1. WFH Bangkok 2004 Factor VIII and von Willebrand Factor (VWF) VWF and Inhibitor Antibodies

2. WFH Bangkok 2004 Main Clinical Difference Between FVIII Concentrates Immune tolerance is the key area where there are clinical differences between factor VIII concentrates, in: –Inhibitor reactivity variation between concentrates –Inhibitor development in PUPs –Immune tolerance induction

3. WFH Bangkok 2004 Inhibitors acc. to Preparation Type PreparationTypeInh/Total Incidence (%) NotesRef. KoatePd + VWF0/370Zanon 1999 BPL 8YPd + VWF1/372.7 2.7 % at 200 ED Yee 1997 FVIII-HPSDPd + VWF5/568.9Guerois 1995 Hemofil MPd – VWF5/539.4 At least 25 % moderates Addiego 1992 FVIII-LFBPd + VWF7/6311 Rothschild 2003 FVIII-HPSDPd + VWF8/6612.1 Goudemand 1998 MonoclatePd – VWF7/3818.4 Severes & moderates Lusher 1991 Pd: Plasma-derived

4. WFH Bangkok 2004 Inhibitors acc. to Preparation Type PreparationTypeInh/Total Incidence (%) NotesRef. RecombinateRec14/5028 38.7 % at ED 91 Rothschild 1998 RecombinateRec7/2528 5/7 tolerant to pd FVIII Zanon 1999 RecombinateRec14/4928.5Lusher 1993 KogenateRec19/6430Lusher 2000 Kogenate & Recombinate Rec27/8631 Rothschild 2003 ReFactoRec32/10132 Courter 2001 Pd: Plasma-derived

5. WFH Bangkok 2004 Inhibitor Reactivity In vitro and in vivo evidence now suggests that VWF may affect the reactivity of FVIII inhibitors with FVIII –Berntop et al (1996): Concentrates rich in VWF neutralise inhibitors and yield higher FVIII:C recovery than concentrates containing no, or only traces of, VWF –Amano et al (1995): Inhibitory titres are higher in concentrates with low or absent VWF –Suzuki et al (1996): C2 inhibitors are less inhibitory to FVIII complexed with VWF –Sukhu et al (2000): Statistically significant lower inhibitory titres titres found with VWF-rich concentrates (both intermediate and high purity) vs. monoclonal and recombinant concentrates –Gensana et al (2001): Inhibitor neutralising capacity is significantly higher for FVIII/VWF than for monoclonal FVIII –Kallas et al (2001): Lower neutralisation of FVIII in the presence of VWF –Mancuso et al (2000): Lower inhibitor neutralising capacity with FVIII/VWF than with monoclonal or recombinant FVIII

6. WFH Bangkok 2004 Structure of Factor VIII and Binding of VWF VWF binds the N-terminal part of the A3 domain and the C-terminal part of the C2 domain

7. WFH Bangkok 2004 C2C1A3A1 AR1 A2 AR2 B AR3 FXFIXa VWF Heavy chainLight chain VWF VWF VWF PL PL Adopted in part from Saenko et al. Haemophilia 2002 IIa, Xa Factor VIII Functional Binding Sites Targeted by Inhibitory Antibodies

8. WFH Bangkok 2004 VWF C2C1A3A1 AR1 A2 AR2 B AR3 Inhibitor Activity Modulated by VWF The presence of VWF appears to influence the way anti-factor VIII antibodies can react with the factor VIII molecule, preventing antibody binding and/or protecting parts of factor VIII which are important for coagulation interaction PL

9. WFH Bangkok 2004 Inhibitor Activity Modulated by VWF The hypothesis that Inhibitor reactivity with FVIII is partially blocked by VWF in concentrates containing high levels of VWF is supported by (among others): –Inhibitors from some patients react with light-chain epitopes (region C2, AR-A3-C1) (Scandella et al, 1997) –The C2 region binds VWF and phosphatidyl serine (Shima et al.1995; Saenko and Scandella 1995) – Anti-FVIII antibodies inhibit FVIII binding to VWF and to phospholipid (Shima et al, 1995) and are less inhibitory to FVIII combined with VWF (Suzuki et al, 1996)

10. WFH Bangkok 2004 Clinical consequences for VWF-containing concentrates –Prevent / reduce inhibitor development in PUPs FVIII-VWF concentrates may protect against inhibitor development and be the treatment of choice for PUP’s and minimally exposed patients –Immune Tolerance Induction in PTPs FVIII-VWF concentrates by avoiding antibody reaction may be the first choice in treatment for bleeding and ITI Inhibitor Activity Modulated by VWF in Concentrates

11. WFH Bangkok 2004 Inhibitor Activity Modulated by VWF in Concentrates Clinical evidence appears to support biochemical observations In PUPs In PTPs

12. WFH Bangkok 2004 *GTH: Gesellschaft für Thrombose und Hämostase (Society for Thrombosis and Haemostasis) Auerswald, G. GTH Congress 2003 Severe 92 29 Moderate 58 4 Mild 24 1 Inhibitor 39,5 % recFVIII 24,5 % pdFVIII Inhibitor incidence in severe haemophilia A 161 Haemophilia A PUPs Inhibitors GTH* PUP Study Results

13. WFH Bangkok 2004 Inhibitor Incidence in PUPs GTH PUP StudyBonn Rothschild C et al. French Multi-centre Recombinant Factor VIII 12/49 (39.5 %)14/45 (31.1 %)27/86 (31.4 %) Plasma-derived Factor VIII 17/43 (24.5 %)20/94 (21.3 %)7/63 (11.1 %) These studies show there is a trend towards less inhibitors in PUPs who received a plasma-derived FVIII concentrate containing VWF

14. WFH Bangkok 2004 ITI: A Treatment Option in Inhibitor Patients After treating acute bleeding, the priority is to Eradicate inhibitor with ITI (Immune Tolerance Induction) with –Bonn protocol –Mälmo protocol

15. WFH Bangkok 2004 Immune Tolerance Induction – ITI Choice of product –Inhibitor reactivity and FVIII concentrate –VWF modulation of antibody recognition by VWF-containing concentrates implies potential effectivity in ITI

16. WFH Bangkok 2004 ITI in Frankfurt Period Preparation Type 1979-1993 Plasma-derived only FVIII-VWF After 1993 No VWF MAb-purified or recombinant After 1993 Plasma-derived only FVIII-VWF ITI Cases (N)21142 ITI Success (n)1942 Success Rate (%) 9155100

17. WFH Bangkok 2004 ITI in Bonn and Frankfurt Period Preparation Type Before 1990 Only plasma- derived 1990 – 1999 Mainly recombinant 1999-2001 Mainly plasma- derived ITI Cases (N)512942 ITI Success (n)441629 Success Rate (%) 865571

18. WFH Bangkok 2004 ITI – Clinical Summary ITI results at the Bonn and Frankfurt centres have been less successful since the introduction of very high-purity FVIII concentrates depleted in VWF in 1990* This is not supported by results from the North American IT Registry (DiMichele and Kroner, 1999) However, only the Bonn and Frankfurt data are based on a reasonably large patient population, identical treatment protocol and success criteria throughout the studied period * Brackmann HH. 1999, 2001; Kreuz W et al. 2001; Kreuz W. 2002

19. WFH Bangkok 2004 ITI – Clinical Observations Germany In the past ITI was usually commenced with the concentrate used at the time of inhibitor development ITI is initiated with the product and batch demonstrating the least reactivity with the patient’s inhibitors A change of concentrate is made when patients appear to have become refractory to ITI Changing from non VWF to VWF-containing products has achieved ITI success in some cases (Kreuz et al, 1996)

20. WFH Bangkok 2004 ITI Conclusions Inhibitors react differently with different types of factor concentrates Several studies, mainly in vitro, indicate a modulating role of VWF against inhibitors directed against the C2 region of FVIII Inhibitor testing against a panel of concentrates may facilitate selection of a least neutralized concentrate thereby improving haemostatic effect and cost/efficacy in ITI