Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1997-06-24
1999-03-16
Patterson, Jr., Charles L.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
435212, 435226, C12N 1557
Patent
active
058832416
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a novel human metalloproteinase, to homologues and fragments thereof, to means for producing the metalloproteinases, and to means for regulating their production and activity in vivo.
A number of physiologically important processing events are mediated by metalloproteinases, which under certain circumstances contribute to pathologies as diverse as inflammation and cancer, and it has been suggested that such enzymes would provide targets for therapeutic intervention. Thus, by varying the production of the enzyme, or inhibiting or enhancing its activity in vivo it should be possible to achieve a therapeutic effect.
In one example, tumour necrosis factor-alpha (TNF-.alpha.) is a potent pro-inflammatory and immunomodulatory mammalian cytokine produced primarily by activated monocytes and macrophages. It is initially expressed as a 233-amino-acid membrane-anchored precursor (pro-TNF-.alpha.) which is proteolytically processed to yield the mature, 157-amino-acid cytokine. Evidence has been obtained which indicates that at least one metalloproteinase-like enzyme mediates pro-TNF-.alpha. cleavage, but to date the enzyme(s) responsible for this in vivo are Gearing, A. J. et al ibid 370, 555-557 (1994); McGeehan, G. M. et al, ibid 370, 558-561 (1994)!. A number of known matrix metalloproteinase papers and International Patent Specification Publication No. WO 95/06031!. These compounds were originally designed to selectively inhibit matrix metalloproteinases such as collagenase with primary functions unrelated to pro-TNF-.alpha. cleavage. Where new inhibitors have been described these have apparently been selected on the basis of their effect on TNF-.alpha. secretion seen in cell-based assays.
In another example, L-selectin shedding is thought to be a pro-inflammatory Science, 258, 964-969 (1992)!. Some inhibitors of L-selectin proteolysis have been identified, but these have been obtained using cell based assays Chem., 271, 7019-7024 (1996)!.
In general, in order to obtain compounds capable of selectively regulating the action of a metalloproteinase implicated in human disease, for example as in the above TNF-.alpha. and L-selectin instances it would be clearly advantageous to have the enzyme unequivocally identified and obtainable in an isolated, purified and unambiguous characterised form.
Through the use of a cloning and screening approach, we have been able to identify human DNA which is responsible for coding part of one such metalloproteinase. This DNA has the sequence described in SEQ I.D. No: 1 below and may be of use (1) in the generation of a gene coding for the metalloproteinase, (2) in the production of the metalloproteinase, (3) in the provision of means to regulate the activity of the metalloproteinase in vivo, and (4) in the provision of means to detect and measure a metalloproteinase in a biological system, e.g. in serum, synovial fluid or a tissue extract.
Thus according to one aspect of the invention we provide DNA comprising the nucleotide sequence of SEQ I.D. No: 1: ##STR1## and homologues and fragments thereof.
It will be appreciated that the nucleotide sequence of SEQ I.D. No: 1 also includes control sequences, such as a polyadenylation sequence, providing for expression of the sequence in a host cell.
One particular DNA fragment according to the invention is the isolated human metalloproteinase-encoding nucleotide sequence of SEQ I.D. No: 2: ##STR2## and homologues and fragments thereof.
In the sequences herein standard one letter codes are used to represent nucleotides or amino acids as appropriate.
DNA according to the invention may be obtained using conventional molecular biology procedures, for example by probing a human genomic or cDNA library with one or more labelled oligonucleotide probes containing for example fifteen or more contiguous nucleotides designed using the nucleotide Molecular Biology", Ausubel, F. M. et al (eds), Greene Publishing Associates and Wiley-Interscience, New York (1987)!.
Where the term homologue is used herein in relation to a particul
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Docherty Andrew James Penrose
Slocombe Patrick Marcel
Celltech Therapeutics Limited
Conlin David G.
Patterson Jr. Charles L.
Resnick David S.
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