Abstract Th0018: Rational Engineering of δ-Plasmin-Antibody Complexes as Direct-Acting Thrombolytics

Plasminogen activators (PAs) are the primary choice in the clinic for thrombolytic therapy for conditions such as stroke, deep vein thrombosis or pulmonary embolism. A large dose of PA is usually administered to account for its rapid clearance from the blood leading to excess conversion of plasminogen to plasmin (Pln). Although excess Pln dissolves blood clots rapidly, it also depletes various endogenous inhibitors and precursors of the thrombogenic pathway. Therefore, the success of a tPA-based thrombolytic therapy relies on intentional disruption of vascular homeostasis, which can lead to adverse events such as hemorrhage and death. It is unlikely to address the problems with PAs as the side effects are mechanism dependent. Our aim is to develop a direct acting thrombolytic that is orthogonal to the natural hemostatic process. Our designs are based on enginnered Pln-antibody complexes. Considering that the primary limitation for the clinical translation of Pln is its short half-life (<1 sec) due to rapid inhibition by alpha 2-antiplasmin (α2AP), our approach is focused on structure-based design concepts to construct Pln-antibody complexes that are resistant to α2AP inhibition. As a potential safety mechanism, our design also enables turning off the α2AP resistance after clot-dissolution, to prevent potential side effects. Our approach involves insertion of an epitope in Pln catalytic domain, which when bound to the corresponding antibody sterically blocks inhibition by α2AP. Subsequently, introduction of a peptide (with the sequence of the epitope) into the system in excess displaces the antibody to reinstate α2AP-dependent Pln inhibition. Based on molecular models of Pln-α2AP Michaelis-Menten complex, we created four variants of δ-Pln with epitopes at different sites at the α2AP binding interface. We also engineered a Fab that binds to the Pln variants with sub-nanomolar affinities. In addition to biochemical assays, we showed using crystal structures that Fab binding does not affect enzyme activity. We also demonstrated that Fab-Pln complexes acquire high resistance to α2AP inhibition in vitro, and have significantly higher half-lives in normal pooled plasma. Fab-Pln complexes also have higher efficacy at dissolving in vitro blood clots.