Data are n (%) unless noted. P values by two-tailed Mann-Whitney test for numerical variables and Fisher’s exact test for categorical variables. n=106 neoantigensA Non-responders (n = 8) Patient: n = 0 clones 02 08 0 4060 Weeks after surgery1000.0010.010.11101003 Total Mean n = 0 clones49 n = 0 clones18 n = 0 clones19 23 28 n = 0 clones n = 0 clones20 02 08 0 4060 1000 20 80 4060 1000 20 80 4060 10002 08 0 4060 10002 08 0 4060 10002 08 0 4060 10002 08 0 4060 100n = 0 clonesPercentage of al l blood T cell sn=230neoantigensNon-immunogenic ImmunogenicAll patients (n = 16) Responders (n = 8) 0204060 P = 0.7Non-synonymous mutations (no.) 051015Neoantigens in autogene cevumeran (no. ) 2025 Responder sN on-responder sP = 0.4 Responder sN on-responder sB C n=8 88 8 D Adjusted P for de novo expansio n11 0-610-210-4 Atezolizumab Autogene cevumeran mFOLFIRINOX Surgery0246810Immunogenic neoantigens (No.)Responders (n = 8) Non-responders (n = 8) Clones:76%24% 89%11% n = 3 clones 0.0001 Extended Data Fig. 2 | Immunogenic autogene cevumeran neoantigens. (A) Frequency of immunogenic autogene cevumeran (vaccine) neoantigens in all patients ( left ) and in vaccine responders ( right ). (B) Number of immunogenic vaccine neoantigens per patient. ( C) Number of non-synonymous mutations (left ) and vaccine neoantigens ( right ) in vaccine responders and non-responders. ( D) Vaccine-expanded T cell clones by CloneTrack in non-responders. n = neoantigens or patients as noted. P values by two-tailed Mann-Whitney test ( C) and modified two-tailed Fisher’s exact test ( D). ArticleB Autogene cevumeran responders (n = 8) Patient: n = 10 clones 02 08 0 4060 Weeks after surgery100Percentage of al l blood T cell s 0.0010.010.11101001 Total Mean n = 0 clones56 n = 8 clones10 n = 0 clones11 25 29 n = 0 clones n = 2 clones n = 2 clones Autogene cevumeran non-responders (n = 8)n = 0 clones14 02 08 0 4060 10002 08 0 4060 10002 08 0 4060 10002 08 0 4060 10002 08 0 4060 10002 08 0 4060 10002 08 0 4060 100 Patient: n = 0 clones 02 08 0 4060 Weeks after surgery1000.0010.010.11101003 Total Mean n = 4 clones49 n = 6 clones18 n = 8 clones19 23 28 n = 1 clone n = 0 clonesAdjusted P for atezolizumab expansio n1 10-610-210-4 n = 2 clones20 02 08 0 4060 10002 08 0 4060 10002 08 0 4060 10002 08 0 4060 10002 08 0 4060 10002 08 0 4060 10002 08 0 4060 100n = 0 clones Atezolizumab Autogene cevumeran mFOLFIRINOX SurgeryPercentage of al l blood T cell sA Autogene cevumeran non-responders (n = 8) Atezolizumab Autogene cevumeran mFOLFIRINOX SurgeryWeeks after surgeryPercentage of all blood T cellsAtezolizumab-specific T cell clonal expansion by CloneTrack Autogene cevumeran responders (n = 8) Atezolizumab responde r C Autogene cevumeran responders (n = 8) 79 22 Atezolizumab-expanded T cell clonesAutogene cevumeran - expanded T cell clonesAutogene cevumeran non-responders (n = 8) 21 3 Atezolizumab-expanded T cell clonesAutogene cevumeran- expanded T cell clonesClones : Clones :0.0001 0.0001* ** 02 08 0 40 60 1003* 4* 9 18* 19 20 23 28Patient * 02 08 0 40 60 1000.0010.010.1110100Patient 156 10 11 14 * 25 29 0.0001 Extended Data Fig. 3 | Autogene cevumeran–expanded T cell clones do not overlap with atezolizumab-expanded T cell clones. ( A) Cumulative percentage of atezolizumab-expanded T cells by CloneTrack in autogene cevumeran (vaccine) responders and non-responders. * = altered treatment schedules for patients 3, 4, 14 and 18. ( B) Atezolizumab-expanded T cell clones by CloneTrack in vaccine responders and non-responders. Red box = atezolizumab responder. Blue line = trajectory of an individual atezolizumab-expanded clone. Black line = geometric mean trajectory of all atezolizumab-expanded clones; error bars = geometric standard deviation. Red line = cumulative percentage of all atezolizumab-expanded clones. Black rectangle/triangle = time of surgery; blue rectangle/triangle = time of atezolizumab; green rectangle/triangle = times of autogene cevumeran (vaccine); yellow bars = mFOLFIRINOX cycles. ( C) Venn diagrams show overlap of vaccine-expanded clones (as identified in Fig. 2a) with atezolizumab-expanded clones in vaccine responders and non-responders. n = number of clones or individual patients as noted. P values by modified two- tailed Fisher’s exact test ( B). Extended Data Fig. 4 | See next page for caption. ArticleExtended Data Fig. 4 | Autogene cevumeran–expanded T cell clones contain immunodominant neoantigen-specific T cells. (A, B ) Autogene cevumeran (vaccine) induced IFNγ production by ex vivo IFNγ ELISpot (assay schematic, Fig. 1e) and clonal expansion by CloneTrack (assay schematic, B, top ). Briefly, for ELISpot, we analyzed each patient’s PBMCs for specific T cells against all individual vaccine neoantigens post-atezolizumab/pre-vaccine, 1-3 weeks post-vaccine priming doses, and 5-6 weeks post-mFOLFIRINOX. Each patient’s PBMCs were stimulated overnight with separate pools of overlapping peptides (15 amino acids long), each pool representing one of up to 20 neoantigens in vaccines, or with anti-CD3 antibody as a positive control, followed by measurement of IFNγ production by ELISpot. PBMCs incubated with media alone were used as a negative control. To track T cell clones, we identified vaccine-expanded T cell clones with CloneTrack. To identify if expanded clones contained immunodominant neoantigen-specific clones, in 4 of 8 responders, we stimulated vaccine-expanded PBMCs collected 1-3 weeks post-vaccine priming doses in vitro with computationally predicted minimal neopeptide pools (8-14 amino acids long) from 6 neoantigens that generated the highest per-patient magnitude response by ex vivo IFNγ ELISpot. We then purified CD8 + T cells that either expressed or did not express the degranulation marker CD107a, identified clones with greater proportion of CD107a+ versus CD107a- cells as in vitro neoepitope-activated clones, and examined overlap of in vitro neoepitope-activated to in vivo vaccine-expanded clones (Venn diagrams). For select patients, we further validated neoepitope-specificity by TCR cloning. (A ) Flow cytometry gating strategies. ( B) Ex vivo IFNγ ELISpot and T cell clonal expansion by CloneTrack in n = 3 of 4 patients tested (fourth patient in Fig. 2d). (B, left ) Normalized ex vivo IFNγ ELISpot spot count per 1 x 10 6 PBMCs for each immunogenic neoantigen in patients 10, 11 and 5. In patient 5, one neoantigen induced a high magnitude T cell response, while patients 10 and 11 had polytopic high magnitude responses against 8 and 3 vaccine neoantigens respectively. Black lines = individual neoantigens; coloured lines = neoantigen pools. ( B, middle ): T cell clonal expansion in patients 10, 11 and 5 by CloneTrack. Green line = trajectory of an individual expanded clone. Black line = geometric mean trajectory of all expanded clones; error bars = geometric standard deviation. Red line = cumulative percentage of all expanded clones. Black triangle = time of surgery; blue triangle = time of atezolizumab; green triangles = times of autogene cevumeran (vaccine); yellow bars = mFOLFIRINOX cycles. Dotted black line = detection threshold. (B, right ): In vitro neoantigen-specific activation. Flow cytometry = CD107a expression on CD8 + T cells stimulated with neopeptides or control (DMSO). Dot plots = number of CD107a+ versus CD107a- cells per T cell clone. Each circle = individual T cell clone. Green/blue squares = clones also detected by CloneTrack. Diagonal: CD107a+ clone frequency = CD107a- clone frequency. Venn diagram = vaccine-expanded and in vitro neoantigen-specific clonal overlap. For patient 11, immunodominant neoantigen-specific clones resided in a lower magnitude vaccine-expanded clonal pool. ( B, right, bottom ) TCR cloning, patient 10: 4-1BB expression on putative neoantigen-specific TCR-transduced CD8 + T cells cultured with HLA-matched, neopeptide-pulsed antigen presenting cells (HLA-transduced K562 cells). n = number of clones. P values by modified two-tailed Fisher’s exact test ( B, middle ), and by one-tailed binomial test with Bonferroni correction ( B, right ). Extended Data Fig. 5 | See next page for caption.