Molecular cloning, characterization, genomic organization and promoter analysis of the α1,6-fucosyltransferase gene (fut8) expressed in the rat hybridoma cell line YB2/0

<p>Abstract</p> <p>Background</p> <p>The rat hybridoma cell line YB2/0 appears a good candidate for the large-scale production of low fucose recombinant mAbs due to its lower expression of <it>fut8 </it>gene than other commonly used rodent cell lines. However, important variations of the fucose content of recombinant mAbs are observed in production culture conditions. To improve our knowledge on the YB2/0 fucosylation capacity, we have cloned and characterized the rat <it>fut8 </it>gene.</p> <p>Results</p> <p>The cDNAs encoding the rat α1,6-fucosyltransferase (FucT VIII) were cloned from YB2/0 cells by polymerase chain reaction-based and 5' RNA-Ligase-Mediated RACE methods. The cDNAs contain an open reading frame of 1728 bp encoding a 575 amino acid sequence showing 94% and 88% identity to human and pig orthologs, respectively. The recombinant protein expressed in COS-7 cells exhibits a α1,6-fucosyltransferase activity toward human asialo-agalacto-apotransferrin. The rat <it>fut8 </it>gene is located on chromosome 6 q and spans over 140 kbp. It contains 9 coding exons and four 5'-untranslated exons. FISH analysis shows a heterogeneous copy number of <it>fut8 </it>in YB2/0 nuclei with 2.8 ± 1.4 mean copy number. The YB2/0 <it>fut8 </it>gene is expressed as two main transcripts that differ in the first untranslated exon by the usage of distinct promoters and alternative splicing. Luciferase assays allow defining the minimal promoting regions governing the initiation of the two transcripts, which are differentially expressed in YB2/0 as shown by duplex Taqman QPCR analysis. Bioinformatics analysis of the minimal promoter regions upstream exons E-2 and E-3, governing the transcription of T1 and T2 transcripts, respectively, evidenced several consensus sequences for potential transcriptional repressors. Transient transfections of Rat2 cells with transcription factor expression vectors allowed identifying KLF15 as a putative repressor of T1 transcript in Rat2 cells.</p> <p>Conclusion</p> <p>Altogether, these data contribute to a better knowledge of <it>fut8 </it>expression in YB2/0 that will be useful to better control the fucosylation of recombinant mAbs produced in these cells.</p

The European AntibotABE Framework Program and Its Update: Development of Innovative Botulinum Antibodies

The goal of the AntiBotABE Program was the development of recombinant antibodies that neutralize botulinum neurotoxins (BoNT) A, B and E. These serotypes are lethal and responsible for most human botulinum cases. To improve therapeutic efficacy, the heavy and light chains (HC and LC) of the three BoNT serotypes were targeted to achieve a synergistic effect (oligoclonal antibodies). For antibody isolation, macaques were immunized with the recombinant and non-toxic BoNT/A, B or E, HC or LC, followed by the generation of immune phage-display libraries. Antibodies were selected from these libraries against the holotoxin and further analyzed in in vitro and ex vivo assays. For each library, the best ex vivo neutralizing antibody fragments were germline-humanized and expressed as immunoglobulin G (IgGs). The IgGs were tested in vivo, in a standardized model of protection, and challenged with toxins obtained from collections of Clostridium strains. Protective antibody combinations against BoNT/A and BoNT/B were evidenced and for BoNT/E, the anti-LC antibody alone was found highly protective. The combination of these five antibodies as an oligoclonal antibody cocktail can be clinically and regulatorily developed while their high “humanness” predicts a high tolerance in humans.Peer reviewe

Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody

Botulinum neurotoxins (BoNTs) cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and naturally-occurring botulism cases, well-tolerated antibodies by humans that neutralize BoNTs are relevant. In our previous work, we showed the neutralizing potential of macaque (Macaca fascicularis)-derived scFv-Fc (scFv-Fc ELC18) by in vitro endopeptidase immunoassay and ex vivo mouse phrenic nerve-hemidiaphragm assay by targeting the light chain of the botulinum neurotoxin type E (BoNT/E). In the present study, we germline-humanized scFv-Fc ELC18 into a full IgG hu8ELC18 to increase its immunotolerance by humans. We demonstrated the protection and prophylaxis capacity of hu8ELC18 against BoNT/E in a mouse model. A concentration of 2.5 ng/mouse of hu8ELC18 protected against 5 mouse lethal dose (MLD) in a mouse protection assay and complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18 in mouse paralysis assay. Furthermore, hu8ELC18 protected mice from 5 MLD if injected up to 14 days prior to intraperitoneal BoNT/E administration. This newly-developed humanized IgG is expected to have high tolerance in humans.Peer reviewe

The antimicrobial peptide parabutoporin competes with p47(phox) as a PKC-substrate and inhibits NADPH oxidase in human neutrophils

We investigated parabutoporin (PP), an antimicrobial scorpion peptide, to understand its inhibition on NADPH oxidase in human PMN. We show that PP is a good substrate for all PKC-isotypes, implicated in the activation of NADPH oxidase, and acts as a potent competitive inhibitor of in vitro p47(phox)-phosphorylation by PKC-alpha, -beta I, -beta II and -delta, but not PKC-sigma. In PMN, PP also inhibits the PMA-stimulated phosphorylation of p47(phox) and its subsequent translocation. In contrast, PP affects the PKC-independent activation to a much lesser degree. This indicates that PP inhibits the activation of NADPH oxidase at submicromolar concentrations in a strongly PKC-dependent manner. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.status: publishe

Inhibition of platelet glycoprotein Ib and its antithrombotic potential

The platelet receptor glycoprotein (GP)Ib-IX-V complex plays a dominant role in the first steps of platelet adhesion and arterial thrombus formation. Through its interaction with the multimeric plasma protein von. Willebrand factor (VWF), which is bound to the damaged subendothelial structures, GPIb-IX-V tethers the platelets from the flowing blood thereby slowing them down. This step is a prerequisite for the collagen receptors to participate in firm adhesion resulting in the formation of a first platelet layer which is the basis for further thrombus formation. Recently, other ligands for GPIb-IX-V besides the extensively studied VWF have been identified, such as : a-thrombin, coagulation factor XII (FXII), high molecular weight kininogen (HMWK), factor XI (FXI), integrin Mac-I and P-selectin. In this review, the interaction of GPIb-IX-V with its different ligands is described and the anticipated or demonstrated in vivo effects are discussed

Paratope and epitope mapping of the antithrombotic antibody 6B4 in complex with platelet glycoprotein Ibalpha

The monoclonal antibody 6B4 has a potent antithrombotic effect in nonhuman primates by binding to the flexible loop, also known as the beta-switch region (amino acids 230-242), of glycoprotein Ibalpha (GPIbalpha). This interaction blocks, in high shear stress conditions, the specific interaction between GPIbalpha and von Willebrand factor suppressing platelet deposition to the damaged vessel wall, a key event in the pathogenesis of arterial thrombosis. To understand the interactions between this antibody and its antigen at the amino acid level, we here report the identification of the paratope and epitope in 6B4 and GPIbalpha, respectively, by using computer modeling and site-directed mutagenesis. The docking programs ZDOCK (rigid body docking) and HADDOCK (flexible docking) were used to model the interaction of 6B4 with GPIbalpha and to delineate the respective paratope and epitope. 6B4 and GPIbalpha mutants were constructed and assayed for their capacity to bind GPIbalpha and 6B4, respectively. From these data, it is found that the paratope of 6B4 is mainly formed by five residues: Tyr(27D), Lys(27E), Asp(28), and Glu(93) located in light chain CDR1 and -3, respectively, and Tyr(100C) of the heavy chain CDR3. These residues form a valley, where the GPIbalpha flexible loop can bind via residues Asp(235) and Lys(237). The experimental results were finally used to build a more accurate docking model. Taken together, this information provides guidelines for the design of new derivatized lead compounds with antithrombotic properties.status: publishe

Development of monoclonal antibodies that inhibit platelet adhesion or aggregation as potential anti-thrombotic drugs

Cardiovascular disease is the major cause of mortality in Western countries. Platelets play a crucial role in the development of arterial thrombosis and other pathophysiologies leading to clinical ischemic events. In the damaged vessel wall, platelets adhere to the subendothelium through an interaction with von Willebrand factor (VWF), which forms a bridge between subendothelial collagen and the platelet receptor glycoprotein (GP) Ib/IX/V. This reversible adhesion allows platelets to roll over the damaged area, decreasing their velocity and resulting in strong platelet activation. This leads to the conformational activation of the platelet GPIIb/IIIa receptor, fibrinogen binding and finally to platelet aggregation. As each interaction (collagen-VWF, VWF-GPIb and GPIIb/IIIa-fibrinogen) plays an essential role in primary haemostasis, loss of either of these interactions results in a bleeding diathesis, implying that interfering with these interactions might result in an anti-thrombotic effect. Whereas GPIIb/IIIa antagonists indeed are effective anti-thrombotics, it has been suggested that drugs which block the initial steps of thrombus formation (collagen-VWF or VWF-GPIb interaction) might have advantages over the ones that merely inhibit platelet aggregation. In this review we will discuss and compare the development of monoclonal antibodies (moAbs) that inhibit platelet adhesion or platelet aggregation. The effect of the moAbs in in vitro experiments, in in vivo models and in clinical trials will be described. Benefits, limitations, current applications and the future perspectives in the development of antibodies for each target will be discussed.status: publishe

Development of a high-throughput ELISA assay for platelet function testing using platelet-rich plasma or whole blood

Platelets play an essential role in the development of cardiovascular diseases and are the target of several agents that can inhibit their function. Despite the existence of a wide array of techniques to study platelet function, an assay to evaluate several platelet signalling pathways in a high-throughput fashion, combined with minimal blood volume and handling is still needed. We have developed a sensitive assay in the form of a sandwich ELISA where monoclonal antibodies against P-selectin or alphaIIbbeta3 and GPIbalpha were used to capture and detect platelets, respectively, in the presence of five different agonists [ADP, TRAP (thrombin receptor agonist), U46619 (thromboxane A2 analogue), collagen-related-peptide, and arachidonic acid]. Binding of platelets to the antibodies increased dose-dependently with the concentration of either agonist, while binding of ADP-activated platelets was abrogated when inhibitors of platelet activation were concomitantly added. The test showed good sample reproducibility in 15 healthy donors with conserved platelet response to agonists throughout the assay. Healthy subjects could be identified as normal-, hypo- or hyper-responders for each agonist, which for most cases (73%) was confirmed upon retesting. Finally, we demonstrated that the platelet ELISA assay can not only be used in platelet-rich plasma but also in whole blood; it now awaits large scale studies to assess its full screening and diagnostic values.status: publishe