Phosphatidylserine mediated enhancement of the therapeutic efficacy of B domain deleted recombinant factor VIII
Miclea, Razvan D
MetadataShow full item record
Factor VIII (FVIII) is an important cofactor in the blood coagulation cascade. Deficiency or dysfunction of FVIII causes hemophilia A, a life threatening bleeding disorder. Replacement therapy using FVIII is the first line therapy in the management of the disease. Despite the advantages of replacement therapies with recombinant FVIII, the efficacy of current treatment is compromised, as 15 to 30% of the patients develop inhibitory anti-FVIII antibodies that neutralize the activity of the protein. A rational design of FVIII preparations that are less immunogenic represents the main objective of this dissertation. The novelty of this work is the rational development strategies of novel FVIII formulations: understanding the factors that govern physical stability and biological activity of FVIII, and engineering specific interactions between the protein and lipid based structures that will interfere with the degradation, elimination and processing of FVIII by the immune system. Aggregation is the main source of physical instability in proteins. In chapter 2.1, we investigated the role of the heterogeneity of the protein on the propensity of FVIII to aggregate. Taking into account that the B domain is the main cause of heterogeneity in FVIII preparations and since B domain did not play any known role in the biological relevant interactions of this protein, a truncated form of FVIII lacking the B domain (B domain deleted recombinant FVIII - BDDrFVIII) is used as a therapeutic preparation. We expressed BDDrFVIII and the biophysical and biochemical characterization of the protein revealed that despite a low degree of heterogeneity, the truncated protein has a tendency to aggregate, similar to that reported for full length recombinant FVIII. As protein products are expressed in various mammalian cell lines that cause modifications in the glycosylation pattern, we addressed the role of glycosylation on the aggregation, activity and biological relevant interactions of FVIII (chapter 2.2). Our studies established that removal of carbohydrates alter the biological activity of FVIII and led to exposure of hydrophobic patches on the protein surface and to partial aggregation, possibly mediated by the peptide region 2303-2332 located in the C2 domain of FVIII. Furthermore, the peptide 23030-2332 (i) participates in the molecular recognition of the membrane of activated platelets, mediated by the head group of phosphatidylserine (PS), (ii) participates in the receptor mediated elimination of FVIII from circulation by the low density lipoprotein receptor related protein and (iii) is an immunodominant epitope, as antibodies against this region are frequently found in patients' plasma. Thus, we hypothesized that specific interactions between FVIII and analogs of PS or PS containing liposomes could delay the aggregation process and most importantly, reduce the immunogenicity of this protein. Biophysical and biochemical studies demonstrated that PS improved the stability of FVIII, while preserving its biological activity (chapters 3 and 4). Immunogenicity studies carried out in hemophilia A murine model showed a significant decrease in antibody titers for FVIII in the presence of analogs of PS (chapter3) , and PS containing liposomes (chapter 4), compared to the free protein alone. Pharmacokinetic analysis indicated that stealth PS containing liposomes do not alter the systemic exposure of FVIII in murine models (chapter 4.2). Overall, our results indicated that the specific interactions of the protein with PS may be exploited to generate less immunogenic formulations of FVIII. The systemic exposure to FVIII preparations could be improved further by preventing the proteolytic degradation following administration. It has been reported that the topology of liposomal FVIII is such that a large surface of the protein molecule exposed to the external milieu and thus, it is susceptible to degradation by proteases. To decrease the solvent exposure of the protein, we developed a complex of FVIII with cochleate cylinders that are packed lipidic structures with no aqueous space (chapter 5). Multiple biophysical and biochemical studies indicated that both heavy and light chains were properly shielded from the bulk water, while the in vitro and in vivo biological activity of the protein was preserved. In addition, we developed a procedure to generate cochleate cylinders with particle size below 600 nm. The results are promising and demonstrated the ability to develop FVIII - novel cochleate cylinders for protein delivery applications.