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

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide, with a 5-year survival rate under 10%. Advanced HCC is relatively immune-resistant, showing low T cell infiltration and modest tumor mutational burden (TMB). Immune checkpoint inhibitors (ICIs) targeting PD-1 have shown only modest response rates (approximately 12-18%) as monotherapy. Personalized therapeutic cancer vaccines (PTCVs) offer a potential solution by inducing tumor-specific immunity and enhancing responses to ICIs. PTCVs target mutation-associated neoantigens (MANAs), abnormal proteins unique to tumor cells. Preclinical studies showed that MANA-targeting PTCVs induced tumor-specific T cell responses and inhibited tumor growth. Initial clinical trials demonstrated the induction of neoantigen-specific immune responses, although the efficacy in less immunotherapy-responsive tumors like HCC remained unclear. This study investigated the combination of a PTCV and pembrolizumab in patients with advanced HCC who had progressed after multityrosine kinase inhibitor (mTKI) treatment. The PTCV (GNOS-PV02) contained up to 40 neoantigens identified by sequencing of each patient's tumor DNA, RNA, and germline DNA; it was co-formulated with interleukin-12 (IL-12) as an adjuvant and delivered by intradermal injection followed by electroporation. The primary endpoints were safety and immunogenicity. Secondary endpoints included efficacy and feasibility.

Immune checkpoint inhibitors, particularly anti-PD-1 therapies, have shown promise in treating various cancers. However, their efficacy in hepatocellular carcinoma (HCC) remains limited, with response rates typically ranging from 12% to 18%. This modest efficacy highlights the need for innovative therapeutic strategies to enhance antitumor immunity in HCC. Preclinical and early clinical studies have shown that personalized cancer vaccines targeting neoantigens can elicit robust tumor-specific T cell responses. These neoantigens, unique to tumor cells, represent promising targets for immunotherapy. Combining a personalized cancer vaccine with an immune checkpoint inhibitor like pembrolizumab could potentially overcome the immune resistance observed in HCC, leading to improved clinical outcomes. This strategy leverages the ability of the vaccine to prime new tumor-specific T cell responses, while the immune checkpoint inhibitor removes the inhibitory signals that prevent these T cells from effectively attacking tumor cells. Several studies have reported promising results using different types of neoantigen vaccines in various cancer types, providing further support for this approach.

This was a single-arm, open-label, multicenter phase 1/2 study of 36 patients with advanced HCC who had progressed on or were intolerant to first-line mTKI therapy. Patients received a personalized therapeutic cancer vaccine (PTCV) consisting of a DNA plasmid (GNOS-PV02) encoding up to 40 neoantigens identified via next-generation sequencing of their tumor samples, along with a plasmid encoding IL-12 as an adjuvant. The PTCV was administered intradermally with electroporation every 3 weeks for the first four doses, then every 9 weeks until 2 years, and every 12 weeks thereafter. Pembrolizumab (200 mg IV every 3 weeks) was administered concurrently for up to 2 years. The primary endpoints were safety and immunogenicity, assessed using CTCAE v5.0 and IFN-γ ELISpot assays, respectively. Secondary endpoints included objective response rate (ORR) per RECIST 1.1, progression-free survival (PFS), and overall survival (OS). Exploratory endpoints included analyses of tumor and immune biomarkers (TMB, AFP, CD8 infiltration, PD-L1 expression, T-cell inflamed gene expression profile) and TCR sequencing to assess clonal expansion and tumor infiltration. Circulating tumor DNA (ctDNA) analysis was performed in a subset of patients to monitor molecular responses. Further analysis included intracellular staining of PBMCs and TCR sequencing of tumor biopsies to confirm neoantigen specificity and assess clonal expansion and trafficking.

The treatment was well-tolerated, with the most frequent treatment-related adverse events (TRAEs) being injection-site reactions (41.6% of patients). No dose-limiting toxicities or grade ≥3 TRAEs were observed. The modified intention-to-treat (mITT) ORR per RECIST 1.1 was 30.6% (11/36 patients), including 3 complete responses (CRs) and 8 partial responses (PRs). The disease control rate was 55.6%. The median PFS was 4.2 months, and the median OS was 19.9 months. Clinical responses were significantly associated with survival. Neoantigen-specific T cell responses were detected in 19 of 22 (86.4%) evaluable patients by ELISpot assays, with responses increasing post-treatment. Multiparametric cellular profiling revealed active, proliferative, and cytolytic vaccine-specific CD4 and CD8+ effector T cells. TCRβ sequencing demonstrated vaccine-enriched T cell clone expansion and tumor infiltration. Single-cell analysis revealed post-treatment T cell clonal expansion of cytotoxic T cell phenotypes, and reactivity against vaccine-encoded neoantigens was confirmed. An exploratory analysis showed a positive correlation (P=0.025) between the number of neoantigens in the vaccine and clinical response. ctDNA analysis showed a strong correlation with imaging in monitoring objective responses. A ctDNA decrease at week 9 was significantly associated with longer survival (P=0.01). Analysis of a single patient showed evidence of tumor escape due to neoantigen loss in a new metastatic lesion.

This phase 1/2 trial provides evidence that a personalized neoantigen vaccine combined with pembrolizumab has clinical activity in advanced HCC. The observed ORR of 30.6% is significantly higher than the response rates reported for anti-PD-1 monotherapy in this patient population (12-18%), suggesting that the addition of the PTCV enhances the efficacy of pembrolizumab. The immunological data strongly support this conclusion, showing the induction of new neoantigen-specific T cell responses and significant T cell clonal expansion and infiltration into tumors after vaccination. The correlation between the number of neoantigens in the vaccine and clinical response highlights the importance of targeting a broad range of neoantigens for optimal efficacy. The single-patient case study demonstrating tumor escape due to neoantigen loss underscores the challenges posed by tumor heterogeneity and the need for adaptive therapeutic strategies. The safety profile of the combination therapy was favorable, with injection site reactions being the most common adverse event.

This study demonstrates the feasibility and clinical activity of a personalized neoantigen vaccine combined with pembrolizumab in patients with advanced HCC. The improved response rate compared to anti-PD-1 monotherapy warrants further investigation in larger, randomized controlled trials. Future research should focus on addressing tumor heterogeneity and developing adaptive strategies to overcome tumor escape mechanisms.

The study's limitations include its small sample size and single-arm design, which limits definitive conclusions about the PTCV's efficacy. The lack of a control arm makes it difficult to definitively attribute the observed responses solely to the vaccine. The study was conducted in a rapidly evolving clinical landscape, and newer first-line treatments for advanced HCC may offer superior outcomes.