WO2012045334A1

WO2012045334A1 - Biologically active il-10 fusion proteins

Info

Publication number: WO2012045334A1
Application number: PCT/EP2010/064856
Authority: WO
Inventors: Frans Van Dalen; Ruud Hendrikus Petrus Wilbers; Lotte Bregje Westerhof; Arjen Schots
Original assignee: Synthon Bv
Priority date: 2010-10-05
Filing date: 2010-10-05
Publication date: 2012-04-12

Abstract

The present invention relates to a biologically active interleukin-10 fusion protein, in which two IL-10 monomers, or variants thereof, are respectively fused to the C-terminus of two immunoglobulin Fc parts. The Fc parts associate with one another, which enables the two IL-10 monomers (or variants thereof) to act as a dimer having anti-inflammatory activity. The present invention further relates to a method of expressing the fusion protein in plant cells (such as tobacco cells) by introducing an amino acid spacer into the interdomain region of at least one of the IL-10 monomers. The amino acid spacer enables reduced aggregation of the fusion protein, as compared to expression of the IL-10 dimer, in the plant cells.

Description

BIOLOGICALLY ACTIVE IL-10 FUSION PROTEINS

FIELD OF THE INVENTION

The present invention relates to a biologically active interleukin-10 (IL-10) fusion protein having anti-inflammatory activity, methods of expressing such fusion proteins, pharmaceutical compositions containing such fusion proteins, and methods of using such fusion proteins.

BACKGROUND OF THE INVENTION

Cytokines are the soluble mediators of innate and adaptive immunity and constitute the mechanisms by which leukocytes communicate with each other and other cells. Cytokines are produced in response to microbes, other antigens and various direct and indirect stimuli and stimulate diverse reactions involved in immunity. The biological functions of cytokines include T-cell activation, proliferation and cytotoxicity, augmentation of neutrophils & macrophages and the promotion of growth and differentiation of B-cells and multilineage bone marrow stem-cell precursors. Mosmann and coworkers (D.F. Fiorentino et al., J. Exp. Med. 1989, 170(6), 2081- 95) were the first to describe a cytokine that was produced by T helper 2 (Th2) cell clones and inhibited interferon-γ (IFN-γ) synthesis by Thl cell clones. This so-called "cytokine synthesis inhibiting factor" is now better known as interleukin-10 (IL-10). The last couple of years, more and more knowledge has become available about IL-10 expression and function, which also enables a better understanding of the pathogenesis of several diseases. Anti-inflammatory interleukin-10 is a master immuno modulating cytokine with great therapeutic potential for several autoimmune and inflammatory diseases. For an overview of structure, activity and function of IL-10 see "Interleukin-10", F.M. Marincola ed., Landes Bioscience USA 2006, ISBN 1-58706-285-2.

Human IL-10 is a non-covalently linked homodimeric cytokine with a molecular mass of approximately 37 kDa. IL-10 is a domain swapped dimer, the structural integrity of which depends on the intertwining of two peptide chains - one from each monomer (Josephson et al, JBC Vol. 275, No. 18, 2000, pp. 13552-13557). Each monomer consists of 160 amino acids (-18.5 kDa). The monomer shows a substantially reduced activity in murine mast cell proliferation (MC/9) bioassays (R. Syto et al, Biochemistry, 1998, 37, 16943-16951 and R. Menassa et al, J. Biotech., 2004, 108, 179-183).

The interleukin-10 receptor (IL-10R) by which IL-10 exerts its activity is mainly expressed by cells of the immune system, but also by other cell populations like fibroblasts, however only a few copies of the receptor are found on the surface of those cells. IL-10R is a heterodimeric receptor, composed of two different chains, IL-10R1 and IL-10R2. Both receptor chains are very similar and have an L-shaped structure. IL-10R1 is supposed to be the ligand-binding subunit and IL-10R2 is in principal involved in signal transduction. Cell signaling can only occur if both receptors bind to IL-10 (See, e.g., Josephson et al, JBC Vol. 275, No. 18, 2000, pp. 13553, left column). A model for IL-10/IL-10R interaction has been proposed by Josephson et al.

(Immunity, Vol. 14, 35-46, July, 2001). In solution, IL-10 and soluble IL-10R1 (sIL-lORl) form a complex that contains two IL-10 dimers (parallel to each other) and four sIL-lORl molecules. It was then suggested that two IL-10R1 's could be replaced by two IL-10R2 chains, because both receptors could share the same binding site. In 2005, Pletnev et al. (BMC Struct. Biol.

2005;5:10.) revealed an alternative binding site for IL-10R2 by homology modeling. The intermediate IL-10/IL-10R1 complex is the same, but reveals two binding sites for IL-10R2, involving both IL-10 and IL-10R1.

The biological effects of IL-10 are pleiotropic and act primarily on antigen presenting cells and lymphocytes, furthermore both innate and adaptive immune responses are influenced. IL-10 inhibits the production of IL-12 by activated macrophages and dendritic cells and in turn inhibits IFN-γ production by lymphocytes. IFN-γ acts as an inducer of both innate and cell-mediated immunity by activating macrophages and promoting the development of Thl cells. Thus, inhibition of IL-12 and IFN-γ by IL-10 promotes the development of a type 2 cytokine response. IL-10 also inhibits the expression of costimulatory and class II MHC molecules on macrophages and dendritic cells, thereby inhibiting T cell activation and cell-mediated immune responses. In contrast to this, proliferation and differentiation of B-cells is induced by IL-10, promoting a humoral immune response. Furthermore, IL-10 is involved in isotype switching to IgA antibodies together with TGF-β. IL-10 is an anti-inflammatory cytokine that inhibits the expression of proinflammatory cytokines. Like IL-1, IL-6, IL-8, IL-12 and TNF-a, IL-10 inhibits the production of chemokines that are involved in inflammation and has inhibitory effects on the production of reactive oxygen species (ROS) and nitric oxide (NO). However, IL-10 not only inhibits the production of proinflammatory molecules, but IL-10 also induces the production of anti-inflammatory molecules, such as the IL-1 receptor antagonist (IL-1RA) and soluble TNF receptors.

The crucial role of IL-10 in immunoregulation has led to its use in clinical trials for the treatment of several autoimmune and inflammatory diseases. So far, recombinant human IL-10 (rhIL-10) has been evaluated in the treatment of Crohn's disease, rheumatoid arthritis, psoriasis, hepatitis C & HIV infection and for the inhibition of therapy associated cytokine release in organ transplantation and Jarisch-Herxheimer reaction (an acute systemic inflammatory response). In phase I clinical trials, safety, tolerance, pharmacokinetics, pharmacodynamics, immunological and hematological effects of single or multiple doses of IL-10 administered by intravenous or subcutaneous injection have been investigated on healthy volunteers. These studies showed that IL-10 is well tolerated without serious effects at doses of up to 25 μg/kg. However, to date clinical trials with IL-10 have provided disappointing results. Problems in the clinical use of IL- 10 mainly result from low stability (short half- life) and lack of suitable targeting to disease sites.

Recombinant human IL-10 has been produced using various expression systems, e.g., rice seed (Fujiwara et al., Protein Expression and Purification 72 (2010) 125-130), CHO cells (Syton et al, Biochemistry 1998, 37, 16943-16951), E. coli (Josephson et al, JBC Vol. 275, No. 18, 2000, pp. 13552-13557), and non-food plants such as tobacco (WO9967401).

SUMMARY OF THE INVENTION

An aspect of the invention relates to a fusion protein comprising a first IL-10 monomer fused to the C-terminus of a first immunoglobulin Fc part, and a second IL-10 monomer fused to the C-terminus of a second immunoglobulin Fc part, wherein the first and second

immunoglobulin Fc parts are bound together.

The first and second Fc parts can be bound together by covalent disulfide bonds and/or non-covalent interactions. The N-terminus of at least one of the first and second IL-10 monomers can be fused to the C-terminus of the corresponding immunoglobulin Fc part by a peptide linker or a peptide bond. The first and second IL-10 monomers can be human IL-10 monomers.

At least one of the IL-10 monomers can contain an amino acid spacer positioned such that the amino acid spacer prevents domain swapping between the first and second IL-10 monomers. The amino acid spacer can be a peptide comprising 2-10 amino acids, such as 4-8 amino acids, and preferably is an a 6-amino acid linker consisting of Gly-Gly-Gly-Ser-Gly-Gly, and the amino acid spacer can be located between helices D and E of the interdomain region.

At least one of the first and second immunoglobulin Fc parts can be a human

immunoglobulin Fc part, such as a human IgA or human IgG Fc part. The human IgA Fc parts can be Ser245-Tyr479 of a human IgA antibody. The first and second immunoglobulin Fc parts can be the constant heavy chain domains of an intact antibody. The first and second

immunoglobulin parts can be identical.

The fusion protein can decrease TNFa expression by lipopolysaccharide-stimulated macrophages. The fusion protein can also induce proliferation of MC/9 murine mast cells.

Another aspect of the invention relates to a method for the recombinant production an antiinflammatory fusion protein, comprising (i) expressing in plant cells, preferably tobacco cells such as Nicotiana benthamiana cells, a fusion protein comprising a first IL-10 monomer fused to the C-terminus of a first immunoglobulin Fc part and a second IL-10 monomer fused to the C- terminus of a second immunoglobulin Fc part, wherein the first and second immunoglobulin Fc parts are bound together, at least one of the IL-10 monomers is modified to prevent the at least one IL-10 monomer from dimerizing with another IL-10 monomer, and the fusion protein decreases TNFa expression by lipopolysaccharide-stimulated macrophages, and (ii) optionally extracting said fusion protein from said plant cells, as well as fusion proteins obtainable by this method.

Yet another aspect of the invention relates to a method for the recombinant production an anti-inflammatory fusion protein, comprising (i) expressing in plant cells, preferably tobacco cells such as Nicotiana benthamiana cells, a fusion protein comprising a first IL-10 monomer fused to the C-terminus of a first immunoglobulin Fc part and a second IL-10 monomer fused to the C-terminus of a second immunoglobulin Fc part, wherein the first and second

immunoglobulin Fc parts are bound together, and the fusion protein decreases TNFa expression by lipopolysaccharide-stimulated macrophages, and (ii) optionally extracting said fusion protein from said plant cells, as well as fusion proteins obtainable by this method.

Still another aspect of the invention relates to a pharmaceutical composition comprising a fusion protein according to the invention.

A further aspect of the invention relates to a fusion protein or pharmaceutical invention according to the invention for use in medicine, preferably for use in the treatment of Multiple Sclerosis or an inflammatory condition in a human.

A still further aspect of the invention relates to an expression vector comprising DNA encoding a fusion protein according to the invention, and also to a plant cell, preferably a tobacco cell such as a Nicotiana benthamiana cell, comprising the expression vector.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the structures of native human IL-10 monomeric unit (1A), native human IL-10 in homodimeric form (IB), and variant IL-10 (1C).

Figure 2 shows a comparison between the amino acid sequences of native human IL-10 (2A, hIL-10), mouse IL-10 (2B, mIL-10), and variant IL-10 (2C, vIL-10).

Figure 3 shows the levels of extractable native human IL-10 (hIL-10) and variant IL-10 (vIL-10) transiently expressed in tobacco at various times post-transfection. Figure 4A shows the inhibitory effect of commercially obtained purified recombinant human IL-10 (rhuIL-10), plant expressed human IL-10 (hIL-10) and plant expressed variant IL- 10 on the TNF-a expression of LPS stimulated RAW264.7 macrophages. This shows that the variant IL-10 which preferably forms a monomer has no significant anti-inflammatory effect in this test. Figure 4B shows the activity of the human IL-10 and variant IL-10 fused to the C- terminal end of Fc-a (Fca-hIL-10, resp. Fca-vIL-10) produced in planta compared to human IL- 10 (hIL-10) in the same test. This shows that fusion of variant IL-10 to the C-terminal end of Fca substantially restores anti-inflammatory activity.

Figure 5 shows the structure of the Fca IL-10 fusion constructs: Native human IL-10 fused to N-terminus of human Fca (5 A; hIL- 10-Fca), variant IL- 10 fused to the N-terminus of Fca

(5B; vIL-10-Fca), native human IL-10 fused to C-terminus of Fca (5C; Fca-hIL-10), and variant IL-10 fused to the C-terminus of Fca (5D; Fca-vIL-10).

Figure 6 shows non-reduced and reduced Western blots of the various fusion constructs as shown in figure 5 and variant IL-10 transiently expressed in tobacco.

Figure 7 shows levels of extractable Fca-hIL-10 and Fca-vIL-10 fusion constructs compared to vIL-10 transiently expressed in tobacco at two days after transfection.

Figure 8 shows the vectors used in transformation. 8 A shows the pRAP35 vector, 8B shows pHYG Vector.

Figure 9 shows a summary of results obtained with a fusion protein according to an embodiment of the invention in Nicotiana benthamiana.

Figure 10 shows the effects of hIL-10, vIL-10 and the Fca-vIL-10 fusion protein in a murine mast cell proliferation (MC/9) bioassay. This shows comparable activity of vIL-10 and Fca-vIL-10 fusion protein in this test. vIL-10 and Fca-vIL-10 require higher concentration levels in this test than hIL-10. For each of the four fusion proteins in Figure 10, the left bar is 25ng/ml and the right bar is 3ng/ml.

BRIEF DESCRIPTION OF THE SEQUENCES

Seq. Id. No.: l aa sequence of hIL-10 preceded by an 18 aa signal peptide. Seq. Id. No. :2 aa sequence of vIL-10 preceded by an 18 aa signal peptide.

Seq. Id. No. :3 aa sequence of Fca-hIL-10 preceded by an 19 aa signal peptide.

Seq. Id. No. :4 aa sequence of Fca-vIL-10 preceded by an 19 aa signal peptide.

Seq. Id. No. :5 aa sequence of hIL-10-Fca preceded by an 20 aa signal peptide.

Seq. Id. No. :6 aa sequence of vIL-10-Fca preceded by an 20 aa signal peptide.

Seq. Id. No. :7 nt sequence of hIL-10.

Seq. Id. No. :8 nt sequence of vIL-10.

Seq. Id. No. :9 nt sequence of Fca-hIL-10.

Seq. Id. No. :10 nt sequence of Fca-vIL-10.

Seq. Id. No. :11 nt sequence of hIL-10-Fca.

Seq. Id. No. :12 nt sequence of vIL-10-Fca.

Seq. Id. No. :13 nt sequence of transformation vector pRAP35.

Seq. Id. No. : 14 nt sequence of transformation vector pHYG.

DETAILED DESCRIPTION OF THE INVENTION

Although the human IL-10 monomer has been shown to have some biological activity, namely the ability to induce proliferation in a MC/9 cell line (Josephson et al, JBC Vol. 275, No. 18, 2000, pp. 13552-13557), the human IL-10 monomer does not have anti-inflammatory activity. This is in contrast to the human IL-10 dimer, which is known to have anti-inflammatory activity. Unfortunately, the human IL-10 dimer aggregates in planta, making it difficult to extract an anti-inflammatory IL-10 compound from a plant expression system.

It has been discovered, however, that fusion proteins containing IL-10 monomers fused to the C-terminus of two immunoglobulin heavy chain constant domains bound together restores anti-inflammatory activity, despite the fact that the fusion protein is not a native human IL-10 dimer and in contrast to human IL-10 monomers which lack anti-inflammatory activity. It has also been discovered that fusion proteins in which the interdomain region of at least one of the IL-10 monomers contains an amino acid spacer to prevent native dimerization can be expressed in a plant expression system without the aggregation observed with native IL-10 dimer expression.

Compounds

Accordingly, the present invention relates to fusion proteins comprising IL-10 monomers fused to the C-terminus of corresponding immunoglobulin Fc parts, in which the

immunoglobulin Fc parts are bound together. The fusion proteins of the present invention have anti-inflammatory activity, e.g., the fusion proteins can decrease TNFa expression by

lipopolysaccharide-stimulated macrophages, despite the fact that the fusion protein is not a native IL- 10 dimer.

The term "native IL-10 dimer" means the IL-10 domain-swapped dimer in which four helices from one monomer (A-D) associate/intertwine with two helices of the other monomer (E and F). See, e.g., Josephson et al, JSC Vol. 275, No. 18, 2000, pp. 13552, right column. The term "IL-10" is used interchangeably with the term "native IL-10 dimer," unless otherwise stated.

The term "IL-10 monomer" means a monomer subunit of the native IL-10 dimer. The helices of the IL-10 monomer are not associated/intertwined with a second IL-10 monomer. The IL-10 monomer can be a naturally-occurring or recombinant IL-10 monomer or a modified versions thereof. For example, the IL-10 monomer can contain amino acid additions, deletions, and substitutions that do not interfere with the monomer's function. In an embodiment, the IL-10 monomers contain an amino acid spacer, in which case the IL-10 monomer is referred to as a "variant IL-10 monomer," a "vIL-10 monomer," a "modified IL-10 monomer," and a "modIL-10 monomer." These terms are used interchangeably to refer to the spacer-containing IL-10 monomers. For the purposes of this application, "vIL-10 monomers" refers only to variant IL-10 monomers and not to viral IL-10 monomers (viral IL-10 monomers will contain an abbreviation more clearly identifying the originating virus, such as cmv for cytomegalovirus and ebv for Epstein-Barr virus). Preferably, the IL-10 monomers (naturally-occurring or modified versions thereof) are human IL-10 monomers, which are also referred to as hIL-10 monomers.

Alternatively, the IL-10 monomers can be of non-human origin provided that the non-human IL- 10 monomers shows biological activity (e.g., lack of anti-inflammatory activity; ability to induce cell proliferation) comparable to human IL-10 monomers. For example, suitable non-human IL- 10 monomers include viral IL-10 analogues, such as produced by Epstein-Barr virus (ebvIL-10) and Human cytomegalovirus (cmvIL-10).

The term "immunoglobulin Fc part" refers means at least one heavy chain constant domain of an antibody, such as the CH4 (only present in IgM), CH3, CH2, or CHI heavy chain domain of an antibody. Preferably, the first and second Fc parts are identical. In embodiments, the "immunoglobulin Fc part" can be the CH3-CH2 heavy chain constant domains of an antibody. In other embodiments, the "immunoglobulin Fc part" can be the heavy chain constant domains of an intact antibody. In these other embodiments, the fusion protein is an antibody having IL-10 monomers fused to the C-terminus of each heavy chain constant domain, e.g., to the C-terminus of each CH3 domain. The Fc parts can be derived from a human immunoglobulin heavy chain of any isotype such as IgA, IgE, IgD, IgG, and IgM. Included in these are all subclasses (IgAl, IgA2, IgGl, IgG2, IgG3, IgG4) and allotypes. Preferably, the immunoglobulin Fc parts are heavy chain constant domains of a human antibody, such as a human IgG or human IgA antibody. More preferably, the immunoglobulin Fc parts are heavy chain constant domains of a human IgA. When the fusion protein is an antibody having IL-10 monomers fused to the C- terminus of each heavy chain constant domain, preferably the antibody is a human antibody, such as an IgG or IgA antibody, and more preferably the antibody is a human IgA antibody. For further details on structure, properties and sequences of human immunoglobulins see: M.-P. Lefranc and G. Lefranc, "7¾e Immunoglobulin FactsBook", Academic Press, London, 2001, ISBN 0-12-441351-X.

The phrase "immunoglobulin Fc parts are bound together" means that the two Fc parts are held together, such as by covalent disulfide bonds and/or non-covalent interactions. In embodiments, the Fc parts are bound together by a disulfide bond connecting the CH2 domains of each Fc part. In embodiments, the Fc parts are bound together in an intact antibody.

The phrase "IL-10 monomers fused to the C-terminus of immunoglobulin Fc parts" means that each IL-10 monomer is linked to the C-terminus of a corresponding Fc part. In an embodiment, the N-terminus of each IL-10 monomer is covalently linked, such as through a peptide linker or a peptide bond, to the C-terminus of a corresponding Fc part. Preferably, each N-terminus of the IL-10 monomers is fused to each C-terminus of the Fc parts, respectively, by a 19 amino acid glycine-serine linker, which provides flexibility to the fusion protein.

The phrase "amino acid spacer" means a peptide that is introduced (e.g., by recombinant DNA technology) into the peptide chain of an IL-10 monomer that substantially prevents the IL- 10 monomer from dimerizing with a second IL-10 monomer. As explained above, the native IL- 10 is a domain-swapped dimer in which helices A-D from one monomer associate/intertwine with helices E and F of the other monomer. The combination of helices A-E can therefore be referred to as the "interdomain region" of an IL-10 monomer. Thus, the amino acid spacer can be a peptide introduced into the interdomain region of an IL-10 monomer, such as between the D and E helices of an IL-10 monomer, to prevent domain swapping between the spacer-containing IL-10 monomer and a second IL-10 monomer. In embodiments, at least one IL-10 monomer of the fusion proteins of the invention contains an amino acid spacer. For example, both IL-10 monomers of the fusion protein can contain an amino acid spacer. In embodiments, the amino acid spacer is a 6-amino acid peptide consisting of Gly-Gly-Gly-Ser-Gly-Gly located between N 116 and Kl 17 of human IL- 10 (as shown in Figure 2) in at least one IL- 10 monomer, preferably in both IL-10 monomers.

In a preferred embodiment the fusion protein comprises first and second human IL-10 monomers respectively bound to first and second Fc parts, each Fc part comprising the CH3- CH2 heavy chain constant domains (Ser245-Tyr479) of a human IgA antibody. In this embodiment, the N-terminus of the first monomer is fused to the C-terminus of the first Fc part, and the N-terminus of the second monomer is likewise fused to the C-terminus of the second Fc part. The Fc parts are bound together, at least in part by a disulfide bond between the CH2 regions thereof. In another preferred embodiment, the fusion protein also contains an amino acid spacer between helices D and E of each IL-10 monomer.

Production Methods

Native human IL-10 (hIL-10) and mouse IL-10 (mIL-10) were expressed in Nicotiana benthamiana plants using a transient expression system. Agrobacterium tumefaciens mediated transformation of tobacco plants resulted in relatively low expression levels for native human IL- 10. hIL-10/Green fluorescent protein (GFP) and mIL-10-GFP fusion proteins were also expressed in tobacco. Using confocal microscopy, it was observed that hIL-10 aggregates in planta, whereas mIL-10 does not aggregate in planta. hIL-10 aggregates were found which were up to 20 um in size. It is believed that the formation of aggregates resulted in a decreased recovery of native human IL-10 when expressed in planta (see Figure 9).

In an effort to identify the cause of native human IL-10 aggregation in planta, a variant IL-

10 monomer was expressed. The variant IL-10 monomer was made by introducing a 6 amino acid glycine-serine linker in between a-helical domains D and E of human IL-10, as described by Josephson et al. (K. Josephson et al, J. Biol. Chem. 2000, 275(18), 13552-13557).

Expression of the variant IL-10 - GFP fusion protein in tobacco did not lead to significant aggregation in planta, possibly due to a decrease in intermolecular interactions (i.e., interactions between helices A-D of one IL-10 monomer with helices E and F of another IL-10 monomer). It is therefore believed that the aggregation observed in planta for native human IL-10 was due to domain swapping between multiple monomers thereof resulting in poorly soluble polymeric structures. Furthermore, comparing the accumulation levels of the native human IL-10 and the variant human IL-10 in tobacco over time, it was observed that the accumulation levels of the variant human IL-10 are approximately 10-fold higher than the accumulation levels of the native human IL-10 (Figure 3). Based on QPCR, the higher accumulation levels of the variant human IL-10 was not caused by higher mR A levels.

It was found that the variant human IL-10 was also present to some extent in dimeric form when extracted from the tobacco (see Figure 6). Nevertheless, the variant human IL-10 showed substantially reduced aggregation as compared to native human IL-10.

It has now been found that variant human IL-10 monomer is not able to reduce TNF-a expression in RAW264.7 macrophages after LPS stimulation, indicating that variant IL-10 monomer does not have anti-inflammatory properties (Figure 4).

On the other hand, it has been found that anti-inflammatory activity of variant IL-10 monomers can be restored by forcing the monomers into a pseudo-dimeric form through fusion of the monomers to corresponding Fca parts of an IgA immunoglobulin (Figure 5), thereby taking advantage of the dimerization abilities of antibody heavy chains. To this end, four constructs were created in which native hIL-10 and variant IL-10 monomer were fused to the Ser245-Tyr479 Fc part of a human IgA heavy chain in a C-terminal-to-N-terminal and N- terminal-to-C-terminal manner. Fusion was done using a 19 amino acid glycine-serine linker. The fusion proteins were preceded by a murine secretion signal peptide. The amino acid sequences of the constructs are shown in Seq. Id. Nos.: 3-6.

Analysis of the four constructs indicated that construct in which the variant human IL-10 monomer was fused to the C-terminal end of the human IgA Fc part was the optimal fusion protein. In particular, accumulation data for this optimal fusion protein were similar to the variant IL-10 monomer itself and dimerization of the Fc parts was optimal. Additionally, the accumulation data for this optimal fusion protein was substantially better than the accumulation data for the construct in which the native human IL-10 was fused to the C-terminal end of the human IgA Fc part (see Figure 7). Moreover, the fusion of the IL-10 monomer to the N-terminal end of Fca increased levels of higher molecular forms were observed. Notably, the optimal fusion protein was capable of reducing TNFa expression by LPS stimulated macrophages, indicating anti-inflammatory biological activity. This is in contrast to IL-10 monomers, which lack anti-inflammatory biological activity. Furthermore, accumulation differences between the constructs in which the native human IL-10 and the variant IL-10 monomer were fused to the C- terminal end of the IgA Fc part was comparable to that observed for native human IL-10 and variant IL-10 monomer themselves. This indicates that the productive advantage in planta of variant IL-10 (i.e. lack of aggregation), as observed for the GFP fusion proteins also is present in the fusion proteins according to the present invention.

Pharmaceutical formulations & therapeutic uses

Pharmaceutical formulations containing fusion proteins of the present invention can be prepared according to standard techniques. Such pharmaceutical formulations may be, for example, solid or liquid dosage forms, systemic or directed dosage forms, or even oral dosage forms such as in the form of the plant expression system.

The ability of the fusion proteins of the present invention to decrease TNFa expression by LPS-stimulated macrophages corresponds to an ability to inhibit inflammation in a human subject. Additionally, it is know that IL-10 can be useful for treatment Multiple Sclerosis (MS). Accordingly, pharmaceutical formulations containing fusion proteins of the present invention are suitable for use in treating or preventing anti-inflammatory conditions in a human subject and for treating MS in a human subject.

EXAMPLES

Example 1: Fca - IL-10 and IL-10 - Fca fusion proteins transiently expressed in tobacco

Construction of DNA constructs and transfection vectors

The native open reading frame (ORF) of hIL-10 was amplified from the cDNA library MegaMan™ Human Transcriptome (Stratage) with the primers hIL-lOf

5 ' CC ATGGGC ATGC AC AGCTC AGC ACTGCTCT 3' and hIL-lOr 5'CGGTACCT

CAGTTTCGTA TCTTCATTGTCA 3'. The glycine-serine spacer (Gly-Gly-Ser-Gly-Gly-Gly) was introduced into the inter-domain linker region of IL-10 (between residues Asnl 16 and Lysl 17) by overlap extension PCR using the above mentioned primers in combination with vIL- lOf 5'AAACGGTGGCGGATCTGGGGGTAAGAGCAAGGCCGTGGAGCAGG TGAA 3' and vIL-lOr 5'CTTACCCCCAGATCCGCCACCGTTTTCACAGGGA AGAAATCGATGA 3'. After translation, the addition of the Ncol restriction site resulted in the addition of two extra amino acids at the N-terminus, methionine and glycine, which were cleaved off together with the native signal peptide upon post-translational processing. The FCa-hIL-10 fragment consists of the murine IgA heavy chain signal peptide followed by the human IgA heavy chain Ca domains 2 & 3, a 19 amino acid glycine-serine linker and native human IL-10. FCa-vIL-10 was created by exchanging the C-terminal part of hIL-10 using the unique Clal restriction site just at the end of helix D. The IL-10-FCa fragments consist of the native human IL-10 signal peptide followed by either h/vIL-10, a 19 amino acid glycine-serine linker and finally the human IgA heavy chain Ca domains 2 & 3. For all cloning procedures general enzymatic digestion and subsequent ligation and electroporation techniques were used.

All open reading frames (ORF's) were placed under the control of a double 35S cauliflower mosaic virus promoter with an alfalfa mosaic viral leader and nopaline synthase terminator ' and 3' respectively, as donated by pRAP35 in which all genes were cloned with NcoI/BspHI (same restriction overhang, but site is lost after ligation) and Kpnl. All expression cassettes were digested from pRAP35 with Ascl and Pad and ligated into a modified version of pMDC32, renamed pHYG. pHYG resulted from the removal of the gateway recombination sequences by digestion with EcoRI and Hindlll and replacement with an oligo fragment including Ascl and Pad restriction site sequences for insertion of the expression cassettes. The pHYG plant expression vectors were transformed into Agrobacterium tumefaciens (strain MOG101) by electroporation.

Transformation of tobacco plants

A. tumefaciens was cultured overnight at 28°C and 200 rpm in 5 ml of LB medium containing 50 μg/ml kanamycin and 25 μg/ml rifampicin. Optical density (OD) of the culture was measured at 600 nm and was used to inoculate 100 ml of LB medium containing 200 μΜ acetosyringone and 50 μg/ml kanamycin with x μΐ of culture using the following formula x = 80000/(1028*OD). OD was measured again after 16 hours and the bacterial culture was centrifuged for 15 min. at 4000 rpm. The medium was discarded and the pellet was resuspended in 45*OD ml of MMA infiltration medium containing 200 μΜ acetosyringone. After 1-2 hours incubation at room temperature, the two youngest fully expanded leaves of 6-week old tobacco plants (Nicotiana benthamiana) were infiltrated. Infiltration was performed by injecting

Agrobacterium suspension into the abaxial side of the Nicotiana benthamiana leaf with a 1 ml syringe. Infiltrated plants were maintained in a controlled greenhouse compartment (UNIFARM, Wageningen) and infiltrated leaves were harvested at selected time points. Protein extraction

Infiltrated leafs were snap-frozen in liquid nitrogen and then homogenized in liquid nitrogen and resuspended in ice-cold extraction buffer (50 mM sodium phosphate (pH=8), 100 mM NaCl, 10 mM EDTA, 0.1% Tween-20, 2% w/v polyvinylpyrrolidone (Serva)). Samples were centrifuged for 10 min at 4°C, and the supernatant was collected. The obtained crude extracts were subsequently desalted over a G25 Sephadex column and filtered through 0.22 μι^η syringe driven filter units (Millipore Corporation). The total soluble protein (TSP) content was determined according to the BCA method (Pierce) using bovine serum albumin (BSA) as a standard.

Apoplast isolation Apoplastic fluids were isolated to prepare samples for biological activity assays having less contaminating plant proteins or bacterial compounds. Infiltrated leafs were submerged in cold extraction buffer (see above, but without polyvinylpyrrolidone). By applying vacuum, the buffer was infiltrated into the apoplast. The surface of the leaves were blotted on pieces of tissue to remove excess of buffer. The leaves were placed in a 20-ml syringe and centrifuged at 4°C for 30 min at -3000 x g, while collecting the apoplastic fluid in a 50 ml tube. Apoplastic fluid was filtered through 0.22 μιη syringe driven filter units (Millipore Corporation).

ELISA

The quantification of plant-produced IL-10 in tobacco leaf crude extracts and apoplast fluid was achieved with commercially available ELISA kits. The human interleukin-10 ELISA kit Ready-Set-Go! (eBioscience) was used according to the supplier's protocol. The OD value was measured at 450 nm with the Anthos ELISA plate reader 2001 and converted into micrograms of IL-10 per gram of leaf material by reference to an ELISA standard curve constructed with recombinant IL-10 standards. Using the data from the BCA method, the IL-10 accumulation was corrected for TSP. Results are shown in Figures 3, 7 and 9.

Western blot analysis

Plant extracts and apoplast fluids were further analyzed by SDS-PAGE and western blotting. Recombinant human IL-10 (R&D Systems) was used as a control. After separation by non-reducing/reducing SDS-PAGE on a NuPAGE® 12% Bis-Tris gel (Invitrogen), proteins were stained by Coomassie Brilliant Blue staining (Sigma) or were transferred to an

InvitrolonTM PVDF membrane (Invitrogen). Then the membrane was blocked in PBST-BL (containing 0.1% Tween-20 and 5% non-fat dry milk powder) for 1 hour at room temperature, followed by 5 washing steps of 5 min. in PBST. The membrane was incubated for overnight at 4°C with a anti-IL-10 monoclonal antibody in PBST (1 : 1000 purified Rat anti-hIL-10 IgGl (JES3-19F1, BioLegend). The membrane was washed again as described above, followed by a 1 hour incubation at room temperature with 1 :5000 Goat Anti-Rat IgG:HRP polyclonal antibody (AbD Serotec) in PBST. The membrane was washed again as described before. Finally, the SuperSignal West Dura/Femto substrate (Pierce) was used to detect IL-10. Results are shown in Figures 6 and 9.

Confocal laser scanning microscopy To investigate the subcellular localization of human IL-10, GFP fusions with IL-10 were agroinfiltrated as described above. Leafs were taken from the plant and small sections were examined from the abaxial side using a Zeiss LSM510 confocal laser scanning microscope in combination with an argon ion laser supplying a 488 nm wavelength. Results are shown in Figure 9.

Example 2: Bioassay: MC/9 proliferation assay

MC/9 cells were obtained from the American Type Culture Collection and cultured in DMEM medium supplemented with 10% fetal calf serum (FCS), 50 U/mL of penicillin, 50 ug/mL of streptomycin, 50 μΜ β-mercaptoethanol and 5 ng/ml mouse IL-3. Cells were kept at 37°C with 5% C02 and medium was renewed every 2-3 days. For proliferation assays cells were harvested 3 days after the last IL-3 supplement and seeded in 96 well plates at a density of 2xl0⁴ cells/well using culture medium without IL-3. Cells were incubated overnight at 37°C/5% C02 to starve. Cells were then treated with apoplastic fluids or the recombinant human IL-10 control (R&D Systems) at several concentrations. After three days of culture, proliferation was assessed with the CellTiter 96® AQueous One Solution (Promega). Results are shown in figure 10.

Example 3: Bioassay: Inhibition of TNF-a expression of RAW264.7 macrophages

The monocyte/macrophage cell line RAW264.7 was purchased from the American Type Culture Collection. The RAW264.7 cells were maintained at 37°C with 5% C02 in IMDM containing 4 mM L-glutamine, 25 mM HEPES and supplemented with 10% fetal calf serum, 50 U/ml penicillin and 50 μg/ml streptomycin. RAW264.7 cells were harvested by gently disrupting the monolayer with a cell scraper and sub-cultured every 2-3 days. For cell-based assays cells were harvested and seeded in 96 well plates at a density of 5xl0⁴ cells/well in DMEM medium supplemented with 10% fetal calf serum (FCS), 50 U/mL of penicillin and 50 ug/mL of streptomycin. Cells were incubated overnight at 37°C/5% C02 to recover. Cells were then pre- treated with crude extracts, apoplastic fluids or the recombinant IL-10 control (R&D Systems) for 1 hour, and subsequently stimulated with 10 ng/ml of lipopolysaccharide (Sigma). After overnight incubation supernatants were analysed with the mouse TNF-a ELIS A kit Ready-Set- Go! (eBioscience) according to the supplier's protocol. Results are shown in Figures 4 and 9.

SEQUENCE LISTING

Yellow (underlined): signal peptides

Purple (italic underlined ): IL-10 spacer

Green (bold): IL-10 part of fusion constructs

Blue (italic): Linker between IL-10 and Fca; replaces tail section of Fca

Seq. Id. No.: l hIL-10

MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRV TFF QMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLG ENLKTLRLRLRRCHRFLPCENKSKAVEQVK AFNKLQEKGIYKAMSEFDIFINYIEAYM TMKIRN

Seq. Id. No.:2 vIL-10

MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRV TFF QMKDQLDNLLLKESLLEDF GYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHV SLG ENLKTLRLRLRRCHRFLPCENGGG^GGKSKAVEQVK AFNKLQEKGIYKAMSEFDIFIN YIEAYMTMKIRN

Seq. Id. No.:3 FCa-hIL-10

MKHLWFFLLLVAAPRWLSAASRTSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTG LRDASGATFTWTPSSG SAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPE LKTPLTANIT SGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQE LPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWK GDTFSCMVGHEALPLAFTQKTI DRLAGKJTHWVSVVMAEVDGTCYEZGGGGSGGGGSGGGGSGGGGrSSPGQGTQSEN SCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQAL

SEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAV

EQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

Seq. Id. No.:4 Fca-vIL-10

MKHLWFFLLLVAAPRWLSAASRTSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTG LRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPE LKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQE LPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWK GDTFSCMVGHEALPLAFTQKTI DRLAGKPTHWVSVVMAEVDGTCYEZGGGGSGGGGSGGGGSGGGG7SSPGQGTQSEN SCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQAL SEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGG^G GKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

Seq. Id. No.:5 hIL-10-FCa

MGMHSSALLCCLVLLTGVRASPGOGTQSENSCTHFPGNLPNMLRDLRDAFSRV KTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIK AHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEF

DIFINYIEAYMTMKIRNEIGGGG^GGGG^GGGG.SGGGGr.SRTSPSCCHPRLSLHRPALE DLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQ PWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGF SPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWK GDTFSC MVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY

Seq. Id. No.:6 vIL-10-FCa

MGMHSSALLCCLVLLTGVRASPGOGTQSENSCTHFPGNLPNMLRDLRDAFSRV KTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIK AHVNSLGENLKTLRLRLRRCHRFLPCENGGG^GGKSKAVEQVKNAFNKLQEKGIY KAMSEFDIFINYIEAYMTMKIRN£:LGGGG5GGGG5GGGG5GGGGreRTSPSCCHPRLSL HRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSV LPGCAQPW HGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTL TCLARGFSPI DVLVRWLQGSQELPRE YLTWASRQEPSQGTTTFAVTSILRVAAEDW GDTFSCMVGHEALPLAFTQKTIDRLAGI PTHVNVSVVMAEVDGTCY

Underlined: restriction sites (Ncol, Kpnl or BspHI/Ncol overhang)

Seq. Id. No.:7 hIL-10

ccatgggcatgcacagctcagcactgctctgttgcctggtcctcctgactggggtgagggccagcccaggccagggcacccagt ctgagaacagctgcacccacttcccaggcaacctgcctaacatgcttcgagatctccgagatgccttcagcagagtgaagactttctttcaaa tgaaggatcagctggacaacttgttgttaaaggagtccttgctggaggactttaagggttacctgggttgccaagccttgtctgagatgatcca gttttacctggaggaggtgatgccccaagctgagaaccaagacccagacatcaaggcgcatgtgaactccctgggggagaacctgaaga ccctcaggctgaggctacggcgctgtcatcgatttcttccctgtgaaaacaagagcaaggccgtggagcaggtgaagaatgcctttaataag ctccaagagaaaggcatctacaaagccatgagtgagtttgacatcttcatcaactacatagaagcctacatgacaatgaagatacgaaactg asstacc

Seq. Id. No.:8 vIL-10

Ccatgggcatgcacagctcagcactgctctgttgcctggtcctcctgactggggtgagggccagcccaggccagggcacccag tctgagaacagctgcacccacttcccaggcaacctgcctaacatgcttcgagatctccgagatgccttcagcagagtgaagactttctttcaa atgaaggatcagctggacaacttgttgttaaaggagtccttgctggaggactttaagggttacctgggttgccaagccttgtctgagatgatcc agttttacctggaggaggtgatgccccaagctgagaaccaagacccagacatcaaggcgcatgtgaactccctgggggagaacctgaag accctcaggctgaggctacggcgctgtcatcgatttcttccctgtgaaaacggtggcggatctgggggtaagagcaaggccgtggagcag gtgaagaatgcctttaataagctccaagagaaaggcatctacaaagccatgagtgagtttgacatcttcatcaactacatagaagcctacatg acaatgaagatacgaaactgaggtacc

Seq. Id. No.:9 FCa-hIL-10

ccatgaagcacctctggttcttcctcctcctcgtcgccgccccccgttggctctccgccgctagccgtacgagccccagctgctgc caccccaggctgtccctgcacaggcccgctctcgaggacctgctgctgggctccgaggccaacctgacctgcaccctgaccggcctgag ggacgcctccggcgctaccttcacctggaccccctcctccggcaagtccgctgtccaaggcccacctgagcgtgatctctgcggctgctac tccgtgtccagcgtgctgcccggctgcgcccagccctggaaccacggcgagaccttcacctgcaccgccgctcaccccgagcttaagac ccccctgaccgccaacatcaccaagtccggcaacaccttccgccccgaggtgcacctgctgccccctccctccgaggagctggccctga acgagctggtgaccctgacctgcctcgccaggggcttctccccgaaggacgtgcttgtgcgctggctccagggctcccaggagctgcccc gcgagaagtacctgacctgggcctccaggcaggagccttcccagggcaccaccaccttcgccgtgacctccatcctgagggtggccgcc gaggactggaagaagggcgacaccttcagctgcatggtgggtcacgaggctctgcccctggccttcacccaaaagaccatcgaccgcct ggctggcaagcccacccacgtgaacgtgtccgtggtgatggccgaggtggacggcacctgctacgagctcggtggtggtggttccggtg gtggtggttccggtggtggtggttccggtggtggtggtactagtagcccaggccagggcacccagtctgagaacagctgcacccacttccc aggcaacctgcctaacatgcttcgagatctccgagatgccttcagcagagtgaagactttctttcaaatgaaggatcagctggacaacttgttg ttaaaggagtccttgctggaggactttaagggttacctgggttgccaagccttgtctgagatgatccagttttacctggaggaggtgatgcccc aagctgagaaccaagacccagacatcaaggcgcatgtgaactccctgggggagaacctgaagaccctcaggctgaggctacggcgctg tcatcgatttcttccctgtgaaaacaagagcaaggccgtggagcaggtgaagaatgcctttaataagctccaagagaaaggcatctacaaag ccatgagtgagtttgacatcttcatcaactacatagaagcctacatgacaatgaagatacgaaactgaggtacc

Seq. Id. No.: 10 FCa-vIL-10

ccatgaagcacctctggttcttcctcctcctcgtcgccgccccccgttggctctccgccgctagccgtacgagccccagctgctgc caccccaggctgtccctgcacaggcccgctctcgaggacctgctgctgggctccgaggccaacctgacctgcaccctgaccggcctgag ggacgcctccggcgctaccttcacctggaccccctcctccggcaagtccgctgtccaaggcccacctgagcgtgatctctgcggctgctac tccgtgtccagcgtgctgcccggctgcgcccagccctggaaccacggcgagaccttcacctgcaccgccgctcaccccgagcttaagac ccccctgaccgccaacatcaccaagtccggcaacaccttccgccccgaggtgcacctgctgccccctccctccgaggagctggccctga acgagctggtgaccctgacctgcctcgccaggggcttctccccgaaggacgtgcttgtgcgctggctccagggctcccaggagctgcccc gcgagaagtacctgacctgggcctccaggcaggagccttcccagggcaccaccaccttcgccgtgacctccatcctgagggtggccgcc gaggactggaagaagggcgacaccttcagctgcatggtgggtcacgaggctctgcccctggccttcacccaaaagaccatcgaccgcct ggctggcaagcccacccacgtgaacgtgtccgtggtgatggccgaggtggacggcacctgctacgagctcggtggtggtggttccggtg gtggtggttccggtggtggtggttccggtggtggtggtactagtagcccaggccagggcacccagtctgagaacagctgcacccacttccc aggcaacctgcctaacatgcttcgagatctccgagatgccttcagcagagtgaagactttctttcaaatgaaggatcagctggacaacttgttg ttaaaggagtccttgctggaggactttaagggttacctgggttgccaagccttgtctgagatgatccagttttacctggaggaggtgatgcccc aagctgagaaccaagacccagacatcaaggcgcatgtgaactccctgggggagaacctgaagaccctcaggctgaggctacggcgctg tcatcgatttcttccctgtgaaaacggtggcggatctgggggtaagagcaaggccgtggagcaggtgaagaatgcctttaataagctccaag agaaaggcatctacaaagccatgagtgagtttgacatcttcatcaactacatagaagcctacatgacaatgaagatacgaaactgaggtacc Seq. Id. No.: 11 hIL-10-Fca

ccatgggcatgcacagctcagcactgctctgttgcctggtcctcctgactggggtgagggccagcccaggccagggcacccagt ctgagaacagctgcacccacttcccaggcaacctgcctaacatgcttcgagatctccgagatgccttcagcagagtgaagactttctttcaaa tgaaggatcagctggacaacttgttgttaaaggagtccttgctggaggactttaagggttacctgggttgccaagccttgtctgagatgatcca gttttacctggaggaggtgatgccccaagctgagaaccaagacccagacatcaaggcgcatgtgaactccctgggggagaacctgaaga ccctcaggctgaggctacggcgctgtcatcgatttcttccctgtgaaaacaagagcaaggccgtggagcaggtgaagaatgcctttaataag ctccaagagaaaggcatctacaaagccatgagtgagtttgacatcttcatcaactacatagaagcctacatgacaatgaagatacgaaacga gctcggtggtggcggttctggcggaggtggcagcggcggtggcggatctggtggcggtggaactagccgtacgagccccagctgctgc caccccaggctgtccctgcacaggcccgctctcgaggacctgctgctgggctccgaggccaacctgacctgcaccctgaccggcctgag ggacgcctccggcgctaccttcacctggaccccctcctccggcaagtccgctgtccaaggcccacctgagcgtgatctctgcggctgctac tccgtgtccagcgtgctgcccggctgcgcccagccctggaaccacggcgagaccttcacctgcaccgccgctcaccccgagcttaagac ccccctgaccgccaacatcaccaagtccggcaacaccttccgccccgaggtgcacctgctgccccctccctccgaggagctggccctga acgagctggtgaccctgacctgcctcgccaggggcttctccccgaaggacgtgcttgtgcgctggctccagggctcccaggagctgcccc gcgagaagtacctgacctgggcctccaggcaggagccttcccagggcaccaccaccttcgccgtgacctccatcctgagggtggccgcc gaggactggaagaagggcgacaccttcagctgcatggtgggtcacgaggctctgcccctggccttcacccaaaagaccatcgaccgcct ggctggcaagcccacccacgtgaacgtgtccgtggtgatggccgaggtggacggcacctgctactaaggtacc

Seq. Id. No.: 12 vIL-10-FCa

ccatgggcatgcacagctcagcactgctctgttgcctggtcctcctgactggggtgagggccagcccaggccagggcacccagt ctgagaacagctgcacccacttcccaggcaacctgcctaacatgcttcgagatctccgagatgccttcagcagagtgaagactttctttcaaa tgaaggatcagctggacaacttgttgttaaaggagtccttgctggaggactttaagggttacctgggttgccaagccttgtctgagatgatcca gttttacctggaggaggtgatgccccaagctgagaaccaagacccagacatcaaggcgcatgtgaactccctgggggagaacctgaaga ccctcaggctgaggctacggcgctgtcatcgatttcttccctgtgaaaacggtggcggatctgggggtaagagcaaggccgtggagcagg tgaagaatgcctttaataagctccaagagaaaggcatctacaaagccatgagtgagtttgacatcttcatcaactacatagaagcctacatga caatgaagatacgaaacgagctcggtggtggcggttctggcggaggtggcagcggcggtggcggatctggtggcggtggaactagccg tacgagccccagctgctgccaccccaggctgtccctgcacaggcccgctctcgaggacctgctgctgggctccgaggccaacctgacct gcaccctgaccggcctgagggacgcctccggcgctaccttcacctggaccccctcctccggcaagtccgctgtccaaggcccacctgag cgtgatctctgcggctgctactccgtgtccagcgtgctgcccggctgcgcccagccctggaaccacggcgagaccttcacctgcaccgcc gctcaccccgagcttaagacccccctgaccgccaacatcaccaagtccggcaacaccttccgccccgaggtgcacctgctgccccctccc tccgaggagctggccctgaacgagctggtgaccctgacctgcctcgccaggggcttctccccgaaggacgtgcttgtgcgctggctccag ggctcccaggagctgccccgcgagaagtacctgacctgggcctccaggcaggagccttcccagggcaccaccaccttcgccgtgacctc catcctgagggtggccgccgaggactggaagaagggcgacaccttcagctgcatggtgggtcacgaggctctgcccctggccttcaccc aaaagaccatcgaccgcctggctggcaagcccacccacgtgaacgtgtccgtggtgatggccgaggtggacggcacctgctactaaggt acc

Seq. Id. No.: 13 pRAP35 vector

gcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcg ggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaat tgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagctggcgcgccaagcttgaattaattctactccaaaaat atcaaagatacagtctcagaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcctcggattccattgcccagct atctgtcactttattgtgaagatagtggaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcc tctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagt ggattgatgtgataattccgcatgggtcaaagattcaaatagaggacctaacagaactcgccgtaaagactggggacagttcatacagagtc tcttacgactcaatgacaagaagaaaatcttcgtcaacatggtggagcacgacacacttgtctactccaaaaatatcaaagatacagtctcag aagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcctcggattccattgcccagctatctgtcactttattgtgaa gatagtggaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgacagtggtccc aaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgatatctcca ctgacgtaagggatgacgcacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttggagagggtttttatttttaa ttttctttcaaatacttccaccatggggtacccgggagatcttcgatcccgatcgttcaaacatttggcaataaagtttcttaagattgaatcctgtt gccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggttttta tgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgccggtgtcatctatg ttactagatcgggaattgccaagctaattcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttct tagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaat aaccctgataaatgcttcaataatgggaccgactcgaattcttaattaacaattcactggccgtcgttttacaacgtcgtgactgggaaaaccct ggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaac agttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagt acaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcat ccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgtgacgaaagggcct cgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatt tgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtat tcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaag atcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgat gagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaat gacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgat aacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgcct tgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgca aactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgct cggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggt aagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcac tgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcct ttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcc tttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttcc gaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcacc gcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagtta ccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacc tacagcgtgagcattgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggag agcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatg ctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttc ctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgag tcagtgagcgaggaagcggaaga

Seq. Id. No.: 14 pHYG Vector

aattcggcgcgcctacgcgtaaggacgagctctgaggtacctctagattaattaaaagcttggcactggccgtcgttttacaacgtc gtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcac cgatcgcccttcccaacagttgcgcagcctgaatggcgaatgctagagcagcttgagcttggatcagattgtcgtttcccgccttcagtttaaa ctatcagtgtttgacaggatatattggcgggtaaacctaagagaaaagagcgtttattagaataacggatatttaaaagggcgtgaaaaggttt atccgttcgtccatttgtatgtgcatgccaaccacagggttcccctcgggatcaaagtactttgatccaacccctccgctgctatagtgcagtcg gcttctgacgttcagtgcagccgtcttctgaaaacgacatgtcgcacaagtcctaagttacgcgacaggctgccgccctgcccttttcctggc gttttcttgtcgcgtgttttagtcgcataaagtagaatacttgcgactagaaccggagacattacgccatgaacaagagcgccgccgctggcc tgctgggctatgcccgcgtcagcaccgacgaccaggacttgaccaaccaacgggccgaactgcacgcggccggctgcaccaagctgttt tccgagaagatcaccggcaccaggcgcgaccgcccggagctggccaggatgcttgaccacctacgccctggcgacgttgtgacagtga ccaggctagaccgcctggcccgcagcacccgcgacctactggacattgccgagcgcatccaggaggccggcgcgggcctgcgtagcc tggcagagccgtgggccgacaccaccacgccggccggccgcatggtgttgaccgtgttcgccggcattgccgagttcgagcgttccctaa tcatcgaccgcacccggagcgggcgcgaggccgccaaggcccgaggcgtgaagtttggcccccgccctaccctcaccccggcacaga tcgcgcacgcccgcgagctgatcgaccaggaaggccgcaccgtgaaagaggcggctgcactgcttggcgtgcatcgctcgaccctgtac cgcgcacttgagcgcagcgaggaagtgacgcccaccgaggccaggcggcgcggtgccttccgtgaggacgcattgaccgaggccgac gccctggcggccgccgagaatgaacgccaagaggaacaagcatgaaaccgcaccaggacggccaggacgaaccgtttttcattaccga agagatcgaggcggagatgatcgcggccgggtacgtgttcgagccgcccgcgcacgtctcaaccgtgcggctgcatgaaatcctggccg gtttgtctgatgccaagctggcggcctggccggccagcttggccgctgaagaaaccgagcgccgccgtctaaaaaggtgatgtgtatttga gtaaaacagcttgcgtcatgcggtcgctgcgtatatgatgcgatgagtaaataaacaaatacgcaaggggaacgcatgaaggttatcgctgt acttaaccagaaaggcgggtcaggcaagacgaccatcgcaacccatctagcccgcgccctgcaactcgccggggccgatgttctgttagt cgattccgatccccagggcagtgcccgcgattgggcggccgtgcgggaagatcaaccgctaaccgttgtcggcatcgaccgcccgacga ttgaccgcgacgtgaaggccatcggccggcgcgacttcgtagtgatcgacggagcgccccaggcggcggacttggctgtgtccgcgatc aaggcagccgacttcgtgctgattccggtgcagccaagcccttacgacatatgggccaccgccgacctggtggagctggttaagcagcgc attgaggtcacggatggaaggctacaagcggcctttgtcgtgtcgcgggcgatcaaaggcacgcgcatcggcggtgaggttgccgaggc gctggccgggtacgagctgcccattcttgagtcccgtatcacgcagcgcgtgagctacccaggcactgccgccgccggcacaaccgttct tgaatcagaacccgagggcgacgctgcccgcgaggtccaggcgctggccgctgaaattaaatcaaaactcatttgagttaatgaggtaaa gagaaaatgagcaaaagcacaaacacgctaagtgccggccgtccgagcgcacgcagcagcaaggctgcaacgttggccagcctggca gacacgccagccatgaagcgggtcaactttcagttgccggcggaggatcacaccaagctgaagatgtacgcggtacgccaaggcaaga ccattaccgagctgctatctgaatacatcgcgcagctaccagagtaaatgagcaaatgaataaatgagtagatgaattttagcggctaaagga ggcggcatggaaaatcaagaacaaccaggcaccgacgccgtggaatgccccatgtgtggaggaacgggcggttggccaggcgtaagc ggctgggttgtctgccggccctgcaatggcactggaacccccaagcccgaggaatcggcgtgacggtcgcaaaccatccggcccggtac aaatcggcgcggcgctgggtgatgacctggtggagaagttgaaggccgcgcaggccgcccagcggcaacgcatcgaggcagaagca cgccccggtgaatcgtggcaagcggccgctgatcgaatccgcaaagaatcccggcaaccgccggcagccggtgcgccgtcgattagga agccgcccaagggcgacgagcaaccagattttttcgttccgatgctctatgacgtgggcacccgcgatagtcgcagcatcatggacgtggc cgttttccgtctgtcgaagcgtgaccgacgagctggcgaggtgatccgctacgagcttccagacgggcacgtagaggtttccgcagggcc ggccggcatggccagtgtgtgggattacgacctggtactgatggcggtttcccatctaaccgaatccatgaaccgataccgggaagggaa gggagacaagcccggccgcgtgttccgtccacacgttgcggacgtactcaagttctgccggcgagccgatggcggaaagcagaaagac gacctggtagaaacctgcattcggttaaacaccacgcacgttgccatgcagcgtacgaagaaggccaagaacggccgcctggtgacggt atccgagggtgaagccttgattagccgctacaagatcgtaaagagcgaaaccgggcggccggagtacatcgagatcgagctagctgattg gatgtaccgcgagatcacagaaggcaagaacccggacgtgctgacggttcaccccgattactttttgatcgatcccggcatcggccgttttct ctaccgcctggcacgccgcgccgcaggcaaggcagaagccagatggttgttcaagacgatctacgaacgcagtggcagcgccggaga gttcaagaagttctgtttcaccgtgcgcaagctgatcgggtcaaatgacctgccggagtacgatttgaaggaggaggcggggcaggctgg cccgatcctagtcatgcgctaccgcaacctgatcgagggcgaagcatccgccggttcctaatgtacggagcagatgctagggcaaattgcc ctagcaggggaaaaaggtcgaaaaggtctctttcctgtggatagcacgtacattgggaacccaaagccgtacattgggaaccggaacccg tacattgggaacccaaagccgtacattgggaaccggtcacacatgtaagtgactgatataaaagagaaaaaaggcgatttttccgcctaaaa ctctttaaaacttattaaaactcttaaaacccgcctggcctgtgcataactgtctggccagcgcacagccgaagagctgcaaaaagcgcctac ccttcggtcgctgcgctccctacgccccgccgcttcgcgtcggcctatcgcggccgctggccgctcaaaaatggctggcctacggccagg caatctaccagggcgcggacaagccgcgccgtcgccactcgaccgccggcgcccacatcaaggcaccctgcctcgcgcgtttcggtgat gacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcaggg cgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtatactggcttaactatgcggca tcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccg cttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaa tcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttcca taggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgt ttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgct ttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccg ctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagca gagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgc tgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagca gattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggat tttggtcatgcattctaggtactaaaacaattcatccagtaaaatataatattttattttctcccaatcaggcttgatccccagtaagtcaaaaaata gctcgacatactgttcttccccgatatcctccctgatcgaccggacgcagaaggcaatgtcataccacttgtccgccctgccgcttctcccaa gatcaataaagccacttactttgccatctttcacaaagatgttgctgtctcccaggtcgccgtgggaaaagacaagttcctcttcgggcttttcc gtctttaaaaaatcatacagctcgcgcggatctttaaatggagtgtcttcttcccagttttcgcaatccacatcggccagatcgttattcagtaagt aatccaattcggctaagcggctgtctaagctattcgtatagggacaatccgatatgtcgatggagtgaaagagcctgatgcactccgcataca gctcgataatcttttcagggctttgttcatcttcatactcttccgagcaaaggacgccatcggcctcactcatgagcagattgctccagccatcat gccgttcaaagtgcaggacctttggaacaggcagctttccttccagccatagcatcatgtccttttcccgttccacatcataggtggtccctttat accggctgtccgtcatttttaaatataggttttcattttctcccaccagcttatataccttagcaggagacattccttccgtatcttttacgcagcggt atttttcgatcagttttttcaattccggtgatattctcattttagccatttattatttccttcctcttttctacagtatttaaagataccccaagaagctaatt ataacaagacgaactccaattcactgttccttgcattctaaaaccttaaataccagaaaacagctttttcaaagttgttttcaaagttggcgtataa catagtatcgacggagccgattttgaaaccgcggtgatcacaggcagcaacgctctgtcatcgttacaatcaacatgctaccctccgcgaga tcatccgtgtttcaaacccggcagcttagttgccgttcttccgaatagcatcggtaacatgagcaaagtctgccgccttacaacggctctcccg ctgacgccgtcccggactgatgggctgcctgtatcgagtggtgattttgtgccgagctgccggtcggggagctgttggctggctggtggca ggatatattgtggtgtaaacaaattgacgcttagacaacttaataacacattgcggacgtttttaatgtactgaattaacgccgaattaattcggg ggatctggattttagtactggattttggttttaggaattagaaattttattgatagaagtattttacaaatacaaatacatactaagggtttcttatatg ctcaacacatgagcgaaaccctataggaaccctaattcccttatctgggaactactcacacattattatggagaaactcgagcttgtcgatcga cagatccggtcggcatctactctatttctttgccctcggacgagtgctggggcgtcggtttccactatcggcgagtacttctacacagccatcg gtccagacggccgcgcttctgcgggcgatttgtgtacgcccgacagtcccggctccggatcggacgattgcgtcgcatcgaccctgcgcc caagctgcatcatcgaaattgccgtcaaccaagctctgatagagttggtcaagaccaatgcggagcatatacgcccggagtcgtggcgatc ctgcaagctccggatgcctccgctcgaagtagcgcgtctgctgctccatacaagccaaccacggcctccagaagaagatgttggcgacct cgtattgggaatccccgaacatcgcctcgctccagtcaatgaccgctgttatgcggccattgtccgtcaggacattgttggagccgaaatccg cgtgcacgaggtgccggacttcggggcagtcctcggcccaaagcatcagctcatcgagagcctgcgcgacggacgcactgacggtgtc gtccatcacagtttgccagtgatacacatggggatcagcaatcgcgcatatgaaatcacgccatgtagtgtattgaccgattccttgcggtcc gaatgggccgaacccgctcgtctggctaagatcggccgcagcgatcgcatccatagcctccgcgaccggttgtagaacagcgggcagtt cggtttcaggcaggtcttgcaacgtgacaccctgtgcacggcgggagatgcaataggtcaggctctcgctaaactccccaatgtcaagcac ttccggaatcgggagcgcggccgatgcaaagtgccgataaacataacgatctttgtagaaaccatcggcgcagctatttacccgcaggaca tatccacgccctcctacatcgaagctgaaagcacgagattcttcgccctccgagagctgcatcaggtcggagacgctgtcgaacttttcgatc agaaacttctcgacagacgtcgcggtgagttcaggctttttcatatctcattgccccccgggatctgcgaaagctcgagagagatagatttgta gagagagactggtgatttcagcgtgtcctctccaaatgaaatgaacttccttatatagaggaaggtcttgcgaaggatagtgggattgtgcgtc atcccttacgtcagtggagatatcacatcaatccacttgctttgaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggtgggg gtccatctttgggaccactgtcggcagaggcatcttgaacgatagcctttcctttatcgcaatgatggcatttgtaggtgccaccttccttttctac tgtccttttgatgaagtgacagatagctgggcaatggaatccgaggaggtttcccgatattaccctttgttgaaaagtctcaatagccctttggtc ttctgagactgtatctttgatattcttggagtagacgagagtgtcgtgctccaccatgttatcacatcaatccacttgctttgaagacgtggttgga acgtcttctttttccacgatgctcctcgtgggtgggggtccatctttgggaccactgtcggcagaggcatcttgaacgatagcctttcctttatcg caatgatggcatttgtaggtgccaccttccttttctactgtccttttgatgaagtgacagatagctgggcaatggaatccgaggaggtttcccga tattaccctttgttgaaaagtctcaatagccctttggtcttctgagactgtatctttgatattcttggagtagacgagagtgtcgtgctccaccatgtt ggcaagctgctctagccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactgga aagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtg tggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacg

Claims

P1517PC00 CLAIMS

1. A fusion protein, comprising:

a first IL-10 monomer fused to the C-terminus of a first immunoglobulin Fc part; and a second IL-10 monomer fused to the C-terminus of a second immunoglobulin Fc part, wherein the first and second immunoglobulin Fc parts are bound together.

2. The fusion protein of claim 1 , wherein said first and second Fc parts are bound together by covalent disulfide bonds and/or non-covalent interactions.

3. The fusion protein of claims 1-2, wherein the N-terminus of at least one of said first and second IL-10 monomers is fused to the C-terminus of the corresponding immunoglobulin

Fc part by a peptide linker or a peptide bond.

4. The fusion protein of claim 1-3, wherein said first and second IL-10 monomers are human IL-10 monomers.

5. The fusion protein of claims 1-4, wherein at least one of the IL-10 monomers contains an amino acid spacer positioned such that the amino acid spacer prevents domain swapping between the first and second IL-10 monomers.

6. The fusion protein of claim 5, wherein said amino acid spacer is a peptide comprising 2-10 amino acids, such as 4-8 amino acids, and preferably is an a 6-amino acid linker consisting of Gly-Gly-Gly-Ser-Gly-Gly, and wherein said amino acid spacer is located between helices D and E of the interdomain region.

7. The fusion protein of claims 1-6, wherein at least one of the first and second

immunoglobulin Fc parts is a human immunoglobulin Fc part.

8. The fusion protein of claim 7, wherein at least one of the first and second human

immunoglobulin Fc parts is a human IgA or human IgG Fc part.

9. The fusion protein of claim 8, wherein human IgA Fc parts are each Ser245-Tyr479 of a human IgA antibody.

10. The fusion protein of claims 1-9, wherein said first and second immunoglobulin Fc parts are the constant heavy chain domains of an intact antibody.

11. The fusion protein of claims 1-10, wherein said first and second immunoglobulin parts are identical.

12. The fusion protein of claims 1-11, wherein said fusion protein decreases TNFa expression by lipopolysaccharide-stimulated macrophages.

13. The fusion protein of claims 1-12, wherein said fusion protein induces proliferation of MC/9 murine mast cells.

14. A method for the recombinant production an anti-inflammatory fusion protein, said method comprising:

expressing in plant cells, preferably tobacco cells such as Nicotiana benthamiana cells, a fusion protein, comprising:

a first IL-10 monomer fused to the C-terminus of a first immunoglobulin Fc part; and

a second IL-10 monomer fused to the C-terminus of a second immunoglobulin Fc part,

wherein the first and second immunoglobulin Fc parts are bound together, wherein at least one of the IL-10 monomers is modified to prevent the at least one IL-10 monomer from dimerizing with another IL-10 monomer, and

wherein the fusion protein decreases TNFa expression by lipopolysaccharide- stimulated macrophages; and

optionally extracting said fusion protein from said plant cells.

15. A method for the recombinant production an anti-inflammatory fusion protein, said method comprising:

wherein the first and second immunoglobulin Fc parts are bound together, and wherein the fusion protein decreases TNFa expression by lipopolysaccharide- stimulated macrophages; and

optionally extracting said fusion protein from said plant cells.

16. A fusion protein obtainable by the methods of claims 14 or 15.

17. A pharmaceutical composition, comprising the fusion protein of claims 1-13 or 16.

18. A fusion protein according to claims 1-13 or 16 or a pharmaceutical composition according to claim 17, for use in medicine, preferably for use in the treatment of Multiple Sclerosis or an inflammatory condition in a human.

19. An expression vector comprising DNA encoding the fusion protein according to claims 1- 13 or 16.

20. A plant cell, preferably a tobacco cell such as a Nicotiana benthamiana cell, comprising the expression vector of claim 19.