PANCS-spec-Binders: A system for rapidly discovering isoform- or epitope-specific binders

Proteins that bind to a target protein of interest, termed "binders", are essential components of biological research reagents and therapeutics. Target proteins present multiple binding surfaces with varying interaction potential. "Hot spots", or high-potential surfaces, are experimentally identified as the most probable binding sites in de novo discovery campaigns. However, hot spots and their default binding modes do not always confer the desired specificity. Related proteins or isoforms often share similar hot spots, resulting in promiscuous binding. Interaction with a hot spot may also fail to elicit the intended biological outcome. Consequently, methods that direct de novo binder discovery toward targets with defined specificity are critically needed. We recently developed phage-assisted noncontinuous selection of binders (PANCS-Binders), a selection platform with unparalleled speed and sequence-function fidelity that enables routine de novo binder discovery within days. However, because PANCS-Binders selections enrich variants based primarily on affinity, secondary screening is unlikely to identify binders to lesser hot spots because of the high likelihood of convergence. These alternative binding surfaces with weaker inherent interactions may possess desirable specificity profiles. Here, we develop PANCS-spec-Binders, which incorporates simultaneous selection and counterselection to control the specificity of enriched binders. We demonstrate PANCS-spec-Binders in two proof-of-concept applications: 1) discovery of isoform-selective binders that bind HRAS with >100-fold higher affinity than the highly related KRAS isoform, and 2) discovery of epitope-specific binders that either target or avoid the LIR interaction region of LC3B. PANCS-spec-Binders enables rapid identification of binders with defined specificity within days.