Preparation of antibody or an antibody fragment-targeted immunoliposomes for systemic administration of therapeutic or diagnostic agents and uses thereof
The present invention provides methods of preparing an antibody- or antibody fragment-targeted cationic immunoliposome or polymer 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 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. The present invention also provides cationic immunoliposome or polymer complexes produced by such methods and compositions comprising such complexes. The present invention also provides methods for treating various diseases and disorders, including cancers, by administering the complexes and compositions of the invention to a patient.
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 that does not comprise maleimido-phenylbutyrate-lipid (MPB-lipid) 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 an siRNA to form said antibody- or antibody fragment-targeted-cationic immunoliposome complex,
wherein the cationic immunoliposome complex is 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 an anti-HER-2 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 about 1:20 to about 1:40 (w:w).
10. The method of claim 1 , wherein said cationic liposome comprises a mixture of, dioleoyltrimethylammonium phosphate with dioleoylphosphatidylethanolamine and cholesterol, a mixture of dioleoyltrimethylammonium phosphate with cholesterol, a mixture of dimethyldioctadecylammonium bromide with dioleoylphosphatidylethanolamine and cholesterol, a mixture of dimethyldioctadecylammonium bromide with dioleoylphosphatidylethanolamine, a mixture of dimethyldioctadecylammonium bromide with cholesterol, or a mixture of dioleoyltrimethylammonium phosphate with dioleoylphosphatidylethanolamine.
11. The method of claim 1 , wherein said cationic immunoliposome is mixed with said siRNA at a ratio in the range of about 1:3.5 to about 1:14 (μg siRNA:nmol liposome.
12. The method of claim 1 , wherein said cationic immunoliposome is mixed with said siRNA at a molar ratio of about 1:7 (μg siRNA:nmol liposome).
13. The method of claim 1 , wherein said cationic immunoliposome is mixed with said siRNA at a molar ratio of about 1:5 (μg siRNA:nmol liposome).
14. The method of claim 1 , wherein said siRNA is an anti-HER-2 siRNA.
15. The method of claim 14 , wherein a sense strand of said anti-HER-2 siRNA comprises the sequence:
5′-d(TITIT)-2′OMe(GCGGUGGUU)-d(GICIT) (SEQ ID NO:9).
16. The method of claim 14 , wherein said anti-HER-2 siRNA comprises the sequences:
TITIT gcggugguu GICIT
(SEQ ID NO: 9)
AGAGACGCCACCAACCGUA.
(SEQ ID NO: 16)
17. A cationic immunoliposome complex prepared by a method comprising:
(a) preparing an antibody or antibody fragment;
(b) mixing said antibody or antibody fragment with a cationic liposome that does not comprise maleimido-phenylbutyrate-lipid (MPB-lipid) 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 an siRNA to form said antibody- or antibody fragment-targeted-cationic immunoliposome complex,
wherein said cationic immunoliposome complex is 50-400 nm in size.
18. An antibody- or antibody fragment-targeted cationic immunoliposome complex comprising a cationic liposome that does not comprise maleimido-phenylbutyrate-lipid (MPB-lipid), an antibody or antibody fragment, and an siRNA, 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, wherein said cationic immunoliposome complex is 50-400 nm in size.
19. The cationic immunoliposome complex of claim 18 , wherein said siRNA is encapsulated within said cationic liposome.
20. The cationic immunoliposome complex of claim 18 , wherein said siRNA is associated with an inner or outer monolayer of said cationic liposome.
21. The cationic immunoliposome complex of claim 18 , wherein said antibody fragment is a single chain Fv fragment.
22. The cationic immunoliposome complex of claim 18 , wherein said antibody fragment is an anti-transferrin receptor single chain Fv (TfRscFv).
23. The cationic immunoliposome complex of claim 18 , wherein said antibody or antibody fragment is an anti-HER-2 antibody or an anti-HER-2 antibody fragment.
24. The cationic immunoliposome complex of claim 18 , wherein said cationic liposome comprises a mixture of one or more cationic lipids and one or more neutral or helper lipids.
25. The cationic immunoliposome complex of claim 18 , wherein said antibody or antibody fragment and said cationic liposome are present at a ratio in the range of about 1:20 to about 1:40 (w:w).
26. The cationic immunoliposome complex of claim 18 , wherein said cationic liposome comprises a mixture of dioleoyltrimethylammonium phosphate with dioleoylphosphatidylethanolamine and cholesterol, a mixture of dioleoyltrimethylammonium phosphate with cholesterol, a mixture of dimethyldioctadecylammonium bromide with dioleoylphosphatidylethanolamine and cholesterol, a mixture of dimethyldioctadecylammonium bromide with dioleoylphosphatidylethanolamine, a mixture of dimethyldioctadecylammonium bromide with cholesterol, or a mixture of dioleoyltrimethylammonium phosphate with dioleoylphosphatidylethanolamine.
27. The cationic immunoliposome complex of claim 18 , wherein said siRNA and said cationic immunoliposome are present at a ratio in the range of about 1:3.5 to about 1:14 (μg siRNA:nmol liposome).
28. The cationic immunoliposome complex of claim 18 , wherein said siRNA and said cationic immunoliposome are present at a molar ratio of about 1:7 (μg siRNA:nmol liposome).
29. The cationic immunoliposome complex of claim 18 , wherein said siRNA and said cationic immunoliposome are present at a molar ratio of about 1:5 (μg siRNA:nmol liposome).
30. The cationic immunoliposome complex of claim 18 , wherein said siRNA is an anti-HER-2 siRNA.
31. The cationic immunoliposome complex of claim 30 , wherein a sense strand of said anti-HER-2 siRNA comprises the sequence:
5′-
(SEQ ID NO: 9)
d(TITIT)-2′OMe(GCGGUGGUU)-d(GICIT).
32. The cationic immunoliposome complex of claim 30 , wherein said anti-HER-2 siRNA comprises the sequences:
TITIT gcggugguu GICIT;
(SEQ ID NO: 9)
and
AGAGACGCCACCAACCGUA.
(SEQ ID NO: 16)
33. An antibody- or antibody fragment-targeted cationic immunoliposome complex comprising a cationic liposome, an antibody or antibody fragment, and an anti-HER-2-siRNA, wherein said antibody or antibody fragment is complexed with said cationic liposome but is not chemically conjugated to said cationic liposome, and wherein the sense strand of said anti-HER-2 siRNA comprises the sequence:
5′-d(TITIT)-2′OMe(GCGGUGGUU)-d(GICIT) (SEQ ID NO:9).
34. The cationic immunoliposome complex of claim 33 , wherein said anti-HER-2 siRNA comprises the sequences:
TITIT gcggugguu GICIT;
(SEQ ID NO: 9)
and
AGAGACGCCACCAACCGUA.
(SEQ ID NO: 16)
35. The cationic immunoliposome complex of claim 33 , further comprising a K[K(H)KKK] 5 -K(H)KKC (HoKC) (SEQ ID NO: 20) peptide associated with said complex.
36. The cationic immunoliposome complex of claim 33 , wherein said antibody fragment is a single chain Fv fragment.
37. The cationic immunoliposome complex of claim 33 , wherein said antibody fragment is an anti-transferrin receptor single chain Fv (TfRscFv).
38. The cationic immunoliposome complex of claim 33 , wherein said antibody or antibody fragment is an anti-HER-2 antibody or an anti-HER-2 antibody fragment.
39. The cationic immunoliposome complex of claim 33 , wherein said antibody or antibody fragment and said cationic liposome are present at a ratio in the range of about 1:20 to about 1:40 (w:w).
40. The cationic immunoliposome complex of claim 33 , wherein said cationic liposome comprises a mixture of dioleoyltrimethylammonium phosphate with dioleoylphosphatidylethanolamine and cholesterol, a mixture of dioleoyltrimethylammonium phosphate with cholesterol, a mixture of dimethyldioctadecylammonium bromide with dioleoylphosphatidylethanolamine and cholesterol, a mixture of dimethyldioctadecylammonium bromide with dioleoylphosphatidylethanolamine, a mixture of dimethyldioctadecylammonium bromide with cholesterol, or a mixture of dioleoyltrimethylammonium phosphate with dioleoylphosphatidylethanolamine.
41. The cationic immunoliposome complex of claim 33 , wherein said anti-HER-2-siRNA and said cationic immunoliposome are present at a ratio in the range of about 1:3.5 to about 1:14 (μg siRNA:nmol liposome).
42. The cationic immunoliposome complex of claim 33 , wherein said anti-HER-2-siRNA and said cationic immunoliposome are present at a molar ratio of about 1:7 (μg siRNA:nmol liposome).
43. The cationic immunoliposome complex of claim 33 , wherein said anti-HER-2-siRNA and said cationic immunoliposome are present at a molar ratio of about 1:5 (μg siRNA:nmol liposome).
44. The cationic immunoliposome complex of claim 17 , wherein said siRNA and said cationic immunoliposome are present at a ratio in the range of about 1:3.5 to about 1:7 (μg siRNA:nmol liposome).
45. The cationic immunoliposome complex of claim 18 , wherein said siRNA and said cationic immunoliposome are present at a ratio in the range of about 1:3.5 to about 1:7 (μg siRNA:nmol liposome).
46. An antibody- or antibody fragment-targeted cationic immunoliposome complex comprising a cationic liposome that does not comprise maleimido-phenylbutyrate-lipid (MPB-lipid), an antibody or antibody fragment, and siRNA, wherein said antibody or antibody fragment is complexed with said cationic liposome but is not chemically conjugated to said cationic liposome and said antibody or antibody fragment does not comprise a lipid tag, and wherein said siRNA and said cationic immunoliposome are present at a ratio in the range of about 1:3.5 to about 1:5 (μg siRNA:nmol liposome).
47. The cationic immunoliposome complex of claim 46 , wherein said siRNA is an anti-HER-2 siRNA comprising the sequence:
5′-d(TITIT)-2′OMe(GCGGUGUU)-d(GICIT) (SEQ ID NO:9).
48. The cationic immunoliposome complex of claim 47 , wherein said anti-HER-2 siRNA comprises the sequences:
TITIT gcggugguu GICIT;
(SEQ ID NO: 9)
and
AGAGACGCCACCAACCGUA.
(SEQ ID NO: 16)
49. The cationic immunoliposome complex of claim 46 , wherein said antibody fragment is a single chain Fv fragment.
50. The cationic immunoliposome complex of claim 46 , wherein said antibody fragment is an anti-transferrin receptor single chain Fv (TfRscFv).
51. The cationic immunoliposome complex of claim 46 , wherein said antibody or antibody fragment is an anti-HER-2 antibody or an anti-HER-2 antibody fragment.
52. The cationic immunoliposome complex of claim 46 , wherein said antibody or antibody fragment and said cationic liposome are present at a ratio in the range of about 1:20 to about 1:40 (w:w).
53. The cationic immunoliposome complex of claim 46 , wherein said cationic liposome comprises a mixture of dioleoyltrimethylammonium phosphate with dioleoylphosphatidylethanolamine and cholesterol, a mixture of dioleoyltrimethylammonium phosphate with cholesterol, a mixture of dimethyldioctadecylammonium bromide with dioleoylphosphatidylethanolamine and cholesterol, a mixture of dimethyldioctadecylammonium bromide with dioleoylphosphatidylethanolamine, a mixture of dimethyldioctadecylammonium bromide with cholesterol, or a mixture of dioleoyltrimethylammonium phosphate with dioleoylphosphatidylethanolamine.
54. The cationic immunoliposome complex of claim 46 , wherein said siRNA and said cationic immunoliposome are present at a molar ratio of 1:5 (μg siRNA:nmol liposome).
55. An antibody- or antibody fragment-targeted cationic immunoliposome complex comprising a cationic liposome, an antibody or antibody fragment, and siRNA, wherein said antibody or antibody fragment is complexed with said cationic liposome but is not chemically conjugated to said cationic liposome and said antibody or antibody fragment does not comprise a lipid tag, and wherein said siRNA and said cationic immunoliposome are present at a ratio in the range of about 1:3.5 to 1:5 (μg siRNA:nmol liposome) said complex further comprising a K[K(H)KKK] 5 -K(H)KKC (HoKC) (SEQ ID NO: 20) peptide associated with said complex.
56. The cationic immunoliposome complex of claim 55 , wherein said siRNA is an anti-HER-2 siRNA comprising the sequence:
5′-d(TITIT)-2′OMe(GCGGUGGUU)-d(GICIT) (SEQ ID NO:9).
57. The cationic immunoliposome complex of claim 55 , wherein said anti-HER-2 siRNA comprises the sequences:
TITIT gcggugguu GICIT;
(SEQ ID NO: 9)
and
AGAGACGCCACCAACCGUA.
(SEQ ID NO: 16)
58. The cationic immunoliposome complex of claim 55 , wherein said antibody fragment is a single chain Fv fragment.
59. The cationic immunoliposome complex of claim 55 , wherein said antibody fragment is an anti-transferrin receptor single chain Fv (TfRscFv).
60. The cationic immunoliposome complex of claim 55 , wherein said antibody or antibody fragment is an anti-HER-2 antibody or an anti-HER-2 antibody fragment.
61. The cationic immunoliposome complex of claim 55 , wherein said antibody or antibody fragment and said cationic liposome are present at a ratio in the range of about 1:20 to about 1:40 (w:w).
62. The cationic immunoliposome complex of claim 55 , wherein said cationic liposome comprises a mixture of dioleoyltrimethylammonium phosphate with dioleoylphosphatidylethanolamine and cholesterol, a mixture of dioleoyltrimethylammonium phosphate with cholesterol, a mixture of dimethyldioctadecylammonium bromide with dioleoylphosphatidylethanolamine and cholesterol, a mixture of dimethyldioctadecylammonium bromide with dioleoylphosphatidylethanolamine, a mixture of dimethyldioctadecylammonium bromide with cholesterol, or a mixture of dioleoyltrimethylammonium phosphate with dioleoylphosphatidylethanolamine.
63. The cationic immunoliposome complex of claim 55 , wherein said siRNA and said cationic immunoliposome are present at a molar ratio of 1:5 (μg siRNA:nmol liposome).
64. The cationic immunoliposome complex of claim 33 , wherein said cationic immunoliposome complex is 50-400 nm in size.
65. The cationic immunoliposome complex of claim 46 , wherein said cationic immunoliposome complex is 50-400 nm in size.
66. The cationic immunoliposome complex of claim 55 , wherein said cationic immunoliposome complex is 50-400 nm in size.