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 convenient site for covalent attachment of ETA to other proteins in the production of chimeric toxins. The mutation was then introduced into a variant of ETA that is impaired in receptor binding, termed ETA-60EF61, that has the dipeptide Glu-Phe inserted between residues 60 and 61. The resulting double mutant, ETA-60EF61 Cys161, was conjugated to three different monoclonal antibodies via a thioether linkage, and the immunotoxins were tested for cytotoxicity with cells in culture. Each immunotoxin was extremely potent against cells that expressed surface determinants for the monoclonal antibodies but had little cytotoxicity for cells that did not bind the antibodies. For comparison, we also conjugated ricin A chain to each of the three monoclonal antibodies and found that the resulting immunotoxins were at least two-orders of magnitude less potent than the corresponding immunotoxins made with ETA-60EF61Cys161. This study demonstrates that ETA-60EF61Cys161 makes potent and specific immunotoxins and may potentially be useful in selectively eliminating subpopulations of cells in vitro and in vivo.
Chaudry
G J
GJ
Molecular and Cell Biology Program, University of Texas at Dallas, Richardson 75083.
Fulton
R J
RJ
Draper
R K
RK
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D001427:Bacterial Toxins; D001483:Base Sequence; D002352:Carrier Proteins; D002470:Cell Survival / Q000187:drug effects*; D003001:Cloning, Molecular; D003545:Cysteine; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity*; D014644:Genetic Variation; D006801:Humans; D000922:Immunotoxins / Q000633:toxicity*; D007739:L Cells; D008715:Methionine; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000235:genetics; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity; D015183:Restriction Mapping; D014407:Tumor Cells, Cultured; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D002352:Carrier Proteins; D003545:Cysteine; D005098:Exotoxins; D000922:Immunotoxins; D008715:Methionine; D009838:Oligodeoxyribonucleotides; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C027553:Pseudomonas exotoxin binding protein, mouse