Commun. 13, 3289 (2022). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. © The Author(s), under exclusive licence to Springer Nature America, Inc. 2022 NATuRE MEDICINE | VOL 28 | AUGUST 2022 | 1619–1629 | www.nature.com/naturemedicine 1629 Articles Nature MediciNeMethods Ethics statement. The research presented in this paper complies with all the relevant ethical regulations. Animal studies were performed according to institutional animal care and use (IACUC)-approved protocol at the California National Primate Research Center at the University of California, Davis. The clinical study and all related analyses were carried out in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines and was approved by the appropriate institutional review board (IRB) or ethics committees at each participating site (University of Chicago Comprehensive Cancer Center, Chicago, IL; Columbia University Medical Center, New Y ork, NY; The Ohio State University Medical Center, Columbus, OH; Virginia Cancer Specialists, Fairfax, V A; Memorial Sloan Kettering Cancer Center, New Y ork, NY; Peter MacCallum Cancer Centre, Melbourne, VIC, Australia; Mayo Clinic, Phoenix, AZ; Mayo Clinic, Rochester, MN; The University of Texas MD Anderson Cancer Center, Houston, TX). All patients provided written, informed consent. Further details can be found at https://clinicaltrials.gov/ct2/show/NCT03639714. NHP study. Animal selection and dosing . Study was performed according to IACUC-approved protocol at the California National Primate Research Center at the University of California, Davis. Animals did not receive any prior treatment with immune-modulatory antibodies or vaccination against SIV and had no prior exposure to SIV . Animal groups and dosing are outlined in Supplementary Table 1. Briefly, both ChAd68 and samRNA were administered as bilateral i.m. injections into the quadriceps muscle. All ChAd68 doses were at 10 12 VP with 2 ml total injection volume (1 ml per leg). All samRNA doses were at 300 μg with 1 ml total injection volume (500 μl per leg). Anti-CTLA-4 (ipilimumab) was administered as either an i.v. injection of 5 mg kg−1 or a flat dose of 50 mg per animal injected subcutaneously bilaterally near each vaccine site draining lymph node. Initial weights, doses of ipilimumab and treatment groups are shown below. Animals 37576 and 42300 were excluded from the study after week 20 with no further immune monitoring or immunizations due to recurrent issues with anemia and low-yield blood draws. SIV model antigen cassettes. A model antigen cassette containing six previously published SIV viral epitopes 29 (SIV Gag CM9 (CTPYDINGQM), SIV Env TL9 (TVPWPNASL), SIV Env CL9 (CAPPGYALL), SIV Tat TL8 (TTPESANL), SIV Gag LW9 (LSPRTLNAW) and SIV Pol SV9 (SGPKTNIIV)) was designed. Each minimal epitope is embedded in a 25 amino acid sequence with 8 amino acids flanking each minimal epitope sequence. The cassette also contains class II helper epitopes. The cassette was synthesized by GenScript and designed to allow cloning into plasmids containing ChAd and samRNA vectors. The ChAd vector is an E1, E3 deleted virus, and the cassette expression was flanked by a cytomegalovirus promoter and an SV40 polyadenylation signal. The ChAd plasmid was digested with PacI and then transfected into HEK293 cells to generate virus. The virus for the NHP studies was made by CsCl gradient centrifugation and for the good manufacturing practice studies was purified by anion exchange chromatography. The same cassette was cloned into BstB1–PacI sites of a pBAC-VEE alphavirus-based plasmid that was used to make the samRNA RNA by in vitro transcription (TriLink) and then formulated into lipid nanoparticles (Arbutus). Test articles (NHP study). Anti-CTLA-4 monoclonal antibody (mAb) (ipilimumab) was provided by Bristol Myers Squibb. ChAd68-MAG was generated by Gritstone Oncology (non-GLP) and prepared at 5 × 10 11 VP ml−1 in Adenovirus Reference Material buffer (10 mM Tris, pH 8.0, 25 mM NaCl and 2.5% glycerol buffer). samRNA was generated by TriLink Biotechnologies and formulated in lipid nanoparticles by Arbutus. Isolation of PBMCs and serum (NHP study). For immune monitoring, 10–20 ml blood was collected into vacutainer tubes containing heparin and maintained at room temperature until isolation. PBMCs were isolated by density centrifugation using lymphocyte separation medium and Leucosep separator tubes. PBMCs were counted using the Attune NxT (Thermo Fisher Scientific) and resuspended at a concentration of 4 × 10 6 cells ml−1 in complete RPMI. IFN-γ ELISpot assay (NHP study). IFN-γ enzyme-linked immunospot (ELISpot) assays were performed using precoated 96-well plates (MAbtech, Monkey IFNγ ELISpot PLUS, ALP) following the manufacturer’s protocol. Then, 10 5 PBMCs per well were plated in triplicate with 10 µg ml−1 peptide stimuli (GenScript) and incubated overnight in complete RPMI (RPMI + 10% FBS). A human hepatitis B virus S-antigen peptide not contained in the cassette (WLSLLVPFV , GenScript) was used as a negative control for each sample. Plates were washed with PBS and incubated with anti-monkey IFN-γ mAb biotin (MAbtech) for 2 h, followed by an additional wash and incubation with Streptavidin-ALP (MAbtech) for 1 h. After final wash, plates were incubated for ten minutes with BCIP/NBT (MAbtech) to develop the immunospots and dried overnight at 37°C. Spots were imaged and enumerated using AID reader (Autoimmun Diagnostika). For data processing and analysis, samples with replicate well variability (variability = variance/(median + 1)) greater than 10 and median greater than 10 were excluded 44. Spot values were adjusted based on the well saturation according to the formula45:AdjustedSpots =RawSpots +2∗(RawSpots ∗saturation/ (100−saturation ) Each sample was background corrected by subtracting the average value of the negative control peptide wells. Data are presented as SFU per 106 PBMCs. Wells with well saturation values greater than 50% were labeled as too numerous to count, and their value was set to the maximum measured SFU for that experiment. ICS assay (NHP study). Freshly isolated PBMCs were distributed at 10 6 per well into V-bottom 96-well plates. Cells were pelleted and resuspended in 100 μl complete RPMI containing 1 μg ml−1 anti-CD28 mAb (CD28.2, BD Biosciences), 1 μg ml−1 anti-CD49d mAb (9F10, BD Biosciences) and 10 μg ml−1 pooled peptide antigens or human hepatitis B virus S-antigen peptide (WLSLLVPFV) as a negative control stimulant (GenScript). After 1 h of stimulation, Brefeldin A (Biolegend) was added to a final concentration of 5 μg ml−1 and incubated for an additional 16 h. Following stimulation, cells were washed with PBS and stained with fixable viability dye (eBioscience). Extracellular staining was performed in FACS buffer (PBS + 2% FBS + 2 mM EDTA) with anti-CD3, anti-CD4, anti-CTLA-4, anti-PD1 and anti-CD8 antibodies. Cells were washed, fixed and permeabilized with Transcription Factor fixation/permeabilization kit (eBioscience), and then intracellular staining was performed in permeabilization buffer. Samples were stained for IFN-γ, TNF-α, granzyme B and CD69. Samples were collected on the Attune NxT (Thermo Fisher Scientific). Gating strategy was as follows (Supplementary Fig. 1): lymphocytes (SSC-A versus FSC-A), single cells (SSC-H versus SSC-A), viable cells (SSC-A versus LD-506), CD4 + or CD8+ (CD4-phycoerythrin (PE) versus CD8-e450), cytokine+ (TNF-α-BV421 versus IFN-γ-APC-R700). Tetramer staining assay (NHP study). Following overnight rest at 4°C, isolated PBMCs were distributed into 96-well V-bottom plates at a density of 106 cells per 100 µl and stained with a viability dye (Live/Dead e506, eBioscience) in PBS for 20 min at 4°C. TCR recycling was inhibited by treatment with 50 nM Dasatinib for 1 h at 4°C. Cells were stained for 1 h at 4°C in FACS buffer with Mamu-A*01 tetramers for each of the peptide antigens with a unique combination of two fluorophores per antigen (produced in-house). Cells were co-stained for CD3 (A700, SP34.2, BD Biosciences), CD8 (e450, SK1, BD Biosciences), CD45RA (FITC, 5H9, BD Biosciences) and CCR7 (SB780, 3D12, eBioscience). Samples were collected on the Attune NxT (Thermo Fisher Scientific). The gating strategy was as follows (Supplementary Fig. 1): lymphocytes (SSC-A versus FSC-A), single cells (SSC-H versus SSC-A), viable cells (SSC-A versus LD-506), CD3 + cells (SSC-A versus CD3-A700), CD8+ (SSC-A versus CD8-e450), tetramer double positive (tetramer-UV405 versus tetramer-UV605; tetramer-UV405 versus tetramer-PE; tetramer-PE versus tetramer-UV675; tetramer-PE-Cy7 versus tetramer-UV405) and memory populations (CD45RA-FITC versus CCR7-SB780). Cytotoxicity (T cell killing) assay (NHP study). Freshly isolated PBMCs for each sample were divided into two portions. T-cell enrichment was performed on one portion using the Miltenyi NHP T-cell isolation kit (130-092-143), following manufacturer’s protocol and using MS columns (Miltenyi) to deplete non-T cells. The second portion (referred to as PBMCs) was stained with carboxyfluorescein succinimidyl ester (CFSE) at 1 mM (eBioscience). The CFSE-labeled PBMCs were then loaded with 50 mg ml −1 SIV peptides or a negative control peptide (WLSLLVPFV) by incubation for 1 h at 37˚C in RPMI + 1% FBS. Isolated T cells and peptide-loaded target PBMCs were then counted and plated in a 20:1 ratio (T cells to target cells) in a V-bottom plate. Then, 400,000 T cells and 20,000 target cells were plated per well. Target cells only were also plated as a control. Cells were incubated for 6 h at 37˚C in RPMI + 10% FBS. Following incubation, cells were stained with 7-AAD (Thermo Fisher Scientific) and immediately analyzed by flow cytometry using a Cytoflex LX (Beckman). CFSE + target cells were gated and 7-AAD+ cells measured (as a percentage of CFSE+ cells). Remnant T cells were also analyzed by flow cytometry to confirm T cell isolation. Samples were collected on the Attune NxT (Thermo Fisher Scientific). The gating strategy was as follows (Supplementary Fig. 1): lymphocytes (SSC-A versus FSC-A), single cells (SSC-H versus SSC-A), 7-AAD +CFSE+ (7-AAD versus CFSE). Anti-ChAd68 neutralizing antibody assay (NHP and clinical study). Dilutions of heat-inactivated human and NHP sera were evaluated for the ability to neutralize a ChAd virus expressing the reporter protein beta-galactosidase (ChAd-LacZ). The reciprocal of the dilution that gives 50% or greater neutralization is used to assign a neutralization titer. In brief, pre- and post-vaccination serum samples were heat inactivated at 56˚C for 30 min. Sera samples were then diluted twofold in serum-free media (DMEM) starting at 1:20 dilution down to 1:1,280. If needed, additional dilutions were made to get a titer within the range of the assay. The diluted samples were then mixed with an equal volume of media containing diluted virus sufficient to infect 293 A cells at an approximate multiplicity of infection (m.o.i.) of 1. The diluted sera and virus were incubated for 1 h at 37˚C before adding onto 293 A cells plated at 2.5 ×10 4 cells/well on a 96-well tissue culture plate 1 day earlier. The cells and neutralizing mix were incubated for 1 h at 37˚C and then an equal volume of media with 20% FBS was added. The cells and virus were then incubated for an additional 18–20 h before lysis and measurement of NATuRE MEDICINE | www.nature.com/naturemedicine Articles Nature MediciNeB-galactosidase activity. Media was aspirated and the cells lysed in 100 µl lysis buffer from Thermo Fisher Scientific (Galacto-Star System). The cells were then freeze thawed twice (alternating −80˚C and 37˚C), mixed, and then 50 µl was mixed with an equal volume of Galacto-Star substrate (Thermo Fisher Scientific) for B-galactosidase measurement. Wells with media alone were used to determine expression in the absence of sera (100% B-galactosidase activity) and wells with no virus were used to determine the background of the assay. The percent inhibition was calculated for each dilution and plotted to generate a curve of percent neutralization versus dilution. A titer was assigned either as a limiting dilution titer, which is the reciprocal dilution that gives 50% or greater inhibition, or as interpolated from the dilution curve. Phase 1 clinical trial. Study design . The study protocol is available in the Supplementary Information. The main objectives of this phase 1/2 study (NCT03639714) were to evaluate the safety and tolerability, immunogenicity and early clinical activity to determine and evaluate the RP2D of i.m. administration of GRT-C901 (ChAd68) and GRT-R902 (self-amplifying RNA formulated in lipid nanoparticles; samRNA), an individualized neoantigen cancer vaccine, in combination with i.v. nivolumab and s.c. ipilimumab, in patients with metastatic NSCLC, MSS-CRC, GEA and metastatic urothelial cancer. The phase 1 portion of the study was designed to find an RP2D using an adaptive dose-escalation method and modified toxicity probability interval-2 (mTPI-2) among the planned doses; DL1 through DL4 are outlined in Fig. 2a, with the goal of determining the highest vaccine dosage with a dose-limiting toxicity (DLT) rate below 30%. 46 All patients received 1012 VP ChAd68 prime and monthly i.v. anti-PD-1 antibody nivolumab administrations (480 mg every 4 weeks). Patients at DL1 and DL2 received 30 µg and 100 µg samRNA boosts, respectively. Patients at DL3 and DL4 received 100 µg and 300 µg samRNA, respectively, in addition to s.c. ipilimumab (30 mg) with each vaccination. ChAd68 boost was administered to eight patients with varying intervals at a dose of 10 12 VP (Extended Data Table 1). The sample size was 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 ranged from 11 to maximum of 24 patients which is dependent on occurrence of DLTs. This study was carried out in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines and was approved by the appropriate IRB or ethics committees at each participating site (University of Chicago Comprehensive Cancer Center, Chicago, IL; Columbia University Medical Center, New Y ork, NY ,; The Ohio State University Medical Center, Columbus, OH; and Virginia Cancer Specialists, Fairfax, V A). All patients provided written, informed consent. Further details can be found at https://clinicaltrials.gov/ct2/show/NCT03639714. Per protocol, patients had the potential to receive study treatment concurrent with ongoing standard-of-care chemotherapy. The first patient enrolled on 24 September 2018 and the last patient enrolled on 25 November 2019 for the phase 1 portion of the study. As of the data cutoff date of 31 January 2022, a total of 29 patients (i.e., 14 patients in phase 1 and 13 patients in phase 2) have been treated, and the study is still ongoing to evaluate the efficacy of the RP2D using overall response rate, duration of response, clinical benefit rate, deepening of response, progression of survival and OS. Of the 14 phase 1 patients reported on in this study, patients G8, G9 and G10 received continued standard-of-care chemotherapy with study treatment. G8 received fluorouracil (5-FU) and bevacizumab (initiated 21 months before study treatment), and G9 and G10 received fluoropyrimidine/irinotecan (FOLFIRI) (initiated 12 and 8 months before study treatment). Four clinical sites enrolled and treated patients for the phase 1 part of the study: University of Chicago Comprehensive Cancer Center, Columbia University Medical Center, The Ohio State University Medical Center and Virginia Cancer Specialists. There is no data safety monitoring board for this study. A study committee consisting of the investigators and study sponsor meets regularly to assess the safety profile of the study treatment and make recommendations for dose escalation. Patient data were collected via electronic data capture and analyzed as described in the Statistics and reproducibility section. Study population. Inclusion criteria . Inclusion criteria for vaccine production stage . Patients must meet the following inclusion criteria to be eligible for vaccine production stage: 1. Provide a signed and dated Informed Consent Form (ICF) document before initiation of study-specific procedures. 2. Patients with the indicated advanced or metastatic solid tumor as follows: a. NSCLC who are planned for or have received no more than one cycle of systemic treatment with cytotoxic, platinum-based chemotherapy (patients receiving anti-PD-(L)1 monotherapy are eligible) b. GEA who are planned for or have received no more than one cycle of systemic treatment with cytotoxic, platinum-based chemotherapy c. metastatic urothelial cancer who are planned for or have received no more than one cycle of systemic treatment with cytotoxic, platinum-based chemotherapy d. CRC-MSS who are receiving first-line systemic therapy or who are planned for or have received no more than one cycle of second-line sys- temic therapy including a fluoropyrimidine and oxaliplatin or irinotecan 3. Availability of formalin-fixed, paraffin-embedded (FFPE) tumor specimen for neoantigen assessment. 4. Measurable disease according to RECIST v1.1. 5. Lesions amenable to biopsy. 6. ≥12 years of age (patients 12–17 years of age must weigh >40 kg). 7. Life expectancy of > 6 months per the investigator. 8. ECOG performance status of 0 or 1 (or equivalent for patients 12–17 years of age based on Karnofsky or Lansky performance scale). 9. Patient has adequate organ function using values obtained within 4 weeks before starting routine therapy as defined below. The sponsor may consider patients eligible despite having a laboratory value that does not meet inclu-sion criteria provided the Investigator reports the patient is otherwise in good health and the history and kinetics of the change in the laboratory value do not raise a substantial safety concern over including the patient in the study or compromise the integrity of the study data. The sponsor and investigator must agree to include any patients with a laboratory value below the defined criteria below: a. Peripheral white blood cell (WBC) ≥3,000/mm 3 b. Absolute lymphocyte count ≥800/mm3 c. Absolute neutrophils count (ANC) ≥ 1,500/mm3 d. Platelets ≥100,000/mm3 e. Hemoglobin ≥ 9 g dl−1 f. Albumin ≥ 3 g dl−1 g. Calculated creatinine clearance >50 ml min−1 using Cockcroft–Gault equation h. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤3× upper limit of normal (ULN) i. Total bilirubin ≤1.5× ULN or direct bilirubin ≤1× ULN (patients with Gilbert’s disease may be included if their total bilirubin is ≤3.0 mg dl −1) j. International normalized ratio or prothrombin time (PT) or partial thromboplastin time (PTT) ≤1.5× ULN 10. For NSCLC only, a history of smoking, defined as more than five packs, or 100 lifetime cigarettes. 11. For women of childbearing potential, willing to undergo pregnancy testing and agreeing to use at least one highly effective contraceptive method during the study treatment period and for 5 months after last investigational study treatment. Inclusion criteria for study treatment stage . Patients must sign a second ICF and meet the following inclusion criteria to be eligible for the study treatment stage: 12. If patient did not participate in the vaccine production part of this study, patient has been enrolled in the screening protocol GO-003. 13. Provide a signed and dated ICF document before initiation of any treatment-related study-specific procedures. 14. Patient has received routine therapy as follows: i. Patient with NSCLC, metastatic urothelial cancer or GEA who has received systemic treatment with cytotoxic, platinum-based chemo-therapy, has measurable disease according to RECIST v1.1 and: a. has chemotherapy and has an assessment of SD, partial response or complete response: will either receive study treatment in addition to continued routine therapy, or will receive study treatment after stop-ping routine therapy, per Investigator discretion; or b. has experienced disease progression: will receive study treatment as next line of therapy (i.e., without chemotherapy); or c. is intolerant to chemotherapy: will receive study treatment as next line of therapy (i.e. without chemotherapy) ii. Patient with CRC-MSS who has received second-line systemic therapy including a fluoropyrimidine and oxaliplatin or irinotecan, has measur - able disease according to RECIST v1.1 and: a. has assessment of SD, partial response or complete response: will receive study treatment in addition to continued routine therapy per investigator discretion; or b. has experienced disease progression: will receive study treatment as next line of therapy; or c. is intolerant to chemotherapy: will receive study treatment as next line of therapy 15. Patient agrees to undergo research biopsies before study treatment and ap- proximately 16 weeks after first vaccine administration (if patient has lesions amenable to biopsy). 16. ECOG performance status of 0 to 2 (or equivalent for patients 12–17 years of age based on Karnofsky or Lansky performance scale). 17. Patient has adequate organ function as defined below. The sponsor may consider patients eligible despite having a laboratory value that does not meet inclusion criteria provided the investigator reports the patient is otherwise in good health and the history and kinetics of the change in the laboratory value do not raise a substantial safety concern over including the patient in the study or compromise the integrity of the study data. The sponsor and NATuRE MEDICINE | www.nature.com/naturemedicine Articles Nature MediciNeinvestigator must agree to include any patients with a laboratory value below the defined criteria below. a. Peripheral WBC ≥2,000/mm3 b. Absolute lymphocyte count ≥500/ mm3 c. ANC ≥1,000/mm3 d. Platelets ≥75,000/mm3 e. Hemoglobin ≥ 9 g dl−1 f. Calculated creatinine clearance >40 ml min−1 using Cockcroft–Gault equation g. ALT and AST ≤3× ULN unless liver metastases are present in which case patients are included if ALT and AST ≤5 × ULN h. Total bilirubin ≤1.5× ULN or direct bilirubin ≤1× ULN (patients with Gilbert’s disease may be included if their total bilirubin is ≤ 3.0 mg dl−1). i. International normalized ratio and PT and PTT ≤1.5× ULN, unless patient is receiving anti-coagulant therapy, in which case patients are eligible if PT and PTT is within therapeutic range of intended use of anti-coagulants. Exclusion criteria.