Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-01-21
2002-11-12
Nolan, Patrick J. (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C530S387100, C530S388100
Reexamination Certificate
active
06479245
ABSTRACT:
The invention relates to a monoclonal antibody which specifically binds the activated coagulation factor VII (FVIIa), and to its use.
Blood coagulation is a complex system in which proteins are involved in the form of proteases, accelerators and inhibitors. Most of the proteases are present in a non-activated state. When coagulation is triggered, their proforms are converted into the activated state, resulting in the factors being activated in a cascade-like manner and the reaction thereby being amplified. The so-called intrinsic and extrinsic coagulation pathways differ fundamentally. When tissue is injured, the extrinsic cascade is initiated by thromboplastin (TF=tissue factor) becoming exposed on cell surfaces and binding the coagulation factor VII (FVII) or FVIIa. FVII is either activated autocatalytically on TF or by way of proteases such as thrombin or FXa. The TF/VIIa complex activates FX to give FXa, with the subsequent activation of prothrombin in turn taking place on phospholipid surfaces in the presence of calcium. This reaction is accelerated by FVa and leads, by way of the resulting thrombin, to the formation of fibrin and thereby wound closure.
Although the blood coagulation factors are normally present in a non-activated state, small quantities of FVIIa have been detected in the plasma of healthy individuals. This mechanism is possibly used so that it is physiologically possible to react very rapidly to very small tissue injuries when TF becomes exposed. A correlation of circulating, elevated FVIIa levels might play a role in pathophysiological reactions and induce these reactions, that is lead, for example, to an increased risk of thrombosis.
Coagulation tests which, because of the way they have been conceived, also measure traces of FVIIa, that is are unable to differentiate between FVII and FVIIa, are presently available for quantitatively determining FVII. A far more specific test system for determining FVIIa has been introduced in the form of the so-called rTF-FVIIa test. This system operates particularly reliably when no other activated factors, such as FXa or FIIa, or only small quantities of these factors are present. However, when higher concentrations of activated factors are present, the system can falsely indicate that FVIIa levels are elevated.
Apart from quantitatively determining factor VIIa in body fluids, particularly in plasma, it is also of great interest to determine FVII- and/or FVIIa-containing coagulation products. For example, so-called prothrombin complex concentrates (PPSB) are administered to patients who are suffering from deficiencies in the corresponding factors (FII/FVII/FIX/FX, etc.). Although it has not been possible to demonstrate that the presence of traces of FVIIa increases the risk of thromboembolic complications, efforts are made to ensure that the content of FVIIa in non-activated PPSB concentrates is as low as possible. The analysis in this regard is consequently of considerable interest. In addition, complex concentrates which are already activated are employed for certain indications, with it being necessary to quantify the activated factors carefully in this case as well.
Apart from the rTF-FVIIa assay, which measures the activity of the FVIIa, it is also desirable to have a system for detecting FVIIa antigen. The invention was consequently based on the object of providing a method for detecting FVIIa on an antigen basis.
This object is achieved by a monoclonal antibody which binds the activated factor VII specifically.
In order to prepare this antibody, mice were immunized with recombinant, activated factor VII. The mouse spleen cells were then fused with the murine myeloma cell line Sp2/0-Ag14. Polyethylene glycol 4000 was used as the fusion reagent. The cells were distributed on 24-well culture plates. The medium employed was Dulbecco's mod. Eagle's medium containing 10% fetal calf serum, and HAT medium was employed for the selection. After about 2 weeks, the growing cell lines were transferred to the wells of a 48-well plate and coded. The culture supernatant was then taken from approx. 2400 cell lines which had been grown and tested by ELISA for the presence of mouse IgG.
392 mouse IgG-positive cell lines were tested for specificity using immobilized factor VII and activated factor VII (ELISA). Of the tested cell lines, 1 cell line, having the code number 1069/1373, was identified as being specific for the activated factor VII. This cell line has been deposited in the German Sammlung für Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, on Nov. 11, 1997 [Collection of Microorganisms and Cell Cultures] under No. DSM ACC 2332. The specificity of the antibody formed by this cell line was confirmed in the so-called BIAcore system. The purified monoclonal antibody is of the IgG 1 type.
The novel monoclonal antibody was further characterized by testing its ability to inhibit activated factor VII in a coagulation test. In this connection, it was found that the activity of the activated factor VII was inhibited by incubation with monoclonal antibody (Mab) 1069/1373 in a concentration-dependent manner. SDS-PAGE carried out on factor VII and activated factor VII, with subsequent transfer to nitrocellulose and incubation with Mab 106911373, confirmed that it was only activated factor VII, and not factor VII itself, which was bound and which led to corresponding labeling of the band when POD-coupled goat anti-mouse antibody and an appropriate substrate were added.
An additional feature of Mab 106911373 is that it is, in particular, free, activated factor VII which is recognized; i.e. there is no binding of activated factor VlI which is complexed, for example, with antithrombin III (ATIII). The following experiment clarifies this property:
Complexes such as these are prepared in-vitro by incubating activated factor VII with an excess of antithrombin III/heparin at 4° C. for several hours. Depending on the extent to which the formation of the complex between activated factor VII and antithrombin III is complete, the activated factor VII is either markedly reduced or not detectable at all in the corresponding activity test. In this experiment, the activity of the activated factor VII was observed to decrease by more than 90% as compared with a control. A signal which was altered in a corresponding manner was found in the antigen detection system. This makes it clear that it is only free activated factor VII, and not the protease inhibitor complex, which is recognized. Mab 1069/1373 is outstandingly suitable for qualitatively and quantitatively detecting activated factor VII in solutions, such as body fluids, or in dissolved coagulation preparations or intermediates which arise during the preparation of blood coagulation factors. Example 1 (see below) describes the setting up of an appropriate ELISA test. In addition to this, the novel monoclonal antibody 1069/1373 is also suitable for detecting the binding of activated factor VII to cell surfaces and tissues. Known methods, such as the direct reaction of the Mab with activated factor VII or an indirect detection using a second (anti-mouse) antibody which is directed against the Mab, can be employed for the detection. Antigen-binding fragments of the novel monoclonal antibody which contain the activated factor VII-binding regions, such as F(ab2) or F(ab), can also be used for this purpose. Because of its inhibitory potential, a corresponding, humanized monoclonal antibody can, apart from the Mab and its fragments, be particularly advantageously used prophylactically and/or therapeutically, in particular for preventing or treating thrombotic events. A humanized monoclonal antibody of this nature comprises the activated factor VII-binding hypervariable regions of the novel monoclonal antibody and the framework regions of the variable and constant regions of the light and heavy chains of a human antibody.
In addition, the Mab can also be used for removing activated factor VII from solutions. For example, an affinity gel on w
Feussner Annette
Lang Wiegand
Röder Joachim
Römisch Jürgen
Aventis Behring GmbH
DeCloux Amy
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
Nolan Patrick J.
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