Antibody translocation into the cytosol: Mechanisms and engineering of endosomal escape

The development of cytosol-penetrating immunoglobulin G (IgG) antibodies that autonomously enter target cells and reach the cytosol is challenging despite their potential to enable the therapeutic targeting of intracellular antigens and delivery of functional payloads. Inefficient endosomal escape after receptor-mediated internalization underlies the inability of IgGs to reach the cytosol as it requires the energetically unfavorable passage of a large hydrophilic macromolecule across the hydrophobic endosomal membrane. In this review, we first summarize passive and active strategies for cytosolic antibody delivery and then critically examine active approaches that use rational engineering to embed intrinsic endosomal escape motifs in antibodies, emphasizing both the mechanistic foundations and current limitations. To develop next-generation cytosol-penetrating antibodies, we propose a reaction coordinate-based mechanistic framework that defines distinct energy barriers at each sequential stage of endosomal escape: dissociation from receptors within endosomes, endosomal membrane engagement, membrane-bound intermediates, cooperative clustering-induced membrane destabilization, and translocation across the lipid bilayer. This framework provides actionable design principles for generating cytosol-penetrating antibodies with improved intracellular delivery efficiency and translational potential. Finally, we discuss additional considerations associated with endosomal escape motif engineering, including receptor selection for cellular uptake, immunogenicity and developability risks, and quantitative assessment of cytosolic antibody delivery.