Directed evolution of a picomolar-affinity, high-specificity antibody targeting phosphorylated tau

Abstract

Antibodies are essential biochemical reagents for detecting protein post-translational modifications (PTMs) in complex samples. However, recent efforts in developing PTM-targeting antibodies have reported frequent nonspecific binding and limited affinity of such antibodies. To address these challenges, we investigated whether directed evolution could be applied to improve the affinity of a high-specificity antibody targeting phosphothreonine 231 (pThr-231) of the human microtubule-associated protein tau. On the basis of existing structural information, we hypothesized that improving antibody affinity may come at the cost of loss in specificity. To test this hypothesis, we developed a novel approach using yeast surface display to quantify the specificity of PTM-targeting antibodies. When we affinity-matured the single-chain variable antibody fragment through directed evolution, we found that its affinity can be improved >20-fold over that of the WT antibody, reaching a picomolar range. We also discovered that most of the high-affinity variants exhibit cross-reactivity toward the nonphosphorylated target site but not to the phosphorylation site with a scrambled sequence. However, systematic quantification of the specificity revealed that such a tradeoff between the affinity and specificity did not apply to all variants and led to the identification of a picomolar-affinity variant that has a matching high specificity of the original phosphotau antibody. In cell- and tissue-imaging experiments, the high-affinity variant gave significantly improved signal intensity while having no detectable nonspecific binding. These results demonstrate that directed evolution is a viable approach for obtaining high-affinity PTM-specific antibodies and highlight the importance of assessing the specificity in the antibody engineering process.

Keywords: affinity; anti-PTM antibody; antibody; antibody specificity; immunochemistry; neurodegeneration; phospho-specific; post-translational modification; protein phosphorylation; tau protein (tau).