Effect of CpG-DNA inclusion in pH-sensitive polymer-liposome vaccines on their immune-inducing activities

Abstract

Event Abstract Back to Event Effect of CpG-DNA inclusion in pH-sensitive polymer-liposome vaccines on their immune-inducing activities Yuta Yoshizaki1*, Eiji Yuba1*, Naoki Sakaguchi2*, Kazunori Koiwai2*, Atushi Harada1 and Kenji Kono1* 1 Osaka prefecture university, Applied chemistry, Graduate school of engineering, Japan 2 Terumo Corp., Ltd., Japan Introduction: Cancer immunotherapy is regarded as a minimally invasive and next-generation cancer therapy. For achievement of cancer immunotherapy, effective antigen carriers to deliver antigen into the cytosol of dendritic cells (DCs), which induces antigen-specific cellular immunity, are required. Our previous study showed that pH-sensitive polymer modified-liposomes are effective antigen carriers to deliver antigen into cytosol of DCs[1]. Because activated DCs lead efficient antigen presentation and induce strong immunity, not only to deliver antigen, but also to activate DCs are important. We recently reported pH-sensitive polymer-modified liposomes containing cationic lipids and cationic lipid-inclusion promoted their immune-inducing effect[2]. To obtain more effective antigen carriers, toll-like receptor (TLR) ligands was additionally introduced into cationic lipid-incorporated pH-sensitive polymer-modified liposomes (Fig. 1).CpG-DNA is selected as the ligand of TLR9, which is one of TLRs expressed in endosomes of DCs to detect bacterial DNA containing unmethylated CpG motifs. In this study, the effect of CpG-DNA inclusion to liposomes on their immune-inducing effects was examined. Materials and Methods: Mixed thin membrane of egg yolk phosphatidylcholine (EYPC), 3,5-didodecyl oxybenzamidine (TRX) as a cationic lipid and 3-methylglutarylated poly(glycidol)s (MGlu-HPG) as a pH-sensitive polymer was dispersed in PBS containing ovalbumin (OVA) as a model antigen. For CpG-DNA inclusion to liposomes, two complexation methods were compared. One is “Pre-mix”: mixed thin membrane was dispersed by mixture of OVA/CpG-DNA and another is “Post-mix”: CpG-DNA was added to pre-formed liposomes. DC2.4 cells, a murine dendritic cell line, were treated with liposomes and then, intracellular distribution of FITC-labeled CpG-DNA, the expression of MHC and CD80 molecules on cells, and the production of cytokines from cells were analyzed by CLSM, FCM using immunofluorescent staining and by ELISA, respectively. These liposomes were subcutaneously administered to mice bearing E.G7-OVA tumor and their antitumor effects was evaluated. Results and Discussion: In the case of DC2.4 cells treated with CpG-DNA solution, most of FITC-labeled CpG-DNA adsorbed onto the cell surface. In contrast, in the cases of cells treated with Pre-mix and Post-mix liposome, CpG-DNA was located at inside of the cells: most of them at endosomes, suggesting that CpG-DNA formed complexes with liposomes and delivered to inside of cells together. Post-mix showed higher up-regulation of CD80 molecules on DC2.4 cells than Pre-mix, while Pre-mix induced higher production of cytokines than Post-mix (Fig. 2). Post-mix exhibited higher cellular immune response in vivo and antitumor effects than Pre-mix, suggesting that higher up-regulation of CD80 on DCs by Post-mix is crucial to induce stronger cancer immunity than that of Pre-mix. Conclusion: In this study, the effects of CpG-DNA conjugation methods to cationic lipid/pH-sensitive polymer-modified liposomes on their immune-inducing effects was evaluated. Both Pre-mix and Post-mix delivered CpG-DNA into inside of DCs and activated them. For induction of antigen-specific immunity in vivo, Post-mix was superior, which induce higher up-regulation of co-stimulatory molecules, CD80 than Pre-mix. Therefore, the delivery of CpG-DNA by Post-mix is promising as effective antigen carriers for cancer immunotherapy. This work was supported by JSPS KAKENHI Grant Number 15J12180.