CFTR Modulation – 25 Years of NACFC Progress Marcus A. Mall, M.D. Division of Pediatric Pulmonology and CF Center Department of Pediatrics University of Heidelberg, Germany

NACFC Motivation Courtesy of Jeff Wine

Early NACFC Milestones: Unraveling the Cause of Cystic Fibrosis Closed Open Time Current Cl - Courtesy of David Sheppard Gibson RL et al., Am J Respir Crit Care Med 2003

Most CF is Caused by a Processing Defect of  F508 CFTR Courtesy of Martina Gentzsch CFTR  F508 Lukacs GL et al., New Engl J Med 2003  F508

CFTR Dysfunction Causes Airway Surface Liquid Depletion Normal CF Ion transport defect Airway surface dehydration Mucociliary dysfunction Mucus obstruction Infection Inflammation Lung damage Courtesy of Ric Boucher Ratjen F, New Engl J Med 2006 Human Bronchial Epithelial Cultures (HBE)

Different Mechanisms of CFTR Dysfunction and Proof of Concept for Mutation-Specific Therapies Rowe SM et al., New Engl J Med 2005 Courtesy of M Gentzsch Low temperature correction Potentiation of channel gating Courtesy of D Sheppard Translational readthrough Placebo Gentamicin Wilschanski M et al., NEJM 2003

High-Throughput Screening Speeding Up CF Drug Discovery >10,000 Primary assays/day High-throughput screening CFTR Modulator Drug SAR based Medicinal Chemistry Prioritize hits Screening Assay Courtesy of Vertex Pharmaceuticals

2011 Clinical Studies with CFTR Modulators Rowe SM et al., New Engl J Med 2005 CFTR potentiator VX-770: Phase III study in CF patients with the G551D CFTR mutation CFTR corrector VX-809: Phase IIa study in CF patients with the  F508 CFTR mutation Ataluren (PTC124): Phase III study in CF patients with CFTR nonsense mutations – ongoing –

Results from First Phase III Clinical Study with CFTR Modulator Efficacy and Safety of VX-770 in Subjects with CF and the G551D Mutation See talk by BW Ramsey: W23 (Saturday, 2:30 PM) – Poster #211

Potentiator VX-770 Restores G551D CFTR Function and Improves Airway Surface Hydration in Vitro VX-770 Control Unpublished data provided by Vertex 0.2 sec Intensity Control VX-770 Cilia beat frequency (representative tracings) Airway surface liquid ASL Van Goor F et al., PNAS 2009 See poster by B Woodworth: #114

VX-770 Phase III Study Design Randomized, double-blind, placebo-controlled Recruitment: 161 subjects Key inclusion criteria –G551D mutation on at least one CFTR allele –Aged ≥ 12 years –FEV 1 40% to 90% predicted Run-in Screening Randomization (1:1) Or 2-yr Follow-up VX mg q12h Open-label rollover study Placebo Day Week 24 VX mg q12h VX-770 Treatment period Extension period Primary analysis Elborn JS, 34th ECFC (NCT )

VX-770 Phase III Study Endpoints Primary Endpoint Absolute change from baseline in % predicted FEV 1 through week 24 Secondary Efficacy Endpoints Absolute change from baseline in sweat Cl – Absolute change from baseline in CFQ ‑ R respiratory domain Absolute change in weight Time to first pulmonary exacerbation Absolute change from baseline in % predicted FEV 1 through week 48 Safety Assessment Adverse events (clinical laboratory values, ECGs) Elborn JS, 34th ECFC (NCT )

Change from Baseline in Sweat Chloride Treatment effect through Week 24 – 47.9 mmol/L P < Treatment effect through Week 48 – 48.1 mmol/L P < Elborn JS, 34th ECFC 2011 Vertex press release June 10, 2011

Absolute Change in FEV 1 % Predicted Treatment effect through Week % P < Treatment effect through Week % P < Elborn JS, 34th ECFC 2011 Vertex press release June 10, 2011

Time to First Pulmonary Exacerbation Week 24 Hazard Ratio 0.40 P = Week 48 Hazard Ratio 0.46 P = Hazard Ratio: 0.45 ( 0.28, 0.73) P= PLACEBOVX-770 Event-Free Rate At Week PLACEBOVX-770 Event-Free Rate At Week 48 Placebo VX-770 Proportion of event-free subjects Study day Modified Fuchs’ criteria Elborn JS, 34th ECFC 2011 Vertex press release June 10, 2011

Change from Baseline in CFQ-R Respiratory Domain * MCID, minimal clinically important difference (Quittner et al 2009) MCID = 4* Treatment effect through Week P < Treatment effect through Week P < Elborn JS, 34th ECFC 2011;Vertex press release June 10, 2011

Change from Baseline in Weight Treatment effect at Week kg P < Treatment effect at Week kg P = Elborn JS, 34th ECFC 2011 Vertex press release June 10, 2011 See talk by M Drumm: S1 (Thursday 2:00 PM)

Safety Summary Through Week 48 Adverse event, n (%) Placebo (N = 78) VX-770 (N = 83) Subjects with any serious adverse event33(42.3)20(24.1) Pulmonary exacerbation (physician determined)26(33.3)11(13.3) Hemoptysis4(5.1)1(1.2) Hypoglycemia02(2.4) Serious adverse events occurring in > 1 subject in either group Adverse event, n (%) Placebo (N = 78) VX-770 (N = 83) Any adverse event78 (100)82 (99) Adverse events leading to study discontinuation4 (5)1 (1) Elborn JS, 34th ECFC 2011 Vertex press release June 10, 2011

Summary of VX-770 Phase III Study in CF Patients with the G551D Mutation Rapid onset and sustained improvement in lung function (primary endpoint: absolute change in % predicted FEV 1 ) Parallel improvement in CFTR function and lung function Sustained improvements in other outcomes including risk of exacerbation, respiratory symptoms and weight gain No important safety concerns For more information on other VX-770 studies, see posters by R Ahrens, E McKone & P Flume: #s 203, 204 & 206, respectively

Questions Arising from the First CFTR Modulator Studies

How Much CFTR Function Is Required to Ameliorate or Prevent CF? Sweat test nasal PD Hirtz S et al., Gastroenterology 2004 Knowles MR et al., Human Gene Therapy 1995 Wilschanski M et al., Am J Respir Crit Care Med 2006  ~20%  ~35% I sc rectal biopsies  ~30%

Effect of VX-770 on CFTR Function in CF Patients with the G551D Mutation Accurso FJ et al, New Engl J Med 2010 Sweat test Nasal PD See talk by SM Rowe: S14 (Friday 10:30 AM) See poster by JP Clancy: #202

Which Outcome Measures Will Capture Therapeutic Benefits of CFTR Modulation? Restoration of mutant CFTR function Mucociliary clearance Inflammation Microbiology Lung function Lung structure Clinical outcomes

The G551D Observational (GOAL) Study Day 1 Pre-Dose First dose of VX month after day 1 3 months after day 1 6 months after day 1 Core Study MeasuresAdditional Sub-Study Measures Clinical outcome Sweat chloride Quality of life CFQ-R SNOT-20 Biomarker collection Serum Plasma DNA Urine Sputum Clinical outcome Sweat chloride Quality of life CFQ-R SNOT-20 Biomarker collection Serum Plasma DNA Urine Sputum VX-770 not prescribed yes no MCC/Rheology – visits 2, 5 Radionuclear mucociliary clearance Micro-rheology Bulk rheology MCC/Rheology – visits 2, 5 Radionuclear mucociliary clearance Micro-rheology Bulk rheology Sputum Inflammation & Microbiome – visits 2,5 Induced sputum Inflammatory mediators Sputum Inflammation & Microbiome – visits 2,5 Induced sputum Inflammatory mediators Sweat Rate – visits 1 to 5 Sweat evaporimetry Exploratory sweat outcomes Sweat Rate – visits 1 to 5 Sweat evaporimetry Exploratory sweat outcomes Intestinal pH – visits 2, 3 Intestinal pH by radiofrequency transmitter Intestinal pH – visits 2, 3 Intestinal pH by radiofrequency transmitter Visit 2 Visit 3 Visit 4 Visit 5 Visit 1b Visit 1 Decision made to start VX-770? (before end of study enrollment) Decision made to start VX-770? (before end of study enrollment) Courtesy of Steven Rowe See talks by C De Boeck & N Yang: S14 & S19 (Friday 10:30 AM & Saturday 10:30 AM, respectively) See posters by F Ratjen, T Altes & T Gonska: #s 201, 205 & 213, respectively

Relationship Between Timing of CFTR Modulator Treatment and Clinical Benefit? Progression of CF Lung Disease BirthChildhoodAdult ReversibleIrreversible Normal airway Mucus & Air trapping Airway wall thickening Bronchiectasis Adapted from Ramsey BW, PATS 2007 Window of opportunity See talk by SM Stick: S5 (Thursday, 2:00 PM) See posters by T Nguyen, L Mott & M Rosenfeld: #s 88, 332 & 335 CF newborn screening

CF Lung Disease Starts in Infancy Sly PD et al, Am J Respir Crit Care Med 2009 Bronchial dilatation (19%) Normal Bronchial thickening (45%) Air trapping (67%)

Early “Causal Therapy” Prevents Decline of Lung Function in COPD Fletcher C & Peto R, Br Med J 1977

Progress in Correcting  F508 CFTR CFTR  F508 Courtesy of Martina Gentzsch

VX-809 Corrects  F508 CFTR in Primary Human Bronchial Epithelial Cultures VX-809 Cl-  F508 Cl- Van Goor F et al., PNAS 2011  F508 HBE  F508 Activity

Results of Phase IIa Study of VX-809 in CF Patients Homozygous for  F508 CFTR Clancy JP et al. Thorax 2011 No significant differences in: Adverse events Nasal PD Lung function Patient-reported outcomes VX-809 versus placebo for 28 days (n=89)  Combination of VX VX-770 for  F508 ongoing See talk by SM Rowe: S15 (Saturday, 10:30 AM); See talk by M Boyle: W23 (Saturday, 2:30 PM) – poster #212

CFF 2010 Strategic Planning Meeting on Development of Next Generation  F508 CFTR Correctors Recommendations : Development of new lead compounds that rescue  F508 CFTR activity to critical threshold (goal:  50% of normal CFTR activity) Conduct two independent HTS discovery assays (functional and mechanistic) Use human airway epithelia as early as possible in the discovery process Increase the number of compounds tested in primary screening assays See poster by F Liang: #190

Recent Results from CFTR Folding Consortia:  F508 Interferes with at Least Two Steps in CFTR Folding NBD1 Folding  F508 Domain Assembly  F508 F508 NBD1 NBD2 Serohijos AWR et al., PNAS 2008 Mendoza JL et al., J Bioenerg Biomembr 2007 See talks by R Ford, P Thibodeau: S4 (Thursday, 2:00 PM) See poster by A Aleksandrov: #17

wt CFTR MSD2 R NBD1 folded ΔF508 Cell Surface Expression Is Synergistically Rescued by NBD1 and NBD1-CL4 Interface Stabilization - WT folded ΔF508-CFTR +R1S +R1070W R MSD2 ΔF508 NBD1-R1S 1070W R1S CFTR surface density (%) non-native ΔF508-CFTR R MSD2 ΔF508 NBD1 - ΔF508 ΔF508-CFTR +R1070W R MSD2 ΔF508 NBD1 1070W - ΔF508-CFTR +R1S R MSD2 ΔF508 NBD1-R1S R1S - WM Rabeh & GL Lukacs

ΔF508 Function Is Synergistically Rescued by NBD1 and NBD1-CL4 Interface Stabilization + CFTR Inh-172 Wild-type  F508  F508 + suppressor mutation  F508 + R1070W  F508 + suppressor mutation + R1070W NBD1 and assembly defects corrected See talk by JL Mendoza: W2 (Thursday 10:00) – Poster #4 and talk by PJ Thomas: W12 (Friday 2:00 PM)

Correctors That Target Both Steps May Rescue Surface Expression and Function of  F508 CFTR NBD1 Folding Domain Assembly Corrector A + Corrector B  F508

See talk by M Pregel: S15 (Saturday, 10:30 AM) Courtesy of Pfizer’s CF Program Combinations of Correctors Can Produce Synergistic Rescue of  F508 CFTR Function

Ongoing Efforts to Identify the Next Generation  F508 CFTR Correctors and more to follow

Effects of CFTR Modulators on Rare CFTR Mutations More than 1,800 CFTR mutations Most are rare Functional consequences often unknown

VX-770 Increases Open Probability and Cl - Transport of Rare CFTR Gating Mutants in Vitro ATP G1349D G551D,S S1255P G178R G970R G1244E Mutant CFTR expressed in Fischer rat thyroid cells Courtesy of Vertex Pharmaceuticals See talk by F Van Goor: W12 (Friday 2:00 PM) – Poster #10

Increased Folding and Channel Activity of a Rare Folding Mutation V232D- CFTR by Corrector Cor 4a Caldwell RA et al., Am J Physiol Lung Cell Mol Physiol 2011

CFTR Modulators Provide Opportunity for Personalized Medicine for CF Patients with Rare CFTR Mutations Identify individual CFTR mutations by genotyping Determine molecular phenotype of rare CFTR mutations (Mutation class) and test effects of CFTR modulators in vitro Study consequence on CFTR function and response to CFTR modulator therapy in vivo + +  Identify CF patients with rare CFTR mutations who may benefit from specific CFTR modulators  CFTR2 Project See talks by GR Cutting and PR Sosnay: Plenary 2 (Friday 9:00 AM)

Alternative Targets to Compensate for CFTR Dysfunction

Targeting the Epithelial Sodium Channel (ENaC) to Restore Airway Surface Liquid in CF Tarran R et al., J. Biol. Chem CFTR ENaC Normal CF Normal CF

Increased ENaC Activity Produces ASL Depletion and CF-Like Lung Disease in Mice Wild-type  ENaC-Tg Mall M et al., Nat. Med Inflammation βENaC-Tg Mucus obstruction βENaC-Tg Wild-type  ENaC-Tg ASL See posters by A Livraghi-Butrico & T Ono: #177 & 187, respectively

Increased Potency on ENaC Decreased Rate of Absorption by HBE Greater Durability, Increased Water Retention by Airway Epithelial Cells (8 hours) Courtesy of A Hirsh & R Johnson, Parion Sciences New Generation Durable ENaC Blocker P643 Enhanced Mucociliary Clearance in Large Animal Model **

P643 Reduces Airway Mucus Obstruction and Inflammation in  ENaC-Tg Mice with Chronic Lung Disease Vehicle P643  ENaC-Tg Wild-type Zhe Zhou et al. Vehicle Amiloride  ENaC-Tg Wild-type Amiloride study P643 study

The Calcium-Activated Chloride Channel TMEM16A as Possible Drug Target in CF HBE Microarrays RNA (+/- IL-4) Courtesy of Luis Galietta TMEM16A See posters by G Veit, JP Clancy, W Namkung, S Zhang & E Sondo:#s 75, 93,107, 109 & 118 Identification of TMEM16A activators by high-throughput screening

Cystic Fibrosis Foundation Therapeutics Pipeline

CF Research and Drug Development Strategy – Leading the Way for Other Rare Diseases Genetic defect Basic research Clinical research Patients Industry Patient organizations + Abnormal protein Disease mechanisms Rare diseases more than 6,000 rare diseases 75% affect children 80% have genetic basis often misdiagnosed no causal therapies

Drugs Developed for CF May Have Benefits for Patients with Common Lung Diseases ASL Depth Mucociliary Transport CFTR Activity CFTR function is reduced in cigarette smokers HBE in vitro Cantin A et al., Am J Respir Crit Care Med 2006 Clunes LA et al., FASEB J 2011 Hogg J et al., New Engl J Med 2004 See poster by P Sloane: #245

Conclusion 25 years of dedicated research have built a sound foundation for development of drugs that target CF at root cause Exemplary drug development program has translated basic research findings into several small molecules that improve activity of mutant CFTR in patients First phase III study with CFTR modulator VX-770 showing significant clinical benefits in patients with the G551D mutation Robust pipeline to develop and enhance efficacy of CFTR modulators that rescue activity of other mutations including  F508 to critical threshold Process and results of CF research may be applicable to other diseases

THANK YOU! CF Researchers CF Foundation Therapeutics Development Network and Other Clinical Trial Networks (ECFS-CTN) CF Care Teams People with CF and Their Families