WO2016179472A2 - Modulation of natural killer cell tolerance - Google Patents

Abstract

Embodiments of the disclosure concern methods and compositions related to reducing, inhibiting, reversing or preventing immune tolerance associated with antibody therapy in a subject. In specific embodiments, the disclosure concerns reducing, inhibiting, reversing or preventing natural killer (NK) cell tolerance occurring as a result of antibody-dependent cell- mediated cytotoxicity. Certain aspects utilize inhibitors of certain cell surface molecules on NK cells, including TIM-3, CCR7, and B7-H1.

Description

MODULATION OF NATURAL KILLER CELL TOLERANCE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Serial No.

62/158,221, filed May 7, 2015, the contents of which are incorporated by reference herein in its entirety.

BACKGROUND

Natural killer (NK) cells play a prominent role in mediating the anti-tumor response to targeted monoclonal antibody (mAb) therapy. FcyRIIIA (CD16), expressed on host NK cells, is able to engage the constant (Fc) domains of tumor associated mAbs and induce antibody- dependent cell-mediated cytotoxicity (ADCC) (Strome et al., 2007, Oncologist 12: 1084-1095). Although mAb therapies have been successfully used for the treatment of various malignancies such as anti-CD20 against B cell lymphoma and anti-EGFR against head and neck squamous cell carcinoma, patients often develop resistance to therapy, which has resulted in high relapse rates (Stolz and Schuler, 2009, Leuk. Lymphoma 50:873-885; Perez-Callejo et al, 2015, Cancer Treat. Rev. 41:680-689; Zarour, 2011, Eur J Immunol 41 : 1510-1515).

Thus, there is a need in the art for compositions and methods for overcoming tolerance to antibody therapy. The present invention provides this long-felt, unmet need in the art.

SUMMARY

Described herein are compositions and methods for modulating the immune system to reduce, inhibit, reverse or prevent immune tolerance. The methods can be used to reduce, inhibit, reverse or prevent immune tolerance by natural killer (NK) cells, such as when the tolerance results from antibody-dependent cell-mediated cytotoxicity (ADCC) induced by an antibody- based therapy administered to an individual, such as anti-tumor antibody for an individual with cancer. Also described herein are methods for modulating the immune system in order to initiate, increase or enhance immune tolerance.

In various embodiments, the compositions and methods target cell surface molecules on NK cells to prevent or restore functional suppression of the NK cells. In various embodiments, the NK cell surface molecules are at least one of TIM-3, B7-H1, and/or CCR7. The targeting of TIM-3, B7-H1, and/or CCR7 may be of any kind, but in certain aspects contacting TIM-3, B7- Hl, and/or CCR7 with at least one inhibitor agent restores NK cell function. Such compositions and methods allow the NK cells to become functionally responsive after being rendered functionally unresponsive upon antibody treatment of a disease or disorder.

In specific embodiments, antibody treatment results in antibody opsonization of cells (such as cancer cells). Following this, Fc:FcyR interactions between the NK cells and the antibody-opsonized target cells results in FcyR crosslinking, cytokine production, release of perforin/granzyme, and so forth. Inhibition of at least one of TIM-3, B7-H1, and/or CCR7 reduces, inhibits, reverses or prevents NK tolerance by inhibiting at least one step in this process.

In other embodiments, the compositions and methods of the disclosure can be used to initiate, increase or enhance immune tolerance in patients in need thereof, e.g., for the treatment of autoimmunity, inflammatory disease, or to prevent rejection of transplanted tissues, organs and cells. Such methods encompass agents that enhance ligation of CD 16 on NK cells to initiate, increase or enhance NK cell tolerance in individuals in need of a reduced immune response.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

Figure 1, comprising Figure 1A and Figure IB, provides the results of an exemplary experiment showing protein expression of NK cells. Whole PBMCs were stimulated with G001 (monomeric Fc, 10 μg/ml), Rituximab (anti-CD20 Ab, 10 μg/ml), 4542 (multimerized anti-CD20 Ab, 10 μg/ml; "Stradobody™"), G045 (multimerized Fc, 10 μg/ml or 100 ug/ml;

"Stradomer™"), IVIG (100 μg/ml) for different lengths of time (6 hours and 24 hours).

Following stimulation, percent positive (FIG. 1A) and mean fluorescence intensities (MFI, FIG. IB) for CD137, TIM-3, B7-H1, and CCR7 were quantified on NK cells (CD3 CD56⁺) by flow cytometry. Legend: □ Donor 1; Δ Donor 2; O Donor 3. Figure 2 depicts a schematic of a model of NK cell-mediated tolerance. Subsequent to NK cell activation and cancer cell lysis through ADCC, NK cells up regulate molecules involved in immune tolerance (i.e., TIM-3, B7-H1, and CCR7). This mode of regulation is exerted by NK cells within the microenvironment and also within secondary lymphoid organs by which tolerogenic NK cells migrated to in a CCR7-dependent manner.

Figure 3 depicts a schematic illustrating an example of an antibody (left) and an ADCC mechanism of action (right).

Figure 4 illustrates a molecular schematic of TIM-3. Modified figure from Freeman et al, 2010, Immunol. Rev. 235, 172-89.

Figure 5, comprising Figure 5A and Figure 5B, depicts the results of example experiments. Figure 5A depicts an inter-array correlation between antibody-stimulated and non- stimulated NK cells and Figure 5B provides a principal component analysis (PCA) of antibody- stimulated and non-stimulated NK cells.

Figure 6 shows differentially expressed genes associated with T cell development pathway at 4 and 24 hours post-stimulation. Circled: HAVCR2.

Figure 7, comprising Figure 7A through Figure 7C, depicts the results of example experiments. Figure 7A and Figure 7B are a set of graphs depicting the results of experiments assessing mean fluorescence intensity (MFI) (Figure 7A) and percent expression (Figure 7B) of CD137 (left) and TIM-3 (right) on isolated NK cells after stimulation with 1) media only, 2) G001, 3) G045, or 4) IVIG. Figure 7C illustrates CD 137 (top) and TIM-3 (bottom) expression histograms from a representative donor (gray: isotype) * denotes p-value <0.05.

Figure 8, comprising Figure 8A and Figure 8B, depicts the results of experiments assessing the expression of CD 137 and TIM-3 following treatments of PBMCs. Figure 8A shows the results of treating PBMCs with IVIG or Rituximab (anti-CD20) and Figure 8B shows the results of treating PBMCs with G001 or G045. Mean fluorescence intensity (MFI) (left) and percent expression (right) of CD 137 (top) and TIM-3 (bottom) gated on the NK cell fraction within treated PBMCs. * denotes p-value <0.05 Figure 9, comprising Figure 9A through Figure 9C, depicts the results of a NK cell transwell assay. Mean fluorescence intensity (Figure 9A) and percent expression (Figure 9B) of CD137 (Left) and TIM-3 (Right) after stimulation with media only or immobilized IVIG (bottom compartment). Figure 9C illustrates CD 137 (Top) and TIM-3 expression histograms from a representative donor (gray: isotype, blue: top compartment, red: bottom compartment). * denotes p-value <0.05

Figure 10 depicts the results of experiments assessing the expression of CD137 (top) and TIM-3 (bottom) after stimulation with 1) media only, 2) G001, 3) G045, or 4) IVIG in the presence of 1) no cytokine, 2) IL-2, 3) IL-15, 4) IL-12, 5) IL-18, 6) IL-4, 7) IL-6, or 8) IL-10.

Figure 11 depicts the results of experiments showing redirected ADCC (against P815 targets) and natural cytotoxicity (against K562). Resting (left) and G045-stimulated (right) NK cells cultured with P815 in the presence of isotype control antibody (open circles) or anti-CD 16 (open squares) for redirected ADCC, and cultured with K562 (open triangles) for natural cytotoxicity. X-axis represents the Effector to Target ratio, Y-axis represents % cell cytotoxicity.

Figure 12 depicts the results of experiments assessing the expression of Ceacam-1 (left) and TIM-3 (right) on HT29 cells. Black line: unstained; Gray filled: isotype control; Red line: Marker. MFI = Marker expression - isotype.

Figure 13 shows photographs of cell culture wells. Unstimulated (top row) or ConA- stimulated (bottom row) CD8+ T-cells co-cultured with NK cells previously stimulated with 1) G001, 2) Rituximab, 3) 4542, 4) G045 10 ug/ml, 5) G045 100 ug/ml, or 6) IVIG.

Figure 14 is a schematic showing an example timeline for blood sample collection from patients undergoing mAb-based therapies for the treatment of cancer.

Figure 15 is a schematic showing an example model of NK-mediated immune suppression. (1) Fc:FcyR interactions on NK cells triggers NK cell activation and cell lysis by ADCC, however, (2) ensuing TIM-3:TIM-3 ligand interactions may lead to the activation of a feedback mechanism shutting off pro-inflammatory processes, not only to itself, but (3) to potential bystander effector cells as well as a result of FcyR signaling. DETAILED DESCRIPTION

The invention is based, at least in part, on the discovery that certain cell surface molecules on NK cells are involved in the development and persistence of immune tolerance. Thus, in some embodiments, the invention includes compositions and methods of administering inhibitor agents for reducing, inhibiting, reversing or preventing NK cell-mediated tolerance, such as tolerance to immunotherapy that can occur in an individual upon antibody treatment for a disease or disorder. In other embodiments, the invention includes compositions and methods of administering activator agents for initiating, increasing or enhancing NK tolerance, for use when the subject being treated for a disease or disorder is in need of a reduced immune response through increased immune tolerance. In other embodiments, the invention includes methods of using combinations of agents, as well as kits, and pharmaceutical compositions of the agents described herein.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

As used herein, each of the following terms has the meaning associated with it in this section.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The terms "inhibit" and "inhibition," as used herein, means to reduce, suppress, reverse, diminish, block or prevent an activity, mechanism or function by at least about 10% relative to a control value. Preferably, the activity is reduced by about 20%, 30%, 40%, or 50% compared to a control value, more preferably by 75%, and even more preferably by 95%.

The terms "patient," "subj ect," "individual," and the like are used interchangeably herein, and refer to any animal, preferably a mammal, and most preferably a human, including a human in need of therapy for, or susceptible to, a disease or disorder or its sequelae. A "disease" is a state of health of subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

In contrast, a "disorder" in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder.

A disease or disorder is "treated" if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a patient, or both, is reduced.

"Cancer," as used herein, refers to the abnormal growth or division of cells. Generally, the growth and/or life span of a cancer cell exceeds, and is not coordinated with, that of the normal cells and tissues around it. Cancers may be benign, pre-malignant or malignant. Cancer occurs in a variety of cells and tissues, including the oral cavity (e.g., mouth, tongue, pharynx, etc.), digestive system (e.g., esophagus, stomach, small intestine, colon, rectum, liver, bile duct, gall bladder, pancreas, etc.), respiratory system (e.g., larynx, lung, bronchus, etc.), bones, joints, skin (e.g., basal cell, squamous cell, meningioma, etc.), breast, genital system, (e.g., uterus, ovary, prostate, testis, etc.), urinary system (e.g., bladder, kidney, ureter, etc.), eye, nervous system (e.g., brain, etc.), endocrine system (e.g., thyroid, etc.), and hematopoietic system (e.g., lymphoma, myeloma, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, etc.).

An "effective amount" or "therapeutically effective amount" of a compound is that amount of a compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered.

A "therapeutic" treatment is a treatment administered to a subject who exhibits a signs or symptom of a disease or disorder, for the purpose of reducing, diminishing or eliminating a sign or symptom of the disease or disorder.

As used herein, "treating a disease or disorder" means to reduce diminish or eliminate the frequency and/or severity of a sign and/or symptom of a disease or disorder experienced by a subject.

The term "antibody," as used herein, refers to an immunoglobulin molecule such as IgG, IgM, IgA, IgD and IgE, which is able to specifically bind to a specific epitope of an antigen. Antibodies can be intact immunoglobulins derived from natural sources, or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), Fv, Fab, Fab', F(ab)2 and F(ab')2, single domain antibodies (DABs), single chain antibodies (scFv), heavy chain antibodies, such as camelid antibodies, and humanized antibodies (Harlow et al, 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

By the term "specifically binds," as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen.

However, such cross reactivity does not itself alter the classification of an antibody as specific. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A" and the antibody, will reduce the amount of labeled A bound to the antibody.

"Isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in its normal context in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural context is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

I. Methods for Inhibiting NK Cell Tolerance

The invention includes methods for reducing, inhibiting, reversing or preventing tolerance to immunotherapy that can occur in an individual upon antibody treatment for a disease or disorder. In particular embodiments, tolerance occurs following antibody treatment because natural killer (NK) cells upregulate molecules that are involved in immune tolerance. The methods of the disclosure circumvent such mechanisms by directly or indirectly targeting the expression and/or activity of the molecules that are upregulated.

Thus, embodiments of the invention provide methods of administering inhibitor agents to reduce or prevent a decrease in therapeutic efficacy of antibody therapy. In some embodiments, the prevention may be total prevention, such as allowing for the efficacy of the antibody therapy to maintain a level substantially similar to initial clinical outcomes. In other embodiments, the prevention may be partial prevention, and the efficacy of the antibody therapy may decrease over time but not as much as compared with the decrease that would occur if the methods of the invention were not practiced. The efficacy of the antibody therapy before, during or after administering the inhibitor agent can be measured in a variety of ways, such as by measuring tumor load, by detecting the presence or absence of one or more symptoms of the disease or disorder, by detecting the presence or absence of one or more signs of the disease or disorder, by measuring the size of one or more particular tumors, by measuring survival, by measuring disease-free survival, by measuring progression-free survival, by measuring mortality, by measuring quality of life and/or by measuring the activity of NK cells.

Thus, the methods of the invention restore, increase, or potentiate NK cell responses and activities, including the improvement of a clinical response, such as the clinical response of a subject to antibody therapy.

The upregulation of molecules associated with tolerance on NK cells can occur through any mechanism, although in some embodiments it occurs through crosslinking of FcyR following Fc:FcyR interactions. In some embodiments, the methods of the invention that target the upregulated molecules associated with tolerance may do so at any time in the process of tolerance development, including, by way of non-limiting examples, before tolerance induction, after tolerance induction, before FcyR engagement by the antibody-opsonized cancer cells, or after before FcyR engagement by the antibody-opsonized cancer cells. Thus, in some

embodiments, the methods of the invention that target the upregulated molecules associated with tolerance may do so at any time in the process of ADCC.

In some embodiments, the methods of the invention are useful for circumventing the natural mechanisms for maintaining immune homeostasis, such as, for example, mechanisms that reduce, diminish, or turn off inflammation. In these embodiments, the methods impact events that follow Fc:FcyR interactions on NK cells that lead to NK cell activation and cell lysis by ADCC. In specific embodiments, methods and compositions target cell surface molecules on NK cell surfaces, such as by targeting and interfering with their interactions with one or more ligands.

In some embodiments, the individual subjected to the method of administering at least one inhibitor agent of the invention is also given at least one other agent that reduces, inhibits, reverses or prevents antibody-induced NK cell-mediated tolerance. In some embodiments, the individual subjected to the method of administering at least one inhibitor agent of the invention is also given at least one other agent for treating the individual's disease or disorder. In some embodiments, the at least one other agent directly or indirectly impacts antibody-mediated NK cell-mediated tolerance. In some embodiments, the at least one other agent does not impact antibody-mediated NK cell-mediated tolerance.

Some embodiments of the invention include compositions and methods that enhance treatment efficacy of antibody therapy and may be given at any time to an individual with respect to the antibody administration.

In various embodiments, the methods of the invention include administration of at least one inhibitor agent that reduces, inhibits, reverses or prevents NK cell-mediated tolerance, which may be administered to the subject before antibody administration, at the same time as antibody administration, and/or after antibody administration to the subject.

In various embodiments, the subject receiving antibody therapy has a disease or disorder treatable by antibody therapy. In some embodiments, the disease or disorder is cancer. The individual may have primary cancer, metastatic cancer, recurrent cancer, or cancer of any kind. The cancer may be of the lung, skin, brain, breast, colon, liver, kidney, prostate, pancreas, thyroid, endometrium, spleen, gall bladder, blood, rectum, ovary, testicle, intestine, lymph system, and so forth. In cases wherein the individual is being treated with antibodies for a disease or disorder other than cancer, the disease or disorder may by any disease or disorder treatable by antibody therapy, including, but not limiting to, cardiovascular disease, macular degeneration, osteoporosis, Alzheimer's disease, dyslipidemia, hypercholesterolemia, asthma, thrombotic thrombocytopenic purpura, organ transplant rejection, graft versus host disease, osteoporosis, scleroderma, muscular dystrophy, and so forth.

Although the individual may be receiving antibody treatment, or will be receiving antibody treatment, or had been receiving antibody treatment, or a combination thereof, the antibody treatment may be of any kind. In various embodiments, the antibody administered to the subject is at least one selected from the group consisting of the following: 3F8, 8H9, Abagovomab, Abciximab, Abituzumab, Abrilumab, Actoxumab, Adalimumab, Adecatumumab, Aducanumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD518, Alemtuzumab,

Alirocumab, Altumomab pentetate, Amatuximab, Anatumomab mafenatox, Anetumab ravtansine, Anifrolumab, Anrukinzumab (IMA-638), Apolizumab, Arcitumomab,

Ascrinvacumab, Aselizumab, Atezolizumab, Atinumab, Atlizumab (tocilizumab),

Atorolimumab, Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Begelomab,

Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab,

Biciromab, Bimagrumab, Bimekizumab, Bivatuzumab mertansine, Blinatumomab, Blosozumab, Bococizumab, Brentuximab vedotin, Briakinumab, Brodalumab, Brolucizumab,

Brontictuzumab,Canakinumab, Cantuzumab mertansine, Cantuzumab ravtansine, Caplacizumab, Capromab pendetide, Carlumab, Catumaxomab, cBR96-doxorubicin immunoconjugate, Cedelizumab, Certolizumab pegol, Cetuximab, Ch.14.18, Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan, Codrituzumab, Coltuximab ravtansine, Conatumumab, Concizumab, CR6261, Crenezumab, Dacetuzumab, Daclizumab, Dalotuzumab, Dapirolizumab pegol, Daratumumab, Dectrekumab, Demcizumab, Denintuzumab mafodotin, Denosumab, Derlotuximab biotin, Detumomab, Dinutuximab, Diridavumab, Dorlimomab aritox, Drozitumab, Duligotumab, Dupilumab ,Durvalumab, Dusigitumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Eldelumab, Elgemtumab, Elotuzumab, Elsilimomab, Emactuzumab, Emibetuzumab, Enavatuzumab, Enfortumab vedotin, Enlimomab pegol, Enoblituzumab, Enokizumab, Enoticumab, Ensituximab, Epitumomab cituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evinacumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FBTA05, Felvizumab, Fezakinumab, Ficlatuzumab, Figitumumab, Firivumab, Flanvotumab, Fletikumab,

Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galiximab, Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin, Gevokizumab,

Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, Guselkumab, Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Idarucizumab, Igovomab, IMAB362, Imalumab, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, Indusatumab vedotin, Infliximab,

Inolimomab, Inotuzumab ozogamicin, Intetumumab, Ipilimumab, Iratumumab, Isatuximab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lambrolizumab, Lampalizumab,

Lebrikizumab, Lemalesomab, Lenzilumab, Lerdelimumab, Lexatumumab, Libivirumab, Lifastuzumab vedotin, Ligelizumab, Lilotomab satetraxetan, Lintuzumab, Lirilumab,

Lodelcizumab, Lokivetmab, Lorvotuzumab mertansine, Lucatumumab, Lulizumab pegol, Lumiliximab, Lumretuzumab, Mapatumumab, Margetuximab, Maslimomab, Matuzumab, Mavrilimumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mirvetuximab soravtansine, Mitumomab, Mogamulizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox, Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox, Narnatumab, Natalizumab, Nebacumab, Necitumumab, Nemolizumab, Nerelimomab,

Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab merpentan, Obiltoxaximab,

Obinutuzumab, Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab,

Olokizumab, Omalizumab, Onartuzumab, Ontuxizumab, Opicinumab, Oportuzumab monatox, Oregovomab, Orticumab, Otelixizumab, Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Panitumumab, Pankomab, Panobacumab, Parsatuzumab,

Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, Polatuzumab vedotin, Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO 140, Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranibizumab, Raxibacumab, Refanezumab, Regavirumab, Reslizumab, Rilotumumab,

Rinucumab, Rituximab, Robatumumab, Roledumab, Romosozumab, Rontalizumab,

Rovelizumab, Ruplizumab, Sacituzumab govitecan, Samalizumab, Sarilumab, Satumomab pendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab, SGN-CD19A, SGN-CD33A, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Sofituzumab vedotin, Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Suvizumab, Tabalumab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomab paptox, Tarextumab, Tefibazumab, Telimomab aritox, Tenatumomab,

Teneliximab, Teplizumab, Teprotumumab, Tesidolumab, Tetulomab, TGN1412, Ticilimumab (tremelimumab), Tildrakizumab, Tigatuzumab, TNX-650, Tocilizumab (atlizumab),

Toralizumab ,Tosatoxumab, Tositumomab, Tovetumab, Tralokinumab, Trastuzumab,

Trastuzumab emtansine, TRBS07, Tregalizumab, Tremelimumab, Trevogrumab, Tucotuzumab celmoleukin, Tuvirumab, Ublituximab, Ulocuplumab, Urelumab, Urtoxazumab, Ustekinumab, Vandortuzumab vedotin, Vantictumab, Vanucizumab, Vapaliximab, Varlilumab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Volociximab,

Vorsetuzumab mafodotin, Votumumab, Zalutumumab, Zanolimumab, Zatuximab, Ziralimumab, and Zolimomab aritox.

As described herein, in order to characterize pathways involved in molecular resistance to targeted mAbs, expression profiles of NK cells cultured on immobilized IgG were examined at defined time-points after exposure. Such studies indicated that at least HAVCR2, a gene encoding T-cell immunoglobulin domain and mucin domain-3 (TIM-3), is significantly upregulated on NK cells stimulated with aggregated antibodies, Fc aggregates, and/or multimerized Fc domains. Antibody aggregation can result from a variety of events, including but not limited to, binding to target cells having one or more target, binding to proteins with one or more target binding epitopes, and/or multimeric proteins, idiotype-anti-idiotype reactions, etc. This upregulation was under the secondary control of pro and anti-inflammatory cytokines, suggesting that the milieu where Fc:FcyR interactions on NK cells occurs, influences TIM-3 expression. In some embodiments of the invention, the decrease in the therapeutic efficacy of antibody (including monoclonal antibody) therapies are a direct or indirect result of upregulation of TIM-3 on NK cells upon antibody stimulation. In specific embodiments, TIM-3 engagement renders NK cells functionally unresponsive and/or tolerogenic. Thus, embodiments of the invention include methods of reducing or inhibiting TIM-3 to prevent a decrease in therapeutic efficacy of an antibody.

II. Methods for Enhancing NK Cell-Mediated Tolerance

In certain embodiments, the invention includes methods comprising administering an activator agent for initiating, increasing or enhancing NK cell-mediated tolerance, for use when the subject being treated for a disease or disorder is in need of stimulating or increasing immune tolerance. Some subjects may, for example, have a disease or disorder for which the subject would benefit from a decrease in the natural immune response. By way of example, such diseases and disorders include, but are not limited to, autoimmune diseases, inflammatory diseases, and organ, tissue, and cell transplantation. In some embodiments, the subject is administered at least one agent that inhibits the expression or activity of CD 16. In some embodiments, the subject is administered at least one activator agent that increases the expression or activity of at least one of TIM-3, B7-H1, and CCR7. In some embodiments, the subject is administered at least one activator agent that increases the expression or activity of a TIM-3 ligand, such as ceacam-1 or galectin-9. In some embodiments, the subject is administered at least one activator agent that increases the expression or activity of a B7-H1 ligand, such as PD-1 or CD80. In some embodiments, the subject is administered at least one activator agent that increases the expression or activity of a CCR7 ligand, such as CCL19 or CCL21. In certain embodiments, the at least one activator agent for initiating, increasing or enhancing immune tolerance is anti-CD16 antibody. In other embodiments, the at least one activator agent for increasing or stimulating immune tolerance is a cytokine and/or an antibody aggregate, and/or an Fc aggregate, and/or and Fc multimer, such as but not limited to G045.

In various embodiments, the disease or disorder is an inflammatory disorder or an autoimmune disorder, such as at least one selected from the group consisting of Addison's disease, diabetes mellitus Type 1, Graves' disease, endometriosis, celiac disease, Crohn's disease, colitis, rheumatoid arthritis, fibromyalgia, Myasthenia gravis, multiple sclerosis, allergic asthma, pelvic inflammatory disease, allergy, inflammatory bowel disease, ulcerative colitis, cardiovascular disease, macular degeneration, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, psoriasis, plaque psoriasis, ankylosing spondylitis, juvenile idiopathic arthritis, multiple sclerosis, asthma, thrombotic thrombocytopenic purpura, organ transplant rejection, tissue transplant rejection, cell transplant rejection, graft versus host disease, osteoporosis, and scleroderma.

In some embodiments, the subject is a transplant recipient. In cases wherein the individual will receive or has received an organ transplant, a tissue transplant, or a cell transplant, the source of the respective organ, tissue, or cell may be from a live person or a cadaver. The individual may have received any kind of organ transplant of, including for example, but not limited to, the heart, kidneys, liver, lungs, pancreas, intestine, or thymus, for example. The individual may have received any kind of tissue transplant of, including for example, but not limited to, bones, tendons, cornea, skin, heart valves, nerves or veins. Thus, at least one agent for increasing stimulating immune tolerance may be given to the individual prior to, during, and/or subsequent to transplantation.

III. Inhibitor Compositions

Some embodiments of the invention use at least one inhibitor agent that reduces, inhibits, reverses or prevents NK cell-mediated tolerance, such as tolerance associated with antibody therapy. In specific embodiments, the at least one inhibitor agent reduces or inhibits the activity and/or expression of at least one NK cell cell-surface molecule. In some embodiments, the cell- surface molecule is part of a pathway for the induction of NK cell-mediated tolerance associated with antibody therapy. In various embodiments, the NK cell surface molecules that are directly or indirectly targeted include TIM-3, B7-H1, or CCR7. In some embodiments, the at least one inhibitor agent directly binds the molecule on the NK cell surface, so as to interfere with, inhibit or prevent the binding of a ligand of TIM-3, B7-H1 , and/or CCR7. In other cases, the at least inhibitor one agent directly binds a ligand that binds the cell surface molecule on the NK cell surface, so as to interfere with, inhibit or prevent the binding of the ligand to the molecule on the NK cell surface.

The skilled artisan will understand that the inhibitor agents of the invention can inhibit in a number of ways. In various embodiments, the inhibitor agents and methods of the invention inhibit by diminishing the amount of target polypeptide, the amount of target mRNA, the amount of target activity, the amount of target binding activity, or a combination thereof. Thus, based upon the disclosure provided herein, a decrease in the level of the target encompasses the decrease in target expression, including transcription, translation, or both. The skilled artisan will also appreciate, once armed with the teachings of the present invention, that a decrease in the level of the target includes a decrease in target activity (e.g., binding activity, etc.). Thus, decreasing the level or activity of the target includes, but is not limited to, decreasing transcription, translation, or both, of a nucleic acid encoding the target; and it also includes decreasing any activity of a target polypeptide as well. In some embodiments, the at least one inhibitor agent that reduces, inhibits, reverses or prevents NK cell-mediated tolerance, such as that associated with antibody therapy is at least one selected from the group consisting of a protein, peptide, peptidomemetic, antibody, ribozyme, small molecule chemical compound, a nucleic acid (DNA or RNA), an antisense nucleic acid molecule (e.g., siRNA, miRNA, etc.), or combinations thereof. The agent has inhibitory activity towards a particular molecule, in at least certain embodiments.

In certain embodiments, the TIM-3 molecule on the surface of NK cells is the target of the inhibitor agent. In some embodiments, one can inhibit the activity of TIM-3 and/or expression of TIM-3. In other embodiments, one can target for inhibition a ligand for TIM-3, such as ceacaml and/or galectin-9. Thus, in some embodiments, an individual is administered an inhibitor of the activity and/or expression of ceacam 1 and/or galectin-9.

In some embodiments, the B7-H1 molecule on the surface of NK cells is the target of the inhibitor agent. In some embodiments, one can inhibit the activity and/or expression of B7-H1. In other embodiments, one can target a ligand for B7-H1, such as PD-1 and/or CD80. Thus, in some embodiments, an individual is administered an inhibitor of the activity and/or expression of PD-1 and/or CD80.

In some embodiments, the CCR7 molecule on the surface of NK cells is the target of the inhibitor agent. In some embodiments, one can inhibit the activity and/or expression of CCR7. In other embodiments, one can target a ligand for CCR7, such as CCL19 and/or CCL21. Thus, in some embodiments, an individual is administered an inhibitor of the activity and/or expression of CCL19 and/or CCL21.

IV. Activator Compositions

Some embodiments of the invention use at least one activator agent that initiates, increases or activates NK cell-mediated tolerance. In specific embodiments, the at least one activator agent increases or activates the activity and/or expression of at least one NK cell cell- surface molecule. In some embodiments, the cell-surface molecule is part of a pathway for the induction of NK cell-mediated tolerance, such as that associated with antibody therapy. The skilled artisan will understand that the activator agents of the disclosure can activate in a number of ways. In various embodiments, the activator agents and methods of the invention activate directly or indirectly by increasing the amount of target polypeptide, the amount of target mRNA, the amount of target activity, the amount of target binding activity, or a combination thereof. Thus, based upon the disclosure provided herein, an increase in the level of the target encompasses the increase in target expression, including transcription, translation, or both. The skilled artisan will also appreciate, once armed with the teachings of the present invention, that an increase in the level of the target includes an increase in target activity (e.g., binding activity, etc.). Thus, increasing the level or activity of the target includes, but is not limited to, increasing transcription, translation, or both, of a nucleic acid encoding the target; and it also includes increasing any activity of a target polypeptide as well.

In some embodiments, the at least one activator agent that initiates, increases or enhances NK cell-mediated tolerance is at least one selected from the group consisting of a protein, peptide, peptidomemetic, antibody, ribozyme, small molecule chemical compound, a nucleic acid (DNA or RNA), an antisense nucleic acid molecule (e.g., siRNA, miRNA, etc.), or combinations thereof. The activator agent has activating activity towards a particular molecule, in at least some embodiments. In some embodiments, the activator agent has an inhibitory activity towards a regulator (i.e., negative regulator) of one or more targets described herein.

In various embodiments, the NK cell surface molecules that are directly or indirectly targeted include TIM-3, B7-H1, or CCR7.

In certain embodiments, the TIM-3 molecule on the surface of NK cells is the target of the activator agent. In some embodiments, one can increase the activity of TIM-3 and/or expression of TIM-3. In other embodiments, one can target a ligand for TIM-3, such as ceacaml and/or galectin-9. Thus, in some embodiments, an individual is administered an activator of the activity and/or expression of ceacam 1 and/or galectin-9.

In some embodiments, the B7-H1 molecule on the surface of NK cells is the target of the activator agent. In some embodiments, one can increase the activity and/or expression of B7-H1. In other embodiments, one can target a ligand for B7-H1 , such as PD-1 and/or CD80. Thus, in some embodiments, an individual is administer an activator of the activity and/or expression of PD-1 and/or CD80.

In some embodiments, the CCR7 molecule on the surface of NK cells is the target of the activator agent. In some embodiments, one can activate the activity and/or expression of CCR7. In other embodiments, one can target a ligand for CCR7, such as CCL19 and/or CCL21. Thus, in some embodiments, an individual is administered an activator of the activity and/or expression of CCL19 and/or CCL21.

In some embodiments, the CD 16 molecule on the surface of NK cells is the target of the activator agent. In some embodiments, one can activate the activity and/or expression of CD 16.

V. Therapeutic Applications and Pharmaceutical Preparations

Therapeutic compositions and methods involving the use of agents that reduce, inhibit, reverse or prevent NK cell-mediated tolerance are provided for treating diseases or disorders being treated with antibody therapy. In other various embodiments, agents that initiate, increase or enhance NK cell-mediated tolerance are provided for individuals with a disease or disorder where NK cell-mediated tolerance would be beneficial, such as, but not limited to, an autoimmune disorder, inflammatory disorder, or an organ, tissue or cell transplant.

Any agent that reduces, inhibits, reverses or prevents NK cell-mediated tolerance can be formulated in a pharmaceutical composition. Any agent that initiates, increases or enhances NK cell-mediated tolerance can be formulated in a pharmaceutical composition. Pharmaceutical compositions of the present invention comprise an effective amount of one or more cell or gene delivery compositions or additional agent dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference).

The amount of composition administered to a subject, such as a patient with a disease or disorder, can be determined by the skilled practitioner by considering physical and physiological factors such as body weight, severity of condition, the type of disease or disorder being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions comprise, for example, at least about 0.1% of an active compound. In other embodiments, the active compound comprises between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose comprises from about 1 to about 1000 mg/kg/body weight (this such range includes intervening doses) or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 μg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., is administered.

In some embodiments, solutions of therapeutic compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

In some embodiments, the therapeutic compositions are administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. An exemplary composition for such purpose may comprise a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.

Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well-known parameters.

Additional formulations that are suitable for oral administration are also provided. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

The therapeutic compositions may include pharmaceutical preparations. Administration of therapeutic compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Topical administration may be particularly advantageous for the treatment of skin diseases or disorders, such as skin cancers, to prevent chemotherapy -induced alopecia or other dermal hyperproliferative disorder, for example. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable

compositions that include physiologically acceptable carriers, buffers or other excipients. In further aspects, aerosol delivery can be used. The volume of the aerosol can ran from about 0.01 ml to about 5.0 ml.

In some embodiments, the therapeutic compositions comprise at least one inhibitor agent that reduces, inhibits, reverses or prevents NK cell-mediated tolerance associated with antibody therapy. The agent may be of any kind, such as a protein, peptide, peptidomemetic, antibody, ribozyme, small molecule chemical compound, a nucleic acid (DNA or RNA), an antisense nucleic acid molecule (e.g., siRNA, miRNA, etc.), or combinations thereof. The agent has inhibitory activity towards a particular molecule, in at least certain embodiments.

In some embodiments, the therapeutic compositions comprise at least one activator agent that initiates, increases or enhances NK cell-mediated tolerance. The agent may be of any kind, such as a protein, peptide, peptidomemetic, antibody, ribozyme, small molecule chemical compound, a nucleic acid (DNA or RNA), an antisense nucleic acid molecule (e.g., siRNA, miRNA, etc.), or combinations thereof. The activator agent has activating activity towards a particular molecule, in at least some embodiments.

In some embodiments, the inhibitor agent or activator agent comprises an antibody. As used herein, the term "antibody" is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally, IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting. The term "antibody" is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Means for preparing and characterizing antibodies are also well known in the art

(See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988;

incorporated herein by reference).

In some embodiments, a monoclonal antibody is employed. Monoclonal antibodies (MAbs) are recognized to have certain advantages, e.g., reproducibility and large-scale production, and their use is generally preferred. In other embodiments, "humanized" antibodies are also contemplated, as are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof.

In some embodiments, antibodies to TIM-3 are utilized in the methods of the invention. An example of a TIM-3 antibody is described in U.S. Patent Application Publication No.

20150218274. Inhibitors of TIM-3 are also described in U.S. Patent Application Publication No. 20100247521.

In some embodiments, the at least one inhibitor agent that reduces, inhibits, reverses or prevents NK cell-mediated tolerance associated with antibody therapy is a nucleic acid. In some cases, the nucleic acid may be utilized to reduce expression of a particular molecule, such as a molecule upregulated in an NK cell following antibody stimulation, for example HAVCR2 (the gene that encodes TIM-3). In some embodiments, the at least one agent that reduces, inhibits, reverses or prevents NK cell-mediated tolerance associated with antibody therapy is a nucleic acid that targets TIM-3 mRNA. Nucleic acid compositions that target TIM-3 mRNA may be of any length, so long as at least part of the composition hybridizes sufficiently and specifically to the TIM-3 mRNA molecule. The compositions may target any unique region of the TIM-3 mRNA. In specific embodiments, the nucleic acid compositions target a particular domain of the TIM-3 mRNA. RNA interference (RNAi) may be utilized for any target of the disclosure and may utilize any type of RNAi molecule, including at least using siRNA or shRNA. As an example, a representative TIM-3 mRNA is at the NCBI GenBank database under Accession Number JX049979.

Various aspects of the present invention include the use of the methods and compositions in combination with other therapies.

VI. Combination Therapy

It is an aspect of this invention that in some embodiments, the inhibit agents and methods for reducing, inhibiting, reversing or preventing NK cell-mediated tolerance associated with antibody therapy in a subject can be used in combination with at least one other agent or method of therapy. In some embodiments, the at least one other agent or method of therapy is another anti-cancer agent or anti-cancer therapy. In some embodiments, the at least one other agent is a CD137 agonist, such as but not limited to, and agonist antibody the binds to CD137. In some embodiments, the at least one other agent is the antibody that invoked or had the capacity to invoke the NK cell-mediated tolerance. In other embodiments, the at least one other agent is not the antibody that invoked or had the capacity to invoke the NK cell-mediated tolerance.

Treatment with the at least one agent that reduces, inhibits, reverses or prevents NK cell- mediated tolerance may precede or follow the other agent or therapy by an interval ranging from minutes to weeks. In some embodiments, where the at least one other agent is administered, a significant period of time will not transpire between the time of each delivery, such that the agents used in combination would be able to exert an combined effect on the cell. For example, it is contemplated that one may administer two, three, four or more doses of the at least one other agent substantially simultaneously (i.e., within less than about a minute) with the therapeutic agents of the present disclosure. In other aspects, the other at least one therapeutic agent or method is administered within about 1 minute to about 48 hours or more prior to and/or after administering the at least one agent that reduces, inhibits, reverses or prevents NK cell-mediated tolerance, or prior to and/or after any amount of time not set forth herein. In certain other embodiments, the at least one agent that reduces, inhibits, reverses or prevents NK cell-mediated tolerance of the present invention may be administered within from about 1 day to about 21 days prior to and/or after administering the at least one other agent, method or therapeutic modality. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several weeks (e.g., about 1 to 8 weeks or more) lapse between the respective administrations.

It is expected that treatment cycles will be repeated as necessary. It also is contemplated that various standard therapies, including surgical methods, may be applied in combination with the compositions and methods described here. These therapies include, but are not limited to, additional drug therapy, chemotherapy, radiotherapy, immunotherapy, gene therapy and surgery.

A. Chemotherapy

Cancer therapies also include a variety of combination therapies with both chemical- and radiation-based treatments. Chemotherapies include, but are not limited to, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate, or any analog or derivative variant of the foregoing. In some embodiments, the compositions and methods of the invention for treating a subject with the at least one inhibitor agent that reduces, inhibits, reverses or prevents NK cell-mediated tolerance associated with antibody therapy are used in combination with chemotherapy.

B. Radiotherapy

Other factors that cause DNA damage and have been used extensively include what are commonly known as γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV- irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells. The terms

"contacted" and "exposed," when applied to a cell, are used herein to describe the process by which a therapeutic construct and a chemo therapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing or stasis, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing. In some embodiments, the compositions and methods of the invention for treating a subj ect with the at least one inhibitor agent that reduces, inhibits, reverses or prevents NK cell-mediated tolerance associated with antibody therapy are used in combination with radiotherapy.

C. Immunotherapy

Immunotherapeutics, generally, rely on the use of immune modulators, immune effector cells and molecules to reduce, prevent or eliminate the signs and/or symptoms of a disease or disorder. In certain embodiments, immune modulators, immune effector cells and molecules target and destroy cancer cells. The immune effector may be, for example, an antibody specific to a molecule on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionucleotide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

Immunotherapy, thus, could be used as part of a combined therapy, in conjunction with gene therapy. Generally, the tumor cell bears a molecule or marker that is amenable to targeting, i.e., is not present on the majority of other non-tumor cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include carcino embryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55. In some embodiments, the compositions and methods of the invention for treating a subj ect with the at least one inhibitor agent that reduces, inhibits, reverses or prevents NK cell-mediated tolerance associated with antibody therapy are used in combination with immunotherapy.

D. Nucleic Acid-based Therapy

In some embodiments, the composition of the invention that reduces, inhibits, reverses or prevents NK cell-mediated tolerance associated with antibody therapy is a nucleic acid. In other embodiments, the at least one other agent is a nucleic acid that is administered before, after, or at the same time as a composition of the present invention. Delivery of at least one agent that reduces, inhibits, reverses or prevents NK cell-mediated tolerance in combination with a nucleic acid encoding a therapeutic gene product will have a combined therapeutic effect, such as an anti-hyperproliferative effect on target tissues.

RNA interference (RNAi) is a powerful gene-silencing process that holds great promise in the field of cancer therapy. The evolving understanding of the molecular pathways important for carcinogenesis has created opportunities for cancer therapy employing RNAi technology to target the key molecules within these pathways. Major targets for siRNA therapy include oncogenes and genes that are involved in angiogenesis, metastasis, survival, antiapoptosis and resistance to chemotherapy.

Many gene products involved in carcinogenesis have already been explored as targets for RNAi intervention, and RNAi targeting of molecules crucial for tumor-host interactions and tumor resistance to chemo- or radiotherapy has also been investigated. In most of these studies, the silencing of critical gene products by RNAi technology has generated significant antiproliferative and/or proapoptotic effects in cell-culture systems or in preclinical animal models. siRNA can be introduced into the cells by using either chemically synthesized siRNA oligonucleotides (oligos), or vector-based siRNA (shRNA), which allows long lasting and more stable gene silencing. Nanoparticles and liposomes are commonly used carriers, delivering the siRNA with better transfection efficiency and protecting it from degradation. In combination with standard chemotherapy, siRNA therapy can also reduce the chemoresistance of certain cancers, demonstrating the potential of siRNA therapy for treating many malignant diseases. In some embodiments, the compositions and methods of the invention for treating a subject with the at least one inhibitor agent that reduces, inhibits, reverses or prevents NK cell- mediated tolerance associated with antibody therapy are used in combination with a nucleic acid.

E. Surgery

Approximately 60% of subjects with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the compositions or methods of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.

In some embodiments, the compositions and methods of the invention for treating a subject with the at least one inhibitor agent that reduces, inhibits, reverses or prevents NK cell- mediated tolerance associated with antibody therapy are used in combination with surgery.

VII. Kits

Any of the compositions described herein may be part of a kit. In a non-limiting example, at least one inhibitor agent that reduces, inhibits, reverses or prevents NK cell-mediated tolerance associated with antibody therapy, may be part of a kit. In some embodiments, at least one activator agent that initiates, increases or enhances NK cell-mediated tolerance may be part of a kit. In some embodiments, the kit comprises an inhibitor of TIM-3. In one embodiment, the inhibitor of TIM-3 is an antibody.

The components of the kits may be packaged either in aqueous media or in lyophilized form. In some embodiment, the kit contains a contain, such as, but not limited to, at least one vial, test tube, flask, bottle, syringe or other container, into which a component of the kit may be placed. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a container. The kits of the present invention also will typically include a means for containing the agent(s) and any reagent container(s) in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

When the components of the kit are provided in one and/or more liquid solutions, the liquid solution can be an aqueous solution, such as a sterile aqueous solution. The composition may also be formulated into a syringeable composition. In which case, the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into a subject, and/or applied to and/or mixed with the other components of the kit. However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure. EXAMPLE 1 : INHIBITION OF NK CELL TOLERANCE UPON ANTIBODY-DEPENDENT

CELL-MEDIATED CYTOTOXICITY

The present disclosure is based, at least in part, on the finding that following antibody- dependent cell-mediated cytotoxicity (ADCC), natural killer (NK) effector cells become tolerogenic and express cell surface markers that can be manipulated to alter their function.

To assess this, expression profiling of NK cells stimulated with immobilized human IgGl was performed at distinct time intervals (4 hours and 24 hours). These data revealed a set of cell surface inhibitory molecules that are differentially expressed following activation (Table I).

Tabic I: NK cell gene exi pression profile

Average

GeneED ProtemlD 4H change p-vaise 24H dhsaige p-vakte

ExRression

HAVCR2 ΊΊ -3 0.280 3.S4E-01 1.202 1.61E-03 9.276

CD274 B7-H ! 0.666 6.01 E-02 0.259 4.32E-0I 4.606

CCR7 CCR7 0.006 9.91E-01 2.107 2.33E-03 5.701

Table 1. Gene expression profile of NK cells. NK cells were stimulated with or without immobilized human IgGi at different time intervals (4 hours and 24 hours; H=hours). Changes in expression and average expression are represented as log base 2 values.

Next, protein expression of several of these markers was validated in peripheral blood mononuclear cells (PBMC) treated with either: (1) anti-CD20 antibodies, (2) anti-CD20 Stradobodies™ (anti-CD20-like molecules bearing multimerized Fc and Fab2 fragments), or (3) fully recombinant multimerized Fc fragments alone (Stradomers™).

Following treatment with anti-CD20 antibodies, with anti-CD20 Stradobodies™, or with Stradomers™, CD3 CD56⁺NK cells up-regulated CD137, TIM-3, B7-H1, and CCR7 (FIG. 1). Importantly, these effects were observed not only after treatment with anti-CD20 antibodies or treatment with anti-CD20 Stradobodies™, but also after treatment with Stradomers™ (which cause Fc receptor (FcR) crosslinking in the absence of Fab2 binding), consistent with the explanation that the observed phenomenon is Fc-dependent. The data described herein are the first report showing that FcR ligation induces up-regulation of TIM-3, B7-H1, and CCR7 on the surface of CD3 CD56⁺NK cells.

Based on these data, an example of a model is provided in Figure 2 in which, following ADCC, involved NK cells up-regulate select molecules that have the potential to induce tolerance in both NK and non-NK cell populations. In addition, in a specific embodiment CCR7 expression on the surface of these NK cells modulates migration to regional lymph nodes, thereby allowing the NK cells to induce centrally-mediated effects.

An emerging body of evidence suggests that immune checkpoint blockade enhances treatment of cancer, and that dual (and possibly triple) modality therapy will yield the best therapeutic results. For example, a recent clinical study demonstrates that combined blockade of PD-1 and CTLA-4, yields better therapeutic effects than inhibition of either of these pathways in isolation in the treatment of cancer (Postow et al, New England Journal of Medicine 2015). The data described herein suggest that targeted antibody therapies, such as anti-CD20, might induce a tolerogenic population of NK cells that express TIM-3, B7-H1, and CCR-7. Therefore, in one embodiment of the disclosure, blockade of these pathways, each alone and in any combination, before, during or after the time of therapeutic antibody administration, enhances therapeutic efficacy by preventing NK cell-mediated tolerance.

For example, in a subject being treated with an anti-tumor antibody, the blockade of: (1) TIM-3 and/or a TIM-3 ligand, such as ceacam 1 and/or galectin-9, (2) B7-H1 and/or a B7-H1 ligand, such as PD-1 and/or CD80, and/or (3) CCR7 and/or and/or a CCR7 ligand, such as CCL19 and/or CCL21, can be used, each alone or any in any combination, to reduce, inhibit, reverse or prevent NK cell-mediated tolerance following ADCC. Blockade of each of the foregoing molecules can be accomplished, for example, by administering to the patient an inhibitor, such as but not limited to an antibody, that interferes with the ability of the molecule to interact with the molecule's cognate receptor (or ligand) and/or inhibits its biological activity. Such blockade reduces, inhibits, reverses or prevents NK cell-mediated tolerance.

In some embodiments, the present discovery can also be used to induce NK cell-mediated tolerance for the treatment of diseases or disorders where NK cell-mediated tolerance can be beneficial to the subject, such as, by way of non-limiting examples, autoimmunity, inflammatory disease, and to prevent rejection of transplants in patients with transplanted organs, tissues or cells. For example, anti-CD 16 antibodies can be administered to patients in order to induce ligation of CD 16 (low-affinity FcR) on NK cells, thereby inducing NK cell-mediated tolerance in patients in need of such treatment.

EXAMPLE 2: FC:FCYR INTERACTIONS ON HUMAN NK CELLS UPREGULATE TIM-3: A DRUGGABLE TARGET TO OVERCOME NK TOLERANCE

Natural killer (NK) cells are innate, bone marrow-derived lymphocytes that are capable of directly responding to and eliminating virally -infected, stressed, and malignant cells (Vivieret al, 2008, Nat. Immunol. 9:503-510). Although functionally analogous to cytotoxic CD8+ T cells, NK cells require no prior antigen stimulation (Orange and Ballas, Clin. Immunol. 118: 1- 10). NK cells consist of about -10% of the total circulating lymphocyte population and are divided up into two subsets based on the expression of various cell surface molecules. The minor subset is phenotypically defined as CD56^{Bn ht}CD16^" while the major subset is defined as CD56^DimCD16⁺. CD 16, or FcyRIIIA, is a moderate affinity Fc receptor for which the ligand is the constant (Fc) region of immunoglobulin G (IgG) (Vivier et al., 2008, Nat. Immunol. 9:503- 510; Orange and Ballas, 2006, Clin. Immunol. 118: 1-10).

Antibody-dependent cell-mediated cytotoxicity (ADCC): In the context of monoclonal antibody (mAb) therapy for the treatment of cancer, the variable (Fab) domain of therapeutic antibodies targets a specific antigen or molecule present on the tumor. For example, anti-CD20 antibodies target CD20⁺ B cells for the treatment of B cell lymphoma. When NK cells encounter antibody-opsonized tumor cells, Fc:FcyR interactions result in FcyR crosslinking, leading to cytokine production, and release of perforin/granzyme. The end result of this process is target cell cytotoxicity, which is commonly referred to as ADCC (Figure 3) (Strome et al, 2007, Oncologist 12: 1084-1095; Iannello et al, 2005, Cancer Metastasis Rev. 24, 487-499).

ADCC inducing mAbs may cause cancer relapse and resistance to antibody treatment: In many cases, cancer patients undergoing mAb therapy as a single agent or in combination with other therapeutic agents, e.g. chemotherapy, achieve remission. However, there are instances where patients fail to completely respond to initial mAb therapy, relapse, or even develop secondary resistance (Stolz and Schuler, 2009, Leuk. Lymphoma 50:873-885; Perez-Callejo et al, 2015, Cancer Treat. Rev. 41 :680-689; Zarour, 2011, Eur J Immunol 41 : 1510-1515). More specifically, about 30% of patients with B-cell non-Hodgkin lymphoma fail anti-CD20-based therapies due to secondary resistance mechanisms (Stolz and Schuler, 2009, Leuk. Lymphoma 50:873-885).

Tolerogenic NK cells - a potential regulator of antibody-mediated immune responses: There have been extensive reports indicating that NK cells have the ability to suppress the immune response and induce tolerance (MacDonald, 2005, Nat. Immunol. 6, 868-9; Zhang et al, 2006, Cell. Mol. Immunol. 3, 241-254; Deniz et al, 2008, J. Immunol. 180, 850-7; Olson et al, 2010, Blood 115, 4293-4301 ; Cook and Whitmire, 2013, J. Immunol. 190, 641-9; Fu et al, 2014, Immunology 141, 483-9; Wirsdorfer et al, 2015, Clin. Sci. 128, 825-838). In addition, antibodies have been linked in cultivating 'immunoregulatory' NK cells (Chong et al, 2013, PLoS One 8, e60862; Gregoire-Gauthier et al, 2015, Biol. Blood Marrow Transplant. 21, 821- 828). One mouse study involving intravenous immunoglobulin (IVIG) in the treatment of graft versus host disease (GvHD) demonstrated that NK cell were essential in preventing disease incidence (Gregoire-Gauthier et al, 2015, Biol. Blood Marrow Transplant. 21, 821-828). In fact, if NK cells were depleted prior to IVIG treatment, GvHD incidence was exacerbated compared to the saline treated group. Moreover, FcyRIII null mice had better anti-tumor responses during anti-4-lBB therapy which further alludes to the NK cells involvement in immune suppression (Sallin et al, 2014, Cancer Immunol. Immunother. 63, 947-58).

TIM-3, a potential immunoregulatory molecule on NK cells: T-cell immunoglobulin and mucin domain containing 3 (TIM-3) is a molecule found highly expressed on NK cells (Khademi et al, 2004, J. Immunol. 172, 7169-76) (FIG. 4). TIM-3 is found expressed on dendritic cells, monocytes, macrophages, and T lymphocytes (Anderson et al, 2007, Science 318, 1141-1143; Jones et al, 2008, J. Exp. Med. 205, 2763-2779; Nakayama et al, 2009, Blood 113, 3821- 3830). Moreover, TIM-3 has been described as having the capacity to regulate innate and adaptive immune responses (Freeman et al, 2010, Immunol. Rev. 235, 172-89) and mediate the induction of peripheral tolerance (Sanchez-Fueyo et al, 2003, Nat. Immunol. 4: 1093-1101 ; Sabatos et al, 2003, Nat. Immunol. 4: 1102-1110). These immunoregulatory responses are generated through engagement with TIM-3 ligands, such as Galectin-9 (Zhu et al, 2005, Nat. Immunol. 6, 1245-1252) and the more recently discovered Ceacam-1 (Huang et al, 2014, Nature 517, 386-390).

In specific embodiments, the upregulation of TIM-3 subsequent to Fc-mediated stimulation renders NK cells more susceptible to functional suppression after engagement with TIM-3 ligands. Moreover, the upregulation of TIM-3 on NK cells will also increase the likelihood of inducing peripheral tolerance through the engagement of TIM-3 ligands, such as Ceacam-1 on T cells. In addition, studies have shown that NK cells from patients with gastrointestinal stromal tumors (Komita et al., 2015, Oncol. Rep. 34, 2099-105), lung adenocarcinoma (Xu et al, 2015, Int. Immunopharmacol. doi: 10.1016/j .intimp.2015.09.017), and melanoma (da Silva et al, 2014, Cancer Immunol. Res. 2, 410-22) exhibited increased TIM- 3 expression on NK cells, which was associated with poor prognosis. Treatment of these patients with mAb therapy renders NK cells more susceptible to functional suppression and result in poorer outcomes, in particular embodiments. Therefore, it is useful to characterize the mechanism by which antibodies regulate TIM-3 on NK cells and how these regulatory mechanisms affect NK cells during an immune response.

The data described in the Examples is significant for a variety of reasons. Monoclonal antibodies have been a vital part of the clinician's armamentarium for the battle against cancer. Furthermore, NK cells are one of, if not the most important, effector cell population mediating tumor regression during mAb therapy. However, the Achilles' heel of mAb therapy is the development of tolerance where patients become unresponsive and often resistant to treatment. In order to address this problem, one can define druggable molecular targets that mediate NK cell-mediated tolerance after exposure to antibody-opsonized tumors. The data herein demonstrated that TIM-3, a molecule involved in the induction of peripheral tolerance, is upregulated in the presence of opsonized target cells. Therefore, blocking TIM-3 : TIM-3 ligand interactions on NK cells following targeted mAb therapy is an approach to enhancing treatment efficacy, in various embodiments of the disclosure.

EMBODIMENT 1 : A genomics based approach to define how Fc:FcyR engagement induces the upregulation of TIM-3 on NK cells.

Transcriptomics studies revealed that HAVCR2, a gene encoding TIM-3, was upregulated on NK cells following antibody stimulation. In order to confirm that TIM-3 upregulation was induced by direct, FcyR crosslinking, both isolated NK cells and NK cells in peripheral blood mononuclear cells (PBMCs) were studied. The data showed that direct, crosslinking interactions with antibody can mediate the upregulation of TIM-3 on NK cells. In order to complete this embodiment, one can block CD 16, to confirm that this is the receptor required for induction of TIM-3. These studies will reveal that direct, crosslinking Fc:FcyR interactions are required for the upregulation of TIM-3 on NK cells.

See Table 1 : Gene expression profile of NK cells. NK cells were stimulated with or without immobilized human IgGi at different time intervals (4 hours and 24 hours; H=hours). Changes in expression and average expression are represented as log base 2 values.

In Embodiment 1 A, the inventors utilized enriched NK cells and NK cells in peripheral blood mononuclear cells (PBMCs) and confirmed the type of antibody stimulation needed to induce TIM-3 upregulation i.e., monomeric vs. crosslinking. In Embodiment IB, the inducing signal required for TIM-3 upregulation on NK cells was determined as a direct effect of antibody stimulation. In Embodiment 1 C, CD 16 blocking assays are performed to confirm that antibodies require this FcyR for the induction of TIM-3. These studies reveal that direct, crosslinking Fc:FcyR interactions are necessary for the upregulation of TIM-3 on NK cells.

The studies in this embodiment are significant. TIM-3 is an immunoregulatory molecule involved in the induction of peripheral tolerance. It is found highly expressed on NK cells (Khademi et al., 2004, J. Immunol. 172, 7169-76) and upon antibody stimulation, HAVCR2, the gene encoding TIM-3 becomes significantly upregulated. Although NK cells are known mediators of ADCC and crucial effectors cells for tumor resolution, the upregulation of TIM-3 and its ability to inhibit immune responses implicates NK cells as a factor contributing to the decrease in clinical efficacy of mAb therapies. The following studies verify TIM-3 protein expression on antibody-stimulated NK cells and how antibodies contribute to TIM-3 upregulation, in certain aspects.

Initial studies were performed to determine the gene expression profile of human NK cells after antibody stimulation. RNA was extracted from non-stimulated (resting) and immobilized IgGl -stimulated NK cells at 4 and 24 hours post-stimulation and gene expression was quantified by microarray (Illumina Direct Hybridization Microarray). Because it has been previously shown that human NK cells upregulate 4- IBB (CD 137) upon antibody stimulation Lin et al, 2008, Blood 112, 699-707), the inventors utilized the expression of CD 137 on NK cells as a phenotype for validating successful antibody stimulation. In all subsequent studies, CD137 expression is used to verify and validate the antibody-mediated stimulation of the NK cells. Microarray analysis of the gene expression dataset was compiled to construct an inter- array correlation heat map and was examined by principal component analysis indicating a differential gene expression profile between antibody stimulated and resting NK cells (Figure 5). Ingenuity Pathway analysis (IP A) was subsequently used to identify key gene network pathways differentially regulated upon antibody stimulation. After NK cells were stimulated, key genes associated within the T cell development gene network were strongly perturbed (Figure 6). Interestingly, HAVCR2, a gene encoding T-cell immunoglobulin and mucin-containing domain- 3 (TIM-3), was significantly upregulated upon antibody stimulation. At 24 hours of antibody stimulation, HAVCR2 was 2.4-fold greater on antibody-stimulated NK cells than non-stimulated NK cells.

Embodiment 1 A. Determination of the type of antibody interaction necessary for TIM-3 upregulation on NK cells.

The studies described herein have shown that HAVCR2, the gene encoding TIM-3, is upregulated on NK cells upon antibody stimulation. The gene expression results were validated by quantifying the level of TIM-3 expression at the protein level on isolated NK cells. This was accomplished by flow cytometry using a commercially purchased fluorophore-conjugated anti- human TIM-3 antibody. During these studies, different forms of antibody-mediated stimulation were used to deduce the minimal requirements necessary for TIM-3 upregulation. The different types of stimulation included: 1) IVIG, non-specific pooled IgG, 2) Rituximab, a CD20-specific antibody, 3) GOOl, a recombinant Fc monomer, and 4) G045, a recombinant Fc multimer. GOOl and G045 can be obtained from Gliknik, Inc. Furthermore, in order to recapitulate this effect in a more natural setting and to resemble a patient being treated with therapeutic mAbs, TIM-3 levels on NK cells were measured on stimulated PBMCs.

It was found that the upregulation of TIM-3 on NK cells was dependent on Fc-mediated crosslinking. Therefore, on isolated NK cells, TIM-3 upregulation was only observed with G045 stimulation because the multimeric nature of its Fc domains are capable of crosslinking. The inventors were unable to observe TIM-3 upregulation on NK cells stimulated with either GOOl or IVIG as neither of these antibody forms are able to induce crosslinking (Figure 7). The inventors were able to recapitulate these results on NK cells from stimulated PBMCs. In addition, NK cells from PBMCs stimulated with Rituximab exhibited TIM-3 upregulation since CD20⁺ B cells were present (Figure 8). Anti-CD20 opsonized B cells were able to resemble multimeric Fc stimulation capable of crosslinking molecules that can engage Fc similar to that observed with G045. Therefore, Fc-mediated crosslinking is useful for TIM-3 upregulation on NK cells.

Tumors opsonized with targeted mAbs bearing functional Fc fragments, can enhance NK cell expression of CD 137 and ligation of CD 137 on these Fc activated NK cells, enhances their antitumor effects. To test whether aggregated Fc fragments induce other molecules on the NK cells surface that can be targeted to enhance NK cell function, the expression patterns of human NK cells stimulated with coated human IgGl was analyzed. These studies revealed upregulation of HAVCR2 mRNA on Fc treated NK cells. HAVCR2 encodes for the T cell immunoglobulin domain and mucin domain three (TIM-3) protein.

TIM-3 protein expression was analyzed on NK cells in PBMC stimulated with mAb opsonized tumors, mAb alone, homodimeric Fc, intravenous immunoglobulin (IVIG) or a fully recombinant human IgGl Fc multimer called GL-2045. Exposure of NK cells to GL-2045 or opsonized tumors induced surface expression of both CD137 and TIM-3, while the other conditions failed to mediate similar responses. TIM-3 expression on these Fc-stimulated NK cells was not reliant on other cell types and was contact dependent. Collectively, these data demonstrate that Fc interactions with FcRs induce contact-dependent TIM-3 expression on NK cells.

Despite the fact that both CD 137 and TIM-3 are both upregulated on NK cells, following exposure to aggregated Fc, their patterns of expression and regulation are distinct. Specifically, while a percentage of cells co-express TIM-3 and CD 137, the majority of cells express either one or the other. In addition, following exposure to multimerized Fc, TIM-3 expression is enhanced by exposure to IL-2, IL-15, IL-12, and IL-10, while CD137 levels are augmented by co-culture with IL-18. These differences in expression and regulation may have therapeutic relevance as they suggest that following exposure to tumors decorated with targeted mAbs, a TIM-3 blockade may act in synergy with agonistic mAbs against CD 137 to enhance anti -tumor NK cell function.

Embodiment IB. Testing to determine that antibody stimulation directly mediates the upregulation of TIM-3 on NK cells.

Transwell assays were used to determine the potential direct and indirect effects of antibody stimulation on the upregulation of TIM-3. In some of the wells, IVIG was immobilized in the lower chamber to induce antibody-mediated stimulation. Isolated NK cells were then added to both the lower and upper chambers. The expression of TIM-3 was quantified by flow cytometry and it was determined if the upregulation of TIM-3 was a direct or indirect result of antibody stimulation.

TIM-3 upregulation by indirect antibody stimulation would have implied that a soluble mediator was facilitating this effect. If so, TIM-3 would have been found upregulated in both the lower and upper chambers in immobilized antibody stimulation conditions. However, only direct antibody stimulation was observed to induce the upregulation of TIM-3 since only NK cells within the lower compartment demonstrated this result (Figure 9).

Embodiment 1C. Testing to define the receptor necessary for TIM-3 upregulation on NK cells after antibody stimulation.

CD 16 blocking assays can be used to determine that the upregulation of TIM-3 is dependent upon the engagement of FcyRs on NK cells. Previous reports indicate that the anti- human CD16 antibody (Clone: 3G8) (Tamm and Schmidt, 1996, J. Immunol. 157, 1576-1581) recognizes the Fc recognition domain on CD16 and is able to block the binding of antibodies. Fab fragments may be generated from clone 3G8 and used as a blocking reagent for this assay. The reason for generating Fab fragments from clone 3G8 rather than using whole antibody as the CD 16 blocking reagent is to avoid non-specific NK cell activation. The bivalent nature of antibodies could potentially crosslink CD 16 and mediate NK cell activation. In addition, the Fc domain of clone 3G8, which is of the mouse IgGl isotype, has an inherent affinity for human FcyRIIIA (Jonsson and Daeron, 2012, M. Mast cells and company. Front. Immunol. 3, 1-18). One can test for the upregulation of TIM-3 after G045 stimulation in the presence or absence of blocking Fab fragments from clone 3G8.

In specific embodiments, blocking CD 16 with clone 3G8 Fab fragments will abrogate G045-mediated upregulation of TIM-3 on NK cells. Therefore, in at least certain cases the upregulation of TIM-3 on antibody-stimulated NK cells is dependent upon FcyRIIIA binding.

EMBODIMENT 2: Characterize how TIM-3 on antibody-activated NK cells responds to secondary stimuli (i.e., cytokines). Previous studies have shown that cytokine stimulation can regulate the expression of TIM-3 on NK cells (Gleason et al, 2012, Blood 119:3064-3072; Ndhlovu et al., 2012, 1 19:3734-3743). In Embodiment 2A, it was investigated how specific cytokines influence TIM-3 expression on NK cells exposed to aggregated and multimerized Fc domains. It was determined how specific cytokines influence TIM-3 expression on NK cells exposed to aggregated and multimerized Fc domains. The data showed that combinatorial stimulation with both cytokine and antibody potentiated the upregulation of TIM-3. Using this information as a backdrop, in Embodiment 2B, the signaling pathways that contribute to TIM-3 expression on NK cells are defined. These studies can provide the mechanism by which TIM-3 becomes regulated on NK cells.

The studies in Embodiment 2 are significant. During mAb therapy for the treatment of cancer, not only will NK cells react against antibody-opsonized tumor cells and mount ADCC, they will also be subjected to a plethora of stimuli, including cytokines, especially within the tumor microenvironment where ADCC predominantly occurs. Previous investigations have shown that cytokines capable of inducing NK cell proliferation, such as IL-2 and IL-15, as well as pro-inflammatory cytokines, such as IL-12 and IL-18, mediated TIM-3 upregulation on the surface of NK cells (Gleason et al, 2012, Blood 1 19:3064-3072; Ndhlovu et al, 2012, 1 19:3734-3743). The data described herein indicate that TIM-3 also becomes upregulated upon Fc:FcyR stimulation. Furthermore, by identifying key molecules within the cytokine pathways inducing TIM-3 expression, and comparing them to the genes regulated upon antibody stimulation, one can identify redundant and/or convergent signaling nodes related to TIM-3 modulation. Therefore, an understanding of the interplay between antibody and cytokine stimulation is useful to characterize the mechanism governing TIM-3 expression on NK cells.

Embodiment 2A. To determine how TIM-3 expression is modulated after combinatorial antibody-cytokine stimulation on NK cells.

Freshly isolated NK cells were used and soluble G001 , G045, and IVIG (see

Embodiment 1A) were utilized as different forms of antibody stimulation. In addition, NK cells were co-cultured with various cytokines: IL-2, IL-15, IL-12, IL-18, IL-4, IL-6, and IL-10. The degree of TIM-3 expression regulated by antibody and cytokine stimulation on NK cells was evaluated by flow cytometry.

It was observed that NK cells stimulated with G045 (recombinant Fc multimer) upregulated TIM-3 (as shown previously in Embodiment 1). Moreover, it was determined that G045 stimulation in combination with certain cytokines (e.g., IL-2, IL-15, IL-12, IL-10, and IL- 18) further increased TIM-3 expression levels (Figure 10).

Embodiment 2B. To investigate signal transduction pathways regulating TIM-3 expression on antibody activated NK cells.

One can use freshly enriched NK cells and stimulate them with G045. In addition, NK cells can be co-cultured with cytokines that regulate TIM-3 as determined from Embodiment 2A. In some culture wells, blocking reagents (e.g., STAT or NF-kB inhibitors) can be added to inhibit certain aspects of cytokine signaling. Subsequently, TIM-3 expression can be evaluated by flow cytometry, for example.

In specific aspects, blocking specific STAT or NF-kB signaling pathways will abrogate the upregulation of TIM-3 that would normally be seen in the absence of the blocking reagents. Moreover, depending on the results, one can deduce redundant or converging pathways in regards to Fc:FcyR signaling.

EMBODIMENT 3: Testing of the functional role of TIM-3 on NK cells after antibody stimulation.

Homeostatic levels of TIM-3 are postulated to be immunosuppressive on NK cells.

However, the consequences of TIM-3 upregulation on FcR activated NK cells are unknown. Redirected ADCC assays may be performed to determine the suppressive effects of TIM-3 signaling on NK cell function. TIM-3 ligand bearing cell lines may also be used to test the ability of antibody-stimulated NK cells to mediate cell cytotoxicity. Furthermore, TIM-3 and/or TIM-3 ligand blocking antibodies may be used to restore and/or potentiate NK cell-mediated cytotoxicity. In order to study the tolerogenic effects of TIM-3 upregulation on NK cells, one can assess how other lymphocyte populations (i.e., CD8⁺ T cells) respond to antibody-stimulated NK cells. As a second part of this embodiment, one can determine the NK cell phenotype from samples obtained from cancer patients undergoing mAb therapy. These studies can provide the foundation for clinical relevance and justify that blocking TIM-3 on NK cells may restore and/or potentiate NK cell responses thereby potentially improving clinical responses.

In Embodiment 3A, redirected ADCC assays are performed to determine the suppressive effects of TIM-3 signaling on NK cell function. In Embodiment 3B, TIM-3 ligand bearing cell lines are also used to test the ability of antibody-stimulated NK cells to mediate cell cytotoxicity. TIM-3 blocking and/or TIM-3 ligand blocking antibodies are used to restore and/or potentiate NK cell-mediated cytotoxicity. In Embodiment 3C, to examine the tolerogenic effects of TIM-3 upregulation on NK cells, one can determine how other lymphocyte populations (i.e., CD8⁺ T cells) respond to antibody-stimulated NK cells. In Embodiment 3D, one can determine the NK cell phenotype from samples obtained from cancer patients undergoing mAb therapy. These studies show that blocking TIM-3 on NK cells restores and/or potentiates NK cell responses thereby improving clinical responses.

The studies in Embodiment 3 are significant. Several lines of evidence suggest that TIM- 3 is involved in regulating the immune response. TIM-3 signaling has been implicated in suppressing effector cell function and in the induction of peripheral tolerance. It was discovered that upon antibody stimulation, NK cells upregulate TIM-3. Therefore, in one embodiment, one can improve mAb-based therapies by blocking TIM-3 on NK cells. One can determine whether TIM-3 signaling on antibody-activated NK cells leads to its functional suppression. Furthermore, blocking the interaction between TIM-3 and its ligands potentiates NK cell responses, in specific embodiment. In addition, one can characterize the tolerogenic effects of antibody-stimulated NK cells on bystander effector populations. Lastly, a clinical correlate can further support therapeutically targeting TIM-3 on NK cells during mAb therapy as a means to improve clinical responses.

Embodiment 3 A. To test the suppressive effect of TIM-3 signaling on NK cell function.

Redirected ADCC assays may be performed using resting and G045 -stimulated NK cells. mFcyR+ P815 cells, a mouse mastocytoma cell line, may be used as targets. In conventional ADCC assays, the experimental Ab opsonizes the target cell through Fab-mediated engagement of a specific target cell epitope leaving the exposed Fc domains to engage and crosslink FcyRs on NK cells. However, in redirected ADCC assays, the experimental Ab opsonizes the target cell via Fc-mediated engagement of the mFcyRs expressed on the target cell leaving the Fab domains exposed. Depending on the specificity of the experimental Ab used, this would allow

engagement and subsequent crosslinking of a desired protein on the surface of NK cells.

Nevertheless, both conventional and redirected ADCC assays are used to assess the cytolytic potential of NK cells. In this study, anti-CD16 may be added in the presence or absence of anti- TIM-3. A standard chromium release assay may be used to measure target cell lysis. In certain embodiments, NK cells cultured in the presence of P815, anti-CD16 and anti- TIM-3 will not be able to mount a redirected ADCC response as high as NK cells cultured with P815 and anti-CD16. Moreover, in at least some cases G045 -stimulated NK cells are less responsive after TIM-3 crosslinking compared to resting NK cells. The parameters for this assay may be worked out using standard practices, e.g. Effector: Target ratio, concentration of experimental antibodies, etc. The inventors have been able to successfully achieve a redirected ADCC response against P815 using an anti-CD 16 antibody and a natural cytotoxicity response against K562 cells using resting and G045 -stimulated NK cells (Figure 11).

Embodiment 3B. To test the restoration of NK cell function by blocking TIM-3:TIM-3 ligand interactions.

Conventional ADCC assays may be performed against an EGFR⁺ HT29 colorectral carcinoma cell line. In addition to EGFR expression, HT29 cells also express Ceacam-1, a TIM- 3 ligand (Figure 12). Freshly isolated NK cells may be stimulated with G045. After stimulation, NK cells may be cocultured with HT29 cells and anti-EGFR-mediated ADCC may be evaluated. In some ADCC experiments, one can block the interaction between TIM-3 and its ligand by using either anti-TIM-3 or anti-Ceacam-1.

In specific embodiments, resting NK cells will have better ADCC responses compared to G045 stimulated NK cells due to differences in TIM-3 expression. Furthermore, blocking the interaction between TIM-3 and its ligand, can potentiate ADCC responses.

Embodiment 3C. To test the tolerogenic effect of TIM-3 upregulation on antibody- stimulated NK cells.

For this tolerance study, NK and CD8⁺ T cells were isolated from the same donor. CD8⁺ T cells were loaded with CFSE and activated with either CD3/CD28 beads or PHA. Once activated, they were co-cultured with either resting or G045-stimulated NK cells. Cells can be collected at different time points and T cell proliferation can be measured by flow cytometry.

In specific embodiments, the proliferation of activated T cells can be suppressed by the presence of TIM-3⁺ NK cells (Figure 13). Moreover, inhibition of proliferation can be more pronounced when activated T cells are cultured with G045-stimulated NK cells compared to resting NK cells. Embodiment 3D. To characterize TIM-3 on NK cells from cancer patients undergoing mAb therapy.

Blood samples can be collected from cancer patients receiving mAb therapy at four different time points (Figure 14). NK cells may be isolated and the expression of TIM-3 may be quantified by flow cytometry. Baseline TIM-3 levels may be acquired from the sample collected prior to mAb administration.

In specific embodiments, TIM-3 can become upregulated on NK cells from patients undergoing antibody therapy. While the upregulation of TIM-3 may not be evident immediately following mAb therapy, an upregulation is more likely be observed after about 2 days to about 2 weeks post-treatment.

There has been much deliberation over mechanisms contributing to the failure of mAb therapies. The data presented herein are consistent with the explanation that these outcomes are due to the induction of regulatory mechanisms that diminish or turn off inflammation. As NK cells become activated by antibody, they must have a way to turn off this activation in order to return back to a state of immune homeostasis. Although not wishing to be bound by any particular theory, the data disclosed herein are consistent with the explanation that: (1) Fc:FcyR interactions on NK cells trigger NK cell activation and cell lysis by ADCC, (2) however, ensuing TIM-3 :TIM-3 ligand interactions lead to the activation of a feedback mechanism shutting off pro-inflammatory processes, not only to itself, (3) but also to potential bystander effector cells as well as a result of protracted FcyR signaling (Figure 15).

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed is:

1. A method of inducing the expression of at least one NK cell surface molecule, the method comprising the step of contacting at least one NK cell with at least one Fc aggregate formed ex vivo or in vivo upon binding to a target cell, wherein the at least one NK cell surface molecule is selected from the group consisting of TIM-3, B7-H1, and CCR-7.

2. The method of claim of claim 1, further comprising contacting the at least one NK cell with an effective amount of at least one inhibitor agent that reduces, inhibits, prevents or reverses natural killer (NK) cell-mediated tolerance.

3. The method of claim 2, wherein the at least one inhibitor agent comprises at least one selected from the group consisting of an inhibitor of TIM-3, an inhibitor of a TIM-3 ligand, an inhibitor of B7-H1, an inhibitor of a B7-H1 ligand, an inhibitor of CCR7, and an inhibitor of a CCR7 ligand.

4. The method of claim 3, wherein the at least one inhibitor agent comprises an inhibitor of a TIM-3 ligand and the TIM-3 ligand is at least one selected from the group consisting of ceacam-1 and galectin-9.

5. The method of claim 3, wherein the at least one inhibitor agent comprises an inhibitor of a B7-H1 ligand and the B7-H1 ligand is at least one selected from the group consisting of PD-1 and CD-80.

6. The method of claim 3, wherein the at least one inhibitor agent comprises an inhibitor of a CCR7 ligand and the CCR7 ligand is at least one selected from the group consisting of CCL19 and CCL21.

7. The method of claim 2, wherein the at least one inhibitor agent is at least one selected from the group consisting of a protein, peptide, peptidomemetic, antibody, ribozyme, small molecule chemical compound, a nucleic acid (DNA or RNA), and an antisense nucleic acid molecule.

8. The method of claim of claim 1, further comprising contacting the at least one NK cell with an effective amount of at least one activator agent that initiates, increases or enhances NK cell-mediated tolerance.

9. The method of claim 8, wherein the at least one activator agent comprises at least one selected from the group consisting of an activator of TIM-3, an activator of a TIM-3 ligand, an activator of B7-H1, an activator of a B7-H1 ligand, an activator of CCR7, an activator of a CCR7 ligand, and an activator of CD 16.

10. The method of claim 9, wherein the at least one activator agent comprises an activator of a TIM-3 ligand and the TIM-3 ligand is at least one selected from the group consisting of ceacam-1 and galectin-9.

11. The method of claim 9, wherein the at least one activator agent comprises an activator of a B7-H1 ligand and the B7-H1 ligand is at least one selected from the group consisting of PD-1 and CD-80.

12. The method of claim 9, wherein the at least one activator agent comprises an activator of a CCR7 ligand and the CCR7 ligand is at least one selected from the group consisting of CCL19 and CCL21.

13. The method of claim 8, wherein the at least one activator agent is at least one selected from the group consisting of a protein, peptide, peptidomemetic, antibody, ribozyme, small molecule chemical compound, a nucleic acid (DNA or RNA), and an antisense nucleic acid molecule.

14. A method of diminishing natural killer (NK) cell-mediated tolerance in a subject in need thereof, comprising the step of administering to the subject an effective amount of at least one inhibitor agent that reduces, inhibits, prevents or reverses natural killer (NK) cell- mediated tolerance.

15. The method of claim 14, wherein the at least one inhibitor agent comprises at least one selected from the group consisting of an inhibitor of TIM-3, an inhibitor of a TIM-3 ligand, an inhibitor of B7-H1, an inhibitor of a B7-H1 ligand, an inhibitor of CCR7, and an inhibitor of a CCR7 ligand.

16. The method of claim 15, wherein the at least one inhibitor agent comprises an inhibitor of a TIM-3 ligand and the TIM-3 ligand is at least one selected from the group consisting of ceacam-1 and galectin-9.

17. The method of claim 15, wherein the at least one inhibitor agent comprises an inhibitor of a B7-H1 ligand and the B7-H1 ligand is at least one selected from the group consisting of PD-1 and CD-80.

18. The method of claim 15, wherein the at least one inhibitor agent comprises an inhibitor of a CCR7 ligand and the CCR7 ligand is at least one selected from the group consisting of CCL19 and CCL21.

19. The method of claim 15, wherein the at least one inhibitor agent is at least one selected from the group consisting of a protein, peptide, peptidomemetic, antibody, ribozyme, small molecule chemical compound, a nucleic acid (DNA or RNA), and an antisense nucleic acid molecule.

20. A method of enhancing antibody therapy in a subject in need thereof, comprising the step of administering to the subject an effective amount of at least one inhibitor agent that reduces, inhibits, prevents or reverses natural killer (NK) cell-mediated tolerance.

21. The method of claim 20, wherein the at least one inhibitor agent comprises at least one selected from the group consisting of an inhibitor of TIM-3, an inhibitor of a TIM-3 ligand, an inhibitor of B7-H1, an inhibitor of a B7-H1 ligand, an inhibitor of CCR7, and an inhibitor of a CCR7 ligand.

22. The method of claim 21, wherein the at least one inhibitor agent comprises an inhibitor of a TIM-3 ligand and the TIM-3 ligand is at least one selected from the group consisting of ceacam-1 and galectin-9.

23. The method of claim 21, wherein the at least one inhibitor agent comprises an inhibitor of a B7-H1 ligand and the B7-H1 ligand is at least one selected from the group consisting of PD-1 and CD-80.

24. The method of claim 21, wherein the at least one inhibitor agent comprises an inhibitor of a CCR7 ligand and the CCR7 ligand is at least one selected from the group consisting of CCL19 and CCL21.

25. The method of claim 20, wherein the at least one inhibitor agent is at least one selected from the group consisting of a protein, peptide, peptidomemetic, antibody, ribozyme, small molecule chemical compound, a nucleic acid (DNA or RNA), and an antisense nucleic acid molecule.

26. The method of claim 21, wherein the antibody therapy is for at least one disease or disorder selected from the group consisting of cancer, cardiovascular disease, macular degeneration, osteoporosis, Alzheimer's disease, dyslipidemia, hypercholesterolemia, asthma, thrombotic thrombocytopenic purpura, organ transplant rejection, graft versus host disease, osteoporosis, scleroderma, and muscular dystrophy.

27. The method of claim 21, wherein the at least one inhibitor agent that reduces, inhibits, reverses or prevents NK cell-mediated tolerance is given to the subject in at least one time period selected from the group consisting of before antibody administration, during antibody administration, and after antibody administration.

28. The method of claim 12, wherein the disease or disorder is cancer and the individual is administered an additional treatment for cancer.

29. The method of claim 18, wherein the additional treatment for cancer comprises at least one selected from the group consisting of surgery, radiation, chemotherapy, hormone therapy, and immunotherapy.

30. A method of treating a disease or disorder with an antibody, comprising the steps of: a) administering to a subject in need thereof an effective amount of the antibody; and b) administering to the subject an effective amount of at least one inhibitor agent that reduces, inhibits, reverses or prevents NK cell-mediated

tolerance.

31. The method of claim 30, wherein the at least one inhibitor agent comprises at least one selected from the group consisting of an inhibitor of TIM-3, an inhibitor of a TIM-3 ligand, an inhibitor of B7-H1, an inhibitor of a B7-H1 ligand, an inhibitor of CCR7, and an inhibitor of a CCR7 ligand.

32. The method of claim 31, wherein the at least one inhibitor agent comprises an inhibitor of a TIM-3 ligand and the TIM-3 ligand is at least one selected from the group consisting of ceacam-1 and galectin-9.

33. The method of claim 31, wherein the at least one inhibitor agent comprises an inhibitor of a B7-H1 ligand and the B7-H1 ligand is at least one selected from the group consisting of PD-1 and CD-80.

34. The method of claim 31, wherein the at least one inhibitor agent comprises an inhibitor of a CCR7 ligand and the CCR7 ligand is at least one selected from the group consisting of CCL19 and CCL21.

35. The method of claim 30, wherein the at least one inhibitor agent is at least one selected from the group consisting of a protein, peptide, peptidomemetic, antibody, ribozyme, small molecule chemical compound, a nucleic acid (DNA or RNA), and an antisense nucleic acid molecule.

36. The method of claim 30, wherein the antibody therapy is for at least one disease or disorder selected from the group consisting of cancer, cardiovascular disease, macular degeneration, osteoporosis, Alzheimer's disease, dyslipidemia, hypercholesterolemia, asthma, thrombotic thrombocytopenic purpura, organ transplant rejection, graft versus host disease, osteoporosis, scleroderma, and muscular dystrophy.

37. The method of claim 30, wherein the at least one inhibitor agent that reduces, inhibits, reverses or prevents NK cell-mediated tolerance is given to the subject in at least one time period selected from the group consisting of before antibody administration, during antibody administration, and after antibody administration.

38. The method of claim 30, wherein the disease or disorder is cancer and the individual is administered an additional treatment for cancer.

39. The method of claim 38, wherein the additional treatment for cancer comprises at least one selected from the group consisting of surgery, radiation, chemotherapy, hormone therapy, and immunotherapy.

40. A method of initiating, increasing or enhancing NK cell-mediated tolerance in a subject in need thereof, comprising the step of administering to the subject an effective amount of at least one activator agent that initiates, increases or enhances NK cell- mediated tolerance.

41. The method of claim 40, wherein the at least one activator agent comprises at least one selected from the group consisting of an activator of TIM-3, an activator of a TIM-3 ligand, an activator of B7-H1, an activator of a B7-H1 ligand, an activator of CCR7, an activator of a CCR7 ligand, and an activator of CD 16.

42. The method of claim 40, wherein the at least one activator agent comprises an activator of a TIM-3 ligand and the TIM-3 ligand is at least one selected from the group consisting of ceacam-1 and galectin-9.

43. The method of claim 40, wherein the at least one activator agent comprises an activator of a B7-H1 ligand and the B7-H1 ligand is at least one selected from the group consisting of PD-1 and CD-80.

44. The method of claim 40, wherein the at least one activator agent comprises an activator of a CCR7 ligand and the CCR7 ligand is at least one selected from the group consisting of CCL19 and CCL21.

45. The method of claim 40, wherein the at least one activator agent is at least one selected from the group consisting of a protein, peptide, peptidomemetic, antibody, ribozyme, small molecule chemical compound, a nucleic acid (DNA or RNA), and an antisense nucleic acid molecule.

46. The method of claim 40, wherein the subject has a disease or disorder.

47. The method of claim 46, wherein the disease or disorder is a least one selected from the group consisting of an autoimmune disease or disorder, an inflammatory disease or disorder, or an organ, tissue or cell transplantation.

48. The method of claim 46, wherein the disease or disorder is at least one selected from the group consisting of Addison's disease, diabetes mellitus Type 1, Graves' disease, endometriosis, celiac disease, Crohn's disease, colitis, rheumatoid arthritis, fibromyalgia, Myasthenia gravis, multiple sclerosis, allergic asthma, pelvic inflammatory disease, allergy, inflammatory bowel disease, ulcerative colitis, cardiovascular disease, macular degeneration, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, psoriasis, plaque psoriasis, ankylosing spondylitis, juvenile idiopathic arthritis, multiple sclerosis, asthma, thrombotic thrombocytopenic purpura, organ transplant rejection, tissue transplant rejection, cell transplant rejection, graft versus host disease, osteoporosis, and scleroderma.

49. A composition, comprising: a) at least one inhibitor agent that reduces, inhibits, reverses or prevents NK cell-mediated tolerance; and b) a pharmaceutically acceptable carrier.

50. The composition of claim 49, wherein the at least one agent is at least one selected from the group consisting of an inhibitor of TIM-3, an inhibitor of a TIM-3 ligand, an inhibitor of B7-H1, an inhibitor of a B7-H1 ligand, an inhibitor of CCR7, and an inhibitor of a CCR7 ligand.

51. The composition of claim 49, wherein the at least one inhibitor agent is at least one selected from the group consisting of a protein, peptide, peptidomemetic, antibody, ribozyme, small molecule chemical compound, a nucleic acid (DNA or RNA), and an antisense nucleic acid molecule.

52. A composition, comprising: a) at least one activator agent that initiates, increases or enhances NK cell-mediated tolerance; and b) a pharmaceutically acceptable carrier.

53. The composition of claim 52, wherein the at least one activator agent comprises at least one selected from the group consisting of an activator of TIM-3, an activator of a TIM-3 ligand, an activator of B7-H1, an activator of a B7-H1 ligand, an activator of CCR7, an activator of a CCR7 ligand, and an activator of CD 16.

54. The composition of claim 52, wherein the at least one activator agent is at least one selected from the group consisting of a protein, peptide, peptidomemetic, antibody, ribozyme, small molecule chemical compound, a nucleic acid (DNA or RNA), and an antisense nucleic acid molecule.

55. A kit comprising the composition of any one of claims 49-54.