Mechanisms of Topical Bruton Tyrosine Kinase Inhibitor PRN473 P1572 in Immune‑Mediated Models of Skin Disease Yan Xing, Katherine A

E‑Poster Mechanisms of Topical Bruton Tyrosine Kinase Inhibitor PRN473 P1572 in Immune‑Mediated Models of Skin Disease Yan Xing, Katherine A. Chu, Jyoti Wadhwa, Wei Chen, Jiang Zhu, Jin Shu, Matthew C. Foulke, Natalie Loewenstein, Philip Nunn, Kolbot By, Pasit Phiasivongsa, David M. Goldstein, and Claire L. Langrish Principia Biopharma Inc, A Sanofi Company, South San Francisco, CA, USA • In vitro, PRN473 inhibited B‑cell activation and FcR signaling in monocytes and mast cells, and IgE‑Mediated PCA Mouse Reaction INTRODUCTION MATERIALS AND METHODS in the absence of cytotoxic effects and limited functional effects in basophils, epidermal growth factor receptor (EGFR), and cells absent BTK signaling (Table 1A) • Significant dose‑dependent inhibition of mouse PCA IgE‑mediated reaction was also observed with single and multiday • PRN473 demonstrated durable BTK inhibition at 4 h that was partially sustained through 18 h applications of PRN473 ≥1%, approaching positive controls with higher strengths up to 4% PRN473 (Figure 6) Bruton Tyrosine Kinase In Vitro Selectivity and Functional Assays (Table 1B) • Multiday (3 days) treatment achieved similar levels of inhibition with respect to single administration (data not shown) • Bruton tyrosine kinase (BTK) is a promising target in immunology because of its expression in • PRN473 1 µM was tested against a panel of 230 kinase targets in the KINOMEscan™ panel B cells and innate immune cells, providing essential downstream signaling for B‑cell receptors, Biochemical studies were performed to characterize the potency, selectivity, biochemical Table 1. Preclinical Target Specificity and BTK Occupancy for PRN473 Figure 6. Dose‑Dependent Inhibition in IgE Antibody‑Mediated Mouse Passive Fc receptors, and other innate cell pathways (Figure 1)1,2 • on‑rate and off‑rate, and reversibility of the interaction between PRN473 and BTK Cutaneous Anaphylaxis (PCA) With Single Application of PRN473 • Selective inhibition of BTK function and occupancy and off‑target effects were evaluated in A. Cell‑Based Target Specificity Assays Figure 1. BTK Plays a Critical Role in Innate and Adaptive Immunity cell‑based assays and human whole blood (HWB) 1.4 3h before IgE On‑Target Activity IC50 ± SD, nM 1.2 INNATE IMMUNITY ADAPTIVE IMMUNITY In Vivo PRN473 Treatment BTK Ramos B cell occupancy 30 ± 15 ) PRN473 was applied topically at single and multiday doses of 0.25%‑4% before IgG/IgE 610 1.0 • ** challenges FcγR IgG‑induced TNFα production in human monocytes 76 ± 40 0.8 • Positive controls were topical betamethasone, oral prednisolone, and antihistamines ** FcεR IgE mast cell degranulation and β‑hexaminidase release 89 Mast cell Vehicle control was used as a comparator (t‑test with P < 0.05) 0.6 BTK Natural killer cell B cell T Cell • Dye Density (OD ** Megakaryocyte BTK BTK 9 FcεR IgE mast cell degranulation and histamine release 175 0.4 IgG‑Mediated Passive Rat Arthus Model ** ** • The passive Arthus reaction is an acute IgG antibody challenge model dependent on FcγR BCR B cell activation in HWB 274 ± 95 0.2 activation in macrophages and neutrophils 0.0 BTK BTK occupancy in whole blood 364 ± 112 Microglia Platelets Basophil Neutrophil Eosinophil Monocyte Plasma cell • Female Sprague‑Dawley rats (n = 5) were administered topical gel application with vehicle No treatment Betamethasone 4% PRN473 1% PRN473 BTK BTK BTK BTK BTK BTK control; betamethasone diproprionate 0.05% (positive control); or PRN473 doses of 0.25%, FcεR IgE‑induced basophil CD63 activation in HWB 1130 ± 510 Gel Vehicle Diphenhydramine 2% PRN473 0.5% PRN473 0.5%, 1%, or 2% for 3 or 16 h before IgG‑mediated skin challenge **P < 0.001 versus vehicle. - Single‑day or multiday (3 days) evaluations were performed Off‑Target Activity (>5000) Macrophage Expression, functional inhibition BTK BTK Inflammatory reactions were measured by reduction in intradermal extravasation diameter and BTK Expression, no/limited functional effects • TCR T cell activation in whole blood >7900 Systemic Pharmacology optical dye density at OD610nm of Evans blue dye assessed after the challenge N/A EGFR EGFR signaling reporter assay >5000 • Systemic BTK occupancy evaluated with topical PRN473 in Arthus rat and PCA mouse models resulted in low to no The role of innate immune cells is underappreciated in immune‑mediated dermatological IgE‑Mediated Passive Cutaneous Anaphylaxis (PCA) systemic accumulation, suggesting that localized exposure to PRN473 provided the major role for the efficacy (data • not shown) diseases (Figure 2)3,4 9 N/A Cytotoxicity HCT116 cell (no BTK) cytotoxicity >20,000 Mouse Model - No measurable systemic BTK occupancy in the Arthus rat model with single or multiday doses of topical PRN473 - In lesional skin, innate cells such as mast cells, eosinophils, and neutrophils activate and The PCA model is an IgE FcεR‑mediated model, with mechanisms similar to those observed in 2% or with single application of PRN473 2%‑4% in PCA mouse model accumulate, often correlating with tissue damage and disease severity • B. BTK Target Engagement – Occupancy in Human B Cells human allergic disease - <50% systemic BTK occupancy with multiday topical PRN473 2%‑4% in the PCA mouse model - These skin‑resident and infiltrating immune cells can be targeted topically or systemically - Measures IgE‑dependent responses via rapid mast cell activation, degranulation, and Time Ramos B cell BTK Occupancy ± SD release of inflammatory mediators 4 h 91% ± 3 Figure 2. Multiple BTK‑Dependent Cells Are Active in Skin • Female BALB/c mice (n = 5/group) were administered a single topical gel application 3 h 18 h 54% ± 6 CONCLUSIONS Inflammation3,4 before IgE‑mediated skin challenge with vehicle control, bethamethasone diproprionate 0.05% and diphenhydramine (positive control), or PRN473 doses of 0.5%, 1%, 2%, or 4% BCR, B‑cell receptor; BTK, Bruton tyrosine kinase; EGFR, epidermal growth factor receptor; HWB, human whole blood; Ig, immunoglobulin; TNFα, tumor necrosis factor alpha. Preclinical studies of PRN473 provide a strong biological basis for targeting skin innate immune • Thirty minutes after challenge, mice were euthanized, and skin samples were collected to • Epidermis assess the density of dye extravasation in the skin cell responses with a rapid onset of action with once‑daily topical administration and minimal T cell Langerhans cell systemic exposure Keratinocytes IgG‑Mediated Passive Rat Arthus Reaction Systemic Pharmacology: BTK Occupancy Significant dose‑dependent inhibition of IgG‑mediated passive Arthus reaction in rats was • PRN473 effectively inhibited IgG (FcγR)‑ and IgE (FcεR)‑mediated signaling equivalent to topical • Spleens were collected terminally and evaluated after the challenge to assess systemic effects observed with single and multiday topical PRN473 ≥0.5% strengths (Figure 5) • Dermis B cell T cell or oral corticosteroids and prevented downstream immune effects locally in the skin - Equivalent inhibition to topical betamethasone was observed with 1% and 2% PRN473 Basophil - Inhibition was maintained when PRN473 was administered 16 h before challenge, reinforcing Macrophage Dendritic cell extended anti‑inflammatory activity with once‑daily application • Overall, dose‑dependent efficacy was demonstrated with topical PRN473 in preclinical models of RESULTS immune‑mediated skin diseases, supporting future clinical studies Mast cell Plasma cell Neutrophil Eosinophil Natural killer cell Figure 5. Dose‑Dependent Inhibition in IgG Antibody‑Mediated Preclinical Selectivity and Functionality of PRN473 - Email for more information: clinicaltrials @ principiabio.com Arthus Reaction Rat Model Single (A) and Multiday (B) Application of • PRN473 exhibited selectivity to 6 of 230 kinases with >90% inhibition at 1 µM (Figure 4 left panel) and durable in vitro occupancy with BTK, TXK, TEC, and BMX, but not BLK and ITK PRN473 Topical BTK Inhibitor PRN473 (Figure 4 right panel) A. Single Topical Application PRN473 is a topically administered covalent inhibitor of BTK designed with Tailored Covalency® REFERENCES • 1.6 3h before IgG 16 h before IgG to show durable, reversible BTK occupancy (Figure 3)5,6 1. Crofford LJ, et al. Exp Rev Clin Immunol. 2016:12:763‑773. 5. Bradshaw JM, et al. Nat Chem Biol. 2015;11:525‑531. Figure 4. Preclinical Optimization of PRN473 for Selectivity, 1.4 The longer BTK residence time optimizes prolonged, localized efficacy in rats and mice with ) 2. Rip J, et al. Crit Rev Immunol. 2018:38:17‑62. 6. Bisconte A, et al. J Immunol (AAI). 2015;194(1 suppl):139.6. • 610 1.2 low systemic exposure5 Potency, and Durability 3. Quaresma JAS. Clin Microbiol Rev. 2019;32:e00034. 7. Herter JM, et al. Br J Pharmacol. 2018;175:429‑439. 1.0 * 4. Richmond JM, Harris JE. Cold Spring Harb Perspect Med. 8. Goodale EC, et al. Vet Dermatol. 2020;31:291‑e71. Mechanistically, PRN473 inhibits IgE (FcεR)‑mediated activation of mast cells and basophils, • 0.8 2014;4:a015339. 7 Durable Occupancy ** 9. Chang BY, et al. Arthritis Res Ther. 2011;13:R115. IgG (FcγR)‑mediated activation of monocytes, and neutrophil migration Selectivity Profile (Kinase Panel) ** ** ** Over Time 0.6 ** ** PRN473 was also demonstrated to be efficacious with excellent tolerability when given orally in ** • Kinase PRN473 100 0.4 the treatment of canine pemphigus foliaceus8 Dye Density (OD Assay IC50 ± SD, nM 0.2 ACKNOWLEDGMENTS BTK 1.8 ± 0.2 80 0.0 These studies were sponsored by Principia Biopharma Inc, A Sanofi Company, South San Francisco, CA, USA. Editorial support was provided by ® TXK 3.4 ± 1.0 Second City Science and funded by Principia Biopharma Inc, A Sanofi Company. The authors directed development of the presentation and are Figure 3. PRN473 Tailored Covalency B. Multiday (3 days) Topical Applications fully responsible for all content and editorial decisions TEC 2.0 ± 0.2 60 BMX 1.8 ± 0.3 BTK 1.4 3h before IgG 16h before IgG NON‑COVALENT COVALENT 40 TXK 1.2 BINDING REGION BINDING REGION BLK 2.2 ± 0.6 ) TEC 610 DISCLOSURES ITK 300 ± 10 1.0 20 BMX * All authors report employment with and stock ownership from Principia Biopharma Inc, A Sanofi Company.

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