Immunostimulatory properties and mechanisms of action of encapsulated methylated cpg oligodeoxynucleotides

Immunostimulatory oligodeoxynucleotides (ODN) containing unmethylated CpG motifs are powerful stimulators of innate as well as adaptive immune responses, exerting their activity through the triggering of the endosomally localized TLR9 by a poorly understood mechanism. The immunopotency and broad range of activity of CpG ODN makes it a promising immunotherapeutic for the treatment and prevention of cancer and other diseases. However, rapid degradation of ODN by serum nucleases, low levels of accumulation in target tissue and lack of specificity for and poor uptake into target cells following systemic administration pose significant hurdles for the clinical application of CpG ODN. This thesis describes the immunostimulatory properties of CpG ODN encapsulated in liposomal nanoparticles (LN), a delivery system that overcomes many of the problems impeding the clinical development of "free" ODN. In particular, it is shown that LN delivery of CpG ODN specifically targets the ODN for uptake by immune cells in vivo, providing a basis for significantly enhanced immunostimulatory activity, including more potent innate and adaptive immune responses, that ultimately improve anti-tumour efficacy. A particular focus of this thesis concerns previous observations that methylated sequences in ODN (mCpG ODN) are immunologically inert. It is shown that encapsulation of mCpG ODN in LN results in immunostimulatory activity that is equal to or greater than that observed for LN formulations of the equivalent unmethylated form as judged by various immune parameters and anti-tumour efficacy. Further, it is shown that both LN-mCpG ODN and LN-CpG ODN exert their immunostimulatory effects via TLR9 based on preliminary in vitro results and confirmed by studies performed in TLR9 knockout animals. The mechanisms responsible for the differentiation between both CpG ODN and mCpGODN and how encapsulation endows immunostimulatory potential are explored. It is shown that discrimination occurs upstream of TLR9 and that the lack of immunological activity of free mCpG ODN is not due to differences in uptake, trafficking to endosomal compartments or ability to bind to TLR9, when compared with CpG ODN, but rather due to its ability to colocalize with TLR9 in the endosomal compartment. It is proposed that whereas the uptake of free CpG ODN results in the induction of the Src Family Kinase signalling cascade which mediates the migration of TLR9 from the ER to the late endosome, the uptake of free mCpGODN does not. However, it is suggested that encapsulation bypasses the methylation specific recognition of CpG ODN, allowing for the activation of SFK signalling resulting in subsequent co-localization of TLR9 and mCpG ODN in the endosome thus initiating immunostimulatory activity.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat