1. 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

2. 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

3. 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: ; 1Reid PA, et al. Am J Respir Crit Care Med. 2015;19:233-6; 3Levy H, et al. Pediatr Pulmonol. 2007;42:
CF, cystic fibrosis.

4. 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.

5. 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 < 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:

6. 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

7. 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.

8. 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.

9. 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%

10. 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

11. 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)

12. 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

13. 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:

14. 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

15. 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.

16. 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

17. 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)