The application of antibodies as therapeutics is restricted to a small number of extracellular targets since antibodies cannot cross the cell membrane. To target intracellular proteins, we developed a self-assembling protein carrier (Hex carrier) that binds the conserved Fc region of antibodies and delivers them intracellularly. The carrier is comprised of a self-assembling hexamer barrel with an Fc binding domain (SPAB) fused to each monomer of the barrel. Characterization of serum stability of the Hex carrier fully loaded with Immunoglobulin G (IgG) showed that antibodies loaded on the carrier were exchanging with the excess IgG present in simulated serum conditions. In order to use Hex for <i>in vivo</i> drug delivery applications, antibodies needed to be conjugated to Hex to prevent exchange with serum IgG. The SPAB domain contains two phenylalanine residues directly facing the Fc region in the binding interface between the two proteins. To create a covalent linkage between the Fc region and SPAB domain, a non-canonical amino acid analog of phenylalanine, para-azido-phenylalanine (pAzF), was incorporated into the Hex carrier. Upon exposure to UV light, pAzF forms a reactive group that inserts into nearby C-H and N-H bonds of the protein backbone. Incorporation of pAzF was confirmed by ESI mass spectrometry and the Hex proteins containing pAzF (pHex) retained their binding function to the Fc region of antibodies. Upon crosslinking of IgG to pHex, antibody exchange with serum IgG was significantly reduced compared to the original Hex proteins and a non-crosslinked pHex control. Finally, the crosslinked pHex carriers showed significantly higher delivery of fluorescently labeled IgG inside of cells compared to Hex without pAzF. Ongoing work on the pHex proteins is examining the biodistribution and pharmacokinetics of pHex carriers in a mouse model and future work will test the delivery of a functional antibody to a relevant disease target.

Site-specific conjugation of antibodies to a protein carrier for intracellular antibody delivery

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.