AU598300B2 - Succinylated interleukin-2 for pharmaceutical compositions - Google Patents

Description

n W.dW9O .WoRLP 'JON, TREATY (PCT) INTERNATIONAL APPLICATION PUBLISHED U:, (51) International Patent Classificaf ion 4 ()Inlernational. Pttblitatil; Nunibr: WO 015i 1 A61K 37/02' Al (43) International Publication"'atc: I10 March 1988 (10.03,88) (21) International ApplidationtNimlier. PCT/US87/02l 2 (81) Designiated States: AT (European patent), AU, BE*(Eu.

ropean, patent), CH (European patent), DE (Euro- Infertiational Filing Daio: 25 August 1987 (25.0$.87) 'pean patent), DK, Fl, FR (European patent), OB (European patent), IT (European patent), JP, ILUI (European patent), NL (European patent), NO, Sr.

(31) Priority Application Numbert 903,668 (European patent).

(32) Priority Date: 4$Sepernber 1986 (04.09.86) PgIse 0,r--c'rity.Cpuntry: us With internationalvgorch report.

(71) Applicant:, CET-VJS CORPORATION (US/US]; 1400 Fifty-Thi1rd Street, Emeryville, CA 94608 (US).

Inventors: KATRE, Nandihi .6107 Jordan Avenue #403, El Cerrifo, CA 94530 KNAUF, Michael, J. 2391 'rinceton Drive, San Bruno, CA 94066 .28AR18 (74)'Agent: HALLUIN, Albert, Cetus Corporation, 1400 ASRLA Fifty-Third Street Emq;yville, CA 94608

UTRLA

2 4 MAR 1988 PATENT

OFFCEI

SUCCINYLATED INTERLEUKCIN-2 FOR PHARMACEUTICAL COMPOSITIONS (57) Abstract A pharmaceutical cbrnpositton is prepared wherein biologically active conjpgatedl interleukin-2 is dissolved in an aqueous carrier medilum without the presence of a solubilizing agent. The unconjugated IL-2, which is not water soluble at pH 6-8 without such solubilizing agent, is selectively conjugated to one or more succinyl groups by reaction with succirtic anhydride.

This docu'Ment contains the' arendments made Under Section 49 and is correct for, Prim~ing.

WO,88/01511 PCT/US87/02132 EXAMPLE II i WO 88/01511 PCT/US87/02132 1 4 8 SUCCINYLATED INTERLEUKIN-2 FOR PHARMACEUTICAL COMPOSITIONS .This invention relates to a chemical modification of biologically active interleukin-2 (IL-2) Which alters the chemical 5 and/or physiological properties of the IL-2. More specifically, this invention, relates to selective succinylation of IL-2 to render it soluble at physiological pH.

Many heterologous proteins produced in microbial host cells, including IL-2, are found as insoluble material in refractile bodies. In addition, IL-2 is hydrophobic in nature and tends to stick to materials and to itself aggregate) rather than remain in solution. Also, the recombinant IL-2 from E. coll is unglycosylated, whereas its native counterpart is a water-soluble, glycosylated molecule,, Modification of the IL-2 which might alter its solubility properties would be desirable to facilitate formulation of IL-2 fotherapeutic use-' In addition, modifications may reduce or eliminate aggregation of the IL-2 when it is introduced in vivo, thereby rdducing its immunogenicity.

The use of polypeptides such as IL-2 in circulatory systems 'for the purpose of producing a particular physiological response is well -known *in the ,medicinal arts. A limitation to the potential therapeutic benefit deriyed from the clinical use of polypeptides is their ability to elicit .an immune response in the circulatory system. This immune response may be caused by aggregates in the material prior to injection as described by R. Illig (1970), J. Clin.

SEndrocr., 31, 679-688,, Moore (1978), J. Clin. Endrocrinol. Metab., 46, 20-27 and W. Moore and P. Leppert (1980), Clin. Endrocrinol.

ietab. 51, 691-697.

Modification of these potentially useful therapeutic polypeptides so as to preclude 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 aforementioned disadvantages.

WO 88/01511 PCT/US87/02132 2 U.S. Patent .No. 4,179,337 discloses conjugating of -watersoluble polypeptides such as enzymes and insulin to PEG or PPG. U,S.

Patent No. 4,002,531 discloses a different method of conjugating enzymes to PEG through an aldehyde derivatiye, W087/00056 published January 15, 1987 discloses conjugation of interferon-., interleukin-2 or an -immunotoxin with PEG .or polyoxyethylated polyols to obtain increased solubility and reduced ilnmunogenicity of the protein. Also, EP 154,316 published September 11, 1985 to Takeda Chemical Industries, .Ltd., discloses and claims chemically modified lymphokines such as IL- 2 containing PEG bonded directly to at least one primary amino group of a lymphokine.

The properties of various succinylated proteins are described in Holcenberg, J.S. et al., J. Biol. Chem., 250, 4165-4170 (1975) Holcenberg, J. S. et al., Cancer Research, 39, 3145-3151 (1979), and Rulter, D, A. and Wade, Br. J. Exp. Path., 52, 610-614 (1971) None of these articles discusses any solubility changes in the protein as a result of succinylation.

U.S Patent No. 4,414,147 describes rendering interferon less hydrpphobic by conjugating it to an anhydride of a dicarboxylic acid such *as succinic anhydride. While the interferon-s specifically .exemplified .therein is made less hydrophobic, it appears also to lose substantial specific activity upon succinylation, It is not a priori possible to predict which selected proteins would be favorably responsive to derivatization with which chemical moieties due to the vast difference in the pharmacokinetics and physical properties of various proteins.

Accordingly, the present invention provides for succinylating interleukin-2, which is not ordinarily soluble in water under ambient conditions at pharmaceutically acceptable pH' ranges, to render it soluble in aqueous .buffer under such conditions. This succinylation also avoids addition of cextraneous solubilizing additives such as' detergents or denaturants to keep the protein in solution. The succinylated IL-2 retains the biological activity of the unmodified IL-2, both initially and over time. The in vivo half- I I 3 life may be modulated by selecting appropriate conditions such as degree of succinylatioh.

More specifically, the present invention is directed to a biologically active conjugated interleukln-2 (IL-2) wherein the IL-2 is covalently and selectively conjugated to one or more succinyl moieties, and wherein said IL-2 in its unconjugated form is normally hydrophobic and not soluble in an aqueous carrier medium at pH 6-8 in the absence of a solubilizing agent.

The invention also provides a pharmaceutical composition comprising a non-toxic, inert, pharmaceutically acceptable aqueous cprrier medium in which the succinylated IL-2 herein is dissolved.

Preferably, the IL-2 is recombinart interleukin-2.

Another aspect of the invention resides in a process for preparing a S biologically active, succinylated IL-2 comprising: reacting biologically active, normally hydrophobic, water-insoluble interleukin-2 with succinic anhydride, whereby the Interleukin-2 is selectively conjugated to one or more succinyl moieties; and isolating the succinylated interleukin-2.

A further aspect relates to a process for preparing a pharmaceutical composition comprising: reacting biologically active, normally hydrophobic, water-insoluble interleukin-2 with succinic anhydride, whereby the interleukin-2 is selectively conjugated to one or more succinyl moieties; isolating the succinylated interleukin-2; and formulating said succinylated interleukin-2 in a non-toxic, inert, pharmaceutically acceptable aqueous carrier medium.

In a final aspect, the invention herein provides a sustained release formulation comprising a conjugated, biologically active interleukin-2 that is covalently and selectively conjugated to one or more succinyl moieties, wherein said interleukin-2 in its ncii-succinylated form is normally hydrophobic and not soluble in an aqueous carrier medium at pH 6-8 in the absence of A solubilizing agent. A sustained release formulation, i.e., microencapsulated form, of the succinylated protein described above, is useful for pharmaceutical treatment of animals for, shipping fever.

As used herein, the term "normally hydrophobic, water insoluble" as describing the interleukin-2 refers to those interleukin-2 molecules which Tbl TCW/788v -7 3a are insoluble or not readily soluble in water or an aqueous medium under ambient conditions of room temperature and atmospheric pressure at a pH of between about 6 and 8, at about neutral or physiological pH. The modification herein acts to increase the solubility of such interleukin-Z proteins wherein 0 0 a 6* 0 4 *660 a.

eq 0 a. a a 6 a TCW/788V WO 88/01511 PCT/US87/02132 4 they are subjected to such physiological conditions,. For purposes here-in, solubi.lity may be, tested by turbidity, as measu'red by spectrophotometric means, S value, as measured by ultracentrifugatfon, Wherein the monomeric protein sedimentation rate rather than th'much, greater aggregate sedimentation rate signals solubility', and apparent native molecular weight, as measured by size' exclusion ,chrompatography,. wherein the soluble interleukin-2 is closer to this value than the insoluble, interleukin-2. The exact numbers which woul1d indicate solubility for each of these tests will depend on the, type of buffer in which the interleukin-2 is formulated, the-pH of the buffer, an! the ionic strength of 'the buffer.

The interleukin-2 'herein 'may be obtained from tissue -cultures or by recombinant techniques, and from any mammialian source such, gs, mouse, rat, rabbit, primate, :pi g, and human.

Preferably,, the interleukin-2 is derived from a human source, and more preferably is, a recombinant, human protein.* The term 1 recombinant interleukin-2,"' designated as IL-2, ~preferably 'huma6 refers to fnterleukin-2 having -comparable bio-logipca1 activity to native IL-2 prepared by recombinant DNA :-technI'ques as d~escribed, 'by Taniguchi et al., Nature, 302:305- (+1983)- and 'Devos, Nucleic Acids Research, 11:4307-33(9). I general, the gene, coding, for IL-2 is excised from its native plasmid Sand: insert.d into, a cloning, vector' to, be cloned and then into an expr'essi~n -vector, Which l's used 'to, transform a host -organism, 2- preferably 'a mi.croorganifsm, ;an4. most -preferably F. colib The lhos~t -organism expresses, the foreign, gene to produce IL"2. under expression ;cono~ttions,.

More' pt'eferab.1y the 1442 is, mu~tein as described in, U.S.

*PAtent 4,518;5.84, in whfch,, the cysteirie normally qccurring9 at 15,,,.of the wi 1d-type or n~ative. molecul ar chas been rieplaced. by t- -neutral amino Acid.. such at seri ne 6or al anine. P1ternativel'y or dbnjun ti vely, thq'L2rue' rh-ay be~ one 'In which the methta'nirve 'nirmialy occurri ng, at po§ ii on 104. of ,the,wi 1d.-type or nativd molecule his been-rn bla'ed, a neutraI ain acid suchi as alanine. Finally, WO 88/01511 PCT/US87/02132 the IL-2 employed may have one or more of the first five N-terminal amino acids of the native IL-2 deleted.

Preferably, the IL-2 is a protein produced by a microorganism or by yeast which has been 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 at positions 58 and 105, and has biological activity which is common to native human IL-2. Substantial identity of amino acid sequences means the sequences are identical or differ by one or more amino acid alterations (additions, substitutions) which do not cause an adverse functional dissimilarity between the synthetic protein and native human IL-2. Examples of IL-2 proteins with such properties include those described by Taniguchi et al., supra; Devos, supra; European Patent Application Nos. 91,539 and 88,195; and U.S. Patent 4,518,584, supra. Most preferably, the IL-2 is ser 125 IL-2, des-alalserl 25 IL-2, des-alalIL-2, des-alalalal 0 4 IL-2, or des-alalalal 04 ser 125 11-2, where *"des-alal" indicates that the N-terminal" alanyl residue of the IL-2 has been deleted.

The precise chemical structure of the IL-2 herein will depend on a number of factors. As ionizable amino and carboxyl groups are present in the molecule, a particular IL-2 may be obtained as an acidic or basic salt, or in neutral form. All such preparations which retain their bioactivity when placed in suitable environmental conditions are included in the definition of IL-2 herein. Further, the primary amino acid sequence of the IL-2 may be augmented by derivatization using sugar moteties (glycosylation) or by other supplementary molecules such as lipids, phosphate, acetyl groups .and the like, more commonly by conjugation with saccharides. Certain aspects of such augmentation are accomplished through posttranslational processing systems of the producing host; other such mqdifications may be introduced, in. vitro. In any even.t, such Smodifications. are included in the definition of IL-2 herein .so long as the..'bioactivity of. the IL-2 is not. destroyed. It is .expected, of 35" course, that such modifications may quantitatively or qualiotativ.el.y So WO 88/01511 PCTUS87/02132 6 affect the bioactivity by either enhancing or diminishing the activity of the IL-2 in the various assays.

The hydrophobic recombinant IL-2 produced from transformed host cells containing recombinant DNA precipitates inside the cell as opposed to being .soluble in the cell culture medium. The intracellularly produced IL-2 must be separated from the cellular debris and recovered from the cell before it can be formulated into a purified biologically active material. European Patent Publication 0206828 published December 30, 1986 discloses a process for isolating such a refractile material. In this process the cell membrane of the transformed host microorganism is disrupted, greater than 99% by weight of the salts is removed from the disruptate, the desalted disruptate is redisrupted, a material, preferably a sugar such as sucrose, is added to the disruptate to cieate a density or viscosity gradient in the liquid within the disruptate, and the refractile material is separated from the cellular debris by high-speed centrifugation, at about 10,000 to 40,000 x g. Preferably, the salts are removed from the disruptate by diafiltration or centrifugation and sucrose is added to increase the density of the liquid' to about' 1,1 to 1..3 g/m1.

After the Centrifugation step, the pellet containing the refractile bodies is solubilized with a denaturant such as sodium dodecyl sul.fate, the resulting suspension is centrifuged, and the supernate containing the protein is treated to isolate the protein.

The protein is separated from the supernate by appropriate 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 like its native counterpart. Such ;ox-dation is described in U.S. Patent No,. 4,550,787 to Z. Shaked.et al. The.oxidation may also be carried out by reacting an., aqueou's.'soluti,on, containing a solubilized form of the ,protein at a pH. between abo.ut 5.5 and 9 in the p;esence of air with at least ah effectiveamodnt'of an oxidation promoter containing a CU 2 cation, as 'describpd in Patent No. 4,572,798 to K. Koths et al. The WO 88/01511 PCT/US87/02132 7 preferred oxidation promoter or oxidant is CuCl 2 or (ophenanthroline) 2 Cu 2 After oxidation, the protein may optionally be desalted and purified further by RP-HPLC,, dilution/diafiltration, S 200 gel filtration chromatography, and ultrafiltration techniques before succinylation as described further hereinbelow. The succinylation may be carried out, however, at any step after the heterologous IL-2 has been isolated in sufficiently pure form to be biologically a-.ive for therapeutic purposes. The point at which the modification will occur will depend on the ultimate purity of the IL-2 required for the final pharmaceutical formulation and use.

The term "selectively conjugated" as used herein to apply to the IL-2 refers to those IL-2 proteins which are covalently bonded via one or two of the amino acid residues of the protein, depending mainly On the reaction conditions and the ultimate use. While the residues may be any reactive amino acids on the IL-2, such as one or two cysteines or the N-terminal amino acid group, preferably the reactive amino acid is. lysine, which is linked, to the succinic anhydride through its free e-amino group.

According to the process' of this invention, the IL-2, which is normally hydrophobic and water insoluble, is rendered soluble in an aqueous carrier medium, preferably at a pH of about 5 to 8, more preferably abou- 6-8 and most preferably, 6.5-7.8, without use of solubilizing agents, by modifying the IL-2 through conjugation to succinyl moTeties. If the IL-2 is reacted through its lysine residues, the pH of the reaction is preferably about 7 to 9, more preferably The success of such a modification, including retention 'of substantial biological activity, cannot be predicted from earlTer succinylation of interferon-,..

The succinyl moiety(ies) to which the IL-2 is attached is/are derived from; succinic anhydride., The succinic anhydride rselectively reacts Lwith free am.ino or other reactive groups on the IL- 2. -It will .be understood, however, that the total amount of succinic .anhydride employed and the time for reaction, to obtain optimum S* results,. will 6-depend .on the specific properties desired, to avoid 1_ WO 88/01511 PCT/US87/02132 8 having the succinic acid react with too many or too few particularly active" groups on the IL-2. The exact amount of succinic anhydride employed depends on the solvent employed (an organic solvent may require less succinic anhydride), the protein concentration, the pH of the reaction medium, the salt present in the reaction medium, and the specific properti-es desired. It is preferred, however, to use at least 2 moles, more preferably from about 2 to 60 moles, of succinic anhydride per mole of IL-2. The final amount to be employed is a balance to maintain optimum activity and solubility (achieved using higher amounts of succinic anhydride), while at the same time optimizing, if possible, the half-life of the IL-2 (the half-life decreases with higher amounts of succinic anhydride). Preferably, at least about 50% of the biological activity of the IL-2 is retained, and most preferably 100% is retained. It is noted that while the 'monosubstituted succinyl-IL-2 has improved solubility without added solubilizers at pH 7, storage at 40C for three days or physical agitation at room temperature causes visible precipitation of the IL-2 protein The covalent succinylation reaction takes place' by contacting the IL-2 with succinic anhydride, preferably at about pH 9, more preferably 7-9, and most preferably 8-9 if the reactive groups on the IL-2 are lysine groups. The reaction may take place in any 'solvent which will not adversely affect the protein. For example, dimethyl formamide may be employed if the protein is not affected adversely. 'Preferably, the reaction takes place at room temperature in an aqueous, solution containing a buffer with a solubilizing agent such as sodium dodecyl sulfate.

-The succinlc acid may be added all at once to the solution of- the oIL-2, but preferably it is added at intervals of, five minutes as -shown "in "the examples, hereinbelow. The bioactivity of the *succ.inylated IL-2 may; be monitored at the various time points during the reaction.

°o "'''After the reaction, the succinylatejd IL-2 is isolated from the reaction mixtur.e. This may be accomplished by separating the IL-2 I I WO 88/01511 PCT/US87/02132 9 and succinylated IL-2 species from other species such as the solubilizing agent, and then, if the mono- or di-succinylated IL-2 is prepared, separating the IL-2 species such as by use of a TSK-DEAE ion exchange HPLC and desalting the separated IL-2 species. If more highly modified species are produced, after the solubilizing agent is removed, the pH may be lowered to neutral pH, if not done earlier, and the sample desalted and concentrated.

The IL-2 thus succinylated is then formulated in a nontoxic, inert, pharmaceutically acceptable aqueous carrier medium, preferably at a pH of about 3 to 8, more preferably 6-8. For in vitro applications, the modes of administration and formulation are not critical. Aqueous formulations compatible with the culture or perfusion medium will generally be used. When used in vivo for therapy, the sterile product will consist of a mixture of IL-2 dissolved in an aqueous buffer in an amount which will provide a ,pharmaceutically acceptable pH when the mixture is reconstituted. A water-soluble carrier such as mannitol may optionally be added to the medium.. The currently formulated unmodified IL-2 is stable fbr at least six months at 4°C.

The dosage level of IL-2 in the formulation will depend on the jn vivo efficacy data obtained after preclinical testing and will depend mainly op the ultimate use.

If the formulation is lyophilized, the lyophilized mixture may be reconstituted by injecting into the vial a conventional parenteral aqueous injection such as, distilled water, The reconstituted formulation prepared as described above is.

sui:table for *parenteral administrati.on to humans or other mammals in therapeutically effective amounts amounts which eliminate or .reduce :the patient's pathological condition) to provide therapy 4 thereto. IL-2 therapy "is appropriate for a varfety of immunomodulatory indications .such as T cell mutagenesis, induction) of cytotoxic T cells, augmentation of natural killer cell acti.vity, .induction. of IFN-gamma; -restoration or enhancement of cell-ular immunity .'treatmient .of immune. deficient -conditions and augmentation of cell mediated anti-tumor activity.

WO 88/01511 PCT/US87/02132 In an alternative to direct administration of IL-2, the IL-2 may be administered in an adoptive immunotherapy method, together with isolated, .lymphokine-activated lymphocytes, in a pharmaceuticallTy acceptable carrier, where the lymphocytes are reactive to tumor when administered with the IL-2 to humans suffering from the tumor. This method is described more fully by S. Rosenberg et al., New England Journal of Medicine (1985)., 313:1485-1492.

In one specific application to animals, the succinylated IL- 2 herein may be employed to enhance weight gain in livestock or domestic animals. The administration of an effective amount of human IL-2 to such animals, preferably for a prolonged period of time and at a dose of about 103 to 105 units/kg/day, is described more fully in European Patent Publication No. 0219979, published April 29, 1987.

In another specific application to animals, the succinylated IL-2 herein may be employed to prevent and ameliorate in animals symptoms of stress and, in particular, of the malaise associated with livestock animals on feedlot, a symptomology commonly known as "shipping fever." The administration of an effective amount of human interleukin-2 to livestock, preferably cattle, to protect against such stress-induced syndromes is described more fully in European Patent Publication No. 0219979, published April 29, 1987.

Briefly, the regime of administration for shipping fever will depend on the conditions of shipment and the feedlot. It is preferred that administration be begun at least as early as arrival on the feedlot and be continued over a period of, for example, 1-8 or more days, as required either by multiple injections or by means of sustained release as described below. Total amounts in the range of 103-105 units/kg/day are generally used.

For other livestock stress-related or respiratory distress .syndromes, the regime and amounts administered will depend on the nature, and size of, the animal pig, goat, sheep, etc.) and on the severity of the symptoms.

Preferably, .the composition is administered for these veterinary .purposes as a susta ined releaose 'formulati'on. Such WO 88/01511 PCT/US87/02132 11 formulations "are of considerable variety, as is understood by those skilled in. the art. An exemplary composition for parenteral administration .using sustained release is an injectable microcapsule :.formulation :that- with a single injection will deliver recombinant human succihylated IL-2 at a controlled rate (0.07 to 7.0 mg/total dose) for a duration of 15 to 30 days for stress-induced syndromes, and at a controlled rate in the range of about 103 to 105 units/kg/day for enhancing.weight gain. (Pure human IL-2 has a specific activity of about 3-6 x 106 U/mg, so the above total stress-induced syndromes dose converts to about 2 x 105 to about 4 x 107 units.) The microcapsule formulation is a free-flowing powder consisting of spherical particles 20 to 100 um in diameter that can be injected intramuscularly or subcutaneously with a conventional hypodermic -needle, and the microcapsules consist of 0.5 to 5% human succinylated IL-2 encapsulated in poly (DL-lactide-co-glycolide) (DL- PLG) excipient, a bhi degradable, biocompatible polyester. Alternative standard formulations for sustained release are also usable.

The dose and dosage regimen of the IL-2 will depend, for example, on the pharmacokinetics of the drug, the nature of the disease, the degree of succinylation, the characteristics of the IL-2, the patient and the patient's history. For example, different modified IL-2 proteins are expected to have different pharmacokinetic and therapeutic properties which are advantageous for different routes of administration. A long-acting drug might only be administered every 3-4 days., every week or once every two weeks. The clearance 'rate can be varied to give ultimate flexibility to fit the particular need- of the patient by changi.ng, the degree of succinylation.

For .example, the mono- and di-succinyulated IL-2 proteins have the same., clearance rate as the )dified IL-2. The more highly succinyla.ted IL-2 proteins, however, have a faster clearance rate than the.unmodified IL-2.

SIn-.the -following examples, .which illustrate the invention further, all parts .and percentages are by weight unless otherwise noted, and all temperatures are in degrees Celsius.

WO 88/01511 PCT/US87/02132 12 EXAMPLE I Preparation of Succinylated IL-2 A. Mono- and Di-Succinyl IL-2.

RP-HPLC purified recombinant des-alanyl, ser1 25 IL-2 (where the cysteine at position 125 is replaced by serine and the N-terminal alanyl residue is deleted), prepared as described in U.S. Patent Nos.

4,518,584 and 4,530,787, supra, was employed 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 0.2 moles of succinic anhydride (in dimethylformamide) per mole of IL-2. After t+rough mixing, the solutions were stirred at room temperature (23*C) for 5 minutes. Then 0.2 moles of succinic anhydride per mole of IL-2 was added. This addition was repeated nine more times at 5-minute intervals. The reaction mixture was applied to a 1 x 45 cm desalting column to separate IL-2 and succinylated IL-2 species from low molecular weight species. The desalting column was run in 10 mM Na borate pH 9 containing no SDS and served also to remove most of the SDS from the protein. The two species of succinylated IL-2 were separated from each other and from the unmodified IL-2 by applying the reaction mixture to a DEAE ion exchange HPLC column, with pH adjusted to 7.

Then the purified pools were desalted and concentrated using a microconcentrator.

B. Highly Succinylated IL-2.

To a 5 mg/ml IL-2 solution containing 0.1 M sodium borate, pH 9 and 0.1% SDS (the IL-2 being the post-diafiltered des-alalserl 2 IL-2 from the production process described above) was added with stirring at room temperature 10 moles of succinic anhydride (in DMF) per mole of IL-2 at 5-mi.nute intervals, for a total of either three or six additions. Aliquots of the reaction mixture at various time points during the reaction were assayed for IL-2 bioactivity (cell proliferation) by the methods generally described in Gillis, et al., J. Immunol., 120, 2027-2032 (1978). The SDS was removed as described above, the pH was adjusted to 7.3, and the sample was concentrated to 0.7 mg/ml using a microconcentrator.

WO 88/01511 PcT/US7/02 132 13 EXAMPLE II Characterization of Succinylated IL-2 Species A. Size Characterization of Modified IL-2 Products from Reactions with Varying Succinic Anhydride to IL-2 Molar Ratios.

SDS-PAGE of the products from the reaction described in Example I indicated that the two preparations of highly succinylated IL-2 showed slightly increased molecular weights over unmodified IL-2. The mono- and di-succinylated IL-2 species had molecular weights very near to that of unmodified IL-2.

B. Bioactivity of Succinylated IL-2 as a Function of the Extent of Modification.

Fractions from the aforementioned elutions of highly succinylated IL-2 reactions of Example IB were assayed by the IL-2 cell proliferation bioassay described in Example I. The results show that all IL-2 fractions are bioactive. Large variations in specific bioactivity of IL-2 cause the bioactivity of the succinylated IL-2 to be about ±30% of the bioactivity of the unmodified IL-2.

C. Solubility of Succinylated IL-2 Compared to Unmodified IL-2.

After the reaction and subsequent Sephadex chromatography resulting in SDS removal, the pH of all the IL-2 preparations was lowered to 6.5-7. The unmodified IL-2 in low SDS precipitated at pH 5-7. The mono-succinylated IL-2 exhibited improved solubility without added solubilizing agents, but storage at 4 0 C for three days or physical agitation at room temperature caused visible precipitation of the mono-succinylated IL-2. However, the highly succinylated IL-2 was completely soluble at 0.7 mg/ml and the solution was stable over weeks at 4*C. The solubility of the di-succinyl IL-2 was not determined.

D. Pharmacokinetics of Succinylated IL-2 Compared to Unmodified IL-2.

1. Mono- and Di-Succinylated IL-2 Pharmacokinetic data of unmodified IL-2 and the mono- and di-succinylated IL-2 were obtained after intravenous administration of WO 88/01511 PCT/US87/02132 14 12.5 ug of protein in D5W dextrose in water) in each mouse in a total of 8 mice, four per group.

Each mouse from two groups of four female balb/c mice was injected with one of the three samples into the tail vein and all were bled retro-orbitally at 1.5 min. At various later times after injection 100 ul blood samples were removed retro-orbitally into heparinized capillary tubes. Plasma was prepared immediately bycentrifugation (1 min.) and an aliquot was diluted into assay medium for bioassay as described in Example I.

The results indicated that, within experimental error, plasma clearance of mono- and di-succinyl IL-2 was the same as that of unmodified IL-2.

2. Highly Succinylated IL-2 Three cannulated male Sprague-Dawley rats were injected intravenously for the succinylated IL-2 (two rats for unmodified IL- The rats. were bled from the cannula at each time point and plasma was prepared and diluted into medium for biQassay as described above. Highly succinylated TL-2 was found to clear faster than unmodified IL-2.

In summary, the present invention is seen to provide a succinylated IL-2 and pharmaceutical composition containing such IL-2, wherein a biologically active specific IL-2 selectively conjugatea to one or more suctinyl moieties and thereby made soluble or more soluble in an aqueous medium at physiological pH is dissolved in such medium. The conjugation serves not only to solubilize the normally hydrophobic water-insoluble protein in water at pH 6-8, but also in some. cases alters its physiological half-life. Without the conjugation, the IL-2 must be solubilized by addition of solubilizing agents such as detergents or denaturants, by raising the pH in combination with addition of a stabilizer, or by lowering the pH.

0

Claims (13)

1. A biologically active, conjugated interleukin-2 that is covalently and selectively conjugated to one or more succinyl moieties, wherein said interleukin-2 in its uncoilugated form is normally hydrophobic and not soluble in an aqueous carrier medium at pH 6-8 in the absence of a solubilizing agent.

2. A pharmaceutical composition comprising a non-toxic, inert, pharmaceutically acceptable aqueous carrier medium in which is disso' ed the interleukin-2 of claim 1.

3. A composition according to claim 2 wherein the medium is at a pH of about 5-8 and contains a buffer. s

4. A composition according to claim 2 or 3 wherein the S interleukin-2 is a recombinant interleukin-2 from a human source.

5. A composition according to any one of claims 2-4 wherein the S: Interleukin-2 is an interleukin-2 mutein that has the amino acid(s) that are at position(s) 104 and/or 125 in the native protein substituted with a neutral amino acid.

6. A process for preparing a biologically active, succinylated interleukln-2 comprising: reacting biologically active, normally hydrophobic, water-insoluble interleukin-2 with succinic anhydride, whereby the interleukin-2 is selectively conjugated to one or more succinyl moieties; and isolating the succinylated interleukin-2.

7. A process for preparing a pharmaceutical composition comprising: reacting biologically active, normally hydrophobic, water-insoluble interleukin-2 with succinic anhydride, whereby the S Interleukin-2 is selectively conjugated to one or more succinyl moieties; isolating the succinylated interleukin-2; and formulating said succinylated interleukin-2 in a non-toxic, inert, pharmaceutically acceptable aqueous carrier medium.

8. A process according to claim 6 or 7 wherein in step a total of at least 2 moles of succinic anhydride is employed per mole of interleukin-2.

9. A process according to .claim 7 or 8 wherein the succinylated interleukin-2 is formulated at a pH of 6-8 and the interleukin-2 is from a human source. TCN/788v n p. i;_iii :ii.

I 16 A sustained release formulation comprising a conjugated, biologically active interleukin-2 that is covalently and selectively conjugated to one or more succinyl moieties, wherein said interleukin-2 in its non-succinylated form is normally hydrophobic and not soluble in an aqueous carrier medium at pH 6-8 in the absence of a solubilizing agent.

11. A biologically active, conjugated interleukin-2 that is covalently and selectively conjugated to one or more succinyl moieties, substantially as hereinbefore described with reference to Example I or II but excluding any comparative examples therein.

12. A pharmaceutical composition comprising a non-toxic, inert, pharmaceutically acceptable aqueous carrier medium in which is dissolved the interleukin-2 of claim 1, substantially as hereinbefore described with S0 reference to Example II.

13. A process for preparing a biologically active, succinylated interleukin-2, substantially as herein described with reference to Example I. DATED this THIRTEENTH day of MARCH 1990 Cetus Corporation 0 Patent Attorneys for the Applicant SPRUSON FERGUSON i t L1 TCW/788v INTERNATIONAL SEARCH REPORT International Application No PCT/US 87/02132 1. CLASSIFICATION OF SUBJECT MATTER (it sevelal cl~Asiflc3tiom symools apply, idicate aill) According to International Patent Ciasaificalion (IPC) or to both National Ciassflcatlon and IPC PC 4 A 61. K 37/02 1I. FIELDS SEARCHED Minimym Documentation Searched Classification System I Classification Symbols 4 A 61 K ICi C 12 P Documentation Searched other than Minimum Documentation to the Extent that such Documents are Inciuded In the Fieids Searched Ill. DOCUMENTS CONSIDERED TO BE RELEVANT' Categoryl Citation ot Document,"1 with Indication, where appropriate, of the relevant passages 12 Relevant to Claim No,.13 UA, 4414147 (KLIBANOV) 8 November 1984 see'column 5, lines 25-46; column 7, 1-4 Ilineq 26-41 cited in the application Y EP, A, 0154316 (TAKEDA) 11 September 1985 see page 22, line 10 page 23, line 1-4 18 cited in the application SRqicil categorjer of cited documents. 'f later document published after the internatOiol riling date document defining the general, stats ~of Jhe art Which It not or Priority date and not In conflict with the appiication but ,consid red to be of-panRcular relevance cited to understand the principle 'or theory underlying the Invention earlier document but published on or ahter the lntprnailal document of particurr elevance,, this claimed Invention fiing date cannot be considered ncol or cannot be considered to L" 'document which may throw doubts on pribrity cicim(s) or Involve an Inventive step Which is cited to .atabist~ too., publication date of another document of particular relevance;' the claimed invention citationi or other special reason -(mi xpecifsedL cannot be considered to Involve an inventilv step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means Monts, such combination being obvious to a person skiiled "Is" documentpoublished prior to the International fi'ling date but in' the art. later then the Priority date climed document member of the same patent family IV. Date of the Actual Comiletion of the"International Search Date of Mailing of this lnternmtlonxL Search Rerr h Oktober 1987 ~u International Searching Authority Signature of Authorized f EUROPEAN PATENT OFFICE M. VAN MQ4 Form PCTIISA/91O (second sheell (January 13) ii mmm~ I ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL APPLICATION NO. PCT/US 87/02132 (SA 18501) This Annex lists the patent family members relating to the patent documents cited in the above-mentioned international search report. The members are as contained in the European Patent Office EDP file on 16/11/87 The European Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent document Publication Patent family Publication cited in search date member(s) date report US-A- 4414147 08/11/83 None EP-A- 0154316 11/09/85 WO-A- 8503934 12/09/85 WO-A- 8503868 12/09/85 For more details about this annex see Official Journal of the European Patent Office, No. 12/82