Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-10-22
2002-06-18
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S005000, C435S006120, C435S007800, C435S007230, C435S007240, C435S007400, C435S007920, C435S007940, C435S015000, C435S021000, C435S029000, C435S188000, C435S091500, C436S518000, C436S548000, C436S547000, C436S543000, C424S001210, C424S010400, C023S296000
Reexamination Certificate
active
06406869
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for determining the level of kinase activity in a biological sample and, in particular, to a method of identifying drugs that inhibit tyrosine kinases, or drugs that inhibit receptors associated with tyrosine kinases.
BACKGROUND OF THE INVENTION
Protein phosphorylation is a common regulatory mechanism used by cells to selectively modify proteins carrying regulatory signals from outside the cell to the nucleus. The proteins that execute these biochemical modifications are a group of enzymes known as protein kinases. They may further be defined by the substrate residue that they target for phosphorylation. One group of protein kinases are the tyrosine kinases (TK's), which selectively phosphorylate a target protein on its tyrosine residues. Some tyrosine kinases are membrane-bound receptors and upon activation by a ligand, can autophosphorylate as well as modify substrates. The initiation of sequential phosphorylation by ligand stimulation is a paradigm that underlies the action of such effectors as, for example, epidermal growth factor (EGF), insulin, platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF). The receptors for these ligands are tyrosine kinases and provide the interface between the binding of a ligand (hormone, growth factor) to the activation of one or more biochemical pathways. Ligand binding to a receptor tyrosine kinase activates its intrinsic enzymatic activity. Tyrosine kinases can also be cytoplasmic, non-receptor type enzymes and act as a downstream component of a signal transduction pathway.
The profound cellular effects mediated by tyrosine kinases, for example, their putative role in angiogenesis [Giroux, S. et al. Curr. Biol. 9: 369 (1999)] and lymphoid development [Nosaka, T., et al. Science 270: 800, (1995)] coupled with the implication that mutant or defective tyrosine kinase variants may be involved in tumorigenesis [Jeffers, M., et al. PNAS 94: 11445, (1997)], have made them attractive targets for the development of new therapeutic molecules.
Traditional methods for measuring the phosphorylation state of cellular proteins have relied on
32
P-orthophosphate incorporation, for example, by exposing cells cultured in the presence of
32
P-orthophosphate to the appropriate ligand or activator. Alternatively, phosphorylated tyrosine residues can be detected in immunoassays, for example, immunoprecipitation or blotting using a radiolabeled antiphosphotyrosine antibody. The fact that the techniques for detecting radioactive isotope (i.e. blotting, immunoprecipitation, gel electrophoresis) are very time consuming, however, minimizes the appeal of these methodologies for high throughput screening.
More recent methods utilize a standard enzyme-linked immunosorbent assay (ELISA) for measuring kinase activity. These methods utilize purified heterologous substrate protein or synthetic substrate peptides anchored to a microtiter plate. After exposure of the substrate molecule to a sample containing the appropriate kinase, the level of phosphorylation is evaluated with antiphosphotyrosine antibodies to quantitate the amount of phosphorylated protein bound to the plate. The obvious limitation of this type of assay is that the activity of a kinase specific for the particular substrate used, is the only activity detected. Additionally, methods such as protein tyrosine kinase enzyme assays are unable to eliminate as potential drug candidates, inhibitors which are not cell permeable and, therefore, are not good choices for therapeutic agents.
Hirth et al., U.S. Pat. No. 5,763,198, for example, describes an ELISA-type assay in which a substrate-specific antibody is used as an anchoring molecule to isolate a protein substrate from a cell lysate preparation and immobilize it on a solid phase support. Hirth's method then determines the level of kinase activity by evaluating the tyrosine phosphorylation state of the protein substrate bound to the solid phase using an anti-phosphotyrosine antibody as the detecting molecule. Other methods for measuring tyrosine kinase activity, particularly tyrosine kinase receptor activity, are described in WO95/04136, EP 0 730 740 B1, and U.S. Pat. No. 5,599,681.
The availability of an efficient, high throughput assay of kinase activity, particularly, one that is cell-based, is highly desirable because it provides the means, not only to characterize the phosphorylation status of a cell, but to identify agonists and antagonists useful as therapeutic agents for the treatment of a wide variety of human diseases.
SUMMARY OF THE INVENTION
Because the method of the present invention uses a capture agent capable of capturing any molecule containing a phosphorylated tyrosine, it can bind any tyrosine kinase substrate which has been phosphorylated. The method is, therefore, not limited to the measurement of a single tyrosine kinase. The method can be used with any kinase molecule without the necessity of having to use different capture agents for different kinases. Furthermore, the method of the present invention has the advantage of detecting cellular signaling events downstream of the receptor, thereby providing a means for detecting in a single test a drug's ability to inhibit more than one therapeutic target.
In one aspect, the invention relates to a method for measuring tyrosine kinase activity in a biological sample by contacting the sample with a solid phase coated with a first anti-phosphotyrosine antibody to capture phosphorylated proteins contained in the biological sample, and determining the amount of phosphorylated protein captured using a second labeled anti-phosphotyrosine antibody as the detecting molecule. The biological sample may be a purified homogeneous tyrosine kinase, a cell fraction, such as a cytosol or cell membrane preparation, cell or tissue extracts or bodily fluids, such as serum, plasma, urine or the like.
In another aspect, the invention relates to a method for measuring tyrosine kinase activity in a biological sample which employs a lanthanide ion as the reporter group on the labeled detecting antibody. By virtue of their unique fluorescence properties, use of lanthanide ions such as samarium (Sm), dysprosium (Dy), europium (Eu) and terbium (Tb) as labels make the method of the invention particularly well suited for highthroughput screening.
In yet another aspect, the invention features a method of screening for molecules, including agonist/antagonist small molecules, that modulate tyrosine kinase activity in whole cells, cell fractions, purified tyrosine kinase preparations or biological fluids. Samples are incubated with potential agonists or antagonists, and assayed for tyrosine kinase activity in accordance with the method described herein. Where the biological sample is a preparation of whole cells, the cells are fractionated, and the cytosol or cell membrane fractions are assayed for tyrosine kinase activity in accordance with the method of the present invention. The method can be used to evaluate cell activation through a general mechanism or through specific receptor activation by directly measuring the level of phosphorylated proteins within a biological sample that results from cell activation.
In yet another aspect, the invention relates to a kit for use in measuring the level of tyrosine kinase activity in a biological sample. The kit provides a solid support, for example a microtiter plate or beads, which is coated with an anti-phosphotyrosine antibody which captures phosphorylated proteins contained within the sample. A labeled anti-phosphotyrosine antibody is also provided to detect the amount of phosphorylated protein bound to the solid phase.
REFERENCES:
patent: 4281061 (1981-07-01), Zuk et al.
patent: 5599681 (1997-02-01), Epstein et al.
patent: 5616726 (1997-04-01), Mitsuda et al.
patent: 5759787 (1998-06-01), Strulovici
patent: 5763198 (1998-06-01), Hirth et al.
patent: 5958719 (1999-09-01), Ullrich et al.
patent: 6066462 (2000-05-01), Goueli
patent: 0 730 740 (1998
Damaj Bassam
Glickman J. Fraser
Inglese James
Chin Christopher L.
Cook Lisa V.
Heslin Rothenberg Farley & & Mesiti P.C.
Pharmacopeia Inc.
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