krn-7000 and octaarginine

Alpha-galactosylceramide (GC) represents a potentially new class of adjuvant because GC strongly induces interferon (IFN) gamma production from natural killer T (NKT) cells, leading to the induction of strong antitumor immunity. Interleukin (IL)-12 is another stimulating signal that induces IFN-γ production by NKT cells. We report herein on an investigation of the effect of recombinant IL-12 on NKT cell activation, when used in combination with GC-loaded octaarginine modified liposomes (GC-Lip). IFN-γ production from splenocytes simulated with GC-Lip was dose dependently enhanced in the presence of IL-12 in vitro. In contrast, IFN-γ production in vivo was enhanced at a low dose of IL-12. Enhanced IFN-γ production was observed in the case of low doses (0.5 μg and 2.5 μg) of GC-Lip but not a high dose (5 μg), that is, the IL-12 combination enhanced NKT cell activation at a 10-fold lower GC dose. The use of the above combination also enhanced the expansion of the NKT cell population. These findings indicate that in vivo IFN-γ production is inversely correlated with the dose of IL-12 during dual signal stimulation of NKT cells via both GC-Lip and IL-12, indicating that the dose of GC-Lip can be reduced without weakening NKT cell activation.

Topics: Animals; Dose-Response Relationship, Drug; Drug Delivery Systems; Female; Galactosylceramides; Interferon-gamma; Interleukin-12; Liposomes; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Natural Killer T-Cells; Oligopeptides; Spleen; Vaccines

Alpha-galactosylceramide (αGC), a lipid antigen present on CD1d molecules, is predicted to have clinical applications as a new class of adjuvant, because αGC strongly activates natural killer T (NKT) cells which produce large amounts of IFN-γ. Here, we incorporated αGC into stearylated octaarginine-modified liposomes (R8-Lip), our original delivery system developed for vaccines, and investigated the effect of nanoparticulation. Unexpectedly, the systemic administered R8-Lip incorporating αGC (αGC/R8-Lip) failed to improve the immune responses mediated by αGC compared with soluble αGC in vivo, although αGC/R8-Lip drastically enhanced αGC presentation on CD1d in antigen presenting cells in vitro. Thus, we optimized the αGC/R8-Lip in vivo to overcome this inverse correlation. In optimization in vivo, we found that size control of liposome and R8-modification were critical for enhancing the production of IFN-γ. The optimization led to the accumulation of αGC/R8-Lip in the spleen and a positive therapeutic effect against highly malignant B16 melanoma cells. The optimized αGC/R8-Lip also enhanced αGC presentation on CD1d in antigen presenting cells and resulted in an expansion in the population of NKT cells. Herein, we show that R8-Lip is a potent delivery system, and size control and R8-modification in liposomal construction are promising techniques for achieving systemic αGC therapy.

Topics: Animals; Antigen-Presenting Cells; Antigens, CD1d; Antineoplastic Agents; Cell Line; Cell Line, Tumor; Female; Galactosylceramides; Interferon-gamma; Liposomes; Liver; Lung; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Nanoparticles; Natural Killer T-Cells; Oligopeptides; Spleen