Immunostimulatory TLR7 agonist-nanoparticles together with checkpoint blockade for effective cancer immunotherapy.

Mono- or dual-checkpoint inhibitors for immunotherapy have changed the paradigm of cancer care; however, only a minority of patients responds to such treatment. Combining small molecule immuno-stimulators can improve treatment efficacy, but they are restricted by poor pharmacokinetics. In this study, TLR7 agonists conjugated onto silica nanoparticles showed extended drug localization after intratumoral injection. The nanoparticle-based TLR7 agonist increased immune stimulation by activating the TLR7 signaling pathway. When treating CT26 colon cancer, nanoparticle conjugated TLR7 agonists increased T cell infiltration into the tumors by > 4× and upregulated expression of the interferon γ gene compared to its unconjugated counterpart by ~2×. Toxicity assays established that the conjugated TLR7 agonist is a safe agent at the effective dose. When combined with checkpoint inhibitors that target programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), a 10-100× increase in immune cell migration was observed; furthermore, 100 mm3 tumors were treated and a 60% remission rate was observed including remission at contralateral non-injected tumors. The data show that nanoparticle based TLR7 agonists are safe and can potentiate the effectiveness of checkpoint inhibitors in immunotherapy resistant tumor models and promote a long-term specific memory immune function.

Conjugation of a Small-Molecule TLR7 Agonist to Silica Nanoshells Enhances Adjuvant Activity.

Stimulation of Toll-like receptors (TLRs) and/or NOD-like receptors on immune cells initiates and directs immune responses that are essential for vaccine adjuvants. The small-molecule TLR7 agonist, imiquimod, has been approved by the FDA as an immune response modifier but is limited to topical application due to its poor pharmacokinetics that causes undesired adverse effects. Nanoparticles are increasingly used with innate immune stimulators to mitigate side effects and enhance adjuvant efficacy. In this study, a potent small-molecule TLR7 agonist, 2-methoxyethoxy-8-oxo-9-(4-carboxybenzyl)adenine (1V209), was conjugated to hollow silica nanoshells (NS). Proinflammatory cytokine (IL-6, IL-12) release by mouse bone-marrow-derived dendritic cells and human peripheral blood mononuclear cells revealed that the potency of silica nanoshells-TLR7 conjugates (NS-TLR) depends on nanoshell size and ligand coating density. Silica nanoshells of 100 nm diameter coated with a minimum of ∼6000 1V209 ligands/particle displayed 3-fold higher potency with no observed cytotoxicity when compared to an unconjugated TLR7 agonist. NS-TLR activated the TLR7-signaling pathway, triggered caspase activity, and stimulated IL-1ß release, while neither unconjugated TLR7 ligands nor silica shells alone produced IL-1ß. An in vivo murine immunization study, using the model antigen ovalbumin, demonstrated that NS-TLR increased antigen-specific IgG antibody induction by 1000× with a Th1-biased immune response, compared to unconjugated TLR7 agonists. The results show that the TLR7 ligand conjugated to silica nanoshells is capable of activating an inflammasome pathway to enhance both innate immune-stimulatory and adjuvant potencies of the TLR7 agonist, thereby broadening applications of innate immune stimulators.