646. Covalently-Closed, Dumbbell-Shaped Double-Stem-Loop DNA Constructs (dSLIM) with Specific Immunomodulatory Function

Linear, single-stranded oligonucleotide sequences (ODN) with unmethylated cytosine-guanine (CpG) motifs are immunomodulatory since they resemble bacterial DNA and serve as “danger signals”. These CpG-ODNs promote predominately a TH1-response, with secretion of IL-12 and IFN-γ, and B-cell, natural killer (NK)-cell and dendritic cell (DC) activation. They have a wide potential utility as therapeutic agents, i.e. for cancer gene therapy and as immunomodulators for the treatment of allergic diseases. Distinct groups of CpG-ODNs were characterized differing in structure and function: one group promotes B-cell proliferation, monocyte stimulation and secretion of IgM, IL-12 and IL-6; another activates γδT- and NK-cells to express CD69 and secrete IFN-γ, a third stimulating DC to produce IFN-α/IFN-β. Phosphorothioate (PS) modifications, usually introduced to enhance stability, result in several side-effects, like prolongation of the blood clotting time, non-specific binding to various proteins and acute toxicities in primates. A possibility to avoid PS-modifaction is to protect the open ends of linear phosphorodiester (PO)-ODN via generation of a short covalently-closed dumbbell-like structure. Here, we analyzed the stimulatory potential of a new class of molecules, double-stem-loop immunomodulators (dSLIM) with phosphorodiester (PO) backbones and a dumbbell-like structure: dSLIM-30L1 with CG motifs in both loops. We found a general stimulatory pattern of dSLIM-30L1 with enhanced surface expression of CD80/B7.1, CD40, HLA-DR and CD54/ICAM-1, which was concentration-dependent and restricted to a cell line of the B-lineage, while no such effect was detected in T-, NK-cells or monocytes. Interestinly, the level of expression was strictly dependent on molecule structure, size and backbone chemistry (PS vs. PO): Lin-30L1, a linear PS-ODN, had a similar effect compared to dSLIM-30L1, whereas changes in structure or CG content diminished their potential, testing molecules with only one loop, with CG motifs only in one loop, with a smaller size and a linear PO-ODN. Interestingly, dSLIM-30L1 significantly increased IL-12p40 production by B-cells, and IFN-γ production by NK-cells. Using total PBMCs, we found induction of a specific cytokine pattern by dSLIM-30L1 with levels of IFN-γ, IFN-α and IL-12p40 which is distinct from the pattern induced by the linear, PS-based lin-30L1. Furthermore, the PO-based dSLIM had significantly less prolonged blood clotting time compared to PS-based lin-30L1.