Supplementary Appendix

Transcription

1 Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Shima M, Hanabusa H, Taki M, et al. Factor VIII mimetic function of humanized bispecific antibody in hemophilia A. N Engl J Med 2016;374: DOI: /NEJMoa

2 SUPPLEMENTARY APPENDIX CONTENTS INVESTIGATORS... 2 SUPPLEMENTARY METHODS... 3 SUPPLEMENTARY DESCRIPTION... 4 SUPPLEMENTARY FIGURES... 5 Figure S1. Schematic Illustrations of the Action of FVIIIa and Emicizumab as a Cofactor Promoting the Interaction between FIXa and FX Figure S2. Median Annualized Bleeding Rates for Each Cohort during Weekly Emicizumab Administration Figure S3. Laboratory Values for Platelets, D-Dimer, and TAT during Weekly Emicizumab Administration Figure S4. Plasma Emicizumab Concentration and APTT (All Patients in the Study + In Vitro Addition to Commercial FVIII-Deficient Plasma) Figure S5. Plasma Emicizumab Concentration and TG Peak Height (All Patients in the Study + In Vitro Addition to Commercial FVIII-Deficient Plasma) Figure S6. Plasma Emicizumab Concentration and TG Peak Height with Low-Tissue Factor Trigger (In Vitro Addition to Commercial FVIII-Deficient Plasma) SUPPLEMENTARY TABLES Table S1. Treatment with Clotting Factor Concentrates before Enrollment in the Study Table S2. Episodic Use of Clotting Factor Concentrates during Emicizumab Administration Table S3. Annualized Clotting Factor Dose for Episodic Treatment before and during Emicizumab Administration SUPPLEMENTARY REFERENCES

3 INVESTIGATORS Keiji Nogami (Nara Medical University, Kashihara, Japan), Hideji Hanabusa (Ogikubo Hospital, Tokyo, Japan), Masashi Taki (St. Marianna University School of Medicine, Kawasaki, Japan), Tadashi Matsushita (Nagoya University, Nagoya, Japan), Tetsuji Sato (University of Occupational and Environmental Health, Kitakyushu, Japan), and Katsuyuki Fukutake (Tokyo Medical University, Tokyo, Japan). 2

4 SUPPLEMENTARY METHODS Refer to the protocol and statistical analysis plan. 3

5 SUPPLEMENTARY DESCRIPTION The previous nonclinical study with an acquired hemophilia A model in nonhuman primates indicated that 61 g (first dose) or 36 g (at 16 hours thereafter) of plasma emicizumab per milliliter exerted similar hemostatic activity against on-going bleeds to the estimated levels in porcine factor VIII: 25 U (first dose) or 7.4 U (at 16 hours thereafter, trough) per deciliter. S1 From these data, a factor for the conversion of micrograms of emicizumab per milliliter to units of equivalent factor VIII hemostatic activity per deciliter was estimated to be 0.2 to 0.4 (around 0.3). 4

6 SUPPLEMENTARY FIGURES Figure S1. Schematic Illustrations of the Action of FVIIIa and Emicizumab as a Cofactor Promoting the Interaction between FIXa and FX. A. FVIIIa consists of the A1 subunit, the A2 subunit and the light chain (A3, C1 and C2 subunits), and has multiple contacts with FIXa S2-S5, FX S6,S7, and the phosphatidylserine-exposed phospholipid membrane. Since FIXa and FX also have binding capability to phosphatidylserine-exposed phospholipid membrane, FVIIIa is considered to support the interaction between FIXa and FX on the membrane and to promote FIXa-catalyzed FX activation. B. Emicizumab binding to FIXa and FX would promote the interaction between FIXa and FX on phosphatidylserineexposed phospholipid membrane and exert FVIII-mimetic activity. The illustrations summarize the hypothesized mechanisms of action of emicizumab only and do not necessarily indicate precise molecular structures and positions. FIXa, activated factor IX; FVIII, factor VIII; FVIIIa, activated factor VIII; FX, factor X. 5

7 Figure S2. Median Annualized Bleeding Rates for Each Cohort during Weekly Emicizumab Administration. The annualized bleeding rates for each patient were calculated by the number of bleeding episodes that required treatment with coagulation factor products during the 6 months before study enrollment and during emicizumab administration. Panel A: all bleeding; Panel B: joint bleeding. 6

8 Figure S3. Laboratory Values for Platelets, D-Dimer, and TAT during Weekly Emicizumab Administration. Patients received a 1 mg per kilogram loading dose followed by weekly injections of 0.3 mg per kilogram (Cohort 1, blue circles), a 3 mg per kilogram loading dose followed by weekly injections of 1 mg per kilogram (Cohort 2, red circles), or weekly injections of 3 mg per kilogram (Cohort 3, green circles). Panel A: platelets; Panel B: D-dimer; Panel C: TAT. * Considered to be a result of bleeding, ** considered to be a result of dehydration, *** considered to be an effect of the blood collection technique. TAT, thrombin-antithrombin complex. 7

9 Figure S4. Plasma Emicizumab Concentration and APTT (All Patients in the Study + In Vitro Addition to Commercial FVIII-Deficient Plasma). Relationship between plasma emicizumab concentration and APTT in plasma samples collected from patients with (red open squares) and without (black open circles) FVIII inhibitors in the study is shown. Plasma emicizumab concentration was imputed by 0.05 g per milliliter if below the limit of quantification. A concentration-response curve from an in vitro experiment in which emicizumab was added to commercial FVIIIdeficient plasma at each concentration was overlaid (blue closed circles). The method of the in vitro experiment was previously described S1 except that the analyzer used in this study was CA-7000 (Sysmex Corporation, Kobe, Japan). APTT, activated partial thromboplastin time; FVIII, factor VIII. 8

10 Figure S5. Plasma Emicizumab Concentration and TG Peak Height (All Patients in the Study + In Vitro Addition to Commercial FVIII-Deficient Plasma). Relationship between plasma emicizumab concentration and FXIa-triggered TG peak height in plasma samples collected from patients with (red open squares) and without (black open circles) FVIII inhibitors in the study is shown. Plasma emicizumab concentration was imputed by 0.05 g per milliliter if below the limit of quantification. TG peak height was imputed by 2 nmol per liter if undetectable. A concentration-response curve from an in vitro experiment in which emicizumab was added to commercial FVIII-deficient plasma at each concentration was overlaid (blue closed circles). The method of the in vitro experiment was previously described. S8 FVIII, factor VIII; FXIa, activated factor XI; TG, thrombin generation. 9

11 Figure S6. Plasma Emicizumab Concentration and TG Peak Height with Low-Tissue Factor Trigger (In Vitro Addition to Commercial FVIII-Deficient Plasma). Relationship between plasma emicizumab concentration (left panel) or FVIII activity (right panel) and low tissue factor-triggered TG peak height in an in vitro experiment in which emicizumab or rhfviii was added to commercial FVIII-deficient plasma at each concentration S9 are shown. The method of the in vitro experiment was previously described. S1 Data represent mean values, error bars represent standard deviations (measured in triplicate). FVIII, factor VIII; rhfviii, recombinant human factor VIII; TG, thrombin generation. 10

12 SUPPLEMENTARY TABLES Table S1. Treatment with Clotting Factor Concentrates before Enrollment in the Study. FVIII Presence of Patient Age Inhibitor Target Treatment before Enrollment Cohort No. (yr) Titer (BU/ml) Joints Prophylaxis* Episodic Yes None apcc Yes None apcc / rfviia C Yes None apcc / rfviia Yes None apcc / rfviia Non-inhibitor Yes FVIII FVIII Non-inhibitor Yes FVIII FVIII Yes None apcc Yes None apcc / rfviia C Yes None apcc / rfviia Yes None apcc Non-inhibitor Yes FVIII FVIII Non-inhibitor Yes FVIII FVIII Yes apcc apcc / rfviia No apcc apcc / rfviia C Yes rfviia rfviia Non-inhibitor Yes FVIII FVIII Non-inhibitor No FVIII Non-inhibitor No FVIII FVIII * All patients without FVIII inhibitors had been treated with secondary prophylaxis. apcc, activated prothrombin complex concentrates; BU, Bethesda unit; FVIII, factor VIII; rfviia, recombinant activated factor VII; yr, year. 11

13 Table S2. Episodic Use of Clotting Factor Concentrates during Emicizumab Administration. Number of Clotting Factor Doses Weight Bleeding Sites Needed to Treat Each Bleeding Episode Patient No. (kg) (Number of Episodes) Clotting Factor 1 Dose 2 Doses 3 Doses C Joint (2), Muscle (1) rfviia C Joint (2) rfviia C Joint (14) FVIII C Muscle (1) rfviia C Other (1) FVIII FVIII, factor VIII; rfviia, recombinant activated factor VII. 12

14 Table S3. Annualized Clotting Factor Dose for Episodic Treatment before and during Emicizumab Administration. 6 Months before During Emicizumab Clotting Factor Statistics Enrollment Administration N 6 6 FVIII (IU) Mean±SD Median 75,411±112,813 34,493 37,162±87,944 0 Range 12, , ,602 N 9 9 apcc (IU)* Mean±SD Median 248,214±158, ,944 0±0 0 Range 12, , N 7 7 rfviia (mg)* Mean±SD Median 305± ±118 0 Range * Patient C was treated with apcc or rfviia for episodic use before enrollment. However, data on 70% of the doses given to this patient for each bleeding episode were missing. Therefore, this patient s data were not included in this analysis. apcc, activated prothrombin complex concentrates; FVIII, factor VIII; rfviia, recombinant activated factor VII; SD, standard deviation. 13

15 SUPPLEMENTARY REFERENCES S1. Muto A, Yoshihashi K, Takeda M, et al. Anti-factor IXa/X bispecific antibody (ACE910): hemostatic potency against ongoing bleeds in a hemophilia A model and the possibility of routine supplementation. J Thromb Haemost 2014;12: S2. Lenting PJ, Donath MJ, van Mourik JA, Mertens K. Identification of a binding site for blood coagulation factor IXa on the light chain of human factor VIII. J. Biol Chem. 1994;269: S3. Fay PJ, Koshibu K. The A2 subunit of factor VIIIa modulates the active site of factor IXa. J. Biol. Chem. 1998;273: S4. Soeda T, Nogami K, Nishiya K, et al. The factor VIIIa C2 domain (residues ) interacts with the factor IXa Gla domain in the factor Xase complex. J. Biol Chem. 2009;284: S5. Griffiths AE, Rydkin I, Fay PJ. Factor VIIIa A2 subunit shows a high affinity interaction with factor IXa: contribution of A2 subunit residues to the interaction with factor IXa. J Biol Chem. 2013;288: S6. Lapan KA, Fay PJ. Localization of a factor X interactive site in the A1 subunit of factor VIIIa. J. Biol. Chem. 1997;272: S7. Takeyama M, Wakabayashi H, Fay PJ. Factor VIII light chain contains a binding site for factor X that contributes to the catalytic efficiency of factor Xase. Biochemistry. 2012;51: S8. Sampei Z, Igawa T, Soeda T, et al. Identification and multidimensional optimization of an asymmetric bispecific IgG antibody mimicking the function of factor VIII cofactor activity. PLoS One 2013;8:e S9. Soeda T, Kitazawa T, Muto A, et al. In vitro characterization of ACE910, a humanized bispecific antibody to factors IXa and X. Haemophilia 2014;20:Suppl 3:77. abstract. 14