Abstract
Anthrax toxin protective antigen (PA) delivers its effector proteins into the host cell cytosol through formation of an oligomeric pore, which can assume heptameric or octameric states. By screening a highly directed library of PA mutants, we identified variants that complement each other to exclusively form octamers. These PA variants were individually nontoxic and demonstrated toxicity only when combined with their complementary partner. We then engineered requirements for activation by matrix metalloproteases and urokinase plasminogen activator into two of these variants. The resulting therapeutic toxin specifically targeted cells expressing both tumor associated proteases and completely stopped tumor growth in mice when used at a dose far below that which caused toxicity. This scheme for obtaining intercomplementing subunits can be employed with other oligomeric proteins and potentially has wide application.
Highlights
Anthrax toxin protective antigen (PA) forms heptameric or octameric oligomers after proteolytic activation
We focused on the portion of the codon table that would lead to charge reversal
This result was observed, with a pair of complementary proteins (7.5 g of PA-L1-GN ϩ 7.5 g of PA-U2-D512K ϩ 7.5 g of lethal factor (LF)) completely inhibiting tumor growth, whereas the individual proteins had no effect, allowing tumors to grow at the same rate as those treated with PBS (Fig. 6A)
Summary
Anthrax toxin protective antigen (PA) forms heptameric or octameric oligomers after proteolytic activation. Anthrax toxin protective antigen (PA) delivers its effector proteins into the host cell cytosol through formation of an oligomeric pore, which can assume heptameric or octameric states. The resulting therapeutic toxin targeted cells expressing both tumor associated proteases and completely stopped tumor growth in mice when used at a dose far below that which caused toxicity This scheme for obtaining intercomplementing subunits can be employed with other oligomeric proteins and potentially has wide application. We prepared a library of PA variants having the D512K substitution together with random mutations in several residues on the complementary face of PA63 within the oligomers (see Fig. 1B) and screened for a (re)gain of function Screening of this library successfully identified mutations that complement D512K. We applied the octamer strategy to create a tumor-targeting agent that had high specificity and efficacy
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