Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Patent
1997-10-28
1999-10-26
Mertz, Prema
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
424 852, 435 6952, 4352523, 435325, 4353201, 435 712, 435471, 536 235, 530351, C07K 1454, C12N 510, C12N 1524, A61K 3820
Patent
active
059729023
DESCRIPTION:
BRIEF SUMMARY
DESCRIPTION
The present invention relates to antagonists of human interleukin-6 (hIL-6), that are totally incapable of binding the receptor chain (that is to say with gp 130) responsible for transducing the signal associated with this cytokine.
As is known, in known hIL-6 antagonists binding with gp 130 is actually weakened, but not totally abolished. This circumstance may result, in certain cells that are particularly sensitive to the action of the cytokine, in the known hIL-6 antagonists working as weak agonists. For this reason they have no therapeutic use in the formulation of drugs for the treatment of diseases such as multiple myeloma, rheumatoid arthritis, lupus erythematosus and osteoporosis.
There is therefore a need in this specific sector for human interleukin-6 antagonists whose use is not associated with the above risk.
The use of human interleukin-6 antagonists according to the present invention allows all the problems indicated above to be overcome, and also offers other advantages which will become clear from the following.
Subject of the present invention are therefore antagonists of human interleukin-6 (hIL-6), characterised in that they are totally incapable of binding with gp 130 and consist of an amino acid sequence chosen from the group comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.
Another subject of the present invention are: isolated and purified molecules of DNA coding for the interleukin-6 antagonists indicated above; recombinant DNA molecules comprising the DNA molecules mentioned above operatively bound to a sequence controlling expression in said recombinant DNA molecules; a unicellular host transformed using the recombinant DNA molecules, the unicellular host being selected from the group comprising bacteria, yeasts and other fungi, animal cells and plant cells. Further subjects of the present invention are the use of human interleukin-6 antagonists of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12 and of recombinant DNA containing DNA encoding such antagonists for the preparation of pharmaceutical compounds and the use of said antagonists as active principles in the preparation of drugs for the treatment of multiple myeloma, rheumatoid arthritis, lupus erythematosus and osteoporosis.
Up to this point a general description has been given of the present invention. With the aid of the following examples, a more detailed description of a form of embodiment of the invention will now be given, in order to give a clearer understanding of the aims, characteristics, advantages and methods of preparation.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the absence of interaction between antagonists according to the invention and gp 130.
FIG. 2 shows the biological antagonism of the antagonists according to the invention compared to wild type interleukin-6 on human hepatoma cells.
FIG. 3 shows the absence of gp130 interaction of the mutant Sant 7 according to the present invention.
EXAMPLE 1
Present Invention
In all the mutagenesis reactions the plasmid pT7.7/IL-6/DFRD/Hind was used as a template. This plasmid is a derivative from the plasmid pT7.7 (Studier, F. W. and Moffat, B. A., J. Mol. Biol. 189, 113-130, 1986) and contains the coding region of human IL-6 (hIL-6) cloned between sites NdeI and ClaI of the pT7.7 polylinker. The human IL-6 gene is controlled by the T7 RNA polymerase promoter, present in pT7.7. Mutations were introduced into four codons in the region coding for human IL-6 cloned into pT7.7/IL-6/DFRD/Hind (see SEQ ID NO:1), creating the following amino acid substitutions: Tyr31Asp, Gly35Phe, Ser118Arg and Val121Asp. The published international application No. W095/00852 by the same Applicant, claiming Italian priority date 20.06.93, teaches that the introduction of the four amino acid substitutions indicated above into wild-type human IL-6 drastically reduces the biological activity of the cytokine modified in this way, without altering its ability to bind to the hIL-6 receptor itself, thus generating IL-6 DFRD (see SEQ ID NO:8), a
REFERENCES:
EMBO Journal, vol. 13, No. 24, 1996, Eynsham, Oxford, GB, pp. 5863-5870, XP002012981. Savino, R. et al., Rational design of a receptor super-antagonists of human Interleukin-6.
EMBO Journal, vol. 14, No. 9, 1995, Eynsham, Oxford, GB, pp. 1942-1951, XP000565718. Paonessa, G. et al., "Two distinct and independent sites on iL-6 trigger gp130 dimer formation and signalling".
Proc. of the Amer. Assoc. for Cancer Res. Annual Meeting, vol. 37, No. 0, 1996, pp. 410-411, XP000601653. Borsellino, N. et al., "Effects of an iL-6 receptor super-antagonist (iL-6R Sant 7) on the growth and sensitivity to etoposide (VP-16) of the hormone-Resistant and iL-6-secreting prostate tumor PC-3 cell line."
Ciliberto Gennaro
Paonessa Giacomo
Savino Rocco
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