NO171295B - PROCEDURE FOR PREPARING A PHARMACEUTICAL PREPARATION IN THE FORM OF A BIOLOGICALLY ACTIVE CONJUGATED PROTEIN - Google Patents

Description

The present invention relates to chemical modification of biologically active proteins which change the chemical and/or physiological properties of these proteins. More specifically, the invention relates to the selective conjugation of lipophilic water-soluble proteins to polymers to render them protein-soluble at physiological pE values.

According to this, the present invention relates to a method for producing a pharmaceutical preparation.

In particular, the invention relates to a method for the production of a biologically active conjugated protein selected from p-interferon (IFN-p), interleukin-2 (IL-2) and immunotoxins where the protein is covalently conjugated to a water-soluble polymer selected from polyethylene glycol homopolymers and polyoxyethylated polyols wherein the homopolymer is optionally substituted at one end with an alkyl group and the polyol is unsubstituted.

Many heterologous proteins produced in microbial host cells are found as insoluble material in refractile bodies. Examples of heterologous proteins that form refractile bodies in commonly found culture conditions include interleukin-2 (IL-2), interferon-p (IFN-<p>), feline leukemia virus (FeLV) envelope protein, human growth hormone (hGH), bovine growth hormone (bGH), porcine growth hormone (pGH) as well as certain proteins that are coated or fused with a virus such as FMD virus. In addition, many of these proteins are hydrophobic in nature and tend to stick to materials and to themselves (that is, aggregate) rather than remaining in solution. Furthermore, many of these recombinant proteins are not glycosylated while their native counterparts are water-soluble, glycosylated molecules. Modifications of these proteins that can alter their solubility properties would be desirable to increase the production yields of these proteins as well as to facilitate their formulation for therapeutic use. In addition, modifications can reduce or eliminate aggregation of the proteins when these are introduced in vivo to thereby reduce immunogenicity.

The use of polypeptides in circulatory systems to promote a particular physiological response is well known in the medical art. A limitation to the potential therapeutic benefit thereby arising from the clinical use of polypeptides is their ability to elicit an immune response in the circulatory system. This immune response can be caused by aggregates in material before injection as described by R. Illig.

(1970), "J.Clin. Endrocr.", 31, 679-688, W. Moore (1978), "J. Cl in. Endrocrinol. Metab.", 46, 20-27 and W. Moore and P. Leppert (1980), "J. Clin. Endocrinol. Metab.", 51, 691-697. This response involves the production of antibodies against the polypeptides by the circulatory system into which they are injected. This antibody production can reduce or eliminate the desired biological function of the polypeptide, sometimes by causing a reduced residence time in the circulatory system (reduced half-life) or by modifying the molecule by means of antibody-polypeptide interaction.

Modification of these potentially useful therapeutic polypeptides to prevent or at least reduce an immune response while still maintaining desired physiological activities of the polypeptide would allow the use of these polypeptides in the mammalian circulatory system without the above-mentioned drawbacks. In addition and due to the reduced half-life of the circulating polypeptide, smaller amounts of polypeptide will be required for the desired therapeutic effect than has previously been possible.

The problems of immunity and short half-life in circulation as indicated above, and other undesirable properties of certain proteins, are well known, and various modifications of polypeptides have been proposed to solve the problems. These include modification of proteins with essentially straight-chain polymers such as polyethylene glycol (PEG) or polypropylene glycol (PPG). For example, US-PS 4,261,973 describes the conjugation of immunogenic allergen molecules with non-immunogenic water-soluble polymers such as PEG, to reduce the immunogenicity of the allergen. US-PS 4,301,144 describes the conjugation of hemoglobin to PEGF, a copolymer of ethylene glycol with propylene glycol, or ethers, esters or dehydrated products of such polymers to increase the oxygen carrying capacity of the hemoglobin molecule. EP publ. 98,110, discloses that conjugation of a polypeptide or glycoprotein to a polyoxyethylene-polyoxypropylene copolymer increases the length of the physiological activity. Preferably, the polypeptide or glycoprotein is an enzyme or a native interferon which is difficult to dissolve. US-PS 4,179,337 describes the conjugation of water-soluble polypeptides such as enzymes and insulin to PEG or PPG to reduce the immunogenicity of the polypeptide while retaining a significant proportion of its desired physiological activity. US-PS 4,002,531 describes another method for conjugating enzymes to PEG via an aldehyde derivative.

US-PS 4,055,635 describes pharmaceutical preparations comprising a water-soluble complex of a proteolytic enzyme bound covalently to a polymeric material such as polysaccharides.

US-PS 3,960,830 describes peptides attached to a polyalkylene glycol polymer such as polyethylene glycol.

US-PS 4,088,538 describes a reversibly soluble enzymatically active polymeric enzyme product comprising an enzyme covalently bound to an organic polymer such as polyethylene glycol.

US-PS 4,415,665 describes a method for conjugating an organic ligand containing at least one primary or secondary amino group, at least one thiol group and/or at least one aromatic hydroxy group (described in column 3, lines 19-36) to a polymer carrier with at least one hydroxyl group (described in column 2, lines 42 to 66).

US-PS 4,495,285 discloses a non-immunogenic plasminogen activator whose amino acid side chains are linked to a polyalkylene glycol via a coupling agent.

US-PS 4,12,989 describes an oxygen-carrying material containing hemoglobin or a derivative thereof covalently linked via an amine bond to polyethylene or polypropylene glycol.

US-PS 4,496,689 describes a covalently attached complex of alpha-1-proteinase inhibitor with a polymer such as PEG or methoxy polyethylene glycols.

US-PS 3,619,371 describes a polymer matrix with a biologically active material chemically bound thereto.

US-PS 3,788,948 describes the use of organic cyanate compounds to bind proteins to polymers.

US-PS 3,876,501 describes the activation of water-soluble carbohydrates with cyanogen bromide to thereby improve their binding to enzymes and other proteins.

US-PS 4,055,635 describes pharmaceutical preparations of a proteolytic enzyme bound covalently to a polymeric material.

EP-PS 152,847 describes an enzyme conjugate preparation comprising an enzyme conjugate, a calcium salt and a polyethylene glycol.

JP publ. 5792435 describes modified polypeptides in which all or part of the amino groups are substituted with a polyethylene oxide. DE publ. 2312615 describes the conjugation of polymers to compounds containing hydroxy or amino groups. EP-PS 147,761 describes a covalent conjugate of alpha-1 proteinase inhibitor and a water-soluble polymer, where the polymer may be polyethylene glycol.

TJS-PS 4,414,147 describes a method for making interferon less hydrophobic by conjugating it to an anhydride of a dicarboxylic acid such as poly(ethylene succinic anhydride).

In addition to these patents and patent publications, there are various articles that describe the concept of using activated PEG or PPG as a modifier for proteins such as enzymes, IgG and albumin. For example, Inada et al., "Biochem and Biophys. Res. Comm.", 122, 845-850 describe

(1984) modified water-soluble lipoprotein lipase to render it soluble in organic solvents such as benzene by using cyanuric chloride to conjugate with PEG. Takahashi et al., "Biochem. and Biophys. Res. Comm.", 121, 261-265 (1984) describe the modification of horseradish peroxidase using cyanuric chloride triazine with PEG to produce the water soluble enzyme which is active and soluble in benzene. Suzuki et al., in "Biochem. Biophys. Acta," 788, 248-255 (1984), have described suppression of aggregation of IgG using cyanuric acid chloride-activated PEG. Abuchowski et al. describes in "Cancer Biochem. Biophys.", 7, 175-186 (1984) that modification of asparaginases from E. coli and Vibrio succinogenes using PEG activated by succinimidyl succinate increases the half-life and reduces the immunogenicity of the proteins. Davis et al describe in "Biodical Polymers", (New Tork: Academl Press, 1980), pp. 441-451 that enzymes which are normally insoluble can be solubilized by PEG linkage without further details. Several other articles describe modifications of enzymes such as urcase, streptokinase, catalase, arginase and asparaginase with PEG activated by succinimidyl succinate or cyanuric chloride to increase the half-life and to reduce the immunogenicity of the protein. However, none of these references describe details of how to use a polymer modification process to water-solubilize recombinant proteins such as IL-2 and IFN-β which are hydrophobic and therefore resist formulation in aqueous medium at physiological pH values. Furthermore, it is not a priori possible to predict which selected proteins would be responsive to treatment by polymers due to the large difference in the pharmacokinetic and physical properties of different proteins. Furthermore, none of the references describe reducing or eliminating aggregation of the protein, a phenomenon that elicits an immune response when the protein is introduced in vivo.

EP-PS 154,316 describes and claims chemically modified lymphokines such as IL2 containing PEG linked directly to at least one primary amino group of a lymphokine.

Accordingly, the present invention provides for the modification of those proteins selected from interferon, interleukin-2 and immunotoxins which are normally insoluble in water under ambient conditions under pharmaceutically acceptable pH values to render them soluble in aqueous buffer under such conditions. This modification can be an imitation of glycosylation of the protein to thereby surprisingly make the protein soluble as the native glycosylated protein is soluble. This modification also avoids the addition of external solubilizing additives such as detergents or denaturants to keep the protein in solution. The modified protein retains the biological activity of the unmodified protein, both initially and over time.

As a secondary benefit, the modification under certain conditions increases the physiological half-life of the protein and may reduce its immunogenicity by reducing or eliminating aggregation of the protein or by masking antigenic determinants. It has also been found that this prolonged half-life is related to the effectiveness of the protein. The in vivo half-life can be modulated by choosing suitable conditions and polymers.

In accordance with this, the invention as indicated in the introduction concerns a method for the production of a pharmaceutical preparation and this method is characterized by the fact that it comprises: a) the production of a water-soluble polymer with a molecular weight preferably between 350 and 40,000 with at least one terminal reactive group where the polymer is selected from polyethylene glycol homopolymers and polyoxyethylated polyols, the homopolymer being optionally substituted at one end with an alkyl group and the polyol being unsubstituted; b) conjugating a biologically active normally hydrophobic, water-insoluble protein selected from β-interferon, interleukin-2 and an immunotoxin with the reactive group of the polymer, in a ratio of 0.1 to 1000 moles of polymer per mol protein, to provide a water-soluble, biologically active, conjugated protein; and c) formulating the protein at pH 6-8 in a non-toxic, inert, pharmaceutically acceptable, aqueous carrier medium;

provided that when the protein is an IFN-β or an IL-2 and when the polymer is a polyethylene glycol, then the polymer is conjugated indirectly to the protein via a group comprising a carboxylic acid ester, a carboxylic acid or an amide bond.

Preferably, the polymer is unsubstituted polyethylene glycol (PEG), monomethyl PEG (mPEG) or polyoxyethylated glycerol (POG), and preferably is linked to the protein via an amide bond formed from the 4-hydroxy-3-nitrobenzenesulfonate ester or N-hydroxysuccinimide ester of a PEG -, mPEG or POG carboxylic acid.

This also includes a method of the type mentioned at the outset for producing a biologically active conjugated protein, and this method includes: a) producing a water-soluble polymer with at least one terminal reactive group where the polymer is selected from polyethylene glycol homopolymers and polyoxyethylated polyols, the homopolymer possibly is substituted at one end with an alkyl group and the polyol is unsubstituted; and b) conjugating a biologically active, normally hydrophobic, water-insoluble protein selected from ε-interferon, interleukin-2, and an immunotoxin, with the reactive group of the polymer to provide a water-soluble, biologically active, conjugated protein, provided that when the protein is an IFN-β or an IL-2, and when the polymer is a polyethylene glycol, then the polymer is conjugated indirectly to the protein via a group comprising a carboxylic acid ester, a carboxylic acid or an amide bond. Figure 1 shows densitometric sweeps of 14$ SDS-polyacrylamide gels for molecular weight analyzes of PEG modified (PEGylated) IL-2 obtained from turnover of 0, 10, 20, 50 and 100 mol of activated PEG (PEG<*>) per moles of IL-2. Figure 2 shows the solubility of PEGylated IL2 compared to unmodified IL-2 at two different pH values by absorbance sweep from 200 to 650 nm. Figure 3 shows the pharmacokinetics of PEGylated and unmodified IL2 after intravenous injection in mice. Figure 4 shows the pharmacokinetics of PEGylated and unmodified IL2 after subcutaneous injection in mice. Figure 5 shows densitometry sweeps of 14% non-reducing SDS-polyacrylamide gel for molecular weight analysis of PEGylated IFN-P obtained at turnovers of 0, 10, 20 and 50 mol PEG<*> per mol

IFN-β.

Figure 6 shows the solubility of PEGylated IFN-β compared to unmodified IFN-β at two different pH values by absorbance sweep from 200 to 650 nm.

As used herein, the term "generally hydrophobic, water insoluble" in connection with proteins refers to those proteins which are insoluble or not completely soluble in water or in an aqueous medium under ambient temperatures at room temperature and atmospheric pressure at a pH value between 6 and 8, that is, at a neutral or physiological pH value. The modifications herein have the effect of increasing the solubility of such proteins when subjected to such physiological conditions. For the purposes of the present application, solubility can be tested by 1) turbidity, measured spectrophotometrically; 2) The S value measured by ultracentrifugation where the monomeric protein sedimentation rate instead of the much stronger aggregate sedimentation rate indicates solubility; and 3) apparent native molecular weight, as measured by size exclusion chromatography, where the soluble protein is closer to this value than the insoluble protein. The exact number that will indicate solubility for each of these samples depends on the type of buffer in which the protein is formulated, the buffer's pH value and the buffer's ionic strength.

Interferon-p and interleukin-2 can here be obtained from tissue cultures or by recombinant technology, and from any mammalian source such as mouse, rat, rabbit, primate, pig and human. Preferably, such proteins are obtained from a human source, and preferably recombinant human proteins.

The term "recombinant β<->interferon", called IFN-β, preferably human IFN-β, refers to fibroblast interferon with comparable biological activity to native IFN-β produced by known recombinant DNA techniques. In general, the gene encoding interferon is excised from the native plasmid and inserted into a cloning vector to be cloned and then into an expression vector, which is used to transform a host organism, preferably a microorganism, and most preferably E. coil. The host organism expresses the foreign interferons under certain conditions for the production of IFN-β. More particularly, IFN-β is a mutein as described in US-PS 4,588,585, where the cysteine usually occurs at position 17 in the wild-type or native molecule is replaced with a neutral amino acid such as serine or alanine. Preferably, IFN-β is mutein in IFN-Pseri7.

The term "recombinant interleukin-2", called IL-2, preferably human IL-2, refers to interleukin-2 with comparable biological activity to native IL-2 produced by recombinant DNA techniques as described for example by Taniguchi et al. , "Nature", 302: 305-310 (1938) and Devos, "Nucleic Acids Research", 11:4307-4323 (1983). In general, the gene encoding IL-2 is excised from the native plasmid and inserted into a cloning vector to be cloned, and then into an expression vector used to transform a host organism, preferably a microorganism and most preferably E. coli. The host organism expresses the foreign gene to produce IL-2 under expression conditions.

More particularly, this IL-2 is a mutein as described in US-PS 4,518,564, where the cysteine normally occurs in position 125 of the wild-type or native molecule is replaced by a neutral amino acid such as serine or alanine. Alternatively or conjunctively, the IL-2 mutein can be one in which methionine normally occurs in position 104 in the wild type or the native molecule is replaced by a neutral amino acid such as alanine.

Preferably, IL2 is a protein produced by a microorganism or by a yeast transformed with the human cDNA sequence of IL-2 which encodes a protein with an amino acid sequence at least substantially identical to the amino acid sequence of native human IL-2, including the disulfide bond of the cysteines in positions 58 and 105, and has biological activity in common with native human IL-2. Substantial identity between amino acid sequences means that the sequences are identical or differ from each other by one or more amino acid changes (omissions, additions, substitutions) that do not cause any negative functional dissimilarity between the synthetic protein and native human IL-2. Examples of IL proteins with such properties are those described by Taniguchi et al., supra; Devos, supra; EP-PS 91,539 and 88,195, US-PS 4,518,584, supra. Most preferably, IL-2 is ser125, des-ala^ser^25H—2, des-ala^IL-2, des-alaialaiQ4lL-2 f or des-alaial<a>io4se<r>125IL~2" where "des- ala^" suggests that the N-terminal alanyl residue in IL2 is omitted.

The exact chemical structure of the proteins described here depends on a number of factors. Since ionizable amino and carboxyl groups are present in the molecule, a particular protein can be obtained as an acidic or basic salt, or in neutral form. All such preparations which retain their bioactivity when placed in suitable surroundings are included under the definition of the protein given here. Furthermore, the primary amino acid sequence of the protein can be improved by derivatization using sugar moieties (glycosylation) or by other supplementary molecules such as lipids, phosphate, acetyl groups and the like, more generally by conjugation with saccharides. Certain aspects of such enhancement are accomplished by post-translational processing of the producing host; other such modifications can be introduced in vitro. In any case, such modifications are included in the definition of protein as long as the bioactivity of the protein is not destroyed. It is of course expected that such modifications quantitatively or qualitatively affect the bioactivity either by improving or by reducing the protein's activity in the various analyses.

Often, hydrophobic recombinant proteins such as IL-2 and IFN-β, produced from transformed host cells containing recombinant DNA, precipitate inside the cell rather than being soluble in the cell culture medium. The intracellularly produced protein must be separated from cellular debris and recovered from the cell before it can be formulated into a purified biologically active material. In a process for isolating such a refractile material, the cell membrane of the transformed host microorganism is broken down, more than 99% by weight of salts removed from the disruptate, desalted dlsruptate redisrupted, a material, preferably a sugar such as sucrose, added to the disruptate to form a density or viscosity gradient in the liquid in the disruptate, and where refractile material is separated from cellular debris by high-speed centrifugation, i.e. 10,000 to 40,000 g. Preferably, salts are removed from the disruptate by diafiltration or centrifugation, and sucrose is added to increase the density of the liquid to 1.1 to 1.3 g/ml.

After the centrifugation step, the pellet containing refractile substances is solubilized with a denaturant such as sodium dodecyl sulfate, the resulting suspension is centrifuged, and the supernatant containing the protein is treated to isolate the protein. The protein is separated from the supernatant by suitable means such as reverse phase high pressure liquid chromatography, RP-HPLC, and/or gel filtration chromatography. After such separation, the protein is preferably oxidized to ensure the production of high yields of recombinant protein in a configuration most similar to its native counterpart. Such oxidation is described in US-PS 4,530,787. The oxidation can also be carried out by reacting an aqueous solution containing a solubilized form of the protein at a pH value between 5.5 and 9 in the presence of air with at least an effective amount of an oxidation promoter containing a Cu<+2> cation, as described in US-PS 4,572,798. The preferred oxidation promoter or oxidizing agent is CuCl2 or (o-phenanthroline)2 Cu<+2.> After oxidation, the protein can optionally be desalted and further purified by RP-HPLC, dilution/diafiltration, S-200 gel filtration chromatography and ultrafiltration techniques before modification with activated polymer as further described below. However, the polymer modification can be carried out at any stage after the heterologous protein is isolated in sufficiently pure form to be biologically active for therapeutic purposes. The point at which the modification occurs depends on the ultimate purity of the protein required for the final pharmaceutical formulation and use.

The term "immunotoxin" as used herein to describe the third class of proteins refers to a conjugate of an antibody and a cytotoxic moiety. The cytotoxic part of the immunotoxin includes a cytotoxic drug or an enzymatically active toxin of bacterial or plant origin or an enzymatically active fragment ("A chain") of such a toxin. Examples of enzymatically active toxins and fragments thereof are the diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginose), ricin A chain, abrin A chain, modekin A chain, oc-sarkin, Aleurites fordii- proteins, diantin proteins, Phytolacca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, kursin, crotin, saponaria officinalls inhibitor, gelonin, mitogellin, restrictosin, phenomycin and anomycin. Ricin A chain, non-binding active fragments of diphtheria toxin, abrin A chain and PAPII are preferred. Most preferred is the ricin A chain which is modified by reaction with the polymer.

The antibodies used in the immunotoxin are preferably monoclonal antibodies directed against a specific pathological condition such as cancer in the form of breast, prostate, colon or ovarian cancer, manoma, myeloma and so on.

Conjugates of the antibody and the cytotoxic moiety can be obtained using a number of bifunctional protein modifying reagents. Examples of such are N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), iminothiolate (IT), bifunctional derivatives of imidoesters such as disuccinimidyl substrate, aldehydes such as glutaraldehyde, bis-azido compounds such as (p-azidobenzoyl)hexanediamine, bis-diazonium derivatives such as bis-(p-diazonium-benzoyl)-ethylenediamine, diisocyanates such as tolylene-2,6-diisocyanate and bis-active fluorine compounds such as 1,5-difluoro-2,4-dinitrobenzene.

The term "selectively conjugated" as used herein to fit the proteins indicated as proteins that are covalently bound via one or more amino acid residues in the proteins depends mainly on the reaction conditions, the final use, the molecular weight of the polymer and the particular protein used. While the residues can be any reactive amino acid on the protein such as one or two cysteines or the N-terminal amino acid group, preferably the reactive amino acid is lysine which is bound to the reactive group of the activated polymer through its free c-amino group, or glutamine or aspartic acid which is bound to the polymer via an amine bond.

In a preferred embodiment, the protein is covalently linked via one or two of the amino acid residues in the protein, preferably lysines, for maximum biological activity. In another preferred embodiment, the protein is covalently bound via ten of the amino acid residues in the protein, preferably lysines, where higher substitutions generally increase the circulatory lifetime of the protein.

According to the method of the invention, the three types of proteins described above, which are usually hydrophobic and water-insoluble, are made soluble in an aqueous carrier medium, preferably at a pH value of 5-8, preferably 6-8 and most preferably 6.5 -7,8, without the use of solubilizing agents but by modifying the proteins by conjugation to a specified polymer. If the protein is reacted via the lysine residues, the pH value for the reaction is preferably between 7 and 9 and preferably 8-9. The success of such a modification of these proteins cannot be predicted from the previous use of polymer modification of water-soluble enzymes and hormones.

The polymer to which the protein is attached is a homopolymer of polyethylene glycol (PEG) or a polyoxyethylated polyol, provided in all cases that the polymer is soluble in water at room temperature. Examples of polyoxyethylated polyols are, for example, polyoxyethylated glycerol, polyoxyethylated sorbitol, polyoxyethylated glucose and the like.

The glycerol skeleton for polyexoethylated glycol is the same skeleton that occurs naturally, for example, in animals and humans in mono-, di- and triglycerides. Therefore, this branching will not necessarily be seen as a foreign substance in the body.

The polymer need not have any particular molecular weight, but it is preferred that the molecular weight is between 300 and 100,000 and most preferably between 350 and 40,000, e.g. depending on the particular protein used.

Preferably, the PEG homopolymer is unsubstituted, but it can also be substituted at one end with an alkyl group. Preferably, the alkyl group is a C 1-4 alkyl group, and most preferably it is a methyl group. The most preferred is that the polymer is an unsubstituted homopolymer of PEG, a monomethyl substituted homopolymer of PEG or a polyoxyethylated glycerol, and has a molecular weight of from 350 to 40,000.

Furthermore, the protein is conjugated via a terminal reactive group on the polymer. The polymer with the reactive group(s) is referred to here as activated polymer. The reactive group reacts selectively with free amino or other reactive groups on the protein. However, it will be understood that the type and amount of reactive group chosen, as well as the type of polymer used to achieve optimal results, will depend on the protein used to avoid the reactive group reacting with too many particularly active groups on the protein. As this is not necessarily completely unavoidable, it is recommended that in general from 0.1 to 1000 mol and preferably 2 to 200 mol of activated polymer is used per mol of protein, depending on the protein concentration. Especially for IL-2, the amount of activated polymer used is preferably no more than 50 mol per mol IL-2, and most preferably the value is from 2 to 20 mol per mol IL-2, depending on the specific proportions that are finally determined, for example, the final value is a balance to maintain optimal activity with the same operating time, if possible the half-life of the protein. Preferably, at least 50# of the biological activity of the protein is retained and most preferably 100#.

The covalent modification reaction can take place by any suitable method that is generally used for reactive biologically active substances with inert polymers, especially at pH 5-9, if the reactive groups on the protein are lysine groups. In general, the process involves the preparation of an activated polymer (at least one terminal hydroxy group) and then reacting the protein with the activated polymer to provide the two solubilizing proteins suitable for formulation.

The above-mentioned modified reaction can be carried out by several methods which can use one or more steps.

Examples of suitable modifying agents that can be used to prepare the activated polymer are a one-step reaction that includes cyanuric acid chloride (2,4,6-trichloro-S-triazine) and cyanuric acid fluoride.

In a preferred embodiment, the modification reaction takes place in two steps where the polymer is first reacted with an acid anhydride such as succinic or glutaric anhydride, thereby forming a carboxylic acid, after which the carboxylic acid is then reacted with a compound capable of reacting with carboxylic acid to form an activated polymer with a reactive ester group capable of reacting with the protein. Examples of such compounds are N-hydroxysuccinimide, 4-hydroxy-3-nitrobenzenesulphonic acid and the like, and preferably N-hydroxysuccinimide or 4-hydroxy-3-nitrobenzenesulphonic acid. For example, monomethyl substituted PEG can be reacted at an elevated temperature, preferably 100-110°C for four hours with glutaric anhydride. The monomethyl PEG-glutaric acid thus produced is then reacted with N-hydroxysuccinimide in the presence of a carbodiimide reagent such as dicyclohexyl or isopropyl carbodiimide to thereby give the activated polymer, methoxypolyethylene glycol-N-succinimidyl glutarate, which can be reacted with the protein. This method is described in detail by Abuchowski et al., "Cancer Biochem. Biophys.", 7, 175-186 (1984). In another example, monomethyl substituted PEG is reacted with glutaric anhydride followed by reaction with 4-hydroxy-3-nitrobenzenesulfonic acid (HNSA) in the presence of dicyclohexylcarbodiimide to thereby give the activated polymer. (HNSA) is described in Bhatnagar et al., "Peptides: Synthesis-Structure-Function, Proceedings of the Seventh American Peptide Symposium", Rich, et al. (ed.)

(Pierce Chemical Co., Rockford IL, 1981), pp. 97-100 and in NItecki et al., "HIgh-Technology Route to Virus Vaccines"

(American Society for Microbiology: 1986) called "Novel Agent for Coupling Synthetic Peptides to Carriers and Its Application".

As ester bonds are chemically and physiologically less stable than amide bonds, it may be preferred to use chemical transformations in the conjugation reaction which give carboxylic acids or amides without the simultaneous formation of esters.

The proteins modified in this way are then formulated in a non-toxic, inert pharmaceutically acceptable aqueous carrier,

preferably at a pH from 3 to 8, and preferably 6 to 8. For in vitro applications such as for immunotoxins used for diagnostic purposes, the administration method and -formulas-

none not significant. Aqueous formulations compatible with the culture or perfusion medium will generally be used. When used in in vivo therapy, the sterile product will consist of a mixture of protein dissolved in an aqueous buffer in an amount which gives a pharmaceutically acceptable pH value when the mixture is reconstituted. A water-soluble carrier such as mannitol can optionally be added to the medium. The commonly formulated unmodified IL-2 is stable for at least six months at 4°C.

The dosage level of protein in the formulation will depend on in vivo efficacy data obtained after preclinical testing and will depend mainly on the protein used and the end use.

If the formulation is lyophilized, the lyophilized mixture can be reconstituted by injecting into the ampoule a conventional parenteral aqueous injection such as distilled water.

The reconstituted formulation prepared as described above is suitable for parenteral administration to humans or other mammals in therapeutically effective amounts (that is, amounts that eliminate or reduce the patient's pathological condition to provide therapy, the type of therapy being dependent on the type of protein. For for example, IL-2 therapy is appropriate for a number of immunomodulatory indications such as T-cell mutagenesis, induction of cytotoxic T-cells, increase of natural killer cell activity, induction of IFN-nr, establishment or improvement of cellular immunity (for example treatment of immunodeficient conditions) , and enhancement of cell-mediated antitumor activity.

In an alternative direct administration of IL2, this can be administered in an adoptive immunotherapy method together with isolated lymphokine-activated lymphocytes, in a pharmaceutically acceptable carrier where the lymphocytes are reactive against tumor administered with IL-2 to humans suffering from the tumor. This method is described by S. Rosenberg et al., "New England Journal of Medicine" (1985), 313:1485-1492.

IFN-P therapy is suitable for anti-cancer, antiviral and anti-psoriasis treatment. Specific cancers against which IFN-β has shown some effectiveness include lymphoma, myeloma, hairy cell leukemia and certain viral diseases including venereal warts and rhinoviruses.

Immunotoxin therapy is appropriate for diseases against which the intended antibody is effective, usually cancer. In particular, immunotoxins are targeted for silk cancer types such as breast cancer.

Dosage and dosage range for the immunotoxin will depend, for example, on the drug's pharmacokinetics, the type of cancer (primary or metastatic) and its population, type and length of polymer, the particular immunotoxin's characteristics, for example the therapeutic index, further the patient and the patient's history. The dose and dosage range for IL-2 and IFN-β will similarly depend, for example, on the pharmacokinetics of the drug, the nature of the disease, the different characteristics of IL-2 or IFN-β, the patient and the patient's history. For example, different modified IL-2 proteins are expected to have different pharmacokinetic and therapeutic properties that are advantageous for different routes of administration. A long-acting drug only needs to be administered every three to four days, every week or once every two weeks. The degree of clarification can be varied to give the ultimate flexibility for adaptation to the particular needs of a patient by changing, for example, the type of polymer and the size of the bound polymer.

In the following examples showing the invention, all parts and percentages are by weight unless otherwise stated, and further all temperatures are given in °C.

EXAMPLE 1

Preparation of PEGylated interleukin-2 (IL-2)

A. Preparation of PEG ester.

A linear, monomethyl substituted ester of PEG with a molecular weight of 5000 can be obtained by first reacting commercially available monomethyl PEG-5000 with glutaric anhydride at 100 to 110°C for four hours by a method similar to that of Abuchowski et al., "Cancer Biochem. Biophys. ", 7, 175-186 (1984). The resulting PEG glutarate is reacted with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide, as described by Abuchowski et al., supra, on page 176. The resulting product is methoxypolyethylene glycol N-succinimidyl glutar, hereinafter referred to as PEG<*>.

In a similar manner, succinic anhydride was reacted with monomethyl PEG-5000 and the resulting PEG succinate was reacted with a hydroxysuccimide. The resulting product is methoxy polyethylene glycol N-succinimidyl succinate.

In an alternative step and by a corresponding method, a PEG carboxylic acid ester HNSA is prepared using HNSA instead of a hydroxysuccinimide. This ester preparation is described by Bhatnagar et al., supra and Nitecki et al., supra. The PEG carboxylic acid ester HNSA can be used as the activated PEG by the procedures described here and in the following examples.

B. Conjugation of PEG<*> to IL-2.

RP-HPLC-purified recombinant des-alanyl, ser^25 IL-2 (where cysteine at position 125 is replaced by serine and the N-terminal alanyl residue is omitted), prepared as described in US-PS 4,518,584 and 4,530,787 , supra. or the post-diaphrated des-ala^, ser^gs IL-2 from the manufacturing process as described above, was used for this example.

To 0.5 mg of this purified IL-2 in 1.0 ml of buffer (sodium borate, pH 9; 0.1* SDS) was added freshly prepared aqueous PEG<*> in molar ratios of 0, 2.5, 5, 10, 20, 50 and 100 mol PEG<*> per moles of IL-2. After thorough mixing, the solution was stirred at room temperature, approximately 23°C, for 30 minutes. Each reaction mixture was run on a Pharmacia Sephadex G-25 column to separate IL-2 and PEG-IL-2 from low molecular weight species. The Sephadex G-25 column was run in 1 10 mM Na borate pH 9 which did not contain SDS and it also served to remove most of the SDS from the protein. Alternatively, most of the unmodified IL-2 and SDS were removed by adding the reaction mixture to a mixed cation retarder resin (Bio-Rad AG11A8). The level of residual SDS in the PEGylated IL-2 samples, as measured by an acridine orange test as described by R. Sokoloff and R. Frigon, "Anal. Biochem.", 118, 138-141 (1981), was 3-7 I SDS per mg of protein.

C. Purification of modified IL-2.

Using hydrophobic exchange chromatography (Bio-Rad; Biogelphenyl-5-PW), purified PEGylated IL-2 was obtained. A linear reduced salt gradient (solvent A is 1.7 M(NH4)2SO4 in 50 mM Na phosphate pH 7; 100-0* A for 15 min gave good separation of PEGylated IL2 and unmodified IL-2. Addn. of 10% ethanol to solvent B and maintaining the column in an ice bath greatly increased the recovery and dissolution of PEGylated IL-2 Fixed amounts of the fractions were assayed for IL-2 bioactivity (cell proliferation) by the methods generally described by Gillis , S., et al., "J. Immunol.", 120, 2027-2032 (1978).

EXAMPLE II

Characterization of PEGylated IL-2.

Å. Size characterization of modified IL2 products from

from reactions with varying PEG<*>: IL-2 ratios.

SDS-PAGE (14* of the products from the reactions described in Example IA, containing 0, 10, 20, 50 or 100 mol PEG* per mol IL-2 indicated an increasing degree of modification with increasing molar ratio PEG<*>: IL- 2. Densitometer sweeps of different gel tracks were obtained using the Shimadzu Dual Wavelength Scanner (CS-930) as shown in Figure 1. The 10 PEG<*>/IL-2 and 20 PEG<*>/IL-2 samples showed a discrete species with an apparent molecular mass of about 25 kd in addition to small amounts of unmodified IL-2 At 50 PEG<*>/IL-2 and 100 PEG<*>/IL-2 there was a blur in the high molecular weight region, which is characteristic of extensively PEGylated proteins, and there was no unmodified IL-2.

Size exclusion of PEG-IL-2 solutions on a TSK-250 column (Bio-Rad; 25 x 0.4 cm in PBS) provided further evidence of increasing modification with increasing ratios of PEG<*>:IL-2.

B. Bioactivity of PEGylated IL-2 as a function of

the degree of modification.

Fractions of the aforementioned biogel phenyl column eluates in the IL-2 PEGylation reactions containing 0, 2.5, 5, 10, 20, 50 or 100 mol PEG<*> per mol IL-2 were analyzed by the IL-2 cell profiling bioassay described in I.C. The results are increasing in Table I. As more amino groups were modified, IL-2 was increasingly slowly inactivated. In reactions conducted at a molar ratio of 100 PEG<*>/IL-2, the specific activity of the modified IL-2 product was significantly reduced to only 10* of the unmodified IL-2. C. Solubility of PEGylated IL-2 compared to Unmodified IL-2.

After the modification reaction and subsequent Sephadex G-25 chromatography resulting in SDS removal, the pH of PEGylated IL-2 was reduced to 6.5-7. The unmodified IL-2 in low-SDS precipitated at pH 5-7. The modified IL-2 from the reaction carried out at low molar ratios relative to the amounts of PEG<*>/IL-2 in Table I had some turbidity due to unmodified IL-2 which can then be removed by AGllA8 resin or biogel phenyl (HPLC ) chromatography. The dissolution of modified IL-2 from higher molar ratios of PEG<*>/IL-2 remained clear for some time. The pH-adjusted solutions were ultracentrifuged (50,000 revolutions per minute SW60 rotor, 12 hours at 5°C). The supernatants were removed and stored. Analysis of equal amounts of both residues and supernatants by SDS-PAGE showed that the residues were unmodified IL-2 while the supernatants contained PEGylated IL-2. This dramatic difference in solubility between PEGylated and unmodified IL-2 in aqueous media at neutral pE 1 in the absence of SDS or other denaturants is shown by the absorbance sweeps (Eewlett-Packard 8450A Spectrophotometer) in Figure 2. It unmodified IL-2 at pH 7 precipitates out of solution as shown by a loss of the characteristic spectrum.

Purified PEGylated IL-2 (after the HPLC-phenyl column) was completely soluble at pH 7 in aqueous buffer without detergents or denaturants. The purified PEGylated IL-2 remained in solution and retained its bioactivity during the time they were tested (at least five months). The PEGylated IL-2 soluble at neutral pH without SDS had the following specific activities compared to unmodified IL-2 in the presence of 0.1* SDS:

NK (natural killer; described in US-PS 4,518,584) and LAK (lymphokine-activated killer; described in Grimm et al., "J.Exp. Med.", 155:1823-41 (1982) activities of 10 PEG<* >/IL-2 and 20 PEG<*>/IL-2 were identical to those of Unmodified IL-2 Addition of free PEG (4K daltons) in a 20-fold molar excess over unmodified IL-2 did not affect NK or LAK activities D. Stability of PEGylated IL-2 as a function of pH.

PEGylated IL-2 from a modification reaction using 50 mol PEG<*> per mol IL-2 was incubated at different pH values at room temperature for three hours and then analyzed by 14* SDS-PAGE for hydrolysis of the amide-linked PEG from the IL-2 polypeptide. PEGylated IL-2 was threefold diluted in 10* acetonitrile containing 0.1* trifluoroacetic acid at pH 2.5, and also incubated at pH 7.5, 10 and 11. Alkaline pH values were obtained by addition of NaOH to sodium borate buffer at pH 9. No evidence of hydrolysis was obtained below pH 11 under these conditions. However, at pH 11 the PEGylated IL-2 was susceptible to hydrolysis. The observation that PEGylated IL-2 is stable at pH 2.5 for three hours at room temperature, i.e. 20-23°C, is particularly useful in light of US-PS., 4,569,790 which describes a method for recovering IL- 2 involving an SP-HPLC step carried out under similar conditions.

E, Pharmacokinetics of PEGylated IL-2 compared to Unmodified IL-2 in mice.

1. Intravenous administration.

Pharmacokinetic data of unmodified IL-2 and two preparations of PEGylated IL-2 were obtained after intravenous administration of 12.5 µg protein in D5W (5* dextrose in water) in each mouse in a total of 36 mice. Samples used for injection, 100 µl bolus, are identified below and had the following activities.

Sample A was made free of aggregated material by injection into D5W containing 0.1* SDS final concentration. The PEGylated IL-2 samples, B and C, contained no SDS because they are completely soluble without aggregation under normal aqueous conditions.

Each mouse from three groups of twelve female Balb/C mice was injected with one of the three samples in the tail vein and all were bled retroorbally at 1.5 minutes. At various later times after injection, 100 µl blood samples were removed retroorbitally into heparinized capillary tubes. Plasma was prepared immediately by centrifugation for 1 minute, and a fixed amount was diluted into assay medium for bioassay as described under I-C. Figure 3 shows the pharmacokinetics of unmodified IL-2 and two preparations of PEGylated IL samples. The half-lives for the original dilution of IL-2 from Figure 3 (at 50* bioactivity) are as follows:

Thus, PEGylation of IL-2 1 results in a fivefold increase in circulating half-life in mice using 10 PEG<*>/IL-2, as measured by cell proliferation assay, and more specifically a 17-fold increase in circulating half-life using 50 PEG<* ->IL-2.

When Unmodified IL-2, PEGylated IL-2 (from 10 mol PEG<*>/mol IL-2 reaction) and PEGylated IL-2 (from 50 mol PEG<*>/mol IL-2 reaction mixture) were injected intravenously in 12 mice per each type of IL-2, is the percentage recovery of the bioactivity of total injected units of IL-2 after 1.5 minutes as indicated below:

These results show a dramatic increase in percent recovery of the IL-2 bioactivity with the degree of modification of PEG<*> with 100* recovery occurring at the modification level of 50 mol PEG<*> per moles of IL-2.

2. Subcutaneous administration.

Pharmacokinetic data of unmodified and PEGylated IL-2 were obtained after subcutaneous administration in 48 mice at 12.5 pg protein in sterile water. Samples used for scapular subcutaneous injection (single 100 µl bolus) 1 mouse including unmodified IL-2, PEGylated IL-2 (from 20 mol PEG1*/mol IL-2 reaction mixtures) and PEGylated IL-2 (from 50 mol PEG< *>/mol IL-2 reaction mixtures). All three tests occurred at 0.125 mg/ml. The unmodified IL-2 sample contained 0.1* SDS due to its insolubility in aqueous solution at neutral pH.

At various time points, 100 µl samples were removed retroorbitally into heparinized tubes as previously indicated. Plasma was prepared and quantity provided for bioanalysis. The 45 minute time slot had 16 minutes for each of the three tests. All other times had 2-5 mice per try. Figure 4 shows the pharmacokinetics of unmodified and PEGylated IL-2 after subcutaneous injection in mice. Not only was the maximum percentage of total injected IL-2 bioactivity found in plasma much higher for PEGylated molecules as indicated above, but the clearance rate for PEGylated IL-2 was significantly reduced (see Figure 4).

F. Immune response in rabbits after injections of PEGylated

IL-2 and unmodified IL-2.

This study had three groups with four rabbits in each group.

Group A were rabbits injected with Unmodified postdia-f Utrated des-alanyl, ser^25 IL-2 from the manufacturing process as described above (lot LP 304). Group B were rabbits injected with PEG/IL-2 prepared from lot LP 304 using a 20-fold excess of PEG<*> as described above. Group C were rabbits injected with PEG/IL-2, also prepared from LP 304 using a 50-fold excess of PEG<*>. Each IL-2 preparation was diluted 1 sterile water before injection.

White female New Zealand rabbits with approx. 2.2 kg body weight, each was injected intramuscularly at two sites at 0.5 ml (1-2 x 10^ units) per place with the relevant IL-2 or pegylated IL-2.

At different time points, all rabbits were bled from the marginal ear vein or middle ear artery. The blood was allowed to clot and centrifuged to obtain serum. Equal amounts of sera were diluted 5-fold in IL-2 assay medium and bioanalyzed by the cell proliferation assays from example I.C. The pharmacokinetic profiles of IL-2 and PEGylated IL-2 in circulating blood after intramuscular injection were similar to those obtained for mice.

One week after the injections, all rabbits were given a second series of injections, Intramuscular, with suitable IL-2 or PEGylated IL-2. The injected units were as above.

Three weeks after the first injections, all rabbits were treated with the relevant Unmodified IL-2 or PEGylated IL-2 1 1-2 x 1<4> units per kilo. Antigen-specific antibody response was measured in sera (obtained as described above) at regular intervals by ELISA assays using horseradish peroxidase-linked goat rabbit IgG as labeled reagent and orthophenylenediamine as substrate. The absorbance was measured at 492 nm. The antigens were coated on two types of ELISA plates, polystyrene and polyvinyl. The antigens tested against sera were unmodified IL-2 (LP 304), PEG/IL-2 (20-fold excess PEG<*>) and PEG/IL-2 (50-fold excess PEG<*>). The results five weeks after the first injections were as follows: Group A. These rabbits had all developed IL-2-specific IgM, seen at dilutions of 10<4->10<5> in ELISAs. TWO, A3 and A4, of the four rabbits also had high IL-specific non-IgG at up to 10<4> dilutions. Rabbit A2 had a somewhat lower level of IgG. Rabbit Al had the least IL-2-specific IgG.

Group B. These rabbits did not develop detectable IL-2-specific IgG. All had IL-2-specific IgM, detected at 10<2> dilutions in ELISA analyses. These were repeated using PEG/IL-2 as the antigen on the ELISA plates, with the same result.

Group C. These rabbits had no detectable IL-2-specific IgG. All had IL-2-specific IgM, detected up to 10<2> dilutions in ELISA analyzes carried out with PEG/IL-2 as antigen.

These studies indicate that the antigen-specific IgG response is reduced in those cases where PEG/IL-2 is antigenic, while antigen-specific IgG develops over time with unmodified IL-2.

G. Efficacy studies of PEGylated IL-2 in Balb/c mice by

use of met A.

In this experiment mice were dosed daily, PEG/11-2 was very effective against met A sarcon in doses where unmodified IL-2 had only a slight effect.

66 Balb/c mice were all injected subcutaneously in the back of the neck with 6 x 10<5> methacolanten-A (met A) mouse fibrosarcoma tumor cell lines, obtained from Sloan-Kettering as a cell suspension from ascites fluid of Balb/c mice. The mice were segregated into three groups with corresponding numbers of large, medium and small tumors (5-100 mm). The three groups were then injected intraperitoneally with test material. Group Å received 0.5 ml PBS containing 0.01 mg/ml PEG (monomethyl 5000). Group B received 0.5 ml of tissue culture medium containing 10* calf serum + 3 µg of the PEG<*>/IL-2 obtained using a 20-fold excess of PEG<*> relative to IL-2. Group C received tissue culture medium containing 10* calf serum + 3 Mg of unmodified postdiaphUtrated des-ala^, ser125*Jj~2 as described above.

The three groups were injected daily for 7 days. The mice were weighed and tumor volumes measured on four days.

On day 8, the mice treated with formulated IL-2 showed 64* tumor growth inhibition. On the 9th day, however, the inhibition was only 40* and the tumors grew rapidly. The group was euthanized to limit suffering. Mice treated with PEG<*>/IL-2 showed 94* tumor growth inhibition both on day 8 and day 9. These mice were also killed early.

EXAMPLE III

Preparation of PEGylated IL-2 with PEG molecular weight 350

A. Preparation of PEG ester.

A linear, monomethyl substituted ester of PEG with a molecular weight of 350 was obtained by the following method.

A total of 10 g of commercial monomethyl PEG-350 was heated to 110°C. To this was added 14.3 g of succinic anhydride. The mixture was stirred overnight at 110°C and then cooled. Benzene was added and the benzene solution was filtered. The reagent was isolated from the filtrate.

Two grams of the resulting PEG-350 succinate was mixed with 25 ml of dimethylformamide, 3.531 g of dlcyclohexylcarbodiimide and 6.914 g of HNSA, prepared as described in Example 1. The mixture was stirred at room temperature for 48 hours in the dark and filtered. 1 liter of ether was slowly added to the filtrate to precipitate the reagents. A total of 150 mg of crude mixture was added to 1 ml of H2O, centrifuged and decanted. The supernatant was applied to a Sephadex G-25 column in water and the appropriate fractions were collected and lyophilized. The resulting purified product is then called PEG<*>.

B. Conjugation of PEG<*> to IL-2.

Des-alaiseri 25lL-2 prepared as described in US-PS 4,518,584 and 4,572,798, supra, was used for this example. To 0.4 mg of this purified IL-2 in 1.0 ml buffer (0.1 M sodium borate, pH 9, 0.1* SDS) was added freshly prepared aqueous PEG<*> in a molar ratio of 10 mol PEG* per moles of IL-2. After thorough mixing, the solutions were stirred at 32°C for 15 minutes, 1 hour, 5 hours and 20 hours. At each time point, 125 µl of solution was removed and added to 40 µl lmg/ml c-NH 2 -caproic acid and stored at 4°C.

C. Characterization of PEGylated IL-2.

SDS-PÅGE (14*, reducing) analysis of the products from point B showed that there was a significant degree of modification that occurred at 15 minutes.

PEG-350 IL-2 was active as tested by the in vitro IL-2 cell proliferation bioassay, mentioned under I.C. D. Pharmacokinetics of PEGylated IL-2 compared to Unmodified IL-2 in mice.

PEG-350 IL-2 and Unmodified IL-2 were injected intravenously into 1 mouse for pharmacokinetic analysis in a manner similar to that described in II.E. The results are shown in Table II.

EXAMPLE IV

Preparation of PEGylated IL-2 with PEG

molecular weight 400 and 1000

IL-2 derivatives of linear dihydroxy (unsubstituted) PEG of molecular weight 400 and 1000 were prepared generally by the method described in Example II using unsubstituted PEG-400 and PEG-1000.

EXAMPLE V

Production of PEGylated IL-2 with PEG molecular weight 10,000, 20,000 and 35,000

An IL-2 derivative of linear, monomethyl substituted PEG with a molecular weight of 10,000 was obtained generally by following the method described in Example I.A. using commercial PEG 10,000. IL-2 derivatives of dihydroxy PEG with molecular weight 20,000 and 35,000 were obtained by following a method similar to Abuchowski et al., supra, (using base in a solvent at room temperature instead of at elevated temperature using commercial PEG 20,000 and PEG 35000. The resulting modified IL-2 proteins were bioactive, as analyzed by the cell proliferation assay described above.

EXAMPLE VI

Preparation of PEGylated interferon-p

(IFN-P) using PEG-5000

Preparation of activated PEG ester and conjugation of the activated PEG ester to RP-HPLC-purified recombinant IFN-β where the cysteine residue in position 17 is replaced with a serine residue (ser17IFN-P), as described in US-PS 4,518,584, was carried out essentially as described for IL-2 in Example I.A. and I.B., in reactions containing 0, 10, 20 or 50 mol PEG<*> per moles of IFN-β. Separation of PEGylated IFN-β from unmodified IFN-β can be achieved by molecular exclusion chromatography using a Sephacryl S-200 column in 50 mM sodium acetate pH 5 with 0.1* SDS. Equal amounts of S-200 fractionation were assayed for IFN-β antiviral activity using the cytopathic effect, CPE, assay method as described generally by V.E. Stewart, "The Interferon System", Sprlnger-Verlag, pp. 17-18 (1979) and found to be active as described in Example VII. The CPE assay works on the principle that interferon protects cells treated with it from the effects of viruses. Cells that are more resistant to viruses will survive, while those that are sensitive to viruses will notice the cytopathic effect and die.

EXAMPLE VII

Characterization of PEGylated IFN<p>,

modified with PEG-5000.

A. Size characterization of modified IFN-β<-> products

from reactions with varying PEG<*>: IFN-β molar ratios.

SDS-PAGE (14*, non-reducing) of the products from the reactions described in Example VI, containing 0, 10, 20 or 50 mol PEG1* per mol IFN-β, shows, as is the case for IL-2, an increasing degree of modification with increasing PEG*: IFN-β molar ratio. Densitometric sweeps, Figure 5, show a decrease in the amount of unmodified IFN-β running at a molecular weight of 20,000 with increasing PEG<*>:IFN-β molar ratio. A discrete species with an apparent molecular weight of 30-35,000 was present after PEG-modlf in the serum of IFN-P at all three molar ratios tested (10 PEG<*>/IFN-p, 20 PEG<*>/IFN-P and 50 PEG<*>/IFN-P). An increase in higher molecular mass species, probably more strongly modified IFN-p<-> molecules, was obvious 1 the reaction carried out at 50 mol PEG<*> per moles of IFN-β.

B. Blo activity for PEGylated IFN-β compared to

unmodified IFN-β.

Fractions of S-200 the separation of PEGylated reactions containing 0, 10, 20 or 50 mol PEG<*> per mol of IFN-β was analyzed for antiviral activity as described in Example III. The bioactivities of PEGylated IFN-β obtained from all three modification reactions were comparable to unmodified IFN-β as shown in Table III. C. Solubility of PEGylated IFN-β compared to unmodified IFN-β.

After the modification reaction and S-200 fractionation, SDS was removed using Sephadex G-25 chromatography similar to that described in Example I, all of the PEGylated IFN-β and unmodified IFN-β were each adjusted to pH 7. While PEGylated IFN-β -p remained in solution as indicated by absorbance sweeps from 200 to 650 nm, unmodified IFN-p precipitated out of solution at pH 7, Figure 6. Both modified and unmodified IFN-p were soluble at pH 9. Similar results were obtained for all PEGylated IFN-β<-> samples investigated. D. Pharmacokinetics of PEGylated IFN-β compared to unmodified IFN-β.

The in vivo half-life was improved similar to that of IL-2 in rats and mice using PEGylated IFN-β versus unmodified IFN-β.

EXAMPLE VIII

Preparation and characterization of PEGylated ricin A

A. Preparation of PEGylated ricin A chain.

A soluble recombinant ricin A which does not require solubilization to undergo purification and to show cytotoxicity was prepared according to the procedure described as follows. When the coding sequence for ricin A was placed in direct reading frame with the DNA-encoding leader sequence of phoA to give a putative fusion peptide such that the leader sequence is a terminal part of a leader/ricln A chimera, the thus arranged ricin A sequences resulted in the soluble cytotoxic material.

Expression vectors containing genes for the precursor proteins contained in pRT3 (ATCC No. 67,027, March 7, 1986) pRT17 (ATCC No. 67,026, March 7, 1986) and pRT38 (ATCC No. 67,025, March 7, 1986) or their mutagenized forms was constructed. Transformation of the host cells with these expression vectors resulted in solubilization of the encoded precursor protein. The arg-arg-modified precursor was digested with trypsin; The A and B parts of the precursors were produced as separate proteins as described here.

In the phoA expression system, the essential component is the terminated phoA leader sequence upstream to be proximal to, and out of frame with, the ricin A coding sequence, in which the ricin A is initiated ATG codon. The two coding sequences must of course be provided with a compatible bacterial promoter which was the phoA promoter already associated with the leader. In addition, the production was improved in the presence of a positive retroregulatory sequence which was the positive retroregulatory sequence associated with the crystal protein of B. thuriniensis. This was provided on bacterial transport vectors that included replicons and selectable markers.

The vectors were then used to transform a suitable prokaryotic host which was grown under conditions suitable for the particular host chosen, most often under conditions where the promoter placed in control of the expression system was under pressure. The production of ricin A was then induced by providing conditions which effected expression under the control of the selected promoter and the production allowed to occur for a sufficient time to effect the desired accumulation of the product. The protein product was then isolated by breaking up the cells and cellular debris was removed. Prepared ricin A was then further purified using standard techniques known in the art as applied to freely soluble proteins. However, the efficiency of the extraction and purification was improved by treating partially clarified extracts with phenyl sepharose. The solubility of ricin A in the sonicate (once separated from the membrane or other associated material) was demonstrated by its ability to remain in the supernatants when the sonicate was subjected to high-speed centrifugation, 100,000 x g for 30 minutes, to spin down insoluble proteins.

A total of 2 ml of this soluble ricin A (at 98.0 mg/ml) was reduced again by adding 3 µl of fresh p<->mercapto-methanol to 0.1* and incubating at room temperature overnight. The 2 ml of reduced ricin A was applied to a G-25 column from Pharmacia, equilibrated with 0.10 M NAP0 4 pH 8.0, followed by 0.5 ml of buffer to provide a 2.5 ml sample volume. The next 3.0 ml of eluate (buffer was used) was collected as desalted ricin A.

1.0 ml of desalted ricin A (in an amount of about 6 mg) was transferred to a 1.5 ml microtube. To this ricin A was added 4.5 mg of N-hydroxysuccinimide ester of polyethylene glycol 2000 (activated PEG), obtained as in Example I.A. The 4.5 mg of activated PEG represented an 11-fold excess of activated PEG 1 relative to ricin A.

The activated PEG was dissolved in the ricin Å solution by gentle mixing. At various times, 100 µl aliquots of the reaction mixture were desalted by the following procedure G-25 columns to remove unreacted activated PEG and to stop the PEGylation reaction:

100 µl PEG/ricin A added

2.4 ml M NaPO 4 pH 8.0 added

1.1 ml of 0.10 M NaPO 4 pH 8.0 added and the eluate collected as desalted ricin A.

The time points were 10 min., 20 min., 30 min., 45 min., 1 hour, 2 hours, 3 hours, 4 hours and 5 hours.

The reaction mixture was kept at ls from time 0.

A 15* minigel was run to determine the degree of PEGylation. The results suggested that the PEGylation appeared to have worked well and that the reaction occurred rapidly.

A new sample of PEGylated ricin A was prepared for conjugation to an antibody. About. 40 mg of ricin A as described above was mixed with approx. 10 ml tris buffer, pH 8.5 with 1* p<->mercapto-ethanol. This was concentrated to approx. 5 ml and desalted in an EDTA buffer on two G-25 columns to thereby give 6.37 mol of total eluate. A total of 24.6 mg of N-hydroxysuccinimide ester of PEG (about 2000) was added to this ricin A and the mixture was allowed to react for 15 minutes on ice. (These amounts are a 10-fold excess of the activated PEG).

The PEGylated ricin A was desalted over three G-25 columns, thereby giving a final eluate volume of 9.70 ml. β-mercapto-ethanol was added to 5 mM, approx. 0.05*. The PEGylated ricin A mixture was stored at 4<*>C.

A total of 50 µg of PEGylated ricin A mixture was injected into a preparative "Zorbax G-250" size distribution column at a flow rate of 1 mm/min. using a buffer 50 mM (NH 4 ) 2 SO 4 , 40 mM NaPO 4 , pH 6.5.

The first peak was PEGylated ricin A as determined by a 13.5* minigel assay on 5 ml fractions from a preparative fractionation.

The quantity obtained in the first sample was concentrated to approx. 2 ml and then purified by HPLC on a "Zorbax GF-250" column by monitoring the absorbance at 280 nm. Fractions 15-23 in each sample were pooled as PEGylated ricin A.

The molecular weight for PEGylated ricin A was then determined by running a molecular weight standard (BioRad) on HPLC, under conditions corresponding to the purification method described above. A linear regression was set up which indicated that the PEGylated ricin A had an apparent molecular weight of approx. 22 K (1-mer), 44 K (2-mer) and 59 K (higher mer).

B. Characterization of PEGylated ricin A.

Aliquots from the 2 mg PEGylated ricin A Zorbaz GG-250 trial were tested for bioactivity in a reticulocyte assay (translation) using a kit manufactured by Promega Brotec. The samples given were fraction 28 (high molecular weight), 34 (2-mer), 40 (1-mer) and some crude PEGylated ricin A/unmodified ricin A mixtures. Rabbit reticulocyte cell-free translation system assays measure protein synthesis by incorporation of radioactive methionine.

The results shown in Table IV showed that only the purified 1-mer showed inhibition approaching that of free ricin A. The 2-mer and higher mer fractions showed greatly reduced inhibition as measured at elevated inhibitory doses at 50* (ID50). Thus, it appears that it may be necessary for PEGylated ricin to produce only 1-mers and to remove higher mers from the mixture.

The solubility advantage of the PEGylation was noted, the concentration of PEGylated ricin A to 20 mg/ml that was achieved would not be possible using non-PEGylated ricin A.

C. Conjugation of PEGylated ricin A to antibody.

A breast monoclonal antibody designated 520C9 was deposited under the number HB8696 on January 8, 1985 at ATCC. This antibody was reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) at room temperature and then cooled, after which sufficient 2-iminothiolan, IT, was added to give 2.5 IT molecules per antibody molecule.

A total of 166 µl of propylene glycol was added to 0.84 ml of the IT-derivatized antibody. The 2.32 ml of PEGylated ricin A chain as described above was added to initiate the conjugation reaction. The mixture was incubated at room temperature for two hours.

The conjugation reaction mixture was applied to a "Zorbax-GF-250" size (gel filtration) HPLC column using an elution buffer of 0.15 M NaPO 4 , pH 8.0. Overall 78% recovery of purified immunoconjugate was obtained from the column.

D. Characterization of Conjugate.

1. Ribosomal translation assay.

The collected conjugate was filter sterilized under a sterile hood into a sterile tube from which amounts of sterile were pipetted into various sterile micro test tubes. A 100 µl amount of final sterilized immunotoxin was incubated with purified extracts from rabbit reticulocytes, 35 g methionine, mRNA and other factors for mRNA synthesis. The analysis was measured without and with an inhibitor of ribosomal translation. The dose response curve measured protein synthesis. Control samples included a conjugate of non-PEGylated immunotoxin, ricin A chain and PEGylated ricin A chain. The results were as follows:

This analysis indicated that the ID50 for PEGylated conjugate and non-PEGylated conjugate was 15.85 and 7.94 ng/ml, respectively. Due to the fact that the accuracy of this analysis is no better than approx. +/- 100*, the inhibition for both is identical. It is obvious that the addition of antibody alone to ricin A chain reduces the ID50 compared to unconjugated ricin A - Thus, PEGylation does not impair ricin A activity more than the addition of antibody. PEGylation of the immunotoxin instead of the toxin itself is expected to improve the results.

2. In vitro cytotoxicity assay.

40,000 sample breast cancer cells (from the generally available cell line SKBR-3) in 1 ml medium were added to a set of 8 ml glass vials followed by the addition of PEGylated and non-PEGylated conjugate dilution (in phosphate buffered saline,

PBS, containing 100 pg/ml bovine serum albumin, BSA). After incubation at 37°C for 22 hours, the medium was aerated, the monolayers were washed with PBS and methionine-free medium, supplemented with 35 g of methionine, was added. The vials were further incubated for 2 hours at 37°C, the medium was removed and the cells were washed twice with 2 ml of 10* trichloroacetic acid containing 1 mg/ml methionine. The cells were dried, scintillation ion fluid was added and the radioactivity was counted in a scintillation counter. Cytotoxicity was expressed as the tissue culture inhibitory dose of conjugate resulting in 50* control (untreated) protein synthesis (TCID 50*).

The results were as follows:

The accuracy of this analysis is also no better than +/-100* and thus the TCID values are both identical. In terms of cellular intoxication, the rate-limiting step does not seem to involve ricin A chain activity but rather something else, very possibly conjugate binding, translocation and/or intracellular phenomena.

EXAMPLE IX

Preparation and characterization of IL-2 modified with polyoxyethylated glycerol, POG.

A. Production of active P0G-IL-2.

POG with a molecular weight of 5000 was generally synthesized from polysclences. To 10 g of POG, 2.28 g of glutaric anhydride was added, a 10-fold excess in relation to POG. This mixture was stirred for 2 hours at 110°C and cooled. The whole was dissolved in 20 ml of CHCl3 and very slowly in 500 ml of ether with vigorous stirring. The product was collected and rinsed with ethyl ether to give approx. 90** POG glutarate product. This product was reacted with N-hydroxysuccinimide as described in Example I.Å. thereby obtaining the active ester POG-glutaryl N-hydroxysuccinimide (POG<*>). Then 20 ml of 0.25 mg/ml IL-2 in 0.1 M sodium borate, pH 9, 0.1* as described in example I.B, was reacted with 5 mg of POG<*> at room temperature for 30 minutes. 10 ml of the reaction mixture was concentrated to 2 ml and applied to a Sephadex G-25 column of 1 10 mM sodium borate pH 9. This was adjusted to pH 7 (soluble) and concentrated. To 2 ml of the concentrate was added a solvent consisting of 1.7 M (NH 4 ) 2 SO 4 » 50 mM sodium phosphate, pH 7. This was then applied to a phenyl-TSK column at 0°C. SDS-PAGE, 14* and reducing, of the fractions indicated good separation.

The other 10 ml of POG-IL-2 was concentrated to 3 ml after adjustment to pH 5. The whole was applied to a Sephacryl S-200 column. SDS-PAGE, 14* and reducing, of the fractions showed a homogeneous POG-IL-2 product.

B. Characterization of POG-IL-2.

The fractions from the S-200 separation of the reaction mixture were analyzed for blo activity as described in Example I.C. The results are indicated in Table V.

Depositions

The plasmids with sequences used to produce the ricin A chain and the hybridomas that produced the antibody 520C9 have been deposited at ATCC. The deposit number and deposit data for the samples are:

According to the contract with ATCC, the applicant wants the expert solution in accordance with the Budapest Convention.

As a summary, the present invention provides a pharmaceutical preparation in which a biologically active specific protein is selectively conjugated to a protein selectively conjugated to a PEG homopolymer or a polyoxyethylated polyol and thereby made soluble or more soluble in an aqueous medium at physiological pH values by dissolution in such a medium. The conjugates not only serve to solubilize the normally hydrophobic water-insoluble protein in water at pH 6-8, but also increase the physiological half-life and may reduce immunogenicity by reducing or eliminating aggregation of the normally hydrophobic protein or by shielding its antigenic determinants. Without conjugation, the protein must be made soluble by adding solubilizing agents such as detergents or denaturants by raising the pH value in combination with the addition of a stabilizer or by reducing the pH value.

Claims (1)

Process for the production of a pharmaceutical preparation, characterized in that it comprises: a) production of a water-soluble polymer with a molecular weight preferably between 350 and 40,000 with at least one terminal reactive group where the polymer is selected from polyethylene glycol homopolymers and polyoxyethylated polyols, the homopolymer being optionally substituted in one end with an alkyl group and the polyol is unsubstituted; b) conjugating a biologically active normally hydrophobic, water-insoluble protein selected from β<->interferon, interleukin-2 and an immunotoxin with the relative group of the polymer, in a ratio of 0.1 to 1000 moles of polymer per mol protein, to provide a water-soluble, biologically active, conjugated protein; and c) formulating the protein at pH 6-8 in a non-toxic, inert, pharmaceutically acceptable, aqueous carrier medium; provided that when the protein is an IFN-β or an IL-2 and when the polymer is a polyethylene glycol, then the polymer is conjugated indirectly to the protein via a group comprising a carboxylic acid ester, a carboxylic acid or an amide bond.