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Intermolecular complementation achieves high-specificity tumor targeting by anthrax toxin

Nature Biotechnology volume 23, pages 725–730 (2005)Cite this article

Abstract

Anthrax toxin protective antigen (PrAg) forms a heptamer in which the binding site for lethal factor (LF) spans two adjacent monomers1,2. This suggested that high cell-type specificity in tumor targeting could be obtained using monomers that generate functional LF-binding sites only through intermolecular complementation. We created PrAg mutants with mutations affecting different LF-binding subsites and containing either urokinase plasminogen activator (uPA) or matrix metalloproteinase (MMP) cleavage sites. Individually, these PrAg mutants had low toxicity as a result of impaired LF binding, but when administered together to uPA- and MMP-expressing tumor cells, they assembled into functional LF-binding heteroheptamers. The mixture of two complementing PrAg variants had greatly reduced toxicity in mice and was highly effective in the treatment of aggressive transplanted tumors of diverse origin. These results show that anthrax toxin, and by implication other multimeric toxins, offer excellent opportunities to introduce multiple-specificity determinants and thereby achieve high therapeutic indices.

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Figure 1: Schematic representation of intermolecular complementation by mutated PrAg proteins.
Figure 2: PrAg proteins with different LF-binding subsite mutations can complement LF binding and toxicity.
Figure 3: Efficient killing of human melanoma A2058 cells requires the intermolecular complementation of the two groups of PrAg proteins.
Figure 4: Potent intermolecular complementation-dependent tumoricidal activity of the engineered PrAg proteins.

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Acknowledgements

We thank Dana Hsu for assistance with toxin purification.

Author information

Authors and Affiliations

  1. Microbial Pathogenesis Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Shihui Liu, Jeffrey G Kuremsky & Stephen H Leppla

  2. Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Vivien Redeye, Marissa Kuhnen, Alfredo Molinolo & Thomas H Bugge

Authors
  1. Shihui Liu
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  2. Vivien Redeye
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  3. Jeffrey G Kuremsky
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  7. Stephen H Leppla
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Correspondence to Thomas H Bugge or Stephen H Leppla.

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Supplementary information

Supplementary Fig. 1

Toxicity of the engineered PrAg proteins. (PDF 853 kb)

Supplementary Table. 1

Properties and maximum tolerated doses of PrAg proteins when injected intraperitoneally at days 0, 3, and 6. (PDF 37 kb)

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Liu, S., Redeye, V., Kuremsky, J. et al. Intermolecular complementation achieves high-specificity tumor targeting by anthrax toxin. Nat Biotechnol 23, 725–730 (2005). https://doi.org/10.1038/nbt1091

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