Engineering a Hyperstable Synthetic Miniprotein for B7-H3 Targeting.

Small protein ligands, such as miniproteins, have the potential to provide the efficient physiological transport and rapid clearance required for radioligand therapy. Yet, binders require single-digit nanomolar affinity for potent targeting and high stability for radiolabeling and physiological integrity. High affinity and stable single-domain miniproteins are also modular candidates for multispecific applications in cell engagers and avid constructs. Previous work demonstrated computationally designed 40-amino acid β⍺ββ miniproteins as effective scaffolds for ligand discovery. Herein, to yield the desired single-digit nanomolar affinity for B7-H3, an important immune regulator and cell surface antigen upregulated in numerous tumors, we used structural modeling to guide experimental evolution of the lead molecule. We performed constrained diversification at six sites on a β-sheet, structurally distinct from the originally engineered ⍺-helical paratope, as well as nine sites within the ⍺-helix, and sorted for strong binding to B7-H3 via yeast display and flow cytometry with detergent-solubilized cell lysate. Top variants discovered from this campaign yielded affinities of 8.4 and 11 nM, with the most performant variant, named βm.1, resulting in 1.2 ± 0.2 nM affinity. βm.1 had four β-sheet mutations relative to parental and Tm,app ≥91 °C, with reversible unfolding. The lead variant, βm.1, putatively engages B7-H3 at the same domain I epitope as the parental lead but with improved engagement via a T27F mutation and three homologous mutations. βm.1 is a hyperstable, high affinity miniprotein B7-H3 binder with compelling potential for radioligand therapy and multispecific applications.