Example responses are shown in Extended Data Fig. 2a–d. Clinical results were generally consistent across sex, etiologic disease subgroups and time on first-line TKI treat - ment at baseline (Extended Data Fig. 3). Anti-PD-1 agents have been studied extensively in both the first-line and second-line advanced HCC settings. Across registrational clinical trials of pembrolizumab, nivolumab, durvalumab and tislelizumab, the ORR ranged from 12% to 18% (refs. 4 –10,27). For the protocol develop - ment, sample size estimation and prespecified statistical hypothesis testing, we used the comparator pembrolizumab ORR of 16.9% based on KN-240 (ref. 28), which was consistent with the 17.0% ORR for pem-brolizumab monotherapy observed in the KN-224 phase 2 study 9. The observed ORR of 30.6% (11 of 36 patients) achieved statistical sig - nificance with a one-sided P value of 0.031 (one-sided 90% confidence interval (CI), 20.4–100%) versus the prespecified historical control. We performed circulating tumor DNA (ctDNA) analyses using day 0 (baseline), week 3 and week 9 on-treatment samples from 13 patients. A molecular response was defined as a >50% reduction in the ctDNA level from baseline22, 29. Changes in ctDNA levels broadly tracked with magnetic resonance imaging scans in monitoring objec- tive responses (CR and PR). As shown in Extended Data Fig. 4a, the difference in the percentage change in ctDNA levels between patients with disease control (CR, PR or SD) and those with PD reached signifi- cance at week 9 (P = 0.006). The ctDNA analysis detected a stronger molecular response relative to imaging in two patients (Extended Data Fig. 4b,c). Patient 9 (determined to have SD by imaging) had a 95% reduction in ctDNA level, and patient 19 (determined to have PR by imaging) had a 100% reduction in ctDNA level. Both patients had durable responses lasting for more than 12 months. A ctDNA decrease –100–50050100 –73 –100–100–100–69–62–57–45–44–44–34–20–19–16–15–9–3–2015561116242528383839485783Best change in target lesion from baseline (%) aBest overall response by RECIST 1.1 CR/PR SD PDORR (mITT) = 30.6% CR PR SD PD8.3% 22.2% 25.0%38.9% Unevaluable 5.6% n = 36b c d –100–50050100150200 Time on treatment (weeks)Change in target lesion from baseline (%)Dosing (weeks)Tumor imagingBaseline then Q9w until week 54; thereafter, Q12w 0369121518212427… Y1 Y2 Pembrolizumab GNOS-PV02 + pIL12 (1) (2)Y3 Y4 Y5 …Q3w ...Q9w ….Q12w 1531441351261171081827364554637281909990a SequencingImmunization M anufacturing PTCV plasmid Neoantigen IDTumor biopsy 1 2 3 456a Fig. 2 | Clinical response. a, Manufacturing process for GNOS-PV02 and clinical trial design. In patients without disease progression, (1) treatment with pembrolizumab may continue every 3 weeks (Q3w) for 2 years per label recommendation; (2) treatment with GNOS-PV02 + pIL12 may continue Q3w for four doses, followed by Q9w until year 2 (Y2) and Q12w beyond 2 years. b , Pie chart with the percentage ORR, CR, PR, SD and PD according to RECIST 1.1 (n = 36, mITT). c, Waterfall plot showing the best overall response achieved by the 34 evaluable patients of the GT-30 trial at the time of data cutoff (August 18, 2023). aPR patient with a primary liver lesion and two lung metastases who achieved secondary resectability owing to tumor shrinkage and remained tumor-free for 18.2 months after the first treatment dose. d , Spider plot showing changes in the target lesion from baseline for the 34 evaluable patients of the GT-30 trial at the time of data cutoff (August 18, 2023). aThe same as in c . Nature Medicine | Volume 30 | April 2024 | 1044–1053 1047 Article https://doi.org/10.1038/s41591-024-02894-yat week 9 was significantly associated with longer survival (P = 0.01) (Extended Data Fig. 4d). Biomarker analysis of the observed clinical responses We next evaluated potential biomarkers of the observed clinical responses. All patients enrolled in GT-30 had a low TMB (fewer than five mutations per megabase), with a median TMB of 2.0 mutations per megabase. The TMB was similar in patients achieving CR/PR and those with SD/PD (Extended Data Fig. 5a). Similarly, while we observed a numerically higher ORR (35% versus 25%) in patients with baseline α-fetoprotein (AFP) levels of <400 ng ml−1 (n = 26) relative to patients with baseline AFP levels of >400 ng ml−1 (n = 8), there was no signifi - cant difference in disease control rate (58% versus 63%), mPFS (4.1 versus 6.2 months) or mOS (24.4 versus 15.6 months) (Table 1 and Supplementary Fig. 1). We next evaluated the relationship between baseline CD8 infiltration (assessed by the mRNA expression level of CD8A), pretreatment tumor CD274 (PD-1 ligand 1) and KDR mRNA expression levels, and the T cell-inflamed gene expression profile of 15 biomarkers and the response to treatment (Extended Data Fig. 5b). These biomarkers broadly characterize an immune-inflamed phe - notype and have previously shown potential utility in distinguish - ing responders from nonresponders to anti-PD-1-based therapies in HCC30,31. However, we did not observe any relationship between these pretreatment markers and the response to the PTCV plus anti-PD-1 therapy. In contrast to the lack of differentiation based on pretreatment tumor biomarkers, an exploratory post hoc analysis demonstrated a positive correlation ( P = 0.025) between the number of neoantigens included in the PTCV and the clinical response achieved. Sequencing of the patients’ tumors identified a median of 30 vaccine-targetable neo - antigens (range 4–67). Among patients receiving a vaccine encoding ≥30 neoantigens, 7 of 17 (41.2%) had an objective response. Conversely, among those receiving a vaccine encoding <30 neoantigens, only 4 of 17 patients (23.5%) had an objective response. There was a significant difference in the number of targeted neoantigens between the CR/PR group and the SD/PD group (Extended Data Fig. 5c). We next evaluated pretreatment versus on-treatment (week 9) tumor biopsy samples to determine whether changes in the expression levels of biomarkers associated with T cell activation and infiltration, consistent with a PTCV-mediated effect, could explain the observed clinical response in responders (CR/PR, n = 9) and nonresponders (SD/PD, n = 13). The expression of the T cell biomarkers CD8A, CD8B, CCL5, CXCR6, LCK and TIGIT was significantly increased in responders but not in nonresponders (Extended Data Fig. 6a,b). Although these analyses were exploratory, the results are broadly consistent with the proposed mechanism of action for PTCV-induced clinical responses. In contrast to anti-PD-1 monotherapy, which reinvigorates preexist - ing antitumor immunity, therapeutic cancer vaccines can prime new antitumor immune responses, providing a potential rationale for why inflamed and noninflamed tumors responded similarly to the therapy. Furthermore, the relationship between the number of neoantigens included in the PTCV and the clinical response achieved suggests that features of tumor antigenicity or of the vaccine itself drive clinical benefit with PTCV plus anti-PD-1 therapy. Vaccination elicits neoantigen-specific responses In our study, predicted neoepitopes were selected for inclusion in the PTCVs using a pipeline for called variants based on an in silico analysis of the exome and transcriptome sequencing data from each patient (Methods). While the tumor RNA-sequencing (RNAseq) data confirmed that the nonsynonymous somatic variants included in the PTCVs were expressed, we did not experimentally confirm the processing and pres - entation of the predicted epitopes in the tumor. Twenty-two patients with available peripheral blood mononuclear cell (PBMC) samples were evaluated for the presence of vaccine-induced neoantigen-specific responses before and after treatment using the ex vivo interferon-γ (IFNγ) enzyme-linked immunosorbent spot (ELISpot) assay. The criteria for ELISpot positivity are described in Methods. In almost all patients, treatment was associated with an increase in the magnitude of cumulative PTCV neoantigen-specific T cell responses (P < 0.0001) (Fig. 3a).Table 2 | Overall summary of GT-30 TRAEs TRAEsa (n = 27/36) Grade 1/2 Injection-site reactions Pain, erythema, pruritus or swelling 15 (41.6%) Gastrointestinal Gastroesophageal reflux 2 (5.6%) Diarrhea 1 (2.8%) Abdominal pain (upper) 1 (2.8%) Nausea 1 (2.8%) Dry mouth 1 (2.8%) Skin Rash 4 (11.1%) Pruritus 2 (5.6%) Dermatitis 1 (2.8%) Alopecia 1 (2.8%) Skin disorder 1 (2.8%) Dry skin 1 (2.8%) Endocrine Hypothyroidism 2 (5.6%) Musculoskeletal Pain in extremities, musculoskeletal pain or stiffness 3 (8.3%) Arthralgia 1 (2.8%) Metabolism Decreased appetite 2 (5.6%) Hypophosphatemia 1 (2.8%) Polydipsia 1 (2.8%) General Fatigue 4 (11.1%) Pyrexia 1 (2.8%) Chills 1 (2.8%) Anemia 1 (2.8%) Dysesthesia 1 (2.8%) Lethargy 1 (2.8%) Somnolence 1 (2.8%) Infusion-related reaction 1 (2.8%) Chest discomfort 1 (2.8%) Hepatitis 1 (2.8%) Elevated liver function test results 1 (2.8%) Immune-related Immune-mediated nephritis 1 (2.8%) Pneumonitis 1 (2.8%) ICI hepatitis 1 (2.8%) Use of systemic steroids 7 (19.4%) aTRAEs were determined by the investigator for those events deemed as possibly, probably or definitely related to the PTCV, IL-12, electroporation and/or ICI. Nature Medicine | Volume 30 | April 2024 | 1044–1053 1048 Article https://doi.org/10.1038/s41591-024-02894-yPTCV treatment was also associated with an increase in the num- ber of encoded neoantigens eliciting an immune response. In 19 of 22 patients (86.4%), the number of vaccine-encoded neoantigens with T cell reactivity was higher after than before treatment (Fig. 3b ). Two patients with PD, treated with PTCVs encoding 4 and 11 neoepitopes, did not yield any detectable ELISpot responses either before or after treatment; one patient with SD (20 neoantigens) had a reduced number of reactive epitopes detected after treatment relative to their pretreat - ment baseline. Individual epitope analyses across the cohort revealed PTCV encoded neoantigen-specific T cell responses to a median of 64.0% (range 0–100.0%) epitopes after treatment compared to 11.8% (range 0–85.3%) epitopes before treatment. PTCV immunization resulted in a significant increase in positive epitopes in both clinically responding and nonresponding patients (Fig. 3c ). A positive correlation was observed between the total number of neoantigens included in the PTCV and the number of positive neo - antigen responses detected by ELISpot assays (P = 0.0007, Spearman correlation coefficient) (Extended Data Fig. 7a). We evaluated the magnitude of IFNγ response by quartiles and observed that patients in the top quartile had a trend toward longer OS compared to patients in the bottom quartile (mOS 30.2 versus 15.7 months) (Extended Data Fig. 7b). Patients with CR or PR showed a trend toward a greater mag - nitude of IFNγ response (Extended Data Fig. 7c). Immune responses were observed against neoepitopes with predicted high binding affinity (kd <500 nM), as well as against those with predicted medium or low binding affinity (kd 500–2,000 nM), to human leukocyte antigen (HLA) class I molecules (Extended Data Fig. 7d). Neoantigen-specific responses were confirmed in a subset of four responding patients (one with CR, three with PR) through intracel - lular staining of PBMCs stimulated with patient-specific neoepitope pools in vitro. Upon neoantigen stimulation, both CD4+ and CD8+ populations presented an increased activation profile as determined by the individual expression of the CD69, Ki67, CD107a, IFNγ and tumor necrosis factor (TNF) markers (Fig. 3d). Boolean gating confirmed an increasing trend of active (CD69 +CD107a+) (Fig. 3e) and proliferative (Ki67+) (Fig. 3f) polyfunctional CD4+ and CD8+ T cells with cytolytic capabilities (GZMA+PRF1+) after stimulation. Taken together, these data indicate that vaccination is capable of eliciting polyfunctional neoantigen-specific CD4+ and CD8+ responses with cytolytic potential. We next characterized T cell clonal expansion, trafficking, neoantigen specificity, and clonal and subclonal genetic profiles as exploratory endpoints. Vaccination enriches T cell clone expansion and infiltration Complementarity-determining region 3 (CDR3) regions of the T cell receptor β-chain (TCRβ) were sequenced from paired pretreatment and posttreatment (weeks 9–12) PBMC and tumor biopsy samples in 14 patients with available paired tumor biopsy samples. Although anti-PD-1 therapy is not known to modulate the diversity of tumor-reactive T cell clones32,33, we hypothesized that the addition of the PTCV to anti-PD-1 therapy would lead to both an increase in abundance and a broadening of the circulating HCC-reactive T cell clonal repertoire, which would subsequently traffic to the tumor microenvironment. Consistent with this hypothesis, we observed significant T cell clonal expansion in 14 of 14 (100%) patients in both the peripheral blood and tumor using a differential abundance statistical framework (Fig. 3g). The median number of new or expanded T cell clones in the periphery was 47 (range 24–132), of which a median of 21 (range 6–71) T cell clones were also new or expanded in the posttreatment tumor. The median increase in the cumulative frequencies of the significantly expanded clones was 1.94% (range 0.35–8.70%) (Supplementary Fig. 2a,b). We identi - fied an increase in both the abundance and number of expanded T cell clones within the tumor after treatment, which was also identified in the peripheral blood after treatment (Fig. 3h,i). Importantly, we observed higher frequencies and numbers of T cell clones newly present in the tumor after vaccination (Supplementary Fig. 3a,b). Additionally, we found significantly increased TCR clonality (P = 0.035) (Fig. 3j) but no significant change in TCR repertoire richness in the tumor (P = 0.216) (Fig. 3k). These data suggest that therapy with PTCV results in the expan - sion of T cells in the periphery, with T cells trafficking to the tumor. Vaccination drives effector T cell memory clonal expansion To characterize the vaccine-induced T cell response further, we per - formed single-cell RNAseq/TCR sequencing (TCRseq) of peripheral blood at the 12-week postvaccination time point. In four samples obtained from three patients (patients 6 (SD), 7 (CR) and 8 (PR)), we assigned PBMCs to 14 clusters based on transfer learning from refer - ence PBMC data and analysis of canonical marker genes (Extended Data Fig. 8a–d)34–36. Clusters demonstrating the highest expression of genes associated with cytotoxicity, including chemokine C–C motif ligand 5 (CCL5) and granzyme K (GZMK), GZMB or granulysin (GNLY), were CD4+ effector memory T (TEM), CD4+ cytotoxic T lymphocyte (CTL), CD8+ TEM and CD8+ proliferating cells, as well as γδ T and natural killer (NK) cells. In 31,843 of 39,439 cells, a TCR sequence was identified by paired single-cell immune repertoire sequencing. Across all single-cell clusters with an associated TCR, clonally expanded T cell populations, which we defined as more than five cells that shared the same TCR, were most strongly associated with the CD8+ proliferating (odds ratio, 5.84; 95% CI, 4.07 to 8.43; P < 0.001), CD8+ TEM (odds ratio, 5.58; 95% CI, 5.15 to 6.06; P < 0.001) and CD4+ CTL (odds ratio, 4.14; 95% CI, 3.13 to 5.48; P < 0.001) clusters by Fisher’s exact test (Extended Data Fig. 8e). For each patient, we identified all T cells in the single-cell dataset with a TCRβ that had been identified as clonally expanded in bulk TCRseq of prevaccination and postvaccination peripheral blood Fig. 3 | GNOS-PV02 drives polyfunctional antitumor neoantigen-specific T cell immunity. a, Vaccine-induced responses assessed by IFNγ ELISpot assays without cytokine stimulation (n = 22). Cumulative magnitudes were collected from positive epitopes before and after treatment. The postvaccination response is the ‘best’ (highest magnitude) response for each patient across time points. SFU, spot-forming units. b , Total neoantigens (gray bars) and positive neoantigens before (black bars) and after (red bars) vaccination in each patient’s PTCV assessed by IFNγ ELISpot. c , Percentage of positive responding epitopes by groups. The definition of a neoantigen-specific ELISpot response can be found in Methods. d , Representative density plots (patient 22) of the T cell markers CD69, Ki67, CD107a, IFNγ and TNF upon stimulation with patient-specific PTCV epitope pools. e ,f, Polyfunctionality assessed by Boolean gating of CD4 + or CD8+ cytokine-producing populations. T cell activation (CD69 and CD107a; e) and proliferation (Ki67; f ) were assessed together with the double-positive expression of GZMA and perforin 1 (PRF1) to evaluate the cytolytic potential of neoantigen-reactive T cells. Four patients (patients 7, 11, 18 and 22) were analyzed in d–f. g, T cell clones expanded in the periphery and the new or expanded clones enriched in the matched tumor sample for each patient (n = 14). Total PBMC and tumor-associated T cell expansion were calculated by comparing posttreatment to pretreatment PBMC or tumor samples (differential abundance statistical analysis). h , Cumulative frequencies of peripherally expanded TCR rearrangements in tumor biopsy samples. i , Expanded clone numbers in tumor biopsy samples. j ,k, TCR clonality (j ) and repertoire richness (k ) in tumor biopsy samples (n = 14). PD (red), SD (gray), and CR/PR (blue). Error bars correspond to the upper s.e.m. of each group. Simpson clonality reports the distribution of TCR rearrangements in a sample, in which 0 indicates an even distribution of frequencies and 1 indicates an asymmetric distribution. TCR repertoire richness reports the mean number of unique rearrangements. Lower numbers indicate focused TCR diversity. Filled symbols in c , e and f and open circles in h and i represent individual patients; 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 the maximum value. Significance between groups was evaluated by a two-tailed Mann–Whitney test (c ); significance within groups was evaluated by a two-tailed Wilcoxon rank test (a , h–k). Nature Medicine | Volume 30 | April 2024 | 1044–1053 1049 Article https://doi.org/10.1038/s41591-024-02894-y(Extended Data Fig.