Abstract

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—an intrinsically multidimensional challenge. We develop 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 crosslinking kinetics ( k obs = 0.18 min −1 , t 1/2 = 3.8 min) and improved tumor suppression compared with envafolimab and atezolizumab. Similarly, we engineered a fast-acting covalent interleukin-18 (IL-18) ( k obs = 0.54 min −1 , t 1/2 = 1.3 min) and a covalent miniprotein targeting the receptor binding domain (RBD) of SARS-CoV-2, demonstrating applicability across protein modalities.