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We significantly acknowledge the patients for his or her participation and the Biobank of the University of Navarra for its collaboration. Authors thank Virginia Villar for her help with sample administration. We also thank Thomas Blankenstein’s lab for offering MC703 cells. Retroviral particles have been generated utilizing Plat-A cell-mediated transfection.

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RNA was purified from tumor biopsy and preserved at -80 °C. Briefly, complementary DNA libraries had been constructed from High high quality RNA (1 μg) utilizing TruSeq stranded Total RNA Sample Prep Kit. Library was subjected to paired-end sequencing utilizing the NovaSeq platform (10 Gb 100PE 100Mreads/sample). Alignments had been carried out using the STAR methodology to human genome construct hg38.

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By utilizing these mutations, we predicted HLA class I-restricted potential neoAgs encoded by mutated genes, observing a median of 98 neoAgs. Finally, to show recognition of processed antigens within the context of tumor cells, we prepared transfectants of MC703 cells expressing the neoAgs used for vaccination. We demonstrated that T cells induced by DNA vaccines, mainly for these similar to patient CAM69, specifically recognized tumor cells expressing the neoAgs however not control cells . As shown in Figure 5D, these T cells specifically recognized neoAg-expressing tumor cells.

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The inserts contain a sign peptide from MHC class I, adopted by chosen 15-mer neoAgs situated in tandem and the MHC class I trafficking area sequence . Inserts encoding the neoAgs have been obtained from Genscript and cloned into MSCV-IRES-Thy1.1 DEST and pBK-T-SFV-b12A utilizing BglII&SalI and Apa I websites, respectively. MSCV-IRES-Thy1.1 DEST was a present from Anjana Rao (Addgene plasmid # 17442). Plasmids pMSCV-TMGP69-IRES-Thy1.1, pMSCV-TMGP73-IRES-Thy1.1 encoding the neoAgs and control plasmid (MSCV-IRES-Thy1.1 DEST) had been used to prepare retrovirus for transduction experiments. A needle tumor biopsy was extracted at analysis and preserved at -80°C in RNAlater.

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