WO2013088136A1 - Ligand for dngr-1 receptor - Google Patents

Abstract

F-actin is identified as a ligand for DNGR-1. The invention consequently provides materials and methods for the regulation of the immune system by blocking or otherwise exploiting the interaction between DNGR-1 and F-actin, e.g. for modulation of immune responses and targeting of antigens to antigen presenting cells. Also provided are methods for identifying agents capable of modulating the interaction between DNGR- and F-actin.

Description

Ligand for DNGR-1 receptor

Field of the Invention

The invention relates to the regulation of the immune system, and in particular to methods of modulating immune responses via the DNGR-1 receptor.

Background to the Invention

Recognition of pathogen associated molecular patterns (PAMPs) by innate immune receptors triggers inflammation and, when coupled to antigen encounter, favours the initiation of adaptive immunity by dendritic cells (DC) (Iwasaki and

Medzhitov, 2010) . However, inflammation can also be driven by sterile injury to tissues in the presumed absence of PAMPs (Chen and Nunez, 2010; Kono and Rock, 2008; Rock and Kono, 2008; Rock et al . , 2010) . In this case, damaged cells may act as a source of sterile pro-inflammatory signals, which, by analogy to PAMPs, have been termed "damage associated molecular patterns" (DAMPs) (Seong and Matzinger, 2004). Many DAMPs are thought to be intracellular components that play a housekeeping role in healthy cells but are leaked or exposed by damaged cells, most notably after loss of membrane integrity associated with primary or secondary necrosis (Chen and Nunez, 2010; Kono and Rock, 2008; Rock and Kono, 2008; Rock et al . , 2010) . Exposure of DAMPs by dead or damaged cells in most instances appears designed to initiate a controlled inflammatory response that induces tissue repair (Chen and Nunez, 2010; Rock et al., 2010) . However, in some cases, DAMPs might substitute for PAMPs in activating DC and inducing adaptive immunity to dead cell-associated antigens (Matzinger, 1994) . In this scenario, DAMPs would act as an alternative to PAMPs and help provide an explanation for PAMP-independent immunity such as observed in response to tumors or to allografts (Matzinger, 1994, 2002) . In addition, DAMPs could also act in concert with PAMPs during infection, serving as a sign of pathogen-induced damage to tissues and thereby providing the immune system with information about pathogenicity (Lazzaro and Rolff, 2011; Vance et al., 2009). Finally, acute DAMP release after trauma can result in a life- threatening sepsis-like syndrome while persistent innate immune stimulation by DAMPs might underlie chronic

inflammation and contribute to diseases such as cancer, atherosclerosis, type II diabetes and neurodegeneration (Chen and Nunez, 2010; Rock et al., 2010) . Therefore, much recent interest has revolved around defining the universe of DAMPs and DAMP receptors .

One approach to identify DAMPs has been to validate putative candidates in immunological assays. DAMPs identified in this manner include mitochondrial formyl peptides, cellular RNA and DNA, uric acid, heat shock proteins, HMGB1, calreticulin and others (Chen and Nunez, 2010; Rock et al . , 2010). This approach can sometimes be marred by issues such as PAMP contamination of the putative DAMP and is not necessarily accompanied by identification of the DAMP receptors. An alternative approach is to identify the latter and then search for their DAMP ligands, much as was used to identify many of the PAMPs recognized by toll-like receptors (TLRs) (Uematsu and Akira, 2008) . Such an approach has led to the recent identification of nuclear protein SAP130 as a DAMP recognized by Mincle on necrotic cells (Yamasaki et al., 2008) .

We have recently identified DNGR-1, also known as CLEC9A, as an innate immune receptor for a DAMP that is exposed when cells die (Sancho et al., 2009). Like Mincle, DNGR-1 is a member of the C-type lectin superfamily and its C-type lectin- like domain (CTLD) can be used as a soluble probe to detect ligand in cell staining experiments (Sancho et al., 2009) . Such experiments revealed that the ligand for DNGR-1 is a pre^¬ formed component of healthy cells that can be revealed independently of cell death by cell fixation and

permeabilization (Sancho et al . , 2009) . Further

experimentation showed that the ligand is protease sensitive, as well as acid and heat labile, and present throughout the cytoplasm but excluded from the nucleus (Sancho et al . , 2009) . Interestingly, expression of DNGR-1 is highly restricted to a sub-type of cross-presenting DC in both mouse and human suggesting that the receptor may play a prominent role in regulation of CD8⁺ T cell responses (Caminschi et al . , 2008; Huysamen et al . , 2008; Poulin et al., 2010; Sancho et al . , 2008). Consistent with that possibility, the receptor is non- redundant in mice for efficient cross-priming of cytotoxic T- cells against dead cell associated antigens, making it the first reported innate immune receptor to bridge DAMP sensing to the induction of T cell immunity (Sancho et al., 2009). However, further dissection of the role of DNGR-1 in DC function and in regulation of immune responses has been hampered by the failure to identify the DNGR-1 ligand.

Summary of the Invention

The present inventors have now identified F-actin as a ligand for DNGR-1. This finding enables the interaction between DNGR-1 and its ligand to be blocked, which may find use in the modulation of immune responses.

The invention therefore provides a method of inhibiting (i.e. reducing or preventing) interaction between F-actin and DNGR- 1, comprising contacting F-actin with a binding agent capable of binding to F-actin and inhibiting (i.e. reducing or preventing) interaction between F-actin and DNGR-1. The binding agent may therefore be regarded as an antagonist of the interaction between F-actin and DNGR-1.

Such methods may be applied in vitro or in vivo.

When applied in vivo, they may be used to inhibit (i.e. reduce or prevent) an undesirable immune response in a subject. Thus the invention further provides a method of inhibiting an immune response in a subject comprising administering to the subject a binding agent capable of binding to F-actin and inhibiting (i.e. reducing or preventing) interaction between F-actin and DNGR-1.

The invention further provides a binding agent capable of binding to F-actin and inhibiting interaction between F-actin and DNGR-1 for use in a method of medical treatment, for example in the inhibition of an immune response.

The invention further provides the use of a binding agent capable of binding to F-actin and inhibiting interaction between F-actin and DNGR-1 in the preparation of a medicament for the inhibition of an immune response.

Antagonists of the interaction between F-actin and DNGR-1 may be particularly useful in the treatment of inflammation (e.g. acute or chronic inflammation) , and diseases characterised by inflammation, undesirable T cell activity (e.g. undesirable CTL activity), and/or high levels of cell death. These include inflammatory conditions and autoimmune diseases. Some autoimmune diseases are associated with defects in clearance of apoptotic cells, which undergo secondary necrotic death. It is believed that immune responses against self antigens associated with these cells may contribute to the pathogenesis of these conditions, and that cells expressing DNGR-1 may be implicated in the generation of such responses by binding to a ligand exposed in dead and dying cells (especially those undergoing primary or secondary necrotic cell death) . Thus antagonists of the interaction between DNGR-1 and F-actin may be used to prevent activation of DNGR-1 and may thus inhibit or prevent stimulation of immune responses against antigens associated with these cells. Consequently they may be used for the prophylaxis or treatment of conditions characterised by an undesirable immune response against such cell-associated antigens .

Thus the subject to whom the binding agent or antagonist is administered may be suffering from, or at risk of, inflammation or an inflammatory or autoimmune condition, especially a condition characterised by undesirable T cell activity, e.g. CTL activity, and/or a condition characterised by high levels of cell death. Such conditions include:

- inflammation (e.g. acute or chronic inflammation);

- autoimmune diseases, including rheumatoid arthritis and other types of chronic or acute arthritis or arthropathies with an immune component, systemic lupus erythematosus (which is known to involve particularly high levels of cell death), scleroderma, Sjogren syndrome, autoimmune (particularly Type I) diabetes, thyroiditis, and other organ-specific immune diseases, including psoriasis;

- neurologic diseases, including multiple sclerosis,

myasthenia gravis, and other neurologic immune-mediated diseases. Also included are gastrointestinal diseases, including Crohn's disease, colitis, celiac disease and hepatitis;

- cardiovascular diseases, which are now recognised to have a significant immune-mediated component, including

atherosclerosis, cardiomyopathy, rheumatic fever,

endocarditis, vasculitis, and other immune-mediated

cardiovascular diseases;

- immune-mediated respiratory diseases, including emphysema, respiratory airways infections, chronic obstructive pulmonary disease and other immune-mediated respiratory diseases;

- allergic processes and hypersensitivity reactions (type I, II, III, and IV), including asthma, rhinitis, and other immune-mediated hypersensitivity reactions;

- transplant or graft rejection and graft versus host disease, as occurs during or subsequent to, for example, organ transplant, tissue graft, blood transfusion, bone marrow transplant ;

- immunopathological responses to infectious agents, including septic shock syndromes;

- degenerative processes, such as neurodegenerative processes, that implicate immune competent cells such as microglia. The binding agent may be an antibody specific for F-actin, e.g. an antibody capable of binding to F-actin but without substantial affinity for other forms of actin, such as G- actin. Such an antibody can be regarded as a blocking antibody, i.e. an antibody capable of binding to F-actin and blocking or inhibiting its interaction with DNGR-1.

Particularly when used in vivo, it may be desirable that the binding agent is not capable of binding to Fc receptors.

Thus, when the binding agent is an antibody, it may be desirable that it does not comprise an Fc region capable of binding to Fc receptors. This may reduce the risk of inadvertent activation of DNGR-1 by co-localisation of DNGR-1 and F-actin at the surface of an antigen presenting cell which expresses both DNGR-1 and Fc receptors.

Alternatives to antibodies are increasingly available. So- called "affinity proteins" or "engineered protein scaffolds" can routinely be tailored for affinity against a particular target. They are typically based on a non-immunoglobulin scaffold protein with a conformationally stable or rigid core, which has been modified to have affinity for the target. Such molecules are clearly envisaged for use as binding agents in the present invention.

Other types of binding agent capable of binding specifically to F-actin may also be used, such as nucleic acids, (e.g. aptamers), carbohydrates (e.g. oligo- or polysaccharide), small molecules, etc..

The finding that F-actin is a physiological ligand for DNGR-1 also makes available new methods for targeting antigens to antigen presenting cells (e.g. DCs) which express DNGR-1.

Thus, in a further aspect, the invention provides a method for targeting an antigen to an antigen presenting cell, comprising contacting the antigen presenting cell with a composition comprising the antigen, wherein the antigen is associated with F-actin, and wherein the antigen presenting cell expresses DNGR-1. By "associated" is meant "physically associated" as explained in more detail below.

The method may be applied in vitro or in vivo. The antigen presenting cell will typically be a DC, and preferably is capable of cross-presenting extracellular antigen via MHC class I molecules. By "extracellular" is meant that the antigen has been taken up by the cell from its extracellular environment, typically by endocytosis or phagocytosis. Such antigens may also be referred to as "exogenous" antigens. It will be appreciated that the antigen presenting cell (e.g. DC) may also present such antigens via MHC class II molecules.

The method may further comprise contacting the antigen presenting cell with an adjuvant, as described in more detail below . As explained elsewhere in this specification, F-actin may have the ability to induce DNGR-1 signalling, and so may be useful to stimulate or enhance an immune response against a target antigen, especially to promote cross-presentation of the target antigen to CD8+ T cells by DNGR-1 + APCs. Thus the invention further provides a method for stimulating an immune response against a target antigen in a subject, comprising administering F-actin and the target antigen to the subject. The F-actin and the target antigen may be provided in the same composition, or may be in separate compositions to be administered together or separately. When present in the same composition, the F-actin and the target antigen may be physically associated.

In the above methods, it may be desirable that an adjuvant is administered in conjunction with the antigen and F-actin, as described in more detail below.

The above methods may comprise a single administration, or a sequence of two or more administrations separated by suitably- determined intervals of time. For example, the method may comprise a priming step (i.e. a first administration) followed by one or more boosting steps (a subsequent administration or administrations). For example, a first administration and second administration may be separated by one or more days, one or more weeks, or one or more months, preferably between two weeks and one month. Subsequent administrations may be provided after one or more weeks or months .

Immune responses stimulated via DNGR-1 targeting involve activation of T cells, which may be CD8+ T cells or CD4+ T cells. Activation may, but need not, be accompanied by proliferation, depending on the particular response. Antigen presenting cells (and in particular dendritic cells)

expressing DNGR-1 can induce activation, proliferation and effector differentiation of both CD8+ T cells and CD4+ T cells, and may stimulate activation of both types of T cell in any given immune response.

Under certain conditions, it is believed that DC are capable of stimulating regulatory T cell (Treg) activation and/or proliferation, and/or differentiating naive CD4+ T cells into Treg cells. Treg cells are characterised by the expression of the Foxp3 (Forkhead box p3 ) transcription factor. Most Treg cells are CD4+ and CD25+, and can be regarded as a subset of CD4+ T cells (although a small population may be CD8+) . Thus the immune response which is to be stimulated by a method of the invention may comprise inducing Treg cells in response to an antigen. Thus the method may comprise administering to the subject a composition comprising the antigen, wherein the antigen is associated with F-actin. The antigen may be administered with an adjuvant which promotes proliferation of Treg cells and/or differentiation of naive CD4+ T cells into Treg cells .

Insofar as this method involves stimulating proliferation and/or differentiation of Treg cells in response to a specific antigen, it can be considered to be a method of stimulating an immune response. However, given that Treg cells may be capable of modulating the response of other cells of the immune system against an antigen in other ways, e.g.

inhibiting or suppressing their activity, the effect on the immune system as a whole may be to modulate (e.g. suppress or inhibit) the response against that antigen. Thus the methods of this aspect of the invention can equally be regarded as methods of modulating (e.g. inhibiting or suppressing) an immune response against an antigen.

In practice, then, these methods of the invention may be used therapeutically or prophylactically to inhibit or suppress an undesirable immune response against a particular antigen, even in a subject with pre-existing immunity or an on-going immune response to that antigen. This may be particularly useful (for example) in the treatment of autoimmune disease.

Under certain conditions, it may also be possible to tolerise a subject against a particular antigen by targeting the antigen to an antigen presenting cell expressing DNGR-1. The invention thus provides a method for inducing tolerance in a subject towards an antigen, comprising administering to the subject a composition comprising the antigen, wherein the antigen is associated with F-actin, and wherein the antigen is administered in the absence of an adjuvant.

Tolerance in this context typically involves depletion of immune cells which would otherwise be capable of responding to that antigen, or inducing a lasting reduction in

responsiveness to an antigen in such immune cells. Induction of Treg cells can also be regarded as induction of tolerance, however .

The invention further provides a composition comprising an antigen, wherein the antigen is associated with F-actin. The composition may be a pharmaceutical composition, e.g. a vaccine, containing the antigen and its associated binding agent in combination with a pharmaceutically acceptable carrier. It may be formulated for any suitable route of administration, including but not limited to intravenous, intramuscular, intraperitoneal, nasal, subcutaneous,

intradermal, etc..

The invention further provides a composition comprising an antigen for use in a method of medical treatment, wherein the antigen is to be administered in conjunction with F-actin, e.g. wherein the composition further comprises F-actin, e.g. where the antigen is associated with F-actin.

Also provided is a composition comprising an antigen, for use in stimulating an immune response against the antigen, wherein the antigen is to be administered in conjunction with F-actin, e.g. wherein the composition further comprises F-actin, e.g. wherein the antigen is associated with F-actin.

Also provided is the use of a composition comprising an antigen in the preparation of a medicament for stimulating an immune response against the antigen, wherein the antigen is to be administered in conjunction with F-actin, e.g. wherein the composition further comprises F-actin, e.g. wherein the antigen is associated with F-actin.

The invention further provides a composition comprising F- actin for use in a method of medical treatment, wherein the F- actin is to be administered in conjunction with a target antigen, e.g. wherein the composition further comprises the target antigen, e.g. where the F-actin is associated with the target antigen.

Also provided is a composition comprising F-actin, for use in stimulating an immune response against a target antigen, wherein the F-actin is to be administered in conjunction with the target antigen, e.g. wherein the composition further comprises the target antigen, e.g. wherein the F-actin is associated with the target antigen.

Also provided is the use of a composition comprising F-actin in the preparation of a medicament for stimulating an immune response against a target antigen, wherein the F-actin is to be administered in conjunction with the target antigen, e.g. wherein the composition further comprises the target antigen, e.g. wherein the F-actin is associated with the target antigen .

Also provided is a therapeutic kit comprising a target antigen and F-actin.

DNGR-1⁺ dendritic cells may be implicated in at least Thl, Th2, and Thl7-type immune responses. Thus the methods of the invention may be applied to stimulation of various types of immune response against any antigen. However these cells are believed to be particularly important in the generation of CTL responses, so the immune response to be stimulated may be a CTL response. The method may comprise determining production and/or proliferation of CTLs, which are typically T cells expressing CD8 and are capable of cytotoxic activity against cells displaying their cognate antigen in the context of MHC class I molecules.

Nevertheless, targeting of antigen to DNGR-1+ dendritic cells can result in proliferation of CD4+ T cells as well as, or instead of, CTLs . Thus the method may additionally or alternatively comprise determining production and/or

proliferation of CD4+ T cells, which may be helper T cells (of Thl, Th2 or Thl7 type) or Treg cells. However, some Treg cells may not express CD4, e.g. CD8+ Treg cells.

It will therefore be understood that the methods and

compositions described above may be used for the prophylaxis and/or treatment of any condition in which it is desirable to induce a CTL response, such as cancer, or infection by an intracellular parasite or pathogen, such as a viral infection

It may be desirable also to administer further

immunostimulatory agents in order to achieve maximal CTL stimulation and proliferation, and/or stimulation and proliferation of other T cell types. These may include agents capable of activating dendritic cells and stimulating their ability to promote T cell activation. Such an agent may be referred to as an adjuvant. The adjuvant may comprise an agonist for CD40 (such as soluble CD40 ligand, or an agonist antibody specific for CD40), an agonist of CD28, CD27 or OX40 (e.g. an agonist antibody specific for one of those

molecules), a CTLA-4 antagonist (e.g. a blocking antibody specific for CTLA-4), and/or a Toll-like receptor (TLR) agonist, and/or any other agent capable of inducing dendritic cell activation. A TLR agonist is a substance which activates a Toll-like receptor. Preferably, the TLR agonist is an activator of TLR3, TLR4, TLR5, TLR7 or TLR8. A suitable TLR agonist is MPL (monophosphoryl lipid A), which binds TLR4. Other TLR agonists which may be used are LTA ( lipoteichoic acid, which binds TLR2 ; Poly I:C (polyinosine-polycytidylic acid), which binds TLR3 ; flagellin, which binds TLR5 ; resiquimod (R-848; 1- [ 4-amino-2- (ethoxymethyl ) imidazo [ 4, 5- c] quinolin-l-yl] -2-methylpropan-2-ol 1- [ 4-amino-2- (ethoxymethyl ) imidazo [4, 5-c] quinolin-l-yl] -2-methylpropan-2- ol ) or polyU RNA which bind TLR7 in mice and are believed to bind TLR8 in humans, and CpG (DNA CpG motifs), which binds

TLR9 ; or any other component which binds to and activates a TLR. For more details, see Reis e Sousa, Toll-like receptors and dendritic cells. Seminars in Immunology 16:27, 2004. When the dendritic cells are of human origin, TLR3 and/or TLR8 agonists may be particularly suitable.

Adjuvants which may not work via TLRs include 5' triphosphate RNA and β-glucans such as curdlan ( β-l, 3-glucan) . Pro^¬ inflammatory cytokines such as TNF-oi or IL-1 may also be used as adjuvants.

Without wishing to be bound by theory, it is believed that different adjuvants may affect the immune response in different ways, for example, by promoting stimulation and/or proliferation of different T cell types. Antigen presenting cells expressing DNGR-1 can stimulate both CD4+ T cells and CD8+ T cells, and the nature of the CD4+ response in

particular may be affected by the adjuvant used. Thus, despite F-actin possessing DNGR-1 agonist activity, it may still be desirable to administer an adjuvant in conjunction with F-actin, especially where it is desirable to influence the nature of the immune response obtained, e.g. the

particular T cell response to be generated. For example, use of poly I:C appears to favour generation of a

Thl-type CD4+ response. Curdlan appears to stimulate a Thl7- type CD4+ response.

Certain adjuvants promote stimulation of Treg cells. These include IL-2 and retinoic acid, and in particular all-trans retinoic acid (ATRA) , also known as trenitoin. Thus, when the immune response to be stimulated is a Treg response (which may in practice suppress responses of other components of the immune system against a particular antigen) it may be

appropriate to use a Treg-promoting adjuvant. It may also be possible to stimulate Treg cell activation or differentiation without administration of an adjuvant.

The compositions of the invention may be administered with or formulated for administration with the adjuvant, either sequentially or simultaneously, in the same or separate compositions. Thus the compositions of the invention may, but need not, comprise an adjuvant.

Without wishing to be bound by theory, and as explained above, it is believed that administration of the antigen in the absence of an adjuvant may result in the development of tolerance to the antigen. That is to say, the immune system is induced not to respond to future administrations of the same antigen. This may (but need not) involve the generation of Treg cells which are capable of active suppression of the response. Thus further administrations of an antigen to a subject who has been tolerised to that antigen should result in a lesser immune response than in a subject who is naive for that antigen (i.e. whose immune system has not previously been exposed to the antigen) . The magnitude of the immune response may be assessed by any appropriate criteria, such as

appearance of inflammation, swelling, cell proliferation (e.g. of Thl, Th2 or Thl7 CD4+ T cells, or CTLs) or inflammatory cytokine production (e.g. IL-1, IL-4, IL-12, IFN-gamma, TNF- alpha) . In certain embodiments, the tolerised individual will display substantially no immune response to that antigen.

In the above-described compositions and methods, the antigen is associated with F-actin, i.e. physically associated with F actin. Such association may be via covalent or non-covalent (e.g. electrostatic or van der Waals) interactions.

Preferably the antigen is covalently coupled to F-actin. For example, the antigen may be coupled to F-actin via a suitable coupling reagent. The skilled person is well aware of suitable methods and reagents which may be used for such coupling reactions.

Alternatively, the antigen may be part of the same peptide chain as a subunit of the F-actin, i.e. the actin subunit and the antigen form a fusion protein. The fusion protein may contain a linker sequence between the antigen and the actin subunit .

Alternatively, the antigen and F-actin may each be associated with a carrier such as a liposome or nanoparticle . For example, F-actin may be provided on the surface of a liposome which contains or is separately coupled to the antigen, or F- actin and the antigen may be provided in association with (e.g. coupled to) a nanoparticle.

The antigen is a peptide antigen. The term "peptide" refers to the nature of the antigen, i.e. that it is formed from amino acids linked by peptide bonds, and should not be taken to imply any particular size or length. Typically the peptide antigen will be at least 8 amino acids in length, and may be up to 30 amino acids in length, up to 50 amino acids in length, up to 100 amino acids, up to 200 amino acids, or even longer and may have residues coupled to the amino acids, such as glycan chains. For example, it may be 25 to 35 amino acids in length.

Without wishing to be bound by any particular theory, the peptide antigen should be capable of binding to a MHC class II or MHC Class I molecule, or should be capable of being processed within an antigen-presenting cell (such as a dendritic cell) to give rise to one or more peptides capable of binding to a MHC class II molecule or MHC Class I. It has recently been suggested that short epitope peptides of around

8 amino acids in length may induce less sustained CTL reactivity than longer peptides (e.g. around 30 amino acids in length) (Bijker, M.S. et al. J. Immunol. 179(8), 5033-5040 (2007) ) . MHC class I molecules typically bind peptides of 8 or 9 amino acids in length, while MHC class II molecules can bind peptides from 8 amino acids up to 20 amino acids, up 30 amino acids, or even longer.

The antigen is not F-actin. More generally, the antigen is not actin. That is to say, its sequence is not identical with a corresponding portion of an actin monomer, or an actin monomer endogenous to the individual to which the antigen is to be administered. For example, it may have less than 95% amino acid sequence identity with any portion of corresponding length of an endogenous actin sequence, e.g. less than 90%, less than 80%, less than 70%, less than 60% or less than 50% identity with any corresponding length of endogenous actin sequence and preferably even less. By "corresponding length" is meant a portion of the same length when optimally aligned and taking account of any insertions or deletions necessary to make the optimal alignment .

The antigen may be any protein or fragment thereof against which it is desirable to raise an immune response, in particular a CTL response, but also a Thl7 response or a Treg response. These may include antigens associated with, expressed by, displayed on, or secreted by cells against which it is desirable to stimulate a CTL response, including cancer cells and cells containing intracellular pathogens or parasites. For example, the antigen may be, or may comprise, an epitope peptide from a protein expressed by an

intracellular pathogen or parasite (such as a viral protein) or from a protein expressed by a cancer or tumour cell. Thus the antigen may be a tumour-specific antigen. The term

"tumour-specific" antigen should not be interpreted as being restricted to antigens from solid tumours, but to encompass antigens expressed specifically (or preferentially) by any cancerous, transformed or malignant cell. It may be particularly desirable to raise a Treg response against an antigen to which the subject exhibits, or is at risk of developing, an undesirable immune response. For example, it may be a self antigen against which an immune response occurs in an autoimmune disease. Examples of autoimmune diseases in which specific antigens have been identified as potentially pathogenically significant include multiple sclerosis (myelin basic protein), insulin-dependent diabetes mellitus (glutamic acid decarboxylase), insulin- resistant diabetes mellitus (insulin receptor), coeliac disease (gliadin), bullous pemphigoid (collagen type XVII), auto-immune haemolytic anaemia (Rh protein) , auto-immune thrombocytopenia (GpIIb/IIIa) , myaesthenia gravis

(acetylcholine receptor), Graves' disease (thyroid-stimulating hormone receptor), glomerulonephritis, such as Goodpasture's disease (alpha3 ( IV) NCI collagen), and pernicious anaemia (intrinsic factor) . Alternatively the target antigen may be an exogenous antigen which stimulates a response which also causes damage to host tissues. For example, acute rheumatic fever is caused by an antibody response to a Streptococcal antigen which cross-reacts with a cardiac muscle cell antigen. Thus these antigens, or particular peptide fragments or epitopes thereof may be suitable antigens for use in the present invention.

Depletion of Treg cells or impairment of Treg cell function has been shown to result in autoimmune disease in murine models. Disease caused in test animals include arthritis (e.g. rheumatoid arthritis), inflammatory bowel disease, gastritis, pernicious anaemia, thyroiditis, insulitis, diabetes, sialoadenitis, adrenalitis, autoimmune

orchitis/oophoritis, glomerulonephritis, chronic obstructive pulmonary disease and experimental autoimmune encephalitis and multiple sclerosis. Induction of a regulatory T cell type 1 response has also been shown to reduce the development of atherosclerosis in murine models (Mallat Z. et al. Circulation 108:1232-7, 2003). Treg activity has also been shown to be significant in the rate at which allografts are rejected. Depletion of Treg cells or impairment of function accelerates the rate of rejection, while infusion of test animals with syngeneic lymphocytes enriched in Treg cells has been shown to prolong graft survival.

The methods of the present invention may therefore find use in the treatment of any of these conditions.

The invention further provides a method of screening for a substance capable of modulating the interaction between DNGR-1 and F-actin, the method comprising contacting a test substance with

(i) a first substance comprising an extracellular domain of DNGR-1 or a portion thereof sufficient to bind to F-actin; and

(ii) a second substance comprising F-actin;

and determining interaction between (i) and (ii) .

To facilitate the analysis, either the first or second substance may be immobilised on (e.g. covalently linked to) a solid support. Thus the method may comprise providing the first or second substance immobilised on a solid support, contacting the solid support with a sample (typically a liquid sample) containing the second or first substance respectively, and determining the amount of second or first substance associated with the support.

One or more washing steps may be included between the contacting and determining steps to reduce or minimise the amount of second or first substance non-specifically

associated with the support. Binding between the first and second substances may be determined directly, or indirectly For example, the second substance may be labelled, e.g. with radioactive or spectrophotometrically detectable probe (e.g. fluorescent probe) . Alternatively the method may involve contacting the solid support with a further binding agent capable of detecting a complex between the first and second agents, such as an antibody specific for the substance not attached to the support. The binding agent may itself be labelled . The amount of binding detected will thus depend on the ability of the test agent to modulate binding.

The skilled person will be aware of numerous techniques assay formats which would be suitable, including ELISA, surface plasmon resonance, etc..

The method may comprise the steps of determining interaction between DNGR-1 and F-actin in the presence and the absence of the test substance, and selecting the test substance if the interaction is different in the presence and absence of the test substance. Modulation may involve an increase or a decrease in binding, i.e. the test agent may enhance or inhibit binding between F-actin and DNGR-1. It may be particularly desirable to screen for (and select) agents capable of inhibiting (i.e. reducing or preventing) the interaction between F-actin and DNGR-1.

The test agent may be any suitable molecule, including protein (such as an antibody), carbohydrate, small molecule (e.g.

having a molecular mass of less than 500 Da), etc..

The method may comprise testing a plurality of test agents, which may or may not be structurally related to one another. A high throughput format is preferably used. The plurality of test agents may be a library of small molecules or protein molecules, for example a plurality of analogues or variants of a known small molecule or protein. A library may comprise 10³, 10⁴, 10⁵, 10^s, 10⁷, 10⁸, 10⁹ or more different test agents.

Having identified or selected a suitable test agent, it may be desirable to confirm its ability to inhibit binding of F-actin to cells expressing DNGR-1 and/or to inhibit DNGR-1 activity induced by F-actin. This may include contacting the test agent with a cell expressing DNGR-1 and with F-actin, and determining binding of F-actin to DNGR-1 and/or to the cell. It may comprise the step of contacting the test agent with a cell expressing DNGR-1 and with F-actin, and determining signalling via DNGR-1 and/or activation of the cell. The cell may be a DC which naturally expresses DNGR-1. Alternatively, the cell may be engineered to express DNGR-1 or a chimera comprising a DNGR-1 CTLD operably linked to a heterologous intracellular signalling domain. An example is the ΰϋ3ζ-ΝΚ¾Ρ1- CLEC9a chimera described in WO2009/013484.

The inventors' findings suggest that F-actin may be used as a marker of conditions characterised by cell damage or cell death, e.g. necrosis and/or immunogenic cell death.

Consequently, the invention further provides a method of determining whether a subject is suffering from or at risk of developing a condition characterised by high levels of cell damage or cell death, particularly immunogenic cell damage or cell death, comprising the steps of:

(i) providing a biological sample from said subject;

(ii) determining the presence or amount of F-actin in said sample; and

(iii) correlating the presence of amount of F-actin in said sample with the existence or risk of developing such a condition .

The method may comprise contacting the sample with a binding agent having a binding site capable of binding to F-actin. The binding agent may be immobilised, for example on a solid support, such as a microtiter plate or a bead.

The determination step may comprise determining the fractional occupancy of the binding sites. Determination may be direct, for example using a secondary binding agent which is also specific for F-actin and is capable of binding to a complex formed between the primary binding agent and F-actin.

Alternatively it may be indirect, e.g. by determining empty binding sites, or by using a competitor for the binding sites, which may be labelled. Suitable techniques and materials are well known to the skilled person.

The correlation step may involve comparison with one or more control values obtained from comparable or equivalent samples derived from normal or unaffected subjects. It may be desirable to compare the result obtained with a plurality of such control values. An amount of F-actin outside the normal range may be considered to indicate the existence, or risk of developing, a relevant condition. This may represent, for example, a value which is more than one standard deviation, more than two standard deviations, more than three standard deviations, or even more, away from the mean normal value. Since F-actin is an intracellular antigen, the method may comprise determining extracellular F-actin, i.e. F-actin which has been released from lysed or permeabilised cells, or F- actin which is associated with lysed or permeabilised cells or debris from such cells. The sample may be any suitable biological sample. For example, it may be a biological fluid such as blood, plasma or serum, in which case the method may comprise determining F-actin present in the fluid component of the sample. Alternatively, it may be a tissue sample. The skilled person is well aware of suitable techniques which may be used, depending on the nature of the sample. Such techniques include gel electrophoresis (e.g. 2-dimensional gel electrophoresis), western blotting, enzyme-linked immunosorbent assay (ELISA) , immunohistochemistry,

spectroscopy and surface plasmon resonance.

The invention will now be described in more detail, by way of example and not limitation, by reference to the accompanying drawings and examples.

Description of the Drawings

Fig. 1: DNGR-1 recognizes a ligand in lysates of mammalian and insect cells. Lysates from human (HeLa; A) or insect (Sf9, S2;

B) cells were serially diluted as indicated, spotted onto NC membranes using a dot blot apparatus and probed with mDNGR-1 ECD or hDNGR-l-Fc or the respective control reagents (mDectin- 1 CTLD or hDC-SIGN-Fc) , as indicated. Data are from one representative experiment of at least 17 (HeLa cells) and 9

(insect cells ) .

Fig. 2: Biochemical characterization of DNGR-1L. (A) HeLa cell lysate was either boiled (left panel) or not (right panel) and treated with 5% SDS or left untreated as indicated. Samples were analyzed by dot blot with FLAG-mDNGR-1 ECD . One out of two independent experiments is shown. (B) HeLa cell lysates were prepared by Triton X-100 (lane 1) or Dig/DDM (lane 2) lysis, subjected to native PAGE and blotted under native conditions onto NC membranes. Immobilized proteins were detected using FLAG-mDNGR-1 ECD. The latter was also used in the native PAGE as control for the detection of FLAG-tagged protein (lane 3) . The arrow indicates the bottom of the gel pocket. Data are representative of 5 independent experiments. (C) HeLa cell lysate (0.5ml) was fractionated by size exclusion chromatography. Absorbance profile (280nm) of eluted proteins (left panel) and dot blot analysis for DNGR-1L of selected fractions (right panel) from one out of 2

experiments. Elution volumes of several molecular weight standards are marked by arrows. Fig. 3: Affinity purification of DNGR-IL enriches for components of the actin cytoskeleton . (A, B) The indicated volumes of lysate of HeLa (A) or S2 (B) cells were incubated with FLAG-mDNGR-1 ECD or FLAG-mDectin-1 pre-absorbed onto anti-FLAG beads. After centrifugation, post-pull-down supernatants (SN) containing unbound material were serially diluted and analyzed for the presence of DNGR-IL by dot blot. Input lysates before pull-down are shown for comparison (A, B left panels) . Proteins recovered after elution of beads with FLAG-peptide were analyzed by SDS-PAGE (A, B right panels) . Position of the FLAG-tagged proteins is indicated by arrows. Data shown are from one of four experiments with HeLa cells and one of two experiments with S2 cells. (C) Table

representing pooled results of mass spectrometric analysis of DNGR-1 pull-downs for HeLa cells (left) or S2 cells (right).

Fig. 4: Polymerization of G-actin is sufficient to generate DNGR-IL. (A) Dot blot analysis of DNGR-IL in HeLa cell lysate diluted in either G-buffer + 5μΜ latrunculin A (Lat.) or F- buffer + 5μΜ phalloidin (Phall.). {B, C) Dot blot of muscle (B) or non-muscle (C) actin probed with FLAG-mDNGR-1 ECD.

12.5μΜ muscle actin and 0.6μΜ non-muscle actin, reconstituted in G-buffer, were diluted 2-fold in G- or F-buffer in the presence of latranculin A or phalloidin as indicated. Data in A, B and C are representative of 2, 6 and 2 experiments, repectively. (D) Non-muscle (NM) F-actin with or without phalloidin (Phall.) was added to BWZ-mDNGR-1- ζ cells at the depicted final concentration. F-buffer with and without phalloidin served as controls. Histogram shows absorbance after addition of β-galactosidase substrate to lysed cells. Data represent one of three independent experiments.

Fig. 5: The DNGR-IL is protease sensitive. HeLa cell lysate was treated for lh at 37°C with different concentrations of papain or trypsin or left untreated as indicated. Samples were analyzed by (A) dot blot for the presence of DNGR-IL or (B) SDS-PAGE and SYPRO Ruby staining to assess proteolysis. One out of two representative experiments is shown.

Fig. 6: Plectin is not a DNGR-IL. HeLa cells were transfected with siRNAs specific for Plectin or with control siRNA or left untreated. Lysates of transfected or untransfected controls were analyzed by (A) Western blot probed with Plectin-specific antibody and (B) dot blot probed with mDNGR-l-ECD . One out of two representative experiments is shown.

Fig. 7: Actin filament bundling by cx-actinin increases detection by DNGR-1. F-actin was generated from non-muscle G- actin as in Fig. 4 and used at a concentration of 0.6μ in the presence or absence of 0.13μΜ -actinin and transferred onto NC membranes (dot blot) . Serial dilutions of F-actin and oi- actinin (negative control) were spotted at a starting concentration of 0.6μΜ and 0.13μΜ, respectively. Membranes were probed with FLAG-mDNGR-1 ECD to reveal DNGR-IL. One out of two representative experiments is shown.

Fig. 8: DNGR-1 reporter assay. Freeze-thawed BM1-OVA mouse embryonic fibroblasts (MEFs) that were treated for lh with ΙΟΟηΜ Jasplakinolide were added to BWZ-mDNGR-1-ζ cells at depicted ratios. Live BM1-OVA MEFs and RIO medium served as controls. Histogram shows absorbance after addition of β- galactosidase substrate to lysed cells.

Fig. 9: SYK activation. Non-muscle (NM) F-actin with or without phalloidin (Phall.) was added to B3 Z-mDNGR-l-Syk cells at the depicted final concentrations. F-buffer and RIO medium served as controls. Histogram shows absorbance after addition of β-galactosidase substrate to lysed cells.

Fig. 10: F-actin pelleting assay. 1.2μΜ non-muscle (NM) F- actin was incubated with 2-fold dilutions of hDNGR-l-Fc starting with 4μg or with 2μg of hDC-SIGN-Fc. Samples were pelleted by ultracentrifugation . Western blots developed with Cy5-con ugated anti-human IgG Fc-fragment show the presence of hDNGR-l-Fc or hDC-SIGN-Fc in the supernatant and pellet fractions after ultracentrifugation . l g hDNGR-l-Fc in the absence of F-actin served as a control.

Detailed Description of the Invention

DNGR-1

DNGR-1 (also known as Clec9a - see for example WO2009/01348 ) is a C-type lectin expressed on dendritic cells. As used in this specification, the term DNGR-1 is intended to embrace the human protein, the murine protein, their homologues

(especially orthologues) in other species, and variants and derivatives thereof which retain DNGR-1 activity. Such variants and derivatives preferably have at least about 30% sequence identity, more preferably at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to the human protein sequence shown below, or at least about 35% identity, more preferably at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity to the extracellular domain (CTLD) of the human protein sequence shown below.

(The transmembrane portion is shown in italics and the CTLD is underlined . )

In particular, conservative substitutions in the DNGR-1 sequence (as compared to the reference sequences) may be particularly well tolerated, without substantial effect on function . F-actin

Actin is found in all eukaryotic cells and is one of the most highly conserved proteins known. It is a component of the cytoskeleton and also forms part of the contractile apparatus of muscle cells.

Higher eukaryotes have several different isoforms of actin, each classified as alpha, beta or gamma. Any given organism may have more than one isoform of any particular class. For example, mammals express at least 6 different actin isoforms, each encoded by separate genes, while lower eukaryotes may have fewer isoforms. Most yeasts, for example, have only one

In general, alpha and gamma2 isotypes are found in muscle (alpha-skeletal, alpha-aortic smooth, alpha-cardiac and gamma2-enteric smooth) while beta and gammal isotypes are found in non-muscle cells (beta-cytoplasmic and gammal- cytoplasmic) .

Actin molecules have ATPase activity. They possess a deep nucleotide-binding cleft capable of binding either ATP or ADP and of hydrolysing ATP to ADP.

The monomeric (of globular) forms of actin molecules, of any isoform is generally designated "G-actin". G-actin is capable of polymerising into strands, and the polymerised form is designated "F-actin".

Actin filaments consist of two parallel actin strands in a helical configuration, approximately 7 nm in diameter and with a pitch (i.e. distance along the axis for one complete turn) of approximately 37 nm.

Polymerisation of G-actin into F-actin can be achieved by incubation of the monomeric form in suitable buffer containing physiological salt concentrations and ATP (such as the "F- buffer" described in the examples below) . In addition, F- actin filaments of defined length (circa 100 subunits) can be made by subjecting F-actin to the action of actin-severing and capping proteins such as gelsolin.

F-actin can be stabilised by various molecules which bind to it and inhibit depolymerisation, such as phalloidin,

jasplakinolide or tropomyosin.

For the purposes of the present specification, the term "F- actin" can be taken to refer to any substance containing two or more actin subunits associated covalently or non-covalently in a conformation which mimics that of the actin subunits in physiological F-actin strands or filaments. The substance may comprise any suitable number of associated monomers, e.g. 5 or more, 10 or more, 20 or more, 50 or more, or 100 or more monomer units. For example, it may comprise between 2 and 100 monomer units or more, e.g. 2-10 monomer units, 10-50 monomer units, 50-100 monomer units.

Each of the monomer units present in the substance may have bound ADP. Alternatively, each may have bound ATP.

Alternatively, the substance may comprise a mixture of monomer units each having bound either ATP or ADP.

The substance may consist of a single F-actin strand, a filament, or a plurality of filaments. The free end(s) may or may not be capped, e.g. with F-actin capping protein.

The substance will be capable of binding to the extracellular domain of DNGR-1, and may be capable of inducing intracellular signalling via Syk as a result of such binding.

The substance may additionally comprise an F-actin stabilising agent, such as phalloidin, jasplakinolide or gelsolin, or a plurality of F-actin stabilising agents.

F-actin may consist of one F-actin isoform, or may be composed of a mixture of different F-actin isoforms. For example, it may be composed entirely of alpha, beta or gamma subunits, or it may be a mixture thereof. It may be composed entirely of non-muscle (NM) isoforms (NM F-actin) or may be entirely composed of muscle isoforms (muscle actin) .

Each subunit may have at least 80% identity to one of th sequences, for example, at least 85%, at least 90%, or a least 95% sequence identity to one of these sequences.

When F-actin is used as a targeting agent to direct an antigen to DNGR-1⁺ DCs, the antigen will typically be covalently or non-covalently linked to the F-actin. For example, the antigen may be expressed as a fusion protein with one of the actin subunits or may be chemically cross-linked or conjugated to the F-actin, or to one or more of the subunits .

The association between the antigen and the F-actin may be indirect, in that they are not directly bound to one another For example, the antigen may be associated with an F-actin binding agent such as phalloidin, an antibody specific for F actin, or the peptide Lifeact (a 17-mer peptide having the sequence MGVADLIKKFESISKEE ) . Thus the antigen may be covalently associated with the F-actin binding agent, which in turn binds (covalently or, more usually, non-covalently) to F- actin.

Substitutions and sequence identity

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

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

According to another classification, the amino acid classes are small hydrophilic, acid/acid amide/hydrophilic, basic, small hydrophobic and aromatic, wherein small hydrophilic amino acids are Ser, Thr, Pro, Ala and Gly;

acid/acidamide/hydrophilic amino acids are Asn, Asp, Glu and Gin; basic amino acids are His, Arg and Lys; small hydrophobic amino acids are Met, lie, Leu and Val; and aromatic amino acids are Phe, Tyr and Trp

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

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

Alternatively a specific pairwise alignment program may be used. A suitable example is 'lalign' (implementing the algorithm of Huang and Miller; Adv. Appl . Math. (1991) 12:337- 357) using default parameters.

Binding agents

Any suitable molecule having a sufficiently high affinity and specificity for F-actin may be used as a binding agent. The molecule may be a protein, nucleic acid (e.g an aptamer) , carbohydrate (e.g. oligo- or polysaccharide), small molecule, etc. Particularly preferred binding agents are antibodies against F-actin and functional fragments thereof.

The binding agent preferably has a binding affinity (affinity constant) for F-actin of at least 10⁵M^_1, at least 10⁶M^_1, at least 10⁷M^_1, preferably at least 10⁸M^_1, more preferably at least 10^{9 _1}. The binding agent preferably has an affinity at least 2x, and preferably at least 5x, at least lOx, at least 50x, at least lOOx, at least lOOOx or at least lOOOOx greater for F-actin than for any monomeric (G-actin) isoform from the same species .

It is well known that fragments of a whole antibody can perform the function of binding antigens. Examples of functional binding fragments are (i) the Fab fragment

consisting of VL, VH, CL and CHI domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S. et al . , Nature 341, 544-546 (1989)) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment

comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site {Bird et al,

Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879- 5883, 1988); (viii) bispecific single chain Fv dimers

(PCT/US92/09965) and (ix) "diabodies", multivalent or

multispecific fragments constructed by gene fusion

(WO94/13804; P. Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993) .

As antibodies can be modified in a number of ways, the term "antibody" should therefore be construed as covering any specific binding substance having an binding domain with the required specificity. Thus, this term covers the antibody fragments described above, as well as derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or synthetic. Chimaeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimaeric antibodies are described in EP-A- 0120694 and EP-A-0125023.

It will be appreciated that those binding agents described herein which are capable of binding to DNGR-1 are generally required to bind the extracellular domain of DNGR-1 in order to exert the required effect. Reference to a binding agent capable of binding DNGR-1 should therefore be construed accordingly, unless the context requires otherwise.

Antibodies specific for F-actin should have at least 10-fold higher affinity for F-actin than for any monomeric (G-actin) isoform from that species, and ideally 100, 1000, 10000-fold or even greater affinity for F-actin than for G-actin.

Blocking antibodies against F-actin, intended to inhibit interaction with DNGR-1, preferably lack a Fc domain capable of binding to Fc receptors. This may reduce the chance of F- actin bound by such antibodies becoming co-localised with DNGR-1 itself at the surface of APCs expressing both Fc receptor and DNGR-1. Thus, it may be desirable to use a Fab fragment, Fd fragment, Fv fragment, dAb fragment , isolated CDR, F(ab')2 fragment, or scFv molecule.

Alternatives to antibodies are increasingly available. So- called "affinity proteins" or "engineered protein scaffolds" can routinely be tailored for affinity against a particular target. They are typically based on a non-immunoglobulin scaffold protein with a conformationally stable or rigid core, which has been modified to have affinity for the target.

Modification may include replacement of one or more surface residues, and/or insertion of one or more residues at the surface of the scaffold protein. For example, a peptide with affinity for the target may be inserted into a surface loop of the scaffold protein or may replace part or all of a surface loop of the scaffold protein. Suitable scaffolds and their engineered equivalents include: - BPTI, LAC-DI, ITI-D2 (Kuitz domain scaffolds);

- ETI-II, AGRP (Knottin) ;

- thiredoxin (peptide aptamer) ;

- Fn3 (AdNectin) ;

- lippcalin (BBP) (Anticalin) ;

- ankyrin repeat (DARPin);

- Z domain of protein A (Affibody) ;

- gamma-B-crystallin/ubiquitin (Affilin) ;

- LDLR-A-domain (Avimer) .

See, for example, Gebauer, M and Skerra, A, Current Op. Chem. Biol. 2009, 13: 245-255, and Friedman, M and Stahl, S,

Biotechnol . Appl . Biochem. (2009) 53: 1-29, and references cited therein.

For the purposes of the present invention, such alternatives to antibodies are preferably also not capable of binding to Fc receptors. As such, they typically do not contain an Fc domain capable of binding to Fc receptors.

In certain aspects of the invention, it is desirable to crosslink an antigen (e.g. a protein or peptide antigen) to a binding agent as described. The skilled person is well aware of suitable methods and reagents. Where the binding agent is a protein, the antigen may be coupled via a sulphydryl group of the binding agent. The sulphydryl group may normally be free, or it may normally be part of a disulphide bond in which case it may be exposed by selective reduction of the binding agent. For example, an antibody can be mildly reduced selectively in the hinge region using the reducing agent mercaptoethanosulfonate . Then, the antigen is activated using sulpho-SMCC, an hetero-bifunctional cross-linking reagent that reacts with the tertiary amines of the protein,

generating groups reactive with free sulphydryls. Then, the antibody and the activated antigen are incubated together resulting in the protein being conjugated to the monovalent antibody. Alternatively, if a suitably immunogenic peptide sequence from the antigen is known, such a peptide containing a cysteine with a free sulphydryl can be synthesized and coupled to sulpho-SMCC activated antibody, which will remain bivalent and with several peptides bound per molecule of antibody .

Subjects

Typically the subject is vertebrate, preferably a mammal. The subject may be a human, other primate, or a domestic, laboratory or livestock animal, such as a mouse, rat, guinea pig, lagomorph (e.g. rabbit), cat, dog, pig, cow, horse, sheep or goat. In certain embodiments , the subject is human.

Pharmaceutical compositions

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

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included . For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH,

isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.

Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Administration is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners.

Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

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

Examples

DNGR-l-b±nds to a ubiquitous protein-based ligand conserved from insects to man

In order to choose an appropriate reagent for purifying the DNGR-1 ligand (DNGR-1L), we tested different versions of the extracellular domain of DNGR-1. Similar to the CTLD originally used to demonstrate DNGR-1 binding to dead cells (Sancho et al . , 2009), a more stable full-length extracellular domain (ECD) of mouse DNGR-1 with an N-terminal FLAG tag,

specifically stained irradiated cells that had lost membrane integrity and become permeable to TO-PRO-3 dye. A human DNGR- 1-Fc fusion protein was also able to specifically stain dead cells, unlike a control human DC-SIGN-Fc reagent. Both the mDNGR-1 ECD and the hDNGR-l-Fc were chosen for subsequent studies.

The DNGR-IL has previously been detected exclusively on dead cell corpses or in fixed and permeabilized cells (Sancho et al . , 2009) . We therefore first tested whether the ligand could be solubilized from cell extracts. By dot blot analysis, ligand could be detected in lysates of HeLa cells using either the mDNGR-1 ECD or the hDNGR-l-Fc, but not the control reagents, mDectin-1 (CTLD) or hDC-SIGN-Fc (Fig. 1A) .

Consistent with our previous conclusions derived from staining of cell corpses (Sancho et al . , 2009), the ligand was protein- based as its detection was markedly reduced by treatment of the lysates with trypsin or papain (Fig. 5) . To determine the evolutionary conservation of DNGR-IL, we tested additional species, including insects and yeast. Notably, specific signal was detected in lysates of Sf9 cells from Spodoptera

frugiperda and of S2 cells from Drosophila melanogaster (Fig. IB) . In contrast, mDectin-1 did not detect ligand in lysates of insect or human cells (Fig. IB), consistent with the ability of Dectin-1 to recognize fungal β-glucans (Brown and Gordon, 2001) . We conclude that DNGR-1 recognizes either many different ligands or a universal ligand expressed in most cell types and highly conserved from insects to Man. The DNGR-IL is protein-based and can be solubilized from cell extracts for the purpose of biochemical characterization. The DNGR-IL is a large structure that requires purification under native conditions

Attempts to separate cell lysates by SDS-PAGE and to detect the ligand after Western transfer all failed, suggesting that structural integrity of the ligand is important for detection (data not shown) . We therefore utilized the dot blot assay to assess the biochemical properties of the ligand and design an appropriate purification scheme. As shown in Fig. 2A, detection of the ligand was abrogated or markedly decreased by boiling or addition of SDS, explaining earlier failures and indicating that the ligand has to be separated under native conditions. However, when we attempted to separate it by native PAGE on 4-20% gradient gels, Far Western blotting with the mDNGR-1 ECD revealed that the ligand had failed to enter the gel and remained trapped in the well (Fig. 2B) . These results suggested that the ligand is a very high molecular weight structure under native conditions that cannot be resolved by gel electrophoresis. This was confirmed by gel filtration analysis, which showed that the ligand elutes primarily in the void volume and in the first few fractions, which correspond to molecular weights greater than 450 kDa (Fig. 2C) . We conclude that the DNGR-IL is likely to be a very large protein complex that is destroyed by denaturation and heat and cannot be purified using traditional chromatographic separation methods .

Affinity purification of DNGR-IL followed by mass spectrometry reveals proteins associated with the actin cytoskeleton

In an attempt to characterize the ligand by enrichment, we tested whether it could be affinity isolated from mammalian and insect cell lysates by mDNGR-1 ECD. This approach was successful, as measured by significant depletion of ligand from the post-incubation supernatant, provided the amount of mDNGR-1 ECD was carefully matched to the amount of input lysate (Fig 3A, B) . When the bound material was subsequently eluted using an excess of FLAG peptide and resolved by SDS- PAGE, a number of discrete bands, in addition to a large band corresponding to the mDNGR-1 ECD itself (marked) , could be revealed by gel staining with SYPRO Ruby dye (Fig. 3A, B) . Notably, several proteins appeared similar between pull-downs from HeLa and S2 cells but were absent in eluates from control pull-downs carried out with the mDectin-1 reagent {Fig. 3A,

B) .

We cut up each lane into multiple slices and analyzed them by mass spectrometry. Analysis of the results from 4 experiments with HeLa cells revealed that multiple actin-associated proteins were consistently found in the DNGR-1 but not the Dectin-1 precipitates. These proteins included cytolinkers such as plectin, actin filament crosslinkers such as spectrin, actin motor proteins such as myosins, as well as actin itself (Fig. 3C) . Proteins associated with microtubules were not especially prevalent in the DNGR-1 precipitates, indicating selectivity for the actin cytoskeleton (Fig. 3C and data not shown) . We selected the proteins with the highest number of unique peptide hits across experiments and tested their putative identity as DNGR-IL. The validation included carrying out loss-of function experiments such as siRNA-mediated knockdown in HeLa cells followed by probing for DNGR-IL by dot blot (see Fig. 6 for an example with plectin), as well as gain-of-function experiments such as procuring the protein in question and assessing its binding to DNGR-1 (data not shown) .

All of those experiments failed to identify a ligand for DNGR- 1 (data not shown) . Furthermore, some of the top candidates found in the HeLa cell pull-downs (e.g., plectin, spectrin) were not identified in pull-downs from S2 cells even though the latter still contained predominantly actin-associated proteins (Fig. 3C) . We therefore contemplated the possibility that the enrichment for actin cytoskeleton-associated proteins in the DNGR-1 pull-downs might be because DNGR-1 binds to the actin cytoskeleton itself. The DNGR-1 ECD labels the actin cytoskeleton across species

To test the above hypothesis, we performed detailed analysis of the intracellular localization of DNGR-IL in different cells using confocal fluorescence microscopy. Staining of various cells and tissues with mDNGR-1 ECD revealed a

remarkably discrete pattern. DNGR-IL showed apical

polarisation in the pseudo-stratified columnar epithelium of Drosophila imaginal wing discs and, in that tissue, could additionally be seen on contractile rings around the cleavage furrow of mitotic cells undergoing cytokinesis. In Drosophila ovaries, DNGR-IL co-localized with striations in the

surrounding muscle sheet. In HeLa cells, DNGR-IL was

prominently detected on stress fibers and lamellipodia .

Notably, in HeLa cells infected with vaccinia virus, mDNGR-1 ECD additionally labelled the virus-induced actin tails that propel progeny virions onto neighboring cells. This overall staining pattern is very reminiscent of actin filaments and, consistent with that notion, fluorescent phalloidin co- staining revealed perfect co-localization of DNGR-IL and F- actin. (Data not shown.) We conclude that the intracellular distribution of the DNGR-IL overlaps directly with that of the actin cytoskeleton in multiple cell types and tissues across species .

Polymerization of actin generates DNGR-IL

We pursued the hypothesis that the staining pattern observed was due to the ability of DNGR-1 to bind to F-actin. To support this notion, we examined the effect of actin

polymerization and depolymerization on DNGR-IL detection. HeLa cell extracts incubated in low salt buffer (G-buffer) and latrunculin to depolymerize actin filaments and stabilize G- actin, respectively, contained markedly less ligand than the same lysates incubated in physiological salt buffer (F-buffer) plus phalloidin, conditions that stabilize F-actin (Fig. 4A) . Notably, a preparation of purified rabbit muscle actin, which contains mainly a-actin, spontaneously generated DNGR-IL when incubated in F-buffer but not when incubated in G-buffer with latrunculin (Fig. 4B) . Non-muscle actin (β- and γ-actin) from human platelets also failed to generate DNGR-1L in G-buffer but did so in F-buffer (Fig. 4C) . As an independent means of assessing the presence of DNGR-1L, we used a cell-based assay in which β-galactosidase reports signaling by a ϋΝ6Κ-1-0ϋ3ζ chimeric receptor upon ligand binding (Sancho et al., 2009) . β-galactosidase activity was observed when F-actin was added to reporter cells at concentrations above ΙμΜ but was abruptly lost when the actin concentration was dropped 2.5-fold (Fig. 4D) . Pre-addition of phalloidin allowed stimulation of the reporter cells by concentrations of F-actin below ΙμΜ (Fig. 4D) . These results are consistent with the well-established rapid depolymerization of actin below a critical concentration (which is normally ΙΟΟηΜ but increased in the presence of actin depolymeri zing proteins in serum - see Discussion) , and the ability of phalloidin to inhibit filament depolymerization (Alberts et al . , 2008) .

Figure 8 shows that pre-exposure of cells to the F-actin stabilising agent jasplakinolide prior to disruption by freeze thawing increases signalling when the disrputed cells are added to cells expressing the chimeric ΟΝΏ-1-003ζ reporter.

Non-muscle actin, with or without phalloidin stabilisation, induces Syk signalling in wild type mouse cells expressing and DNGR-1 (Figure 9).

We conclude that preparations of G-actin do not contain DNGR- 1L but the latter is generated upon actin polymerization into filaments.

The DNGR-1 ECD binds F-actin

Finally, we assessed whether DNGR-1 is able to bind directly to actin filaments. Non-muscle or muscle actin was polymerized in vitro using F-buffer together with fluorescent phalloidin to stabilize F-actin and permit filament visualization. The preparation was subsequently incubated with mDNGR-1 or mDectin-1, labelled with Cy3-conjugated anti-FLAG mAb and ultracentrifuged . The pellet was resuspended in F-buffer and adsorbed onto poly-D-lysine-coated glass slides before vizualization by total internal reflection (TIRF) microscopy. mDNGR-1 but not Dectin-1 decorated non-muscle actin filaments along their entire length. Pixel intensity analysis confirmed the exact overlap of the phalloidin and DNGR-IL signals.

Identical results were obtained when using the preparation of rabbit muscle actin or the hDNGR-l-Fc vs. the hDC-SIGN-Fc reagents. (Data not shown.) Figure 10 shows that hDNGR-l-Fc co-pellets with F-actin filaments under ultracentrifugation . We conclude that DNGR-1 specifically and directly binds to actin filaments.

DISCUSSION

DAMPs and their receptors provide an important alternative to PAMP-based mechanisms for initiation of inflammation and adaptive immunity. DNGR-1 is a dedicated DAMP receptor selectively expressed in DC that controls cross-priming of cytotoxic T cells to dead cell-associated antigens (Sancho et al . , 2009) . Identification of the DNGR-1 ligand has proven elusive and has hampered research into the mechanism of action of this receptor and into the connection between DAMP release and adaptive immunity. Here, we identify F-actin as a universal DNGR-1 ligand conserved from invertebrates to mammals .

The conclusion that F-actin acts as the ligand for DNGR-1 rests on three lines of evidence. First, the conclusion that DNGR-1 binds F-actin can account for all the data we have gathered on the properties of the DNGR-1 ligand, such as its large mass and susceptibility to denaturation . Furthermore, it explains the abundance of actin-binding proteins in the mass spectrometry analysis of DNGR-1 affinity isolates. Second, the intracellular distribution of DNGR-IL overlaps precisely with that of the actin cytoskeleton in all cell types tested, from Drosophila to Man. Although many actin-associated proteins will give a staining pattern that overlaps with the actin cytoskeleton in any given cell, none of these proteins are present in every single one of the structures labelled by DNGR-1 (muscle, apical surface of Drosophila wing disc epithelium, contractile rings, stress fibers and vaccinia actin tails) . For example, although muscle F-actin is

decorated with myosin, there is no myosin in vaccinia virus actin tails. Similarly, of the proteins we identified by mass spectrometry, spectrin is confined to the cortical

cytoskeleton and not found in contractile rings or vaccinia virus actin tails and plectin is not found on stress fibers. As such, the only common protein to all the structures visualized by DNGR-1 staining is actin itself. Third, we show that incubation of purified G-actin under conditions that favor polymerization is both necessary and sufficient to generate ligand and we reveal that F-actin polymers assembled in vitro can be labeled with DNGR-1 along their entire length. However, short of obtaining an actual structure of DNGR-1 directly bound to F-actin, we cannot formally exclude the possibility that DNGR-1 binds to an F-actin-associated protein. We believe this possibility to be exceedingly unlikely as the actin preparations used in this study were highly purified and therefore contaminants in trace amounts would not be able to account stoichiometrically for the extent of binding along the filaments observed by TIRF microscopy.

The above considerations do not negate the possibility that F- actin binding proteins do, nevertheless, contribute to ligand detection by DNGR-1. Single actin filaments are 5-8 nm in diameter but have a tendency to self-assemble into thicker bundles (Pollard and Cooper, 2009) . The structures visualized by TIRF microscopy are 200-300 nm in diameter. Because this corresponds to the diffraction limit of light microscopy, it cannot be ascertained whether they represent single filaments that cannot be resolved apart or fibers composed of multiple filaments. Supporting the latter, the images clearly show heterogeneous staining with areas of greater staining intensity that could correspond to filament bundles. Actin filament bundling and network formation could facilitate DNGR- 1 binding if the DNGR-1 dimer can dock on two adjacent binding sites on separate actin filaments, a model that will need to be validated by mapping of the binding sites and modeling of the interaction. Notably, bundling or network formation is promoted by the action of F-actin crosslinking proteins such as a-actinin and fimbrin or filamin and spectrin,

respectively. These proteins are ubiquitous and most F-actin in cells is therefore present in the form of either bundles (e.g. stress fibers) or networks (e.g., lamellipodia, cortical actin) rather than single filaments. Thus, actin crosslinking proteins can be expected to contribute to DNGR-1 detection simply by their ability to create higher ordered actin filament assemblies. Consistent with that notion, addition of a-actinin to in vitro polymerized F-actin markedly increases ligand detection even though a-actinin itself does not act as a ligand (Fig. S5) . This means that even trace amounts of F- actin crosslinking or bundling proteins in actin preparations may contribute to ligand detection upon polymerization in vitro. By the same token, contaminating actin in purified preparations of actin-binding proteins could give the misleading impression that those proteins act as DNGR-1 ligands (unpublished observations) .

The identification of F-actin as a ligand for DNGR-1 opens the door to further studies on the role of this interaction in the regulation of immunity. Although DNGR-1 is necessary for cross-priming cytotoxic T cells against antigens borne by dead cells, the exact mechanism involved remains unclear. In the absence of a known ligand, DNGR-l/Dectin-1 chimeras have been used to analyze the potential of DNGR-1 to signal for myeloid cell activation in response to β-glucans, with mixed results

(Huysamen et al., 2008; Zelenay et al . , 2011). The

identification of the ligand, assuming it acts as a receptor agonist, now offers the possibility to study DNGR-1 signalling and its effect on DC biology in a more physiological context. In addition, mapping of the binding sites on DNGR-1 and on F- actin may lead to new strategies to target, block or stimulate the receptor, which could have applications in a variety of immunomodulation protocols. Finally, the identification of the DNGR-1 ligand as F-actin raises fascinating questions as to the maintenance of cytoskeletal integrity in cells undergoing demise, how the kinetics of cytoskeletal exposure vary depending on insult, how and in which DC sub-cellular compartment the receptor encounters its ligand and, finally, how F-actin recognition by DNGR-1 is coordinated with signals from other innate immune receptors to dictate DC function.

Actin is a very ancient protein that is extremely conserved across species because actin evolution has been constrained by the vast number of interactions that it needs to maintain with actin-binding proteins (Erickson, 2007; Pollard and Cooper, 2009) . It is therefore not surprising that DNGR-1 is able to recognize F-actin in cells from both insects and mammals. The abundance and relative stability of F-actin, as well as its presence in all eukaryotic cells, makes it an ideal DAMP. It is therefore tempting to speculate that F-actin release may act more generally as a universal sign of cell damage, engaging DAMP receptors other than DNGR-1. Indeed, actin release from dead cells has long been known to be a marker of tissue damage that correlates with extent of injury (Dahl et al . , 2003; Lee and Galbraith, 1992) . Plasma contains two abundant actin-binding proteins, Gc protein (also known as vitamin D-binding protein) and gelsolin, that act to sever F- actin (gelsolin) and sequester G-actin (gelsolin and Gc protein) (Lee and Galbraith, 1992) . Although it is generally assumed that the function of these proteins is to prevent actin polymerization from leading to blood vessel occlusion (Lee and Galbraith, 1992), it may be that an additional purpose is to dampen any pro-inflammatory effects of innate immune F-actin recognition. Consistent with that possibility, the gelsolin/Gc protein actin-scavenger system can be overwhelmed by massive cell injury, a condition that often leads to a sepsis-like syndrome in patients suffering from severe trauma (Dahl et al., 2003; Lee and Galbraith, 1992) . The identification of F-actin as the ligand for DNGR-1 may therefore open the door for future studies on a more general role of this DAMP in the triggering of inflammation and immunity from insects to man.

EXPERIMENTAL PROCEDURES

Reagents

Purified rabbit muscle actin was a kind gift from Richard Treisman (London Research Institute) . Non-muscle actin and a- actinin were purchased from Cytoskeleton Inc. Unlabeled and A488-conjugated Phalloidin were from Invitrogen. Latrunculin A and latrunculin B were from Invitrogen and Calbiochem, respectively. Recombinant hFc-tagged hDNGR-1 and hDC-SIGN were from R&D Systems. Rat-anti-mDNGR-1 antibody (7H11) and monomeric FLAG-mDectin-1 CTLD have been described previously (Sancho et al . , 2009; Sancho et al . , 2008). 7H11 was

conjugated to Alexa 488 (A488) fluorophore using the Alexa Fluor 488 Monoclonal Antibody Labeling Kit from Invitrogen. cDNAs encoding mDNGR-1 CTLD or the CTLD plus the neck region of the long form of mDNGR-1 (mDNGR-l-ECD ) were generated by PCR amplification and cloned in frame into the p3XFLAG-CMV™-9 expression vector (Sigma). Forward primer sequences for the two constructs were: mDNGR-l-CTLD : 5'- TTTCCCGCGGCCGCGCCTTGTC, mDNGR-l-ECD: 5 ' -TTTCCCGCGGCCGCGAAGTTCT ; the reverse primer sequence 5 ' -CCCTTTTCTAGATCAGATGCAG was used for both

constructs. Constructs were expressed in 293-F cells and supernatant containing FLAG-tagged mDNGR-1 proteins was used directly or after affinity purification using anti-FLAG (M2) beads (Sigma) and elution with an excess of 3xFLAG peptide (Sigma) . Cells

HeLa cells were grown in MEM low bicarbonate medium containing 10% FCS and 2mM glutamine and split every 2-3 days. BWZ cells stably expressing the extracellular domain of mouse DNGR-1 fused to the transmembrane region from NKRP1B and the intracellular tail of Οϋ3ζ (BWZ-mDNGR-1- ζ cells; (Sancho et al . , 2009)) were grown in RPMI 1640 containing 10% FCS, 2mM glutamine, 50 μΜ 2-mercaptoethanol , 100 units/ml penicillin, 100 μg/ml streptomycin (complete RPMI medium) . Sf9 cells were grown in Sf-900 II medium ( Invitrogen ) , supplemented with 100 units/ml penicillin, 100 pg/ml streptomycin, 50 g/ml

gentamycin (Sigma) and 0.25μg/ml amphotericin B (Fungizone, Invitrogen), in roller bottles on an orbital shaker (250rpm) at 27-28°C under atmospheric C0₂ pressure. S2 cells (kind gift from Nic Tapon, London Research Institute) were grown in

Schneider's Drosophila medium (Invitrogen) at 25°C under atmospheric C0₂ pressure. 293-F cells were grown in protein- free Freestyle 293 Expression medium (Invitrogen) as per manufacturer's instructions. All media and medium supplements were from Invitrogen except MEM (London Research Institute) and FCS (Source Bioscience).

Solubilisation and precipitation of DNGR-IL

15xl0⁶ cells were lysed in 1ml 0.5% Triton X-100 in 50mM Tris- buffered saline (TBS) buffer containing protease inhibitor mix (Roche) for 30 min on ice. In a few experiments, lysis was perfomed with 1% Digitonin (Dig) /1% n-dodecyl^-D-maltoside (DDM) in TBS. Lysates were centrifuged, insoluble material was discarded and the supernatant was used immediately for downstream applications such as dot blot or pull-downs. In some instances, cells were "pre-extracted" for 1 min in 0.05%

Triton X-100 in 60mM PIPES, 25mM HEPES, lOmM EGTA, ImM MgAc, 5μΜ Phalloidin; pre-extracted cells were pelleted by

centrifugation and then subjected to the lysis protocol detailed above. Similar results were obtained with or without pre-extraction (data not shown) . For DNGR-1L pull-down, different volumes (0.1 - 0.75ml) of cell lysate (corresponding in the case of HeLa cells to -30- 200 μg of total protein) were added to 20-40μ1 of packed anti- FLAG (M2) beads (Sigma) pre-incubated with FLAG-mDNGR-1 ECD or FLAG-mDectin-1 CTLD. Unbound material (post pull-down supernatant) was separated from bound material by

centrifugation and stored for further analysis by dot blot to check for depletion of DNGR-1L. Beads were washed 6 times for 5min with lysis buffer and eluted with 40μ1 of 400 g/ml 3xFLAG peptide. Eluted material was separated by SDS-PAGE and protein bands were visualized by SYPRO Ruby (Invitrogen) staining. Gel lanes were manually cut from top to bottom into 1mm thick slices and the excised protein gel pieces were placed in a well of a 96-well microtiter plate and destained with 50% v/v acetonitrile and 50 mM ammonium bicarbonate, reduced with 10 mM DTT, and alkylated with 55 mM iodoacetamide . After alkylation, proteins were digested with 6 ng/ L Trypsin

(Promega, UK) overnight at 37 °C. The resulting peptides were extracted in 2% v/v formic acid, 2% v/v acetonitrile. The digest was analyzed by nano-scale capillary LC-MS/MS using a nanoAcquity UPLC (Waters, UK) to deliver a flow of

approximately 300 nL/min. A C18 Symmetry 5 μιη, 180 μιη x 20 mm μ-Precolumn (Waters, UK) , trapped the peptides prior to separation on a C18 BEH130 1.7 μιη, 75 μιτι x 250 mm analytical UPLC column (Waters, UK) . Peptides were eluted with a gradient of acetonitrile. The analytical column outlet was directly interfaced via a modified nano-flow electrospray ionisation source, with a hybrid linear quadrupole fourier transform mass spectrometer (LTQ Orbitrap XL/ETD, ThermoScientific, San Jose, USA) . Data dependent analysis was carried out, using a resolution of 30,000 for the full MS spectrum, followed by eight MS/MS spectra in the linear ion trap. MS spectra were collected with an automatic target gain control of 5xl0⁵ and a maximum injection fill time of 100 ms over a m/z range of 300- 2000. MS/MS scans were collected using an automatic gain control value of 4xl0⁴ and a threshold energy of 35 for collision induced dissociation. LC-MS/MS data were then searched against a protein database (UniProt KB) using the Mascot search engine programme (Matrix Science, UK) . Database search parameters were set with a precursor tolerance of 5 ppm and a fragment ion mass tolerance of 0.8 Da. One missed enzyme cleavage was allowed and variable modifications for oxidized methionine, carbamidomethyl cysteine, pyroglutamic acid, phosphorylated serine, threonine and tyrosine were included. MS/MS data were validated using the Scaffold programme

(Proteome Software Inc., USA). All data were additionally interrogated manually.

Detection of DNGR—1 ligand by dot blot

Cell lysates, fractions from size exclusion chromatography, mDNGR-1 ECD pull-downs or actin samples were spotted onto a nitrocellulose (NC) membrane (Whatman) pre-soaked in PBS or G- buffer using a Bio-Dot microfiltration apparatus (Bio-Rad) according to the manufacturer's instructions. NC membranes were left overnight in blocking buffer (PBS + 5% milk + 0.05% Tween 20) . DNGR-IL was revealed after sequential probing of the dot blot with either FLAG-mDNGR-1 ECD (supernatant from transfected 293-F cells diluted 1:10 in blocking buffer) or hDNGR-l-Fc (I g/ml) or the respective control reagents, followed by HRP-conjugated mouse anti-FLAG (M2) antibody (Sigma) or HRP-conjugated rabbit-anti-hFc antibody (Stratech Scientific) . After each step, membranes were washed 6 times for 5 min with washing buffer (PBS + 0.05% Tween 20). Signal was revealed using the SuperSignal West Pico Chemiluminescent substrate kit (Thermo Scientific).

Native PAGE and gel filtration chromatography

For native PAGE, proteins within the lysate were separated on a 4-20% Tris-Glycine gel (Invitrogen) and transferred to NC membranes using methanol-free transfer buffer. Blotted membranes were blocked overnight and probed for DNGR-IL using FLAG-mDNGR-1 ECD and HRP-anti-FLAG antibody as described for dot blotting. For gel filtration, 0.5ml cell lysate was injected onto a Superose 6 column, which had been equilibrated in PBS, and proteins were separated by fast protein liquid chromatography (FPLC) at a flow rate of 500μ1/ιηίη using the Akta system (GE Healthcare) . Eluted proteins were collected in 0.5ml fractions and analyzed by dot blot for the presence of DNGR- 1L .

Polymerization of actln and TIRF microscopy

Rabbit muscle G-actin (kind gift from Richard Treisman, London Research Institute) was reconstituted in G-buffer (5mM

Tris/HCl pH 8.0 + 0.2m CaCl₂) at a concentration of 5mM. Non- muscle actin was reconstituted in G-buffer as per

manufacturer's instructions to give a G-actin stock

concentration of 23μΜ. For dot blot experiments, muscle actin in G-buffer was either incubated with ΙΟΟμΜ latrunculin B or polymerized in F-buffer (lOmM Tris-HCl pH 7.5 + 50mM KC1 + 2 mM MgCl₂ and 1 mM ATP) in the presence of 5μΜ phalloidin. Non- muscle actin in G-buffer was left untreated or polymerized in F-actin buffer without phalloidin for use in dot blot experiments and reporter cell assays. For destabilization of actin filaments, latrunculin B (25μΜ) was added to Ο.βμΜ non- muscle F-actin; a-actinin was added to non-muscle F-actin for bundle formation. For TIRF microscopy, Ο.ΟβμΜ F-actin was co- labeled with A488-labelled phalloidin and FLAG-mDNGR-1 ECD or FLAG-mDectin-l-CTLD or hDNGR-l-Fc or hDC-SIGN-Fc in F-actin buffer. Filaments were incubated for lh at room temperature, labeled with Cy3-con ugated mouse-anti-FLAG or Cy5-conjugated rabbit anti-human Fc antibody and spun down by

ultracentrifugation (lOOOOOg, 30min, 4°C). Stained filaments were resuspended in 200μ1 F-actin buffer to a final

concentration of 0.06 μΜ actin and allowed to adsorb for lhr onto glass bottom microwell dishes (MaTek corporation) . TIRF images were acquired using a Cell^NR (Olympus) system equipped with 150x NA 1.45 TIRFM objective (Olympus). Image analysis was performed using ImageJ. Background was subtracted in each case as a function of the minimum intensity in the image. Flow cytometry

HeLa cells were UVC irradiated (240 mJ/cm²) and cultured for 12-24 h to induce secondary necrosis. UV-treated and untreated (control) HeLa cells were stained with FLAG-mDNGR-1- CTLD, FLAG-mDNGR-1 ECD or Fc-hDNGR-1 (2 g/ml) followed by

A488-conjugated 7H11 (10 g/ml) or Cy5-conjugated rabbit-anti- human Fc (Stratech Scientific; lOpg/ml), respectively. Samples were counterstained with TO-PRO-3 to distinguish live and dead cells. Samples were run on a FACS Calibur (Becton Dickinson) and data were analyzed using FlowJo software (Treestar) .

Confocal microscopy

HeLa cells were cultured on fibronectin-coated coverslips overnight at 37°C, fixed in 4% paraformaldehyde/PBS for 15 min and washed and permeabilized in 0.1% Triton-XlOO/PBS for 4 min. After washing with PBS, cells were left in blocking buffer (1% BSA + 2% FCS in PBS) overnight and stained with FLAG-mDNGR-1 ECD, followed by 7H11 mAb and A546-conjugated goat-anti-rat antibody ( Invitrogen ) , as well as the nuclear dye DRAQ5 (eBioscience) . For vaccinia virus infection, HeLa cells cultured on fibronectin-coated glass coverslips were infected with the Western Reserve strain at a multiplicity of infection of 2 in serum-free MEM, as described (Arakawa et al . , 2007) . 1 hour post infection (hpi), the medium was replaced with medium containing 10% FCS. Infected cells were fixed with 4% paraformaldehyde at 8 hpi. In some experiments,

A488-phalloidin (1:400) was added to stain the actin

cytoskeleton . Coverslips were mounted using Fluoromount-G (Southern Biotech) and images were collected using a laser scanning confocal microscope (LSM 510; Zeiss).

For confocal imaging of Drosophila tissues, ovaries and wing imaginal discs were isolated from female flies and larvae, respectively. Ovaries were dissected in PBS, fixed for 20 mins in 4% paraformaldehyde + PBS, washed for 30 minutes in

PBS/0.1% Triton X-100 and blocked for 15 minutes in 5% normal goat serum in PBS/0.1% Triton X-100. FLAG-mDNGR-1 ECD was added to the samples and incubated overnight at 4 C. Imaginal discs were processed as above, except they were dissected on ice, fixed in 4% formaldehyde + PBS for 30 min, and blocked using 0.1% BSA (Sigma) . All samples were further stained with Cy3-con ugated mouse-anti-FLAG antibody (1:200) and 1 pg/ml

DAPI (Invitrogen) for 2 hrs at room temperature before mounting in Vectashield (Vector labs). In some experiments, A488-phalloidin (1:400) was also added to stain the actin cytoskeleton . Samples were imaged on a Leica SP5 confocal microscope.

DNGR-1L reporter assay

The cell reporter assay for DNGR-1 ligand has been described previously (Sancho et al . , 2009). Briefly, BWZ-mDNGR-1-ζ cells were plated in 96 well plates (IxlO⁵ cells/well) in the presence of the indicated stimuli. After overnight culture, cells were washed once in PBS and LacZ activity was measured by lysing cells in CPRG (Roche ) -containing buffer. 1-4 hours later O.D. 595 was measured using O.D. 655 as a reference.

Plectin knock-down

HeLa cells were seeded at 2xl0⁵ cells/well in 12-well plates in

MEM medium without antibiotics. Cells were transfected with a pool of three plectin-specific siRNAs (Hs_PLECl_7, Hs_PLECl_8 and Hs_PLECl_9; Qiagen) at 5nM each or 5nM control siRNA (All Stars negative control siRNA; Qiagen) using the HiPerFect transfection reagent (Qiagen) . Cell lysates were prepared

48hrs after initial transfection and analyzed for plectin content by Western Blot (WB) and for DNGR-1L by dot blot as described above. WBs were developed with mouse-anti-Plectin monoclonal antibody (1:200, clone 10F6; Insight Biotechnology) followed by HRP-conjugated goat-anti-mouse antibody (1:5000;

Invitrogen ) .

DNGR-1L reporter assay

The cell reporter assay for DNGR-1 ligand has been described previously (Sancho et al . , 2009). Briefly, BWZ-mDNGR-1-ζ cells were plated in 96 well plates (IxlO⁵ cells/well) in the presence of the indicated stimuli. Detection of the DNGR-1- ligand upon alteration of the actin cytoskeleton was evaluated using BM1-OVA mouse embryonic fibroblasts (MEFs) that were treated for Ih with ΙΟΟηΜ Jasplakinolide . To induce exposure of the DNGR-l-ligand, MEF pellets were shock frozen in liquid nitrogen for 30 seconds to induce necrotic cell death and added to BWZ-mDNGR-1- ζ cells at indicated ratios. After overnight culture, cells were washed once in PBS and LacZ activity was measured by lysing cells in CPRG (Roche)

SYK activation

To determine agonist activity of F-actin, B3Z cells expressing wild type mouse DNGR-1 and Syk were stimulated with NM actin at indicated concentrations.

F-actin pelleting assay

1.2μΜ non-muscle (NM) F-actin was incubated for 30min at room temperature with 2-fold dilutions of hDNGR-l-Fc starting from 4ug or with 2]ig of hDC-SIGN-Fc. Actin filaments were pelleted by ultracentrifugation at 150 000 g for 1.30h at room temperature. Supernatants and pellets (resuspended in water) were subsequently analyzed for the presence of hDNGR-l-Fc or hDC-SIGN-Fc by Western Blot developed with Cy5-conjugated rabbit anti-human IgG Fc-fragment (1:2000) .

While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth are

considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention. All documents cited herein are expressly incorporated by

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Submitted .

Claims

Claims

1. A method of inhibiting interaction between F-actin and DNGR-1, comprising contacting F-actin with a binding agent capable of binding to F-actin and inhibiting interaction between F-actin and DNGR-1.

2. A method of inhibiting an immune response in a subject comprising administering to the subject a binding agent capable of binding to F-actin and inhibiting interaction between F-actin and DNGR-1.

3. A binding agent capable of binding to F-actin and inhibiting interaction between F-actin and DNGR-1 for use in a method of medical treatment

4. A binding agent capable of binding to F-actin and inhibiting interaction between F-actin and DNGR-1 for use in the inhibition of an immune response.

5. A method according to claim 1 or claim 2 or a binding agent according to claim 3 or claim 4 wherein the condition is inflammation or is characterised by inflammation, undesirable T cell activity and/or high levels of cell death.

6. A method or binding agent according to claim 5 wherein the condition is acute or chronic inflammation, an autoimmune disease, neurologic disease, cardiovascular diseases, immune- mediated respiratory disease, allergic or hypersensitivity reaction, transplant or graft rejection, graft versus host disease, immunopathological response to an infectious agent or a degenerative process.

7. A method or binding agent according to any one of the preceding claims wherein the binding agent is an antibody specific for F-actin.

8. A method or binding agent according to any one of the preceding claims wherein the binding agent is not capable of binding to Fc receptors.

9. A method for targeting an antigen to an antigen

presenting cell, comprising contacting the antigen presenting cell with a composition comprising the antigen, wherein the antigen is associated with F-actin, and wherein the antigen presenting cell expresses DNGR-1.

10. A method according to claim 9 wherein the antigen presenting cell is a dendritic cell.

11. A method according to claim 10 wherein the dendritic cell is capable of cross-presenting extracellular antigen via MHC class I molecules.

12. A method for stimulating an immune response against a target antigen in a subject, comprising administering F-actin and the target antigen to the subject.

13. A method according to claim 12 wherein the F-actin and the target antigen are provided in the same composition.

14. A method according to claim 13 wherein the F-actin is physically associated with the target antigen.

15. A method according to any one of claims 12 to 14 wherein the immune response comprises activation of T cells.

16. A method according to claim 15 wherein the T cells are CD8+ T cells

17. A method according to claim 16 wherein the T cells are regulatory T cells (Treg) .

18. A method for inducing tolerance in a subject towards an antigen, comprising administering to the subject a composition comprising the antigen, wherein the antigen is associated with F-actin, and wherein the antigen is administered in the absence of an adjuvant.

19. A composition comprising an antigen, wherein the antigen is associated with F-actin.

20. A composition according to claim 19 further comprising a pharmaceutically acceptable carrier.

21. A composition comprising an antigen for use in a method of medical treatment, wherein the antigen is to be

administered in conjunction with F-actin.

22. A composition according to claim 21 wherein the

composition comprises F-actin.

23. A composition according to claim 21 wherein the antigen is associated with F-actin.

24. A composition comprising an antigen, for use in

stimulating an immune response against the antigen, wherein the antigen is to be administered in conjunction with F-actin.

25. A composition according to claim 24 wherein the

composition further comprises F-actin.

26. A composition according to claim 25 wherein the antigen is associated with F-actin.

27. A composition comprising F-actin for use in a method of medical treatment, wherein the F-actin is to be administered in conjunction with a target antigen.

28. A composition according to claim 27 wherein the

composition further comprises the target antigen.

29. A composition according to claim 28 wherein the F-actin is associated with the target antigen.

30. A composition comprising F-actin, for use in stimulating an immune response against a target antigen, wherein the F- actin is to be administered in conjunction with the target antigen.

31. A composition according to claim 30 wherein the

composition further comprises the target antigen.

32. A composition according to claim 31 wherein the F-actin is associated with the target antigen.

33. A therapeutic kit comprising a target antigen and F- actin .

34. A method of screening for a substance capable of modulating the interaction between DNGR-1 and F-actin, the method comprising contacting a test substance with

(i) a first substance comprising an extracellular domain of DNGR-1 or a portion thereof sufficient to bind to F-actin; and

(ii) a second substance comprising F-actin;

and determining interaction between (i) and (ii) .

35. A method according to claim 34 wherein the first or second substance is immobilised on a solid support.

36. A method according to claim 35 comprising the steps of providing the first or second substance immobilised on a solid support, contacting the solid support with a sample containing the second or first substance respectively, and determining the amount of second or first substance associated with the support .

37. A method according to any one of claims 34 to 36 comprising the steps of determining interaction between DNGR-1 and F-actin in the presence and the absence of the test substance, and selecting the test substance if the interaction is different in the presence and absence of the test

substance .

38. A method according to any one of claims 34 to 37 wherein the agent is selected if it is capable of inhibiting the interaction between F-actin and DNGR-1.

39. A method according to any one of claims 34 to 38 further comprising the step of confirming the ability of the agent to inhibit binding of F-actin to cells expressing DNGR-1 and/or to inhibit DNGR-1 activity induced by F-actin.

40. A method according to claim 39 comprising contacting the test agent with a cell expressing DNGR-1 and with F-actin, and determining binding of F-actin to DNGR-1 and/or to the cell.

41. A method according to claim 39 comprising contacting the test agent with a cell expressing DNGR-1 and with F-actin, and determining signalling via DNGR-1 and/or activation of the cell.

42. A method of determining whether a subject is suffering from or at risk of developing a condition characterised by high levels of cell damage or cell death, particularly immunogenic cell damage or cell death, comprising the steps of :

(i) providing a biological sample from said subject;

(ii) determining the presence or amount of F-actin in said sample; and

(iii) correlating the presence of amount of F-actin in said sample with the existence or risk of developing such a condition .

43. A method according to claim 42 comprising contacting the sample with a binding agent having a binding site capable of binding to F-actin.