Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1995-06-06
2001-08-07
Ungar, Susan (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S388100, C530S388200, C530S388240, C530S388800, C435S326000
Reexamination Certificate
active
06271352
ABSTRACT:
TECHNICAL FIELD
This invention relates to monoclonal antibodies, a method of producing such antibodies, hybridoma cells capable of producing the antibodies and uses of the antibodies. Furthermore, the invention relates to the prognostic and diagnostic use of PAI-1 determinations in e.g. plasma samples, and to measurement of uPA:PAI-1 complexes and uses thereof.
BACKGROUND ART
The fusion of mouse myeloma cells with spleen cells from immunized mice (Köhler and Milstein, Nature (1975), 256, 496-497) was the first indication that it is possible to obtain continuous cell lines which produce homogenous (so-called “monoclonal”) antibodies. Since then, a large number of attempts have been made to produce various hybrid cells (so-called “hybridomas”) antibodies. Since then, a large number of attempts have been made to produce various hybrid cells (so-called “hybridomas”) and to employ the antibodies formed by these cells for various scientific investigations (cf. Current Topics in Microbiology and Immunology, volume 81—“Lymphocye Hybridomas”, F. Melchers et. al., Springer-Verlag (1978) and references therein; C. J. Barnstable et al., Cell, (1978), 14, 9-20; P. Parham, W. F. Bodmer, Nature (1978), 276, 397-399; Handbook of Experimental Immunology, 3rd edition, vol. 2, D. M. Wier, editor, Blackwell, 1978, Chapter 25, Chem. Eng. News, 15-17 (1979); Kennett, R. H., McKearn, J. T., and Bechtol, K. B. (1980) Monoclonal Antibodies. Aybridomas: A New Dimension in Biological Analysis (Plenum, N.Y.)). These reports describe the principal techniques for the production of monoclonal antibodies by hybridomas.
Monoclonal antibodies against human plasminogen activators (urokinase-type (u-PA) and tissue-type (t-PA)) and produced by hybridomas have been prepared and have been used for purification, identification, and immunochemical localization of the activators and their proenzymes (Kaltoft, K., Nielsen, L. S. , Zeuthen, J., and Danø, K. (1982) Proc. Natl. Acad. Sci. USA, 79, 3720-3723; Nielsen, L. S., Hansen, J. G., Andreasen, P. A., Skriver, L., Danø, K., and Zeuthen, J. (1983) The EMBO Journal, 2, 115-119; Nielsen, L. S., Hansen, J. G., Skriver, L., Wilson, E. L., Kaltoft, K., Zeuthen, J., and Danø, K. (1982), J. Histochem. Cytochem., 30, 1165-1170). Andreasen, P. A., Nielsen, L. S., Grøndahl-Hansen, J., Skriver, L., Zeuthen, J., Stephens, R. W., and Danø, K. (1984), The EMBO Journal, 3, 51-56). It has recently been shown that inhibitors of plasminogenn activators play an important role in the regulation of the plasmin mediated proteolysis. Such inhibitors have been identified in a variety of tissues, body fluids and cultured cell lines (Holmberg, L, Lecander, I., Persson, B., and {dot over (A)}stedt, B. (1978), Biochim. Biophys. Acta, 544 128-137; Seifert, S. C. and Gelehrter, T. D. (1978) Proc. Natl. Acad. Sci. USA, 75, 6130-6133; Chmielewska, J., R{dot over (a)}nby, M., and Wiman, B. (1983) Thromb. Res., 31, 427-431; Emeis, J. J., Van Hindsbergh, V. W. M., Verheijen, J. H. and Wijngaards, G. (1983) Biochem. Biophys. Res. Commun., 110, 391-398; Golder, J. P. and Stephens, R. W. (1983) Eur. J. Biochem., 136, 517-522; Loskutoff, D. J., van Mourik, J. A., Erickson, L. A., and Lawrence, D. (1983). Proc. Natl. Acad. Sci. USA, 80, 2956-2960; Philips, M., Juul, A. G., and Thorsen, S. (1984)Biochim. Biophys. Acta, 802, 99-110; Vassalli, J. D., Dayer, J. M., Wohlwend, A. and Belin, D. (1984) J. Exp. Med., 159, 1653-1668; Erickson, L. A., Ginsberg, M. H., and Loskutoff, D. J., (1984), J. Clin. Invest., 74, 1465-1472; Cwikel, B. J., Barouski-Miller, P. A., Coleman, P. L., and Gelehrter, T. D. (1984), J. Biol. Chem., 259, 6847-6851; {dot over (A)}stedt, B., Lecanders, I., Brodin, T., Lundblad, A., and Löw, K. (1985), Thrombos.Haemost, 53, 122-125; J. Biol Chem. (1985), 260, 7029-7034).
The mutual relationship of these inhibitors is at present not fully clarified, although recent evidence indicates that at least three immunologically dissimilar types of plasminogen activator inhibitors exist. These include (1) protease nexin, (2) plasminogen activator inhibitor purified from placenta ({dot over (A)}stedt, B., Lecander, I., Brodin, T., Lundblad, A., and Löw, K., (1985) Thromb. Haemost. 53, 122-125), and (3) plasminogen activator inhibitors that inhibit u-PA and t-PA and which typically have been obtained from human endothelial cells, human blood platelets, and rat hepatoma cells (HTC), in the following referred to as endothelial type plasminogen activator inhibitor (e-PAI).
An inhibitor with remarkable similarities to e-PAI has been found in human plasma (Thorsen, S. and Philips, M. (1984) Biochim. Biophys. Acta 802, 111-118).
Monoclonal antibodies against placental plasminogen activator inhibitor have been prepared and such antibodies have been used for the purification of said inhibitor ({dot over (A)}stedt, B., Lecander, I., Brodin, T., Lundblad, A., and Löw, K., (1985) Thromb. Haemost. 53, 122-125).
SUMMARY OF THE INVENTION
The present invention provides monoclonal antibodies against the endothelial type plasminogen activator inhibitor and immunologically similar inhibitors.
The term “immunologically similar inhibitors” denotes plasminogen activator inhibitors which cross-react with polyclonal or monoclonal antibodies raised against inhibitors derived from any of the sources mentioned in connection with the above definition of endothelial type plasminogen activator inhibitor.
The provision of these antibodies makes it possible to study the role of plasminogen activator inhibitors in plasmin mediated proteolysis including fibrinolysis and the mutual relationship of the above mentioned plasminogen activator inhibitors. Moreover, such monoclonal antibodies are useful for the purification of plasminogen activator inhibitor by means of immunoadsorption chromatography, for removal of the inhibitor from body fluids and other biological materials by means of immunoadsorption, for neutralization of the inhibitory activity of the plasminogen activator inhibitor and for the detection, identification and quantification, e.g. by the ELISA technique, of plasminogen activator inhibitor in body fluids, normal or malignant cells and tissues, and other biological materials.
The invention also provides a method of producing the above mentioned antibodies. This method comprises fusing myeloma cells with antibody-producing cells obtained from mammals which have been immunized with endothelial type plasminogen activator inhibitor or immunologically similar inhibitors or with antibody-producing cells which in vitro has been immunized with said plasminogen activator inhibitor, and selecting the hybridomas producing antibodies against the above mentioned inhibitors. Thus the hybridomas are produced by a derivation of the method of Köhler and Milstein mentioned above. The antibody-producing cells used are preferably spleen cells or lymph node cells. The particular species of mammals from which the myeloma and antibody producing cells are derived is not critical insofar as it is possible to fuse the cells of the one species with another, e.g. mouse to rat, rat to human, or mouse to human.
It is preferred, however, to use the same species of animal as a source of both myeloma and anti plasminogen activator inhibitor antibody-producing cells. One preferred cell line for the practice of this invention is a fused cell hybrid between a plasminogen activator inhibitor primed mouse spleen cell and a mouse myeloma cell.
The hybridomas resulting from the fusion are systematically examined for production of antibodies which selectively react with plasminogen activator inhibitor.
It should be noted that monoclonal antibodies raised against a single antigen may be distinct from each other depending on the determinant that induced their formation; but for any given hybridoma (clone), all of the antibodies it produces are monospecific for a particular antigenic determinant in the plasminogen activator inhibitor molecule.
The invention also relates to hybridoma cells capable of producing monoclonal antibodies against the endothelial type plasmin
Andreasen Peter
Brunner Nils
Dano Keld
Nielsen Lars S.
Cooper Iver P.
Fonden til Fremme af Eksperimentel Cancerforskning
Ungar Susan
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