Design and Evaluation of Stable Cysteine-Modified Monobody Scaffolds for Mirror-Image Synthesis

Mirror-image proteins (d-proteins) are promising therapeutic molecules with high biological stability and low immunogenicity. We recently developed a novel d-monobody scaffold variant with reduced immunogenicity. This variant incorporates two cysteine substitutions that enable the chemical synthesis of d-monobodies via native chemical ligation. In this study, the structure–activity relationship of monobody scaffold variants was investigated to identify more suitable positions for cysteine modifications. Several monobody variants with different cysteine substitution patterns and additional cysteine-selective modifications were designed and synthesized. Comprehensive functional analysis of the synthetic monobody derivatives led to the identification of a favorable monobody scaffold with potent target binding and high thermal stability. The optimized monobody scaffold with a cysteine cross-linker was used to develop d-monobody with additional functional groups.