IgE-immunotoxins. II. IgE-ricin A-chain

Abstract

In order to develop a reagent capable of killing cells with high-affinity IgE Fc receptors, such as mast cells and basophils, ricin A-chain (the toxic portion of ricin) was conjugated to rat IgE myeloma protein, IR 162, via derivatization of the IgE by n- -succinimidyl-3-(2-pyridyldithio)propionate (SPDP) thus creating an IgE-immunotoxin. Monensin (10−7−10−8 M), a carboxylic ionophore, facilitated IgE-ricin A-chain (3 × 10−7 M) toxicity in a dose-related fashion with significant reductions in [3H]leucine incorporation compared to cells exposed only to monensin. This enhanced toxicity could be inhibited by the addition of both anti-ricin A-chain or anti-IgE, suggesting that different routes of intracellular processing may play a role in determining the toxicity of the IgE-ricin A-chain conjugate. Ricin B-chain (5 × 10−7 and 5 × 10−8 M) added to free ricin A-chain (10−6−10−8 M) reproducibly facilitated toxicity, and this toxicity could be inhibited (30–90%) by lactose (50 mM). Ricin B-chain also facilitated IgE-ricin A-chain (2.75 × 10−8 M) toxicity; however, this toxicity was not affected by lactose. The data suggest that ricin B-chain potentiates the cytosolic access of internalized IgE-immunotoxin and that the binding and internalization of the toxin was mediated via the IgE Fc receptor. A second type of IgE-ricin A-chain conjugate was synthesized whereby both IgE and ricin A-chain were derivatized with SPDP. RBL cells were killed in a dose-dependent manner by this IgE-ricin A-chain conjugate (2.5 × 10−6−2.5 × 10−9 M) without requiring the addition of monensin or ricin B-chain. These data indicate that the intracellular route and processing of internalized immunotoxin is critical to eliciting toxicity.