De Novo Design of High-Affinity HER2-Targeting Protein Minibinders

Human Epidermal Growth Factor Receptor 2 (HER2) is a key therapeutic target in breast cancer. However, the application of existing anti-HER2 antibody drugs is limited by such issues as large molecular weight and poor stability. In this study, a series of small protein minibinders targeting HER2 domain IV were de novo designed using the RFdiffusion method. Candidate molecules were selected through a combination of ProteinMPNN and AlphaFold2 screening, and their binding capabilities were further evaluated using <i>Escherichia coli</i> surface display coupled with flow cytometry analysis. By integrating molecular dynamics simulations, confocal fluorescence imaging, and isothermal titration calorimetry (ITC) experiments, a highly efficient minibinder (0_703_6) with nanomolar affinity and a smaller molecular size was finally identified. Compared with the existing drug molecules, the identified minibinder exhibited approximately threefold higher affinity and a threefold reduction in molecular size. This study provides strong support for the development of novel, stable, and easily expressible HER2-targeted therapeutic molecules and also offers new insights into the rapid development of robust breast cancer drugs that may serve as ideal alternatives to monoclonal antibodies.