Polymer-Drug Conjugates for STING Pathway Activation to Enhance Cancer Immunotherapy

Introduction:: Immune checkpoint blockade is revolutionizing treatment of diverse cancer types; however, these treatments only benefit a minority of cancer patients. Low response rates are often correlated to immunogenically “cold” tumors, which lack sufficient tumor antigen-specific CD8+ T-cell infiltration. Therefore, there is a need for therapeutics that shift tumors towards an immunogenically “hot” phenotype. One promising strategy is to activate the stimulator of interferon genes (STING) pathway, which triggers a type I interferon (IFN-I)-driven inflammatory response that enhances T cell priming, activation, and tumor infiltration. Although STING agonists are currently being explored in clinical trials, most are limited to intratumoral routes of administration, which is not ideal for all patients and tumor types. Small molecule STING agonists have recently been developed for systemic administration; however, these have suboptimal pharmacological properties and activate STING indiscriminately with potential to lead to systemic inflammation and toxicity that limits safety and efficacy. Therefore, we aim to develop a polymeric delivery platform for systemic administration of a novel, chemically-modified diamidobenzimidazoles (diABZI) STING agonist that enhances pharmacokinetics, biodistribution, and tumor-targeting with environmentally-responsive drug release to enhance efficacy and safety in treatment of breast cancer.

Materials and Methods:: We synthesized a small molecule STING diABZI prodrug with a cathepsin-cleavable linker for enzyme-mediated intracellular release and a DBCO reactive handle for conjugation to azide groups on carrier platforms. Polymeric carrier platforms consisting of poly(dimethyl acrylamide-co-azide ethyl methacrylate) at various molecular weights were synthesized through reversible addition-fragmentation chain-transfer polymerization and characterized through NMR and GPC. The diABZI STING agonist was conjugated to the carriers via click chemistry and conjugation efficiency was measured via UV-Vis spectrophotometry. Prodrug and conjugate activity was evaluated using interferon reporter assays and ELISA. Using an orthotopic E0771 breast cancer model, tumor and organ accumulation of intravenously-administered, fluorescently-labeled polymer-diABZI conjugates was evaluated through IVIS imaging and plasma half-life was determined by measuring polymer fluorescence in the blood as a function of time. Cell uptake in the tumor microenvironment was measured through flow cytometry. Therapeutic efficacy was evaluated in an orthotopic EO771 breast cancer model, comparing systemically administered 100kDa and 25kDa conjugates to a commercially-available diABZI STING agonist. Mice exhibiting complete responses were rechallenged with EO771 cells on day 60 post-primary inoculation.


Results, Conclusions, and Discussions:: We have developed polymer-drug conjugates utilizing a novel diABZI STING agonist that can be covalently linked to polymer backbones via click chemistry (Figure 1A) and have shown that drug conjugation to poly(dimethyl acrylamide) carriers improves drug circulation half-life, tumor biodistribution, and therapeutic efficacy. Conjugation of a the diAZBI prodrug to 25 kDa and 100 kDa poly(dimethyl acrylamide-co-azide ethyl methacrylate) resulted in a decreased activity in vitro (Figure 1B) compared to the parent compound, most likely due to the dependence on endocytosis and enzymatic drug release. Drug half-life of fluorescently-labeled conjugates was increased as a function of polymer molecular weight (Figure 1C), with a half-life of approximately 1 hour for the 25kDa conjugate and 4 hours for the 100kDa conjugate. IVIS imaging using an EO771 murine breast model demonstrated polymer accumulation of the 25kDa conjugate in the kidneys, liver, and tumor and accumulation of the 100kDa conjugate in the tumor and liver (Figure 1D-G). By minimizing renal clearance, the high molecular weight conjugate circulates longer and can accumulate in the tumor through the enhanced permeation and retention effect. Both conjugates demonstrated improved therapeutic response compared to the free diABZI STING agonist in the EO771 model with approximately 50% complete remission (Figure 1H-K). Mice exhibiting complete responses to the 100kDa platform were resistant to tumor rechallenge. Further analysis of polymer-STING agonist conjugates is expected to demonstrate reprogramming of the tumor-immune microenvironment and mitigated off-target STING activation and systemic inflammation.


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