PDGF-AB, preparation process and pharmaceuticals containing them

Details PDGF-AB, preparation process and pharmaceuticals containing them PDGF-AB, preparation process and pharmaceuticals containing them

1997-02-24

1999-10-19

Duffy, Patricia A.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

435 697, 43525233, 514 12, 530399, 530402, 530408, 530412, 530417, C12D 2104

Patent

active

059687789

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

1. Field of the Invention
The invention relates to a process for the preparation of PDGF-A and biologically active PDGF-AA and PDGF-AB.
2. Brief Description of Related Art
Platelet derived growth factor (PDGF) is a major mitogen in serum which promotes growth of fibroblasts and smooth muscle cells in vitro. In vivo, PDGF is stored in the a granules of the platelets and released after stimulation of the platelets. Highly purified PDGF is a basic protein which displays a considerable heterogeneity with regard to its molecular weight (27,000 to 31,000 d). The reasons for this heterogeneity are aging, processing and the existence of various isoforms of type AA, AB or BB. The biological activity of all these forms is destroyed by reduction of the disulfide bridges. Human PDGF from platelets consists principally of AB heterodimers (Heldin, C.-H. & Westermark, B. (1984) Cell, 37, 9-20; Deuel, T. F., Tong, B. D. & Huang, J. S. (1985) Current Topics in Cellular Regulation, 26, 51-61; and Ross, R., Raines, E. W. & Bowen-Pope, D. F. (1986) Cell, 46, 155-169.).
Amino-acid sequencing in conjunction with DNA sequencing of the genes have shown that A and B are homologous (Betsholtz et al. Nature 320, 695-699, 1986). PDGF-B is virtually identical to the transformed gene product P.sub.28.sup.v-sis of simian sarcoma virus (SSV). Corresponding homodimers of type BB have been detected in SSV-transformed cells and showed properties similar to those of PDGF from platelets. However, these BB dimers were secreted to only a small extent from the infected cells. PDGF-AA forms are, by contrast, secreted efficiently by producing cells (Heldin et al. Nature, 319, 511-514, 1986). There is an increasing amount of evidence that the three isoforms, AA, AB and BB, assume different functions. Thus, for detailed investigations, it was necessary to develop a process for producing larger quantities of PDGF-A, PDGF-B, PDGF-AA and PDGF-AB.
Compare EP-A-0 288 307 for the use of PDGF for wound treatment.


SUMMARY OF THE INVENTION

According to one embodiment, the invention relates to PDGF-A (monomer) or biologically active (growth-stimulating) PDGF-AA (dimer), which can be prepared in such a way that DNA which encodes the following fusion protein: following amino-acid sequence (i) (SEQ ID NO:1) absent, in which any desired amino acid has been replaced by any other desired amino acid or in which any desired additional amino acid has been provided at any desired point, or the N terminus has been deleted or supplemented by up to 14 amino acids; according to (a), (b) and/or (c), chemically cleaved, and a monomer of the amino-acid sequence according to (I) (a) or a corresponding monomer in which any desired amino acid can be absent, any desired amino acid can be replaced by any other desired amino acid, any desired additional amino acid can be provided at any desired point, and/or the C terminus or the N terminus can be deleted or supplemented by up to 14 amino acids, is liberated, where appropriate, furthermore and then
According to another embodiment, the invention relates to biologically active (growth-stimulating) PDGF-AB, which can be prepared by employing in stage (III) as detailed above PDGF-A as detailed above in stage (II) and PDGF-B of the following amino-acid sequence (ii) (SEQ ID NO:2) or (iii) (SEQ ID NO:3)


20 30 40 50 60 (ii) IAECKTRTEVFEISRRLIDRTNANFLVWPPCVEVQRCSGCCNNRNVQC - 70 80 90 100 110 RPTQVQLRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCETVAAARPVTRSPLN - 1 10 20 30 40 50 60 (iii) SLGSLTIAEPAMIA ECKTRTEVFEISRRL IDRTNANFLVWPPCV EVQRCSGCCNNRNVQ C - 70 80 90 100 110 RPTQVQLRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCETVAAARPVT amino acid is absent, in which any desired amino acid is replaced by any other desired amino acid, in which any desired additional amino acid is provided at any desired point and/or in which the C terminus or the N terminus is deleted or supplemented by up to 14 amino acids, and the process as detailed above in stages (III) to (VII) is carried out, and PDGF-AB is obtained.
Accordi

REFERENCES:
Hoppe, J. et al., Biochemistry, 28(7): 2956-2960, 1989.
Wang, J.Y. et al., J. Biol. Chem., 259 (17): 10645-10648, 1984.
Devare, S. G. et al., Cell, 36(1): 43-50, 1984.

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