A MERS-CoV Receptor Decoy

 描述(申请人提供)：中东呼吸综合征冠状病毒(MERS-CoV)是一种新出现的人类健康威胁，病死率约为35%。MERS冠状病毒利用细胞表面蛋白二肽基肽酶4(DPP4)进入细胞并感染细胞。在我们的第一阶段研究中，使用瞬时植物表达系统，我们产生了人DPP4与人免疫球蛋白Fc序列IgG1、IgA1和IgA2的融合，以产生“受体诱骗”来阻止MERS-CoV的细胞感染。我们发现DPP4-Fc结合在MERS-CoV S蛋白的S1结构域上，并且DPP4-Fc对MERS-CoV细胞感染的抑制作用比可溶性DPP4更强。我们表明，基于IgA1的DPP4-Fc融合可能比基于IgG1的融合更有效。此外，我们还证明，通过修改人DPP4与MERS-CoV刺突蛋白接触处的单一氨基酸，可以将结合和病毒中和能力提高10倍以上。DPP4-Fc还有望具有优越的药代动力学，因为Fc具有较长的循环半衰期，并能够被输送到MERS-CoV感染部位的呼吸道粘膜表面。在第二阶段的研究中，我们将构建新的DPP4-Fc结构，以提高DPP4‘与MERS冠状病毒刺突蛋白的S亲和力，并通过突变活性位点来消除DPP4’S多肽酶的活性。我们将优化新的构建结构，以提高在植物中的表达，并用类似人的N-连接多糖生产DPP4-Fc。新的DPP4-Fc融合将通过ELISA对MERS-CoV尖峰蛋白结合进行评级，并测试其中和敏感细胞中MERS-CoV感染的能力。表现最好的DPP4-FC变种将在小鼠MERS模型中进行保护效果测试，评估腹膜内和鼻腔给药途径。我们将扩大生产和提纯我们的铅分子，以提纯7克DPP4-FC，这将用于在大鼠身上进行为期两周的重复给药安全性/毒理学试点研究。我们将对DPP4-Fc进行安全性评价和血清中DPP4-Fc的定量，以获得药物动力学参数。在这些研究中，我们还将筛查抗DPP4-Fc抗体。

 DESCRIPTION (provided by applicant): Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly emerging human health threat with a ~35% case fatality rate. MERS-CoV uses dipeptidyl peptidase 4 (DPP4), a cell surface protein, to enter and infect cells. During our Phase I study, using a transient plant expression system, we produced fusions of human DPP4 and the human immunoglobulin Fc sequences of IgG1, IgA1 and IgA2, to produce "receptor decoys" to block cellular infection with MERS-CoV. We demonstrated that DPP4-Fc binds to the S1 domain of MERS-CoV S protein, and that DPP4-Fc is a more potent inhibitor of MERS-CoV cellular infection than soluble DPP4. We showed that a DPP4-Fc fusion based on IgA1 might possibly be more effective than one based on IgG1. In addition, we demonstrated that binding and virus neutralization could be improved by more than 10-fold by modifying a single amino acid where human DPP4 and MERS-CoV spike protein contact. DPP4-Fc is also expected to have superior pharmacokinetics, as Fc will confer a long circulating half-life and the ability to be delivered to airway mucosal surfaces, the site of MERS-CoV infection. In a phase II study, we will produce new DPP4-Fc constructs to improve DPP4's affinity for MERS-CoV spike protein, and eliminate DPP4's peptidase activity by mutating the active site. We will optimize new constructs to improve expression in plants and to produce DPP4-Fc with human-like N-linked glycans. New DPP4-Fc fusions will be ranked for MERS-CoV spike protein-binding by ELISA and tested for the ability to neutralize MERS-CoV infection in susceptible cells. The best performing DPP4-Fc variants will be tested for protective efficacy in a mouse MERS model, evaluating both intraperitoneal and intranasal routes of administration. We will scale up production and purification of our lead molecule to purify 7 grams of DPP4-Fc, which will be used for a pilot 2-week repeat-dose safety/toxicology study in rats. We will evaluate safety and quantify DPP4-Fc in serum to obtain pharmacokinetic parameters. We will also screen for anti-DPP4-Fc antibodies in these studies.