Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Animal cell – per se – expressing immunoglobulin – antibody – or...
Patent
1995-06-01
1998-10-06
Feisee, Lila
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Animal cell, per se, expressing immunoglobulin, antibody, or...
435 74, 435 76, 435 7021, 4351722, 53038826, 5303911, 5303913, C07K 1640, C12N 512, G01N 33577
Patent
active
058175148
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to monoclonal antibodies capable of preferentially binding one of human 0.sup.6 -methylguanine-DNA-methyltransferase (MGMT) and active site alkylated derivatives thereof, methods of obtaining such antibodies and use of such antibodies.
Human MGMT is an important DNA repair enzyme which can transfer alkyl groups from 0.sup.6 -alkylguanine and 0.sup.4 -alkylthymine residues in DNA to a cysteine residue (Cys 167) at the active site of the enzyme. Such alkylated nucleotide residues, which can give rise to transition mutations in DNA sequences by virtue of their miscoding behaviour, can be produced by carcinogenic alkylating agents such as N-alkylnitrosoureas, e.g. N-methyl-nitrosourea (NMU) and N-ethylnitrosourea (NEU), and N,N-dialkylnitrosamines.
Various human genes associated with tumour formation have been found to carry point mutations, for example ras, p53 and ERCC-3. These mutations are widely believed to be crucial in the activation (oncogenes) or suppression (tumour suppressor genes) of these genes and much evidence supports point mutations arising through the action of alkylating agents on DNA being involved in the etiology of certain human cancers. Thus, it has been shown that a single dose of NMU produces mammary tumours in rats as a result of a G. C to A. T transition mutation in codon 12 of the rat H-ras oncogene. This point mutation can also be formed in vivo by substituting 0.sup.6 -methylguanine for guanine in DNA. As 0.sup.6 -methylguanine is one of the alkylated nucleotides formed by action of alkylating carcinogens on DNA and it can produce the G. C to A. T transition mutation, it is thought to be involved in the formation of mammary tumours in rats exposed to NMU (Zarbl et al. Nature (1985) 315, 382-385; Mitra et al. Proc. Natl. Acad. Sci. USA (1989) 86, 8650-8654).
N,N-dialkylnitrosamines are environmental alkylating agents and have also been shown to be potent tumour promoters in experimental animals. Hence, they are considered likely causative agents of human cancers through their ability to produce mutagenic DNA base adducts in vivo (Bartsch et al., Relevance of N-Nitroso compounds to Human Cancer: Exposures and Mechanisms, IARC Scientific Publication no. 84. Lyon: International Agency for Research on Cancer (1987); O'Neill, et al., Relevance To Human Cancer of N-Nitroso Compounds, Tobacco and Mycotoxins. ibid. no. 105 (1991); P. D. Lawley, in Chemical Carcinogens Vol I, Ed, C. E. Searle (ACS Monograph 182, American Chemical Society, Washington, 1984); Singer and Grunberger, Molecular Biology of Mutagens and Carcinogens (Plenum Press, New York, 1983)).
Since MGMT can specifically repair lesions in DNA produced by alkylating agents, the level of this enzyme in cells is believed to be a crucial factor in determining the sensitivity of cells towards the tumour-inducing mutagenic action of alkylating carcinogens (Lindahl et al., Annu. Rev. Biochem. (1988) 57, 133; A. E. Pegg, Cancer Res. (1990) 50 6119-6129).
A MGMT repair enzyme was first identified in bacteria. cDNAs coding for such a repair enzyme have been cloned and characterised from bacteria yeast, mice, rats and humans. MGMT thus appears to be ubiquitous protein. However, there is considerable variation in the content of MGMT between species and cell types. In E. coli, two proteins are known to repair 0.sup.6 -methylguanine lesions in DNA, i.e. the ada product which is inducible and the constitutive ogt product. Bacteria carrying the ada-phenotype are sensitive to killing by alkylating agents such as NMU. Only one MGMT protein has been reported in mammals.
In addition to having implications for tumour induction, human MGMT also has implications for tumour treatment with chemotherapeutic alkylating agents such as the bifunctional chloroethylating agent 1,3-bis (2-chloroethyl)-1-nitrosurea (BCNU). The chemotherapeutic effect of such drugs depends upon formation of lethal cross-links in DNA, which may be disrupted by the suicidal repair action of MGMT. Tumour cell lines which are devoid of this enzyme (me
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Ayi Teck Choon
Li Benjamin Fuk Loi
Feisee Lila
Johnson Nancy A.
National University of Singapore
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