CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/417,444 filed November 4, 2016, the disclosure of which is incorporated herein by reference in its entirety; including any drawings and sequence listings.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled "NKp46-9 PCT_ST25 txt", created October 31 , 2017, which is 19 KB in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to natural ligands of NKp46 and compositions and methods for modulating NK-cell activation through NKp46 receptors.

BACKGROUND OF THE INVENTION

Natural killer (NK) cells are innate lymphocytes that belong to the innate immune system. These cytotoxic lymphocytes have been originally described as being spontaneously capable to kill tumor cells without any prior activation. NK cells also participate in the clearance of pathogen-infected cells through their cytotoxic properties and proinflammatory cytokine-secretion such as the production of IFN-γ. Recent researches have shown that NK cells can act as regulatory cells and shape the adaptive immunity by acting on macrophages, dendritic cells and T cells.

NK cell effector activities are tightly controlled by a fine balance of inhibitory and activating signals delivered by surface receptors. Inhibitory receptors on NK cells such as inhibitory isoforms of Killer cell immunoglobulin receptors (KIRs) and NKG2 receptors, among others, are essential to prevent lysis of self-cells that express self-molecules such as constitutively expressed MHC-I self-molecules. For example, a decrease in MHC-I expression reduces the number of inhibitory signals deliver to NK cells render them more prone to be activated. NK cell activation results from engagement of activating receptors on NK cells such as the activating isoforms of Ly49 and Killer cell immunoglobulin receptors (KIRs), the natural cytotoxicity receptors (NCRs), activating NKG2 receptors, e.g., NKG2D, and CD16 that can induce NK cell activation by initiating distinct signaling pathways. The NCR family is composed of three molecules: NKp30 (NCR3, CD337) and NKp44 (NCR2, CD336) in humans and NKp46 (NCR1 , CD335) which is expressed in all mammals and highly conserved between human and mouse. NKp46 is mainly expressed by NK cells except for a small population of T lymphocytes and innate lymphoid cells (ILC3) in mucosa.

Activating receptors can recognize two types of ligands, self-molecules whose expression is induced upon cellular stress, or exogenous molecules produced by microbes during infection. For example, NCRs have been described to bind several but not all hemagglutinin and hemagglutinin neuraminidases of the influenza virus, Sendai virus, Newcastle disease virus, ectromelia virus and vaccine virus. NKp46 could also recognize PfEMPI of Plasmodium falciparium and an unknown ligand from Fusobacterium nucleatum. Despite the finding that the cell surface transmembrane protein B7-H6 is a ligand for NKp30 and that the three NCRs can bind to different heparan sulfates sequences, the identification of cellular-derived surface ligands for the NCR family still remains to be elucidated. It has been reported that NKp30 recognizes the nucleic factor HLA-B-associated transcript BAT3 that can be expressed in the cytoplasm of tumor and apoptotic cells. Similarly, NKp44 can recognize the proliferating cell nuclear antigen (PCNA) and the mixed-lineage leukemia protein 5 (MLL5)-related NKp44L which are normally expressed in the nucleus of healthy cells but can be found in the cytoplasm of tumors cells. NKp46 has been described to bind the intracellular filamentous cytoskeletal protein vimentin expressed on the surface Mycobacterium tuberculosis-infected monocytes. It has also has been reported that NKp46 could recognize a surface protein on healthy pancreatic β cells but the identification of this cellular ligand still awaits identification.

Complement Factor P (CFP), also referred to as "Properdin", is a glycoprotein circulating as dimers, trimers and tetramers of a single chain glycoprotein in blood serum. It has been reported that addition of purified properdin can restore alternative pathway activation in properdin-deficient sera in a dose-dependent manner (Schwaeble and Reid, 1999, Immunology Today 20(1 ) : 17-21 ). Recently, it has been published that mice infected with lethal doses of Nm can be cured by intraperitoneal injection of recombinant (Youssif et al. (2014) Proc. Nat. Acad. Sci. USA 1 1 1 (14):5301 -5306 and PCT patent application WO2013/033518).

Identification and characterization of cell surface receptors for NKp46 is important for understanding of the mechanisms of NKp46 regulation of immune functions and for the development of new therapies for the treatment of diseases and disorders related to these mechanisms.

SUMMARY OF THE INVENTION It is shown herein that Complement factor P (CFP) binds to human NKp46. It is further shown herein that Complement factor P triggers NKp46 signalling. It is further shown herein that NKp46 is required for survival of mice in response to invasive N. meningitidis infection.

One object of the invention to provide natural ligands of NKp46. Another object of the invention is to provide compositions, e.g. compositions comprising NKp46-expressing effector cells (e.g. NKp46+ NK cells), or compositions that enhance or stimulate the activity of NKp46+ NK cells, for use in targeting pathogenic CFP-opsonized cells, notably for the treatment of a disease (e.g. cancer, bacterial or viral infection) in an individual whose diseased cells (e.g. cancer cells, bacterial cells, virus-infected cells) bear CFP at their surface.

It is another object of the invention to provide molecules that modulate natural ligands of NKp46 ligands and methods of use thereof to modulate immune responses. In one embodiment, provided are molecules that interfere with the NKp46-CFP interaction to downregulate immune responses (e.g. via NKp46). In another embodiment, provided are molecules that enhance the NKp46-CFP interaction to enhance NK cell activation toward pathogenic cells (via NKp46 triggering in NK cells).

One object of the invention is to provide NKp46 ligands or compositions that modulate NKp46 ligands, and methods of use thereof, to reduce NK cell activity, e.g. towards NKp46-ligand opsonized cells in inflamed tissue.

It is one object of the invention to provide compositions and methods for the treatment of cancers, infectious disease, inflammatory responses, autoimmune disorders, and transplant rejection by targeting NKp46 or CFP. In any of the treatment methods, the method may by characterized by a step of administering an agent to an individual having a disease or disorder.

It is another object of the invention to provide molecules that inhibit, reduce or block NKp46-CFP interaction, and methods of use thereof, to reduce NK cell activation. Such molecules can be useful, for example in the treatment of inflammation or autoimmune disorders, e.g., inflammatory disease (for example, inflammatory vascular disease) in which properdin or complement is involved in disease pathology and/or any inflammatory disease in which NKp46-expressing cells are contributing to disease pathology.

Another object of the invention is to provide NKp46 ligands, and compositions that modulate NKp46 ligands, and methods of use thereof, to increase NK cell activity, e.g. toward NKp46-ligand opsonized disease cells (e.g. cancer cells, bacterial cells, virus- infected cells). It is one object of the invention to provide molecules that increase NKp46 ligands or enhance or promote the NKp46-CFP interaction, and methods of use thereof, to increase NK cell activation, e.g. for the treatment of cancer or infectious disease.

It is another object of the invention to provide methods for identifying agents (e.g. protein, antibody or small molecule agents) that modulate NKp46 or CFP activity, for example that modulate (e.g., inhibit or enhance) the NKp46-CFP interaction.

In one aspect, provided are methods for identifying agents (e.g. protein, antibody or small molecule agents) that enhance the NKp46-CFP interaction and/or enhance the activity of NKp46-expressing NK cells, e.g., that enhances the recognition and/or lysis of a target cell by an NKp46+ cell. In one embodiment, the method comprises identifying an agent(s) (e.g. an antibody) that mediates opsonization of target cells (e.g. cancer cells, infected cells, bacterial cells) with CFP. In one embodiment, the method comprises identifying an agent(s) that mediates recruitment of NKp46-expressing immune effector cells (e.g. NK cells) to the site of target cells that express CFP (e.g. cancer cells, infected cells, bacterial cells); for example the agent can promote the co-localization or aggregation of NKp46-expressing immune effector cells (e.g. NK cells) and the target cells. In one embodiment, the method comprises identifying an agent that mediates recruitment of NKp46-expressing immune effector cells is a multispecific binding protein (e.g. a bispecific antibody) that binds CFP and a cell surface receptor on NKp46-expressing cells (NK cells). In one embodiment, the method comprises identifying an agent that mediates recruitment of NKp46-expressing immune effector cells is a multispecific binding protein (e.g. a bispecific antibody) that comprises a CFP polypeptide or fragment thereof (that binds NKp46) and an antigen binding domain (e.g. an antibody variable region(s) or hypervariable region) that binds a cell surface receptor on a disease cell (e.g. that binds a cancer antigen, a bacterial antigen, a viral antigen); such an agent can be useful to promote NK cell cytotoxicity toward a disease causing cell that is otherwise not sufficiently CFP opsonized to elicit NK cell cytotoxicity.

Agents that enhance the interaction between NKp46 and CFP can be used to enhance NK cell activity, e.g. to direct an NK cell to recognize, be activated in the presence of, or lyse a target cell associated with (e.g. opsonized by) CFP. Such methods and compositions can be useful for the treatment or prevention of infectious disease or proliferative disease (e.g. cancer).

It is another object of the invention to provide methods for identifying neutralizing anti-NKp46 antibodies and small molecules. It is another object of the invention to provide methods for identifying neutralizing anti-NKp46 ligand antibodies and small molecules. In one embodiment, the neutralizing anti-NKp46 antibody is an antibody that binds NKp46 and inhibits the interaction between NKp46 and CFP. It is still another object of the invention to provide biomarkers for assessing the effectiveness of immunotherapies, e.g. immunotherapies that have the ability to modulate (e.g. enhance) NK cell activity. Such immunotherapies may typically be useful for the treatment or prevention of an autoimmune or inflammatory disease/disorder, infectious disease or cancer in a subject. In one embodiment, the immunotherapy is an antibody that binds to an antigen (e.g. a cancer antigen) on a target cell to be depleted (e.g. via ADCC mediated by NK cells). In one embodiment, the antibody is capable of recruiting complement factors, e.g. the antibody comprises an Fc domain (e.g. a human Fc domain) capable of binding to C1 q (e.g. human C1 q).

It is another object of the invention to provide compositions and methods for assisting in the diagnosis of an autoimmune or inflammatory disease/disorder, infectious disease or cancer in a subject, or assessing the propensity for a subject to develop an autoimmune or inflammatory disease/disorder, or cancer.

It is a further object of the invention to provide compositions and methods for determining the severity or prognosis of an autoimmune or inflammatory disease/disorder, infectious disease or cancer in a subject having or suspected of having an autoimmune or inflammatory disease/disorder, or cancer.

It is another object of the invention to provide compositions and methods for determining (e.g. predicting, assessing, etc.) the efficacy of a treatment for an autoimmune or inflammatory disease/disorder, infectious disease or cancer.

It is another object of the invention to provide compositions and methods for selecting a subject for treatment for an autoimmune or inflammatory disease/disorder, or cancer.

It is another object of the invention to provide compositions and methods for measuring pharmacokinetic and pharmacodynamics parameters of NKp46 therapies, such as NKp46-expressing NK cell compositions, soluble proteins that mimic NKp46 ligand or therapies that promote the activity or number of NKp46-expressing NK cells, or therapies that promote NKp46-NKp46 ligand interactions.

The inventors have shown that NKp46 binds to properdin, also known as Complement Factor P (CFP), the only positive regulator of the complement cascade. Binding of CFP to NKp46 triggers NKp46 signaling in NK cells. While it is shown that CFP can bind alone to NKp46, it will be appreciated that CFP can also bind NKp46 as part of a complex of proteins that collectively act as a receptor for NKp46. The binding or association of CFP can enhance or increase the binding of NKp46 (e.g., as may be expressed at the surface of an NK cell) to a cell opsonized with CFP. The inventors' findings reveal how NKp46 mediates the interaction of NK cells with the complement system as a cooperative innate immune defense and may represent a novel and attractive therapeutic possibility to treat disease in which target cells can be eliminated by the NK cells, e.g. cancers, infectious disease, multidrug resistant bacterial strains. The model used to study the mechanism of NKp46-mediated control of disease by NK cells was the gram-negative bacterium Neisseria meningitidis (Nm) which causes Invasive Meningococcal Disease. Neisseria meningitidis is an emergent public health problem responsible for meningitidis and septicemia in human and sepsis in mice. Using a depleting antibody, genetically-engineered NK cell and NK cell receptor deficient mice, we first showed that NK cell were required for mice survival to Nm infection through the expression of the activating NK cell receptor NKp46. While NKp46 did not directly recognize Nm, recombinant soluble NKp46 molecules can bind to serum-opsonized Nm. Complement system deficiencies are associated with increased susceptibility and severity to meningococcal infection. In particular, Complement Factor P (CFP)-deficient patients are selectively predisposed to lethal meningococcal infection caused by Nm. CFP, the only positive regulator of the complement system identified so far, has been reported to bind to certain pathogens, to apoptotic and necrotic cells, thus providing a platform for de novo convertase assembly and complement activation. We showed here that serum purified and recombinant CFP can bind to soluble NKp46 molecule and also trigger NKp46-expressing cells, demonstrating that CFP is a ligand for the NK cell receptor NKp46. Our findings thus show that NK cells can interact with a component of the complement system and provide a conceptual framework for the role of NK cells and NKp46 in immunity.

In one embodiment, the present invention provides compositions and methods of use thereof for inducing or enhancing immune stimulatory responses by modulating activating immune cell signalling, optionally NKp46 signalling. The methods are particularly useful for treating or preventing cancers and infectious disease.

In one embodiment, the present invention provides compositions and methods of use thereof for treating or preventing diseases, e.g. cancers and infectious disease, in an individual having pathogenic cells characterized by CFP opsonization. In one embodiment, the disease is a bacterial infection (e.g. infection with a bacterium selected from the group consisting of Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Listeria monocytogenes, Group B streptococci, Escherichia coli, and Mycobacteri), wherein bacterial cells are opsonized with CFP. In one embodiment, the disease is a cancer, wherein cancer cells are opsonized with CFP. In one embodiment, an amount of the composition is administered to an individual that results in NK cell mediated lysis of CFP-opsonized pathogenic cells (e.g. tumor cells, bacterial cells or infected cells). In one embodiment, the composition for use in treating disease is a composition that causes an increase in NK cell activity, for example an increase in activation and/or cytotoxicity of NKp46-expressing NK cells toward a target cell (e.g., a pathogenic cell). In one embodiment, the composition for use in treating disease comprises a cell composition comprising NKp46-expressing effector cells. In one embodiment, the composition for use in a method of treating disease comprises an agent (e.g. an antibody) that acts as agonist at an activating receptor on the surface of an NKp46-expressing NK cell. In one embodiment, the composition for use in treating disease comprises an agent (e.g. an antibody) that binds a cancer antigen on the surface of a cancer cell and comprises an Fc domain that can be bound by CD16 on the surface of an NKp46- expressing NK cell. In one embodiment, the composition for use in treating disease comprises an agent (e.g. an antibody) that acts as antagonist at an inhibitory receptor on the surface of an NKp46-expressing NK cell.

In one embodiment, compositions and methods of use are for increasing NK cell activity, e.g. for increasing NK cell mediated lysis of a target cell (e.g. a cancer cell, an infected cell or a bacterial cell), wherein the compositions and methods can comprise enhancing CFP-NKp46 binding, for example by providing or increasing the presence of CFP on the surface of a disease cell (e.g. a cancer cell, a bacterial cell, an infected cell), optionally for increasing the amount of CFP on a target cell available for binding to NKp46. In one embodiment, a composition for increasing NK cell activity comprises CFP (as a monomeric or multimeric protein) or a fragment thereof that is capable of binding NKp46.

In one embodiment, provided is a receptor complex comprising CFP and a NKp46 polypeptide, optionally an isolated receptor complex, optionally further comprising a linking agent (e.g. an antibody or protein) that links CFP and NKp46. Optionally the complex comprises one or more further proteins.

In one embodiment, a composition for increasing NK cell activity is an agent that mediates, causes, increases, stabilizes or otherwise enhances the CFP-NKp46 interaction, thereby enhancing NK cell activation and/or lysis of a target cell. In one embodiment, the agent is a multispecific antigen binding protein (e.g. a bispecific antigen binding protein, a bispecific antibody) that binds CFP on a target cell (e.g. CFP as found with a protein complex on the surface of a CFP-opsonized cell, a protein complex comprising CFP) and NKp46.

In one embodiment, the agent is a multispecific antigen binding protein that binds to CFP on a target cell and a protein other than NKp46 (e.g. an NK cell activating receptor other than NKp46) expressed on the surface of immune effector cells. An exemplary an NK cell activating receptor other than NKp46 is CD16. Such agent can bring effector cells into proximity with target cells opsonized with CFP such that NK cell activation and/or target cell lysis can be mediated by NKp46 and/or activating receptor(s) other than NKp46. Such agent may be useful to redirect NK (including NK cells that express NKp46 or do not express NKp46) and/or other effector cells to a CFP-opsonized target cell.

In one embodiment, the present invention provides compositions and methods of use thereof for inhibiting immune responses, e.g. NK cell-mediated cytotoxicity, by interfering with CFP-NKp46 interaction. The compositions and methods are particularly useful for treating or preventing cancers and infectious disease.

In one embodiment, a composition for inhibiting immune responses (or decreasing NK cell activity) is an agent that inhibits the interaction of CFP with the NKp46 protein of the surface of a cell (e.g. an NK cell), thereby inhibiting immune cell (e.g., an NK cell) activation and/or lysis of a CFP-opsonized cell. In one embodiment, the agent is an antibody that binds CFP on a cell (e.g. CFP as found with a protein complex on the surface of a CFP-opsonized cell, a protein complex comprising CFP) that competes with human NKp46 for binding to CFP. In one embodiment, the agent is an antibody that binds human NKp46 on a cell (e.g. an NK cell) and that competes with CFP for binding to NKp46.

Also provided are methods and assays for identifying agents that modulate the interaction between CFP and NKp46. Such assays can be used advantageously to screen or test antibodies or other antigen binding agents to increase, decrease NK cell or immune effector cell activity. In one embodiment of such an assay, the method comprises: (i) bringing NKp46 (e.g. as a cell expressing NKp46 or as a soluble NKp46 protein) into contact with CFP (e.g. as a cell opsonized with CFP or as a soluble CFP protein) in the presence of a test agent (e.g. a plurality of test agents), and (ii) assessing binding of CFP to NKp46. A decrease in binding (compared to negative control) indicates that the agent interferes with binding of CFP and NKp46. An increase in binding (compared to negative control) indicates that the agent enhances the interaction of CFP and NKp46.

In one embodiment provided is a method of identifying agents that decrease NK cell activity, comprising: (i) bringing NKp46 (e.g. as a cell expressing NKp46 or as a soluble NKp46 protein) into contact with CFP (e.g. as a cell opsonized with CFP or as a soluble CFP protein) in the presence of a test agent (e.g. a plurality of test agents), (ii) assessing binding of CFP to NKp46. Optionally the method further comprises: (iii) selecting (e.g. for use in therapy, for production of a batch of therapeutic agent, for further processing or evaluation) an agent that interferes with binding of CFP and NKp46. In one embodiment, the agent is for use in treatment or prevention of an autoimmune or inflammatory condition. In one embodiment, the agent comprises an antibody. In one embodiment, the agent (e.g. an antibody) binds NKp46. In one embodiment provided is a method of binding (e.g. for targeting, detecting, modulating, etc.) to NKp46 on the surface of an NK cell, optionally a method for modulation NKp46 on the surface of an NK cell, the method comprising bringing a cell expressing NKp46 (e.g., an NK cell) into contact with CFP (e.g., a soluble CFP or CFP-comprising protein, optionally complexed with other molecules).

In one embodiment provided is a method of binding (e.g. for targeting, detecting modulating, etc.) a CFP protein on the surface of a target cell (e.g. infected cell, cancer cell, etc.), the method comprising bringing the target cell into contact with an NKp46 polypeptide (e.g., a NKp46 polypeptide comprising the amino acid sequence of SEQ ID NOS: 1 or a fragment thereof, for example a NKp46-lg fusion protein comprising any of the foregoing). In one embodiment provided is a method of detecting a CFP protein on the surface of a target cell (e.g. a pathogenic cell, infected cell, bacterial cell, cancer cell, etc.), the method comprising bringing the target cell into contact with an NKp46 polypeptide comprising a detectable moiety, wherein detection of binding of NKp46 to the target cell indicates presence of (or opsonization with) CFP. In one embodiment provided is a method of targeting an agent of interest (e.g. a detectable moiety, a toxic moiety, a drug) to a target cell (e.g. infected cell, cancer cell, etc.), the method comprising bringing the target cell into contact with a NKp46 polypeptide or fragment thereof associated with, or conjugated or fused to the agent of interest.

These aspects are more fully described in, and additional aspects, features, and advantages will be apparent from, the description of the invention provided herein. Any of the methods can further be characterized as comprising any step described in the application, including notably in the "Detailed Description of the Invention"). The invention further relates to methods of identifying, testing and/or making proteins described herein. The invention further relates to agents obtainable by any of present methods. The disclosure further relates to pharmaceutical or diagnostic formulations containing at least one of the agents disclosed herein. The disclosure further relates to methods of using the subject agents in methods of treatment or diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. NK cells mediate protection against Nm infection.

WT control mice treated or not with anti-NK1.1 mAb (a, b) and genetically- engineered NKp46 ⁺ cell-deficient mice {NKp46 ^iCRE/+ R26 ^!slDTA/+ ) and control littermates (c, d) were infected i.p. with Nm. Survival at the indicated dose (a, c) and bacterial load after infection with 2x10 ⁶ bacteria (b, d) were monitored over time. Survival: in (a): n=17-19, Log Rank test, dose stratified, p=0.00696. Survival in (c): n=16, Log Rank test, dose stratified, p=0.0206. Bacterial load in (b, d) Two-way Anova, Bonferroni's multiple comparisons test.

Figure 2. The activating receptor NKp46 is required for NK cell mediated protection against Nm infection.

(a, b) NCR1 ^GFP/GFP and control littermates were infected i.p. with Nm. Survival at the indicated dose and bacterial load after infection with 2x10 ⁶ bacteria were monitored over time. Survival in (a): n=29, Log Rank test, dose stratified, p=0.0181. Bacterial load in (b) Two-way Anova, Bonferroni's multiple comparisons test, (c, d) FACS profiles of NCR-Fc staining on Nm bacteria in indicated conditions. In grey full histogram: Nm bacteria only. Data are representative of 3 independent experiments. One-Way Anova, Bonferroni's multiple comparisons test.

Figure 3. Complement factor P binds to NKp46.

(a-e) FACS staining of CFP HIS-tagged binding to indicated NCR-Fc molecules coated on beads as depicted in (a). In (b, c) binding is expressed as a fold increase of the mean fluorescence intensity over the control without CFP. In (d), the experiment was performed with a range of CFP concentration and in (e), in the presence of anti-NKp46 mAb or isotype control mAb. (f, g) Flow sensograms of the indicated NCR-Fc constructs with serum purified CFP (f) or at the indicated concentrations of HuCFP-HIS (g) flowed over a flow cell with similar response units of immobilized NCR-Fcs. (h-k) Binding of recombinant HuCFP HIS-tagged to indicated cells as depicted in (h). In (j), CFP binding to primary human bulk NK cells and in (k), to purified Ncr1 ^GFP/GFP and Ncr1 ^{m m} NK cells. Data are representative of 2 independent experiments in (d, e, g, j, k) and at least 3 in (b ,c, i). One- Way Anova, Bonferroni's multiple comparisons test.

Figure 4. NKp46-expressing NK cells participate to the therapeutic benefit effect of CFP delivery for Nm infection control in vivo.

(a-c) WT mice were treated as depicted in (a). In (b) bacterial load after 24 hours and in (c) after 48 hours of infection. n=6 representative of 2 independent experiments, (d-f) _Ncr1 wT/wr _{a nd Ncr1} GFP/GFP |j _{ttermates were} treated as depicted in (d). In (e), bacterial load after 6 hours and in (f) after 24 hours of infection. n=5-8. Two Way Anova, Bonferroni's multiple comparisons test.

DESCRIPTION OF THE INVENTION

Introduction

Natural killer (NK) cells are innate lymphocytes involved in antitumoral and antimicrobial immune responses. NK cells recognized their targets by detecting the diminution of major histocompatibility complex class I molecules and through the direct recognition of microbial- and non-microbial-derived activating ligands. The definition of these activating ligands is still not fully complete, in particular, for the major activating NK cell receptor NKp46. We show here that NKp46 binds to Complement Factor P (CFP), the only positive regulator of the complement cascade. CFP binding to NKp46 triggers NKp46 signaling in NK cells and is critical for NK cell mediated protection against the CFP- dependent Neisseria meningitidis invasive bacterial infection. These findings reveal that NK cells can interact with the complement system as a cooperative innate immune defense and may represent a novel and attractive therapeutic possibility to mediate NK cell lysis of pathological cells.

Definitions

As used herein, "a" or "an" may mean one or more. As used in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more.

Where "comprising" is used, this can optionally be replaced by "consisting essentially of" or by "consisting of".

The term "antibody," as used herein, refers to polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chains, antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM. Several of these are further divided into subclasses or isotypes, such as lgG1 , lgG2, lgG3, lgG4, and the like. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 1 10 or more amino acids that is primarily responsible for antigen recognition. The terms variable light chain (V _L ) and variable heavy chain (V _H ) refer to these light and heavy chains respectively. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are termed "alpha," "delta," "epsilon," "gamma" and "mu," respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. IgG are the exemplary classes of antibodies employed herein because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting. In one embodiment, an antibody is a monoclonal antibody. Encompassed also are humanized, chimeric, human, or otherwise-human-suitable antibodies. "Antibodies" also includes any fragment or derivative of any of the herein described antibodies.

The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a "complementarity-determining region" or "CDR" (e.g. residues 24-34 (L1 ), 50-56 (L2) and 89-97 (L3) in the light-chain variable domain and 31 -35 (H1 ), 50-65 (H2) and 95-102 (H3) in the heavy-chain variable domain; Kabat et al. 1991 ) and/or those residues from a "hypervariable loop" (e.g. residues 26-32 (L1 ), 50-52 (L2) and 91 -96 (L3) in the light-chain variable domain and 26-32 (H 1 ), 53-55 (H2) and 96-101 (H3) in the heavy-chain variable domain; Chothia and Lesk, J. Mol. Biol 1987;196:901 -917), or a similar system for determining essential amino acids responsible for antigen binding. Typically, the numbering of amino acid residues in this region is performed by the method described in Kabat et al., supra. Phrases such as "Kabat position", "variable domain residue numbering as in Kabat" and "according to Kabat" herein refer to this numbering system for heavy chain variable domains or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of CDR H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.

By "framework" or "FR" residues as used herein is meant the region of an antibody variable domain exclusive of those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into the contiguous regions separated by the CDRs (FR1 , FR2, FR3 and FR4).

The term "specifically binds to" means that an antibody can bind in a competitive binding assay to the binding partner, e.g. NKp46 or CFP, as assessed using either recombinant forms of the proteins, epitopes therein, or native proteins present on the surface of isolated target cells. Competitive binding assays and other methods for determining specific binding are further described below and are well known in the art.

When a first molecule (e.g. CFP, NKp46, another antibody) is said to "compete with" a second molecule (e.g. CFP, NKp46, another antibody) for binding to a target protein (e.g. CFP, NKp46, another antibody), it means that the first molecule competes with the second molecule in a binding assay using either recombinant target protein or surface expressed target protein. For example, if a test antibody reduces the binding of NKp46 to a CFP polypeptide or CFP-expressing cell in a binding assay, the antibody is said to "compete" respectively with NKp46.

The term "affinity", as used herein, means the strength of the binding of an antibody to an epitope. The affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen. The affinity constant K _a is defined by 1/Kd. Methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. One standard method well known in the art for determining the affinity of mAbs is the use of surface plasmon resonance (SPR) screening (such as by analysis with a BIAcore™ SPR analytical device).

The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a cell or cell composition, a biological macromolecule, or an extract made from biological materials. The term "therapeutic agent" refers to an agent that has biological activity.

A "humanized" or "human" antibody refers to an antibody in which the constant and variable framework region of one or more human immunoglobulins is fused with the binding region, e.g. the CDR, of an animal immunoglobulin. Such antibodies are designed to maintain the binding specificity of the non-human antibody from which the binding regions are derived, but to avoid an immune reaction against the non-human antibody. Such antibodies can be obtained from transgenic mice or other animals that have been "engineered" to produce specific human antibodies in response to antigenic challenge (see, e.g., Green et al. (1994) Nature Genet 7:13; Lonberg et al. (1994) Nature 368:856; Taylor et al. (1994) Int Immun 6:579, the entire teachings of which are herein incorporated by reference). A fully human antibody also can be constructed by genetic or chromosomal transfection methods, as well as phage display technology, all of which are known in the art (see, e.g., McCafferty et al. (1990) Nature 348:552-553). Human antibodies may also be generated by in vitro activated B cells (see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275, which are incorporated in their entirety by reference).

A "chimeric antibody" is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity. The terms "Fc domain," "Fc portion," and "Fc region" refer to a C-terminal fragment of an antibody heavy chain, e.g., from about amino acid (aa) 230 to about aa 450 of human γ (gamma) heavy chain or its counterpart sequence in other types of antibody heavy chains (e.g., α, δ, ε and μ for human antibodies), or a naturally occurring allotype thereof. Unless otherwise specified, the commonly accepted Kabat amino acid numbering for immunoglobulins is used throughout this disclosure (see Kabat et al. (1991 ) Sequences of Protein of Immunological Interest, 5th ed., United States Public Health Service, National Institute of Health, Bethesda, MD).

The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a term well understood in the art, and refers to a cell-mediated reaction in which non- specific cytotoxic cells that express Fc receptors (FcRs) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. Non-specific cytotoxic cells that mediate ADCC include natural killer (NK) cells, macrophages, monocytes, neutrophils, and eosinophils.

The terms "isolated", "purified" or "biologically pure" refer to material that is substantially or essentially free from components which normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.

The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.

The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (nonrecombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

Within the context herein, the term antibody that "binds" a polypeptide or epitope designates an antibody that binds said determinant with specificity and/or affinity.

The term "identity" or "identical", when used in a relationship between the sequences of two or more polypeptides, refers to the degree of sequence relatedness between polypeptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity" measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms"). Identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1 , Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991 ; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

Methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well-known Smith Waterman algorithm may also be used to determine identity.

In the context herein, "treatment" or "treating" refers to preventing, alleviating, managing, curing or reducing one or more symptoms or clinically relevant manifestations of a disease or disorder, unless contradicted by context. For example, "treatment" of a patient in whom no symptoms or clinically relevant manifestations of a disease or disorder have been identified is preventive or prophylactic therapy, whereas "treatment" of a patient in whom symptoms or clinically relevant manifestations of a disease or disorder have been identified generally does not constitute preventive or prophylactic therapy.

As used herein, the terms "cancer antigen" and "tumor antigen" are used interchangeably and refer to antigens that are differentially expressed by cancer cells and can thereby be exploited in order to target cancer cells. Cancer antigens are antigens which can potentially stimulate apparently tumor-specific immune responses. Some of these antigens are encoded, although not necessarily expressed, by normal cells. These antigens can be characterized as those which are normally silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation and those that are temporally expressed such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses. The cancer antigens are usually normal cell surface antigens which are either over- expressed or expressed at abnormal times. Ideally the target antigen is expressed only on proliferative cells (e.g., tumour cells), however this is rarely observed in practice. As a result, target antigens are usually selected on the basis of differential expression between proliferative and healthy tissue. Antibodies have been raised to target specific tumour related antigens including: Receptor Tyrosine Kinase- like Orphan Receptor 1 (ROR1 ), Cripto, CD4, CD20, CD30, CD19, CD38, CD47, Glycoprotein NMB, CanAg, Her2 (ErbB2/Neu), CD22 (Siglec2), CD33 (Siglec3), CD79, CD138, CD171 , PSCA, L1 -CAM, PSMA (prostate specific membrane antigen), BCMA, CD52, CD56, CD80, CD70, E-selectin, EphB2, Melanotransferin, Mud 6 and TMEFF2. Examples of cancer antigens also include B7-H3, B7-H4, B7-H6, PD-L1 , MAGE, MART- 1/Melan-A, gp100, adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC)-C017-1A/GA733, Killer-lg Like Receptor 3DL2 (KIR3DL2), protein tyrosine kinase 7(PTK7), receptor protein tyrosine kinase 3 (TYRO-3), nectins (e.g. nectin- 4), carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, amll , prostate specific antigen (PSA), T-cell receptor/CD3-zeta chain, MAGE-family of tumor antigens, GAGE-family of tumor antigens, anti-Mullerian hormone Type II receptor, delta-like ligand 4 (DLL4), DR5, BAGE, RAGE, LAGE-1 , NAG, GnT-V, MUM-1 , CDK4, MUC family, VEGF, VEGF receptors, Angiopoietin-2, PDGF, TGF-alpha, EGF, EGF receptor, a member of the human EGF-like receptor family such as HER-2/neu, HER-3, HER-4 or a heterodimeric receptor comprised of at least one HER subunit, gastrin releasing peptide receptor antigen, Muc-1 , CA125, ανβ3 integrins, α5β1 integrins, al^3-integrins, PDGF beta receptor, SVE-cadherin, IL-8, hCG, IL-6, IL-6 receptor, IL-15, a-fetoprotein, E-cadherin, a- catenin, β-catenin and γ-catenin, p120ctn, PRAME, NY-ESO-1 , cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, imp-1 , P1A, EBV- encoded nuclear antigen (EBNA)-1 , brain glycogen phosphorylase, SSX-1 , SSX-2 (HOM- MEL-40), SSX-1 , SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2, although this is not intended to be exhaustive.

As used herein, the term "bacterial antigen" includes, but is not limited to, intact, attenuated or killed bacteria, any structural or functional bacterial protein or carbohydrate, or any peptide portion of a bacterial protein of sufficient length (typically about 8 amino acids or longer) to be antigenic. Examples include gram-positive bacterial antigens and gram- negative bacterial antigens. In some embodiments the bacterial antigen is derived from a bacterium selected from the group consisting of Helicobacter species, in particular Helicobacter pylons; Borrelia species, in particular Borrelia burgdorferi; Legionella species, in particular Legionella pneumophilia; Mycobacteria s species, in particular M. tuberculosis, M. avium, M. intracellulare, M. kansasii, M. gordonae; Staphylococcus species, in particular Staphylococcus aureus; Neisseria species, in particular N. gonorrhoeae, N. meningitidis; Listeria species, in particular Listeria monocytogenes; Streptococcus species, in particular S. pyogenes, S. agalactiae; S. faecalis; S. bovis, S. pneumoniae; anaerobic Streptococcus species; pathogenic Campylobacter species; Enterococcus species; Haemophilus species, in particular Haemophilus influenzae; Bacillus species, in particular Bacillus anthracis; Corynebacterium species, in particular Corynebacterium diphtheriae; Erysipelothrix species, in particular Erysipelothrix rhusiopathiae; Clostridium species, in particular C. perfringens, C. tetani; Enterobacter species, in particular Enterobacter aerogenes, Klebsiella species, in particular Klebsiella 1S pneumoniae, Pasteurella species, in particular Pasteurella multocida, Bacteroides species; Fusobacterium species, in particular Fusobacterium nucleatum; Streptobacillus species, in particular Streptobacillus moniliformis; Treponema species, in particular Treponema pertenue; Leptospira; pathogenic Escherichia species; and Actinomyces species, in particular Actinomyces israeli.

As used herein, the term "viral antigen" includes, but is not limited to, intact, attenuated or killed whole virus, any structural or functional viral protein, or any peptide portion of a viral protein of sufficient length (typically about 8 amino acids or longer) to be antigenic. Sources of a viral antigen include, but are not limited to viruses from the families: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., Ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses); Bunyaviridae (e.g., Hantaan viruses, bunya viruses, phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviruses and rotaviruses); Bornaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), Hepatitis C; Norwalk and related viruses, and astroviruses).

As used herein, "NK cells" refers to a sub-population of lymphocytes that is involved in non-conventional immunity. NK cells can be identified by virtue of certain characteristics and biological properties, notably the expression of surface antigens NKp46 (NK cell specific) and/or CD56 for human NK cells, the absence of the alpha/beta or gamma/delta TCR complex on the cell surface, the ability to bind to and kill cells that fail to express "self" MHC/HLA antigens by the activation of specific cytolytic machinery, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response. Any of these characteristics and activities can be used to identify NK cells, using methods well known in the art. Any subpopulation of NK cells will also be encompassed by the term NK cells. Within the context herein "active" NK cells designate biologically active NK cells, including NK cells having the capacity of lysing target cells or enhancing the immune function of other cells. NK cells can be obtained by various techniques known in the art, such as isolation from blood samples, cytapheresis, tissue or cell collections, etc. Useful protocols for assays involving NK cells can be found in Natural Killer Cells Protocols (edited by Campbell KS and Colonna M). Human Press, pp. 219-238 (2000).

"NKp46" refers to a protein or polypeptide encoded by the Ncr1 gene or by a cDNA prepared from such a gene. Any naturally occurring isoform, allele or variant is encompassed by the term NKp46 polypeptide (e.g., an NKp46 polypeptide 90%, 95%, 98% or 99% identical to SEQ ID NO 1 , or a contiguous sequence of at least 20, 30, 50, 100 or 200 amino acid residues thereof). The 304 amino acid residue sequence of human NKp46 (isoform a) is shown as follows:

MSSTLPALLC VGLCLSQRIS AQQQTLPKPF IWAEPHFMVP KEKQVTICCQ GNYGAVEYQL HFEGSLFAVD RPKPPERINK VKFYIPDMNS RMAGQYSCIY RVGELWSEPS NLLDLVVTEM YDTPTLSVHP GPEVISGEKV TFYCRLDTAT SMFLLLKEGR SSHVQRGYGK VQAEFPLGPV TTAHRGTYRC FGSYNNHAWS FPSEPVKLLV TGDIENTSLA PEDPTFPADT WGTYLLTTET GLQKDHALWD HTAQNLLRMG LAFLVLVALV WFLVEDWLSR KRTRERASRA STWEGRRRLN

TQTL

(SEQ ID NO: 1 ).

SEQ ID NO: 1 corresponds to NCBI accession number NP_004820, the disclosure of which is incorporated herein by reference. The human NKp46 mRNA sequence is described in NCBI accession number NM_004829, the disclosure of which is incorporated herein by reference. Complement Factor P (CFP), also referred to as "Properdin", is a glycoprotein circulating as dimers, trimers and tetramers of a single chain glycoprotein in blood serum. Each subunit is composed mainly of 6 thrombospondin-like repeat domains. CFP stabilizes the inherently labile C3 and C5 convertase complexes (C3bBb and C3bnBb), and thus acts as a positive regulator of the alternative pathway of complement. The functional activity of the polymers increases with their size with the tetramer being 10 times as active as the dimer. The CFP protein and gene sequences are known in the art. Natural CFP protein may be purified from normal serum. Recombinant CFP protein may be expressed and purified using conventional techniques. The CFP protein may or may not be purified. The CFP may be a monomer, dimer, trimer, tetramer, or a combination thereof. For example, amino acid sequences of human CFP are described in GenBank accession number P27918 and also shown below in SEQ ID NO: 2:

MITEGAQAPR LLLPPLLLLL TLPATGSDPV LCFTQYEESS GKCKGLLGGG VSVEDCCLNT AFAYQKRSGG LCQPCRSPRW SLWSTWAPCS VTCSEGSQLR YRRCVGWNGQ CSGKVAPGTL EWQLQACEDQ QCCPEMGGWS GWGPWEPCSV TCSKGTRTRR RACNHPAPKC GGHCPGQAQE

SEACDTQQVC PTHGAWATWG PWTPCSASCH GGPHEPKETR SRKCSAPEPS QKPPGKPCPG LAYEQRRCTG LPPCPVAGGW GPWGPVSPCP VTCGLGQTME QRTCNHPVPQ HGGPFCAGDA TRTHICNTAV PCPVDGEWDS WGEWSPCIRR NMKSISCQEI PGQQSRGRTC RGRKFDGHRC AGQQQDIRHC YSIQHCPLKG SWSEWSTWGL CMPPCGPNPT RARQRLCTPL LPKYPPTVSM VEGQGEKNVT FWGRPLPRCE ELQGQKLWE EKRPCLHVPA CKDPEEEEL

(SEQ ID NO : 2)

Use of agents that provide or enhance NKp46+ NK cell activity

Agents that enhance or provide NKp46+ NK cell activity can be particularly useful to treat cancer or infectious disease characterized by CFP-opsonized disease cells. Providing exogenously administered NKp46+ immune effector cells (e.g. NKp46+ NK cells) or enhancing the activity of endogenous NKp46+ NK cells in an individual can be useful to enhance the ability of NK cells in a context where CFP opsonization of disease cells (e.g. cancer cells, bacterial cells, virally infected cell or generally any other pathogenic cells) provides the potential for such cells to control disease in an individual. In some individuals, natural NKp46+ NK cell activity may not suffice to control disease, for example due to insufficient numbers of NK cells or insufficient cytotoxicity of NK cells toward disease cells for example due to lack of sufficient activating signalling (e.g. disease cells do not bear sufficient ligands for activating NK cell receptors) or due to inhibitory signalling (by inhibitory NK cell receptors); in such individuals enhancement of NKp46+ NK cell activity can be beneficial. The disease cells can be naturally opsonized with CFP, or may be opsonized with CFP following a treatment that induces or increases CFP opsonization. For example, a treatment comprising an agent that enhances or provides NKp46+ NK cell activity can be administered subsequent to or in combination with a treatment that comprises exogenous administration of CFP (e.g., as a therapeutic agent). In another example, a treatment comprising an agent that enhances or provides NKp46+ NK cell activity can be administered subsequent to or in combination with a treatment that comprises an antibody comprising an Fc domain that binds human C1 q.

Examples of cancer that can treated with agents that enhance or provide NKp46+ NK cell activity include, inter alia, hematologoical, e.g., leukemias and lymphomas, and solid tumors, e.g. carcinomas and sarcomas. Malignant tumors which may be treated are classified herein according to the embryonic origin of the tissue from which the tumor is derived. Carcinomas are tumors arising from endodermal or ectodermal tissues such as skin or the epithelial lining of internal organs and glands. Sarcomas, which arise less frequently, are derived from mesodermal connective tissues such as bone, fat, and cartilage. The leukemias and lymphomas are malignant tumors of hematopoietic cells of the bone marrow. Leukemias proliferate as single cells, whereas lymphomas tend to grow as tumor masses. Malignant tumors may show up at numerous organs or tissues of the body to establish a cancer. Cancers that can be treated include, but are not limited to, bladder, brain, breast, cervical, colo-rectal, esophageal, kidney, liver, lung, nasopharangeal, pancreatic, prostate, skin, stomach, uterine, ovarian, and testicular cancers.

Examples of infectious disease include disease caused by bacteria or viral infection, inter alia, meningitis, such as bacterial meningitis, viral meningitis, fungal meningitis, parasitic meningitis, or noninfectious meningitis, or for example infection with Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Listeria monocytogenes, Group B streptococci, or Escherichia coli.

One example of providing NKp46+ NK cell activity is to administer a composition comprising NKp46-expressing human immune effector cells. Thus one example of a composition that can be administered to an individual having a cancer or infectious disease characterized by CFP-opsonized disease cells is a composition comprising NKp46- expressing human effector cells (e.g. NKp46-expressing human NK cells). The cells may be purified or isolated. A cell composition may or may not comprise other cells in addition to the effector cells. The NKp46-expressing effector cells can for example be autologous cells derived from the individual, expanded and/or activated ex vivo, and then re-infused to the individual. Alternatively, such cells can be allogeneic cells derived from a donor, expanded and/or activated in vitro (or ex vivo), and infused to an individual. In another example, the cells are effector cell lines, e.g., NK cell lines. While NK cells naturally express NKp46, the most widely used human NK cell lines may not necessarily express NKp46 and therefore can be engineered to express NKp46. Furthermore, NK cell lines can be engineered to express NKp46 at higher levels that the levels typically observed in normal human NK cells in healthy individuals. For example, a widely used NK cell line, the NK-92 cell line (available from the ATCC under reference CRL-2407), can be made to express NKp46 polypeptide at its surface. In another example, a KHYG-1 NK cell line (available from the German Collection of Microorganisms and Cell Cultures (DSMZ) as reference ACC-725) can be transfected with human NKp46 and thus made to express NKp46. Optionally, but by no means required, the NK cells can be selected (e.g. by cell sorting, by FACS using an anti- NKp46 antibody) so that substantially all NK cells in the composition express NKp46.

In embodiments where the NK cells are allogeneic, they can optionally be selected to be alloreactive NK cells, optionally NK cells that lack one or more inhibitory cell surface receptors bound by an HLA molecule of an individual who is to be treated with the cells. Such cells can be derived from a donor, more particularly an alloreactive donor, selected for having mismatch with the recipient for at least one antigen of the three major HLAs, optionally those of the HLA-C and HLA-B groups. For example, if the recipient presents a group 1 HLA-C allotype, the donor has a group 2 HLA-C allotype. Reciprocally, for a recipient having a group 2 HLA-C allotype, the donor is selected such that it presents a group 1 HLA-C allotype. In an additional example, for a recipient having a group Bw4 HLA-B allotype, the donor is selected such that it does not present a group Bw4 HLA-B allotype. Reciprocally, for a recipient who does not have a group Bw4 HLA-B allotype, the donor is selected such that it presents a group Bw4 HLA-B allotype. Alloreactive cells and methods for obtaining and administering them are described in PCT application no. WO2005/009466, the disclosure of which is incorporated herein by reference. As discussed in PCT application no. WO2005/009466, when donor-recipient haplo-mismatched pairs were also KIR ligand mismatched, 100% of the donors tested had (at least some) alloreactive NK cells in their repertoires. Therefore, in on embodiment, in a population of NK cells from an alloreactive donor, 5-50 % of NK cells are alloreactive.

Alloreactive NK cells are prepared from a donor by different techniques which are known by the skilled person. More particularly, these cells can be obtained by different isolation and enrichment methods using peripheral blood mononuclear cells (lymphoprep, leucapheresis, etc.). These cells can be prepared by Percoll density gradients, by negative depletion methods or by FACS sorting methods. These cells can also be isolated by column immunoadsorption using an avidine-biotin system or by immunoselection using microbeads grafted with antibodies. It is also possible to use combinations of these different techniques, optionally combined with plastic adherence methods. For example, the alloreactive NK cells can be prepared by providing blood mononuclear cells depleted of T cells from the donor, activating said cells with phytohemagglutinin (PHA) and culturing said cells with interleukin (IL)-2 and irradiated feeder cells. Optionally, the population of NK cells can be tested for the alloreactivity against the recipient cells. Optionally, said NK cells can be cloned and each clone is tested for the alloreactivity against the recipient cells. Optionally, the NK cell clones presenting the alloreactivity are pooled. The alloreactivity is tested by standard ⁵¹ Cr release cytotoxicity against recipient PHA lymphoblasts, or Epstein-Barr virus transformed B lymphoblastoid cell lines.

Generally, autologous and/or allogeneic NK cells can be prepared by a method comprising: a) providing NK cells from an appropriate donor (e.g. the individual to be treated, an allogeneic donor, an alloreactive donor); b) activating said NK cells, optionally with a cytokine, optionally IL-2; c) collecting the active NK cells resulting from step b). Optionally, said method comprises an additional step of testing the alloreactivity of the NK cells collected from step c) against the recipient cells. Alternatively, the alloreactive NK cells can be prepared by a method comprising : a) providing NK cells from an alloreactive donor; b) isolating or cloning said NK cells; c) activating said NK cells with IL-2; d) testing the alloreactivity of the NK cells resulting from step c) against the recipient cells; and, optionally, e) pooling the alloreactive NK cells. In any method, NK cells can be further expanded in vivo or in vitro.

The efficient amount of effector cells (e.g. NK cells, a composition of substantially pure or isolated NK cells) administered to an individual can for example be between about 1 x 10 ⁴ to about 1 x 10 ⁹ cells/kg of recipient's body weight, optionally between about 0.05 x 10 ⁶ and about 1 x 10 ⁸ cells/kg of recipient's body weight. Subranges of pure alloreactive NK cells are also provided, for example, about 0.05 x 10 ⁶ to 5 x 10 ⁶ cells/kg of recipient's body weight, about 5 x 10 ⁶ to 10 x 10 ⁶ cells/kg of recipient's body weight, about 1 x 10 ⁷ to 5 x 10 ⁷ cells/kg of recipient's body weight, about 5 x 10 ⁷ to 1 x10 ⁸ cells/kg of recipient's body weight.

Another example of a composition that can be administered to an individual having a cancer or infectious disease characterized by CFP-opsonized pathogenic cells is a composition comprising an immunotherapeutic agent (e.g. a monoclonal antibody) that directly or indirectly increases the activity or number of NKp46-expressing NK cells. For example, a composition can comprise a cytokine that enhances NK cell activity. In another example, a composition can comprise an immunotherapeutic agent (e.g. a monoclonal antibody) that binds (e.g. and leads to activation of) an activating receptor (including co- activatory receptors) expressed by NKp46-expressing NK cells (e.g., activating receptors CD16, TMEM173 also known as Stimulator of interferon genes (STING), CD40, CD27, OX- 40, NKG2D, NKG2E, NKG2C, KIR2DS2, KIR2DS4, glucocorticoid-induced tumor necrosis factor receptor (GITR), CD137, NKp30, NKp44, or DNAM-1 , 2B4 (CD224). A further example of a composition that can be administered to an individual having a cancer or infectious disease characterized by CFP-opsonized pathogenic cells is a composition comprising an immunotherapeutic agent (e.g. a monoclonal antibody) that binds and inhibits an inhibitory NK cell and/or T cell receptor, or a natural ligand of such an inhibitory receptor (e.g. an inhibitory Siglec family member, LAG-3, PD-1 (CD279) or its natural ligand PD-L1 , TIM-3, TIGIT or CD96).

A further example of a composition that can be administered to an individual having a cancer or infectious disease characterized by CFP-opsonized pathogenic cells is a composition comprising an immunotherapeutic agent (e.g. an antibody, a multi-specific protein or antibody) that binds both NKp46 on an NK cell and a CFP-opsonized pathogenic cell; optionally, the agent which enhances the NKp46-CFP interaction.

A further example of a composition that can be administered to an individual having a cancer or infectious disease characterized by CFP-opsonized pathogenic cells is a composition comprising antibody or other Fc domain-containing protein capable of binding, via its Fc domain to CD16 on NK cells. Such antibody can act as an CD16 agonist in NK cells, can optionally provide enhanced cytotoxicity together with NKp46 signalling via CFP binding, and can inducing ADCC toward a cell to which it is bound, e.g. via CD16 expressed by an NK cell. For treatment of cancer, such antibody or other protein will typically comprise a domain (e.g. a hypervariable domain) that binds to an antigen of interest, e.g. an antigen present on a tumor cell (tumor antigen), and an Fc domain or portion thereof, and will exhibit binding to the antigen via the antigen binding domain and to Fey receptors (e.g. CD16) via the Fc domain. For treatment of infection (e.g. viral infection, bacterial infection), such antibody or other protein will typically comprise a domain (e.g. a hypervariable domain) that binds to an antigen of interest, e.g. an antigen present on an infected cell or on a pathogen (e.g. a viral or bacterial antigen), and an Fc domain or portion thereof, and will exhibit binding to the antigen via the antigen binding domain and to Fey receptors (e.g. CD16) via the Fc domain. In one embodiment, ADCC activity will be mediated at least in part by CD16. In one embodiment, the additional therapeutic agent is an antibody having a native or modified human Fc domain, for example a Fc domain from a human lgG1 or lgG3 antibody. The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a term well understood in the art, and refers to a cell-mediated reaction in which non-specific cytotoxic cells that express Fc receptors (FcRs) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. Non-specific cytotoxic cells that mediate ADCC include natural killer (NK) cells, macrophages, monocytes, neutrophils, and eosinophils. The term "ADCC-inducing antibody" refers to an antibody that demonstrates ADCC as measured by assay(s) known to those of skill in the art. Such activity is typically characterized by the binding of the Fc region with various FcRs. Without being limited by any particular mechanism, those of skill in the art will recognize that the ability of an antibody to demonstrate ADCC can be, for example, by virtue of it subclass (such as lgG1 or lgG3), by mutations introduced into the Fc region, or by virtue of modifications to the carbohydrate patterns in the Fc region of the antibody. Examples of antibodies that induce ADCC include rituximab (for the treatment of lymphomas, CLL, trastuzumab (for the treatment of breast cancer), alemtuzumab (for the treatment of chronic lymphocytic leukemia) and cetuximab (for the treatment of colorectal cancer, head and neck squamous cell carcinoma). Examples of ADCC-enhanced antibodies include but are not limited to: GA-101 (hypofucosylated anti- CD20), margetuximab (Fc enhanced anti-HER2), mepolizumab, MEDI-551 (Fc engineered anti-CD19), obinutuzumab (glyco-engineered/hypofucosuylated anti-CD20), ocaratuzumab (Fc engineered anti-CD20), XmAb ^® 5574/MOR208 (Fc engineered anti-CD19).

Agents that bind disease cells and recruit NK cells

In other embodiments, agents that induce CFP deposition on disease causing cells and/or that mediate recruitment of NKp46-expressing NK cells to disease causing cells (e.g. tumor cells, bacterial cells, etc.) is a multispecific binding protein (e.g. a bispecific antibody) can be used to treat cancer or infectious disease, irrespective of whether or not the disease is characterized by CFP opsonized cells. For example, an agent (e.g. a protein) that comprises (i) a CFP polypeptide or fragment thereof that binds NKp46 and (ii) an antigen binding domain (e.g. an antibody variable region(s) or hypervariable region) that binds a cell surface antigen on disease causing cells (e.g. that binds a cancer antigen, a bacterial antigen, a viral antigen) can be constructed. Such an agent can be referred to as a multi- specific (or bi-specific) binding protein that binds both human NKp46 and an antigen on a disease cell (e.g. a cancer antigen, a bacterial antigen, a viral antigen). Such an agent will bind to disease causing cells via its antigen binding domain (e.g. an antibody variable region(s) or hypervariable region) that binds a cell surface antigen on disease causing cells, and the CFP domain will bind to NKp46 on NK cells, thereby promote NK cell recognition and/or cytotoxicity toward the disease causing cell, even where the disease causing cell is otherwise not sufficiently CFP opsonized to elicit NK cell cytotoxicity. An antigen binding domain can be any domain comprising a three-dimensional structure capable of immunospecifically binding to an epitope, e.g., on a cancer antigen, a bacterial antigen or a viral antigen. Thus, in one embodiment, said domain can comprise a hypervariable region, optionally a V _H and/or V _L domain of an antibody chain, optionally at least a V _H domain, optionally a scFv (e.g., a V _H and a V _L domain separated by a flexible linker peptide). In another embodiment, the antigen binding domain may comprise at least one complementarity determining region (CDR) of an antibody chain. In another embodiment, the binding domain may comprise a polypeptide domain from a non-immunoglobulin scaffold.

Combination therapies

The agent that enhances or provides NKp46+ NK cell activity and the agents that recruits NKp46 cells, such as the multi-specific binding protein) may each be used as monotherapies, or combined treatments with one or more other therapeutic agents, including agents normally utilized for the particular therapeutic purpose for which the agent is being administered. The additional therapeutic agent will normally be administered in amounts and treatment regimens typically used for that agent in a monotherapy for the particular disease or condition being treated. Such therapeutic agents include, but are not limited to anti-cancer agents and chemotherapeutic agents. In one embodiment, the agent that enhances or provides NKp46+ NK cell activity can be used in combination with a second or additional second therapeutic agent, wherein the second or additional second therapeutic agent causes, increases or promotes the deposition (e.g. opsonization) of CFP on a target cell. In one embodiment, the agent is a CFP protein. In another embodiment, the agent is an Fc domain-comprising protein, optionally an antibody (e.g. a human IgG isotype, lgG1 , lgG3, etc.) or antibody fragment, that specifically binds to an antigen (e.g. a cancer antigen) on a target cell to be depleted. Optionally the antibody is capable of binding human C1 q and/or recruiting complement factors, and in turn recruiting CFP, to a target cell (e.g. a disease cell). In one embodiment, such an agent that enhances CFP opsonization can be used advantageously in combination with a therapy that enhances NK cell activity in an individual, for example administration to an individual of an NKp46-expressing effector cell (e.g. a native or engineered NK cell), an agent that acts as agonist at an activating receptor on the surface of an NK cell, or an agent that acts as antagonist at an inhibitory receptor on the surface of an NK cell.

In the treatment methods, the agent that enhances or provides NKp46+ NK cell activity and the second therapeutic agent can be administered separately, together or sequentially, or in a cocktail. In some embodiments, the antigen-binding compound is administered prior to the administration of the second therapeutic agent. For example, the agent that enhances or provides NKp46+ NK cell activity can be administered approximately

0 to 30 days prior to the administration of the second therapeutic agent. In some embodiments, the agent that enhances or provides NKp46+ NK cell activity is administered from about 30 minutes to about 2 weeks, from about 30 minutes to about 1 week, from about

1 hour to about 2 hours, from about 2 hours to about 4 hours, from about 4 hours to about 6 hours, from about 6 hours to about 8 hours, from about 8 hours to 1 day, or from about 1 to 5 days prior to the administration of the second therapeutic agent. In some embodiments, the agent that enhances or provides NKp46+ NK cell activity is administered concurrently with the administration of the therapeutic agents. In some embodiments, the agent that enhances or provides NKp46+ NK cell activity is administered after the administration of the second therapeutic agent. For example, the agent that enhances or provides NKp46+ NK cell activity can be administered approximately 0 to 30 days after the administration of the second therapeutic agent. In some embodiments, the agent that enhances or provides NKp46+ NK cell activity is administered from about 30 minutes to about 2 weeks, from about 30 minutes to about 1 week, from about 1 hour to about 2 hours, from about 2 hours to about 4 hours, from about 4 hours to about 6 hours, from about 6 hours to about 8 hours, from about 8 hours to 1 day, or from about 1 to 5 days after the administration of the second therapeutic agent.

Use of agents that inhibit the NKp46-CFP interaction

Agents that inhibit the NKp46-CFP interaction can reduce NKp46+ NK cell activity, and can be particularly useful to treat inflammatory disease characterized by CFP-opsonized host cells or tissue. For example, antibodies inhibit the NKp46-CFP interaction can inhibit the CFP-mediated activation of NKp46-expressing cells, e.g. they can inhibit the NKp46 signaling pathway, optionally with or without blocking the binding to NKp46 of natural or endogenous ligands other than CFP. Agents (e.g. antibodies) that inhibit the NKp46-CFP interaction are thus referred to as "neutralizing" or "inhibitory" or "blocking" agents (or, e.g., antibodies). Such agents are useful, inter alia, for decreasing the activity of NKp46- expressing immune cells, e.g. for the treatment or prevention of conditions where decreasing NK cell activity can ameliorate, prevent, eliminate, or in any way improve the condition or any symptom thereof. A range of cellular assays can be used to assess the ability of the antibodies to antibodies to inhibit the NKp46-CFP interaction. Furthermore, any of a large number of assays, including molecular, cell-based, and animal-based models can be used to assess the ability of anti-NKp46 antibodies to modulate NKp46-expressing cell activity. An antibody may advantageously be a human, humanized or chimeric antibody, e.g. comprising a human constant regions and optionally further human variable domain framework segments. Assays include, without limitation, any of the assays in the "Examples" section herein. Exemplary anti-NKp46 antibodies include antibody Bab281 (Beckman Coulter, inc.) , or antibodies having the VH and VL variable region sequences of mAbsl , 2, 3, 4, 6 and 9 below, or having a VH CDR1 , 2 and 3 of the VH of mAbl , 2, 3, 4, 6 or 9 and a VL CDR1 , 2 and 3 of the VL of the respective mAbl , 2, 3, 4, 6 or 9, as shown in Table 1 below.

Table 1

Antibody SEQ ID Amino acid sequence NO

mAbl VH 3 QVQLQQSGPELVKPGASVKMSCKASGYTFTDYVINWGKQRSGQGLEWIGEI

YPGSGTNYYNEKFKAKATLTADKSSNIAYMQLSSLTSEDSAVYFCARRGRY GLYAMDYWGQGTSVTVSS mAbl VL 4 DIQMTQTTSSLSASLGDRVTI SCRASQDI SNYLNWYQQKPDGTVKLLIYYT

SRLHSGVPSRFSGSGSGTDYSLTINNLEQEDIATYFCQQGNTRPWTFGGGT KLEIK mAb2 VH 5 EVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGY

ITYSGSTSYNPSLESRI SITRDTSTNQFFLQLNSVTTEDTATYYCARGGYY GSSWGVFAYWGQGTLVTVSA mAb2 VL 6 DIQMTQSPASLSASVGETVTITCRVSENIYSYLAWYQQKQGKSPQLLVYNA

KTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPWTFGGGT KLEIK mAb3 VH 7 EVQLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKSLEWIGGI

SPNIGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRGGS FDYWGQGTTLTVSS mAb3 VL 8 DIVMTQSPATLSVTPGDRVSLSCRASQSI SDYLHWYQQKSHESPRLLIKYA

SQSISGI PSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGT KLELK mAb4 VH 9 QVQLQQSAVELARPGASVKMSCKASGYTFTSFTMHWVKQRPGQGLEWIGYI

NPSSGYTEYNQKFKDKTTLTADKSSSTAYMQLDSLTSDDSAVYYCVRGSSR GFDYWGQGTLVTVSA mAb4 VL 10 DIQMIQSPASLSVSVGETVTITCRASENIYSNLAWFQQKQGKSPQLLVYAA

TNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGIYYCQHFWGTPRTFGGGT KLEIK mAb6 VH 11 QVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWMHWAKQRPGQGLEWIGHI

HPNSGISNYNEKFKGKATLTVDTSSSTAYVDLSSLTSEDSAVYYCARGGRF DDWGAGTTVTVSS mAb6 VL 12 DIVMTQSPATLSVTPGDRVSLSCRASQSI SDYLHWYQQKSHESPRLLIKYA

SQSISGI PSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFLMYTFGGG TKLEIK mAb9 VH 13 DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGY

ITYSGSTNYNPSLKSRI SITRDTSKNQFFLQLNSVTTEDTATYYCARCWDY ALYAMDCWGQGTSVTVSS mAb9 VL 14 DIQMTQSPASLSASVGETVTITCRTSENIYSYLAWCQQKQGKSPQLLVYNA

KTLAEGVPSRFSGSGSGTHFSLKINSLQPEDFGIYYCQHHYDTPLTFGAGT KLELK In one embodiment, the aforementioned CDRs are according to Kabat. In one embodiment, the aforementioned CDRs are according to Chotia numbering. In one embodiment, the aforementioned CDRs are according to IMGT numbering. In one embodiment, the VH and/or VL comprise human framework (FR) sequences, optionally human FR1 , FR2, FR3 and FR4 amino acid sequences. In another aspect, an antibody that inhibits the NKp46-CFP interaction competes with a monoclonal antibody of mAbsl , 2, 3, 4, 6 and/or 9 for binding to a NKp46 polypeptide.

In one embodiment, an antibody is a monospecific antibody (binds, via its variable region(s) to a single target protein, binds solely to NKp46). In one embodiment, an antibody that inhibits the NKp46-CFP interaction does not have substantial specific binding to human Fey receptors, e.g., any one or more of CD16A, CD16B, CD32A, CD32B and/or CD64). Such antibodies may comprise constant regions of various heavy chains that are known to lack or have low binding to Fey receptors. Alternatively, antibody fragments that do not comprise (or comprise portions of) constant regions, such as F(ab')2 fragments, can be used to avoid Fc receptor binding. Fc receptor binding can be assessed according to methods known in the art, including for example testing binding of an antibody to Fc receptor protein in a BIACORE assay. Also, generally any antibody IgG isotype can be used in which the Fc portion is modified (e.g., by introducing 1 , 2, 3, 4, 5 or more amino acid substitutions) to minimize or eliminate binding to Fc receptors (see, e.g., WO 03/101485, the disclosure of which is herein incorporated by reference). Assays such as cell based assays, to assess Fc receptor binding are well known in the art, and are described in, e.g., WO 03/101485.

In one embodiment, the antibody can comprise one or more specific mutations in the Fc region that result in "Fc silent" antibodies that have minimal interaction with effector cells. Silenced effector functions can be obtained by mutation in the Fc region of the antibodies and have been described in the art: N297A mutation, the LALA mutations, (Strohl, W., 2009, Curr. Opin. Biotechnol. vol. 20(6):685-691 ); and D265A (Baudino et al., 2008, J. Immunol. 181 : 6664-69) see also Heusser et al., WO2012/065950, the disclosures of which are incorporated herein by reference. In one embodiment, an antibody comprises one, two, three or more amino acid substitutions in the hinge region. In one embodiment, the antibody is an lgG1 or lgG2 and comprises one, two or three substitutions at residues 233-236, optionally 233-238 (EU numbering). In one embodiment, the antibody is an lgG4 and comprises one, two or three substitutions at residues 327, 330 and/or 331 (EU numbering). Examples of silent Fc lgG1 antibodies are the LALA mutant comprising L234A and L235A mutation in the lgG1 Fc amino acid sequence. Another example of an Fc silent mutation is a mutation at residue D265, or at D265 and P329 for example as used in an lgG1 antibody as the DAPA (D265A, P329A) mutation (US 6,737,056). Another silent lgG1 antibody comprises a mutation at residue N297 (e.g. N297A, N297S mutation), which results in aglycosylated/non-glycosylated antibodies. Other silent mutations include: substitutions at residues L234 and G237 (L234A/G237A); substitutions at residues S228, L235 and R409 (S228P/L235E/R409K _! T,M,L); substitutions at residues H268, V309, A330 and A331 (H268QA 309L/A330S/A331 S); substitutions at residues C220, C226, C229 and P238 (C220S/C226S/C229S/P238S); substitutions at residues C226, C229, E233, L234 and L235 (C226S/C229S/E233P/L234V/L235A; substitutions at residues K322, L235 and L235 (K322A/L234A/L235A); substitutions at residues L234, L235 and P331 (L234F/L235E/P331 S); substitutions at residues 234, 235 and 297; substitutions at residues E318, K320 and K322 (L235E/E318A/K320A/K322A); substitutions at residues (V234A, G237A, P238S); substitutions at residues 243 and 264; substitutions at residues 297 and 299; substitutions such that residues 233, 234, 235, 237, and 238 defined by the EU numbering system, comprise a sequence selected from PAAAP, PAAAS and SAAAS (see WO201 1/066501 ).

In one embodiment, the antibody can comprise one or more specific mutations in the Fc region that result in improved stability of an antibody of the disclosure, e.g. comprising multiple aromatic amino acid residues and/or having high hydrophobicity. For example, such an antibody can comprise an Fc domain of human lgG1 origin, comprises a mutation at Kabat residue(s) 234, 235, 237, 330 and/or 331 . One example of such an Fc domain comprises substitutions at Kabat residues L234, L235 and P331 (e.g., L234A/L235E/P331 S or (L234F/L235E/P331 S). Another example of such an Fc domain comprises substitutions at Kabat residues L234, L235, G237 and P331 (e.g., L234A/L235E/G237A/P331 S). Another example of such an Fc domain comprises substitutions at Kabat residues L234, L235, G237, A330 and P331 (e.g., L234A/L235E/G237A/A330S/P331 S). In one embodiment, the antibody comprises an Fc domain, optionally of human lgG1 isotype, comprising: a L234X-I substitution, a L235X ₂ substitution, and a P331 X ₃ substitution, wherein X-i is any amino acid residue other than leucine, X ₂ is any amino acid residue other than leucine, and X ₃ is any amino acid residue other than proline; optionally wherein X-i is an alanine or phenylalanine or a conservative substitution thereof; optionally wherein X ₂ is glutamic acid or a conservative substitution thereof; optionally wherein X ₃ is a serine or a conservative substitution thereof. In another embodiment, the antibody comprises an Fc domain, optionally of human lgG1 isotype, comprising: a L234X-I substitution, a L235X ₂ substitution, a G237X ₄ substitution and a P331X ₄ substitution, wherein X-i is any amino acid residue other than leucine, X ₂ is any amino acid residue other than leucine, X ₃ is any amino acid residue other than glycine, and X ₄ is any amino acid residue other than proline; optionally wherein X-i is an alanine or phenylalanine or a conservative substitution thereof; optionally wherein X ₂ is glutamic acid or a conservative substitution thereof; optionally, X ₃ is alanine or a conservative substitution thereof; optionally X ₄ is a serine or a conservative substitution thereof. In another embodiment, the antibody comprises an Fc domain, optionally of human lgG1 isotype, comprising: a L234X-I substitution, a L235X ₂ substitution, a G237X ₄ substitution, G330X ₄ substitution, and a P331X ₅ substitution, wherein X-i is any amino acid residue other than leucine, X ₂ is any amino acid residue other than leucine, X ₃ is any amino acid residue other than glycine, X ₄ is any amino acid residue other than alanine, and X ₅ is any amino acid residue other than proline; optionally wherein X-i is an alanine or phenylalanine or a conservative substitution thereof; optionally wherein X ₂ is glutamic acid or a conservative substitution thereof; optionally, X ₃ is alanine or a conservative substitution thereof; optionally, X ₄ is serine or a conservative substitution thereof; optionally X ₅ is a serine or a conservative substitution thereof. In the shorthand notation used here, the format is: Wild type residue: Position in polypeptide: Mutant residue, wherein residue positions are indicated according to EU numbering according to Kabat.

Agents that inhibit the NKp46-CFP interaction can be useful, for example, for treating or preventing inflammatory and autoimmune disorders, e.g. any inflammatory condition caused by CFP-opsonized cells, such as in an individual who has inflammation following an infection with a bacterial or viral pathogen, e.g. bacterial meningitis, viral meningitis, fungal meningitis, parasitic meningitis, or noninfectious meningitis, or for example for treating or preventing septic shock, septicemia, graft-versus-host disease. Examples of inflammatory and autoimmune disorders may include, inter alia, inflammatory vascular disease, arthritis, systemic lupus erythematosus, autoimmunity to central nervous system antigens, autoimmune diabetes, inflammatory bowel disease, autoimmune carditis, idiopathic inflammatory myopathy or autoimmune hepatitis.

Detection, Diagnosis, Pharmacodynamics, and Patient Selection

A. Methods of Detection

Methods of detecting CFP (and/or co-ligands that form a protein complex with CFP) and/or NKp46 in a biological sample are also provided. The detection of CFP (and/or co- ligands) and NKp46 proteins in a biological sample obtained from a subject is made possible by a number of conventional methods including but not limited to electrophoresis, chromatography, mass spectroscopy and immunoassays. A preferred method includes immunoassays whereby CFP or NKp46 proteins are detected by their interaction with a CFP-specific, or respectively NKp46-specific, antibody. In other examples, CFP can be detected using a composition comprising a NKp46 polypeptide or a CFP-binding fragment thereof. In other examples, NKp46 can be detected using a composition comprising a CFP polypeptide or a NKp46-binding fragment thereof. In one preferred method, a NKp46 polypeptide or fragment thereof that retains the ability to bind CFP is used to bind and/or detect the presence and/or levels of CFP, including but not limited to detecting CFP opsonization on pathogenic cells. An exemplary NKp46 polypeptide comprises an amino acid sequence of SEQ ID NO 1 , or a fragment thereof (e.g. a fragment of at least 50, 100 or more contiguous amino acids), or an amino acid sequence at least 70%, 80%, 90%, 95% amino acid identity to any of the foregoing. Such compounds can be used to detect the presence of proteins in either a qualitative or quantitative manner.

Exemplary immunoassays that can be used for the detection of NKp46 and/or CFP and co-ligands include, but are not limited to, radioimmunoassays, ELISAs, immunoprecipitation assays, Western blot, fluorescent immunoassays, and immunohistochemistry.

A biological sample that may contain CFP or co-ligand proteins thereof can be obtained from an individual. In one embodiment, the sample is a tumor or tumor-adjacent tissue sample. If the biological sample is of tissue or cellular origin, the sample can be solubilized in a lysis buffer optionally containing a chaotropic agent, detergent, reducing agent, buffer, and salts. The sample can be a biological fluid sample taken from a subject, e.g. a sample of urine, blood, serum, plasma, tears, saliva, cerebrospinal fluid, tissue, lymph, synovial fluid, or sputum etc. In one preferred embodiment, the biological fluid is whole blood, or more preferably serum or plasma. Serum is the component of whole blood that is neither a blood cell (serum does not contain white or red blood cells) nor a clotting factor. It is the blood plasma with the fibrinogens removed. Accordingly, serum includes all proteins not used in blood clotting (coagulation) and all the electrolytes, antibodies, antigens, hormones, and any exogenous substances (e.g., drugs and microorganisms). The sample can be diluted with a suitable diluent before contacting the sample to the antibody.

Generally, a sample obtained from a subject can be contacted with the antibody that specifically binds CFP or co-ligand. Optionally, the antibody can be fixed to a solid support to facilitate washing and subsequent isolation of the complex, prior to contacting the antibody with a sample. Examples of solid supports include glass or plastic in the form of, e.g., a microtiter plate, a stick, a bead, or a microbead. Antibodies can also be attached to a probe substrate or ProteinChip® array and can be analyzed by gas phase ion spectrometry as described above.

Immuoassays for the detection of CFP or co-ligand proteins include the ability to contact a biological sample with an antibody specific to a CFP or co-ligand protein under conditions such that an immunospecific antigen- antibody interaction may occur, followed by the detection or measurement of this interaction. The binding of the antibody to CFP or co- ligand proteins may be used to detect the presence and altered production of CFP or co- ligand proteins.

An exemplary immunoassay is ELISA. ELISA typically includes the use of two different CFP or co-ligand-specific antibodies: a capture antibody and a detection antibody. In some embodiments an antibody or antigen binding fragment thereof that recognizes CFP or co-ligand is used to capture most or all of the CFP or co-ligand in the sample. A detection antibody that can recognize most or all of the CFP or co-ligand can be used to determine the total level of CFP or co-ligand in the biological sample. In some embodiments, the detection antibody recognizes a different domain or epitope than the capture antibody.

In some embodiments, anti-CFP receptor antibodies, and methods of detecting CFP can be used to detect the presence and altered production of CFP or opsonization of a cell of interest by CFP.

Detecting CFP (and/or co-ligands that form a protein complex with CFP) in a biological sample (e.g., on cells in the biological sample) from an individual can be useful in methods to assess or predict whether cells in the biological sample are susceptible of undergoing NK cell-mediated lysis, for example in an individual harboring sufficient NKp46- expressing NK cell numbers and/or activity, for example in an individual receiving or who is a candidate to receive treatment with a therapeutic agent that enhances NK cell-mediated activity. In one embodiment, the therapeutic agent is an NK cell composition. In one embodiment, the therapeutic agent is an agent that acts as an agonist at an activating receptor on an NK cell. In one embodiment, the therapeutic agent is an agent that acts as an antagonist at an inhibitory receptor on an NK cell. In one embodiment, the therapeutic agent is an agent that is capable of binding a cancer antigen, a viral antigen or a bacterial antigen and is capable of mediating ADCC. In one embodiment, the NK cells are NKp46-expressing NK cells.

In other aspects, methods of detecting NKp46-expressing NK cells in a biological sample are also provided. Detecting NKp46 (e.g. NKp46-expressing NK cells) in a biological sample from an individual can be useful in methods to assess whether an individual has immune effector cells (e.g. NK cells) capable of depleting target cells bearing CFP, e.g. cancer cells, infected cells, pathogens, bacterial cells, e.g. Neisseria meningitides, that are opsonized with CFP.

B. Diagnosis

A disease or disorder in an individual can be detected by quantifying the amount of one or more CFP or co-ligands thereof in a biological sample of the individual, wherein an elevated or reduced amount of CFP or co- ligand in the individual's biological sample compared to a control (single or more preferably pooled or averaged values of normal individuals in same assay) is indicative of a disorder or disease state. In one embodiment the CFP opsonization of a cell type of interest (e.g. in a normal tissue, e.g. non-tumoral or non-infected self-tissue) is indicative of a immunological disorder, for example an autoimmune disorder or inflammation, for example an immunological disorder mediated at least in part by NK cells, e.g. NKp46-expressing NK cells. In one embodiment decreased or lack of CFP opsonization of a cell type of interest (e.g. a pathological cell, a cancer cell, a microbial cell, a bacterial cell, an infected cell, in tumor tissue) is indicative of a disorder characterized by insufficient NK cell activation. In one embodiment the CFP opsonization of a cell type of interest (e.g. a pathological cell, a cancer cell, a microbial cell, a bacterial cell, an infected cell, in tumor tissue) is indicative of a disorder characterized by susceptibility to disease control by NKp46 cells.

A biological sample includes tissue or biological fluid such as a fluid from the individual, for example, blood, plasma, synovial fluid or lymph, or tumor tissue or tumor- adjacent tissue. A control refers to a biological sample from an individual not experiencing the disease or disorder being tested for. In some embodiment, the receptor or co-ligand is a membrane bound, transmembrane or membrane associated receptor or co-ligand. Therefore, in some embodiment, the biological sample is cells obtained from the subject.

The amount of CFP or co-ligand in a sample can be determined using conventional techniques such as chromatography, electrophoresis, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assays, mass spectrometry, spectrophotometry, or a combination thereof.

The severity of a disorder or a disease can be detected or assessed by quantifying the level of CFP or co-ligand in an individual's biological sample and correlating the amount of CFP in the individual's biological sample with amount CFP or co-ligand expression indicative of different stages of a disorder or disease. The amounts of CFP or co-ligand that correlate to different stages of disease or disorder or different levels of severity can be predetermined by quantifying CFP or co-ligand levels in patients at different stages of, or with different severity of, a disease or disorder.

C. Pharmacodynamic Markers

The effectiveness of treatments using agents that provide and/or modulate NKp46+ NK cell activity (e.g. effector cell infusions that comprise NKp46-expressing effector cells, agents that act as agonists or antagonists of NKp46-expressing NK cells) can be determined by assaying a sample obtained from a subject receiving treatment with the agent that modulates NKp46+ NK cell activity or that act as agonist or antagonist of NK cell function for changes in levels of CFP and/or co-ligands on the surface of cells or tissues of interest. For example, baseline levels of CFP in a biological sample obtained from a subject can be determined prior to treatment with a receptor agonist or antagonist. After or during treatment with the agent that acts as agonist or antagonist of NK cell function, biomarker (e.g. CFP and/or co-ligand) levels in biological samples from the subject can be monitored.

A change in biomarker level, for example a decline in CFP opsonization (lower level of CFP on cells of interest in a tissue characterized by inflammation), relative to baseline levels can indicate that the treatment is effective in reducing one or more symptoms of an inflammatory disorder. Alternatively, the cytokine levels in blood samples from a subject undergoing treatment can be compared to predetermined levels of biomarkers determined from subjects without the disease or condition being treated. In some embodiments the level of only one biomarker is monitored. In other embodiments, the levels of 2, 3, 4, 5 or more biomarkers are monitored.

The effectiveness of treatments can also be determined by assaying a sample obtained from a subject receiving treatment with the receptor agonist or antagonist for changes in the expression levels of genes, including, but not limited to, those encoding serum proteins, preferably proinflammatory cytokines and/or chemokines, as well as secreted factors, cell surface receptors, and transcription factors that are characteristic of inflammation or immune cell activation. Methods of measuring gene expression are well known in the art and include, but are not limited to, quantitative RT-PCR and microarray analysis.

Exemplary markers that can be monitored to determine the effectiveness of treatment include, but are not limited to, one or more of IL-Ιβ, TNF-a, TGF-beta, IFN-γ, IL-10, IL-17, IL-6, IL-23, IL-22, IL-21 , and MMPs.

D. Patient Selection

Methods of determining the level of CFP and/or co-ligand may also allow the selection of patients most likely to respond to a therapy that provides, enhances or inhibits the activity of NKp46-expressing effector cells. For example, as shown herein, CFP alone, or in a complex, mediates an immune inhibitory signal via NKp46. CFP opsonization of pathogenic cells permits NKp46-expressing NK cells to deplete the pathogenic cells, e.g. N. meningitidis cells or cancer cells, and thus to control disease mediated by the pathogenic cells, e.g., N. meningitidis infection or cancer. Individuals having such disease and/or such CFP-opsonised pathogenic cells could be selected for treatment with an NKp46-expressing cell therapy (e.g. infusion of native or engineered effector cells that express NKp46), an agent that acts as agonist at an activating receptor on the surface of an NKp46-expressing NK cell (including but not limited to an agent that binds an antigen at the surface of the pathogenic cells and that mediates ADCC (and/or that binds CD16 on an NK cell via its Fc domain)), or an agent that acts as antagonist at an inhibitory receptor on the surface of an NKp46-expressing NK cell.

Similarly, some patients with pathogenic or other disease cells, e.g. N. meningitidis cells or cancer cells may have low levels of CFP (and/or other receptor proteins) on the pathogenic cells. Such subjects could be selected for treatment (and/or predicted to be good responders for such treatment) with an agent that mediates or increase CFP deposition (e.g. opsonization) on the disease cells or pathogenic cells. In one embodiment, the agent that mediates or increase CFP deposition is an agent (e.g. an antibody) that binds an antigen at the surface of the disease or pathogenic cells and mediates complement deposition via its Fc domain, e.g. an antibody that mediates complement-dependent cytotoxicity and/or whose Fc domain binds C1 q, e.g. an antibody of human lgG1 or lgG3 isotype. In one embodiment, the subjects can be treated with the agent that mediates or increase CFP deposition (e.g. opsonization) on the pathogenic cells, in combination with an NKp46-expressing cell therapy, an agent that acts as agonist at an activating receptor on the surface of an NKp46- expressing NK cell, or an agent that acts as antagonist at an inhibitory receptor on the surface of an NKp46-expressing NK cell.

In some embodiments, patients with increased CFP or co-ligand levels may be more likely to respond to treatment that enhances the NKp46+ NK cell activity, e.g., an NKp46- expressing cell therapy, an agent that acts as agonist at an activating receptor on the surface of an NKp46-expressing NK cell, an agent that acts as antagonist at an inhibitory receptor on the surface of an NKp46-expressing NK cell, or an antibody that binds an antigen at the surface of the disease or pathogenic cells and mediates complement deposition via its Fc domain and/or whose Fc domain binds C1 q. The effectiveness of the treatment that enhances the NKp46+ NK cell activity can be predicted by pre-screening target patients for CFP on pathogenic cells.

In some embodiments, patients with increased CFP or co-ligand levels and who also have sufficient or increased NKp46+ NK activity (e.g. increased number of NKp46+ NK cells and/or increased reactivity of such cells) may be more likely to respond to treatment that enhances the NKp46+ NK cell activity, e.g., an agent that acts as agonist at an activating receptor on the surface of an NKp46-expressing NK cell, or an agent that acts as antagonist at an inhibitory receptor on the surface of an NKp46-expressing NK cell. The effectiveness of the treatment that enhances the NKp46+ NK cell activity can be predicted by pre-screening target patients for number of NKp46+ NK cells and/or increased reactivity of such cells. For example, number of NKp46+ NK cells can be accessed via FACS. Reactivity of NKp46+ NK cells can be assessed, for example, by bringing cells into contact with a solid support (e.g. plate) comprising immobilized anti-NKp46 antibodies such that the antibodies mediate NKp46 crosslinking and activation on cells. In another aspect, inflammatory or autoimmune diseases/disorders may be associated with inappropriate CFP opsonization of host cells in inflamed tissues. Patients with inflammatory or autoimmune diseases/disorders may have cells bearing (e.g., inappropriately opsonized with) CFP (and/or other receptor proteins). Such subjects could be selected for treatment with an NKp46 antagonist, such as a recombinant CFP binding fragment that binds NKp46 or an anti-NKp46 antibody that interferes with the binding of CFP to NKp46, to decrease NKp46 signalling and reduce the autoimmune or inflammatory response.

Thus, in some embodiments, CFP on self-cells, can be an indicator of an inflammatory or autoimmune diseases/disorder. Subjects with CFP levels on host cells in inflamed tissue that is higher than a control can be selected for CFP antagonist therapy (and/or predicted to be good responders for such therapy), for example CFP binding fragment that binds NKp46 or an anti-NKp46 antibody that interferes with the binding of CFP to NKp46.

Patients can also be monitored for the efficacy of a treatment by screening the patients for levels of one or more biomarkers, including levels of one or more CFPs of ligands thereof, during the course of treatment. The dosage, frequency, or a combination thereof can be increased or decreased accordingly.

Identification of new modulators of the CFP-NKp46 interaction

Agents that interfere with or enhance the CFP-NKp46 interaction can be useful in a variety of application. For example, these compositions can be used to measure CFP-NKp46 interactions, to measure the inhibition or enhancement of CFP-NKp46 interactions, for the identification of CFP receptor expressing cells, for the identification of new cell types, and/or for studies into the molecular mechanisms of action of CFP and NKp46.

Additionally, bioactive agents may be screened for NKp46 agonistic or antagonistic activity, for example toward CFP-opsonized cells or in the presence of CFP. Accordingly, methods of screening for additional bioactive agents that function enhance, promote or cause an CFP-NKp46 interaction also provided. In another embodiment candidate bioactive agents are screened for their ability to interfere with or enhance the CFP-NKp46 interaction. The assays preferably utilize human NKp46 receptors and human CFP, although other Nkp46 and CFP may also be used.

Bioactive agents that may be screened for NKp46 agonistic or antagonistic activity, including for example to inhibit or enhance the CFP-NKp46 interaction, include, but are not limited to, proteins, small organic molecules, carbohydrates (including polysaccharides), polynucleotides and lipids. Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection. In addition, positive controls, i.e. the use of agents known to cause NKp46 agonistic or antagonistic activity may be used.

Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons, more preferably between 100 and 2000, more preferably between about 100 and about 1250, more preferably between about 100 and about 1000, more preferably between about 100 and about 750, more preferably between about 200 and about 500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, proteins, antibodies, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

Additional candidate agents include peptidomimetics. Peptidomimetics can be made as described, e.g., in WO 98/156401. Peptidomimetics, as used herein, refers to molecules which mimic peptide structures. Peptidomimetics have general features analogous to their parent structures, polypeptides, such as amphiphilicity. Peptidomimetics have been developed in a number of classes, such as peptoids, azapeptides, urea- peptidomimetics, sulphonamide peptides, oligoureas, oligocarbamates, Ν,Ν'-linked oligoureas, oligopyrrolinones, oxazolidin-2- ones, azatides, and hydrazino peptides.

Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Libraries of antibody variable domains can be produced through phage display techniques. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs. In a preferred embodiment, the candidate bioactive agents are organic chemical moieties or small molecule chemical compositions, a wide variety of which are available in the art.

In an exemplary assay, NKp46 and CFP proteins (e.g., independently as soluble proteins or as proteins bound to a surface such as a cell surface or solid support) are used to identify antibodies, small molecules, and other modulators of the NKp46-CFP interaction.

In another exemplary assay, NK expressing NKp46 or other cells made to express NKp46 can be used to identify antibodies, small molecules, and other modulators that are agonists or antagonists of NKp46, in the presence of CFP or CFP-opsonized cells.

In another exemplary assay, NK expressing NKp46 or other cells made to express NKp46 can be used to identify antibodies, small molecules, and other modulators that enhance the activity (e.g. the cytolytic activity) of NKp46-expressing NK cells, towards CFP- opsonized target cells (e.g. bacterial cells, virus infected cells, cancer cells).

In another exemplary assay, NK expressing NKp46 or other cells made to express NKp46 can be used to identify antibodies, small molecules, and other modulators that inhibit the activity (e.g. the cytolytic activity) of NKp46-expressing NK cells, towards CFP-opsonized target cells (e.g. healthy, non-tumoral or non-infected host cells, or host cells from inflamed tissues).

In one embodiment of such an assay, the method comprises: (i) bringing NKp46 (e.g. as a cell expressing NKp46 or as a soluble NKp46 protein) into contact with CFP (e.g. as a cell opsonized with CFP or as a soluble CFP protein) in the presence of a test agent (e.g. a plurality of test agents), and (ii) assessing binding of CFP to NKp46. A decrease in binding (compared to negative control) indicates that the agent interferes with binding of CFP and NKp46. An increase in binding (compared to negative control) indicates that the agent enhances the interaction of CFP and NKp46.

In one embodiment provided is a method of identifying agents that decrease NK cell activity, comprising: (i) bringing NKp46 (e.g. as a cell expressing NKp46 or as a soluble NKp46 protein) into contact with CFP (e.g. as a cell opsonized with CFP or as a soluble CFP protein) in the presence of a test agent (e.g. a plurality of test agents), (ii) assessing binding of CFP to NKp46. Optionally the method further comprises: (iii) selecting (e.g. for use in therapy, for production of a batch of therapeutic agent, for further processing or evaluation) an agent that interferes with binding of CFP and NKp46. In one embodiment, the agent is for use in treatment or prevention of an autoimmune or inflammatory condition. In one embodiment, the agent comprises an antibody. In one embodiment, the agent (e.g. an antibody) binds NKp46. Optionally, step (i) comprises a step of causing CFP opsonization of a cell (e.g. a target or disease cell). In one embodiment provided is a method of identifying agents that enhance NK cell activity, comprising: (i) bringing NKp46 (e.g. as a cell expressing NKp46 or as a soluble NKp46 protein) into contact with CFP (e.g. as a cell opsonized with CFP or as a soluble CFP protein) in the presence of a test agent (e.g. a plurality of test agents), (ii) assessing binding of CFP to NKp46. Optionally the method further comprises: (iii) selecting (e.g. for use in therapy, for production of a batch of therapeutic agent, for further processing or evaluation) an agent that does not substantially interfere with binding of CFP and NKp46. In one embodiment, the agent is for use in treatment or prevention of cancer or infectious disease. In one embodiment, the agent comprises an antibody. In one embodiment, the agent (e.g. an antibody) binds NKp46; optionally the agent binds NKp46 and a protein (e.g. cancer antigen, viral antigen, bacterial antigen) on a target cell. In one embodiment, the agent (e.g., an antibody) binds CFP (e.g., a CFP monomer, CFP multimer and/or a protein complex comprising CFP).

In one embodiment provided is a method of assessing NK cell activity towards target cells (e.g. tumor cells, bacterial cells, infected cells), comprising: (i) bringing an NKp46- expressing NK cell composition into contact with a target cell opsonized with CFP in the presence of a test agent (e.g. a plurality of test agents), (ii) assessing the activity of the NK cells towards the target cell. Optionally, assessing the activity of the NK cell towards the target cell comprises assessing effector cell-mediated cytotoxicity towards the target cell, e.g. target cell lysis, a marker of cytotoxicity/cytotoxic potential, e.g. CD107 and/or CD137 expression (mobilization) on the effector cell. Optionally the method further comprises: (iii) selecting (e.g. for use in therapy, for production of a batch of NK cells, for further processing or evaluation) NK cells (or a preparation of NK cells) that results in an increase in NK cell cytotoxicity (e.g. an increase in target cell lysis, an increase in CD107 and/or CD137 expression (mobilization) on the effector cell).

In one embodiment provided is a method of identifying agents that enhance NK cell activity towards target cells (e.g. tumor cells, bacterial cells, infected cells), comprising: (i) bringing an effector cell expressing NKp46 (e.g., an NKp46-expressing NK cells) into contact with a target cell opsonized with CFP in the presence of a test agent (e.g. a plurality of test agents), (ii) assessing the activity of the NKp46-expressing effector cell towards the target cell. Optionally, assessing the activity of the NKp46-expressing effector cell towards the target cell comprises assessing effector cell-mediated cytotoxicity towards the target cell, e.g. target cell lysis, a marker of cytotoxicity/cytotoxic potential, e.g. CD107 and/or CD137 expression (mobilization) on the effector cell. Optionally the method further comprises: (iii) selecting (e.g. for use in therapy, for production of a batch of therapeutic agent, for further processing or evaluation) an agent that causes an increase in cytotoxicity (e.g. an increase in target cell lysis, an increase in CD107 and/or CD137 expression (mobilization) on the effector cell). In one embodiment, the agent comprises an antibody, optionally an antibody that binds an antigen on the target cell, optionally an antibody that binds an activating or inhibitory receptor on an NK cell.

In one embodiment provided is a method for identifying an Fc domain-containing antigen binding protein (e.g. an antibody agent) having enhanced ability to mediate NK cell lysis of target cells (e.g. via NKp46 signalling in NK cells), the method comprises: (i) bringing test antigen binding protein (e.g. a plurality of such proteins) into contact with a target cell in the presence of CFP (e.g. in serum), and (ii) assessing CFP opsonization on target cells. A finding that the antigen binding protein causes or enhances CFP opsonization on target cells (e.g. causes a high level of opsonization, compared to reference level) indicates that the antigen binding protein has enhanced ability to mediate NK cell lysis of target cells (e.g. via NKp46 signalling in NK cells). Optionally the method further comprises: (iii) selecting (e.g. for use in therapy optionally further in combination with a therapy that enhances NK cell activity, for production of a batch of antigen binding protein, for further processing or evaluation) an agent that causes or enhances CFP opsonization on target cells. Optionally an antigen binding protein obtained by the method is administered to an individual having a disease (e.g. cancer, infectious disease) in combination with a therapy that enhances NK cell activity in an individual, for an NK cell composition (e.g. a native or engineered NK cell composition), an agent that acts as agonist at an activating receptor on the surface of an NK cell, or an agent that acts as antagonist at an inhibitory receptor on the surface of an NK cell.

Where the test agent is an antibody, for example for the identification of antibodies that interfere with and/or inhibit the CFP-NKp46 interaction, any of a variety of techniques known in the art can be used. Typically, they are produced by immunization of a non-human animal, preferably a mouse, with an immunogen, e.g., an immunogen comprising a NKp46 polypeptide, preferably a human NKp46 polypeptide. The NKp46 polypeptide may comprise the full length sequence of a human NKp46 polypeptide, or a fragment or derivative thereof, typically an immunogenic fragment, i.e., a portion of the polypeptide comprising an epitope exposed on the surface of cells expressing a NKp46 polypeptide. Such fragments typically contain at least about 7 consecutive amino acids of the mature polypeptide sequence, even more preferably at least about 10 consecutive amino acids thereof. Fragments typically are essentially derived from the extra-cellular domain of the receptor. In a preferred embodiment, the immunogen comprises a wild-type human NKp46 polypeptide in a lipid membrane, typically at the surface of a cell. In a specific embodiment, the immunogen comprises intact cells, particularly intact human cells, optionally treated or lysed. In another preferred embodiment, the polypeptide is a recombinant NKp46 polypeptide. The step of immunizing a non-human mammal with an antigen to obtain antibodies may be carried out in any manner well known in the art for stimulating the production of antibodies in a mouse (see, for example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988), the entire disclosure of which is herein incorporated by reference). Antibodies may also be produced by selection of combinatorial libraries of immunoglobulins, as disclosed for instance in (Ward et al. Nature, 341 (1989) p. 544, the entire disclosure of which is herein incorporated by reference).

The identification of one or more antibodies that bind(s) to NKp46 and that compete with NKp46 for binding to CFP, can be readily determined using any one of a variety of immunological screening assays in which antibody competition can be assessed. Many such assays are routinely practiced and are well known in the art (see, e. g., U. S. Pat. No. 5,660,827, issued Aug. 26, 1997, which is specifically incorporated herein by reference). For example, a simple competition assay may be employed in which the test antibodies are admixed (or pre-adsorbed) and applied to a sample containing NKp46 polypeptides, followed by addition of CFP (or vice versa). Protocols based upon western blotting and the use of BIACORE analysis are suitable for use in such competition studies.

EXAMPLES

Materials and Methods

Mice, experimental infection and assessment

Ncr1 ^iCm/+ R26 ^ISIDTA/+ , NCR1 ^GFP/GFP and control littermates were bred and maintained under specific pathogen-free conditions. Female and male mice of 6-12 weeks were used for the experiments. Experimental meningococcal infections were performed by intraperitoneal injection of N. meningitidis MC58 strain supplemented with human transferrin delivery. Survival was scored. Bacteria load in blood was also evaluated by plating serial dilutions on GCB medium (Difco) supplemented with Kellog supplements and results were expressed as colony forming units (CFU) per milliliter. For CFP delivery therapy, 100ng of MoCFP-HIS tagged was delivered 6 hours before infection (see ref).

Nm opsonization

5x10 ⁷ Nm were incubated at 37° for 30 minutes in PBS CaCI2 Mg plus 20% of human serum, CFP- or control C3-depeleted serum. Nm were then incubated with 10 μg ml of NKp-Fc precomplexed with Fab'2 anti-human Fc at 37° for 30 minutes.

NCR-Fc and CFP-HIS Fusion Proteins NCR-Fes: The sequences encoding the extracellular portion of mouse NKp46, human NKp46 and NKp30 proteins were amplified by PCR from cDNA isolated from mouse and human NK cells. The sequence encoding the Fc portion of human lgG1 containing a single substitution (N297Q) was amplified by PCR from cDNA of a designed construction vector. The N297Q mutation led to avoid Fc binding to Fc receptors.

CFP-HIS: The sequences encoding mouse and human CFP were amplified by PCR from cDNA isolated from mouse splenocytes and human PBMC respectively. The HIS Tag sequence was inserted directly into the reverse primers.

PCR-generated fragments were cloned into a mammalian expression vector. Sequencing of the constructs revealed that the cDNAs of all immunoglobulin fusion proteins were in frame with the human Fc genomic DNA and were identical to the reported sequences. The same observation has been done for the recombinant CFP-HIS proteins.

CHO cells were then stably nucleofected with the vectors containing the moNKp46- Fc, huNKp46-Fc, NKp30-Fc, moCFP-HIS or huCFP-HIS cDNAs. Bulk CHO cells were then subcloned in order to obtain good producers of recombinant proteins. Each selected subclone was then cultivated in serum free CD CHO complete medium (GIBCO) during 8 days and supernatants were collected.

Proteins were purified using rProtein A Sepharose Fast Flow (GE Healthcare life science) for the NCR-Fc proteins and Ni-NTA agarose (Qiagen) for the CFP-HIS proteins. Proteins were then separated by size exclusion chromatography on a Superdex 200 increase 10/300 GL column coupled to a UV detector (Akta Pure3, GE Healthcare).

Measure of CFP-HIS tagged binding to NKp46-Fc by flow cytometry

Beads were coupled to protein A-A488 in PBS Ca++Mg++ for 2 hours. After extensive wash, beads were incubated PBS 2% BSA for 1 hour. Beads were then incubated overnight with 20ug/ml of NCR-Fc in PBS 2% BSA. Beads were extensively washed and NCR-Fc were covalently coupled to protein A by 6 U/mL microbial transglutaminase (MTGase, Zedira, Darmstadt, Germany) for 1 hour at 37 °C according to. Beads were then washed and incubated with indicated amount of CFP-HIS tagged in 96 wells V shape bottom plate in veronal buffer 0.06M NaCI pH 7.5 at 37°C for one hour. When indicated, anti- HuNKp46 mAb (BAB281 ) or isotype control was added. After washing, beads were treated with 1 % paraformaldehyde for 15 minutes. Beads were then incubated with 5ug/ml of biotinylated anti-HIS Tag mAb for 30 minutes at 37°C. After washing, CFP-HIS/NCR-Fc coupled beads was revealed with a streptavidin A-APC. Samples were run on a Canto II cytometry analyser (BD Biosciences). SPR experiments

a. Immobilization of NKp46 and NKp30 recombinant proteins

HumanNKp46-Fc, MouseNKp46-Fc and HumanNKp30-Fc recombinant proteins were immobilized covalently to carboxyl groups at the surface of C1 Sensor Chips (GE Healthcare) The chip surface was activated with EDC/NHS (N-ethyl-N'-(3- dimethylaminopropyl) carbodiimidehydrochloride and N-hydroxysuccinimide (Biacore GE Healthcare)). Recombinant proteins were diluted to 10 μg ml in coupling buffer (10 mM acetate, pH 5.2) and injected until the appropriate immobilization level was reached (i.e. 700 to 900 RU). Deactivation of the remaining activated groups was performed using 100 mM ethanolamine pH 8 (Biacore GE Healthcare).

b. Binding assessment

Binding study was done following classical kinetic wizard (as recommended by the manufacturer). Serial dilutions of soluble analytes (recombinant CFP) ranging from 9 to 600 nM were injected over the NKp46-Fc immobilized proteins and allowed to dissociate for 10 min before regeneration. The entire sensorgram sets were fitted using the 1 :1 kinetic binding model. In all experiments NHS/EDC activated and ethanolamine deactivated flow cell 1 served as reference for blank subtraction.

c. Antibody blocking activity

To evaluate the blocking activity of anti-NKp46 antibodies, HumanNKp46-Fc and MouseNKp46-Fc chips were saturated by a first injection of Anti-NKp46 antibodies Bab281 and 29A1 .4 respectively at 10 μg mL, followed by injection of CFP proteins at 10 μg mL. Binding of the CFP proteins in the presence of anti-NKp46 antibodies was compared to the binding of CFP on nude NKp46 recombinant molecules. CFP binding to NKp46+ cells

HuDOMsp46, MoDOMsp46, DOMsp30, KHYG1 and KHYG1 CI3C4 NKp46 ^igh cells were incubated 1 hour with 10ug/ml of CFP-HIS tagged at 37°C. CFP-HIS binding was revealed as described above. For CFP-HIS binding to human primary cells, human NK cell bulk was generated as described in. respectively). For CFP-HIS binding to mouse primary cells, NK cells were enriched with the NK Cell Isolation Kit II (Miltenyi Biotec).

CFP stimulation of NKp46 reporter cells

CFP was coated at 20ug/ml on anti-HIS mAb-coated 96 wells U bottom plate. After washing, HuDOMsp46, DOMsp30 and parental D01 1.10 were added and cultured for 24 hours, cell supernatants were assayed for the presence of mouse IL-2 in a standard CTLL-2 survival assay using Cell Titer-Glo Luminescent Cell Viability Assay (Promega). DOMSP30, DOMSP46, or DO.1 1 .10 (30,000 cells/well in 96-well plates) were incubated on anti-NKp30 or anti-NKp46 mAb-coated plates or with tumor cell lines (30,000 cells/well in 96-well plates).

Example 1 : NKp46-expressing NK cells are required for mice survival to Nm invasive bacterial infection

We studied the extracellular gram ^" bacterial agent causing Invasive Meningococcal Disease known as Neisseria meningitidis {Nm), which is an emergent public health problem responsible for meningitidis and septicemia in human and sepsis in mice. Wild-type (WT) mice treated with anti-NK1 .1 mAbs to deplete NK cells and ILC1 prior to infection with the MC58 Nm bacterial strain exhibited increased mortality as compared to control group (Fig. 1 a). We also scored 8 to 20 fold more bacteria in their blood (Fig. 1 b). NKp46 (NCR1 , CD335) is a natural cytotoxic receptor (NCR) expressed on all mature NK cells, ILC1 and NCR ⁺ ILC3 and is highly conserved across mammalian species. We thus assessed the requirement of NKp46 ⁺ cells for mice survival to Nm using Ncr1-' ^Cre mice crossed to R26- ^,S,DTA (Rosa-DTA) x ^' mice which contain essentially no NKp46 ⁺ cells ^{(re )} . In absence of NKp46 ⁺ cells, mice were more sensitive to Nm infection than control littermates and were less capable to clear Nm (Fig. 1 c, d). These results demonstrated that NK cells or ILC1 -defined thereafter as NKp46 ⁺ ILCs- participate to the control of Nm infection in mice.

NK cell activation results from engagement of activating receptors. Among them, NKp46 has been previously described to bind to several microbial-derived ligands. We sought to test whether NKp46 was directly involved in the mechanisms controlling Nm infection. NKp46-deficient mice (referred as NCR1 ^GFP/GFP ) were more sensitive to Nm infection than control littermates at medium infectious doses and died significantly faster at higher Nm challenge (Fig. 2a). The lack of Nm control was associated with increased bacteria burden in the blood of NCR1 ^GFP/GFP mice as compared to control mice (Fig. 2b). These results demonstrated that the protection conferred by NKp46 ⁺ ILCs cells during Nm infection was dependent on NKp46 expression.

Example 2: Soluble NKp46 molecules bind to serum opsonized-A/m

We next dissected the mechanism by which NKp46 can contribute to the control of

Nm. Using recombinant soluble NCR-Fc molecules, we assessed the binding of NKp46 to Nm bacteria by flow cytometry. The lack of reliable reagents for NCR-Fc prompted us to generate recombinant soluble NCR-Fc molecules by fusing the extracellular domain of NKp46 or control NCR NKp30 with a mutated Ig-Fc portion. Soluble NKp46-Fc proteins did not directly bind to Nm bacteria similarly to NKp30-Fc (Fig. 2c). Since Nm is an extracellular bacterium targeted by complement factors in vivo, we thus assessed the binding of NKp46- Fc proteins on Nm opsonized by serum. We detected NKp46-Fc binding on serum opsonized-/Vm but not with control molecules (Fig. 2c). Human Complement Factor P (CFP)- deficiencies are selectively predisposed to lethal meningococcal infection by Nm. CFP is indeed critical for the lysis of Nm owing to the activation of the alternative pathway and the formation of the terminal membrane attack complex (MAC). NKp46-Fc binding signal was lost when bacteria were opsonized with a CFP-depleted serum (Fig. 2d, e). Altogether, these data showed that NKp46 can recognize Nm bacteria opsonized by seric factors in the presence of CFP.

Example 3: Soluble NKp46 molecules bind to recombinant CFP

Since both CFP and NKp46 were involved in the control of Nm infection, we next tested whether NKp46 can directly interact with CFP. To this aim we also generated recombinant human and mouse CFP-HIS tagged proteins. We set up a flow cytometry assay in which NCR-Fc molecules were covalently bound on beads and incubated with CFP-HIS tagged proteins (Fig. 3a). NKp30-Fcs control molecule did not interact with human or mouse CFP but we observed interactions of NKp46-Fcs with CFP cross-species as recombinant human CFP bound both human and mouse NKp46-Fcs and mouse CFP bound, albeit at a lesser extent, both human and mouse NKp46-Fcs (Fig. 3b, c). These results were consistent with the high conservation of both NKp46 and CFP across mammals with 62% and 77% identity at the protein level, respectively, between human and mouse. A dose-response of CFP binding to NKp46 was observed for a physiological range of CFP (5 to 50 pg/ml) (Fig. 3d). Importantly, the CFP-NKp46 interaction was inhibited by blocking anti-NKp46 mAbs (Fig. 3e). These results thus strongly suggested that CFP can be a ligand for NKp46.

Example 4: Surface plasmon resonance analysis confirms NKp46-Fc binding to CFP

Surface plasmon resonance experiments showed that serum purified CFP bound to human and mouse NKp46-Fcs and not to control NKp30-Fc (Fig. 3f). The kinetic data performed with increasing concentration of recombinant CFP showed similar binding properties and avidity to human and mouse NKp46-Fcs as opposed to NKp30-Fc coated biosensor (Fig. 3g). Of note, since native CFP is described to be naturally presents in serum as oligomers assembled into dimers (P2), trimers (P3) and tetramers (P4), we confirmed the binding of those purified fractions to NKp46-Fc coated biosensor. Altogether, these data demonstrated that natural and recombinant CFP can bind to NKp46-Fc molecules.

Example 5: CFP binds on NKp46-expressing cells We next assessed the binding of recombinant CFP to cell surface NKp46 (Fig. 3h-k). Using the IL-2-producing D01 1.10 mouse T cell hybridoma which express a chimeric receptor formed by the NKp46 extracellular domain fused to 003ζ (DOMsp46), we observed a binding of CFP to human and mouse NKp46-expressing DOMsP cells (HuDOMsP46 and MoDOMsP46 cells) but not on DOMsP cells expressing NKp30 (DOMsp30 cells) (Fig. 3i). We also observed CFP binding on KHYG1 -NKp46 cells human cell line genetically modified to express high amount of NKp46 but not on KHYGH1 parental cells that express low levels of NKp46. We also detected the binding of CFP on NKp46-expressing human bulk NK cells but not on T cells (Fig. 3j). Finally, CFP can bind at low level to Ncr1 ^WT/WT enriched mouse NK cells and not to Ncr1 ^GFP/GFP NKp46 negative control NK cells (Fig. 3k). These results thus showed that cells engineered to express NKp46 and primary NKp46-expressing NK cells can bind soluble CFP.

Example 6: NKp 6 ⁺ primary K cells mediate therapeutic benefit of CFP delivery for Nm bacterial control in vivo

CFP has been shown to act as a soluble pattern recognition molecule binding to a variety of pathogens, apoptotic and necrotic stressed cells. Neither CFP coated on apoptotic cells nor tumor cells engineered to express membrane CFP could activate NKp46-reporter cells. As NKp46 is a potent NK cell activating receptor, we investigated the impact of CFP on NK cell activation. We did not observe IFN-γ production or CD107a expression after a direct NK cell stimulation with coated CFP. We did also not measure a costimulatory effect of CFP when co-added to several NK cell stimuli. In the same line, we did not observe increased mortality or bacterial burden in WT mice infected with Nm and treated with a blocking anti- IFN-γ mAb as compared to control group. Providing CFP neither improved survival of Ncr1 ^WTWT as compared to Ncr1 ^GFP/GFP NK cells. We thus thought to analyze the phenotype of NK cells that have been generated in the absence of CFP. We did not observe abnormal development or functional defect of NK cell derived from CFP-deficient mice as compared to control. Therefore, even if CFP is a ligand for NKp46, none of the classical assays performed to measure NK cell activation scored positive after CFP stimulation suggesting another outcome for CFP binding to NKp46.

We thus sought to evaluate the biological relevance of CFP-NKp46 interaction in vivo. CFP is critical for immunity against Nm in humans. Along this line, WT mice can be rescued from lethal Nm infection via the delivery of recombinant CFP. We next tested whether the injection of purified CFP can control the clearance of Nm in absence of NKp46+ ILCs using a depleting anti-NK1.1 mAb (Fig. 4a). Delivery of CFP in WT mice protected mice from Nm infection as they exhibited 16 to 84% reduced bacteremia as compared to untreated group at 24 and 48 hours post infection, respectively, as previously described (Fig. 4b-d). Anti-NK1 .1 mAb treatment impaired mice survival in control group as expected, and CFP delivery enable the rescue of 33% of Nm infected mice from death. However, CFP treatment did not allowed Nm clearance in surviving mAb-treated mice as compared to control group (Fig. 4b-d). These results thus showed that a part of the therapeutic benefit of CFP delivery required NKp46 ⁺ ILCs cells. We further assess the requirement of NKp46 for the benefit of CFP treatment (Fig. 4e-h). All the mice survived to this infectious Nm dose and _Ncr1 wT/wr NKp46-sufficient mice have cleared 50 to 100% of bacteria at 6 and 24 hours post infection, respectively, as compared to nontreated mice. However, CFP treatment of /Vcri ^GFP/GFP NKp46-deficient mice only enable the clearance of 4 to 28% bacteria at 6 and 24 hours post infection, respectively, as compared to control group (Fig. 4h). Altogether these results demonstrated that a significant part of the therapeutic benefit effect of recombinant properdin delivery in a murine model of Nm infection was dependent on NKp46 ⁺ ILCs cells and NKp46.

Here, we identified that the Complement Factor P (CFP), the only positive regulator of the complement cascade, can bind to NKp46. CFP-NKp46 interaction is critical for NK cell mediated protection against the CFP-dependent Neisseria meningitidis invasive bacterial infection. Our results suggest a model in which NKp46 acts as a docking site for soluble CFP and may constitute a major determinant for local alternative pathway activation. These data uncover the role of a new molecular dialog between NK cells and complement, two partners of the innate immune system, as a novel cooperative mechanism for immune defense against bacteria.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law), regardless of any separately provided incorporation of particular documents made elsewhere herein.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate). The description herein of any aspect or embodiment of the invention using terms such as "comprising", "having," "including," or "containing" with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that "consists of", "consists essentially of", or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.