A high-throughput selection system for fast-acting covalent protein drugs.

Covalent protein drugs offer therapeutic potential but are limited by slow target engagement and the absence of high-throughput selection platforms. Rapid covalent binding requires coordinated optimization of affinity, stability, and warhead geometry, which is an intrinsically multidimensional challenge. We developed a yeast display platform coupled with chemoselective modification that enables selection of fast-acting covalent proteins without increasing intrinsic warhead reactivity. Using this system, we engineered a covalent programmed death-ligand 1 (PD-L1) antagonistic nanobody with rapid cross-linking kinetics [observed rate constant (kobs) = 0.18 min-1, half-life (t1/2) = 3.8 min] and improved tumor suppression compared with envafolimab and atezolizumab. Similarly, we engineered a fast-acting covalent interleukin-18 (kobs = 0.54 min-1, t1/2 = 1.3 min) and a covalent miniprotein targeting the receptor binding domain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), demonstrating applicability across protein modalities.