Purpose: CpG oligodeoxynucleotide (CpG ODN) is a ligand of Toll like receptor 9 (TLR9) which exist in the endosome of B cells and plasmacytoid dendritic cells (pDC). D35 is a kind of CpG ODN which mainly consists of natural phosphodiester backbone and has high IFN-alpha inducing activity through the activation of TLR9 signaling. Therefore, D35 is expected to have anti-tumor effect. However, the anti-tumor effect was very limited in vivo probably because of its inefficient uptake into the target cells in vivo. To improve this point, we attempted to develop optimal delivery system for D35. In this study, we established a lipid nanoparticle which works as an effective carrier for D35 to induce anti-tumor immunity in vivo. We also elucidated the mechanism of anti-tumor effect.
Methods: D35 containing lipid nanoparticle (D35-LNP) was made by mixing D35 and lipids (cationic lipids, non-charged phospholipids, cholesterol and polyethylene glycol (PEG) modified lipids) with microfluidics (Nanoassemblr Benchtop system (Precision Nano Systems)). D35-LNP was incubated with human peripheral blood mononuclear cells (PBMC), and the amount of IFN-alpha in medium was measured by ELISA. Mice (C57BL/6) were inoculated MC38 cells (Colon carcinoma). After 9 days of tumor inoculation, D35-LNP was intravenously injected into the tumor bearing mice every 2 days. The anti-tumor effect was evaluated by measuring tumor size. In addition, to elucidate the involvement of CD8 positive T cells, CD8 positive T cells was depleted in mice by injecting anti-CD8 antibody intraperitoneally. The liver toxicity after 24 hr of D35-LNP injection was assessed by AST and ALT levels in plasma. We also examined Lipopolysaccharide (LPS) injected mice as a positive control for liver toxicity.
Results: D35-LNP significantly increased the IFN-alpha production of human PBMC compared to D35 in vitro. Even at the one-twelfth of the dose, D35-LNP induce higher IFN-alpha production compared to D35, suggesting that lipid nano particulation of D35 effectively deliver D35 into the endosome where TLR9 was located, and release D35 to bind to TLR9. In line with this result, the tumor growth was significantly delayed in D35-LNP injected mice, but not in D35 injected mice, suggesting that D35-LNP effectively activated anti-tumor immune response in vivo. To analyze the mechanisms of this anti-tumor response, we depleted CD8 positive T cells. The depletion of CD8 positive T cells effectively blocked the anti-tumor effect of D35-LNP, suggesting that the CD8 positive T lymphocytes played a key role to suppress the tumor growth. Finally, we assessed the liver toxicity after D35-LNP injection. In this experiment, we also assessed LPS injection as positive control of liver toxicity. In the mice of LPS injection, AST and ALT levels were significantly increased. On the other hand, we did not observe the increase of AST and ALT levels in D35-LNP injected mice. Therefore, D35-LNP injection did not induce liver toxicity compared to LPS injection.
Conclusion: D35-LNP induced the production of IFN-alpha more efficient than D35. In in vivo experiment, D35-LNP effectively suppressed the tumor growth. The anti-tumor effect of D35-LNP was disappeared in mice with depleted CD8 positive T cells, indicating that the anti-tumor effect of D35-LNP was depended on the presence CD8 positive T cells. These results suggested that D35-LNP injection activated anti-tumor immune response initiation (probably through Th1 type immune response) by IFN-alpha production, finally resulted in the activation of tumor killing CD8 positive T lymphocytes. Importantly, the administration of D35-LNP did not increase AST and ALT, indicating that liver toxicity was not induced. According to the above results, lipid nano particulation of D35 is a promising approach to induce anti-tumor immune response in vivo.
This research was supported by AMED under Grant Number JP18cm0106310h0003.
Yoshihiko Tanimoto– Osaka University
Yasunari Haseda– Osaka University
Shohei Koyama– Osaka University
Yasuo Yoshioka– Osaka University
Yoshiaki Okada– Osaka University
Daiki Omata– Faculty of Pharma-Science, Teikyo University
Kazuo Maruyama– Faculty of Pharma-Science, Teikyo University, Tokyo
Taiki Aoshi– Osaka University
Ryo Suzuki– Dr., Teikyo University