Patent EA-005248-B1 (EA)
Title: Method for down-regulating gdf-8 activity
Inventor: HALKIER TORBEN
IPC: C12N5/10
Filed: 2000-07-20
Abstract: 1. A method for in vivo down-regulation of growth differentiation factor 8 (GDF-8) activity in an animal, including a human being, the method comprising effecting presentation to the animal's immune system of an immunogenically effective amount of at least one GDF-8 analogue wherein is introduced at least one modification in the GDF-8 amino acid sequence which has as results that - immunization of the animal with the analogue induces production of antibodies against the GDF-8 polypeptide, and - a substantial fraction of GDF-8 B-cell epitopes are preserved, and - a foreign T helper epitope (TH epitope) is introduced corresponding to any one of residues 18-41, 49-69, or 79-104 in SEQ ID NO: 11 or 12 by means of addition, deletion, insertion or substitution, or introduced corresponding to an equivalent sequence from a GDF-8 polypeptide of different origin than human, bovine, porcine, chicken or turkey, or a foreign TH epitope is inserted or substituted into any one of the loop areas or the flexible termini in the GDF-8 polypeptide, wherein a number of aminoacid additions, deletions, insertions or substitutions is not more than 60. 2. The method according to claim 1, which further comprises that - at least one first moiety is introduced which effects targeting of the modified molecule to an antigen presenting cell (APC) or a B-lymphocyte, and/or - at least one second moiety is introduced which stimulates the immune system, and/or - at least one third moiety is introduced which optimises presentation of the modified GDF-8 polypeptide to the immune system. 3. The method according to claim 1 or 2, wherein the modification includes introduction as side groups, by covalent or non-covalent binding to suitable chemical groups in GDF-8 or a subsequence thereof, of the foreign TH epitope and/or of the first and/or of the second and/or of the third moiety. 4. The method according to any one of claims 1-3, wherein the modification includes amino acid substitution and/or deletion and/or insertion and/or addition. 5. The method according to claim 4, wherein the modification results in the provision of a fusion polypeptide. 6. The method according to claim 4 or 5, wherein introduction of the amino acid substitution and/or deletion and/or insertion and/or addition results in a substantial preservation of the overall tertiary structure of GDF-8. 7. The method according to any one of claims 1-6, wherein the modification includes duplication of at least one GDF-8 B-cell epitope and/or introduction of a hapten. 8. The method according to any one of claims 1-7, wherein the foreign TH epitope is immunodominant in the animal. 9. The method according to any one of claims 1-8, wherein the foreign TH epitope is promiscuous, such as a natural promiscuous TH epitope and an artificial MHC-II binding peptide sequence. 10. The method according to claim 9, wherein the natural TH epitope is selected from a Tetanus toxoid epitope such as P2 or P30, a diphtheria toxoid epitope, an influenza virus hemagluttinin epitope, and a P. falciparum CS epitope. 11. The method according to any one of claims 2-10, wherein the first moiety is a substantially specific binding partner for a B-lymphocyte specific surface antigen or for an APC specific surface antigen, such as a hapten or a carbohydrate for which there is a receptor on the B-lymphocyte or the APC, such as mannan or mannose. 12. The method according to any one of claims 2-11, wherein the second moiety is selected from a cytokine, a hormone, and a heat-shock protein. 13. The method according to claim 12, wherein the cytokine is selected from, or is an effective part of, interferon gamma (IFNgamma), Flt3L, interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 9 (IL-4), interleukin 6 (IL-6), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 15 (IL-15), and granulocyte-macrophage colony stimulating factor (GM-CSF), and wherein the heat-shock protein is selected from the group consisting of HSP70, HSP90, HSC70, GRP94, and calreticulin (CRT), or an effective part thereof. 14. The method according to any one of claims 2-13, wherein the third moiety is of lipid nature, such as a palmitoyl group, a myristyl group, a farnesyl group, a geranyl-geranyl group; a GPI-anchor, and an N-acyl diglyceride group. 15. The method according to any one of the preceding claims, wherein the GDF-8 subsequence or the GDF-8 analogue is derived from the C-terminal, active form of GDF-8, such as subsequence or analogue derived from a bovine, porcine, human, chicken, sheep, or turkey GDF-8 polypeptide. 16. The method according to claim 15, wherein the GDF-8 polypeptide has been modified by substituting at least one amino acid sequence in SEQ ID NO: 11 or 12 with at least one amino acid sequence of equal or different length which contains a foreign TH epitope, wherein substituted amino acid sequences are comprised in residues 1-12, 18-91, 43-48, 49-69, or 79-104 in SEQ ID NO: 11 or 12, or wherein the GDF-8 polypeptide has been modified by inserting at least one amino acid sequence which contains a foreign TH epitope, wherein insertion is performed anywhere in positions 1-12, 18-30, 42-51, 82-86, and 105-109 in SEQ ID NO: 11 or 12. 17. The method according to any one of the preceding claims, wherein presentation to the immune system is effected by having at least two copies of the GDF-8 polypeptide, the subsequence thereof or the modified GDF-8 polypeptide covalently of non-covalently linked to a carrier molecule capable of effecting presentation of multiple copies of antigenic determinants. 18. The method according to any one of the preceding claims, wherein an effective amount of the GDF-8 polypeptide or the GDF-8 analogue is administered to the animal via a route selected from the parenteral route such as the intradermal, the subdermal, the intracutaneous, the subcutaneous, and the intramuscular routes; the peritoneal route; the oral route; the buccal route; the sublinqual route; the epidural route; the spinal route; the anal route; and the intracranial route. 19. The method according to claim 18, wherein the effective amount is between 0.5 mug and 2,000 mug of the GDF-8 polypeptide, the subsequence thereof or the analogue thereof. 20. The method according to claim 18 or 19, which includes at least one administration of the GDF-8 polypeptide or analogue per year, such as at least 2, at least 3, at least 4, at least 6, and at least 12 administrations per year. 21. The method according to any one of claims 18-20, wherein the GDF-8 polypeptide, the subsequence thereof, or the modified GDF-8 polypeptide optionally has been formulated with a pharmaceutically and immunologically acceptable carrier and/or vehicle and has been formulated with an adjuvant which facilitates breaking of autotolerance to autoantigens, such as an adjuvant selected from the group consisting of an immune targeting adjuvant; an immune modulating adjuvant such as a toxin, a cytokine and a mycobacterial derivative: an oil formulation; a polymer; a micelle forming adjuvant; a saponin; an immunostimulating complex matrix (an ISCOM matrix); a particle; DDA; aluminium adjuvants; DNA adjuvants; gamma-inulin: and an encapsulating adjuvant. 22. The method according to any one of claims 19-21, wherein the GDF-8 polypeptide or analogue is contained in a virtual lymph node (VLN) device. 23. The method according to any one of claims 1-17, wherein presentation of modified GDF-8 to the immune system is effected by introducing nucleic acid(s) encoding the modified GDF-8 into the animal's cells and thereby obtaining in vivo expression by the cells of the nucleic acid(s) introduced. 24. The method according to claim 23, wherein the nucleic acid(s) introduced is/are selected from naked DNA, DNA formulated with charged or uncharged lipids, DNA formulated in liposomes, DNA included in a viral vector, DNA formulated with a transfection-facilitating protein or polypeptide, DNA formulated with a targeting protein or polypeptide, DNA formulated with Calcium precipitating agents, DNA coupled to an inert carrier molecule, DNA formulated with chitin or chitosan, and DNA formulated with an adjuvant. 25. The method according to claim 23 or 24, wherein the nucleic acids are administered intraarterially, intravenously, or by the routes defined in claim 18. 26. The method according to claim 24 or 25, wherein the nucleic acid(s) is/are contained in a VLN device and/or are formulated as defined in claim 21. 27. The method according to any one of claims 24-26, which includes at least one administration of the nucleic acids per year, such as at least 2, at least 3, at least 4, at least 6, and at least 12 administrations per year. 28. A method for increasing the muscle mass of an animal, the method comprising down-regulating GDF-8 activity according to the method of any one of claims 1-27 to such an extent such that the muscle mass is increased at least 5% when compared to animals which exhibit normal GDF-8 activity, such as at least 10, 15, 20, 25, 30, 35, 40, and 45%. 29. A GDF-8 analogue which is derived from an animal GDF-8 polypeptide wherein is introduced a modification as defined in any one of claims 1-17. 30. An immunogenic composition comprising an immunogenically effective amount of an GDF-8 analogue according to claim 29, the composition further comprising a pharmaceutically and immunologically acceptable carrier and/or vehicle and optionally an adjuvant. 31. An immunogenic composition according to claim 30, wherein the adjuvant is selected from the group consisting of the adjuvants of claim 21. 32. A nucleic acid fragment which encodes a GDF-8 analogue according to claim 29. 33. A vector carrying the nucleic acid fragment according to claim 32, such as a vector capable of autonomous replication. 34. The vector according to claim 33 which is selected from the group consisting of a plasmid, a phage, a cosmid, a minichromosome, and a virus. 35. The vector according to claim 33