Drug – bio-affecting and body treating compositions – Lymphokine – Interleukin
Reexamination Certificate
2000-04-21
2004-06-08
Mertz, Prema (Department: 1646)
Drug, bio-affecting and body treating compositions
Lymphokine
Interleukin
C424S184100, C424S198100, C530S350000
Reexamination Certificate
active
06746669
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to improvements in therapy and prevention of conditions characterized by an elevated level of eosinophil leukocytes, i.e. conditions such as asthma and other chronic allergic diseases. More specifically, the present invention provides a method for down-regulating interleukin 5 (IL5) by enabling the production of antibodies against IL5 thereby reducing the level of activity of eosinophils. The invention also provides for methods of producing modified IL5 useful in this method as well as for the modified IL5 as such. Also encompassed by the present invention are nucleic acid fragments encoding modified IL5 as well as vectors incorporating these nucleic acid fragments and host cells and cell lines transformed therewith. The invention also provides for a method for the identification of IL5 analogues which are useful in the method of the invention as well as for compositions comprising modified IL5 or comprising nucleic acids encoding the IL5 analogues.
BACKGROUND OF THE INVENTION
Asthma is a common disease of the airways, affecting about 10% of the population. The present treatments is primarily based on the administration of steroids and represents a market value exceeding well over a billion dollars. For yet unknown reasons the incidence and morbidity of asthmatics have increased worldwide over the past two decades. Today, an improved understanding of the immunological mechanisms involved in asthmatic conditions combined with an explosive development in biotechnology provides a new basis for the development of alternative and perhaps better strategies for treatment.
A general feature in the pathogenesis of asthma and other chronic allergic diseases has proven to be elevated numbers of eosinophils, especially in the bronchial mucosa of the lungs. Upon activation eosinophils secrete a number of mediators that are actively involved in the inflammatory airway response. In the activation of eosinophils, interleukin 5 (IL5) plays an important role.
IL5 is a cytokine found in many mammalian species and among others both the human and the murine gene for IL5 have been cloned (Tanabe et al., 1987, Campbell et al., 1988). The human gene consists of four exons with three introns positioned at chromosome 5 and codes for a 134 amino acid residue precursor, including a 19 amino acid N-terminal leader sequence which has the amino acid sequence set forth in SEQ ID NO: 62. Posttranslational cleavage generates the mature 115 amino acid residue protein (SEQ ID NO: 1). The murine IL5 (mIL5) gene similarly codes for a 133 amino acid residue pre-cursor with a 20 amino acid leader sequence which has the amino acid sequence set forth in SEQ ID NO: 64. The processed mature mIL5 is thus 113 amino acid residues long (SEQ ID NO: 12), missing two N-terminal amino acid residues by alignment with human IL5. The amino acid sequences of hIL5 and mIL5 are 70% identical compared to 77% at nucleotide level of the coding regions (Azuma et al., 1986). Higher similarity was reported within human primates; 99% identity is reported for the coding regions of the human and the Rhesus monkey nucleotide sequences (Villinger et al., 1995).
The human amino acid sequence has two potential N-glycosylation sites and the murine three. Human IL5 has been shown to be both N-glycosylated as well as O-glycosylated at Thr 3. Studies of hIL5 has demonstrated that the glycosylation is not necessary for the biological activity even though the stability seems to be affected by de-glycosylation (Tominaga et al., 1990; Kodama et al., 1993).
Structure of IL5
The active IL5 is a homo-dimer and the 3-dimensional structure of recombinant hIL5 has been determined by X-ray crystallography (Milburn et al., 1993). The 2 monomers are organised in an antiparallel manner and covalently bound by two interchain disulfide bridges (44-87′ and 87-44′), thus engaging all 4 cysteines of the 2 monomers.
The secondary structure of the monomers consists of 4 &agr;-helices (A-D) intermitted by 3 linking regions (loops) including two short stretches of &bgr;-sheets. This 4&agr; helix bundle is known as the “common cytokine fold”, which has also been reported for IL-2, IL-4, GM-CSF, and M-CSF. But all these are monomers and the homodimer-structure in which the D-helix completes the 4&agr; helix motif of the opposite monomer is unique to IL5.
The native monomers alone has been shown to be biologically inactive (for reviews see Callard & Gearing, 1994; Takutsu et al., 1997). It is nevertheless possible to produce a modified recombinant biologically active monomer by inserting 8 additional amino acid residues in loop 3, connecting the helices C and D. This enables helix D to complete the 4 helix structure within one polypeptide chain and thus enable the monomer to interact with its receptor (Dickason & Huston, 1996; Dickason et al., 1996).
The IL5 receptor is primarily present on eosinophils and it is composed of an &agr;-chain and a &bgr;-chain. The &agr;-chain of the receptor is specific for IL5 and the &bgr;-chain, which assure high-affinity binding and signal transduction, is shared with the hetero-dimer receptors for IL-3 and GM-CSF. The sharing of a receptor component could be the reason for the cross-competition seen between IL5, IL-3 and GM-CSF (for review, see Lopez et al., 1992). However, it was recently demonstrated that the regulation of the IL5R is distinct from the regulation of the IL-3R and the GM-CSFR, further indicating a highly specialised role of IL5 in the regulation of the eosinophilic response (Wang et al., 1998).
The C-terminal part of IL5 seems to be important in both binding to the IL5R and for the biological activity, since removal of more than two C-terminal amino acid residues results in a decline in both the binding affinity to the IL5 R and in the biological activity in an IL5 bioassay (Proudfoot et al., 1996). Other residues have also been found to be important for binding to the receptor, such as Glu12, which is involved in binding to the &bgr;-chain, while the Arg90 and Glu109 residues are involved in the binding to the &agr;-chain of the receptor. In general, binding to the IL5R seems to occur in regions overlapping helices A and D, where helix D is primarily responsible for the binding to the specific IL5R &agr;-chain (Graber et al., 1995; Takastsu et al., 1997).
IL5's Homology to Other Proteins
The two 4-helix domain motifs seen in the homodimer has strikingly similar secondary and tertiary structure as compared to the cytokine fold found in GM-CSF and M-CSF, IL-2, IL-4 and human and porcine growth hormone (Milburn et al., 1993). However, even though striking similarities are also observed in the intron/exon organisation and position of cysteines (Tanabe et al., 1987; Cambell et al., 1988) suggesting a phylogenetic relationship with IL-2, IL-4 and GM-CSF, no significant homology with any of these or other cytokines is observed from the amino acid sequence.
Biological Activity of IL5
IL5 is mainly secreted by fully differentiated Th2 cells, mast cells and eosinophils (Cousins et al., 1994; Takutsu et al., 1997). It has been shown to act on eosinophils, basophiles, cytotoxic T lymphocytes and on murine B cells (Callard & Gearing, 1994; Takutsu et al., 1997). The effects of IL5 on human cells are still a matter of controversy. Augmentation of immunoglobulin synthesis under certain circumstances and binding to a variety of human B cell lines have been demonstrated. Even though mRNA for the hIL5R has been found in human B-cells, the actual presence of the receptor on these cells has still to be verified (Baumann & Paul, 1997; Huston et al., 1996).
The actions of IL5 on eosinophils include chemotaxis, enhanced adhesion to endothelial cells, activation and terminal differentiation of the cells. Furthermore it has been demonstrated that IL5 prevents mature eosinophils from apoptosis (Yamaguchi et al., 1991). These findings have contributed to the present concept of IL5 as being the most important cytokine for eosinophil differentiation (Corrigan & Kay, 1996; Karlen e
Birch & Stewart Kolasch & Birch, LLP
Mertz Prema
Pharmexa A/S
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