Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Patent
1996-12-19
2000-10-10
Draper, Garnette D.
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, 424 852, C07K 1454, A61K 3820
Patent
active
061303186
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to novel hIL-4 mutant proteins, to processes for preparing them, and to their use as medicaments, in particular in association with overshooting, falsely regulated immune reactions and autoimmune diseases.
2. Description of Related Art
PCT WO 93/10235 already discloses therapeutic agents which are or which contain antagonists or partial agonists of hIL-4, with the antagonists or partial agonists being hIL-4 mutant proteins.
Human interleukin 4 hIL-4) is one of the many cytokines which induce and coordinate the proliferation, maturation, survival and differentiation of lymphoid and myeloid cells. In particular, hIL-4 is involved in the IgE-mediated immune reaction and directly accelerates the proliferation of thymocytes and activated T cells. A high-affinity IL-4 receptor protein of Mr 140,000 has been identified which, according to its cDNA sequence, consists of 800 amino acid residues. This protein belongs to a recently described group of receptors which are designated the haematopoietin receptor superfamily.
Based on the cloned cDNA, the amino acid sequence of the mature IL-4 consists of 129 residues. The cDNA has been expressed in E. coli and yeast. Recombinant IL-4 having high biological activity can be isolated from these sources.
Very recently, a monoclonal antibody has been disclosed which exhibits antagonistic properties towards human interleukin 4. This antibody contains a Fab fragment and is produced by a human/human hybridoma cell line. A hybridoma cell line from the spleen cells of a rat which was immunized against (non-)glycosylated human IL-4 also produces monoclonal antibodies against hIL-4.
The role of interleukin 4 in allergic processes provides grounds for hoping that substances which inhibit interleukin 4-mediated processes, or compete with hIL4, might interrupt the disease-triggering reaction chain.
DE 41 37 333 A1 describes hIL-4 mutant proteins in which the amino acid(s) occurring naturally in the wild type at one or more of positions 120, 121, 122, 123, 124, 125, 126, 127 or 128 has/have been replaced with one or more, respectively, of the other possible natural amino acids. These hIL-4 mutant proteins are antagonists or partial agonists of human IL-4.
SUMMARY OF THE INVENTION
The present invention now relates to novel hIL-4 mutant proteins which are antagonists or partial agonists of human interleukin 4 and in which further modifications of the hIL-4 protein have been carried out in addition to the replacement(s) at positions 121, 124 or 125. These modifications are carried out in order to increase the stability of the hIL-4 mutant proteins, in order to extend the biological half life or in order to facilitate the preparation and purification process.
For this, the amino acids which naturally occur in the wild type are deleted, or replaced by other amino acids, at one or more positions, or else additional amino acids are inserted, at the C terminus or at the N terminus as well, or else one or more of the amino acids is/are substituted by various non-protein polymers, for example polyethylene glycol and its derivatives, or by glycosyl residues.
Within the context of the invention, amino acids are generally designation.
Non-protein polymers are understood, for example, as being polyethylene glycol, polypropylene glycol or polyoxyalkylenes, as described in U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 or 4,179,337.
Glycosylation is understood as being the linking of a carbohydrate skeleton to the side chain of an asparagine residue ("N-glycosylation") or the coupling of a sugar, preferably N-acetylgalactosamine, galactose or xylose to serine, threonine, 4-hydroxyproline or 5-hydroxylysine (O-glycosylation).
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in more detail with reference to the drawing, wherein:
FIG. 1 is a map of vector RPR9-IL 4-Y124 4327.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Preference is given to hIL-4 mutant proteins in which a
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Apeler Heiner
Beunink Jurgen
Dorschug Michael
Hanko Rudolf
Horlein Hans-Dietrich
Bayer Aktiengellschaft
Draper Garnette D.
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