弥漫性中线脑胶质瘤（DMG）是一种儿童脑肿瘤，预后极差。嵌合抗原受体（CAR）T细胞疗法最近在DMG中显示出一些成功，但可能需要针对多个肿瘤特异性靶点以避免抗原逃脱。我们开发了第二代CAR，针对DMG中HLA-A∗02:01限制的组蛋白3K27M表位，该表位是先前肽疫苗和T细胞受体模拟物的靶标。这些CAR T细胞对用H3.3K27M肽脉冲处理的细胞表现出特异性、可调节的结合。然而，我们无法观察到scFv结合、CAR T细胞激活或对H3.3K27M阳性患者来源模型的细胞毒作用。尽管使用了敏感的免疫肽组学技术，我们无法在这些患者来源模型上检测到H3.3K27M26-35-HLA-A∗02:01肽。有趣的是，从DMG中检测到与HLA-A∗02:01和其他I类分子结合的其他非突变肽，包括一个包含K27M突变并与H3 K27M26-35-HLA-A∗02:01肽重叠的新的限制于HLA-A3的肽。这些结果表明，在HLA-A∗02:01背景下靶向H3 K27M26-35突变可能不是一种可行的免疫疗法策略，因为它缺乏呈递。这些发现应该为DMG中的未来研究和临床试验提供信息。© 2023 The Authors.

Diffuse midline glioma (DMG) is a childhood brain tumor with an extremely poor prognosis. Chimeric antigen receptor (CAR) T cell therapy has recently demonstrated some success in DMG, but there may a need to target multiple tumor-specific targets to avoid antigen escape. We developed a second-generation CAR targeting an HLA-A∗02:01 restricted histone 3K27M epitope in DMG, the target of previous peptide vaccination and T cell receptor-mimics. These CAR T cells demonstrated specific, titratable, binding to cells pulsed with the H3.3K27M peptide. However, we were unable to observe scFv binding, CAR T cell activation, or cytotoxic function against H3.3K27M+ patient-derived models. Despite using sensitive immunopeptidomics, we could not detect the H3.3K27M26-35-HLA-A∗02:01 peptide on these patient-derived models. Interestingly, other non-mutated peptides from DMG were detected bound to HLA-A∗02:01 and other class I molecules, including a novel HLA-A3-restricted peptide encompassing the K27M mutation and overlapping with the H3 K27M26-35-HLA-A∗02:01 peptide. These results suggest that targeting the H3 K27M26-35 mutation in context of HLA-A∗02:01 may not be a feasible immunotherapy strategy because of its lack of presentation. These findings should inform future investigations and clinical trials in DMG.© 2023 The Authors.