Lassa fever (LF) is an often-fatal viral hemorrhagic fever (VHF) endemic in West Africa. A combination of three broadly neutralizing human monoclonal antibodies (BNhMAbs) derived from West African patients who survived LF (Arevirumab-3) rescued 100% of nonhuman primates (NHPs), even after delay in initiation of treatment to 8 days post-infection. Recently, we solved the crystal structure of the pre-fusion form of the Lassa virus glycoprotein complex (GPC) bound to a BNhMAb, the first structure for the virion configuration of the GPC of any arenavirus. Additional newly solved structures for GPC:BNhMAb complexes and derivation of escape mutants have informed the design and evaluation of optimally formulated BNhMAb cocktails targeting independent neutralizing epitopes. We plan to expand upon this extensive body of work to support completion of pre-clinical evaluation of Arevirumab-3 and functionally enhanced derivatives with improved pharmacological properties toward first-in-human clinical studies with this novel class of LF therapeutics. In studies proposed under Specific Aim 1, we will complete the pre-clinical evaluation of Arevirumab-3. Completion of Chemistry, Manufacturing and Control Data (CMC), preclinical pharmacology and toxicology in animal models of LF, including prophylactic interval and recrudescence evaluations, will enable the filing of an Investigational New Drug (IND) application toward clinical studies with Arevirumab-3 The protective activity of non-neutralizing LASV huMAbs in animal models of LF will be evaluated in studies proposed in Specific Aim 2. The protective efficacy of non-neutralizing LASV huMAbs will be evaluated and down-selected in guinea pigs. If non-neutralizing protective huMAbs (NNPhuMAbs) are identified, the activity of this class of antibodies will be further evaluated in NHP models of LF, either individually or in rationally designed cocktails. In studies proposed under Specific Aim 3, we will design enhanced immunotherapeutics via Fc effector and computational optimization, bi-specific antibody engineering (BsAbs), “designer” production cell lines that attach specific glycans, and surveillance data inputs. Enhancement of immunotherapeutic cocktails with Fc engineering, evaluation of BsAbs, machine learning algorithms, and CRISPR/Cas9-based generation of NS0 or CHO production cell lines with designer modifications will define characteristics of next generation LF immunotherapeutics. Surveillance of circulating and emerging clinical LASV strains will educate the design of LF immunotherapeutics with robust clinical characteristics and sustained potency. Under Specific Aim 4, we propose studies to valuate in vivo protection and PK of optimized BNhMAbs, NNPhMAbs, and BsAbs. In vivo evaluation of optimized LASV antibodies in established models of LF will educate advanced pre-clinical evaluation of second generation immunotherapeutics with improved pharmacological properties. The studies proposed in Project 1 are expected to result in the filing of an IND application for Arevirumab-3 in Year 3 and a Pre-IND for a 2nd generation therapeutic at the conclusion of the 5-year performance period.