The immunostimulatory activity of unmethylated and methylated CpG oligodeoxynucleotide is dependent on their ability to colocalize with TLR9 in late endosomes.

TLR9 recognizes CpG motifs present in pathogenic DNA and triggers potent immune responses. It is generally accepted that TLR9 distinguishes pathogenic DNA based, in part, on methylation status, where TLR9 binds unmethylated but not methylated CpG. However, we showed that methylated CpG induces potent TLR9-mediated responses when delivered in lipid nanoparticles. In this article, we report that methylation dictates the ability of free CpG DNA to colocalize with TLR9 in late endosomes. However, when delivered in lipid nanoparticles, CpG DNA and TLR9 colocalize, regardless of methylation status. Therefore, it is proposed that the ability of immune cells to distinguish unmethylated pathogenic from methylated mammalian DNA is controlled by a mechanism that regulates TLR9 mobilization and colocalization rather than a differential binding affinity.

Synthetic methylated CpG ODNs are potent in vivo adjuvants when delivered in liposomal nanoparticles.

Although it is well documented that the immunological activity of cytosine-guanine (CpG) motifs is abrogated by 5' methylation of the cytosine residue, encapsulation within stabilized lipid nanoparticles endows these methylated cytosine-guanine- (mCpG-) containing oligonucleotides (ODNs) with potent immunostimulatory activity in murine animal models. Surprisingly, not only do liposomal nanoparticulate (LN) mCpG ODN possess immunostimulatory activity, their potency is found to be equivalent and often greater than the equivalent unmethylated form, as judged by a number of ex vivo innate and adaptive immune parameters and anti-tumor efficacy in murine models. Preliminary data indicate that both methylated and unmethylated CpG ODN act through a common receptor signaling pathway, specifically via toll-like receptor (TLR) 9, based on observations of up-regulated TLR9 expression, induction of nitric oxide and dependence on endosomal maturation. This is confirmed in TLR9 knockout animals which show no immunostimulatory activity following treatment with LN-mCpG ODN. These data, therefore, indicate that the mCpG DNA is fully competent to interact with TLR9 to initiate potent immune responses. Furthermore, this work implicates an as yet unidentified mechanism upstream of TLR9 which regulates the relative activities of free methylated versus unmethylated CpG ODN that is effectively bypassed by particulate delivery of CpG ODN.

Lipid-based delivery of CpG oligonucleotides enhances immunotherapeutic efficacy.

There has been significant interest in the potential of cytosine-guanine (CpG) containing oligodeoxynucleotides (ODN) as an immunotherapy for malignant, infectious and allergic diseases. While human trials have yielded promising results, clinical use of free CpG ODN still faces several challenges which limit their effectiveness. These include suboptimal in vivo stability, toxicity, unfavorable pharmacokinetic/biodistribution characteristics, lack of specificity for target cells and the requirement for intracellular uptake. To overcome these challenges, optimized lipid-based delivery systems have been developed to protect the CpG ODN payload, modify their circulation/distribution so as to enhance immune cell targeting and facilitate intracellular uptake. Ultimately, lipid-mediated delivery has the capacity to increase the immunopotency of CpG ODN and enhance their prophylactic or therapeutic efficacy in a range of diseases. Lipid-encapsulation provides a feasible strategy to optimize the immunostimulatory activity and immunotherapeutic efficacy of CpG ODN, thereby allowing their full clinical potential to be realized.

The combination of stabilized plasmid lipid particles and lipid nanoparticle encapsulated CpG containing oligodeoxynucleotides as a systemic genetic vaccine.

BACKGROUND: DNA vaccines offer unique potential for generating protective and therapeutic immunity against infectious and malignant diseases. Unfortunately, rapid degradation and poor cellular uptake has significantly limited the efficacy of 'naked' plasmid DNA vaccines. We have previously described stabilized plasmid lipid particles (SPLP) as effective nonviral gene delivery vehicles for the transfection of tumours at distal sites following intravenous administration. Based on their low toxicity and favourable transfection profile following systemic administration, we investigate SPLP as gene delivery vehicles for the generation of a systemically administered genetic vaccine. METHODS: The uptake of SPLP and their ability to transfect splenic antigen presenting cells (APC) following systemic administration is assessed through fluorescently-labelled SPLP in combination with phenotype markers and a very sensitive flow cytometry-based assay for the detection of the transgene, beta-galactosidase. The priming of antigen-specific adaptive and humoural immune responses following vaccination with SPLP alone or in combination with liposomal nanoparticle encapsulated CpG-ODN containing oligodeoxynucleotides (LN CpG-ODN) is characterized through the use of antigen-specific cytotoxicity assays, interferon-gamma secretion assays and enzyme-linked immunosorbant assay. RESULTS: We demonstrate that SPLP are taken up by and transfect APC in the spleen following intravenous administration and that, in the presence of a strong immunostimulatory signal provided by LN CpG-ODN, are able to prime transgene-specific humoural and cellular immune responses. CONCLUSIONS: SPLP represent an effective candidate for the nonviral delivery of a systemic genetic vaccine when combined with additional immune stimulation provided by LN CpG-ODN.

Lipid-based delivery of CpG oligodeoxynucleotides for cancer immunotherapy.

The anti-tumor activity of CpG-containing oligodeoxynucleotides (ODNs) has been well established in numerous animal models and confirmed in a number of early clinical trials. While the use of chemical modifications has effectively reduced the sensitivity of ODNs to nuclease degradation and a number of human trials have yielded promising results, the clinical utility of free CpG ODN still faces several significant challenges that must be addressed to achieve optimal potency and therapeutic activity. These include unfavorable pharmacokinetic/biodistribution characteristics, lack of specificity for target cells and poor intracellular uptake. To overcome these challenges, lipid-based delivery systems have been developed to protect the CpG ODN payload, modify their circulation/distribution characteristics, enhance immune cell targeting and facilitate intracellular uptake. In preclinical cancer models, lipid-mediated delivery has demonstrated the capacity to increase the immunopotency of CpG ODNs and dramatically enhance their anti-tumor efficacy as monotherapies, vaccine adjuvants and combination therapies with monoclonal antibodies or chemotherapy. This review will focus on investigating CpG ODNs as a cancer immunotherapeutic and the promising enhancement in efficacy that can be achieved through the use of lipid nanoparticles as delivery vehicles.

The effect of circulation lifetime and drug-to-lipid ratio of intravenously administered lipid nanoparticles on the biodistribution and immunostimulatory activity of encapsulated CpG-ODN.

The encapsulation of conventional drugs in lipid nanoparticles (LNs) has been extensively utilized to enhance therapeutic activity by altering their pharmacokinetic (PK) and biodistribution (BD) properties. We have previously shown that the immunostimulatory activity of unmethylated cytidine-guanosine (CpG)-containing immunostimulatory oligodeoxynucleotides (ODN) is greatly enhanced when encapsulated in an LN (LN CpG-ODN). Here, we investigate the effect of circulation lifetime (determined by lipid composition) and drug-to-lipid (D/L) ratio of intravenously (i.v.) administered LN CpG-ODN on PK, BD, and cellular uptake and correlate these parameters with the immunostimulatory activity. Results from these studies show that despite significant differences in the circulation lifetime and the D/L ratio, the immune response is similar with respect to immune cell activation and cytolytic activity in the spleen and the blood compartments. Our findings indicate that the benefits of liposomal nanoparticles for the delivery of immunomodulatory drugs such as CpG-ODN are defined by a different paradigm than that for conventional drugs.

Effects of intravenous and subcutaneous administration on the pharmacokinetics, biodistribution, cellular uptake and immunostimulatory activity of CpG ODN encapsulated in liposomal nanoparticles.

We have previously demonstrated that the immune response to an unmethylated cytidine-guanosine (CpG)-containing oligonucleotide (ODN) is greatly enhanced when encapsulated in a lipid nanoparticle (LN-CpG ODN). In this study, the pharmacokinetics, biodistribution and cellular uptake of LN-CpG ODN following intravenous (i.v.) and subcutaneous (s.c.) administration was characterized and correlated with immunostimulatory activity. It is shown that, despite dramatic differences in tissue distribution profiles and considerable differences in uptake by CD11c-positive, CD11b-positive, Mac-3-positive and CD45R/B220-positive cells following i.v. and s.c. administration, the resultant immune response is very similar with respect to levels of cellular activation (DX5, Mac-3, CD11b, CD45/B220, CD4, CD8 and CD11c) and cytolytic activity of immune cells [natural killer (NK) cells and monocytes/macrophages] in the spleen and blood compartments. Some differences in response kinetics and antibody-dependent cellular cytotoxicity (ADCC) activity were noted in the peripheral blood NK cell population. Analyses of particle biodistribution and cell types involved in uptake leads to the conclusion that the inherent ability of antigen-presenting cells (APCs) to sequester LN-CpG ODN results in efficient uptake of the particle, even when present at very low concentrations, leading to similar responses following i.v. and s.c. administration. These results contrast with the behavior of free CpG ODN, for which distinctly different immune responses are observed following i.v. or s.c. administration.