Engineering isoform-selective miniprotein binders to the fetal form of the insulin receptor

The fetal form of the insulin receptor (IRA) is predominantly expressed in many cancers, where IR signaling promotes progression of the disease. Alternatively, the adult IR isoform (IRB) is predominantly expressed in normal tissues. Previous attempts to target IR indiscriminately via dual IR/IGF1R tyrosine kinase inhibitors frequently failed due to on-target off-tumor effects. However, an IRA-specific therapeutic has the potential to specifically target cancer while sparing normal tissues. Furthermore, an IRA-specific binder would advance research regarding this disease-associated isoform. Yet, isoform-specific IR binders have not been developed. Herein, we engineer six high-affinity, IRA-specific protein binders from a synthetic miniprotein scaffold. These miniprotein variants have IRA KDs ranging from 0.98-7.6 nM with no appreciable IRB binding at 100 nM ligand. Five of the six variants are synergistic agonists. One variant (V1) is a passive, IRA-specific binder with a KD = 6.3 nM and no IRB binding at 1 μM ligand as well as no IGF1R binding at 100 nM, which provides compelling potential as a targeted therapeutic. Additionally, we present the discovery process for these molecules, a yeast display directed evolution campaign utilizing a combination of sorting techniques. Retrospective analysis of the discovery process revealed the importance of alternating selections for target affinity and isoform specificity to generate binders with a favorable combination of these properties.