Nature Medicine | Volume 30 | April 2024 | 1044–1053 1044 nature medicine Articlehttps://doi.org/10.1038/s41591-024-02894-y Personalized neoantigen vaccine and pembrolizumab in advanced hepatocellular carcinoma: a phase 1/2 trial 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 . Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer and is a leading cause of cancer-related death worldwide1. Despite recent advancements in systemic therapy for advanced HCC, the 5-year survival rate remains <10% (ref. 2 ). Advanced HCC is a relatively immune-resistant tumor type generally characterized by low T cell infil - tration and a modest tumor mutational burden (TMB)3. Immune check - point inhibitors (ICIs) targeting programmed cell death protein 1 (PD-1) have response rates of approximately 12–18% as a monotherapy4–10.Received: 28 December 2022 Accepted: 1 March 2024 Published online: 7 April 2024 Check for updates e-mail: mark.yarchoan@jhmi.edu; sardesai@geneostx.comA list of authors and their affiliations appears at the end of the paper Nature Medicine | Volume 30 | April 2024 | 1044–1053 1045 Article https://doi.org/10.1038/s41591-024-02894-yinto established tumors and can induce tumor clearance in combina- tion with anti-PD-1 therapy in less immunotherapy-responsive tumor types, such as HCC, has not been established. We conducted a 36-patient, single-arm, open-label, multicenter phase 1/2 study of a PTCV in combination with pembrolizumab (a PD-1 inhibitor) in patients with advanced HCC previously treated with a mul - tityrosine kinase inhibitor (mTKI). The PTCV consisted of a DNA plas - mid encoding up to 40 neoantigens (GNOS-PV02) identified through sequencing of each patient’s tumor DNA and RNA, as well as their ger - mline DNA, as described previously24. GNOS-PV02 is coformulated with a second DNA plasmid encoding the cytokine interleukin-12 (IL-12) as a vaccine adjuvant (pIL12) and administered by intradermal injection fol - lowed by in vivo electroporation. Intradermal injection of pIL12 results in only local and transient production of IL-12 at the injection site and facili - tates the localized induction of cellular responses to the expressed anti - gens25,26. The primary study endpoints were safety and immunogenicity. Results Safety, feasibility and clinical responses The baseline demographic and clinical characteristics of the study population are shown in Table 1 . The first and last patients were enrolled on June 16, 2020, and June 14, 2023, respectively. The trial is ongoing. The median number of vaccinations at the data cutoff date was 5 (range 1–18), and the median duration of treatment was 6.1 months. At the data cutoff date (August 18, 2023), 25 patients had discontinued the study therapy (Fig. 1 ). The most common reason for discontinuation was disease progression (n = 22). All 36 patients had their personalized vaccine product available for dosing at the time they were eligible to receive second-line therapy (Fig. 2a). Treatment-related adverse events (TRAEs) observed by the cut - off date are listed in Table 2. Overall, the treatment was safe and well tolerated, with an adverse event profile similar to that reported for pembrolizumab monotherapy in HCC, except for an increase in local vaccine injection-site reactions. Low-grade TRAEs were observed in 27 patients (75.0%), and there were no grade ≥3 TRAEs. Three patients (8.3%) experienced an immune-related adverse event (irAE) requiring systemic steroid treatment (grade 2 nephritis, grade 2 pneumonitis and grade 2 hepatitis). One patient (2.8%) discontinued pembrolizumab owing to an adverse event, but no patients discontinued PTCV therapy because of an adverse event. At the time of data analysis, 34 of the 36 patients had undergone at least one on-treatment restaging scan and were evaluable for response according to Response Evaluation Criteria in Solid Tumors (RECIST) 1.1. Two patients discontinued therapy owing to unrelated severe adverse Mutations within the tumor genome cause tumors to express abnormal proteins that are not found in any normal host cell, called mutation-associated neoantigens (MANAs)11,12. Advancements in next-generation sequencing facilitate the development of person - alized immunotherapies targeting MANAs for an individual cancer patient13. Patients with preexisting immunity to tumor neoantigens often have robust responses to ICIs14, providing an initial rationale for combining ICIs with therapies that induce neoantigen-specific immunity. In preclinical studies, therapeutic cancer vaccines targeting MANAs induced tumor-specific T cell responses and impeded tumor growth15–18. Initial clinical trials of personalized therapeutic cancer vac - cines (PTCVs) have demonstrated the induction of neoantigen-specific immune responses in patients19–22. Recently, data from the phase 2b randomized KEYNOTE-942 study provided initial evidence of clinical efficacy in a highly immune-sensitive tumor type in the absence of measurable disease23. However, whether vaccine-induced T cells traffic Table 1 | GT-30 baseline patient demographic and clinical characteristics Characteristics (n = 36) n (%) Median age, years (range) 66.5 (40–83) Sex Female 11 (30.6%) Male 25 (69.4%) Race White 21 (58.4%) Asian 8 (22.2%) Other (Black and Pacific Islander) 7 (19.4%) ECOG performance status 0 25 (69.4%) 1 11 (30.6%) Child–Pugh score A 36 (100%) BCLC stage B 18 (50.0%) C 18 (50.0%) Etiology HBV 8 (22.2%) HCV 12 (33.3%) HBV + HCV 1 (2.8%) Nonviral 15 (41.7%) Prior treatment Sorafenib 2 (5.6%) Sorafenib + lenvatinib 1 (2.8%) Lenvatinib 33 (91.6%) PVI 7 (19.4%) Wnt/β-catenin mutation 10 (27.8%) Baseline AFP, ng ml−1 ≥400 8 (22.2%) <400 28 (77.8%) Targetable neoantigensa ≤20 10 (27.8%) 21–40 16 (44.4%) 41–67 10 (27.8%) Data cutoff date: August 18, 2023. n = 36 patients. BCLC, Barcelona Clinic Liver Cancer; ECOG, Eastern Cooperative Oncology Group; HBV, hepatitis B virus; HCV, hepatitis C virus; PVI, portal vein invasion. aEach patient’s vaccine included up to 40 neoantigens.Survival follow-up (n = 6)On treatment (n = 11) Discontinued treatment (n = 25) Progression (n = 22) Pursued potentially curative surgery (n = 1) Unrelated SAE (n = 2)Initiated study treatment (mITT, n = 36)Enrollment Excluded (n = 16) Failed primary screening eligibility (n = 4) Withdrew consent (n = 2)Failed secondary screening eligibility (n = 7)Insuﬀicient tumor biopsy material (n = 3)Assessed for eligibility (n = 52) 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. Nature Medicine | Volume 30 | April 2024 | 1044–1053 1046 Article https://doi.org/10.1038/s41591-024-02894-yevents (one after the first dose, one after the third dose of therapy) and were deemed unevaluable, but both patients were included in the modified intention-to-treat (mITT) analysis as nonresponders. By inves - tigator review, the objective response rate (ORR) (confirmed + uncon - firmed, mITT) per RECIST 1.1 was 30.6% (11 of 36 patients), with 8.3% (3 of 36) of patients achieving a complete response (CR) and 22.2% (8 of 36) of patients achieving a partial response (PR). The disease con - trol rate was 55.6% (20 of 36 patients) (Fig. 2b–d ). Of the 11 patients with an objective response, 9 patients (including the 3 patients with CR) had their response confirmed at the next regularly scheduled on-treatment imaging scan. Two patients with PR had their target lesions continue to show further tumor reduction over subsequent imaging time point(s), confirming a durable response (−44% and −59%), but were categorized as unconfirmed PR owing to the emergence of new nontarget lesions (one of these patients is described as a case study in ‘Vaccine-induced immune editing leads to tumor escape’ below). At the data cutoff, the median follow-up was 21.5 months. Initial response assessment was performed at 9 weeks; among patients who had a response, the median time to the response was 9.3 weeks (range 8–46 weeks). One patient with initially unresectable HCC achieved secondary resectability after five PTCV doses. The median progression-free survival (mPFS) was 4.2 months, and the median over - all survival (mOS) was 19.9 months. The median duration of response was not reached. Clinical response (CR/PR versus stable disease (SD)/ progressive disease (PD)) was significantly associated with survival (PFS and OS) (Extended Data Fig. 1).