Claims for Patent: 9,453,052

Summary for Patent: 9,453,052

Title:	Cyclotides as immunosuppressive agents
Abstract:	The present invention relates to a pharmaceutical composition comprising a cyclotide for use in immune suppression as well as to a method for immune suppression comprising the step of administering an effective amount of a pharmaceutical composition comprising such a cyclotide to a subject in need thereof. The present invention also relates to a pharmaceutical composition comprising a cyclotide for use in treating or preventing a disorder selected from the group consisting of (i) an autoimmune disorder; (ii) a hypersensitivity disorder; and (iii) a lymphocyte-mediated inflammation. Likewise, the present invention also relates to a method for treating or preventing a disorder selected from the group consisting of (i) an autoimmune disorder; (ii) a hypersensitivity disorder; and (iii) a lymphocyte-mediated inflammation. The present invention further relates to a method of screening for and/or selecting an immunosuppressive cyclotide or a mutation which results in a mutated cyclotide having an induced or enhanced immunosuppressive activity. The present invention further relates to a method of producing an immunosuppressive cyclotide or an immunosuppressive pharmaceutical composition. The present invention further relates to a mutated cyclotide having immunosuppressive activity and a pharmaceutical composition comprising the same.
Inventor(s):	Gruber; Christian Werner (Vienna, AT), Gruendemann; Carsten (Freiburg, DE)
Assignee:	Universitatsklinikum Freiburg (Freiburg, DE) Medizinische Universitat Wien (Vienna, AT)
Application Number:	14/366,427
Patent Claims:	1. A mutated cyclotide having immunosuppressive activity, said cyclotide comprising an amino acid sequence of formula IL wherein formula II comprises: Xxx.sub.1-Leu-Pro-Val-Cys-Gly-Glu-Xxx.sub.2-Cys-Xxx.sub.3-Gly-Gly-Thr-Cys- -Asn-Thr-Pro-Xxx.sub.1-Cys-Xxx.sub.1-Cys-Xxx.sub.1-Trp-Pro-Xxx.sub.1-Cys-T- hr-Arg-Xxx.sub.1 (SEQ ID NO: 17); wherein each Xxx.sub.1 position independently comprises any amino acid, non-natural amino acid or peptidomimetic; wherein Xxx.sub.2 comprises an amino acid non-natural amino acid or peptidomimetic but not Lys; wherein Xxx.sub.3 comprises any amino acid, non-natural amino acid or peptidomimetic but not Ala or Lys; wherein the mutated cyclotide comprises one or more mutations from the naturally occurring kalata B1 cyclotide (SEQ ID NO:1), wherein said cyclotide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence SEQ ID NO:7, and wherein the amino acid sequence of said cyclotide carries a mutation at least one amino acid position corresponding to an amino acid position which has been mutated in any one of the mutated cyclotides as depicted in SEQ ID NO: 5, 6 or 7. 2. The mutated cyclotide of claim 1, wherein said mutation is G.fwdarw.K at position 1, T.fwdarw.K at position 20, and/or N.fwdarw.K at position 29. 3. A cyclotide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, and 7. 4. A mutated cyclotide having immunosuppressive activity, said cyclotide comprising an amino acid sequence of formula IL wherein formula II comprises: Xxx.sub.1-Leu-Pro-Val-Cys-Gly-Glu-Xxx.sub.2-Cys-Xxx.sub.3-Gly-Gly-Thr-Cys- -Asn-Thr-Pro-Xxx.sub.1-Cys-Xxx.sub.1-Cys-Xxx.sub.1-Trp-Pro-Xxx.sub.1-Cys-T- hr-Arg-Xxx.sub.1 (SEQ ID NO:17); wherein each Xxx.sub.1 position independently comprises any amino acid, non-natural amino acid or peptidomimetic; wherein Xxx.sub.2 comprises an amino acid non-natural amino acid or peptidomimetic but not Lys; wherein Xxx.sub.3 comprises any amino acid, non-natural amino acid or peptidomimetic but not Ala or Lys; wherein the cyclotide comprises one or more mutations from the naturally occurring kalata B1 cyclotide (SEQ ID NO:1), wherein the cyclotide comprises an amino acid sequence having one or more of amino residues at amino acid positions 1, 18, 20, 22, 25, and/or 29 of SEQ ID NO:17 representing a different amino acid from the same position of SEQ ID NO:1, and wherein said at least one amino acid position corresponds to: (i) amino acid position 29 of the mutated cyclotide SEQ ID NO: 5; (ii) amino acid position 1 of the mutated cyclotide SEQ ID NO. 6; (iii) amino acid position 20 of the mutated cyclotide SEQ ID NO. 6; or (iv) amino acid position 20 of the mutated cyclotide as depicted in SEQ ID NO: 7. 5. A mutated cyclotide having immunosuppressive activity, said cyclotide comprising an amino acid sequence of formula IL wherein formula II comprises: Xxx.sub.1-Leu-Pro-Val-Cys-Gly-Glu-Xxx.sub.2-Cys-Xxx.sub.3-Gly-Gly-Thr-Cys- -Asn-Thr-Pro-Xxx.sub.1-Cys-Xxx.sub.1-Cys-Xxx.sub.1-Trp-Pro-Xxx.sub.1-Cys-T- hr-Arg-Xxx.sub.1 (SEQ ID NO: 17); wherein each Xxx.sub.1 position independently comprises any amino acid, non-natural amino acid or peptidomimetic; wherein Xxx.sub.2 comprises an amino acid non-natural amino acid or peptidomimetic but not Lys; wherein Xxx.sub.3 comprises any amino acid, non-natural amino acid or peptidomimetic but not Ala or Lys; and wherein the mutated cyclotide comprises one or more mutations from the naturally occurring kalata B1 cyclotide (SEQ ID NO:1), and wherein said mutation is G.fwdarw.K, T.fwdarw.K, S.fwdarw.K, N.fwdarw.K, or D.fwdarw.K. 6. The mutated cyclotide of claim 1, wherein the amino acid sequence of said cyclotide is radio labelled, fluorescence-labelled or biotin-labelled. 7. A pharmaceutical composition comprising the mutated cyclotide of claim 1 and a pharmaceutically acceptable carrier, excipient or diluent. 8. The composition of claim 7, wherein said pharmaceutical composition further comprises one or more additional immunosuppressants. 9. The composition of claim 8, wherein said additional immunosuppressant comprises Cyclosporine A, Muromonab-CD3 or Basiliximab. 10. A method for immunosuppression, the method comprising administering an effective amount of a non-grafted mutated cyclotide to a subject in need thereof, said cyclotide comprising an amino acid sequence of formula IL wherein formula II comprises: Xxx.sub.1-Leu-Pro-Val-Cys-Gly-Glu-Xxx.sub.2-Cys-Xxx.sub.3-Gly-Gly-Thr-Cys- -Asn-Thr-Pro-Xxx.sub.1-Cys-Pro-Xxx.sub.1-Cys-Thr-Arg-Xxx.sub.1 (SEQ ID NO: 17); wherein each Xxx.sub.1 position independently comprises any amino acid, non-natural amino acid or peptidomimetic; wherein Xxx.sub.2 comprises any amino acid, non-natural amino acid or peptidomimetic but not Lys; wherein Xxx.sub.3 comprises any amino acid, non-natural amino acid or peptidomimetic but not Ala or Lys; and wherein the mutated cyclotide comprises one or more mutations from the naturally occurring kalata B1 cyclotide (SEQ ID NO:1). 11. The method of claim 10, wherein said mutation is X.fwdarw.K, wherein X is any amino acid. 12. The method of claim 10, wherein said cyclotide has an anti-proliferative effect on (an) immune cell(s) and/or suppresses/reduces the effector function(s) of (an) immune cell(s). 13. The method of claim 10, wherein said cyclotide is administered so that cytostatic but no cytotoxic activity occurs. 14. The method of claim 10, wherein said cyclotide is administered in an amount to reach a serum concentration in the range of 1 to 50 .mu.M. 15. The method of claim 10, wherein said pharmaceutical composition further comprises one or more additional immunosuppressants. 16. The method of claim 15, wherein said additional immunosuppressant comprises Cyclosporine A, Muromonab-CD3 or Basiliximab. 17. The method of claim 10, for the treatment of a subject suffering from a disorder selected from the group consisting of an autoimmune disorder, a hypersensitivity disorder, and a lymphocyte-mediated inflammation. 18. The method of claim 17, wherein said autoimmune disorder is selected from the group consisting of Multiple Sclerosis, Psoriasis, Systemic Lupus Erythematosus, Sjogren's syndrome, Rheumatoid Arthritis, Idiopathic Thrombocytopenic Purpura, Diabetes, Vasculitis, and Crohn's disease. 19. The method of claim 17, wherein said lymphocyte-mediated inflammation comprises a T cell-mediated inflammation. 20. The method of claim 17, wherein said lymphocyte-mediated inflammation comprises Keratoconjunctivitis sicca or Dry Eye Syndrome (DES). 21. The method of claim 10, wherein: the proliferation of (an) immune cell(s); the effector function(s) of (an) immune cell(s); the degranulation/cytotoxicity of (an) immune cell(s); the expression of a cytokine surface receptor on (an) immune cell(s); the proliferation of (primary) activated lymphocytes; the proliferation of peripheral blood mononuclear cells (PBMC); secretion/production of IL-2, IFN-gamma and/or TNF-alpha; degranulation/cytotoxicity of CD107a+ CD8+ PBMCs; and/or expression of IL-2 surface receptor CD25 is/are suppressed. 22. The method of claim 10, wherein said cyclotide suppresses/reduces secretion/production of IL-2, IFN-gamma and/or TNF-alpha; suppresses/reduces degranulation/cytotoxicity of CD107a+ CD8+ PBMCs; and/or suppresses/reduces expression of IL-2 surface receptor CD25. 23. The method of claim 10, wherein the anti-proliferative effect or suppression/reduction is mediated in an IL-2-, IFN-gamma- and/or TNF-alpha-depending manner and/or can be antagonized by IL-2. 24. A method of producing an immunosuppressive pharmaceutical composition comprising mixing a mutated cyclotide of claim 1 with a pharmaceutically acceptable carrier. 25. The method of claim 10, said cyclotide comprising an amino acid sequence having one or more of amino residues at amino acid positions 1, 18, 20, 22, 25, and/or 29 of SEQ ID NO:17 representing a different amino acid from the same position of SEQ ID NO:1. 26. The method of claim 10, said cyclotide comprising an amino acid sequence that is: at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:5, 6, or 7; or at least 90% identical to an amino acid sequence encoded by a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs:5, 6, or 7; wherein the amino acid sequence of said cyclotide carries a mutation at least one amino acid position corresponding to an amino acid position which has been mutated in any one of the mutated cyclotides as depicted in SEQ ID NO: 5, 6 or 7. 27. The method of claim 10, said cyclotide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence SEQ ID NO:7. 28. The method of claim 10, said cyclotide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence SEQ ID NO:7. 29. The method of claim 14, wherein said cyclotide is administered in an amount to reach a serum concentration in the range of 3 to 10 .mu.M. 30. The method of claim 14, wherein said cyclotide is administered in an amount to reach a serum concentration in the range of 4 to 9 .mu.M. 31. The method of claim 14, wherein said cyclotide is administered in an amount to reach a serum concentration in the range of 5 to 9 .mu.M. 32. A method for immunosuppression, the method comprising administering an effective amount of a mutated cyclotide of claim 1 to a subject in need thereof. 33. A method for immunosuppression, the method comprising administering an effective amount of a mutated cyclotide of claim 2 to a subject in need thereof. 34. A method for immunosuppression, the method comprising administering an effective amount of a cyclotide of claim 3 to a subject in need thereof. 35. A method for immunosuppression, the method comprising administering an effective amount of a mutated cyclotide of claim 4 to a subject in need thereof. 36. A method for immunosuppression, the method comprising administering an effective amount of a mutated cyclotide of claim 5 to a subject in need thereof.

Details for Patent 9,453,052

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Centocor Ortho Biotech Products, L.p.	ORTHOCLONE OKT3	muromanab-cd3	Injection	103463	09/14/1992		2031-12-22
Novartis Pharmaceuticals Corporation	SIMULECT	basiliximab	For Injection	103764	05/12/1998		2031-12-22
Novartis Pharmaceuticals Corporation	SIMULECT	basiliximab	For Injection	103764	01/02/2003		2031-12-22