Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Patent
1993-08-31
1996-04-30
Allen, Marianne P.
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
530350, 530399, 435 694, 43525233, 4352551, 4353201, 536 2351, A61K 3818, C12N 1518, C07K 14475
Patent
active
055125450
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to protease-resistant mutants of the B chain of human platelet-derived growth factor (PDGF).
BACKGROUND OF THE INVENTION
PDGF is a mitogen which is released from the alpha-granules of platelets following activation. The mitogenic activity of this growth factor is restricted to cells of mesothelial origin, glial cells and fibroblasts, but excluding arterial endothelium cells.
PDGF isolated from blood is predominantly a disulphide-linked heterodimer having a molecular weight of 28000-31000 and is composed of two chains, the A chain (PDGF-A) and the B chain (PDGF-B). The mature A and B chains show 60% homology and the 8 cysteine residues in each chain are conserved. The PDGF B chain (SEQ ID NO: 1) is essentially homologous to the v-sis oncogene product, p28.sup.sis, derived from simian sarcoma virus. The major form of PDGF found in human serum is the heterodimer of the A and B chains, but homodimers are also present in small quantities. Homodimers of PDGF-BB are found in porcine.serum and PDGF-AA is produced by several human tumour cell Lines. The reduced monomeric forms of PDGF are biologically inactive.
PDGF receptors are composed of two sub-units, alpha and beta. The beta-receptor sub-unit can only bind the B chain of PDGF, but the alpha-receptor sub-unit binds both the A and the B chains and therefore it is possible to mediate the different biological functions of PDGF via the isoforms and the porportion of alpha-and beta-receptor sub-units on cells.
There is no structural model of PDGF and the residues responsible for binding to the receptor have not been defined. Circular dichroism measurements have shown a high content of random structure and a low alpha helical content (Vogel and Hoppe 1989, Biochemistry 28, 2961-2966). The minimal v-sis transforming domain has been shown to span the 89 residues indentical to PDGF and containing all 8 conserved cysteines (King et al 1985, P.N.A.S., 82, 5259-5299). Chimeras of PDGF-A and B chains have been used to define a region from residues 24-63 (numbering from the mature N-terminus) to be responsible for the transforming ability of PDGF-BB (LaRocheHe et al, 1990, Science, 248, 1541-1544). More recently, using single amino acid deletions, the receptor binding domain has been shown to be between residues 25 and 37 from the mature N-terminus. (Giese et al, 1990, M.C.B., 10, 5496-5501).
PDGF has been implicated in a number of diseases involving abnormal cell proliferation, inflammation, fibrosis, atherosclerosis and neoplasia. In addition, PDGF is thought to be an important component of the wound healing process. There is therefore considerable interest in the therapeutic potential of PDGF antagonists and agonists. The production of large quantities of PDGF is therefore desirable and the simplest way to achieve this is by recombinant DNA technology.
Recombinant PDGF is known and has been described in several prior publications for example U.S. Pat. No. 4769328 and U.S. Pat. No. 4801542, describe biologically active PDGF analogues expressed in yeast.
U.S. Pat. No. 4766073 discloses dimetic proteins substantially homologous to the A-chain or the B-chain of PDGF or portions thereof, and an A-B heterodimer, expressed in yeast. In addition a portion of the DNA sequence may encode a portion of the A-chain while another may encode a portion of the B-chain.
U.S. Pat. No. 4845075 discloses dimeric proteins which have substantially the same biological activity as PDGF. The proteins consist of dimers of polypeptides which are substantially homologous to PDGF-B and the specification suggests that it may be advantageous to truncate the protein or to change the amino acid residues. In particular, Cys residues may be substituted by other amino acid residues.
More specifically a truncated form of PDGF is disclosed. The Lys-Arg at the alpha factor B-chain boundary was removed, and it was proposed that proteolytic processing would occur at the internal Arg-Arg site at position 27-28. Mitogenic activity was observed with this cons
REFERENCES:
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patent: 4769328 (1988-09-01), Murray et al.
patent: 4801542 (1989-01-01), Murray et al.
patent: 4845075 (1989-07-01), Murray et al.
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Brown David
Clements John M.
Cook Anne L.
Craig Stewart
Edwards Richard M.
Allen Marianne P.
British Biotech Pharmaceuticals Limited
Pfizer Limited
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