Dendritic Cell Myeloma Fusions Stimulate Anti-Tumor Immunity: Results from Pre-Clinical Studies and a Clinical Trial.

Dendritic cell (DC)-tumor fusions effectively present a broad array of tumor associated antigens in the context of DC derived costimulation. Vaccination with fusion cells induces tumor specific immunity in animal models and clinical studies. We have examined the antigen presenting characteristics of DCs fused with myeloma cells. Immature DCs were generated by culturing adherent mononuclear cells with GM-CSF and IL-4 for 1 week. Maturation was induced by exposure to TNFa for 48 hours. Compared to immature DCs, patient derived mature DC had decreased CD14 expression, increased expression of CD80 and CD83 and increased mean flourescent intensity of CD86. Using polyethyelene glycol (PEG), monocyte derived immature and mature DCs were fused to myeloma cells derived from human cell lines and patient derived bone marrow specimens. Fusion cells were isolated by flow cytometric gating of cells that co-expressed unique DC and tumor antigens. For both immature and mature DC populations, cell fusion was associated with marked upregulation of costimulatory and maturation markers. Mean expression of CD86 was 98% in both fusion cell populations and CD83 was observed in 86% and 84% of immature and mature DC/myeloma fusions, respectively. Immature and mature DC/myeloma fusions prominently express IL-12 (mean 48% and 50%, respectively) and the chemokine receptor, CCR7 (mean 33% and 46%, respectively), necessary for the migration to sites of T cell traffic in the draining lymph node. DC/myeloma fusions induce IFNγ expression by autologous T cells. In 5 serial studies, immature and mature DC/myeloma fusion cells prominently stimulate cytotoxic T lymphocyte mediated lysis of tumor targets (mean 45% and 57%, respectively). Based on these findings we have initiated a clinical trial in which successive cohorts of patients with multiple myeloma undergo vaccination with myeloma cells fused with autologous mature DCs administered in conjunction with GM-CSF. Patients with clinically stable disease who demonstrate at least 20% marrow involvement with plasma cells are potentially eligible. DCs are generated from adherent mononuclear cells collected by leukapheresis that are cultured with GM-CSF, IL-4 and TNFa. Myeloma cells are obtained from short-term culture of bone marrow aspirate specimens. DCs and myeloma cells are co-cultured in the presence of PEG and fusion cells are quantified by identifying cells that co-express DC (CD86, CD83) and myeloma (CD38, CD138) antigens. To date, 6 patients have been enrolled of which 4 have been vaccinated; 3 with 1x10(6) and 1 with 2 x 10(6) fusion cells. Mean yields of DC, tumor, and fusion cells were 7.64 x10⁷, 1.87 x10⁷, and 6.12 x10⁶ cells, respectively. In contrast to myeloma cells, DC and fusion cells preparations prominently induced allogeneic T cell proliferation with mean stimulation indices of 47 and 50, respectively. Toxicities judged to be potentially vaccine related have been mild and include muscle aches/stiffness, transient fever, pruritis, rash and fatigue. Vaccination resulted in the induction of tumor specific immunity as determined by increased percentage of CD4+ and CD8+ T cells expressing IFNγ following exposure to autologous tumor lysate. The effect of vaccination on clinical markers of disease is being monitored.