et al. BTN3A1 governs antitumor responses by coordinating αβ and γδ T cells. Science 369, 942–949 (2020). Acknowledgements We thank the patients who participated in the GT-30 trial and their families, the investigators, nurses, site staff and the GT-30 clinical trial team supporting the study. We would like to thank A. Winans (ctDNA sample processing) and D. Norton (ctDNA management) for their contributions. Author contributions M.Y., I.C., A.P.-P., J.P., D.B.W. and N.Y.S. designed the clinical study and developed the clinical protocol. M.Y., E.J.G., T.U.M., S.R., J.P., I.C., and A.P.-P. contributed to clinical oversight, patient recruitment and treatment and evaluated the clinical response and safety data. N.C., A.P.-P., S.R., J.P. and N.Y.S. contributed to vaccine design and production. R.P.-L., J.Y., N.C., D.B.W., A.P.-P. and N.Y.S. contributed to the experimental design, execution and analysis of the immune monitoring and biomarker analytical assays. D.H.S., L.T.K., E.J.F. and E.M.J. contributed to the single-cell analysis. G.B., J.N. and J.L. Nature Medicine Article https://doi.org/10.1038/s41591-024-02894-ycontributed to the ctDNA analysis. J.T.C. contributed to the statistical analysis. M.Y., R.P.-L., J.Y. and N.Y.S. drafted the manuscript. All authors reviewed the manuscript, provided edits and approved the final version. Competing interests M.Y. has received grant/research support (to Johns Hopkins) from Bristol-Myers Squibb, Incyte and Genentech; has received honoraria from Genentech, Exelixis, Eisai, AstraZeneca, Replimune and Hepion; and has equity in Adventris. T.U.M. currently serves, or at any point in the past has served, on advisory and/or data safety monitoring boards for Rockefeller University, Regeneron, AbbVie, Merck, Bristol-Meyers Squibb, Boehringer Ingelheim, Atara, AstraZeneca, Genentech, Celldex, Chimeric, DrenBio, Glenmark, Simcere, Surface, G1 Therapeutics, NGM Bio, DBV Technologies, Arcus, Fate, Ono, Larkspur, Avammune and Astellas and has research grants from the National Institutes of Health (National Cancer Institute), the Cancer Research Institute, Regeneron, Genentech, Bristol-Myers Squibb, Merck and Boehringer Ingelheim. E.J.G. has served as a member of scientific advisory boards for AbbVie, Abbott Diagnostics, Aligos, Arbutus, Arrowhead, Assembly, Avalia, ClearB Therapeutics, Dicerna, Enanta, Gilead Sciences, GlaxoSmithKline, Intellia, Janssen, Merck, Novartis, Genentech-Roche, Vaccitech, Ventorx, Vir Biotechnology and Virion Therapeutics. He is a speaker for AbbVie, Abbott Diagnostics, Gilead Sciences and Intellia. R.P.-L., J.Y., N.C., S.R., J.P., A.P.-P. and N.Y.S. are either current or previous Geneos employees. A.P.-P. and N.Y.S. are listed as inventors on several pending applications related to the vaccine platform presented in this study. D.H.S. and L.T.K. have no disclosures to report. E.J.F. is on the scientific advisory board of Viosera Therapeutics/Resistance Bio and is a consultant for Mestag Therapeutics and Merck. E.M.J. has received personal fees from Achilles, Dragonfly, the Parker Institute for Cancer Immunotherapy, the Cancer Prevention and Research Institute of Texas, Surge, HDT Bio, Neuvogen, NeoTx, Mestag and Medical Home Group. She has research grants from Lustgarten, Genentech, Bristol-Meyers Squibb and Break Through Cancer outside the submitted work. She receives other support from Abmeta and Adventris. G.B., J.L. and J.N. are full-time employees of Personalis, Inc. I.C. has a consultant role with Geneos; she has served as an advisor and/or a member of the data safety review board for Coherus, Exscientia AI, Riboscience and the University of Utah Therapeutic Accelerator Hub. J.T.C. is a paid statistical consultant for Geneos Therapeutics. D.B.W. has received grant funding, participates in industry collaborations, has received speaking honoraria and has received fees for consulting, including serving on scientific review committees. Remunerations received by D.B.W. include direct payments and equity/options. D.B.W. also discloses the following associations with commercial partners: Geneos (consultant/advisory board), AstraZeneca (advisory board, speaker), Inovio (board of directors, consultant), BBI/Sumitomo Dainippon (advisory board), Flagship (consultant), Pfizer (advisory board) and Advaccine (consultant). Additional information Extended data is available for this paper at https://doi.org/10.1038/s41591-024-02894-y . Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41591-024-02894-y . Correspondence and requests for materials should be addressed to Mark Yarchoan or Niranjan Y. Sardesai. Peer review information Nature Medicine thanks Lars Zender and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Ulrike Harjes, in collaboration with the Nature Medicine team. Reprints and permissions information is available at www.nature.com/reprints. Nature Medicine Article https://doi.org/10.1038/s41591-024-02894-y Extended Data Fig. 1 | Association of clinical response with survival. a. Probability of progression-free survival in patients with partial and complete response (n = 11) and patients with stable disease and progression (n = 23). b. Probability of overall survival in patients with partial and complete response (n = 11) and patients with stable disease and progression (n = 23). Significance was evaluated by the Log-rank (Mantel-Cox) test. Nature Medicine Article https://doi.org/10.1038/s41591-024-02894-y Extended Data Fig. 2 | Representative clinical responses. a, Tumor imaging scans of GT-30 patients #17, #7, and #18 categorized as CR, CR and PR, respectively at various treatment time-points. Red arrows point at the tumor location. b-d , Change in target lesion from baseline (%) in three patients (Pt) #17 (CR), Pt #7 (CR), and Pt #18 (PR) evaluated by RECIST 1.1. Nature Medicine Article https://doi.org/10.1038/s41591-024-02894-yExtended Data Fig. 3 | Clinical results across sex, etiologic disease subgroups, and TKI treatment. No apparent correlation of etiology (a-c), gender (male, n = 24; female, n = 10) (d ), and time on TKI at baseline (PD, n = 14; SD, n = 9; PR/CR, n = 11) (e ) with BOR. Note: 1 CR patient had a previous history of both HCV and HBV infection and is shown marked with an (*) in both (b) and (c). d, e, Data are presented as mean ± SEM. Significance was tested by a two-tailed, Mann-Whitney statistical analysis. Nature Medicine Article https://doi.org/10.1038/s41591-024-02894-y Extended Data Fig. 4 | Change in ctDNA levels from baseline is predictive of clinical outcome to vaccine (n = 13). a, Percent change in ctDNA at weeks 3 and 9 relative to baseline levels in patients with disease control (CR/PR/SD, n = 8) vs patients with progressive disease (PD, n = 5). Each circle indicates an individual patient per group. Data are presented as mean ± SEM. All P-values were evaluated by a two-tailed, Mann-Whitney test, and P-values below 0.05 were considered statistically significant. b , Percent change in ctDNA (molecular response) at Week 9 relative to baseline levels shown as best overall response evaluated by RECIST 1.1. c , Change in target lesion from baseline (%) evaluated by RECIST 1.1 in two patients who had a deeper molecular response at 95% and 100% reduction in ctDNA relative to their objective response. d , Probability of survival in patients with (n = 5) or without (n = 8) reduction in ctDNA at week 9. Significance was evaluated by the Log-rank (Mantel-Cox) test. Nature Medicine Article https://doi.org/10.1038/s41591-024-02894-yExtended Data Fig. 5 | Observed clinical responses are associated with number of neoantigens included in the PTCV , but not known biomarkers of pembrolizumab response. a, Responses stratified by tumor mutational burden (TMB). b , Biomarkers of the T-cell inflamed gene expression profile (GEP) are evaluated at pre-treatment between available non-responders (SD/PD; n = 23) and responders (CR/PR; n = 11) RNA sequencing data (tumor biopsy). c, Association between clinical responses and the number of neoantigens included in the PTCV. SD/PD (n = 23) and CR/PR (n = 11) subjects were evaluated. Graphs indicate mean ± standard error of the mean (SEM) of data from individual subjects (circles). Data are presented as mean ± SEM. Significance was evaluated by a two-tailed (a, c), one-tailed (b), Mann-Whitney statistical analysis. Nature Medicine Article https://doi.org/10.1038/s41591-024-02894-yExtended Data Fig. 6 | Change in T cell biomarker gene expression in the tumor upon PTCV treatment. T cell activation biomarkers are evaluated in pair-matched tumor biopsy samples from (a) responder (CR/PR; n = 9) and (b) non-responder (SD/PD; n = 15) patients treated with PTCV. Significance was tested by a two-tailed, Wilcoxon matched-pairs signed rank statistical analysis. Nature Medicine Article https://doi.org/10.1038/s41591-024-02894-yExtended Data Fig. 7 | Association of clinical response with survival. a, A two- tailed, Spearman correlation between positive epitopes versus the total number of neoantigens included in each patient’s PTCV. b , Interquartile survival analysis by magnitude (best response post-PTCV; IFNγ SFU) of evaluable patients. Six patients per group; Log-rank (Mantel-Cox) test. c , T cell reactivity evaluated by IFNγ-ELISpot stratified by responder or non-responder groups. Symbols represent individual patients (CR/PR, n = 6; SD/PD, n = 15 per group), the box extends from the 25th to the 75th percentile, the line inside the box is the median, and the whiskers extend from the minimum to maximum values. Significance was tested by a two-tailed, Mann-Whitney statistical analysis. d , Cumulative number of targetable neoantigens encoded in the vaccines of the 22 pts whose samples were evaluated by IFNγ ELISpot assay. The neoantigens are stratified by the predicted MHC Class I binding affinity as high (< 500 nM), medium (500–1000nM), and low (1000–2000 nM). While the majority of the neoantigens encoded in the PTCVs were categorized as high-affinity antigens, the medium and low-affinity antigens also yielded positive responses in a similar proportion. Nature Medicine Article https://doi.org/10.1038/s41591-024-02894-y Extended Data Fig. 8 | See next page for caption. Nature Medicine Article https://doi.org/10.1038/s41591-024-02894-yExtended Data Fig. 8 | Peripheral blood T cell responses to PTCV are primarily driven by clonal expansion of T cell effector memory cells clusters. a, Uniform Manifold Approximation and Projection (UMAP) of single-cell transcriptomes of 39,435 T cells from peripheral blood samples (n = 4) obtained at 12 weeks post-vaccination from 3 patients (#6, #7, and #8), colored by cluster. b , Barplot showing the number of cells occupying each cluster. c , Dotplot showing gene expression CD3E, CD3G, CD8A, CD8B, CCL5, IDO1, CD69, NKG7, LCK, IFNG, CXCR6, CD27, PDCD1, and TIGIT. Size of the dot indicates the percent expression among cells assigned to each cluster and the color represents the average expression across all cells within the cluster. d , Heatmaps showing scaled expression of the top 5 marker genes identified for each subset. e , UMAP indicating areas of clonal expansion within the 31,842 cells identified as having a TCR by paired single-cell TCR-sequencing. Colors indicate the degree of clonal expansion of each TCR clonotype. f , UMAPs indicating single cells with a TCRβ identified as clonally expanded after vaccination by bulk TCRβ sequencing of pre- and post-treatment peripheral blood, by patient. g , Transcriptional phenotype of cells with a TCRβ identified as clonally expanded by bulk TCR sequencing of pre- and post-treatment peripheral blood, represented as a percentage of total number of single cells per patient. Nature Medicine Article https://doi.org/10.1038/s41591-024-02894-y Extended Data Fig.