Structure-Guided Design of Broadly Neutralizing Lassa Virus BiSpecific Antibodies

“广谱中和拉萨病毒双特异性抗体的结构导向设计” 摘要 拉沙热(LF)是一种经常致命的病毒性出血热(VHF)，在西非流行 造成严重的社会和经济混乱。缺乏经批准的治疗方法或疫苗，记录在案 啮齿动物宿主的地理扩展、病毒的采购和武器化的简便性以及 最近新出现的拉萨病毒(LASV)毒株支持加强防备的建议 如果是这样。我们分离并鉴定了113个人类单抗(HMAbs)，这是第一个大规模的 描述了人类针对LASV的抗体。我们发现，最有效的中和hMAb靶标 需要糖蛋白复合体中每个单体的GP1和GP2亚基的四元表位 (GPC)三聚体。LASV在基因上是多样化的，在西非有四个不同的谱系。某些hMAb 中和了所有4个LASV血统。在致死性LF模型中挑战远交种豚鼠(GP) 向下选择用于非人灵长类(NHP)模型研究的广中和hMAb(BNhMAbs)， 食蟹猴。3个BNhMAb的组合，每个都具有广泛的中和活性和识别能力 在LASV GPC上的不同表位，即使在延迟治疗至8%后仍挽救了100%的NHP 感染后几天，动物表现出严重的血液和代谢失调。 3BNhMAb鸡尾酒对来自NHP的LASV毒株的致死攻击具有100%的保护作用 谱系II和IV。我们现在建议利用BNhMAbs与GPC络合的结构信息来 设计跨越两个高度保护性表位的人类双特异性抗体(BsAbs)，从而减少 提供针对LF的卓越保护所需的分子数量。LASV的初步结果- 挑战GP建议在GPC的基础上用双特异性靶向第四系中和表位 抗体可以产生更好的保护作用，即使剂量比以前对个体测试的低10倍 BNhMAbs(初步结果)。我们建议的项目符合RFA-AI-17-026在 LASV BsAbbs是基于先前确定的、特征良好的候选治疗性hMAb 抗NIAID列出的新出现的病原体LASV。该项目将解决RFA-AI的一个特殊兴趣- 17-026用于免疫疗法，以防止面对感染或中毒 直接威胁、对免疫受损个人的保护或暴露后治疗以抑制 感染和疾病。在里程碑1中，我们将向下选择针对碱基、中和和帽中和的BsAbbs LASV GPC表位。在里程碑2中，单抗和联合单抗的剂量和给药间隔研究 将对GP和NHP的治疗进行评估。里程碑3化学、制造和控制数据(CMC) 将为领导BsAbs而设立。在里程碑4中，我们将执行临床前药理学和 BsAbs的毒理学研究。在拟议的计划结束时，我们将完成对 一流的免疫治疗性BsAb用于LF的预防和暴露后治疗。 此应用程序包含Zalgen Labs及其分包商要求的专有/特权信息 除非出于审查和评估的目的，否则不得向政府以外的人发布。

“Structure-Guided Design of Broadly Neutralizing Lassa Virus BiSpecific Antibodies” ABSTRACT Lassa fever (LF) is an often-fatal viral hemorrhagic fever (VHF) that is endemic in West Africa where it causes significant social and economic disruption. The lack of an approved therapeutic or vaccine, recorded geographic expansion of rodent reservoirs, ease of procurement and weaponization of the virus, and the recent emergence of new Lassa virus (LASV) strains support recommendations for enhanced preparedness for LF. We isolated and characterized 113 human monoclonal antibodies (hMAbs), the first large panel of human antibodies against LASV described. We found that the most potent neutralizing hMAbs target quaternary epitopes that require both GP1 and GP2 subunits of each monomer in the glycoprotein complex (GPC) trimer. LASV is genetically diverse with four distinct lineages present in West Africa. Some hMAbs neutralized all 4 LASV lineages. Challenge of outbred guinea pigs (GP) in a model of lethal LF informed the down-selection of broadly neutralizing hMAbs (BNhMAbs) for studies in a nonhuman primate (NHP) model, Cynomolgus macaques. A combination of 3 BNhMAbs, each with broad neutralizing activity and recognition of distinct epitopes on the LASV GPC, rescued 100% of NHPs even after delay in the start of treatment to 8 days post-infection, a time when the animals displayed severe hematological and metabolic dysregulation. The 3 BNhMAb cocktail conferred 100% protection in NHP against lethal challenge with LASV strains from lineages II and IV. We now propose to utilize the structural information of BNhMAbs complexed with GPC to engineer human bi-specific antibodies (BsAbs) that span two highly protective epitopes, thereby reducing the number of molecules required to confer superior protection against LF. Preliminary results in LASV- challenged GP suggest that targeting quaternary neutralizing epitopes in the base of GPC with a bi-specific antibody results in superior protection, even at 10-fold lower doses than previoulsy tested for individual BNhMAbs (Preliminary Results). Our proposed project meets the strict requirements of RFA-AI-17-026 in that the LASV BsAbs are based on previously identified, well-characterized, candidate therapeutic hMAbs against an NIAID listed emerging pathogen, LASV. The project will address a particular interest of RFA-AI- 17-026 for immunotherapeutics that would “enable prevention of infection or intoxication in the face of an immediate threat, protection of immunocompromised individuals, or post-exposure treatment to suppress infection and disease.” In Milestone 1 we will down-select BsAbs targeting base, middle, and cap neutralizing epitopes on LASV GPC. In Milestone 2 dose and dosing interval studies with mono and combination BsAb therapy in GP and NHP will be evaluated. In Milestone 3 Chemistry, Manufacturing and Control Data (CMC) will be established for leading BsAbs. In Milestone 4 we will perform Preclinical Pharmacology and Toxicology of BsAbs. At the conclusion of the proposed program we will complete preclinical evaluation of a first-in-class immunotherapeutic BsAb for the prophylactic and post-exposure treatment of LF. This application contains proprietary/priviledged information that Zalgen Labs and its subcontractors request not be released to persons outside the Government, except for the purposes of review and evaluations.