Nature-inspired design of motif-specific antibody scaffolds

Aberrant changes in post-translational modifications (PTMs) such as phosphorylation underlie a majority of human diseases. However, detection and quantification of PTMs for diagnostic or biomarker applications often requires monoclonal PTM-specific antibodies, which are challenging to generate using traditional antibody-generation platforms. Here we outline a general strategy for producing synthetic PTM-specific antibodies by engineering a motif-specific ‘hot spot’ into an antibody scaffold. Inspired by a natural phosphate-binding motif, we designed antibody scaffolds with hot spots specific for phosphoserine, phosphothreonine, or phosphotyrosine. Crystal structures of the phospho-specific antibodies revealed two distinct modes of phosphoresidue recognition. Our data suggest that each hot spot functions independently of the surrounding scaffold, as phage display antibody libraries using these scaffolds yielded >50 phospho- and target-specific antibodies against 70% of target peptides. Ultimately, our motif-specific scaffold strategy may provide a general solution for the rapid, robust development of monoclonal anti-PTM antibodies for signaling, diagnostic and therapeutic applications.