Department of Chemistry, University of California-Berkeley, Berkeley, CA 94720.
Division of Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.
Proc Natl Acad Sci U S A. 2024 Sep 24;121(39):e2406680121. doi: 10.1073/pnas.2406680121. Epub 2024 Sep 19.
Antibody-derived T-cell receptor (TCR) agonists are commonly used to activate T cells. While antibodies can trigger TCRs regardless of clonotype, they bypass native T cell signal integration mechanisms that rely on monovalent, membrane-associated, and relatively weakly binding ligand in the context of cellular adhesion. Commonly used antibodies and their derivatives bind much more strongly than native peptide major histocompatibility complex (pMHC) ligands bind their cognate TCRs. Because ligand dwell time is a critical parameter that tightly correlates with physiological function of the TCR signaling system, there is a general need, both in research and therapeutics, for universal TCR ligands with controlled kinetic binding parameters. To this end, we have introduced point mutations into recombinantly expressed α-TCRβ H57 Fab to modulate the dwell time of monovalent Fab binding to TCR. When tethered to a supported lipid bilayer via DNA complementation, these monovalent Fab'-DNA ligands activate T cells with potencies well-correlated with their TCR binding dwell time. Single-molecule tracking studies in live T cells reveal that individual binding events between Fab'-DNA ligands and TCRs elicit local signaling responses closely resembling native pMHC. The unique combination of high on- and off-rates of the H57 R97L mutant enables direct observations of cooperative interplay between ligand binding and TCR-proximal condensation of the linker for activation of T cells, which is not readily visualized with pMHC. This work provides insights into how T cells integrate kinetic information from TCR ligands and introduces a method to develop affinity panels for polyclonal T cells, such as cells from a human patient.
抗体衍生的 T 细胞受体 (TCR) 激动剂常用于激活 T 细胞。虽然抗体可以触发 TCR，而无需考虑克隆型，但它们绕过了依赖单价、膜相关和相对较弱结合配体的天然 T 细胞信号整合机制，这些机制在细胞黏附的情况下发生。常用的抗体及其衍生物的结合强度远高于天然肽主要组织相容性复合物 (pMHC) 配体与其同源 TCR 的结合强度。由于配体停留时间是与 TCR 信号转导系统生理功能密切相关的关键参数，因此在研究和治疗中都普遍需要具有受控动力学结合参数的通用 TCR 配体。为此，我们已经在重组表达的 α-TCRβ H57 Fab 中引入点突变，以调节单价 Fab 与 TCR 结合的停留时间。当通过 DNA 互补作用连接到支持的脂质双层时，这些单价 Fab'-DNA 配体以与其 TCR 结合停留时间高度相关的效力激活 T 细胞。活 T 细胞中的单分子跟踪研究表明，Fab'-DNA 配体与 TCR 之间的单个结合事件引发的局部信号反应与天然 pMHC 非常相似。H57 R97L 突变体的高结合和解离速率的独特组合使我们能够直接观察到配体结合和 T 细胞激活的链接体 TCR 近端凝聚之间的协同相互作用，而用 pMHC 则不易观察到这种作用。这项工作深入了解了 T 细胞如何整合来自 TCR 配体的动力学信息，并介绍了一种用于多克隆 T 细胞（例如来自人类患者的细胞）开发亲和力面板的方法。
