WO2003100419A1

WO2003100419A1 - Methods for using the cd163 pathway for modulating an immune response

Info

Publication number: WO2003100419A1
Application number: PCT/NL2003/000395
Authority: WO
Inventors: Louis Boon; Petrus Johannes Simons; David Speijer; Ruprecht Jules Joost Van Neerven
Original assignee: Bioceros B.V.; Macrozyme B.V.
Priority date: 2002-05-27
Filing date: 2003-05-27
Publication date: 2003-12-04
Also published as: AU2003243058A8; AU2003243058A1

Abstract

The invention relates to the field of immunology, gene therapy and medicine. More specifically, the invention relates to the identification of a molecule capable of interacting with a soluble and/or cell bound form of CD163 and as a result of said interaction an immune response is either instigated or suppressed in an organism. Furthermore it relates to the preparation of a pharmaceutical composition comprising said molecule and/or antagonist I and/or agonist I thereof, and/or an isolated CD163 and/or an antagonist II or agonist II thereof, for the therapeutic or prophylactic treatment of an individual with an immune response disorder, e.g. inflammation, cancer, or infection.

Description

Title: Methods for using the CD163 pathway for modulating an immune response.

The invention relates to the field of immunology, gene therapy and medicine. More specifically, the invention relates to the identification of a molecule capable of interacting with a cell bound and/or soluble form of CD 163 and as a result of said interaction an immune response is either instigated or suppressed in an organism. Furthermore it relates to the preparation of a pharmaceutical composition comprising said CD163-ligand molecule and/or antagonist I and/or agonist I thereof, and/or an isolated CD 163 and/or an antagonist II or agonist II thereof, for the therapeutic or prophylactic treatment of an individual with an immune related disorder, e.g inflammation, cancer or infection.

Antigen presenting cells (APC) of the myeloid lineage such as monocytes, macrophages, and dendritic cells, are key regulators in innate and acquired immune responses. They are capable of capturing and processing antigens, and presenting them to T lymphocytes. The complete activation of T-cells is dependent on the interaction of costimulating molecules on APC (e.g. CD80, CD86, CD40) with their counterparts on T-cells (CD28, CTLA-4, CD40L). The cytokines secreted by APC are regulated by the activity of a range of receptors, known as pattern recognition receptors (PRR). These receptors are a key feature of the innate immune response because they help discriminate between self and infectious non-self. The PRR include the Toll-like receptors, mannose receptor, and scavenging receptors (Linehan et al.. 2000, Imler and Hoffmann 2001). These receptors recognize conserved pathogen-associated molecular patterns, which are shared by large groups of microorganisms, and may also recognize endogenous ligands induced during inflammatory responses. Upon activation via costimulatory molecules as CD40 or via PRR, monocytes, macrophages and dendritic cells are further capable of secreting pro-inflammatory cytokines like for instance tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) or, depending on the signal, anti-inflammatory cytokines like for instance interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta). Depending on the profile of cytokines produced, immune response generated against a given pathogen are either stimulated or dampened. Further, when activated through PRR the APC are capable of initiating type 1 (inflammatory) versus type 2 (antibody mediated) immune responses (Kopp and Medzhitov 1999, Imler and Hoffmann 2001, Re and Strominger 2001), largely dependent on the amount of IL-12 they produce.

One of these pattern recognition receptors, human CD163 (also recognized as M130 or RM3/1) was identified as a membrane protein that is expressed on macrophages and on monocytes (Morganelli et al. 1988, Zwadlo et al. 1987, Law et al. 1993, Pulford et al. 1992, Sulahian et al. 2000). cDNAs encoding murine (Schaer et al. 2001, 2002) as well as human CD163 (Law et al.. 1993, Hδgger et al. 1998, Ritter et al. 1999) have been identified and have shown that CD 163 is a member of the cystine-rich scavenger receptor (SRCR) protein superfamily type B. The CD163 gene encodes a 130-155 kDa transmembrane protein. In addition, several isoforms have been described that differ in the length of the cytoplasmic region (45, 84 and 89 amino acids respectively), suggesting that these isoforms may have differential signaling and function (Hδgger et al. 1998, Law et al. 1993, Ritter et al. 1999). CD 163 can be removed from the cell membrane, for example upon treatment in vitro using the potent inflammatory mediators phorbol 12-mysristatel3- acetate (PMA) or lipopolysaccharide (LPS) (Droste et al. 1999, Hintz et al. 2002). This CD 163 downregulation is the result of protease-mediated shedding of the receptor, rather than endocytosis of CD 163, resulting in a soluble protein named sCD163 (sCD163). An ELISA to detect sCD163 in human serum has been published and reagents are available (Sulahian et al. 2001). Using this assay, it was shown that sCD163 is present in serum from healthy volunteers and therefore also exists in vivo. In addition, plasma levels of CD 163 were shown to be upregulated after cardiac surgery (Sulahian et al. 2001, and WO 01/73435, US

20010041177) , as well as after infusion of LPS into healthy human volunteers (WO 01/73435, US 20010041177). Both these situations resemble acute inflammation, and TNF-alpha, IL-6 and cortisol are also detectable in plasma. Levels of sCD163 are also increased in arthritis (Matsushita et al., 2002), Gaucher disease (Mδller et al. 2002-b), and in the serum of patients with myelomonocytic leukemias and infections (Mδller 2002-a). This indicates that sCDl63 acts as an acute phase protein during an inflammatory response. A potential anti-inflammatory effect of sCD163 was demonstrated (Hδgger et al. 2001, Frings et al. 2002). These experiments showed that sCD163 inhibited phorbol-ester induced human T-celll proliferation in vitro and thus may attenuate immune responses in vivo. In contrast, we disclose herein that sCDl63 has an immunomodulatory effect which can be mediated in part by binding either to a CD163-ligand present on the cell surface of antigen specific T lymphocytes or to it's soluble form (sCD163-ligand). Preferably, such an immunomodulatory effect comprises stimulation of an immune response.

CD 163 has recently been shown to be involved in hemoglobin (Hb) metabolism ( ristiansen et al., 2001). Efficient removal of free Hb is essential for health because of the oxidative and toxic properties of the iron-containing hae in Hb. The elevated expression of CD163 on tissue macrophages is therefore in line with its physiological important function in Hb catabolism of macrophages. Free Hb released into the serum is complexed by the acute phase protein haptoglobin and the complexes of haptoglobin and Hb are scavenged by CD 163 that only recognizes the complex and not the components alone (Kristiansen et al., 2001). Upon binding to CD163 the haptoglobin- hemoglobin complexes are endocytosed by the macrophages, whereupon the haem present in Hb is converted into bilirubin and iron. This was confirmed by Schaer et al. (Schaer et al. 2002), who disclose the notion that this action of CD 163 may be a direct anti-inflammatory action of corticosteroids. This is in line with the finding that haem oxygenase-1 is induced by hemoglobin as well as by IL-10, and mediates the anti-inflammatory effect of IL-10 and hemoglobin (Otterbein et al. 1995, Lee and Chau 2002). These data link macrophage haem metabolism to anti-inflammatory responses, and also suggest an important role for CD 163 in anti-inflammatory responses. Further, immunohistochemical analysis has shown that CD163-positive macrophages accumulate in inflammatory sites during the healing phase of both acute and chronic inflammation, suggesting a role for CD163-positive macrophages with the healing phase of the inflammatory response (Zwadlo et al. 1987).

Freshly isolated human monocytes have a low level expression of CD163. Upon in vitro culture of such monocytes in the presence of macrophage colony- stimulating factor (M-CSF) the monocytes differentiate into macrophages. This is accompanied by up-regulation of CD163 mRNA and protein (Buechler et al., 2000). When monocytes are cultured in the presence of granulocyte/macrophage colony stimulating factor (GM-CSF) and IL-4, factors that induce differentiation into dendritic cells, CD 163 expression is downregulated (Buechler et al., 2000). Interestingly, dendritic cells are the central cells involved in initiating immune responses in vivo, and therefore the rationale of the low expression levels of CD 163 on these cells may be related to the strong immunostimulatory capacity of these cells.

Although (gluco)corticosteroids are the most widely used immunosuppressive and anti-inflammatory agent in current clinical medicine, their pharmacological activities involved in modulation of the immune system are poorly understood. Unlike the role of CD 163 in Hb catabolism, scavenger receptor cysteine-rich (SRCR) proteins are typically associated with immune function and are therefore expressed on cells from the immune system. Interestingly, the expression of CD 163 on monocytes and macrophages is upregulated in vitro by anti-inflammatory mediators such as IL-10 and (gluco)corticoisteroids. Further, in vivo administration of corticosteroids to human volunteers resulted in an increase both in antigen density and the number of cells that express CD 163, 6 hours after administration (Zwadlo- Klarwasser et al. 1990). On the other hand, pro -inflammatory mediators like LPS, interferon-gamma (IFN-gamma) and TNF-alpha suppress expression of CD 163 on these cells.

Even though many of the findings described above suggest an anti- inflammatory role for membrane CD 163, stimulation of macrophages by crosslinking CD 163 with the mAb (mAb) EDHU-1 has been reported to induce the production of the pro-inflammatory cytokines IL-1 beta, IL-6 and GM-CSF (Ritter et al. 2001, van den Heuvel et al. 1999). CD163 signal transmission upon stimulation depends on a protein tyrosine kinase (PTK) activity resulting in calcium mobilization and inositol triphosphate generation. Since the cytoplasmic domains of CD 163 and its isoforms contain no known PTK motif, the molecular mechanisms by which CD 163 signal transduction is mediated are not understood. In addition to the PTK-dependent signal another non characterized signal pathway has been described that is independent of PTK activity. More recently, interaction with the regulatory subunit of casein kinase II and protein kinase C with the cytoplasmic tail of CD 163 on macrophages have been observed (Ritter et al., 2001).

Surprisingly, we disclose herein, that ligation of CD 163 by another CD 163- specific mAb, RM 3/1, downregulates the production of the pro-inflammatory cytokine TNF-alpha in response to LPS, whilst upregulating the anti- inflammatory cytokine IL-10. This therefore proves that immune responses will be dampened when CD163 binds to its ligand(s). We further demonstrate herein that apart from its role in Hb catabolism, CD163 (i.e. membrane bound (mCD163) and/or soluble (sCD163) form) has immunoregulatory properties mediated in part through binding/interaction with a novel ligand (referred to herein as CD163-ligand) besides haptoglobin-hemoglobin complexes.

In one embodiment the invention thus provides an agonist of CD163/CD163- ligand signalling in the form of antibody RM 3/1. The invention thus provides the use of antibody RM 3/1 or a functionally related molecule for the dampening of an immune response in an individual. A functionally related molecule is apart ofRM 3/l that is capable of recognizing the same epitope as RM 3/1 suitable parts are those that retain a variable part of antibody RM 3/1 such as FAB fragments or single chain variable fragments thereof. A functional equivalent ofRM 3/1 is also a different antibody comprising the same epitope recognition capabilities as RM 3/1. Such antibodies may be selected from CD163 binding antibodies by competition assays with RM 3/1. Those which compete with binding to CD 163 with RM 3/1 are likely to have the same effect as RM 3/1. It is of course not excluded that among the other CD163 binding antibodies there are those that have similar effects as RM 3/1 such antibodies are also part of the invention. An antibody of the invention is preferably a human or humanized antibody. At least part of the variable domain ofRM 3/l or functionally related molecule may be grafted on a human antibody backbone to generate a humanized version ofRM 3/l or the related molecule.

Apoptosis is defined as genetically programmed cell death, which provides a counterbalance to mitosis in the regulation of tissue growth and homeostasis. Apoptosis is characterized by cell shrinkage, retention of organelles and nuclear chromatin condensation, which is accompanied by nuclear fragmentation. In many cell lineages, apoptosis is featured by blebbing of plasma membranes, which leads to detachment of membrane-enclosed apoptotic bodies. Apoptotic bodies can contain organelles and even nuclear fragments. (Millset al. 1999, Willingham et al. 1999, Savill et al. 2002).

It generally accepted that macrophages are the most important cells in the body, which recognize and remove apoptotic cells and their related products. However, non-professional phagocytes, like dendritic cells, fibroblasts, hepatocytes, epithelial and endothehal cells, are also described to mediate clearance of apoptotic cells. (Fadok et al. 2001). Many surface receptors on macrophages are believed to contribute to the initial recognition and binding of apoptotic cells. These include lectins and integrins, the class A and class B scavenger receptors (CD 36, SRA, CD 68, LOX-1), complement receptors, the phosphathidylserine (PtdSer) receptor and the endotoxin receptor CD14 (Fadok et al. 2001, Savill et al. 2002). After recognition and binding of apoptotic cells, rapid engulfment of dying cells by macrophages is promoted. There is growing evidence from many studies that the net effect of all these interactions results in anti-inflammatory and immunosuppressive responses from macrophages ( Fadok et al. 1998, McDonald et al. 1999, Voll et al. 1997, Byrne et al. 2002). This is examplified by the induction of anti-inflammatory factors, like IL-10 and TGF-beta, and inhibition of pro-inflammatory factors, like IL-1 beta and TNF-alpha, from

LPS-stimulated macrophages, which have been previously exposed to apoptotic cells. In addition, the antitumor activity of macrophages is seriously impaired after exposure of macrophages to apoptotic tumor cells (Reiter et al. 1999).

Primary necrosis is defined as accidental or murderous cell death resulting from e.g. exposure to toxins, hypoxia or temperature extremes. Necrosis is characterized by cell swelling, leading to bursting of cells, and consequently the release of free organelles and cellular contents. (Willingham et al. 1999, Savill et al. 2002). Secondary or post-apoptotic necrosis is the eventual disintegration of apoptotic cells when the clearance of apoptotic cells is impaired (Savill et al. 2002, Nauta et al. 2003).

In contrast to apoptotic cells, primary necrotic cells are a toxic and pro- inflammatory activator of macrophages, due to the release of cellular contents (Fadok et al. 2001, Savill et al. 2002, Reiter et al. 1999). For this reason, it postulated that autoimmunity could be induced in situations of defective apoptotic cell removal, which results as stated above in secondary necrotic cells. (Savill et al. 2002, Nauta et al. 2003)

In summary, the rapid uptake of intact apoptotic cells by macrophages and the coinciding anti-inflammatory/immunosuppressive local micro-environment are of great in vivo importance to prevent damaging of neighboring cells and triggering of unwanted inflammatory (auto)immune responses. On the other hand, apoptotic tumor cells could induce anti- inflammatory/immunosuppressive responses as a mechanism to escape immune surveillance.

The novel ligand for CD163 disclosed herein interacts with membrane CD163, induces the production of IL-10, which in turn induces the production of haem oxygenase 1 and upregulates the expression of CD163, which enables the CD163 expressing cells to efficiently remove Hp-Hb complexes from the circulation. In addition, the resulting induction of haem oxygenase-I will exert a direct anti-inflammatory effect. Provided is thus the use of a CD 163 ligand for inducing haem-oxygenase-I in a subject.

For example CD 163 binds to apoptosis-related proteins on the cell surface of apoptotic cells, such as proteins belonging to the histone family. As described above, apoptotic cells need to be removed from the body to prevent them from leaking their contents and inducing necrosis. It is known that the binding of apoptotic cells by macrophages leads to an inhibition of the production of pro- inflammatory cytokines such as TNF-alpha, and to the induction of IL-10 and other anti-inflammatory factors (Fadok et al., 1998, McDonald et al. 1999, Voll et al. 1997, Byrne et al. 2002 ). We disclose that interaction of CD163 on the macrophage/monocyte with these apoptosis related proteins (e.g proteins belonging to the histone family) may be a key event in the inhibition of the production of pro-inflammatory cytokines such as TNF-alpha, and to the induction of IL-10 and other anti-inflammatory factors during the uptake and degradation of the apoptotic cells. The invention thus further provides a method to interfere with apoptosis related events in a subject, comprising providing said subject with a means for modulating CD163 / CD163-ligand signalling in said subject. Said means may comprise an agonist of CD163 / CD163-ligand signalling and/ or an antagonist thereof. Such means is preferably a soluble CD 163, a soluble CD163-ligand, a CD 163 specific antibody, a CD163-ligand specific antibody or a histone or functional part, derivative and/or analogue of such a molecule. The invention further provides a method for detecting apoptotic cells comprising providing said cells with a CD 163 molecule or functional part, derivative and/ or analogue thereof. The invention further provides a method for detecting apoptic cells comprising providing said cells with a histone specific antibody or functional part, derivative and/or analogue thereof. The invention further provides a method for detecting a histone, particularly histone H2A, H2B and H4, characterized in that a CD 163 molecule, or a functional part, derivative and /or analogue thereof is used.

It was demonstrated (Wu et al. 2002) that nucleosomal histones become separated from DNA within a few hours during apoptosis, and detectable in cell lysates prepared by using a non-ionic detergent. Here we disclose that apoptotic cells express molecules (within hours) that interact with CD 163 from macrophages. These molecules were identified as nucleosomal histones: H2A, H2B and H4. We disclose that the interaction of CD 163 on macrophages with histones on apoptotic cells play a role in the induction of anti-inflammatory and immunosuppressive responses observed during the subsequent phagocytosis and elimination of apoptotic cells.

Inflammation is a major hallmark of a wide range of diseases such as autoimmune diseases, inflammatory diseases, organ rejection, and infectious diseases. Inflammation is characterized by an influx of cells of the immune system, the release of cytokines and other inflammatory mediators such as histamine, leukotriens and prostaglandins, resulting in fever and tissue destruction. Inflammatory processes include: the local reactions and resulting morphologic changes; the destruction or removal of the injurious material; and the responses that lead to repair and healing. The typical signs of inflammation are redness, heat or warmth, swelling, pain, and occasionally inhibited or lost function. In the present invention it was found that by manipulating the CD163 / CD163-ligand signalling pathway it is possible to modulate an immune response in a subject. The immune response plays a role in many different diseases. Those vary from typical immune system diseases such as auto-immune diseases and infections etc. to diseases where the immune system has a more hidden role such as leptin metabolism. The immune system is affected for example in disturbed fat metabolism, where changes in leptin levels directly influence immune function.

Pharmaceutical agents used to modulate inflammation in a host should ideally be endogenous substances, such as therapeutical proteins. These therapeutical proteins should not be recognized as foreign agents (i.e. no neutralizing antibodies will be formed, no nephrotoxicity is expected from endogenous proteins and no complexes are expected to be formed due to an antibody response against the therapeutical protein). Although many pharmaceutical compounds are currently used to treat inflammatory diseases, there is a need for more effective compounds (immunosuppressive molecules) with lowered toxicity profiles.

Currently used pharmacological therapies in immune related diseases in many cases only provide temporal relief. In addition drugs used to combat inflammation are not very selective, targeting non-inflammatory cells as well as inflammatory cells and often have moderate to serious side effects after chronic treatment, especially in children. Many patients become resistant to the drugs (e.g. glucocorticoid) used and high doses are associated with unpleasant side effects. Hence, there is a strong need for safer, more selective and more efficacious therapies which display a long-term clinical benefit to patients suffering from immune related disorders.

Inflammation often accompanies and is a response to infection or other injury, however, chronic and autoimmue inflammation represent undesirable pathological conditions in which infection is not typically present. In these latter diseases one wishes to inhibit inflammation.

In infection and cancer on the other hand, one wishes to stimulate the inflammatory response to generate a more aggressive immune responses to the infectious agent and/or the tumor cell. Cancer and infectious disease are significant health problems throughout the world. Although advances have been made in detection and therapy of these diseases, no vaccine or other universally successful method for prevention or treatment is currently available.

Infection is defined by the invasion and multiplication of foreign microorganisms such as viruses, bacteria, fungi including yeast, and parasites, in the body. Infections are generally harmful to the host, resulting in local cellular injury. A local infection may persist and spread by extension to become an acute, subacute or chronic clinical infection or disease state.

Many of the frequently occurring infectious diseases of today are caused by bacteria, which have gained resistance to drugs previously effective in treating diseases caused by these bacteria, or by viruses. Only a small number of antiviral drugs are currently available for treatment of virus infections. A complication to the development of such drugs is that mutant strains of virus which are resistant to currently available antiviral drugs develop readily.

As with the antiviral agents, the development of anticancer agents for treating cancer effectively has also been problematic. Cancer therapy currently relies on a combination of early diagnosis and aggressive treatment, which may include radiotherapy, chemotherapy or hormone therapy. However, the toxicity of such treatments limits the use of presently available anticancer agents. The high mortality rate for many cancers indicates that improvements are needed in cancer prevention and treatment.

Many tumors escape surveillance by our immune system. In cancer patients there is clearly a quantitative and/or qualitative defect in the immune system's specific mechanisms to eliminate tumor cells. It is clear that there is still a great need to find ways to generate and/or enhance protective anti- tumor responses involving cellular and humoral immunity. In fact, tumor cells can produce anti-inflammatory cytokines such as IL-10 and TGF-beta, and certain tissues in mammals with cancer of the immune and circulatory systems express significantly reduced levels of the pro-inflammatory cytokine TNF-alpha.

As described above, immune related disorders may be due to either immune heightening such as in the case of auto-immune diseases or immune dampening such as in the case of cancer and infection. The present invention through the identification of a ligand for CD 163 (membrane bound and/or soluble/secreted/shed form) provides an efficacious method to modulate undesirable immune responses through CD163 or its ligand, termed CD163- ligand.

The invention provides a method for identifying a molecule with immune modulatory activity capable of interacting with CD163 comprising providing CD 163 or a functional part, derivative and/or analogue thereof and under suitable conditions detecting a molecule capable of interacting with said CD163 and determining whether said molecule is capable of modulating an immune response. CD 163 as used herein refers to the membrane bound and/or its soluble (i.e. secreted/shed) form. For instance, said CD163 is a receptor or functional fragment thereof present on cells of myeloid lineage (monocytes, macrophages, dendritic cells) and/or cells of lymphoid lineage and its soluble form (sCD163) is the shed product of this receptor. A molecule as used herein can be any substance be it nucleic acid, amino acid, a carbohydrate or a lipid comprising moiety (or combinations thereof), or any other moiety that can interact with CD 163 and as a result of said interaction an immune response is modulated (i.e. instigated or suppressed). It is understood that said molecule with immune modulatory activity can be cell/membrane bound and/or soluble. It is also understood that said molecule can block the active site of CD 163 and in doing so can modulate an immune response. The active site as used herein is the binding/adherence site on CD 163 for a particular substance (e.g. a functional site such as a receptor-binding cavity). It should be noted that CD163-ligand does not necessarily bind to the same region of the CD163 molecule as haptoglobin-hemoglobin complexes do. In a preferred embodiment of a method of the invention said molecule with immune modulatory activity is present on cells of lymphoid lineage (e.g. T-cells, B cells) or endothelium and comprises a CDl63-ligand. In an even more preferred embodiment of the invention, the expression of CD163-ligand on the cell membrane is enhanced after induction of apoptosis, and said CD163-ligand is a member of the histone family, more particularly comprising histones H2A, H2B and H4.

For example, a CD163-ligand can bind to membrane bound CD 163 (mCD163) thus inhibiting the production of pro-inflammatory cytokines such as TNF- alpha, and inducing the production of anti-inflammatory cytokines such as IL- 10, by CD163 bearing cells, serving to suppress an immune response. On the other hand, a CD163-ligand (e.g. sCD163-ligand) can be used to neutralize CD163 (e.g. sCD163) in situations where sCD163 is increased. This can at least in part reduce the immunostimulatory effects of sCD163, and serve to suppress an immune response.

An immune response is a physiological response of an organism to agents (e.g. infectious agents, proteins, tumor cells etc.) that pose a threat to said organism. For example an immune response can involve activation and production of factors by leucocytes comprising B lymphocytes and T lymphocytes, NK cells, granulocytes, monocytes, macrophages and dendritic cells. Two types of immune responses are well recognized, namely the innate immune response and the adaptive immune response. An adaptive immune response is highly antigen-specific and can generate long lived immune memory. Cells involved in the adaptive immune response (henceforth called antigen-specific immune response) such as B lymphocytes and T lymphocytes recognize their antigens through highly specific cell surface receptors. Innate immune responses are responses involving granulocytes and monocytes, macrophages and dendritic cells and NK cells. These cells recognize frequently encountered antigens with germline encoded PRR, or the constant region of antibodies, thus providing a first line of defense before the acquired immune system is able to mount a response. PRR when they bind their ligand transmit signals into the immune cell which can lead to the release of biological mediators that can instruct the adaptive (acquired) as well as innate arm of the immune response.

Modulation as used herein can refer to up -regulation or down-re ulation of an immune response, for example by activation and/or suppression of gene(s) which are essentially capable of initiation and/or progression and/or suppression and/or repression of an immune response and/or symptoms of said immune response. Said modulation can be mediated by positive (i.e. up- regulation) or negative (i.e. down-regulation) regulation of gene transcription, and/or by the modification of a gene and/or gene product (e.g. post- translational modification). A functional part of CD163 is defined as a part of CD 163 which has the same properties (e.g CD163-Iigand binding property) as CD 163 in kind but not necessarily in amount. A functional derivative of CD 163 is defined as CD 163 which has been altered such that the ligand- binding properties of the altered CD 163 are essentially the same in kind, but not necessarily in amount. Suitable derivatives can be generated through using codon degeneracy, for example by conservative amino acid substitution. A functional analogue of CD 163 is a homologue and/or functional equivalent of CD 163 which can be derived from a different species and/or generated synthetically.

In a preferred embodiment of a method of the invention said CD 163 comprises sCD163. sCD163 is a secreted or shed form of the membrane bound CD163. In another preferred embodiment of a method of the invention said molecule with immune modulatory activity comprises a proteinaceous molecule, functional derivative, functional fragment and/or analogue thereof. A proteinaceous molecule as used herein can be any amino acid comprising moiety. In one embodiment said proteinaceous molecule comprises a CD163-ligand. A CD 163- ligand can be a proteinaceous molecule encoded by a nucleic acid of a cell. A functional part of said CD163-ligand is defined as a part of a CD163-ligand which has the same immunomodulatory properties in kind as a CD163-ligand but not necessarily in amount. By immunomodulatory properties is meant the capability to induce or inhibit an immune response in a hosT-cell. A functional derivative of a CD163-ligand is defined as a CD163-ligand which has been altered such that the properties (e.g. immunomodulatory properties) of said altered CD163-Iigand are essentially the same in kind, but not necessarily in amount. Suitable derivatives can be generated through using codon degeneracy, for example by conservative amino acid substitution. A functional analogue of said CD163-ligand is a homologue and/or functional derivative (i.e. functional equivalent) of said CD163-ligand which can be derived from a different species and/or generated synthetically. As used herein, the term "functional equivalent" means that the amino acid of a proteinaceous molecule according to the invention can be modified by means of one or more substitutions, deletions, or additions, the net effect of which does not result in a functional dissimilarity in kind not necessarily in amount between the amino acid of a proteinaceous molecule according to the invention and the modified form.

In a preferred embodiment the invention provides an isolated and/or recombinant and/or synthetic molecule obtainable by a method according to the invention. In one aspect the invention provides a substantially isolated or purified CD163-ligand or a recombinant (i.e. a modified form generated through genetic engineering approaches) or a synthetic (i.e. artificially generated as opposed to naturally occurring, for example by chemical synthesis) form thereof having substantially similar immunomodulatory activity. As used herein, the term "substantially isolated or purified" refers to a molecule according to the invention that is removed from its natural environment, isolated or separated, and is essentially free from components with which it is naturally associated. In terms of the invention the phrase "substantially similar immunomodulatory activity" means that said natural, recombinant or synthetic CD163-ligand polypeptide according to the invention, or any oligopeptide thereof, is similarly immunologically active in kind not necessarily in amount, that is it is capable of inducing a specific immune response in a mammal or a cell. Preferably said molecule is of mammalian or avian origin. It is understood that said CD163-ligand can be cell/membrane bound (e.g. a cell surface protein or receptor), for example on cells of lymphoid lineage, endothehal cells and/or myeloid lineage or soluble (e.g. freely circulating), and can be involved in apoptosis. For instance the soluble form of the CD163-ligand can arise as a result of shedding of the membrane bound form, or be produced as a soluble molecule. Preferably said CD163-ligand soluble form (sCDl63-ligand) comprises at least part of the extracellular and/or cytoplasmic domain, more preferably the CDl63-binding domain of a cell/membrane bound CD163-ligand (mCD163-ligand). An APC can be any cell (e.g. macrophages, endothelium, dendritic cells, langerhans cells of the skin etc.) which carries on its surface antigenic peptides bound to major histocompatibility complex (e.g. MHC Class I or Class II) molecules and presents the antigen in this context to cells of the immune system. It is also understood that a molecule of the present invention can inhibit or induce signal transduction into APC. Preferably, binding of said molecule to CD 163 results in inhibition of the production of pro -inflammatory cytokines as TNF- alpha whilst increasing the production of anti-inflammatory cytokines such as IL-10 by APC (e.g. macrophages)

In another preferred embodiment the invention provides a CD163-ligand molecule obtainable by a method according to the invention. Preferably said CD163-ligand molecule comprises a protein belonging to the histone family (e.g H2A, H2B or H4) or functional fragment thereof. For instance, said CD163-ligand can be a protein belonging to the histone family or functional part thereof induced on cells of lymphoid lineage and/or endothehal cells and/or cells of myeloid lineage (monocytes, macrophages, dendritic cells) after induction of apoptosis and its soluble form (sCD163-ligand) is the shed and/or soluble product of this protein, which may be capable of freely circulating in body fluids.

In another aspect the invention provides a CD163-ligand molecule according to the invention coupled to a moiety. For instance a CD163-ligand molecule or functional fragment thereof, such as a proteinaceous molecule of the invention can be linked to a second or subsequent moiety to form a fusion protein. In terms of the invention said moiety can serve to confer additional properties to the proteinaceous molecule (e.g. increased longevity and stability, improved intracellular targeting) or to improve its biological activity (i.e. imunomodulatory activity and/or pharmacokinetics). Suitable moieties can include a molecule comprising at least part of an immunoglobulin chain (e.g. a constant region of said chain), a molecule with immunoregulatory activity like a cytokine (e.g. IL-10, IL-12, granulocyte colony stimulating factor (GCSF), IFN-gamma, etc.), a toxic moiety, and a protection molecule like polyethylene glycol (PEG). It is understood that by using a nucleic acid encoding a molecule, or a fragment thereof, of the present invention any combination of fusion protein(s) can also be generated using recombinant techniques known in the art. For example a fusion protein of CD163-ligand (e.g. sCD163-ligand) coupled to the constant region of an immunoglobulin (e.g. sCD163-ligand-Fc) can be used to neutralize CD 163 (e.g. sCD163) and/or to cross link membrane bound CD 163 to inhibit the production of pro-inflammatory cytokines and enhance the production of anti-inflammatory cytokines by CD163 expressing cells. This can at least in part reduce the immunostimulatory effects of sCDl63, and serve to stimulate an immune response. Such a fusion protein has an advantage over a monovalent sCD163-ligand alone in that it has a longer life in circulation and as a result of its bivalency may have an additive effect on inhibiting immune responses. In another aspect the invention provides use of a molecule according to the invention to modulate an immune response. Said immune response can comprise an innate and/or an antigen specific (adaptive) immune response. Preferably said immune response comprises an antigen specific (adaptive / acquired immune) response.

In inflammatory diseases such as autoimmune diseases, allergy, asthma, transplant rejection the activation of the immune system is either unwanted or excessive. These diseases are highly specific for the antigens that are recognized by the immune system such as auto-antigens (autoimmune diseases), allo-antigens (transplant rejection), allergens (allergy) or infectious agents (certain infections).

In yet another aspect the invention provides use of a CD163-ligand molecule and/or functional derivative and/or functional fragment and/or analogue thereof according to the invention for modulating an immune response wherein said modulation comprises inhibition of an immune response. As a way of illustration but not as a way of limitation by modulating the activity of CD163 and/or a CD163-ligand of the present invention (such as sCD163- ligand), one is capable of suppressing an immune response in auto-immune disease like rheumatoid arthritis, diabetes, multiple sclerosis, systemic lupus erythematosous, psoriasis, autoimmune thyroidits, inflammatory diseases, transplantation diseases, infectious diseases (i.e. septic shock), etc. This can be achieved in a number of ways.

The capability to interfere with CD163 / CD163-ligand signalling is one of the hallmarks of the present invention. The invention provides several means for manipulating this signalling. The invention provides both agonist and antagonists of said signalling. Agonists are capable of at least in part mimicking CD163/CD163-ligand signalling (either in the CD163 positve cell or in the CD 163-ligand positive cell). The effect of such agonists is thus to dampen an immune response. Antagonists are capable of at least in part preventing CD163/CD163-ligand signalling and are thus capable of stimulating an immune response when compared to the situation in the absence of the antagonist. Agonists can be divided into two groups. Agonists of group I are capable of binding CD 163-ligand whereas agonists of group II are capable of binding CD163. Antagonists are likewise dividable into two groups. Group I is capable of binding to CD 163 ligand, whereas antagonists of group II are capable of binding to CD 163.

One way to inhibit an immune response is to stimulate the production of IL-10 and inhibit the production of pro -inflammatory cytokines. For example, by providing an excess of sCD 163-ligand, and/or an excess of sCD 163-ligand coupled to a moiety as mentioned previously, and/or an excess of an an agonist II, and /or an agonist II coupled to a moiety, one can directly stimulate the production of IL-10 and inhibit the production of pro-inflammatory cytokines by CDl63-bearing cells.

Another way to inhibit an immune response is by neutralizing sCD163- induced immune stimulation in situations in which sCD163 levels are increased. For example by providing an excess of sCD 163-ligand, and/or an excess of sCD 163-ligand coupled to a moiety as mentioned previously, and/or an excess of an antagonist of CD 163 (termed antagonist II), and/or an excess of an antagonist II coupled to a moiety, one can neutralize the stimulating effects of sCD163 in situations where sCD163 levels are increased. The sCD 163-ligand can engage the sCD163 shed from cell membranes under disease conditions, and this can effectively restore the ability of membrane bound CD 163 to interact with the cell bound CD 163-ligand on T-cells or other CD 163-ligand bearing cells, thus dampening the immune response. It is understood that a CD 163-ligand molecule with immune modulatory activity according to the invention (e.g. sCD 163-ligand) can induce or prevent signal transduction into CD163-bearing cells, like macrophages. Interaction of a membrane bound and/or sCD 163-ligand molecule of the invention with membrane bound CD 163 modulates activation and cytokine production of a cell bearing CD163 (e.g. an APC, like a macrophage) by enhancing the production of anti-inflammatory cytokines (like for instance IL-10) whilst inhibiting the production of pro-inflammatory cytokines (like for instance TNF-alpha). This may or may not depend on the valency of the binding. Said molecule of the invention (e.g. sCD 163-ligand) or an agonist II to CD163 of the invention is particularly suitable for the inhibition of immune responses in inflammatory diseases.

A molecule of the invention (e.g. sCD 163-ligand) or an antagonist II to CD163 of the invention is also particularly suitable for the inhibition of immune responses in diseases in which serum sCD163 levels are increased, by neutralizing the stimulatory effect of sCD163.

In another aspect the invention provides an antagonist of a CD 163-ligand molecule according to the invention. Said antagonist, further referred to as antagonist I, is a substance that at least in part tends to nullify the action of a molecule of the invention, whether said molecule is membrane bound or soluble. Preferably said antagonist I completely blocks the binding of CD 163- ligand to CD 163. Preferably said antagonist I is an antibody or a functional part, derivative and/or analogue thereof. A functional part of an antibody is defined as a part which has the same kind of binding properties in kind, not necessarily in amount (e.g. a Fab fragment). A functional derivative of an antibody is defined as an antibody which has been altered such that the binding properties of said antibody are essentially the same in kind, not necessarily in amount. A derivative can be provided in many ways, for instance through conservative amino acid substitution. A non-limiting example of a suitable derivative is a single chain antibody. A non-limiting example of a suitable analogue is a synthetic antibody selected from a recombinant antibody library. In a preferred embodiment an antibody of the invention is a mAb or a functional part, derivative and/or analogue thereof. A mAb of the invention can be generated by standard immunization and cell fusion techniques or by combinatorial library approaches. It is also understood that said antagonist I of the present invention can comprise a chimeric antibody, a humanized antibody or a fully human antibody. It is also understood that said antagonist I can comprise an antibody with a neutralizing and/or blocking function.

The invention further provides an antagonist I coupled to a moiety. In terms of the invention said moiety can serve to confer additional properties to said antagonist I (e.g. increased longevity and stability, improved intracellular targeting) or to improve its biological activity (i.e. imunomodulatory activity and/or pharmacokinetics). Suitable moieties can include a molecule comprising at least part of an immunoglobulin chain (e.g. a constant region of said chain), a molecule with immunoregulatory activity like a cytokine (e.g. IL-10, IL-12, GCSF, IFN-gamma, etc.), a toxic moiety, and a protection molecule like polyethylene glycol (PEG). It is understood that by using a nucleic acid encoding an antagonist I, or a fragment thereof, any combination of fusion protein(s) can also be generated using recombinant techniques known in the art.

The invention as disclosed herein thus provides a method(s) using antibodies of an isolated and/or recombinant and/or synthetic proteinaceous molecule according to the invention to induce or prevent signalling through CD163 and/or CD 163-ligand. An antibody of the present invention can bind to mCD 163-ligand and/or sCD 163-ligand and can either block binding of the CD 163-ligand to CD 163 or can induce or enhance signalling through the CD 163-ligand. Thus an antibody of the present invention can have immunomodulatory effects, either by blocking of signalling through CD 163, or by directly affecting signalling through CD 163-ligand. It is understood that an antibody of the present invention can bind to mCD 163-ligand and/or sCD163- ligand and have no immunomodulatory activity (i.e. will not influence signalling through the CD163-ligand). It is also understood that an antibody of the present invention can induce the mCD 163-ligand to be internalised, shed from the membrane, or secreted. Moreover an antibody of the present invention can be coupled to an effector molecule (e.g. a cytokine) to increase the immunomodulatory capacity of said antibody. Additionally, an antibody of the present invention can be coupled to a toxic moiety, allowing the reduction or a depletion of cells expressing the CD 163-ligand.

In another aspect the invention provides use of an antagonist I to modulate an immune response. An antagonist I CD163-ligand-specific antibody of the present invention can bind the CD 163-ligand on CD 163-ligand bearing cells and can inhibit the binding of CD 163-ligand to CD 163, thus neutralize the CD 163-ligand induced induction of anti-inflammatory cytokine production in CD163-bearing cells. Furthermore, an antagonist I CD163-ligand-specific antibody of the present invention can induce or enhance the signalling that is induced by signal transduction through the CD 163-ligand, or prevent the interaction of CD163-ligand with CD163 or sCD163. This can counteract (i.e. at least in part nullify) the CD 163-ligand mediated dampening of immune responses. Also an antagonist I CD163-ligand-specific antibody of the present invention can induce shedding or endocytosis of CD 163-ligand, thus decreasing CD 163-ligand expression on CD 163-ligand bearing cells, hence serving to induce an immune response.

Therefore an antagonist I can be used to modulate an immune response, preferably in a CD 163 related pathway, wherein said modulation comprises augmentation (i.e. amplification) of an immune response. For example, an antagonist I can be used to stimulate immune responses in cancer, in infections, and in other diseases in which no proper immune response is mounted. With an antagonist I (e.g. CD163-ligand-specific antibody) of the present invention, and/or an antagonist II (e.g. sCD163-specific antibody) of the invention, and/or a molecule according to the invention (e.g. sCD163 or sCD163-Fc), it is now for instance possible to efficiently block the immune dampening effect of interaction of CD 163-ligand with membrane CD 163. This serves to boost an organisms immune response against tumor cells or where there is an insufficient response against infectious pathogens. In this case, the membrane bound form of the CD163-ligand (m CD 163-ligand) is prevented from binding to CD163 or sCD163, and is therefore able to prevent the production of anti-inflammatory cytokines such as IL-10, whilst enabling the production of pro-inflammatory cytokines such as TNF-alpha by CD 163 expressing cells such as macrophages, resulting in fewer persistent tumours and/or infections. Furthermore, antagonist I antibodies of a CD 163-ligand may mediate direct activation of CD 163-ligand expressing immune cells like T and/or B cells, resulting in fewer persistent tumours or infections.

The invention also provides an agonist of a CD163-ligand molecule according to the invention. Said agonist, further referred to as agonist I, is a substance that tends to at least in part induce a negative signal through CD163-ligand into the cell expressing CD 163-ligand, resulting in modulation of an immune response. Preferably said agonist I is an antibody or a functional part, derivative and/or analogue thereof. A functional part of an antibody is defined as a part which has the same kind of binding properties in kind, not necessarily in amount (e.g. a FAB fragment). A functional derivative of an antibody is defined as an antibody which has been altered such that the binding properties of said antibody are essentially the same in kind, not necessarily in amount. A derivative can be provided in many ways, for instance through conservative amino acid substitution. A non-limiting example of a suitable derivative is a single chain antibody. A non-limiting example of a suitable analogue is a synthetic antibody selected from a recombinant antibody library. In a preferred embodiment an antibody of the invention is a mAb or a functional part, derivative and/or analogue thereof. A mAb of the invention can be generated by standard immunization and cell fusion techniques or by combinatorial library approaches. It is also understood that said agonist I can comprise a chimeric antibody, a humanized antibody or a fully human antibody.

An agonist I can bind a molecule of the invention, for instance mimicking the effects of binding of sCD163 to a CD 163-ligand molecule of the invention. It is understood that an agonist I can bind with a higher avidity to a molecule of the invention. This can give rise to at least a similar or even a superior signaling through a CD163-ligand (e.g. membrane bound). Such a signal will result in suppression of an immune response. Preferably, an agonist I does not completely inhibit the binding of CD163-ligand to CD163 (e.g mCD163, sCD163).

In another aspect the invention provides an agonist I coupled to a moiety. For instance linked to a second or subsequent moiety to form a fusion protein. In terms of the invention said moiety can serve to confer additional properties to the agonist I (e.g. increased longevity and stability, improved intracellular targeting) or to improve its biological activity (i.e. immunomodulatory activity and/or pharmacokinetics). Suitable moieties can include a molecule comprising at least part of an immunoglobulin chain (e.g. a constant region of said chain), a molecule with immunoregulatory activity like a cytokine (e.g. IL-10, IL-12, GCSF, IFN-gamma, etc.), a toxic moiety, and a protection molecule like polyethylene glycol (PEG). It is understood that by using a nucleic acid encoding an agonist I, or a fragment thereof, any combination of fusion protein(s) can also be generated using recombinant techniques known in the art.

Additionally the invention provides use of an agonist I to modulate an immune response. Preferably said modulation comprises suppression of an immune response. It is understood that an agonist I through interacting with a CD163- ligand can serve to inhibit an immune response, for instance an antigen specific (adaptive) immune response. An agonist I can by itself or in association with CD163 deliver a signal (e.g. inhibitory signal) through the CD 163-ligand on CD 163-ligand bearing cells, and this can inactivate the immune system.

An agonist I according to the invention is particularly efficacious for the prophylactic or therapeutic treatment of immunological diseases, such as for instance autoimmune diseases, inflammatory diseases, transplant rejection, or infectious diseases. Such an agonist I can at least in part prevent activation of CD163-ligand bearing cells, such as T-cells or other cells of the immune system, resulting in dampening of an immune response. Preferably said immune response comprises an antigen specific (adaptive immune) response. Furthermore such agonist I may at least in part prevent or overcome endocytosis or shedding and or secretion of CD 163-ligand from CD 163-ligand bearing cells thereby increasing the net amount of CD 163-ligand present on CD163-ligand bearing cells.

The invention further provides an isolated and/or recombinant and/or synthetic CD163, a functional part, derivative and/or analogue thereof. Preferably said CD163 comprises a sCDl63. In a preferred embodiment of the invention said sCDl63 comprises a C-terminus and/or N-terminus identical to the natural or PMA- induced shed CD163 molecule. Preferably said N- terminal end comprises APGWANSSAGSGRIWMDHVSCR, and said C- terminus comprises EAEFGQGTGPIWLNEVK. The invention provides a substantially isolated or purified CD 163 or a recombinant (i.e. a modified form generated through genetic engineering approaches) or a synthetic (i.e. artificially generated as opposed to naturally occurring, for example by chemical synthesis) form thereof having substantially similar immunomodulatory activity. As used herein, the term "substantially isolated or purified" refers to CD163 according to the invention removed from its natural environment, isolated or separated, and is essentially free from components with which it is naturally associated. In terms of the invention the phrase "substantially similar immunomodulatory activity" means that said natural, recombinant or synthetic CD 163 polypeptide according to the invention, or any oligopeptide thereof, is similarly immunologically active in kind not necessarily in amount. A functional part of CD 163 is defined as a part of CD 163 which has the same properties as CD 163 in kind but not necessarily in amount. A functional derivative of CD163 is defined as CD163 which has been altered such that the CD163-ligand-binding properties of the altered CD 163 are essentially the same in kind, but not necessarily in amount. Suitable derivatives can be generated through using codon degeneracy, for example by conservative amino acid substitution. A functional analogue of CD 163 is a homologue and/or functional equivalent of CD 163 which may be derived from a different species and/or generated synthetically.

The invention further provides an isolated CD 163 coupled to a moiety. For instance an isolated CD163, a functional part, derivative and/or analogue thereof according to the invention can be linked to a second or subsequent moiety to form a fusion protein. In terms of the invention said moiety can serve to confer additional properties to said isolated CD 163 molecule (e.g. increased longevity and stability, improved intracellular targeting) or to improve its biological activity (i.e. imunomodulatory activity and/or pharmacokinetics). Suitable moieties can include a molecule comprising at least part of an immunoglobulin chain (e.g. a constant region of said chain), a molecule with immunoregulatory activity like a cytokine (e.g. IL-10, IL-12, GCSF, IFN-gamrαa, etc.), a toxic moiety, and a protection molecule like polyethylene glycol (PEG). In a preferred embodiment of the present invention said moiety comprises a constant region of an immunoglobulin.

Said CD 163 is preferably linked to the constant region of an immunoglobulin by the C-terminus EAEFGQGTGPIWLNEVK to prevent proteolytic cleavage of the CD163-Fc molecule.

In another aspect the invention provides an antagonist of CD163. An antagonist, further referred to as antagonist II of CD163, can at least in part neutralize or block CD163 (e.g. sCD163). Preferably, an antagonist II can completely block the interaction of CD 163 with CD 163-ligand. By neutralizing CD163, the binding/interaction of a molecule of the invention (e.g. CD163- ligand) with CD163 (e.g. sCD163) is at least in part reduced. Neutralizing CD 163 can serve to counteract the dampening effects of CD 163 on immune responses, like T-celll responses. For instance an antagonist II can serve to augment an immune response through neutralization of sCD163, by preventing the interaction of CD163 with the CD163-ligand. Preferably said antagonist II is an antibody or functional part, derivative and/or analogue thereof. It is also understood that said antagonist can comprise an antibody with a neutralizing and/or blocking function.

The invention also provides an antagonist II coupled to a moiety. In terms of the invention said moiety can serve to confer additional properties to said antagonist II of CD163 (e.g. increased longevity and stability, improved intracellular targeting) or to improve its biological activity (i.e. imunomodulatory activity and/or pharmacokinetics). Suitable moieties can include a molecule comprising at least part of an immunoglobulin chain (e.g. a constant region of said chain), a molecule with immunoregulatory activity like a cytokine (e.g. IL-10, IL-12, GCSF, IFN-gamma, etc.), a toxic moiety, and a protection molecule like polyethylene glycol (PEG). For example, said antagonist II can comprise an antibody which can be coupled to a toxic moiety, allowing the reduction or a depletion of cells expressing CD163. In the same manner a toxic moiety can also be coupled to sCD163 or a fragment thereof, to reduce or eliminate its target cells.

The invention further provides an agonist of CD 163, further referred to as agonist II. Preferably said agonist II is an antibody or functional part, derivative and/or analogue thereof. It is understood that said agonist II can serve to inhibit an immune in a similar manner to a CD 163-ligand molecule of the invention, through its ability to stimulate the production of anti- inflammatory cytokines such as IL-10 whilst inhibiting the production of pro- inflammatory cytokines such as TNF-alpha in CD 163 expressing cells. The invention further provides an agonist II coupled to a moiety. In terms of the invention said moiety can serve to confer additional properties to said agonist II of CD 163 (e.g. increased longevity and stability, improved intracellular targeting) or to improve its biological activity (i.e. imunomodulatory activity and/or pharmacokinetics). Suitable moieties can include a molecule comprising at least part of an immunoglobulin chain (e.g. a constant region of said chain), a molecule with immunoregulatory activity like a cytokine (e.g. IL-10, IL-12, GCSF, IFN-gamma, etc.), a toxic moiety, and a protection molecule like polyethylene glycol (PEG).

Furthermore the invention provides a method to detect the presence of a CD 163-ligand molecule according to the invention in a sample comprising contacting the sample with a binding molecule for said CD 163-ligand molecule according to the invention to form a complex, further comprising detecting said complex in the sample. For example said binding molecule is any entity, be it nucleic acid, amino acid, a carbohydrate or a lipid comprising moiety (or combinations thereof), than can bind a molecule of the invention. Preferably said entity is coupled/linked to a moiety which enables the detection of a molecule of the invention in a sample.

Preferably said binding molecule comprises a CD163-ligand-binding antibody according to the invention. More preferably, said antibody is an antagonist I and/or agonist I according to the invention. For example said antagonist I or agonist I can have a moiety attached, which can be for example recognized by a detection/determining substance (e.g. a label). For instance a visually detectable or direct label (e.g. radioactive label, enzyme label, fluorescent label chemiluminescent label, bioluminescent label, gold label etc.). Examples of commonly used enzyme labels are horseradish peroxidase, alkaline phosphatase and beta-galactosidase etc. Also preferred is that said binding molecule comprises sCD163 and/or mCD163.

In yet another aspect the invention provides a method to determine the binding activity of a CD163-ligand molecule according, to the invention in a sample comprising detecting the presence of a molecule using a method according the invention, further determining the levels (i.e. amount) of binding molecule-molecule (i.e. complex) in the sample. It is therefore an object of the present invention to monitor an immune response comprising detecting the presence of and/or monitoring the binding activity of a CD 163-ligand molecule (e.g. sCD 163-ligand) in a sample. Screening technologies are known in the art for example proteomic technologies.

The invention provides a method for the production of a diagnostic kit comprising a method to detect the presence of a CD 163-ligand molecule according to the invention in a sample and/or to determine the binding activity of a CD163-Iigand molecule according to the invention in a sample. Suitable basis for a diagnostic kit are known in the art. In one aspect the invention provides a nucleic acid, functional part, functional derivative and/or analogue thereof encoding a CD 163-ligand molecule according to the invention. A functional part of a nucleic acid of the invention is a part of said nucleic acid whose encoded product is capable of modulating an immune response, preferably in a CD163 related pathway, in an organism. A functional derivative of a nucleic acid of the invention is any nucleic acid produced from or related to said nucleic acid, which retains the same properties as said nucleic acid in kind not necessarily in amount. An analogue of a nucleic acid of the invention can be for example an allelic variant.

In another aspect the invention provides a nucleic acid functional part, functional derivative and/or analogue thereof encoding an antagonist I of a CD 163-ligand molecule according to the invention. In another aspect the invention provides a nucleic acid, functional part, functional derivative and/or analogue thereof encoding an agonist I of a CD163-ligand molecule according to the invention. In yet another aspect the invention provides a nucleic acid encoding an isolated CD 163 according to the invention. In yet another aspect the invention provides a nucleic acid encoding an antagonist II or an agonist II of an isolated CD 163 according to the invention.

In yet another aspect the invention provides a vector comprising a nucleic acid according to the invention. Suitable vectors are known to one of skill in the art, for example plasmid vectors, viral vectors etc. In another aspect the invention provides a cell comprising a vector according to the invention. Preferably a mammalian or avian cell. Furthermore the invention provides a gene delivery vehicle comprising a vector according to the invention. A gene delivery vehicle as used herein is any vehicle that can deliver a nucleic acid of the invention to an organism, for the purpose of modulating an immune response in an organism. The invention also provides the use of a gene delivery vehicle of the invention for the preparation of a medicament. The invention provides a method for the production of a CD 163-ligand molecule according to the invention, or an antagonist I of a molecule according to the invention or an agonist I of a molecule according to the invention, or CD 163 according to the invention, or an antagonist II of CD 163 according to the invention or an agonist II of CD 163 according to the invention in an organism comprising inserting into the genome of said organism one or more copies of a nucleic acid according to the invention. An organism in the context of the present invention can be for example a micro-organism (e.g. Archaea, Bacteria, Cyanobacteria, Microalgae, Fungi, Yeast, Viruses, Protozoa, Rotifers, Nematodes, Micro- Crustaceans, Micro-Molluscs, Micro-Shellfish, Micro-insects etc.), a plant, a non-human animal and a plant or animal cell (e.g. artificial cell, cell culture or protoplast etc.).

In yet another aspect the invention provides use of an isolated CD 163 according to the invention and/or an antagonist II of CD 163 according to the invention to modulate an immune response. For instance, said CD163 according to the invention and/or an antagonist II can be linked to a second or subsequent moiety to form a fusion protein. In terms of the invention said moiety can serve to confer additional properties to said CD163 molecule (e.g. increased longevity and stability, improved intracellular targeting) or to improve its biological activity (i.e. imunomodulatory activity and/or pharmacokinetics). Suitable moieties can include a molecule comprising at least part of an immunoglobulin chain (e.g. a constant region of said chain), a molecule with immunoregulatory activity like a cytokine (e.g. IL-10, IL-12, GCSF, TNF-alpha, IFN-gamma, etc.), a toxic moiety, and a protection molecule like polyethylene glycol (PEG).

For example, the use of a fusion protein of CD163 (e.g. sCD163) coupled to the constant region of an immunoglobulin (e.g. CD163-Fc) can have several advantages over unmodified CD 163. For instance, a CD163-Fc has an increased half life in circulation. Additionally a CD163-Fc can be generated as a monovalent (i.e. one CD163 coupled to the constant region of an immunoglobulin chain) molecule, or more preferred as a bivalent (i.e. one or two CD163 moieties coupled to the constant region of an immunoglobulin chain) molecule. A bivalent CD163-Fc molecule is likely to block a molecule of the invention (e.g. CD163-ligand) with a higher avidity than a monovalent sCD163. For instance, an isolated CD163 according to the invention and/or an isolated CD 163 coupled to a moiety, can upon binding to a CD 163-ligand molecule of the invention, preferably a membrane bound molecule, at least in part block the binding to mCD163 with CD163-ligand, resulting in inducing/stimulating/augmenting of an immune response. Said CD163 and/or CD 163 coupled to a moiety can for instance bind monovalently, bivalently and/or multivalently to a molecule according to the invention (i.e. CD 163- ligand). Preferably said immune response comprises an immune response to a cancer cell or an infectious agent such as a virus, a parasite, a fungus, or a bacterium. In the case of an immune response which comprises an immune response to a cancer cell or an infectious agent such as a virus, a parasite, a fungus, or a bacterium, CD163 and/or CD163 coupled to a moiety can be used to suppress unwanted and uncontrolled tumor growth and also eradicate infections with pathogens through its stimulating effect on immune responses.

An antagonist II of CD163 can at least in part prevent CD163 from binding/interacting with a CD 163-ligand molecule of the invention thereby preventing the delivery of a signal though CD 163 resulting in the production of anti-inflammatory cytokines. For instance, said antagonist II of CD 163 can be a blocking antibody, or a functional derivative thereof. An antagonist II of CD163 of the present invention is particularly suitable for inducing/stimulating/augmenting an immune response in an organism, (e.g. mammal), in which the immune system is not efficiently stimulated, as is the case in cancer and infection.

It should be noted that substances that can remove CD 163 from the cell surface either by downregulating CD 163 gene expression or by active removal of the molecule from the surface have similar immunological effects as antagonists II and are thus equivalents of antagonist II. Such equivalents are also part of the invention, as well as method and uses

Further, in diseases with increased serum levels of sCD163, an antagonist II according to the invention is capable of binding to sCDl63 and of at least in part neutralizing the immune stimulating effect of sCD163. With an antagonist II, it is now for instance possible to efficiently decrease unwanted high levels of circulating sCD163 in patients, suffering from inflammatory diseases without harmful side-effects.

In another aspect the invention provides use of sCD163 and/or sCD163 coupled to a moiety and/or an antagonist II and/or an antagonist II coupled to a moiety to modulate an immune response. Said immune response can comprises an innate and/or an antigen-specific (adaptive) immune response. Preferably said immune response comprises an antigen-specific (adaptive) immune response. In the case of an immune response which comprises an insufficient immune response as seen in cancer and infections, sCDl63 and/or sCD163 coupled to a moiety and/or an antagonist II and/or an antagonist II coupled to a moiety can be used to stimulate immune responses to effectively remove tumor cells and eradicate infection.

In the case of diseases with increased serum levels of sCD163, excessive immune responses can be dampened by an excess of antagonist II and/or an antagonist II coupled to a moiety that efficiently neutralize sCD163. The invention further provides an agonist II that is capable of at least interacting (e.g. binding) with mCD163, modulating the activation of a cell bearing CD 163, resulting in the enhanced production of anti-inflammatory cytokines like IL-10, and decreased production of pro-inflammatory cytokinessuch as TNF-alpha. For instance, said agonist II of CD163 can be a blocking antibody, or a functional derivative thereof. Preferably, said agonist II is the antibody RM3/1, an antibody that has the same functional effect on CD 163 expressing cells, or an antibody that recognizes the same epitope as the RM3/1 antibody. Thus by modulating the activation of CD 163 bearing cells an immune response is dampened. Preferably an agonist II does not completely block interaction of CD 163 with CD 163-ligand. It should be noted however, that an agonist II may completely block the interaction of CD 163 with CD 163-ligand, but is discriminated from an antagonist II on the basis of its functional effect on CD163 expressing cells (ie. stimulating production of IL-10 whilst inhibiting production of TNF-alpha).

In another aspect the invention provides use of an agonist II of CD 163 and/or an agonist II coupled to a moiety to modulate an immune response. Said immune response can comprises an innate and/or an antigen-specific

(adaptive) immune response. Preferably said immune response comprises an antigen-specific (adaptive) immune response. Even more preferably, modulation of an immune response with an agonist II comprises dampening/suppression/inhibition of an immune response. With an agonist II, it is now for instance possible to treat diseases characterized by excessive immune activation, such as for instance autoimmune diseases, inflammatory diseases, or transplant rejection.

In another aspect the invention provides a pharmaceutical composition comprising a CD 163-ligand molecule according to the invention and/or an antagonist I of a CD 163-ligand molecule according to the invention and/or an agonist I of a CD 163-ligand molecule according to the invention and/or an isolated CD 163 according to the invention and/or an antagonist II of an isolated CD 163 and/or an agonist II of an isolated CD 163 and/or a cell according to the invention and/or a gene delivery vehicle according to the invention. Suitable basis for pharmaceutical compositions are known in the art. Pharmaceutically acceptable carriers are well known in the art and include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. One example of such an acceptable carrier is a physiologically balanced culture medium containing one or more stabilizing agents such as stabilized, hydrolyzed proteins, lactose, etc. The carrier is preferably sterile.

In yet another aspect the invention provides a pharmaceutical composition according to the invention for augmenting or suppressing an immune response. Depending on the use (i.e. to enhance or suppress an immune response) a therapeutic or a prophylactic effective amount of a second substance (i.e. anti-inflammatory/immunosuppressive substance like cyclosporin A, FK506, sulfasalazine, antihistamines, bronchodilators, leukotrien inhibitors, (gluco)-corticosteroids, anti-TNF-alpha antibodies, anti- CD40 antibodies, CpGs, or immunostimulatory substances like type I interferon, GCSF, IFN-gamma or other cytokines, adjuvants to induce inflammation) can be added to said pharmaceutical composition. Said pharmaceutical composition can also be used in combination with various cancer treatments, treatments for infectious diseases (e.g. antibacterial, antifungal or antiviral substances), or vaccines comprising tumor antigens and/or antigens derived from infectious agents.

In another aspect the invention provides use of a CD 163-ligand molecule according to the invention and/or an antagonist I of a CD 163-ligand molecule according to the invention and/or an agonist I of a CD 163-ligand molecule according to the invention and/or an isolated CD 163 according to the invention and/or an antagonist II of an isolated CD 163 according to the invention and/or an agonist II of an isolated CD 163 according to the invention and/or a cell according to the invention and/or a gene delivery vehicle according to the invention in the preparation of a pharmaceutical composition for the therapeutic or prophylactic treatment of a disease, treatable by modulating an immune response.

On one hand an isolated CD 163 according to the invention and/or an isolated CD163 of the invention coupled to a moiety (e.g. the constant region of an immunoglobulin (e.g. sCD163-Fc)) and/or an antagonist I of CD 163-ligand and/or an antagonist I of CD 163-ligand coupled to a moiety and/or an antagonist II of CD 163 and/or an antagonist II of CD 163 coupled to a moiety can be used to prevent the interaction of mCD163 with CD163-Iigand, serving to stimulate an immune response. This is particularly useful for the therapeutic or prophylactic treatment of an individual with an insufficient immune response disorder, like cancer, infections and other disease characterised by insufficient immune responses.

On the other hand a CD163-ligand molecule of the invention and/or a CD163- ligand molecule of the invention coupled to a moiety (e.g. the constant region of an immunoglobulin (e.g. sCD163-ligand-Fc)) and/or an agonist I of a CD163- ligand molecule of the invention and/or an agonist I of a CD 163-ligand molecule of the invention coupled to a moiety and/or an agonist II of CD 163 and/or an agonist II of CD 163 coupled to a moietycan serve to suppress an immune response. This is particularly useful for the therapeutic or prophylactic treatment of an individual with an immune response disorder, like inflammatory diseases, autoimmune diseases, transplantation, allergy, asthma etc. In another aspect the invention provides use of a CD 163-ligand molecule according to the invention and/or an antagonist I of a CD 163-ligand molecule according to the invention and/or an agonist I of a CD 163-ligand molecule according to the invention and/or an isolated CD 163 according to the invention and/or an antagonist II of an isolated CD 163 according to the invention and/or an agonist II of an isolated CD 163 according to the invention and/or a cell according to the invention and/or a gene delivery vehicle according to the invention in the preparation of a pharmaceutical composition, wherein said disease comprises an autoimmune disease (e.g. rheumatoid arthritis, etc.), inflammatory disease (e.g. inflammatory bowel disease etc.) allergy, asthma, cancer, infectious diseases (e.g. sepsis etc.), transplantation related diseases, host versus graft related diseases, cardiovascular disease and/or neurological diseases. It is understood that all diseases associated with increased serum SCD163 levels are also included.

The invention provides a method of treatment of a immune related disease comprising a CD 163-ligand molecule according to the invention and/or an antagonist I of a CD163-ligand molecule according to the invention and/or an agonist I of a CD 163-ligand molecule according to the invention and/or an isolated CD 163 according to the invention and/or an antagonist II of an isolated CD 163 according to the invention and/or an agonist II of an isolated CD 163 according to the invention and/or a cell according to the invention and/or a gene delivery vehicle according to the invention according to the invention with a carrier to a suitable recipient. A suitable recipient for example is a mammal, preferably human. Brief description of the drawings

Figure 1. Purification of human soluble CD 163.

Human monocytes were isolated as described in example 1. After a 4 day culture in the presence of 200 nM dexamethasone, shedding of CD 163 was induced by treating the cells with 50 nM PMA for 1 hour in serum-free PBS. The supematants were collected and sCD163 was purified from these supematants by conventional column chromatography methods as described in detail in example 1. Lane 1 is a Mw marker, lane 2 is the supernatant before purification, and lane 3 is purified human soluble CD 163.

Figure 2.

Detection of a ligand to CD 163 present on apoptotic T cells.

Apoptosis was indeed in the human T-cell line Jurkat E6-1 by treating the cells with anti-CD95 antibody (clone CH11; 20 ng/ml). The cells were collected at several time points, treated with sCD163-biotin for 30 minutes, and binding of sCD163-biotin was detected using streptavidin-phycoerythrin, followed by detection in a flowcytometer. The closed curves represent cells not treated with sCD163-biotin, and the open curves represent cells treated with SCD163- biotin. Figure 3.

Induction of low Mw proteins after induction of apoptosis in Jurkat E6-1 T cells.

Apoptosis was indeed in the human T-cell line Jurkat E6-1 by treating the cells for

4 hours with anti-CD95 antibody (clone CH11; 20 ng/ml). As a control, untreated cells were used. The cells were lysed in the non-ionic deteregent Nonidet P40 (NP40), and run on an SDS-PAGE gel. Upon induction of apoptosis (Lane 2), three distinct bands at approximately 10 to 20 kDa were induced that were not present in cells that were not treated with anti-CD95 antibody (lane 1).

Figure 4. Detection of CD163-ligand molecules in a western blot of apoptotic Jurkat E6- 1 cells.

Apoptosis was induced in the human T-cell line Jurkat E6-1 by treating the cells for 4 hours with anti-CD95 antibody (clone CHll; 20 ng/ml). The cells were lysed in the non-ionic deteregent Nonidet P40 (NP40), and run on an SDS-PAGE gel. After transfer of the proteins to nitrocellulse, the blots were treated with sCD163-biotin (lanes 2) or left untreated (lanes 3). Lanes 1 represents a Mw marker. The binding of sCD163-biotin was detected using streptavidin-HRP to detect the biotin moiety (panel A) as well as using anti- CD163-HRP to detect the sCD163 (panel B) Figure 5.

N- and C-terminus of human sCD163.

The figure is a schematic representation of the membrane bound CD 163 protein as encoded by the full length CD 163 gene, indicating the N- and C- terminus of sCD163. To determine the N- and C-terminus of sCD163, peptide mass fingerprinting was performed on purified sCD163 as described in detail in example 17. The most C-terminal peptide detected was EAEFGQGTGPIWLNEVK, and the most N-terminal peptide detected was APGWANSSAGSGRIWMDHVSCR.

Figure 6.

Human CD163-Fc construct.

A schematic representation of the construct is shown in panel A. Panel B shows a Western blot of the human CD163-Fc construct as described in example 18. After 10% SDS-PAGE reduced proteins were transferred to nitrocellulose. Human CD163-Fc was revealed by incubation with anti-human IgG HRP followed by staining with 4-chloro-l-naphtol. Lane 1 control Sf9 cell- lysate, lane 2 human CD163-Fc transfected Sf9 cell-lysate, lane 3 molecular weight markers.

Figure 7.

Murine CD163-Fc construct.

A schematic representation of the construct is shown in panel A. Panel B shows a western blot of the murine CD163-Fc construct expressed as described in example 18. After 10% SDS-PAGE reduced proteins were transferred to nitrocellulose. Murine CD163-Fc was revealed by incubation with anti-murine IgG HRP followed by staining with 4-chloro-l-naphtol. Lane 1 molecular weight markers, lane 2 murine CD163-Fc transfected Sf9 cell- lysate, lane 3 control Sf9 cell-lysate.

Figure 8. Cathepsins B and D remove CD 163 from the cell surface of CD163-expressing cells.

To study the effect of isolated proteinases on cell surface CD 163, monocytes were isolated from buffy coats as described in example 1, and cultured for 4 days in the presence of dexamethasone to induce high levels of CD 163 expression. The monocytes/macrophages were collected and incubated in the presence of PMA or without cathepsin B, cathepsin C, cathepsin D, cathepsin G, or elastase and incubated for 1 hour at 37°C in PBS, 0.1% BSA. Expression of CD 163, Mannose receptor, CD 14, CD36, CD86 and HLA-DR was assessed by FACS analysis. Clearly, cathepsin B and cathepsin G were able to remove CD 163 from the cell membrane, but not other membrane proteins such as CD14, CD36, or HLA-DR.

Figure 9. Induction of IL-10 and inhibition of TNF-alpha by anti-CD163 antibody RM3/1

Purified peripheral blood monocytes were cultured with 200 nM dexamethasone for 4 days. Subsequently, these matured macrophage subpopulations (lxl06/ml) were stimulated with various concentrations of LPS in the absence (open bars) or in the presence of anti-CD163 antibody RM3/1 (black bars; 10 microgram IgGl/ml). After 24 hour LPS treatment TNF-alpha, and IL-10 levels were measured in culture supe atants. Results are presented as mean +/- standard deviation from two separate donors. The figure shows that IL-10 production is enhanced, whilst TNF-alpha production is decreased when the cells are treated with RM3/1 in the presence of LPS. Examples

Example 1. Identification of putative CD163-ligand molecules using (s)CD163

Generation of sCD 163 from human monocytes

To isolate sCD163 peripheral blood mononuclear cells (PBMC) were isolated from a buffy coat by density centrifugation on lymphoprep (Nycomed, Oslo, Norway), followed by three washes. This was followed by gradient centrifugation on Percoll (Pharmacia, Uppsala, Sweden) to isolate monocytes. The monocytes were resuspended in RPMI (BioWhittaker, Walkersville, MD) supplemented with 5% human pool serum (BioWhittaker, Walkersville, MD) and gentamycin, and cultured for 72 hours in the presence of 200 nM dexamethasone (Sigma, St. Louis, MO) to enhance CD163 expression. Upon induction of cell surface CD 163, shedding of CD 163 was induced by washing the cells in serum free PBS, resuspending them in PBS in the presence of 50 nM PMA (Sigma, St. Louis, MO). After a 1 hour incubation at 37°C, supematants were collected to measure sCD163 levels in SDS-PAGE and western blotting analysis using commercially available anti-CD 163 mAbs. In addition, removal of CD 163 from the cell surface was assessed by FACS analysis using commercially available anti-CD163 mAbs. sCD163 was diluted 5-fold with 20 mM Sodium phosphate buffer (pH 7.0), and sCD163 was purified from these supematants by conventional column chromatography methods (SP sepharose followed by Q sepharose column).

A SP Sepharose-column (SP Sepharose fast flow, Amersham Biosciences, Uppsala, Sweden) was equilibrated with 20 mM Sodium phosphate buffer (pH 7.0), the sample was loaded at 0.5 ml/min, and the flow through that contained the sCD163 was collected in several fractions. The sCDl63 containing fractions were pooled, and loaded at 0.5 ml/min onto a Q Sepharose-column (Q Sepharose fast flow Amersham Biosciences, Uppsala, Sweden) that was previously equilibrated with 20 mM sodium phosphate buffer (pH 7.0). sCD163 was eluted from the column by a 0-100% gradient of 20 mM sodium phosphate buffer / 1 M NaCl (pH 7.0) (figure 1).

Biotinylation of sCD163 sCD163-containg fractions were collected from sCD163 purified as described above, and fractions containing sCD163 as evidenced by SDS-PAGE analysis and western blotting were pooled and dialysed against 0.1 M NaHCO3 (pH 8.6). N-hydroxysuccinimide-biotin (Perbio Science, Etten-Leur, the

Netherlands) was dissolved in DMSO, and was added to the CD163 in a 1:10 w/w ratio. The mixture was incubated at room temperature for 4 hours, and dialysed extensively against PBS. BSA was added to a final concentration of 0.2% and NaN₃ was added to a final concentration of 0.1%. Biotin labeling of sCD163 was confirmed by western blotting using streptavidin-HRP (Jackson Immunoresearch laboratories, West Grove, PA).

sCD163-biotin binds to apoptotic cells

To study binding of sCD163 to a putative ligand, initial experiments were performed on peripheral blood mononucuclear cells. PBMC isolated as described above were incubated 15-30 minutes at 37°C in PBS 0.1% BSA in the presence or absence of sCD163-biotin (1:5 - 1:10 dilution), followed by washing and incubation in the presence of streptavidin-PE (1:100, Jackson Immunoresearch laboratories, West Grove, PA). Cells were washed and analysed by FACS analysis (FACScan, Becton & Dickinson, Erembodegem, Belgium). This showed that sCD163-biotin bound to a minor fraction of lymphocytes present in the PBMC. Surprisingly, the sCD163-biotin (but not sterptavidin-PE by itself) bound to cells that had a forward scatter-side scatter profile resembling (pre)apoptotic cells. Similar results were found by using the human T-cell line Jurkat E6-1 (ATCC, Rockville, MD). To obtain more apoptotic Jurkat E6-1 cells, apoptosis was induced by treating the cells with anti-CD95 antibody (clone CH11; 20 ng/ml, Campro Scientific, Veenendaal, the Netherlands). As can be seen in figure 2, the number of early apoptotic cells increased in the first 6 hours of incubation. Simultaneously, sCD163-biotin binding increased to reach >50% of these early apoptotic Jurkat E6-1 cells.

Identification of CD163-lisand molecules on apoptotic Jurkat E6-1 T-cells When these cells were lysed in the non-ionic deteregent Nonidet P40 (NP40), three distinct bands at approximately 10 to 20 kDa were induced, all three of which bound sCD163 (detected with anti-CD163), as well as CD163-biotin (detected with streptavidin-HRP) in western blots (figure 4 a + b).

Mass analyses of CD 163-ligand proteins on apoptotic Jurkat E6-1 T-cells The three 10-20 kDa protein bands of interest (see figure 3) were cut from the gel after Coomassie staining. For MALDI analysis, protein-containing gel slices were S-alkylated with iodoacetamide, digested with trypsin (Roche Molecular Biochemicals, sequencing grade), and extracted according to Shevchenko et al.. (Shevchenko et al., 1996). Only peptides eluted with 20 mM NH HCO3 were used in the analysis. After drying in a vacuum centrifuge, peptides were dissolved in 10 μl of a solution containing 1% formic acid and 60% acetonitrile. Eluted peptides were mixed 1:1 (v/v) with a solution containing 52 M α-cyano-4-hydroxycinnamic acid (Sigma-Aldrich Chemie BV) in 49% ethanol/49% acetonitril/2% TFA and 1 mM Ammoniumacetate (0.5 μl with 0.5 μl). Prior to dissolving, the α-cyano-4-hydroxycinnamic acid was washed briefly with acetone. The mixture was spotted on target and allowed to dry at room temperature. Reflectron MALDI-TOF spectra were acquired on a Micromass M@LDI (Wythenshawe, UK). The resulting peptide spectra were used to search several Non-Redundant Protein Databases. For further MSMS analysis, a few μl of several peptide solutions was concentrated on a C 18 ZipTip (Millipore), eluted in 5 microliter 60% acetonitrile/ 1% HCOOH and analysed on a Micromass Q-TOF mass spectrometer. Low-energy collision- induced dissociation (CID) experiments were performed using argon as a collision gas. Homology searches with fragmented peptides were performed using the BLASTx program with default settings and dbEST database (at http://www.ncbi.nlm.nih.gov:80/blast/Blast.cgi and http://www.ncbi.nlm.nih.gov/dbEST/) and with the MASCOT routine (Matrixscience) .

Results: database search with MASCOT routine (Matrixscience).

Sample T (top band) = datafile EXT0878: Histone H2B, 3 MSMS spectra hits. 40 S ribosomal protein, 1 hit and Histone H2A, 1 hit. Sample M (middle band) = datafile EXT0879: Histone H2A, 2 hits. Sample B (band below) = datafile EXT0880: Profilin 1. 3 hits and Histone H4, 2 hits.

Taken together, the data presented above demonstrates that the 3 bands consist mostly of human H2B, H2A and H4.

In conclusion, the experiments described here show that sCD163 is capable to bind to ligands present on the membranes of early apoptotic cells other than haptoglobin-hemoglobin complexes. The expression of these ligands may be very important for the removal of early apoptotic cells from the body, thus preventing the presence of necrotic cells and tissue damage leading to inflammation.

Example 2. cDNA cloning, expression and purification of recombinant CD163-ligand Ligand molecules to human CD 163, such as histones 2A (H2A, H2B, and H4), can be cloned using PCR primers based on known nucleotide sequences, or (partial) amino acid sequences of CD 163-ligand molecules as described in example 1. PCR reactions can be performed on mRNA derived from T-cells with known CD 163-ligand expression, cloned into a plasmid vector, sequenced, amplified and expressed in several expression systems. Cloning and expression is carried out using standard techniques known to one of skill in the art. CD 163-ligand molecules are expressed and purified by affinity purification using sCD163 or anti-CD 163-ligand (e.g. anti-H2A, -H2B or -H4 mAb) Mabs or by conventional chromatography methods.

Example 3. Generation of a soluble CD163-ligand and a CD163-ligand- Fc fusion protein.

CD 163-ligand and/or fractions thereof, and/or a sCD163-ligand-Fc fusion protein can be generated to be used as therapeutic molecule. CD163- ligand or truncated versions thereof comprising the CD163-binding site are generated and expressed as described in example 1. From these constructs, a highly soluble CD 163-ligand (sCD 163-ligand) that can be expressed at high levels is isolated. Further, the sCD 163-ligand is expressed as fusion protein containing the extracellular part of the membrane molecules fused to the constant region of a human immunoglobulin, for example of IgG4. Other isotypes such as IgGl, IgG2a, IgG2b, and/or IgG3 may also be used for this purpose. For the cloning of the human CH4/hinge-CH3 region PCR primers are designed based on the sequence by Ellison et al.. (Ellison et al., 1982). To allow successful linkage of this fragment to the extracellular domains (ED) of CD 163-ligand, a small linker of 6 amino acids was introduced. In this small linker the BamHI cloning site was incorporated to allow the in-frame fusion of the CD 163-ligand ED. The human IgG4 Fc-region is cloned from stimulated human B cells. Total RNA is isolated and by RT-PCR the Fc-region is cloned and sequence verified. For expression, the Fc region is subcloned in the baculovirus expression vector pVL1393. By DNA sequence analysis the construct is checked. Using suitable primers starting from activated human T-cells the CD 163-ligand or a functional part thereof is amplified by RT-PCR (see example 2). After sequence analysis the correct clones are cloned using BamHI in the already mentioned pVL1393 expression vector containing human Fc. About 3xl0⁶ Sf9 insecT-cells are plated in a 25 cm² culture flask and co-transfected with 2 mg CD163- ligand -Fc transfer vector together with 0.5 mg wild-type linearized baculovirus. After 3 days the virus is re-amplified on fresh Sf9 cells, followed by several new rounds to obtain recombinant baculovirus stocks for expression. By Western blot analysis expression of both proteins is confirmed.

Example 4. In vitro immunomodulatory effect of sCD163- ligand and sCD163-ligand -Fc

The immunomodulatory effect of SCD163- ligand and sCD163- ligand -Fc can be studied by performing a range of immunological assays. Mixed lymphocyte reactions (MLR), as well as antigen-specific T-cell proliferation assays against antigens as tetanus toxoid, Candida albicans, or house dust mite are performed in the absence or presence of sCD 163-ligand and/or sCD163-ligand-Fc. Such experiments can be used to demonstrate that sCD163- ligand and/or sCD163- ligand -Fc inhibit the proliferation of allospecific (in the MLR) as well as antigen-specific T-cell proliferation. Further, modulation of stimulation of CD 163 expressing monocytes and/or macrophages using CD 163-ligand and/or CD163-ligand-Fc can be studied using human monocytes (e.g cultured in the presence of glucocorticoids to induce CD163 expression). The CD 163- expressing monocytes are incubated in the presence or absence of a monocyte /macrophage stimulus such as LPS in the presence or absence of (s)CD163- ligand and/or (s)CD163-ligand-Fc. After 24 hours, supematants of these cultures are collected, and the levels of cytokines produced (such as TNF- alpha, IL-lbeta, IL-10 and others) are determined, showing that the monocytes/macrophages are induced by (s)CD 163-ligand and/or (s)CD163- ligand-Fc to produce less pro-inflammatory cytokines, whilst enhancing the production of anti-inflammatory cytokines such as IL-10. Further, cells are collected and the expression of a range of cell surface molecules (e.g. CD14,CD40, MR, CD83 etc) is studied using FACS analysis, to determine if the expression levels of a number of relevanT-cell surface markers is influenced as a result of treatment with (s)CD163-ligand and/or (s)CD163- ligand-Fc.

Example 5. In vivo immunosuppressive effect of (s)CD163-ligand and/or (s)CD163-ligand-Fc.

The immunosuppressive activity of (s)CD 163-ligand and/or (s)CD163-ligand-Fc can be tested in a range of animal models for inflammatory disease. For this, human (s)CD 163-ligand and/or (s)CD163-ligand-Fc and/or murine (s)CD163- ligand and/or (s) CD 163-ligand- Fc may be used. For example, (s) CD 163-ligand and/or (s)CD 163-ligand- Fc can be tested in a mouse model for multiple sclerosis, namely acute Experimental Allergic Encephalomyehtis (EAE). EAE can be induced with in SJL/J mice a synthetic peptide which is encephalitogenic in SJL/J. When mice are treated with (s)CD 163-ligand and/or (s)CD163-ligand-Fc, efficacy can be shown by decreased clinical symptoms of EAE as well as by histological analysis. Similar experiments can be performed by those skilled in the art in animal models for arthritis, allergy, asthma, transplantation, inflammatory bowel disease, and other inflammatory diseases.

Example 6. In vitro neutralization of immunostimulatory effect of sCD163 by sCD163-ligand and CD163-ligand-Fc

The immunostimulatory effect of soluble sCD163 can be demonstrated by MLR as well as by antigen-specific T-celll proliferation assays (See example 19). Similar assays can be performed in the absence or presence of (s)CD163-ligand and/or (s)CDl63-ligand-Fc. Thus, the immunostimulatory effect of sCD163 can be overcome by the addition of (s) CD 163-ligand and/or (s)CD163-ligand-Fc to the cultures, most likely as a result of preventing the blocking of membrane bound CD 163-ligand by sCD163. This can be proven to be correct in FACS experiments, in which labeled sCD163 is added to CD 163-ligand expressing T- cells in the absence or presence of (s)CD163-ligand and/or (s)CD163-ligand-Fc.

Example 7. Treatment of human inflammatory diseases with (s)CD163- ligand and/or (s)CD163-ligand-Fc

A variety and myriad number of autoimmune and inflammatory diseases are indications for treatment with (s)CD 163-ligand and/or (s)CD 163-ligand- Fc molecules of the invention. These include but are not limited to rheumatoid arthritis, diabetes, multiple sclerosis, systemic lupus erythematosous, psoriasis, autoimmune thyroiditis, allergy, asthma, inflammatory bowel disease, septic shock, transplant rejection, atherosclerosis, other cardiovascular diseases, and Alzheimer's disease.

Example 8. Generation of antibodies to CD163-ligand

Polyclonal antiserum against CD 163-ligand (for example histone2A, histone 2B and/or histone 4) is raised by immunization of rabbits with recombinant human CD 163-ligand or fractions thereof (e.g 4 injections/animal; 20-200 μg/injection). After the final booster the animals are bled to determine the titer of the polyclonal antiserum. To obtain monoclonal antibodies 6-8 wk old Balb/c mice are immunized with recombinant human CD 163-ligand or fractions thereof (for example 4 times with 2 wk intervals with 10-100 μg/injection dissolved in Freunds complete adjuvans for the first injection, and Freunds incomplete adjuvans for subsequent immunizations). Splenocytes are isolated and fused with a fusion cell line (Sp2/0 myeloma cells), followed by limiting dilution. Growing clones are screened using for example an enzyme-linked immunosorbant assay (ELISA). Therefore 96 wells plates are coated with CD 163-ligand or with a control protein. The culture supernatant is added, followed by washing and addition of a labeled anti-mouse antibody for detection. After limited dilution cloning of CD 163- ligand-specific antibody producing hybridomas, stable hybridomas are obtained. From each clone cell supernatant are collected and by affinity chromatography using protein A sepharose columns (Pharmacia, Uppsala, Sweden) monoclonal antibodies are purified. Based on their blocking abilities and/or their functional activities, such antibodies can be subdivided into agonist I, antagonist I and/or non- blocking, non agonist antibodies.

Example 9. In vitro immunomodulatory effect of agonist I and antagonist I antibodies to CD163-ligand

The immunomodulatory effect CD163-ligand-specific agonist I and/or antagonist I antibodies can be studied by performing a range of immunological assays. Mixed lymphocyte reactions (MLR), as well as antigen-specific T-cell proliferation assays against antigens as tetanus toxoid, Candida albicans, or house dust mite are performed in the absence or presence of CD163-ligand- specific agonist I and/or antagonist I antibodies. Such experiments can be used to demonstrate that CD163-ligand-specific antagonist I antibodies stimulate the proliferation of allospecific (in the MLR) as well as antigen-specific T-cell proliferation. Likewise, such experiments can be used to demonstrate that CDl63-ligand-specific agonist I antibodies inhibit the proliferation of allospecific (in the MLR) as well as antigen-specific T-cell proliferation. Further, supematants of these cultures can be collected, and the levels of cytokines produced (such as TNF-alpha, IL-1 beta, IL-10 and others) are determined, showing that the monocytes/macrophages are induced by the blocking effect of CD163-ligand-specific antagonist I antibodies to produce more pro-inflammatory cytokines like TNF-alpha, whilst inhibiting the production of anti-inflammatory cytokines such as IL-10. Further, cells are collected and the expression of a range of cell surface molecules (e.g. CD14,CD40, MR, CD83 etc) is studied using FACS analysis, to determine if the expression levels of a number of relevanT-cell surface markers is influenced as a result of treatment with CD163-ligand-specific antagonist I antibodies. Also, inhibitory effects of agonist I antibodies on the activation of T-cells can be studied by activating T-cells with the cytokines as IL-2 and/or IL-15, by stimulation with mitogens as PMA, by stimulation with monoclonal antibodies (mAb) (for example with a combination of anti-CD28 and anti-CD3 mAb), or in the absence of other stimuli. Activation of T-cells can be evaluated by measuring T-cell cytokines in the supematants, T-cell proliferation and/or the expression of activation markers.

Example 10. In vivo immunostimulatory effect of antagonist I CD163- ligand-specific antibodies

The immunostimulatory effect of (mouse-specific and/or species crossreactive) antagonist I CD 163-ligand specific antibodies can be tested in a range of animal models for cancer and/or infection known to those skilled in the art. One can study survival as well as histology to determine said immunostimulatory activity of antagonist I antibodies, to show that antagonist I antibodies enhance survival in cancer and/or infection through the stimulation of an adequate immune response to the tumor and/or infectious agent.

Example 11. Treatment of cancer and/or infection using antagonist I antibodies to CD163-ligand molecules.

A number of human cancers and/or infection by viruses, bacteria, fungi and/or parasites in which insufficient immune responses are raised to adequately remove the tumor celll and/or the infectious agent are indications for treatment with antagonist I CD163-ligand-specific antibodies.

Example 12. Effect of agonist I CD163- ligand -specific antibodies on degranulation of human basophils As CD 163-ligand can be present on basophils and masT-cells, the effect of CDl63-ligand-specific agonist I antibodies on basophils that are triggered to degranulate either as a result of crosslinking of the high affinity receptor for IgE by anti IgE, or by stimulation with complement factor C5a can be studied. It can be demonstrated that the upregulation after activation as described above of two cell surface markers, namely CD203C (97A6) and CD63, is inhibited by agonist I CDl63-ligand-specific antibodies, showing that agonist I antibodies prevent the degranulation of basophils.

Example 13. In vivo immunosuppressive effect of agonist I CD163- ligand-specific antibodies

The immunosuppressive activity of (mouse-specific and/or species crossreactive) agonist I CD163-ligand-specific antibodies can be tested in a range of animal models for inflammatory disease. For example, agonist I CD163-ligand-specific antibodies can be tested in a mouse model for multiple sclerosis, namely acute Experimental Allergic Encephalomyehtis (EAE). EAE can be induced with in SJL/J mice a synthetic peptide which is encephalitogenic in SJL/J. When mice are treated with agonist I CD 163- ligand-specific antibodies, efficacy can be shown by decreased clinical symptoms of EAE as well as by histological analysis. Similar experiments can be performed by those skilled in the art in animal models for arthritis, allergy, asthma, transplantation, inflammatory bowel disease, and other inflammatory diseases.

Example 14. Treatment of autoimmune and inflammatory diseases with agonist I CD163-ligand-specific antibodies

A variety and myriad number of autoimmune and inflammatory diseases are indications for treatment with agonist I CD163-ligand-specific antibodies of the invention. These include but are not limited to rheumatoid arthritis, diabetes, multiple sclerosis, systemic lupus erythematosous, psoriasis, autoimmune thyroiditis, allergy, asthma, inflammatory bowel disease, septic shock, transplant rejection, atherosclerosis, other cardiovascular diseases, and Alzheimer's disease .

Example 15. Identification of CD163-ligand expressing cells

Using the CD163-ligand-specific antibodies described in example 8, the expression of CD 163-ligand on a wide variety of cell types can be studied, for example by fluorescence activated cell sorter (FACS) analysis, and by immunohistochemistry. The expression of CD 163-ligand can be shown on a number of cell types, including but not limited to lymphoid, myeloid and endothehal cells.

Example 16. Assay to measure CD163-ligand in biological fluids or culture supematants A method was developed in which an anti-CD163-ligand mAb or sCD163 is used to capture SCD163- ligand. After this step, the sCD163- ligand can be detected with another anti-CD 163- ligand mAb or sCD163 that are labeled to allow detection. This assay can be used as a diagnostic procedure, or can be used to screen compound for their effect on removal of CD 163- ligand from the cell membrane of CD163- ligand -expressing cells.

Example 17. Determination of C- and N-terminus of sCD163.

To determine the exact C- and N-terminus of sCD163 shed from monocytes peripheral blood mononuclear cells (PBMC) were isolated from sixteen buffy coats by density centrifugation on lymphoprep, followed by percoll density centrifugation. The monocytes were collected from the interphase, washed and suspended in RPMI, 5% human pool serum, and gentamycin as antibiotic. The monocytes were cultured for 48 hours in the presence of 200 nM dexamethasone. After 48 h, the cells were washed and resuspended in PBS (10⁷/ml). Shedding of membrane CD163 was induced by 50 nM PMA for 1.5 hours at 37C. Cells were centrifuged and the supernatant containing sCD163 was removed and purified by ion exchange chromatography (IEX). For the determination of the C- and N- terminus, the sCD163 was subjected to electrophoresis on a denatured, reduced 7.5% SDS-PAGE gel, stained with coomassie blue, and the 130-155 kDa band (corresponding to the band of 110- 130 kDa on the western blot made of a non-reduced SDS-PAGE) was excised for analysis of the N- and C-terminus by peptide mass fingerprinting. This analysis indicated that the 130-155 kDa (110-130 kDa under non-reducing circumstances) sCD163 was truncated at the C-terminus as well as at the N- terminus, resulting in a C-terminus of EAEFGQGTGPIWLNEVK (C-terminal residue: lysine) and a N-terminus of APGWANSSAGSGRIWMDHVSCR (N- terminal residue: alanine) (figure 5). It should be noted however, that additional C- or N-terminal amino acids may be present but were not identified in the analysis.

Example 18. Generation of sCD163-Fc fusion proteins

A cDNA encoding human CD 163 was cloned from dexamethasone stimulated monocytes using PCR primers (SEQ 1 and SEQ 2) based on the literature. From this full-length clone, truncated variants were generated to generate a sCD163 (sCD163). The sCD163 was expressed in several expression systems, and purified. A recombinant SCD163-FC fusion protein was generated to be used as therapeutic molecule (figure 6 a+b). For this molecule the extracellular domain of human CD163 was cloned using PCR primers (SEQ 3 and SEQ 4). CD163 was expressed as fusion protein containing the extracellular part of the membrane molecule fused to the constant re ion of a human antibody, in this case IgGl. Other isotypes such as IgG4, IgG2a, IgG2b, and/or IgG3 may however also be used. For the cloning of a human Fc region PCR primers (SEQ 5 and SEQ 6) were designed based on the published sequence. To allow successful linkage of this fragment to the extracellular domain of CD 163, a small linker was introduced. In this small linker the Xbal cloning site was incorporated to allow the in-frame fusion of the CD 163 ED with the human IgGl Fc part. The human IgGl Fc-region was cloned from stimulated human PBMC cells. Total RNA was isolated and by RT-PCR the Fc-region was cloned and sequence verified. Using the Xbal site a clone encoding for the human CD 163 Fc fusion protein was obtained (SEQ 11).

Similarly, a murine CD 163 fusion protein was cloned (figure 7 a+b). cDNA encoding for the extracellular part of mCD163 was obtained by RT-PCR on spleen cells from dexamethasone treated mice using PCR primers (SEQ 7 and SEQ 8). A murine IgGl Fc region was cloned by RT-PCR using PCR primers (SEQ 9 and SEQ 10). As template RNA was isolated from a murine hybridoma cell line expressing a IgGl mAb. After sequence verification a correct clone was obtained by fusing the mCD163 part with the mlgGl part using BamHI (SEQ 12).

For expression, both the human and the murine fusion protein were subcloned in the baculovirus expression vector pVL1393. About 3xl0⁶ Sf9 insecT-cells were plated in a 25 cm² culture flask and co-transfected with 2 μg pVL1393 transfer vector containing the recombinant insert together with 0.5 μg wild- type linearized baculovirus. After 3 days the virus was re-amphfied on fresh Sf9 cells, followed by several new rounds to obtain recombinant baculovirus stocks for expression. By Western blot analysis expression of both proteins was confirmed (Figures 6b and 7b).

Example 19. In vitro immunostimulatory effect of (s)CD163 and/or (s)CD163-Fc

The immunostimulatory effect of (s)CD163 and/or (s)CD163-Fc can be demonstrated by performing a range of immunological assays. Mixed lymphocyte reactions (MLR), as well as antigen-specific T-cell proliferation assays against antigens as tetanus toxoid, Candida albicans, or house dust mite are performed in the absence or presence of (s)CD163 and/or (s)CD163-Fc. Such experiments can be used to demonstrate that (s)CD163 and/or (s)CD163- Fc stimulate the proliferation of allospecific (in the MLR) as well as antigen- specific T-cell proliferation. Further, supematants of these cultures can be collected, and the levels of cytokines produced (such as TNF-alpha, IL-lbeta, IL-10 and others) are determined, showing that the monocytes/macrophages are induced by the blocking effect of (s)CD163 and/or (s)CD163-Fc to produce more pro-inflammatory cytokines like TNF-alpha, whilst inhibiting the production of anti-inflammatory cytokines such as IL-10. Further, cells are collected and the expression of a range of cell surface molecules (e.g. CD14,CD40, MR, CD83 etc) is studied using FACS analysis, to determine if the expression levels of a number of relevanT-cell surface markers is influenced as a result of treatment with (S)CD163 and/or (s)CD163-Fc.

Example 20. In vivo immunostimulatory effect of (s)CD163 and/or

(s)CD163-Fc

The immunostimulatory effect of (murine and/or human) (s)CD163 and/or

(s)CD163-Fc can be tested in a range of animal models for cancer and/or infection known to those skilled in the art. One can study survival as well as histology to determine said immunostimulatory activity of (s)CD163 and/or (s)CD163-Fc, to show that (S)CD163 and/or (s)CD163-Fc enhance survival in cancer and/or infection through the stimulation of an adequate immune response to the tumor and/or infectious agent.

Example 21. Treatment of cancer and/or infection using (s)CD163 and/or (s)CD163-Fc

A number of human cancers and or infection by viruses, bacteria, fungi and/or parasites in which insufficient immune responses are raised to adequately remove the tumor celll and/or the infectious agent are indications for treatment with (s)CD163 and/or (s)CD163-Fc.

Example 22. Screening assay to identify factors that induce or prevent CD163 shedding from cultured monocytes / macrophages

It is known that PMA as well as LPS can induce shedding of CD 163 from the cell membranes. This process is inhibited by proteinase inhibitors, especially by metalloproteinase inhibitors as TIMP-3 or TAPI-0. To study the effect of other groups of isolated proteinases (aspartic, serine, and cystein endoproteinases) on cell surface CD 163 expression, monocytes were isolated from buffy coats. Monocytes were isolated from buffy coats as described in example 1. The monocytes were resuspended in IMDM (BioWhittaker, Walkersville, MD) supplemented with 5% human pool serum and gentamycin, and cultured for 4 days in the presence of dexamethasone (200 nM, Sigma, St. Louis, MO) to induce high levels of CD163 expression. The monocytes/macrophages were collected and incubated in the presence of PMA or without cathepsin B, cathepsin C, cathepsin D, cathepsin G, or elastase and incubated for lh at 37 C in PBS, 0.1% BSA. Expression of CD163, CD14, and HLA-DR was assessed by FACS analysis. As is shown in figure 8 cathepsin B and cathepsin G were able to remove CD 163 from the cell membrane, but not other membrane proteins such as CD14, CD36, or HLA-DR. One can use this method, also extendible to test for the presence of sCD163 in supematants using ELISA and/or western blotting, to screen for additional compounds that remove CD 163 from the cell surface (ie detecting factors that actively remove CD163 and/or compounds that downregulate the expression of CD163).

Example 23. Generation of antibodies to CD163

Polyclonal antiserum against (s)CD163 can be raised by immunization of rabbits with recombinant human (s)CD163 (e.g 4 injections/animal; 20-200 μg/injection). After the final booster the animals are bled to determine the titer of the polyclonal antiserum. To obtain monoclonal antibodies 6-8 wk old Balb/c mice arre immunized with human sCD163 (for example 4 times with 2 wk intervals with 10-100 μg/injection dissolved in Freunds complete adjuvans for the first injection, and Freunds incomplete adjuvans for subsequent immunizations). Splenocytes are isolated and fused with a fusion cell line such as Sp2/0 myeloma cells, followed by limiting dilution. Growing clones are screened using for example an ELISA. Therefore 96 wells plates are coated with sCD163 or with a control protein. The culture supernatant is added, followed by washing and addition of a labeled anti-mouse antibody for detection. After limited dilution cloning of sCD163-specific antibody producing hybridomas stable hybridomas are obtained. From each clone cell supernatant is collected and by affinity chromatography using protein A sepharose columns (Pharmacia, Uppsala, Sweden) monoclonal antibodies are purified. Monoclonal antibodies are further characterized by FACS analysis to determine binding of sCD163-specific mAbs to soluble as well as to membrane bound CD163. Based on their blocking abilities and/or their functional activities, such mAb can be subdivided into agonist II, antagonist II and/or non-blocking, non agonist mAb.

Example 24. Induction of anti-inflammatory cytokines and inhibition of pro-inflammatory cytokines using CD163-specific agonist II antibodies in vitro.

To study the possible role of cell surface CD 163 in enhancing inflammatory responses, the effect of CD 163 antibodies on LPS-induced activation of macrophages was studied. To this aim monocytes were isolated from buffy coats and cultured in the presence or absence of dexamethasone as described in example 1.

These CD163-expressing macrophages were stimulated with bacterial lipopolysaccharide (LPS, E.Coli strain 055:B5, Sigma, 100 ng/ml) in the absence or in the presence of anti-CD163 mAb RM3/1 (RDI Research Diagnostics, Flanders, NJ) to mimic the interaction of CD 163 with its putative ligand. After a 24 hour incubation, supematants were collected and levels of pro- (TNF-alpha) and anti-inflammatory (IL-10) cytokines from these cultures were determined by enzyme-linked immunosorbent assay (ELISA) (CLB, Amsterdam, the Netherlands) after 24 hour incubation. Surprisingly, rather than increasing TNF-alpha production, addition of anti-CD 163 antibodies dampened LPS-induced TNF-alpha responses from macrophages in two independent donors. In contrast, IL-10 levels were augmented under these conditions (figure 9). This was the case for dexamethasone treated macrophages that had a very high CD 163 expression, and also to a lesser extent for control macrophages, that expressed lower levels of CD 163.

However, CD 163 has been shown to be enzymatically (metalloproteinase) cleaved from the cell membrane of dexamethasone-stimulated macrophages after exposure to LPS (Hintz et al. 2002) The CD 163 positive macrophages described here were incubated with anti-CD 163 mAb prior to LPS addition. After the incubation with LPS, however, CD163 was still present on the cell membrane, indicating that shedding of CD163 molecules induced by LPS is inhibited by CD 163 ligation. Thus, CD 163 ligation may have anti- inflammatory activity by preventing proteinase -induced tissue damage as well as by shifting to a more anti-inflammatory profile of cytokine production.

Example 25. In vivo immunosuppressive effect of CD163-specific agonist II antibodies

The immunosuppressive activity of CD163-specific agonist II antibodies can be tested in a range of animal models for inflammatory disease. For this, species crossreactive human and/or mouse-specific CD163-specific agonist II antibodies may be used. For example, CD163-specific agonist II antibodies can be tested in a mouse model for multiple sclerosis, namely acute Experimental Allergic Encephalomyehtis (EAE). EAE can be induced with in SJL/J mice a synthetic peptide which is encephalitogenic in SJL/J. When mice are treated with CDl63-specific agonist II antibodies, efficacy can be shown by decreased clinical symptoms of EAE as well as by histological analysis. Similar experiments can be performed by those skilled in the art in animal models for arthritis, allergy, asthma, transplantation, inflammatory bowel disease, and other inflammatory diseases.

Example 26. Treatment of human inflammatory diseases with CD163- specific agonist II antibodies

A variety and myriad number of autoimmune and inflammatory diseases are indications for treatment with CD163-specific agonist II antibodies of the invention. These include but are not limited to rheumatoid arthritis, diabetes, multiple sclerosis, systemic lupus erythematosous, psoriasis, autoimmune thyroiditis, allergy, asthma, inflammatory bowel disease, septic shock, transplant rejection, atherosclerosis, other cardiovascular diseases, and Alzheimer's disease.

Example 27. In vitro immunostimulatory effect of CD163-specific antagonist II antibodies

The immunostimulatory effect of (s)CD163 and/or (s)CD163-Fc can be demonstrated by performing a range of immunological assays. Mixed lymphocyte reactions (MLR), as well as antigen-specific T-cell proliferation assays against antigens as tetanus toxoid, Candida albicans, or house dust mite are performed in the absence or presence of (s)CD163 and/or (s)CD163-Fc. Such experiments can be used to demonstrate that (s)CD163 and/or (s)CD163- Fc stimulate the proliferation of allospecific (in the MLR) as well as antigen- specific T-cell proliferation. Further, supematants of these cultures can be collected, and the levels of cytokines produced (such as TNF-alpha, IL-lbeta, IL-10 and others) are determined, showing that the monocytes/macrophages are induced by the blocking effect of (s)CD163 and/or (s)CD163-Fc to produce more pro-inflammatory cytokines like TNF-alpha, whilst inhibiting the production of anti-inflammatory cytokines such as IL-10. Further, cells are collected and the expression of a range of cell surface molecules (e.g. CD14, CD40, MR, CD83 etc) is studied using FACS analysis, to determine if the expression levels of a number of relevanT-cell surface markers is influenced as a result of treatment with (s)CD163 and/or (s)CD163-Fc.

Example 28. In vivo immunostimulatory effect of CD163-specifϊc antagonist II antibodies

The immunostimulatory effect of (murine and/or human) (s)CD163 and/or (s)CD163-Fc can be tested in a range of animal models for cancer and/or infection known to those skilled in the art. One can study survival as well as histology to determine said immunostimulatory activity of (s) CD 163 and/or (s)CD163-Fc, to show that (s)CD163 and/or (s)CD163-Fc enhance survival in cancer and/or infection through the stimulation of an adequate immune response to the tumor and/or infectious agent.

Example 29. Treatment of human cancer and/or infections with CD163-specific antagonist II antibodies

A number of human cancers and/or infection by viruses, bacteria, fungi and/or parasites in which insufficient immune responses are raised to adequately remove the tumor celll and/or the infectious agent are indications for treatment with (s)CD163 and/or (s)CD163-Fc.

Table 1

SEQ 1 sense 5'-gcgc gatatctagaccaccatggtgctacttgaagactctggat-3'

SEQ 2 antisense 5'-gcgcgatatcgcggccgctcagtgtggctcagaatggcct-3'

SEQ 3 sense 5'-gcgcaagcttgtcgacgatatccaccatggtgctacttgaagactctgga-3'

SEQ 4 antisense 5'-gcgctctagacttcacttcattgagccatatcgg-3'

SEQ 5 sense 5'-gcgctctagaggatcccccgggctgcaggagcccaaatcttgtgacaaaact-3'

SEQ 6 antisense 5'-gcgcgcggccgctcatttacccggagacagggagaggct-3'

SEQ 7 sense 5'-gcgcaagcttgtcgacgatatccaccatgggtggacacagaatggttct-3'

SEQ 8 antisense 5'-gcgcggatcccttaatttcattgagccatatggg-3'

SEQ 9 sense 5'-gcgcggatcccccgggctgcaggtgcccagggattgtggttgtaa-3' SEQ 10 antisense 5'-gcgcagatctgatatcgcggccgctcatttaccaggagagtgggaga-3'

SEQ 11 hCD163-hIgGl

MVLL EDS GSA DFRR HFV NLS

10 20 30 40 50 60 atggtgctac ttgaagactc tggatctgct gacttcagaa gacattttgt caacctgagt taccacgatg aacttctgag acctagacga ctgaagtctt ctgtaaaaca gttggactca

PFTI TVV LLL SACF VTS SLG

70 80 90 100 110 120 cccttcacca ttactgtggt cttacttctc agtgcctgtt ttgtcaccag ttctcttgga gggaagtggt aatgacacca gaatgaagag tcacggacaa aacagtggtc aagagaacct

GTDK ELR LVD GENK CSG RVE

130 140 150 160 170 180 ggaacagaca aggagctgag gctagtggat ggtgaaaaca agtgtagcgg gagagtggaa ccttgtctgt tcctcgactc cgatcaccta ccacttttgt tcacatcgcc ctctcacctt

VKVQ EEW GTV CNNG WSM EAV

190 200 210 220 230 240 gtgaaagtcc aggaggagtg gggaacggtg tgtaataatg gctggagcat ggaagcggtc cactttcagg tcctcctcac cccttgccac acattattac cgacctcgta ccttcgccag

SVIC NQL GCP TAIK APG WAN

250 260 270 280 290 300 tctgtgattt gtaaccagct gggatgtcca actgctatca aagcccctgg atgggctaat agacactaaa cattggtcga ccctacaggt tgacgatagt ttcggggacc tacccgatta SSAG SGR IWM DHVS CRG NES

310 320 330 340 350 360 tccagtgcag gttctggacg catttggatg gatcatgttt cttgtcgtgg gaatgagtca aggtcacgtc caagacctgc gtaaacctac ctagtacaaa gaacagcacc cttactcagt

ALWD CKH DGW GKHS NCT HQQ

370 380 390 400 410 420 gctctttggg attgcaaaca tgatggatgg ggaaagcata gtaactgtac tcaccaacaa cgagaaaccc taacgtttgt actacctacc cctttcgtat cattgacatg agtggttgtt

DAGV TCS DGS NLEM RLT RGG

430 440 450 460 470 480 gatgctggag tgacctgctc agatggatcc aatttggaaa tgaggctgac gcgtggaggg ctacgacctc actggacgag tctacctagg ttaaaccttt actccgactg cgcacctccc

N M C S G R I E I K F Q G R W G T V C D

490 500 510 520 530 540 aatatgtgtt ctggaagaat agagatcaaa ttccaaggac ggtggggaac agtgtgtgat ttatacacaa gaccttctta tctctagttt aaggttcctg ccaccccttg tcacacacta

D N F N I D H A S V I C R Q L E C G S A

550 560 570 580 590 600 gataacttca acatagatca tgcatctgtc atttgtagac aacttgaatg tggaagtgct ctattgaagt tgtatctagt acgtagacag taaacatctg ttgaacttac accttcacga

V S F S G S S N F G E G S G P I W F D D 610 620 630 640 650 660 gtcagtttct ctggttcatc taattttgga gaaggctctg gaccaatctg gtttgatgat cagtcaaaga gaccaagtag attaaaacct cttccgagac ctggttagac caaactacta L I C N G N E S A L W N C K H Q G W G K

670 680 690 700 710 720 cttatatgca acggaaatga gtcagctctc tggaactgca aacatcaagg atggggaaag gaatatacgt tgcctttact cagtcgagag accttgacgt ttgtagttcc tacccctttc

HNCD HAE DAG VICS KGA DLS

730 740 750 760 770 780 cataactgtg atcatgctga ggatgctgga gtgatttgct caaagggagc agatctgagc gtattgacac tagtacgact cctacgacct cactaaacga gtttccctcg tctagactcg

LRLV DGV TEC SGRL EVR FQG

790 800 810 820 830 840 ctgagactgg tagatggagt cactgaatgt tcaggaagat tagaagtgag attccaagga gactctgacc atctacctca gtgacttaca agtccttcta atcttcactc taaggttcct

E W G T I C D D G W D S Y D A A V A C K

850 860 870 880 890 900 gaatggggga caatatgtga tgacggctgg gacagttacg atgctgctgt ggcatgcaag cttaccccct gttatacact actgccgacc ctgtcaatgc tacgacgaca ccgtacgttc

QLGC PTA VTA IGRV NAS KGF

910 920 930 940 950 960 caactgggat gtccaactgc cgtcacagcc attggtcgag ttaacgccag taagggattt gttgacccta caggttgacg gcagtgtcgg taaccagctc aattgcggtc attccctaaa

G H I W L D S V S C Q G H E P A V W Q C

970 980 990 1000 1010 1020 ggacacatct ggcttgacag cgtttcttgc cagggacatg aacctgctgt ctggcaatgt cctgtgtaga ccgaactgtc gcaaagaacg gtccctgtac ttggacgaca gaccgttaca KHHE WGK HYC NHNE DAG VTC

1030 1040 1050 1060 1070 1080 aaacaccatg aatggggaaa gcattattgc aatcacaatg aagatgctgg cgtgacatgt tttgtggtac ttaccccttt cgtaataacg ttagtgttac ttctacgacc gcactgtaca

SDGS DLE LRL RGGG SRC AGT

1090 1100 1110 1120 1130 1140 tctgatggat cagatctgga gctaagactt agaggtggag gcagccgctg tgctgggaca agactaccta gtctagacct cgattctgaa tctccacctc cgtcggcgac acgaccctgt

VEVE IQR LLG KVCD RGW GLK

1150 1160 1170 1180 1190 1200 gttgaggtgg agattcagag actgttaggg aaggtgtgtg acagaggctg gggactgaaa caactccacc tctaagtctc tgacaatccc ttccacacac tgtctccgac ccctgacttt

EADV VCR QLG CGSA LKT SYQ

1210 1220 1230 1240 1250 1260 gaagctgatg tggtttgcag gcagctggga tgtggatctg cactcaaaac atcttatcaa cttcgactac accaaacgtc cgtcgaccct acacctagac gtgagttttg tagaatagtt

VYSK IQA TNT WLFL SSC NGN

1270 1280 1290 1300 1310 1320 gtgtactcca aaatccaggc aacaaacaca tggctgtttc taagtagctg taacggaaat cacatgaggt tttaggtccg ttgtttgtgt accgacaaag attcatcgac attgccttta

E T S L W D C K N W Q W G G L T C D H Y

1330 1340 1350 1360 1370 1380 gaaacttctc tttgggactg caagaactgg caatggggtg gacttacctg tgatcactat ctttgaagag aaaccctgac gttcttgacc gttaccccac ctgaatggac actagtgata EEAK ITC SAH REPR LVG GDI

1390 1400 1410 1420 1430 1440 gaagaagcca aaattacctg ctcagcccac agggaaccca gactggttgg aggggacatt cttcttcggt tttaatggac gagtcgggtg tcccttgggt ctgaccaacc tcccctgtaa

PCSG RVE VKH GDTW GSI CDS

1450 1460 1470 1480 1490 1500 ccctgttctg gacgtgttga agtgaagcat ggtgacacgt ggggctccat ctgtgattcg gggacaagac ctgcacaact tcacttcgta ccactgtgca ccccgaggta gacactaagc

DFSL EAA SVL CREL QCG TVV

1510 1520 1530 1540 1550 1560 gacttctctc tggaagctgc cagcgttcta tgcagggaat tacagtgtgg cacagttgtc ctgaagagag accttcgacg gtcgcaagat acgtccctta atgtcacacc gtgtcaacag

SILG GAH FGE GNGQ IWA EEF

1570 1580 1590 1600 1610 1620 tctatcctgg ggggagctca ctttggagag ggaaatggac agatctgggc tgaagaattc agataggacc cccctcgagt gaaacctctc cctttacctg tctagacccg acttcttaag

QCEG HES HLS LCPV APR PEG

1630 1640 1650 1660 1670 1680 cagtgtgagg gacatgagtc ccatctttca ctctgcccag tagcaccccg cccagaagga gtcacactcc ctgtactcag ggtagaaagt gagacgggtc atcgtggggc gggtcttcct

TCSH SRD VGV VCSR YTE IRL

1690 1700 1710 1720 1730 1740 acttgtagcc acagcaggga tgttggagta gtctgctcaa gatacacaga aattcgcttg tgaacatcgg tgtcgtccct acaacctcat cagacgagtt ctatgtgtct ttaagcgaac VNGK TPC EGR VELK TLG AWG

1750 1760 1770 1780 1790 1800 gtgaatggca agaccccgtg tgagggcaga gtggagctca aaacgcttgg tgcctgggga cacttaccgt tctggggcac actcccgtct cacctcgagt tttgcgaacc acggacccct

S L C N S H W D I E D A H V L C Q Q L K

1810 1820 1830 1840 1850 1860 tccctctgta actctcactg ggacatagaa gatgcccatg ttctttgcca gcagcttaaa agggagacat tgagagtgac cctgtatctt ctacgggtac aagaaacggt cgtcgaattt

CGVA LST PGG ARFG KGN GQI

1870 1880 1890 1900 1910 1920 tgtggagttg ccctttctac cccaggagga gcacgttttg gaaaaggaaa tggtcagatc acacctcaac gggaaagatg gggtcctcct cgtgcaaaac cttttccttt accagtctag

WRHM FHC TGT EQHM GDC PVT

1930 1940 1950 1960 1970 1980 tggaggcata tgtttcactg cactgggact gagcagcaca tgggagattg tcctgtaact acctccgtat acaaagtgac gtgaccctga ctcgtcgtgt accctctaac aggacattga

ALGA SLC PSE QVAS VIC SGN

1990 2000 2010 2020 2030 2040 gctctaggtg cttcattatg tccttcagag caagtggcct ctgtaatctg ctcaggaaac cgagatccac gaagtaatac aggaagtctc gttcaccgga gacattagac gagtcctttg

QSQT LSS CNS SSLG PTR PTI

2050 2060 2070 2080 2090 2100 cagtcccaaa cactgtcctc gtgcaattca tcgtctttgg gcccaacaag gcctaccatt gtcagggttt gtgacaggag cacgttaagt agcagaaacc cgggttgttc cggatggtaa P E E S A VA C I E S G Q L R L V N G G

2110 2120 2130 2140 2150 2160 ccagaagaaa gtgctgtggc ctgcatagag agtggtcaac ttcgcctggt aaatggagga ggtcttcttt cacgacaccg gacgtatctc tcaccagttg aagcggacca tttacctcct

G R C A G R V E I Y H E G S W G T I C D

2170 2180 2190 2200 2210 2220 ggtcgctgtg ctgggagagt agagatctat catgagggct cctggggcac catctgtgat ccagcgacac gaccctctca tctctagata gtactcccga ggaccccgtg gtagacacta

DSWD LSD AHV VCRQ LGC GEA

2230 2240 2250 2260 2270 2280 gacagctggg acctgagtga tgcccacgtg gtttgcagac agctgggctg tggagaggcc ctgtcgaccc tggactcact acgggtgcac caaacgtctg tcgacccgac acctctccgg

I N A T G S A H F G E G T G P I W L D E

2290 2300 2310 2320 2330 2340 attaatgcca ctggttctgc tcattttggg gaaggaacag ggcccatctg gctggatgag taattacggt gaccaagacg agtaaaaccc cttccttgtc ccgggtagac cgacctactc

MKCN GKE SRI WQCH SHG WGQ

2350 2360 2370 2380 2390 2400 atgaaatgca atggaaaaga atcccgcatt tggcagtgcc attcacacgg ctgggggcag tactttacgt taccttttct tagggcgtaa accgtcacgg taagtgtgcc gacccccgtc

QNCR HKE DAG VICS EFM SLR

2410 2420 2430 2440 2450 2460 caaaattgca ggcacaagga ggatgcggga gttatctgct cagaattcat gtctctgaga gttttaacgt ccgtgttcct cctacgccct caatagacga gtcttaagta cagagactct LTSE ASR EAC AGRL EVF YNG

2470 2480 2490 2500 2510 2520 ctgaccagtg aagccagcag agaggcctgt gcagggcgtc tggaagtttt ttacaatgga gactggtcac ttcggtcgtc tctccggaca cgtcccgcag accttcaaaa aatgttacct

AWGT VGK SSM SETT VGV VCR

2530 2540 2550 2560 2570 2580 gcttggggca ctgttggcaa gagtagcatg tctgaaacca ctgtgggtgt ggtgtgcagg cgaaccccgt gacaaccgtt ctcatcgtac agactttggt gacacccaca ccacacgtcc

QLGC ADK GKI NPAS LDK AMS

2590 2600 2610 2620 2630 2640 cagctgggct gtgcagacaa agggaaaatc aaccctgcat ctttagacaa ggccatgtcc gtcgacccga cacgtctgtt tcccttttag ttgggacgta gaaatctgtt ccggtacagg

I P M W V D N V Q C P K G P D T L W Q C

2650 2660 2670 2680 2690 2700 attcccatgt gggtggacaa tgttcagtgt ccaaaaggac ctgacacgct gtggcagtgc taagggtaca cccacctgtt acaagtcaca ggttttcctg gactgtgcga caccgtcacg

PSSP WEK RLA SPSE ETW ITC

2710 2720 2730 2740 2750 2760 ccatcatctc catgggagaa gagactggcc agcccctcgg aggagacctg gatcacatgt ggtagtagag gtaccctctt ctctgaccgg tcggggagcc tcctctggac ctagtgtaca

DNKI RLQ EGP TSCS GRV EIW

2770 2780 2790 2800 2810 2820 gacaacaaga taagacttca ggaaggaccc acttcctgtt ctggacgtgt ggagatctgg ctgttgttct attctgaagt ccttcctggg tgaaggacaa gacctgcaca cctctagacc HGGS WGT VCD DSWD LDD AQV

2830 2840 2850 2860 2870 2880 catggaggtt cctgggggac agtgtgtgat gactcttggg acttggacga tgctcaggtg gtacctccaa ggaccccctg tcacacacta ctgagaaccc tgaacctgct acgagtccac

VCQQ LGC GPA LKAF KEA EFG

2890 2900 2910 2920 2930 2940 gtgtgtcaac aacttggctg tggtccagct ttgaaagcat tcaaagaagc agagtttggt cacacagttg ttgaaccgac accaggtcga aactttcgta agtttcttcg tctcaaacca

QGTG PIW LNE VKSR GSP GLQ

2950 2960 2970 2980 2990 3000 caggggactg gaccgatatg gctcaatgaa gtgaagtcta gaGGATCCCC CGGGCTGCAG gtcccctgac ctggctatac cgagttactt cacttcagat ctCCTAGGGG GCCCGACGTC

E P K S C D K T H T C P P C P A P E L L 3010 3020 3030 3040 3050 3060 GAGCCCAAAT CTTGTGACAA AACTCACACA TGCCCACCGT GCCCAGCACC TGAACTCCTG

CTCGGGTTTA GAACACTGTT TTGAGTGTGT ACGGGTGGCA CGGGTCGTGG ACTTGAGGAC

G G P S V F L F P P K P K D T L M I S R 3070 3080 3090 3100 3110 3120

GGGGGACCGT CAGTCTTCCT CTTCCCCCCA AAACCCAAGG

ACACCCTCAT GATCTCCCGG

CCCCCTGGCA GTCAGAAGGA GAAGGGGGGT TTTGGGTTCC TGTGGGAGTA CTAGAGGGCC TPEV TCV VVD VSHE DPE VKF

3130 3140 3150 3160 3170 3180 ACCCCTGAGG TCACATGCGT GGTGGTGGAC GTGAGCCACG AAGACCCTGA GGTCAAGTTC

TGGGGACTCC AGTGTACGCA CCACCACCTG CACTCGGTGC TTCTGGGACT CCAGTTCAAG

N WY V D G V E V H N A K T K P R E E Q 3190 3200 3210 3220 3230 3240

AACTGGTACG TGGACGGCGT GGAGGTGCAT AATGCCAAGA

CAAAGCCGCG GGAGGAGCAG

TTGACCATGC ACCTGCCGCA CCTCCACGTA TTACGGTTCT

GTTTCGGCGC CCTCCTCGTC

Y N S T Y R V V S V L T V L H Q D W L N 3250 3260 3270 3280 3290 3300

TACAACAGCA CGTACCGGGT GGTCAGCGTC CTCACCGTCC

TGCACCAGGA CTGGCTGAAT ATGTTGTCGT GCATGGCCCA CCAGTCGCAG GAGTGGCAGG

ACGTGGTCCT GACCGACTTA

G K E Y K C K V S N KA L P A P I E K T 3310 3320 3330 3340 3350 3360 GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT CGAGAAAACC

CCGTTCCTCA TGTTCACGTT CCAGAGGTTG TTTCGGGAGG GTCGGGGGTA GCTCTTTTGG

I S KA K G Q P R E P Q V Y T L P P S R 3370 3380 3390 3400 3410 3420 ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA CCACAGGTGT ACACCCTGCC CCCATCCCGG

TAGAGGTTTC GGTTTCCCGT CGGGGCTCTT GGTGTCCACA TGTGGGACGG GGGTAGGGCC

D E L T K N Q V S L T C L V K G F Y P S

3430 3440 3450 3460 3470 3480 GATGAGCTGA CCAAGAACCA GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT CTATCCCAGC

CTACTCGACT GGTTCTTGGT CCAGTCGGAC TGGACGGACC AGTTTCCGAA GATAGGGTCG

D I AV E W E S N G Q P E N NY K T T P 3490 3500 3510 3520 3530 3540

GACATCGCCG TGGAGTGGGA GAGCAATGGG CAGCCGGAGA

ACAACTACAA GACCACGCCT

CTGTAGCGGC ACCTCACCCT CTCGTTACCC GTCGGCCTCT

TGTTGATGTT CTGGTGCGGA

P V L D S D G S F F L Y S K L T V D K S 3550 3560 3570 3580 3590 3600

CCCGTGCTGG ACTCCGACGG CTCCTTCTTC CTCTACAGCA

AGCTCACCGT GGACAAGAGC GGGCACGACC TGAGGCTGCC GAGGAAGAAG GAGATGTCGT

TGGAGTGGGA CCTGTTCTCG

R W Q Q G NV F S C S V M H E A L H N H 3610 3620 3630 3640 3650 3660 AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT GCACAACCAC

TCCACCGTCG TCCCCTTGCA GAAGAGTACG AGGCACTACG TACTCCGAGA CGTGTTGGTG

Y T Q K S L S L S P G K 3670 3680 3690 3696 TACACGCAGA AGAGCCTCTC CCTGTCTCCG GGTAAA ATGTGCGTCT TCTCGGAGAG GGACAGAGGC CCATTT

SEQ 12 mCD163-mIgGl

MGGH RMV LLG GAGS PGC KRF 10 20 30 40 50 60 atgggtggac acagaatggt tcttcttgga ggtgctggat ctcctggttg taaaaggttt tacccacctg tgtcttacca agaagaacct ccacgaccta gaggaccaac attttccaaa

V H L G F F V VAV S S L L S A S AV T 70 80 90 100 110 120 gtccatctag gtttctttgt tgtggctgtg agctcacttc tcagtgcctc tgctgtcact caggtagatc caaagaaaca acaccgacac tcgagtgaag agtcacggag acgacagtga

NA P G E M K K E L R L A G G E N N C S 130 140 150 160 170 180 aacgctcctg gagaaatgaa gaaggaactg agactggcgg gtggtgaaaa caactgtagt ttgcgaggac ctctttactt cttccttgac tctgaccgcc caccactttt gttgacatca

G R V E L K I H D K W G T V C S N G W S 190 200 210 220 230 240 gggagagtgg aacttaagat ccatgacaag tggggcacag tgtgcagtaa cggctggagc ccctctcacc ttgaattcta ggtactgttc accccgtgtc acacgtcatt gccgacctcg

M N E V S V V C Q Q L G C P T S I KA L 250 260 270 280 290 300 atgaatgaag tgtccgtggt ttgccagcag ctgggatgcc caacttctat taaagccctt tacttacttc acaggcacca aacggtcgtc gaccctacgg gttgaagata atttcgggaa

G WA N S S A G S G Y I W M D KV S C T 310 320 330 340 350 360 ggatgggcta actccagcgc cggctctgga tatatctgga tggacaaagt ttcttgtaca cctacccgat tgaggtcgcg gccgagacct atatagacct acctgtttca aagaacatgt

G N E S A L W D C K H D G W G K H N C T 370 380 390 400 410 420 gggaatgagt cagctctttg ggactgcaaa catgatgggt ggggaaagca taactgtacc cccttactca gtcgagaaac cctgacgttt gtactaccca cccctttcgt attgacatgg

H E K D A G V T C S D G S N L E M R L V 430 440 450 460 470 480 catgaaaaag atgctggagt gacctgctca gatggatcta atttggagat gagactggtg gtactttttc tacgacctca ctggacgagt ctacctagat taaacctcta ctctgaccac

N S A G H R C L G R V E I K F Q G K W G 490 500 510 520 530 540 aacagtgcgg gccaccgatg cttaggaaga gtagaaataa agttccaggg aaagtggggg ttgtcacgcc cggtggctac gaatccttct catctttatt tcaaggtccc tttcaccccc

T V C D D N F S K D H A S V I C K Q L G 550 560 570 580 590 600 acggtgtgtg acgacaactt cagcaaagat cacgcttctg tgatttgtaa acagcttgga tgccacacac tgctgttgaa gtcgtttcta gtgcgaagac actaaacatt tgtcgaacct

C G S A I S F S G S A K L G A G S G P I 610 620 630 640 650 660 tgtggaagtg ccattagttt ctctggctca gctaaattgg gagctggttc tggaccaatc acaccttcac ggtaatcaaa gagaccgagt cgatttaacc ctcgaccaag acctggttag

W L D D L A C N G N E S A L W D C K H R 670 680 690 700 710 720 tggctcgatg acctggcatg caatggaaat gagtcagctc tctgggactg caaacaccgg accgagctac tggaccgtac gttaccttta ctcagtcgag agaccctgac gtttgtggcc

G W G K H N C D HA E D V G V I C L E G 730 740 750 760 770 780 ggatggggca agcataactg tgaccatgct gaggatgtcg gtgtgatttg cttagaggga cctaccccgt tcgtattgac actggtacga ctcctacagc cacactaaac gaatctccct

A D L S L R L V D G V S R C S G R L E V 790 800 810 820 830 840 gcagatctga gcctgagact agtggatgga gtgtccagat gttcaggaag attggaagtg cgtctagact cggactctga tcacctacct cacaggtcta caagtccttc taaccttcac

R F Q G E W G T V C D D N W D L R D A S 850 860 870 880 890 900 agattccaag gagaatgggg gaccgtgtgt gatgataact gggatctccg ggatgcttct tctaaggttc ctcttacccc ctggcacaca ctactattga ccctagaggc cctacgaaga

VV C K Q L G C P T A I S A I G R V NA 910 920 930 940 950 960 gtggtgtgca agcaactggg atgtccaact gccatcagtg ccattggtcg agttaatgcc caccacacgt tcgttgaccc tacaggttga cggtagtcac ggtaaccagc tcaattacgg

S E G S G Q I W L D N I S C E G H E A T 970 980 990 1000 1010 1020 agtgagggat ctggacagat ttggcttgac aacatttcat gcgaaggaca tgaggcaact tcactcccta gacctgtcta aaccgaactg ttgtaaagta cgcttcctgt actccgttga

L W E C K H Q E W G K HY C H H R E D A 1030 1040 1050 1060 1070 1080 ctttgggagt gtaaacacca agagtgggga aagcattact gtcatcatag agaagacgct gaaaccctca catttgtggt tctcacccct ttcgtaatga cagtagtatc tcttctgcga

G V T C S D G A D L E L R L V G G G S R 1090 1100 1110 1120 1130 1140 ggtgtgacat gttctgatgg agcagatctg gaacttagac ttgtaggtgg aggcagtcgc ccacactgta caagactacc tcgtctagac cttgaatctg aacatccacc tccgtcagcg

C A G I V E V E I Q K L T G K M C S R G 1150 1160 1170 1180 1190 1200 tgtgctggca ttgtggaggt ggagattcag aagctgactg ggaagatgtg tagccgaggc acacgaccgt aacacctcca cctctaagtc ttcgactgac ccttctacac atcggctccg

W T L A D A D VV C R Q L G C G S A L Q 1210 1220 1230 1240 1250 1260 tggacactgg cagatgcgga tgtggtttgc agacagcttg gatgtggatc tgcgcttcaa acctgtgacc gtctacgcct acaccaaacg tctgtcgaac ctacacctag acgcgaagtt

T Q A K I Y S K T G A T N T W L F P G S 1270 1280 1290 1300 1310 1320 acccaggcta agatctactc taaaactggg gcaacaaata cgtggctctt tcctggatct tgggtccgat tctagatgag attttgaccc cgttgtttat gcaccgagaa aggacctaga

C N G N E T T F W Q C K N W Q W G G L S 1330 1340 1350 1360 1370 1380 tgtaatggaa atgaaactac tttttggcaa tgcaaaaact ggcagtgggg cggcctttcc acattacctt tactttgatg aaaaaccgtt acgtttttga ccgtcacccc gccggaaagg

C D N F E E A KV T C S G H R E P R L V 1390 1400 1410 1420 1430 1440 tgtgataatt tcgaagaagc caaagttacc tgctcaggcc acagggaacc cagactggtt acactattaa agcttcttcg gtttcaatgg acgagtccgg tgtcccttgg gtctgaccaa

G G E I P C S G R V E V K H G D V W G S 1450 1460 1470 1480 1490 1500 ggaggagaaa tcccatgctc tggtcgtgtg gaagtgaaac acggagacgt gtggggctcc cctcctcttt agggtacgag accagcacac cttcactttg tgcctctgca caccccgagg

V C D F D L S L E A A S V V C R E L Q C 1510 1520 1530 1540 1550 1560 gtctgtgatt ttgacttgtc tctggaagct gccagtgtgg tgtgcaggga attacaatgt cagacactaa aactgaacag agaccttcga cggtcacacc acacgtccct taatgttaca

G T V V S I L G G A H F G E G S G Q I W 1570 1580 1590 1600 1610 1620 ggaacagtcg tctctatcct agggggagca cattttggag aaggaagtgg acagatctgg ccttgtcagc agagatagga tccccctcgt gtaaaacctc ttccttcacc tgtctagacc

G E E F Q C S G D E S H L S L C S V A P 1630 1640 1650 1660 1670 1680 ggtgaagaat tCCAGTGTAG TGGGGATGAG TCCCATCTTT CACTATGCTG

AGTGGCGCCC ccacttctta aGGTCACATC ACCCCTACTC AGGGTAGAAA GTGATACGAG

TCACCGCGGG

PLDR TCT HSR DVSV VCS RYI

1690 1700 1710 1720 1730 1740 CCGCTAGACA GAACTTGTAC CCACAGCAGG GATGTCAGCG TAGTCTGCTC ACGATACATA GGCGATCTGT CTTGAACATG GGTGTCGTCC CTACAGTCGC ATCAGACGAG TGCTATGTAT

D I R L A G G E S S C E G R V E L K T L

1750 1760 1770 1780 1790 1800 GATATTCGTC TGGCAGGCGG CGAGTCCTCC TGTGAGGGAA GAGTGGAGCT CAAGACACTC

CTATAAGCAG ACCGTCCGCC GCTCAGGAGG ACACTCCCTT CTCACCTCGA GTTCTGTGAG

G A W G P L C S S H W D M E D A H V L C

1810 1820 1830 1840 1850 1860 GGAGCCTGGG GTCCCCTCTG CAGTTCTCAT TGGGACATGG AAGATGCTCA TGTCTTATGT CCTCGGACCC CAGGGGAGAC GTCAAGAGTA ACCCTGTACC TTCTACGAGT ACAGAATACA

Q Q L K C G V A Q S I P E G A H F G K G 1870 1880 1890 1900 1910 1920 CAGCAGCTGA AGTGTGGGGT TGCCCAATCT ATTCCAGAAG GAGCACATTT TGGGAAAGGA

GTCGTCGACT TCACACCCCA ACGGGTTAGA TAAGGTCTTC CTCGTGTAAA ACCCTTTCCT

A G Q V W S H M F H C T G T E E H I G D 1930 1940 1950 1960 1970 1980

GCTGGTCAGG TCTGGAGTCA CATGTTCCAC TGCACTGGAA

CTGAGGAACA TATAGGAGAT

CGACCAGTCC AGACCTCAGT GTACAAGGTG ACGTGACCTT GACTCCTTGT ATATCCTCTA C L M T A L G A P T C S E G Q V A S V I

1990 2000 2010 2020 2030 2040 TGCCTCATGA CTGCTCTGGG TGCGCCGACG TGTTCCGAAG GACAGGTGGC CTCTGTCATC

ACGGAGTACT GACGAGACCC ACGCGGCTGC ACAAGGCTTC CTGTCCACCG GAGACAGTAG

C S G N Q S Q T L L P C S S L S P V Q T 2050 2060 2070 2080 2090 2100

TGCTCAGGAA ACCAATCCCA GACACTATTG CCATGTAGTT

CATTGTCTCC AGTCCAAACA

ACGAGTCCTT TGGTTAGGGT CTGTGATAAC GGTACATCAA

GTAACAGAGG TCAGGTTTGT

T S S T I P K E S E V P C I A S G Q L R 2110 2120 2130 2140 2150 2160

ACAAGCTCTA CAATTCCAAA GGAGAGTGAA GTTCCCTGCA

TAGCAAGTGG CCAGCTTCGC TGTTCGAGAT GTTAAGGTTT CCTCTCACTT CAAGGGACGT

ATCGTTCACC GGTCGAAGCG

L V G G G G R C A G R V E V Y H E G S W 2170 2180 2190 2200 2210 2220 TTGGTAGGTG GAGGTGGTCG CTGCGCTGGA AGAGTGGAGG TCTACCACGA GGGCTCTTGG

AACCATCCAC CTCCACCAGC GACGCGACCT TCTCACCTCC AGATGGTGCT CCCGAGAACC

G T V C D D N W D M T D A N VV C K Q L 2230 2240 2250 2260 2270 2280 GGCACCGTCT GTGATGACAA TTGGGATATG ACTGATGCCA ATGTGGTGTG CAAGCAGCTG

CCGTGGCAGA CACTACTGTT AACCCTATAC TGACTACGGT TACACCACAC GTTCGTCGAC

D C G V A I N A T G S A Y F G E G A G A

2290 2300 2310 2320 2330 2340 GACTGTGGCG TGGCAATTAA CGCCACTGGC TCTGCTTACT TCGGGGAAGG AGCAGGAGCT

CTGACACCGC ACCGTTAATT GCGGTGACCG AGACGAATGA AGCCCCTTCC TCGTCCTCGA

I W L D E V I C T G K E S H I W Q C H S 2350 2360 2370 2380 2390 2400

ATCTGGCTAG ACGAAGTCAT CTGCACTGGG AAAGAGTCTC

ATATTTGGCA GTGCCATTCA

TAGACCGATC TGCTTCAGTA GACGTGACCC TTTCTCAGAG

TATAAACCGT CACGGTAAGT

H G W G R H N C R H K E D A G V I C S E 2410 2420 2430 2440 2450 2460

CATGGCTGGG GACGCCATAA CTGCAGGCAC AAAGAAGATG

CAGGTGTTAT CTGCTCCGAG GTACCGACCC CTGCGGTATT GACGTCCGTG TTTCTTCTAC

GTCCACAATA GACGAGGCTC

F M S L R L T N E A H K E N C T G R L E 2470 2480 2490 2500 2510 2520 TTCATGTCTC TGAGGCTGAC CAACGAAGCC CACAAAGAAA ACTGCACAGG TCGCCTTGAA

AAGTACAGAG ACTCCGACTG GTTGCTTCGG GTGTTTCTTT TGACGTGTCC AGCGGAACTT

V F Y N G T W G S I G S S N M S P T T V

2530 2540 2550 2560 2570 2580 GTGTTTTACA ATGGTACATG GGGCAGTATT GGCAGTAGCA ATATGTCTCC AACCACTGTG CACAAAATGT TACCATGTAC CCCGTCATAA CCGTCATCGT TATACAGAGG TTGGTGACAC

G V V C R Q L G C A D N G T V K P I P S 2590 2600 2610 2620 2630 2640 GGGGTGGTGT GCCGTCAGCT GGGCTGTGCA GACAACGGGA CTGTGAAACC CATACCTTCA

CCCCACCACA CGGCAGTCGA CCCGACACGT CTGTTGCCCT GACACTTTGG GTATGGAAGT

D K T P S R P M W V D R V Q C P K G V D 2650 2660 2670 2680 2690 2700

GACAAGACAC CATCCAGGCC CATGTGGGTA GATCGTGTGC

AGTGTCCAAA AGGAGTTGAC

CTGTTCTGTG GTAGGTCCGG GTACACCCAT CTAGCACACG TCACAGGTTT TCCTCAACTG

T L W Q C P S S P W K Q R Q A S P S S Q

2710 2720 2730 2740 2750 2760 ACTTTGTGGC AGTGCCCCTC GTCACCTTGG AAACAGAGAC AGGCCAGCCC CTCCTCCCAG TGAAACACCG TCACGGGGAG CAGTGGAACC TTTGTCTCTG TCCGGTCGGG GAGGAGGGTC

E S W I I C D N K I R L Q E G H T D C S

2770 2780 2790 2800 2810 2820 GAGTCCTGGA TCATCTGTGA CAACAAAATA AGACTCCAGG AAGGGCATAC AGACTGTTCT

CTCAGGACCT AGTAGACACT GTTGTTTTAT TCTGAGGTCC TTCCCGTATG TCTGACAAGA

G R V E I W H K G S W G T V C D D S W D

2830 2840 2850 2860 2870 2880 GGACGTGTGG AGATCTGGCA CAAAGGTTCC TGGGGAACAG TGTGTGATGA CTCCTGGGAT CCTGCACACC TCTAGACCGT GTTTCCAAGG ACCCCTTGTC ACACACTACT GAGGACCCTA

L N D A K V V C K Q L G C G Q A V KA L 2890 2900 2910 2920 2930 2940 CTTAATGATG CTAAGGTTGT ATGTAAGCAG TTGGGCTGTG GCCAAGCTGT GAAGGCACTA

GAATTACTAC GATTCCAACA TACATTCGTC AACCCGACAC CGGTTCGACA CTTCCGTGAT

K E AA F G P G T G P I W L N E I K G S 2950 2960 2970 2980 2990 3000

AAAGAAGCAG CATTTGGTCC AGGAACTGGG CCCATATGGC

TCAATGAAAT TAAGGGATCC

TTTCTTCGTC GTAAACCAGG TCCTTGACCC GGGTATACCG AGTTACTTTA ATTCCCTAGG PGLQ VPR DCG CKPC ICT VPE

3010 3020 3030 3040 3050 3060 CCCGGGCTGC AGGTGCCCAG GGATTGTGGT TGTAAGCCTT GCATATGTAC AGTCCCAGAA

GGGCCCGACG TCCACGGGTC CCTAACACCA ACATTCGGAA CGTATACATG TCAGGGTCTT

V S S V F I F P P K P K D V L T I T L T 3070 3080 3090 3100 3110 3120

GTATCATCTG TCTTCATCTT CCCCCCAAAG CCCAAGGATG

TGCTCACCAT TACTCTGACT

CATAGTAGAC AGAAGTAGAA GGGGGGTTTC GGGTTCCTAC

ACGAGTGGTA ATGAGACTGA

P KV T C V V V D I S K D D P E V Q F S 3130 3140 3150 3160 3170 3180

CCTAAGGTCA CGTGTGTTGT GGTAGACATC AGCAAGGATG

ATCCCGAGGT CCAGTTCAGC GGATTCCAGT GCACACAACA CCATCTGTAG TCGTTCCTAC

TAGGGCTCCA GGTCAAGTCG

W FV D D V E V H T A Q T Q P R E E Q F 3190 3200 3210 3220 3230 3240 TGGTTTGTAG ATGATGTGGA GGTGCACACA GCTCAGACGC AACCCCGGGA GGAGCAGTTC

ACCAAACATC TACTACACCT CCACGTGTGT CGAGTCTGCG TTGGGGCCCT CCTCGTCAAG

N S T F R S V S E L P I M H Q D W L N G 3250 3260 3270 3280 3290 3300 AACAGCACTT TCCGCTCAGT CAGTGAACTT CCCATCATGC ACCAGGACTG GCTCAATGGC

TTGTCGTGAA AGGCGAGTCA GTCACTTGAA GGGTAGTACG TGGTCCTGAC CGAGTTACCG

K E F K C R V N S A A F P A P I E K T I

3310 3320 3330 3340 3350 3360 AAGGAGTTCA AATGCAGGGT CAACAGTGCA GCTTTCCCTG CCCCCATCGA GAAAACCATC

TTCCTCAAGT TTACGTCCCA GTTGTCACGT CGAAAGGGAC GGGGGTAGCT CTTTTGGTAG

S K T K G R P KA P Q VY T I P P P K E 3370 3380 3390 3400 3410 3420

TCCAAAACCA AAGGCAGACC GAAGGCTCCA CAGGTGTACA

CCATTCCACC TCCCAAGGAG

AGGTTTTGGT TTCCGTCTGG CTTCCGAGGT GTCCACATGT

GGTAAGGTGG AGGGTTCCTC

Q M A K D KV S L T C M I T D F F P E D 3430 3440 3450 3460 3470 3480

CAGATGGCCA AGGATAAAGT CAGTCTGACC TGCATGATAA

CAGACTTCTT CCCTGAAGAC GTCTACCGGT TCCTATTTCA GTCAGACTGG ACGTACTATT

GTCTGAAGAA GGGACTTCTG

I T V E W Q W N G Q P A E N Y K N T Q P 3490 3500 3510 3520 3530 3540 ATTACTGTGG AGTGGCAGTG GAATGGGCAG CCAGCGGAGA ACTACAAGAA CACTCAGCCC

TAATGACACC TCACCGTCAC CTTACCCGTC GGTCGCCTCT TGATGTTCTT GTGAGTCGGG

I M D T D G S Y F V Y S KL N V Q K S N

3550 3560 3570 3580 3590 3600 ATCATGGACA CAGATGGCTC TTACTTCGTC TACAGCAAGC TCAATGTGCA GAAGAGCAAC TAGTACCTGT GTCTACCGAG AATGAAGCAG ATGTCGTTCG AGTTACACGT CTTCTCGTTG

W E A G N T F T C S V L H E G L H N H H 3610 3620 3630 3640 3650 3660 TGGGAGGCAG GAAATACTTT CACCTGCTCT GTGTTACATG AGGGCCTGCA CAACCACCAT

ACCCTCCGTC CTTTATGAAA GTGGACGAGA CACAATGTAC TCCCGGACGT GTTGGTGGTA

T E K S L S H S P G K 3670 3680 36903693 ACTGAGAAGA GCCTCTCCCA CTCTCCTGGT AAA TGACTCTTCT CGGAGAGGGT GAGAGGACCA TTT

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Claims

1) A method for identifying a molecule with immune modulatory activity capable of interacting with CD163 comprising providing CD163 or a functional part, derivative and/or analogue thereof and under suitable conditions detecting a molecule capable of interacting with said CD 163 and determining whether said molecule is capable of modulating an immune response.

2) A method according to claim 1 wherein said CD163 comprises a sCD163.

3) A method according to claim 1 or claim 2 wherein said CD 163-ligand molecule with immune modulatory activity is cell bound and/or soluble. 4) A method according to anyone of claims 1-3, wherein said CD163-ligand molecule comprises a proteinaceous molecule, functional derivative, functional fragment and/or analogue thereof. 5) An isolated and/or recombinant and/or synthetic CD 163-ligand molecule obtainable by a method according to anyone of claims 1-4. 6) A CD163-ligand molecule according to claim 5 which comprises a histone or functional fragment thereof.

7) A CD 163-ligand molecule according to claim 5 or claim 6 coupled to a moiety.

8) A CD 163-ligand molecule according to claim 7, wherein said moiety comprises a constant region of an immunoglobulin.

9) Use of a CD163-ligand molecule according to anyone of claims 5-8 to modulate an immune response.

10) Use according to claim 9 wherein said modulation comprises suppression of an immune response. 11) An antagonist of CD 163/CD 163-ligand signalling. 12) An antagonist according to claim 11 which is an antibody or functional part, derivative and/or analogue thereof.

13) An antagonist according to claim 11 or claim 12 coupled to a moiety.

14) An antagonist I according to claim 11-13, capable of specifically binding a CD 163-ligand molecule.

15) An antagonist I according to claim 14, wherein said antagonist I comprises sCD163.

16) An antagonist II according to any one of claims 11-13, capable of specifically binding a CD 163 molecule. 17) An agonist of CD 163/CD 163-ligand signalling.

18) An agonist according to claim 17 which is an antibody or functional part, derivative and/or analogue thereof.

19) An agonist according to claim 17 or claim 18 coupled to a moiety.

20) An agonist I according to any one of claims 17-19, capable of specifically binding a CD 163-ligand molecule.

21) An agonist II according to any one of claims 17-19, capable of specifically binding a CD163 molecule.

22) An agonist II according to claim 21, wherein said agonist comprises a soluble CD 163 ligand. 23) An agonist II according to claim 22, wherein said soluble CD163 ligand is derived from a histone.

24) Use of an agonist according to any of claims 17-23, for at least in part suppressing an immune response.

25) Use an antagonist according to any one of claims 11-16 for augmenting an immune response.

26) An isolated and/or recombinant and/or synthetic CD 163, a functional part, derivative and/or analogue thereof.

27) An isolated CD 163 according to claim 26, wherein said CD 163 comprises a sCD163. 28) An isolated CD 163 according to claim 26 or claim 27 coupled to a moiety. 29) An isolated CD163 according to claim 28 wherein said moiety comprises a constant region of an immunoglobulin.

30) An immunoglobulin capable of specifically binding a CD 163-ligand.

31) An immunoglobulin according to claim 30, wherein said CD163-ligand comprises a histone.

32) A method to detect the presence of a CD163-ligand molecule according to any one of claims 5-8 in a sample comprising; contacting the sample with a binding molecule for said molecule to form a complex; further comprising detecting said complex in the sample. 33) A method according to claim 32, wherein said binding molecule comprises an antagonist I according to claim 14 or 15, an agonist I according to claim 20. 34) A method according to claim 32 or claim 33, wherein said binding molecule comprises membrane bound CD 163 and/or a sCD163. 35) A method to determine the binding activity of a CD163-ligand molecule according to anyone of claims 5-8 in a sample comprising detecting the presence of a molecule using a method according to anyone of claims 32-34, further determining the levels of binding molecule-molecule in said sample. 36) A nucleic acid or a functional part, functional derivative and/or analogue thereof encoding a CD 163-ligand molecule according to anyone of claims 5- 8.

37) A nucleic acid or a functional part, functional derivative and/or analogue thereof encoding an agonist or an antagonist according to any one of claims 11-23.

38) A nucleic acid or a functional part, functional derivative and/or analogue thereof encoding an isolated CD 163 according to anyone of claims 26-29.

39) A nucleic acid or a functional part, derivative and/or analogue thereof encoding an immunoglobulin according to claim 30 or claim 31. 40) A vector comprising a nucleic acid according to anyone of claims 36-39. 41) A cell comprising a vector according to claim 40.

42) A gene delivery vehicle comprising a vector according to claim 41.

43) A method for the production of a molecule according to anyone of claims 5- 8, or an agonist or antagonist according to any one of claims 11-23 in an organism comprising inserting into the genome of said organism one or more copies of a nucleic acid according to anyone of claims 36-39.

44) Use of an isolated CD 163 according to anyone of claims 26-29, an immunoglobulin according to claim 30 or claim 31 or an CD 163-ligand according to any one of claims 5-8, for modulating an immune response. 45) Use according to claim 44, wherein said immune response comprises an antigen specific immune response.

46) A pharmaceutical composition comprising a molecule according to anyone of claims 5-8, an antagonist or agonist according to any one of claims 11- 23, an isolated CD163 according to anyone of claims 26-29, a gene delivery vehicle according to claim 42, a cell according to claim 41 and/or an immunoglobulin according to claim 30 or claim 31.

47) Use of a pharmaceutical composition according to claim 46 for augmenting or suppressing an immune response.

48) Use of a composition comprising a molecule according to anyone of claims 5-8, an antagonist or agonist according to any one of claims 11-23, an isolated CD 163 according to anyone of claims 26-29, a gene delivery vehicle according to claim 42, a cell according to claim 41 and/or an immunoglobulin according to claim 30 or claim 31 for the preparation of a pharmaceutical composition for the treatment and/or prophylactic treatment of a disease, treatable by modulating an immune response.

49) Use according to claim 48, wherein said disease comprises an autoimmune disease, allergy, asthma, inflammatory disease, cancer, Alzheimer, infectious diseases, host versus graft related diseases, cardiovascular disease and/or neurological diseases and/or a disease associated with elevated serum sCD163 levels. 50) A method of treatment of an immune related disease comprising administrating a pharmaceutical composition according to claim 47 comprising a carrier to a suitable recipient.

51) A method according to claim 50, further comprising providing a further treatment for said disease.