POS0224 SELECTIVITY OF CLINICAL JAK INHIBITORS AND THE IMPACT ON NATURAL KILLER (NK) CELL FUNCTIONAL RESPONSES
Authors: P. Gonzalez-Traves, L. Simpson, B. Murray, A. Meng, J. A. Di Paolo, E. Grant, G. Min-Oo
Journal: Annals of the Rheumatic Diseases (2021)

Background: Janus kinase (JAK) inhibitors (JAKinibs) show similar efficacy in rheumatoid arthritis (RA). However, in vitro studies have shown differences in JAK selectivity profiles for baricitinib (BARI), tofacitinib (TOFA), upadacitinib (UPA) and filgotinib (FIL). 1,2 These lead to distinct pharmacologic profiles in cellular signaling assays that may impact clinical efficacy or safety 1 . NK cells are innate lymphocytes important in anti-pathogen responses and immune surveillance, which function via production of cytokines and cell killing 3 . NK cell proliferation and IFNγ production are JAK-dependent pathways and may be modulated by JAKinibs. Clinical findings show transient decreases in NK cell numbers in patients treated with JAKinibs, but the link to safety is unclear 4 Objectives: To extend upon findings in proximal cell signaling assays, we compared the selectivity and potency of clinical JAKinibs on NK cell function by assessing proliferation mediated by IL-15 (JAK1/3) and IFN-γ production driven by IL-12 (JAK2/TYK2)+IL-18. Methods: NK cells were isolated from healthy donor PBMC, incubated in vitro with 8 concentrations of each evaluated JAKinib (TOFA, BARI, FIL, FIL metabolite, UPA) and stimulated with IL-15 for proliferation or IL-12/18 for IFNγ production. Proliferation was assessed by Cell Trace dye dilution after 6 days and IFNγ production by intracellular flow cytometry 4hrs post-stimulation. Half maximal inhibitory concentration (IC 50 ) values were calculated for CD56 bright , CD56 dim , and total NK cells. Steady-state pharmacologic profile over a clinical dosing interval was modeled using concentration-time profiles from JAKinib population pharmacokinetic data in RA subjects under the therapeutic dose 5-7 . For each JAKinib, the time above IC 50 and average daily inhibition of IFNγ or proliferation were calculated for each NK cell population in each donor. Results: Cellular assays in purified NK cells showed dose-dependent inhibition of IL-15-induced proliferation by all JAKinibs with TOFA showing the highest average inhibition and time above IC 50 (35-60% inhibition for 8-15 hrs; TOFA&gt;UPA&gt;BARI≈FIL). The differences between JAKinibs are in line with differences in pSTAT inhibition downstream of IL-15 1 . Interestingly, IL-12/18-induced production of IFNγ, which is mediated via JAK2/TYK2 (IL-12) and non-JAK dependent pathways (IL-18), showed weaker inhibition for all compounds. Moreover, all JAKinibs showed &lt;25% average inhibition of IFNγ production over 24hrs and did not show any time above IC 50 for IFNγ production or pSTAT4 inhibition at clinical doses. CD56 dim and CD56 bright sub-populations of NK cells are proposed to have distinct functions and unique expression of surface receptors. Analysis of the IC 50 for pSTAT4 and IFNγ production showed ~2-10-fold weaker inhibition by JAKinibs in CD56 bright NK cells, suggesting less dependence on JAK-dependent signals in CD56 bright NK cells than CD56 dim NK cells. Conclusion: NK cell proliferation depends on JAK1 and JAK3-mediated signaling and is differentially inhibited at clinical doses of distinct JAKinibs. In contrast, functional responses downstream of JAK2/TYK2-dependent IL-12/18 were not substantially inhibited by any of the JAKinibs studied. Inhibition of functional and proliferative responses in purified NK cells aligned well with proximal pSTAT inhibition. JAKinib modulation of NK cell proliferation, but not response to IL-12, reflects unique pharmacologic profiles of the drugs studied and could be one component underlying clinical safety observations, including increased risk of viral infections or malignancy 4 . References: [1]Traves PG et al. Ann Rheum Dis 2021 (in press) [2]McInnes IB, et al. Arthritis Res Ther 2019;21:183. [3]Cooper MA, Fehniger TA, Caligiuri MA. Trends Immunol 2001 Nov;22(11):633-40. [4]Winthrop KL. Nat Rev Rheumatol 2017; 13(4):234-243 [5]Zhang X, et al. CPT Pharmacometrics Syst Pharmacol 2017;6(12):804-13. [6]CDER. Application Number: 203214Orig1s000. NDA 203214: Tofacitinib. [7]Klunder B et al. Clin Pharmacokinet 2019;58(8):1045-58. Disclosure of Interests: Paqui Gonzalez-Traves Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Laura Simpson Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Bernard Murray Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Amy Meng Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Julie A. Di Paolo Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Ethan Grant Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Gundula Min-Oo Shareholder of: Gilead Sciences, Employee of: Gilead Sciences