Background

Ocular neovascularization is a hallmark of retinal diseases including neovascular age-related macular degeneration and diabetic retinopathy, two leading causes of blindness in adults. Neovascularization is driven by the interaction of soluble vascular endothelial growth factor (VEGF) ligands with transmembrane VEGF receptors (VEGFR), and inhibition of the VEGF pathway has shown tremendous clinical promise. However, anti-VEGF therapies require invasive intravitreal injections at frequent intervals and high doses, and many patients show incomplete responses to current drugs due to the lack of sustained VEGF signaling suppression.

Methods

We synthesized insights from structural biology with molecular engineering technologies to engineer an anti-VEGF antagonist protein. Starting from the clinically approved decoy receptor protein aflibercept, we strategically designed a yeast-displayed mutagenic library of variants and isolated clones with superior VEGF affinity compared to the clinical drug. Our lead engineered protein was expressed in the choroidal space of rat eyes via nonviral gene delivery.

Results

Using a structure-informed directed evolution approach, we identified multiple promising anti-VEGF antagonist proteins with improved target affinity. Improvements were primarily mediated through reduction in dissociation rate, and structurally significant convergent sequence mutations were identified. Nonviral gene transfer of our engineered antagonist protein demonstrated robust and durable expression in the choroid of treated rats one month post-injection.

Conclusions

We engineered a novel anti-VEGF protein as a new weapon against retinal diseases and demonstrated safe and noninvasive ocular delivery in rats. Furthermore, our structure-guided design approach presents a general strategy for discovery of targeted protein drugs for a vast array of applications.

中文翻译：

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血管内皮生长因子拮抗剂的结构引导分子工程治疗视网膜疾病

背景

眼部新生血管形成是视网膜疾病的标志，包括新生血管性年龄相关性黄斑变性和糖尿病性视网膜病变，这是成人失明的两个主要原因。新生血管形成是由可溶性血管内皮生长因子 (VEGF) 配体与跨膜 VEGF 受体 (VEGFR) 的相互作用驱动的，抑制 VEGF 通路已显示出巨大的临床前景。然而，抗 VEGF 疗法需要以频繁的间隔和高剂量进行侵入性玻璃体内注射，并且由于缺乏持续的 VEGF 信号抑制，许多患者对当前药物的反应不完全。

方法

我们利用分子工程技术综合了结构生物学的见解，以设计一种抗 VEGF 拮抗剂蛋白。从临床批准的诱饵受体蛋白阿柏西普开始，我们战略性地设计了一个酵母展示的变体诱变文库和分离的克隆，与临床药物相比，具有优越的 VEGF 亲和力。我们的先导工程蛋白通过非病毒基因递送在大鼠眼睛的脉络膜空间中表达。

结果

使用结构知情的定向进化方法，我们鉴定了多种具有改善的靶亲和力的有前途的抗 VEGF 拮抗剂蛋白。改善主要是通过降低解离率来介导的，并且确定了结构上显着的收敛序列突变。我们的工程拮抗蛋白的非病毒基因转移在注射后一个月在治疗大鼠的脉络膜中表现出稳健和持久的表达。

结论

我们设计了一种新型抗 VEGF 蛋白作为对抗视网膜疾病的新武器，并在大鼠中展示了安全且无创的眼部给药。此外，我们的结构导向设计方法提出了一种发现靶向蛋白质药物的通用策略，用于广泛的应用。