ArticleExtended Data Fig. 5 | Autogene cevumeran activates neoantigen-specific polyfunctional effector CD8+ T cells. (A) Uniform manifold approximation and projection (UMAP) plots of single peripheral blood T cells by single-cell RNA/TCR sequencing in n = 4 patients (patients 1, 10, 11 and 29) stratified by lineage (CD8 vs. CD4, left ), patient ( middle ), and vaccine-expanded clones (right ; expanded clones identified in Fig. 2a). T cells purified post-vaccine priming doses at time points indicated in Fig. 2b (inverted triangles). (B, C ) UMAP plots of single peripheral blood CD8 + T cells in patients 1, 10, 11 and 29 stratified by CD8+ T cell naïve ( SELL , CCR7, IL7R, BCL2, PECAM1, TCF7, BACH2, LEF1 ), dysfunctional ( TIGIT, TOX, LAG3, ENTPD1, CXCL13, HAVCR2, GZMB ), memory ( EOMES, GZMK, CXCR3, TCF1, ID3, STAT4, CCR7, SELL ) and effector (PRF1, GZMB, GNLY, IFNγ, EOMES, ZEB2, E2F7, TBX21, PDCD1, CXCR3, FAS ) transcriptional phenotypes ( B) and select individual phenotype defining markers ( C). (D) Cytokine (IFNγ, TNFα) production in CD8+ and CD4+ T cells after post-vaccine bulk PBMC ex vivo rechallenge with pools of overlapping long neopeptides. ( E) Cytokine (IFNγ, TNFα) production and degranulation (CD107a) by CD8 + T cells after bulk PBMC ex vivo rechallenge with predicted minimal neopeptides. Flow cytometry gating strategies as in Extended Data Fig. 4a. ( F) Peripheral blood CD8 + T cell proliferation (Ki67) and activation (PD-1, LAG-3, TIM-3, HLA-DR) pre- and serially post-atezolizumab, vaccine and mFOLFIRINOX by flow cytometry in n = 7 of 8 responders and n = 7 of 8 non-responders with available data. n = number of patients. Percentage of all blood T cells0.0010.010.1110 Vaccine priming Chemo Vaccine boosterResponders (n = 7) Patient: 1Non-responders (n = 1) 20 6 10 11 14 25 29 Vaccine priming ChemoVaccine booster Vaccine priming Chemo Vaccine booster Vaccine priming ChemoVaccine booster Vaccine priming Chemo Vaccine booster Vaccine priming Chemo Vaccine booster Vaccine priming Chemo Vaccine boosterVaccine primingChemo Vaccine booster n = 28 clones61%25% 14% n = 7 clones 43%28.5% 28.5% n = 9 clones 11% 22% 67% n = 2 clones100% n = 10 clones 40%10% 50% n = 3 clones67%33% n = 6 clones16.7% 66.7%16.7% n = 15 clonesExpanded Non-expanded Undetectabl e53%7% 40%Boost:BA 0.0001 Extended Data Fig. 6 | Post-mFOLFIRINOX autogene cevumeran booster re-expands primed T cell clones. ( A) CloneTrack-identified autogene cevumeran (vaccine) expanded T cell clones with vaccine priming, mFOLFIRINOX chemotherapy (chemo) and vaccine booster. Data shown for patients that had detectable vaccine-expanded clones and received the vaccine booster. ( B) Percentage of vaccine-expanded T cell clones that are detectable and re-expand with vaccine booster in responders. n = number of patients or clones. ArticleD Cycles of mFOLFIRINO XNumber of cycles 0612 Responders (n = 8) Non-responders (n = 8) ET cell infiltration DAPICD8 CD3 100 μmResponders Non-responder s Patient 1P atient 6P atient 19 Patient 28C Atezolizumab response 06 12 18 24 30050100 MonthsP= 0.3 Responde r Non-responder9 77766351100 At risk RFS (%)Responders (n = 9) Non-responder s(n = 7) 100 μm 100 μm 100 μmCD3+ cells/mm2 08001000 600 400 200T cells CD3+ CD8- cells/mm2 08001000 600 400 200CD4+ T cellsCD3+ CD8+ cells/mm2 08001000 600 400 200CD8+ T cells Responders (n = 8) Non-responders (n = 8)B n = 16 biomarker-evaluable patientsctDN A+ctDN A- 25% 75%(n =4) (n =12)CA19-9 +CA19 -9 - 81% (n =13)19% (n =3) 050100150200250300350CA-19-9 (U/m l)Responders (n= 7) Non-responders (n= 6) Upper limit of norma lP = 0.03n = 13 with study-specified treatment schedules andeligible for comparison Atezolizumab Autogene cevumeran priming doses mFOLFIRINOX dose 1SurgeryP = 0.03 P = 0.03P = 0.04 TreatmentA CA19-9 positivit y at diagnosis n = 16 biomarker-evaluable patientsctDNA positivit y at diagnosis 06 12 18 24 30050100 < 2.5 cm (n = 8) > 2.5 cm (n = 8) MonthsAt risk < 2.5 cm > 2.5 cm8 82 5 46 7141 81P= 0.08Primary tumor sizeRFS (%) Extended Data Fig. 7 | Autogene cevumeran responders evidence lower post-vaccination serum CA19-9, equivalent chemotherapy doses and comparable intratumoural T cells. (A, B ) Fraction of biomarker-evaluable patients with detectable CA19-9 ( A) or circulating tumour DNA (ctDNA) ( B) in the peripheral blood at diagnosis. Longitudinal serum CA19-9 levels in autogene cevumeran (vaccine) responders and non-responders ( A, bottom ). Circle = mean, error bars = standard error of the mean ( C) Recurrence-free survival (RFS) stratified by atezolizumab response ( left ) and median primary tumour size ( right ). (D) Number of cycles of mFOLFIRINOX (chemotherapy) in vaccine responders and non-responders. ( E) Intratumoural T cell infiltration in autogene cevumeran responders and non-responders. n = individual patients. P values by two-tailed Mann Whitney test ( A), and log-rank test ( C). Extended Data Fig. 8 | Autogene cevumeran responders and non-responders have equivalent humoral and cellular responses to an unrelated mRNA vaccine. (Top lef t ) Anti-SARS-CoV-2 spike IgG in sera of vaccine responders and non-responders before (pre), after priming (post-prime) and after booster (post-boost early and late) doses of COVID-19 mRNA vaccine (Pfizer-BioNTech BNT162b2 or Moderna Spikevax). ( Top right ) Serial PBMCs pre- and post- COVID-19 mRNA vaccination were stimulated with SARS CoV-2 peptide pools. SARS CoV-2-specific IFNγ and/or TNFα production by all T cells was measured by flow cytometry. Composite data ( top right ) with representative gating ( bottom ) are shown. Flow cytometry pre-gated on CD3 + CD56− cells. Circle = mean, error bars = standard error of the mean; n = individual patients; data shown for all patients with available samples. P values by two-tailed Wilcoxon matched-pairs signed rank test. Article012345Tregs(%of CD4 5+) TreatmentP = NS Atezolizumab Autogene cevumeran priming dose sm FOLFIRINOX doses Surgery Atezolizumab Autogene cevumeran priming dose sm FOLFIRINOX doses SurgeryTreatmentMonocytes Eosinophils Neutrophil s Basophil sInnate immune cellsCells(%of CD45+) 020406080 Individual patientsResponders (n= 7) Non-responders (n= 6) Treatment Atezolizumab Autogene cevumeran priming dose s mFOLFIRINOX dosesSurgeryCD8+T cells NKT cell s B cellsAdaptive immune cells CD4+T cell sCells (% of CD45+) 020406080P = NS P = NS P = NS P = NS P = NSA B CResponder s(n= 7) Non-responders (n= 6)Regulatory T cell s Dendritic cell sCells (% of CD45 +) 020406080 051015P = NSNK cell s 012345 Responders (n= 7) Non-responders (n= 6) 020406080Cells (%of WB C) 051015 012345 Responders (n= 7) Non-responders (n= 6)P = NS P = NSP = NS P = NS Individual patient sCells (% of CD45+) 020406080 Extended Data Fig. 9 | Autogene cevumeran responders and non-responders have equivalent frequencies of circulating immune cells. ( A-C ) Longitudinal frequencies of regulatory T cells ( A), innate ( B) and adaptive ( C) immune cells in the peripheral blood of autogene cevumeran responders and non-responders during vaccination. Analyses in n = 13 patients with identical study-specified treatment schedules and thus eligible for direct comparison. Circle = mean, error bars = standard error of the mean; n = individual patients. P values by two-tailed Mann Whitney test. A B 00.51.01.52.0Entropy (nat), SResponder s (n = 8) Non-responder s (n = 8) True positive rate False positive rate00.20.40.60.81.0 0 0.2 0.4 0.6 0.8 1.0P = 0.01nreact = 22 ntotal = 79Neoantigen Quality AUC = 0.68Less clonal More clonal2.5 P = 0.03 C 0 500FAM amplitude (TP53 R175H ) HEX amplitude ( TP53 WT)01000 10002000300040005000 1500 2000 2500 3000 3500Liver lesion 010002000300040005000 Positive control 010002000300040005000 gDNA control Extended Data Fig. 10 | Tumour clonality and neoantigen quality in biomarker-evaluable patients, and mutated TP53 in a disappearing liver lesion. (A) Shannon entropy ( S) of tumour clones in autogene cevumeran (vaccine) responders and non-responders. ( B) Receiver operating curve indicating the ability of neoantigen quality as a continuous variable to classify vaccine neoantigens as an inducer or non-inducer of an IFNγ ELISpot response. Dotted line = all neoantigens included in individualized mRNA vaccines. (C) Digital droplet PCR showing number of wild-type (WT) TP53, or R175H mutated TP53 droplets in liver lesion from patient 29 (Fig. 4), with positive and negative (gDNA) controls. n = individual patients ( A) or neoantigens ( B). P values by two-tailed Mann-Whitney test. ArticleExtended Data Table 1 | Primary Safety Endpoint The number of patients with grade 3 or higher adverse events (AEs) due to atezolizumab and autogene cevumeran required to stop trial.Number of tr eated patien ts Number of pa tients with grad e 3 or higher AEsto stop tria l 3-10 ≥3 11-1 6≥ 4 17-2 0≥ 5 Extended Data Table 2 | Repertoire rescaling for CloneTrack # cells ∈T cell clone Rescaled # cells ∉ T cell clone Rescaled # cells in samp le Baseline sample /uni230A/2/uni230B− /uni230A/2/uni230B Compara tive sample − β ≥ γ α γ α