Multiple genetic events and subsequent clonal evolution drive carcinogenesis, making disease elimination with single-targeted drugs difficult. The multiplicity of gene mutations derived from clonal heterogeneity therefore represents an ideal setting for multiepitope tumor vaccination. Here, we used next generation sequencing exome resequencing to identify 962 nonsynonymous somatic point mutations in B16F10 murine melanoma cells, with 563 of those mutations in expressed genes. Potential driver mutations occurred in classical tumor suppressor genes and genes involved in proto-oncogenic signaling pathways that control cell proliferation, adhesion, migration, and apoptosis. Aim1 and Trrap mutations known to be altered in human melanoma were included among those found. The immunogenicity and specificity of 50 validated mutations was determined by immunizing mice with long peptides encoding the mutated epitopes. One-third of these peptides were found to be immunogenic, with 60% in this group eliciting immune responses directed preferentially against the mutated sequence as compared with the wild-type sequence. In tumor transplant models, peptide immunization conferred in vivo tumor control in protective and therapeutic settings, thereby qualifying mutated epitopes that include single amino acid substitutions as effective vaccines. Together, our findings provide a comprehensive picture of the mutanome of B16F10 melanoma which is used widely in immunotherapy studies. In addition, they offer insight into the extent of the immunogenicity of nonsynonymous base substitution mutations. Lastly, they argue that the use of deep sequencing to systematically analyze immunogenicity mutations may pave the way for individualized immunotherapy of cancer patients. Cancer Res; 72(5);

Adoptive transfer of dendritic cells (DCs) transfected with in vitro-transcribed, RNA-encoding, tumor-associated antigens has recently entered clinical testing as a promising approach for cancer immunotherapy. However, pharmacokinetic exploration of RNA as a potential drug compound and a key aspect of clinical development is still pending. While investigating the impact of different structural modifications of RNA molecules on the kinetics of the encoded protein in DCs, we identified components located 3 of the coding region that contributed to a higher transcript stability and translational efficiency. With the use of quantitative reverse transcription-polymerase chain reaction (RT-PCR) and eGFP variants to measure transcript amounts and protein yield, we showed that a poly(A) tail measuring 120 nucleotides compared with a shorter one, an unmasked poly(A) tail with a free 3 end rather than one extended with unrelated nucleotides, and 2 sequential ␤-globin 3 untranslated regions cloned head to tail between the coding region and the poly(A) tail each independently enhanced RNA stability and translational efficiency. Consecutively, the density of antigen-specific peptide/MHC complexes on the transfected cells and their potency to stimulate and expand antigen-specific CD4 ؉ and CD8 ؉ T cells were also increased. In summary, our data provide a strategy for optimizing RNA-transfected DC vaccines and a basis for defining release criteria for such vaccine preparations. IntroductionAntigen-encoding RNA 1,2 has the advantages of a genetic vaccine (delivery of all epitopes of the whole antigen, easy manufacturing, standardized purification) and the added value of a safe pharmaceutical characterized by transient expression and lack of integration into the genome of the treated host. The combination of this versatile antigen delivery molecule with dendritic cells (DCs) as the most potent antigen-presenting cells is regarded as an attractive approach to induce cellular and potentially therapeutic immune responses in patients with cancer. Reports demonstrated convincingly that the use of RNA results in efficient induction of antigen-specific immune responses in vitro and in animal models 1,[3][4][5][6][7] and paved the way for trials in humans. Early clinical trials showed feasibility, lack of toxicity, and promising efficacy based on immunologic and clinical read-outs. [8][9][10][11][12] At this early phase of clinical development, antigen-specific RNA has the status of a drug compound requiring detailed exploration.Basic pharmacologic issues that must be addressed in drug development include the pharmacokinetics of the compound of interest within the system of its physiological activity after administration. In the quoted clinical trials, this system would be represented by immature or mature DCs. A key objective of such investigations is better understanding of the impact of the structural features of the drug formulation on its pharmacologic properties. Neither of these questions has thus far been addressed for antigen-e...