Multivalent hyaluronic acid bioconjugates improve sFlt-1 activity in vitro

Abstract

Anti-VEGF drugs that are used in conjunction with laser ablation to treat patients with diabetic retinopathy suffer from short half-lives in the vitreous of the eye resulting in the need for frequent intravitreal injections. To improve the intravitreal half-life of anti-VEGF drugs, such as the VEGF decoy receptor sFlt-1, we developed multivalent bioconjugates of sFlt-1 grafted to linear hyaluronic acid (HyA) chains termed mvsFlt. Using size exclusion chromatography with multiangle light scattering (SEC-MALS), SDS-PAGE, and dynamic light scattering (DLS), we characterized the mvsFlt with a focus on the molecular weight contribution of protein and HyA components to the overall bioconjugate size. We found that mvsFlt activity was independent of HyA conjugation using a sandwich ELISA and in vitro angiogenesis assays including cell survival, migration and tube formation. Using an in vitro model of the vitreous with crosslinked HyA gels, we demonstrated that larger mvsFlt bioconjugates showed slowed release and mobility in these hydrogels compared to low molecular weight mvsFlt and unconjugated sFlt-1. Finally, we used an enzyme specific to sFlt-1 to show that conjugation to HyA shields sFlt-1 from protein degradation. Taken together, our findings suggest that mvsFlt bioconjugates retain VEGF binding affinity, shield sFlt-1 from enzymatic degradation, and their movement in hydrogel networks (in vitro model of the vitreous) is controlled by both bioconjugate size and hydrogel network mesh size. These results suggest that a strategy of multivalent conjugation could substantially improve drug residence time in the eye and potentially improve therapeutics for the treatment of diabetic retinopathy.

Introduction

Diabetic retinopathy is one of the most detrimental consequences of diabetes, which imposes enormous healthcare costs in the U.S. and worldwide, and affects over 25 million American adults. Within two decades of their initial diagnosis, all patients with diabetes type I and 60% of patients with diabetes type II will exhibit symptoms of DR [1]. The main mediator of retinal neovascularization in diabetic retinopathy is vascular endothelial growth factor (VEGF), which is upregulated by hypoxic neurons in the retina [2], [3], [4]. Elevated intravitreal levels of VEGF results in the aberrant growth of blood vessels with loose cell-cell junctions leading to areas of vision loss caused by pooled blood and edema in and around the macula [5]. The reference-standard treatment for DR is pan-retinal laser photocoagulation (PLP), which forestalls retinal neovascularization by destroying ischemic neurons and reducing the number of VEGF-producing cells in the retina [6]. While this therapy can reduce the risk of disease progression and severe vision loss, the best outcomes for improved visual acuity occur when PLP is used in combination with anti-angiogenic therapies [7], as intravitreal VEGF remains elevated even after treatment by PLP [8].

The most commonly used anti-VEGF therapies for DR are Lucentis (ranibizumab, Genentech, a 48 kDa humanized antibody fragment) [9], Avastin (bevacizumab, Genentech, a 150 kDa humanized antibody) [10], and Eylea (aflibercept, Regeneron, a 110 kDa VEGF-receptor fusion protein) [11]. Due to limited drug retention in the vitreous as a result of their small molecular size, a single administration of these drugs provides only a limited therapeutic benefit for a finite period of time. In order to maintain an effective drug dose in the vitreous to inhibit VEGF-mediated retinal neovascularization, patients require monthly injections. Consequently, low patient compliance to these treatment protocols continues to be the most significant factor limiting the ability of anti-VEGF treatments to maintain improvements in visual acuity [12], [13]. Therefore, novel approaches are required to enhance the half-life of anti-VEGF therapies within the vitreous, which would in turn reduce the frequency of required intravitreal drug injections and improve patient quality of life.

In this work, we describe a novel multivalent drug bioconjugate composed of the anti-angiogenic VEGF decoy receptor sFlt-1, and hyaluronic acid (HyA), a naturally occurring biopolymer present in high concentrations throughout the body and in particular within the vitreous of the eye [14]. The overall goal of this study was to create high-molecular weight multivalent bioconjugates of sFlt (mvsFlt) that were capable of binding and inhibiting VEGF₁₆₅ activity in vitro, improving protein stability, and increasing mvsFlt residence time over unconjugated sFlt. For the latter, we modeled the vitreous in vitro using a cross-linked hyaluronic acid hydrogel and observed that mvsFlt bioconjugates diffused slower and had reduced mobility in comparison to unconjugated sFlt. We anticipate that the future clinical use of multivalent conjugates of sFlt may significantly increase the intravitreal drug residence time and inhibit retinal angiogenesis over a longer period of time.