DC vaccine generation
Peripheral blood mononuclear cells (PBMCs) were obtained from the patients with HCC through leukapheresis. Dendritic cells were generated from blood monocytes. Peripheral blood mononuclear cells isolated by Ficoll–Paque PLUS density gradient centrifugation were resuspended in RPMI1640 medium supplemented with autologous heat-inactivated plasma, and then incubated in CellSTACK Culture Chambers. After 0.5–1hours incubation at 37 C in a 5% CO2 incubator, non-adherent cells were removed by gentle washes. The adherent monocytes were cultured in X-VIVO15 supplemented with 100 ng ml -1 of granulocyte macrophage- colony-stimulating factor and 300 ng ml -1 of interleukin (IL)-4 for 5 days.
On day 5, nonattached immature DCs were collected and pulsed with CTP-fused human AFP, MAGE-1, and GPC-3 recombinant proteins at a final concentration of 5 mg ml -1 each. Antigen-pulsed DCs were matured in the presence of cytokine cocktail, IL -6, IL-1b, tumour necrosis factor (TNF)-a, prostaglandin E2 (PGE2), interferon (IFN)-g, OK432, and poly I:C (Sigma) for 2 days.
On day 7, the DCs were collected, washed, and resuspended in 2.0 ml of cryopreserving solution containing 5% dimethyl sulfoxide. Finally, fully equipped DCs were packed into a sterile glass vial (5 x 107 cells per vial), sealed with a snap-cap, and then stored at an ultralow freezer until administration.
TAA-pulsed DC vaccine was injected subcutaneously (SC) into the thigh near the inguinal lymph nodes. Toll-like receptor-7 (TLR-7) agonist (imiquimod) was applied topically around the injection site for 2 consecutive days before injection. Patients received six DC vaccines over 14 weeks (four treatments every 2 weeks and then two treatments every 4 weeks).
Dose and administration of DC vaccines
For the first three patients, 5×107 antigen-pulsed DCs were administered per injection, and there was no DLT. Thus, 5 x 107 cells per injection was defined as the TD. At the same dose, there was no DLT in the next nine patients. Although two patients experienced grade 3 haematologic adverse events (i.e., persistent leukopenia, neutropenia, and lymphopenia from the screening test in one patient and thrombocytopenia probably related to HCC progression in other patient), the cases were assessed as no adverse drug reactions and thus no DLT. All patients received six injections of DC vaccine.Safe evaluation
During the treatment period, a total of 144 adverse events occurred among the 12 patients, and there were no grade 3 or 4 adverse events except for the aforementioned grade 3 haematologic adverse events in two patients. Approximately 90% of adverse events (129 of 144) were assessed as adverse drug reactions. The most common adverse drug reactions were injection site pain (12 patients), fever (8 patients), myalgia (7 patients), headache (5 patients), and fatigue (4 patients). However, most adverse drug reactions were self- limited and resolved within 1 or 2 days. Four patients experienced a total of serious adverse events including HCC progression (five events in three patients) and menorrhagia (one event in one patient), none of which were assessed as drug-related adverse reactions and thus did not stop the DC vaccine treatment.
DC vaccination augmented TAA-reactive T-cell response
Eleven of 12 patients (91.6%) showed an increase in TAA-reactive lymphocyte population after DC vaccination. Each TAA showed comparable capacity to induce lymphocyte proliferation, and the TAA- reactive T-cell populations significantly increased in proportion to the number of repeated vaccinations except at week 24. Particularly, it is worth noting that nine recurrence-free patients showed greater TAA- specific T-cell proliferation when compared with the three patients with recurrence after TAA-pulsed DC vaccinations.
Clinical efficacy evaluation
Nine of 12 patients were free of recurrence up to 24 weeks after DC vaccination. Three patients experienced tumour recurrence; cumulative recurrence rates at weeks 18 and 24 were 16.7% (2 of 12) and 25% (3 of 12), respectively.
Tumour recurrences occurred at lymph nodes (one patient) or liver (two patients). All three patients with tumour recurrence had prior history of HCC treatment including TACE and PEI.
The median levels of serum AFP were comparable between baseline and week 24 (6.6 (range, 2.6–5470) vs 5.6 (range, 1.8– 36 600) ng ml -1). The median serum level of PIVKA-II increased during the study period (18.5 (range, 9–2040) vs 21.5 (range, 13– 35080) nAU ml -1).
In the 4-year follow-up study, an additional 4 (total 7, 58.3%) of 12 patients showed recurrence. The median time of TTP was 38.4 months in the DC-vaccination group and 10.8 months in the control group (hazard ratio (HR) with immunotherapy, 0.41; 95% confidence interval (CI), 0.18–0.95). With respect to recurrence during the entire study and follow-up period, 5-year RFS rates were 41.7% for patients who received DC vaccine therapy (n = 12), while 12.9% for the historical controls (n = 31). In addition, the 1-, 2-, and 5-year cumulative RFS rates were 75%, 69%, and 41.7%, respectively, for patients who received DC vaccine therapy (n=12), compared with 58%, 19.3%, and 16.1%, respectively, for the historical controls.
Adjuvant DC vaccine for HCC was safe and well tolerated in phase I/IIa study, and preliminary efficacy data are encouraging to warrant further clinical study in patients with HCC after primary treatments.