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Modular Pore-Forming Immunotoxins with Caged Cytotoxicity Tailored by Directed Evolution.

Published version
Peer-reviewed

Type

Article

Change log

Authors

Soskine, Misha 
Huang, Gang 
Albuquerque, Inês S 
Bernardes, Gonçalo JL  ORCID logo  https://orcid.org/0000-0001-6594-8917

Abstract

Immunotoxins are proteins containing a cell-targeting element linked to a toxin that are under investigation for next-generation cancer treatment. However, these agents are difficult to synthesize, chemically heterogeneous, expensive, and show toxicity toward healthy cells. In this work, we describe the synthesis and characterization of a new type of immunotoxin that showed exquisite selectivity toward targeted cells. In our construct, targeting molecules were covalently attached or genetically fused to oligomeric pore-forming toxins. The activity of the immunotoxin was then caged by fusing a soluble protein to the transmembrane domain and activated via cleavage with furin, which is a protease that is overexpressed in many cancer cells. During the several coupling steps, directed evolution allowed the efficient synthesis of the molecules in E. coli cells, as well as selection for further specificity toward targeted cells. The final construct showed no off-target activity, while acquiring an additional degree of specificity toward the targeted cells upon activation. The pore-forming toxins described here do not require internalization to operate, while the many protomeric subunits can be individually modified to refine target specificity.

Description

Keywords

Animals, Cell Line, Tumor, Cell Membrane, Cnidarian Venoms, Directed Molecular Evolution, Drug Design, Escherichia coli, Folic Acid, Furin, Humans, Immunotoxins, Mutagenesis, Pore Forming Cytotoxic Proteins, Proteolysis, Recombinant Fusion Proteins, Salmonella typhi, Sea Anemones, Tetrahydrofolate Dehydrogenase

Journal Title

ACS Chem Biol

Conference Name

Journal ISSN

1554-8929
1554-8937

Volume Title

13

Publisher

American Chemical Society (ACS)
Sponsorship
The Royal Society (uf110046)
European Research Council (676832)