OA19456A

OA19456A - Liposome encapsulated affinity drug.

Info

Publication number: OA19456A
Application number: OA1201700055
Authority: OA
Inventors: Clet Niyikiza; Jose VARGHESE
Original assignee: L.E.A.F Holdings Group Llc.
Priority date: 2014-08-14
Filing date: 2015-08-14
Publication date: 2020-10-23

Abstract

The disclosure provides a liposomal antifolate composition comprising a liposome including an interior space, a bioactive antifolate agent disposed within said interior space, a steric stabilizer attached to an exterior of the liposome, and a targeting moiety comprising a protein with specific affinity for at least one folate receptor, said targeting moiety attached to at least one of the steric stabilizer and the exterior of the liposome.

Description

Antifolate drugs, as discussed above, were designed as folate mimetic molécules that woik by interfering with the action of folates once inside a cell, depriving cells of the DNA piecursors they need to replicate and proliferate. Because cancer cells arc fast growing cells with 5 a high demand for DNA precursors in the form of folates, they take up antifolate drugs in the same manner as folates and are as a resuit susceptible to the effects of antifolates. However, fast growing normal cells, such as cells that line the gastrointestinal (GI) tract and cells of the bone mairow such as, for example, neutrophils, divide rapidly as well using folates supplied primarily via RFCs. Normal cells arc therefore also susceptible to the toxic effects of antifolates because the RFCs mediated transport mechanism which most antifolates are designed to use to infiltrate and kill cancer cells is the same mechanism that normal cells use to supply themselves with folates. As a resuit, treatment of cancers using very promising and effective antifolates has been a difficult challenge in the clinical care of patients because of the high likelihood of the treatment causing collateral damages to fast-growing normal cells, thereby causing antifolate- related severe and potentially life-threatening toxicities.

As discussed above, antifolates as a class are used for their antiproliférative effect in the treatment of cancer to inhibit cell growth and division, which causes cancer cells to die. The fast replicating cancer cells require increased amount of folates when compared to most normal cells. This led to the clinical development of antifolates as anticancer agents almost 70 years ago.

However, though antifolate-based thérapies were shown to be effective for cancer treatment, the clinical development of antifolates has been problematic and often derailed in view of a compelhng clinical dilemma. This dilemma stems from two competing clinical dynamics. On one hand, antifolates are designed to be folate mimic molécules with most of them intended to reach cancer cells using RFCs as the preferred cross-membrane transport mechanism. On the other hand, fast renewing tissues in the body such as the bone marrow or intestinal track tissue cells are, like cancer cells, also highly folate-dependent and use also RFCs as the primary crossmembrane folate cell supply mechanism. The net resuit of these two clinical dynamics is that bone man-ow and gastrointestinal (GI) tract cells hâve been the most prévalent sites of patients’ life-threatening antifolate-related toxicities. Some of these toxicities hâve included mucositis, diarrhea, anémia, neutropenia, and low white blood counts. Such toxicities, alone or in combination, were in a number of instances blamed for patient death from antifolate-based • ¹² tieatment. The conséquence is that to date many effective promising antifolates continue to fail during their development, not because of a lack of effectiveness against cancer cells, but instead because of patient safety concerns. The few that hâve managed to reach the stage of becoming medicines hâve limited use in clinical practice again due to safety concerns.

Antifolates as a class remain a promising treatment modality for cancer despite the associated nsk of severe and even life-threatening toxicities for patients. The challenge is to figuie out a way to deliver these highly effective antifolates in a manner that avoids damage to normal cells.

Prior efforts hâve generally focused on using RFCs to deliver an anticancer agent.

However, the présent inventors exploit another pathway that is especially prévalent in cancer cells involving folate receptors, including, but not limited to, for example, folate receptor alpha, folate receptor beta and/or folate receptor delta. It has been observed in cancer biology that cancer cells preferentially express folate receptor alpha in contrast to normal cells in order to effxciently uptake folates for the sustainment of their fast réplication and prolifération needs.

Cancer cells are very efficient at supplying themselves with folates contained in the blood stream as compared to normal cells. One way that cancer cells do this is by their overexpression of folate receptors, such as, for example, folate receptor alpha. As cancer progresses, tumor cell surface folate receptor alpha levels tend to increase, most likely due to increasing needs for folate supply.

Because of its high affinity to folate receptor alpha, folie acid was conventionally investigated as a targeting moiety for delivering anti-cancer or cytotoxic molécules to cancer cells with the intent to preferentially deliver a cytotoxic drug to cancer cells, either conjugated to a liposome containing the cytotoxic drug or conjugated to the cytotoxic drug itself. This approach has not led to improved patient safety in large part because, as recognized by the inventors, this approach fails to appreciate a key biological différence in exploiting folate pathways as an approach to deliver a cytotoxic to cancer cells while reducing and/or minimizing exposure of normal cells to the cytotoxic drug; with folie acid as the targeting ligand, normal cells were not bemg spared from toxicity since such a targeted drug was still being taken up by normal cells via RFCs. In other words, a targeted drug using folie acid as the targeting moiety is biologically no different than a regular untargeted antifolate because a drug of such construct binds to both folate receptor alpha and RFCs just like any other folate mimic molécule that is

W ¹³ indiscriminately taken up by both cancer and normal cells. Therefore, using folie acid as the targeting moiety does not provide the sélective delivery of cytotoxic agents to cancer cells while avoiding normal cells. Thus, with folie acid as the targeting moiety, drug related toxicity remained a concern in patient care. As a resuit, leading experts suggested that trying to exploit 5 folate receptors as a means for sélective targeting of cancer cell may be ineffective, guiding the efforts of those skilled in the art away from attempting to exploit folate receptors.

Targeting an antifolate to a folate receptor with a targeting moiety has not been attempted to date. Because antifolates mimic folates, one would not consider exploiting the folate pathways to deliver an antifolate in a targeted way. It would be considered redundant since the reduced 10 folate carrier already transport folate into the cells. From this understanding, it was inherently logical to conclude that because an antifolate mimics a folate, an antifolate drug will be taken up effectively by a folate receptor by a cell and further assistance using, for example, an antibody would not be necessary. A counter-intuitive approach was taken by the current inventors. Because it was important to shield antifolates from being taken up by normal cells via RFCs in 15 order to reduce or prevent antifolate-related toxicity, the inventors found that this goal could be achieved by, among other things, exploiting a cancer spécifie morphology which has been unappreciated as useful to the field of antifolate research: the loss of polarity by tumor tissue cells.

Disruption of cell polarity and tissue disorganization is a hallmark of advanced épithélial 20 tumors. As illustrated in Figure 1 A, normal simple epithelium generally comprises a monolayer of individual cells that display a distinct apical- basal polarity. Cells are tightly packed and connected to each other by the apical junctional complexes (Figure 1A-101), which separate apical and basolateral membrane domains. In normal tissue where polarity is preserved, folate receptor alpha is attached at the apical surface of cells situated away from, and out of direct contact with folates in the blood circulation (Figure 1A-102). Figure IB illustrâtes how cells in high-grade épithélial tumors display loss of apical-basal polarity and overall tissue disorganization, puttmg folate receptor alpha in direct contact with folates in the blood circulation (1B-103). This feature of tumor tissue cells, was believed by the inventors to hâve greater sigmficance for antifolate based thérapies than conventional thinking had appreciated.

The inventors discovered that this held a significant potential to rehabilitate antifolates as

V ¹⁴ anticancer thérapies while reducing and/or even minimizing associated severe and sometime life threatening toxicities associated with antifolates.

In this regard, the inventors designed a Chemical entity to deliver an antifolate agent in a manner that selectively targets folate receptors that are highly expressed in cancer cells, such as, 5 for example, folate receptor alpha, beta and delta while avoiding RFCs (the folate pathway used by normal cells), to selectively expose the antifolate to tumor tissue cells while reducing or avoiding exposure of antifolates to normal cells. This is made possible by recognizing that following loss of polarity, tumor tissue cells not only overexpress and expose folate receptors, such as folate receptor alpha but also that folate receptors in cancer cells are in direct contact with blood circulation, both of which are not the case for the normal tissues. This approach may also extend to other cell surface folate receptors (e.g. folate receptor beta, folate receptor delta, etc.) because of their structural and functional similarities to folate receptor alpha.

The disclosure relates in general to liposome compositions useful for delivering a variety of bioactive agents, such as, for example, antifolates, methods of making the liposomal compositions and methods for treating patients using the liposomal compositions. There is spécial utility in providing an antifolate encapsulating liposome that is targeted to folate receptors but which is not specifically targeted to reduced folate carriers.

More specifically, the disclosure is based on the discovery that a neutral or anionic liposome (i.e., a non-cationic liposome) with affinity and specificity to a folate receptor or more 20 than one folate receptor containing one or more bioactive agent such as, for example, an anticancer (antineoplastic) agent is surprisingly effective against cells presenting and expressing folate receptors on their cell surface.

In an example embodiment, a liposomal antifolate composition is provided. The liposomal antifolate composition may comprise a liposome including an interior space; a 25 bioactive antifolate agent disposed within the interior space; a PEG molécule attached to an exterior of the liposome; and a targeting moiety comprising a protein with spécifie affinity for at least one folate receptor, the targeting moiety attached to at least one of the PEG and the exterior of the liposome.

The term attach or attached refers, for example, to any type of bonding such as covalent 30 bonding, ionic bonding (e.g., avidin-biotin) bonding by hydrophobie interactions, and bonding via functional groups such as maleimide, or linkers such as PEG. For example, a détectable

W ¹⁵ marker, a steric stabilizer, a liposome, a liposomal component, an immunostimulating agent may be attached to each other directly, by a maleimide functional group, or by a PEG-malemide group.

The liposomes m some example embodiments include a steric stabilizer that may increase 5 their longevity m circulation. The basic concept is that one or more steric stabilizers such as a hydrophilic polymer (Polyethylene glycol (PEG)), a glycolipid (monosialoganglioside (GM1)) or others occupies the space immediately adjacent to the liposome surface and exclude other macromolecules from this space. Consequently, access and binding of blood plasma opsonins to the liposome surface are hindered, and thus interactions of macrophages with such liposomes, or 10 any other clearing mechanism, are inhibited and longevity of the liposome in circulation is enhanced. In example embodiments, the steric stabilizer or the population of steric stabilizers may be a PEG or a combination comprising PEG. In an example embodiment, the steric stabilizer may be a PEG with a number average molecular weight (Mn) of 200 to 5000 daltons. These PEGs can be of any structure such as linear, branched, star or comb structure and aie 15 commercially available.

The liposomes contained in the liposome composition of various example embodiments can be any liposome known or later disco vered in the art. In general, the liposomes of the example embodiments may hâve any liposome structure, e.g., structures having an inner space sequestered from the outer medium by one or more lipid bilayers, or any microcapsule that has a 20 semi-permeable membrane with a lipophilie central part where the membrane sequesters an intenor. A lipid bilayer can be any arrangement of amphiphilic molécules characterized by a hydrophilic part (hydrophilic moiety) and a hydrophobie part (hydrophobie moiety). Usually amphiphilic molécules in a bilayer are arranged into two dimensional sheets in which hydrophobie moieties are oriented inward the sheet while hydrophilic moieties are oriented 25 outward. Amphiphilic molécules forming the liposomes of the example embodiments can be any known or later discovered amphiphilic molécules, e.g., lipids of synthetic or natural origin or biocompatible lipids. Liposomes of the example embodiments may also be formed by amphiphilic polymers and surfactants, e.g., polymerosomes and niosomes. For the purpose of this disclosure, without limitation, these liposome-forming materials also are referred to as 30 lipids.

φ 16

The liposome composition may be a liquid or it may be dry, such as, for example, in the form of a dry powder or a dry cake. The dry powder or dry cake may hâve undergone primary drying undei, foi example, lyophilization conditions or optionally, it may hâve undergone both primary drying only or both primary drying and secondary drying. In the dry form, the powder or 5 cake may, for example, hâve between 1% to 6% moisture, for example, such as between 2% to 5% moisture or between 2% to 4% moisture. One example method of drying is lyophilization (also called freeze-drying, or cyrodessication). Any of the compositions and methods of the disclosure may involve the liposomes, lyophilized liposomes or liposomes reconstituted from lyophihzed liposomes. In lyophilization, lyoprotectants or cryoprotectants, molécules protect freeze-dned material may be used. These molécules are typically polyhydroxy compounds such as sugars (mono-, di-, and polysaccharides), polyalcohols, and their dérivatives, glycérol, or polyethyleneglycol, trehalose, maltose, sucrose, glucose, lactose, dextran, glycérol, and aminoglycosides. The lyoprotectants or cryoprotectants may, for example, comprise up to 10% or up to 20% of a solution outside the liposome or inside the liposome or both outside and inside 15 the liposome.

The liposomes of the example embodiments may, for example, hâve a diameter of in the range of 30-150 nm (nanometer). In other example embodiments, the liposome may, for example, hâve a diameter in the range of 40-70 nm.

The liposomes of the example embodiments may, for example, preferably be anionic or 20 neutral. That is, the liposome should not be cationic. The détermination of the charge (i.e., anionic, neutral or cationic) may be made by measuring the zêta potential of the liposome. In an example embodiment, the zêta potential of the liposome is less than or equal to zéro. In another example embodiment, the zêta potential of the liposome is in a range of 0 to -150 mV. In another example embodiment, the zêta potential should be in the range of -30 to -50 mV.

The properties of liposomes are influenced by the nature of lipids used to make the liposomes. A wide variety of lipids hâve been used to make liposomes. These include cationic, anionic and neutral lipids. Cationic lipids are used to make cationic liposomes which are commonly used as gene transfection agents. The positive charge on cationic liposomes enables interaction with the négative charge on cell surfaces. Following binding ofthe cationic liposomes to the cell, the liposome is transported inside the cell through endocytosis. However, cationic liposomes will bind to both normal cells and tumor cells. Because the example embodiments are φ 17 intended to specifically and selectively target tumor cells while substantially sparing normal cells, the use of catiomc lipids is not preferred. Using a mixture of, for example, neutral lipids such as HSPC and anionic lipids such as PEG-DSPE results in the formation of anionic liposomes which are less likely to non-specifically bind to normal cells. Spécifie binding to 5 tumor cells can be achieved by using a tumor targeting antibody such as, for example, a folate receptor antibody, including, for example, folate receptor alpha antibody, folate receptor beta antibody and/or folate receptor delta antibody.

As an example, at least one (or some) of the lipids is/are amphipathic lipids, defined as having a hydrophilic and a hydrophobie portions (typically a hydrophilic head and a hydrophobie 10 tail). The hydrophobie portion typically orients into a hydrophobie phase (e.g., within the bilayer), while the hydrophilic portion typically orients toward the aqueous phase (e.g., outside the bilayer). The hydrophilic portion may comprise polar or charged groups such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxy and other like groups. The hydrophobie portion may comprise apolar groups that include without limitation 15 long Chain saturated and unsaturated aliphatic hydrocarbon groups and groups substituted by one or more aromatic, cyclo-aliphatic or heterocyclic group(s). Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids and sphingolipids.

Typically, for example, the lipids aie phospholipids. Phospholipids include without limitation phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and the like. It is to be understood that other lipid membrane components, such as cholestérol, sphingomyelin, cardiolipin, etc. may be used.

In an example embodiment, the lipids may be anionic and neutral (including zwitterionic and polar) lipids including anionic and neutral phospholipids. Neutral lipids exist in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, 25 for example, dioleoylphosphatidylglycerol (DOPG), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholestérol, cerebrosides and diacylglycerols. Examples of zwitterionic lipids include without limitation dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), and dioleoylphosphatidylserine (DOPS). An anionic lipid is a lipid that is negatively charged at physiological pH. These lipids include without limitation phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dode- canoyl phosphatidylethanolamines, φ 18

N-succmyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

Collectively, anionic and neutral lipids are referred to herein as non-cationic lipids. Such 5 lipids may contain phosphorus but they are not so limited. Examples of non-cationic lipids include lecithin, lysolecithin, phosphatidylethanoiamine, lysophosphatidylethanolamine, dioleoylphosphati- dylethanolamine (DOPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidy 1-ethanolamine (DSPE), palmitoyloleoyl-phosphatidylethanolamine (POPE) palmitoyloleoylphosphatidylcholine (POPC) 10 egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleyolphosphatidylglycerol (POPG), 16-0-monomethyl PE, 16-0- dimethyl PE, 18-1trans PE, palmitoyloleoyl-phosphatidylethanolamine (POPE), l-stearoyl-215 oleoylphosphatidyethanolamine (SOPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, and cholestérol.

Liposomes of «ample embodiments may be assembled using any liposomal assembly method using liposomal components (also referred to as liposome components). Liposomal 20 components include, for «ample, lipids such as DSPE, HSPC, cholestérol and dérivatives of these components. Other suitable hpids are commerc,ally available for example, by Avant, Polar Lipids, Inc. (Alabaster, Alabama, U.S.A.). A partial listing of available negatively or ueutrally chai-ged lipids suitable for making anionic liposomes, may be, for example, at least one of the following: DLPC, DMPC, DPPC, DSPC, DOPC, DMPE, DPPE, DOPE, DMPA-Na, DPPA-Na, 25 DOPA-Na, DMPG-Na, DPPG-Na, DOPG-Na. DMPS-Na, DPPS-Na, DOPS-Na. DOPEGlutaryl.(Na)i, Tetramyristoyl Cardiolipin.(Na)_!, DSPE-mPEG-2000-Na, DSPE-mPEG5000*Na, and DSPE-Maleimide PEG-2000»Na.

Dérivatives of these lipids may, for example, include, at least, the bonding (preferably covalent bonding) of one or more steric stabilizers and/or functional groups to the liposomal 30 component after which the steric stabilizers and/or functional groups should be considered part of the liposomal components. Functional groups comprises groups that can be used to attach a ¹⁹ liposomal component to another moiety such as a protein. Such functional groups include, at least, maleimide. These steric stabilizers include at least one from the group consisting of polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinyl pyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoIine); poly(2-ethyl-25 oxazoline); phosphatidyl polyglycerol; poly[N-(2-hydroxypropyl) methacrylamide] ; amphiphilic poly-N-vinylpyrrolidones; L-amino-acid-based polymer; andpolyvinyl alcohol.

Because a liposomal components may include any molecule(s) (i.e., chemical/reagent/protein) that is bound to it, the liposomal components may, for example, include, at least, DSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG; HSPC-maleimide;

cholestérol; cholesterol-PEG; and cholesterol-maleimide. In a preferred embodiment, the liposomal components that make up the liposome comprises DSPE; DSPE-FITC; DSPEmaleimide; cholestérol; and HSPC.

ht an example embodiment, at least one component of the lipid bilayer is functionalized (or reactive). As used herein, a functionalized component is a component that comprises a 15 reactive group that can be used to crosslink reagents and moieties to the lipid. If the lipid is functionalized, any liposome that it forms is also functionalized.

In example embodiments, the reactive group is one that will react with a crosslinker (or other moiety) to form crosslinks. The reactive group may be located anywhere on the lipid that allows it to contact a crosslinker and be crosslinked to another moiety (i.e., steric stabilizer, 20 targeting moiety, etc.). In some embodiments, it is in the head group of the lipid, including for example a phospholipid. An example of a reactive group is a maleimide group. Maleimide groups may be crosslinked to each other in the presence of dithiol crosslinkers such as but not limited to dithiolthrietol (DTT).

It is to be understood that the example embodiments contemplate the use of other 25 functionalized lipids, other reactive groups, and other crosslinkers. In addition to the maleimide groups, other examples of reactive groups include but are not limited to other thiol reactive groups, amino groups such as primary and secondary amines, carboxyl groups, hydroxyl groups, aldéhyde groups, alkyne groups, azide groups, carbonyls, halo acetyl (e.g., iodoacetyl) groups, imidoester groups, N-hydroxysuccinimide esters, sulfhydryl groups, pyridyl disulfide groups, 30 and the like.

Functionahzed and non-functionalized lipids are available from a number of commercial sources including Avanti Polar 5 Lipids (Alabaster, Ala.).

The liposomes of example embodiments may further comprise an immunostimulatory agent, a détectable marker, or both disposed on its exterior. For example, immunostimulatory agent or détectable marker may be ionically bonded or covalently bonded to an exterior of the liposome, including, for example, optionally to the steric stabilizer.

Immunostimulatory agents, also known as immunostimulants, immunostimulators, haptens and adjuvants, are substances that stimulate the immune system by inducing activation or incieasing activity of any of its components.

These immunostimulatory agents can include one or more of a hapten, an adjuvant, a protein immunostimulating agent, a nucleic acid immunostimulatiug agent, and a Chemical immunostimulating agent. Many adjuvants contain a substance designed to stimulate immune responses. such as lipid A. Bortadella pertussis or Mycobacterium tnberculosis derived proteins. Certain adjuvants are commercial!, available as. for example, Freund’s Incomplète Adjuvant and Complété Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N J.); AS-2 (SmtthKline Beecham, Philadelphia, Pa.); aluminmn salts such as aluminmn hydroxide gel (alum) or aluminmn phosphate; salts of calcium, iron or zinc- an insoluble suspension of acylated tyrosine; acylated sugars; cationically or amonically derivatized polysaccharides; polyphosphazenes; biodégradable microspheres; monophosphoryl lipid A and quil A. Cytokines, sueh as GM-CSF, mterleukin-2, -7, -12, and other like growth factors, may also be used as adjuvants. In a prefeiTed embodiment, the immunostimulant may be at least one selected fromthe group consisting of fluorescein, DNP, beta glucan, beta-l,3-glucan, bela-1,6glucan.

A détectable marker may, for example, include, at least, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a métal chelator, an enzyme, a dye, an ink, a magnetic compound, a biocatalyst or a pigment that is détectable by any suitable means known in the art, e.g., magnetic résonance imaging (MRI), optical imaging, fluorescent/luminescent imaging, or nuclear imaging techniques.

The immunostimulatory agent and/or détectable marker may be attached to the exterior by co-incubating it with the liposome. For example, the immunostimulatory agent and/or détectable marker may be associated with the liposomal membrane by hydrophobie interactions

W 21 by an ionic bond such as an avidin/biotin bond or a métal chélation bond (e.g., Ni-NTA). Alternatively, the immunostimulatory agent or détectable marker may be covalently bonded to the extenor of the liposome such as, for example, by being covalently bonded to a liposomal component or to the steric stabilizer which is the PEG.

One example reagent is fluorescein isothiocyanate (FITC) which, based on our expenments, may surpnsingly serve as both an immunostimulant and a détectable marker.

Example embodiments also provide for a liposome that encloses an interior space. In an example embodiment, the interior space may comprise, but is not limited to, an aqueous solution. The interior space may comprise a bioactive agent, such as, for example, an antifolate agent and 10 an aqueous pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may comprise, for example, trehalose. In an example embodiment, the trehalose may, for example, be présent at about 5% to 20% weight percent of trehalose or any combination of one or more lyopiotectants or cryoprotectants at a total concentration of 5% to 20%. The interior space may, foi example, comprise a citrate buffer at a concentration of between 5 to 200 mM. The citrate 15 buffer may buffer the interior space at a _PH of between 2.8 to 6. Independent of the trehalose or citrate concentration, the pharmaceutically acceptable carrier may comprise a total concentration of sodium acetate and calcium acetate of between 50 mM to 500 mM

In an example embodiment, the bioactive antifolate agent may, for example, be a water soluble bioactive agent. That is, the bioactive agent may form an aqueous solution. According to 20 example embodiments, each liposome may comprise an interior space that contains less than 200,000 molécules of the bioactive agent. For example, in an example embodiment, the liposome may comprise between 10,000 to 100,000 of a bioactive antifolate agent.

In an example embodiment, the bioactive agent can be at least one from the group consisting of pemetrexed, lometrexol, methotrexate, ralitrexed, aminopterin, pralatrexate, 25 lometrexol analogs thereof, thiophene analog of lometrexol, furan analog of lometrexol, trimetrexed, LY309887; and GW 1843U89. In another embodiment, the bioactive agem can be at least one from the group consisting of proguanil, pyrimethamine, trimethoprim and 6-Substituted Pyrrolo and Thieon[2,3-d]pyrrolopyrimidine class of GARFT inhibitors. In one preferred embodiment, the bioactive antifolate agent is pemetrexed. In another example embodiment, the 30 bioactive antifolate agent is lometrexol.

W 22

The pH of a solution comprising the bioactive agent may, for example, be set, for example, to from 5 to 8 or from 2 to 6.

According to the example embodiments, the liposomes contained in the liposome composition ofthe examples can also be targeting liposomes, e.g., liposomes including one or 5 more targeting moieties or biodistribution modifiers on the surface of the liposomes. Example embodiments of targeting liposomes may, for example, be called immunoliposomes. A targeting moiety can be any agent that is capable of specifically binding or interacting with a desired target. In an example embodiment, a targeting moiety may be a moiety that binds with specificity and affinity to a folate receptor, such as, for example, folate receptor alpha, folate receptor beta 10 and/or folate receptor delta. Folate receptors are distinct and different from reduced folate carriers and exploit different pathways to the interior of the cells. The targeting moiety, according to example embodiments, specifically and preferentially binds to and/or internalizes into, a target cell in which the liposome-entrapped entity exerts its desired effect. A target cell may, for example, be a cancer cell, a tumor cell and/or a metastatic cell. In an example 15 embodiment, the liposome carrying a targeting moiety is internalized by a target cell.

In any of the example embodiments of this disclosure, the targeting moiety may be a protein which an antigen binding sequence of an antibody. In an example embodiment, the protein may, for example, hâve a three-dimensional structure of, at least, the antigen bmding site of an antibody. One example of such a protein as a targeting moiety is an antibody. However a 20 complété antibody is not necessary. For example, a protein which is a targeting moiety of any of the example embodiments may comprise one or more complementary determining régions (CDRs) of antibody origin. Examples of suitable proteins that can serve as targeting moieties include at least one selected from the group consisting of an antibody, a humanized antibody, an antigen binding fragment of an antibody, a single chain antibody, a single-domain antibody, à bi25 spécifie antibody, a synthetic antibody, a pegylated antibody and a multimeric antibody. An antibody may hâve a combination of these characteristics. For example, a humanized antibody may be an antigen binding fragment and may be pegylated and multimerized as well. Antibodies to folate receptor alpha are commercially available.

An example antibody that may be employed is a murine antibody against folate receptor 30 alpha. The sequence is described in U.S. patent US5646253. For example, based on the sequences disclosed, the gene was synthesized and placed into a transient expression vector and

W 23 the antibody was produced in HEK-293 transient expression System. The antibody can be a complété antibody, a Fab, or any of the varions antibody variations discussed.

Each of the liposomes may comprise, for example from 30 to 250 targeting moieties, such as. for example, from 30-200 targeting moieties. Alternatively, each of the liposomes may 5 comprise less than 220 targeting moieties such as, for example, less than 200 moieties. The targeting moieties can be attached, such as, for example, by being covalently bonded to the outside of the liposome. The molécules that are on the outside of the liposome may. for example, comprise, at least, a lipid, a steric stabilizer, a maleimide, a cholestérol and the like. In an example embodiment, the targeting moiely may be covalently bound via a maleimide functional io group to at least one selected from the group consisting of a liposomal componem and a steric stabilizer such as a PEG molécule. It is possible that ail the targeting moieties are bound to one component such as PEG. It is also possible that the targeting moieties are bound to different components. For example, some targeting moieties may be bound to the lipid components or cholestérol, some targeting moieties may be bound to the steric stabilizer (e.g PEG) and still 15 other targetmg moieties may be bound to a détectable marker or to another targetmg moiety.

In an example embodiment, the targeting moiety has affinity and specificity for at least one or more antigen where the antigen is selected from the group consisting of folate receptor alpha, folate receptor beta, and folate receptor delta. In an example embodiment, the targeting moiety has spécifie affinity (t.e., affinity and specificity) for at !east two antigens selected from 20 the group consisting of folate receptor alpha, folate receptor beta, and folate receptor delta In another exam_pIe embodiment. the targeting moiety has spécifie affinity for three antigens which are, for example, folate receptor alpha; folate receptor beta; and folate receptor delta. The targeting moiety may hâve affinity and specificity to an epitope of the antigen because sometimes a targetmg motety does not bmd the compta antigen but just an epitope of many 25 epitopes in an antigen. In an example embodiment, the targeting moiety has spécifie affinity for an epitope on a tumor cell surface antigen that is présent on a tumor cell but absent or inaccessible on a non-tumor cell. For example, in some situations, the tumor antigen may be on the surface of both normal cells and malignant cancer cells but the tumor epitope may only be exposed in a cancer cell. As a further example, a tumor antigen may expérience a confirmation 30 change in cancer causing cancer cell spectfic epuopes to be présent. A targeting moiety with spécifie affinity to epitopes described above aie useful and envisioned in the example

W 24 embodiments. In these embodiments, the tumor cell with cancer cell spécifie epitopes may be a cancer cell. Examples of such tumor cell surface antigens include, at least, folate receptor alpha, folate receptor beta and folate receptor delta.

Example embodiments relate to a liposomal antifolate composition comprising: a 5 medium comprising a liposome including an interior space; an aqueous bioactive antifolate agent disposed within said interior space; a targeting moiety comprising a protein with spécifie affinity for at least one folate receptor, said targeting moiety disposed at an the exterior of the liposome. In the example embodiments, the medium is an aqueous solution. In an example embodiment, the interior space, the exterior space (i.e., the medium), or both the interior space and the 10 medium contains one or more lyoprotectants or cryoprotectants which are listed above. In an example embodiment, the cryoprotectants mannitol, trehalose, sorbitol, and sucrose are preferred.

As discussed above, the liposomes of example embodiments may comprise a steric stabihzer that can increase their longevity in circulation. The basic concept is that one or more 15 steric stabilizers such as a hydrophilic polymer (Polyethylene glycol (PEG)), a glycolipid (monosialoganglioside (GM1)) or others occupies the space immediately adjacent to the liposome surface and exclude other macromolecules from this space. Consequently, access and binding of blood plasma opsonins to the liposome surface aie hindered, and thus interactions of macrophages with such liposomes, or any other clearing mechanism, are inhibited and longevity 20 of the liposome in circulation is enhanced.

For any of the example embodiments which incorporate a steric stabilizer. the steric stabihzer may be at least one from the group consisting of polyethylene glycol (PEG), poly-Llysine (PLL), monosialoganglioside (GM1), polyfvinyl pyrrolidone) (PVP), poly(acrylamide) (PAA), poly(2-methyl-2-oxazoline), poly(2-ethyl-2-oxazoline), phosphatidyl polyglycerol.

poly[N<2-hydroxypropyl) methacrylamide], amphiphilic poly-N-vinylpyrrolidones. L-aminoacid-based polymer, and polyvinyl alcohol. In exemple embodiments, the steric stabihzer or the population of steric stabilizer is PEG. In an example embodiment, the steric stabilizer is a PEG with a number average molecular weight (Mn) of 200 to 5000 dallons. These PEGs can be of any structure such as hnear, branched, star or comb structure and are commercially available.

According to example embodiments, the liposome composition may be provided as a pharmaceutical composition containing the example liposome composition of the example

W ²⁵ embodiments and a carrier, e.g., pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carries are normal saline, isotonie dextrose, isotonie sucrose, Ringer s solution, and Hanks' solution. A buffer substance can be added to provide pH optimal for storage stability. For example, pH between about 6.0 and about 7.5, more preferably pH 5 about 6.5, is optimal for the stability of liposome membrane lipids, and provides for excellent rétention of the entrapped entities. Histidine, hydroxyethylpiperazine-ethylsulfonate (HEPES), morpholipoethylsulfonate (MES), succinate, tartrate, and citrate, typically at 2-20 mM concentration, are exemplary buffer substances. Other suitable carriers include, e.g., water, buffered aqueous solution, 0.4% NaCl, 0.3% glycine, and the like. Protein, carbohydrate, or 10 polymeric stabilizers and tonicity adjusters can be added, e.g., gelatin, albumin, dextran, or polyvinylpyrrolidone. The tonicity of the composition can be adjusted to the physiological level of 0.25-0.35 mol/kg with glucose or a more inert compound such as lactose, sucrose, mannitol, or dextrin. These compositions may be sterilized by conventional, well known sterilization techniques, e.g., by filtration. The resulting aqueous solutions may be packaged for use or 15 filtered under aseptie conditions and lyophilized, the lyophilized préparation being combined with a stérile aqueous medium prior to administration.

The pharmaceutical liposome compositions can also contain other pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium 20 acetate, sodium lactate, sodium chloride. potassium chloride, calcium chloride, etc. Additionally the liposome suspension may include lipid-protective agents which protect lipids against freeradical and lipid-peroxidative damages on storage. Lipophilie free-radical quenchers. such as alpha-tocopherol and water-soluble iron-specific chelators, such as feiTioxamine, are suitable.

The concentration of the liposomes of example embodiments in the fluid pharmaceutical 25 formulations can vary widely, i.e., from less than about 0.05% usually or at least about 2-10% to as much as 30 to 50% by weight and will be selected primarily by fluid volumes, viscosities, etc., m accordance with the particular mode of administration selected. For example, the concentration may be increased to lower the fluid load associated with treatment. This may be particularly désirable in patients having atherosclerosis-associated congestive heart failure or severe hypertension. Altematively, liposome pharmaceutical compositions composed of initatmg lipids may be diluted to low concentrations to lessen inflammation at the site of administration.

W ²⁶

Example embodiments relate to a method of delivering a bioactive agent, such as, for example, an antifolate, to a tumor expressing folate receptor on its surface. An example method comprises the step of administering at least one of any of the compositions comprising a liposome in this disclosure in an amount to deliver a therapeutically effective dose of the bioactive antifolate agent to the tumor.

The amount of liposome pharmaceutical composition administered will dépend upon the particular therapeutic entity entrapped inside the liposomes, the disease state being treated, the type of liposomes being used, and the judgment of the clinician. Generally the amount of liposome pharmaceutical composition administered will be sufficient to deliver a therapeutically 10 effective dose of the particular therapeutic entity.

The quantity of liposome pharmaceutical composition necessary to deliver a therapeutically effective dose can be determined by routine in vitro and in vivo methods, common in the art of drug testing. See, for example, D. B. Budman, A. H. Cal vert, E. K. Rowmsky (editors). Handbook of Anticancer Drug Development, LWW, 2003. Therapeutically 15 effective dosages for various therapeutic entities are well known to those of skill in the art; and according to the example embodiments a therapeutic entity delivered via the pharmaceutical liposome composition and provides at least the same or higher activity than the activity obtained by administering the same amount of the therapeutic entity in its routine non-liposome formulation. Typically the dosages for the liposome pharmaceutical composition of the example 20 embodiments may, for example, range between about 0.005 and about 500 mg of the therapeutic entity per kilogram of body weight, most often, between about 0.1 and about 100 mg therapeutic entity/kg of body weight.

An effective amount is a dosage of the agent sufficient to provide a medically désirable resuit. The effective amount will vary with the desired outcome, the particular condition being 25 treated or prevented, the âge and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent or combination therapy (if any), the spécifie route of administration and like factors within the knowledge and expertise of the health practitioner. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to Sound medical judgment.

³θ F°^r sample, if the subject has a tumor, an effective amount may be that amount that reduces the tumor volume or load (as for example determined by imaging the tumor). Effective * 27 amounts may also be assessed by the presence and/or frequency of cancer cells in the blood or other body fluid or tissue (e.g., a biopsy). If the tumor is impacting the normal functioning of a tissue or organ, then the effective amount may be assessed by measuring the normal functioning of the tissue or organ. In some instances the effective amount is the amount required to lessen or 5 eliminate one or more, and preferably ail, symptoms.

The example embodiments provide pharmaceutical compositions. Pharmaceutical compositions are stérile compositions that comprise a sample liposome and preferably antifolate agent(s), preferably in a pharmaceutically-acceptable carrier.

The term pharmaceutically-acceptable carrier may, for example, refer to one or more 10 compatible solid or liquid Hier, diluents or encapsulating substances which are suitable for administration to a human or other subject contemplated by the example embodiments.

The term carrier dénotés an organic or inorganic ingrédient, natural or synthetic with whtch liposome compositions are combined to facilitate administrai.™. The components ofthe pharmaceutical compositions are comingled in a manner that precludes interaction that would 15 substantially impair their desired pharmaceutical efficiency. Suitable buffering agents include acetic acid and a sait (1-2% W/V); citric acid and a sait (1-3% W/V); boric ac,d and a sait (0.52.5% W/V); and phosphoric acid and a sait (0.8-2% W/V). Suitable preservatives include benzalkonium chloride (0.003-0.03% W/V); chlorobutanol (0.3-0.9% W/V); and parabens (0 01 0.25% W/V).

Unless otherwise stated herein. a variety of administration routes are available The particular mode selected will dépend, of course, upon the particular active agent selected, the particular condition being treated and the dosage required for therapeutic efficacy. The methods provided, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of a desired response without causing clinically unacceptable adverse effects. Possible administration routes include injections, by parentéral routes such as intramuscular, subeutaneous, intravenous. intraarterial, intraperitoneal, intraarticular, intraepidural. intrathecal, intravenous, intramuscular, intra sternal injection or infusion or others, as well as oral, nasal, mucosal, sublingual, intratracheal, ophthalmic, rectal, vaginal, ocular, topical, transdermal, pulmonary, inhalation.

M In an example embodiment. the liposome pharmaceutical composition may, for example, be prepared as an infusion composition, an injection composition, a patenterai composition, or a

W 28 topical composition, either as a liquid solution or suspension. However, solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The composition may, for example, also be formulated into an enteric-coated tablet or gel capsule according to known methods in the art.

Foi the delivery of liposomal drugs formulated according to example embodiments, to tumors ofthe central nervous system, a slow, sustained intracranial infusion ofthe liposomes directly into the tumor (a convection-enhanced delivery, or CED) may be of particular advantage. See Saito, et al., Cancer Research, vol. 64, p. 2572-2579, 2004; Mamot, et al., J. Neuro-Oncology, vol. 68, p. 1-9, 2004. The compositions may, for example, also be directly applied to tissue surfaces. Sustained release, pH dépendent release, or other spécifie Chemical or environmental condition mediated release administration is also specifically included in the example embodiments, e.g., by such means as depot injections, or erodible implants. A few spécifie examples are listed below for illustration.

For oral administration, the compounds may, for example, be formulated readily by combining the liposomal compositions with pharmaceutically acceptable carriers well known in the art. Such carriers enable formulation as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, films, suspensions and the like, for oral ingestion by a subject to be treated. Suitable excipients may, for example, include, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose préparations such as, for example, maize starch, wheat starch, rice starch, ’ 20 potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxyméthylcellulose, and/or polyvinylpyrrolidone (PVP). Optionally the oral formulations may also be formulated in saline or buffers for neutralizing internai acid conditions or may be administered without any carriers.

Pharmaceutical préparations which can be used orally include push-fit capsules made of 25 gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the liposomal composition suspended in suitable liquids, such as aqueous solutions, buffered solutions, fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Ail formulations for oral administration should be in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

V 29

For administration by inhalation, the compositions may be conveniently delivered in the form of an aérosol spray présentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, ichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aérosol 5 the dosage unit may be determined by providing a valve to deliver a metered amount.

When it is désirable to deliver the compositions systemically, they may be formulated for parentéral administration by injection, e.g., by bolus injection or continuons infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers. Pharmaceutical parentéral formulations include aqueous solutions of the 10 ingrédients. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Altematively, suspensions of liposomes may be prepared as oil-based suspensions. Suitable lipophilie solvents or vehicles include fatty oils such as sesarne oil, or synthetic fatty acid esters, such as ethyl oleate or triglycérides.

Altematively, the liposomal compositions may be in powder form or lyophilized form for constitution with a suitable vehicle, e.g., stérile pyrogen-free water, before use.

The compositions may also be formulated in rectal or vaginal compositions such as suppositories or rétention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

^{20 The example embod}iments contemplate administration of agents to subjects having or at risk of developing a cancer including for example a solid tumor cancer, using the compositions and liposomes of example embodiments. In an example embodiment, the cancer may, for example, be distinguished by the expression of folate receptors on its cell surface. The folate receptor may, for example, include folate receptor alpha, folate receptor beta or folate receptor delta. The example embodiments contemplate that the compositions are able to deliver higher quantities of the bioactive agents, alone or in combination, to these subjects without excessive delivery to normal cells (i.e., cells not expressing folate receptors).

Any cancers that express folate receptors may be treated. It should be noted that some cancers may express folate receptors in an early stage while the majority of cancers may express 30 folate receptors at late stages. The cancer may be carcinoma, sarcoma or melanoma. Carcinomas include without limitation to basal cell carcinoma, biliary tract cancer, bladder cancer, breast

W 30 cancer, cervical cancer, choriocarcinoma, CNS cancer, colon and rectum cancer, kidney or rénal cell cancer, larynx cancer, liver cancer, small cell lung cancer, non-small cell lung cancer (NSCLC, including adenocarcinoma, giant (or oat) cell carcinoma, and squamous cell carcinoma), oral cavity cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer 5 (including basal cell cancer and squamous cell cancer), stomach cancer, testicular cancer, thyroid cancer, uterine cancer, rectal cancer, cancer of the respiratory System, and cancer of the urinary System.

Sarcomas are mesenchymal neoplasms that arise in bone (osteosarcomas) and soft tissues (fibrosarcomas). Sarcomas include without limitation liposarcomas (including myxoid liposarcomas and pleiomorphic liposarcomas), leiomyosarcomas, rhabdomyosarcomas, malignant peripheral nerve sheath tumors (also called malignant schwannomas, neurofibrosarcomas, or neurogenic sarcomas), Ewing's tumors (including Ewing's sarcoma of bone, extraskeletal (i.e., not bone) Ewing's sarcoma, and primitive neuroectodermal tumor), synovial sarcoma, angiosarcomas, hemangiosarcomas, lymphangiosarcomas, Kaposi’s sarcoma, 15 hemangioendothelioma, desmoid tumor (also called aggressive fibromatosis ), dermatofibrosarcoma protuberans (DFSP), malignant fibrous histiocytoma (MFH), hemangiopencytoma, malignant mesenchymoma, alveolar soft-part sarcoma, epithelioid sarcoma, clear cell sarcoma, desmoplastic small cell tumor, gastrointestinal stromal tumor (GIST), and chondrosarcoma.

Melanomas are tumors arising from the melanocytic System of the skin and other organs.

Examples of melanoma include without limitation lentigomaligna melanoma, superficial spreading melanoma, nodular melanoma, and acral lentiginous melanoma.

The cancer may be a solid tumor lymphoma. Examples include Hodgkin's lymphoma, Non-Hodgkin's lymphoma, and B cell lymphoma.

A The cancer may be without limitation bone cancer, brain cancer, breast cancer, colorectal cancer, connective tissue cancer, cancer ofthe digestive System, endométrial cancer, esophageal cancer, eye cancer, cancer ofthe head and neck, gastric cancer, intra-epithelial neoplasm, melanoma neuroblastoma, Non-Hodgkin's lymphoma, non-small cell lung cancer, prostate cancet, retinoblastoma, or rhabdomyosarcoma.

The example embodiments may be practiced in any subject that is likely to benefit from dehvery of agents as contemplated herein. Human subjects are preferred subjects in example

W ³¹ embodiments but subjects may also include animais such as Household pets (e.g., dogs, cats, rabbits, fenets, etc.), livestock or farm animais (e.g., cows, pigs, sheep, chickens and other poultry), horses such as thoroughbred horses, laboratory animais (e.g., mice, rats, rabbits, etc.), mammal and the like. Subjects also include fish and other aquatic species.

The subjects to whom the agents are delivered may be normal subjects. Alternatively they may hâve or may be at risk of developing a condition that can be diagnosed or that can benefit from delivery of one or more particular agents. In an example embodiment, such conditions include cancer (e.g., solid tumor cancers or non-solid cancer such as leukemias). In a more pieferred embodiment, these conditions include cancers involving cells that express folate receptors on their cell surface.

Tests for diagnosing the conditions embraced by the example embodiments are known in the art and will be familiar to the ordinary medical practitioner. The détermination of whether a cell type expresses folate receptors can be made using commercially available antibodies. These laboratory tests include without limitation microscopie analyses, cultivation dépendent tests (such as cultures), and nucleic acid détection tests. These include wet mounts, stain-enhanced microscopy, immune microscopy (e.g., FISH), hybridization microscopy, particle agglutination, enzyme-hnked immunosorbent assays, urine screening tests, DNA probe hybridization, sérologie tests, etc. The medical practitioner will generally also take a full history and conduct a complété physical examination in addition to running the laboratory tests listed above.

A subject having a cancer may, for example, be a subject that has détectable cancer cells.

A subject at risk of developing a cancer may, for example, be a subject that has a higher than normal probability of developing cancer. These subjects include, for instance, subjects having a genetic abnormality that has been demonstrated to be associated with a higher likelihood of developing a cancer, subjects having a familial disposition to cancer, subjects exposed to cancer causing agents (i.e., carcinogens) such as tobacco, asbestos, or other Chemical toxins, and subjects previously treated for cancer and in apparent remission.

In an example embodiment, the methods may selectively deliver a liposomal antifolate composition to the tumor at a rate which is higher, e.g. at least two-fold greater, than a cell not expressing folate receptor.

Example embodiments relate to a method of making any of the compositions of this disclosure. In an example embodiment, the method involves forming a mixture comprising: (1)

W 32 liposomal components; (2) the bioactive antifolate agent in aqueous solution; and (3) the targeting moiety. The mixture may then be homogenized to form liposomes in said aqueous solution. Further, the mixture may be extruded through a membrane to form liposomes enclosing the bioactive antifolate agent in an aqueous solution. It is understood the liposomal components 5 comprise any lipid (including cholestérol) of this disclosure including functionalized lipids and lipids attached to targeting moieties, détectable labels, and steric stabilizers, or any subset of ail of these. It is further noted that the bioactive antifolate in aqueous solution may comprise any reagents and Chemicals discussed for the interior or exterior of the liposome including, for example, buffers, salts, cryoprotectants and the like.

^{10 The meth}°^{d may further com}P^rise the optional step of lyophilizing the composition after said removing step to form a lyophilized composition. As stated above, the bioactive antifolate agent in aqueous solution may comprise cryoprotectants which may be any cryoprotectants are hsted in this disclosure. If the composition is to be lyophilized, a cryoprotectant may be preferred.

Further, after the lyophilizing step, the method can comprise the optional step of reconstituting said lyophilized composition by dissolving the lyophilized composition in a solvent after said lyophilizing step. Methods of reconstitution are well known. One preferred solvent is water. Other preferred solvents include saline solutions and buffered solutions.

While certain example embodiments are discussed, it should be understood that liposomes can be made by any method that is known or will become known in the art. See, for example, G. Gregoriadis (éditer), Liposome Technology, vol. 1-3, Ist édition, 1983; 2nd édition, 1993, CRC Press, 45 Boca Raton, Fia. Examples of methods suitable for making liposome composition include extrusion, reverse phase évaporation, sonication, solvent (e.g., éthanol) injection, microfluidization, detergent dialysis, ether injection, and dehydration/rehydration. The 25 size of liposomes can be controlled by controlling the pore size of membranes used for low pressure extrusions or the pressure and number of passes utilized in microfluidisation or any other suitable methods.

In general, the bioactive antifolate agent is contained inside, that is, in the inner (interior) space of the liposomes. In an example embodiment, the substituted ammonium is partially or substantially completely removed from the outer medium suiTounding the liposomes. Such removal can be accomplished by any suitable means known to one skilled in the art e e

W 33 dilution, ion exchange chromatography, size exclusion chromatography, dialysis, ultrafiltration, précipitation, etc. Therefore, one optional step may comprise a step of: removing bioactive antifolate agent in aqueous solution outside ofthe liposomes after said extruding step.

Another example embodiment relates to a targeted liposomal composition that selectively 5 targets folate receptors comprising: a liposome including an interior space, a bioactive agent disposed within said interior space, a steric stabilizer molécule attached to an exterior of the liposome, and a targeting moiety comprising a protein with spécifie affinity for at least one folate receptor, said targeting moiety attached to at least one of the steric stabilizer and the exterior of the liposome.

The components of this example embodiment may be the same as described for other embodiments of this disclosure. For example, the bioactive agent, the steric stabilizer which may be PEG, are as described in other parts of this disclosure.

In example embodiment, the bioactive agent of the example embodiment may be ellipticme; paclitaxel or any other bioactive agents listed in this disclosure. Agents related to 15 elliptictae or paclitaxel are also envisioned. These include, at least, taxanes snch as docetaxel and cabazitaxel.

The example embodiments further contemplate in vitro applications of the compositions and methods. In vitro use may be, for example, in the use such as cell culturing and tissue engineering where sélective treatment of a subpopulation of cells are desired. For example during the culture of stem cells from a norma! patient or a patient suffermg from cancer, the ceUs can be treated with a sample composition or sample liposome as discussed to address cancerous subpopulations of cells. The cancerous subpopnlation may arise because the donor origiually has cancer or because the cells spontaneously transform during in vitro procedures.

According to example embodiments, the liposomes and liposome compositions can be 25 provided ta a kit comprising a container with the liposomes, and optionally, a container with the entity and an instruction, e.g., procedures or information related to using the liposome composition in one or more applications. Such instruction can be provided via any medium, e.g., hard paper copy, electronic medium, or access to a database or website containing the instruction.

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EXAMPLES

The following examples are intended to illustrate but not to limit the invention in any manner, shape, or form, either explicitly or implicitly. While they are typical of those that might be used, other procedures, méthodologies, or techniques known to those skilled in the art may 5 alternatively be used.

Using the procedures of this disclosure including the procedures in the Example section, example compositions and example liposomes such as the liposomal antifolate composition are constructed. The example compositions comprise example liposomes. Both example composition and example liposome are used in the experiments described in the examples 10 section and throughout this disclosure are spécifie embodiments of the disclosure and are not meant to define the full scope of the disclosure.

Example 1: Production of folate receptor alpha targeted liposomes containing Pemetrexed and a Hapten

Production of Pemetrexed Liposomes

Pemetrexed disodium heptahydrate (ALIMTA) is highly water soluble with a solubility of 100 mg/ml at neutral pH. Pemetrexed is encapsulated in liposomes by the following procedure. First, the hpid components of the liposome membrane are weighed out and combined as a concentrated solution in éthanol at a température of around 65°C. In this example, the lipids 20 used are hydrogenated soy phosphitidyl choline, cholestérol, DSPE-PEG-2000 ( 1,2-distearoylsn-glyceiO-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), PEG-DSPEmalemide and PEG-DSPE-FITC. The molar ratio of HSPC: Cholestérol: PEG-DSPE is approximately 55:40:5. Next, Pemetrexed is dissolved in an aqueous buffer at a concentration of 100 mg/ml. The drug solution is heated to 65°C. The ethanolic lipid solution is injected into the 25 Pemetrexed solution using a small bore needle. During this step the drug solution is well stirred using a magnetic stirrer. The mixing is performed at an elevated température (63°C -72°C) to ensure that the lipids are in the liquid crystalline State (as opposed to the gel State that they attain at températures below the lipid transition température Tm = 51°C -54°C). As a resuit, the lipids are hydrated and form multiple bilayer (multilamellar) vesicles (MLV) containing pemetrexed in 30 the aqueous core.

W 35

Downsizing of MLV’s Using Filter Extrusion

The MLVs aie fragmented into unilamellar (single bilayer) vesicles of the desired size by high-pressure extrusion using three passes through stacked (track-etched polycarbonate) membranes. The membranes used during the first pass hâve a pore size of 200nm. The membranes used during the second pass hâve a pore size of lOOnm followed by 80 nm pore size membranes as the final pass. During extrusion the température is maintained above the Tm to ensure plasticity of the lipid membranes. As a resuit of the extrusion, large and heterogeneous in size and lamellarity MLVs turn into small, homogenous (80-100 nm) unilamellar vesicles (ULV) that sequester the drug in their interior. A Malvern Zetasizer Nano ZS instrument (Southborough, MA) with back scattering detector (90°) was used for measuring the hydrodynamic size (diameter) at 25°C in a quartz micro cuvette. The samples were diluted 50fold in formulation matrix before analysis.

Our results show that liposomes down sized using filter extrusion had an average particle size of 85 nM with a PDI of 0.007 and a zêta potential of -43.7. As an alternative to filter extrusion, high pressure microfluidization can also be used to down size liposomes. We hâve been able to produce liposomes having a size from 40 nm and up, such as between 30-150 nm (data not shown) or even smaller than 30 nm, and particularly between 40 nm and 120 nm using methods such as high pressure filter extrusion or microfluidization alone or in combination.

Tangential Flow Filtration (TFF) and Drug formulation

After the ULV’s containing Pemetrexed hâve been produced, the extra-liposomal Pemetrexed is removed using dialysis or tangential flow diafiltration against a suitable buffer. Although any buffer solution can be used, in this example the buffer used was 5 mM Sodium Citrate, 60mM Sodium Chloride, pH 6.1. Upon completion of Dialysis, filter sterilize using 0.22 25 micron filter.

Thiolation of Anti-Folate receptor alpha antibody

In order to conjugale the antibody to the PEG-DSPE-malemide moieties on the liposome, the antibody needs to be thiolated. In this example, thiolation of the antibody is achieved using

Traut’s reagent ( Themo Fisher Scientific). The antibody is added to freshly prepared 14 mM

Trauts reagent and 5 mM EDTA in phosphate buffered saline at a pH of 8.1. After incubation with gentle stirring for 60 minutes the thiolated antibody is separated from excess Trauts reagent by dialysis against 200 volumes of 25 mM HEPES pH 7.0, 60 mM NaCl for a minimum of 4 hours.

Çonjugation of Thiolated Antibody to the Pemetrexed Liposomes

The amount of thiolated antibody to be used is calculated based on the desired number of antibodies per liposomes. A 2 fold excess of of each préparation of thiolated antibody is added to diafiltered stenle liposomes. The reaction vessel is overlaid with Nitrogen gas and incubated overnight with slow stirring at room température of 4°C. The çonjugation reaction is stopped by blocking unreacted maleimide groups by adding a stock aqueous lOOmM L-Cysteine-HCl solution to a final concentration of 15 mM in the reaction mixture. Free thiolated antibody is then separated from the antibody conjugated liposomes by using size exclusion chromatography.

Example 2: Cell Unes used for Experiments

Cells lines used in the experiments are commercially available from sources such as the

ATCC (American Type Culture Collection of Manassas, Virginia, U.S.A). The cell lines, their ATCC accession numbers and growth conditions are listed below.

Calu-3 (ATCC HTB-55); EMEM (Cat. # 30-2003); 10% HI FBS; 1% Pen/Strep; 1% LGlutamine.

KB; EMEM (Cat. # 30-2003); 10% HI FBS; 1% Pen/Strep; 1% L-Glutamine.

CCD841 (ATCC CRL-1790); EMEM (Cat. # 30-2003); 10% HI FBS; 1% Pen/Strep; 1% L-Glutamine.

Hs578Bst (ATCC HTB-125); Hybri-Care Medium pH 7.0 (Cat.# 46-X); 30 ng/ml mouse EGF; 10% HI FBS; 1% Pen/Strep; 1% L-Glutamine.

NCLH2087 (ATCC CRL-5922); RPML1640 (Cat. # 30-2001); 5% HI FBS; 1%

Pen/Strep; 1% L-Glutamine.

NCI-H2452 (ATCC CRL-5946); RPML1640 (Cat. # 30-2001); 10% HI FBS; 1% Pen/Strep; 1% L-Glutamine.

OVCAR-3 (ATCC HTB-161); RPMI-1640 (Cat. # 30-2001); 20% HI FBS; 1%

Pen/Strep; 1% L-Glutamine.

SKBR3; McCoy 5A Medium; 10% HI FBS; 1% Pen/Strep; 1% L-Glutamine.

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SL0003 (ATCC PTA-6231); F-12K Medium; 10% HIFBS; 1% Pen/Strep; 1% LGlutamine.

A549 (ATCC CCL-185); F-12K Medium; 10% HI FBS; 1% Pen/Strep; 1% L-Glutamine.

Example 3----Determining binding specificity of one sample construct

The level of folate receptor alpha on the cell surface was measured by flow cytometry with a monoclonal antibody conjugated with a fluorochrome. A shift to the right after binding of an antibody (see, for example, Figure 6, line 606) compared to the line before antibody treatment (see, for example, Figure 6, line 602 and 604) indicates the détection of receptor by flow cytometry. The more the histogram (e.g., Figure 6, line 606) shifts to the right relative to the untreated cells (see, for example, Figure 6, line 602 and 604) the higher the levels of receptors are on the cell surface. The plots demonstrate high levels of folate receptor alpha on cancer cells, but almost undetectable levels on normal cells.

The example liposome which is part of the example liposomal composition constructed, 15 binds to the cell surface to cells that are folate receptor alpha positive, but not cells which are folate receptor alpha négative. The example liposome contains antibody to folate receptor alpha. When the example liposome is incubated for a short period (30 minutes) with folate receptor alpha+ cells, you can detect the example liposome on the cell surface by measuring the level of FITC integrated in the liposome by flow cytometry. A shift of the peak of the histogram indicates that the example liposome is detected on the cell surface. The more the peak shifts to the right, the more example liposome is detected on the cells.

In this experiment we determined the binding of example liposome to cells to access their affmity and specificity. Bnefly the example liposome which comprises a détectable label, were coincubated with cells and the cells were analyzed by flow cytometry. The following data shows .5 that the example liposome binds to folate receptor alpha positive cancer cells, but not folate receptor alpha négative, normal cells.

Fi gui e 3 is a schematic depicting rhe measurement of folate receptor alpha on the cell surface.

Figure 6 is a représentative histog -ams of KB cancer cell lines expressing high surface “ 0 levels of folate receptor alpha as measured by flow cytometry. In Figure 6, label 602 = no antibody; label 604 = isotype control; label 606 = anti-folate receptor alpha APC.

Figure 7 is a représentative histograms of OVCAR-3 cancer cell line expressing high surface levels of folate receptor alpha as measured by flow cytometry. In Figure 7, label 702 = no antibody; label 704 = anti-folate receptor alpha APC.

Figure 8 is a représentative histograms of NCIH2452 cancer cell line expressing high surface levels of folate receptor alpha as measured by flow cytometry. In Figure 8, label 802 = no antibody; label 804 = isotype control; label 806 = anti-folate receptor alpha APC.

Figure 9 is a représentative histogram of normal cell line derived from colon epithelia expressing low surface levels of folate receptor alpha. In Figure 9, label 902 = no antibody; label 904 = isotype control; 906 = anti-folate receptor alpha APC.

Figure 2 is a schematic depicting an example liposome binding to the cell surface.

Figure 10 is a représentative histograms of SL0003 (lung) cell line. The lung cancer cell line binds high levels of the example liposome. Label 1002 = untreated cells. Label 1004 = the example liposome treated cells.

Figure 11 is a représentative histograms of CCD841 (normal colon) cell line, folate receptor alpha-negative cell line bind little example liposome. Label 1102 = untreated cells. Label 1104 = example liposome treated cells.

Figure 12 shows composite data derived from lung (SL0003) and ovarian (OVCAR-3) cells demonstrating high levels of example liposome binding on the cell surface compared to normal cells derived from colon (CCD841) and breast (Hs578), P<0.05. Shown are surface levels of example liposome detected at 30 minutes or 4 hours of incubation at 37°C.

In these experiments, the assays were performed as follows:

Cell were collected and washed in 0.2% Bovine sérum albumin in PBS (FACS buffer.) Cell were lesuspended in 100 pl volume in FACS buffer. 5 μΐ of anti-folate receptor alpha monoclonal conjugated with APC was added (cat# FAB5646A; R&D Systems). The cells were incubated for 30 min in the dark at 4°C. 100 μΐ of FACS buffer was added to wash the cells and then the cells were evaluated by flow cytometry (FL4). For measuring the example lyposome on cell surface. Cell weie collected, counted, and washed in 0.2% Bovine sérum albumin in PBS (FACS buffer ) 20,000 cells were resuspendedlOO μΐ volume in FACS buffer. 2 μΐ of the example liposome was added to the cells. The cells were incubated at 37°C for 30 minutes, washed with FACS buffer, and evaluated by flow cytometry.

W 39

Example 4---RhodoRed Experiment on Sample Compositions and Sample Liposomes

Figure 4 Schematic depicting sample liposome in the cell. Sample liposome was labeled with pH-RhodoRed, which fluoresces in the presence of reduced pH, such as in the endolysosome of the cell. Internalization is seen as a shift to the right of the peak relative to untreated 5 cells.

Figuie 13 shows that RhodoRed-labeled cells of the example liposome is internalized by ovarian cancer cells. This is évident because peak 1504 (cells treated with the example composition/example liposome) is shifted to the right of the untreated peak 1502. Similarily, in Figure 14, we see thaï relative to the untreated peak 1502, the treated peak with increasing .0 amounts of example liposome, begin to shift right as seen in peaks 1504, 1506, 1508, 1510 and

1512 refemng to 10 pl, 20 μΐ, 30 μΐ, 40 μΐ, and 50 μΐ respectively (see Figure 17). The same data is plotted m a bar chart in Figure 17. In Figure 17 a control pH-RhodoRed-labeled liposome lacking anti-folate receptor a (FOLR1) was assessed for comparison. Liposomes lacking antiFOLRlwere not internalized by KB cells. In contrast, pH-RhodoRed labeled sample liposome was internalized by KB cells. This data in Figure 17, as shown, is the resuit of 18 hour incubation at 37°C, dose response, also quantified in Figure 14. Folate Receptor alpha négative normal cell Unes (breast cell; left panel and colon; right panel) did not internalize pH-RhodoRed labeled sample liposome. Figures 15 shows that internalization is minimal in normal breast cells. Peak 1704 is only slightly shifted relative to peak 1702. Figure 16 shows that internalization is minimal in normal colon cells as peak 1802 and 1804 were not substantially shifted.

To fuither evaluate the internalization, SL0003 lung cancer cells were assessed for MAP kinase activation levels by PhosFlow. Schematic depicting sample liposome inside the cell achvating kinases is shown in Figure 5. Figure 18A and Figure 18B shows the effect of pemetrexed on the réduction of basal levels of phosphorylation of p38 at 30 minutes post^{2 5} treatment. Figure 18A shows untreated cells with 52.5% phosphyrylated p38. When the cells were treated with the example composition comprising the example liposome in Figure 18B, this percentage is reduced to 8.95%. Figure 19 shows the quantification of phosphorylated levels of p38 in cancer cells and normal cells at 30 minutes post-treatment. The sample composition and sample liposome, labeled as “Targeted Liposome,” affects p38 activation in SL0003 lung cancer 30 cells.

Φ

We interpret the data as follows: Sample liposome enters cells that are FOLR1 positive, sample liposomes were labeled with a dye (RhodoRed) that can only be detected by flow cyometry if it enters the cell. Varions cells were incubated with differing amounts of RhodoRedlabeled sample liposome and the level of fluorescence was measured by flow cytometry (FL2.) 5 RhodoRed labeled sample liposomes enters cancer cells that express FOLR1, but not normal cells that are FOLR1 négative. Shown are ovarian cancer cells with the peak shifting to right indicating the drug has entered the cell (Figure 13). The same experiment was done with FOLR1 high KB cells with titrated amounts of RhodoRed-labeled sample liposome (Figure 14). Thèse data are quantified in Figure 17. Sample liposomes entered KB cells when treated with high concentrations but the control liposomes that lacked anti-FOLRl antibody were not able to enter the cell.

A second measure of sample liposome entering the cell is intracellular activation pathways. Cells respond to ligands binding to their receptors by activating kinases, in this case p38. The activated kinases can be measured by flow cytometry. The cells are incubated with 15 sample liposome for 30 minutes and then lysed with a mild detergent. The activated p38 kinase is detected with an antibody by flow cytometry. A shift under the red line gâte indicates a higher level of phosphorylated p38 (See Figure 18A and 18BG). Cancer cells may hâve a higher basal level of activated kinases. In this case, pemetrexed reduces p38 activation similarly to sample liposome demonstrating that the pemetrexed inside sample liposome is active (Figure 19).

²⁰ Experimental conditions are as follows:

For Figures 4, 13, 14, 15, 16 and 17: Measuring uptake of RhodoRed -labeled sample liposome. Cells (OvCAR-3, KB, normal colon and normal breast) were plated cell at 7,000 cell/well the night before the experiment. The next day, cells were treated with the following 1) No drug 2) RhodoRed-labeled anti-FOLRl monoclonal antibdy (MABFRAH H/L) Ab - (lui); 3) 25 sample liposome (Liposome FOLR1 Ab conjugated - (5ul); 4) Non targeted pH Rhodored liposome - (5ul)/ Liposomes with no anti-FOLRl. The cells were incubated at 37°C for 18 hours and 24 hours. 100 μΐ of ice cold FACS buffer was added and the cells were evaluated by flow cytometry (FL2).

In addition, for Figure 17, measuring p38 phosphorylation (PhosFlow) SL0003, normal 30 colon cells, and normal breast cells were seeded at 10,000 cells/well. Cells were treated with: Fresh Pemetrexed (50 μΜ, 10 μΜ), LEAF-001 (Liposome 070715 MPF; 5traut/50 Mab,

P ⁴¹

15traut/50Mab, 45traut/50Mab) (13.33X dilution), anti-folate receptor alpha (MABFRA H/L .01 mg/ml) (13.33X dilution) to détermine the effect of the antibody in sample liposome, or no treatment. The cells were gently mixed and quickly placed in incubator. At each time point (30 minutes- 4 hours), the plates were removed from the incubator and immediately fixed with formaldéhyde for a final concentration of 2%. The plates were incubated for 5 minutes at room température. 25 pl of media was removed. 25 pl of FACS buffer was added. 100 pl of cell lysis buffer (FACS buffer, 0.2% triton X-100, 0.3% formaldéhyde) were added. Cells were collected into 1.5 ml centrifuge tubes and vortexed for 3 minutes to lyse the cells. The PE-conjugated antiP38 (BD Pharmingen) antibody or PE-conjugated isotype control was added and the cells were incubated for 30 minutes in the dark at 4oC. 200 pl FACS buffer was added to wash. The tubes were spun and the supernatant was carefully removed. The cells were read on the flow cytometer (in FL2).

Example 5 MTS Assay ^{15 The MTS} (^(4’5-dim^ethylthi^azol-2-yl)-5-(3-cai'boxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazohum) assay is a well-known colorimétrie assay for assessing cell metabolic activity. The cell lines were used for the assays and their growth conditions are as follows:

(a) Calu-3; EMEM, 10% HI FBS, 1% Pen/Strep, IX L-glutamine;

(b) KB: EMEM, 10% HI FBS, 1% Pen/Strep, IX L-glutamine;

’° (c) NCI-H2087: RPMI, 5% HI FBS, 1% Pen/Strep, IX L-glutamine;

(d) NCI-H2452: RPMI, 10% HI FBS, 1% Pen/Strep, IX L-glutamine;

(e) SKBR3: McCoy’s, 10% HI FBS, 1% Pen/Strep, IX L-glutamine;

(f) CHO: FreeStyle CHO, 5% HI FBS, l%Pen/Strep, IX L-glutamine;

(g) A549: F-12K, 10% HI FBS, l%Pen/Strep, IX L-glutamine; and (h) SL0003: F-12K, 10% HI FBS, l%Pen/Strep, IX L-glutamine.

The night before, the cells are seeded according to the amount of cells required for each cell line in 96 well tissue culture plate. Final volume in each well is 100 pL (see Table of cell lines for reference; ail cell lines obtained from ATCC.). Cell line used and assay conditions are as follows:

(1) Calu-3: 10000 cells per well. Stock is 3.1x107ml: diluted 3.55mL of cell to 7.45mL of completed media. Transfer 100 pl to each well (pl refers to microliter).

(2) KB: 3000 cells per well. Stock is 2.0xl0⁵/ml: diluted 1.65mL of cell to 9.35mL of completed media. Transfer 100 μΐ to each well.

(3) NCI-H2087: 3000 cells per well. Stock is 3.7xl0⁵/ml: diluted 892 μΐ of cell to lO.lmL of completed media. Transfer 100 μΐ to each well.

(4) NCI-H2452: 5000 cells per well. Stock is 5.0xl0⁴/ml: no need for dilution (5) SKBR3: 4000 cells per well. Stock is 5.5xl0⁵/ml: diluted 800 μΐ cell to 10.2mL of completed media. Transfer 100 μΐ to each well.

(6) CHO: 3000 cells per well. Stock is 3.6xl0⁵/ml: diluted ImL cell to 11 mL of completed media. Transfer 100 μΐ to each well.

(7) A549: 3000 cells per well. Stock is 2.3x10⁵/ml: diluted 1.43mL cell to 9.57mL of completed media. Transfer 100 μΐ to each well.

(8) SL0003: 3000 cells per well. Stock is 2.3xl0⁵/ml: diluted 1.43mL cell to 9.57mL of completed media. Transfer 100 μΐ to each well.

The seeded cells are incubated at 37°C and 5% CO2 ovemight. The next day, the drugs were prepared in the cell-specific cell culture media and titrated 2-fold diluted concentrations added to the cells. The préparations are as follows:

Pemetrexed heptahydrate (5 mM stock). Top dilution lOuM: Add 2 μΐ of stock to 998 μΐ of completed media.

Example liposome/Liposome FOLR-1 Ab (0.4mg/ml = 666.67 μμΜ). Top dilution 10 μΜ: Add 9 μΐ of stock to 591 μΐ of completed media.

Liposome Lot 0707F (stock is 2mM). Top dilution 10 μΜ: Add 3 μΐ of stock to 597 μΐ of completed media.

On day 4, the effect on cellular prolifération was measured with MTS assay. 10 μΐ of reagent (Celltiter 96® Aqueous One Solution) were added to each well. This is a colormetric assay that turns a deep purple when there is extensive cellular prolifération. The plates were incubated for 2 hours at 37oC and the absorbance was measured at 490nm. Percent inhibition of cell growth was calculated using the untreated cell absorbance values set at 100% for each cell line.

Example 6____Sample Liposome Effect on Cellular Prolifération

Figure 20 shows folate receptor alpha surface levels on cancer cells correlate with susceptibility to sample liposome growth inhibition. Shown are the levels of inhibition in A; p=0.05. To further assess the effect of sample liposome on cell cycle, SL0003 (lung cancer) cells were treated with pemetrexed or sample liposome for 4 days.

Figure 21 is a chart showing cell lines derived from patient with lung or breast cancer were treated with titrated concentrations of pemetrexed or the example liposome. Cells were incubated for 90 hours at 37°C and cellular viability and number were assessed by MTS. Shown are results from 10 mM pemetrexed compared to sample liposome with estimated 10 mM pemetrexed. Sample liposome demonstrates a similar· efficacy as pemetrexed.

Cells were lysed and the DNA labeled with propidium idodine to quantify cell cycle (Figure 23A) Pemetrexed treatment induces cells to accumulate in S phase (Figure 23B) Composite data demonstrating that sample liposome induces the same effect on cell cycle as pemetrexed with an accumulation of cells in S phase is shown in Figure 24.

This data shows that pemetrexed is an effective chemotherapy by stopping cancer cells from dividing. We tested whether the pemetrexed contained within sample liposome was effective m inhibiting cells from dividing. Several cancer cell lines were treated with either 10 mM pemetrexed or sample liposome with an estimated matched concentration of pemetrexed for 4 days. The cells were then assessed for numbers of cells that divided. The data show that sample liposome and pemetrexed hâve similar effects on each of the cell lines.

The FOLR1 expression on the cell surface (see Figure 20) correlates with susceptibility to sample liposome. We used a second measure of the effects of pemetrexed on the ability of cells to divide. Cells treated with pemetrexed cannot produce new DNA and become trapped in the S phase of the cell cycle. Sample liposome has the same effect as pemetrexed.

Example 7----Evaluation of the effect of sample liposome on cell cycle

SL0003 (lung cancer) cells were prepared as described in the Example describing MTS assays (Example 5).

Foi this assay, the cells were cultured for 4 or 5 days (shown is day 5.). More specifically, SC0003 cells (lung adenocarcinoma) were seeded in 96 well plates and treated the next day with titrated concentrations of LEAF-001 or pemetrexed. At day 5, cells were lysed

V 44 with FACS buffer, 0.2% triton X-100, 0.3% formaldéhyde. The DNA was stained with

Propidium Idodine for 30 minutes was labeled with Propidium lodide to evaluate cell cycle The cells were washed and evaluated by flow cytometry (FL2). Figure 22A depicts a schematic showing the cell cycle. The experimental results are shown in Figure 22B. By 5 inhibiting the formation of precursor purine and pyrimidine nucléotides, pemetrexed prevents the formation of DNA and RNA, which are required for the growth and survival of both normal cells and cancer cells.

Example 8----Sample liposome reduces the toxicity of pemetrexed on bone marrow-derived 10 neutrophils.

CD34+ cells were induced to differentiate into neutrophils with IL-3, stem cell factor, and G-CSF. By day 2, there is a dramatic increase in mature neutrophils depicted in the oval (Figure 26A). In the presence of pemetrexed (2-50 mM), neutrophil différentiation is inhibited Figure 26B; n=4 donors).

Mac-1 expression on neutrophils in drawn circles in Figure 25A and Figure 25B is shown in Figure 25. As can be seen in Figure 27, sample liposome (at 10 mM pemetrexed) reduces the toxicity of pemetrexed on neutrophil différentiation (n=3 donors.) Cells were treated with a calculated estimation of sample liposome at 10 mM pemetrexed for two days. Numbers of diffeientiated neutrophils were assessed by flow cytometry as shown in Figure 26A and 26B.

The circle dénotés maturing neutrophils expressing Mac-1 and CD 15.

One ofthe side effects from pemetrexed treatment is the réduction of neutrophils in the bloodstream. This is the resuit of CD34+ stem cells not differentiating, or developing, into mature neutrophils in the bone marrow. We measured the effect of sample liposome on neutrophil différentiation compared to the same dose of pemetrexed (lOmM.) Stem cells from 4 25 donors were purchased and treated with a panel of growth factors to induce neutrophil différentiation. CD34+ cells that were also treated with pemetrexed failed to develop into mature neutrophils.

The level of a molécule called Mac-1 is elevated on more mature neutrophils. This molécule is elevated on cells in the circles drawn on the plots. A shift to the red indicates 30 increased levels on the cells.

V ⁴⁵

In contrast, sample liposome was able to reduce this toxicity by allowing more cells to develop into neutrophils. See, e.g., Figure 27.

Experiments were performed as follows: CD34+ stem cells were obtained from ATCC.

CD34+ cells were thawed at 37°C for 1 minute. While on ice, the cells were transferred to cold stem cell medium (“StemSpan SFEM” - Stem Cell Tech, cat.# 9650), 10% heat activated fêtai bovine sérum (HI FBS.) Each vial contained approximated 5x105 cell/ml. The cells were placed in 96 well tissue culture plates ~ 35,000 cell/well.

The neutrophils GROWTH media contained 100 ng/ml of stem cell factor human (SCFSigma H8416, lot# MKBT8036V), 20 ng/ml of granulocyte colony- stimulation factor, human (G-CSF- Sigma H5541, lot # SLBC9602V), 10 ng/ml of IL3 recombinant human (Sigma SRP3090, lot #1008AFC13) in StemSpam media as above.

The cells were also treated as follows 1) StemSpam media alone with no growth cytokines; 2) StemSpam media + growth cytokines, 3) 50, 10 or 2 μΜ pemetrexed, 4) sample liposome (équivalent to 10 μΜ pemetrexed), or 5) anti-folate alpha Ab (1.01 mg/ml) - 5ug/ml

Cells were incubated for 1-5 days and assayed at each time point for mature neutrophils by flow cytometry with antibodies to CD15, Mac-1, and CD34. The cells shown in the circle on the plots are maturing neutrophils expressing Mac-1 and CD34.

Example 9 Results and Discussion

Folate receptor alpha expression is restricted to spécifie organs beyond the fetal/embryonic stage in humans in noncancerous States. As shown in Figure IA, in the setting of normal polarity, normal simple epithelium comprises a monolayer of individual cells that display a distinct apical- basal polarity. Cells are tightly packed and connected to each other by the apical junctional complexes, which separate apical and basolateral membrane domains (Figure

IA label 101). In normal tissue where polarity is preserved, folate receptor alpha is attached at the apical surface of cells situated away from, and out of direct contact with folates in the blood circulation (Figure IA label 102). By contrast, disruption of cell polarity and tissue disoiganization is a hallmark of advanced épithélial tumors. Figure IB shows how cells in highgiade épithélial tumors display loss of apical-basal polarity and overall tissue disorganization, putting folate receptor alpha in direct contact with folates in the blood circulation (Figure 1 B, label 103). In addition, tumor tissue cells in general express higher levels of folate receptor alpha

W 46 than normal cells that happen to express this receptor. This differentiating feature of tumor tissue cells from normal épithélial cells is at the core of the design of the new Chemical entity designed to lehabilitate antifolates as anticancer thérapies while minimizing associated severe and sometime life-threatening toxicities. Such Chemical entity delivers an antifolate agent in a mannei that selectively targets specifically folate receptor alpha, not with folie acid but with a folate receptor alpha spécifie targeting moiety to bypass RFCs. This approach limits the exposure of the antifolate to tumor tissue cells only due to loss of cell polarity, because these tumor tissue cells oveiexpress folate receptor alpha during the time this receptor is in direct contact with blood circulation. This is not the case for limited normal tissues where folate receptor alpha is 10 expressed, because the receptor is not in direct contact with circulating blood.

From this point on, folate receptor alpha can also be used interchangeably with folate receptor alpha that describes the gene encoding the folate receptor alpha protein. Both terms are used interchangeably to describe the folate receptor alpha protein. In addition, the new Chemical entity will be referred, for purpose of illustration, the example liposome (also referred to as the 15 targeted liposome). Methods for making the example compositions and example liposomes are disclosed throughout the spécification and at least in Example 1. The discussion below refers to some expenments performed on a few example compositions and a few example liposomes. It is not meant to define ail possible example compositions and ail possible example liposomes.

Fi gui e 2 illustrâtes the example liposome and how it binds to a cell that expresses folate 20 receptor alpha. In addition to being a hapten, FTIC serves as an imaging agent that allows visualization of binding of the example liposome to the folate receptor alpha on the surface of a folate receptor alpha-expressing cell while Figure 3 illustrâtes the construct designed on one hand to document binding to the folate receptor alpha and, on the other hand, to quantify the number of folate alpha receptors exposed on the cell surface.

²⁵ Figure 4 illustrâtes intemalization of the example liposome into a folate receptor alpha expressing cell using RhodoRed. The exercise is to demonstrate that the example liposome is intemalized independent of bioactive agent payload. Figure 5 illustrate further the effect of intemalization of the example liposome on the cell prolifération using p38 protein kinase pathways as a read out of the cellular response to stress.

The next sériés of illustrations (Figures 6-11) describe the experiments and results showing first that the folate receptor alpha targeting antibody used binds preferentially folate

W 47 receptor alpha. In these experiments, the level of folate receptor alpha on the cell surface was measured by flow cytometry with a monoclonal antibody conjugated with a fluorochrome. A shift to the light indicates the détection of receptor by flow cytometry. The more the histogram shifts to the right, the higher the levels of receptors are on the cell surface. The plots demonstrate 5 high levels of folate receptor alpha on cancer cells (Figures 6-8), but almost undetectable levels on normal cells (Figure 9).

The example liposome can hâve an antibody targeting preferentially folate receptor alpha. When the example liposome is incubated for a short period (30 minutes) with folate receptor alpha positive cells, you can detect example liposome on the cell surface by measuring the level 10 of FITC integrated in the example liposome by flow cytometry. A shift of the histogram line to the right indicates that the example liposome is detected on the cell surface. The more the histogram shifts to the right, the more the example liposome drug is detected on the cells. The experiments show that the example liposome binds to folate receptor alpha expressing lung cancer cells (Figure 10) but not to normal colon épithélial cells (Figure 11).

The example liposome binding experiments described above were repeated using multiple cancer cell lines overexpressing folate receptor alpha (lung-SL0003 and ovarianOVCAR-3) and normal cells derived from colon (CCD841) and breast (Hs578) tissues. Figure 12 shows that the composite data derived from lung (SL0003) and ovarian (OVCAR-3) cancer cells, which hâve high levels of cell surface folate receptor alpha, demonstrate significantly 20 higher levels of the example liposome binding on the cell surface compared to normal cells derived from colon (CCD841) and breast (Hs578) (with a p-value <0.05). Data shown in Figure 12 comprise surface levels of the example liposome detected at 30 minutes and at 4 hours of incubation at 37 degrees Celsius.

Another sériés of experiments was conducted to show that upon binding to folate receptor 25 alpha expressing cells, the example liposome is further taken into the cells (internalized). This was assessed in two ways:

First, the example liposome liposomes were labeled with a dye (RhodoRed) that can only be detected by flow cytometry if it enters the cell (Figure 4). Various cells were incubated with diffenng amounts of RhodoRed-labeled example liposome and the level of fluorescence was 30 measured by flow cytometry (FL2). Shown in Figure 13 are ovarian cancer cells with a shift to right indicating the example liposome has entered the cell. RhodoRed labeled example liposome enters cancer cells that express folate receptor alpha (Figure 13-14), but not normal cells that are folate receptor alpha négative (Figure 15-16).

The same experiment was specifically conducted in high folate receptor alpha expressing KB cells this time with titrated amounts of RhodoRed-labeled the example liposome. As shown in Figure 17, the example liposome entered KB cells when treated with high concentrations but the control liposomes that lacked anti-folate receptor alpha antibody were not able to enter the cell.

Taken together, these results from the RhodoRed experiments provide evidence that the technology used in the design construct of the example liposome is such that the example liposome as a delivery system, armed with a folate receptor targeting moiety other than folie acid or its analogues, enters cancer cells expression folate receptor alpha regardless of its liposome bioactive agent payload. Furthermore, the same experiments demonstrate preferential targeting of folate receptor alpha expressing cancer cells by the example liposome while limiting exposure of normal cells to the bioactive agent payload.

A second measure of the example liposome entering the cell was based on assessing intracellular activation pathways. Cells respond to stress from ligands binding to their receptors or mtemahzation by activating p38 protein kinase pathways (Figure 20). The activated kinases can be measured by flow cytometry. The cells were incubated with the example liposome for 30 minutes and then lysed with a mild detergent. The activated p38 kinase was detected with an antibody by flow cytometry. Cancer cells may hâve a higher basal level of activated kinases (Figuie 18A). A shift under the control line gâte indicates a higher level of phosphorylated p38. In this case, pemetrexed reduces p38 activation similarly to at two different concentration (10 μΜ and 50 μΜ). The example liposome reduces phosphorylated p38 more substantially demonstrating that the pemetrexed inside the example liposome is active (Figure 19).

Another sériés of experiments was conducted to show that the example liposome inhibits cellular prolifération in similar degree to free pemetrexed at matched concentrations as pemetrexed is an effective chemotherapy in stopping cancer cells from dividing. B y inhibiting the foimation of precursor purine and pyrimidine nucléotides, pemetrexed prevents the formation of DNA and RNA, which are required for the growth and survival of both normal cells and cancer cells. This was done in two ways:

First, we tested whether the pemetrexed contained within the example liposome was effective in inhibiting cells from dividing, also referred to as cell prolifération. Several cancer cell Unes were treated with either 10 mM pemetrexed or the example liposome with an estimated matched concentiation of pemetrexed for four days. The cells were then assessed for numbers of cells that divided. The results demonstrated that there is a corrélation between cell growth inhibition and folate receptor alpha expression on the cancer cell surface (Figure 20). The results further showed that not only was there a susceptibility of folate receptor alpha expressing cancer cells to the example liposome but also that the example liposome and pemetrexed hâve similar effccts on each of the cell Unes (Figure 21).

To fuithei assess the effect of the example liposome on cell cycle, a second approach was used to measure the effects of pemetrexed on the ability of cells to divide. The rationale was that cells treated with pemetrexed cannot produce new DNA and become trapped in the S phase of the cell cycle (Figures 22a and 22b). Cell Unes derived from patient with lung or breast cancer were treated with titrated concentrations of pemetrexed or the example liposome. Cells were incubated for 90 houis at 37 degrees Celsius and cellular viability and number were assessed by MTS. Cells were lysed and the DNA labeled with propidium iodine to quantify cell cycle (Figure 23 a). The data showed that pemetrexed treatment induces cells to accumulate in S phase (Figure 23b). Furthermore, SC0003 cells (lung adenocarcinoma) were seeded in 96 well plates and treated the next day with titrated concentrations of example liposome or pemetrexed. On day 5, the cells were fixed and lysed and the DNA was labeled with Propidium lodide to evaluate cell cycle. The results demonstrated that the example liposome induces the same effect on cell cycle as pemetrexed on each of the cell lines as measured by accumulation of cells in S phase (Figure 24).

Anothei experiment was conducted to assess the impact of the example liposome on bone mairow cells. The rationale is that one of the side effects from an antifolate treatment, such as pemetrexed containing treatment, is the réduction of neutrophils in the bloodstream, leading to severe and sometime life threatening infections. This is due to CD34+ stem cells not differentiating, or developing, into mature neutrophils in the bone marrow. We measured the effect of the example liposome on neutrophil différentiation compared to the same dose of pemetiexed (10 mM) Stem cells from four human donors were purchased and treated with a panel of growth factors to induce neutrophil différentiation. More specifically, CD34+ stem cells

W 50 were induced to differentiate into neutrophils with IL-3, stem cell factor, and G-CSF. Cells were treated with 10 mM pemetrexed for two days or with a calculated estimation of the example liposome at 10 mM pemetrexed for two days. Numbers of differentiated neutrophils were assessed by flow cytometry.

The results showed that in absence of pemetrexed, there is a dramatic increase in mature neutrophils by day 2, as depicted in the oval area of Figure 25 and Figure 26A. In the presence of pemetrexed (2-50 mM), however, neutrophil différentiation is inhibited (Figure 26B; n=4 donors) demonstrating that CD34+ stem cells treated with pemetrexed failed to develop into mature neutrophils. In contrast to free premetrexed, the example liposome was able to reduce this 10 toxicity by allowing more CD34+ stem cells to develop and mature into differentiated neutrophils when compared to pemetrexed at similar pemetrexed concentration (Figure 27).

Taken together, these results from the experiments conducted provide evidence that the technology used in the design construct of the example liposome is such that the example liposome as a delivery system of a bioactive agent/payload, armed with a folate receptor 15 targeting moiety other than folie acid or its analogues, enters tumor cells expressing folate receptor alpha the cells regardless of its liposome bioactive agent payload, préserves efficacy of the bioactive agent in folate receptor alpha expressing cancer cells and minimizes exposure of noimal cells to the toxic effects of an antifolate agent payload such as a pemetrexed payload, thereby offermg the opportunity to reintroduce in the clinical setting otherwise very efficacious 20 but toxic agents, such as antifolates as a class, without typically associated severe and sometime life-threatening toxicities.

Although the invention has been described with reference to the presently preferred embodiment, it should be understood that varions modifications can be made without departing fiom the spint ofthe invention. Accordingly, the scope of the invention should be determined 25 with reference to the appended claims, along with the full scope of équivalents to which such claims are entitled. The disclosures of ail cited articles and references, including patent applications and publications, are incorporated herein by reference for ail purposes.

Claims

1. A liposomal antifolate composition comprising:

a liposome including an interior space;

a bioactive antifolate agent disposed within said interior space;

a PEG attached to an exterior of the liposome; and a targeting moiety comprising a protein with spécifie affinity for at least one folate receptor, said targeting moiety attached to at least one of the PEG and the exterior of the liposome.

2. The liposomal antifolate composition of claim 1, wherein said PEG has a number average molecular weight (M_n) of 200 to 5000 daltons.

^^3. The liposomal antifolate composition of claim 61 wherein the medium is an aqueous solution comprising at least one cryoprotectants selected from the group consisting of mannitol; trehalose; sorbitol; and sucrose.

64. The liposomal antifolate composition of claim 61 further comprising:

a steric stabilizer attached to the exterior of the liposome, wherein the targeting moiety is attached to at least one of the steric stabilizer and the exterior of the liposome.

65. The liposomal antifolate composition of claim 64 wherein the steric stabilizer is at least one selected from the group consisting of polyethylene glycol (PEG); poly-Llysine (PLL); monosialoganglioside (GM1); poly(vinyl pyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[A-(2-hydroxypropyl) methacrylamide]; amphiphilic poly-TV-vinylpyrrolidones; L-amino-acid-based polymer; and polyvinyl alcohol.

66. The liposomal antifolate composition of claim 65 wherein said PEG has a number average molecular weight (Mn) of 200 to 5000 daltons.

67. The liposomal antifolate composition of claim 61 further comprising at least one of an immunostimulatory agent, a détectable marker and a maleimide disposed on at least one of the steric stabilizer and an exterior of the liposome.

68. The liposomal antifolate composition of claim 67 wherein the at least one of an immunostimulatory agent and a détectable marker is covalently bonded to at least one of the steric stabilizer and the exterior of the liposome.

69. The liposomal antifolate composition of claim 67 wherein immunostimulating agent is at least one selected from the group consisting of protein immunostimulating agent; nucleic acid immunostimulating agent; Chemical immunostimulating agent; hapten; and adjuvant.

70. The liposomal antifolate composition of claim 67 wherein the immunostimulating agent is fluorescein isothiocyanate (FITC).

71. The liposomal antifolate composition of claim 67 wherein the immunostimulating agent is at least one selected from the group consisting of: fluorescein; DNP; beta glucan; beta-l,3-glucan; and beta-l,6-glucan.

72. The liposomal antifolate composition of claim 67 wherein the détectable marker is at least one selected from the group consisting of fluorescein and fluorescein isothiocyanate (FITC).

73. The liposomal antifolate composition of claim 67 wherein the immunostimulatory agent and the détectable marker is the same.

74. The liposomal antifolate composition of claim 61 wherein the liposome has a diameter in the range of 30-150 nm.

75. The liposomal antifolate composition of claim 74 wherein the liposome has a diameter in the range of 40-70 nm.

76. The liposomal antifolate composition of claim 61 wherein the liposome is an anionic liposome or a neutral liposome.

77. The liposomal antifolate composition of claim 76 wherein the zêta potential of the liposome is less than or equal to zéro.

78. The liposomal antifolate composition of claim 76 wherein the zêta potential of the liposome is in a range of 0 to -150 mV.

79. The liposomal antifolate composition of claim 76 wherein the zêta potential of the liposome is in the range of -30 to -50 mV.

80. The liposomal antifolate composition of claim 61 wherein the liposome is formed from liposomal components.

The liposomal antifolate composition of claim 80 wherein said liposomal component comprises at least one of an anionic lipid and a neutral lipid.

82. The liposomal antifolate composition of claim 80 wherein the liposomal component is selected from the group consisting of: DSPE; DSPE-PEG; DSPE-maleimide; HSPC; HSPC-PEG; HSPC-maleimide; cholestérol; cholesterol-PEG; and cholesterolmaleimide.

83. The liposomal antifolate composition of claim 80 wherein the liposome is formed from liposomal components and the liposomal components comprise at least one selected from the group consisting of: DSPE; DSPE-FITC; DSPE-maleimide; cholestérol; and HSPC.

84. The liposomal antifolate composition of claim 61 wherein the liposome encloses an aqueous solution.

85. The liposomal antifolate composition of claim 84 wherein the liposome encloses a bioactive antifolate agent and an aqueous pharmaceutically acceptable carrier.

86. The liposomal antifolate composition of claim 85 wherein the pharmaceutically acceptable carrier comprises trehalose.

87. The liposomal antifolate composition of claim 86 wherein the pharmaceutically acceptable carrier comprises 5% to 20% weight percent of trehalose.

88. The liposomal antifolate composition of claim 85 wherein the pharmaceutically acceptable carrier comprises citrate buffer at a concentration of between 5 to 200 mM and a pH of between 2.8 to 6.

89. The liposomal antifolate composition of claim 85 wherein the pharmaceutically acceptable carrier comprises a total concentration of sodium acetate and calcium acetate of between 50 mM to 500 mM.

^

90. The liposomal antifolate composition of claim 61 wherein the bioactive antifolate agent is water soluble.

91. The liposomal antifolate composition of claim 61 wherein each liposome comprises 5 less than 200,000 molécules of the bioactive antifolate agent.

92. The liposomal antifolate composition of claim 91 wherein each liposome comprises between 10,000 to 100,000 of the bioactive antifolate agent.

10

93. The liposomal antifolate composition of claim 61 wherein the bioactive antifolate agent is pemetrexed.

94. The liposomal antifolate composition of claim 61 wherein the bioactive antifolate agent is lometrexol.

95. The liposomal antifolate composition of claim 61 wherein the bioactive antifolate agent is at least one selected from the group consisting of methotrexate; ralitrexed; aminopterin; pralatrexate; lometrexol; trimetrexed; LY309887; and GW 1843U89.

20

96. The liposomal antifolate composition of claim 61 wherein the bioactive antifolate agent is at least one selected from at least one from the group consisting of proguanil; pyrimethamine; trimethoprim and 6-Substituted Pyrrolo and Thieon[2,3-d]pyrrolopyrimidine class of GARFT inhibitors.

25

97. The liposomal antifolate composition of claim 61 wherein the bioactive antifolate agent is at a pH of 5-8.

98. The liposomal antifolate composition of claim 61 wherein the bioactive antifolate agent is at a pH of 2-6.

99. The liposomal antifolate composition of claim 61 wherein the targeting moiety is covalently bound via a maleimide functional group to at least one selected from the group consisting of a liposomal component and a steric stabilizer molécule.

^

100. The liposomal antifolate composition of claim 61 wherein the targeting moiety has spécifie affinity for at least one selected from the group consisting of: folate receptor alpha; folate receptor beta; and folate receptor delta.

101. The liposomal antifolate composition of claim 61 wherein the targeting moiety has spécifie affinity for at least two selected from the group consisting of: folate receptor alpha; folate receptor beta; and folate receptor delta.

102. The liposomal antifolate composition of claim 61 wherein the targeting moiety has spécifie affinity for folate receptor alpha; folate receptor beta; and folate receptor delta.

103. The liposomal antifolate composition of claim 61 wherein the targeting moiety has spécifie affinity for an epitope on a tumor cell surface antigen that is présent on a tumor cell but absent or inaccessible on a non-turnor cell.

104. The liposomal antifolate composition of claim 103 wherein said tumor cell is a malignant cell.

105. The liposomal antifolate composition of claim 103 wherein the tumor cell surface antigen is at least one selected from the group consisting of: folate receptor alpha; folate receptor beta; and folate receptor delta.

106. The liposomal antifolate composition of claim 61 wherein the targeting moiety is protein comprising an antigen binding sequence of an antibody.

107. The liposomal antifolate composition of claim 106 wherein the antigen binding sequence of an antibody comprises one or more complementary determining régions of antibody origin.

108. The liposomal antifolate composition of claim 106 wherein said protein comprises an antibody.

3. The liposomal antifolate composition of claim 1, further comprising at least one of an immunostimulatory agent, a détectable marker and a maleimide disposed on at least one of the PEG and an exterior of the liposome.

4. The liposomal antifolate composition of claim 3, wherein the at least one of an immunostimulatory agent and a détectable marker is covalently bonded to at least one of the PEG and the exterior of the liposome.

5 39. The liposomal antifolate composition of claim 1 wherein the targeting moiety has spécifie affinity for an epitope on a tumor cell surface antigen that is présent on a tumor cell but absent or inaccessible on a non-tumor cell.

40. The liposomal antifolate composition of claim 39 wherein said tumor cell is a

10 malignant cell.

41. The liposomal antifolate composition of claim 39 wherein the tumor cell surface antigen is at least one selected from the group consisting of: folate receptor alpha; folate receptor beta; and folate receptor delta.

42. The liposomal antifolate composition of claim 1 wherein the targeting moiety is protein comprising an antigen binding sequence of an antibody.

43. The liposomal antifolate composition of claim 42 wherein the antigen binding

20 sequence of an antibody comprises one or more complementary determining régions of antibody origin.

44. The liposomal antifolate composition of claim 42, wherein said protein comprises an antibody.

45. The liposomal antifolate composition of claim 42 wherein the targeting moiety is at least one selected from the group consisting of an antibody; a humanized antibody; an antigen binding fragment of an antibody; a single chain antibody; a single-domain antibody; a bi-specific antibody; a synthetic antibody; a pegylated antibody; and a

30 multimeric antibody.

46. The liposomal antifolate composition of claim 1 wherein each liposome comprises up to 200 targeting moieties.

^^

47. The liposomal antifolate composition of claim 1 wherein each liposome comprises from 30 to 200 targeting moieties.

48. A composition as claimed in claim 1 for use in a method of delivering a bioactive 5 antifolate agent to a tumor expressing folate receptor on its surface.

49. The composition as claimed in claim 48 wherein said tumor is in a subject and said composition is administered by a method selected from the group consisting of: infusion;

10 injection;

parentéral administration; and topical administration.

50. The composition as claimed in claim 49 wherein said subject is a human.

51. The composition as claimed in claim 48 wherein the composition is for use in a method wherein a liposomal antifolate composition is selectively delivered to the tumor at a rate which is 2 folds more than a cell not expressing folate receptor.

20

52. A method of preparing a composition of claim 16 comprising:

forming a mixture comprising:

liposomal components;

the bioactive antifolate agent in aqueous solution;

the targeting moiety;

25 homogenizing the mixture to form liposomes in said aqueous solution; and extruding the mixture through a membrane to form liposomes enclosing the bioactive antifolate agent in an aqueous solution.

53. The method of claim 52 further comprising a step of:

30 removing excess bioactive antifolate agent in aqueous solution outside of the liposomes after said extruding step.

54. The method of claim 53, further comprising a step of:

| lyophilizing said composition after said removing step to form a lyophilized composition.

55. The method of claim 54, further comprising a step of:

reconstituting said lyophilizing composition by dissolving said lyophilizing composition in a solvent after said lyophilizing step.

56. The method of claim 52 wherein the mixture comprises at least one selected from the group consisting of mannitol; trehalose; sorbitol; and sucrose.

57. The method of claim 52 wherein one or more liposomal components further comprises a steric stabilizer.

58. The method of claim 57 wherein the steric stabilizer is at least one selected from the group consisting of polyethylene glycol (PEG); poly-L-lysine (PLL);

monosialoganglioside (GM1); poly(vinyl pyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[V-(2-hydroxypropyl) methacrylamide]; amphiphilic poly-Avinylpyrrolidones; L-amino-acid-based polymer; and polyvinyl alcohol.

59. The method of claim 58, wherein said PEG has a number average molecular weight (Mn) of 200 to 5000 daltons.

60. The method of claim 55, wherein said solvent is an aqueous solvent.

61. A liposomal antifolate composition comprising:

a medium comprising a liposome including an interior space;

an aqueous bioactive antifolate agent disposée! within said interior space;

a targeting moiety comprising a protein with spécifie affinity for at least one folate receptor, said targeting moiety disposed at an the exterior of the liposome.

62. The liposomal antifolate composition of claim 61 wherein the medium is an aqueous solution.

5. The liposomal antifolate composition of claim 3 wherein immunostimulating agent is at least one selected from the group consisting of protein immunostimulating agent; nucleic acid immunostimulating agent; Chemical immunostimulating agent; hapten; and adjuvant.

6. The liposomal antifolate composition of claim 3 wherein the immunostimulating agent is fluorescein isothiocyanate (FITC).

7. The liposomal antifolate composition of claim 3 wherein the immunostimulating agent is at least one selected from the group consisting of: fluorescein; DNP; beta glucan; beta-l,3-glucan; and beta-l,6-glucan.

^h

^^8. The liposomal antifolate composition of claim 1 wherein the targeting moiety has spécifie affinity for folate receptor alpha; folate receptor beta; and folate receptor delta.

8. The liposomal antifolate composition of claim 3 wherein the détectable marker is at least one selected from the group consisting of fluorescein and fluorescein isothiocyanate (FITC).

5

·⁰⁹· The liposomal antifolate composition of claim 106 wherein the targeting moiety is at least one selected from the group consisting of: an antibody; a humanized antibody;

an antigen binding fragment of an antibody; a single chain antibody; a single-domain antibody; a bi-specific antibody; a synthetic antibody; a pegylated antibody; and a multimeric antibody.

110. The liposomal antifolate composition of claim 61 wherein each liposome comprises up to 200 targeting moieties.

111. The liposomal antifolate composition of claim 110 wherein each liposome comprises from 30 to 200 targeting moieties.

112. A composition as claimed in claim 61 for use in a method of delivering a bioactive antifolate agent to a tumor expressing folate receptor on its surface.

113. The composition as claimed in claim 112 wherein said tumor is in a subject and said composition is administered by a method selected from the group consisting of: infusion; injection; parentéral administration; and topical administration.

114. The composition as claimed in claim 113 wherein said subject is a human.

115. The composition as claimed in claim 112 wherein the composition is for use in a method wherein a liposomal antifolate composition is selectively delivered to the tumor at a rate which is 2 folds more than a cell not expressing folate receptor.

116. A method of preparing a composition of claim 80 comprising: forming a mixture comprising:

the liposomal components;

the bioactive antifolate agent in aqueous solution;

the targeting moiety;

homogenizing the mixture to form liposomes in said aqueous solution; and extruding the mixture through a membrane to form liposomes enclosing the bioactive antifolate agent in an aqueous solution.

17. The method of claim 116 further comprising a step of:

removing excess bioactive antifolate agent in aqueous solution outside of the liposomes after said extruding step.

118. The method of claim 117, further comprising a step of:

lyophilizing said composition after said removing step to form a lyophilized composition.

119. The method of claim 118, further comprising a step of:

120. The method of claim 116 wherein the mixture comprises at least one selected from the group consisting of mannitol; trehalose; sorbitol; and sucrose.

121. The method of claim 116 wherein one or more liposomal components further comprises a steric stabilizer.

122. The method of claim 121 wherein the steric stabilizer is at least one selected from the group consisting of polyethylene glycol (PEG); poly-L-lysine (PLL);

monosialoganglioside (GM1); poly(vinyl pyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[A-(2-hydroxypropyl) methacrylamide]; amphiphilic poly-M vinylpyrrolidones; L-amino-acid-based polymer; and polyvinyl alcohol.

123. The method of claim 122, wherein said PEG has a number average molecular weight (Mn) of 200 to 5000 daltons.

124. The method of claim 119, wherein said solvent is an aqueous solvent.

125. A targeted liposomal composition that selectively targets folate receptors comprising: a liposome including an interior space;

a bioactive agent disposed within said interior space;

a steric stabilizer molécule attached to an exterior of the liposome; and | a targeting moiety comprising a protein with spécifie affinity for at least one folate receptor, said targeting moiety attached to at least one of the steric stabilizer and the exterior of the liposome.

126. The targeted liposomal composition of claim 125 wherein the steric stabilizer is at least one selected from the group consisting of polyethylene glycol (PEG); poly-Llysine (PLL); monosialoganglioside (GM1); poly(vinyl pyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[?/-(2-hydroxypropyl) methacrylamide]; amphiphilic poly-V-vinylpyrrolidones; L-amino-acid-based polymer; and polyvinyl alcohol.

127. The targeted liposomal composition of claim 126, wherein said PEG has a number average molecular weight (Mn) of 200 to 5000 daltons.

128. The targeted liposomal composition of claim 125, wherein the bioactive agent comprises at least one of the group consisting of ellipticine; paclitaxel; pemetrexed; methotrexate; ralitrexed; aminopterin; pralatrexate; lometrexol; trimetrexed; LY309887; GW 1843U89; proguanil; pyrimethamine; trimethoprim and 6-Substituted Pyrrolo and Thieon[2,3-d]pyrrolopyrimidine class of GARFT inhibitors.

129. A composition as claimed in claim 125 for use in a method of delivering a bioactive antifolate agent to a tumor expressing folate receptor on its surface.

130. The composition as claimed in claim 129 wherein said tumor is in a subject and said composition is administered by a method selected from the group consisting of: infusion; injection; parentéral administration; and topical administration.

131. The composition as claimed in claim 130 wherein said subject is a human.

132. The composition as claimed in claim 129 wherein the composition is for use in a method wherein a liposomal antifolate composition is selectively delivered to the tumor at a rate which is 2 folds more than a cell not expressing folate receptor.

33. A method of preparing a composition of claim 125 wherein said liposome is formed from liposomal components comprising:

forming a mixture comprising:

liposomal components;

the bioactive agent in aqueous solution;

the targeting moiety;

134. The method of claim 133 further comprising a step of:

135. The method of claim 134, further comprising a step of:

136. The method of claim 135, further comprising a step of:

137. The method of claim 133 wherein the mixture comprises at least one selected from the group consisting of mannitol; trehalose; sorbitol; and sucrose.

138. The method of claim 133 wherein one or more liposomal components further comprises a steric stabilizer.

139. The method of claim 138 wherein the steric stabilizer is at least one selected from the group consisting of polyethylene glycol (PEG); poly-L-lysine (PLL);

monosialoganglioside (GM1); poly(vinyl pyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[A-(2-hydroxypropyl) methacrylamide]; amphiphilic poly-Avinylpyrrolidones; L-amino-acid-based polymer; and polyvinyl alcohol.

140. The method of claim 139, wherein said PEG has a number average molecular weight (Mn) of 200 to 5000 daltons.

5

141. The method of claim 136, wherein said solvent is an aqueous solvent.

142. A kit for providing a composition of claim 80 comprising:

the liposomal components, an instruction for using the composition to encapsulate a bioactive agent, and

9. The liposomal antifolate composition of claim 3 wherein the immunostimulatory agent and the détectable marker is the same.

10. The liposomal antifolate composition of claim 1, wherein the liposome has a diameter in the range of 30-150 nm.

11. The liposomal antifolate composition of claim 10, wherein the liposome has a diameter in the range of 40-70 nm.

12. The liposomal antifolate composition of claim 1, wherein the liposome is an anionic 15 liposome or a neutral liposome.

13. The liposomal antifolate composition of claim 12, wherein the zêta potential of the liposome is less than or equal to zéro.

20

14. The liposomal antifolate composition of claim 12, wherein the zêta potential of the liposome is in a range of 0 to -150 mV.

15. The liposomal antifolate composition of claim 12, wherein the zêta potential of the liposome is in the range of -30 to -50 mV.

16. The liposomal antifolate composition of claim 1 wherein the liposome is formed from liposomal components.

17. The liposomal antifolate composition of claim 16 wherein said liposomal component 30 comprises at least one of an anionic lipid and a neutral lipid.

18. The liposome of claim 16 wherein said liposomal component is at least one selected from the group consisting of: DSPE; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholestérol; cholesterol-PEG; and cholesterol-maleimide.

19. The liposomal antifolate composition of claim 16 wherein the liposomal components comprise at least one selected from the group consisting of: DSPE; DSPE-PEG-FITC;

DSPE-PEG-maleimide; cholestérol; and HSPC.

20. The liposomal antifolate composition of claim 1 wherein the liposome encloses an aqueous solution.

21. The liposomal antifolate composition of claim 1 wherein the liposome encloses a 10 bioactive antifolate agent and an aqueous pharmaceutically acceptable carrier.

22. The liposomal antifolate composition of claim 21 wherein the pharmaceutically acceptable carrier comprises trehalose.

15

23. The liposomal antifolate composition of claim 21 wherein the pharmaceutically acceptable carrier comprises 5% to 20% weight percent of trehalose.

24. The liposomal antifolate composition of claim 21 wherein the pharmaceutically acceptable carrier comprises citrate buffer at a concentration of between 5 to 200 mM

20 and a pH of between 2.8 to 6.

25. The liposomal antifolate composition of claim 21 wherein the pharmaceutically acceptable carrier comprises a total concentration of sodium acetate and calcium acetate of between 50 mM to 500 mM.

26. The liposomal antifolate composition of claim 1 wherein the bioactive antifolate agent is water soluble.

27. The liposomal antifolate composition of claim 1 wherein each liposome comprises 30 less than 200,000 molécules of the bioactive antifolate agent.

28. The liposomal antifolate composition of claim 27 wherein each liposome comprises between 10,000 to 100,000 molécules of the bioactive antifolate agent.

p9. The liposomal antifolate composition of claim 1 wherein the bioactive antifolate agent is pemetrexed.

30. The liposomal antifolate composition of claim 1 wherein the bioactive antifolate agent is lometrexol.

31. The liposomal antifolate composition of claim 1 wherein the bioactive antifolate agent is at least one selected from the group consisting of methotrexate; ralitrexed;

aminopterin; pralatrexate; lometrexol; thiophene analog of lometrexol; furan analog of lometrexol; trimetrexed; LY309887; and GW 1843U89.

32. The liposomal antifolate composition of claim 1 wherein the bioactive antifolate agent is at least one selected from at least one from the group consisting of proguanil;

pyrimethamine; trimethoprim and 6-Substituted Pyrrolo and Thieon[2,3-d]pyrrolopyrimidine class of GARFT inhibitors.

33. The liposomal antifolate composition of claim 1 wherein the bioactive antifolate agent is at a pH of 5-8.

34. The liposomal antifolate composition of claim 1 wherein the bioactive antifolate agent is at a pH of 2-6.

35. The liposomal antifolate composition of claim 1 wherein the targeting moiety is covalently bound via a maleimide functional group to at least one selected from the group consisting of a liposomal component and a PEG molécule.

36. The liposomal antifolate composition of claim 1 wherein the targeting moiety has spécifie affinity for at least one selected from the group consisting of: folate receptor alpha; folate receptor beta; and folate receptor delta.

37. The liposomal antifolate composition of claim 1 wherein the targeting moiety has spécifie affinity for at least two selected from the group consisting of: folate receptor alpha; folate receptor beta; and folate receptor delta.

10 optionally, in a separate container, the bioactive agent.