Up to 15 predicted epitopes 3. Any driver or suppressor genes identified by WES Identifying Defined Epitopes 12, 14 Whole exome sequencing is performed on a patient’s tumor DNA as well as their matched normal DNA, extracted from the patient’s PBL. RNA sequencing is performed on the patient's tumor sample only. Sequencing is perform ed on an Illumina Nextseq500 or Nextseq550. The output from the sequencer is de-multiplexed and converted to fastq format using Illumina's bcl2fastq program. Reads are then trimmed for quality and to remove and adapter sequence with Trimmomatic software (USADELLAB.org). Once trimmed, Exome reads are aligned to hg19 genome using novoalign from novocraft (novocraft.c om) to create initia l starting bams. RNA- Seq reads are aligned to hg19 using STAR (https://github.com/alexdobin/STAR) 2pass alignment process. Both RNA-Seq and Exome bam files are preprocessed according to the GATK best practices protocol. Ex ome SNVs are called using Strelka, Somatic Sniper, Varscan2, and Mutect, and In/Dels are called using Strelk a and Varscan2. For single nucleotide variants (SNVs) cutoff criteria for evaluation of a variant is Tumor, and normal coverage of 10 or greater, Tumor variant read count of 4 or greater, tumor variant frequency of 7% or greater and two or greater callers calling that variant. For neoantigens arise from insertions/deletions (In/Dels), the criteria are the same except there are no caller criteria. RNA variants are called with Varscan with no cutoffs. Somatic variants are annotated using Annovar against three separate reference databases (Refgene, Ensembl, UCSC). All variants that pass cutoff criteria and those found in COSMIC regardless of cutoff criteria have ne oepitopes generated using an in-house python script. This script produces 25mers with 12 aa flanking the mutation on either side where possible. In the event, it is not possible due to the mutation be located closer than 12 aa from the beginning or end of a transcript, the maximum numb er of aa that can flank are used. For In/Del mutations the corresponding change is made to the cDNA sequence, and then 12 aa before the mutation (where possible) are extracted as we ll as all amino acids beyond the mutation up until the first stop codon is encountered. If no stop codon is encountered, the neoepitope will encompass all sequence up to the end of the cDNA transcript. For each non-synonymous substitution mutation identified by next-generation sequencing, both a “minigene” construct encoding the corresponding amino acid change flanked by 12 amino acids of the wild-type protein sequence or a crude 25 mer peptide are made. Multiple minigenes (10-15) are strung together to generate one TMG construct. These minigene constructs are codon optimized, synthesized, and cloned in-frame into a modi fied pcDNA3.1 vector. This modified vector contains a signal sequence and a DC-LAMP tra fficking sequence to enhance processing and presentation, in addition to a poly-A tail to enha nce mRNA stability. The nucleotide sequence of DOO70*VLVYHULILHGE\VWDQGDUG6DQJHUVHTXHQFLQJ *HQVFULSW  $SSUR[LPDWHO\RQHȝJRIWKH linearized plasmid is used for the generation of IVT RNA using the mmessage mmachine T7 Ultra kit (Life Technologies) as directed by th e manufacturer. RNA purity and concentrations are Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 59 assessed using a NanoDrop spectrophotometer. R NA is then aliquoted into microtubes and stored at -80 degrees Celsius until use. Crude pe ptides are pooled at a final concentration of approximately 3 mg/mL per peptide (10-18 peptides per pool). To isolate TILs, surgically resected tumors are cut into approximately 1-2 mm fragments and placed individually into wells of a 24-well plate containing 2 mL of complete media (CM) containing high dose IL-2 (6000 IU/mL, Chiron) . CM consisted of RPMI supplemented with 10 percent in-house human serum, two mM L- JOXWDPLQHP0+(3(6DQGWHQȝJP/ gentamicin. In some cases, after the initial outgrowth of TIL (between 2-4 weeks), selected cultures are rapidly expanded in gas permeable G-Rex100 flasks using irradiated PBMC at a ratio of 1 to 100 in 400 mL of 50/50 medium, su pplemented with 5 percent human AB serum, 3000 IU/mL of IL-2, and 30 ng/mL of OKT3 antibody (Miltenyi Biotec). 50/50 media consisted of a 1-to-1 mixture of CM with AIM-V media. All cells are cultured at 37 degrees Celsius with 5 percent CO2. Monocyte-derived, immature dendritic cells are generated using the plastic adherence method. Apheresis samples are thawed, washed, set to 5-10e6 cells/mL with neat AIM-V media (Life Technologies) and then incu bated at approximately 1e6 cells/cm2 in an appropriate sized tissue culture flask and incubated at 37 degrees Celsius, 5 percent CO2. After 90 minutes, non-adherent cells are collected, and the flasks are vigorously washed with AIM-V media, and then incubated with AIM-V media fo r another 60 min. The flasks are then washed again with AIM-V media and then the adherent cells are incubated with DC media. DC media FRPSULVHGRI530,FRQWDLQLQJ SHUFHQWKXPDQVHUXP8P/SH QLFLOOLQDQGȝJP/ streptomycin, 2 mM L-glutamine, 800 IU/mL GM-CSF (Leukine) and 200 U/mL IL-4 (Peprotech). On days 2-3, fresh DC media is added to the cultures. Fresh or freeze/thawed DCs are used in experiments on day 4-6 after culture initiation. DCs are harvested, washed 1x with PBS, and then resuspended in Opti-MEM (Life Technologies) at 10-30e6 cells/mL. IVT RNA (8 ȝJ LVDOLTXRWHGWRWKHERWWRPRI DPPJDSHOHFWURSRUDWLRQFX YHWWHDQGȝORI$3&VDUH added directly to the cuvette. The final RNA conc entration used in electroporations is thus 80 ȝJP/(OHFWURSRUDWLRQVLVFDUULHGRXWXVLQJD%7; -830 square wave electroporator. DCs are electroporated with 150 V, 10 ms, and 1 pulse. Fo llowing electroporation, cells are immediately transferred to polypropylene tubes containing DC media supplemented with the appropriate cytokines. Transfected cells are incubated ov ernight (12-14 hours) at 37 degrees Celsius, 5 percent CO2. Cells are washed 1x with PBS prio r to use in co-culture assays. For peptide pulsing, DCs are harvested and then resuspende d at 0.5e6 cells/mL with DC media containing the appropriate cytokines. Long peptides (usual ly 25mers, Genscript) are dissolved with DMSO DQGSXOVHGRQWRWKH$3&VDWDSSUR[LPDWHO\ȝJP/DQGLQFXEDWH GRYHUQLJKWDWGHJUHHV Celsius with 5 percent CO2. The following day, APCs are washed 1x prior co-culture with T cells. For co-culture with APCs, approximately 3.5e4 to 7e4 DCs are used per well of a 96-well plate. Prior to processing the ELISPOT plates, cells are harvested from the plate and processed for flow cytometry analysis described below. All co-cultures are performed in the absence of exogenously added cytokines. For IFN- Ȗ(/,6327DVVD\V(/,,3SODWHV 0LOOLSRUH0$,36:8  are pre-WUHDWHGZLWKȝORIHWKDQRO SHUZHOOIRUPLQZDVKHG[ ZLWK3%6DQGWKHQ FRDWHGZLWKȝORIȝJP/,)1 -ȖFDSWXUHDQWLERG\ 0DEWHFKFORQH  -D1K) and incubated overnight in the fridge. Prior to co-culture, the plates are washed 3x with PBS, followed by blocking with 50/50 media for at least 1 h at room temperature (RT). After 20-22 hours of co- culture, cells are harvested from the ELISPOT plat es into a standard 96-well round bottom plate for flow cytometry analysis, and then the ELISPOT plates are washed 6x with PBS + 0.05% Tween-20 (PBS- 7 DQGWKHQLQFXEDWH GIRUKRXUVDW57ZLWKȝOZHOORIDȝPILOWHUHG Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 60ȝJP/ELRWLQ\ODWHGDQWL -human IFN- ȖGHWHFWLRQ antibody solution (Mabtech, clone: 7-B6-1, diluent consisted of 1X PBS suppl emented with 0.5% FBS). The plate is then washed 3x with PBS-T, followed by a 1- KRXULQFXEDWLRQZLWKȝOZHOORIVWUHSWDYLGLQ -ALP (Mabtech, diluted 1:3000 with above diluent). The plate is then wash ed 5x with PBS followed by development with ȝOZHOORIȝPILOWHUHG%&,3 1%7VXEVWUDWHVROXWLRQ .3 /,QF 7KHUHDFWLRQLV stopped by rinsing thoroughly with cold tap wa ter. ELISPOT plates are scanned and counted using an ImmunoSpot plate read er and associated software (Cellular Technologies, Ltd). Expression of the T-cell activati on markers OX40 and 4-1BB is a ssessed by flow cytometry at approximately 18-24h post-stimulation. OX40 is predominantly expressed by activated effector CD4+ T cells, while 4-1BB can be expressed by both activated CD4+ an d CD8+ T cells. Cells that were harvested from the ELISPOT plate are pelleted, washed with FACS buffer (1X PBS supplemented with 1 percent FBS and 2 mM EDTA ), and then stained with the appropriate antibodies for approximately 30 minutes, at 4 degr ees Celsius in the dark. Cells are washed at least once with FACS buffer prior to acquisiti on on a BD FACSCanto II flow cytometer. The number of live T-cell events collected is usually ranged between 2e3-8e3. Any positive reactivity (3-fold over background of DC only) identified by ELISPOT, 41BB or OX40 is considered as a TIL reactivity against a mutated epitope. To identify the specific antigen, individual peptides comprising each positive TMG or peptide pool are used for co-culture with the reactive TIL. Once identified, an HPLC purified mutated and wild type peptides are synthesized and used to validate immunogenic peptide integrity. A defined ne oantigen is considered as a mutated peptide recognized by TIL and showed no reactivity to the wild type epitope. Any defined epitope identified will be included in the vaccine backbone. The average turnaround time from TIL harvest to identification of immunogenic epitopes is 7-14 weeks ( Table A-1 ). Table A-1. Turnaround time for defi ned neoantigen discovery pipeline. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 61 Selecting Predicted Epitopes A patients HLAs are predicted from the exome tumor sample, the exome normal sample and the tumor RNA using PHLAT (https://sites.google.com/site/phlatfortype/). If there are differences in allelic predictions the top two most frequently predicted locus for each HLA are used. The 25mers previously generated are then run th rough netMHCpan-3.0 for each class I HLA and minimal epitopes of 8,9,10,11,12 mer lengths are genera ted. All predicted binders not containing the mutated amino acid are removed. All 25mers ar e also ran through netchop-3.1 and scores for proteosomal cleavage for each amino acid are recorded. In order to determine which variants should be screened several steps are taken filtering is performed: 1. Retained variants must possess at least one tr anscript which has evidence of coding for a protein. 2. Retained variants must be found to be presen t in at least two of the annotation databases (NCBI, UCSC, Ensembl). The rationale behind this is to remove genes or transcripts with less support 3. Retained variants must not been seen in our sequencing data at a rate greater than 2.5%. This excludes known driver genes as descri bed in Wellcome Sanger’s cancer genome census list. For MHC Ranking we created an observed positive rate table from the analysis of > 8000 screened 25mers. A combina tion of filters was app lied to the data and the observed positive rate for all 25mers that remain after that filtering. x Filters: a. Exome pass 1,0 = yes, no b. RNA seen 1,0 = yes, no-filter c. Gene expression quartile >= 1,2,3,4 d. netMHCpan3.0 rank <= 5, 4, 3, 2, 1, .75, .50, .25, 0.1 e. netCHOP Cterm score>= 0, .1, .2, .3, .4, .5, .6, .7, .8, .9 f. netCHOP 20S score >= 0, .1, .2, .3, .4, .5, .6, .7, .8, .9 g. # of minimals that meet last 3 criteria >= 1, 2, 3, 4, 5 Taking all possible combinations there are ~45000 different filtering combinations. Each 25mer is analyzed to see what filtering groups it fits into and the highest observed positive rate from these groups is then assigned to the 25mer. After all retained 25mers have an observed positive rate. The variants with evidence in RNA-seq data are taken 1 st sorted by the observed rate and ranked in descending order. After that the same is done to the remaining 25mers. The first 15 ranked 25mers will be include d in the vaccine backbone. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 62 APPENDIX B: PERFORMANCE STATUS CRITERIA ECOG Performance Status Scale Grade Descriptions 0 Normal activity. Fully active, able to carry on all pre-disease performance without restriction.