Introduction • Atopic dermatitis (AD) is a chronic, pruritic skin disease characterized by type 2 immune-mediated infl ammation and skin barrier dysfunction1 • In a recent large-scale RNA-sequencing-based transcriptomic study of AD, psoriasis, and matched control samples, it was found that the type 2 cytokine interleukin 13 (IL13) was the most distinctive marker for AD2 — Increased expression levels of IL13 were found in both lesional and nonlesional AD skin — Expression levels of IL13 in lesional AD skin correlated with disease severity • In contrast, expression of the type 2 cytokine IL4 was detectable in 40% of the AD skin samples and at very low expression levels • IL-13 has been shown to modulate the expression of infl ammatory mediators, such as chemokines, and skin barrier markers related to the pathophysiology of AD3-8 • Tralokinumab is a fully human IgG4 monoclonal antibody in Phase 3 development for AD that specifi cally neutralizes IL-139 Methods Study population Participants • Adult patients with a history of AD for at least 3 years — The same AD cohort as reported by Tsoi et al from their large-scale transcriptomic study of AD2 • Adult volunteers without personal or familial history of allergic atopic and chronic infl ammatory diseases Inclusion criteria • Dermatologist-confi rmed diagnosis of AD (diagnosed on the basis of a skin examination by experienced dermatologists according to standard criteria for AD [American Academy of Dermatology consensus criteria]) Exclusion criteria • Any other chronic skin disease • Systemic treatment with immune-e� cient medication • Topical treatment within 1 week prior to material sampling Results Atopic dermatitis disease biomarkers strongly correlate with IL-13 levels, are regulated by IL-13, and are modulated by tralokinumab in vitro Stephan Weidinger,1 Maxim A.X. Tollenaere,2 Katharina Drerup,1 Thomas Litman,2 Hanne Norsgaard2 1Department of Dermatology and Allergy, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany; 2Skin Research, LEO Pharma A/S, Ballerup, Denmark Objectives • To examine the correlation of IL13 expression with AD disease biomarkers by use of RNA-sequencing data from AD lesional skin samples • To investigate IL-13-mediated regulation of AD disease biomarkers and their modulation by tralokinumab in primary cultures of human keratinocytes and dermal fi broblasts Conclusions • IL13 expression levels correlate strongly with disease severity and with biomarkers related to the pathophysiology of AD • The expression of several AD disease biomarkers is regulated by IL-13 and is normalized in a dose-dependent manner by tralokinumab in cultures of human keratinocytes and dermal fi broblasts • These fi ndings support the rationale for neutralizing excessive levels of IL-13 in AD by utilizing monoclonal antibodies targeting IL-13, such as tralokinumab References 1. Weidinger S et al. Nat Rev Dis Primers 2018;4:1 2. Tsoi LC et al. J Invest Dermatol 2019;139:1480–1489 3. Purwar R et al. J Invest Dermatol 2006;126:1043–1051 4. Fukuda K et al. J Allergy Clin Immunol 2003;111:520–526 5. Matsumura S et al. J Dermatol Sci 2015;78:215–223 6. Kim BE et al. Clin Immunol 2008;126:332–337 7. Pellerin L et al. J Allergy Clin Immunol 2013;131:1094–1102 8. Berdyshev E et al. JCI Insight 2018;3:pii 98006 9. Popovic B et al. J Mol Biol 2017;429:208–219 Disclosures • Stephan Weidinger is a speaker, advisory board member, and/or investigator for: AbbVie; Galderma; Incyte; Kymab; La Roche-Posay; LEO Pharma; Lilly; Novartis; Pfi zer; and Regeneron and Sanofi -Genzyme • Maxim A.X. Tollenaere, Thomas Litman, and Hanne Norsgaard are employees of LEO Pharma • Katharina Drerup has nothing to disclose Acknowledgments • Re-analysis of RNA-sequencing data and in vitro studies were performed by LEO Pharma • This presentation is sponsored by LEO Pharma • Medical writing support was provided by Stephanie Rippon, PhD, and Amy Graham, PhD, from McCann Health, funded by LEO Pharma Table 1. Patient samples included in the study AD Controls (healthy) Number of individuals (male/female) 27 (12/15) 38 (16/22) Age, years, mean (SD) 34.07 (10.96) 32.63 (11.64) Objective SCORAD,* mean (SD) 31.11 (10.96) - FLG mutation carriers 5 1 *Objective SCORAD does not include the subjective items daily pruritus and sleeplessness. FLG, fi laggrin; objective SCORAD, objective component of SCORing Atopic Dermatitis; SD, standard deviation. Figure 1. Study design In silico bioinformatics analysis of RNA-sequencing data2 to examine the correlation between IL13 expression and AD disease biomarkers Validate correlations in vitro using disease-relevant cell cultures Can IL-13-driven expression of AD disease biomarkers be reverted by tralokinumab? Human keratinocytes Human dermal fibroblasts Figure 2. IL13 expression strongly correlates with key AD disease biomarkers: A) Positive correlation between IL13 expression and infl ammatory mediators and B) Negative correlation between IL13 expression and skin barrier markers 2 7 6 5 4 3 A B IL13 –5 –4 –3 –2 –1 0 1 2 C C L2 2 IL13 –6 210–1–2–3–4–5 C C L2 2 7 6 5 4 3 IL13 –4 210–1–2–3 C C L1 7 7 –5 –3 –1 1 3 5 Pearson r=0.79 Pearson r=0.69 IL13 –4 210–1–2–3 N TR K 1 5 –7 –5 –3 –1 1 3 Pearson r=0.82 IL13 –4 210–1–2–3 FL G 2 14 9 10 11 12 13 Pearson r=-0.61 IL13 –4 210–1–2–3 EL O V L6 7 4 5 6 Pearson r=-0.73 IL13 –4 210–1–2–3 FL G 16 10 11 12 13 14 15 Pearson r=-0.55 IL13 –4 210–1–2–3 LO R 10 5 6 7 8 9 Pearson r=-0.59 Pearson r=0.81 Graph axes represent Log2 (CPM) values. CCL, C-C motif chemokine ligand; CPM, counts per million; ELOVL, elongation of very long chain fatty acids; IL, interleukin; LOR, loricrin; NTRK, neurotrophic tyrosine kinase. Figure 3. Tralokinumab inhibits IL-13-induced expression of the chemokine CCL-2 in human dermal fi broblasts: A) CCL-2 protein concentration in supernatant; B) Percentage inhibition of IL-13-induced CCL-2 protein secretion; C) CCL2 mRNA expression; and D) Percentage inhibition of IL-13-induced CCL2 mRNA expression LL O D CCL-2 protein in supernatant IgG4 isotype control Tralokinumab C C L- 2 p g /m l � S D A Tralokinumab (nM) Tralokinumab (nM) 150 0 50 100 150 0 50 100 Antibody (nM) 0.01 1 100 % in h ib it io n � S D B C D % in h ib it io n � S D Antibody (nM) 0.01 1 100 0 400 300 200 100 C o n tr o l 3010 31 0 .30 .1 0 .0 3 0 .0 1 0 .0 0 30 B a se lin e le ve l CCL2 mRNA Fo ld -C C L2 e xp re ss io n � S D 0 10 8 6 4 2 C o n tr o l 3010 31 0 .30 .1 0 .0 3 0 .0 1 0 .0 0 30 rhIL-13 rhIL-13 IC 50 : 335 pM IC 50 : 467 pM Cells were stimulated with 2 ng/mL (0.16 nM) rhIL-13. N=1 experiment. 1 nM of antibody=0.15 µg/mL. Mean change � SD. IC 50 , half maximal inhibitory concentration; LLOD, lower limit of detection; mRNA, messenger RNA; rhIL-13, recombinant human IL-13. Figure 4. Tralokinumab inhibits IL-13-induced expression of infl ammatory mediators in human keratinocytes: A) CCL-2 protein concentration in supernatant; B) Percentage inhibition of IL-13-induced CCL-2 protein secretion; C) CCL2 mRNA expression; D) Percentage inhibition of IL-13-induced CCL2 mRNA expression; E) NTRK1 mRNA expression; and F) Percentage inhibition of IL-13-induced NTRK1 mRNA expression Fo ld -C C L2 e xp re ss io n � S EM * Fo ld -N TR K 1 e xp re ss io n � S EM * LL O D CCL-2 protein in supernatant IgG4 isotype control Tralokinumab C C L- 2 p g /m l � S D A Tralokinumab (nM) 150 0 –50 50 100 Antibody (nM) 0.01 1 100 % in h ib it io n � S D B C D 0 200 150 100 50 C o n tr o l 3010 31 0 .30 .1 0 .0 3 0 .0 1 0 .0 0 30 rhIL-13 IC 50 : 217 pM B a se lin e le ve l CCL2 mRNA Tralokinumab (nM) 150 0 –50 50 100 Antibody (nM) 0.01 1 100 % in h ib it io n � S EM * 0 30 20 10 C o n tr o l 3010 31 0 .30 .1 0 .0 3 0 .0 1 0 .0 0 30 rhIL-13 IC 50 : 209 pM E F B a se lin e le ve l NTRK1 mRNA Tralokinumab (nM) 150 0 –50 50 100 Antibody (nM) 0.01 1 100 % in h ib it io n � S EM * 0 2000 500 1000 1500 20 10 C o n tr o l 3010 31 0 .30 .1 0 .0 3 0 .0 1 0 .0 0 30 rhIL-13 IC 50 : 246 pM *n=3. Cells were stimulated with 10 ng/mL (0.8 nM) rhIL-13. N=3 experiments (� SEM) and N=1 on protein measurement. 1 nM of antibody=0.15 µg/mL. SEM, standard error of the mean. Figure 5. Tralokinumab restores expression of skin barrier markers decreased by IL-13 in human keratinocytes: A) LOR mRNA expression; B) Percentage inhibition of IL-13-induced LOR mRNA suppression; C) FLG mRNA expression; D) Percentage inhibition of IL-13-induced FLG mRNA suppression; E) FLG2 mRNA expression; and F) Percentage inhibition of IL-13-induced FLG2 mRNA suppression Fo ld -F LG e xp re ss io n � S D Fo ld -F LG 2 e xp re ss io n � S D LOR mRNA IgG4 isotype control Tralokinumab Fo ld -L O R e xp re ss io n � S D A Tralokinumab (nM) 150 0 –50 50 100 Antibody (nM) 1 10 100 % in h ib it io n � S D B C D C o n tr o l 10 05 025 12 .5 6. 25 3. 13 1. 5 6 0 .7 80 C o n tr o l 10 0502 5 12 .5 6. 25 3. 13 1. 56 0 .7 80 C o n tr o l 10 0502 5 12 .5 6. 25 3. 13 1. 56 0 .7 80 rhIL-13 IC 50 : 11.4 nM B a se lin e le ve l B a se lin e le ve l FLG mRNA Tralokinumab (nM) 150 0 –50 50 100 Antibody (nM) 1 10 100 % in h ib it io n � S D rhIL-13 IC 50 : 20.1 nM E F B a se lin e le ve l FLG2 mRNA Tralokinumab (nM) 150 0 –50 50 100 Antibody (nM) 1 10 100 % in h ib it io n � S D 0.0 1.5 0.5 1.0 0.0 1.5 0.5 1.0 0.0 1.5 0.5 1.0 rhIL-13 IC 50 : 12.6 nM Cells were stimulated with 50 ng/mL (4 nM) rhIL-13. N=1 experiment. 1 nM of antibody=0.15 µg/mL. 39th Annual Fall Clinical Dermatology Conference, October 17–20, 2019, Las Vegas, NV, USA. Data previously presented at the 28th European Academy of Dermatology and Venereology Congress, October 9–13, 2019, Madrid, Spain