Alexander Leithner, Oskar Staufer, Tanmay Mitra, Falk Liberta, Salvatore Valvo, Mikhail Kutuzov, Hannah Dada, Jacob Spaeth, Weijie Zhou, Felix Schiele, Sophia Reindl, Herbert Nar, Stefan Hoerer, Maureen Crames, Stephen Comeau, David Young, Sarah Low, Edward Jenkins, Simon J. Davis, David Klenerman, Andrew Nixon, Noah Pefaur, David Wyatt, Omer Dushek, Srinath Kasturirangan, Michael L. Dustin
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Small-angle X-ray scattering (SAXS) and modeling of the conformational states of isolated TcEs and TcE–antigen complexes predicted close contacts (≤13 nm) for Formats A and B and far contacts (≥18 nm) for Formats C and D. In supported lipid bilayer (SLB) model interfaces, Formats A and B recruited, whereas Formats C and D repelled, CD2–CD58 interactions. Formats A and B also excluded bulky Quantum dots more effectively. SAXS also revealed that TcE–antigen complexes formed by Formats A and C were less flexible than complexes formed by Formats B and D. Functional data with Her2-expressing tumor cells showed cytotoxicity, surface marker expression, and cytokine release following the order A > B = C > D. In a minimal system for IS formation on SLBs, TcE performance followed the trend A = B = C > D. Addition of close contact requiring CD58 costimulation revealed phospholipase C-γ activation matching cytotoxicity with A > B = C > D. Our findings suggest that when adhesion is equivalent, TcE potency is determined by two parameters: contact distance and flexibility. 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引用次数: 0
摘要
双特异性T细胞接合物(TcEs)将T细胞受体与癌细胞上的肿瘤相关抗原连接起来,形成细胞毒性免疫突触(IS)。膜与膜之间的紧密接触(≤13 nm)被认为是TcE功能的关键机制。为了研究这一点并确定潜在的其他机制,我们比较了四种基于免疫球蛋白g1 (IgG1)的靶向CD3ε和Her2的TcE格式(A - d),旨在产生不同的膜间距离(A <;B & lt;C & lt;D)。小角度x射线散射(SAXS)和分离的TcEs和tce抗原复合物的构象态建模预测了格式A和B的密切接触(≤13 nm)和格式C和D的远接触(≥18 nm)。在支持脂质双分子层(SLB)模型界面中,格式A和B被吸收,而格式C和D排斥CD2-CD58相互作用。格式A和B也更有效地排除了体积庞大的量子点。SAXS还显示,格式A和C形成的tce抗原复合物比格式B和d形成的复合物更不灵活。表达her2的肿瘤细胞的功能数据显示,细胞毒性、表面标记物表达和细胞因子释放顺序依次为A >;B = C >;D.在slb上最小IS地层系统中,TcE性能遵循a = B = C >;D.需要CD58共刺激的密切接触者的增加显示磷脂酶C-γ激活与A >的细胞毒性相匹配;B = C >;D.我们的研究结果表明,当粘附相等时,TcE效力由两个参数决定:接触距离和柔韧性。近/远接触形成轴和低/高柔韧性形成轴都显著影响TcE效能,这解释了B型(近接触/高柔韧性)和C型(远接触/低柔韧性)的相似效能。
Solution structure and synaptic analyses reveal determinants of bispecific T cell engager potency
Bispecific T cell engagers (TcEs) link T cell receptors to tumor-associated antigens on cancer cells, forming cytotoxic immunological synapses (IS). Close membrane-to-membrane contact (≤13 nm) has been proposed as a key mechanism of TcE function. To investigate this and identify potential additional mechanisms, we compared four immunoglobulin G1-based (IgG1) TcE Formats (A–D) targeting CD3ε and Her2, designed to create varying intermembrane distances (A < B < C < D). Small-angle X-ray scattering (SAXS) and modeling of the conformational states of isolated TcEs and TcE–antigen complexes predicted close contacts (≤13 nm) for Formats A and B and far contacts (≥18 nm) for Formats C and D. In supported lipid bilayer (SLB) model interfaces, Formats A and B recruited, whereas Formats C and D repelled, CD2–CD58 interactions. Formats A and B also excluded bulky Quantum dots more effectively. SAXS also revealed that TcE–antigen complexes formed by Formats A and C were less flexible than complexes formed by Formats B and D. Functional data with Her2-expressing tumor cells showed cytotoxicity, surface marker expression, and cytokine release following the order A > B = C > D. In a minimal system for IS formation on SLBs, TcE performance followed the trend A = B = C > D. Addition of close contact requiring CD58 costimulation revealed phospholipase C-γ activation matching cytotoxicity with A > B = C > D. Our findings suggest that when adhesion is equivalent, TcE potency is determined by two parameters: contact distance and flexibility. Both the close/far-contact formation axis and the low/high flexibility axis significantly impact TcE potency, explaining the similar potency of Format B (close contact/high flexibility) and C (far contact/low flexibility).
期刊介绍:
The Proceedings of the National Academy of Sciences (PNAS), a peer-reviewed journal of the National Academy of Sciences (NAS), serves as an authoritative source for high-impact, original research across the biological, physical, and social sciences. With a global scope, the journal welcomes submissions from researchers worldwide, making it an inclusive platform for advancing scientific knowledge.