Department of Biotechnology and BOKU Core Facility Biomolecular and Cellular Analysis, BOKU - University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
Department of Biotechnology, BOKU - University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
ACS Synth Biol. 2021 May 21;10(5):1184-1198. doi: 10.1021/acssynbio.1c00010. Epub 2021 Apr 12.
CD19 is among the most relevant targets in cancer immunotherapy. However, its extracellular domain (ECD) is prone to aggregation and misfolding, representing a major obstacle for the development and analysis of CD19-targeted therapeutics. Here, we engineered stabilized CD19-ECD (termed SuperFolder) variants, which also showed improved expression rates and, in contrast to the wild type protein, they could be efficiently purified in their monomeric forms. Despite being considerably more stable, these engineered mutants largely preserved the wild type sequence (>98.8%). We demonstrate that the variant SF05 enabled the determination of the monovalent affinity between CD19 and a clinically approved FMC63-based CAR, as well as monitoring and phenotypic characterization of CD19-directed CAR-T cells in the blood of lymphoma patients. We anticipate that the SuperFolder mutants generated in this study will be highly valuable tools for a range of applications in basic immunology and CD19-targeted cancer immunotherapy.
CD19 是癌症免疫治疗中最相关的靶点之一。然而，它的细胞外结构域（ECD）容易聚集和错误折叠，这是开发和分析 CD19 靶向治疗药物的主要障碍。在这里，我们设计了稳定的 CD19-ECD（称为 SuperFolder）变体，这些变体还显示出更高的表达率，与野生型蛋白相比，它们可以以单体形式有效地进行纯化。尽管这些工程突变体的稳定性大大提高，但它们在很大程度上保留了野生型序列（>98.8%）。我们证明，变体 SF05 能够确定 CD19 与一种临床批准的基于 FMC63 的嵌合抗原受体（CAR）之间的单价亲和力，以及监测和表型分析淋巴瘤患者血液中 CD19 定向 CAR-T 细胞。我们预计，本研究中产生的 SuperFolder 突变体将成为基础免疫学和 CD19 靶向癌症免疫治疗中一系列应用的非常有价值的工具。
