Polyethylene glycol-interferon conjugates, their preparation and pharmaceutical compositions containing them

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Polyethlene glycol-interferon conjugates, their preparation and pharmarceuticalcompositions containing them

F. Hoffmann-La Roche AG C.106645 14

INTRODUCTION

Interferon (IFN) alpha belongs to a family of proteins which exhibit antiviral,antiproliferative and immunomodulatory activities. A number of these proteinshave been expressed in Escherichia coli. To-date, IFN alpha has been approvedfor the treatment of a variety of diseases, i.e., hairy-cell leukemia, AIDS-relatedKarposi sarcoma, chronic hepatitis B, chronic hepatitis C (non-A/non-B),condylomata acuminata, to name a few.

The reported elimination half-life for IFN alpha ranges from 4-10 or more hours,with peak serum concentration at 3-8 hrs following IM or SC injection. Thefrequent administration necessary for sustained efficacy in interferonmonotherapy results in several dose-dependent side effects ranging from flu-likesymptoms to more pronounced manifestations (fatigue, anorexia, weight loss,transient leukopenia and some psychiatric adverse events such as depression,instability, insomnia, anxiety and suicidal behavior).

The covalent attachment of polyethylene glycol to intprfprnn alfa-2a to producePEGASYS has resulted in prolonged serum half-life thus reducing administrationfrequency as well as possible side effects. For example, Adagen (PEG-adenosine deaminase from Enzon, Inc.) has a half-life of 357 hrs compared to 20hrs for the unpegylated protein.

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120902/3 - 1 -

Interferon, in particular interferona2a, is a pharmaceutically active proteinwhich has antiviral and antiproliferative activity. For example interferon is used totreat hairy cell leukemia and Kaposi’s sarcoma, and is active against hepatitis. Inorder to improve stability and solubility, and reduce immunogenicity,pharmaceutically active proteins such as interferon may be conjugated to thepolymer polyethylene glycol (PEG). A method for the preparation of PEG reagentswith high purity is described and the introduction of two polyethylene glycol chainsinto one amino group of a protein by linking them with a disubstituted cyanuricchloride is disclosed in EP A 0 400 472. Furthermore, PEG interferon alphaconjugates where the PEG-group is directly linked via an urethane to the interferonare disclosed in WO 96 13090. PEG-G-CSF and PEG-IFN coni conjugates aredisclosed in WO 95/21629 and WO 96/11953, and polyethylene glycol proteinconjugates comprising a protein pegylated with linear polyethylene glycol aredisclosed in EP 510 356.

The bioavailability of protein therapeutics are often limited dueto their short plasma half-life, thus preventing them from attainingtheir maximum clinical potency. In recent years, PEG conjugatedbiomolecules have been shown to possess clinically useful properties(Inada et al., J. Bioact. and Compatible Polymers 5, 343 (1990);

Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9,249 (1992); Katre, Advanced Drug Delivery Systems 10, 91 (1993)).Among these are better physical and thermal stability, protectionagainst susceptibility to enzymatic degradation, increased solubility,longer in vivo circulating half-life, decreased clearance and enhancingpotency. It has been reported that branched PEG conjugates exhibitincreased pH luad thermal stability and . greater stability towardsproteolytic digestion than linear PEG conjugates. (Monfardini et al.,Bioconjugate Chem. 6, 62 (1995)). Other properties of PEG proteinsare reduced immunogenicity and antigenicity, as well as reducedtoxicity. Another effect of PEGylation of certain proteins may bereduced in vitro activity'accompanied .by enhanced in vivo activity.This has been observed in G-CSF (Satake-Ishikawa et al., CellStructure and Function 17, 157-160 (1992)), IL-2 (Katre et al., Proc.Natl. Acad. Sci. USA 84, 1487 (1987)), TNF-α (Tsutsumi et al., Jpn. J.Cancer Res. 85, 9 (1994)), IL-6 (Inoue et al., J. Lab. Clin. Med. 124, 529(1994)) and CD4-IgG (Chamow et al., Bioconj. Chem. 5, 133 (1994)),among others. - 2 -

It has been now observed that in the case of interferon,PEGylation reduces in vitro antiviral activity but increasesantiproliferative activity in human tumor cells. However the new PEGinterferon conjugate of this invention has surprising properties in that

5 the antiproliferative activity of the PEG interferon is much higherthan that not only of interferon but of other PEG interferonconjugates. Although the antiproliferative activity of the conjugate ismuch increased over other PEG interferon-α conjugates, yet thereduction in antiviral activity is similar. In addition, the PEG 10 interferon-α conjugate of this invention is non-immunogenic, it elicitsvirtually no antibody formation. In contrast, other PEG interferon-aconjugates do elicit limited antibody formation.

Accordingly, the invention is a new class of PEG derivatives of 15 interferona (IFNa). The conjugate of this invention has a branchedPEG structure, as can be seen below. The branched PEG has theadvantage of allowing the attachment of 2 linear PEG molecules at asingle site, thus doubling the attached PEG mass without multiple sitesof PEGylation. 20

Compared to unmodified IFNa (i.e. IFNa without a PEG attached),the conjugate has an increased circulating half-life and plasmaresidence time, reduced immunogenicity, decreased clearance, andincreased antiproliferative activity, concomitant with decreased in 25 vitro antiviral activity. Compared with other PEG-IFNa conjugates,the conjugate of this invention has a much greater antiproliferativeactivity, disproportionate to the enhancement or reduction that occursin its other characteristics, and virtually no immunogenicity. 30 The physiologically active PEG-IFNa conjugate species of this invention has the formula: - 3 -

I

II

ROCH2CH2(OCH2CH2)n—Ο—C —NH Ο

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—X— IFNa

O O

ROCH2CH2(OCH2CH2)n'—O—C—NH

The conjugate of this invention has the same uses as IFNa, forexample, antiproliferative uses. In particular, the PEG interferon-aconjugates of this invention are useful to treat immunomodulatorydisorders such as neoplastic diseases, for example, hairy cell leukemia,CML, and Kaposi's sarcoma, and infectious diseases, in the same wayIFNas (especially IFNa2a) are used to treat these diseases. However,the conjugate of this invention has improved properties includingsuperior stability, greater solubility, enhanced circulating half-life andplasma residence times. In addition, these conjugates have anti-proliferative activity which is superior to IFNa. Also as noted theconjugate shows a surprising dissociation of antiviral and anti-proliferative effects. This property is additionally useful to enhance adesired activity of a conjugate, while decreasing or eliminating anundesired activity. For example, if an undesired side effect isassociated with the antiviral activity, eliminating this activity wouldeliminate the side effect, while retaining the antiproliferative activity.Therefore, the present invention also comprises the pharmaceuticalcompositions on the basis of the compounds of formula I or their saltsand to methods for producing them.

The pharmaceutical compositions of the present invention usedin the control or prevention of illnesses comprises an interferonconjugate of the general formula I and a therapeutically inert, nontoxic and therapeutically acceptable carrier material. The pharma-ceutical compositions to be used can be formulated and dosed in a

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-4- 120902/2 fashion consistent with good medical practice taking into consideration the disorder to be treated, the condition ofthe individual patient, the site delivery ofthe proteinconjugate, the method of administration and other factors known to practitioners.

SUMMARY OF THE INVENTION

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X—. IFNa wherein R and R1 are independently Ci-C6-alkyl; X is NH or Ο; n and n' areintegers having a sum of from 600 to 1500; and the average molecular weight ofthe polyethylene glycol units in said conjugate is from 26,000 daltons to 66,000daltons. The conjugate of formula I has a branched structure, in that two PEGmoieties are attached to the protein via a single linkage.

The numbers n and n' are selected such that the resultingconjugate of Formula I has a physiological activity of IFNa, whichactivity may represent the same as, more than, or a fraction of thecorresponding activity of unmodified IFNa. n and n' (n and n' may bethe same or different) represent the number of ethylene glycol unitsin the PEG. A single PEG unit of OCH2CH2 has a molecular weight ofabout 44 daltons. The molecular weight of the conjugate (excludingthe molecular weight of the IFNa) depends on the numbers n and n'.The sum of n and n‘ for the conjugate of Formula I is from 600 to - 5 - 1500, producing a conjugate having a total average molecular weightof PEG units of from about 26,000 to 66,000 and preferably fromabout 35,000 to 45,000 daltons, and especially about 39,000 to 45,000daltons, with 40,000 daltons especially preferred. A preferred sum ofn and n‘ is from about 800 to 1200, with the average sum being fromabout 850 to 1000, and a preferred sum being about 910. Either of nor n‘ may individually be 420 or 520, or both may be 420 or 520, orboth may be 455. The preferred ratio of n to n‘ is from about 0.5 to1.5, with an especially preferred ratio of from about 0.8 to about 1.2. A molecular weight of "about" a certain number means that it iswithin a reasonable range of that number as determined byconventional analytical techniques.

Also preferred is a conjugate of Formula I where IFNa isIFNa2a, a conjugate where R and R' are methyl, a conjugate where X isNH, and a conjugate where n and n‘ are individually or both either420 or 520. Such a conjugate having all the above characteristics isespecially preferred. R and R' may be any lower alkyl, by which is meant an alkylgroup having from one to six carbon atoms such as methyl, ethyl,isopropyl, etc. Branched alkyls are included. A preferred alkyl ismethyl. With regard to the two PEG groups of Formula I, R and R'may be the same or different.

By IFNa (interferon a) and its species IFNa2a is meant thenatural or recombinant protein, preferably human, as obtained fromany conventional source such as tissues, protein synthesis, cell culturewith natural or recombinant cells. Any protein having the activity ofIFNa , such as muteins or otherwise modified proteins, is encompassed. Obtaining and isolating IFNa from natural orrecombinant sources is well known (Pestka, Arch. Biochem. Biophys.221, 1 (1983)). A preferred IFNa is IFNa2a, which as stated above, isobtained by known methods (Pestka, Sci. Am. 249, 36 (1983);

European Patent No. 43 980)). - 6 -

The physiologically active conjugate of Formula I has IFNaactivity, by which is meant any fraction or multiple of any knownIFNa activity, as determined by various assays known in the art. Inparticular, the conjugates of this invention have IFNa activity asshown by antiproliferative activity against tumor cells and antiviralactivity against cells infected with a virus. These are known activitiesof IFNa. Such activity in a conjugate can be determined by assayswell known in the art, for example the assays described below (seealso Rubinstein et al., J. Virol. 37, 755 (1981); Borden et al., Cane. Res.42, 4948 (1982)). Part of this invention is a conjugate of Formula Iwhich has greater antiproliferative activity and less antiviral activitythan unmodified IFNa.

The conjugate of Formula I is produced by covalent linkage ofIFNa to PEG which has been activated by replacement of the PEGhydroxyl with a linking group, forming a reagent which is an N-hydroxy succinimide ester derivative of PEG (in particularmonomethoxy PEG) of Formula II. The reagent may be obtained byconventional methods (Monfardini et al., supra). Linkage is via anamide or ester bond. In a preferred conjugate, linkage is via an amidebond (X is NH). Part of this invention is a method for increasing theantiproliferative activity of IFNa while reducing the antiviral activityof the IFNa, by linking the IFNa as described above to a reagent ofFormula II to produce a PEG-IFN conjugate. X represents the attachment site on IFNa by which the PEGreagent of Formula Π is covalently attached to the IFNa. The reagentsattach to primary amino groups (XH = NH2) on for example lysine or tothe N-terminus of the IFNa. The reagents can also attach to ahydroxyl (XH = OH) on for example serine. - 7 -

ROCHjCH 2(OCH2CH2) n —O—C—NH (CH2)4 ROCH2CH2(OCH2CH2)n-.

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+ XH-IFNa

0II

ROCH2CH2(OCH2CH2)n —0 — C —NH

(L k + HON. R'OCH2CH2(OCH2CH2)n·—0“C—N HII0

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•X— IFNa

O 5 The reagent of formula Π (PEG2-NHS), in which a total of 2 mono-methoxy PEG (m-PEG) chains are linked to lysine, one each atthe a and ε amino groups via carbamate (urethane) bonds and havingthe lysine carboxyl group activated to a succinimidyl ester, may beobtained by conventional methods, according to known procedures 10 (Monfardini et al,, supra) applicable to a reagent with R as lower alkyl,and a desired n. The reagent may be obtained from ShearwaterPolymers, Inc. (Huntsville, Alabama). The preferred average MW ofthe PEG obtained is about 20,000 daltons, providing a total PEG massof about 40,000 daltons in PEG2-NHS (other MWs may be obtained by 15 varying n for the PEG-alcohol starting materials for the reagent ofFormula II, by conventional methods).

The reagent of formula II may be conjugated to IFNa byconventional methods. Specifically, the reagent of Formula II - 8 - primarily reacts with one or more of the primary amino groups (forexample N-terminus and lysine side chains) of IFNa (for exampleIFNa2a) to form an amide linkage between the IFNa and the polymerbackbone of PEG. The PEGylation reaction can also take place betweenPEG2-NHS and the free (if any) hydroxyl groups (for example serine)of IFNa to form an ester linkage. The reaction mechanism is shownabove. The reaction conditions are conventional to a skilled person,and are provided in detail below. The PEG reagent is combined withIFNa under mildly basic conditions at low temperature underconditions suitable for a nucleophilic substitution which will producethe conjugate of Formula I. This is also shown in the above reactionmechanism.

Attaching the reagents to IFNa may be accomplished byconventional methods. PEGs of any selected MW of this inventionmay be used. Reaction conditions may be selected to provide theclaimed conjugate with one reagent attached. The conjugate ofFormula I, which has a single reagent of Formula II attached, isseparated from unmodified IFNa and conjugates having attachedmore than one reagent molecule by conventional methods.

Purification methods such as cation exchange chromatography may beused to separate conjugates by charge difference, which effectivelyseparates conjugates into their various molecular weights. Thecontent of the fractions obtained by cation exchange chromatographymay be identified by molecular weight using conventional methods,for example, mass spectroscopy, SDS-PAGE, or other known methodsfor separating molecular entities by molecular weight. A fraction thenis accordingly identified which contains the conjugate of Formula Ipurified free from unmodified IFNa and from conjugates having morethan one reagent attached. In addition, the reagents of Formula IIrelease one lysine per reagent upon acid hydrolysis, so that thenumber of lysines in the hydrolysis indicates the number of PEGsattached to the protein, thus the number of reagent moleculesattached to a conjugate may be verified. - 9 -

The following Examples are provided to illustrate the inventionand do not limit it in any way. IFNa2a is used in these examples.Other species of IFNa may also be conjugated to PEG by the methodsexemplified.

DESCRIPTION OF THE DRAWINGS

Figure 1: Antitumor activity of the PEG2-IFNalpha2a in nude miceimplanted subcutaneously with human renal A498 cells. All animalsreceived a subcutaneous implant of 2 x 106 human renal A498 cellson Study Day -33. On Study Day 0 PEG-IFNalpha2a treatment wasinitiated. The indicated amount (30, 60, 120 or 300 gg) of PEG2-IFNalpha2a was administered subcutaneously under the opposite flank ofthe tumor, 1 time per week for a four week period.

Figure 2: Antitumor activity of IFNalpha2a in nude mice implantedsubcutaneously with human renal A498 cells. All animals received asubcutaneous implant of 2 x 106 human renal A498 cells on StudyDay -33. On Study Day 0 IFNalpha2a treatment was initiated. Theindicated amount (10, 20, 40 or 100 gg) of IFNalpha2a wasadministered subcutaneously under the opposite flank of the tumor, 3times per week for a four week period.

Figure 3: Antitumor activity of PEG2-IFNalpha2a in nude miceimplanted subcutaneously with human renal ACHN cells. All animalsreceived a subcutaneous implant of 2 x 106 human renal ACHN cellson Study Day -25. On Study Day 0 PEG2-IFNalpha2a treatment wasinitiated. The indicated amount (30, 60, 120 or 300 gg) of PEG2-IFNalpha2a was administered subcutaneously under the oppositeflank of the tumor, 1 time per week for a five week period.

Figure 4: Antitumor activity of IFNalpha2a in nude mice implantedsubcutaneously with human renal ACHN cells. All animals received asubcutaneous implant of 2 x 106 human renal ACHN cells on StudyDay -25. On Study Day 0 IFNalpha2a treatment was initiated. The - 10 - indicated amount (10, 20, 40 or 100 gg) of IFNalpha2a wasadministered subcutaneously under the opposite flank of the tumor, 3times per week for a five week perid.

Figure 5: Antitumor activity of PEG2-IFNalpha2a in nude miceimplanted subcutaneously with human renal G402 cells. All animalsreceived a subcutaneous implant of 2 x 106 human renal G402 cells onStudy Day -45. On Study Day 0 PEG2-IFNalpha2a treatment wasinitiated. The indicated amount (30, 60, 120 or 300 gg) of PEG2-IFNalpha2a was administered subcutaneously under the oppositeflank of the tumor, 1 time per week for a five week period.

Figure 6: Antitumor activity of IFNalpha2a in nude mice implantedsubcutaneously with human renal G402 cells. All animals received asubcutaneous implant of 2 x 106 human renal G402 cells on Study Day-45. On Study Day 0 IFNalpha2a treatment was initiated. Theindicated amount (10, 20, 40 or 100 gg) of IFNalpha2a wasadministered subcutaneously under the opposite flank of the tumor, 3times per week for a five week period.

Example 1

Preparation of conjugate of Formula I

Materials

Interferona2a was prepared by known methods (Pestka, supra).Polyethylene glycol (PEG) reagent of formula II was purchased fromShearwater Polymers, Inc. (Huntsville, Ala). Fractogel® EMD CM650(S) resin, with particle sizes 25-40gm, were supplied by EMSeparations (Gibbstown, MA). Concentrated (10X) phosphate bufferedsaline (PBS), pH 7.3, was purchased from BioWhittaker (Walkersville,MD). Sodium dodecyl (laurel) sulfate/polyacrylamide gelelectrophoresis (SDS-PAGE) pre-cast gels and electrophoresis unitswere obtained from NOVEX (San Diego, CA). Concentrated Fast Stainfor protein staining of PEG conjugates on SDS-PAGE was purchased - 11 - from Zoion Research, Inc. (Newton, MA). The LAL endotoxin assay kitwas purchased from Associates of Cape Cod, Inc. (Woods Hole, MA).

All other reagents used were of the highest quality available. Thejugular cannulated rats and BDF-1 mice were supplied by CharlesRiver Laboratories (Wilmington, MA).

Experimental Procedures

A. Small scale preparation of conjugate of Formula I

Two hundred-eight milligrams (5.2gmol) of the reagent ofFormula II (average MW of 40,000 daltons) were added to 50 mg(2.6gmol) of IFNa in 10ml of lOOmM borate, pH 8.0. Final proteinreagent molar ratio was 1:2. The reaction mixture was stirred at 4°Cfor 2 hours. The reaction was stopped by adjusting the pH to 4.5 withglacial acetic acid.

The reaction mixture was diluted 50-fold with water, filteredthrough a 0.2μ filter and applied onto an Amicon column packed with100ml (3.2x13cm) Fractogel EMD CM 650(S), at a flow rate of20ml/min. The column was previously equilibrated with lOmMammonium acetate, pH 4.5. The column effluent was monitored byUV absorbance at 280nm. The column was then washed with theequilibration buffer until UV absorbance returned to baseline. PEG-IFN conjugates having more than one reagent of Formula II attached(PEG-IFN oligomers) were eluted with 40 mM ammonium acetate, pH4.5 and the conjugate of Formula I was eluted with 0.12M NaCI in the40 mM ammonium acetate buffer. The unmodified IFN remaining inthe column was eluted with 0.5M NaCI in the same buffer. Thecolumn was regenerated by a 1.0M NaCI wash followed by theequilibration buffer wash. The pooled fractions of the conjugate ofFormula I were concentrated in an Amicon stirred cell concentratorfitted with a YM10 membrane to approximately lmg/ml concentration. - 12 -

The Fractogel CM 650(S) cation exchange resin used forpurification, adsorbed the PEG and unmodified IFN effectively. Thestrength of adsorption was dependent upon the degree of PEGylation.The conjugates bound less tightly than the unmodified IFN. The PEG-IFN oligomers were eluted with 40mM ammonium acetate, while theconjugate of Formula I eluted with 0.12M NaCL The unmodified IFNeluted with 0.5M NaCI. All preparations contained &lt;5EU/mgendotoxins. The resulting preparation contained &gt;99% of conjugate ofFormula I and was free of unmodified IFN.

B. Large-Scale Preparation of conjugate of Formula I

Six thousand two hundred and forty milligrams (156 gmol) ofthe reagent of Formula II (average molecular weight of 40,000daltons) was dissolved in 63 ml of ImM HCI at 4°C and quickly addedto 125 ml of a solution containing 1000 mg (52 pmol) of interferon in50 mM borate buffer, pH 9.0. The final protein/reagent ratio was 1:3and the final reaction mixture protein concentration was 5.3 mg/ml.The reaction mixture was stirred for 2 hours at 4°C. The reaction wasstopped by adjusting the pH to 4.5 with glacial acetic acid.

The reaction mixture was diluted 10-fold with water andapplied onto a column packed with 600 ml Fractogel EMD CM 650(M)previously equilibrated with 20mM sodium acetate, pH, 4.5 at a linearvelocity of 1.3cm/min. The column was washed with theequilibration buffer followed by 10 mM NaCI to remove excessreagent, reaction byproducts and PEG-IFN oligomers. The conjugate ofFormula I was eluted with the equilibration buffer containing 200mMNaCI. The unmodified interferon still adsorbed to the column wasremoved by washing with 0.75 M NaCI in the equilibration buffer. Theconjugate of Formula I, which was eluted at 0.3-0.5mg/ml was furtherconcentrated and diafiltered into the final formulation buffer, 20 mMsodium acetate, pH, 5.0, containing 150mM NaCI. The overall yield ofthe conjugate of Formula I was 40-45%. 13 -

The purified PEG-IFN from the large-scale preparation consistsof &gt;99% conjugate of Formula I. The average molecular weight of theconjugate of Formula I of this example is 62,000 daltons, including themolecular weight of IFNa2a which is 19,241 daltons, and the averagemolecular weight of the reagent which is between 40,000 and 45,000daltons, about 43,000 daltons.

Example 2

Characterization of conjugate of Formula I

Protein Determination

Protein concentrations were determined using an A280 value of1.0 for a lmg/ml solution of IFNa a2a. SDS-PAGE Analysis

The conjugate was analyzed by sodium dodecyl (lauryl)sulfate/polyacrylamide (8-16%) gel electrophoresis, under reducingconditions, according to the methods of Laemmli (Nature 227, 680(1970)). SDS-PAGE containing PEG-conjugates were stained forprotein using Fast Stain (Zoion Research, Inc.) according to themanufacturer's instructions.

Determination of Endotoxin Levels

Endotoxin levels were determined using the LAL method,according to the manufacturer's instructions. All preparationscontained &lt;5 EU/mg endotoxins. - 14 -

Example 3

In Vitro Bioactivities of conjugate of Formula I

Antiviral Activity in Bovine Kidney Cells

The in vitro antiviral activity of EFNa2a and the conjugate ofFormula I as prepared in Example l.A. were determined in a cellculture bioassay employing Madin-Darby bovine kidney (MDBK) cellschallenged with vesicular stomatitis virus (Rubinstein et al., supra).The antiviral activities are listed in Table 1, along with theircorresponding residual activities as a percentage of the starting IFN.

Table 1

Anti-Viral Activities

Samples PEG Type Total PEGMass(kDa) # LysModified Specific Activity (U/mg) Residual Activity (%) IFN&lt;x2a - * - 2.00 x108 100 Conjugate ofFormula I Branched 40 1 1.40 x107 7

In Vitro Antiproliferative Activity in Human Tumor Cells

The in vitro antiproliferative activities were assayed in humanDaudi (Burkitt's Lymphoma) cells, as described by Borden et al.Human Daudi cells were maintained as stationary suspension culturesin RPMI 1540 supplemented with 10% fetal bovine serum and 2 mMglutamine (Grand Island Biologicals, Grand Island, NY). The cells werescreened and found to be free of mycoplasma. Cells (2 x 104) wereadded to wells of microtiter plates (Costar, MA) in 100 μΐ of medium.Various concentrations of IFN and the conjugate of Formula I asprepared in Example l.A. were added to the wells in a volume of100 μΐ. The plates were incubated at 37°C in 5% CO2 for 72 hours. - 15 -

Cells were pulsed with 0.25gCi/well of3H-thymidine (New EnglandNuclear, Boston, MA), sixteen hours before cell harvesting. The cellswere harvested onto glass filters and counted in a liquid scintillationcounter. The results were expressed as % inhibition calculated usingthe formula: % Inhibition = [ (A - B / A] x 100, where; A = cpm in control culture (cells incubated in medium alone) B = cpm in experimental culture

Samples were run in quadruplicate and standard deviation wasless than 20% of the mean of all cases. Experiments were run at leasttwice with comparable results.

The antiproliferative activities (IC50) of IFN and the conjugateare listed in Table 2. The data indicate that there is a 28-fold increasein antiproliferative activity for the conjugate of Formula I, ascompared to that of IFN.

Table 2

In Vitro antiproliferative activities in human Daudi (Burkitt'slymphoma) cell lines.

Antiproliferative ActivitySample IC50 (ng/ml) Increase IFNa2a 0.56 lx

Conjugate of Formula I 0.02 28x - 16 -

Example 4

Pharmacokinetics

Female Sprague Dawley rats, surgically implanted with jugularcannulas, with an average body weight of 240 - 260g were housedindividually, allowed free access to food and water and maintained ina 12 hour light -dark cycle. Within 4-6 hours after arrival, jugularcannulas were flushed with PBS. The following day, after flushingwith 0.15 - 0.2ml PBS, 2xl06 units of IFNa in 0.2 - 0.4ml PBS wereinjected, followed by injection of 0.15 - 0.2ml PBS to assure that alldrug was washed into the animal. Thus each animal received adosage of 8xl06 IFNa units/kg body weight.

Blood samples were drawn at 5, 15 and 30 minutes, as well as, 1, 3, 5, 12 and 24 hours after injection of IFN and, the conjugate ofFormula I. At all time points, after discarding the first 0.15 - 0.2mlof blood, an aliquot of 0.5ml blood was withdrawn using a freshsyringe via the jugular cannula. The samples were discharged intoserum separating tubes at room temperature. Once all the sampleswere collected for the time points, the tubes were centrifuged at14,000 x g in a refrigerated Eppendorf centrifuge for 10 minutes. Theseparated serum was transferred into 1.5ml microfuge tubes andfrozen at -80°C, until ready for bioassay. Serum samples were dilutedappropriately and the antiviral activity at each time-point wasdetermined as described. From the plot of time vs. activity, theterminal half-life of the conjugate of Formula I and IFNa weredetermined and listed in Table 3, which also include plasma residencetimes. - 17 -

Table 3

Terminal Half-Lives (ti/2) and Mean Plasma Residence Time 5 Sample ll/2 (hours) Plasma ResidenceTime (hours) IFNa2a 2.1 1.0 10 Conjugate of Formula I 15.0 20.0

Terminal ll/2 estimated by log linear regression.

Example 5 15

Immunogenicitv

Normal BDF-1 mice (ten per group) were injectedintraperitonially once per day five times per week with various 20 interferon preparations having 300,000 units of antiviral activity.Some mice were also injected with aggregated form of IFNa2a which ismore immunogenic than the monomer form. Blood samples weretaken 19 days following the last injection and the serum wasevaluated for neutralizing antibodies. 25 As seen in Table 4, mice injected with IFNa2a produced neutralizingantibodies and this response was greatly increased in mice injectedwith interferon aggregates. No antibodies were detectable in themajority of animals injected with the conjugate of this invention. - 18 -

Table 4

Immunogenicitv

Antibody (INU/ml) *

Treatment Median Range IFNa2a 2,400 217-8,533 IFNa2a Aggregates 42,667 8,000-768,000 Conjugate of Formula I 0 0-1,133 * Interferon neutralizing units/ml

Example 6

Antitumor activity In Vivo

The in vivo antitumor activity of a conjugate of Formula I(PEG2-IFNalpha2a) and unmodified IFNalpha2a were evaluated bydetermining their ability to reduce the size of various human tumorcells implanted subcutaneously into mice. Results are shown inFigures 1-6.

Procedure: Athymic nude mice (Harlan) received asubcutaneous implant under the left rear flank of 2 χ 106 humanrenal A498 cells (Figures 1 and 2), human renal ACHN cells (Figures 3and 4), or human renal G402 cells (Figures 5 and 6). 3 to 6 weekswere allowed for the tumors to become established, as indicated. Thesize criteria for acceptance into the study was 0.05 to 0.50 cubiccentimers. The mice were given total weekly doses of PEG2-IFNalpha2a or unmodified IFNalpha2a of 30, 60, 120 or 300 pg. In the case of PEG2-IFNalpha2a the mice were treated one time perweek (Monday) with 30, 60, 120 or 300 pg of PEG2-IFNalpha2a pertreatment. In the case of unmodified IFNalpha2a the mice were - 19 - treated three times per week (Monday, Wednesday, Friday) with 10,20, 40 or 100 pg of IFNalpha2a per treatment. The duration oftreatment was 4 to 5 weeks depending on tumor aggressiveness.Tumor volumes were measured every Monday prior to treatments.

Results: PEG2-IFNalpha2a showed a marked reduction in A498tumor size as compared to unmodified IFNalpha2a for all weeklydosage levels tested, at 7 days, 14 days, 21 days and 28 days after thebeginning of treatment (Figures 1 and 2). Treatment continued forfour weeks. Seven days after treatment was discontinued three micein each group were sacrified. In the three mice treated with PEG2-IFNalpha2a no residual tumor was observed. In mice treated withunmodified IFNalpha2a the A498 tumor weight was 1.28 grams, 0.62grams, and 1.60 grams respectively in each of three mice. The A498tumor weight was 2.32 grams, 2.37 grams, and 1.94 grams in each ofthree control mice. At 80 days after the end of the four weektreatment period the existence of tumors was determined bypalpation in seven mice. All seven mice were free of tumor tissue bypalpation. PEG2-IFNalpha2a showed a significant reduction in ACHN tumorsize as compared to unmodified IFNalpha2a for weekly dosage levelsof 60, 120, and 300 pg, at 14 days, 21 days, 28 days and 35 days(Figures 3 and 4). PEG2-IFNalpha2a showed a significant reduction in G402 tumorsize as compared to unmodified IFNalpha2a for weekly dosage levelsof 60 and 120 pg, at 14 days, 21 days, 28 days and 35 days (Figures 5and 6).