摘要：SummarySystematic control ofin vivobehavior of protein-based therapeutics is considered highly desirable for improving their clinical outcomes. Modulation of biochemical properties including molecular weight, surface charge, and binding affinity has thus been suggested to enhance their therapeutic effects. However, establishing a relationship between the binding affinity and tumor localization remains a debated issue. Here we investigate the influence of the binding affinity of proteins on tumor localization by using four repebodies having different affinities to EGFR. Biochemical analysis and molecular imaging provided direct evidence that optimal affinity with balanced target binding and dissociation can facilitate deep penetration and accumulation of protein binders in tumors by overcoming the binding-site-barrier effect. Our findings suggest that binding kinetics-based protein design can be implicated in the development of fine-tuned protein therapeutics for cancers.Graphical AbstractDisplay OmittedHighlights•High binding affinity limits the tumor localization of protein bindersin vivo•Moderate-affinity binders can exhibit better tumor localization than higher binders•Binding kinetics of binders play a central role in controlling tumor localization•Exploring the optimal affinity of binders can enhance their therapeutic potentialBiochemistry; molecular medicine; cancer
