# US 9,931,419 B2 · Methods and compositions for liposomal formulation of antigens and uses thereof

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- Number: US 9,931,419 B2
- Document ID: US9931419B2
- Status: Granted
- Kind: B2
- Application number: 15/181,028
- Filed: 2016-06-13
- Published: 2017-06-08
- Granted: 2018-04-03
- Earliest claimed: 2009-03-09
- Assignees: Molecular Express, Inc.; The Regents of the University of California
- Applicants: Molecular Express, Inc.; The Regents of the University of California
- Inventors: Gary Fujii; Francis C. Szoka, Jr.; Douglas S. Watson
- Web page: https://patentgrep.com/patent/US9931419B2

## CPC

- A61K 47/6913: {the liposome being modified on its surface by an antibody}
- A61K 9/1272: comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers (lipids as modifying agents {A61K47/543})
- A61K 39/07: Bacillus
- A61K 39/12: Viral antigens
- A61K 39/145: Orthomyxoviridae, e.g. influenza virus
- A61K 39/385: Haptens or antigens, bound to carriers
- A61K 47/543: {Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine}
- A61K 47/554: {the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid}
- A61K 47/6911: {the form being a liposome}
- A61P 37/04: Immunostimulants
- 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
- 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}
- C07J 43/003: {not condensed}
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- A61K 2039/55555: {Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers}
- A61K 2039/55572: {Lipopolysaccharides; Lipid A; Monophosphoryl lipid A}
- A61K 2039/60: {characteristics by the carrier linked to the antigen}
- A61K 2039/6018: {Lipids, e.g. in lipopeptides}
- A61K 2039/627: {characterised by the linker}
- C12N 2740/16134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/16134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2770/00011: Details
- C12N 2770/00034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07J: STEROIDS (seco-steroids C07C)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## 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

### Claim 1

A composition comprising:
a) an aqueous vehicle; and
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
wherein said sterol derivative comprises a reactive group selected from the group consisting of imidoesters, N-hydroxysuccinimidyl (“NHS”) esters, maleimides, alkyl halides, aryl halides, α-haloacyls, pyridyl disulfides, carbodiimides, glyoxals, amines, hydrazides, and arylazides.

### Claim 2 (depends on 1)

A composition according to claim 1, wherein the said sterol derivative comprises a maleimide reactive group.

### Claim 3 (depends on 1)

A composition according to claim 1, wherein the sterol derivative comprises a covalent linkage between the sterol derivative and the reactive group, the covalent linkage having the structure St-R3-RG, wherein:
St is a ring atom of the sterol derivative;
R3 is C0-18 straight or branched chain alkyl, or C0-12 straight or branched chain alkyl-(alkylene oxide)n-C0-12 straight or branched chain alkyl, wherein n is on average between 40 and 1000;
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, —C1-6 alkoxy, —NO2, —NH2, —OH, —CH2OH, —CONH2, and —C(O)(OR4) where R4 is H or C1-3 alkyl; and
RG is the maleimide reactive group.

### Claim 4 (depends on 2)

A composition according to claim 2, wherein said relative percentages are 70%-85% (i):5%-15% (ii):10%-15% (iii).

### Claim 5 (depends on 2)

A composition according to claim 2, wherein said relative percentages are about 75% (i), about 10% (ii), and about 15% (iii).

### Claim 6 (depends on 2)

A composition according to claim 2, wherein said sterol derivative has the following structure:
wherein:
one of R1 or R2 is —CH2—CH2—C(O)—RG and the other of R1 or R2 is H, or
R1 is —CH2—CH2—CH2—C(H)(CH3)2 and R2 is —C(O)—CH2—CH2—C(O)—RG,
wherein RG is the maleimide reactive group.

### Claim 7 (depends on 2)

A composition according to claim 2, wherein said liposomes are substantially between 50 and 500 nm in diameter.

### Claim 8 (depends on 7)

A composition according to claim 7, wherein said liposomes are substantially between 50 and 200 nm in diameter.

### Claim 9 (depends on 8)

A composition according to claim 8, wherein said liposomes are substantially between 50 and 150 nm in diameter.

### Claim 10 (depends on 2)

A composition according to claim 2, wherein said liposomes further comprise one or more components selected from the group consisting of monophosphoryl lipid A, resiquimod, flagellin, CpG, and α-galactosylceramide.

### Claim 11 (depends on 2)

A composition according to claim 2, wherein the lipid in (ii) is DMPG.

### Claim 12 (depends on 2)

A composition according to claim 2, wherein said sterol derivative(s) is/are derivative(s) of cholesterol, cholesteryl chloroformate, stigmasterol, sitosterol, ergosterol, lanosterol, desmosterol, or campesterol.

### Claim 13 (depends on 3)

A composition according to claim 3, wherein said relative percentages are 70%-85% (i):5%-15% (ii):10%-15% (iii).

### Claim 14 (depends on 3)

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

### Claim 15 (depends on 3)

A composition according to claim 3, wherein said sterol derivative has the following structure:
wherein:
one of R1 or R2 is —CH2—CH2—C(O)—RG and the other of R1 or R2 is H, or
R1 is —CH2—CH2—CH2—C(H)(CH3)2 and R2 is —C(O)—CH2—CH2—C(O)—RG,
wherein RG is the maleimide reactive group.

### Claim 16 (depends on 3)

A composition according to claim 3, wherein said liposomes are substantially between 50 and 500 nm in diameter.

### Claim 17 (depends on 16)

A composition according to claim 16, wherein said liposomes are substantially between 50 and 200 nm in diameter.

### Claim 18 (depends on 17)

A composition according to claim 17, wherein said liposomes are substantially between 50 and 150 nm in diameter.

### Claim 19 (depends on 3)

A composition according to claim 3, wherein said liposomes further comprise one or more components selected from the group consisting of monophosphoryl lipid A, resiquimod, flagellin, CpG, and α-galactosylceramide.

### Claim 20 (depends on 3)

A composition according to claim 3, wherein the lipid in (ii) is DMPG.

### Claim 21 (depends on 3)

A composition according to claim 3, wherein said sterol derivative(s) is/are derivative(s) of cholesterol, cholesteryl chloroformate, stigmasterol, sitosterol, ergosterol, lanosterol, desmosterol, or campesterol.