Characterization of the B Cell Epitopes Associated with a Truncated Form of Pseudomonas Exotoxin (PE38) Used to Make Immunotoxins for the Treatment of Cancer Patients

Abstract

Recombinant immunotoxins composed of an Ab Fv fragment joined to a truncated portion of Pseudomonas exotoxin A (termed PE38) have been evaluated in clinical trials for the treatment of various human cancers. Immunotoxin therapy is very effective in hairy cell leukemia and also has activity in other hemological malignancies; however, a neutralizing Ab response to PE38 in patients with solid tumors prevents repeated treatments to maximize the benefit. In this study, we analyze the murine Ab response as a model to study the B cell epitopes associated with PE38. Sixty distinct mAbs to PE38 were characterized. Mutual competitive binding of the mAbs indicated the presence of 7 major epitope groups and 13 subgroups. The competition pattern indicated that the epitopes are discrete and could not be reproduced using a computer simulation program that created epitopes out of random surface residues on PE38. Using sera from immunotoxin-treated patients, the formation of human Abs to each of the topographical epitopes was demonstrated. One epitope subgroup, E1a, was identified as the principal neutralizing epitope. The location of each epitope on PE38 was determined by preparing 41 mutants of PE38 in which bulky surface residues were mutated to either alanine or glycine. All 7 major epitope groups and 9 of 13 epitope subgroups were identified by 14 different mutants and these retained high cytotoxic activity. Our results indicate that a relatively small number of discrete immunogenic sites are associated with PE38, most of which can be eliminated by point mutations.

Recombinant immunotoxin engineered for low immunogenicity and antigenicity by identifying and silencing human B-cell epitopes

“…We are unable to test the immunogenicity of HA22-LR-LO10 in humans so instead we assessed its antigenicity; that is, the binding of HA22-LR-LO10 to preexisting antibodies made against native RIT, as a surrogate measure of the immune response (9). We previously showed in mice that immunogenicity and antigenicity are closely related (13). We performed a competition assay in which we measured the concentration of RIT that reduced the level of patient serum antibodies reacting with HA22 by 50% (18).…”

“…1). Both domains II and III contain B-cell epitopes (13). The B-cell epitopes in domain II were removed when we found that all of domain II, except for the furin-processing site, could be excised without greatly diminishing the cytotoxic activity of the RIT (17).…”

“…In previous studies we identified and removed mouse-specific B-cell epitopes by immunizing mice with RITs and isolating hybridomas that made mAbs to the immunotoxins (13). We used these mAbs to show that PE38 contained seven major epitopes, and identified their location on the protein surface through a series of point mutations (14).…”

“…3 show that we identified six human B-cell epitopes in domain III using 41 Fvs. In the previous mouse study we used 24 mAbs to identify five epitopes in domain III (13).…”

“…The pharmacokinetics and cytotoxic and antitumor activities were measured as previously described (13,16,21) using mAb IP12 for the blood levels. Animal experiments were performed under National Cancer Institute Animal Care and Use Committee-approved protocols.…”