Protein engineering of diphtheria-toxin-related interleukin-2 fusion toxins to increase cytotoxic potency for high-affinity IL-2-receptor-bearing target cells

We have used site-directed insertion and point mutagenesis inan attempt to increase the cytotoxic potency and receptor-bindingaffinity of the diphtheria-toxin-related interleukin-2 (IL-2)fusion toxins. Previous studies have demonstrated that boththe DAB₄₈₆-IL-Z and DAB₃₈₉-IL-2 forms of the fusion toxin consistof three functional domains: the N-tenninal fragment-A-assodatedADP-ribosyltransferase, the hydrophobk-membrane-associatingdomains, and the C-terminal receptor-binding domain of humanIL-2. By insertion mutagenesis we have increased the apparentflexibility of the polypeptide chain between the membraneassociatingdomains and the receptor-binding domain of this fusion toxin.In comparison to DAB₄₈₆-IL-2, the cytotoxic potency of the insertionmutants was increased by 17-fold for high-affinity IL-2-receptor-bearingcell lines in vitro. Moreover, competitive displacement experimentsusing ¹²⁵I]rIL-2 demonstrate that the increase in cytotoxicpotency correlates with an increase in receptor-binding affinityfor both the high and intermediate forms of the IL-2 receptor.