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
1989-08-21
1992-12-15
Kepplinger, Esther L.
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, 530806, 530832, 435 79, 436518, 436817, C07K 1300
Patent
active
051718456
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a novel protein of about 17 KD, with angiogenic action, to the method of isolating it from mammalian milk, to therapeutic compositions in which it is present, to a method of detection and/or determination and to immunological reagents for detecting and/or determining mammalian angiogenins, homologs thereof and fragments thereof.
Following the pioneering work of J. FOLKMAN (q.v. J. Exp. Med., 133, 275 (1971)), who demostrated that the growth of a tumor requires a substantial blood supply, this being provided by the continuous growth of new blood vessels, he suggested that this growth results from the presence of a diffusible substance which he called "Tumor Angiogenesis Factor" (TAF). Several proteins which stimulate angiogenesis were isolated (FOLKMAN et al., Science, (1987), 235, 442). Among these substances, angiogenin, a protein of human origin, was purified by VALLEE's team and its gene was cloned. In 1985, J. W. FETT et al. (Biochem., (1985), 24, 5480-5486) isolated human angiogenin from cancerous human intestinal cells in culture. The intestinal cells HT 29 secrete human angiogenin in the culture medium. 0.5 .mu.g/liter of human angiogenin was isolated from these serum-free culture media. VALLEE's team determined the concentration of angiogenin first by Bradford's method (Anal. Biochem., 1975, 72, 248-254, a stain fixing method using SAB as the standard) and then by the method described in Biochem., 1986, 25, 3527-3732, by SHAPIRO et al. The amino acid sequence was also specified by STRYDOM et al. (BIOCHEMISTRY, (1985), 24, p. 5486-5494).
Human angiogenin is a protein with a molecular weight of 14,400 D.
The angiogenin isolated from human tumoral cells consists of a single protein chain comprising 123 amino acids and having the following sequence: ##STR1##
The C-terminal amino acid is proline; three disulfide bridges link the cysteines 26-81, 39-92 and 57-107.
The sequence of human angiogenin is 35% homologous with that of human pancreatic ribonuclease, in particular with regard to the amino acids essential for ribonucleolytic activity (q.v. BIOCHEMISTRY, (1985), 24, 5494-5499, KURACHI et al.). The activity of human angiogenin is considerable since 50 ng, i.e. 3.5 picomoles, are capable of causing vascularization of the rabbit cornea and 35 femtomoles are capable of inducing vascularization of the chicken embryo.
It is known to prepare human angiogenin by cloning. Cloning of the gene coding for human angiogenin makes it possible to prepare satisfactory amounts via suitable expression systems. However, such methods are expensive.
It has proved necessary to look for a compound which has properties similar to those of human angiogenin and whose method of preparation is simpler and less expensive.
One object of the present invention is consequently to provide a novel protein which has properties similar to those of human angiogenin and is obtained by inexpensive means which are easy to carry out and afford high quantitative yields.
Another object of the invention is to provide a novel method of obtaining the said protein which does not have the disadvantages of the methods of the prior art; in fact, this novel method makes it possible to obtain large amounts of the protein in question at very low cost, which has important advantages in the context of the industrial manufacture of pharmaceutical compositions containing this protein.
Yet another object of the invention is to provide pharmaceutical compositions containing the said protein.
Another object of the invention is to provide an agent for detecting and determining mammalian angiogenins, homologs thereof and fragments thereof in biological fluids.
Another object of the invention is to provide a kit for detecting and determining the above-mentioned proteins in the said fluids.
The present invention relates to a protein which has a sequence comprising 125 amino acids and has formula I below: ##STR2## whose sequence has 81 amino acids in common with human angiogenin and whose molecular weight is about 17 KD.
The molecul
REFERENCES:
patent: 4440860 (1984-04-01), Klagsbrun
patent: 4529590 (1985-07-01), LeVeen et al.
Yagi ey al., Biol. Abstr. 82(2):12599, 1986.
Bond et al., Biochemistry 27:6282-6287, 1988.
Biochemistry, vol. 24, No. 20, 24 Sep., 1985, pp. 5480-5486, American Chemical Society; J. W. Fett et al.: "Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells".
Biochemistry, vol. 24, No. 20, 24 Sep. 1985, pp. 5486-5494, American Chemical Society; D. J. Strydom, et al.: "Amino acid sequence of human tumor derived angiogenin".
Biochemistry, vol. 24, No. 20, 24, Sep. 1985, pp. 5494-5499, American Chemical SOciety K. Kurachi et al.: "Sequence of the cDNA and gene for angiogenin, a human angiogenesis factor".
Biochemistry, vol. 26, No. 16, 11 Aug., 1987, pp. 5141-5146, American Chemical Society; R. Shapiro, et al.: "Isolation of angiogenin from normal human plasma".
Montreuil Jean
Spik Genevieve
Tarjar Andre
Kepplinger Esther L.
Wortman Donna C.
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