Cyclotides as immunosuppressive agents

• US 10,357,537 B2
• Filed: 10/30/2018
• Issued: 07/23/2019
• Est. Priority Date: 12/22/2011
• Status: Active Grant

• Current Assignee: Medizinische Uni Wien, Universitätsklinikum Freiburg
• Original Assignee: Medizinische Uni Wien
• Inventors: Gruber, Christian Werner, Gruendemann, Carsten
• Primary Examiner(s): Lieb, Jeanette M
• Application Number:
  US16/174,428
• Publication Number:
  US 20190046607A1
• Time in Patent Office: 266 Days
• Field of Search: None
• CPC Class Codes: A61K 38/12 Cyclic peptides , e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C A61K38/043 - A61K38/046 take precedence
• CPC Class Codes: A61K 38/16 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof enzyme inhibitors A61K38/005
• CPC Class Codes: A61K 38/168 from plants
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61K 51/08 Peptides, e.g. proteins , carriers being peptides, polyamino acids, proteins
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/415 from plants
• CPC Class Codes: G01N 33/5023 on expression patterns
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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.

Claims

1. Method of screening for and/or selecting an immunosuppressive cyclotide, the method comprising:

(i) contacting activated immune cells with a cyclotide or a plant extract containing a cyclotide and determining the proliferative activity of said activated immune cells,

wherein a reduced proliferative activity as compared to a control is indicative for the immunosuppressive activity of the cyclotide or plant extract containing a cyclotide;

or

(ii) administering to an animal model a pharmaceutically effective amount of a cyclotide or a plant extract containing a cyclotide and determining one or more parameters of the immune system,

wherein the reduction of the one or more parameters of the immune system as compared to a control is indicative for the immunosuppressive activity of the cyclotide or plant extract containing a cyclotide.

2. The method of claim 1, comprising introducing a mutation into the cyclotide prior to:

contacting the activated immune cells with the cyclotide or plant extract;

or

administering to the animal model a pharmaceutically effective amount of the cyclotide or plant extract.

3. The method of claim 2, wherein the mutated cyclotide demonstrates:

a reduced proliferative activity in activated immune cells as compared to the non-mutated cyclotide;

or

a reduction of one or more parameters of the immune system in the animal model as compared to the non-mutated cyclotide.

4. The method of claim 1, further comprising isolating and/or identifying the immunosuppressive cyclotide from the plant extract.

5. The method of claim 1, wherein the cyclotide is or comprises a non-grafted cyclotide.

6. The method of claim 1 (i), wherein the control comprises:

determining the proliferative activity of activated immune cells that have not been contacted with the cyclotide or the plant extract containing a cyclotide;

or

determining the proliferative activity of activated immune cells that have been contacted with a known cyclotide that does not have immunosuppressive activity.

7. The method of claim 1 (ii), wherein the control comprises:

determining the one or more parameters of the immune system in the animal model that have not been administered the cyclotide or the plant extract containing a cyclotide;

or

determining the one or more parameters of the immune system in the animal model that have been administered a known cyclotide that does not have immunosuppressive activity.

8. The method of claim 1 (i), wherein activated immune cells comprise activated human peripheral blood mononuclear cells (PBMCs) or human T-lymphocyte.

9. The method of claim 8, wherein the activated human PBMCs comprises CD107a⁺ CD8⁺ PBMCs.

10. The method of claim 1 (ii), wherein the parameter of the immune system is selected from the group consisting of secretion/production of IL-2, secretion/production of IFN-gamma, secretion/production of TNF-alpha, degranulation/cytotoxicity of CD107a+ CD8+ PBMCs, and expression of IL-2 surface receptor CD25.

11. The method of claim 1 (ii), wherein the animal model comprises an animal model for an autoimmune disease.

12. The method of claim 11, wherein the animal model for an autoimmune disease comprises the Experimental Autoimmune Encephalitis (EAE) mouse model for Multiple Sclerosis (MS).

13. The method of claim 1, wherein the cyclotide comprises a head-to-tail cyclized peptide comprising six conserved cysteine residues capable of forming three disulphide bonds arranged in a cyclic cystine knot (CCK) motif.

14. The method of claim 1, wherein said cyclotide comprises a cyclic backbone having the structure of formula I, wherein formula I comprises the amino acid sequence:

Cyclo(C[X₁ . . . Xa] C[XI1 . . . XIb] C[XII1 . . . XIIc] C[XIII1 . . . XIIId] C[XIV1 . . . XIVe] C[XV1 . . . XVf]);

wherein C is cysteine;

wherein each of [X1 . . . Xa], [XI1 . . . XIb], [XII1 . . . XIIc], [XIII1 . . . XIIId], [XIV1 . . . XIVe], and [XV1 . . . XVf] represents one or more amino acid residues, wherein each one or more amino acid residues within or between the sequence residues may be the same or different; and

wherein a, b, c, d, e, and f represent the number of amino acid residues in each respective sequence and each of a to f may be the same or different and range from 1 to about 20.

15. The method of claim 14, wherein a is 3 to 6, b is 4 to 8, c is 3 to 10, d is 1, e is 4 to 8, and/or f is 5 to 13.

16. The method of claim 1, wherein the cyclotide is or comprises a mutant or variant of kalta B.

17. The method of claim 1, wherein said cyclotide comprises an amino acid sequence of formula II;

wherein formula II comprises;

18. The method of claim 1, wherein said cyclotide comprises an amino acid sequence that is at least 90% identical to any amino acid sequence of SEQ ID NOs:

1, 2, 3, 4, 5, 6, and 7.

19. The method of claim 1, wherein said cyclotide reduces secretion/production of IL-2, reduces secretion/production of IFN-gamma, reduces secretion/production of TNF-alpha, suppresses/reduces degranulation/cytotoxicity of CD107a+CD8+ PBMCs, and/or suppresses/reduces expression of IL-2 surface receptor CD25.

20. The method of claim 1, 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.