Simplified and improved method for preparing an antibody or an antibody fragment targeted immunoliposome for systemic administration of a therapeutic or diagnostic agent
A method of preparing an antibody- or antibody fragment-targeted cationic immunoliposome or polymer complex comprises the steps of (a) preparing an antibody or antibody fragment; (b) mixing said antibody or antibody fragment with a cationic liposome to form a cationic immunoliposome or with a cationic polymer to form a polyplex; and (c) mixing said cationic immunoliposome or said polyplex with a therapeutic or diagnostic agent to form said antibody- or antibody fragment-targeted cationic immunoliposome or polymer complex.
1. A method of preparing an antibody- or antibody fragment-targeted cationic immunoliposome complex comprising:
a) preparing an antibody or antibody fragment;
b) mixing said antibody or antibody fragment with a cationic liposome to form a cationic immunoliposome, wherein said antibody or antibody fragment is complexed with said cationic liposome, but is not chemically conjugated to said cationic liposome, and wherein said antibody or antibody fragment does not comprise a lipid tag; and
c) mixing said cationic immunoliposome with a therapeutic or diagnostic agent to form said antibody- or antibody fragment-targeted cationic immunoliposome complex wherein said antibody- or antibody fragment-targeted cationic immunoliposome complex is about 50-400 nm in size.
2. The method of claim 1 , wherein an antibody is mixed with said cationic liposome.
3. The method of claim 1 , wherein an antibody fragment is mixed with said cationic liposome.
4. The method of claim 3 , wherein said antibody fragment is a single chain Fv fragment.
5. The method of claim 4 , wherein said antibody fragment is an anti-transferrin receptor single chain Fv (TfRscFv).
6. The method of claim 1 , wherein said antibody or antibody fragment is an anti-HER-2 antibody or antibody fragment.
7. The method of claim 1 , wherein said antibody fragment comprises a cysteine moiety at a carboxy terminus prior to being mixed with said cationic liposome.
8. The method of claim 1 , wherein said cationic liposome comprises a mixture of one or more cationic lipids and one or more neutral or helper lipids.
9. The method of claim 1 , wherein said antibody or antibody fragment is mixed with said cationic liposome at a ratio in the range of 1:5 to 1:40 (w:w).
10. The method of claim 1 , wherein said cationic liposome comprises a mixture of dioleoyltrimethylammonium phosphate with dioleoylphosphatidylethanolamine and/or cholesterol; or a mixture of dimethyldioctadecylammonium bromide with dioleoylphosphatidylethanolamine and/or cholesterol.
11. The method of claim 1 , wherein said antibody or antibody fragment and liposome are mixed with a therapeutic or diagnostic agent at a ratio in the range of 1:8 to 1:20 (μg of agent:nmole of total lipid).
12. The method of claim 11 , wherein said agent is a therapeutic agent and comprises a gene, plasmid DNA, or nucleic acid.
13. The method of claim 12 , wherein said agent is a gene encoding a tumor suppressor protein.
14. The method of claim 13 , wherein said tumor suppressor protein is p53.
15. The method of claim 11 , wherein said agent is a diagnostic agent.
16. The method of claim 1 , which further comprises formulating said complex as a pharmaceutically acceptable formulation for in vivo administration.
17. The method of claim 16 , wherein said complex is combined with a pharmacologically acceptable carrier.
18. The method of claim 16 , wherein said complex is formulated for intravenous administration.
19. The method of claim 1 , wherein said complex is optimized to target tumor cells.
20. The method of claim 1 , wherein said complex is optimized to target non-tumor cells.
21. The method of claim 1 , wherein said mixing in (b) and (c) are conducted in a solution comprising water.