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Erhardseifried - One of the best experts on this subject based on the ideXlab platform.

  • Sustained Expansion and Transgene Expression of Coagulation Factor VIII–Transduced Cord Blood–Derived Endothelial Progenitor Cells
    Arteriosclerosis Thrombosis and Vascular Biology, 2003
    Co-Authors: Christianherder, Torstentonn, Robertoostendorp, Svenbecker, Ulrichkeller, Christianpeschel, Manuelgrez, Erhardseifried
    Abstract:

    Objective— Although hemophilia A seems particularly suitable for gene therapy because even low amounts of plasma Coagulation Factor VIII (FVIII) provide a significant clinical benefit to the patients, the ideal target cell for recombinant FVIII expression and gene therapy approaches remains to be identified. In this study, we tested the capacity of cord blood–derived endothelial progenitor cells (CBECs) for FVIII expression on stable lentiviral transduction. Methods and Results— CD34+ endothelial progenitor cells (EPCs) from cord blood were differentiated into CBECs. Endothelial phenotype was characterized, and lentiviral transduction of early-passage CBECs with a vector encoding FVIII and EGFP did not alter their functional properties and proliferative potential. CBEC could be expanded by 5 to 9 orders of magnitude, thus allowing the expansion of up to 1015 FVIII-secreting CBECs, starting from as little as 106 CD34+ cells. CBECs proved to be highly suitable for FVIII secretion, with 0.35 to 0.39 IU FVIII...

Erhard Seifried - One of the best experts on this subject based on the ideXlab platform.

Christianherder - One of the best experts on this subject based on the ideXlab platform.

  • Sustained Expansion and Transgene Expression of Coagulation Factor VIII–Transduced Cord Blood–Derived Endothelial Progenitor Cells
    Arteriosclerosis Thrombosis and Vascular Biology, 2003
    Co-Authors: Christianherder, Torstentonn, Robertoostendorp, Svenbecker, Ulrichkeller, Christianpeschel, Manuelgrez, Erhardseifried
    Abstract:

    Objective— Although hemophilia A seems particularly suitable for gene therapy because even low amounts of plasma Coagulation Factor VIII (FVIII) provide a significant clinical benefit to the patients, the ideal target cell for recombinant FVIII expression and gene therapy approaches remains to be identified. In this study, we tested the capacity of cord blood–derived endothelial progenitor cells (CBECs) for FVIII expression on stable lentiviral transduction. Methods and Results— CD34+ endothelial progenitor cells (EPCs) from cord blood were differentiated into CBECs. Endothelial phenotype was characterized, and lentiviral transduction of early-passage CBECs with a vector encoding FVIII and EGFP did not alter their functional properties and proliferative potential. CBEC could be expanded by 5 to 9 orders of magnitude, thus allowing the expansion of up to 1015 FVIII-secreting CBECs, starting from as little as 106 CD34+ cells. CBECs proved to be highly suitable for FVIII secretion, with 0.35 to 0.39 IU FVIII...

Christopher B. Doering - One of the best experts on this subject based on the ideXlab platform.

  • The 3.2 Å structure of a bioengineered variant of blood Coagulation Factor VIII indicates two conformations of the C2 domain.
    Journal of thrombosis and haemostasis : JTH, 2019
    Co-Authors: Ian W Smith, Christopher W. Coyle, Gabriela Denning, Ernest T. Parker, Pete Lollar, Christopher B. Doering, Anne E. D’aquino, Andrew Fedanov, Harold Trent Spencer, Paul Clint Spiegel
    Abstract:

    BACKGROUND Coagulation Factor VIII represents one of the oldest protein-based therapeutics, serving as an effective hemophilia A treatment for half a century. Optimal treatment consists of repeated intravenous infusions of blood Coagulation Factor VIII (FVIII) per week for life. Despite overall treatment success, significant limitations remain, including treatment invasiveness, duration, immunogenicity, and cost. These issues have inspired research into the development of bioengineered FVIII products and gene therapies. OBJECTIVES To structurally characterize a bioengineered construct of FVIII, termed ET3i, which is a human/porcine chimeric B domain-deleted heterodimer with improved expression and slower A2 domain dissociation following proteolytic activation by thrombin. METHODS The structure of ET3i was characterized with X-ray crystallography and tandem mass spectrometry-based glycoproteomics. RESULTS Here, we report the 3.2 A crystal structure of ET3i and characterize the distribution of N-linked glycans with LC-MS/MS glycoproteomics. This structure shows remarkable conservation with the human FVIII protein and provides a detailed view of the interface between the A2 domain and the remaining FVIII structure. With two FVIII molecules in the crystal, we observe two conformations of the C2 domain relative to the remaining FVIII structure. The improved model and stereochemistry of ET3i served as a scaffold to generate an improved, refined structure of human FVIII. With the original datasets at 3.7 A and 4.0 A resolution, this new structure resulted in improved refinement statistics. CONCLUSIONS These improved structures yield a more confident model for next-generation engineering efforts to develop FVIII therapeutics with longer half-lives, higher expression levels, and lower immunogenicity.

  • Development and characterization of recombinant ovine Coagulation Factor VIII.
    PloS one, 2012
    Co-Authors: Philip M Zakas, H. Trent Spencer, Bagirath Gangadharan, Graça Almeida-porada, Christopher D. Porada, Christopher B. Doering
    Abstract:

    Animal models of the bleeding disorder, hemophilia A, have been an integral component of the biopharmaceutical development process and have facilitated the development of recombinant Coagulation Factor VIII (fVIII) products capable of restoring median survival of persons with hemophilia A to that of the general population. However, there remain several limitations to recombinant fVIII as a biotherapeutic, including invasiveness of intravenous infusion, short half-life, immunogenicity, and lack of availability to the majority of the world's population. The recently described ovine model of hemophilia A is the largest and most accurate phenocopy. Affected sheep die prematurely due to bleeding-related pathogenesis and display robust adaptive humoral immunity to non-ovine fVIII. Herein, we describe the development and characterization of recombinant ovine fVIII (ofVIII) to support further the utility of the ovine hemophilia A model. Full-length and B-domain deleted (BDD) ofVIII cDNAs were generated and demonstrated to facilitate greater biosynthetic rates than their human fVIII counterparts while both BDD constructs showed greater expression rates than the same-species full-length versions. A top recombinant BDD ofVIII producing baby hamster kidney clone was identified and used to biosynthesize raw material for purification and biochemical characterization. Highly purified recombinant BDD ofVIII preparations possess a specific activity nearly 2-fold higher than recombinant BDD human fVIII and display a differential glycosylation pattern. However, binding to the carrier protein, von Willebrand Factor, which is critical for stability of fVIII in circulation, is indistinguishable. Decay of thrombin-activated ofVIIIa is 2-fold slower than human fVIII indicating greater intrinsic stability. Furthermore, intravenous administration of ofVIII effectively reverses the bleeding phenotype in the murine model of hemophilia A. Recombinant ofVIII should facilitate the maintenance of the ovine hemophilia A herd and their utilization as a relevant large animal model for the research and development of novel nucleic acid and protein-based therapies for hemophilia A.

  • enhanced biosynthesis of Coagulation Factor VIII through diminished engagement of the unfolded protein response
    Journal of Biological Chemistry, 2011
    Co-Authors: Harrison C Brown, Bagirath Gangadharan, Christopher B. Doering
    Abstract:

    Human and porcine Coagulation Factor VIII (fVIII) display a biosynthetic efficiency differential that is being exploited for the development of new protein and gene transfer-based therapies for hemophilia A. The cellular and/or molecular mechanism(s) responsible for this phenomenon have yet to be uncovered, although it has been temporally localized to post-translational biosynthetic steps. The unfolded protein response (UPR) is a cellular adaptation to structurally distinct (e.g. misfolded) or excess protein in the endoplasmic reticulum and is known to be induced by heterologous expression of recombinant human fVIII. Therefore, it is plausible that the biosynthetic differential between human and porcine fVIII results from differential UPR activation. In the current study, UPR induction was examined in the context of ongoing fVIII expression. UPR activation was greater during human fVIII expression when compared with porcine fVIII expression as determined by ER response element (ERSE)-luciferase reporter activity, X-box-binding protein 1 (XBP1) splicing, and immunoglobulin-binding protein (BiP) up-regulation. Immunofluorescence microscopy of fVIII expressing cells revealed that human fVIII was notably absent in the Golgi apparatus, confirming that endoplasmic reticulum to Golgi transport is rate-limiting. In contrast, a significant proportion of porcine fVIII was localized to the Golgi indicating efficient transit through the secretory pathway. Overexpression of BiP, an integral UPR protein, reduced the secretion of human fVIII by 50%, but had no effect on porcine fVIII biosynthesis. In contrast, expression of BiP shRNA increased human fVIII expression levels. The current data support the model of differential engagement of UPR by human and porcine fVIII as a non-traditional mechanism for regulation of gene product biosynthesis.

  • Lentiviral vector platform for production of bioengineered recombinant Coagulation Factor VIII.
    Molecular therapy : the journal of the American Society of Gene Therapy, 2010
    Co-Authors: H. Trent Spencer, Gabriela Denning, Richard E. Gautney, Boro Dropulic, Andre Roy, Lajos Baranyi, Bagirath Gangadharan, Ernest T. Parker, Pete Lollar, Christopher B. Doering
    Abstract:

    Patients with hemophilia A present with spontaneous and sometimes life-threatening bleeding episodes that are treated using blood Coagulation Factor VIII (fVIII) replacement products. Although effective, these products have limited availability worldwide due to supply limitations and product costs, which stem largely from manufacturing complexity. Current mammalian cell culture manufacturing systems yield around 100 µg/l of recombinant fVIII, with a per cell production rate of 0.05 pg/cell/day, representing 10,000-fold lesser production than is achieved for other similar-sized recombinant proteins (e.g. monoclonal antibodies). Expression of human fVIII is rate limited by inefficient transport through the cellular secretory pathway. Recently, we discovered that the orthologous porcine fVIII possesses two distinct sequence elements that enhance secretory transport efficiency. Herein, we describe the development of a bioengineered fVIII product using a novel lentiviral-driven recombinant protein manufacturing platform. The combined implementation of these technologies yielded production cell lines that biosynthesize in excess of 2.5 mg/l of recombinant fVIII at the rate of 9 pg/cell/day, which is the highest level of recombinant fVIII production reported to date, thereby validating the utility of both technologies.

  • identification of porcine Coagulation Factor VIII domains responsible for high level expression via enhanced secretion
    Journal of Biological Chemistry, 2004
    Co-Authors: Christopher B. Doering, Ernest T. Parker, John F Healey, Rachel T Barrow, Pete Lollar
    Abstract:

    Blood Coagulation Factor VIII has a domain structure designated A1-A2-B-ap-A3-C1-C2. Human Factor VIII is present at low concentration in normal plasma and, comparably, is produced at low levels in vitro and in vivo using transgenic expression techniques. Heterologous expression of B domain-deleted porcine Factor VIII in mammalian cell culture is significantly greater than B domain-deleted human or murine Factor VIII. Novel hybrid human/porcine Factor VIII molecules were constructed to identify porcine Factor VIII domains that confer high level expression. Hybrid human/porcine Factor VIII constructs containing the porcine Factor VIII A1 and ap-A3 domains expressed at levels comparable with recombinant porcine Factor VIII. A hybrid construct containing only the porcine A1 domain expressed at intermediate levels between human and porcine Factor VIII, whereas a hybrid construct containing the porcine ap-A3 domain expressed at levels comparable with human Factor VIII. Additionally, hybrid murine/porcine Factor VIII constructs containing the porcine Factor VIII A1 and ap-A3 domain sequences expressed at levels significantly higher than recombinant murine Factor VIII. Therefore, the porcine A1 and ap-A3 domains are necessary and sufficient for the high level expression associated with porcine Factor VIII. Metabolic radiolabeling experiments demonstrated that high level expression was attributable to enhanced secretory efficiency.

Jan Voorberg - One of the best experts on this subject based on the ideXlab platform.

  • Limited promiscuity of HLA-DRB1 presented peptides derived of blood Coagulation Factor VIII.
    PloS one, 2013
    Co-Authors: Simon D. Van Haren, Alexander B. Meijer, Aleksandra Wroblewska, Eszter Herczenik, Paul H. P. Kaijen, Anja Ten Brinke, Aleksandra Ruminska, Jan Voorberg
    Abstract:

    The formation of inhibitory antibodies directed against Coagulation Factor VIII (FVIII) is a severe complication in the treatment of hemophilia A patients. The induction of anti-FVIII antibodies is a CD4+ T cell-dependent process. Activation of FVIII-specific CD4+ T cells is dependent on the presentation of FVIII-derived peptides on MHC class II by antigen-presenting cells. Previously, we have shown that FVIII-pulsed human monocyte-derived dendritic cells can present peptides from several FVIII domains. In this study we show that FVIII peptides are presented on immature as well as mature dendritic cells. In immature dendritic cells half of the FVIII-loaded MHC class II molecules are retained within the cell, whereas in LPS-matured dendritic cells the majority of MHC class II/peptide complexes is present on the plasma membrane. Time-course studies revealed that presentation of FVIII-derived peptides was optimal between 12 and 24 hours after maturation but persisted for at least 96 hours. We also show that macrophages are able to internalize FVIII as efficiently as dendritic cells, however FVIII was presented on MHC class II with a lower efficiency and with different epitopes compared to dendritic cells. In total, 48 FVIII core-peptides were identified using a DCs derived of 8 different donors. Five HLA-promiscuous FVIII peptide regions were found – these were presented by at least 4 out of 8 donors. The remaining 42 peptide core regions in FVIII were presented by DCs derived from a single (30 peptides) or two to three donors (12 peptides). Overall, our findings show that a broad repertoire of FVIII peptides can be presented on HLA-DR.

  • uptake of blood Coagulation Factor VIII by dendritic cells is mediated via its c1 domain
    The Journal of Allergy and Clinical Immunology, 2012
    Co-Authors: Eszter Herczenik, Maartje Van Den Biggelaar, Alexander B. Meijer, Aleksandra Wroblewska, Simon D. Van Haren, Paul H. P. Kaijen, Anja Ten Brinke, Luisa Martinezpomares, Jan Voorberg
    Abstract:

    Background The uptake and processing of blood Coagulation Factor VIII (FVIII) by antigen-presenting cells and the subsequent presentation of FVIII-derived peptides to CD4 + T cells direct the immune response to FVIII in patients with hemophilia A. Multiple receptors including mannose receptor and low-density lipoprotein receptor–related protein-1 (LRP1) have been implicated in FVIII uptake. Objective This work studies the involvement of receptor candidates in FVIII uptake by dendritic cells (DCs). Furthermore, we explore FVIII residues that mediate endocytosis. Methods FVIII uptake was performed with human monocyte–derived and murine bone marrow–derived DCs. To investigate FVIII endocytosis, competition assays with soluble receptor ligands, binding studies with recombinant receptor fragments, and small-interfering RNA–induced gene silencing were performed. In addition, FVIII-targeting monoclonal antibodies KM33 and VK34 were used. To confirm in vitro results, hemophilic E17 knockout mice were pretreated with antibodies prior to FVIII injections and anti-FVIII titers were determined. Results Upon treatment of DCs with mannan or LRP ligand α2-macroglobulin, we observed only a minor decrease in FVIII internalization. In addition, small interfering RNA–mediated knockdown of LRP, mannose receptor, or DC-SIGN expression in monocyte-derived dendritic cells did not prevent FVIII uptake. Binding studies using Fc chimeras revealed that LRP, DC-SIGN, and mannose receptor can bind to FVIII; however, we did not observe a critical role for these receptors in FVIII uptake. Previous studies have shown that human antibodies targeting the C1 (KM33) and A2 (VK34) domains of FVIII interfere with binding to endocytic receptors. Preincubation of FVIII with VK34 did not influence FVIII uptake; however, KM33 completely inhibited FVIII endocytosis by both monocyte-derived dendritic cells and bone marrow-derived dendritic cells. Accordingly, anti-FVIII antibody titers were greatly reduced following the preadministration of KM33 in vivo . Conclusion Together, our observations emphasize the physiological significance of KM33-targeted residues within the C1 domain in the uptake of FVIII by DCs in vitro and in vivo .

  • hla dr presented peptide repertoires derived from human monocyte derived dendritic cells pulsed with blood Coagulation Factor VIII
    Molecular & Cellular Proteomics, 2011
    Co-Authors: Simon D. Van Haren, Jan Voorberg, Eszter Herczenik, Anja Ten Brinke, Koen Mertens, Alexander B. Meijer
    Abstract:

    Activation of T-helper cells is dependent upon the appropriate presentation of antigen-derived peptides on MHC class II molecules expressed on antigen presenting cells. In the current study we explored the repertoire of peptides presented on MHC class II molecules on human monocyte derived dendritic cells (moDCs) from four HLA-typed healthy donors. MHC class II-bound peptides could be routinely recovered from small cultures containing 5 × 106 cells. A fraction of the identified peptides were derived from proteins localized in the plasma membrane, endosomes, and lysosomes, but the majority of peptides that were presented on MHC class II originate from other organelles. Subsequently, we studied the antigen-specific peptide repertoire after endocytosis of a soluble antigen. Blood Coagulation Factor VIII (FVIII) was chosen as the antigen since our current knowledge on MHC class II presented peptides derived from this immunogenic therapeutic protein is limited. Analysis of the total repertoire of MHC class II-associated peptides revealed that per individual sample 20–50 FVIII-derived peptides were presented on FVIII-pulsed moDCs. Repertoires of FVIII-derived peptides eluted from moDCs derived from a panel of four HLA typed donors revealed that some MHC class II-presented FVIII peptides were presented by multiple donors, whereas the presentation of other FVIII peptides was donor-specific. In total 32 different core peptides were presented on FVIII-pulsed moDCs from four HLA-typed donors. Together our findings provide an unbiased approach to identify peptides that are presented by MHC class II on antigen-loaded moDCs from individual donors.

  • Phage Display Technology: A Tool to Explore the Diversity of Inhibitors to Blood Coagulation Factor VIII
    Seminars in Thrombosis and Hemostasis, 2000
    Co-Authors: Jan Voorberg, Edward Norbert Van Den Brink
    Abstract:

    Hemophilia A is a X-linked bleeding disorder that is caused by the functional absence of blood Coagulation Factor VIII. The bleeding tendency in hemophilia A patients can be corrected by the administration of plasma-derived or recombinant Factor VIII concentrates. A serious complication in hemophilia care is the development of Factor VIII neutralizing antibodies (inhibitors) that arise as a consequence of Factor VIII replacement therapy. The majority of Factor VIII inhibitors are directed toward epitopes located within the A2, A3, and C2 domains of Factor VIII. In this article, we summarize current knowledge on the epitope specificity of Factor VIII inhibitors. In addition, we will discuss recent information on the molecular characteristics of human anti-Factor VIII antibodies generated by phage display technology