Personalized neoantigen vaccine and pembrolizumab in advanced hepatocellular carcinoma: a phase 1/2 trial

Abstract

Programmed cell death protein 1 (PD-1) inhibitors have modest efficacy as a monotherapy in hepatocellular carcinoma (HCC). A personalized therapeutic cancer vaccine (PTCV) may enhance responses to PD-1 inhibitors through the induction of tumor-specific immunity. We present results from a single-arm, open-label, phase 1/2 study of a DNA plasmid PTCV (GNOS-PV02) encoding up to 40 neoantigens coadministered with plasmid-encoded interleukin-12 plus pembrolizumab in patients with advanced HCC previously treated with a multityrosine kinase inhibitor. Safety and immunogenicity were assessed as primary endpoints, and treatment efficacy and feasibility were evaluated as secondary endpoints. The most common treatment-related adverse events were injection-site reactions, observed in 15 of 36 (41.6%) patients. No dose-limiting toxicities or treatment-related grade ≥3 events were observed. The objective response rate (modified intention-to-treat) per Response Evaluation Criteria in Solid Tumors 1.1 was 30.6% (11 of 36 patients), with 8.3% (3 of 36) of patients achieving a complete response. Clinical responses were associated with the number of neoantigens encoded in the vaccine. Neoantigen-specific T cell responses were confirmed in 19 of 22 (86.4%) evaluable patients by enzyme-linked immunosorbent spot assays. Multiparametric cellular profiling revealed active, proliferative and cytolytic vaccine-specific CD4⁺ and CD8⁺ effector T cells. T cell receptor β-chain (TCRβ) bulk sequencing results demonstrated vaccination-enriched T cell clone expansion and tumor infiltration. Single-cell analysis revealed posttreatment T cell clonal expansion of cytotoxic T cell phenotypes. TCR complementarity-determining region cloning of expanded T cell clones in the tumors following vaccination confirmed reactivity against vaccine-encoded neoantigens. Our results support the PTCV's mechanism of action based on the induction of antitumor T cells and show that a PTCV plus pembrolizumab has clinical activity in advanced HCC. ClinicalTrials.gov identifier: NCT04251117 .

Fig. 1. Patient flowchart.

The CONSORT (Consolidated Standards of Reporting Trials) diagram shows the flow of patients as of August 18, 2023. SAE, severe adverse event.

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.

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).

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; LTR, 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.

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.

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.

Extended 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.

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.

Extended 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.

Extended 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.

Extended 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.

Extended 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.

Extended Data Fig. 9. Subcluster analysis of CD8 TEM cluster in single cell sequencing of peripheral blood T cells at week 12 post-vaccination.

a, Uniform Manifold Approximation and Projection (UMAP) of single-cell transcriptomes of 3861 CD8 TEM 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 CD8 TEM subcluster. c, Dot plot showing gene expression CD3E, CD8A, CCL5, CD69, NKG7, LCK, CD27, PDCD1, LAG3, TIGIT, GZMK, GZMB, GZMA, PRF1, and GNLY. across CD8 TEM subclusters. d, Heatmaps showing scaled expression of the top 10 marker genes identified for each subset. e, UMAP indicating areas of clonal expansion in 2,738 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 matching clonally expanded TCRs identified by bulk sequencing of pre- and post-treatment peripheral blood, divided by patient. g, CD8 TEM subcluster identities of single cells with a TCRB matching clonally expanded TCRs identified by bulk sequencing of pre- and post-treatment peripheral blood, represented as percentage of total number of single cells per patient.

Extended Data Fig. 10. Patient #11 – Case Study.

a, Change in target lesion from baseline (%) evaluated by RECIST 1.1 and tumor imaging scans at various treatment time points. Liver lesion (red arrows); Adrenal lesion (yellow arrows). b, TCR frequencies (%) of 25 expanded T cell clones at pre- or post-treatment in the periphery or in the target lesion (liver). c, PBMCs (3 × 10^5/per well) were stimulated with vaccine-encoded epitopes at the concentration of 10 μg/mL for 18–24 hours. Cells were evaluated for the presence of vaccine-induced neoantigen-specific responses prior to and post-personalized GNOS-PV02 vaccination using an interferon IFNγ ELISPOT assay without cytokine stimulation. The bar indicates the mean SFU of n = 3 individual technical replicates ± SD per group. d, Neoantigen-specific T cell activation was evaluated by stimulating patient-derived PBMCs (week 9) with DMSO, PROS1, or OBSCN peptides ex vivo by intracellular cytokine staining. e, Venn diagram of neoantigens identified in the liver (day 0) and adrenal lesions (week 54) by RNA/DNA sequencing. f, Monitoring of the dynamic expression of liver- or adrenal-specific targets collected over 50 weeks by ctDNA analysis. g, T cell infiltration/activation biomarkers are evaluated at pre-treatment and post-treatment (liver lesion), and from the adrenal lesion (week 54). The T cell suppressor, IDO1, is separated from the other markers with a dashed line. The significance between evaluated stacked gene expression groups was tested by a two-tailed, Mann-Whitney statistical analysis.