The development of antibody responses against therapeutic proteins such as Factor VIII (FVIII) is a major clinical complication that compromises the safety and efficacy of protein based therapeutics. Our goal is to improve upon the therapeutic efficacy of proteins by developing non-immunogenic forms of the molecules. FVIII is a multi-domain protein that plays a critical role in blood coagulation cascade. The deficiency and dysfunction of FVIII causes Hemophilia A, a bleeding disorder. Administration of recombinant FVIII is the first line of therapy for Hemophilia A (HA) patients. The safety and efficacy of this replacement therapy is severely compromised by the development of inhibitory antibody responses that occur in 15-30% of HA patients treated with FVIII. We have established that the antibody response to FVIII is reduced significantly by complexing the protein with phosphatidylserine (FVIII-PS) or phosphatidylinositol (FVIII-PI). To exploit this novel finding for developing a more therapeutically effective form of FVIII, and to determine how such lipid modifications may be used with other therapeutic proteins, it is imperative to determine the mechanisms by which the reduction of immunogenicity is achieved. Therefore, in Aim 1 we propose to establish whether the decrease in the antibody response is due to the lipid complexes'ability to alter antigen processing and presentation of the protein, resulting in active induction of immunological tolerance in HA mice. Alternatively we will determine whether it is due to a passive process that results from lipid shielding of immunogenic determinants. These studies will reveal the robustness, specificity, safety and durability of the reduced antibody responses observed, and will establish the degree to which the lipid complexes reduce or eliminate an already-established anti-FVIII antibody response. Because the frequency of administration increases immunogenic response, long acting lipid-FVIII complexes that reduce the frequency of administration are useful.
In Aim 2, the effect of lipid headgroup and PEG modification upon the pharmacokinetics (PK) of FVIII will be investigated. PK properties, such as circulation half-life, mean residence time and exposure of FVIII following iv or sc injection, will be established by quantifying and comparing the FVIII levels achieved with lipidic FVIII formulations to those observed with FVIII alone. The haemostatic efficacy of FVIII is the key endpoint of clinical significance.
Aim 3 will establish how lipid complexation affects the pharmacodynamics and therapeutic efficacy of the lipid- complexed FVIII by using an in vivo standardized tail vein transection assay. In SA 4, the translational utility of reduction in immunogenicity and PK of FVIII-lipid complex will be tested in a primate model, Macaque mulatta. These studies are expected to elucidate the mechanistic basis by which lipidic FVIII complexes decrease immunogenicity, and improve pharmacokinetics and therapeutic efficacy in animal models, so that this observation may be extended to clinical trial, and applied also to the design, formulation and delivery of other therapeutic proteins, so as to overcome issues that negatively impact their clinical use.

Public Health Relevance

Development of inhibitory antibody against FVIII is a major clinical complication that impacts safety and efficacy of the replacement therapy for Hemophilia A, a bleeding disorder. The proposed research is relevant to public health because the proposal is focused on developing lipidic FVIII as therapeutic preparations that are less immunogenic and long acting FVIII to improve safety and therapeutic efficacy of FVIII replacement therapy. The experimental design involves an interdisciplinary approach involving biophysics, immunology, pharmacokinetics and pharmacodynamics.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL070227-12
Application #
8644844
Study Section
Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
Program Officer
Link, Rebecca P
Project Start
2002-04-01
Project End
2015-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
12
Fiscal Year
2014
Total Cost
$391,340
Indirect Cost
$117,714
Name
State University of New York at Buffalo
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
038633251
City
Buffalo
State
NY
Country
United States
Zip Code
14260
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Kosloski, Matthew P; Pisal, Dipak S; Mager, Donald E et al. (2013) Allometry of factor VIII and informed scaling of next-generation therapeutic proteins. J Pharm Sci 102:2380-94
Gaitonde, Puneet; Ramakrishnan, Radha; Chin, Jamie et al. (2013) Exposure to factor VIII protein in the presence of phosphatidylserine induces hypo-responsiveness toward factor VIII challenge in hemophilia A mice. J Biol Chem 288:17051-6
Fathallah, Anas M; Bankert, Richard B; Balu-Iyer, Sathy V (2013) Immunogenicity of subcutaneously administered therapeutic proteins--a mechanistic perspective. AAPS J 15:897-900
Peng, Aaron; Pisal, Dipak S; Doty, Amy et al. (2012) Phosphatidylinositol induces fluid phase formation and packing defects in phosphatidylcholine model membranes. Chem Phys Lipids 165:15-22

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