinal, and suprachoroidal) administration. For example, a composition containing one or more VEGF antagonist polypeptides provided herein (and / or nucleic acid sequences designed to express one or more VEGF antagonist polypeptides provided herein) can be administered locally by an intravitreal injection to a mammal (e.g., a human).Atorney Docket No. 44807-0507W01 / Pl 8657-02

[0104] This document also provides methods for using one or more VEGF antagonist polypeptides provided herein (e.g., one or more polypeptides (e.g., one or more chimeric polypeptides) that each include (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains). In some cases, mammal having, or at risk of developing, neovascularization (e.g., ocular neovascularization) can be administered or instructed to self-administer one or more (e g., one, two, three, four, or more) VEGF antagonist polypeptides. For example, one or more VEGF antagonist polypeptides can be administered to a mammal (e.g., a human) having, or at risk of developing, neovascularization to treat the mammal. In some cases, this document provides methods and materials for using one or more VEGF antagonist polypeptides to treat a mammal (e.g., a human) having a disease or disorder associated with ocular vascularization. For example, one or more VEGF antagonist polypeptides can be administered to a mammal (e.g., a human) having a disease or disorder associated with ocular vascularization to treat the mammal. In some cases, mammal having, or at risk of developing, macular edema can be administered or instructed to self-administer one or more (e.g., one, two, three, four, or more) VEGF antagonist polypeptides. For example, one or more VEGF antagonist polypeptides can be administered to a mammal (e.g., a human) having, or at risk of developing, macular edema to treat the mammal.

[0105] Any type of mammal having, or at risk of developing, neovascularization (e.g., ocular neovascularization) and / or macular edema can be treated as described herein. Examples of mammals that can be treated with one or more VEGF antagonist polypeptides provided herein (e.g., one or more polypeptides (e.g., one or more recombinant polypeptides) that each include (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 lg3 domains, and (d) one or more Fc domains) as described herein include, without limitation, humans, non-human primates (e.g., monkeys), dogs, cats, horses, cows, pigs, sheep, rabbits, mice, and rats. For example, a human having, or at risk of developing, neovascularization and / or macular edema can be treated with one or more VEGF antagonist polypeptides as described herein. In some cases, a human having, or at risk of developing, neovascularization and / or macular edema can have been previously treated with one or more anti-VEGF therapies such as ranibizumab (e.g., LUCENTIS®, CIMERLI®, andAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0106] ongavia), aflibercept (e.g., EYLEA® HD, yesafili, and opuviz), and bevacizumab (e.g., AVASTIN®). In some cases, a human having, or at risk of developing, neovascularization and / or macular edema that is treated with one or more VEGF antagonist polypeptides as described herein can be resistant to one or more anti-VEGF therapies such as ranibizumab (e.g., LUCENTIS®, CIMERLI®, and ongavia), aflibercept (e.g., EYLEA® HD, yesafili, and opuviz), and bevacizumab (e.g., AVASTIN®).

[0107] In some cases, the methods provided herein can include identifying a mammal (e.g., a human) as having, or at risk of developing, neovascularization (e.g., ocular neovascularization). Any appropriate method can be used to identify a mammal as having, or at risk of developing, ocular neovascularization. For example, imaging techniques

[0108] (e.g., fluorescein angiography, indocyanine green (ICG) angiography, optical coherence tomography (OCT), optical coherence tomography angiography (OCTA)andZor indirect ophthalmoscopy (e.g., a funduscopic examination) can be used to identify a human or other mammal as having ocular neovascularization.

[0109] When the methods and materials provided herein are used to treat a mammal (e.g., a human) having a disease or disorder associated with vascularization (e.g., ocular neovascularization), the mammal can have any type of disease or disorder associated with vascularization. Examples of diseases and disorders associated with ocular neovascularization include, without limitation, age-related macular degeneration (AMD; e.g., NVAMD), polypoidal choroidal vasculopathy, DR, ocular histoplasmosis, myopic degeneration, angioid streaks, choroidal neovascularization (e.g., idiopathic choroidal neovascularization), retinal neovascularization, and retinopathy of prematurity (ROP).

[0110] In some cases, the methods provided herein can include identifying a mammal (e.g., a human) as having, or at risk of developing, macular edema. Any appropriate method can be used to identify a mammal as having, or at risk of developing, macular edema. For example, OCT can be used to identify a human or other mammal as having macular edema.

[0111] When the methods and materials provided herein are used to treat a mammal (e g., a human) having macular edema, the mammal can have any type of macular edema. Examples of types of macular edema include, without limitation, diabetic macular edema and macular edema due to retinal vein occlusion.Atorney Docket No. 44807-0507W01 / Pl 8657-02

[0112] In some cases, one or more (e.g., one, two, three, four, or more) VEGF antagonist polypeptides provided herein (e.g., one or more polypeptides (e.g., one or more chimeric polypeptides) that each include (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) and / or nucleic acid sequences designed to express one or more VEGF antagonist polypeptides provided herein can be used to reduce the number of blood vessels (e.g., within one or both eyes of a treated mammal). For example, one or more VEGF antagonist polypeptides provided herein (and / or nucleic acid sequences designed to express one or more VEGF antagonist polypeptides provided herein) can be administered to a mammal (e.g., to one or both eyes of mammal such as a human) in need thereof (e.g., a human having neovascularization such as ocular neovascularization) to reduce the number of blood vessels within one or both eyes of the mammal. In some cases, the methods and materials described herein can be effective to reduce the number of blood vessels in one or both eyes of a mammal having ocular neovascularization by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.

[0113] In some cases, one or more (e.g., one, two, three, four, or more) VEGF antagonist polypeptides provided herein (e.g., one or more polypeptides (e.g., one or more chimeric polypeptides) that each include (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) and / or nucleic acid sequences designed to express one or more VEGF antagonist polypeptides provided herein can be used to reduce the amount of vascular permeability within one or both eyes of a treated mammal (e.g., thus reducing the amount of leakage into the macula within one or both eyes of a treated mammal). For example, one or more VEGF antagonist polypeptides provided herein (and / or nucleic acid sequences designed to express one or more VEGF antagonist polypeptides provided herein) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having macular edema) to reduce the amount of vascular permeability within one or both eyes of the mammal (e.g., to reduce the amount of leakage into the macula within one or both eyes of the mammal). In some cases, the methods and materials described herein can be effective to reduce the amount of vascular permeability within one or both eyes of the mammal (e.g., to reduce the amount of leakageAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0114] into the macula within one or both eyes of the mammal) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.

[0115] In some cases, one or more (e.g., one, two, three, four, or more) VEGF antagonist polypeptides provided herein (e.g., one or more polypeptides (e.g., one or more chimeric polypeptides) that each include (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) and / or nucleic acid sequences designed to express one or more VEGF antagonist polypeptides provided herein can be used to reduce the amount of fluid in the macula (e.g., within one or both eyes of a treated mammal). For example, one or more VEGF antagonist polypeptides provided herein (and / or nucleic acid sequences designed to express one or more VEGF antagonist polypeptides provided herein) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having macular edema) to reduce the amount of fluid in the macula within one or both eyes of the mammal. In some cases, the methods and materials described herein can be effective to reduce the amount of fluid in the macula of one or both eyes of a mammal having macular edema by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, the methods and materials described herein can be effective to reduce a central subfield thickness (e.g., as measured using a Heidelberg Spectralis SD-OCT) to less than about 315 pm.

[0116] An effective amount of a composition e.g., a pharmaceutical composition) containing one or more VEGF antagonist polypeptides provided herein (e.g., one or more polypeptides (e.g., one or more recombinant polypeptides) that each include (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) and / or nucleic acid sequences designed to express one or more VEGF antagonist polypeptides provided herein can be any amount that can treat a mammal (e.g., a human) without producing significant toxicity to the mammal. For example, an effective amount of one or more VEGF antagonist polypeptides provided herein can be from about 2 mg per dose to about 8 mg per dose (e.g., from about 2 mg to about 7 mg, from about 2 mg to about 6 mg, from about 2 mg to about 5 mg, from about 2 mg to about 4 mg, from about 2 mg to about 3 mg, from about 3 mg to about 8 mg, from about 4 mg to about 8 mg, from about 5 mg to about 8 mg, from about 6 mg to about 8 mg, from about 7 mg toAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0117] about 8 mg, from about 3 mg to about 7 mg, from about 4 mg to about 6 mg, from about 3 mg to about 5 mg, from about 4 mg to about 6 mg, or from about 5 mg to about 7 mg per dose). The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition may require an increase or decrease in the actual effective amount administered.

[0118] The frequency of administration of a composition (e.g., a pharmaceutical composition) containing one or more VEGF antagonist polypeptides provided herein (e.g., one or more polypeptides (e.g., one or more recombinant polypeptides) that each include (a) one or more VEGFR- 1 lg2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 lg3 domains, and (d) one or more Fc domains) and / or nucleic acid sequences designed to express one or more VEGF antagonist polypeptides provided herein can be any frequency that can treat a mammal (e.g., a human) without producing significant toxicity to the mammal. For example, the frequency of administration can be from about once every 4 weeks to once every 16 weeks or longer. The frequency of administration can remain constant or can be variable during the duration of treatment. A course of treatment with a composition containing one or more VEGF antagonist polypeptides provided herein (and / or nucleic acid sequences designed to express one or more VEGF antagonist polypeptides provided herein) can include rest periods. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition may require an increase or decrease in administration frequency.

[0119] An effective duration for administering a composition e.g., a pharmaceutical composition) containing one or more VEGF antagonist polypeptides provided herein (e.g., one or more polypeptides (e.g., one or more recombinant polypeptides) that each include (a) one or more VEGFR- 1 lg2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) and / or nucleic acid sequencesAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0120] designed to express one or more VEGF antagonist polypeptides provided herein can be any duration that treat a mammal (e.g., a human) without producing significant toxicity to the mammal. For example, the effective duration can vary from several weeks, to months, or years. In some cases, an effective duration for the treatment of a mammal (e.g., a human) having, or at risk of developing, neovascularization (e.g., ocular neovascularization) and / or macular edema can be the duration of the life of the mammal. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the condition being treated.

[0121] In some cases, one or more VEGF antagonist polypeptides provided herein (e.g., one or more polypeptides (e.g., one or more recombinant polypeptides) that each include (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) and / or nucleic acid sequences designed to express one or more VEGF antagonist polypeptides provided herein can be used as the sole active agent(s) to treat a mammal (e.g., a human) having, or at risk of developing, neovascularization (e.g., ocular neovascularization) and / or macular edema.

[0122] In some cases, one or more VEGF antagonist polypeptides provided herein (e.g., one or more polypeptides (e.g., one or more recombinant polypeptides) that each include (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) and / or nucleic acid sequences designed to express one or more VEGF antagonist polypeptides provided herein can be administered to a mammal (e.g., a human) having, or at risk of developing, neovascularization (e.g., ocular neovascularization) together with one or more (e.g., one, two, three, four, or more) additional agents / therapies used to treat neovascularization (or to treat one or more symptoms of neovascularization). In some cases, an agent that can be used to treat neovascularization (or to treat one or more symptoms of neovascularization) can be anti-VEGF agent. Examples of agents that can be used to treat neovascularization (or to treat one or more symptoms of neovascularization) include, without limitation, bevacizumab (e.g., AVASTIN®), ranibizumab (e.g., LUCENTIS®, CIMERLI®, and ongavia), aflibercept (e.g., EYLEA® HD, yesafdi, and opuviz), , and any combinations thereof. In cases where one orAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0123] more VEGF antagonist polypeptides provided herein (and / or nucleic acid sequences designed to express one or more VEGF antagonist polypeptides provided herein) are used in combination with one or more additional agents used to treat neovascularization (or to treat one or more symptoms of neovascularization), the one or more additional agents can be administered at the same time (e.g., in a single composition containing one or more VEGF antagonist polypeptides provided herein and the one or more additional agents) or independently. For example, one or more VEGF antagonist polypeptides provided herein (and / or nucleic acid sequences designed to express one or more VEGF antagonist polypeptides provided herein) can be administered first, and the one or more additional agents administered second, or vice versa. An example of a therapy that can be used to treat retinal neovascularization is panretinal scatter photocoagulation. Examples of therapies that can be used to treat choroidal neovascularization include, without limitation, photodynamic therapy and photobiomodulation. In cases where one or more VEGF antagonist polypeptides provided herein (and / or nucleic acid sequences designed to express one or more VEGF antagonist polypeptides provided herein) are used in combination with one or more additional therapies used to treat neovascularization (or to treat one or more symptoms of neovascularization), the one or more additional therapies can be performed at the same time or independently of the administration of one or more VEGF antagonist polypeptides provided herein (and / or nucleic acid sequences designed to express one or more VEGF antagonist polypeptides provided herein). For example, one or more VEGF antagonist polypeptides provided herein (and / or nucleic acid sequences designed to express one or more VEGF antagonist polypeptides provided herein) can be administered before, during, or after the one or more additional therapies are performed.

[0124] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.Atorney Docket No. 44807-0507W01 / Pl 8657-02

[0125] EXAMPLES

[0126] Example 1: Engineered bispecific receptor decoy polypeptides enhance inhibition of ocular neovascularization through simultaneous blockade of vascular endothelial growth factor ligands A and C

[0127] This Example describes the design and generation of receptor decoy traps that serve as bispecific antagonist polypeptides capable of binding all VEGFR-1 and VEGFR-2 ligands, including VEGF-A and VEGF-C. This Example also demonstrates that such engineered bispecific receptor decoy polypeptides can block angiogenesis and can be used to treat neovascularization such as ocular neovascularization.

[0128] RESULTS

[0129] Engineering bispecific VEGF receptor decoy polypeptides

[0130] To generate bi specific receptor decoy polypeptides which serve as decoys that trap both VEGF-A and VEGF-C, the immunoglobulin (Ig)-like domain 2 of human VEGFR-1 (which engages all VEGF-A isoforms, VEGF-B, P1GF-1, and P1GF-2) was combined with the Ig-like domains 2-3 of human VEGFR-2 (which collectively engage all VEGF-A isoforms, VEGF-C, and VEGF-D) (Figure IB). These receptor fragments were dimerized through fusion to the Fc domain of a human IgGl antibody. Two fusion polypeptides were designed: one with the VEGFR-1 binding domain at the N-terminal end of the protein (12-Fc); and one with the VEGFR-2 binding domains at the N-terminal end of the protein (21-Fc) (Figures 1C and 7, Table 1). In both geometries, the receptor decoys were designed to have a 2:1 binding stoichiometry of VEGF ligand to receptor decoy, allowing each protein to bind and inhibit two dimeric VEGF ligands at a time.

[0131] Table 1. Amino acid sequences of recombinantly produced vascular endothelial growth factor ligands and receptor decoy polypeptides.

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[0141] Fusion proteins were expressed recombinantly through transient transfection of human embryonic kidney (HEK) 293F cells and purified via protein G affinity chromatography followed by size-exclusion chromatography (SEC). Both 12-Fc and 21-Fc eluted from SEC as monodisperse peaks with only a small aggregation shoulder, similar to the FDA-approved drug aflibercept (Figure ID). Under reducing SDS-PAGE conditions, the purified receptor decoy proteins showed diffuse band patterns and a slightly greater-than- expected molecular weight, two characteristic patterns of glycosylated proteins. When run using nonreducing conditions, receptor decoy proteins were observed as dimers due to disulfide bonding between the Fc monomers (Figure IE).

[0142] Bispecific receptor decoy polypeptides bind all VEGFR-1 and VEGFR-2 ligands

[0143] The capacity of aflibercept and the engineered receptor decoy polypeptides to bind VEGF ligands in vitro was interrogated via biolayer interferometry titrations (Figure 2A, Table 2). VEGF-E, which is only expressed in the context of viral infection and is not known to contribute to ocular neovascularization, was excluded from these characterization studies. Binding properties were compared to those of the Fc-fused parental VEGFR-1 D23 and VEGFR-2 D23 receptor fragments for context (Figure 9, Table 3).

[0144] Table 2. Equilibrium and kinetic binding parameters for receptor decoy protein interactions with vascular endothelial growth factor ligands, as measured by biolayer interferometry.

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[0152] Table 3. Binding parameters for VEGFR-1 D23 and VEGFR-2 D23 with multiple vascular endothelial growth factor ligands, as measured by biolayer interferometry.

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[0156]

[0157] Both 12-Fc (equilibrium dissociation constant (KD)=3.3 nM) and 21-Fc (KD=4.2 nM) bound VEGF-A165 with similar affinities to aflibercept (KD=2.3 nM) (Figures 2B and 8, Table 2). These affinities were also in agreement with those of VEGFR-1 D23 Fc (2.04 nM) and VEGFR-2 D23 Fc (2.5 nM) (Figures 9 A, 9H, and 91, Table 3). Similarly, 12-Fc (KD=5.04 nM), 21-Fc (KD=7.41 nM), and aflibercept (KD=2.07 nM) showed nearly identical affinities for the VEGF-Am isoform (Figures 2C and 8, Table 2), which were slightly tighter than those of the Fc-fused parental VEGFR-1 D23 (KD=21.2 nM) and VEGFR-2 D23 (KD=12.5 nM) receptor fragments (Figures 9B, 9H, and 91, Table 3). Additionally, the VEGF-B binding affinities of 12-Fc (KD=18.4 nM) and 21-Fc (KD=30.5 nM) were virtually identical to that of aflibercept (KD=16.5 nM) (Figures 2D and 8, Table 2). VEGFR-1 D23 Fc bound to VEGF-B with similar affinity compared to the receptor decoy polypeptides (KD=18.9 nM), whereas VEGFR-2 D23 showed weaker binding to VEGF-B (KD=148 nM) (Figures 9C, 9H, and 91, Table 3). Notable differences in affinity between the engineered receptor decoy polypeptides and aflibercept were observed for P1GF ligands. 12-Fc showed ~10-fold lower P1GF-1 (KD=21.8 nM) and P1GF-2 (KD=26.8), and 21-Fc showed ~100-fold lower P1GF-1 (KD=211 nM) and P1GF-2 (KD=148 nM) binding compared to afliberceptAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0158] affinity for P1GF-1 (KD=5.33 nM) and P1GF-2 (KD=2.59 nM) (Figures 2E, 2F, and 8, Table 2). As anticipated, the parental VEGFR-1 D23 Fc showed similar P1GF-1 (KD=4.48 nM) and P1GF-2 (KD=5.75 nM) affinities to those of aflibercept, whereas VEGFR-2 D23 Fc showed very weak binding to P1GF-1 (KD=646 nM) and P1GF-2 (KD>1000 nM) (Figures 9D, 9E, 9H, and 91, Table 3). Although the second Ig-like binding domain of VEGFR-1 constitutes the minimal binding domain for all VEGFR-1 ligands, this domain alone has been shown to bind P1GF-1 and P1GF-2250-fold weaker than VEGFR-1 D2-3 (Christinger, J Biol Chem 279:10382-10388 (2004)).

[0159] Importantly, whereas aflibercept did not bind to VEGF-C (Papadopoulos, Angiogenesis 15:171-185 (2012); and Cursi efen, J Clin Invest 113:1040-1050 (2004)), both 12-Fc and 21-Fc demonstrated high affinity binding to VEGF-C, with KD values of 12.9 nM and 3.5 nM, respectively (Figures 2G and 8, Table 2), validating the design approach. The parental VEGFR-2 D23 Fc showed a KD value of 4.02 nM for VEGF-C, slightly lower affinity than 12-Fc and slightly higher affinity than 21-Fc, and VEGFR-1 D23 Fc did not bind to VEGF-C (Figures 9F, 9H, and 91, Table 3). Similarly, whereas aflibercept did not bind to VEGF-D (Cursiefen, J Clin Invest 113:1040-1050 (2004)) 12-Fc and 21-Fc showed binding to VEGF-D (KD>1000 nM for both constructs), albeit much less potent compared to VEGF-D binding (Figures 2H and 8, Table 2). The parental VEGFR-2 D23 Fc bound VEGF-D with a KD value of 35.14 nM and VEGFR-1 D23 Fc did not bind to VEGF-C (Figures 9G, 9H and 91, Table 3).

[0160] Consistently across ligand binding characterization assays, it was observed that higher affinities were observed when the binding domain implicated in binding a ligand was at the N-terminal end of the protein rather than proximal to the Fc (e.g., 12-Fc bound tighter to the VEGFR-1 -binding P1GF-1 whereas 21-Fc bound tighter to the VEGFR-2-biding VEGF-C). Thus, the distinct geometries of the 2 engineered receptor decoy polypeptides may lead to differences in binding site accessibility and consequent differences in ligand affinities. Overall, binding studies demonstrated that, in contrast with aflibercept, the 2 engineered receptor decoy polypeptides engage all VEGFR-1 and VEGFR-2 ligands.

[0161] Bispecific receptor decoy polypeptides bind two ligands simultaneously and compete withAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0162] VEGF receptors for ligand binding

[0163] The ability of 12-Fc and 21-Fc to engage 2 ligands simultaneously was interrogated using biolayer interferometry -based competition assays. In one configuration, titrated amounts of VEGF-C were pre-incubated with a constant amount of the engineered bispecific receptor decoy polypeptides to occupy the VEGFR-2 D23 binding domain. These complexes were then interrogated for their ability to bind immobilized VEGF-A through the available VEGFR-1 D2, normalized to the binding of the receptor decoy protein alone. Both receptor decoy polypeptides simultaneously bound VEGF-A and VEGF-C, although there was a significant difference in the maximum signal observed, with 21-Fc reaching a higher maximum response (Rmax) than 12-Fc (Figure 3A, Table 4). Simultaneous binding was also observed when VEGFR-1 D2 was pre-blocked with P1GF-1 and binding of immobilized VEGF-C through the available VEGFR-2 D23 was measured. In this orientation, 12-Fc reached a higher Rmax than 21-Fc (Figure 3B, Table 4). Collectively, these simultaneous binding experiments revealed that when the N-terminal binding domain is occupied, the Fc- proximal binding domain of the bispecific receptor decoy protein becomes more accessible, allowing for greater extent of binding to the second ligand. This observation suggests that potential positive cooperativity between the binding sites within the receptor decoy protein in certain molecular geometries.

[0164] Table 4. Binding parameters for receptor decoy protein interactions with multiple vascular endothelial growth factor ligands, as measured by biolayer interferometry.

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[0167] To confirm that 12-Fc and 21-Fc successfully blocked the interaction of the two main angiogenic ligands, VEGF-A and VEGF-C, with full-length VEGF receptors, biolayerAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0168] interferometry -based competition studies were performed that measured ligand / receptor binding in the presence of the engineered receptor decoy polypeptides. It was found that both engineered receptor decoy polypeptides, like aflibercept, prevented VEGF-A from interacting with VEGFR-2 (Figure 3C, Table 5) and VEGFR-1 (Figure 10A, Table 5).

[0169] Moreover, the engineered receptor decoy polypeptides, but not aflibercept, blocked VEGF-C from interacting with VEGFR-2 (Figure 3D, Table 5) and VEGFR-3 (Figure 10B, Table 5). Notably, the potency of the bispecific receptor decoy polypeptides and aflibercept were identical for all ligand / receptor competition studies, revealing the robust inhibitory effects of the engineered receptor decoy polypeptides.

[0170] Table 5. Competitive binding parameters for receptor decoy protein interactions with vascular endothelial growth factor ligands, as measured by biolayer interferometry.

[0171]

[0172] Bispecific receptor decoy polypeptides inhibit key angiogenic signaling pathways VEGF ligands, particularly VEGF-A and VEGF-C, promote the proliferation, migration, and survival of endothelial cells through activation of VEGFR-2 and downstream signaling pathways. To investigate the effectiveness of the engineered receptor decoy polypeptides, VEGFR-2 signaling pathways were examined in human microvascular endothelial cells (HMECs). HMECs were chosen as a model system over the more commonly used human umbilical vein endothelial cells (HUVECs) due to their higher responsiveness to VEGF-C in cell survival assays (Figure 11). Both 12-Fc and 21-Fc inducedAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0173] equivalent inhibition of VEGF-A-induced phosphorylation of VEGFR-2 and downstream signaling effectors protein kinase B (Akt, in the phosphoinositide 3 -kinase (PI3K) pathway) and extracellular signal-regulated protein kinase (ERK1 / 2, in the mitogen-activated kinase (MAPK) pathway) compared to aflibercept, reducing activity to basal levels (Figure 4A). By contrast, only 12-Fc and 21-Fc inhibited VEGF-C-induced phosphorylation of VEGFR-2, Akt, and ERK1 / 2 (Figure 4B), demonstrating their superior breadth of inhibition compared to the clinically approved drug aflibercept.

[0174] To explore whether the bispecific receptor decoy polypeptides can inhibit synergistic activity between multiple VEGF ligands, fixed, subsaturating concentrations of VEGF-A and VEGF-C were administered and the antagonistic activities of the engineered receptor decoys was evaluated. Treatment of HMECs showed with both VEGF-A and VEGF-C led to greater phosphorylation of signaling molecules compared to treatment with either ligand individually, and 12-Fc and 21-Fc but not aflibercept reduced signaling to basal levels (Figure 4C). Taken together, these results indicate that 12-Fc and 21-Fc inhibit angiogenic signaling pathways activated by VEGF-A, VEGF-C, or a combination of the 2 ligands in human endothelial cells.

[0175] Bispecific receptor decoy polypeptides inhibit endothelial cell proliferation, migration and survival

[0176] Having demonstrated the inhibitory activities of 12-Fc and 21-Fc on VEGF ligand / receptor interactions and signaling, it was sought to establish that the bispecific receptor decoy polypeptides blocked the pro-angiogenic activities of VEGF ligands in various functional assays. Saturating doses for VEGF ligands were determined by generating dose response curves for proliferation, migration, and survival in the absence of any added inhibitor (Figures 1 IB and 12). VEGF ligands were then administered at the determined doses in the presence of receptor decoy polypeptides (either 12-Fc, 21-Fc, or aflibercept).

[0177] In HMEC proliferation assays, both the engineered receptor decoy polypeptides and aflibercept inhibited VEGF-A-induced proliferation with low nanomolar half-maximal inhibitory concentration (ICso) values (Figures 5A and 13A-13C left, Table 6). However, only the engineered receptor decoy polypeptides, and not aflibercept, inhibited VEGF-C-Attorney Docket No. 44807-0507W01 / Pl 8657-02

[0178] driven proliferation, again exhibiting low nanomolar IC50 values (Figures 5 A and 13A-13C right, Table 6). Similarly, whereas all 3 tested VEGF receptor decoy polypeptides potently (low nanomolar IC50 values) inhibited VEGF-A-induced HMEC migration (Figures 5B and 14A-14C left, Table 6), only the engineered receptor decoys inhibited VEGF-C-induced HMEC migration (Figures 5B and 14A-14C right, Table 6). HMEC survival studies demonstrated a similar trend, wherein 12-Fc, 21-Fc, and aflibercept all potently (low nanomolar IC50 values) inhibited VEGF-A-induced survival, but only 12-Fc and 21-Fc inhibited VEGF-C-driven survival (Figure 5C, Table 6). Across these functional studies, 12- Fc and 21-Fc showed similar potency, in agreement with their equally potent competition with VEGF / VEGFR interactions (Figures 3C, 3D, 9, and Table 5).

[0179] Table 6. Fitted inhibition parameters for receptor decoy protein inhibition of human microvascular endothelial cell proliferation, migration, and survival.

[0180] >

[0181] >

[0182] >

[0183]

[0184] Atorney Docket No. 44807-0507W01 / Pl 8657-02

[0185]

[0186] It was also examined whether the bispecific receptor decoy polypeptides could inhibit HMEC proliferation and survival driven by a combination of VEGF-A and VEGF-C. For both proliferation (Figure 5D, Table 6) and survival (Figures 5E and 15A-15C, Table 6) studies, aflibercept only partially inhibited ligand-driven response, whereas 12-Fc and 21-Fc mediated significantly more inhibition through blockade of both VEGF-A and VEGF-C. Interestingly, despite less complete inhibition of proliferation and survival, aflibercept exhibited a lower ICsothan the engineered receptor decoy polypeptides. This finding was consistent with the more potent competition observed with VEGF-A versus VEGF-C for VEGFR-2 binding (Figures 3C and 3D, Table 5) and the more potent inhibition of proliferation and survival responses driven by VEGF-A versus VEGF-C independently (Figures 5A and 5C, Table 6). Overall, cell-based assays demonstrated the powerful anti- angiogenic effects of the engineered receptor decoy polypeptides and highlighted their broader spectrum inhibition of VEGF ligands compared to the clinical therapeutic aflibercept.

[0187] Bispecific receptor decoy polypeptides demonstrate minimal binding to extracellular matrix In order to translate the in vitro findings into in vivo models of neovascularization, key properties associated with bioavailability were characterized. The isoelectric point (pl) of a protein serves as an indicator of the pharmacokinetic profile and thus bioavailability of a protein. Protein therapeutics exhibiting a high pl (i.e., carrying a large positive charge), exhibit poor bioavailability due to nonspecific interactions with negatively charged proteoglycans in the extracellular matrix (ECM), which result in deposition of protein near the injection site. The binding domains of VEGFR-1, particularly Ig-like domain 3, exhibit very high positive charges (pl=9.4), which has hampered the development of VEGFR-1 - based antagonist polypeptides. The FDA-approved drug aflibercept has a much lower pl (8.0), and whereas 12-Fc and 21-Fc have a theoretical pl value of 8.5. The engineeredAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0188] receptor decoy polypeptides showed comparable pl to aflibercept when run on an isoelectric focusing (IEF) gel. By contrast, an Fc-fused chimera of VEGFR-1 domains 1-3 (denoted VEGFR-1 Dl-3 Fc) was poorly resolved in a streak at a pl greater than 8.2 (Figure 16A).

[0189] To confirm these results, an enzyme-linked immunosorbent assay (ELISA) was performed to assess ECM binding to 12-Fc, 21-Fc, aflibercept, VEGFR-1 Dl-3 Fc, and VEGFR-2 D23 Fc (Figure 16B, top). In accordance with the observed pl values, VEGFR-1 Dl-3 Fc bound nonspecifically to ECM, whereas minimal binding was observed for the engineered receptor decoy polypeptides, aflibercept, and VEGFR-2 D23 Fc (Figure 16B, bottom). Collectively, these studies demonstrated that 12-Fc and 21-Fc exhibit similarly low ECM binding to the approved drug aflibercept, motivating in vivo investigation of these engineered receptor decoy polypeptides.

[0190] Engineered bispecific receptor decoy polypeptides significantly reduce angiogenesis in vivo, outperforming current clinical antagonist protein

[0191] Motivated by the promising anti-angiogenic activity of the engineered VEGF receptor decoy polypeptides in cellular models, it was sought to evaluate the efficacy of the novel molecules in vivo. Motivated by the promising anti -angiogenic activity of the engineered VEGF receptor decoy polypeptides in cellular models, it was sought to evaluate the efficacy of the novel molecules in vivo. Human VEGFR-1 and VEGFR-2 show ~80% amino acid sequence identity to their murine counterparts and binding studies with aflibercept have demonstrated its cross-reactivity with mouse VEGF ligands (Papadopoulos, Angiogenesis 15:171-185 (2012)). It was therefore hypothesized that the engineered receptor decoy polypeptides would also cross-react with murine ligands, most importantly VEGF-A and VEGF-C. It was observed near-identical affinities of the engineered receptor decoy polypeptides for the human and mouse VEGF-Aiss and VEGF-C (Figures 17A-17E, Table 2), enabling exploration of the molecules in mouse models of ocular neovascularization.

[0192] To this end the ability of 12-Fc and 21-Fc to inhibit neovascularization in a murine model of oxygen-induced retinopathy (OIR) was interrogated. This model is relevant to retinopathy of prematurity and diabetic retinopathy, ischemic retinopathies in humans.

[0193] VEGF- A and VEGF-C have both been shown to play prominent roles in the development ofAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0194] neovascularization and OIR in mice, rendering this model an excellent system to investigate the benefit of the new treatments that simultaneously inhibit both VEGF ligands. On day 7 post-birth (P7), litters of mice and mothers were placed in a closed chamber containing 75% oxygen. On P12, mice were returned to room air, and pups were given an intravitreous injection of VEGF antagonist protein in one eye and saline control in the fellow eye. Mice were euthanized 5 days later, and retinas were dissected and briefly stained with a fluorescent lectin. This brief incubation with the lectin stains endothelial cells in new vessels and hyaloid vessels but not pre-existent retinal vessels reducing background and allowing precise measurement of the area of neovascularization, which was normalized to retinal area.

[0195] Aflibercept was titrated in the OIR mouse model in order to determine a dose at which there was no statistically significant suppression of retinal neovascularization. In untreated mice with OIR, there was modest variability in area of retinal neovascularization between mice, but little variability between eyes in the same mouse; therefore all comparisons were made between eyes injected with antagonist protein and contralateral control eyes. Aflibercept significantly decreased neovascularization area at higher doses but not at the 1 pg dose; thus, aflibercept was compared to the engineered bispecific receptor decoy polypeptides at the 1 pg dose (Figure 18). Eyes injected with aflibercept at this dose had no significant difference in mean area of retinal neovascularization compared with saline-injected fellow eye controls (Figure 6A). However, eyes injected with 1 pg 12-Fc showed a dramatic reduction in both the percentage of neovascularization in the retina and the mean area of retinal neovascularization compared with fellow eye controls (p<0.01) (Figures 6B, 6D, and 19A). Eyes injected with 1 pg 21-Fc showed similar results, with significantly reduced percentage of neovascularization in the retina and mean area of neovascularization relative to fellow eye controls (p<0.01) (Figures 6C, 6E, and 19B).

[0196] To further probe the potential clinical value of the engineered receptor decoy polypeptides, their performance in a murine model of CNV, which is relevant to patients with NV AMD, was investigated. Six to eight- week-old C57BL / 6I mice were 6 to 8-week subjected to laser injury of Bruch’s membrane to induce neovascularization, and mice were treated with intraocular injections of receptor decoy polypeptides in one eye and saline in the fellow eye. Mice were euthanized 7 days later, and eyes were dissected and stained with aAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0197] fluorescent lectin (Figures 6F-6I). It was found that 12-Fc and 21-Fc performed strikingly better than aflibercept in this model as well; whereas the mean area of CNV was similar in the eyes injected with aflibercept compared with saline-injected fellow eyes, eyes injected with the engineered receptor decoys showed significant reduction in mean area of CNV compared with saline-injected fellow eyes (p< 0.001) (Figure 6J).

[0198] Altogether, these findings demonstrate that the increased anti-angiogenic activity of the bispecific VEGF receptor decoy polypeptides led to more effective suppression of pathological neovascularization compared to a standard-of-care antagonist protein in a well-validated animal models of OIR and CNV.

[0199] Together, these results demonstrate that VEGF antagonist polypeptides provided herein (e.g., VEGF antagonist polypeptides that each include (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains) can simultaneously target both VEGFR-1 and VEGFR-2 ligands (e.g., can simultaneously target VEGF-A and VEGF-C) and can inhibit ocular neovascularization. These results also demonstrate that such VEGF antagonist polypeptides can be used to treat eye diseases associated with ocular neovascularization, including NVAMD and DR.

[0200] METHODS

[0201] Cell lines

[0202] HEK293-F cells (Thermo Fisher Scientific) were cultivated in Freestyle 293 Expression Medium (Thermo Fisher Scientific) supplemented with 0.2 U / mL penicillinstreptomycin (Gibco). Human adult dermal microvascular endothelial cells (Lifeline Cell Technologies) were cultivated in EGM-2MV media (Lonza). HMECs were cultured in T75 flasks coated with 0.1% gelatin in PBS solution. All cell lines were maintained at 37°C in a humidified atmosphere with 5% CO2. HEK 293F cells were rotated continuously at 125 rpm.Atorney Docket No. 44807-0507W01 / Pl 8657-02

[0203] Protein expression and purification

[0204] Human VEGF-A165 (Sequence ID P15692-4: amino acids 27-191) and VEGF-C (Sequence ID P49767: amino acids 103-227), were expressed and purified using a human embryonic kidney cell (HEK) 293F expression system (Thermo Fisher Scientific). Genes encoding VEGF-A165 and VEGF-C with an N-terminal hexahistidine tag followed by a GGS linker were cloned into the gWiz mammalian expression vector (Genlantis) and constructs were verified by sequence analysis.

[0205] A total of 1 mg of maxi-prepped (Qiagen), endotoxin-free plasmid DNA and 615 mg of poly(beta-amino ester), 4-4-6, per L of cells were independently diluted to 0.05 and 3 mg / mL in 25 mM magnesium acetate buffer (Sigma- Aldrich) pH 5.2 respectively, then combined by vortexing and incubated at room temperature for 15 minutes. The resulting DNA / 4-4-6 nanoparticles were subsequently added dropwise to the diluted HEK 293-F cells in a shaking flask and the HEK293F cells were incubated, with orbital shaking at 125 rpm, at 37°C with 5% CO2. Transfected cells were harvested after 5 days and secreted protein was captured from the supernatant via Ni-NTA (Expedeon) agarose affinity chromatography. The growth factors were further purified to >98% homogeneity with a Superdex 200 sizing column on a fast protein liquid chromatography (FPLC) instrument (Cytiva Life Sciences) equilibriated in HEPES-buffered saline (HBS, 150 mM NaCL in 10 mM HEPES pH 7.3). Size and purity (>99%) were verified by SDS-PAGE analysis.

[0206] Aflibercept (sequence obtained from drugbank.ca), engineered decoy polypeptides, VEGFR-1 D23 Fc, and VEGFR-2 D23 were expressed through transient transfection of HEK 293F cells, as described above for the VEGF growth factors. For aflibercept, VEGFR-1 domain 2 (amino acids 129-231) were fused to VEGFR-2 domain 3 (amino acids 226-327) followed by the human IgGl Fc domain. For 12-Fc and 21-Fc, the same amino acid cutoffs were used for their VEGFR-1 (P17948-1, amino acids 132-225 and VEGFR-2 (P35968-1, amino acids 119-327) domain 1 and domain 2 / 3 binding domains respectively. For 12-Fc, VEGFR-1 D2 was fused N-terminally to VEGFR-2 D23, which was fused to the IgGl Fc domain. For 21-Fc, VEGFR2 D23 was fused N-terminally to VEGFR1 D2, which was fused to the IgGl Fc domain. As receptor controls, VEGFR-1 D23 (P17948-1, amino acids 129-330) and VEGFR-2 D23 (P35968-1, amino acids 119-327) were similarly fused to IgGl Fc.Atorney Docket No. 44807-0507W01 / Pl 8657-02

[0207] P329 and L234A / L235A (LALA) mutations were introduced into all Fc-fused receptor decoy constructs to prevent Fc effector activity. All sequences are provided in Table 1.

[0208] Secreted polypeptides were captured from the supernatant via Protein G agarose (Thermo Scientific) affinity chromatography, then purified using an FPLC as above.

[0209] Biolayer interferometry binding studies

[0210] Binding of VEGF receptor decoy polypeptides to VEGF-A165 (produced in-house), VEGF-A121 (Peprotech), VEGF-C (produced in-house), P1GF-1 (Peprotech), P1GF-2 (R&D Systems), VEGF-B (Aero biosystems), VEGF-D (Peprotech), murine VEGF-A165 (Fujifilm) and murine VEGF-C (Kingfisher Biotech) was measured on an OctetRED96 system (Sartorius). All polypeptides were diluted in lx PBS pH 7.2 containing 0.1% BSA (PBSA) and filtered through a 0.45 um filter. 12-Fc, 21-Fc, and Aflibercept (100 nM) were immobilized on anti-human capture (AHC) biosensors (Sartorius) for 120 seconds and baseline measurements were generated in PBSA. Binding kinetics were measured by submerging coated biosensors into serially diluted VEGF ligands for 300 seconds (association), followed by PBSA for 300 seconds (dissociation). Biosensors were regenerated in 0.1 M glycine pH 2.75 in between analytes, and antagonists were re-loaded onto the biosensors for each analyte. Curves were fitted using the Octet Data Analysis HT Software version 7.1 (Sartorius) assuming a 1:1 binding model to determine the association rates, dissociation rates, and equilibrium dissociation constant (KD) values. Experiments were repeated twice with similar results.

[0211] To assess whether the VEGF receptor decoy polypeptides were competitive with full-length VEGF receptors for VEGF-A / VEGF-C binding, biotinylated full-length VEGFR-1. VEGFR-2, and VEGFR-3 extracellular domains (Sino Biological) were loaded onto streptavidin biosensors in 0.45 pm filtered PBSA (phosphate-buffered saline containing 0.1% BSA). Competitor VEGF antagonist polypeptides were serially diluted into PBSA containing saturating concentrations of either VEGF-A or VEGF-C (100 nM) and incubated at room temperature for 30 minutes. Binding kinetics were measured by submerging biosensors in these mixtures for 300 seconds (association) and subsequently into wells containing only PBSA for 300 seconds (dissociation). Tips were regenerated in 0.1 M glycine pH 2.7. ExtentAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0212] of binding in presence of the competitor polypeptides were determined by total response measured after 295 seconds of association. Signal was normalized to that of the VEGF-A or VEGF-C only samples. Data were analyzed in Prism software (GraphPad) using a three-parameter inhibitor versus response regression and half maximal inhibitory concentration (ICso) values were determined where applicable. Experiments were repeated twice with similar results.

[0213] To assess the ability of the engineered VEGF receptor decoy polypeptides to simultaneously bind two ligands, biotinylated VEGF-A or VEGF-C (100 nM) were immobilized on streptavidin biosensors (Sartorius) in 0.45 pm filtered PBSA (phosphate-buffered saline containing 0.1% BSA).

[0214] To determine whether the VEGF receptor decoy polypeptides could bind a second ligand if the VEGFR-2-based binding domain was occupied first, the streptavidin tips loaded with VEGF-A were used. VEGF-C (produced in-house) was serially diluted into PBSA containing saturating concentration of each of the VEGF antagonists (100 nM) and incubated at room temperature for 30 minutes to allow for specific pre-blocking of VEGFR-2 D2-3. Binding kinetics were measured by submerging biosensors in these mixtures for 300 seconds (association) and subsequently into wells containing only PBSA for 300 seconds (dissociation). Tips were regenerated in 0.1 M glycine pH 2.7. Extent of binding in presence of the VEGF antagonist polypeptides was determined by total response measured after 295 seconds of association. Signal was normalized to that of the antagonist-only samples. Data were analyzed in Prism software (GraphPad) using a sigmoidal dose-response curve.

[0215] Experiments were repeated twice with similar results.

[0216] To determine whether the VEGF receptor decoy polypeptides could bind a second ligand if the VEGFR-1 -based binding domain was occupied first, the streptavidin tips loaded with VEGF-C were used. P1GF-1 (produced in-house) was serially diluted into PBSA containing saturating concentration of each of the VEGF antagonists (300 nM) and incubated at room temperature for 30 minutes to allow for specific pre-blocking of VEGFR-1 D2. Binding kinetics were measured by submerging biosensors in these mixtures for 300 seconds (association) and subsequently into wells containing only PBSA for 300 seconds (dissociation). Tips were regenerated in 0.1 M glycine pH 2.7. Extent of binding in presenceAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0217] of the VEGF antagonist polypeptides was determined by total response measured after 295 seconds of association. Signal was normalized to that of the antagonist-only samples. Data were analyzed in Prism software (GraphPad) using a sigmoidal dose-response curve.

[0218] Experiments were repeated twice with similar results.

[0219] Immunoblotting

[0220] HMECs were seeded in 6-well plates and grown to confluence in EGM-2MV (Lonza). Confluent cells were serum starved in EBM-2 (Lonza) with 0.1% FBS for 3 hours, then were washed with PBS prior to treatment.

[0221] Shortly before treatment, VEGF-A (50 ng / mL), VEGF-C (100 ng / mL), or VEGF-A and VEGF-C (10 ng / mL, 31 ng / mL) polypeptides were pre-incubated in the presence or absence of 100 nM antagonist polypeptides for 30 minutes to allow for complexation. EBM-2 with 0.1% FBS was added to bring the protein solutions to the proper volume, and the different treatments were added to the cells and allowed to sit for 12 minutes at 37°C.

[0222] After treatment, cells were quickly washed in ice-cold PBS and lysed in RIPA buffer (Thermo Fisher Scientific) with added protease and phosphatase inhibitors (Thermo Fisher Scientific). Protein content of cell lysates was determined using a BCA assay kit (Thermo Fisher Scientific) and equal protein quantities (20 pg) were run on an SDS-PAGE gel. Gels were transferred onto poly(vinylidene fluoride) (PVDF) membranes using the iBlot 2 Dry Blotting System (Thermo Fisher Scientific) and blocked for 5 minutes at room temperature using EveryBlot blocking buffer (BioRad). Blots were probed with the appropriate primary antibody for 1 hour at room temperature in EveryBlot blocking buffer. After washing for 30 minutes in TBST (lx Tris-buffered saline with 0.01% Tween-20), blots were probed with HRP-conjugated anti-rabbit antibody for 1 hour at room temperature in EveryBlot blocking buffer. SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) was used for chemiluminescent detection, and images were taken with an Azure 280 gel imager.Atorney Docket No. 44807-0507W01 / Pl 8657-02

[0223] xCELLigence Real-Time Cell Analysis: Cell Proliferation and Transwell Migration Cell proliferation and cell migration experiments were carried out by using the xCELLigence Real-Time Cell Analysis (RTCA) DP instrument (Agilent) in a humidified incubator at 37 °C and 5% (vol / vol) CO2.

[0224] Cell proliferation experiments were performed by using modified 16-well plates (E-plate; Agilent). First, VEGF-A (100 ng / mL; 2X), VEGF-C (200 ng / mL; 2X) or VEGF-A and VEGF-C (20 ng / mL, 62 ng / mL; 2X) were pre-incubated with a titration of antagonist polypeptides for 30 minutes to allow for complexation. Protein complexes were then added to 100 pL of “EGM-2MV-weak” cell-free assay media (EBM-2 basal medium supplemented with ascorbic acid, hydrocortisone, GA-1000, and 5% fetal bovine serum from EGM-2MV SingleQuots (Lonza)) and added to the plate wells. 100 pL of HMEC cell suspension in assay media was seeded into the wells (8,000 cells per well) and allowed to setle to the bottom of the plates at room temperature for 15 minutes, also serving to bring the protein complexes to their final IX concentrations (VEGF-A: 50 ng / mL, VEGF-C: 100 ng / mL; VEGF-A + VEGF-C: 10 ng / mL & 31 ng / mL). E-plates were locked in the RTCA DP device in the incubator and impedance for each well was measured and expressed as a cell index value (CI). CI measurements were taken every 15 minutes for 72 hours total. Two replicates of growth factor / antagonist concentrations were used for each experiment.

[0225] Transwell migration studies were performed using modified 16-well plates (CIM-16; Agilent) with each well consisting of an upper and lower chamber separated by a microporous membrane with integrated impedance electrodes in the lower chamber.

[0226] Impedance electrodes on the bottom of the membrane were coated with 10 ug / mL bovine plasma fibronectin (Sigma-Aldrich) before being aspirated in order to encourage cell migration through the membrane. VEGF-A (50 ng / mL) or VEGF-C (50 ng / mL) were preincubated with a titration of antagonist polypeptides for 30 minutes to allow for complexation, before being brought to 160 pL in EGM-2MV weak. VEGF ligand / antagonist complex solutions were added to the lower chamber before attaching the upper chamber. 200 pL of HMEC cell suspension (30,000 cells per well) was then seeded into the upper chamber wells and allowed to settle at room temperature for 15 minutes. CIM-16 plates were locked in the RTCA DP device in the incubator and impedance for each well was measured andAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0227] expressed as a cell index value (CI). CI measurements were taken every 5 minutes for 24 hours total. Two replicates of growth factor / antagonist concentrations were used for each experiment. All data have been recorded by the supplied RTCA software (vs. 2.8.1).

[0228] Cel! survival assays

[0229] HMECs were seeded in 48-well plates (Greiner) coated with 0.1% gelatin (Millipore Sigma) in PBS. Once cells reached confluency, they were washed with PBS before being starved in Opti-MEM (Invitrogen) containing VEGF-A (50 ng / mL), VEGF-C (100 ng / mL), or VEGF-A and VEGF-C (10 ng / mL, 31 ng / mL) with serial dilutions of antagonists. Prior to addition to the cells, VEGF ligands and antagonists were allowed to incubate for 30 minutes at room temperature. After 48 hours in a 37°C humidified incubator, WST-1 (water-soluble tetrazolium salt, Sigma-Aldrich) was added to each well to a final concentration of 10%. WST-1 was allowed to incubate at 37°C for 1-4 hours, and absorbance of each well was measured at 440 nm on a Varioskan LUX (Thermo Fisher Scientific) plate reader.

[0230] Extracellular matrix binding assay

[0231] 96-well Matrigel matrix thin-layer flat bottom plates (Coming) were washed with phosphate-buffered saline (PBS) - Tween (0.1%) to rehydrate the Matrigel layer and wash away any debris. These plates were then incubated with blocking buffer (1% bovine serum albumin in PBS) at room temperature for 1.5 hours. After blocking, the plates were washed three times with 300 pl PBS-Tween. Serial dilutions of bispecific receptor decoy polypeptides (produced in-house), aflibercept (produced in-house), VEGFR-1 DI -3 Fc chimera (R&D Systems), and VEGFR-2 D23 Fc (produced in-house) in blocking buffer were then added to the plate, which was then incubated at 37 DC for 1.5 hours. Plates were then washed three times with PBS-Tween again. After washing, 50 pl of HRP-conjugated goat anti-human Fc antibody (1 : 10,000 dilution, Sigma- Aldrich) was added and the plate was incubated at room temperature for 1.5 hours. After another set of three washes, 50 pl of TMB solution (Sigma-Aldrich) was added and the plate was incubated at room temperature for 15 minutes. The reaction was stopped by adding 50 pl of IM hydrochloric acid (HC1), and reactive colors were analyzed at an optical wavelength of 450 nm using a Varioskan LUX (Thermo Fisher Scientific) plate reader.Atorney Docket No. 44807-0507W01 / Pl 8657-02

[0232] Measurement of pl values

[0233] The theoretical pl values for the bi specific receptor decoy polypeptides as well as aflibercept was calculated using the Expasy tool ProtParam. The measurement of the actual pl values of the different recombinant polypeptides was performed using an isoelectronic focusing (lEF) gel. To measure the isoelectronic point, 5 pg of each protein sample was loaded into the wells of a pH 3-10 Criterion IEF gel (Biorad) alongside an IEF standard ladder (Biorad). The gel was run in a Criterion gel electrophoresis cell (Biorad), with the upper buffer chamber containing lx IEF cathode buffer (Biorad) and the lower buffer chamber containing lx IEF anode buffer (Biorad). The gel was run at 100 V constant voltage for 1 hour, then 250 V for 1.5 hours on a PowerPac power supply (Biorad). The gel was visualized with Bio-Safe Coomassie stain (Biorad).

[0234] Oxygen-induced ischemic retinopathy (OIR) studies in mice

[0235] Ischemic retinopathy was induced in neonatal wild-type C57BL / 6 mice. Seven-day-old (P7) mice and their mothers were placed in an airtight incubator and exposed to an atmosphere of 75 ± 3% oxygen for 5 days. They were returned to room air on P12 and given a 1 pL intravitreous injection containing aflibercept, 12-Fc, or 21-Fc in one eye and a 1 pL intravitreous of HBS in the fellow eye to serve as control. At Pl 7, mice were euthanized and their eyes were fixed in 10% phosphate-buffered formalin (Thermo Fisher Scientific) for 4h at room temperature. The entire retinas were carefully dissected intact from the eye cup, washed and incubated with fluorescein isothiocyanate (FITC)-conjugated Griffonia simplicifolia (GSA)-lectin (1 : 100, Vector Lab) at room temperature for 45 min. This brief incubation with GSA-lectin selectively stains endothelial cells participating in neovascularization and not those in pre-existent vessels. This staining protocol was chosen to better highlight neovascularization with no background from pre-existent vessels, which facilitates quantification. GSA-lectin also stains hyaloid vessels. Slides were mounted with Aquamount solution (Polysciences). Retinas were flat mounted, digital photographs were obtained with a Zeiss fluorescent microscope at 5X magnification, and images were merged into a single image to show the entire retina using the photomerge option of Photoshop CS5.4. An observer masked with respect to treatment group measured the area ofAtorney Docket No. 44807-0507W01 / Pl 8657-02

[0236] neovascularization per retina by image analysis. Image-Pro Plus software (Media Cybernetics) was used to measure the area of each retinal neovascularization lesion. The area of each retina was also measured, and results are reported as percentage area of neovascularization per retinal area.

[0237] Within each group, data points did not show a normal distribution and therefore the nonparametric Mann-Whitney test was used for statistical comparisons between each experimental group and its corresponding vehicle control group using GraphPad Prism® software vl0.2.

[0238] Mouse model of laser-induced choroidal neovascularization

[0239] Choroidal neovascularization was produced in mice by rupture of Bruch’s membrane with laser photocoagulation. Six to eight-week-old C57BL / 6J mice were anesthetized with ketamine hydrochloride (100 mg / kg body weight) and xylazine (4 mg / kg body weight) and the pupils were dilated with 1% tropicamide and 2.5% phenylephrine. Eyes were covered with 2.5% goniosol (Akom) to avoid corneal drying. An OcuLight GL diode laser with slit lamp delivery system (Iridex, wavelength 630-650 nm) was used with parameters 75 pm spot size, 100 millisecond duration, and 120 mW power. A piece of cover slip was used as a contact lens to view the retina and Bruch’s membrane was ruptured in the 9, 12, and 3 o’clock positions 2 disc diameters from the margin of the optic nerve. Mice were then given an intraocular injection of 1.08 pM anti-VEGF agent in one eye (12-Fc, 21-Fc, or aflibercept) in one eye and PBS in the fellow eye. One group of mice were given an injection of PBS in one eye and no injection in the fellow eye. After 7 days, mice were euthanized and the eyes were fixed in 10% buffered formalin for 3 hours for at room temperature. Anterior segments, vitreous and retinas were removed and the eyecups were incubated in FITC-conjugated GSA-lectin at 4°C overnight. After three washes with PBST, eyecups were mounted, images were obtained with a florescence microscope and CNV areas were measured by image analysis using ImagePro Plus software.

[0240] Quantification and statistical analyses

[0241] GraphPad Prism vl0.2 was used for all graphical representations and statistical analyses. The sample size, number of replicates, mice, definition of center, and dispersionAttorney Docket No. 44807-0507W01 / Pl 8657-02

[0242] and precision measures, and type of analysis performed are described in each of the above sections and / or in the figure legends where applicable. Note that for all studies, significance is defined as p < 0.05 (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). Full statistical analyses are presented in Table 7.

[0243] Table 7. Statistical analysis for all applicable figures.

[0244]

[0245] Atorney Docket No. 44807-0507W01 / Pl 8657-02

[0246] <

[0247]

[0248] Atorney Docket No. 44807-0507W01 / Pl 8657-02

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[0250]

[0251] Attorney Docket No. 44807-0507W01 / Pl 8657-02

[0252]

[0253] Example 2: Treating ocular neovascularization

[0254] A human identified as having ocular neovascularization is administered one or more VEGF antagonist polypeptides provided herein (e.g., one or more polypeptides (e.g., one or more recombinant polypeptides) that each include (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains). In some cases, the administered polypeptides can reduce the number of blood vessels within one or more eyes of the treated mammal.

[0255] Example 3: Treating a Disease or Disorder Associated with Ocular Neovascularization A human identified as having a disease or disorder associated with ocular neovascularization (e.g., NVAMD and / or DR) is administered one or more VEGF antagonist polypeptides provided herein (e.g., one or more polypeptides (e.g., one or more recombinant polypeptides) that each include (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains).Attorney Docket No. 44807-0507W01 / Pl 8657-02

[0256] Example 4: Treating macular edema

[0257] A human identified as having macular edema (e.g., diabetic macular edema or macular edema due to retinal vein occlusion) is administered one or more VEGF antagonist polypeptides provided herein (e.g., one or more polypeptides (e.g., one or more recombinant polypeptides) that each include (a) one or more VEGFR-1 Ig2 domains, (b) one or more VEGFR-2 Ig2 domains, (c) one or more VEGFR-2 Ig3 domains, and (d) one or more Fc domains). In some cases, the administered polypeptides can reduce the amount of fluid within the macula one or more eyes of the treated mammal.

[0258] OTHER EMBODIMENTS

[0259] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No. 44807-0507W01 / Pl 8657-02WHAT IS CLAIMED IS:

1. A vascular endothelial growth factor (VEGF) antagonist polypeptide comprising (a) a VEGFR-1 Ig2 domain, (b) a VEGFR-2 Ig2 domain, (c) a VEGFR-2 Ig3 domain, and (d) a Fc domain.

2. The VEGF antagonist polypeptide of claim 1, wherein said VEGF antagonist polypeptide simultaneously binds VEGF-A and VEGF-C.

3. The VEGF antagonist polypeptide of any one of claims 1-2, wherein said (a) comprises, consists of, or consists essentially of SEQ ID NO:1.

4. The VEGF antagonist polypeptide of any one of claims 1-2, wherein said (b) comprises, consists of, or consists essentially of SEQ ID NO:2.

5. The VEGF antagonist polypeptide of any one of claims 1-2, wherein said (c) comprises, consists of, or consists essentially of SEQ ID NO:3.

6. The VEGF antagonist polypeptide of any one of claims 1-2, wherein said (d) comprises, consists of, or consists essentially of SEQ ID NO:4.

7. The VEGF antagonist polypeptide of any one of claims 1-6, wherein said VEGF antagonist polypeptide comprises said (a), followed by said (b), followed by said (c), followed by said (d).

8. The VEGF antagonist polypeptide of claim 1, wherein said VEGF antagonist polypeptide comprises, consists of, or consists essentially of SEQ ID NO:5.Attorney Docket No. 44807-0507W01 / Pl 8657-029. The VEGF antagonist polypeptide of any one of claims 1-6, wherein said VEGF antagonist polypeptide comprises said (b), followed by said (c), followed by said (a), followed by said (d).

10. The VEGF antagonist polypeptide of claim 1, wherein said VEGF antagonist polypeptide comprises, consists of, or consists essentially of SEQ ID NO:6.

11. A nucleic acid encoding the VEGF antagonist polypeptide of any one of claims 1-10.

12. A composition comprising the VEGF antagonist polypeptide of any one of claims 1-10 or the nucleic acid of claim 11.

13. The composition of claim 12, wherein said composition comprises a pharmaceutically acceptable carrier.

14. A method for treating a mammal having ocular neovascularization, wherein said method comprises administering the VEGF antagonist polypeptide of any one of claims 1-10 or the nucleic acid of claim 11 to the mammal.

15. A method for treating a mammal having a disease or disorder associated with ocular neovascularization, said method comprising administering to said mammal the VEGF antagonist polypeptide of any one of claims 1-10 or the nucleic acid of claim 11.

16. The method of claim 15, wherein the disease or disorder associated with ocular neovascularization is neovascular age-related macular degeneration (NVAMD), polypoidal choroidal vasculopathy, diabetic retinopathy (DR), ocular histoplasmosis, myopic degeneration, angioid streaks, choroidal neovascularization, retinal neovascularization, or retinopathy of prematurity (ROP).Attorney Docket No. 44807-0507W01 / Pl 8657-0217. A method for treating a mammal having macular edema, wherein said method comprises administering the VEGF antagonist polypeptide of any one of claims 1-10 or the nucleic acid of claim 11 to the mammal.

18. The method of claim 17, wherein the macular edema is diabetic macular edema or macular edema due to retinal vein occlusion.

19. The method of any one of claims 14-18, wherein said mammal is a human.

20. The method of any one of claims 14-19, wherein said mammal has been previously treated with an anti-VEGF therapy.

21. The method of claim 20, wherein said mammal is resistant to said anti-VEGF therapy.

22. The use of a composition comprising the VEGF antagonist polypeptide of any one of claims 1-10 or the nucleic acid of claim 11 to treat ocular neovascularization or macular edema.

23. A polypeptide of any one of claims 1-10 or a nucleic acid of claim 11 for use in the preparation of a medicament to treat ocular neovascularization or macular edema.

24. A polypeptide of any one of claims 1-10 or a nucleic acid of claim 11 for use in treating ocular neovascularization or macular edema.

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