Preclinical and clinical characterization of the RORγt inhibitor JNJ-61803534

The nuclear receptor retinoid-related orphan receptor gamma t (RORγt) plays a critical role in driving Th17 cell differentiation and expansion, as well as IL-17 production in innate and adaptive immune cells. The IL-23/IL-17 axis is implicated in several autoimmune and inflammatory diseases, and biologics targeting IL-23 and IL-17 have shown significant clinical efficacy in treating psoriasis and psoriatic arthritis. JNJ-61803534 is a potent RORγt inverse agonist, selectively inhibiting RORγt-driven transcription versus closely-related family members, RORα and RORβ. JNJ-61803534 inhibited IL-17A production in human CD4+ T cells under Th17 differentiation conditions, but did not inhibit IFNγ production under Th1 differentiation conditions, and had no impact on in vitro differentiation of regulatory T cells (Treg), nor on the suppressive activity of natural Tregs. In the mouse collagen-induced arthritis model, JNJ-61803534 dose-dependently attenuated inflammation, achieving ~ 90% maximum inhibition of clinical score. JNJ-61803534 significantly inhibited disease score in the imiquimod-induced mouse skin inflammation model, and dose-dependently inhibited the expression of RORγt-regulated genes, including IL-17A, IL-17F, IL-22 and IL-23R. Preclinical 1-month toxicity studies in rats and dogs identified doses that were well tolerated supporting progression into first-in-human studies. An oral formulation of JNJ-61803534 was studied in a phase 1 randomized double-blind study in healthy human volunteers to assess safety, pharmacokinetics, and pharmacodynamics. The compound was well tolerated in single ascending doses (SAD) up to 200 mg, and exhibited dose-dependent increases in exposure upon oral dosing, with a plasma half-life of 164 to 170 h. In addition, dose-dependent inhibition of ex vivo stimulated IL-17A production in whole blood was observed, demonstrating in vivo target engagement. In conclusion, JNJ-61803534 is a potent and selective RORγt inhibitor that exhibited acceptable preclinical safety and efficacy, as well as an acceptable safety profile in a healthy volunteer SAD study, with clear evidence of a pharmacodynamic effect in humans.

In vitro human Treg differentiation Total CD4 + T cells were isolated as described above, and seeded in a 96-well plate at 2×10 5 cells per well. Titrated JNJ-61803534 was added to each well, followed by addition of anti-CD3/CD28 beads at final concentration of 4x10 6 /mL, and medium only for Th0 or cocktail for Treg differentiation at the final concentration of anti-IL-4 at 10 µg/mL, anti-IFN at 10 µg/mL, anti-IL-12 10 µg/mL, TGF-β1 10 ng/mL and IL-2 200 U/mL. Cells were cultured at 37°C and 5% CO2 for 6 days, then cell pellets were collected and lysed and FOXP3 gene expression was measured using Quantigene 2.0 assay.

In vitro whole blood assay
For human blood assay, heparinized whole blood was collected from healthy volunteers and diluted 1:1 with RP RPMI1640 (R&D), then was added to a 96-well plate at 200 µl per well. DMSO or titrated JNJ-61803534 was added to the blood samples at 2 µL/well for a final DMSO concentration of 0.2% and incubated at 37°C for 1 hr. A 10 µL stimulation cocktail, was added to each well at the final concentrations: 1 µg/mL anti-CD28 (eBioscience), 50 ng/mL IL-23 (R&D systems), 10 ng/mL IL-1β (R&D systems). The blood was then transferred to an anti-CD3 pre-coated plate (1 µg/mL BD Biosciences) and incubated at 37°C for 2 days. For mouse blood assay, heparinized mouse blood was diluted 1:4 (5-fold dilution) in RPMI1640, stimulated with pre-coated anti-CD3 and soluble anti-CD8 (4 µg/mL each; R&D) and IL-23 (50 ng/mL; R&D) at 37°C for 42 hrs. Plasma was collected after incubation and human or mouse IL-17A levels were determined by ELISA, using commercial ELISA kits (R&D).

RNA extraction, quantitative RT-PCR
Frozen ear samples from IMQ model were homogenized in RNAzol®RT (Sigma-Aldrich) and were mixed with 200 μL RNase-free water and incubated for 10 min at room temperature. Samples were then centrifuged and ~500 μL supernatant was collected and mixed with 200 μL 75 % ethanol then transferred to one well of a RNeasy 96 well plate (Qiagen). Total RNA was prepared as described in the manual of the RNeasy 96 Kit. The RNAs were reverse transcribed into cDNA using the Superscript II reverse transcriptase (Life Technologies), then real time PCR reactions (Taqman) were performed on an ABI Prism 7900HT Sequence Detection System using predesigned qPCR Assays (Integrated DNA Technologies, Inc.) containing validated probe/primer forward/primer reverse combinations for the individual genes: mIL-17A (TCCACCGCAATGAAGACCCTGATAG; AGACTACCTCAACCGTTCCA; GAGCTTCCCAGATCACAGAG); mIL-17F (ACCCGTGAAACAGCCATGGTCAA; TGGAGAAACCAGCATGAAGTG; TGGAGAAACCAGCATGAAGTG); mIL-22 (AGCCGTACATCGTCAACCGCA; AGCTTGAGGTGTCCAACTTC; GGTAGCACTGATCTTTAGCACTG). For calculation of relative changes in gene expression the ddCT algorithm (1) was applied and TATA-Box binding protein (TBP) was used for normalization.

Flow cytometry analysis
Single cell suspensions were prepared by incubating ear samples from IMQ model with 0.05 mg/mL DNase and 0.4 mg/mL Liberase (Roche) in RPMI 37°C for 1 h. Cells were stained with fluorescenceconjugated antibodies against surface markers for different cells types. Intracellular staining for IL-17A and IL-22 was performed on cells incubated with a leukocyte activation cocktail containing brefeldin A, PMA and ionomycin for 4 h and stained with anti-TCR antibodies. The stained cells were acquired with the flow cytometer Canto II (BD Biosciences), and the data was analyzed using the FlowJo version 9.7.5 (Treestar). The following mouse antibodies were used for staining: anti-CD45.2-APC-ef780, anti- TCR-FITC, anti-CD11b-Biotin, anti-F4/80-PE, purchased from eBioscience; anti-CD3-PerCp, anti-CD4-V450, anti-CD19-Biotin, anti-Gr1-APC, purchased from BD Biosciences; anti-IL-17A-APC (eBioscience) and anti-IL-22-PE (Biolegend).  Supple Figure S1. Effect of JNJ-61803534 on Treg function for its suppression on the proliferation of effector T cells. Data are presented as mean ± SD, n=3.

Suppl
Suppl  Table S2. Plasma exposure and percent inhibition of ex vivo IL-17A production at various time points after oral administration of 100 mg/kg JNJ-61803534. Exposure levels measured in undiluted plasma (in ng/mL and µM) and calculated for 5-fold dilution (in µM) are presented as mean ± SD (n=10) Suppl Figure Table S5. Pharmacokinetic results of JNJ-61803534 after administration of a single dose in healthy volunteers. (a) under fasted condition; (b) under fasted and fed conditions. All presented as Mean (SD), except tmax as median (range). Cmax = Maximum observed plasma concentrations during a dosing interval; Tmax = Time to reach the maximum observed plasma concentration; AUC216h = area under the plasma concentration-time curve from time 0 through 216 hours; AUClast = Area under the plasma concentration-time curve from time 0 to time of the last quantifiable concentration; AUC∞= Area under the plasma concentration-time curve from time 0 to infinite time, calculated as the sum of AUClast and Clast/z, in which Clast is the last observed quantifiable concentrations; t1/2 = Elimination half-life associated with the terminal slope (z) of the semilogarithmic drug concentration-time curve, calculated as 0.693/z; z = First-order rate constant associated with the terminal portion of the curve, determined as the negative slope of the terminal log-linear phase of the drug concentration-time curve; CL/F = Total clearance of drug after extravascular administration, uncorrected for absolute bioavailability, calculated as: dose (D)/AUC; Vdz/F = Apparent volume of distribution after extravascular administration, uncorrected for absolute bioavailability.