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Initial characterization of an immunotoxin constructed from domains II and III of cholera exotoxin

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Abstract

Immunotoxins are antibody–toxin fusion proteins under development as cancer therapeutics. In early clinical trials, immunotoxins constructed with domains II and III of Pseudomonas exotoxin (termed PE38), have produced a high rate of complete remissions in Hairy Cell Leukemia and objective responses in other malignancies. Cholera exotoxin (also known as cholix toxin) has a very similar three-dimensional structure to Pseudomonas exotoxin (PE) and when domains II and III of each are compared at the primary sequence level, they are 36% identical and 50% similar. Here we report on the construction and activity of an immunotoxin made with domains II and III of cholera exotoxin (here termed CET40). In cell viability assays, the CET40 immunotoxin was equipotent to tenfold less active compared to a PE-based immunotoxin made with the same single-chain Fv. A major limitation of toxin-based immunotoxins is the development of neutralizing antibodies to the toxin portion of the immunotoxin. Because of structure and sequence similarities, we evaluated a CET40 immunotoxin for the presence of PE-related epitopes. In western blots, three-of-three anti-PE antibody preparations failed to react with the CET40 immunotoxin. More importantly, in neutralization studies neither these antibodies nor those from patients with neutralizing titers to PE38, neutralized the CET40-immunotoxin. We propose that the use of modular components such as antibody Fvs and toxin domains will allow a greater flexibility in how these agents are designed and deployed including the sequential administration of a second immunotoxin after patients have developed neutralizing antibodies to the first.

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Notes

  1. (for results sections) Amino acids 270–634 of CET encompass domains II, III and a small sub-domain termed, Ib. For simplicity, domain Ib is not routinely mentioned (also see “Discussion”).

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Acknowledgments

This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.

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Authors and Affiliations

  1. Laboratory of Molecular Biology, CCR, NCI, NIH, HHS, 37 Convent Dr, Room 5124, Bethesda, MD, 20892, USA

    Robert Sarnovsky, Tara Tendler, Matheusz Makowski, Maureen Kiley, Antonella Antignani, Roberta Traini, Jingli Zhang, Raffit Hassan & David J. FitzGerald

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  1. Robert Sarnovsky
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  2. Tara Tendler
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Correspondence to David J. FitzGerald.

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262_2009_794_MOESM1_ESM.eps

Supplemental Fig 1. Fractions of HB21-CET40 eluted from TSK G3000 column. Fractions 19-30 are shown after migration through a 4-20% Tris–glycine precast gel under reducing and non-reducing conditions. Fractions 28 and 29, marked with an asterisk, were used for experiments described in this paper (EPS 14682 kb)

262_2009_794_MOESM2_ESM.jpg

Supplemental Fig 2. The monoclonal antibody M40-1 was mixed with twice the desired final immunotoxin concentration for 1 hr at room temp and then added to each well of DLD-1 cells in a 96-well format. After a 48-hr incubation, cell viability was assessed using the WST-1 reagent. Each bar represents a replicate of 5 with the error bar indicating one SD. Comparisons of immunotoxin activity with and without antibody incubations are indicated with thin black lines (JPG 84 kb)

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Sarnovsky, R., Tendler, T., Makowski, M. et al. Initial characterization of an immunotoxin constructed from domains II and III of cholera exotoxin. Cancer Immunol Immunother 59, 737–746 (2010). https://doi.org/10.1007/s00262-009-0794-4

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  • DOI: https://doi.org/10.1007/s00262-009-0794-4

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