The present invention is based on the discovery that proteins produced in insect cell cultures are glycosylated in a unique manner that causes them to be selectively imported by cells that express mannose receptors on their membranes, particularly macrophages. Proteins that are selectively imported into cells containing mannose receptors are provided, as well as pharmaceutical compositions containing such proteins and methods for producing such proteins. Application of the present invention to produce proteins useful for treating lysosomal storage disorders is also disclosed. Engineering of cells to express mannose receptors so that they will selectively import proteins produced in insect cells is also taught, as well as a protein targeting system using such cells and proteins. Finally, an improved elution buffer for the purification of proteins produced in insect cells from a Concanavalin A column is provided.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that proteins produced in insect cells by standard baculovirus expression systems are glycosylated in a unique way which makes them susceptible to uptake by macrophages via mannose receptors that are present on macrophage membranes but which are not normally present on the membranes of other cells. Accordingly, the invention provides a method for targeting a protein for uptake by macrophages by expression of the protein in insect cells, preferably using a baculovirus expression system.

Pharmaceutical compositions comprising baculovirus-expressed proteins as a component which are designed for delivery to macrophages are also provided by the present invention.

The therapeutic activity of proteins which may be used to treat lysosomal storage disorders is attributed primarily to the lysosomal activity of such proteins in macrophages. Therefore these proteins particularly benefit from the targeting achieved by the present invention. Such proteins include Cathepsin A/Protective Protein (PPCA), lysosomal neuraminidase, .beta.-galactosidase and all other proteins identified in Table 1.

The present invention also provides a method for causing a cell to import a protein produced in insect cells through expression of a mannose receptor on the membrane of the cell. This method may be achieved, for example, by engineering the cell to express a mannose receptor or by providing mannose receptor protein to the cell.

The present invention further provides a targeting system whereby a desired protein is targeted for delivery into a desired cell by expressing the protein in a baculovirus expression system and causing the cell to express a mannose receptor on its membrane.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered an unexpected advantage of using insect cells to produce proteins. As taught herein, proteins produced via expression in insect cells have a glycosylation pattern which renders them susceptible to selective uptake by cells which express mannose receptors on their membranes, such as macrophages. Accordingly, the present invention provides a simple method for producing a protein of interest in a form which will be selectively taken up by macrophages or other cells having mannose receptors on their membranes.

For purposes of the present invention, the method used to express a protein of interest in an insect cell is not critical; any method which results in expression of the protein of interest in an insect cell may be used, as long as such method does not disturb glycosylation of the protein by the insect cell. For example, an insect cell may be stably transformed with a gene encoding the protein of interest and propagated under conditions which cause the gene to be expressed. See, e.g., Express Insect.TM. Vector Set Non-Viral Insect Expression System, Invitrogen Cat. No. 12249017. The Express INsect Non-Viral Insect Expression System is designed for continuous expression of heterologous proteins in Lepidopteran insect cells. It is a plasmid-based, non-lytic system for stable expression. The system uses Gateway Cloning Technology to create the Expression Clone easily. Alternatively, traditional restriction endonuclease cloning can be used. More preferably, the protein of interest may be produced in an insect cell using a Baculovirus expression system. While any insect cell that is useful for foreign protein expression may be used, Spodoptera frugiperda or Tricoplusia ni cells are preferred, particularly the Spodoptera frugiperda cell lines designated Sf9 and Sf21.

Once a protein of interest is expressed in an insect cell, it may be purified for use in accordance with the present invention by conventional methods which preserve the glycosylation pattern of the expressed protein or enhance the occurrence of exposed mannose residues. A protein of interest produced from stably transformed insect cells may be purified using standard techniques such as ammonium sulfate precipitation and column chromatography (cation and anion exchange (resource S and Q columns); gel filtration (Sephacryl 200 high resolution) Amersham-Pharmacia Biotech). See, e.g. Calhoun et al., Proc. Natl. Acad. Sci. USA 82: 7364-7368 (1985). A protein of interest produced using a baculovirus expression system may be purified using these same techniques (see Examples 2 and 3). Preferably, the protein is purified on a Concanavalin A-Sepharose column ("Con-A column"; Cat. No. 17-0440-03, Amersham-Pharmacia Biotech). Rather than eluting the protein from the Con-A column with a standard elution buffer containing methyl-.alpha.-D-gluco-pyranoside, proteins produced by insect cells are eluted more efficiently with an elution buffer containing methyl-.alpha.-D-manno-pyranoside due to their glycosylation pattern. The use of a mannoside-containing buffer to elute proteins produced by insect cells from a Con-A column represents another aspect of the present invention.

As taught by the present invention, proteins produced via expression in insect cells exhibit a unique glycosylation pattern that includes exposed mannose residues. As a result, proteins expressed in insect cells are recognized by mannose receptors and taken up by cells which express mannose receptors on their membranes. This particularly includes macrophages, immature dendritic cells, certain endothelial cells, tracheal smooth muscle cells, retinal pigmental epithelium, kidney mesangial cells, Kaposis sarcoma cells, and cells engineered to express mannose receptors on their membranes.

In one aspect, the present invention provides a method for targeting a protein to macrophages by producing it in an insect cell. Proteins produced via insect cell expression have exposed mannose residues and are selectively imported into macrophages, which naturally express mannose receptors.

The present invention finds particular use in enzyme replacement therapy (ERT) for lysosomal storage disorders (LSDs). Macrophages and the reticulo endothelial system represent a prominent site of pathology for many LSDs. The present invention provides a way to target these sites of pathology via ERT using proteins produced in insect cells.

The effectiveness of treating LSDs by targeting macrophages and the reticulo endothelial system is supported by the study of an LSD in a transgenic mouse model. In this study, transgenic mice suffering from a deficiency in Protective protein/cathepsin A (PPCA) were transplanted with bone marrow from transgenic mice overexpressing PPCA exclusively in tissue macrophages and microglia. The treatment, which only recovered PPCA in macrophages and microglia, completely corrected systemic pathology in the transgenic mice. Hahn et al, Proc. Natl. Acad. Sci. 95: 14880-14885 (1998).

LSDs may be treated according to the present invention by producing the appropriate protein(s) in an insect cell culture and administering a therapeutically effective amount to the subject who is suffering from a deficiency of this protein. Thus, for example, a subject suffering from Galactosialidosis would be treated with protective protein/cathepsin A (PPCA), neuraminidase, and/or .beta.-Galactosidase produced in insect cells, a subject suffering from sialidosis would be treated with .alpha.-neuraminidase produced in insect cells, and a subject suffering from GM1-gangliosidosis would be treated with .beta.-galactosidase produced in insect cells. With respect to Galactosialidosis, treatment with both PPCA and neuraminidase is preferred over treatment with PPCA alone based on experimental results showing that neuraminidase activity in PPCA deficient macrophages treated with a mix of baculovirus-expressed neuraminidase and PPCA was ten-fold higher than in PPCA deficient macrophages treated with PPCA alone.

Pharmaceutical compositions comprising proteins produced via insect cell expression are also contemplated by the present invention. Such compositions will comprise a protein or an active fragment or derivative thereof which is therapeutically active in a cell which expresses mannose receptors, particularly a macrophage, in a therapeutically effective amount combined with a pharmaceutically acceptable carrier. The components of the pharmaceutical composition other than the therapeutically active protein will vary depending upon the properties of the therapeutically active protein, the route of administration, the desired form of the composition (e.g. tablet, capsule, pill, injectible, etc.), and other variables recognized by those of skill in the art.

Accordingly, the subject invention contemplates treating LSDs resulting from an enzyme deficiency by administering a pharmaceutical composition containing a pharmaceutically effective amount of the missing or deficient enzyme or an active fragment or derivative thereof. By active fragments is meant any part of the enzyme which is derived from the intact whole enzyme and still retains biological activity. Likewise, derivatives refer to enzymes which have been chemically modified or genetically engineered to effect minor changes, for example amino acid substitutions, which maintain biological activity.

The active ingredients of the pharmaceutical compositions comprising recombinant enzyme are contemplated to exhibit excellent and effective therapeutic activity in replacing the enzymatic deficiency found in the associated LSD. Thus, the active ingredients of the therapeutic compositions including recombinant enzyme exhibit enzymatic activity when administered in therapeutic amounts from about 0.1 ug to about 2000 ug per kg of body weight per day. The dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The active compound may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, intravenous, intranasal, intradermal, subcutaneous, or suppository routes. Depending on the route of administration, the active ingredients of a recombinant pharmaceutical composition may be required to be coated in a material to protect said ingredients from the action of enzymes, acids or other natural conditions.

The active proteins may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Coatings for enzyme preparations are useful to reduce degradation of the enzyme when administered as a therapeutic agent. Coatings also reduce the immunogenicity of the enzyme to help prevent undesirable side effects of administering such a therapeutic agent. A particularly useful coating to provide these characteristics is polyethylene glycol.

Sterile injectable solutions are prepared by incorporating the active enzymes in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

When recombinant protein is suitably protected as described above, the active compound may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparation should contain at least 1% of active enzyme. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage is obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral unit dosage form contains between about 10 ug and 1000 ug of active enzyme.

The tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum agragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, lactose or saccharin or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the unit dosage. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health impaired as herein disclosed in detail.

The principal active ingredient is compounded for convenient and effective administration in pharmaceutically effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 10 ug to about 1000 ug. Expressed in proportions, the active enzyme is generally present in from about 10 ug to about 1000 ug/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

In another aspect of the invention, an agent is targeted to macrophages or other cells having mannose receptors by conjugating the agent to a protein produced via expression in insect cells. Such an agent may comprise any substance which can be conjugated with a protein produced in an insect cell, including but not limited to a small molecule, a peptide, and a polypeptide, particularly an antibody.

In yet another aspect of the invention, a cell is rendered receptive to uptake of proteins produced via expression in insect cells by causing the cell to express mannose receptors on its membrane. One may cause the cell to express mannose receptors, for example, by engineering the cell to contain a gene capable of expressing a mannose receptor. Alternatively, the cell may be contacted with mannose receptor protein under conditions which induce incorporation of the mannose receptor protein into the membrane of the cell.
 

Claim 1 of 7 Claims

1. A method of treating a subject suffering from a lysosomal storage disorder other than Fabry Disease caused by a deficiency of a specific protein comprising: (a) producing said protein in an insect cell culture such that said protein will be selectively imported into macrophages when administered to said subject, and (b) administering a therapeutically effective amount of said protein to said subject.

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