Heparin-binding VEGFR1 variants as long-acting VEGF inhibitors for treatment of intraocular neovascular disorders

Neovascularization is a key feature of ischemic retinal diseases and the wet form of age-related macular degeneration (AMD), all leading causes of severe vision loss. Vascular endothelial growth factor (VEGF) inhibitors have transformed the treatment of these disorders. Millions of patients have been treated with these drugs worldwide. However, in real-life clinical settings, many patients do not experience the same degree of benefit observed in clinical trials, in part because they receive fewer anti-VEGF injections. Therefore, there is an urgent need to discover and identify novel long-acting VEGF inhibitors. We hypothesized that binding to heparan-sulfate proteoglycans (HSPG) in the vitreous, and possibly other ocular structures, may be a strategy to promote intraocular retention, ultimately leading to a reduced burden of intravitreal injections. We designed a series of VEGF receptor 1 variants and identified some with strong heparin-binding characteristics and ability to bind to vitreous matrix. Our data indicate that some of our variants have longer duration and greater efficacy in animal models of intraocular neovascularization than current standard of care. Our study represents a systematic attempt to exploit the functional diversity associated with heparin affinity of a VEGF receptor.

The development of a neovascular supply or angiogenesis serves crucial homeostatic roles, since the blood vessels carry nutrients to tissues and organs and remove catabolic products (1). However, uncontrolled growth of blood vessels can promote or facilitate numerous disease processes, including tumors and intraocular vascular disorders (1). Although numerous angiogenic factors were initially identified and characterized (2), work performed in many laboratories has established vascular endothelial growth factor (VEGF) as a key regulator of normal and pathological angiogenesis as well as vascular permeability (3–5). Alternative exon splicing results in the generation of multiple isoforms that differ in their affinity for heparin, including VEGF₁₂₁, VEGF₁₆₅, and VEGF₁₈₉. VEGF₁₂₁ lacks significant heparin binding. While VEGF₁₆₅ has a single, exon-7-encoded, heparin-binding domain, VEGF₁₈₉ has two heparin-binding domains encoded respectively by exon-6 and exon-7 (6, 7). Much experimental evidence documents the key role of the heparin-binding VEGF isoforms in the establishment of biochemical gradients required for angiogenesis (8–10). VEGF is a member of a gene family that also includes PlGF, VEGF-B, VEGF-C, and VEGF-D. Three related receptor tyrosine kinases (RTKs) have been reported to bind VEGF ligands: VEGFR1 (11, 12), VEGFR2 (13), and VEGFR3 (14). VEGF binds both VEGFR1 and VEGFR2, while PlGF and VEGF-B interact selectively with VEGFR1. VEGFR3 and its two ligands, VEGF-C and VEGF-D, are primarily implicated in lymphangiogenesis (15, 16). Each member of this RTK class has seven immunoglobulin (Ig)-like domains in the extracellular portion (17). There is agreement that VEGFR2 is the main signaling receptor for VEGF (16), although VEGFR1 binds VEGF with substantially higher affinity than VEGFR2 (17).VEGF inhibitors have become a standard of therapy in multiple tumors and have transformed the treatment of intraocular neovascular disorders such as the neovascular form of age-related macular degeneration (AMD), proliferative diabetic retinopathy, and retinal vein occlusion, which are leading causes of severe vision loss and legal blindness (3, 5, 18). Currently, three anti-VEGF drugs are widely used in the United States for ophthalmological indications: bevacizumab, ranibizumab, and aflibercept (3). Bevacizumab is a full-length IgG antibody targeting VEGF (19). Even though bevacizumab was not developed for ophthalmological indications, it is widely used off-label due to its low cost. Ranibizumab is an affinity-matured anti-VEGF Fab (20). Aflibercept is an IgG-Fc fusion protein (21), with elements from VEGFR1 and VEGFR2, that binds VEGF, PIGF, and VEGF-B (22). Importantly, after a 5-y treatment with ranibizumab or bevacizumab, about half of neovascular AMD patients had good vision, i.e., visual acuity 20/40 or better, an outcome that would not have been possible before anti-VEGF agents were available (23). However, in real-life clinical settings, many patients receive fewer anti-VEGF injections than in clinical trials, and it has been hypothesized that this correlates with less satisfactory visual outcomes (24). Therefore, there is a need to develop agents with longer duration after intraocular injection, thus reducing the frequency of injections, and a number of approaches to this end have been attempted (25, 26). Aflibercept (Eylea) was approved based on clinical trials showing that every 8-wk administration of the dose of 2 mg could match the efficacy of monthly ranibizumab (0.5 mg). However, despite the prediction that a switch to aflibercept would reduce the number of intravitreal injections, recent studies suggest that it is not the case (27). Another approach aiming to reduce frequency of injections is the use of high doses of brolucizumab, a recently Food and Drug Administration (FDA)-approved high-affinity single-chain variable fragments (scFV) targeting VEGF (28). However, an increased incidence of retinal vasculitis and intraocular inflammation that adversely affected vision has been observed in a subset of patients treated with brolucizumab (29). A refillable slow-release device system implanted in the vitreous that enables continuous delivery of ranibizumab has been developed (30). The phase II data reported are promising, but this approach requires surgery and complications such as bleeding have been noted (30). Therefore, there is still an unmet medical need for intravitreal anti-VEGF agents with improved half-life.In 1996, in the course of structure–function studies aiming to identify VEGF-binding elements in VEGFR1, we found that deletion of Ig-like domain (D) 2, but not of other Ds, abolished VEGF or PIGF binding (31). Replacing D2 of VEGFR3 with D2 of VEGFR1 conferred on VEGFR3 the ligand specificity of VEGFR1 and abolished the ability of the mutant VEGFR3 to interact with VEGF-C (31). Subsequent studies documented the interaction between D2 and VEGF (or PlGF) by X-ray crystallography (32–34). However, D3 was important for optimal VEGF binding (31, 32). These initial studies led to the design of a construct comprising the first three lg-like Ds of VEGFR1, fused to an Fc-lgG (Flt-1-3-IgG) (31). Flt-1-3-lgG showed a potent ability to neutralize VEGF in vitro and in several in vivo models of physiological and pathological angiogenesis (35–40). However, the half-life of this molecule following systemic administration was relatively short due to the presence of clusters of basic residues in D3, which resulted in binding to heparan-sulfate proteoglycans (HSPG) and sequestration in the extracellular matrix of various tissues.In 2002, Holash et al. (22) described an IgG fusion construct comprising VEGFR1 D2 and VEGFR2 D3, which has much lower heparin affinity than VEGFR1 D3. This molecule, known today as aflibercept, ziv-aflibercept, or Eylea, was reported to have a significantly longer systemic half-life than Flt(1-3-lgG) (22). These pharmacokinetic characteristics, combined with high binding affinity for VEGF and the ability to bind PlGF and VEGF-B, led to the prediction that aflibercept would be a more effective antitumor agent than other VEGF inhibitors (22, 41). However, aflibercept has gained FDA approval only for second line treatment of colorectal cancer, while bevacizumab and the anti-VEGFR2 antibody ramucirumab received several FDA approvals in multiple cancer types (3, 5), suggesting that the above-mentioned characteristics did not provide a therapeutic advantage. Clearly, aflibercept has had its major clinical impact as an intravitreal treatment for ocular vascular disorders.We hypothesized that heparin-binding mediated by D3 (or other Ig-like domain) of VEGFR1 (12), while a disadvantage for systemic administration, might confer significant advantages for intravitreal and potentially other local administration.