Combination therapy of renal cell carcinoma or breast cancer patients with dendritic cell vaccine and IL-2: results from a phase I/II trial

Abstract

Background: Ten cancer patients (Six renal cell carcinoma and four breast cancer patients) were treated in a phase I/II study with a vaccine composed of autologous dendritic cells (DCs) and IL-2 to evaluate the DC vaccine-related toxicity and antigen-specific immune alteration.

Methods: Cancer patients were treated twice with autologous CD34+ hematopoietic stem cell-derived, GM-CSF/IFN-γ-differentiated DCs pulsed with autologous tumor lysate and KLH, by 4-week interval. Following each subcutaneous injection of therapeutic DCs, low-dose (200 MIU) IL-2 was introduced for 14 consecutive days as an immune adjuvant. To determine the DC vaccine-induced immunological alterations, the KLH-specific lymphocyte proliferation, number of IFN-γ secreting T cells (ELISPOT assay), NK activity and the cytokine modulation were measured.

Results: Cultured-DCs expressing HLA-DR, CD11c, CD83, and B7.1/B7.2 produced IL-12p70. After vaccination, the patients tolerated it. Clinical response was observed in one RCC patient as stable disease. However DC-vaccine related antigen-specific immune responses including peripheral blood lymphocyte proliferation and the number of IFN-r secreting cells were induced in six patients without clear correlation with clinical responses. Also NK activity was induced significantly in six patients after vaccination. DC vaccine-related decrease of TGF-β level or increase of IL-12p70 level and decline of CD4+CD25+ T cells were observed in three patients. However only in the RCC patient whose disease stabilized, combination of stimulatory as well as inhibitory immune alterations including induction of IFN-γ secreting T cell with reduction of CD4+ CD25+ T cell were correlated with clinical responses.

Conclusion: Data indicated that DC vaccine combined with IL-2 is well tolerated without major side effects. DC vaccine induced the specific immunity against introduced antigen. Combinatorial alterations of immunological parameters indicating antigen-specific immune induction along with reduction of inhibitory immunity were correlated with clinical responses in DC vaccine treated patients.

Figure 1

Vaccination and immune monitoring Protocol.

Figure 2

Therapeutic-DC Generation from the Autologous HSCs. DCs were differentiated from the patient's CD34⁺ hematopoietic stem cells as described in materials and methods. (A) Differentiated cells on day 14 have many dendrites on the surface. Scant intracellular organelles are observed by Wright Giemsa stain × 1000, Transmission electronic microscope (TEM) and Scanning electron microscope (SEM) × 2200. (B) Phenotype alterations of day 14 cultured cells were analyzed by flow cytometry (Green line: control, Blue solid line: cultured cells). The cultured cells expressed mature myeloid-DC markers including CD11c and CD83. (C) Cytokines secreted into the culture supernatant (day 7 and day 14 after HSC culture setting) were measured by ELISA. Amount of cytokines were expressed as pg/10⁶ cultured cells. Stars indicate the statistical significance comparing day 7 vs. day 14 cytokine secretion by p < 0.05.

Figure 3

NK Activity and Cytotoxic NK Cell Proportion. (A) NK activity was measured as described in materials and methods using PBMC (effector) obtained at day 0 (baseline), during (4W) and after the vaccination (8W). Stars indicate the statistical significance comparing day 0 vs. after the vaccination by p < 0.05. (B) Alteration of NK cell (0W: White bar) and after the vaccination (8W: Black bar) was measured by flow cytometry. Numbers indicate the CD16-FITC⁺ and CD56-PE^dim cytotoxic NK cell proportion.

Figure 4

Antigen-Specific Lymphocyte Proliferation. The proliferative response was determined by³H-thymidine incorporation (DPM). Data plotted as the relative change from pre-vaccination baseline. Stars are indicated the statistical significance (*p < 0.05; **p < 0.001) comparing pre (White bar) vs. post-vaccination (Black bar).

Figure 5

IFN-γ Secreting T Cells was measured by ELISPOT assay. ELISPOT assay counting IFN-γ secreting T cells was performed on before (Pre: White bar) and after the vaccination (Post: Black bar). PBMCs were incubated with KLH for 20 h at 37°C to induce the Ag-specific IFN-γ secretion. Stars are indicated the statistical significance (*p < 0.05) comparing before vs. after vaccination.

Figure 6

Cytokine Responses. DC vaccine induced cytokine (A) IFN-γ (B) TGF-β (C) IL-12 secretion was determined by ELISA. Plasma level of IFN-γ was ranged from 1.31 pg/ml to72.788 pg/ml, of TGF-β was from 41.71 pg/ml to 7305.01 pg/ml, and of IL-12 was from 0.697 pg/ml to 927.789 pg/ml. Data plotted as the relative changes from pre-vaccination baseline. Stars are indicated the statistical significance (*p < 0.05; **p < 0.001) comparing before (Pre: White bar) vs. after vaccination (Post: Black bar).