1. Manuel Carcao MD, MSc Paediatric Hematologist Associate Professor Hospital for Sick Children University of Toronto Toronto, Canada Prevention of Inhibitors in Hemophilia How to reduce inhibitors

2. Wish to thank the Organizers, the Lebanese Society of Haematology, the WFH and Bayer for bringing me to Lebanon

3. Haemophilia at the start of 20th century A terrible disease with no treatment

4. Tremendous Progress in Hemophilia Care during the 20 th Century Discovery of Cryoprecipitate National and International Hemophilia Organizations Recombinant factors Hemophilia treatment centers Virally inactivated safe factor concentrates “Prophylaxis superior to on demand therapy” Discovery of the FVIII and FIX genes Immune Tolerance Induction Home Care

5. Where are we now in Haemophilia Care  We have safe factor concentrates  But expensive and hence much of the world has little access to these products  Inhibitors continue to occur with no reduction in their incidence

6. Inhibitors Antibodies formed against factor concentrates Inactivate / neutralize / inhibit the activity of factor → Patients become refractory to conventional factor replacement → need expensive and less effective alternative bypassing agents Light chainHeavy chain

7. Economic Clinical Inhibitor development is a huge problem

8. Clinical  More difficult to treat bleeds  ↑ susceptibility to Life-threatening bleeds e.g. ICH  Worse joint disease  Worse quality of life Inhibitor development is a huge problem

9. Economic Inhibitor development is a huge problem Bypassing agents $$$ Immune tolerance therapy (to eradicate inhibitors) $$$$$

10. Why Do Inhibitors Develop? Boy with Severe Haemophilia A “Produces no FVIII” Immune system does not see endogenous FVIII No immune central tolerance

11. Why Do Inhibitors Develop? Receives exogenous FVIII Boy with Severe Haemophilia A “Produces no FVIII”

12. Why Do Inhibitors Develop? FVIII Receives exogenous FVIII Boy with Severe Haemophilia A “Produces no FVIII”

13. Inhibitors develop early in life (usually) Median ≈11 Exposure Days; age 1.5 yrs 20-40% in severe haemophilia A

14. NON-GENETIC POTENTIALLY MODIFIABLE GENETIC NON-MODIFIABLE Multifactorial Risk Factors for Inhibitor Development

15. GENETIC NON-MODIFIABLE Type of Hemophilia: A >>>B Hemophilia Severity: Severe (20-40%) > Moderate (5-10%) >Mild (1-5%) Mutation: Null (25-50%); non-null (<10%) Ethnicity: 2X higher in African ancestry Family History of an inhibitor:  risk by 2-3X Inherited polymorphisms for immune-modulating genes

16. NON-GENETIC POTENTIALLY MODIFIABLE Choice of Factor concentrate: Unclear if a risk factor Intense exposure to factor early on in life: ↑ risk (2-3X) Early start of Prophylaxis: ↓ risk by 40-60%

17. 1) Ben2) Nathan A Case of 2 brothers with severe hemophilia A

18. Born 2007 13 mo of age: Diagnosed I-22 inversion (SHA) 13 mo  21 mo: On demand (6 Exposures to rFVIII for soft tissue bleeds) 21 mo of age: Tongue bleed (5 Consecutive days) 2 wks later: Inhibitor (9 BU) 1) Ben Intense exposure

19. Born 2007 13 mo of age: Diagnosed I-22 inversion (SHA) 13 mo  21 mo: On demand (6 Exposures to rFVIII for soft tissue bleeds) 21 mo of age: Tongue bleed (5 Consecutive days) 2 wks later: Inhibitor (9 BU) 1) Ben Intense exposure Bypassing therapy & ITT X 1.5 yrs & Port

20. Born 2009 Diagnosed at birth Age 8 mo: no exposures Parents asked - “Is there anything that we can do to try to reduce Nathan’s chances of developing an inhibitor?” 2) Nathan

21. Interventions to reduce inhibitors Which factor to use? How can we expose him in the least immunogenic way?

22. Interventions to reduce inhibitors Which factor to use? Is there a factor that has less inhibitors?

23. Plasma derived (PD) FVIII Recombinant (r) FVIII Type of Factor

24. Plasma derived (PD) FVIII Recombinant (r) FVIII Type of Factor Some studies suggest lower incidence of inhibitors

25. Plasma derived (PD) FVIII Recombinant (r) FVIII Type of Factor Some studies suggest lower incidence of inhibitors But confounding variables Differences in how Inhibitor surveillance Different populations - different inhibitor risks Reporting bias – studies showing low inhibitor incidence more likely to be reported

26. Iorio et al. JTH, 2010 Systematic literature review of inhibitor development in Hemophilia A Pd-FVIII (1,167 pts) rFVIII (927 pts) 24 studies included

27. Iorio et al. JTH, 2010 Systematic literature review of inhibitor development in Hemophilia A Inhibitor development Pd-FVIII (1,167 pts) rFVIII (927 pts) P value HTI and LTI in severe pts 16% (10-23%) 34% (29-40%) <0.001 HTI (≥ 5 BU) in severe pts 9% (4-19%) 18% (14-23%) 0.009 24 studies included

28. Iorio et al. JTH, 2010 Systematic literature review of inhibitor development in Hemophilia A Inhibitors Pd-FVIII (1,167 pts) rFVIII (927 pts) P value All in severe pts 16% (10-23%) 34% (29-40%) <0.001 ≥ 5 BU in severe pts 9% (4-19%) 18% (14-23%) 0.009 24 studies included Most of the difference explained by Testing frequency “more recent studies (usually with rFVIII) have tested more frequently and consequently find more inhibitors” Follow-up time “if patients are followed for a longer time more inhibitors will be found” “factor type was not a statistically significant determinant of inhibitor development”

29. Iorio et al. JTH, 2010 Systematic literature review of inhibitor development in Hemophilia A Inhibitors Pd-FVIII (1,167 pts) rFVIII (927 pts) P value All in severe pts 16% (10-23%) 34% (29-40%) <0.001 ≥ 5 BU in severe pts 9% (4-19%) 18% (14-23%) 0.009 24 studies included Most of the variability explained by Testing frequency “more recent studies have tested more and find more inhibitors” Follow-up time When taken into account factor type not statistical significance “..not possible to prove or disprove the hypothesis that there is a higher risk of inhibitor development associated with the use of recombinant vs. Plasma derived FVIII”

30. Systematic literature review of inhibitor development Sippet Study (Survey of Inhibitor development in plasma-product exposed toddlers) Is ongoing (150 of 300 patients already enrolled) Randomized head to head comparison

31. Interventions to reduce inhibitors How can we expose him in the least immunogenic way Early prophylaxis & avoidance of intense exposure

32. Can introduction of early prophylaxis reduce risk of inhibitor? 1.Is there an explanation as to why early start of prophylaxis may reduce inhibitor incidence? 2.Is there any evidence that it does?

33. Danger FVIII  Risk Inflammation Severe bleed (ICH) Surgery 1. Possible explanation “The Danger Signal Theory” Signal 1 Signal 2 Baseline genetic risk  Cytokine production  Activation of Antigen Presenting Cells  Activation of T lymphocytes  Antibody prod. by B lymphocytes Matzinger P. The danger model: a renewed sense of self. Science 2002; 296:301-5.

34. Danger FVIII No Danger Signal FVIII  Risk  Risk Prophylaxis Inflammation Severe bleed (ICH) Surgery 1. Possible explanation “The Danger Signal Theory” Signal 1 Signal 2 Baseline genetic risk

35. 2. EVIDENCE: early introduction of prophylaxis  inhibitor risk 1.Morado 2005 Spain 2.Santagostino 2005Italy 3.Gouw 2007 Multi-Europe + Canada 4.Gouw 2007Netherlands 5.Ragni 2009 USA 6.Kurnik 2010 Germany 7.MacLean 2011 UK

36. 1.Morado 2005 Spain 2.Santagostino 2005Italy 3.Gouw 2007 Multi-Europe + Canada 4.Gouw 2007Netherlands 5.Ragni 2009 USA 6.Kurnik 2010 Germany 7.MacLean 2011 UK Largest Interventional 2. EVIDENCE: early introduction of prophylaxis  inhibitor risk

37. #3) Gouw et al (CANAL study) (2007) Retrospective cohort study 366 consecutive severe hemophilia A patients born 1990-2000 14 centers (13 Europe + 1 Canada) 87 developed inhibitors (24%) Median age at INH. development: 15 mo (14 ED) Gouw SC et al. Blood. 2007;109(11):4648-4654. A large study

38. #3) Gouw et al (CANAL study) (2007) Main Findings 1) Intense exposure  risk of inhibitor (≥ 5 CED) at 1 st exposure= 3.3 X (≥ 5 CED) during 1 st 50 ED= 2.0 X 2) Early start of prophylaxis  risk of inhibitor by 60% Gouw SC et al. Blood. 2007;109(11):4648-4654.

39. #7) Kurnik et al. (2010) (Bremen & Munich) New Early Start (Low dose) Prophylaxis Traditional (High dose) Prophylaxis Prophylaxis regimen Start very early 25 U/kg 1/wk No Ports 40–50 U/kg; 3/wk start at or after 1 st joint/severe bleed # of patients26 consecutive30 consecutive Kurnik K et al. Haemophilia 2010; 16:256-62. Interventional PILOT Study to explore whether early start of prophylaxis would reduce inhibitor development

40. Median age at New Early Start (Low dose) Prophylaxis Traditional (High dose) Prophylaxis Start of prophylaxis 10.7 mo19 mo EDs before prophylaxis 130 Inhibitor incidence 1 / 26 (3.8%) [0 HTI] Kurnik K et al. Haemophilia 2010; 16:256-62. Findings #7) Kurnik et al. (2010) (Bremen & Munich)

41. Median age at New Early Start (Low dose) Prophylaxis Traditional (High dose) Prophylaxis Start of prophylaxis 10.7 mo19 mo EDs before prophylaxis 130 Inhibitor incidence 1 / 26 (3.8%) [0 HTI] 14 / 30 (47%) [ 8 HTI] Kurnik K et al. Haemophilia 2010; 16:256-62. Findings #7) Kurnik et al. (2010) (Bremen & Munich)

42. Early start of prophylaxis protects against inhibitor development It permits children to be exposed to FVIII without any danger signals for inhibitor development Conclusions from these studies

43. At 8 months of age Parents asked is there anything that we can do to reduce Nathan’s chances of developing an inhibitor What happened to Nathan?

44. 8 mo: Started on Early Low dose “Prophylaxis” with PD FVIII 25 U/kg every 2 wks - peripheral IV 18 mo:  to 1 / wk prophylaxis 25 mo:  to 2 / wk prophylaxis Now 34 mo: Has had > 75 ED No Inhibitor What happened to Nathan?

45. Why did he not get an inhibitor? –Choice of Factor? –Early Prophylaxis? –Luck? We will never know from one case alone! What happened to Nathan?

46. Summary 1.Inhibitor Development is a major problem 2.Reducing inhibitors is critical 3.Non genetic inhibitor risk factors are important and may be modifiable 4.We must try to reduce inhibitor development

47. Thank-you for your attention Thank-you for the kind invitation to come to Beirut

48. Do I believe that exposing children with severe hemophilia A at very young ages to factor in the least immunogenic way will result in less inhibitors? YES

49. Multiple PD FVIII Single PD FVIII Single rFVIII Inhibitor Risk in PUPs: High titer (≥5 BU) only Wight J, Paisley S. Haemophilia 2003;9:318-35

50. Multiple PD FVIII Single PD FVIII Single rFVIII Inhibitor Risk in PUPs: High titer (≥5 BU) only Wight J, Paisley S. Haemophilia 2003;9:318-35 No difference

51. Often very small studies with few patients (some as low as n= 19 pts) Mostly retrospective Reporting bias – studies showing low incidence more likely to be reported Single PD FVIII studies

52. Larger Studies Goudemand J. et al. Blood 2006;107:46-51 148 patients followed in France Type of factorPts% Inhibitor% HTI rFVIII (Recombinate & Kogenate) 6232%15% FVIII-LFB (VWF containing-pdFVIII) 8610%5% P value - adjusted for race, family history & age at 1 st exposure 0.049NS

53. Other recent large studies Chalmers et al. Haemophilia 2007 348 PUPS with severe hemophilia A Gouw et al. (CANAL) Blood 2007 366 PUPS with severe hemophilia A

54. Other recent large studies Chalmers et al. Haemophilia 2007 348 PUPS with severe hemophilia A Gouw et al. (CANAL) Blood 2007 366 PUPS with severe hemophilia A No statistically increased risk of inhibitors with rFVIII vs. Pd FVIII (after adjusting for other risk factors)

55. Type of Factor Recombinant (r) FVIII Plasma derived (PD) FVIII

56. 40 separate slides – must reduce by 10

57. Written by David Lillicrap June 11, 2010 After studying FVIII Ab generation for the past several years in many hemophilic mice, the lessons we have learnt are - most severe hemophiliacs will not have central tolerance for FVIII - ie. they don't make FVIII and thus won't delete FVIII-specific T cells or generate Tregs in the thymus. Because of this, you need to provide these patients with FVIII so that they can generate peripheral tolerance to the protein. This requires controlled "non-inflammatory" presentation of FVIII by APCs to FVIII-specific CD4+ cells that will either die by apoptosis, become anergic (functionally useless) or become FVIII-specific Tregs. It's the generation, expansion and maintenance of this latter T cell population that likely keeps us from generating FVIII inhibitors. You are much more likely to achieve this in a controlled low dose prophylaxis setting than at the time of a surgical intervention where coexistent high doses of FVIII and inflammation may favor immunogenicity and FVIII-specific T effector cell generation.

58. #1) Morado et al (2005) 10 year retrospective review 50 PUPS with Severe Hemophilia A - 19 treated on-demand - 31 treated on prophylaxis Main findings 15/19 PUPS treated on demand  inhibitors Mean age at inhibitor = 21 mo 0/31 PUPS placed on Prophylaxis  inhibitors Mean age at start of prophylaxis = 34 mo Morado M et al. Haemophilia. 2005;11(2):79-83.

59. #1) Morado et al (2005) 10 year retrospective review 50 PUPS with Severe Hemophilia A - 19 treated on-demand - 31 treated on prophylaxis Main findings 15/19 PUPS treated on demand  inhibitors Mean age at inhibitor = 21 mo 0/31 PUPS placed on Prophylaxis  inhibitors Mean age at start of prophylaxis = 34 mo Morado M et al. Haemophilia. 2005;11(2):79-83. Problem with study: By time pts were started on prophylaxis those who were destined to develop inhibitors had already developed them. Analysis flawed!

60. #3) Gouw et al (CANAL study) (2007) Main Findings of the Canal Study Intense therapy at 1 st exposure = 3.3 fold  risk Intense therapy during first 50 ED = 2 fold  risk Early start of prophylaxis (min. 1/wk)  risk of inhibitor by 60% Gouw SC et al. Blood. 2007;109(11):4648-4654. Analysis: Cox Regression (Kaplan Meier type) analysis done To adjust for time varying issues of when prophylaxis is commenced in relation to exposure days Much larger study with better analysis!

61. Inhibitor Risk in PUPs: All Inhibitors Wight J, Paisley S. Haemophilia 2003;9:318-35 Multiple PD FVIII Single PD FVIII Single rFVIII

62. #2) Santagostino E et al. (2005) Case-control study 108 PUPS with severe hemophilia A 60 inhibitor POS. patients (Cases) 48 inhibitor NEG. patients (Controls) Main Findings Cases: Only 7/60 (12%) had been placed on prophylaxis Controls: Most [ 34/48 (71%)] had been placed on prophylaxis Santagostino E et al. Br J Haematol. 2005;130(3):422-427

63. #2) Santagostino E et al. (2005) Main problem Median age at inhibitor detection: 23 mo Median age at start of prophylaxis: 35 mo Santagostino E et al. Br J Haematol. 2005;130(3):422-427 Similar problems to Morado study By time pts were started on prophylaxis those who were destined to develop inhibitors had already developed them. Sub-analysis helped but did not completely resolve statistical issues

64. #2) Santagostino E et al. (2005) Main problem Median age at inhibitor detection: 23 mo Median age at start of prophylaxis: 35 mo Santagostino E et al. Br J Haematol. 2005;130(3):422-427 Similar problems to Morado study By time pts were started on prophylaxis those who were destined to develop inhibitors had already developed them. Sub-analysis helped but did not completely resolve the analytical issues Conclusion: Pts who started prophylaxis had an 80% lower incidence of inhibitors

65. #4) Gouw et al (2007) [4 PUP studies] Retrospective cohort study of 4 rFVIII registration studies involving 236 PUPS (FVIII <2%) 67 (28%) developed inhibitors (median 10 ED; 16 mo) Main findings Intense exposure  risk of inhibitor (≥ 5 CED) at 1 st exposure= 2.1 X (≥ 5 CED) during 1 st 50 ED= 1.6 X Unable to comment on effect of prophylaxis as few pts on prophylaxis Gouw SC et al. JTH 2007; 5: 1383-90.

66. #5) Ragni et al (2009) Case–control study (single center – USA) 77 PUPS (severe hemophilia A) 20 Inhibitor POS. 57 age-matched Inhibitor NEG. controls Main finding Patients who developed inhibitors had received (prior to inhibitor development) much greater intensity exposure (p < 0.005) Ragni M et al. Haemophilia 2009; 15: 1074-82.

67. #5) Ragni et al (2009) “Given the potential benefits of prophylaxis in reducing inhibitor formation … it is critical to direct efforts at early prevention through early institution of prophylaxis” Ragni M et al. Haemophilia 2009; 15: 1074-82.

68. #6) Maclean et al (2011) Retrospective case control study from 13 UK haemophilia centers of 156 PUPS born (1982- 2007) 78 inhibitor POS. 78 inhibitor NEG. [age & center matched controls] All Followed for at least 50 ED Maclean P et al. Haemophilia 2011; 17:282-7.

69. Main Findings 1) High intensity treatment during 1 st 50 ED  inhibitor risk ≥ 5 CED: 2.7 fold (CI: 1.4–5.4) ≥ 10 CED: 5.5 fold (CI: 1.5–20) (e.g. ICH ) 2) To few patients started prophylaxis to find a statistically significant effect of prophylaxis Maclean P et al. Haemophilia 2011; 17:282-7. #6) Maclean et al (2011)

70. Why Do Inhibitors Develop? Severe hemophilia (20-30%) Mild / Mod Hemophilia (3-10%) No endogenous factor Some endogenous factor (conformationally altered) Entire Exogenous factor = foreign protein Small part of exogenous factor = foreign protein

71. A Population map of the world The world is changing

72. YES

73. Woof

74. YES Woof Thanks for your attention!

75. Clinical Evidence for decreased inhibitors with Pd-FVIII (% developing inhibitors) Goudemand et al, Blood 2006: PUPS with SHA (France) Chalmers et al, Haemophilia 2007: PUPS with SHA (UK) rFVIII32% (26 / 86 pts)27% (46 / 172 pts) Pd VWF- FVIII 10% (6 / 62 pts)14% (18 / 132 pts) Both retrospective studies

76. Clinical Evidence for decreased inhibitors with Pd-FVIII (% developing inhibitors) Goudemand et al, Blood 2006: PUPS with SHA (France) Chalmers et al, Haemophilia 2007: PUPS with SHA (UK) rFVIII32% (26 / 86 pts)27% (46 / 172 pts) Pd VWF- FVIII 10% (6 / 62 pts)14% (18 / 132 pts) Both retrospective studies No randomized head to head comparison (Sippet study)

77. Overall incidence of Inhibitor development (PUP studies) 1998 19971998 1998 2001 Gruppo Lusher Rothschild Lusher Courter Product Rec Kog Rec Kog Refacto # of pts72 64 52 97 92 Incidence 31% 28% 29% 28% 35% Rec = Recombinate Kog = Kogenate No statistical difference in inhibitor incidence

78. Specific FVIII products A few PD FVIII products reported to be associated with high incidence of inhibitors – Belgium – Netherlands Modifications in the manufacturing +/or viral inactivating process led to Neo-antigen development A few PD FVIII reported to be associated with a low incidence of inhibitors

79. Product (Study Period); Author (Country), date of publication # of pts % Inhibitor % with HTI Octanate (00-06); Klukowska (Poland), 06 248% Emoclot (87-03); Gringeri (Italy), 06 7110%7% FVIII-LFB (88-01); Goudemand (France), 05 8610%6% OctaVI (89-99); Glomstein (Norway), unpub. 1911%n.a. Fandhi (97-99); Rokicka-Milewska (Poland), 00 195%n.a. 8Y (85-04); Brown (UK), unpub. 744%3% OVERALL 2698%6% Information courtesy of Dr A. Gringeri, Sept 06 European studies on Inhibitor development in SHA PUPs treated with pdFVIII

80. Product (Study Period); Author (Country), date of publication # of pts % Inhibitor % with HTI Octanate (00-06); Klukowska (Poland), 06 248% Emoclot (87-03); Gringeri (Italy), 06 7110%7% FVIII-LFB (88-01); Goudemand (France), 05 8610%6% OctaVI (89-99); Glomstein (Norway), unpub. 1911%n.a. Fandhi (97-99); Rokicka-Milewska (Poland), 00 195%n.a. 8Y (85-04); Brown (UK), unpub. 744%3% OVERALL 2698%6% Information courtesy of Dr A. Gringeri, Sept 06 Reporting bias European studies on Inhibitor development in SHA PUPs treated with pdFVIII

81. Product (Study Period); Author (Country), date of publication # of pts % Inhibitor % with HTI Octanate (00-06); Klukowska (Poland), 06 248% Emoclot (87-03); Gringeri (Italy), 06 7110%7% FVIII-LFB (88-01); Goudemand (France), 05 8610%6% OctaVI (89-99); Glomstein (Norway), unpub. 1911%n.a. Fandhi (97-99); Rokicka-Milewska (Poland), 00 195%n.a. 8Y (85-04); Brown (UK), unpub. 744%3% OVERALL 2698%6% European studies on Inhibitor development in SHA PUPs treated with pdFVIII Information courtesy of Dr A. Gringeri, Sept 06

82. Overall conclusions regarding Type of and Inhibitor risk 1.No consensus as to whether rFVIII is more immunogenic than pd FVIII 2.Some pd FVIII may be less immunogenic - Some much more immunogenic 3.No difference in inhibitor risk between rFVIII products 4.There is bias in reporting

83. Recent experiences of pdFVIII vs. rFVIII Shirahata et al (2011; Hemophilia) Japan experience of inhibitor development in 153 pts (PUPS) with severe hemophilia A Inhibitor incidence pdFVIIIrFVIII 29.7%25% No difference

84. Recent experiences of pdFVIII vs. rFVIII Strauss et al (2011; Hemophilia) Israeli experience of inhibitor development in 292 pts (PUPS) with severe hemophilia A Inhibitor incidence pdFVIIIrFVIII All inhibitors 8.8% (22/249)32.5% (14/43) HTI 8.8% (22/249)28% (12/43) Not clear that all severeAll severe

85. ICH is a risk factor for inhibitors Strauss et al (2011; Hemophilia) Israeli experience of inhibitor development in 292 pts (PUPS) with severe hemophilia A 9/11 patients who suffered an ICH developed an inhibitor