T 细胞接合双特异性药物在治疗癌症和传染病方面具有巨大的临床潜力。双特异性分子对靶标和 T 细胞 CD3 的结合亲和力和动力学对效力和特异性具有重大影响，但控制这些关系的规则尚未完全了解。使用 ImmTAC（针对癌症的免疫动员单克隆 TCR）分子作为模型，我们探讨了改变靶标和 CD3 的亲和力对重定向 T 细胞反应的效力和特异性的影响。这类双特异性药物，例如 tebentafusp，最近在一项随机 3 期临床试验中显示出生存益处1，通过亲和力增强的 T 细胞受体结合细胞表面人类白细胞抗原 (HLA) 呈递的特定靶肽，并可以重定向 T使用抗 CD3 效应器部分激活细胞。数据显示，将强亲和力 TCR 与中等亲和力抗 CD3 相结合可实现最佳 T 细胞激活，而靶向结构域和效应结构域的强亲和力可显着减少细胞因子的最大释放。此外，通过优化分子两部分的亲和力，可以提高选择性。这些结果可以基于有限信号的动力学校对进行有效建模。该模型解释了实验观察结果，即分子两端的强结合导致活性降低，通过非常稳定的靶标双特异性效应复合物导致 CD3 进入非信号暗状态。这些发现对于设计具有最佳活性和特异性生物物理参数的基于抗 CD3 的双特异性药物具有重要意义。© 作者 2023。由牛津大学出版社代表英国免疫学会出版。

T cell engaging bispecifics have great clinical potential for the treatment of cancer and infectious diseases. The binding affinity and kinetics of a bispecific molecule for both target and T cell CD3 have substantial effects on potency and specificity, but the rules governing these relationships are not fully understood. Using ImmTAC (Immune mobilizing monoclonal TCRs Against Cancer) molecules as a model, we explored the impact of altering affinity for target and CD3 on the potency and specificity of the re-directed T cell response. This class of bispecifics, exemplified by tebentafusp which has recently shown survival benefit in a randomized phase 3 clinical trial1, bind specific target peptides presented by human leukocyte antigen (HLA) on the cell surface via an affinity-enhanced T cell receptor and can redirect T cell activation with an anti-CD3 effector moiety. The data reveal that combining a strong affinity TCR with an intermediate affinity anti-CD3 results in optimal T cell activation, while strong affinity of both targeting and effector domains significantly reduces maximum cytokine release. Moreover, by optimising the affinity of both parts of the molecule, it is possible to improve the selectivity. These results could be effectively modelled based on kinetic proof-reading with limited signalling. This model explained the experimental observation that strong binding at both ends of the molecules leads to reduced activity, through very stable target-bispecific-effector complexes leading to CD3 entering a non-signalling dark-state. These findings have important implications for the design of anti-CD3 based bispecifics with optimal biophysical parameters for both activity and specificity.© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Immunology.