PT  - JOURNAL ARTICLE
AU  - Franke, Katka
AU  - Mülfarth, Ronja Isabelle
AU  - Martin, Michael
AU  - Aguilar-Rosas, Salvador
AU  - Pieterse, Mark
AU  - Langelaar, Jamie
AU  - Esch, Wim van
AU  - Schreurs, Renee van
AU  - Olsman, Hugo
AU  - Made, Lisa van der
AU  - Löwenberg, Bob
AU  - Plasterk, Ronald
AU  - Krüger, Janna
AU  - Heidenreich, Regina
AU  - Kloosterman, Wigard
TI  - 1350 Neo-open reading frame peptides as a source of highly immunogenic neoantigens for mRNA therapeutic cancer vaccines
AID  - 10.1136/jitc-2023-SITC2023.1350
DP  - 2023 Nov 01
TA  - Journal for ImmunoTherapy of Cancer
PG  - A1504--A1504
VI  - 11
IP  - Suppl 1
4099  - http://jitc.bmj.com/content/11/Suppl_1/A1504.short
4100  - http://jitc.bmj.com/content/11/Suppl_1/A1504.full
SO  - J Immunother Cancer2023 Nov 01; 11
AB  - Background Neoantigens expressed and presented by tumor cells can be recognized by the immune system and facilitate destruction of tumors by cytotoxic CD8+ T cells. The main source of neoantigens has been single nucleotide variations with only one amino acid difference from a wildtype protein. We have instead focused on a class of neoantigens that arise from insertions and deletions (indels) or other gene re-arrangements, leading to formation of neo-open reading frame peptides (NOPs). NOPs are stretches of novel amino acid sequences containing numerous potentially highly immunogenic epitopes and therefore provide a valuable source of neoantigens with the capability to improve efficacy of cancer vaccines.Methods The analysis of The Cancer Genome Atlas (TCGA) database and in-house sequenced human tumor samples was used to predict expression of shared neoantigens resulting from indels and other gene re-arrangements. Several selected candidates underwent thorough in vitro experimental validation to confirm tumor cell presentation and immunogenicity of NOPs. Additionally, we tested immunogenicity of mouse surrogate NOPs identified via the same pipeline in the melanoma cell line B16F10 by encoding ten different NOP sequences in an mRNA vaccine.Results We identified several different NOP sequences with predicted expression in human tumor samples that were selected for further validation. From each tested NOP, one or multiple epitopes were binding in vitro to either HLA-A*02:01, HLA-A*01:01, or HLA-A*24:02 allele. Presentation of epitopes on the tumor cell surface was confirmed by eluting the epitopes from HLA alleles followed by mass spectrometry analysis. We also detected presence of antigen specific CD8+ T cells in blood of healthy donors by tetramer staining. In vitro expanded NOP specific CD8+ T cell clones were able to recognize target positive tumor cell lines as measured by increased activation and degranulation markers. In vivo immunogenicity assessment after vaccination of mice with a mRNA vaccine encoding B16F10-derived NOPs showed large number (up to 60%) of polyfunctional CD8+ T cells, and to lower extent also CD4+ T cells after in vitro re-stimulation of splenocytes with NOP peptide pools. Deconvolution confirmed that 3/10 of NOPs induced CD8+ T cell responses and 2/10 NOPs mounted CD4+ T cells responses.Conclusions The neo-open reading frame peptides represent a class of experimentally validated, highly immunogenic neoantigens, suitable to induce robust in vivo immune responses when encoded in an mRNA vaccine format.