Abstract 4891: Identification of recurrent high-affinity MHC class I restricted neo-epitopes in neuroblastoma using ProTECT
Authors: Arjun A. Rao, Jugmohit Toor, Sarvind Tripathi, Jacob Pfeil, Nikolaos Sgourakis, Sofie Salama, David Haussler
Journal: Cancer Research (2017)

Abstract Introduction: T-cells are trained to differentiate between cell-surface MHC-displayed peptide sequences from self- and non-self proteins and act on the latter. The numerous mutations often associated with cancers can occur in coding regions of the genome and modify the sequence of wild-type proteins, potentially creating targets for immunotherapies. We have developed an analysis pipeline ProTECT (Prediction of T-cell Epitopes for Cancer Therapy) to identify and rank neo-epitopes in terms of immunogenicity. Running ProTECT on a set of Neuroblastomas patients predicted hotspot mutations that bind well to high-frequency MHC alleles – combinations that would potentially benefit a large subset of NBL patients. Methods: ProTECT accepts paired tumor and normal DNA sequencing fastq files, and tumor RNA sequencing fastqs. Mutations are called using a panel of callers [1-4], and are annotated[5] to identify coding mutations. Prediction of self-MHC:mutated-peptides is carried out[6] and the final binding predictions are ranked using an in-house algorithm. Summary: Running ProTECT on 6 Neuroblastoma samples (NBL) from the TARGET (Therapeutically Applicable Research to Generate Effective Treatments) project revealed 2 well-known hotspot mutations in NBL (NRAS Q61K and ALK R1275Q) that bind to common MHC alleles (A*01:01 and B*15:01 respectively). We also found 2 closely related mutations in ALK F1174L and F1174I that are predicted to C*04:01 and C*07:02. We carried out in-vitro refolding and crystallization assays [7] for the five highest-ranking mutant NRAS and ALK R1275Q predictions. Properly conformed MHC trimers were verified by a monodisperse peak after anion exchange chromatography. SDS gel electrophoresis and Mass-spec confirmed bound peptide for 4/5 tested predictions and 3 of these were used to set up hanging-drop crystallization trials in various conditions. Positive hits were obtained for one (AQDIYRASY::HLA-B*15:01) and the structure was obtained at 1.7A. The structure suggested the binding of the 10-mer (AQDIYRASYY) to the MHC and this was shown to bind better than the 9-mer using Differential Scanning Fluorimetry[9]. We will run ProTECT on the remaining 100+ TARGET NBL trios, and on relevant cohorts within The Cancer Genome Atlas (TCGA). We aim to reveal clinically relevant hotspot-mutation:MHC pairs. Conclusion: We have described a pipeline for identification and ranking of therapeutically relevant neo-epitopes. We have predicted potential targets for NBL that have been validated in-vitro. References 1. Cibulskis, K. et al. Nat Biotech 31, 213-219 (2013) 2. Radenbaugh, A. J. et al. PLoS ONE 9, e111516 (2014) 3. Larson, D. E, et.al. Bioinformatics 28 (3) (2012) 4. Saunders, C. T, et.al Bioinformatics 28 (14) (2012) 5. Cingolani, P. et al. Fly 6, 80-92 (2012) 6. Vita, R. et al. Nucl. Acids Res. (2014) 7. Garboczi, D. N. et.al. PNAS 89 (8) (1992) 9. Lance M. Hellman et.al. J Imm Meth 432:95-101 (2016) Citation Format: Arjun A. Rao, Jugmohit Toor, Sarvind Tripathi, Jacob Pfeil, Nikolaos Sgourakis, Sofie Salama, David Haussler. Identification of recurrent high-affinity MHC class I restricted neo-epitopes in neuroblastoma using ProTECT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4891. doi:10.1158/1538-7445.AM2017-4891