Abstract

Toll-Like receptors are important targets in the search for new treatments against cancer. We have developed NZ-TLR9, a local, long-acting formulation of an oligodeoxynucleotide TLR9 agonist based on the innovative NanoZolid formulation technology, which aims to activate local plasmacytoid dendritic cells leading to the activation of tumor-specific T cells and the induction of systemic anti-tumoral immunity. The advantageous characteristics of NZ-TLR9 were determined by an in-vitro release assay prior to determining the local and systemic pharmacokinetic profile of the TLR9 agonist depots in-vivo. We have demonstrated the effect of NZ-TLR9 on tumor growth, survival, intratumoral immune cells, and plasma biomarkers in-vivo in various rodent cancer models. After optimising the formulation, we selected NZ-TLR9 as the candidate with the best profile. A single intratumoral injection of NZ-TLR9 in CT26 tumors of mice was well tolerated and resulted in a significant decrease of tumor growth and increase in survival. This efficacy was further improved by combining aPD1 treatment with NZ-TLR9 monotherapy. Combining local NZ-TLR9 and systemic aPD1 treatment was highly efficacious against both treated and non-treated tumors in bilateral CT26, and bilateral EMT6 mouse models. Local treatment with NZ-TLR9 resulted in an increase of intratumoral cytotoxic T cells and activated dendritic cells. We demonstrated that an intra-tumoral injection of NZ-TLR9 resulted in high local, but low systemic TLR9 agonist levels, and that NZ-TLR9 depots could release the TLR9 agonist for at least six weeks in-vivo.In conclusion, we have developed a novel, highly efficacious and well-tolerated TLR9 agonist depot formulation offering a sustained release of the active agent which, in combination with systemic aPD1, may overcome resistance to checkpoint inhibitor therapy.