Engineered pore-forming proteins for the intracellular delivery of macromolecular therapeutics

Macromolecules such as proteins and nucleic acids can potentially be used as powerful therapeutics that reconstitute lost cellular functions or inhibit aberrant functions with high specificity. However, while many macromolecules require cytoplasmic access for function, they are prohibited from voluntarily crossing the cell membrane. The bacterial pore-forming protein Perfringolysin O (PFO) can efficiently permeablize the cell membrane to allow the passage of macromolecular payloads, but severe cytotoxicity limits its therapeutic window. We have thus engineered a protein binder against PFO that sterically inhibits pore formation when bound to its target, but allows normal function once it is dissociated. By fusing this PFO binder with a monoclonal antibody against EGFR, a validated cancer target, we successfully created a bi-specific antibody that can simultaneously attenuate PFO and deliver it to EGFR-expressing cells with high specificity. Combined with fine-tuning of PFO´s affinity for its native cell-surface receptor cholesterol, the therapeutic window of delivery was increased by four orders of magnitude in vitro compared to wild-type PFO. Gelonin, a membrane-impermeable protein toxin, was used as the payload. In addition, this antibody/PFO system was well tolerated in vivo, and mediated the intracellular delivery of gelonin in a subcutaneous tumor model. In overall, we demonstrate an engineering strategy that can successfully modulate the balance between safety and efficacy for an otherwise cytotoxic pore-forming protein, for therapeutic applications as an intracellular delivery system for macromolecular drugs.