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

Abstract 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.