(502d) Constrained Combinatorial Libraries of Gp2 Proteins Enhance Discovery of Synthetic PD-L1 Ligands

Molecular binding ligands are valuable for targeted therapy, diagnostics, and industrial biotechnology. The 45-amino acid Gp2 domain is a scaffold for evolution of synthetic binding ligands via diversification of two solvent-exposed loops. We hypothesized that discovery of novel Gp2 domains would be enhanced by sitewise amino acid constraint, including cysteine pairs, within de novo combinatorial library design. We comparatively evaluated eight libraries, systematically varied to compare sitewise constraints, for binder discovery via yeast display against a panel of targets. A conserved cysteine pair at the termini of the first diversified paratope loop increased binder discovery 16-fold (p < 0.001). Yet conservation of two other cysteine pairs, within the second loop or an interloop pair, did not aid discovery thereby indicating site-specific impact. Gp2 variants from the loop one cysteine pair library were 3.3 ± 2.1-fold (p = 0.005) more stable to protease – as assessed via a high-throughput yeast display assay – than non-constrained variants. Sitewise constraint of non-cysteine residues – guided by previously evolved binders, natural Gp2 homology, computed stability, and structural analysis – did not benefit discovery. A panel of binders to programmed death ligand 1 (PD-L1), a key target in cancer immunotherapy, were discovered from the loop 1 cysteine constraint library. Affinity maturation via loop walking resulted in stable Gp2 domains (T_m = 73 ± 2 ºC) with strong, specific cellular PD-L1 affinity (K_d = 6 - 9 nM). The lead domain binds to a similar epitope to the clinical antibody atezolizumab. Further biochemical analysis will be presented as well as characterization of positron emission tomography (PET) imaging of PD-L1 in tumor xenograft models.