H3K27M, a driver mutation with T- and B-cell neoepitope characteristics, defines an aggressive subtype of diffuse glioma with poor survival. We functionally dissect the immune response of one patient who was treated with an H3K27M peptide vaccine and subsequently entered complete remission. The vaccine robustly expanded class II HLA-restricted peripheral H3K27M-specific T cells. Using functional assays, we characterized 34 clonally unique H3K27M-reactive T cell receptors and identified critical, conserved motifs in their CDR3 regions. Using detailed HLA mapping, we further demonstrate that diverse HLA-DQ, and -DR alleles present immunogenic H3K27M epitopes. Furthermore, we identified and profiled H3K27M-reactive B cell receptors from activated B cells in the cerebrospinal fluid. Our results uncover the breadth of the adaptive immune response against a shared clonal neoantigen across multiple HLA allelotypes and support the use of class II-restricted peptide vaccines to stimulate tumor-specific T and B cells harboring receptors with therapeutic potential.

Cellular immunotherapies, such as those utilizing T lymphocytes expressing native or engineered T cell receptors (TCRs), have already demonstrated therapeutic efficacy. However, some high-affinity TCRs have also proved to be fatal due to off-target immunotoxicity. This process occurs when the immune system acts against epitopes found on both tumor cells and healthy tissues. Moreover, some TCRs can be cross-reactive to epitopes with highly dissimilar sequences. To address this issue, we developed ARDitox, a novel in silico method based on computational immunology and artificial intelligence (AI) for predicting and analyzing potential off-target binding. We tested the performance of ARDitox in silico on different cases found in the literature where TCRs were used to target cancer-related antigens, as well as on a set of TCRs targeting a viral epitope. ARDitox was able to identify previously reported cross-reactive epitopes in line with the data available in the literature. In addition, we investigated a TCR targeting an HLA-A*02:01-restricted immunodominant epitope from the glioblastoma-associated antigen NLGN4X, identifying a cross-reactive ADH1A epitope that would not be detected in murine models. In conclusion, our in silico approach is a powerful tool that identifies potential off-target epitopes, complementing preclinical studies in developing safer cell therapies targeting tumor(-associated) antigens.