Antibody-Mediated Neutralization of Perfringolysin O for Intracellular Protein Delivery

Abstract

Perfringolysin O (PFO) is a member of the cholesterol-dependent cytolysin (CDC) family of bacterial pore-forming proteins, which are highly efficient in delivering exogenous proteins to the cytoplasm. However, the indiscriminate and potent cytotoxicity of PFO limits its practical use as an intracellular delivery system. In this study, we describe the design and engineering of a bispecific, neutralizing antibody against PFO, which targets reversibly attenuated PFO to endocytic compartments via receptor-mediated internalization. This PFO-based system efficiently mediated the endosomal release of a co-targeted gelonin construct with high specificity and minimal toxicity in vitro. Consequently, the therapeutic window of PFO was improved by more than 5 orders of magnitude. Our results demonstrating that the activity of pore-forming proteins can be controlled by antibody-mediated neutralization present a novel strategy for utilizing these potent membrane-lytic agents as a safe and effective intracellular delivery vehicle.

Cytosolic delivery of siRNA by ultra-high affinity dsRNA binding proteins

“…This result was expected, as we have previously shown that C225.2/PFO T490A,L491V predominantly permeablizes endosomal membranes following EGFR-mediated internalization (17). Thus, delivery of siRNA is maximal when it can co-localize with PFO T490A,L491V in endosomes most efficiently.…”

Intracellular Delivery by Membrane Disruption: Mechanisms, Strategies, and Concepts

“…Multifunctional nanocarriers that switch to a membrane disrupting state are already being developed for endosomal escape purposes 6 . Similarly, conjugation with membrane-active peptides 191 or pore-forming toxins 194 can be harnessed to produce nanoscale cargo with more potent cell penetration properties. If membrane-perturbing nanoparticles can be made switchable by light or other environmental stimuli, they may confer the level of control required for reversible permeabilization at discrete locations on the cell surface.…”

Inhibition of Pore-Forming Proteins

“…PFPs also play an important role in a wide range of recent advances in nanotechnology [230,231]. By gaining new findings about their pore-forming mechanisms, we could use them more effectively in developing technologies, such as molecular sensing and detection [232,233,234,235], DNA sequencing [236,237,238], monitoring of chemical and biochemical reactions, development of biocompatible nanotransistors [239], and novel drug delivery systems and targeted killing of cells [179,230,240], as well as develop new applications. As the technology proceeds, the potential space for applications of PFP inhibitors will also grow.…”

“…The mAb enabled targeted delivery, whereas the adnectin ensured inactivity of PFO in the extracellular environment. After endocytosis, the adnectin dissociated from PFO, which lead to pore formation on membranes of endocytic compartments and the release of co-targeted protein with therapeutic effect [179]. Protein fusions can also aim for improved stability, such as scFv fused to the human antibody light chain constant κ domain (fusion called scAb), that bound to the PA 83 of the anthrax toxin and as such acted as a competitor of the cellular receptor for PA 83 binding [133].…”