Therapeutic agents which are antagonists or partial agonists of

Details Therapeutic agents which are antagonists or partial agonists of Therapeutic agents which are antagonists or partial agonists of

1994-05-06

1998-03-03

Walsh, Stephen

Drug, bio-affecting and body treating compositions

Lymphokine

Interleukin

435 721, 435 6952, 4352523, 4353201, 530351, 514 12, C07K 1454, A61K 3820

Patent

active

057231184

DESCRIPTION:

BRIEF SUMMARY
Therapeutic agents which are antagonists or partial agonists of human interleukin 4, or contain these antagonists or partial agonists, and mutant hIL-4 proteins, as well as processes for their preparation
Broad sectors of the population suffer nowadays from allergies. The increasing pollution of the air and the increase in number of allergy-eliciting substances which are diffused within the environment make it probable that the number of cases will increase still further in the future, particularly in children. For this reason, it is urgently necessary to develop medicaments which can intervene in the course of allergic processes.
Human interleukin 4 (hIL-4) is one of the numerous cytokines which induce and coordinate the proliferation, the maturation, the survival and the differentiation of lymphoid and myeloid cells (1-3). In particular, hIL-4 participates in the IgE-mediated immune reaction and directly accelerates the proliferation of thymocytes and activated T cells. It has been possible to identify a high-affinity IL-4 receptor protein of Mr 140,000 which, according to its cDNA sequence, consists of 800 amino acid residues (4). This protein belongs to a recently described group of receptors which are designated the haematopoietin receptor superfamily (5).
Based on the cloned cDNA (6), the amino acid sequence of the mature IL-4 consists of 129 residues. The cDNA has been expressed in E. coli (7, 8) and yeast (9). Recombinant IL-4 possessing a high level of biological activity can be isolated from these sources.
The role of interleukin 4 in allergic processes makes it seem likely that substances which inhibit interleukin-4-mediated processes, or compete with hIL-4, will interrupt the disease-eliciting reaction chain.
It is, therefore, the object of the invention to make available therapeutic agents whose active constituents are antagonists or partial agonists of human interleukin 4.
Very recently, a monoclonal antibody has become known which exhibits antagonistic properties towards human interleukin 4 (10). This antibody contains a Fab fragment and is produced by a human-human hybridoma cell line. In addition, a hybridoma cell line from the spleen cells of a rat immunized against (non-)glycosylated human IL-4 produces monoclonal antibodies against hIL-4 (11).
The stated object was achieved, in accordance with the present invention, by making available therapeutic agents which are antagonists or partial agonists of hIL-4, or which contain these antagonists or partial agonists, and are characterized in that the antagonists or partial agonists are mutant hIL-4 proteins. The choice of the therapeutic agents according to the invention has the advantage that "genetic engineering" can be used, in a targeted manner, to prepare proteins which, due to their similarity to the wild-type hIL-4, compete with the latter for occupation of the hIL-4 receptor.
In addition, it is an object of the invention to make available mutant hIL-4 proteins as well as processes for their preparation.
hIL-4 can be produced as a recombinant protein (rhIL-4) by genetic manipulation, e.g. in E. coli. The protein which is formed under these circumstances can be solubilized, renatured and isolated. The rhIL-4 then possesses a high level of specific biological activity, which can be determined, for example, by measuring the DNA synthesis/proliferation of activated T cells, or the CD23 expression of activated B cells (see, e.g., Kruse, N. et al. (1991) FEBS Lett. 286, 58-60; Kikutani, H. et al. (1986) Cell 47, 657-665).
In accordance with the invention, a process has now been designed by which mutant proteins of the hIL-4 wild type can be produced, which proteins possess the properties of hIL-4 antagonists or partial hIL-4 agonists. The antagonists of hIL-4, in particular, offer the possibility of specifically inhibiting the effect of hIL-4. For this purpose
cDNA, which contains a DNA region which encodes the mature region of hIL-4, is subjected to a targeted oligonucleotide mutagenesis (site-directed mutagenesis) such that a selected diffe

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