Evolution-guided design of mini-protein for high-contrast in vivo imaging

Available online 15 July 2025

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Traditional development of small protein scaffolds has relied on display technologies and mutation-based engineering, which limit sequence and functional diversity, thereby constraining their therapeutic and application potential. Protein design tools have significantly advanced the creation of novel protein sequences, structures, and functions. However, further improvements in design strategies are still needed to more efficiently optimize the functional performance of protein-based drugs and enhance their druggability. Here, we extended an evolution-based design protocol to create a novel minibinder, BindHer, against the human epidermal growth factor receptor 2 (HER2). It not only exhibits super stability and binding selectivity but also demonstrates remarkable properties in tissue specificity. Radiolabeling experiments with 99mTc, 68Ga, and 18F revealed that BindHer efficiently targets tumors in HER2-positive breast cancer mouse models, with minimal nonspecific liver absorption, outperforming scaffolds designed through traditional engineering. These findings highlight a new rational approach to automated protein design, offering significant potential for large-scale applications in therapeutic mini-protein development.

Graphical abstractThrough the application of an evolution-guided design approach, we have successfully automated the creation of mini-protein aimed at selectively targeting tumors. This strategy also significantly reduces nonspecific absorption by liver tissues, presenting a promising opportunity for the scalable development of therapeutic mini proteins.