EP1871412A2 - Soluble vox2 protein as immunoregulatory factor - Google Patents

Abstract

The invention provides soluble derivatives of the vOX2 protein encoded by Kaposi’s sarcoma-associated herpesvirus (KSHV), and their use for the treatment of inflammatory disorders such as autoimmune diseases, allergy and graft rejection.

Description

Soluble Inununoregrulat-ory Factor

Field of the Invention

The invention relates to iπimunoregulatory molecules, and in particular to soluble derivatives of the vOX2 protein encoded by Kaposi's sarcoma-associated herpesvirus (KSHV).

Background to the Invention

The physiological function of the mammalian immune system is to defend the host against microbial infection. Defence is mediated by the early reactions of innate immunity and the delayed responses of adaptive immunity. The earliest antiviral component of the immune system that is activated is the inflammatory response, which is characterized by the release of chemotactic mediators from injured cells, including endothelial cells, to recruit leukocytes (primarily neutrophils, the most abundant cellular components of the immune system) from the peripheral blood to the site of the inflammatory reaction. Neutrophils play an essential role in the innate immune response to infection by eliminating microbes and infected cells by ingesting them during the process of phagocytosis. This process is accompanied by an effector ^λrespiratory burst' within the phagosome, involving a sudden increase in oxidative metabolism that results in the production of toxic reactive oxygen intermediate metabolites (ROIs; such as O₂ ^', ^'OH, H₂O₂). Although ROIs are generated to eliminate a pathogen intracellularly, they inevitably diffuse through cell membranes into the site of inflammation where they have the capacity to damage tissues, thereby augmenting immune pathogenesis .

Among the most important pro-inflammatory intercellular mediators of the inflammatory response are the chemokines, interleukin 8 (IL-8) and macrophage chemoattractant protein-1 (MCP-I). Accumulating evidence shows IL-8 and MCP-I as major mediators of acute (neutrophilic) and chronic (macrophage/monocytic) inflammation, respectively (Akahoshi et al., 1994, Frangogiannis et al., 2002, Hatano et al., 1999, Smith et al., 1997, Villiger et al., 1992). The CXC-chemokine IL-8 is a pro-survival signal for leukocytes; numerous reports show that various cells including monocytes, T lymphocytes, neutrophils, fibroblasts, endothelial cells and epithelial cells, express IL-8 mRNA and produce IL-8 protein rapidly. However, it is primarily produced by monocyte/macrophage lineage cells and recruits predominantly neutrophils (reviewed in (Baggiolini, 2001, Mukaida et al., 1998)). MCP-I is a pro-inflammatory chemokine involved in cell mobilization and stimulation that acts predominantly on lymphocytes, monocytes, mast cells, and eosinophils (Mukaida et al., 1998). This chemokine is expressed by various cell types including monocytes, smooth muscle cells, and human vascular endothelial cells (HUVECs) in response to different stimuli such as IL-I, tumor necrosis factor (TNF) , and immunoglobulin (Ig) G (Rollins et al., 1990, Sica et al., 1990, Yang et al., 2004). TNF-α and IgG complexes are known stimulators of ROI generation as well (Satriano et al., 1993).

As viruses have co-evolved with their hosts, in turn they have evolved strategies to evade the immune response. Kaposi's sarcoma-associated herpesvirus (KSHV) is the most recently identified herpes virus infecting humans (Chang et al., 1994), and the aetiological agent of Kaposi's sarcoma (KS), and perhaps primary effusion lymphoma and multicentric Castleman' s disease (for review see (Sarid et al., 1999)). From a genome of approximately 90 KSHV open reading frames (ORFs) , at least 16 specify proteins that could modulate host immune responses (Russo et al., 1996). The inflammatory response might be considered a pivotal target for immune modulation, to augment virus infection and dissemination. Indeed, KSHV encodes three CXC chemokine modulators (Boshoff et al., 1997).

Another protein produced during the lytic cycle of KSHV is encoded by the open reading frame designated K14 (Jenner et al., 2001) . This protein shows homology with the mammalian 0X2 protein (also called CD200) , which belongs to the Ig superfamily of membrane-associated glycoproteins and is expressed on the surface of many cell types, including endothelial cells, B cells, T cells, neuronal cells and tonsil follicles (Morris & Beech, 1987, Wright et al . , 2001).

The role of cellular 0X2 is believed to be one of regulating the activity of myeloid lineage cells, perhaps acting as an accessory signal (Wright et al., 2000). Elevated expression of 0X2 increases renal and skin allograft survival in mice and rats (Gorczynski et al . , 1998, Gorczynski et al., 1999), suggesting that 0X2 delivers a tolerizing signal (reviewed in (Gorczynski, 2001) . Signal delivery of 0X2 is likely through binding the 0X2 ligand (receptor) , whose is expression is restricted to myeloid lineage cells (Gorczynski et al., 2000, Wright et al., 2003)

As a result of this homology, the viral protein is normally referred to as vOX2. However, this is simply a convenient notation. The two proteins share only approximately 40% sequence identity at the amino acid level, so it cannot be assumed that the viral protein mimics any of the activities of the cellular protein. Furthermore, the viral protein also has homology with the neural cell adhesion molecule (NCAM), Thy-1 and Ll, so vOX2 has also been referred to as vNCAM and v-adh. S-ummary o£ the Invention.

The results of studies to date on the function of vOX2 are contradictory. Chung et al. (2002) report that vOX2 protein expressed as a soluble GST fusion protein can stimulate primary monocytes, macrophages and dendritic cells and induce them to produce inflammatory cytokines. In that study, vOX2 expressed on the surface of B lymphocytes was also shown to be capable of stimulating inflammatory cytokine production by monocytic cells. The authors suggest that this pro-inflammatory activity may result in recruitment of lymphocytes to a site of KSHV infection, providing further cells for infection by the virus and so promoting dissemination of the virus throughout the system of an infected host. By contrast, Foster-Cuevas et al. (2004) concluded that membrane-expressed vOX2 down-regulates macrophage activation. They suggest that Chung's results may be due to misfolding of their soluble recombinant protein, but this would not explain the contradictory results obtained with cell-surface expressed protein in the two studies. Thus, to date, the biological activity of vOX2 remains unknown.

The present inventors have found that soluble vOX2 is capable of exerting anti-inflammatory effects on the innate arm of the immune system. Accordingly, in its most general form, the present invention provides the use of soluble vOX2 as an antiinflammatory agent.

In a first aspect, the invention provides the use of soluble vOX2 to inhibit an inflammatory immune response comprising contacting a population of cells of the immune system with soluble vOX2. All such uses are included within the scope of the invention, whether performed in vitro, ex vivo, or in vivo. The cell population typically comprises cells of the innate immune system, particularly phagocytes such as neutrophils, monocytes and/or macrophages, optionally in combination with a variety of other cells of the immune system including T lymphocytes (including CD4⁺ and CD8⁺ T lymphocytes) and B lymphocytes .

Without wishing to be bound by any particular theory, soluble v0X2 is believed to exert anti-inflammatory effects on phagocytic cells such as monocytic cells (such as macrophages) and on neutrophils. For example, it can specifically reduce the production of MCP-I and IL-8 by the macrophage-like cell line U937 treated with interferon gamma, and can also inhibit the oxidative burst of neutrophils.

Thus the methods may comprise the step of determining the extent of inhibition of the inflammatory immune response. This may be achieved by measuring inflammatory cytokine or chemokine production especially by macrophages (e.g. MCP-I and/or IL-8), and/or production of reactive oxygen intermediates, such as O₂ or OH radicals or H₂O₂. Of course, when the methods are performed in vivo, any suitable symptom of inflammation may be used to monitor the effect of the vOX2 protein, such as tissue infiltration by inflammatory cells, swelling, redness, etc..

The anti-inflammatory properties displayed by soluble v0X2 make it suitable for the treatment of inflammatory disorders or other conditions in which an inappropriate or undesirable immune response is responsible for patient symptoms and/or disease pathogenesis, including autoimmune diseases, allergies and graft rejection. Such conditions include rheumatoid arthritis, psoriasis, inflammatory bowel disease, multiple sclerosis, asthma, chronic obstructive pulmonary disease (COPD) , contact and allergic dermatoses, inflammatory eye disease, transplant rejection, vascular inflammation (cardiac disease) and inflammatory neurologic syndromes.

The therapeutic methods described in this specification may involve any suitable method of delivering soluble vOX2 to the site of inflammation. For example, a soluble vOX2 protein may be administered directly to a patient in need thereof. Alternatively a nucleic acid encoding soluble vOX2 may be administeed to a patient so that the protein is synthesised and secreted by the patient's own cells. In a further alternative, a cell capable of expressing and secreting soluble vOX2 may be administered to the patient.

Thus the present invention provides a soluble vOX2 protein, a nucleic acid encoding soluble vOX2, or a cell capable of expressing and secreting soluble vOX2, for use in a method of medical treatment.

The invention further provides a soluble vOX2 protein, a nucleic acid encoding soluble vOX2, or a cell capable of expressing and secreting soluble vOX2, for use in the preparation of a medicament for the prophylaxis or treatment of an inflammatory disorder.

The invention further provides a method of prophylaxis or treatment of an inflammatory disorder, comprising administering an effective amount of a soluble vOX2 protein, a nucleic acid encoding soluble vOX2, or a cell capable of expressing and secreting soluble vOX2, to a patient in need thereof. The invention further provides a pharmaceutical composition comprising a soluble vOX2 protein, a nucleic acid encoding soluble vOX2, or a cell capable of expressing and secreting soluble vOX2, in combination with a pharmaceutically acceptable carrier.

The vOX2 protein for use in the methods of the invention is typically obtained by recombinant expression and secretion, preferably from a eukaryotic cell.

The vOX2 protein may be multivalent. That is to say, it comprises two or more vOX2 moieties, in a divalent, trivalent, tetravalent or a higher-order complex. Without wishing to be bound by any particular theory, multivalent vOX2 may be capable of cross-linking receptors on the surface of target cells more effectively than monovalent vOX2 and so be capable of exerting its anti-inflammatory activity more effectively than monovalent protein.

Individual vOX2 moieties may be associated covalently or non- covalently in a vOX2 complex. The association may be direct (i.e. between vOX2 moieties) or indirect (via heterologous components linked to the vOX2 moieties) .

For example, the vOX2 moieties may be chemically cross-linked to one another.

Alternatively, the vOX2 moieties may be expressed as a fusion protein comprising two (or more) vOX2 moieties in a single polypeptide chain, optionally separated by a suitable polypeptide linker. Alternatively, the vOX2 moieties may be linked to heterologous (i.e. non-vOX2) components which interact with one another to cause association. Thus the complex may comprise first and second vOX2 moieties, linked to respective first and second heterologous components, wherein the first and second heterologous components associate with one another. The first and second heterologous components may be the same or different, and may associate with one another via covalent or non-covalent interactions .

Preferably each vOX2 moiety is expressed as a fusion protein with its respective heterologous component. Examples of heterologous components include oligoitierisation domains of transcription factors, such as leucine zipper motifs which associate with one another via hydrophobic interactions. However a particularly preferred example of a heterologous component is an antibody Fc region. A vOX2-Fc fusion protein in its native conformation will generally be dimeric, comprising two polypeptide chains, each chain consisting of one Fc moiety and one vOX2 moiety, the polypeptide chains being covalently associated by disulphide bonds between the Fc moieties.

The heterologous component may be capable of modulating the pharmacokinetic properties of the vOX2 molecule. For example, it may be able to modulate bioavailability, solubility, stability, half-life in vivo, etc.. Fusion proteins comprising an effector molecule linked to an antibody Fc region (also known as immunoadhesins) generally have increased solubility, stability and half-life as compared to the effector molecule alone. (Although it is normally desirable to increase these properties of the vOX2 moiety, it will be appreciated that it may sometimes be desirable to decrease some or all of these properties. A suitable heterologous component may be chosen accordingly.) It may also be desirable to link the vOX2 moiety to a heterologous components which modulates its pharmacokinetic properties in a desired manner but does not mediate association of vOX2 moieties into a multivalent complex.

In a further aspect the present invention provides a soluble anti-inflammatory agent comprising a complex of at least two vOX2 moieties .

As described above, the complex may comprise first and second vOX2 moieties, linked to respective first and second heterologous components, wherein the first and second heterologous components associate with one another.

Where the heterologous components are proteins or peptides, they are preferably expressed as fusion proteins with their respective vOX2 moieties. The invention therefore provides nucleic acids encoding such fusion proteins, the fusion proteins preferably being capable of being secreted by a host cell. Also provided are expression vectors comprising such nucleic acids and host cells comprising such expression vectors.

Where the heterologous components are the same (i.e. identical heterologous components associate with one another) , it will generally be possible to form multivalent complexes by expressing protein products from just one such nucleic acid construct.

Where the heterologous components are different (i.e. one heterologous component associates with another of a different type), it will be necessary to express protein products from nucleic acids encoding each type of fusion protein. The invention therefore provides a composition comprising a first nucleic acid encoding a fusion protein comprising a first vOX2 moiety and a first heterologous component, and a second nucleic acid encoding a fusion protein comprising a second vOX2 moiety and a second heterologous component, wherein, when expressed as proteins, the first and second heterologous components associate with one another. The fusion proteins are typically capable of being secreted by a suitable host cell. The first and second nucleic acids may be on separate expression vectors or the same expression vector. The invention also provides a host cell comprising said first and second nucleic acids.

In preferred embodiments, the heterologous components are antibody Fc regions. Thus the present invention provides a soluble vOX2-Fc fusion protein.

The invention also provides a nucleic acid encoding a soluble vOX2-Fc fusion protein capable of being secreted by a suitable host cell. Thus the nucleic acid typically encodes a signal peptide at the N-terminus of the fusion protein, to enable the fusion protein to be secreted by a host cell in which it is expressed

The invention also provides an expression vector comprising a nucleic acid encoding a soluble vOX2-Fc fusion protein.

Typically the vector comprises suitable transcriptional and translational regulatory sequences operably linked to a sequence encoding the fusion protein, to enable transcription and translation of the protein by the host cell.

The invention also provides a host cell containing an expression vector as described herein. The host cell is preferably a eukaryotic host cell, such as a mammalian cell, insect cell or yeast cell. In some embodiments, mammalian cells (such as CHO cells or human cells) may be preferred.

The invention also provides a pharmaceutical composition containing a fusion protein, a nucleic acid, an expression vector or a host cell as described in relation to the second aspect of the invention, in combination with a pharmaceutically acceptable carrier .

Brief Description of the Drawings figure 2: Production and purification of vOX2-Fc. A. Production of vOX2~Fc in stably transfected CHO cells. Western blot analysis of supernatants of engineered CHO cells harvested and analysed by with airti-human IgG-HRP antibody and visualized with ECL blotting reagents (Amersham) . Lane 1, untransfected normal CHO cells; Lane 2, CHO cells transfected with a recombinant pDR2ΔEFlα plasmid derivative, expressing an irrelevant Fc-fusion protein, KSHV complement control protein (KCP:Fc); Lane 3, CHO cell line (CHO-15"69) , stably transfected with pvOX2-Fc The positions of molecular size markers are indicated at the left of the blot. B. Protein purification and polishing: Western blot analysis of affinity purified and ^polished' recombinant vOX2~Fc. Lane 1, untransfected normal CHO cells; Lane 2, Recombinant vOX2- Fc purified from the CHO cell line (CHO-15"69) , stably transfected with pvOX2-Fc. Protein purification was performed by affinity chromatography (Hi Trap Protein A column) and size exclusion chromatography (Superdex 200). Purified vOX2-Fc protein was analysed by western blot with an anti-human IgG-HRP antibody and visualized with ECL blotting reagents (Amersham) .

Figure 2 : Inhibi tion of inflamma tory chemokine production in U937 monocytic cells by recombinant vOX-2 : Fc protein . A . MCP-I levels. B. IL-8 levels. U937 cells were cultured at a density of Ix 10^s for 48 hours in the presence or absence of recombinant IFN-Y (5ng) and vOX~2:Fc, (lOμg/ml) before cytokine quantification from the culture fluid by the Luminex assay.

Figure 3: Inhibition of oxidative burst, but not engulfment, in primary human peripheral blood neutrophils by recombinant vOX- 2: Fc protein. A. Oxidative burst. B. Engulfment. These components of phagocytic activity were measured in whole blood flow cytorαetrically by the commercial Bursttest and Phagotest assays (OPREGEN Pharma; BD Biosciences) , gating on granulocytes, for which 5000 events were acquired. Prior to performing the assay, whole, heparinised blood was incubated (90 minutes, 37°C, 5% CO₂) with either recombinant vOX2~Fc (10 μg/ml) , or purified human IgG (10 μg/ml), or left untreated. White blood cell differential counts were performed on each blood sample at the time of collection, which enabled the assays to be performed at an E. coli: neutrophil ratio of 6:1, and the data to be normalised to mean fluorescence intensity (MFI) for each subject. The cumulative data are shown for the study of 13 whole blood samples from 11 healthy subjects in three separate experiments.

Figure 4: In vivo inhibition of the carrageenan-induced acute inflammatory response by recombinant vOX-2 : Fc . Acute inflammation of the footpad of BALB/c mice was induced by administration of the seaweed extract carrageenan into one hind paw of each animal. Four groups of mice received intraperitoneal (IP) injection of either recombinant vOX2-Fc (80μg/mouse; group ■) , purified human IgG (80μg/mouse; group A), Dexamethasone (500μg/mouse; group •) or PBS (group ♦) 30 minutes before carrageenan administration, and then again at 6-, 12-, and 24-hours post-administration of carrageenan. The extent of inflammation was assessed from, footpad thickness measurements and calculated as the difference in depth between the carrageenan- inoculated hind footpad compared with the uninoculated hind footpad of the same animal. The data are pooled from three independent, single blinded experiments and represent means +/- SE for each group (24 mice per group for PBS and vOX2-Fc treatments, eight mice received purified human IgG and 16 were treated with Dexamethasone) . Group comparison data are presented in the Table.

Figures 5 and 6: Effect of vOX2-Fc on development and severity of collagen-induced, arthritis in DBA mice. In each figure, Panel A shows mean arthritic score for each group of 10 mice. Panel B shows percentage incidence of arthritis within each group. Figure 5 shows development of disease up to day 37. Figure 6 shows results of the same experiment up to day 42.

Figure 7; The amino acid sequence of the vOX2-Fc fusion protein used in this study. vOX2 sequence is shown in normal font and underlined. This represents amino acids 78-307 of the full- length sequence proposed by Russo et al. The sequence AADPI (normal font, not underlined) is a linker region. Immunoglobulin γl Fc sequence is shown in italic font. The immunoglobulin hinge region (PKSCDKPHTCP) is shown in italics and underlined.

Detailed Description of the Invention

V0X2 vOX2 is a protein encoded by the open reading frame designated K14 of the KSHV virus (Jenner et al. , 2001). An exemplary sequence for the vOX2 protein, taken from Russo et al. (PNAS 93:14862 (1996)), is as follows: 1 MIHTFFDCPGRRVVEGGVISSYFLIGAPGRTAIKTEEGVSALQN

78 LPPTVLPVAGTGSVVRPVVCAPPWTTPSASRGSMSSLFISLPWVAFIWLALLGAVGGA

RVQGPMRGSAALTCAITPRADIVSVTWQKRQLPGPVNVATYSHSYGVVVQTQYRHKAN

ITCPGLWNSTLVIHNLAVDDEGCYLCIFNSFGGRQVSCTACLEVTSPPTGHVQVNSTE

DADTVTCLATGRPPPNVTWAAPWNNASSTQEQFTDSDGLTVAWRTVRLPRGDNTTPSE

307 TM GICLITWGNESISIPASIQGPLAHDLPAAQGTLAGVAITLVGLFGIFALHBCRRKQGG ASPTSDDMDPLSTQ

The viral protein is a membrane-bound protein comprising an N- terminal extracellular domain, a transmembrane domain (underlined) , and a C-terminal intracellular domain. The open reading frame contains two potential initiation methionines, at amino acids 1 and 78. Methionine 78 is generally thought most likely to be used as the initiation codon in vivo.

The function of vOX2 in vivo is controversial. However, the present inventors have now found that soluble vOX2 displays antiinflammatory properties and consequently may be used to inhibit inflammatory responses both in vivo and in vitro.

The soluble vOX2 proteins used in the methods of the invention may be multivalent. That is to say, they may comprise two or more vOX2 moieties. Multivalent vOX2 may be capable of cross- linking receptors on the surface of target cells more effectively than monovalent vOX2. vOX2 moieties may be associated with one another either covalently or non-covalently, although covalent association may be preferred for reasons of stability and possibly also activity.

The two vOX2 moieties may be coexpressed as a fusion protein. To produce a fusion protein, a nucleic acid expression vector is constructed comprising coding sequences for each moiety in one continuous open reading frame, so that the two moieties can be translated as part of the same polypeptide chain.

Typically, a flexible peptide linker is included between the two moieties to allow the two components to interact freely with one another without steric hindrance. The skilled person Is perfectly capable of designing a suitable linker. Conventionally, such linkers are between 12 and 20 amino acids in length, and have a high proportion of small and hydrophilic amino acid residues (e.g. glycine and serine) to provide the required flexibility without compromising aqueous solubility of the molecule .

Alternatively, the vOX2 moieties may be engineered to increase their affinity for one another. This may be achieved in various ways. For example, cysteine residues may be introduced to enable the two moieties to form disulphide bonds with one another.

As a further alternative, interaction between the two vOX2 moieties may be promoted by linking each one to a heterologous component, wherein the two heterologous components are capable of associating with one another. Where the heterologous components are polypeptides, they may be expressed as fusion proteins with the vOX2 moieties. Preferred heterologous components are polypeptides comprising antibody Fc sequences, and preferably one or more antibody Fc domains (e.g. CH2, CH3 and/or CH4 domains (if appropriate) of IgG, IgM, etc.) . Preferably the hinge sequence normally located between the CHl and CH2 domains is also included. The hinge region contains cysteine residues which form disulphide bonds between the heavy chains of the intact native antibody. Thus if the hinge regions are present in the vOX2-Fc molecules described herein, similar bonds will be formed to stabilise the interactions between the chains. Human IgGl is a preferred fusion partner.

The skilled person will be aware of alternative heterologous components which may be used to increase or stabilise the interaction between vOX2 moieties. These include leucine zipper polypetides, which dimerise via hydrophobic interactions.

Although recombinant methods are preferred, vOX2 moieties may also be covalently linked by chemical means. Bifunctional and polyfunctional chemical linker molecules suitable for conjugating or cross-linking polypeptide molecules to one another are well known to the skilled person.

Whether multivalent or monovalent, each vOX2 moiety of the soluble vOX2 proteins described herein preferably comprises the extracellular domain of the vOX2 protein (i.e. the sequence from amino acid 78 to 309, including or not including the signal peptide as desired) , or a fragment thereof sufficient to demonstrate an anti-inflammatory effect. By anti-inflammatory effect is meant a statistically significant reduction in any one or more of IL-8 or MCP-I production by U937 cells, oxidative burst by neutrophils, inflammation in the carrageenan model, or disease incidence or severity in collagen-induced arthritis model using DBA mice, as assessed using any one of the assays described in the Examples .

Preferably the vOX2 moiety comprises at least 50 amino acids of the ECD sequence shown above, and may comprise at least 100 , 150 or 200 amino acids of the ECD sequence shown above.

However, the skilled person will appreciate that certain changes may be made to this amino acid sequence while substantially retaining the anti-inflammatory activity of the soluble protein. Thus the vOX2 protein or moiety may comprise a sequence of at least 50, 100, 150 or 200 amino acids having at least 80% identity with that sequence over the relevant overlap, preferably at least 85% sequence identity, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to that sequence, as long as it displays the required anti-inflammatory activity.

In particular, conservative substitutions in the vOX2 sequence

(as compared to the reference sequences) may be particularly well tolerated, without substantial effect on function.

A conservative substitution may be defined as a substitution within an amino acid class and/or a substitution that scores positive in the BLOSUM62 matrix.

According to one classification, the amino acid classes are acidic, basic, uncharged polar and nonpolar, wherein acidic amino acids are Asp and GIu; basic amino acids are Arg, Lys and His; uncharged polar amino acids are Asn, GIn, Ser, Thr and Tyr; and non-polar anno acids are Ala , GLy , VaI , Leu, lie , Pro , Phe , Met , Trp and Cys .

According to another classification, the ammo acid classes are small hydrophilic, acid/acid amide/hydrophilic, basic, small hydrophobic and aromatic, wherein small hydrophilic ammo acids are Ser, Thr, Pro, Ala and GIy; acid/acidamide/hydrophilic ammo acids are Asn, Asp, GIu and GIn; basic ammo acids are His, Arg and Lys; small hydrophobic ammo acids are Met, lie, Leu and VaI; and aromatic ammo acids are Phe, Tyr and Trp

Substitutions which score positive in the BLOSUM62 matrix are as follows :

Percent (%) ammo acid sequence identity with respect to a reference sequence is defined as the percentage of ammo acid residues m a candidate sequence that are identical with the ammo acid residues m the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. % identity values may be determined by WU-BLAST-2 (Altschul et al., Methods m Enzymology, 266:460-480 (1996)). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11. A % ammo acid sequence identity value is determined by the number of matching identical residues as determined by WU-BLAST-2, divided by the total number of residues of the reference sequence (gaps introduced by WU-BLAST-2 into the reference sequence to maximize the alignment score being ignored), multiplied by 100.

Percent (%) amino acid similarity is defined in the same way as identity, with the exception that residues scoring a positive value in the BLOSUM62 matrix are counted. Thus, residues which are non-identical but which have similar properties (e.g. as a result of conservative substitutions) are also counted.

In a similar manner, percent (%) nucleic acid sequence identity with respect to a reference nucleic acid is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the reference nucleic acid sequence. The identity values used herein may be generated by the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.

The soluble vOX2 proteins described in this specification are preferably expressed from eukaryotic cells such as yeast cells, insect cells such as Sf9 cells, and mammalian cells such as CHO (Chinese Hamster Ovary) cells.

Preferably the protein is correctly folded and therefore able to bind to CD200 receptor (CD200R) . Suitable assays are described by Foster-Cuevas et al. (2004) and references cited therein. Preferably at least 20% of the vOX2 moieties in a preparation of the vOX2 protein are able to bind to CD200 receptor; more preferably at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the vOX2 moieties in a preparation of the vOX2 protein are able to bind to CD200 receptor. Affinity purification techniques using CD200 receptor may be used to achieve the desired level of binding activity in a preparation of soluble vOX2 protein.

Therapeutic uses of soluble vOX2 protein

The present inventors have shown that soluble vOX2 has antiinflammatory properties and in particular can down-modulate reactions mediated by the innate arm of the immune system, e.g. by neutrophils and macrophages. The protein can specifically reduce the production of MCP-I and IL-8 by the macrophage-like cell line U937 treated with interferon gamma, and can also inhibit the oxidative burst of neutrophils. It is also capable of reducing inflammation induced by carrageenan in a murine model, and, importantly, has a profound effect on the development and severity of collagen-induced arthritis in DBA mice. This result is particularly surprising.

Soluble vOX2 can therefore be used in the treatment of inflammation, inflammatory disorders, or other disorders in which an inappropriate or undesirable immune response involving the innate immune system is responsible for disease symptoms and/or pathogenesis. Such conditions include autoimmune diseases, allergies and graft rejection. Thus, for example, soluble vOX2 may be used to treat rheumatoid arthritis, psoriasis, inflammatory bowel disease, multiple sclerosis, asthma, chronic obstructive pulmonary disease (COPD) , contact and allergic dermatoses, inflammatory eye disease, transplant rejection, vascular inflammation (cardiac disease) and inflammatory neurologic syndromes. Soluble vOX2 protein may be administered directly to subjects in pharmaceutical compositions.

Alternatively, nucleic acids encoding soluble vOX2 protein may be administered to subjects such that soluble vOX2 protein is expressed and secreted from the subject's own cells. Typically the nucleic acids will be part of one or more expression vectors, which may be administered as naked nucleic acid or in a delivery vehicle such as viral vector (e.g. an adenoviral, lentiviral or retroviral vector) .

As an alternative, cells which have been engineered to secrete soluble vOX2 protein may be administered to a subject. Preferably the cells are syngeneic or histocompatible with the subject. For example, cells may be removed from a subject, transfected with one or more suitable vectors, and readministered to the subject. Additionally or alternatively, the secretor cells may be encapsulated, e.g. in a biologically inert polymer, to prevent interaction between the implanted cells and the host immune system.

The skilled person will be capable of designing suitable nucleic acid expression vectors for therapeutic uses (as well as for other uses described in this specification) . The vectors will typically contain appropriate regulatory sequences, including promoter sequences, terminator fragments, enhancer sequences, marker genes and other sequences, depending upon the particular form of soluble vOX2 protein which is to be administered (see above) . The vectors may be intended to integrate into a host cell chromosome, or may exist and replicate independently of the host chromosomes as an episome. Preferred, subjects for treatment by the methods of the invention are mammals. Preferred subjects are primates (including humans), rodents (including mice and rats) , and other common laboratory, domestic and agricultural animals, including but not limited to rabbits, dogs, cats, horses, cows, pigs, sheep, goats, etc..

The complexes, polypeptides, nucleic acids and cells described herein can be formulated in pharmaceutical compositions. These compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular and intraperitoneal routes.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Whatever the nature of the active agent that is to be given to an individual (e.g. a cell, polypeptide, nucleic acid molecule, other pharmaceutically useful agent according to the present invention) , administration is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington' s Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

Alternatively, targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons; for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.

A composition may be administered alone or in combination with. other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Materials and Methods

Cloning- of vOX2-Fc Fusion protein

KSHV vOX2 protein was expressed as the amino-terminal domain of a fusion protein in frame with a C-terminal fragment of the Fc region of human IgGi. The second of two potential methionine residues (residue 1 or residue 78) was selected as the initiation codon, based on our own studies (data not shown) and previously published analyses (Chung et al., 2002, Neipel et al., 1997, Russo et al., 1996). ORF Kl4 was PCR amplified and TA cloned into the pCR2.1-TOPO vector (Invitrogen) . The gene was amplified from, the BC-I KSHV-infected primary effusion lymphoma cell line (Cesarman et al., 1995) with the PCR primers: sense, 5'-GCT CTA GAT GTC TAG CCT CTT CAT TTC ATT AC-3'; antisense, 5'-TAT GCG GCC GCG GCC GCG GGA AGG TCA TGG GC-3'. These amplification primers included restriction sites (Not I and Xba I) enabling subcloning of the gene into the expression vector. Sequencing of both strands of the K14 insert confirmed that no errors had been introduced by PCR and the gene was subcloned into the pDR2ΔEFlα expression vector (see (Spiller et al., 2003)) to create pvOX2-

Fc. Subcloning involved digestion of the K14 insert from ρCR2.1- TOPO with Xba I and Not I and ligation into Spe I and Wot I digested pDR2ΔEFlα. The pvOX2-Fc plasrαid was sequenced over the site at which K14 was cloned to ensure that the ligation had generated an in-frame fusion of the viral and IgG₁ genes. The amino acid sequence of the resulting vOX2-Fc fusion protein is shown in Figure 7.

Cells and cell culture The human monoblastic cell line U937 was cultured at 37⁰C, 5% CO₂ in RPMI 1640 medium (BioWhittaker) supplemented with 10% heat- inactivated fetal bovine serum (FBS), L-glutamine (2 πiM) , penicillin-streptomycin. (1%), non-essential amino acids (0.1 mM) . To differentiate U937 cells into a macrophage-like phenotype, they were cultured for 48 hours in the presence of recombinant interferon (IFN) -Y (5 ng/ml; R & D Systems).

Heparinised venous blood was collected from healthy adult volunteers, after obtaining their informed consent. White blood cell and differential counts were performed with a Sysmex SE, 9500 haematology analyser (Sysmex Corp., Japan). In some assays, polymorphonuclear cells were isolated from whole blood by dextran sedimentation and hypotonic lysis of red blood cells, followed by differential density gradient centrifugation through Ficoll (Sigma) .

To create a vOX2-Fc protein-producing cell line, pvOX2-Fc was transfected into CHO cells. Stable cell lines were generated in medium containing Hygromycin B (500 μg/ml) and screened for recombinant protein production by immunofluorescence and western blot with anti-human IgG-FITC (Sigma) or anti-human IgG-HRP (Sigma) antibodies, respectively. The most productive cell line for vOX2-Fc protein production was designated CHO-15"69.

Protein production and purification

CHO-15"69 cell supernatants were harvested and vOX2-Fc was affinity purified on a protein A column (Hi Trap, Amersham) using the AKTA protein purifier system (Amersham) . To remove truncated vOX:Fc and Fc protein fragments, this protein, was then ^polished' by gel filtration on a Superdex 200 size exclusion column (Amersham) . Protein purity was assessed by polyacrylamide gel electrophoresis and analyses of the gels by staining with

Cαorαassie Brilliant Blue R250 (Bio-Rad Laboratories), or Sypro Ruby (Analgene) , and western blot detection with anti-human IgG- HRP and anti-bovine Ig-HRP (Sigma) . All protein bands were excised and identified by either mass spectrometry (MS) or matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS. Human IgG was purified by the same protocol for use as a negative control in some experiments.

Oxidative burst assay

The oxidative burst component of the phagocytic process was measured in whole blood peripheral polymorphonuclear monocytes with the commercial flow cytometric-based Bursttest assay

(OPREGEN Pharma; BD Biosciences, Oxford, UK) . This assay allows the quantitative determination of leukocyte activity without confounding it by prior purification of the cells. The Bursttest assay relies on unlabelled opsonized E.coli bacteria as the particulate stimulus and dihydrorhodamine (DHR) 123 as a fluorogenic substrate of oxidative activity. Briefly, heparinised whole blood was incubated (90 minutes, 37°C, 5% CO₂) with either recombinant vOX2-Fc (10 μg/ml) or purified human IgG (10 μg/ml) and again (7 minutes, 37⁰C, 5% CO₂) with opsonized E. coli cells (6 cells per leukocyte). A sample without stimulus serves as a negative (background) control. DHR 123 was then added and the cells incubated (10 minutes, 37°C, 5% CO₂) . The reaction was stopped by the addition of lysing buffer, which partially fixes leukocytes and lyses erythrocytes. Finally, to exclude aggregation artifacts of bacteria or platelets, cellular DNA was stained immediately prior to flow cytometric analysis. Cells were analysed by flow cytometry (FACSCalibur, BD Biosciences) and 5000 granulocyte events acquired to obtain the percentage and number of cells having produced reactive oxygen radicals, as well as the extent of their enzymatic activity, as measured by the mean fluorescence intensity. Phagocytosis assay

The engulfment aspect of phagocytosis was measured in whole blood peripheral polymorphonuclear monocytes with the commercial flow cytoπietric-based Phagotest kit (OPREGEN Pharma; BD Biosciences) . Like the Bursttest, this assay system allows the quantitative determination of leukocyte engulfment activity without confounding it by prior purification. Briefly, heparinised whole blood was incubated (90 minutes, 37°C, 5% CO₂) with either recombinant vOX2-Fc (10 μg/ml) or purified human IgG^' (10 μg/ml) and again (10 minutes, 37°C, 5% CO₂) with opsonized FITC-labeled E. coli cells (6 cells per leukocyte). Ingestion was terminated by incubation at 4⁰C and the addition of quenching solution. This solution allows flow cytometric discrimination between attached and internalised bacteria by quenching the FITC fluorescence of surface bound bacteria, but leaving the fluorescence of internalised particles unaltered. After washing steps, erythrocytes were removed by hypotonic lysis. Finally, cellular DNA was stained, as described for the Bursttest assay. Cells were analysed by flow cytometry (FACSCalibur, BD Biosciences) and 5000 granulocyte events acquired.

Measurement of MCP-I and IL-8 Protein

U937 cells (lxlO⁶/ml) were either untreated, or treated with rIFN-γ (5 ng/iαl) in the presence or absence of recombinant vOX2- Fc protein (10 μg/ml) and incubated for 48 hours (37°C, 5% CO₂) . The culture fluids were then, collected and the concentration of cytokines and chemokines quantified by the Luminex assay.

In vivo stndy A murine model of acute inflammation was tested to assess the in vivo anti-inflammatory effects of recombinant vOX2-Fc protein. In this model, acute inflammation of the footpad of BALB/c mice (Jackson Laboratories) was induced by administration of the seaweed extract carrageenan into one hind paw of each animal, as described previously (Leung et al., 2001). In each experiment, four groups of BALB/c mice received intra-peritoneal (IP) injection of either recombinant vOX2-Fc (80 μg/mouse) , purified human IgG (80 μg/mouse) , Dexamethasone (500 μg/mouse) or phosphate buffered saline (PBS) 30 minutes before carrageenan administration. The extent of inflammation was determined from footpad thickness measurements and calculated as the difference in depth between the carrageenan-inoculated hind footpad compared with the uninoculated hind footpad of the same animal. These measurements were taken 6-, 12-, and 24~hours post-administration of carrageenan. Three independent single blinded experiments were performed.

Statistical analyses

The differences between treatment groups in the Bursttest, Phagotest and cytokine assays were determined by the non- parametric Mann Whitney test. These differences were considered significant if P ≤0.05. For the in vivo study, data were analysed by GLM Repeated Measures, followed by Tukey, as a multiple comparison test (SPSS) between the four treatment groups. To analyses the differences between groups at each time point (6, 12, 24h) , the Mann Whitney test was performed. Differences were considered significant if P ≤0.05.

Results

Cloning, production and purification of recombinant vOX~2 :Fc pxotein The full-length KSHV ORFK14 gene was cloned into the ρDR2ΔEFlα eukaryotic vector from which vOX2 protein was produced as a fusion with the C-terminal domain of human IgGi Fc. Advantages of expressing vOX2 as a fusion protein with this IgG₁ domain include (i) it provides an affinity tag for protein purification, and (ii) it can increase he half-life of a recombinant protein by up to 10-fold (Harris et al., 2002). Recombinant vOX-2-.Fc protein was then produced in the cell line CHO-15"69 (Figure IA) and purified from the culture fluid by protein A affinity and size exclusion chromatography (Fig. IB) . Size exclusion chromatography was selected since multiple bands of recombinant vOX2-Fc were purified by protein A affinity chromatography alone (Fig. IB) . The identity of these bands was determined by MS or MALDI-TOF MS to be vOX2-Fc dimers, monomers and truncations, and Fc alone (data not shown) . Following size exclusion chromatography, a single band of recombinant vOX2-Fc was obtained (Fig. IB) .

XnhiJbi.ti.on of myeloid cell inflammatory responses in vitro by vOX-2;Fc protein

To determine if vOX~2:Fc protein modulates myeloid cell inflammatory responses, the cytokine production profile was evaluated from macrophage-like U937 cells exposed to this recombinant protein. U937 cells were either untreated, or treated with rlFN-y in the presence and absence of v0X-2:Fc protein and assayed for inflammatory cytokine production. Treatment with v0X2-Fc reduced significantly the production of MCP-I, by about 30% (Figure 2A), and IL-8 by approximately 50%, (Figure 2B) . This effect was specific, since the production of other inflammatory cytokines including eotaxin, MIP-Ia, MIPlβ_r RANTES, IL-lβ, IL-6 and TNFα was unaffected (data not shown) .

Further investigation of the modulation of myeloid cell activity by vOX-2:Fc revealed that the protein significantly down- regulates, by at least 25%, the oxidative burst activity of primary human neutrophils (Figure 3A) . This inhibition was specific to this aspect of phagocytosis, since the engulfment activity of these cells was unaffected (Figure 3B) .

Inhibition of the acute inflammatory response in vivo by recombinant vOX-2 :Fc

Since recombinant vOX2-Fc protein effected anti-inflammatory activities in vitro, it's in vivo potential in this regard was determined. Thus, to examine the effect of vOX2-Fc on the development of experimental acute inflammation, BALB/c mice were treated with recombinant vOX2-Fc protein (80 μg) , which was administered IP 30 minutes before induction of acute inflammation by footpad inoculation with carrageenan. The extent of footpad inflammation was determined 6-, 12- and 24-hours later (see Methods) . Each of three control groups of mice were injected with either purified human IgG (80 μg) , Dexamethasone (500 μg) or PBS, in place of vOX2-Fc (Figure 4) . Overall, treatment with recombinant vOX2-Fc significantly suppressed the acute inflammatory responses, as determined by footpad thickness measurements, according to analysis by GLM Repeated Measures, followed by Tukey test: PBS vs. vOX2-Fc, P<0.0001; IgG vs. vOX2- Fc, P <0.005; Dexamethasone vs. vOX2-Fc, P <0.001. As expected, there was no significant difference between the inflammatory responses in the PBS and IgG treatment groups. To analyse the inhibitory impact between groups of vOX2-Fc at each time point (6,12 and 24 hrs) , the non-parametric Mann Whitney test was performed (Table 1) . These analyses indicated that recombinant vOX2-Fc protein significantly suppressed experimentally induced acute inflammation when compared with the PBS treatment group for the duration of the study (24 hours) and for at least 12 hours, when compared with treatment with human IgG. Histopathological analyses of the footpads support these analyses. Thus, the data showed a statistically-significant decrease in severity of acute inflammation and inflammatory cell recruitment at the site of inflammation as compared with untreated mice.

Table 1. Statistical analyses for the inhibition of inflammation induced by carrageenan treatment of mouse footpads . Measurements for the vOX2-Fc treatment group were compared with those of each of the control groups by the Mann Whitney non-parametric analysis. See the legend to Figure 4 for the experimental methods .

rnhϋbxtion of collagen-induced azthritis in DBA mice The effects of vOX2-Fc on collagen-induced arthritis in DBA mice was examined in a blinded study. Two other Fc fusion proteins were used as controls. These were an Fc fusion of the complement regulatory protein KCP, and an Fc fusion of a triple lysine mutant of the KCP protein (KCPmut) which lacks complement regulatory activity. Mice were immunised with type II collagen and complete Freund' s adjuvant on day 0, and challenged with type II collagen in saline on day 21. The first signs of disease are expected on day 27. Extensive disease is expected by day 35.

Groups of mice (10 in each group) received lOOμg of the relevant Fc fusion protein in PBS on each of days -1 and 0. Each group received 50μg of protein in PBS on day 3 and every third day thereafter up to and including day 27. A further control group of mice received PBS only. Results up to day 37 are shown in

Figure 5. The data is presented as mean score for each group at the relevant time point. Results up to day 42 (except for the KSP: Fc control) are shown in Figure 6.

The data indicates that vOX2-Fc significantly inhibits the development and severity of arthritis in this model.

Discussion

KSHV is the most recently described human oncogenic virus (Chang et al., 1994). It is the aetiologic agent of Kaposi's sarcoma (KS) , which is a complex tumour affecting AIDS patients and elderly Mediterranean men and is the most common tumour occurring in Africa. The pathogenesis of KS is highly complex, and recent evidence points to KSHV inducing transcriptional reprogramming of infected endothelial cells to a lymphatic phenotype, thereby promoting lymphangiogenesis, and hence tumourigenesis (Hong et al., 2004, Wang et al . , 2004) . The viral genes responsible for this transcriptional reorganisation have yet to be identified definitively, but could include a G-protein coupled receptor (Bais et al., 2003). Nevertheless, almost 20% of the KSHV genome encompasses genes that have immunomodulatory activity. They include genes specifying inhibition of MHC I surface expression (Coscoy & Ganem, 2000), modulation of the T cell immunological synapse (Coscoy & Ganerα, 2001) , inhibition of complement activation (Spiller et al., 2003), interruption of the interferon signaling cascade (Zhu et al., 2002), chemokine signaling {Boshoff et al., 1997) and B cell receptor signaling (Choi et al., 2000) .

The function of the vOX2 product of one of the putative immunomodulatory KSHV genes (ORF K14) is controversial, having been attributed with both macrophage activating (Chung et al., 2002) and inhibitory (Foster-Cuevas et al., 2004) activities.

To evaluate the function of vOX2 in the present study, it was expressed as an N-terminal fusion protein with the C-terminal crystallisable fragment (Fc) domain of human IgGi. We assessed the anti-inflammatory properties of vOX2-Fc in vitro by measuring its effects on phagocytic engulfment, oxidative burst and proinflammatory cytokine release in primary cells or cell lines and found that it reduces oxidative burst activity in neutrophils and down regulates the production of the pro-inflammatory chemokines IL-8 and MCP-I. Importantly, the impact of vOX2-Fc was also evaluated in a model in which acute inflammation of the footpad of mice is induced by administration of the seaweed extract carrageenan. Administration of recombinant vOX2-Fc significantly inhibited this inflammatory effect, providing incontrovertible evidence for the inhibition of acute inflammatory responses, particularly neutrophil influx, by this recombinant protein. Our findings reveal that the recombinant vOX2-Fc protein delivers a negative immunomodulatory signal to phagocytes, thereby inhibiting the innate inflammatory response. The vOX2-Fc fusion protein is also capable of inhibiting the development of collagen-induced arthritis in a murine model. The vOX2-Fc protein inhibited ROI production by human peripheral blood neutrophils, following their stimulation by bacterial cells. ROI homeostasis is critical for proper cellular function, since low levels are vital for many cell signaling events. Under physiological conditions, a balance exists between the level of ROIs produced during normal cellular metabolism and the level of endogenous antioxidants, which serve to protect tissues from oxidative damage (McCord, 1993) . Inhibition of oxidative burst in neutrophils by v0X2-Fc was dependent on pre-incubating v0X2-Fc with the cells for at least two hours. This dependency upon preincubation of cells with other immune modulators has been reported previously (Chen et al., 2004, Lehn et al., 1989). Also of note, IgG complexes are known stimulators of ROI production (Satriano et al., 1999), which provides further support for our IgG control studies that the inhibitory activities of v0X2-Fc are not due to the Fc component of the molecule .

Oxidative stress has been implicated in inflammatory reactions, and even a transient increase in ROI levels is an important mediator of vascular biology, affecting cell growth, apoptosis, migration, inflammation and secretion (reviewed in (Fattman et al., 2003, Touyz & Schiffrin, 2004). Furthermore, overproduction of ROIs has been associated with the pathogenesis of many diseases, such as cardiovascular diseases, neurological disorders, renal failures and pulmonary diseases (reviewed in (Bowler & Crapo, 2002, Delanty & Dichter, 1998, Fukai et al., 2002) ) .

ROIs could also represent a second messenger system for gene activation (reviewed in (Droge, 2002)), which may be of significance for production of cherαokines such as MCP-I and IL- during tissue injury (DeForge et al., 1993, Satriano et al., 1993, Vlahopoulσs et al., 1999). Indeed, chemokine release was a second myeloid lineage function negatively regulated by vOX2-Fc. We found that monocytic U937 cells, which had been differentiated by IFN-γ treatment, were significantly inhibited in their ability to produce MCP-I and IL-8 (Figure 2), both of which are proinflammatory (Akahoshi et al., 1994, Baggiolini, 2001, Endo et al., 1994, Frangogiannis et al., 2002, Hatano et al., 1999, Poddar et al., 2001, Withanage et al., 2004, Yla-Herttuala et al., 1991) .

Since proinflammatory chemokines and toxic oxygen metabolites augment the pathogenesis of cardiovascular, arthritic, neoplastic and neurodegenerative disorders, vOX2~Fc offers insight into the development of novel therapies against these diseases.

While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention. All references cited herein are expressly incorporated by reference. References

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Claims

Claims :

1. A method of inhibiting an inflammatory immune response comprising contacting a population of cells of the immune system with soluble vOX2.

2. A method according to claim 1, wherein said contacting is performed in vivo, in vitro or ex vivo.

3. A method according to claim 1 or claim 2 wherein the cell population comprises cells of the innate immune system.

4. A method according to claim 3, wherein the cell population comprises phagocytes such as neutrophils, monocytes and/or macrophages.

5. A method according to claim 3 or claim 4, wherein the cell population further comprises T lymphocytes and/or B lymphocytes.

6. A method according to any one of claims 1 to 6 further comprising determining the extent of inhibition of the inflammatory immune response.

7. A method according to claim 6 comprising determining inflammatory cytokine or chemokine production.

8. A method according to claim 7 wherein the inflammatory cytokine or chemokine is MCP-I and/or IL-8.

9. A method according to claim 6 comprising determining production of reactive oxygen intermediates .

10. A method according to claim 6 as dependent on claim 1 wherein the method is performed in vivo, and a symptom of inflammation such as tissue infiltration by inflammatory cells, swelling, redness, etc. is used to monitor the extent of inhibition of the inflammatory immune response.

11. A method according to any one of claims 1 to 10 wherein the vOX2 protein comprises two or more vOX2 moieties.

12. A method according to claim 11 wherein the vOX2 moieties are linked to heterologous components which interact with one another to cause association of the vOX2 moieties.

13. A method according to claim 12 wherein each vOX2 moiety is expressed as a fusion protein with its respective heterologous component .

14. A method according to claim 12 or claim 13 wherein the heterologous components are antibody Fc regions.

15. A method according to claim 12 or claim 13 wherein the heterologous components are oligomerisation domains of transcription factors.

16. A method according to claim 15 wherein the heterologous components are leucine zipper motifs.

17. A method according to any one of claims 1 to 16 which is performed in vivo for the prophylaxis or treatment of an autoimmune diseases, allergy or graft rejection.

18. A method according to claim 17 which is performed in vivo for the prophylaxis or treatment of rheumatoid arthritis, psoriasis, inflammatory bowel disease, multiple sclerosis, asthma, chronic obstructive pulmonary disease (COPD), contact or allergic dermatosis, inflammatory eye disease, transplant rejection, vascular inflammation (cardiac disease) or an inflammatory neurologic syndrome.

19. A method according to claim 17 or claim 18 comprising administering soluble v0X2 protein, a nucleic acid encoding soluble v0X2 protein, or a cell capable of expressing and secreting soluble vOX2 protein to a subject in need thereof.

20. A soluble vOX2 protein for use in a method of medical treatment.

21. A soluble vOX2 protein for the prophylaxis or treatment of an inflammatory disorder.

22. A method of prophylaxis or treatment of an inflammatory disorder, comprising administering an effective amount of a soluble vOX2 protein to a patient in need thereof.

23. A pharmaceutical composition comprising a soluble vOX2 protein in combination with a pharmaceutically acceptable carrier.

24. A soluble vOX2 protein or a method according to claim 21 or claim 22 wherein the inflammatory disorder is an autoimmune diseases, allergy or graft rejection.

25. A soluble vOX2 protein or a method according to claim 24 wherein the inflammatory disorder is rheumatoid arthritis, psoriasis, inflammatory bowel disease, multiple sclerosis, asthma, chronic obstructive pulmonary disease (COPD) , contact or allergic dermatosis, inflammatory eye disease, transplant rejection, vascular inflammation (cardiac disease) or an inflammatory neurologic syndrome.

26. A soluble vOX2 protein, method or pharmaceutical composition according to any one of claims 20 to 25 wherein the vOX2 protein comprises two or more vOX2 moieties.

27. A soluble vOX2 protein, method or pharmaceutical composition according to claim 26 wherein the vOX2 moieties are linked to heterologous components which interact with one another to cause association of the vOX2 moieties.

28. A soluble vOX2 protein, method or pharmaceutical composition according to claim 21 wherein each vOX2 moiety is expressed as a fusion protein with its respective heterologous component .

29. A soluble vOX2 protein, method or pharmaceutical composition according to claim 27 or claim 28 wherein the heterologous components are antibody Fc regions .

30. A soluble vOX2 protein, method or pharmaceutical composition according to claim 27 or claim 28 wherein the heterologous components are oligomerisation domains of transcription factors.

31. A soluble vOX2 protein, method or pharmaceutical composition according to claim 30 wherein the heterologous components are leucine zipper motifs.

32. A nucleic acid encoding soluble vOX2 for use in a method of medical treatment.

33. A nucleic acid encoding soluble vθX2 for the prophylaxis or treatment of an inflammatory disorder.

34. A method of prophylaxis or treatment of an inflammatory disorder, comprising administering an effective amount of a nucleic acid encoding soluble vOX2 to a patient in need thereof.

35. A pharmaceutical composition comprising a nucleic acid. encoding soluble vOX2 in combination with a pharmaceutically acceptable carrier.

36. A nucleic acid or a method according to claim 33 or claim 34 wherein the inflammatory disorder is an autoimmune diseases, allergy or graft rejection.

37. A nucleic acid or a method according to claim 36 wherein the inflammatory disorder is rheumatoid arthritis, psoriasis, inflammatory bowel disease, multiple sclerosis,^' asthma, chronic obstructive pulmonary disease (COPD) , contact or allergic dermatosis, inflammatory eye disease, transplant rejection, vascular inflammation (cardiac disease) or an inflammatory neurologic syndrome.

38. A nucleic acid, method or pharmaceutical composition according to any one of claims 32 to 37 wherein the soluble vOX2 protein is capable of being secreted from a host cell in which it is expressed.

39. A nucleic acid, method or pharmaceutical composition according to any one of claims 32 to 38 wherein the nucleic acid encodes a fusion protein comprising two or more vOX2 components optionally separated by a linker.

40. A nucleic acid, method or pharmaceutical composition according to any one of claims 32 to 38 wherein the nucleic acid encodes a fusion protein comprising a soluble vOX2 component linked to a heterologous component, wherein two such heterologous components are capable of associating with one another to mediate association of the vOX2 moieties.

41. A nucleic acid, method or pharmaceutical composition according to claim 39 or claim 40, wherein the fusion protein is capable of being secreted from a host cell in which it is expressed.

42. A nucleic acid, method or pharmaceutical composition according to claim 40, wherein the heterologous component is an antibody Fc region.

43. A cell capable of expressing and secreting soluble vOX2 for use in a method of medical treatment.

44. A cell capable of expressing and secreting soluble vOX2, for the prophylaxis or treatment of an inflammatory disorder.

45. A method of prophylaxis or treatment of an inflammatory disorder, comprising administering an effective amount of a cell capable of expressing and secreting soluble vOX2, to a patient in need thereof.

46. A pharmaceutical composition comprising a cell capable of expressing and secreting soluble vOX2, in combination with a pharmaceutically acceptable carrier.

47. K cell or a method according to claim 44 or claim 45 wherein the inflammatory disorder is an autoimmune diseases, allergy or graft rejection.

48. A cell or a method according to claim Al wherein the inflammatory disorder is rheumatoid arthritis, psoriasis, inflammatory bowel disease, multiple sclerosis, asthma, chronic obstructive pulmonary disease (COPD) , contact or allergic dermatosis, inflammatory eye disease, transplant rejection, vascular inflammation (cardiac disease) or an inflammatory neurologic syndrome.

49. A cell, method or pharmaceutical composition according to any one of claims 43 to 48 wherein the soluble vOX2 protein is as defined in any one of claims 26 to 31.

50. A composition comprising a first nucleic acid encoding a fusion protein comprising a first vOX2 moiety and a first heterologous component, and a second nucleic acid encoding a fusion protein comprising a second vOX2 moiety and a second heterologous component, wherein, when expressed as proteins, the first and second heterologous components associate with one another.

51. A composition according to claim 50, wherein the fusion proteins are capable of being secreted from a host cell in which they are expressed.

52. A composition according to claim 50 or claim 51 wherein the first and second nucleic acids are on separate expression vectors .

53. A composition according to claim 50 or claim 51 wherein the first and second nucleic acids are on the same expression vector.

54. A composition according to any one of claims 50 to 53 wherein the heterologous components are oligomerisation domains of transcription factors.

55. A composition according to claim 54 wherein the heterologous components are leucine zipper motifs .

56. A composition according to any one of claims 50 to 55, for use in a method of medical treatment.

57. A composition according to any one of claims 50 to 55 for the prophylaxis or treatment of an inflammatory disorder.

58. A method of prophylaxis or treatment of an inflammatory disorder, comprising administering an effective amount of first and second nucleic acids as described in any one of claims 50 to 55 to a patient in need thereof.

59. A pharmaceutical composition comprising a composition according to any one of claims 50 to 55 in combination with a pharmaceutically acceptable carrier.

60. A composition or a method according to claim 59 wherein the inflammatory disorder is an autoimmune disease, allergy or graft rejection.

61. A composition or a method according to claim 60 wherein the inflammatory disorder is rheumatoid arthritis, psoriasis, inflammatory bowel disease, multiple sclerosis, asthma, chronic obstructive pulmonary disease (COPD) , contact or allergic dermatosis, inflammatory eye disease, transplant rejection, vascular inflammation (cardiac disease) or an inflammatory neurologic syndrome.

62. A kit comprising first and second nucleic acids as described in any one of claims 50 to 55.

63. A kit according to claim 62, wherein each nucleic acid is in combination with a pharmaceutically acceptable carrier.

64. A kit according to claim 62 or claim 63 wherein said first and second nucleic acids are present on respective first and second expression vectors.

65. A host cell comprising a nucleic acid as described in any one of claims 38 to 42, or first and. second nucleic acids as described in any one of claims 50 to 55.

66. A cell according to claim 65 for use in a method of medical treatment.

67. A cell according to claim 65 for the prophylaxis or treatment of an inflammatory disorder.

68. A method of prophylaxis or treatment of an inflammatory disorder, comprising administering an effective amount of a cell according to claim 65 to a patient in need thereof.

69. A pharmaceutical composition comprising a cell according to claim 65 in combination with a pharmaceutically acceptable carrier.

70. A cell or a method according to claim 67 or claim 68 wherein the inflammatory disorder is an autoimmune diseases, allergy or graft rejection.

71. A cell or a method according to claim 72 wherein the inflammatory disorder is rheumatoid arthritis, psoriasis, inflammatory bowel disease, multiple sclerosis, asthma, chronic obstructive pulmonary disease (COPD) , contact and allergic dermatosis, inflammatory eye disease, transplant rejection, vascular inflammation (cardiac disease) or an inflammatory neurologic syndrome.

72. A soluble anti-inflammatory agent comprising a complex of at least two vOX2 moieties .

73. A soluble anti-inflammatory agent according to claim 72 comprising a fusion protein comprising at least two vOX2 moieties, optionally separated by a linker.

74. A soluble anti-inflammatory agent according to claim 72 comprising first and second vOX2 moieties, linked to respective first and second heterologous components, wherein the first and second heterologous components associate with one another.

75. A soluble anti-inflammatory agent according to claim 74 wherein the heterologous components are fusion proteins with their respective vOX2 moieties .

76. A soluble anti-inflammatory agent according to claim 74 or claim 75 wherein the heterologous components are antibody Fc regions .

77. A soluble anti-inflammatory agent according to claim 74 or claim 75 wherein the heterologous components are oligomerisation domains of transcription factors.

78. A soluble anti-inflammatory agent according to claim 77 wherein the heterologous components are leucine zipper motifs.

79. A nucleic acid encoding a fusion protein as described in any one of claims 73 or 75 to 78.

80. An expression vector comprising a nucleic acid according to claim 79.

81. A host cell comprising a nucleic acid according to claim 79 or an expression vector according to claim 80.