Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification
Reexamination Certificate
2000-02-24
2004-01-06
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
C530S350000, C514S002600, C514S021800, C424S198100, C424S278100, C536S023100, C435S069100, C435S320100
Reexamination Certificate
active
06673609
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to polypeptides having part or all of the primary structural conformation of erythropoietin and having an improved in vivo half-life and biological activity due to a modified glycosylation profile. The present invention also provides DNA sequences encoding the amino acid sequence of said polypeptides operatively linked to regulatory elements which allow for the expression of said DNA sequence in eukaryotic host cells as well as vectors comprising such DNA sequences. The present invention also relates to host cells comprising the aforementioned DNA sequences and vectors and their use for the production of the aforedescribed polypeptides. Furthermore, the present invention relates to pharmaceutical and diagnostic compositions comprising the aforementioned polypeptides, DNA sequences and vectors. The present invention also relates to the use of the aforedescribed polypeptides, DNA sequences and vectors for the preparation of pharmaceutical compositions for treating all kinds of anaemia caused by a lack of erythropoietin.
2. Description of the Related Art
The erythrocyte is by far the most common type of cell in the blood. When mature, it is packed full of hemoglobin and contains practically none of the usual cell organelles. In an erythrocyte of an adult mammal, even the nucleus, endoplasmic reticulum, mitochondria, and ribosomes are absent, having been extruded from the cell in the course of its development. The erythrocyte, therefore, cannot grow or divide; the only possible way of making more erythrocytes is by means of stem cells. Furthermore, erythrocytes have a limited life span of about 120 days in humans. Worn-out erythrocytes are phagocytosed and digested by macrophages in the liver and spleen, which remove more than 10
11
senescent erythrocytes in every human being per day. A lack of oxygen or a shortage of erythrocytes stimulates cells in the kidney to synthesize and secrete increased amounts of erythropoietin into the blood-stream. The erythropoietin in turn stimulates the production of more erythrocytes. Since the change in the rate of release of new erythrocytes into the blood-stream is observed as early as 1 or 2 days after an increase in erythropoietin levels in the blood-stream, the hormone must act on cells that are very close precursors of the mature erythrocytes. The cells that respond to erythropoietin can be identified by culturing bone marrow cells in a semisolid matrix in the presence of erythropoietin. In a few days colonies of about 60 erythrocytes appear, each founded by a single committed erythrocyte progenitor cell. This cell is known as an erythrocyte colony-forming cell, or CFC-E, and gives rise to mature erythrocytes after about six division-cycles or less. The CFC-Es do not yet contain hemoglobin, and they are derived from an earlier type of progenitor cell whose proliferation does not depend on erythropoietin. CFC-Es themselves depend on erythropoietin for their survival as well as for proliferation: if erythropoietin is removed from the cultures, the cells rapidly undergo programmed cell death. Erythropoietin as other colony stimulating factor is a glycoprotein that acts at low concentration (about 10
−12
M) by binding to specific cell-surface receptors. These receptors belong to a large receptor family, the so-called “cytokine receptor family”, whose members are usually composed of two or more subunits, one of which is frequently shared among several receptor types. Mature human erythropoietin is a glycoprotein with a molecular weight of 34 to 38 kD and consists of 166 amino acids (AS) and the glycosyl residue accounts for about 40% of the molecular weight. Since erythropoietin is required for the renewal of erythrocytes, this hormone is essential for the quality of life, especially of patients, which suffer from anaemia and hypoxia, due to reduced numbers of red blood cells which can be caused by, e.g., dialysis or through reduction of erythroid precursor cells as a consequence of therapies based on the suppression of cellular proliferation or by inborne or aquired insufficiency of erythropoietin production. The identification of the human gene encoding erythropoietin made it possible to recombinantly express this protein in heterologous host cells and to provide sufficient amounts of recombinant human erythropoietin (rhuEpo) for the treatment of the diseases mentioned. However, apart from the primary structure of the protein the structure of the sugar side chains of the molecule is of particular importance for the interaction of Epo within the organism. For example, desialylated Epo shows no effect upon application in animals. It nevertheless binds to the receptor and stimulates precursor cells. The activity decrease of asialo-Epo in vivo can be explained by the fact that it is removed in the liver via receptors with a specificity for galactosyl residues which are susceptible in desialylated Epo.
The wildtype Epo, which has been used therapeutically, has in some patients the effect of increasing the blood pressure, which is disadvantageous in therapy. It is to be assumed that Epo also is integrated in the blood pressure regulation. Therefore, it is desirable to have proteins with the physiological effect of Epo at one's disposal which do, however, not show these undesired properties but which nevertheless stimulate the differentiation and division rate of precursor cells to erythrocytes. A further side effect of Epo found in some patients is the stimulation of the megakaryocytes for the formation of thrombocytes. Therefore, there is potential danger of thrombosis during the therapy with Epo, which then has to be discontinued immediately. In this case, a higher specificity of the Epo used would be desirable.
Thus, the technical problem underlying the present invention is to provide rhuEpo having an improved biological activity and in-vivo half-life compared to naturally occurring and rhuEpo so far available.
BRIEF SUMMARY OF THE INVENTION
The solution to the above technical problem is achieved by providing the embodiments characterized in the claims.
Accordingly, the invention relates to a polypeptide having part or all of the primary structural conformation of erythropoietin possessing the biological property of causing bone marrow cells to increase production of reticulocytes and red blood cells and to increase haemoglobin synthesis or iron uptake, said polypeptide being the product of eukaryotic expression of an exogenous DNA sequence and having the following physiochemical properties:
(i) the amino acid sequence comprises the amino acid sequence given in SEQ ID No. 1 or any fragment or derivative thereof by way of amino acid deletion, substitution, insertion, addition and/or replacement of the amino acid sequence given in SEQ ID No. 1, wherein at least one of the consensus N-linked glycosylation sites is modified to other than a consensus N-linked glycosylation site;
(ii) it is glycosylated;
(iii) greater 5% of the N-glycan structures are sulfated; and
(iv) the ratio Z* of the total N-glycan charge Z to the number of N-glycosylation sites is greater than 170.
REFERENCES:
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Boissel, J.-P. et al. (993) Erythropoietin structure-function relationships. Mutant proteins that test a model of tertiary structure. J. Biol. Chem. vol. 268, pp. 15983-15993.*
Higuchi, M. et al. (1992) Role of sugar chains in the expression of the biological activity of human erythropoietin. J. Biol. Chem. vol. 267, pp. 7703-7709.*
Conradt, H. S. et al. (1997) The stryctural Variety of natural glycoproteins requires the construction of novel cell factories for the biotechnological production of improved recombinant human therapeutics. pp. 1-22.*
Fibi et al.; N- and O-Glycosylation Muteins of Recombinant Human Erythropoietin Secreted From BHK-21 Cells; Blood; vol. 84, No. 5; 1995; pp. 1229-1236; XP-002053700.
Takeuchi et al
Fibi Mathias
Hermentin Peter
Aventis Pharma Deutschland GmbH
Carlson Karen Cochrane
Heller Ehrman White and McAuliffe
Liu Samuel Wei
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