Molecular determinants of the factor VIII/von Willebrand factor complex revealed by BIVV001 cryo-electron microscopy.

Interaction of factor VIII (FVIII) with von Willebrand factor (VWF) is mediated by the VWF D'D3 domains and thrombin-mediated release is essential for hemostasis after vascular injury. VWF-D'D3 mutations resulting in loss of FVIII binding are the underlying cause of von Willebrand disease (VWD) type 2N. Furthermore, the FVIII-VWF interaction has significant implications for the development of therapeutics for bleeding disorders, particularly hemophilia A, in which endogenous VWF clearance imposes a half-life ceiling on replacement FVIII therapy. To understand the structural basis of FVIII engagement by VWF, we solved the structure of BIVV001 by cryo-electron microscopy to 2.9 Å resolution. BIVV001 is a bioengineered clinical-stage FVIII molecule for the treatment of hemophilia A. In BIVV001, VWF-D'D3 is covalently linked to an Fc domain of a B domain-deleted recombinant FVIII (rFVIII) Fc fusion protein, resulting in a stabilized rFVIII/VWF-D'D3 complex. Our rFVIII/VWF structure resolves BIVV001 architecture and provides a detailed spatial understanding of previous biochemical and clinical observations related to FVIII-VWF engagement. Notably, the FVIII acidic a3 peptide region (FVIII-a3), established as a critical determinant of FVIII/VWF complex formation, inserts into a basic groove formed at the VWF-D'/rFVIII interface. Our structure shows direct interaction of sulfated Y1680 in FVIII-a3 and VWF-R816 that, when mutated, leads to severe hemophilia A or VWD type 2N, respectively. These results provide insight on this key coagulation complex, explain the structural basis of many hemophilia A and VWD type 2N mutations, and inform studies to further elucidate how VWF dissociates rapidly from FVIII upon activation.

The von Willebrand factor D'D3 assembly and structural principles for factor VIII binding and concatemer biogenesis.

D assemblies make up half of the von Willebrand factor (VWF), yet are of unknown structure. D1 and D2 in the prodomain and D'D3 in mature VWF at Golgi pH form helical VWF tubules in Weibel Palade bodies and template dimerization of D3 through disulfides to form ultralong VWF concatemers. D'D3 forms the binding site for factor VIII. The crystal structure of monomeric D'D3 with cysteine residues required for dimerization mutated to alanine was determined at an endoplasmic reticulum (ER)-like pH. The smaller C8-3, TIL3 (trypsin inhibitor-like 3), and E3 modules pack through specific interfaces as they wind around the larger, N-terminal, Ca2+-binding von Willebrand D domain (VWD) 3 module to form a wedge shape. D' with its TIL' and E' modules projects away from D3. The 2 mutated cysteines implicated in D3 dimerization are buried, providing a mechanism for protecting them against premature disulfide linkage in the ER, where intrachain disulfide linkages are formed. D3 dimerization requires co-association with D1 and D2, Ca2+, and Golgi-like acidic pH. Associated structural rearrangements in the C8-3 and TIL3 modules are required to expose cysteine residues for disulfide linkage. Our structure provides insight into many von Willebrand disease mutations, including those that diminish factor VIII binding, which suggest that factor VIII binds not only to the N-terminal TIL' domain of D' distal from D3 but also extends across 1 side of D3. The organizing principle for the D3 assembly has implications for other D assemblies and the construction of higher-order, disulfide-linked assemblies in the Golgi in both VWF and mucins.

Mapping the interaction between factor VIII and von Willebrand factor by electron microscopy and mass spectrometry.

Association with the D'D3 domain of von Willebrand factor (VWF) stabilizes factor VIII (FVIII) in the circulation and maintains it at a level sufficient to prevent spontaneous bleeding. We used negative-stain electron microscopy (EM) to visualize complexes of FVIII with dimeric and monomeric forms of the D'D3 domain. The EM averages show that FVIII interacts with the D'D3 domain primarily through its C1 domain, with the C2 domain providing a secondary attachment site. Hydrogen-deuterium exchange mass spectrometry corroborated the importance of the C1 domain in D'D3 binding and implicates additional surface regions on FVIII in the interaction. Together, our results establish that the C1 domain is the major binding site on FVIII for VWF, reiterate the importance of the a3 acidic peptide in VWF binding, and suggest that the A3 and C2 domains play ancillary roles in this interaction.

Mortality and morbidity in oesophageal atresia.

BACKGROUND: Several classification systems exist to predict mortality in oesophageal atresia, the most widely quoted of these being over 20 years old. No classification system exists to predict morbidity. We sought to test whether these classification systems remain relevant and to determine whether they can be useful to predict morbidity. In addition, we aimed to identify independent risk factors for predicting mortality and morbidity. METHODS: Neonates presenting with oesophageal atresia over a 20-year period (1990-2010) were retrospectively reviewed. Discriminative statistical analysis compared the performance of current classification systems. Stepwise logistic regression analysis of the influence of perioperative risk factors on mortality and duration of ventilatory support and intensive care unit stay were performed. RESULTS: All classification systems predicted mortality in this series of 248 neonates. Birth weight, cardiac anomalies and pre-operative pneumonia were independent risk factors for predicting mortality in oesophageal atresia. The Waterston classification is the most useful classification for predicting post-operative morbidity in terms of length of hospital stay and time spent ventilated. CONCLUSION: Despite advances in the neonatal care of the very low birth weight infant and those with congenital cardiac disease, these conditions remain relevant in predicting mortality and morbidity in oesophageal atresia.

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.

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.

Laparoscopic hernia repair in children: does recreating the open operation improve outcomes? A systematic review.

PURPOSE: The use of laparoscopy for paediatric inguinal hernia repairs has increased significantly over the past 2 decades. However, there is significant variation in the reported recurrence rates in the literature, with many studies reporting higher rates than the open operation. This may be explained by the range of different techniques currently included under the term laparoscopic inguinal hernia repair. The purpose of this study is to determine whether dividing the hernia sac before ligation improves surgical outcomes following a paediatric laparoscopic inguinal hernia repair compared to ligation alone. METHODS: A systematic review of the literature was performed following PRISMA guidelines of all studies reporting the outcomes following paediatric laparoscopic inguinal hernia repair where the technique was recorded as laparoscopic suture ligation alone (LS) or laparoscopic sac division and suture ligation (LSDS). Studies were assessed for risk of bias and exclusion criteria included reported follow-up of less than 6 months. RESULTS: A total of 8518 LS repairs and 6272 LSDS repairs were included in the final analysis. LSDS repair was associated with a significantly lower recurrence rate (odds ratio 0.51, 95% CI 0.36-0.71, p = 0.001). There was no significant difference in the rates of testicular ascent or atrophy. CONCLUSION: Recreating the open operation by hernia sac division followed by suture ligation significantly reduces the risk of hernia recurrence.

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.

Laparoscopic inguinal herniotomy: Recreating the open operation optimises outcomes.

BACKGROUND: Recent evidence suggests simple laparoscopic inguinal herniorrhaphy is associated with higher rates of recurrence and testicular ascent. We instigated a standardised approach to laparoscopic inguinal herniotomy (LIH), with circumferential sac division and 'purse-string' closure (4/0 monofilament polypropylene). An active follow-up programme was pursued. We reviewed our outcomes of this technique and compared them to an open herniotomy (OIH) cohort. METHODS: LIH patients were identified prospectively (2017-2021): OIH retrospectively from 2016. Risk factors for complications were defined: extremely to very preterm (< 32 weeks), emergency presentation with incarceration, and redo surgery for recurrence. Data are presented as median [IQR]. Comparisons used Fisher's exact and Mann-Whitney U tests: significance defined as p < 0.05. RESULTS: 192 inguinal herniae in 140 patients were included in the LIH group and 214 herniae in 179 patients in the OIH group. Groups were similar in age and gender. The LIH group had a significantly larger proportion of cases that were premature, had emergency surgery, or had redo surgery after previous OIH. Follow-up was 24.4 months [10.8-33.6] vs. 66.4 [64.5-68.5] (LIH vs. OIH). Hernia recurrence occurred in 2/192 (1.0%) vs. 4/214 (1.9%) (LIH vs. OIH), p = 0.69. There was one known case of testicular ascent after OIH but none in the LIH group. CONCLUSIONS: Recreation of the open herniotomy laparoscopically appears to confer excellent outcomes, with low rates of recurrence despite a high proportion of patients having known risk factors. Further long-term data on rates of testicular ascent after active follow-up are required.

Endoscopic vs laparoscopic paediatric gastrostomies: Time to change our practice?

BACKGROUND: Gastrostomy insertion is a common procedure for paediatric surgeons, with the percutaneous endoscopic gastrostomy (PEG) technique long favoured for its simplicity and speed. However, there is growing evidence to suggest that primary laparoscopic balloon gastrostomy (LBG) insertions may have lower complication rates. This study aimed to determine the relative safety and healthcare resource burden of PEG and LBG. METHODS: A retrospective review of all primary gastrostomy insertions (2011-2019). Primary outcome measures included return to theatre for emergency laparotomy and healthcare burden (total gastrostomy-related admissions, length of stay and total theatre utilisation). RESULTS: 338 PEGs and 277 LBGs were inserted with a minimum follow-up period of six months. Following PEG insertion 12/338(3.6%) children required an emergency laparotomy for gastrostomy-related complications. This compared to 2/277(0.7%) following LBG insertion (ARR2.8% (95%CI0.6-5.0), p < 0.0267). When considering all gastrostomy related admissions, there was no significant difference in total theatre utilisation (PEG = 85 [IQR58-117] minutes, LBG = 86 [IQR75-105] minutes, p = 0.12). However, PEGs were found to have an overall longer length of stay 4 [IQR3-7] vs 3 [IQR2-4] days. CONCLUSIONS: LBGs carry a significantly lower rate of major complications and are not associated with an increased healthcare burden. LBG should be considered as the first line method of gastrostomy insertion in children.

Management of suspected thyroglossal duct cysts.

AIM OF STUDY: The aim of this study was to evaluate management of children with an anterior midline neck swelling by establishing 1) whether a preoperative ultrasound scan (USS) was appropriately requested, performed and reported; 2) whether there was preoperative infection; 3) whether a Sistrunk procedure was performed; 4) the rate of thyroglossal duct cyst (TGDC) recurrence following simple excision vs. Sistrunk procedure. METHODS: A single centre retrospective study of children who underwent surgery for anterior midline neck swelling between April 2000 and May 2015 at our institution was performed. These were identified using a clinical coding system, and data were collected from electronic medical records, radiology, and histopathology reports. Recurrence rates between simple excision and Sistrunk groups were compared using Chi-square test. MAIN RESULTS: 227 patients were identified (115 male, 112 female). 169 (74%) had a preoperative USS. The presence of a thyroid gland was stated in 79% of USS reports. This increased to 92% when the requesting surgeon had specifically asked about this. 48 (21%) patients underwent simple excision, while 175 (77%) had a Sistrunk procedure. Recurrence was significantly more likely following simple excision than a Sistrunk procedure (29% vs 6.9%; P<0.0001). Of 25 TGDC recurrences, 9 (36%) had an inconclusive or alternative histopathological diagnosis at first operation. CONCLUSION: Preoperative USS should be performed in all patients with an anterior midline neck swelling. Appropriate requesting increases likelihood of a report confirming (or otherwise) the presence of a thyroid gland. A Sistrunk procedure is the operation of choice in all children presenting with an anterior midline neck swelling. The surgeon cannot reliably differentiate a TGDC from alternative pathology intraoperatively. LEVEL OF EVIDENCE: Treatment study: level IV.

Recombinant factor VIII Fc fusion protein drives regulatory macrophage polarization.

The main complication of replacement therapy with factor in hemophilia A (HemA) is the formation of inhibitors (neutralizing anti-factor VIII [FVIII] antibodies) in ∼30% of severe HemA patients. Because these inhibitors render replacement FVIII treatment essentially ineffective, preventing or eliminating them is of top priority in disease management. The extended half-life recombinant FVIII Fc fusion protein (rFVIIIFc) is an approved therapy for HemA patients. In addition, it has been reported that rFVIIIFc may induce tolerance to FVIII more readily than FVIII alone in HemA patients that have developed inhibitors. Given that the immunoglobulin G1 Fc region has the potential to interact with immune cells expressing Fc receptors (FcRs) and thereby affect the immune response to rFVIII, we investigated how human macrophages, expressing both FcRs and receptors reported to bind FVIII, respond to rFVIIIFc. We show herein that rFVIIIFc, but not rFVIII, uniquely skews macrophages toward an alternatively activated regulatory phenotype. rFVIIIFc initiates signaling events that result in morphological changes, as well as a specific gene expression and metabolic profile that is characteristic of the regulatory type Mox/M2-like macrophages. Further, these changes are dependent on rFVIIIFc-FcR interactions. Our findings elucidate mechanisms of potential immunomodulatory properties of rFVIIIFc.

Computed tomography in the assessment of suspected large bowel obstruction.

BACKGROUND: The aim of this study was to assess the efficacy of computed tomography (CT) scanning in the diagnosis of acute large bowel obstruction. METHODS: Forty-four patients (22 men; 22 women, ages 39-94 years, mean 71 years) with clinical features and abdominal radiographic findings suggesting acute large bowel obstruction (LBO) or pseudo-obstruction were examined with CT. Supine scans were obtained with i.v. contrast medium (unless contraindicated), but (in the majority) without oral contrast. Additional prone and/or decubitus scans were obtained in 33 patients when clarification of a possible transition point on the supine scan was required. CT diagnosis of LBO was made by finding a transition point +/- mass. Final diagnosis was confirmed by surgery, further imaging and/or clinical course. RESULTS: Twenty-two patients had proven mechanical acute LBO of whom 18 had an obstructing carcinoma; 22 patients had no mechanical obstruction. Sensitivity, specificity, Positive Predictive Value, Negative Predictive Value of CT for diagnosis of mechanical LBO were each 91%. Positive and negative likelihood ratios were 10.1 and 0.1, respectively. There were two false-negative CT scans, although one of these was reported as showing segmental mural thickening. A mass was identified on 14 of 17 patients with true-positive CT, subsequently found to have carcinoma. CONCLUSION: Computed tomography with additional selective prone and/or decubitus scanning is highly effective in the diagnosis of mechanical LBO. It is suggested that it replace contrast enema as the initial imaging method.

Evaluation of recombinant factor VIII Fc (Eloctate) activity by thromboelastometry in a multicenter phase 3 clinical trial and correlation with bleeding phenotype.

: The aim of this study was to compare the hemostatic efficacy of recombinant factor VIII Fc (rFVIIIFc) (Eloctate) and Advate by ex-vivo rotation thromboelastometry (ROTEM) of whole blood and to explore potential ROTEM parameters that may be more predictive of a patient's bleeding tendency than plasma FVIII activity. Thirteen clinical sites were selected to perform ROTEM on freshly collected blood samples from 44 patients in the phase 3 study for rFVIIIFc, including 16 patients undergoing sequential pharmacokinetic assessment of Advate and rFVIIIFc. Equivalent hemostatic activity was observed for rFVIIIFc and Advate in postinfusion samples, followed by improvements for rFVIIIFc in clotting time, clot formation time and alpha angle (α) for a longer duration than Advate, consistent with the pharmacokinetic improvements reported previously for rFVIIIFc. Our study did not demonstrate a statistical correlation between a patient's ROTEM activity at baseline or at trough and the occurrence of spontaneous bleeds while on prophylactic therapy. However, an association was observed between postinfusion clotting time and the occurrence of one or more spontaneous bleeds vs. no bleeds over a follow-up period of 1 year (P = 0.003). How well a patient's whole blood clotting deficiency is corrected after a dose of FVIII may be an indicator of subsequent bleeding tendency in patients with otherwise equivalent FVIII peak and trough levels. The technical challenges of standardizing the ROTEM, largely overcome in the current study, may however preclude the use of this method for widespread assessment of global hemostasis unless additional assay controls or normalization procedures prove to be effective.

Monomeric Fc fusions: impact on pharmacokinetic and biological activity of protein therapeutics.

The delivery of therapeutic proteins by noninvasive routes of administration has been a challenging goal, hence current modes of delivery generally require injections. However, we have recently shown that a naturally occurring receptor, the neonatal Fc receptor (FcRn) can be utilized to carry aerosolized therapeutic proteins conjugated to a portion of its respective ligand (Fc domain of immunoglobulin G) across epithelial cells of the lung to effectively deliver biologically active molecules to the bloodstream. First-generation dimeric Fc fusion molecules were successfully transported by the pulmonary route and biologic activity was demonstrated in both non-human primates and human volunteers. Continuing efforts to improve transport efficiency have led to the development of an alternate configuration of Fc fusion proteins with improved characteristics. These second generation Fc fusion molecules are monomeric with respect to the therapeutic protein and dimeric with respect to the Fc region, and have been termed Fc fusion 'monomers'. Several different Fc fusion monomers have demonstrated improved transport efficiency, achieving high bioavailabilities for pulmonary delivery in non-human primates. While the traditional dimeric Fc fusion molecule generally increases the half-life compared with the unconjugated effector molecule, the monomer configuration has been shown to result in an even greater extension of the circulating half-life, which improves pharmacokinetic parameters for protein therapeutics, whether administered by pulmonary delivery or injection. Finally, many of the Fc monomer fusions have enhanced biologic activity compared with the dimeric configuration. Because of these many advantages, the monomer configuration promises to be an enabling advance to achieve clinically relevant, noninvasive delivery with potentially less frequent administration regimens for a broad range of protein therapeutics. In addition, molecules that are comprised of heterodimeric subunits or multi-subunit complexes can also be constructed as Fc fusions that result in a molecule with enhanced pharmacokinetics and greater bioactivity. Several examples of novel Fc fusion proteins, both monomer and heterodimer are described herein.

Extension of in vivo half-life of biologically active molecules by XTEN protein polymers.

XTEN™ is a class of unstructured hydrophilic, biodegradable protein polymers designed to increase the half-lives of therapeutic peptides and proteins. XTEN polymers and XTEN fusion proteins are typically expressed in Escherichia coli and purified by conventional protein chromatography as monodisperse polypeptides of exact length and sequence. Unstructured XTEN polypeptides have hydrodynamic volumes significantly larger than typical globular proteins of similar mass, thus imparting a bulking effect to the therapeutic payloads attached to them. Since their invention, XTEN polypeptides have been utilized to extend the half-lives of a variety of peptide- and protein-based therapeutics. Multiple clinical and preclinical studies and related drug discovery and development efforts are in progress. This review details the most current understanding of physicochemical properties and biological behavior of XTEN and XTENylated molecules. Additionally, the development path and status of several advanced drug discovery and development efforts are highlighted.

Recombinant Factor IX Fc Fusion Protein Maintains Full Procoagulant Properties and Exhibits Prolonged Efficacy in Hemophilia B Mice.

INTRODUCTION: Hemophilia B is an inherited X chromosome-linked disorder characterized by impaired blood clotting owing to the absence of functional coagulation factor IX. Due to the relatively short half-life of factor IX, patients with hemophilia B require frequent factor IX infusions to maintain prophylaxis. We have developed a recombinant factor IX (rFIX) fused to the Fc region of IgG (rFIXFc) with an extended half-life in animals and humans. MATERIALS AND METHODS: Procoagulant properties of rFIXFc and rFIX (BENEFIX®) were compared to determine the effect of the Fc region on rFIXFc hemostatic function. Specifically, we assessed rFIXFc activation, intermolecular interactions within the Xase complex, inactivation by antithrombin III (AT) and thrombin generation potential compared with rFIX. We also assessed the acute and prophylactic efficacy profiles of rFIXFc and rFIX in vivo in hemophilia B mouse bleeding models. RESULTS AND CONCLUSIONS: The activation by factor XIa or factor VIIa/tissue factor, inhibition by AT, interaction profiles with phospholipids, affinities for factor VIIIa within the context of the Xase complex, and thrombin generation profiles were similar for rFIXFc and rFIX. Xase complexes formed with either molecule exhibited similar kinetic profiles for factor Xa generation. In acute efficacy models, mice infused with rFIXFc or rFIX were equally protected from bleeding. However, in prophylactic efficacy models, protection from bleeding was maintained approximately three times longer in rFIXFc-dosed mice than in those given rFIX; this prolonged efficacy correlates with the previously observed half-life extension. We conclude that rFIXFc retains critical FIX procoagulant attributes and that the extension in rFIXFc half-life translates into prolonged efficacy in hemophilia B mice.

Comparative field study: impact of laboratory assay variability on the assessment of recombinant factor IX Fc fusion protein (rFIXFc) activity.

Due to variability in the one-stage clotting assay, the performance of new factor IX (FIX) products should be assessed in this assay. The objective of this field study was to evaluate the accuracy of measuring recombinant FIX Fc fusion protein (rFIXFc) activity in clinical haemostasis laboratories using the one-stage clotting assay. Human haemophilic donor plasma was spiked with rFIXFc or BeneFIX® at 0.80, 0.20, or 0.05 IU/ml based on label potency. Laboratories tested blinded samples using their routine one-stage assay and in-house FIX plasma standard. The mean spike recoveries for BeneFIX (n=30 laboratories) were 121 %, 144 %, and 168 % of expected at nominal 0.80, 0.20, and 0.05 IU/ml concentrations, respectively. Corresponding rFIXFc spike recoveries were 88 %, 107 %, and 132 % of expected, respectively. All BeneFIX concentrations were consistently overestimated by most laboratories. rFIXFc activity was reagent-dependent; ellagic acid and silica gave higher values than kaolin, which underestimated rFIXFc. BeneFIX demonstrated significantly reduced chromogenic assay activity relative to one-stage assay results and nominal activity, while rFIXFc activity was close to nominal activity at three concentrations with better dilution linearity than the typical one-stage assay. In conclusion, laboratory- and reagent-specific assay variabilities were revealed, with progressively higher variability at lower FIX concentrations. Non-parallelism against the FIX plasma standard was observed in all one-stage assays with rFIXFc and BeneFIX, leading to significant overestimation of FIX activity at lower levels and generally high inter-laboratory variability. Compared to the accuracy currently achieved in clinical laboratories when measuring other rFIX products, most laboratories measured rFIXFc activity with acceptable accuracy and reliability using routine one-stage assay methods and commercially available plasma standards.

A single chain variant of factor VIII Fc fusion protein retains normal in vivo efficacy but exhibits altered in vitro activity.

Recombinant factor VIII Fc (rFVIIIFc) is a fusion protein consisting of a single B-domain-deleted (BDD) FVIII linked recombinantly to the Fc domain of human IgG1 to extend half-life. To determine if rFVIIIFc could be further improved by maintaining the heavy and light chains within a contiguous single chain (SC), we evaluated the activity and function of SC rFVIIIFc, an isoform that is not processed at residue R1648. SC rFVIIIFc showed equivalent activity in a chromogenic assay compared to rFVIIIFc, but approximately 40% activity by the one-stage clotting assay in the presence of von Willebrand Factor (VWF), with full activity in the absence of VWF. Moreover, SC rFVIIIFc demonstrated markedly delayed thrombin-mediated release from VWF, but an activity similar to that of rFVIIIFc upon activation in FXa generation assays. Therefore, the apparent reduction in specific activity in the aPTT assay appears to be primarily due to delayed release of FVIII from VWF. To assess whether stability and activity of SC rFVIIIFc were affected in vivo, a tail vein transection model in Hemophilia A mice was utilized. The results demonstrated similar pharmacokinetic profiles and comparable efficacy for SC rFVIIIFc and rFVIIIFc. Thus, while the single chain configuration did not promote enhanced half-life, it reduced the rate of release of FVIII from VWF required for activation. This impaired release may underlie the observed reduction in the one-stage clotting assay, but does not appear to affect the physiological activity of SC rFVIIIFc.