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
1999-11-26
2003-10-28
Scheiner, Laurie (Department: 1648)
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...
C530S387300, C530S388100, C530S388150, C424S141100, C424S142100, C424S146100, C435S325000, C435S338000
Reexamination Certificate
active
06639057
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a monoclonal antibody which reacts specifically to human telomerase catalytic subunit (hereinafter referred to as hTERT). In addition, the present invention relates to a diagnosis method, diagnosis agent, and therapeutic agent using this monoclonal antibody for diseases in which telomerase is involved, such as cancer.
This application is based on patent application No. Hei 10-98486 filed in Japan, the content of which is incorporated herein by reference.
BACKGROUND ART
The ends of the chromosomes of eukaryotic cells like those of animal cells are called telomeres, and they have a higher-order structure comprising characteristic repetitive DNA sequences and proteins which are bonded thereto. The telomere structure is considered to have an important role in the stabilization of chromosomes. In cells in which the telomere has become shortened, deletions of chromosome and fusion of the ends of chromosomes with each other are frequently observed. In replication of straight chain DNA, since the RNA primer of the 5′ terminal end cannot be replaced by DNA, the chromosome becomes shorter by the primer portion at each replication. Actually, when human somatic cells are cultured and successively passaged, the telomere becomes shortened and the cells finally die due to instability of the chromosomes as mentioned above. In this way, the length of the telomere is known as one factor which regulates the limited division potential which cells have and is known to be closely related to the aging of cells, the immortalization of cells, and the like.
In contrast, the telomeres of protozoa and yeasts which are single cells do not shorten even when they are repeatedly propagated. In these organisms, an RNA dependent DNA polymerase, called telomerase, which lengthens one chain of the telomere repeating sequence, is active, and fixedly maintains the length of the telomere which is shortened along with division. Conventionally, measurement of the telomerase enzyme activity has been difficult because of its low sensitivity but a method (Telomeric Repeat Amplification Protocol; TRAP Method) has been developed which amplifies the reaction products of telomerase by PCR (Kim, N. W., et al., Science, 266, 2011, 1995), and it is possible to measure the telomerase activity of various cells and tissues of higher animals including humans.
Cells derived from human cancer are different from normal cells in that they can proliferate without limit in vitro, and the telomeres do not shorten even with repeated cell division. The results of an investigation of telomerase activity in various human tissues using the above-mentioned TRAP method indicate that telomerase activity, which is not detected in almost all normal human tissues with the exception of reproductive cells and some bone marrow cells, was characteristically detected in cancer tissues (Shay, J. W., et al., Eur. J. Cancer, 33, 787, 1997). These results indicate that the acquisition of a mechanism which avoids shortening of telomeres via telomerase is important in the development of human cancer. Since telomerase is characteristically expressed by cancer cells and concerned with their unlimited proliferation activity, it is expected that a pharmaceutical which blocks the activity will be a highly selective anti-cancer pharmaceutical.
It is presumed that telomerase is a complex comprising a plurality of units, and the genes for the two structural units, namely, hTR (the human telomerase RNA), which is the RNA molecule which forms the template for extending a strand of the telomere DNA, and hTERT (Human Telomerase Reverse Transcriptase), which is the enzyme subunit which catalyzes the polymerase reaction, have been cloned (Feng, J. et al., Science 269, 1236, 1995; Nakamura, T. M., et al., Science 211, 955, 1997). The relationship between the expression of these units and telomerase activity has been analyzed and it has been reported that the telomerase activity characteristically detected in human cancer tissue is correlated with the expression of the hTERT protein, i.e., the expression of telomerase activity in cancer is regulated by the hTERT protein (Nakamura, T. M., et al., Science 277, 955, 1997; Nakayama, J., et al., Nature Genetics 18, 65, 1998). In addition, it has been shown that, by the introduction of the hTERT gene into normal human cells having dividing life, the life of those cells was extended and it has become clear that hTERT functions as a molecule which regulates cell aging and the immortalization of cells (Bodnar, A. G., et al., Science 279, 349, 1998). In this way, it is expected that analysis of the function of the hTERT protein will provide important information in development of pharmaceuticals not only for cancer but also for disease associated with aging.
As a means for investigating the expression and function of specific proteins in cells and tissues, antibodies having high affinity and specificity to antigen are extremely important in the functional analysis of proteins. As antibodies to the hTERT protein, polyclonal antibodies produced using rabbits are known, and it has been reported that it is possible to detect telomerase activity in a sample obtained by immune precipitation of an extract of human cancer cells (HeLa S3) (Harrington, L., et al., Genes & Dev. 11. 3109, 1997). However, because the amount of hTERT protein expressed is extremely small, it is not possible to detect the hTERT protein expressed within the human cancer cells (HeLe S3) with the Western blotting method using the reported polyclonal antibodies.
DISCLOSURE OF THE INVENTION
The present invention provides a monoclonal antibody which can specifically and efficiently recognize hTERT protein which is the catalytic subunit of telomerase, and provides a human chimeric antibody, a CDR grafted antibody, a single chain antibody, and a disulfide stabilized antibody which contain the monoclonal antibody, and thereby providing detecting and assaying methods for the hTERT protein.
The present invention also provides diagnosis methods, diagnosis agents, and therapeutic agents which use this monoclonal antibody for diseases in which telomerase is involved such as cancer.
The present invention relates to the following items (1) to (37).
(1) A monoclonal antibody which recognizes a human telomerase catalytic subunit.
An object of the present invention is providing superior monoclonal antibodies having high reactivity for the purpose of detecting hTERT protein within cells.
It is sufficient for the monoclonal antibody of the present invention to be one which recognizes human telomerase reverse transcriptase (hereinafter referred to as hTERT), which is the catalytic subunit of telomerase.
Examples of the monoclonal antibodies include antibodies produced by hybridoma, and genetic recombinant antibodies produced by transformants which are transformed by an expression vector containing the antibody gene.
In more detail, it is possible to obtain an anti-hTERT monoclonal antibody by preparing hTERT protein, which is the catalytic subunit of telomerase, or a peptide or the like chemically synthesized based on the amino acid sequence of the hTERT protein [Science, 277, 955(1997)] as an antigen; inducing antibody-producing cells having antigenic specificity from animals immunized with the antigen; fusing them with bone marrow tumor cells to produce hydbridomas; culturing the hybridoma or inducing ascitic cancer in an animal by administering the hybridoma to the animal separating the anti-hTERT monoclonal antibody from culture medium or the ascites and then purifying.
(2) A monoclonal antibody according to the above-mentioned item (1), wherein said monoclonal antibody is obtainable by immunizing an animal with a partial peptide of the human telomerase catalytic subunit, the partial peptide having an amino acid sequence designated as one of SEQ ID NOs: 1, 2, 3, and 6.
(3) A monoclonal antibody according to the above-mentioned item (1), wherein said monoclonal antibody reacts specifically with the amino acid sequence of
Anazawa Hideharu
Furuya Akiko
Hanai Nobuo
Mikuni Osamu
Shibata Kenji
Kyowa Hakko Kogyo Co. Ltd.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Scheiner Laurie
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