RESET: A reversible TCR-coupled antigen receptor with superior targeting sensitivity and pharmacologically controlled anti-tumor activity

Chimeric antigen receptor (CAR) T cells are effective cancer therapies, particularly in indications with high, stable, and tumor-specific antigen expression. Other settings may require improved targeting sensitivity, controllable targeting selectivity, and/or additional potency enhancements to achieve robust efficacy. Here, we describe a novel receptor architecture called RESET (rapamycin-enabled, switchable endogenous T-cell receptor) that combines: (i) cell surface antigen targeting; (ii) small-molecule regulation; and (iii) the signaling proficiency and inherent sensitivity of native T-cell receptors. RESET-T cells outperformed both constitutive and drug-regulated CAR-T cells and show hallmarks of TCR activation that suggest improved fidelity to native T-cell responses. Pharmacological control then increases safety through toggling T-cell activation between active and resting states and may mitigate T- cell exhaustion caused by continuous antigen exposure. This convergence of drug-regulated targeting and natural immune receptor signal transduction may better replicate the kinetics and physiology of a classical T-cell responses and potentiate more successful and safer immunotherapies. Overall design: T cells from N=4 healthy donors were transduced with RESET, DARIC, CAR, TCR, or untransduced control were cultured either alone or with A549 cells engineered to overexpress the antigen of interest (CD33 or MAGEA4-HLA-A:0201) in the presence or absence of 1nM rapamycin for 18h. Following coculture, T cells were isolated from tumor cells using MACS sorting, tested for purity using flow cytometry, then RNA was isolated and evaluated by RNA-seq.

嵌合抗原受体（chimeric antigen receptor, CAR）T细胞是一类有效的癌症治疗手段，尤其适用于抗原表达水平高、稳定且具有肿瘤特异性的适应症。在其他治疗场景中，需优化靶向敏感性、实现可控的靶向选择性，并辅以额外的效力增强策略，方可获得稳定可靠的治疗效果。本研究介绍了一种名为RESET（雷帕霉素调控型可切换内源性T细胞受体，rapamycin-enabled, switchable endogenous T-cell receptor）的新型受体架构，其整合了三大核心模块：(i) 细胞表面抗原靶向功能；(ii) 小分子调控能力；(iii) 天然T细胞受体的信号转导效率与固有敏感性。RESET-T细胞的表现优于组成型表达型与药物调控型CAR-T细胞，且呈现出T细胞受体（T cell receptor, TCR）激活的特征性标志物，提示其对天然T细胞应答的模拟保真度有所提升。通过在激活与静息状态之间切换T细胞的活化状态实现药理学调控，不仅可提升治疗安全性，还能缓解因持续抗原暴露引发的T细胞耗竭。这种将药物调控靶向与天然免疫受体信号转导相结合的策略，能够更精准地复现经典T细胞应答的动力学特征与生理过程，助力开发更高效且更安全的免疫治疗手段。实验整体设计：从N=4名健康供体中分离T细胞，分别转染RESET、DARIC、CAR、TCR载体，同时设置未转染的空白对照组；将上述细胞单独培养，或与经基因工程改造以过表达目标抗原（CD33或MAGEA4-HLA-A:0201）的A549细胞共培养，培养体系中添加或不添加1nM雷帕霉素，共培养时长为18小时。共培养结束后，采用磁珠分选（magnetic-activated cell sorting, MACS）技术从肿瘤细胞中分离T细胞，通过流式细胞术（flow cytometry）检测细胞纯度；随后提取RNA并利用RNA测序（RNA sequencing, RNA-seq）进行转录组分析。