Control Clin. Trials 10:1-10, 1989 Abbreviated Title: Personalized DC Vaccine Version Date: March 26, 2018 4413 FIGURES AND TABLES FIGURE 1: A P ROCESS FOR THE IDENTIFICATION OF IMMUNOGENIC SOMATIC MUTATIONS Abbreviated Title: Personalized DC Vaccine Version Date: March 26, 2018 45 FIGURE 2: TLR M IX INDUCES THE SECRETION OF IL-12 P70, TNFDAND MULTIPLE CHEMOKINES Monocyte derived dendr itic cells were stimulated with TLR mix ( PolyI:C, R848, and IFN J), 3T3CD40L cells, LPS or a cytokine mix (Il-1 E, IL-6, TNF Dand PGE2), Non-treated cells (NT) were used as negative control . Sixteen hours post incubation th e level of costimulatory molecules was evaluated by flow cytometry (A, B) and the secretion of mul tiple cytokines and chemokines was measured by multiplex assay (C, D). Abbreviated Title: Personalized DC Vaccine Version Date: March 26, 2018 46 FIGURE 3: TLR M IX ALONE IS SUFFICIENT TO INDUCE POTENT DC M ATURATION Monocyte derived dendr itic cells were stimulated with TLR mix ( PolyI:C, R848, and IFNg), K463H+TLR mix and K602C14 cells + TLRmix. As a negative control, we used DCs alone or DCs stimulated with K562 cells. S ixteen hours post incubation the level of costimulatory molecules was evaluated by flow cytometry (A) and the secretion of multiple cytokines was measured by multiplex assay (B). Abbreviated Title: Personalized DC Vaccine Version Date: March 26, 2018 47 FIGURE 4: L ONG PEPTIDES ,MINIMAL EPITOPES AND TMG MRNA USED TO EVALUATE ANTIGEN PRESENTATION FOR CD4 AND CD8 C ELLS Abbreviated Title: Personalized DC Vaccine Version Date: March 26, 2018 48 FIGURE 5: L ONG PEPTIDES AND MINIMAL EPITOPES ARE SUPERIOR TO TMG MRNA IN PRESENTING ANTIGENS TO CD4 AND CD8 C ELLS DCs were transfected with the T MG constructs or loaded with lon g peptide and minimal epitopes for 12 hours. DCs were co-culture d with PBMC transduced with TC R’s recognizing the corresponding antigens for 18 hours, and antigen recognition wa s evaluated for Class-I epitopes (A) and Class-II epitopes (B) by flow cytometry for 41BB expres sion. Irrelevant TMG and DMSO were used as negative cont rol for TMG transfection and pep tide loading, respectively. Abbreviated Title: Personalized DC Vaccine Version Date: March 26, 2018 49 FIGURE 6: P EPTIDE LOADED , TLR S TIMULATED DC’ SEFFICIENTLY PRESENT BOTH CD4 AND CD8 E PITOPES DCs were loaded with long peptides for 2, 4, and 10 hours and matured using a TLR mix. DCs were washed and co-cultured wit h PBMC transduced with TCRs recognizing the corresponding antigens for 18 hours (A); antige n recognition was evaluated by flow cytometry for 41BB expression (B). Abbreviated Title: Personalized DC Vaccine Version Date: March 26, 2018 50 FIGURE 7: O VERVIEW OF THE METHOD USED TO EVALUATE THE ACTIVATION OF NEOANTIGEN SPECIFIC TC ELLS Abbreviated Title: Personalized DC Vaccine Version Date: March 26, 2018 51 FIGURE 8: DC SMATURED WITH TLR M IX ARE CAPABLE OF STIMULATING NAÏVE , ANTIGEN -EXPERIENCED AND TERMINALLY DIFFERENTIATED EFFECTOR CELLS Apheresis samples from a colorectal cancer patient were thawed and then incubated in an appropriately sized tissue cultu re flask. Adherent monocytes we re differentiated into DCs, loaded with neoantigen peptide pools and matured using a TLR mi x. Non-adherent cells were collected, and memory, naïve and t erminally differentiated effe ctor memory cells (TEMRA) were sorted, and in vitr o stimulated with mat ure peptide-pulsed autologous DCs. Ten days after the first stimulation T cells w ere re-stimulated with DCs loade d with all peptide pools, and sorted based on 41BB and OX40 expression to enrich for neoantigen-spec ific cells. Sorted cells were stimulated by DCs loaded with a ll peptide pools and tested for upregulation of 41BB (data not shown). Memory (A), TEMRA (B), and naïve(C) T cells were then t ested for neoantigen specific recognition by 41BB upregulation f ollowing co-culture with DCs loaded with single peptides derived from the most reactive peptide pools. Abbreviated Title: Personalized DC Vaccine Version Date: March 26, 2018 52 FIGURE 9: P ROCESS FOR EPITOPE SELECTION AND SYNTHESIS Abbreviated Title: Personalized DC Vaccine Version Date: March 26, 2018 53 TABLE 1: D EFINED MUTATED ANTIGENS RECOGNIZED BY TIL I DENTIFIED AT THE SURGERY BRANCH Abbreviated Title: Personalized DC Vaccine Version Date: March 26, 2018 5414 APPENDICES APPENDIX 1: C ELLINFUSION INSTRUCTIONS Necessary equipment: xPrimary IV tubing (2) (DO NOT USE AN INLINE FILTER FOR CELLS) xSecondary IV tubing (1) xNS (sodium chloride 0.9%) xIV infusion pump xGloves Steps: Key Points: 1. Patient RN will be informed of the approximate time of cell arrival at the bedside.