1. (A) Mutations within neoepitopes lead to structural alterations across the peptide backbone, as illustrated with structural snapshots from the simulations of the mutant and WT Tnpo3.1 epitope bound to H-2Kd. (B) Summary of structural differences for highly DAI ranked nonamers. Differences were quantified by superimposing average peptide conformations from the molecular dynamics simulations and computing RMSDs for all common atoms. Green and red bars indicate epitopes that led to either positive or negative immunological responses, respectively. The yellow bar shows the results for control calculations for an immunogenic HBV core epitope. (C) The numbers in the legend give the mean RMSF for the all amino acids of each peptide; those at the right give the value for only the C-terminal α carbon. Mutated amino acids are indicated by lower case in the x-axis. (D) Effects of mutations on the conformational stability of all nonamers, calculated as the difference between the mean RMSF of the mutant and the WT peptide. (E) Fluctuations at the peptide C-terminal ends and immunogenicity. The dashed vertical line shows the mean value for all mutant nonamers. The yellow bar shows the C-terminal stability of the HBV core epitope control. Bioinformatics pipeline and summary statistics Genomic and bioinformatic profiling of mutational neoepitopes reveals new rules to predict anticancer immunogenicity Fei Duan 1*, Jorge Duitama 2*, Sahar Al Seesi 2*, Cory M. Ayres 3, Steven A. Corcelli 3, Arpita P. Pawashe 1, Tatiana Blanchard 1, David McMahon 1, John Sidney 4, Alessandro Sette 4, Brian M. Baker 3, Ion I. Mandoiu 2, and Pramod K. Srivastava 1 1 Department of Immunology and Carole and Ray Neag Comprehensive Cancer Center, University of Connecticut School of Medicine 2 Department of Computer Science and Engineering, University of Connecticut 3 Department of Chemistry and Biochemistry and Harper Cancer Research Institute, University of Notre Dame 4 LaJolla Institute of Allergy and Immunology * Authors contributed equally to this work Abstract The mutational repertoire of cancers creates the neoepitopes that make cancers immunogenic. Here, we introduce two novel tools that identify, with relatively high accuracy, the small proportion of neoepitopes (among the hundreds of potential neoepitopes) that protect the host through an antitumor T cell response. The two tools consist of (a) the numerical difference in NetMHC scores between the mutated sequences and their unmutated counterparts, termed the differential agretopic index (DAI), and (b) the conformational stability of the MHC I– peptide interaction. Mechanistically, these tools identify neoepitopes that are mutated to create new anchor residues for MHC binding, and render the overall peptide more rigid. Surprisingly, the protective neoepitopes identified here elicit CD8-dependent immunity, even though their affinity for Kd is orders of magnitude lower than the 500- nM threshold considered reasonable for such interactions. We applied our method to multiple independent tumors. The pipeline, including the DAI algorithm, was first derived empirically on the data from Meth A tumor cell line, and then tested on the CMS5 cell line. Antitumor activity predicted from the DAI algorithm was significantly stronger in CMS5 than in Meth A. This variation is most likely a reflection of the immuno-suppressive mechanisms unique to the Meth A tumor and thus unrelated to the merits of the DAI algorithm per se. The DAI algorithm has since been tested in yet another mouse tumor, the B16 melanoma, and data on T cell responses in this line as well, are consistent with significant superiority of DAI over NetMHC alone. Future and Ongoing Work: (1) Extending the analysis to MHC II-restricted epitopes of CD4 T cells (2) Currently developing a human pipeline to predict epitopes from paired tumor/normal exomes and tumor RNA-Seq Conclusions and ongoing work Structural stability as a correlate with immunogenicity (A-B) Tumor-protective activity of the mutated epitopes with top NetMHC (A) or top DAI (B) scores for CMS5 and Meth A. Area under the curve (AUC) for each individual tumor growth curve was calculated and normalized by setting the naive group to a value of 100, shown by a horizontal red line (C) Examples of tumor growth curves in untreated mice (naive) and mice immunized with indicated mutant peptides from CMS5 or their WT counterparts. Stau1.2 is a representative neoepitope that does not elicit protective immunity, whereas Atxn10.1 and Alkbh6.2 are representative neoepitopes that do. Each line shows the kinetics of tumor growth in a single mouse. The experiments were performed three times. Landscape of protective tumor immunity elicited by tumor specific epitopes F. Duan, J. Duitama, S. Al Seesi, C. Ayres, S. Corcelli, A. Pawashe, T. Blanchard, D. McMahon, J. Sidney, A. Sette, B. Baker, I.I. Mandoiu, and P.K. Srivastava, Genomic and bioinformatic profiling of mutational neo-epitopes reveals new rules to predict anti-cancer immunogenicity, Journal of Experimental Medicine 211, pp. 2231-2248, 2014 J. Duitama, P.K. Srivastava, and I.I. Mandoiu, Towards Accurate Detection and Genotyping of Expressed Variants from Whole Transcriptome Sequencing Data, BMC Genomics 13(Suppl 2):S6, 2012 References L d