METHODS AND COMPOSITIONS FOR LIPOSOMAL FORMULATION OF ANTIGENS AND USES THEREOF

• US 20100226973A1
• Filed: 03/09/2010
• Published: 09/09/2010
• Est. Priority Date: 03/09/2009
• Status: Active Grant

• Current Assignee: Molecular Express, Inc., Regents of the University of California (University of California)
• Original Assignee: Molecular Express, Inc.
• Inventors: WATSON, Douglas S., SZOKA, Francis C. JR., FUJII, Gary
• Granted Patent: US 8,765,171 B2
• Time in Patent Office: Days
• US Class Current: 424/450
• CPC Class Codes: A61K 2039/55555 Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
• CPC Class Codes: A61K 2039/55572 Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
• CPC Class Codes: A61K 2039/60 characteristics by the carrier linked to the antigen
• CPC Class Codes: A61K 2039/6018 Lipids, e.g. in lipopeptides
• CPC Class Codes: A61K 2039/627 characterised by the linker
• CPC Class Codes: A61K 39/07 Bacillus
• CPC Class Codes: A61K 39/12 Viral antigens
• CPC Class Codes: A61K 39/145 Orthomyxoviridae, e.g. influenza virus
• CPC Class Codes: A61K 39/385 Haptens or antigens, bound to carriers
• CPC Class Codes: A61K 47/543 Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• CPC Class Codes: A61K 47/554 the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
• CPC Class Codes: A61K 47/6911 the form being a liposome
• CPC Class Codes: A61K 47/6913 the liposome being modified on its surface by an antibody
• CPC Class Codes: A61K 9/1272 with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids with cholesterol as the only non-phosphatidyl surfactant A61K9/127; lipids as modifying agent A61K47/543
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: C07J 41/0055 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
• CPC Class Codes: C07J 43/003 not condensed
• CPC Class Codes: C07J 9/00 Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
• CPC Class Codes: C12N 2740/16134 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 2760/16134 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 2770/00011 : Details
• CPC Class Codes: C12N 2770/00034 : Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 7/00 : Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00

Abstract

The present invention relates to liposomal vaccine compositions, methods for the manufacture thereof, and methods for the use thereof to stimulate an immune response in an animal. These compositions comprise dimyristoylphosphatidylcholine (“DMPC”); either dimyristoylphosphatidylglycerol (“DMPG”) or dimyristoyltrimethylammonium propane (“DMTAP”) or both DMPC and DMTAP; and at least one sterol derivative providing a covalent anchor for one or more immunogenic polypeptide(s) or carbohydrate(s).

Claims

1. A composition comprising one or more immunogenic polypeptides or carbohydrates of interest, the composition comprising:

a) an aqueous vehicle;

b) liposomes comprising

(i) dimyristoylphosphatidylcholine (“

DMPC”

),

(ii) one or more lipids selected from the group consisting of dimyristoylphosphatidylglycerol (“

DMPG”

), and dimyristoyltrimethylammonium propane (“

DMTAP”

), and

(iii) at least one sterol derivative,

at relative percentages of 50%-98% (i);

1%-25% (ii);

1%-25% (iii); and

c) said one or more immunogenic polypeptide(s) or carbohydrates covalently linked to between 1% and 100% of said at least one sterol derivative.

2. A composition according to claim 1, wherein said relative percentages are 70%-85% (i):

5%-15% (ii);

10%-15% (iii).

3. A composition according to claim 1, wherein said relative percentages are about 75% (i), about 10% (ii), and about 15% (iii).

4. A composition according to claim 1, wherein said sterol derivative has the following structure:

5. A composition according to claim 1, wherein said liposomes are substantially between 50 and 500 nm in diameter.

6. A composition according to claim 5, wherein said liposomes are substantially between 50 and 200 nm in diameter.

7. A composition according to claim 5, wherein said liposomes are substantially between 50 and 150 nm in diameter.

8. A composition according to claim 1, wherein said one or more immunogenic polypeptide(s) are covalently linked to between about 5% and about 10% of said at least one sterol derivative.

9. A composition according to claim 1, wherein said liposomes further comprise one or more components selected from the group consisting of monophosphoryl lipid A, resiquimod, flagellin, CpG, and a-galactosylceramide.

10. A composition according to claim 1, wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivative through a lysine residue on said immunogenic polypeptide(s).

11. A composition according to claim 1, wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivative through a cysteine residue on said immunogenic polypeptide(s).

12. A composition according to claim 1, wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivative through a aspartate or glutamate residue on said immunogenic polypeptide(s).

13. A composition according to claim 1, wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivative through a serine or threonine residue on said immunogenic polypeptide(s).

14. A composition according to claim 1, wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivative through a lysine residue on said immunogenic polypeptide(s).

15. A composition according to claim 1, wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivative through an N-terminal amine on said immunogenic polypeptide(s).

16. A composition according to any of claims 1, wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivative through a C-terminal carboxyl on said immunogenic polypeptide(s).

17. A composition according to claim 1, wherein at least one of said immunogenic carbohydrate(s) are covalently linked to said one or more sterol derivative through a hydroxyl on said immunogenic carbohydrate(s).

18. A composition according to claim 1, wherein said covalent linkage to said immunogenic polypeptide comprises an (alkylene oxide)_n moiety having an average length n of between 40 and 1000.

19. A composition according to claim 1, wherein said covalent linkage to said immunogenic polypeptide has the structure —

R3-X, wherein;

R3 is C_0-12 straight or branched chain alkyl, or C_0-6 straight or branched chain alkyl-(alkylene oxide), C_0-6 straight or branched chain alkyl, wherein

n is on average between 40 and 1000;

each said straight or branched chain alkyl optionally comprises from 1-3 chain heteroatoms and one or more substituents independently selected from the group consisting of halogen, trihalomethyl, —

C_1-6 alkoxy, —

NO₂, —

NH₂, —

OH, —

CH₂OH, —

CONH₂, and —

C(O)(OR4) where R4 is H or C_1-3 alkyl; and

X is said immunogenic polypeptide or carbohydrate.

20. A composition according to claim 4, wherein R1 is —

CH₂—

CH₂—

C(O)—

X, wherein X is said immunogenic polypeptide, and R2 is H.

21. A composition according to claim 4, wherein R1 is —

CH₂—

CH₂—

CH₂—

C(H)(CH₃)₂, and R2 is —

C(O)—

CH₂—

CH₂—

C(O)—

X, wherein X is said immunogenic polypeptide.

22. A composition according to claim 1, wherein the lipid in (ii) is DMPG.

23. A composition according to claim 1, wherein said sterol derivative(s) is/are derivative(s) of cholesterol, cholesteryl chloroformate, stigmasterol, sitosterol, ergosterol, lanosterol, desmosterol, or campesterol.

48. A method for immunizing an animal with one or more immunogenic polypeptides or carbohydrates of interest, comprising:

delivering to said animal by a parenteral or enteral route an effective amount of a liposomal composition of claim 1.

49. A method according to claim 48, wherein said animal is a mammal.

50. A method according to claim 48, wherein said animal is a human.

51. A method according to claim 48, wherein said liposomal composition is delivered via an enteral route of administration.

52. A method according to claim 48, wherein said liposomal composition is delivered via an parenteral route of administration

53. A method according to claim 48, wherein the method comprises delivering multiple doses of an immunogenic polypeptide or carbohydrate to said animal according to a prime-boost immunization protocol.

55. A method according to claim 53, wherein the prime-boost immunization protocol is a heterologous prime-boost protocol.

24. A method for preparing compositions comprising one or more immunogenic polypeptides or carbohydrates of interest, comprising:

(a) covalently coupling one or more immunogenic polypeptides or carbohydrates to one or more sterol derivatives to provide one or more conjugated sterol derivatives; and

(b) combining

(i) dimyristoylphosphatidylcholine (“

DMPC”

),

(ii) one or more lipids selected from the group consisting of dimyristoylphosphatidylglycerol (“

DMPG”

), and dimyristoyltrimethylammonium propane (“

DMTAP”

), and

(iii) one or more sterol derivatives, wherein between 1% and 100% of said sterol derivative(s) is(are) said conjugated sterol derivative(s) to provide a lipid mixture; and

(c) preparing liposomes from said lipid mixture.

25. A method for preparing compositions comprising one or more immunogenic polypeptides or carbohydrates of interest, comprising:

(a) combining

(i) dimyristoylphosphatidylcholine (“

DMPC”

),

(ii) one or more lipids selected from the group consisting of dimyristoylphosphatidylglycerol (“

DMPG”

), and dimyristoyltrimethylammonium propane (“

DMTAP”

), and

(iii) at least one sterol derivative to provide a lipid mixture;

(b) preparing liposomes from said lipid mixture; and

(c) covalently coupling one or more immunogenic polypeptides or carbohydrates to said at least one sterol derivative, wherein said one or more immunogenic polypeptide(s) or carbohydrate(s) are covalently linked to between 1% and 100% of said at least one sterol derivative.

26. A method according to claim 25, wherein preparing liposomes from said lipid mixture comprises:

drying said lipid mixture;

hydrating said dried lipid mixture in an aqueous vehicle; and

sonicating said hydrated lipid mixture to form liposomes.

27. A method according to claim 25, wherein said relative percentages are 70%-85% (i):

5%-15% (ii);

10%-15% (iii).

28. A composition according to claim 25, wherein said relative percentages are about 75% (i), about 10% (ii), and about 15% (iii).

29. A method according to claim 25, wherein said sterol derivative has the following structure:

30. A method according to claim 25, wherein said liposomes are substantially between 50 and 500 nm in diameter.

31. A method according to claim 30, wherein said liposomes are substantially between 50 and 200 nm in diameter.

32. A method according to claim 30, wherein said liposomes are substantially between 50 and 150 nm in diameter.

33. A method according to claim 25, wherein said one or more immunogenic polypeptide(s) are covalently linked to between about 5% and about 10% of said at least one sterol derivative.

34. A method according to claim 25, wherein said liposomes further comprise one or more components selected from the group consisting of monophosphoryl lipid A, resiquimod, flagellin, CpG, and a-galactosylceramide.

35. A method according to claim 25, wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivative through a lysine residue on said immunogenic polypeptide(s).

36. A method according to claim 25, wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivative through a cysteine residue on said immunogenic polypeptide(s).

37. A method according to claim 25, wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivative through a aspartate or glutamate residue on said immunogenic polypeptide(s).

38. A method according to claim 25, wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivative through a serine or threonine residue on said immunogenic polypeptide(s).

39. A method according to claim 25, wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivative through a lysine residue on said immunogenic polypeptide(s).

40. A method according to claim 25, wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivative through an N-terminal amine on said immunogenic polypeptide(s).

41. A method according to claim 25, wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivative through a C-terminal carboxyl on said immunogenic polypeptide(s).

42. A method according to claim 25, wherein at least one of said immunogenic carbohydrate(s) are covalently linked to said one or more sterol derivative through a hydroxyl on said immunogenic carbohydrate(s).

43. A method according to claim 25, wherein said covalent linkage to said immunogenic polypeptide comprises an (alkylene oxide)_n moiety having an average length n of between 40 and 1000.

44. A method according to claim 25, wherein said covalent linkage to said immunogenic polypeptide has the structure —

R3-X, wherein;

R3 is C_0-12 straight or branched chain alkyl, or C_0-6 straight or branched chain alkyl-(alkylene oxide)_n-C_0-6 straight or branched chain alkyl, wherein

n is on average between 40 and 1000;

each said straight or branched chain alkyl optionally comprises from 1-3 chain heteroatoms and one or more substituents independently selected from the group consisting of halogen, trihalomethyl, —

C_1-6 alkoxy, —

NO₂, —

NH₂, —

OH, —

CH₂OH, —

CONH₂, and —

C(O)(OR4) where R4 is H or C_1-3 alkyl; and

X is said immunogenic polypeptide or carbohydrate.

45. A method according to claim 29, wherein R1 is —

CH₂—

CH₂—

C(O)—

X, wherein X is said immunogenic polypeptide, and R2 is H.

46. A method according to claim 29, wherein R1 is —

CH₂—

CH₂—

CH₂—

C(H)(CH₃)₂, and R2 is —

C(O)—

CH₂—

CH₂—

C(O)—

X, wherein X is said immunogenic polypeptide.

47. A method according to claim 25, wherein the lipid in (ii) is DMPG.

56. A method according to claim 25, wherein said sterol derivative(s) is/are derivative(s) of cholesterol, cholesteryl chloroformate, stigmasterol, sitosterol, ergosterol, lanosterol, desmosterol, or campesterol.

57. A functionalized sterol derivative, comprising:

a reactive group attached directly to a sterol derivative ring atom via a covalent linkage having the structure St-R5-X, wherein;

St is a ring atom of the sterol derivative;

R5 is C_0-18 straight or branched chain alkyl, or C_0-12 straight or branched chain alkyl-(alkylene oxide)_n-C_0-12 straight or branched chain alkyl, wherein n is on average between 40 and 1000, wherein each said straight or branched chain alkyl comprises from 0-4 chain heteroatoms and one or more substituents independently selected from the group consisting of halogen, trihalomethyl, —

C_1-6 alkoxy, —

NO₂, —

NH₂, —

OH, —

CH₂OH, —

CONH₂, and —

C(O)(OR6) where R6 is H or C_1-3 alkyl; and

X is a reactive group selected from the group consisting of an amine linking group, a sulfhydryl linking group, a carboxyl linking group, a photoaffinity linking group, an arginine linking group, and a carbonyl linking group.

58. A functionalized sterol derivative according to claim 57, wherein said functionalized sterol derivative is formulated in liposomes, said liposomes comprising:

(i) dimyristoylphosphatidylcholine (“

DMPC”

),

(ii) one or more lipids selected from the group consisting of dimyristoylphosphatidylglycerol (“

DMPG”

), and dimyristoyltrimethylammonium propane (“

DMTAP”

), and

(iii) at least one sterol derivative,

at relative percentages of 50%-98% (i);

1%-25% (ii);

1%-25% (iii), wherein between 1% and 100% of said at least one sterol derivative(s) is(are) said functionalized sterol derivative.

59. A functionalized sterol derivative according to claim 58, wherein said relative percentages are 70%-85% (i):

5%-15% (ii);

10%-15% (iii).