Iwamoto Naoya, Sato Yukino, Manabe Asako, Inuki Shinsuke, Ohno Hiroaki, Nonaka Motohiro, Oishi Shinya
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
Laboratory of Medicinal Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan.
ACS Med Chem Lett. 2023 Oct 9;14(11):1596-1601. doi: 10.1021/acsmedchemlett.3c00342. eCollection 2023 Nov 9.
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 ADA production was observed following immunization with the mirror-image anti-GFP monobody (d-monobody). These results suggest that the synthetic d-monobody is a non-antibody protein scaffold with low immunogenic properties.
镜像蛋白（D-蛋白）因其高蛋白水解稳定性和低免疫原性，是药物研发中很有前景的支架。功能性D-蛋白的制备需要简便且可重复的过程，以便其应用于治疗性生物制品。在本研究中，我们设计并合成了一种新型单域抗体变体，其具有两个半胱氨酸取代，可通过顺序天然化学连接促进合成过程，并通过形成二硫键提高蛋白质稳定性。该模型研究中的合成抗绿色荧光蛋白单域抗体对目标增强型绿色荧光蛋白表现出良好的结合亲和力。向小鼠体内注射合成抗绿色荧光蛋白单域抗体（L-单域抗体）会诱导抗药物抗体（ADA）产生，而用镜像抗绿色荧光蛋白单域抗体（D-单域抗体）免疫后未观察到ADA产生。这些结果表明，合成的D-单域抗体是一种具有低免疫原性的非抗体蛋白支架。
