Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1991-04-24
2001-04-24
Graser, Jennifer (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S173300, C435S242000, C435S810000, C436S513000, C436S548000, C436S169000, C436S813000, C436S164000, C436S169000, C436S170000, C436S518000, C436S805000, C530S350000, C530S806000, C530S388260, C530S388800, C536S023200, C536S023700
Reexamination Certificate
active
06221620
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
The present invention relates, in general, to monoclonal antibodies and, in particular, to monoclonal antibodies that are specific for thymidylate synthase.
2. Background Information
The fluoropyrimidines are an important group of antineoplastic agents that are widely used in the treatment of gastrointestinal tumors, breast tumors, and epithelial tumors of the upper aerodigestive tract (Danenberg, P. V. (1977)
Biochim. Biophys. Acta Acta
473, 73-92; Santi, D. V., McHency, C. S. and Sommer, M. (1974)
Biochemistry
13, 471-480; Moertel, C. G. (1978)
N. Engl. J. Med.
299, 1049-1052). Thymidylate synthase (TS; EC 2.1.1.45) catalyzes the methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine monphosphate (dTMP). The de novo synthesis of dTMP is an essential step in the synthesis of pyrimidine nucleotides and DNA biosynthesis (Santi, D. V. and Danenberg, P. V. (1984) In
Folates and Pterins,
eds. Blakley, R. L. and Benkovic, S. J. (Wiley, New York), pp. 345-398; Heidelberger, C. (1975) In
Handbook of Experimental Pharmacology.
Vol. 38, eds. Sartorelli, A. C. and Johns, D. G. (Springer Verlag, New York), pp. 193-231). TS enzyme inhibition is one of the main biochemical events underlying the antineoplastic action of the fluoropyrimidines 5-fluorouracil (5-FU) and fluorodeoxyuridine (FUdR). Once metabolized to FdUMP, this metabolite forms a tight-binding covalent complex with TS in the presence of 5-10-methylenetetrahydrofolate (CH
2
H
4
PteGlu).
The TS enzyme exists as a dimer of identical subunis, each 36 kD in size, and has been purified and well characterized from various sources. A striking degree of homology exists between the amino acid sequence of human TS and TS from other sources such as bacteria, parasite and yeast (Hardy, L. W., Finer-Moore, J., Montfort, W., Jones, M., Santi, D. V. and Stroud, R. M. (1987)
Science
235, 448-4553). Since TS provides the sole de novo source of thymidylate, which is crucial for DNA synthesis, the maximal cellular TS activity occurs during the S phase of the cell cycle and is 20-fold higher in rapidly proliferating cells than in nondividing cells (Conrad, A. H. and Ruddle, F. H. (1972)
J. Cell. Sci.
10, 471-486; Navalgund, L. G., Rossann, C., Muench, A. J. and Johnson, L. F. (1980)
J. Biol. Chem.
255, 7386-7390; Johnson, L. F. (1984) In
Recombinant DNA and Cell Proliferation,
eds. Stein, G. S. and Stein, J. L. (Academic Press, New York), pp. 25-47). Moreover, the activity of the TS enzyme increases acutely as a cell passes from late-G
1
to early-S phase of the cycle (Jenh, C. H., Rao, L. G. and Johnson, L. F. (1985)
J. Cell. Physiol.
122, 149-154).
The clinical importance of TS has recently been noted by several investigators who have demonstrated in vivo as well as in vitro that TS enzyme levels in neoplastic cells rise rapidly when cells are exposed to 5-fluorouracil (Spears, C. P., Antranik, A. H., Moran, R. G., Heidelberger, C. and Corbett, T. H. (1982)
Cancer Res.
42, 450-456; Washtein, W. L. (1984)
Mol. Pharmacol.
25, 171-177; Chu, E., Zinn, S., Boarman, D. and Allegra, C. J. (1990) Cancer Res. 50, 5834-40; Keyomarsi, K. and Moran, R. G. (1988)
J. Biol. Chem.
263, 14402-14409; Scanlon, K. J. and Kashani-Sabet, M. (1988)
Proc. Natl. Acad. Sci.
USA 85, 650-653; Swain, S. M., Lippman, M. E., Egan, E. F., Drake, J. C., Steinberg, S. M. and Allegra, C. J. (1989)
J. Clin. Oncol.
7, 890-896). Thus, the ability of a tumor to acutely overexpress the TS enzyme may play a key role in the development of tumor resistance and may represent an important protective mechanism in response to this drug. Understanding the biochemical and molecular mechanisms involved in antimetabolite resistance both in tumor cell lines and individual patients is critical in assessing and improving chemotherapeutic approaches.
The quantitation and detection of TS in human tissues has traditionally been performed by enzymatic biochemical assays that either measure catalytic activity or measure the amount of radiolabeled FdUMP binding to TS following extraction of the enzyme from cells and tissues (Lockshin, A. and Danenberg, P. V. (1981)
Biochem. Pharmacol.
30, 247-257; Lockshin, A. and Danenberg, P. V. (1979)
J. Biol. Chem.
254, 12285-12288). These assays have several limitations when applied to the measurement of TS activity in human tissue samples. While the assays have the required sensitivity for quantitating enzyme in vitro pure populations of malignant cells in culture, they lack adequate sensitivity to measure the lower levels of enzyme activity in human tumors. Previous investigations from our laboratory measuring the TS levels in human breast tumor biopsy specimens revealed that large quantities of tumor (>50 mg) were required to carry out these studies (Scanlon, K. J. and Kashani-Sabet, M. (1988)
Proc. Natl. Acad. Sci.
USA 85, 650-653). Both the catalytic and FdUMP binding assays also require that the enzyme is active; therefore only fresh or frozen tissue can be assayed, thus limiting the assay to prospective studies with the caveat that no enzyme degradation has occurred during the preparation of the samples. In addition, the biochemical assay does not discriminate between areas of the tumor with differing morphologies nor can it measure TS on a cell-to-cell basis. Since tissues and cell preparations are a composite of a heterogenous population, any measurement of TS enzyme using biochemical techniques is confounded by the degree of contamination by cells other than those of interest.
The advent of monoclonal antibody (MoAb) technology has created the opportunity to use monoclonal antibodies as tools in studying the cellular distribution, tissue expression and pharmacokinetics of biologically important molecules. The availability of a monoclonal antibody directed against human TS might provide the adequate sensitivity and specificity needed to overcome those problems inherent in the biochemical assay. In addition, a monoclonal antibody to TS may provide a method for the immunological quantitation of the enzyme in a variety of human tissues and enable analysis to be undertaken on a cell-by-cell basis. This study presents the derivation and characterization of monoclonal antibodies to TS. These antibodies provide a basis for the immunological detection and quantitation of the TS enzyme in human cells, and describe the immunohistochemical localization of the TS enzyme in human colon carcinomas.
The instant invention presents the derivation and characterization of monoclonal antibodies to thymidylate synthase. These antibodies provide a basis for the immunological detection and quantitation of the T.S.enzyme in human cells, and enable the immunohistochemical localization of the T.S. enzyme in human colon carcinomas.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide hybridomas capable of producing monoclonal antibodies specific for antigenic sites on thymidylate synthase.
It is another object of the present invention to provide monoclonal antibodies that recognize antigenic epitopes of thymidylate synthase.
Yet another object of the present invention is to provide a method of immunological detection and quantitation of thymidylate synthase in tissue cells, including tumors (for example, colon carcinomas) in normal and malignant tissue.
It is a further object of the present invention to provide a diagnostic method for the immunological quantitation and detection of thymidylate synthase that does not require large quantities of tissue.
It is yet another object of the present invention to provide a diagnostic method for the immunological quantitation and detection of thymidylate synthase where the tissue architecture will be intact.
It is a further object of the present invention to provide a qualitative method by which to detect changes in the amount of thymidylate synthase present individual cells.
Yet another object of the present invention is to provide a method of detecting thymidylate synthase with adequate sensitivity for both in vitro and i
Allegra Carmen J.
Chabner Bruce A.
Johnston Patrick G.
Liang Chi-Ming
Daniels-Cook Lisa V.
Graser Jennifer
The United States of America as represented by the Department of
Townsend and Townsend / and Crew LLP
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