8f). Of 92 TCRβ sequences found to be clonally expanded by bulk sequencing, 64 sequences were identified in 1,041 cells within the single-cell dataset, of which 84.4% (879 of 1,041) were from patient 8, 10.8% (112 of 1,041) were from patient 7 and 4.8% (50 of 1,041) were from patient 6. The single-cell cluster most strongly associ - ated with vaccine expansion by Fisher’s exact test was CD8+ TEM (odds 0 PretreatmentPosttreatment5001,0001,5002,0005,000100 75 50 25 0 Pre-PTCVPost-PTCVPre-PTCVPost-PTCVPre-PTCVPost-PTCV10,000IFNγ (SFU per 106 cells)P < 0.0001 Positive epitopes (%)CR/PR SDPDP = 0.892 P = 0.269 P = 0.031 P = 0.187 P = 0.008 Patient ID (n = 14)No. of T cell clones after vaccination (new & expanded)Detected in PBMC Found in tumorCumulative frequency in tumor No. of clones in tumor TCR clonality TCR clonalityP = 0.0171P = 0.0079P = 0.035P = 0.216 TCR repertoire richness (unique V(D)J rearrangements)TCR repertoire richness (unique V(D)J rearrangements)0 10 20 30 40150 100 50 0 4 814 321 16 15 14 5 13 1 10 23 12 6 4 2 9 19 18 22 11 8 7203 Neoantigens in GNOS-PV02Patient ID (n = 22)Positive neoantigens (pretreatment) Positive neoantigens (posttreatment)Total neoantigensin GNOS-PV02 0246 1.0 0.8 0.6 0.4 0.2 0 1.0 0.8 0.6 0.4 0.2 0 UnstimulatedGNOS-PV02UnstimulatedGNOS-PV02UnstimulatedGNOS-PV020.8 0.6 0.4 0.2 0 8 6 4 2 0% CD4+CD69+ CD107A+ % CD8+CD69+ CD107A+ % CD8+Ki67+ % CD8+CD69+CD107A+ GZMA+PRF1+% CD4+Ki67+ % CD8+Ki67+ GZMA+PRF1+ 56711121517192022 0.3 600.5 0.6 120400 300 200 100 0100 80 600.4 0.2 00.4 0.3 0.2 0.1 040 20 00.2 0.1 0 PretreatmentPosttreatmentPretreatmentPosttreatmentPretreatmentPosttreatmentPretreatmentPosttreatmentPretreatmentPosttreatmentPretreatmentPosttreatmentCR/PR SD PD0246DMSOCD8+ (BUV805) CD4+ (BUV395)6.68%105 105104 104103 103–1030 0 1051041030 1.81% 1.68% 0.84% 0.57% 6.52% 1.31% 11.6%1.73% 0.91%1.46%2.33%6.41% 14.3% 5.13% 3.80% 1.86%4.37%5.76%14.3%GNOS-PV02 DMSO GNOS-PV02 CD69+ (BV421) Ki67+ (AF700) CD107a+ (APC) IFNγ+ (BV786) TNF+ (PE/Cy7)a bc d e f g h i j k105 105 105 105105 105105105105 105106104 104 104 104104104104104104104 104 104 104 104104104 104104104104 104103 103 103 103103 103103103103 103–103 –103 –103 –103–103–103–103–103–103–103–103 –103–103–103–103–1030 0 0 00 0 000 0 1051041030105106104103–1030 105105105105105105 106106 105 105 1041031031031031031031030 103 103103 103103 00000 00 0 0 0 0 1051041030104104 105103103 00 Nature Medicine | Volume 30 | April 2024 | 1044–1053 1050 Article https://doi.org/10.1038/s41591-024-02894-yratio, 107.40; 95% CI, 88.75 to 130.91; P < 0.001), which comprised 87.3% (909 of 1,041) of vaccine-expanded TCRβ (Extended Data Fig. 8g and Supplementary Fig. 4a). On a per-patient basis, CD8+ TEM was also the most highly represented cluster, being found in 88% (775 of 879), 88% (99 of 112) and 70% (35 of 50) of vaccine-expanded TCRβ for patients 8, 7 and 6, respectively. We then subdivided the CD8+ TEM cluster into subclusters and identified seven subclusters, among which three (CD8+ TEM_3, CD8+ Fig. 4 | Postvaccination expanded TCR clones identified in the tumor are reactive to PTCV-encoded antigens. a, Most frequent TCRs identified by TCRseq and RNAseq in a patient (before vaccination versus week 9 after vaccination, pairwise scatterplots). Different superscript letters show selected high-frequency new T cell clones detected in PBMCs after vaccination and their abundance in the tumor. Orange, green, and gray circles represent expanded, contracted and not significantly changed T cell clones, respectively. b , CDR3 sequences of the three TCRs (from patient 8; TCR 1, TCR 2 and TCR 3) selected for cloning and their frequency (freq.) in the tumor before (pre-Vax) and after (post-Vax) vaccination. Selected cloned TCRs were present in high frequency only in the peripheral blood and tracked into the tumor after treatment. c, UMAP (Uniform Manifold Approximation and Projection) and stacked barplot indicating the single-cell cluster identities and number of cells for each of the three TCRs selected for cloning. d , Patient-specific clonal TCR sequences were gene optimized and inserted into the pMXs-IRES-GFP retroviral plasmid vector containing the viral packaging signal, transcriptional and processing elements, and the GFP reporter gene. MuLV, murine leukemia virus; Mo-MuLV, Moloney MuLV; L TR, long terminal repeat; Amp R, ampicillin resistance. e , TCR-engineered T cells (GFP+) from unvaccinated patient-derived PBMCs were stimulated for 6 h with epitope pools or the nonspecific epitope CTA1 (10 µg ml−1), and CD69 expression was evaluated by flow cytometry. Peptide pool 1 included the most reactive epitopes measured by ELISpot, whereas pool 2 (consisting of peptides corresponding to epitopes 21–40) served as an internal negative control. 1.41% 1.30% 1.16%5.19% 4.84% 7.44%22.7% 19.7% 36.0%5.56% 5.01% 8.08%TCR 1 TCR 2 TCR 3 CD4+ (BUV395) CD69+ (BV421) MuLV 5’ LTRpsi+ (packaging signal)Mo-MuLV (pol region and splice acceptor)AmpRIRES GFPMuLV 3’ LTRTCR insertConstruct IDCDR3 sequenceTumor freq. pre-VaxTumor freq. post-Vax aTCR 1 CASSAIGTPSGEQFF 0 0.0023 bTCR 2 CATTSGSPAGELFF 0 0.0061 cTCR 3 CAGRLAGASGELFF 0 0.0002 CD4+ proliferating CD8+ TEM CD8+ proliferating200 150 100 50 0No. of single cellsUnselected TCR 3TCR 2TCR 1 No. of single cells CD4+ proliferatingCD8+ TEMCD8+ proliferating TCR 1 1 161 29 TCR 2 0 23 1 TCR 3 0 10 0PBMC TumorPosttreatment TCRβ sum (clone frequency) Pretreatment TCRβ sum (clone frequency)10–2 10–3 10–4 10–5 10–510–510–410–310–2 10–410–310–210–410–310–2ab ca b c Unstimulated CTA1 Pool 2 Pool 1 4.64% 4.67% 4.02%12.2% 12.1% 14.3%21.4% 23.2% 28.2%12.4% 12.3% 14.9%TCR 1 TCR 2 TCR 3 CD8+ (BUV805) CD69+ (BV421)0–103103104105 0 1041050 1041050 1041050 104105 TCR 3 TCR 2 TCR 1a b c de –103103104105 0–103103104105 0 –103103104105 0 –103103104105 0 –103103104105 0 0 1041050 1041050 1041050 104105 Nature Medicine | Volume 30 | April 2024 | 1044–1053 1051 Article https://doi.org/10.1038/s41591-024-02894-yTEM_5 and CD8+ TEM_6) displayed high expression of multiple genes associated with cytotoxicity, including GZMB and NKG7, and three (CD8+ TEM_1, CD8+ TEM_2 and CD8+ TEM_4) had increased expression of GZMK (a preexhaustion marker)37 (Extended Data Fig. 9). Cytotoxic sub - clusters accounted for 85% (776 of 901) of vaccine-expanded CD8+ TEM, with CD8+ TEM_5 and CD8+ TEM_3, which comprised 47.2% (429 of 909) and 37.1% (337 of 909) of CD8+ TEM, respectively, being the two largest clusters represented. In contrast, the GZMK-expressing preexhausted subclusters CD8+ TEM_2, CD8+ TEM_1 and CD8+ TEM_4 were less numerous (Supplementary Fig. 4b). Expanded TCR clones are reactive to PTCV-encoded antigens Lastly, we sought to validate the neoantigen-specific activity of tumor-infiltrating T cells in two representative patients. The first patient had 42 significantly expanded clones in the periphery, of which 27 were found in the tumor sample after treatment (Figs. 3g and 4a). Three of the most frequent TCR sequences (Fig. 4b ) from T cell clones newly present in the tumor after vaccination were found primarily in the CD8+ TEM cluster in postvaccination peripheral blood single-cell sequencing (Fig. 4c). These three TCR sequences were selected and cloned into the pMXs-IRES (internal ribosome entry site)-GFP (green fluorescent pro - tein) retroviral plasmid vector for further studies (Fig. 4d). To character - ize the neoantigen-specific cellular response driven by treatment with GNOS-PV02, we stimulated TCR-engineered T cells from patient-derived PBMCs with the patient’s PTCV-specific neoantigen pools. We found T cell activation (CD69+) associated with pool 1 (consisting of peptides corresponding to neoantigens 1–20), which included the most reac - tive epitopes measured by ELISpot; pool 2 (consisting of peptides cor- responding to neoantigens 21–40) served as an internal control for specificity and showed similar levels as the nonspecific epitope (CTA1) control (Fig. 4e). In the second representative patient, we were able to map the new T cells/TCRs to a specific vaccine-encoded epitope. From IFNγ ELISpot analysis, we first identified a strongly immunogenic epitope (ATP1A1-ALB) encoded in the patient’s personalized vaccine (Supplementary Fig. 5a). Patient-derived PBMCs were subjected to in vitro stimulation for T cell enrichment and expansion and then stimu - lated with ATP1A1-ALB peptides. We found both CD4+ and CD8+ T cells with specific polyfunctional responses (CD69+, Ki67+, CD137+, IFNγ+, IL-2+) against ATP1A1-ALB (Supplementary Fig. 5b). High-frequency TCRs were identified by TCRseq/RNAseq (33 clones expanded in the periphery, of which 15 were found in the tumor) and engineered (Sup - plementary Fig. 5c,d). Engineered TCRs were stimulated with a pool of epitopes containing all the neoantigens in the patient’s PTCV. Similar to the first patient, we observed CD4+ and CD8+ T cell specificity against the patient-specific neoantigens (Supplementary Fig. 5e) relative to the unstimulated or nonspecific peptide (CTA1)-stimulated controls. These data validate the postvaccination infiltration and increase in the frequency of T cells in the tumor with specificity to vaccine-encoded neoantigens. Vaccine-induced immune editing leads to tumor escape Tumor immune editing and escape are key mechanisms of cancer pro - gression and metastatic dissemination. We used paired tumor biopsy samples to investigate the mechanisms of tumor escape in a patient with nondurable PR (patient 11) treated with a PTCV encoding 29 neo - antigens plus pembrolizumab. At the first restaging interval at week 9, the patient had a target liver lesion reduction of −36%. By week 18, the response in the target liver lesion intensified, eventually reaching a −59% reduction, but a new lesion was observed in the adrenal gland (Extended Data Fig. 10a). TCR/tumor-infiltrating lymphocyte (TIL) analysis of week 9 versus screening biopsy samples revealed the clonal expansion and infiltra- tion frequencies of 25 T cell clones after vaccination (Extended Data Fig. 10b). IFNγ ELISpot analysis detected T cell responses to 9 of 29 epitopes and strong responses to 4 of 29 vaccine epitopes (Extended Data Fig. 10c), none of which were recognizable as driver mutations. Flow cytometry analysis showed a high frequency of antigen-specific, activated (CD69+, Ki67+) CD8+ and CD4+ T cells (Extended Data Fig. 10d). Sequencing of the new adrenal lesion identified 25 neoantigens, includ - ing 16 shared with the primary liver lesion (Extended Data Fig. 10e). However, all four vaccine neoantigens with the strongest ELISpot responses were absent in the adrenal lesion, consistent with neoanti- gen loss resulting from immune editing and clonal escape. The ctDNA analysis results were consistent with CR of the liver-specific tumor clones by week 21, persisting through week 57, but showed an increasing level of the adrenal-specific ctDNA over time (Extended Data Fig. 10f). Tumor tissue biomarker analysis of week 0 versus week 9 liver tumor samples showed robust CD8+ T cell infiltration, whereas the adrenal lesion had low CD8+ T cell density resembling that of the pretreatment liver tumor (Extended Data Fig. 10g). These data are consistent with tumor immune escape in the setting of tumor heterogeneity and the loss of passenger neoantigens targeted by the PTCV, which supports the proposed mechanism of action of PTCV-mediated antitumor immunity. Discussion HCC is characterized by a modest response to anti-PD-1 monotherapy. New personalized immunotherapies that induce tumor-specific T cell responses may sensitize tumors to ICI therapy11,12,14 ,38–40. We show that treatment with a PTCV containing up to 40 tumor neoantigens in com- bination with pembrolizumab is feasible and associated with clinical responses in a subset of patients with advanced HCC. Although the single-arm design and small sample size of the current study limit the ability to attribute clinical efficacy to the PTCV definitively, the observed response rate (30.6%) is higher than that in historical clinical trials of anti-PD-1 monotherapy in HCC (12–18%)4–10. Immunological analysis confirmed both the induction of new T cell responses to vaccine-encoded antigens and an expansion of the TCR repertoire in both the peripheral blood and tumor. Anti-PD-1 mono - therapy can reverse T cell dysfunction in existing neoantigen-specific T cell clones but is not known to induce new neoantigen-specific T cell clones32,33. Therefore, the present study provides evidence that a PTCV can enhance responses to anti-PD-1 therapy through the induction of neoantigen-specific T cells in the peripheral blood and tumor. Single-cell sequencing analysis showed that the PTCV-expanded T cells clustered predominantly with the CD8+ TEM populations, followed by CD8+ proliferating populations, and displayed high expression of mark - ers consistent with a cytotoxic (GZMB, NKG7) phenotype and, less fre - quently, with a preexhausted (GZMK) phenotype. These immunologic data support a PTCV’s contribution to anti-PD-1 efficacy. The present trial builds upon recent clinical updates with neoantigen-specific cancer vaccines across a variety of tumor types and vaccine platforms. One notable difference between the vaccine plat- form used in the present study and other neoantigen vaccine platforms is that other vaccine platforms have generally targeted a more limited selection of neoantigens. Our vaccination approach aimed to include all targetable neoantigens in each patient’s vaccine—drivers and passen- gers, truncal and branch, shared and private, and with a broader range of predicted binding affinity to major histocompatibility complex mol - ecules. We reasoned that vaccines that encode for a larger repertoire of tumor-derived neoantigens may lead to the priming of a broader set of immune responses, increasing the likelihood of effective tumor control. We observed that an increased number of epitopes encoded in the PTCV correlated with an increased number of reactive epitopes after vaccination. We detected de novo responses not only to predicted high-affinity epitopes but also to medium- and low-affinity epitopes at similar rates. Collectively, these data are consistent with preclinical findings18,24 that contemporary vaccines can prime immune responses to a broader range of neoantigens than previously envisioned. A recent study of an mRNA-based neoantigen vaccine (autogene cevumeran) in the adjuvant treatment of pancreatic ductal Nature Medicine | Volume 30 | April 2024 | 1044–1053 1052 Article https://doi.org/10.1038/s41591-024-02894-yadenocarcinoma showed that patients with a vaccine response (50% of all patients) achieved improved recurrence-free survival compared to those who did not mount a T cell response to the vaccine neoanti - gens41. We also observed a trend toward longer OS among patients who had a stronger vaccine-driven response relative to those who had a weaker response. However, in our study, 90.1% of the evaluated patients (20 of 22) had neoantigen-specific T cells detected by IFNγ ELISpot assays, limiting comparisons to only n = 2 patients who did not have such responses. Our on-treatment PBMC and tumor tissue biomarker data showed that patients with a clinical response of CR or PR demonstrated increased levels of T cell activity markers relative to baseline. Thus, other factors such as the tumor microenvironment and immune fitness may have a confounding role in ultimately driving the clinical response42. Our analysis of a single patient with early acquired resistance to therapy in the GT-30 study provides initial support for neoantigen loss and tumor heterogeneity as important barriers to PTCV therapy. We envision that, in the future, longitudinal sampling of tumor DNA from multiple lesions coupled with rapidly synthesized therapeutic vaccines with evolved neoantigen panels can be used to address tumor heterogeneity and control newly unresponsive lesions. The major limitations of the present study include the small sample size and the single-arm study design. Although the combination of a PTCV and pembrolizumab met the prespecified endpoint for clinical response rate as compared to a historical control of pembrolizumab monotherapy, we cannot exclude the possibility that study population differences affected the response rates observed in the present study. Prospective, randomized clinical trials are needed to confirm that the PTCV can improve clinical outcomes as compared to pembrolizumab alone. An additional limitation is that this study was conducted in the setting of a rapidly evolving HCC clinical landscape, limiting the clinical applicability of these findings. Since the initiation of the present study, multiple ICI combinations have demonstrated improved survival ben- efits versus mTKI therapy in patients with advanced HCC in the first-line setting (including bevacizumab plus atezolizumab43, durvalumab plus tremelimumab27, and camrelizumab plus rivoceranib44), with similar response rates as were observed in the present study. Additionally, new triplet regimens are under investigation and may prove superior to cur - rent therapeutic options. However, many patients are not candidates for bevacizumab-based combinations, and a particular challenge with anti-vascular endothelial growth factor-sparing combinations in HCC is the potential for irAEs. For example, the combination of ipilimumab (an anti-CTL-associated antigen 4 antibody) and nivolumab (an anti-PD-1 antibody) is approved with a 32% response rate in second-line HCC treat - ment45, which is similar to what was observed in the present study; how - ever, more than half of the patients experienced serious irAEs. Therefore, there is a particular need for new immunotherapies that can enhance anti-PD-1 responses without increasing the risk of irAEs. Except for an increase in injection-site reactions, the overall safety profile of the PTCV plus anti-PD-1 combination was similar to that of pembrolizumab alone despite driving tumor-directed TILs and clinical responses. Therefore, a PTCV could eventually be integrated into the current armamentarium of effective systemic therapies for HCC. In summary, our data indicate that the development of PTCVs is feasible and can induce clinical responses in combination with anti-PD-1 therapy in advanced HCC. Online content Any methods, additional references, Nature Portfolio reporting sum- maries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contri - butions and competing interests; and statements of data and code avail - ability are available at https://doi.org/10.1038/s41591-024-02894-y . References 1. Llovet, J. M. et al. Hepatocellular carcinoma. Nat. Rev. Dis. Primers 7, 6 (2021).2. Cancer Facts & Figures 2022 www.cancer.org/content/dam/ cancer-org/research/cancer-facts-and-statistics/annual-cancer- facts-and-figures/2022/2022-cancer-facts-and-figures.pdf (American Cancer Society, 2022). 3.