Chemistry: analytical and immunological testing – Involving production or treatment of antibody – Monoclonal antibody
Reexamination Certificate
2000-06-21
2003-11-18
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving production or treatment of antibody
Monoclonal antibody
C435S070200, C435S070210, C436S512000, C436S513000, C436S547000, C530S388100, C530S388150, C530S866000
Reexamination Certificate
active
06649421
ABSTRACT:
The invention relates to antibodies against VASP (vasodilator-stimulated phosphoprotein) which only bind VASP as antigen when VASP is present in phosphorylated form, to hybridoma cells for their preparation, and to the use of the antibodies or antibody fragments as diagnostic agents and/or therapeutic agents.
Diseases of the vascular system are responsible for a large number of chronic and life-threatening diseases, such as cardiac infarction, stroke, arterial occlusion disease and many forms of kidney failure.
The endothelial cells, which, inter alia, control the blood coagulation system, the functional state of the thrombocytes, the migration of inflammatory and tumor cells into the vascular wall, the state of contraction and growth of smooth muscle cells and consequently also blood pressure and vascular wall structure and also neovascularization, are of particular importance for regulating the vascular system. Many of these vascular wall functions are disturbed in serious vascular diseases, a situation which can in the end lead to cardiac infarction, stroke and many forms of kidney failure.
The endothelium forms the important substances prostacyclin (PGI
2
) and nitrogen monoxide (NO), which is also known as endothelium-derived relaxing factor (EDRF), which substances inhibit both the thrombocytes and the vascular muscle cells. The endothelial functions are controlled by said endothelial factors such as prostacyclin (PGI
2
) or EDRF, e.g. NO.
Consequently, in order to treat diseases which are associated with an endothelial dysfunction in a specific manner, it is necessary to develop biochemical parameters which enable an endothelial dysfunction to be diagnosed and its course to be controlled.
It is desirable to be able to recognize endothelial dysfunctions as early as possible, that is at a stage at which irreversible damage, such as atherosclerotic lesions, caused by endothelial dysfunctions has still not manifested itself.
Identification of an endothelial dysfunction at an early stage makes it possible to develop new therapeutic approaches which can bring about reversible treatment of the endothelial dysfunction.
Methods which are known for determining in-vivo endothelial functions are invasive detection methods, such as quantitative angiography, or else non-invasive, image-providing methods. Disadvantages of these methods are that these investigations are carried out directly on the patient, and are difficult to quantify and very expensive.
It is therefore also desirable to have available biochemical/immuno-biological methods which make it possible to determine endothelial functions rapidly and simply in biological material ex vivo, for example in cell samples or blood samples by means of routine investigations in the laboratory. Endothelial functions are those functions which can be regulated by endothelial factors such as prostacyclin (PGI
2
) or EDRF, e.g. NO.
It is an object of the invention to provide reagents for the evaluation and modulation of endothelial function. According to this and other objects of the invention, an antibody is provided which is directed to VASP (Vasodilator-stimulated phosphoprotein) and binds VASP only when VASP is phosphorylated.
In one aspect of the invention an antibody is disclosed which binds VASP only when VASP is phosphorylated at position serine 239 (phosphoserine 239 VASP). In another aspect an antibody is provided which binds VASP only when VASP is phosphorylated at position serine 157 (phosphoserine 157 VASP).
Different embodiments of the invention include polyclonal antibodies, monoclonal antibodies and antibody fragments. Other embodiments include a monoclonal antibody produced by the hybridoma cell line 16C2, and particularly Mab 16C2.
It is a further object of the invention to provide hybridoma cells which are useful in manufacturing the antibodies of the invention. Further to this object of the invention hybridoma cell line is provided which produces a monoclonal antibody against VASP which binds VASP only when it is phosphorylated. In one embodiment, the hybridoma cell line 16C2 (DSM ACC2330) is provided. p It is yet another object of the invention to provide methods of evaluating endothelial function. According to this object of the invention methods are provided for determining the phosphorylation status of VASP. In one aspect of the invention biological material is contacted with an antibody against VASP (vasodilator-stimulated phosphoprotein), which binds VASP as antigen only when VASP is phosphorylated.
In another aspect of the invention a quantitative method is provided which further involves quantifying the amount of VASP antibody which binds the biological material. A specific embodiment includes a Western blotting method, which entails resolving VASP by eletrophoresis and contacting, the VASP with an antibody against VASP, which binds VASP only when it is phosphorylated. Another embodiment is a flow cytometry method, which involves contacting a sample with an antibody against VASP which binds VASP only when it is phosphorylated.
In yet another aspect of the invention, methods of diagnosis are provided. A representative method entails contacting a sample with an antibody against VASP which binds VASP only when it is phosphorylated and evaluating the phosphorylation state of VASP. In one embodiment, this method involves quantitatively determining the phosphorylation of VASP in the sample. In another embodiment, methods are described where the antibody binds phosphoserine 239 VASP or phosphoserine 157 VASP. In still another embodiment, the sample is human thrombocytes or human whole blood.
It is yet another object of the invention to provide methods for detecting markers of endothelial function. In one aspect, a method for detecting substances which affect the level of cGMP and/or cAMP is provided which involves testing a sample from patient who has been exposed to a substance of affecting cGMP and/or cAMP levels, contacting the sample with an antibody against VASP which binds VASP only when it is phosphorylated and evaluating the phosphorylation state of VASP relative to a control sample.
In yet one more aspect, a method is detailed for detecting endothelial dysfunction which involves contacting a sample from a patient with an antibody against VASP which binds VASP only when it is phosphorylated and evaluating the phosphorylation state of VASP relative to a normal control sample.
It is a further object of the invention to provide a convenient kit for accomplishing the diagnostic methods disclosed herein. Further to this object a diagnostic kit is provided which contains an antibody against VASP which binds VASP only when it is phosphorylated.
It is still another object of the invention to provide methods of treating patients suffering from endothetial dysfunction. According to this and other a therapeutic objects of the invention a method of treatment is disclosed where a patient in need of treatment is administered a therapeutically effective amount of an antibody against VASP which binds VASP only when it is phophorylated.
Human VASP (vasodilator-stimulated phosphoprotein), which is phosphorylated in thrombocytes andvascular wall cells in response to hypotensive (vasodilatory) hormones and drugs, has recently been discovered, isolated and characterized from the point of view of molecular genetics (Haffner et al., EMBO J. 14, 19-27, 1995).
VASP is an important component of the ANF/NO/cGMP/cGMP protein kinase signal pathway, which is very important physiologically, pathophysiologically and pharmacologically, and also of the cAMP/cAMP protein kinase signal pathway (U. Walter, Blick 1/97 Würzburg University, pp. 79-81,1997). VASP is expressed in almost all human and animal cells, with particularly high concentrations being found in thrombocytes, vascular smooth muscle cells and fibroblasts. In cultured cells, VASP is associated with focal contacts (cell/matrix contact sites), cell/cell contacts, microfilaments and dynamic membrane regions (e.g. leading edge) (Walter et al., Agents and Actions 45S, 255-268, 1995).
The
Eigenthaler Martin
Hoschuetzky Heinz
Walter Ulrich
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Nguyen Bao-Thuy L.
Vasopharm Biotech GmbH
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