Improvement in Inflammatory Biomarkers in Patients with Cystic Fibrosis Homozygous for the F508del-CFTR Mutation Treated with Lumacaftor and Ivacaftor James C. Sullivan1; Frank J. Accurso2; Gautham Marigowda1; Jack Beusmans1; David Geho1; Eileen Zhang1; Richard B. Moss3; David Waltz1 1Vertex Pharmaceuticals Incorporated, Boston, MA, United States; 2Department of Pediatrics, University of Colorado Denver School of Medicine, Children’s Hospital Colorado, Aurora, CO, United States; 3Stanford University School of Medicine, Palo Alto, CA, United States Presented at the 39th European Cystic Fibrosis Conference, Basel, Switzerland, 8-11 June 2016

Author Disclosures JCS, GM, JB, DG, EZ, and DW are employees of Vertex Pharmaceuticals Incorporated and may own stock or stock options in that company FA has no disclosures RBM received consultancy fees and his institution received financial support from Vertex Pharmaceuticals Incorporated for participation in clinical trials

Impaired Mucociliary Function and Inflammation in Cystic Fibrosis Normal airway CF airway In CF, impaired mucociliary escalator function and mucus clearance render the lungs prone to infection and proinflammatory stimuli Increased levels of biomarkers of inflammation in CF, such as calprotectin and C-reactive protein, are associated with pulmonary exacerbation (PEx) events and increase further as lung function deteriorates1-2 CF chronic airway infections are associated with repeated PEx and progressive loss of pulmonary function Figure from Lyczak JB, et al. Clin Microbiol Rev. 2002;15:194-222; 1Reid PA, et al. Am J Respir Crit Care Med. 2015;19:233-6; 3Levy H, et al. Pediatr Pulmonol. 2007;42:256-62. CF, cystic fibrosis.

Lumacaftor/Ivacaftor Improves Membrane Delivery and Function of F508del-CFTR Patients with CF homozygous for F508del-CFTR have impaired membrane delivery and function of CFTR LUM (a CFTR corrector) increases delivery of CFTR to cell surface IVA (a CFTR potentiator) increases channel-open probability of CFTR CFTR, cystic fibrosis transmembrane conductance regulator; Cl−, chloride; IVA, ivacaftor; LUM, lumacaftor.

Phase 3 Trials (TRAFFIC/TRANSPORT) of LUM/IVA Showed Benefit in Patients Aged ≥12 Years With CF Homozygous for F508del Mutation Placebo LUM 600 mg qd/IVA 250 mg q12h LUM 400 mg q12h/IVA 250 mg q12h This slide is to illustrate that the two factors that are associated with inflammation – namely reduced lung function and incidence of PEx – are improved by LUM/IVA treatment as per TT clinical trials, setting up next slide *, P < 0.025 for difference between each LUM/IVA group and the placebo group Both lung function and Pex are known to be associated with inflammation What effects on systemic inflammation might be associated with these improvements? FEV1, forced expiratory volume in 1 second; ppFEV1, percent predicted FEV1; q12h, every 12 hours; qd, once daily. Wainwright CE, et al. N Engl J Med. 2015;373:220-31.

Objectives To investigate whether: LUM/IVA treatment affects the inflammatory state in patients with CF Levels of inflammatory markers are associated with measures of lung function at baseline and over the course of LUM/IVA treatment

Methods and Study Population Analysis population included 889 patients from TRAFFIC/TRANSPORT who provided optional baseline and week 24 blood biomarker samples 84% of 1054 patients who completed treatment Baseline characteristics were similar to the overall study population In this retrospective analysis, levels of calprotectin, CRP, IL-8, IL-18, and IgG were measured using an immunoassay approach (Myriad RBM; Austin, TX) Upper limits of normal (ULN) were inferred from an assessment from 100 volunteers, with ULN defined as the 97.5 percentile White blood cell counts (total and differential) were assessed as part of standard clinical laboratory assessment Change from baseline at week 24 for each marker was compared between placebo and each active treatment group by t test using log-transformed values Associations between variables were assessed by linear regression In all cases, statistical significance was defined as α=0.05, following Bonferroni correction for multiple comparisons CRP, C-reactive protein; IgG, immunoglobulin G; IL, interleukin.

Serum Biomarkers of Inflammation Decreased Significantly in Patients on Treatment Relative to Placebo * * * * * * * These graphs will ultimately only have the LUM 400mg q12h arm. Levels of calprotectin, CRP, IgG, and IL-8 showed significant reductions in active treatment groups relative to placebo at week 24 (P<9e-6; denoted by *) No significant effect was observed for IL-18 (P>0.05) Cal, calprotectin; CI, confidence interval.

Fewer Subjects on LUM/IVA Were Above the Normal Range at Week 24 Baseline Week 24 LUM 400 mg q12h/IVA LUM 600 mg qd/IVA Placebo Reduction in % of patients with >ULN observations across all biomarkers 58% 33% 8%

Biomarkers of Inflammation Were Correlated With ppFEV1 ● Placebo × LUM 400 mg q12h/IVA ▫ LUM 600 mg qd/IVA Baseline (P<0.0001) Assay limit of detection r =0.26 Significant association between baseline ppFEV1 and levels of calprotectin, CRP, IL-8, and IgG (P<0.0001) but not IL-18

Biomarkers of Inflammation Were Correlated With ppFEV1 ● Placebo × LUM 400 mg q12h/IVA ▫ LUM 600 mg qd/IVA Baseline (P<0.0001) On-Treatment Changes (P<0.0001) Assay limit of detection r =0.33 r =0.39 r =0.26 r =0.23 Absolute Change in ppFEV1 (95% CI in shaded area) Significant association between baseline ppFEV1 and levels of calprotectin, CRP, IL-8, and IgG (P<0.0001) but not IL-18 Significant association between changes in ppFEV1 and changes in calprotectin, CRP, and IL-8 (P<0.0001)

Changes in Inflammatory Biomarkers Were Associated With Changes in Neutrophils and Monocytes Total white blood cell count (WBC) decreased by ≈10% on treatment relative to the placebo group (P<0.0001)a This decrease was driven by 13% and 15% decreases in monocyte and neutrophil cell counts (P<0.0001)a These decreases were positively correlated with changes in calprotectin, CRP, and IL-8 aPercentage change from baseline in the 400 mg LUM q12h/IVA group as compared to placebo

Conclusions Modulating CFTR with LUM/IVA: Reduces PEx1 Improves lung function measures Changes in lung function were correlated with changes in inflammatory markers Our analyses of biomarker data provide evidence supportive of the effectiveness of LUM/IVA on PEx Previous studies showed that calprotectin and CRP levels reflect disease activity and correlate with time to next PEx event2-3 Reductions in inflammation associated with LUM/IVA treatment were also associated with reduced neutrophil, monocyte, and overall WBC counts, further supporting a generalized reduction in inflammation Reduced levels of circulating inflammatory markers suggest an improvement in the inflammation characteristic of CF 1Wainwright CE, et al. N Engl J Med. 2015;373:220-31; 2Reid PA, et al. Am J Respir Crit Care Med. 2015;19:233-6; 3Levy H, et al. Pediatr Pulmonol. 2007;42:256-62.

Acknowledgements The authors thank the patients and their families, the study investigators, and the study coordinators for their roles in the study This study was sponsored by Vertex Pharmaceuticals Incorporated Editorial coordination and support were provided by Dhrupad Patel, PharmD, who is an employee of Vertex Pharmaceuticals Incorporated and may own stock or stock options in that company Medical writing and editorial support were provided by Jeremy Kennard, PhD, and Mary Kacillas. JK and MK are employees of Infusion Communications, which received funding from Vertex Pharmaceuticals Incorporated

Pearson Correlation Coefficients Changes in Inflammatory Biomarkers Were Associated with Changes in Neutrophil and Monocyte Counts Neutrophils and monocyte cell counts: Decreased on treatment relative to the placebo group Are associated with changes in calprotectin, CRP, and IL-8 Pearson Correlation Coefficients Treatment Effect (%)a P Value Cal CRP IL-8 IgG IL-18 Total WBC -9.7 2.4e-4 0.41 0.39 0.16 0.17 <0.01 Neutrophils -15.4 7.8e-5 0.45 0.43 0.19 -0.03 Monocytes -12.5 7.9e-6 0.23 0.10 0.11 -0.05 Basophils 1.8 0.76 0.20 0.18 0.07 0.05 Eosinophils 10.6 0.04 -0.01 -0.04 -0.11 Lymphocytes -1.1 0.58 -0.07 a Percentage change from baseline in the 400 mg LUM q12h/IVA group as compared to placebo.

Changes in Inflammatory Biomarkers Were Associated with Changes in Neutrophils and Monocytes ● Placebo × LUM 400 mg q12h/IVA ▫ LUM 600 mg qd/IVA r = 0.38 r = 0.18 r = 0.46 r = 0.29 r = 0.39 r = 0.16

Biomarkers of Inflammation Were Correlated With ppFEV1 Baseline At Baseline, calprotectin, CRP, IL-8, and IgG levels were correlated with ppFEV1 (P<0.0001) Change on treatment On-treatment changes in calprotectin, CRP, and IL-8 levels were correlated with on-treatment changes in ppFEV1 (P<0.0001)