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bioRxiv · 10.1101/2024.08.16.608260

Anchor Point Engineering on Anticalin Scaffolds for Enhanced Particle Adhesion to CTLA-4 Under Shear Stress

Sun, Y.·Li, J.·Vanella, R.·Liu, H.·Liu, Z.·Nash, M.
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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-4s 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 provides a heuristic for choosing surface immobilization points of targeting proteins such that they withstand high mechanical forces.

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Sun, Y., Li, J., Vanella, R., Liu, H., Liu, Z., Nash, M.. 2024-08-17. Anchor Point Engineering on Anticalin Scaffolds for Enhanced Particle Adhesion to CTLA-4 Under Shear Stress. https://doi.org/10.1101/2024.08.16.608260

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