Interferon conjugates

PEG-IFNa conjugate: physiologically active, process for its preparation, pharmaceutical compositions containing it and their use

Interferon, in particular interferon a2a, is a pharmaceutically active protein which has antiviral and antiproliferative activity. For example, interferon is used to treat hairy cell leukemia and Kaposi's sarcoma, and is active against hepatitis. In order to improve stability and solubility, and to reduce

Immunogenicity, pharmaceutically active proteins such as interferon can be conjugated to the polyethylene glycol (PEG) polymer.

The bioavailability of protein therapeutic agents is frequently limited by their short shelf life in plasma, thus preventing them from reaching their maximum clinical activity. In recent years, PEG-conjugated biomolecules have been shown to have clinically useful properties [Inada et coil., J. Bioact. and Compatible Polymers 5, 343 (1990); Delgado and coil.,

Critical Reviens in Therapeutic Drug Carrier Systems 9, 249 (1992); Katre, Advanced Drug Delivery Systems 10, 91 (1993)]. These include better physical and thermal stability, better protection against sensitivity to enzymatic degradation, increased solubility, longer circulation half-life in vivo,

010488 decreased clearance and increased activity. Branched PEG conjugates have been reported to have increased temperature and pH stability and greater stability to proteolytic digestion than linear PEG conjugates [Monfardini et al. , Bioconjugate Chem. 6, 62 (1995)]. Other properties of PEG-conjugated proteins are reduced immunogenicity and antigenicity, as well as reduced toxicity. Another effect of PEG conjugation (also hereinafter referred to as PEGylation) of certain proteins may be reduced in vivo activity accompanied by increased in vivo activity. This has been found, among others, for G-CSF (granulocyte growth factor) (Satake-Ishikawa et al., Cell, Structure and Function 17, 157-160 (1992)],.

IL-2 [Katre et al., Proc. Natl. Acad. ' Sci. USA 84, 1487 (1987)], TNF-oc [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)].

It has now been shown that in the case of interferon, PEGylation reduces the antiviral activity in vitro but increases the antiproliferative activity in human tumor cells. However, the new interferon-PEG conjugate of the present invention has surprising properties, in that the antiproliferative activity of interferon-PEG is much greater than that not only of interferon but also of other interferon-PEG conjugates. . Although the antiproliferative activity of the conjugate is greatly increased compared to that of other PEG-interferon conjugates and, the decrease in antiviral activity is similar. In addition, the conjugate PEGinterferon and of the invention is non-immunogenic, it practically does not cause the formation of antibodies. In contrast, other PEG-interferon a conjugates elicit limited antibody formation.

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Consequently, the invention relates to a new class of PEG derivatives of interferon a (IFNa). The conjugate of the present invention has a branched PEG structure, as can be seen below. The branched structure offers the advantage of allowing the attachment of two linear PEG molecules at a single site, which doubles the mass of PEG attached without multiple PEGylation sites.

Compared with unmodified IFNa (i.e., IFNa without PEG attached), the conjugate has an increased circulating half-life and increased plasma residence time, reduced immunogenicity, decreased clearance and increased antiproliferative activity, along with decreased in vitro antiviral activity. Compared with other PEG-IFNa conjugates, the conjugate of the present invention has a much stronger antiproliferative activity, out of proportion to the increase or decrease occurring for its other characteristics, and has practically no immunogenicity.

The physiologically active PEG-IFNa conjugate of the present invention corresponds to the formula:

O

II

ROCH₂CH₂(OCH₂CH₂) n — O —C —NH

R'OCH₂CH₂(OCH₂CH₂) laughed

<img file="OA10488A_D0001.tif" />

IFNa

The conjugate of the present invention has the same uses as IFNa, for example, antiproliferative uses. In particular, the PEGinterferon a conjugates of the present invention can be used to treat immunomodulatory disorders such as

010488 neoplastic diseases, for example hairy cell leukemia, chronic myeloid leukemia (CML) and Kaposi's sarcoma, and infectious diseases, in the same way that IFNa (especially IFNa2a) are used for the treatment of these diseases . However, the conjugate of the present invention has improved properties, including greater stability, greater solubility, extended half-life in circulation and increased residence times in plasma. In addition, these conjugates have a proliferative activity which is greater than that of IFNa. As noted, the conjugate also exhibits a surprising dissociation of antiviral and antiproliferative effects. This property is further useful for stimulating a desired activity of a conjugate, while decreasing or eliminating an undesirable activity. For example, if an undesirable side effect is associated with antiviral activity, eliminating this activity would eliminate the side effect, while retaining antiproliferative activity. Consequently, the present invention also includes pharmaceutical compositions based on the compounds of formula I or their salts, and methods for their preparation.

The pharmaceutical compositions of the present invention, used to combat or prevent diseases, include an interferon conjugate of general formula I and a therapeutically inert, non-toxic and therapeutically acceptable vehicle. The pharmaceutical compositions to be used can be formulated and dosed in a manner consistent with good medical practice, taking into consideration the disorder to be treated, the condition of the individual patient, the site where the protein conjugate is brought, the mode administration and other factors known to the practitioner.

The claimed conjugate is a physiologically active PEG-IFNa conjugate, of formula

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Ο

II

HOCI l₂CH_?(OCH_?CH₂) N-O-C-Nl-I

<img file="OA10488A_D0002.tif" />

ROCH₂CH₂(OCH₂CH₂) ri-

<img file="OA10488A_D0003.tif" />

- X— IFNcc in which R and R 'each represent, independently of one another, a lower alkyl group; X is NH or O (X is at least one of the functional groups, in the IFNa molecule, chosen from NH NH and OH); net n are whole numbers whose sum ranges from 600 to 1,500; and the average molecular weight of the polyethylene glycol units in said conjugate ranges from about 26,000 Da to about 60,000 Da. The conjugate of formula I has a branched structure, since two PEG fragments are linked to the protein by a single link.

The numbers n and n 'are chosen such that the resulting conjugate of formula I has a physiological activity of IFNa, which activity can be the same as, or can be greater than, the activity of unmodified IFNa or represent a fraction of it. n and n '(n and n' may be the same or different) represent the number of ethylene glycol units in the PEG. A single OCH motif₂CH₂ of PEG has a molecular weight of about 44 Da. The molecular weight of the conjugate (excluding the molecular weight of IFNa) depends on the numbers n and n '. The sum of n and n 'for the conjugate of formula I ranges from 600 to 1,500, to give a conjugate having a total average molecular weight of PEG units ranging from approximately 26,000 to 66,000, preferably approximately 35,000 at 45,000 Da, and in particular around 39,000 to 45,000 Da, the molecular mass of

000 Da being particularly preferred. The preferred sum of * · n and n 'is from about 800 to 1,200, the average sum going to 10488 from about 850 to 1,000 and the preferred sum being about 910. n or n' can individually represent 420 or 520, or both may represent 420 or 520, or both may each represent 455. The preferred ratio of n to n 'is from about 0.5 to 1.5, a particularly preferred ratio ranging from about 0, 8 to about 1.2. A molecular weight of about a certain number means that it is within a reasonable range around that number, as determined by conventional analytical techniques.

Also preferred is a conjugate of formula I in which IFNa is IFNa2a, a conjugate in which R and R 'represent the methyl group, a conjugate in which X is NH, and a conjugate in which n and n' represent individually or both 420 or 520. Such a conjugate having all of the above characteristics is particularly preferred.

R and R 'can be any lower alkyl group, by which is meant an alkyl group having from 1 to 6 carbon atoms, such as methyl, ethyl, isopropyl, etc. Branched alkyl groups are included. A preferred alkyl group is the methyl group. As regards the two PEG groups of formula I, R and R ′ may be the same or different.

By IFNa (interferon a.), And in particular IFNa2a, is meant the natural or recombinant protein, preferably human, as obtained from any conventional source such as tissues, protein synthesis, culture of natural or recombinant cells. Any protein with IFNa activity, such as a mutein or a protein modified in some other way, is encompassed. Obtaining and isolating IFNa from natural or recombinant sources is well known [Pestka, Arch. Biochem. Biophys. 221, 1 (1983)]. A preferred IFNa is IFNa2a which, as indicated above, is obtained by known methods [Pestka, Sci. Am. 249, 36 (1983); EP-B-43 980)].

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The physiologically active conjugate of formula I has an IFNa activity, by which is meant a fraction or a multiple of any known IFNa activity, as determined by various tests known in the art. In particular, the conjugates of the present invention have IFNa activity, as indicated by antiproliferative activity against tumor cells, and antiviral activity against cells infected with a virus. These are activities known to IFNa. Such activity in a conjugate can be determined by tests well known in the art / for example, the tests described below [see also Rubinstein et coil., J. Virol. 37, 755 (1981); Borden et coil., Cane. Res. 42, 4948 (1982)]. Part of the present invention is a conjugate of formula I having stronger antiproliferative activity and less antiviral activity than unmodified IFNa.

The conjugate of formula I is obtained by attachment by a suitable bond of IFNa to the PEG, which has been activated by replacement of a hydroxy group of the PEG by a linking group, to give a reagent which is a N-hydroxyester succinimide derivative of PEG (in particular monomethoxy-PEG) of formula II. The reagent can be obtained by conventional methods (Monfardini et coil., Ref. Cit.). The attachment is effected by an amide or ester bond. In a preferred conjugate, attachment takes place via an amide bond (X is NH). The invention also relates to a method for increasing the antiproliferative activity of IFNa, while reducing the antiviral activity of IFNa, by attachment of IFNa, as described above, to a reagent of formula II, for obtaining a PEG-IFN conjugate.

X represents the attachment site on IFNa, by which the PEG reagent of formula II is covalently linked to IFNa. Reagents bind to primary amino groups (XH = NH₂), for example, on lysine, or at the N-terminus of IFNa. Reagents can also

010488 stain with a hydroxy group (XH = OH) present for example on serine.

O

II

ROCI-l₂CH₂(OCH₂CII₂) n— O — C — NH

<img file="OA10488A_D0004.tif" />

ROC1 Î₂CII₂(OCH₂CII₂)₍₁-Q-C

R OCI / CI I₂(OCH₂CI I₂)_not--o-c-Nil

II o

<img file="OA10488A_D0005.tif" />

The reagent of formula II (PEG2-NHS), in which two monomethoxy-PEG chains (m-PEG) in total are linked to lysine, each at the level of amino groups a and c, by carbamate (urethane) bonds and of which the carboxy group of lysine is activated into a succinimidyl ester, can be obtained by conventional methods, according to known methods (Monfardini et coil., ref. cit.) applicable to a reagent in which R represents a lower alkyl group, and n is a desired number. The reagent can be obtained from Shearwater Polymers Inc. (Huntsville, Alabama, USA). The preferred average molecular weight of the PEG obtained is approximately 20,000 Da, which gives a total PEG mass of approximately 40,000 Da in PEG2-NHS (other molecular weights can be obtained by varying n for the products of

010488 starting PEG-alcohol for the reagent of formula II, by conventional methods.

The reagent of formula II can be conjugated to IFNa by conventional methods. In particular, the reagent of formula II reacts mainly with one or more of the primary amino groups (for example the N-terminal end and the side chains of lysine) of IFNa (for example IFNa2a), to form a amide bond between IFNa and the polymer backbone of PEG. The PEGylation reaction can also take place between PEG2-NHS and the free hydroxy groups (if they exist) (e.g. serine) of IFNa to form an ester bond. The reaction mechanism is shown above. The reaction conditions are conventional for a person skilled in the art and are given in detail below. The PEG reagent is combined with IFNa under moderately basic conditions, at low temperature, under conditions suitable for nucleophilic substitution, leading to the formation of the conjugate of formula I. This is also indicated in the reaction mechanism above .

The attachment of the reagents to IFNa can be carried out by conventional methods. PEGs of any selected molecular weight of the present invention can be used. The reaction conditions can be chosen to give the claimed conjugate with an attached reagent. The conjugate of formula I, to which a single reagent of formula II is attached, is separated, by conventional methods, from unmodified IFNa and conjugates to which are attached several reagent molecules. Purification methods, such as cation exchange chromatography, can be used to separate conjugates by charge difference, which effectively separates conjugates according to their various molecular weights. The content of the fractions obtained by cation exchange chromatography can be identified by molecular mass, by means of

010488 conventional methods, for example mass spectroscopy, polyacrylamide-SDS gel electrophoresis (SDS-PAGE) or other known methods for the separation of molecular entities according to molecular weight. A fraction is therefore identified which contains the purified conjugate of formula I, free of unmodified IFNa and of conjugates to which more than one reagent is attached. In addition, the reagents of formula II release one lysine residue per reagent, during acid hydrolysis, so that the number of lysine residues in the hydrolysis indicates the number of PEGs attached to the protein, which makes it possible to verify the number of reagent molecules attached to a conjugate.

The invention is illustrated with the aid of the descriptive and nonlimiting examples below. IFNa2a is used in these examples. Other IFNa species can also be conjugated to PEG by the methods given as examples. DESCRIPTION OF THE DRAWINGS

Figure 1: antitumor activity of the PEG2IFNoc2a conjugate in nude mice in which human A498 renal cells have been implanted subcutaneously. All animals received a subcutaneous implant of g

2x10 human A498 kidney cells on day -33 of the study. On day zero of the study, treatment with the PEG-IFNa2a conjugate was started. The indicated amount (30, 60, 120 or 300 Mg) of PEG2-IFNa2a was administered subcutaneously, under the flank opposite the tumor, once a week for a period of 4 weeks.

Figure 2: Anti-tumor activity of IFNa2a in nude mice which have been implanted subcutaneously with human A498 renal cells. All animals received a subcutaneous implant of 2x10 ° human A498 kidney cells on day -33 of the study. On day zero of the study, treatment with IFNa2a was started. The indicated amount (10, 20, 40 or 100 μg) of IFNa2a was administered subcutaneously under the flank opposite the tumor,

010488 three times a week for a period of 4 weeks.

Figure 3: anti-tumor activity of PEG2-IFNa2a in nude mice in which human ACHN renal cells have been implanted subcutaneously. All animals 6 received a subcutaneous implant of 2x10 human ACHN renal cells on day 25 of the study. On day zero of the study, treatment with PEG2-IFNa2a was started. The indicated amount (30, 60, 120 or 300 µg) of PEG2-IFNa2a was administered subcutaneously under the flank opposite the tumor, once a week for a period of 5 weeks.

Figure 4: anti-tumor activity of IFNa2a in nude mice to which human ACHN renal cells have been implanted subcutaneously. All animals received a subcutaneous implant of 2x10 ^ human ACHN renal cells on day 25 of the study. On day zero of the study, treatment with IFNa2a was started. The indicated amount (10, 20, 40 or 100 µg) of IFNa2a was administered subcutaneously under the flank opposite the tumor, three times a week for a period of 5 weeks.

FIG. 5: anti-tumor activity of PEG2-IFNa2a in nude mice in which human G402 renal cells have been implanted subcutaneously. All animals g received a subcutaneous implant of 2x10 human G402 kidney cells on day -45 of the study. On day zero of the study, treatment with PEG2-1FNa2a was started. The indicated amount (30, 60, 120 or 300 µg) of PEG2-IFNa2a was administered subcutaneously under the flank opposite the tumor, once a week for a period of 5 weeks.

Figure 6: antitumor activity of IFNa2a in nude mice in which human G402 renal cells have been implanted subcutaneously. All animals received a subcutaneous implant of 2x10 ^ renal cells

G402 humans at day -45 of the study. On day zero of

010488 the study, treatment with IFNa2a was started. The indicated amount (10, 20, 40 or 100 µg) of IFNa2a was administered subcutaneously under the flank opposite the tumor, three times a week for a period of 5 weeks.

Example 1 Preparation of the conjugate of formula I Materials

Interferon a2a has been prepared by known methods (Petska, ref. Cit.). The polyethylene glycol reagent (PEG) of formula II was purchased from Shearwater Polymers Inc. (Huntsville, Ala, USA). The Fractogel® EMD CM 650 (S) resin, with particle sizes of 25-40 µm, was supplied by EM Separations (Gibbstown, MA, USA). The concentrated phosphate buffered saline (STP) (10x), pH 7.3, was purchased from BioWhittaker (Walkersville, MD, USA). The precast gels for electrophoresis in sodium polyacrylamide / dodecyl (lauryl) sulfate gel (SDS-PAGE) and the electrophoresis units were obtained from NOVEX (San Diego, CA, USA). The fast concentrated dye for protein staining of PEG conjugates on SDS-PAGE was purchased from Zoion Research Inc. (Newton, MA, USA). The LAL endotoxin test kit was purchased from Associates o Cape Cod Inc. (Woods Hole, MA, USA). All other reagents used were of the best quality available. BDF-1 mice and rats with jugular cannulas were supplied by Charles River Laboratories (Wilmington, MA, USA).

Experimental methods

A. Small-scale preparation of the conjugate of formula I

208 mg (5.2 umol) of the reagent of formula II (average molecular weight: 40,000 Da) were added to 50 mg (2.6 μοίθε) of IFNa in 10 ml of 100 mM borate buffer, pH 8.0 . The final protein / reagent molar ratio was 1: 2. The reaction mixture was stirred at 4 ° C for 2 hours.

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The reaction was terminated by adjusting the pH to 4.5 using glacial acetic acid.

The reaction mixture was diluted by the factor 50 with water, the dilution was filtered through a 0.2 μιη filter and the filtrate was injected into an Amicon column (3.2 x 13 cm) packed with Fractogel EMD CM 650 (S), at a flow rate of 20 ml / min. The column was previously equilibrated with 10 mM ammonium acetate, pH 4.5. The column effluent was monitored by UV absorption at 280 nm. The column was then washed with the equilibration buffer, until the UV absorption returned to baseline. PEG-IFN conjugates with more than one reagent of formula II attached (PEG-IFN oligomers) were eluted with 40 mM ammonium acetate, pH 4.5, and the conjugate of formula I was eluted with 0.12 M NaCl in 40 mM ammonium acetate buffer. The unmodified IFN remaining in the column was eluted with 0.5 M NaCl in the same buffer. The column was regenerated by washing with 1.0 M NaCl, followed by washing with the equilibration buffer. The combined fractions of the conjugate of formula I were concentrated to approximately 1 mg / ml in an agitated Amicon cell concentrator provided with a YM10 membrane.

The cation exchange resin Fractogel CM 650 (S), used for purification, effectively adsorbed PEG and unmodified IFN. The adsorption force depended on the degree of PEGylation. The conjugates were less strongly bound than the unmodified IFN. PEG-IFN oligomers were eluted with 40 mM ammonium acetate, while the conjugate of formula I was eluted with 0.12 M NaCl. Unmodified IFN was eluted with 0.5 M NaCl. . All preparations contained less than 5 EU / mg endotoxin. The resulting preparation contained more than 99% conjugate of formula I and was free of unmodified IFN.

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B. Large-scale preparation of the conjugate of formula I

6.24 g (156 nmol) of the reagent of formula II (average molecular weight: 40,000 Da) were dissolved in 63 ml of 1 mM HCl, at 4 ° C, and added quickly to 125 ml of a solution containing 1 g (52 nmol) of interferon in 50 mM borate buffer, pH 9.0. The final protein / reagent ratio was 1: 3, and the final protein concentration in the reaction mixture was 5.3 mg / ml. The reaction mixture was stirred for 2 hours at 4 ° C. The reaction was terminated by adjusting the pH to 4.5 with glacial acetic acid.

The reaction mixture was diluted by a factor of 10 with water, and injected into a column packed with 600 ml of Fractogel EMD CM 650 (M), previously equilibrated with 20 mM sodium acetate , pH 4.5, at a linear speed of 1.3 cm / min. The column was washed with equilibration buffer and then with 10 mM NaCl, to remove excess reagent, reaction by-products and PEG-IFN oligomers. The conjugate of formula I was eluted with the equilibration buffer containing 200 mM NaCl. The unmodified interferon, still adsorbed on the column, was removed by washing with 0.75 M NaCl in the equilibration buffer. The conjugate of formula I, which was eluted at 0.3-0.5 mg / ml, was further concentrated and subjected to diafiltration in the final formulation buffer, composed of 20 mM sodium acetate, pH 5, 0, containing 150 mM NaCl. The overall yield of the conjugate of formula I was 40-45%.

The purified PEG-IFN from the large-scale preparation consists of more than 99% of conjugate of formula I. The average molecular mass of the conjugate of formula I of this example is 62,000 Da, comprising the molecular mass of IFNa2a , which is 19,241 Da, and the average molecular weight of the reagent, which is between 40,000 and 45,000 Da, or approximately 43,000 Da.

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Example 2 Characterization of the Conjugate of Formula I Determination of Proteins

Protein concentrations were determined using an A value_28Q. 1.0 for a 1 mg / ml solution of IFNa2a.

SDS-PAGE analysis

The conjugate was analyzed by electrophoresis in polyacrylamide / dodecyl (lauryl) sodium sulfate gel (8-16%), under reducing conditions, according to the method of Laemmli [Nature 227, 680 (1970)]. For protein determination, SDS-PAGE gels containing PEG conjugates were stained with Fast Stain dye (Zoion Research Inc.) according to the supplier's directions. Determination of endotoxin levels

Endotoxin levels were determined using the LAL method, according to the supplier's instructions. All preparations contained less than 5 EU / mg endotoxin.

Example 3

In vitro biological activities of the conjugate of formula I Antiviral activity in bovine renal cells

The in vitro antiviral activity of IFNa2a and the conjugate of formula I, as prepared in Example IA, was determined in a cell culture bioassay using infected Madin-Darby bovine kidney cells (MDBK). with vesicular stomatitis virus (Rubinstein et coil., cit. ref.). The antiviral activities are given in Table I, together with their corresponding residual activities, as a percentage of the starting IFN.

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Table 1

Antiviral activities

<td>Samples</td><td>Type of PEG</td><td>Total mass of PEG (KDa)</td><td>Number of Lys residues modified</td><td>Specific activity (U / mg)</td><td>Residual activity (%)</td>

<td>IFNa2a</td><td> -</td><td> -</td><td> -</td><td>2 OOxlO⁸</td><td> 100</td>

<td>Conjugate of formula I</td><td>ramified</td><td> 40</td><td> 1</td><td>1,40xl0⁷</td><td> 7</td>

Antiproliferative activity in vitro on human tumor cells

Antiproliferative activities in vitro were tested on human Daudi cells (Burkitt lymphoma), as described by Borden et coil. Human Daudi cells were maintained in the form of cultures in stationary suspension in RPMI 1640 medium supplemented with 10% fetal bovine serum and 2 mM glutamine (Grand Island Biologicals, Grand Island, NY, USA). The cells were analyzed and found to be free from mycoplasmas. In each well of microtiter plates (Costar, MA), 2x10 cells were introduced into 100 ql of medium. Various wells of IFN and the conjugate of formula I, as prepared in Example IA, were introduced into the wells in a volume of 100 Rl. Plates were incubated for 72 hours at 37 ° C in an atmosphere containing 5% CO ^ · Sixteen hours before cell collection, cells were labeled with 0.25 qCi / well of³H-thymi'dine (New England, Boston, MA, USA). The cells were harvested on glass filters and subjected to counting in a scintillation counter in liquid medium. The results were expressed as a percentage of inhibition, calculated using the following formula: inhibition,% = [(A - B / A)] x 100, where:

A = cpm in the control culture (cells incubated in the medium alone)

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B = cpm in experimental culture.

The samples were subjected to 4 tests in parallel, and the standard deviation was less than 20% of the average of all cases. The tests were carried out at least twice, with comparable results.

The antiproliferative activities (ΩΙ ^ θ) of IFN and of the conjugate are given in table 2. The data indicate an increase by factor 28 of the antiproliferative activity for the conjugate of formula I, in comparison with that of IFN.

Table 2

Antiproliferative activities in vitro on the human Daudi cell line (Burkitt lymphoma)

Sample

IFNa2a

Antiproliferative activity

CI5₀ (ng / ml) Increase

0.56 Ix

Conjugate of formula I

0,02

28x

Example 4 Pharmacokinetics

Sprague-Dawley rats, weighing on average 240,260 g, on which jugular cannulas had been surgically implanted, were kept in individual cages, with a 12 hour light-dark cycle, and with free access to food and at the water. Within 4-6 hours of arrival, STP was passed through the jugular cannulas of the rats.

The next day, after passing 0, ΙΞΟ, 2 ml of STP through the cannulas, 2x10 ^ units of IFNa were injected into 0.20.4 ml of STP, then 0.15-0.2 ml was injected of STP to ensure that all of the drug had been injected into the animals. Each animal therefore received a dose of 8χ10^δ IFNa / kg bodyweight units.

Blood samples were taken 5, 15 and minutes as well as 1, 3, 5, 12 and 24 hours after the injection of IFN and the conjugate of formula I. At all times,

010488 after having removed the first 0.15-0.2 ml of blood, 0.5 ml of blood was withdrawn using a new syringe, by the jugular cannula. The samples were transferred to serum separation tubes at room temperature. After collecting all the samples corresponding to the different times, the tubes were centrifuged at 14,000 g for 10 minutes in an Eppendorf refrigerated centrifuge. The separated serum was transferred to 4.5 ml microcentrifuge tubes, and frozen at -80 ° C, until used for the bioassay. The serum samples were suitably diluted, and the antiviral activity at all times was determined as described. From the curve of the variation of activity as a function of time, the maximum half-lives of the conjugate of formula I and of IFNa have been determined, and they are given in table 3, which also gives the times. of plasma stay.

Table 3

Maximum half-lives (t)

1/2 and average plasma residence time

Sample

IFNa2a t (hours) —1/2 —-------- 2.1

Plasma residence time (hours)

1,0

Conjugate of formula I

The maximum t value 1/2 logarithmic area.

15.0 been assessed

20.0 by linear regression Example 5 Immunogenicity

Five times a week, normal BDF-1 mice (10 per group) were injected intraperitoneally once a day with various interferon preparations containing 300,000 units of antiviral activity. Some mice were also injected with an aggregated form of IFNa2a, more immunogenic than the monomeric form. Blood samples were taken 19 days after the last injection, and the

010488 neutralizing antibodies in serum. As can be seen in Table 4, mice injected with IFNa2a produced neutralizing antibodies, and this response was greatly increased in mice injected with interferon aggregates. No antibody was detectable in the majority of animals to which the conjugate of the present invention had been injected.

Table 4

immunogenicity

Antibody (UNI / ml) *

<td>Treatment</td><td>Average</td><td>Beach</td>

<td>IFNa2a</td><td> 2 400</td><td> 217 - 8 533</td>

<td>IFNa aggregates: 2a</td><td> 42 667</td><td> 8 000 - 768 000</td>

<td>Conjugate of formula I</td><td> 0</td><td> 0-1 133</td>

<td>* Neutralization units</td><td colspan="2">interferon / ml.</td>

Example 6

In vivo anti-tumor activity

The anti-tumor activity in vivo was evaluated of a conjugate of formula I (PEG2-IFNa2a) and of unmodified IFNa2a by determining their ability to reduce the size of various human tumor cells implanted subcutaneously in mice. The results are shown in Figures 1 to 6.

Procedure: nude athymic mice (Harlan) received under the left rear flank a subcutaneous implant of 2x10⁶ human A498 kidney cells (Figures 1 and 2), human ACHN kidney cells (Figures 3 and 4) or human G402 kidney cells (Figures 5 and 6). The tumors were allowed to grow for 3 to 6 weeks, as indicated. The size criterion for acceptance in.

the study was 0.05-0.50 cm. Mice were administered weekly total doses of 30, 60, 120 or 300 pg of

PEG2-IFNa2a or unmodified IFNa2a. In the case of PEG2IFNa2a, the mice were treated once a week (the

010488 Monday) with 30, 60, 120 or 300 ng of PEG2 FNa2a per treatment. In the case of unmodified IFNa2a, the mice were treated three times a week (Monday, Wednesday, Friday) with 10, 20, 40 or 100 pg of IFNa2a per treatment. The duration of treatment was 4 to 5 weeks, depending on the aggressiveness of the tumors. The tumor volumes were measured every Monday before treatment.

Results: the PEG2-IFNa2a conjugate showed a marked reduction in the size of the A498 tumors, compared with the unmodified IFNa2a, for all the weekly doses tested, 7 days, 14 days, 21 days and 28 days after the start of the treatment (Figures 1 and 2). The treatment was continued for 4 weeks. Seven days after stopping treatment, three mice were sacrificed in each group. In the three mice treated with PEG2-IFNa2a, no residual tumor was observed. In mice treated with unmodified IFNa2a, the weight of A498 tumors was 1.28 g, 0.62 g and 1.60 g, respectively, in each of the three mice. The weight of A498 tumors was

2.32 g, 2.37 g and 1.94 g in each of the three control mice. Eighty days after the start of the 4-week treatment period, tumors were examined by palpation in seven mice. The seven mice were all devoid of tumor tissue on palpation.

The PEG2-IFNa2a conjugate showed a significant reduction in the size of ACHN tumors, compared with unmodified IFNa2a, for the weekly doses of 60, 120 and 300 pg, after 14, 21, 28 and 35 days ( Figures 3 and 4).

The PEG2-IFNa2a conjugate showed a significant reduction in the size of G402 tumors, compared with the unmodified IFNa2a, for the weekly doses of 60 and 120 ng, after 14, 21, 28 and 35 days (FIGS. 5 and 6).

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