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.

BACKGROUND:Foreign injectable sulphate calcium has good biocompatibility, injectability and in situimmobilization, moulding based on adaptation to the shape of bone defects, but the price is expensive. OBJECTIVE:To explore the optimal fabricating parameters of bone repair materials with α-calciumsulfate hemihydrates as the main component, and to study the performance and characterization METHODS:α-Calciumsulfate hemihydrates powder was mixed with sodium hyaluronate at liquid-solid-ratios of 0.2, 0.25, 0.3, 0.35, 0.4 mL/g using vapor-heat method to prepare injectable bone materials. Performance, setting time and compressive strength of the injectable bone was detected. The best liquid-solid-ratio was 0.3 mL/g.α-Calcium sulfate hemihydrates powder was mixed with calcium sulfate dihydrate powder (1%, 2%, 3% mass fractionas) to fabricate injectable bone materials. Performance, setting time and compressive strength of the injectable bone was also detected; meanwhile, the biosafety of the injectable bone was determined. Theinjectable bone material that was made at the liquid-solid-ratio of 0.3 mL/g and by 2% calcium sulfate dihydrate was implanted into Ba-ma swine models of thoracic bone defects. At the time points of 8, 16 and 24 weeks after implantation, histological observation was done. RESULTS AND CONCLUSION: The injectable bone material was made at the liquid-solid-ratio of 0.3 mL/g and by 2% calcium sulfate dihydrate. The initial and final setting time was 4.0-5.0 minutes and 8.0-9.0 minutes, respectively. The compressive strength of the injectable bone reached (8.93±0.23) MPa. These findings indicate that the injectable boen material has good performance, initial setting time and compressive strength meeting the requirements of clinical application and good biosafety. Animal experiments show that the injectable bone can provide space for new bone in creeping substitution way by auto-degradation, with osteogenic activity.