Anticalin is a non-antibody protein scaffold with potential as an alternative to monoclonal antibodies for targeted drug delivery to cytotoxic T-lymphocyte antigen 4 (CTLA-4) positive T-cells. In this context, one limiting factor is the ability of the anticalin:CTLA-4 complex to resist mechanical forces exerted by local shear stress. Here, we used single-molecule AFM force spectroscopy (AFM-SMFS) to screen residues along the anticalin backbone and determine the optimal anchor point that maximizes binding strength of the anticalin:CTLA-4 complex. We used non-canonical amino acid incorporation by amber suppression combined with click chemistry to attach an Fgβ peptide at internal residues of the anticalin. We then used Fgβ as a handle to pick up and mechanically dissociate anticalin from CTLA-4 from eight different anchoring residues using an AFM cantilever tip. By quantifying the unbinding energy landscape for each pulling geometry, we found that pulling on the anticalin from residue 60 or 87 resulted in significantly higher rupture forces and a decrease in koff by 2-3 orders of magnitude over a force range of 50-200 pN. Five of the six internal pulling points tested were significantly more stable than N- or C-terminal anchor points, rupturing at up to 250 pN at loading rates of 0.1-10 nN/sec. Anisotropic network modelling, along with molecular dynamics (MD) simulations using the Gō-MARTINI approach explain the mechanism underlying the geometric dependency of mechanostability. These results suggest that optimization of attachment residue position for therapeutic and diagnostic cargo can provide large improvements in binding strength, allowing affinity maturation without requiring genetic mutation of binding interface residues.