Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Patent
1994-07-27
1997-09-09
Ulm, John
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lymphokines, e.g., interferons, interlukins, etc.
424 851, 530363, 435 695, 4352523, 4353201, 43525421, 4352542, 435325, 435360, 4353651, C12N 1527, C07K 1453
Patent
active
056658630
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to new polypeptides having human granulocyte colony stimulating activity, to their preparation and to pharmaceutical compositions containing them.
BACKGROUND OF THE INVENTION
The present invention relates especially to chimeric polypeptides composed of a biologically active portion consisting of all or part of G-CSF or of a variant of G-CSF, and an essentially proteinaceous stabilizing structure endowing it with new biological properties.
Human G-CSF is a secreted polypeptide of 174 amino acids having a molecular weight of approximately 18 kD. It was isolated initially from a cancer cell line (EP 169,566), and its gene has been cloned, sequenced and expressed in different cell hosts by genetic engineering techniques (EP 215,126, EP 220,520). An mRNA potentially coding for a form of G-CSF having 177 amino adds has, moreover, been detected [Nagata S. et al., EMBO J. 5 (1986) 575-581]. G-CSF possesses the capacity to stimulate the differentiation and proliferation of bone marrow stem cells to granulocytes. As such, it possesses the capacity to stimulate the body's protective capacities against infection by promoting the growth of polymorphonuclear neutrophils and their differentiation ending in maturity. It is thus capable of activating the body's prophylactic functions, and may be used in different pathological situations in which the number of neutrophils is abnormally low or in which the immune system needs to be strengthened. Such situations arise, for example, following cancer chemotherapy treatments, in transplantation, and especially bone marrow transplantation, or in leukopenic states.
One of the drawbacks of currently available G-CSF lies in the fact that it is rapidly degraded by the body once administered. This is all the more noticeable for the fact that G-CSF is generally used at low doses. Furthermore, the use of larger doses has not been able to permit therapeutic capacities of this molecule to be improved, and may induce adverse side effects. These phenomena of elimination and degradation in vivo hence constitute at present an obstacle to exploitation of the biological activity of the G-CSF as a pharmaceutical agent.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention enables these drawbacks to be remedied. The present invention provides, in effect, new molecules enabling the biological properties of G-CSF to be optimally exploited from a therapeutic standpoint. The Applicant demonstrated, in effect, that optimal G-CSF activity was manifested when the G-CSF was present at a low dose and for a prolonged time. The Applicant has now produced molecules capable of maintaining G-CSF activity in the body for a sufficiently long time. Furthermore, the Applicant has shown that it is possible to express, in cell hosts at high levels, genetic fusion generating chimeras possessing new pharmacokinetic properties and the desirable biological properties of G-CSF. In particular, hybrid polypeptides of the invention retain their affinity for G-CSF receptors, and are sufficiently functional to lead to proliferation and to cell differentiation. The molecules of the invention possess, moreover, a distribution and pharmacokinetic properties which are especially advantageous in the body, and enable their biological activity to be developed therapeutically.
A subject of the present invention hence relates to recombinant polypeptides containing an active portion consisting of all or part of G-CSF, or of a variant of G-CSF, and an essentially proteinaceous stabilizing structure.
For the purposes of the present invention, the term variant of G-CSF denotes any molecule obtained by modification of the sequence lying between the residues Thr586 and Pro759 of the sequence presented in FIG. 1, retaining G-CSF activity, that is to say the capacity to stimulate the differentiation of target cells and the formation of granulocyte colonies. This sequence corresponds to that of mature G-CSF described by Nagata et al. [EMBO J. 5 (1986) 575-581 ]. Modification is understood to mean an
REFERENCES:
Obayashi et al. (1991), Proc. of the Soc. of Exptal. Biol & Med., vol. 196, No. 2, pp. 164-169.
Williams et al. (1991) Int. J. of Cell Cloning vol. 9, pp. 542-547.
Williams et al (1990) Exp. Hematol. (N.Y.) vol. 18. (6), p. 615.
Mertz Prema
Rhone-Poulenc Rorer S.A.
Ulm John
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