1c). Finally, the ability of the boosted antigen-specific CD8 T cells to kill target cells presenting SIV epitopes was determined. CD8 T cells from all six animals showed cytotoxic activity against their cognate targets, with CD8 T cells from four of six animals achieving killing of > 70%, even at the lower effector-to-target ratio of 10:1 (Extended Data Fig. 1d). Individualized vaccine regimen is safe and well tolerated. After establishing the potency and longevity of T cell responses induced by this vaccine in NHPs, the vaccine was assessed as an individual - ized, heterologous regimen expressing patient-specific neoantigens in an ongoing phase 1/2 clinical study in patients with advanced cancers (Extended Data Fig. 2a; NCT03639714). The primary objectives for the phase 1 portion of this study were assessment of safety, tolerability and RP2D. A single priming dose of ChAd68 was administered i.m. followed by multiple i.m. samRNA boosts with escalating doses of samRNA (Fig. 2a). Eligible patients included those with metastatic MSS-CRC, NSCLC or GEA who were begin - ning treatment with standard-of-care chemotherapy during vaccine manufacturing and had tissue available for neoantigen prediction, normal organ function and no contraindications for treatment with immunotherapy (Methods). As of data cutoff, enrollment in phase 1 had been completed, and the study is ongoing. For each patient meeting a threshold for cumulative predicted neoantigen score 30 (Extended Data Fig. 2a), the 20 highest ranking mutations were selected for inclusion in a codon-optimized vaccine expres - sion cassette. The same expression cassette was inserted into both patient-specific vaccine vectors for manufacturing. Patient-specific vaccines were successfully manufactured for 15 patients enrolled for vaccine manufacturing, confirming manufacturing feasibility (a secondary endpoint) of this individualized vaccination approach (one patient subsequently elected not to receive study treatment). Fourteen patients received vaccination and monthly intravenous (i.v.) anti-PD-1 antibody nivolumab administration (480 mg every 4 weeks). Patients at dose level 3 (DL3) and DL4 also received s.c. ipi - limumab (30 mg) with each vaccination (Fig. 2a and Table 1). Four patients (G1, G8, G9 and G10) received continued standard-of-care chemotherapy with study treatment, and eight patients received a single ChAd68 boost ≥24 weeks after the prime vaccination (Extended Data Table 1). Planned dosing intervals and timepoints with blood draws for immunogenicity are shown in Extended Data Fig. 2b. The median age of the 14 phase 1 patients was 59 years; tumor types included metastatic MSS-CRC (n = 7), GEA (n = 6) or NSCLC (n = 1); and patients had received one or two prior lines of therapy (Table 1 and Extended Data Table 1). The individualized vaccine regimen was safe and well tolerated, with no dose-limiting toxicities. The primary safety endpoint of this vaccine regimen was reflective of a viral-based vaccine regimen with TRAEs consistent with a vaccine-induced immune response (Fig. 2b). TRAEs >10% included pyrexia (86%), fatigue (43%), musculoskeletal and injec - tion site pain (both 36%) and diarrhea (29%). Serious TRAEs were largely consistent with the known safety profile of CPI therapy and included one count each of pyrexia, duodenitis, increased transami - nases and hyperthyroidism (Fig. 2b). Based on the totality of the safety and immunogenicity data (secondary endpoint; see next sec - tion) from the phase 1 part of the study, the primary endpoint of RP2D was determined to be 10 12 VP ChAd68 and 30 µg samRNA. Vaccine induces neoantigen-specific CD8+ T cells. The key secondary endpoint of immunogenicity was assessed for neoantigen-specific T cells. To preferentially stimulate CD8 T cells NATuRE MEDICINE | VOL 28 | AUGUST 2022 | 1619–1629 | www.nature.com/naturemedicine 1620 Articles Nature MediciNevia major histocompatibility complex (MHC) class I presenta - tion in immune assays30,35,36, individual patient minimal epitope (8–11mer) peptide pools were designed to include the 40 highest ranked predicted neoepitopes (CD8 pool; Supplementary Table 2). Because this exploratory minimal epitope pool design does not include all potential neoantigens per mutation and can dispro - portionately favor some mutations with high prediction scores, additional patient-specific peptide pools consisting of overlapping 15mers spanning each vaccine cassette were also used to interrogate T cell responses more broadly (15mer pool; Supplementary Table 2). Posttreatment PBMC samples were available for 13 of 14 patients (all except patient G5). Stimulation of PBMCs with patient-specific peptide pools resulted in IFN-γ responses detectable by ex vivo ELISpot following heterologous prime boost vaccination in 100% of patients (13/13), with a ChAd68 boost further increasing peripheral T cell levels in 67% of patients (4/6) with evaluable post-boost sam - ples (Fig. 2c and Extended Data Fig. 3a). Only 15% of patients (2/13) had responses above LOD to their cognate neoantigen peptide pool at baseline, which were increased following vaccine administra - tion (Fig. 2c). Similarly to observations in NHPs, vaccine-induced T cell responses were long-lived, and T cell levels of 700–5,000 SFU/10 6 cells were maintained for >52 weeks in three patients with available longitudinal samples (Extended Data Fig. 3a,b). Notably, readministration of ChAd68 resulted in elevated T cell lev - els detectable 12–24 weeks after ChAd68 boost (patients G1, G3, G8 and G11; Fig. 2b and Extended Data Fig. 3a). Dose escalation 0a ~1 yearShown in panel c 4 12 20 32 33 Shown in panel b Shown in panel d80 106 107 108ChAd68 (1012 VP) samRNA (300 µg) Pol SV9 Gag LW9 Env CL9 Env TL9 Gag CM9 Tat TL8 02456781011121314151617181920212223242526272829303132 0245678101112131415161718192021222324252627282930313202,5005,0007,50010,000IFN-/uni03B3 (SFU per 106 cells)b ChAd68 prime + samRNA boosts 02,5005,0007,50010,000IFN-/uni03B3 (SFU per 106 cells)ChAd68 prime + samRNA boosts + ipilimumab Weeks after ChAd68 primec ChAd68 boost 32 3305,00010,00015,000IFN-/uni03B3 (SFU per 106 cells)+ Ipilimumab – Ipilimumab 106 107010,00020,00030,000IFN-/uni03B3 (SFU per 106 cells)samRNA boost Weeks after ChAd68 primed + Ipilimumab – Ipilimumab Fig. 1 | Heterologous vaccination with ChAd68 and samRNA induces broad, durable CD8+ T cell responses in NHPs that are detectable long-term and can be boosted ≥2 years after prime. a, Schematic of study treatment schedule. b, Antigen-specific T cell response (IFN-γ SFU per 106 cells) at each measured timepoint for duration of study for each group (mean ± standard error of the mean for each antigen stacked), n = 6 per group up to week 20 (n = 5 for group without ipilimumab at weeks 24–107; Supplementary T able 1). c, Antigen-specific T cell response at weeks 32 and 33 (before and after ChAd68 boost) for each group (n = 5 without ipilimumab, n = 6 with ipilimumab), average response per animal (SFU per 10 6 cells) for all antigens stacked. d, Antigen-specific T cell response at weeks 106 and 107 (before and after samRNA boost) for each group (n = 5 without ipilimumab, n = 6 with ipilimumab), average response per animal (SFU per 10 6 cells) for all antigens stacked. NATuRE MEDICINE | VOL 28 | AUGUST 2022 | 1619–1629 | www.nature.com/naturemedicine 1621 Articles Nature MediciNe Phase 1: dose selection based on safety data Adaptive design (n = 14); all patients received nivolumab Dose level 4: GRT-C901 (IM: 1012 VP) + 30 mg ipilimumab GRT-R901 (IM: 300 µg) + 30 mg ipilimumab Dose level 3: GRT-C901 (IM: 1012 VP) + 30 mg ipilimumab GRT-R901 (IM: 100 µg) + 30 mg ipilimumab Dose level 2: GRT-C901 (IM: 1012 VP) GRT-R901 (IM: 100 µg) Dose level 2: GRT-C901 (IM: 1012 VP) GRT-R901 (IM: 30 µg)a b Grade 1/2 Grade 3/4 0 5 10 15PyrexiaDuodenitisTransaminases increasedHyperthyroidismPyrexiaFatigueMuskoskeletal painInjection site painDiarrheaDecreased appetiteArthralgiaInjection site reactionRashHypotension Number of events c G1G2G3G4G6G7G8G9G10G11G12G13G1401002003004005005001,0001,5002,000IFN-/uni03B3 (SFU per 106 cells) G1G2G3G4G6G7G8G9G10G11G12G13G140100200300400500 LOD5001,0001,5002,000 G1 G3 G8G1102004006008001,0002,0004,0006,000 LODLODULOQ Patient ID DMSO CD8 pool 15mer pool05001,0001,500 LODTotal PBMC CD8enr (CD4 depl) CD4enr (CD8 depl) DMSO CD8 pool 15mer pool02004006008001,000 LODTotal PBMC CD4enr (CD8 depl)CD8enr (CD4 depl)d G2 G8 CD4-APCeF780CD8-PerCP-Cy5.5Treatment-related serious AE by patientTreatment-related AEs >10% 90.1% 0.11% 7.00% 2.77%1.18% 0.02% 3.60% 95.2%G1 70.1% 1.27% 23.9% 4.69%5.71% 14.5%0.30% 79.5% IFN/uni03B3 (SFU per 106 cells) IFN/uni03B3 (SFU per 106 cells)CD8enr (CD4 depl) CD4enr (CD8 depl) 0 1041051060 10410510600 103103104105106 –1030103104105106 –103 1050 103105 0103104105106 –1030103104105106 –103 Fig. 2 | First-in-human data show the vaccine regimen is safe and induces CD8+ T cell responses against predicted patient-specific cancer neoantigens. a, Phase 1 study schematic outlining dose escalation. b, Safety data: TRAEs >10% and all treatment-related serious adverse events (AEs) are shown (n = 14 patients). Graph shows counts of grade 1/2 (blue bars) and 3/ 4 (orange bars) events. c, Patient PBMC ex vivo IFN-γ ELISpot responses to patient-specific peptide pools (Supplementary T able 2) are shown for baseline (left panel), maximum response following ChAd prime and samRNA boosts (middle panel) and maximum response post ChAd boost (right panel). Graphs show mean SFU per 10 6 PBMCs ± standard deviation (s.d.) for triplicate ELISpot wells. Assay limit of detection (LOD) and upper limit of quantitation (ULOQ) are indicated by dotted lines. d, Flow plot showing relative CD8 and CD4 T cell frequencies in CD4- or CD8-depleted (depl) PBMC populations. Corresponding ELISpot data (mean SFU of technical replicates ± s.d.) are shown for whole PBMCs (black bars), CD8 enr PBMCs (navy blue bars) and CD4enr PBMCs (orange bars) stimulated with dimethylsulfoxide (DMSO), patient-specific CD8 or 15mer pools for patients G2 and G8. NATuRE MEDICINE | VOL 28 | AUGUST 2022 | 1619–1629 | www.nature.com/naturemedicine 1622 Articles Nature MediciNeof samRNA suggested that more consistent boosts in T cell responses were observed in patients receiving 30-µg and 100-µg doses of sam - RNA (Extended Data Fig. 3c). Prolonged samRNA dosing intervals of 8–16 weeks occurring due to logistical constraints during coro - navirus disease 2019 (COVID-19) restrictions revealed interest - ing insights into the kinetics of samRNA-boosted T cell responses (Extended Data Fig. 3a,b). In patient G1 (GEA, concomitant che - motherapy), T cells expanded 1.5-fold during an 8-week interval between samRNA boosts and remained consistently >700 SFU/10 6 cells during a 16-week period following samRNA administration at week 52. A sevenfold increase in CD8 T cell responses between samRNA doses at weeks 24 and 36 was observed in patient G3 (NSCLC). Patient G2 (GEA) T cell responses showed ~1.5-fold contractions and expansions of the T cell response during a 9-week samRNA dosing interval between weeks 4 and 13. These data sug - gested that lower doses and longer boost intervals may be beneficial in driving T cell responses with this new samRNA platform. This led to the selection of 30 µg samRNA as the RP2D, a primary end - point (see previous section) and longer boost intervals (8 weeks) being assessed in the ongoing phase 2 part of this study. CD8 + T cells recognize neoantigens in multiple HLA alleles. Responses to cognate CD8 pools and 15mer pools were analyzed for CD8 versus CD4 T cell bias in five patients with available samples (G1, G2, G8, G11 and G14). As expected based on pep - tide length, IFN-γ responses following CD8 pool stimulation were driven by CD8 T cells, whereas responses to 15mer pool stimula - tion were either CD8-driven or mixed CD4/CD8 T cell responses (Fig. 2d, Extended Data Fig.