Abstract Background: We established a platform for the design of patient-individual peptide vaccination cocktails by combination of whole exome sequencing of tumor and normal tissue with in silico epitope prediction algorithms for individual patient HLA types. Accumulation of somatic mutations is one characteristic feature of malignanT-cells. These single-nucleotide variants (SNVs) can lead to altered amino acid sequences of the translated proteins, which in turn can be presented by malignant cells as antigenic peptides on HLA molecules. A peptide vaccination to induce neoantigen-specific T-cell responses, therefore, is a promising and versatile immunotherapeutic approach for the treatment of malignant diseases. Such approaches were predominantly applied in malignancies with high mutational load in adult patients until now. We developed a vaccine design platform based on sequencing data generated from a cohort of acute lymphoblastic leukemia (ALL) patients and tested individualized peptide vaccines in pediatric patients suffering from a variety of tumors with low mutational profiles. Methods: Nonsynonymous mutations were identified by whole-exome and transcriptome sequencing of patient leukemic blasts and healthy reference tissue. HLA binding peptides harboring the altered amino acids were subsequently predicted in silico by algorithms SYFPEITHI, NetMHC and NetMHCpan for the patients’ individual HLA type. Individual peptides for treatment attempts were produced by chemical synthesis and vaccination cocktails were formulated. The vaccination schedule was 16 vaccinations over 33 weeks using GM-CSF and Imiquimod as adjuvants. Some patients received simultaneous checkpoint blockade treatment with pembrolizumab or nivolumab. Response to the vaccination was monitored by detection of T-cells recognizing the vaccinated peptides occurring over time in peripheral blood of the patients. Monitoring was performed for each vaccination time point by pre-stimulation with the peptides and subsequent intracellular cytokine staining (ICS) of T-cells and FACS analysis. Results: Whole-exome sequencing was performed for 25 patients to identify ALL-specific SNVs using a comparative bioinformatics pipeline. We found an average of 39.2 mutations per patient on DNA level, with an average validation rate of 47% by RNA sequencing. Based on these data, an average of 35.1 HLA binders could be predicted per patient. We applied our platform for 6 patients with various malignancies based on compassionate need and designed individual peptide vaccines. In all cases validated mutations could be identified and epitope prediction was performed for HLA Class I and II binders. In 6/6 patients a de novo induced T-cell response against the vaccinated mutated HLA-binding peptides was detectable. Combination therapy with PD-1 blockade and peptide vaccination was well tolerated. T-cell responses were predominantly, but not exclusively, CD4+-restricted. Encouraged by these findings, we started a phase I/II clinical trial in patients with primary/relapsed ALL with the aim to prevent relapse and show safety and immunogenicity of the personalized peptide vaccine. Conclusions: We established a platform for the design of patient-individual peptide vaccination cocktails by combination of whole exome sequencing of tumor and normal tissue with in silico epitope prediction algorithms for individual patient HLA types. Whole-exome sequencing of pediatric ALL patients is feasible and yields a small amount of tumor-specific mutations per patient. However, these few mutations are sufficient to predict HLA-binding peptides that are immunogenic when vaccinated and elicit specific T-cell responses in patients. The universal character of individualized peptide vaccination allows for application in virtually any malignant disease, as well as combination therapy approaches. The concept is now translated to clinical application in a phase I/II clinical trial in ALL, started in 2016 (NCT03559413). Citation Format: Armin Rabsteyn, Christina Kyzirakos, Christopher Schroeder, Marc Sturm, Christopher Mohr, Jakob Matthes, Magdalena Feldhahn, Nicolas Casadei, Martin Ebinger, Stefan Stevanovic, Peter Bauer, Oliver Kohlbacher, Cecile Gouttefangeas, Juergen Schaefer, Hans-Georg Rammensee, Rupert Handgretinger, Peter Lang. Personalized peptide vaccination based on patient-individual tumor-specific variants induces T-cell responses in pediatric patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B124.