Therapeutic DC vaccination with IL-2 as a consolidation therapy for ovarian cancer patients: a phase I/II trial

Abstract

While ovarian cancer (OvCa) responds well to surgery and conventional chemotherapy, a high recurrence rate of advanced OvCa is observed. In this phase I/II study, 10 OvCa patients with minimal residual disease were treated with autologous dendritic cells (DCs) and IL-2 to evaluate the safety and feasibility of this therapeutic strategy and to characterize the antigen-specific immune alterations induced through this treatment. Approximately 4 months after initial debulking and chemotherapy, patients received two subcutaneous doses of autologous monocyte-derived DCs pulsed with autologous tumor lysate and keyhole limpet hemocyanin (KLH) at 4-week intervals. After each DC inoculation, low-dose (200 mIU) IL-2 was introduced for 14 consecutive days as an immune adjuvant. The vaccination was well tolerated. In three out of 10 patients, the inclusion status after the initial therapy showed the maintenance of complete remission (CR) after DC vaccination for 83, 80.9 and 38.2 months without disease relapse. One patient with stable disease (SD) experienced the complete disappearance of tumor after DC vaccination, and this status was maintained for 50.8 months until tumor recurrence. In two patients with partial response (PR) was not responding to DC vaccination and their disease recurred. In the three patients with disease free long-term survival, significant immune alterations were observed, including increased natural killer (NK) activity, IFN-γ-secreting T cells, immune-stimulatory cytokine secretion and reduced immune-suppressive factor secretion after DC vaccination. Thus, in patients with NED status and increased overall survival, DC vaccination induced tumor-related immunity, potentially associated with long-term clinical responses against OvCa.

Figure 1

Schedule of vaccination and immune monitoring.

Figure 2

DCs were differentiated from the PBMCs. (a) On day 9, the differentiated cells showed many dendrites on the surface. Scant intracellular organelles were observed using Wright–Giemsa stain (×400). (b) Phenotypic alterations in the cultured cells. Flow cytometric immunophenotyping of the cultured cells was performed on day 9. (c) The cytokines secreted into the culture supernatant were measured using ELISA. The cytokine amounts are expressed as pg/10⁷ cultured cells. DC, dendritic cell; ELISA, enzyme-linked immune sorbent assay; PBMC, peripheral blood mononuclear cell.

Figure 3

(a) NK activity was measured in the lymphocytes (effector) obtained from patients at pre- and post-vaccination (0 week vs. 8 weeks). The radioactivity released from the lymphocytes cocultured with Na₂⁵¹CrO₄-labeled K562 cells (target) was measured to calculate the %NK activity. Asterisks represent the statistical significance compared with pre-vaccination (*P<0.05 and **P<0.01). (b) CD16⁺CD56^dim cytotoxic NK cell proportions were measured at pre- and post-vaccination. The data represent the % of CD16⁺CD56^dim cells among the total population of PBMCs. NK, natural killer; PBMC, peripheral blood mononuclear cell.

Figure 4

Lymphocyte proliferation assay after DC vaccination. Peripheral blood lymphocytes were stimulated with KLH (50 µg/ml) for 4 days in a 96-well plate and loaded with 1 µCi ³H-thymidine/well for 18 h. The proliferative response was determined through ³H-thymidine incorporation (DPM) using a liquid scintillation counter. The values are presented as the mean±s.e.m. to indicate the relative change from the pre-vaccination baseline. The asterisks represent a statistically significant induction compared with pre-vaccination (*P<0.05 and **P<0.01). The cross represents a statistically significant reduction compared with pre-vaccination (⁺P<0.05). KLH, keyhole limpet hemocyanin; DPM, disintegration per minute; s.e.m., standard error of mean.

Figure 5

ELISPOT assay for IFN-γ-secreting T cells was performed with CD3⁺ T cells derived from PBMCs obtained from the patients at pre and post-vaccination (0 week vs. 8 weeks). T cells (2×10⁵ cells) were incubated with KLH (10 µg/ml) for 20 h at 37 °C to induce antigen-specific IFN-γ secretion. The values represent the IFN-γ-secreting T-cell spots from each patient and are shown as the mean±s.e.m. of duplicate samples. The asterisks represent a statistically significant increase compared with pre-vaccination (*P<0.05 and **P<0.01). The crosses represent a statistically significant reduction compared with pre-vaccination (⁺P<0.05). ELISPOT, enzyme-linked immunospot; PBMC, peripheral blood mononuclear cell; s.e.m., standard error of mean.

Figure 6

In vivo cytokine (IL-12 and TGF-β) release in the plasma was determined through ELISA. Plasma was obtained from the peripheral blood at pre- and post-vaccination (0 week vs. 8 weeks). The data are plotted as the relative cytokine release at pre-vaccination (baseline) and are presented as the mean±s.e.m. of duplicate samples. The asterisks represent a statistically significant induction compared with pre-vaccination (P<0.05). The crosses represent a statistically significant reduction compared with pre-vaccination (⁺P<0.05). ELISA, enzyme-linked immune sorbent assay; s.e.m., standard error of mean.