Engineering antibody fusion proteins for targeted intracellular therapeutic delivery

Newswise — Intracellular (IC) proteins make up a significant portion of the proteome, leading to an interest in achieving immune modulation via IC targeted therapeutics. Most IC 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. Fusing a therapeutic to an antibody presents a promising solution, but antibodies lack a good path into the cell. We recently discovered a multiparatopic antibody that binds multiple epitopes on the immune checkpoint protein programed death-ligand 1 (PD-L1) to induce its downregulation. This platform promotes endocytosis through clustering receptors together on the surface and enhancing internalization. We sought to exploit this mechanism for IC delivery by introducing endosomalytic peptides to induce escape of the multiparatopic antibody into the cytosol. We inserted two different endosomalytic peptides (EPs) into the downregulating antibody, one before and after the hinge region. We found that these EPs do not hinder downregulation and observed decreased lysosomal accumulation of the antibodies.

To determine the efficacy of this modified antibody as a vehicle for an IC therapeutic, we fused Cas9 to the C-terminus of the heavy chain (HC) of our anti-PD-L1 multiparatopic antibody. We further demonstrated robust PD- L1 downregulation and endosomal escape for these fusion proteins. These data generate proof-of-concept results, establishing a promising new vehicle for IC delivery, with wide-ranging applications including gene therapy, signaling modulation, and genetic engineering.