High-affinity mirror-image monobody targeting MCP-1 generated via TRAP display and chemical protein synthesis

Abstract

Engineered protein scaffolds function as binders against various target molecules with high affinity and specificity comparable to conventional IgG antibodies. However, biologically produced protein drugs are generally degraded by proteases and often exhibit immunogenicity. To increase protease resistance and decrease immunogenicity of peptides and proteins, mirror-image peptide/protein binders consisting of D-amino acids have been developed so far mainly through the mirror-image phage display technique. Here, we develop a mirror-image protein binder derived from a monobody, one of the most promising protein scaffolds, using two notable technologies: chemical protein synthesis and TRAP (transcription-translation coupled with association of puromycin linker) display, an improved and streamlined 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 (KD = 1.3 nM) against the pharmaceutically important monocyte chemoattractant protein-1 (MCP-1) D-enantiomer. By symmetry, the chemically synthesized D-monobody demonstrates strong and enantio-selective binding against L-MCP-1 and possesses pharmaceutically favorable properties such as resistance to proteolytic degradation, minimal immune response, and a potent inhibitory effect on MCP-1 binding to its cell membrane receptor. The production of a high-affinity mirror-image monobody elevates the value of mirror-image peptide/protein binders as a new modality in drug discovery.