No abstract

Linear, single-stranded oligonucleotides (ODN) with non-methylated 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 and have a broad potential as therapeutic agents, i.e. for cancer gene therapy and 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 and IL-6; another activates plasmacytoid DC to produce IFN-α/-β and thus γδT-cells and NK-cells to express CD69 and secrete IFN-γ. And a third group exhibits combined properties of stimulating IL-6 and IgM secretion from B cells as well as IFN-α production from pDCs. 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. In addition, a recent publication showed severe side-effect like significant organ enlargements and morphological changes in mice [Heikenwalder et al., Nat Med. 10:187, 2004]. We generated short covalently-closed dumbbell-like structures (dSLIM) to stabilize the DNA without the otherwise necessary PS-modification. Moreover, the covalently closed constructs do not signal for apoptosis, as high intracellular concentrations of open DNA ends would do. These dSLIM molecules are stable in serum and during long-term storage regarding both DNA integrity and biological function. Their broad activity, like increasing surface expression of CD80/B7.1, CD40, HLA-DR/MHC-II and CD54/ICAM-1 and enhancing production of a wide range of cytokines (IL-6, IFN-α, IFN-γ, IL-12, IL-2), was strictly dependent on molecule structure and size. Increasing or decreasing of stem size lead to reduced potency of the dumbbell-shaped dSLIM molecule. This was observed for a decreased size of the loops as well. But the most intriguing result was the significantly reduced toxicity of dSLIM compared to PS-ODN: After repeated injection of dSLIM or PS-ODN, respectively, into mice the mice receiving PS-ODN developed enlargement of liver, spleen and lymph nodes whereas dSLIM did not induce such changes. In addition, damage of liver and spleen - such as necrotic hepatocytes or hyperplasia - was observed in PS-ODN treated but not in dSLIM treated mice. Nevertheless, both dSLIM and PS-ODN induced a comparable IL-12 production in these mice in vivo. The significant differences of side-effect were true for PS-ODN and dSLIM molecules with various nucleotide sequences. In conclusion, we present the new class of potent immunomodulators (dSLIM) with significantly reduced side-effects.

Cytosine-guanine (CpG) motifs containing oligonucleotides (ODN) are commonly used for immunomodulatory purpose in cancer therapy and for the treatment of allergic diseases 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, In addition their broad potential includes activation of B-cell proliferation, monocyte stimulation and secretion of IgM and IL-6, and stimulation of plasmacytoid DC to produce IFN-α/-β and thus γδT-cells and NK-cells to express CD69 and secrete IFN-γ. Usually phosphorothioate (PS) modifications are to enhance the stability, but these are leading to several side-effects, like severe organ enlargements, morphological changes and immunosuppression in mice. We designed immunomodulatory molecules based on short covalently-closed dumbbell-like structures (dSLIM) to stabilize the DNA without the otherwise necessary PS-modification. To evaluate the anti-tumor effect of the dSLIM molecules we developed an in vitro anti-tumor assay. This assay uses supernatant from dSLIM-activated human PBMCs for incubation with tumor cells in vitro. We observed increased apoptosis and necrosis by the HT-29 tumor cell line after incubation with supernatant from dSLIM-treated PBMC which was significantly higher than the effect of supernatant from non-treated PBMC. In addition, supernatant from dSLIM-treated PBMC increased the expression of HLA-ABC on the tumor cells, a pre-requisite for tumor cell recognition by the immune system. These effects were confirmed with human HEK293 and murine Renca cell lines. Analyzing the effect with neutralizing antibodies to various apoptosis-related cytokines, we observed a crucial role of IFN-γ but not IFN-α or TNFα. To investigate the anti-tumor effects of dSLIM in vivo, we employed a SKH1 murine model which is prone to spontaneous development of papillomas. Using chemicals for initiation and weekly promotion of de novo papilloma development we compared groups of weekly s.c. or i.p. dSLIM injections, respectively, with the PBS control group. The number of papilloma developing mice was significantly lower in the dSLIM groups and the total number of papillomas on all mice was reduced by approximately 50%. In conclusion, we showed that dSLIM immunomodulators exhibit potent anti-tumor effects in vitro and in vivo.