CD6-ALCAM Pathway is Elevated in Patients with Severe Asthma 1Manali Mukherjee, 1Nan Zhao, 1Katherine Radford, 2Sole Gatto, 2Adam Pavlicek, 3Jeanette Ampudia, 3Cherie Ng, 3Stephen Connelly & 1Parameswaran Nair 1Division of Respirology, Department of Medicine, McMaster University & St Joseph’s Healthcare Hamilton, Ontario, Canada; 2Monoceros Biosystems LLC, San Diego, CA USA; 3Equillium, Inc, La Jolla, CA USA Introduction Figure 1 Figure 3 Clinical Need: 6 1.5 • A subset of severe asthma patients have persistent airway inflammation despite high dose A *** B *** Eos (%) Non-eos (%) p-value *** n *** n o n 11 11 ---- o inhaled and/or oral corticosteroid therapy i 1.0 ) ) i 4 s s M M s s K K Sex (M) 7 (63) 5 (45) 0.3 • e Steroid insensitivity in a portion of these patients may be driven by non-classical T2 e r r P P p p F F 0.5 x x 2 inflammatory pathways. 2 Atopic (n,%) 8 (72.7) 5 (45.4) 0.4 2 e e g g e o o 6 l l 3 ( ( The CD6-ALCAM pathway: D Age 53.55 (15.6) 56.00 (18.6) 0.74 D 0.0 C • CD6 is a co-stimulatory receptor on T-cells and certain innate lymphoid cells that binds 0 C BMI 28.10 (3.7) 27.27 (7.2) 0.74 activated leukocyte cell adhesion molecule (ALCAM) on antigen presenting cells and -0.5 FEV1% 76.67 (17.5) 71.87 (23.8) 0.6 y te re y te re h a e h a e endothelial and epithelial tissues 6 lt r v 8 lt r v a e e a e e FEV1/FVC 0.65 (0.036) 0.67 (0.04) 0.71 C e d * S D e d **** S H o * H o • M M ** The CD6-ALCAM pathway plays an integral role in modulating T cell activation and n n Blood eosinophil 0.55 (0.44) 0.28 (0.29) 0.11 o i o 6 ) ) i s s trafficking and is central to immune mediated inflammation. M 4 M s s Sp eosinophil % 28.92 (18.86) 0.75 (0.79) 0 e K K e r r P P • The pathway is implicated in T2 (T 2), non-T2 (T 1/T 17) and innate lymphoid cells (group p p h h h F F 4 x Sp neutrophil % 28.31 (16.78) 63.10 (30.07) 0.04 x 2 2 e e g g a 2) driven responses responses, demonstrating effects in multiple disease models including o o 4 l 2 l 8 FEG (median) 3 (0-3) 0 (0-1) 0.0001 ( ( D D 2 C allergic asthma. C ICS (median) 1000 (250-2000) 1000 (0-3200) 0.9 Objective: 0 0 OCS (median) 0 (0-7.5) 0 (0-7.5) >0.9 • Examine the CD6-ALCAM axis as a potential target for severe asthma. y te re y te re h a e h a e lt r v lt r v Irritants, pollutants, a e e a e e Type 2 Inflammation Non-Type 2 / Th17 Inflammation e d S e d S Table 1. Sputum was collected from 22 characterized asthmatics on inhaled/oral microbes, and viruses H o H o Allergens M M corticosteroids (not on any anti-T2 biologics). Patients were categorized by >3% or <3% Patients with severe asthma had significantly increased expression of (A) CD6 with sputum eosinophils (sp-Eos; n=11/group) concomitant increases in T cell markers (B) CD3, (C) CD4, and (D) CD8 compared to TSLP IL-25 IL-33 IL-6 CXCL8 moderate and non-asthma patients. ( ***p<0.001, *p<0.05) CD6 IL-13 CD6 CD6 TGF-βV GM-CSF CD6 ✱ P=0.04 ✱ P=0.03 Th2 IL-23 A B IL-4, IL-5, IL-13 ILC2 Th17 Th1 10000 cell cell cell 10000 IFN-γ TNF Figure 2 8000 IL-4 IL-5 IL-6, IL-17, IL-8 8000 Teff 1.2 4 1.5 ** * IL-3, IL-4, ** ** 6000 n IL-5, IL-9 n 3 6000 n B cell o o IgE i 1.0 i ) ) ) o s i A B s C M M M s 0.8 s Mast Eosinophil ALCAM Neutrophil s e K s K K e 2 r r e P cell P P r p p 4000 F F F 0.5 x p x 4000 2 2 2 e x e g g g 1 e F o o o G l 0.4 l l 4 7 ( ( ( N 1 L I 0.0 Sp ALCAM (pg/mL) Sp F L I I 0 2000 2000 Sp ALCAM (pg/mL) Sp Figure Legend: CD6 is expressed on Th1, Th2, Th17 and ILC2 effector cells that secrete multiple proinflammatory cytokines 0.0 -1 -0.5 y te re y te re y te re h a e h a e h a e implicated in asthma pathogenesis. ALCAM is expressed on antigen presentation cells that activate effector cells. ALCAM is lt r v lt r v lt r v 0 0 a e e a e e a e e e d S e d S e d S H o H o H o also expressed on endothelial and smooth muscle cells where it plays a role in trafficking of CD6+ effector cells. Adapted 8 M 6 M 3 M Eos Non-eos Eos Neutro Pauci ** **** ** * **** ** from Israel E, et al., N Engl J Med. 2017;377(10):965-976. D n E F n 6 n o o i o 2 ) ) ) i i s 4 s s M M M s (A) Analysis of sputum ALCAM levels revealed significantly higher concentrations in s s e K K K 4 e e r r r P P P p p p F F F 1 x x 2 2 x 2 eosinophilic vs non-eosinophilic patients (3.8±3 vs. 1.2±1.3ng/mL; p=0.04). (B) When non- e 2 e e g g g 2 Methods 3 o o o 3 A l l l R ( ( ( 1 1 C L 0 L eosinophilic patients were categorized by neutrophilic (n=5) or paucigranulocytic (n=6), I I C 0 0 • To examine CD6 expression and other genes associated with the pathway, we analyzed a publicly -2 -1 ALCAM levels were significantly higher in eosinophilic vs. paucigranulocytic patients y te re y te re y te re h a e h a e h a e available gene expression microarray dataset from BAL cells of non-asthma, moderate asthma and lt r v lt r v lt r v (0.6±0.9 ng/mL; p=0.03). a e e a e e a e e e d S e d S e d S 1-3 H o H o H o severe asthma patients derived from two multi-center observational studies BOBCAT and MAST . 8 M 6 M M G *** H **** Patients with severe asthma 4 n **** *** n o Global microarray normalization using the vsn package . Log2 intensities are shown in figures 1 and i 6 o i ) 4 had significantly increased s ) s s M M Conclusions 5 s e K r e 2. Differential expression was calculated with limma package . K r P p P cytokines, including, (A) IL17F, p x F 4 F 2 x e 2 2 e g g 1 o 2 R o l l ( (B) IFNG, (C) IL4, (D) CCR3, (F) 1 ( X 2 2 • To examine ALCAM levels, sputum was collected from 22 characterized asthmatics on inhaled B 0 The data indicate that the CD6-ALCAM pathway is elevated in severe asthmatics and may be a L T corticosteroids. Patients were categorized by >3% or <3% sputum eosinophils (sp-Eos; n=11/group). I IL13, (G) TBX21 and (H) IL21R, 0 -2 viable target in the treatment of severe, uncontrolled asthma. The ability to detect ALCAM in y te re e e compared to moderate and h a e y t r The supernatant processed from plugs (dispersed in phosphate buffered saline), was further diluted lt r v th ra e a e e l e v sputum may provide a non-invasive approach to further examine this pathway. Blockade of e d S a d e H o e o S M H non-asthma patients. 1:5 and ALCAM levels measured by ELISA (R&D Biosystems). M CD6 is currently being tested in a clinical trial for the treatment of severe asthma. Clinical Trial ( ***p<0.001, *p<0.05) Identifier: NCT04007198 References: 1Sun et al., Sci. Signal. 2015 Dec 1;8(405):ra122; 2Jia et al., J Allergy Clin Immunol. 2012 Sept; 130(3): 647–654.e10; 3Simpson et al., Nat Immunol. 2014 December ; 15(12): 1162–1170; 4 Huber W et al., Bioinformatics 2002;18 Suppl 1:S96-104; 5 Ritchie et al., 2015 Nucleic Acids Res. 2015 Apr 20;43(7):e47.