Structure-Guided Design of Broadly Neutralizing Lassa Virus BiSpecific Antibodies

“广泛中和拉沙病毒双特异性抗体的结构导向设计” 摘要 拉沙热（LF）是一种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破坏。缺乏批准的治疗或疫苗，记录 啮齿动物水库的地理扩张，易于采购和病毒武器化，以及 最近出现的拉沙病毒新毒株支持加强防范的建议 对于LF。我们分离并鉴定了113种人单克隆抗体（hMAbs），这是第一个大的 描述了抗LASV的人抗体。我们发现最有效的中和hMAbs靶向 需要糖蛋白复合物中每个单体的GP 1和GP 2亚基的四级表位 (GPC)三聚体。LASV在遗传上是多样化的，在西非有四个不同的谱系。一些hMAb 中和了所有4种LASV谱系。在致死性LF模型中对远交豚鼠（GP）的攻击告知了 在非人灵长类动物（NHP）模型中向下选择用于研究的广泛中和hMAb（BNhMAb）， 食蟹猴。3种BNhMAb的组合，每种都具有广泛的中和活性和识别 不同的表位的LASV GPC，拯救100%的NHP，即使在治疗开始延迟至8 在感染后30天，动物表现出严重的血液学和代谢失调。 3BNhMAb混合物在NHP中赋予100%的保护，以抵抗来自 血统II和IV。我们现在提出利用与GPC复合的BNhMAbs的结构信息， 工程化跨越两个高度保护性表位的人双特异性抗体（BsAbs），从而减少 提供抗LF的上级保护所需的分子数量。LASV的初步结果- 挑战的GP表明，在GPC的基础上用双特异性抗体靶向第四中和表位， 抗体导致上级保护，即使在比先前测试的个体低10倍的剂量下， BNhMAb（初步结果）。我们提出的项目符合RFA-AI-17-026的严格要求， LASV BsAb基于先前鉴定的、充分表征的候选治疗性hMAb， 针对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对于免疫治疗，将“能够预防感染或中毒，在面对一个 直接威胁，保护免疫功能低下的个体，或暴露后治疗，以抑制 感染和疾病”。在里程碑1中，我们将向下选择靶向碱基、中间体和帽中和的BsAb LASV GPC上的表位。在里程碑2单药和联合BsAb的剂量和给药间隔研究中 将评估GP和NHP中的治疗。在里程碑3化学、生产和控制数据（CMC）中 将为领先的BsAb建立。在里程碑4中，我们将进行临床前药理学研究， BsAbs的毒理学。在拟议的计划结束时，我们将完成临床前评估的一个 用于LF的预防性和暴露后治疗的一流免疫抑制剂BsAb。 本申请包含Zalgen Labs及其分包商要求的专有/特权信息 不得向政府以外的人公布，除非是为了审查和评估的目的。

“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.