Protein coupling to lipid vesicles

• US 5,000,960 A
• Filed: 01/19/1989
• Issued: 03/19/1991
• Est. Priority Date: 03/13/1987
• Status: Expired due to Term

• Current Assignee: Novavax Inc.
• Original Assignee: Micro-Pak, Inc.
• Inventors: Wallach, Donald F. H.
• Primary Examiner(s): Lovering, Richard D.
• Application Number:
  US07/300,079
• Time in Patent Office: 789 Days
• Field of Search: 264/4.3, 428/402.2, 424/85.8, 424/420, 424/450, 424/7.1, 436/829, 514/963
• US Class Current: 424/1.21
• CPC Class Codes: A61K 47/62 the modifying agent being a protein, peptide or polyamino 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: A61K 9/1277 Processes for preparing; Proliposomes
• CPC Class Codes: C11D 17/0021 Aqueous microemulsions
• CPC Class Codes: C11D 17/0026 Structured liquid compositions, e.g. liquid crystalline phases or network containing non-Newtonian phase
• CPC Class Codes: Y10S 424/812 Liposome comprising an antibody, antibody fragment, antigen, or other specific or nonspecific immunoeffector
• CPC Class Codes: Y10S 436/829 Liposomes, e.g. encapsulation
• CPC Class Codes: Y10T 428/2984 Microcapsule with fluid core [includes liposome]

Abstract

A new method of coupling proteins and other targeting molecules to lipid vesicles has been developed. A bifunctional agent forms a covalent bond without damaging the lipid structure and permits retention of protein activity.

Claims

1. A method of coupling targeting molecules to nonphospholipid lipid vesicles while retaining the targeting specificity of said targeting molecules comprising the steps of:

selecting a targeting molecule having a free NH₂ group thereon,

reacting said targeting molecule with a bifunctional agent comprising N-hydroxysuccinimidyl 3-(2-pyridyldithio)propionate, in a manner such that said bifunctional agent reacts with said NH₂ group on said targeting molecule to incorporate a free SH group onto said targeting molecule,

forming a nonphospholipid lipid vesicle having a steroid with a free SH group as one of its structural components, and

reacting said lipid vesicle having a free SH group thereon and said targeting molecule with said free SH group,

thereby coupling said targeting molecule to said lipid vesicle.

2. The method of claim 1 wherein said steroid with a free SH group comprises thiocholesterol.

3. The method of claim 1 wherein said nonphospholipid lipid vesicle comprises polyoxyethylene fatty esters, polyoxyethylene fatty acid ethers, diethanolamines, long-chain acyl amino acid amides, long-chain acyl amides, polyoxyethylene sorbitan mono and tristearates and oleates, polyoxyethylene glyceryl monostearates and monooleates, and glyceryl monostearates and monooleates.

4. The method of claim 3 wherein said nonphospholipid lipid vesicle comprises a paucilamellar lipid vesicle.

5. The method of claim 4 wherein said nonphospholipid lipid vesicle further comprises a charge-producing agent as a structural component.

6. The method of claim 1 wherein said targeting molecule is selected from a group consisting of peptide hormones.

7. The method of claim 1 wherein said targeting molecule comprises a protein.

8. The method of claim 7 wherein said protein is selected from a group consisting of lectins, and immunoglobulins.

9. The method of claim 8 wherein said lectin is selected from a group consisting of concanavalin A, and wheat germ agglutinin.

10. The method of claim 8 wherein said immunoglobulin is selected from a group consisting of monoclonal antibodies, chimeric antibodies, and portions and fragments thereof.

11. A method of delivering a specified material to a particular location or tissue in a human or animal body comprising the steps of:

encapsulating said specified material in a nonphospholipid lipid vesicle, said lipid vesicle containing a steroid having a free SH group as one of its structural components,

selecting a proteinaceous targeting molecule which reacts with said particular location or tissue,

reacting said proteinaceous targeting molecule with a bifunctional agent comprising N-hydroxysuccinimidyl 3-(2-pyridyldithio)propionate, in a manner such that said bifunctional agent reacts with free NH₂ groups on said targeting molecule and provides a free SH group, thereby forming a modified targeting molecule,

reacting a plurality of said modified targeting molecules with said lipid vesicle so that at least one of said modified targeting molecules is bound to the surface of said lipid vesicle by an S--S bond,

introducing said lipid vesicle having said modified targeting molecule bound thereto into said human or animal body, and

allowing said targeting molecule to react with its target, thereby bringing said encapsulated material to said specified location or tissue.

12. The method of claim 11 wherein said nonphospholipid lipid vesicle comprises a paucilamellar lipid vesicle.

13. The method of claim 11 wherein said proteinaceous targeting molecule is selected from a group consisting of immunoglobulins.

14. The method of claim 13 wherein said immunoglobulin is selected from a group consisting of monoclonal antibodies, chimeric antibodies, and fragments and portions thereof.

15. A nonphospholipid lipid vesicle having a targeting molecule attached thereto comprising a nonphospholipid lipid vesicle containing a steroid having free SH groups as one of its structural components, said targeting molecule being attached to said lipid vesicle by an S--S bond between a free SH group on said targeting molecule and a free SH group on said lipid vesicle.

16. The lipid vesicle of claim 15 wherein said lipid vesicle comprises a paucilamellar lipid vesicle.

17. The lipid vesicle of claim 15 wherein said targeting molecule is selected from a group consisting of proteins, and peptide hormones.

18. The lipid vesicles of claim 17 targeting molecule is a protein selected from a group consisting of lectins, immunoglobulins, monoclonal antibodies, and chimeric antibodies.

19. The lipid vesicles of claim 15 wherein said targeting molecule is attached to said lipid vesicle by a bifunctional agent comprising N-hydroxysuccinimidyl 3-(2-pyridyldithio)propionate, in a manner such that said bifunctional agent attaches to a free NH₂ group on said targeting molecule and supplies a free SH group thereto.

20. The lipid vesicle of claim 15 wherein said vesicle has a diagnostic agent encapsulated therein.

21. The lipid vesicle of claim 20 wherein said targeting molecule comprises an immunoglobulin.

22. The lipid vesicle of claim 21 wherein said immunoglobulin is selected from a group consisting of monoclonal antibodies, chimeric antibodies, and portions and fragments thereof.

23. The lipid vesicle of claim 20 wherein said diagnostic agent is selected from a group consisting of immunodiagnostic agents, visualization agents, high density particles and magnetic particles.