Spatial architecture of complex antibody formats dictates pharmacokinetics via FcRn-recycling
Complex antibody formats are advancing in the biotherapeutic landscape to address the need for multi-specificity, tailored valency, and novel mechanisms of action. However, understanding how non-canonical architectures impact fundamental pharmacokinetic pathways remains a critical challenge. While canonical IgGs experience steric hindrance at the endosomal membrane during FcRn engagement, whether -- and how strongly -- this constraint impacts FcRn-mediated recycling across the expanding diversity of non-canonical antibody architectures has remained unknown. By systematically evaluating a diverse panel of rationally designed antibody geometries utilizing orthogonal in vitro assays we demonstrate that spatial layout profoundly influences FcRn-recycling efficiency; inverting the antibody structure by positioning the Fab-domains to the C-terminus (invertbodies) maximizes recycling, whereas crowding the N-terminus severely impairs it. In contrast, non-specific endocytosis was only modestly and inconsistently affected by Fab number, dependent on the Fv-sequence, but not by the spatial architecture itself. Notably, antibody architecture and affinity-enhancing Fc-FcRn mutations act independently yet cooperatively, ultimately revealing a physiological recycling ceiling. These FcRn-recycling dynamics translate with high fidelity to in vivo clearance (CL) in a humanized FcRn mouse model. Across the antibody panel, architecture drove a near 15-fold variance in CL, an effect that collapsed in FcRn-knockout mice. As proof of concept, we applied the invertbody design to a variant of the marketed antibody golimumab, achieving a three-fold reduction in clearance without compromising potency. These findings reveal the magnitude and predictability with which the antibody architecture governs FcRn-recycling and non-specific CL, establishing a rational, generalizable framework to optimize the pharmacokinetics of biotherapeutics.