Using extension‐based <scp>mRNA</scp> display to design antibody‐like proteinogenic peptides for human <scp>PD</scp>‐<scp>L1</scp>

Many peptide drugs rely on nonproteinogenic amino acids and chemical modifications for improved activity and proteolytic stability. However, these features also make drug production expensive and challenging to scale. Here, we engineered small, linear, proteinogenic peptides that bind human programmed death-ligand 1 (hPD-L1) with high affinity and stability using mRNA display affinity maturation. The resulting peptides, SPAM2 and SPAM3, have antibody-like affinities for hPD-L1 (dissociation constants between ~250 and 300 pM) and are selective for hPD-L1. Both SPAM2 and SPAM3 compete with hPD-L1 ligands known to interact with the programmed cell death protein 1 site and are stable in human serum. SPAM3 bound human glioma D423 cells with high affinity in flow cytometry experiments comparable to that of a clinical therapeutic antibody. These results support the use of affinity maturation selections to dramatically enhance the biophysical properties of linear, proteinogenic peptides for translational applications.