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Host cell protein (HCP) contaminant clearance is a significant concern during downstream process development for biopharmaceuticals. Protein A chromatography as a capture step for monoclonal antibodies and Fc fusion proteins can clear a large proportion of these impurities from cell culture harvest. Nevertheless, remaining levels of this process-related impurity class do present significant constraints on the rapid development of effective and robust polishing steps. Conventionally, an intermediate pH wash is employed between column loading and elution to minimize HCP levels after Protein A chromatography. A significant mechanistic finding presented in this work is that HCP contaminants that persist following Protein A capture predominantly comprise species that associate with the product in preference to direct interaction with the chromatographic resin. This suggests that the development of improved column wash techniques to maximize HCP clearance ought to focus on disrupting protein-HCP interactions rather than Protein A-HCP interactions. A higher wash pH to preserve product--Protein A binding along with the use of additive combinations to disrupt interactions between HCPs and the product are investigated. This strategy was successfully applied to develop a broadly applicable wash condition that has the potential for eliminating the need for product specific optimization of wash conditions. A combination of 1 M urea and 10% isopropanol in the wash buffer were successfully applied as a platform wash condition for Protein A chromatography. Use of this (and other similar) wash conditions are anticipated to aid the rapid development of effective downstream processes for monoclonal antibodies and Fc fusion proteins resulting in their rapid introduction into clinical trials.

International audience; Replacement therapy for patients with hemophilia A using plasma-derived or recombinant factor VIII (FVIII) is complicated by the short half-life of the FVIII products and by the occurrence of neutralizing antibodies in a substantial number of patients. In the recent years, enormous efforts have been invested to develop new generations of coagulation factors with extended half-lives. Presumably, the use of long-lasting FVIII products should reduce the frequency of administration to the patients and drastically improve their quality of life. The question of their immunogenicity remains however unanswered as yet. The present review proposes a summary of the different strategies developed to enhance the half-life of FVIII, including fusion of FVIII to the Fc fragment of the human IgG1 or to human serum albumin, or attachment of polyethylene glycol. Based on the available literature, we hypothesize on the potential benefits or risks associated with each of the latter strategies in terms of immunogenicity of the newly derived hemostatic drugs.

SummaryThe Phase 3 A-LONG and Kids A-LONG studies demonstrated the prolonged half-life of rFVIIIFc compared with rFVIII, and the safety and efficacy of rFVIIIFc in subjects with severe haemophilia A. Eligible subjects from A-LONG and Kids A-LONG continued rFVIIIFc treatment by enrolling in ASPIRE, an ongoing extension study. Based on combined data from the primary studies and ASPIRE interim data, the safety and efficacy of rFVIIIFc in subjects requiring surgery were evaluated. Perioperative dosing regimens were determined by investigators with guidance based on pharmacokinetic data and recommendations from a clinical dosing committee. In addition to dosing frequency, factor consumption, blood loss, transfusions, bleeding episodes, and haemostatic response were assessed. Across studies, 21 subjects underwent 23 evaluable major surgeries, including 19 orthopaedic surgeries; 41 subjects underwent 52 minor surgeries, including 30 dental procedures. No major and 10 minor surgeries were performed in paediatric subjects. Of the major (n = 22) and minor (n = 32) surgeries assessed for haemostatic response, all were rated as excellent or good by the investigator/surgeon. During most major surgeries (95.7 %), haemostasis was maintained with one rFVIIIFc infusion. Blood loss in major surgeries was consistent with similar surgeries in subjects without haemophilia. Across studies, rFVIIIFc was well tolerated; no subject developed an inhibitor.

Protein bioconjugates can be synthesized by using chemical reactions, enzymatic reactions or genetic engineering technologies. Naturally occurred protein fusion event is used on purpose in the development of better biopharmaceuticals by applying genetic engineering methodologies. This review will mainly focus on the types of fusion proteins produced with the use of recombinant DNA technology, by combining genes or parts of genes from the same or different organisms, in order to be used in pharmaceutical applications for several purposes. Main concerns for the development of better biopharmaceuticals include quality, efficacy, safety, immunogenicity and toxicity issues. Extending half-life of the drug to increase patient compliance, targeting the drugs to reduce toxicity, improving the manufacturing environment to reduce the costs and revealing protein interaction technologies to find novel and superior drugs are the main aims of fusion protein production. Here, related tags and examples of fusion methods for different purposes will be explained precisely.

Introduction Llama single domain antibody fragments (VHH), which can pass endothelial barriers, are being investigated for targeting amyloid plaque load in Alzheimer's disease (AD). Contrary to conventional human or murine antibodies consisting of IgG or F(ab′)2 antibody fragments, VHH are able to effectively pass the blood brain barrier (BBB) in vitro . However, in earlier in vivo studies, anti-amyloid VHH showed poor BBB passage due to their short serum half-lives. It would be of interest to develop a VHH based protein with elongated serum half-life to enhance BBB passage, allowing the VHH to more easily reach the cerebral amyloid deposits. Methods To increase serum persistence, the Fc portion of the human IgG1 antibody (hinge plus CH2 and CH3 domains) was fused to the C-terminus of the VHH (VHH-pa2H-Fc). To determine the pharmacokinetics and biodistribution profile of the fusion protein, the chelator p-SCN-Bz-DTPA was linked to the protein and thereafter labeled with radioactive indium-111 ( 111 In). Double transgenic APPswe/PS1dE9 and wild type littermates were injected with 20μg VHH-pa2H-Fc-DTPA- 111 In (10-20MBq). Pharmacokinetics of the tracer was determined in blood samples at 10 intervals after injection and imaging using microSPECT was performed. The biodistribution of the radioactivity in various excised tissues was measured at 48h after injection. Results We succeeded in the expression of the fusion protein VHH-pa2H-Fc in HEK293T cells with a yield of 50mg/L growth medium. The fusion protein showed homodimerization – necessary for successful Fc neonatal receptor recycling. Compared to VHH-pa2H, the Fc tailed protein retained high affinity for amyloid beta on human AD patient brain tissue sections, and significantly improved serum retention of the VHH. However, at 48h after systemic injection of the non-fused VHH-DTPA- 111 In and the VHH-Fc-DTPA- 111 In fusion protein in transgenic mice, the specific brain uptake of VHH-Fc-DTPA- 111 In was not improved compared to non-fused VHH-DTPA- 111 In. Conclusion Using VHH-Fc conjugates increases the blood half-life of the protein. However, purely extending the time window for brain uptake does not increase BBB passage. Nevertheless, VHH-Fc holds promise for therapeutic applications where a sustained systemic circulation of VHH is advantageous.

Hemophilia care has improved dramatically over the past 50 years, evolving from plasma concentrates, to purified plasma proteins, to recombinant clotting factors. These collective developments allowed for home delivery of on-demand and prophylactic treatment, resulting in the reduction of hemophilia morbidity and mortality and improved quality of life. Although efficacious in treating bleeding, conventional factor products' half-lives require frequent venipuncture, which remains a significant burden to patients. Despite the remarkable advances in hemophilia care, no improvements have, until now, been made to the pharmacokinetic properties of factor products. Multiple strategies have more recently been employed to generate novel bioengineered products that, with great hope, represent the next wave of progress in hemophilia care. The use of these products will undoubtedly raise important discussion about choosing conventional factor over new long-acting factor products. Incorporation of these therapies into clinical care is accompanied by unanswered safety questions that will likely be evaluated only in postmarketing surveillance analysis. Further, these products may change current treatment paradigms with unclear cost repercussions and feasibility. This paper will review efraloctocog alfa (FVIII-Fc) and its role in the treatment of hemophilia A.

The field of therapeutic antibodies has been revolutionized over the past decade, led by the development of novel antibody-modification technologies. Besides the huge success achieved by therapeutic monoclonal antibodies, a diversity of antibody derivatives have emerged with hope to outperform their parental antibodies. Here we review the recent development of methodologies to modify immunoglobulin domains and their therapeutic applications. The innovative genetic and chemical approaches enable novel and controllable modifications on immunoglobulin domains, producing homogeneous therapeutics with new functionalities or enhanced therapeutic profiles. Such therapeutics, including antibody-drug conjugates, bispecific antibodies, and antibody/Fc fusion proteins, have demonstrated great prospects in the treatment of cancer, auto-immune diseases, infectious diseases, and many other disorders.

Proteins and peptides have a significant and rapidly expanding role as medicines, due to their high affinity, specificity, low toxicity, and ability to alter protein–protein interactions. There are currently over 220 widely launched therapeutic proteins and peptides, compared to 64 antibody-derived drugs. The recombinant and synthetic engineering strategies have advanced from those used to generate the first approved molecules such as insulin over 30 years ago. The aim of this chapter is to review, with the exception of antibodies, the novel modalities and technologies employed in both recombinant engineering and synthetic chemistry to design novel therapeutic proteins and peptides.

Screening protein molecules for a particular biological activity sometimes requires the purification of hundreds of proteins. Often many constructs, from several 100 to a 1000, are screened before the lead candidate is found. What is needed is a rapid and reliable way to purify these proteins. Here we describe the simultaneous purification of 10 fusion proteins utilizing a novel instrument, the BioOptix 10.

Inhibitor formation is among the most serious complications of hemophilia treatment. With the US FDA licensure of the novel long-lasting recombinant factor VIII (FVIII) Fc fusion protein, Eloctate, which prolongs FVIII half-life, we propose an innovative approach to prevent inhibitor formation. In this paper, we describe a multicenter, Phase II, single-arm, 48-week trial, the INHIBIT trial, to determine if Eloctate, begun before a bleed and continued as once weekly prophylaxis, will reduce inhibitor formation in children with hemophilia A. We hypothesize that avoiding ‘danger,’ that is, immune activation by a bleed at first factor exposure and prolonging FVIII half-life will prevent inhibitors and promote FVIII-specific T-cell tolerance. If successful, this approach will suggest a new paradigm in clinical practice.

The platform technology of fragment crystallizable (Fc) fusion, in which the Fc region of an antibody is genetically linked to an active protein drug, is among the most successful of a new generation of bioengineering strategies. Immunogenicity is a critical safety concern in the development of any protein therapeutic. While the therapeutic goal of generating Fc-fusion proteins has been to extend half-life, there is a critical mass of literature from immunology indicating that appropriate design of the Fc component has the potential to engage the immune system for product-specific outcomes. In the context of Fc-fusion therapeutics, a review of progress in understanding Fc biology suggests the prospect of engineering products that have an extended half-life and are able to modulate the immune system.

SummaryBackground Routine prophylaxis with replacement factor VIII (FVIII) – the standard of care for severe hemophilia A – often requires frequent intravenous infusions (three or four times weekly). An FVIII molecule with an extended half-life could reduce infusion frequency. The A-LONG study established the safety, efficacy and prolonged pharmacokinetics of recombinant FVIII Fc fusion protein (rFVIIIFc) in previously treated adolescents and adults with severe hemophilia A. Objective In this post hoc analysis, we investigated the relationship between subjects' prestudy (FVIII) and on-study (rFVIIIFc) regimens. Methods We analyzed two subgroups of subjects: prior prophylaxis and on-study individualized prophylaxis (n = 80), and prior episodic treatment and on-study weekly prophylaxis (n = 16). Subjects' prestudy dosing regimens and bleeding rates were compared with their final rFVIIIFc regimens and annualized bleeding rates (ABRs) in the last 3 months on-study. Dosing regimen simulations based on population pharmacokinetics models for rFVIII and rFVIIIFc were performed. Results As compared with their prestudy regimen, 79 of 80 (98.8%) subjects on individualized rFVIIIFc prophylaxis decreased their infusion frequency. Overall ABRs were low, with comparable factor consumption. Longer dosing intervals, including 5-day dosing, were associated with higher baseline von Willebrand factor antigen levels. Simulated dosing regimens predicted a greater proportion of subjects with steady-state FVIII activity trough levels of ≥ 1 IU dL−1 (1%) with rFVIIIFc than with equivalent rFVIII regimens. Conclusion These results suggest that patients on rFVIIIFc prophylaxis can reduce their infusion frequency as compared with their prior FVIII regimen while maintaining low bleeding rates, affording more patients trough levels of ≥ 1 IU dL−1 than with rFVIII products requiring more frequent dosing regimens.

Summary In the phase 3 B-LONG [Recombinant Factor IX Fc Fusion Protein (rFIXFc) in Subjects with Haemophilia B] study, rFIXFc dosed every 1–2 weeks was safe and efficacious in previously treated subjects with haemophilia B. To date, there are no evaluations of transitioning from conventional to long-acting factor IX (FIX) prophylaxis. This post-hoc analysis of B-LONG subjects compared prophylaxis with other FIX products and rFIXFc. Pre- and on-study data were analysed to assess dosing regimen, weekly FIX consumption and annualized bleeding rates (ABRs). Population pharmacokinetics models were used to generate FIX activity profiles with rFIXFc and recombinant FIX prophylaxis. Thirty-nine subjects, previously treated prophylactically, were evaluated. Prior to study, most subjects (69·2%) received twice-weekly FIX infusions; on study, subjects infused rFIXFc once every 1–2 weeks with c. 30–50% reductions in weekly consumption. On-study estimated mean ABRs were lower than pre-study estimated mean ABRs. Models predicted that rFIXFc administered 50 iu/kg weekly and 100 iu/kg every 10 d would maintain steady-state FIX trough levels ≥1 iu/dl in 95·4% and 89·2% of subjects, respectively. These results indicate that patients receiving rFIXFc prophylaxis can markedly reduce infusion frequency and FIX consumption, have a greater likelihood of maintaining FIX activity >1 iu/dl and experience fewer bleeding episodes compared with prior FIX prophylaxis.

Protein A chromatography is commonly used for the affinity capture of antibodies directly from harvested cell culture fluid (HCCF). The highly specific interaction between the protein A ligand and the Fc portion of antibodies and Fc fusion molecules enables significant purification factors to be

This protocol describes protein production in mammalian cells by transient transfection. It assumes the expression construct contains either a 6-HIS or Fc fusion tag to allow recovery of the protein by affinity chromatography. The method is one of the simplest available for protein expression in eukaryotic cells, requires little specialized equipment, and has a reasonably high rate of success.

Therapeutic Fc fusion proteins , Therapeutic Fc fusion proteins , کتابخانه مرکزی دانشگاه علوم پزشکی تهران

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.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.As compared with recombinant factor IX, rFIXFc exhibited a prolonged terminal half-life (82.1 hours) (P0.001). 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.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.).

SummaryDue 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.

Endostatin is a potent anti-angiogenesis compound with efficacy in treating solid tumors and other diseases. However, its clinical application has been hampered by the susceptibility to proteolytic degradation during cell culture production. Here we describe a simple and effective strategy for stabilizing a CHO cell-derived human endostatin Fc fusion. Mass spectrometry analysis of the prominent clipped species revealed that the cleavage sites are located at the N-terminal zinc binding region, which is known to be critical for the structural stability of the molecule. Accordingly, we tested the effect of zinc supplementation on stabilizing the molecule and found that micromolar concentrations of zinc chloride significantly reduced the level of clipping. The protective effect appeared to be mediated via direct interaction between zinc and endostatin, as zinc protects purified endostatin spiked into conditioned medium. Interestingly, copper which is known to have high affinity to endostatin, also prevents degradation. The method provides a robust process for manufacturing Fc-endostatin.

Allometric scaling has been applied to the pharmacokinetics (PK) of factor VIII (FVIII), but published relationships are based on relatively small subsets of available data. Numerous next-generation forms of FVIII are being developed (e.g., Fc fusion, PEGylated, and liposomal formulations) and traditional PK scaling of these products would not incorporate the wealth of existing knowledge for current FVIII therapy in humans. We conducted a meta-analysis and developed allometric relationships of FVIII from over 100 PK studies collected from literature. Normalized Wajima curves were used to relate mean FVIII profiles between species. An “informed scaling” approach was derived for predicting first-in-human PK parameters and demonstrated with a case study for an Fc fusion FVIII. NCA values for FVIII PK were well described by the allometric equations CL = 6.59 W0.85 and Vss = 65.0 W0.97. A subset of studies characterized by two-compartment modeling showed strong linearity in scaling of total clearance (CL) and central volume, but more variability in distributional CL and peripheral volume. Wajima curves for FVIII superimposed across species and the disposition of Fc fusion FVIII in humans was well predicted by “informed scaling.” This approach might be generally applicable for predicting human PK of next-generational therapeutics. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2380–2394, 2013

Rhipicephalus sanguineus, the common brown dog tick, produces several chemokine-binding proteins which are secreted into the host in its saliva to modulate the host response during feeding. Two of these demonstrate very restricted selectivity profiles. Here, we describe the characterization of the third, which we named Evasin-4. Evasin-4 was difficult to produce recombinantly using its native signal peptide in HEK cells, but expressed very well using the urokinase-type plasminogen activator signal peptide. Using SPR, Evasin-4 was shown to bind most CC chemokines. Investigation of the neutralization properties by inhibition of chemokine-induced chemotaxis showed that binding and neutralization did not correlate in all cases. Two major anomalies were observed: no binding was observed to CCL2 and CCL13, yet Evasin-4 was able to inhibit chemotaxis induced by these chemokines. Conversely, binding to CCL25 was observed, but Evasin-4 did not inhibit CCL25-induced chemotaxis. Size-exclusion chromatography confirmed that Evasin-4 forms a complex with CCL2 and CCL18. In accordance with the standard properties of unmodified small proteins, Evasin-4 was rapidly cleared following in vivo administration. To enhance the in vivo half-life and optimize its potential as a therapeutic agent, Fc fusions of Evasin-4 were created. Both the N- and C-terminal fusions were shown to retain binding activity, with the C-terminal fusion showing a modest reduction in potency.

AIMS: Glycoprotein VI (GPVI) is a key platelet receptor which mediates plaque-induced platelet activation and consecutive atherothrombosis, but GPVI is also involved in platelet-mediated atheroprogression. Therefore, interference in GPVI-mediated platelet activation has the potential to combine short-term and long-term beneficial effects, specificity and safety especially regarding bleeding complications. METHODS AND RESULTS: We investigated the effects of the soluble dimeric GPVI receptor fusion protein, Revacept, an antagonist of collagen-mediated platelet activation, in an animal model of atherosclerosis: twenty week old rabbits, which had been fed on a cholesterol-rich diet for 8 weeks, received Revacept (8 mg/kg) or control twice weekly for 4 weeks. Pharmacokinetics indicated a slight accumulation of the drug in the serum after repeated dosing of Revacept for 3 weeks. A significant improvement of endothelial dysfunction after 0.06 and 0.6 µg/min acetylcholine and a significant decrease of vessel wall thickening were found after Revacept treatment. Accordingly, aortic vessel weight was reduced, and plaque sizes, macrophage and T-cell invasion tended to be reduced in histological evaluations. Bleeding time was determined after tail clipping in mice. Revacept alone or in combination with widely used anti-platelet drugs revealed a high safety margin with no prolongation of bleeding times. CONCLUSION: Repeated doses of Revacept led to a significant improvement of endothelial dysfunction and vascular morphology in atherosclerotic rabbits. Furthermore, no influence of Revacept on bleeding time alone or in combinations with various anti-platelet drugs was found in mice. Thus, the inhibition of collagen-mediated platelet interaction with the atherosclerotic endothelium by Revacept exerts beneficial effects on morphology and vascular function in vivo and seems to have a wide therapeutic window without influencing the bleeding time.

Introduction: The completed Phase 3 A-LONG ([NCT01181128][1]) and Kids A-LONG ([NCT01458106][2]) studies established the safety and efficacy of rFVIIIFc among adults/adolescents and children with severe hemophilia A, respectively. Long-term safety and efficacy of rFVIIIFc are being evaluated in the ongoing ASPIRE extension study ([NCT01454739][3]). For people with hemophilia, frequent bleeding into the same joint (a target joint) may contribute to hemophilic arthropathy (chronic joint disease). Here we report longitudinal data from subjects with target joints at entry into A-LONG and Kids A-LONG throughout ASPIRE. Methods: Subjects with ≥1 target joint (major joint with ≥3 bleeding episodes in a 6-mo period) at entry into the parent study (A-LONG or Kids A-LONG) with available prestudy (pre-parent study) and on-study data were evaluated. There are 4 treatment groups in ASPIRE for subjects ≥12 y: individualized prophylaxis (IP), weekly prophylaxis (WP), modified prophylaxis (MP; for subjects not achieving optimal prophylaxis, as intended by treating physician, with IP or WP), or episodic treatment. Subjects

Introduction: The eradication of inhibitors using immune tolerance induction (ITI) remains the mainstay of therapy in patients with severe hemophilia A who develop inhibitors. The long-acting recombinant factor VIII Fc fusion protein, rFVIIIFc (Eloctate™), which is safe and effective in the prevention and treatment of bleeding events, may promote tolerance to FVIII as shown in preclinical animal models and an inhibitor prone child, as Fc suppresses immunoregulatory Tcells to proteins to which Fc is attached. We therefore previously hypothesized rFVIIIFc would provide effective ITI, specifically shortening and simplifying ITI, and have previously described successful inhibitor eradication in three patients. Long-term follow-up data after successful ITI in patients with severe hemophilia remains limited. In the International Immune Tolerance Induction study, at 1-year follow-up, 6 of 66 subjects who had achieved tolerance demonstrated evidence of relapse at a median of 9.5 months. Of these 6 subjects, 1 had a measurable inhibitor titer and 5 had reduced FVIII recovery. We aim to provide follow-up data on our cohort of patients who had successful inhibitor eradication utilizing rFVIIIFc for ITI. Methods: Immune tolerance induction was initiated in three patients with severe hemophilia A and anti-VIII >5 B.U., in two as initial ITI (Pt. 1, 3), and one as salvage (Pt. 2) after failing to achieve ITI with standard rFVIII due to poor compliance. Follow-up was scheduled every 6-8 weeks, with planned determination of FVIII half-life once the anti-FVIII fell to 6 hours. Once a t½ >6 hours was documented, incremental reduction to rFVIIIFc occurred. Patients continued to be followed by their local HTC as per standard of care. Results: ITI was initiated with rFVIIIFc at a dose of 100-200 IU/kg rFVIIIFc every other day or three times weekly per MD discretion. The time to initial anti-FVIII 6 hours, at weeks 18 and 17, respectively, after initiation of ITI. Patient 3 has improved but is not yet fully tolerized, as evidenced by 57% recovery and a t½ of approximately 7 hours. Anti-VIII inhibitor titers remain negative at 15, 16 and 15 months, from the initiation of ITI in patients 1, 2, and 3 respectively. Patients 1 and 2 have been able to decrease their post ITI prophylaxis dosing regimen to 80 IU/kg and 65 IU/kg three times a week while maintaining a FVIII trough of >1%. No patients were maintained on bypassing prophylaxis during ITI and no patients have experienced hemarthroses or other major bleeding event since the initiation of ITI. Discussion: Immune tolerance induction was successful in three children with inhibitors using rFVIIIFc, including a child previously failing rFVIII ITI. The time to anti-FVIII=0 was 4-12 weeks, significantly shorter than with current rFVIII ITI. At a mean duration of follow up of 15.3 months, all patients achieved an anti-VIII inhibitor titer of 0 B.U. Repeat pharmacokinetics studies will be available at planned subsequent follow-up visit. To date, these data indicate that rFVIIIFc safely and effectively induced immune tolerance to FVIII in three children with inhibitors, and has provided durable and continuing immune tolerance to FVIII. Whether rFVIIIFc ITI will be successful and durable in a larger cohort of children with severe hemophilia A will require prospective studies. A prospective observational study of rFVIIIFc ITI pre- and post-ITI T cell responses in children with hemophilia and inhibitors, the Hemophilia Inhibitor Response to Eloctate (HIRE) Study, has begun recruitment. Disclosures Ragni: SPARK: Research Funding; Shire: Consultancy; Novo Nordisk: Research Funding; Genentech: Research Funding; CSL Behring: Research Funding; Biomarin: Consultancy; Biogen: Consultancy, Research Funding; Baxalta: Research Funding; Alnylam Pharmaceuticals: Consultancy, Research Funding; Tacere Benitec: Consultancy; Vascular Medicine Institute: Research Funding; OPKO: Research Funding. Malec:Vascular Medicine Institute: Research Funding; Biogen: Research Funding; Baxalta: Research Funding; Biogen: Consultancy. Journeycake:CSL: Consultancy; Biogen: Consultancy; Baxalta/Shire: Consultancy.

Introduction: The phase 3 Kids B-LONG study (NCT01440946) demonstrated the safety, efficacy, and pharmacokinetics of prophylactic recombinant factor IX Fc fusion protein (rFIXFc) for the prevention and treatment of bleeding episodes in children with severe hemophilia B. The ongoing rFIXFc extension study B-YOND (NCT01425723) is evaluating long-term safety and efficacy of rFIXFc in children and adults with hemophilia B. The safety and efficacy data for pediatric subjects from the second B-YOND interim data cut (11 Sep 2015) are reported here. Methods: The Kids B-LONG study enrolled males aged Results: At the time of the second B-YOND interim data cut, 27 subjects had completed Kids B-LONG and enrolled in B-YOND ( Conclusion: These data confirm the long-term safety of rFIXFc with maintenance of low ABRs and extended-interval prophylaxis in children with hemophilia B. This research was funded by Biogen and Sobi. Biogen and Sobi reviewed and provided feedback on the abstract. The authors had full editorial control of the abstract and provided their final approval of all content. Disclosures Ducore: Octapharama: Membership on an entity's Board of Directors or advisory committees; Baxalta (Shire): Membership on an entity's Board of Directors or advisory committees; LFB: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Biogen: Membership on an entity's Board of Directors or advisory committees. Fischer:Baxter: Consultancy, Research Funding, Speakers Bureau; NovoNordisk: Consultancy, Research Funding, Speakers Bureau; Octapharma: Speakers Bureau; Biogen: Consultancy; Biotest: Consultancy, Speakers Bureau; Baxalta/Baxter: Consultancy, Research Funding, Speakers Bureau; Wyeth: Research Funding; Pfizer: Consultancy, Research Funding, Speakers Bureau; CSL Behring: Consultancy, Speakers Bureau; Bayer: Consultancy, Research Funding, Speakers Bureau; Freeline: Consultancy. Kulkarni:Kedrion: Membership on an entity's Board of Directors or advisory committees; BPL: Membership on an entity's Board of Directors or advisory committees; Biogen: Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxter: Membership on an entity's Board of Directors or advisory committees, Research Funding. Nolan:Sobi: Research Funding; Biogen: Research Funding. Perry:Biogen: Consultancy, Honoraria; Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees. Yuan:Biogen: Employment, Equity Ownership. Ramirez-Santiago:Biogen: Employment, Equity Ownership. Ferrante:Sobi: Employment. Lethagen:Sobi: Employment.

Background Thrombolytic therapy with recombinant tissue plasminogen activator (rtPA) remains the only approved medication for acute ischemic stroke, but incurs significant bleeding risks. Therefore, approaches to combine lower doses of thrombolytic therapy with other effective drugs aim at improving efficacy and reducing bleeding rates. We examined the safety and therapeutic effects of various dosings of rtPA, either alone or combined with glycoprotein VI-Fc fusion protein (GPVI-Fc, Revacept) on experimental stroke in mice. Methods and results The effect of filament-induced intracerebral thrombus formation and embolization was investigated after a one-hour occlusion of the middle cerebral artery. In accordance with previous studies, treatment with 10 mg/kg rtPA significantly improved functional outcome, cerebral infarct size and edema, but also resulted in markedly increased intracranial bleeding volumes. In contrast, low doses of rtPA (0.1 or 0.35 mg/kg body weight) did not change outcome parameters. However, addition of 1 mg/kg Revacept to 0.35 mg/kg rtPA led to improved reperfusion compared to rtPA alone. Moreover, these combined treatments resulted in improved grip strength, compared to the respective dose of rtPA alone. Infarct-surrounding edema improved after combined treatments, but not after respective single rtPA dosings. Intracranial bleeding volumes were below controls after all low-dose rtPA therapies, given either alone or combined with Revacept. Conclusions In contrast to using the equally effective full dose of rtPA, intracranial bleeding was not increased by low-dose rtPA combined with Revacept. Therefore, addition of Revacept to low-dose rtPA does not incur safety risks, but improves efficacy of treatment.

Among the growing number of therapeutic proteins on the market, there is an emergence of biotherapeutics designed from our comprehension of the physiological mechanisms responsible for their peripheral and tissue pharmacokinetics. Most of them have been optimized to increase their half-life through glycosylation engineering, polyethylene glycol conjugation or Fc fusion. However, our understanding of biological drug behaviors is still its infancy compared to the huge amount of data regarding small molecular weight drugs accumulated over half a century. Unfortunately, therapeutic proteins share few resemblances with these drugs. For instance drug-targeted-mediated disposition, binding to glycoreceptors, lysosomal recycling, large hydrodynamic volume and electrostatic charge are typical critical characteristics that cannot be derived from our anterior knowledge of classical drugs. However, the numerous discoveries made in the two last decades have driven and will continue to drive new options in biochemical engineering and support the design of complex delivery systems. Most of these new developments will be supported by novel analytical methods for assessing in vitro or in vivo metabolism parameters.

P. pastoris has previously been genetically engineered to generate strains that are capable of producing mammalian-like glycoforms. Our objective was to investigate the correlation between sialic acid content and pharmacokinetic properties of recombinant TNFR2:Fc fusion proteins generated in glycoengineered P. pastoris strains.TNFR2:Fc fusion proteins were generated with varying degrees of sialic acid content. The pharmacokinetic properties of these proteins were assessed by intravenous and subcutaneous routes of administration in rats. The binding of these variants to FcRn were also evaluated for possible correlations between in vitro binding and in vivo PK.The pharmacokinetic profiles of recombinant TNFR2:Fc produced in P. pastoris demonstrated a direct positive correlation between the extent of glycoprotein sialylation and in vivo pharmacokinetic properties. Furthermore, recombinant TNFR2:Fc produced in glycoengineered Pichia, with a similar sialic acid content to CHO-produced etanercept, demonstrated similar in vivo pharmacokinetic properties to the commercial material. In vitro surface plasmon resonance FcRn binding at pH6.0 showed an inverse relationship between sialic acid content and receptor binding affinity, with the higher affinity binders having poorer in vivo PK profiles.Sialic acid content is a critical attribute for modulating the pharmacokinetics of recombinant TNFR2:Fc produced in glycoengineered P. pastoris.

Current factor IX (FIX) products display a half-life (t1/2) 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 IgG1, 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 t1/2 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.

Hemophilia B, the deficiency or complete absence of coagulation factor IX (FIX), affects an estimated 80,000 people throughout the world. Some of these individuals are managed with prophylaxis, which involves the intravenous infusion of FIX concentrate two to three times weekly to prevent bleeding. Because FIX prophylaxis remains underutilized, patients with hemophilia B are at risk for bleeding that may be severe and potentially life- or limb-threatening, and they may experience arthropathy resulting from recurrent hemarthroses.This review focuses on recent advances in therapeutic protein fusion technology as they apply to FIX deficiency. The National Library of Medicine Medline database was searched for articles containing the term 'Fc fusion proteins'.Genetically engineered recombinant FIX fused to the Fc portion of immunoglobulin significantly extends FIX half-life, thereby decreasing the frequency of prophylactic infusions. This in turn may increase the adoption of, and adherence to, prophylaxis, leading to better outcomes for hemophilia B patients.

Therapeutic antibodies have captured substantial attention due to the relatively high rate at which these products reach marketing approval, and the subsequent commercial success they frequently achieve. In the 2000s, a total of 20 antibodies (18 full-length IgG and 2 Fab) were approved by the Food and Drug Administration (FDA) or European Medicines Agency (EMA). In the 2010s to date, an additional 3 antibodies (denosumab, belimumab, ipilimumab) have been approved and one antibody-drug conjugate (brentuximab vedotin) is undergoing regulatory review and may be approved in the US by August 30, 2011. However, a less heralded group of antibody-based therapeutics comprising proteins or peptides fused with an Fc is following the success of classical antibodies.

The success of Fc-fusion bio-therapeutics has spurred the development of other Fc-fusion products for treating and/or vaccinating against a range of diseases. We describe a method to modulate their function by converting them into well-defined stable polymers. This strategy resulted in cylindrical hexameric structures revealed by tapping mode atomic force microscopy (AFM). Polymeric Fc-fusions were significantly less immunogenic than their dimeric or monomeric counterparts, a result partly owing to their reduced ability to interact with critical Fc-receptors. However, in the absence of the fusion partner, polymeric IgG1-Fc molecules were capable of binding selectively to FcγRs, with significantly increased affinity owing to their increased valency, suggesting that these reagents may prove of immediate utility in the development of well-defined replacements for intravenous immunoglobulin (IVIG) therapy. Overall, these findings establish an effective IgG Fc-fusion based polymeric platform with which the therapeutic and vaccination applications of Fc-fusion immune-complexes can now be explored.