(164p) Engineering Antibody Fusion Proteins for Targeted Intracellular Therapeutic Delivery

Intracellular proteins make up a significant portion of the proteosome, leading to an 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. and can target 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 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 and degradation. We sought to exploit this mechanism for intracellular delivery by introducing endosomalytic peptides to induce escape of the multiparatopic antibody into the cytosol. We inserted two different endosomalytic peptides into the downregulating antibody, one before and after the hinge region. 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 this modified antibody as a vehicle for an intracellular 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 results offer proof-of-concept establishing a promising new vehicle for intracellular delivery, with wide-ranging applications including gene therapy, signaling modulation, and genetic engineering.