Drug – bio-affecting and body treating compositions – Liposome comprising an antibody – antibody fragment – antigen,...
Reexamination Certificate
1996-04-18
2001-04-10
Nolan, Patrick J. (Department: 1644)
Drug, bio-affecting and body treating compositions
Liposome comprising an antibody, antibody fragment, antigen,...
C530S388220, C530S387300, C530S387100
Reexamination Certificate
active
06214388
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of liposomes. In particular, the present invention relates to liposomes specifically targeted to characteristic markers on target cells and which contain up to 4 mole percent of a hydrophilic polymer which results in an unexpected high rate of cellular incorporation.
BACKGROUND OF THE INVENTION
A number of pharmaceutical agents and potential pharmaceutical agents suffer from poor aqueous solubility, high levels of antigenicity, toxicity, or rapid degradation in serum which can hamper the development of suitable clinical formulations. One solution to these problems has been to encapsulate the pharmaceutical agent in a delivery vehicle that is soluble in aqueous solutions and that shields the agent from direct contact with tissues and blood. In particular, formulations based on liposome technology are of significant interest. Liposomes are vesicles comprised of concentrically ordered phospholipid bilayers which encapsulate an aqueous phase. They form spontaneously when phospholipids are exposed to aqueous solutions and can accommodate a variety of bioactive molecules.
Liposomes have proved a valuable tool as an in vivo delivery system for enhancing the efficacy of various pharmacologically active molecules (Ostro et al.
Liposomes from Biophysics to Therapeutics
, Dekker, New York, pp. 1-369 (1987)). Animal studies have shown that liposomes can decrease the toxicity of several antitumor and antifungal drugs, leading to clinical trials with promising results (Sculier et al.
Eur. J. Cancer Clin. Oncol.,
24: 527-538; Gabizon, et al. Eur. J. Cancer Clin. Oncol., 25: 1795-1803 (1989); Treat et al.,
J. Natl. Cancer Inst.,
82: 1706-1710 (1990); Lopez-Berestein et al.
J. Infect. Dis.,
151: 704-710 (1985); Presant et al.
Cancer,
62: 905-911 (1988)). In addition, liposomes have been shown to be efficient carriers of antiparasitic drugs for treating intracellular infections of the reticuloendothelial system (RES), in activating macrophage cells to become tumoricidal, in models of metastasis, and in enhancing the immune response to encapsulated antigens, thus facilitating the formulation of artificial vaccines (
Liposomes in the Therapy of Infectious Diseases and Cancer
, Lopez-Berestein & Fidler, eds. Liss, New York (1989); Alving et al.
Immunol. Lett.,
25: 275-280 (1990)).
All these effects stem from the capacity of macrophage cells in the liver and spleen (mononuclear phagocytic system MPS or reticuloendothelial system RES) to remove the majority of liposomes from the blood circulation within minutes (
Liposomes as Drug Carriers
, Gregoriadis, ed., Wiley, New York. (1988)). Such rapid clearance of liposomes however, has limited their prospects as an in vivo delivery system for transporting drugs to sites of disease beyond the RES.
Recent reports have described the use of various polymers to increase serum half-life of liposomes. In particular, it has been recognized that formulations of liposomes containing either mono-sialoganglioside (GM,) or lipid derivatives of polyethylene glycol avoid MPS removal and significantly increase serum half-life (Allen et al.
FEBS Lett.,
223: 42-46 (1987); Klibanov et al.,
FEBS Lett.,
268: 235-237 (1990); Blume et al.
Biochim, Biophys. Acta.,
1029: 91-97 (1990); Allen et al.
Biochim. Biophys. Acta,
1066: 29-36 (1991); Papahadjopoulos et al.,
Proc. Natl. Acad. Sci. USA,
88: 11460-11464 (1991); Senior et al.
Biochim. Biophys. Acta.,
1062: 77-82 (1991); Allen et al.
Biochim. Biophys. Acta.,
1068: 133-141 (1991)).
Many reports have demonstrated that rapid removal of circulating liposomes in vivo by cells of the mononuclear phagocytic system (MPS) can be overcome by incorporation of lipids derivatized with the hydrophilic polymer polyethylene glycol (PEG). These liposomes are referred to as sterically stabilized or “stealth” liposomes. With PEG having a molecular weight in the range of 1000 to 5000, prolonged circulation and reduced MPS uptake is achieved (Woodle & Lasic.
Biochim. Biophys. Acta
1113:171-199 (1992)). However, this reduction in clearance by the MPS is also associated with a reduction in uptake by a variety of cells (Lee, K. D. et al.
Biochim. Biophys. Acta
1103:185-197 (1992)). In addition, the presence of hydrophilic polymers on the surface of the liposome appears to interfere with specific ligand recognition by targeting moieties conjugated to the liposome. Presumably this occurs due to steric hinderance of the active site of the targeting moiety by the long chain PEG molecules. (Klibanov et al.
Biochim. Biophys. Acta
1062:148-148 (1991)).
Finally, while most therapeutic agents transported by liposomes must enter the cytoplasm of the target cell in order to express their biological activity, it is generally appreciated that most liposomes are either not actually internalized by the target cells, or, where uptake does occur, it is generally via an endocytotic pathway. Thus actual drug to the target cell typically entails release from the liposome (e.g. through disruption of the liposome itself or through “leakage”) in the vicinity of the target cell and then subsequent uptake (either through diffusion, endocytosis, phagocytosis, or active transport) of the therapeutic agent from solution by the target cell. Indeed immunoliposomes have been designed to actually induce destabilization and fragmentation of the liposome once the targeting antibody has bound a target, thereby freeing the liposome contents (see, U.S. Pat. No. 4,957,735). Even these “target-sensitive” liposomes, lose a considerable amount of the therapeutic agent in solution before it can be taken up by the target cell. Alternatively, if the liposome is internalized by an endocytotic process, it is ultimately incorporated in a lysosome where strong acid conditions exist that can degrade a number of therapeutic agents (e.g. proteins).
Thus, delivery of effective doses of therapeutic agents to the cytoplasm of the target cell is hampered by low residence times in serum, ineffective targeting when residence times are increased, considerable loss of the therapeutic agent in solution before it may be taken up by the target cell, and degradation of the therapeutic in the endosomic/lysosomic pathway. Clearly, it would be desirable to obtain a liposome with increased serum half-life, capable of specifically targeting particular cells, and also capable of being internalized into the cytoplasm by the target cells thereby avoiding loss of the therapeutic agent or degradation by the endosomic/lysosomic pathway.
SUMMARY OF THE INVENTION
The present invention provides novel immunoliposomes optimized for delivering therapeutic agents to the cytoplasm of a target cell. These immunoliposomes exhibit increased half-life in blood, are capable of specifically targeting particular cells, and are capable of being internalized into the cytoplasm by the target cells thereby avoiding loss of the therapeutic agents or degradation by the endolysosomal pathway.
Thus, in one preferred embodiment this invention provides for immunoliposomes that optimize internalization of a therapeutic agent into the cytoplasm of a cell bearing a characteristic cell surface marker. These immunoliposomes comprise an Fab′ domain of an antibody wherein the Fab′ domain specifically binds the characteristic marker, an amphipathic vesicle-forming lipid that forms a liposome, a polyethylene glycol derivatized lipid wherein the polyethylene glycol has an average molecular weight of between about 750 D and 5000 D, more preferably between about 1200 D and about 3000 D, most preferably about 1900 D, and a therapeutic agent contained within the liposome. The derivatized lipid is present at up to about 1.2 mole percent, more preferably at up to about 2.4 mole percent, and most preferably at up to about 3.6 mole percent of total lipid. Preferred characteristic markers include growth factor receptors. Particularly preferred are growth factor receptors including HER1, HER2, HER3 and HER4 with HER2 being most preferred. The Fab′ domai
Benz Christopher C.
Hong Keelung
Kirpotin Dmitri
Papahadjopoulos Demetrios P.
Park John W.
Nolan Patrick J.
The Regents of the University of California
Townsend and Townsend / and Crew LLP
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