No abstract

Methods We report stimuli-responsive Toll-Like Receptor (TLR) agonist prodrugs that rely upon the irregular metabolism (Warburg Effect and efflux potential) common among different types of cancers to activate immune cells. Beginning with Coley’s Toxins in the late 1800’s, TLR agonists have exhibited anticancer properties, and more recently have been shown to act synergistically with anti-CTLA-4 or anti-PD-L1 checkpoint blockades. Specifically, TLR7/8 agonists deplete MDSCs, activate CD8+ T-cells, M1 macrophages and pDCs; and enhance abscopal effects across animal models for melanoma, breast, and prostate cancers. Despite these profound effects, the severe systemic inflammatory toxicity elicited by TLR agonists presents a significant barrier to using them as drugs. To address this, we developed stimuli-responsive TLR agonist prodrugs targeted to cancer cell metabolism. Results Our prodrugs are inactive until conversion by complementary stimuli selected from α-mannosidase, β-galactosidase, or β-glucuronidase with concomitant efflux via MDR1 and comparable processes. We observe conversion and efflux in several cancer types, including B16 melanoma, 4T1 breast, and TRAMP prostate cancers, resulting in immunogenicity as measured by activation of primary BMDCs. In-vivo, prodrugs do not induce systemic inflammation and perform differently than parent TLR agonist in a 4T1 neoadjuvant resection metastasis model. Conclusion These results suggest that stimuli-responsive TLR agonist prodrugs target the action of TLR agonists to cancer cell metabolism and mitigate systemic inflammatory effects relative to the parent TLR agonist.

In nature, trehalose plays many roles, from bacterial signaling and immune cell activation through C-Type Lectin Receptors (CLRs) activated by trehalose-6,6′-dimycolate (cord factor), to the excipient properties of trehalose, which stabilizes organisms against extreme environmental conditions. To-date, our molecular understanding of trehalose has enabled the creation of synthetic trehalose analogues that are superior to the natural systems: trehalose-6,6′-dibehenate is an optimally active motif that retains CLR agonism, and polymers of 4,6-O-(4-vinylbenzylidene)-α,α-trehalose far out-perform the natural excipient abilities of trehalose. Here we report our results from combining these benefits to create synthetic copolymers of 4,6-O-(4-vinylbenzylidene)-trehalose and 4′,6′-O-(4-vinylbenzylidene)-trehalose-6-behenate by Reversible Addition-Fragmentation Chain Transfer Polymerization. The resulting alkylated trehalose copolymers are well defined (PDI < 1.4) with incorporation of behenic acid dispersed throughout the entire length of polymer as measured by DOSY-NMR. Overall, the copolymers retain the excipient properties of trehalose and enhance their ability to act as CLR agonists. A systematic exploration of the macromolecular parameters that drive CLR agonism (DP, PDI, and end-group chemistry), particularly through Dectin-1 and MINCLE, will be presented. These results suggest that alkylated trehalose copolymers could find use as dual vaccine adjuvants and stabilizing excipients.