Leithner Alexander, Staufer Oskar, Mitra Tanmay, Liberta Falk, Valvo Salvatore, Kutuzov Mikhail, Dada Hannah, Spaeth Jacob, Zhou Weijie, Schiele Felix, Reindl Sophia, Nar Herbert, Hoerer Stefan, Crames Maureen, Comeau Stephen, Young David, Low Sarah, Jenkins Edward, Davis Simon J, Klenerman David, Nixon Andrew, Pefaur Noah, Wyatt David, Dushek Omer, Kasturirangan Srinath, Dustin Michael L
The Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences (NDORMS), University of Oxford, Roosevelt Drive, Oxford OX3 7FY, United Kingdom.
INM - Leibniz Institute for New Materials, Campus D2 2, 66117 Saarbrücken, Germany.
Proc Natl Acad Sci U S A. 2025 Jun 3;122(22):e2425781122. doi: 10.1073/pnas.2425781122. Epub 2025 May 30.
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).
双特异性T细胞衔接器（TcE）将T细胞受体与癌细胞上的肿瘤相关抗原相连，形成细胞毒性免疫突触（IS）。膜与膜之间的紧密接触（≤13纳米）被认为是TcE发挥功能的关键机制。为了研究这一点并确定潜在的其他机制，我们比较了四种靶向CD3ε和Her2的基于免疫球蛋白G1（IgG1）的TcE形式（A - D），其设计目的是产生不同的膜间距离（A < B < C < D）。小角X射线散射（SAXS）以及对分离的TcE和TcE - 抗原复合物构象状态的建模预测，形式A和B会形成紧密接触（≤13纳米），而形式C和D会形成远距离接触（≥18纳米）。在支持脂质双层（SLB）模型界面中，形式A和B能招募CD2 - CD58相互作用，而形式C和D则排斥这种相互作用。形式A和B也能更有效地排除体积较大的量子点。SAXS还显示，形式A和C形成的TcE - 抗原复合物比形式B和D形成的复合物灵活性更低。对表达Her2的肿瘤细胞的功能数据表明，细胞毒性、表面标志物表达和细胞因子释放遵循A > B = C > D的顺序。在SLB上形成IS的最小系统中，TcE的性能遵循A = B = C > D的趋势。添加需要紧密接触的CD58共刺激后，显示磷脂酶C - γ激活与细胞毒性相匹配，顺序为A > B = C > D。我们的研究结果表明，当黏附相当时，TcE的效力由两个参数决定：接触距离和灵活性。紧密/远距离接触形成轴和低/高灵活性轴都会显著影响TcE的效力，这解释了形式B（紧密接触/高灵活性）和C（远距离接触/低灵活性）效力相似的原因。
