Engineering Mechanostable Anticalin Scaffolds to Enhance Particle Adhesion and Targeting of CTLA-4 Under Shear Stress

Achieving high binding strength and efficient delivery of molecular cargo to cells expressing biomarker targets is a significant challenge in drug delivery. Here, we investigated how altering the surface immobilization residue (i.e., anchor point) within a non-antibody binding scaffold called Anticalin can enhance particle adhesion to the immune checkpoint protein CTLA-4 on mammalian cells under shear stress. By introducing bio-orthogonal clickable amino acids into Anticalin at various positions and applying tension to the protein complex using single-molecule AFM force spectroscopy and bead-based adhesion assays, we elucidate the relationship between anchor point position and mechanostability of the Anticalin:(CTLA-4) complex. Multi-regression analysis of the physicochemical properties of the anchor points revealed that the distance from the anchor point on Anticalin to CTLA-4's center of mass was a major determinant of binding strength under shear flow. These results demonstrate how anchor point engineering can enhance particle adhesion and cellular delivery to CTLA-4 targets and provide a heuristic for choosing surface immobilization points of targeting proteins such that they withstand high mechanical forces.