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

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 (kobs = 0.18 min-1, t1/2 = 3.8 min) and improved tumor suppression compared with envafolimab and atezolizumab. Similarly, we engineered a fast-acting covalent interleukin-18 (IL-18) (kobs = 0.54 min-1, t1/2 = 1.3 min) and a covalent miniprotein targeting the receptor binding domain (RBD) of SARS-CoV-2, demonstrating applicability across protein modalities.