biostudies-literatureIwamoto NTakeda Science FoundationJapan Agency for Medical Research and DevelopmentMinistry of Education, Culture, Sports, Science and TechnologyAstellas Foundation for Research on Metabolic DisordersTokyo Biochemical Research Foundation1596-1601https://www.ebi.ac.uk/biostudies/studies/S-EPMC10641909biostudies-literatureUnknown14(11)Mirror-image proteins (d-proteins) are promising scaffolds for drug discovery because of their high proteolytic stability and low immunogenic properties. Facile and reproducible processes for the preparation of functional d-proteins are required for their application in therapeutic biologics. In this study, we designed and synthesized a novel monobody variant with two cysteine substitutions that facilitate the synthetic process via sequential native chemical ligations and improve protein stability by disulfide bond formation. The synthetic anti-GFP monobody in this model study exhibited good binding affinity to the target enhanced green fluorescent protein. In vivo administration of the synthetic anti-GFP monobody (l-monobody) to mice induced antidrug antibody (ADA) production, whereas no ACS medicinal chemistry lettersDesign and Synthesis of Monobody Variants with Low Immunogenicity.PMC10641909JP20ak0101144JP18H02555JP22K19376JP22H02747JP20K21252JP22KJ1842Iwamoto NOishi SNonaka MManabe ASato YOhno HInuki SfalseDesign and Synthesis of Monobody Variants with Low Immunogenicity.Mirror-image proteins (d-proteins) are promising scaffolds for drug discovery because of their high proteolytic stability and low immunogenic properties. Facile and reproducible processes for the preparation of functional d-proteins are required for their application in therapeutic biologics. In this study, we designed and synthesized a novel monobody variant with two cysteine substitutions that facilitate the synthetic process via sequential native chemical ligations and improve protein stability by disulfide bond formation. The synthetic anti-GFP monobody in this model study exhibited good binding affinity to the target enhanced green fluorescent protein. In vivo administration of the synthetic anti-GFP monobody (l-monobody) to mice induced antidrug antibody (ADA) production, whereas no 2023-01-01T00:00:00Z2023 Nov2025-04-04T09:36:58.89Z2025-04-04T09:36:58.89ZS-EPMC106419093797493910.1021/acsmedchemlett.3c00342