David R. Light

FcRn Rescues Recombinant Factor VIII Fc Fusion Protein from a VWF Independent FVIII Clearance Pathway in Mouse Hepatocytes

We recently developed a longer lasting recombinant factor VIII-Fc fusion protein, rFVIIIFc, to extend the half-life of replacement FVIII for the treatment of people with hemophilia A. In order to elucidate the biological mechanism for the elongated half-life of rFVIIIFc at a cellular level we delineated the roles of VWF and the tissue-specific expression of the neonatal Fc receptor (FcRn) in the biodistribution, clearance and cycling of rFVIIIFc. We find the tissue biodistribution is similar for rFVIIIFc and rFVIII and that liver is the major clearance organ for both molecules. VWF reduces the clearance and the initial liver uptake of rFVIIIFc. Pharmacokinetic studies in FcRn chimeric mice show that FcRn expressed in somatic cells (hepatocytes or liver sinusoidal endothelial cells) mediates the decreased clearance of rFVIIIFc, but FcRn in hematopoietic cells (Kupffer cells) does not affect clearance. Immunohistochemical studies show that when rFVIII or rFVIIIFc is in dynamic equilibrium binding with VWF, they mostly co localize with VWF in Kupffer cells and macrophages, confirming a major role for liver macrophages in the internalization and clearance of the VWF-FVIII complex. In the absence of VWF a clear difference in cellular localization of VWF-free rFVIII and rFVIIIFc is observed and neither molecule is detected in Kupffer cells. Instead, rFVIII is observed in hepatocytes, indicating that free rFVIII is cleared by hepatocytes, while rFVIIIFc is observed as a diffuse liver sinusoidal staining, suggesting recycling of free-rFVIIIFc out of hepatocytes. These studies reveal two parallel linked clearance pathways, with a dominant pathway in which both rFVIIIFc and rFVIII complexed with VWF are cleared mainly by Kupffer cells without FcRn cycling. In contrast, the free fraction of rFVIII or rFVIIIFc unbound by VWF enters hepatocytes, where FcRn reduces the degradation and clearance of rFVIIIFc relative to rFVIII by cycling rFVIIIFc back to the liver sinusoid and into circulation, enabling the elongated half-life of rFVIIIFc.

Dimethyl fumarate increases fetal hemoglobin, provides heme detoxification, and corrects anemia in sickle cell disease

Sickle cell disease (SCD) results from a point mutation in the β-globin gene forming hemoglobin S (HbS), which polymerizes in deoxygenated erythrocytes, triggering recurrent painful vaso-occlusive crises and chronic hemolytic anemia. Reactivation of fetal Hb (HbF) expression ameliorates these symptoms of SCD. Nuclear factor (erythroid derived-2)-like 2 (Nrf2) is a transcription factor that triggers cytoprotective and antioxidant pathways to limit oxidative damage and inflammation and increases HbF synthesis in CD34+ stem cell-derived erythroid progenitors. We investigated the ability of dimethyl fumarate (DMF), a small-molecule Nrf2 agonist, to activate γ-globin transcription and enhance HbF in tissue culture and in murine and primate models. DMF recruited Nrf2 to the γ-globin promoters and the locus control region of the β-globin locus in erythroleukemia cells, elevated HbF in SCD donor-derived erythroid progenitors, and reduced hypoxia-induced sickling. Chronic DMF administration in SCD mice induced HbF and increased Nrf2-dependent genes to detoxify heme and limit inflammation. This improved hematological parameters, reduced plasma-free Hb, and attenuated inflammatory markers. Chronic DMF administration to nonanemic primates increased γ-globin mRNA in BM and HbF protein in rbc. DMF represents a potential therapy for SCD to induce HbF and augment vasoprotection and heme detoxification.

VLA-4 blockade by natalizumab inhibits sickle reticulocyte and leucocyte adhesion during simulated blood flow

Very Late Antigen‐4 (VLA‐4, α4β1‐integrin, ITGA4) orchestrates cell‐cell and cell‐endothelium adhesion. Given the proposed role of VLA‐4 in sickle cell disease (SCD) pathophysiology, we evaluated the ability of the VLA‐4 blocking antibody natalizumab to inhibit SCD blood cell adhesion. Natalizumab recognized surface VLA‐4 on leucocytes and reticulocytes in whole blood from SCD subjects. SCD reticulocytes were positive for VLA‐4, while VLA‐4 staining of non‐SCD reticulocytes was undetectable. Titrations with natalizumab revealed the presence of saturable levels of VLA‐4 on both SCD reticulocytes and leucocytes similar to healthy subject leucocytes. Under physiological flow conditions, the adhesion of SCD whole blood cells and isolated SCD leucocytes to immobilized vascular cell adhesion molecule 1 (VCAM‐1) was blocked by natalizumab in a dose‐dependent manner, which correlated with cell surface receptor binding. Natalizumab also inhibited >50% of whole blood cell binding to TNF‐α activated human umbilical vein endothelial cell monolayers under physiological flow at clinically relevant concentrations (10 to 100 μg/ml). This indicates that VLA‐4 is the dominant receptor that drives SCD reticulocyte and mononuclear cell adhesion to VCAM‐1 and that the VLA‐4 adhesion to VCAM‐1 is a significant contributor to SCD blood cell adhesion to endothelium. Thus, VLA‐4 blockade may be beneficial in sickle cell disease.

Evaluation of the toxicology, pharmacokinetics, and local tolerance of recombinant factor IX Fc fusion protein in animals.

INTRODUCTION Recombinant factor IX Fc fusion protein (rFIXFc) is a recombinant coagulation factor composed of a single molecule of recombinant factor IX (rFIX) covalently fused to the Fc domain of human immunoglobulin G1 (IgG1) with no intervening sequence. An extensive nonclinical program was performed to support the clinical development of rFIXFc for treatment of people with hemophilia B. MATERIALS AND METHODS Repeat-dose toxicology studies of rFIXFc were performed in 2 relevant species: Sprague Dawley rats (4-week study) and cynomolgus monkeys (5- and 27-week studies). Assessments included in-life observations, electrocardiograms (monkeys only), laboratory evaluations (including hematology and blood chemistry), postmortem analyses, local tolerance, and pharmacokinetics (PK). Allometric scaling was performed with PK data from multiple species, including humans. Local tolerance (single-dose study) and thrombogenic potential (Wessler stasis model) of rFIXFc were tested in New Zealand White rabbits. RESULTS There were no significant local or systemic toxicity findings in the repeat-dose studies. Allometric scaling data suggested that animal rFIXFc PK results are predictive of human PK parameters. There were no findings from the local tolerance study in rabbits; thrombogenic activity was less than that elicited by rFIX and a prothrombin complex concentrate, and similar to vehicle control. CONCLUSIONS rFIXFc was well tolerated in toxicology studies and demonstrated a low thrombogenic potential. These results are consistent with phase 1/2a and phase 3 clinical studies of rFIXFc in people with hemophilia B.

Evaluation of the toxicology and pharmacokinetics of recombinant factor VIII Fc fusion protein in animals

INTRODUCTION Recombinant factor VIII Fc fusion protein (rFVIIIFc) is a novel recombinant factor VIII with a prolonged half-life, developed for the treatment of hemophilia A. Studies that evaluated the toxicological effects of rFVIIIFc in 2 pharmacologically relevant species, cynomolgus monkeys and Sprague Dawley rats, are reported here. MATERIALS AND METHODS In repeat-dose toxicology studies, rats and monkeys received 0, 50, 250, or 1000 IU/kg rFVIIIFc every other day for 4 weeks. In a high-dose tolerance study, monkeys received 1 rFVIIIFc dose of 3000, 10,000, or 20,000 IU/kg. Evaluations included in-life observations, laboratory and post-mortem evaluations, pharmacokinetics, and local tolerance. Allometric scaling, using data from 4 animal species and humans, was used to evaluate the relationship between animal and human pharmacokinetics. RESULTS rFVIIIFc was well tolerated with no adverse toxicological findings directly attributable to rFVIIIFc. As expected, antibodies to this fully human protein developed in rats and monkeys in a time-dependent fashion following repeated dosing, leading to increased clearance in both species. There were no local reactions (infusion site) or evidence of thrombosis at high doses in rats and monkeys. Allometric scaling demonstrated more rapid clearance in small animals compared with humans and a volume of distribution (steady state) proportional to body weight across species, suggesting that animal pharmacokinetics are predictive of human pharmacokinetics. CONCLUSIONS Repeated doses of rFVIIIFc in 2 relevant animal species and high doses of rFVIIIFc in monkeys were well tolerated. These results supported the clinical safety of rFVIIIFc observed in phase 1/2a and phase 3 clinical trials.