Intracellular trafficking of furin enhances cellular intoxication by recombinant immunotoxins based on Pseudomonas exotoxin A.

Abstract

Furin is a mammalian serine protease with important roles in cellular homeostasis and disease. It cleaves and activates numerous endogenous and exogenous substrates, including the SARS-CoV-2 viral spike protein and protein toxins such as diphtheria toxin and Pseudomonas exotoxin A (PE). Recombinant immunotoxins (RITs) are toxin conjugates used as cancer therapeutics that connect tumor-directed antibodies with toxins for targeted cell killing. RITs based on PE have shown success in treating a variety of cancers, but often suffer from safety and efficacy concerns when used clinically. We have explored furin as a potential limiting factor in the intoxication pathway of PE-based RITs. Although the furin has widely recognized importance in RIT intoxication, its role is incompletely understood. Circumstantial evidence suggests that furin may act as a transporter for RITs in addition to its role of activation by cleavage. Here, we describe the creation of a CRISPR-engineered furin-deficient HEK293 cell line, ΔFur293. Using ΔFur293 and derivatives that express mutant forms of furin, we confirm the importance of furin in the PE RIT intoxication pathway and show that furin trafficking has a significant impact on RIT efficacy. Our data support the hypothesis that furin acts as a transporter during RIT intoxication and suggest furin as a target for modification to improve the effectiveness of RITs.