Lipidic microparticles linked to multiple proteins

The invention provides lipidic microparticles stably associated with at least two different targeting moieties, which targeting moieties are attached to linker molecules comprising a hydrophilic domain and a hydrophobic domain. The targeting moieties can be antibodies, antibody fragments, hormones, growth factors, enzymes, or nucleic acid binding proteins, or other proteins. The targeting moieties can be chemically conjugated to the linker molecules, or they can be fused by recombinant techniques.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

FIELD OF THE INVENTION

The present invention relates to the field of cationic lipid:DNA complexes ( CLDC ). In particular, the present invention relates to lipid:nucleic acid complexes that contain (1) hydrophilic polymer; (2) nucleic acid that has been condensed with organic polycations; and (3) hydrophilic polymer and nucleic acid that has been condensed with organic polycations. The lipid:nucleic acid complexes of this invention show high transfection activity in vivo following intravenous injection and an unexpected increase in shelf life, as determined by in vivo transfection activity.

The present invention further relates to the field of lipidic microparticles, such as liposomes, lipid:DNA complexes, lipid:drug complexes, and microemulsion droplets, attached to proteins. In particular, the invention relates to lipidic microparticles with attached proteins which have been first conjugated to linker molecules having a hydrophilic polymer domain and a hydrophobic domain capable of stable association with the microparticle, or proteins which have been engineered to contain a hydrophilic domain and a lipid moiety permitting stable association with a lipidic microparticle.

BACKGROUND OF THE INVENTION

Liposomes that consist of amphiphilic cationic molecules are useful non-viral vectors for gene delivery in vitro and in vivo (reviewed in Crystal, Science 270: 404-410 (1995); Blaese et al., Cancer Gene Ther. 2: 291-297 (1995); Behr et al., Bioconjugate Chem. 5: 382-389 (1994); Remy et al., Bioconjugate Chem. 5: 647-654 (1994); and Gao et al., Gene Therapy 2: 710-722 (1995)). In theory, the positively charged liposomes complex to negatively charged nucleic acids via electrostatic interactions to form lipid:nucleic acid complexes. The lipid:nucleic acid complexes have several advantages as gene transfer vectors. Unlike viral vectors, the lipid:nucleic acid complexes can be used to transfer expression cassettes of essentially unlimited size. Since the complexes lack proteins, they may evoke fewer immunogenic and inflammatory responses. Moreover, they cannot replicate or recombine to form an infectious agent and have low integration frequency.

There are a number of publications that demonstrate convincingly that amphiphilic cationic lipids can mediate gene delivery in vivo and in vitro, by showing detectable expression of a reporter gene in culture cells in vitro (Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-17 (1987); Loeffler et al., Methods in Enzymology 217: 599-618 (1993); Felgner et al., J. Biol. Chem. 269: 2550-2561 (1994)). Because lipid:nucleic acid complexes are on occasion not as efficient as viral vectors for achieving successful gene transfer, much effort has been devoted in finding cationic lipids with increased transfection efficiency (Behr, Bioconjugate Chem. 5: 382-389 (1994); Remy et al., Bioconjugate Chem. 5: 647-654 (1994); Gao et al., Gene Therapy 2: 710-722 (1995)). Lipid:nucleic acid complexes are regarded with enthusiasm as a potentially useful tool for gene therapy.

Several groups have reported the use of amphiphilic cationic lipid:nucleic acid complexes for in vivo transfection both in animals, and in humans (reviewed in Gao et al., Gene Therapy 2: 710-722 (1995); Zhu et al., Science 261: 209-211 (1993); and Thierry et al., Proc. Natl. Acad. Sci. USA 92: 9742-9746 (1995)). However, the technical problems for preparation of complexes that have stable shelf-lives have not been addressed. For example, unlike viral vector preparations, lipid:nucleic acid complexes are unstable in terms of particle size (Behr, Bioconjugate Chem. 5: 382-389 (1994); Remy et al., Bioconjugate Chem. 5: 647-654 (1994); Gao et al., Gene Therapy 2: 710-722 (1995)). It is therefore difficult to obtain homogeneous lipid:nucleic acid complexes with a size distribution suitable for systemic injection. Most preparations of lipid:nucleic acid complexes are metastable. Consequently, these complexes typically must be used within a short period of time ranging from 30 minutes to a few hours. In recent clinical trials using cationic lipids as a carrier for DNA delivery, the two components were mixed at the bed-side and used immediately (Gao et al., Gene Therapy 2: 710-722 (1995)). The structural instability along with the loss of transfection activity of lipid:nucleic acid complex with time have been challenges for the future development of lipid-mediated gene therapy.

Liposomes consisting of amphiphilic cationic molecules are not, of course, the only form of lipidic microparticles and gene therapy is not the only utility for such particles. Lipidic microparticles have also been used for delivery of drugs and other agents to target sites. Targeting of the microparticles is typically achieved through use of a protein attached to the surface of the microparticle, which may, for example be a ligand for cell surface receptor on a cell type of interest. Conversely, the protein may be an antibody which specifically recognizes an antigen on a cell type of interest, such as diseased cells carrying specific markers. Additionally, proteins can be attached for purposes other than targeting. For example, liposomes can contain prodrugs which slowly seep from the liposome into the circulation. An enzyme attached to the liposome can then convert the prodrug into its active form.

Current methods for effecting the attachment of proteins to lipidic microparticles have been of two types. The first type requires introducing a linker molecule bearing an active group (one which reacts with a functional group of the protein) into the microparticle composition prior to conjugation of the activated particle with the protein of interest. The disadvantages of methods of this type are: often uncontrollable, incomplete reaction of the protein with the linker; the presence of excess linker on the resulting conjugate, potentially adverse effect of the linker on the ability of the particle, and the inability to incorporate components reactive with the links into the composition of the particle.

The second group of methods employs the steps of (a) attachment of a hydrophobic moiety, such as a hydrocarbon chain, to the protein molecule, (b) dissolving the components of the lipidic microparticle, along with the conjugate of step (a) in the presence of a detergent, and (c) removing the said detergent, effecting the formation of the lipidic particle incorporating the protein conjugate (Torchilin, Immunomethods 4-244-258 (1994); Laukkanen et al., Biochemistry 33:11664-11670 (1994)). These methods have a number of disadvantages, including the imposition of severe limitations on the range of methods by which the particle can be formed, (e.g. the detergent removal technique is required) and by which the drug or other agent can be loaded into the microparticle. Moreover, step (b) requires the dissolution of the microparticle. These methods are therefore unable to attach a protein to a premade particle without first destroying it. The presence of detergent in these methods is unavoidable because without a detergent the hydrophobically modified protein is insoluble in aqueous medium

The insertion into liposomes of hydrophilic polymer-lipid linked to a small (5 amino acid) oligopeptide or small oligosaccharide has been reported. (Zalipsky et al., Bioconjugate Chem. 8:111-118 (1997). The peptide and oligosaccharide employed were, however, of a size (molecular weight, 500-3,000 Da) smaller than, or comparable to, the linker itself (molecular weight 2,750 Da). This study therefore provides no guidance for inserting into liposomes or other lipidic microparticles proteins, such as antibodies, or fragments thereof, conjugated to linkers significantly smaller than the protein. In view of the hydrophilic nature of antibodies and other proteins, the art has taught that the larger, protein portion of such a conjugate prevents the hydrophobic linking moiety from stable association with a lipidic microparticle.

SUMMARY OF THE INVENTION

The present invention provides a novel method of preparing cationic lipid:nucleic acid complexes that have increased shelf life. In one embodiment, these complexes are prepared by contacting a nucleic acid with an organic polycation, to produce a condensed or partially condensed nucleic acid. The condensed nucleic acid is then combined with an amphiphilic cationic lipid plus a neutral helper lipid such as cholesterol in a nolar ratio from about 2:1 to about 1:2, producing the lipid:nucleic acid complex. Optionally, a hydrophilic polymer is subsequently added to the lipid:nucleic acid complex. Alternatively, the hydrophilic polymer is added to a lipid:nucleic acid complex comprising nucleic acid that has not been not condensed. These lipid:nucleic acid complexes have an increased shelf life, e.g., when stored at 22 C. or below, as compared to an identical lipid:nucleic acid complex in which the nucleic acid component has not been contacted with the organic polycation and/or in which the lipid:nucleic acid complex has not been contacted with a hydrophilic polymer.

In a particularly preferred embodiment, the polycation is a polyamine, more preferably a polyamine such as sperrnidine or spermine.

In another preferred embodiment, the lipid:nucleic acid complexes are prepared by combining a nucleic acid with an amphiphilic cationic lipid and then combining the complex thus formed with a hydrophilic polymer. This lipid:nucleic acid complex has an increased shelf life, e.g., when stored at 22 C. or below as compared to an identical complex that has not been combined with the hydrophilic polymer.

In one embodiment, the hydrophilic polymer is selected from the group consisting of polyethylene glycol (PEG), polyethylene glycol derivatized with phosphatidyl ethanolamine (PEG-PE), polyethylene glycol derivatized with tween, polyethylene glycol derivatized with distearoylphosphatidylethanolamine (PEG-DSPE), ganglioside G M1 and synthetic polymers.

In one embodiment, the lipid:nucleic acid complex is lyophilized.

In any of the methods and compositions of this invention, the nucleic acid can be virtually any nucleic acid, e.g., a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA), and peptide nucleic acid (PNA) etc., and is most preferably a DNA. In a particularly preferred embodiment, the DNA is an expression cassette capable of expressing a polypeptide in a cell transfected with the lipid:nucleic acid complex.

In one embodiment the lipid:nucleic acid complexes are formed by first forming a liposome, and then combining the formed liposome with condensed or partially condensed nucleic acid to form a lipid;nucleic acid complex. Optionally, the lipid:nucleic acid complex is subsequently contacted with a hydrophilic polymer. The liposomes can alternatively be combined with an uncondensed nucleic acid to form a lipid:nucleic acid complex to which a hydrophilic polymer (e.g., PEG-PE) is later added. A lipid:nucleic acid complex prepared by the combination of nucleic acid and a liposome contacted with a hydrophilic polymer can be subsequently combined with additional hydrophilic polymer. In a preferred embodiment, the lipid and nucleic acid are combined in a ratio ranging from about 1 to about 20, more preferably from about 4 to about 16, and most preferably from about 8 to about 12 nmole lipid: g nucleic acid. The lipid and hydrophilic polymer are combined in a molar ratio ranging from about 0.1 to about 10%, more preferably from about 0.3 to about 5% and most preferably from about 0.5% to about 2.0% (molar ratio of hydrophilic polymer to cationic lipid of the complex).

It will be appreciated that a targeting moiety (e.g., an antibody or an antibody fragment) can be attached to the lipid and/or liposome before or after formation of the lipid:nucleic acid complex. In a preferred embodiment, the targeting moiety is coupled to the hydrophilic polymer (e.g., PEG), where the targeting moiety/hydrophilic polymer is subsequently added to the lipid:nucleic acid complex. This provides a convenient means for modifying the targeting specificity of an otherwise generic lipid:nucleic acid complex.

In a particularly preferred embodiment, the method of increasing the shelf life of the lipid:nucleic acid complex includes the steps of combining an expression cassette with spermidine or spermine with an amphiphilic cationic lipid plus a helper lipid such as cholesterol, and a Fab fragment of an antibody attached to a spacer, e.g., polyethylene glycol, so that the complex has increased shelf life when stored at about 4 C.

In one particularly preferred embodiment, the method of increasing the shelf life of the lipid:nucleic acid complex includes the steps of combining an expression cassette with spermidine or spermine with an amphiphilic cationic lipid, and a Fab fragment of an antibody attached to a polyethylene glycol derivative. In another particularly preferred embodiment, includes the steps of combining an expression cassette with an amphiphilic cationic lipid, and a Fab fragment of an antibody attached to a polyethylene glycol derivative so that the complex has increased shelf life when stored at about 4 C.

This invention also provides for a method of transfecting a nucleic acid into a mammalian cell, the method comprising contacting the cell with any one of the lipid:nucleic acid complexes prepared as described above. In one embodiment, the method uses systemic administration of a lipid:nucleic acid complex into a mammal. In a preferred embodiment, the method of transfecting uses intravenous administration of the lipid:nucleic acid complex into a mammal. In a particularly preferred embodiment, the method comprises contacting a specific cell that expresses a ligand that recognizes the Fab fragment.

In yet another embodiment, this invention also provides for pharmaceutical composition comprising the lipid:condensed nucleic acid complex described above. The pharmaceutical compositions comprise a therapeutically effective dose of the lipid:nucleic acid complex and a pharmaceutically acceptable carrier or excipient.

In yet another embodiment, the invention also provides a kit for preparing a lipid:nucleic acid complex, the kit comprising a container with a liposome; a container with a nucleic acid; and a container with a hydrophilic polymer, wherein the liposome and the nucleic acid are mixed to form the lipid:nucleic acid complex and wherein the lipid:nucleic acid complex is contacted with the hydrophilic polymer. In a preferred embodiment, the hydrophilic polymer is derivatized with a targeting moiety, preferably an Fab fragment. In another preferred embodiment, the nucleic acid is condensed.

This invention also provides for a lipid:condensed nucleic acid complex prepared using the method of increasing shelf life using nucleic acid condensed with an organic polycation, as summarized above.

The invention further provides a method for making lipidic microparticles bearing attached proteins. The method employs proteins which have been conjugated to linker molecules which will stably associate with lipidic microparticles. The invention therefore permits the attachment of proteins to the surface, for example, of lipidic microparticles which have been preformed.

EXAMPLES

The invention is illustrated by the following examples. These examples are offered to illustrate, but not to limit the present invention.

Preparation of Stable Lipid:Plasmid DNA Complexes For In Vivo Gene delivery

A. Materials and methods

DOPE was purchased from Avanti (Alabaster, Ala.). Highly purified Cholesterol was obtained from Calbiochem (San Diego, Calif.). DDAB and dextran (M.W. 40,000) were purchased from Sigma (St. Louis, Mo.). DDAB was recrystalized once from acetone-methanol solution. D-luciferin was obtained from Boehringer Mannheim. PEG-PE was a gift from Sequus Pharmaceuticals (Menlo Park, Calif.). DC-Chol, MMCE and DOGS were obtained from UCSF Gene Transfer Vehicle Core of Gene Therapy Center. ESPM, DOTAP, POEPC, DOEPC, DMEPC and DODAP were gifts from Avanti (Alabaster, Ala.). Chloroform solution of each lipid was stored under argon in sealed ampules at 40 C. Other reagents of possible highest grade were purchased and used without further purification.

2. Preparation of liposomes

Small cationic liposomes were prepared in 5% (w/v) dextrose solution in the following fashion. DDAB or other cationic lipids in chloroform was mixed with DOPE or/and cholesterol in a desired molar ratio, and the solvent was removed slowly under reduced pressure at 50 C. on a rotary evaporator. The dry lipid film was hydrated with 5% dextrose solution prewarmed to 50 C. and the container was sealed under argon. The hydrated lipid suspension was sonicated in a bath sonicator (Lab Supplies, Hicksville, N.Y.) for 5-10 min at 50 C. The final concentration of liposomes was 5 mM cationic lipid and the size of liposomes was measured by dynamic light scattering to be 195 65 nm. Sonicated liposomes were stored under argon at 4 C. until use.

3. Luciferase reporter system

Plasmid, pCMV/IVS-luc , was constructed as follows. A fragment containing the CMV promoter and synthetic IgE intron was excised from pBGt2.CAT using Spe I and Hind III and cloned into pBSIIKS . The cDNA encoding the modified firefly luciferase (luc ) including SV40 late poly (A) signal was cut from the pGL3-Basic Vector (Promega) with Hind III and Sal I and was put into the pBS-CMV-IVS clone downstream of the splice. Plasmids were purified using alkaline lysis procedures adopted and devised by Qiagen Corp. (Chatsworth, Calif.). Plasmid purity was measured by the ratio of absorbance at 260 nm vs 280 nm, and stored in buffer containing 10 mM Tris-Cl and ImM EDTA at pH 8.0 at concentrations of 1-2 mg/ml.

4. Preparation of transfection complexes

Prior to the transfection experiments, the optimal DNA/liposome ratio for forming complexes which were not large aggregates was determined by mixing fixed amount plasmid to various amount of liposomes. In general, the transfection complexes were formed by pipetting plasmid into liposome suspension of equal volume and mixing rapidly. Routinely, liposomes containing 8-12 nmole of DDAB could complex with 1 g plasmid without forming visible large aggregates. Such complexes have excess positive charge, but still tend to aggregate with time during storage at 4 C. and lose transfection activity in 4 days. For in vitro experiments, which called for much dilute complexes, cationic lipid:plasmid DNA complexes ( CLDC ) at 5 nmole DDAB per g DNA were used. To keep the lipid:plasmid DNA complexes from forming large aggregates and losing transfecting activity with time, two approaches were taken: (1) incorporating a small amount of PEG-PE (approx. 1% mole ratio) into lipid:plasmid DNA complexes within a few minutes after their preparation; and/or (2) condensing plasmid with polyamines (e.g., 0.05 to 5.0 nmole of spermidine per g DNA) prior to mixing with liposomes. The optimal amount of the polyamines was determined by titrating polyamines to DNA before forming large aggregates. The size of these complexes was estimated by dynamic light scattering to be in the range of 410 150 nm.

5. Assay of reporter gene expression

Purified luciferase was purchased from Boehringer Mannheim as a standard for calibrating the luminometer and constructing a control standard for the relative specific activity of luciferase. Reporter gene expression in a tissue extract was presented in nanogram quantities by converting relative light unit measured from a luminometer into weight unit according to a standard curve. Luciferase expressed in cells or tissues was extracted with chemical cell lysis. Effective lysis buffer consisted of 0.1 M potassium phosphate buffer at pH 7.8, 1% Triton X-100, 1 mM DTT and 2 mM EDTA.

Female CD1 mice (4-6 weeks old, weighing approx. 25 g) were obtained from Charles River Laboratory. Mice received lipid:plasmid DNA complexes by tail vein injection and were sacrificed 24 h later. The anesthetized animals were perfused with cold phosphate-buffered saline (PBS) via heart puncture. Each tissue was dissected and washed in PBS, and then homogenized in 6 ml round-bottomed culture tube containing 500 l of lysis buffer. The samples were kept at room temperature for 20 min with occasional mixing. The homogenized samples were centrifuged for 10 min at 3000 rpm in an Eppendorf centrifuge. Luciferase activity of each tissue was measured by mixing 100 l of the reconstituted luciferase substrate (Promega, Madison, Wis.) with 20 l of the supernatant of tissue homogenate in the injection system of a luminometer. Peak light emission was measured for 10 sec. at 20 C. Relative light units of each sample were converted to the amount of luciferase in the tissue extract by comparing with a standard curve which was established for each set of experiments. The protein content of the extract was determined using protein assay kits (BioRad, Richmond, Calif.). Background was the count of lysis buffer only.

SK-BR-3 cells (Park et al., Proc. Natl. Acad. Sci. USA 92: 1327-1331 (1995)) were cultured in McCoy's 5 A medium supplemented with 10% heat-inactivated bovine calf serum and in 5% CO 2 . SK-BR-3 cells in monolayer culture were plated at 50,000 cells per well in 12-well plates and incubated overnight. Each well received 0.5 1 g of pCMV/IVS-luc within 20 min of complex formation. Cells were harvested after 24 hr of incubation with complexes at 37 C. Luciferase activity in cells was determined as described above.

1. Optimizing the helper lipid

The use of cationic liposomes for in vitro gene transfer has become widespread since Feigner et al. published their study (Feigner et al., Proc. Nati. Acad. Sci. USA 84: 7413-17 (1987)). It was established later (Feigner et al., J. Biol. Chem. 269: 2550-2561 (1994)) that DOPE is by far the most efficient helper lipid for in vitro gene transfection and this result has confirmed by several laboratories (Farhood et al., in Gene Therapy for Neoplastic Diseases , pp 23-55 (Huber & Lazo eds., 1994); Zhou et al., Biochim. Biophys. Acta 1189: 195-203 (1994)). It has been suggested, on the basis of in vitro studies, that DOPE may facilitate the cytoplasmic delivery via membrane fusion once positively charged lipid:plasmid DNA complexes are bound to the cell membrane (Zhou et al., Biochim. Biophys. Acta 1189: 195-203 (1994)). Even though Friend et al. did not obtain any morphological evidence that the DOTMA/DOPE lipid:plasmid DNA complexes fuse directly with the plasma membrane, they do not exclude the possibility of fusion events (Friend et al., Biochim. Biophys. Acta 1278: 41-50 (1996)). They suggested that the complexes are endocytosed and the cationic lipids disrupt the endosomal/lysosomal membranes and then facilitate an escape of the DNA complexes into the cytoplasm and eventually into the nucleus.

Contrary to most expectations, the helper role of DOPE established from in vitro studies is not evident for in vivo gene delivery following i.v. injection of the complexes. When DOPE was included in DDAB cationic liposomes, the in vivo gene transfection was inhibited. This DOPE-dependent inhibition is shown in FIG. 1 . Cholesterol, not DOPE, was found to be effective as helper lipid for in vivo gene delivery. There was a ten-fold reduction in luciferase expression in mouse lungs when half of the cholesterol was replaced with DOPE. The in vivo results of DDAB and other cationic liposomes are not consistent with the general assumption that DOPE is a suitable helper lipid. On the contrary, DOPE in cationic lipid:plasmid DNA complexes attenuates the in vivo transfection to such a great degree that DOPE is considered as an inhibitory agent in formulations for in vivo gene delivery. Cholesterol has been chosen for in vivo studies in recent published reports (Liu et al., J. Biol. Chem. 270: 24864-70 (1995); Solodin et al., Biochemistry 34: 13537-44 (1995)) in which the authors do not elaborate on how and why they selected different helper lipids for their experimental designs, i.e. DOPE for in vitro and cholesterol for in vivo studies. Stabilization of anionic and neutral liposomes in blood by cholesterol has been known for a long time (Mayhew et al., Cancer Treat. Rep. 63: 1923-1928 )1979)). It is therefore obvious that for systemic gene delivery, one has to consider the stability of lipid:plasmid DNA complexes in blood, various components of which are known to react with macromolecular complexes. In fact, the preliminary study of various formulations of lipid:plasmid DNA complexes using freeze-fracture electron microscopy has shown that the cholesterol-containing complexes were structurally more stable than the DOPE-containing complexes in the presence of serum.

Using DDAB/Chol lipid:plasmid DNA complexes (8 nmole DDAB/ g DNA) for in vivo transfection experiments, detectable luciferase expression in the lung of 25 g mouse required a DNA dose ranging from 30 g to 60 g. Routinely 40 60 g plasmid DNA per mouse gave consistent gene expression. The amount of DDAB usually associated with 80 g DNA (or more) per mouse was found to be too toxic to the animal. The expression of luciferase in various tissues is shown in FIG. 2 . As observed before (Zhu et al., Science 261: 209-211 (1993); Liu et al., J. Biol, Chem. 270: 24864-70 (1995); Solodin et al., Biochemistry 34: 1353744 (1995)), maximal expression was found in lung tissue. For 60 g plasmid injected, 1-2 ng luciferase per mg tissue protein was routinely obtained. FIG. 3 shows the duration of reporter gene expression in lung tissue. Expression of luciferase decreased rapidly and reached undetectable levels in 2 weeks. Zhu et al. reported that following i.v. injection of DOTMA/DOPE (1:1) plasmid complexes into adult mice, the expression of the reporter gene (CAT) is widespread among various tissues and the maximum expression is from complexes with a ratio of 1 I g plasmid to 8 nmole total lipids (Zhu et al., Science 261: 209-211 (1993)). However, at this ratio (corresponding to 1 g plasmid to 4 nmole cationic lipid), DDAB/Chol lipid:plasmid DNA complexes tended to aggregate and did not produce measurable gene expression in this investigation.

Since different reporter genes have been used among different laboratories, it has been difficult to attribute the variations in the efficiency of in vivo gene delivery to changes in the formulation of liposomes. For a direct comparison of the results in the literature, the relative light units of luciferase activity measured from a luminometer was converted to a standard of purified luciferase. By doing so, the peak transfection activity of DDAB/Chol formulations was 3 orders of magnitude higher than values reported recently in comparable experiments (Thierry et al., Proc. Natl. Acad. Sci . USA 92: 9742-9746 (1995)). Given that same reporter gene along with same promoter in the experimental design, the difference in expression may reflect the selection of liposome formulation. In fact, DDAB/Chol was one of the most efficient gene delivery vehicle among many formulations from 18 different cationic lipids which was screened recently. Preliminary results of expression in mouse lung following i.v. injection indicated that DOTMA/Chol, DOTAP/Chol, MMCE/Chol and ESPM/Chol gave 10-100% transfection activity of DDAB/Chol, DOGS/Chol, POEPC/Chol, LYSPE/DOPE and DC-Chol/DOPE gave 1-10% of DDAB/Chol. DOEPC/Chol, DMEPC/Chol, DODAP/Chol and DDAB/DOPE did not give any measurable activity.

In parallel with the transfection studies, the morphology of these complexes in serum and in cell medium was examined by freeze-fracture electron microscopy. When examined in 50% mouse serum (10 minute incubation time), non-stabilized, one day old CLDC are as small as they are in buffer at low ionic strength (100-250 nm) but show very few protrusions. Six day old, non stabilized CLDC incubated in 50% mouse serum appeared as densely packed aggregates of spherical particles, with a high number of attached particles. Such formulations have lost all of their in vivo transfection activity within 4 days. Residual fibrillar protrusions are not observed.

PEG-PE stabilized CLDC incubated in 50% mouse serum were small (100-200 nm) even at six days. Similarly, CLDC prepared with condensed DNA were also quite small even after six days of storage. Specifically, the CLDC were shaped like map pins that were structurally stable in the presence of serum.

After incubation in cell meditim (RPMI-1640 with 10% FCS), non-stabilized six day old CLDC were morphologically similar to those incubated in mouse serum, as described above. These complexes, however, were more loosely packed and showed no fibrillar protrusions. Similar morphology was observed with PEG-PE stabilized CLDC and condensed DNA CLDC incubated in cell medium.

2. Increasing shelf life for transfection activity

The relationship between structural stability and transfection activity of lipid:plasmid DNA complexes has not been detailed in the published reports so far. Screening procedures have been established to avoid large aggregates of lipid:plasmid DNA complexes by changing the ratio of DNA to lipid from net negatively charged to positively charged. Lipid:plasmid DNA complexes of each particular cationic lipid at various ratios of DNA/lipid were prepared and the resulting stable and metastable formulations were used for in vivo transfection. Complexes which contained 8 to 12 nmole of cationic lipid per g DNA were found to have the highest in vivo transfection activity. However, the transfection activity of these complexes decreased with time. Without modifying the procedures of forming the lipid:plasmid DNA complexes, there was a visible aggregation within a few days, and the transfection activity decreased by more than a thousand fold to almost background levels after one month's storage at 4 C. (FIG. 4 ). Therefore, formulation of stabilized lipid:plasmid DNA complexes was undertaken, which could maintain high in vivo transfection activity during storage.

Inserting PEG-PE (1% of total lipid) into the freshly formed lipid:plasmid DNA complexes not only could prevent the complexes from aggregating during storage, but the PEG-PE containing complexes also exhibited reasonably high transfection activity in vivo, only slightly lower activity as compared to the complexes without PEG-PE (FIG. 4 ). Incorporation of PEG-PE into the complexes is evident in view of the dose-related inhibition of the transfection activity with increasing percentage of PEG-PE (results not shown). Unexpectedly, storage of the complexes containing PEG-PE at 4 C. slowly restored the original activity, as shown in FIG. 4 . The mechanistic aspects of the inhibition effect on transfection by PEG-PE, as well as the recovery of the activity following storage at low temperature, are not known at present time.

In addition to the role of PEG-PE in increasing the shelf life of lipid:nucleic acid complexes, condensing nucleic acid with polyamines also gave a similar unexpected increase in shelf life of the complexes. The lipid:plasmid DNA complexes formed with condensed DNA were stable at a lower ratio of lipid to DNA without aggregation. FIG. 4 shows the level of in vivo transfection activity of such preparation, and its fate during storage. Again, an unexpected increase of the transfection activity was found in aged polyamine-treated lipid:plasmid DNA complexes, when compared to that of the samples which were not pretreated with polyamines and used immediately after complexes were formed. A different approach to obtain stable cationic lipid/DNA complexes by complexing plasmid with lipid in lipid-detergent micelles was published recently (Hofland et al., Proc. Natl. Acad. Sci. USA 93: 7305-7309 (1996)). However, only 30% of the transfection efficiency was maintained by such complexes in 15% serum for in vitro, and no in vivo results were reported.

Finally, conditions have been established for the stabilization of lipid:plasmid DNA complexes by lyophilization. Liposomes composed of DDAB/Chol suspended by sonication in 5% (w/v) of dextran in water, when mixed with DNA in 1:10 ratio ( g DNA per nmole DDAB) as described in methods, could be lyophilized without loss of activity. The final concentration of dextran in which lipid:plasmid DNA complexes were formed was 8% (w/v). The lyophilized preparations were reconstituted by adding distilled water and their transfection activity in the lungs of mice after i.v. injection was measured by luciferase reporter gene expression. Freezing and thawing the reconstituted preparation did not affect the activity (usually 1-2 ng luciferase protein per mg tissue protein).

Several of the cationic lipid:plasmid DNA complexes described herein are stable and can give consistent in vivo transfection activity (ranging from 0.5 to 2 ng luciferase per mg tissue protein) even after long storage at 4 C. or lyophilization. Formulations containing cholesterol as the helper lipid generate much higher in vivo transfection efficiency. Stabilizing the complex structure by PEG-PE maintains the complex activity in storage and may prolpng the circulation time in blood for targeting to specific tissues. Condensing the DNA with polyamines before lipid complexation enhances in vitro storage and levels of activity in vivo. The methodical approach for producing stable formulations of lipid:plasmid DNA complexes exhibiting high transfection activity in vivo confers advantages for establishing pharmaceutically acceptable preparations, and therefore facilitates liposome based gene therapy.

In Vitro Transfection of Lipid:Plasmid DNA Complexes with Targeting Ligands

A. Preparation of Fab fragments

Cloned rhuMAbHER2 sequences for heavy and light chain were co-expressed in E. coli as previously described (Carter et al., Biotechnology 10: 163-167 (1992)). The antibody fragment, rhuMAbHER2-Fab , was recovered from E. coli fermentation pastes by affinity chromatography with Streptococcal protein G (Carter et al., Biotechnology 10:163-167 (1992)), typically yielding Fab with 60-90% containing reduced free thiol (Fab -SH).

B. Preparation of liposomes

Condensed DNA was complexed with three different lipid compositions, using the methods described above in Example 1, with the following modifications. The first complex was made with DDAB/DOPE (1/1), which produced cationic liposomes complexed with DNA only, as described above. The second complex was made with DDAB/DOPE (1/1) with 1% PEG-PE derivatized with maleimide at the ultimate position of PEG, producing CLDC with the steric stabilization component added after complexation with the DNA. The third complex was made with DDAB/DOPE (1/1) with 1% PEG-PE derivatized with the Fab fragment of a humanized anti-Her-2 antibody attached to the ultimate position of PEG via the free thiol group to the maleimide residue. This produced CLDC with the targeting ligand attached to the steric stabilization component added after the complexation with the DNA.

C. Transfection and results

Cells were transfected as described above in Example 1, but without storage of the lipid:plasmid DNA complex. Two cell lines were used in this Example. The first cell line was MCF-7; cells of this cell line do not overexpress the HER-2 receptor. These cells were cultured in DME H-21 with 10% bovine calf serum and in 5% CO 2 . The second cell line was SK-BR3 cells, cells of which overexress the HER-2 receptor, cultured in McCoy's SA medium with bovine calf serum in 5% CO 2 . In both cases, the cells ( 5 10 4 cells per well) were transfected and incubated with 12 g plasmid DNA complexed with lipid as described above (PCMV/IVS-luc , luciferase reporter gene described above) for 4 hours at 37 C. The supernatant was then aspirated, fresh medium was added and the cells were incubated for 24 hours at 37 C. Cells were then harvested by washing with PBS (Ca/Mg free) and then suspended in lysis buffer for the luciferase assay, as described above.

FIG. 5A shows that transfection of non-target cells, not over-expressing the HER-2 receptor, was inhibited by the addition of PEG-PE, even in the presence of the targeting ligand conjugated at the tip of PEG via the terminal maleimide residue. FIG. 5B shows that transfection of target cells overexpressing the HER-2 receptor was also inhibited by the addition of PEG-PE, but the transfection activity was restored and augmented when the PEG-PE was conjugated to a targeting ligand, which recognizes the HER-2 receptor.

Comparison of FIGS. 5A and 5B indicates that the targeted immuno-CLDC were active in transfecting target cells much more efficiently than non-target cells. This result occurs because the addition of the ligand-carrying stabilizing agent (PEG-PE) conjugated to anti-HER-2-Fab ), which inhibits the transfection of non-target cells ( FIG. 5A ) but augments transfection of the target cells (FIG. 5 B).

Preparation of the Linker Maleimido-propionylantido-PEG2000-distearoylphosphatidylethanolamine (Mal-PEG-DSPE).

Conjugation of Mal-PEG-DSPE with Fab Fragment of an Antibody Reactive Against HER2 Oncoprotein.

300 nmol of Mal-PEG-DSPE in 0.5 ml of chloroform were placed in a glass test-tube and the solvent was removed in vacuum. The dry residue was dissolved in 1 ml of MES-20 buffer (20 mM morpholinoethane sulfonic acid, 144 mM sodium chloride, 2 mM ethylenediamine tetraacetic acid, and NAOH to pH 6.0). 2.5 ml of solution containing 0.57 mg/ml of Fab fragments of a recombinant humanized monoclonal antibody against extracellular domain of HER2 oncoprotein (rhuMAbHER2, Genentech, Inc.) was added to the Mal-PEG-DSPE solution, and the pH was carefully adjusted to 7.2-7.4 with diluted NaOH. The mixture was incubated under argon at room temperature for 2.5 hours, and the reaction was stopped by addition of 0.2 M cysteine hydrochloride to a final concentration of 5 mM. Fifteen minutes after the addition of cysteine, the reaction mixture was dialyzed against HEPES-buffered saline (20 mM hyrdoxyethylpiperazino ethanesulfonic acid, 144 mM NaCl, NaOH to pH 7.2), concentrated by ultrafiltration through a YM-10 membrane (Amicon) under pressure, and sterilized by filtration through a 0.2 m cellulose acetate filter. The reaction products were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), with Coomassie Blue staining. Total protein was determined the dye binding assay (Bio-Rad). The assay revealed 62% conversion of the original protein (M.w. 46,000) into slower-moving product (M.w. 49,000) consistent with the expected conjugate. Total protein recovery in the products was 98%.

Conjugation of Mal-PEG-9SPE with A Single Chain Fv Antibody Reactive Against HER2 Oncoprotein.

150 nmol of Mal-PEG-DSPE were dissolved in 0.5 ml of MES-20 and reacted with 0.5 ml of solution containing 0.7 mg/ml of single-chain Fv antibody C6.5Cys reactive against extracellular domain of HER2 oncoprotein. The antibody was prepared as described by Schier et al. ( Immunotechnology 1:73-81 (1995)). The reaction and products assay were conducted as described in the Example above. Total protein recovery was 86%. Approximately 52% of the recovered protein (M.w. 27,000) was in the form of a product with higher molecular weight (M.w. 29,000-30,000), consistent with the expected conjugate.

Preparation of Immunoliposomes with Conjugated Anti-HER2 Fab Fragments and Loaded with A Fluorescent Ph-sensitive Indicator

Small (100 mn) unilamellar liposomes containing entrapped pH-sensitive fluorescent indicator 8-hydroxypyrene trisulfonic acid were prepared from a mixture of 1-palmitoyl-2oleoyl-phosphatidylcholine (Avanti), cholesterol (Calbiochem), and methoxypolyoxyethyleneglycol (M.w. 1,900)-derivatized distearoyl phosphatidylethanolamine (Sygena) in the molar ratio of 30:20:3 as described by Kirpotin et al. ( Biochemistry, 36:66-75 (1997)), and sterilized by filtration through 0.2 m cellulose acetate filter. 0.26 ml of liposome preparation containing 2 mol of phospholipids was mixed with 0.106 ml of a solution containing 100 g of the anti-HER2 Fab -PEG-DSPE conjugate prepared according to Example 4, above, and incubated overnight at 37 C. Following incubation, the liposomes were separated from unbound material by gel-filtration on a column with Sepharose 4B (Pharmacia), using HEPES-buffered saline as eluent. The liposomes were eluted in the void volume of the column. The amount of liposome-bound protein was determined by the Bio-Rad dye binding assay, and the liposome concentration was measured by total phosphorus using molybdate method (Morrison, Anal. Biochem., 7:218-224 (1964). SDS-PAGE of the liposomes (see Example 13, below) revealed the presence of anti-HER2 Fab -PEG-DSPE conjugate, but no free anti-HER2 Fab in the liposome preparation. Liposome-associated protein was quantified by SDS-PAGE (see Example 13) and binding of the added Fab -PEG-DSPE conjugate with the liposomes was expressed as percentage of the output protein/phospholipid ratio over the input protein/phospholipid ratio. The binding of Fab -PEG-DSPE conjugate to the liposomes was 80%. The leakage of HPTS from the liposomes during incubation with the protein-PEG-DSPE conjugate to the liposomes was less than 2%.

Preparation of Immunoliposomes with Conjugated Anti-HER2 scFv antibodies and loaded with a fluorescent pH-sensitive indicator Using the procedure of Example 6, the conjugate of anti-HER2 single chain Fv C6.5Cys with Mal-PEG-DSPE, obtained according to Example 5, was incubated with HPTS-loaded liposomes at the input ratio of 15.6 g of protein per 1 mol of liposome phospholipid. After separation of unbound material by gel-filtration on Sepharose 4B, the liposomes were assayed as described in Example 6. The output protein/phospholipid ratio was 14.4 g/ mol, which indicated 92.3% binding of the conjugate to the liposomes.

Uptake of the Liposomes by HER2-Overexpressing Cells.

HER2-overexpressing human breast cancer cells (SK-BR-3) were grown in McCoy 5A medium supplemented with 10% fetal calf serum, 50 U/ml of penicillin, and 50 U/mi of streptomycin at 37 C. and 5% CO 2 . Twenty four hours prior to assay, the cells were harvested by treatment with 5 mM EDTA in phosphate buffered saline, and plated into 24-well cell culture plates at a density of 200,000 cells/well in 1 ml of cell culture medium. Liposomes were added to the cell culture medium in the wells (in triplicates) to achieve a final concentration of 25 M of liposome phospholipids. The plates were then incubated 4 hours with gentle agitation at 37 C. and 5% CO 2 . After incubation the media were aspirated from the wells, the cell layers were rinsed four times with 1 ml of phosphate buffered saline, harvested into 1 ml of 5 mM EDTA in phosphate buffered saline, and the amounts of cell-bound and endocytosed liposomes were determined by fluorometry as described in Kirpotin et al., Biochemistry, 36:66-75 (1997). For comparison, incubations were also performed with the liposomes conjugated to anti-HER2 Fab and scFv via Mal-PEG-DSPE linkers pre-included into the liposome composition (Kirpotin et al., Ibid). The results are summarized in the following table:

Total cell-associated liposomes, nmol Endocytosed Proteins per phospholipid/10 6 liposomes, Liposomes liposome cells % of total No antibody 0 0.0059 0.00036 0 anti-HER2 Fab , 34 0.744 0.086 86 7.8 conjugation to pre-incorporated linker anti-HER2 scFv, 37 0.311 0.025 59.3 4.3 conjugation to pre-incorporated linker anti-HER2 Fab , 43 1.304 0.054 95.9 3.2 according to Example 4 anti-HER2 scFv, 39 0.576 0.035 60.4 0.9 according to Example 5 As evidenced by these data, target cell binding and internalization of the liposomes prepared according to the present invention was at least equal, and often superior to, that of the similar liposomes prepared according to the best prior method.

Preparation Of Anti-HER2 Immunoliposomal Doxorubicin by Modification of Premanufactured Liposomal Doxorubicin with Anti HER2 Fab -PEG-DSPE Conjugate at 55 C.

0.38 ml of commercially available liposomal doxorubicin (Doxil , Sequus Pharmaceuticals, Inc.) containing 2 mg/ml of doxorubicin was mixed with 0.26 ml of the preparation of anti HER2 Fab -PEG-DSPE conjugate obtained according to Example 6, incubated at 55 C. for 20 min., and quickly cooled down in ice-water. Unbound material and low-molecular components were removed by gel-filtration of the incubation products through a column with Sepharose 4B (Pharmacia). The liposomes were collected in the void volume of the column, and assayed for protein using SDS-PAGE, for phospholipid using the molybate method, and for doxorubicin by spectrophotometry after solubilization in acidified isopropanol (E 1% 480 208). Found: approx. 45 Fab /liposome (77% binding of the added conjugate). The leakage of doxorubicin from liposomes was not observed (doxorubicin content prior to incubation, 145.9 g/ mol phospholipid; after incubation, 155.8 g/mol phospholipid).

Preparation of Anti-HER2 Immunoliposomal Doxorubicin by Modification of Premanufactured Liposomal Doxorubicin with Anti-HER2 scFv-PEG-DSPE Conjugate at 55 C.

The modification was performed as described in Example 9, using 0.4 ml of C6.5Cys-PEG-DSPE conjugate preparation (Example 5) and 0.31 ml of Doxil . Found: 48 proteins/liposome (quantitative binding of the conjugate to liposomes); drug leakage 3.7% (doxorubicin content prior to modification, 145.9 g/ mol phospholipid; after modification, 140.5 g/ mol phospholipid).

Preparation of Anti-HER2 Immunoliposomal Doxorubicin by Modification of Doxil with Anti-HER2 Fab -PEG-DSPE Conjugate at 37 C.

The modification was performed as described in Example 9 above, using 0.31 ml of Doxil and 0.212 ml of anti-HER2 Fab -PEG-DSPE preparation (Example 4), but the incubation was overnight at 37 C. Found: 46 Fab /liposome (82% binding of the added conjugate to liposomes); drug leakage was not observed (doxorubicin prior to modification 145.9 g/ mol phospholipid; after modification, 146.0 g/tmol phospholipid). Transition temperature of the lipid constituent of Doxil (hydrogenated soy phosphatidylcholine) is close to 55 C. Thus, modification is equally effective when the liposome lipids are in the gel state.

Preparation of anti-HER2 Immunoliposomal Doxorubicin by Modification of Doxil with anti-HER2 scFv-PEG-DSPE Conjugate at 37 C.

The modification was performed as described in Example 11, above, using 0.31 ml of Doxil and 0.4 ml of C6.5Cys-PEG-DSPE conjugate preparation (Example 5). Found: 49 proteins/liposome (quantitative binding of the conjugate to liposomes). Drug leakage was not detected (doxorubicin prior to modification 145.9 g/ mol phospholipid; after modification, 150.3.0 g/ mol phosholipid). Thus, modification of the liposomes with scFv-PEG-DSPE conjugate was equally effective when the liposome lipids were in the gel state.

Quantitation of Antibody Conjugate in the Liposomes and Conjugation Products Prepared According to Examples 6-12.

The amount of protein-PEG conjugate in the conjugation product and in the liposomes was assayed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE) under non-reducing conditions according to Laemmli (1974). Typically, 5-20 L aliquots of analytical sample were mixed with 6 sample buffer containing SDS and track dye (bromophenol blue), incubated 1 min. at 60 C., and applied onto a polyacrylamide gel (dimensions 10 10 075 cm) with a concentration of 10-12%, and cross-linker content of 2.6%. The separation was effected in a vertical slab gel electrophoresis apparatus at constant current of 30 mA. The protein bands were developed by Coomassie Blue staining using conventional methods. The conjugate formed a distinct band with lower electrophoretic mobility than the original protein. For quantitation of protein, the bands were excised, and the dye was extracted into 50% aqueous dimethylforrnamide at 100 C. for 30 min. The amount of extracted dye was quantified by spectrophotometry at 595 nm, and the protein amount per band was determined by comparison to a standard curve produced from the similarly processed bands of concomitantly run standard amounts of corresponding protein 9(Fab or scFv).

Delivery of Doxorubicin to HER2-Overexpressing Cancer Cells by anti-HER2 Immunoliposomes Prepared According to Examples 9-12.

HER2-overexpressing human breast cancer cells (SK-BR-3) were grown and plated as described in Example 8, above. Preparations of anti-HER2 immunoliposomal doxorubicin (Examples 9-12 above) were added to the cell culture medium in the wells (in triplicates) to achieve final 200 M concentration of liposome phospholipids (0.030 0.001 mg/ml of doxorubicin). The plates were then incubated 4 hours with gentle agitation at 37 C. and 5% CO 2 . After incubation the liquid was aspirated from the wells, the cell layers were rinsed 3 times with 1 ml each time of phosphate buffered saline, and the cells were harvested into 0.5 ml of 5 mM EDTA in phosphate buffered saline, pelleted by centrifugation, and extracted with 0.3N HCl/50% ethanol mixture. The amount of doxorubicin in ethanol-HCI extracts was determined by spectrofluorometry (excitation wavelength, 470 nm; emission wavelength 590 nm) and normalized to the quantity of plated cells. For comparison, incubations were performed also with the liposomes conjugated to anti-HER2 scFv (C6.5Cys) via Mal-PEG-DSPE linkers pre-incorporated into the liposome lipid matrix (Kirpotin et al., 1997). To assess the specificity of binding, in some wells the cells were preincubated with 5 g of the free anti-HER2 bivalent monoclonal antibody (anti-HER2MAb). The results are summarized in the following table:

Preparation of Lipid-DNA Complex Microparticles with Conjugated Antibody Fragments

A suspension of lipid-DNA microparticles (measuring 410 150 nm in size by dynamic laser scattering) composed of plasmid DNA (pCMA/IVS-Luc ; 10 g/mL), dimethyl dioctadecylammonium bromide (DDAB, 60 nmol/mL), and dioleoyl phosphatidylethanolarmine (DOPE, 60 mnol/mL) in 5% aqueous dextrose, was prepared as described by Hong et al. (FEBS Lett. 400:233-237, 1997). Fab -PEG-DSPE conjugate was prepared by co-incubation of Mal-PEG-DSPE and anti-HER2 antibody Fab fragments at a molar ratio of 4:1, at a concentration of the protein of 0.3 mg/mL in aqueous physiological buffer, at pH 7.2 for 2 hours. Lipid-DNA microparticles with conjugated anti-HER2 Fab fragments were prepared by incubation of the lipid-DNA microparticles with the conjugate in the amount of 0.5 mol. % relative to total particle lipid content for at least 30 min. at room temperature. Control particles with linker alone (non-targeted control) were prepared in the similar manner, but non-conjugated, -mercaptoethanol-quenched Mal-PEG-DSPE was substituted for the Fab -PEG-DSPE conjugate.

Targeted DNA Transfection of the Cells by Lipid-DNA Microparticles with Conjugated Antibody Fragments

Transfection activity of pCMV/IVS-Luc DNA-lipid microparticles prepared as in Example 15, above was studied in the cultures of human breast cancer cells: SK-BR-3 (overexpressing the target antigen, HER2 oncoprotein) and MCF-7 (the line with low expression of HER2). Expression of the reporter gene (luciferase) was determined by luminometry after 24-hour exposure of the cells to lipid-DNA complexes (1 g of DNA per 50-100,000 cells) in 10% serum-supplemented growth medium, and served as the measure of transfection efficiency. The detailed description of this experimental procedure is given in Hong et al., FEBS Lett. 400:233-237 (1997). Anti-HER2 Fab conjugated DNA-lipid microparticles prepared according to this invention were about 25-times more efficient for the plasmid delivery to target-positive SK-BR-3 cells than matching non-targeted particles. In the target-negative MCF-7 cells, targeted and nontargeted DNA-lipid particles had equal efficiency. Thus, antibody-modified lipid-DNA particles prepared according to the invention, are capable of target-specific delivery of functional DNA into human cancer cells.