1 CCL2 Inhibition in Idiopathic Pulmonary Fibrosis: a Phase 2 Trial of Carlumab

3 Ganesh Raghu, Fernando J. Martinez, Kevin K. Brown, Ulrich Costabel, Vincent Cottin, Athol U.

4 Wells, Lisa Lancaster, Kevin F. Gibson, Tarik Haddad, Prasheen Agarwal, Michael Mack, Bidisha

5 Dasgupta, Ivo P. Nnane, Susan K. Flavin, Elliot S. Barnathan

6 7 Online Data Supplement 8 9 METHODS 10 11 Patients 12 13 Key exclusion criteria were: evidence of other interstitial pneumonias or any other significant respiratory

14 disorder; partial pressure of oxygen in arterial blood <55 mmHg (sea level) or 50 mmHg (altitude) at rest

15 on room air; clinically significant pulmonary hypertension requiring vasodilator therapy/chronic

16 anticoagulation therapy; FEV1/FVC <0.7 at screening; an increase in FEV1 ≥12% post-bronchodilator;

17 current treatment with prohibited investigational medications including sildenafil, interferon-γ,

18 mycophenolate, or endothelin receptor antagonists.

20 Other exclusion criteria included current or recent treatment with an investigational/experimental

21 medication; suspected active or history of latent tuberculosis; current listing for lung transplantation; or

22 predicted life expectancy <1 year. Pregnant women were excluded and those of childbearing potential

23 were required to use adequate birth control from screening through 12 months after the last study

24 agent infusion.

26 Concomitant medications

27 Concomitant medication use (see Table 1) was reviewed at each study visit. During the study, patients

28 were permitted, but not required, to be managed on 1 or more classes of medications that was

1 referenced in the ATS/ERS consensus statement of 2000 as “conventional treatment options”, including

2 corticosteroids, immunosuppressive/cytotoxic agents (e.g., azathioprine or cyclophosphamide), and

3 anti-fibrotic agents (e.g., D-penicillamine and colchicine). Other treatments described in the ATS/ERS

4 guidelines as “potential alternative treatments” (e.g., N-acetylcysteine) were also permitted at the

5 discretion of the investigator. Stable doses must have been established for all concomitant medications

6 before and maintained throughout the treatment period. The initiation of new concomitant medications

7 for IPF was strongly discouraged through week 52.

8 9 Statistics 10 11 Hypothesis testing for continuous endpoints in lung function parameters was based on the analysis of

12 variance model to compare treatment groups. Non-lung function parameters were analyzed using non-

13 parametric methods. Time-to-event data were analyzed using the cox-proportional hazard model.

14 Missing FVC data at week 52 were imputed based on a linear mixed model that took into account the

15 available FVC assessments.

16 17 Prespecified subgroup analyses for the primary endpoint were performed for age (< or ≥ median), race

18 (Caucasian/non-Caucasian), sex, investigational site region (Europe/North America), baseline FVC (

19 median), high-risk category (yes/no), baseline use of oral corticosteroids (yes/no), baseline use of proton

20 pump inhibitors (yes/no), baseline DLCO (< or ≥ median), baseline 6MWD (< or ≥ median), baseline

21 SGRQ total score (< or ≥ median), respiratory-related hospitalizations or emergency room visits in the

22 last year (none or ≥1), and baseline CCL2 (< or ≥ median).

24 The planned sample size of 120 total patients (30 per group) to be enrolled at approximately 25 sites

25 globally, provided approximately 79% statistical power to detect a difference of 7% in the annual rate of

26 percent change in FVC between the placebo group and any carlumab group, assuming a 20% drop-out

27 rate and a variance of 0.02, at the 0.05 level of significance. This was computed assuming a population

1 standard deviation of 12% in the annual change from baseline in FVC, which was obtained using data

2 from an in-house study of infliximab treatment in patients with chronic pulmonary sarcoidosis, another

3 interstitial lung disease [1]. Similar declines in lung function have also been reported in trials of IPF

4 patients [2, 3]. The FVC of an IPF patient was assumed to range from 1.5L to 3.5L and thus

5 corresponded to a standard deviation of 180-420 mL in absolute change in FVC in IPF patients [4].

7 PHARMACOKINETICS, IMMUNOGENICITY, AND PHARMACODYNAMICS

8 Methods

9 All patients in the study had serum samples drawn pre-infusion at all visits, as well as end-of-infusion at

10 weeks 0 and 48 to evaluate carlumab PK. Patients in the PK subset analysis had additional blood samples

11 taken on Day 0 (pre-infusion, end-of-infusion, and 4 hours post-infusion), Day 7, and any timepoint

12 between Days 14 and 21. Also, for the week 48 (last) infusion, blood samples were taken on the day of

13 the infusion (pre-infusion, end-of-infusion, and 4 hours post-infusion), 7 days after the week 48 infusion,

14 any timepoint between 14 and 21 days after the week 48 infusion, and any timepoint between 35 and

15 56 days after the last infusion.

17 Serum samples were collected from all patients at weeks 0, 24, 52, and 72 to determine antibodies to

18 Carlumab. Carlumab concentration in serum was measured using an ELISA-based immunoassay. The PK

19 parameters of carlumab were calculated using non-compartmental methods using WinNonlin 5.2.1.

21 Serum samples for PD analyses of relevant markers in serum, urine, peripheral blood mononuclear cells,

22 exhaled breath condensates, and induced sputum were collected at weeks 0, 4, 12, 24, 36, and 52.

24 Results

1 After carlumab administration, the exposure (Cmax and AUCtau) increases in a dose-proportional manner

2 (Supplemental Figure S1). The combined incidence of antibodies to carlumab was 7.6%. There was no

3 significant difference in the change from baseline to weeks 24 or 52 in CRP or TGFβ concentrations (two

4 PD markers).

6 The mean serum concentration of carlumab peaked following the infusion after the week 0 (see

7 Supplemental Table S2), week 4, and week 48 infusions, and declined exponentially after the last

8 infusion at week 48, until carlumab was undetectable in most patients at week 72. Steady state appears

9 to have been achieved after the second dose of carlumab across all dose levels. The mean t1/2 of

10 carlumab ranged from 6.29 to 9.01 days, the CL ranged from 5.50 mL/day/kg to 9.94 mL/day/kg, and the

11 Vz ranged from 70.80 mL/kg to 88.13 mL/kg following the first IV infusion of carlumab across all

12 treatment groups (Supplemental Table S2). The PK parameters after the last IV infusion appear to be

13 similar to estimates after the first IV infusion of carlumab.

15 Two PD markers, C-reactive protein (CRP) and Transforming Growth Factor beta (TGFβ), were measured

16 at baseline, week 24, and week 52 for all patients. CRP is a marker of inflammation that is related to

17 CCL2 expression [5] and TGFβ is well characterized as a marker of fibrosis [6].

25 4

1 Table S1- Subgroup analyses for baseline demographic characteristics, Rate of change from baseline in FVC through Week 52; modified ITT 2 patients

Carlumab Placebo 1 mg/kg 5 mg/kg 15 mg/kg Modified ITT patients 28 33 32 32 Modified ITT patients with baseline value for the FVC 28 33 32 32 Age

Carlumab Placebo 1 mg/kg 5 mg/kg 15 mg/kg 95% CI (-0.0330; (-0.0135; (-0.0222; (-0.0177; -0.0045) 0.0066) 0.0153) -0.0007) p-valuea 0.218 0.268 Race Caucasian 28 32 32 32 Rate of change from baseline -0.02 -0.02 -0.02 -0.01 95% CI (-0.0265; (-0.0234; (-0.0307; (-0.0207; -0.0080) -0.0073) -0.0139) -0.0040) p-valuea 0.426 0.443 Investigational site region Europe 6 8 5 6 Rate of change from baseline -0.02 -0.01 -0.02 -0.01 95% CI (-0.0351; (-0.0228; (-0.0413; (-0.0258; -0.0010) 0.0074) -0.0056) 0.0096) p-valuea 0.673 0.437 North America 22 25 27 26 Rate of change from baseline -0.02 -0.02 -0.02 -0.01 95% CI (-0.0281; (-0.0272; (-0.0317; (-0.0228; -0.0064) -0.0081) -0.0125) -0.0036) p-valuea 0.516 0.587 FVC(L) < median 13 14 18 17 Rate of change from baseline -0.01 -0.01 -0.03 -0.01

Carlumab Placebo 1 mg/kg 5 mg/kg 15 mg/kg 95% CI (-0.0257; (-0.0213; (-0.0353; (-0.0193; -0.0023) -0.0021) -0.0175) -0.0022) p-valuea 0.103 0.663 ≥ median 15 19 14 15 Rate of change from baseline -0.02 -0.02 -0.02 -0.01 95% CI (-0.0334; (-0.0307; (-0.0327; (-0.0295; -0.0059) -0.0054) -0.0034) 0.0011) p-valuea 0.877 0.604 DLCO (mL/min/mmHg) < median 12 15 15 19 Rate of change from baseline -0.02 -0.01 -0.03 -0.02 95% CI (-0.0388; (-0.0219; (-0.0421; (-0.0284; -0.0100) 0.0013) -0.0175) -0.0066) p-valuea 0.578 0.462 ≥median 16 17 17 12 Rate of change from baseline -0.01 -0.02 -0.02 -0.01 95% CI (-0.0240; (-0.0315; (-0.0276; (-0.0188; -0.0001) -0.0082) -0.0048) 0.0070) p-valuea 0.625 0.497 6MWD (meters) < median 12 10 19 20 Rate of change from baseline -0.02 -0.01 -0.02 -0.02 95% CI (-0.0361; (-0.0258; (-0.0351; (-0.0286; -0.0067) 0.0031) -0.0138) -0.0080)

Carlumab Placebo 1 mg/kg 5 mg/kg 15 mg/kg ≥median 16 23 13 12 Rate of change from baseline -0.02 -0.02 -0.02 0.00 95% CI (-0.0272; (-0.0271; (-0.0331; (-0.0176; -0.0029) -0.0068) -0.0060) 0.0108) p-valuea 0.628 0.226 SGRQ total score < median 14 17 19 11 Rate of change from baseline (SE) -0.01 -0.02 -0.02 -0.01 95% CI (-0.0258; (-0.0300; (-0.0268; (-0.0216; 0.0017) -0.0067) -0.0036) 0.0079) p-valuea 0.730 0.619 ≥median 13 15 13 20 Rate of change from baseline -0.02 -0.01 -0.03 -0.02 95% CI (-0.0342; (-0.0239; (-0.0446; (-0.0259; -0.0088) -0.0011) -0.0202) -0.0059) p-valuea 0.232 0.500 High Risk Category No 9 12 11 12 Rate of change from baseline -0.02 -0.02 -0.02 -0.01 95% CI (-0.0412; (-0.0347; (-0.0315; (-0.0227; -0.0070) -0.0038) 0.0005) 0.0080) p-valuea 0.476 0.162 Yes 19 21 21 20 Rate of change from baseline -0.01 -0.01 -0.03 -0.02

Carlumab Placebo 1 mg/kg 5 mg/kg 15 mg/kg 95% CI (-0.0241; (-0.0225; (-0.0357; (-0.0251; -0.0027) -0.0040) -0.0165) -0.0054) p-valuea 0.088 0.800 Use of proton pump inhibitors at screening No 13 18 20 16 Rate of change from baseline -0.01 -0.01 -0.03 -0.01 95% CI (-0.0268; (-0.0251; (-0.0362; (-0.0208; -0.0006) -0.0042) -0.0156) 0.0007) p-valuea 0.158 0.672 Yes 15 15 12 16 Rate of change from baseline -0.02 -0.02 -0.02 -0.02 95% CI (-0.0340; (-0.0296; (-0.0320; (-0.0296; -0.0064) -0.0032) -0.0027) -0.0022) p-valuea 0.785 0.670 Baseline Use of Oral Corticosteroids No 24 28 26 27 Rate of change from baseline -0.02 -0.02 -0.02 -0.01 95% CI (-0.0261; (-0.0282; (-0.0294; (-0.0217; -0.0067) -0.0112) -0.0109) -0.0040) p-valuea 0.586 0.598 Yes 4 5 6 5 Rate of change from baseline -0.02 0.01 -0.03 -0.01 95% CI (-0.0448; (-0.0068; (-0.0469; (-0.0285; 0.0000) 0.0333) -0.0139) 0.0118)

Carlumab Placebo 1 mg/kg 5 mg/kg 15 mg/kg p-valuea 0.584 0.378 95% CI (-0.0241; (-0.0334; (-0.0243; (-0.0275; 0.0094) 0.0208) 0.0217) 0.0013) p-valuea 0.686 0.617 CCL2 < median 14 22 13 13 Rate of change from baseline -0.02 -0.02 -0.02 -0.02 95% CI (-0.0317; (-0.0266; (-0.0307; (-0.0310; -0.0030) -0.0047) -0.0030) -0.0011) p-valuea 0.963 0.906 ≥ median 14 11 18 19 Rate of change from baseline -0.02 -0.01 -0.03 -0.01 95% CI (-0.0291; (-0.0275; (-0.0373; (-0.0197; -0.0054) -0.0021) -0.0164) -0.0004) p-valuea 0.241 0.360 1

2 a Treatment comparisons are based on linear contrasts within each subgroup

3 CI, confidence interval; DLCO, diffusing capacity of the lung for carbon monoxide; FVC, forced vital capacity; ITT, intent-to-treat; SE, standard error; 4 SGRQ, Saint George’s Respiratory Questionnaire; 6MWD, 6-minute walk distance; CCL2, CC-chemokine ligand (CCL) 2

1 Table S2. Carlumab pharmacokinetic parameters after first dose at week 0; treated patients* in 2 the PK sub-study

Carlumab 1 mg/kg 5 mg/kg 15 mg/kg Cmax (ug/mL) Mean (SD) 23.72 (7.448) 169.53 (144.862) 450.38 (163.406) AUC(0-28d) (ug.day/mL) Mean (SD) 137.98 (81.242) 829.91 (118.561) 2531.49 (849.129) t1/2 (day) Mean (SD) 6.29 (1.491) 9.01 (1.053) 8.30 (1.506) CL (mL/day/kg) Mean (SD) 9.94 (5.125) 5.50 (0.857) 6.03 (1.845) Vz (mL/kg) Mean (SD) 88.13 (44.318) 70.80 (5.829) 71.86 (22.243) 3 4 Cmax, maximum observed serum concentration; AUC, area under the serum (or other biological fluids) 5 concentration versus time curve; t1/2, half-life; CL, total systemic clearance of drug after intravenous 6 administration; Vz, volume of distribution during terminal phase; SD, standard deviation. 7 *Number of patients with appropriate data for calculation of PK parameters in each dose level varied 8 from 6-8. 9 10 11 12 13

1 Supplemental References 2 3 1. Baughman RP, Drent M, Kavuru M, Judson MA, Costabel U, du Bois R, Albera C, Brutsche M, 4 Davis G, Donohue JF, Müller-Quernheim J, Schlenker-Herceg R, Flavin S, Lo KH, Oemar B, Barnathan ES, 5 Sarcoidosis Investigators. Infliximab therapy in patients with chronic sarcoidosis and pulmonary 6 involvement. Am J Respir Crit Care Med 2006: 174: 795-802. 7 2. Richeldi L, Cottin V, Flaherty KR, Kolb M, Inoue Y, Raghu G, Taniguchi H, Hansell DM, Nicholson 8 AG, Le Maulf F, Stowasser S, Collard HR. Design of the INPULSIS trials: two phase 3 trials of nintedanib in 9 patients with idiopathic pulmonary fibrosis. Respir Med 2014: 108: 1023-1030. 10 3. du Bois RM, Weycker D, Albera C, Bradford WZ, Costabel U, Kartashov A, King TE, Jr., Lancaster 11 L, Noble PW, Sahn SA, Thomeer M, Valeyre D, Wells AU. Forced vital capacity in patients with idiopathic 12 pulmonary fibrosis: test properties and minimal clinically important difference. Am J Respir Crit Care 13 Med 2011: 184: 1382-1389. 14 4. Nathan SD, Barnett SD, Moran B, Helman DL, Nicholson K, Ahmad S, Shorr AF. Interferon 15 gamma-1b as therapy for idiopathic pulmonary fibrosis. An intrapatient analysis. Respiration 2004: 71: 16 77-82. 17 5. Montecucco F, Steffens S, Burger F, Pelli G, Monaco C, Mach F. C-reactive protein (CRP) induces 18 chemokine secretion via CD11b/ICAM-1 interaction in human adherent monocytes. J Leukoc Biol 2008: 19 84: 1109-1119. 20 6. Selman M, Pardo A, Kaminski N. Idiopathic pulmonary fibrosis: aberrant recapitulation of 21 developmental programs? PLoS Med 2008: 5: e62.

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1 Supplemental Figure Legend:

2 Figure S1: Mean (± SD) Serum concentration versus Time Profiles

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32