Single-Chain Fab Chain-Exchange (scFab-PACE) converts targeted prodrugs into functional T cell engagers on tumor cells.

T-cell bispecific antibodies (TCBs) have demonstrated significant clinical potential; however, their widespread application remains limited by the lack of truly tumor-specific targets. Conditionally active TCBs (proTCBs) are being designed to mitigate safety risks by becoming activated preferentially at the tumor site. Our previously published Prodrug-Activating Chain Exchange (PACE) approach utilized two functionally inactive antibody derivatives that, upon co-accumulation on a tumor cell, undergo a chain-exchange reaction to reconstitute an active CD3 binder. Here, we present a second-generation PACE approach that overcomes two key limitations of the original concept. First, we refined the PACE prodrug design by including an intact Fc domain, enabling FcRn binding to improve pharmacokinetic properties. Second, we moved the conditional effector domain into one of the Fab arms and covalently connected it with a flexible linker, yielding a single-chain Fab (scFab). These new scFab-PACE prodrugs consist of a targeting arm as well as a scFab effector arm composed of an Fd region and a light chain from two separate binders, rendering the scFab inactive. The Fd/light chain interface of the scFab is destabilized by repulsive charges introduced into the framework regions of the variable domains, which leads to chain-exchange and binder activation upon accumulation on target cells. We demonstrate that the scFab linker reduces in-solution chain exchange, which aims to reduce the risk of nonspecific prodrug activation in the periphery. We demonstrate the feasibility of this approach for the generation of proTCBs using HER2-targeting scFab-PACE precursors with an inactive CD3 binder in the effector arm. These prodrugs selectively gain CD3 binding functionality upon accumulation on HER2-expressing tumor cells in a HER2-density-dependent manner. Furthermore, the scFab-PACE technology enables the design of prodrugs containing two distinct conditionally active binding entities. We demonstrate this feature by generating prodrugs that conditionally engage both CD3 and CD28, allowing for target-specific activation of T cell signal 1 and signal 2 by one distinct pair of prodrugs.