Effects of human plasma proteins on maturation of monocyte-derived dendritic cells.

Dendritic cells (DC) are a promising tool for vaccine therapy due to their unique properties as antigen presenting cells and their ability to prime naïve T cells. Increasing evidence suggests that maturation stage of DC critically influences the fate of the immune response. Generation of monocyte-derived DC for clinically applicable immunotherapy requires the use of well-defined components and stringent culture conditions. An alternative strategy is to use human autologous serum. However, its constituents are not stable and reflect the inflammatory condition of the donor. In order to investigate whether DC properties are influenced by proteins present in the plasma, we matured human monocyte-derived DC with four main plasma components: fibrinogen, fibronectin, plasminogen or C-reactive protein. These purified proteins were added at various concentrations on day 6 after the initial differentiation induced by IL-4 and GM-CSF. The maturation was assessed by phenotyping of maturation-associated marker (CD83) and co-stimulatory molecule CD86 as well as IL-12 production. Functional properties of DC were assessed by endocytic activity and mixed leukocyte culture. Our results indicate that fibrinogen had DC-maturation effect comparable to poly-I:C, TNF-alpha and PGE(2) as a positive control, but it failed to induce IL-12 production. The other plasma proteins had no effect on DC maturation. CRP at high concentration had rather inhibitory effect on DC induced lymphocyte function. We conclude that none of the tested plasma components and acute phase proteins sufficiently induce fully competent mature DC. This finding is important for the preparation of human DC-based vaccines supplemented by autologous sera.

A phase I trial of DNA vaccination with a plasmid expressing prostate-specific antigen in patients with hormone-refractory prostate cancer.

Prostate-specific antigen (PSA) is a serine protease secreted at low levels by normal luminal epithelial cells of the prostate and in significantly higher levels by prostate cancer cells. Therefore, PSA is a potential target for various immunotherapeutical approaches against prostate cancer. DNA vaccination has been investigated as immunotherapy for infectious diseases in patients and for specific treatment of cancer in certain animal models. In animal studies, we have demonstrated that vaccination with plasmid vector pVAX/PSA results in PSA-specific cellular response and protection against tumour challenge. The purpose of the trial was to evaluate the safety, feasibility and biological efficacy of pVAX/PSA vaccine in the clinic. A phase I trial of pVAX/PSA, together with cytokine granulocyte/macrophage-colony stimulating factor (GM-CSF) (Molgramostim) and IL-2 (Aldesleukin) as vaccine adjuvants, was carried out in patients with hormone-refractory prostate cancer. To evaluate the biologically active dose, the vaccine was administered during five cycles in doses of 100, 300 and 900 microg, with three patients in each cohort. Eight patients were evaluable. A PSA-specific cellular immune response, measured by IFN-gamma production against recombinant PSA protein, and a rise in anti-PSA IgG were detected in two of three patients after vaccination in the highest dose cohort. A decrease in the slope of PSA was observed in the two patients exhibiting IFN-gamma production to PSA. No adverse effects (WHO grade >2) were observed in any dose cohort. We demonstrate that DNA vaccination with a PSA-coding plasmid vector, given with GM-CSF and IL-2 to patients with prostate cancer, is safe and in doses of 900 microg the vaccine can induce cellular and humoral immune responses against PSA protein.

Development of a technology platform for large-scale clinical grade production of DC.

BACKGROUND: Clinical studies require protocols where a sufficient number of well-characterized highly immunogenic DC are produced according to good manufacturing practice (GMP) guidelines. METHODS: In the present study, using leukapheresis products from 10 cancer patients, we validated an elutriation technology for large-scale clinical grade production of monocyte-derived DC. RESULTS: The elutriation method gave a very high purity (mean+/-SD) (86+/-5.3%) and recovery (66+/-10.4%) of monocytes. Specifically for the two monocyte-rich fractions (3 and 4,) the recovery was 42+/-13% of viable cells that could be further differentiated into immature DC in hydrophobic culture bags using GM-CSF and IL-4. The immature DC exhibited<1% CD83+ expression and >98% phagocytic activity. Maturation with TNF-alpha or poly I:C resulted in DC with expression of CD80+, CD86+ and HLA-DR+ (>99%) and CD83+ (80+/-11.9%), as well as producing IL-12p70 and lacking phagocytic activity (<5%). This cell product can be cryopreserved with cell viability >85% and cell recovery >80% after thawing. DISCUSSION: The elutriation procedure, when optimized and if the monocyte content of the starting material exceeds 5%, does not require further selection or depletion using affinity approaches.