Abstract Introduction: Cancer neoantigens result from mutations accumulated in an individual tumor. These mutations are usually private and yield high-affinity T-cells as they are not limited by mechanisms of self-tolerance. Neoantigens presented in the context of MHC class I can be recognized by anti-tumor CD8+ T-cells. Methods: Three consecutive patients with confirmed pancreatic ductal adenocarcinoma (PDAC) had DNA and RNA isolated from areas of high tumor cellularity. Comparative normal DNA was isolated from peripheral blood mononuclear cells (PBMC). Nonsynonymous mutations were identified by tumor/normal whole exome sequencing and expression in the tumor was verified by cDNA capture. HLA typing was inferred from exome sequencing. Candidate neoantigens were identified using multiple epitope prediction algorithms for MHC class I. Matched patient PBMC were cultured in serum-free media for 48 hours with synthetic mutated short peptides to determine the baseline immune response by interferon-gamma (IFNγ) enzyme-linked immunospot assay. Results: A KRAS driver mutation was identified in all three patients. Using KRAS variant allele frequency (VAF) as a surrogate for tumor purity, we achieved greater than 40% tumor purity for all three samples. This provides an adequate level of purity to proceed with neoantigen identification. In these samples, the mean number of nonsynonymous mutations identified was 27 (range: 18-32; DNA VAF cutoff 10%). Of these nonsynonymous mutations, a mean of 8.6 mutations (range: 7-10) were expressed (RNA VAF cutoff >10% and FPKM >1). Following confirmation of mutation expression, a mean of 5 mutations (range: 4-6) were predicted to bind to MHC class I using multiple prediction algorithms (binding affinity <500nM). IFNγ immunospot assay revealed a baseline immune response to one neoantigen in each patient (Patient A - SDHA: 75 spot forming units per millions cells (SFU/M) vs Negative: 12.5 SFU/M, p=0.01; Patient B - ZRANB1: 588 SFU/M vs Negative: 17.5 SFU/M, p<0.001; Patient C - ATAD3C: 340 SFU/M vs Negative: 75 SFU/M, p=0.002). Conclusion: Utilizing advanced genomic and bioinformatics tools we have successfully characterized the cancer neoantigens in a series of three consecutive patients with PDAC. From this data, we plan to open a phase I clinical trial to assess the safety and immunogenicity of a neoantigen DNA vaccine. Citation Format: Darren R. Cullinan, Michael McLellan, Xiuli Zhang, Timothy M. Nywening, Chris A. Miller, S. Peter Goedegebuure, William G. Hawkins, William E. Gillanders. Development of a personalized pancreatic cancer vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5640.