Patent EA-004793-B1 (EA)
Title: Chimeric interleukin-6 soluble receptor/ligand protein, analogs thereof and uses thereof
Inventor: REVEL MICHEL
IPC: A61P1/16
Filed: 1998-07-09
Abstract: 1. A chimeric glycosylated soluble interleukin-6 receptor (sIL-6R)-interleukin-6 (IL-6) protein (sIL-6R/IL-6), comprising a fusion protein product between the naturally occurring form of sIL-6R and naturally occurring form of IL-6. 2. A chimeric sIL-6R/IL-6 protein according to claim 1, wherein said sIL-6R is fused to IL-6 via a peptide linker molecule. 3. A chimeric sIL-6R/IL-6 protein according to claim 2, wherein said linker is a non-immunogenic linker of about 3 amino acid residues. 4. A chimeric sIL-6R/IL-6 protein according to claim 3, wherein said linker is a tripeptide of the sequence E-F-M (Glu-Phe-Met). 5. A chimeric sIL-6R/IL-6 protein according to claim 2, wherein said linker is a peptide of 13 amino acid residues of sequence E-F-G-A-G-L-V-L-G-G-Q-F-M (Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met). 6. A chimeric sIL-6R/IL-6 protein according to any one of claims 1-4, being the herein designated sIL-6RdeltaVal/IL-6 having a tripeptide linker of sequence E-F-M between the C-terminal Val-356 of sIL-6R and the N-terminal Pro-29 of IL-6R, said chimeric protein having the sequence set forth in Fig. 3. 7. A chimeric sIL-6R/IL-6 protein according to any one of claims 1, 2, and 5, being the herein designated sIL-6RdeltaVal/L/IL-6 having a 13 amino acid peptide linker of sequence E-F-G-A-G-L-V-L-G-G-Q-F-M between the C-terminal Val-356 of sIL-6R and the N-terminal Pro-29 of IL-6R, said chimeric protein having the sequence set forth in Fig. 3, wherein the tripeptide of sequence E-F-M between positions 357-359 of Fig. 3 is replaced by said 13 amino acid peptide sequence. 8. A chimeric sIL-6R/IL-6 protein according to claim 1 being the herein designated IL-6/sIL-6R having the entire sequence of IL-6 preceeding the sIL-6R sequence with a 14 amino acid peptide linker of sequence G-G-G-G-D-P-G-G-G-G-G-G-P-G (SEQ ID NO: 6) between the C-terminal Met-212 of IL-6 and the VAL-112 of sIL-6R, said chimeric protein having the sequence set forth in Fig. 11. 9. A chimeric sIL-6R/IL-6 protein according to any one of claims 1-8, wherein said protein is produced in mammalian cells in a fully processed form. 10. A chimeric sIL-6R/IL-6 protein according to claim 9, wherein said protein is produced in human cells. 11. A chimeric sIL-6R/IL-6 protein according to claim 9, wherein said protein is produced in CHO cells. 12. A DNA sequence encoding a chimeric sIL-6R/IL-6 protein and biologically active analogs thereof according to any one of claims 1-11. 13. A DNA vector comprising a DNA sequence encoding a chimeric sIL-6R/IL-6 protein and biologically active analogs thereof according to any one of claims 1-11, said vector being suitable for expression of said chimeric protein in mammalian cells. 14. A DNA vector according to claim 13, wherein said vector is suitable for expression of said chimeric protein in human cells. 15. A DNA vector according to claim 13, wherein said vector is suitable for expression of said chimeric protein in CHO cells. 16. A DNA vector according to any one of claims 13-15, wherein said vector is the herein designated plasmid pcDNA sIL-6R/L/IL-6 comprising a pcDNA3 vector containing the DNA sequence encoding the chimeric sIL-6R/IL-6 protein under the control of a cytomegalovirus (CMV) promoter. 17. Transformed mammalian cells containing a DNA vector according to any one of claims 13-16 which are capable of expressing the sIL-6R/IL-6 chimeric protein sequence carried by said vector and of fully processing the expressed protein and secreting it into the culture medium in which said cells are grown. 18. Transformed cells according to claim 17 wherein in said cells are the herein described human embryonal kidney cells 293 (HEK293) transfected by the pcDNA sIL-6R/I1-6 vector, said cells being capable of expressing the sIL-6R/IL-6 chimeric protein, fully processing said protein and secreting said protein into the culture medium in which said cells are grown in the form of an about 85 kDa glycoprotein. 19. A method for producing a chimeric protein according to any one of claims 1-11, comprising growing transformed cells according to claim 17 or 18 under conditions suitable for expression, processing and secretion of said protein or analogs into the culture medium in which said cells are grown; and purifying said protein or analogs from said culture medium. 20. A method according to claim 19, wherein the purification is carried out by immunoaffinity chromatography using monoclonal antibodies specific for sIL-6R. 21. The use of a chimeric sIL-6R/IL-6 protein according to any one of claims 1-11, salts of any one thereof, and mixtures thereof, as an inhibitor of cancer cells. 22. The use of a chimeric protein according to any one of claims 1-11, as an inhibitor of highly malignant melanoma cells. 23. The use of a chimeric sIL-6R/IL-6 protein according to any one of claims 1-11, salts of any one thereof, and mixtures thereof, as an active ingredient for eliciting engraftment of human hematopoietic cells in bone marrow transplantation. 24. The use of a chimeric sIL-6R/IL-6 protein or analogs according to any one of claims 1-11, salts of any one thereof, and mixtures thereof, as an active ingredient for protecting liver against hepatotoxic agents. 25. The use of a chimeric sIL-6R/IL-6 protein or analogs according to any one of claims 1-11, salts of any one thereof, and mixtures thereof for use in the preparation of a medicament for treating mammalian cancers by way of inhibition of mammalian cancer cells. 26. The use of a chimeric sIL-6R/IL-6 protein or analogs according to any one of claims 1-11, salts of any one thereof, and mixtures thereof for use in the preparation of a medicament for enhancement of bone marrow transplantation by way of eliciting engraftment of human hematopoietic cells in bone marrow transplantation. 27. The use of a chimeric sIL-6R/IL-6 protein or analogs according to any one of claims 1-11, salts of any one thereof, and mixtures thereof for use in the preparation of a medicament for increasing haemopoiesis. 28. The use of a chimeric sIL-6R/IL-6 protein or analogs according to any one of claims 1-11, salts of any one thereof, and mixtures thereof for use in the preparation of a medicament for treating liver. 29. The use of a chimeric sIL-6R/IL-6 protein or analogs according to any one of claims 1-11, salts of any one thereof, and mixtures thereof for use in the preparation of a medicament for treating neurological disorders. 30. A pharmaceutical composition comprising as active ingredient a chimeric sIL-6R/IL-6 protein or analog thereof according to any one of claims 1-11, and a pharmaceutically acceptable carrier, diluent or excipient. 31. A pharmaceutical composition according to claim 30 for the treatment of cancers. 32. A pharmaceutical composition according to claim 30 for the enhancement of bone marrow transplantation. 33. A pharmaceutical composition according to claim 30 for increasing haemopoiesis. 34. A pharmaceutical composition according to claim 30 for the treatment of liver. 35. A pharmaceutical composition according to claim 30 for the treatment of neurological disorders.