Dendritic Cell Vaccines

We have an active IND to prepare monocyte-derived dendritic cells (DCs) loaded with HLA-A2 restricted peptide antigens for the treatment of malignant melanoma. Immature DCs (a and c) are derived from autologous monocytes by culturing for 6 days in the presence of IL-4 and GM-CSF. The DCs are then matured overnight using a cytokine cocktail (IL-1, IL-6, TNF and PGE2), and the mature DCs (b and d) are loaded with peptides, washed and frozen using a mechanical controlled-rate freezer. The DCs are frozen in a solution that is compatible with injection into human subjects, and are ready to be injected after thawing and drawing up into a syringe.

Antigen/adjuvant mixtures

We also prepare vaccines that are emulsions of either whole recombinant proteins or HLA-A2 restricted peptides in Montanide ISA-51 adjuvant (an oil-based adjuvant commonly used for vaccines). All emulsions are tested for stability by floating a drop on water -- a good emulsion will not mix with water, even if left for several days.

Immune Monitoring Assays

Assays can be performed either using mononuclear cells directly purified from peripheral blood, or using cells that have been pre-stimulated with antigen for 1 week to enhance sensitivity. Assays include:

• cell proliferation (3H-thymidine uptake, CFSE staining)
• cytokine production (ELISPOT, ELISA, cytometric bead array, intracellular cytokine staining)
• cytotoxicity (51Cr release assay)
• cell phenotyping (8-color flow cytometry)
• MHC tetramer staining

ELISPOT assay

MHC tetramer staining

Intracellular cytokine staining

MHC Tetramer Synthesis

We currently syntheize HLA-A2 tetramers only. Other Class I HLA types or mouse MHC tetramers may be available in the future if there is sufficient demand.