5. Patient has received prior therapy consisting of anti-CTLA-4, anti-PD-1, anti-PD-L1 or any other antibody or drug specifically targeting T cell co-stimulation or checkpoint pathways. Patients with NSCLC and GEA who have been, or are currently being, treated with anti-PD-(L)1 monotherapy are eligible. 6. Immunosuppression that is expected to be present at the time of study treat- ment after vaccine production and coming from: a. Concurrent, recent (≤4 weeks) or anticipated treatment with systemic corticosteroids (>10 mg daily prednisone equivalent) or other immu- nosuppressive medications such as OKT3, ATG/ALG, methotrexate, tacrolimus, cyclosporine, azathioprine or rapamycin. Inhaled or topical steroids, physiologic corticosteroid replacement therapy for adrenal or pituitary insufficiency, steroid pretreatment for chemotherapy (e.g., pemetrexed), antihistamines, nonsteroidal anti-inflammatory drugs and aspirin are permitted in the absence of active autoimmune disease b. Conditions such as common variable hypogammaglobulinemia or ra- diation exposure such as large field radiotherapy 7. History of allogeneic tissue/solid organ transplant. 8. Patients who have had a history of life-threatening TRAEs with prior im- munotherapy or have not recovered from prior cancer therapy-induced AEs (i.e., any AE that remains ≥ grade 2 or that has not returned to baseline with the exception of peripheral neuropathy, alopecia and hypothyroidism that is controlled with hormone replacement therapy). 9. Active, known or suspected autoimmune disease. a. Patients with type I diabetes mellitus, hypothyroidism, adrenal or pitui-tary insufficiency that is controlled with hormone replacement therapy, skin disorders (such as vitiligo, psoriasis or alopecia) not requiring sys-temic treatment, or conditions not expected to recur in the absence of an external trigger are permitted. Replacement therapy (e.g., thyroxine, insulin or physiologic corticosteroid replacement therapy for adrenal or pituitary insufficiency) is not considered a form of systemic treatment. 10. History of other cancer within 2 years with the exception of basal or squa- mous cell carcinoma of the skin, superficial bladder cancer or carcinoma in situ of the breast, cervix, prostate or other neoplasm that has undergone potentially curative therapy with no evidence of disease recurrence following agreement with sponsor. 11. Any severe concurrent non-cancer disease (including active systemic infec- tion and/or uncontrolled hypertension) that, in the judgment of the investiga- tor, would make the patient inappropriate for the current study. 12. Active tuberculosis or recent (<2 week) clinically substantial infection, or evidence of active hepatitis B or hepatitis C. 13. Known history of positive test for HIV or known acquired immunodeficiency syndrome. 14. History of pneumonitis requiring systemic steroids for treatment (with the exception of prior resolved in-field radiation pneumonitis). 15. Patient with known central nervous system (CNS) metastases and/or carcino- matous meningitis. 16. Myocardial infarction within 6 months of study initiation, active cardiac ischemia, myocarditis or New Y ork Heart Association grade III or IV heart failure. 17. Pregnant, planning to become pregnant or nursing. 18. Medical, psychiatric, cognitive or other conditions that compromise the patient’s ability to understand the patient information, give informed consent, comply with the study protocol or complete the study. Exclusion criteria for the study treatment stage. Patients meeting any of the of the following exclusion criteria will not be eligible for the study treatment stage: 19. Immunosuppression from: • concurrent, recent (≤ 4 weeks) or anticipated treatment with systemic corticosteroids (>10 mg daily prednisone equivalent) or other immuno- suppressive medications such as OKT3, ATG/ALG, methotrexate, tacroli- mus, cyclosporine, azathioprine or rapamycin. Inhaled or topical steroids, physiologic corticosteroid replacement therapy for adrenal or pituitary insufficiency, steroid pretreatment for chemotherapy (e.g., pemetrexed), antihistamines, nonsteroidal anti-inflammatory drugs and aspirin are permitted in the absence of active autoimmune disease • conditions such as common variable hypogammaglobulinemia or expo-sures such as large field radiotherapy 20. Patients who have had history of life-threatening TRAEs with prior immuno-therapy or who have not recovered from prior cancer therapy-induced AEs (i.e., any AE that remains ≥ grade 3, or that has not returned to baseline with the exception of alopecia, neuropathy and hypothyroidism if controlled by hormone replacement). Only applicable to those AEs not covered by other inclusion and exclusion criteria (e.g., ANC or ALT values). 21. Active, known or suspected autoimmune disease. Patients with type I diabetes mellitus, hypothyroidism if requiring hormone replacement, skin disorders (such as vitiligo, psoriasis or alopecia) not requir - ing systemic treatment, or conditions not expected to recur in the absence of an external trigger are permitted. Replacement therapy (e.g., thyroxine, insu-lin or physiologic corticosteroid replacement therapy for adrenal or pituitary insufficiency) is not considered a form of systemic treatment. 22. Received radiation therapy within 2 weeks before first dose of study treatment. 23. Any severe concurrent non-cancer disease (including active infection and/or uncontrolled hypertension) that, in the judgment of the investigator, would make the patient inappropriate for the current study (study treatment may be delayed until patient has recovered from disease (such as an infection) or has stabilized). 24. Active tuberculosis or recent (<2 week) clinically substantial infection, or evidence of active hepatitis B or hepatitis C. 25. History of pneumonitis requiring systemic steroids for treatment (with the exception of prior resolved in-field radiation pneumonitis). 26. Patient with uncontrolled CNS metastases or brain stem metastasis, patient receiving steroids for CNS disease and/or carcinomatous meningitis; patients with irradiated CNS metastases should be discussed on a case-by-case basis with the sponsor before enrollment. 27. Myocardial infarction, active cardiac ischemia, myocarditis or New Y ork Heart Association grade III or IV heart failure since inclusion in the study. 28. Pregnant, planning to become pregnant or nursing. 29. Treatment with botanical preparations (e.g. herbal supplements or traditional Chinese medicines) intended for general health support or to treat the disease under study within 2 weeks before treatment that may interfere with study treatment per investigator discretion. 30. Medical, psychiatric, cognitive or other conditions that compromise the patient’s ability to understand the patient information, give informed consent, comply with the study protocol or complete the study. Individual patient-specific vaccine cassettes. Patient tissue next-generation sequencing, analysis and neoantigen prediction using the EDGE model were performed as previously described 30. Twenty neoantigens were selected for each patient, as summarized in Supplementary Table 2. The 20 neoantigen epitopes were designed with flanking sequences so that each are 25 amino acids in length and arranged as a cassette to minimize generation of new junction epitopes. A class II helper domain consisting of universal class II epitopes PADRE and tetanus toxoid were included at the C terminus of the cassette. The cassettes were codon optimized for maximum protein expression in humans and for ease of synthesis. The cassettes were synthesized from overlapping oligonucleotides (Integrated NATuRE MEDICINE | www.nature.com/naturemedicine Articles Nature MediciNeDNA Technologies (IDT)), that cover both DNA strands of the cassette sequence plus sequences in the plasmid backbones and assembled into either ChAd or samRNA plasmid backbones by Gibson assembly (Codex DNA). The assembled plasmids were then transformed into bacteria and screened by Sanger sequencing for correct clones which served as the templates for good manufacturing practice ChAd68 production or for samRNA transcription. ChAd68 plasmids were linearized with PacI and then transfected with TransIt Lenti (Mirus Bio) into 293F (Thermo Fisher Scientific) cells to initiate virus production. The virus was amplified through successive rounds of infection before the final production run at scale. The virus was purified at 48–72 h after infection and purified by anion exchange chromatography (Satorius) and formulated in 5 mM Tris, pH 7.9, 1 mM MgCl 2 and 75 mM NaCl. VP titers were determined by absorbance at 260 nm after SDS disruption and infectious unit titers were determined by immunostaining with an anti-Ad antibody (Abcam). samRNA plasmid was used as a template for RNA transcription using T7 polymerase, and the RNA was capped using a vaccine-capping enzyme and mRNA CAP 2′-O-methyl transferase (NEB). The RNA concentration was measured by Ribogreen quantitation and then formulated into lipid nanoparticles (Genevant). Patient PBMC isolation and storage. Patient demographics and dosing schedules are shown in Table 1 and Extended Data Table 1. Due to COVID-19 restrictions, several blood draws were missed. Ad hoc dosing and blood draw timepoints were added with IRB approval for several patients. Whole blood for immunogenicity testing was collected before administration of the first dose (ChAd68 prime) to assess baseline levels and at 14 and 28 days (2 and 4 weeks) after ChAd68 prime. Subsequent planned sample collection post samRNA boosts include days 7, 14 and 28 after dose 2 (samRNA boost 1) and at days 7 and/or 28 after each additional samRNA boost. Two patients (G2 and G8) consented to an optional leukapheresis procedure (week 19 for G2, week 25 for G8). PBMCs from whole blood or leukopak were isolated at local PBMC processing sites according to standardized protocols. Briefly, cells were isolated using density gradient centrifugation on Ficoll Paque Plus (GE Healthcare), washed with D‐PBS (Corning), counted and cryopreserved in CryoStor CS10 (STEMCELL Technologies) at 5 × 10 6 cells ml−1 for whole blood and 2 ×107 cells ml−1 for leukopak. Cryopreserved cells were stored in liquid N2, shipped in cryoports and transferred to storage in liquid N2 upon arrival. Cryopreserved cells were thawed and washed twice in OpTmizer T Cell Expansion Basal Medium (Gibco) with Benzonase (EMD Millipore) and once without Benzonase. Cell counts and viability were assessed using the Guava ViaCount reagents and module on the Guava EasyCyte HT cytometer (EMD Millipore). Cells were rested overnight before use in functional assays. Peptides (clinical study). Custom-made, recombinant, lyophilized peptides specific for each patient were produced by GenScript and reconstituted at 5 mg ml −1 per peptide in sterile DMSO (VWR International), aliquoted and stored at −80˚C (Supplementary Table 2). Control peptides to assess responses to infectious disease antigens from cytomegalovirus, Epstein–Barr virus and influenza (CEF peptide pool) were purchased from JPT Peptide Technologies. IVS cultures. Neoantigen-reactive T cells from patient samples were expanded in the presence of cognate peptides and low-dose IL-2 as described previously 30. Briefly, thawed PBMCs were rested overnight and stimulated in the presence of minimal epitope peptide pools (10 µg ml −1 per peptide, 40 patient-specific peptides per pool) or control peptides (CEF) in ImmunoCult-XF T Cell Expansion Medium (IC media; STEMCELL Technologies) with 10 IU ml −1 recombinant human IL-2 (R&D Systems) for 14 days in 48- or 24-well tissue culture plates. Cells were seeded at 1-2 ×10 6 cells/well and fed every 2–3 days by replacing two thirds of the culture media with recombinant human IL-2. IFNγ ELISpot assay (clinical study). Detection of IFN-γ-producing T cells was performed by ex vivo ELISpot assay47. Briefly, cells were harvested, counted and resuspended in media at 4 × 106 cells ml−1 (ex vivo PBMCs) or 2 × 106 cells ml−1 (IVS-expanded cells) and cultured in the presence of DMSO (VWR International), phytohemagglutinin-L (Sigma-Aldrich), CEF peptide pool or cognate peptides in ELISpot Multiscreen plates (EMD Millipore) coated with anti-human IFN-γ capture antibody (Mabtech). Following 18–24 h incubation in a 5% CO 2, 37˚C, humidified incubator, supernatants were collected, cells were removed from the plate and membrane-bound IFNγ was detected using anti-human IFN-γ detection antibody (Mabtech), Vectastain Avidin peroxidase complex (Vector Labs) and AEC Substrate (BD Biosciences). ELISpot plates were allowed to dry, stored protected from light and sent to Zellnet Consulting for standardized evaluation 45. Data are presented as SFU per million cells. ICS (clinical study). PBMCs or IVS-expanded PBMCs were stimulated with either cognate minimal (8–11mer) or long overlapping (15mer) peptides, DMSO (vehicle control; VWR) or PMA/ionomycin cell stimulation cocktail (positive control; Affymetrix) in the presence of anti-human CD28/CD49d antibody (BD Biosciences), anti-human CD107α-BV421 (BioLegend), BD GolgiStop (BD Biosciences) and Brefeldin A (BioLegend) over a period of 18 h. Following overnight incubation, cells were stained with live/dead ZombieRed (BioLegend) and surface marker antibodies (CD8-PerCP-Cy5.5, CD3-BV605, CCR7-PE-Cy7 from BioLegend; CD4-APC-eF780, CD45RA-SuperBright702 from eBioscience; PD-1-BUV737 from BD Biosciences) before fixation and permeabilization with FIX & PERM Cell Permeabilization Kit (Thermo Fisher Scientific). Following permeabilization, cells were stained for intracellular cytokines using anti-human IFN-γ-APC, TNF-α-BV785, and IL-2-PE antibodies (BioLegend) before data acquisition on a BD LSRFortessa flow cytometer (BD Biosciences). HLA class I fluorophore-conjugated tetramer generation and staining (clinical study). HLA class I tetramers were generated from Flex-T biotinylated HLA monomers (BioLegend) or MBL International with target peptides loaded onto the monomers by UV exchange according to the manufacturer’s instructions. Successful loading of target peptides by UV exchange was assessed by ELISA (BioLegend, according to manufacturer’s instructions). Alternatively, biotinylated monomers were generated in-house and prefolded with target peptides. pHLA monomers were assembled into tetramers using fluorophore-conjugated Streptavidin (PE from BioLegend; APC from eBioscience). Using generated fluorophore-labeled peptide-MHC tetramers, PBMCs were tetramer stained to help identify antigen-specific T cells. Patient PBMC samples were thawed and resuspended in a 96-well V-bottom plate with FACS buffer (PBS, 10% FBS [v/v]). Before staining, samples were incubated with 50 nM Dasatinib (Sigma-Aldrich) in a 5% CO 2, 37˚C incubator for 30 min. Subsequently, HLA class I fluorophore-labeled tetramers loaded with target peptides of interest were added directly to each corresponding PBMC sample. In instances where multiple tetramers needed to be added to the same sample, a cocktail was made with equal amounts of each tetramer, then mixed and added at the same time. Samples were incubated at room temperature for 1 h in the dark. Following incubation, samples were washed in FACS buffer and stained with an antibody master mix containing purified anti-human HLA-A,B,C antibody, anti-human CD8-PerCP-Cy5.5, anti-human CCR7-PE-Cy7, anti-human CD3-BV421 (all BioLegend), anti-human CD45RA-SB702, anti-human CD4-APC-eF780 (both eBioscience) and Live/Dead Green (Thermo Fisher Scientific). Sample were mixed and incubated at 4˚C in the dark protected from light for 20 min. Samples were washed once with FACS buffer and then resuspended in FACS buffer prior to acquisition on a BD LSRFortessa flow cytometer (BD Biosciences). Flow cytometry analyses (clinical study). Samples acquired on flow cytometers were analyzed using FlowJo software (FlowJo). Gating strategies for human samples are as follows (Supplementary Figs 2 and 3). For CD4 versus CD8 T cell assessments: lymphocytes (SSC-A versus FSC-A), single cells (FSC-H versus FSC-A), viable cells (FSC-A versus LD-ZombieRed), CD3 + cells (FSC-A versus CD3-BV605), CD4+ and CD8+ (CD4-APCeF780 versus CD8-PerCP-Cy5.5). For tetramer studies: lymphocytes (SSC-A versus FSC-A), single cells (FSC-H versus FSC-A), viable cells (FSC-A versus LD-AF488), CD3 + cells (FSC-A versus CD3-BV421), CD8+Tet+ (CD8-PerCP-Cy5.5 versus tetramer-PE or tetramer-APC), memory phenotypes (CD45RA-SB702 versus CCR7-PE-Cy7). For ICS studies: lymphocytes (SSC-A versus FSC-A), single cells (FSC-H versus FSC-A), viable cells (FSC-A versus LD-ZombieRed), CD3 + cells (FSC-A versus CD3-BV605), CD4+ and CD8+ (CD4-APCeF780 versus CD8-PerCP-Cy5.5), CD8+ or CD4+ cytokine+ cells (CD8-PerCP-Cy5.5 versus CD107a-BV421, IFN-γ-APC, TNF-ɑ-BV786, IL-2-PE; CD4-APCeF780 versus CD107a-BV421, IFN-γ-APC, TNF-ɑ-BV786, IL-2-PE). Polyfunctionality analyses were performed using Boolean gating (FlowJo) and graphed using SPICE software v6 (ref. 48). Results are represented as the percentage of positive cell populations (frequency of parent). Data are shown as background subtracted where indicated. MSD U-plex assay. Detection of secreted IL-2, TNF-α and granzyme B (GRZB) in ex vivo ELISpot supernatants was performed using a U-plex assay MSD U-PLEX Biomarker assay. Assays were performed according to the manufacturer’s instructions. Data were acquired on the MESO SECTOR S 600 instrument and DISCOVERY WORKBENCH assay analysis software. Analyte concentrations (pg ml −1) were calculated using serial dilutions of known standards for each cytokine. For graphical data representation, values below the minimum range of the standard curve were represented as zero. Triplex FluoroSpot assay. A multiplexed FluoroSpot assay was performed according to manufacturer’s instructions (MABtech) on a subset of patient samples for the detection of IFN-γ , IL-2 and granzyme B-producing T cells. Briefly, patient PBMCs were thawed and rested overnight at 2 × 106 cells ml−1. The following day, cells were harvested, counted and resuspended at 4 × 106 cells ml−1 before plating. Precoated FluoroSpot plates were washed with PBS and blocked with culture media before patient-specific peptide pools and cells were added (2 × 105 PBMCs per well). After incubating in a 5% CO2, 37˚C humidified incubator for 18–24 h, secreted IFN-γ , IL-2 and granzyme B were detected using anti-human IFN-γ -BAM/anti-BAM-490, biotinylated anti-human granzyme B/ Streptavidin-550 and anti-human-IL-2-W ASP/anti-W ASP-650 antibody pairs followed by fluorescence enhancer. Plates were imaged and enumerated on an AID iSpot reader (Autoimmun Diagnostika). Data are presented as SFU per million cells. NATuRE MEDICINE | www.nature.com/naturemedicine Articles Nature MediciNeSingle-cell TCR sequencing and digital gene expression. Single-cell library preparation and sequencing was completed according to 10x Genomics v1 5’ Chromium Single Cell V(D)J protocol CG000186 Rev A. The only deviation in library preparation was in V(D)J library step 5.4 with five cycles of PCR. All sequencing was completed on the Illumina NovaSeq at a loading concentration of 450 pM with 5% PhiX. The 10x Genomics Cell Ranger version 3.1 was used for V(D)J and Digital Gene Expression library analysis and was current at time of research. Adaptive TCR sequencing of RNA later-preserved tumor samples. Tumor DNA was extracted via Qiagen AllPrep DNA/RNA kit and quantified with both Qubit high sensitivity dsDNA assay (Thermo Fisher Scientific) and Agilent Genomic DNA tape on the 4200 TapeStation system. Between 300 and 1,000 ng extracted DNA was sent to Adaptive Biotechnologies for T cell receptor beta sequencing, and results were returned to us via the immunoSEQ Analyzer 3.0. TCR sequencing analysis. Significant tumor TCR expansion was determined via a binomial test with Benjamini–Hochberg correction and P value <0.05, analyzing TCRs with five cells or more in either timepoint. IVS TCR expansion was analyzed by filtering 10× V(D)J results for each timepoint for TCRs (1) with a full alpha/beta pair, (2) found above an abundance threshold and (3) not found in the CEF IVS. The proportion of the resulting TCRs was then tracked from baseline to on treatment, and the polyfunctional activation of these expanded TCR populations was also analyzed by searching in the transcriptome for non-zero expression of IFN-γ, granzyme B, TNF and IL-2. The expanded TCRs were cross-referenced with those expanded in the tumor biopsies, and overlap was statistically significant by Fisher test (P < 0.001). cfDNA collection and isolation. Whole blood was collected in two 10-ml cell-free DNA (cfDNA) BCT (Streck) starting at the time of the prime, and subsequent draws were collected at dosing visits. Due to COVID-19 restrictions, several blood draws were missed. Whole blood underwent a double-spin protocol to first separate plasma from WBCs and red blood cells before a second spin to remove any remaining cellular debris. The separated plasma was frozen and shipped to Gritstone Bio and stored at −80°C until extraction. cfDNA was extracted from the entire plasma volume of a single draw using the Apostle MiniMax cfDNA Isolation kit (ApostleBio) and quantified using the Qubit 1x dsDNA High Sensitivity Assay (Thermo Fisher Scientific). ctDNA sequencing and analysis. Custom hybrid capture panels were designed for each patient enrolled in the study. Each patient panel was designed to capture the EDGE-predicted neoantigens and all predicted coding (nonsynonymous) mutations present in the original screening biopsy specimen. Target mutations for each patient were curated and sent for customized design to IDT as xGen Lockdown Probes. Each capture pool contained up to nine patients per pool. Patient-matched genomic DNA was fragmented before library preparation using the NEB FS module (NEB). Shotgun libraries for cfDNA (up to 30 ng) and the fragmented, patient-matched genomic DNA (20–30 ng) were prepared using the KAPA HyperPrep (KAPA Biosystems) kit using a customized pool of duplex adapters containing unique molecular identifiers for duplex sequencing (IDT). Shotgun libraries were captured with patient-specific probes overnight using the IDT xGen Hybridization and Wash kit. Enriched libraries were sequenced on an Illumina NovaSeq to a minimum mean raw depth of 80,000×. Briefly, unique molecular identifiers were clipped from the raw sequencing reads before alignment to hg38 using BW A-MEM. Using fgbio, aligned reads were grouped by position and duplex identity. Consensus reads were created using a duplex of 3× (three supporting reads from each strand) and realigned to hg38. Variant calling was performed using FreeBayes and filtered for the patient-specific variants. Variant allele frequency was converted to mutated haploid genomic equivalents per milliliter plasma (hGE ml −1) using the extracted plasma volume and total cfDNA yield from the extraction. Percent change in ctDNA was calculated as the change of the median mutated hGE ml −1 from the baseline sample. Patient tumor biopsy whole-exome and transcriptome analysis and TCGA data comparison. Pretreatment GRANITE patient FFPE biopsy-derived DNA and RNA and matched whole blood-derived DNA were whole-exome sequenced (IDT xGen V1) and analyzed as described previously 38. Relevant tumor types for genomic comparison from TCGA Pan-Cancer Atlas dataset included gastric (n = 405 stomach adenocarcinoma), colon adenocarcinoma (n = 332, MSS-CRC (281) and MSI-CRC (51)), lung adenocarcinoma (n = 498), lung squamous cell carcinoma (n = 463, LUSC) and melanoma (n = 436, skin cutaneous melanoma). For RNA expression analyses, all GRANITE and TCGA sample RNA-seq by Expectation-Maximization expected counts were normalized utilizing DESeq2 (ref. 49). Normalized counts were used to determine patient PD-L1 RNA expression and the IFN-γ RNA expression signatures, which were calculated as the average of 28 IFN-γ-associated gene specific z-scores across all GRANITE and TCGA samples (n = 2,494) 50. Tumor mutational burden was calculated for each GRANITE patient as the total of all somatic variants called >4% variant allele frequency divided by the exome bait panel size (39 Mb). For TCGA samples, total mutations called were divided by exome bait panel size used (38 Mb) 51 (Supplementary Table 4).Transduction, culture and live sorting of single HLA-expressing A375 NucLight Red cell lines. A375 cells were obtained from ATCC (CRL-1619). Endogenous class I HLAs were knocked out using CRISPR-Cas9 ribonucleoprotein complexes. HiFi S.p. Cas9 Nuclease V3 (1081060), Cas9 Electroporation Enhancer (1075915) and custom guide RNAs were obtained from IDT. Ribonucleoproteins were electroporated into A375 cells using the Neon transfection system from Thermo Fisher Scientific with a voltage of 1,400 and two pulses for 20 ms. CRISPR-treated A375 cells were stained using a 1:100 anti-human HLA-A,B,C antibody from Biolegend (311405), and HLA-null cells were sorted using the Cell Sorter SH800S (SONY Biotechnology) (Supplementary Fig. 4). Single-allele A375 cell lines were generated with HLA sequences obtained from the coding sequence of the human genome (HLA-B*08:01:01:01, HLA-B*57:01:01:01, HLA-A*30:04:01:01, HLA-A*03:01:01:01, HLA-C*04:01:01:01). HLAs were cloned into the pCDH-EF1α-MCS1 lentiviral expression backbone vector from System Biosciences (CD502A-1). Lentivirus was generated for each HLA and titer determined using the Lenti-X qRT-PCR titration kit from Takara Bio (631235). Single HLAs were transduced into A375 HLA-null cells at an m.o.i. of 100 with 8 ug ml −1 polybrene (EMD Millipore, TR-1003). Cells were spinoculated at 800g for 45 min at room temperature at a density of 8 × 105 cells per ml. Fresh media was added and cells were incubated overnight. HLA-positive cells were sorted after at least 48 h using the method described above. Fluorescent single-allele A375 cells were generated with NucLight Red (IncuCyte, 4476) at an m.o.i. of 3 with 8 μg ml −1 polybrene (EMD Millipore, TR-1003). Cells were spinoculated at 800g for 45 min at room temperature at a density of 8e5 cells ml−1. Fresh media was added and cells were incubated overnight. Red fluorescent and HLA-positive cells were sorted after at least 48 h using the method described above. IncuCyte killing assays. Single HLA-expressing A375 NucLight Red cell lines were seeded in 96- or 48-well plates at concentrations of 2.5 × 10 4 or 3.5 × 104 cells per well in DMEM with 10% heat-inactivated FBS. The plates were placed in the IncuCyte S3 (Essen Biosciences) and 24 h after the seeding, the effector cells were plated at a concentration of 2.5 × 105 or 3.5 × 105 cells per well in a 96- or 48-well plate, respectively, for an effector to target ratio of 10:1. Individual peptides (GenScript; Supplementary Table 2) were added to the treated wells for a final concentration of 10 µg ml −1, and DMSO was used for the control wells. The plates were imaged with the IncuCyte for up to 72 h, after which the data were analyzed using the IncuCyte S3 2018 analysis software. Viability of A375 was assessed by red cell count, and relative viability was calculated relative to DMSO co-culture control wells. Statistics and reproducibility. For the clinical study, the sample size is not driven by a statistical power due to the nature of an adaptive dose-escalation method. Therefore, the sample size of the phase 1 portion of the study ranges from 11 to maximum of 24 patients, which is dependent on occurrence of DLTs. No data were excluded from the analyses, and experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. For the NHP study, the number of animals per group is n = 3/sex, which is the minimum number of animals needed to do simple statistics. The total number of animals used in this study is considered the minimum required to properly characterize the effect of the test articles (i.e., vaccine and anti-CTLA-4) and compare their route of administration (i.e., s.c. versus i.v.) and has been designed such that it does not require an unnecessary number of animals to accomplish its objectives. For both the NHP and clinical study, descriptive statistics are described throughout. Where applicable, statistical analyses applied for specific methods are outlined in the relevant methods sections. Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. Data availability Deidentified individual participant clinical data that underlie the results reported in this article are available for transfer. Interested investigators can obtain and certify the data transfer agreement and submit requests to the principal investigator (K.J.). Investigators and institutions who consent to the terms of the data transfer agreement form, including, but not limited to, the use of these data for the purpose of a specific project and only for research purposes, and to protect the confidentiality of the data and limit the possibility of identification of participants in any way whatsoever for the duration of the agreement, will be granted access. Gritstone bio will then facilitate the transfer of the requested deidentified data. This mechanism is expected to be via a Gritstone Secure File Transfer Service, but Gritstone bio reserves the right to change the specific transfer method at any time, provided appropriate levels of access authorization and control can be maintained. Source data are provided with this paper. Pan-Cancer Atlas data sets were obtained from the cBioPortal (https://www.cbioportal.org/) and the UCSC Xena TCGA Pan-Cancer Atlas data hub (https://pancanatlas.xenahubs.net), and raw data are available through the Genomic Data Commons (https://gdc.cancer.gov/). Epitope selection and patient-specific neoantigen selection used a previously published algorithm 30. Source data are provided with this paper.