Purpose:

Immunotherapy for hepatocellular carcinoma (HCC) shows considerable promise in improving clinical outcomes. HepaVac-101 represents a single-arm, first-in-human phase I/II multicenter cancer vaccine trial for HCC (NCT03203005). It combines multipeptide antigens (IMA970A) with the TLR7/8/RIG I agonist CV8102. IMA970A includes 5 HLA-A*24 and 7 HLA-A*02 as well as 4 HLA-DR restricted peptides selected after mass spectrometric identification in human HCC tissues or cell lines. CV8102 is an RNA-based immunostimulator inducing a balanced Th1/Th2 immune response.

Patients and Methods:

A total of 82 patients with very early- to intermediate-stage HCCs were enrolled and screened for suitable HLA haplotypes and 22 put on study treatment. This consisted in a single infusion of low-dose cyclophosphamide followed by nine intradermal coadministrations of IMA970A and CV8102. Only patients with no disease relapse after standard-of-care treatments were vaccinated. The primary endpoints of the HepaVac-101 clinical trial were safety, tolerability, and antigen-specific T-cell responses. Secondary or exploratory endpoints included additional immunologic parameters and survival endpoints.

Results:

The vaccination showed a good safety profile. Transient mild-to-moderate injection-site reactions were the most frequent IMA970A/CV8102-related side effects. Immune responses against ≥1 vaccinated HLA class I tumor-associated peptide (TAA) and ≥1 vaccinated HLA class II TAA were respectively induced in 37% and 53% of the vaccinees.

Conclusions:

Immunotherapy may provide a great improvement in treatment options for HCC. HepaVac-101 is a first-in-human clinical vaccine trial with multiple novel HLA class I– and class II–restricted TAAs against HCC. The results are initial evidence for the safety and immunogenicity of the vaccine. Further clinical evaluations are warranted.

Translational Relevance

Development of a therapeutic cancer vaccine for hepatocellular carcinoma (HCC) may greatly contribute to change the therapeutic scenario for such a disease, which still represents a highly unmet medical need. Indeed, HCC is the third most common cancer and the accounts for 8.2% of all cancer-related deaths globally, second only to lung cancer, with more than 800,000 new cases and deaths per year. Surgery (e.g., resection and transplantation) is the most effective treatment for patients with early-stage HCC on an intention-to-treat perspective, leading to a 60% to 80% 5-year survival. In more advanced disease stages, locoregional or systemic therapies show poor efficacy with dramatically lower and highly variable survival rates. In this framework, the herein reported newly developed therapeutic cancer vaccine, based on novel HCC-specific TAAs and tested in the Hepavac-101 phase I clinical trial, may have an enormous translational relevance.

Hepatocellular carcinoma (HCC) frequently arises in livers that are chronically inflamed because of underlying viral or nonviral disease and are thus considered potentially immunogenic. Therefore, immunotherapeutic approaches have been suggested as promising options for HCC (1–4).

Because of its central roles in host defense and the maintenance of self-tolerance, the liver has a strong intrinsic immunosuppressive microenvironment, which may impede effective immune responses against HCC. Furthermore, liver cancer cells have a low expression of tumor antigens and a lack of mutated HLA ligands (5), which lead to reduced T-cell activation and tumor infiltration, resulting in inefficient control of tumor growth and metastasis with poor clinical outcomes. Thus, immunotherapeutic strategies against HCC need to counteract the mechanisms underlying the immunosuppressive tumor microenvironment (TME), including immune evasion, effector T-cell dysfunction, alterations of immune checkpoint molecule expression, and dysregulation of cytokine profiles.

Treatment with checkpoint inhibitor monotherapy failed to show superior overall survival compared with sorafenib as a first-line therapy in advanced HCC (6). Only very recently, a combination of the anti-PD-L1 antibody atezolizumab (Tecentriq) and the anti-VEGF antibody bevacizumab (Avastin) was evaluated in the IMbrave 150 clinical trial and approved by FDA as a first-line treatment in unresectable HCC. However, it yields only limited improvement of progression-free survival (PFS) and overall survival (OS; ref. 7). A potential explanation for the limited success of anti-PD-L1 checkpoint inhibition is that HCC tumor cells frequently express low levels of PD-L1, if any (8). A combination of anti-PD-1 (nivolumab) and anti-CTLA-4 (ipilimumab) antibodies was evaluated in the CheckMate040 clinical trial and approved by FDA as a second-line treatment in patients with advanced HCC who were previously treated with sorafenib (9). Against this background, therapeutic approaches based on immune modulatory strategies (e.g., chemotherapy and checkpoint inhibitors), active immunization with cancer vaccines, or combinations thereof could prove highly effective (10, 11), but are a challenging field of research.

Previous attempts to design effective immunotherapeutic strategies for the treatment of HCC and their clinical translation have proven unsuccessful (12–18). The few completed HCC vaccine clinical trials have shown only limited indications of efficacy. This is in line with the poor efficacy of other cancer vaccine trials reported from several tumor types. Indeed, modest immunologic effects have been reported, correlating with so far very limited clinical benefits (19). The hitherto only FDA-approved therapeutic cancer vaccine is Provenge, indicated for patients with metastatic castration-resistant prostate cancer. However, it showed only marginal improvements in OS compared with the control arm (20) and its approval in the European Union has meanwhile been withdrawn.

A small number of tumor-associated antigens (TAA) has been identified in HCC, and variable TAA-directed T-cell responses have been observed (21). Most TAAs are not specific to HCC and only a few have been tested in cancer vaccine trials in humans (22).

An innovative strategy was pursued in the “Cancer Vaccine Development for Hepatocellular Carcinoma: HepaVac” project (www.hepavac.eu), which was funded by the European Union within the 7th framework programme (22, 23). During this project, an off-the-shelf vaccine was designed, comprising a selection of newly identified HLA class I– and class II–restricted tumor-associated peptides (TUMAP) that are naturally presented on HLA molecules of cells from either primary HCC tumors or CIITA-transduced hepatoma cell lines. The HLA ligandome of HCC was discovered using MS-MS (24, 25). Novel TAAs were selected on the basis of a broad presentation on HCC tissues and low or no presentation on normal tissues.

Following immunologic validation in HLA-matched healthy donor peripheral blood mononuclear cells (PBMC), a multiepitope, multi-HLA peptide selection (hereafter referred to as IMA970A) was evaluated during the HepaVac-101 study. The evaluation criteria were safety and immunogenicity in patients with very early- to intermediate-stage HCC who had already undergone surgical and/or locoregional treatments and were eligible for treatment based on their matching individual HLA allele background. Vaccination was preceded by a single low-dose administration of cyclophosphamide (CY), intended to diminish regulatory T cells (Treg) and related immunoinhibitory effects (26). The study treatment consisted of IMA970A and a novel RNA-based immunomodulator (CV8102; RNAdjuvant), which is a noncoding, long-chain RNA molecule that was able to induce balanced, long-lasting immune responses and to support strong antitumor activity in preclinical models (27).

Target discovery and immunologic validation of HLA class I vaccine candidate peptides

Sample collection

Tissue samples from 126 patients with HCC were available and samples from HLA-A*02–positive (n = 17) and HLA-A*24–positive (n = 15) individuals were selected for downstream analyses (Fig. 1A). Furthermore, PBMCs were isolated from venous blood of healthy subjects, using fully standardized procedures, and were cryopreserved until analysis. Informed consent was obtained from all patients and healthy blood donors and documented in writing, in accordance with applicable laws and regulations, including the Declaration of Helsinki.

Figure 1.

Workflows for vaccine discovery, target selection, and immunogenicity testing. A, Mass spectrometry–based assessment of available paired HCC tumor and adjacent liver samples along with the strategy for selection of tumor-exclusive/overexpressed vaccine candidate peptides. B, Example of the results for the HLA-A*02–restricted peptide VMAPFTMTI, derived from APOB, leveraging a comprehensive in-house database. Top, quantitative transcriptomics data indicating overexpression on tumor tissues. Bottom, signal intensities derived from mass spectrometry HLA-ligandome analyses showing almost exclusive presentation on cancer. C, Example of immunogenicity testing via in vitro priming of candidate peptide-specific CD8+ T-cell immune responses: ALDH1L1-derived HLA-A*02–restricted peptide VMAPFTMTI (left); APOB-derived HLA-A*24–restricted peptide (right).

Figure 1.

Workflows for vaccine discovery, target selection, and immunogenicity testing. A, Mass spectrometry–based assessment of available paired HCC tumor and adjacent liver samples along with the strategy for selection of tumor-exclusive/overexpressed vaccine candidate peptides. B, Example of the results for the HLA-A*02–restricted peptide VMAPFTMTI, derived from APOB, leveraging a comprehensive in-house database. Top, quantitative transcriptomics data indicating overexpression on tumor tissues. Bottom, signal intensities derived from mass spectrometry HLA-ligandome analyses showing almost exclusive presentation on cancer. C, Example of immunogenicity testing via in vitro priming of candidate peptide-specific CD8+ T-cell immune responses: ALDH1L1-derived HLA-A*02–restricted peptide VMAPFTMTI (left); APOB-derived HLA-A*24–restricted peptide (right).

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LC/MS

HLA molecules from cryopreserved tissue samples were obtained as described previously (28, 29). HLA class II–bound peptides were immunoprecipitated as described in Supplementary Materials and Methods (30).

Selection of vaccine candidate peptides

The selection of candidate peptides for in vitro priming was based on multiple criteria and generally conformed to a well-established strategy (25) that was used alike already for vaccine design in several preceding clinical trials (31–33). The criteria for peptide selection were as follows: (i) frequent presentation on HCC, (ii) strong overexpression and/or overpresentation on HLA ligand level on HCC in comparison with normal liver tissue, (iii) lack of/or very low expression and absence/or low-level presentation on HLA ligand level on other normal tissues. In addition, favorable characteristics for chemical synthesis were considered. Respective peptides of interest selected for immunogenicity testing were < 0.7% of all initially characterized peptides.

Assessment of in vitro immunogenicity

PBMCs of 4 HLA-A*02–positive and 4 HLA-A*24–positive healthy control donors were assessed for precursor T cells that were specific for the IMA970A peptides as described previously (31).

Clinical trial conduct

Study design and setting

HepaVac-101 was a single-arm, open-label, multicenter, first-in-human phase I/II study to investigate an off-the-shelf vaccination, consisting of the multipeptide component IMA970A plus the RNA-based adjuvant CV8102, following a single prevaccination infusion of low-dose CY in patients with very early-, early-, and intermediate-stage HCC (Supplementary Materials and Methods; ref. 34). This trial is registered with ClinicalTrials.gov Identifier: NCT03203005 and EudraCT number: 2015-003389-10.

The study was conducted between 2017 and 2019 at six specialized oncologic centers in five European countries (Antwerp University Hospital, Edegem, Belgium; University Hopital Tübingen, Germany; Istituto Nazionale Tumori IRCCS - Fondazione G. Pascale, Naples, Italy; Ospedale Sacro Cuore Don Calabria, Negrar, Italy; Universidad de Navarra, Pamplona, Spain; University of Birmingham, United Kingdom).

Study treatment and vaccination schedule

The study treatment consisted of the multi-TUMAP selection IMA970A and the RNA-based adjuvant CV8102 as vaccine, and the immunomodulator CY. IMA970A is a combination of 16 synthetic TUMAPs with the ability to elicit HLA class I (CD8+ cytotoxic lymphocytes) and class II (CD4+ helper cells) T-cell responses. One HBV peptide was included as control (Table 1). Each vaccine dose consisted of 400 μg per peptide (i.e., a total peptide dosage of 6.8 mg per injection) dissolved in a sodium bicarbonate solution and administered in a total volume of 420 μL. CV8102 (RNAdjuvant, developed by CureVac AG) is a novel RNA-based immunostimulatory adjuvant (35, 36). Patients received nine intradermal injections of IMA970A plus CV8102. The first four vaccinations (visits 1–4) were applied at weekly intervals (days 1, 8, 15, and 22), and the subsequent five vaccinations (visits 5–9) at 3 weekly intervals (days 43, 64, 85, 106, and 127). All vaccine components were coadministered by intradermal vaccination at the same injection site, preferably using a consistent vaccination site with all repeated vaccinations (the skin of the inner side of the thighs or the upper arms) aiming for targeting the same draining lymph nodes. IMA970A was always injected first, followed by CV8102 injected in close proximity about 10 minutes later. Separate injections were required because of issues with in use stability of the mixed product. Feasibility of separate injections of IMA970A and CV8102 was supported by preclinical in vivo experiments performed with an unrelated HPV-16 E7 peptide, demonstrating the induction of antigen-specific T-cell responses (data not shown). For sequential injections, the vaccination site was marked to assure precise administration at the same location.

Table 1.

IMA970A multipeptide antigen composition.

#Source proteinHLALength (aa)Sequence
ACSS3 (acyl-CoA synthetase short-chain family member 3) A*02:01 ILDDNMQKL 
ALDH1L1 (aldehyde dehydrogenase 1 family member L1) A*02:01 KLQAGTVFV 
APOB (apolipoprotein B) A*02:01 VMAPFTMTI 
AXIN2 (axin-related protein 2) A*02:01 KLSPTVVGL 
C1QTNF3 (C1q and tumor necrosis factor-related protein 3) A*02:01 VLADFGARV 
IGF2BP3 (insulin-like growth factor 2 mRNA-binding protein 3) A*02:01 KIQEILTQV 
QAR (glutaminyl-tRNA synthetase) A*02:01 KMDPVAYRV 
HBV Control peptide (hepatitis B virus) A*02:01 10 FLPSDFFPSV 
AFIM2 (acyl-CoA synthetase short-chain family member 3) A*24:02 AYKPGALTF 
10 APOB (apolipoprotein B) A*24:02 DYIPYVFKL 
11 DAP3 (death-associated protein 3) A*24:02 AYPAIRYLL 
12 MANE (mannosidase) A*24:02 SYTKPEKW 
13 SLC35B1 (solute carrier family 35 member B1) A*24:02 YYGILQEKI 
14 IGF2BP3 (insulin-like growth factor 2 mRNA-binding protein 3) DR 14 KLYIGNLSENAAPS 
15 MET (hepatocyte growth factor receptor) DR 17 TFSYVDPVITSISPKYG 
16 MTT (mitochondrial tricarboxylate transporter) DR 18 LKMENKEVLPQLVAVTS 
17 SLC25A13 (calcium-binding mitochondrial carrier protein Aralar2) DR 21 GLYLPLFKPSVSTSKAIGGGP 
#Source proteinHLALength (aa)Sequence
ACSS3 (acyl-CoA synthetase short-chain family member 3) A*02:01 ILDDNMQKL 
ALDH1L1 (aldehyde dehydrogenase 1 family member L1) A*02:01 KLQAGTVFV 
APOB (apolipoprotein B) A*02:01 VMAPFTMTI 
AXIN2 (axin-related protein 2) A*02:01 KLSPTVVGL 
C1QTNF3 (C1q and tumor necrosis factor-related protein 3) A*02:01 VLADFGARV 
IGF2BP3 (insulin-like growth factor 2 mRNA-binding protein 3) A*02:01 KIQEILTQV 
QAR (glutaminyl-tRNA synthetase) A*02:01 KMDPVAYRV 
HBV Control peptide (hepatitis B virus) A*02:01 10 FLPSDFFPSV 
AFIM2 (acyl-CoA synthetase short-chain family member 3) A*24:02 AYKPGALTF 
10 APOB (apolipoprotein B) A*24:02 DYIPYVFKL 
11 DAP3 (death-associated protein 3) A*24:02 AYPAIRYLL 
12 MANE (mannosidase) A*24:02 SYTKPEKW 
13 SLC35B1 (solute carrier family 35 member B1) A*24:02 YYGILQEKI 
14 IGF2BP3 (insulin-like growth factor 2 mRNA-binding protein 3) DR 14 KLYIGNLSENAAPS 
15 MET (hepatocyte growth factor receptor) DR 17 TFSYVDPVITSISPKYG 
16 MTT (mitochondrial tricarboxylate transporter) DR 18 LKMENKEVLPQLVAVTS 
17 SLC25A13 (calcium-binding mitochondrial carrier protein Aralar2) DR 21 GLYLPLFKPSVSTSKAIGGGP 

Preconditioning with the Treg-depleting agent CY (Baxter Healthcare Ltd) was performed by a single intravenous infusion at a low dose of 300 mg/m2 body surface area during visit C, 3 ± 1 days preceding the first vaccination (Supplementary Materials and Methods).

Immunomonitoring

Whole blood sampled at the study sites was transferred to specifically qualified local laboratories, where PBMC isolation and cryoconservation was carried out according to established standards. Afterward, samples were shipped to a central laboratory for immunologic assays (Supplementary Materials and Methods; ref. 37). In brief, assays were performed in pooled samples from different timepoints and vaccine-induced (VI) T-cell responses to the administered peptides were individually monitored for each patient without presensitization of PBMCs (ex vivo). VI responses against HLA class I peptides were assessed by multimer staining without prior restimulation of CD8 T cells. For assessment of vaccine-induced responses against HLA class II peptides, cytokine secretion of CD8-negative cells was assessed after restimulation with the relevant peptide for 6 hours. Furthermore, VI T cells were assessed regarding their memory phenotype (CD8) and functionality (CD4) as described previously (37). CD4+ T cells were assigned to different subsets (Th1, Th2, suppressive, and activated) as well as categorized according to their functionality pattern.

Clinical efficacy and safety variables

Furthermore, preliminary clinical efficacy analyses were performed to assess clinical outcomes concerning PFS according to RECIST 1.1 (38) and modified RECIST for HCC (39–41) among patients with measurable and nonmeasurable disease, respectively, as well as relapse-free survival among patients with nondetectable disease at baseline (Supplementary Materials and Methods).

Data availability statement

The data generated in this study are available within the article and its Supplementary Data files.

Target discovery and immunologic validation of HLA class I vaccine candidate peptides

Of the 126 samples with primary HCC histology available in May 2015, 25.4% were obtained from HLA-A*02– and HLA-A*24–positive individuals and selected for subsequent analysis (Fig. 1A). HLA-bound peptides were eluted from tissues after immunoaffinity chromatography and characterized using MS-MS. We identified 7.262 unique TUMAPs bound to HLA-A*02 and 3.297 bound to HLA-A*24.

We then performed analyses of the data obtained from HCC sample materials, employing a comprehensive in-house database encompassing HLA-ligand profiles from over 1,300 different tissue samples (including more than 20 different malignancies and more than 50 benign tissue samples from over 40 different sources) and quantitative transcriptomics data. On the basis of this, 34 HLA-A*02– and 36 HLA-A*24–eluted peptides were selected as vaccine candidates. Figure 1B provides example results for the HLA-A*02–restricted peptide VMAPFTMTI, derived from apolipoprotein B (APOB), showing the signal intensities in HLA ligandomics as well as RNA expression profiles.

The selected TUMAPs, constituting HLA ligands either highly overexpressed or exclusively presented on cancer tissues according to mass spectrometry, were assessed for their immunogenicity via in vitro priming of candidate peptide-specific CD8+ T cells, using established protocols and artificial antigen-presenting cells (42) to confirm a human T-cell repertoire in healthy donors, which is essential for vaccine development. An example of the results is shown in Fig. 1C. On the basis of this, 32 HLA-A*02– and 35 HLA-A*24–restricted peptides were selected for assessment. We omitted immunogenicity assessment of peptides that were already extensively characterized for vaccine development, such as the IGF2BP3-derived HLA ligand KIQEILTQV (33). Thus, all the selected peptides for IMA970A could be shown to be immunogenic through specific precursor T cells established in PBMCs, respectively. Results for the validation of the selected peptides are provided in Supplementary Table S1.

Target discovery of HLA class II vaccine candidate peptides in CIITA-transfected HCC cell lines

To generate an effective and long-lasting adaptive immune response against tumors, we aimed to also include vaccine peptides for targeting helper CD4+ T-cell responses in the patients. Four HCC cell lines with no HLA class II surface expression were transfected with the AIR-1–encoded transcriptional activator CIITA. AIR-1 was previously shown to induce the expression of HLA class II genes and consequently the presentation of HLA class II–associated peptides (43–45). The cell lines Alex (DRB1*15:01) and Hep3B (DRB1*01:07/DRB1*13:01), which display a stable HLA class II–positive phenotype, were selected because of their expression of common HLA class II alleles in a White European population and used for immunoaffinity chromatography and peptide characterization via MS-MS, contributing supplementary vaccine candidate peptides.

Vaccine formulation and development

After the validation of the vaccine candidate peptides through ex vivo priming in HLA-matched healthy donors, the respective synthetic peptides were analyzed for their physicochemical properties relevant for GMP manufacturing (Supplementary Table S2). The final drug product (a multipeptide selection named IMA970A) contains 7 HLA-A*02–restricted TUMAPs, 5 HLA-A*24–restricted TUMAPs, one HLA A*02 HBV marker peptide as a control for the induction of an immune response against a known epitope, and four promiscuous HLA-DR–restricted long peptides (Table 1).

CV8102, which is a noncoding, uncapped RNA complexed with a small immunostimulatory arginine-rich cationic peptide dissolved in water (35), was selected for adjuvanting the IMA970A vaccine. The peptides and adjuvant were injected separately in close proximity.

Patient recruitment and analysis sets

Overall, 82 patients were enrolled and underwent HLA typing during Screening 1 (Fig. 2A and B), but 49 patients did not fulfil the initial eligibility criteria (mostly either lacking the required HLA*02:01 and/or HLA*24:02 allotype or having advanced disease) and/or refused clinical trial participation retroactively after initial screening measures. The remaining 33 eligible patients entered Screening 2 and are referred to as the full analysis set. Among these, 11 patients did not undergo study drug treatment due to reasons including deviations from the eligibility criteria, withdrawal of consent, or disease progression and worsening clinical condition, leaving 22 patients to be treated with at least one dose of study medication. They constitute the safety analysis set (SAF). One SAF patient had received CY pretreatment but withdrew informed consent prior to the first vaccination, leaving 21 patients available for the immunogenicity analyses (IRE analysis population) and efficacy evaluation as per protocol (PP analysis population). A second SAF patient was lost to follow-up after visit 9 (premature discontinuation).

Figure 2.

Study design, patient recruitment, standard and study-specific procedures and treatment allocation flowchart. A, The study design with details on the timing of interventions and clinical evaluations. B, Flowchart of patient enrollment and progression throughout the trial.

Figure 2.

Study design, patient recruitment, standard and study-specific procedures and treatment allocation flowchart. A, The study design with details on the timing of interventions and clinical evaluations. B, Flowchart of patient enrollment and progression throughout the trial.

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Baseline demographic and disease characteristics

Demographic and disease characteristics for the 22 SAF patients are shown in Supplementary Table S3. All patients had confirmed HCC, consistent with the inclusion criteria. An overview of standard treatments administered before the study or during follow-up is provided in Supplementary Table S4. No standard treatment was scheduled after Screening 2, except for 1 patient listed for liver transplantation, which was permitted according to the study eligibility criteria. This patient underwent the only orthotopic liver transplantation after trial treatment in the SAF patient set during the follow-up period. Throughout the treatment phase, no concomitant treatments were administered, so all vaccinations were applied as a monotherapy.

Safety and tolerability of study treatment

Treatment-emergent adverse events (TEAE), defined as events that occurred from the time of the first application of study treatment (CY administration at visit C) until study completion at visit 10/end of visit (EOV), occurred in 95.5% of patients. TEAEs organized by maximum severity are provided in Table 2. Overall, 21 patients in the SAF group had at least one TEAE documented. The most common TEAEs (by preferred terms) were local injection site reactions such as erythema (n = 16, 72.2%), edema (n = 10, 45.5%), pruritus (n = 6, 27.3%), pain (n = 4, 18.2%), warmth (n = 3, 13.6%), and induration (n = 2, 9.1%). Common (potential) systemic reactions to the vaccination were fatigue (n = 9, 40.9%), pyrexia (n = 5, 22.7%), and influenza-like illness (n = 4, 18.2%). Other TEAEs were relatively rare, but included nausea and vomiting (n = 3; 13.6%) and ascites, diarrhea, hypertension, oropharyngeal pain, and skin hyperpigmentation (n = 2; 9.1%). CY-related TEAEs comprised influenza-like illness, neutropenia, fatigue, gastroenteritis, pyrexia, and neutropenia (n = 4, 18.2% each) with a maximum of grade 2 and being transient (recovered/resolved). Most drug-related TEAEs were of severity grade 1 or 2, but grade 3 or 4 TEAEs were reported in 2 patients and included: non-serious amylase increases (grade 3) and lipase increase (grade 4) in 1 patient; and influenza-like illness (grade 3) in another patient. Four patients (19.0%) experienced treatment-emergent severe adverse events (SAE) after the start of the study. Two SAEs occurring in 2 patients (9.1%) were regarded as study drug related, including pyrexia (grade 1; CY: possible, IMA970A/CV8102: probable) and lipase increase (grade 4; IMA970A/ CV8102: possible; Table 2). Of note, the latter was not life threatening or disabling from a clinical point of view. Pancreatitis was ruled out, but it was assigned grade 4 based on exceeding a numerical threshold [>5 × upper limit of normal (ULN)] and attributed as a “medically important event.” Abnormalities in laboratory values likely reflected concomitant conditions or the study disease, as they were mostly already present at baseline (e.g., elevated bilirubin and other cholestasis-indicating parameters). Furthermore, no obvious trends were observed during the study.

Table 2.

TEAEs by maximum severity (safety population; n = 22).

All gradesGrade 1Grade 2Grade 3Grade 4
Preferred TermNo.%No.%No.%No.%No.%
Any TEAEa 21 96 21 96 11 50 18 
Injection site erythema 16 72 14 63     
Injection site oedema 10 46 10 46 —      
Fatigue 41 32     
Injection site pruritus 27 18     
Pyrexiab 23 18     
Influenza-like illness 18   
Injection site pain 18 14     
Injection site warmth 14 14 —      
Nausea 14     
Vomiting 14     
Ascitesc     
Conjunctivitis     
Diarrhea —      
Hypertension —    
Injection site induration —      
Oropharyngeal pain     
Skin hyperpigmentation —      
Amylase increased —  —    
Cholangitis —  —    
Lipase increasedd,e —  —    
Upper respiratory tract infection —  —    
All gradesGrade 1Grade 2Grade 3Grade 4
Preferred TermNo.%No.%No.%No.%No.%
Any TEAEa 21 96 21 96 11 50 18 
Injection site erythema 16 72 14 63     
Injection site oedema 10 46 10 46 —      
Fatigue 41 32     
Injection site pruritus 27 18     
Pyrexiab 23 18     
Influenza-like illness 18   
Injection site pain 18 14     
Injection site warmth 14 14 —      
Nausea 14     
Vomiting 14     
Ascitesc     
Conjunctivitis     
Diarrhea —      
Hypertension —    
Injection site induration —      
Oropharyngeal pain     
Skin hyperpigmentation —      
Amylase increased —  —    
Cholangitis —  —    
Lipase increasedd,e —  —    
Upper respiratory tract infection —  —    

Note: The table shows TEAEs regardless of relatedness to study treatment and irrespective of severity (all grades) as well as grade 1 or 2 TEAEs occurring in at least 2 patients (incidence ≥ 9%) and all grade 3 or 4 TEAEs, sorted by decreasing frequency. Maximum severity was graded according to the U.S. NCI's Common Terminology Criteria for Adverse Events.

aIf a patient had more than one TEAE, the patient was counted only once, including the event with the maximum severity (as appropriate), within each preferred term and once with the maximum severity within the summary of any TEAE that occurred.

bCY: possible; IMA970A: probable; CV8102: probable.

cCY: unrelated; IMA970A: unrelated; CV8102: unrelated.

dAs the grading of lipase increase according to CTCAE 4.0 is solely driven by numerical threshold exceedance (grade 4: > 5 × ULN), the patient's condition was not necessarily life-threatening or disabling from a clinical point of view.

eCY: unrelated; IMA970A: possible; CV8102: possible.

Study drugs and treatment modifications due to adverse events

The study treatment was mostly administered as prespecified in the protocol. All SAF patients received low-dose CY. After one dropout, 21 patients received vaccinations at visits 1 to 7, and 20 patients at visits 8 and 9. The upper left or right medial arm were the administration sites. These were usually maintained during the vaccination period. IMA970A (420 μL) was generally administered intradermally as recommended separately from CV8102, which required dose reduction in 1 patient (from 50 to 25 μg from visit 4 onward) due to flu-like symptoms. No therapy-limiting AEs were observed.

Immunogenicity of the vaccine

A moderate immunogenicity of IMA970A TUMAPs was detected ex vivo based on class I and class II immunomonitoring using two-dimensional tetramer staining, memory phenotyping (class I; Fig. 3A) and intracellular cytokine staining (class II). Because of low sample quality, 2 of the 21 vaccinated patients were not evaluable for Immunomonitoring. In total, 36.8% of the evaluable patients (7/19) responded to at least one HLA-matching class I TUMAP (with a total of 11 responses), and 21.1% of the evaluable patients (4/19) responded to more than one class I TUMAP (Fig. 3BD), according to prespecified criteria. Similarly, a VI immune response against at least one class II TUMAP was observed in 10 of 19 patients (52.6%; with a total of 18 responses) and 5/19 (26.3%) patients had a VI immune response to more than one class II TUMAP (Fig. 3EG).

Figure 3.

Vaccine-induced HLA class I and class II TUMAP responses. A, Top, representative data of a VI HLA class I TUMAP response shown as C1QTNF3-specific cells among all CD8+ T cells for pre- and post-vaccination timepoint pools. Bottom, memory phenotype of C1QTNF3-specific cells (blue) and all CD8+ T cells (gray) for pre- and post-vaccination timepoint pools. B and C, Immune responder rates (B) and multiresponder rates (C) for HLA class I TUMAPs. D, Number of HLA class I TUMAPs with vaccine-induced HLA class I TUMAP responses per patient. E and F, Immune responder rates (E) and multiresponder rates (F) for HLA class II TUMAPs. G, Number of HLA class II TUMAPs with vaccine-induced HLA class II TUMAP responses per patient. H, Absolute and relative number of patients with either no response, only CD4+ responses (HLA class II TUMAPs), only CD8+ response (HLA class I TUMAPs) or CD8+CD4+ responses (HLA class I and class II TUMAPs).

Figure 3.

Vaccine-induced HLA class I and class II TUMAP responses. A, Top, representative data of a VI HLA class I TUMAP response shown as C1QTNF3-specific cells among all CD8+ T cells for pre- and post-vaccination timepoint pools. Bottom, memory phenotype of C1QTNF3-specific cells (blue) and all CD8+ T cells (gray) for pre- and post-vaccination timepoint pools. B and C, Immune responder rates (B) and multiresponder rates (C) for HLA class I TUMAPs. D, Number of HLA class I TUMAPs with vaccine-induced HLA class I TUMAP responses per patient. E and F, Immune responder rates (E) and multiresponder rates (F) for HLA class II TUMAPs. G, Number of HLA class II TUMAPs with vaccine-induced HLA class II TUMAP responses per patient. H, Absolute and relative number of patients with either no response, only CD4+ responses (HLA class II TUMAPs), only CD8+ response (HLA class I TUMAPs) or CD8+CD4+ responses (HLA class I and class II TUMAPs).

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Notably, 68.4% of patients (13/19) showed a T-cell response against the vaccine (i.e., against at least one IMA970A class I TUMAP). A total of 21.1% (4/19) of patients showed both CD8+ and CD4+ T-cell responses, while 15.8% (3/19) showed only CD8+ and 31.6% (6/19) only CD4+ responses (Fig. 3H).

The sum frequency of all IMA970A class I TUMAP-specific CD8+ T cells was weakly induced after vaccination. In line with these findings, a memory shift of all HLA-matching IMA970A class I TUMAP-specific CD8+ T cells [sum memory cell induction factor (MCIF) ≥ 2.0] was only observed in 2 of 19 patients after vaccination (Fig. 4A and B). Notably, 6 of 14 (42.9%) HLA-A*02–positive patients showed a VI response to the HBV marker peptide included in the vaccine (Supplementary Fig. S1A). However, alike the TUMAP-specific T-cell responses, such responses to the HBV marker peptide remained of very limited magnitude but did show a more pronounced memory shift (Supplementary Fig. S2). Functionality analyses showed that VI class II TUMAP responses were predominantly of a Th1 phenotype in 9/18 cases (50.0%). However, 8 of 18 class II TUMAPs that mounted VI responses (44.4%) induced a Th2 phenotype (Fig. 4A and B), making up a noteworthy fraction of IMA970A class II TUMAP responses. A total of 53.8% (14/26) of the VI functional CD4 patterns were polyfunctional, that is, expressing at least two functional markers or cytokines (Supplementary Fig. S1B).

Figure 4.

Quantity and quality of VI HLA class I and class II TUMAP responses. A and B, Summary of key parameters determining the overall response to HLA class I and class II TUMAP vaccination for individual patients. The frequency and phenotype of the sum of HLA-matching class I TUMAP-specific CD8+ T cells is shown for all evaluable patients and assay timepoint pools. Assay time points are abbreviated as follows: 0 = Pre2; 1 = Post1; 2 = Post2; 3 = Post3. The number of VI immune responses against HLA-matching class I TUMAPs per patient and the MCIF of the sum of HLA-matching class I TUMAP-specific CD8+ T cells at peak memory response is shown. An MCIF ≥ 2 is considered to be a memory shift. The number and response phenotype of HLA class II TUMAPs with VI immune responses per patient is shown in the bottom panel.

Figure 4.

Quantity and quality of VI HLA class I and class II TUMAP responses. A and B, Summary of key parameters determining the overall response to HLA class I and class II TUMAP vaccination for individual patients. The frequency and phenotype of the sum of HLA-matching class I TUMAP-specific CD8+ T cells is shown for all evaluable patients and assay timepoint pools. Assay time points are abbreviated as follows: 0 = Pre2; 1 = Post1; 2 = Post2; 3 = Post3. The number of VI immune responses against HLA-matching class I TUMAPs per patient and the MCIF of the sum of HLA-matching class I TUMAP-specific CD8+ T cells at peak memory response is shown. An MCIF ≥ 2 is considered to be a memory shift. The number and response phenotype of HLA class II TUMAPs with VI immune responses per patient is shown in the bottom panel.

Close modal

Clinical outcomes

The associations between TUMAP responses and clinical outcomes, that is, radiographic progressive disease (PD) or relapse, are provided for each patient in Supplementary Table S5. Of the patients with at least one HLA class I TUMAP response, 5 of 7 (71.4%) remained event free during the study, as did 3 of 4 in the subgroup of HLA class I multi-TUMAP responders. Of the patients who did not respond to HLA class I TUMAPs, 7 of 12 (58.3%) experienced PD or relapse and the others remained event free. Furthermore, of the 4 patients with more than one HLA class I and II TUMAP responses, half remained event free, whereas 4 of 6 patients without HLA class I and II TUMAP responses relapsed or showed PD. Two additional patients remaining disease free for the study duration were not evaluable regarding VI TUMAP responses.

In advanced stages, all currently available HCC treatments are palliative, including immunotherapy with checkpoint inhibitors. Single-agent treatment has thus far only shown limited improvements in clinical outcomes (46). However, combination therapies might yield relevant improvements (7, 47) and have not been widely explored yet. For example, a therapeutic cancer vaccine inducing an immune response against HCC-specific TAAs might have a relevant impact on the course of HCC treatment, especially if administered in the earlier stages of disease and combined with treatments such as checkpoint inhibitors. Cancer vaccine clinical trials conducted thus far have yielded unsatisfactory results, probably due to the lack of specific and sufficiently immunogenic TAAs, the administration of vaccines in advanced stages of disease, and the lack of appropriate combination treatments (48, 49). It is therefore reasonable to assume that both a lack of duly confirming vaccine antigens as natural HLA ligands and the immune-suppressive TME that may partially or totally abrogate the antitumor activity of cellular immune responses may have contributed to vaccine futility (50).

The European Union–funded HepaVac project aimed at overcoming these limitations. Novel HCC-specific HLA class I and class II TUMAPs were identified in primary liver cancer tissues and HCC cell lines and immunologically validated. A vaccine including 16 HLA class I and II TUMAPs was developed (IMA970A) and administered together with CV8102 RNA adjuvant in the first-in-human phase I/II clinical trial HepaVac-101, which was conducted in five European countries. To limit the presumed effects of the immune suppressive tumor environment, the vaccine was investigated in patients who either showed no evidence of a residual tumor burden or had malignancies controlled with previous locoregional treatment.

The results show that the vaccine formulation was safe and well tolerated, with a limited number of transient drug-related grade 3 or 4 TEAEs in 2 patients. They included nonserious amylase and lipase increases as well as influenza-like illness. None of the TEAEs were life threatening or disabling from a clinical point of view. Observed abnormalities in laboratory values likely reflected concomitant conditions or the underlying HCC, being mostly present already at baseline (e.g., elevated bilirubin and other cholestasis-indicating parameters). HepaVac-101 treatment induced a moderate vaccine-specific immune response. A total of 36.8% of the evaluable patients (7/19) responded to at least one class I TUMAP while 21.1% (4/19) responded to more than one class I TUMAP. Similarly, 52.6% of the patients responded to at least one class II TUMAP and 26.3% responded to more than one class II TUMAP. In total, 68.4% of patients showed a T-cell response against the vaccine. Of them, 21.1% showed both CD8 and CD4 T-cell responses, while 15.8% showed only CD8 and 31.6% only CD4 responses.

HLA class II TUMAP responses were predominantly of a Th1 phenotype in 50.0% of cases and of a Th2 phenotype in 44.4% of cases. Moreover, 53.8% of the VI functional CD4 patterns were polyfunctional, that is, expressing at least two functional markers or cytokines.

Overall, the magnitude of immunogenicity of the HepaVac-101 vaccine proved limited. However, these results are well comparable with several other peptide-based cancer vaccine trials performed previously in HCC (12, 16, 17, 51–56). To address this issue, plans are underway to modify the formulation to improve the immunogenicity of the vaccine. Concerning the Th1/Th2 ratio, it should not represent a primary issue, given the low magnitude of the responses witnessed here. Obviously Th2 cells are more relevant in providing protection against extracellular pathogens, such as bacteria and a variety of parasites, and are also involved in asthmatic reactions. Therefore, a different adjuvant will be selected for a new vaccine formulation. This will allow mixing with peptides and simultaneous administration, aiming for a more pronounced Th1 polarization of CD4+ Th cells. In addition, an immune checkpoint inhibitor will be administered in the study to enhance T-cell responses elicited by the vaccine (57). In the GAPVAC-101 trial in patients with newly diagnosed glioblastoma, HLA-A*02:01–positive or HLA-A*24:02–positive patients were treated with a warehouse-based vaccine (APVAC1) targeting nonmutated antigens followed by a second vaccine (APVAC2) preferentially targeting neoepitopes (36). The immunogenicity of the HepaVac-101 treatment was lower than that of the APVAC1 treatments, which induced at least one HLA class I TUMAP response in 12 of 13 (92.3%) and at least two class I TUMAP responses in 10 of 13 (76.9%) immune response evaluable patients. However, a direct comparison is not feasible because in the GAPVAC-101 trial, the HLA class I peptides contained in the APVAC1 vaccine were personalized to each patient and the presence of specific precursor T cells in the treated patients was one of the selection criteria for the peptides. Anyhow, more GAPVAC patients also mounted a VI response toward the vaccinated HBV-derived HLA class I viral control peptide (8 of 11 or 72.7% of HLA-A*02:01+ patients) and toward one or more class II TUMAPs (9 of 13 or 69.2% of patients).

Conclusions

The results of the HepaVac-101 clinical trial show a favorable safety profile and moderate immunogenicity for the IMA970A vaccine combined with the CV8102 adjuvant. The preliminary evidence generated here provides a basis for further evaluation of the IMA970A vaccine. The final aim may still be achievable: to develop a cancer vaccine with improved therapeutic efficacy in patients with HCC who currently face an adverse prognosis.

M.W. Löffler reports grants from European Union, 7th Framework Program (EU FP7, grant no. 602893) during the conduct of the study; personal fees from Boehringer Ingelheim outside the submitted work; and being listed as a co-inventor on several patents owned by Immatics Biotechnologies, involving peptides for use in immunotherapies. A. Mayer-Mokler reports employment of Immatics Biotechnologies GmbH and owning shares of Immatics N.V. P.A. Ascierto reports grants and personal fees from BMS, Roche-Genentech, Sanofi, and Pfizer/Array; and personal fees from MSD, Novartis, Merck-Serono, Pierre-Fabre, AstraZeneca, Sun Pharma, Idera, Sandoz, Immunocore, 4SC, Italfarmaco, Nektar, Boehringer-Ingelheim, Eisai, Regeneron, Daiichi Sankyo, Oncosec, Nouscom, Lunaphore, Seagen, iTeos, Medicenna, and Bio-Al Health outside the submitted work. Y.T. Ma reports personal fees from Eisai, Roche, AstraZeneca, Ipsen, and Faron outside the submitted work. B. Sangro reports grants from European Commission during the conduct of the study; personal fees from Adaptimmune, AstraZeneca, Bayer, Boston Scientific, Eisai, Incyte, Ipsen, and Roche; and grants and personal fees from BMS, Sirtex Medical, and Terumo outside the submitted work. J. Ludwig reports employment of Immatics Biotechnologies GmbH and owning shares of Immatics N.V. D.D. Alcoba reports employment of Immatics Biotechnologies GmbH and owning shares of Immatics N.V. C. Flohr reports at the time of the study employment of Immatics Biotechnologies GmbH and owning shares of Immatics N.V. K. Aslan reports employment of Immatics Biotechnologies GmbH. R. Mendrzyk reports employment of Immatics Biotechnologies GmbH and owning shares of Immatics N.V. H. Schuster reports employment of Immatics Biotechnologies GmbH and owning shares of Immatics N.V. R. Heidenreich reports employment of CureVac AG. C. Gouttefangeas reports grants from European Commission FP7-Health during the conduct of the study and grants from Enterome and RhoVac ApS outside the submitted work; in addition, C. Gouttefangeas has a patent for WO2009138236 issued, a patent for WO2009138236 issued, a patent for WO2011073215 issued, and a patent for WO2022017895 issued. M. Iñarrairaegui reports grants from European Commission during the conduct of the study and personal fees from BMS outside the submitted work. U. Gnad-Vogt reports grants from Hepavac project funded by the European Commission during the conduct of the study; and other support from CureVac AG outside the submitted work. C. Reinhardt reports grants from European Union during the conduct of the study and personal fees from Immatics outside the submitted work. T. Weinschenk reports grants from European Union during the conduct of the study, and T. Weinschenk is co-inventor of patents related to the work which are owned by Immatics (employer). H. Singh-Jasuja reports personal fees and other support from Immatics during the conduct of the study and outside the submitted work; in addition, H. Singh-Jasuja has a patent for peptides included in HepaVac-101 pending. H.-G. Rammensee reports grants from European Union during the conduct of the study. No disclosures were reported by the other authors.

M.W. Löffler: Conceptualization, data curation, formal analysis, supervision, funding acquisition, validation, investigation, visualization, writing–original draft, project administration, writing–review and editing. S. Gori: Clinical trial conduct. F. Izzo: Clinical trial conduct. A. Mayer-Mokler: Conceptualization, validation, writing–review and editing. P.A. Ascierto: Clinical trial conduct. A. Königsrainer: Clinical trial conduct. Y.T. Ma: Conceptualization, clinical trial conduct. B. Sangro: Conceptualization, clinical trial conduct. S. Francque: Conceptualization, clinical trial conduct. L. Vonghia: Clinical trial conduct. A. Inno: Clinical trial conduct. A. Avallone: Clinical trial conduct. J. Ludwig: Conceptualization, writing–review and editing, clinical trial conduct. D.D. Alcoba: Formal analysis, writing–review and editing. C. Flohr: Methodology. K. Aslan: Validation. R. Mendrzyk: Validation. H. Schuster: Validation. M. Borrelli: Clinical trial conduct. D. Valmori: Conceptualization, writing–review and editing. T. Chaumette: Conceptualization, writing–review and editing. R. Heidenreich: Conceptualization, validation, methodology, writing–review and editing. C. Gouttefangeas: Conceptualization, validation, methodology, writing–review and editing. G. Forlani: Investigation. M. Tagliamonte: Validation, investigation, writing–review and editing. C. Fusco: Clinical trial conduct. R. Penta: Clinical trial conduct. M. Iñarrairaegui: Conceptualization, clinical trial conduct. U. Gnad-Vogt: Conceptualization, writing–review and editing, clinical trial conduct. C. Reinhardt: Conceptualization, writing–review and editing. T. Weinschenk: Conceptualization, methodology, writing–review and editing. R.S. Accolla: Conceptualization, methodology, writing–review and editing. H. Singh-Jasuja: Conceptualization, data curation, funding acquisition, writing–review and editing. H.-G. Rammensee: Conceptualization, supervision, writing–review and editing. L. Buonaguro: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, validation, investigation, visualization, writing–original draft, project administration, writing–review and editing.

The study was funded by FP-7 HEPAVAC (grant no. 602893; all authors, except S. Gori, F. Izzo, P.A. Ascierto, A. Inno, A. Avallone, M. Borrelli). We thank all the participants who volunteered for this study and their families as well as the members of data and safety monitoring board for their dedication and their diligent review of the data, as well as support by Derek Handley concerning proofreading of the final article version. Serena Salerno and Lisa Mazzone served as project Managers from 2014 to 2016 (S. Salerno) and from 2016 to 2018 (L. Mazzone). Luigi Bolondi (Bologna University, Italy), Pedro Romero (Lausanne University, Switzerland), Tim Greten (NCI, NIH), Stefan Endres (Ludwig Maximilian University, Germany) served as Members of the data safety monitoring board (DSMB).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data