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.