WO2012123570A1 - Control of phagocytosis - Google Patents

Abstract

The present invention relates to a method to treat uncontrolled phagocytosis, especially diseases caused by uncontrolled phagocytosis such as Hemophagocytic lymphohistiocytosis (HLH), or for inhibiting unwanted phagocytosis to boost immunogenic properties of specific cancer therapies. The invention relates further to a sialoadhesin binding antibody for use as a medicament to treat uncontrolled phagocytosis, and to a pharmaceutical composition comprising a sialoadhesin binding antibody.

Description

CONTROL OF PHAGOCYTOSIS

Field of the invention

The present invention relates to a method to treat uncontrolled phagocytosis, especially diseases caused by uncontrolled phagocytosis such as Hemophagocytic lymphohistiocytosis (HLH) or to prevent unwanted phagocytosis by immunosuppressive macrophages in cancer immunotherapy. The invention relates further to a sialoadhesin binding antibody for use as a medicament to treat uncontrolled phagocytosis, and to a pharmaceutical composition comprising a sialoadhesin binding antibody.

Background of the invention

The process of phagocytosis plays an important role in the clearance of microbial pathogens and apoptotic cells. Macrophages are a major component of the mononuclear phagocyte system that consists of closely related cells of bone marrow origin, including blood monocytes and tissue macrophages (Gordon & Taylor, 2005). Phagocytosis by macrophages is an essential step in innate immunity for protection against foreign particles, pathogens, effete and dead cells by clearing them from their environment (Jutras & Desjardins, 2005). Moreover, phagocytosis is at the crossroads with the adaptive immune response. Upon encounter of infected cells, extracellular pathogens, antigens or particles, macrophages efficiently internalize them into phagosomes, where proteolytic processing of antigens produces antigenic peptides, that are subsequently presented by class II major histocompatibility complex (MHC-II) molecules to MHC-ll-restricted CD4+ T cells (Ramachandra et al., 2009). On the other hand, endogenous antigens, e.g. in virus-infected macrophages, are processed and subsequently presented by class I major histocompatibility complex (MHC-I) molecules to MHC-l-restricted CD8+ T cells. A third possibility is referred to as MHC-I cross-processing, to distinguish this mechanism from conventional MHC-I processing of cytosolic antigens. In this case exogenous antigens, for example antigens derived from extracellular, phagosomal or intravacuolar microbes, are presented by MHC-I molecules to MHC-l-restricted CD8+ T cells. Uncontrolled phagocytosis can lead to diseases, such as but not limited to Hemophagocytic lymphohistiocytosis (HLH). Moreover, phagocytosis by tumor-induced immunosuppressive macrophages can counteract experimental treatments such as cancer vaccination, especially in those cases where vaccination is performed with apoptotic bodies and/or apoptopic cell associated antigens (Miyake et al.,2007).

Although it is known that macrophages are responsible for phagocytosis, the key factors controlling phagocytosis are unknown. Macrophages are functionally heterogenous, which is reflected by their phenotypical diversity. Depending on their tissue site and activation status, a range of macrophages, from resting resident to fully activated inflammatory macrophages, can be found (Varin and Gordon, 2009), each expressing various receptors, enabling them to exercise their function (Taylor et al., 2005).

Recently it was demonstrated that Siglec-11 -expressing microglia have an impaired capacity to phagocytose apoptotic neuronal material compared to microglia that do not express this receptor (Wang & Neuman, 2010). It was suggested that Siglec-1 1 signalling via its immunoreceptor tyrosine-based inhibitory motifs (ITIMs) might antagonize the phagocytosis- associated immunoreceptor tyrosine-based activating motifs (ITAMs) Syk signalling pathways (Ziegenfuss et al, 2008). Besides Siglec-1 1 , Siglec-5 is also implied in impaired phagocytosis of apoptitic bodies (Rapoport et al., 2005). It was observed that cross-linking Siglec-5 lead to a decreased phagocytosis capacity, in this case of apoptotic bodies.

Sialoadhesin (Sn, Siglec-1 , CD169) is the prototypic member of the family of sialic acid binding immunoglobulin-like lectins (siglecs) (Crocker & Varki, 2001), and therefore, in view of the role of Siglec-5 and Siglec 1 1 , it may be hypothesized that Sn is a candidate regulator of phagocytosis. However, all present evidence is teaching away from the view that Sn plays an essential role in phagocytosis. Indeed, Sn was originally described as a non-phagocytic receptor in cell-cell interactions (Crocker et al., 1994; van den Berg et al., 1992; Crocker et al., 1995). Contrary to Siglec 1 1 , Sn does not contain ITIMs or ITAMs, motives that are considered essential in the anti-phagocytic function of Siglec-1 1. Discordantly and in contrast to most other siglecs, Sn is devoid of tyrosine-based motifs that are implicated in signal transduction and endocytosis, nor associates with the DAP-12 adaptor implicated in both positive and negative immunoregulation and endocytosis (Crocker & Redelinghuys, 2008; O'Reilly & Paulson, 2009). Currently, there is little evidence that Sn mediates signalling functions via its transmembrane tail or via its short cytoplasmic region, which lacks obvious signalling motifs and is poorly conserved between mammalian species, which suggests a primary role in cell- cell and cell-matrix interactions relating to homeostasis and immunity (Crocker & Redelinghuys, 2008; Crocker et al., 2007; Munday et al., 1999).

On the other hand, Sn was eventually shown to have endocytic (vs phagocytic) activity and mediate uptake of sialylated bacterial and viral pathogens through a clathrin-mediated mechanism (Jones et al., 2003; Vanderheijden et al., 2003). Of late, it was shown that porcine alveolar macrophages (PAM) expressing Sn internalized porcine Sn together with a Sn- specific monoclonal antibody (mAb) into the cell (Vanderheijden et al, 2003; Revilla et al., 2009; Delputte et al., 201 1). This internalization was mediated by Sn and not by other macrophage receptors, since isotype-matched control mAbs were not internalized and since F(ab')₂ fragments, lacking the Fc domain, are internalized into the cell similarly to the intact mAb. In addition, antibody-induced internalization was also observed in non-macrophage cell lines that express recombinant pSn, but that do not express Fc receptors. Furthermore, it was shown that a Sn-specific immunotoxin and an immunoconjugate of the antigen HSA linked to a Sn-specific mAb were also internalized into PAM together with pSn (Delputte et al., 201 1). However, recently Ducreux et al (2009) clearly demonstrated that PEGylated anti-Sn monoclonal antibodies, although showing a higher inhibitory potency in solid-phase red blood cell assays, were not impairing phagocytosis of latex particles, indicating again that Sn is not an essential player in phagocytosis.

Surprisingly we found that Sn cross-linking by an Sn binding antibody causes a significant reduction in phagocytic capacity of PAM against microspheres compared to control groups, as assayed by Flow cytometry (FC) or confocal microscopy (CM). With increasing antibody dose, phagocytosis dropped markedly compared to both controls, starting from 1.5 μg/ml and a small dose-dependent difference in the number of microspheres phagocytosed per cell was observed. In addition, it was observed that Sn cross-linking at the highest doses caused a drop in phagocytosis that was maintained over time, whether Sn cross-linking was allowed for 1 hour or continuously. Moreover, in both cases it was assessed that Sn cross-linking caused a drop in the number of microspheres that were phagocytosed per cell over time. Yet, using confocal microscopy, it was established that phagocytosis was not entirely blocked upon antibody treatment. Furthermore, it was seen that the number of PAM phagocytosing microspheres increased over time for all control groups, which is a result of keeping PAM in culture for a longer period of time and results in more mature PAM that have a higher phagocytic capacity (Pensati et al, 1979). However, this increase in the number of PAM phagocytosing microspheres was not observed in PAM after Sn cross-linking.

The observed downregulation of microsphere phagocytosis in PAM is of utmost importance, since the main function of PAM is the removal of particulate antigens and microorganisms from the alveolar space (Schneberger et al, 201 1), while simultaneously dampening potentially damaging immune/inflammatory responses. In cases where excessive macrophage phagocytosis is involved in the pathology of a disease, such as but not limited to HLH, ligand binding to Sn will be beneficial due to the effect of Sn cross-linking on phagocytosis.

Summary of the invention

The present invention provides a method for inhibiting phagocytosis in Sn expressing cells, in particular macrophages and inflammatory monocytic cells, comprising administering a sialoadhesin binding moiety. Said method is particularly useful for treating diseases characterised by uncontrolled phagocytosis or for inhibiting unwanted (physiologically) normal phagocytosis such as cancer vaccination using apoptotic tumor cells. A further aspect of the invention is a sialoadhesin binding moiety, and especially an antibody, for use in treatment of uncontrolled phagocytosis. Uncontrolled phagocytosis, as used here, is every form of phagocytosis resulting in unwanted effects. Indeed, in some cases, such as in cancer vaccination using apoptotic cell associated antigens, one wants to downregulate the normal phagocytosis by tumor-associated, immunosuppressive macrophages to increase the efficiency of the vaccination. Preferably, said uncontrolled phagocytosis is causing a disease, even more preferably said uncontrolled phagocytosis is causing Hemophagocytic lymphohistiocytosis (HLH), Macrophage activation syndrome (MAS), or acute liver failure.

A sialoadhesin binding antibody can be any sialoadhesin binding antibody known to the person skilled in the art such as but not limited to polyclonal antibodies, monoclonal antibodies, heavy chain antibodies (hcAb), single domain antibodies (sdAb), minibodies (Tramontano et al., 1994), variable domain of camelid heavy chain antibody (VHH) variable domain of the new antigen receptor (VNAR) and engineered CH2 domains (nanoantibodies; Dimitrov, 2009). It further includes peptides with antibody like characteristics, such as single chain antiparallel coiled protein (alphabodies; WO2010066740). Another aspect of the invention is a pharmaceutical composition, comprising a sialoadhesin binding antibody as described above. Preferably, said sialoadhesin binding antibody is the monoclonal antibody 7D2 or 41 D3 or a humanized derivative thereof.

Brief description of the figures

Figure 1. Flow cytometric analysis of the effect of mAb 41 D3 (black), isotype-matched control mAb 13D12 (grey) or negative control PBS (white) on macrophage viability, expressed as the percentage of cells that are alive upon treatment. Cells were stained with PI, which stains nuclei of dead cells. (A) Cells were incubated with the indicated amount of mAb for 24 hours. (B) Cells were incubated with 50 μg/ml mAb for the indicated time. Data represent the means ± SD of 3 independent experiments.

Figure 2. Chemiluminescent analysis of the effect of mAb 41 D3 (black) or isotype-matched control mAb 13D12 (grey) on ROI production by macrophages. After appropriate treatment, cells were triggered to produce ROI with 10 μg/ml PMA and ROI production was determined as lucigenin CL. (A) Cells were incubated with the indicated amount of mAb for 24 hours. (B) Cells were incubated with 50 μg/ml mAb for the indicated time. All data were corrected for background CL, are expressed as fold induction compared to PMA and represent the means ± SD of 3 independent experiments.

Figure 3. (A - F) Flow cytometric analysis of the effect of mAb 41 D3 (black), isotype-matched control mAb 13D12 (grey) or negative control PBS (dashed line) on the phagocytosis of beads by viable macrophages. After appropriate treatment, 20 fluorescent microspheres per cell were added to the macrophages, phagocytosis was allowed for 1 hour and cells were stained with PI. Data are expressed as the percentage of viable cells that are associated with beads (A, C, E) or the MFI per viable cell (B, D, F). (A & B) Cells were incubated with the indicated amount of mAb for 24 hours. (C & D) Cells were incubated with 50 μςΛηΙ mAb for the indicated time. (E & F) Cells were incubated with 50 μςΛηΙ mAb for 1 hour and phagocytosis was measured after the indicated time. (G - H) Confocal microscopical analysis of the effect of mAb 41 D3 or isotype-matched control mAb 13D12 on the phagocytosis of beads by macrophages. Cells were treated with 50 μg/ml mAb for 24 hours, subsequently 20 fluorescent microspheres per cell were added, phagocytosis was allowed for 1 hour. As a control, the phagocytosis assay was performed with untreated macrophages that were kept on ice. (G) Data show the percentage of macrophages without beads (black) and with internalized and/or surface bound beads (dark grey). (H) Data show the number of surface bound (light grey) and internalized (white) beads per 50 macrophages counted. Data represent the means ± SD of 3 independent experiments.

Figure 4. Flow cytometric analysis of the effect of mAb 41 D3 (black), isotype-matched control mAb 13D12 (grey) or negative control PBS (white or dashed line) on uptake and processing of OVA-DQ by viable macrophages. After appropriate treatment, 10 μg/ml OVA-DQ, which becomes fluorescent upon proteolytic cleavage, was added to the cells and uptake and processing was allowed for 2 hours. Cells were stained with PI, which stains nuclei of dead cells, and the number of viable cells taking up and processing OVA-DQ (A & B) or the amount of OVA-DQ taken up and processed per viable cell, expressed as MFI, (C & D) was measured. (A & C) Cells were incubated with the indicated amount of mAb for 24 hours. (B & D) Cells were incubated with 50 μg/ml mAb for the indicated time. Data represent the means ± SD of 3 independent experiments.

Figure 5. Flow cytometric analysis of the effect of mAb 41 D3 (black) or isotype-matched control mAb 13D12 (grey) on cell surface expression of MHC I and MHC II molecules. After appropriate treatment macrophages were stained with mAb PT85A (MHC I) or MSA3 (MHC II), followed by lgG_2a-specific goat-anti-mouse-AlexaFluor488. Subsequently, cells were stained with PI and the MFI of viable cells expressing MHC I (A & B) or MHC II (C & D) molecules on their cell surface was determined. (A) Cells were incubated with the indicated amount of mAb for 48 hours. (C) Cells were incubated with the indicated amount of mAb for 24 hours. (B & D) Cells were incubated with 50 μg/ml mAb for the indicated time. Data represent the means ± SD of 5 (A) or 4 (B, C, D) independent experiments.

Figure 6. Analysis of the effect of Sn cross-linking on the expression of I L- 1 β , IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IFN-γ and TNF-a in the cell culture supernatant by macrophages as determined by multiplex ELISA. (A) Cytokine concentration (pg/ml) measured at the indicated times post treatment with 50 μg/ml mAb 41 D3 (black) or isotype-matched control mAb 13D12 (grey). (B) Cytokine production assessed 12 hours post treatment with 50 μg/ml mAb 41 D3 (black), 50 μg/ml isotype-matched control mAb 13D12 (dark grey), 0.1 EU/ml endotoxin (negative control; white) or 1 μg/ml LPS (positive control; light grey). Data are expressed as fold induction compared to negative control. All data represent the means ± SD of 3 independent experiments.

Figure 7: Flow cytometric analysis of crosslinking Sn on phagocytosis of fluorescent beads in the absence or presence of anti-sialoadhesin mAB 7D2. Phagocytotic index is the Mean Fluorescence Intensity of beads associated cells x % phagocytosis. For the relative phagocytic index (rel Phi) values are normalized to phagocytosis at 37°C.

Detailed description

A first aspect of the present invention relates to a sialoadhesin (Sn) binding moiety for inhibiting phagocytosis in macrophages. A "sialoadhesin binding moiety" binds specifically to sialoadhesin. The term "binds specifically" as used herein is intended to indicate that a sialoadhesin binding moiety interacts preferentially with sialoadhesin and does not significantly interact with other proteins or other molecules. Examples of such other molecules include but are not limited to SIGLEC-5 (sialic acid binding Ig-like lectin 5; CD170) and SIGLEC-11 (sialic acid binding Ig-like lectin 1 1). In particular, a sialoadhesin binding moiety binds to an extracellular portion of sialoadhesin expressed by a cell. Further, a sialoadhesin binding moiety binds specifically with sialoadhesin present in the cell membrane of a target cell.

In one embodiment, a sialoadhesin binding moiety is an antibody. The term "antibody" refers to polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies, as well as antigen binding antibody fragments and molecules having antigen binding functionality.

The term "antibody" includes an intact immunoglobulin having four polypeptide chains, two heavy (H) chains and two light (L) chains linked by disulfide bonds. The term "antibody" also includes sialoadhesin binding antibody fragments illustratively including, but not limited to, such fragments as a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv fragment, a domain antibody (dAb), heavy chain antibodies (hcAb), minibodies (Tramontano et al., 1994), a variable domain of camelid heavy chain antibody (VHH), a variable domain of the new antigen receptor (VNAR) and engineered CH2 domains (nanoantibodies; Dimitrov, 2009). It further includes peptides and scaffolds with antibody like characteristics, such as single chain antiparallel coiled protein (alphabodies; WO2010066740).

An anti-sialoadhesin antibody and/or sialoadhesin binding antibody fragment of the present invention is capable of binding sialoadhesin. A preferred sialoadhesin binding moiety binds sialoadhesin with greater affinity than it binds another member of the Siglec family. A preferred sialoadhesin binding moiety is characterized by specific binding activity for sialoadhesin of at least about 1 x 10⁵ M^"1. In further embodiments, a preferred sialoadhesin binding moiety has a specific binding affinity for sialoadhesin of at least about 1 x 10⁶ M^"1. In still further embodiments, a preferred sialoadhesin binding moiety has a specific binding affinity for sialoadhesin of at least about 1 x 10⁷ M^"1.

Anti-sialoadhesin antibodies and sialoadhesin binding antibody fragments may be provided by any method, illustratively including, but not limited to, immunization, isolation and purification, enzymatic cleavage of an intact immunoglobulin, screening of phage display libraries, chemical synthesis of a desired sialoadhesin binding peptide or protein, production by recombinant nucleic acid technology. Combinations of such methods may also be used.

An anti-sialoadhesin antibody can be made by immunization using as an antigen a full length sialoadhesin or a peptide fragment of sialoadhesin. Such proteins and peptides may be, illustratively a human, pig, sheep, rat, mouse, monkey, ape, or other sialoadhesin protein or peptide. Exemplary human, porcine and mouse sialoadhesin protein and nucleic acid sequences included herein are respectively identified by GenBank Accession number NM_023068 Gl:89142743 (human); GenBank Accession number AF509585.1 Gl:31323698 (porcine); and GenBank Accession number NM_011426 Gl:226958331 (mouse). In a specific aspect of the invention, the antibody is non-conjugated, i.e. not directly coupled or linked to another molecule or compound. Extracellular portions of sialoadhesin from various species have been characterized, as have sialic acid binding sites, as exemplified in Nath, D. et al, J 1995; Vinson, M. et al., 1996; Hartnell, A. et al., 2001 ; and Vanderheijden, N. et al., 2003. For example, an extracellular portion of human sialoadhesin extends from amino acid 1 -1642, an extracellular portion of porcine sialoadhesin extends from amino acid 1 - 1643 and an extracellular portion of mouse sialoadhesin extends from amino acid 1 -1638, each with reference to the sequences described herein. A sialoadhesin fragment used as an antigen in preparation of a sialoadhesin binding antibody preferably includes one or more Ig-like domains.

Antigens may be prepared by any of various methods, including isolation from natural sources, recombinant production or by chemical synthetic techniques. Sialoadhesin proteins and peptides for use as antigens in preparation of a sialadhesin binding antibody are similarly prepared by any of various techniques.

A peptide portion of sialoadhesin or other antigen may be made more immunogenic if desired by linkage to a carrier molecule such as bovine serum albumin or keyhole limpet hemocyanin. Such a linkage may be accomplished by any of various techniques, illustratively including, but not limited to, conjugation and expression of a fusion protein.

Recombinantly expressed proteins and peptides, such as, but not limited to, sialoadhesin and sialoadhesin fragments, may be tagged to allow for easier isolation. For instance, such proteins and peptides may be Fc-tagged.

Antibodies, antigen binding fragments and methods for their generation are known in the art and such antibodies, antigen binding fragments and methods are described in further detail, for instance, in Antibody Engineering, Kontermann, R. and Dubel, S. (Eds.), Springer, 2001 ; Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988; Ausubel, F. et al., (Eds.).

The term "antigen" in the context of making a sialoadhesin binding moiety refers to sialoadhesin or an antigenic portion thereof. In a particular embodiment, an antigenic portion of sialoadhesin includes a portion of sialoadhesin present external to a cell expressing sialoadhesin. An antibody which is a sialoadhesin binding moiety may be made using a native sialoadhesin, such as exemplified by amino acid sequences included herein, and/or peptide fragments thereof, as an antigen. An antibody which is a sialoadhesin binding moiety may also be made using a sialoadhesin homologue, modified sialoadhesin and/or fragment thereof as an antigen.

In a specific embodiment, a sialoadhesin binding moiety is a monoclonal antibody 41 D3, or a humanized derivative thereof.

Monoclonal antibody 41 D3 (mAb 41 D3) is a mouse monoclonal anti-porcine sialoadhesin antibody. Methods to humanize murine antibodies are known to the person skilled in the art and have been described, amongst others, by Bernett et al. (2010), Zettlitz et al. (2010) and Mader and Kunert (2010). Monoclonal antibody 41 D3 is described in Vanderheijden, N. et al., 2003,; and in Duan, X. et al., 1998,. A hybridoma producing monoclonal antibody 41 D3 was deposited with the CNCM (Collection Nationale de Cultures de Microorganisms) at the Institute Pasteur, 28, Rue du Docteur Roux, F-75724 Paris Cedex 15 and given Accession number 1-2719.

In a further specific embodiment, a sialoadhesin binding moiety is mouse monoclonal antibody 7D2 (mAb 7D2) which binds human sialoadhesin. MAb 7D2 was raised against an Fc fusion protein containing the N-terminal four domains of human sialoadhesin. MAb 7D2 is further described in Hartnell, A. et al., 2001 and is commercially available.

Another specific example of a sialoadhesin binding moiety is mouse anti-porcine sialoadhesin monoclonal antibody MCA2316 described, for example, in Bullido, R., 1997, and commercially available.

A sialoadhesin binding moiety is a sialoadhesin ligand in a further embodiment of the present invention. As noted above, sialoadhesin is a sialic acid-binding immunoglobulin-like lectin. Sialoadhesin binds sialic acid, and in particular, a2-3 sialic acid residues and some a2-6 and a2-8 sialic acid residues. Such sialic acid residues illustratively include Siaa2-3Gap - 3GalNAc; Siaa2-3Gap-3GlcNAc; and Siaa2-3Gap -4GlcNAc, Siaa2-6Gap -3GalNAc and Siaa2- 8Neu5Aca2-3Gap-3GalNAc. Thus, in an embodiment in which a sialoadhesin binding moiety is a sialoadhesin ligand, a sialoadhesin binding moiety preferably includes a sialylated organic structure such as, but not limited to, a sialylated protein or peptide, lipid, and/or carbohydrate, and/or a sialyl like synthetic carbohydrate. In a further embodiment, a sialoadhesin binding moiety includes a natural sialylated ligand for sialoadhesin. A natural sialylated ligand for sialoadhesin is a sialylated structure which occurs naturally and binds sialoadhesin in vivo. Natural sialylated ligands illustratively include CD43, galactose-type C- type lectin 1 , and MUCI antigen. A natural sialylated ligand of sialoadhesin may be isolated from a natural source or recombinantly produced for inclusion in a conjugate composition according to the present invention.

Sialoadhesin is a cell adhesion molecule found on the surface of certain cells of the immune system, in particular on macrophages and activated monocytes. Hence a further aspect of the present invention relates to a sialoadhesin (Sn) binding moiety for inhibiting phagocytosis in Sn expressing cells, especially macrophages. In cases where excessive macrophage phagocytosis is involved in the pathology of a disease, such as but not limited to HLH or macrophage activation syndrome (MAS), ligand binding to Sn will be beneficial due to the effect of Sn binding on phagocytosis. In a specific aspect, the method of the invention aims to inhibit, reduce, or prevent uncontrolled or unwanted phagocytosis.

As used herein, the term "phagocytosis" is a specific form of endocytosis involving the vesicular internalization of solid particles. It is therefore distinct from other forms of endocytosis such as pinocytosis, the vesicular internalization of various liquids, and receptor-dependent endocytosis. Phagocytosis (literally, cell-eating) is the process by which cells ingest large objects or molecular aggregates. The membrane folds around the object, and the object is sealed off into a large vacuole known as a phagosome. The phagosome is usually delivered to the lysosome, an organelle involved in the breakdown of cellular components, which fuses with the phagosome. The contents are subsequently degraded and either released extracellularly via exocytosis, or released intracellular^ to undergo further processing. The term hemophagocytosis describes the pathologic finding of activated macrophages, engulfing erythrocytes, leukocytes, platelets, and their precursor cells.

By "inhibiting phagocytosis" is meant a down-regulation in phagocytosis by at least about 10%, or up to 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or up to 100% compared to the level of phagocytosis observed in absence of intervention. Thus in the context of decreasing phagocytosis of circulating cells or particles, inhibiting phagocytosis means a down-regulation in phagocytosis compared to level of phagocytosis observed in absence of the intervention or method as described herein.

In some instances, macrophages may show enhanced and/or uncontrolled phagocytic activity which results in (excess) uptake of self, resulting in disease. Hence, with the term "uncontrolled phagocytosis" is meant every form of phagocytosis resulting in unwanted effects or resulting in elimination of essential or healthy cells.

Examples of diseases characterized by uncontrolled phagocytosis are macrophage activation syndrome, hemophagocytic lymphohistiocytosis and immune thrombocytopenia (ITP). "Macrophage activation syndrome" (MAS) is a severe and acute clinical event occurring with fever, hepatosplenomegaly, and pancytopenia due to uncontrolled phagocytosis of blood cells and precursors. "Hemophagocytic lymphohistiocytosis" (HLH), also called "Hemophagocytic syndrome" (HPS) is characterized by a dysregulated activation and proliferation of macrophages, leading to uncontrolled phagocytosis of platelets, erythrocytes, lymphocytes, and their hematopoietic precursors throughout the reticuloendothelial system. This life- threatening disease combines non-specific clinical signs (fever, cachexia, hepatomegaly, enlargement of spleen and lymph nodes) as well as typical laboratory findings (bi- or pancytopenia, abnormal hepatic tests, hypofibrinemia, elevation of serum LDH, ferritinemia and triglyceride levels). Diagnosis is confirmed by cytological or pathological examination of bone marrow or tissue specimens. Most commonly used treatments include high-dose glucocorticoids, and cyclosporine.

"Acute liver failure" embraces a number of conditions whose common thread is severe injury of hepatocytes or massive necrosis. Monocytes and macrophages are central players in the complex process of initiation, propagation, and resolution of acute liver injury. Loss of hepatocyte function sets in motion a multiorgan response, and death may occur even when the liver has begun to recover. Altered mental status (hepatic encephalopathy) and coagulopathy in the setting of an acute hepatic disease define acute liver failure. The term "fulminant hepatic failure" is generally applied to patients in whom hepatic encephalopathy develops within 8 weeks of the onset of illness, whereas "subfulminant hepatic failure" is used to describe a minority of patients in whom hepatic encephalopathy develops after a longer illness, up to 26 weeks in duration (also called late-onset hepatic failure). "Acute liver failure" is used as the umbrella term, since it encompasses all these clinical presentations (William M. Lee, 1993).

In a further embodiment, the present invention relates to cancer treatment. More specific, the present invention relates to an enhanced induction of a tumor specific immunological response, especially a T cell immune response, comprising the use of a sialoadhesin binding moiety. In said embodiment, the sialoadhesin binding moiety will be administered to the subject together with certain cancer therapies including chemotherapy, radiotherapy, and/or cancer vaccination with apoptotic bodies.

In cancer, there are 2 settings in which phagocytosis of apoptotic cell fragments could be a clinically relevant phenomenon. The first setting involves conventional treatment with chemotherapy or radiotherapy. It is assumed that the ensuing apoptosis and subsequent tumor regression is accompanied by a massive release of apoptotic cell fragments into the tissue environment, fragments which are taken up by cells of the mononuclear phagocyte system (macrophages, dendritic cells). Uptake of apoptotic bodies by macrophages is usually considered a "silent" removal, i.e. no active immune response or tissue inflammation results from this process. In contrast, recent experimental reports have validated the concept of immunogenic cell death. Briefly, cancer cell apoptosis induced by specific chemotherapeutic agents or radiotherapy results in the release of specific molecular "eat-me" signals (calreticulin exposure) and "danger" signals (HMGB1 , ATP), both of which targeted at patrolling dendritic cells (Apetoh et al., 2007; Ghiringhelli et al., 2009). When taking up apoptotic cell material, dendritic cells will induce a protective anti-tumoral T-cell response only when responsive to these danger signals. Accordingly, chemotherapy-treated patients in which the sensing of these danger signals is impaired have worse relapse-free survival rates.

This suggests that redirecting uptake of apoptotic tumor fragments by DCs rather than macrophages could improve long-term outcome after conventional cancer treatments. In this setting the addition of a sialoadhesin targeted biological to regular chemotherapy and/or radiotherapy is evaluated in terms of progression-free survival.

In said specific embodiment of the invention, the subject to be treated with an anti-sialoadhesin binding moiety is a cancer patient receiving chemotherapy, i.e. receiving a chemotherapeutic agent, and/or receiving radiotherapy. More specific, the present invention provides a sialoadhesin binding moiety, more particular an antibody, and a chemotherapeutic agent for use in treating cancer by inhibiting phagocytosis of apoptotic bodies and/or by inducing a tumor specific immune response. A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. It is usually used to refer to antineoplastic drugs. Chemotherapeutic agents fall under the following classes: alkylating agents, DNA-crosslinkers, topoisomerase inhibitors and anti-metabolites. In a preferred embodiment, the chemotherapeutic agent results in the release of "immunogenic apoptotic bodies", said agents including but not limited to anthracyclins (e.g. mitoxanthrone, doxorubicin) and oxaliplatin. We define cell death as 'immunogenic' if dying cells that express a specific antigen elicit a protective immune response. It has recently been shown that calreticulin exposure -a key prerequisite for immunogenic cell death- requires induction of an endoplasmatic reticulum stress response by the chemotherapeutical agent. Accordingly, chemotherapeutics that do not induce immunogenic cell death (e.g. cisplatin) can become immunogenic when ER stress is co- induced artificially (Martins I., 2011).

Furthermore, tumor derived apoptotic bodies can also be induced by radiotherapy. The radiotherapy may be external beam radiation, internal radiation therapy, or conformal radiation therapy, in which a computer is used to shape the beam of radiation to match the shape of the tumor. The radiation used in radiotherapy may come from a variety of sources, including an x- ray, electron beam, or gamma rays. The doses and timing of administration of the radiation during radiotherapy can and will vary depending on the location and extent of the cancer.

The second setting involves the use of cancer vaccination, specifically immunization with apoptotic cells or cell fragments/bodies. Cancer vaccination, regardless of its incarnation, is still an investigational approach. Tumor cell vaccines have shown modest, albeit encouraging results in clinical trials. One way to try to improve efficacy is therapeutic vaccination with dendritic cells (DC). Various strategies are being explored in manufacturing DC vaccines ex vivo, e.g., monocyte-derived DC (MoDC) loaded with cancer-associated antigens. Therapies that would however allow direct administration of cancer-associated antigens, such as cancer cell derived apoptotic bodies (AB), would allow for a less labor intensive process. One major drawback of direct injection of AB into humans is that the majority of these AB are being phagocytosed by macrophages, such as marginal zone macrophages (MZM), which results in removal of the apoptotic bodies without proper induction of cancer-specific immune responses. Such silent removal of self antigens is essential for avoiding immune-responses to self. Depletion of resident macrophages appears to promote apoptotic body vaccination efficiency, suggesting that in some instances, such as apoptotic body cancer vaccination, a block of such an unwanted phagocytosis by macrophages could be beneficial. (Henry F. et al., 1997). Since complete elimination of macrophages to circumvent unwanted phagocytosis carries an increased risk for potentially fatal infections, temporarily blocking the capacity of macrophages to phagocytose apoptotic bodies without killing these cells, would allow cancer cell derived AB to reach and be taken up by DC, which in turn can initiate tumor-specific immune responses. Therefore, it is also an aim of the present invention to provide a sialoadhesin binding moiety, more specifically an antibody, for inhibiting (unwanted) phagocytosis of cancer cell derived apoptotic bodies or apoptotic cell-associated fragments. In said embodiment, the tumor derived apoptotic bodies are prepared in vitro and administered to the subject. The apoptotic bodies and the sialoadhesin binding moiety can be administered to the subject simultaneously or serially.

As used herein, the term "apoptotic bodies" refers to cells with early hallmarks of apoptosis, apoptotic cells and fragments thereof, and the typical small pieces which are observed during apoptosis (apobodies). These bodies are surrounded by a sealed membrane and contain none or variable amounts of nucleus and other intracellular content. The term apoptosis was coined in a now-classic paper by Kerr, Wyllie, and Currie in 1972 as a means of distinguishing a morphologically distinctive form of cell death which was associated with normal physiology. Apoptosis was distinguished from necrosis, which was associated with acute injury to cells. Apoptosis is characterized by nuclear chromatin condensation, cytoplasmic shrinking, dilated endoplasmic reticulum, and membrane blebbing. Mitochondria remain unchanged morphologically. The simplest way to observe this phenomenon in vitro is to use a cell permeant DNA-staining fluorescent dye such as Hoechst 33342, which allows a striking visualization of the chromatin condensation.

Biochemical correlates of these morphological features have emerged during the subsequent years of study of this phenomenon. The first and most dramatic is DNA fragmentation. When DNA from apoptotically dying cells was subjected to agarose gel electrophoresis, ladders with -200 bp repeats were observed, corresponding with histone protection in the nucleosomes of native chromatin. Subsequent pulsed field gel techniques have revealed earlier DNA cleavage patterns into larger fragments. Since even a few double stranded DNA breaks will render the cell unable to undergo mitosis successfully, such DNA fragmentation can be regarded as a biochemical definition of death. However, in some apoptotic systems (e.g., Fas killing of tumor cells) artificially enucleated cells lacking a nucleus still die, showing that the nucleus is not always necessary for apoptotic cell death. The changes in the apoptotic cell which trigger phagocytosis by non-activated macrophages have been investigated by several groups. Macrophages appear to recognize apoptotic cells via several different recognition systems, which seem to be used preferentially by different macrophage subpopulations. There is good evidence that apoptotic cells lose the normal phospholipid asymmetry in their plasma membrane, as manifested by the exposure of normally inward-facing phosphatidyl serine on the external face of the bilayer. Macrophages can recognize this exposed lipid headgroup via an unknown receptor, triggering phagocytosis.

Another biochemical hallmark of apoptotic death which increasingly appears general is the activation of caspases, which are cysteine proteases related to ced-3, the "death gene" of the nematode Caenorhabditis elegans. Caspases seem to be widely expressed in an inactive proenzyme form in most cells. Their proteolytic activity is characterized by their unusual ability to cleave proteins at aspartic acid residues, although different caspases have different fine specificities involving recognition of neighboring amino acids. Active caspases can often activate other pro-caspases, allowing initiation of a protease cascade. While several protein substrates have been shown to be cleaved by caspases during apoptotic death, the functionally important substrates are not yet clearly defined. Persuasive evidence that these proteases are involved in most examples of apoptotic cell death has come from the ability of specific caspase inhibitors to block cell death, as well as the demonstration that knockout mice lacking caspase 3, 8 and 9 fail to complete normal embryonic development.

Apoptotic death can be triggered by a wide variety of stimuli, and not all cells necessarily will die in response to the same stimulus. Among the more studied death stimuli is DNA damage (by irradiation or drugs used for cancer chemotherapy), which in many cells leads to apoptotic death via a pathway dependent on p53. Some hormones such as corticosteroids lead to death in particular cells (e.g., thymocytes), although other cell types may be stimulated. Some cells types express Fas, a surface protein which initiates an intracellular death signal in response to crosslinking. In other cases cells appear to have a default death pathway which must be actively blocked by a survival factor in order to allow cell survival.

Apoptosis can be triggered by intrinsic and extrinsic stimuli. In intrinsic apoptosis, the death- inducing stimulus involves cellular damage or malfunction brought about by stress, ultraviolet (UV) or ionizing radiation, oncogene activation, toxin exposure, etc. Extrinsic apoptosis is triggered by binding of extracellular ligands to specific transmembrane receptors, primarily members of the tumor necrosis factor receptor (TNFR) family (S.H. Kaufmann and W.C. Earnshaw, 2000).

Various factors induce apoptosis, but the major factors can be classified as: (i) extracellular factors and (ii) intracellular factors depending on whether the conditions outside the cell or an injury within the cell determine the cell's faith to die. Extracellular factors act through cell surface death receptors or granzyme/perforin system. Intracellular factors cause the mitochondrial activation. However, mitochondrial activation also is triggered by extracellular factors. Hence, the mitochondria have a central role in the apoptosis.

As for extracellular factors, apoptosis is induced by the interaction of death receptors, namely, Fas (APO-1 , CD95) and TNF receptor-1 (TNFR-1), DR-3 (TRAMP), DR-4 (TRAIL-R1) and DR- 5 (TRAIL-R2), with their ligands. These cell surface receptors are located on the cell membrane, and they are members of the TNFR family. Ligands of death receptors cause oligomerization and thus activation of receptors. Oligomerization of the receptors is followed by binding of specific adapter proteins (FADD, TRADD) to their receptor, which in turn leads to the activation of the caspase system. Fas is found in lymphoid cells, hepatocytes, some tumor cells, pulmonary cells and even myocardial cells. The ligand is termed as Fas ligand (FasL). FasL is a member of the TNF family. It is found in cytotoxic T lymphocytes and natural killer cells. As they receive the death alert when they bind to their ligands, Fas and TNFR-1 undergo a series of protein-protein interactions. First, they initiate the interaction with the death domains TNFR-1-associated death domain (TRADD) and Fas-associated death domain (FADD), which are naturally bound to them. Death domains initiate the cascade of caspases by activating pro-caspase-8. Caspase-8 is activated by autoactivation following oligomerization. Active caspase-8 executes the apoptotic process in two ways: (i) direct cleavage and activation of caspase-3 and (ii) cleavage of Bid, a pro-apoptotic protein. Cleaved BID becomes activated and translocates from the cytosol to the mitochondria, where it induces cytochrome c release. TNFR-1 and DR-3 cause activation of caspase-8 using FADD and TRADD as adaptor proteins. FADD (but not TRADD) also appears as an adaptor molecule for both DR-4 and DR-5 and, hence, has been even proposed as a universal adaptor for death receptors. As a regulatory system, the proteins that inhibit these death domains also are available within the cell. For instance, caspase-8 commonly referred to as FLICE (FADD-like ICE) inhibits FLIP (FLICE-like inhibitory protein) at high concentrations. J3ome examples of clinical conditions characterized by an increase in FasL include toxic epidermal necrolysis (Lyell syndrome), esophageal cancer, metastatic colon cancer, hepatocellular carcinoma, multiple myeloma, sarcoma, non-Hodgkin lymphoma, melanoma and nasal lymphoma. Apoptosis, apart from the activation of cell death receptors, is initiated by the induction of p53 as a response to DNA damage caused by genotoxic agents as previously mentioned. Induced p53 initiates apoptosis by activating Bax, a pro-apoptotic member of the Bcl-2 family. In fact, there is a close link between DNA damage and Bcl-2 family members (e.g. Bid, as a pro- apoptotic member). p53 also initiates apoptosis by inducing cell surface death receptors such as Fas and DR5. Apoptosis also is initiated as a result of damage in the mitochondria, the plasma membrane or the genome caused by reactive oxygen radicals (oxidative stress). Deficiency of growth factors also induces apoptosis and can be considered as another factor acting extracellularly. It has been shown that cultured cells undergo apoptosis if they are exposed to serum starvation. This mechanism eventuates with the activation of p53, which is an apoptosis-inducing nuclear protein. Failure to phosphorylate Bad, a pro-apoptotic member of the Bcl-2 protein family, induces apoptosis by the release of cytochrome c from the mitochondria to the cytosol. Another factor inducing apoptosis is the activation of caspase system in target cells (infected cell with virus and cancer cells) in response to the release of granzyme B from cytotoxic T lymphocytes. Ceramide, which is another plasma membrane- related activator of apoptosis, is a product of the activation of membrane-bound acid sphingomyelinase and thought to be involved in signal transduction in response to the damaged plasma membrane. Irradiation, hyperthermia, hydrogen peroxide and ultraviolet radiation lead to the activation of sphingomyelinase and hence to the increase of ceramide. Accumulation of ceramide has been observed in primary cultures of neurons undergoing apoptosis because of choline deficiency. However, ceramide was proposed not to be an initiator of apoptosis considering the occurrence of ceramide accumulation as a late event in apoptosis. Ceramide may have various functions depending on the cell type. In terms of the intracellular factors, the mitochondrion is the key organelle. The mitochondria are the intersection point of signals of both death and survival such as death receptors, growth factors and cytokines. The opposing forces, death and survival, are directed to the mitochondrial activation, the release of cytochrome c from the mitochondria to the cytosol, which is an irreversible event during the progression of apoptosis. The major factor in mitochondrial activation is the Bcl-2 protein family. Pro-apoptotic and anti-apoptotic members of this family exert their effects on the mitochondria either to induce (initiation of apoptosis) or to inhibit (inhibition of apoptosis) the release of cytochrome c to the cytosol (Ulukaya et al., 201 1). Other methods, as described in "Methods for inducing apoptosis" (Roberts KM, et al., 2004) can also be used. In a specific embodiment, the apoptotic bodies can be administered to the subject in combination with an adjuvant. Adjuvants for immunization include but are not limited to defined Toll-like receptor ligands such as MPLA, Polyl:C or R848, or complex formulations such as Freund's adjuvant. The role of the adjuvant is to trigger proper activation of the antigen- presenting cell at the time of antigen uptake. Antigen encounter in the absence of adjuvant can result in the induction of tolerance rather than active immunity.

The types of cancer to be treated by the methods of the present invention especially include solid tumor cancers. The method is not restricted to a tumor from a particular organ. Examples of cancers to be treated are lung cancer, prostate cancer, colorectal cancer, myeloma, kidney cancer, melanoma, colon cancer, ovarian cancer, breast cancer, and renal cell cancer. In the case of a cancer patient receiving chemotherapy and/or radiotherapy, the method of the invention will especially be applicable for cancers whereby the presence of sialoadhesin- expressing macrophages is documented in the tumor bed.

In a further embodiment, the present invention relates to a pharmaceutical composition, comprising, preferably consisting of a sialoadhesin binding moiety, and a pharmaceutically acceptable carrier, excipient and/or additive as known in the art. Preferably said sialoadhesin binding moiety is freely occurring i.e. not bound to another moiety or molecule. More specific, the pharmaceutical composition further comprises a chemotherapeutic agent. Even more specific, the pharmaceutical composition further comprises apoptotic bodies as described herein, and optionally an adjuvant. Preferably said sialoadhesin binding moiety is an antibody. Preferably the antibody is non-conjugated, i.e. not coupled or linked to other molecules. The composition of the invention is suitable for administration directly to the subject to be treated. Formulations typically comprise at least one active ingredient (e.g. the anti-Sn antibody), as defined above, together with one or more acceptable carriers. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. Optionally, the composition of the invention further comprises a buffering agent. Supplementary active ingredients can also be incorporated into the composition.

As used herein "acceptable carrier" includes any and all solvents and dispersion media, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art.

It is understood by the skilled artisan that the preferred dosage would be individualized to the patient following good laboratory practice (GLP) and good clinical practice (GCP). In compliance with GCP, the particular dosing regimen to be employed shall be primarily determined by first-in-human/phase I toxicity and pharmacokinetic studies, followed by dose- finding phasel/ll clinical trials using pre-specified clinical endpoints as well as biomarkers as surrogates for clinical effect. The therapeutic agent (e.g. the antibody or a composition comprising said therapeutic agent) should be delivered in a sufficient dose as defined herein. Preferred means of administration are subcutaneous (sc), intravenous, interstitial, or intramuscular. Administration may, depending on the case, also be done by organ perfusion, catheterization through blood vessels to the target organ, or through direct injection into an organ.

The different aspects of the present invention are illustrated by, but not limited to the examples detailed hereafter. EXAMPLES

Materials and methods to the examples

Cells and antibodies

Primary PAM were obtained from 4- to 6-week-old conventional Belgian Landrace pigs from a PRRSV-negative herd by post mortem bronchoalveolar lavage and were frozen in liquid nitrogen as described by Wensvoort et al. (1991). The cells were cultivated in RPMI-1640 (Gibco), supplemented with 10 % (v/v) fetal bovine serum (FBS; Greiner), 2 mM L-glutamine (BDH Chemicals Ltd.), 1 % (v/v) non-essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco) and a mixture of antibiotics in a humidified 5 % C0₂ atmosphere at 37 °C.

Primary mouse alveolar macrophages were obtained by lavage of mouse lungs in situ with 1 to 2 ml PBS containing 1 mM EDTA. Cells were then pelleted by centrifugation, resuspended in RPMI-1640, supplemented with 10% FBS and a mixture of antibiotics and incubated in a humidified 5% C0₂ atmosphere at 37°C. Mouse alveolar macrophage cell lines J774 and RAW264.7 were obtained from ATCC and maintained in DMEM supplemented with 10% FBS and a mixture of antibiotics in a humidified 5% C0₂ atmosphere at 37°C. Human monocyte cell lines THP-1 and U937 were obtained from ATCC and maintained in RPMI-1640, supplemented with 10% FBS and a mixture of antibiotics in a humidified 5% C0₂ atmosphere at 37°C. Cells differentiated into macrophages following PMA-treatment.

For cross-linking of Sn, the Igd mouse monoclonal anti-porcine Sn antibody (mAb) 41 D3 was used (Duan et al., 1998; Vanderheijden et al., 2003). Isotype-matched irrelevant mAb 13D12, directed against pseudorabies virus glycoprotein gD (Nauwynck & Pensaert, 1995), was used as a negative control. Class I Major Histocompatibility Antigen (MHC I) was visualized using the lgG_2a mouse mAb PT85A (VMRD, Pullman, WA), directed against the heavy chain of MHC I. Class II Major Histocompatibility Antigen (MHC II) was detected using the lgG_2a mouse mAb MSA 3 (Hammerberg & Shurig, 1986). A mouse mAb, 13H4, directed against the PCV2 capsid protein (Lefebvre et al., 2008), was used as an isotype-matched irrelevant control mAb for both. For all experiments, mAb 41 D3, 13D12, MSA3 and 13H4 were purified using protein G column chromatography (GE Healthcare) following the manufacturers' instructions.

Generation of sialoadhesin-specific antibodies and nanobodies

Antibodies recognizing human sialoadhesin were generated by immunizing mice with MEF (Mouse Embryonic Fibroblast) cells expressing ectopic human sialoadhesin. Antibodies recognizing murine sialoadhesin were raised in rats by immunization with purified recombinant mouse sialoadhesin (R&D Systems). Hybridoma supernatants were screened in ELISA against purified recombinant human or murine sialoadhesin (R&D Systems) and CHO cells stably expressing human or mouse sialoadhesin.

Nanobodies (VHH) recognizing human and murine sialoadhesin were generated by immunization of a llama (Lama glama) with HEK-T cells transiently expressing human sialoadhesin. The VHH genes were cloned into a phage display vector using published protocols (Ghassabeh et al. , 2010). Panning of the phage display library and screening of single clones was done against recombinant human and mouse Sn (R&D Systems).

Sialoadhesin expression

To assess the expression of sialoadhesin and tissue distribution of sialoadhesin positive cells, cryosections (4 μηι) were made from human or murine lung tumors and lymph nodes. Sections were mounted on poly-lysine coated glass slides. Sections were fixed in cold acetone and blocked with blocking reagent (Abeam). Sections were then stained with mAb 7D2 (Abeam) and mAb 3D6.1 12 (AbD Serotec) or their respective isotype controls (mouse lgG1 and rat lgG2a) for 1 h at room temperature. The stained sections were mounted with Fluorsave (Calbiochem) and observed by fluorescence microscopy (Olympus).

Sialoadhesin treatment

To cross-link Sn mAb 41 D3 was used, as previously described (Genini et al. , 2008). In order to assess the dose-dependent effect of the antibody treatment, all experiments were performed in a similar way: 10⁶ macrophages were treated with 0, 0.15, 0.5, 1 .5, 5, 15 or 50 μg/ml mAb 41 D3 or isotype control and were subsequently incubated in a humidified 5 % C0₂ atmosphere at 37 °C for 24 hours. When determining the time-dependent effect of the antibody treatment, all experiments were performed as follows: 24 hours after seeding, 10⁶ macrophages were treated with 50 μg/ml mAb 41 D3 or isotype control, and were further incubated in a humidified 5 % C0₂ atmosphere at 37 °C for 6, 12, 24, 48 or 72 hours. In order to determine the time- dependent effect of a short-term antibody treatment, all experiments were performed akin: 24 hours after seeding, 10⁶ macrophages were treated with 50 μg/ml mAb 41 D3 or isotype control, and were further incubated in a humidified 5 % C0₂ atmosphere at 37 °C for 1 hour. Cells were then shifted to 4 °C for 15 minutes, washed twice, resuspended in ice-cold medium and subsequently incubated in a humidified 5 % C0₂ atmosphere at 37 °C until 6, 12, 24, 48 or 72 hours after starting the mAb treatment.

Viability staining

Macrophages were treated with mAb 41 D3, isotype control or phosphate buffered saline (PBS; negative control) 24 hours post seeding. After appropriate treatment, cells were shifted to 4 °C for 15 minutes, washed twice and resuspended in ice-cold PBS. Live-dead staining was performed by adding a live/dead dye such as propidium iodide (PI; Molecular Probes, Eugene, OR) or 7-AAD (Miltenyi) which stain nuclei of (dead) cells with a permeated plasma membrane, to a final concentration of 0.015 mM, followed by a ten minute incubation at 4 °C, after which cells were analysed immediately with a Becton-Dickinson (BD; San Jose, CA) FACSCanto or MACSQuant (Miltenyi) The percentage of viable cells was determined by subtracting the percentage of PI or 7AAD stained cells from the unstained population.

Chemiluminescent detection of Reactive Oxygen Intermediate (ROI) production

The release of ROI in the supernatant of elicited macrophages was determined using lucigenin (bis-N-methylacridinium nitrate; Invitrogen) as chemiluminigenic probe (adapted from Boyen et al., 2006), since it interacts with superoxide anion radicals (0₂ ^" ), which results in a chemiluminescent (CL) response that can be measured. Lucigenin was dissolved in dimethylsulfoxide (DMSO; Merck) to a concentration of 100 mM and was stored in aliquots at - 70 °C. Prior to use, the lucigenin stock was diluted in Hank's Balanced Salt Solution (HBSS; Gibco) to a final assay concentration of 400 μΜ. Phorbol 12-myristate 13-acetate (PMA; Sigma) was used as triggering agent in the chemiluminescent assay. A 1 mg/ml PMA stocksolution in DMSO was used and stored as aliquots at -20 °C. The final assay concentration of PMA was 10 μg/ml. The CL assays were performed in white clear bottom chimney 96-well plates (Greiner Bio-One) at 37°C, using a FluoroScan Ascent fluorometer (Labsystems, Helsinki, Finland). The plates were not stirred during the assay. Measuring of the CL reactions of isolated PAMs was performed as follows. Macrophages were treated with mAb 41 D3 or isotype control 72 hours post seeding. After appropriate treatment, the culture supernatant was replaced by 175 μΙ lucigenin solution and background CL (of cells without stimulating agent) was recorded for 10 minutes. The CL was measured as light units (LU). Each well was read for 1 second per minute. After determining the spontaneous CL, cells were triggered with PMA in the presence of mAb 41 D3, isotype control or HBSS (negative control) in a final volume of 25 μΙ and the oxidative activity of the cells was recorded for one hour. From this data, the area under curve (RLU produced in one hour) was determined for all conditions and data were corrected for spontaneous CL. Data are expressed as fold induction relative to the negative control (10 μg/ml PMA).

Phagocytosis assay

Flow cytometry (FC) - To assess the effect of Sn cross-linking on phagocytosis of particulate antigens, yellow-green fluorescent carboxylate-modified microspheres with a diameter of 1 μηι were used (FluoSpheres, Molecular Probes). Twenty-four hours post seeding macrophages were treated with mAb 41 D3, isotype control or PBS (negative control) for 1 hour (pulsed Ab treatment) or 24 hours (continuous Ab treatment). Twenty microspheres per cell were added to the macrophages after appropriate treatment, and uptake was allowed for 1 hour. Thereafter, cells were shifted to 4 °C for 15 minutes, washed twice to wash away unbound microspheres, resuspended in ice-cold PBS and analysed using FC. The percentage of cells that had beads bound to their surface and/or had internalized beads was determined. As a measure for the number of beads associated (bound and/or internalized) per cell, the median fluorescence intensity (MFI) was used.

Confocal microscopy (CM) - To determine how many of the associated microspheres are bound to the cell surface and how many are internalized, phagocytosis of microspheres was analysed using confocal microscopy. The phagocytosis assay was performed as earlier described on cells that were treated for 24 hours with 50 μg/ml mAb 41 D3 or isotype control. As a control untreated cells were put on ice for 15 minutes prior to adding the microspheres, and were kept on ice during the entire assay. After the last wash step in the phagocytosis assay, cells were fixed with 4 % (w/v) paraformaldehyde (PF; Sigma, St. Louis, MO) in PBS, washed twice and permeabilized for 10 minutes using 0.1 % (w/v) saponin (Sigma) in PBS (PBS-S). Next, cells were incubated for one hour at 4 °C with TexasRed-X phalloidin (Invitrogen, Carlsbad, CA) diluted in PBS-S to stain cortical actin, washed twice with PBS-S and incubated with 10 μg/ml Hoechst 33342 (Invitrogen) in PBS for 10 minutes to stain all nuclei. Subsequently, cells were washed two more times and embedded in a glycerine-PBS solution (0.9/0.1 v/v) containing 2.5 % 1 ,4-diazabicyclo(2.2.2)octane (DABCO), mounted onto microscope slides and analysed on a laser-scanning spectrum confocal system (TCS SP2; Leica Microsystems) linked to a LEICA DM IRBE fluorescence microscope using an Argon 488 nm and a Gre/Ne 543 nm laser for excitation. Z-section images were taken using a 63x oil objective (N.A. 1.40-0.60) at room temperature and by using confocal acquisition software (LEICA TCS SP2 confocal software package, Leica Microsystems), which was also used for producing overlay images. At least 50 cells were analysed per sample and for each cell the number of internalized and surface bound beads was counted.

Detection of uptake and processing of soluble antigen

Macrophages were treated with mAb 41 D3, isotype control or PBS (negative control) 24 hours post seeding. To determine the effect of Sn cross-linking on soluble antigen uptake and processing, 10 μg/ml DQ Ovalbumin (OVA-DQ; Molecular Probes) was added to the macrophages after appropriate treatment. After two hours of incubation, cells were shifted to 4 °C for 15 minutes, washed twice, resuspended in ice-cold PBS and analysed using FC. The percentage of cells that took up and processed OVA-DQ was determined by subtracting the percentage of non-fluorescent macrophages from the total population. As a measure for the amount of OVA-DQ that was taken up and processed, the MFI of cells that took up and processed OVA-DQ was used. Immunofluorescence staining of surface expression of MHC I and MHC II

To determine the effect of Sn cross-linking on surface expression of MHC I and MHC II, macrophages were immunostained for extracellular MHC I and MHC II. Macrophages were treated with mAb 41 D3 or isotype control 48 hours post seeding for the MHC I experiment and 24 hours post seeding for the MHC II experiment. After appropriate treatment, cells were shifted to 4 °C for 15 minutes, washed twice and incubated for one hour at 4 °C with primary mAb PT85A, mAb MSA3 or isotype-matched control antibody 13H4 diluted in PBS containing 10 % heat-inactivated goat serum (PBS-G). Next, cells were washed two times and subsequently incubated with lgG_2a-specific AlexaFluor488-labelled goat-anti-mouse mAb (Molecular Probes) diluted in PBS-G. The secondary Ab needed to be isotype-specific in order to prevent cross-reaction with mAb 41 D3 or 13D12 (Igd isotype control), both previously used to cross-link Sn. Afterwards, cells were washed two times, resuspended in ice-cold PBS and analysed using FC. The percentage of cells that express MHC I or MHC II on their surface was determined by subtracting the percentage of non-fluorescent macrophages from the total population. As a measure for the amount of MHC I or MHC II expressed, the MFI of cells that express these molecules was used.

Detection of cytokine and chemokine production

To determine the effect of Sn cross-linking on cytokine and chemokine production, cell supernatant was collected after treatment and used for quantification of various cytokines and the chemokine IL-8. Endotoxin levels of mAb 41 D3 and 13D12 were less than 0.1 endotoxin units/ml (EU/ml), as determined using the Genscript ToxinSensor Chromogenic LAL Endotoxin Assay Kit (Genscript Corporation, Piscataway, NJ). The manufacturer's protocol was followed and the EU/ml were determined in comparison to a standard curve prepared with lipopolysaccharide (LPS) from Escherichia coli 011 1 :B4 (Sigma). Twenty-four hours after seeding, 10⁶ macrophages were treated with 50 μg/ml 41 D3 or isotype control and further incubated in a humidified 5% C0₂ atmosphere at 37 °C for 0, 6, 12 or 24 hours. In addition, cells were treated with 1 μg/ml lipopolysaccharide (LPS) from E. coli 011 1 :B4 as a positive control or with 0.1 EU/ml as a control for residual LPS in the mAb solutions, and further incubated in a humidified 5% C0₂ atmosphere at 37 °C for 12 hours. At each timepoint, cell supernatant was collected, centrifuged 5 minutes at 4 °C at 400 x g in order to discard cells and debris, aliquoted and stored at -70 °C until further use.

IFN-alfa bioassay - IFN-a levels in the cell supernatant were determined in a cytopathic effect reduction test with Madin-Darby Bovine Kidney (MDBK) cells and vesicular stomatitis virus (VSV) as described earlier (Van Reeth et al., 2002). Briefly, twofold dilutions of samples were added to MDBK cells in 96-well plates. A laboratory standard using recombinant porcine IFN-a (kind gift from C. La Bonnardiere, INRA, Jouy en Josas, France) was run as an internal control. Following overnight incubation, cells were challenged with VSV and two days later the antiviral effect of the samples was determined. One unit of IFN-a was defined as the reciprocal of the dilution producing 50 % inhibition of cytopathogenic effect (CPE). The limit of detection (LOD) for this test was 20 U/ml.

ΙΙ_-1 β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IFN-γ and TNF-a concentrations in the cell supernatant of all samples were quantified by multiplex ELISA following shipment on dry ice to Aushon BioSystems (Billerica, MA) for analysis using the SearchLight Chemiluminescent Porcine Cytokine Array (Aushon BioSystems). As an internal control, recombinant cytokines were used to run laboratory standards. All samples were tested twice at three dilutions (1 :2, 1 :50, 1 :1000). Calculated concentrations were baseline LOD-corrected per cytokine or chemokine per plate. Data are expressed as measured (pg/ml) or as fold induction relative to the negative control (0.1 EU/ml treated PAM).

Flow cytometry

For all FC assays, live-dead staining was performed as mentioned before and cells were analysed immediately with a BD FACSCanto or Miltenyi MACSQuant. Ten thousand cells were analysed per sample and three (viability assay) or four (all other FC assays) parameters were stored for further analysis: forward light scatter (FSC), sideward light scatter (SSC) and fluorescence emission centered at 530 nm (FL-1 for FluoSpheres, OVA-DQ and AlexaFluor488) and 585 nm (FL-2 for PI) or 670 nm (FL-3 for 7-AAD). Data were analysed with BD FACSDiva software and macrophages were gated on FSC and SSC to remove debris from the analysis and on FL-2 to exclude non-viable cells from the analysis (for all FC assays except the viability assay).

Statistical analysis

For each experiment, PAM were obtained from the same batch and each experiment was independently performed for at least three times. Results are expressed as mean ± standard deviations (SD). Statistical data analysis was performed using the GraphPad PRISM software package (v 5.0, La Jolla, CA) using a paired t-test (all dose-dependent assays and MHC I and MHC II expression time-dependent assays) or a repeated measures ANOVA with a Bonferroni post test (all other time-dependent assays). P values < 0.05 were considered significantly different. Experimental set up in human and murine cell lines

Phagocytosis experiments are performed with human and murine monocytic cell lines (THP-1 and MM-1 , respectively) induced with IFN-alpha or transfected with a sialoadhesin-containing vector to express sialoadhesin or induced with PMA to differentiate towards macrophages. In addition, macrophages differentiated from human peripheral blood monocytes after treatment with IFN-alpha are used, as well as primary murine and human alveolar macrophages isolated from broncho-alveolar lavage fluid or resected lung tissue.

Macrophages and THP-1 cells are cultured at 37°C and 5% C0₂ in RPMI 1640 medium complemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 1 mM sodium pyruvate and 50 nM beta-mercaptoethanol. MM-1 cells are cultured in RPMI complemented with FCS and P/S. Surface sialoadhesin expression is induced by adding 500 U/ml human or murine IFN-alpha (PBL Biomedical Laboratories, Piscataway, NJ to the culture medium for 2 to 3 days. Differentiation of THP-1 and MM-1 to macrophages is induced by the addition of 200 nM PMA (Phorbol 12-myristate 13-acetate; Sigma) to the culture medium for 2 to 3 days. After induction, cells are washed once with fresh medium and cultured one day further in suspension (at 1 E6 cells/ml) in siliconized glass recipients. Cells are then stimulated with antibodies or nanobodies at a concentration of 5 μg/ml and further incubated at 37°C, 5% C0₂. The following antibodies are used for human Sn: mAb 7D2 (Abeam), and nanobodies raised against human sialoadhesin. For mouse Sn: mAb 3D6.1 12, mAb MOMA-1 (both AbD Serotec) and mAb Ser4 (from dr. P. Crocker) raised against murine sialoadhesin. Negative controls include PBS, lgG1 and lgG2a isotype control antibodies and non-Sn binding nanobodies. The phagocytosis experiment is performed 24 h later as follows: green fluorescent polystyrene beads (1 μΜ diameter; Molecular Probes) are added to the cells (20 beads/cell) and cells are further incubated at 37°C to allow phagocytosis. After 1 h, cells are cooled on ice for 15 min, washed twice with cold PBS and analysed by flow cytometry to detect phagocytosis of fluorescent beads. Propidium iodide or 7- AAD is used to discriminate live from dead cells. The percentage of live cells associated with beads is determined from the FL-1 fluorescence signal. Effective internalization of the beads is confirmed by confocal microscopy.

Example 1 : Sn cross-linking has no effect on macrophage viability

To assess the effect of Sn cross-linking on macrophage viability, flow cytometry was performed upon live-dead staining of mAb 41 D3 treated macrophages using PI. As a control, macrophages were treated with irrelevant, isotype-matched mAb 13D12 or PBS (negative control). Viability was evaluated in response to different doses (0, 0.15, 0.5, 1.5, 5, 15, 50 μg/ml; Figure 1A) or at different times post start of the treatment (6, 12, 24, 48, 72 hours; Figure 1 B). No differences in macrophage viability were detected for mAb 41 D3 treated macrophages versus isotype or negative control treated groups in response to all studied concentrations. When no mAb was added, viability was 92.9 ± 1.1 % (data not shown). In addition, treatment of macrophages with the highest mAb dose did not have any cytotoxic effect up to 72 hours after starting the treatment. Overall, cell viability was in the range of 80.8 - 96.3 % with the average being 92.4 %.

Example 2: Effect of mAb 41 D3 treatment on reactive oxygen intermediate (ROI) production

An important effector function of macrophages in the innate immune response is the production of toxic mediators, such as reactive oxygen intermediates, upon encounter of pathogens. To determine the effect of Sn cross-linking on ROI production by PAM, a lucigenin CL assay was performed. As a control, macrophages were treated with mAb 13D12 (isotype control) or HBSS (negative control). The analysis was done in response to different doses (0, 0.15, 0.5, 1.5, 5, 15, 50 μg/ml; Figure 2A) or at different times post start of the treatment (6, 12, 24, 48, 72 hours; Figure 2B). After appropriate treatment, macrophages were triggered to produce ROI with 10 μg/ml PMA and ROI production was quantified using lucigenin CL. No treatment-related differences in ROI production could be seen for all mAb doses tested (Figure 2A). Though, when looking at the time-dependent curve (Figure 2B), it was observed that the isotype control treatment tended to cause an increased ROI production by PAM compared to the negative control, whereas mAb 41 D3 treated PAM tended to have an equal or lesser ROI production compared to the negative control. At 24 (p<0.02) and 72 (p<0.01) hours post treatment, the observed increase in ROI production was statistically significant compared to both mAb 41 D3 and negative control treated cells. It can be concluded that Sn cross-linking has no effect on ROI production, yet, treatment with the isotype-matched control mAb tends to cause an increase in ROI production.

Example 3: Sn treatment of macrophages significantly decreases macrophage phagocytosis

Another important effector function of macrophages in the innate immune response is the phagocytosis of pathogens, infected cells, effete or dead cells and particulate or soluble antigens. To determine whether Sn cross-linking has the ability to alter macrophage phagocytosis of particulate antigens, the effect of mAb 41 D3 treatment on PAM phagocytosis of beads was assessed by flow cytometry. As a control, macrophages were treated with mAb 13D12 (isotype control) or PBS (negative control). When the analysis was done in response to different mAb doses (0, 0.15, 0.5, 1.5, 5, 15, 50 μg/ml; Figure 3A&B) it was observed that with increasing mAb 41 D3 dose, phagocytosis decreased markedly compared to the isotype control and PBS treated macrophages (0 μg/ml; negative control), which had an association level of 63.5 ± 5.5 % (data not shown). Starting from 1.5 μg/ml, this difference was statistically significant (p<0.003). The highest measured dose, 50 μg/ml, yielded 26.1 % PAM that were associated with fluorescent beads, whereas the isotype control maintained the same level association at all doses tested (67.6 ± 4.4 %). Thus, mAb 41 D3 has a dose-dependent effect on the number of macrophages phagocytosing beads. As a measure for the number of beads that are associated with each macrophage, the MFI of the macrophages under study was also analysed (Figure3B). When looking at the effect of the treatment dose, the MFI tended to decrease a little for mAb 41 D3 treated macrophages compared to the isotype control and PBS treated macrophages (14648 ± 2128; data not shown). This indicates that there is a small, yet unsignificant, dose-dependent difference in the number of beads associated after Sn cross- linking.

When performing the analysis at different times (6, 12, 24, 48, 72 hours) post start of the treatment with the highest mAb dose, 50 μg/ml, the same decrease in phagocytosis upon treatment with mAb 41 D3 was seen compared to both controls (Figure 3C). At all timepoints, the difference in phagocytosis between mAb 41 D3 treated cells and controls was statistically significant (p<0.01) and was in the range of 33.9 - 52.4 %, with an average difference of 43.4 %. To assess whether this effect was also observed shortly after the start of the mAb treatment, the assay was repeated allowing only one hour of mAb treatment. A similar decrease in phagocytosis was observed (data not shown). Over time, a slight increase in phagocytosis by both control groups, but not by the mAb 41 D3 treated group, was observed. Similarly, when looking at the MFI of macrophages that were associated with fluorescent beads, it was observed that the MFI of mAb 41 D3 treated cells did not change much over all timepoints, whereas the MFI of controls increased over time (Figure 3D). Six hours after the start of the antibody treatment, MFI levels were similar for mAb 41 D3 or control treated PAM. From then on, the MFI of control cells went up, whereas it remained the same for mAb 41 D3 treated cells. Starting from 48 hours of mAb 41 D3 treatment, this difference became significant (p<0.001). Based on these results it can be concluded that mAb 41 D3 has an effect on macrophage phagocytosis that is maintained over time. Not only are there less cells associated with beads at all timepoints in response to mAb 41 D3 treatment, there is also a decreased number of beads that are associated per cell, starting from 48 hours post treatment.

Keeping previous results in mind, we wondered whether the negative effect of mAb 41 D3 treatment on macrophage phagocytosis would also be maintained when the mAb was washed away after 1 hour of treatment. Similar results were obtained as discussed above (Figure 3E). Based on these observations it can be concluded that mAb 41 D3 causes a decrease in macrophage phagocytosis that is maintained over time, even if the mAb is washed away after one hour of treatment, after which it is no longer present in the culture medium.

Since the flow cytometric analysis did not discriminate between beads that are ingested by macrophages and those that are bound to the plasma membrane, the effect of mAb 41 D3 treatment on PAM phagocytosis was assessed using confocal microscopy. The number of macrophages that associated with beads and the number of beads that were internalized or that were present on the cell surface was counted. In this way we hoped to find out whether the observed decrease in bead association was due to a decrease in cell surface binding and/or due to a decrease in phagocytosis of beads. The analysis was done in response to 50 μg/ml mAb 41 D3 or isotype control mAb, 24 hours post start of the treatment. As a control, the phagocytosis assay was performed with untreated macrophages that were kept on ice at all times, to prevent internalization of the beads. The previously observed decrease in phagocytosis of macrophages upon mAb 41 D3 treatment was confirmed (Figure 3G), as the number of macrophages associated with beads decreased from 69.5 ± 3.1 to 34.4 ± 4.1 upon Sn cross-linking. This was mainly due to a reduction in internalized beads, since the number of internalized beads per 50 macrophages decreased from 61 ± 5 to 29 ± 14 upon mAb 41 D3 treatment, whereas the number of surface bound beads decreased from 31 ± 7 to 22 ± 5 (Figure 3H). These observations also show that internalization was not fully blocked upon mAb 41 D3 treatment. The number of beads bound to the cell surface upon Sn cross-linking (22 ± 5) was higher than the number of beads associated when the phagocytosis experiment was performed at 4 °C (10 ± 4), which corresponds to the background surface binding. In addtion, there was no mAb treatment-related difference in the number of beads bound to the cell surface. Thus, the observed decrease in bead association is not due to a decreased capacity of macrophages to bind beads on their cell surface, yet results from a decreased capacity to internalize beads.

Example 4: Cross-linking Sn has no effect on the uptake and processing of soluble antigen

Another important characteristic of macrophages is their ability to take up soluble and particulate antigens by different mechanisms. Uptake of these antigens is not only important in steady state and innate host defense, it is also an important factor related to the antigen- presenting capacity of a macrophage, since uptake of antigens often results in processing and antigen presentation at the cell surface. To test the effect of Sn cross-linking on uptake and processing of soluble antigen by PAM, these cells were incubated with the self-quenching protein conjugate OVA-DQ. OVA is a well characterized substrate for proteases that is generally used as a probe for antigen processing and presentation (Rock et al., 1994). It has been demonstrated that OVA is internalized via mannose receptors (Janicka et al., 1994), and that OVA-DQ acquires fluorescence after dequenching upon proteolytic degradation, permitting the analyses of both antigen uptake and processing (Santambroggio et al., 2000). To determine the effect of mAb 41 D3 treatment of PAM on soluble antigen uptake and processing, the number and MFI of cells that took up and processed OVA-DQ was quantified by flow cytometry. The evaluation was done in response to different doses (0, 0.15, 0.5, 1.5, 5, 15, 50 μg/ml; Figure 4A) or at different times post start of the treatment (6, 12, 24, 48, 72 hours; Figure 4B). As a control, macrophages were treated with mAb 13D12 (isotype control) or PBS (negative control). No difference in the number of PAM that took up and processed OVA-DQ was detected under all treatment conditions (Figure 4A&B). Overall, the percentage of PAM that took up and processed OVA-DQ was 99.2 % ± 0.5 %. When looking at the MFI of the macrophages that have taken up and are processing OVA-DQ, there was no dose-, nor time-dependent effect of 41 D3 treatment on MFI (Figure 4C&D). It can be noted that there is a slight increase in MFI over time (Figure 4D), for all treatment conditions evaluated. When no mAb was added, the MFI of cells was 13236 ± 1597 (data not shown). The results show that mAb 41 D3 treatment has no effect on the number of macrophages that take up and process soluble antigen, as well as on the amount of antigen processed.

Example 5: 41 D3 treatment of macrophages has no effect on MHC I and MHC II surface expression

MHC molecule expression on the cell surface of macrophages is yet another important factor associated with the antigen-presenting capacity of a macrophage. To test the effect of Sn cross-linking on the expression of MHC I and MHC II molecules on the PAM cell surface, cells were immunostained for MHC I and MHC II surface expression upon appropriate treatment with mAb 41 D3, and the number and MFI of cells expressing MHC I and MHC II molecules on their surface was quantified using flow cytometry, analysing live macrophages only. The assessment was done in response to different doses (0, 0.15, 0.5, 1.5, 5, 15, 50 μg/ml; Figure 5A&B) or at different times post start of the treatment (6, 12, 24, 48, 72 hours; Figure 5C&D). As a control, macrophages were treated with mAb 13D12 (isotype control). No differences in the number of PAM expressing MHC I and MHC II at their cell surface was seen under all treatment conditions (data not shown). In total, the percentage of PAM that expressed MHC I or MHC II molecules on their cell surface was 99.8 ± 0.3 % and 97.8 ± 1.8 % respectively. When looking at the MFI of the macrophages that expressed MHC I or MHC II at their cell surface (Figure 5), we observed no treatment-related differences in MHC I or MHC II surface expression. When no mAb was added (0 μg/ml mAb dose; negative control), the MFI of the macrophages that expressed MHC I or MHC II on their cell surface was 1330 ± 249 for MHC I and 469 ± 37 for MHC II (data not shown). For MHC I, a decrease in MFI over time was observed, for all treatment conditions evaluated (Figure 5B). These data indicate that cross- linking Sn has no effect on the expression of MHC I and MHC II on the cell surface. Example 6: Effect of Sn cross-linking on cytokine and chemokine expression

The ability of macrophages to produce cytokines and chemokines is of great importance in innate as well as adaptive immunity. To determine the effect of mAb 41 D3 treatment on cytokine expression by PAM, macrophages were treated for 0, 6, 12 or 24 hours with 50 μg/ml mAb 41 D3 or isotype-matched control mAb 13D12 and secreted cytokine were assessed by a bioassay (for IFN-a) and a multiplex ELISA (for ΙΙ_-1 β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IFN-γ and TNF-a). The residual endotoxin amount in the antibody solutions was 0.046 EU/ml for mAb 41 D3 and 0.039 EU/ml for mAb 13D12, as determined by a LAL-test. As a control for cytokine induction by residual endotoxins present in the antibody solutions, PAM were stimulated with 0.1 EU/ml for 12 hours. As a positive control, PAM were stimulated with 1 μg/ml LPS for 12 hours to trigger cytokine production. None of the conditions tested induced IFN-a production (data not shown). PAM treated with 1 μg/ml LPS for 12 hours showed an IFN-a level that was below the detection limit two out of three times. The third time, the IFN-a level was 23 U/ml, barely above the detection limit of 20 U/ml.

IL-Ιβ, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IFN-γ, TNF-a Multiplex ELISA - At the start of the treatment (t = 0) cytokine levels were below the limit of detection (LOD) for all conditions tested (Figure 6A), except for TNF-a, which was detected, barely above the detection limit, one out of three times. All assayed cytokines could be detected in the cell culture supernatant upon treatment at all other timepoints, except for IL-2, where the level was below the detection limit for all but 1 condition. This is not unexpected, since IL-2 is known to be produced mainly by activated CD4⁺ T lymphocytes (Malek and Castro, 2010). No mAb treatment-related differences in cytokine production were observed at all timepoints, apart from IL-4 where a marginal but significant decrease was observed at 6 hours post mAb 41 D3 treatment, compared to the isotype-matched control treatment. In addition, there was a small but significant increase in chemokine IL-8 production 24 hours post treatment. Expression levels of cytokines IL-4, IL-10, IL-12p70, IFN-γ, TNF-a and chemokine IL-8 were just above the detection limit at all times (Figure 6B), whereas I L-1 β and IL-6 were strongly induced. When looking at the expression of cytokines IL-4, IL-6, IL-10, IL-12p70, IFN-γ and TNF-a or chemokine IL-8 in function of time, longer treatments led to higher cytokine levels. When comparing the effect of background endotoxin levels (0.1 EU/ml) with the effect of both mAb treatments, no differences could be found for I L- 1 β , IL-2 and IFN-γ (Figure 6B). A slightly lower production was observed for IL-4, IL-6, IL-8, IL-12p70 and TNF-a, whereas IL-10 production was slightly upregulated. However, none of these differences were significant. The stimulatory capacity of LPS on PAM was significantly evident when looking at the induction of cytokine production by 1 μg/vΓ^\ LPS (Figure 6B). It was observed that IL-Ιβ (> 1.4 fold), IL-2 (> 1.4 fold) and IL-4 (> 1.5 fold) production was weakly induced and IL-12p70 (> 3.3 fold), IL-8 (> 3.4 fold) and IL-10 (> 5.9 fold) production was mildly induced, whereas IFN-γ (> 19.8 fold), IL-6 (> 25 fold) and TNF-a (> 299 fold) production was strongly induced in response to LPS stimulation at a concentration of 1 μg/ml, compared to the background control (0.1 EU/ml). This induction was statistically significant (p<0.05) for IL-4, IL-8, IL-10, IL-12p70, IL-6, IFN-γ and TNF-a. Example 7: Inhibition of unwanted phagocytosis of apoptotic bodies

Animals and cell lines

C57BL6, OT1 and OT2 mice are purchased from The Jackson Laboratory (Maine, USA) and housed in a pathogen-free animal facility at Ugent. A549, LL/2, U937, THP-1 , RAW264.7, J774 are all purchased from ATCC. THP-1 and U937 are transfected with a huSn-expressing vector using retroviral infection. RAW264.7 and J774 are transfected with a mSn-expressing vector using retroviral infection.

Primary murine macrophages are obtained by lung lavage and are maintained in culture in RMPI supplemented with 10% FCS. A549 and RAW264.7 are maintained in DMEM supplemented with 10% FCS, LL/2 is maintained in DMEM supplemented with 10% FCS and 10 mM sodium pyruvate, U937 and J774 are maintained in RPMI supplemented with 10% FCS, THP-1 wt and THP-1 huSn are maintained in RPMI supplemented with 10% FCS and 0.05 mM 2-mercaptoethanol. Differentiation of THP-1 and U937 is induced by treatment with 200 nM PMA for 72 hours.

Tumor model

C57BL6 mice are injected s.c. with 10E5 tumor cells or instillated with 10E6 tumor cells of an appropriate cancer cell line and/or tumor, depending on the type of tumor to be studied (melanoma, lung cancer, breast cancer, myeloma,... ), to induce tumor generation and growth. Tumor volume is monitored three times a week.

Alternatively, rats may be used as model for different tumor types.

Apoptotic body preparation and purification

Apoptosis is induced in in vitro cancer cell cultures (LL/2, A459 and OVA expressing cells), for example given by treatment with UV-B radiation (10 Gy), sodium butyrate (5 mM for 3 days), staurosporine (1 μΜ for 24 hours), chemotherapeutic agents (5 μΜ doxorubicin or 100 nM etoposide for 24 hours) or another apoptosis inducing agent. Light microscopy is used to identify shrinkage and blebbing during apoptosis. Apoptotic bodies are collected by centrifugation at 4000g for 10 min and stored at 4°C until use. Purified apoptotic bodies are characterized, eg. stained with annexin V-PE and 7-AAD and determined as annexin V-PE- positive/7-AAD -negative. For phagocytosis of apoptotic bodies, cancer cell cultures are labeled with a cell tracker dye such as CMFDA, CFSE, PKH26 (10 uM, Molecular Probes) according to the manufacturer's instructions prior to induction of apoptosis.

In vitro inhibition of phagocytosis ofAB by cross/inking Sn

To determine the effect of crosslinking Sn on phagocytosis of tumor apoptotic cells, phagocytosis of fluorescent beads or labeled AB in the absence or presence of anti- sialoadhesin is assessed by microscopy and flow cytometry. For microscopy, cells are cytospun on a microscope slide, fixed in cold acetone and counterstained with DAPI.

Samples are mounted with Fluorsave and imaged with a fluorescence microscope. Murine primary macrophages or macrophage cell lines THP-1 wt, THP-1 huSn, RAW264.7 and J774 are treated with PBS, a sialoadhesin-specific antibody (7D2, 3D6.112, MOMA-1 or Ser4) or a mock control antibody at different concentrations for 24 hours.

Differentiated THP-1 cells and Sn-transfected THP-1 cells treated with 5 μg/ml 7D2 show reduced phagocytosis of fluorescent beads compared to isotype-treated or nontreated macrophages (Figure 7). Results are confirmed by microscopy.

In vivo inhibition of AB uptake

To determine the anatomical distribution and clearance of apoptotic bodies by macrophages in vivo, mice (or rats) are injected subcutaneously or instillated with mixture of 10E7 fluorescent- labeled apoptotic bodies and either a sialoadhesin-specific antibody (ABmAb⁺) or a mock control antibody (ABmAb^") at different concentrations. Alternatively, the antibody is coadministered systemically (intraperitoneal). Distribution of injected AB is analyzed by immunofluorescence microscopy, confocal microscopy and flow cytometry. For microscopy, at different time points after administration, tissues are snap frozen and embedded in OCT before cryosectioning. 4 μηι cryosections are prepared and counterstained with DAPI, in combination with anti-F4/80, and/or CD3 and/or CD11 c antibodies (to localize macrophages, T-cell zones, dendritic cells resp). Stained sections are mounted with Fluorsave. For flow cytometry, draining lymph nodes are prelevated, teased apart with forceps and digested with 100U/ml collagenase type III and DNAse for 1 hour at 37°C to obtain single cell suspension. Cells are stained with a combination of markers for DCs (CD11 c, MHCII) and macrophages (F4/80, CD169). Cells subsets positive for the cell tracker are considered to have engulfed AB. Flow cytometry is used to confirm that AB are engulfed by Sn+ macrophages.

The location of AB in the T cell rich regions, the decrease of the number of double positive Sn+ macrophages and an increased number of CD1 1c+ cells that are positive for the cell tracker dye indicate that a sialoadhesin-specific antibody blocks clearance of AB by macrophages in the lymph nodes and induces increased uptake of AB by DCs.

Impact on tumor specific immune response

Method 1

To measure the effect of anti-sialoadhesin on antigen specific T cell activation and acquisition of effector function. IFN gamma production is assayed in supernatant of antigen stimulated, primed lymph node cells. To this end, C57BL6 mice are injected sc with a mixture of OVA- expressing tumor AB and either a sialoadhesin-specific antibody (ABmAb⁺) or a mock control antibody (ABmAb^") at different concentrations. Again, the antibody can be administered with the apoptotic bodies, or systemically (i.p.). Apoptosis in OVA-expressing cells is induced by treatment with chemotherapeutic agents as described above.

Draining lymph node cells from OVA-expressing tumor bearing mice are harvested 14 days after tumor cell injection. Lymph node cells are restimulated in vitro with 50 μg/ml OVA in the culture medium. Secretion of IFN-gamma in the supernatant is assayed by standard cytokine ELISA (Ready-Set-Go, eBioscience) after an overnight culture.

An increased IFN-gamma secretion following OVA-stimulation in the presence of SN-specific antibodies compared to the presence of mock control antibodies indicates a positive effect of anti-sialoadhesin on T cell inflammatory activation.

Method 2

To assess proliferation of OVA-specific CD8+ and CD4+ T cells, OT-I and OT-II cells are labeled with 10 μΜ 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and 5x10E6 cells are injected i.v. into a lateral tail vein. 24 hours later, the mice are s.c. injected with a mixture of chemo-induced AB from 10E6 OVA-expressing tumor cells and either 5 ug/ml sialoadhesin-specific antibody (ABmAb⁺) or mock control antibody (AbmAb). For proliferation, draining lymph nodes cells are collected 5 days after T cell transfer, stained with anti-CD8 or anti-CD4 mAb, and T cell proliferation is analyzed by flow cytometry. An increase of OT-I and OT-II proliferation in the presence of sialoadhesin specific antibodies indicates a positive effect on the tumor specific immune response. Method 3

A similar IFN-gamma production assay is performed on supernatant of mediastinal lymph node cells harvested from mice instillated with a mixture of 5x10E6 OVA-expressing apoptotic tumor cells and either 5 μg/ml sialoadhesin-specific antibody (ABmAb⁺) or mock control antibody (AbmAb).

Higher levels of IFN-gamma in supernatant of OVA-restimulated primed lymph node cells isolated from mice injected with AB and a sialoadhesin specific antibody as compared to supernatant from mice injected with a mixture of AB and mock antibody suggest that T cell activation is more efficient after vaccination with AB in the presence of sialoadhesin-specific antibodies.

Method 4

Proliferation of OTI and OTII cells in response to vaccination with 5x10E6 OVA-expressing tumor cells in the presence of either 5 μg/ml sialoadhesin-specific antibody (ABmAb⁺) or mock control antibody (AbmAb) is also assessed in mediastinal lymph node harvested from mice instillated with OVA-expressing apoptotic tumor cells.

An increased proliferation of both OTI and OTII cells in the mediastinal lymph nodes in mice that received sialoadhesin-specific antibodies as compared to proliferation measured in lymph node cells from mice that received mock control antibody indicates a positive effect on the tumor specific immune response.

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Claims

1. A sialoadhesin (Sn) binding antibody for inhibiting phagocytosis in Sn expressing cells, in particular macrophages and inflammatory monocytes.

2. The sialoadhesin binding antibody according to claim 1 , for use in inhibiting phagocytosis of apoptotic bodies, in particular tumor derived apoptotic bodies.

3. The sialoadhesin binding antibody according to claim 2, wherein the apoptotic bodies are administered to the subject or wherein the apoptotic bodies are induced by chemotherapy or radiotherapy in the subject.

4. The sialoadhesin binding antibody according to claim 1 , wherein the phagocytosis is uncontrolled.

5. The sialoadhesin binding antibody according to claim 4, wherein the uncontrolled phagocytosis comprises hemophagocytosis.

6. The sialoadhesin binding antibody according to claim 4, wherein the hemophagocytosis comprises phagocytosis of erythrocytes, leukocytes, platelets and/or lymphocytes.

7. The sialoadhesin binding antibody according to any one of claims 1 to 6, wherein said antibody is the monoclonal antibody 41 D3, or a humanized derivative thereof, or the monoclonal antibody 7D2.

8. The sialoadhesin binding antibody according to any one of claims 1 to 7, for use in treating a disease.

9. The sialoadhesin binding antibody according to claim 8, wherein said disease is Hemophagocytic lymphohistiocytosis, Macrophage activation syndrome, cancer, immune thrombocytopenia, or acute liver failure.

10. The sialoadhesin binding antibody according to claim 9, wherein cancer is a solid tumor cancer.

1 1. A pharmaceutical composition comprising a sialoadhesin binding antibody and a pharmaceutically acceptable carrier, excipient and/or additive.

12. The pharmaceutical composition according to claim 1 1 , wherein said sialoadhesin binding antibody is the monoclonal antibody 41 D3, or a humanized version thereof, or the monoclonal antibody 7D2.

13. A pharmaceutical composition according to claims 1 1 or 12, further comprising a chemotherapeutic agent, or apoptotic bodies optionally in combination with an adjuvant.