Generating a mirror-image monobody targeting MCP-1 via TRAP display and chemical protein synthesis.

Biologically produced protein drugs are generally susceptible to degradation by proteases and often exhibit immunogenicity. To address this issue, mirror-image peptide/protein binders consisting of D-amino acids have been developed so far through the mirror-image phage display technique. Here, we develop a mirror-image protein binder derived from a monobody, one of the promising protein scaffolds, utilizing two notable technologies: chemical protein synthesis and TRAP display, an improved version of mRNA display. A sequential workflow of initial screening followed by affinity maturation, facilitated by TRAP display, generates an L-monobody with high affinity (K = 1.3 nM) against monocyte chemoattractant protein-1 (MCP-1) D-enantiomer. The chemically synthesized D-monobody demonstrates strong and specific binding to L-MCP-1 and exhibits pharmaceutically favorable properties such as proteolytic resistance, minimal immune response, and a potent inhibitory effect on MCP-1-induced cell migration. This study elevates the value of mirror-image peptide/protein binders as an alternative modality in drug discovery.