(T1430-03-16) Evaluation of a Toll-Like Receptor 7/8 Antagonist for Anti-Inflammatory Immunotherapy

Purpose: Immunotherapy serves to modulate the host immune system to fight foreign pathogens or suppress autoimmunity. An emerging immunotherapy strategy is to stimulate the innate immune system through targeting pattern recognition receptors, such as toll-like receptors (TLRs). Stimulation of TLRs leads to activation of the transcription factor NF-κB and subsequent expression of inflammatory genes, cytokines, and costimulatory molecules designed to mount a potent immune response.¹ These classes of receptors respond to a wide variety of both extracellular and intracellular pathogen signals. Specifically, TLRs 7 and 8 are located within the endosome of many immune cells and respond chiefly to viral mRNA. Thus, the development of drugs that mimic mRNA structure can lead to selective TLR7/8 agonists/antagonists that trigger a potent innate immune response.¹ In this work, we assess a novel TLR7/8 antagonist, 621, for it’s ability to create an anti-inflammatory immune response in vitro and it’s potential to maintain or increase this response during inflammatory challenges using stimulation by lipopolysaccharide (LPS) or TLR7/8 agonist resiquimod. Furthermore, we assessed 621’s potential in reducing inflammation and disease progression in DSS-colitis model, a mouse model of inflammatory bowel disease. We hypothesize that antagonizing TLR 7 and 8 will induce a robust anti-inflammatory response that can mitigate inflammatory stimuli and diseases.

Methods: Initial in vitro evaluation of toll-like receptor 7/8 antagonist 621 was performed by measuring the release of the anti-inflammatory cytokine IL-10 and the pro-inflammatory cytokine TNF-α from both bone marrow-derived dendritic cells (BMDCs) and splenocytes. The ratio IL-10 to TNF-α was compared by TLR stimulation by 621 alone or by co-stimulation from bacterial liposaccharide (LPS) or resiquimod and compared to another TLR7/8 agonist, 558. Flow cytometry analysis was performed to quantify expression of BMDC cell surface marker expression following 621 or 558 treatments. A DSS-induced colitis model was employed in C57BL/6 female immunocompetent mice alongside daily intraperitoneal (IP) injections of 621 or 558 to assess the inflammatory protection and disease progression. Mouse weight and colon length were measured. Immunophenotyping of systemic immune cells from harvested spleens was performed via flow cytometry. Colon-specific cytokines and myeloperoxidase (MPO) levels were assessed from homogenized colon biopsies and quantified via enzyme-linked immunosorbent assays (ELISAs). Colon morphology was assessed following hematoxylin and eosin (H&E) histology staining.

Results: Dosing of 1 µM 621 led to an increased IL-10/TNF-α ratio in BMDCs and splenocytes both with or without LPS or resiquimod stimulation in vitro and the addition of either LPS or resiquimod further stimulated production of IL-10 from 621-stimulated cells without an increase in TNF-α (Figure 1). Expression of costimulatory markers CD40, CD80, or CD86 on BMDCs were not increased after 621 treatment. In the DSS colitis mouse disease model, 621-dosed mice lost less weight than PBS-dosed mice (p < 0.01), while no difference in colon length was observed (p >0.05). IP administration of 621 to DSS mice led to increased levels of systemic Foxp3+, IL-10+, and TGF-β1+ CD4+ T-cells and IL-10 expressing macrophages (Figure 2). Colon inflammatory cytokines (TNF-α & IL-1β) significantly decreased while IL-10 levels significantly increased from 621 treatment compared to PBS-dosed DSS mice (p < 0.01). Colon MPO levels in DSS mice were significantly reduced with either 558 or 621 administration (p < 0.01). Furthermore, administration of 621 led to visual improvements in colon morphology following DSS treatment.

Conclusion: Initial discovery and evaluation of potential new immunomodulatory drugs is crucial to develop increasingly potent and targeted immunotherapies. Here, we show evaluation of a TLR7/8 antagonist, 621, and its ability to stimulate high levels of the anti-inflammatory cytokine IL-10 from immune cells, even when faced with an inflammatory challenge. IP injections of 621 led to a suppression of inflammatory cytokines in the colon and promoted systemic anti-inflammatory T-cell phenotypes and a reduction of DSS-induced colitis in mice. This data suggests that 621, and potentially other TLR antagonists, may be promising candidates for anti-inflammatory immunotherapies. Ongoing work and the larger goal of this study will be the formulation of 621 into a mucosal binding nanoparticle for enhanced protection, bioavailability, and endosomal delivery for improved TLR7/8 delivery.

References: Luchner, M., Reinke, S., & Milicic, A. (2021). TLR Agonists as Vaccine Adjuvants Targeting Cancer and Infectious Diseases. Pharmaceutics, 13(2), 142.


Figure 1. IL-10/TNF-α ratio from BMDCs stimulated with 621 or 558 and with LPS or resiquimod inflammatory challenge. n=5. **** p<0.0001


Figure 2. Immunophenotyping of immune cells harvested from DSS colitis mice. n=5. **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05