Engineering Affinity and Specificity of Antibodies Targeting Protein Modifications

Antibodies targeting protein post-translational modifications (PTMs) are an essential tool in scientific research and clinical investigations. Due to the transient and heterogeneous nature of protein PTMs, high specificity and affinity of PTM-specific antibodies are critical for their sensitive and reproducible detection. However, recent studies have revealed an alarming degree of non-specific binding found in these antibodies, and whether the affinity meets the required detection sensitivity is unclear. To address these challenges, we investigated whether directed evolution could be applied to improve the affinity of a high-specific antibody targeting phospho-threonine 231 of the human microtubule-associated protein tau. We developed a novel approach using yeast surface display to quantify the specificity of PTM-specific antibodies. This approach enabled us to measure its affinity and reveal its cross-reactivity towards the non-targets when we affinity-matured the single-chain variable antibody fragment through directed evolution. We 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. The yeast surface display approach allows specificity measurement when the sequence of the antibody is known, which makes it useful for screening antibody variants. However, for many antibody users, the assay is not readily applicable for measuring specificity, since the antibody sequence information may be unavailable. Therefore, we developed another assay that enables PTM-specificity measurement for existing, commercially available antibodies. The assay relies on multi-color flow cytometry in human embryonic kidney (HEK) cells. We defined a specificity parameter, which measures the fraction of non-specific signal in PTM-specific antibodies, and successfully applied the HEK cell-based assay to measure the specificity parameter. We validated the assay using antibodies with known specificity profiles and used it to measure the specificity of 7 widely used phospho-tau antibodies (AT270, AT8, AT100, AT180, PHF-6, TG-3, and PHF-1) among others. We anticipate that the quantitative approach and parameter introduced here will be widely adopted as a standard for reporting the specificity for phospho-tau antibodies, and potentially for PTM-specific antibodies in general