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1350 Neo-open reading frame peptides as a source of highly immunogenic neoantigens for mRNA therapeutic cancer vaccines
  1. Katka Franke1,
  2. Ronja Isabelle Mülfarth2,
  3. Michael Martin1,
  4. Salvador Aguilar-Rosas1,
  5. Mark Pieterse1,
  6. Jamie Langelaar1,
  7. Wim van Esch1,
  8. Renee van Schreurs1,
  9. Hugo Olsman1,
  10. Lisa van der Made1,
  11. Bob Löwenberg1,
  12. Ronald Plasterk1,
  13. Janna Krüger2,
  14. Regina Heidenreich2 and
  15. Wigard Kloosterman1
  1. 1CureVac Netherlands B.V., Amsterdam, Netherlands
  2. 2CureVac, Tübingen, Baden-Württemberg, Germany
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


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.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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