Acquired Drug Resistance Enhances Imidazoquinoline Efflux by P-Glycoprotein.

Multidrug-Resistant (MDR) cancers attenuate chemotherapeutic efficacy through drug efflux, a process that transports drugs from within a cell to the extracellular space via ABC (ATP-Binding Cassette) transporters, including P-glycoprotein 1 (P-gp or ABCB1/MDR1). Conversely, Toll-Like Receptor (TLR) agonist immunotherapies modulate activity of tumor-infiltrating immune cells in local proximity to cancer cells and could, therefore, benefit from the enhanced drug efflux in MDR cancers. However, the effect of acquired drug resistance on TLR agonist efflux is largely unknown. We begin to address this by investigating P-gp mediated efflux of TLR 7/8 agonists. First, we used functionalized liposomes to determine that imidazoquinoline TLR agonists Imiquimod, Resiquimod, and Gardiquimod are substrates for P-gp. Interestingly, the least potent imidazoquinoline (Imiquimod) was the best P-gp substrate. Next, we compared imidazoquinoline efflux in MDR cancer cell lines with enhanced P-gp expression relative to parent cancer cell lines. Using P-gp competitive substrates and inhibitors, we observed that imidazoquinoline efflux occurs through P-gp and, for Imiquimod, is enhanced as a consequence of acquired drug resistance. This suggests that enhancing efflux susceptibility could be an important consideration in the rational design of next generation immunotherapies that modulate activity of tumor-infiltrating immune cells.

Comparing the immunogenicity of glycosidase-directed resiquimod prodrugs mediated by cancer cell metabolism.

We have recently developed an enzyme-directed immunostimulant (EDI) prodrug motif, which is metabolized to active immunostimulant by cancer cells and, following drug efflux, activates nearby immune cells, resulting in immunogenicity. In this study, we synthesized several EDI prodrugs featuring an imidazoquinoline immunostimulant resiquimod (a Toll-like receptor 7/8 agonist) covalently modified with glycosidase enzyme-directing groups selected from substrates of ß-glucuronidase, α-mannosidase, or ß-galactosidase. We compared the glycosidase-dependent immunogenicity elicited by each EDI in RAW-Blue macrophages following conversion to active immunostimulant by complementary glycosidase. At a cellular level, we examined EDI metabolism across three cancer cell lines (B16 melanoma, TC2 prostate, and 4T1 breast cancer). Comparing the relative immunogenicity elicited by each EDI/cancer cell combination, we found that B16 cells produced the highest EDI prodrug immunogenicity, achieving >95% of that elicited by unmodified resiquimod, followed by TC2 and 4T1 cells (40% and 30%, respectively). Immunogenicity elicited was comparable for a given cell type and independent of the glycosidase substrate in the EDIs or differences in functional glycosidase activity between cell lines. Measuring drug efflux of the immunostimulant payload and efflux protein expression revealed that EDI/cancer cell-mediated immunogenicity was governed by efflux potential of the cancer cells. We determined that, following EDI conversion, immunostimulant efflux occurred through both P-glycoprotein-dependent and P-glycoprotein-independent transport mechanisms. Overall, this study highlights the broad ability of EDIs to couple immunogenicity to the metabolism of many cancers that exhibit drug efflux and suggests that designing future generations of EDIs with immunostimulant payloads that are optimized for drug efflux could be particularly beneficial.

An Enzyme-Directed Imidazoquinoline Activated by Drug Resistance.

Drug efflux and enzymatic drug degradation are two cellular mechanisms that contribute to drug resistance in many cancers. Herein, we report the synthesis and in vitro activity of a pro-immunostimulant that exploits both processes in tandem to selectively confer cancer-mediated immunogenicity. We demonstrate that an imidazoquinoline pro-immunostimulant is inactive until it is selectively metabolized to an active immunostimulant by an endogenous α-mannosidase enzyme expressed within multidrug-resistant cancer cells. Following conversion, the immunostimulant is transported to the extracellular space via drug efflux, resulting in the activation of model bystander immune cells. Taken together, these results suggest that enzyme-directed immunostimulants can couple immunogenicity to these mechanisms of drug resistance. We name this process bystander-assisted immunotherapy, and envision that it could be advanced to treat drug-resistant diseases that rely on enzymatic degradation or drug efflux to persist.