Immunoliposomes that optimize internalization into target cells

• US 20010016196A1
• Filed: 02/28/2001
• Published: 08/23/2001
• Est. Priority Date: 11/09/1994
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Hong, Keelung, Benz, Christopher C., Park, John W., Kirpotin, Dmitri, Papahadjopoulos, Demetrois
• Granted Patent: US 7,135,177 B2
• Time in Patent Office: Days
• US Class Current: 424/178.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 47/6849 the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• CPC Class Codes: A61K 47/6913 the liposome being modified on its surface by an antibody
• CPC Class Codes: A61K 9/127 Liposomes
• CPC Class Codes: A61K 9/1271 Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers liposome as conjugate A61K47/6911
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/77 Internalization into the cell

Abstract

The present invention provides for immunoliposomes that optimizes internalization of a drug into target cells bearing a characteristic cell surface marker. The immunoliposomes comprise an Fab′ domain of an antibody that specifically binds the characteristic marker, an amphipathic vesicle-forming lipid, and a polyethylene glycol derivatized lipid. The invention also provides for growth-inhibiting immunoliposomes that lack growth-inhibiting therapeutic agents and yet are capable of inhibiting the growth and proliferation of target cells.

Claims

1. A growth-inhibiting immunoliposome that specifically binds to a cell bearing a characteristic cell surface marker, said immunoliposome comprising:

a Fab′

domain of an antibody where said Fab′

domain specifically binds said marker; and

an amphipathic vesicle-forming lipid, said lipid forming a liposome containing no growth-inhibiting therapeutic agent;

wherein said immunoliposome inhibits the proliferation of said cell.

2. The immunoliposome of

claim 1

, wherein said marker is a growth factor receptor.

3. The immunoliposome of

claim 2

, wherein said marker is a growth factor receptor selected from the group consisting of HER1, HER2, HER3 and HER4.

4. The immunoliposome of

claim 3

, wherein said marker is HER2.

5. The immunoliposome of

claim 4

, wherein said Fab′

domain is a humanized Fab′

domain of an anti-HER2 monoclonal antibody.

6. The immunoliposome of

claim 1

, wherein said liposome further comprises a maleimide derivatized phosphatidylethanolamine (M-PE) which forms a thioether linkage to said Fab′

domain of an antibody.

7. The immunoliposome of

claim 1

, wherein Fab′

is attached to said liposome through a polyethyleneglycol (PEG) linker.

8. The immunoliposome of

claim 1

, wherein said vesicle forming lipid comprises a lipid selected from the group consisting of a phospholipid, a glycolipid, a sphingolipid, and a sterol.

9. The immunoliposome of

claim 1

further comprising a polyethylene glycol derivatized lipid wherein the polyethylene glycol has an average molecular weight of between about 750 D and 5000 D.

10. The immunoliposome of

claim 9

, wherein said Fab′

is attached to said polyethylene glycol.

11. The immunoliposome of

claim 1

, wherein the average diameter of said liposome ranges from about 50 nm to about 500 nm.

12. The immunoliposome of

claim 9

wherein the average diameter of said liposome is about 100 nm.

13. The immunoliposome of

claim 8

wherein:

said Fab′

domain of an antibody is rhuMAbHER2

said Fab′

domain of an antibody is conjugated to maleimide derivatized phosphatidylethanolamine (M-PE);

said vesicle forming lipid is phosphatidylcholine (PC) and cholesterol (Chol); and

said derivatized lipid is polyethylene glycol derivatized phosphatidylethanolamine (PEG-PE) wherein said polyethylene glycol has a molecular weight of about 1900 D;

where the ratio PC;

Chol;

M-PE is about 150;

100;

3 and said PEG-PE is present in an amount up to about 3.6 mole percent of total lipid.

15. The method of

claim 12

, wherein said marker is a growth factor receptor selected from the group consisting of HER1, HER2, HER3 and HER4.

16. The method of

claim 13

, wherein said Fab′

domain is a humanized Fab′

domain of a monoclonal antibody.

17. The method of

claim 12

, wherein said liposome further comprises a maleimide derivatized phosphatidylethanolamine (M-PE) which forms a thioether linkage to said antibody.

18. The method of

claim 12

, wherein said vesicle forming lipid comprises a lipid selected from the group consisting of a phospholipid, a glycolipid, a sphingolipid, and a sterol.

19. The method of

claim 12

, wherein said immunoliposome further comprises a polyethylene glycol derivatized lipid wherein said polyethylene glycol has an average molecular weight of between about 750 D and 5000 D.

20. The method of

claim 12

, wherein the average diameter of said liposome ranges from about 50 nm to about 500 nm.

21. The method of

claim 12

, wherein:

said Fab′

domain of an antibody is rhuMAbHER2;

said Fab′

domain of an antibody is conjugated to maleimide derivatized phosphatidylethanolamine (M-PE);

said vesicle forming lipid comprises phosphatidylcholine (PC) and cholesterol (Chol); and

said derivatized lipid is polyethylene glycol derivatized phosphatidylethanolamine (PEG-PE) wherein said polyethylene glycol has a molecular weight of about 1900 D;

where the ratio PC;

Chol;

M-DPE is 150;

100;

3 and said PEG-PE is present in an amount up to 3.6 mole percent of total lipid.

23. The immunoliposome of

claim 20

, wherein said marker is a growth factor receptor selected from the group consisting of HER1, HER2, HER3 and HER4.

24. The immunoliposome of

claim 21

, wherein said Fab′

domain is a humanized Fab′

domain of a monoclonal antibody.

25. The immunoliposome of

claim 20

, wherein said liposome further comprises a maleimide derivatized phosphatidylethanolamine (M-PE) which forms said thioether linkage to Fab′

domain of an antibody.

26. The immunoliposome of

claim 20

, wherein said vesicle forming lipid is selected from the group consisting of a phospholipid, a glycolipid, a sphingolipid, and a sterol.

27. The immunoliposome of

claim 20

, wherein the average diameter of said liposome ranges from about 50 nm to about 500 nm.

28. The immunoliposome of

claim 20

, wherein said therapeutic agent is selected from the group consisting of daunomycin, idarubicin, mitoxantrone, mitomycin, cisplatin and other Platinum II analogs, vincristine, epirubicin, aclacinomycin, methotrexate, etoposide, doxorubicin, cytosine arabinoside, fluorouracil and other fluorinated pyrimidines, purines, or nucleosides, bleomycin, mitomycin, plicamycin, dactinomycin, cyclophosphamide and derivatives thereof, thiotepa, BCNU, taxol, taxotere and other taxane derivatives and isolates, camptothecins, polypeptides, a nucleic acid, a nucleic acid having a phosphorothioate internucleotide linkage, and a nucleic acid having a polyamide internucleotide linkage.

29. The immunoliposome of

claim 20

, wherein:

said Fab′

domain of an antibody is rhuMAbHER2;

said vesicle forming lipid comprises phosphatidylcholine (PC) and cholesterol (Chol);

said derivatized lipid is polyethylene glycol derivatized phosphatidylethanolamine (PEG-PE) wherein said polyethylene glycol has a molecular weight of about 1900 D;

said antibody is conjugated to maleimide derivatized phosphatidylethanolamine (M-PE);

wherein said ratio of PC;

Chol;

M-DPE is 150;

100;

3;

6 and said PEG-PE is present in an amount less than 3.6 mole percent of total lipid.

31. The method of

claim 28

, wherein said internalizing comprises transporting said liposome directly into the cytoplasm of said cell.

32. The method of

claim 28

, wherein said marker is a growth factor receptor selected from the group consisting of HER1, HER2, HER3 and HER4.

34. The method of

claim 28

, wherein said vesicle forming lipid is selected from the group consisting of a phospholipid, a glycolipid, a sphingolipid, and a sterol.

35. The immunoliposome of

claim 28

, wherein said therapeutic agent is selected from the group consisting of daunomycin, idarubicin, mitoxantrone, mitomycin, cisplatin and other Platinum II analogs, vincristine, epirubicin, aclacinomycin, methotrexate, etoposide, doxorubicin, cytosine arabinoside, fluorouracil and other fluorinated pyrimidines, purines, or nucleosides, bleomycin, mitomycin, plicamycin, dactinomycin, cyclophosphamide and derivatives thereof, thiotepa, BCNU, taxol, taxotere and other taxane derivatives and isolates, camptothecins, polypeptides, a nucleic acid, a nucleic acid having a phosphorothioate internucleotide linkage, and a nucleic acid having a polyamide internucleotide linkage.

36. The immunoliposome of

claim 28

, wherein:

said Fab′

domain of an antibody is rhuMAbHER2;

said vesicle forming lipid comprises phosphatidylcholine (PC) and cholesterol (Chol);

said derivatized lipid is polyethylene glycol derivatized phosphatidylethanolamine (PEG-PE) wherein said polyethylene glycol has a molecular weight of about 1900 D;

said Fab′

domain of an antibody is conjugated to maleimide derivatized phosphatidylethanolamine (M-PE);

wherein said ratio of PC;

Chol;

M-DPE is 150;

100;

3;

6 and said PEG-PE is present in an amount up to 3.6 mole percent of total lipid.

14. A method of inhibiting growth of a cell bearing a characteristic cell surface marker, said method comprising contacting said cell with a growth-inhibiting immunoliposome, said immunoliposome comprising:

a Fab′

domain of an antibody where said Fab′

domain specifically binds said marker; and

an amphipathic vesicle-forming lipid, said lipid forming a liposome containing no growth-inhibiting therapeutic agent.

22. An immunoliposome that optimizes internalization of a therapeutic agent into the cytoplasm of a cell bearing a characteristic cell surface marker, said immunoliposome comprising:

a Fab′

domain of an antibody wherein said Fab′

domain specifically binds said marker;

an amphipathic vesicle-forming lipid, said vesicle-forming lipid forming a liposome;

a polyethylene glycol derivatized lipid wherein the polyethylene glycol has an average molecular weight of between about 750 D and 5000 D, the derivatized lipid being present at up to about 3.6 mole percent of the total lipid; and

a therapeutic agent contained within said liposome.

30. A method of optimizing internalization of a therapeutic agent into the cytoplasm of a cell bearing a characteristic cell surface marker, said method comprising the steps of

a) contacting said cell with an immunoliposome, said immunoliposome comprising:

i) an Fab′

domain of an antibody where said Fab′

domain specifically binds said marker;

ii) an amphipathic vesicle-forming lipid, said lipid forming a liposome;

iii) a polyethylene glycol derivatized lipid wherein the polyethylene glycol has an average molecular weight of between about 750 D and 5000 D, the derivatized lipid being present at less than 3.6 mole percent of the total lipid; and

iv) a therapeutic agent contained within said liposome; and

b) internalizing said liposome itself into said cell.

33. The method of

claim 30

, wherein said Fab′

domain is a humanized Fab′

domain of a monoclonal antibody.

37. An immunoliposome that is internalized into the cytoplasm of a cell bearing a characteristic cell surface marker, said immunoliposome comprising:

a Fab′

domain of an antibody attached to a polyethylene glycol derivatized lipid, wherein said Fab′

domain specifically binds said marker;

an amphipathic vesicle-forming lipid, said vesicle-forming lipid forming a liposome;

a polyethylene glycol derivatized lipid;

wherein when said immunoliposome is contacted with said cell, said immunoliposome is internalized into the cytoplasm of said cell.

38. The immunoliposome of

claim 37

, wherein said polyethylene glycol has an average molecular weight of between about 750 D and 5000 D.

39. The immunoliposome of

claim 37

, wherein said marker is a growth factor receptor selected from the group consisting of HER1, HER2, HER3 and HER4.

40. The immunoliposome of

claim 39

, wherein said Fab′

domain is a humanized Fab′

domain of a monoclonal antibody.

41. The immunoliposome of claim of

claim 37

, wherein:

said Fab′

attached to a polyethylene glycol derivatized lipid is rhuMAbHER2 attached to the distal terminus of PEG derivatized distearoylphosphatidylethanolamine (DSPE);

said polyethylene glycol derivatized lipid is PEG-PE said vesicle forming lipid comprises phosphatidylcholine (PC) and cholesterol (Chol);

wherein said ratio of PC;

Chol is about 3;

2, said PEG-PE is ranges from about 10 mole percent to about 12 mole percent of total phospholipid, and said Fab′

attached to a polyethylene glycol derivatized lipid ranges from about 0.6 to about 1.0 mole percent of total phospholipid.

42. A method of internalizing a liposome into the cytoplasm of a cell bearing a characteristic cell surface marker, said method comprising contacting said cell with an immunoliposome comprising:

an Fab′

domain of an antibody attached to a polyethylene glycol derivatized lipid, wherein said Fab′

domain specifically binds said marker;

an amphipathic vesicle-forming lipid, said vesicle-forming lipid forming a liposome;

a polyethylene glycol derivatized lipid;

wherein said contacting results in the internalization of said liposome into the cytoplasm of said cell.

43. The method of

claim 42

, wherein said polyethylene glycol has an average molecular weight of between about 750 D and 5000 D.

44. The method of

claim 42

, wherein said marker is a growth factor receptor selected from the group consisting of HER1, HER2, HER3 and HER4.

45. The method of

claim 42

, wherein said Fab′

domain is a humanized Fab′

domain of a monoclonal antibody.

46. The method of claim of

claim 42

, wherein:

said Fab′

attached to a polyethylene glycol derivatized lipid is rhuMAbHER2 attached to the distal terminus of PEG derivatized distearoylphosphatidylethanolamine (DSPE);

said polyethylene glycol derivatized lipid is PEG-PE said vesicle forming lipid comprises phosphatidylcholine (PC) and cholesterol (Chol);

wherein said ratio of PC;

Chol is about 3;

2, said PEG-PE is ranges from about 10 mole percent to about 12 mole percent of total phospholipid, and said Fab′

attached to a polyethylene glycol derivatized lipid ranges from about 0.6 to about 1.0 mole percent of total phospholipid.