Elsevier

Journal of Controlled Release

Volume 184, 28 June 2014, Pages 20-27
Journal of Controlled Release

A new adjuvant delivery system ‘cyclic di-GMP/YSK05 liposome’ for cancer immunotherapy

https://doi.org/10.1016/j.jconrel.2014.04.004Get rights and content

Abstract

Cyclic dinucleotides are of importance in the field of microbiology and immunology. They function as second messengers and are thought to participate in the signal transduction of cytosolic DNA immune responses. One such dinucleotide, cyclic di-GMP (c-di-GMP), stimulates the immune system. It is thought that c-di-GMP is recognized by ATP dependent RNA helicase (DDX41) in the cytosol, forms a complex with the Stimulator of interferon genes protein (STING), triggers a signal via the tank binding kinase 1-interferon regulatory factor 3 (TBK1-IRF3) pathway and induces the production of type I interferons. Therefore c-di-GMP can be thought of as a new class of adjuvant. However, because c-di-GMP contains two phosphate groups, this prevents its use as an adjuvant because it cannot pass through the cell membrane, even though the target molecule of c-di-GMP is located in the cytoplasm.

Our group has been developing a series of liposomal drug delivery systems and recently investigated YSK05 which is a synthetic, pH sensitive lipid that has a high fusogenicity. We utilized this lipid as a carrier to transport c-di-GMP into the cytosol to then use c-di-GMP as an adjuvant. Based on screening experiments, YSK05/POPE/cholesterol = 40/25/35 was found to induce IFN-β in Raw264.7 cells. The induction of IFN-β from c-di-GMP liposomes was inhibited by adding BX795, a TBK1 inhibitor, indicating that the production of IFN-β caused the activation of the STING-TBK1 pathway. C-di-GMP liposomes also showed significantly higher levels of expression of CD80, CD86 and MHC class I. The c-di-GMP/YSK05 liposome facilitated antigen specific cytotoxic T cell activity and the inhibition of tumor growth in a mouse model. These findings indicate that c-di-GMP/YSK05 liposomes could be used, not only to transfer c-di-GMP to the cytosol and induce an innate immune system but also as a platform for investigating the mechanism of immune sensing with cyclic dinucleotides in vitro and in vivo.

Introduction

Cancer immunotherapy is a frequently used approach in the treatment of cancer [1]. Cancer specific peptides or proteins are used as antigens to induce an immune system and to kill cancer cells. Sipuleucel-T (Provenge®) is a cancer vaccine for prostate cancer that has been approved by the FDA and prostatic acid phosphatase is the antigen in this system [2]. In addition, antigen nonspecific cancer immunotherapy in which an adjuvant is involved, is also of current interest, because stimulation of the innate immune system aids in this type of therapy. A tumor-specific immune response starts by recognizing the cancer antigen by antigen presenting cells (e.g. dendritic cells). The captured antigen is present on MHC class I and II molecules and the stimulated dendritic cells then elicit the formation of anticancer effector T cells with the help of co-stimulatory molecules, such as CD80 and CD86. Antigen educated T cells then kill the cancer cells [3], [4]. An adjuvant can help to induce the expression of MHC classes I and II and also CD80 and CD86 [5]. Many efforts have been made in this area using cancer vaccines and the results show that the stimulation of innate immune system with a suitable adjuvant is also important for achieving cancer immunotherapy. An adjuvant can reduce the amount of antigen produced and induce the desired immune responses. Because of this, the development of efficient adjuvants is an important issue in the field of cancer immunotherapy.

Cyclic dinucleotides function as second messengers in microbes and animals. For a long time, the key molecule involved in the innate immune response stimulated by cytosolic dsDNA was unknown. In 2013, however, it was revealed that cytosolic dsDNA binds to cGAMP synthase (cGAS) and produces cyclic G(2′–5′)pA(3′–5′)p as the second messenger and cyclic G(2′–5′)pA(3′–5′)p binds to stimulator of interferon genes protein (STING) and induce the production of type I interferons. Therefore cGAS is a DNA sensor and cyclic G(2′–5′)pA(3′–5′)p is a cytosolic DNA sensor [6], [7], [8]. Cyclic di-GMP (c-di-GMP) is also a cyclic dinucleotide and is a cyclic guanosine dimer that contains two (3′–5′) phosphate linkages and functions as a co-factor of cellulose synthase in Gluconabactor xylinum [9], [10]. C-di-GMP also controls biofilm formation, exopolysaccharide production, toxin production and related outcomes, therefore c-di-GMP can be considered to be a second messenger in bacteria [11], [12], [13]. In addition, our group recently reported that c-di-GMP has the ability to stimulate the innate immune system [14] and, given the fact that c-di-GMP binds to DDX41 in the cytosol, can form a complex with STING, send the signal via the TBK1-IRF3 pathway and activate type I interferons [15], [16]. Therefore the receptor for c-di-GMP is DDX41 in the cytosol is different from known adjuvants such as polyIC (toll like receptor 3 on the cell surface) and CpG oligonucleotides (toll like receptor on endosome). It has recently been reported that type I interferons help to stimulate CD8 positive T cells to present the tumor associate antigen [17]. This is because c-di-GMP is considered to be a new class of useful adjuvants and some researchers have attempted to use this compound as a vaccine.

Recently, several groups have attempted to use c-di-GMP to prevent bacterial infections [14], [18], [19], [20], [21], [22], [23], problems were encountered when c-di-GMP was used as a drug. C-di-GMP contains two phosphate groups and this prevents it from passing through the cell membrane, even though the target molecule of c-di-GMP is located in the cytoplasm [24]. Clearly, a suitable drug delivery system (DDS) is needed to permit c-di-GMP to penetrate the cell membrane and pass into the cytoplasmic region, if it is to be used as an adjuvant.

Liposomes are lipid bilayered nanoparticles encapsulating small molecules, such as DNAs and RNAs. Liposomes are readily taken up by cells and pharmacological findings indicate that they are relatively safe for use, both in vitro and in vivo also for these kinds of small molecules. Our group has been investigating functional liposomes (referred to as MENDs) for the delivery of plasmid DNA, siRNA and related molecules [25], [26]. Liposomes have already been used as vehicles for adjuvants [27], have been employed in clinical trials such as AS01 and good results have been reported [28]. We recently developed a synthetic lipid named YSK05. YSK05 was designed to have optimal functionality at pH = 6.4 and to possess a high fusogenic activity, with a high endosomal escape ability which is well known to promote better gene silencing [29]. Therefore we hypothesize that a c-di-GMP/YSK05 liposome system could be used to enhance the immunostimulating activity of c-di-GMP and related compounds. The c-di-GMP/YSK05 liposome is taken up well by cells and large amount of c-di-GMP can be released to the cytosolic region as the result of the high fusogenic property of YSK05 liposome. The high level of the released c-di-GMP readily recognizes DDX41, activates the TBK1-IRF3 pathway and produces large amounts of type I interferons which can function as an effective adjuvant. Here, we report on attempts to prepare YSK05 liposomes that contain encapsulated c-di-GMP, for use as an efficient adjuvant system for cancer immunotherapy (Fig. 1).

Section snippets

Materials

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), N-[1-(2,3-dioleoyloxy)propyl]-N, N, N-trimethylammonium chloride (DOTAP) and cholesterol (Chol) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). Egg phosphatidylcholine (EPC) was purchased from NOF Corp. (Tokyo, Japan). Lipofectamine 2000 and Carboxyfluorescein diacetate succinimidyl ester (CFSE) were

Optimization of c-di-GMP encapsulating liposome

We evaluated the induction of IFN-β by the stimulation with the c-di-GMP/YSK05 liposome system compared to other liposomes such as cationic liposomes, anionic liposomes, neutral liposomes and Lipofectamine2000 which is a conventional transfection reagent. The induction of IFN-β was quantified by ELISA and none of the liposomes showed IFN-β production except for the c-di-GMP/YSK05 liposomes (data not shown). The YSK05 lipid is a synthetic lipid developed in our laboratory and has properties that

Conclusions

The findings reported herein show that c-di-GMP/YSK05 liposomes were taken up by antigen presenting cells, that the encapsulated c-di-GMP was released to the cytosol, and that this process resulted in the stimulation of the induction of IFN-β via the STING-TBK1-IRF3 pathway. The large induction of IFN-β can mainly be attributed to the high fusogenic properties of the YSK05 lipid. The activity of the vaccinated c-di-GMP/YSK05 liposomes was inhibited by treatment with the TBK1 inhibitor, in other

Acknowledgment

This study was supported by grants from the Special Education and Research Expenses of the Ministry of Education, Culture, Sports, Science and Technology of Japan (MH, 26830101). This study was also supported by Grant for Basic Science Research Projects from the Sumitomo Foundation. C-di-GMP was kindly provided by Yamasa Corporation.

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