背景：晚期非小细胞肺癌（NSCLC）是最常见的肺癌类型，预后不佳。采用工程化T细胞受体（TCR）靶向肿瘤睾丸抗原（如黑色素瘤相关抗原3（MAGE-A3））的细胞免疫治疗是治疗NSCLC的一种潜在方法。然而，对MAGE-A3抗原和相应抗原特异性TCR的T细胞免疫反应的系统分析仍然缺乏。方法：本研究采用体外人工抗原呈递细胞（APC）系统、单细胞转录组和TCR V(D)J测序以及机器学习的方法，全面筛选了与HLA-A2限制性的MAGE-A3肿瘤表位相关的TCR，并对其进行了表征。此外，我们筛选和验证了具有杀伤能力的肿瘤反应性TCR。结果：我们通过体外人工APC系统确定了能够有效诱导CD8+ T细胞活化和细胞毒性的MAGE-A3的HLA-A2限制性T细胞表位。一组HLA-A2阳性的NSCLC捐献者证明，在使用来自候选MAGE-A3表位的四聚体进行测定时，NSCLC患者与健康对照组相比，特异于表位的CD8+ T细胞数量增加，尤其是表位Mp4（MAGE-A3：160-169，LVFGIELMEV）。统计和基于机器学习的分析表明，MAGE-A3-Mp4表位特异性CD8+ T细胞克隆主要处于效应和增殖状态。重要的是，人工表达MAGE-A3-Mp4特异性TCR的T细胞表现出强烈的MAGE-A3+肿瘤细胞识别和杀伤效应。对候选TCR的交叉反应风险分析显示其与MAGE-A3-Mp4表位的结合稳定性高，交叉反应风险低。结论：该研究鉴定了一系列候选TCRs，可能适用于靶向HLA-A2限制性的MAGE-A3肿瘤抗原的TCR-T设计。©作者。

Background: Advanced non-small cell lung cancer (NSCLC) is the most common type of lung cancer with poor prognosis. Adoptive cell therapy using engineered T-cell receptors (TCRs) targeting cancer-testis antigens, such as Melanoma-associated antigen 3 (MAGE-A3), is a potential approach for the treatment of NSCLC. However, systematic analysis of T cell immune responses to MAGE-A3 antigen and corresponding antigen-specific TCR is still lacking. Methods: In this study, we comprehensively screened HLA-A2 restricted MAGE-A3 tumor epitopes and characterized the corresponding TCRs using in vitro artificial antigen presentation cells (APC) system, single-cell transcriptome and TCR V(D)J sequencing, and machine-learning. Furthermore, the tumor-reactive TCRs with killing potency was screened and verified. Results: We identified the HLA-A2 restricted T cell epitopes from MAGE-A3 that could effectively induce the activation and cytotoxicity of CD8+ T cells using artificial APC in vitro. A cohort of HLA-A2+ NSCLC donors demonstrated that the number of epitope specific CD8+ T cells increased in NSCLC than healthy controls when measured with tetramer derived from the candidate MAGE-A3 epitopes, especially epitope Mp4 (MAGE-A3: 160-169, LVFGIELMEV). Statistical and machine-learning based analyses demonstrated that the MAGE-A3-Mp4 epitope-specific CD8+ T cell clones were mostly in effector and proliferating state. Importantly, T cells artificially expressing the MAGE-A3-Mp4 specific TCRs exhibited strong MAGE-A3+ tumor cell recognition and killing effect. Cross-reactivity risk analysis of the candidates TCRs showed high binding stability to MAGE-A3-Mp4 epitope and low risk of cross-reaction. Conclusions: This work identified candidate TCRs potentially suitable for TCR-T design targeting HLA-A2 restricted MAGE-A3 tumor antigen.© The author(s).