Background Systemic lupus erythematosus (SLE), a chronic autoimmune disease, is characterized by high clinical and molecular heterogeneity and the presence of autoantibodies with various specificities, including those against DNA and RNA. While steroids, such as prednisolone, are a part of the standard-of-care for SLE, alternative treatments that overcome steroid resistance and minimize the risk of numerous short- and long-term side effects associated with their use remain an important unmet need. Toll-like receptors 7 and 8 (TLR7/8), two endosomal receptors expressed in various immune cells, play key roles in innate immunity through recognition of single-stranded RNA and driving downstream signaling via the nuclear factor kappa B (NFkB) and interferon regulatory factor (IRF) pathways. Activation of the NFkB pathway via TLR7/8 along with inhibition of plasmacytoid dendritic cell (pDC) apoptosis via TLR7 are reported to contribute to steroid resistance.

Objectives In vitro determination of the potency and modes of action of afimetoran in whole blood (WB) of healthy volunteers (HVs) and patients with SLE, as well as probing the modes of action for the steroid-sparing potential of afimetoran.

Methods Afimetoran was evaluated in vitro for its ability to inhibit TLR7- and TLR8-mediated interleukin 6 (IL-6) induction in WB. Briefly, WB samples from HVs and patients with SLE were treated with afimetoran for 1 hour before the addition of the TLR7 or TLR8 agonists. Following overnight incubation, supernatants were collected, and IL-6 was quantified by ELISA. The same assay was used to determine the pharmacodynamic activity of afimetoran in WB from HVs in a phase 1 multiple ascending dose study (NCT03634995). The potency of afimetoran to inhibit TLR7 agonist-induced CD69 expression on B cells and TLR8 agonist-induced CD319 expression on monocytes from WB was assessed using flow cytometry. The steroid-sparing potential of afimetoran was evaluated in vitro in combination with prednisolone through inhibition of IL-6 induction in WB from HVs and reversal of TLR7 agonist-induced resistance to prednisolone-induced pDC apoptosis, which was measured by flow cytometry staining of annexin V.

Results Afimetoran equipotently inhibited TLR7- and TLR8-mediated induction of IL-6 in WB from HVs and patients with SLE. Afimetoran also equipotently inhibited surface expression of CD69 on B cells and CD319 on monocytes with single-digit nM IC50 values. Afimetoran robustly and durably inhibited TLR7- or TLR8-mediated induction of IL-6 in HV WB samples from a phase I multiple ascending dose clinical trial. When combined with prednisolone, afimetoran had a synergistic effect in inhibiting TLR7- and TLR8-mediated induction of IL-6 in WB of HVs and reversed TLR7-mediated resistance to prednisolone-induced pDC apoptosis.

Conclusion WB-based data show that afimetoran is an equipotent TLR7/8-dual antagonist with sub-nM to nM potency in blocking TLR7- and TLR8-mediated functions. WB- and cell-based evidence demonstrates that afimetoran in combination with prednisolone synergistically inhibits induction of IL-6, a NFkB-dependent cytokine, and reverses TLR7-mediated resistance to prednisolone-induced pDC apoptosis, thus supporting the steroid-sparing potential of afimetoran in patients with SLE.

Acknowledgements Medical writing support was provided by Gleb Baida, PhD, of Spark Medica Inc, and editorial support was provided by Agata Shodeke of Spark Medica Inc.

Disclosure of Interests Qihong Zhao Employee of: Bristol Myers Squibb, Alaric Dyckman Employee of: Bristol-Myers Squibb, Gary Schieven Employee of: Genesis Research and Development Institutes (current employee)

Bristol-Myers Squibb (Former employee), Hongchen Qiu Employee of: Bristol-Myers Squibb, Rosemary Zhang Employee of: Bristol-Myers Squibb, Mary Ellen Cvijic Shareholder of: Bristol-Myers Squibb (as an employee), Employee of: Bristol-Myers Squibb, Melanie Harrison Shareholder of: Bristol-Myers Squibb (as an employee), Employee of: Bristol-Myers Squibb, Manoj Chiney Shareholder of: Bristol-Myers Squibb, Employee of: current employee of Gilead Sciences.

Bristol Myers Squibb (Former), Diane Shevell Shareholder of: holds stock in Novartis, Employee of: former Bristol Myers Squibb employee; current employee of Novartis, Ihab Girgis Shareholder of: hold stocks in Bristol Myers Squibb, Employee of: current employee of CSL Behring; former Bristol Myers Squibb employee, Frederic Baribaud Shareholder of: hold stocks in Bristol Myers Squibb, Employee of: Bristol Myers Squibb employee, Kristina Chadwick Shareholder of: holds stock in Bristol Myers Squibb, Employee of: Bristol Myers Squibb employee, Shailesh Dudhgaonkar Shareholder of: holds stock in Bristol Myers Squibb, Employee of: Bristol Myers Squibb employee.

Keywords: Systemic lupus erythematosus, Cytokines and chemokines, Targeted synthetic drugs

DOI: 10.1136/annrheumdis-2023-eular.2549