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Induction of T-cell responses against mutation-specific peptides from malignant pediatric brain tumor samples
Induction of T-cell responses against mutation-specific peptides from malignant pediatric brain tumor samples
Medulloblastoma is the most common malignant brain tumor in childhood and adolescence and constitutes an important cause for cancer-related death in pediatric patients. Although standard therapy including surgery, chemotherapy and radiation can cure up to 80 % of average-risk patients, they imply severe cognitive long-term adverse effects and are unsatisfactory in advanced tumors. Therefore, alternative treatment strategies need to be established. Immunotherapeutic approaches like peptide vaccination and adoptive T-cell transfer (ATT) aim at enhancing self-protection through detection and elimination of malignant cells. Tumor-specific neoepitopes are promising targets for ATT as they are expressed exclusively by cancer tissue. Moreover, administration of mutation-derived peptide vaccines allows augmenting the endogenous immune response through abundant presentation of tumor antigen. In this proof-of-concept study we demonstrate a highly individualized approach where patient-specific neoepitopes are determined and tested for immunogenicity. Primary tumor samples from two pediatric medulloblastoma patients were analyzed in this project. Tumor-specific mutations were identified by next generation sequencing of tumor tissue and whole blood. Variants were confirmed by deep sequencing. In order to identify neoepitope peptides presented by the patients’ human leucocyte antigen (HLA) molecules, HLA binding affinity was predicted in silico by netMHC database. Respective peptides were synthesized and blood cells from healthy donors matching the patients’ HLA types were used to provide T lymphocytes and dendritic cells for antigen presentation. After seven restimulations in vitro, CD8+ cytotoxic T-cell reactivity against neoepitopes was assessed via flow-cytometric analysis of Interferon gamma and Tumor Necrosis Factor alpha release. A successful de novo T-cell response was induced for 9 of 19 tested peptides. In this proof-of-principle study we show that induction of a T-cell response against medullobastoma-derived neoantigens is feasible despite low mutational burden and low immunogenicity. In the future, this strategy can be used to synthesize individualized peptide cocktails for peptide vaccination or identify medulloblastoma-specific T-cell receptors for ATT. Long-term aims of this study are the identification of medulloblastoma/T-cell interaction and improvement of current treatment options for pediatric patients with advanced medulloblastoma.
medulloblastoma, neoantigen, T-cell response, peptide vaccination, adoptive T-cell transfer
Paul, Milan
2020
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Paul, Milan (2020): Induction of T-cell responses against mutation-specific peptides from malignant pediatric brain tumor samples. Dissertation, LMU München: Faculty of Medicine
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Abstract

Medulloblastoma is the most common malignant brain tumor in childhood and adolescence and constitutes an important cause for cancer-related death in pediatric patients. Although standard therapy including surgery, chemotherapy and radiation can cure up to 80 % of average-risk patients, they imply severe cognitive long-term adverse effects and are unsatisfactory in advanced tumors. Therefore, alternative treatment strategies need to be established. Immunotherapeutic approaches like peptide vaccination and adoptive T-cell transfer (ATT) aim at enhancing self-protection through detection and elimination of malignant cells. Tumor-specific neoepitopes are promising targets for ATT as they are expressed exclusively by cancer tissue. Moreover, administration of mutation-derived peptide vaccines allows augmenting the endogenous immune response through abundant presentation of tumor antigen. In this proof-of-concept study we demonstrate a highly individualized approach where patient-specific neoepitopes are determined and tested for immunogenicity. Primary tumor samples from two pediatric medulloblastoma patients were analyzed in this project. Tumor-specific mutations were identified by next generation sequencing of tumor tissue and whole blood. Variants were confirmed by deep sequencing. In order to identify neoepitope peptides presented by the patients’ human leucocyte antigen (HLA) molecules, HLA binding affinity was predicted in silico by netMHC database. Respective peptides were synthesized and blood cells from healthy donors matching the patients’ HLA types were used to provide T lymphocytes and dendritic cells for antigen presentation. After seven restimulations in vitro, CD8+ cytotoxic T-cell reactivity against neoepitopes was assessed via flow-cytometric analysis of Interferon gamma and Tumor Necrosis Factor alpha release. A successful de novo T-cell response was induced for 9 of 19 tested peptides. In this proof-of-principle study we show that induction of a T-cell response against medullobastoma-derived neoantigens is feasible despite low mutational burden and low immunogenicity. In the future, this strategy can be used to synthesize individualized peptide cocktails for peptide vaccination or identify medulloblastoma-specific T-cell receptors for ATT. Long-term aims of this study are the identification of medulloblastoma/T-cell interaction and improvement of current treatment options for pediatric patients with advanced medulloblastoma.