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.

Herein we report the synthesis and activity of an enzyme-directed immunostimulant with immune cell activation mediated by β-galactosidase, either exogenously added, or on B16 melanoma cells. Covalent attachment of a β-galactopyranoside to an imidazoquinoline immunostimulant at a position critical for activity resulted in a pro-immunostimulant that could be selectively converted by β-galactosidase into an active immunostimulant. The pro-immunostimulant exhibited β-galactosidase-directed immune cell activation as measured by NF-κB transcription in RAW-Blue macrophages or cytokine production (TNF, IL-6, IL-12) in JAWSII monocytes. Conversion of the pro-immunostimulant into an active immunostimulant was also found to occur using β-galactosidase-enriched B16 melanoma cells. In co-culture experiments with either immune cell line, β-galactosidase-enriched B16 cells effected activation of bystander immune cells.

Synthetic agonists of innate immune cells are of interest to immunologists due to their synthesis from well-defined materials, optimized activity, and monodisperse chemical purity. These molecules are used in both prophylactic and therapeutic contexts from vaccines to cancer immunotherapies. In this review we highlight synthetic agonists that activate innate immune cells through three classes of pattern recognition receptors: NOD-like receptors, RIG-I-like receptors, and C-type lectin receptors. We classify these agonists by the receptor they activate and present them from a chemical perspective, focusing on structural components that define agonist activity. We anticipate this review will be useful to the medicinal chemist as a guide to chemical motifs that activate each receptor, ultimately illuminating a chemical space ripe for exploration.

Imidazoquinoline Toll-Like Receptor (TLR) agonists such as Imiquimod are effective immunotherapeutics in the treatment of basal skin cell carcinoma, and more recently, melanoma. However, direct injection of imidazoquinoline TLR agonists causes systemic inflammation and autoimmune side-effects thereby limiting clinical use to topical applications. Here we use a metabolic caging strategy to confine the immunostimulatory effects of imidazoquinoline TLR agonists to tumor microenvironment. We have covalently attached a galactopyranoside metabolic cage to the primary amino group, critical for activity, of the TLR7 agonist Imiquimod. Addition of the metabolic cage abrogates TLR agonism as measured by NFκB transcription in RAW-Blue cells or inflammatory cytokines produced by primary murine Bone Marrow Derived Dendritic Cells (BMDCS). The galactopyranoside cage is efficiently removed via enzymatic cleavage from exogenously added β-galactosidase resulting in activation of immune cells that is comparable to native agonist. Enzymatic cleavage also occurs using β-galactosidase that is overexpressed in B16 melanoma cells. This results in increased immunostimulatory effects in co-cultures of BMDCs and B16 melanoma cells relative to BMDCs co-cultured with healthy melanocytes. We anticipate this will provide a general framework upon which TLR agonists can be targeted to the tumor microenvironment.

Acquired drug resistance is a longstanding challenge that reduces the efficacy of chemotherapeutic drugs. In contrast, the efficacies of modern immunotherapies do not directly correlate to the prevalence of chemo-resistant phenotypes. Here we improve upon this paradigm by targeting the action of immunotherapeutics to two general mechanisms of acquired drug resistance: irregular metabolism and drug efflux mediated by the ABC superfamily of transport proteins. In chemo-resistant prostate cancer cells, we demonstrate that these two mechanisms act in concert to selectively metabolize our newly developed class of enzyme-directed prodrug to the Toll-Like Receptor 7 immunotherapeutic Imiquimod. Following metabolism, liberated Imiquimod undergoes drug efflux to the extracellular space where it activates bystander immune cells in local proximity. In-vitro, we characterize this process of Bystander-Assisted ImmunoTherapy (BAIT) using an AT3B-1 chemo-resistant prostate cancer model with RAW-Blue and JAWSII reporter immune cell lines. Co-culture of AT3B-1 cancer cells with reporter immune cells results in immunogenicity mediated selectively by cancer cells. This results in enhanced NF-κB transcription, expression of cell surface markers, and secreted cytokines indicative of a cell-mediated immune response. Our prodrug is non-immunogenic with healthy cells alone and the enzyme-directing groups are stable for over 3 days in serum. Taken together, our results demonstrate that BAIT co-opts common mechanisms of drug resistance to elicit immunogenicity mediated by cancer cells themselves. We anticipate that BAIT will find use as a new mechanism of action that exploits drug resistant phenotypes to generate an immune response.