Cytosol delivery of immunoglobulin G (IgG) antibodies would unlock direct engagement of intracellular proteins and broaden antibody based therapeutic payload strategies, yet remains difficult because internalized IgGs rarely escape endosomes. To address this bottleneck, we created a second generation cytosol penetrating antibody (cytotransmab, CT), in2CT41, by embedding an endosomal acidity activated escape module into an IgG1 kappa scaffold. This R W E motif, composed of Arg Trp pairs together with a Glu patch positioned within the CH3 and CL domains, is designed to remain inert at neutral pH but to promote selective endosomal membrane destabilization under acidic conditions, thereby enabling cytosolic access.
In2CT4.1 achieves efficient cytosolic accumulation at nanomolar concentrations and exhibits approximately threefold higher endosomal escape efficiency than a first generation CT. The platform’s modularity was demonstrated by introducing an alpha tubulin specific variable domain to generate a cytosolic alpha tubulin targeting CT. Moreover, in2CT41 supports functional intracellular delivery of protein cargo, as shown by transport of the diphtheria toxin catalytic domain into EGFR overexpressing tumor cells, which resulted in near complete tumor growth suppression in a xenograft model. Together, these results establish in2CT41 as a versatile IgG platform for cytosolic protein targeting and payload delivery with broad relevance to targeted cancer therapy and other applications.