HK1165725B - Polymer conjugates of interferon-beta with enhanced biological potency - Google Patents

Description

Polymer conjugates of interferon-beta with enhanced biological utility

The present application is a divisional application of an inventive patent application having an application date of 23/12/2003, application No. 200380109034.4, entitled "polymer conjugate of interferon- β having enhanced biological utility".

Background

Technical Field

The present invention belongs to the field of protein biochemistry and medicine. In particular, the present invention provides a method for producing conjugates by coupling a cytokine (e.g., interferon-beta) to a water-soluble polymer (e.g., poly (ethylene glycol) and derivatives thereof), the conjugates having increased potency compared to polymer conjugates of the same cytokine synthesized by standard synthetic methods. The invention also provides conjugates made according to the methods, compositions comprising the conjugates, kits comprising the conjugates and compositions, and methods of preventing, diagnosing, and treating various medical and veterinary conditions using the conjugates and compositions. The invention also provides methods for reductive alkylation under certain conditions to identify polymer attachment sites.

Prior Art

The following description of related art includes an explanation of the present disclosure which is not part of the prior art. A cytokine is a secreted regulatory protein that controls cell survival, growth, differentiation, and/or effector function in an endocrine, paracrine, or autocrine manner (see Nicola, N.A. (1994) in: Guideboost to Cytokines and Their Receptors, Nicola, N.A., ed., pp.1-7, Oxford University Press, New York). Cytokines have many potential therapeutic uses due to their potency, specificity, small size, and relative ease of production in recombinant organisms.

Cytokines and recombinant proteins have two major obstacles to development as therapeutics: its short half-life in the circulation and its potential antigenicity and immunogenicity. Herein and in the field, the term "antigenicity" means the ability of a molecule to bind to an existing antibody, while the term "immunogenicity" means the ability of a molecule to evoke an immune response in vivo, whether or not the response involves the formation of antibodies ("humoral response") or the stimulation of a cellular immune response.

In order to achieve maximum circulation activity and reduce problems with bioavailability and degradation when administering recombinant therapeutic proteins, intravenous (i.v.) administration is often used. However, the half-life of small proteins is usually very short after i.v. administration (see e.g. Mordenti, J., et al, (1991) Pharm Res 8: 1351-1469; Kuwabara, T., et al, (1995) Pharm Res 12: 1466-1469). In general, healthy kidneys retain blood with a hydrodynamic radius greater than that of serum albumin (i.e., Stokes radius is aboutAnd proteins having a molecular weight of about 66,000 daltons, 66 kDa. however, smaller proteins, including cytokines such as granulocyte colony stimulating factor ("G-CSF"), interleukin-2 ("interleukin 2"), interferon- α ("interferon- α") and interferon- γ ("interferon- γ"), are rapidly eliminated from the bloodstream by filtration through the glomeruli (Brenner, B.M., et al, (1978) Am J Physiol 234: F455-F460; Venkatachalam, M.A., et al, (1978) Circ Res 43: 337-agnhammar, p., et al, (1994) Blood 84: 4078-; wadhwa, M., et al, (1999) Clin Cancer Res 5: 1353-; hjelm Skog, a. -l., et al, (2001) Clin Cancer Res 7: 1163-1170; li, j, et al, (2001) Blood 98: 3241-3248; baser, r.l., et al, (2002) Blood 99: 2599 and 2602; schellekens, h., (2002) Clin Ther 24: 1720-1740).

There have been many studies focused on modifying recombinant proteins by covalent attachment of poly (ethylene glycol) ("PEG") to ameliorate the above disadvantages (evaluated by Sherman, M.R., et al, (1997) in: Poly (ethylene glycol): Chemistry and Biological Applications, Harris, J.M., et al, eds., pp.155-169, American Chemical Society, Washington, D.C.; Roberts, M.J., et al, (2002) Adv DrugDeliv Rev 54: 459-476). Attachment of PEG to proteins has been shown to stabilize proteins in vivo, improve their bioavailability and/or reduce their immunogenicity. (PEG is covalently attached to a protein or other substrate, referred to herein and in the art as "PEGylation") in addition, PEGylation can significantly increase the hydrodynamic radius of the protein. When a small protein, such as a cytokine, is coupled to a single long chain PEG (e.g., having a molecular weight of at least about 18kDa), the resulting conjugate has a hydrodynamic radius greater than serum albumin, which is significantly slowed down by clearance from the circulatory system via the glomeruli. Combined effects of pegylation: reduced proteolysis, reduced immune recognition, and reduced renal clearance rates all confer substantial advantages to pegylated proteins as therapeutics.

Attempts have been made since the 1970 s to improve the safety and efficacy of various pharmaceutical proteins using covalently attached polymers (see, e.g., Davis, f.f., et al, U.S. patent No.4,179,337). Some examples include: coupling PEG or poly (ethylene oxide) ("PEO") to adenosine deaminase (EC 3.5.4.4) for the treatment of severe combined immunodeficiency disease (Davis, S., et al, (1981) ClinExp Immunol 46: 649-; coupled to superoxide dismutase (EC 1.15.1.1) to treat inflammatory conditions (Saifer, M., et al, U.S. Pat. Nos. 5,006,333 and 5,080,891) and to urate oxidase (EC1.7.3.3) to remove excess uric acid from blood and urine (Kelly, S.J., et al, (2001) J Am Soc Nephrol 12: 1001-1009; Williams, L.D., et al, PCT publication No. WO 00/07629A 3 and U.S. Pat. No.6,576,235; Sherman, M.R., et al, PCT publication No. WO 01/59078A 2).

PEOs and PEGs are polymers composed of covalently linked ethylene oxide units. Such polymers have the following general structure:

R₁-(OCH₂CH₂)_n-R₂

wherein R is₂May be hydroxy (or a reactive derivative thereof), and R₁May be hydrogen (as in dihydroxypeg ("PEG diol"), methyl (as in monomethoxypeg ("mPEG"), or other lower alkyl groups, such as in isopropoxy PEG or tert-butoxy PEG. The parameter n of the general formula of PEG is the number of ethylene oxide units in the polymer, referred to herein and in the art as the "degree of polymerization". Polymers of the same general formula (wherein R₁Is C_1-7Alkyl groups), also known as oxirane derivatives (Ya sukohchi, t., et al, U.S. Pat. No.6,455,639). PEGs and PEOs can be linear, branched (Fuke, I., et al (1994) JControl Release 30: 27-34) or star-shaped (Merrill, E.W, (1993) J Biomate Sci Polym Ed 5: 1-11). PEGs and PEOs are amphoteric compounds, i.e., they are soluble in water and certain organic solvents, and they adhere to lipid-containing materials, including the cell membranes of enveloped viruses and animal and bacterial cells. Certain Oxiranes (OCH)₂CH₂) And random or block or alternating copolymers of propylene oxide having the structure:

such copolymers have properties very similar to PEG, and may be suitable replacements for PEG in certain applications (see, e.g., Hiratani, h., U.S. patent No.4,609,546 and Saifer, m., et al, U.S. patent No.5,283,317). The terms "polyalkylene oxide" and the abbreviation "PAOs" herein mean such copolymers, PEG or PEO and poly (oxyethylene-oxymethylene) copolymers (Pitt, c.g., et al, U.S. patent No.5,476,653). The terms "polyalkylene glycol" and the abbreviation "PAGs" are used herein to refer generally to polymers suitable for use in the conjugates of the invention, and specifically to PEGs that contain a single reactive group ("monofunctional activated PEGs").

Covalent attachment of PEG or other polyalkylene oxides to proteins requires conversion of at least one terminal group in the polymer to a reactive functional group. This process is commonly referred to as "activation" and the product is then referred to as "activated PEG" or activated polyalkylene oxide. The monomethoxy PEGs most commonly used in this method are capped (resulting in a "methoxy") with a non-reactive, chemically stable methyl group for the oxygen on one end and a functional group reactive with an amino group on the protein molecule on the other end. Less frequently so-called "branched" mPEGs are used, which contain two or more methoxy groups distal to a single activated functional group. An example of a branched PEG is di-mPEG-lysine, in which PEG is coupled to two amino groups, and the carboxyl group of lysine is mostly activated by esterification with N-hydroxysuccinimide (Martinez, A., et al, U.S. Pat. No.5,643,575; Greenwald, R.B., et al, U.S. Pat. No.5,919,455; Harris, J.M., et al, U.S. Pat. No.5,932,462).

Typically the activated polymer can be reacted with a biologically active compound having nucleophilic functional groups (as attachment sites). One nucleophilic functional group that is typically the attachment site is the amino group of a lysine residue. Solvent accessible alpha-amino groups, carboxylic acid groups, guanidine groups, imidazole groups, suitably activated carbonyl groups, oxidized carbohydrate moieties and thiol groups may also serve as attachment sites.

Prior to attachment to the protein, the hydroxyl groups of PEG have been activated with cyanuric acid chloride (Abuchowski, A., et al, (1977) J Biol Chem 252: 3582-3586; Abuchowski, A., et al, (1981) Cancer Treat Rep 65: 1077-1081). However, there are disadvantages to this approach, such as the toxicity of cyanuric chloride and its non-specific reactivity towards proteins with non-ammoniacal functional groups, such as cysteine or tyrosine residues that are accessible to some solvents important for its function. To overcome these and other disadvantages, additional activated PEGs may be introduced, such as succinimidyl succinate derivatives of PEG ("SS-PEG") (Abuchowski, A., et al, (1984) Cancer biochem Biophys 7: 175-186), succinimidyl carbonate derivatives of PAG ("SC-PAG") (Saifer, M., et al, U.S. Pat. No.5,006,333), and aldehyde derivatives of PEG (Royer, G.P., U.S. Pat. No.4,002,531).

Typically, a few chains (e.g., 5 to 10) of one or more PAGs, such as one or more PEGs (molecular weight about 5kDa to about 10kDa) can be coupled to a target protein via primary amino groups (the amino group of a lysine residue and the alpha amino group of the N-terminal amino acid). More recently, conjugates have been synthesized containing higher molecular weight (e.g., 12kDa, 20kDa or 30kDa) single-chain mPEG. There is a direct correlation between the half-life of conjugate plasma and increased molecular weight and/or increased chain number of coupled PEGs (Knauf, M.J., et al, supra; Katre, N.V. (1990) J lmmunol 144: 209-; Clark, R., et al, (1996) JBiolChem 271: 21969-. On the other hand, increasing the number of chains of PEG coupled to each protein molecule increases the chance of modifying the amino groups of the essential regions of the protein (especially the receptor-binding protein) and thus impairing the biological function of the protein. For larger proteins containing many amino groups, and for enzymes with low molecular weight substrates, increasing the reaction time while decreasing the specific activity is acceptable because it results in a net increase in the biological activity of the PEG-containing conjugate in vivo. However, for some smaller proteins that function by interacting with cell surface receptors (e.g., cytokines), studies have reported that higher degrees of substitution reduce functional activity and even the advantage of longer blood half-lives are not sufficiently offset (Clark, R., et al, supra).

Thus, in order to prolong the biological activity and reduce immunogenicity of therapeutic proteins (e.g., enzymes), polymer conjugation techniques have been well established (see, e.g., U.S. provisional application No. 60/436,020, application date 2002, 26/12, and U.S. provisional application nos. 60/479,913 and 60/479,914, both of which have application dates 2003, 6/20, incorporated herein by reference in their entirety). A class of therapeutic proteins that would particularly benefit from reduced immunogenicity is interferon beta, especially interferon beta-1 b ("IFN-. beta. -1 b; SEQ ID NO: 1) (The IFNB Multiple Sclerosis study Group (1996) Neurology 47: 889-894). However, conjugation of polymers to regulatory proteins that act through specific binding of cell surface receptors will typically: (1) interfering with the binding; (2) significantly reducing the signaling efficacy of cytokine agonists; and (3) significantly diminishing the competitive efficacy of cytokine antagonists. Examples of published conjugates of reduced receptor-binding activity include granulocyte colony stimulating factor ("G-CSF") (Kinstler, 0., et al, PCT publication No. WO 96/11953; Bowen, S., et al, supra); human growth hormone ("hGH") (Clark, R., et al, supra); hGH antagonists (Ross, R.J.M., et al, (2001) J Clin Endocrinol Metab 86: 1716-1723; and interferon- α (Bailon, P., et al, (2001) bioconjugate Chem 12: 195-202; Wylie, D.C., et al, (2001) Pharm Res 18: 1354-1360; and Wang, Y. -S., et al, (2002) Adv Drug Deliv 54: 547-570) and the like, in the extreme case of polymer conjugates coupled with interleukin-15 ("IL-15"), which convert the IL-2 type growth factor into a cell proliferation inhibitor (Pettit, D.K., et al, (1997) J Biol Chem 272: 2-8), without being bound by theory, the mechanism of this PEGylated undesirable effect may involve steric hindrance in the interaction of the receptor groups and the PEG 2316, or both.

Thus, there is a need for methods of producing conjugates of PAGs (e.g., PEG and/or PEO) inclusive, particularly conjugates between water-soluble polymers and receptor binding proteins, that retain substantial biological activity (e.g., at least about 40%), nearly complete biological activity (e.g., at least about 80%), or substantially complete biological activity (e.g., at least about 90%). The introduction of conjugates to animals for prophylactic, therapeutic or diagnostic purposes will provide increased solubility, stability and bioavailability of the polymeric component in vivo and greatly enhance potency or utility compared to conventional polymer conjugates.

Summary of The Invention

The present invention meets the above needs and provides methods for preparing conjugates of water-soluble polymers, such as poly (ethylene glycol) and derivatives thereof, with biologically active ingredients, particularly receptor-binding proteins, particularly therapeutically or diagnostically biologically active ingredients, such as cytokines, including interferon beta, most particularly interferon-beta-1 b. The invention also provides conjugates produced by the method. The conjugates of the invention may have increased stability (i.e., longer shelf life and longer half-life in vivo) compared to the corresponding unconjugated bioactive component. Furthermore, the conjugates of the invention can increase receptor-binding activity, which can be measured or used in vitro, and increase potency, which can be measured in vitro or in vivo, compared to conjugates having the same bioactive component and the polymer randomly attached to a solvent accessible site on the backbone of the polypeptide bond. In addition, the invention also provides compositions comprising the conjugates, kits containing the conjugates and compositions, and methods of using the conjugates and compositions in various therapeutic and diagnostic protocols.

In one embodiment, the invention provides a method of increasing cytokine potency. Some methods according to this aspect of the invention include, for example: selectively coupling one or more synthetic water-soluble polymers to an amino-terminal amino acid of the cytokine, wherein the amino-terminal amino acid is distal to one or more receptor-binding domains of the cytokine. The present invention provides a method for enhancing the potency of cytokines, comprising: for example, one or more synthetic water-soluble polymers are selectively conjugated at or near one or more cytokine glycosylation sites, wherein the one or more glycosylation sites are distal to one or more receptor-binding domains of the cytokine.

Suitable polymers for use in such methods of the invention include, but are not limited to: one or more polyalkylene glycols (including, but not limited to, one or more poly (ethylene glycols), one or more monomethoxy-poly (ethylene glycols) and one or more monohydroxypoly (ethylene glycols)), one or more polyalkylene oxides, one or more polyethylene oxides, one or more polyalkene alcohols such as polyvinyl alcohol, one or more polycarboxylates, one or more poly (vinylpyrrolidone), one or more poly (oxyethylene-oxymethylene) s, one or more poly (amino acids), one or more polyacryl-morpholines, one or more copolymers of one or more amides and one or more alkylene oxides, one or more dextrans and one or more hyaluronic acids. Polymers suitable for use in the methods of the invention typically have a molecular weight between about 1kDa and about 100kDa, inclusive, or more specifically between about 8kDa and about 14kDa, inclusive; a molecular weight between about 10kDa and about 30kDa, inclusive; a molecular weight between about 18kDa and about 22kDa, inclusive; or about 20kDa or about 30 kDa.

Various cytokines and analogs that mimic or antagonize the biological effects of the corresponding cytokine mediated by specific cell-surface receptors are suitable for use in preparing the present conjugates. Such compounds include cytokines having four helical bundle structures (including, but not limited to, granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), Leukemia Inhibitory Factor (LIF), erythropoietin (Epo), thrombopoietin (Tpo), Stem Cell Factor (SCF), Flt3 ligand, oncostatin M (OSM), interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12(p35 subunit), IL-13, IL-15, IL-17, interferon-alpha (IFN-alpha), Interferon beta (IFN- β) (particularly IFN- β -1b), interferon consensus sequences and muteins, variants, analogs and derivatives thereof, and cytokines having a β -sheet or β -barrel structure (including, but not limited to: tumor necrosis factor alpha (TNF-alpha), IL-1 alpha, IL-1 beta, IL-12 (subunit p 40), IL-16, Epidermal Growth Factor (EGF), insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), acidic FGF, FGF-4, and keratinocyte growth factor (KGF; FGF-7), as well as muteins, variants, analogs and derivatives thereof.

Particularly preferred cytokines suitable for use in the present invention include interleukin 2; interferon- α; interferon-beta; IGF-1; EGF and bFGF. Also particularly suitable for use are competitive antagonists of the aforementioned cytokines with muteins, variants and derivatives of such cytokines.

In certain embodiments, the one or more polymers are alpha amino groups covalently coupled (particularly via a secondary amine linkage) to the amino-terminal amino acid of the cytokine. In other embodiments, the one or more polymers are chemically reactive side chain groups (e.g., hydroxyl, sulfhydryl, guanidino, imidazolyl, amino, carboxyl, or aldehyde derivatives) covalently coupled to an amino-terminal amino acid on the cytokine. In other embodiments, coupling of the polymer to the cytokine at the amino-terminal amino acid of the cytokine or at or near one or more glycosylation sites may mimic the beneficial effects of glycosylation. In related embodiments, coupling of a polymer to a cytokine at or near one or more glycosylation sites of the cytokine may mimic the beneficial effects of hyperglycosylation, where "hyperglycosylation" means the covalent attachment of simple or complex carbohydrate moieties other than those naturally occurring structures.

The invention also provides conjugates produced by the methods of the invention. The conjugates of the invention comprise a selected cytokine or a selected antagonist thereof (e.g., as described above) coupled to one or more synthetic water-soluble polymers (e.g., as described above), wherein the one or more polymers are coupled to the amino-terminal amino acid of the cytokine and wherein the amino-terminal amino acid is distal to one or more receptor-binding domains of the selected cytokine. In addition, the conjugates of the invention comprise a selected cytokine or a selected antagonist thereof (e.g., as described above) coupled to one or more synthetic water-soluble polymers (e.g., as described above), wherein the one or more polymers are coupled to one or more glycosylation sites of the selected cytokine and wherein the one or more glycosylation sites are located distal to one or more receptor-binding domains of the cytokine. When the polymer is conjugated to an agonist of the invention, it is preferred that the polymer attachment site is distal to all of the receptor-binding domains. When polymers are conjugated to certain antagonists of the invention, it is preferred that the polymer attachment site be distal to some of the receptor binding domains necessary for binding, but not necessarily distal to all of the receptor-binding domains necessary for signaling by the agonist. The invention also provides compositions, particularly pharmaceutical compositions, comprising one or more conjugates of the invention and one or more other ingredients, such as one or more pharmaceutically acceptable diluents, excipients or carriers. The invention also provides kits comprising one or more conjugates, compositions, and/or pharmaceutical compositions of the invention.

The invention also provides methods of preventing, diagnosing, or treating a physical disorder in an animal (e.g., a mammal, such as a human) suffering from or susceptible to the physical disorder. The method comprises, for example: an effective amount of one or more of the conjugates, compositions, or pharmaceutical compositions of the invention is administered to an animal. Physical conditions that may be suitably treated or prevented according to the methods of the present invention include, but are not limited to: cancers (e.g., breast, uterine, ovarian, prostate, testicular, lung, leukemia, lymphoma, colon, gastrointestinal, pancreatic, bladder, kidney, bone, neural, head and neck, skin, sarcoma, carcinoma, adenocarcinoma, and bone marrow); infectious diseases (e.g., bacterial diseases, fungal diseases, parasitic and viral diseases (e.g., viral hepatitis, diseases caused by cardiotropic virus, HIV/AIDS, and the like), and genetic diseases (e.g., anemia, neutropenia, thrombocytopenia, hemophilia, dwarfism, and severe combined immunodeficiency ("SCID")), autoimmune disorders (e.g., psoriasis, systemic lupus erythematosus, and rheumatoid arthritis), and neurodegenerative diseases (e.g., multiple sclerosis ("MS") in various forms and stages, such as relapsing-remitting MS, primary progressive MS, and secondary progressive MS; Creutzldfet-Jakob Disease; Alzheimer's Disease; and the like).

In related embodiments, the invention also provides methods for determining the amount of polymer attached to the amino terminus of a protein having an N-terminal serine residue in a reductively alkylated synthetic polymer-protein conjugate. Methods according to this aspect of the invention include, for example: (a) reacting the conjugate with a sufficient amount of an oxidizing agent for a sufficient time to cleave the polymer from the serine residue of the protein; and (b) measuring an increase in the proportion of unconjugated protein in the preparation. Proteins suitable for use according to the method include, but are not limited to: cytokines including interferon beta (especially interferon beta-1 b, preferably having the amino acid sequence of SEQ ID NO: 1) and megakaryocyte growth and development factor (megakaryocyte growth and development factor). Oxidizing agents useful in certain processes of the invention are periodates, including but not limited to: sodium metaperiodate, potassium metaperiodate, lithium metaperiodate, calcium periodate, barium periodate, and periodic acid. Suitable methods for measuring the increase in the proportion of unconjugated protein in the preparation include any protein and peptide analysis method known in the art, including, for example: size exclusion chromatography, reverse phase chromatography, colloidal electrophoresis, capillary electrophoresis, ultracentrifugation, ultrafiltration, light scattering, and mass spectrometry.

In other related embodiments, the invention provides methods for selectively oxidatively cleaving an N-terminal serine residue of a biologically active protein without oxidizing an amino acid residue essential for the function of the biologically active protein. Some of the methods of the invention comprise, for example: (a) adjusting the hydrogen ion concentration of the biologically active protein solution to a pH of from about 5 to about 10, more preferably to a pH of from about 7 to about 8; (b) mixing a solution of a biologically active protein with periodate (from about 0.1 moles to about 10 moles periodate, or more preferably from about 0.5 moles to about 5 moles periodate per mole of biologically active protein); and (c) reacting the mixture for at least one hour, preferably at a temperature of between about 2 ℃ and about 40 ℃. Proteins suitable for use according to the method include, but are not limited to: cytokines (including interferon beta (especially interferon beta-1 b, which preferably has the amino acid sequence of SEQ ID NO: 1).

In other embodiments, the present invention provides methods of increasing the bioavailability of a prepared interferon beta, and in particular of a prepared interferon-beta-1 b, comprising removing one or more inhibitory components of an interferon beta (or interferon-beta-1 b) preparation. In accordance with this aspect of the invention, one or more inhibitory components may be removed from the preparation by a variety of art-known protein and peptide processing, purification and/or analytical methods including, but not limited to, one or more chromatographic methods, such as size-exclusion chromatography, reverse-phase chromatography, hydrophobic interaction chromatography and affinity chromatography. Determination of the biological potency (i.e., whether the potency is increased, decreased, or unaffected relative to the interferon beta stock solution) of a given interferon beta preparation may be accomplished by any in vitro or in vivo assay well known to those skilled in the art. For example, cell culture assays that respond to interferon beta can be used to determine the biological effectiveness of interferon beta preparations. Non-limiting examples of suitable such cell culture assays include antiproliferative assays, antiviral assays, signaling assays, and gene activation assays, examples of which are well known to those of ordinary skill in the art.

Other preferred embodiments of the present invention will become apparent to those skilled in the art upon review of the following drawings and description of the invention and the appended claims.

Brief Description of Drawings

FIGS. 1 to 8 show the molecular patterns of various cytokines and growth factors created by RasMol software based on crystallographic data (Sayle, R.A., et al, (1995) Trends Biochem Sci 20: 374-376). Except for certain specific residues (in "ball-and-stick" format), each pattern is expressed in "band" or "cartoon (carton)" format. Such formats were selected using RasMol software. The black band portion represents the functional domain of cytokines and growth factors, which is reported to be associated with the binding of their receptors. In each structure, the coding for a protein database ("PDB") is indicated (see Laskowski, R.A., (2001) Nucleic Acids Res 29: 221-.

FIG. 1a shows a model of interferon- α -2a, in which four lysine residues (Lys 31, Lys 121, Lys131, and Lys 134) are reported as the Roche's PEG-interferon productThe primary sites of pegylation of (a) are shown in the format of "sphere-and-rod" (based on Bailon, p., et al, supra). Regions involved in binding to the receptor ("binding sites 1 and 2") were also identified. All four lysine residues PEGylated in Pegasys are located in the region of binding site 1. (PDB code 1ITF)

FIG. 1b shows a model of interferon- α -2b, wherein Schering-Plough' sThe residues of the pegylated primary sites of (His 34, Lys 31, Lys 121, Tyr 129, and Lys131) are shown in a "ball-and-rod" format (based on Wylie, d.c., et al, supra). Such amino acid residues are located in the region of binding site 1.

FIG. 1c shows a model of interferon- α -2b, in which the amino-terminal cysteine residue ("Cys 1"), according to the invention, is targeted for PEGylation, shown in the format of "ball-and-stick". Cys 1 is remote from binding sites 1 and 2.

FIG. 1d shows the same model as for interferon- α -2b of FIG. 1c, in which a single-chain 20-kDa PEG is attached to the N-terminal cysteine residue ("Cys 1"). The PEG structure was determined using Lee, l.s., et al, (1999) Bioconjug Chem 10: the procedure described in 973-981 was modified to produce and have the same ratio as the protein.

FIG. 2 shows a molecular model of human interferon beta-1 a (see SEQ ID NO: 2), wherein several lysine residues (Lys 19, Lys 33, Lys 99 and Lys 134) are also indicated to be located within or adjacent to the receptor-binding domain. Furthermore, the glycosylation site (Asn 80) as well as the N-terminal methionine residue ("Met I") are shown in the format of "ball-and-rod" (based on the data of Karpusas, M., et al, (1997) Proc Natl Acad Sci USA 94: 11813-11818; Karpusas, M., et al, (1998) Cell Mol Life Sci 54: 1203-1216; Runkel, L., et al, (2000) Biochemistry 39: 2538-2551). Met 1 is remote from the binding sites 1 and 2, while several lysine residues are located within the receptor-binding domain. (PDB, accession number 1AUI) Interferon-beta-1 b (see SEQ ID NO: 1) differs from Interferon-beta-1 a in that it lacks the N-terminal methionine residue and the carbohydrate moiety, but has a serine residue (Cys 17 of SEQ ID NO: 2) substituted for the unpaired cysteine residue.

FIG. 3 shows a molecular model of human granulocyte-macrophage colony-stimulating factor ("GM-CSF"), in which three lysine residues (Lys 72, Lys107, and Lys 111) located within the receptor-binding domain, as well as the first amino acid residue ("Arg 4") near the amino-terminus that is visually accessible in the crystal structure, are shown in a "ball-and-rod" format (based on data from Rozwarski, D.A., et al, (1996) Proteins 26: 304-313). The amino-terminal region of GM-CSF is located distally to binding sites 1 and 2. (PDB code 2GMF)

FIG. 4 shows a molecular model of human interleukin-2, in which amino acid residues involved in each of the three receptors (α, β, and γ) are expressed in the format of "ball-and-rod", and several lysine residues within or near the receptor-binding domain are also expressed in the format of "ball-and-rod". The amino-terminal most proximal amino acid residue in the crystal Structure that is visualized is serine 6 ("Ser 6"), which is located distal to the receptor-binding domain (based on Bamborough, P., et al, (1994) Structure 2: 839-851; Pettit, D.K., et al, supra). (PDB No. 3I NK)

FIG. 5 shows a "cartoon" version of the molecular model of human epidermal growth factor ("EGF"), except for residues involved in receptor binding and two lysine residues (Lys 28 and Lys 48) adjacent to the receptor-binding region. The disulfide bonds within the chain are shown by dashed lines. The model was established based on the crystal structure in which the amino acid residue closest to the amino terminus that can be visualized is cysteine 6 ("Cys 6") (based on the data of Carpenter, G., et al, (1990) J Biol Chem 265: 7709-. The non-visible amino terminal soft part of EGF (residues 1-5) within the crystal structure does not appear to be located in the receptor-binding region. (PDB No. 1JL9)

FIG. 6 shows a "cartoon" format of a basic fibroblast growth factor ("bFGF") molecular model in which residues involved in binding to the receptor and heparin are presented in a "ball-and-rod" format (based on Schlesinger, J., et al, (2000) Mol Cell 6: 743-750 data). The first 12 amino acid residues from the amino terminus are not involved in receptor binding. (PDB No. 1FQ9)

FIG. 7 shows a "cartoon" version of the molecular model for insulin-like growth factor-1 ("IGF-1"), except for the residues involved in receptor binding (23-25 and 28-37), and glutamic acid residue 3 ("Glu 3"), which is the amino-terminal amino acid residue that is visually closest to the crystal structure. Two lysine residues were identified, one adjacent to the receptor-binding domain (Lys 27) and the other distant from the receptor-binding domain (based on the data of Brzowski, A.M., et al, (2002) Biochemistry 41: 9389-. The IGF-1 amino terminus is distal to the receptor-binding domain. (PDB No. 1GZR)

FIG. 8 shows a molecular model of interferon-gamma ("interferon-gamma"), which is a homodimer. To illustrate the interaction of the dimeric peptide bonds, one monomer ("chain A") is shown in the format of a "band" and the other is shown in the format of a "backbone" ("chain B"). Lysine residues are present along polypeptide bonds (shown in the format of light colored "spheres and rods"), including regions involved in the interface between monomers or regions adjacent to amino acid residues involved in receptor binding. The amino-terminal region of interferon-gamma is far from the interface of dimerization, but glutamine 1(Gln 1) is involved in receptor binding. (Thiel, D.J., et al, (2000) Structure 8: 927-936; PDB codes IFG9)

FIG. 9 shows unpegylated interferon- α -2b ("IFN"), monopegylated interferon- α -2b ("PEG") by cation exchange chromatography of a reaction mixture containing interferon, 20-kDa mPEG-aldehyde, and a reducing agent₁-IFN ") and dimeric pegylated interferon α -2b (" PEG₂-IFN ") fraction.

FIG. 10 is a size exclusion chromatographic analysis of the reaction mixture separated as shown in FIG. 9 and a selected fraction collected from the ion-exchange column the results of which are shown in FIG. 9.

FIG. 11 shows cation exchange chromatography fractionation of a reaction mixture containing human interleukin 2, 20-kDa mPEG-aldehyde, and a reducing agent. Under the specified elution conditions, the non-pegylated IL-2 residue did not elute from the column, unlike interferon- α -2b shown in FIG. 9.

Figure 12 shows size exclusion chromatographic analysis of the reaction mixture separated as shown in figure 11 and the selected fractions eluted through the column.

FIG. 13 shows an electrophoretic analysis of a pegylated interleukin-2 ("PEG-I L-2") reaction mixture and its chromatogram presented in the cation-exchange column chromatography fraction of FIG. 11.

FIG. 14 depicts the reaction of 20kDa mPEG aldehyde with sodium cyanoborohydride (NaBH) at various concentrations ("1 x", "2 x", or "4 x")₃CN) as reducing agent, size exclusion HPLC resolved interferon- β -1b ("IFN") in the conjugate₁-IFN ") or a two-chain (" PEG)₂-IFN ") can be resolved from PEG-unconjugated interferon (" Mock pegylated IFN ").

FIG. 15 shows that sodium periodate oxidatively cleaves about 90% of PEG synthesized by reductive alkylation under various conditions₁Interferon- β sodium dodecyl sulfate ("SDS") resolves residual PEG from cleavage products including formaldehyde and interferon that decomposes from N-terminal serine to Aldehyde ("IFN Aldehyde") by size exclusion HPLC in the presence of size exclusion HPLC₁-interferon- β.

FIG. 16 depicts reverse phase chromatography of PEG from Mock PEGylated interferon- β, unbound PEG, unbound SDS, and minor components of the reaction mixture₁-interferon- β.

FIG. 17 depicts PEG-enriched reverse phase ("RP") chromatography of PEGylation reaction mixtures and preparative RP columns₁Results for interferon- β (fraction 51) or the control pegylated interferon- β (fraction 53) fraction.

FIG. 18 depicts PEG-enriched PEG reaction mixtures and preparative RP column fractions₁Results of electrophoretic analysis of interferon- β (fraction 51) or PEG conjugates containing more than one chain (fraction 49) gels were stained with fluorescent dyes and photographed under UV illumination.

FIG. 19 depicts the results of electrophoretic analysis of the sample of FIG. 18, except that the gel contains BaCl₂、I₂And KI reagent to stain PEG. The staining intensity of the gel photograph was measured as in fig. 18. An absorption peak of residual free 20-kDa PEG was detected in the reaction mixture.

FIG. 20 depicts reverse phase chromatography of interferon- β -1b samples, untreated (top curve) or subjected to NaIO at a concentration of 0.5mM₄Reaction by which the main and minor N-terminal serine residues are decomposed to give aldehyde derivatives (middle curve), or by NaIO₄Oxidized and reacted with 9-fluorenylmethylcarbazate ("Fmoc-carbazate"). Minor components ("absorption peak a") contain oxidized methionine residues. The similar increase in the retention time of absorption peak a and the major components after oxidation reflects the cleavage of the N-terminal serine residue of each absorption peak to aldehyde. Under such conditions with NaIO₄No increase in the percentage of absorption peak a was detected after the reaction. Increase of retention time and absorbance after reaction with Fmoc-carbazate indicated formation of Fmoc conjugate from the oxidized form of absorption peak a as well as the major component.

FIG. 21 shows PEG synthesized by reaction of 20-kDa PEG-carbazate and interferon- β aldehyde derivative₁Interferon- β protein and NaIO at a concentration of 0.125mM₄The increasing proportion of the conjugate was examined after 0.5, 1 or 2 hours of reaction at room temperature, showing that more than 1 hour was required to completely convert the N-terminal serine to the aldehyde under such conditions. PEG₁Interferon- β failed to resolve completely with the 20-kDa PEG-carbazate in a size-exclusion column.

FIG. 22 shows diluted PEG₁Interferon- β (reverse phase chromatography purified fraction 51, characteristic of which is shown in figures 17-19) has greater antiproliferative capacity on human Burkitt's lymphoma cells (Daudi cells) than the diluted interferon- β stock solution₁Interferon- β the concentration required to inhibit growth of 50% of such cells was about 40 pg/ml, which is about one-sixth of the stock solution of interferon- β purified Mock pegylated interferon- β (shown in fraction 53 of the reverse phase chromatography of fig. 17) the concentration required to inhibit growth of 50% of such cells was about 80 pg/ml, which is about one-third of the stock solution of interferon- β.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below.

Definition of

About: as used herein, the term "about" as used herein means + -10% of the stated value (e.g., "about 50℃" includes temperature ranges of 45℃ to 55℃, inclusive; likewise "about 100 mM" includes concentration ranges of 90mM to 110mM, inclusive).

Amino acid residues: the term "amino acid residue" as used herein means a particular amino acid, typically an amino acid involved in the dehydration of two peptide bonds, located in the backbone or side chain of a polypeptide, and also when the amino acid is involved in only one peptide bond, e.g., the end point of a polypeptide bond located in a straight line. Amino acid residues are represented in the art using either a three-letter code or a single-letter code.

Antagonists: the term "antagonist" as used herein means a compound, molecule, moiety or complex that reduces, substantially reduces or completely inhibits the biological and/or physiological effects of a given cytokine on a cell, tissue or organism, which is modulated via the receptor for that cytokine. Antagonists can achieve this effect in a variety of ways, including (but not limited to): competes with the agonist for binding sites or receptors on the cell surface; and an agonist to reduce, substantially reduce, or completely inhibit the ability of the agonist to bind to a cell surface receptor; bind to and induce a change in the conformation of a cell surface receptor, such that the receptor changes structure without binding to an agonist (or can only bind with reduced or substantially reduced affinity and/or efficiency); inducing a physiological change in a cell, tissue or organism (e.g., increasing an intracellular signaling complex; increasing an inhibitor of transcription; decreasing cell surface ligand receptor expression; etc.) such that the physiological signal induced upon agonist binding or agonist binding to the cell is reduced, substantially reduced, or completely inhibited; and other mechanisms by which antagonists achieve their activity are well known to those skilled in the art. Those skilled in the art will appreciate that an antagonist may have a structure similar to the ligand it antagonizes (e.g., the antagonist may be a mutein, variant, fragment or derivative of an agonist), or may have a completely unrelated structure.

The biological active ingredients are as follows: the term "bioactive component" herein means a compound, molecule, moiety or complex that has a particular biological activity in vivo, in vitro or ex vivo on a cell, tissue, organ or organism and is capable of binding one or more polyalkylene glycols to form a conjugate of the invention. Preferred bioactive ingredients include (but are not limited to): proteins and polypeptides, such as those described herein.

Combining: the term "binding" herein means covalent binding or attachment, such as chemical coupling, or non-covalent binding, such as: ionic interactions, hydrophobic interactions, hydrogen bonding, and the like. The covalent bond may be, for example: esters, ethers, phosphates, thioesters, thioethers, carbamates, amides, amines, peptides, imines, hydrazones, hydrazides, carbon-sulfur bonds, carbon-phosphorus bonds, and the like. The term "binding" is used broadly herein and includes, for example, "coupling", "conjugation", and "attachment".

Conjugate/conjugation: "conjugate" herein means a product obtained by covalently attaching a polymer such as PEG or PEO to a bioactive ingredient such as a protein or glycoprotein. "conjugation" means the formation of a conjugate as defined in the preceding sentence. Any method of coupling a polymer to a bioactive material commonly used by those skilled in the art can be used in the present invention.

Coupling: the term "coupled" herein means attached via covalent bonds or strong non-covalent interactions, typically and preferably covalent bonds. Any method of coupling bioactive materials commonly used by those skilled in the art can be used in the present invention.

Cytokines: the term "cytokine" is defined herein as a secreted regulatory Protein that can control cell survival, growth, differentiation, and/or effector function in an endocrine, paracrine, or autocrine manner (see Nicola, N.A., supra; Kossikoff, A.A., et al, (1999) Adv Protein Chem 52: 67-108). By this definition, cytokines include: interleukins, colony-stimulating factors, growth factors, and other peptide factors made by various cells, including but not limited to those specifically disclosed or exemplified herein. Similar to their cytokine affinities, polypeptide hormones, as well as growth factors, can initiate their regulatory functions via binding to specific receptor proteins on the surface of target cells.

Diseases, disorders, conditions: the term "disease" or "disorder" herein means any undesirable condition of a human or animal, including: tumors, cancer, allergies, addiction, autoimmunity, infection, poisoning, or impaired optimal mental or biological function. "conditions" as used herein includes diseases and disorders, and also refers to physiological conditions. For example, fertility is a physiological state but not a disease or disorder. Compositions of the invention are useful for preventing pregnancy by reducing fertility and are therefore described as treating a condition (fertility), but not a disorder or disease. Other conditions are known to those of ordinary skill in the art.

Effective amount: the term "effective amount" herein means an amount of a given conjugate or composition that is necessary or sufficient to produce the desired biological effect. An effective amount of a given conjugate or composition of the invention should be that amount which achieves the selected result, and that amount can be determined by conventional methods well known to those skilled in the art, using methods known in the art and/or described herein, without undue experimentation. For example, an effective amount to treat a deficiency of the immune system must result in activation of the immune system, resulting in an antigen-specific immune response upon antigen exposure. This term is also synonymous with "sufficient amount". The effective amount for any particular use may vary depending on factors such as the disease or condition it is treating, the particular composition being administered, the route of administration, the size of the subject, and/or the severity of the disease or condition. Effective amounts of a particular conjugate or composition of the invention can be determined experimentally by one of ordinary skill in the art without undue experimentation.

One, a, or an: the terms "a", "an" or "an" herein mean "at least one" or "one or more", unless specifically stated otherwise.

PEG: "PEG" as used herein includes all ethylene oxide polymers, including linear or branched or multi-armed and end-capped or hydroxyl terminated polymers. "PEG" includes polymers known in the art: poly (ethylene glycol), methoxy poly (ethylene glycol) or mPEG or poly (ethylene glycol) -monomethyl ether, alkoxy poly (ethylene glycol), poly (ethylene oxide) or PEO, alpha-methyl-omega-hydroxy-poly (oxy-1, 2-ethanediyl), and polyethylene oxide, as well as other names for ethylene oxide polymers used in this field.

Pegylation, and Mock pegylation: as used herein, "PEGylation" means any method of covalently coupling PEG to a biologically active target molecule, particularly a receptor-binding protein. The conjugation product is what is referred to as a "pegylated" product. As used herein, "Mock PEGylation" means that the protein portion of the PEGylation reaction mixture is not covalently attached to PEG. However, the Mock pegylated product may be altered during the reaction or subsequent purification steps, such as being reductively alkylated due to exposure to a reducing agent during pegylation and/or removing one or more inhibitors, compounds, etc. during processing and/or purification steps.

Polypeptide: the term "polypeptide" as used herein means a molecule consisting of monomers (amino acids) linearly linked via amide bonds (also known as peptide bonds). Meaning the molecular chain of amino acids and not a specific length of the product. Thus, polypeptides within this definition include peptides, dipeptides, tripeptides, oligopeptides, and proteins. The term also refers to the post-translational modification products of the polypeptide, such as glycation, hyperglycosylation, acetylation, phosphorylation, and the like. Polypeptides may be derived from natural biological sources or made by recombinant DNA techniques, but are not necessarily translated from a specified nucleic acid sequence. It may be produced in any manner including chemical synthesis.

Proteins and glycoproteins: the term protein herein means a polypeptide, generally about 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids in size. Generally, proteins have a defined three-dimensional structure, although they do not necessarily have such a structure and are considered to have a folded structure, peptides and polypeptides, in contrast, do not generally have a defined three-dimensional structure, but rather have a large number of different conformations, known as unfolded structures. However, peptides may also have a well-defined three-dimensional structure. The term glycoprotein herein means a protein coupled to at least one carbohydrate moiety via a side chain of an oxygen-or nitrogen-containing amino acid residue, such as a serine residue or an aspartic acid residue.

A far end: the term "distal" (e.g., "distal N-terminal amino acid" or "distal glycosylation site") as used herein refers to a structure in which one or more attachment sites for one or more polymers in a protein are located distal to, or spatially outside, one or more receptor-binding regions or domains of the protein (as assessed by molecular modeling). Coupling of the polymer at a distal attachment site (typically the N-terminal amino acid, referred to as a "distal N-terminus" or "RN" receptor-binding protein for a receptor-binding protein) or one or more carbohydrate moieties or glycosylation sites of a glycoprotein (thus a receptor-binding protein referred to as a "distal glycosylation" or "RG" receptor-binding protein) does not result in substantial steric hindrance of the protein's binding to its receptor. Thus, the amino-terminal amino acid or glycosylation site of a cytokine is "distal" to one or more receptor-binding domains when the cytokine is coupled (e.g., covalently attached) to the amino-terminal amino acid or glycosylation site, respectively, without substantially interfering with the ability of the cytokine to bind to its receptor, particularly a cell-surface receptor. It has been determined that a given cytokine may contain more than one receptor-binding domain. In this case, the amino-terminal amino acid or glycosylation site of the cytokine may be located distal to one of the domains or more than one of the domains, provided that the coupling amino-terminal amino acid or glycosylation site does not substantially interfere with the binding of one or more receptor-binding domains of the cytokine to its receptor, and is still considered "distal to one or more receptor-binding domains". Whether the coupling substantially interferes with the ability of the protein to bind to its receptor can be readily determined using techniques well known to those skilled in the art for determining ligand-receptor binding.

As shown in figure 1d herein, PEG is a highly extended and elastic polymer that occupies a large volume in solution relative to proteins of similar molecular weight. Although the amino acid residues to which PEG is attached may be very remote from one or more receptor-binding sites, the polymer moiety may interfere with receptor binding to some extent. The chance of this interference increases with the molecular weight and the volume occupied by the polymer in solution. In any event, the directional attachment of PEG to one or more sites remote from the receptor-binding region will interfere less with cytokine function than random pegylation.

Methods for assessing ligand-receptor binding include (but are not limited to): competitive binding assays, radioreceptor binding assays, cell-based assays, surface plasmon resonance assays, dynamic light scattering, ultracentrifugation, and ultrafiltration.

Substantially, substantially: as used herein, a linkage of a protein "does not substantially" interfere with the ability of the protein to bind to its receptor if the rate and/or amount of binding of the conjugated protein is not less than about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% or more of the binding rate and/or amount of its corresponding unconjugated cytokine.

Treatment: the term "treatment" or "therapeutic" herein means prophylaxis and/or treatment. When, for example, in reference to an infectious disease, the term herein may refer to prophylactic treatment to increase the resistance of a host to infection by a pathogen, in other words to reduce the likelihood of the host becoming infected with the pathogen in the future and showing symptoms of the disease infected by the pathogen, as well as to treatment following infection of a host to render the host resistant to infection, for example: reduce or eliminate infection or prevent exacerbations.

SUMMARY

The present invention provides methods of synthesizing polymer conjugates of receptor-binding proteins that unexpectedly retain high receptor-binding activity relative to identical receptor-binding protein polymer conjugates in which one or more polymers are randomly attached. The present inventors confirmed that the cytokine pegylation target site bound to its receptor was involved or not involved via structural analysis using X-ray crystallography and nuclear magnetic resonance, analysis of mutations, and molecular modeling software. Depending on the protein classification, such cytokines are receptor binding proteins. Synthetic strategies that select for attachment of the polymer of interest to regions of the receptor-binding protein that are not involved in receptor interactions can avoid certain troublesome steric hindrances, and the resulting polymer conjugates can retain very high potency. Those in which the amino-terminal residue of the receptor binding protein is very distant from one or more receptor-binding regions or domains are defined herein as "distal N-terminal" or "RN" receptor binding proteins; it comprises all cytokines or antagonists thereof whose amino terminal amino acid is located distal to the receptor-binding site or site of the protein.

In other embodiments of the invention, conjugates are made that comprise covalently coupling one or more synthetic polymers (e.g., one or more polyethylene glycols) to cytokines that are remote from one or more receptor-binding regions or domains at the natural glycosylation site. According to this aspect of the invention, when the synthetic polymer is coupled to the glycosylation site region, the biologically active component of the conjugate (e.g., a cytokine) will exhibit well-preserved receptor-binding activity. This subset of receptor binding proteins is referred to as "RG" receptor binding proteins. When hydrophilic or amphoteric polymers are selectively coupled to or near this "distal glycosylation" site, particularly when the target protein is a non-glycosylated form of a naturally glycosylated protein, then the polymer may mimic the superior effects of the natural carbohydrate, such as aggregation, stability, and/or solubility. Thus, attachment of a polymer at or near the glycosylation site is referred to herein as "pseudoglycosylation". Thus, the present invention provides a method of synthesizing conjugates in which a site-selectively coupled synthetic polymer effectively replaces the natural carbohydrate moiety. The results of pseudoglycosylation may improve solubility, reduce aggregation, and retard clearance from the bloodstream compared to other, unglycosylated protein forms. This solution is therefore particularly advantageous for the preparation of conjugates and protein compositions, since prokaryotic organisms in general do not glycosylate the proteins they express, which proteins are produced in prokaryotic host cells, for example bacteria, such as Escherichia coli, using recombinant deoxyribonucleic acid technology.

Similarly, selective pegylation of the carbohydrate portion of a glycoprotein can result in a "pseudo-hyperglycosylation" of the glycoprotein. This method is described, for example, in PCT publication No. WO96/40731 to C.Bona et al, which is incorporated herein by reference in its entirety. The method can be particularly advantageous for the preparation of conjugates and protein compositions if the protein comprises a native glycosylation signal or a glycosylation signal introduced by recombinant deoxyribonucleic acid technology, which is a protein produced by recombinant deoxyribonucleic acid technology in eukaryotic host cells (e.g., yeast, plant cells, and animal cells, including mammalian and insect cells), since eukaryotic organisms generally glycosylate the proteins they express. Such pseudoglycosylated as well as pseudohyperglycosylated RG receptor binding proteins are within the scope of the present invention.

Thus the invention also encompasses "RN" receptor-binding protein polymer conjugates that retain substantial, nearly complete, or essentially complete receptor-binding activity, as well as pseudoglycosylated or pseudohyperglycosylated "RG" receptor-binding protein polymer conjugates that retain substantial, nearly complete, or essentially complete receptor-binding activity. A cytokine is considered herein to "retain substantial, nearly complete, or essentially complete receptor-binding activity" when conjugated to one or more water-soluble polymers according to the methods of the invention, provided that the conjugated cytokine does not substantially interfere with the ability of the protein to bind to its receptor, i.e., the rate and/or amount of binding of the conjugated protein to its corresponding receptor is not less than about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% or more of the unconjugated form of the corresponding protein. Also included within the scope of the present invention are receptor binding protein polymer conjugates classified as "RN" as well as "RG" receptor binding proteins. Two examples of the latter are interferon beta (especially interferon-beta-Ib) and interleukin 2.

In other embodiments, the invention provides methods of synthesizing receptor-binding protein polymer conjugates that unexpectedly retain high receptor-binding activity relative to the same receptor-binding protein polymer conjugates in which one or more polymers are randomly attached. The invention also provides conjugates made by the method, as well as compositions comprising one or more such conjugates of the invention, which may further comprise one or more additional components or reagents, such as one or more buffer salts, one or more saccharide excipients, one or more carrier proteins, one or more enzymes, one or more detergents, one or more nucleic acid molecules, one or more polymers such as unconjugated PEG or polyalkylene glycols, and the like. The invention also provides kits comprising the conjugates and/or compositions of the invention.

The invention also provides pharmaceutical or veterinary compositions comprising a conjugate of the invention and at least one pharmaceutical or veterinarily acceptable excipient or carrier. The invention also provides methods of using the compositions to treat or prevent various physical disorders, the methods comprising administering to an animal suffering from or susceptible to a physical disease or disorder an effective amount of one or more conjugates or compositions of the invention.

Furthermore, the present invention provides stable receptor-binding proteins and methods for producing them in industrial cell culture, thus unexpectedly obtaining a high efficiency product that substantially retains both biological activity and combined effects during prolonged action for industrial use. Such high potency of the conjugates of the invention is reflected in very high biological throughput, very high recombinant protein expression, and increased efficiency of other bioprocesses.

In other embodiments, the invention provides ready-to-use methods for increasing the biological potency of interferon beta (particularly interferon-beta-1 b) formulations. The method according to this aspect of the invention may comprise, for example, removing one or more inhibitory components from the prepared interferon beta (or interferon beta-1 b). In accordance with this aspect of the invention, one or more inhibitory components may be removed from the preparation by a variety of art-known protein and peptide processing, purification and/or analytical methods including, but not limited to, one or more chromatographic methods, such as size-exclusion chromatography, reverse-phase chromatography, hydrophobic interaction chromatography and affinity chromatography. In practice, the determination of the biological potency (i.e., whether its potency is increased, decreased, or unaffected relative to a cytokine interferon beta stock solution) of a given interferon beta preparation can be accomplished by any in vitro or in vivo assay known to those skilled in the art. For example, cell culture assays that respond to interferon beta can be used to determine the biological effectiveness of interferon beta preparations. Non-limiting examples of suitable such cell culture assays include antiproliferative assays, antiviral assays, signaling assays, and gene activation assays, examples of which are well known to those of ordinary skill in the art.

In related embodiments, the invention also provides methods of determining the amount of polymer attached to the amino terminus of a protein having an N-terminal serine residue in a reductively alkylated synthetic polymer-protein conjugate. Methods according to this aspect of the invention include, for example: (a) reacting the conjugate with a sufficient amount of an oxidizing agent for a sufficient time to cleave the polymer from the serine residue of the protein; and (b) measuring an increase in the proportion of unconjugated protein in the formulation. Proteins suitable for use according to the method include, but are not limited to: cytokines, including interferon beta (especially interferon-beta-1 b, preferably having the amino acid sequence of SEQ ID NO: 1) and megakaryocyte growth and genesis factors (Guerra, P.I., et al, (1998) Pharm Res 15: 1822-1827, incorporated herein by reference in its entirety.) oxidizing agents useful in certain methods of the invention are periodates, including, but not limited to, sodium metaperiodate, potassium metaperiodate, lithium metaperiodate, calcium periodate, barium periodate, and periodic acid.

In related other embodiments, the invention provides methods for selectively oxidatively cleaving an N-terminal serine residue of a biologically active protein without oxidizing an amino acid residue essential for the function of the biologically active protein. Some of the methods of the invention comprise, for example: (a) adjusting the hydrogen ion concentration of the biologically active protein solution to a pH of from about 5 to about 10, more preferably to a pH of from about 7 to about 8; (b) the biologically active protein solution is mixed with periodate (about 0.1 to about 10 moles, or more preferably about 0.5 to about 5 moles of periodate per mole of biologically active protein; and (c) the mixture is reacted for at least one hour, preferably at a temperature of between about 2 ℃ to about 40 ℃. proteins suitable for use in accordance with the method include, but are not limited to, cytokines including interferon beta (especially interferon beta-1 b, preferably having the amino acid sequence of SEQ ID NO: 1).

Method of producing a composite material

The present inventors have discovered that the directional attachment of a polymer to the amino-terminal amino acid of an "RN" receptor-binding protein or to the vicinity of the glycosylation site of an "RG" receptor-binding protein ensures that the polymer is attached to the distal site of one or more protein receptor-binding regions or functional domains, thereby minimizing steric hindrance of receptor interaction by the attached polymer molecule. Thus, linking proteins according to the methods of the invention preserves a higher percentage of receptor-binding activity than if the polymer were attached to the moiety involved in binding to its receptor or to an adjacent moiety. This principle can lead to an unexpectedly high retention of receptor binding activity, which can be illustrated by a receptor binding protein selected from the group consisting of: basic fibroblast growth factor ("bFGF" or "FGF-2"), epidermal growth factor ("EGF"), insulin-like growth factor-1 ("IGF-1"), interferon-alpha ("IFN- α"), interferon beta ("IFN β"), including but not limited to IFN- β -1b), granulocyte-macrophage-colony stimulating factor ("GM-CSF"), monocyte-colony stimulating factor ("M-CSF"), Flt3 ligand, stem cell factor ("SCF"), interleukin 2, 3, 4,6, 10, 12, 13, and 15, transforming growth factor- β ("TGF- β"), human growth hormone ("hGH"), prolactin, placental prolactin hormone, ciliary neurotrophic factor ("CNTF"), leptin, and these receptor-binding-protein antagonists that mimic or are receptor-binding proteins for these proteins Analogues of the texture. In contrast, selective attachment of a large polymer to the amino terminus of interferon- γ would not be expected to preserve a large portion of the cytokine activity, since this coupling would be expected to interfere with the binding of the active dimer to its receptor (based on the data of Walter, M.R., et al, (1995) Nature 376: 230-235).

In related embodiments of the invention, the polymer is coupled to the amino-terminal residue of a mutein of a receptor binding protein (a natural protein competitive antagonist whose function is to bind to one or more of the same receptors without triggering signal transduction). Examples are polymer conjugates of hGH antagonists containing the point mutation G120R (Sundstrom, M., et al, (1996) Jbiol Chem 271: 32197-32203) and polymer conjugates of prolactin antagonists containing the point mutation G129R (Goffin, V., et al, (1997) JMammary Gland Biol Neoplasia 2: 7-17; Chen, W.Y., et al, (1999) Clin Cancer Res 5: 3583-3593; Chen, W.Y., PCT Publication No. WO 99/58142A 1). Selective point mutations, truncations or deletions may make antagonists of other receptor binding proteins (see, e.g., Tchelet, A., et al, (1997) Mol Cell Endocrinol 130: 141-152; Peterson, F.C., (1998) Identification of Motifs Associated with the Lactogenic and micropropic Actions of Humangrowth Hormine, Ph.D. Dispersion, Ohio State University, UMI # 9822357).

In other embodiments of the "RG" receptor-binding proteins of the present invention, the methods of the present invention attach one or more synthetic polymers proximal to the natural site of the carbohydrate moiety attached to the receptor-binding glycoprotein. Resulting in "pseudo-glycosylation" of such receptor-binding proteins (e.g., when expressed by recombinant deoxyribonucleic acid techniques in E.coli or other prokaryotic cells that do not undergo post-translational glycosylation) or in the glycoprotein form thereof (e.g., naturally-produced glycoproteins or glycoproteins produced by eukaryotic host cells (e.g., yeast, plant cells, and animal cells including mammalian and insect cells) that undergo post-translational glycosylation). Examples are polymer conjugates of interferon alpha and beta, erythropoietin ("Epo"), and interleukin-2. Attachment of synthetic polymers to or near the natural glycosylation site can be performed by any method known in the art, including the mutagenesis method of R.J. Goodson, et al ((1990) Biotechnology 8: 343-86346) and the method of R.S. Larson, et al ((2001) bioconjugateg Chem 12: 861-869), which involves first oxidizing the saccharide; the entire disclosures of such references are incorporated herein by reference.

Modification of the amino terminus of certain proteins has been disclosed in the prior art (see, e.g., Dixon, H.B.F. (1984) J Protein Chem 3: 99-108). For example, N-terminally modified proteins have been reported to stabilize certain proteins against aminopeptidase (Guerra, P.I., et al, supra), to improve protein solubility (Hinds, K., et al, (2000) bioconjugate Chem 11: 195-201), to reduce the N-terminal amino electrovalence, or to improve the homogeneity of the resulting conjugate (Kinstler, 0., et al European Patent Application No. EP 0822199A 2; Kinstler, 0., et al, (2002) Adv Drug Deliv Rev 54: 477-485), and the like. Alternative methods for coupling polymers to the alpha amino group of the N-terminal cysteine or histidine residue (improved procedures known in the art as "native chemical ligation") have been disclosed (Roberts, M.J., et al, PCT publication No. WO 03/031581A2 and U.S. patent application publication No. 2003/0105224A1, incorporated herein by reference in their entirety). However, the existence of receptor-binding protein "RN" and "RG" subclasses, generally applicable methods for selecting members of these classes, and methods of making and using the receptor-binding protein polymer conjugates as a means to preserve unexpectedly high functional activity of "RN" receptor-binding proteins have not been recognized or described in the art.

Thus, it would be advantageous to determine whether a given cytokine has an N-terminus and/or glycosylation site that is remote from the receptor-binding site of the ligand. The ability to predict whether a given cytokine is an "RN" or "RG" ligand prior to coupling of the ligand to the polymer can substantially reduce the experimentation required to produce polymer-ligand conjugates (e.g., cytokines or antagonists thereof conjugated to polymers such as PEGs) in which the antigenicity and immunogenicity of the conjugate is reduced relative to the unconjugated ligand without substantially reducing the receptor-binding and physiological activity of the conjugated ligand.

Accordingly, in other embodiments, the invention provides methods for identifying and selecting receptor-binding protein ligands (e.g., cytokines and antagonists thereof) that have an N-terminal and/or glycosylation site(s) remote from the receptor-binding site of the protein ligand (i.e., methods for identifying and selecting "RN" or "RG" proteins). In certain embodiments of the invention, the optimal location for coupling one or more polymers (e.g., one or more PEGs) can be determined using molecular modeling, e.g., using molecular modeling software to view the three-dimensional structure of a protein (cytokine or antagonist thereof) to predict where one or more polymers can attach to the protein without substantially losing biological or receptor-binding activity of the protein (see also Schein, c.h., supra). Similar approaches have been described, such as coupling PEG with G-CSF in an attempt to improve its resistance to protein hydrolysis (see published U.S. application No. 2001/0016191 a1, issued to t.d. osslund, incorporated herein by reference in its entirety). Suitable molecular modeling software for use in the present invention, such as RASMOL software (Sayle et al, supra) and other programs for generating macromolecular structure databases deposited in protein databases (PDB; see Laskowski, r.a., supra), are software known in the art and are well known to those of ordinary skill in the art. Using such molecular modeling software, the three-dimensional structure of a polypeptide, such as a cytokine or antagonist thereof, can be analyzed or predicted with high confidence based on the crystallography of the ligand and its receptor. In this manner, one of ordinary skill in the art can readily determine which ligands are "RN" or "RG" ligands suitable for use in the present invention.

In the practice of the present invention, a convenient route for covalently coupling a water-soluble polymer to the alpha amino group of the N-terminal amino acid residue of a protein is to reductively alkylate the Schiff base formed by a polymer bearing a single aldehyde group, see, for example, G.P. Royer (U.S. Pat. No.4,002,531), but not J.M. Harris, et al (U.S. Pat. No.5,252,714), which is not suitable for synthesizing long-acting receptor-binding proteins that retain substantial receptor-binding activity, since the latter inventor only applies for polymers derivatized with aldehyde groups at both ends, which are cross-linking agents.

The Amino group that mediates reductive alkylation of the schiff base of PEG-monoaldehydes with the Amino acid alpha to the N-terminal Amino acid of receptor-binding Proteins, but away from its lysine residue, can be accomplished in a variety of ways, based on the Proteins Amino Acids and Peptides as Ions and nucleotides Ions ((1943), reinhold publishing Corporation, New York), disclosed in j.t. edsall, chapter 4 and 5, incorporated herein by reference in its entirety. The acidic dissociation constant ("pKa") of the alpha amino group of the N-terminal amino acid of the polypeptide is expected to be less than 7.6; whereas the pKa value of the amino group of a lysine residue of the polypeptide is expected to be about 9.5. Edsall (1943, supra) clearly indicates that aldehydes bind to the amino group of an amino acid only when its isoelectric point is basic.

Thus, based on the present disclosure and data readily available in the art, one skilled in the art will recognize that (1) selective reaction of aldehydes with protein alpha amino groups prefers a pH range below 9.5 (approximately the pKa of the protein amino group); (2) if the reaction pH is less than 7.6 (about the pKa of the alpha amino group of the protein), the reaction rate of the aldehydes and amino groups will decrease; (3) if the reaction pH is less than 7.6, the reaction rate of the aldehyde and the alpha amino group will be slower than that of the amino group, and (4) the selectivity of the reaction of the aldehyde and the alpha amino group at a pH below 6.6 will be improved. Since the latter values are approximately one pH unit below the pKa of the alpha amino group and three pH units below the pKa of the amino group, about 10% of the alpha amino group and about 0.1% of the amino group will be in a reactive, unprotonated state. Thus, at pH6.6, the unprotonated alpha amino moiety is 100 times that of the unprotonated amino group. Thus, further lowering of the pH, e.g. to 5.6, gives a very small increase in selectivity, where theoretically 1% of the alpha amino groups and 0.01% of the amino groups will be in a reactive, unprotonated state. Thus, in certain embodiments of the invention, a protein ligand (particularly an "RN" or "RG" ligand, including cytokines and antagonists thereof) is present at a pH of from about 5.6 to about 7.6; a pH of about 5.6 to about 7.0; a pH of about 6.0 to about 7.0; a pH of about 6.5 to about 7.0; a pH of about 6.6 to about 7.6; a pH of about 6.6 to about 7.0; or conjugated to one or more polymers via a mixture of formed ligand and one or more reactive polymers at a pH of about 6.6. Thus, the method of the invention differs significantly from known techniques in which the pH of the alpha amino coupled polymer at the N-terminal amino acid residue of the ligand is about 5(Kinstler, O., et al, (2002) supra; EP 0822199A 2; U.S. Pat. Nos. 5,824,784 and 5,985,265; Roberts, M.J., et al, (2002), spura; Delgado, C., et al, U.S. application publication No. 2002/0127244A 1), while the pH of the amino group of the lysine residue of the backbone of the polymer coupled to the ligand polypeptide is 8.0(Kinstler, O., et al, EP 0822199A 2; U.S. Pat. Nos. 5,824,784 and 5,985,265). Similarly, the present method differs significantly from the enzymatic method of coupling poly (ethylene glycol) alkylamine derivatives to certain proteins using transglutaminase at pH7.5 (Sato, H., (2002) Adv Drug Deliv Rev 54: 487-504).

The Schiff base formed by reduction with a mild reducing agent, such as sodium cyanoborohydride or pyridine borane (Cabacungan, J.C., et al, (1982) Anal Biochem 124: 272-278) forms a secondary amine linkage that preserves the positive charge of the α amino group at the N-terminus of the protein at physiological pH. This bond, which retains the same charge as the native protein, preserves its biological activity more than other chemical ligation chemistries, which neutralize the charge, for example by forming an amide bond (Burg, J., et al, PCT publication No. WO 02/49673A 2; Kinstler, 0., et al, European patent application No. EP 0822199A 2; Kinstler, 0., et al, (1996) Pharm Res, 13: 996. multidot. 1002; Kita, Y., et al, supra) or a carbamate bond (Gilbert, C.W., et al, US patent No.6,042,822; Grace, M., et al, (2001) Jlnterferon Cytokine Res 21: 1103-, Young, S., et al, (2002) currrm Des 8: 2139: 2157).

Alternative methods for selectively coupling polymers to N-terminal amino acid residues are well known to those skilled in the art. Including coupling of hydrazides, hydrazines, semicarbazides, or other internal amine-containing polymers to the N-terminal serine or threonine residues that are oxidatively cleaved to aldehydes with periodate (Dixon, H.B.F., supra; Geoghegan, K.F., U.S. Pat. No.5,362,852; Gaertner, H.F., et al, (1996) bioconjugate Chem 7: 38-44; Drummond, R.J., et al, U.S. Pat. No.6,423,685).

Suitable polymers

In certain embodiments of the invention, it is desirable to minimize intra-and intermolecular cross-linking of polymers, such as PEG, during the reaction of coupling the polymers to the bioactive components to produce the conjugates of the invention. This can be accomplished using polymers activated with only one terminus ("monofunctional activated PEGs" or "monofunctional activated PAGs") or polymer formulations that are difunctional activated (in the case of linear PEGs, referred to as "difunctional PEG diols") or multifunctional activated polymers in a percentage of less than about 30% or more preferably less than about 10% or most preferably less than about 2% (w/w). The use of fully or almost fully monofunctional activated polymers can minimize the formation of: intramolecular cross-linking within a single protein molecule, a "dumbbell" structure in which one-chain polymers link two protein molecules, and larger aggregates or gels.

The activated forms of the polymers suitable for use in the methods and compositions of the present invention may comprise any linear or branched, monofunctional activated form of the polymers known in the art. Such as those having a molecular weight (excluding activating group mass) of between about 1kDa and about 100 kDa. Suitable molecular weight ranges include, but are not limited to, about 5kDa to about 30 kDa; about 8kDa to about 14 kDa; about 10kDa to about 20 kDa; about 18kDa to about 60 kDa; about 18kDa to about 22 kDa; about 12kDa to about 30kDa, about 5kDa, about 10kDa, about 20kDa or about 30 kDa. In the case of linear PEGs, the molecular weight of about 10kDa, about 20kDa or about 30kDa corresponds to a degree of polymerization (n) of about 230, about 450 or about 680 ethylene oxide monomer units, respectively. It is worth mentioning that before confirming the existence of the receptor binding protein "RN" and "RG" classes, the advantages of coupling therapeutic proteins to relatively high molecular weight (i.e., > 20-30kDa) polymers have been disclosed (Saifer, M., et al, PCT publication No. WO 89/01033A 1, published in Feb.9, 1989, incorporated herein by reference in its entirety).

In other embodiments of the invention, receptor-binding protein conjugates that retain a very high percentage of biological activity can be prepared in vitro, e.g., cell culture, according to the methods of the invention via coupling to monofunctional activated polymers of about 1kDa, about 2kDa, or about 5 kDa. For such in vitro applications, a lower molecular weight range is preferred.

If desired, the linear polymer may have a reactive group at one or both ends, thereby creating a "reactive polymer". In certain embodiments of the invention, N-hydroxysuccinimide esters of monopropionic acid derivatives of PEG may be satisfactorily used, as disclosed in j.m. harris, et al, U.S. patent No.5,672,662, which is incorporated herein by reference in its entirety, or other N-hydroxysuccinimide-activated PEG-monocarboxylic acids. In certain other embodiments, monosuccinimidyl carbonate derivatives of PEG ("SC-PEG"), described in m.saifer, et al, U.S. patent nos. 5,006,333; 5,080,891, respectively; 5,283,317 and 5,468,478, or mono-p-nitrophenyl carbonate derivatives of PEG, disclosed in S.J. Kelly, et al, supra; l.d. williams, et al PCT publication nos. WO00/07629 a2, l.d. williams, et al, U.S. patent No.6,576,235 and m.r. sherman, et al, PCT publication No. WO 01/59078 a 2. In addition, other types of reactive groups can be used to synthesize protein polymer conjugates. Such derivatives include (but are not limited to): monoaldehyde derivatives of PEG (Royer, g.p., U.S. patent No.4,002,531; Harris, j.m., et al, U.S. patent No.5,252,714), monoamines, mono-tribromophenyl carbonate, monocarbonyl-imidazole, mono-trichlorophenyl carbonate, mono-trifluorophenyl carbonate, monohydrazide, monohydrazine, monohemihydrazine, monohydrazinoformate, monothiohemicarbazide, monoiodoacetamide, monomaleimide, mono-orthopyridyl disulfide, mono-oxime, mono-phenylglyoxal, mono-thiazolidine-2-glycopeptide, mono-thioester, mono-thiol, mono-triazine, and monovinylsulfone derivatives of PEG. In other embodiments, cytokines, chemokines, growth factors, polypeptide hormones, and antagonists thereof may be conjugated to one or more polymers, as described in co-pending application U.S. patent application No. 10/669,597, which is incorporated herein by reference in its entirety.

Bioactive ingredients

As noted above, the conjugates of the invention comprise a PAG or PAO, and in particular a chain of PEG, covalently attached to one or more bioactive components. The biologically active ingredient to which one or more polymers (or chains thereof) are covalently attached is referred to herein as a "conjugated biologically active ingredient" or a "modified biologically active ingredient". Such terms are to be distinguished from "unconjugated bioactive component", "starting bioactive component", or "unmodified bioactive component", which is intended to mean a bioactive component that is not covalently attached to a polymer. It is to be understood, however, that a "unconjugated", "unmodified" or "starting" bioactive component, which is referred to as a "Mock pegylated" bioactive component, will still be considered "unconjugated", "unmodified" or "starting" in accordance with the present invention because it is "unconjugated", "unmodified" or "starting" in terms of attachment to the conjugate, as compared to the wild-type or native molecule, may contain other non-polymer conjugations or modifications.

The term "stabilizing" a biologically active ingredient (or "stabilization method" or "stabilized biologically active ingredient") herein indicates that the biologically active ingredient has been stabilized according to the methods of the invention (i.e., the biologically active ingredient has been covalently attached to a polymer according to the methods of the invention). The stabilized bioactive component will exhibit certain altered biochemical and biophysical properties when compared to the non-stabilized bioactive component (i.e., the bioactive component of the non-covalently attached polymer). The altered biochemical and biophysical parameters, particularly with respect to receptor-binding proteins, include reduced susceptibility to proteolytic degradation and preservation of the activity of the receptor-binding protein, particularly under certain harsh environmental or experimental conditions. In certain embodiments of the invention, altered biochemical and biophysical parameters may include, for example, increased half-life in the living circulation, increased bioavailability, increased duration of action in vitro, and the like.

Any receptor-binding protein (typically a cytokine) having biological (i.e., physiological, biochemical or pharmaceutical) activity associated with the amino terminus of the molecule or the distal portion of a glycosylation site, either naturally or mutationally introduced, may be suitable as a starting component of the present invention. The bioactive components include (but are not limited to): peptides, polypeptides, proteins, and the like. Biologically active ingredients also include fragments, muteins and derivatives of the peptides, polypeptides, proteins and the like, particularly fragments, muteins and derivatives thereof having biological (i.e., physiological, biochemical or pharmaceutical) activity.

Suitable peptides, polypeptides and proteins, glycoproteins and the like that are suitable for use as the bioactive components of the invention include any peptide, polypeptide or protein, etc., having one or more available amino, thiol or other groups distal to the receptor-binding region or bioactive component region and optionally attached to a polymer. The peptides, polypeptides, proteins, glycoproteins, and the like comprise cytokines, which may have any of a variety of structures (Nicola, n.a., supra; Schein, c.h., supra).

For example, suitable peptides, polypeptides and proteins of interest include, but are not limited to, cytokine classes (long and short subclasses) comprising structures with four α -helical bundles (see Schein, c.h., supra). Various four-helix bundle containing proteins suitable for use in the present invention include (but are not limited to): interleukins, such as IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12(p35 subunit), IL-13, IL-15 and IL-17; colony-stimulating factors, such as macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF; Rozwarski, D.A., et al, (1996) Proteins 26: 304-313); interferons such as interferon- α, interferon- β (including but not limited to interferon- β -1b), and interferon consensus (consensus IFNs); leukemia Inhibitory Factor (LIF); erythropoietin (Epo); thrombopoietin (Tpo); megakaryocyte Growth and Development Factor (MGDF); stem Cell Factor (SCF), also known in the art as graying factor (Morrissey, P.J., et al, (1994) Cell Immunol 157: 118-131; McNiece, I.K., et al, (1995) J Leukoc Biol 58: 14-22); oncostatin m (osm); phospholipase-activating protein (PLAP); a neurotrophic factor; and peptidomimetics thereof. Although prolactin and growth hormone are conventional hormones that circulate extensively in the body, unlike cytokines, which are usually produced close to their target cells, prolactin and growth hormone belong to the same structural class as cytokines with four α -helical bundles (Nicola, n.a., supra; Goffin, v., et al, supra) and as such are polymer-coupled and suitable targets for producing conjugates according to the invention.

Long-chain β -sheet or β -barrel structural class of receptor binding proteins (see Schein, c.h., supra) are also suitable for use in preparing the conjugates and compositions of the invention. Such include (but are not limited to): the tumor necrosis factor family of cytokines, such as TNF- α, TNF- β, and Fas ligand, which exhibit a β -colloidal rolling structure; IL-1 (including IL-1a and IL-1 β) and the FGF (including basic fibroblast growth factor (bFGF), acidic FGF, FGF-4 and keratinocyte growth factor (KGF; FGF-7)) family, which exhibit beta-clover folding (Schein, C.H., supra; Schlesinger, J., et al, supra); IL-12; IL-16; epidermal growth factor (EGF; Lu, H. -S., et al, supra); and Platelet Derived Growth Factors (PDGFs), transforming growth factors (including transforming growth factor-alpha and transforming growth factor-beta (TGF-beta)), and nerve growth factors, which exhibit cystine-network junction structures.

Other structures of proteins advantageously used in the conjugates and compositions of the invention are classified as disulfide-rich mixed α/β cytokines and growth factors (see Schein, c.h., supra), including but not limited to: the EGF family, which carries a β -serpentine structure; IL-8; RANTES; neutrophil activating peptide-2 (NAP-2); stromal cell derived factor-1 a (SDF-1 a); monocyte chemoattractant proteins (MCP-1, MCP-2, and MCP-3); eotaxin proteins (e.g., eotaxin-1, eotaxin-2, and eotaxin-3); myeloid progenitor inhibitory factor-1 (MPIF-1); neuronal chemotactic protein, macrophage Migration Inhibitory Factor (MIF); growth-associated oncogene/melanoma growth stimulating activity (GRO-a/MGSA); a growth regulator; and insulin-like growth factors (e.g., IGF-1 and IGF-2). A related structural classification of proteins for use in the conjugates and compositions of the invention are cytokines with mosaic structure, including growth factors such as IL-12 and hepatocyte growth factor (Nicola, n.a., supra).

Other proteins of interest include (but are not limited to): growth hormone (especially human growth hormone (hGH; see Tchelet, A., et al, (1997) Mol Cell Endocrinol 130: 141-, j.c., et al, (2000) Ann RheumDis 59(Supp 11): i109-i114) which are not suitable candidates for N-terminal polymer conjugation within the scope of the present invention, since the light and heavy chain amino-terminal regions are involved in antigen recognition and are not RN receptor binding proteins.

Particularly useful bioactive components for preparing the polymer conjugates of the invention are interferon- α, interferon β, interleukin 2, IL-4, IL-10, hGH, prolactin, insulin, IGF-1, EGF, bFGF and erythropoietin (Epo). Also particularly useful are muteins and fragments of these biologically active ingredients, especially those that bind to receptors for the corresponding wild-type or intact polypeptide, whether or not the binding induces a biological or physiological effect. In certain embodiments, muteins and fragments of biologically active ingredients can act as antagonists of the corresponding ligand, which can reduce, substantially reduce, or completely inhibit the binding of the ligand to its receptor and/or the activity of the ligand on the target cell, tissue, and/or organism. Other antagonists, which may or may not be structural analogs, muteins, variants or derivatives of the ligand of interest, are also suitable for use in preparing the conjugates of the invention. In essence, whether a given mutein, fragment, variant, derivative or antagonist antagonizes the biological and/or physiological effect of a given ligand can be determined without undue experimentation using various techniques known and/or described herein for determining the biological/physiological effect of the ligand itself.

The structures (primary, secondary, tertiary, and quaternary) of such and other polypeptides of interest that are advantageously employed in the present invention are known in the art and are well known to those of ordinary skill in the art, especially in view of the structures provided herein and the references cited therein, which are incorporated herein in their entirety by reference.

Conjugates

The present invention provides stable conjugates of bioactive components, particularly cytokines, for use in a variety of applications. The conjugates of the present invention have many advantages over previously known techniques, as evidenced by comparison to the following non-limiting and exemplary art-known conjugates.

Hiratani (European patent No. EP 0098110B 1 and U.S. Pat. No.4,609,546) discloses conjugates of ethylene oxide and propylene oxide copolymers ("PEG-PPG", members of the general PAGs) and proteins, including interferons and interleukins, in which regions involved in protein receptor binding are not avoided. Interferons α, β and γ in such references are equivalent targets for coupling PAGs, differing in the present invention: interferon-gamma is not a suitable target for N-terminal coupling because the amino terminus is within the cytokine receptor-binding region. Furthermore, Hiratani revealed conjugates synthesized with only 1kDa to 10kDa PAGs, whereas the method of the invention prefers the use of coupling water-soluble, synthetic polymers with molecular weights above 10kDa for therapeutic use. Similarly, n.v. katre ((1990) supra) revealed coupling a larger number of strands of 5-kDa mPEG to human recombinant interleukin-2 to increase the lifetime of the conjugate in mouse and rabbit blood streams. However, this reference does not disclose or appreciate the advantages provided by the present invention of coupling a smaller number of longer chain PEGs or coupling a single chain of high molecular weight PEG to the amino terminus of interleukin 2.

Shaw (U.S. patent No.4,904,584 and PCT publication No. WO 89/05824 a2) discloses methods of introducing, substituting, or deleting lysine residues of a protein of interest (particularly Epo, G-CSF, and interleukin 2) to induce site-selective attachment of an ammonia-reactive polymer. However, unlike the present disclosure, such references do not disclose that ammonia-reactive polymers can react with ammonia other than the amino group of a lysine residue in any protein of interest, and are clearly distinguished from the present disclosure.

Nitecki et al, (U.S. patent No.4,902,502) disclose multi-pegylated interleukin 2 conjugates prepared from various PEG chloroformate derivatives that are expected to react with the amino group of a lysine residue. In contrast to the methods of the present invention, however, the reference does not disclose a method for avoiding lysine residues involved in receptor binding in the pegylated interleukin 2 protein, nor does it recognize the advantage of avoiding this site.

Katre, et al, (U.S. patent No.5,206,344) discloses PEG-IL-2 conjugates in which PEG is coupled to the amino group of a lysine residue, to an unpaired sulfhydryl group of a native cysteine residue at position 125 (counted from the amino terminus), or to a sulfhydryl group of a cysteine residue introduced by mutation between the first to twentieth residues from the amino terminus of interleukin 2. The muteins disclosed in the' 344 patent include "des-ala-1" interleukin 2, i.e., a mutein in which the amino-terminal alanine is deleted and which is not pegylated. In contrast to the present disclosure, however, the' 344 patent does not disclose any method of avoiding coupling of PEG to amino acid residues involved in binding to the receptor, nor does it recognize any advantages of this method. Consistent with this concept, and in contrast to the present invention, the broad range of attachment points proposed in the' 344 patent does not suggest that coupling PEG to the amino terminus of interleukin 2 would be particularly advantageous.

S.p. monkarsh, et al, (1997) Anal Biochem 247: 434-440 and Harris, j.m., et al, eds., poly (ethylene glycol): chemistry and Biological Applications, pp.207-216, American Chemical Society, Washington, D.C., revealed that the reaction of interferon- α -2a with a three-fold molar excess of activated PEG (molecular weight 5,300 daltons) produced eleven positional isomers of mono-PEG-interferon, corresponding to eleven lysine residues of interferon- α -2 a. PEG-interferon production without PEG coupling to the alpha amino group at the amino terminus of interferon. The eleven position isomers reported in such references show antiviral activity in the cultured cells ranging from 6% to 40% of unmodified interferon and antiproliferative activity in the cultured cells ranging from 9% to 29% of unmodified interferon. In contrast to the conjugates prepared by the method of the present invention, this result clearly shows that random pegylation of lysine residues by such investigators interferes with the regulatory function of interferon- α -2a via its receptor. Furthermore, unlike the conjugates of the present invention, such references do not report whether the conjugate contains an N-terminally pegylated interferon.

O Nishimura et al, (U.S. patent statutory invention registration patent No. H1662) disclose interferon-alpha, interferon-gamma and interleukin 2 conjugates prepared by reductive alkylation of activated "polyethylene glycol methyl ether aldehydes" and sodium cyanoborohydride at pH7.0 (interferon conjugate) or pH7.15 (interleukin 2 conjugate). However, the conjugates prepared by this method lost up to 95% of the biological activity of the unmodified protein, apparently due to the presence of multiple sites for attachment of the polymer to the amino groups of lysine residues (see FIGS. 1 and 4 of the present invention).

D.k.pettitt, et al, (1997) J Biol Chem 272: 2312-2318 disclose interleukin-15 ("IL-15") polymer conjugates. However, the conjugated IL-15 reported in this reference not only loses its interleukin 2-like growth-promoting ability (due to coupling of the polymer to lysine residues of the protein involved in receptor binding), but also shows antagonistic rather than agonistic effects. Such authors conclude that selective inhibition of IL-15 binding to one of many cell surface receptors, possibly due to polymer conjugation, not only reduces receptor binding, but can reverse the biological effects of the protein. Avoiding coupling of polymers to portions of receptor-binding proteins involved in receptor interactions, the present invention avoids the undesirable consequences of polymer coupling.

Hakimi, et al, (U.S. patent nos. 5,792,834 and 5,834,594) disclose carbamate-linked PEG conjugates of proteins comprising: interferon-alpha, interleukin 2, interleukin-1 ("IL-1"), and I L-1-receptor antagonists, which have been reported to reduce immunogenicity, increase solubility, and increase protein biological half-life. In such references PEG is coupled to "various free amino groups", the reference does not disclose N-terminal PEGylation and does not disclose that the N-terminal alpha amino group may or should be PEGylated. Such patents also state that: the conjugates disclosed herein "have at least a portion" of the biological activity of the original starting protein, thus indicating that a significant amount of the biological activity may also be lost. This result is consistent with the non-directional pegylation method of the present invention. In contrast to the present invention, such patents do not disclose any attempt to alter the selectivity of the pegylation process to improve the retention of the biological activity of the conjugate (disclosed in the present invention).

Kinstler, et al, (european patent application No. EP 0822199 a2) discloses a method of reacting poly (ethylene glycol) with the amino-terminal amino acid alpha amino group of a polypeptide, particularly the amino-terminal amino acid alpha amino group of two proteins manufactured by Amgen, inc. The text shows that "acid to a pH sufficient to selectively activate the alpha amino group" is an essential feature of the process. In contrast, the present inventors have found that lowering the pH reduces the reactivity of the amino group with PEG aldehydes, and that the alpha amino group is more reactive when unprotonated (i.e., pH greater than its pKa). Thus, the inventors found that there was no pH "sufficiently acidic to selectively activate the alpha amino group" of any RN cytokine conjugate of the invention. The reactivity of the N-terminal alpha amino group and aldehydes in pH dependence is demonstrated in J.T.Edsal1 (supra) and R.S.Larsen et al ((2001) bioconjugate Chem 12: 861-869) and is more compatible with the inventors' experience. Furthermore, Kinstler et al report the use of N-terminal PEGylated polypeptides to increase the homogeneity of the resulting conjugates and to protect the amino terminus from protease degradation, but do not disclose that N-terminal PEGylation preserves most of the receptor-binding activity of certain receptor-binding proteins (see, e.g., PCT publication No. WO 96/11953; European patent No. EP 0733067 BI, and U.S. Pat. Nos. 5,770,577, 5,824,784, and 5,985,265, all of which are Kinstler, O.B., et al).

The European application of Kinstler et al (EP 0822199A 2) also generalizes the advantages of all N-terminal PEGylation of polypeptides, but differs from the experience of the present inventors. In particular, since the amino terminus of the antibody molecule is adjacent to the antigen binding region of the antibody protein (Chapman, A.P. (2002) Adv Drug Deliv Rev 54: 531-545), the N-terminally PEGylated antibody was unexpectedly deleterious to biological activity compared to randomly PEGylated lysine residues as disclosed in Larsen, R.S., et al, supra. Similarly, N-terminally PEGylated receptor binding proteins other than "RN" receptor binding proteins, such as interferon-gamma (see FIG. 8), are expected to be more receptor interaction inhibitory than receptor binding proteins with randomly PEGylated lysine residues.

Thus, as noted above, unlike the methods disclosed in Kinstler et al, the conjugates of the invention are prepared at a pH of from about 5.6 to about 7.6; a pH of about 5.6 to about 7.0; a pH of about 6.0 to about 7.0; a pH of about 6.5 to about 7.0; a pH of about 6.6 to about 7.6; a pH of about 6.6 to about 7.0; or one or more cytokines or antagonists thereof (selected as RN receptor binding proteins) and one or more polymers (forming a mixture between the ligand and the one or more polymers) at a pH of about 6.6. In contrast, Kinstler et al, which attaches ligands at a pH below 5.5, found by the present inventors to be a suboptimal or inferior pH for conjugating polymers and ligands distal to the N-terminal amino acid and/or glycosylation site to make formulations.

Pepinsky, b, et al (PCT publication No. WO 00/23114 and U.S. patent application publication No. 2003/0021765 a1) disclose glycosylated interferon beta-1 a polymer conjugates that are more active than non-glycosylated interferon beta-1 b in an antiviral assay. When Pepinsky et al coupled 5-kDa or 20-kDa mPEG to the amino terminus of interferon-beta-1 via reductive alkylation, PEGylation was observed to have no effect on antiviral efficacy, while coupling higher molecular weight PEGs reduced or eliminated this effect. This reference also discloses that polyalkylene glycols can be coupled to interferon beta-1 a via a variety of different site coupling groups, including amino-terminal, carboxy-terminal, and glycosylated protein carbohydrate moieties. However, this approach is not general: "such studies indicate that despite the conservation between interferon beta-1 a and interferon beta-1 b sequences, it is a different biochemical entity, and therefore knowledge of interferon beta-1 b cannot be applied to interferon beta-1 a, and vice versa". In contrast, the present invention discloses the common features between "RN" and "RG" receptor binding proteins as defined herein. According to the present invention, interferon beta-1 a and interferon beta-1 b are "RN" receptor binding proteins. In addition, interferon beta-1 b is "RG" receptor-binding protein. Accordingly, in contrast to the method of WO 00/23114, the method of the present invention is useful for preparing stable, biologically active interferon beta-1 b and interferon beta-1 a conjugates.

Wei, et al, (U.S. Pat. No.6,077,939) disclose a method of coupling a water soluble polymer, particularly PEG, to the N-terminal alpha carbon atom of a polypeptide, particularly erythropoietin, wherein the amine at the alpha carbon of the N-terminal amino acid is first transaminated to an alpha carbonyl group and then reacted with a PEG derivative to form an oxime or hydrazone bond. Since the goal disclosed in this reference is with respect to developing a general approach for proteins and does not contemplate preserving receptor-binding activity (which can be achieved by selecting some amino termini as sites for pegylation of certain receptor-binding proteins). Thus, contrary to the disclosures of Wei, et al, the present invention does not require removal of the N-terminal alpha amino group, but rather preserves the charge of the N-terminal alpha amino group at neutral pH via formation of a secondary amino bond between the protein and the polymer.

Gilbert et al, (U.S. Pat. No.6,042,822; European patent No. EP 1039922131) disclose the necessity of a mixture of isomers at the PEG-interferon- α -2b position, with particularly satisfactory isomers having PEG coupled to the histidine residue of interferon- α -2b (particularly histidine-34) and showing instability of the coupling of PEG to histidine-34. Since histidine-34 is located on the surface of interferon- α -2b in a region that must be in close contact with interferon receptors to initiate signaling (see fig. 1b of the present specification), the disclosure of labile linkages between PEG and histidine-34 in such references appears to be critical to the function of the PEG-interferon conjugates disclosed herein. Fairly pure histidine-linked protein polymer conjugates are described in s.lee et al, U.S. patent No.5,985,263. In contrast, the present invention shows that one preferred conjugate is a PEG-interferon conjugate, wherein the PEG is stably attached at a site distal to the receptor-binding domain of the interferon component.

Bailon, et al, ((2001) bioconjugate Chem 12: 195-202) revealed: interferon- α -2a in which interferon is pegylated with a molecule of 40-kDa di-mPEG-lysine per molecule contains four major positional isomers. This reference discloses that almost all PEG is attached via amide bonds to lysines 31, 121, 131 or 134, each of which is within or adjacent to the interferon-. alpha. -2a receptor-binding domain (residues 29-35 and 123-. Bailon et al did not report N-terminal PEGylation. The antiviral activity of the isolated mixture of PEG-interferon positional isomers against vesicular stomatitis virus infection of Madin-Darby bovine kidney cells in vitro was tested to be 7% of that of unconjugated interferon- α -2 a. PEG-interferon conjugates comprising N-terminally unpegylated interferon have a substantial loss of biological activity, which clearly distinguishes Bailon et al from the conjugates of the invention.

Pepinsky et al, ((2001) J Pharinacol Exp Ther 297: 1059-1066) revealed the synthesis of conjugates with (1) glycosylated interferon beta-1 a with an N-terminal methionine residue and (2)20-kDa PEG-aldehyde. The conjugate is monopegylated at the N-terminal methionine and retains intact biological activity in antiviral assays. The authors revealed that their choice of the N-terminal site for pegylation of glycosylated interferon beta-1 a depends on the availability of site-selective pegylation reagents and modeling of the molecule, which acknowledges that "some effects are product specific". Furthermore, in contrast to the present invention, its observations report no generalization to include the class of receptor binding proteins defined herein as "RN" receptor binding proteins.

Burg, et al, (PCT publication No. WO 01/02017A 2) disclose the production of alkoxy PEG conjugates of erythropoietin glycoproteins in which one to three chains of methoxy PEG are reacted with chemically introduced sulfhydryl groups by modifying the amino groups of lysine residues on the surface of the glycoprotein. In contrast to the present invention, however, this reference does not disclose any attempt to link PEG to the N-terminal amino acid free alpha amino group of erythropoietin or to avoid modification of lysine residues in important regions of the interaction of erythropoietin glycoproteins with erythropoietin receptors.

Burg, et al, (PCT publication No. WO 02/49673 a2) disclose the synthesis of N-terminal amide-linked PEG conjugates of native and mutant erythropoietin glycoproteins by: selectively cleavable N-terminal peptide extensions are used, which are cleaved before PEGylation and after reversible citraconylation (cyclization) of all lysine residue amino groups of the glycoprotein. In this document, the idea of the multistep process is to select the free alpha amino group of the N-terminal amino acid for pegylation to produce a homogeneous mono-pegylated conjugate, thereby avoiding the separation of the mono-pegylated conjugate from the multiply pegylated derivative. This method differs from the present invention in a number of important ways, including but not limited to: (1) the Burg et al method is limited to erythropoietin glycoprotein, where the alkoxy PEG is linked via an amide linkage, and the present invention may employ various synthetic polymers to conjugate various bioactive components; (2) the present invention is applicable to both glycosylated and non-glycosylated "RN" and "RG" receptor binding proteins, whereas Burg et al only disclose conjugation of glycoproteins; (3) the present invention encompasses alkoxy PEGs (e.g., mPEG) as well as monofunctional activated hydroxyl PEGs, whereas Burg et al only disclose the use of alkoxy PEGs; and (4) the use of secondary amine linkages between the polymer of the invention and the protein is better than the amide linkages of Burg et al, because the former are more stable and retain the positive charge of the amino group. Burg, et al, (U.S. patent No.6,340,742) similarly worked to disclose the production of amide-linked conjugates of erythropoietin glycoproteins in which one to three-chain alkoxy PEGs were linked to one to three amino groups of the protein. However, in contrast to the present invention, this document does not prefer the alpha amino group of the N-terminal amino acid or the amino group of the region not involved in the interaction with the receptor.

Delgado et al, (U.S. patent No.6,384,195) disclose granulocyte-macrophage colony-stimulating factor conjugates prepared using the reactive polymer tresyl monomethoxypeg ("TMPEG"). This document shows that when TMPEG is contacted with recombinant human GM-CSF, the "modified material contains material that is inactive and more active than the unmodified material". One of ordinary skill in the art will immediately recognize that inactive materials in a mixture of polymer-bioactive ingredient conjugates (particularly therapeutic compositions comprising the conjugates) are undesirable because they have no beneficial effect on the patient in need thereof and may, instead, contribute to risk. The present invention overcomes, at least in part, the limitations of this technology by avoiding the modification of GM-CSF and other receptor binding proteins at sites on the protein involved in receptor-binding activity, thereby reducing or eliminating the synthesis of inactive substances. The present invention also provides methods for isolating and purifying conjugates having different sizes, different electrovalencies, and/or different degrees of electrical shielding of the protein by the polymer (see FIGS. 9-12).

It is noted that U.S. Pat. No.6,384,195 does not describe N-terminally PEGylated GM-CSF, and therefore does not show the advantages of the methods of the present invention. Finally, U.S. patent No.6,384,195 prefers conjugates in which more than one PEG is coupled to each GM-CSF molecule, and does not contemplate GM-CSF molecules (other than to lysine residues) to which PEG molecules are attached. Since up to six PEG molecules are thought to be conjugated to each GM-CSF, the document states that PEG may be attached to all possible lysine residues, ensuring that PEG will be attached at a position that sterically hinders the action of protein access to its cell-surface receptor (see figure 3 of the present specification). In contrast, the present invention indicates that coupling PEG to lysine residues is not necessary unless the lysine residues are very remote from the receptor interaction and therefore the functional receptor-binding protein domain that transmits the signal (agonist) or in order to competitively inhibit signal transduction (antagonist).

Nakamura, et al (PCT publication No. WO 02/32957 a1) disclose that increasing the molecular weight of PEG coupled to the amino group of a lysine residue at position 52 of an erythropoietin glycoprotein increases the erythropoietic effect of the conjugate in vivo and decreases the affinity of the conjugate for the erythropoietin receptor. However, in contrast to the present invention, this reference does not disclose coupling PEG at the amino terminus or near the glycosylation site, nor does it recognize any of its advantages.

Thus, the present invention provides conjugates of bioactive components coupled to synthetic polymers that have significant structural and functional advantages over those previously described.

Composition comprising a metal oxide and a metal oxide

The present invention provides conjugates or complexes comprising one or more bioactive components (suitably one or more cytokines) coupled to one or more stabilizing polymers such as one or more PEGs. Typically, the conjugates are made by the methods of the invention described herein; however, conjugates having the structure and activity described herein, regardless of the method used to produce the conjugate, are contemplated to be equivalent to the methods of the invention and are therefore included within the invention. In related aspects, the invention also provides compositions comprising one or more conjugates or complexes. Compositions according to this aspect of the invention will comprise one or more (e.g., one, two, three, four, five, ten, etc.) of the conjugates or complexes of the invention described above. In certain aspects, the compositions may comprise one or more additional ingredients, such as one or more buffer salts, one or more chaotropic agents, one or more detergents, one or more proteins (e.g., albumin or one or more enzymes), one or more unbound polymers, one or more osmotically active agents, and the like. The compositions of this aspect of the invention may be in any form, including solid (e.g., dry powder) or solution (especially in the form of a physiologically compatible buffered saline solution comprising one or more conjugates of the invention).

A. Pharmaceutical composition

Certain compositions of the invention, particularly pharmaceutical compositions that can be formulated for prophylactic, diagnostic or therapeutic use. The compositions typically comprise one or more conjugates, complexes or compositions of the invention and one or more pharmaceutically acceptable carriers or excipients. The term "pharmaceutically acceptable carrier or excipient" herein means a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or any type of formulating excipient that is tolerable to the recipient animal, including humans or other mammals, into which the pharmaceutical composition is introduced, without adverse effects.

The pharmaceutical compositions of the invention may be administered to a recipient animal via any suitable mode, for example: modes of administration orally, rectally, parenterally, systemically, vaginally, intraperitoneally, topically (powders, ointments, drops, or transdermal patches), buccally, as an oral or nasal spray or inhaler, and the like. The term "parenteral" as used herein is intended to encompass intravenous, intraarterial, intramuscular, intraperitoneal, intracisternal, subcutaneous and intraarticular injection and infusion modes of administration.

The parenteral pharmaceutical compositions provided by the present invention may comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include: water, ethanol, polyols (such as glycerol and the like, propylene glycol, poly (ethylene glycol)), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

The pharmaceutical compositions of the invention may also contain adjuvants such as: preservatives, wetting agents, emulsifiers and dispersants. Prevention of the action of microorganisms can be achieved by the addition of various antibacterial and antifungal agents, for example: paraoxybenzoic acid, benzyl alcohol, chlorobutanol, phenol, sorbic acid, and the like. Penetrants such as sugars, sodium chloride, and the like may also be desirably included. Agents such as aluminum monostearate which delay absorption are included; hydrogels and gelatins can provide prolonged absorption of injectable drug forms.

In some cases, in order to prolong the drug effect, the absorption from subcutaneous or intramuscular injection can be satisfactorily delayed. This can be done using suspensions of crystalline or amorphous materials with poor solubility in body fluids. The rate of absorption of the drug then depends on its rate of dissolution, which depends on its physical form. In addition, prolonged absorption of the parenterally administered drug form can be accomplished by dissolving or suspending the drug in an oil vehicle.

The formation of drug microencapsulated matrices with biodegradable macromolecules such as polypropiolactone-polyolactone can create injectable depot forms. Depending on the ratio of drug to carrier polymer and the nature of the particular carrier polymer, the rate of drug release can be controlled. Examples of other biodegradable polymers include biocompatible poly (n-esters) and poly (anhydrides). Depot injectable formulations can also be prepared with body tissue compatible liposomes or microemulsions to entrap the drug.

The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by the addition of a disinfectant in the form of a sterile solid composition which is dissolved or dispersed in sterile water or other sterile injectable solution prior to use.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In this solid dosage form, the active compound is mixed with at least one pharmaceutically acceptable excipient or carrier, such as: sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as: starch, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as: carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as: agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as: cetyl alcohol and glycerol monostearate, h) adsorbents such as kaolin and bentonite, and i) lubricants such as: talc, calcium stearate, magnesium stearate, solid PEG, sodium lauryl sulfate, and mixtures thereof. Capsules, tablets, pills, and the like may also contain buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard gelatin capsules using excipients such as lactose and high molecular weight PEG.

Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings or chronic control coatings and other coatings well known in the art of pharmaceutical compounding. Optionally compositions containing opacifying agents and releasing the active ingredient only (or preferentially) in certain parts of the gastrointestinal tract, optionally in a sustained release manner. Examples of useful embedding compositions include polymeric materials as well as waxes. The active compound may also be in microencapsulated form, optionally with one or more of the excipients mentioned above.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, for example: water or other solvents, dissolving agents, and emulsifiers, such as: ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (specifically, cottonseed, groundnut, corn, germ, olive oil, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, PEGs, and sorbitan fatty acid esters, and mixtures thereof.

In addition to inert diluents, the oral compositions may also contain adjuvants such as: wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents and flavoring agents.

In addition to the active compounds, suspensions may also contain suspending agents such as: ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide (aluminum metahydroxide), bentonite, agar-agar, tragacanth, and mixtures thereof.

Topical administration includes application to the skin or mucous membranes, including the lungs as well as the ocular surfaces. Compositions for topical administration, including inhalants, may be prepared using dry powders, whether pressurized or non-pressurized. In non-pressurized powder compositions, the active ingredient in finely divided form may be mixed with a larger pharmaceutically acceptable inert carrier comprising particles of a size, for example up to 100 microns in diameter. Suitable inert carriers include sugars such as lactose and sucrose. Suitably, at least 95% by weight of the active ingredient particles have an effective particle size in the range 0.01 to 10 microns.

Alternatively, the pharmaceutical composition may be pressurized and contain a compressed gas, such as nitrogen or a liquid gas propellant. Preferably, the active ingredient is not soluble to any substantial extent in the liquefied propellant. The pressurized composition may also contain a surfactant. The surfactant may be a liquid or solid nonionic surfactant or may be a solid anionic surfactant. The solid anionic surfactant is preferably used in the form of a sodium salt.

Other forms of topical administration are ophthalmic administration forms. In this form of administration, the conjugates or compositions of the invention are delivered in a pharmaceutically acceptable ophthalmic vehicle, and the active compound is maintained in contact with the ocular surface for a time sufficient to allow the compound to penetrate the conjunctiva or cornea of the eye and the internal regions of the eye, such as the anterior chamber, the posterior chamber, the vitreous body, aqueous humor, vitreous humor, cornea, iris/cilia, lens, choroid/retina, and sclera. Pharmaceutically acceptable ophthalmic vehicles are, for example, ointments, vegetable oils, or encapsulating materials.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by stirring the conjugates or compositions of the invention with a suitable non-irritating excipient or carrier such as cocoa butter, PEG or a suppository wax which is solid at room temperature but melts to a liquid at the rectal or vaginal temperature and releases the drug.

The pharmaceutical compositions of the present methods of treatment may also be administered in the form of liposomes. Liposomes are generally known in the art to be derived from phospholipids or other lipid materials. Liposomes are formed by dispersing mono-or multilamellar hydrated liquid crystals in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid can be used to form the liposomes. In addition to one or more of the conjugates or compositions of the invention, the pharmaceutical compositions in liposome form may also contain one or more stabilizers, preservatives, excipients, and the like. Preferred lipids are phospholipids and phosphatidylcholines (lecithins), both natural and synthetic. Methods of forming liposomes are known in the art (see, e.g., Zalipsky, s., et al, U.S. patent No.5,395,619). Liposomes comprising phospholipids can be coupled to PEG, most commonly phosphatidylethanolamine coupled to monomethoxy-PEG, with advantageous properties including extended life span in mammalian blood circulation (Fisher, d., U.S. patent No.6,132,763).

B. Use of

The methods, conjugates, and compositions of the invention can be advantageously used to maintain or enhance the biological activity of a biological component without interfering with the ability of the biological component to bind to its receptor. Certain of the methods of the invention can deliver one or more conjugates and compositions to a cell, tissue, organ, or organism. In particular, the present invention provides for the controlled delivery of one or more conjugate, complex or composition components to a cell, tissue, organ or organism, thereby allowing a user to modulate the release of an active amount of a particular component to a cell, tissue, organ or organism both temporally and spatially.

The methods of the invention generally involve one or more activities. For example, one of the methods of the present invention comprises: (a) preparing one or more conjugates or compositions of the invention; and (b) contacting one or more cells, tissues, organs or organisms with one or more conjugates or compositions of the invention under conditions conducive to binding of the one or more conjugates or compositions to the cells, tissues, organs or organisms. Once the biologically active component of the conjugates and/or compositions of the invention has bound to (or in some cases internalized into) a cell, tissue, organ or organism, this component can continue to perform its intended biological function. For example, a peptide component can bind to a receptor or other component on or within a cell, tissue, organ, or organism; participating in metabolic reactions in cells, tissues, organs or organisms; performing, up-regulating or activating, or down-regulating or inhibiting the activity of one or more enzymes in a cell, tissue, organ or organism; providing a structural component lost to a cell, tissue, organ or organism; providing one or more nutrients required by a cell, tissue, organ or organism; inhibiting, treating, reversing, or ameliorating the progression or symptoms of one or more diseases or disorders of the body; and the like.

The conjugates and compositions of the invention are useful in other embodiments for industrial cell culture, due to the surprisingly high potency of the biologically active components of the conjugates to substantially retain their biological activity and to increase the combined effect over a sustained period of action even under conditions of industrial use. Such surprisingly high potency of the present conjugates can lead to high biological yields, very high recombinant protein expression levels, and increased efficiency of other bioprocesses.

C. Dosage regimen

The conjugates, complexes or compositions of the invention may be administered to a cell, tissue, organ or organism in vitro, in vivo or ex vivo to deliver one or more bioactive components (i.e., one or more cytokines or antagonists thereof). One of ordinary skill in the art will recognize that an effective amount of a given active compound, conjugate, complex or composition can be determined empirically and that the pure form or, if present, a pharmaceutically acceptable formulation or prodrug form can be used. The compounds, conjugates, complexes or compositions of the invention can be administered to an animal (including mammals, e.g., humans) in need thereof in a veterinary or pharmaceutical composition in combination with one or more pharmaceutically acceptable excipients. The therapeutically successful dose for any particular patient will depend on a variety of factors including: the type and extent of cellular response achieved; the identity and/or activity of the particular compound, conjugate, complex, or composition used; patient age, body weight or surface area, overall health, gender, and diet; time of administration, route of administration, and rate of excretion of the active compound; the duration of the treatment; other pharmaceutical products in combination or concomitant with a particular compound, conjugate, complex or composition; and other factors well known to those of ordinary skill in the art familiar with medicine and medical science. For example, one of ordinary skill in the art can start with a given compound, conjugate, complex or composition of the invention at a lower dose than that required to achieve the desired therapeutic effect and slowly increase the dose until the desired effect is achieved.

The dosage regimen may also be arranged in a patient-specific manner to provide a predetermined concentration of a given active compound in the blood, which may be determined by techniques acceptable and conventional in the art, such as size-exclusion, ion-exchange or reverse-phase high performance liquid chromatography ("HPLC"), bioassay or immunoassay, and the like. Thus, the patient dosage regimen can be adjusted to achieve a relatively constant blood level (as measured by HPLC or immunoassay) according to conventional and familiar methods well known to those of ordinary skill in the art of medicine and medical science.

D. Diagnostic and therapeutic uses

Diagnostic use of the conjugates of the invention cells or tissues with high binding capacity for cytokines, such as cancer, can be targeted in animals, especially humans, by administering the conjugates or compositions of the invention, wherein the conjugate (or one or more components, i.e. the bioactive component and/or the synthetic polymer) is labeled or comprises one or more detectable labels for detection according to methods known in the art, such as optical, radiological, fluorescent or resonance detection. For example, most non-small cell lung cancers express very high concentrations of epidermal growth factor receptor (Bunn, P.A., et al, (2002) Semin Oncol29(Suppl 14): 38-44). Thus, in another aspect of the invention, the conjugates and compositions of the invention are useful in methods of diagnosis or treatment, such as diagnosis, treatment or prevention of a disorder in the body of various animals (particularly mammals such as humans) susceptible to or suffering from the disorder. In this method, the goal of treatment is to delay or prevent the progression of the disease, and/or to cure, induce remission, or maintain remission of the condition, and/or to reduce or minimize side effects of other treatment regimens.

Thus, the conjugates, complexes and compositions of the invention are useful for protecting, inhibiting or treating a condition of the body, such as an infection or disease. The term "protection" of a bodily disease as used herein includes "prevention", "inhibition" and "treatment". "preventing" comprises administering a complex or composition of the invention prior to induction of a disease or condition of the body, and "inhibiting" comprises administering a conjugate or composition prior to the onset of a clinical manifestation of the disease, with "preventing" and "inhibiting" of a condition of the body generally being directed to an animal that has a predisposition to, or is predisposed to, the condition, but has not yet experienced the disease. However, "treating" a disorder of the body comprises administering a therapeutic conjugate or composition of the invention after the manifestation of the disease. It is understood that human and veterinary medicine are not readily able to distinguish between "preventing" and "inhibiting" physical conditions. In many cases, the last induction event or events may be unknown or latent and cannot be noticed by the patient or physician before it occurs. Thus, in general, the term "prevention" as opposed to "treatment" herein encompasses "prevention" as well as "inhibition" as defined herein. The term "protection" herein encompasses "prevention" according to the methods of the present invention. Methods according to this aspect of the invention may include one or more steps that allow a clinician to achieve the above-described therapeutic goals. One of the methods of the present invention may comprise, for example: (a) identifying an animal (preferably a mammal, such as a human) suffering from or susceptible to a physical disorder; and (b) administering to the animal an effective amount of one or more of the conjugates, complexes or compositions described herein, such that administration of the conjugate, complex or composition prevents, delays or diagnoses the development of, or cures or induces the alleviation of, a physical disorder in the animal.

An animal "susceptible to a physical disorder" is defined herein as an animal that does not exhibit the majority of the apparent physical disorder, but is genetically, physiologically, or at risk of developing such a disorder. Identification of an animal susceptible to, at risk of, or having a given physical condition (e.g., a mammal, including a human being) in the methods of the invention can be accomplished by a skilled clinician according to methods known in the standard techniques, including, for example: radioactive assays, biochemical assays (e.g., determining the relative amounts of specific peptides, proteins, electrolytes, etc. in a sample from an animal), surgical procedures, genetic screening, family history, physical palpation, pathological or histological tests (e.g., microscopic evaluation of tissue or fluid samples or smears, immunoassays, etc.), test bodily fluids (e.g., blood, serum, plasma, cerebrospinal fluid, urine, saliva, semen, and the like), imaging (e.g., radio, fluorescence, optical, resonance (e.g., using nuclear magnetic resonance ("NMR") or electron spin resonance ("ESR")), etc.

The bodily conditions that can be prevented, diagnosed or treated with the conjugates, complexes, compositions and methods of the invention include any bodily condition that can be prevented, diagnosed or treated with the bioactive component (typically a cytokine or antagonist thereof) of the conjugate or composition. Such conditions include (but are not limited to): various cancers (e.g., breast, uterine, ovarian, prostate, testicular, leukemia, lymphoma, lung, neural, skin, head and neck, bone, colon and other gastrointestinal, pancreatic, bladder, kidney and other cancers, sarcomas, adenocarcinomas, and bone marrow); iatrogenic diseases; infectious diseases (e.g., bacterial diseases, fungal diseases, viral diseases (including those caused by hepatitis, cardiotropic virus, HIV/AIDS, and the like), parasitic diseases, and the like); genetic diseases (e.g., cystic fibrosis, amyotrophic lateral sclerosis, muscular dystrophy, gaucher's disease, glycogen storage disease type II, severe combined immunodeficiency disorders, dwarfism and the like), anemia, neutropenia, thrombocytopenia, hemophilia and other blood disorders; neurodegenerative diseases (e.g., multiple sclerosis ("MS") including, but not limited to, relapsing-remitting MS, primary progressive MS, secondary progressive MS, and the like), Creutzfeldt-Jakob disease, Alzheimer's disease, and the like); enzymatic disorders (e.g., gout, uremia, hypercholesterolemia, and the like); unknown or multifocal etiology (e.g., cardiovascular disease, hypertension, inflammatory bowel disease, and the like); autoimmune disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis, psoriasis, and the like) and other medically important disorders well known to those skilled in the art. The conjugates, complexes, compositions, and methods of the invention are also useful for preventing disease progression, e.g., chemically preventing progression from a pre-malignant condition to a malignant condition.

The treatment methods of the invention thus employ one or more conjugates, complexes or compositions of the invention, or one or more pharmaceutical compositions of the invention, which can be administered to an animal in need thereof via a variety of routes, including: oral, rectal, parenteral (including: intravenous, intraarterial, intramuscular, intraperitoneal, intracisternal, subcutaneous and intraarticular injection and infusion), systemic, vaginal, intraperitoneal, topical (powders, ointments, drops or transdermal patches), buccal, oral or nasal spray or inhalation. In the present invention, an effective amount of the conjugate, complex or composition can be administered to a cell or an animal suffering from or susceptible to a particular disorder in vitro, ex vivo or in vivo, thereby preventing, delaying, diagnosing or treating the disorder in the animal. By "effective amount of a conjugate (or complex or composition)" herein is meant a biologically active amount of a biologically active ingredient (i.e., a cytokine or antagonist thereof) carried by the administered conjugate (or complex or composition) of the invention that prevents, delays, diagnoses, treats, or cures a physical condition in the animal to which the conjugate, complex or composition is administered. One skilled in the art will recognize that an effective amount of a conjugate, complex or composition of the invention, as determined experimentally according to standard methods well known in medicine and medical arts; see, e.g., Beers, m.h., et al, eds. (1999) Merck Manual of Diagnosis & Therapy, 17th edition, Merck and co., Rahway, NJ; hardman, J.G., et al, eds. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th edition, McGraw-Hill Medical Publishing Division, New York; sight, t.m., et al, eds. (1997) Avery's Drug Treatment, 4th edition, Adis International, Auckland, New Zealand; katzung, B.G, (2000) Basic & Clinical Pharmacology, 8th edition, Lange medical Books/McGraw-Hill, New York; (ii) a This reference, as well as the cited references, are hereby incorporated by reference in their entirety.

It is understood that the total daily, weekly or monthly dosage of the conjugates, complexes and compositions of the invention when administered to a human patient will be determined by a physician within the sound judgment of medicine. For example, the appropriate dosage to achieve satisfactory results for administration of certain conjugates, complexes or compositions of the invention will depend on the specific biologically active compound used, and the dosage will be readily determinable by one skilled in the art, or using only routine experimentation. According to this aspect of the invention, the conjugate, complex or composition may be administered in a single dose, in divided doses, for example: once or twice daily, or once or twice weekly, or once or twice monthly, etc. The appropriate dosage regimen for a wide variety of different modes of administration (e.g., parenteral, subcutaneous, intramuscular, intraocular, intranasal, etc.) can also be readily determined using only routine experimentation, or by one of skill in the art depending on the identity of the biologically active ingredient (i.e., cytokine or antagonist thereof) of the conjugate, complex or composition.

In other applications, the conjugates, complexes and compositions of the invention can specifically target diagnostic or therapeutic agents to cells, tissues, organs or organisms that express receptors that bind, integrate or ingest the biologically active components (i.e., cytokines or antagonists thereof) of the conjugates, complexes and compositions. Methods according to this aspect of the invention may include, for example: contacting a cell, tissue, organ or organism with one or more conjugates, complexes or compositions of the invention additionally comprising one or more diagnostic or therapeutic agents, such that the conjugate, complex or composition can bind to or be taken up into the cell, tissue, organ or organism, thereby delivering the diagnostic or therapeutic agent to the cell, tissue, organ or organism. The diagnostic or therapeutic agent according to this aspect of the invention may be, but is not limited to, at least one agent selected from the group consisting of nucleic acids, organic compounds, proteins or peptides, antibodies, enzymes, glycoproteins, lipoproteins, chemical elements, lipids, sugars, isotopes, carbohydrates, imaging agents, detectable probes, or any combination thereof, which may be detectably labeled as described herein. The therapeutic agents of this aspect of the invention may exert a therapeutic effect on a target cell (or tissue, organ or organism) selected from (but not limited to): a gene or protein that corrects a defect, a drug action, a toxic action, a growth stimulating effect, a growth inhibitory effect, an anabolic effect, a catabolic effect, an anabolic effect, an antiviral effect, an antifungal effect, an antibacterial effect, a hormonal effect, a neurohumoral effect, a cell differentiation stimulating effect, a cell differentiation inhibitory effect, a neuromodulatory effect, an anticancer effect, an anti-tumor effect, an insulin stimulating or inhibiting effect, a bone marrow stimulating effect, a pluripotent stem cell stimulating effect, an immune system stimulating effect, and any other well-known therapeutic effect provided by delivery of a therapeutic agent to a cell (or tissue, organ or organism) via a delivery system according to this aspect of the invention.

The additional therapeutic agent may be selected from, but is not limited to, well-known and novel compounds and compositions, including antibiotics, steroids, cytotoxic agents, vasoactive drugs, antibodies, and other therapeutic agents. Non-limiting examples of such agents include antibiotics and other drugs used to treat bacterial shock, such as: gentamicin, tobramycin, nafcillin, parenteral cephalosporins, and the like; adrenocortical steroids and analogs thereof, such as: dexamethasone to mitigate endotoxin-induced cellular injury; vasoactive drugs such as alpha adrenergic receptor blockers (e.g., phenoxybenzamine), beta adrenergic receptor agonists (e.g., isoproterenol), and dopamine.

The conjugates, complexes and compositions of the invention can also be used to diagnose diseases and monitor therapeutic responses. In certain methods the conjugates, complexes, and compositions of the invention may comprise one or more detectable labels (e.g., as described elsewhere herein). Such detectably labeled conjugates, complexes, and compositions of the invention in this method can be used to detect cells, tissues, organs, or organisms that express the receptor or take up the biologically active component of the conjugate, complex, or composition (i.e., the cytokine or antagonist thereof). In one example of such a method, cells, tissues, organs, or organisms are contacted with one or more of the conjugates, complexes, and compositions of the invention under conditions that facilitate binding or uptake of the conjugate by the cell, tissue, or organism (e.g., binding of the conjugate to a cell-surface receptor or endocytosis or diffusion of the conjugate to a cell), and then binding or incorporation of the conjugate to the cell is detected using label-specific detection methods (e.g., fluorimetry, observation of fluorescently labeled conjugate; magnetic resonance imaging, observation of magnetically labeled conjugate; radiographic imaging, observation of radiolabeled conjugate, etc.). Other uses of the detectably labeled conjugate can include, for example, administering one or more labeled forms of the conjugate of the invention in an amount effective to image structures within a cell, tissue, organ, or organism, or an animal (including a human), and measuring detectable radiation associated with the cell, tissue, organ, or organism (or animal). Methods for detecting a wide variety of markers and their use in diagnostic and therapeutic imaging are well known to those skilled in the art and are described elsewhere herein.

In another aspect, the conjugates and compositions of the invention are useful for modulating the concentration or specific activity of a receptor for a bioactive component in a conjugate, the receptor being located on the surface of a cell expressing the receptor. By "modulating" the activity of a given receptor is meant that the conjugate, when bound to the receptor, can activate or inhibit a physiological activity (e.g., an intracellular signaling cascade) modulated via the receptor. While not binding to a particular mechanism to explain the activity modulated by the conjugates of the invention, the binding of the conjugate to the receptor via the bioactive component antagonizes the physiological activity of the cellular receptor, thereby blocking the binding of the native agonist (e.g., unconjugated bioactive component) and preventing the activation of the receptor by the native agonist, without inducing substantial activation of the physiological activity of the receptor itself. The method according to this aspect of the invention may comprise one or more steps, such as contacting the cell (in vitro or in vivo) with one or more conjugates of the invention under conditions in which the conjugate (i.e. the biologically active component portion of the conjugate) binds to, but does not substantially activate, a receptor for the biologically active component on the surface of the cell. The method will be useful for a variety of diagnostic, as well as therapeutic, uses that will be recognized by those skilled in the art.

Reagent kit

The invention also provides kits comprising the conjugates and/or compositions of the invention. The kit typically comprises a carrier, e.g., a carton, tube, etc., having one or more containers, e.g., vials, tubes, ampoules, bottles, syringes, and the like, closed, wherein a first container contains one or more conjugates and/or compositions of the invention. Kits of this aspect of the invention may further comprise one or more other components (e.g., reagents and compounds) necessary for performing the specified uses of one or more conjugates and compositions of the invention, such as one or more components useful for diagnosing, treating or preventing a particular disease or physical condition (e.g., one or more other therapeutic compounds or compositions, one or more diagnostic agents, one or more carriers or excipients, and the like), one or more other conjugates or compositions of the invention, and the like.

One of ordinary skill in the art will recognize that the methods and uses described herein can be suitably modified and adapted without departing from the scope of the invention or any embodiment thereof. Having now described the invention in detail, it will be apparent that the same is by way of illustration and not by way of limitation with reference to the following examples.

Examples

Example 1: PEG-interferon-alpha conjugates

Interferon-alpha is a commercially important pharmaceutical protein, which is more than twenty billion dollars in the world market in 2001, primarily for the treatment of hepatitis C virus ("HCV") infected patients. There are three to four million people infected with chronic hepatitis C in the United states and about 10,000 HCV-related deaths per year (Chander, G., et al, (2002) Hepatology 36: 5135-5144). There are two companies that are primarily responsible for development and marketing in improving useful interferon-alpha (Schering-Plough Corp. and F. Hoffmann-La Roche AG), conjugates of interferon-alpha and monomethoxy poly (ethylene glycol) or "mPEG." have been developed and marketed. In each case, mPEG is linked to each molecule of interferon- α only at one attachment point. In each case, the product contains a mixture of positional isomers with significantly reduced receptor-binding activity compared to the unmodified interferon. In each case, the increased bioavailability and duration of action of the conjugate in vivo compensates for the reduced in vitro bioactivity of PEG conjugation, which can be improved to the clinical efficacy of once weekly injections, compared to three weekly injections of unmodified protein, for the treatment of chronic infection with HCV (Manns, M.P., et al, (2001) Lancet 358: 958-za 965).

In the PEG-interferon- α -2a conjugate of f.hoffmann-La Roche, two-stranded 20-kDa mPEG is coupled to a single lysine linker (so-called "branched PEG") that is linked predominantly to one of Lys 31, Lys 121, Lys131 or Lys 134 (Bailon, p., et al, supra), with all of the more Lys located within or adjacent to the interferon- α -2a receptor-binding domain (see binding site 1 of fig. 1 a).

In the PEG-interferon- α -2b conjugate of Schering-Plough Corp., a single-chain 12-kDa mPEG is primarily coupled to a histidine residue at position 34 (His 34; Wylie, D.C., et al, supra; Gilbert, C.W., et al, U.S. patent No.6,042,822; Wang, Y. -S., et al, supra) located in a region of interest for binding to the receptor (see FIG. 1 b). The PEG attachment sites (Lys 121, Tyr 129 and Lys131) of the other Schering-Plough products are also located at or near the binding site 1 (FIG. 1 b).

In contrast to these two commercial products, the conjugates of the invention have a single-chain, water-soluble synthetic polymer, preferably PEG or mPEG, attached to the N-terminal amino acid residue, which is remote from the receptor-binding region of the protein (see Cys-1 and the spatial relationship of the binding sites, FIGS. 1c and 1d), revealing that interferon- α is an "RN" cytokine. Figures 9 and 10 show cation-exchange and size exclusion chromatography, respectively, of exemplary PEG-interferon-alpha conjugates of the invention. The reaction mixture contains interferon- α -2b preceded by an amino-terminal Cys-1 residue, the Cys-1 residue being the first residue in the native sequence. The reactive PEG was 20-kDa PEG-aldehyde and its concentration was 0.2 mM. The reducing agent was sodium cyanoborohydride, at a final concentration of 14 mM. The progress of the reaction was monitored periodically by size-exclusion chromatography during the incubation at 4 ℃. Although interferon- α is sufficiently soluble to be pegylated under the conditions described, other cytokines such as interferon β are less soluble and require the presence of a surfactant to be pegylated, as is interferon- α of c.w. gilbert et al, (U.S. patent No.5,711,944), and interferon α and β of r.b. greenwald, et al, (U.S. patent No.5,738,846).

The cation-exchange column used for fractionation (shown in FIG. 9) was ToyoPearl MD-G SP (1X6.8 cm; Tosoh Biosep, Montgomery-ville, Pa.), developed with a linear gradient of 0-0.4M NaCl in 20mM sodium acetate buffer, pH4.6, at a flow rate of 0.5 ml/min. The size-exclusion column used to obtain the data of FIG. 10 was200(HR 10/30; Amersham Biosciences, Piscataway, NJ) with 20mM sodium acetate buffered at 0.5 ml/min with 150mM NaClThe wash (pH4.6) elution, other appropriate ion-exchange and size exclusion chromatography media and fractionation conditions are well known to those skilled in the art, amino-terminal amino acid analysis of the purified mono-PEG-interferon- α -2b of the present invention by automated Edman degradation showed > 90% of the PEG attached to the N-terminal residue, this analysis was performed by Commonweloth Biotechnologies, Inc. (Richmond, Va.).

Example 2: PEG-interleukin-2 conjugates

Interleukin-2 ("interleukin 2") is a cytokine with immunomodulatory activity against certain cancers, including renal cell cancers and malignant melanoma. However, clinical efficacy is poor, and is only partially or completely effective in a small fraction of patients (Weinreich, D.M., et al, (2002) J lmmunother 25: 185-187). Interleukin 2 has a short half-life in the bloodstream and a low rate of remission induction in cancer patients. Attempts to make interleukin 2 more suitable by random pegylation of lysine residues have not been optimized (Chen, S.A., et al, (2000) J Pharrnacol Exp Ther 293: 248-259). Attempts to selectively attach PEG to the glycosylation site of interleukin 2 (Goodson, r.j., et al, supra) or to the non-essential cysteine (Cys 125) or to the cysteine contained within residues 1 to 20 of the interleukin 2 mutein (Katre, n., et al, U.S. patent No.5,206,344) did not produce a suitable clinical product.

FIG. 4 shows the distribution of lysine residues in the receptor-binding region of interleukin 2, showing that many surface-accessible lysine residues are located in the receptor-binding region. Actually, Lys-35 and Lys-43 were confirmed to be essential for the interaction of the alpha-receptor with interleukin 2, suggesting a mechanism for inactivating interleukin 2 by PEGylating lysine residues. FIG. 4 also shows that the N-terminal region of interleukin 2 is remote from the receptor-binding domain of the protein, revealing that interleukin 2 has an "RN" cytokine structure. Our conclusion is that interleukin 2 is an "RN" cytokine compatible with the observations made by H.Sato, et al, ((2000) bioconjugateg Chem 11: 502-509) that uses an enzymatic transglutamaterial reaction to link one or two strands of 10-kDa mPEG to one or two glutamine residues ("Q") on the N-terminal overhang sequence AQQIVM of the author introduced interleukin 2 muteins. Sato et al reported that conjugates of muteins pegylated near the amino terminus by transglutaminic reaction retained more biological activity than conjugates prepared by randomly pegylating the lysine residue of interleukin 2 muteins. Similar methods for PEGylating other proteins can be found in Sato, H. (2002) supra. Based on the spatial separation of the amino-terminal end of interleukin 2 from the receptor-binding region of the protein, as shown in FIG. 4, it can be appreciated that the glycosylation site residue Thr-3 (not shown) causes interleukin 2 to be a "RG" receptor-binding protein as defined herein. Thus, interleukin 2 is a RN cytokine as well as an RG cytokine.

FIGS. 11 and 12 show cation-exchange and size exclusion chromatography, respectively, of a PEG-interleukin 2 conjugate of the invention, which is selectively PEGylated (reductively alkylated) at the N-terminus, as in example 1. The conditions of the fractionation are consistent with the description of fig. 9 and 10. FIG. 13 shows polyacrylamide gel electrophoretic analysis of the same conjugate in the presence of sodium dodecyl sulfate ("SDS-PAGE") before and after ion exchange chromatography purification, as shown in FIG. 11. Bis-Tris buffer gradient containing 4-12% total acrylamide in the gel (catalog # NP0335, Invitrogen, Carlsbad, Calif.). The samples, each containing about 1-2 micrograms of protein, were heated at 90 ℃ for 10 minutes prior to analysis. The gel was operated at a fixed voltage 117 and 120 for about 135 minutes under cooling. For partial gelationRuby protein gel dyes (Molecular Probes, Eugene, OR) staining and other fractions PEG staining was performed using modified C.E.Childs ((1975) Microchem J20: 190-. The two-absorption peak of the purified mono-PEG-interleukin of the invention (fig. 11) was subjected to amino-terminal amino acid analysis by automated Edman degradation showing that > 90% of the PEG was attached to the N-terminal residue. The assay was performed by Commonwelth Biotechnologyes, inc. (Richmond, VA).

Example 3: members and non-members of the "RN" receptor binding protein family

FIGS. 2, 3, and 5-8 show the surface distribution of the receptor-binding proteins interferon beta, granulocyte-macrophage colony-stimulating factor ("GM-CSF"), epidermal growth factor ("EGF"), basic fibroblast growth factor ("bFGF", also known in the art as "FGF-2"), insulin-like growth factor-1 ("IGF-1"), and interferon-gamma ("interferon-gamma") relative to the lysine residues of their receptor-binding regions, and whether such proteins are "RN" cytokines and growth factors. In addition, FIG. 2 shows that interferon-beta is an "RG" cytokine.

FIG. 2 shows the distribution of lysine residues in binding site 1 and binding site 2 regions of interferon β, whereas the amino terminus of the polypeptide chain is remote from the protein receptor-binding region, demonstrating that interferon β is an RN cytokine.

FIG. 3 shows the distribution of lysine residues in the binding site 1 (binding to the alpha receptor) and binding site 2 (binding to the beta receptor) regions of GM-CSF, while the amino terminus of the polypeptide chain is remote from the protein receptor-binding region, demonstrating that GM-CSF is an RN cytokine.

FIG. 5 shows the distribution of lysine residues along the epidermal growth factor ("EGF") polypeptide chain, including lysine residues at or near the receptor-binding region of the protein, while the amino terminus of the polypeptide chain is remote from the receptor-binding region of the protein.

Figure 6 shows that several lysine residues of basic fibroblast growth factor ("bFGF") are involved in binding to the receptor or heparin, both of which are required for bFGF signaling (Schlessinger, j., et al, supra). The amino terminus of bFGF is remote from the bFGF heparin-binding region and sufficiently remote from the receptor binding site so that bFGF becomes an RN growth factor.

FIG. 7 shows that several lysine residues of insulin-like growth factor-1 ("IGF-1") are located within or adjacent to the receptor-binding domain of the polypeptide, whereas the amino-terminus of IGF-1 is remote from the receptor-binding domain, indicating that IGF-1 is an RN growth factor.

FIG. 8 shows that interferon-gamma ("interferon-gamma") is a homodimer in which two polypeptide chains have extensive interactions. Several lysine residues on each polypeptide are adjacent to amino acid residues on interferon-gamma involved in binding to the receptor or in the dimerization interface. The format of the "ball-and-bar" of amino acid residue Gln-1 reflects evidence of the functional importance of the N-terminal residue. (the crystal structure of this figure essentially includes an additional methionine residue (labeled "Met 0") that is not present in the native protein.) since the interferon- γ N-terminal residue is remote from the dimerization interface, N-terminal PEGylation may avoid the inhibitory effect of lysine PEGylation on interferon- γ homodimerization. On the other hand, coupling the polymer to the amino terminus can inhibit the interaction of the dimer with its receptor, especially when a long chain polymer is attached.

Interferon-gamma, IL-10 and stem cell factor are examples of homodimeric cytokines (Walter, M.R., et al, supra; Josephson, K., et al, (2000) J Biol Chem 275: 13552-13557; McNiece, I.K., et al, supra). Receptor-binding protein dimerization is a particular subject of the identification of N-terminally monopegylated conjugates, since different possible molecular structures may be present in similar or identical size and shape conjugate preparations. For example, one di-pegylated monomer and one non-pegylated monomer (PEG)₂-proteins₁+ protein₁) The constituent dimers would be difficult or impossible to evaluate by size-exclusion chromatography or sedimentation coefficient, light scattering or diffusion coefficient based analysis of dimeric conjugates with monomers pegylated by the di-N-terminus (PEG)₁-proteins₁)₂The constituent dimers are distinguished, and the receptor-binding capacity of two such conjugates containing an average of one PEG per protein monomer may be quite different.

When long chain β -sheet receptor binding proteins form homotrimers, such as tumor necrosis factor α ("TNF- α"), PEG₃-proteins₃Chemical modification of TNF near the amino terminus has been shown to inactivate cytokines (Utsumi, T., et al, (1992) Mol Immunol 29: 77-81), and TNF- α cannot retain substantial activity when the N-terminal residue is selectively pegylated with an agent.

In order to characterize cytokine conjugates that function as oligomeric forms, a combinatorial analytical approach is required. Amino-terminal sequence analysis can detect the presence of monomers having a free N-terminal alpha amino group, and electrophoretic analysis of dissociated monomers (e.g., SDS-PAGE or capillary electrophoresis) can reveal the presence of non-pegylated and multi-pegylated monomers in the receptor-bound protein. Without this evidence, it cannot be clearly stated that monopegylated conjugates of proteins forming homodimers as well as homotrimers are equally synthesized.

Such embodiments, and in particular those schematically illustrated in FIGS. 1-8, provide a basic understanding of the steric hindrance of a PEGylated receptor binding protein to protein-receptor interactions in regions within or adjacent to the receptor-binding domain of such bioactive agents. If PEG is coupled to the monomer interaction required region, then highly extended and elastic PEG chain (see figure 1d) occupied large volume will also be in the space of preventing some receptor binding protein monomer binding functionality of homodimers or homotrimers. Thus, targeted pegylation at a site remote from the receptor-binding region of the receptor binding protein can reduce the interference of pegylation with the intermolecular interactions required for its function. It is expected that PEGylated receptor binding proteins would be advantageous to perform the method according to the invention. The resulting conjugates have the benefits of improved solubility, increased bioavailability, increased stability, and reduced immunogenicity, while unexpectedly retaining high biological activity.

Example 4: PEGylation of interferon-beta-1 b by reductive alkylation

In one series of embodiments, conjugates of synthetic interferon- β -1b ("interferon- β -1 b; SEQ ID NO: 1) and monomethoxyPEG (" mPEG ") were made by reductively alkylating 20-kDa or 30-kDa mPEG-aldehyde using borane-pyridine complex as a reducing agent (Cabacungan, J.C., et al, supra.) interferon- β -1b, which does not contain a carrier protein, dissolved in a solution at a concentration of about 1.9 mg/ml, and which contains about 3 mg/ml SDS, obtained from Chiron corporation (Emeryville, Calif.)Sold in the form of preparations by Berlex laboratories (American subsidiary of Schering AG), also known asSold as a formulation from schering borane-pyridine complex (Aldrich 17, 975-2, Milwaukee, WI) is a 60% (v/v) aqueous acetonitrile solution of 20-kDa mPEG n-propanal ("PEG-aldehyde"; NOF Corporation, Tokyo) diluted to 450mM borane dissolved in 1mM HCl at a concentration of 30 mg/ml after dissolution, 0.1 ml of PEG-aldehyde solution is added to 0.7 ml of interferon- β -1b solution and mixed, 0.05 ml of 100mM acetic acid buffer (pH4.6) is added to the reaction mixture to bring the final pH of the reaction mixture to 5, 200mM acetic acid, 200mM Na are added to another reaction mixture containing 0.1 ml of PEG-aldehyde solution and 0.7 ml of interferon- β -1b solution₂HPO₄And 0.05 ml of a mixture of 68mM NaOH to a final pH of 6.4. To three 0.85-ml aliquots of these mixtures were added 0.1 ml of water, 1.5M NaCl or 10 mg/ml SDS, respectively. The diluted borane-pyridine complex was then added to each reaction mixture to yield a final concentration of 23mM borane. Each of the resulting reaction mixtures was divided into two tubes and reacted at 4 ℃ or room temperature for 2 days. An aliquot of the reaction mixture was treated with Superose in 10mM Tris, 150mM NaCl (pH8.3) containing 0.3 mg/ml SDS at a flow rate of 0.5 ml/min^TM12 columns (Amersham biosciences HR 10/30; Piscataw)ay, NJ) was analyzed by size exclusion HPLC the absorbance of the eluate was monitored at 214nm and the major product was the monopegylated interferon- β -1b (PEG) when the protein was dosed at a ratio of about 2 moles PEG/mole₁Interferon- β -1b) PEG at all test conditions (pH5 or pH 6.4; 4 ℃ or room temperature; presence or absence of NaCl or other SDS)₁The yield of interferon- β -1b was between 65% and 72%.

NaBH was used in other experiments₃CN as reducing agent and sample analysis was performed using size exclusion HPLC on a Superdex200HR 30/10 column (Amersham Biosciences) eluting with 10mM acetate, 150mM NaC l (pH4.6) (containing 1 mg/ml SDS), at a flow rate of 0.5 ml/min. the results of this experiment are shown in FIG. 14.20-mPEG input concentrations of approximately 0.1mM, 0.2mM, and 0.4mM (referred to as "1X", "2X", and "4X", respectively, in FIG. 14) and the reaction mixture is reacted at room temperature for 3 days. the control sample (bottom tracer) reacts only with the reducing agent. similar results are obtained when the same sample is chromatographed under the same conditions (except for SDS removed from the elution buffer), no unpegylated interferon- β -1b is detected in the eluate. the method of example 4 in which a 20-kDa mPEG-propionaldehyde was substituted for 20-mPEG-propionaldehyde, similar results are obtained using a similar method for the substitution of 10-mPEG-20-mPEG-propionaldehyde, and in addition, the results are obtained using a similar method for the production of interferon-36-terminal serine-alanine conjugates.

Example 5: determination of the extent of N-terminal PEGylation by oxidative cleavage

The ratio of PEG coupling to the amino group of the N-terminal serine residue α of the protein, rather than to the amino group of an accessible lysine residue, can be assessed using novel methods including oxidative cleavage of alkylated serine residues₁Reactive mixture of interferon- β -1b as the major species (approximately 70% of total protein)The material was dialyzed against 1 mg/ml SDS in acetate buffer (pH 4.6). Then 10mM Na was added₃PO₄The pH was adjusted to 7.4. Part of this solution is in the absence of sodium periodate at 4 ℃ or at a final concentration of 0.1 to 10mM NaIO₄The reaction was carried out for up to 20 hours. The reaction mixture after the reaction was made Superose^TM6 column chromatography using 10mM acetate buffer, 150mM NaCl (pH4.6) (containing 1 mg/ml SDS) similar results were obtained with Superose 12 column, which has the advantage of allowing resolution of the unmodified interferon- β -1 b.214nm absorbance absorption peak from the "salt absorption peak" corresponding to PEG₁Plot of the area of interferon- β -1b against periodate concentration, showing a drastic reduction in this area up to about 1mM periodate concentration and a residual PEG at 1mM to 10mM periodate concentration₁Interferon- β -1b maintained almost a fixed level similar analysis after 0.2, 2 or 7 hours of treatment with > 3mM periodate indicated that oxidative cleavage of serine-linked PEG was essentially complete within 2 hours residual PEG conjugates contained only lysine-linked PEG, which was stable up to 10mM periodate treatment the ratio of conjugates present after oxidation with periodate was similar to the ratio of pegylation sites other than amino termini estimated by Edman degradation.

Interferon- β -1b was coupled to 20-kDa PEG-aldehyde under various conditions described in example 4 using borane-pyridine complex as a reducing agent Monopegylated interferon- β -1b was purified by Superose 12 column chromatography using 20mM sodium phosphate buffer, 150mM NaCl (pH7.4) containing 0.3 mg/ml SDS at a flow rate of 0.5 ml/min₁Interferon- β -1b conjugate reacted for 2 hours at room temperature in the absence or presence of 3mM sodium periodate FIG. 15 shows untreated and oxidatively degraded PEG₁-drySuperose 12 column chromatography of interferon- β -1b samples eluted with 10mM Tris, 150mM NaCl (pH8.3) containing 0.3 mg/ml SDS at a flow rate of 0.5 ml/min₁The present example method also measures the extent of N-terminal reductive alkylation of proteins other than interferon- β -1b, where the proteins have N-terminal serine or threonine residues, and likewise, oxidative cleavage by reductive alkylation of polymers attached to N-terminal serine or threonine residues can be achieved using periodates other than sodium periodate, including, but not limited to, sodium metaperiodate (also referred to herein and in the art as sodium periodate), potassium metaperiodate, lithium metaiodate, calcium metaiodate, and barium periodate.

Polymer conjugates synthesized by reductive alkylation of other cytokines suitable for use in the method of example 5 include interleukin-1-alpha (Geoghegan, K.F., et al, supra) and megakaryocyte growth and genesis factors (Guerra, P.I., et al, supra).

Example 6: purification of the conjugate by reverse phase chromatography and removal of free PEG and SDS

Hershenson et al (U.S. Pat. No.4,894,330) purified bacterial cell culture-expressed interferon- β -1b using reverse phase ("RP") chromatography the inventors isolated the individual pegylated compounds synthesized in example 4 from the unmodified protein using the method of Hershenson et al, this step also resolved free PEG from protein absorption peaks and most of the SDS, FIG. 16 shows Jupiter^TMC4300A column (15 cm. times.4.6 mm; Phenomenex; Torrance, Calif.) analytical chromatography with 20% acetonitrile plus 0.04% trifluoroacetic acid to 80%Acetonitrile plus 0.1% trifluoroacetic acid gradient elution 0.1 ml of the reaction mixture was loaded and 0.5 ml fractions were collected at a flow rate of 1 ml/min.an absorbance at 280nm (solid line) detected the absorption peak of unmodified interferon- β -1b exposed to the PEGylation reagent ("Mock PEGylation") and the absorption peak of PEG conjugates containing one or more chains of PEG per molecular protein in this experiment, the column was maintained at 40 ℃ and chromatography at room temperature gave similar qualitative results with different retention times.

The results of the SDS assay in the collected fractions are shown as open triangles. Stock solutions of SDS assay reagent contained 1 mg/ml of the carbocyanine dye Stains-Al1(Sigma, # E-9379; 3,3 ' -diethyl-9-methyl-4, 5,4 ', 5 ' -dibenzothiocarbocyanine) dissolved in 50% (v/v) aqueous isopropanol (Rusconi, F., et Al, (2001) Anal biochem 295: 31-37). This working reagent was prepared using 2 ml of stock solution previously stirred plus 2 ml of N, N-dimethylformamide and 41 ml of water. The addition of SDS to the reagent resulted in a change in the specific SDS spectrum and a reduction in the absorption peak at 510-515nm and an absorption peak at 439 nm. Each 2mcL fraction from the RP chromatography column was added to 250mcL of the working reagent in a 96-well microtiter plate and the change in absorbance at 439nm was monitored with a SpectraMax 250 plate reader (Molecular Devices, Sunnyvale, CA).

The results of analyzing PEG in the collected fractions are shown in the filled circles of fig. 16. The PEG assay reagents were prepared by stirring 1 volume of 20% (w/v) barium chloride (dissolved in 1 equivalent of HC1) and 4 volumes of 4 mg/ml iodine in 1% (w/v) potassium iodide prior to use. To a 96-well microtiter plate containing 90mcL of water was added 10mcL of each RP column fraction (and PEG standard) followed by 100mcL of PEG assay reagents. After mixing the sample and the reagent, the reaction was carried out at room temperature for 15 minutes, and the absorbance at 508nm was measured by a SpectraMax plate reader. FIG. 16 shows a scheme for RP chromatography to separate Mock pegylated proteins from one or more strand-inclusive 20-kDa PEG conjugates, and to resolve unbound PEG as well as SDS from the conjugates. Similar results were obtained with conjugates containing other molecular weight PEGs (e.g., 10-kDa or 30-kDa PEG) and with other acid-stable linkages between PEG and interferon-beta-1 b.

Example 7: chromatographic and electrophoretic analysis of fractions purified by preparative reverse phase HPLC

Reverse phase chromatography conditions were similar to those described in example 6, and larger samples (0.3 or 0.5 ml) of the pegylation reaction mixture were analyzed using the same Jupiter C4 column with a modified gradient. When larger samples were loaded, a wide variety of interferons (Mock pegylated, PEG)₁Interferon- β -1b or conjugates with more than one chain of PEG) with less resolution than in fig. 16, however, fractions highly enriched in Mock pegylated or mono-pegylated proteins were obtained by re-chromatography of a small sample of partially purified fractions with the same RP column (fig. 17). chromatographic analysis was performed using EZChrom Elite Software (Scientific Software, inc., Pleasanton, CA.) from this analysis, the preparation shown in the upper curve (fraction 53 of the preparative RP column) contained about 70-80% Mock pegylated interferon- β -1b, while the preparation shown in the middle curve (fraction 51) contained about 99% PEG₁Interferon- β -1 b. bottom curve of fig. 17 shows the chromatogram of the reaction mixture from which the fractions are derived, where about 19% of the absorbance is related to Mock pegylated protein, about 61% and PEG₁Interferon absorption peaks correlation, about 12% and labeled PEG₂Correlation of interferon absorption peaks, and PEG at 5-6% vs. elution ratio₂Fast form correlation of interferon. Similar qualitative results were obtained from reverse phase columns from other manufacturers.

The same reaction mixture (RP chromatography analysis, FIG. 17) as well as the two RP chromatographically purified PEG-interferon fractions were analyzed by polyacrylamide gel electrophoresis in the presence of SDS ("SDS-PAGE"). This result is shown in fig. 18 and 19. The reaction of the samples with and without reducing agent (Invitrogen, # NP 0004; Carlsbad, CA) was repeated for 10 min at ambient temperature or 102 ℃ and electrophoresis at 120V for 140 min on 4-12% Bis-Tris gels (Invitrogen, # NP0321B). The samples shown in the traces of fig. 18 and 19 were neither reduced nor heated. For protein gelsRuby Stain # S-12000, Eugene, OR), 302nm illumination and photographs with orange/red visible filters (Molecular Probes, # S-6655) (FIG. 18). digital imaging was analyzed using 1D image analysis software of Kodak (Rochester, NY). horizontal axis represents the shift (100 units) relative to the dye front, vertical axis represents the relative intensity of fluorescent protein staining. the baseline values of each row have been shifted vertically to illustrate the results presented.the bottom trace represents a mixture of standard proteins (Mark12TM, # LC5677, Invitrogen) in which the absorption peaks 1 to 9 are identified as proteins with the following Molecular weights (expressed in kDa): 200, 116, 97.1, 66.3, 55.4, 36.5, 31.0, 21.5 and 14.4. the second trace shows a reaction mixture for electrophoretic analysis, the percentage of protein associated with each interferon-351 b in this mixture is the percentage of PEG-3564% staining pattern of protein₁-an interferon; 20% PEG₂Interferon and about 2% greater than PEG₂-a form of interferon. The third trace from the bottom shows an inclusion of 33. + -. 1% PEG₁-an interferon; 64. + -. 1% PEG₂Interferon and about 6% greater than PEG₂-an RP chromatography column fraction in the form of interferon. The top curve shows an inclusion of 95. + -. 1% PEG₁Interferon, about 2% Mock pegylated interferon and about 2% greater than PEG₂-fractions in the form of interferon. The above percentages are the average and standard deviation of four replicates of each sample. Similar qualitative results were obtained for the 10kDa and 30kDa PEGs.

FIG. 19 shows the result of SDS-PAGE analysis, which is the same as that of FIG. 18, except that 20% (w/v) BaCl was used as the gel₂1 equivalent of HC1 combined with 4 volumes of 4 mg/ml I₂1% (w/v) KI to PEG staining. The bottom trace represents the mixture of standard proteins before staining (pre-stain) (see Blue P1us2TM, Invitrogen # LC5625) with apparent molecules of proteins with absorption peaks 1 to 9The amounts are as follows (in kDa): 204. 111, 68.8, 51.5, 40.2, 28.9, 20.7, 14.9 and 0.6. Quantitative analysis of the PEG-stained gel showed that about 99% of PEG and PEG from fraction 51 of the RP column₁Interferon-related (apical curve), whereas the fraction enriched in diPEG conjugate (second curve from apical) is depleted of PEG by about 71%₂25. + -. 1% PEG in addition to interferon₁Interferon and about 3% greater than PEG₂To estimate the relative amount of interferon- β -1b in each different form stained with PEG, PEG was added₂The area under the interferon absorption peak divided by 2. In the reaction mixture (second curve from the bottom), about 50% of the PEG staining was associated with unbound PEG, and about 35% was associated with PEG₁Interferon related, about 14% and PEG₂Interferon related and about 1% and greater than PEG₂-form-related interferon.

Example 8: selective N-terminal oxidation and coupling to Low molecular weight Hydrazoformic acid of Interferon-beta-1 b

Coupling mPEG to the amino terminus of interferon- β -1b using the alternate method significantly increases the selectivity of the attachment site from about 90% to about 100% for reductive alkylation (described in examples 4 and 5). The first step of the PEGylation method is based on a principle similar to oxidative cleavage of reductively alkylated PEG-interferon- β -1b described in example 5. this method utilizes periodate to specifically cleave N-terminal serine or threonine residues to aldehydes as reported in H.B.F.Dixon (supra) and K.F.Geoghegan et al, ((1992) Bioconjug Chem 3: 138. 146; Geoghegan, K.F., U.S.Patent No.5,362,852; Drummond, R.J., et al, U.S. Pat. 6,423,685. U.S. Ser. No. β -1b, where the maximum degree of oxidation of the N-terminal serine residue is 3mM, e.g., IO 3₄After 2 hours of treatment at room temperature, the absorbance absorption peak of the protein was broadened by Superose6 column size-exclusion chromatography. Subsequent analysis on the Superose 12 column eluted with 10mM acetate, 150mM NaCl (pH4.6) containing 0.3 mg/ml SDS, which clearly removed oxidized proteinThe resolution was deduced to be in the form of protein monomers and dimers. The nature of the two absorption peaks was confirmed by SDS-PAGE as described in example 7.

The reverse phase chromatography analysis further found that: preferential oxidation of the N-terminal serine can be achieved with minimal oxidation of at least one essential methionine residue. L.S.Lin et al, ((1996) Pharrn Biotechnol 9: 275-301) and L.Lin ((1998) Dev Biol Stand 96: 97-104) showed that RP chromatography resolved the preparation of interferon-. beta.1B into a major component ("absorption peak B") and an earlier eluting minor component ("absorption peak A"). Lin ((1998) supra) further illustrates that the absorption peak A contains interferon-. beta. -1b, in which the functionally active methionine (Met 61 of BETASERON) is oxidized to the sulfoxide. The present inventors have found conditions under which N-terminal serine can be almost completely oxidized while minimizing the oxidation reaction of Met 61, which is reflected in the percentage of absorption peak A of RP chromatography. The oxidation of Met 61 as determined by RP chromatography may serve as a surrogate marker for the oxidation of other methionine residues (Met 35 and Met 116 of BETASERON) of interferon-beta-1 b.

Study of pH and reaction (0.25mM NaIO)₄The relationship between the 4 ℃ C. time and the degree of oxidation of Met 61 is summarized in Table 1.

Table 1: the effect of pH and periodate exposure time on the degree of methionine oxidation was determined from the region of the absorption peak A after reverse phase chromatography.

Confirmation of the aldehyde formed by the N-terminal oxidation of interferon- β -1b can be facilitated by its attachment to 9-fluorenylmethylcarbazate ("Fmoc-carbazate", also known in the art as "Fmoc-hydrazide") (Fluka 46917; Zhang, R. -E., et al, (1991) Anal Biochem 195: 160-A light detector. With NaIO of various concentrations₄Interferon- β -1b was oxidized by treatment at pH7.8 for various periods of time (0.5 to 2 hours at room temperature, or up to several days under cooling.) in the experiment shown in FIG. 20, the protein was 0.5mM NaIO at room temperature₄The reaction was carried out for 1 hour. The reaction was terminated by the addition of glycerol. After 30 minutes at room temperature, acetic acid was added to a final concentration of 19mM to lower the pH. To each ml of the resulting mixture, 182mcL of 15mM Fmoc-carbazate in methanol was added to give a final concentration of 2.3mM Fmoc-carbazate. The reaction mixture was allowed to react overnight at 4-8 ℃ before reverse phase chromatography.

FIG. 20 illustrates interferon- β -1b and 0.5mM NaIO₄Comparison of the results for the control sample (upper curve) and the oxidized sample (middle curve, open circles) shows an increase in the principal components and retention time after oxidation of absorption peak A (reflecting the presence of about 5% of interferon- β -1b with an oxidized methionine residue) by 0.2 to 0.3 minutes₄The oxidized methionine after the reaction was less than 1%.

The results of the bioassay described in example 11 provide additional evidence that controlled oxidation with 0.1 to 0.3mM periodate (e.g., up to 2 hours under cooling) preserves the integrity of important biologically active amino acid residues of the protein.

As shown in the lower solid triangular curve of figure 20, the major component as well as absorption peak a' (the N-terminal aldehyde derivative of absorption peak a) shifted to longer residence times, providing evidence of Fmoc adduct formation. In addition, the ratio of the absorbance at 278nm to 214nm (shifted absorption peak) was increased by 50%. For both forms of protein, the increase in retention time due to the formation of the corresponding N-terminal aldehydes (0.2-0.3 min) was much less than the increase in retention time due to the formation of the corresponding Fmoc derivatives (1.0-1.2 min).

Example 9: synthesis of PEG-carbazate adducts of N-terminally oxidized interferon-beta-1 b

Interferon- β -1b, which is selectively oxidized at the amino terminus (as in example 8), can also be coupled to a carbazate derivative of PEG using the methods described in R.J. Drummond et al (PCT publication No. WO 99/45026; U.S. Pat. No.6,423,685) and S.Zalipsky et al (PCT publication No. WO 92/16555A 1 and Harris, J.M, et al, eds., (1997) Chemistry and Biological Applications of Poly (ethylene glycol), pp.318-341, Washington, D.C., American Chemical Society.) PEG-carbazate is the product of the reaction of hydrazine with a para-carbonate derivative of 20-PEG ("NPC-PEG", NOF Corporation.) the protein lacks high iodate or 0.125mM NaIO at room temperature₄After 0.5, 1 or 2 hours of reaction in the presence, the sample was diluted with 4 volumes of 20-kDa PEG-carbazate in 10mM acetate buffer, 150mM NaCl (pH4.6) (containing 1 mg/ml SDS), and reacted at room temperature for 1 day. The samples were subjected to size-exclusion chromatography using Superose 12 column, eluting with 10mM Tris, 150mM NaCl (pH8.3) containing 0.3 mg/ml SDS. As shown in FIG. 21, the protein was oxidized for up to 2 hours prior to reaction with PEG-carbazate, resulting in a sustainable reduction in the concentration and sustained increase of unmodified protein (elution retention time of about 25 minutes) with PEG₁The ratio of absorbance associated with the interferon- β -1b conjugate (retention time for elution about 20 minutes)₁Interferon- β -1b was more than 80% similar results were obtained using monocarbazate derivatives of 10-kDa or 30kDa peg.

Example 10: bioassay of reverse phase HPLC-purified monopegylated interferon-beta-1 b

Antiproliferative assays of interferon- β -1a and various muteins were performed using human Daudi Burkitt's lymphoma cells (ATCC # CCL-231, Manassas, Va.)Runkel et al ((2000) Biochemistry 39: 2538) -2551) FIG. 22 depicts the results of an assay of antiproliferative activity of untreated interferon- β -1b and partially purified conjugates of N-terminally monopegylated conjugates of RP chromatography on Daudi cells grown in supplemented RPMI 1640 medium (Gibco #11875-093, Grand Island, NY) containing 10% (v/v) fetal bovine serum (Irvine Scientific #3000, Santa Ana, CA) inoculated ten thousand cells into each well of a 48-well plate containing 250mcL of culture medium and allowed to incubate at 37 ℃ and 5% CO₂Growth for 4 hours thereafter, equal volumes of pre-heated medium or medium diluted interferon- β -1b or PEG conjugate were mixed, cells were counted in a Coulter counter (Model ZI, Miami, FL) over a 3 day period, and the number of cells diluted with medium alone increased to 590 +/-24% (s.d.) at time zero, whereas the number of cells increased to 283 +/-8% at time zero under conditions of maximal inhibition of growth of interferon- β -1b or its PEG conjugate, so that the maximal percent growth inhibition observed in this experiment was 48%. the data for the two interferon- β -1b preparations at various concentrations in FIG. 22 were expressed as a percentage of inhibition of cell growth.

Figure 22 shows the results using interferon- β -1b stock solution dilutions and fractions from preparative reverse phase chromatography experiments (described in example 7) containing almost pure mono-PEG conjugates (as shown in figure 17-19, fraction 51), or almost pure Mock pegylated interferon- β -1b (figure 17, column shown, fraction 53). Triplicate samples were diluted with 1. mu.g/ml supplemented fetal bovine serum and culture broth filter sterilized through a 0.2-micron filter. From each initial dilution, 32 ng/ml and subsequent serial dilutions were prepared. From the data in fig. 22, the concentration at which each preparation inhibited 50% cell growth ("IC 50") was calculated. The results show that the mono-pegylated interferon-beta-1 b (IC 50= c.40pg/ml) was approximately 6-fold more potent than the unmodified interferon-beta-1 b (IC 50= c.250pg/ml). A series of experiments similar to FIG. 22 tested conjugates of PEG of various sizes with mean increases in potency against proliferation ranging from about 2.5-fold (10 kDa PEG) to about 5-fold (30 kDaPEG).

Surprisingly, the Mock PEGylated formulation shown in FIG. 22 had an IC50 of about 80 pg/ml, intermediate to the interferon-beta-1 b stock solution and the mono-PEGylated formulation. Without wishing to be bound by any theory or particular mechanism, a reasonable explanation is: the increase in the antiproliferative capacity of Mock PEGylated formulation indicates that reverse phase chromatography removed some of the inhibitory material present in the interferon-beta-1 b solution. This interpretation is consistent with the results of Superose6 column size-exclusion chromatography (SDS in elution buffer, described in example 4), which revealed that absorbance peaks at 214nm and 280nm eluted between interferon-. beta.1 b and the "salt peak" elution positions. Fraction 49 of the RP column, which contained a mixture of PEG 2-and PEG 1-Interferon-. beta.1 b (see FIGS. 18 and 19), was assayed by a bioassay similar to the experiment set forth in FIG. 22. Like the Mock PEGylated sample, the multi-pegylated sample had an antiproliferative capacity that was greater than the interferon-beta-1 b stock solution, but less than the mono-pegylated conjugate. In other experiments, a six-fold to ten-fold increase in potency of the monopegylated interferon-beta-lb was observed. Similar increases in potency were observed using 10, 20 and 30kDa PEG carbazate adducts (described in example 9).

The Daudi cells obtained with the conjugate of the invention have an antiproliferative capacity similar to the specific activity of the antiviral assays reported in the three interferon- β pharmaceutical forms, according to the respective packages,(IFN- β -1b) has an activity of 32X106 IU/mg,(Biogen's preparation of IFN- β -1a) the activity was 200X106 IU/mg and(Serono's preparation of IFN- β -la) was 270X106 IU/mg, accordingly, FIG. 22 illustrates that mono-pegylatedThe potency in the betaseroon antiproliferative assay was increased by a minimum of six-fold, showing that the potency of the N-terminally pegylated betaseroon of the method of the invention reached that of the commercially available glycosylation preparations AVONEX and REBIF.

It was previously mentioned that the solubility of unglycosylated interferon- β (expressed in E.coli) could be improved by using acidic solutions (Hanisch, W.H. et al, U.S. Pat. No.4,462,940) or by adding SDS (Thomson, J.W., U.S. Pat. No.4,816,440). without wishing to be bound by theory, the mechanism by which PEGylation could increase the antiproliferative efficacy of interferon- β -1b in vitro was by reducing its tendency to self-associate in the culture broth₂Potency ratio of samples of interferon- β -1b PEG₁Interferon- β -1b is poor, indicating that the positive effect of reduced agglutination can be masked by the negative effect of excess pegylation on the ability of the cytokine to bind to its receptor and/or to trigger signaling of antiproliferative activity.

Example 11: bioassay of selectively oxidized interferon-beta-1 b

The antiproliferative activity of interferon- β -1b, oxidized to various degrees, on Daudi cells was determined as described in example 10 test samples included a stock solution of interferon- β -1b and samples treated with 0.1, 0.3, or 3mM periodate at 4 ℃ for several days, samples were diluted as described in example 10, and equal volumes of Daudi cell suspension were mixed 4 hours after seeding the cells, which were 5% CO at 37 ℃₂Growth was continued for 2 days and then counted using a Coulter counter. 0.075-0.5mM NaIO under test conditions₄Treatment, whether interferon- β -1b antiproliferative activity is unaffected or increased.3 day antiproliferative assays (e.g., example 10) gave similar results.As described in example 9, conjugation of selectively oxidized interferon- β -1b to PEG-hydrazinoformate further enhanced the antiproliferative capacity.Final product of selectively oxidized interferon- β -1b to PEG-hydrazide gave similar results to that of PEG-hydrazinoformate.conversely, treatment of interferon- β -1b with higher concentrations of periodate (e.g., 1-3mM) inhibited or completely abolished Daudi anti-proliferative effects of cells. Such high concentration NaIO₄The induction of protein dimerization was detected by Superose 12 column size exclusion HPLC (described in example 5) and the induction of methionine oxidation by reversed phase chromatography (described in example 6) (results not shown).

The biological activity of the conjugates of the invention can be determined using known anti-proliferative and anti-viral techniques based on various cell lines or primary cultures (cells bearing the cell-surface receptor for interferon β) additionally, the interferon β response can be monitored, including the induction of neopterin (Pepinsky, R.B., et al, supra), β₂Microglobulin (Pepinsky, R.B., et al, supra), or 2 '-5' -oligoadenylate synthetase (Bruchelt, G., et al, (1992) EurJ. Clin Chemin Clin biochem 30: 521-42) or reporter gene proteins operably linked to Interferon β -inducible protein promoters other methods for determining the biological activity of Interferon β polymer conjugates include signaling assays as well as gene activation assays (e.g., Pungor, E., et al, (1998) J Interferon Cytokine Res 18: 1025-1030).

The invention has been described with reference to certain embodiments and certain examples. The methods of the invention are equally applicable to certain receptor-binding peptides and proteins other than cytokines or antagonists thereof, as well as to the use of other finishing agents. The scope of the invention is therefore not limited by the described embodiments but only by the scope of the claims. Those of ordinary skill in the art will immediately recognize that other embodiments may be practiced without departing from the scope of the present invention. All such variations are considered a part of the present invention.

All publications, patents, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. Which is incorporated herein by reference as if each individual application, patent or patent application was specifically or individually indicated to be incorporated by reference.

Claims (36)

1. A method of increasing the in vitro bioavailability of non-glycosylated interferon beta, comprising selectively coupling a poly (ethylene glycol) (PEG) to the amino-terminal amino acid of the interferon beta in the presence of Sodium Dodecyl Sulfate (SDS) to obtain a modified interferon beta,

wherein the amino-terminal amino acid is distal to the receptor-binding domain of interferon beta,

wherein the PEG has a molecular weight of 10kDa to 30kDa, wherein the increased in vitro bioefficacy is increased antiproliferative potency as measured in a cell culture assay in response to interferon-beta, and

wherein the coupling comprises

(a) (ii) mixing said interferon- β and an aldehyde derivative of said PEG to form a schiff base, and (ii) reducing said schiff base with a mild reducing agent under conditions such that a secondary amine bond is formed, wherein the in vitro bioefficacy of said modified interferon- β is 2.5-fold to 6-fold greater than the in vitro bioefficacy of said interferon- β prior to said coupling, and wherein said selective coupling results in 90% conjugation of said PEG to the amino-terminal amino acid; or

(b) (ii) oxidizing the N-terminal serine or threonine residue of said interferon- β with periodate to an aldehyde under conditions that minimize oxidation of at least one essential methionine residue of said interferon- β, and (ii) coupling said aldehyde to a carbazate derivative of said PEG, wherein the in vitro bioefficacy of said modified interferon- β is 6-10 times the in vitro bioefficacy of said interferon- β prior to said coupling.

2. The method of claim 1, wherein the interferon-beta has the amino acid sequence of SEQ ID NO: 1.

3. The method of claim 1, wherein in (a) the PEG is covalently coupled to the alpha amino group of the amino-terminal amino acid.

4. The method of claim 3, wherein the covalent coupling of the PEG to the alpha amino group is via a secondary amine linkage.

5. The method of claim 1, wherein the PEG has a molecular weight of 18kDa to 22 kDa.

6. The method of claim 5, wherein the PEG has a molecular weight of 20 kDa.

7. The method of claim 1, wherein the PEG has a molecular weight of 30 kDa.

8. A composition comprising a conjugate produced by the method of claim 1 and a detergent.

9. A pharmaceutical composition comprising one or more conjugates produced by the process of claim 1 and one or more pharmaceutically acceptable excipients.

10. A composition comprising a detergent and a conjugate, wherein the conjugate comprises an unglycosylated interferon-beta covalently coupled at its amino-terminal amino acid to PEG,

wherein the PEG has a molecular weight of 10kDa to 30kDa,

wherein the coupling between the interferon beta and the PEG comprises

(a) Forming a modified interferon beta by: (i) mixing unmodified, non-glycosylated interferon- β and an aldehyde derivative of said PEG to form a schiff base, and (ii) reducing said schiff base with a mild reducing agent under conditions such that a secondary amine bond is formed, wherein the in vitro bioefficacy of said modified interferon- β is 2.5-fold to 6-fold greater than the in vitro bioefficacy of said unmodified, non-glycosylated interferon- β, and wherein 90% of said PEG is conjugated to the amino-terminal amino acid of said modified interferon- β; or

(b) Forming a modified interferon beta by: (i) oxidizing an N-terminal serine or threonine residue of an unmodified, non-glycosylated interferon- β with periodate to an aldehyde under conditions that minimize oxidation of at least one essential methionine residue, and (ii) coupling the aldehyde to a carbazate derivative of the PEG, wherein the in vitro bioefficacy of the modified interferon- β is 6-10 times greater than the in vitro bioefficacy of the unmodified, non-glycosylated interferon- β,

wherein the in vitro bioefficacy is an antiproliferative potency measured in a cell culture assay responsive to interferon-beta.

11. The composition of claim 10, wherein the interferon beta has the amino acid sequence of SEQ ID NO: 1.

12. The composition of claim 10, wherein the PEG in (a) is covalently coupled to the alpha amino group of the amino-terminal amino acid of the interferon beta.

13. The composition of claim 12, wherein the PEG is covalently coupled to the alpha amino group via a secondary amine linkage.

14. The composition of claim 10, wherein the PEG has a molecular weight of 18kDa to 22 kDa.

15. The composition of claim 14, wherein the PEG has a molecular weight of 20 kDa.

16. The composition of claim 10, wherein the PEG has a molecular weight of 30 kDa.

17. A kit comprising the pharmaceutical composition of claim 9.

18. Use of the composition of any one of claims 8 and 10-13 in the manufacture of a medicament for preventing, diagnosing, or treating an interferon-beta responsive physical disorder in an animal suffering from or susceptible to said physical disorder.

19. Use of the pharmaceutical composition of claim 9 in the manufacture of a medicament for the prevention, diagnosis, or treatment of an interferon-beta responsive physical disorder in an animal suffering from or susceptible to said physical disorder.

20. The use of claim 18, wherein the animal is a mammal.

21. The use of claim 19, wherein the animal is a mammal.

22. The use of claim 20 or 21, wherein the mammal is a human.

23. The use of claim 18, wherein the interferon beta responsive physical disorder is selected from the group consisting of: cancer, infectious disease, neurodegenerative disorders, autoimmune disorders, and genetic diseases.

24. The use of claim 23, wherein the cancer is selected from the group consisting of: carcinomas, sarcomas, adenomas, and myelomas.

25. The use of claim 23, wherein the cancer is selected from the group consisting of: neural cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, testicular cancer, lung cancer, leukemia, lymphoma, gastrointestinal cancer, pancreatic cancer, bladder cancer, kidney cancer, bone cancer, head and neck cancer, and skin cancer.

26. The use of claim 23, wherein the cancer is colon cancer.

27. The use of claim 23, wherein the interferon-beta responsive infectious disease is selected from the group consisting of: viral hepatitis, cardiotropic virus, and HIV/AIDS-induced diseases.

28. The use of claim 23, wherein the interferon beta responsive neurodegenerative disease is multiple sclerosis.

29. The use of claim 28, wherein the multiple sclerosis is selected from the group consisting of: relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis.

30. The use of claim 19, wherein the interferon beta responsive physical disorder is selected from the group consisting of: cancer, infectious disease, neurodegenerative disorders, autoimmune disorders, and genetic diseases.

31. The use of claim 30, wherein the cancer is selected from the group consisting of: carcinomas, sarcomas, adenomas, and myelomas.

32. The use of claim 30, wherein the cancer is selected from the group consisting of: neural cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, testicular cancer, lung cancer, leukemia, lymphoma, gastrointestinal cancer, pancreatic cancer, bladder cancer, kidney cancer, bone cancer, head and neck cancer, and skin cancer.

33. The use of claim 30, wherein the cancer is colon cancer.

34. The use of claim 30, wherein the interferon-beta responsive infectious disease is selected from the group consisting of: viral hepatitis, cardiotropic virus, and HIV/AIDS-induced diseases.

35. The use of claim 30, wherein the interferon-beta responsive neurodegenerative disorder is multiple sclerosis.

36. The use of claim 35, wherein the multiple sclerosis is selected from the group consisting of: relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis.