Abstract
Anthrax lethal toxin (LT) is an A-B type toxin secreted by Bacillus anthracis, consisting of the cellular binding moiety, protective antigen (PA), and the catalytic moiety, lethal factor (LF). To target cells, PA binds to cell-surface receptors and is then proteolytically processed forming a LF-binding competent PA oligomer where each LF binding site is comprised of three subsites on two adjacent PA monomers. We previously generated PA-U2-R200A, a urokinase-activated PA variant with LF-binding subsite II residue Arg200 mutated to Ala, and PA-L1-I210A, a matrix metalloproteinase-activated PA variant with subsite III residue Ile210 mutated to Ala. PA-U2-R200A and PA-L1-I210A displayed reduced cytotoxicity when used singly. However, when combined, they formed LF-binding competent heterogeneous oligomers by intermolecular complementation, and achieved high specificity in tumor targeting. Nevertheless, each of these proteins, in particular PA-L1-I210A, retained residual LF-binding ability. In this work, we screened a library containing all possible amino acid substitutions for LF-binding site to find variants with activity strictly dependent upon intermolecular complementation. PA-I207R was identified as an excellent replacement for the original clockwise-side variant, PA-I210A. Consequently, the new combination of PA-L1-I207R and PA-U2-R200A showed potent anti-tumor activity and low toxicity, exceeding the performance of the original combination, and warranting further investigation.
Highlights
Since proteolytic activation of PA on the cell-surface is absolutely required for the action of the toxin, modification of the cleavage site amino acid sequence can create tumor-selective PA variants that are only activated in the presence of tumor-associated proteases
Two such PA variants are PA-L1, which is activated by matrix metalloproteinases (MMPs)[12,13], and PA-U214–16, which is activated by urokinase plasminogen activator
We screened a PA variant library containing all possible substitutions at residues R200, R178, I207, I210, and K214 to find PA variants with activities that were strictly dependent on intermolecular complementation and on the presence of the two distinct tumor-associated proteases to achieve highly specific tumor targeting
Summary
Since proteolytic activation of PA on the cell-surface is absolutely required for the action of the toxin, modification of the cleavage site amino acid sequence can create tumor-selective PA variants that are only activated in the presence of tumor-associated proteases. Two such PA variants are PA-L1, which is activated by matrix metalloproteinases (MMPs)[12,13], and PA-U214–16, which is activated by urokinase plasminogen activator (uPA). Subsite II is on the counterclockwise-side monomer and comprises residue R200 Mutations at these sites disrupt LF binding and result in diminished toxicity. We screened a PA variant library containing all possible substitutions at residues R200, R178, I207, I210, and K214 to find PA variants with activities that were strictly dependent on intermolecular complementation and on the presence of the two distinct tumor-associated proteases to achieve highly specific tumor targeting
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