Post-translational modification (PTM) of proteins is a key epigenetic mechanism in regulating protein function. Modifications at specific protein sites are often implicated in diseased states. Therefore, robust and sensitive detection of PTM is highly desired in basic scientific studies, and has translational value. Unfortunately, many antibodies targeting PTMs with high specificity characterized to date have relatively low affinities (Kd &gt; 100 uM). Recently, structural studies provided valuable insights on interaction of antibodies against phosphorylated epitopes. These studies revealed that antibodies targeting PTMs interact with both the phosphate group as well as the amino acid residues of the target epitope. Such dual-mode recognition may pose a limit on the affinity and specificity of antibodies, since affinity gains may occur primarily by strengthening only one of the dual interactions. Based on this information, we asked whether it is possible to engineer the affinity of PTM-targeting antibodies, using human tau (microtubule associated protein tau, MAPT) as a model target protein. We developed a novel method that enables ratiometric imaging of antibody specificity, and applied it to assess the impact of affinity maturation on the specificity. A single chain variable region fragment of a phospho-tau antibody was subjected to three rounds of directed evolution using yeast surface display to improve its affinity. The resulting single-chain antibody fragments showed picomolar monovalent affinity. Quantitative specificity analysis revealed that affinity-matured clones have significant cross reactivity towards the non-phosphorylated epitope. By applying further selection pressure to enhance specificity, we successfully identified picomolar affinity clones with no detectable off target binding to non-phosphorylated or irrelevant phospho-epitopes. These results demonstrate that antibodies with extremely high affinity and specificity against PTM sites can be engineered. The new methods developed may be useful for setting a quantitative standard for characterizing PTM-specific antibodies, and providing essential guidelines on how to use these antibodies.

Impact of affinity maturation on the specificity of antibodies targeting protein post-translational modification sites

253rd National Meeting (2017)

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