FIELD OF THE INVENTION

This invention relates to methods of treating or preventing cancers such solid tumors, as well as other disease relating to excessive or insufficient immune responses, arising from the discovery of natural ligands of NKp44 that mediate immunosuppression.

BACKGROUND OF THE INVENTION

NK cells are innate lymphoid cells (ILCs) involved in various immune processes ranging from the direct elimination of pathogens or tumor cells to the release of cytokines and chemokines and the regulatory interactions with different immune cells. In order to fulfill this variety of functions, NK cells use an array of receptors which sense microenvironmental stimuli and mediate appropriate responses. Several NK receptors are capable of regulating different NK cell functions. For example, the Natural Cytotoxicity Receptors (NCRs) NKp44, NKp30, and NKp46, play an important role in human NK cell-mediated recognition and killing of virally infected and tumor cells, and also induce the release of a number of cytokines and chemotactic factors. In addition, NKp30 and NKp46 mediate regulatory interactions occurring between NK and different leukocytes, including dendritic cells (DCs), neutrophils, eosinophils, macrophages, and T cells. NCRs were identified and molecularly characterized in‘90s. Since then, numerous studies attempted to identify their ligands as this information is crucial for a better exploitation of the NK cell potential in the therapy of tumors, infections, or immune- mediated diseases. In spite of many efforts, so far, the panel of the NCR ligands has been only partially defined. A reason of these difficulties is related to the fact that study models of receptor-ligand interaction for the NCR are rather limited, and among human NCR only NKp46 is expressed on rat and mouse NK cells. Moreover, although the NCRs belong to the Ig superfamily, they greatly differ in their molecular structure, implying that their ligands may be rather heterogeneous. In addition, each NCR may interact with different ligands, not necessarily displaying similar molecular features. Thus, for example, NKp44 and NKp46 have been shown to bind viral hemagglutinins. On the other hand, molecularly unrelated cellular ligands have been shown to bind NKp30 (B7H6 and BAT3/BAG6) and NKp44 (21 spe-MLL5 and PDGF-DD). To further complicate this issue, NCRs may interact with ligands by different modalities and even opposite functional outcomes. For example, NKp44 appears to differently modulate NK cell function by cis- or trans-interactions with heparan-sulphate proteoglycans (present at the NK or target cell surface). In addition, it has been reported to transduce inhibitory signals upon interaction with Proliferating Cell Nuclear Antigen (PCNA). NKp30 triggers cytotoxicity and cytokine release upon binding B7H6 or BAT3 at the cell surface. Besides NCRs, other activating receptors, including NKG2D and DNAM-1 , contribute to trigger the NK cell-mediated cytotoxicity. Ligands specific for these receptors have been extensively characterized: MICA/B and ULBPs molecules are recognized by NKG2D, while PVR and Nectin-2 (belonging to the Nectin family), are ligands for DNAM-1. Soluble ligands, endowed with suppressive capability, have been described for NKG2D and DNAM-1 activating receptors (sMICA, sULBPs, and sPVR) and for NKp30 (sB7H6 and sBAT3/BAG6). Regarding other NCRs, NKp46 has recently been shown to bind an extracellular molecule: the Complement Factor P (Narni-Mancinelli et al. (2017) Science Immunol. 2(10)). In the case of the NKp46- CFP interaction, however, CFP may actually represent an important tool for innate responses to pathogens rather than inducing an inhibitory loop.

Identification and characterization of cell surface ligands for NCRs is important for understanding of the mechanisms of NCR 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 human NKp44 recognizes an extracellular ligand known as Nidogen-1 (NID1 ) protein (also known as Entactin). It is further shown herein that the NKp44/NID1 interaction results in a reduced NKp44-mediated induction of cytokine release by human NK cells. Further analysis of the proteomic changes induced in NK cells by the exposure to human NID1 revealed a substantial modulation of different molecules and pathways involved in important biological processes. It is further shown herein that NID1 , besides being released in the extracellular space, can also be detected by NKp44-Fc proteins and anti-NI D1 antibodies at the surface of cells (e.g. NID1-releasing cells).

The present disclosure provides natural ligands of NKp44, notably NID1 polypeptides, e.g. full-length soluble NID1 polypeptide, soluble NID polypeptide fragments, membrane-bound NID polypeptides, and NKp44-binding fragments of any of the foregoing (e.g. a NID1 fragment comprising a NKp44 binding domain).

In some embodiments, the technology described herein relates to antibodies and antibody fragments (e.g. comprising an antigen-binding portion of an antibody which binds a NID1 polypeptide). In some embodiments, the technology described herein relates to NKp44 polypeptides and fragments thereof (e.g. as isolated, purified and/or soluble polypeptides) which bind a NID1 polypeptide. Among the agents that bind NID1 , included, inter alia, are molecules that bind NID1 and deplete NID1 -expressing cells, molecules that bind NID1 on NID1 -expressing cells and inhibit the amount of NID1 released from cells, as well as molecules that bind NID1 and modulate the interaction between NID1 and NKp44. Also provided are methods of use thereof to modulate, optionally enhance, immune responses (e.g. an anti- tumor immune response, immune response against a pathogen (e.g. virus) or pathogen- infected cell) in an individual. In some embodiments, the NID1 polypeptide is a soluble NID1 polypeptide (sNID1 ). Soluble NID1 can for example be a NID1 protein that has been released from cells and is available for binding by NKp44, optionally NID1 that is not associated with the basement membrane. Soluble NID1 may be a full NID1 protein or a NID1 fragment (e.g., cleaved by the action of cathepsin-S, ADAMTS1 and/or other enzymes such as matrix metalloproteases, optionally MMP2 or MMP9).

Besides being released, Nidogen-1 (NID1 ) polypeptides can be associated at the cell surface. Notably, in many tumors, NID1 is released from the cells, resulting in the possible inhibition of NKp44 on NK and/or other ILC, which may in turn cause decreased host immunity against the tumor cell and promote tumor evasion and progression. In some embodiments, the methods and compositions described herein relate to inhibition of sNID1 , e.g. reducing the level and/or activity of sNID1 that is available to interact with cellular receptors. In some embodiments, inhibition of sNID1 can be a reduction of unbound sNID1 , e.g. sNID1 not bound to a receptor and/or available to be bound by the antibody or soluble NKp44 reagents described herein). In some embodiments, inhibition of sNID1 can be a reduction of the level of sNID1 , e.g. the level of sNID1 in tumor tissue and/or circulation.

Accordingly, one object of the invention is to provide use of the compositions and agents provided herein to enhance or stimulate the activity (e.g. cytotoxic activity, proliferation, development and/or maturation, etc.) of NKp44-expressing lymphocytes, notably NKp44- expressing ILC, such as NK cells or ILC3 cells. In one embodiment, the compositions and agents provided herein are used to restore or enhance the expression of the MYSM1 protein in ILC (e.g. NK cells) and/or to promote ILC (e.g. NK cells) development and/or maturation. The composition can be used for the treatment of disease in an individual, optionally wherein the disease is a cancer, optionally a solid cancer, optionally wherein the disease is an infectious disease (e.g. viral infection). The composition can be used to restore or enhance immunity (e.g. anti-tumor immunity) in an individual having soluble NID1 (sNID1 ). The composition can be used to relieve NID1 -mediated suppression of NK cell activity in an individual. The composition can be used to enhance NK cell-mediated targeting (e.g. lysis) of disease promoting cells, notably tumor cells, tumor-promoting cells in the tumor environment, pathogens or pathogen-infected cells (e.g. virally infected cells) notably for the treatment of a disease (e.g. cancer, infectious disease) in an individual. In one embodiment, the disease promoting cells bear at their surface membrane-bound NID1 (mNID1 ). In one embodiment, the individual is an individual having soluble NID1 , optionally further wherein soluble NID is present (e.g., has been determined to be present) in circulation and/or in the tumor environment, optionally in tumor tissue and/or tumor adjacent tissue.

The compositions and agents provided herein can be advantageously used for the treatment or prevention of cancer in an individual, optionally in an individual having a solid tumor, optionally a lung cancer (e.g. non-small cell lung carcinoma), colon cancer, endometrial cancer, melanoma, ovarian cancer or breast cancer. In one embodiment, the compositions and agents provided herein are used to treat or prevent metastatic cancer, or to prevent metastases and/or disease progression in an individual having a cancer.

It is one object to provide compositions and methods for relieving or reducing sNID1- mediated suppression or NK cell activity and/or anti-tumor immunity by targeting sNID1. It is another object of the invention to provide compositions and methods for the treatment of cancers by targeting NID1. In any of the treatment methods, the method may be characterized by a step of administering an agent that targets NID1 to an individual having a disease or disorder, e.g. a cancer.

In one aspect, provided is a composition (e.g. an NKp44 protein or protein fragment, a non-NKp44 protein agent, an antibody, an antibody fragment) that binds NID1. In one aspect, the composition that reduces the level and/or activity of sNID1 that is available to interact with a NKp44 polypeptide. In one embodiment, the agents disclosed herein are useful in a method of reducing the amount of sNID1 in circulation and/or in a tissue (e.g. tumor tissue or tumor adjacent tissue) in an individual. In one embodiment, the agents are useful in a method of reducing the release of NID1 , optionally a method of reducing the release of, or the amount of released sNID1 , in the tumor environment (e.g. tumor tissue and/or tumor-adjacent tissue).

In one embodiment, provided are agents that interfere with the NKp44-sNID1 interaction. Such agents can enhance immune responses. In one embodiment, provided are agents that interfere with the NKp44-sNID1 interaction, to relieve sNID1-mediated suppression of immune responses (e.g. anti-tumor immune response). In one embodiment, the agent is a protein, an Fc protein, an antibody or antibody fragment that binds sNID1 and interferes with the interaction between sNID1 and NKp44. In one embodiment, the agent lacks binding to one or more human Fey receptors, optionally the agent lacks binding to human CD16, optionally the agent lacks binding to one or more of, or each of, human CD16A, CD16B, CD32A, CD32B and/or CD64 polypeptides. Optionally the agent is an antibody or Fc protein comprising an Fc domain of human lgG1 , lgG2, lgG3 of lgG4 isotype comprising an amino acid modification (e.g. one or more substitutions) that decreases the binding affinity of the antibody for one or more of, or each of, human CD16A, CD16B, CD32A, CD32B and/or CD64 polypeptides. In one embodiment, the agent is a non-depleting agent.

One object of the disclosure is to provide compositions that inhibit, reduce or block the NKp44-NID1 interaction, particularly the NKp44-sNID1 interaction, and methods of use thereof, to increase NK cell activity. In one embodiment, the composition is a protein (e.g. antibody or antibody fragment) that binds to the sNID1 polypeptide. It is one object of the invention to provide methods of use of such compositions to increase NK cell activation, optionally to increase NK cell activation toward target cells, (e.g., disease cells, cancer cells), optionally further for the treatment of cancer.

In another embodiment, provided are agents (e.g. depleting agents) that bind mNID1 (e.g., NID1 as expressed at the surface of cells) and are capable of mediating the depletion of cells expressing mNID1 (e.g., NID1-releasing cells; cells present in tumor tissue or tumor adjacent tissue, cancer cells, or cancer-promoting cells). Such molecules can be useful to eliminate mNID1 -expressing cells that secrete sNID1 into the extracellular space (e.g. mNID1- expressing cells in cancer or cancer-adjacent tissues), thereby in turn decreasing sNID1 in the tumor environment and/or in circulation. The molecules can thereby also relieve the sNID1- mediated suppression of NK cell activity. In one embodiment, the molecule is or comprises a protein, for example a NKp44 protein fragment, an Fc protein, an anti-NI D1 antibody or antibody fragment, and bi-specific or multi-specific antibody that binds mNID1. In one embodiment, the molecule further binds sNID1 and interferes with the interaction between sNID1 and NKp44. In another embodiment, the molecule does not interfere with the interaction between NID1 (e.g. sNID1 and/or mNID1 ) and NKp44. In one embodiment, the molecule is administered to an individual in an amount effective to deplete mNID1 -expressing cells that release sNID1 into the extracellular space (e.g. mNID1 -expressing cells in cancer tissues or cancer cells).

In one embodiment, the agents disclosed herein are useful in a method of reducing the amount of circulating sNID1 in an individual. In one embodiment, the agents are useful in a method of reducing the release of NID1 , optionally a method of reducing the release of, or the amount of released sNID1 , in the tumor environment (e.g. tumor tissue and/or tumor-adjacent tissue).

In another aspect, provided is a composition comprising a NKp44 ligand (e.g., a sNID1 polypeptide or NKp44-binding fragment thereof; an antibody that binds NKp44 and inhibits NKp44 signaling) or compositions that comprises such, and methods of use thereof, to reduce NK cell activity and/or to cause immunosuppression, e.g. in vitro, in vivo or in inflamed tissues, for treatment of inflammatory disorders or autoimmune disorders.

In another aspect, assessing presence or levels of NID1 , particularly sNID1 , in an individual (e.g. in a biological sample from an individual) provides an indicator (e.g., a biomarker) to assess immunosuppression, for example, to assess the immunosuppressive state of anti-tumor NK cells mediated by sNID1. The presence of sNID1 , or elevated levels of sNID1 , can be indicative for example of an inhibited or suppressed lymphocyte (e.g. NK cell) cell activity, for example a state of inhibition of lymphocyte (e.g. NK cell) cytotoxicity, activation, maturation, or homing toward cancer cells. A finding that sNID1 is present in a biological sample from an individual at significant levels or at elevated levels (e.g. compared to a control value, for example from healthy individuals or from individuals who are not immunosuppressed) can be indicative of immunosuppression in the individual and/or can be used to assess the responsiveness to a cancer therapy, optionally an immunotherapy. In many cases individuals having cancer show resistance to treatment with anti-cancer treatments that have immune modulating effects (including chemotherapeutic agents, radiotherapy, immunotherapy or immunotherapeutic agents). Consequently, assessing markers of immunosuppression prior to treatment may be useful to predict, assess and/or monitor responsiveness to such anti-cancer treatments. It is thus another object to provide detection of sNID1 as a biomarker for assessing, predicting or monitoring the effectiveness of therapies, optionally cancer therapies. In one embodiment, the cancer therapy is a chemotherapeutic agent, radiotherapy, an immunotherapy, optionally an immunotherapeutic agent that has the ability to modulate (e.g. enhance) T cell and NK cell activity. In one embodiment, the immunotherapy is an agent (e.g. comprising a cell, an antibody or antibody fragment, a protein, a nucleic acid, or a small molecule organic compound) that modulates the activity of tumor infiltrating and/or cancer adjacent tissue NK cells, optionally further tumor infiltrating and/or cancer adjacent tissue T cells. In one embodiment, the immunotherapeutic agent (e.g. an antibody or protein agent) binds (e.g. and activates) an activating receptor (including co- activating receptors) expressed by NK and/or T 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). Optionally, the immunotherapeutic agent binds and inhibits an inhibitory NK cell and/or T cell receptor, or a natural ligand of such an inhibitory receptor (e.g. a Siglec family member, LAG-3, PD-1 (CD279) or its natural ligand PD-L1 , TIGIT or CD96). Optionally, the immunotherapeutic agent binds inhibits a polypeptide that is associated with lymphocyte (e.g. T and/or NK cell) exhaustion, inhibition or suppression, e.g. the exhaustion marker TIM-3, or polypeptides such as indoleamine-2, 3-dioxygenase (IDO), CD39 and CD73 which are associated with production of immunosuppressive metabolites. In one embodiment, the immunotherapeutic agent activates CD16, optionally wherein the agent is an agent (e.g. a full-length lgG1 antibody, a bispecific antibody) comprising an Fc domain that is bound by FcyR (e.g. human CD16) and which is capable of inducing ADCC via CD16 (e.g. via CD16 on NK cells). 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).

It is a further object of the invention to provide compositions and methods for determining the severity or prognosis of cancer in a subject having or suspected of having cancer. The presence of sNID1 , or elevated levels of sNID1 , can be indicative for example of an increased severity, increased risk of progression or increased risk of metastasis in an individual having a 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 a cancer.

It is another object of the invention to provide compositions and methods for selecting a subject for treatment for cancer.

It is another object of the invention to provide compositions and methods for measuring pharmacokinetic and pharmacodynamics parameters of NKp44 therapies, such as NKp44-expressing NK cell compositions, soluble proteins that mimic NKp44 ligand or therapies that promote the activity or number of NKp44-expressing NK cells, or therapies that promote NKp44-NKp44 ligand interactions.

It is another object of the invention to provide methods for identifying agents (e.g. protein, antibody or small molecule agents) that modulate NKp44 or NID1 activity, for example that modulate (e.g., inhibit or enhance) the NKp44-NID1 interaction.

In one aspect, provided are methods for identifying agents (e.g. protein, antibody or small molecule agents) that inhibit the NKp44-sNID1 interaction and/or enhance the activity of NK cells, e.g., that enhances the recognition and/or lysis of a target cell by an NKp44+ NK cell. In one embodiment, the method comprises identifying an agent(s) (e.g. an antibody or antibody fragment, an Fc protein, an NKp44-derived protein or peptide, or a composition that comprises any of the foregoing) that binds sNID1 and that inhibits the NKp44-sNID1 interaction.

Also provided are methods and assays to detect the interaction of NKp44 with NID-1. Said NID1 may be for example an isolated sNID1 protein or may be a NID1 protein expressed at the surface of a cell (e.g. a NID1 -releasing cell). Such assays can be used in medical or biological research, in diagnostics or prognostic methods, or advantageously to screen or test antibodies or other antigen binding agents to increase or decrease NK cell or immune effector cell activity. In one embodiment provided is a method comprising: (i) bringing NKp44 (e.g. as a cell expressing NKp44 or as a NKp44 protein in solution) into contact with NID1 (e.g. as a cell expressing NID1 at its surface, or as a soluble NID1 protein), (ii) assessing (e.g. detecting) the interaction of the NKp44 and NID1 polypeptides. Optionally, assessing the interaction comprises assessing (e.g. detecting) binding of NID1 to NKp44. In one embodiment, the NKp44 polypeptide is a membrane-bound polypeptide (e.g. expressed at the surface of a cell). In another embodiment, the NKp44 polypeptide is a soluble NKp44 polypeptide (e.g. a NKp44 fragment fused to an Fc domain). In one embodiment, the NID1 polypeptide is a soluble NID1 polypeptide. In another embodiment, the NID1 polypeptide is a membrane-bound polypeptide (e.g. expressed at the surface of a cell).

Also provided are methods and assays for identifying agents that modulate the interaction between NID1 and NKp44. In one embodiment of such an assay, the method comprises: (i) bringing NKp44 (e.g. as a cell expressing NKp44 or as a NKp44 protein in solution) into contact with NID (e.g. as a cell expressing NID1 at its surface, or as a soluble NID1 protein) in the presence of a test agent (e.g. a plurality of test agents), and (ii) assessing binding of NID1 to NKp44. A decrease in binding (compared to negative control) indicates that the agent interferes with binding of NID1 and NKp44. An increase in binding (compared to negative control) indicates that the agent enhances the interaction of NID1 and NKp44.

In one embodiment provided is a method of identifying agents that increase NK cell activity, comprising: (i) bringing NKp44 (e.g. as a cell expressing NKp44 or as a NKp44 protein in solution) into contact with NID1 (e.g. as a cell expressing NID1 at its surface, or as a soluble NID1 protein) in the presence of a test agent (e.g. a plurality of test agents), (ii) assessing binding of NID1 to NKp44. 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 NID1 and NKp44. In one embodiment, the agent is for use in treatment or prevention of a cancer. In one embodiment, the agent comprises an antibody. In one embodiment, the agent (e.g. an antibody) binds NID1.

In one embodiment, provided is a receptor complex comprising a NID1 polypeptide and a NKp44 polypeptide, optionally an isolated receptor complex, optionally further comprising a linking agent (e.g. an antibody or protein) that links NID1 and NKp44. Optionally the complex comprises one or more further proteins. Optionally, the NKp44 polypeptide is a fragment of NKp44 that comprises a NID1 binding domain.

In one embodiment provided is a method of binding to (e.g. for targeting, detecting, modulating, etc.) NKp44 on the surface of an NK cell, optionally a method for modulation NKp44 on the surface of an NK cell, the method comprising bringing a cell expressing NKp44 (e.g., an NK cell) into contact with a NID1 polypeptide (e.g., a soluble NID1 or NID1-comprising protein, optionally complexed with other molecules).

In one embodiment provided is a method of binding (e.g. for targeting, detecting modulating, etc.) a NID1 polypeptide on the surface of a target cell (e.g. a cancer cell, etc.), the method comprising bringing the target cell into contact with an NKp44 polypeptide (e.g., a NKp44 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a NID1 -binding fragment thereof, for example a NKp44-lg fusion protein comprising any of the foregoing).

In other aspects, an object of the invention is to provide use of NID1 compositions (e.g. a soluble NID1 protein or an NKp44-binding fragment thereof) to reduce the activity (e.g. proinflammatory or tissue-destructive activity, proliferation, development and/or maturation, etc.) of NKp44-expressing lymphocytes, notably NKp44-expressing ILC, such as NK cells or ILC3 cells. Such NID1 compositions can be used for treatment or prevention of an autoimmune or inflammatory disease in an individual, wherein the NID1 composition is administered to the individual having an autoimmune or inflammatory disease, e.g. an autoimmune or inflammatory disease characterized by disease-promoting NKp44-expressing lymphocytes, notably NKp44-expressing ILC, such as NK cells or ILC3 cells. The NID1 composition can be used to cause or enhance NID1 -mediated suppression of NK cell activity in an individual. Examples of autoimmune or inflammatory disease include systemic lupus erythematosus, Wegener's granulomatosis, autoimmune hepatitis, Crohn's disease, scleroderma, ulcerative colitis, Sjogren's syndrome, Type 1 diabetes mellitus, uveitis, myocarditis, rheumatic fever, ankylosing spondylitis, rheumatoid arthritis, multiple sclerosis, and psoriasis.

In one embodiment provided is a method of detecting a NID1 protein on the surface of a target cell (e.g. a NID-releasing cell, a cancer cell, etc.), the method comprising bringing the target cell into contact with an NKp44 polypeptide comprising a detectable moiety, wherein detection of binding of NKp44 to the target cell indicates presence of NID1 , optionally wherein detection of binding of NKp44 to the target cell indicates a NID1-releasing cell.

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 shows results of analysis of culture supernatant from HEK293T cells by two- dimensional electrophoresis (2-DE). Concentrated HEK293T-SN was analyzed by two- dimensional electrophoresis (2-DE). After blotting, membranes were probed with NKp44Fc (Figure 1A), NKp46Fc (Figure 1 B), or NKp30Fc (Figure 1 C) followed by HRP-conjugated anti- human IgG mAb. HEK293T-SN-biot was subjected to the same procedure and the membrane was incubated with HRP-conjugated Neutravidin (Figure 1 D). In parallel, HEK293T-SN (600 pg) was separated by 2-DE and proteins were stained with“blue silver” colloidal Coomassie (Figure 1 E). Numbers indicate the spots selected for mass spectrometry analysis; in panels (A), (D), and (E) spot 26 is highlighted with a black circle.

Figure 1 F shows reactivity of NKp44Fc and additional chimeric NK receptors with culture supernatant from HEK293T cells (a) ELISA plates were coated with concentrated HEK293T-SN followed by incubation with the indicated Fc molecules and detection with HRP- conjugated anti-human IgG mAb. (b) ELISA plates were coated with NKp44Fc or NKp30Fc, followed by incubation with HEK293T-SN labeled with biotin (HEK293T-SN-biot), or HEK293T- SN as negative control, and HRP-conjugated Streptavidin. Graphs represent the absorbance at 405 nm after normalization to background (nonspecific binding of the HRP-conjugated secondary reagent). In (a) and (b) data are medians ± interquartile range of three independent experiments performed in triplicate (a) or duplicate (b). ^**** p<0.0001 (A), ^** p0.0022, ns=0.0606 (B) by two-tailed Mann-Whitney test.

Figure 1 G shows Western blot analysis of HEK293T-SN and immunoblotting with NKp44Fc and additional chimeric NK receptors. Concentrated SN from HEK293T cells (HEK293T-SN) cultured in protein-free medium was concentrated, analyzed by SDS-PAGE on a 7.5% polyacrylamide gel, and immunoblotted with the indicated Fc molecules followed by HRP-conjugated anti-human IgG mAb. MW markers (kDa) are indicated on the right. One representative experiment of three is shown.

Figure 2 shows the specific recognition of NID1 by NKp44Fc chimeric receptor. Figure 2A shows concentrated HEK293T-SN immunoprecipitated with the indicated Fc molecules and analyzed in 7.5% SDS-PAGE under non-reducing conditions; in parallel, concentrated HEK293T-SN was loaded as a positive control. After blotting, the membrane was probed with mouse anti-NI D1 mAb followed by HRP-conjugated anti-mouse IgG mAb. One representative experiment of three is shown. Figure 2B shows concentrated HEK293T-SN and rNID1 analyzed in SDS-PAGE on a 7.5% polyacrylamide gel under non-reducing conditions and, after blotting, probed with the indicated Fc molecules or with anti-NI D1 mAb, followed by HRP- conjugated anti-human or anti-mouse IgG secondary reagent, respectively. Molecular weight (MW) markers (kDa) are indicated on the right. One representative experiment of five is shown. Figure 2C shows ELISA plates coated with rNID1 and incubated with different concentrations of the indicated Fc molecules followed by HRP-conjugated anti-human IgG mAb. Graph represents absorbance at 405 nm after normalization to background (nonspecific binding of the secondary reagent). Data are medians of triplicates ± interquartile range and are the pooled results of three independent experiments. ^**** p<0.0001 by two-tailed Mann- Whitney test.

Figure 3A shows the effect of rNID1 pretreatment on NKp44-induced IL-2 production in Bw-NKp44 cells. Bw-NKp44 and Bw-NKp30 cells were left untreated or were pre-treated with rNID1 at the indicated concentrations for 1 h at 37°C and subsequently incubated on anti- NKp44 or anti-NKp30 mAb-coated plates, respectively, for 20 h. IL-2 release in the SN was evaluated by ELISA. The background (from GAM-stimulated cells) was subtracted for each value. Data are medians of duplicates ± interquartile range and are the pooled results of three independent experiments. ^** p=0.022, ns (not significant)=0.584, by two-tailed Mann-Whitney test.

Figure 3B shows NID1 mRNA and protein expression in Bw cell line and transfectants. (a) NID1 mRNA expression was assessed by RT-PCR in Bw cells and in the corresponding NID1 transfectants. Primers specific for b-actin were utilized as positive control. PCR products were run on 1.5% agarose gel and visualized by ethidium bromide staining. One representative experiment of three is shown (b) SN derived from Bw cells and the corresponding NID1 transfectants were analyzed in SDS-PAGE on a 7.5% polyacrylamide gel; membrane was probed with mouse anti-NI D1 mAb followed by HRP-conjugated anti-mouse IgG mAb. MW markers (kDa) are indicated on the right. One representative experiment of two is shown (c) ELISA plates were coated with mouse anti-NID1 mAb followed by incubation with SN obtained from Bw cells and the corresponding NID1 transfectants. NID1 was detected using a goat anti-NID1 Ab followed by a HRP-conjugated anti-goat IgG Ab. Graph represents absorbance at 405 nm after normalization to background (nonspecific binding of goat anti- NID1 + secondary reagent). Data are medians of triplicates ± interquartile range and are the pooled results of three independent experiments. ^**** p<0.0001 by two-tailed Mann-Whitney test.

Figure 4 shows NKp44Fc recognizes NID1 released by NID1 -transfected K562 cells. Figure 4A shows NID1 mRNA expression as assessed by RT-PCR in wild type or NID1- transfected K562 cells. Primers specific for b-actin were utilized as positive control. PCR products were run on 1.5% agarose gel and visualized by ethidium bromide staining. One representative experiment of three is shown. Figures 4B and C shows results of ELISA plates coated with mouse anti-NI D1 mAb (Figure 4B) or with the indicated Fc molecules (Figure 4C), followed by incubation with concentrated SN obtained from untransfected or NID1 -transfected K562 cells cultured in protein-free medium. NID1 was detected using a goat anti-NI D1 Ab followed by a HRP-conjugated anti-goat IgG Ab. Graphs represent absorbance at 405 nm after normalization to background (nonspecific binding of goat anti-NI D1 + secondary reagent). Data are medians of triplicates ± interquartile range and are the pooled results of three independent experiments. ^**** p<0.0001 by two-tailed Mann-Whitney test. Figure 4D shows concentrated SN (30 pi for each sample) derived from K562 cells and the corresponding NIID1 transfectants cultured in protein-free medium were analyzed in SDS-PAGE on a 7.5% polyacrylamide gel; membrane was probed with NKp44Fc molecule or with mouse anti-NID1 mAb followed by the appropriate HRP-conjugated secondary mAb. MW markers (kDa) are indicated on the right. One representative experiment of two is shown.

Figure 4E shows the effect of soluble NID1 on NKp44-induced IL-2 production in Bw- NKp44 cells. Bw-NKp4 and Bw-NKp30 cells were incubated on anti-NKp44 or anti-NKp30 mAb-coated plates, respectively, for 20 h at 37°C in the presence or absence of SN derived from untransfected or NID1 -transfected Bw cells. IL-2 release in the SN was evaluated by ELISA. The background (from GAM-stimulated cells) was subtracted for each value. Data are medians of duplicates ± interquartile range and are the pooled results of three independent experiments. ^** p=0.0022, ns=0.8983 by two-tailed Mann Whitney test. Figure 5 shows the effects of soluble NID1 on NKp44-induced function in Bw-NKp44 cells and human polyclonal NK cells. Figure 5A shows Bw-NKp44 and Bw-NKp30 cells incubated on anti-NKp44 or anti-NKp30 mAb-coated plates, respectively, for 20 h at 37°C in the absence or in the presence of SN derived from untransfected or NID1 -transfected K562 cells. IL-2 release in the SN was evaluated by ELISA. The background (from GAM-stimulated cells) was subtracted for each value. Data are medians of duplicates ± interquartile range and are the pooled results of four independent experiments. ^*** p=0.0002, ns=0.1044, by two-tailed Mann Whitney test. Figure 5B shows polyclonal NK cell lines incubated with SN derived from untransfected or NID1 -transfected K562 cells and cultured for 20 h at 37°C on plates coated with anti-NKp44, -NKp30, or -NKp46 mAbs. IFN-g release in the SN was evaluated by ELISA. Data are medians of six independent experiments ± interquartile range performed with NK cells from three donors. ^* p=0.0156, ns=0.1094 (NK + anti-NKp30) and 0.0983 (NK + anti- NKp46) by one-tailed Wilcoxon test. Figure 5C shows polyclonal NK cell lines incubated with medium or with SN derived from untransfected or N I D1 -transfected K562 cells. After 20 h cells were utilized in a redirected killing assay against the FcyR+ P815 target cell line in the absence or in the presence of the indicated mAbs (E:T ratios 2:1 and 1 :1 ). Data are medians of duplicates ± interquartile range and are the pooled results of six experiments performed with NK cells derived from three donors ^* p=0.0313 (2:1 ratio) and 0.0156 (1 :1 ratio) by one-tailed Wilcoxon test.

Figure 6 shows results of analysis of NID1 surface expression in HEK293T cell line and in NID1 cell transfectants. HEK293T cells were stained with a NID1-specific mAb followed by PE-conjugated anti-mouse lgG1 mAb. The figure shows HEK293T, K652, K562-NID1 , Bw, and Bw-NIDI cells stained with a NID1-specific mAb or with NKp44Fc followed by the appropriate isotype-matched PE-conjugated secondary mAb. Samples were analyzed by flow cytometry. Grey profiles represent cells stained with anti-NID1 or with NKp44Fc, while white profiles correspond to isotype control. One representative experiment of four is shown.

Figures 7A to D shows the functional effects of cell surface-associated NID1 or plastic-bound rNID1 in Bw-NKp44 and in NK cells. Figure 7A shows Bw-NKp44 cells cultured alone or in the presence of untransfected or N I D1 -transfected K562 cells for 20 h at 37°C. IL-2 release in the SN was evaluated by ELISA. Data are medians of duplicates ± interquartile range and are the pooled results of three independent experiments. ns=0.5714 by two-tailed Mann-Whitney test. Figure 7B shows Bw-NKp44 cells incubated on plates coated with rNID1 , anti-NID1 + rNID1 , or anti-His + rNID1 for 20 h at 37°C. IL-2 release in the SN was evaluated by ELISA. Data are medians of duplicates ± interquartile range and are the pooled results of two independent experiments. ns=0.8286 (-) or ns=0.6571 (anti-NI D1 , anti-His) by two-tailed Mann-Whitney test. Figure 7C shows polyclonal NK cell lines evaluated for their cytolytic activity in a 4-h ⁵¹ Cr release assay against Bw and Bw-NID1 cells at the indicated E:T ratios. Data are medians of duplicates ± interquartile range and are the pooled results of fifteen experiments performed with NK cells derived from five donors. ^* p=0.0277 by one-tailed Wilcoxon test. Figure 7D shows polyclonal NK cell lines incubated on plates coated with rNID1 for 20 h at 37°C. IFN-g release in the SN was evaluated by ELISA. Data are medians of eight independent experiments ± interquartile range performed with NK cells derived from three donors. ns=0.2305 by one-tailed Wilcoxon test.

Figure 7E shows Venn diagrams showing protein profile overlap between rNID1- and anti-NKp44 mAb-stimulated NK cells (a) Venn diagram of total proteins identified in NK cells exposed to the indicated controls (CTR, GAM) or stimuli (NID1 , anti-NKp44 mAb). Venn diagram shows common and exclusive proteins. Numbers represent the distinct proteins in the respective overlapping and not— overlapping areas (b) Venn diagram of up- and down-regulated proteins in NK cells stimulated with rNID1 or anti- NKp44 mAb. Numbers present in not-overlapping areas indicate proteins exclusively identified in the NIDIvsCTR or NKp44vsGAM dataset.

Figure 8 shows proteomic analysis of NK cells stimulated with rNID1 or anti-NKp44 mAb. Figures 8A and B show volcano plot representation of differentially expressed proteins. Plots represent stimulated (rNID1 ) vs. not stimulated (CTR) NK cells (Figure 8A) or stimulated (anti-NKp44 mAb) vs. not stimulated (GAM) NK cells (Figure 8B). Black dots represent proteins that display both large magnitude fold-changes (x-axis, proteins up-regulated after treatment are shown on the right) as well as high statistical significance (-Iog10 of P value, y- axis). The black line shows where FDR = 0.05 and sO = 0.1. Red asterisks represent proteins (with the indicated gene names) whose fold-change is < 2 (log2 = 1 ) or the P> 0.05 and which are present in the two datasets. Gray squares represent non statistical significant proteins. Figures 8C and D show Gene Ontology (GO) categorization of proteins significantly modulated by rNID1 and anti-NKp44 mAb stimuli according to biological processes (Figure 8C) or cellular component (Figure 8D). On the x axis, the percentage of proteins associated with the indicated GO terms is shown.

Figure 9. Analysis of NID1 surface expression and NKp44Fc reactivity on a panel of human cell lines. The indicated cell lines (SH-SY-5Y neuroblastoma; A172 glioblastoma; C-32 melanoma; A2774 ovarian adenocarcinoma; JEG-3 placental choriocarcinoma; A549 lung carcinoma) were stained with a NID1 -specific mAb or with NKp44Fc followed by the appropriate isotype-matched PE-conjugated secondary mAb. Samples were analyzed by flow cytometry. Grey profiles represent cells stained with anti-NID1 or with NKp44Fc, while white profiles correspond to isotype control. One representative experiment of three is shown.

DESCRIPTION OF THE INVENTION In this study we have identified and characterized an extracellular ligand of the natural cytotoxicity receptor (NCR) known as NKp44. Analysis of the culture supernatant of the HEK293T cell line that was reactive with a NKp44-Fc fusion protein (NKp44Fc) and the use of 2-DE, combined with high-resolution mass spectrometry, revealed NID1 as an NKp44-reactive molecule. Interaction between NID1 and NKp44 was validated both by immunoprecipitation and by ELISA experiments, indicating that NKp44 recognizes NID1 in its native conformation. Soluble NID1 could down-regulate NKp44-mediated NK cell activation; in addition, plastic- bound NID1 induced significant changes in the NK cell proteomic profile, suggesting a possible effect on various NK cell functions.

NID1 glycoprotein is an essential component of the basement membrane (BM) that plays a role both in BM assembly and stabilization, and in the adhesion between cells and extracellular matrix (ECM). Therefore, NKp44-NID1 interactions are likely to occur in tissues, in particular in the mucosae, which contain both NK cells and ILC3 cells, expressing NKp44. The engagement of NKp44 in ILC3 may induce TNF-a production and synergize with IL-1 , IL-7, and IL-23 to induce secretion of IL-22 (a cytokine typically produced by ILC3s). In a recent study, the transcriptome analysis of NKp44-stimulated ILCs revealed a “genome wide regulating effect” (Glatzer et al. Immunity 2013; 38:1223-35). This finding is in line with our present proteomic study showing that NK cell stimulation both via mAb-mediated NKp44 cross- linking and by plastic-bound NID1 could induce significant changes in a relevant number of proteins. Consistent with the multiple ligand specificities of NKp44 and its ability to mediate different functional responses, NKp44 cross-linking and NID1 -induced stimulation resulted in protein changes that were only partially overlapping. However, the GO analysis suggested some common functional effects induced by the two stimuli. Based on this analysis, NID1 recognition appeared to influence different biological processes related not only to immunologic functions but also to cell metabolism, proliferation, and development. In this context, it is of note that among the limited number of proteins up-regulated by both stimuli, the highest score was associated with MYSM1 , a molecule that has been proposed to play a role in NK cell development/maturation. Thus, the finding that MYSM1 was induced by both stimulation with rNID1 and mAb-mediated NKp44 cross-linking suggests a new NKp44- induced cell function and offers insights into the role of NKp44-NID1 interaction in the process of NK cell (and/or ILC) maturation and differentiation.

Functional experiments disclosed herein revealed that soluble NID1 may play a role as a decoy ligand. Indeed, the addition of rNID1 to cell cultures resulted in inhibition of NKp44- mediated responses in Bw-NKp44 cells. An inhibitory effect was displayed also by soluble NID1 released from cell transfectants. Importantly, released NID1 could significantly decrease NKp44-induced IFN-g secretion (and, to a lesser extent, cytotoxicity) in normal, polyclonal human NK cells. Remarkably, the NID1 -mediated inhibition was specific for NKp44, although high concentrations of rNID1 had a slight inhibitory effect also in Bw-NKp30 cells. On the other hand, the supernatant from NID1 -releasing cell transfectants did not inhibit NKp30 function in Bw-NKp30 cells. In addition, it could not inhibit NKp30- and NKp46-mediated responses in normal, polyclonal NK cells (see Figure 5). Finally, rNID1 did not react with NKp30Fc in ELISA (see Figure 2C). The release of NID1 in extracellular fluids may thus represent a regulatory mechanism that can act on NKp44+ NK cells at specific sites or in the blood stream.

Recent studies have highlighted the presence of soluble NID1 in samples from different human tumors, including in ovarian cancer patients (Li et al. Jpn J Clin Oncol 2015; 45:176-82) or NSCLC patients (Willumsen et al., Neoplasia N Y N 2017; 19:271-8) or gastrointestinal tumor samples (Ulazzi et al. Mol Cancer 2007; 6:17), breast cancer and melanoma (Aleckovic et al., Genes Dev 2017; 31 :1439-55). Thus, NID1 appears to be released in the extracellular environment of various tumor types. In the present disclosure, the inhibitory NID1-NKp44 interaction however provides a suppressive mechanism exploited by tumors to prevent the NK cell-mediated attack.

NID1 is generally released as a whole molecule. Therefore, the inhibitory effect on NK cells may be mediated by the intact molecule. In other embodiments, NID1 may be modified in the ECM by the action of extracellular proteases, such as Cat-S and ADAMTS1 (A Disintegrin and Metalloproteinase with ThromboSpondin motifs) (Martino-Echarri et al. Int J Cancer 2013; 133:2315-24). In addition, NID1 can interact with different BM/ECM proteins including laminin, collagen type IV, and perlecan (Ho et al. Microsc Res Tech 2008; 71 :387-95; Kruegel et al. Cell Mol Life Sci CMLS 2010; 67:2879-95). Thus, it is possible that the tissue microenvironment and the molecular context may further influence the functional outcome of the NID1-NKp44 interaction, and NID1 can be associated with other proteins or co-receptors.

Importantly, we show that NID1 , besides being released in the extracellular space, can be also detected at the cell surface of NID1 -releasing cells (see Figure 6). In this context, it has been reported that NID1 may associate through the interaction of its RGD sequence with a3b1 or anb3 integrins (Dedhar et al. J Biol Chem 1992; 267:18908-14; Yelian et al. J Cell Biol 1993; 121 :923-9). Notably, we could detect surface NID1 on a panel of different NKp44Fc- binding human tumor cell lines (Figure 9). Although it has been reported that NID1 up- regulation occurs in several tumor types, information was still lacking on whether NID1 is associated to the tumor cell surface. We assessed the possible functional outcome of the interaction between cell surface-associated NID1 on NKp44+ responding cells. In the Bw- NKp44 model, no induction of IL-2 production could be observed. In NKp44+ NK cells, neither induction of IFN-g production upon exposure to rNID1 -coated plates nor increase of cytotoxic activity against NID1 -expressing target cells could be detected. On the other hand, as discussed in the Examples, using a proteomic approach, we found that surface-bound rNID1 could induce changes in proteins involved in different biological processes. Such proteomic changes may be induced also by cell surface-associated NID1.

Taken together, provided are new opportunities for a more effective exploitation of NK cells and/or other ILCs that express activating receptors that are bound and inhibited by NID (e.g. NKp44-expressing ILCs), for therapeutic purposes, including but not limited to the immunotherapy of tumors.

Definitions

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 110 or more amino acids that is primarily responsible for antigen recognition. The terms variable light chain (Vi_) 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” includes full-length antibodies but 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 (H1 ), 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. NKp44 or NID1 , 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. NID1 , NKp44, another antibody) is said to“compete with” a second molecule (e.g. sNID1 , NKp44, another antibody) for binding to a target protein (e.g. NID1 , NKp44, 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 NKp44 to a NID1 polypeptide or NID1 -expressing cell in a binding assay, the antibody is said to“compete” respectively with NKp44.

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“deplete” or“depleting”, with respect to cells (e.g. NID1 -expressing cells) means a process, method, or compound that results in killing, elimination, lysis or induction of such killing, elimination or lysis, so as to negatively affect the number of such cells present in a sample or in a subject. “Non-depleting”, with reference to a process, method, or compound means that the process, method, or compound is not depleting.

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 g (gamma) heavy chain or its counterpart sequence in other types of antibody heavy chains (e.g., a, d, e and m 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,“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 and/or CD56 for human NK cells (as well as NKp44), and 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).

“NKp44” refers to a protein or polypeptide encoded by the Ncr2 gene or by a cDNA prepared from such a gene, see, e.g., UniprotKB identifier 095944. Any naturally occurring isoform, allele or variant is encompassed by the term NKp44 polypeptide. An NKp44 polypeptide may for example be 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 amino acid residue sequence of human NKp44 (isoform 1 ) is shown as follows:

MAWRALHPLL LLLLLFPGSQ AQSKAQVLQS VAGQTLTVRC QYPPTGSLYE KKGWCKEASA LVCIRLVTSS KPRTMAWTSR FTIWDDPDAG FFTVTMTDLR EEDSGHYWCR IYRPSDNSVS KSVRFYLVVS PASASTQTSW TPRDLVSSQT QTQSCVPPTA GARQAPESPS TIPVPSQPQN STLRPGPAAP IALVPVFCGL LVAKSLVLSA LLVWWGDIWW KTMMELRSLD TQKATCHLQQ VTDLPWTSVS SPVEREILYH TVARTKISDD DDEHTL (SEQ ID NO: 1 ).

SEQ ID NO: 1 corresponds to Uniprot accession number 095944, the disclosure of which is incorporated herein by reference.

Nidogen-1 (NID1 ), also referred to as“entactin”, is a sulfated glycoprotein widely distributed in basement membranes and serves as the linker between other basement membrane proteins, namely, collagen type IV, perlecan, and laminin. It is synthesized by cells as a 1247 amino acid (aa) precursor with a 28 aa signal sequence and a 1219 aa mature protein. NID1 comprises three globular domains (G1 , G2, and G3) connected by two rod- shaped domains. Nidogen-1 has been found to be a substrate for cathepsin-S (Cats) and at least two MMPs, MMP-7 and MMP-19. Degradation of nidogen-1 by Cats results in impaired binding between nidogen-1 and type IV collagen, laminin, and perlecan and can thereby affect the properties of the basement membrane. Nidogen-1 degraded by Cats may reflect loss of basement membrane integrity (e.g. as may be associated with cancer having an invasive phenotype). The NID1 protein and gene sequences are known in the art. Natural NID1 protein may be purified from biological samples. Recombinant NID1 protein may be expressed and purified using conventional techniques. The NID1 protein may or may not be purified. Exemplary amino acid sequences of human NID1 are described in Uniprot accession number P14543. The amino acid sequence of the mature human NID1 protein (not including the 28 amino acid leader sequence MLASSSRIRA AWTRALLLPL LLAGPVGC SEQ ID NO: 7) is shown below in SEQ ID NO: 2:

LSRQELFPFG PGQGDLELED GD DFVSPALELS GALRFYDRSD IDAVYVTTNG IIATSEPPAK ESHPGLFPPT FGAVAPFLAD LDTTDGLGKV YYREDLSPSI TQRAAECVHR GFPEISFQPS SAVWTWESV APYQGPSRDP DQKGKRNTFQ AVLASSDSSS YAIFLYPEDG LQFHTTFSKK

ENNQVPAVVA FSQGSVGFLW KSNGAYNIFA NDRESVENLA KSSNSGQQGV WVFEIGSPAT

TNGWPADVI LGTEDGAEYD DEDEDYDLAT TRLGLEDVGT TPFSYKALRR GGADTYSVPS

VLSPRRAATE RPLGPPTERT RSFQLAVETF HQQHPQVIDV DEVEETGVVF SYNTDSRQTC

ANNRHQCSVH AECRDYATGF CCSCVAGYTG NGRQCVAEGS PQRVNGKVKG RIFVGSSQVP IVFENTDLHS YWMNHGRSY TAISTIPETV GYSLLPLAPV GGIIGWMFAV EQDGFKNGFS ITGGEFTRQA EVTFVGHPGN LVIKQRFSGI DEHGHLTIDT ELEGRVPQIP FGSSVHIEPY TELYHYSTSV ITSSSTREYT VTEPERDGAS PSRIYTYQWR QTITFQECVH DDSRPALPST QQLSVDSVFV LYNQEEKILR YALSNSIGPV REGSPDALQN PCYIGTHGCD TNAACRPGPR TQFTCECSIG FRGDGRTCYD IDECSEQPSV CGSHTICNNH PGTFRCECVE GYQFSDEGTC VAWDQRPIN YCETGLHNCD IPQRAQCIYT GGSSYTCSCL PGFSGDGQAC QDVDECQPSR CHPDAFCYNT PGSFTCQCKP GYQGDGFRCV PGEVEKTRCQ HEREHILGAA GATDPQRPIP PGLFVPECDA HGHYAPTQCH GSTGYCWCVD RDGREVEGTR TRPGMTPPCL STVAPPIHQG PAVPTAVIPL PPGTHLLFAQ TGKIERLPLE GNTMRKTEAK AFLHVPAKVI IGLAFDCVDK MVYWTDITEP SIGRASLHGG EPTTI IRQDL GSPEGIAVDH LGRNIFWTDS NLDRIEVAKL DGTQRRVLFE TDLVNPRGIV TDSVRGNLYW TDWNRDNPKI ETSYMDGTNR RILVQDDLGL PNGLTFDAFS SQLCWVDAGT NRAECLNPSQ PSRRKALEGL QYPFAVTSYG KNLYFTDWKM NSVVALDLAI SKETDAFQPH KQTRLYGITT ALSQCPQGHN YCSVNNGGCT HLCLATPGSR TCRCPDNTLG VDCIEQK

(SEQ ID NO : 2)

The inventors have shown that NKp44 binds to NID1. Binding of soluble NID1 (sNID1 ) as may be released by cells, binds to NKp44 and inhibits NKp44 signaling in NK cells. While it is shown that NID1 can bind alone to NKp44, it will be appreciated that NID1 can also bind NKp44 as part of a complex of proteins that collectively act as a receptor for NKp44.

Detection, Diagnosis, Pharmacodynamics, and Patient Monitoring and Selection

Methods of Detection

Methods of detecting sNID1 (and/or co-ligands that form a protein complex with sNID1 ) in a biological sample are also provided. The detection of sNID1 (and/or co-ligands) 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 sNID1 proteins are detected by their interaction with a NID1 -specific reagent such as antibody. The NID1 -specific reagent can for example be an antibody that specifically binds a full-length sNID1 (e.g. as secreted from cells) or a fragment thereof. Optionally a fragment is a NKp44-binding fragment of full-length sNID1 (e.g. as cleaved by an enzyme such as Cats or an MMP. The antibody can for example be characterized by binding a NKp44-binding sNID1 fragment preferentially over full length sNID1 , or of full-length sNID1 preferentially over a sNID1 cleavage fragment, e.g. the antibody has binding affinity for a NKp44-binding form sNID1 that is substantially greater (e.g., 100x, 500x, 1000x, 10,000x, or more) that its binding affinity for another form of NID1. In other examples, NID1 can be detected using a composition comprising a NKp44 polypeptide or a NID1 -binding fragment thereof. In one exemplary method, a NKp44 polypeptide or fragment thereof (e.g. a fragment fused to a detectable moiety) that retains the ability to bind NID1 is used to bind and/or detect the presence and/or levels of sNID1 , including but not limited to identify NID1 -expressing and/or -releasing cells (e.g. in tumor tissues or tumor adjacent tissues). An exemplary NKp44 polypeptide comprises an amino acid sequence of SEQ ID NO 1 , or a fragment of an extracellular domain 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 NID1 proteins and/or NID1 -expressing cells in either a qualitative or quantitative manner.

Exemplary immunoassays that can be used for the detection of NID1 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 NID1 (e.g. sNID1 ) 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 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 NID1. 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 NID1 include the ability to contact a biological sample with an antibody specific to a NID1 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 NID1 may be used to detect the presence and altered production of NID1. An exemplary immunoassay is ELISA. ELISA typically includes the use of two different NID1 or co-ligand-specific antibodies: a capture antibody and a detection antibody. In some embodiments an antibody or antigen binding fragment thereof that recognizes NID1 is used to capture most or all of the NID1 in the sample. A detection antibody that can recognize most or all of the NID1 can be used to determine the total level of NID1 in the biological sample. In some embodiments, the detection antibody recognizes a different domain or epitope than the capture antibody.

In some embodiments, anti-sNID1 receptor antibodies, and methods of detecting sNID1 can be used to detect the presence and altered (e.g. increased) production of sNID1.

Detecting NID1 in a biological sample (e.g., as sNID1 and/or as mNID1 on cells in the biological sample) from an individual can be useful in methods to assess or predict whether the individual has a disease or condition characterized by immunosuppression, optionally a disease or condition characterized by insufficient NK and/or ILC activity, for example in an individual receiving or who is a candidate to receive treatment with a therapeutic agent that enhances anti-tumor immunity, including but not limited to agents that inhibit immunosuppression or that enhance NK and/or ILC cell-mediated activity. Such agents can be tested to assess their efficacy in individuals having (or not) detectable sNID1 or mNID1- expressing cells, and can thereby be administered (or not) as a function of NID1 status in the individual. In one embodiment, the therapeutic agent is an NK cell or ILC cell composition. In one embodiment, the therapeutic agent is an agent that acts as an agonist at an activating receptor on an NK and/or ILC cell. In one embodiment, the therapeutic agent is an agent that acts as an antagonist at an inhibitory receptor on an NK and/or ILC cell. In one embodiment, the therapeutic agent is an agent that is capable of binding a cancer antigen and is capable of mediating ADCC. In one embodiment, the NK cells are NKp44-expressing NK cells.

Diagnosis

A disease or disorder in an individual (e.g. a cancer) characterized by immunosuppression, including insufficient anti-tumor NK and/or ILC cell activity, can be detected by quantifying the amount of sNID1 or co-ligands thereof in a biological sample of the individual, wherein an elevated amount of sNID1 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 characterized by immunosuppression. In one embodiment, sNID1 (e.g. at elevated levels compared to a control, for example a healthy individual or a non-immunosuppressed individual) in circulation or in a tissue of interest (e.g. tumor tissue or tumor adjacent tissue) is indicative of a disorder characterized by immunosuppression, optionally low and/or insufficient NK cell activation. In one embodiment, mNID1 -expressing cells (e.g. NID1 -releasing cells) in a tissue of interest (e.g. tumor tissue or tumor adjacent tissue) is indicative of a disorder characterized by immunosuppression, optionally low and/or insufficient NK cell activation. In one embodiment the sNID1 and/or mNID1 -expressing cells is indicative of a disorder characterized by susceptibility to disease control by NK cells, optionally NKp44-expressing 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 NID1 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 NID1 or co-ligand in an individual's biological sample and correlating the amount of NID1 in the individual's biological sample with amount NID1 or co-ligand expression indicative of different stages of a disorder or disease. The amounts of NID1 or co-ligand that correlate to different stages of disease or disorder or different levels of severity can be predetermined by quantifying NID1 or co-ligand levels in patients at different stages of, or with different severity of, a disease or disorder.

Pharmacodynamic Markers

The effectiveness of treatments using immunotherapeutic agents can be determined by assaying a sample obtained from a subject receiving treatment with the immunotherapeutic agent for changes in levels of sNID1 and/or co-ligands, or for presence or levels of mNID1- expressing cells in tissues of interest. For example, baseline levels of sNID1 in a biological sample obtained from a subject can be determined prior to treatment. After or during treatment with the immunotherapeutic agent (e.g. an agent that acts as agonist or antagonist of NK or ILC cell function, sNID1 levels in biological samples from the subject can be monitored.

A change in biomarker level, for example a decline in sNID1 , relative to baseline levels can indicate that the treatment is effective in reducing immunosuppression. 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.

Patient Selection Methods of determining the level of NID1 (e.g. sNID1 ) and/or co-ligand may also allow the selection of patients most likely to respond to a therapeutic agent, optionally a therapy that provides, enhances or inhibits immune cell activity, notably NK cell and/or ILC cell activity. For example, as shown herein, sNID1 mediates immune suppression via binding to NKp44. sNID1 can thereby decrease the ability of NK and/or other NKp44-expressing cells to control disease, notably cancer. sNID1 can therefore indicate insufficient anti-tumor immunity, particularly a deficiency in NK cell activity.

Individuals having sNID1 and/or NID1-releasing cells could be selected for treatment with an immunotherapeutic agent that reduces the level and/or activity of sNID1. In another example, individuals having sNID1 and/or NID1-releasing cells (or having high or increased level of sNID1 and/or NID1-releasing cells) could be selected for treatment with an immunotherapy and/or a chemotherapeutic agent that is effective in enhancing anti-tumor immunity in patients having such immunosuppression (e.g. in patients having sNID1 and/or NID1-releasing cells). Optionally the immunotherapy enhances NK and/or ILC cell activity. A further example could be selection for treatment with a composition comprising cytotoxic NK and/or ILC cells.

Examples of immunotherapeutic agents include agents (e.g. a monoclonal antibody) that bind an antigen expressed at the surface of a malignant cell, optionally wherein the agent is a depleting agent that mediates ADCC, CDC and/or ADCP. Other examples of immunotherapeutic agents include agents (e.g. a monoclonal antibody) that bind (e.g. and leads to activation of) an activating receptor (including co-activating receptors) expressed by NK and/or T cells. Examples of activating receptors are 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). Yet further examples of immunotherapeutic agents include agents (e.g. a monoclonal antibody) that bind and inhibit 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).

Identification of new modulators of the NID1-NKp44 interaction

Agents that interfere with or enhance the NID1-NKp44 interaction can be useful in a variety of application. For example, these compositions can be used to measure NID1-NKp44 interactions, to measure the inhibition or enhancement of NID1-NKp44 interactions, for the identification of NID1 -expressing cells, for the identification of new cell types, and/or for studies into the molecular mechanisms of action of NID1 and NKp44.

Bioactive agents that may be screened for the ability to inhibit or enhance the NID1- NKp44 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 NKp44 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, N,N'-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, NKp44 and NID1 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 NKp44-NID1 interaction.

In another exemplary assay, NK expressing NKp44 or other cells made to express NKp44 can be used to identify antibodies, small molecules, and other modulators that are agonists or antagonists of NKp44, in the presence of NID1 or NID1 -expressing cells.

In another exemplary assay, NK cells expressing NKp44 or other cells made to express NKp44 can be used to identify antibodies, small molecules, and other modulators that enhance the activity (e.g. the cytolytic activity) of NKp44-expressing NK cells, towards target cells (e.g. cancer cells).

In another exemplary assay, NK expressing NKp44 or other cells made to express NKp44 can be used to identify antibodies, small molecules, and other modulators that inhibit the activity (e.g. the cytolytic activity) of NKp44-expressing NK cells, towards target cells.

In one embodiment of such an assay, the method comprises: (i) bringing NKp44 (e.g. as a cell expressing NKp44 or as a soluble NKp44 protein) into contact with NID1 (e.g. as a cell expressing NID1 or as a soluble NID1 protein) in the presence of a test agent (e.g. a plurality of test agents), and (ii) assessing binding of NID1 to NKp44. A decrease in binding (compared to negative control) indicates that the agent interferes with binding of NID1 and NKp44. An increase in binding (compared to negative control) indicates that the agent enhances the interaction of NID1 and NKp44.

In one embodiment provided is a method of identifying agents that increase NK cell activity, comprising: (i) bringing NKp44 (e.g. as a cell expressing NKp44 or as a soluble NKp44 protein) into contact with a composition comprising a NID1 polypeptide (e.g. sNID1 ) or fragment thereof, (ii) assessing binding of the composition to NKp44. In another embodiment provided is a method of identifying agents that increase NK cell activity, comprising: (i) bringing NKp44 (e.g. as a cell expressing NKp44 or as a soluble NKp44 protein) into contact with a composition comprising a NID1 polypeptide (e.g. sNID1 ) or fragment thereof, (ii) assessing whether the composition increases NK cell activity. 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 NID1 to NKp44 and/or increases NK cell activity. In one embodiment, the agent is for use in treatment or prevention of cancer.

In one embodiment provided is a method of assessing NK cell (or other NKp44- expressing cell) activity towards target cells (e.g. tumor cells), comprising: (i) bringing an NKp44-expressing cell (e.g. NK cell) composition into contact with a target cell in the presence of sNID1 , (ii) assessing the activity of the NKp44-expressing cell (e.g. NK cell) towards the target cell. Optionally, assessing the activity of the NKp44-expressing cell towards the target cell comprises assessing a parameter or marker of cell maturation and/or development, optionally, assessing MYSM1 expression. Optionally, assessing the activity of the NKp44- expressing cell towards the target cell comprises assessing cytokine production and/or any measure of 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.

Where the test agent is an antibody, for example for the identification of antibodies that interfere with and/or inhibit the NID1-NKp44 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 NID1 polypeptide, preferably a human NID1 polypeptide. The NID1 polypeptide may comprise the full length sequence of a human NID1 polypeptide, or a fragment or derivative thereof, typically an immunogenic fragment, i.e., a portion of the polypeptide comprising an epitope exposed on sNID1 polypeptide, optionally which is not present on mNID1 polypeptides (e.g., as expressed by cells). 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.

Use of agents that alleviate the immunosuppressive effect of sNID-1 for treatment of cancer or infectious disease

Agents that alleviate the immunosuppressive effect of sNID-1 and/or restore and/or enhance NKp44+ NK cell activity can be particularly useful to treat cancer and/or restore anti- tumor immunity in individuals having a cancer, or to treat an infectious disease and/or restore immunity to a pathogen or pathogen-infected cell. Optionally, the cancer is characterized by a solid tumor, optionally an invasive and/or metastatic cancer, particularly a cancer characterized by the presence of detectable NID1 , for example as may occur during loss of basement membrane integrity. Loss of basement membrane integrity is typically associated with invasive and/or metastatic disease. Agents that block the interaction of NID1 with NKp44 and/or reduce the amount of sNID1 present in an individual can be useful to enhance or restore the activity of NK cells, notably NKp44-expressing NK cells.

Additionally, because the immunosuppressive effect of sNID1 may attenuate the anti- tumor response mediated by immunotherapeutic agents, agents that block the interaction of NID1 with NKp44 and/or reduce the amount of sNID1 present in an individual can be useful to enhance the therapeutic effect of an immunotherapy, e.g. an immunotherapy that directly or indirectly enhances the activity of T cell and/or NK cells. Reducing the amount of sNID1 present in an individual can be achieved by any of a variety of methods. In one example, sNID1 as may be found in circulation and/or tissues can be sequestered, destroyed or otherwise eliminated, for example by targeting with an agent such as a protein or antibody that binds sNID1 , optionally the agent binds sNID1 and targets sNID1 for degradation by proteolysis. In another example, NID1 releasing cells, for example cells that express at their surface membrane-bound NID1 (mNID1 ) can be sequestered, removed, destroyed or otherwise eliminated, for example by targeting with a composition comprising an agent such as a protein or antibody that binds mNID1. In another example, the immunosuppressive effective of sNID1 on NKp44-expressing immune cells is blocked with an agent such as a protein or antibody that interferes with the interaction between NKp44 and sNID1.

In one aspect of any embodiment herein, an agent, antibody or protein is in purified or at least partially purified form. In one aspect of any embodiment herein, the antibody is in isolated form.

In one aspect of any embodiment herein, an agent, antibody or protein may be characterized as specifically binding to sNID1 and mNID1. In one aspect of the invention, the antibody may be characterized as specifically binding to sNID1 but not binding to mNID1.

In one aspect of any embodiment herein, the agent is an antibody selected from a full- length antibody, an antibody fragment, and a synthetic or semi-synthetic antibody-derived molecule.

In one aspect of any embodiment herein, the agent is an antibody selected from a fully human antibody, a humanized antibody, and a chimeric antibody.

In one aspect of any embodiment herein, the agent is a fragment of an antibody comprising a constant or Fc domain selected from lgG1 , lgG2, lgG3 and lgG4, optionally modified compared to a naturally occurring constant or Fc domain.

In one aspect of any embodiment herein, the agent is or comprises an antibody fragment selected from a Fab fragment, a Fab' fragment, a Fab'-SH fragment, a F(ab)2 fragment, a F(ab')2 fragment, an Fv fragment, a Heavy chain Ig (a llama or camel Ig), a VHH fragment, a single domain FV, and a single-chain antibody fragment.

In one aspect of any embodiment herein, the agent is or comprises a synthetic or semisynthetic antibody-derived molecule selected from a scFV, a dsFV, a minibody, a diabody, a triabody, a kappa body, an IgNAR; and a multispecific (e.g. bispecific, tri-specific) antibody.

Agents that inhibit the NKp44-sNID1 interaction can restore NKp44+ NK cell activity, and can be particularly useful to treat disease characterized by sNID1 protein, optionally sNID1 in circulation, optionally sNID1 in tumor tissue and/or tumor-adjacent tissue. For example, antibodies or other agents (e.g. a composition or protein comprising a sNID1 -binding fragment of NKp44) that inhibit the NKp44-sNID1 interaction can inhibit the effect of sNID1 on NKp44. Optionally the antibody or other agent reduces the sNID1 -mediated inhibition of NKp44- expressing cell activity, e.g. the antibodies can restore the NKp44 signaling pathway. Agents (e.g. antibodies) that inhibit the NKp44-sNID1 interaction can thus be referred to as “neutralizing” or“inhibitory” or“blocking” agents (or, e.g., antibodies). Such agents are useful, inter alia, for restoring, enhancing or potentiating the activity of NKp44-expressing immune cells, e.g. for the treatment or prevention of conditions where potentiating or increasing 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 inhibit the NKp44-sNID1 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- NKp44 antibodies to modulate NKp44-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.

In one embodiment, an agent or composition that inhibits the NKp44-sNID1 interaction is a protein that comprises a sNID1 -binding fragment of a human NKp44 protein. Optionally, the protein further comprises an Fc domain, optionally a dimeric Fc domain that binds to human FcRn polypeptides.

In one embodiment, an agent or composition that inhibits the NKp44-sNID1 interaction comprises an antibody that specifically binds to a sNID1 protein. In one embodiment, the antibody is capable of binding to human sNID1 and human mNID1. In one embodiment, the antibody binds to human sNID1 but not to human mNID1.

In one embodiment, an antibody or other agent that inhibits the NKp44-sNID1 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 or other protein that comprises an Fc domain 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 WO2011/066501 ).

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/P331S or (L234F/L235E/P331S). Another example of such an Fc domain comprises substitutions at Kabat residues L234, L235, G237 and P331 (e.g., L234A/L235E/G237A/P331S). 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 L234Xi substitution, a L235X ₂ substitution, and a P331X ₃ substitution, wherein Xi 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 Xi 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 L234Xi substitution, a L235X ₂ substitution, a G237X ₄ substitution and a P331 X ₄ substitution, wherein Xi 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 Xi 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 L234Xi substitution, a L235X ₂ substitution, a G237X ₄ substitution, G330X ₄ substitution, and a P331Xs substitution, wherein Xi 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 Xi 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 Xs 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.

In some examples, reducing the level and/or activity of sNID1 that is available to interact with NKp44 can in one example be achieved by targeting NID1 -releasing cells with an agent such as a protein or antibody that binds mNID1 on the surface of a NID1 -releasing cell and prevents the generation and/or secretion of NID1 by the cell. While such an agent (e.g. protein or antibody) may additionally be depleting towards mNID1 -expressing cells (e.g. it may comprise an Fc domain that mediates ADCC, CDC, ADCP), the agent that prevents the release of sNID1 from cells need not be depleting towards mNID1 -expressing cells. The agent may or may not interfere with the interaction between NKp44 and sNID1. A non-depleting agent may have the advantage of decreased toxicity to healthy cells. For example, in one embodiment, an antibody or other agent that prevents the release of NID1 from cells 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 agents may comprise constant regions of various heavy chains that are known to lack or have low binding to Fey receptors.

The affinities and binding properties of the anti-NID1 antibodies for an FcyR can be determined using in vitro assays (biochemical or immunological based assays) known in the art for determining antibody-antigen or Fc-FcyR interactions, i.e., specific binding of an antigen to an antibody or specific binding of an Fc region to an FcyR, respectively, including but not limited to ELISA assay, surface plasmon resonance assay, immunoprecipitation assays.

Examples of cancer that can be particularly advantageously treated with agents described herein (e.g. an agent that restores NKp44+ NK cell activity; an antibody or antigen binding domain that binds NID1 ) include, inter alia, 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. Malignant tumors may show up at numerous organs or tissues of the body to establish a cancer. Examples of cancers that can be treated include, but are not limited to bladder, skin, breast, cervical, head and neck, colon or bowel (e.g. colorectal), esophageal, kidney, liver, lung, prostate, skin, stomach, uterine, ovarian, endometrial, and testicular cancers. Further examples include neuroblastoma and glioblastoma.

Examples of infectious disease that can be particularly advantageously treated with immune response-enhancing agents described herein (e.g. an agent that restores NKp44+ NK cell activity; an antibody or antigen binding domain that binds NID1 ) include, inter alia, infections caused by infection by viruses, bacteria, protozoa, molds or fungi. Such viral infectious organisms include, but are not limited to, hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, herpes simplex type I (HSV-1 ), herpes simplex type 2 (HSV-2), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papilloma virus, cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus, polio virus and human immunodeficiency virus type I or type 2 (HIV-1 , HIV-2). Bacteria constitute another preferred class of infectious organisms including but are not limited to the following: Staphylococcus; Streptococcus, including S. pyogenes ; Enterococci; Bacillus, including Bacillus anthracis, and Lactobacillus; Listeria; Corynebacterium diphtheriae ; Gardnerella including G. vaginalis ; Nocardia; Streptomyces; Thermoactinomyces vulgaris ; Treponema; Camplyobacter, Pseudomonas including P. aeruginosa ; Legionella; Neisseria including N. gonorrhoeae and N. meningitides ; Flavobacterium including F. meningosepticum and F. odoraturn ; Brucella; Bordetella including B. pertussis and B. bronchiseptica ; Escherichia including E. coli, Klebsiella; Enterobacter, Serratia including S. marcescens and S. liquefaciens ; Edwardsiella; Proteus including P. mirabilis and P. vulgaris ; Streptobacillus; Rickettsiaceae including R. fickettsfi, Chlamydia including C. psittaci and C. trachomatis, Mycobacterium including M. tuberculosis, M. intracellulare, M. folluiturn, M. laprae, M. avium, M. bovis, M. africanum, M. kansasii, M. intracellulare, and M. lepraernurium ; and Nocardia. Protozoa may include but are not limited to, leishmania, kokzidioa, and trypanosoma. Parasites include but are not limited to, chlamydia and rickettsia.

An antibody or antigen binding domain that binds NID1 may be produced by a variety of techniques known in the art. In one example, the antigen binding domain comprises a NKp44 polypeptide (e.g. an extracellular domain of the NKp44 polypeptide or a fragment thereof that binds human NID1 ). In another embodiment, an antibody or antibody fragment comprising an antigen binding domain (e.g. immunoglobulin hypervariable region) that binds NID1 is generated, for example by selection from a library antigen binding domains or by immunization of a non-human animal. The step of immunizing a non-human mammal with an antigen 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).

Once antibodies are identified that are capable of binding a NID1 polypeptide and/or having other desired properties, they will also typically be assessed, using standard methods including those described herein, for their ability to bind to other polypeptides. Ideally, the antibodies only bind with substantial affinity to the NID1 polypeptide (sNID1 and/or mNID1 , as a function of the particular desired mode of action), and do not bind at a significant level to unrelated polypeptides. However, it will be appreciated that the antibodies can also be characterized as being suitable if affinity for NID1 (or sNID1 or mNID1 ) is substantially greater (e.g., 100x, 500x, 1000x, 10,000x, or more) than it is for a particular other polypeptide).

Upon production of antibodies, e.g., via immunization and production of antibodies in a vertebrate or cell or via phage display, particular selection steps may be performed to identify and/or isolate antibodies as claimed. In this regard, the disclosure also relates to methods of producing such antibodies, comprising: (a) providing a plurality of antibodies that bind sNID1 ; and (b) selecting antibodies from step (a) that are capable of interfering with the interaction between sNID1 and a NKp44 polypeptide (e.g. a NKp44 polypeptide expressed by a cell; an NKp44 polypeptide in solution or bound to a solid support). The disclosure also relates to methods of producing such antibodies, comprising: (a) providing a plurality of antibodies that bind mNID1 ; and (b) selecting antibodies from step (a) that are capable of mediating depletion of cells that express mNID1.

The anti-NID1 antibodies can for example be prepared as depleting antibodies that mediate lysis of mNID1 -expressing cells by immune effector cells, e.g., NK and/or T cells. In one example, a bispecific anti-NID1 antibody comprises a first antigen binding domain that bind mNID1 and a second antigen binding domain that binds a protein (e.g., an activating receptor such as CD3, CD8, CD16, NKp44, NKp30 NKG2D etc.) expressed by an immune effector cell. The bispecific antibody will direct the effector cell to lyse the mNID1 -expressing cell.

In one example, the anti-NID1 antibodies can be prepared as depleting antibodies that additionally or alternatively mediate ADCC through the incorporation of Fc domain that bind to human Fey receptors. Such antibodies, whether monospecific or multispecific, may comprise constant regions of various heavy chains that are known to bind, or to have high binding affinity for, human Fey receptors, particularly CD16. One such example is a human lgG1 constant region. Also, any antibody isotype can be used in which the Fc portion is modified to enhance or increase binding to Fc receptors, as further discussed herein.

In another embodiment, the anti-NID1 antibodies can for example be prepared as non- depleting antibodies that interfere with the interaction between NKp44 and sNID1. The ability to interfere with the interaction between NKp44 and sNID1 can be tested by the methods disclosed herein. Such antibodies may comprise constant regions of various heavy chains that are known not to bind, or to have low binding affinity for, human Fey receptors, particularly CD16. One such example is a human lgG4 constant region. Another example is an antibody of any isotype (e.g. lgG1 , lgG2, lgG3 or lgG4) that comprises an amino acid substitution to reduce binding to human Fey receptors, particularly CD16.

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 by surface plasmon resonance (e.g. in a BIACORE assay).

The DNA encoding an antibody that binds an epitope present on NID1 polypeptides is isolated from the hybridoma and placed in an appropriate expression vector for transfection into an appropriate host. The host is then used for the recombinant production of the antibody, or variants thereof, such as a humanized version of that monoclonal antibody, active fragments of the antibody, chimeric antibodies comprising the antigen recognition portion of the antibody, or versions comprising a detectable moiety.

DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e. g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. As described elsewhere in the present specification, such DNA sequences can be modified for any of a large number of purposes, e.g., for humanizing antibodies, producing fragments or derivatives, or for modifying the sequence of the antibody, e.g., in the antigen binding site in order to optimize the binding specificity of the antibody. Recombinant expression in bacteria of DNA encoding the antibody is well known in the art (see, for example, Skerra et al., Curr. Opinion in Immunol., 5, pp. 256 (1993); and Pluckthun, Immunol. 130, p. 151 (1992).

The identification of one or more antibodies that bind(s) to NID1 and/or inhibit(s) the interaction with NKp44 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, which is incorporated herein by reference). For example, where the test antibodies to be examined are obtained from different source animals, or are even of a different Ig isotype, a simple competition assay may be employed in which the control and test antibodies are admixed (or pre-adsorbed) and applied to a sample containing NID1 polypeptides.

Fragments and derivatives of antibodies (which are encompassed by the term “antibody” or“antibodies” as used in this application, unless otherwise stated or clearly contradicted by context) can be produced by techniques that are known in the art.“Fragments” comprise a portion of the intact antibody, generally the antigen binding site or variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F (ab') 2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a“single- chain antibody fragment” or“single chain polypeptide”), including without limitation (1 ) single- chain Fv molecules (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety and (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multispecific antibodies formed from antibody fragments. Included, inter alia, are a nanobody, domain antibody, single domain antibody or a“dAb”.

In certain embodiments, the DNA of a hybridoma producing an antibody, can be modified prior to insertion into an expression vector, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous non- human sequences (e.g., Morrison et al., PNAS pp. 6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In that manner, “chimeric” or“hybrid” antibodies are prepared that have the binding specificity of the original antibody. Typically, such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody.

Thus, according to another embodiment, the antibody is humanized. “Humanized” forms of antibodies are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F (ab') 2, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from the murine immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of the original antibody (donor antibody) while maintaining the desired specificity, affinity, and capacity of the original antibody.

In some instances, Fv framework residues of the human immunoglobulin may be replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in either the recipient antibody or in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of the original antibody and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details see Jones et al., Nature, 321 , pp. 522 (1986); Reichmann et al, Nature, 332, pp. 323 (1988); Presta, Curr. Op. Struct. Biol., 2, pp. 593 (1992); Verhoeyen et Science, 239, pp. 1534; and U.S. Patent No. 4,816,567, the entire disclosures of which are herein incorporated by reference.) Methods for humanizing the antibodies are well known in the art.

Another method of making “humanized” monoclonal antibodies is to use a XenoMouse (Abgenix, Fremont, CA) as the mouse used for immunization. A XenoMouse is a murine host according that has had its immunoglobulin genes replaced by functional human immunoglobulin genes. Thus, antibodies produced by this mouse or in hybridomas made from the B cells of this mouse, are already humanized. The XenoMouse is described in United States Patent No. 6,162,963, which is herein incorporated in its entirety by reference.

Human antibodies may also be produced according to various other techniques, such as by using, for immunization, other transgenic animals that have been engineered to express a human antibody repertoire (Jakobovitz et al., Nature 362 (1993) 255), or by selection of antibody repertoires using phage display methods. Such techniques are known to the skilled person and can be implemented starting from monoclonal antibodies as disclosed in the present application. Once an antigen-binding compound is obtained it may be assessed for its ability to mediate a biological activity, e.g., mediating depletion of a cell, induce ADCC or CDC towards, inhibiting the activity of, proliferation of cells, modulating (e.g. enhancing) the activity of NK cells, reducing the amount and/or activity of sNID1 , inhibiting production or secretion of NID1 from mNID1 -expressing target cells, and/or inhibiting the binding between sNID1 and NKp44.

An agent that alleviates the immunosuppressive effect of sNID1 and/or restore and/or enhance NKp44+ NK cell activity, may each be used in the treatment of cancer as monotherapies, or advantageously as combined treatments with one or more other therapies or therapeutic agents, including agents and therapies 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 therapies and therapeutic agents include, but are not limited to radiotherapy, anti-cancer agents and chemotherapeutic agents.

In one embodiment, the agent that alleviates the immunosuppressive effect of sNID1 and/or restore and/or enhance NKp44+ NK cell activity can particularly advantageously be used in combination with am immunotherapy (e.g. an immunotherapy or immunotherapeutic agent) that is designed to provide, stimulate and/or enhance an anti-cancer immune response. Since sNID1 may be limiting the efficacy of such immunotherapy, use of such immunotherapy in combination with an agent that alleviates the immunosuppressive effect of sNID1 can be advantageous. Examples of immunotherapeutic agents include agents (e.g. a monoclonal antibody) that bind an antigen expressed at the surface of a malignant cell, optionally wherein the agent is a depleting agent that mediates ADCC, CDC and/or ADCP, optionally wherein the agent is a multispecific agent that further specifically binds a protein at the surface of T and/or NK cells (e.g. CD3, CD16). Examples of immunotherapeutic agents include agents (e.g. a monoclonal antibody) that bind (e.g. and leads to activation of) an activating receptor (including co-activating receptors) expressed by NK and/or T cells. Examples of activating receptors are 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). Yet further examples of immunotherapeutic agents include agents (e.g. a monoclonal antibody) that bind and inhibit 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). In another example, the immunotherapeutic agent is a chemotherapeutic agent that has an immunomodulatory, particularly an immunostimulatory effect.

In the treatment methods, the agent that alleviates the immunosuppressive effect of sNID1 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 alleviates the immunosuppressive effect of sNID1 can be administered approximately 0 to 30 days prior to the administration of the second therapeutic agent. In some embodiments, the agent that alleviates the immunosuppressive effect of sNID1 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 alleviates the immunosuppressive effect of sNID1 is administered concurrently with the administration of the therapeutic agents. In some embodiments, the agent that alleviates the immunosuppressive effect of sNID1 is administered after the administration of the second therapeutic agent. For example, the agent that alleviates the immunosuppressive effect of sNID1 can be administered approximately 0 to 30 days after the administration of the second therapeutic agent. In some embodiments, the agent that alleviates the immunosuppressive effect of sNID1 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.

Exemplary Embodiments

1. A method of assessing the interaction of a NKp44 polypeptide with a ligand thereof, comprising the steps of: (i) bringing a NKp44 polypeptide into contact with a NID1 polypeptide and (ii) detecting the binding of NKp44 to the NID1 polypeptide.

2. The method of embodiment 1 , wherein NKp44 protein is brought into contact with NID1 in the presence of a test agent, optionally a plurality of test agents.

3. The method of embodiment 2, wherein step (ii) comprises assessing whether the test agent modulates binding of NKp44 to NID1.

4. The methods of embodiments 1-3, wherein the NID1 polypeptide is a soluble NID1 polypeptide (sNID1 ).

5. The methods of embodiments 1-3, wherein the NID1 polypeptide is a membrane- bound NID1 polypeptide (mNID1 ). 6. The methods of embodiments 1-5, wherein the NKp44 polypeptide is expressed at the surface of a cell.

7. A composition comprising an agent that binds NID1 , for use in the prevention or treatment of a cancer or infectious disease in an individual.

8. The composition of embodiment 7, wherein the agent binds sNID1 and inhibits binding of NKp44 to sNID1.

9. The composition of embodiments 7 or 8, wherein the agent that binds mNID1 at the surface of a NID1-releasing cell.

10. The composition of embodiments 7-9, wherein the agent is a non-depleting antigen binding protein.

1 1. The composition of embodiments 7-9, wherein the agent is a depleting antigen binding protein.

12. The composition of embodiments 9-11 , wherein the agent decreases sNID1 release by NID1-releasing cells.

13. The composition of any one of embodiments 7 to 12, wherein the method comprises administering to the individual the agent in an amount sufficient to decrease sNID1 -mediated immunosuppression.

14. The composition of any one of embodiments 7 to 13, wherein the method comprises administering to the individual the agent in an amount sufficient to decrease sNID1 -mediated suppression of NKp44-expressing cells.

15. The composition of any one of embodiments 7 to 8 or 10 to 14, wherein the method comprises administering to the individual the agent in an amount sufficient to deplete NID1- releasing cells.

16. The composition of any one of embodiments 7 to 15, wherein the method comprises administering to the individual the agent in an amount sufficient to decrease sNID1 in circulation or in the tumor environment. 17. The composition of any one of embodiments 7 to 16, wherein the individual has a solid tumor.

18. The composition of any one of embodiments 7 to 17, wherein the individual has a cancer selected from the group consisting of: bladder cancer, skin cancer, breast cancer, cervical cancer, head and neck cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, prostate cancer, stomach cancer, uterine cancer, ovarian cancer, endometrial cancer and testicular cancer.

19. The composition of any one of embodiments 7 to 18, wherein the individual has an immunosuppressive state, optionally a state characterized by a deficiency in NK cell activity.

20. The composition of any one of embodiments 7 to 19, wherein the individual has sNID1 in circulation and/or tumor tissue, optionally at an increased level compared to a healthy individual.

21. The composition of any one of embodiments 7 to 20, wherein the individual has tumor or tumor adjacent tissue characterized by presence of NID1-releasing cells.

22. A composition comprising an agent that binds NID1 , for use in a method of assessing immunosuppression in an individual having a cancer.

23. The composition of embodiment 22, wherein the method comprises detecting sNID1 , wherein the presence of, or elevated levels of, sNID1 indicates that the individual has disease characterized by immunosuppression.

24. The composition of embodiment 22, wherein the method comprises detecting NID1- releasing cells, wherein the presence of, or elevated numbers of, NID1 -releasing cells indicates that the individual has disease characterized by immunosuppression.

25. A composition comprising an agent that binds NID1 , for use in the treatment of a cancer in an individual having detectable and/or elevated levels of sNID1.

26. A composition comprising an agent that binds NID1 , for use in the treatment of a cancer or an infectious disease in an individual having disease characterized by immunosuppression.

27. The composition of any one of embodiments 25-26, wherein the agent inhibits binding of NKp44 to sNID1. 28. The composition of any one of embodiments 25-27, wherein the agent is a depleting antigen binding protein.

29. The composition of any one of embodiments 25-28, wherein the agent decreases sNID1 release by NID1-releasing cells.

30. The composition of any one of embodiments 7-29, wherein the agent is an antibody or antibody fragment.

31. The composition of any one of embodiments 7-29, wherein the agent comprises a NKp44 polypeptide.

32. The composition of embodiment 31 , wherein the agent the NKp44 polypeptide is a NID1 -binding fragment of NKp44, optionally fused to an Fc domain.

33. The composition of any one of embodiments 7-32, wherein the agent binds a full length sNID1 protein and/or a cleaved sNID1 protein.

34. The composition of embodiment 33, wherein the agent specifically binds a Cats cleavage site on NID1.

35. The agent of embodiments 26-34, wherein the individual has sNID1 in circulation.

36. The agent of embodiments 26-34, wherein the individual has tumor or tumor adjacent tissue characterized by presence of NID1-releasing cells.

37. A composition comprising an agent that reduces the level and/or activity of sNID1 that is available to interact with a NKp44 polypeptide.

38. The composition of embodiment 37, wherein the agent is an antibody or antibody or antibody fragment that binds sNID1 , optionally wherein the antibody competes with NKp44 polypeptide for binding to a sNID1 polypeptide.

39. The composition of embodiment 37, wherein the agent comprises a sNID1 -binding fragment of NKp44.

40. The composition of embodiments 37 to 39, wherein the agent lacks an Fc domain or comprises an Fc domain that is modified to have reduced or to lack to human CD16A, CD16B, CD32A, CD32B and/or CD64 polypeptides. 41. The composition of any one of the above embodiments, wherein the agent is a protein, optionally an antibody or antibody fragment, having a bivalent Kd of less than 10 ⁹ M for binding to an NID1 polypeptide.

42. The composition of embodiments 37-41 , for use as a medicament, optionally for use in reverting or reducing immunosuppression.

43. The composition of embodiment 37-42, for the treatment of cancer or infectious disease.

44. A pharmaceutical composition comprising a composition according to any one of the above embodiments, and a pharmaceutically acceptable carrier.

45. A NKp44 polypeptide or fragment thereof, for use in detecting NID1 on the surface of a cell, optionally for detecting a NID1-releasing cell, optionally wherein the cell is a cell present in tumor or tumor-adjacent tissue.

46. A kit comprising the NKp44 polypeptide or fragment thereof of embodiment 45, optionally further comprising a labeled secondary antibody that specifically recognizes the NKp44 polypeptide or fragment thereof.

47. A method for detecting NID1 at the surface of a cell, the method comprising binding a cell into contact with a NKp44 polypeptide, and detecting binding of the NKp44 polypeptide to the cell, wherein a detection of binding indicates that the cell bears NID1 at its surface.

48. A method for detecting NID1 polypeptide in a biological sample, the method comprising binding a biological sample into contact with a NKp44 polypeptide, and detecting binding of the NKp44 polypeptide to the NID1 polypeptide.

49. The method of any of embodiments 45-48, wherein the cell or biological sample from an individual having a cancer.

50. An in vitro method for assessing disease in an individual having a cancer, the method comprising bringing a biological sample from an individual into contact with an agent that is capable of specifically binding a sNID1 polypeptide, and detecting presence and/or levels of the sNID1 polypeptide, wherein a detection of presence of and/or increased levels of sNID1 indicates that the individual has an immunosuppressed state and/or that the individual has sNID1 capable of mediating immunosuppressive activity.

51. A method of treatment or prevention of a disease in an individual comprising:

a) determining whether tumor or tumor adjacent tissues bear cells having detectable NID1 at their surface, optionally at increased levels compared to a control, and

b) upon a determination that tumor or tumor adjacent tissues bear cells having detectable NID1 at their surface, optionally at increased levels compared to a control, administering to the individual an agent that enhances the activity or number of lymphocytes, optionally NK cells.

52. A method of treatment or prevention of a disease in an individual comprising:

a) determining whether the individual has sNID1 , optionally at increased levels compared to a control, and

b) upon a determination that the individual has sNID1 , optionally at increased levels compared to a control, administering to the individual an agent that enhances the activity or number of lymphocytes, optionally NK cells.

53. The method of embodiments 51-52, wherein the disease is cancer.

54. The method of any of embodiments 51-53, wherein the agent is an agent that reduces the level and/or activity of sNID1 that is available to interact with NKp44.

55. The method of embodiment 54, wherein the agent is an agent that inhibits the interaction between a NKp44 polypeptide and NID1.

56. A composition comprising a NID1 protein or NKp44-binding fragment thereof, for use in the treatment of an autoimmune or inflammatory disease in an individual.

EXAMPLES

Materials and Methods

Antibodies

The following monoclonal antibodies (mAbs), produced in our laboratory, were used in this study: BAB281 (lgG1 , anti-NKp46), AZ20 (lgG1 , anti-NKp30) and Z231 (lgG1 , anti- NKp44). The following commercial antibodies were also used: mouse anti-Nidogen-1 mAb (lgG1 , clone 302117, R&D, MAB2570) and goat anti-NI D1 polyclonal Ab (R&D, AF2570); mouse anti-NKp46 (lgG1 , clone 29A1.4.9, Miltenyi Biotec, 130-095-1 18); goat anti-human IgG Horseradish Peroxidase (HRP)-conjugated mAb, (Southern Biotech, 2040-05) goat anti-mouse Ig HRP-conjugated mAb (Southern Biotech, 1031-05), mouse anti-goat IgG HRP-conjugated mAb (Southern Biotech, 6164-05), and goat anti-mouse lgG1 phycoerythrin (PE)-conjugated mAb (Southern Biotech, 1070-09); goat anti-human IgG PE-conjugated mAb (Jackson ImmunoResearch, 109-116-170); rabbit polyclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Ab (ThermoFisher, PA1 -16780); mouse anti-His mAb (lgG1 , clone BMG-His-1 , Roche Diagnostics, 1 922 416).

Cell lines

The following cell lines, purchased from American Type Culture Collection (ATCC), were used in this study: HEK293T (human embryonic kidney), K562 (human erythroleukemia), Bw1547 (Bw, murine thymoma), SH-SY-5Y (human neuroblastoma), A172 (human glioblastoma), C-32 (human melanoma), A2774 (human ovarian adenocarcinoma), JEG-3 (human placental choriocarcinoma), A549 (human lung carcinoma). Cell lines were maintained either in DMEM (HEK293T) or RPMI1640 medium supplemented with 10% (v/v) fetal calf serum (FCS), L-glutamine, and antibiotics (penicillin and streptomycin). For some experiments, HEK293T and K562 cell lines were also cultured in protein-free CD medium (ThermoFisher, 1 1279023). Stably transfected K562 cells were selected and cultured in the presence of G418 sulphate (Calbiochem, 345810) at the final concentration of 1.2 mg/ml. All cell lines were periodically tested and were mycoplasma free.

Preparation of soluble chimeric receptors

For preparation of NKp44Fc, NKp30Fc, and NKp46Fc soluble receptors, the sequences coding for the extracellular portion of the different receptors were subcloned in the pRB1-2B4Fcmut vector (Dr. M Falco, Istitute G. Gaslini, Genova) in frame with the sequence coding for the human lgG1 portion, that was mutagenized in order to obtain a mutated Fc that does not bind to Fc receptors. These constructs were transfected into HEK293 cell line utilizing JetPEI (Polyplus, 101-10) following manufacturer’s instructions; after 48-72 h, cells were selected with 0.5 mg/ml G418 sulphate, in order to obtain stably transfected cells, and subcloned by limiting dilution. SN were collected from cell clones cultured either in DMEM/10% Ultra-low IgG FCS (ThermoFisher) or in Ex-CELL ACF CHO medium (Sigma, C5467) and the soluble Fc molecules were purified by affinity chromatography utilizing Protein A Sepharose 4 Fast Flow (G-Biosciences, 786-283). Purified recombinant proteins were checked by SDS- PAGE followed by silver staining and by ELISA utilizing mAbs specific for the different receptors. Preparation of cell culture supernatants

Cell culture supernatants (SN) were obtained from wild type or transfected K562 or Bw cells. After 48-72 h culture in medium supplemented with 1% (v/v) FCS or in protein-free CD medium, SN were collected and utilized for subsequent experiments either as they were or following concentration (up to 30-fold) with Amicon UltraceM OK (Millipore, UFC901024). Protein content in SN derived from culture in protein-free CD medium was determined using Bradford Protein Assay (Bio-Rad, 5000006).

Metabolic labeling

HEK293T cells were cultured in protein-free CD medium in the presence of ManAz (N-azidoacetylmannosamine-tetraacylated) (ThermoFisher, 88904) at the final concentration of 40 mM. After 72 h, SN was collected, concentrated (up to 24-fold) with Amicon UltraceMOK, and incubated with Biotin-PEG3-Phosphine (200 pM final concentration, ThermoFisher, 88901 ) O/N at room temperature, in order to label secreted glycoproteins containing azido- sugars. Labeled SN was dialyzed in PBS using D-Tube Dialyzer Maxi, MWCO 6-8 kDa (Novagen, 71509-3).

Enzyme-linked immunosorbent assay (ELISA)

Direct ELISA: 100 pi of concentrated SN derived from HEK293T cells cultured in protein-free CD medium were coated on ELISA plates O/N at 4°C. Next, wells were saturated with PBS/3% (w/v) bovine serum albumin (BSA) for 3 h at r.t, washed in PBS, and incubated with Fc molecules at different concentrations ranging from 20 to 2.5 pg/ml or with 1 pg/ml anti- NID1 mAb followed by the appropriate HRP-conjugated secondary reagent. Similar experiments were carried out on ELISA plates coated with 5 pg/ml recombinant human Nidogen-1 (rNID1 ) (R&D, 2570-ND-050; source: Mouse myeloma cell line, NSO-derived Leu29-Lys11 14 NID-1 protein with an N-terminal 9-His tag) in PBS.

Indirect (sandwich) ELISA: A similar procedure was applied. Briefly, ELISA plates were coated with Fc molecules (5 pg/ml in PBS), saturated, and incubated with 100 pi HEK293T-SN-biot followed by HRP-conjugated Streptavidin (Southern Biotech, 7100-05). Alternatively, ELISA plates were coated with mouse anti-NID1 mAb or Fc molecules (5 pg/ml in PBS), saturated, and incubated with 100 pi SN derived from wild type or NID1 -transfected K562 or Bw cells cultured in medium with 1% (v/v) FCS or in protein-free CD medium. Next, a polyclonal goat anti-NI D1 Ab was added, followed by a HRP-conjugated anti-goat Ig mAb.

In all experiments a final incubation with the HRP substrate ABTS (2,2'-azino-di-(3- ethylbenzthiazoline sulfonic acid) (Roche Diagnostics, 102 946) was performed. The colorimetric signal was measured with a microplate reader (TECAN Sunrise) at an optical density (OD) of 405 nm. Western blot analysis

Samples (6-28 pg SN obtained in protein-free medium, 30 pi SN obtained in medium/1 % FCS, 160 ng rND1 ) were run on 7,5% or 10% polyacrylamide gels under non- reducing conditions and transferred to Immobilon-P PVDF membranes (Millipore, IPVH00010). For some experiments, gels were prepared with 7.5% TGX Stain-Free Acrylamide Solutions (Bio-Rad, 161-0181 ). Membranes were blocked with 5% BSA in Tris-buffered-saline containing 0.05% Tween-20 (TBS-T) and probed with anti-NI D1 mAb, a rabbit polyclonal anti-GAPDH Ab, or Fc molecules (7 pg/ml) followed by the appropriate HRP-conjugated secondary reagent. The SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher, 34080) was used for detection. Images were acquired with ChemiDoc Touch Imaging System (Bio-Rad) and analyzed with Image Lab software (Bio-Rad).

Two-dimensional Electrophoresis (2-DE) and Western blot

Concentrated HEK293T-SN and HEK293T-SN-biot (300 pg for Western blot and 600 pg for preparative gels) were solubilized in the reduction/alkylation solution containing 8 M urea, 4% CHAPS, 5 mM tributylphosphine (TBP), 20 mM iodoacetamide (IAA), 40 mM Tris, and 0.1 mM EDTA for 1 h. To prevent over-alkylation during the isoelectro focusing (IEF) step, excess of IAA was neutralized by adding an equimolar amount of DTT. Finally, samples were dissolved in the focusing/re-hydration solution, i.e. 7 M urea, 2 M thiourea, 4% CHAPS, and 15 mM dithioerythritol (DTE) and a 0.6% (v/v) carrier ampholyte cocktail, containing 40% of the pH 3.5-10 and 60% of the pH 4-8 intervals (BDH Biochemical, 44430 2F) and loaded onto home-made non-linear pH 3-10 strips. After IEF runs, the strips were equilibrated in 6 M Urea, 50 mM Tris-HCI pH 8.8, 2% (w/v) SDS, 30% (v/v) glycerol, and traces of bromophenol blue; the proteins were separated using a SDS-PAGE (T% 8-16) and transferred onto nitrocellulose membranes (Protran BA85, Whatman, 10402588) with a semidry system. The membranes were saturated with 3% w/v polyvinyl-pyrrolidone (PVP) in TBS and incubated overnight separately with NKp44Fc, NKp30Fc, or NKp46Fc in 3% w/v BSA in TBS-Tween 0.15% v/v (TBS-T). Membranes were then rinsed in TBS-T and incubated with anti-human IgG HRP- conjugated mAb. HEK293T-SN-biot was subjected to the same procedure and immunoblotted with Neutravidin-HRP (ThermoFisher, 31001 ). For preparative experiments, SDS-gels were stained with“blue silver” colloidal Coomassie. Images were digitalized using ChemiDoc Touch (Bio-Rad) and analyzed with PDQuest software (Bio-Rad).

2-DE spot identification

Spots excised from 2D-PAGE were fully discolored and digested with trypsin. All mass spectrometric measurements were performed using a LTQ linear ion trap mass spectrometer (Thermo Electron) coupled to a HPLC Surveyor (Thermo Electron) equipped with a Jupiter C18 column 250 mm x 1 mm (Phenomenex). Protein identification was performed using SEQUEST software and searched against a Human protein database. Peptide MS/MS assignments were filtered following very high stringent criteria: Xcorr >1.9 for the singly charged ions, Xcorr >2.2 for doubly charged ions, Xcorr >3.7 for triply charged ions, peptide probability <0.01 , delta Cn >0.1 and Rsp <4.26.

Immunoprecipitation

Immunoprecipitation experiments were carried out using 12 pg NKp44Fc, NKp30Fc, or DNAM-I Fc molecules linked to 50 pi Dynabeads Protein G (ThermoFisher, 10003D) and incubated O/N at 4 °C with 400 pi concentrated HEK293T SN (corresponding to 500 pg proteins). Samples were eluted using non-reducing sample buffer (10% glycerol, 2% SDS, 62.5 mM Tris-HCI pH 6.8, 0.01% bromophenol blue) for 5’ at 60 °C and subsequently analyzed by SDS-PAGE using TGX Stain-Free Acrylamide Solutions; membrane was probed with mouse anti-NI D1 mAb followed by HRP-conjugated anti-mouse Ig mAb.

RT-PCR analysis

Total RNA was extracted using RNAeasy Mini Kit (Qiagen, 74104) from the following cells: K562, K562-NID1 , Bw, Bw-NID1. Oligo(dT)-primed cDNA was prepared by standard technique using a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, 04379012001 ) following manufacturer’s instructions. Amplifications were performed for 30 cycles utilizing Platinum TAQ DNA Polymerase (ThermoFisher, 10966034) with an annealing T of 58°C (b-actin) or 62°C (NID1 ). Primers used were: b-actin for 5’

ACT CCAT CAT GAAGT GT GACG (SEQ ID NO: 3) and b-actin rev 5’

CAT ACT CCTGCTT GCT GAT CC (SEQ ID NO: 4); NID1 for 5’ CTCCATTGGGCCTGTGAGG (SEQ ID NO: 5) and NID1 rev 5’ AGACACGGGGGC GTCATC (SEQ ID NO: 6). PCR products (249 bp fragment for b-actin and 795 bp for NID1 ) were separated by electrophoresis on a 1.5% (w/v) agarose gel and visualized by ethidium bromide staining.

Stable cell transfectants

K562 cell line was transfected with pcDNA3.1-NID1 construct (Geneart, ThermoFisher) using JetPEI following manufacturer’s instructions. After 72 h cells were cultured in medium containing 1.2 mg/ml G418 sulphate. At the end of the selection period, surviving cells were sub-cloned by limiting dilution.

Bw-NIDI cells were prepared by retrovirus gene transfer. NID1 ORF cDNA was sub- cloned in pMXs-IG (IRES-GFP) retrovirus vector (kindly provided by Dr. Kitamura, Tokyo, Japan). The pMXs-IG-NID1 construct was transiently transfected into Plat-E packaging cell line in order to generate viral particles that were used to infect Bw cells. NID1 -positive cells were sorted according to GFP expression; subsequently, NID1 -positive cells were sub-cloned by limiting dilution. Bw-NKp44/DAP12 cells (Bw-NKp44) were obtained by a similar approach, utilizing pMXS-IG vector, in which NKp44 ORF and DAP12 ORF cDNAs were sub-cloned in the two available cloning sites (GFP-encoding sequence was replaced by DAP12 ORF cDNA). NKp44-expressing cells were sorted and sub-cloned by limiting dilution.

For the recovery of SN from K562 and BW cells and transfectants, cell culture was carried out either in RPMI/1% FCS or in protein-free CD medium and SN was collected after 48-72 h. In the latter case, SN were concentrated up to 10-fold using Amicon UltraceM OK.

Functional assays on Bw cells

1x10 ⁵ Bw cells were pre-treated 1 h at 37°C with 20 or 7.5 pg/ml rNID1 or with 100 pi SN obtained from wild type or transfected K562 and Bw cells cultured in RPMI/1% FCS, and subsequently plated on 96-well cell culture plates (1X105 cells/well) coated with 5 pg/ml goat anti-mouse IgG (GAM, MP Cappel, 55481 ) alone or with GAM plus anti-NKp44 or -NKp30 mAbs. For co-culture experiments, Bw cells were pre-incubated 1 h at 37°C with wild type or NID1 -transfected K562 and Bw cells at an effector/target (E/T) ratio of 1 :1 and then transferred on mAb-coated plates. In other experiments, Bw-NKp44 cells were incubated on plates in which rNID1 was coated either directly or through anti-NI D1 or anti-His mAbs. For all these assays, after 20 h at 37°C, SN were collected and analyzed for their IL-2 content by ELISA using the Mouse IL-2 ELISA Ready-SET-Go (eBioscience, BMS88-7024-77) according to manufacturer’s instructions. Each sample was run in duplicate.

Generation of polyclonal NK cell lines

NK cells from healthy donors were purified from peripheral blood using the RosetteSep™ NK Cell Enrichment Cocktail (StemCell Technologies, 15025). Those populations displaying more than 95% of CD56+CD3-CD14- NK cells were selected. Polyclonal NK cell lines were obtained by culturing purified NK cells at appropriate dilutions on irradiated feeder cells in the presence of 100 U/mL rhlL-2 (Proleukin, Novartis) and 1 ,5 ng/mL phytohemagglutinin (PHA, Gibco Ltd, 10576-015) in round-bottomed 96-well microtiter plates. After 3/4 weeks of culture the expanded NK cells were used for NK cell stimulation experiments.

Functional assays on NK cells

Evaluation of NK cell-mediated cytotoxicity against Bw and Bw-NID-1 transfected cells or against the P815 FcyR+ murine cell line (redirected killing assay) was done in a 4-h 51 Cr- release test. In the redirected killing assay mAbs were added at the final concentration of 1.25 pg/ml (anti-NKp30 and anti-NKp46 mAbs) or 0.5 pg/ml (anti-NKp44 mAb) (appropriate concentration was determined after titration to induce specific functional response in NK cells). When indicated, K562-SN or K562-NID1-SN was added to NK cells (50% v/v) 1 h before the onset of the test.

For the IFN-g secretion assay NK cells (10X10 ⁴ cells/well) were cultured overnight in 96-well microtiter plates pre-coated with GAM either in the absence or in the presence of anti- NKp44, -NKp30, -NKp46 mAbs. When indicated, K562-SN or K562-NID1-SN was added at the onset of culture. The culture SN were then collected and analyzed for the presence of IFN-g using the IFN gamma Human ELISA Kit (ThermoFisher, EHIFNG).

Imaging flow cytometry

HEK293T cells were incubated with anti-NI D1 mAb followed by PE-conjugated anti- lgG1 mAb. Prior to analysis, cells were stained with the nuclear dye Hoechst 33342 (ThermoFisher, 62249) (1 :1000 dilution). Cells were acquired with a 12 channel MultiMag system ImageStreamX Mark II imaging flow cytometer (IFC) (Merck) using INSPIRE acquisition software (Amnis Corporation). Three ImageStream channels were used: channel 1 for the brightfield, channel 3 for NID1-PE (excited by a 488-nm laser), channel 7 for Hoechst44432 (excited by a 405-nm laser). In order to collect only on focus-single-cells, we first restricted our attention on the brightfield (BF) parameters analysis. Single cells were selected using a biparametric dot plot representing BF aspect ratio (i.e. width/height) versus BF cell area then, by mean of the BF gradient RMS (root mean square), which measures the sharpness quality of an image, we gated only on focus cells. For each staining condition, 4,000 raw images were collected using a 40x objective. To avoid spectral overlaps, single-color compensation controls (500 cells each) were gathered and a compensation matrix was generated. The raw image files were then analyzed using IDEAS 6.0.3 software (Amnis Corporation) setting compensation on the basis of the calculated matrix. To verify the correspondence between data derived from traditional cytometry and IFC, we compared NID expression with its negative control (i.e. cells labeled only with secondary PE-conjugated mAb) by mean of their MFI (mean fluorescence intensity). To grant a better visualization of these data, we merged the two files.

Flow cytometry

Cells were incubated with the primary mAbs or Fc molecules (20 pg/ml) for 30 minutes at 4°C, washed, and stained with the appropriate PE- isotype-matched secondary mAbs for 30’ at 4°C. All samples were analyzed using a FACSCalibur flow cytometer and CellQuest Pro software (BD Biosciences). Proteomic analysis of polyclonal NK cell populations

Polyclonal IL-2-activated NK cells derived from four different healthy donors were cultured for 20 h at 37°C in FCS- and IL-2-deprived medium in the absence or in the presence of rNID1 (20 pg/ml) directly coated on the plate or of anti-NKp44 mAb coated via GAM. Next, for subsequent proteomic analysis, cells were processed by in-StageTip (iST) method73. Briefly, the pellets were lysed, reduced, alkylated in a single step using a buffer containing 2% (w/v) SDC (sodium deoxycholate), 10 mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride), 40 mM CAA (chloroacetamide), 100 mM Tris HCI pH 8.0, and loaded into StageTip. The lysates were diluted with 25 mM Tris pH 8.5 containing 1 pg of trypsin. Samples were acidified with 100 pi of 1% (v/v) TFA (Trifluoroacetic Acid) and washed three times with 0.2 % (v/v) TFA. Elutions were performed with 60 pi of 5% (v/v) ammonium hydroxide, 80% (v/v) ACN.

Samples were loaded from the sample loop directly into a 75-pm ID x 50 cm 2 pm, 100 A C18 column mounted in the thermostated column compartment and the peptides were separated with increasing organic solvent at a flow rate of 250 nl/min using a non-linear gradient of 5-45 % solution B (80% CAN and 20% H20, 5% DMSO, 0.1 % FA) in 180 min. Eluting peptides were analyzed using an Orbitrap Fusion Tribrid mass spectrometer (ThermoFisher Scientific). Orbitrap detection was used for both MS1 and MS2 measurements at resolving powers of 120 K and 30 K (at m/z 200), respectively. Data dependent MS/MS analysis was performed in top speed mode with a 2 sec. cycle time, during which precursors detected within the range of m/z 375-1500 were selected for activation in order of abundance. Quadrupole isolation with a 1.8 m/z isolation window was used, and dynamic exclusion was enabled for 30s. Automatic gain control targets were 2.5 x 105 for MS1 and 5 x 104 for MS2, with 50 and 54 ms maximum injection times, respectively. The signal intensity threshold for MS2 was 1 x 10 ⁴ . HCD was performed using 28% normalized collision energy. One microscan was used for both MS1 and MS2 events.

MaxQuant software74, version 1.6.0.1 , was used to process the raw data, setting a false discovery rate (FDR) of 0.01 for the identification of proteins, peptides and PSM (peptide- spectrum match), a minimum length of 6 amino acids for peptide identification was required. Andromeda engine, incorporated into MaxQuant software, was used to search MS/MS spectra against Uniprot human database (release UP000005640_9606 February 2017). In the processing the variable modifications are Acetyl (Protein N-Term) Oxidation (M), Deamidation (NQ); on the contrary the Carbamidomethyl (C) was selected as fixed modification. The intensity values were extracted and statistically evaluated using the ProteinGroup Table and Perseus software. Algorithm MaxLFQ was chosen for the protein quantification with the activated option‘match between runs’ to reduce the number of the missing proteins. Statistical analyses

Data were analyzed using GraphPad Prism 6, R, or Perseus software. Details of statistical analysis for each experiment are indicated in the corresponding figure legend.

Example 1 : Identification of Nidogen-1 as an extracellular ligand for NKp44 activating NK receptor.

The aim of this study was the identification of putative extracellular ligands for NKp44. To this end, we analyzed the HEK293T cells as possible source of such ligands since these cells were found to bind NKp44Fc chimeric receptor at their cell surface. This data suggested that HEK293T cells could synthesize a putative NKp44-ligand. Importantly, these cells could be cultured extensively in protein-free medium, thus facilitating the analysis of proteins released in the culture supernatant.

Thus, HEK293T cells were cultured in protein-free medium and the supernatants (HEK293T-SN) were collected, concentrated, and coated on ELISA plates. Direct ELISA was performed using NKp44Fc, NKp30Fc, NKp46Fc, and DNAM-1 Fc soluble chimeric receptors (Fc molecules). As shown in Figure 1 F panel (a), NKp44Fc strongly bound to HEK293T-SN- coated wells. Regarding the other Fc molecules tested, only NKp46Fc showed some reactivity (although significantly weaker than NKp44Fc), while NKp30Fc and DNAM-1 Fc displayed no binding. These data suggested that NKp44 could recognize ligand(s) released by HEK293T cells.

To gain further information on such putative NKp44 soluble ligand(s), a metabolic labeling of HEK293T cells was performed in the presence of azido-sugars. As a result of this procedure, cells synthetize glycoproteins characterized by modified glycosylated residues, capable of linking covalently to biotin, enabling the detection of glycosylated proteins. The supernatant of labeled HEK293T cells (HEK293T-SN-biot) was analyzed by ELISA on plates coated with NKp44Fc or NKp30Fc molecules. As shown in Figure 1 F panel (b), HEK293T-SN- biot reacted with NKp44Fc- (and not with NKp30Fc-) coated wells, indicating that NKp44Fc can bind glycosylated protein(s) secreted by HEK293T cells.

The NKp44Fc-reactive glycoprotein(s) were further analyzed by Western blot. Proteins from concentrated HEK293T-SN were separated by SDS-PAGE and immunoblotted with NKp44Fc. As shown in Figure 1G, NKp44Fc, but not other Fc molecules analyzed (NKp30Fc, NKp46Fc, DNAM-1 Fc), recognized a band of approximately 180 KDa under non-reducing conditions.

In order to characterize the high molecular weight glycoprotein(s) recognized by NKp44Fc, concentrated HEK293T-SN was resolved by two-dimensional electrophoresis (2- DE) and analyzed by Western blot with NKp44Fc, NKp30Fc, or NKp46Fc (Fig 1A-C). Concentrated HEK293T-SN-biot was subjected to the same procedure and immunoblotted with Neutravidin-HRP (for the detection of glycoproteins) (Figure 1 D). In parallel, a preparative 2-D gel was stained with Blue Coomassie to visualize all proteins and excise the spots of interest (Figure 1E). Following 2-DE and immunoblotting, NKp44Fc was found to recognize different proteins present in HEK293T-SN (Figure 1A). Thus, 27 spots, visualized in NKp44Fc blot, were excised, digested, and analyzed by high resolution mass spectrometry. Thanks to the comparison with blots stained with NKp46Fc and NKp30Fc soluble receptors (Figure 1B, 1C) it was possible to subtract the background and further restrict the analysis to 17 spots. Finally, by the comparative analysis with the blot stained with Neutravidin (Figure 1 D), we could further select 7 spots recognized by NKp44Fc and corresponding to glycosylated proteins. We focused on spot n. 26 because it corresponded to high MW proteins. Among the proteins identified in this spot, Nidogen-1 (NID1 ) displayed a predicted MW compatible with the electrophoretic mobility of the glycoprotein(s) that had been detected by NKp44Fc in mono- dimensional SDS-PAGE (i.e. approximately 180 kDa) (see Figure 1G).

The direct and specific binding of NKp44 to NID1 was confirmed by different experimental evidences. Thus, immunoprecipitation experiments showed that NKp44Fc (but not NKp30Fc nor DNAM-1 Fc used as controls) was able to immunoprecipitate NID1 protein from concentrated HEK293T-SN (Figure 2A). In addition, the ability of NKp44Fc to bind purified NID1 was investigated by Western blot. Recombinant human Nidogen-1 (rNID1 ) and HEK293T-SN were run in SDS-PAGE in parallel and immunoblotted with NKp44Fc (and NKp30Fc or DNAMI Fc as controls). Figure 2B shows that NKp44Fc was able to recognize both rNID1 and NID1 released from HEK293T-SN. In contrast, NKp30Fc and DNAM-1 Fc did not display any reactivity. Finally, the interaction between NKp44 and rNID1 was assessed by ELISA. As shown in Figure 2C, different concentrations of NKp44Fc bound to rNID1 -coated wells, while NKp30Fc, NKp46Fc, and DNAM-1 Fc did not. This experiment shows that soluble NKp44Fc can specifically bind to NID1 in its native conformation.

Example 2: Effect of soluble NID1 on NKp44-mediated cell activation

In order to investigate the potential effect of NID1-NKp44 interaction, we took advantage of a model available in our lab based on the use of murine Bw5147 (Bw) cell transfectants expressing either the NKp44/DAP12 receptor complex (Bw-NKp44) or the chimeric receptor NKr30-Oϋ3z (Bw-NKp30). In this model, cell activation induced by mAb- mediated cross-linking of NKp44 or NKp30 results in the release in culture SN of IL-2, which can be measured by ELISA. Since soluble ligands of different activating receptors have been shown to interfere with receptor function, we asked whether rNID1 pretreatment of Bw-NKp44 cells had a similar inhibitory effect. As shown in Figure 3, different rNID1 concentrations inhibited the NKp44-induced IL-2 production. As control, Bw-NKp30 cells were also analyzed. A minor inhibition of NKp30-induced IL-2 production could be detected only at the highest rNID1 concentration.

Next, we asked whether also NID1 released from cells could inhibit NKp44-mediated cell activation. To this end, the NID1 construct was transfected in NID1-negative cells. The culture supernatant of such transfected cells was assessed in functional assays. The human K562 cell line was used as recipient, since it does not express NID1 mRNA (Figure 4A). Stable transfection with the NID1 construct resulted in NID1 transcript expression and protein secretion in cell culture supernatants (Figures. 4A-B). In ELISA, NKp44Fc reacted with K562- NID1-SN and not with K562-SN (Figure 4C) and in Western blot experiments it specifically reacted with the band corresponding to NID1 protein, while it didn’t bind to any band of K562- SN (Figure 4D). In order to investigate the potential functional consequences of NID1-NKp44 interaction we used again Bw-NKp44 and Bw-NKp30. Bw-NKp44 cells were stimulated with anti-NKp44 mAb in the absence or in the presence of K562-SN or K562-NID1-SN. As shown in Figure 5A, K562-SN had no effect, while K562-NID1-SN inhibited NKp44-induced IL-2 release. In contrast, K562-NID1-SN didn’t induce any inhibitory effect in Bw-NKp30 cells stimulated via NKp30, as compared to K562-SN. A similar inhibitory effect on NKp44-induced IL-2 production was also detected with culture SN from NID1 -transfected Bw cells (Figures 3B and 4E).

We next investigated whether NID1 released by NID1 -expressing cells could exert an inhibitory effect on NKp44-mediated activation of normal, polyclonal NK cells. To this end, NK cells isolated from PB were cultured in the presence of IL-2 to induce the expression of NKp44 (absent on resting PB-NK cells). NK cells were then stimulated with anti-NKp44, anti-NKp30, or anti-NKp46 mAbs either in the absence or in the presence of K562-SN or K562-NID1-SN. After stimulation, SN of NK cells were collected and analyzed by ELISA for their IFN-g content. As shown in Figure 5B, a significant reduction of NKp44-induced IFN-g production was observed in the presence of K562-NID1-SN, as compared to K562-SN. On the other hand, neither K562-NID1-SN nor K562-SN inhibited IFN-g secretion triggered via NKp46 or NKp30. Finally, we investigated whether NID1 -containing SN could affect NKp44-induced NK cell cytotoxicity. To this end, NK cells were analyzed in a redirected killing assay either in the absence or in the presence of K562-SN or K562-NID1-SN. K562-NID1-SN (but not K562-SN) reduced anti-NKp44-triggered killing of P815 target cells, while it was ineffective on NKp30- and NKp46-mediated cytotoxic activity (Figure 5C).

Collectively, these results show that NID1 may interfere with target cell recognition via NKp44, exerting a regulatory effect on NKp44-induced NK cell activation.

Example 3: NID1 surface expression and functional effects on NKp44 Having demonstrated that soluble NID1 is able to interfere with NKp44-mediated NK cell activation, we next asked whether NID1 could be expressed also at the cell surface and be recognized as surface-associated molecule with possible functional outcomes. To this end, we assessed the cellular expression and distribution of NID1 in HEK293T, a cell line capable of binding NKp44Fc at its surface (see above). Thus, HEK293T cells were stained with a NID1- specific mAb and analyzed by imaging flow cytometry. This analysis revealed a weak but detectable surface expression of NID1. Single cells stained with anti-NI D1 mAb, while the histogram profiles showed NID1 staining on the basis of mean fluorescence intensity of all cells analyzed. The presence of NID1 protein at the cell surface suggested that it could be accessible for a direct interaction with NKp44. Thus, we analyzed by flow cytometry the surface expression of NID1 and, in parallel, the reactivity of NKp44Fc on HEK293T, K562, and K562-NID1 cells. As shown in Figure 6, anti-NI D1 mAb stained HEK293T and K562-NID1 cells, but not K562 cells. Consistently, NKp44Fc bound to HEK293T and K562-NID1 but not to K562 cells. Comparable results were obtained with Bw-NID1 cell transfectants. Taken together, the data indicated that NID1 can be expressed at the cell surface and recognized by NKp44(Fc) receptor.

On the basis of these results, we further investigated the possible effect of NID1 expressed at the cell surface on NKp44-induced NK cell activation. To this end, Bw-NKp44 cells were cultured in the presence of K562 or K562-NID1 cells or in rNID1-coated plates. After 20 h, IL-2 was measured in culture SN. As shown in Figure 7A, neither K562 nor K562-NID1 cells could induce increases of IL-2 production by Bw-NKp44 cells. Similarly, no increments in IL-2 production occurred upon culture of Bw-NKp44 in rNID1 -coated plates (Figure 7B). Comparable results were obtained when rNID1 coating was performed through anti-NI D1 (or anti-His) mAb.

We then analyzed whether cell surface NID1 had any effect on NK cells expressing NKp44 upon activation. To this end, IL-2-activated polyclonal NK cells were obtained from several healthy donors and used in functional assays. Since, in NK cells, NKp44 triggers cytotoxicity, we analyzed the susceptibility to NK cell-mediated killing of NID1 + or NID1- cells in a cytolytic assay. Because NK cells express different activating receptors that recognize ligands on human K562 cells, this assay was performed using murine Bw cell transfectants (see Figure 3B). As shown in Figure 7C, Bw-NID1 and Bw cells didn’t display significant differences in their susceptibility to NK-mediated cytolysis.

We also assessed the possible effect of plastic-bound rNID1 on IFN-g release from NK cells cultured in rNID1-coated plates. As shown in Figure 7D, exposure to NID1 failed to induce significant increments of IFN-g production by NK cells.

Example 4: Effect of NID1 on NK cell proteome The above data suggest that NID1 exposed at the target cell surface or bound to a solid support could not significantly modify the cytokine release or cytotoxicity in NKp44+ cells. Thus, to gain insight on the possible effects of surface-exposed NID1 on NK cells, we used a proteomic approach. To this end, NKp44+ polyclonal NK cell populations expanded from four healthy donors were cultured for 20 h on plates coated with rNID1 (NID1 ) (or w/o coating as control, CTR). In parallel, NK cells were also cultured on plates coated with goat anti-mouse IgG (GAM) + anti-NKp44 mAb (NKp44) (or GAM alone as control, GAM). NK cells were then collected and lysed; total cell lysates were analyzed by high-resolution mass spectrometry.

Data processing through the MaxQuant software allowed the identification of a total of 6903 proteins (of which 5682 were quantified using a Label-Free Quantitation approach). Most of the proteins (5317) could be identified in all of the experimental conditions analyzed (i.e. CTR, NID1 , GAM, and NKp44), while few proteins were exclusive of specific conditions (Figure 7E, panel (a)). An initial analysis of the whole data set (i.e. without any restriction for statistical significance and fold change threshold) indicated that both stimuli could modify the NK cell proteomic profile and could induce concordant expression changes (i.e. up- or down- regulation) in a substantial number of proteins (Figure 7E, panel (b).

In order to analyze the statistically significant regulated proteins and to define the possible relationships between rNID1 and anti-NKp44 stimulation, two volcano plots were generated from the NIDIvsCTR and NKp44vsGAM data sets, and proteins were selected on the basis of a two-sample t-test (FDR=0.05 S0=0.1 ) (Fig 8A-B). By this selection, 112 proteins resulted modulated by rNID1 (64 up- and 44 down-regulated, as compared to CTR), and 129 proteins were modulated by anti-NKp44 mAb (41 up- and 88 down-regulated, as compared to GAM). 15 modulated proteins were common to the two conditions (indicated in red in Figure 8A-B). The GO analysis indicated that a major fraction of NID1 -modulated proteins were involved in the regulation of cell metabolism, but substantial numbers of proteins was also involved in processes related to cell proliferation, signal transduction, endo/exocytosis, immune response, indicating that NID1 stimulation could induce effective functional responses on NK cells (Figure 8C). Interestingly, NID1- and NKp44-modulated proteins appeared to be similarly involved in the same biological processes, and were similarly distributed among the main cellular components, with the highest percentage of proteins located in membrane compartments (Figure 8C-D). In order to further compare the two stimuli, we drilled down through the GO biological processes/molecular functions and built a functional heatmap depicting the quantitative modulation of the NK cell proteotype in response to NID1 or anti- NKp44 stimuli. This analysis indicated that part of the effects on the modulation of biological processes is shared by the two stimuli.

Our data suggest that NID1 can bind NKp44 and induce still unnoticed functional responses on NK cells. The GO analysis provides substantial information on these new putative functions, which, however, remain incompletely defined. In this context, the evaluation of the proteins modulated by both stimuli provides guidance. Within this group one of the most up-regulated proteins is represented by MYSM1 (see Figure 8). Interestingly, MYSM1 was recently demonstrated to be essential for NK cell maturation and differentiation (Alsultan et al. Blood 2013; 122:3844-5; Nandakumar et al., Proc Natl Acad Sci USA 2013; 110:E3927-3936), suggesting that the NKp44-NID1 interaction may be relevant to these processes and play a key role for the homeostasis of the mature NK cell population.

Example 5: Analysis of NID1 surface expression and NKp44 reactivity on a panel of human cell lines.

Different human cell lines including SH-SY-5Y neuroblastoma, A2774 ovarian adenocarcinoma, JEG3 placental chorio carcinoma and A549 lung carcinoma were stained with a NID1 -specific mAb or with NKp44Fc, followed by the appropriate isotype matched PE- conjugated secondary reagent mAb. Samples were analysed by flow cytometry. As shown in Figure 9, NID1 was expressed in the neuroblastoma, ovarian adenocarcinoma, placental chorio carcinoma and lung carcinoma. Grey profiles represent cells stained with anti-NI D1 or with NKp44Fc, while white profiles correspond to isotype control. One representative experiment of three is shows.

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.