CORE B (Antibody Optimization Core) Abstract In the absence of protective vaccines and known correlates of immunity, passive transfer of monoclonal antibodies (mAbs) is a promising strategy to target, control and clear infections. Although genomic sequencing and B cell cloning technologies revolutionized our capacity to generate libraries of mAbs against nearly any target of interest, in many cases these mAbs fall short, and fail to achieve sufficient protective immunity, in pre- clinical or clinical studies. Fortunately, new antibody engineering strategies now allow us to enhance mAb- mediated protective immunity through optimization of mAb functionality. Evolution of the mAb antigen-binding (Fv) and constant regions (Fc) can improve overall mAb performance. This Core, the Antibody Optimization Core (Core B) will support all three CETR Research Projects by providing high-throughput functional profiling of mAb functionality, as well as engineering approaches to optimize the effectiveness of both Fab and Fc mAb domains. This optimization will be engineered through: (i) use of yeast surface display to evolve Fv affinity for antigen targets; (ii) generation of point mutations in the Fc domain known to alter affinity for Fcg receptors (FcgR) and in turn affect antibody-dependent cell-mediated cytoxicity); and (iii) alterations in Fc glycosylation via expression in specialized cell lines with (a) CRISPR/Cas9-mediated knockout or (b) lentiviral-mediated knockin of enzymes governing carbohydrate synthesis, or (c) point mutations in the Fc domain that abolish core fucosylation. We will assess a range of immune functions mediated by the engineered mAbs using both high-throughput systems serology and cell-based assays, and work with all three Research Projects to determine performance of optimized antibodies against authentic viruses in cell culture and in animal models. Core B will also integrate with Core C (Computational Analysis and Modeling Core) to interpret our results and reveal correlations between mAb features and measures of protection determined by the Projects. We will work with Core A (Administrative Core) to ensure that data are curated and shared among all projects and other stakeholders. Together, we will discover and deliver optimized, highly efficacious mAb therapeutics against three major families of viruses. We further seek to understand what findings are general rules and what strategies are specific to each virus family, to provide systems for antibody choice and optimization that do not yet exist, and to build a broadly applicable platform for mAb discovery and delivery against novel pathogens as well.