Tac (CD25) is expressed on multiple hematological malignancies and is a target for cancer therapies. LMB-2 is an extremely active anti-Tac recombinant immunotoxin composed of an Fv that binds to Tac and a 38kDa fragment of Pseudomonas exotoxin A (PE38). While LMB-2 has shown high cytotoxicity towards Tac-expressing cancer cells in clinical trials, its efficacy was hampered by the formation of anti-drug antibodies against the immunogenic bacterial toxin and by dose-limiting off-target toxicity. To reduce toxin immunogenicity and non-specific toxicity, we introduced six point mutations into domain III that were previously shown to reduce T cell immunogenicity and deleted domain II from the toxin, leaving only the 11aa furin cleavage site which is required for cytotoxic activity. While this strategy has been successfully implemented for mesothelin and CD22 targeting immunotoxins, we found that removal of domain II significantly lowered the cytotoxic activity of αTac immunotoxins. To restore cytotoxic activity in the absence of PE domain II, we implemented a combined rational design and screening approach to isolate highly active domain II deleted toxin variants. The domain II deleted variant with the highest activity contained an engineered disulfide-bridged furin cleavage site designed to mimic its native conformation within domain II. We found that this approach restored five-fold of the cytotoxic activity and dramatically improved the maximum tolerated dose. Both of these improvements led to significantly increased anti-tumor efficacy in vivo. We conclude that the next generation anti-Tac immunotoxin is an improved candidate for targeting Tac expressing malignancies.
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