Research Towards a LongerActing FactorVIII

1
Research Towards aLonger-Acting Factor VIII
CURE
Convenience
t1/2 Improvement
Optimal Therapy
Bruce M. Ewenstein, MD, PhD Global Medical
Director Baxter BioScience

• Prophylaxis

Pathogen Safety

• Plasma-Albumin Free Method

2
Molecular Mechanism of FVIII Clearance
Ananyeva et al., 2001 Saenko EL and Ananyeva NM,
2006
3
Potential Targets for FVIIIHalf-Life Extension

• Indirect modifications
• Interference with FVIII receptor mediated
  clearance
• Formulations with improved stability
• PEGylated liposomes
• Modification of VWF as the FVIII carrier protein
• VWF mutant
• Biochemical modificationof VWF

• Direct modifications
• FVIII Mutants
• Resistant to degradation
• Resistant to inactivation
• Resistant to clearance
• Biochemical modification of FVIII
• PEGylation
• Modifications of glycosylation

4
Concepts for a Next-Generation Hemophilia A
Therapy With Improved Pharmacokinetics

• Baxter is developing 4 approaches for half-life
  extension of Factor VIII (FVIII), pursuing both
  direct and indirect modification

TARGET
APPROACH
rVWF recombinant von Willebrands factor.
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PEGylationDerivatization of Proteins With
Synthetic Polyethylene Glycol

• Knowns
• Enhanced solubility
• Decreased proteolysis
• Altered distribution and absorption
• Enhanced storage stability
• Increased half-life

• Unknowns
• Clearance mechanism of large PEG-protein
  conjugates
• Effects of long-term exposure
• Impact on immunogenicity

mPEG2-SBA (Succinimidyl butyrate)
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PEGylated rFVIII Showed ImprovedPK Parameters
Compared With rFVIII Half-life Increased 2-fold
PEGylated rFVIII In Vivo Evaluation in
FVIII-Deficient Mice
Application of PEG-rFVIII or rFVIII (200 IU
FVIII/kg)
10.00
PEG-rFVIII (200 U/kg)
rFVIII (200 U/kg)
AUC (mUhr/mL)/(mU/kg)
1.00
FVIII (IU/mL) SD
126
65
0.10
n5
0.01
0
10
20
30
Hours Post-infusion
Calculation with MS Excel. AUC area under the
curve.
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VWF as a Target for the Prolongation of the
Half-Life of FVIII

• The survival of FVIII in the circulation is
  dependent on its chaperone VWF
• VWF rapidly forms a tight complex with FVIII with
  high affinity
• FVIII-VWF complex formation may prevent FVIII
  from interacting with lower affinity binding
  partners, such as FIXa, phospholipids and LRP
• FVIII bound to VWF is protected from enzymatic
  (in)activation
• Correlation between pre-infusion from VWF levels
  andhalf-life of FVIII in severe hemophilia A1,2

1. Fijnvandraat et al., Br J Haematol.
199591474-6. 2. Vloet et al., Thromb
Haemost. 200078365-9.
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rVWF Largest Protein Ever Successfully PEGylated
VWF multimer
-S-S-
-S-S-
-S-S-
-S-S-
-S-S-
-S-S-
-S-S-
-S-S-
-S-S-
-S-S-
PEGylation
PEGylated VWF multimer
-S-S-
-S-S-
-S-S-
-S-S-
-S-S-
-S-S-
-S-S-
-S-S-
-S-S-
-S-S-
Immunoblot withanti-PEG pAb
Coomassie staining
kDa

• SDS-PAGE (reduced)
• Increase in MW
• after PEGylation

• Multimer distribution
• (2.5 agarose)
• Broadening of all multimer bands in
  PEGylated rVWF

MW marker
MW marker
PEG-rVWF 5K
Native rVWF
Native rVWF
PEG-rVWF 5K
PEG-rVWF 20K
PEG-rVWF 20K
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Substantial Half-life Increase for rVWF After
PEGylation
PEG-rVWF in FVIII-deficient KO mice
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PEG-rVWF Demonstrated Substantial Half-life
Increase of FVIII
PEG-rVWF in FVIII-Deficient KO Mice
Dose (200/300 U/kg)
PEG-rVWF / rFVIII
100.0
rVWF / rFVIII
AUC 102
10.0
FVIII ( from Umax)
1.0
AUC 40
n5
0.1
0
5
10
15
20
25
30
35
Hours Post-infusion
(mUh/mL)/(mU/kg).
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Polysialic Acid (PSA) Technology as an
Alternative to PEGylation

• Features of PSA
• ?-2,8-linked polysialic acid
• Negatively chargedhydrophilic polymer
• Natural constituent of human tissue
• Non-immunogenic
• Biodegradable (to sialic acid)
• Chemical derivatization of proteins with PSA
• Preferred process conjugation with Lys residues
  by reductive amination

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Polysialylation Prolonged VWFHalf-life and
Sustained the Secondary Rise of FVIII
rVWF-polysialic acid conjugate PK in VWD mice
PSA-rVWF (80 IU VWFAg/kg)
rVWF (70 IU VWFAg/kg)
rVWF-PSA
rVWF-PSA
10.0
0.6
rVWF
rVWF
AUC 66
1.0
0.4
AUC 68
VWFAg (U/mL)
FVIII (U/mL)
0.2
0.1
AUC 47
AUC 50
FVIII base level in VWD mice
0.0
0.0
0
5
10
15
20
25
0
5
10
15
20
25
Hours Post-infusion
Hours Post-infusion
(mUh/mL)/(mU/kg). VWD von Willebrand disease.
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Factor VIII Levels and Bleeding

• Patients with severe hemophilia A
• N 64 (51 HA, 13 HB)
• Individual PKs defined
• Time FVIII lt1 IU/dL calculated
• Comparison made with joint bleeds over 6 years
• For each additional hour per week FVIII lt1, 1.9
  increase in annual hemarthroses

Ahnstrom et al.,Haemophilia 200410689-97
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Clinical Implications

• Modeled data from completed ADVATE clinical
  studies
• For each additional hour per week with FVIII lt1,
  annual bleed rate increased

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Open Questions

• Is chemical modification of very large proteins
  feasible, reproducible, and scalable?
• Is modified rVWF susceptible to specific
  proteolytic degradation by ADAMTS13?
• Do modified rFVIII and rVWF retain their
  efficacy in vivo?
• Does PEGylation impact the immunogenicity of rVWF
  and rFVIII?

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Immunogenicity of PEG-FVIII and FVIII in New
Hemophilia Model
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Recombinate Expansion
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Recombinate Expansion
1992 - 1996
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Recombinate Expansion
1992 - 1996
1997 - 2001
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Recombinate Expansion
1992 - 1996
1997 - 2001
2001 - 2007
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Recombinate Expansion
1992 - 1996
1997 - 2001
2001 - 2007
Pending/In Process Registrations
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Acknowledgments

• The Half-life Scientific and Technical Team
• Peter Turecek
• Hans Peter Schwarz
• Birgit Reipert
• Eva Maria Muchitsch
• Bernhard Baumgartner
• Herbert Gritsch
• Katalin Varàdi
• Fritz Scheiflinger
• Wolfgang Mundt
• Artur Mitterer
• Hartmut Ehrlich
• gt150 Team members of Baxter RD currently working
  on our hemophilia project