Vaccine engineering

Patent metadata

Field	Value
Publication number	US-9289488-B2
Application number	US-201113816461-A
Country	US
Kind code	B2
Filing date	Aug 12, 2011
Priority date	Aug 12, 2010
Publication date	Mar 22, 2016
Grant date	Mar 22, 2016

Abstract

The method and constructs disclosed and claimed herein allow to control peptide conformation by modulation of the peptide lipidation pattern, spacer and liposome composition, or via co-administration with small molecules. Accordingly, this technology can be applied to the rational design of liposomal vaccines and for the generation of safer and more efficacious therapies for a range of human disease, in particular those based on misfolded proteins.

First claim

The invention claimed is:

1. A method of preparing a modulated immunogenic construct for generating an immune response in an animal or a human, wherein said immunogenic construct comprises a peptide molecule and a hydrophobic moiety covalently bound to the C- and/or N-terminal part of the peptide molecule, which peptide molecule is anchored into the lipid membrane of a liposomal carrier via said hydrophobic moiety, said method comprising:
a. modulating the secondary and/or quaternary structure and the extent of β-sheet structure and/or β-sheet aggregation of the liposome-bound peptide molecule, comprising one or more of;
i. modulating the amount of metal ions capable of disrupting peptide-liposome or peptide-peptide stabilizing interactions in the liposomal solution or composition,
ii. changing the net surface potential of the liposome by modulating the proportion of anionic or cationic lipids in the lipid bilayer of the liposome,
iii. varying the total number of hydrophobic moieties covalently bound to the N- and/or C-terminus of the peptide molecule such that there is a disparity in the number of hydrophobic moieties between the N- and the C-terminus, and
iv. adding small molecules to a liposomal solution or composition which are capable of either disaggregating β-sheet structures or promoting aggregation to form β-sheet structures, and

b. determining the extent of β-sheet structure and/or β-sheet aggregation of the liposome-bound antigenic peptide; and
c. obtaining the modulated immunogenic construct,
wherein the peptide molecule is a peptide derived from an amyloid protein, a tau protein, a prion protein, an alpha-synuclein protein, or a huntington protein.

2. A method of modulating the secondary and/or quaternary structure of an antigenic peptide molecule, which antigenic peptide molecule has a hydrophobic moiety covalently bound to its C- and/or N-terminus and is anchored into the lipid membrane of a liposomal carrier via said hydrophobic moiety, comprising
a. modulating the amount of metal ions capable of disrupting peptide-liposome or peptide-peptide stabilizing interactions in the liposomal solution or composition; and/or
b. changing the net surface potential of the liposome by modulating the proportion of anionic or cationic lipids in the lipid bilayer of the liposome; and/or
c. varying the total number of hydrophobic moieties covalently bound to the N- and/or C-terminus of the peptide molecule such that there is a disparity in the number of hydrophobic moieties between the N- and the C-terminus; and/or
d. adding small molecules to a liposomal solution or composition which are capable of either disaggregating β-sheet structures or promoting aggregation to form β-sheet structures,
wherein said modulation of the secondary and/or quaternary structure leads to an increase or decrease of the extent of β-sheet structures and/or β-sheet aggregation of the liposome-bound antigenic peptide molecule, and
wherein the antigenic peptide molecule is a peptide derived from an amyloid protein, a tau protein, a prion protein, an alpha-synuclein protein, or a huntington protein.

3. The method of claim 1 , wherein said stabilizing compound is selected from:
a. heparin, myo-inositol, glycogen or ellagic acid; and/or
b. the net negative surface potential of the liposome is increased from >+35 mV to at least <0mV, to at least <−10 mV, or to at least <−40 mV, in PBS; or
c. the net negative surface potential of the liposome is decreased from <−10 mV to at least >0mV, to at least >+10 mV, or to at least >+35 mV, in PBS.

4. The method of claim 3 , wherein the decrease in net negative surface potential is accomplished by exchanging a proportion of anionic lipids in the lipid bilayer of the liposome with an equal proportion of cationic lipids,
wherein said anionic lipids are selected from the group consisting of:
i. diacyl-phospholipids with headgroups phosphatidyl glycerol, phosphatidyl serine, phosphatidyl inositol, L-α-phosphatidylinositol-4-phosphate or phosphatidic acid;
ii. diether-phospholipids with headgroups phosphatidyl glycerol, phosphatidyl serine, phosphatidyl inositol, L-α-phosphatidylinositol-4-phosphate or phosphatidic acid;
iii. lyso-phospholipids with headgroups phosphatidyl glycerol, phosphatidyl serine or phosphatidic acid;
iv. cardiolipin, dilyso-cardiolipin, monolyso-cardiolipin;
v. glycolipids, and lipopolysaccharides;
wherein said cationic lipids are selected from the group consisting of:
i). diacyl-phospholipids with headgroups 3-trimethylammonium-propane, 3-dimethylammonium-propane, 3-ethylphosphocholine or 3-phosphatidylethanolamine;
ii) diether-phospholipids with headgroups 3-trimethylammonium-propane, 3-dimethylammonium-propane, 3-ethylphosphocholine or 3-phosphatidylethanolamine;
iii) lyso-phospholipids with headgroups 3-trimethylammonium-propane, 3-dimethylammonium-propane, 3-ethylphosphocholine or 3-phosphatidylethanolamine;
iv. D-erythro-sphingosine, dimethyldioctadecylammonium bromide, N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl) ammonium bromide, N,N,N-trimethyl-2-bis[(1-oxo-9-octadecenyl)oxy]-(Z,Z)-1-propanaminium methyl sulfate and 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride.

5. The method of claim 1 , wherein
a. the number of hydrophobic moieties covalently bound to the N-terminus of the peptide molecule exceeds the number of hydrophobic moieties covalently bound to the C-terminus of the peptide molecule by at least one, at least two, at least three, at least four moieties; or
b. the number of hydrophobic moieties covalently bound to the C-terminus of the peptide molecule exceeds the number of hydrophobic moieties covalently bound to the N-terminus of the peptide molecule by at least one, at least two, at least three, at least four moieties.

6. The method of claim 1 , wherein the secondary and/or quaternary conformation of the peptide molecule is modulated by disaggregating the β-sheet aggregates, which is accomplished by adding metal ions which are capable of disrupting peptide-liposome or peptide-peptide stabilizing interactions, wherein the metal ions are Cu(II) or Zn(II).

7. An immunogenic construct comprising a peptide molecule anchored into the membrane of a liposome via hydrophobic moieties covalently bound to the N-terminus, C-terminus, or both of the peptide molecule such that there is a disparity between the number of hydrophobic moieties between the N-terminus and C-terminus, or a composition comprising said immunogenic construct,
wherein said immunogenic construct is modulated to have an increased extent of β-sheet structure and/or β-sheet aggregation of between 50%-100%, as compared prior to modulation,
wherein the number of hydrophobic moieties covalently bound to the N-terminus of the peptide molecule exceeds the number of hydrophobic moieties covalently bound to the C-terminus of the peptide molecule by at least three, or by at least four moieties, or the number of hydrophobic moieties covalently bound to the C-terminus of the peptide molecule exceeds the number of hydrophobic moieties covalently bound to the N-terminus of the peptide molecule by at least three, or by at least four moieties, and
wherein the peptide molecule is a peptide derived from an amyloid protein, a tau protein, a prion protein, an alpha synuclein protein, or a huntingtin protein.

8. The immunogenic construct or composition of claim 7 , wherein the hydrophobic moiety is covalently bound to the C-terminal, N-terminal, or both the C-terminal and N-terminal part of the peptide molecule, wherein said immunogenic construct has been modulated to show an enhanced extent of β-sheet structure, β-sheet aggregation, or both, and comprises at least two features selected from the gr

Assignees

• Hickman David
• Lopez-Deber Maria Pilar
• Muhs Andreas
• Ac Immune Sa

Inventors

• Hickman David
• Lopez-Deber Maria Pilar
• Muhs Andreas

Classifications

• C07K16/18

  against material from animals or humans · CPC title

• A61K2039/6018

  Lipids, e.g. in lipopeptides · CPC title

• A61K2039/55555

  Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers · CPC title

• A61K2039/64

  characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units · CPC title

• G01N2440/10

  acylation, e.g. acetylation, formylation, lipoylation, myristoylation, palmitoylation · CPC title