Lower Inhibitor Development in Hemophilia A Mice following Administration of Recombinant Factor VIII-O-Phospho-L-serine Complex

Abstract. Factor VIII (FVIII) is an important cofactor in blood coagulation cascade. It is a multidomain protein that consists of six domains, NH 2 -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.

Section: In Vivo Characterization Of Fviii-pimentioning

confidence: 99%

Section: Measurement Of Inhibitory Anti-fviii Antibody Titersmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Phosphatidylinositol Containing Lipidic Particles Reduces Immunogenicity and Catabolism of Factor VIII in Hemophilia A Mice

Peng

Straubinger

Balu‐Iyer

2010

“…Plasma was collected as before and stored immediately at −80°C. Inhibitory antibody titers were measured by the Nijmegen modification of the Bethesda assay (38,39). Clotting times determined from various dilutions (1:2 to 1:1,024) of normal coagulation control plasma were used to create a calibration curve.…”

Section: Immunogenicity Studiesmentioning

confidence: 99%

PEGylation of a Factor VIII–Phosphatidylinositol Complex: Pharmacokinetics and Immunogenicity in Hemophilia A Mice

Peng

Kosloski

Nakamura

et al. 2011

Abstract. 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.

“…It has been established that the presentation of an antigen to dendritic cells in a tolerogenic manner can promote peripheral tolerance via induction of regulatory T cells (32). We have shown that administration of therapeutic proteins such as FVIII in the presence of an immune-modulatory lipid phosphatidyl serine (PS) reduces development of inhibitory antibody responses against FVIII and promotes a hyporesponsiveness by tolerogenic presentation to dendritic cells (33)(34)(35)(36). As the preexposure of FVIII in the presence of PS induced tolerance/hyporesponsiveness, we proposed a novel strategy called reverse vaccination to induce tolerance and thereby reduce immunogenicity of therapeutic proteins given sc.…”

Section: Strategies To Mitigate Immunogenicitymentioning

confidence: 99%

Immunogenicity of Subcutaneously Administered Therapeutic Proteins—a Mechanistic Perspective

Fathallah

Bankert

Balu‐Iyer

2013

Abstract. The administration of therapeutic proteins via the subcutaneous route (sc) is desired for compliance and convenience, but could be challenging due to perceived immunogenic potential or unwanted immune responses. There are clinical and preclinical data supporting as well as refuting the generalized notion that sc is more immunogenic. We provide a mechanistic perspective of immunogenicity of therapeutic proteins administered via the sc route and discuss strategies and opportunities for novel therapeutic approaches to mitigate immunogenicity.KEY WORDS: biotechnology; immunogenicity; mitigation of immunogenicity; protein therapeutics; subcutaneous administration. IMMUNOGENICITY OF PROTEINS GIVEN VIA SC ROUTEThe subcutaneous (sc) route of administration of therapeutic proteins is desired over the intravenous (iv) route due to patient comfort and compliance. In addition, it could also prolong half-life of the therapeutic in circulation (1,2). However, this route of administration could be problematic due to a perceived potential for unwanted immunogenicity (3). As most of the vaccines are given via the sc route, it is expected that the sc route is more immunogenic than the iv route. However, a recent comparative clinical study of sc vs iv administration of abatacept, a fusion protein of Fc of human IgG and extracellular domain of CTLA-4, showed that the efficacy and immunogenicity are comparable between the two routes of administration (4,5). However, the immunogenic potential of chronic administration and long-term effects are not often adequately addressed during clinical trials (6). Few preclinical studies have shown that the sc route of administration does not increase immunogenicity (7-9). For example, the relative immunogenicity of Betaseron, interferon beta, is less for sc administration compared to iv administration (9). Based on clinical experience of head-to-head comparison and a few preclinical studies, one could argue that the generalization that the sc route is more immunogenic than the iv route is not universally valid.A comparative immunogenicity study of three brands of insulin in type 1 diabetics showed an increase in incidence of anti-insulin titer development, across brands, in patients selfadministering via sc route as compared to iv administration in hospital in the same cohort (10). In the therapeutic use of erythropoietin-α, incidence of immunogenicity increased with the change from iv to sc. It is appropriate to mention here that elimination of human serum albumin from the formulation as well as stopper material change (11) coincided with changes in route of administration, and thus, the effect of the sc route of administration on immunogenicity is not unambiguous for erythropoietin. There are several examples from preclinical relative immunogenicity studies that show the sc route is more immunogenic than the iv route. The sc administration of FVIII showed significantly higher total antibody titers compared to hemophilia A mice that were given FVIII via the iv route (12). A similar ob...