采用T细胞受体工程T细胞（TCR-Ts）的癌症免疫疗法代表了一种有希望的治疗选择。然而，现有的临床前安全评估技术对于大多数实验室来说不完善或无法获取。在本研究中，通过以下五个步骤评估了TCR-T的非靶向反应：（1）确定TCR-T识别所需的靶向氨基酸，接着进行（2）在人类蛋白质组中进行候选交叉反应肽的计算搜索，并对其进行（3）反应性筛选。通过短mRNA（4）和完整蛋白（5）评估被识别肽的自然加工和呈递。筛选TCR-T是否识别非意向的HLA等位基因，为了作为体内非靶向反应的替代，使用了同基因性的HLA-A*02:01转基因小鼠模型。验证结果表明，在研究识别候选非靶向目标时，研究完整的蛋白质的识别极其重要，临床应用的1G4 TCR对非意向的HLA等位基因具有迄今未知的反应性，这对于患者选择非常重要。这种广泛适用的策略应有助于评估候选治疗TCR并指导临床决策。© 2023. Springer Nature Limited.

Cancer immunotherapy using T cell receptor-engineered T cells (TCR-Ts) represents a promising treatment option. However, technologies for pre-clinical safety assessment are incomplete or inaccessible to most laboratories. Here, TCR-T off-target reactivity was assessed in five steps: (1) Mapping target amino acids necessary for TCR-T recognition, followed by (2) a computational search for, and (3) reactivity screening against, candidate cross-reactive peptides in the human proteome. Natural processing and presentation of recognized peptides was evaluated using (4) short mRNAs, and (5) full-length proteins. TCR-Ts were screened for recognition of unintended HLA alleles, and as proxy for off-target reactivity in vivo, a syngeneic, HLA-A*02:01-transgenic mouse model was used. Validation demonstrated importance of studying recognition of full-length candidate off-targets, and that the clinically applied 1G4 TCR has a hitherto unknown reactivity to unintended HLA alleles, relevant for patient selection. This widely applicable strategy should facilitate evaluation of candidate therapeutic TCRs and inform clinical decision-making.© 2023. Springer Nature Limited.