A VARIANT OF EXOTOXIN-A THAT FORMS POTENT AND SPECIFIC CHEMICALLY CONJUGATED IMMUNOTOXINS

To introduce a free sulfhydryl into Pseudomonas aeruginosa exotoxin A (ETA), methionine 161 in domain I of the toxin was changed to cysteine by site-directed mutagenesis. The free sulfhydryl provides a convenie nt site for covalent attachment of ETA to other proteins in the produc tion of chimeric toxins. The mutation was then introduced into a varia nt of ETA that is impaired in receptor binding, termed ETA-60EF61, tha t has the dipeptide Glu-Phe inserted between residues 60 and 61. The r esulting double mutant, ETA-60EF61Cys161, was conjugated to three diff erent monoclonal antibodies via a thioether linkage, and the immunotox ins were tested for cytotoxicity with cells in culture. Each immunotox in was extremely potent against cells that expressed surface determina nts for the monoclonal antibodies but had little cytotoxicity for cell s that did not bind the antibodies. For comparison, we also conjugated ricin A chain to each of the three monoclonal antibodies and found th at the resulting immunotoxins were at least two-orders of magnitude le ss potent than the corresponding immunotoxins made with ETA-60EF61Cys1 61. This study demonstrates that ETA-60EF61Cys161 makes potent and spe cific immunotoxins and may potentially be useful in selectively elimin ating subpopulations of cells in vitro and in vivo.