Leveraging Monoclonal Antibody Engineering to Elucidate Fc Receptor-Mediated Activation in Monocytes 2675

Abstract Description Antibody Fc-mediated activation of innate immune cells via Fc-receptors (FcRs) is central to antibody defense against pathogens. Multiple FcRs are expressed on innate immune cells such as monocytes; however, the specific contributions of each FcR and the influence of their combined engagement in driving effector pathways remain unclear. Defining these pathways is essential for designing monoclonal antibody (mAb) therapies for infectious diseases and cancer. To identify the pathways activated by antibodies in monocytes, we developed a panel of mAbs based on the SARS-CoV-2 mAb, CR3022, with Fc-domain point mutations that alter binding affinities for FcγR1, Fcγ2A, and FcγR3A. Wild type IgG1 binds all FcγRs, SDIESA LS and SEHFST LS show reduced affinities for FcγR2A and FcγR3A, respectively. LALA LS exhibits high binding affinity for FcγR1 only, and PAAD LS does not bind to any FcγRs. Using these antibodies bound to SARS-CoV-2 Spike protein, we activated THP-1 monocytes and performed capped small RNA-seq to analyze differential gene expression and transcription factor binding site enrichment linked to FcR engagement. Preliminary data reveal distinct pathways are induced by mAbs with varying FcγR binding profiles, including those related to phagocytosis, cytoskeleton organization, cell migration, cytokine signaling, and antiviral responses. Additional experiments using FcγR knockout cells will further define each FcR’s role in pathway activation. Funding Sources NIH: T32GM152310 NIH:5R21AI176078-02 Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)