Catenacci10, Andrew Allen1, Andrew R. Ferguson1 and Karin Jooss1 ✉ Checkpoint inhibitor (CPI) therapies provide limited benefit to patients with tumors of low immune reactivity. T cell-inducing vaccines hold promise to exert long-lasting disease control in combination with CPI therapy. Safety, tolerability and recom- mended phase 2 dose (RP2D) of an individualized, heterologous chimpanzee adenovirus (ChAd68) and self-amplifying mRNA (samRNA)-based neoantigen vaccine in combination with nivolumab and ipilimumab were assessed as primary endpoints in an ongoing phase 1/2 study in patients with advanced metastatic solid tumors (NCT03639714). The individualized vaccine regi- men was safe and well tolerated, with no dose-limiting toxicities. Treatment-related adverse events (TRAEs) >10% included pyrexia, fatigue, musculoskeletal and injection site pain and diarrhea. Serious TRAEs included one count each of pyrexia, duodenitis, increased transaminases and hyperthyroidism. The RP2D was 10 12 viral particles (VP) ChAd68 and 30 µg sam- RNA. Secondary endpoints included immunogenicity, feasibility of manufacturing and overall survival (OS). Vaccine manu- facturing was feasible, with vaccination inducing long-lasting neoantigen-specific CD8 T cell responses. Several patients with microsatellite-stable colorectal cancer (MSS-CRC) had improved OS. Exploratory biomarker analyses showed decreased circu-lating tumor DNA (ctDNA) in patients with prolonged OS. Although small study size limits statistical and translational analy - ses, the increased OS observed in MSS-CRC warrants further exploration in larger randomized studies. NATuRE MEDICINE | VOL 28 | AUGUST 2022 | 1619–1629 | www.nature.com/naturemedicine 1619 Articles Nature MediciNecan lead to diminished immune responses to antigens delivered via adenoviral-based vaccines20,25, preexisting anti-vector immunity can be circumvented by using non-human adenoviral species, such as chimpanzee adenovirus (ChAd). RNA virus-based vaccines have been tested in humans in a range of cancer and infectious disease indications 26,27, and samRNA vectors are of particular interest due to their ability to express high and durable antigen levels28. In this study, the quality of antigen-specific T cell responses induced by a heterologous vaccine regimen consisting of a ChAd vector (ChAd68) and a fully synthetic Venezuelan equine encepha - litis virus-based samRNA vector was assessed in non-human pri - mates (NHPs) and patients with advanced cancers in combination with CPIs. The potency and longevity of the immune response against simian immunodeficiency virus (SIV) model antigens 29 and the impact of subcutaneous (s.c.) anti-CTLA-4 administra - tion were assessed in NHPs. Immunogenicity against individual - ized (patient-specific) predicted cancer neoantigens30 and in-depth analysis of T cell responses elicited by vaccination are being assessed in an ongoing first-in-human phase 1/2 clinical trial (GRANITE, NCT03639714) in patients with metastatic MSS-CRC, non-small cell lung cancer (NSCLC) or gastroesophageal adenocarcinoma (GEA). Primary endpoints of the phase 1 part of the study were safety and tolerability and RP2D. Secondary and exploratory end - points include immunogenicity, OS, feasibility of manufactur - ing patient-specific vaccines and changes in ctDNA. Induction of neoantigen-specific CD8 T cell responses was observed in all patients, including patients with MSS-CRC whose tumors lacked immune reactivity before vaccination. Decreases in ctDNA and stabilization of disease and/or reduction in tumor burden in these patients suggest early signs of potential therapeutic benefit in this CPI-refractory patient population 31–33. Results Vaccination induces durable CD8+ T cell responses in NHPs. To assess the potency and durability of the immune responses induced by this heterologous vaccine regimen preclinically in NHPs, a model antigen cassette encoding six well-defined NHP-specific MAMU-A*01-restricted SIV antigens 29 was inserted into the ChAd68 and samRNA vaccine platforms. MAMU-A*01-positive rhesus macaques were immunized intramuscularly (i.m.) with ChAd68 prime (n = 11), followed by i.m. samRNA boosts at 4, 12 and 20 weeks, with (n = 6) or without (n = 5) concomitant admin - istration of s.c. anti-CTLA-4 antibody ipilimumab (Fig. 1a and Supplementary Table 1). ChAd68 induced immune responses against all six antigens (mean 923 and 313 spot-forming units (SFU)/10 6 cells at week 4 ± s.c. ipilimumab, respectively; Fig. 1b). Primed T cell responses were boosted following administration of samRNA, with T cell responses still detectable 32 weeks after prime in both groups. Concomitant administration of s.c. ipilimumab resulted in maintenance of an overall higher T cell response (mean 843 and 277 SFU/10 6 cells ± s.c. ipilimumab, respectively; Fig. 1b). ChAd68 readministration at week 32 boosted T cell responses ~15-fold and ~12-fold ± concomitant ipilimumab, respectively (Fig. 1c). Immunization resulted in the generation and maintenance of antigen-specific effector memory T cells (Extended Data Fig. 1a), which have been demonstrated to be predictive of providing thera - peutic benefit to patients with cancer34. In both therapeutic and prophylactic vaccine settings, the gen - eration and maintenance of long-lived memory responses is highly desirable to maintain disease control or provide protection from future infections. To investigate whether antigen-specific memory T cells were detectable in NHPs up to 2 years after the ChAd68 prime vaccination, six animals that had not received additional treatment and remained available from the initial study were assessed. Tetramer analysis of peripheral blood mononuclear cells (PBMCs) 80 weeks after ChAd68 prime identified the presence of antigen-specific central memory T cells (Extended Data Fig. 1b). Accordingly, interferon-γ (IFN-γ ) ELISpot at week 106 revealed positive responses to four or more of the SIV antigens in all six animals, and samRNA adminis - tration at week 106 resulted in an ~8-fold and ~17-fold increase in antigen-specific T cell response detectable at week 107 ± s.c. ipilim - umab, respectively (Fig.