Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1996-11-27
1999-06-29
Fitzgerald, David L.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 695, 4353201, 435325, 435348, 435419, 4352523, 43525411, 530351, 536 234, 424 851, 514 2, C07K 1900, C12N 1562, A61K 3819
Patent
active
059167734
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to hybrid hematopoietic factors for the stimulation of hematopoiesis. In particular, to hybrid molecules of formula GM-CSF-L-EPO or EPO-L-GM-CSF comprising complete GM-CSF and EPO molecules or fragments thereof fused by means of a linking peptide (hereinafter simply indicated by the definition linker) L having a variable length in amino acids. Such hybrid molecules exhibit a higher specificity of action on erythroid differentiation if compared to that of an equimolar mixture of not-linked GM-CSF and EPO molecules.
STATE OF THE ART
Hematopoiesis is a multi-step cell proliferation and differentiation process starting from a pool of multipotent stem cells. These cells can proliferate and differenziate into hematopoietic progenitors in reply to different stimuli.
A group of proteins named growth factors or Colony Stimulating Factors (CSFs) or cytokines control survival, proliferation and differentiation of stem and progenitor cells. Furthermore, they affect several functional activities of mature terminal cells. In brief, at the level of immature cells, CSFs assure the self-renewal of the staminal pool and activate the first stage of hematopoietic differentiation; in the middle stage, when cell proliferation is associated to a progressive acquisition of characteristics of mature cells, they enormously enhance the number of differentiating cells; in the terminal stage they control the circulation and the activation of mature cells.
For many CSFs, the target cell is known. Interleukin 3 (IL3) acts on multipotent (CFU-GEMM), myeloid (CFU-GM), erythroid (BFU-E) and megakaryocytic (CFU-MK) progenitors, whereas GM-CSF exerts its effects on early progenitors. Erythropoietin (EPO), G-CSF, interleukin 5 (IL5) and M-CSF are specific for end-stage progenitors of the erythroid (CFU-E), granulocytic (CFU-G), eosinophilic (CFU-Eo) and monocytic (CFU-M) lineage, respectively. The importance of these molecules is justified by their potential clinical use.
In particular, the EPO administration, which causes an increase of blood cells, is useful in case of anaemia, aplasia or marrow hypoplasia induced by chemotherapy or radiant theraphy and in hemodialysed patients with chronic renal failure.
The administration of GM-CSF, as shown by some preliminary clinical studies, is useful when the organism absolutely needs to produce granulocytes, for example under AIDS conditions or during anti-blastic theraphy in case of neoplasia or transplantation. The interaction of several growth factors at the level of single cells can have sinergetic or antagonist additive effects. For example, severe anemia responds well to the combination of GM-CSF and IL3 with EPO (Clark S. C., Kamen R., 1987, Science, 236:1229).
Since current blood available is not sufficient for covering transfusion needs and as the risk of haematic contact diseases like AIDS, B and C hepatitis, etc. is higher and higher, it is justified the industrial interest for the production of recombinant hematopoietic factors or new molecules with hemopoietic activity.
Patent application WO 92/06116 discloses recombinant hematopoietic hybrid growth factors comprising an early factor and a late factor. As early factors are disclosed IL-3 and GM-CSF, whilst as late factors it discloses EPO, G-CSF, IL5 and M-CSF. Said patent application describes only hybrid growth factors IL3:EPO, EPO:IL3 and IL3:G-CSF, wherein the two components of each hybrid factor can be fused together directly or by means of a linker.
Although said patent application also quotes the GM-CSF molecule (pag.7), no description or indication is given with reference to the behaviour and use of said molecule in combination with EPO.
The authors of patent application WO 92/06116 assume that the early factor (IL3) down-regulates the expression of the receptors for the late fator (EPO) and therefore it leads to decrease the receptors for the late factor. On the other hand, Testa U., et al. (Blood, 81, 1442-1456, 1993) found that the early factor acts positively up-stimul
REFERENCES:
patent: 5567611 (1996-10-01), Ralph et al.
Testa et al. "Cascade Transactivation of Growth Factor Receptors in Early Human Hematopoiesis" Mar. 15, 1993 pp. 1442-1456.
Clark et al. "The Human Hematopoietic Colony-Stimulating Factors" Jun. 5, 1987, pp. 1229-1237.
Valtieri et al. "Erythropoietin Alone Induces Erythroid Burst Formation by Human Embryonic but Not Adult BFU-E in Unicellular Serum-Free Culture" Jul. 1989 pp. 460-470.
Felgner "Cationic Liposome-Mediated Transfection" pp. 21-37.
Urlaub et al. "Isolation of Chinese Hamster Cell Mutants Deficient in Dihydrofolate Reductase Activity" Jul. 1980 pp. 4216-4221.
C. Peschle "Stringently Purified Human Hematopoietic Progenitors/Stem Cells: Analysis of Cellular/Molecular Mechanisms Underlying Early Hematopoiesis" Apr. 12, 1993, pp. 356-370.
Weich, N.S., et al. (1993) Exp. Hematol. 21: 647-55.
Carloni Cristina
Coscarella Annamaria
De Santis Rita
Mele Antonio
Fitzgerald David L.
Menarini Ricerche S.p.A.
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