Enzymatic conversion of prodrugs at disease sites offers specific targeting of different pathophysiology and decreased off-target toxicity. To date, the use of exogenous enzymes directed by antibodies has been burdened by dose-limiting toxicities due to off-target activation of prodrug. The enzyme-fusion proteins remain in circulation, complicating administration schedules and necessitating inhibition of enzymes persisting in the blood. Split-enzymes, unlike their full counterparts, are inactive as individual fragments in circulation, but refold after binding to their respective target. The resulting site-activated approach could provide a promising solution to mitigate off-target activation of prodrugs. In one composition, individual fragments of a binary split beta-lactamase are fused to HER2 binding proteins. As the two unique binder-enzyme fragment fusion proteins engage HER2, the induced proximity of the two fragments drives folding, resulting in an active beta-lactamase capable of hydrolyzing an array of prodrugs. Molecular modeling, in tandem with the generation of a molecular library, enabled screening of various fusion protein formats incorporating different binders and orientations of enzyme fragments. Ultimately, a DARPin-directed fragment in combination with a Fab-directed fragment exhibit the anticipated target-engaged complementation of the split beta-lactamase fragments upon binding HER2. Various characterization studies validate the specificity of the system to HER2, dependence of enzymatic activity on the presence of both fragments, influence of individual fusion concentrations on activity, and epitope specificity of both fusion proteins. Reconstitution of an active enzyme using this system has been shown to facilitate specific release of beta-lactam prodrugs, exerting cytotoxic effects on target-expressing cells. Furthermore, <i>in vivo</i> studies have demonstrated the feasibility of this approach using a comparable surrogate system producing a bioluminescent signal upon target binding in xenograft mice.

Split-enzyme activation induced by target binding: Applicability to targeted drug delivery and <i>in vivo </i>molecular imaging

2019 Fall National Meeting

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Split-enzyme activation induced by target binding: Applicability to targeted drug delivery and in vivo molecular imaging