Lipid encapsulation promotes co-localization of methylated CpG ODN and TLR9 in late endosomes: A new model for the immunostimulatory activity of CpG DNA

3802 Over the past several years, there has been significant interest in the development of immunostimulatory CpG oligonucleotides (CpG ODN) as a cancer immunotherapeutic. Toll-like receptor 9 (TLR9) recognizes CpG motifs present in pathogenic DNA and CpG ODN, triggering potent innate and adaptive immune responses. We have previously shown that encapsulation in lipid nanoparticles (LN) effectively protects the CpG ODN payload and enhances delivery and uptake by target immune cells, resulting in dramatically enhanced immunostimulatory activity and anti-tumor efficacy a number of animal models. Based on these data, Tekmira Pharmaceuticals is developing INX-0167, a phosphodiester CpG ODN encapsulated in a LN as an oncology therapeutic.
 It is generally accepted that TLR9 distinguishes immunogenic, pathogenic DNA from non-stimulatory vertebrate DNA based, in part, on its methylation status, where TLR9 specifically binds and is activated by unmethylated but not methylated CpG (mCpG) motifs and consistent with this premise, numerous studies have shown that CpG methylation effectively abrogates immunostimulatory activity. However, we have shown that mCpG is capable of inducing TLR9-mediated immune responses when delivered in LN that are equal or greater than those induced by the equivalent unmethylated CpG ODN, thus implicating an alternate mechanism by which the relative immunostimulatory activity of CpG and mCpG DNA is regulated.
 We show here that methylation status, rather than impacting binding affinity, determines the ability of “free” CpG DNA to co-localize with TLR9 in the late endosomal compartment of antigen-presenting cells. While free methylated and unmethylated CpG ODN are taken up and traffic to the endosome similarly, only unmethylated ODN promotes effective trafficking of TLR9 to the late endosomal compartment allowing for co-localization and interaction with its CpG ligand. However, when delivered in LN form, CpG DNA mobilizes and co-localizes with TLR9 regardless of methylation status, a property that is even shared with empty LN. We demonstrate that colocalization following exposure to either free unmethylated CpG or LN CpG or mCpG ODN is mediated by a src-family kinase (SFK) signaling pathway, inhibition of which effectively abrogates immunostimulatory activity. It is therefore proposed that immune cells distinguish unmethylated, pathogenic DNA from methylated, mammalian DNA based on the ability of TLR9 to co-localize with its ligand rather than a differential affinity of TLR9 for CpG and mCpG DNA. Therefore, the lack of immunological activity of free mCpG ODN is due to a failure to localize with TLR9 in the late endosomal compartment. Ultimately, we confirm this hypothesis by studies in which pre-dosing with empty LN to induce TLR9 mobilization to the endosomal compartment endows free mCpG ODN with immunostimulatory activity.

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