Protein Processing Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, USA.
Computational Biomolecular Magnetic Resonance Core, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA.
Commun Biol. 2024 Aug 31;7(1):1072. doi: 10.1038/s42003-024-06770-w.
T-cell therapies based on chimeric antigen receptor (CAR) targeting of a tumor-specific antigen offer hope for patients with relapsed or refractory cancers. CAR hinge and transmembrane regions link antigen recognition domains to intracellular signal transduction domains. Here, we apply biophysical methods to characterize the structure and dynamic properties of the CD28 CAR hinge (CD28H) used in an FDA-approved CD19 CAR for the treatment of B-lineage leukemia/lymphoma. By using nuclear Overhauser effect spectroscopy (NOESY), which detects even transiently occupied structural motifs, we observed otherwise elusive local structural elements amidst overall disorder in CD28H, including a conformational switch from a native β-strand to a 3-helix and polyproline II helix-like structure. These local structural motifs contribute to an overall loosely formed extended geometry that could be captured by NOESY data. All FDA-approved CARs use prolines in the hinge region, which we find in CD28, and previously in CD8α, isomerize to promote structural plasticity and dynamics. These local structural elements may function in recognition and signaling events and constrain the spacing between the transmembrane and antigen recognition domains. Our study thus demonstrates a method for detecting local and transient structure within intrinsically disordered systems and moreover, our CD28H findings may inform future CAR design.
基于嵌合抗原受体 (CAR) 靶向肿瘤特异性抗原的 T 细胞疗法为复发或难治性癌症患者带来了希望。CAR 的铰链和跨膜区将抗原识别域与细胞内信号转导域连接起来。在这里，我们应用生物物理方法来表征 FDA 批准的用于治疗 B 细胞谱系白血病/淋巴瘤的 CD19 CAR 中使用的 CD28 CAR 铰链 (CD28H) 的结构和动态特性。通过使用核 Overhauser 效应光谱 (NOESY)，它可以检测到即使是短暂占据的结构模体，我们观察到在 CD28H 的整体无序中存在其他难以捉摸的局部结构元素，包括从天然 β-折叠到 3-螺旋和多脯氨酸 II 螺旋样结构的构象转换。这些局部结构元素有助于形成整体松散的扩展构象，这可以通过 NOESY 数据来捕获。所有 FDA 批准的 CAR 都在铰链区使用脯氨酸，我们在 CD28 中发现，以前在 CD8α 中发现，脯氨酸异构化以促进结构可塑性和动力学。这些局部结构元素可能在识别和信号事件中发挥作用，并限制跨膜和抗原识别域之间的间隔。因此，我们的研究证明了一种在固有无序系统中检测局部和瞬态结构的方法，此外，我们对 CD28H 的发现可能为未来的 CAR 设计提供信息。
