From the Protein Interactions Group, Cancer and Inflammation Program, Center for Cancer Research, NCI, National Institutes of Health, Frederick, Maryland 21702 and.
From the Protein Interactions Group, Cancer and Inflammation Program, Center for Cancer Research, NCI, National Institutes of Health, Frederick, Maryland 21702 and.
J Biol Chem. 2013 Aug 30;288(35):25154-25164. doi: 10.1074/jbc.M113.484154. Epub 2013 Jul 18.
Most of the therapeutic antibodies approved for clinical use are full-size IgG1 molecules. The interaction of the IgG1 Fc with the neonatal Fc receptor (FcRn) plays a critical role in maintaining their long half-life. We have hypothesized that isolated Fc domains could be engineered to functionally mimic full-size IgG1 (nanoantibodies) but with decreased (10-fold) size. Here, we report for the first time the successful generation of a soluble, monomeric CH3 domain (mCH3). In contrast to the wild-type dimeric CH3, the mCH3 exhibited pH-dependent binding to FcRn similar to that of Fc. The binding free energy of mCH3 to FcRn was higher than that of isolated CH2 but lower than that of Fc. Therefore, CH3 may contribute a larger portion of the free energy of binding to FcRn than CH2. A fusion protein of mCH3 with an engineered antibody domain (m36.4) also bound to FcRn in a pH-dependent fashion and exhibited significantly higher neutralizing activity against HIV-1 than m36.4-Fc fusion proteins. The m36.4-mCH3 fusion protein was monomeric, stable, soluble, and expressed at a high level in Escherichia coli. We also found that engineering an additional disulfide bond in mCH3 remarkably increased its thermal stability, whereas the FcRn binding was not affected. These data suggest that mCH3 could not only help in the exploration of the dual mechanisms of the CH3 contribution to Fc functions (dimerization and FcRn interactions) but could also be used for the development of candidate therapeutics with optimized half-life, enhanced tissue penetration, access to sterically restricted binding sites, and increased therapeutic efficacy.
大多数已批准用于临床的治疗性抗体都是全尺寸 IgG1 分子。IgG1 Fc 与新生儿 Fc 受体（FcRn）的相互作用在维持其长半衰期方面起着关键作用。我们假设可以对分离的 Fc 结构域进行工程改造，使其在功能上模拟全尺寸 IgG1（纳米抗体），但大小减小（10 倍）。在这里，我们首次成功生成了一种可溶性、单体 CH3 结构域（mCH3）。与野生型二聚体 CH3 不同，mCH3 表现出 pH 依赖性与 FcRn 的结合，类似于 Fc。mCH3 与 FcRn 的结合自由能高于 CH2，但低于 Fc。因此，CH3 与 FcRn 的结合可能比 CH2 贡献更大的结合自由能部分。mCH3 与工程化抗体结构域（m36.4）的融合蛋白也以 pH 依赖性方式与 FcRn 结合，对 HIV-1 的中和活性明显高于 m36.4-Fc 融合蛋白。m36.4-mCH3 融合蛋白为单体，稳定，可溶，在大肠杆菌中高表达。我们还发现，在 mCH3 中构建额外的二硫键可显著提高其热稳定性，而 FcRn 结合不受影响。这些数据表明，mCH3 不仅可以帮助探索 CH3 对 Fc 功能的双重贡献机制（二聚化和 FcRn 相互作用），还可以用于开发具有优化半衰期、增强组织穿透性、可进入空间受限结合位点和提高治疗效果的候选治疗药物。
