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

T-cell engaging antibodies (TCBs) have significant clinical potential; however, their application can be limited by a narrow therapeutic window. TCB prodrugs (proTCBs) that become preferentially activated at tumors may mitigate safety risks. Our previously published prodrug-activating chain exchange (PACE) approach utilized two inactive antibody derivatives that, upon coaccumulation on tumor cells, undergo chain-exchange reactions to reconstitute active CD3 binders. Here, we present a second-generation PACE approach. We refined the PACE prodrugs by including Fc domains to improve pharmacokinetic properties, and placed the conditional effector domain into linker-connected single-chain Fab (scFab) arms. These scFab-PACE prodrugs harbor a tumor-targeting arm and an inactive scFab effector arm composed of an Fd region and light chain from two separate binders. The Fd/light chain interface of the scFab harbors repulsive charges which triggers chain-exchange and binder activation upon accumulation on target cells. The scFab linker minimizes in-solution chain exchange, which reduces the risk of nonspecific prodrug activation. We demonstrate the feasibility of this approach for the generation of proTCBs using HER2-targeting scFab-PACE precursors with a CD3 binder prodrug as the effector. These prodrugs gain CD3-binding functionality upon accumulation on tumor cells in a HER2-density-dependent manner. ScFab-PACE also enables the combination of dual prodrug functionalities. We demonstrate this feature by generating prodrugs that conditionally engage both CD3 and CD28, allowing for target-specific simultaneous activation of T cell signal 1 and signal 2.