WO2004044006A1 - Conjugates of interleukin-10 and polymers - Google Patents

Conjugates of interleukin-10 and polymers Download PDF

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WO2004044006A1
WO2004044006A1 PCT/DK2003/000774 DK0300774W WO2004044006A1 WO 2004044006 A1 WO2004044006 A1 WO 2004044006A1 DK 0300774 W DK0300774 W DK 0300774W WO 2004044006 A1 WO2004044006 A1 WO 2004044006A1
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polypeptide
il
polypeptide conjugate
introduced
cysteine residue
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PCT/DK2003/000774
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French (fr)
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Jennifer T. Jones
Torben Lauesgaard Nissen
Claus M. Krebber
Kim Vilbour Andersen
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Maxygen, Inc.
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K17/00Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02Peptides being immobilised on, or in, an organic carrier
    • C07K17/08Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/54Interleukins [IL]
    • C07K14/5428IL-10
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Abstract

Conjugate comprising an interleukin-10 (IL-10) polypeptide and a polymer. The IL-10 has at least one cyteine residue introduced (by substitution) in a position selected form 89 positions. The polymer (e.g. PEG) is attached to cysteine residue of the polypeptide. The conjugate exhibits immunosuppressive activity on T cells, B cells or antigen presenting cells. The conjugate should exhibit a reduced immunostimulatory activity compared to hIL-10. Use of said conjugate for treating inflammatory or autoimmune disease.

Description

CONJUGATES OF INTERLEUKIN-10 AND POLYMERS

FIELD OF THE INVENTION

The present invention relates to new polypeptides exhibiting interleukin 10 (IL- 10) activity, to conjugates between a polypeptide exhibiting IL-10 activity and a non- polypeptide moiety, to methods of preparing such polypeptides or conjugates, and the use of such polypeptides or conjugates in therapy, in particular for the treatment of inflammatory diseases such as psoriasis and Crohn's disease.

BACKGROUND OF THE INVENTION

IL-10 was initially described as an activity in the supernatants of activated T- helper type 2 (Th2) clones that could inhibit the production of cytokines, especially interferon gamma (IFN-gamma) by T-helper type 1 (Thl) clones (Fiorentino et al., JExp Med 1989;

170:2081-2095), thus the original name "cytokine synthesis inhibitory factor" (CSIF). IL-10 is now known to be an extremely pleiotrophic cytokine that induces both immunostimulatory and immunosuppressive activities which may vary depending on the cell types involved and other events in immune regulation. Detailed information on the biology of IL-10 may be found in a review by Moore et al. (Annu. Rev. Immunol. Vol. 19:683-765 (2001)). Other than CSIF, IL-10 has also been described under the names mast cell growth factor III (MCGF-III) and B-cell derived T-cell growth factor (B-TCGF).

Among its immunosuppressive activities, IL-10 inhibits monocyte and macro- phage synthesis of IL-1-alpha, IL-beta, IL-6, IL-8, IL-12, TNF-alpha, GM-CSF, and reactive oxygen and nitrogen intermediates. IL-10 inhibits dendritic cell stimulation of Thl IFN-gamma production, antigen-presenting cell (APC) B7 expression, and antigen presentation to Thl but not Th2 cells, while inducing IL-1 receptor antagonist production in neutrophils. IL-10 also suppresses epidermal Langerhans cell APC functions, chemokine expression by monocytes, and the bactericidal response of macrophages to IFN-gamma. IL-10 treated dendritic cells in- duce peptide antigen and alloantigen specific tolerance.

Studies have demonstrated that IL- 10 inhibits the immune function of other cell types as well. Thus, IL-10 inhibits NK cell production of IFN-gamma, ICAM-1 expression on activated vascular endothelial cells, and T independent responses of B cells. Overall, studies have shown that the predominant effect of IL-10 is to suppress multiple immune responses through individual actions on T cells, B cells, APCs, and other cell types.

In addition to its immunosuppressive activities, IL-10 is also known to have a number of immunostimulatory activities. As indicated above, IL-10 exerts many of its anti- inflammatory actions by counteracting the biological effects of IFN-gamma, but a recent study of IL- 10 in patients with Crohn' s disease showed that high doses of IL- 10 actually induce formation of IFN-gamma by LPS (lipopolysaccharide) stimulated whole blood cells. (Fedorak et al, Gastroenterόlogy (2000) 119:1473-82). In another study of IL-10 in patients with chronic active Crohn's disease, it was shown that subcutaneous (s.c.) administration of 20 microg/kg IL-10 (but not 1-10 microg/kg) induced formation of neopterin by phytohaemagglutinin- induced whole blood cells (Tilg et al, Gut (2002)50:191-195). Neopterin is mainly produced by monocytes/macrophages under the control of IFN-gamma, and thus is a valuable in vitro and in vivo marker for monitoring cell mediated immune function and IFN-gamma activity. There are a large number of studies in which there has been a general failure to correlate the presence or absence of IL-10 with allograft survival or rejection. One interpretation of these results is that the presence or absence of other cytokines such as IL-4, IFN-gamma or IL-12 could affect the final immune outcome. Another view, however, is that IL-10 could be acting in a pro-inflammatory fashion and actually contributing to graft rejection. Indeed, IL-10 can induce the expression of E-selectin on vascular endothelium, which would be expected to promote and sustain inflammatory responses.

Thus, various studies show that IL-10 may have immunostimulatory or immunosuppressive effects depending on the assay, cell types involved, or other concomitant immune events. The ability to manipulate the immune responses to IL-10 in either a stimulatory or sup- pressive direction would be important for determining which aspects of IL-10 activity are critical for normal T cell development and channeling Thl and Th2 responses, and would be of enormous practical value in regulating immune responses, e.g. for use in for disease therapy. For example, the immunosuppressive activities of IL-10 are potentially of great interest for treatment of psoriasis, since this disease may be caused by unregulated cutaneous Thl re- sponses. Likewise, due to its anti-inflammatory activities, IL-10 has great potential in treatment of inflammatory diseases such as Crohn's disease. Unfortunately, the immunostimulatory activities of IL-10 have limited its success in clinical trials so far. Ding et al. (J. Exp. Med. 191(2): 213-223 (2000)) and US 6,428,985 describe that the amino acid isoleucine at position 87 of hIL-10 is required for its immunostimulatory function, and that substitution of this isoleucine residue with alanine eliminates immunostimulatory activity while maintaining the immunosuppressive activity. The corresponding residue in viral IL- 10 (vIL- 10), which lacks the immunostimulatory activity of hlL- 10, is alanine, and it was also found that replacing alanine with isoleucine in vIL-10 resulted in an increased immunostimulatory activity in the viral molecule.

Mature human IL-10 (hIL-10) consists of 160 amino acid residues (SEQ ID NO:2 in the appended sequence listing), is biologically active as a homodimer and is derived from a precursor consisting of 178 amino acid residues (SEQ ID NO:l in the appended sequence listing). The DNA sequences encoding these proteins have been reported {Human Cytokines, Handbook of Basic and Clinical Research, Volume II, Eds. Aggarwal and Gutterman, 1996, pp. 19-42). The same publication discloses the IL-10 receptor, its amino acid sequence and underlying DNA sequences, a bioassay for IL-10, methods of purifying IL-10, as well as fur- ther information of relevance to IL-10.

11-10 has four cysteine residues that form two intramolecular disulfide bonds, and an unoccupied glycosylation site. The three-dimensional structure of IL-10 has been reported (Walter et al., Biochemistry 1995, 34:12118-12125; Zdanov et al., Structure 1995, 3:591-601; Zdanov et al., Protein Sci. 1996, 5:1955-1962). According to Zdanov et al. (1995), human IL- 10 comprises the following structural elements:

N-terminal coil N10-G17

Helix A N18-D41

Strand AB Q42-L48

Helix B K49-G58

Junction BC Y59

Helix C L60-N82

Loop CD Q83-D86

Helix D I87-C108

Loop DE H109-K117

Helix E S118-L131

Junction EF Q132

Helix F E133-R159

Viral homologues of IL-10 have been detected in the genomes of Epstein-Barr virus (EBV) and equine herpesvirus 2 (Moore et al., Science 1990; 248:1230-1234) (Vieira et al., Proc Nat Acad Sci USA 1991; 88:1172-1176) (Rode et al., Virus Genes 1993; 7:111-116).

IL-10 has been suggested as an anti-inflammatory agent for treatment of inflammatory and autoimmune diseases such as inflammatory bowel disease, rheumatoid arthritis, uveitis, etc. It has also been suggested in connection with transplantation, immunodeficiencies and parasitic infections. As noted above, however, the immunostimulatory activities of IL-10 have hampered its use for immunosuppressive purposes. A further concern in therapy with recombinant human proteins, including IL-10, is the need for frequent administration by injec- tion, as well as a potential for development of neutralizing antibodies in response.to therapy with such molecules.

WO 01/58950 discloses certain IL-10 variants with substitutions to introduce one or more sites for attachment of a polyethylene glycol (PEG) moiety or a glycosylation site. WO 02/26265 discloses "mono-PEG" IL-10, defined as 1-9 PEG molecules attached to the - terminal, or a lysine or histidine residue, of one subunit of IL-10 via a linker. WO 99/03887 discloses cysteine variants of members of the growth hormone supergene family, including a general disclosure of cysteine-added variants of IL-10 at any of the three amino acids comprising the N-linked glycosylation site, in the region proximal to the A helix, distal to the E helix, in the A-B, B-C, C-D loop or D-E loop, and proximal to the first amino acid or distal to the final amino acid of the mature protein, although no such cysteine-added IL-10 variants are actually exemplified.

Although certain IL-10 variants, including PEGylated IL-10, have been described in the literature, little experimental data on such variants has been published, and no such products are currently available. Thus, a clinical need exists for a product that provides thera- peutically relevant IL-10 activity, in particular a product that exhibits the immunosuppressive activity of hIL-10 without its immunostimulatory activity, and which in addition may be ad- ministered at less frequent intervals and/or which has a reduced immunogenic potential as compared to rhIL-10. The present invention is directed to such products.

BRIEF DISCLOSURE OF THE INVENTION

The present invention provides novel IL-10 polypeptides and conjugates comprising one or more cysteine residues introduced into a parent IL-10 polypeptide, which may be conjugated to a polymer moiety such as PEG. Some such polypeptides and conjugates of the invention exhibit improved properties that address the clinical needs described above, in particular an improved immunosuppressive activity with reduced immunostimulatory activity and/or an increased in vivo half-life. Accordingly, one aspect of the invention relates to a polypeptide conjugate comprising (a) a polypeptide exhibiting IL-10 activity comprising an amino

1 acid sequence that differs from the amino acid sequence shown in SEQ ID NO:2 in 1-15 amino acid residues and which comprises at least one cysteine residue introduced in a position selected from those disclosed below, and (b) at least one polymer moiety attached to a cysteine residue of the polypeptide. In another aspect, the invention relates to a polypeptide exhibiting IL-10 activity, wherein the polypeptide comprises a cysteine residue introduced in at least one of the positions disclosed below.

In a further aspect, the invention relates to a composition comprising the polypeptide conjugate or the polypeptide defined above and at least one pharmaceutically acceptable carrier or excipient, as well as methods for preparing such polypeptides, conjugates and compositions.

In a still further aspect, the invention relates to a nucleotide sequence which encodes the polypeptide defined above, an expression vector comprising such a nucleotide sequence, and a host cell comprising the expression vector or nucleotide sequence. Finally, the invention also relates to a method for preventing or treating an inflammatory or autoimmune disease, comprising administering to a patient in need thereof a polypeptide conjugate, a polypeptide or a composition of the invention, as well as use of the polypeptides or conjugates as a medicament and for preparing a pharmaceutical composition for the prevention or treatment of an inflammatory or autoimmune disease.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless otherwise defined herein or below in the remainder of the specification, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs.

The term "conjugate" is intended to indicate a heterogeneous molecule formed by the covalent attachment of one or more polypeptides, typically a single polypeptide, to at least one polymer molecule. The term covalent attachment means that the polypeptide and the polymer molecule are either directly covalently joined to one another, or else are indirectly covalently joined to one another through an intervening moiety or moieties, such as a bridge, spacer, or linkage moiety or moieties. Although in some cases more than one polymer moiety may be bound to a single attachment group (a single amino acid residue), e.g. via a linker, normally an individual attachment group will be bound to a single polymer moiety. Preferably, the conjugate is soluble at relevant concentrations and conditions, i.e. soluble in physiological fluids such as blood. The term "non-conjugated polypeptide" may be used about the polypeptide part of the conjugate. The term "polypeptide" may be used interchangeably herein with the term "protein".

The "polymer molecule" is a molecule formed by covalent linkage of two or more monomers, wherein none of the monomers is an amino acid residue, except where the polymer is human albumin or another abundant plasma protein. The term "polymer" or "polymer moi- ety" may be used interchangeably with the term "polymer molecule". The term is intended to cover carbohydrate molecules, although in the present context the term is not intended to cover the type of carbohydrate molecule which is attached to the polypeptide by in vivo N- or O- glycosylation. Except where the number of polymer molecule(s) is expressly indicated, every reference to "a polymer", "a polymer molecule", "the polymer" or "the polymer molecule" contained in a polypeptide of the invention or otherwise used in the present invention shall be a reference to one or more polymer molecule(s).

The term "attachment group" is intended to indicate an amino acid residue group of the polypeptide capable of coupling to the relevant polymer molecule. For instance, in particular for polymer conjugation to polyethylene glycol (PEG), frequently used attachment groups include the ε-amino group of a lysine residue, the N-terminal amino group, and the sulfhydryl group of a cysteine residue. Other polymer attachment groups include a free carbox- ylic acid group (e.g. that of the C-terminal amino acid residue or of an aspartic acid or glutamic acid residue), suitably activated carbonyl groups, oxidized carbohydrate moieties and mercapto groups. In the present application, amino acid names and atom names (e.g. CA, CB, NZ,

N, O, C, etc.) are used as defined by the Protein Data Bank (PDB) (Berman et al., "The Protein Data Bank", Nucleic Acids Res., 28(1): 235-242 (2000); www.rcsb.org/pdbΛ. which is based on the IUPAC nomenclature (IUPAC Nomenclature and Symbolism for Amino Acids and Pep- tides (residue names, atom names etc.), Eur. J. Biochem., 138, 9-37 (1984) together with their corrections in Eur. J. Biochem., 152, 1 (1985). The term "amino acid residue" is primarily intended to indicate an amino acid residue contained in the group consisting of the 20 naturally occurring amino acids, i.e. alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Tip or W), and tyrosine (Tyr or Y) residues. The terminology used for identifying amino acid positions/substitutions is illustrated as follows: T13 indicates position number 13 occupied by a threonine residue in a reference amino acid sequence, e.g. SEQ ID NO:2. T13C indicates that the threonine residue of position 13 has been substituted with a cysteine residue. Unless otherwise indicated, the numbering of amino acid residues made herein is made relative to the amino acid sequence of ma- ture hIL-10 as shown in SEQ ID NO:2. Alternative substitutions are indicated with a "/", e.g. G95S/T means an amino acid sequence in which the glycine residue in position 95 is substituted with a serine or a threonine residue. Multiple substitutions are indicated with a "+", e.g. S93N+G95S/T means an amino acid sequence which comprises a substitution of the serine residue in position 93 with an asparagine residue and a substitution of the glycine residue in position 95 with a serine or threonine residue.

Unless otherwise indicated, the term "human IL-10" or "hIL-10" as used herein is intended to refer to the mature sequence containing 160 amino acid residues as shown in SEQ ID NO:2. It will be clear, however, that any amino acid substitutions, insertions, additions or deletions described herein with reference to the mature sequence shown in SEQ ID NO:2 may be performed in the same manner in the precursor sequence shown in SEQ ID NO: 1.

Further, although the invention will primarily be discussed with reference to alterations in the amino sequence of human IL-10 as shown in SEQ ID NO:2, it will be understood that corresponding alterations may also be performed in an equivalent position in other IL-10 polypeptides, e.g. other mammalian IL-10 polypeptides or a viral IL-10 polypeptide. An "equivalent position" or a "position corresponding to" is intended to indicate a position in the amino acid sequence of a given IL-10 polypeptide which is homologous (i.e. corresponding in position in either primary or tertiary structure) to the relevant position in the amino acid sequence shown in SEQ ID NO:2. The "equivalent position" is conveniently determined on the basis of an alignment of members of the IL-10 protein sequence family, e.g. using the ClustalW program, version 1.8, June 1999, using default parameters (Thompson et al., Nucleic Acids Res. 1994, 22:4673-4680), or from the PFAM families database version 4.0 (http://pfam.wustl.edu/") (Nucleic Acids Res. 1999; 27(l):260-2) by use of GENEDOC version 2.5 (Nicholas et al., "GeneDoc: Analysis and Visualization of Genetic Variation", EMB- NET.NEWS Vol. 4, No. 2 (31 Julyl997)).

The term "nucleotide sequence" is intended to indicate a consecutive stretch of two or more nucleotide molecules. The nucleotide sequence may be of genomic, cDNA, RNA, semi-synthetic or synthetic origin, or any combination thereof.

"Cell", "host cell", "cell line" and "cell culture" are used interchangeably herein and all such terms should be understood to include progeny resulting from growth or culturing of a cell. "Transformation" and "transfection" are used interchangeably to refer to the process of introducing DNA into a cell. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it increases the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. However, since en- hancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous.

The term "introduce" refers to introduction of an amino acid residue comprising an attachment group for a polymer molecule, in particular by substitution of an existing amino acid residue, or alternatively by insertion of an additional amino acid residue within the sequence or addition of the residue at the N- or C-terminal. The term "remove" refers to removal of an amino acid residue comprising an attachment group for a polymer molecule, in particular by substitution of the amino acid residue to be removed by another amino acid residue, or alternatively by deletion (without substitution) of the amino acid residue to be removed. In general, when substitutions are performed in relation to a parent polypeptide, they are preferably "conservative substitutions", in other words substitutions performed within groups of amino acids with similar characteristics, e.g. small amino acids, acidic amino acids, basic amino acids, hydrophilic amino acids, polar amino acids, hydrophobic amino acids, sulfur-containing amino acids, aliphatic amino acids and aromatic amino acids. Conservative sub- stitutions may for example be chosen from among the conservative substitution groups listed in the table below. 1 Alanine (A) Glycine (G) Serine (S) Threonine (T)

2 Aspartic acid (D) Glutamic acid (E)

3 Asparagine (N) Glutamine (Q)

4 Arginine (R) Histidine (H) Lysine (K)

5 Isoleucine (I) Leucine (L) Methionine (M) Valine (V)

6 Phenylalanine (F) Tyrosine (Y) Tryptophan (W)

Additional groups of amino acids can also be formulated, e.g. based on similar function, chemical structure or composition. For example, an aliphatic grouping may comprise glycine, alanine, valine, leucine and isoleucine. Other conservative substitution groups include: hydrophilic amino acids: serine, threonine, asparagine and glutamine; hydrophobic amino acids: leucine, isoleucine and valine; sulfur-containing: cysteine and methionine. Further, substitution of a cysteine for a serine or threonine is also considered to be a conservative substitution in the context of the present invention.

The term "immunogenicity" as used in connection with a given substance is in- tended to indicate the ability of the substance to induce a response from the immune system. The immune response may be a cell or antibody mediated response (see, e.g., Roitt: Essential Immunology (8 Edition, Blackwell) for further definition of immunogenicity). Normally, reduced antibody reactivity will be an indication of reduced immunogenicity. The reduced immunogenicity may be determined by use of any suitable method known in the art, e.g. in vivo or in vitro.

The term "functional in vivo half-life" is used in its normal meaning, i.e. the time at which 50% of the biological activity of the polypeptide conjugate is still present in the body or target organ, or the time at which the activity of the polypeptide conjugate is 50% of the initial value. As an alternative to determining functional in vivo half-life, "serum half-life" may be determined, i.e. the time in which 50% of the polypeptide conjugate molecules circulate in the plasma or bloodstream prior to being cleared. Alternative terms to serum half-life include "plasma half-life", "circulating half-life", "serum clearance", "plasma clearance" and "clearance half-life". The polypeptide conjugate is cleared by the action of one or more of the reticu- loendothelial systems (RES), kidney, spleen or liver, by receptor-mediated degradation, or by specific or non-specific proteolysis, in particular by the action of receptor-mediated clearance and renal clearance. Normally, clearance depends on size (relative to the cutoff for glomerular filtration), charge, attached carbohydrate chains, and the presence of cellular receptors for the protein. The functionality to be retained is normally selected from IL-10 immunosuppressive activity and IL-10 receptor-binding activity. The functional in vivo half-life and the serum half- life may be determined by any suitable method known in the art.

The term "increased" as used about the functional in vivo half-life or serum half- life is used to indicate that the relevant half-life of the conjugate or polypeptide is statistically significantly increased relative to that of a reference molecule, such as a non-polymer conjugated human IL-10 (e.g. Tenovil®), determined under comparable conditions. For instance, the relevant half-life may increased by at least about 25%, such as by at least about 50%, e.g. by at least about 100%, 200%, 500% or 1000%. The term "renal clearance" is used in its normal meaning to indicate any clearance taking place by the kidneys, e.g. by glomerular filtration, tubular excretion or tubular elimination. Renal clearance depends on physical characteristics of the conjugate, including size (diameter), symmetry, shape/rigidity and charge. Reduced renal clearance may be established by any suitable assay, e.g. an established in vivo assay. Typically, renal clearance is de- termined by administering a labelled (e.g. radioactive or fluorescent labelled) polypeptide conjugate to a subject and measuring the label activity in urine collected from the subject. Reduced renal clearance is determined relative to a corresponding reference polypeptide, e.g. the corresponding non-conjugated polypeptide, under comparable conditions. Preferably, the renal clearance rate of the conjugate is reduced by at least 50%, preferably by at least 75%, and most preferably by at least 90% compared to a relevant reference polypeptide.

A molecular weight of about 67 kDa is generally considered to be a cutoff value for renal clearance, although this can vary depending on e.g. the diameter and shape of the molecule. Significantly, a high "apparent molecular weight" or "apparent size", as determined e.g. by gel permeation chromatography (GPC) or SDS-PAGE, can be obtained by means of e.g. PEGylation even if the actual molecular weight of the conjugate as determined by mass spectrometry is much lower. A conjugated protein can therefore, depending on the nature and distribution of the polymer moieties, have an apparent weight of above about 67 kDa and thus a reduced or substantially eliminated renal clearance, even when the actual molecular weight of the conjugate is far lower than 67 kDa. The term "exhibiting IL-10 activity" is intended to indicate that the polypeptide or polypeptide conjugate has one or more of the functions of human IL-10, in particular hIL-10 with the amino acid sequence shown in SEQ ID NO:2, including the capability to bind to an IL-10 receptor and to suppress the production of interferon-gamma or TNF-alpha. The IL-10 activity is conveniently assayed using one or more of the assays described below. The polypeptide or conjugate "exhibiting" IL-10 activity is considered to have such activity when it displays a measurable function, e.g. a measurable immunosuppressive activity or receptor-binding activity. The polypeptide exhibiting IL-10 activity may also be termed "IL-10 molecule" herein for the sake of simplicity, even though such polypeptides are in fact variants of IL-10.

The term "parent IL-10" or "parent polypeptide" is intended to indicate the molecule to be modified in accordance with the present invention. The parent IL-10 is normally hIL-10 or a variant thereof, in particular with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2. A "variant" is a polypeptide which differs in one or more amino acid residues from a parent polypeptide, normally in up to 15 amino acid residues, such as up to 12, 10, 8 or 6 residues, e.g. in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues.

The term "pharmaceutical composition" means a composition suitable for pharmaceutical use in a subject, including an animal or human. A pharmaceutical composition generally comprises an effective amount of an active agent and at least one pharmaceutically ac- ceptable carrier or excipient.

The term "effective amount" or "effective dose" means a dosage or amount sufficient to produce a desired result, i.e. prevention, treatment or other improvement in relation to a given condition or disease. The desired result may comprise an objective or subjective improvement in the recipient of the dosage or amount. Generally, the nomenclature used hereafter and the laboratory procedures in cell culture, molecular genetics, molecular biology, nucleic acid chemistry and protein chemistry described below are those well known and commonly employed by those of ordinary skill in the art. Standard techniques, such as described in Sambrook et al., Molecular Cloning - A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2001 and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994, supplemented through 1999), are used for recombinant nucleic acid methods, nucleic acid synthesis, cell culture methods, etc. Generally, oligonucleotide synthesis and purification steps are performed according to specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references which are provided throughout this document. The procedures therein are believed to be well known to those of ordinary skill in the art and are provided for the convenience of the reader. Description of preferred embodiments

As stated above, in a first aspect the invention relates to a polypeptide conjugate comprising a polypeptide exhibiting IL-10 activity with an amino acid sequence that differs from the amino acid sequence of SEQ ID NO:2 in at least one introduced cysteine residue selected from those specified herein, and having at least one polymer molecule attached to a cysteine residue of the polypeptide. A related aspect is directed to IL-10 polypeptides altered as described herein by introduction of one or more cysteine residues.

By introducing at least one cysteine residue comprising an attachment group for the polymer molecule it is possible to specifically adapt the polypeptide so as to make the molecule more susceptible to conjugation to the polymer molecule of choice, to optimize the conjugation pattern (e.g. to ensure an optimal distribution of polymer molecules on the surface of the IL-10 molecule and to ensure that only the attachment groups intended to be conjugated are present in the molecule) and thereby to obtain a new conjugate molecule which has IL-10 activity and in addition one or more improved properties as compared to IL-10 molecules available today.

While the IL-10 polypeptide may be of any origin, e.g. mammalian IL-10 or alternatively viral IL-10, it is preferably of human origin.

In addition to the amino acid alterations disclosed herein aimed at introducing cysteine attachment sites for the polymer molecule, it will be understood that the amino acid sequence of the polypeptide of the invention may if desired contain other alterations that need not be related to introduction or removal of attachment sites, i.e. other substitutions, insertions, additions or deletions. These may, for example, include truncation of the N- and/or C-terminus by one or more amino acid residues, or addition of one or more extra residues at the N- and/or C-terminus, e.g. addition of a methionine residue at the N-terminus. Also, such other alterations may include introduction of one or more glycosylation sites, in particular N-glycosylation sites, as described below.

The conjugate of the invention may have one or more of the following improved properties as compared to hIL-10: increased immunosuppressive activity, reduced or eli i- nated immunostimulatory activity, increased functional in vivo half-life, increased serum half- life, reduced renal clearance, reduced receptor-mediated clearance and reduced immunogenicity. Sites for introduction of a cysteine residue, typically by substitution in place of a native residue, may be selected on the basis of a number of different considerations, for example accessibility of the residue, whether the residue is located e.g. in a helix or a loop, the nature of the residue to be replaced by a cysteine (e.g. considerations of charge and hydrophilic- ity), location of the residue in relation to the receptor-binding site, etc. Further details on such considerations are provided below.

In order to avoid too much disruption of the structure and function of the parent IL-10 molecule, the total number of amino acid residues to be altered in accordance with the present invention, e.g. as described in the subsequent sections herein, (as compared to the amino acid sequence shown in SEQ ID NO:2) will generally not exceed 15. The exact number of amino acid residues and the type of amino acid residues to be introduced or removed depends in particular on the desired nature and degree of conjugation (e.g. the identity of the polymer molecule, how many polymer molecules it is desirable or possible to conjugate to the polypeptide, where conjugation is desired or should be avoided, etc.). Typically, the polypep- tide of the invention will comprise an amino acid sequence which differs in up to 12 amino acid residues from the amino acid sequence shown in SEQ ID NO:2, more typically in up to 10 amino acid residues, e.g. in up to 6 or 8 amino acid residues, such as in up to 3 or 4 a ino acid residues.

IL-10 structural analysis Experimental three-dimensional structures of human IL- 10 have been reported by

Zdanov et al., Structure 3:591-601, (1995); Zdanov et al., Protein Sci. 10:1955-62 (1996); Walter et al., Biochemistry 34:12118-25 (1995); Josephson et al., Immunity 14:35-46, (2001); and Josephson et al., Structure 10: 981-987, (2002).

Josephson et al. (2001) report the complex between IL-10 and IL-10R1 as a fully symmetric dimeric complex containing an intertwined dimer of IL-10 binding two copies of IL-10R1. The reported structure contains only the coordinates of one of the IL-10 monomers, and the symmetric part can be generated by applying the appropriate symmetry operation using e.g. the software Swiss-Pdb Viewer v.3.7 (Guex et al., Electrophoresis 18:2714-2723 (1997)). The X-ray structure of human IL-10 bound to a soluble form of the high-affinity receptor IL-10R1 (Josephson et al., 2001) was used to determine surface exposure of amino acid residue side chains in the IL-10 dimer alone as well as for the IL-10 dimer bound to IL-10 receptor 1 (IL-10R1). The coordinates for this structure are available from the Protein Data Bank (PDB) (Bernstein et al., J. Mol. Biol. (1977) 112, p. 535) and electronically available via The Research Collaboratory for Structural Bioinformatics PDB at http://www.pdb.org/ under accession code U7V. Determination of the degree of surface exposure was performed as described in WO 01/58950.

Analysis of the isolated IL-10 dimer

By performing fractional accessible surface area (ASA) calculations on the isolated IL-10 dimer molecule (removing the IL-10R1 molecules from the structure), it was determined that the following residues have more than 25% of their side chain exposed to the surface (only residues in one monomer are listed since the dimer is symmetrical): SI 1, T13, H14, P16, G17, N18, P20, N21, R24, D25, R27, D28, S31, R32, K34, T35, F36, Q38, M39, K40, Q42, L43, D44, N45, L46, K49, E50, S51, E54, K57, G58, Y59, L60, Q70, E74, E75, P78, Q79, N82, D84, P85, D86, K88, A89, H90, N92, S93, E96, K99, T100, L103, R104, R106, R107, H109, R110, N116, K117, K119, E122, Q123, K125, N126, A127, N129, K130, Q132, E133, K134, D144, 1145, N148, E151, A152, T155, M156, 1158, R159, N160. The following residues in the isolated dimer were determined to have more than

50% of their side chain exposed to the surface: SI 1, T13, H14, P16, G17, N18, P20, N21, R24, D25, D28, S31, R32, K34, T35, M39, Q42, D44, N45, K49, E50, E54, K57, G58, Y59, E74, N82, P85, D86, K88, A89, H90, N92, E96, K99, T100, L103, R110, K117, K119, Q123, N126, N129, K130, Q132, E133, K134, N148, T155, R159, N160. Since the first detected residue in this structure of IL-10 is Serl 1, residues Serl to

AsnlO are regarded as having 100% surface accessibility.

Analysis of IL-10 when bound to IL-10R1

By performing fractional ASA calculations on the IL-10 dimer molecule in the structure including the two IL-10R1 molecules it was determined that the following residues in IL-10 have more than 25% of their side chain exposed to the surface: SI 1, T13, H14, P16, G17, N18, N21, R24, D25, D28, S31, R32, T35, F36, M39, K40, Q42, L43, N45, K49, E50, S51, E54, K57, G58, Y59, L60, Q70, E74, E75, P78, Q79, N82, D84, P85, D86, K88, A89, H90, N92, S93, E96, K99, T100, L103, R104, R106, R107, H109, R110, N116, K117, K119, E122, Q123, K125, N126, A127, N129, K130, Q132, E133, K134, N148, A152, T155, M156, R159, N160.

The following IL-10 residues were determined to have more than 50% of their side chain exposed to the surface when IL-10 was bound to IL-10R1 : SI 1, T13, H14, P16, G17, N18, N21, D25, D28, S31, R32, T35, M39, Q42, K49, E50, E54, K57, G58, Y59, E74, N82, P85, D86, K88, A89, H90, N92, E96, K99, T100, L103, R110, K117, K119, Q123, N126, N129, K130, Q132, E133, K134, R159, N160.

In this case as well, the first 10 residues of IL-10, from Serl to AsnlO, are con- sidered to be 100% surface exposed.

Receptor-binding site

The receptor-binding site (also referred to as the "receptor interface") can be defined in a number of ways. Here we define the site as including those residues having the accessible surface area (ASA) of the complete residue changed by more than 5 A2 when compar- ing the isolated IL-10 dimer with the complex of IL-10 and IL-10R1. These residues defined to be in the receptor-binding site are: P20, L23, R24, R27, D28, K34, T35, Q38, M39, D41, Q42, D44, N45, L46, K138, S141, E142, D144, 1145, N148, Y149, E151, T155, 1158 and R159.

Introduction ofcysteines

Introduction of a free cysteine residue in the IL-10 structure is preferably per- formed by substitution of an existing residue with a cysteine residue. To preserve activity of the IL-10 variant molecule, it is preferable not to alter interior residues that might prevent proper folding of the molecule. It is therefore preferable to change residues having their side chain surface at least partially exposed, preferably those with more than 25% of the side chain surface exposed, and more preferably those with more than 50% of the side chain surface ex- posed. For subsequent PEGylation or other chemical modification of the introduced cysteine residue, a similar degree of surface exposure is also preferable. Further, in order not to alter the interaction with the receptor molecules it is preferable not to alter any of the residues in the receptor-binding site as defined above.

Another consideration is that certain residues are more preferable than others for substitution with a cysteine residue. Preferred residues are selected mainly based on their hy- drophilic/charged properties. It is thus preferable to introduce a cysteine as a substitution for a hydrophilic uncharged residue, in particular Ser, Thr, Asn or Gin, other possibilities being His or Tyr. Of these residues, Ser and Thr are most preferred, in particular Ser. It is less preferable to substitute other uncharged residues, i.e. Gly, Ala, Leu, Val, He, Met, Phe, Trp or Pro. In the event a Cys residue is substituted for any of these residues, Gly and Ala are most preferred, and Phe, Tip and Pro are least preferred among these. Least preferable is substitution of Cys for a charged residue (Asp, Glu, Lys or Arg). In one embodiment, the polypeptide or conjugate of the invention has an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO:2 in that at least one cysteine residue has been introduced in a position selected from the group consisting of: SI, P2, G3, Q4, G5, T6, Q7, S8, E9, N10, Sll, T13, H14, P16, G17, N18, P20, N21, R24, D25, R27, D28, S31, R32, K34, T35, F36, Q38, M39, K40, Q42, L43, D44, N45, L46, K49, E50, S51, E54, K57, G58, Y59, L60, Q70, E74, E75, P78, Q79, N82, D84, P85, D86, K88, A89, H90, N92, S93, E96, K99, TlOO, L103, R104, R106, R107, H109, R110, N116, K117, K119, E122, Q123, 125, N126, A127, N129, K130, Q132, E133, K134, D144, 1145, N148, E151, A152, T155, M156, 1158, R159 and N160; preferably from those residues not believed to belong to the receptor interface, i.e. from the group consisting of SI, P2, G3, Q4, G5, T6, Q7, S8, E9, N10, Sll, T13, H14, P16, G17, N18, N21, D25, S31, R32, F36, K40, L43, K49, E50, S51, E54, K57, G58, Y59, L60, Q70, E74, E75, P78, Q79, N82, D84, P85, D86, K88, A89, H90, N92, S93, E96, K99, TlOO, L103, R104, R106, R107, H109, Rl 10, Nl 16, Kl 17, Kl 19, E122, Q123, K125, N126, A127, N129, K130, Q132, E133, K134, D144, A152, M156 and N160.

In another embodiment, the polypeptide or conjugate of the invention has an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO:2 in that at least one cysteine residue has been introduced in a position selected from the group consisting of: SI, P2, G3, Q4, G5, T6, Q7, S8, E9, N10, Sll, T13, H14, P16, G17, N18, P20, N21, R24, D25, D28, S31, R32, K34, T35, M39, Q42, D44, N45, K49, E50, E54, K57, G58, Y59, E74, N82, P85, D86, K88, A89, H90, N92, E96, K99, TlOO, L103, Rl 10, Kl 17, Kl 19, Q123, N126, N129, K130, Q132, E133, K134, N148, T155, R159 and N160; preferably from those residues not believed to belong to the receptor interface, i.e. from the group consisting of SI, P2, G3, Q4, G5, T6, Q7, S8, E9, N10, SI 1, T13, H14, P16, G17, N18, N21, D25, S31, R32, K49, E50, E54, K57, G58, Y59, E74, N82, P85, D86, K88, A89, H90, N92, E96, K99, TlOO, L103, Rl 10, Kl 17, K119, Q123, N126, N129, K130, Q132, E133, K134 andN160.

In a further embodiment, the polypeptide or conjugate of the invention has an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO:2 in that at least one cysteine residue has been introduced in a helix position selected from the group consisting of: N18, P20, N21, R24, D25, R27, D28, S31, R32, K34, T35, F36, Q38, M39, K40, K49, E50, S51, E54, K57, G58, L60, Q70, E74, E75, P78, Q79, N82, K88, A89, H90, N92, S93, E96, K99, TlOO, L103, R104, R106, R107, K119, E122, Q123, K125, N126, A127, N129, K130, E133, K134, D144, 1145, N148, E151, A152 T155, M156, 1158 and R159; pref- erably from those residues not believed to belong to the receptor interface, i.e. from the group consisting of N18, N21, D25, S31, R32, F36, K40, K49, E50, S51, E54, K57, G58, L60, Q70, E74, E75, P78, Q79, N82, K88, A89, H90, N92, S93, E96, K99, TlOO, L103, R104, R106, R107, K119, E122, Q123, K125, N126, A127, N129, K130, E133, K134, D144, A152 and M156.

In a further embodiment, the polypeptide or conjugate of the invention has an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO:2 in that at least one cysteine residue has been introduced in a helix position selected from the group consisting of: N18, P20, N21, R24, D25, D28, S31, R32, K34, T35, M39, K49, E50, E54, K57, G58, E74, N82, K88, A89, H90, N92, E96, K99, TlOO, L103, R107, Kl 19, Q123, N126, N129, K130, Q132, E133, K134, N148, T155 and R159; preferably from those residues not believed to belong to the receptor interface, i.e. from the group consisting of: N18, N21, D25, S31, R32, K49, E50, E54, K57, G58, E74, N82, K88, A89, H90, N92, E96, K99, TlOO, L103, R107, Kl 19, Q123, N126, N129, K130, Q132, E133 and K134. In a further embodiment, the polypeptide or conjugate of the invention has an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO:2 in that at least one cysteine residue has been introduced in a non-helix position selected from the group consisting of: SI, P2, G3, Q4, G5, T6, Q7, S8, E9, N10, Sll, T13, H14, P16, G17, Q42, L43, D44, N45, L46, Y59, D84, P85, D86, HI 09, R110, Ni l 6, K117, Q132 and N160; pref- erably from those residues not believed to belong to the receptor interface, i.e. from the group consisting of: SI, P2, G3, Q4, G5, T6, Q7, S8, E9, N10, Sll, T13, H14, P16, G17, L43, Y59, D84, P85, D86, H109, Rl 10, Nl 16, Kl 17, Q132 and N160.

In a further embodiment, the polypeptide or conjugate of the invention has an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO:2 in that at least one cysteine residue has been introduced in a non-helix position selected from the group consisting of: SI, P2, G3, Q4, G5, T6, Q7, S8, E9, N10, Sl l, T13, H14, P16, G17, Q42, D44, N45, Y59, P85, D86, Rl 10, Kl 17, Q132 and N160; preferably from those residues not believed to belong to the receptor interface, i.e. from the group consisting of: SI, P2, G3, Q4, G5, T6, Q7, S8, E9, N10, Sll, T13, H14, P16, G17, Y59, P85, D86, R110, K117, Q132 and N160.

In a still further embodiment, the polypeptide or conjugate of the invention may have an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO:2 in that at least one cysteine residue has been introduced in a helix position selected from the group consisting of: P20, N21, M22, L23, R24, D25, L26, R27, D28, A29, F30, K34, T35, F36, F37, Q38, M39, K40, S51, L52, L53, E54, D55, F56, L60, G61, Q63, A64, L65, S66, E67, M68, 169, Q70, F71, Y72, L73, E74, E75, V76, M77, P78, Q79, A80, E81, N82, A89, H90, V91, N92, S93, L94, G95, E96, N97, L98, K99, TlOO, L101, R102, L103, R104, L105, R106, Kl 19, A120, V121, E122, Q123, V124, K125, N126, A127, F128, Q132, E133, K134, G135, 1136, Y137, K138, A139, F143, D144, 1145, F146, 1147, N148, Y149, 1150, E151, A152, Y153, M154 and T155. Among this group, residues not located in the receptor interface are preferred, i.e. N21, M22, D25, L26, A29, F30, F36, F37, K40, S51, L52, L53, E54, D55, F56, L60, G61, Q63, A64, L65, S66, E67, M68, 169, Q70, F71, Y72, L73, E74, E75, V76, M77, P78, Q79, A80, E81, N82, A89, H90, V91, N92, S93, L94, G95, E96, N97, L98, K99, TlOO, L101, R102, L103, R104, L105, R106, Kl 19, A120, V121, E122, Q123, V124, K125, N126, A127, F128, Q132, E133, K134, G135, 1136, Y137, A139, F143, F146, 1147, 1150, A152, Y153 and Ml 54. Further preferred are residues that are not located in the receptor interface and whose side chains are surface-exposed, i.e. N21, D25, F36, K40, S51, E54, L60, Q70, E74, E75, P78, Q79, N82, A89, H90, N92, S93, E96, K99, TlOO, L103, R104, R106, Kl 19, E122, Q123, K125, N126, A127, Q132, E133, K134 and A152.

Since IL-10 is biologically active as a homodimer, while potential amino acid alterations are described herein with reference to the IL-10 monomer, it will be clear that the dimeric form of the IL-10 variants according to the invention will normally consist of two identical IL-10 variant monomers each having the same amino acid alterations, and that the same will apply to any attached polymer moieties. It is also possible, however, to produce dimeric IL-10 in the form of a single-chain polypeptide in which the two monomers are joined by a polypeptide linker. In this case, it is possible to introduce different amino acid alterations into the two monomeric subunits forming the dimer, or to have one monomeric subunit with the amino acid sequence of the native IL-10 (typically hIL-10) and a second subunit with an altered amino acid sequence. Single-chain multimeric polypeptides of this type are described in more detail in WO 02/36626.

Generally, activation of the receptor is associated with receptor-mediated clearance (RMC) such that binding of a polypeptide to its receptor without activation does hot lead to RMC, while activation of the receptor leads to RMC. The clearance is due to intemalisation of the receptor-bound polypeptide with subsequent lysosomal degradation. Reduced RMC may be achieved by designing the conjugate so as to be able to bind and activate a sufficient number of receptors to obtain optimal in vivo biological response and avoid activation of more recep- tors than required for obtaining such response. This may be reflected in reduced in vitro activity and/or increased off-rate. In one embodiment, the conjugates of the invention have a reduced in vitro activity compared to that of non-conjugated hIL-10. Typically, reduced in vitro activity reflects reduced efficacy/efficiency and/or reduced potency and may be determined by any suitable method for determining any of these properties. Examples of suitable assays for determining IL-10 activity in vitro include the following:

• Immunosupressive activity may be measured as a function of inhibition of interferon gamma (IFN-γ) or TNF-alpha production in human peripheral blood mononuclear cells (human PBMC). • Immunostimulatory activity can be measured as the proliferative effect on the murine mast cell line MC/9 (ATCC #CRL-8306). This assay utilizes thymidine incorporation to measure IL-10 dependent cell proliferation.

• The receptor-binding affinity of hIL-10 and hIL-10 variants can be measured e.g. by displacement of 125I-labeled hIL-10 from cells expressing an IL-10 receptor, in particular IL-10R1, or alternatively IL-10R1 and IL-10R2. These cells can e.g. be a cell line such as

MC/9, cultured primary cells such as PBMC or cells such as BaF/3 modified to express one or more of the IL-10 receptors. Alternatively, receptor-binding affinity can be measured using e.g. the Biacore® technology (Biacore AB, Sweden).

• In vitro assays can also be performed to measure IL- 10-mediated down regulation of pro- inflammatory cytokines (such as TNF-α, IL-1 and IL-6), down regulation of MHC Class II antigens (HLA-DR, -DP and -DQ) and inhibition of LPS-stimulated TNF-α production Further details regarding these assay methods are provided in the examples below; see in particular Examples 9, 10 and 11.

It is contemplated that a substantially reduced in vitro activity, compared to the activity of non-conjugated hIL-10 (SEQ ID NO:2), will be advantageous in terms of both a long plasma half-life and a high degree of immunosuppressive activity. Thus, in a preferred embodiment, the in vitro activity of a conjugate of the invention is in the range of about 0.1-50% of the bioactivity of hIL-10, as determined by at least one of the assays described herein. In this embodiment, the in vitro activity of the conjugate of the invention may thus be as low as about 0.1% of that of hIL-10, but will typically be somewhat higher, e.g. at least about 0.2% or at least about 0.5%, and more typically at least about 1%, such as at least about 2%. Similarly, the in vitro activity in this embodiment will typically be reduced by at least about 50% compared to that of hIL-10, for example reduced by at least about 60% or 70%, such as by at least about 75%, 80% or 85%. For instance, the in vitro activity of the conjugate may be in the range of about 0.2-40% of that of hIL-10, such as 0.5-30% or 1-25%.

It is further contemplated that amino acid alterations, in particular substitutions, in the helix regions of hIL-10, i.e. in an amino acid residue selected from amino acid position N18-D41 (helix A), K49-G58 (helix B), L60-N82 (helix C), I87-C108 (helix D), SI 18-L131 (helix E) and E133-R159 (helix F) will result in a reduced receptor-mediated clearance and thus an increased in vivo half-life when the resulting polypeptides are conjugated to polyethylene glycol. Within these helices, preferred residues for introduction of a cysteine residue include those whose side chains have a relatively high degree of surface exposure and that are not located in a receptor-binding interface.

Preferably, the off-rate between the polypeptide conjugate and its receptor is increased by a magnitude resulting in the polypeptide conjugate being released from its receptor before any substantial intemalisation of the receptor-ligand complex has taken place. The off- rate may e.g. be determined using the Biacore® technology. The in vitro RMC may be deter- mined by labelling (e.g. radioactive or fluorescent labelling) the polypeptide conjugate, stimulating cells comprising the receptor for the polypeptide, washing the cells, and measuring label activity. Alternatively, the conjugate may be exposed to cells expressing the relevant receptor. After an appropriate incubation time the supernatant is removed and transferred to a well containing similar cells. The biological response of these cells to the supernatant is determined relative to a non-conjugated polypeptide or another reference polypeptide, and this is a measure of the extent of the reduced RMC.

Normally, reduced in vitro activity of the conjugate is obtained as a consequence of its modification by a polymer molecule. However, in order to further reduce in vitro activity or for other reasons it may be of interest to modify the polypeptide part of the conjugate fur- ther. For instance, in one embodiment at least one amino acid residue located at or near a receptor-binding site of the polypeptide may be substituted with another amino acid residue as compared to the corresponding wild-type polypeptide so as to obtain reduced in vitro activity. The amino acid residue to be introduced by substitution may be any amino acid residue capable of reducing in vitro activity of the conjugate, although in the context of the present invention it will normally not be desired to introduce a cysteine residue in or near a receptor-binding site. Conjugation to a cysteine residue

The polymer molecule to be coupled to a polypeptide variant of the invention to result in a polypeptide conjugate may be any suitable polymer molecule, such as a natural or synthetic homopolymer or heteropolymer, typically with a molecular weight in the range of about 500-50,000 Da, such as in the range of about 1000-40,000 Da or 1000-30,000 Da, e.g. in the range of about 1000-20,000 Da, 2000-15,000 or 3000-12,000 Da or 4000-10,000. For example, the polymer molecule, preferably a polyethylene glycol such as mPEG, may have a molecular weight of about 2 kDa, about 5 kDa, about 10 kDa, about 12 kDa, about 15 kDa or about 20 kDa. When used about polymer molecules herein, the word "about" indicates an approximate average molecular weight and reflects the fact that there will normally be a certain molecular weight distribution in a given polymer preparation.

Examples of homopolymers include a polyol (i.e. poly-OH), a polyamine (i.e. poly-NH2) and a polycarboxylic acid (i.e. poly-COOH). A heteropolymer is a polymer which comprises one or more different coupling groups, for example a hydroxyl group and an amine group.

Examples of suitable polymer molecules include polyalkylene oxide (PAO), including polyalkylene glycol (PAG), such as polyethylene glycol (PEG) and polypropylene glycol (PPG), branched PEGs, poly- vinyl alcohol (PVA), poly-carboxylate, ρoly-(vinylpyrolidone), polyethylene-co-maleic acid anhydride, polystyrene-co-malic acid anhydride, dextran, including carboxymethyl-dextran, or any other biopolymer suitable for reducing immunogenicity and/or increasing functional in vivo half-life and/or serum half-life. Another example of a polymer molecule is human albumin or another abundant plasma protein, although protein polymers are less preferred compared to synthetic polymers such as PEG. Generally, polyalkylene glycol-derived polymers are biocompatible, non-toxic, non-antigenic, non-immunogenic, water soluble, and are easily excreted from living organisms. Such polymers are therefore preferred.

In particular, PEG is the preferred polymer molecule to be used, since it has only few reactive groups capable of cross-linking compared to e.g. polysaccharides such as dextran. In particular, monofunctional PEG, such as monomethoxypolyethylene glycol (mPEG), is of interest since its coupling chemistry is relatively simple (only one reactive group is available for conjugating with attachment groups on the polypeptide). Consequently, the risk of CToss-linking is eliminated, the resulting conjugated polypeptide variants are more homogeneous and the reaction of the polymer molecules with the polypeptide is easier to control.

To effect covalent attachment of the polymer molecule(s) to the polypeptide variant, the hydroxyl end groups of the polymer molecule are provided in activated form, i.e. with reactive functional groups (examples of which include primary amino groups, hydrazide (HZ), thiol, succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide (SSA), suc- cinimidyl proprionate (SPA), succinimidy carboxymethylate (SCM), benzotriazole carbonate (BTC), N-hydroxysuccinimide (NHS), aldehyde, nitrophenylcarbonate (NPC), and tresylate (TRES)). Suitably activated polymer molecules are commercially available, e.g. from Shear- water Corp., Huntsville, AL, USA. Alternatively, the polymer molecules can be activated by conventional methods known in the art, e.g. as disclosed in WO 90/13540. Specific examples of activated linear or branched polymer molecules are described in the Shearwater Corp. Catalog 2001 ("Polyethylene Glycol and Derivatives for Biomedical Applications", incorporated herein by reference). Specific examples of activated PEG polymers include the following linear PEGs: NHS-PEG (e.g. SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG- PEG, and SCM-PEG), and NOR-PEG), BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI- PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branched PEGs such as PEG2-NHS and those disclosed in US 5,932,462 and US 5,643,575, both of which references are incorporated herein by reference. Furthermore, the following publications, incorporated herein by reference, disclose useful polymer molecules and/or PEGylation chemistries: US 5,824,778, US 5,476,653, WO 97/32607, EP 229,108, EP 402,378, US 4,902,502, US 5,281,698, US 5,122,614, US 5,219,564, WO 92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO 94/28024, WO 95/00162, WO 95/11924, WO95/13090, WO 95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439 508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131, US 5,736,625, WO 98/05363, EP 809 996, US 5,629,384, WO 96/41813, WO 96/07670, US 5,473,034, US 5,516,673, EP 605 963, US 5,382,657, EP 510 356, EP 400 472, EP 183 503 and EP 154 316.

Specific examples of activated PEG polymers particularly preferred for coupling to cysteine residues include the following linear PEGs: vinylsulfone-PEG (VS-PEG), preferably vinylsulfone-mPEG (VS-mPEG); maleimide-PEG (MAL-PEG), preferably maleimide- PEG (MAL-mPEG) and orthopyridyl-disulfide-PEG (OPSS-PEG), preferably orthopyridyl- disulfide-mPEG (OPSS-mPEG). Typically, such PEG or mPEG polymers will have a size of about 3-4 kDa, about 5 kDa, about 10 kD, about 12 kDa or about 20 kDa.

The conjugation of the polypeptide variant and the activated polymer molecules is conducted by use of any conventional method, e.g. as described in the following references (which also describe suitable methods for activation of polymer molecules): Harris and Zalip- sky, eds., "Poly(ethylene glycol) Chemistry and Biological Applications", AZC, Washington; R.F. Taylor, (1991), "Protein immobilisation. Fundamental and applications", Marcel Dekker, N.Y.; S.S. Wong, (1992), "Chemistry of Protein Conjugation and Crosslinking", CRC Press, Boca Raton; G.T. Hermanson et al., (1993), "Immobilized Affinity Ligand Techniques", Aca- demic Press, N.Y.). For PEGylation to cysteine residues the polypeptide is usually treated with a reducing agent, such as dithiothreitol (DDT), prior to PEGylation. The reducing agent is subsequently removed by any conventional method, such as by desalting. Conjugation of PEG to a cysteine residue typically takes place in a suitable buffer at pH of about 6-9 at temperatures varying from about 4°C to 25°C for periods up to about 16 hours. It will be understood that the PEGylation is designed so as to produce the optimal molecule with respect to the number of PEG molecules attached, the size and form (e.g. whether they are linear or branched) of such molecules, and where in the polypeptide variant such molecules are attached. In particular, the molecular weight of the polymer to be used, as well as the number of polymer molecules to be attached, will be chosen taking into considera- tion the desired effect(s) to be achieved. In the present context, a preferred embodiment of the conjugate of the invention has up to four, typically up to three, e.g. one or two, PEG moieties with a molecular weight of about 5 kDa attached to each IL-10 polypeptide.

Normally, the polymer conjugation is performed under conditions aiming at reacting most or all available polymer attachment groups with polymer molecules. The molar ratio of activated polymer molecules to polypeptide molecules used for conjugation will typically be up to about 1000: 1 , in particular up to about 200: 1 , e.g. up to about 100: 1 , although lower ratios such as about 10: 1 or 5: 1 or even equimolar ratios may also be used in certain cases.

It is also contemplated according to the invention to couple the polymer molecules to the polypeptide variant through a linker. Suitable linkers are well known to the skilled person. See, for example, WO 02/26265.

Subsequent to the conjugation, residual activated polymer molecules may be blocked according to methods known in the art, e.g. by addition of primary amine to the reaction mixture, and the resulting inactivated polymer molecules removed by a suitable method. Introduction of glycosylation sites

In addition to having introduced cysteine residues that may be conjugated to at least one polymer molecule such as PEG, the polypeptides and conjugates of the invention may also be conjugated to one or more oligosaccharide moieties. This may be performed either in vivo or in vitro, although glycosylation will normally be obtained in vivo. Normally, the in vivo glycosylation site is an N-glycosylation site, but also an O-glycosylation site may also be of interest. In order to achieve in vivo glycosylation of an IL-10 molecule comprising one or more glycosylation sites the nucleotide sequence encoding the polypeptide must be inserted in a gly- cosylating eukaryotic expression host. The expression host cell may be selected from fungal (filamentous fungal or yeast), insect or animal cells or from transgenic plant cells. In one embodiment the host cell is a mammalian cell, such as a CHO cell, a BHK or HEK cell, e.g. HEK 293, or an insect cell, such as an SF9 cell, or a yeast cell, e.g. Saccharomyces cerevisiae or Pichiapasto is, or any of the host cells mentioned hereinafter. Suitable methods of in vitro coupling are described, for example, in WO

87/05330 and in Aplin et al., CRC Crit Rev. Biochem., pp. 259-306, 1981. In vitro coupling of oligosaccharide moieties to protein- and peptide-bound Gin-residues can also be carried out by rransglutaminases (TGases), e.g. as described by Sato et al., 1996 Biochemistry 35, 13072- 13080 or in EP 725145. For in vivo N-glycosylation, the attachment site for the oligosaccharide moiety comprises the sequence N-X'-S/T/C-X", wherein X' is any amino acid residue except proline, X" any amino acid residue that may or may not be identical to X' and preferably is different from proline, N is asparagine, and S/T/C is either serine, threonine or cysteine, preferably serine or threonine, and most preferably threonine). Although the asparagine residue of the N- glycosylation site is the one to which the sugar moiety is attached during glycosylation, such attachment cannot be achieved unless the other amino acid residues of the N-glycosylation site is present.

When introduction of an N-glycosylating site is desired, this can be performed by way of a substitution selected from the group consisting of: P2N+Q4S, P2N+Q4T, G3N+G5S, G3N+G5T, Q4N+T6S, Q4N, G5N+Q7S, G5N+Q7T, T6N, T6N+S8T, Q7N+E9S, Q7N+E9T, S8N+N10S, S8N+N10T, E9N, E9N+S11T, S11N+T13S, SUN, H14N+P16S, H14N+P16T, P16N+N18S, P16N+N18T, P20S, P20T, P20N+M22S, P20N+M22T, L23S, L23T, R24N+L26S, R24N+L26T, D25N+R27S, D25N+R27T, R27N+A29S, R27N+A29T, D28N+F30S, D28N+F30T, S31N+V33S, S31N+V33T, R32N+K34S, R32N+K34T, K34N+F36S, K34N+F36T, T35N+F37S, T35N+F37T, Q38N+K40S, Q38N+K40T, M39N+D41S, M39N+D41T, K40N+Q42S, K40N+Q42T, Q42N+D44S, Q42N+D44T, L43N+N45S, L43N+N45T, D44N+L46S, D44N+L46T, L47S, L47T, L46N+L48S, L46N+L48T, K49N, K49N+S51T, E50N+L52S, E50N+L52T, S51N+L53S, S51N+L53T, E54N+F56S, E54N+F56T, K57N+Y59S, K57N+Y59T, G58N+L60S, G58N+L60T, S66N+M68S, S66N+M68T, Q70N+Y72S, Q70N+Y72T, E74N+V76S, E74N+V76T, P78N+A80S, P78N+A80T, Q79N+E81S, Q79N+E81T, Q83N+P85S, Q83N+P85T, P85N+I87S, P85N+I87T, D86N+K88S, D86N+K88T, K88N+H90S, K88N+H90T, A89N+V91 S, A89N+V91T, H90N+N92S, H90N+N92T, L94S, L94T, S93N+G95S, S93N+G95T, E96N+L98S, E96N+L98T, K99S, K99T, K99N+L101S, K99N+L101T, T100SN+R102S, T100N+R102T, R102N+R104S, R102N+R104T, L103N+L105S, L103N+L105T, R107N+H109S, R107N+H109T, H109N+F111S, H109N+F111T, P113N+E115S, P113N+E115T, K117N+K119S, K117N+K119T, E122N+V124S, E122N+V124T, Q123N+K125S, Q123N+K125T, K125N+A127S, K125N+A127T, F128S, F128T, A127N+N129S, A127N+N129T, L131S, L131T, K130N+Q132S, K130N+Q132T, Q132N+K134S, Q132N+K134T, E133N+G135S, E133N+G135T, K134N+I136S, K134N+I136T, D144N+F146S, D144N+F146T, I145N+I147S, I145N+I147T, I150S, I150T, E151N+Y153S, E151N+Y153T, T155N+K157S, T155N+K157T, M156N+I158S, M156N+I158T, K157N+R159S and K157N+R159T.

Preferably, introduction of a glycosylation site is performed by way of a substitution selected from the group consisting of: P2N+Q4S, P2N+Q4T, G3N+G5S, G3N+G5T, Q4N+T6S, Q4N, G5N+Q7S, G5N+Q7T, T6N, T6N+S8T, Q7N+E9S, Q7N+E9T, S8N+N10S, S8N+N10T, E9N, E9N+S11T, S11N+T13S, SUN, H14N+P16S, H14N+P16T, P16N+N18S, P16N+N18T, P20S, P20T, P20N+M22S, P20N+M22T, L23S, L23T, R24N+L26S, R24N+L26T, D25N+R27S, D25N+R27T, D28N+F30S, D28N+F30T, S31N+V33S, S31N+V33T, R32N+K34S, R32N+K34T, K34N+F36S, K34N+F36T, Q38N+K40S, Q38N+K40T, M39N+D41S, M39N+D41T, Q42N+D44S, Q42N+D44T, D44N+L46S, D44N+L46T, L47S, L47T, K49N, K49N+S51T, E50N+L52S, E50N+L52T, E54N+F56S, E54N+F56T, K57N+Y59S, K57N+Y59T, G58N+L60S, G58N+L60T, Q70N+Y72S, Q70N+Y72T, E74N+V76S, E74N+V76T, Q79N+E81S, Q79N+E81T, P85N+I87S, P85N+I87T, D86N+K88S, D86N+K88T, K88N+H90S, K88N+H90T, A89N+V91 S, A89N+V91T, H90N+N92S, H90N+N92T, S93N+G95S, S93N+G95T, E96N+L98S, E96N+L98T, K99N+L101S, K99N+L101T, T100SN+R102S, T100N+R102T, L103N+L105S, L103N+L105T, R107N+H109S, R107N+H109T, H109N+F111S, H109N+F111T, K117N+K119S, K117N+K119T, E122N+V124S, E122N+V124T, F128S, F128T, A127N+N129S, A127N+N129T, L131S, L131T, K130N+Q132S, K130N+Q132T, Q132N+K134S, Q132N+K134T, E133N+G135S, E133N+G135T, D144N+F146S, D144N+F146T, I150S, I150T, T155N+K157S and T155N+K157T.

In further preferred embodiments, introduction of a glycosylation site is performed by way of a substitution selected from the group consisting of: Q4N, T6N, E9N, SI IN, P20S, P20T, L23S, L23T, L47S, L47T, K49N, L94S, L94T, K99S, K99T, F128S, F128T, L131S, L131T, I150S and I150T; or a substitution selected from the group consisting of: Q4N, T6N, E9N, SUN, L23S, L23T, L47S, L47T, K49N, F128S, F128T, L131S, L131T, I150S and I150T.

Expression of polypeptides Persons skilled in the art will be familiar with available vectors, expression control sequences and hosts, and will be able to select from among suitable vectors, expression control sequences and hosts without undue experimentation.

The recombinant vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chro- mosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the nucleotide sequence encoding the polypeptide of the invention is operably linked to additional segments required for transcription of the nucleotide sequence. The vector is typically derived from plasmid or viral DNA. A number of suitable expression vectors for expression in the host cells mentioned herein are commercially available or described in the literature. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma vims, adenoviras and cytomegalovirus. Specific vectors are, e.g., ρCDNA3.1 (+)\Hyg (Invitrogen, Carlsbad, CA, USA) and pCI-neo (Stratagene, La JoUa, CA, USA). Useful expression vectors for yeast cells include the 2μ plasmid and derivatives thereof, the POT1 vector (US 4,931,373), the ρJSO37 vector described in Okkels, Ann. New York Acad. Sci. 782, 202-207, 1996, and pPICZ A, B or C (Invitrogen). Useful vectors for insect cells include pVL941, pBG311 (Gate et al, Cell, 45, pp. 685-98 (1986)), pBluebac 4.5 and pMelbac (both available from Invitrogen). Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pBR322, pET3a and pET12a (both from Novagen Inc., WI, USA), wider host range plasmids, such as RP4, phage DNAs, e.g. the numerous derivatives of phage lambda, e.g. NM989, and other DNA phages, such as Ml 3 and filamentous single stranded DNA phages.

Other vectors for use in this invention include those that allow the nucleotide sequence encoding the polypeptide to be amplified in copy number. Such amplifiable vectors are well known in the art. They include, for example, vectors able to be amplified by DHFR ampli- fixation (see, e.g., US 4,470,461 and Kaufman and Sharp, Mol. Cell. Biol., 2, pp. 1304-19 (1982)) and glutamine synthetase ("GS") amplification (see, e.g., US 5,122,464 and EP 338 841).

The recombinant vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. An example of such a sequence (when the host cell is a mammalian cell) is the SV40 origin of replication. When the host cell is a yeast cell, suitable sequences enabling the vector to replicate are the yeast plasmid 2μ replication genes REP 1-3 and origin of replication.

The vector may also comprise a selectable marker, e.g. a gene whose product complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or the Schizosaccharomyces pombe TPI gene (described by P.R. Russell, Gene 40, 1985, pp. 125-130), or one which confers resistance to a drug, e.g. ampicillin, kanamycin, tet- racyclin, chloramphenicol, neomycin, hygromycin or methotrexate. For Saccharomyces cere- visiae, selectable markers include ura3 and leu2. For filamentous fungi, selectable markers include amdS,pyrG, arcB, niaD and sC. The term "control sequences" is defined herein to include all components which are necessary or advantageous for the expression of the polypeptide of the invention. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader sequence, polyadenylation sequence, propeptide sequence, promoter, enhancer or upstream activating sequence, signal pep- tide sequence, and transcription teiminator. At a minimum, the control sequences include a promoter.

A wide variety of expression control sequences may be used in the present invention. Such useful expression control sequences include the expression control sequences asso- ciated with structural genes of the foregoing expression vectors as well as any sequence known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

Examples of suitable control sequences for directing transcription in mammalian cells include the early and late promoters of SV40 and adenovirus, e.g. the adenovirus 2 major late promoter, the MT-1 (metallothionein gene) promoter, the human cytomegalovirus immediate-early gene promoter (CMV), the human elongation factor lα (EF-lα) promoter, the Dro- sophila minimal heat shock protein 70 promoter, the Rous Sarcoma Vims (RSV) promoter, the human ubiquitin C (UbC) promoter, the human growth hormone terminator, SV40 or adenovi- rus Elb region polyadenylation signals and the Kozak consensus sequence (Kozak, M. JMol Biol 1987 Aug 20;196(4):947-50).

In order to improve expression in mammalian cells a synthetic intron may be inserted in the 5' untranslated region of the nucleotide sequence encoding the polypeptide. An example of a synthetic intron is the synthetic intron from the plasmid pCI-Neo (available from Promega Corporation, WI, USA).

Examples of suitable control sequences for directing transcription in insect cells include the polyhedrin promoter, the P10 promoter, the Autographa californica polyhedrosis virus basic protein promoter, the baculovirus immediate early gene 1 promoter, the baculovirus 39K delayed-early gene promoter, and the SV40 polyadenylation sequence. Examples of suit- able control sequences for use in yeast host cells include the promoters of the yeast α-mating system, the yeast triose phosphate isomerase (TPI) promoter, promoters from yeast glycolytic genes or alcohol dehydrogenase genes, the ADH2-4c promoter, and the inducible GAL promoter. Examples of suitable control sequences for use in filamentous fungal host cells include the ADH3 promoter and terminator, a promoter derived from the genes encoding Aspergillus oryzae TAKA amylase triose phosphate isomerase or alkaline protease, an A. niger α-amylase, A. niger or A. nidulans glucoamylase, A. nidulans acetamidase, Rhizomucor miehei aspartic proteinase or lipase, the TPI1 terminator and the ADH3 terminator. Examples of suitable control sequences for use in bacterial host cells include promoters of the lac system, the trp system, the TAC or TRC system, and the major promoter regions of phage lambda. The nucleotide sequence of the invention encoding a polypeptide exhibiting

IL-10 activity, whether prepared by site-directed mutagenesis, synthesis, polymerase chain reaction (PCR) or other methods, may optionally also include a nucleotide sequence that encodes a signal peptide. The signal peptide is present when the polypeptide is to be secreted from the cells in which it is expressed. Such signal peptide, if present, should be one recognized by the cell chosen for expression of the polypeptide. The signal peptide may be homologous (e.g. be that normally associated with hIL-10) or heterologous (i.e. originating from another source than hIL-10) to the polypeptide or may be homologous or heterologous to the host cell, i.e. be a signal peptide normally expressed from the host cell or one which is not normally expressed from the host cell. Accordingly, the signal peptide may be prokaryotic, e.g. derived from a bacterium such as E. coli, or eukaryotic, e.g. derived from a mammalian, or insect or yeast cell.

The presence or absence of a signal peptide will, e.g., depend on the expression host cell used for the production of the polypeptide to be expressed (whether it is an intracellu- lar or extracellular polypeptide) and whether it is desirable to obtain secretion. For use in filamentous fungi, the signal peptide may conveniently be derived from a gene encoding an As- pergillus sp. amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase or protease or a Humicola lanuginosa lipase. The signal peptide is preferably derived from a gene encod- ing A. oryzae TAKA amylase, A. niger neutral α-amylase, A. niger acid-stable amylase, or A. niger glucoamylase. For use in insect cells, the signal peptide may conveniently be derived from an insect gene (cf. WO 90/05783), such as the Lepidopteran manduca sexta adipokinetic hormone precursor, (cf. US 5,023,328), the honeybee melittin (Invitrogen), ecdysteroid UDPglucosyltransferase (egt) (Murphy et al., Protein Expression and Purification 4, 349-357 (1993) or human pancreatic lipase (hpl) (Methods in Enzymology 284, pp. 262-272, 1997). A preferred signal peptide for use in mammalian cells is that of hIL-10 or the murine Ig kappa light chain signal peptide (Coloma, M (1992) J. Imm. Methods 152:89-104). For use in yeast cells suitable signal peptides include the α-factor signal peptide from S. cereviciae (cf. US 4,870,008), a modified carboxypeptidase signal peptide (cf. L.A. Vails et al, Cell 48, 1987, pp. 887-897), the yeast BAR1 signal peptide (cf. WO 87/02670), the yeast aspartic protease 3 (YAP3) signal peptide (cf. M. Egel-Mitani et al, Yeast 6, 1990, pp. 127-137), and the synthetic leader sequence TA57 (WO98/32867). For use in E. coli cells a suitable signal peptide is the signal peptide ompA.

Any suitable host may be used to produce the polypeptide or polypeptide part of the conjugate of the invention, including bacteria, fungi (including yeasts), plant, insect, mammal, or other appropriate animal cells or cell lines, as well as transgenic animals or plants. Examples of bacterial host cells include gram-positive bacteria such as strains of Bacillus, e.g. B. brevis or B. subtilis, Pseudomonas or Streptomyces, or gram-negative bacteria, such as strains of E. coli. The introduction of a vector into a bacterial host cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), using competent cells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thome, 1987, Journal of Bacteriology 169: 5771-5278). Examples of suitable filamentous fungal host cells include strains of Aspergϊllus, e.g. A. oryzae, A. niger, iA. nidulans, Fusarium or Trichoderma. Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and re- generation of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and US 5,679,543. Suitable methods for transforming Fusarium species are described by Malardier et al, 1989, Gene 78: 147-156 and WO 96/00787. Examples of suitable yeast host cells include strains of Saccharomyces, e.g. S. cerevisiae, Schizosaccharomyces, Klyveromyces, Pichia, such as P. pastoris or P. methanolica, Hansenula, such as H. polymorpha or Yarrowia. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al, 1983, Journal of Bacteriology 153: 163; Ηinnen et al, 1978, Proc NatAcad Sci USA 75: 1920: and as disclosed by Clontech Laboratories, Inc, Palo Alto, CA, USA (in the product protocol for the Yeastmaker™ Yeast Transformation System Kit).

Examples of suitable insect host cells include a Lepidoptora cell line, such as Spodoptera frugiperda (S 9 or Sf21) or Trichoplusioa ni cells (High Five) (US 5,077,214). Transformation of insect cells and production of heterologous polypeptides therein may be performed as described by Invitrogen.

Examples of suitable mammalian host cells include Chinese hamster ovary (CHO) cell lines, (e.g. CHO-K1; ATCC CCL-61), Green Monkey cell lines (COS) (e.g. COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-1651)); mouse cells (e.g. NS/O), Baby Hamster Kidney (BHK) cell lines (e.g. ATCC CRL-1632 or ATCC CCL-10), and human cells (e.g. HEK 293 (ATCC CRL-1573)), as well as plant cells in tissue culture. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland. Methods for introducing exogeneous DNA into mammalian host cells include calcium phosphate-mediated transfection, electroporation, DEAE-dextran mediated transfection, liposome-mediated transfection, viral vectors and the transfection method described by Life Technologies Ltd, Paisley, UK using Lipofectamin 2000. These methods are well known in the art and e.g. described by Ausbel et al (eds.), 1996, Current Protocols in Molecular Biology, John Wiley & Sons, New York, USA. The cultivation of mam- malian cells is conducted according to established methods, e.g. as disclosed in: Animal Cell Biotechnology, Methods and Protocols, Edited by Nigel Jenkins, 1999, Human Press Inc, To- towa, New Jersey, USA and Harrison MA and Rae IF, General Techniques of Cell Culture, Cambridge University Press 1997.

In the production methods of the present invention, the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient me- dium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates. The resulting polypeptide may be recovered by methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. The polypeptides may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusmg, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification (2nd Edition), Janson and Ryden, editors, Wiley, New York, 1998).

Pharmaceutical compositions and administration

In a further aspect, the present invention comprises a composition comprising a polypeptide or polypeptide conjugate as described herein and at least one pharmaceutically acceptable carrier or excipient, as well as use of the polypeptides and conjugates of the invention for preparing a pharmaceutical composition.

The polypeptides, conjugates and compositions according to the invention are contemplated for use in the prevention and/or treatment of inflammatory and autoimmune dis- eases and conditions, including psoriasis, inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, rheumatoid arthritis, allergic inflammation, inhibition of some types of tumor growth, improved graft acceptance after transplantation, and chronic hepatitis C infection.

The polypeptides and conjugates of the invention will be administered to patients in a "therapeutically effective" dose, i.e. a dose that is sufficient to produced the desired effects in relation to the condition for which it is administered. The exact dose will depend on the disorder to be treated, and will be ascertainable by one skilled in the art using known techniques. It will be apparent to those of skill in the art that an effective amount of a polypeptide or conjugate of the invention depends, inter alia, upon the disease, the dose, the administration schedule, whether the polypeptide or conjugate is administered alone or in conjunction with other therapeutic agents, the serum half-life of the compositions, the general health of the patient, and the frequency of administration.

The polypeptide or conjugate of the invention will normally be administered in a composition including one or more pharmaceutically acceptable carriers or excipients. The polypeptide or conjugate can be formulated into pharmaceutical compositions in a manner known er se in the art to result in a polypeptide pharmaceutical that is sufficiently storage- stable and is suitable for administration to humans or animals. The pharmaceutical composition may be formulated in a variety of forms, including as a liquid or gel, or lyophilized, or any other suitable form. The preferred form will depend e.g. upon the particular indication being treated and will be apparent to one of skill in the art. A variety of aqueous carriers can be used, e.g. buffered saline, such as PBS, and the like. The compositions may also contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions or to enhance stability, such as pH adjusting and buffering agents, toxicity adjusting agents, etc., e.g. as described in more detail below.

Drugform

The polypeptide or conjugate of the invention can be used "as is" and/or in a salt form thereof. Suitable salts include, but are not limited to, salts with alkali metals or alkaline earth metals, such as sodium, potassium, calcium and magnesium, as well as e.g. zinc salts. These salts or complexes may by present as a crystalline and/or amorphous structure.

Excipients

"Pharmaceutically acceptable" means a carrier or excipient that at the dosages and concentrations employed does not cause any untoward effects in the patients to whom it is administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 19th edition, A. R. Gennaro, Ed., Mack Publishing Company [1995]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000] ; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press [2000]).

Mix of drugs

The polypeptide, conjugate or pharmaceutical composition of the invention may be administered alone or in conjunction with other therapeutic agents. These agents may be incorporated as part of the same pharmaceutical composition or may be administered separately from the polypeptide conjugate of the invention, either concurrently or in accordance with another treatment schedule. In particular, the polypeptide, conjugate or composition of the invention may be used in conjunction with other agents suitable for the prevention or treatment of inflammatory or autoimmune diseases, for example Remicade® (infliximab), Antegren® (natalizumab), methotrexate, cyclosporine, budesonide or other corticosteroids, tumor necrosis factor-alpha (TNF-α) antibodies or antagonists, Azulfidine® (sulfasalazine), 5-aminosalicylic acid (5-ASA), 4-aminosalicylic acid (4-ASA) and granulocyte colony stimulating factor (G- CSF). In addition, the polypeptide, conjugate or pharmaceutical composition of the invention may be used as an adjuvant to other therapies.

Patients A "patient" for the purposes of the present invention includes both humans and other mammals. The methods are thus applicable to both human therapy and veterinary applications, but are in particular directed to human therapy.

Administration route

The administration of the formulations of the present invention can be performed in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, in- tracerebrally, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, intraocularly, or in any other acceptable manner. Such administration routes and appropriate formulations are generally known to those of skill in the art.

Parenterals

An example of a pharmaceutical composition is a solution designed for parenteral administration. Although in many cases pharmaceutical solution formulations are provided in liquid form, appropriate for immediate use, such parenteral formulations may also be provided in frozen or in lyophilized form. In the former case, the composition must be thawed prior to use. The latter form is often used to enhance the stability of the active compound contained in the composition under a wider variety of storage conditions, as it is recognized by those skilled in the art that lyophilized preparations are generally more stable than their liquid counterparts. Such lyophilized preparations are reconstituted prior to use by the addition of one or more suitable pharmaceutically acceptable diluents such as sterile water for injection or sterile physiological saline solution.

In case of parenterals, they are prepared for storage as lyophilized formulations or aqueous solutions by mixing, as appropriate, the polypeptide having the desired degree of purity with one or more pharmaceutically acceptable carriers, excipients or stabilizers typically employed in the art (all of which are termed "excipients"), for example buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants and/or other miscellaneous additives. Buffering agents help to maintain the pH in the range which approximates physiological conditions. They are typically present at a concentration ranging from about 2 mM to about 50 mM. Suitable buffering agents for use with the present invention include both organic and inorganic acids and salts thereof such as citrate buffers (e.g., monosodium citrate- disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fu- marate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodium glyconate mixture, glu- conic acid-sodium hydroxide mixture, gluconic acid-potassium glyuconate mixture, etc.), ox- alate buffer (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.) and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). Additional possibilities are phosphate buffers, histidine buffers and trimethylamine salts such as Tris. Preservatives are added to retard microbial growth, and are typically added in amounts of about 0.2%- 1% (w/v). Suitable preservatives for use with the present invention include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldi- methylbenzyl ammonium chloride, benzalkonium halides (e.g. benzalkonium chloride, bromide or iodide), hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.

Isotonicifiers are added to ensure isotonicity of liquid compositions and include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. Polyhydric alcohols can be present in an amount between 0.1% and 25% by weight, typically 1% to 5%, taking into account the relative amounts of the other ingredients.

Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can be polyhydric sugar alcohols (see above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, his- tidine, alanine, omithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribi- tol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (i.e. <10 residues); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvi- nylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran. Stabilizers are typically present in the range of from 0.1 to 10,000 parts by weight based on the active protein weight.

Non-ionic surfactants or detergents (also known as "wetting agents") may be present to help solubilize the therapeutic agent as well as to protect the therapeutic polypeptide against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the polypeptide. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188 etc.), Pluronic® polyols, poly- oxyethylene sorbitan monoethers (Tween®-20, Tween®-80, etc.).

Additional miscellaneous excipients include bulking agents or fillers (e.g. starch), chelating agents (e.g. EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E) and cosolvents.

The active ingredient may also be entrapped in microcapsules prepared, for example, by coascervation techniques or by interfacial polymerization, for example hydroxy- methylcellulose, gelatin or poly-(methylmethacylate) microcapsules, in colloidal drag delivery systems (for example liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

Parenteral formulations to be used for in vivo administration must be sterile. This is readily accomplished using well-known techniques, for example by filtration through sterile filtration membranes.

Sustained release preparations

Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the polypeptide or conjugate, the matrices having a suitable form such as a film or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels, poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, and poly-D-(-)-3-hydroxybutyric acid.

Pulmonary delivery

The polypeptide or conjugate of the invention, alone or in combination with other suitable components, can also be made into aerosol formulations (e.g., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise the polypeptide or conjugate dissolved in water at a concentration of, e.g., about 0.01 to 25 mg of conjugate per ml of solution. The aerosol formulation may also include, e.g., one or more of a buffer, a simple sugar (e.g. for protein stabilization and regulation of osmotic pressure), a sugar alcohol and a surfactant. Formulations for powder inhalers will comprise a finely divided dry powder containing the polypeptide or conjugate and may also include a bulking agent such as lactose, sorbitol, sucrose or mannitol in an amount which facilitates dispersal of the powder from the device, e.g., 50% to 90% by weight of the formulation. Mechanical devices designed for pulmonary delivery of therapeutic products include, but are not limited to, nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those of skill in the art.

All references cited herein are hereby incorporated by reference in their entirety for all purposes. The invention is further described in the non-limiting examples below.

EXAMPLE 1

Construction and cloning of synthetic genes encoding ML- 10 and hIL-10 cysteine variants for expression in E. coli The DNA sequence encoding mature human IL-10 is synthesized using standard DNA procedures. The nucleic acid sequence is modified towards codon usage commonly found in E. coli. Variants of human IL-10 are synthesized utilizing the same methods as above. Each variant contains, initially, a single amino acid substitution. Cysteine is always the amino acid replacing the wild type amino acid. Cysteine is substituted at the following positions: P20, N21, M22, L23, R24, D25, L26, R27, D28, A29, F30, K34, T35, F36, F37, Q38, M39, K40, S51, L52, L53, E54, D55, F56, L60, G61, Q63, A64, L65, S66, E67, M68, 169, Q70, F71, Y72, L73, E74, E75, V76, M77, P78, Q79, A80, E81, N82, A89, H90, V91, N92, S93, L94, G95, E96, N97, L98, K99, TlOO, L101, R102, L103, R104, L105, R106, K119, A120, V121, E122, Q123, V124, K125, N126, A127, F128, Q132, E133, K134, G135, 1136, Y137, K138, A139, F143, D144, 1145, F146, 1147, N148, Y149, 1150, E151, A152, Y153, M154, T155. Once variants having desired properties are identified, e.g. using the assays described below, variants having multiple advantageous mutations, e.g. with 2 or 3 introduced cysteine residues, may be synthesized and tested.

In addition to IL-10 or variant IL-10 coding sequences, the DNA fragments may be synthesized to contain a fusion protein partner, a protease cleavage site (such as factor Xa) a linker and/or a 6-HIS tag at the 3 'end of the IL-10 sequence. The DNA fragments will contain restriction digestion sites at the 5' and 3' ends. These fragments are ligated to the vector via the digestion sites using standard DNA techniques. EXAMPLE 2

Construction and cloning of synthetic genes encoding hIL-10 and ML- 10 variants for expression in mammalian cells The DNA sequence encoding mature human IL-10 is synthesized using standard DNA procedures. Variants of human IL-10 are synthesized to introduce one or more potential N- linked glycosylation sites utilizing the same methods as above. Glycosylation site(s) are introduced by way of substitution selected from the group consisting of P2N+Q4S, P2N+Q4T, G3N+G5S, G3N+G5T, Q4N+T6S, Q4N, G5N+Q7S, G5N+Q7T, T6N, T6N+S8T, Q7N+E9S, Q7N+E9T, S8N+N10S, S8N+N10T, E9N, E9N+S1 IT, SI 1N+T13S, SI IN, H14N+P16S, H14N+P16T, P16N+N18S, P16N+N18T, P20S, P20T, P20N+M22S, P20N+M22T, L23S, L23T, R24N+L26S, R24N+L26T, D25N+R27S, D25N+R27T, R27N+A29S, R27N+A29T, D28N+F30S, D28N+F30T, S31N+V33S, S31N+V33T, R32N+K34S, R32N+K34T, K34N+F36S, K34N+F36T, T35N+F37S, T35N+F37T, Q38N+K40S, Q38N+K40T, M39N+D41 S, M39N+D41T, K40N+Q42S, K40N+Q42T, Q42N+D44S, Q42N+D44T, L43N+N45S, L43N+N45T, D44N+L46S, D44N+L46T, L47S, L47T, L46N+L48S, L46N+L48T, K49N, K49N+S51T, E50N+L52S, E50N+L52T, S51N+L53S, S51N+L53T, E54N+F56S, E54N+F56T, K57N+Y59S, K57N+Y59T, G58N+L60S, G58N+L60T, S66N+M68S, S66N+M68T, Q70N+Y72S, Q70N+Y72T, E74N+V76S, E74N+V76T, P78N+A80S, P78N+A80T, Q79N+E81S, Q79N+E81T, Q83N+P85S, Q83N+P85T, P85N+I87S, P85N+I87T, D86N+K88S, D86N+K88T, K88N+H90S, K88N+H90T, A89N+V91S, A89N+V91T, H90N+N92S, H90N+N92T, L94S, L94T, S93N+G95S, S93N+G95T, E96N+L98S, E96N+L98T, K99S, K99T, K99N+L101S, K99N+L101T, T100SN+R102S, T100N+R102T, R102N+R104S, R102N+R104T, L103N+L105S, L103N+L105T, R107N+H109S, R107N+H109T, H109N+F111S, H109N+F111T, P113N+E115S, P113N+E115T, K117N+K119S, K117N+K119T, E122N+V124S, E122N+V124T, Q123N+K125S, Q123N+K125T, K125N+A127S, K125N+A127T, F128S, F128T, A127N+N129S, A127N+N129T, L131S, L131T, K130N+Q132S, K130N+Q132T, Q132N+K134S, Q132N+K134T, E133N+G135S, E133N+G135T, K134N+I136S, K134N+I136T, D144N+F146S, D144N+F146T, I145N+I147S, I145N+I147T, I150S, I150T, E151N+Y153S, E151N+Y153T, T155N+K157S, T155N+K157T, M156N+I158S, M156N+I158T, K157N+R159S and K157N+R159T. In addition to the IL-10 or variant IL-10 coding sequence, the DNA fragments will contain a signal sequence at the 5' end of the gene for translocation of the expressed protein into the cell culture supernatant and, at the 3 'end, may contain a linker and a 6-HIS tag. The fragments will contain restriction digestion sites at the 5' and 3' ends. These fragments are ligated 5 via the digestion sites into a pcDNA mammalian expression vector (Invitrogen) using standard DNA techniques.

EXAMPLE 3

Expression ofhIL-10 and ML- 10 cysteine variants in E. coli l o Transformation of E. coli with plasmids containing hlL- 10 or hlL- 10 cysteine variants, isolation of transformants containing the plasmids, and subsequent periplasmic expression of the proteins is performed using standard techniques described in the literature. The protein may be expressed as a fusion with another protein, such as maltose binding protein. The fusion partner can aid in achieving higher expression levels and in maintaining solubility of the pro-

15 tein. A protease cleavage site is engineered between the fusion partner and the IL-10 variant, allowing the fusion partner to be cleaved from IL-10 and the variants thereof. The fusion partner is removed from the mixture by specific chromatography such as that described by Salek- Ardakani, et al (2002) Cytokine 17:1-13.

Expression of hIL-10 or hIL-10 cysteine variants by E. coli is verified by Western blot

20 analysis using a polyclonal antibody against hIL-10, an HRP secondary antibody conjugate and chemiluminescent detection reagents.

EXAMPLE 4

Generation of stable CHO-K1 ML- 10 and glycoslylated ML- 10 producing cell lines

25 The day before transfection the CHO-K1 cell line (ATCC #CC1-61) is seeded in a fresh tissue culture flask in DMEM/F-12 medium (Gibco # 31330-038) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. The following day (at nearly 100% conflu- ency) the transfection is prepared. DMEM medium without supplements is aliquoted into a 14 ml polypropylene tube. Fugene 6 (Roche) is added directly into the medium followed by incu-

30 bation for 5 min. at room temperature. In the meantime, plasmid containing hIL-10 or modified hIL-10 is aliquoted into another 14 ml polypropylene tube. After incubation, the Fugene 6 mix is to be added directly to the DNA solution and incubated for 15 min. at room temperature. After incubation the whole volume is added drop-wise to the cell medium. The next day the medium is exchanged with fresh medium containing G418, neomycin antibiotic. Every few days the selection medium is renewed until the primary transfection pool reaches 80-90% confluency. The primary transfection pool is sub-cloned by dropping single cells into 96-well plates using FACS. After a cell population of a significant size has grown from a single cell, expression of hlL- 10 or hlL- 10 variants by CHO-K1 is verified by Western blot analysis. IL-10 protein is detected using a polyclonal antibody against hIL-10 or an antibody against the 6-HIS tag. An HRP conjugated secondary antibody can also be used. Protein is to be visualized with chemiluminescent detection reagents.

EXAMPLE 5

Purification of ML- 10 and variants thereof from E. coli

Purification of hIL-10 and variants from E. coli periplasm is performed using standard techniques. Briefly, cells are harvested by centrifugation, resuspended in 30 mM Tris-Cl, 20% sucrose at pH 8.0, 1 mM EDTA and incubated on ice 5-10 min. with agitation. The cell sus- pension is then centrifuged (8000g x 20 min.), the supernatant is removed and the pellet is re- suspended in cold 5 mM MgSO4. This suspension is incubated for 10 min. at 4°C with agitation. This suspension is centrifuged (8000g x 20 min.) at 4°C. The periplasmic fraction, containing the IL-10 protein, is the supernatant resulting from the second centrifugation. Proteins containing the 6-HIS tag are purified from the periplasmic fraction using standard metal che- late or immobilized metal affinity chromatography (IMAC). Kits for this type of purification are available from many vendors (such as Qiagen). Purification is carried out in the presence of 2-5 mM beta-mercapto-ethanol to avoid dimerization of IL-10 variants. Endotoxin is removed from E. coli produced proteins using standard methods.

An example of IL-10 expression and purification is described in Salek-Ardakani et al., (2002) Cytokine 17:1-13.

EXAMPLE 6

Purification of ML- 10 and variants thereof from CHO-K1 supematants

Purification of hIL-10 and variants from the CHO-K1 supernatant is performed as follows. Cell culture supematants are collected 24-48 h after having been subcultured into fresh media. The greatest amount of protein is usually collected from an 80-90% confluent flask. Cell culture media is filtered (pore size 0.4 μm or smaller) to remove cells and cell debris. Pro- teins containing the 6-HIS tag are purified from the supernatant using standard metal chelate or immobilized metal affinity chromatography (IMAC). Kits for this type of purification are available from many vendors (such as Qiagen).

EXAMPLE 7

Cysteine PEGylation ofhIL-10

Free cysteine residues are introduced by gene synthesis into hIL-10 at defined positions as described above. After expression and purification, PEG groups are attached to the purified proteins via the introduced cysteines.

PEGylation using OPSS coupling chemistry

One method of PEGylation involves OPSS coupling chemistry. In this method, cys- teine-containing protein variants are suspended in 5 mM sodium succinate, 4% mannitol, 0.01% Tween® 20, pH 6.0, and reduced by incubation in 20 mM dithiothreitol (DTT) for 30 min. at room temperature. The protein is then desalted on NAP25 gel filtration columns

(Pharmacia) in buffer A (50 mM sodium phosphate, 1 mM EDTA, pH 8.1). mPEG-OPSS (or- thopyridyldisulfide monofunctional PEG, Shearwater Corp.) is suspended in buffer A to a concentration of 2 mg/ml and added in equal volume to the reduced and desalted hIL-10 variant and incubated for 60 min. with gentle shaking at room temperature. Following this incubation, the mixture is concentrated using Vivaspin20 columns (VivaScience) and the remaining free mPEG is removed by gel filtration using Sephacryl® S-100 columns (Pharmacia) equilibrated in buffer A. Finally, PEGylated hIL-10 variants are diafiltered into 5 mM sodium succinate, 4% mannitol, pH 6.0 using Vivaspin 6 columns (VivaScience) and Tween® 20 is added to a final concentration of 0.01%.

PEGylation using the MAL coupling chemistry

In another method, PEGylation is achieved using MAL coupling chemistry. In this method, cysteine-containing protein variants are suspended in 5 mM sodium succinate, 4% mannitol, 0.01% Tween® 20, pH 6.0 and reduced by incubation in 20 mM DTT for 30 min. at room temperature. The protein is then desalted on NAP25 gel filtration columns (Pharmacia) in buffer A (50 mM sodium phosphate, 1 mM EDTA, pH 8.1). mPEG-MAL (mPEG-maleimide, Shearwater Corp.) is dissolved in buffer A to a concentration of 0.5 mg/ml and added in equal volume to reduced and desalted hIL-10 variant and incubated for 120 min. with gentle shaking at room temperature. Ammonium sulphate is added to a concentration of 0.9 M and the PEGylated protein is applied onto a Resource™ phenyl column (Pharmacia) equilibrated in buffer B (20 mM sodium phosphate, 0.9 M ammonium sulphate, pH 6.6). The column is washed with 5 column volumes of buffer B and the bound PEGylated protein is eluted in a linear gradient from 0-50% buffer C (20 mM sodium phosphate, pH 6.6) over 30 column volumes. Eluted PEGylated protein is diafiltered into 5 mM sodium succinate, 4% mannitol, pH 6.0 using Vivaspin 6 columns (VivaScience) and Tween® 20 is added to a final concentration of 0.01%.

EXAMPLE 8 Identification and quantification of non-conjugated and conjugated ML-IO and variants thereof

SDS-Polyacryl Amide Gel Electrophoresis

The purified, concentrated IL-10 is analyzed by SDS-PAGE. A single band having an apparent molecular weight of approximately 18.7 kDa should predominate for non-conjugated hIL-10. Additional larger bands should predominate in the case of PEGylated or glycosylated IL-10. The presence of a single larger band suggests a uniform population of PEGylated or glycosylated protein. Multiple bands suggest incomplete conjugation or a mixed population of PEGylated or glycosylated proteins.

Absorbance

An estimate of the IL-10 protein concentration is determined by measuring the absorbance of the protein at 280 run. The theoretically calculated extinction coefficient of IL-10 is 0.35, this can be utilized to estimate the concentration (Gill and von Hippel (1989) Anal. Biochem. 182:319-326).

Amino Acid Analysis

A more accurate protein determination can be obtained by amino acid analysis. Amino acid analysis performed on a purified IL-10 can show that the experimentally determined amino acid composition is in agreement with the expected amino acid composition based on the DNA sequence. EXAMPLE 9

In vitro immunosupressive biological activity of non-conjugated and conjugated ML- 10 and variants thereof

Immunosupressive activity of IL-10 and variants of IL-10 is measured as a function of inhibition of interferon gamma (IFN-γ) or TNF-α production in human peripheral blood mono- nuclear cells (PBMC). hIL-10 suppresses IFN-γ and TNF-α synthesis in activated human PBMC but does not alter proliferation. PBMC is purified from buffy coat preparations from healthy donors. Cells are activated using phytohaemaglutinin (PHA), IL-2 or LPS. After activation hIL-10 or hIL-10 variants are added at various concentrations for 24-48 h. IFN-γ and TNF-α secretion into the cell culture supernatant can be measured using a two-antibody capture ELISA (such as sold by R&D Systems). Proliferation of PBMC can be measured by the incorporation of 3H-thymidine.

Different IL-10 variants are expected to have differed abilities to inhibit IFN-γ and TNF-α production. The concentration of IL-10 needed to reduce IFN-γ and TNF-α production to one half the maximum amount can be determined (IC50).

EXAMPLE 10

In vitro immunostimulatory biological activity of non-conjugated and conjugated ML- 10 and variants thereof Immunostimulatory activity of hIL-10 can be measured as the proliferative effect on the murine mast cell line MC/9 (ATCC #CRL-8306). MC/9 cells are cultured according to ATCC specifications. For measurement of immunostimulatory activity, cells are incubated overnight in fresh media. Cells are incubated in individual wells in the presence of various concentrations of hIL-10 or hIL-10 variants for 24-48 h. The protein can be present in a com- plex mixture such as in CHO supematants or as purified protein. 3H-thymidine is added to the cells and allowed to incorporate into dividing cells for 6-12 h, after which time the cells are harvested and incorporated cpm is measured.

Alternatively, immunostimulation in the form of proliferation can be measured on thymocytes purified and cultured from mice. EXAMPLE 11

Receptor-binding affinity of non-conjugated and conjugated ML- 10 and variants thereof

The affinity of hIL-10 and hIL-10 variants for IL-10R1 can be measured e.g. by displacement of 125I-labeled hIL-10 from cells expressing IL-10R1 and/or IL-10R2. These cells can be a cell line, such as MC/9, or cultured primary cells, such as PBMC. Alternatively, cells, such as BaF/3, can be genetically modified to express one or more of the receptors. Affinity can also be measured using BIAcore® (BIAcore Pharmacia Biosensor, Uppsala, Sweden). This biosensor system relies on surface plasmon resonance, which uses polarized light to detect changes in optical resonance that occur when molecules associate or dissociate from one an- other. In this method, IL-10R1 or IL-10R2 is covalently attached to a sensor chip composed of a carboxylated dextran layer linked to a gold film coated on a glass slide and the IL-10 ligand flows over the chip. Measurement of the changes in optical resonance allows for precise determination of affinity, including off- and on-rate. Additional methods for binding measurements include ELISA methods and also FACS. It may be useful to assess binding not only to IL-10R1, but also to IL-10R2, the complex of IL-10R1 and I1-10R2, and/or IL-22R.

EXAMPLE 12

Generation of variants containing two or conjugated PEG groups

In order to create a longer half-life IL- 10 protein, the molecule should be of a large enough molecular weight to reduce both receptor mediated and renal clearance. In order to achieve a sufficient increase in apparent molecular weight, it may be advantageous to have 2 or more PEG groups, each having a molecular weight of e.g. about 5000, attached to each IL- 10 monomer. Once positions are identified that are suitable for PEGylation (that is, they retain the desired amount of activity), variants are created that combine useful cysteine substitutions and PEGylation is carried out on those variants. It is anticipated that the desired number of PEG 5000 groups per monomer will be 1-3, e.g. 2 or 3.

EXAMPLE 13 Selection of PEGylated hIL-10 variants with differing affinities for IL-10R1 An IL-10 variant with reduced receptor-binding affinity that retains immunosuppressive activity may be desirable. PEGylated hIL-10 variants with different affinities for IL- 10R1 are identified and selected, e.g. by determining receptor-binding affinity as described above. To determine the optimal affinity, molecules with a range of affinities towards IL-10R1 (e.g. 10-100% of rhIL-10) may be tested in animal models of disease.

EXAMPLE 14

In vivo half-life of non-conjugated and conjugated hIL-10 and variants thereof

An important aspect of the invention is the prolonged biological half-life that can be obtained by PEGylation, and optionally also glycosylation, of hIL-10. The half-life is measured as follows. Healthy mice (e.g. 5 per treatment) are injected intravenously via the tail vein with 10-1000 μg/kg of conjugated and non-conjugated hIL-10. At 1 and 30 min., 1, 2, 4, 6, 24 and 48 h after the injection, mice are sacrificed and blood is collected. The blood samples can be stored at room temperature for 1.5 h followed by isolation of serum by centrifugation (4° C, 18000 x g for 5 min.). The serum samples can be stored at -80°C until analysis. The amount of IL-10 in the serum samples is quantified by an IL-10 ELISA (R&D Systems). Additionally, IL- 10 activity may be measured using the in vitro assays described above.

EXAMPLE 15 In vivo biological activity in a marine model ofCrohn 's disease

Specific IL-10 variants with reduced immunostimulatory properties, longer half- lives and altered renal and receptor clearance modalities are tested for efficacy in vivo using one or more established murine models of Crohn's disease. In one model, colitis, which resembles inflammatory bowel disease or Crohn's disease, is induced in mice by injection of 2,4,6- trinitrobenzene sulfonic acid (TNBS) (Kanai et al, Gasxroenterology (2001) 121 :875-888 and Duchmann et al, Eur. J. Immunology (1996) 26:934-938). In another model, the SAMP/Yit mouse strain is used. This strain spontaneously develops chronic intestinal inflammation with a resemblance to human Crohn's disease (Kosiewicz et al, J. Clin. Invest. (2001) 107:695-702). In a third model, 6-12 weeks after CD45RBhi CD4 T-cells from wild type mice are purified and injected into SCID mice inflammation of the large intestine, cecum, colon and rectum develops (Powrie et al., Int. Immunol. (1993) 5:1461-1471). Once chronic intestinal inflammation is established in the mice, purified IL-10 variants are given. Injections of IL-10 variants are given either IV or SC. Injections are given 1-2 x weekly for up to 8 weeks at dosages up to 200 μg/kg-

The mice are sacrificed and examined histologically to assess the extent of inflammatory disease. FACS is used to analyze inflammatory and TH1/TH2 markers on cell populations. Levels of cytokines and other molecules whose levels are modulated by IL-10 are analyzed in serum, cultured whole blood, activated PBMC and/or other relevant cellular populations. Cytokines modulated by IL-10 include, but are not limited to, IFN-γ and TNF-α. Efficacy is measured as a decrease in inflammatory index and/or a reduction in TH2 cytokines.

EXAMPLE 16

In vivo biological activity in a murine model of psoriasis

Specific IL-10 variants with reduced immunostimulatory properties, longer half- lives and altered renal and receptor clearance modalities are tested for efficacy in vivo using an established murine model of psoriasis. In one model, human psoriatic plaque ketatome skin samples are orthotropically transplanted onto severe combined immunodeficiency (SCID) mice (Nickoloff et al. (1995) Amer. J. Pathol. 146:580-588). In another model, non-lesional skin is transplanted onto SCID mice and psoriasis is induced by injecting SP activated lymphomono- nuclear cells or autologous immunocytes activated with nerve growth factor (NGF) (Ray- chaudhuri, et al. (2001) British J. Derm. 144:931-939). Once psoriasis is established in the mice (2-4 weeks after transplantation), purified IL-10 variants are given to the mice. Injections of IL-10 variants are given either IV, SC or directly into the psoriatic plaques. Injections are given 1-2 x weekly for up to 8 weeks at dosages up to 200 μg/kg. Throughout the experiment, plaques are examined by light microscopy for phenotypic changes. At various points during the experiment, mice are sacrificed and punch biopsies (4 mm) are taken from each xenograph. The xenographs are examined histologically and for psoriasis markers within the plaques.

These markers may include, but are not limited to: CD4 + and CD8+ T cells expressing natural killer receptors, and specific cytokines (IFN-γ, TNF-α, IL-8 and NGF). Levels of cytokines and other molecules whose levels are modulated by IL-10 can be analyzed in serum. Efficacy is assessed by the reduction in immunostimulatory cytokines as well as histological disease reduction.

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclo- sure that various changes in form and detail can be made without departing from the true scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. For example, all the techniques and apparatus described above may be used in various combinations. All publications, patents, patent applications, and/or other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and/or other document were individually indicated to be incorporated herein by reference in its entirety for all purposes.

Claims

1. A polypeptide conjugate, comprising (a) a polypeptide exhibiting IL-10 activity comprising an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO:2 in 1-15 amino acid residues and comprising at least one introduced cysteine residue, and (b) at least one polymer moiety attached to a cysteine residue of the polypeptide.
2. The polypeptide conjugate of claim 1 , wherein at least one cysteine residue has been introduced in a position selected from the group consisting of: SI, P2, G3, Q4, G5, T6,
Q7, S8, E9, N10, Sll, T13, H14, P16, G17, N18, P20, N21, R24, D25, R27, D28, S31, R32, K34, T35, F36, Q38, M39, K40, Q42, L43, D44, N45, L46, K49, E50, S51, E54, K57, G58, Y59, L60, Q70, E74, E75, P78, Q79,N82, D84, P85, D86, K88, A89, H90, N92, S93, E96, K99, TlOO, L103, R104, R106, R107, H109, Rl10, Nl16, Kl17, Kl19, E122, Q123, K125, N126, A127, N129, K130, Q132, E133, K134, D144, 1145, N148, E151, A152, T155, M156, 1158, R159 andN160
3. The polypeptide conjugate of claim 1 , wherein at least one cysteine residue has been introduced in a position selected from the group consisting of: SI, P2, G3, Q4, G5, T6, Q7, S8, E9, 10, Sl l, T13, H14, P16, G17, 18, N21, D25, S31, R32, F36, K40, L43,
K49, E50, S51, E54, K57, G58, Y59, L60, Q70, E74, E75, P78, Q79, N82, D84, P85, D86, K88, A89, H90, N92, S93, E96, K99, TlOO, L103, R104, R106, R107, H109, Rl 10, Nl 16, Kl 17, Kl 19, E122, Q123, K125, N126, A127, N129, K130, Q132, E133, K134, D144, A152, M156 and N160.
4. The polypeptide conjugate of claim 1 , wherein at least one cysteine residue has been introduced in a position selected from the group consisting of: SI, P2, G3, Q4, G5, T6, Q7, S8, E9, N10, Sl l, T13, H14, P16, G17, N18, P20, N21, R24, D25, D28, S31, R32, K34, T35, M39, Q42, D44, N45, K49, E50, E54, K57, G58, Y59, E74, N82, P85, D86, K88, A89, H90, N92, E96, K99, TlOO, L103, Rl lO, K117, K119, Q123, N126, N129,
K130, Q132, E133, K134, N148, T155, R159 and N160.
5. The polypeptide conjugate of claim 1 , wherein at least one cysteine residue has been introduced in a position selected from the group consisting of: SI, P2, G3, Q4, G5, T6, Q7, S8, E9, N10, Sll, T13, H14, P16, G17, N18, N21, D25, S31, R32, K49, E50, E54, K57, G58, Y59, E74, N82, P85, D86, K88, A89, H90, N92, E96, K99, TlOO, LI 03, Rl 10, Kl 17, Kl 19, Q123, N126, N129, K130, Q132, E133, K134 and N160.
6. The polypeptide conjugate of claim 1 , wherein at least one cysteine residue has been introduced in a position selected from the group consisting of: N18, P20, N21, R24, D25, R27, D28, S31, R32, K34, T35, F36, Q38, M39, K40, K49, E50, S51, E54, K57, G58, L60, Q70, E74, E75, P78, Q79, N82, K88, A89, H90, N92, S93, E96, K99, TlOO,
L103, R104, R106, R107, Kl 19, E122, Q123, K125, N126, A127, N129, K130, E133, K134, D144, 1145, N148, E151, A152 T155, M156, 1158 and R159.
7. The polypeptide conjugate of claim 1 , wherein at least one cysteine residue has been introduced in a position selected from the group consisting of: N18, N21, D25, S31,
R32, F36, K40, K49, E50, S51, E54, K57, G58, L60, Q70, E74, E75, P78, Q79, N82, K88, A89, H90, N92, S93, E96, K99, TlOO, L103, R104, R106, R107, K119, E122, Q123, K125, N126, A127, N129, K130, E133, K134, D144, A152 and M156.
8. The polypeptide conjugate of claim 1 , wherein at least one cysteine residue has been introduced in a position selected from the group consisting of: N18, P20, N21, R24, D25, D28, S31, R32, K34, T35, M39, K49, E50, E54, K57, G58, E74, N82, K88, A89, H90, N92, E96, K99, TlOO, L103, R107, Kl 19, Q123, N126, N129, K130, Q132, E133, K134, N148, T155 and R159.
9. The polypeptide conjugate of claim 1 , wherein at least one cysteine residue has been introduced in aposition selected from the group consisting of: N18, N21, D25, S31, R32, K49, E50, E54, K57, G58, E74, N82, K88, A89, H90, N92, E96, K99, TlOO, L103, R107, Kl 19, Q123, N126, N129, K130, Q132, E133 and K134.
10. The polypeptide conjugate of claim 1, wherein at least one cysteine residue has been introduced in a position selected from the group consisting of: SI, P2, G3, Q4, G5, T6, Q7, S8, E9, NIO, Sl l, T13, H14, P16, G17, Q42, L43, D44, N45, L46, Y59, D84, P85, D86, H109, RllO, N116, K117, Q132 and N160.
11. The polypeptide conjugate of claim 1 , wherein at least one cysteine residue has been introduced in a position selected from the group consisting of: SI, P2, G3, Q4, G5, T6,
Q7, S8, E9, N10, Sll, T13, H14, P16, G17, L43, Y59, D84, P85, D86, H109, Rl lO, N116, K117, Q132 andN160.
12. The polypeptide conjugate of claim 1, wherein at least one cysteine residue has been introduced in a position selected from the group consisting of: SI, P2, G3, Q4, G5, T6,
Q7, S8, E9, N10, Sll, T13, H14, P16, G17, Q42, D44, N45, Y59, P85, D86, Rl lO, K117, Q132 and N160.
13. The polypeptide conjugate of claim 1 , wherein at least one cysteine residue has been introduced in a position selected from the group consisting of: SI, P2, G3, Q4, G5, T6,
Q7, S8, E9, N10, SI 1, T13, H14, P16, G17, Y59, P85, D86, Rl 10, Kl 17, Q132 and N160.
14. The polypeptide conjugate of claim 1, wherein at least one cysteine residue has been introduced in a position selected from the group consisting of: P20, N21, M22, L23,
R24, D25, L26, R27, D28, A29, F30, K34, T35, F36, F37, Q38, M39, K40, S51, L52, L53, E54, D55, F56, L60, G61, Q63, A64, L65, S66, E67, M68, 169, Q70, F71, Y72, L73, E74, E75, V76, M77, P78, Q79, A80, E81, N82, A89, H90, V91, N92, S93, L94, G95, E96, N97, L98, K99, TlOO, LI 01, R102, L103, R104, L105, R106, Kl 19, A120, V121, E122, Q123, V124, K125, N126, A127, F128, Q132, E133, K134, G135, 1136,
Y137, K138, A139, F143, D144, 1145, F146, 1147, N148, Y149, 1150, E151, A152, Y153, M154 and T155.
15. The polypeptide conjugate of claim 1, wherein at least one cysteine residue has been introduced in a position selected from the group consisting of: N21, M22, D25, L26,
A29, F30, F36, F37, K40, S51, L52, L53, E54, D55, F56, L60, G61, Q63, A64, L65, S66, E67, M68, 169, Q70, F71, Y72, L73, E74, E75, V76, M77, P78, Q79, A80, E81, N82, A89, H90, V91, N92, S93, L94, G95, E96, N97, L98, K99, TlOO, L101, R102, L103, R104, L105, R106, K119, A120, V121, E122, Q123, V124, K125, N126, A127, F128, Q132, E133, K134, G135, 1136, Y137, A139, F143, F146, 1147, 1150, A152, Yl 53 and Ml 54.
16. The polypeptide conjugate of claim 1, wherein at least one cysteine residue has been introduced in a position selected from the group consisting of: N21, D25, F36, K40, S51, E54, L60, Q70, E74, E75, P78, Q79, N82, A89, H90, N92, S93, E96, K99, TlOO, L103, R104, R106, Kl 19, E122, Q123, K125, N126, A127, Q132, E133, K134 and A152.
17. The polypeptide conjugate of any of the preceding claims, wherein the at least one introduced cysteine residue is introduced by substitution.
18. The polypeptide conjugate of any of the preceding claims, which exhibits immunosup- pressive activity on T cells, B cells or antigen presenting cells.
19. The polypeptide conjugate of any of the preceding claims, which exhibits a reduced immunostimulatory activity compared to hIL-10, e.g. measured as the proliferative effect on a murine mast cell line MC/9 (ATCC #CRL-8306).
20. The polypeptide conjugate of any of the preceding claims, wherein the polymer moiety is selected from the group consisting of a polyalkylene glycol, a polyvinylalcohol (PVA), a poly-carboxylic acid and a poly-(vinylpyrrolidone).
21. The polypeptide conjugate of any of the preceding claims, wherein the polymer is a polyethylene glycol selected from a linear polyethylene glycol and a branched polyethylene glycol.
22. The polypeptide conjugate of any of the preceding claims, comprising 1-4 polymer moieties each attached to a cysteine residue, e.g. 1-3 or 1-2 polymer moieties.
23. The polypeptide conjugate of any of the preceding claims, wherein each attached polymer moiety has a molecular weight of about 1-20 kDa, typically about 2-15 kDa, such as about 3-12 kDa or 4-10 kDa.
24. The polypeptide conjugate of claim 23, comprising one, two or three attached PEG moieties with a molecular weight of about 5 kDa.
25. The polypeptide conjugate of any of the preceding claims, further comprising at least one introduced glycosylation site.
26. The polypeptide conjugate of any of the preceding claims, having an in vitro activity in the range of about 0.1-50% of the activity of human IL-10.
27. A polypeptide exhibiting IL-10 activity, the polypeptide having an amino acid sequence as defined in any of claims 1-17.
28. A nucleotide sequence which encodes the polypeptide of claim 27.
29. An expression vector comprising the nucleotide sequence of claim 28.
30. A host cell comprising the expression vector of claim 28 or the nucleotide sequence of claim 29.
31. A composition comprising the polypeptide conjugate of any of claims 1 -26 or the poly- peptide of claim 27 and at least one pharmaceutically acceptable carrier or excipient.
32. A method for preventing or treating an inflammatory or autoimmune disease, comprising administering to a patient in need thereof the polypeptide conjugate of any of claims 1-26, the polypeptide of claim 27 or the composition of claim 31.
33. The method of claim 32, wherein the disease is selected from the group consisting of psoriasis, Crohn's disease, ulcerative colitis, inflammatory bowel disease and rheumatoid arthritis.
34. Use of the polypeptide conjugate of any of claims 1 -26 or the polypeptide of claim 27 as a medicament.
35. Use of the polypeptide conjugate of any of claims 1 -26 or the polypeptide of claim 27 for preparing a pharmaceutical composition for the prevention or treatment of an inflammatory or autoimmune disease.
36. Use according to claim 35, wherein the disease is selected from the group consisting of psoriasis, Crohn's disease, ulcerative colitis, inflammatory bowel disease and rheumatoid arthritis.
PCT/DK2003/000774 2002-11-14 2003-11-11 Conjugates of interleukin-10 and polymers WO2004044006A1 (en)

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