4a). Active vaccination against tumor neoantigens should lead to expansion of preexisting and priming of de novo tumor-specific T cell responses. To assess the breadth of responses with limited available PBMC samples, 4 minipools cov - ering the 20 patient-specific vaccine neoantigens (Supplementary Table 2) were used to partially deconvolute T cell responses in 12 of 13 patients. Positive responses to four different minipools indicate responses to at least four different mutations. Antigen-specific expansion of T cells in short in vitro stimulation (IVS) cultures 30,37 confirmed expansion of preexisting and priming of de novo T cell responses after treatment (Extended Data Fig. 4b). All 12 patients tested had detectable T cell responses to multiple mutations at post - treatment timepoints (Fig. 3a). Voluntary leukapheresis and pooled posttreatment timepoints enabled full deconvolution of T cell responses against all 40 minimal epitopes included in the CD8 pool for patients G1, G2 and G8, revealing positive responses to ≥50% of vaccine mutations in multiple HLA class I alleles for all three patients (Fig. 3b,c and Extended Data Fig. 5). These data indicate that partial deconvolution via minipools (often necessitated due to limitations in sample availability) likely underestimates the true breadth of neoantigen-specific responses induced by this heter - ologous vaccine. Peptide-HLA (pHLA) tetramers were generated to assess the breadth and memory phenotype of antigen-specific T cells. pHLA binding was detected in the context of multiple HLA class I alleles for patients G1, G2 and G8. Tetramer-positive CD8 T cells were characterized by 1.3- to 1.5-fold higher frequencies of effector memory and up to 20-fold lower frequencies of naive T cells when compared to total CD8 T cells (Fig. 3d and Extended Data Fig. 6a). ChAd68 boost in patient G1 resulted in a 10-fold increase in tetramer positive cells, with post-boost tetramer-positive CD8 T cells consisting of ≥93% effector memory cells in the context of both class I alleles tested (Fig. 3e and Extended Data Fig. 6b). CD8 + T cells are polyfunctional and kill target cells. In addition to driving broad T cell responses, a potent vaccine regimen should induce cytotoxic T cells that are able to kill target cells presenting cognate epitopes in the context of MHC class I. Secretion of cyto - toxic markers assessed by Meso Scale Discovery (MSD) and tri - plex FluoroSpot was confirmed in eight patients (Extended Data Fig. 6c,d). Notably, readministration of ChAd68 resulted in increased granzyme B secretion following CD8 pool stimulation in four of six patients with evaluable post-ChAd68 boost timepoints (Extended Data Fig. 6e). To assess whether this cytotoxic signature was driven by CD8 T cells, cytokine secretion was analyzed via intra - cellular cytokine staining (ICS). Stimulation of ex vivo PBMCs with CD8 pool resulted in detectable frequencies of IFN-γ +, CD107α+, tumor necrosis factor α (TNF-α)+ and interleukin-2 (IL-2)+ CD8 T cells in patients G2 and G8, which were increased ~2-fold to ~700-fold following IVS expansion (Fig. 4a and Extended Data Fig. 7a). Boolean gating confirmed the presence of quadruple-, triple-, and double-positive polyfunctional CD8 T cells for both patients (Fig. 4b , Extended Data Fig. 7b). Polyfunctional CD4 T cell responses following cognate 15mer pool stimulation were also detectable in patients G2 and G8 following IVS culture (Extended Data Fig. 7c). The ability of antigen-specific CD8 T cells to kill target cells presenting neoantigen was determined. Co-culture of expanded neoantigen-specific patient T cells with single HLA allele-expressing target cells pulsed with cognate neoantigen pep - tides resulted in a 2.5-fold to 6.4-fold decrease in live target cells over time compared to controls, indicating antigen-specific T cell-driven killing of target cells presenting cognate pHLA for both patients G2 and G8 (Fig. 4c and Extended Data Fig. 7d). T cells are increased in tumors following vaccination. To exert anti-tumor effects and clinical benefit in patients, vaccine-induced T cells need to infiltrate tumors and lyse cells presenting their cognate pHLA antigen through recognition via the T cell recep - tor (TCR). Paired TCR sequencing and gene expression profiling via single-cell RNA sequencing was performed on baseline and on-treatment PBMC timepoints from patients with available cor - responding tumor biopsies (G1 and G3; Extended Data Fig. 8a). Neoantigen-reactive CD8 T cells were increased 1.6-fold to 6.4-fold in on-treatment samples compared to baseline (Extended Data Fig. 8b). T able 1 | Baseline and disease characteristics by DL DL1 (n = 3)DL2 (n = 2)DL3 (n = 3)DL4 (n = 6)T otal (n = 14) Age (yr), median (range)72 (59–74)70 (63–76)51 (50–53)55 (38–61)59 (38–76) Female gender, n (%)1 (33) 1 (50) 1 (33) 1 (17) 4 (29) ECOG, n (%) 0 2 (67) 1 (50) 1 (33) 3 (50) 7 (50) 1 1 (33) 1 (50) 2 (67) 3 (50) 7 (50) Number of prior therapies, n (%) 1 2 (67) 1 (50) 1 (33) 2 (33) 6 (43) 2 1 (33) 1 (50) 2 (67) 4 (67) 8 (57) Prior anti-PD(L)1 therapy, n (%) Y es 1 (33) 0 (0) 0 (0) 0 (0) 1 (7) No 2 (67) 2 (100) 3 (100) 6 (100) 13 (93) Tumor types, n (%) MSS-CRC 0 1 (50) 2 (67) 4 (67) 7 (50) GEA 2 (67) 1 (50) 1 (33) 2 (33) 6 (43) NSCLC 1 (33) 0 0 0 1 (7) DL1, GRT-C901/GRT-R902 30 μg + i.v. nivolumab; DL2, GRT-C901/GRT-R902 100 μg + i.v. nivolumab; DL3, GRT-C901/GRT-R902 100 μg + i.v. nivolumab + s.c. ipilimumab; DL4, GRT-C901/ GRT-R902 300 μg + i.v. nivolumab + s.c. ipilimumab. ECOG, Eastern Cooperative Oncology Group. NATuRE MEDICINE | VOL 28 | AUGUST 2022 | 1619–1629 | www.nature.com/naturemedicine 1623 Articles Nature MediciNe a b cAHNAKBICD1BNC2C1orf50CCNHCHP2DNAJC10DYNC1H1LMNB2MUC2MYH14NKAIN1OGTPRUNE2RAB11BSOSTDC1SPG11TAX1BP1TEP1ZNF233Mutated gene ABI1AP1B1CHD9CSMD3CUL4ADNAH2DNAJC11EHBP1L1ERFEXPH5GGA2GLDNKRASNRP1ONECUT2PIGOPLEKHG1SURF6TGM6TTBK1 ADARCENPMCHD6CTR9DHX37FILIP1FKBP15GIN1IGSF23MAP2K2MYO5BPHF8POLR1EQTRT1RPL3LSAP130SLC27A3SUN2YKT6ZBTB46 G1G2G3G4G7G8G9G10G11020406080100Percentage of total response G12G13G14020406080100Mini_4 Mini_3Mini_2 Mini_1Mini pool responses Ex vivo Post-IVS Number of positive epitopes per mutationIndividual peptide deconvolution Patient ID Predicted HLA coverage of positive epitopes 6 HLA-B*44:05 1 HLA-C*02:023 HLA-A*02:01 3 HLA-B*18:01 Total = 13 Total = 194 HLA-A*02:01 4 HLA-A*30:041 HLA-B*08:01 8 HLA-B*57:01 2 HLA-C*06:02 1 HLA-C*04:019 HLA-A*03:01 4 HLA-A*33:01 Total = 14G1 G2 G8d Naive Effector memory Effector memory RA+14.5% 43.5% 40.1%1.85% 57.2%41.8%0.34%0.68% 30.7% 61.4%1.92%6.0% 60.5%35.5%2.63%1.32%Patient G2 leukopak CD45RA-SB702CCR7-PE-Cy7 CD8-PerCP-Cy5.5 FSC-A Tet-APC Tet-PE Tet-PE e 33.9% 58.7%3.54%3.94% CD45RA-SB702CD8-PerCP-Cy5.5 CCR7-PE-Cy7Total CD8+A*30:04 Tet+A*02:01 Tet+B*08:01 Tet+ B*44:05 tet+Tet+ memory Tet+ PE CD8-PerCP-Cy5.5 CCR7-PE-Cy7B*44:05 Tet+ Tet+ memoryG1: pre-ChAd68 boost G1: After-ChAd68 boost 6.01% 93.3%0.39%0.33%0.33%21.5% 0 0 –103 103 104 105 0 –103 103 104 105 0 103 104 105 5K100K150K200K250K0.23% 0.01% 1.85% 14.5% 40.1% 43.5%0.34% 0.68% 41.8% 57.2%1.92% 6.00% 61.4%30.7%2.63% 1.32% 35.5% 60.5% 0.92%9.44%3.54% 3.94% 58.7 33.9%0.39% 0.33% 93.3% 6.01%Central memory0 1 2 3 0 1 2 3 0 1 2 3 4 0 –103105 104 103 –1030105 104 103 –1030105 104 103 –1030105 104 103 –1030 103 104 105 0 –103 103 104 105 0 –103 103 104 105 0 –103 103 104 105105 104 103 –1030105 104 103 –1030105 104 103 –1030105 104 103 –1030 0 –103 103 104 105 0 –103 103 104 105105 104 103 –1030105 104 103 –1030 0 –103 103 104 105 0 –103 103 104 105105 104 103 –1030105 104 103 –1030 CD45RA-SB702 Tet+ PE Fig. 3 | Vaccine induces polyclonal effector memory CD8 T cell responses across multiple neoantigens and HLAs. a, Patient PBMCs from available posttreatment timepoints (G1 W12, G2 W5, G3 W8, G4 W6, G7 W4, G8 W12, G9 W8, G10 W16, G11 W16, G12 W12, G13 W12, G14 W13 and G15 W12) were stimulated overnight in ex vivo or post-IVS IFN-γ ELISpot with patient-specific minipools or DMSO. Graphs show percentage of total response of mean IFN-γ responses to minipool 1 (turquoise), minipool 2 (light blue), minipool 3 (medium blue) and minipool 4 (navy blue) for each patient. b, Number of positive minimal epitopes detected (positive by ex vivo or post-IVS ELISpot) for each mutated gene. Undetectable responses against epitopes tested are indicated by ‘x’. c, Pie charts showing patient-specific predicted HLA class I presentation of positive neoantigen responses shown in panel b. d, Density plots showing recognition of pHLA (A*30:04, A*02:01, B*08:01) and corresponding T cell memory phenotypes for patient G2. Pie charts showing frequencies of naive (CD45RA +CCR7+; purple), central memory (CM; CD45RA−CCR7+; blue), effector memory (EM; CD45RA−CCR7−; green) and T effector memory RA+ (TEMRA; CD45RA+CCR7−; orange) cells in total CD8+ population (left-most chart) and CD8+tetramer+ populations from corresponding dot plots. e, Density plots and corresponding pie charts showing recognition of pHLA (B*44:05) and corresponding T cell memory phenotypes before and after ChAd68 boost for patient G1. NATuRE MEDICINE | VOL 28 | AUGUST 2022 | 1619–1629 | www.nature.com/naturemedicine 1624 Articles Nature MediciNeAnalysis of transcriptional activation signatures confirmed an enrichment for CD8 T cells in on-treatment samples (Extended Data Fig. 8c). TCR analysis revealed a total of 12 and 30 distinct TCR clonotypes (α-β pairs) detected in PBMCs following vaccina - tion for patients G1 and G3, respectively (Extended Data Fig. 8b). Analysis of IFNG, GZMB, TNFA and IL2 transcripts in T cell clo - notypes revealed 12- to 98-fold increases in frequencies of T cells expressing two or more cytokine transcripts in on-treatment sam - ples compared to baseline for both patients (Extended Data Fig. 8d,e and Supplementary Table 3). Baseline and on-treatment tumor biopsy samples from patients G1 and G3 were analyzed for the pres - ence of TCR-β CDR3 sequences via bulk DNA sequencing. Analysis of TCR-β CDR3 sequences obtained independently via single-cell RNA sequencing in PBMCs versus DNA sequencing in tumor samples indicated overlap of putative neoantigen-specific T cells in both the blood and tumor. For patient G1, 341 TCR-β CDR3 clo - notypes were identified across baseline and on-treatment tumor samples with an enrichment of nine clones in the on-treatment biopsy. Two of these nine clonotypes were also found in PBMCs sampled at the time of the on-treatment biopsy (Fig. 4d). In patient G3, a total of 294 TCR-β CDR3 clonotypes were identified via DNA sequencing in both tumor samples, with 40 clonotypes enriched in the on-treatment sample. Five of the 40 TCR-β CDR3 clonotypes enriched in the on-treatment biopsy overlapped with the activated TCR clonotypes in PBMCs at the time of the on-treatment biopsies (Fig. 4d). Representation of clonotypes from neoantigen-specific CD8 T cells in tumor tissue that were also detected in PBMCs pro - vides proof-of-concept that vaccine-induced neoantigen-specific T cells in the periphery are able to infiltrate tumors. Prolonged survival and decreases in ctDNA in MSS-CRC. Multiple measures of activity were assessed, including OS and changes in tumor size via imaging (objective response rate and progression-free survival per RECIST v1.1, all secondary endpoints), as well as transcriptional profiling and changes in ctDNA (both exploratory endpoints). Standard genomic correlates of responsive tumors to CPIs (namely, tumor mutational burden, CD274 (PD-L1) and IFNG RNA sequencing signatures) were analyzed in patient pretreatment biopsies and compared to relevant cancer types from The Cancer Genome Atlas (TCGA). Pretreatment patient genomic data demonstrate that patients with MSS-CRC and GEA in this study had low tumor mutational burden and PD-L1 expression and that these tumors had low immune reactivity (Fig. 5a,b and Supplementary Table 4). Such cold tumors are known to be refrac - tory to CPI therapy. Objective response rate and progression-free survival per RECIST v1.1 show a patient with GEA with a complete response and several patients across tumor types with stable dis - ease (SD) (Extended Data Table 1). Given the observation of SD in several patients with MSS-CRC, we focused on analyzing these seven patients with MSS-CRC for the secondary endpoint of OS and an exploratory endpoint of changes in ctDNA from baseline (Extended Data Table 1 and Supplementary Table 4). For patients with MSS-CRC, 3 of 7 (42.9%) remained alive (median OS of 8.7 months; Extended Data Table 1), with an OS rate at 12 months of 42.9% reflecting multiple patients experiencing durable benefit. Emerging evidence suggests changes in ctDNA levels on-treatment relative to baseline are a useful biomarker to stratify patients as molecular responders or nonresponders and differen - tiate patients with a good prognosis, especially those with SD per RECIST v1.1 criteria 38. Of these patients, 100% (3/3) with treat - ment durations <6 months had increases in ctDNA concentration, whereas 75% of patients (3/4) with treatment durations ≥6 months demonstrated a molecular response (<50% from baseline; Fig. 5c and Supplementary Table 4). Two patients had complete ctDNA clearance while on treatment (G8 and G14; Fig. 5c). Most nota - bly, these decreases in ctDNA were more frequent in patients with prolonged OS (median OS not reached versus 7.8 months; Fig. 5d). Patient G8, a 51-year-old patient with MSS-CRC who received che - motherapy for 21 months, had progressed on first-line chemother - apy before study treatment initiation but continued concomitant standard of care chemotherapy during study treatment (Extended Data Table 1) and showed clear evidence of tumor regression fol - lowing vaccine administration via both ctDNA and radiologic assessments. Computed tomography scans showed size reduction in multiple lung lesions within 16 weeks and persistent reduction in multiple lung and one liver lesion through 48 weeks (Fig. 5e). Fluorodeoxyglucose-positron emission tomography imaging of the stable liver lesions showed a lack of hypermetabolic cells follow - ing study treatment administration, suggesting anti-tumor activity of this vaccine regimen not evidenced by computed tomography imaging (Fig. 5e). In totality, the safety, immunogenicity, OS and ctDNA signals observed in this trial warrant further investigation of this vaccine platform in patient populations that are refractory to CPI monotherapy 31–33. Discussion The quality of antigen-specific T cell responses induced by a het - erologous vaccine regimen, consisting of ChAd68 and samRNA vectors in combination with CPI, was assessed in NHPs and patients with advanced cancers. The potency and longevity of T cell responses to model antigens following vaccination were studied in NHPs, whereas safety, immunogenicity and feasibility of an indi - vidualized patient-specific vaccine were assessed in patients with late-stage cancer. This vaccine regimen was well tolerated and induced neoantigen-specific CD8 T cell responses in all patients. Vaccine-driven T cell responses were (1) polyclonal across multiple mutations and HLA alleles, (2) polyfunctional and (3) able to kill tar - get cells presenting cognate peptide. Of note, more consistent boosts in T cell responses were observed in patients receiving lower doses of samRNA. This could be due to less innate immune activation via mRNA-sensing pattern recognition receptors that might inter - fere with samRNA amplification and antigen expression. Increased Fig. 4 | Neoantigen-reactive CD8 T cells are polyfunctional, kill targets presenting cognate peptides in multiple HLA alleles and are increased in on-treatment tumor samples. a, Representative density plots for patient G2 ICS for CD8+ cells producing IFN-γ, CD107α, TNF-α and IL-2 in ex vivo or IVS-expanded PBMCs stimulated overnight with DMSO or patient-specific CD8 pool. Cells were gated on PBMCs (forward scatter (FSC)-A versus side scatter (SSC)-A), singlets (FSC-A versus FSC-H), live cells (FSC-A versus Live/Dead), CD8 or CD4 T cells (CD8 versus CD4) and cytokine + cells. b, Polyfunctionality was assessed via Boolean gating of CD8+cytokine+ populations (background subtracted). Pie charts show polyfunctionality of ex vivo PBMCs and post-IVS PBMCs. Pie arcs depict individual cytokines in red (CD107α), lime green (IFN-γ), turquoise (TNF-α) and navy blue (IL-2). Pie sections depict frequencies of cells expressing multiple cytokines as outlined in pie category legend. Main functional pie slices are colored red (quadruple positive for CD107α, IFN-γ, TNF-α and IL-2), orange (triple positive for CD107α, IFN-γ and TNF-α) and yellow (double positive for CD107α and IFN-γ). c, T arget cell killing by IVS-expanded PBMCs from patients G2 (B*08:01, B*57:01, A*30:04). Graphs show relative viability based on NucRed count and normalized to effector-to-target ratio of 10:1 DMSO control over time. Data are shown as mean ± s.d. from duplicate wells for targets alone stimulated with DMSO (gray circles) or cognate peptide (black squares) and for 10:1 effector/target co-cultures stimulated with DMSO (light blue circles) or cognate peptide (navy blue squares). d, DNA sequencing of TCR-β chains in tumor samples in baseline and on-treatment samples from patients G1 and G3. TCR-β chains expanded in PBMCs, in both PBMCs and tumors, and contracted are shown. NATuRE MEDICINE | VOL 28 | AUGUST 2022 | 1619–1629 | www.nature.com/naturemedicine 1625 Articles Nature MediciNe a CD107/uni03B1Pie categories IFN-/uni03B3 TNF-/uni03B1 IL-2 + + + + + + + ++ + + + – – – –+ + – – + + – –+ – + – + – + –CD107/uni03B1 IFN-/uni03B3 TNF-/uni03B1 IL-2 – – – – – – –+ + + + – – –+ + – – + + –+ – + – + – +Pie arcs CD107/uni03B1+ IFN-/uni03B3+ TNF-/uni03B1+ IL-2+CD107/uni03B1+ IFN-/uni03B3+ IL-2+ TNF-/uni03B1+ CD107/uni03B1+ IFN-/uni03B3+ TNF-/uni03B1+ CD107/uni03B1+ IFN/uni03B3+CD8-PerCP-Cy5.5 CD8-PerCP-Cy5.5G2: ex vivo G2: post-IVS CytokinebG2: ex vivo G2: post-IVS c Targets only + DMSO Targets only + peptide E:T 10:1 + DMSO E:T 10:1 + peptide0 816 24 32 40 48 56 64 72–4–3–2–1012Relative viabilityG2: HLA-B*08:01 0 816 24 32 40 48 56 64 72–8–6–4–202Relative viabilityG2: HLA-B*57:01 0 816 24 32 40 48 56 64 72–3–2–10123 Time (h)Relative viabilityG2: HLA-A*30:04dIFN/uni03B3-APC IL-2-PECD107/uni03B1-BV421 TNF/uni03B1-BV786 0.61% 11.8%1.66% 32.6%9.0% 47.7%0.58% 36.3% IFN/uni03B3-APC CD107/uni03B1-BV421 TNF/uni03B1-BV786 IL-2-PEDMSO CD8 pool DMSO CD8 pool 0.0008% 0.20% 0.20% 0.32% 0.04% 0.30% 0.016% 0.25% G1 G3On-treatment biopsy TCR-/uni03B2 frequency (%) VAF <1% On-treatment biopsy TCR-/uni03B2 frequency (%) VAF 9%Baseline biopsy TCR-/uni03B2 frequency (%) VAF 10% Baseline biopsy TCR-/uni03B2 frequency (%) VAF 17% Expanded in tumor 7Expanded in tumor and IVS 2 Not significant 334 Expanded in tumor and IVS 5 Expanded in tumor Contracted in tumor Not significant 24311 Proportion of filtered T cells (%) Proportion of filtered T cells (%)Expanded TCR-/uni03B2 in IVS (n = 12) Expanded TCR-/uni03B2 in tumor (n = 9) Expanded TCR-/uni03B2 in IVS (n = 27) Expanded TCR-/uni03B2 in tumor (n = 40)01020304050 012345678 0102030 01234 G1 G1 G3 G3 Baseline On- treatmentBaseline On- treatmentBaseline On- treatmentBaseline On- treatmentPhenotype CD8 CD4 Undetermined Also expanded in tumor 35 10–410–4 10–310–3 10–210–210–410–510–310–3 10–210–2105 105104 104103 1030 0–103105 104 103 0 –103105 104 103 0 –103105 104 103 0 –103 –1031051041030 –103 1051041030 –1031051041030 –103 1051041030 –1031051041030 –103 1051041030 –1031051041030 –1031051041030 –1031051041030 –1031051041030 –1031051041030 –1031051041030 –1031051041030 –1031051041030 –1031051041030 –103 105 104 103 0 –103105 104 103 0 –103 105 104 103 0 –103105 104 103 0 –103 105 104 103 0 –103105 104 103 0 –103105 104 103 0 –103105 104 103 0 –103 105 104 103 0 –103105 104 103 0 –103 105 104 103 0 –103105 104 103 0 –103Comp-PerCP- Cy5-5-A :: CD8Comp-PerCP- Cy5-5-A :: CD8Comp-PerCP- Cy5-5-A :: CD8Comp-PerCP- Cy5-5-A :: CD8 NATuRE MEDICINE | VOL 28 | AUGUST 2022 | 1619–1629 | www.nature.com/naturemedicine 1626 Articles Nature MediciNe a G4 G10 G6 G11 G9 G8 G14–100–500501001,0001,2501,500Percent change in ctDNA % ( mutant hGE ml–1)SD per RECIST for /uni226524 weeksPD per RECIST at <24 weeks Unconfirmed PD per RECIST for /uni226524 weeks0.01 0.1 1 10 100 1,000 10,000050100150200 Tumor mutational burdenPD-L1 RNA-seqLung Sq.Lung Ad. MSS-CRC GEA NSCLC MSS-CRC MSI-CRC Gastric Lung Ad. Lung Sq. Melanoma–4–2024 Study patient populationIFN-/uni03B3 signatureTCGA data 0 20 40 60 800255075100 Weeks after primeProbability of survival No ctDNA decrease (n = 4) ctDNA decrease (n = 3)ctDNA decrease (n = 3) No ctDNA decrease (n = 4)b c d e Lung Liver LiverBaseline 16 weeks 48 weeks PET scan Pretreatment On treatmentNSCLC MSS-CRCGEAMSS-CRC GastricMSI-CRC MelanomaStudy patient population TCGA Fig. 5 | Early signals of clinical efficacy in patients with MSS-CRC in advanced disease setting. a, Scatter plot showing tumor sample RNA sequencing (RNA-seq) for patients before study treatment. PD-L1 normalized RNA-sequencing expected counts (y axis) versus tumor mutational burden (x axis) for patients and relevant TCGA cancer types. b, Patient RNA-sequencing IFN-γ signature average z-scores (x axis) calculated across patients and relevant TCGA cancer types (y axis). TCGA samples: MSS-CRC (n = 286), MSI-CRC (n = 62), gastric (n = 406), lung adenocarcinoma (Lung Ad.)_ (n = 498), lung squamous (Lung Sq.) (n = 463) and melanoma (n = 436). GRANITE patients: MSS-CRC (n = 7), GEA (n = 6) and NSCLC (n = 1). c, Percent change in ctDNA following initiation of study treatment relative to baseline levels shown as best response and colored by RECIST-based response and time on treatment. Patients in blue were all on study treatment >24 weeks with two patients treated beyond RECIST-based progressive disease (PD) that was not confirmed by 24 weeks and continued on study treatment. d, Probability of survival in MSS-CRC patients with (n = 4) or without (n = 3) reduction in ctDNA since ChAd68 prime. Data cutoff 31 January 2022. e, Radiological images showing tumor lesions in the lung and liver of patient G8 before initiating study treatment (baseline) and following study treatment (16 and 48 weeks). Fluorodeoxyglucose-positron emission tomography imaging of tumor lesions in the liver when patient was diagnosed in February 2018 and 7 months after starting study treatment. Arrows indicate relevant target lesions. NATuRE MEDICINE | VOL 28 | AUGUST 2022 | 1619–1629 | www.nature.com/naturemedicine 1627 Articles Nature MediciNefrequencies of vaccine-induced TCR clonotypes were found in on-treatment tumor samples, providing proof-of-concept for tumor infiltration of vaccine-induced T cells. Patients with molecular responses following vaccination showed stabilization and/or reduc - tion of target lesions and prolonged OS, indicating potential early signs of clinical activity in this CPI-refractory patient population. A randomized phase 2/3 study has been initiated to demonstrate the efficacy of this vaccine regimen in patients with MSS-CRC in the first-line metastatic setting (NCT05141721). A vaccine that induces neoantigen-specific CD8 T cells provides a foundation for combination with CPIs, making their combined administration highly desirable. Although the effects of s.c. ipi - limumab were difficult to discern in this small cohort of patients, administration of ipilimumab in addition to a vaccine in NHPs resulted in increased breadth and magnitude of antigen-specific T cell responses in all animals. This may be due to a lower thresh - old for T cell activation against weaker epitopes with ipilimumab compared to vaccine alone. The lower threshold of T cell activation provided by the addition of ipilimumab might allow induction of an overall broader immune response to vaccine-encoded neoantigens and a more robust immune attack of the tumor. Driving broad T cell responses against multiple tumor antigens across multiple HLA alleles, as observed in this study, may limit the impact of common HLA-related immune escape mechanisms 39–41. As a patient’s disease progresses, increased heterogeneity can lead to immune evasion. Treating patients earlier in their disease, such as in the adjuvant set - ting or early in the treatment of metastatic disease, has the potential benefit of completely eradicating tumor cells by vaccine-induced T cells before resistance mutations arise. Also, given the time lag between vaccine administration and T cell expansion, patients with lower initial disease burden and more time to generate a T cell response before disease progression are more likely to receive long-term benefit and durable disease control from our vaccination approach. Increasing evidence suggests that a clear survival benefit (even without a radiologic response) may be a hallmark of immu - notherapies42. Follow-up of this ongoing phase 1/2 study is ongoing, and one phase 2/3 and one phase 2 study are assessing potential clini - cal activity of this vaccine regimen in patients with MSS-CRC in the metastatic and adjuvant treatment settings, respectively. In addition to benefiting patients with cancer, this versatile and potent vaccine can be applied to other indications such as prophylactic vaccination against SARS-CoV-2 (ref. 43) (NCT05148962, NCT04776317). In sum, this versatile heterologous vaccine regimen generates strong, persistent and functional immune responses that have broad appli - cability to a wide range of disease settings and support continued exploration in the clinical setting to bring better treatment options to patients with cancer and people at risk of infection. Online content Any methods, additional references, Nature Research report - ing summaries, source data, extended data, supplementary infor - mation, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/ s41591-022-01937-6. Received: 4 March 2022; Accepted: 6 July 2022; Published online: 15 August 2022 References 1. Lawrence, M. S.