Kureshi Rakeeb, Zhu Angela, Shen Jikui, Tzeng Stephany Y, Astrab Leilani R, Sargunas Paul R, Green Jordan J, Campochiaro Peter A, Spangler Jamie B
Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD USA.
Department of Chemical & Biomolecular Engineering, Johns Hopkins University, Baltimore, MD USA.
Cell Mol Bioeng. 2020 Jul 31;13(5):405-418. doi: 10.1007/s12195-020-00641-0. eCollection 2020 Oct.
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.
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.
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.
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.
眼部新生血管形成是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在内的视网膜疾病的一个标志，这两种疾病是成年人失明的两大主要原因。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）相互作用驱动的，抑制VEGF途径已显示出巨大的临床前景。然而，抗VEGF治疗需要频繁且高剂量地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。
我们将结构生物学的见解与分子工程技术相结合，设计了一种抗VEGF拮抗剂蛋白。从临床批准的诱饵受体蛋白阿柏西普出发，我们精心设计了一个酵母展示的变体诱变文库，并分离出与临床药物相比具有更高VEGF亲和力的克隆。我们的先导工程蛋白通过非病毒基因递送在大鼠眼脉络膜间隙中表达。
使用基于结构的定向进化方法，我们鉴定出了多种具有改善的靶标亲和力的有前景的抗VEGF拮抗剂蛋白。改善主要是通过降低解离速率介导的，并且鉴定出了在结构上具有重要意义的趋同序列突变。我们工程化的拮抗剂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显示出强大而持久的表达。
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，并在大鼠中证明了安全且非侵入性的眼部递送。此外，我们的结构导向设计方法为发现适用于广泛应用的靶向蛋白药物提供了一种通用策略。
