Characterization of a ricin fusion toxin targeted to the interleukin-2 receptor

Fusion toxins are hybrid proteins consisting of peptide ligandslinked through amide bonds to polypeptide toxins. The liganddirects the molecule to the surface of target cells and thetoxin enters the cytosol and induces cell death. Ricin is anexcellent candidate for use in fusion toxins because of itsextreme potency, the extensive knowledge of its atomic structureand the lack of prior immunological exposure in patients. Wesynthesized a baculovirus transfer vector with the polyhedrinpromoter followed sequentially from the 5' end with DNA encodingthe gp67A leader sequence, the tripeptide ADP, IL-2 (interleukin-2),another ADP tripeptide and RTB (ricin toxin B chain) with lectinsitemutations W37S and Y248H. Recombinant baculovirus was generatedin Sf9 insect cells and used to infect Sf9 cells. RecombinantIL-2-RTB[W37S/Y248H] protein (fusion protein of IL-2 with modificationsW37S and Y248H) was recovered at high yields from day 6 insectcell supernatants, partially purified by affinity chromatographyand reassociated with RTA (ricin toxin A chain). The fusiontoxin was soluble, immunoreactive with antibodies to RTB, LL-2and RTA and had a molecular weight of 80 kDa by SDS-PAGE. Themolecule reacted poorly with asialofetuin, but bound stronglyto IL-2 receptor based on selective cytotoxicity to IL-2 receptorbearing cells. The specific cytotoxicity could be blocked withIL-2 but not lactose. Thus, we report a novel targeted fusiontoxin protein with full biological activity.