CpG-oligodeoxynucleotides (CpG ODNs) are known ligands of innate immune receptor, toll-like receptor 9 (TLR9). Activation of TLR9 by its ligands leads to stimulation of an immune cascade represented by secretion of Th1-type cytokine and chemokine milieu, including IFN-α and activation of immune cell surface markers. TLR9 agonists have a number of therapeutic applications. Currently, several TLR9 agonists are in late stage clinical trials in combination with checkpoint inhibitors (CPI) in cancer patients. In these trials, TLR9 agonists are commonly administered once a week for several weeks, suggesting that they may be degraded in the local tissues or dissipated from the site of action rapidly requiring once a week dosing. To improve the stability, delivery, and durability of TLR9 agonist activity with reduced frequency of administration, we encapsulated TLR9 agonists in injectable hydrogels containing poloxamer. TLR9 agonist/hydrogel formulations can be used to slow release of TLR9 agonist following peritumoral/intratumoral or by other routes of administration. In here, we have studied TLR9 agonists with and without encapsulation in hydrogels for tumor growth inhibition and toxicity in syngeneic tumor models in mice.

In CT26 colon cancer model, peritumoral and intratumoral administration of TLR9 agonist in five doses in 12 days without hydrogel encapsulation showed potent tumor growth inhibition and increased survival of tumor bearing mice compared with mice injected with vehicle. Peritumoral administration of encapsulated TLR9 agonist in two different types of hydrogels, one that releases the TLR9 agonist at slower rate or the other that releases little faster at the same total dose in single or two doses, respectively, showed similar levels of tumor growth inhibition up to 11 days as that of the TLR9 agonist dosed five times without hydrogel formulation. Both hydrogel formulations prolonged survival of tumor bearing mice to a similar extent. Moreover, the mice administered with hydrogel encapsulated TLR9 agonist showed lower body weight loss compared with unformulated TLR9 agonist. Benefit of hydrogel encapsulation in tumor bearing mice has been confirmed with a second TLR9 agonist as well. Taken together, the current results demonstrate that hydrogel encapsulated TLR9 agonists are released slowly over a time, resulting in sustained tumor growth inhibition with minimal effect on body weight. The encapsulation of TLR9 agonist in hydrogels may permit less frequent dosing in patients. The hydrogel encapsulation can also be expanded to other therapeutic modalities of oligonucleotides such as antisense, siRNA and gene editing technologies.

Citation Format: Ekambar R. Kandimalla, Jun Jiang, Lokendrakumar C. Bengani, Xinyu Huang, Jason Zhang. Hydrogel encapsulated TLR9 agonists show sustained tumor growth inhibition and prolong survival of CT26 tumor-bearing mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1706.