Cross-species cellular mapping and humanization of Fc? receptors to advance antibody modeling

Fc receptors mediate the effector functions of therapeutic antibodies. Immunoglobulin G (IgG), the predominant antibody in circulation and clinical use, engages diverse Fc gamma (Fc?) receptors that are expressed by multiple immune and structural cells. Here, we provide a comprehensive overview of Fc? receptors and FcRn expression at both the transcriptomic and proteomic level in humans, macaques, and mice. We reveal species-specific differences in both Fc? receptor diversity and cell-specific expression profile that compromise the translation of mouse and macaque preclinical models for antibody research. To overcome this, we generated a new mouse model in which human Fc?RI (hCD64), Fc?RIIA (hCD32A), Fc?RIIB (hCD32B), Fc?RIIIA (hCD16A), Fc?RIIIB (hCD16B) is expressed under control of the relevant human promotors, replacing the murine counterparts (Fc?RI, Fc?RIIB, Fc?RIII, and Fc?RIV). Furthermore, to improve human antibody pharmacokinetics, murine FcRn was replaced by its human analog. We comprehensively mapped the expression of the knock-in receptors, and found that humanization led to much more faithful cell-specific Fcg receptor expression. We validated the functionality of these knock-in human receptors through in vitro and in vivo antibody-dependent cellular cytotoxicity, anaphylaxis and antigen presentation assays. This cross-species Fc? receptor atlas and humanized mouse model with refined Fcg receptor expression and antibody pharmacokinetics will improve the preclinical assessment of antibody-based therapeutics. Overall design: We carried out single-cell RNA sequencing (BD Rhapsody platform; sequenced using NovaSeq6000) on magnetically cell enriched (MagniSort) and fluoresence-activated cell sorted (FACS; BD Symphony S6) splenocytes of C57BL/6J female mice