Slide 1: Insights into the Aryl Hydrocarbon Receptor and miR-124 Axis
Insights into the Aryl Hydrocarbon Receptor and miR-124 Axis May 27th 2026 Stephen Connelly Ph.D., Chief Scientific Officer 1
Slide 2: The Aryl Hydrocarbon Receptor (AhR) as a Signaling Hub
The Aryl Hydrocarbon Receptor (AhR) as a Signaling Hub 2
Slide 3: The Aryl Hydrocarbon Receptor (AhR)
The Aryl Hydrocarbon Receptor (AhR) A ligand-activated transcription factor AhR Ligands Cytoplasm Signaling begins when a 1 The AhR ligand complex then translocates ligand binds to AhR in the to the nucleus and heterodimerizes with AhR cell cytoplasm, forming an the AhR nuclear translocator (ARNT) A
Slide 4: AhR as a Signaling Hub
AhR as a Signaling Hub Top 4 known genomic & non-genomic pathways induced by AhR by functional area Xenobiotic Response Immune Regulation Genes induced: Co-factors: Genes modulated: CYP1A1/A2 STAT1 IL-1β CYP1B1 STAT3 IL-6 NQO1 NF-κβ IL-10 Activated AhR UGT1A1,3,4,6,9 ROR𝛾t IL-22 Complex Barrier…
Slide 5: The AhR/miR-124 Axis
The AhR/miR-124 Axis or . equilliuM ———*
Slide 6: What is Known of the Relationship Between miR-124 and AhR
What is Known of the Relationship Between miR-124 and AhR miR-124 miR-124 Overexpression Inhibition Pre-miR-124 Anti-miR-124 Increased levels Decreased levels Zhao et al. reports an inverse correlation between miR-124 -ve → Feedback and AhR protein levels in tissues from active CD patients AhR → Ove
Slide 7: The Obefazimod (ABX464) Paradox
The Obefazimod (ABX464) Paradox • The Zhao et al. data is at odds with the pre-clinical and clinical data with ABX464, miR-124 where induction of miR-124 reduces inflammation and pro-inflammatory cytokines Induction ABX464 • This model can be reconciled if ABX464 is an AhR modulator inducing miR-124
Slide 8: Approach Taken
Approach Taken 1. In silico modelling: Used computational docking to assess ligand binding potential to PAS-B domain in AhR 2. AhR modulation by ABX464: Used validated AhR-luciferase assay, AhR modulators and an AhR antagonist to compare and confirm activation of AhR through PAS-B binding domain mec
Slide 9: In Silico Modeling of ABX464 and ABX464-N-Glu in AhR
In Silico Modeling of ABX464 and ABX464-N-Glu in AhR 9
Slide 10: AhR PAS-B Ligand Binding Domain
AhR PAS-B Ligand Binding Domain The large hydrophobic binding cleft can accommodate a diverse range of ligands Endogenous Ligands Synthesized in situ in tissues Indoles and tryptophan derivatives “In a 2025 computational drug discovery study targeting Natural Ligands the AhR PAS-B domain, Diet or sy
Slide 11: Pharmacophore Analyses of ABX464
Pharmacophore Analyses of ABX464 ABX464 shares structural similarity to other validated AhR modulators ABX464 / Obefazimod Laquinimod Quinoline core AQ-312 AhR Modulators ✓ Conformationally constrained ✓ Typically planar core ✓ Extended Aromatic heteroaromatic ✓ Moderate to high lipophilicity #68 Ka
Slide 12: Methodology Used for Ligand Docking
Methodology Used for Ligand Docking 3D Experimentally 2D Projection Solved Structure (7ZUB) Ligand Interaction Map (7ZUB) Identify structural templates 1 Assemble key structural templates and identify most appropriate one(s) to generate apo receptor Induced-fit docking protocol 2 Enable receptor fle
Slide 13: Ligand Binding Maps
Ligand Binding Maps ABX464 docks into the lipophilic PAS-B pocket leveraging conserved π-π stacking with key residue Phe295 Indirubin (7ZUB) ABX464 (model) PDB 7ZUB from Gruszczyk et al., Nat. Commun., 2022 13 Figures generated using MOE (MOE 2024.0601)
Slide 14: Ligand Binding Maps
Ligand Binding Maps ABX464 docks into the lipophilic PAS-B pocket leveraging conserved π-π stacking with key residue Phe295 Indirubin (7ZUB) ABX464 (model) PDB 7ZUB from Gruszczyk et al., Nat. Commun., 2022 14 Figures generated using MOE (MOE 2024.0601)
Slide 15: Effects of Glucuronidation of ABX464 on Potential PAS-B Binding
Effects of Glucuronidation of ABX464 on Potential PAS-B Binding Glucuronidation introduces polar steric bulk, disrupts planarity and hinders binding in the PAS-B pocket ABX464 (model) ABX464 Glucuronidated amine 90° 90° Likely Active ABX464-N-Glu 90° 90° Likely Inactive Figures generated using MOE (
Slide 16: Activation of AhR by ABX464
Activation of AhR by ABX464 16
Slide 17: Comparing Potencies of AhR Modulators
Comparing Potencies of AhR Modulators ABX464 is a moderately potent AhR modulator, but ABX464-N-Glu does not bind or modulate AhR AhR-luciferase 24hrAssay 24hr 11.19.25 Raw Data 11.19.25 Raw Data Compound Potency EC50 150 400000 400000 EQ504 EQ504 Compound EC50 (nM) RLU (Luminescence) RLU (Luminesce
Slide 18: AhR Antagonist GNF-351 Inhibits AhR Activation
AhR Antagonist GNF-351 Inhibits AhR Activation GNF-351 inhibits AhR activation by both EQ504 and ABX464 confirming binding to PAS-B domain AhR-luciferase Assay 1000000 ✱✱✱✱ ✱✱✱ GNF-351 (GNF) RLU (Luminescence) 800000 CAS No. : 1227634-69-6 600000 IC50 of 62 nM 400000 200000 1:5 2:1 0 GNF[500] GNF[50
Slide 19: Induction of miR-124 by AhR Activation in Immune Cells
Induction of miR-124 by AhR Activation in Immune Cells 19
Slide 20: Induction of miR-124 in PBMCs
Induction of miR-124 in PBMCs EQ504 & ABX464, but not ABX464-N-Glu, significantly induce miR-124 expression in PBMCs ✱✱ ✱✱✱✱ ✱✱✱✱ 40 ✱ compared with DMSO miR-124 fold change 30 ✱✱ 20 10 0 DMSO EQ504 [500] EQ504 [50] ABX464 [50] EQ504 [5000] ABX464 [5000] ABX464 [500] ABX464-N-Glu [50] ABX464-N-Glu [
Slide 21: Pro-Inflammatory Cytokine Profiling in PBMCs
Pro-Inflammatory Cytokine Profiling in PBMCs EQ504 & ABX464 significantly inhibit key pro-inflammatory cytokines release in PBMCs IL-17a IL-6 ✱ ✱✱✱✱ ✱✱✱ ✱✱✱✱ 150 150 ✱✱ ✱✱✱✱ normalized to DMSO normalized to DMSO IL-17a cytokine IL-6 cytokine 100 100 50 50 0 0 DMSO EQ504 [50] ABX464 [50] ABX464 [5000
Slide 22: Anti-inflammatory Cytokine Profiling in PBMCs
Anti-inflammatory Cytokine Profiling in PBMCs EQ504, but not ABX464, significantly induces IL-22 release in PBMCs IL-22 ✱ 250 normailzed to DMSO 200 IL-22 cytokine 150 100 50 0 DMSO EQ504 [50] ABX464 [50] ABX464 [5000] [nM] Total PBMCs treated with TransAct (CD3/CD28) stimulation for 48hrs, washed a
Slide 23: Effects of AhR Modulation and miR-124 in CD4+ T Cells
Effects of AhR Modulation and miR-124 in CD4+ T Cells Induction of miR-124 in isolated CD4+ T cells by both EQ504 & ABX464 is AhR-dependent ✱✱✱✱ ✱✱✱✱ ✱ 10 compared with DMSO miR-124 fold change 8 6 10:1 4 2 1:10 1:10 0 GNF [500] GNF [500] GNF [500] DMSO EQ504 [50] ABX464 [50] ABX464 [5000] EQ504 [50
Slide 24: Effects on IL-17a Expressing Cells in Isolated CD4+ T Cells
Effects on IL-17a Expressing Cells in Isolated CD4+ T Cells EQ504 & ABX464 significantly reduced number of IL17a+ expressing cells in isolated CD4+ T cells ✱✱✱✱ ✱✱✱✱ 150 IL-17a cytokine expression ✱✱✱✱ normalized to DMSO 100 50 0 EQ504 [50] ABX464 [5000] ABX464 [50] DMSO 5000nM Abivax data, Figure 3
Slide 25: Effects of AhR Modulation and miR-124 in M1 Macrophages
Effects of AhR Modulation and miR-124 in M1 Macrophages Induction of miR-124 in M1 macrophage cells by both EQ504 & ABX464 is AhR-dependent ✱ ✱ ✱ ✱ 50 compared with DMSO miR-124 fold change 40 30 20 10 1:10 10:1 1:10 0 GNF [500] DMSO EQ504 [50] ABX464 [50] ABX464 [5000] EQ504 [50] + GNF-351 [500] GN
Slide 26: Pro-Inflammatory Cytokine Profiling in M1 Macrophages
Pro-Inflammatory Cytokine Profiling in M1 Macrophages EQ504 & ABX464 significantly inhibit key pro-inflammatory cytokines release in M1 cells TNFα IL-6 ✱✱✱✱ ✱✱✱✱ ✱✱✱ ✱✱✱✱ 150 150 ✱✱✱✱ ✱✱✱✱ normalized to DMSO normalized to DMSO TNFα cytokine 100 IL-6 cytokine 100 50 50 0 0 DMSO DMSO EQ504 [50] ABX464
Slide 27: Pro-Inflammatory Cytokine Profiling in M1 Macrophages
Pro-Inflammatory Cytokine Profiling in M1 Macrophages EQ504 significantly inhibits key pro-inflammatory cytokines release in M1 cells IL-23 IL-12 IL-1β ✱✱✱✱ ✱ ✱✱✱✱ 150 150 150 ✱ normalized to DMSO ✱✱✱✱ normalized to DMSO normalized to DMSO IL-1β cytokine IL-12 cytokine IL-23 cytokine 100 100 100 50
Slide 28: Functional Consequence of AhR-antagonism on CYP1A1, miR-124, IL-10 and IL-22
Functional Consequence of AhR-antagonism on CYP1A1, miR-124, IL-10 and IL-22 ✓ Regulatory T Cells (Treg) ✓ Regulatory Macrophages (M2) 28
Slide 29: Effects of AhR Modulation on CYP1A1 and miR-124 in Regulatory T cells
Effects of AhR Modulation on CYP1A1 and miR-124 in Regulatory T cells Induction of CYP1A1 and miR-124 in regulatory T cells by both EQ504 & ABX464 is AhR-dependent CYP1A1 miR-124 ✱ ✱ 10 ✱✱ ✱ ✱✱✱ 15 compared with DMSO CYP1A1 fold change compared with DMSO 8 ✱ ✱ ✱✱ ✱ miR-124 fold change 6 10 4 1:10 5
Slide 30: AhR Antagonism Inhibits IL-10 & IL-22 Expression in Regulatory T Cells
AhR Antagonism Inhibits IL-10 & IL-22 Expression in Regulatory T Cells Induction of IL-10 and IL-22 in regulatory T cells by both EQ504 & ABX464 is AhR-dependent IL-10 IL-22 ✱✱ ✱✱ ✱✱✱✱ ✱ ✱✱ ✱ ✱✱ ✱✱ ✱✱✱✱ 200 300 normalized to DMSO normalized to DMSO 150 IL-22 cytokine IL-10 cytokine 200 100 1:10 10:1
Slide 31: AhR Antagonism Inhibits Decrease of AhR in Regulatory T Cells
AhR Antagonism Inhibits Decrease of AhR in Regulatory T Cells EQ504 & ABX464 decrease AhR expression in regulatory T cells AhR ✱✱✱✱ 150 ✱ normalized to DMSO AhR expression 100 1:10 1:10 10:1 50 0 GNF [500] DMSO EQ504 [50] ABX464 [50] ABX464 [5000] EQ504 [50] + GNF-351 ABX464 [5000] + GNF-351 ABX464
Slide 32: Effects of AhR Modulation on CYP1A1 and miR-124 in M2 Macrophages
Effects of AhR Modulation on CYP1A1 and miR-124 in M2 Macrophages Induction of CYP1A1 and miR-124 in M2 macrophages by both EQ504 & ABX464 is AhR-dependent CYP1A1 miR-124 ✱ ✱✱✱ ✱✱ ✱ ✱✱✱ ✱✱✱✱ ✱✱ ✱✱ ✱ 3 25 compared with DMSO miR-124 fold change compared with DMSO CYP1A1 fold change 20 2 15 10:1 1:10 1
Slide 33: Effects of AhR Modulation on IL-10 and IL-22 in M2 Macrophages
Effects of AhR Modulation on IL-10 and IL-22 in M2 Macrophages Induction of IL-10 and IL-22 in M2 macrophages by both EQ504 & ABX464 is AhR-dependent IL-10 IL-22 ✱ ✱ ✱✱ ✱✱ 4 fold change compared to DMSO fold change compared to DMSO 10 IL-22 relative expression IL-10 relative expression 3 5 2 10:1 1:
Slide 34: Induction of CYP1A1 and miR-124 by AhR Activation in Intestinal Epithelial Cell Lines
Induction of CYP1A1 and miR- 124 by AhR Activation in Intestinal Epithelial Cell Lines 34
Slide 35: Induction of CYP1A1 and miR-124 in Intestinal Epithelial Cell Line
Induction of CYP1A1 and miR-124 in Intestinal Epithelial Cell Line EQ504 & ABX464, significantly induce CYP1A1, but not miR-124 CYP1A1 miR-124 ✱✱ 10 10 miR-124 fold change compared with DMSO CYP1A1 fold change compared with DMSO ✱ ✱ 8 8 ✱ 6 6 4 4 2 2 0 0 EQ504 [1] 50 1000 50 1000 DMSO0 1 100 250 ABX
Slide 36: Effects of AhR Modulation and CYP1A1 in Epithelial Cells
Effects of AhR Modulation and CYP1A1 in Epithelial Cells Induction of CYP1A1 in epithelial cells by both EQ504 & ABX464 is AhR-dependent CYP1A1 ✱✱✱ 6 ✱ compared with DMSO ✱✱ ✱✱ CYP1A1 fold change 4 1:2.5 2 1:25 0 [2500] [2500] EQ504 [100] ABX464 [1000] + GNF2.5uM DMSO + GNF 2.5uM ABX464 [1000] +GNF
Slide 37: Functional Consequence of AhR-antagonism on CYP1A1, miR-124 and CLDN2
Functional Consequence of AhR-antagonism on CYP1A1, miR-124 and CLDN2 ✓ Wound Healing in T84 ✓ TEER Assay in Caco2 37
Slide 38: Effects of EQ504 and ABX464 on Intestinal Cell Wound Healing
Effects of EQ504 and ABX464 on Intestinal Cell Wound Healing EQ504 & ABX464, significantly improve wound healing in T84 intestinal epithelial cells in AhR-dependent manner ✱✱✱ 60 ✱✱✱ Wound Area (% to T0) 1:2.5 40 1:25 ABX464 1000nM EQ504 100nM 20 DMSO 0 EQ504 [100] 2.5uM ABX464 [1000] 2.5uM DMSO GNF
Slide 39: Effects of EQ504 and ABX464 on CYP1A1 Induction in Intestinal Cells
Effects of EQ504 and ABX464 on CYP1A1 Induction in Intestinal Cells Induction of CYP1A1 in T84 epithelial cells by both EQ504 and ABX464 is AhR-dependent CYP1A1 ✱✱ ✱✱✱ 10 compared with DMSO CYP1A1 fold change 8 6 4 1:25 1:2.5 2 0 GNF[2500] GNF[2500] EQ504 [100] 2.5uM ABX464 [1000] 2.5uM DMSO EQ504 [
Slide 40: Effects of EQ504 and ABX464 on Tight Junction Protein CLDN2
Effects of EQ504 and ABX464 on Tight Junction Protein CLDN2 Reduction of CLDN2 in T84 intestinal epithelial cells by both EQ504 & ABX464 is AhR-dependent CLDN2 ✱ 2.5 ✱ ✱✱ ✱✱✱✱ ➢ Elevated CLDN2 is associated with impaired compared with DMSO 1:2.5 epithelial barrier function (“leaky gut”) CLDN2 fold c
Slide 41: Effects of EQ504 and ABX464 on Barrier Integrity in a TEER assay
Effects of EQ504 and ABX464 on Barrier Integrity in a TEER assay EQ504 & ABX464 significantly protect the epithelial barrier integrity in Caco2 in AhR-dependent manner TNF, IFN, IL-6 20 ✱✱ ✱✱✱ ➢ TEER (transepithelial electrical resistance) is a commonly used assay TEER after 72 h (% change to T0)
Slide 42: Effects of EQ504 and ABX464 on CYP1A1 Induction in Intestinal Cells
Effects of EQ504 and ABX464 on CYP1A1 Induction in Intestinal Cells Induction of CYP1A1 in Caco2 epithelial cells by both EQ504 & ABX464 is AhR-dependent 5 TNF, IFN, IL-6 ✱✱ ✱✱✱ 4 compared with DMSO CYP1A1 fold change 3 2 1:2.5 1 1:25 0 A B X 4 6 4 [1 0 0 0 ] E Q 5 0 4 [1 0 0 ] G N F [2500] G N F
Slide 43: Summary and Conclusions
Summary and Conclusions ul or . equilliuM ———*
Slide 44: Summary and Conclusions
Summary and Conclusions 01 Is ABX464 itself an AhR Modulator? • ABX464 is a moderately potent AhR modulator (ca. 400nM) that binds to the canonical PAS-B domain of AhR, but ABX464-N-Gluc does not bind nor modulate AhR 02 Does AhR Modulation Induce miR-124? • AhR modulation by EQ504 and ABX464, but n
Slide 45: The AhR/miR-124 Axis
The AhR/miR-124 Axis Toward a unifying mechanistic framework linking ligand-dependent AhR signaling to miR-124 Ligand Metabolism AhR Ligands AhR Modulation Reduced Negative Expression Feedback AhR Negative Feedback Increased Increased miR-124 Xenobiotic enzymes Modulation of immune & barrier functio
Slide 46: Thank You
Thank You Key Acknowledgements Jeanette Ampudia A.J. Giovannone Mary Casis Phoi Tiet Valeria Marrocco Rosa Quinn Closser 46