Activated-platelet targeting of CD39 as a potential way forward

2016 ◽  
Vol 36 (01) ◽  
pp. 17-25 ◽  
Author(s):  
J. D. Hohmann ◽  
K. Peter
Keyword(s):  
Recombinant Antibody ◽  
Protein A ◽  
Bleeding Complications ◽  
Nucleoside Triphosphate ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Mortality And Morbidity ◽  
Antiplatelet Effect ◽  
Increased Risk ◽  
Major Interest

SummaryAntiplatelet therapy is given to millions of patients and has saved numerous lives. However, it is also associated with complications including fatal bleedings. Clinically used antiplatelet drugs seem to follow the rule of an inherent link of improved anti-thrombotic potency with increased risk of bleeding complications. Therefore, there is an ongoing quest to develop drugs that are able to break this link that has prevented many patients from receiving antiplatelet protection and has resulted in substantial mortality and morbidity. We describe a new antiplatelet approach that is based on an recombinant antibody protein, a drug format that has recently attracted major interest. Two unique components are genetically combined in this molecule: 1) The ecto-nucleoside triphosphate diphosphohydrolase NTPDase CD39, which enzymatically degrades ATP and ADP to AMP, which is then further degraded to adenosine by the endothelially expressed CD73. Thereby, the platelet activating ADP is reduced and replaced by the platelet inhibiting adenosine resulting in a strong antiplatelet effect. 2) A single-chain antibody (scFv) that specifically binds to the activated GPIIb/IIIa receptor and thus allows targeting to activated platelets. The described fusion protein results in strong enrichment of CD39’s antiplatelet effect, resulting in potent inhibition of platelet adhesion and aggregation and thrombosis in mice. The activated platelet targeting allows using a low systemic concentration that does not interfere with normal haemostasis and thus does not cause bleeding time prolongation in mice. Conclusion: We describe a new antiplatelet approach that promises to deliver strong localized antithrombotic effects without associated bleeding problems.

Construction and Expression of a Bifunctional Single-Chain Antibody against Bacillus cereusSpores

1998 ◽  
Vol 64 (7) ◽  
pp. 2490-2496 ◽  
Author(s):  
Kai Koo ◽  
Peggy M. Foegeding ◽  
Harold E. Swaisgood
Keyword(s):  
Monoclonal Antibody ◽  
Recombinant Antibody ◽  
Expression System ◽  
Variable Region ◽  
Hybridoma Cells ◽  
Antigen Specificity ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Western Blots ◽  
Variable Region Genes

ABSTRACT The variable-region genes of monoclonal antibody againstBacillus cereus spores were cloned from mouse hybridoma cells by reverse transcription-PCR. The heavy- and light-chain variable-region genes were connected by a 45-base linker DNA to allow folding of the fusion protein into a functional tertiary structure. For detection of protein expression, a 10-amino-acid strep tag (biotin-like peptide) was attached to the C terminus of recombinant antibody as the reporter peptide. The single-chain antibody construct was inserted into the expression vector and expressed in Escherichia coliunder the control of the T7 RNA polymerase-T7 promoter expression system. The expressed single-chain antibody was detected on Western blots by using a streptavidin-conjugated enzyme system. This small recombinant antibody fragment (ca. 28,000 Da by calculation) hadB. cereus spore binding ability and antigen specificity similar to those of its parent native monoclonal antibody.

scholarly journals Identification of a Novel Marker for Primordial Smooth Muscle and Its Differential Expression Pattern in Contractile vs Noncontractile Cells

1997 ◽  
Vol 137 (4) ◽  
pp. 925-937 ◽  
Author(s):  
Jill E. Hungerford ◽  
James P. Hoeffler ◽  
Chauncey W. Bowers ◽  
Lisa M. Dahm ◽  
Rocco Falchetto ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vessel Wall ◽  
Recombinant Antibody ◽  
Muscle Cells ◽  
Cytoskeletal Proteins ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Differential Expression Pattern ◽  
Extracellular Matrix Components

The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375–392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, Mr = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, Mr = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle α-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle α-actinin. Our results suggest that the 1E12 antigen is a member of the α-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

scholarly journals Treatment Options in Acute Coronary Syndromes: Focus on Fondaparinux Sodium

2010 ◽  
Vol 2 ◽  
pp. CMT.S2197
Author(s):  
Iris Reindl ◽  
Axel Schlitt
Keyword(s):  
Acute Coronary Syndromes ◽  
Coagulation Factor ◽  
Phase Iii ◽  
Bleeding Complications ◽  
Priority Review ◽  
Pivotal Phase ◽  
Mortality And Morbidity ◽  
Conservative Strategy ◽  
Increased Risk ◽  
Coronary Syndromes

Anticoagulants are the mainstay for treating thromboembolism and acute coronary syndromes (ACS). In comparison to heparin newer agents such as bivalirudin and fondaparinux have improved the outcome of ACS in patients managed by using an invasive or conservative strategy, respectively. Using antithrombotic therapy, however, we need to strike a careful balance between reducing ischemic events and running an increased risk of bleeding complications. Fondaparinux is the first selective inhibitor of the coagulation factor Xa that is commercially available for clinical use. This new drug was accepted for priority review by the Food and Drug Administration based on the positive results of two pivotal, phase III trials (OASIS 5 and 6) that evaluated its role in the treatment of patients with acute coronary syndromes. Fondaparinux was associated with a significantly lower rate of bleeding than enoxaparin in the first 9 days, and at 3 and 6 months, respectively, resulting in a lower long-term mortality and morbidity in comparison to enoxaparin. A small, but definite increase in the risk of catheter-related thrombosis has been found among patients undergoing percutaneous coronary interventions, which is ameliorated by administering unfractionated heparin during the procedure.

scholarly journals Mucosal Immunization with an Unadjuvanted Vaccine That Targets Streptococcus pneumoniae PspA to Human Fcγ Receptor Type I Protects against Pneumococcal Infection through Complement- and Lactoferrin-Mediated Bactericidal Activity

2011 ◽  
Vol 80 (3) ◽  
pp. 1166-1180 ◽  
Author(s):  
Constantine Bitsaktsis ◽  
Bibiana V. Iglesias ◽  
Ying Li ◽  
Jesus Colino ◽  
Clifford M. Snapper ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Bactericidal Activity ◽  
Protein A ◽  
Pneumococcal Infection ◽  
Lavage Fluid ◽  
Complement C3 ◽  
Type I ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Content Type

Targeting an antigen to Fc receptors (FcR) can enhance the immune response to the antigen in the absence of adjuvant. Furthermore, we recently demonstrated that intranasal immunization with an FcγR-targeted antigen enhances protection against a category A intracellular mucosal pathogen,Francisella tularensis. To determine if a similar strategy could be applied to the important pathogenStreptococcus pneumoniae, we used an improved mucosal FcR-targeting strategy that specifically targets human FcγR type I (hFcγRI). A humanized single-chain antibody component in which the variable domain binds to hFcγRI [anti-hFcγRI (H22)] was linked in a fusion protein with the pneumococcal surface protein A (PspA). PspA is known to elicit protection against pneumococcal sepsis, carriage, and pneumonia in mouse models when administered with adjuvants. Anti-hFcγRI-PspA or recombinant PspA (rPspA) alone was used to intranasally immunize wild-type (WT) and hFcγRI transgenic (Tg) mice in the absence of adjuvant. The hFcγRI Tg mice receiving anti-hFcγRI-PspA exhibited elevatedS. pneumoniae-specific IgA, IgG2c, and IgG1 antibodies in serum and bronchoalveolar lavage fluid. Neither immunogen was effective in protecting WT mice in the absence of adjuvant, but when PspA was targeted to hFcγRI as the anti-hFcγRI-PspA fusion, enhanced protection against lethalS. pneumoniaechallenge was observed in the hFcγRI Tg mice compared to mice given nontargeted rPspA alone. Immune sera from the anti-hFcγRI-PspA-immunized Tg mice showed enhanced complement C3 deposition on bacterial surfaces, and protection was dependent upon an active complement system. Immune serum also showed an enhanced bactericidal activity directed againstS. pneumoniaethat appears to be lactoferrin mediated.

scholarly journals Rapid Isolation of a Single-Chain Antibody against the Cyanobacterial Toxin Microcystin-LR by Phage Display and Its Use in the Immunoaffinity Concentration of Microcystins from Water

2002 ◽  
Vol 68 (11) ◽  
pp. 5288-5295 ◽  
Author(s):  
Jacqui McElhiney ◽  
Mathew Drever ◽  
Linda A. Lawton ◽  
Andy J. Porter
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Phage Display ◽  
High Performance ◽  
Recombinant Antibody ◽  
World Health ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Cyanobacterial Toxin

ABSTRACT A naïve (unimmunized) human semisynthetic phage display library was employed to isolate recombinant antibody fragments against the cyanobacterial hepatotoxin microcystin-LR. Selected antibody scFv genes were cloned into a soluble expression vector and expressed in Escherichia coli for characterization against purified microcystin-LR by competition enzyme-linked immunosorbent assay (ELISA). The most sensitive single-chain antibody (scAb) isolated was capable of detecting microcystin-LR at levels below the World Health Organization limit in drinking water (1 μg liter−1) and cross-reacted with three other purified microcystin variants (microcystin-RR, -LW, and -LF) and the related cyanotoxin nodularin. Extracts of the cyanobacterium Microcystis aeruginosa were assayed by ELISA, and quantifications of microcystins in toxic samples showed good correlation with analysis by high-performance liquid chromatography. Immobilized scAb was also used to prepare immunoaffinity columns, which were assessed for the ability to concentrate microcystin-LR from water for subsequent analysis by high-performance liquid chromatography. Anti-microcystin-LR scAb was immobilized on columns via a hexahistidine tag, ensuring maximum exposure of antigen binding sites, and the performance of the columns was evaluated by directly applying 150 ml of distilled water spiked with 4 μg of purified microcystin-LR. The procedure was simple, and a recovery rate of 94% was achieved following elution in 1 ml of 100% methanol. Large-scale, low-cost production of anti-microcystin-LR scAb in E. coli is an exciting prospect for the development of biosensors and on-line monitoring systems for microcystins and will also facilitate a range of immunoaffinity applications for the cleanup and concentration of these toxins from environmental samples.

scholarly journals AI179 single-chain antibody recognizes the Myc tag by Western blotting

2019 ◽  
Vol 2 (2) ◽  
pp. e27
Author(s):  
Zsofia Keszei ◽  
Didier Picard
Keyword(s):  
Western Blotting ◽  
Recombinant Antibody ◽  
Human Cells ◽  
Single Chain ◽  
Single Chain Antibody

The recombinant antibody AI179 against the Myc tag detects a Myc-tagged protein exogenously expressed in human cells by Western blotting.

scholarly journals Optimal Neutralization of Centruroides noxius Venom Is Understood through a Structural Complex between Two Antibody Fragments and the Cn2 Toxin

2015 ◽  
Vol 291 (4) ◽  
pp. 1619-1630 ◽  
Author(s):  
Lidia Riaño-Umbarila ◽  
Luis M. Ledezma-Candanoza ◽  
Hugo Serrano-Posada ◽  
Guillermo Fernández-Taboada ◽  
Timoteo Olamendi-Portugal ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Recombinant Antibody ◽  
Current Trend ◽  
Cross Reactivity ◽  
Antibody Fragments ◽  
Crystallographic Structure ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Neutralizing Capacity ◽  
Centruroides Noxius

The current trend of using recombinant antibody fragments in research to develop novel antidotes against scorpion stings has achieved excellent results. The polyclonal character of commercial antivenoms, obtained through the immunization of animals and which contain several neutralizing antibodies that recognize different epitopes on the toxins, guarantees the neutralization of the venoms. To avoid the use of animals, we aimed to develop an equivalent recombinant antivenom composed of a few neutralizing single chain antibody fragments (scFvs) that bind to two different epitopes on the scorpion toxins. In this study, we obtained scFv RU1 derived from scFv C1. RU1 showed a good capacity to neutralize the Cn2 toxin and whole venom of the scorpion Centruroides noxius. Previously, we had produced scFv LR, obtained from a different parental fragment (scFv 3F). LR also showed a similar neutralizing capacity. The simultaneous administration of both scFvs resulted in improved protection, which was translated as a rapid recovery of previously poisoned animals. The crystallographic structure of the ternary complex scFv LR-Cn2-scFv RU1 allowed us to identify the areas of interaction of both scFvs with the toxin, which correspond to non-overlapping sites. The epitope recognized by scFv RU1 seems to be related to a greater efficiency in the neutralization of the whole venom. In addition, the structural analysis of the complex helped us to explain the cross-reactivity of these scFvs and how they neutralize the venom.

scholarly journals Preclinical Efficacy of a Trivalent Human FcγRI-Targeted Adjuvant-Free Subunit Mucosal Vaccine against Pulmonary Pneumococcal Infection

Vaccines ◽  
2020 ◽  
Vol 8 (2) ◽  
pp. 193
Author(s):  
Sudeep Kumar ◽  
Raju Sunagar ◽  
Edmund J. Gosselin
Keyword(s):  
Protein A ◽  
Pneumococcal Infection ◽  
Surface Protein ◽  
Antigen Presenting Cells ◽  
Mucosal Vaccine ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Mhc Ii ◽  
Gamma Receptor ◽  
Antigen Presenting

Lack of safe and effective mucosal adjuvants has severely hampered the development of mucosal subunit vaccines. In this regard, we have previously shown that immunogenicity of vaccine antigens can be improved by targeting the antigens to the antigen-presenting cells. Specifically, groups of mice immunized intranasally with a fusion protein (Bivalent-FP) containing a fragment of pneumococcal-surface-protein-A (PspA) as antigen and a single-chain bivalent antibody raised against the anti-human Fc-gamma-receptor-I (hFcγRI) elicited protective immunity to pulmonary Streptococcus pneumoniae infection. In order to further enhance the immunogenicity, an additional hFcγRI-binding moiety of the single chain antibody was incorporated. The modified vaccine (Trivalent-FP) induced significantly improved protection against lethal pulmonary S. pneumoniae challenge compared to Bivalent-FP. In addition, the modified vaccine exhibited over 85% protection with only two immunizations. Trivalent-FP also induced S. pneumoniae-specific systemic and mucosal antibodies. Moreover, Trivalent-FP also induced IL-17- and IL-22-producing CD4+ T cells. Furthermore, it was found that the hFcγRI facilitated uptake and presentation of Trivalent-FP. In addition, Trivalent-FP also induced IL-1α, MIP-1α, and TNF-α; modulated recruitment of dendritic cells and macrophages; and induced CD80/86 and MHC-II expression on antigen presenting cells.

scholarly journals Generation of High-Affinity Chicken Single-Chain Fv Antibody Fragments for Measurement of the Pseudonitzschia pungens Toxin Domoic Acid

2006 ◽  
Vol 72 (5) ◽  
pp. 3343-3349 ◽  
Author(s):  
William J. J. Finlay ◽  
Iain Shaw ◽  
Joanna P. Reilly ◽  
Marian Kane
Keyword(s):  
Domoic Acid ◽  
Recombinant Antibody ◽  
Variable Region ◽  
Antibody Fragments ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Single Chain Antibody ◽  
High Affinity ◽  
Single Chain Fv ◽  
Recombinant Antibody Fragments

ABSTRACT Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single VH and VL genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules.

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