Drug – bio-affecting and body treating compositions – Lymphokine – Interleukin
Patent
1996-08-14
1998-12-15
Ulm, John
Drug, bio-affecting and body treating compositions
Lymphokine
Interleukin
530351, 514 2, 514 8, 514 12, 930141, 4352523, 43525233, 4353201, 435 6952, 435 711, 435 712, 4351721, 4351723, C07K 1454, A61K 3819
Patent
active
058492836
DESCRIPTION:
BRIEF SUMMARY
DESCRIPTION
The present invention relates to a methodology for selecting superagonists, antagonists and superantagonists of human interleukin-6 (hereinafter referred to also as h IL-6 or IL-6) based on three-dimensional modelling.
As is known, WO 92/21029 to Genentech Inc. teaches a method for determination of agonists or antagonists of growth hormones and ligands with a similar structural conformation. The potential agonists and antagonists are put into contact with a receptor for the hormone and this causes formation of a ternary complex consisting of a molecule of the potential agonist or antagonist and two molecules of such receptor for the hormone to be agonized or antagonized. Dimerization of receptors induced by a ligand molecule allows to conclude that the ligand has two different interaction sites (site 1 and site 2), on which it is possible to operate using mutagenesis to generate agonists or antagonists.
It is known that the ligands in the group of cytokines similar to Interleukin 6 (IL-6), that is Oncostatin M (OSM), Leukemia Inhibitory Factor (LIF), Ciliary Neurotrophic Factor (CNTF), and Interleukin 11 (IL-11), induce the formation of a receptor complex of which the membrane molecule gp 130 is a part. In this receptor complex the specific receptor for each of these cytokines and the membrane molecule gp 130 are always present as common elements. It is thus possible to formulate the hypothesis that site 1 and site 2 bind to two different molecules in this class of hormones: site 1 to the specific receptor and site 2 to gp 130. Identification of the two sites is made possible, as will be seen more clearly from the following, by construction of a three-dimensional model of the receptor complex based on the functional similarity between sequences of the human growth hormone (hGH) receptor and sequences of the receptors for the hormones in question. Isolation of variants that, with respect to the wild type hormone, have a greater affinity for the specific receptor (superagonists or superantagonists) is obtained by construction of filamentous phage libraries, for example M13, carrying the hormone, both in the wild type and mutant version.
According to the invention, the difference between the three-dimensional model, for example of IL-6, adopted here and the one adopted in WO92/21029 leads to identify different residues in helix A and C as constituents of site 2. In fact, for the construction of the IL-6 model according to present invention the structure of a different cytokine instead of growth hormone, was used as template.
Modelling of the human interleukin 6 molecule is performed as follows. It is known, from data available in scientific literature, that the amino acid sequence of human interleukin 6 shows similarities with that of the granulocyte colony stimulating factor (G-CSF). The three-dimensional structure of bovine granulocyte colony stimulating factor (bG-CSF), determined using X-ray crystallography, was used as template to develop a three-dimensional model of human IL-6 from residues 16 to 184. Firstly, the amino acid sequence of human IL-6 was aligned with that of bG-CSF. On the basis of the derived alignment, the amino acid residues in the bG-CSF three-dimensional structure were replaced by the corresponding residues of human IL-6 using molecular modelling program in a computerized interactive graphic unit. In the positions in which alignment involves either deletions or insertions (which suggests a different local structure in the interleukin 6 molecule) adjustments were made by applying the options provided by the molecular modelling program.
This three-dimensional model of interleukin 6, based on the bG-CSF structure, has enabled the identification of the two sites of interaction between human interleukin 6 and its two receptors: the low affinity receptor gp 80 (site 1) and the high affinity signal transducer receptor gp 130 (site 2). The following procedure was used to identify the two sites. From sequence comparison it is known that all the members of the family of hematopoiet
REFERENCES:
Savino, Rocco et al. "Generation of interleukin 6 receptor antagonists by lecular-modeling guided mutagenesis of residues important for gp130 activation." The Embo Journal, vol. 13, No. 6, pp. 1357-1367 (1994).
Savino, Racino et al., "Saturation mutagenesis of the human interleukin 6 receptor binding site: implications for its three-dimensional structure." Proc. Natl. Acad. Sci., vol. 90, pp. 4067-4071, (May 1993).
Ehlers, Marc et al., "Identification of two novel regions of human IL-6 responsible for receptor binding and signal transduction." Journal of Immunology, vol. 153, No. 4, (Aug. 1994).
Cunningham et al. (1989) Science vol. 244, pp. 1081-1084.
Cwirla et al. (1990) PNAS, vol. 87, pp. 6378-6382.
Ciliberto Gennaro
Lahm Armin
Savino Rocco
Toniatti Carlo
Istituto di Ricerche di Biologia Molecolare P. Angeletti S.p.A.
Mertz Prema
Ulm John
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