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WO1996014864A1 - Immunoliposomes optimalisant l'incorporation d'un agent dans des cellules cibles - Google Patents

Immunoliposomes optimalisant l'incorporation d'un agent dans des cellules cibles Download PDF

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Publication number
WO1996014864A1
WO1996014864A1 PCT/US1995/014710 US9514710W WO9614864A1 WO 1996014864 A1 WO1996014864 A1 WO 1996014864A1 US 9514710 W US9514710 W US 9514710W WO 9614864 A1 WO9614864 A1 WO 9614864A1
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Prior art keywords
fab
immunoliposome
lipid
domain
liposome
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PCT/US1995/014710
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English (en)
Inventor
Christopher Benz
Demetrios Papahadjopoulos
John Park
Keelung Hong
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The Regents Of The University Of California
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Priority to AU41556/96A priority Critical patent/AU4155696A/en
Publication of WO1996014864A1 publication Critical patent/WO1996014864A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • the present invention relates to the field of liposomes.
  • 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.
  • 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.
  • 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.
  • 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)).
  • RES reticuloendothelial system
  • liposomes are either not actually internalized by the target cells, or, where uptake does occur, it is generally via an endocytotic pathway.
  • 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.
  • 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. Patent 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).
  • therapeutic agents e.g. proteins
  • 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.
  • 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' domain may be a humanized Fab' domain, more specifically a humanized Fab' domain of an anti-HER2 monoclonal antibody.
  • the growth-inhibiting immunoliposome may further comprise a maleimide-derivatized phosphatidylethanolamine (M-PE) which forms a thioether linkage to the Fab' domain of an antibody.
  • M-PE maleimide-derivatized phosphatidylethanolamine
  • the vesicle forming lipid may include a phospholipid, a glycolipid, a sphingolipid, or a sterol.
  • the immunoliposomes have an average diameter that ranges from about 50 nm to about 500 nm, more preferably about 75 nm to about 300 nm and most preferably is about 100 nm.
  • Therapeutic agents in the liposome may include 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 intemucleotide linkage, and a nucleic acid having a poly amide intemucleotide linkage.
  • the antibody Fab' domain is that of rhuMAbHER2, with the Fab' domain conjugated to maleimide- derivatized phosphatidylethanolamine (M-PE), the vesicle forming lipid is phosphatidylchohne (PC) and cholesterol (Choi) and the polyethylene glycol derivatized lipid is polyethylene glycol derivatized phosphatidylethanolamine (PEG- PE) where the polyethylene glycol component has a molecular weight of about 1900 D, and where the ratio PC:Chol:M-PE is 150:100:3 and the PEG-PE is present in an amount up to about 3.6 mole percent of total lipid.
  • M-PE maleimide- derivatized phosphatidylethanolamine
  • PEG- PE polyethylene glycol derivatized phosphatidylethanolamine
  • This invention also provides for a method of optimizing internalization of a therapeutic agent into a cell bearing a characteristic marker, the method comprising contacting the cell with any of the immunoliposomes summarized above and internalizing the liposome contents into the cytoplasm of the cell.
  • This internalization may be by fusion of the liposome with cell membranes, or early exit from the endocytic vesicles.
  • this invention provides for a growth-inhibiting immunoliposome that specifically binds to a cell bearing a characteristic cell surface marker thereby inhibiting the proliferation or growth of that cell.
  • the immunoliposome comprises an Fab' domain of an antibody where the Fab' domain specifically binds the marker and an amphipathic vesicle-forming lipid that forms a liposome. Unlike typical drug-delivery liposomes, however, this liposome contains no growth-inhibiting therapeutic agent and may contain no therapeutic agent at all.
  • a composition may be identified as not being a growth- inhibiting therapeutic agent when the decrease in proliferation rate of target cells is less than 10 percent, more preferably less than 5 percent, more than the decrease in proliferation rate of the target cells caused by an "empty" liposome containing no agent (other than a neutral buffer or water) at all.
  • 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' domain may be a humanized Fab' domain, more specifically a humanized Fab' domain of an anti-HER2 (anti-pl85HER2) monoclonal antibody.
  • the growth-inhibiting immunoliposome may further comprise a maleimide derivatized phosphatidylethanolamine (M-PE) which forms a thioether linkage to the Fab' domain of an antibody.
  • M-PE maleimide derivatized phosphatidylethanolamine
  • the vesicle forming lipid may include a phospholipid, a glycolipid, a sphingolipid, or a sterol.
  • the growth-inhibiting immunoliposome may also comprise a hydrophilic polymer.
  • Preferred hydrophilic polymers include polyethylene glycol, polypropylene glycol, mono-sialoganglioside (GM,), phosphatidylinositol (PI) or cerebroside sulfate (CS)).
  • Polyethylene glycol when incorporated into the lipid will be incorporated as a polyethylene glycol derivatized lipid, preferably a polyethylene glycol derivatized phospholipid such as PEG-PE.
  • the molecular weight of the polyethylene glycol may range from about 750 D to about 5000 D, more preferably from about 1200 D to about 3000 D, and most preferably is about 1900 D.
  • the liposomes have an average diameter that ranges from about 50 nm to about 500 nm, more preferably about 75 nm to about 300 nm and most preferably is about 100 nm.
  • the antibody Fab' domain is rhuMAbHER2
  • the Fab' domain is conjugated to M-PE
  • the vesicle forming lipid is phosphatidylcholine (PC) and cholesterol (Choi)
  • the polyethylene glycol derivatized lipid is polyethylene glycol derivatized phosphatidylethanolamine (PEG-PE) where the polyethylene glycol component has a molecular weight of about 1900 D, and where the ratio PC:Chol:M-PE is 150: 100:3 and the PEG-PE is present in an amount up to about 3.6 mole percent of total lipid.
  • This invention also provides for a method of inhibiting growth of a cell bearing a characteristic marker, the method comprising contacting the cell with any of the growth-inhibiting liposomes summarized above.
  • this invention also provides for pharmaceutical compositions comprising any of the growth-inhibiting or therapeutic-agent carrying immunoliposomes described above.
  • the pharmaceutical compositions comprise a therapeutically effective dose of the immunoliposome and a pharmaceutically acceptble carrier or excipient.
  • Figure 1 illustrates flow cytometric histograms showing binding of anti-pl85 HER2 immunoliposomes to SK-BR-3 cells.
  • Immunoliposomes bound to SK-BR-3 cells after washing were detected by FITC-labeled goat anti-human IgG, which recognizes rhuMAbHER2 Fab' fragments.
  • SK-BR-3 cells were incubated with conventional immunoliposomes (A), sterically stabilized (6 mole% PEG-PE) immunoliposomes (B), and free rhuMAbHER2-Fab' fragments (C) at equivalent antibody concentrations (3.3 ⁇ g/ml).
  • Figure 2 shows the binding of anti-pl85 HER2 conventional immunoliposomes to BT-474 cells.
  • A BT-474 cells in monolayer culture were treated with conventional immunoliposomes in the presence of competing 12S I-labeled rhuMabHER2 as described in Methods.
  • B Scatchard transformation of the data shown in (A).
  • Figure 3 illustrates the antiproliferative activity of anti-pl85 HER2 immunoliposomes against SK-BR-3 cells.
  • SK-BR-3 cells in monolayer culture were treated with immunoliposomes at antibody doses indicated on the abscissa, and relative cell proliferation determined as described in Methods.
  • Control liposomes lacking antibody were dosed according to liposome concentration, and are plotted at equivalent liposome concentration with the appropriately matched immunoliposome.
  • Figure 4 shows the cytotoxicity of anti-pl ⁇ STM* 2 immunoliposomes containing doxorubicin.
  • HSPC Choi immunoliposomes were loaded with doxorubicin as described in Methods.
  • A SK-BR-3 cells.
  • B WI-38 cells. Cells in culture were treated for 1 hour with: conventional immunoliposomes (triangles); sterically stabilized (2 mole% PEG-PE) immunoliposomes (closed circles); control (irrelevant antibody) sterically stabilized (2 mole% PEG-PE) immunoliposomes (open circles); or free doxorubicin alone (closed squares).
  • Immunoliposomes contained 60-70 ⁇ g antibody/ ⁇ mol phospholipid and 55-80 ⁇ g doxorubicin/ ⁇ mol phospholipid; antibody/doxorubicin ratio was 0.8-1.2. Cells were counted 3 days after treatment as described in Methods.
  • DOX doxorubicin
  • Choi cholesterol
  • PA phosphatidic acid
  • PC phosphatidylcholine
  • PI phosphatidylinositol
  • SM sphinogmyelin
  • M-DPE maleimide derivatized dipalmityolethanolamine
  • PBS phosphate buffered saline
  • LUV large unilamellar vesicles
  • MLV multilamellar vesicles
  • PE phosphatidylethanolamine
  • PEG polyethylene glycol
  • PEG-PE polyethylene glycol derivatized phosphatidylethanolamine .
  • amphipathic vesicle-forming lipid is intended to include any amphipathic lipid having hydrophobic and polar head group moieties, and which by itself can form spontaneously into bilayer vesicles in water, as exemplified by phospholipids, or (b) is stably incorporated into lipid bilayers in combination with phospholipids with its hydrophobic moiety in contact with the interior, hydrophobic region of the bilayer membrane, and its polar head group moiety oriented toward the exterior, polar surface of the membrane.
  • An example of the latter type of vesicle-forming lipid is cholesterol and cholesterol derivatives such as cholesterol sulfate and cholesterol hemisuccinate.
  • the term "specific binding” refers to that binding which occurs between such paired species as enzyme/ substrate, receptor/agonist, antibody/antigen, and lectin/carbohydrate which may be mediated by covalent or non-covalent interactions or a combination of covalent and non-covalent interactions.
  • the binding which occurs is typically electrostatic, hydrogen- bonding, or the result of lipophilic interactions. Accordingly, "specific binding" occurs between a paired species where there is interaction between the two which produces a bound complex having the characteristics of an antibody/ antigen or enzyme/substrate interaction.
  • the specific binding is characterized by the binding of one member of a pair to a particular species and to no other species within the family of compounds to which the corresponding member of the binding member belongs.
  • an antibody preferably binds to a single epitope and to no other epitope within the family of proteins.
  • ligand or “targeting moiety”, as used herein, refer generally to all molecules capable of specifically binding to a particular target molecule and forming a bound complex as described above. Thus the ligand and its corresponding target molecule form a specific binding pair.
  • Examples include, but are not limited to antibodies, lymphokines, cytokines, receptor proteins such as CD4 and CD8, solubilized receptor proteins such as soluble CD4, hormones, growth factors, and the like which specifically bind desired target cells, and nucleic acids which bind corresponding nucleic acids through base pair complementarity.
  • Particularly preferred targeting moieties include antibodies and antibody fragments (e.g. , the Fab' domain).
  • immunosorbome refers to a liposome bearing an antibody or antibody fragment that acts as a targeting moiety enabling the liposome to specifically bind to a particular "target" molecule that may exist in solution or may be bound to the surface of a cell.
  • target molecule is one that is typically found in relative excess (e.g., > 10-fold) and in association with a particular cell type or alternatively in a multiplicity of cell types all expressing a particular physiological condition the target molecule is said to be a "characteristic marker" of that cell type or that physiological condition.
  • a cancer may be characterized by the overexpression of a particular marker such as the HER2 (c-erbB-2lneu) proto-oncogene in the case of breast cancer.
  • hydrophilic polymer refers to long chain highly hydrated flexible neutral polymers attached to lipid molecules. Examples include, but are not limited to polyethylene glycol-, or polypropylene glycol-modified lipids PI or CS, or ganglioside GM,.
  • the term "mole percent" when referring to the percentage of hydrophilic polymer in a liposome is expressed relative to the total lipid in the liposome unless otherwise stated. Thus, for example, in a liposome comprising a ratio of phosphatidylcholine (PC) to cholesterol (Choi) of 150:100, a 4 mole percent of hydrophilic polymer (e.g. PEG) would represent a ratio of PC.Chol.PEG of about 150: 100: 10.
  • proliferation refers to cell division or mitosis. Proliferation may be measured by standard assays such as by uptake of radioactive nucleotides (thymidine) or by direct observation.
  • thymidine radioactive nucleotides
  • the present invention provides immunoliposomes for selective delivery of therapeutic agents to specific tissues in a host and methods of use for those liposomes.
  • the liposomes of this invention employ a composition that optimizes internalization of the liposome into the cytoplasm of the cells of the target tissue.
  • the phrase “optimizes internalization” or “optimal internalization” is used to refer to the delivery of liposome contents such that it achieves maximum delivery to the cytoplasm of the target cell and therefore maximum therapeutic effect.
  • Optimal internalization into die cytoplasm of die cell refers to that condition in which maximal uptake into the cytoplasm of the target cell is achieved while still maintaining a blood half-life significantly greater than the blood half-life of liposomes lacking any hydrophilic polymer and adequate for targeting purposes.
  • this invention relies, in part, on die unexpected discovery tiiat a liposome comprising a hydrophilic polymer (e.g. , PEG-modified lipid) in an amount up to about 3.6 mole percent of total (vesicle-forming) lipid demonstrates an unexpected high rate of internalization into d e cytoplasm of d e target cell while retaining a blood half-life substantially greater tiian mat seen in liposomes lacking a hydrophilic polymer.
  • a hydrophilic polymer e.g. , PEG-modified lipid
  • the immunoliposomes of this invention optimize delivery of therapeutic agents to the cytoplasm of the target cell by maintaining an elevated blood half-life, as compared to a liposome lacking a hydrophilic polymer, by maintaining a high degree of target specificity, and by effective internalization of the liposome itself (carrying merapeutic agent) thereby avoiding considerable loss of me merapeutic agent in solution or degradation of me merapeutic in me endosomic/lysosomic pathway.
  • the liposomes of the present invention are thus particularly useful as vehicles for the delivery of therapeutics to specific target cells.
  • This invention also provides for growth-inhibiting immunoliposomes that may be utilized to inhibit tumor cell proliferation and tiius provide an antitumor activity without encapsulating a growth-inhibiting therapeutic agent.
  • me growm-inhibiting immunoliposomes of the present invention are effective when they contain no therapeutic agent.
  • the growth-inhibiting immunoliposomes of mis invention generally comprise an Fab' domain of an antibody diat specifically binds to a cell bearing a characteristic marker, and an amphipathic vesicle forming lipid.
  • the liposome may be conjugated to me Fab' domain of an anti- HER2 monoclonal antibody.
  • the antibody is me Fab' fragment of me human monoclonal anti-HER2 antibody (rhuMAbHER2-Fab').
  • rhuMAbHER2-Fab' me human monoclonal anti-HER2 antibody
  • liposomal (membrane) anchoring of me monovalent Fab' fragment results in antiproliferative and antitumor activity comparable to bivalent rhuMAbHER2.
  • the antibody rhuMAbHER2-Fab' in solution does not have this property. Without being bound to a particular theory, it is believed diat membrane anchoring of me Fab' fragment in die anti-HER2 immunoliposome confers this antiproliferative property presumably by enabling cross-linking of pl ⁇ S 1 ⁇ 82 on the tumor cell surface.
  • me growm-inhibiting immunoliposomes do not contain a growth-inhibiting agent.
  • a “growm-inhibiting agent” refers to a chemical agent that reduces me growth rate of cells to which it is administered. In me extreme case a growth-inhibiting agent may be cytotoxic to the cell to which it is administered.
  • me growth rate of cells refers to me rate of proliferation of the cells. Increased proliferation rate is typically associated with increased metabolic rate and thus proliferation rates may be assayed by detecting 4864 PC17US95/14710
  • metabolic rates e.g. , by uptake of a labeled metabolic precursor such as tritiated thymidine.
  • increased growth or proliferation rate may be taken as indicating an increased metabolic rate or vice versa.
  • Growth-inhibiting agents are well known to those of skill in the art and include, but are not limited to doxorubicin, ricin A, gelonin. It will be recognized that some compositions (e.g. , antibiotics) may exhibit minor growm inhibitory activity as an incidental consequence of tiieir primary activity. Such compositions are not considered herein to be growm-inhibiting agents.
  • the phrase a "liposome containing no growth-inhibiting therapeutic agent" is intended to capmre me fact that the inhibition of cell growth and proliferation obtained with me growth-inhibiting immunoliposomes of the present invention is a consequence of me liposome/Fab' construct itself and is not a consequence of me liposome contents.
  • a growth inhibiting agent refers to an agent that, when present in the growth inhibiting immunoliposome, results in a decrease in cell proliferation rate at least 10 percent greater than the decrease in cell proliferation rate observed by administration of the same immunoliposomes lacking any therapeutic or growm inhibiting agent.
  • the growth-inhibiting liposomes of this invention will inhibit cell growth and proliferation even when they carry no therapeutic agent and erefore may be administered "empty", one of skill will appreciate that it may be desirable to encapsulate a therapeutic agent omer than a growm-inhibiting merapeutic agent thereby achieving a liposome that shows dual, additive or supradditive activities.
  • an immunoliposome loaded wim an antibiotic will show bo antibiotic activity as well as me ability to inhibit growth and proliferation of me target cells.
  • the growth-inhibiting liposomes and me merapeutic agent-carrying immunoliposomes of the present invention may be utilized to inhibit tumor cell proliferation or to target therapeutics to specific cells in a wide variety of hosts.
  • Preferred hosts include mammalian species such as humans, non-human primates, dogs, cats, cattle horses, sheep, rodents, largomorphs and me like. Liposome Composition
  • me immunoliposomes of me present invention comprise one or more vesicle-forming lipids, an Fab' domain of an antibody which acts as a targeting moiety and, especially in the case of the therapeutic agent delivering immunoliposomes, a hydrophilic polymer.
  • a hydrophilic polymer serves to prevent agglomeration of me liposomes and also to decrease uptake of the liposome by the RES and mereby increase blood half-life
  • die ligand serves to specifically bind me liposomes to a cell or tissue bearing a target (i.e.
  • the low mole percentage of me hydrophilic polymer coupled with me use of me Fab' antibody fragment allows specific targeting of me liposome and unexpectedly results in a high level of internalization of the entire liposome into the cytoplasm of the target cell.
  • the vesicle-forming lipid is preferably one having two hydrocarbon chains, typically acyl chains and a polar head group. Included in this class are the phospholipids, such as phosphatidylcholine (PC), phosphatidylemanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI) and sphingomyelin (SM), where the two hydrocarbon chains are typically between about 14-22 carbon atoms in lengtii and have varying degrees of unsaturation or 14-18 carbon chain saturated phospholipids. Also included in iis class are the glycolipids such as cerebrosides and gangliosides.
  • the major lipid component in the liposomes is phosphatidylcholine.
  • Phosphatidylcholines having a variety of acyl chain groups of varying chain lengm and degree of saturation are available or may be isolated or synthesized by well-known techniques. In general, less saturated phosphatidylcholines are more easily sized, particularly when the liposomes must be sized below about 0.3 microns, for purposes of filter sterilization. Phosphatidylcholines containing saturated fatty acids wim carbon chain lengdis in the range of C 14 to C 22 are preferred.
  • Phosphatidylcholines wim mono or diunsaturated fatty acids and mixtures of saturated and unsaturated fatty acids may also be used.
  • Liposomes useful in the present invention may also be composed of sphingomyelin or phospholipids wim head groups otiier than choline, such as ethanolamine, serine, glycerol and inositol.
  • phospholipids suitable for formation of liposomes useful in the methods and compositions of the present invention include, e.g.
  • phosphatidylcholine phosphatidylglycerol, lecithin, ⁇ , - dipalmitoyl- ⁇ -lecithin, sphingomyelin, phosphatidylserine, phosphatidic acid, N- (2 , 3-di(9-(Z)-octadecenyloxy))-prop- 1 -yl-N ,N ,N-trimethylammonium chloride , phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylinositol, cephalin, cardiolipin, cerebrosides, dicetylphosphate, dioleoylphosphatidylcholine , dipalmitoylphosphatidylcholine , dipalmitoy lphosphatidylglycerol , dioleoy lphosphatidy lglycerol , palmitoy
  • Non-phosphorus containing lipids may also be used in the liposomes of the compositions of the present invention. These include, e.g. , stearylamine, docecylamine, acetyl palmitate, fatty acid amides, and me like. Additional lipids suitable for use in me liposomes of the present invention are well known to persons of skill in the art and are cited in a variety of well known sources, e.g., McCutcheon 's Detergents and Emulsifiers and McCutcheon 's Functional Materials, Allured Publishing Co. , Ridgewood, N . J . , bom of which are incorporated herein by reference.
  • Preferred liposomes will include a sterol, preferably cholesterol, at molar ratios of from 0.1 to 1.0 (cholesterol:phospholipid).
  • Most preferred liposome compositions are phosphatidylcholine/cholesterol , distearoy lphosphatidy lcholine/cholesterol , dipalmitoylphosphatidylcholine/cholesterol, and sphingomyelin/cholesterol. Small amounts (ie. ⁇ 10%) of omer derivatized lipids are often present in liposomes having these compositions.
  • me vesicle- forming lipid may be a relatively fluid lipid, typically meaning that the lipid phase has a relatively low liquid to liquid-crystalline melting temperature, e.g. , at or below room temperature, or a relatively rigid lipid, meaning that me lipid has a relatively high melting temperature, e.g. , up to 60 °C.
  • the more rigid, i.e. , samrated lipids contribute to membrane rigidity in a lipid bilayer structure and also contribute to greater bilayer stability in blood.
  • Omer lipid components, such as cholesterol are also known to contribute to membrane rigidity and stability in lipid bilayer structures.
  • a long chain e.g.
  • C 14 -C 22 samrated lipid plus cholesterol is one preferred composition for delivering merapeutic compositions to target tissues, such as solid tumors, since these liposomes do not tend to release me drugs into the plasma as they circulate through the blood stream.
  • Phospholipids whose acyl chains have a variety of degrees of saturation can be obtained commercially.
  • egg phosphatidylcholine (EPC) can be purchased from Avanti Polar Lipids (Alabaster, AL) and hydrogenated soy phosphatidylcholine (HSPC) can be obtained from Natterman (Cologne, FRG).
  • EPC egg phosphatidylcholine
  • HSPC hydrogenated soy phosphatidylcholine
  • phospholipids can be prepared according to published methods, (see D.M. Small, "The physical chemistry of lipids” (1986) Plenum Press, N.Y., or D.D. Lasic, "Liposomes: from physics to applications” (1993) Elsevier
  • hydrophilic polymers tends to increase the blood half-life of a liposome.
  • a hydrophilic polymer such as polyethylene glycol (PEG)-modified lipids or ganglioside G M , to me liposomes. Addition of such components prevents liposome aggregation during coupling of the targeting moiety to the liposome.
  • PEG polyethylene glycol
  • ganglioside G M ganglioside
  • PEG is incorporated as PEG derivatized phosphatidylemanolamine (PEG-PE) or PEG derivatized distearoyl phosphatidylemanolamine (PEG-DSPE).
  • PEG-PE PEG derivatized phosphatidylemanolamine
  • PEG-DSPE PEG derivatized distearoyl phosphatidylemanolamine
  • a particularly preferred memod of PEG-PE preparation is based on reaction of me PEG with carbonyldiimidazole followed by addition of PE (see, Woodle et al. Proc. Intern. Symp. Control. Rel. Bioact. Mater. , 17: 77-78 (1990), Papahadjopoulos et al., Proc. Natl. Acad. Sci. USA, 88: 11460-11464 (1991), Allen et al., Biochim. Biophys. Acta. , 1066: 29-36 (1991), Woodle et al., Biochim. Biophys. Acta.m, 1105: 193-200 (1992), and Woodle et al., Period. Biol.
  • PEG-PE is available from Avanti Polar lipids (Alabaster, Alabama) or Liposome Technology (Menlo Park, California, USA).
  • Avanti Polar lipids Alignment, Birmingham, California, USA.
  • Liposome Technology Melo Park, California, USA.
  • me liposomes of the present invention are conjugated to the Fab' region of an antibody which acts as a targeting moiety enabling the liposome to specifically bind a target cell bearing the target molecule (e.g., characteristic marker) to which the Fab' antibody fragment is directed.
  • the Fab' region of an antibody represents a monomer comprising the variable regions and the C H 1 region of one arm of an antibody.
  • an “antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in mm define me immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • the basic immunoglobulin (antibody) structural unit is known to comprise a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (V L ) and variable heavy chain (V H ) refer to ese light and heavy chains respectively.
  • Antibodies may exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below me disulfide linkages in the hinge region to produce F(ab)' 2 a dimer of Fab which itself is a light chain joined to V H - C H 1 by a disulfide bond.
  • the F(ab)' 2 may be reduced under mild conditions to break me disulfide linkage in the hinge region thereby converting the F(ab)' 2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, W.E. Paul, ed., Raven Press, N.Y. (1993) for more antibody fragment terminology). While the Fab' domain is 4864 PC17US95/14710
  • the Fab' regions used in the present invention may be derived from antibodies of animal (especially mouse or rat) or human origin or may be chimeric (Morrison et al., Proc Natl. Acad. Sci. USA 81, 6851-6855 (1984) both incorporated by reference herein) or humanized (Jones et al. , Nature 321, 522-525 (1986), and published UK patent application No. 8707252, bom incorporated by reference herein).
  • the Fab' region is selected to specifically bind to a molecule or marker characteristic of the surface of the cells to which it is desired to deliver me contents of me liposome.
  • a molecule is characteristic of cell, tissue, or physiological state when that molecule is typically found in association with that cell type or alternatively in a multiplicity of cell types all expressing a particular physiological condition (e.g. , transformation).
  • a specific characteristic marker is preferably found on me surfaces of cells of a particular tissue or cell type or on the surfaces of tissues or cells expressing a particular physiological condition and on no omer tissue or cell type in the organism.
  • a characteristic cell surface marker will show sufficient tissue specificity if the only non-target tissues are not accessible to the liposome.
  • effective specificity may be achieved by overexpression of the marker in the target tissue as compared to omer tissues.
  • cancers are characterized by me overexpression of cell surface markers such as the HER2 (c- erbB-2, neu) proto-oncogene encoded receptor in me case of breast cancer.
  • HER2 c- erbB-2, neu
  • cell surface markers that provide good characteristic markers for liposomes depending on me particular tissue it is desired to target.
  • cell surface markers include, but are not limited to carbohydrates, proteins, glycoproteins, MHC complexes, and receptor proteins such as HER, CD4 and CD8 receptor proteins as well as omer growth factor receptor proteins.
  • Growth factor receptors are particularly preferred characteristic cell surface markers. Growth factor receptors are cell surface receptors diat specifically bind growth factors and thereby mediate a cellular response characteristic of me particular growth factor.
  • growth factor refers to a protein or polypeptide ligand mat activates or stimulates cell division or differentiation or stimulates biological response like motility or secretion of proteins.
  • Growth factors are well known to those of skill in me art and include, but are not limited to, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor ⁇ (TGF- ⁇ ), fibroblast growth factors (FGF), interleukin 2 (IL2), nerve growth factor (NGF), interleukin 3 (IL3), interleukin 4 (IL4), interleukin 1 (IL1), interleukin 6 (IL6), interleukin 7 (IL7), granulocyte/macrophage colony- stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), erythropoietin and me like.
  • PDGF platelet-derived growth factor
  • EGF epidermal growth factor
  • IGF insulin-like growth factor
  • TGF- ⁇ transforming growth factor ⁇
  • FGF fibroblast growth factors
  • IL2 inter
  • HER receptors comprise protein tyrosine kinases that memselves provide highly specific antibody targets.
  • the PI 85 tyrosine kinase of HER2 provides a most preferred target for me Fab' antibody domain of me utilized in me immunoliposomes of the present invention.
  • the characteristic marker need not be a naturally occurring marker, but rather may be introduced to the particular target cell. This may be accomplished by directly tagging a cell or tissue wim a particular marker (e.g. , by directly injecting die particular target tissue wim a marker, or alternatively, by administering to the entire organism a marker that is selectively incorporated by me target tissue.
  • the marker may be a gene product d at is encoded by a nucleic acid in an expression cassette.
  • the marker gene may be under me control of a promoter that is active only in the particular target cells.
  • introduction of a vector containing the expression cassette will result in expression of the marker in only the particular target cells.
  • One of skill in the art will recognize that there are numerous approaches utilizing recombinant DNA methodology to introduce characteristic markers into target cells.
  • the targeting moiety will specifically bind products or components of a growth factor receptor, in particular products of me HER2 (c-erbB-2, neu) proto-oncogene. It is particularly preferred diat the targeting moiety bind me growth factor receptor-tyrosine kinase encoded by HER2, protein pl85 HER2 , which is commonly overexpressed in breast cancers (Slamon et al., Science, 235: 177-182 (1987).
  • Other suitable targets for the targeting moiety include, but are not limited to EGFR (HERl), HER3, and HER4, combinations of these receptors, and omer markers associated wim cancers.
  • antibodies of interest include, but are not limited to BR96 (Friedman et al., Cancer Res. , 53: 334-339 (1993), e23 to erbB2 (Batra et al, Proc. Natl. Acad. Sci. USA, 89: 5867- 5871 (1992)), PR1 in prostate cancer (Brinkmann et al., Proc. Natl. Acad. Sci. USA., 90: 547-551 (1993)), and Kl in ovarian cancer (Chang et al. Int. J. Cancer, 50: 373-381 (1992).
  • Immunoliposomes of the present invention may be prepared by incorporating the Fab' antibody domain moieties into me liposomes by a variety of techniques well known to those of skill in me art.
  • a biotin conjugated Fab' may be bound to a liposome containing a streptavidin.
  • d e biotinylated Fab' may be conjugated to a biotin derivatized liposome by an avidin or streptavidin linker.
  • a biotinylated monoclonal antibody was biotinylated and attached to liposomes containing biotinylated phosphatidylemanolamine by means of an avidin linker.
  • Fab' molecules per liposome typically about 30 to 125 and more typically about 50 to 100 Fab' molecules per liposome are used.
  • me targeting moiety may be directly conjugated to the liposome.
  • Such means of direct conjugation are well known to tfiose of skill in the art. See for example, G. Gregoriadis, (1984) "Liposome Technology” CRC Press, Boca Raton, Florida and D.D. Lasic, "Liposomes: from physics to applications” (1993) Elsevier, Amsterdam; N.Y. Particularly preferred is conjugation dirough a thioether linkage.
  • M-PE maleimide derivatized phosphatidylemanolamine
  • M-DEP dipalmitoylethanolamine
  • Suitable methods include, e.g. , sonication, extrusion, high pressure/homogenization, microfluidization, detergent dialysis, calcium-induced fusion of small liposome vesicles, and ether-infusion methods, all well known in me art.
  • One method produces multilamellar vesicles of heterogeneous sizes. In this method, me vesicle-forming lipids are dissolved in a suitable organic solvent or solvent system and dried under vacuum or an inert gas to form a thin lipid film.
  • me film may be redissolved in a suitable solvent, such as tertiary butanol, and men lyophilized to form a more homogeneous lipid mixture which is in a more easily hydrated powder-like form.
  • a suitable solvent such as tertiary butanol
  • This film is covered wim an aqueous buffered solution and allowed to hydrate, typically over a 15-60 minute period wim agitation.
  • the size distribution of die resulting multilamellar vesicles can be shifted toward smaller sizes by hydrating me lipids under more vigorous agitation conditions or by adding solubilizing detergents such as deoxycholate.
  • multilamellar liposomes are produced by the reverse phase evaporation method of Szoka & Papahadjopoulos, Proc. Natl. Acad. Sci. USA, 75: 4194-4198 (1978).
  • Unilamellar vesicles are generally prepared by sonication or extrusion. Sonication is generally performed wim a tip somfier, such as a Branson tip sonifier, in an ice bath. Typically, the suspension is subjected to several sonication cycles. Extrusion may be carried out by biomembrane extruders, such as me Lipex Biomembrane Extruder. Defined pore size in me extrusion filters may generate unilamellar liposomal vesicles of specific sizes. The liposomes may also be formed by extrusion dirough an asymmetric ceramic filter, such as a Ceraflow Microfilter, commercially available from the Norton Company, Worcester MA.
  • asymmetric ceramic filter such as a Ceraflow Microfilter
  • the liposomes which have not been sized during formation may be sized to achieve a desired size range and relatively narrow distribution of liposome sizes.
  • a size range of about 0.2-0.4 microns allows the liposome suspension to be sterilized by filtration dirough a conventional filter, typically a 0.22 micron filter.
  • the filter sterilization method can be carried out on a high dirough-put basis if the liposomes have been sized down to about 0.2-0.4 microns.
  • Several techniques are available for sizing liposomes to a desired size. One sizing method is described in U.S. Pat. Nos. 4,529,561 or 4,737,323, incorporated herein by reference.
  • Sonicating a liposome suspension eid er by bath or probe sonication produces a progressive size reduction down to small unilamellar vesicles less than about 0.05 microns in size.
  • Homogenization is another method which relies on shearing energy to fragment large liposomes into smaller ones.
  • multilamellar vesicles are recirculated dirough a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns, are observed.
  • the size of the liposomal vesicles may be determined by quasi-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys.
  • Average liposome diameter may be reduced by sonication of formed liposomes. Intermittent sonication cycles may be alternated wim QELS assessment to guide efficient liposome synthesis.
  • Extrusion of liposome through a small-pore polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing liposome sizes to a relatively well-defined size distribution.
  • me suspension is cycled dirough me membrane one or more times until die desired liposome size distribution is achieved.
  • the liposomes may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in liposome size.
  • the therapeutic agent which may be used is any compound including me ones listed below which can be stably entrapped in liposomes at a suitable loading factor and administered at a therapeutically effective doses (indicated below in parenmeses after each compound, m 2 refers to body surface area).
  • m 2 refers to body surface area.
  • amphipathic antitumor compounds such as me plant alkaloids vincristine
  • doxorubicin 60-75 mg/m 2
  • epirubicin 60-120 mg/m 2
  • daunorubicin 25-45 mg/m 2
  • the water-soluble anti-metabolites such as methotrexate (3 mg/m 2 ), cytosine arabinoside (100 mg/m 2 ) and fluorouracil (10-
  • the antibiotics such as bleomycin (10-20 units/m 2 , mitomycin (20 mg/m 2 ), plicamycin (25-30 ⁇ g/m 2 ) and dactinomycin (15 ⁇ g/m 2 ), and me alkylating agents including cyclophosphamides and derivatives diereof (3-25 mg/kg), thiotepa
  • Omer suitable drugs include aclacinomycin, idarubicin, mitoxantrone, cisplatin and omer Platinum II analogs.
  • the liposomes may also contain the taxanes including taxol, taxotere, dihydroxytaxanes, camptothecines and omer taxane derivatives and isolates.
  • d e liposomes may contain encapsulated tumor-therapeutic peptides (e.g., plant or bacterially derived toxins) and protein drugs such as IL-2 and/ or TNF, and/ or immunomodulators, such as M-CSF, which are present alone or in combination with anti-tumor drugs, such as andiracycline antibiotic drugs.
  • the immunoliposomes may contain fluorinated pyramidine and purine bases or nucleosides.
  • the immunoliposomes may also contain nucleic acids such as oligonucleotides containing namral or modified bases and having a phosphodiester intemucleotide linkage or modified intemucleotide linkages such as a phosphorothioate or polyamide linkages.
  • nucleic acids may be used as antisense or triplex-forming molecules to block transcription and translation dirough binding of DNA or RNA.
  • me nucleic acids may be used to transform cells and to induce me expression of heterologous proteins.
  • the nucleic acid will comprise an expression cassette which includes me nucleic acid sequence encoding me protein to be expressed under me control of a promoter.
  • me transmembrane potential loading method can be used wim essentially any conventional drug which can exist in a charged state when dissolved in an appropriate aqueous medium.
  • me drug will be relatively lipophilic so mat it will partition into me liposome membranes.
  • a transmembrane potential is created across me bilayers of the liposomes or targeting moiety liposome conjugates and me drug is loaded into the liposome by means of me transmembrane potential.
  • the transmembrane potential is generated by creating a concentration gradient for one or more charged species (e.g. , Na + , K + and/or H + ) across me membranes. This concentration gradient is generated by producing liposomes or targeting moiety liposome conjugates having different internal and external media.
  • a transmembrane potential is created across me membranes which has an inside potential which is negative relative to me outside potential, while for a drug which is negatively charged, me opposite orientation is used.
  • One of me requirements for liposome localization in a target tissue is an extended immunoliposome lifetime in the bloodstream following administration.
  • One measure of immunoliposome lifetime in the bloodstream is the blood/RES ratio determined at a selected time after liposome administration.
  • immunoliposomes containing a label e.g. fluorescent marker, electron dense reagent, or radioactive marker
  • a label e.g. fluorescent marker, electron dense reagent, or radioactive marker
  • the time course of retention of immunoliposomes in die blood may also simply be determined by sampling blood at fixed intervals after administration of label-containing liposomes and determining the amount of label remaining in the circulation. The result may be expressed as me fraction of the original dose.
  • Assaying Uptake Into the Cytoplasm of Target Cells and Determining Tissue Distribution Uptake and internalization of immunoliposomes into me cytoplasm of target cells may similarly be determined by administering immunoliposomes containing a label (e.g. fluorescent marker, electron dense reagent, or radioactive marker) and subsequently detecting the presence or absence of that label in the cytoplasm of the target cell.
  • a label e.g. fluorescent marker, electron dense reagent, or radioactive marker
  • an immunoliposome containing a fluorescent marker such as rhodamine conjugated to me lipid constimting me liposome itself
  • the tissues or cells may then be fixed and me fluorescence detected using fluorescence microscopy.
  • an electron-dense label e.g. gold
  • One of skill in the art will recognize that many labels are suitable and me method of detection will reflect me choice of label.
  • the present invention provides for growth-inhibiting immunoliposomes that essentially comprise an empty immunoliposome having an Fab' targeting moiety directed to a cell surface receptor. Particularly preferred are growm factor receptors. Identification of Fab' immunoliposomes that are particularly effective inhibitors of cell proliferation may be accomplished wim routine screening. This involves providing a cell culture where me cells bear a growth factor receptor, or other characteristic cell surface marker, to which the Fab' fragment is directed, contacting me cells in the culture wim the immunoliposome to be tested, and measuring the resulting change in cell proliferation rate. Means of measuring cell proliferation rate are well known to those of skill in the art.
  • proliferation rate may be assessed directly by measuring the change in acmal numbers of cells over a fixed period of time.
  • tumor cells such as SK-BR-3 or BT-474 cells were grown in monolayer culture and then incubated at 37 °C wim varying concentrations of immunoliposomes based on antibody content. After continuous treatment for 4 days, cell monolayers were washed wim PBS and stained with crystal violet dye (0.5% in metiianol) for determination of relative proliferation as previously described (Hudziak, et al, Mol. Cell Biol 9: 1165-1172 (1989) which is incorporated herein by reference).
  • proliferation may be evaluated indirectly by measuring changes in metabolic rate of cells exposed to me immunoliposome to be tested.
  • Numerous means of measuring metabolic rate are well known to those of skill in the art.
  • On particularly preferred approach is to measure the rate of uptake of a labeled metabolic precursor such as tritiated thymidine. Briefly mis is accomplished by administering the [ 3 H]-ti ⁇ ymidine to a test culmre containing the immunoliposome and to a control culmre lacking the immunoliposome. After a fixed period of time, cells are collected and me amount of [ 3 H] -thymidine taken up by the cells is measured utilizing standard techniques (e.g. , scintillation counting). Comparison of the test and control cells indicates changes in metabolic activity and tiierefore proliferation rate.
  • compositions comprising the immunoliposomes of the invention are prepared according to standard techniques and further comprise a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier e.g., normal saline will be employed as the pharmaceutically acceptable carrier.
  • suitable carriers include, e.g. , water, buffered water, 0.4% saline, 0.3% glycine, and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc.
  • These compositions may be sterilized by conventional, well known sterilization techniques.
  • the resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophdized preparation being combined wim a sterile aqueous solution prior to administration.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and me like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
  • auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents and me like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
  • the liposome suspension may include lipid-protective agents which protect lipids against free-radical and lipid-peroxidative damages on storage. Lipophilic free- radical quenchers, such as alphatocopherol and water-soluble iron-specific chelators, such as ferrioxamine, are suitable.
  • the concentration of immunoliposomes, in the pharmaceutical formulations can vary widely, i.e. , from less than about 0.05%, usually at or at least about 2-5% to as much as 10 to 30% by weight and will be selected primarily by fluid volumes, viscosities, etc. , in accordance wim the particular mode of administration selected.
  • the concentration may be increased to lower the fluid load associated wim treatment. This may be particularly desirable in patients having atherosclerosis-associated congestive heart failure or severe hypertension.
  • immunoliposomes composed of irritating lipids may be diluted to low concentrations to lessen inflammation at the site of administration.
  • the amount of immunoliposome administered will depend upon me particular Fab' used, the disease state being treated, die therapeutic agent being delivered, and die judgement of the climcian. Generally the amount of immunoliposomes administered will be sufficient to deliver a therapeutically effective dose of die particular pharmacological agent. The quantity of immunoliposomes necessary to deliver a tiierapeutically effective dose can be determined by uptake assays as described above. Therapeutically effective dosages for various pharmacological agents are well known to tiiose of skill in the art and representative ranges are given for a number of pharmaceuticals above. Typical immunoliposome dosages will generally be between about 0.01 and about 50 mg per kilogram of body weight, preferably between about 0.1 and about 10 mg/kg of body weight.
  • me pharmaceutical compositions are administered parenterally, i.e. , intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. More preferably, me pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection.
  • Particular formulations which are suitable for this use are found in Remington 's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985).
  • the formulations will comprise a solution of the liposomes suspended in an acceptable carrier, preferably an aqueous carrier.
  • an aqueous carriers may be used, e.g. , water, buffered water, 0.9% isotonic saline, and me like.
  • compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered.
  • the resulting aqueous solutions may be packaged for use as is, or lyophilized, me lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and me like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • EPC Egg phosphatidylcholine
  • Lipids (Alabaster, AL); cholesterol (Choi) from Calbiochem (San Diego, CA) N-Tris[hydroxymethyl]-2aminoethanesulfonic acid (TES) from Sigma hydrogenated soy phosphatidylcholine (HSPC) from Natterman (Cologne, FRG) rhodamine-labeled phospholipids from Avanti; desferrioxamine mesylate (desferal) from Ciba-Geigy (Summit, NJ); doxorubicin from Farmitalia, Carlo Erba (Milan, I t a l y ) o r C e t u s ( E m e r y v i l l e , C A ) ; a n d N - [ 4p - maleimidophenyl)butyryl]phosphatidyled ⁇ anolamine (M-PE) fromMolecular Probes (Portland, OR
  • PEG-PE PEG-derivatized phosphatidylethanolamine
  • rhuMAbHER2 sequences for heavy and light chain were co-expressed in E. coli as previously described (Carter et al, Biotechnology 10: 163-167 (1992)).
  • the antibody fragment, rhuMAbHER2-Fab ⁇ was recovered from E. coli fermentation pastes by affinity chromatography with Streptococcal protein G (Carter et al , Biotechnology, 10:163-167 (1992) which is incorporated herein by reference), typically yielding Fab' witii 60-90% containing reduced free tiiiol (Fab'-SH).
  • Fab'-SH reduced free tiiiol
  • rhuMAbH52-Fab' differs from rhuMAbHER2-Fab' only by replacement of the antigen-binding loops, and showed no detectable binding to any known murine or human antigen (Eigenbrot et al Proteins: Structure, Function, and Genetics, 18: 49-62 (1994)). Q Preparation of Liposomes.
  • Liposomes were prepared according to d e reverse phase evaporation method (Szoka & Papahadjopoulos, Proc. Natl. Acad. Sci. USA, 75: 4194-4198 (1978), with lipid composition including EPC:Chol (2:1) or, where stated, HSPC-.Chol (3:2) and PEG-PE (0-6 mole%). Liposomes were subsequently extruded repeatedly under positive pressure wim argon gas through polycarbonate membrane filters of defined pore size sequentially from 0.1 to 0.05 ⁇ m (Olson, et al , Biochim. Biophys. Acta, 55: 9-23 (1979); Szoka et al , Biochem. Biophys.
  • Liposomes widiout encapsulated doxorubicin were prepared in HEPES-NaCl buffer, pH 7.2, 300 mOsm.
  • HSPC/Chol liposomes containing doxorubicin were prepared in 250 mM ammonium sulfate containing 1 mM desferal at pH 5.5. Unencapsulated ammonium sulfate was removed by gel filtration wim G-75 Sephadex.
  • Doxorubicin in powder form was then dissolved in this liposome suspension at 0.1 mg doxorubicin/ ⁇ mole phospholipid(Papahadjopoulos etal. , Proc. Natl. Acad. Sci.
  • Fab' was conjugated to me liposomes after drug loading via tiiioether linkage, as previously described(Martinetfl/. J. Biol Chem., 257: 286-288 (1982). Since maleimide is more stable at lower pH, all procedures were performed at pH 5.5. Unreacted Fab' was separated from immunoliposomes by gel filtration with Sephacryl S-400. The maleimide group on immunoliposomes was deactivated after conjugation by 2-fold excess of mercaptoethanol to M-PE. The amount of Fab' conjugated was determined by BioRad protein assay.
  • liposomes Four types were prepared. "Conventional” liposomes without antibody were composed of phosphatidylcholine and cholesterol only. "Sterically stabilized” liposomes additionally contained PEG-PE. Immunoliposomes were prepared by conjugation of the above with Fab' fragments derived from the humanized antibody rhuMAbHER2 to yield conventional or sterically stabilized immunoliposomes. Fab' fragments rather than intact antibody were used for the following reasons: 1) rhuMAbHER2-Fab' fragments can be expressed as recombinant proteins in E. coli at extremely high efficiency (Carter et al. , supra.
  • SK-BR-3 which express high levels of pl ⁇ STM* 2 , or MCF-7 cells, were exposed to anti-pl ⁇ STM* 2 immunoliposomes for 45 minutes on ice, washed wim PBS, stained wim a secondary anti-human antibody to detect bound immunoliposomes (FITC-labeled goat anti-human IgG), washed wim PBS again, and tiien subjected to flow cytometry (Fig. 1).
  • SK-BR-3 cells bound significant amounts of eitiier conventional or sterically stabilized anti-pl ⁇ STM* 2 immunoliposomes, but not control liposomes lacking Fab'.
  • MCF-7 breast cancer cells, which do not overexpress pl ⁇ STM* 2 showed minimal binding to anti-pl ⁇ 5 HER2 immunoliposomes (data not shown).
  • SK-BR-3 breast cancer cells
  • BT-474 cells in monolayer culmre were simultaneously incubated with 125 I-labeled rhuMAbHER2 or muMAb4D5, at 0.1 nM for l ⁇ hours at 4°C, and increasing concentrations of anti-pl ⁇ S ⁇ * 2 immunoliposomes (Fig. 2).
  • Counts bound were determined by gamma counting.
  • Anti-pl ⁇ STM 112 immunoliposomes efficiently displaced binding of rhuMAbHER2 to both SK-BR-3 cells (data not shown) and BT-474 cells (which also express high levels of pi 85" ⁇ ).
  • tumor cells such as SK-BR-3 or BT-474 cells were grown in monolayer culmre and then incubated at 37 °C with varying concentrations of immunoliposomes based on antibody content. After continuous treatment for 4 days, cell monolayers were washed wim PBS and stained wim crystal violet dye (0.5% in metiianol) for determination of relative proliferation as previously described (Hudziak, et al, Mol Cell Biol 9: 1165-1172 (1989).
  • rhuMAbHER2 While intact (bivalent) rhuMAbHER2 inhibited the growth of pl ⁇ S ⁇ -overexpressing breast cancer cells in monolayer culmre, monovalent Fab' fragments of this antibody (rhuMAbHER2-Fab') are much less effective at inhibiting growth (O'Connell et al. pages 218-239 In Protein Folding In Vivo and In Vitro. , Cleland JL, ed. Washington, D.C., American Chemical Society, (1993)).
  • Example 4 Cvtotoxicitv of Anti-plSS"" 5 * 2 Immunoliposomes Containing Doxorubicin.
  • empty anti-pl ⁇ STM* 2 immunoliposomes displayed antiproliferative activity against pl ⁇ S ⁇ -overexpressing breast cancer cells in culmre, it was possible to greatly augment the antineoplastic effect of the immunoliposomes by loading them with cytotoxic agents, thus producing a targeted drug delivery system.
  • Doxorubicin was used, because of preclinical and clinical evidence suggesting that doxorubicin may be particularly useful against breast cancers overexpressing pl ⁇ STM* 2 , with or without concomitant immunotherapy.
  • SK-BR-3 or WI-3 ⁇ cells in monolayer culmre were incubated with free doxorubicin or doxorubicin-loaded immunoliposomes for 1 hour, and then washed extensively with media. The cells were tiien further incubated at 37 °C for 3 days, after which cell number was estimated by crystal violet staining as described above. Comparison with other assays of cell growth including alamar blue staining, MTT staining, and direct cell counting yielded essentially die same results.
  • Doxorubicin-loaded anti-pl ⁇ S"TM 2 immunoliposomes showed comparable dose-dependent cytotoxicity, with an IC 50 of approximately 0.2 ⁇ g/ml for conventional anti-pl ⁇ 5 HER2 immunoliposomes and approximately 1.0 ⁇ g/ml for sterically stabilized (2 mole% PEG-PE) anti-pl ⁇ 5 HER2 immunoliposomes. These results indicated that anti-pl ⁇ 5 HER2 immunoliposome delivery of doxorubicin to pjg5HER2_ overe ⁇ p ress j n g ce jj s j n Quiture was as efficient a process as the rapid diffusion of free doxorubicin into the cells.
  • Doxorubicin-loaded anti-pl ⁇ STM 2 immunoliposomes were between 10- to 30-fold more cytotoxic than doxorubicin-loaded immunoliposomes bearing irrelevant Fab', which only affected cell growth at relatively high concentrations (>3.3 ⁇ g/ml).
  • WI-38 cells a non-malignant lung fibroblast cell line which expresses minimal levels of pl ⁇ STM* 2 , were also treated with doxorubicin and with doxorubicin-loaded anti-pl ⁇ 5 HER2 immunoliposomes ( Figure 4B). Free doxorubicin again produced significant dose-dependent cytotoxicity against WI-3 ⁇ cells. However, doxorubicin-loaded anti-pl ⁇ 5 HER2 immunoliposomes produced much reduced (20-fold less) cytotoxicity against these cells, and were indistinguishable from doxorubicin-loaded immunoliposomes bearing irrelevant Fab' .
  • liposomes and immunoliposomes were prepared as described in Example 1 with the addition of rhodamine- phosphatidylethanolamine at 1 mole% of the phospholipid components.
  • the resulting rhodamine-labeled liposomes or immunoliposomes were incubated for varying amounts of time at 37 °C with SK-BR-3 cells grown to subconfluence on cover slips.
  • the cells were then fixed with 3% paraformaldehyde, mounted in 90% glycerol/100 mM Tris, pH8.5, containing 0.1% p-phenylenediamine (Sigma) as an antibleaching reagent, and observed with a Leitz Aristoplan fluorescence microscope or a Molecular Dynamics MultiProbe 2001 confocal microscope.
  • immunoliposomes were loaded with colloidal gold particles of 5-15 nm as previously described(Huang et al , Cancer Res. , 52: 5135-5143 (1992); Straubinger et al, Cell, 32: 10639-1079 (19 ⁇ 3). Gold-containing immunoliposomes were incubated at 37 °C witii SK-BR-3 cells grown on cover slips for varying amounts of time, and the cells were then fixed and processed for electron microscopy. Stabilization of liposomes was achieved using tannic acid in the primary fixation(Straubinger et al. , , supra), which provided adequate albeit not optimal preservation of the ultrastructure.
  • the antibody rhuMAbHER2 is rapidly internalized by pl ⁇ S ⁇ -overexpressing tumor cells via receptor-mediated endocytosis (Samp et al , Growth Regul. 1: 72-82 (1991)).
  • SK-BR-3 cells treated with rhodamine-labeled immunoliposomes for different time intervals, fixed and visualized by fluorescence microscopy.
  • SK-BR-3 cells treated witii conventional or sterically stabilized control liposomes lacking Fab' showed neither surface nor internal rhodamine fluorescence, consistent with the inability of control liposomes to bind to these cells.
  • SK-BR-3 cells When treated with conventional anti-pl ⁇ 5 HER2 immunoliposomes, SK-BR-3 cells exhibited intense foci of fluorescence both at the cell surface and intracellularly within 30 minutes of treatment. Confocal fluorescence microscopy confirmed that rhodamine fluorescence was present both at the cell surface and internalized within the cytoplasm of SK-BR-3 cells. In contrast, treatment with sterically stabilized anti-pl ⁇ STM* 2 immunoliposomes containing high PEG-PE concentrations (6 mole % ) resulted in minimal intracellular fluorescence after 30 minutes. Because it appeared that the presence of PEG-PE retarded immunoliposome internalization, sterically stabilized immunoliposomes containing reduced concentrations of PEG-PE were evaluated.
  • Immunoliposomes containing 2 mole% PEG-PE yielded an intermediate degree of intracellular fluorescence after 30 minutes, i.e. less than that seen with conventional immunoliposomes but more than that seen with 6 mole% PEG-PE-containing immunoliposomes.
  • the sterically stabilized immunoliposomes containing 2% PEG-PE did accumulate intracellularly with longer incubation time, such as at 2 hours.
  • anti-pl ⁇ STM* 2 immunoliposomes were internalized within SK-BR-3 cells, the rate of internalization was inversely related to the PEG-PE content of the immunoliposomes .
  • gold particles appeared free within the cytoplasm, not associated with a liposomal capsule or a membrane-bound organelle. Gold particles free within the cytoplasm might have resulted from fusion events between immunoliposomes and the cell membrane. Alternatively, they may have arisen following endocytosis, witii escape of the encapsulated gold particles occurring somewhere along the coated pit pathway.
  • doxombicin did not allow precise delineation of immunoliposomes within tumor tissue.
  • doxombicin was assayed from tissue extracts of treated animals . 24 hours after intraperitoneal injection, doxombicin delivered by sterically stabilized anti-pl ⁇ S 1 ⁇ 112 immunoliposomes had accumulated within tumor xenografts, with lower levels of doxombicin found in surrounding muscle and in blood (Table 2).
  • Anti-Pl ⁇ STM 2 immunoliposome delivery of doxombicin in vivo biodistribution 24 hrs after single ip injection.

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Abstract

La présente invention se rapporte à des immunoliposomes qui optimalisent l'incorporation d'un médicament dans des cellules cibles portant un marqueur de surface cellulaire caractéristique. Ces immunoliposomes comprennent un domaine Fab' d'un anticorps qui lie spécifiquement le marqueur caractéristique, un lipide amphipathique formant vésicule, et un lipide dérivé avec du polyéthylène glycol. L'invention se rapporte également à des immunoliposomes inhibant la croissance cellullaire, qui sont dépourvus d'agents thérapeutiques inhibant la croissance cellulaire mais sont cependant capables d'inhiber la croissance et la prolifération des cellules cibles.
PCT/US1995/014710 1994-11-09 1995-11-08 Immunoliposomes optimalisant l'incorporation d'un agent dans des cellules cibles WO1996014864A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998020857A1 (fr) 1996-11-12 1998-05-22 The Regents Of The University Of California PREPARATION DE FORMULATIONS STABLES DE COMPLEXES DE TYPE LIPIDE-ACIDE NUCLEIQUE POUR UNE ADMINISTRATION EFFICACE $i(IN VIVO)
WO1999065461A3 (fr) * 1998-06-19 2000-02-24 Genzyme Corp Complexes micellaires d'amphiphiles cationiques
EP0912198A4 (fr) * 1996-04-18 2000-04-12 Univ California Immunoliposomes optimisant l'internalisation dans des cellules cibles
US7462703B2 (en) * 2003-01-31 2008-12-09 Max-Delbruck-Centrum Fur Molekulare Medizin Agent for gene transfer
US9085622B2 (en) 2010-09-03 2015-07-21 Glaxosmithkline Intellectual Property Development Limited Antigen binding proteins
CN111289743A (zh) * 2020-03-13 2020-06-16 南京中医药大学 一种具低泄露率、表面可连接大肠杆菌抗体的复合磷脂脂质体及其应用
CN112533946A (zh) * 2018-05-15 2021-03-19 旗舰创业创新六公司 病原体防治组合物及其用途
US11839685B2 (en) * 2008-10-07 2023-12-12 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Composition of matter comprising liposomes embedded in a polymeric matrix and methods of using same

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CANCER RESEARCH, Volume 52, issued 01 October 1992, S.K. HUANG et al., "Microscopic Localization of Sterically Stabilized Liposomes in Colon Carcinoma-bearing Mice", pages 5135-5143. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 89, issued May 1992, P. CARTER et al., "Humanization of an Anti-p185her2 Antibody for Human Cancer Therapy", pages 4285-4289. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 257, Number 1, issued 10 January 1992, F.J. MARTIN et al., "Irreversible Coupling of Immunoglobulin Fragments to Preformed Vesicles", pages 286-288. *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7507407B2 (en) 1994-11-09 2009-03-24 The Regents Of The University Of California Immunoliposomes that optimize internationalization into target cells
EP0912198A4 (fr) * 1996-04-18 2000-04-12 Univ California Immunoliposomes optimisant l'internalisation dans des cellules cibles
EP1655039A3 (fr) * 1996-04-18 2010-01-27 The Regents Of The University Of California Immunoliposomes optimisant l' internalisation dans des cellules cibles
WO1998020857A1 (fr) 1996-11-12 1998-05-22 The Regents Of The University Of California PREPARATION DE FORMULATIONS STABLES DE COMPLEXES DE TYPE LIPIDE-ACIDE NUCLEIQUE POUR UNE ADMINISTRATION EFFICACE $i(IN VIVO)
EP0956001A4 (fr) * 1996-11-12 2006-03-22 Univ California Preparation de formulations stables de complexes de type lipide-acide nucleique pour une administration efficace in vivo
US7462603B2 (en) 1996-11-12 2008-12-09 The Regents Of The University Of California Preparation of stable formulations of lipid-nucleic acid complexes for efficient in vivo delivery
WO1999065461A3 (fr) * 1998-06-19 2000-02-24 Genzyme Corp Complexes micellaires d'amphiphiles cationiques
US7462703B2 (en) * 2003-01-31 2008-12-09 Max-Delbruck-Centrum Fur Molekulare Medizin Agent for gene transfer
US11839685B2 (en) * 2008-10-07 2023-12-12 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Composition of matter comprising liposomes embedded in a polymeric matrix and methods of using same
US9085622B2 (en) 2010-09-03 2015-07-21 Glaxosmithkline Intellectual Property Development Limited Antigen binding proteins
CN112533946A (zh) * 2018-05-15 2021-03-19 旗舰创业创新六公司 病原体防治组合物及其用途
CN111289743A (zh) * 2020-03-13 2020-06-16 南京中医药大学 一种具低泄露率、表面可连接大肠杆菌抗体的复合磷脂脂质体及其应用

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