School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, 950 Atlantic Dr. NW, Atlanta, GA 30332, United States of America.
School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, 950 Atlantic Dr. NW, Atlanta, GA 30332, United States of America.
J Control Release. 2021 Nov 10;339:248-258. doi: 10.1016/j.jconrel.2021.09.026. Epub 2021 Sep 24.
In recent years, protein engineering efforts have yielded a diverse set of binding proteins that hold promise for various therapeutic applications. Despite this, their inability to reach intracellular targets limits their applications to cell surface or soluble targets. To address this challenge, we previously reported a protein carrier that binds antibodies and delivers them to therapeutic targets inside cancer cells. This carrier, known as the Hex carrier, is comprised of a self-assembling coiled coil hexamer at the core, with each alpha helix fused to a linker, an antibody binding domain, and a six Histidine-tag (His-tag). In this work, we designed different versions of the carrier to determine the role of each building block in cytosolic protein delivery. We found that increasing exposure of the Hex coiled coil on the carriers, through molecular design or removing antibodies, increased internalization, pointing to a role of the coiled coil in promoting endocytosis. We observed a clear increase in endosomal disruption events when His-tags were present on the carrier relative to when they were removed, due to an endosomal buffering effect. Finally, we found that the antibody binding domains of the Hex carrier could be replaced with monomeric ultra-stable GFP for intracellular delivery and endosomal escape. Our results demonstrate that the Hex coiled coil, in conjunction with His-tags, could be a generalizable vehicle for delivering small and large proteins to intracellular targets. This work also highlights new biological applications for oligomeric coiled coils and shows the direct and quantifiable impact of histidine residues on endosomal disruption. These findings could inform the design of future drug delivery vehicles in applications beyond intracellular protein delivery.
近年来，蛋白质工程的努力产生了多种具有治疗应用潜力的结合蛋白。尽管如此，它们无法到达细胞内靶标，限制了它们在细胞表面或可溶性靶标上的应用。为了应对这一挑战，我们之前报道了一种能够结合抗体并将其递送到癌细胞内治疗靶标的蛋白质载体。这种载体称为六聚体载体（Hex carrier），由核心的自组装卷曲螺旋六聚体组成，每个α螺旋融合到一个接头、一个抗体结合域和六个组氨酸标签（His-tag）。在这项工作中，我们设计了不同版本的载体，以确定每个构建块在细胞质蛋白递送上的作用。我们发现，通过分子设计或去除抗体增加载体上的 Hex 卷曲螺旋的暴露，增加了内化，这表明卷曲螺旋在促进内吞作用中起作用。我们观察到，当载体上存在 His-tags 时，与去除 His-tags 时相比，内体破坏事件明显增加，这是由于内体缓冲效应。最后，我们发现，Hex 载体的抗体结合域可以被单体超稳定 GFP 取代，用于细胞内递和内体逃逸。我们的结果表明，Hex 卷曲螺旋与 His-tags 一起，可以成为将小分子和大分子蛋白质递送到细胞内靶标的通用载体。这项工作还突出了寡聚卷曲螺旋的新生物学应用，并显示了组氨酸残基对内体破坏的直接和可量化影响。这些发现可以为未来超越细胞内蛋白质递的应用的药物递送载体的设计提供信息。
