scholarly journals Feasibility study of the Fab fragment of a monoclonal antibody against tissue factor as a diagnostic tool

2015 ◽  
Vol 47 (6) ◽  
pp. 2107-2114 ◽  
Author(s):  
RYO TSUMURA ◽  
RYUTA SATO ◽  
FUMIAKI FURUYA ◽  
YOSHIKATSU KOGA ◽  
YOSHIYUKI YAMAMOTO ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Tissue Factor ◽  
Feasibility Study ◽  
Diagnostic Tool ◽  
Fab Fragment

Complete Inhibition of Endotoxin-Induced Coagulation Activation in Chimpanzees with a Monoclonal Fab Fragment against Factor VII/VIIa

1995 ◽  
Vol 73 (02) ◽  
pp. 223-230 ◽  
Author(s):  
Bart J Biemond ◽  
Marcel Levi ◽  
Hugo ten Cate ◽  
Howard R Soule ◽  
Lori D Morris ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Tissue Factor ◽  
Bolus Injection ◽  
Therapeutic Option ◽  
Factor Vii ◽  
Rapid Decline ◽  
Fab Fragment ◽  
Gram Negative ◽  
Fab Fragments

SummaryGram-negative sepsis is oftentimes complicated by activation of coagulation with disseminated intravascular coagulation and microthrombosis. This may contribute to the associated morbidity, multiple organ failure and death. Recent studies have established that the tissue factor-dependent pathway of blood coagulation has a significant participatory role in the initial endotoxin-induced activation of coagulation. Tissue factor (TF), expressed on the surface of activated monocytes and endothelial cells forms cell surface complexes with free circulating factors VII and VIIa. The latter complex proteolytically activates factors X and IX. Recent in vivo experiments have shown that a rapidly neutralizing TF monoclonal antibody prevents and arrests the endotoxin- induced activation of coagulation and similar studies have shown to reduce mortality in baboons. In this study we describe the preparation of a factor VII/VIIa neutralizing monoclonal Fab fragment and characterize its effect on in vivo activation of coagulation during experimental endotoxemia in chimpanzees.Four chimpanzees received a bolus intravenous injection of 4 ng/kg endotoxin in combination with Fab fragments of a factor VII/VIIa neutralizing murine monoclonal antibody (12D10) at a dose of either 50 μg/kg (n = 2) or 100 μfig/kg (n = 2). Four control animals received a bolus injection of endotoxin alone. Administration of the 12D10 Fab fragments, immediately preceding the endotoxin bolus injection, effectively blocked the endotoxin-induced activation of coagulation. Plasma levels of products of in vivo activation, namely F1+2, TAT complexes and FpA remained at baseline values. The administration of 12D10 resulted in a rapid decline in factor VII/VIIa antigen levels which remained below 5 ng/ml for 180-240 min, followed by a rapid return to baseline levels. Endotoxin administration resulted in activation of the fibrinolytic system as reflected by a rapid increase in plasma plasmin- α2-antiplasmin complexes. Administration of 12D10 was without effect on the endotoxin-induced fibrinolytic activation.In conclusion, this study confirmed the importance of the TF: VII complex in the initial, endotoxin-induced activation of coagulation which was completely blocked by neutralizing all free and tissue factor-complexed factor VII/VIIa by a specific monoclonal Fab fragment. Activation of fibrinolysis was not influenced. Therefore, neutralization of factor VII/VIIa might be a promising therapeutic option in preventing endotoxin-induced microthrombosis during Gram-negative sepsis.

The Role of Phospholipids and the Factor VII Gla-Domain in the Interaction of Factor VII with Tissue Factor

1992 ◽  
Vol 67 (06) ◽  
pp. 679-685 ◽  
Author(s):  
Peter Wildgoose ◽  
Tony Jørgensen ◽  
Yutaka Komiyama ◽  
Tomohiro Nakagaki ◽  
Anders Pedersen ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Tissue Factor ◽  
Calcium Binding ◽  
Factor Vii ◽  
Dependent Manner ◽  
Amidolytic Activity ◽  
Fab Fragment ◽  
Calcium Dependent ◽  
Gla Domain

SummaryWhether or not the factor VII Gla-domain is involved in the high-affinity interaction of factor VII and tissue factor via calcium-dependent interactions with surrounding phospholipids is unknown. To investigate this, we have purified the factor VII Gla-peptide (FVII-GP) from digested recombinant human factor VII a and assessed its effect on factor VII: tissue factor interactions. FVII-GP inhibited the activation of factor X by factor Vila in the presence of either soluble or cell surface tissue factor halfmaximally at 0.5 μM and 2.7 μM, respectively. However, FVII-GP failed to inhibit the specific binding of factor Vila to cell-surface tissue factor, and did not inhibit the ability of tissue factor to stimulate the amidolytic activity of factor Vila. Unrelipidated tissue factor apoprotein stimulated the amidolytic activity of factor Vila to the same extent as relipidated tissue factor apoprotein. These findings suggest that the factor VII Gla-domain does not directly interact with tissue factor, but rather is important for calcium binding and concomitant expression of other factor VII epitopes necessary for tissue factor recognition and binding. To test this hypothesis, we have prepared a monoclonal antibody against a putative factor VII epitope that participates in the interaction of factor VII with cell-surface tissue factor (peptide 195-206) and assessed its ability to bind to factor VII in the presence and absence of calcium. Binding of this monoclonal antibody (PW-4) to intact factor VII a was calcium-dependent and could be inhibited in a dose-dependent manner by peptide 195-206. The antibody reacted with Gla-domainless factor Vila, but only 37% as compared to intact factor Vila. In addition, PW4 as well as its Fab’ fragment, inhibited factor Vila binding to cell-surface tissue factor. These studies indicate that the factor VII Gla-domain does not provide structural elements that contribute to the formation of a stable factor VII/VII a-tissue factor binary complex. The factor VII Gla-domain appears to be necessary, however, in binding calcium ions and inducing a calcium-dependent conformational change in factor VII/VII a that expresses one or more neoepitopes that participates in the interaction of factor VII/VII a with the extracellular domain of tissue factor apoprotein.

Preparation of an antifibrin thrombus-specific murine/human chimeric monoclonal antibody fab fragment in escherichia coli

1996 ◽  
Vol 81 (4) ◽  
pp. 477-484 ◽  
Author(s):  
Lijun Xia ◽  
Jianming Gu ◽  
Xiaomin Zhang ◽  
Yue Liu ◽  
Haiying Wan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibody ◽  
Fab Fragment ◽  
Chimeric Monoclonal Antibody

scholarly journals Characterization of a Lipopolysaccharide-Targeted Monoclonal Antibody and Its Variable Fragments as Candidates for Prophylaxis against the Obligate Intracellular Bacterial Pathogen Coxiella burnetii

2014 ◽  
Vol 82 (11) ◽  
pp. 4530-4541 ◽  
Author(s):  
Ying Peng ◽  
Laura Schoenlaub ◽  
Alexandra Elliott ◽  
William J. Mitchell ◽  
Guoquan Zhang
Keyword(s):  
Monoclonal Antibody ◽  
Coxiella Burnetii ◽  
Natural Infection ◽  
Bacterial Pathogen ◽  
Passive Transfer ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Fab Fragment ◽  
Content Type ◽  
Aerosol Infection

ABSTRACTOur previous study demonstrated that treatment ofCoxiella burnetiiwith the phase I lipopolysaccharide (PI-LPS)-targeted monoclonal antibody (MAb) 1E4 significantly inhibitedC. burnetiiinfection in mice, suggesting that 1E4 is a protective MAb. To determine whether passive transfer of antibodies (Abs) can provide protection againstC. burnetiinatural infection, we examined if passive transfer of 1E4 would protect SCID mice againstC. burnetiiaerosol infection. The results indicated that 1E4 conferred significant protection against aerosolizedC. burnetii, suggesting that 1E4 may be useful for preventingC. burnetiinatural infection. To further understand the mechanisms of 1E4-mediated protection and to test the possibility of using humanized 1E4 to preventC. burnetiiinfection, we examined whether the Fab fragment of 1E4 (Fab1E4), a recombinant murine single-chain variable fragment (muscFv1E4), and a humanized single-chain variable fragment (huscFv1E4) retained the ability of 1E4 to inhibitC. burnetiiinfection. The results indicated that Fab1E4, muscFv1E4, and huscFv1E4 were able to inhibitC. burnetiiinfection in mice but that their ability to inhibitC. burnetiiinfection was lower than that of 1E4. In addition, treatment ofC. burnetiiwith Fab1E4, muscFv1E4, or huscFv1E4 can blockC. burnetiiinfection of macrophages. Interestingly, treatment ofC. burnetiiwith huscFv1E4 can significantly reduceC. burnetiiinfectivity in human macrophages. This report provides the first evidence to demonstrate that the humanized variable fragments of an LPS-specific MAb can neutralizeC. burnetiiinfection and appears to be a promising step toward the potential use of a humanized MAb as emergency prophylaxis againstC. burnetiiexposure.

scholarly journals Phenomenon of human T cells rosetting with sheep erythrocytes analyzed with monoclonal antibodies. "Modulation" of a partially hidden epitope determining the conditions of interaction between T cells and erythrocytes

1982 ◽  
Vol 155 (5) ◽  
pp. 1317-1333 ◽  
Author(s):  
A Bernard ◽  
C Gelin ◽  
B Raynal ◽  
D Pham ◽  
C Gosse ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
T Cells ◽  
T Cell ◽  
Peripheral Blood ◽  
Large Fraction ◽  
Rosette Formation ◽  
Fab Fragment ◽  
Activated T Cells ◽  
Neuraminidase Treatment ◽  
Mouse Immunoglobulin

Anti-D66 is a monoclonal antibody able to inhibit E-rosette formation of T cells both at 4 degrees C and at 37 degree C but that does not inhibit T cell rosette formation with neuraminidase or 2-amino-ethylisothiouronium bromide (AET)-pretreated E. As demonstrated by capping experiments, it defines an epitope, D66, that is directly involved in E-rosette formation. D66 is distinct from the epitope defined by 9.6 because 9.6, a previously defined "pan-T" monoclonal antibody, inhibits E(AET) rosette formation and because no cross-blocking occurred between both antibodies fixation. However, 9.6 and D66 are carried by the same molecule, as demonstrated by sequential immunoprecipitation assays performed on two different T cell lines. On the thymocyte surface, also, 9.6 and D66 are most probably carried by the same molecule, as indicated by cocapping and colysostripping experiments. D66 is present at higher densities on thymocytes and activated T cells than on peripheral blood T cells. Investigation of numerous T cell populations, both normal and malignant, showed a straightforward correlation between elevated D66 density and ability to form 37 degrees C stable E-rosettes. Neuraminidase treatment of thymocytes and peripheral blood lymphocytes forming E-rosettes unmasked a large fraction of D66 not readily accessible on their surface. These hidden D66 epitopes appear to be responsible for a surprising observation: the ability of anti-D66 to inhibit E-rosette formation could be totally reversed by fixation on anti-D66 of an antibody to mouse immunoglobulin or an Fab fragment anti-mouse immunoglobulin. This would induce microdisplacement with emergence of hidden D66, as documented by fluorometric studies. Finally, malignant T cells with a differentiative status of mature T cells, but forming no (or low numbers of) E-rosettes, could be induced both to display D66 and to form E-rosettes by neuraminidase treatment.

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