Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Hormones – e.g. – prolactin – thymosin – growth factors – etc.
Patent
1995-11-01
2000-08-15
Chan, Christina Y.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Hormones, e.g., prolactin, thymosin, growth factors, etc.
530350, C07K 14475
Patent
active
061038806
DESCRIPTION:
BRIEF SUMMARY
This is a 371 of PCT/FR94/00219, filed Feb. 25, 1994.
The object of the present invention is HARP family growth factors.
It also relates to preparation methods for these factors using genetic engineering techniques.
It also concerns applications of such factors in therapeutics.
Growth factors named HBBM have been detected during the purification of other growth factors of the FGFS (Fibroblast Growth Factor) group.
The HBBM have in particular been the object of a European patent application 89 101 187 (Publication No EP-326.075) in which their isolation from a brain extract, by a purification process including an extraction at acid pH and chromatography steps, is described. This process led to the isolation of three peptides of 18 kD, 16 kD and 15 kD with the same terminal sequence NH.sub.2 -GLY-LYS-LYS-GLU-LYS-PRO-GLU-LYS-LYS-VAL-LYS-LYS-SER-ASP-CYS-GLY-GLU-TRP-G LN-TRP-SER-VAL-CYS-VAL-PRO (SEQ ID NO:2, residues 4-15).
Application EP-326.075 describes a mitogenic activity of the HBBM on endothelial cells at concentrations of the order of 20 to 50 ng/ml, for the induction of a stimulation effect of 50%. of the maximum stimulation of proliferation, and a minimum effect from 3 ng/ml. At this concentration, the mitogenic effect of the HBBM was comparable to that of the acid form of the FGF (aFGF). This application also states that the three HBBM forms had angiogenesis promotion activity and activities of maintenance of the integrity and tissue cicatrization particularly for skin, bone and nerve tissues, but does not report experimental results.
In a subsequent article (Growth Factors, 4: 97-107, 1991), the inventors returned to the results described in patent EP-326.075 and showed that their new HBBM preparations, also obtained from beef brain, were in fact devoid of mitogenic activity, particularly for endothelial and fibroblast cells, even at concentrations of 1 to 10 .mu.g/ml. The only activity confirmed was a neurotrophic activity giving neurite growth on rat embryo neurons at concentrations of between 80 and 640 ng/ml. The authors of this article thus decided to change the name of this factor to designate it by the new acronym HBNF for "Heparin Binding Neurotrophic Factor". The reason for these differences between the first work described in the patent application and that described subsequently was not clearly understood but the hypothesis advanced was that of a contamination by the growth factor bFGF during the first preparations. Factor bFGF was in fact already well known for its angiogenic and mitogenic activities and had been isolated by a similar procedure from brain.
In order to confirm their results on the absence of mitogenic and angiogenic activities of HBNF, the same authors purified HBNF from the expression product of the gene for this protein in bacteria (Kretschmer et al., Growth Factors, 5: 99-114, 1991). The protein obtained contained 168 amino acids and was purified after maturation in the form of a protein of 136 amino acids, whose sequence was identical to that previously described, beginning thus from the N-terminal end by the amino acids NH.sub.2 -GLY-LYS-LYS-GLU-LYS-PRO-GLU-LYS-LYS-VAL (SEQ ID NO:2, residues 4-13). This protein is devoid of mitogenic and angiogenic activities. Only the neurotrophic activity, comparable to that of the HBNF protein purified from brain, was confirmed.
By a comparable approach, another team of researchers showed that the same protein obtained by expression of the same gene, named HB-GAM by these authors and inserted into a baculovirus vector in insect cells, is also devoid of mitogenic activity (Raulo et al., J. Biol. Chem., 267, 1-9, 1992). The protein thus purified had an N-terminal sequence GLY-LYS-LYS-GLU-LYS (SEQ ID NO:1, residues 4-8) identical to that which these authors had obtained by an extraction from rat brain. This recombinant protein had a neurotrophic activity comparable to the native protein. The authors concluded that only the neurotrophic activity of HB-GAM existed. It was optimal at 200 ng/ml in the test used on the neuron cells cultiv
REFERENCES:
Rudinger, In "Peptide Hormones" (Jun. 1976)ed. J. A. Parsons, University Park Press, Baltimore, pp 1-7.
Courty et al. (1991) J. Cell Biochem. Suppl. 0 (15, part F): 221, abstract No. CF110.
Kretschmer et al. (1991) Growth Factors 5: 99-114.
Li et al. (1990) Science 250: 1690-1694.
Wellstein et al. (1992) J. Biol. Chem. 267: 2582-2587.
Huber et al. (1990) Neurochemical Res. 15 (4): 435-439.
Barritault Denis
Courty Jose
Laaroubi Khalid
Chan Christina Y.
Hayes Robert C.
Valbiofrance
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