Novel PE-DT-fusion toxin enhances internalization and target-dependently restores the cytotoxicity of recombinant immunotoxins.

BACKGROUND: Recombinant immunotoxins (RITs) are fusion proteins of a targeting domain, such as an antibody fragment, and a truncated toxin, including Pseudomonas exotoxin A (PE) or diphtheria toxin (DT). Limiting their possible fusion partners, the targeting domain of DT is typically fused C-terminally and that of PE is fused N-terminally. Among other factors, the activity of immunotoxins depends on the target antigen and the target-specific intracellular trafficking. Because a novel anti-CD138-PE immunotoxin was inactive against multiple myeloma, we hypothesized that rational toxin design would improve trafficking and, thus, cytotoxicity. METHODS: We, therefore, generated several variants with distinct domain sequences including the catalytic (C) units of PE and DT, the furin-cleavage site of PE (fu), the transport (T) domain of DT, and the ER retention sequence KDEL. The lead candidate-a fusion of PE and DT-was combined with antibodies against CD138, CD22, glypican-3, or mesothelin. RESULTS: This final toxin moiety was internalized 4-fold more efficiently over time on average compared to PE-based RITs, regardless of the target antigen or cell type. Improved internalization of DT over PE did not depend on a specific domain of DT. Instead, it was competed dose-dependently by poly-D lysine (PDL), indicating a more unspecific charge effect of DT over PE. The improved internalization frequently translated to enhanced cytotoxicity. Finally, combinatorial treatment with actinomycin D demonstrated synergistic effects with the lead PE-DT-fusion toxin. CONCLUSION: This novel and target-dependently more potent toxin moiety may provide a framework for future immunotoxin design, possibly widening the range of target antigens, including the ongoing efforts to enhance mesothelin- or CD138-targeting immunotoxins against cancer or autoimmune diseases.