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

Abstract

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 β-glucuronidase, α-mannosidase, or β-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.

Acquired drug resistance enhances imidazoquinoline efflux by P-glycoprotein

“…Previously, our own work suggested that IMQ and RSQ undergo drug efflux from a range of MDR or parent cancer cell lines (30,31) and in the present study, we confirm that IMQ, RSQ, and GDQ are in fact substrates of P-gp using an ATPase membrane transport study (Figure 4). We also conclude that, although less potent, IMQ is a better substrate when compared to RSQ and GDQ in most of the MDR-derived cancer cell lines.…”

“…In particular, intratumoral administration of imidazoquinoline immunotherapeutics, which presumably undergo drug efflux, have demonstrated efficacy across a range of cancers both as mono (27) and combination (28) immunotherapies. This has led our group (29)(30)(31) and others (32)(33)(34)(35) to develop drug delivery strategies that liberate imidazoquinolines within cancer cells or the tumor microenvironment. As such, knowing a pathway for imidazoquinoline efflux, and how MDR affects efflux (Figure 1), would be valuable in designing the next generation of TLR agonists targeted to MDR cancers.…”

Trial Watch: experimental TLR7/TLR8 agonists for oncological indications

“…Ryan et al (Washington State University, Pullman, WA, USA) developed an enzyme-directed immunostimulant (EDI) prodrug of resiquimod, which can be metabolized to the active drug by cancer cells and ultimately released into the tumor microenvironment to mediate immunostimulatory effects. 94 This agent exhibited immunostimulatory activity in a panel of transplantable mouse models of melanoma (B16 cells), prostate carcinoma (TC2 cells), and breast carcinoma (4T1 cells). 94 Lu and colleagues (University of Kansas, Lawrence, KS, USA) tested yet another prodrug-based nanocarrier delivery system for resiquimod.…”

“… 94 This agent exhibited immunostimulatory activity in a panel of transplantable mouse models of melanoma (B16 cells), prostate carcinoma (TC2 cells), and breast carcinoma (4T1 cells). 94 Lu and colleagues (University of Kansas, Lawrence, KS, USA) tested yet another prodrug-based nanocarrier delivery system for resiquimod. 92 Specifically, resiquimod was linked with α-tocopherol to generate a prodrug that was further modified with hyaluronic acid into a polymeric nano-suspension (HA-Toco).…”

Nanomedicine and cancer immunotherapy

“…The elicited immunogenicity was compared for different cell types and found to be independent of the glycosidase substrate in the EDI or differences in functional glycosidase expression across each cell line. This study highlights the broad applicability of EDIs to the cancers that exhibit drug efflux, which suggest that EDIs optimized for drug efflux could be particularly beneficial for improving cancer immunotherapy [ 11 ].…”