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1.  O-Phospho-L-Serine, Multi-functional Excipient for B Domain Deleted Recombinant Factor VIII 
The AAPS journal  2007;9(2):E251-E259.
Factor VIII (FVIII) is an important cofactor in the blood coagulation cascade. A deficiency or dysfunction of FVIII causes hemophilia A, a life-threatening bleeding disorder. FVIII circulates in plasma as a heterodimer comprising 6 domains (heavy chain, A1-A2-B and light chain, A3-C1-C2). Replacement therapy using FVIII is the leading therapy in the management of hemophilia A. However, ∼15% to 30% of patients develop inhibitory antibodies that neutralize the activity of the protein. Neutralizing antibodies to epitopes in the lipid binding region of FVIII are commonly identified in patients’ plasma. In this report, we investigated the effect of O-phospho-L-serine (OPLS), which binds to the lipid binding region, on the immunogenicity of B domain deleted recombinant factor VIII (BDDrFVIII). Sandwich enzyme-linked immunosorbent assay (ELISA) studies showed that OPLS specifically bind to the lipid binding region, localized in the C2 domain of the coagulation factor. Size exclusion chromatography and fluorescence anisotropy studies showed that OPLS interfered with the aggregation of BDDrFVIII. Immunogenicity of free-vs BDDrFVIII-OPLS complex was evaluated in a murine model of hemophilia A. Animals administered subcutaneous (sc) injections of BDDrFVIII-OPLS had lower neutralizing titers compared with animals treated with BDDrFVIII alone. Based on these studies, we hypothesize that specific molecular interactions between OPLS and BDDrFVIII may improve the stability and reduce the immunogenicity of BDDrFVIII formulations.
doi:10.1208/aapsj0902028
PMCID: PMC2573386  PMID: 17907766
B domain deleted recombinant factor VIII; O-phospho-L-serine; protein formulation; excipient; physical stability; immunogenicity; inhibitor development
2.  Phosphatidylinositol Containing Lipidic Particles Reduces Immunogenicity and Catabolism of Factor VIII in Hemophilia A Mice 
The AAPS Journal  2010;12(3):473-481.
Factor VIII (FVIII) is an important cofactor in blood coagulation cascade. It is a multidomain protein that consists of six domains, NH2-A1-A2-B-A3-C1-C2-COOH. The deficiency or dysfunction of FVIII causes hemophilia A, a life-threatening bleeding disorder. Replacement therapy using recombinant FVIII (rFVIII) is the first line of therapy, but a major clinical complication is the development of inhibitory antibodies that abrogate the pharmacological activity of the administered protein. FVIII binds to anionic phospholipids (PL), such as phosphatidylinositol (PI), via lipid binding region within the C2 domain of FVIII. This lipid binding site not only consists of immunodominant epitopes but is also involved in von Willebrand factor binding that protects FVIII from degradation in vivo. Thus, we hypothesize that FVIII–PL complex will influence immunogenicity and catabolism of FVIII. The biophysical studies showed that PI binding did not alter conformation of the protein but improved intrinsic stability as measured by thermal denaturation studies. ELISA studies confirmed the involvement of the C2 domain in binding to PI containing lipid particles. PI binding prolonged the in vivo circulation time and reduced catabolism of FVIII in hemophilia A mice. FVIII–PI complex reduced inhibitor development in hemophilia A mice following intravenous and subcutaneous administration. The data suggest that PI binding reduces catabolism and immunogenicity of FVIII and has potential to be a useful therapeutic approach for hemophilia A.
doi:10.1208/s12248-010-9207-z
PMCID: PMC2895449  PMID: 20517659
factor VIII; hemophilia A; inhibitor development; immunogenicity; phosphatidylinositol
3.  Interaction of Dicaproyl Phosphatidylserine With Recombinant Factor VIII and Its Impact on Immunogenicity 
The AAPS journal  2006;8(2):E362-E370.
Replacement therapy with exogenous recombinant factor VIII (rFVIII) to control bleeding episodes results in the development of inhibitory antibodies in 15% to 30% of hemophilia A patients. The inhibitory antibodies are mainly directed against specific and universal immunodominant epitopes located in the C2 domain. Previously we have shown that complexation of O-phospho-L-serine (phosphatidylserine head group) with the phospholipid binding region of the C2 domain can lead to an overall reduction in the immunogenicity of rFVIII. Here, we have investigated the hypothesis that dicaproyl phosphatidylserine, a short-chain water-soluble phospholipid, can reduce the immunogenicity of rFVIII. Circular dichroism and fluorescence spectroscopy studies suggest that dicaproyl phosphatidyl-serine interacts with rFVIII, causing subtle changes in the tertiary and secondary structure of the protein. Sandwich enzyme-linked immunosorbent assay studies indicate that dicaproyl phosphatidylserine probably interacts with the phospholipid binding region of the C2 domain. The immunogenicity of FVIII-dicaproyl phosphatidylserine complexes prepared at concentrations above and below the critical micellar concentrations of the lipid were evaluated in hemophilia A mice. Our results suggest that micellar dicaproyl phosphatidylserine may be useful to reduce the immunogenicity of rFVIII preparations.
doi:10.1208/aapsj080241
PMCID: PMC2574005  PMID: 16796387
Hemophilia A; inhibitor development; aggregation; recombinant human factor VIII; protein folding; factor VIII-DCPS complex
4.  Interaction of dicaproyl phosphatidylserine with recombinant factor VIII and its impact on immunogenicity 
The AAPS Journal  2006;8(2):E362-E370.
Replacement therapy with exogenous recombinant factor VIII (rFVIII) to control bleeding episodes results in the development of inhibitory antibodies in 15% to 30% of hemophilia A patients. The inhibitory antibodies are mainly directed against specific and universal immunodominant epitopes located in the C2 domain. Previously we have shown that complexation of O-phospho-L-serine (phosphatidylserine head group) with the phospholipid binding region of the C2 domain can lead to an overall reduction in the immunogenicity of rFVIII. Here, we have investigated the hypothesis that dicaproyl phosphatidylserine, a short-chain water-soluble phospholipid, can reduce the immunogenicity of rFVIII. Circular dichroism and fluorescence spectroscopy studies suggest that dicaproyl phosphatidyl-serine interacts with rFVIII, causing subtle changes in the tertiary and secondary structure of the protein. Sandwich enzyme-linked immunosorbent assay studies indicate that dicaproyl phosphatidylserine probably interacts with the phospholipid binding region of the C2 domain. The immunogenicity of FVIII-dicaproyl phosphatidylserine complexes prepared at concentrations above and below the critical micellar concentrations of the lipid were evaluated in hemophilia A mice. Our results suggest that micellar dicaproyl phosphatidylserine may be useful to reduce the immunogenicity of rFVIII preparations.
doi:10.1007/BF02854907
PMCID: PMC2574005  PMID: 16796387
Hemophilia A; inhibitor development; aggregation; recombinant human factor VIII; protein folding; factor VIII-DCPS complex
5.  Phosphatidylserine Containing Liposomes Reduce Immunogenicity of Recombinant Human Factor VIII (rFVIII) in a Murine Model of Hemophilia A 
Journal of pharmaceutical sciences  2008;97(4):1386-1398.
Factor VIII (FVIII) is a multidomain protein that is deficient in hemophilia A, a clinically important bleeding disorder. Replacement therapy using recombinant human FVIII (rFVIII) is the main therapy. However, approximately 15-30% of patients develop inhibitory antibodies that neutralize rFVIII activity. Antibodies to epitopes in C2 domain, which is involved in FVIII binding to phospholipids, are highly prevalent. Here, we investigated the effect of phosphatidylserine (PS)-containing liposomes, which bind to C2 domain with high affinity and specificity, upon the immunogenicity of rFVIII. Circular dichroism studies showed that PS-containing liposomes interfered with aggregation of rFVIII. Immunogenicity of free- versus liposomal-rFVIII was evaluated in a murine model of hemophilia A. Animals treated with s.c. injections of liposomal-rFVIII had lower total- and inhibitory titers, compared to animals treated with rFVIII alone. Antigen processing by proteolytic enzymes was reduced in the presence of liposomes. Animals treated with s.c. injections of liposomal-rFVIII showed a significant increase in rFVIII plasma concentration compared to animals that received rFVIII alone. Based on these studies, we hypothesize that specific molecular interactions between PS-containing bilayers and rFVIII may provide a basis for designing lipidic complexes that improve the stability, reduce the immunogenicity of rFVIII formulations, and permit administration by s.c. route.
doi:10.1002/jps.21102
PMCID: PMC2574438  PMID: 17705286
hemophilia A; recombinant FVIII; immunogenicity; inhibitor antibodies; phosphatidylserine liposomes; protein delivery; protein formulation; lipids; immunology
6.  Role of Glycosylation in Conformational Stability, Activity, Macromolecular Interaction and Immunogenicity of Recombinant Human Factor VIII 
The AAPS Journal  2009;11(3):424-431.
Factor VIII (FVIII) is a multi-domain glycoprotein that is an essential cofactor in the blood coagulation cascade. Its deficiency or dysfunction causes hemophilia A, a bleeding disorder. Replacement using exogenous recombinant human factor VIII (rFVIII) is the first line of therapy for hemophilia A. The role of glycosylation on the activity, stability, protein–lipid interaction, and immunogenicity of FVIII is not known. In order to investigate the role of glycosylation, a deglycosylated form of FVIII was generated by enzymatic cleavage of carbohydrate chains. The biochemical properties of fully glycosylated and completely deglycosylated forms of rFVIII (degly rFVIII) were compared using enzyme-linked immunosorbent assay, size exclusion chromatography, and clotting activity studies. The biological activity of degly FVIII decreased in comparison to the fully glycosylated protein. The ability of degly rFVIII to interact with phosphatidylserine containing membranes was partly impaired. Data suggested that glycosylation significantly influences the stability and the biologically relevant macromolecular interactions of FVIII. The effect of glycosylation on immunogenicity was investigated in a murine model of hemophilia A. Studies showed that deletion of glycosylation did not increase immunogenicity.
doi:10.1208/s12248-009-9119-y
PMCID: PMC2758112  PMID: 19499345
factor VIII; glycosylation; hemophilia A; immunogenicity; inhibitor development
7.  Development and characterization of lipidic cochleate containing recombinant factor VIII 
Biochimica et biophysica acta  2007;1768(11):2890-2898.
Hemophilia A, a life threatening bleeding disorder is caused by deficiency of Factor VIII (FVIII). Replacement therapy using rFVIII is the first line therapy for hemophilia A. However, 15-30% of patients develop neutralizing antibody, mainly against the C2, A3 and A2 domains. It has been reported that PS-FVIII complex reduced total and neutralizing anti-rFVIII antibody titers in hemophilia A murine models. Here, we developed FVIII – containing cochleate cylinders, utilizing PS-Ca2+ interactions and characterized these particles for optimal in vivo properties using biophysical and biochemical techniques. Approximately 75% of the protein was associated with cochleate cylinders. Sandwich ELISA, acrylamide quenching and enzymatic digestion studies established that rFVIII was shielded from the bulk aqueous phase by the lipidic structures, possibly leading to improved in vivo stability. Freeze – thawing and rate limiting diffusion studies revealed that small cochleate cylinders with a particles size of 500 nm or less could be generated. The release kinetics and in vivo experiments suggested that there is slow and sustained release of FVIII from the complex upon systemic exposure. In vivo studies using tail clip method indicated that FVIII-cochleate complex is effective and protects hemophilic mice from bleeding. Based on these studies, we speculate that the molecular interaction between FVIII and PS may provide a basis for the design of novel FVIII lipidic structures for delivery applications.
doi:10.1016/j.bbamem.2007.08.001
PMCID: PMC2137893  PMID: 17936245
cochleate cylinders; B Domain Deleted recombinant factor VIII; epitope shielding; Laurdan; protein formulation; acrylamide quenching
8.  Healthy subjects produce both anti-factor VIII and specific anti-idiotypic antibodies. 
Journal of Clinical Investigation  1994;94(4):1496-1505.
Anti-Factor VIII (FVIII) antibodies were prepared by a combination of salt precipitation, gel filtration chromatography, and specific adsorption over insolubilized FVIII from the serum of 10 healthy subjects with normal levels of FVIII. Antibody specificity was confirmed by the capacity to recognize soluble and insolubilized FVIII and to neutralize FVIII cofactor activity in FX activation. Epitope mapping was carried out using a competition ELISA in which affinity-purified human antibodies inhibited the binding of labeled monoclonal antibodies. In most cases, a single region of the A3 domain of the FVIII light chain was recognized by the antibodies, while the reactivity toward heavy chain epitopes differed from one antibody preparation to the other. Sera or IgG fractions of the serum before immunoadsorption over insolubilized FVIII did not bind to FVIII. The IgG fraction that was not retained on the FVIII immunosorbent contained IgG that bound to the variable part of anti-FVIII mouse monoclonal antibodies and inhibited the binding of labeled FVIII; in addition, the IgG fraction inhibited the binding of affinity-purified human antibodies to FVIII, thereby strongly suggesting the presence of anti-idiotypic antibodies. These findings indicate that the presence of anti-FVIII antibodies is a more universal phenomenon than previously thought and that anti-idiotypic antibodies capable of inhibiting the binding of anti-FVIII antibodies to FVIII are produced spontaneously.
PMCID: PMC295292  PMID: 7523452
9.  The Comparative Immunogenicity of Human and Porcine Factor VIII in Hemophilia A Mice 
Thrombosis and haemostasis  2009;102(1):35-41.
Summary
Inhibitory antibodies to factor VIII (fVIII inhibitors) are the most significant complication in the management of hemophilia A. The immunogenicity of fVIII may be driven in part by structural determinants within the fVIII molecule itself. Regions of non-identity between human and porcine fVIII possibly could drive differential immune responses. The goal of this study was to compare the overall antibody response and levels of antibodies to the individual fVIII domains in naïve hemophilia A mice immunized with human or porcine fVIII. Hemophilia A mice were immunized with human or porcine fVIII using a protocol that mimics human clinical use. Inhibitor and total anti-fVIII antibody titers were measured and the domain-specificity of antibodies from 1759 anti-fVIII hybridomas was determined. The overall immunogenicity of human and porcine fVIII was similar but significant differences in domain recognition were discovered. Anti-A2 and anti-C2 antibodies constituted the majority of inhibitors in both the human and porcine fVIII groups, similar to inhibitors that develop in humans. The proportions of anti-A2 or anti-C2 antibodies were not significantly different between the two groups. However, the specific inhibitory activity of anti-A2 antibodies was higher in the human fVIII group. Additionally, proportion of anti-C1 antibodies was significantly higher in the human fVIII group. In contrast, anti-A3 antibodies were more common in the porcine fVIII group. The differential immune response to human and porcine fVIII suggests that it may be possible to reduce the immunogenicity of fVIII by mutagenesis of the A2, A3 and C1 domains.
doi:10.1160/TH08-12-0818
PMCID: PMC3071503  PMID: 19572065
Factor VIII; hemophilia therapy; coagulation inhibitors
10.  Potentiation of Thrombin Generation in Hemophilia A Plasma by Coagulation Factor VIII and Characterization of Antibody-Specific Inhibition 
PLoS ONE  2012;7(10):e48172.
Development of inhibitory antibodies to coagulation factor VIII (fVIII) is the primary obstacle to the treatment of hemophilia A in the developed world. This adverse reaction occurs in 20–30% of persons with severe hemophilia A treated with fVIII-replacement products and is characterized by the development of a humoral and neutralizing immune response to fVIII. Patients with inhibitory anti-fVIII antibodies are treated with bypassing agents including recombinant factor VIIa (rfVIIa). However, some patients display poor hemostatic response to bypass therapy and improved treatment options are needed. Recently, we demonstrated that fVIII inhibitors display widely variable kinetics of inhibition that correlate with their respective target epitopes. Thus, it was hypothesized that for antibodies that display slow rates of inhibition, supplementation of rfVIIa with fVIII would result in improved thrombin generation and be predictive of clinical responses to this novel treatment regimen. In order to test this hypothesis, 10 murine monoclonal antibodies (MAbs) with non-overlapping epitopes spanning fVIII, differential inhibition titers, and inhibition kinetics were studied using a thrombin generation assay. Of the 3 MAbs with high inhibitory titers, only the one with fast and complete (classically defined as “type I”) kinetics displayed significant inhibition of thrombin generation with no improvement upon supplementation of rfVIIa with fVIII. The other two MAbs that displayed incomplete (classically defined as “type II”) inhibition did not suppress the potentiation of thrombin generation by fVIII. All antibodies that did not completely inhibit fVIII activity demonstrated potentiation of thrombin generation by the addition of fVIII as compared to rfVIIa alone. In conclusion, fVIII alone or in combination with rfVIIa corrects the thrombin generation defect produced by the majority of anti-fVIII MAbs better than single agent rfVIIa. Therefore, combined fVIII/rfVIIa therapy may provide better hemostatic control than current therapy in some patients with anti-fVIII inhibitors.
doi:10.1371/journal.pone.0048172
PMCID: PMC3483154  PMID: 23144741
11.  Lipid Binding Region (2303–2332) Is Involved in Aggregation of Recombinant Human FVIII (rFVIII) 
Journal of pharmaceutical sciences  2005;94(6):1288-1299.
Factor VIII (FVIII) is a multi-domain protein that is important in the clotting cascade. Its deficiency causes Hemophilia A, a bleeding disorder. The unfolding of protein domains can lead to physical instability such as aggregation, and hinder their use in replacement therapy. It has been shown that the aggregation of rFVIIII is initiated by small fluctuations in the protein’s tertiary structure (Grillo et al., 2001, Biochemistry 40:586–595). We have investigated the domain(s) involved in the initiation of aggregation using circular dichroism (CD), size exclusion chromatography (SEC), fluorescence anisotropy, domain specific antibody binding, and clotting activity studies. The studies indicated that aggregation may be initiated as a result of conformational change in the C2 domain encompassing the lipid-binding region (2303–2332). The presence of O-phospho-L-Serine (OPLS), which binds to the lipid-binding region of FVIII, prevented aggregation of the protein.
doi:10.1002/jps.20340
PMCID: PMC2583467  PMID: 15858858
recombinant human FVIII (rFVIII); physical instability; multi-domain; lipid-binding region; hemophilia A; inhibitor development
12.  AAV-based Neonatal Gene Therapy for Hemophilia A: Long-Term Correction and Avoidance of Immune Responses in Mice 
Gene therapy  2012;19(12):1166-1176.
Hemophilia A gene therapy has been hampered by immune responses to vector-associated antigens and by neutralizing antibodies or inhibitors to the factor VIII (FVIII) protein; these ‘inhibitors’ more commonly effect hemophilia A patients than those with hemophilia B. A gene replacement strategy beginning in the neonatal period may avoid the development of these immune responses and lead to prolonged expression with correction of phenotype thereby avoiding long-term consequences. Serotype rh10 AAV was developed splitting the FVIII coding sequence into heavy and light chains with the chicken β-actin promoter/CMV enhancer for dual recombinant AAV vector delivery. Coinjection of virions of each FVIII chain intravenously to mice on the second day of life was performed. Mice express sustained FVIII antigen levels of ≥5% to 22 months of life without the development of antibodies to FVIII. Phenotypic correction was manifest in all AAV-FVIII-treated mice as demonstrated by functional assay and reduction in bleeding time. This study demonstrates the use of AAV in a gene replacement strategy in neonatal mice that establishes both long-term phenotypic correction of hemophilia A and lack of antibody development to FVIII in this disease model where AAV is administered shortly after birth. These studies support consideration of gene replacement therapy for diseases that are diagnosed in utero or in the early neonatal period.
doi:10.1038/gt.2011.200
PMCID: PMC3432168  PMID: 22241178
Neonate; Hemophilia A; Gene Therapy; AAV; Immune Response
13.  Identification and Multidimensional Optimization of an Asymmetric Bispecific IgG Antibody Mimicking the Function of Factor VIII Cofactor Activity 
PLoS ONE  2013;8(2):e57479.
In hemophilia A, routine prophylaxis with exogenous factor VIII (FVIII) requires frequent intravenous injections and can lead to the development of anti-FVIII alloantibodies (FVIII inhibitors). To overcome these drawbacks, we screened asymmetric bispecific IgG antibodies to factor IXa (FIXa) and factor X (FX), mimicking the FVIII cofactor function. Since the therapeutic potential of the lead bispecific antibody was marginal, FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX, and the pharmacokinetics was improved by engineering the charge properties of the variable region. Difficulties in manufacturing the bispecific antibody were overcome by identifying a common light chain for the anti-FIXa and anti-FX heavy chains through framework/complementarity determining region shuffling, and by pI engineering of the two heavy chains to facilitate ion exchange chromatographic purification of the bispecific antibody from the mixture of byproducts. Engineering to overcome low solubility and deamidation was also performed. The multidimensionally optimized bispecific antibody hBS910 exhibited potent FVIII-mimetic activity in human FVIII-deficient plasma, and had a half-life of 3 weeks and high subcutaneous bioavailability in cynomolgus monkeys. Importantly, the activity of hBS910 was not affected by FVIII inhibitors, while anti-hBS910 antibodies did not inhibit FVIII activity, allowing the use of hBS910 without considering the development or presence of FVIII inhibitors. Furthermore, hBS910 could be purified on a large manufacturing scale and formulated into a subcutaneously injectable liquid formulation for clinical use. These features of hBS910 enable routine prophylaxis by subcutaneous delivery at a long dosing interval without considering the development or presence of FVIII inhibitors. We expect that hBS910 (investigational drug name: ACE910) will provide significant benefit for severe hemophilia A patients.
doi:10.1371/journal.pone.0057479
PMCID: PMC3585358  PMID: 23468998
14.  Characterization of anti-factor VIII antibody in a patient with acquired hemophilia A 
Blood research  2013;48(1):58-62.
Acquired hemophilia A (AHA) is a bleeding disorder caused by the development of an auto-antibody against endogenous factor VIII (FVIII). In this study, the epitope of the autoantibody was identified in a 67-year-old female patient with AHA. A prolonged activated partial thromboplastin time (77.4 s) that failed to correct in an incubation mixing test (68.2 s), a decreased FVIII activity, and a high FVIII inhibitor (14.6 Bethesda units/mL) were observed. Enzyme-linked immunosorbent assay demonstrated that the antibody belonged to the immunoglobulin G4 subclass. An immunoblotting assay revealed the light chain (A3/C1/C2 domain) of FVIII as the binding region of the antibody. The bleeding experienced by our patient resulted from the interference of FVIII binding to both FIX by anti-A3 antibodies and phospholipids and von Willebrand factor by anti-C2 antibodies. To the best of our knowledge, this is the first study in Korea characterizing an autoantibody in the context of AHA.
doi:10.5045/br.2013.48.1.58
PMCID: PMC3624998  PMID: 23589798
Acquired hemophilia A; Factor VIII autoantibody; Epitope
15.  Effect of route of administration of human recombinant Factor VIII on its immunogenicity in Hemophilia A mice 
Journal of pharmaceutical sciences  2009;98(12):4480-4484.
Factor VIII is a multi-domain glycoprotein and is an essential cofactor in the blood coagulation cascade. Its deficiency or dysfunction causes Hemophilia A, a bleeding disorder. Replacement using exogenous recombinant Factor VIII (FVIII) is the first line of therapy for Hemophilia A. Immunogenicity, the development of binding (total) and neutralizing (inhibitory) antibody against administered protein is a clinical complication of the therapy. There are several product related factors such as presence of aggregates, route and frequency of administration and glycosylation have been shown to contribute to immunogenicity. The effect of route of administration of Factor VIII on antibody development in Hemophilia A is not completely understood. Here we investigated the effect of route of administration (sc or iv) on immunogenicity in Hemophilia A mice. The total and inhibitory titers were determined using ELISA and modified Bethesda Assay respectively. The results indicated that sc is more immunogenic compared to iv route in terms of total antibody titer development (binding antibodies) but no significant differences in inhibitory titer levels could be established.
doi:10.1002/jps.21765
PMCID: PMC2796435  PMID: 19499565
Hemophilia A; Inhibitor development; Immunogenicity; Route of administration
16.  Factor VIII delivered by hematopoietic stem cell-derived B cells corrects the phenotype of hemophilia A mice 
Thrombosis and haemostasis  2011;105(4):676-687.
Summary
The main impediments to clinical application of hematopoietic stem cell (HSC) gene therapy for treatment of hemophilia A are the bone marrow transplant-related risks and the potential for insertional mutagenesis caused by retroviral vectors. To circumvent these limitations, we have adapted a nonmyeloablative conditioning regimen and directed factor VIII (FVIII) protein synthesis to B lineage cells using an insulated lentiviral vector containing an immunoglobulin heavy chain enhancer-promoter. Transplantation of lentiviral vector-modified HSCs resulted in therapeutic levels of FVIII in the circulation of all transplanted mice for the duration of the study (6 months). Immunostaining of spleen cells showed that the majority of FVIII was synthesized by B220+ B cells and CD138+ plasma cells. Subsequent challenge with recombinant FVIII elicited at most a minor anti-FVIII antibody response, demonstrating induction of immune hyporesponsiveness. All transplant recipients exhibited clot formation and survived tail clipping, indicating correction of their hemophilic phenotype. Therapeutic levels of FVIII could be transferred to secondary recipients by bone marrow transplantation, confirming gene transfer into long-term repopulating HSCs. Moreover, short-term therapeutic FVIII levels could also be achieved in secondary recipients by adoptive transfer of HSC-derived splenic B cells. Our findings support pursuit of B cell-directed protein delivery as a potential clinical approach to treat hemophilia A and other disorders correctable by systemically distributed proteins.
doi:10.1160/TH10-11-0725
PMCID: PMC3117307  PMID: 21264447
Hemophilia A; animal models; hematopoietic stem cell gene therapy; B cell-targeted transgene expression; bioengineered factor VIII
17.  Influence of Aggregation on Immunogenicity of Recombinant Human Factor VIII in Hemophilia A Mice 
Recombinant human factor VIII (rFVIII), a multidomain glycoprotein is used in replacement therapy for treatment of hemophilia A. Unfortunately, 15%–30% of the treated patients develop inhibitory antibodies. The pathogenesis of antibody development is not completely understood. The presence of aggregated protein in formulations is generally believed to enhance the immune response. rFVIII has a tendency to aggregate but the effect of such aggregation on the immunogenicity of rFVIII is not known. We have, therefore, characterized aggregated rFVIII produced by thermal stress and evaluated its effect on the immunogenicity of rFVIII in hemophilia A mice. Aggregated rFVIII alone and mixtures of rFVIII with aggregated rFVIII were less immunogenic than native rFVIII. In vitro Th-cell proliferation studies and cytokine analyses conducted on splenocytes obtained from immunized animals suggest that aggregated rFVIII behaves as a unique antigen compared to native monomeric rFVIII. The antigenic properties of the aggregated and native rFVIII were compared using ELISAs (epitope availability) and cathepsin-B (an antigen processing enzyme) digestion. The data suggest significant differences in the antigenic properties of rFVIII and aggregated rFVIII. Overall it appears that aggregated rFVIII does not enhance the immunogenicity (inhibitor development) of rFVIII in hemophilia A mice but rather acts as a distinct antigen.
doi:10.1002/jps.20529
PMCID: PMC2574426  PMID: 16372314
circular dichroism; fluorescence spectroscopy; hemophilia A; inhibitor development; protein aggregation; protein structure; recombinant human Factor VIII; immunogenicity; immunology
18.  Immunogenicity and pharmacokinetic studies of recombinant Factor VIII containing lipid cochleates 
Drug delivery  2010;18(4):246-254.
Replacement therapy using recombinant factor VIII (rFVIII) is currently the most common therapy for hemophilia A, a bleeding disorder caused by the deficiency of FVIII. However, 15–30% of patients develop inhibitory antibodies against administered rFVIII which complicates the therapy. Encapsulation or association of protein with lipidic structures can reduce this immune response. Previously, we developed and characterized rFVIII-containing phosphatidylserine (PS) cochleate cylinders using biophysical techniques. We hypothesized that these structures may provide a reduction in immunogenicity while avoiding the rapid clearance by the reticuloendothelial system (RES) previously observed with liposomal vesicles of similar composition. We investigated in vivo behavior of the cochleates containing rFVIII including immunogenicity and pharmacokinetics in hemophilia A mice. The rFVIII-cochleate complex significantly reduced the level of inhibitory antibody developed against rFVIII following intravenous (i.v.) administration. Pharmacokinetic modeling allowed assessment of in vivo release kinetics. Cochleates acted as delayed release delivery vehicle with an input peak of rFVIII observed around 2 hrs post-injection. rFVIII associated with cochleates showed limited RES uptake and a similar disposition to the free protein upon release from the structure. Incomplete disassociation from the complex limits systemic availability of the protein. Further formulation efforts are warranted to regulate the rate and extent of release of rFVIII from cochleate complexes.
doi:10.3109/10717544.2010.536269
PMCID: PMC3068242  PMID: 21114461
19.  Factor VIII ectopically targeted to platelets is therapeutic in hemophilia A with high-titer inhibitory antibodies 
Journal of Clinical Investigation  2006;116(7):1974-1982.
Inhibitory immune response to exogenously infused factor VIII (FVIII) is a major complication in the treatment of hemophilia A. Generation of such inhibitors has the potential to disrupt gene therapy for hemophilia A. We explore what we believe to be a novel approach to overcome this shortcoming. Human B-domain–deleted FVIII (hBDDFVIII) was expressed under the control of the platelet-specific αIIb promoter in platelets of hemophilic (FVIIInull) mice to create 2bF8trans mice. The FVIII transgene product was stored in platelets and released at the site of platelet activation. In spite of the lack of FVIII in the plasma of 2bF8trans mice, the bleeding phenotype of FVIIInull mice was corrected. More importantly, the bleeding phenotype was corrected in the presence of high inhibitory antibody titers introduced into the mice by infusion or by spleen cell transfer from recombinant hBDDFVIII–immunized mice. Our results demonstrate that this approach to the targeted expression of FVIII in platelets has the potential to correct hemophilia A, even in the presence of inhibitory immune responses to infused FVIII.
doi:10.1172/JCI28416
PMCID: PMC1483176  PMID: 16823491
20.  Long-Term Expression of Human Coagulation Factor VIII in a Tolerant Mouse Model Using the φC31 Integrase System 
Human Gene Therapy  2011;23(4):390-398.
Abstract
We generated a mouse model for hemophilia A that combines a homozygous knockout for murine factor VIII (FVIII) and a homozygous addition of a mutant human FVIII (hFVIII). The resulting mouse, having no detectable FVIII protein or activity and tolerant to hFVIII, is useful for evaluating FVIII gene-therapy protocols. This model was used to develop an effective gene-therapy strategy using the φC31 integrase to mediate permanent genomic integration of an hFVIII cDNA deleted for the B-domain. Various plasmids encoding φC31 integrase and hFVIII were delivered to the livers of these mice by using hydrodynamic tail-vein injection. Long-term expression of therapeutic levels of hFVIII was observed over a 6-month time course when an intron was included in the hFVIII expression cassette and wild-type φC31 integrase was used. A second dose of the hFVIII and integrase plasmids resulted in higher long-term hFVIII levels, indicating that incremental doses were beneficial and that a second dose of φC31 integrase was tolerated. We observed a significant decrease in the bleeding time after a tail-clip challenge in mice treated with plasmids expressing hFVIII and φC31 integrase. Genomic integration of the hFVIII expression plasmid was demonstrated by junction PCR at a known hotspot for integration in mouse liver. The φC31 integrase system provided a nonviral method to achieve long-term FVIII gene therapy in a relevant mouse model of hemophilia A.
Chavez and colleagues report on a gene therapy strategy wherein the uC31 integrase is used to mediate permanent genomic integration of human coagulation factor VIII (hFVIII) cDNA. Plasmids encoding uC31 and hFVIII were administered intravenously to mice defective in murine factor VIII and tolerant to hFVIII. Long-term expression of therapeutic hFVIII levels was observed over 6 months, and bleeding time after tail-clip challenge was significantly decreased.
doi:10.1089/hum.2011.110
PMCID: PMC3327602  PMID: 22077817
21.  PEGylation of a Factor VIII–Phosphatidylinositol Complex: Pharmacokinetics and Immunogenicity in Hemophilia A Mice 
The AAPS Journal  2011;14(1):35-42.
Hemophilia A is an X-linked bleeding disorder caused by the deficiency of factor VIII (FVIII). Exogenous FVIII is administered therapeutically, and due to a short half-life, frequent infusions are often required. Fifteen to thirty-five percent of severe hemophilia A patients develop inhibitory antibodies toward FVIII that complicate clinical management of the disease. Previously, we used phosphatidylinositol (PI) containing lipidic nanoparticles to improve the therapeutic efficacy of recombinant FVIII by reducing immunogenicity and prolonging the circulating half-life. The objective of this study is to investigate further improvements in the FVIII–PI formulation resulting from the addition of polyethylene glycol (PEG) to the particle. PEGylation was achieved by passive transfer of PEG conjugated lipid into the FVIII–PI complex. PEGylated FVIII–PI (FVIII–PI/PEG) was generated with high association efficiency. Reduced activity in vitro and improved retention of activity in the presence of antibodies suggested strong shielding of FVIII by the particle; thus, in vivo studies were conducted in hemophilia A mice. Following intravenous administration, the apparent terminal half-life was improved versus both free FVIII and FVIII–PI, but exposure determined by area under the curve was reduced. The formation of inhibitory antibodies after subcutaneous immunization with FVIII–PI/PEG was lower than free FVIII but resulted in a significant increase in inhibitors following intravenous administration. Passive transfer of PEG onto the FVIII–PI complex does not provide any therapeutic benefit.
doi:10.1208/s12248-011-9309-2
PMCID: PMC3291190  PMID: 22173945
factor VIII; hemophilia A; immunogenicity; inhibitor development; PEGylation
22.  Effect of HLA DR epitope de-immunization of Factor VIII in vitro and in vivo 
Clinical Immunology (Orlando, Fla.)  2011;142(3):320-331.
T cell-dependent development of anti-factor VIII (FVIII) antibodies that neutralize FVIII activity is a major obstacle to replacement therapy in hemophilia A. To create a less immunogenic therapeutic protein, recombinant FVIII can be modified to reduce HLA binding of epitopes based on predicted anchoring residues. Here, we used immunoinformatics tools to identify C2 domain HLA DR epitopes and predict site-specific mutations that reduce immunogenicity. Epitope peptides corresponding to original and modified sequences were validated in HLA binding assays and in immunizations of hemophilic E16 mice, DR3 and DR4 mice and DR3xE16 mice. Consistent with immunoinformatics predictions, original epitopes are immunogenic. Immunization with selected modified sequences lowered immunogenicity for particular peptides and revealed residual immunogenicity of incompletely de-immunized modified peptides. The stepwise approach to reduce protein immunogenicity by epitope modification illustrated here is being used to design and produce a functional full-length modified FVIII for clinical use.
doi:10.1016/j.clim.2011.11.010
PMCID: PMC3288193  PMID: 22222093
Epitope; T cell; Factor VIII; Inhibitors; De-immunization
23.  Transient B Cell Depletion or Improved Transgene Expression by Codon Optimization Promote Tolerance to Factor VIII in Gene Therapy 
PLoS ONE  2012;7(5):e37671.
The major complication in the treatment of hemophilia A is the development of neutralizing antibodies (inhibitors) against factor VIII (FVIII). The current method for eradicating inhibitors, termed immune tolerance induction (ITI), is costly and protracted. Clinical protocols that prevent rather than treat inhibitors are not yet established. Liver-directed gene therapy hopes to achieve long-term correction of the disease while also inducing immune tolerance. We sought to investigate the use of adeno-associated viral (serotype 8) gene transfer to induce tolerance to human B domain deleted FVIII in hemophilia A mice. We administered an AAV8 vector with either human B domain deleted FVIII or a codon-optimized transgene, both under a liver-specific promoter to two strains of hemophilia A mice. Protein therapy or gene therapy was given either alone or in conjunction with anti-CD20 antibody-mediated B cell depletion. Gene therapy with a low-expressing vector resulted in sustained near-therapeutic expression. However, supplementary protein therapy revealed that gene transfer had sensitized mice to hFVIII in a high-responder strain but not in mice of a low-responding strain. This heightened response was ameliorated when gene therapy was delivered with anti-murine CD20 treatment. Transient B cell depletion prevented inhibitor formation in protein therapy, but failed to achieve a sustained hypo-responsiveness. Importantly, use of a codon-optimized hFVIII transgene resulted in sustained therapeutic expression and tolerance without a need for B cell depletion. Therefore, anti-CD20 may be beneficial in preventing vector-induced immune priming to FVIII, but higher levels of liver-restricted expression are preferred for tolerance.
doi:10.1371/journal.pone.0037671
PMCID: PMC3359994  PMID: 22655063
24.  Comparison of Factor VIII Transgenes Bioengineered for Improved Expression in Gene Therapy of Hemophilia A 
Human Gene Therapy  2009;20(5):465-478.
Abstract
Successful gene therapy of hemophilia A depends on the sustained expression of therapeutic levels of factor VIII (fVIII). Because of mRNA instability, interactions with resident endoplasmic reticulum (ER) chaperones, and the requirement for carbohydrate-facilitated transport from the ER to the Golgi apparatus, fVIII is expressed at much lower levels from mammalian cells than other proteins of similar size and complexity. A number of bioengineered forms of B domain-deleted (BDD) human fVIII have been generated and shown to have enhanced expression. Previously, we demonstrated that recombinant BDD porcine fVIII exhibits high-level expression due to specific sequence elements that increase biosynthesis via enhanced posttranslational transit through the secretory pathway. In the current study, high-expression recombinant fVIII constructs were compared directly in order to determine the relative expression of the various bioengineered fVIII transgenes. The data demonstrate that BDD porcine fVIII expression is superior to that of any of the human fVIII variant constructs tested. Mean fVIII expression of 18 units/106 cells/24 hr was observed from HEK-293 cells expressing a single copy of the porcine fVIII transgene, which was 36- to 225-fold greater than that of any human fVIII transgene tested. Furthermore, greater than 10-fold higher expression was observed in human cells transduced with BDD porcine fVIII versus BDD human fVIII-encoding lentiviral vectors, even at low proviral copy numbers, supporting its use over other human fVIII variants in future hemophilia A gene therapy clinical trials.
doi:10.1089/hum.2008.150
PMCID: PMC2828624  PMID: 19222367
25.  Chemical Chaperones Improve Protein Secretion and Rescue Mutant Factor VIII in Mice with Hemophilia A 
PLoS ONE  2012;7(9):e44505.
Inefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking.
doi:10.1371/journal.pone.0044505
PMCID: PMC3433436  PMID: 22973456

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