(553a) Harnessing Multiparatopic Antibodies for Targeted Intracellular Therapeutic Delivery

Intracellular proteins make up a significant portion of the proteome, motivating interest in achieving immune modulation via intracellularly targeted therapeutics. Most intracellular protein-targeted drugs are small molecules, which can easily pass through the cell membrane but are hindered by a lack of specificity and short half-life. Antibodies, thanks to their greater contact surface area, can have superior target specificity and persist longer in the blood, allowing them to engage specific receptors and cell types. Fusing a therapeutic to an antibody presents a promising solution, but antibodies lack an efficient path into the cell.

We engineered a delivery platform based on our recent discovery of a multiparatopic antibody that binds multiple epitopes on the immune checkpoint protein programed death-ligand 1 (PD-L1) to induce its downregulation. This multiparatopic antibody promotes endocytosis through clustering receptors together on the surface and enhancing internalization and degradation, and we exploited this mechanism for intracellular delivery. We introduced endosomalytic peptides into the multiparatopic antibody to induce its escape from the endosome into the cytosol. We inserted two different endosomalytic peptides into the multiparatopic antibody’s heavy chain, both in the hinge region and at the C-terminus. We found that these peptides do not hinder downregulation, and we observed decreased lysosomal accumulation of the antibodies containing these peptides.

To determine the efficacy of these modified antibodies as vehicles for intracellular therapeutic delivery, we fused Cas9 to the C-terminus of the heavy chain (HC) of our anti-PD-L1 multiparatopic antibody using the SpyTag/SpyCatcher system. We further demonstrated robust PD-L1 downregulation and a 25% increase in endosomal escape for these fusion proteins. These results offer proof-of-concept establishing a promising new strategy for intracellular delivery, with wide-ranging applications including gene therapy, signaling modulation, and genetic engineering.