Recombinant factor VIII Fc fusion protein for first-time immune tolerance induction: final results of the verITI-8 study.

Inhibitor development remains a major challenge in factor VIII (FVIII) replacement therapy. verITI-8 is the first prospective study of a recombinant FVIII Fc fusion protein (rFVIIIFc; efmoroctocog alfa) for first-time immune tolerance induction (ITI) in males with severe hemophilia A and high-titer inhibitors (historical peak ≥5 Bethesda units [BU]/mL). In this single-arm, open-label, multicenter study, screening was followed by ITI (rFVIIIFc 200 IU/kg per day until tolerization or maximum of 48 weeks). Those who achieved ITI success entered a tapering period, returning to standard prophylaxis, and then entered follow-up. Primary end point was time to tolerization with rFVIIIFc defined by inhibitor titer <0.6 BU/mL, incremental recovery (IR) ≥66% of expected IR (IR ≥1.32 IU/dL per IU/kg), and half-life (t½) ≥7 hours within 48 weeks. Sixteen patients received ≥1 rFVIIIFc dose. Twelve (75%), 11 (69%), and 10 patients (63%), respectively, achieved negative inhibitor titers, an IR ≥66%, and a t½ ≥7 hours (ie, tolerance) within 48 weeks. Median times in weeks to achieve these markers of success were 7.4 (interquartile range [IQR], 2.2-17.8), 6.8 (IQR, 5.4-22.4), and 11.7 (IQR, 9.8-26.2), respectively. All patients experienced ≥1 treatment-emergent adverse event (TEAE), and 1 reported ≥1 related TEAE (injection site pain). Nine patients experienced ≥1 treatment-emergent serious AE. No thrombotic events, discontinuations because of AEs, or deaths were reported during the study. As the first extended half-life rFVIII with prospective data in ITI, rFVIIIFc offered short time to tolerization with durable responses in almost two-thirds of patients and was well tolerated. This trial was registered at www.clinicaltrials.gov as #NCT03093480.

Phase 3 study of recombinant factor IX Fc fusion protein in hemophilia B.

BACKGROUND: Prophylactic factor replacement in patients with hemophilia B improves outcomes but requires frequent injections. A recombinant factor IX Fc fusion protein (rFIXFc) with a prolonged half-life was developed to reduce the frequency of injections required. METHODS: We conducted a phase 3, nonrandomized, open-label study of the safety, efficacy, and pharmacokinetics of rFIXFc for prophylaxis, treatment of bleeding, and perioperative hemostasis in 123 previously treated male patients. All participants were 12 years of age or older and had severe hemophilia B (endogenous factor IX level of ≤2 IU per deciliter, or ≤2% of normal levels). The study included four treatment groups: group 1 received weekly dose-adjusted prophylaxis (50 IU of rFIXFc per kilogram of body weight to start), group 2 received interval-adjusted prophylaxis (100 IU per kilogram every 10 days to start), group 3 received treatment as needed for bleeding episodes (20 to 100 IU per kilogram), and group 4 received treatment in the perioperative period. A subgroup of group 1 underwent comparative sequential pharmacokinetic assessments of recombinant factor IX and rFIXFc. The primary efficacy end point was the annualized bleeding rate, and safety end points included the development of inhibitors and adverse events. RESULTS: As compared with recombinant factor IX, rFIXFc exhibited a prolonged terminal half-life (82.1 hours) (P<0.001). The median annualized bleeding rates in groups 1, 2, and 3 were 3.0, 1.4, and 17.7, respectively. In group 2, 53.8% of participants had dosing intervals of 14 days or more during the last 3 months of the study. In groups 1, 2 and 3, 90.4% of bleeding episodes resolved after one injection. Hemostasis was rated as excellent or good during all major surgeries. No inhibitors were detected in any participants receiving rFIXFc; in groups 1, 2, and 3, 73.9% of participants had at least one adverse event, and serious adverse events occurred in 10.9% of participants. These events were mostly consistent with those expected in the general population of patients with hemophilia. CONCLUSIONS: Prophylactic rFIXFc, administered every 1 to 2 weeks, resulted in low annualized bleeding rates in patients with hemophilia B. (Funded by Biogen Idec; ClinicalTrials.gov number, NCT01027364.).

Phase 3 study of recombinant factor VIII Fc fusion protein in severe hemophilia A.

This phase 3 pivotal study evaluated the safety, efficacy, and pharmacokinetics of a recombinant FVIII Fc fusion protein (rFVIIIFc) for prophylaxis, treatment of acute bleeding, and perioperative hemostatic control in 165 previously treated males aged ≥12 years with severe hemophilia A. The study had 3 treatment arms: arm 1, individualized prophylaxis (25-65 IU/kg every 3-5 days, n = 118); arm 2, weekly prophylaxis (65 IU/kg, n = 24); and arm 3, episodic treatment (10-50 IU/kg, n = 23). A subgroup compared recombinant FVIII (rFVIII) and rFVIIIFc pharmacokinetics. End points included annualized bleeding rate (ABR), inhibitor development, and adverse events. The terminal half-life of rFVIIIFc (19.0 hours) was extended 1.5-fold vs rFVIII (12.4 hours; P < .001). Median ABRs observed in arms 1, 2, and 3 were 1.6, 3.6, and 33.6, respectively. In arm 1, the median weekly dose was 77.9 IU/kg; approximately 30% of subjects achieved a 5-day dosing interval (last 3 months on study). Across arms, 87.3% of bleeding episodes resolved with 1 injection. Adverse events were consistent with those expected in this population; no subjects developed inhibitors. rFVIIIFc was well-tolerated, had a prolonged half-life compared with rFVIII, and resulted in low ABRs when dosed prophylactically 1 to 2 times per week.

Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives.

Biotherapeutic proteins represent a mainstay of treatment for a multitude of conditions, for example, autoimmune disorders, hematologic disorders, hormonal dysregulation, cancers, infectious diseases and genetic disorders. The technologies behind their production have changed substantially since biotherapeutic proteins were first approved in the 1980s. Although most biotherapeutic proteins developed to date have been produced using the mammalian Chinese hamster ovary and murine myeloma (NS0, Sp2/0) cell lines, there has been a recent shift toward the use of human cell lines. One of the most important advantages of using human cell lines for protein production is the greater likelihood that the resulting recombinant protein will bear post-translational modifications (PTMs) that are consistent with those seen on endogenous human proteins. Although other mammalian cell lines can produce PTMs similar to human cells, they also produce non-human PTMs, such as galactose-α1,3-galactose and N-glycolylneuraminic acid, which are potentially immunogenic. In addition, human cell lines are grown easily in a serum-free suspension culture, reproduce rapidly and have efficient protein production. A possible disadvantage of using human cell lines is the potential for human-specific viral contamination, although this risk can be mitigated with multiple viral inactivation or clearance steps. In addition, while human cell lines are currently widely used for biopharmaceutical research, vaccine production and production of some licensed protein therapeutics, there is a relative paucity of clinical experience with human cell lines because they have only recently begun to be used for the manufacture of proteins (compared with other types of cell lines). With additional research investment, human cell lines may be further optimized for routine commercial production of a broader range of biotherapeutic proteins.

Surviving the heat: heterogeneity of response in Saccharomyces cerevisiae provides insight into thermal damage to the membrane.

Environmental heat stress impacts on the physiology and viability of microbial cells with concomitant implications for microbial activity and diversity. Previously, it has been demonstrated that gradual heating of Saccharomyces cerevisiae induces a degree of thermal resistance, whereas a heat shock results in a high level of cell death. Here, we show that the impact of exogenous nutrients on acquisition of thermal resistance differs between strains. Using single-cell methods, we demonstrate the extent of heterogeneity of the heat-stress response within populations of yeast cells and the presence of subpopulations that are reversibly damaged by heat stress. Such cells represent potential for recovery of entire populations once stresses are removed. The results show that plasma membrane permeability and potential are key factors involved in cell survival, but thermal resistance is not related to homeoviscous adaptation of the plasma membrane. These results have implications for growth and regrowth of populations experiencing environmental heat stress and our understanding of impacts at the level of the single cell. Given the important role of microbes in biofuel production and bioremediation, a thorough understanding of the impact of stress responses of populations and individuals is highly desirable.

Fc-fusion proteins and FcRn: structural insights for longer-lasting and more effective therapeutics.

Nearly 350 IgG-based therapeutics are approved for clinical use or are under development for many diseases lacking adequate treatment options. These include molecularly engineered biologicals comprising the IgG Fc-domain fused to various effector molecules (so-called Fc-fusion proteins) that confer the advantages of IgG, including binding to the neonatal Fc receptor (FcRn) to facilitate in vivo stability, and the therapeutic benefit of the specific effector functions. Advances in IgG structure-function relationships and an understanding of FcRn biology have provided therapeutic opportunities for previously unapproachable diseases. This article discusses approved Fc-fusion therapeutics, novel Fc-fusion proteins and FcRn-dependent delivery approaches in development, and how engineering of the FcRn-Fc interaction can generate longer-lasting and more effective therapeutics.

Manufacturing process used to produce long-acting recombinant factor VIII Fc fusion protein.

Recombinant factor VIII Fc fusion protein (rFVIIIFc) is a long-acting coagulation factor approved for the treatment of hemophilia A. Here, the rFVIIIFc manufacturing process and results of studies evaluating product quality and the capacity of the process to remove potential impurities and viruses are described. This manufacturing process utilized readily transferable and scalable unit operations and employed multi-step purification and viral clearance processing, including a novel affinity chromatography adsorbent and a 15 nm pore size virus removal nanofilter. A cell line derived from human embryonic kidney (HEK) 293H cells was used to produce rFVIIIFc. Validation studies evaluated identity, purity, activity, and safety. Process-related impurity clearance and viral clearance spiking studies demonstrate robust and reproducible removal of impurities and viruses, with total viral clearance >8-15 log10 for four model viruses (xenotropic murine leukemia virus, mice minute virus, reovirus type 3, and suid herpes virus 1). Terminal galactose-α-1,3-galactose and N-glycolylneuraminic acid, two non-human glycans, were undetectable in rFVIIIFc. Biochemical and in vitro biological analyses confirmed the purity, activity, and consistency of rFVIIIFc. In conclusion, this manufacturing process produces a highly pure product free of viruses, impurities, and non-human glycan structures, with scale capabilities to ensure a consistent and adequate supply of rFVIIIFc.

Distinct responses to ozone of abaxial and adaxial stomata in three Euramerican poplar genotypes.

Ozone induces stomatal sluggishness, which impacts photosynthesis and transpiration. Stomatal responses to variation of environmental parameters are slowed and reduced by ozone and may be linked to difference of ozone sensitivity. Here we determine the ozone effects on stomatal conductance of each leaf surface. Potential causes of this sluggish movement, such as ultrastructural or ionic fluxes modification, were studied independently on both leaf surfaces of three Euramerican poplar genotypes differing in ozone sensitivity and in stomatal behaviour. The element contents in guard cells were linked to the gene expression of ion channels and transporters involved in stomatal movements, directly in microdissected stomata. In response to ozone, we found a decrease in the stomatal conductance of the leaf adaxial surface correlated with high calcium content in guard cells compared with a slight decrease on the abaxial surface. No ultrastructural modifications of stomata were shown except an increase in the number of mitochondria. The expression of vacuolar H(+) /Ca(2+) -antiports (CAX1 and CAX3 homologs), ß-carbonic anhydrases (ßCA1 and ßCA4) and proton H(+) -ATPase (AHA11) genes was strongly decreased under ozone treatment. The sensitive genotype characterized by constitutive slow stomatal response was also characterized by constitutive low expression of genes encoding vacuolar H(+) /Ca(2+) -antiports.

Safety and prolonged activity of recombinant factor VIII Fc fusion protein in hemophilia A patients.

Current factor VIII (FVIII) products display a half-life (t(1/2)) of ∼ 8-12 hours, requiring frequent intravenous injections for prophylaxis and treatment of patients with hemophilia A. rFVIIIFc is a recombinant fusion protein composed of a single molecule of FVIII covalently linked to the Fc domain of human IgG(1) to extend circulating rFVIII t(1/2). This first-in-human study in previously treated subjects with severe hemophilia A investigated safety and pharmacokinetics of rFVIIIFc. Sixteen subjects received a single dose of rFVIII at 25 or 65 IU/kg followed by an equal dose of rFVIIIFc. Most adverse events were unrelated to study drug. None of the study subjects developed anti-rFVIIIFc antibodies or inhibitors. Across dose levels, compared with rFVIII, rFVIIIFc showed 1.54- to 1.70-fold longer elimination t(1/2), 1.49- to 1.56-fold lower clearance, and 1.48- to 1.56-fold higher total systemic exposure. rFVIII and rFVIIIFc had comparable dose-dependent peak plasma concentrations and recoveries. Time to 1% FVIII activity above baseline was ∼ 1.53- to 1.68-fold longer than rFVIII across dose levels. Each subject showed prolonged exposure to rFVIIIFc relative to rFVIII. Thus, rFVIIIFc may offer a viable therapeutic approach to achieve prolonged hemostatic protection and less frequent dosing in patients with hemophilia A. This trial was registered at www.clinicaltrials.gov as NCT01027377.

Prolonged activity of a recombinant factor VIII-Fc fusion protein in hemophilia A mice and dogs.

Despite proven benefits, prophylactic treatment for hemophilia A is hampered by the short half-life of factor VIII. A recombinant factor VIII-Fc fusion protein (rFVIIIFc) was constructed to determine the potential for reduced frequency of dosing. rFVIIIFc has an ∼ 2-fold longer half-life than rFVIII in hemophilia A (HemA) mice and dogs. The extension of rFVIIIFc half-life requires interaction of Fc with the neonatal Fc receptor (FcRn). In FcRn knockout mice, the extension of rFVIIIFc half-life is abrogated, and is restored in human FcRn transgenic mice. The Fc fusion has no impact on FVIII-specific activity. rFVIIIFc has comparable acute efficacy as rFVIII in treating tail clip injury in HemA mice, and fully corrects whole blood clotting time (WBCT) in HemA dogs immediately after dosing. Furthermore, consistent with prolonged half-life, rFVIIIFc shows 2-fold longer prophylactic efficacy in protecting HemA mice from tail vein transection bleeding induced 24-48 hours after dosing. In HemA dogs, rFVIIIFc also sustains partial correction of WBCT 1.5- to 2-fold longer than rFVIII. rFVIIIFc was well tolerated in both species. Thus, the rescue of FVIII by Fc fusion to provide prolonged protection presents a novel pathway for FVIII catabolism, and warrants further investigation.

Recombinant factor IX-Fc fusion protein (rFIXFc) demonstrates safety and prolonged activity in a phase 1/2a study in hemophilia B patients.

Current factor IX (FIX) products display a half-life (t(1/2)) of ∼ 18 hours, requiring frequent intravenous infusions for prophylaxis and treatment in patients with hemophilia B. This open-label, dose-escalation trial in previously treated adult subjects with hemophilia B examined the safety and pharmacokinetics of rFIXFc. rFIXFc is a recombinant fusion protein composed of FIX and the Fc domain of human IgG(1), to extend circulating time. Fourteen subjects received a single dose of rFIXFc; 1 subject each received 1, 5, 12.5, or 25 IU/kg, and 5 subjects each received 50 or 100 IU/kg. rFIXFc was well tolerated, and most adverse events were mild or moderate in intensity. No inhibitors were detected in any subject. Dose-proportional increases in rFIXFc activity and Ag exposure were observed. With baseline subtraction, mean activity terminal t(1/2) and mean residence time for rFIXFc were 56.7 and 71.8 hours, respectively. This is ∼ 3-fold longer than that reported for current rFIX products. The incremental recovery of rFIXFc was 0.93 IU/dL per IU/kg, similar to plasma-derived FIX. These results show that rFIXFc may offer a viable therapeutic approach to achieve prolonged hemostatic protection and less frequent dosing in patients with hemophilia B. The trial was registered at www.clinicaltrials.gov as NCT00716716.

Capacity for NADPH regeneration in the leaves of two poplar genotypes differing in ozone sensitivity.

Cell capacity for cytosolic NADPH regeneration by NADP-dehydrogenases was investigated in the leaves of two hybrid poplar (Populus deltoides × Populus nigra) genotypes in response to ozone (O3 ) treatment (120 ppb for 17 days). Two genotypes with differential O3 sensitivity were selected, based on visual symptoms and fallen leaves: Robusta (sensitive) and Carpaccio (tolerant). The estimated O3 flux (POD0 ), that entered the leaves, was similar for the two genotypes throughout the treatment. In response to that foliar O3 flux, CO2 assimilation was inhibited to the same extent for the two genotypes, which could be explained by a decrease in Rubisco (EC 4.1.1.39) activity. Conversely, an increase in PEPC (EC 4.1.1.31) activity was observed, together with the activation of certain cytosolic NADP-dehydrogenases above their constitutive level, i.e. NADP-G6PDH (EC 1.1.1.49), NADP-ME (malic enzyme) (EC 1.1.1.40) and NADP-ICDH (NADP-isocitrate dehydrogenase) (EC1.1.1.42). However, the activity of non-phosphorylating NADP-GAPDH (EC 1.2.1.9) remained unchanged. From the 11th fumigation day, NADP-G6PDH and NADP-ME profiles made it possible to differentiate between the two genotypes, with a higher activity in Carpaccio than in Robusta. At the same time, Carpaccio was able to maintain high levels of NADPH in the cells, while NADPH levels decreased in Robusta O3 -treated leaves. All these results support the hypothesis that the capacity for cells to regenerate the reducing power, especially the cytosolic NADPH pool, contributes to improve tolerance to high ozone exposure.

Biodistribution of recombinant factor IX, extended half-life recombinant factor IX Fc fusion protein, and glycoPEGylated recombinant factor IX in hemophilia B mice.

Extended half-life recombinant FIX (rFIX) molecules have been generated to reduce the dosing burden and increase the protection of patients with hemophilia B. Clinical pharmacology studies with recombinant factor IX Fc fusion protein (rFIXFc) report a similar initial peak plasma recovery to that of rFIX, but with a larger volume of distribution. Although the pegylation of N9-GP results in a larger plasma recovery, there is a smaller volume of distribution, suggesting less extravasation of the latter drug. In this study, we set out to compare the biodistribution and tissue localization of rFIX, rFIXFc, and glycoPEGylated rFIX in a hemophilia B mouse model. Radiolabeled rFIX, rFIXFc, and rFIX-GP were employed in in vivo single-photon emission computed tomography imaging (SPECT/CT), microautoradiography (MARG), and histology to assess the distribution of FIX reagents over time. Immediately following injection, vascularized tissues demonstrated intense signal irrespective of FIX reagent. rFIX and rFIXFc were retained in joint and muscle areas through 5 half-lives, unlike rFIX-GP (assessed by SPECT). MARG and immunohistochemistry showed FIX agents localized at blood vessels among tissues, including liver, spleen, and kidney. Microautoradiographs, as well as fluorescent-labeled images of knee joint areas, demonstrated retention over time of FIX signal at the trabecular area of bone. Data indicate that rFIXFc is similar to rFIX in that it distributes outside the plasma compartment and is retained in certain tissues over time, while also retained at higher plasma levels. Overall, data suggest that Fc fusion does not impede the extravascular distribution of FIX.

The Yin-Yang of the Green Fluorescent Protein: Impact on Saccharomyces cerevisiae stress resistance.

Although fluorescent proteins are widely used as biomarkers (Yin), no study focuses on their influence on the microbial stress response. Here, the Green Fluorescent Protein (GFP) was fused to two proteins of interest in Saccharomyces cerevisiae. Pab1p and Sur7p, respectively involved in stress granules structure and in Can1 membrane domains. These were chosen since questions remain regarding the understanding of the behavior of S. cerevisiae facing different heat kinetics or oxidative stresses. The main results showed that Pab1p-GFP fluorescent mutant displayed a higher resistance than that of the wild type under a heat shock. Moreover, fluorescent mutants exposed to oxidative stresses displayed changes in the cultivability compared to the wild type strain. In silico approaches showed that the presence of the GFP did not influence the structure and so the functionality of the tagged proteins meaning that changes in yeast resistance were certainly related to GFP ROS-scavenging ability (Yang).

Prophylaxis with a recombinant factor VIII Fc in hemophilia A: long-term follow-up on joint health, efficacy, and safety from phase 3 studies in children and adults.

Background: Recurrent joint bleeds are a major cause of morbidity in severe hemophilia. Prophylaxis with efmoroctocog alfa (a recombinant factor VIII Fc fusion protein, [rFVIIIFc]) has demonstrated benefits beyond bleed control, including joint health maintenance. Objectives: To assess long-term efficacy and safety of rFVIIIFc prophylaxis in severe hemophilia A in phase 3 pivotal (A-LONG/Kids A-LONG) and extension (ASPIRE) studies. Methods: Longitudinal analysis included pooled data from A-LONG/Kids A-LONG and ASPIRE. Subgroup analyses investigated outcomes in modified Hemophilia Joint Health Score or Hemophilia Joint Health Score and target joints in subjects with 4 to 5 years follow-up on individualized prophylaxis (IP), and those with the highest annualized bleeding rate (ABR) quartile during Year 1 of IP. Results: Overall, rFVIIIFc consumption remained stable and low ABRs were maintained, with a median treatment duration of 4.2/3.4 years in subjects from A-LONG/Kids A-LONG, respectively. Median overall ABR also remained low (1.0-2.0) in subjects on IP for 4 to 5 years. Sustained improvements in modified Hemophilia Joint Health Score or Hemophilia Joint Health Score were demonstrated over a median follow-up of 3.7 years. In subjects from A-LONG/Kids A-LONG, 99.6% (n = 234)/100% (n = 9) of evaluable baseline target joints were resolved, with no recurrence in 95%/100% of target joints. In IP subjects within the highest ABR quartile in Year 1, continued improvements were observed over a median follow-up of 4.3 years in ABR and joint health, without increased factor consumption. No inhibitors or treatment-related serious adverse events were reported. Conclusion: Previously treated subjects of all ages receiving long-term prophylaxis with rFVIIIFc had sustained clinical benefits, including improved joint health and low ABR.

Post hoc longitudinal assessment of the efficacy and safety of recombinant factor IX Fc fusion protein in hemophilia B.

Long-term efficacy and safety of the extended half-life recombinant factor IX Fc fusion protein (rFIXFc) has been established among previously treated patients with severe hemophilia B in 2 phase 3 trials (B-LONG [#NCT01027364] and Kids B-LONG [#NCT01440946]) and a long-term extension study (B-YOND [#NCT01425723]). In this study, we report post hoc analyses of pooled longitudinal data for up to 6.5 years for rFIXFc prophylaxis. In the B-LONG study, subjects ≥12 years received weekly dose-adjusted prophylaxis (WP; starting dose, 50 IU/kg), individualized interval-adjusted prophylaxis (IP; initially, 100 IU/kg every 10 days), or on-demand dosing. In the Kids B-LONG study, subjects <12 years received 50 to 60 IU/kg every 7 days, adjusted as needed. In the B-YOND study, subjects received WP (20-100 IU/kg every 7 days), IP (100 IU/kg every 8-16 days), modified prophylaxis, or on-demand dosing; switching between treatment groups was permitted. A total of 123 subjects from B-LONG and 30 from Kids B-LONG study were included, of whom 93 and 27, respectively, enrolled in the B-YOND study. The median cumulative duration of treatment was 3.63 years (range, 0.003-6.48 years) in B-LONG/B-YOND and 2.88 years (range, 0.30-4.80 years) in Kids B-LONG/B-YOND group. Annualized bleed rates (ABRs) remained low, annualized factor consumption remained stable, and adherence remained high throughout treatment. Low ABRs were also maintained in subjects with dosing intervals ≥14 days or with target joints at baseline. Complete resolution of evaluable target joints and no recurrence in 90.2% of baseline target joints during follow-up were observed. rFIXFc prophylaxis was associated with sustained clinical benefits, including long-term bleed prevention and target joint resolution, for severe hemophilia B.

Atrial natriuretic peptide-Fc, ANP-Fc, fusion proteins: semisynthesis, in vitro activity and pharmacokinetics in rats.

Atrial natriuretic peptide (ANP) may be a useful molecule for the treatment of cardiovascular diseases due to its potent natriuretic effects. In an effort to prolong the short in vivo half-life of ANP, fusions of the peptide to the Fc domain of IgG were generated using a semisynthetic methodology. Synthetic ANP peptides were synthesized with thioesters at either the N- or C-termini of the peptide and subsequently linked to the N-terminus of recombinantly expressed Fc using native chemical ligation. The linker length between the ANP and Fc moieties was varied among 2, 11, or 16 amino acids. In addition, either one ("monomeric") or two ("dimeric") ANP peptides were linked to Fc to study whether this modification had an effect on in vitro activity and/or in vivo half-life. The various constructs were studied for in vitro activity using a cell-based cGMP assay. The ANP-Fc fusion constructs were between 16- and ∼375-fold weaker than unconjugated ANP in this assay, and a trend was observed where the most potent conjugates were those with longer linkers and in the dimeric configuration. The pharmacokinetics of several constructs were assessed in rats, and the half-life of the ANP-Fc's were found to be approximately 2 orders of magnitude longer than that of the unconjugated peptide. There was no significant difference in terminal half-life between the monomeric and dimeric constructs (2.8-5.5 h), but a trend was observed where the C(max) of the monomeric constructs was approximately 3-fold higher than that of the dimeric constructs, although the origin of this effect is not understood. These novel ANP-Fc fusion constructs hold promise for future therapeutic application in the treatment of cardiovascular diseases.

Prolonged activity of factor IX as a monomeric Fc fusion protein.

Treatment of hemophilia B requires frequent infusions of factor IX (FIX) to prophylax against bleeding episodes. Hemophilia B management would benefit from a FIX protein with an extended half-life. A recombinant fusion protein (rFIXFc) containing a single FIX molecule attached to the Fc region of immunoglobulin G was administered intravenously and found to have an extended half-life, compared with recombinant FIX (rFIX) in normal mice, rats, monkeys, and FIX-deficient mice and dogs. Recombinant FIXFc protein concentration was determined in all species, and rFIXFc activity was measured in FIX-deficient animals. The half-life of rFIXFc was approximately 3- to 4-fold longer than that of rFIX in all species. In contrast, in mice in which the neonatal Fc receptor (FcRn) was deleted, the half-life of rFIXFc was similar to rFIX, confirming the increased circulatory time was due to protection of the rFIXFc via the Fc/FcRn interaction. Whole blood clotting time in FIX-deficient mice was corrected through 144 hours for rFIXFc, compared with 72 hours for rFIX; similar results were observed in FIX-deficient dogs. Taken together, these studies show the enhanced pharmacodynamic and pharmacokinetic properties of the rFIXFc fusion protein and provide the basis for evaluating rFIXFc in patients with hemophilia B.

Genome-Wide Association Study and Gene-Based Analysis of Participants With Hemophilia A and Inhibitors in the My Life, Our Future Research Repository.

Introduction: Up to 30% of individuals with hemophilia A develop inhibitors to replacement factor VIII (FVIII), rendering the treatment ineffective. The underlying mechanism of inhibitor development remains poorly understood. The My Life, Our Future Research Repository (MLOF RR) has gathered F8 and F9 mutational information, phenotypic data, and biological material from over 11,000 participants with hemophilia A (HA) and B as well as carriers enrolled across US hemophilia treatment centers, including over 5,000 whole-genome sequences. Identifying genes associated with inhibitors may contribute to our understanding of why certain patients develop those neutralizing antibodies. Aim and Methods: Here, we performed a genome-wide association study and gene-based analyses to identify genes associated with inhibitors in participants with HA from the MLOF RR. Results: We identify a genome-wide significant association within the human leukocyte antigen (HLA) locus in participants with HA with F8 intronic inversions. HLA typing revealed independent associations with the HLA alleles major histocompatibility complex, class II, DR beta 1 (HLA DRB1*15:01) and major histocompatibility complex, class II, DQ beta 1 (DQB1*03:03). Variant aggregation tests further identified low-frequency variants within GRID2IP (glutamate receptor, ionotropic, delta 2 [GRID2] interacting protein 1) significantly associated with inhibitors. Conclusion: Overall, our study confirms the association of DRB1*15:01 with FVIII inhibitors and identifies a novel association of DQB1*03:03 in individuals with HA carrying intronic inversions of F8. In addition, our results implicate GRID2IP, encoding GRID2-interacting protein, with the development of inhibitors, and suggest an unrecognized role of this gene in autoimmunity.