METHODS AND COMPOSITIONS FOR TREATMENT OF IMMUNE-RELATED DISEASES OR DISORDERS AND/OR THERAPY MONITORING

FIELD OF THE INVENTION

The present invention relates to molecular immunology and cell biology. More specifically, various aspects of the present embodiments provide for methods and compositions for treatment of immune-related diseases or disorders and/or therapy monitoring. In some embodiments, the methods and compositions described herein are directed to treatment and/or therapy monitoring of cancer. In some embodiments, the methods and compositions described herein are directed to treatment and/or therapy monitoring of inflammatory diseases such as infections, allergy, asthma, autoimmune diseases and/or inflammation. Methods for identifying patients who are more likely to be responsive to and benefit from an immunotherapy are also described herein.

BACKGROUND

The immune system protects the body from foreign invaders and diseased cells; but immune disorders, particularly those associated with T-cell tolerance, such as cancers, can wreak havoc. According to the most recent data from the World Health Organization, ten million people around the world were diagnosed with cancer in 2000, and six million died from it. Moreover, statistics indicate that the cancer incidence rate is on the rise around the globe. In America, for example, projections suggest that fifty percent of those alive today will be diagnosed with some form of cancer at some point in their lives.

T-cell tolerance is also implicated in immune suppression that can be desirable, for example, in autoimmune diseases and in organ transplant situations, wherein an overactive immune response can cause great permanent damage to the afflicted individual and or donor organ. More specifically, autoimmune disorders are caused by dysfunctional immune responses directed against the body's own tissues, resulting in chronic, multisystem impairments that differ in clinical manifestations, course, and outcome. Autoimmune diseases are on the rise in the U.S. and around the world. In the U.S. alone, some fifty million are affected, and autoimmune disease is one of the top ten causes of death in women under the age of 65, is the second highest cause of chronic illness, and the top cause of morbidity in women.

Hence, there remains an urgent need for compositions and approaches to treating immune-related disorders or T-cell tolerance mediated immune disorders.

SUMMARY

Embodiments of various aspects described herein are, in part, based on the discovery that TIGIT (T cell Ig and ITIM domain) expression defines a functionally distinct subset of regulatory T cells (Tregs) that selectively suppress pro-inflammatory Th1 and Th17 responses but spare or promote anti-inflammatory Th2 response by inducing the secretion of the soluble effector molecule Fgl2. Further, the inventors discovered that TIGIT+Treg cells can be induced and/or expanded by IL-33. The inventors have also discovered that tumors express TIGIT ligands such as CD112 and CD155, which can induce tumor immune evasion where TIGIT+Tregs infiltrate the tumors and induce suppression of Th1 and/or Th17 responses. Thus, not only can agents that modulate the activity and/or expression of TIGIT, Fgl2, and/or IL-33 be used for treatment of immune related diseases or disorders such as autoimmune disease, infection, chronic inflammation, cancer, asthma, and allergy, but TIGIT, Fgl2 and/or IL-33 can also be used as predictive markers to identify subjects who are more likely to benefit from an immunotherapy that selectively modulates T cell response (e.g., stimulating or suppressing Th1 and/or Th17 responses), e.g., by targeting TIGIT, Fgl2 and/or IL-33. Accordingly, various aspects described herein provide for methods of identifying subjects with an immune-related disease or disorder who are more likely to be responsive to an immunotherapy or a therapy that targets TIGIT, Fgl2 and/or IL-33, as well as monitoring the treatment efficacy. Methods and compositions for treating subjects with an immune-related disease or disorder are also provided herein.

In some immune-related diseases or disorders, e.g., but not limited to cancer and/or infections (e.g., chronic viral infection, intracellular bacterial infection, extracellular bacterial infection, and/or fungal infection), it can be desirable to induce proinflammatory Th1 and/or Th17 responses for a therapeutic effect. Accordingly, these immune-related diseases or disorders can be treated by inhibiting or reducing the expression and/or activity of TIGIT, Fgl2 and/or IL-33.

The TIGIT axis suppresses proinflammatory responses, e.g., via suppression of Th1 and/or Th17 mediated responses. As defined herein, the “TIGIT axis” refers to an immunosuppressive pathway including TIGIT and Fgl2. The inventors have also discovered that TIGIT induces expression and/or activity of a transcription factor CEBPα, which in turn induces Fgl2 expression. Accordingly, in some embodiments, the TIGIT axis can further include CEBPα, and thus the “TIGIT axis” refers to an immunosuppressive pathway including TIGIT, CEBPα, and Fgl2. In some embodiments, the TIGIT axis further includes IL-33, where IL-33 induces or expands the TIGIT+T cells such as TIGIT+Tregs, and thus the “TIGIT axis” refers to an immunosuppressive pathway including IL-33, TIGIT, and Fgl2. IL-33 induces TIGIT expression and/or increases the TIGIT+regulatory T cells (Tregs), where TIGIT induces transcription and secretion of the effector molecule Fgl2 in Tregs, thus resulting in suppression of pro-inflammatory Th1 and/or Th17 cells but not Th2 response.

Accordingly, in some aspects, provided herein are methods of identifying a patient who is diagnosed with cancer and/or infection, and is more likely to be responsive to an anti-TIGIT, anti-Fgl2 and/or anti-IL-33 therapy (or less likely to respond to an intervention designed to stimulate Th1 and/or Th17 cells on its own). Patients whose tumors and/or cells (including, e.g., normal cells and/or diseased cells such as infected cells) express TIGIT ligands (e.g., CD112 and/or CD155), Fgl2 and/or IL-33 would likely have a greater population of TIGIT+, Fgl2+, Fgl2 receptor+ and/or IL-33 receptor+T cells infiltrated therein and thus suppress activity or activation of Th1 and/or Th17 cells. Accordingly, these patients would not be expected to respond effectively to an anti-tumor and/or anti-infection therapy designed to stimulate Th1 and/or Th17 cells on its own, as relative to patients whose tumors and/or tissues (including, e.g., normal tissues or diseased tissues such as infected tissues) (i) lack or express low levels of TIGIT ligands (e.g., CD112 and/or CD155), Fgl2 and/or IL-33; and/or (ii) lack or are low in T cells with these markers. The same patients with (i) tumors and/or cells (including, e.g., normal cells and/or diseased cells such as infected cells) expressing TIGIT ligands (e.g., CD112 and/or CD155), Fgl2 and/or IL-33; and/or (ii) TIGIT+, Fgl2+, Fgl2 receptor+ and/or IL-33 receptor+ tumor or tissue (including, e.g., normal tissue or diseased tissue such as infected tissue) T cells would, however, more likely benefit from anti-TIGIT, anti-Fgl2, and/or anti-IL-33 therapy, which would likely permit either spontaneous Th1 or Th17 responses, or Th1 and/or Th17 responses induced via coadministered agents that stimulate Th1 and/or Th17 cells.

In some embodiments, the patient diagnosed with an infection can have chronic viral infection, intracellular bacterial infection, extracellular bacterial infection, and/or fungal infection.

In some embodiments, patients diagnosed with cancer and/or infection can be identified as more likely to be responsive to an anti-TIGIT and/or anti-IL-33 therapy based on the patients' level of Fgl2 activity or expression in a sample. In some embodiments, patients diagnosed with cancer and/or infection can be identified as more likely to be responsive to an anti-IL-33 therapy and/or anti-Fgl2 therapy based on the patients' level of TIGIT or its ligand (e.g., CD112 and/or CD155) activity or expression in a sample. In some embodiments, patients diagnosed with cancer and/or infection can be identified as more likely to be responsive to an anti-TIGIT and/or anti-Fgl2 therapy based on the patients' level of IL-33 activity or expression in a sample.

By way of example only, some aspects provided herein relate to methods of identifying a patient who is diagnosed with cancer and/or infection and is more likely to be responsive to an anti-TIGIT and/or anti-IL-33 therapy (or less likely to respond to an intervention designed to stimulate Th1 and/or Th17 cells alone), based on the level of Fgl2 activity or expression in the patient's sample. The method comprises (a) measuring the level of Fgl2 activity or expression in a sample from a patient diagnosed with cancer and/or infection; and (b) comparing the level of Fgl2 or expression in the sample with an Fgl2 reference; and: (i) when the level of Fgl2 activity or expression is greater than the Fgl2 reference, the patient is identified to be more likely to be responsive to an anti-TIGIT and/or anti-IL-33 therapy, and less likely to respond to Th1 and/or Th17 stimulation without anti-TIGIT, anti-Fgl2 or anti-IL-33 treatment; or (ii) when the level of Fgl2 activity or expression is the same as or less than the Fgl2 reference, the patient is identified as likely to respond to an alternative, proinflammatory immunotherapy comprising an activator of a proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.

In some embodiments, the patient diagnosed with an infection can have chronic viral infection, intracellular bacterial infection, extracellular bacterial infection, and/or fungal infection.

In this aspect and other aspects described herein, any appropriate modulators of a T cell response pathway that are known in the art can be used in the alternative immunotherapy for patients diagnosed with cancer and/or infection. For example, activators of a proinflammatory T cell response or suppressors of an anti-inflammatory T-cell response pathway can comprise a TIM-3 inhibitor, an anti-galectin-9 molecule, a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a Lag-3 antagonist, a DD1αantagonist, an agonist of an immune checkpoint activating molecule, an antagonist of an immune checkpoint inhibitory molecule, or any combination thereof.

Some aspects provided herein relate to methods of treating a patient diagnosed with cancer and/or infection. An anti-TIGIT, anti-Fgl2 and/or anti-IL33 therapy can be selected for administration to a patient diagnosed with cancer and/or infection, based on the level of Fgl2, IL-33 and/or TIGIT in the patient's sample. In some embodiments, an anti-TIGIT and/or anti-IL-33 therapy can be selected for administration to a patient diagnosed with cancer and/or infection, based on the patient's level of Fgl2 activity or expression in a sample. In some embodiments, an anti-IL-33 therapy and/or anti-Fgl2 therapy can be selected for administration to a patient diagnosed with cancer and/or infection, based on the patient's level of TIGIT activity or expression in a sample. In some embodiments, an anti-TIGIT and/or anti-Fgl2 therapy can be selected for administration to a patient diagnosed with cancer and/or infection, based on the patient's level of IL-33 activity or expression in a sample. In some embodiments, the patient diagnosed with an infection can have chronic viral infection, intracellular bacterial infection, extracellular bacterial infection, and/or fungal infection.

For example, in one aspect, provided herein is a method for treating a patient diagnosed with cancer and/or infection, wherein the method comprises (a) measuring the level of IL-33 activity or expression in a sample from a patient diagnosed with cancer and/or infection; (b) comparing the level of IL-33 activity or expression in the sample with an IL-33 reference, and (c) performing one of the following actions:

- (i) administering to the patient a composition comprising a TIGIT inhibitor and/or an Fgl2 inhibitor, when the level of IL-33 activity or expression is greater than the IL-33 reference;
- (ii) administering an alternative, proinflammatory immunotherapy treatment without the TIGIT inhibitor or Fgl2 inhibitor, when the level of IL-33 activity or expression is the same as or less than the IL-33 reference; or
- (iii) determining if the level of at least one other inhibitory immune regulator in the sample is greater than the level of the corresponding reference, or if the level of at least one activating immune regulator in the sample is less than the level of the corresponding reference, when the level of IL-33 activity or expression is the same as or less than the IL-33 reference.

Examples of inhibitory immune regulator include, but are not limited to Fgl2, TIGIT, ST2, CD155, CD112, PD-1, PD-L1, DD1α, TIM-3, galectin-9, CTLA-4, Lag-3, and any combination thereof.

In accordance with this aspect and other aspects described herein, a TIGIT inhibitor is an agent that directly or indirectly inhibits or reduces the TIGIT-mediated suppression of proinflammatory Th1 and/or Th17 responses. Accordingly, a TIGIT inhibitor can target the TIGIT receptor or its corresponding ligand, or any of TIGIT's upstream molecules. Examples of TIGIT inhibitors include, without limitations, TIGIT−/− immune cells (e.g., T cells), anti-TIGIT molecules, ST2 inhibitors, CD112 inhibitors, CD155 inhibitors, and a combination thereof. A TIGIT inhibitor can be a protein, a peptide, a peptidomimetic, an aptamer, a nucleic acid, an antibody, a small molecule, a vaccine, or any combination thereof. In one embodiment, a TIGIT inhibitor directly binds to TIGIT and inhibits TIGIT-mediated activation of Fgl2 expression or activity. In one embodiment, a TIGIT inhibitor can directly bind to a TIGIT ligand (e.g., CD112 and/or CD155) and inhibit the TIGIT ligand from binding to TIGIT to induce TIGIT-mediated activation of Fgl2 expression or activity. For example, a TIGIT inhibitor can be a soluble TIGIT molecule (e.g., without a transmembrane domain) that binds to a TIGIT ligand (e.g., CD112 and/or CD155), thereby decreasing the concentration of the TIGIT ligand that is available for binding and activating TIGIT, which in turn induces Fgl2 expression or activity.

In accordance with this aspect and other aspects described herein, an Fgl2 inhibitor is an agent that directly or indirectly reduces the expression/secretion and/or activity of Fgl2. Accordingly, an Fgl2 inhibitor can target Fgl2 molecule or its corresponding receptors. Examples of Fgl2 inhibitors include, but are not limited to Fgl2 neutralizing agents, TIGIT inhibitors, and/or ST2 inhibitors. The Fgl2 inhibitors can be a protein, a peptide, a peptidomimetic, an aptamer, a nucleic acid, an antibody, a small molecule, a vaccine, or any combination thereof. In one embodiment, an Fgl2 inhibitor directly binds to Fgl2 and inhibits Fgl2-mediated immunosuppression (e.g., suppression of T cell proliferation and/or production of proinflammatory cytokines, and/or induction of anti-inflammatory cytokines). In one embodiment, an Fgl2 inhibitor can bind to TIGIT or a TIGIT ligand (e.g., CD112 and/or CD155) and inhibit TIGIT-mediated activation of Fgl2 expression or activity. In some embodiments, an Fgl2 inhibitor can be a TIGIT inhibitor as described herein.

In some embodiments where the level of IL-33 and/or Fgl2 activity or expression is the same as or less than the reference, an alternative, proinflammatory immunotherapy treatment without the TIGIT inhibitor or Fgl2 inhibitor to be administered can be a therapy comprising an activator of a proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.

In another aspect, provided herein are methods of treating a patient diagnosed with cancer and/or infection comprising (a) measuring the level of Fgl2 activity or expression in a sample from a patient diagnosed with cancer and/or infection; (b) comparing the level of Fgl2 activity or expression in the sample with an Fgl2 reference; and (c) administering to the patient a composition comprising a TIGIT inhibitor and/or an IL-33 inhibitor when the level of Fgl2 activity or expression is greater than the Fgl2 reference, or administering an alternative, proinflammatory immunotherapy treatment without a TIGIT inhibitor or IL-33 inhibitor when the level of Fgl2 activity or expression is the same as or less than the Fgl2 reference. In some embodiments, the alternative, proinflammatory immunotherapy treatment without a TIGIT inhibitor or IL-33 inhibitor can be a therapy comprising an activator of a proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.

In some embodiments, the patient with an Fgl2 level greater than the Fgl2 reference can be further administered a therapy comprising an activator of a proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.

In accordance with this aspect and other aspects described herein, an IL-33 inhibitor is an agent that directly or indirectly reduces the expression/secretion and/or activity of IL-33. Accordingly, an IL-33 inhibitor can target IL-33 molecule or its corresponding receptors. Examples of IL-33 inhibitors include, but are not limited to ST2 inhibitors or IL-33 neutralizing agents. An IL-33 inhibitor can be a protein, a peptide, a peptidomimetic, an aptamer, a nucleic acid, an antibody, a small molecule, a vaccine, or any combination thereof. In one embodiment, an IL-33 inhibitor directly binds IL-33 and reduces TIGIT expression and/or proliferation of TIGIT+Tregs. For example, an IL-33 inhibitor can be a soluble ST2 receptor (e.g., without a transmembrane domain) that binds to IL-33, thereby decreasing the concentration of IL-33 that is available for functionally interacting with ST2 receptors present on an immune cell (e.g., T cell) or a TIGIT+Treg.

In some aspects described herein, TIGIT, Fgl2 and/or IL-33 can be used as a marker to determine or monitor the efficacy of an anti-TIGIT, anti-Fgl2 and/or anti-IL-33 therapy administered to a patient diagnosed with cancer and/or infection. In some embodiments, Fgl2 can be used as a predictive marker to determine or monitor the efficacy of an anti-TIGIT and/or anti-IL-33 therapy administered to a patient diagnosed with cancer and/or infection. In some embodiments, TIGIT can be used as a predictive marker to determine or monitor the efficacy of an anti-IL-33 therapy and/or anti-Fgl2 therapy administered to a patient diagnosed with cancer and/or infection. In some embodiments, IL-33 can be used as a predictive marker to determine or monitor the efficacy of an anti-TIGIT and/or anti-Fgl2 therapy administered to a patient diagnosed with cancer and/or infection. In some embodiments, the patient diagnosed with an infection can have chronic viral infection, intracellular bacterial infection, extracellular bacterial infection, and/or fungal infection.

As an example, methods of treating a patient diagnosed with cancer and/or infection that has an elevated level of Fgl2 are provided herein. The method comprises: (a) determining a first level of Fgl2 expression or activity in a sample from a patient diagnosed with cancer and/or infection that exhibits an elevated level of Fgl2; (b) administering an agent that inhibits IL-33 activity and/or TIGIT activity; (c) determining a second level of Fgl2 expression or activity after the administering; and (d) comparing the first and second levels of Fgl2 expression or activity, wherein the agent administered in (b) is effective if the second level of Fgl2 expression or activity is lower than the first level, and wherein the agent administered in (b) is ineffective if the second level of Fgl2 expression is the same as or higher than the first level.

By monitoring the effects of the anti-IL-33 and/or anti-TIGIT therapy on the level of Fgl2 expression or activity, one can determine the efficacy of the treatment regimen and adjust the treatment regimen if necessary. Accordingly, in some embodiments, the method can further comprise, when the anti-IL-33 or anti-TIGIT therapy is effective, continuing to administer the agent that inhibits IL-33 activity and/or TIGIT activity. In some embodiments, the method can further comprise, when the anti-IL-33 therapy or the anti-TIGIT therapy is ineffective, administering the agent that inhibits IL-33 activity and/or TIGIT activity at a higher dose. In some embodiments, the method can further comprise, when the anti-IL-33 therapy or the anti-TIGIT therapy is ineffective, discontinuing the anti-IL-33 therapy or the anti-TIGIT therapy. In these embodiments, the method can further comprise administering a therapy comprising an activator of a proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.

Similarly, further aspects provided herein relate to methods of treating a patient diagnosed with cancer and/or infection that exhibits an elevated level of IL-33. The method comprises: (a) determining a first level of TIGIT and/or Fgl2 expression or activity in a sample from a patient diagnosed with cancer and/or infection that has an elevated level of IL-33; (b) administering an agent that inhibits IL-33 activity; (c) determining a second level of TIGIT and/or Fgl2 expression or activity after the administering; and (d) comparing the first and second levels of TIGIT and/or Fgl2 expression or activity, wherein anti-IL-33 therapy is effective if the second level of TIGIT and/or Fgl2 expression or activity is lower than the first level, and wherein anti-IL-33 therapy is ineffective if the second level of TIGIT and/or Fgl2 expression or activity is the same as or higher than the first level.

In some embodiments, the method can further comprise, when the anti-IL-33 therapy is effective, continuing to administer the agent that inhibits IL-33 activity. In some embodiments, the method can further comprise, when the anti-IL-33 therapy is ineffective, administering the agent that inhibits IL-33 activity at a higher dose. In other embodiments, the method can further comprise, when the anti-IL-33 therapy is ineffective, discontinuing the anti-IL-33 therapy. In these embodiments, the method can further comprise administering a therapy comprising an activator of a proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.

Pharmaceutical compositions for treatment of cancer and/or infections are also provided herein. More specifically, a pharmaceutical composition for the treatment of cancer and/or infections can comprise a pharmaceutically-acceptable excipient and at least one of the following therapeutic agents: (a) a TIGIT inhibitor; (b) an IL-33 inhibitor; (c) an ST2 inhibitor; and (d) an Fgl2 inhibitor. In some embodiments, a pharmaceutical composition can comprise a pharmaceutically-acceptable excipient and at least two of the following therapeutic agents: (a) a TIGIT inhibitor; (b) an IL-33 inhibitor; (c) an ST2 inhibitor; and (d) an Fgl2 inhibitor. For example, in some embodiments, the composition can comprise a TIGIT inhibitor and an IL-33 inhibitor, or a TIGIT inhibitor and an ST2 inhibitor. In some embodiments, the composition can comprise a TIGIT inhibitor and an Fgl2 inhibitor. In some embodiments, the composition can comprise an IL-33 inhibitor and an Fgl2 inhibitor, or an ST2 inhibitor and an Fgl2 inhibitor. In some embodiments, the composition can comprise a TIGIT inhibitor, an IL-33 inhibitor and/or an ST2 inhibitor, and an Fgl2 inhibitor.

In some embodiments, the pharmaceutical composition can further comprise an anti-cancer agent and/or anti-infection agent. Examples of an anti-cancer agent include, but are not limited to vaccine, chemotherapy, targeted therapy (e.g., kinase inhibitors), radiation therapy, surgery, immunotherapy, and any combinations thereof. An anti-infection agent can be an agent that kills or inhibits a cellular process, development and/or replication of a target infectious agent. Examples of an immunotherapy for treatment of cancer and/or infection can comprise an agent that increases a proinflammatory T cell response and/or an agent that suppresses an anti-inflammatory T cell response.

In some embodiments, the pharmaceutical composition can be used for treatment of chronic viral infection, intracellular bacterial infection, extracellular bacterial infection, and/or fungal infection.

In yet another aspect, a method of treating a patient diagnosed with cancer and/or infection comprising administering to a patient diagnosed with cancer and/or infection one or more embodiments of the pharmaceutical compositions described herein is also provided. In some embodiments, the patient diagnosed with an infection can have chronic viral infection, intracellular bacterial infection, extracellular bacterial infection, and/or fungal infection. The pharmaceutical composition can be taken alone or in combination with another anti-cancer agent and/or anti-infection agent. Examples of anti-cancer agents include, but are not limited to vaccine, chemotherapy, targeted therapy (e.g., kinase inhibitors), radiation therapy, surgery, immunotherapy, and any combinations thereof. An anti-infection agent can be an agent that kills or inhibits a cellular process, development and/or replication of a target infectious agent. In some embodiments, the method can further comprise administering the patient an immunotherapy. For example, an immunotherapy for treatment of cancer and/or infections can comprise an agent that increases a proinflammatory T cell response and/or an agent that suppresses an anti-inflammatory T cell response.

In some embodiments of this aspect and other related aspects described herein, the patient diagnosed with cancer and/or infection can be previously treated with or is being treated an anti-cancer therapy and/or an anti-infection therapy. Thus, the anti-TIGIT, anti-Fgl2 and/or anti-IL-33 therapy can be used, alone or in combination with another anti-cancer agent and/or anti-infection agent as described herein or known in the art. In some embodiments of this aspect and other related aspects described herein, the methods described herein can further comprise administering to the patient diagnosed with cancer and/or infection a selected therapy (e.g., anti-TIGIT or anti-IL-33 therapy) after they have been identified to be more likely to benefit from one immunotherapy over another.

In some embodiments of this aspect and other related aspects described herein, the TIGIT inhibitor, Fgl2 inhibitor and/or IL-33 inhibitor administered to a patient diagnosed with cancer and/or infection can be constructed to specifically target TIGIT+regulatory T cells (Tregs) that infiltrate the tumor or tissue (including, e.g., normal tissue or diseased tissue such as infected tissue) expressing TIGIT ligands (e.g., CD112 and/or CD155), Fgl2 and/or IL-33. For example, the TIGIT inhibitor, Fgl2 inhibitor and/or IL-33 inhibitor can comprise a cell-targeting moiety against the tumor, tissue and/or cells (including, e.g., normal cells or diseased cells such as infected cells) expressing TIGIT ligands (e.g., CD112 and/or CD155), Fgl2 and/or IL-33.

T cell exhaustion can generally arise during chronic infections and cancer. It is contemplated that TIGIT signaling can play a role in establishing or maintain T cell exhaustion. A further aspect provided herein relates to a method for increasing the differentiation and/or proliferation of functionally exhausted CD8+T cells, or decreasing CD8+T cell exhaustion, in a subject in need thereof. The method comprises administering to the subject in need thereof a pharmaceutical composition comprising a TIGIT antagonist or inhibitor described herein. These methods can be used to treat chronic infections and/or cancer.

In some other immune-related diseases or disorders, e.g., but not limited to inflammatory diseases or disorders such as chronic inflammation and autoimmune diseases, it can be desirable to suppress proinflammatory Th1 and/or Th17 responses for a therapeutic effect, while sparing or promoting a Th2 response. Accordingly, these immune-related diseases or disorders can be treated by enhancing or stimulating the expression or activity of TIGIT, Fgl2 and/or IL-33.

Accordingly, in some aspects, provided herein are methods of identifying a patient diagnosed to have an inflammatory disease or disorder (e.g., autoimmune disease or disorder or chronic inflammation) who is more likely to be responsive to an anti-inflammatory immunotherapy, or a TIGIT agonist, Fgl2 agonist and/or IL-33 agonist therapy. Nonlimiting examples of an inflammatory disease or disorder that would benefit from an anti-inflammatory immunotherapy include, but are not limited to autoimmune disease, parasitic infection, acute inflammation, chronic inflammation, and any combinations thereof. In some embodiments, patients having an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable can be identified as more likely to be responsive to a TIGIT agonist and/or an IL-33 agonist therapy based on the patients' level of Fgl2 activity or expression in a sample. In some embodiments, patients having an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable can be identified as more likely to be responsive to an IL-33 agonist therapy and/or an Fgl2 agonist therapy based on the patients' level of TIGIT activity or expression in a sample. In some embodiments, patients having an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable can be identified as more likely to be responsive to a TIGIT agonist and/or an Fgl2 agonist therapy based on the patients' level of IL-33 activity or expression in a sample.

By way of example only, some aspects provided herein relate to methods of identifying a patient with an autoimmune disease or disorder and/or parasitic infection, who is more likely to be responsive to an anti-inflammatory immunotherapy, or a TIGIT agonist and/or IL-33 agonist therapy, based on the level of Fgl2 activity or expression in the patient's sample. The method comprises (a) measuring the level of Fgl2 activity or expression in a sample from a patient diagnosed to have an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable (e.g., autoimmune disease or disorder and/or parasitic infection); and (b) comparing the level of Fgl2 or expression in the sample with an Fgl2 reference; and (c) (i) identifying the patient to be more likely to be responsive to a TIGIT agonist and/or IL-33 agonist therapy, when the level of Fgl2 activity or expression is lower than the Fgl2 reference; or (ii) identifying the patient to be likely to respond to an alternative, anti-inflammatory immunotherapy comprising an activator of an anti-inflammatory T cell response pathway and/or a suppressor of a proinflammatory T cell response pathway, when the level of Fgl2 activity or expression is the same as or greater than the Fgl2 reference.

In this aspect and other aspects described herein, any appropriate modulators of a T cell response pathway that are known in the art can be used in the alternative anti-inflammatory immunotherapy for patients with an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable. For example, activators of an anti-inflammatory T cell response or suppressors of a proinflammatory T-cell response pathway can comprise a TIM-3 agonist, a galectin-9 molecule, a PD-1 agonist, a PD-L1 agonist, a CTLA-4 agonist, a Lag-3 agonist, a DD1α agonist, an antagonist of an immune checkpoint activating molecule, an agonist of an immune checkpoint inhibitory molecule, or any combination thereof.

Some aspects provided herein relate to methods of treating a patient who is determined to have an inflammatory disease or disorder. In some embodiments where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable (e.g., autoimmune diseases or disorders and/or parasitic infection), a TIGIT agonist, Fgl2 agonist and/or IL-33 agonist therapy can be selected for administration based on the level of Fgl2, IL-33 and/or TIGIT in the patient's sample. In some embodiments where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable (e.g., autoimmune diseases or disorders and/or parasitic infection), a TIGIT agonist and/or IL-33 agonist therapy can be selected for administration based on the patient's level of Fgl2 activity or expression in a sample. In some embodiments where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable (e.g., autoimmune diseases or disorders and/or parasitic infection), an IL-33 agonist therapy and/or Fgl2 agonist therapy can be selected for administration based on the patient's level of TIGIT activity or expression in a sample. In some embodiments where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable (e.g., autoimmune diseases or disorders and/or parasitic infection), a TIGIT agonist and/or Fgl2 agonist therapy can be selected for administration based on the patient's level of IL-33 activity or expression in a sample.

For example, in one aspect, provided herein is a method for treating a patient who is determined to have an inflammatory disease or disorder based on the level of IL-33 activity or expression. In some embodiments where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable (e.g., autoimmune diseases or disorders and/or parasitic infection), the method can comprise (a) measuring the level of IL-33 activity or expression in a sample from a patient who is determined to have this type of an inflammatory disease or disorder; (b) comparing the level of IL-33 activity or expression in the sample with an IL-33 reference; and (c) performing one of the following actions:

- (i) administering to the patient a composition comprising a TIGIT agonist and/or an Fgl2 agonist, when the level of IL-33 activity or expression is lower than the IL-33 reference;
- (ii) administering an alternative, anti-inflammatory immunotherapy treatment without the TIGIT agonist or Fgl2 agonist, when the level of IL-33 activity or expression is the same as or greater than the IL-33 reference; or
- (iii) determining if the level of at least one other activating immune regulator in the sample is greater than the level of the corresponding reference, or if the level of at least one inhibitory immune regulator in the sample is less than the level of the corresponding reference, when the level of IL-33 activity or expression is the same as or greater than the IL-33 reference. Examples of inhibitory immune regulator include, but are not limited to Fgl2, TIGIT, ST2, CD155, CD112, PD-1, PD-L1, DD1α, TIM-3, galectin-9, CTLA-4, Lag-3, and any combination thereof.

In some embodiments where the level of IL-33 activity or expression is the same as or greater than the IL-33 reference, the method can further comprise (a) measuring the level of Fgl2 activity or expression in a sample from the patient, (b) comparing the level of Fgl2 activity or expression in the sample with an Fgl2 reference, and (c) administering to the patient a composition comprising a TIGIT agonist and/or an Fgl2 agonist, when the level of Fgl2 activity or expression is lower than the Fgl2 reference; or administering an alternative, anti-inflammatory immunotherapy treatment without a TIGIT agonist or Fgl2 agonist, when the level of Fgl2 activity or expression is the same as or greater than the reference.

In accordance with this aspect and other aspects described herein, a TIGIT agonist is an agent that directly or indirectly enhances or stimulates the TIGIT-mediated suppression of proinflammatory Th1 and/or Th17 responses. Accordingly, a TIGIT agonist can target the TIGIT receptor or its corresponding ligand, or any of TIGIT's upstream molecules. Examples of TIGIT agonists include, without limitations, TIGIT-expressing or -overexpressing immune cells (e.g., T cells), ST2 agonists, CD112 agonists, CD155 agonists, and/or a combination thereof. The TIGIT agonists can be a protein, a peptide, peptidomimetic, an aptamer, a nucleic acid, an antibody, a small molecule, a vaccine, or any combinations thereof.

In accordance with this aspect and other aspects described herein, an Fgl2 agonist is an agent that directly or indirectly increases the expression/secretion and/or activity of Fgl2. Accordingly, an Fgl2 agonist can target Fgl2 molecule or its corresponding receptors. Examples of Fgl2 agonists include, but are not limited to, Fgl2-expressing or -overexpressing cells (e.g., T cells), Fgl2 soluble molecules, TIGIT agonists, and/or ST2 agonists. The Fgl2 agonists can be a protein, a peptide, a peptidomimetic, an aptamer, a nucleic acid, an antibody, a small molecule, a vaccine, or any combination thereof.

In some embodiments where the level of IL-33 and/or Fgl2 activity or expression is the same as or greater than the reference, an alternative, anti-inflammatory immunotherapy treatment without the TIGIT agonist or Fgl2 agonist to be administered can be a therapy comprising an activator of an anti-inflammatory T cell response pathway and/or a suppressor of a proinflammatory T cell response pathway.

In another aspect, provided herein are methods of treating a patient determined to have an inflammatory disease or disorder based on the level of Fgl2 activity or expression. In some embodiments where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable (e.g., autoimmune diseases or disorders and/or parasitic infection), the method can comprise (a) measuring the level of Fgl2 activity or expression in a sample from a patient determined to have this type of an inflammatory disease or disorder; (b) comparing the level of Fgl2 activity or expression in the sample with an Fgl2 reference; and (c) administering to the patient a composition comprising a TIGIT agonist and/or an IL-33 agonist when the level of Fgl2 activity or expression is lower than the Fgl2 reference, or administering an alternative, anti-inflammatory immunotherapy treatment without a TIGIT agonist or IL-33 agonist when the level of Fgl2 activity or expression is the same as or greater than the Fgl2 reference. In some embodiments, the alternative, anti-inflammatory immunotherapy treatment without a TIGIT agonist or IL-33 agonist can be a therapy comprising an activator of an anti-inflammatory T cell response pathway and/or a suppressor of a proinflammatory T cell response pathway.

In some embodiments, the patient with an Fgl2 level lower than the Fgl2 reference can be further administered a therapy comprising an activator of an anti-inflammatory T cell response pathway and/or a suppressor of a proinflammatory T cell response pathway.

In accordance with this aspect and other aspects described herein, an IL-33 agonist is an agent that directly or indirectly increases the expression/secretion and/or activity of IL-33. Accordingly, an IL-33 agonist can target IL-33 molecule or its corresponding receptors. Examples of IL-33 agonists include, but are not limited to, ST2 agonists or IL-33 soluble molecules. The IL-33 agonists can be a protein, a peptide, a peptidomimetic, an aptamer, a nucleic acid, an antibody, a small molecule, a vaccine, or any combination thereof.

In some aspects described herein, TIGIT, Fgl2 and/or IL-33 can be used as a marker to determine or monitor the efficacy of a TIGIT agonist, Fgl2 agonist and/or IL-33 agonist therapy administered to a patient diagnosed to have an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable (e.g., autoimmune diseases or disorders and/or parasitic infection). In some embodiments, Fgl2 can be used as a predictive marker to determine or monitor the efficacy of a TIGIT agonist and/or IL-33 agonist therapy administered to a patient diagnosed to have an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable. In some embodiments, TIGIT can be used as a predictive marker to determine or monitor the efficacy of an IL-33 agonist therapy and/or Fgl2 agonist therapy administered to a patient diagnosed to have an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable. In some embodiments, IL-33 can be used as a predictive marker to determine or monitor the efficacy of a TIGIT agonist and/or Fgl2 agonist therapy administered to a patient diagnosed to have an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable.

As an example, methods of treating a patient having an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable (e.g., autoimmune diseases or disorders and/or parasitic infection), and a low level of Fgl2 are provided herein. The method comprises: (a) determining a first level of Fgl2 expression or activity in a sample from a patient having an inflammatory disease or disorder and a low level of Fgl2; (b) administering an agent that activates IL-33 activity and/or TIGIT activity; (c) determining a second level of Fgl2 expression or activity after the administering; and (d) comparing the first and second levels of Fgl2 expression or activity, wherein the agent administered in (b) is effective if the second level of Fgl2 expression or activity is greater than the first level, and wherein the agent administered in (b) is ineffective if the second level of Fgl2 expression is the same as or lower than the first level.

By monitoring the effects of the IL-33 agonist and/or TIGIT agonist therapy on the level of Fgl2 expression or activity, one can determine the efficacy of the treatment regimen and adjust the treatment regimen if necessary. Accordingly, in some embodiments, the method can further comprise, when the IL-33 agonist or TIGIT agonist therapy is effective, continuing to administer the agent that activates IL-33 activity and/or TIGIT activity. In some embodiments, the method can further comprise, when the IL-33 agonist therapy or the TIGIT agonist therapy is ineffective, administering the agent that activates IL-33 activity and/or TIGIT activity at a higher dose. In some embodiments, the method can further comprise, when the IL-33 agonist therapy or the TIGIT agonist therapy is ineffective, discontinuing the IL-33 agonist therapy or the TIGIT agonist therapy. In these embodiments, the method can further comprise administering a therapy comprising an activator of an anti-inflammatory T cell response pathway and/or a suppressor of a proinflammatory T cell response pathway.

Similarly, further aspects provided herein relate to methods of treating a patient having an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable (e.g., autoimmune diseases or disorders and/or parasitic infection) and exhibiting a reduced level of IL-33. The method comprises: (a) determining a first level of TIGIT and/or Fgl2 expression or activity in a sample from a patient having this type of an inflammatory disease or disorder (e.g., autoimmune diseases or disorders and/or parasitic infection) with a reduced level of IL-33; (b) administering an agent that activates IL-33 activity; (c) determining a second level of TIGIT or Fgl2 expression or activity after the administering; and (d) comparing the first and second levels of TIGIT and/or Fgl2 expression or activity, wherein IL-33 agonist therapy is effective if the second level of TIGIT and/or Fgl2 expression or activity is greater that the first level, and wherein IL-33 agonist therapy is ineffective if the second level of TIGIT and/or Fgl2 expression is the same as or lower than the first level.

In some embodiments, the method can further comprise, when the IL-33 agonist therapy is effective, continuing to administer the agent that activates IL-33 activity. In some embodiments, the method can further comprise, when the IL-33 agonist therapy is ineffective, administering the agent that activates IL-33 activity at a higher dose. In other embodiments, the method can further comprise, when the IL-33 agonist therapy is ineffective, discontinuing the IL-33 agonist therapy. In these embodiments, the method can further comprise administering a therapy comprising an activator of an anti-inflammatory T cell response pathway and/or a suppressor of a proinflammatory T cell response pathway.

Pharmaceutical compositions for treatment of inflammatory diseases or disorders are also provided herein. In some embodiments, a pharmaceutical composition for treatment of an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable (e.g., autoimmune diseases or disorders and/or parasitic infection) can comprise a pharmaceutically-acceptable excipient and at least one of the following therapeutic agents: (a) a TIGIT agonist; (b) an IL-33 agonist; (c) an ST2 agonist; and (d) an Fgl2 agonist. In some embodiments, a pharmaceutical composition can comprise a pharmaceutically-acceptable excipient and at least two of the following therapeutic agents: (a) a TIGIT agonist; (b) an IL-33 agonist; (c) an ST2 agonist; and (d) an Fgl2 agonist. For example, in some embodiments, the composition can comprise a TIGIT agonist and an IL-33 agonist, or a TIGIT agonist and an ST2 agonist. In some embodiments, the composition can comprise a TIGIT agonist and an Fgl2 agonist. In some embodiments, the composition can comprise an IL-33 agonist and an Fgl2 agonist, or an ST2 agonist and an Fgl2 agonist. In some embodiments, the composition can comprise a TIGIT agonist, an IL-33 agonist and/or an ST2 agonist, and an Fgl2 agonist.

In some embodiments, the pharmaceutical composition can further comprise an additional agent for treatment of an inflammatory disease or disorder (e.g., autoimmune diseases or disorders and/or parasitic infection). For example, the agent can comprise an agent that increases an anti-inflammatory T cell response and/or an agent that suppresses a proinflammatory T cell response.

In yet another aspect, methods of treating a patient determined to have an inflammatory disease or disorder comprising administering to a patient determined to have an inflammatory disease or disorder one or more embodiments of the pharmaceutical compositions described herein are also provided. The pharmaceutical composition can be taken alone or in combination with another agent for treatment of an inflammatory disease or disorder, e.g., an anti-inflammatory agent for treatment of an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses or a shift of balance toward Th2 response is desirable. An exemplary anti-inflammatory agent includes, but is not limited to an immunotherapy. In some embodiments, the method can further comprise administering the patient an immunotherapy for treatment of an inflammatory disease or disorder. For example, for treatment of an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses or a shift of balance toward Th2 response is desirable, the immunotherapy can comprise an agent that activates an anti-inflammatory T cell response and/or an agent that suppresses a proinflammatory T cell response.

In some embodiments of this aspect and other related aspects described herein, the patient having an autoimmune disease or disorder and/or parasitic infection can be previously treated with or is being treated with an anti-inflammatory therapy. Thus, the TIGIT agonist, Fgl2 agonist and/or IL-33 agonist therapy can be used alone or in combination with another anti-inflammatory agent. In some embodiments of this aspect and other related aspects described herein, the methods described herein can further comprise administering to the patient a selected therapy (e.g., TIGIT agonist or IL-33 agonist therapy) after they have been identified to be more likely to benefit from one immunotherapy over another.

In some embodiments of this aspect and other related aspects described herein, the TIGIT agonist, Fgl2 agonist and/or IL-33 agonist administered to a patient can be constructed to specifically target TIGIT+regulatory T cells (Tregs). For example, the TIGIT agonist, Fgl2 agonist and/or IL-33 agonist can comprise a cell-targeting moiety.

Without wishing to be bound by theory, it is contemplated that TIGIT can promote allergy, asthma, and/or atopy, e.g., by inducing the level of expression and/or activity of Fgl2, and thereby shifting the Th1 and/or Th17 vs. Th2 balance in favor of Th2 cytokines. Accordingly, it is also contemplated that other inflammatory diseases or disorders, including, e.g., allergy, asthma, and/or atopy, where a dampening Th2 response is desirable could be treated by downregulating the expression or activity of TIGIT, Fgl2 and/or IL-33 (or suppressing the TIGIT axis signaling). In this aspect, methods and compositions for treatment of cancer and/or infections described herein that stimulate Th1 and/or Th17 responses and thus shift the balance away from Th2 responses can be adapted accordingly for treatment of Th2 cytokine-mediated inflammatory diseases or disorders, including, e.g., allergy/asthma/atopy, where a dampening of the Th2 response is desirable.

In one aspect, provided herein is a method for treating asthma, allergy, and/or atopy. The method comprises administering to a patient diagnosed with asthma, allergy, and/or atopy a composition comprising an anti-Fgl2 therapy. In some embodiments, the method can further comprise identifying a patient diagnosed with asthma, allergy, and/or atopy who is more likely to respond to an anti-Fgl2 therapy, e.g., based on the level of expression and/or activity of TIGIT and/or IL-33. When the level of TIGIT and/or IL-33 activity or expression is greater than the TIGIT and/or IL-33 reference, the patient is identified to be more likely to be responsive to an anti-Fgl2 therapy; or (ii) when the level of TIGIT and/or IL-33 activity or expression is the same as or less than the TIGIT and/or IL-33 reference, the patient is identified as likely to respond to an alternative, Th2-dampening therapy or immunotherapy comprising, e.g., an activator of proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.

In some aspects, provided herein are methods for guiding selection of a treatment for a subject diagnosed with asthma, allergy, and/or atopy. In some embodiments, patients diagnosed with asthma, allergy, and/or atopy can be identified as more likely to be responsive to an anti-TIGIT and/or anti-IL-33 therapy based on the patients' level of Fgl2 activity or expression in a sample. In some embodiments, patients diagnosed with asthma, allergy, and/or atopy can be identified as more likely to be responsive to an anti-IL-33 therapy and/or anti-Fgl2 therapy based on the patients' level of TIGIT activity or expression in a sample. In some embodiments, patients diagnosed with asthma, allergy, and/or atopy can be identified as more likely to be responsive to an anti-TIGIT and/or anti-Fg12 therapy based on the patients' level of IL-33 activity or expression in a sample.

By way of example only, some aspects provided herein relate to methods for guiding selection of a treatment for a subject diagnosed with asthma, allergy, and/or atopy, based on the level of Fgl2 activity or expression in the patient's sample. The method comprises (a) measuring the level of Fgl2 activity or expression in a sample from a patient diagnosed with asthma, allergy, and/or atopy; and (b) comparing the level of Fgl2 or expression in the sample with an Fgl2 reference; and: (i) when the level of Fgl2 activity or expression is greater than the Fgl2 reference, the patient is identified to be more likely to be responsive to an anti-TIGIT and/or anti-IL-33 therapy; or (ii) when the level of Fgl2 activity or expression is the same as or less than the Fgl2 reference, the patient is identified as likely to respond to an alternative, Th2-dampening therapy or immunotherapy. In some embodiments, the alternative, Th2-dampening therapy or immunotherapy can comprise, e.g., an activator of proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.

In some embodiments, the methods can further comprise administering to the patient the selected treatment. Accordingly, methods for treating a patient diagnosed with asthma, allergy and/or atopy are also provided herein.

For example, in addition to using Fgl2 as a diagnostic marker, another aspect provided herein relates to a method for treating a patient diagnosed with asthma, allergy, and/or atopy, wherein the method comprises (a) measuring the level of IL-33 activity or expression in a sample from a patient diagnosed with asthma, allergy, and/or atopy; (b) comparing the level of IL-33 activity or expression in the sample with an IL-33 reference; and (c) performing one of the following actions:

- (i) administering to the patient a composition comprising a TIGIT inhibitor and/or an Fgl2 inhibitor, when the level of IL-33 activity or expression is greater than the IL-33 reference;
- (ii) administering an alternative, Th2-dampening therapy or immunotherapy without the TIGIT inhibitor or Fgl2 inhibitor, when the level of IL-33 activity or expression is the same as or less than the IL-33 reference; or
- (iii) determining if the level of at least one other inhibitory immune regulator in the sample is greater than the level of the corresponding reference, or if the level of at least one activating immune regulator in the sample is less than the level of the corresponding reference, when the level of IL-33 activity or expression is the same as or less than the IL-33 reference.

In some embodiments where the level of IL-33 activity or expression is the same as or less than the IL-33 reference, the method can further comprise (a) measuring the level of Fgl2 activity or expression in a sample from the patient diagnosed with asthma, allergy, and/or atopy, (b) comparing the level of Fgl2 activity or expression in the sample with an Fgl2 reference, and (c) administering to the patient a composition comprising a TIGIT inhibitor and/or an Fgl2 inhibitor, when the level of Fgl2 activity or expression is greater than the Fgl2 reference (e.g., by at least about 30% or more); or administering a Th2-dampening therapy or immunotherapy without a TIGIT inhibitor or an Fgl2 inhibitor, when the level of Fgl2 activity or expression is the same as or less than the reference (e.g., by at least about 30% or more).

In some embodiments where the level of IL-33 and/or Fgl2 activity or expression is the same as or less than the reference (e.g., by at least about 30% or more), an alternative, Th2-dampening therapy or immunotherapy without a TIGIT inhibitor or an Fgl2 inhibitor can be administered. In some embodiments, the alternative, Th2-dampening therapy or immunotherapy can comprise, e.g., an activator of a pro-inflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway as described herein.

In some aspects described herein, TIGIT, Fgl2 and/or IL-33 can be used as a marker to determine or monitor the efficacy of an anti-TIGIT, anti-Fgl2 and/or anti-IL-33 therapy administered to a patient diagnosed with asthma, allergy, and/or atopy. In some embodiments, Fgl2 can be used as a predictive marker to determine or monitor the efficacy of an anti-TIGIT and/or anti-IL-33 therapy administered to a patient diagnosed with asthma, allergy, and/or atopy. In some embodiments, TIGIT can be used as a predictive marker to determine or monitor the efficacy of an anti-IL-33 therapy and/or anti-Fgl2 therapy administered to a patient diagnosed with asthma, allergy, and/or atopy. In some embodiments, IL-33 can be used as a predictive marker to determine or monitor the efficacy of an anti-TIGIT and/or anti-Fgl2 therapy administered to a patient diagnosed with asthma, allergy, and/or atopy.

As an example, methods of treating a patient diagnosed with asthma, allergy, and/or atopy that has an elevated level of Fgl2 are provided herein. The method comprises: (a) determining a first level of Fgl2 expression or activity in a sample from a patient diagnosed with asthma, allergy, and/or atopy that has an elevated level of Fgl2; (b) administering an agent that inhibits IL-33 activity and/or TIGIT activity; (c) determining a second level of Fgl2 expression or activity after the administering; and (d) comparing the first and second levels of Fgl2 expression or activity, wherein the agent administered in (b) is effective if the second level of Fgl2 expression or activity is lower than the first level, and wherein the agent administered in (b) is ineffective if the second level of Fgl2 expression is the same as or higher than the first level.

By monitoring the effects of the anti-IL-33 and/or anti-TIGIT therapy on the level of Fgl2 expression or activity, one can determine the efficacy of the treatment regimen and adjust the treatment regimen if necessary. Accordingly, in some embodiments, the method can further comprise, when the anti-IL-33 or anti-TIGIT therapy is effective, continuing to administer the agent that inhibits IL-33 activity and/or TIGIT activity. In some embodiments, the method can further comprise, when the anti-IL-33 therapy or the anti-TIGIT therapy is ineffective, administering the agent that inhibits IL-33 activity and/or TIGIT activity at a higher dose. In some embodiments, the method can further comprise, when the anti-IL-33 therapy or the anti-TIGIT therapy is ineffective, discontinuing the anti-IL-33 therapy or the anti-TIGIT therapy. In these embodiments, the method can further comprise administering a therapy comprising an activator of a pro-inflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.

Similarly, further aspects provided herein relate to methods of treating a patient diagnosed with asthma, allergy, and/or atopy that exhibits an elevated level of IL-33. The method comprises: (a) determining a first level of TIGIT and/or Fgl2 expression or activity in a sample from a patient diagnosed with asthma, allergy, and/or atopy that exhibits an elevated level of IL-33; (b) administering an agent that inhibits IL-33 activity; (c) determining a second level of TIGIT or Fgl2 expression or activity after the administering; and (d) comparing the first and second levels of TIGIT and/or Fgl2 expression or activity, wherein anti-IL-33 therapy is effective if the second level of TIGIT and/or Fgl2 expression or activity is lower that the first level, and wherein anti-IL-33 therapy is ineffective if the second level of TIGIT and/or Fgl2 expression or activity is the same as or higher than the first level.

In some embodiments, the method can further comprise, when the anti-IL-33 therapy is effective, continuing to administer the agent that inhibits IL-33 activity. In some embodiments, the method can further comprise, when the anti-IL-33 therapy is ineffective, administering the agent that inhibits IL-33 activity at a higher dose. In other embodiments, the method can further comprise, when the anti-IL-33 therapy is ineffective, discontinuing the anti-IL-33 therapy. In these embodiments, the method can further comprise administering a therapeutic agent for treatment of asthma, allergy, and/or atopy. In some embodiments, the therapeutic agent can comprise, e.g., an activator of a pro-inflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.

In yet another aspect, methods of treating a patient diagnosed with asthma, allergy, and/or atopy comprising administering to a patient diagnosed with asthma, allergy, and/or atopy one or more embodiments of the pharmaceutical compositions described herein that provide Th2-dampening therapy are also provided. The pharmaceutical composition can be taken alone or in combination with another agent for treatment of asthma, allergy, and/or atopy. In some embodiments, the method can further comprise administering the patient an immunotherapy for treatment of asthma, allergy, and/or atopy. For example, an immunotherapy for treatment of asthma, allergy and/or atopy can comprise an agent that increases a pro-inflammatory T cell response and/or an agent that suppresses an anti-inflammatory T cell response.

In some embodiments of this aspect and other related aspects described herein, the patient diagnosed with asthma, allergy, and/or atopy can be previously or being treated for the disease or disorder. Thus, the anti-TIGIT, anti-Fgl2 and/or anti-IL-33 therapy can be used, alone or in combination with another anti-asthma, anti-allergy, and/or anti-atopy agent. In some embodiments of this aspect and other related aspects described herein, the methods described herein can further comprise administering to the patient diagnosed with asthma, allergy, and/or atopy a selected therapy (e.g., anti-TIGIT or anti-IL-33 therapy) after they have been identified to be more likely to benefit from one immunotherapy over another.

In some embodiments of this aspect and other related aspects described herein, the TIGIT inhibitor, Fgl2 inhibitor and/or IL-33 inhibitor administered to a patient diagnosed with asthma, allergy, and/or atopy can be constructed to specifically target TIGIT+regulatory T cells (Tregs). For example, the TIGIT inhibitor, Fgl2 inhibitor and/or IL-33 inhibitor can comprise a cell-targeting moiety. In one embodiment, the cell-targeting moiety is a molecule or entity that interacts with a binding site on the surface of a TIGIT+regulatory T cell. For example, the cell-targeting moiety can comprise an antibody against TIGIT. Alternatively, the cell-targeting moiety can comprise a TIGIT ligand as described herein.

In still other aspects, methods for modulating Th17 response based on the level of TIGIT, Fgl2 and/or IL-33 activity or expression are also provided herein. For example, in some embodiments, methods for enhancing Th17 response comprise contacting, modifying and/or engaging Tregs with, or administering to a subject with a deficiency in Th17 response, a TIGIT inhibitor, an Flg2 inhibitor and/or an IL-33 inhibitor. In other embodiments, methods for reducing or suppressing Th17 response comprise contacting, modifying and/or engaging Tregs with, or administering to a subject with an overly active Th17 response, a TIGIT agonist, an Flg2 agonist and/or an IL-33 agonist.

In the methods of various aspects described herein, a reference used for comparison to measured levels of TIGIT, Fgl2 and/or IL-33 activity or expression in a patient's sample is generally a positive control, a negative control, and/or a threshold value. In some embodiments, a reference can correspond to the level of expression or activity of the target molecule (e.g., TIGIT, Fgl2 or IL-33) in a normal healthy subject. In some embodiments, a reference can correspond to the level of expression or activity of the target molecule (e.g., TIGIT, Fgl2 or IL-33) in a normal tissue of the same type or lineage as a tissue biopsy obtained from a target site (e.g., a tumor or an inflammatory tissue) in a patient subjected to at least one aspect of the methods described herein. In some embodiments, a reference can correspond to the level of expression or activity of the target molecule (e.g., TIGIT, Fgl2 or IL-33) in a tissue biopsy with a known level of expression or activity of the target molecule. In some embodiments, a reference can correspond to the level of expression or activity of the target molecule (e.g., TIGIT, Fgl2 and/or IL-33) in a patient's sample taken at a prior time point. In some embodiments, a reference can be a standard numeric level or threshold.

In the methods of various aspects described herein, the sample can be a bodily fluid sample (e.g., blood or urine) or a sample of a tissue at a target site from a patient. For example, for treatment of cancer and/or infections, the sample can be a blood sample or a tumor biopsy from a patient. For treatment of other immune-related diseases or disorders, including, e.g., autoimmune diseases, asthma, allergy and/or atopy, the sample can be a blood sample or a tissue biopsy from a target site to be treated in a patient. Without wishing to be bound by theory, since Fg2 and IL-33 are soluble molecules while TIGIT is a cell surface molecule, Fgl2 and IL-33 can be more easily measured, e.g., from a blood sample, as compared to TIGIT measured, e.g., from a tissue sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F are experimental data showing that TIGIT is expressed on highly suppressive Tregs and promotes Treg conversion. (FIG. 1A) CD4+T cells were purified from Foxp3-GFP.KI mice and the Foxp3+ and Foxp3− cells were sorted. Foxp3+nTregs were stained directly for TIGIT (solid line) or with an isotype control (dotted line) and analyzed by flow cytometry. Foxp3+ induced Tregs (iTregs) were analyzed after 4 days of stimulation with TGF-β. (FIG. 1B) CD4+Foxp3+TIGIT+(ν) or CD4+Foxp3+TIGIT-(⋄) Tregs were sorted from Foxp3-GFP.KI mice and titrated onto Foxp3-GFP-effector T cells stimulated with anti-CD3 and APCs. Proliferation was measured after 72 h by ³H-thymidine incorporation. (Mean±s.d.; * P<0.01; representative experiment of >10 independent experiments). (FIG. 1C) Sorting strategy of ex vivo FACS sorted human effector T cells (CD4+CD25+CD127+) and Tregs (CD4+CD25^highCD127-) sorted into TIGIT+ and TIGIT−. (FIG. 1D) Tregs sorted as outlined in FIG. 1C showed>96% purity in both subsets measured by Foxp3 staining after isolation. (FIG. 1E) Representative suppression assay with human CD4+CD25^highCD127-TIGIT+ and TIGIT−Tregs co-cultured with CFSE-labeled CD25-depleted CD4+ effector T cells for 4 days. (FIG. 1F) Statistical summary of FIG. 1E of six healthy donors (mean±SEM; * P<0.05).

FIGS. 2A-2F are experimental data showing expression profiling of TIGIT+regulatory T cells. Heat map of chemokine (receptor) and cytokine (receptor) (FIG. 2A) or transcription factor (FIG. 2B) genes that are differentially expressed (>1.5-fold) in CD4+Foxp3+TIGIT+ and CD4+Foxp3+TIGIT− Tregs (duplicate samples are shown). (FIG. 2C) Differential expression of a selection of genes from FIG. 2B was determined by quantification of mRNA levels in CD4+Foxp3+TIGIT+ and CD4+Foxp3+TIGIT− Tregs by RT-PCR. Mean±s.d. of at least 3 independent experiments is shown. (FIGS. 2D-2F) Volcano plots comparing the P value versus fold-change for probes from TIGIT+ versus TIGIT−Treg cells. Treg signatures generated from (FIG. 2D) CXCR3+ versus CXCR3− Tregs, (FIG. 2E) WT versus IRF4 KO Tregs and (FIG. 2F) Tregs from GFP-Foxp3 fusion protein reporter mice versus Foxp3-IRES-GFP mice are highlighted as overexpressed or underrepresented. P values form a chi-squared test. P values from a chi-squared test. Genes and Probe IDs included in the signatures are listed in Table 2 in the Examples.

FIGS. 3A-3G are experimental data showing that TIGIT+Tregs are better equipped for suppression. (FIG. 3A) Heat map of surface receptor genes that are differentially expressed (>1.5-fold, duplicate samples) in CD4+Foxp3+TIGIT+ and CD4+Foxp3+TIGIT− Tregs. Quantitative RT-PCR (FIG. 3B) and flow cytometric (FIG. 3C) confirmation for a selection of genes from FIG. 3A and FIG. 3D. (FIG. 3D) Heat map of differentially expressed genes involved in Treg differentiation and function. (FIG. 3E) Volcano plot comparing the P value versus fold-change for probes from TIGIT+ versus TIGIT−Treg cells. The canonical Treg signature is highlighted in red (transcripts upregulated in Treg cells) and green (transcripts downregulated in Treg cells). (FIG. 3F) Foxp3 protein expression was quantified by flow cytometry in mouse Teff (Foxp3−) or Tregs (Foxp3+) and human memory T cells (CD4⁺CD127⁺CD25^med) and Tregs (CD4⁺CD127^lowCD25^high) (n=9; *p<0.05). (FIG. 3G) Relative expression of the indicated genes in CD4+Foxp3+TIGIT+ and CD4+Foxp3+TIGIT− Tregs was determined by quantitative PCR.

FIGS. 4A-4I are experimental data showing that TIGIT ligation induces Fgl2 expression. (FIG. 4A) Foxp3−(Teff) and Foxp3+(Treg) cells were sorted from Foxp3-GFP.KI mice, stimulated with anti-CD3/anti-CD28 in the presence of agonistic anti-TIGIT Ab. After 3 days RNA was extracted and Fgl2 and Il10 mRNA levels were determined by quantitative RT-PCR. (FIG. 4B) Ex vivo human memory T cells (CD4+CD127+CD25^med) and Tregs (CD4+CD127^lowCD25^high) were sorted gating into TIGIT+ and TIGIT−. After isolation, cells were cultured in the presence of agonistic anti-TIGIT or isotype control for 4 days. Fgl2 expression was quantified by RT-PCR (n=6; * P<0.05). (FIG. 4C) Mice were immunized s.c. with MOG_35-55peptide in CFA and treated with anti-TIGIT or isotype control antibody. On day 10 cells were re-stimulated with MOG_35-55peptide for 48 h. Fgl2 concentrations in the supernatants were determined by ELISA. (FIGS. 4D, 4E) CD4+CD25+TIGIT+(closed bars) or CD4+CD25+TIGIT− (open bars) Tregs were sorted from WT, IL-10 KO (FIG. 4D) or Fgl2 KO (FIG. 4E) mice and co-cultured with CD25-effector T cells stimulated with anti-CD3 and APCs at a ratio of 1:8. Where indicated neutralizing anti-IL10 (FIG. 4D) or anti-Fgl2 (FIG. 4E) Ab or the respective isotype control Ab was added to the culture. Proliferation was measured after 72 h by 3H-thymidine incorporation. (FIG. 4F) CD4+Foxp3+TIGIT+ and CD4+Foxp3+TIGIT− Tregs were sorted from Foxp3-GFP.KI mice and mRNA levels for Cebpa were determined by RT-PCR. (FIG. 4G) Cells were isolated and stimulated as in FIG. 4A and on day 3 Cebpa mRNA levels were determined by quantitative RT-PCR. (FIG. 4H) ChIP assays were performed on P815 cells expressing TIGIT using anti-CEBPαantibody or rabbit IgG isotype control. The precipitated chromatin was analyzed by quantitative PCR with primers specific for 3 promoter and 4 intragenic regions of the Fgl2 gene with predicted CEBPαbinding sites. Signals are displayed as % of the total input chromatin. (FIG. 4I) CD4+Foxp3+Treg cells were sorted from Foxp3-GFP.KI mice and transfected with a CEBPα over-expression construct (CEBPα) or the empty vector as control (control) and stimulated with anti-CD3/CD28 Dynabeads. Relative expression of Fgl2 mRNA was determined by RT-PCR 4 days later. (all panels represent mean±s.d)

FIGS. 5A-5I are experimental data showing that TIGIT+regulatory T cells suppress Th1 and/or Th17 but not a Th2 response. (FIG. 5A) Naïve effector T cells, WT Foxp3+TIGIT−, WT Foxp3+TIGIT+Tregs, and Fgl2−/− Foxp3+TIGIT+Tregs were sorted and co-cultured at a ratio of 1:10 under Th1, Th2, or Th17 polarizing conditions. After 3 days mRNA levels were determined by quantitative RT-PCR. On day 5 intracellular cytokines in CD45.1+T effector cells were determined by flow cytometry (values normalized to unsuppressed controls, mean±SEM; * P<0.05, ** P<0.001, paired student's t-test). (FIG. 5B) Human TIGIT+ and TIGIT−Tregs (CD4+CD25^highCD127^neg) were sorted and co-cultured with CFSE-labeled CD25-depleted CD4+T effector cells. Gene expression (qRT-PCR) and intracellular cytokine levels (flow cytometry) were determined on day 4 (mean±SEM; n=6). (FIGS. 5C-5F) CD25-effector OT-II cells and CD25high OT-II Tregs (TIGIT−, TIGIT+ or no Treg control) were transferred i.v. into WT recipients and mice were immunized with OVA in CFA. (FIG. 5C) Expansion of Vβ5⁺ OT-II T cells and (FIG. 5D) proliferation in response to OVA_323-339were determined 10 days later. (FIG. 5E) Intracellular cytokine levels were determined by flow cytometry and (FIG. 5F) cytokine concentration in the culture supernatants was determined by cytometric bead array. (FIGS. 5G-51) CD25-effector OT-II cells and CD25high OT-II Tregs (TIGIT−, TIGIT+ or no Treg control) were transferred i.v. into WT recipients. Mice were then sensitized with OVA (i.p.) on days 0 and 7 and challenged with aerosolized OVA on days 14-17 to induce allergic airway inflammation. (FIG. 5G) Total numbers of Vβ5⁺ OT-II cells in lungs, (FIG. 5H) intracellular cytokine levels from lung-infiltrating CD4+T cells, and (FIG. 5I) total eosinophil numbers in bronchio-alveolar lavage fluid were determined by flow cytometry. Pooled data from two experiments are shown (mean±SEM; n=8).

FIGS. 6A-6F are experimental data showing that TIGIT+regulatory T cells suppress pro-inflammatory responses in vivo. To induce colitis CD45RB^hieffector T cells (CD45.1) were transferred into Rag1−/− mice together with TIGIT+ or TIGIT−Tregs (CD45.2) or no Tregs as controls (Teff:Treg ratio was 4.4:1 for TIGIT+Tregs and 3.6:1 for TIGIT− Tregs). (FIG. 6A) Mice were monitored for weight loss over 10 weeks and (FIG. 6B) total colitis scores were determined by histopathology. (FIGS. 6C-6E) At 10 weeks after transfer mesenteric LNs were harvested and (FIG. 6C) total number of infiltrating CD4+T cells, (FIG. 6D) proportion of Foxp3+Tregs among CD4+T cells, and (FIG. 6E) Foxp3 expression among the transferred Treg population (CD45.2+) were determined by flow cytometry. (FIG. 6F) Mesenteric LN cells were re-stimulated in vitro with 0.5 μg/ml anti-CD3 for 3 days and cytokine secretion was determined by cytometric bead array in supernatants (P<0.05 (*), P<0.01 (**), P<0.005 (***)).

FIGS. 7A-7H are experimental data showing that TIGIT+regulatory T cells display an activated phenotype. (FIG. 7A-7B) Expression of TIGIT in conjunction with the natural Treg markers Neuropilin-1 and Helios was analyzed in (FIG. 7A) murine CD4+CD8-Foxp3+ and (FIG. 7B) human CD4+CD127^lowCD25^highTregs using flow cytometry. (FIG. 7C) CD4+Foxp3+TIGIT+ and CD4+Foxp3+TIGIT− Tregs (CD45.2) were sorted and transferred i.v. into WT recipients (CD45.1). After 20 days TIGIT expression on donor cells (CD45.2) was assessed by flow cytometry in lymph nodes (LN) and spleen. (FIG. 7D) Heat map of the microarray analysis of CD4+Foxp3+TIGIT+ and CD4+Foxp3+TIGIT− Tregs of genes that display differential expression (>1.5-fold); duplicate samples are shown. (FIG. 7E) Volcano plot comparing P value versus fold-change for probes from TIGIT+ versus TIGIT−Treg cells. Genes from the T cell activation/proliferation-responsive genes are highlighted in red (overexpressed) and green (underrepresented). (FIG. 7F) Ex vivo human CD4+T cells were stained for CD45RO, FoxP3, and TIGIT and analyzed by flow cytometry. Flow analysis from one representative healthy donor and pooled data of TIGIT MFI in each T cell subset are depicted (n=6; ANOVA *P<0.05). (FIG. 7G) Ki67 expression in Foxp3+TIGIT+ and Foxp3+TIGIT− cells was determined by intracellular staining and flow cytometry. (FIG. 7H) Naïve Foxp3-GFP.KI mice were administered 1 mg of BrdU/day by i.p. injection over 4 days. On day 5 BrdU incorporation was assessed by flow cytometry. (FIGS. 7G and 7H: representative plots and mean±s.d.).

FIGS. 8A-8C are experimental data showing phenotypic characterization of TIGIT+Tregs. (FIGS. 8A, 8B) Ex vivo human Tregs (CD4+CD127^lowCD25^high) were FACS-sorted gating into TIGIT+ and TIGIT−. (FIG. 8A) RNA was isolated for gene expression analysis by RT-PCR and (FIG. 8B) cell surface expression of T cell markers was determined in both subsets by flow cytometry (n=6). (FIG. 8C) Differential expression of cell surface molecules ICOS, PD-1, KLRG1, and CD103 in murine CD4+Foxp3+TIGIT+ and CD4+Foxp3+TIGIT− Tregs was determined by flow cytometry.

FIGS. 9A-9F are experimental data showing that an anti-TIGIT antibody acts agonistic in vivo. (FIGS. 9A, 9B) Mice were immunized s.c. with MOG_35-55peptide in CFA and treated i.p. with 100 μg of anti-TIGIT or isotype control antibody on days 0, 2, and 4. Spleens and lymph nodes were collected on day 10. (FIG. 9A) Cells were re-stimulated for 48 h in the presence of MOG_35-55peptide, pulsed with 3H-thymidine and proliferation was determined 18 h later (mean±s.d.). (FIG. 9B) Frequencies of TIGIT+Treg, TIGIT+ effector T cells (Teff) and Tregs (Foxp3+) were determined by flow cytometry at the time of sacrifice. (FIGS. 9C-9F) TIGIT suppresses Th1 and Th17 responses in vivo. Mice were immunized s.c. with 50 μg MOG_35-55peptide in CFA, followed by injection of 100 ng pertussis toxin i.v. on day 0 and day 2. In addition, animals were treated with 100 μg anti-TIGIT Ab (open symbols and bars) or an isotype control (filled symbols and bars) i.p. on days 0, 2, 4, 10, and 17. (FIGS. 9C-9D) Mice were monitored daily for EAE. Mean clinical score±SEM (FIG. 9C) and linear regressions (FIG. 9D) are shown (n=14). (FIGS. 9E and 9F) Spleens and draining LN were harvested at disease onset (day 10) and re-stimulated with 30 μg/ml MOG_35-55in vitro. After 48 h supernatants were harvested and analyzed for (FIG. 9E) IFNγ and (FIG. 9F) IL-17 by ELISA.

FIGS. 10A-10B are experimental data showing that TIGIT+regulatory T cells suppress Th1 and/or Th17 but not Th2 differentiation. (FIG. 10A) Naïve CFSE-labeled effector T cells (CD45.1) were transferred into Rag1−/− mice together with WT or Fgl2−/− Tregs (CD45.2) or no Tregs as controls (Teff:Treg ratio 5:1). After 8 days, splenocytes were analyzed for CFSE dilution and the number of undivided (CFSE^high) CD45.1+ cells as well as the total number of lymphocytes/spleen was quantified (mean±SEM). (FIG. 10B) Naïve WT CD4+CD62L+CD25-effector T cells (CD45.1) and WT CD4+Foxp3+TIGIT+, WT CD4+Foxp3+TIGIT-Tregs, and Fgl2−/−CD4+Foxp3+TIGIT+(CD45.2) were sorted and co-cultured at a ratio of 1:10 under Th1, Th2, or Th17 polarizing conditions. On day 5 cells were re-stimulated with PMA/Ionomycin and cytokine levels in CD45.1+T effector cells were determined by flow cytometry. A representative experiment is shown.

FIG. 11 shows that TIGIT+regulatory T cells promote Th2-like responses in vivo. To induce colitis CD45RB^hieffector T cells (CD45.1) were transferred into Rag1−/− mice together with TIGIT+ or TIGIT− Tregs (CD45.2) or no Tregs as controls (Teff:Treg ratio was 4.4:1 for TIGIT+Tregs and 3.6:1 for TIGIT− Tregs). At 10 weeks after transfer mesenteric LNs were harvested and cultured in vitro with 0.5 μg/ml anti-CD3 for 3 days. Cells were then re-stimulated with PMA/Ionomycin for 4 hours and IL-10, IL-4, and IFNγsecretion was determined by intracellular cytokine staining. Plots show quantification of IL-10+, IL-4+ or IFNγ+ cells among CD4+CD45.1+ effector T cells or CD4+CD45.2+ regulatory T cells (mean±s.d.).

FIGS. 12A-12C are data graphs showing expression of TIGIT ligand (CD112 and CD155) on various murine tumors such as colon carcinoma (CT26: FIG. 12A), Lewis lung carcinoma (LLC: FIG. 12B) and melanoma (B16F10: FIG. 12C). RNA was extracted from mouse colorectal carcinoma (CT26), lewis lung carcinoma (LLC) and melanoma (B16F10) for examination of TIGIT and the TIGIT ligands CD155 and CD112 by real-time quantitative PCR. CD155 expression was further confirmed by flow cytometry. No detectable expression of TIGIT receptors was found in murine tumors, but they have displayed TIGIT ligand expression. These data show that TIGIT ligands are expressed on the mouse tumor lines, consistent with a role of the TIGIT pathway in cancer.

FIGS. 13A-13B show that TIGIT is enriched on both CD4 and CD8 T cells that infiltrate tumor (tumor-infiltrating lymphocytes; TILs; right panels), as compared to lymphocytes from the spleen (left panels) or tumor-draining lymph nodes (DLN; middle panels). B16F10 (5×10⁵) cells were inoculated into wild type C57BL/6 mice (n=5) and CT26 (1×10⁶) were inoculated in wild type Balb/c mice (n=7). Representative flow cytometry data showing TIGIT expression is enriched in both CD4 and CD8 tumor infiltrating lymphocytes (TILs) in melanoma. Spleen, tumor draining lymph node (DLN), and TILs were harvested and examined for TIGIT expression. FIG. 13A shows the raw data and quantification of CD4+TIGIT+T cells or CD8+TIGIT+T cells from spleen, DLN, or TILs of a murine melanoma tumor (B16F10). FIG. 13B shows the raw data and quantification of CD4+TIGIT+T cells or CD8+TIGIT+T cells from spleen, DLN, or TILs of a murine colon tumor (CT26). In each figure, upper panels show representative flow data, while bottom panels show summary data. These data show that TIGIT expression is highly enriched on T cells that infiltrate tumor tissue, indicating that targeting TIGIT can have significant effects in tumor tissue but not elsewhere. Thus, there can be fewer systemic effects, and decreased possibility for autoimmune-like toxicities as have been observed with targeting CTLA-4.

FIGS. 14A-14B show that TIGIT+CD4+T cells are predominantly FoxP3+Treg in tumor-bearing mice. B16F10 (5×10⁵) and CT26 (1×10⁶) were inoculated into FoxP3-GFP Knock-in mice on the C57BL/6 (n=5) and Balb/c (n=7) backgrounds, respectively. FIG. 14A corresponds to a murine melanoma tumor model (B16F10). FIG. 14B corresponds to a murine colon tumor model (CT26). In each figure, left panel shows TIGIT and FoxP3 expression on CD4+TILs; middle panel show TIGIT and Tim-3 expression on CD4+FoxP3+TILs; and the right panel (a bar graph) shows summary data for TIGIT expression on FoxP3+ and FoxP3− cells in TILs, spleen, and tumor draining lymph node (DLN). The data in FIGS. 14A-14B show that TIGIT+Treg are highly enriched in tumor tissue and TIGIT+Tregs coexpress Tim-3. These data also show TIGIT expression is selective to FoxP3+Treg in CD4 TILs.

FIGS. 15A-15B show that TIGIT+CD8+ tumor infiltrating lymphocytes (TILs) co-express the T cell inhibitory receptors such as Tim-3 and PD1 (FIG. 15A), and also exhibit exhausted/dysfunctional phenotype, for example, defective IL-2 (FIG. 15B, left panel), TNFa production (FIG. 15B, middle panel), and increased IL-10 production (not shown). B16F10 (5×10⁵) cells were inoculated into wild type C57BL/6 mice. (FIG. 15A) Representative Tim-3 and PD-1 staining on CD8 TILs from B16F10 melanoma. (FIG. 15B) TILs were harvested from B16F10 melanoma tumors and restimulated ex vivo with PMA/ionomycin for 4 hrs prior to intracytoplasmic staining. Expression of IL-2, TNF, and IFNγ on TIGIT CD8+TILs is shown, n=5. These data indicate that TIGIT is found on T cells that co-express other markers of T cell dysfunction/exhaustion such as Tim-3 and PD-1 and show that TIGIT+TILs are defective in IL-2 and TNF production. The TIGIT+CD8+TILs exhibit no significant defects in IFNγ(FIG. 15B, right panel).

FIGS. 16A-16B are experimental data showing roles of TIGIT+Treg in tumor growth and tumor immunity. FIG. 16A is a line graph showing better control of tumor growth in mice bearing melanoma (B16F10) with TIGIT knock-out (KO). 7 week old female C57BL/6 mice were inoculated with B16F10 (5×10⁵). Tumor growth was measured in two dimensions using a caliper. Mean tumor growth is shown. Error bars indicate SEM. Dashed lines indicate linear regression. Difference in slope is statistically significant, p=0.0002. FIG. 16B shows role of TIGIT Treg in tumor immunity. At Day 0, 7 week old female Rag-deficient mice were reconstituted with wildtype CD4 FoxP3-GFP-effectors, wildtype FoxP3-GFP+Treg and wildtype CD8 T cells (WT group) or with wildtype CD4 FoxP3-GFP-effectors, TIGIT− deficient FoxP3-GFP+Treg and wildtype CD8 T cells (KO group). At Day 2, mice were inoculated with B16F10 (5×10⁵). Tumor growth was measured in two dimensions using a caliper. Mean tumor growth is shown. Error bars indicate sem. Dashed lines indicate linear regression. Difference in slope is statistically significant, p=0.0003. Bar group shows mean tumor size at Day 13 from 3 experiments. P=0.0079.

FIGS. 17A-17B are experimental data showing that increase in tumor-specific CD8+T cells from DLN (FIG. 17A) and TILs (FIG. 17B) in mice lacking TIGIT+Tregs.

FIGS. 18A-18B are experimental data showing that TIGIT+Tregs express ST2. (FIG. 18A) Microarray analysis of naïve CD4+Foxp3+TIGIT+ and CD4+Foxp3+TIGIT− Tregs was performed. Signal intensity for probes for the ST2 gene Il1r11 is depicted (duplicate samples). (FIG. 18B) Expression of ST2 and TIGIT was analyzed in naïve CD4+Foxp3+Tregs isolated from spleen. Representative plots from one of >10 independent experiments are shown. Cells are gated on live CD4+Foxp3+Tregs.

FIGS. 19A-19B are experimental data showing expression of an interleukin-1 receptor family member ST2 and TIGIT on T cells in CT26 colon cancer. CT26 (1×10⁶) were inoculated in wild type Balb/c mice. FIG. 19A are representative flow cytometry data showing expression of ST2 and TIGIT on T cells (CD4+FoxP3+; CD4+FoxP3−; CD8+) from TILs (top panels), DLN (middle panels) and spleen (bottom panels), and indicating that ST2 is most highly expressed on TIGIT+Treg in tumor tissue in CT26 colon carcinoma. FIG. 19B is a bar graph showing the number of ST2+T cells from TILs, spleen and DLN, and indicating that ST2+Treg are highly enriched in tumor tissue. Error bars indicate SEM. These data show the role of IL-33/ST2 in promoting TIGIT Treg in tissue.

FIG. 20 is a set of plots showing that IL-33 expands TIGIT⁺ Tregs in vivo. Mice were treated with either 200 ng IL-33 or PBS (control) i.p. for 4 days. On day 5, cells were isolated from spleen, lymph nodes (LN), and lung and expression of ST2 and TIGIT was analyzed in Tregs. Representative plots from one of three independent experiments are shown. Cells are gated on live CD4⁺Foxp3⁺ Tregs.

DETAILED DESCRIPTION

Embodiments of various aspects described herein are, in part, based on the discovery that TIGIT expression defines a functionally distinct subset of regulatory T cells (Tregs) that selectively suppress pro-inflammatory Th1 and Th17 responses but spare or promote an anti-inflammatory Th2 response by inducing the secretion of the soluble effector molecule Fgl2. Further, the inventors discovered that TIGIT+Treg cells can be induced and/or expanded by IL-33. The inventors have also discovered that tumors express TIGIT ligands such as CD112 and CD155, which can induce tumor immune evasion where TIGIT+Tregs infiltrate the tumors and induce suppression of Th1 and/or Th17 responses. Thus, not only can agents that modulate the activity and/or expression of TIGIT, Fgl2, and/or IL-33 be used for treatment of immune related diseases or disorders such as autoimmune disease, infection, chronic inflammation, cancer, asthma, allergy, and atopy, but TIGIT, Fgl2 and/or IL-33 can also be used as predictive markers to identify subjects who are more likely to benefit from an immunotherapy that targets TIGIT, Fgl2 and/or IL-33. Accordingly, various aspects described herein provide for methods of identifying subjects with an immune-related disease or disorder who are more likely to be responsive to an immunotherapy that targets TIGIT, Fgl2 and/or IL-33, as well as monitoring the treatment efficacy. Methods and compositions for treating subjects with an immune-related disease or disorder are also provided herein.

Methods and Compositions for Treating Immune-Related Diseases where an Inhibition of Th2 Response and/or a Shift of Balance Toward a Th1 and/or Th17 Response is Desirable

In some immune-related diseases or disorders, e.g., but not limited to cancer and/or infections, it can be desirable to induce proinflammatory responses, e.g., Th1 and/or Th17 responses, at a target site (e.g., a tumor) for a therapeutic effect. Accordingly, these immune-related diseases or disorders, e.g., but not limited to cancer and/or infections (including, e.g., but not limited to chronic viral infection, intracellular bacterial infection, extracellular bacterial infection, and/or fungal infection), where upregulation of immune response (e.g., Th1 and/or Th17 responses) is desirable, can be treated by inhibiting or reducing the expression or activity of TIGIT, Fgl2 and/or IL-33. However, not every patient, e.g., not every patient with cancer and/or infection, would necessarily benefit from a treatment that inhibits the level of TIGIT, Fgl2 and/or IL-33 expression and/or activity. The inventors discovered that the regulatory T cells in a subject can be separated into distinct populations, TIGIT+ cells with more strongly suppressive phenotypes and TIGIT negative (−) cells, and that Fgl2 and IL-33 are involved in regulation of TIGIT. The TIGIT+cell population and Fgl2 and/or IL-33 expression and/or activity can vary in each individual. Tumors which include high levels of TIGIT+T cell infiltration would be expected to respond poorly to immunotherapies designed to stimulate Th1 and/or Th17-type responses, because the TIGIT axis as defined earlier works to suppress activity and/or activation of Th1/Th7 responses. Only after the TIGIT axis is inhibited would one expect a strong anti-tumor immune response.

Accordingly, in some aspects, provided herein relate to methods of identifying a patient who is diagnosed with cancer and/or infection, and is more likely to be responsive to a Th1 and/or Th17 pro-inflammatory agent or to an anti-TIGIT, anti-Fgl2 and/or anti-IL-33 therapy. In some embodiments, patients diagnosed with cancer and/or infection can be identified as more likely to be responsive to a Th1 and/or Th17 pro-inflammatory agent or to an anti-TIGIT and/or anti-IL-33 therapy based on the patients' level of Fgl2 activity or expression in a sample. In some embodiments, patients with cancer and/or infection can be identified as more likely to be responsive to a Th1 and/or Th17 pro-inflammatory agent or to an anti-IL-33 therapy and/or anti-Fgl2 therapy based on the patients' level of TIGIT activity or expression in a sample. In some embodiments, patients diagnosed with cancer and/or infection can be identified as more likely to be responsive to a Th1 and/or Th17 pro-inflammatory agent or to an anti-TIGIT and/or anti-Fgl2 therapy based on the patients' level of IL-33 activity or expression in a sample.

As used herein, the phrase “more likely to be responsive” generally refers to likelihood of a subject to respond to a treatment. In accordance with one aspect of the discovery that selective suppression by TIGIT+Tregs is Fgl2-dependent and IL-33 can induce or expand the Treg population, by determining the level of TIGIT, Fgl2 and/or IL-33 expression or activity, one can predict the immune response of a subject subjected to an agent that modulates TIGIT, Fgl2 and/or IL-33 expression or activity (e.g., an agent that activates or suppresses a Th1 or Th17 response), which can in turn produce an effect on a disease or condition.

As used herein, an “immune response” being modulated refers to a response by a cell of the immune system, such as a B cell, T cell (CD4 or CD8), regulatory T cell, antigen-presenting cell, dendritic cell, monocyte, macrophage, NKT cell, NK cell, basophil, eosinophil, or neutrophil, to a stimulus. In some embodiments, the response is specific for a particular antigen (an “antigen-specific response”), and refers to a response by a CD4 T cell, CD8 T cell, or B cell via their antigen-specific receptor. In some embodiments, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. Such responses by these cells can include, for example, cytotoxicity, proliferation, cytokine or chemokine production, trafficking, or phagocytosis, and can be dependent on the nature of the immune cell undergoing the response.

By way of example only, one aspect provided herein relates to methods of identifying a patient who is diagnosed with cancer and/or infection, and is more likely to be responsive to a proinflammatory immunotherapy, or an anti-TIGIT and/or anti-IL-33 therapy, based on the level of Fgl2 activity or expression in the patient's sample. The method comprises (a) measuring the level of Fgl2 activity or expression in a sample from a patient who is diagnosed with cancer and/or infection; and (b) comparing the level of Fgl2 or expression in the sample with an Fgl2 reference; and (c) (i) identifying the patient to be more likely to be responsive to a proinflammatory immunotherapy, or an anti-TIGIT and/or anti-IL-33 therapy, when the level of Fgl2 activity or expression is greater than the Fgl2 reference; or (ii) identifying the patient to be likely to respond to an alternative, proinflammatory immunotherapy comprising an activator of a proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway, when the level of Fgl2 activity or expression is the same as or less than the Fgl2 reference. A pro-inflammatory immunotherapy comprises either an agent that directly activates a proinflammatory response, or an agent that suppresses an anti-inflammatory response.

Fgl2, also known as fibroleukin or fibrinogen-like protein 2, is a member of the fibrinogen-related protein superfamily of proteins. Fgl2 was first cloned from human CTLs and is secreted by CD4+ and CD8+T cells or Tregs. Methods for measuring the secreted form of Fgl2 from a sample are known in the art, including, but not limited to mRNA expression using PCR or real-time PCR, protein analysis using western blot, immunoassay, and/or ELISA, and/or sequencing analysis. Thus, in some embodiments, nucleic acid molecules can be isolated from a patient's sample to measure Flg2 mRNA expression, or proteins can be isolated to measure Fgl2 protein expression.

As used herein, the term “expression” refers to the protein or mRNA amount of a target molecule (e.g., TIGIT, Fgl2 or IL-33) in a sample.

As used herein, the term “activity” refers to the ability of a target molecule (e.g., TIGIT, Fgl2, or IL-33) to directly or indirectly produce an immune response in a subject.

As used herein, the term “reference” refers to a pre-determined value for the level of expression or activity of a target molecule to be measured, which can be used in comparison with the expression or activity of the target molecule measured from a patient's sample. In the methods of various aspects described herein, a reference used for comparison to measured levels of TIGIT, Fgl2 and/or IL-33 activity or expression in a patient's sample can be determined from a normal healthy subject, or from a patient who has shown responsiveness to a treatment. In some embodiments, a reference can correspond to the level of expression or activity of the target molecule (e.g., TIGIT, Fgl2 or IL-33) in a normal healthy subject. The term “normal healthy subject” generally refers to a subject who has no symptoms of any diseases or disorders, or who is not identified with any diseases or disorders, or who is not on any medication treatment, or a subject who is identified as healthy by a physician based on medical examinations. In some embodiments, a reference can correspond to the level of expression or activity of the target molecule (e.g., TIGIT, Fgl2 or IL-33) in a normal tissue of the same type or lineage as a tissue biopsy obtained from a target site (e.g., a tumor or an inflammatory tissue) in a patient subjected to at least one aspect of the methods described herein. In some embodiments, a reference can correspond to the level of expression or activity of the target molecule (e.g., TIGIT, Fgl2, or IL-33) at a prior time point in a patient from which a sample is derived or obtained. In some embodiments, a reference can correspond to a threshold level of expression or activity of the target molecule (e.g., TIGIT, Fgl2 or IL-33), above or below which the level of expression or activity of the target molecule (e.g., TIGIT, Fgl2 or IL-33) measured in a patient's sample would indicate the likelihood of a subject to respond to a treatment. In some embodiments, a reference can be a standard numeric level or threshold.

Accordingly, in some embodiments, the Fgl2 reference can correspond to the level of expression or activity of Fgl2 in a normal healthy subject. In some embodiments, the Fgl2 reference can correspond to the level of expression or activity of Fgl2 in a normal tissue of the same type or lineage as a tissue biopsy obtained from a patient. The normal tissue of the same type or lineage can be obtained from the same or a different patient. In some embodiments, the Fgl2 reference can correspond to a threshold level of expression or activity of Fgl2, above which the level of Fgl2 expression activity measured in a patient's sample would indicate the likelihood of the patient diagnosed with cancer and/or infection to respond to a treatment. When the level of Fgl2 activity or expression is greater than the Fgl2 reference, e.g., by at least about 10% or more, including, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more, the patient diagnosed with cancer and/or infection is identified to be more likely to be responsive to an anti-TIGIT and/or anti-IL-33 therapy. In some embodiments, when the level of Fgl2 activity or expression is greater than the Fgl2 reference, e.g., by at least about 1.1-fold or more, including, e.g., at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, or more, the patient diagnosed with cancer and/or infection can be identified to be more likely to be responsive to an anti-TIGIT and/or anti-IL-33 therapy. On the other hand, when the level of Fgl2 activity or expression is substantially the same as or less than the Fgl2 reference, e.g., by at least about 10% or more, including, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, the patient diagnosed with cancer and/or infection is identified as likely to respond to an alternative, proinflammatory immunotherapy comprising an activator of a proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway, e.g., without the need to suppress TIGIT, Fgl2, or IL-33 activity.

As used herein, the term “proinflammatory T cell response” refers to response of T cells to produce proinflammatory factors such as Th1 and/or Th17 cytokines, e.g., but not limited to IFNγ, TNFα, GM-CSF, IL-2, IL-9, IL-17, IL-21, and IL-22. In some embodiments, the term “proinflammatory T cell response” can refer to activation of “stimulatory immune checkpoints,” including, but not limited to CD28, ICOS, 4-1BB, OX40, and/or CD27.

As used herein, the term “anti-inflammatory T cell response” refers to response of T cells to produce anti-inflammatory factors such as Th2 cytokines or immunosuppressive cytokines, e.g., but not limited to IL-4, IL-5, IL-6, IL-10, IL-13, TGFβ, IL-35, and/or IL-27. In some embodiments, the term “anti-inflammatory T cell response” can refer to activation of “inhibitory immune checkpoints,” including, but not limited to PD-1, CTLA-4, BTLA, LAG-3, and/or TIM-3.

In this aspect and other aspects described herein, any appropriate modulators of a T cell response pathway that are known in the art can be used in the alternative, pro-inflammatory immunotherapy for patients with cancer and/or infection. For example, activators of a proinflammatory T cell response or suppressors of an anti-inflammatory T-cell response pathway can comprise a TIM-3 inhibitor, an anti-galectin-9 molecule, a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a Lag-3 antagonist, a DD1α antagonist, an agonist of an stimulatory immune checkpoint molecule, an antagonist of an inhibitory immune checkpoint molecule, or any combination thereof.

Some aspects provided herein relate to methods of treating a patient diagnosed with cancer and/or infection. In some embodiments, the patient diagnosed with an infection can have chronic viral infection, intracellular bacterial infection, extracellular bacterial infection, and/or fungal infection. An anti-TIGIT, anti-Fgl2 and/or anti-IL33 therapy can be selected for administration to a patient diagnosed with cancer and/or infection, based on the level of Fgl2, IL-33 and/or TIGIT in the patient's sample. In some embodiments, an anti-TIGIT and/or anti-IL-33 therapy can be selected for administration to a patient diagnosed with cancer and/or infection, based on the patient's level of Fgl2 activity or expression in a sample. In some embodiments, an anti-IL-33 therapy and/or anti-Fgl2 therapy can be selected for administration to a patient diagnosed with cancer and/or infection, based on the patient's level of TIGIT activity or expression in a sample. In some embodiments, an anti-TIGIT and/or anti-Fgl2 therapy can be selected for administration to a patient diagnosed with cancer and/or infection, based on the patient's level of IL-33 activity or expression in a sample.

- (i) administering to the patient a composition comprising a TIGIT inhibitor and/or an Fgl2 inhibitor, when the level of IL-33 activity or expression is greater than the IL-33 reference;
- (ii) administering an alternative, e.g., proinflammatory immunotherapy, treatment without the TIGIT inhibitor or Fgl2 inhibitor, when the level of IL-33 activity or expression is the same as or less than the IL-33 reference; or
- (iii) determining if the level of at least one other inhibitory immune regulator in the sample is greater than the level of the corresponding reference, or if the level of at least one activating immune regulator in the sample is less than the level of the corresponding reference, when the level of IL-33 activity or expression is the same as or less than the IL-33 reference.

Examples of inhibitory immune regulators include, but are not limited to Fgl2, TIGIT, ST2, CD155, CD112, PD-1, PD-L1, DD1α, TIM-3, galectin-9, CTLA-4, Lag-3, and any combination thereof. Examples of activating immune regulators include, but are not limited to CD28, ICOS, 4-1BB, OX40, CD27, and any combination thereof.

In some embodiments, the IL-33 reference can correspond to the level of expression or activity of IL-33 in a normal healthy subject. In some embodiments, the IL-33 reference can correspond to the level of expression or activity of IL-33 in a normal tissue of the same type or lineage as a tissue biopsy obtained from a patient. The normal tissue of the same type or lineage can be obtained from the same or a different patient. In some embodiments, the IL-33 reference can correspond to the level of expression or activity of IL-33 in a patient's sample obtained at a different or prior time point. In some embodiments, the IL-33 reference can correspond to a threshold level of expression or activity of IL-33, above which the level of IL-33 expression or activity measured in a patient's sample would indicate the likelihood of the patient diagnosed with cancer and/or infection to respond to a treatment. When the level of IL-33 activity or expression is greater than the IL-33 reference, e.g., by at least about 10% or more, including, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more, the patient diagnosed with cancer and/or infection is identified to be more likely to be responsive to a pro-inflammatory immunotherapy and/or a TIGIT inhibitor and/or Fgl2 inhibitor. In some embodiments, when the level of IL-33 activity or expression is greater than the IL-33 reference, e.g., by at least about 1.1-fold or more, including, e.g., at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold or more, the patient diagnosed with cancer and/or infection is identified to be more likely to be responsive to a pro-inflammatory immunotherapy and/or a TIGIT inhibitor and/or Fgl2 inhibitor. On the other hand, when the level of IL-33 activity or expression is substantially the same as or less than the IL-33 reference, e.g., by at least about 10% or more, including, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, the patient diagnosed with cancer and/or infection is identified as likely to respond to a pro-inflammatory immunotherapy, e.g., an activator of a proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway, without the need to suppress the TIGIT axis.

In some embodiments where the level of IL-33 activity or expression is the same as or less than the IL-33 reference, the method can further comprise (a) measuring the level of Fgl2 activity or expression in a sample from the patient, (b) comparing the level of Fgl2 activity or expression in the sample with an Fgl2 reference, and (c) administering to the patient a composition comprising a TIGIT inhibitor and/or an Fgl2 inhibitor, when the level of Fgl2 activity and/or expression is greater than the Fgl2 reference (e.g., by at least about 30% or more, including, e.g., at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 100% or more); or administering a proinflammatory immunotherapy without a TIGIT inhibitor or Fgl2 inhibitor, when the level of Fgl2 activity or expression is the same as or less than the reference (e.g., by at least about 30% or more, including, e.g., at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more).

In some embodiments, when the level of Fgl2 activity and/or expression is greater than the Fgl2 reference, e.g., by at least about 1.1-fold or higher, including, e.g., at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold or higher, the patient can be administered a composition comprising a TIGIT inhibitor and/or an Fgl2 inhibitor.

In some embodiments where the level of IL-33 and/or Fgl2 activity or expression is the same as or less than the reference (e.g., by at least about 30% or more, including, e.g., at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more), a proinflammatory immunotherapy without a TIGIT inhibitor or Fgl2 inhibitor to be administered can be an alternative, proinflammatory immunotherapy treatment comprising an activator of a proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway as described earlier.

As used herein, the term “administering,” or “administration” refer to the placement of an agent (e.g., a pro- or anti-inflammatory immunotherapy agent or an agent that modulates the expression and/or activity of TIGIT, Fgl2 and/or IL-33 into a subject by a method or route which results in at least partial localization of such agents at a desired site, such as a site of inflammation or tumor, such that a desired effect(s) is produced.

As used herein, “modulating” or “modulate” generally means either reducing or inhibiting the expression and/or activity of, or alternatively increasing the expression and/activity of, a target molecule, e.g., TIGIT, Fgl2 and/or IL-33, e.g., as measured using a suitable in vitro, cellular, or in vivo assay. In particular, “modulating” or “modulate” can mean either reducing or inhibiting the expression and/or activity of, or alternatively increasing a (relevant or intended) biological activity and/or expression of, a target molecule, e.g., TIGIT, Fgl2 and/or IL-33, as measured using a suitable in vitro, cellular or in vivo assay (which will usually depend on the target involved), by at least 5%, at least 10%, at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more, inclusive, compared to activity of the target in the same assay under the same conditions but without the presence of an agent. Thus, as used herein, the term “modulating” can refer to an increase or decrease in the expression and/or activity of TIGIT, Fgl2 and/or IL-33 relative to a subject not treated with an agent that modulates the expression and/or activity of TIGIT, Fgl2 and/or IL-33. An “increase” or “decrease” refers to a statistically significant increase or decrease respectively. For the avoidance of doubt, an increase or decrease will be at least 10% relative to a reference, such as at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or more, up to and including at least 100% or more, inclusive, in the case of an increase, for example, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold, at least 100-fold, or more.

As will be clear to the skilled person, “modulating” can also involve effecting a change (which can either be an increase or a decrease) in affinity, avidity, specificity and/or selectivity of a target molecule, e.g., TIGIT, Fgl2 and/or IL-33, for one or more of its ligands, receptors, binding partners, partners for association into a homomultimeric or heteromultimeric form, or substrates; and/or effecting a change (which can either be an increase or a decrease) in the sensitivity of the target molecule, e.g., TIGIT, Fgl2 and/or IL-33, for one or more conditions in the medium or surroundings in which the target molecule is present (such as pH, ion strength, the presence of co-factors, etc.), compared to the same conditions but without the presence of the target molecule, e.g., TIGIT, Fgl2 and/or IL-33. Again, this can be determined in any suitable manner and/or using any suitable assay known per se or described herein, depending on the target involved. “Modulating” can also mean effecting a change (i.e., an activity as an agonist, as an antagonist or as a reverse agonist, respectively, depending on the target molecule, e.g., TIGIT, Fgl2 and/or IL-33, and the desired biological or physiological effect) with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which the target or antigen (or in which its substrate(s), ligand(s) or pathway(s) are involved, such as its signaling pathway or metabolic pathway and their associated biological or physiological effects) is involved. Again, as will be clear to the skilled person, such an action as an agonist or an antagonist can be determined in any suitable manner and/or using any suitable (in vitro and usually cellular or in assay) assay known per se or described herein, depending on the target or antigen involved.

Modulating can, for example, also involve allosteric modulation of the target molecule, such as TIGIT, Fgl2 and/or IL-33; and/or reducing or inhibiting the binding of the target to one of its substrates, receptors, or ligands and/or competing with a natural ligand, receptor or substrate for binding to the target. Modulating can also involve activating the target or the mechanism or pathway in which it is involved. Modulating can for example also involve effecting a change in respect of the folding or confirmation of the target, or in respect of the ability of the target to fold, to change its conformation (for example, upon binding of a ligand), to associate with other (sub)units, or to disassociate. Modulating can for example also involve effecting a change in the ability of the target to signal, phosphorylate, dephosphorylate, and the like.

Thus, TIGIT, Fgl2 and/or IL-33 expression and/or activity is “decreased” or “reduced” if one or more signaling activities or downstream read-outs of TIGIT, Fgl2 and/or IL-33 activity is reduced by a statistically significant amount, such as by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or more, up to and including at least 100%, in the presence of an agent or stimulus relative to the absence of such modulation. As will be understood by one of ordinary skill in the art, in some embodiments, if TIGIT, Fgl2 and/or IL-33 expression and/or activity is decreased or reduced, some downstream read-outs will decrease but others can increase (i.e. things that are normally suppressed by TIGIT, Fgl2 and/or IL-33 expression and/or activity), and the converse would be in those embodiments where TIGIT, Fgl2 and/or IL-33 expression and/or activity is increased.

Conversely, TIGIT, Fgl2 and/or IL-33 expression and/or activity is “increased” if one or more signaling activities or downstream read-outs of TIGIT, Fgl2 and/or IL-33 expression and/or activity is increased by a statistically significant amount, for example by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or more, up to and including at least 100% or more, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold, at least 100-fold, or more, in the presence of an agent or stimulus, relative to the absence of such agent or stimulus.

In some embodiments of this aspect and other aspects described herein, the agents described herein for modulating the expression and/or activity of TIGIT, Fgl2 and/or IL-33 can be administered to a subject by any mode of administration that delivers the agent systemically or to a desired surface, organ, or target, and can include, but is not limited to injection, infusion, instillation, and inhalation administration. To the extent that such agents can be protected from inactivation in the gut, oral administration forms are also contemplated. “Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. In some embodiments, the agents for modulating the expression and/or activity of TIGIT, Fgl2 and/or IL-33 for use in the methods described herein are administered by intravenous infusion or injection.

The phrases “parenteral administration” and “administered parenterally” as used herein, refer to modes of administration other than enteral and topical administration, usually by injection. The phrases “systemic administration,” “administered systemically”, “peripheral administration” and “administered peripherally” as used herein refer to the administration of an agent for modulating expression and/or activity of TIGIT, Fgl2 and/or IL-33 other than directly into a target site, tissue, or organ, such as a tumor site, such that it enters the subject's circulatory system and, thus, is subject to metabolism and other like processes.

In another aspect, provided herein are methods of treating a patient diagnosed with cancer and/or infection comprising (a) measuring the level of Fgl2 activity or expression in a sample from a patient diagnosed with cancer and/or infection; (b) comparing the level of Fgl2 activity or expression in the sample with an Fgl2 reference; and (c) administering to the patient a composition comprising a TIGIT inhibitor and/or an IL-33 inhibitor when the level of Fgl2 activity or expression is greater than the Fgl2 reference (e.g., by at least about 30% or more, including, e.g., at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 100% or more), or administering an alternative, pro-inflammatory immunotherapy treatment without a TIGIT inhibitor or IL-33 inhibitor when the level of Fgl2 activity or expression is the same as or less than the Fgl2 reference (e.g., by at least about 30% or more, including, e.g., at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more). In some embodiments, the alternative, proinflammatory immunotherapy treatment without a TIGIT inhibitor or IL-33 inhibitor can be a therapy comprising an activator of a proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.

In some embodiments, when the level of Fgl2 activity and/or expression is greater than the Fgl2 reference, e.g., by at least about 1.1-fold or more, including, e.g., at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold or more, the patient can be administered a composition comprising a TIGIT inhibitor and/or an IL-33 inhibitor.

In some embodiments, the patient with an Fgl2 level greater than the Fgl2 reference can be further administered with a therapy comprising an activator of a proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway as described earlier.

In some aspects described herein, TIGIT, Fgl2 and/or IL-33 can be used as a predicative marker to determine or monitor the efficacy of an anti-TIGIT, anti-Fgl2 and/or anti-IL-33 therapy administered to a patient diagnosed with cancer and/or infection. In some embodiments, the patient diagnosed with an infection can have chronic viral infection, intracellular bacterial infection, extracellular bacterial infection, and/or fungal infection. In some embodiments, Fgl2 can be used as a predictive marker to determine or monitor the efficacy of an anti-TIGIT and/or anti-IL-33 therapy administered to a patient diagnosed with cancer and/or infection. In some embodiments, TIGIT can be used as a predictive marker to determine or monitor the efficacy of an anti-IL-33 therapy and/or anti-Fgl2 therapy administered to a patient diagnosed with cancer and/or infection. In some embodiments, IL-33 can be used as a predictive marker to determine or monitor the efficacy of an anti-TIGIT and/or anti-Fgl2 therapy administered to a patient diagnosed with cancer and/or infection.

As an example, methods of treating a patient who is diagnosed with cancer and/or infection and has an elevated level of Fgl2 are provided herein. The method comprises: (a) determining a first level of Fgl2 expression or activity in a sample from a patient diagnosed with cancer and/or infection that has an elevated level of Fgl2; (b) administering an agent that inhibits IL-33 activity and/or TIGIT activity; (c) determining a second level of Fgl2 expression or activity after the administering; and (d) comparing the first and second levels of Fgl2 expression or activity, wherein the agent administered in (b) is effective if the second level of Fgl2 expression or activity is lower than the first level, and wherein the agent administered in (b) is ineffective if the second level of Fgl2 expression is the same as or higher than the first level.

Similarly, a further aspect provided herein relates to methods of treating a patient diagnosed with cancer and/or infection that exhibits an elevated level of IL-33. The method comprises: (a) determining a first level of TIGIT and/or Fgl2 expression or activity in a sample from a patient diagnosed with cancer and/or infection and having an elevated level of IL-33; (b) administering an agent that inhibits IL-33 activity; (c) determining a second level of TIGIT or Fgl2 expression or activity after the administering; and (d) comparing the first and second levels of TIGIT and/or Fgl2 expression or activity, wherein anti-IL-33 therapy is effective if the second level of TIGIT and/or Fgl2 expression or activity is lower that the first level, and wherein anti-IL-33 therapy is ineffective if the second level of TIGIT and/or Fgl2 expression is the same as or higher than the first level.

In yet another aspect, methods of treating a patient diagnosed with cancer and/or infection comprising administering to the patient one or more embodiments of the pharmaceutical compositions described herein are also provided. The pharmaceutical composition can be taken alone or in combination with another anti-cancer agent and/or an anti-infection agent. In some embodiments, the patient diagnosed with an infection can have chronic viral infection, intracellular bacterial infection, extracellular bacterial infection, and/or fungal infection.

As used herein, the term “in combination with” or “co-administer” in the context of therapy administration generally refers to administrating a first agent and at least a second agent. The first agent and the second agent can be administered concurrently or simultaneously (e.g., in the same or separate unit dosage forms), or separately at different times. The first agent and the second agent can be administered by the same or different route.

As used herein, an “anti-cancer agent” or “anti-cancer therapy” is generally an agent or a therapy for treatment of cancer, e.g., an agent that kills cancer cells, and/or reduces or prohibits tumor growth and/or progression. Examples of anti-cancer agents include, but are not limited to cancer vaccines, chemotherapy, targeted therapy (e.g., kinase inhibitors), radiation therapy, surgery, immunotherapy, and any combinations thereof. One of skill in the art can readily identify a chemotherapeutic agent for use in treatment of cancer (e.g. see Physicians' Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles of Cancer Therapy, Chapter 85 in Harrison's Principles of Internal Medicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 in Abeloff's Clinical Oncology, 2013 Elsevier; and Fischer D S (ed): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).

As used herein, an “anti-infection agent” or “anti-infection therapy” is generally an agent or a therapy that kills or inhibits a cellular process, development and/or replication of a target infectious agent. Examples of an anti-infection agent or therapy include, but are not limited to anti-viral agent or therapy, anti-bacterial agent or therapy, anti-fungal agent or therapy, and a combination of two or more thereof.

As used herein, an “anti-viral agent” or “anti-viral therapy” is generally an agent or a therapy that kills or inhibits cellular process, development and/or replication of a target virus. For example, an anti-viral agent can be an agent that interferes with one or more viral components and/or interferes with replication or propagation of a virus. Examples of anti-viral agents include, but are not limited to, virus protein specific antibodies, reverse transcriptase inhibitors, protease inhibitors, immunomodulatory agents (e.g., cytokines, various nucleoside analogs, and/or Zn²⁺), plant extracts demonstrated to have an antiviral effect, and any combinations thereof.

As used herein, the term “anti-bacterial agent” or “anti-bacterial therapy” refers to an agent that has bactericidal and/or bacteriostatic activity. The anti-bacterial agent can be naturally occurring or synthetic. In some embodiments, an anti-bacterial agent or therapy can comprise an antibiotic, e.g., to suppress the growth of other microorganisms. Non-limiting examples of anti-bacterial agents include β-lactam antibacterial agents including, e.g., ampicillin, cloxacillin, oxacillin, and piperacillin, cephalosporins and other cephems including, e.g., cefaclor, cefamandole, cefazolin, cefoperazone, cefotaxime, cefoxitin, ceftazidime, ceftriaxone, and cephalothin; carbapenems including, e.g., imipenem and meropenem; and glycopeptides, macrolides, quinolones, tetracyclines, and aminoglycosides. In general, if an antibacterial agent is bacteriostatic, it means that the agent essentially stops bacterial cell growth (but does not necessarily kill the bacteria); if the agent is bacteriocidal, it means that the agent kills the bacterial cells (and may stop growth before killing the bacteria).

As used herein, the term “anti-fungal agent” or “anti-fungal therapy” refers to an agent that is able to exert an inhibitory effect on the growth and/or development of a fungus. Such an effect can be classified as fungicidal, fungistatic, sporocidal, sporostatic, or a combination thereof. Examples of anti-fungal agent or therapy include, but are not limited to polyene-based, imidazole-based, triazole-based, thiazole-based, allylamine-based, echinocandin-based, and a combination of two or more thereof.

In some embodiments, the method can further comprise administering the patient an immunotherapy. As used herein, the term “immunotherapy” refers to a treatment that modifies or affects (e.g., stimulates or suppresses) response and/or number of at least a subset of immune cells. For example, the immunotherapy for treatment of cancer and/or infection can comprise an agent that increases a proinflammatory T cell response and/or an agent that suppresses an anti-inflammatory T cell response.

In some embodiments of this aspect and other related aspects described herein, the patient can be previously treated with or is being treated an anti-cancer therapy and/or anti-infection therapy. Thus, the anti-TIGIT, anti-Fgl2 and/or anti-IL-33 therapy can be used, alone or in combination with another anti-cancer agent and/or anti-infection agent. In some embodiments of this aspect and other related aspects described herein, the methods described herein can further comprise administering to the patient a selected therapy (e.g., anti-TIGIT or anti-IL-33 therapy) after they have been identified to be more likely to benefit from one immunotherapy over another.

As used herein, the term “selected therapy” or “selected treatment” refers to a therapy or treatment selected based on the level and/or activity of a target molecule (e.g., TIGIT, Fgl2 and/or IL-33) as measured in a sample of a subject to be treated according to the methods of various aspects described herein. In accordance with one aspect of the discovery that selective suppression by TIGIT+Tregs is Fgl2-dependent and that IL-33 can induce or expand the Treg population, one can predict the responsiveness of a patient to an agent that modulates TIGIT, Fgl2 and/or IL-33 expression and/or activity, by determining the level of TIGIT, Fgl2 and/or IL-33 expression or activity in the patient's sample, and thus select for the patient an appropriate therapy to which the patient is more likely to respond.

In some embodiments of this aspect and other related aspects described herein, the TIGIT inhibitor, Fgl2 inhibitor and/or IL-33 inhibitor administered to a patient diagnosed with cancer and/or infection can be constructed to specifically target TIGIT+regulatory T cells (Tregs) that infiltrate the tumor or infected tissue. For example, for treatment of a patient diagnosed with cancer, the TIGIT inhibitor, Fgl2 inhibitor and/or IL-33 inhibitor can comprise a tumor-targeting moiety. For treatment of a patient diagnosed with infection, the TIGIT inhibitor, Fgl2 inhibitor and/or IL-33 inhibitor can comprise a targeting moiety against infected tissue.

As used herein, the terms “treat,” “treatment,” “treating,” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder, such as an autoimmune disease, infection or a cancer. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of at least slowing of progress or worsening of symptoms that would be expected in absence of treatment. Beneficial or desired clinical results include, but are not limited to alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. The term “treatment” of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

The term “effective amount” as used herein refers to the amount of an agent for modulating expression and/or activity of TIGIT, Fgl2 and/or IL-33 needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect, i.e., promote or inhibit T cell tolerance, for example. The term “therapeutically effective amount” therefore refers to an amount of an agent for modulating expression and/or activity of TIGIT, Fgl2 and/or IL-33 using the methods as disclosed herein, that is sufficient to effect a particular effect when administered to a subject. An effective amount as used herein would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom of disease (for example but not limited to slow the progression of a symptom of the disease), or reverse a symptom of disease. Thus, it is not possible to specify the exact “effective amount”. However, for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.

Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the agent for modulating expression and/or activity of TIGIT, Fgl2 and/or IL-33), which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

A “cancer” or “tumor” as used herein refers to an uncontrolled growth of cells which interferes with the normal functioning of the bodily organs and systems. A subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject's body. Included in this definition are benign and malignant cancers, as well as dormant tumors, metastases, or micrometastases. Cancers which migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. Hemopoietic cancers, such as leukemia, are able to out-compete the normal hemopoietic compartments in a subject, thereby leading to hemopoietic failure (in the form of anemia, thrombocytopenia and neutropenia) ultimately causing death.

By “metastasis” is meant the spread of cancer from its primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in the distant site are also significant.

Metastases are most often detected through the sole or combined use of magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function studies, chest X-rays and bone scans in addition to the monitoring of specific symptoms.

Examples of cancer include, but are not limited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; as well as other carcinomas and sarcomas; as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.

As used herein, the term “chronic viral infection” refers to a viral infection having the provirus or virus material in the nucleus or cytoplasm of a host cell and which, until induced, has little or no detectable viral RNA or protein. Such infections can persist for many years, or even for the lifetime of the infected individual. Examples of chronic viral infection include, but are not limited to, Hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV) infections, and Herpes viruses.

In some embodiments of various aspects described herein, at least one or more TIGIT antagonist(s), Fgl2 antagonist(s) and/or IL-33 antagonist(s) can be administered to a patient with a bacterial infection. The bacterial infection can be caused by intracellular bacteria and/or extracellular bacteria. Examples of infectious bacteria include: Helicobacterpyloris, Borelia burgdorferi, Chlamydia trachomatis, Legionella pneumophilia, Mycobacteria sps (such as M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus anthracia, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, and Actinomyces israelli. The compositions and methods described herein are contemplated for use in treating infections caused by these bacterial agents.

In some embodiments of various aspects described herein, at least one or more TIGIT antagonist(s), Fgl2 antagonist(s) and/or IL-33 antagonist(s) can be administered to a patient with a viral infection. In some embodiments, the viral infection is a chronic viral infection. Examples of infectious viruses include: Retroviridae (for example, HIV); Picornaviridae (for example, polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (such as strains that cause gastroenteritis); Togaviridae (for example, equine encephalitis viruses, rubella viruses); Flaviridae (for example, dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (for example, coronaviruses); Rhabdoviridae (for example, vesicular stomatitis viruses, rabies viruses); Filoviridae (for example, ebola viruses); Paramyxoviridae (for example, parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (for example, influenza viruses); Bungaviridae (for example, Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and HSV-2, varicella zoster virus, cytomegalovirus (CMV), herpes viruses); Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (such as African swine fever virus); and unclassified viruses (for example, the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1=internally transmitted; class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and astroviruses). The compositions and methods described herein are contemplated for use in treating infections caused by these viral agents.

In some embodiments of various aspects described herein, at least one or more TIGIT antagonist(s), Fgl2 antagonist(s) and/or IL-33 antagonist(s) can be administered to a patient with a fungal infection. The compositions and methods described herein that dampen Th2 responses are contemplated for use in treating infections caused by fungi agents. Examples of fungal infections include but are not limited to: aspergillosis; thrush (caused by Candida albicans); cryptococcosis (caused by Cryptococcus); and histoplasmosis. Thus, examples of infectious fungi include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Candida albicans. The compositions and methods described herein that promote Th17 cell activity or responses are contemplated for use in treating infections with these fungal agents.

CD8+T cell exhaustion: It is contemplated that TIGIT signaling can play a role in establishing or maintain T cell exhaustion. A further aspect provided herein relates to a method for increasing the differentiation and/or proliferation of functionally exhausted CD8+T cells, or decreasing CD8+T cell exhaustion, in a subject in need thereof. The method comprises administering to the subject in need thereof a pharmaceutical composition comprising a TIGIT antagonist or inhibitor described herein.

As used herein, the term “T cell exhaustion” refers to a state of T cell dysfunction. The T cell exhaustion generally arises during many chronic infections and cancer. T cell exhaustion can be defined by poor effector function, sustained expression of inhibitory receptors, and/or a transcriptional state distinct from that of functional effector or memory T cells. T cell exhaustion generally prevents optimal control of infection and tumors. See, e.g., Wherry E J, Nat Immunol. (2011) 12: 492-499, for additional information about T cell exhaustion.

In some embodiments, the subject in need thereof of can be diagnosed with cancer. In some embodiments, the subject diagnosed with cancer has been receiving a cancer therapy, including, e.g., vaccine, chemotherapy, targeted therapy (e.g., kinase inhibitors), radiation therapy, surgery, immunotherapy, or any combination thereof.

In some embodiments, the subject in need thereof can be diagnosed with infection, e.g., but not limited to chronic viral infection, intracellular bacterial infection, extracellular bacterial infection, and/or fungal infection.

In some embodiments, the subject in need thereof can be diagnosed with chronic infection, e.g., chronic viral infection.

When the TIGIT antagonist administered to the subject is determined to be ineffective (e.g., no significant decrease in the level of TIGIT and/or Fgl2 expression and/or activity relative to a reference), the subject can be administered with an alternative therapy that suppresses anti-inflammatory T cell response pathway. Non-limiting examples of such alternative therapy include a TIM-3 antagonist, a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a Lag-3 antagonist, a BTLA antagonist, and any combinations thereof.

Methods and Compositions for Treating Autoimmune Diseases and Other Immune-Related Diseases where an Inhibition of Th1 and/or Th17 Responses and/or a Shift of Balance Toward a Th2 Response is Desirable

In some other immune-related diseases or disorders, e.g., but not limited to, inflammatory diseases or disorders such as parasitic infections and autoimmune diseases, it can be desirable to suppress proinflammatory Th1 and/or Th17 responses for a therapeutic effect, while sparing or promoting a Th2 response. Accordingly, these inflammatory diseases or disorders where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable can be treated by enhancing or stimulating the expression or activity of TIGIT, Fgl2 and/or IL-33 (or promoting the TIGIT axis signaling).

Accordingly, in some aspects, provided herein are methods of identifying a patient diagnosed to have an inflammatory disease or disorder who is more likely to be responsive to an anti-inflammatory immunotherapy, or a TIGIT agonist, Fgl2 agonist and/or IL-33 agonist therapy. Nonlimiting examples of an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable include autoimmune disease, parasitic infection, acute inflammation, chronic inflammation, and any combinations thereof. In some embodiments, patients having an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable can be identified as more likely to be responsive to a TIGIT agonist and/or an IL-33 agonist therapy based on the patients' level of Fgl2 activity or expression in a sample. In some embodiments, patients having an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable can be identified as more likely to be responsive to an IL-33 agonist therapy and/or an Fgl2 agonist therapy based on the patients' level of TIGIT activity or expression in a sample. In some embodiments, patients having an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable can be identified as more likely to be responsive to a TIGIT agonist and/or an Fgl2 agonist therapy based on the patients' level of IL-33 activity or expression in a sample.

By way of example only, some aspects provided herein relate to methods of identifying a patient with an inflammatory disease or disorder, who is more likely to be responsive to a TIGIT agonist and/or IL-33 agonist therapy, based on the level of Fgl2 activity or expression in the patient's sample. In some embodiments, the methods are directed to patients with inflammatory diseases or disorders where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable. The method comprises (a) measuring the level of Fgl2 activity or expression in a sample from a patient diagnosed to have an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable, e.g., autoimmune disease and/or parasitic infection; and (b) comparing the level of Fgl2 or expression in the sample with an Fgl2 reference; and: (i) when the level of Fgl2 activity or expression is lower than the Fgl2 reference, the patient is identified to be more likely to be responsive to a TIGIT agonist and/or IL-33 agonist therapy; or (ii) when the level of Fgl2 activity or expression is the same as or greater than the Fgl2 reference, the patient is identified as likely to respond to an alternative, anti-inflammatory immunotherapy comprising an activator of an anti-inflammatory T cell response pathway and/or a suppressor of a proinflammatory T cell response pathway.

In this aspect and other aspects described herein, any appropriate modulators of a T cell response pathway that are known in the art can be used in the alternative immunotherapy for patients with an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward a Th2 response is desirable, e.g., autoimmune diseases and/or parasitic infection. For example, activators of an anti-inflammatory T cell response or suppressors of a proinflammatory T-cell response pathway can comprise a TIM-3 agonist, a galectin-9 molecule, a PD-1 agonist, a PD-L1 agonist, a CTLA-4 agonist, a Lag-3 agonist, a DD1α agonist, an antagonist of an immune checkpoint activating molecule, an agonist of an immune checkpoint inhibitory molecule, or any combination thereof.

Some aspects provided herein relate to methods of treating a patient who is determined to have an inflammatory disease or disorder. A TIGIT agonist, Fgl2 agonist and/or IL-33 agonist therapy can be selected for administration to a patient with an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward Th2 responses is desirable, based on the level of Fgl2, IL-33 and/or TIGIT in the patient's sample. In some embodiments, a TIGIT agonist and/or IL-33 agonist therapy can be selected for administration to a patient with an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward Th2 responses is desirable, based on the patient's level of Fgl2 activity or expression in a sample. In some embodiments, an IL-33 agonist therapy and/or Fgl2 agonist therapy can be selected for administration to a patient with an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward Th2 responses is desirable, based on the patient's level of TIGIT activity or expression in a sample. In some embodiments, a TIGIT agonist and/or Fgl2 agonist therapy can be selected for administration to a patient with an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward Th2 responses is desirable, based on the patient's level of IL-33 activity or expression in a sample.

For example, some aspects provided herein relate to methods for treating a patient who is determined to have an inflammatory disease or disorder based on the level of IL-33 activity or expression in a sample from the patient. In some embodiments where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward Th2 responses is desirable, the method comprises (a) measuring the level of IL-33 activity or expression in a sample from a patient who is determined to have this type of an inflammatory disease or disorder; (b) comparing the level of IL-33 activity or expression in the sample with an IL-33 reference, and (c) performing one of the following actions:

- (i) administering to the patient a composition comprising a TIGIT agonist and/or an Fgl2 agonist, when the level of IL-33 activity or expression is lower than the IL-33 reference;
- (ii) administering an alternative, anti-inflammatory immunotherapy treatment without the TIGIT agonist or Fgl2 agonist, when the level of IL-33 activity or expression is the same as or greater than the IL-33 reference; or
- (iii) determining if the level of at least one other activating immune regulator in the sample is greater than the level of the corresponding reference, or if the level of at least one inhibitory immune regulator in the sample is less than the level of the corresponding reference, when the level of IL-33 activity or expression is the same as or greater than the IL-33 reference. Examples of inhibitory immune regulator include, but are not limited to, Fgl2, TIGIT, ST2, CD155, CD112, PD-1, PD-L1, DD1α, TIM-3, galectin-9, CTLA-4, Lag-3, and any combination thereof.

In some embodiments where the level of IL-33 activity or expression is the same as or greater than the IL-33 reference, the method can further comprise (a) measuring the level of TIGIT activity or expression, or frequency of TIGIT+T cells in a sample from the patient, and (b) comparing the level of TIGIT activity or expression, or frequency of TIGIT+T cells in the sample with a TIGIT reference. If the level of TIGIT activity or expression, or frequency of TIGIT+T cells is low relative to a TIGIT reference, an increase in TIGIT via the TIGIT axis signaling can help inflammatory conditions where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward Th2 responses is desirable. For example, a composition comprising a TIGIT agonist and/or an Fgl2 agonist can be administered to such a patient when the level of TIGIT activity or expression, or frequency of TIGIT+ cells is lower than the TIGIT reference. However, if the level of TIGIT activity or expression, or frequency of TIGIT+T cells is the same or high relative to a TIGIT reference, an alternative, anti-inflammatory immunotherapy treatment without a TIGIT agonist or Fgl2 agonist can be administered.

In some embodiments where the level of IL-33 activity or expression is the same as or greater than the IL-33 reference, the method can further comprise (a) measuring the level of Fgl2 activity or expression in a sample from the patient, and (b) comparing the level of Fgl2 activity or expression in the sample with an Fgl2 reference. The patient can be administered with a composition comprising a TIGIT agonist and/or an Fgl2 agonist, when the level of Fgl2 activity or expression is lower than the Fgl2 reference; or with an alternative, anti-inflammatory immunotherapy treatment without a TIGIT agonist or Fgl2 agonist, when the level of Fgl2 activity or expression is the same as or greater than the reference.

In some other aspects, provided herein are methods of treating a patient determined to have an inflammatory disease or disorder based on the level of Fgl2 activity or expression in the patient's sample. In some embodiments where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward Th2 responses is desirable, the method comprises (a) measuring the level of Fgl2 activity or expression in a sample from a patient determined to have an inflammatory disease or disorder; (b) comparing the level of Fgl2 activity or expression in the sample with an Fgl2 reference; and (c) administering to the patient a composition comprising a TIGIT agonist and/or an IL-33 agonist when the level of Fgl2 activity or expression is lower than the Fgl2 reference, or administering an alternative, anti-inflammatory immunotherapy treatment without a TIGIT agonist or IL-33 agonist when the level of Fgl2 activity or expression is the same as or greater than the Fgl2 reference. In some embodiments, the alternative, anti-inflammatory immunotherapy treatment without a TIGIT agonist or IL-33 agonist can be a therapy comprising an activator of an anti-inflammatory T cell response pathway and/or a suppressor of a proinflammatory T cell response pathway.

In some aspects described herein, TIGIT, Fgl2 and/or IL-33 can be used as a marker to determine or monitor the efficacy of a TIGIT agonist, Fgl2 agonist and/or IL-33 agonist therapy administered to a patient diagnosed to have an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward Th2 responses is desirable. In some embodiments, Fgl2 can be used as a predictive marker to determine or monitor the efficacy of a TIGIT agonist and/or IL-33 agonist therapy administered to a patient diagnosed to have an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward Th2 responses is desirable. In some embodiments, TIGIT can be used as a predictive marker to determine or monitor the efficacy of an IL-33 agonist therapy and/or Fgl2 agonist therapy administered to a patient diagnosed to have an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward Th2 responses is desirable. In some embodiments, IL-33 can be used as a predictive marker to determine or monitor the efficacy of a TIGIT agonist and/or Fgl2 agonist therapy administered to a patient diagnosed to have an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward Th2 responses is desirable.

As an example, methods of treating a patient having an inflammatory disease or disorder and a low level of Fgl2 are provided herein. In some embodiments where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward Th2 responses is desirable, the method comprises: (a) determining a first level of Fgl2 expression or activity in a sample from a patient having an inflammatory disease or disorder and a low level of Fgl2; (b) administering an agent that activates IL-33 activity and/or TIGIT activity; (c) determining a second level of Fgl2 expression or activity after the administering; and (d) comparing the first and second levels of Fgl2 expression or activity, wherein the agent administered in (b) is effective if the second level of Fgl2 expression or activity is greater than the first level, and wherein the agent administered in (b) is ineffective if the second level of Fgl2 expression is the same as or lower than the first level.

Similarly, a further aspect provided herein relates to methods of treating a patient having an inflammatory disease or disorder that exhibits a reduced level of IL-33. In some embodiments where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward Th2 responses is desirable, the method comprises: (a) determining a first level of TIGIT and/or Fgl2 expression or activity in a sample from a patient having an inflammatory disease or disorder with a reduced level of IL-33; (b) administering an agent that activates IL-33 activity; (c) determining a second level of TIGIT or Fgl2 expression or activity after the administering; and (d) comparing the first and second levels of TIGIT and/or Fgl2 expression or activity, wherein IL-33 agonist therapy is effective if the second level of TIGIT and/or Fgl2 expression or activity is greater that the first level, and wherein IL-33 agonist therapy is ineffective if the second level of TIGIT and/or Fgl2 expression is the same as or lower than the first level.

In yet another aspect, methods of treating a patient determined to have an inflammatory disease or disorder comprising administering to a patient determined to have an inflammatory disease or disorder one or more embodiments of the pharmaceutical compositions described herein are also provided. The pharmaceutical composition can be taken alone or in combination with another agent for treatment of an inflammatory disease or disorder. An exemplary anti-inflammatory agent includes, but is not limited to, an immunotherapy. In some embodiments, the method can further comprise administering the patient an immunotherapy for treatment of an inflammatory disease or disorder. For example, the immunotherapy can comprise an agent that activates an anti-inflammatory T cell response and/or an agent that suppresses a proinflammatory T cell response.

In some embodiments of this aspect and other related aspects described herein, the patient having an inflammatory disease or disorder can be previously treated with or is being treated an anti-inflammatory therapy. Thus, the TIGIT agonist, Fgl2 agonist and/or IL-33 agonist therapy can be used, alone or in combination with another anti-inflammatory agent. In some embodiments of this aspect and other related aspects described herein, the methods described herein can further comprise administering to the patient a selected therapy (e.g., TIGIT agonist or IL-33 agonist therapy) after they have been identified to be more likely to benefit from one immunotherapy over another.

In some embodiments of this aspect and other related aspects described herein, the TIGIT agonist, Fgl2 agonist and/or IL-33 agonist administered to a patient can be constructed or adapted to specifically target TIGIT+regulatory T cells (Tregs). For example, the TIGIT agonist, Fgl2 agonist and/or IL-33 agonist can comprise a cell-targeting moiety.

As used herein, the term “cell-targeting moiety” refers to a molecule or entity that facilitates delivery of an agent to a target cell. For example, a cell-targeting moiety can be a molecule or entity that interacts with a binding site on the surface of a target cell. Thus, the targeting moiety provides specificity or binding affinity for one or more cell types. The molecule on a target cell which is targeted by the targeting moiety can be any selected target, for instance a cell surface receptor. Cell-targeting moieties include, but are not limited to, antibodies, antigen-binding antibody fragments, ligands for a cell-surface receptor, viral surface components, proteins that bind viral surface components, growth factors, lectins, carbohydrates, fatty acids or other hydrophobic substituents, peptides and peptidomimetic molecules. In one embodiment, the cell-targeting moiety is a molecule or entity that interacts with a binding site on the surface of a TIGIT+regulatory T cell.

In some embodiments of this aspect and other related aspects described herein, the TIGIT agonist, Fgl2 agonist and/or IL-33 agonist can be co-administered with an agent, such as a chemokine, that promotes recruitment of TIGIT+regulatory T cells (Tregs) to an inflammatory site or tissue.

In some embodiments of various aspects described herein, the TIGIT agonist, Fgl2 agonist and/or IL-33 agonist can be administered to a patient with an autoimmune disease. “Autoimmune disease” refers to a class of diseases in which a subject's own antibodies react with host tissue or in which immune effector T cells are autoreactive to endogenous self-peptides and cause destruction of tissue. Thus an immune response is mounted against a subject's own antigens, referred to as self-antigens. A “self-antigen” as used herein refers to an antigen of a normal host tissue. Normal host tissue does not include cancer cells.

Accordingly, in some embodiments, the autoimmune diseases to be treated or prevented using the methods described herein, include, but are not limited to: rheumatoid arthritis, Crohn's disease, multiple sclerosis, systemic lupus erythematosus (SLE), autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto's thyroiditis, Goodpasture's syndrome, pemphigus (e.g., pemphigus vulgaris), Grave's disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis, pernicious anemia, idiopathic Addison's disease, autoimmune-associated infertility, glomerulonephritis (e.g., crescentic glomerulonephritis, proliferative glomerulonephritis), bullous pemphigoid, Sjogren's syndrome, insulin resistance, and autoimmune diabetes mellitus (type 1 diabetes mellitus; insulin-dependent diabetes mellitus). Autoimmune disease has been recognized also to encompass atherosclerosis and Alzheimer's disease. In one embodiment of the aspects described herein, the autoimmune disease is selected from the group consisting of multiple sclerosis, type-I diabetes, Hashinoto's thyroiditis, Crohn's disease, rheumatoid arthritis, systemic lupus erythematosus, gastritis, autoimmune hepatitis, hemolytic anemia, autoimmune hemophilia, autoimmune lymphoproliferative syndrome (ALPS), autoimmune uveoretinitis, glomerulonephritis, Guillain-Barre syndrome, psoriasis and myasthenia gravis.

In some embodiments of various aspects described herein, the TIGIT agonist, Fgl2 agonist and/or IL-33 agonist can be administered to a patient having an infection with a pathogen, such as a parasite. In some embodiments of these aspects and all such aspects described herein, the subject has a chronic infection.

The compositions and methods described herein that promote suppression of Th1 and/or Th17 responses and thus shift the balance toward Th2 responses are contemplated for the treatment of infection caused by parasites (e.g., helmiths among others) and intracellular pathogens. Other infectious organisms (such as protists) include: Plasmodium falciparum and Toxoplasma gondii.

Methods for Treating Asthma, Allergy, and/or Atopy

Without wishing to be bound by theory, TIGIT can promote allergy/asthma/atopy, e.g., by inducing the level of expression and/or activity of Fgl2, thereby suppressing the Th1 and/or Th17 versus Th2 balance in favor of Th2 cytokine responses. Accordingly, it is also contemplated that other inflammatory diseases or disorders, including, e.g., allergy/asthma/atopy, where a dampening of the Th2 response is desirable, could be treated by downregulating the expression or activity of TIGIT, Fgl2 and/or IL-33 (or suppressing the TIGIT axis signaling).

In one aspect, provided herein is a method for treating asthma, allergy, and/or atopy. The method comprises administering to a patient diagnosed with asthma, allergy, and/or atopy a composition comprising an anti-Fgl2 therapy. In some embodiments, the method can further comprise identifying a patient diagnosed with asthma, allergy, and/or atopy who is more likely to respond to an anti-Fgl2 therapy, e.g., based on the level of expression and/or activity of TIGIT and/or IL-33. When the level of TIGIT and/or IL-33 activity or expression is greater than the TIGIT and/or IL-33 reference, the patient is identified to be more likely to be responsive to an anti-Fgl2 therapy; or (ii) when the level of TIGIT and/or IL-33 activity or expression is the same as or less than the TIGIT and/or IL-33 reference, the patient is identified as likely to respond to an alternative, Th2-dampening therapy or immunotherapy. In some embodiments, the alternative, Th2-dampening therapy or immunotherapy can comprise, e.g., an activator of proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.

Generally, methods and compositions for treatment of cancer and/or infections described herein that stimulate Th1 and/or Th17 responses and thus shift the balance away from Th2 responses can be adapted accordingly for treatment of allergy, asthma and/or atopy, where a dampening Th2 response is desirable.

Accordingly, in some aspects, provided herein are methods for guiding selection of a treatment for a subject diagnosed with asthma, allergy, and/or atopy. In some embodiments, patients diagnosed with asthma, allergy, and/or atopy can be identified as more likely to be responsive to an anti-TIGIT and/or anti-IL-33 therapy based on the patients' level of Fgl2 activity or expression in a sample. In some embodiments, patients diagnosed with asthma, allergy, and/or atopy can be identified as more likely to be responsive to an anti-IL-33 therapy and/or anti-Fgl2 therapy based on the patients' level of TIGIT activity or expression in a sample. In some embodiments, patients diagnosed with asthma, allergy, and/or atopy can be identified as more likely to be responsive to an anti-TIGIT and/or anti-Fgl2 therapy based on the patients' level of IL-33 activity or expression in a sample.

By way of example only, one aspect provided herein relates to methods for guiding selection of a treatment for a subject diagnosed with asthma, allergy, and/or atopy, based on the level of Fgl2 activity or expression in the patient's sample. The method comprises (a) measuring the level of Fgl2 activity or expression in a sample from a patient diagnosed with asthma, allergy, and/or atopy; and (b) comparing the level of Fgl2 or expression in the sample with an Fgl2 reference; and: (i) when the level of Fgl2 activity or expression is greater than the Fgl2 reference, the patient is identified to be more likely to be responsive to an anti-TIGIT and/or anti-IL-33 therapy; or (ii) when the level of Fgl2 activity or expression is the same as or less than the Fgl2 reference, the patient is identified as likely to respond to an alternative, Th2-dampening therapy or immunotherapy. In some embodiments, the alternative, Th2-dampening therapy or immunotherapy can comprise, e.g., an activator of proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.

Accordingly, in some embodiments, the Fgl2 reference can correspond to the level of expression or activity of Fgl2 in a normal healthy subject. In some embodiments, the Fgl2 reference can correspond to the level of expression or activity of Fgl2 in a normal tissue of the same type or lineage as a blood or tissue biopsy obtained from a patient. The normal tissue of the same type or lineage can be obtained from the same or a different patient. In some embodiments, the Fgl2 reference can correspond to a threshold level of expression or activity of Fgl2, above which the level of Fgl2 expression activity measured in a sample from a patient diagnosed with asthma, allergy, and/or atopy would indicate the likelihood of the patient to respond to a treatment. When the level of Fgl2 activity or expression is greater than the Fgl2 reference, e.g., by at least about 10% or more, including, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more, the patient diagnosed with asthma, allergy, and/or atopy is identified to be more likely to be responsive to an anti-TIGIT and/or anti-IL-33 therapy. In some embodiments, when the level of Fgl2 activity or expression is greater than the Fgl2 reference, e.g., by at least about 1.1-fold or more, including, e.g., at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold or more, the patient diagnosed with asthma, allergy, and/or atopy is identified to be more likely to be responsive to an anti-TIGIT and/or anti-IL-33 therapy. On the other hand, when the level of Fgl2 activity or expression is substantially the same as or less than the Fgl2 reference, e.g., by at least about 10% or more, including, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, the patient diagnosed with asthma, allergy, and/or atopy is identified as likely to respond to an alternative, Th2-dampening therapy or immunotherapy without the anti-TIGIT and/or anti-IL-33 therapy. In some embodiments, the alternative, Th2-dampening therapy or immunotherapy can comprise, e.g., an activator of a proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway, e.g., without the need to suppress TIGIT, Fgl2, or IL-33 activity.

As used herein, the term “Th2-dampening T cell response” refers to a response resulting in reduced production of Th2 cytokines by T cells. In some embodiments, the Th2-dampening T cell response can encompass promoting Th1 and/or Th17 responses.

- (i) administering to the patient a composition comprising a TIGIT inhibitor and/or an Fgl2 inhibitor, when the level of IL-33 activity or expression is greater than the IL-33 reference;
- (ii) administering an alternative, Th2-dampening therapy or immunotherapy without the TIGIT inhibitor or Fgl2 inhibitor, when the level of IL-33 activity or expression is the same as or less than the IL-33 reference; or
- (iii) determining if the level of at least one other inhibitory immune regulator in the sample is greater than the level of the corresponding reference, or if the level of at least one activating immune regulator in the sample is less than the level of the corresponding reference, when the level of IL-33 activity or expression is the same as or less than the IL-33 reference.

In some embodiments, the IL-33 reference can correspond to the level of expression or activity of IL-33 in a normal healthy subject. In some embodiments, the IL-33 reference can correspond to the level of expression or activity of IL-33 in normal blood or a normal tissue of the same type or lineage as a tissue biopsy obtained from a patient. The normal tissue of the same type or lineage can be obtained from the same or a different patient. In some embodiments, the IL-33 reference can correspond to the level of expression or activity of IL-33 in a patient's sample obtained at a different or prior time point. In some embodiments, the IL-33 reference can correspond to a threshold level of expression or activity of IL-33, above which the level of IL-33 expression or activity measured in a sample from a patient diagnosed with asthma, allergy, and/or atopy would indicate the likelihood of the patient to respond to a treatment. When the level of IL-33 activity or expression is greater than the IL-33 reference, e.g., by at least about 10% or more, including, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more, the patient diagnosed with asthma, allergy, and/or atopy is identified to be more likely to be responsive to a TIGIT inhibitor and/or Fgl2 inhibitor. In some embodiments, when the level of IL-33 activity or expression is greater than the IL-33 reference, e.g., by at least about 1.1-fold or more, including, e.g., at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold or more, the patient diagnosed with asthma, allergy, and/or atopy is identified to be more likely to be responsive to a TIGIT inhibitor and/or Fgl2 inhibitor. On the other hand, when the level of IL-33 activity or expression is substantially the same as or less than the IL-33 reference, e.g., by at least about 10% or more, including, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, the patient diagnosed with asthma, allergy, and/or atopy is identified as likely to respond to an alternative, Th2-dampening therapy or immunotherapy. In some embodiments, the alternative, Th2-dampening therapy or immunotherapy can comprise, e.g., an activator of a pro-inflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway, without the need to suppress the TIGIT axis.

In some embodiments where the level of IL-33 activity or expression is the same as or less than the IL-33 reference, the method can further comprise (a) measuring the level of Fgl2 activity or expression in a sample from the patient diagnosed with asthma, allergy, and/or atopy, (b) comparing the level of Fgl2 activity or expression in the sample with an Fgl2 reference, and (c) administering to the patient a composition comprising a TIGIT inhibitor and/or an Fgl2 inhibitor, when the level of Fgl2 activity or expression is greater than the Fgl2 reference (e.g., by at least about 30% or more, including, e.g., at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold or higher); or administering an alternative, Th2-dampening therapy or immunotherapy without a TIGIT inhibitor or Fgl2 inhibitor, when the level of Fgl2 activity or expression is the same as or less than the reference (e.g., by at least about 30% or more).

In some embodiments where the level of IL-33 and/or Fgl2 activity or expression is the same as or less than the reference (e.g., by at least about 30% or more, including, e.g., at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more), a Th2-dampening therapy or immunotherapy without a TIGIT inhibitor or Fgl2 inhibitor can also be administered. In some embodiments, the alternative, Th2-dampening therapy or immunotherapy can comprise, e.g., an activator of a pro-inflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway as described earlier.

In some aspects described herein, TIGIT, Fgl2 and/or IL-33 can be used as a marker to determine or monitor the efficacy of an anti-TIGIT, anti-Fgl2 and/or anti-IL-33 therapy administered to a patient diagnosed with asthma, allergy, and/or atopy. In some embodiments, Fgl2 can be used as a predictive marker to determine or monitor the efficacy of an anti-TIGIT and/or anti-IL-33 therapy administered to a patient diagnosed with asthma, allergy, and/or atopy. In some embodiments, TIGIT can be used as a predictive marker to determine or monitor the efficacy of an anti-IL-33 therapy and/or anti-Fgl2 therapy administered to a patient diagnosed with asthma, allergy, and/or atopy. In some embodiments, IL-33 can be used as a predictive marker to determine or monitor the efficacy of an anti-TIGIT and/or anti-Fgl2 therapy administered to a patient diagnosed with asthma, allergy, and/or atopy.

By monitoring the effects of the anti-IL-33 and/or anti-TIGIT therapy on the level of Fgl2 expression or activity, one can determine the efficacy of the treatment regimen and adjust the treatment regimen if necessary. Accordingly, in some embodiments, the method can further comprise, when the anti-IL-33 or anti-TIGIT therapy is effective, continuing to administer the agent that inhibits IL-33 activity and/or TIGIT activity. In some embodiments, the method can further comprise, when the anti-IL-33 therapy or the anti-TIGIT therapy is ineffective, administering the agent that inhibits IL-33 activity and/or TIGIT activity at a higher dose. In some embodiments, the method can further comprise, when the anti-IL-33 therapy or the anti-TIGIT therapy is ineffective, discontinuing the anti-IL-33 therapy or the anti-TIGIT therapy. In these embodiments, the method can further comprise administering a therapy comprising an activator of a pro-inflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.

In some embodiments, the method can further comprise, when the anti-IL-33 therapy is effective, continuing to administer the agent that inhibits IL-33 activity. In some embodiments, the method can further comprise, when the anti-IL-33 therapy is ineffective, administering the agent that inhibits IL-33 activity at a higher dose. In other embodiments, the method can further comprise, when the anti-IL-33 therapy is ineffective, discontinuing the anti-IL-33 therapy. In these embodiments, the method can further comprise administering a therapeutic agent for treatment of asthma, allergy, and/or atopy. In some embodiments, the therapeutic agent can comprise, e.g., an activator of a pro-inflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.

In yet other aspects, methods of treating a patient diagnosed with asthma, allergy, and/or atopy comprising administering to a patient diagnosed with asthma, allergy, and/or atopy one or more embodiments of the pharmaceutical compositions described herein that provide a Th2-dampening effect are also provided. The pharmaceutical composition can be taken alone or in combination with another agent for treatment of asthma, allergy, and/or atopy. In some embodiments, the method can further comprise administering the patient an immunotherapy for treatment of asthma, allergy, and/or atopy. For example, an immunotherapy for treatment of asthma, allergy, and/or atopy can comprise an agent that increases a pro-inflammatory T cell response and/or an agent that suppresses an anti-inflammatory T cell response.

In some embodiments of this aspect and other related aspects described herein, the patient diagnosed with asthma, allergy, and/or atopy can be previously or currently being treated for the disease or disorder. Thus, the anti-TIGIT, anti-Fgl2 and/or anti-IL-33 therapy can be used, alone or in combination with another anti-asthmatic agent, anti-allergy agent, and/or anti-atopic agent. Examples of anti-asthmatic agents include, but are not limited to beta adrenergic agonists, xanthine derivatives, corticosteroids, antileukotrienes, and any combinations thereof. Exemplary anti-allergy agents and anti-atopic agents include, but are not limited to antihistamines, corticosteroids, and combinations thereof. In some embodiments of this aspect and other related aspects described herein, the methods described herein can further comprise administering to the patient diagnosed with asthma, allergy, and/or atopy a selected therapy (e.g., anti-TIGIT or anti-IL-33 therapy) after they have been identified to be more likely to benefit from one immunotherapy over another.

As used herein, the term “asthma” is intended to cover all types of asthma. Asthma is a chronic lung disease or disorder that inflames and narrows the airways.

As used herein, the term “allergy” refers to a disorder (or improper reaction) of the immune system often also referred to as “atopy.” Allergic reactions can occur when a subject's immune system reacts to environmental substances that are normally harmless to those without allergy. The substances that cause such allergic reactions are known as allergens. In some embodiments, allergy refers to type I (or immediate) hypersensitivity. Allergic reactions occur when there is excessive activation of certain white blood cells (e.g., mast cells and basophils) by immunoglobulin E (IgE). Common allergic reactions include eczema, hives, hay fever, asthma, food allergies, and reactions to the venom of stinging insects such as wasps and bees. Mild allergies like hay fever are highly prevalent in the human population and cause symptoms such as allergic conjunctivitis, itchiness, and runny nose. Allergies can play a role in conditions such as asthma.

TIGIT and Antagonists (Inhibitors) or Agonists Thereof

TIGIT is an immune receptor known as T cell Ig and ITIM (immunoreceptor tyrosine-based inhibitor motif) domain protein. TIGIT is also known as WUCAM and VSTM3. Boles et al. European Journal of Immunology; 39: 695-703; and Levin et al. European Journal of Immunology; 41:902-915. Generally, TIGIT is expressed as a cell surface protein on a variety of immune cells, e.g., regulatory T cells (Tregs), memory T cells, natural killer cells, and follicular T helper cells. TIGIT can directly suppress T cell responses, e.g., but not limited to T cell proliferation and/or proinflammatory cytokine production. In some embodiments, TIGIT+T cells can suppress proliferation of other, TIGIT negative T cells and other immune cells such as antigen presenting cells.

As used herein, the term “TIGIT” generally refers to a TIGIT polypeptide or a TIGIT polynucleotide that is similar or identical to the sequence of a wild-type TIGIT.

In some embodiments, the term “TIGIT” refers to a TIGIT polypeptide having an amino acid sequence that is at least 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to that of a wild-type TIGIT, and is capable of suppressing Th1 and/or Th17 responses. In some embodiments, the TIGIT polypeptide can also increase expression and/or activity of Fgl2 to mediate the suppression of Th1 and/or Th17 responses.

In some embodiments, the term “TIGIT” refers to a TIGIT polynucleotide having a nucleotide sequence that is at least 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to that of a wild-type TIGIT or a portion thereof, and encodes a TIGIT polypeptide as described herein.

The wild-type TIGIT sequences of various species are available on the world wide web from the NCBI, including human, mouse, rat, dog, and chimpanzee. For example, the nucleotide sequence encoding human TIGIT is available at NCBI under Accession No. NM_173799 and its corresponding amino acid sequence is under Accession No. NP_776160.

Where the term “TIGIT” refers to a TIGIT polypeptide, the term “TIGIT polypeptide” also encompasses a portion or fragment of such a TIGIT polypeptide that retains at least about 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) of the immunosuppressive activity of the wild-type TIGIT polypeptide. The term “TIGIT polypeptide” as used herein also encompasses conservative substitution variants of a TIGIT polypeptide that retain at least about 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) of the immunosuppressive activity of the wild-type TIGIT polypeptide. Accordingly, a TIGIT polypeptide refers to any immunosuppressive form of TIGIT, including functional variants of TIGIT. For example, in some embodiments, a TIGIT polypeptide can be a full-length TIGIT. In some embodiments, a TIGIT polypeptide refers to a functional domain or domains of TIGIT that induces immunosuppression and expression and/or activity of Fgl2.

The amino acid identity between two polypeptides can be determined, for example, by first aligning the two polypeptide sequences using an alignment algorithm, such as BLAST® or by other methods well-known in the art.

In various aspects described herein, methods for measuring TIGIT or a fragment thereof from a sample are known in the art, including, but not limited to mRNA expression using PCR or real-time PCR, protein analysis using western blot, immunoassay, and/or ELISA, and/or sequencing analysis. Thus, in some embodiments, nucleic acid molecules can be isolated from a patient's sample to measure TIGIT mRNA expression, or proteins can be isolated to measure TIGIT protein expression.

As used interchangeably herein, the term “antagonist” or “inhibitor” is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native polypeptide disclosed herein. In a similar manner, the term “agonist” is used in the broadest sense and includes any molecule that mimics a biological activity of a native polypeptide disclosed herein. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, antisense oligonucleotides, small organic molecules, recombinant proteins or peptides, etc. Methods for identifying agonists or antagonists of a polypeptide can comprise contacting a polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the polypeptide.

The term “TIGIT antagonist” is used interchangeably with the terms “TIGIT inhibitor” and “anti-TIGIT therapy” and refers to an agent that interferes with the normal functioning of TIGIT, either by decreasing transcription or translation of TIGIT-encoding nucleic acid, or by inhibiting or blocking TIGIT polypeptide activity, or both. Examples of TIGIT antagonists include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, TIGIT-specific aptamers, anti-TIGIT antibodies, TIGIT-binding fragments of anti-TIGIT antibodies, TIGIT-binding small molecules, TIGIT-binding peptides, and other polypeptides that specifically bind TIGIT (including, but not limited to, TIGIT-binding fragments of one or more TIGIT ligands, optionally fused to one or more additional domains), such that the interaction between the TIGIT antagonist and TIGIT results in a reduction or cessation of TIGIT activity or expression. It will be understood by one of ordinary skill in the art that in some instances, a TIGIT antagonist can antagonize one TIGIT activity without affecting another TIGIT activity. For example, a desirable TIGIT antagonist for use in certain of the methods herein is a TIGIT antagonist that antagonizes TIGIT activity in response to one of ligand interaction, CD112 interaction, or CD155 interaction, e.g., without affecting or minimally affecting any of the other TIGIT interactions.

In some embodiments, a TIGIT inhibitor is an agent that directly or indirectly inhibits or reduces the TIGIT-mediated suppression of proinflammatory Th1 and/or Th17 responses. Accordingly, a TIGIT inhibitor can target the TIGIT receptor or its corresponding ligand, or any of TIGIT's upstream molecules. Examples of TIGIT inhibitors include, without limitations, TIGIT−/−immune cells (e.g., T cells), anti-TIGIT molecules, ST2 inhibitors, CD112 inhibitors, CD155 inhibitors, and a combination thereof. A TIGIT inhibitor can be a protein, a peptide, a peptidomimetic, an aptamer, a nucleic acid, an antibody, a small molecule, a vaccine, or any combinations thereof.

In some embodiments, a TIGIT inhibitor is an anti-TIGIT antibody (e.g., an anti-human TIGIT antibody). Anti-TIGIT antibodies are commercially available (e.g., from R&D Systems (Clone 741182; Cat. No. MAB7898), Affymetrix eBioscience (clone MBSA43; Cat. No. 12-9500-41 or 12-9500-42), and Abcam (Cat. No. ab107664)).

In some embodiments, a TIGIT inhibitor can be a fragment or variant of TIGIT itself, e.g., a fragment that binds a TIGIT ligand (e.g., CD112 and/or CD155) but does not transmit an immunosuppressive signal (e.g., via Fgl2 expression). A TIGIT inhibitor of this type can be a dominant negative inhibitor.

In some embodiments, a TIGIT inhibitor is a recombinant soluble TIGIT Fc fusion protein. An exemplary recombinant soluble TIGIT Fc fusion protein can be obtained from R&D Systems (Cat. No. 7267-TG-050).

As used herein, the term “TIGIT agonist” refers to an agent that enhances or stimulates the normal functioning of TIGIT, by increasing transcription or translation of TIGIT-encoding nucleic acid, and/or by inhibiting or blocking activity of a molecule that inhibits TIGIT expression or TIGIT activity, and/or by enhancing normal TIGIT activity (including, but not limited to, enhancing the stability of TIGIT or enhancing binding of TIGIT to one or more target ligands such as CD112 or CD155). For example, the TIGIT agonist can be selected from an antibody, an antigen-binding fragment, an aptamer, an interfering RNA, a small molecule, a peptide, an antisense molecule, and another binding polypeptide. In another example, the TIGIT agonist can be a polynucleotide selected from an aptamer, interfering RNA, or antisense molecule that interferes with the transcription and/or translation of a TIGIT-inhibitory molecule. It will be understood by one of ordinary skill in the art that in some instances, a TIGIT agonist can agonize one TIGIT activity without affecting another TIGIT activity. For example, a desirable TIGIT agonist for use in certain of the methods herein is a TIGIT agonist that agonizes TIGIT activity in response to one of ligand interaction, CD155 interaction, or CD112 interaction, e.g., without affecting or minimally affecting any of the other TIGIT interactions.

In some embodiments, a TIGIT agonist is an agent that directly or indirectly enhances or stimulates the TIGIT-mediated suppression of proinflammatory Th1 and/or Th17 responses. Accordingly, a TIGIT agonist can target the TIGIT receptor or its corresponding ligand, or any of TIGIT's upstream molecules. Examples of TIGIT agonists include, without limitations, TIGIT-overexpressing immune cells (e.g., T cells), ST2 agonists, CD112 agonists, CD155 agonists, and a combination thereof. The TIGIT agonists can be a protein, a peptide, peptidomimetic, an aptamer, a nucleic acid, an antibody, a small molecule, a vaccine, a fusion protein, or any combinations thereof.

In some embodiments, a TIGIT agonist is an agonistic TIGIT antibody (e.g., an agonistic antibody to human TIGIT). Agonistic TIGIT antibody can be provided by ZymoGenetics, Inc. Agonistic TIGIT antibodies against human TIGIT are described by Lozano et al. (Journal of Immunology, 2012; 188: 3869-3875)

TIGIT antagonists or agonists can be obtained from known sources or prepared using known techniques such as recombinant or synthetic technology. The nucleic acid and protein sequences of TIGIT and its ligands of different species (e.g., but not limited to, human, mouse, rat, dog, chimpanzee) are known in the art, e.g., accessible at world wide web from NCBI. Thus, one of skill in the art can generate TIGIT antagonists or agonists based on these sequences using art-recognized molecular technologies such as cloning and expression technologies. For example, a human TIGIT antagonist (e.g., an antibody) can be generated using protein based on the nucleic acid sequence of human TIGIT accessible at NCBI under Accession No. NM_173799 and/or the corresponding amino acid sequence under Accession No. NP_776160, or fragments thereof. In some embodiments, a human TIGIT agonist (e.g., a TIGIT ligand) can be generated based on the nucleic acid sequence of human CD155 accessible at NCBI under Accession No. NM_001135768, NM_001135769, NM_001135770, or NM_006505 and/or the corresponding amino acid sequence under Accession No. NP_001129240, NP001129241, NP001129242, or NP_006496, or fragments thereof. In some embodiments, a human TIGIT agonist (e.g., a TIGIT ligand) can be generated based on the nucleic acid sequence of human CD112 accessible at NCBI under Accession No. NM_001042724 or NM_002856 and/or the corresponding amino acid sequence under Accession No. NP_001036189 or NP_002847, or fragments thereof.

In some embodiments, antagonists or agonists of TIGIT disclosed in the International Patent Publication WO 2009/126688, the content of which is incorporated herein by reference, can be used in various embodiments of the methods and compositions described herein.

Fgl2 and Antagonists (Inhibitors) or Agonists Thereof

Fgl2, also known as fibroleukin or fibrinogen-like protein 2, is a member of the fibrinogen-related protein superfamily of proteins. Fgl2 was first cloned from human CTLs and is secreted by CD4+ and CD8+T cells or Tregs. As used herein, the term “Fgl2” generally refers to an Fgl2 polypeptide or an Fgl2 polynucleotide that is similar or identical to the sequence of a wild-type Fgl2.

In some embodiments, the term “Fgl2” refers to an Fgl2 polypeptide having an amino acid sequence that is at least 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to that of a wild-type Fgl2, and is capable of mediating suppression of Th1 and/or Th17 responses.

In some embodiments, the term “Fgl2” refers to an Fgl2 polynucleotide having a nucleotide sequence that is at least 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to that of a wild-type Fgl2, and encodes an Fgl2 polypeptide as described herein.

The wild-type Fgl2 sequences of various species are available on the world wide web from the NCBI, including human, mouse, rat, dog, and chimpanzee. For example, the nucleotide sequence encoding human Fgl2 is available at NCBI under Accession No. NM_006682 and its corresponding amino acid sequence is under Accession No. NP_00673.

As used herein, the term “Fgl2 polypeptide” also encompasses a portion or fragment of such an Fgl2 polypeptide that retains at least about 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) of the activity of the wild-type FGL2 polypeptide to mediate the suppression of Th1 and/or Th17 responses. The term “Fgl2 polypeptide” as used herein also encompasses conservative substitution variants of an Fgl2 polypeptide that retain at least about 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) of the activity of the wild-type FGL2 polypeptide to mediate the suppression of Th1 and/or Th7 responses.

As used herein, the term “FGL2 antagonist” is used interchangeably with the terms “Fgl2 inhibitor” and “anti-Fgl2 therapy” and refers to an agent that interferes with the normal functioning of Fgl2, either by decreasing transcription or translation of Fgl2-encoding nucleic acid, or by inhibiting or blocking Fgl2 polypeptide activity, or both. Examples of Fgl2 antagonists include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, Fgl2-specific aptamers, anti-Fgl2 antibodies, Fgl2-binding fragments of anti-Fgl2 antibodies, Fgl2-binding small molecules, Fgl2-binding peptides, and other polypeptides that specifically bind Fgl2 (including, but not limited to, Fgl2-binding fragments of one or more Fgl2 ligands, optionally fused to one or more additional domains), such that the interaction between the Fgl2 antagonist and Fgl2 results in a reduction or cessation of Fgl2 activity or expression. It will be understood by one of ordinary skill in the art that in some instances, an Fgl2 antagonist can antagonize one Fgl2 activity without affecting another Fgl2 activity. For example, a desirable Fgl2 antagonist for use in certain of the methods herein is an Fgl2 antagonist that antagonizes Fgl2 activity in response to one of binding partner interactions, e.g., without affecting or minimally affecting any of the other Fgl2 interactions.

In some embodiments, an Fgl2 inhibitor is an agent that directly or indirectly reduces the expression/secretion and/or activity of Fgl2. Accordingly, an Fgl2 inhibitor can target Fgl2 molecule or its corresponding receptors. Alternatively, an Fgl2 inhibitor can bind to TIGIT or a TIGIT ligand (e.g., CD112 and/or CD155) and inhibit TIGIT-mediated activation of Fgl2 expression or activity. Examples of Fgl2 inhibitors include, but are not limited to, Fgl2 neutralizing agents, TIGIT inhibitors, CEBPαinhibitors (i.e., agents that decreases expression and/or activity of CEBPα and inhibits binding of CEBPα to Fgl2 gene), and/or ST2 inhibitors. The Fgl2 inhibitors can be a protein, a peptide, a peptidomimetic, an aptamer, a nucleic acid, an antibody, a fusion construct, a small molecule, a vaccine, a fusion protein, or any combination thereof.

In some embodiments, an Fgl2 inhibitor is an anti-Fgl2 antibody (e.g., an anti-human Fgl2 antibody). Anti-Fgl2 antibodies are commercially available (e.g., from Abcam (Cat. No. ab103584)).

The term “FGL2 agonist” refers to an agent that enhances or stimulates the normal functioning of FGL2, by increasing transcription or translation of FGL2-encoding nucleic acid, and/or by inhibiting or blocking activity of a molecule that inhibits FGL2 expression or FGL2 activity, and/or by enhancing normal FGL2 activity (including, but not limited to, enhancing the stability of FGL2 or enhancing binding of FGL2 to one or more target binding partners). For example, the FGL2 agonist can be selected from an antibody, an antigen-binding fragment, an aptamer, an interfering RNA, a small molecule, a peptide, an antisense molecule, and another binding polypeptide. In another example, the FGL2 agonist can be a polynucleotide selected from an aptamer, interfering RNA, or antisense molecule that interferes with the transcription and/or translation of an Fgl2-inhibitory molecule. It will be understood by one of ordinary skill in the art that in some instances, an Fgl2 agonist can agonize one FGL2 activity without affecting another FGL2 activity. For example, a desirable FGL2 agonist for use in certain of the methods herein is an Fgl2 agonist that agonizes FGL2 activity in response to one of its binding partner interactions, e.g., without affecting or minimally affecting any of the other FGL2 interactions.

In some embodiments, an Fgl2 agonist is an agent that directly or indirectly increases the expression/secretion and/or activity of Fgl2. Accordingly, an Fgl2 agonist can target Fgl2 molecule or its corresponding receptors. Examples of Fgl2 agonists include, but are not limited to, Fgl2 soluble molecules, TIGIT agonists, CEBPα-inducing agents (i.e., agents that increases expression and/or activity of CEBPα and promotes binding of CEBPα to Fgl2 gene), and/or ST2 agonists. The Fgl2 agonists can be a protein, a peptide, a peptidomimetic, a fusion protein, an aptamer, a nucleic acid, an antibody, a small molecule, a vaccine, a fusion construct, or any combination thereof.

In some embodiments, an Fgl2 agonist is a recombinant Fgl2 protein (e.g., a recombinant human Fgl2 protein). Recombinant Fgl2 proteins are commercially available (e.g., from OriGene (Cat. No. TP307557)).

Fgl2 antagonists or agonists can be obtained from known sources or prepared using known techniques such as recombinant or synthetic technology. The nucleic acid and protein sequences of Fgl2 of different species (e.g., but not limited to, human, mouse, rat, dog, chimpanzee) are known in the art, e.g., accessible at NCBI. Thus, one of skill in the art can readily generate Fgl2 antagonists or agonists based on these sequences using art-recognized molecular technologies. For example, a human Fgl2 antagonist (e.g., an antibody) or agonist (e.g., a soluble protein) can be generated based on the nucleic acid sequence of human Fgl2 accessible at NCBI under Accession No. NM_006682 and/or the corresponding amino acid sequence under Accession No. NP_00673 or fragments thereof.

In some embodiments, antagonists or agonists of Fgl2 disclosed in the International Patent Publication WO 2003/074068, the content of which is incorporated herein by reference, can be used in various embodiments of the methods and compositions described herein.

IL-33 and Antagonists (Inhibitors) or Agonists Thereof

IL-33 is interleukin-33 cytokine and is a ligand for ST2 receptor and the co-receptor IL-1 receptor accessory protein (IL-1RAcP). As used herein, the term “IL-33” generally refers to an IL-33 polypeptide or an IL-33 polynucleotide that is similar or identical to the sequence of a wild-type IL-33.

In some embodiments, the term “IL-33” refers to an IL-33 polypeptide having an amino acid sequence that is at least 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to that of a wild-type IL-33, and is capable of inducing TIGIT expression and/or activity, and/or increasing or expanding a TIGIT+Treg population.

In some embodiments, the term “IL-33” refers to an IL-33 polynucleotide having a nucleotide sequence that is at least 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to that of a wild-type IL-33, and encodes an IL-33 polypeptide as described herein.

The wild-type IL-33 sequences of various species and isoforms thereof are available on the world wide web from the NCBI, including human, mouse, rat, pig, and chimpanzee. For example, the nucleotide sequences encoding human IL-33 and isoforms thereof are available at NCBI under Accession Nos. NM_001186569, NM_001196640, and NM_001199641, and their corresponding amino acid sequence are under Accession Nos. NP_254274, NP_001186569, and NP_001186570, respectively.

As used herein, the term “IL-33 polypeptide” also encompasses a portion or fragment of such an IL-33 polypeptide that retains at least about 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) of the activity of the wild-type IL-33 polypeptide to induce TIGIT expression and/or activity and/or increasing or expanding a TIGIT+Treg population. The term “IL-33 polypeptide” as used herein also encompasses conservative substitution variants of an IL-33 polypeptide that retain at least about 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) of the activity of the wild-type IL-33 polypeptide to induce TIGIT expression and/or activity and/or increasing or expanding a TIGIT+Treg population.

In various aspects described herein, methods for measuring IL-33 or a fragment thereof from a sample are known in the art, including, but not limited to mRNA expression using PCR or real-time PCR, protein analysis using western blot, immunoassay, and/or ELISA, and/or sequencing analysis. Thus, in some embodiments, nucleic acid molecules can be isolated from a patient's sample to measure IL-33 mRNA expression, or proteins can be isolated to measure IL-33 protein expression.

The term “IL-33 antagonist” is used interchangeably with the terms “IL-33 inhibitor” and “anti-IL-33 therapy” and refers to an agent that interferes with the normal functioning of IL-33, either by decreasing transcription or translation of IL-33-encoding nucleic acid, or by inhibiting or blocking IL-33 polypeptide activity, or both. Examples of IL-33 antagonists include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, IL-33-specific aptamers, anti-IL-33 antibodies, IL-33-binding fragments of anti-IL-33 antibodies, IL-33-binding small molecules, IL-33-binding peptides, and other polypeptides that specifically bind IL-33 (including, but not limited to, IL-33-binding fragments of one or more IL-33 ligands, optionally fused to one or more additional domains), such that the interaction between the IL-33 antagonist and IL-33 results in a reduction or cessation of IL-33 activity or expression. It will be understood by one of ordinary skill in the art that in some instances, an IL-33 antagonist can antagonize one IL-33 activity without affecting another IL-33 activity. For example, a desirable IL-33 antagonist for use in certain of the methods herein is an IL-33 antagonist that antagonizes IL-33 activity in response to one of binding partner interactions such as ST2, e.g., without affecting or minimally affecting any of the other IL-33 interactions.

In some embodiments, an IL-33 inhibitor is an agent that directly or indirectly reduces the expression/secretion and/or activity of IL-33. Accordingly, an IL-33 inhibitor can target IL-33 molecule or its corresponding receptors. Examples of IL-33 inhibitors include, but are not limited to, ST2 inhibitors or IL-33 neutralizing agents. The IL-33 inhibitors can be a protein, a peptide, a peptidomimetic, an aptamer, a nucleic acid, an antibody, a small molecule, a fusion protein, a vaccine, or any combination thereof.

In some embodiments, an IL-33 inhibitor is an anti-IL-33 antibody (e.g., an anti-human IL-33 antibody). Anti-IL-33 antibodies are commercially available (e.g., from AbD Serotec, a Bio-Rad Company (Cat. No. AHP1482), Biolegend (Clone BL35175; Cat. No. 517201 or 517202), and Abcam (Cat. No. ab72844)).

In some embodiments, an IL-33 inhibitor can be a soluble ST2 receptor or a soluble IL-1RAcP receptor (e.g., without a transmembrane domain) that binds to IL-33, thereby decreasing the concentration of IL-33 that is available for functionally interacting with ST2/IL-1RAcP receptors present on an immune cell (e.g., T cell) or a TIGIT+Treg.

The term “IL-33 agonist” refers to an agent that enhances or stimulates the normal functioning of IL-33, by increasing transcription or translation of IL-33-encoding nucleic acid, and/or by inhibiting or blocking activity of a molecule that inhibits IL-33 expression or IL-33 activity, and/or by enhancing normal IL-33 activity (including, but not limited to, enhancing the stability of IL-33 or enhancing binding of IL-33 to one or more target binding partners such as ST2. For example, the IL-33 agonist can be selected from an antibody, an antigen-binding fragment, an aptamer, an interfering RNA, a small molecule, a peptide, an antisense molecule, and another binding polypeptide. In another example, the IL-33 agonist can be a polynucleotide selected from an aptamer, interfering RNA, or antisense molecule that interferes with the transcription and/or translation of an IL-33-inhibitory molecule. It will be understood by one of ordinary skill in the art that in some instances, an IL-33 agonist can agonize one IL-33 activity without affecting another IL-33 activity. For example, a desirable IL-33 agonist for use in certain of the methods herein is an IL-33 agonist that agonizes IL-33 activity in response to one of its binding partner interactions such as ST2, e.g., without affecting or minimally affecting any of the other IL-33 interactions.

In some embodiments, an IL-33 agonist is an agent that directly or indirectly increases the expression/secretion and/or activity of IL-33. Accordingly, an IL-33 agonist can target IL-33 molecule or its corresponding receptors. Examples of IL-33 agonists include, but are not limited to, ST2 agonists or IL-33 soluble molecules. The IL-33 agonists can be a protein, a peptide, a peptidomimetic, an aptamer, a nucleic acid, an antibody, a small molecule, a fusion protein, a vaccine, or any combination thereof.

In some embodiments, an IL-33 agonist is a recombinant IL-33 protein (e.g., a recombinant human IL-33 protein). Recombinant IL-33 proteins are commercially available (e.g., from Life Technologies (Cat. No. PHC9254); InVivoGen (Cat. No. rhil-33); and R&D Systems (Cat. No. 3625-IL-010)).

IL-33 antagonists or agonists can be obtained from known sources or prepared using known techniques such as recombinant or synthetic technology. The nucleic acid and protein sequences of IL-33 of different species (e.g., but not limited to, human, mouse, pig, chimpanzee) are known in the art, e.g., accessible at NCBI. Thus, one of skill in the art can readily generate IL-33 antagonists or agonists based on these sequences using art-recognized molecular technologies. For example, a human IL-33 antagonist (e.g., an antibody) or agonist (e.g., a soluble protein) can be generated based on the nucleic acid sequence of human IL-33, e.g., accessible at NCBI under Accession No. NM_001186569, NM_001196640, or NM_001199641 and/or the corresponding amino acid sequence under Accession No. NP_254274, NP_001186569, or NP_001186570, or fragments thereof.

In some embodiments, antagonists or agonists of IL-33 disclosed in the International Patent Publication WO 2005/079844, the content of which is incorporated herein by reference, can be used in various embodiments of the methods and compositions described herein.

ST2 and Antagonists (Inhibitors) or Agonists Thereof

ST2 is interleukin 1 receptor-like 1 protein that binds IL-33, and is also known as IL1RL1, IL-1 R4, ST2L, DER4, Fit-1, Ly84, and T1. The ST2 protein has two isoforms: a soluble form (soluble ST2 or sST2) and a membrane bound receptor form (ST2 receptor). As used herein, the term “ST2” generally refers to an ST2 polypeptide or an ST2 polynucleotide that is similar or identical to the sequence of a wild-type ST2.

In some embodiments, the term “ST2” refers to an ST2 polypeptide having an amino acid sequence that is at least 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to that of a wild-type ST2, and is capable of binding IL-33 to induce TIGIT expression and/or activity and/or increase or expand a TIGIT+Treg population.

In some embodiments, the term “ST2” refers to an ST2 polynucleotide having a nucleotide sequence that is at least 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to that of a wild-type ST2 or a portion thereof, and encodes an ST2 polypeptide as described herein.

The wild-type ST2 sequences of various species and isoforms thereof are available on the world wide web from the NCBI, including human, mouse, rat, monkey and dog. For example, the nucleotide sequences encoding human ST2 and isoforms thereof are available at NCBI under Accession Nos. NM_001282408, NM_003853, and NM_016232 and their corresponding amino acid sequences are under Accession Nos. NP_001269337, NP_003847, and NP_057316, respectively.

Where the term “ST2” refers to an ST2 polypeptide, the term “ST2 polypeptide” also encompasses a portion or fragment of such an ST2 polypeptide that retains at least about 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) of the activity of the wild-type ST2 polypeptide to bind IL-33, which in turn induces TIGIT expression and/or activity and/or increases or expands a TIGIT+Treg population. The term “ST2 polypeptide” as used herein also encompasses conservative substitution variants of an ST2 polypeptide that retain at least about 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) of the activity of the wild-type ST2 polypeptide to bind IL-33, which in turn induces TIGIT expression and/or activity and/or increases or expands a TIGIT+Treg population. Accordingly, an ST2 polypeptide refers to any form of ST2 that binds IL-33 and induces immunosuppression (e.g., via TIGIT activity and/or expression), including functional variants of ST2. For example, in some embodiments, an ST2 polypeptide can be a full-length ST2. In some embodiments, an ST2 polypeptide refers to a functional domain or domains of ST2 that binds IL-33, which in turn induces TIGIT expression and/or activity and/or increases or expands a TIGIT+Treg population.

In various aspects described herein, methods for measuring ST2 or a fragment thereof (including sST2 and ST2 receptor or fragments thereof) from a sample are known in the art, including, but not limited to mRNA expression using PCR or real-time PCR, protein analysis using western blot, immunoassay, and/or ELISA, and/or sequencing analysis. Thus, in some embodiments, nucleic acid molecules can be isolated from a patient's sample to measure ST2 mRNA expression, or proteins can be isolated to measure ST2 protein expression.

The term “ST2 antagonist” is used interchangeably with the term “ST2 inhibitor” and refers to an agent that interferes with the normal functioning of ST2, either by decreasing transcription or translation of ST2-encoding nucleic acid, or by inhibiting or blocking ST2 polypeptide activity, or both. Examples of ST2 antagonists include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, ST2-specific aptamers, anti-ST2 antibodies, ST2-binding fragments of anti-ST2 antibodies, ST2-binding small molecules, ST2-binding peptides, and other polypeptides that specifically bind ST2 (including, but not limited to, ST2-binding fragments of one or more ST2 ligands, optionally fused to one or more additional domains), such that the interaction between the ST2 antagonist and ST2 results in a reduction or cessation of ST2 activity or expression. It will be understood by one of ordinary skill in the art that in some instances, an ST2 antagonist can antagonize one ST2 activity without affecting another ST2 activity. For example, a desirable ST2 antagonist for use in certain of the methods herein is an ST2 antagonist that antagonizes ST2 activity in response to one of ligand interaction, IL-33 interaction, or other binding partner interaction, e.g., without affecting or minimally affecting any of the other ST2 interactions.

In some embodiments, an ST2 inhibitor is an agent that directly or indirectly inhibits or reduces the ST2-mediated suppression of proinflammatory Th1 and/or Th17 responses, e.g., by inhibiting or reducing proliferation of TIGIT+Tregs. Accordingly, an ST2 inhibitor can target the corresponding ligand of ST2, or any of ST2's upstream molecules. Examples of ST2 inhibitors include, without limitations, ST2−/− immune cells (e.g., T cells), anti-ST2 molecules, IL-33 inhibitors, and any combination thereof. The ST2 inhibitors can be a protein, a peptide, peptidomimetic, an aptamer, a nucleic acid, an antibody, a small molecule, a vaccine, a fusion protein, or any combinations thereof.

In some embodiments, an ST2 inhibitor is an anti-ST2 antibody (e.g., an anti-human ST2 antibody). Anti-ST2 antibodies are commercially available (e.g., from R&D Systems (Clone 97203; Cat. No. MAB523); Abcam (Cat. No. ab89741); and AdipoGen (Clone ST33868; Cat No. AG-20A-0044)).

In some embodiments, an ST2 inhibitor is a recombinant ST2 Fc fusion protein (e.g., a recombinant mouse ST2 Fc fusion protein). Recombinant ST2 proteins are commercially available (e.g., from R&D Systems (Cat. No. 1004-MP-050)).

In some embodiments, an ST2 inhibitor can be a fragment or variant of ST2 itself, e.g., a fragment that binds IL-33 but does not induce an immunosuppressive signal (e.g., via TIGIT activity and/or expression). An ST2 inhibitor of this type can be a dominant negative inhibitor.

The term “ST2 agonist” refers to an agent that enhances or stimulates the normal functioning of ST2, by increasing transcription or translation of ST2-encoding nucleic acid, and/or by inhibiting or blocking activity of a molecule that inhibits ST2 expression or ST2 activity, and/or by enhancing normal ST2 activity (including, but not limited to, enhancing the stability of ST2 or enhancing binding of ST2 to one or more target ligands such as IL-33). For example, the ST2 agonist can be selected from an antibody, an antigen-binding fragment, an aptamer, an interfering RNA, a small molecule, a peptide, an antisense molecule, and another binding polypeptide. In another example, the ST2 agonist can be a polynucleotide selected from an aptamer, interfering RNA, or antisense molecule that interferes with the transcription and/or translation of an ST2-inhibitory molecule. It will be understood by one of ordinary skill in the art that in some instances, an ST2 agonist can agonize one ST2 activity without affecting another ST2 activity. For example, a desirable ST2 agonist for use in certain of the methods herein is an ST2 agonist that agonizes ST2 activity in response to one of ligand interaction, IL-33 interaction, or other binding partner interaction, e.g., without affecting or minimally affecting any of the other ST2 interactions.

In some embodiments, an ST2 agonist is an agent that directly or indirectly enhances or stimulates the ST2-mediated suppression of proinflammatory Th1 and/or Th17 responses, e.g., by inducing or expanding the TIGIT+cell population. Accordingly, an ST2 agonist can target its corresponding ligand such as IL-33, or any of ST2's upstream molecules. Examples of ST2 agonists include, without limitations, ST2-overexpressing immune cells (e.g., T cells), IL-33 agonists, and a combination thereof. The ST2 agonists can be a protein, a peptide, peptidomimetic, an aptamer, a nucleic acid, an antibody, a small molecule, a vaccine, a fusion protein, or any combinations thereof.

ST2 antagonists or agonists can be obtained from known sources or prepared using known techniques such as recombinant or synthetic technology. The nucleic acid and protein sequences of ST2 of different species (e.g., but not limited to, human, mouse, pig, chimpanzee) are known in the art, e.g., accessible at NCBI. Thus, one of skill in the art can readily generate ST2 antagonists or agonists based on these sequences using art-recognized molecular technologies. For example, a human ST2 antagonist (e.g., an antibody) or agonist (e.g., a soluble protein) can be generated based on the nucleic acid sequence of human ST2, e.g., accessible at NCBI under Accession No. NM_001282408, NM_003853, or NM_016232 and/or the corresponding amino acid sequence under Accession No. NP_001269337, NP_003847, or NP_057316, or fragments thereof.

CD112 and Antagonists (Inhibitors) or Agonists Thereof

CD112 (cluster of differentiation 112) is a single-pass type 1 membrane glycoprotein with two Ig-like C2-type domains and an Ig-like V-type domain. CD112 is also known as PVRL2 (poliovirus receptor-related 2), herpesvirus entry mediator B or nectin 2. Yu et al. Nat. Immunol. 10:48-57 (2009). Generally, CD112 is expressed as a cell surface protein on a variety of cells, e.g., myelomonocytic cells, megakaryocytes, dendritic cells, mast cells, CD34-positive stem cells, endothelial cells, epithelial cells, and neuronal cells, macrophages, and other antigen-presenting cells, e.g., but not limited to cancer cells. CD112 is a ligand for TIGIT with a lower affinity than CD155 (as discussed below).

As used herein, the term “CD112” generally refers to a CD112 polypeptide or a CD112 polynucleotide that is similar or identical to the sequence of a wild-type CD112.

In some embodiments, the term “CD112” refers to a CD112 polypeptide having an amino acid sequence that is at least 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to that of a wild-type CD112, and is capable of binding TIGIT and suppressing Th1 and/or Th17 responses. In some embodiments, the CD112 polypeptide can also increase expression and/or activity of Fgl2 to mediate the suppression of Th1 and/or Th17 responses.

In some embodiments, the term “CD112” refers to a CD112 polynucleotide having a nucleotide sequence that is at least 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to that of a wild-type CD112 polynucleotide or a portion thereof, and encodes a CD112 polypeptide as described herein.

The wild-type CD112 sequences of various species are available on the world wide web from the NCBI, including human, mouse, and monkey. For example, the nucleotide sequence encoding human CD112 is available at NCBI under Accession No. NM_001042724 or NM_002856 and its corresponding amino acid sequence is under Accession No. NP_001036189 or NP_002847.

Where the term “CD112” refers to a CD112 polypeptide, the term “CD112 polypeptide” also encompasses a portion or fragment of such a CD112 polypeptide that retains at least about 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) of the TIGIT binding activity of the wild-type CD112 polypeptide. The term “CD112 polypeptide” as used herein also encompasses conservative substitution variants of a CD112 polypeptide that retain at least about 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) of the TIGIT binding activity of the wild-type CD112 polypeptide. Accordingly, a CD112 polypeptide refers to any form of CD112 that can bind TIGIT, including functional variants of CD112. For example, in some embodiments, a CD112 polypeptide can be a full-length CD112. In some embodiments, a CD112 polypeptide refers to a functional domain or domains (e.g., an extracellular domain) of CD112 that binds TIGIT and induces immunosuppression and expression and/or activity of Fgl2.

The term “CD112 antagonist” is used interchangeably with the term “CD112 inhibitor” and refers to an agent that interferes with the normal functioning of CD112, either by decreasing transcription or translation of CD112-encoding nucleic acid, or by inhibiting or blocking CD112 polypeptide activity, or both. Examples of CD112 antagonists include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, CD112-specific aptamers, anti-CD112 antibodies, CD112-binding fragments of anti-CD112 antibodies, CD112-binding small molecules, CD112-binding peptides, and other polypeptides that specifically bind CD112 (including, but not limited to, CD112-binding fragments of soluble TIGIT, optionally fused to one or more additional domains), such that the interaction between the CD112 antagonist and CD112 results in a reduction or cessation of CD112 activity or expression. It will be understood by one of ordinary skill in the art that in some instances, a CD112 antagonist can antagonize one CD112 activity without affecting another CD112 activity. For example, a desirable CD112 antagonist for use in certain of the methods herein is a CD112 antagonist that antagonizes CD112 binding to TIGIT, e.g., without affecting or minimally affecting any of the other CD112 interactions.

In some embodiments, a CD112 inhibitor is an agent that directly or indirectly inhibits or reduces CD112 binding to TIGIT, which in turn induces suppression of proinflammatory Th1 and/or Th17 responses. Accordingly, a CD112 inhibitor can target the CD112 ligand itself or its corresponding receptor, or any molecule that regulates expression of CD112. Examples of CD112 inhibitors include, without limitations, anti-CD112 molecules, soluble TIGIT molecules, and a combination thereof. A CD112 inhibitor can be a protein, a peptide, a peptidomimetic, an aptamer, a nucleic acid, an antibody, a small molecule, a vaccine, or any combinations thereof.

In some embodiments, a CD112 inhibitor is an anti-CD112 antibody (e.g., an anti-human CD112 antibody). Anti-CD112 antibodies are commercially available (e.g., from BioLegend (Clone TX31; Cat. No. 337402), EMD Millipore (Clone 82.525; Cat. No. MABT62), R&D Systems (Cat. No. AF2229), or Abcam (Clone EPR6717; Cat. No. ab135246)).

In some embodiments, a CD112 inhibitor can be a fragment or variant of CD112 itself, e.g., a fragment that binds TIGIT but does not transmit an immunosuppressive signal (e.g., via Fgl2 expression). A CD112 inhibitor of this type can be a dominant negative inhibitor. For example, a CD112 inhibitor is a recombinant soluble CD112 protein (e.g., with the transmembrane domain substantially removed).

As used herein, the term “CD112 agonist” refers to an agent that enhances or stimulates the normal functioning of CD112, by increasing transcription or translation of CD112-encoding nucleic acid, and/or by inhibiting or blocking activity of a molecule that inhibits CD112 expression or CD112 activity, and/or by enhancing normal CD112 activity (including, but not limited to, enhancing the stability of CD112 or enhancing binding of CD112 to one or more target receptors such as TIGIT). For example, the CD112 agonist can be selected from an antibody, an antigen-binding fragment, an aptamer, an interfering RNA, a small molecule, a peptide, an antisense molecule, and another binding polypeptide. In another example, the CD112 agonist can be a polynucleotide selected from an aptamer, interfering RNA, or antisense molecule that interferes with the transcription and/or translation of a CD112 molecule. It will be understood by one of ordinary skill in the art that in some instances, a CD112 agonist can agonize one CD112 activity without affecting another CD112 activity. For example, a desirable CD112 agonist for use in certain of the methods herein is a CD112 agonist that agonizes CD112 binding to TIGIT, e.g., without affecting or minimally affecting any of the other CD112 interactions.

In some embodiments, a CD112 agonist is an agent that directly or indirectly enhances or stimulates CD112 binding to TIGIT, which in turn induces the TIGIT-mediated suppression of proinflammatory Th1 and/or Th17 responses. Accordingly, a CD112 agonist can target the CD112 ligand itself or its corresponding receptor, or any molecule that modulates expression of CD112. CD112 agonists can be a protein, a peptide, peptidomimetic, an aptamer, a nucleic acid, an antibody, a small molecule, a vaccine, a fusion protein, or any combinations thereof. Exemplary CD112 agonists include recombinant CD112 proteins or peptides.

CD112 antagonists or agonists can be obtained from known sources or prepared using known techniques such as recombinant or synthetic technology. The nucleic acid and protein sequences of CD112 and its ligands of different species (e.g., but not limited to, human, mouse, and monkey) are known in the art, e.g., accessible at world wide web from NCBI. Thus, one of skill in the art can generate CD112 antagonists or agonists based on these sequences using art-recognized molecular technologies such as cloning and expression technologies. For example, a human CD112 antagonist (e.g., an antibody) or agonist (e.g., recombinant protein) can be generated using protein based on the nucleic acid sequence of human CD112 accessible at NCBI under Accession No. NM_001042724 or NM_002856 and its corresponding amino acid sequence is under Accession No. NP_001036189 or NP_002847.

CD155 and Antagonists (Inhibitors) or Agonists Thereof

CD155 (cluster of differentiation 155) is a type 1 membrane glycoprotein with three extracellular immunoglobulin-like domains, D1-D3. In humans, the immunoglobulin-like domain D1 of the CD155 polypeptide binds TIGIT. CD155 is also known as PVR (poliovirus receptor) or nectin-like 5. Yu et al. Nat. Immunol. 10:48-57 (2009). Generally, CD155 is expressed as a cell surface protein on a variety of cells, e.g., endothelial cells, monocytes, epithelia, central nervous system, dendritic cells, macrophages, and other antigen-presenting cells, e.g., but not limited to cancer cells. CD155 is a ligand for TIGIT with a higher affinity than CD112 (as discussed above).

As used herein, the term “CD155” generally refers to a CD155 polypeptide or a CD155 polynucleotide that is similar or identical to the sequence of a wild-type CD155.

In some embodiments, the term “CD155” refers to a CD155 polypeptide having an amino acid sequence that is at least 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to that of a wild-type CD155, and is capable of binding TIGIT and suppressing Th1 and/or Th17 responses. In some embodiments, the CD155 polypeptide can also increase expression and/or activity of Fgl2 to mediate the suppression of Th1 and/or Th17 responses.

In some embodiments, the term “CD155” refers to a CD155 polynucleotide having a nucleotide sequence that is at least 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to that of a wild-type CD155 polynucleotide or a portion thereof, and encodes a CD155 polypeptide as described herein.

The wild-type CD155 sequences of various species are available on the world wide web from the NCBI, including human, mouse, and chimpanzee. For example, the nucleotide sequence encoding human CD155 is available at NCBI under Accession No. NM_001135768, NM_001135769, NM_001135770, or NM_006505 and its corresponding amino acid sequence is under Accession No. NP_001129240, NP_001129241, NP_001129242 or NP_006496.

Where the term “CD155” refers to a CD155 polypeptide, the term “CD155 polypeptide” also encompasses a portion or fragment of such a CD155 polypeptide that retains at least about 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) of the TIGIT binding activity of the wild-type CD155 polypeptide. The term “CD155 polypeptide” as used herein also encompasses conservative substitution variants of a CD155 polypeptide that retain at least about 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) of the TIGIT binding activity of the wild-type CD155 polypeptide. Accordingly, a CD155 polypeptide refers to any form of CD155 that can bind TIGIT, including functional variants of CD155. For example, in some embodiments, a CD155 polypeptide can be a full-length CD155. In some embodiments, a CD155 polypeptide refers to a functional domain or domains (e.g., one or more extracellular domains) of CD155 that binds TIGIT and induces immunosuppression and expression and/or activity of Fgl2.

The term “CD155 antagonist” is used interchangeably with the term “CD155 inhibitor” and refers to an agent that interferes with the normal functioning of CD155, either by decreasing transcription or translation of CD155-encoding nucleic acid, or by inhibiting or blocking CD155 polypeptide activity, or both. Examples of CD155 antagonists include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, CD155-specific aptamers, anti-CD155 antibodies, CD155-binding fragments of anti-CD155 antibodies, CD155-binding small molecules, CD155-binding peptides, and other polypeptides that specifically bind CD155 (including, but not limited to, CD155-binding fragments of soluble TIGIT, optionally fused to one or more additional domains), such that the interaction between the CD155 antagonist and CD155 results in a reduction or cessation of CD155 activity or expression. It will be understood by one of ordinary skill in the art that in some instances, a CD155 antagonist can antagonize one CD155 activity without affecting another CD155 activity. For example, a desirable CD155 antagonist for use in certain of the methods herein is a CD155 antagonist that antagonizes CD155 binding to TIGIT, e.g., without affecting or minimally affecting any of the other CD155 interactions.

In some embodiments, a CD155 inhibitor is an agent that directly or indirectly inhibits or reduces CD155 binding to TIGIT, which in turn induces suppression of proinflammatory Th1 and/or Th17 responses. Accordingly, a CD155 inhibitor can target the CD155 ligand itself or its corresponding receptor, or any molecule that regulates expression of CD155. Examples of CD155 inhibitors include, without limitations, anti-CD155 molecules, soluble TIGIT molecules, and a combination thereof. A CD155 inhibitor can be a protein, a peptide, a peptidomimetic, an aptamer, a nucleic acid, an antibody, a small molecule, a vaccine, or any combinations thereof.

In some embodiments, a CD155 inhibitor is an anti-CD155 antibody (e.g., an anti-human CD155 antibody). Anti-CD155 antibodies are commercially available (e.g., from BioLegend (Clone SKII.4; Cat. No. 337609), Affymetrix eBioscience (Clone 2H7CD155; Cat. No. 12-1550-41), or R&D Systems (Clone 300907; Cat. No. MAB25301)).

In some embodiments, a CD155 inhibitor can be a fragment or variant of CD155 itself, e.g., a fragment that binds TIGIT but does not transmit an immunosuppressive signal (e.g., via Fgl2 expression). A CD155 inhibitor of this type can be a dominant negative inhibitor. For example, a CD155 inhibitor is a recombinant soluble CD155 protein (e.g., with the transmembrane domain substantially removed).

As used herein, the term “CD155 agonist” refers to an agent that enhances or stimulates the normal functioning of CD155, by increasing transcription or translation of CD155-encoding nucleic acid, and/or by inhibiting or blocking activity of a molecule that inhibits CD155 expression or CD155 activity, and/or by enhancing normal CD155 activity (including, but not limited to, enhancing the stability of CD155 or enhancing binding of CD155 to one or more target receptors such as TIGIT). For example, the CD155 agonist can be selected from an antibody, an antigen-binding fragment, an aptamer, an interfering RNA, a small molecule, a peptide, an antisense molecule, and another binding polypeptide. In another example, the CD155 agonist can be a polynucleotide selected from an aptamer, interfering RNA, or antisense molecule that interferes with the transcription and/or translation of a CD155 molecule. It will be understood by one of ordinary skill in the art that in some instances, a CD155 agonist can agonize one CD155 activity without affecting another CD155 activity. For example, a desirable CD155 agonist for use in certain of the methods herein is a CD155 agonist that agonizes CD155 binding to TIGIT, e.g., without affecting or minimally affecting any of the other CD155 interactions.

In some embodiments, a CD155 agonist is an agent that directly or indirectly enhances or stimulates CD155 binding to TIGIT, which in turn induces the TIGIT-mediated suppression of proinflammatory Th1 and/or Th17 responses. Accordingly, a CD155 agonist can target the CD155 ligand itself or its corresponding receptor, or any molecule that modulates expression of CD155. CD155 agonists can be a protein, a peptide, peptidomimetic, an aptamer, a nucleic acid, an antibody, a small molecule, a vaccine, a fusion protein, or any combinations thereof. Exemplary CD155 agonists include recombinant CD155 proteins or peptides.

CD155 antagonists or agonists can be obtained from known sources or prepared using known techniques such as recombinant or synthetic technology. The nucleic acid and protein sequences of CD155 and its ligands of different species (e.g., but not limited to, human, mouse, and monkey) are known in the art, e.g., accessible at world wide web from NCBI. Thus, one of skill in the art can generate CD155 antagonists or agonists based on these sequences using art-recognized molecular technologies such as cloning and expression technologies. For example, a human CD155 antagonist (e.g., an antibody) or agonist (e.g., recombinant protein) can be generated using protein based on the nucleic acid sequence of human CD155 accessible at NCBI under Accession No. NM_001135768, NM_001135769, NM_001135770, or NM_006505 and its corresponding amino acid sequence is under Accession No. NP_001129240, NP_001129241, NP_001129242 or NP_006496. Additionally or alternatively, one of skill in the art can generate CD155 antagonists or agonists based on the crystal structure of CD155 known in the art. See, e.g., Zhang et al. Proc Natl Acad Sci U.S.A. (2008)105: 18284-18289.

Pharmaceutical Compositions for Treatment of Immune-Related Diseases or Disorders

Pharmaceutical compositions for treatment of cancer and/or infections (including, e.g., but not limited to chronic viral infection, intracellular bacterial infection, extracellular bacterial infection, and/or fungal infection) are also provided herein. More specifically, the pharmaceutical composition comprises a pharmaceutically-acceptable excipient and at least one (including, e.g., at least two, at least three or more) of the following therapeutic agents: (a) a TIGIT inhibitor; (b) an IL-33 inhibitor; (c) an ST2 inhibitor; and (d) an Fgl2 inhibitor. For example, in some embodiments, the composition can comprise a TIGIT inhibitor and an IL-33 inhibitor, or a TIGIT inhibitor and an ST2 inhibitor. In some embodiments, the composition can comprise a TIGIT inhibitor and an Fgl2 inhibitor. In some embodiments, the composition can comprise an IL-33 inhibitor and an Fgl2 inhibitor, or an ST2 inhibitor and an Fgl2 inhibitor. In some embodiments, the composition can comprise a TIGIT inhibitor, an IL-33 inhibitor and/or an ST2 inhibitor, and an Fgl2 inhibitor.

In some embodiments, pharmaceutical compositions for treatment of cancer can further comprise an anti-cancer agent. Examples of an anti-cancer agent include, but are not limited to, vaccine, chemotherapy, targeted therapy (e.g., kinase inhibitors), radiation therapy, surgery, immunotherapy, and any combinations thereof.

In some embodiments, pharmaceutical compositions for treatment of extracellular and/or intracellular bacterial infection can further comprise an anti-bacterial agent.

In some embodiments, pharmaceutical compositions for treatment of fungal infection can further comprise an anti-fungal agent.

In some embodiments, pharmaceutical compositions for treatment of chronic viral infections can further comprise an anti-viral agent (e.g., small molecules and/or immunotherapy) as described herein. Examples of anti-viral agents include, but are not limited to, virus protein specific antibodies, reverse transcriptase inhibitors, protease inhibitors, immunomodulatory agents (e.g., cytokines, various nucleoside analogs, and/or Zn²⁺), plant extracts demonstrated to have an antiviral effect, and any combinations thereof.

In some embodiments, pharmaceutical compositions for treatment of asthma, allergy, and/or atopy can further comprise an anti-asthmatic agent, an anti-allergy agent, and/or an anti-atopic agent. Examples of anti-asthmatic agents include, but are not limited to beta adrenergic agonists, xanthine derivatives, corticosteroids, antileukotrienes, and any combinations thereof. Exemplary anti-allergy agents and anti-atopic agents include, but are not limited to antihistamines, corticosteroids, and combinations thereof.

Examples of an immunotherapy for treatment of cancer, infections, and/or asthma, allergy and/or atopy can comprise an agent that increases a proinflammatory T cell response and/or an agent that suppresses an anti-inflammatory T cell response.

Pharmaceutical compositions for treatment of inflammatory diseases or disorders are also provided herein. In some embodiments, the pharmaceutical composition for treatment of an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward Th2 responses is desirable (e.g., autoimmune diseases and/or parasitic infection) can comprise a pharmaceutically-acceptable excipient and at least one (including, e.g., at least two, at least three or more) of the following therapeutic agents: (a) a TIGIT agonist; (b) an IL-33 agonist; (c) an ST2 agonist; and (d) an Fgl2 agonist. For example, in some embodiments, the composition can comprise a TIGIT agonist and an IL-33 agonist, or a TIGIT agonist and an ST2 agonist. In some embodiments, the composition can comprise a TIGIT agonist and an Fgl2 agonist. In some embodiments, the composition can comprise an IL-33 agonist and an Fgl2 agonist, or an ST2 agonist and an Fgl2 agonist. In some embodiments, the composition can comprise a TIGIT agonist, an IL-33 agonist and/or an ST2 agonist, and an Fgl2 agonist.

In some embodiments, the pharmaceutical composition can further comprise an agent for treatment of an inflammatory disease or disorder where an inhibition of Th1 and/or Th17 responses and/or a shift of balance toward Th2 responses is desirable. For example, the agent can comprise an agent that increases an anti-inflammatory T cell response and/or an agent that suppresses a proinflammatory T cell response.

The phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, media, encapsulating material, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in maintaining the stability, solubility, or activity of, an agent for modulating expression and/or activity of TIGIT, Fgl2 and/or IL-33. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) excipients, such as cocoa butter and suppository waxes; (8) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (9) glycols, such as propylene glycol; (10) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (11) esters, such as ethyl oleate and ethyl laurate; (12) agar; (13) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (14) alginic acid; (15) pyrogen-free water; (16) isotonic saline; (17) Ringer's solution; (19) pH buffered solutions; (20) polyesters, polycarbonates and/or polyanhydrides; (21) bulking agents, such as polypeptides and amino acids (22) serum components, such as serum albumin, HDL and LDL; (23) C2-C12 alcohols, such as ethanol; and (24) other non-toxic compatible substances employed in pharmaceutical formulations. Release agents, coating agents, preservatives, and antioxidants can also be present in the formulation. The terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein.

The agents for modulating expression and/or activity of TIGIT, Fgl2 and/or IL-33 can be specially formulated for administration of the compound to a subject in solid, liquid or gel form, including those adapted for the following: (1) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (2) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (3) intravaginally or intrarectally, for example, as a pessary, cream or foam; (4) ocularly; (5) transdermally; (6) transmucosally; or (79) nasally. Additionally, a bispecific or multispecific polypeptide agent can be implanted into a patient or injected using a drug delivery system. See, for example, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed. “Controlled Release of Pesticides and Pharmaceuticals” (Plenum Press, New York, 1981); U.S. Pat. No. 3,773,919; and U.S. Pat. No. 3,270,960.

Further embodiments of the formulations and modes of administration of an agent for expression and/or activity of TIGIT, Fgl2 and/or IL-33 that can be used in the methods described herein are illustrated below.

Parenteral Dosage Forms.

Parenteral dosage forms of an agent for modulating expression and/or activity of TIGIT, Fgl2 and/or IL-33 can also be administered to a subject by various routes, including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, controlled-release parenteral dosage forms, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage forms of the disclosure are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Aerosol Formulations.

An agent for modulating expression and/or activity of TIGIT, Fgl2 and/or IL-33 can be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. An agent for modulating expression and/or activity of TIGIT, Fgl2 and/or IL-33 can also be administered in a non-pressurized form such as in a nebulizer or atomizer. An agent for modulating expression and/or activity of TIGIT, Fgl2 and/or IL-33 can also be administered directly to the airways in the form of a dry powder, for example, by use of an inhaler.

Suitable powder compositions include, by way of illustration, powdered preparations of an agent for modulating expression and/or activity of TIGIT, Fgl2 and/or IL-33 thoroughly intermixed with lactose, or other inert powders acceptable for intrabronchial administration. The powder compositions can be administered via an aerosol dispenser or encased in a breakable capsule which can be inserted by the subject into a device that punctures the capsule and blows the powder out in a steady stream suitable for inhalation. The compositions can include propellants, surfactants, and co-solvents and can be filled into conventional aerosol containers that are closed by a suitable metering valve.

Aerosols for the delivery to the respiratory tract are known in the art. See for example, Adjei, A. and Garren, J. Pharm. Res., 1: 565-569 (1990); Zanen, P. and Lamm, J.-W. J. Int. J. Pharm., 114: 111-115 (1995); Gonda, I. “Aerosols for delivery of therapeutic and diagnostic agents to the respiratory tract,” in Critical Reviews in Therapeutic Drug Carrier Systems, 6:273-313 (1990); Anderson et al., Am. Rev. Respir. Dis., 140: 1317-1324 (1989)) and have potential for the systemic delivery of peptides and proteins as well (Patton and Platz, Advanced Drug Delivery Reviews, 8:179-196 (1992)); Timsina et. al., Int. J. Pharm., 101: 1-13 (1995); and Tansey, I. P., Spray Technol. Market, 4:26-29 (1994); French, D. L., Edwards, D. A. and Niven, R. W., Aerosol Sci., 27: 769-783 (1996); Visser, J., Powder Technology 58: 1-10 (1989)); Rudt, S. and R. H. Muller, J. Controlled Release, 22: 263-272 (1992); Tabata, Y, and Y. Ikada, Biomed. Mater. Res., 22: 837-858 (1988); Wall, D. A., Drug Delivery, 2: 10 1-20 1995); Patton, J. and Platz, R., Adv. Drug Del. Rev., 8: 179-196 (1992); Bryon, P., Adv. Drug. Del. Rev., 5: 107-132 (1990); Patton, J. S., et al., Controlled Release, 28: 15 79-85 (1994); Damms, B. and Bains, W., Nature Biotechnology (1996); Niven, R. W., et al., Pharm. Res., 12(9); 1343-1349 (1995); and Kobayashi, S., et al., Pharm. Res., 13(1): 80-83 (1996), contents of all of which are herein incorporated by reference in their entirety.

The formulations of the agents for modulating expression and/or activity of TIGIT, Fgl2 and/or IL-33 further encompass anhydrous pharmaceutical compositions and dosage forms comprising the disclosed compounds as active ingredients, since water can facilitate the degradation of some compounds. For example, the addition of water (e.g., 5%) is widely accepted in the pharmaceutical arts as a means of simulating long-term storage in order to determine characteristics such as shelf life or the stability of formulations over time. See, e.g., Jens T. Carstensen, Drug Stability: Principles & Practice, 379-80 (2nd ed., Marcel Dekker, NY, N.Y.: 1995). Anhydrous pharmaceutical compositions and dosage forms of the disclosure can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms that comprise lactose and at least one active ingredient that comprises a primary or secondary amine are preferably anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected. Anhydrous compositions are preferably packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials) with or without desiccants, blister packs, and strip packs.

Controlled and Delayed Release Dosage Forms.

In some embodiments of the methods described herein, an agent for modulating expression and/or activity of TIGIT, Fgl2 and/or IL-33 can be administered to a subject by controlled- or delayed-release means. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. (Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000)). Controlled-release formulations can be used to control a compound of formula (I)'s onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a compound of formula (I) is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug.

A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the agents for modulating expression and/or activity of TIGIT, Fgl2 and/or IL-33. Examples include, but are not limited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1, each of which is incorporated herein by reference in their entireties. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, Calif. USA)), multilayer coatings, microparticles, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions. Additionally, ion exchange materials can be used to prepare immobilized, adsorbed salt forms of the disclosed compounds and thus effect controlled delivery of the drug. Examples of specific anion exchangers include, but are not limited to, Duolite® A568 and Duolite® AP143 (Rohm & Haas, Spring House, Pa. USA).

In some embodiments, an agent for modulating expression and/or activity of TIGIT, Fgl2 and/or IL-33 for use in the methods described herein is administered to a subject by sustained release or in pulses. Pulse therapy is not a form of discontinuous administration of the same amount of a composition over time, but comprises administration of the same dose of the composition at a reduced frequency or administration of reduced doses. Sustained release or pulse administrations are particularly preferred when the disorder occurs continuously in the subject, for example where the subject has continuous or chronic symptoms of an infection. Each pulse dose can be reduced and the total amount of the agent for modulating expression and/or activity of TIGIT, Fgl2 and/or IL-33 administered over the course of treatment to the patient is minimized.

The interval between pulses, when necessary, can be determined by one of ordinary skill in the art. Often, the interval between pulses can be calculated by administering another dose of the composition when the composition or the active component of the composition is no longer detectable in the subject prior to delivery of the next pulse. Intervals can also be calculated from the in vivo half-life of the composition. Intervals can be calculated as greater than the in vivo half-life, or 2, 3, 4, 5 and even 10 times greater the composition half-life. Various methods and apparatus for pulsing compositions by infusion or other forms of delivery to the patient are disclosed in U.S. Pat. Nos. 4,747,825; 4,723,958; 4,948,592; 4,965,251 and 5,403,590.

Methods for Modulating Th17 Response

In still another aspect, methods for modulating Th17 response based on the level of TIGIT, Fgl2 and/or IL-33 activity or expression are also provided herein. For example, in some embodiments, methods for enhancing Th17 response comprise contacting Tregs with or administering to a subject with a deficiency in Th17 response a TIGIT inhibitor, a Flg2 inhibitor and/or an IL-33 inhibitor. In other embodiments, methods for reducing or suppressing Th17 response comprise contacting Tregs with or administering to a subject with an over-stimulation in Th17 response a TIGIT agonist, a Flg2 agonist and/or an IL-33 agonist.

As used herein, the term “Th17 response” refers to response of T helper 17 cells (Th17) producing interleukin 17 (IL-17). They are developmentally distinct from Th1 and Th2 cells.

Sample

In accordance with various embodiments described herein, a sample, including any fluid or specimen (processed or unprocessed) or other biological sample, can be subjected to the methods of various aspects described herein.

In some embodiments, the sample can include a biological fluid obtained from a subject. Exemplary biological fluids obtained from a subject can include, but are not limited to, blood (including whole blood, plasma, cord blood and serum), lactation products (e.g., milk), amniotic fluids (e.g., a sample collected during amniocentesis), sputum, saliva, urine, semen, cerebrospinal fluid, bronchial aspirate, perspiration, mucus, liquefied feces, synovial fluid, lymphatic fluid, tears, tracheal aspirate, and fractions thereof. In some embodiments, a biological fluid can include a homogenate of a tissue specimen (e.g., biopsy) from a subject. In one embodiment, a test sample can comprises a suspension obtained from homogenization of a solid sample obtained from a solid organ or a fragment thereof.

In some embodiments, a sample can be obtained from a subject who has or is suspected of having an immune-related disease or disorder, e.g., cancer and/or inflammatory disease or disorder. In some embodiments, the sample can be obtained from a subject who has or is suspected of having cancer, or who is suspected of having a risk of developing cancer. In some embodiments, the sample can be obtained from a subject who has or is suspected of having an inflammatory disease or disorder or who is suspected of having a risk of developing an inflammatory disease or disorder.

In some embodiments, a sample can be obtained from a subject who is being treated for the immune-related disease or disorder. In other embodiments, the sample can be obtained from a subject whose previously-treated disease or disorder is in remission. In other embodiments, the test sample can be obtained from a subject who has a recurrence of a previously-treated disease or disorder. For example, in the case of cancer such as breast cancer, a test sample can be obtained from a subject who is undergoing a cancer treatment, or whose cancer was treated and is in remission, or who has cancer recurrence.

As used herein, a “subject” can mean a human or an animal. Examples of subjects include primates (e.g., humans, and monkeys). Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cattle, cows, horses, pigs, deer, bison, sheep, goats, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, and avian species, e.g., chicken, ducks, geese, turkeys, emu, ostrich. A patient or a subject includes any subset of the foregoing, e.g., all of the above, or includes one or more groups or species such as humans, primates or rodents. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate, e.g., a human. The terms “patient” and “subject” are used interchangeably herein. A subject can be male or female. The term “patient” and “subject” does not denote a particular age. Thus, any mammalian subjects from adult (e.g., young adult, middle-aged adult or senior adult) to pediatric subjects (e.g., infant, child, adolescent) to newborn subjects, as well as fetuses, are intended to be covered. When the term is used in conjunction with administration of a compound or drug, then the subject or patient has been the object of treatment, observation, and/or administration of the compound or drug. The methods and/or pharmaceutical compositions described herein are also contemplated to be used to treat domesticated animals or pets such as cats and dogs.

In one embodiment, the subject or patient is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. In one embodiment, the subject is a human being. In another embodiment, the subject can be a domesticated animal and/or pet.

In some embodiments, the sample can be a blood sample or a sample of a tissue at a target site from a patient. For example, for treatment of cancer, the sample can be a blood sample or a tumor biopsy from a patient. For treatment of inflammatory diseases or disorders, the sample can be a blood sample or a tissue biopsy from an inflammatory site in a patient. Without wishing to be bound by theory, since Fg2 and IL-33 are soluble molecules while TIGIT is a cell surface molecule, Fgl2 and IL-33 can be more easily measured, e.g., from a blood sample, as compared to TIGIT measured, e.g., from a tissue sample.

It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

Embodiments of Various Aspects Described Herein can be Defined in any of the Following Numbered Paragraphs:

1. A method of identifying a patient who is diagnosed with cancer and/or infection and is more likely to be responsive to an anti-TIGIT or anti-IL-33 therapy, the method comprising:
- a. measuring the level of Fgl2 activity or expression in a sample from the patient; and
- b. comparing the level of Fgl2 or expression in the sample with an Fgl2 reference, and:
  - i. when the level of Fgl2 activity or expression is greater than the Fgl2 reference, the patient is identified to be more likely to be responsive to an anti-TIGIT or anti-IL-33 therapy;
  - ii. when the level of Fgl2 activity or expression is the same as or less than the Fgl2 reference, the patient is identified as likely to respond to an alternative, proinflammatory immunotherapy comprising an activator of a proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.
2. The method of paragraph 1, wherein the activator of the proinflammatory T cell response comprises a TIM-3 inhibitor, an anti-galectin-9 molecule, a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a Lag-3 antagonist, an agonist of an immune checkpoint activating molecule, an antagonist of an immune checkpoint inhibitory molecule, or any combination thereof.
3. The method of paragraph 1 or 2, wherein the patient has been receiving an anti-cancer and/or anti-infection therapy.
4. The method of any of paragraphs 1-3, wherein the infection is selected from the group consisting of chronic vial infection, intracellular bacterial infection, extracellular bacterial infection, fungal infection, and a combination of two or more thereof.
5. A method of treating a patient diagnosed with cancer and/or infection, the method comprising:
- a) measuring the level of IL-33 activity or expression in a sample from a patient diagnosed with cancer and/or infection; and
- b) comparing the level of IL-33 activity or expression in the sample with an IL-33 reference, and:
  - i. when the level of IL-33 activity or expression is greater than the IL-33 reference, administering to the patient a composition comprising a TIGIT inhibitor and/or an Fgl2 inhibitor; or
  - ii. when the level of IL-33 activity or expression is the same as or less than the IL-33 reference, either (A) administering an alternative, proinflammatory immunotherapy treatment without the TIGIT inhibitor or Fgl2 inhibitor, or (B) determining if the level of at least one other inhibitory immune regulator in the sample is greater than the level of the corresponding reference, or if the level of at least one activating immune regulator in the sample is less than the level of the corresponding reference.
6. The method of paragraph 5, wherein the at least one inhibitory immune regulator comprises Fgl2, TIGIT, ST2, CD155, CD112, or a combination thereof.
7. The method of paragraph 5, wherein when the level of IL-33 activity or expression is the same as or less than the IL-33 reference, the method further comprises measuring the level of Fgl2 activity or expression in a sample from the patient and comparing the level of Fgl2 activity or expression in the sample with an Fgl2 reference, and:
- a. when the level of Fgl2 activity or expression is greater than the Fgl2 reference, administering to the patient a composition comprising a TIGIT inhibitor and/or an Fgl2 inhibitor; or
- b. when the level of Fgl2 activity or expression is the same as or less than the reference, administering an alternative, proinflammatory immunotherapy treatment without a TIGIT inhibitor or Fgl2 inhibitor.
8. The method of any of paragraphs 5-7, wherein the TIGIT inhibitor is selected from the group consisting of a protein, a peptide, a nucleic acid, an antibody, a small molecule, a vaccine, a TIGIT−/− immune cell (e.g., a T cell), an ST2 inhibitor, a CD112 inhibitor, a CD155 inhibitor, and a combination thereof.
9. The method of any of paragraphs 5-8, wherein the Fgl2 inhibitor is selected from the group consisting of a protein, a peptide, a nucleic acid, an antibody, a small molecule, a vaccine, a TIGIT inhibitor, and a combination thereof.
10. The method of any of paragraphs 5-9, wherein the TIGIT inhibitor or Fgl2 inhibitor is constructed to target TIGIT+regulatory T (Treg) cells.
11. The method of paragraph 5, wherein the alternative, proinflammatory immunotherapy treatment without the TIGIT inhibitor or IL-33 inhibitor is a therapy comprising an activator of a proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.
12. The method of paragraph 5, wherein a patient with an IL-33 level greater than the IL-33 reference is further administered a therapy comprising an activator of a proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.
13. The method of paragraph 11 or 12, wherein the activator of the proinflammatory T cell response comprises a TIM-3 inhibitor, an anti-galectin-9 molecule, a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a Lag-3 antagonist, an agonist of an immune checkpoint activating molecule, an antagonist of an immune checkpoint inhibitory molecule, or any combination thereof.
14. The method of any of paragraphs 5-13, wherein the patient has been receiving an anti-cancer and/or anti-infection agent.
15. The method of any of paragraphs 5-14, wherein the infection is selected from the group consisting of chronic vial infection, intracellular bacterial infection, extracellular bacterial infection, fungal infection, and a combination of two or more thereof.
16. The method of any of paragraphs 5-15, wherein the sample is a blood sample from the patient.
17. The method of any of paragraphs 5-15, wherein the sample is a tissue sample from the patient.
18. The method of any of paragraphs 5-17, wherein the reference corresponds to the level of IL-33 activity or expression in a normal healthy subject.
19. The method of any of paragraphs 5-17, wherein the reference corresponds to the level of IL-33 activity or expression in a normal tissue of the same type or lineage as the sample.
20. The method of any of paragraphs 5-17, wherein the reference corresponds to the level of IL-33 activity or expression in a diseased tissue with a low level of IL-33 expression or activity.
21. The method of any of paragraphs 5-17, wherein the reference is a standard numerical level or threshold.
22. A method of treating a patient diagnosed with cancer and/or infection, the method comprising:
- a) measuring the level of Fgl2 activity or expression in a sample from a patient diagnosed with cancer and/or infection; and
- b) comparing the level of Fgl2 activity or expression in the sample with an Fgl2 reference, and:
  - i. when the level of Fgl2 activity or expression is greater than the Fgl2 reference, administering to the patient a composition comprising a TIGIT inhibitor and/or an IL-33 inhibitor; or
  - ii. when the level of Fgl2 activity or expression is the same as or less than the Fgl2 reference, administering an alternative, proinflammatory immunotherapy treatment without a TIGIT inhibitor or IL-33 inhibitor.
23. The method of paragraph 22, wherein the alternative, proinflammatory immunotherapy treatment without a TIGIT inhibitor or IL-33 inhibitor is a therapy comprising an activator of a proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.
24. The method of paragraph 22 or 23, wherein the patient with an Fgl2 level greater than the Fgl2 reference is further administered a therapy comprising an activator of a proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.
25. The method of paragraph 23 or 24, wherein the activator of the proinflammatory T cell response comprises a TIM-3 inhibitor, an anti-galectin-9 molecule, a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a Lag-3 antagonist, an agonist of an immune checkpoint activating molecule, an antagonist of an immune checkpoint inhibitory molecule, or any combination thereof.
26. The method of any of paragraphs 22-25, wherein the patient has been receiving an anti-cancer agent and/or an anti-infection agent.
27. The method of any of paragraphs 22-26, wherein the infection is selected from the group consisting of chronic vial infection, intracellular bacterial infection, extracellular bacterial infection, fungal infection, and a combination of two or more thereof.
28. A method of treating a patient diagnosed with cancer and/or infection that has an elevated level of Fgl2, the method comprising:
- a) determining a first level of Fgl2 expression or activity in a sample from a patient diagnosed with cancer and/or infection that has an elevated level of Fgl2;
- b) administering an agent that inhibits IL-33 activity and/or TIGIT activity;
- c) determining a second level of Fgl2 expression or activity after said administering; and
- d) comparing said first and second levels of Fgl2 expression or activity, wherein the agent administered in (b) is effective if said second level of Fgl2 expression or activity is lower than said first level, and wherein the agent administered in (b) is ineffective if said second level of Fgl2 expression is the same as or higher than said first level.
29. The method of paragraph 28, wherein the infection is selected from the group consisting of chronic vial infection, intracellular bacterial infection, extracellular bacterial infection, fungal infection, and a combination of two or more thereof.
30. The method of paragraph 28 or 29, further comprising, when said anti-IL-33 or anti-TIGIT therapy is effective, continuing to administer said agent that inhibits IL-33 activity and/or TIGIT activity.
31. The method of paragraph 28 or 29, further comprising, when said anti-IL-33 therapy or said anti-TIGIT therapy is ineffective, administering said agent that inhibits IL-33 activity and/or TIGIT activity at a higher dose.
32. The method of paragraph 28 or 29, further comprising, when said anti-IL-33 therapy or said anti-TIGIT therapy is ineffective, discontinuing said anti-IL-33 therapy or said anti-TIGIT therapy.
33. The method of paragraph 32, further comprising, when said anti-IL-33 therapy or said anti-TIGIT therapy is ineffective, administering a therapy comprising an activator of a proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.
34. The method of paragraph 33, wherein the activator of the proinflammatory T cell response pathway comprises a TIM-3 inhibitor, an anti-galectin-9 molecule, a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a Lag-3 antagonist, an agonist of an immune checkpoint activating molecule, an antagonist of an immune checkpoint inhibitory molecule, or any combination thereof.
35. A method of treating a patient diagnosed with cancer and/or infection that exhibits an elevated level of IL-33, the method comprising:
- a) determining a first level of TIGIT and/or Fgl2 expression or activity in a sample from a patient diagnosed with cancer and/or infection that exhibits an elevated level of IL-33;
- b) administering an agent that inhibits IL-33 activity;
- c) determining a second level of TIGIT or Fgl2 expression or activity after said administering; and
- d) comparing said first and second levels of TIGIT and/or Fgl2 expression or activity, wherein anti-IL-33 therapy is effective if said second level of TIGIT and/or Fgl2 expression or activity is lower that said first level, and wherein anti-IL-33 therapy is ineffective if said second level of TIGIT and/or Fgl2 expression is the same as or higher than said first level.
36. The method of paragraph 35 or 36, wherein the infection is selected from the group consisting of chronic vial infection, intracellular bacterial infection, extracellular bacterial infection, fungal infection, and a combination of two or more thereof.
37. The method of paragraph 35 or 36, further comprising, when said anti-IL-33 therapy is effective, continuing to administer said agent that inhibits IL-33 activity.
38. The method of paragraph 35 or 36, further comprising, when said anti-IL-33 therapy is ineffective, discontinuing said anti-IL-33 therapy.
39. The method of paragraph 35 or 36, further comprising, when said anti-IL-33 therapy is ineffective, administering said agent that inhibits IL-33 activity at a higher dose.
40. A pharmaceutical composition comprising a pharmaceutically-acceptable excipient and at least two of the following therapeutic agents:

a. a TIGIT inhibitor

b. an IL-33 inhibitor;

c. an ST2 inhibitor; and

d. an Fgl2 inhibitor.

41. The pharmaceutical composition of paragraph 40, wherein the composition comprises a TIGIT inhibitor and an IL-33 inhibitor, or a TIGIT inhibitor and an ST2 inhibitor.
42. The pharmaceutical composition of paragraph 40, wherein the composition comprises a TIGIT inhibitor and an Fgl2 inhibitor.
43. The pharmaceutical composition of paragraph 40, wherein the composition comprises an IL-33 inhibitor and an Fgl2 inhibitor, or an ST2 inhibitor and an Fgl2 inhibitor.
44. The pharmaceutical composition of paragraph 40, wherein the composition comprises a TIGIT inhibitor, an IL-33 inhibitor and/or an ST2 inhibitor, and an Fgl2 inhibitor.
45. The pharmaceutical composition of any of paragraphs 40-44, further comprising an anti-cancer agent, an anti-infection agent, and/or an agent for treatment of allergy, asthma and/or atopy.
46. The pharmaceutical composition of paragraph 45, wherein the agent comprises an immunotherapy that increases a proinflammatory T cell response and/or an agent that suppresses an anti-inflammatory T cell response.
47. A method of treating a patient diagnosed with cancer and/or infection comprising administering to the patient a composition comprising at least two of the therapeutic agents selected from the group consisting of a TIGIT inhibitor, an IL-33 inhibitor, an ST2 inhibitor, and an Fgl2 inhibitor.
48. The method of paragraph 47, wherein the infection is selected from the group consisting of chronic vial infection, intracellular bacterial infection, extracellular bacterial infection, fungal infection, and a combination of two or more thereof.
49. The method of paragraph 47 or 48, wherein the composition comprises a TIGIT inhibitor and an IL-33 inhibitor, or a TIGIT inhibitor and an ST2 inhibitor.
50. The method of paragraph 47 or 48, wherein the composition comprises a TIGIT inhibitor and an Fgl2 inhibitor.
51. The method of paragraph 47 or 48, wherein the composition comprises an IL-33 inhibitor and an Fgl2 inhibitor, or an ST2 inhibitor and an Fgl2 inhibitor.
52. The method of paragraph 47 or 48, wherein the composition comprises a TIGIT inhibitor, an IL-33 inhibitor and/or an ST2 inhibitor, and an Fgl2 inhibitor.
53. The method of any of paragraphs 47-52, further comprising administering the patient an anti-cancer agent and/or anti-infection agent.
54. The method of paragraph 53, wherein the anti-cancer and/or anti-infection agent comprises an immunotherapy that increases a proinflammatory T cell response and/or an agent that suppresses an anti-inflammatory T cell response.
55. A method of identifying a patient who is more likely to be responsive to a TIGIT agonist or IL-33 agonist therapy, wherein the patient is diagnosed to have an autoimmune disease or disorder and/or parasitic infection, the method comprising:
- a. measuring the level of Fgl2 activity or expression in a sample from the patient diagnosed to have an autoimmune disease or disorder and/or parasitic infection; and
- b. comparing the level of Fgl2 or expression in the sample with an Fgl2 reference, and:
  - i. when the level of Fgl2 activity or expression is lower than the Fgl2 reference, the patient is identified to be more likely to be responsive to a TIGIT agonist or IL-33 agonist therapy;
  - ii. when the level of Fgl2 activity or expression is the same as or greater than the Fgl2 reference, the patient is identified as likely to respond to an alternative, anti-inflammatory immunotherapy comprising an activator of an anti-inflammatory T cell response pathway and/or a suppressor of a pro-inflammatory T cell response pathway.
56. The method of paragraph 55, wherein the activator of the anti-inflammatory T cell response comprises a TIM-3 agonist, a galectin-9 molecule, a PD-1 agonist, a PD-L1 agonist, a CTLA-4 agonist, a Lag-3 agonist, an antagonist of an immune checkpoint activating molecule, an agonist of an immune checkpoint inhibitory molecule, or any combination thereof.
57. The method of paragraph 55 or 56, wherein the patient has been receiving an immunotherapy.
58. The method of any of paragraphs 55-57, wherein the autoimmune disease or disorder is selected from the group consisting of infection, acute inflammation, chronic inflammation, and any combination thereof.
59. A method of treating a patient who is determined to have an autoimmune disease or disorder and/or parasitic infection, the method comprising:
- a) measuring the level of IL-33 activity or expression in a sample from a patient who is determined to have an autoimmune disease or disorder and/or parasitic infection; and
- b) comparing the level of IL-33 activity or expression in the sample with an IL-33 reference, and:
  - i. when the level of IL-33 activity or expression is lower than the IL-33 reference, administering to the patient a composition comprising a TIGIT agonist and/or an Fgl2 agonist; or
  - ii. when the level of IL-33 activity or expression is the same as or greater than the IL-33 reference, either (A) administering an alternative, anti-inflammatory immunotherapy treatment without the TIGIT agonist or Fgl2 agonist, or (B) determining if the level of at least one other activating immune regulator in the sample is greater than the level of the corresponding reference, or if the level of at least one inhibitory immune regulator in the sample is less than the level of the corresponding reference.
60. The method of paragraph 59, wherein the at least one inhibitory immune regulator comprises Fgl2, TIGIT, ST2, CD155, CD112 or a combination thereof.
61. The method of paragraph 59 or 60, wherein when the level of IL-33 activity or expression is the same as or greater than the IL-33 reference, the method further comprises measuring the level of Fgl2 activity or expression in a sample from the patient and comparing the level of Fgl2 activity or expression in the sample with an Fgl2 reference, and:
- a. when the level of Fgl2 activity or expression is lower than the Fgl2 reference, administering to the patient a composition comprising a TIGIT agonist and/or an Fgl2 agonist; or
- b. when the level of Fgl2 activity or expression is the same as or greater than the reference, administering an alternative, anti-inflammatory immunotherapy treatment without a TIGIT agonist or Fgl2 agonist.
62. The method of any of paragraphs 59-61, wherein the TIGIT agonist is selected from the group consisting of a protein, a peptide, a nucleic acid, an antibody, a small molecule, a vaccine, a TIGIT-overexpressing immune cell (e.g., a T cell), an ST2 agonist, a CD112 agonist, a CD155 agonist, and a combination thereof.
63. The method of any of paragraphs 59-62, wherein the Fgl2 agonist is selected from the group consisting of a protein, a peptide, a nucleic acid, an antibody, a small molecule, a vaccine, a TIGIT agonist, and a combination thereof.
64. The method of any of paragraphs 59-63, wherein the TIGIT agonist or Fgl2 agonist is constructed to target TIGIT+regulatory T (Treg) cells.
65. The method of any of paragraphs 59-64, wherein the alternative, anti-inflammatory immunotherapy treatment without the TIGIT agonist or IL-33 agonist is a therapy comprising an activator of an anti-inflammatory T cell response pathway and/or a suppressor of a proinflammatory T cell response pathway.
66. The method of any of paragraphs 59-65, wherein a patient with an IL-33 level lower than the IL-33 reference is further administered a therapy comprising an activator of an anti-inflammatory T cell response pathway and/or a suppressor of a proinflammatory T cell response pathway.
67. The method of paragraph 65 or 66, wherein the activator of the anti-inflammatory T cell response comprises a TIM-3 agonist, a galectin-9 molecule, a PD-1 agonist, a PD-L1 agonist, a CTLA-4 agonist, a Lag-3 agonist, an antagonist of an immune checkpoint activating molecule, an agonist of an immune checkpoint inhibitory molecule, or any combination thereof.
68. The method of any of paragraphs 59-67, wherein the patient has been receiving an immunotherapy.
69. The method of any of paragraphs 59-68, wherein the sample is a blood sample from the patient.
70. The method of any of paragraphs 59-68, wherein the sample is an inflammatory tissue sample from the patient.
71. The method of any of paragraphs 59-70, wherein the reference corresponds to the level of IL-33 activity or expression in a normal healthy subject.
72. The method of any of paragraphs 59-70, wherein the reference corresponds to the level of IL-33 activity or expression in a normal tissue of the same type or lineage as the sample.
73. The method of any of paragraphs 59-70, wherein the reference corresponds to the level of IL-33 activity or expression in an inflammatory tissue with a high level of IL-33 expression or activity.
74. The method of any of paragraphs 59-70, wherein the reference is a standard numerical level or threshold.
75. The method of any of paragraphs 59-74, wherein the autoimmune disease or disorder is selected from the group consisting of infection, acute inflammation, chronic inflammation, and any combination thereof.
76. A method of treating a patient determined to have an autoimmune disease or disorder and/or parasitic infection, the method comprising:
- a) measuring the level of Fgl2 activity or expression in a sample from a patient determined to have an autoimmune disease or disorder and/or parasitic infection; and
- b) comparing the level of Fgl2 activity or expression in the sample with an Fgl2 reference, and:
  - i. when the level of Fgl2 activity or expression is lower than the Fgl2 reference, administering to the patient a composition comprising a TIGIT agonist and/or an IL-33 agonist; or
  - ii. when the level of Fgl2 activity or expression is the same as or greater than the Fgl2 reference, administering an alternative, anti-inflammatory immunotherapy treatment without a TIGIT agonist or IL-33 agonist.
77. The method of paragraph 76, wherein the alternative, anti-inflammatory immunotherapy treatment without a TIGIT agonist or IL-33 agonist is a therapy comprising an activator of an anti-inflammatory T cell response pathway and/or a suppressor of a proinflammatory T cell response pathway.
78. The method of paragraph 76, wherein the patient with an Fgl2 level lower than the Fgl2 reference is further administered a therapy comprising an activator of an anti-inflammatory T cell response pathway and/or a suppressor of a proinflammatory T cell response pathway.
79. The method of paragraph 77 or 78, wherein the activator of the anti-inflammatory T cell response comprises a TIM-3 agonist, a galectin-9 molecule, a PD-1 agonist, a PD-L1 agonist, a CTLA-4 agonist, a Lag-3 agonist, an antagonist of an immune checkpoint activating molecule, an agonist of an immune checkpoint inhibitory molecule, or any combination thereof.
80. The method of any of paragraphs 76-79, wherein the patient has been receiving an immunotherapy.
81. The method of any of paragraphs 76-80, wherein the autoimmune disease or disorder is selected from the group consisting of infection, acute inflammation, chronic inflammation, and any combination thereof.
82. A method of treating a patient having an autoimmune disease or disorder and/or parasitic infection, and a low level of Fgl2, the method comprising:
- a) determining a first level of Fgl2 expression or activity in a sample from a patient having an autoimmune disease or disorder and/or parasitic infection, and a low level of Fgl2;
- b) administering an agent that activates IL-33 activity and/or TIGIT activity;
- c) determining a second level of Fgl2 expression or activity after said administering; and
- d) comparing said first and second levels of Fgl2 expression or activity, wherein the agent administered in (b) is effective if said second level of Fgl2 expression or activity is greater than said first level, and wherein the agent administered in (b) is ineffective if said second level of Fgl2 expression is the same as or lower than said first level.
83. The method of paragraph 82, further comprising, when said IL-33 agonist or TIGIT agonist therapy is effective, continuing to administer said agent that activates IL-33 activity and/or TIGIT activity.
84. The method of paragraph 82, further comprising, when said IL-33 agonist therapy or said TIGIT agonist therapy is ineffective, administering said agent that activates IL-33 activity and/or TIGIT activity at a higher dose.
85. The method of paragraph 82, further comprising, when said IL-33 agonist therapy or said TIGIT agonist therapy is ineffective, discontinuing said IL-33 agonist therapy or said TIGIT agonist therapy.
86. The method of paragraph 82, further comprising, when said IL-33 agonist therapy or said TIGIT agonist therapy is ineffective, administering a therapy comprising an activator of an anti-inflammatory T cell response pathway and/or a suppressor of a proinflammatory T cell response pathway.
87. The method of paragraph 86, wherein the activator of the anti-inflammatory T cell response comprises a TIM-3 agonist, a galectin-9 molecule, a PD-1 agonist, a PD-L1 agonist, a CTLA-4 agonist, a Lag-3 agonist, an antagonist of an immune checkpoint activating molecule, an agonist of an immune checkpoint inhibitory molecule, or any combination thereof.
88. The method of any of paragraphs 82-87, wherein the autoimmune disease or disorder is selected from the group consisting of infection, acute inflammation, chronic inflammation, and any combination thereof.
89. A method of treating a patient having an autoimmune disease or disorder and/or parasitic infection that exhibits a reduced level of IL-33, the method comprising:
- a) determining a first level of TIGIT and/or Fgl2 expression or activity in a sample from a patient having an autoimmune disease or disorder and/or parasitic infection with a reduced level of IL-33;
- b) administering an agent that activates IL-33 activity;
- c) determining a second level of TIGIT or Fgl2 expression or activity after said administering; and
- d) comparing said first and second levels of TIGIT and/or Fgl2 expression or activity, wherein IL-33 agonist therapy is effective if said second level of TIGIT and/or Fgl2 expression or activity is greater that said first level, and wherein IL-33 agonist therapy is ineffective if said second level of TIGIT and/or Fgl2 expression is the same as or lower than said first level.
90. The method of paragraph 89, further comprising, when said IL-33 agonist therapy is effective, continuing to administer said agent that activates IL-33 activity.
91. The method of paragraph 89, further comprising, when said IL-33 agonist therapy is ineffective, discontinuing said IL-33 agonist therapy.
92. The method of paragraph 89, further comprising, when said IL-33 agonist therapy is ineffective, administering said agent that activates IL-33 activity at a higher dose.
93. The method of any of paragraphs 89-92, wherein the autoimmune disease or disorder is selected from the group consisting of infection, acute inflammation, chronic inflammation, and any combination thereof.
94. A pharmaceutical composition comprising a pharmaceutically-acceptable excipient and at least two of the following therapeutic agents:

a. a TIGIT agonist;

b. an IL-33 agonist;

c. an ST2 agonist; and

d. an Fgl2 agonist.

95. The pharmaceutical composition of paragraph 94, wherein the composition comprises a TIGIT agonist and an IL-33 agonist, or a TIGIT agonist and an ST2 agonist.
96. The pharmaceutical composition of paragraph 94 wherein the composition comprises a TIGIT agonist and an Fgl2 agonist.
97. The pharmaceutical composition of paragraph 94, wherein the composition comprises an IL-33 agonist and an Fgl2 agonist, or an ST2 agonist and an Fgl2 agonist.
98. The pharmaceutical composition of paragraph 94, wherein the composition comprises a TIGIT agonist, an IL-33 agonist and/or an ST2 agonist, and an Fgl2 agonist.
99. The pharmaceutical composition of any of paragraphs 94-98, further comprising an immunotherapy for treatment of an autoimmune disease or disorder and/or parasitic infection.
100. The pharmaceutical composition of paragraph 99, wherein the immunotherapy for treatment of an autoimmune disease or disorder and/or parasitic infection comprises an agent that decrease a proinflammatory T cell response and/or an agent that activates an anti-inflammatory T cell response.
101. The pharmaceutical composition of paragraph 99 or 100, wherein the autoimmune disease or disorder is selected from the group consisting of infection, acute inflammation, chronic inflammation, and any combination thereof.
102. A method of treating a patient determined to have an autoimmune disease or disorder and/or parasitic infection comprising administering to the patient a composition comprising at least two of the therapeutic agents selected from the group consisting of a TIGIT agonist, an IL-33 agonist, an ST2 agonist, and an Fgl2 agonist.
103. The method of paragraph 102, wherein the composition comprises a TIGIT agonist and an IL-33 agonist, or a TIGIT agonist and an ST2 agonist.
104. The method of paragraph 102, wherein the composition comprises a TIGIT agonist and an Fgl2 agonist.
105. The method of paragraph 102, wherein the composition comprises an IL-33 agonist and an Fgl2 agonist, or an ST2 agonist and Fgl2 agonist.
106. The method of paragraph 102, wherein the composition comprises a TIGIT agonist, an IL-33 agonist and/or an ST2 agonist, and an Fgl2 agonist.
107. The method of any of paragraphs 102-106, further comprising administering the patient an immunotherapy for treatment of an autoimmune disease or disorder and/or parasitic infection.
108. The method of paragraph 107, wherein the immunotherapy comprises an agent that decrease a proinflammatory T cell response and/or an agent that activates an anti-inflammatory T cell response.
109. The method of paragraph 107 or 108, wherein the autoimmune disease or disorder is selected from the group consisting of infection, acute inflammation, chronic inflammation, and any combination thereof.
110. A method of guiding selection of a treatment for a patient who is diagnosed with asthma, allergy, and/or atopy, the method comprising:
- a. measuring the level of Fgl2 activity or expression in a sample from the patient; and
- b. comparing the level of Fgl2 or expression in the sample with an Fgl2 reference, and:
  - i. when the level of Fgl2 activity or expression is greater than the Fgl2 reference, the patient is identified to be more likely to be responsive to an anti-TIGIT or anti-IL-33 therapy;
  - ii. when the level of Fgl2 activity or expression is the same as or less than the Fgl2 reference, the patient is identified as likely to respond to an alternative, Th2-dampening therapy or immunotherapy.
111. The method of paragraph 110, wherein the alternative, Th2-dampening therapy or immunotherapy comprises an activator of a proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.
112. The method of paragraph 111, wherein the activator of the proinflammatory T cell response comprises a TIM-3 inhibitor, an anti-galectin-9 molecule, a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a Lag-3 antagonist, an agonist of an immune checkpoint activating molecule, an antagonist of an immune checkpoint inhibitory molecule, or any combination thereof.
113. The method of any of paragraphs 110-112, further comprising administering to the patient the selected therapy.
114. The method of any of paragraphs 110-112, wherein the patient has been receiving a therapy for treatment of asthma, allergy, and/or atopy.
115. A method of treating a patient diagnosed with asthma, allergy, and/or atopy, the method comprising:
- a) measuring the level of IL-33 activity or expression in a sample from a patient diagnosed with asthma, allergy, and/or atopy; and
- b) comparing the level of IL-33 activity or expression in the sample with an IL-33 reference, and:
  - i. when the level of IL-33 activity or expression is greater than the IL-33 reference, administering to the patient a composition comprising a TIGIT inhibitor and/or an Fgl2 inhibitor; or
  - ii. when the level of IL-33 activity or expression is the same as or less than the IL-33 reference, either (A) administering an alternative, Th2-dampening therapy or immunotherapy treatment without the TIGIT inhibitor or Fgl2 inhibitor, or (B) determining if the level of at least one other inhibitory immune regulator in the sample is greater than the level of the corresponding reference, or if the level of at least one activating immune regulator in the sample is less than the level of the corresponding reference.
116. The method of paragraph 115, wherein the at least one inhibitory immune regulator comprises Fgl2, TIGIT, ST2, CD155, CD112, or a combination thereof.
117. The method of paragraph 116, wherein when the level of IL-33 activity or expression is the same as or less than the IL-33 reference, the method further comprises measuring the level of Fgl2 activity or expression in a sample from the patient and comparing the level of Fgl2 activity or expression in the sample with an Fgl2 reference, and:
- a. when the level of Fgl2 activity or expression is greater than the Fgl2 reference, administering to the patient a composition comprising a TIGIT inhibitor and/or an Fgl2 inhibitor; or
- b. when the level of Fgl2 activity or expression is the same as or less than the reference, administering an alternative, Th2-dampening therapy or immunotherapy without a TIGIT inhibitor or Fgl2 inhibitor.
118. The method of any of paragraphs 110-117, wherein the TIGIT inhibitor is selected from the group consisting of a protein, a peptide, a nucleic acid, an antibody, a small molecule, a vaccine, a TIGIT−/− immune cell (e.g., a T cell), an ST2 inhibitor, a CD112 inhibitor, a CD155 inhibitor, and a combination thereof.
119. The method of any of paragraphs 115-118, wherein the Fgl2 inhibitor is selected from the group consisting of a protein, a peptide, a nucleic acid, an antibody, a small molecule, a vaccine, a TIGIT inhibitor, and a combination thereof.
120. The method of any of paragraphs 110-119, wherein the TIGIT inhibitor or Fgl2 inhibitor is constructed to target TIGIT+regulatory T (Treg) cells.
121. The method of any of paragraphs 110-120, wherein the patient has been receiving a therapy for treatment of asthma, allergy, and/or atopy.
122. The method of any of paragraphs 110-121, wherein the sample is a blood sample from the patient.
123. The method of any of paragraphs 110-121, wherein the sample is a tissue sample from the patient.
124. The method of any of paragraphs 110-123, wherein the reference corresponds to the level of IL-33 activity or expression in a normal healthy subject.
125. The method of any of paragraphs 110-123, wherein the reference corresponds to the level of IL-33 activity or expression in a normal tissue of the same type or lineage as the sample.
126. The method of any of paragraphs 110-123, wherein the reference corresponds to the level of IL-33 activity or expression in a diseased tissue with a low level of IL-33 expression or activity.
127. The method of any of paragraphs 110-123, wherein the reference is a standard numerical level or threshold.
128. A method of treating a patient diagnosed with asthma, allergy, and/or atopy that has an elevated level of Fgl2, the method comprising:
- a) determining a first level of Fgl2 expression or activity in a sample from a patient diagnosed with asthma, allergy, and/or atopy that has an elevated level of Fgl2;
- b) administering an agent that inhibits IL-33 activity and/or TIGIT activity;
- c) determining a second level of Fgl2 expression or activity after said administering; and
- d) comparing said first and second levels of Fgl2 expression or activity, wherein the agent administered in (b) is effective if said second level of Fgl2 expression or activity is lower than said first level, and wherein the agent administered in (b) is ineffective if said second level of Fgl2 expression is the same as or higher than said first level.
129. The method of paragraph 128, further comprising, when said anti-IL-33 or anti-TIGIT therapy is effective, continuing to administer said agent that inhibits IL-33 activity and/or TIGIT activity.
130. The method of paragraph 128, further comprising, when said anti-IL-33 therapy or said anti-TIGIT therapy is ineffective, administering said agent that inhibits IL-33 activity and/or TIGIT activity at a higher dose.
131. The method of paragraph 128, further comprising, when said anti-IL-33 therapy or said anti-TIGIT therapy is ineffective, discontinuing said anti-IL-33 therapy or said anti-TIGIT therapy.
132. The method of paragraph 131, further comprising, when said anti-IL-33 therapy or said anti-TIGIT therapy is ineffective, administering a Th2-dampening therapy without the anti-IL-33 or anti-TIGIT therapy.
133. The method of paragraph 132, wherein the Th2-dampening therapy comprises an activator of a proinflammatory T cell response pathway and/or a suppressor of an anti-inflammatory T cell response pathway.
134. The method of paragraph 133, wherein the activator of the proinflammatory T cell response pathway comprises a TIM-3 inhibitor, an anti-galectin-9 molecule, a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a Lag-3 antagonist, an agonist of an immune checkpoint activating molecule, an antagonist of an immune checkpoint inhibitory molecule, or any combination thereof.
135. A method of treating a patient diagnosed with asthma, allergy, and/or atopy that exhibits an elevated level of IL-33, the method comprising:
- a) determining a first level of TIGIT and/or Fgl2 expression or activity in a sample from a patient diagnosed with asthma, allergy, and/or atopy that exhibits an elevated level of IL-33;
- b) administering an agent that inhibits IL-33 activity;
- c) determining a second level of TIGIT or Fgl2 expression or activity after said administering; and
- d) comparing said first and second levels of TIGIT and/or Fgl2 expression or activity, wherein anti-IL-33 therapy is effective if said second level of TIGIT and/or Fgl2 expression or activity is lower that said first level, and wherein anti-IL-33 therapy is ineffective if said second level of TIGIT and/or Fgl2 expression is the same as or higher than said first level.
136. The method of paragraph 135, further comprising, when said anti-IL-33 therapy is effective, continuing to administer said agent that inhibits IL-33 activity.
137. The method of paragraph 135, further comprising, when said anti-IL-33 therapy is ineffective, discontinuing said anti-IL-33 therapy.
138. The method of paragraph 137, further comprising, when said anti-IL-33 therapy is ineffective, administering said agent that inhibits IL-33 activity at a higher dose.
139. A method of treating a patient diagnosed with asthma, allergy, and/or atopy comprising administering to the patient a composition comprising at least two of the therapeutic agents selected from the group consisting of a TIGIT inhibitor, an IL-33 inhibitor, an ST2 inhibitor, and an Fgl2 inhibitor.
140. The method of paragraph 139, wherein the composition comprises a TIGIT inhibitor and an

IL-33 inhibitor, or a TIGIT inhibitor and an ST2 inhibitor.

141. The method of paragraph 139, wherein the composition comprises a TIGIT inhibitor and an Fgl2 inhibitor.
142. The method of paragraph 139, wherein the composition comprises an IL-33 inhibitor and an

Fgl2 inhibitor, or an ST2 inhibitor and an Fgl2 inhibitor.

143. The method of paragraph 139, wherein the composition comprises a TIGIT inhibitor, an IL-33 inhibitor and/or an ST2 inhibitor, and an Fgl2 inhibitor.
144. The method of any of paragraphs 139-143, further comprising administering the patient a therapy for treatment of asthma, allergy, and/or atopy.
145. The method of paragraph 144, wherein the therapy for treatment of asthma, allergy, and/or atopy comprises an immunotherapy that increases a proinflammatory T cell response and/or an agent that suppresses an anti-inflammatory T cell response.
146. A method of treating asthma, allergy, and/or atopy comprising treating a subject in need thereof a composition comprising an Fgl2 antagonist.
147. A method for increasing the differentiation and/or proliferation of functionally exhausted CD8+T cells in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a TIGIT antagonist.
148. A method for decreasing CD8+T cell exhaustion in a subject in need thereof, comprising administering to a subjective a pharmaceutical composition comprising a TIGIT antagonist.
149. The method of paragraph 147 or 148, wherein the subject in need thereof has cancer.
150. The method of paragraph 147 or 148, wherein the subject in need thereof has infection.
151. The method of paragraph 150, wherein the infection is selected from the group consisting of chronic viral infection, intracellular bacterial infection, extracellular bacterial infection, fungal infection, and a combination of two or more thereof.
152. The method of any of paragraphs 148-151, wherein when the TIGIT antagonist is ineffective, administering an alternative therapy that suppresses anti-inflammatory T cell response pathway.
153. The method of paragraph 152, wherein the alternative therapy comprises a TIM-3 antagonist, a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a Lag-3 antagonist, a BTLA antagonist, or any combinations thereof.
154. The method of any of paragraphs 147-153, wherein said subject has been receiving a cancer therapy.
155. The method of paragraph 154, wherein the cancer therapy comprises vaccine, chemotherapy, targeted therapy (e.g., kinase inhibitors), radiation therapy, surgery, immunotherapy, or any combination thereof.
156. The method of any of paragraphs 147-153, wherein said subject has been receiving an anti-infection therapy.

SOME SELECTED DEFINITIONS

As used herein and in the claims, the singular forms include the plural reference and vice versa unless the context clearly indicates otherwise. The term “or” is inclusive unless modified, for example, by “either.” Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” with respect to numerical values means within 5%

As used herein, the term “greater than” in the context of an increase in the activity level and/or expression of a target molecule (e.g., TIGIT, Fgl2, and/or IL-33) relative to its corresponding reference (e.g., a TIGIT reference, an Fgl2 reference and/or an IL-33 reference), the increase can be at least about 30% or more, including, e.g., at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 100% or more. In some embodiments, the increase can be at least about 1.1-fold or more, including, e.g., at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, or more.

The term “antibody” as used herein, whether in reference to an anti-TIGIT, anti-Fgl2, or anti-IL-33 antibody, refers to a full length antibody or immunoglobulin, IgG, IgM, IgA, IgD or IgE molecules, or a protein portion thereof that comprises only a portion of an intact antibody, generally including an antigen binding site of the intact antibody and thus retaining the ability to bind a target, such as an epitope or antigen. Examples of portions of antibodies or epitope-binding proteins encompassed by the present definition include: (i) the Fab fragment, having VL, CL, VH and CH1 domains; (ii) the Fab′ fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CH1 domain; (iii) the Fd fragment having VH and CH1 domains; (iv) the Fd′ fragment having VH and CH1 domains and one or more cysteine residues at the C terminus of the CH1 domain; (v) the Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) the dAb fragment (Ward et al., 341 Nature 544 (1989)) which consists of a VH domain or a VL domain that binds antigen; (vii) isolated CDR regions or isolated CDR regions presented in a functional framework; (viii) F(ab′)2 fragments which are bivalent fragments including two Fab′ fragments linked by a disulfide bridge at the hinge region; (ix) single chain antibody molecules (e.g., single chain Fv; scFv) (Bird et al., 242 Science 423 (1988); and Huston et al., 85 PNAS 5879 (1988)); (x) “diabodies” with two antigen binding sites, comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93/11161; Hollinger et al., 90 PNAS 6444 (1993)); (xi) “linear antibodies” comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al., 8 Protein Eng. 1057 (1995); and U.S. Pat. No. 5,641,870).

“Antibodies” include antigen-binding portions of antibodies such as epitope- or antigen-binding peptides, paratopes, functional CDRs; recombinant antibodies; chimeric antibodies; tribodies; midibodies; or antigen-binding derivatives, analogs, variants, portions, or fragments thereof.

The term “aptamer” refers to a nucleic acid molecule that is capable of binding to a target molecule, such as a polypeptide. For example, an aptamer of the invention can specifically bind to a TIGIT, Fgl2 and/or IL-33 polypeptide, or to a molecule in a signaling pathway that modulates the expression and/or activity of TIGIT, Fgl2 and/or IL-33. The generation and therapeutic use of aptamers are well established in the art. See, e.g., U.S. Pat. No. 5,475,096.

As used herein, the term “fusion protein” refers to a fusion polypeptide comprising a target polypeptide (e.g., TIGIT, Fgl2 or IL-33) and a second, heterologous fusion partner polypeptide. The fusion partner can, for example, increase the in vivo stability of the fusion polypeptide, modulate its biological activity or localization, or facilitate purification of the fusion polypeptide. Exemplary heterologous fusion partner polypeptides that can be used to generate such fusion polypeptides for use in the compositions and methods described herein include, but are not limited to, polyhistidine (His or 6His tag), Glu-Glu tag, glutathione S transferase (GST), thioredoxin, polypeptide A, polypeptide G, an immunoglobulin heavy chain constant region (Fc), and maltose binding polypeptide (MBP), which are particularly useful for isolation of the fusion polypeptides by affinity chromatography. For the purpose of affinity purification, relevant matrices for affinity chromatography, such as glutathione-, amylase-, and nickel- or cobalt-conjugated resins are used. Fusion polypeptides can also include “epitope tags,” which are usually short peptide sequences for which a specific antibody is available. Well known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus hemagglutinin (HA), and c myc tags. In some embodiments, the fusion polypeptides can have a protease cleavage site, such as for Factor Xa or Thrombin, which allows the relevant protease to partially digest the fusion polypeptides and thereby liberate the recombinant polypeptides therefrom. The liberated polypeptides can then be isolated from the fusion polypeptides by subsequent chromatographic separation.

All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

Examples
Example 1. Expression of TIGIT in Subsets of Regulatory T Cells (Treg) Cells

Regulatory T cells (Tregs) are a subset of CD4+T cells that is marked by Foxp3 expression and act as a central component in regulating immune responses to pathogens and in maintaining self-tolerance. Other regulatory populations also contribute to this balance, but Foxp3+Tregs are critical for maintaining immune homeostasis as demonstrated by the devastating multi-organ autoimmune disease caused by genetic deficiencies in Foxp3 (Brunkow et al., 2001; Wildin et al., 2001). Foxp3+Tregs are not all identical, but comprised of multiple, functionally diverse subtypes with distinct phenotypes and specialized functions. Foxp3+Tregs have been previously reported to specialize to selectively regulate specific effector T cell responses and control inflammation at defined anatomical tissue sites (Chaudhry et al., 2009; Cipolletta et al., 2012; Koch et al., 2009; Zheng et al., 2009). Although the transcription factors that differentially induce specialized suppressor functions in Tregs have been identified, the molecules that mediate these selective effector functions remain largely unknown. Identification of cytokines and cell surface molecules that mediate specialization of Treg function would allow the development of therapeutic approaches that target Tregs and selectively regulate specific types of T cell responses.

In conventional T cells, cytokines and co-stimulatory molecules act in concert for their differentiation and acquisition of effector functions. For example, OX40 was shown to augment Th2 responses by increasing IL-4 secretion and to favor the induction of Th9 cells (Flynn et al., 1998; Xiao et al., 2012). Similarly, ICOS regulates TFH expansion and critically contributes to Th17 function by regulating IL-23R expression in an IL-21 and c-Maf-dependent manner (Bauquet et al., 2009). In Tregs, co-inhibitory molecules, such as PD-1 and CTLA-4 promote suppressive function. PD-1 plays an important role in iTreg stability and suppressive function (Francisco et al., 2009). CTLA-4 is essential for Treg function (Wing et al., 2008) and can mediate suppression by enabling Tregs to compete with effector T cells for co-stimulatory signals on APCs and by inducing the production of indoleamine 2,3-dioxygenase (IDO) in APCs, thereby limiting T cell proliferation (Fallarino et al., 2003). While co-stimulatory molecules have been shown to promote effector functions of defined T helper lineages, there are no reports that implicate co-inhibitory molecules in the specialized function of Treg subsets, despite their important role in promoting the suppressive function of Tregs in general.

Recently, the novel co-inhibitory molecule TIGIT has been reported as an inhibitor of autoimmune responses (Joller et al., 2011; Levin et al., 2011). TIGIT can inhibit T cell responses by binding the ligand CD155 on dendritic cells (DCs) and thereby inhibiting IL-12 while inducing IL-10 production (Yu et al., 2009). In addition, TIGIT engagement also directly inhibits T cell activation and proliferation (Joller et al., 2011; Levin et al., 2011; Lozano et al., 2012). Like other co-inhibitory molecules, TIGIT is highly expressed on Tregs (Levin et al., 2011; Yu et al., 2009); however, whether it plays a functional role in these cells has not been explored.

It was sought in this Example to determine whether TIGIT expression defines a functionally distinct Treg subset. To this end, expression of TIGIT was determined in natural as well as in vitro differentiated induced Tregs (nTregs and iTregs, respectively) populations (FIG. 1A). nTregs could be separated into distinct TIGIT+ and TIGIT−populations, while TIGIT was uniformly upregulated in iTregs. To assess whether TIGIT functionally contributes to Treg differentiation, the ability of TIGIT-deficient T cells to differentiate into Foxp3+iTreg in vitro was evaluated. As iTregs express high levels of TIGIT, it was next sought to determine whether TIGIT+Tregs present in vivo might also be generated peripherally. However, TIGIT+Tregs were primarily Neuropilin-1+ and express high levels of Helios, indicating that the majority of TIGIT+Tregs are nTregs (FIGS. 7A and 7B). TIGIT+Tregs also do not appear to be a terminally differentiated lineage as both TIGIT+ and TIGIT− Tregs can convert into the other subset as evidenced by the loss of TIGIT from TIGIT+Tregs upon adoptive transfer and conversely gain of TIGIT expression where TIGIT− Tregs were injected (FIG. 7C).

As nTregs can be separated into distinct TIGIT+ and TIGIT−populations, the functional differences between these two populations were also characterized. To this end, TIGIT+ and TIGIT− nTregs were sorted based on Foxp3-GFP reporter expression and their ability to suppress CD4+Foxp3− effector T cells was compared in vitro. TIGIT+nTreg showed an increased ability to suppress TCR-stimulated proliferation of conventional T cells (FIG. 1B). TIGIT therefore marks a functionally distinct subset of nTregs with superior suppressive capacity.

Next, it was sought to determine whether TIGIT+Tregs are also detected in humans and whether they might represent a similarly potent Treg subset as in mice. TIGIT expression in human CD4+T cells was analyzed and it was found that a large proportion of human Tregs are TIGIT+(FIG. 1C). In vitro suppression assays were then performed to assess whether human TIGIT+ and TIGIT−Tregs also differ in their suppressive capacity. Indeed, increased suppression by TIGIT+Tregs was compared to TIGIT−Tregs (FIGS. 1D-1F), indicating that TIGIT+Tregs are highly suppressive and may also represent a functionally distinct Treg subset in humans. Accordingly, TIGIT expression defines a functionally distinct Treg subset.

Example 2. Characterization of TIGIT+Tregs Display an Activated Phenotype

To determine the differences between TIGIT+ and TIGIT−Tregs, their gene expression patterns were analyzed by microarray profiling. Overall, a total of 472 and 184 genes were over- or under-expressed in TIGIT+ cells relative to their TIGIT− counterparts (with an arbitrary cut-off of at fold change>2 and t-test p<0.05; FIG. 7D, Table 1). These belonged to several functional families including chemokines/cytokines or their receptors, transcription factors, and costimulatory and other surface receptors, as well as molecules typical of activated Treg cells such as Klrg1 and Il10. Overall, TIGIT+Tregs seemed to display a more activated phenotype than their TIGIT− counterparts (FIGS. 7E and 7F). Despite appearing anergic in vitro, Tregs proliferate extensively upon activation in vivo. Therefore, it was sought to determine whether the activated phenotype of TIGIT+Tregs that was observed translates into higher proliferation in vivo. TIGIT+Tregs indeed expressed 3-fold higher levels of Ki67, which serves as an indirect marker for proliferation (FIG. 7G). In addition, TIGIT+Tregs also incorporated four times higher amounts of BrdU than their TIGIT− counterparts when we labeled proliferating cells in vivo (FIG. 2H). The activated phenotype of TIGIT+Tregs characterized by transcriptional profiling therefore translates into a higher rate of proliferation in vivo.

Example 3. Comparison of TIGIT+Tregs with Pro-Inflammatory T Cell Lineages

Tregs are generally known to share a number of features with the effector population they suppress, including the expression of chemokine receptors as well as the transcription factors that induce the development of those effector T cells (Chaudhry et al., 2009; Chung et al., 2011; Koch et al., 2009; Linterman et al., 2011; Zheng et al., 2009). Unexpectedly, the pattern of chemokine receptors expressed by TIGIT+Tregs does not overlap with that of any particular Th effector subset (FIG. 2A), but includes receptors that are expressed by several lineages, mainly by the pro-inflammatory Th1 and Th17 subsets (Ccr2, Ccr5, Ccr6, Cxcr3, and Cxcr6), but to a lesser degree also those expressed by Th2 (Ccr3) or T_FHcells (Cxcr5). Without wishing to be bound by theory, these findings can indicate that TIGIT+Tregs are equipped to target a broad spectrum of effector cells and tissues, for example, in some embodiments, under pro-inflammatory conditions.

Similarly, the transcription factors that are more highly expressed in TIGIT+Tregs do not specifically fall within the fingerprint of a particular effector lineage (FIG. 2B). On the contrary, transcription factors that are expressed at higher levels in TIGIT+Tregs include those that are specific for Th1 (Tbx21) and Th17 cells (Rora, Rorc, Irf4, Ahr), while only minor or no differences could be observed in the expression of the Th2 lineage factor Gata3 and the TFH lineage specific transcription factor Bcl6 (FIG. 3C). Prdm1 was expressed at higher levels in TIGIT+, which is consistent with higher production of IL-10 by TIGIT+Tregs (FIGS. 2A, 2C and 8A).

The expression profile of TIGIT+Tregs was analyzed in relation to signatures of the Treg subsets as described above. TIGIT+Tregs were enriched for a gene set that distinguishes CXCR3+Tregs (FIG. 2D). Cells that express T-bet and were previously reported to be specialized in suppression of Th1 responses (Koch et al., 2012; Koch et al., 2009). IRF4 expression in Tregs is important for control of Th2 responses as demonstrated by the dysregulated Th2 responses observed in mice that lack IRF4 in Foxp3+Tregs (Zheng et al., 2009). Many of the IRF4-dependent genes are upregulated in TIGIT+Tregs (FIG. 2E), which is in line with the increased expression of IRF4 by TIGIT+Tregs (FIG. 2C). Further, it was found that TIGIT+Tregs share features with Tregs from mice in which Foxp3 is modified by an N-terminal fusion with GFP (FIG. 2F). The modification of As previously reported, Foxp3 can lead to modified interaction with Foxp3 cofactors (Bettini et al., 2012; Fontenot et al., 2005). Thus, in these mice the GFP-fusion altered the molecular characteristics of Foxp3, reducing HIF-1α and increasing IRF4 interactions, modifying the Treg transcriptome and resulting in enhanced suppression of Th2/Th17 responses but weaker suppression of Th1 responses (Bettini et al., 2012; Darce et al., 2012). Overall these data indicate that, rather than representing a subset specialized to suppress a specific T effector lineage, TIGIT+Tregs express features of multiple pro-inflammatory Th subsets.

Example 4. Functional Roles of TIGIT+Tregs in Mediating Immune Suppression

Effector as well as regulatory T cell function is shaped through the cytokine environment as well as engagement of co-stimulatory ligands. By analyzing the transcriptional profile for differential expression of membrane receptors, a distinct pattern of co-stimulatory molecules in TIGIT+vs. TIGIT−Tregs was indeed detected. TIGIT+Tregs express higher levels of the co-stimulatory molecule ICOS, but also showed increased expression of a number of co-inhibitory molecules, such as CTLA-4, PD-1 (Pdcd1), Lag3, and Tim3 (Havcr2) (FIGS. 3A-3C, and FIGS. 8A-8C). Co-inhibitory molecules such as CTLA-4 and PD-1 not only serve as markers for T cell activation but also contribute to Treg stability and function (Francisco et al., 2009; Wing et al., 2008). Therefore, increased expression of these molecules by TIGIT+Tregs indicates that they might be better equipped for mediating suppression. Indeed, Treg signature genes and mediators of the suppressive function were found to also be differentially expressed in TIGIT+vs. TIGIT−Tregs (FIGS. 3C-3G and 8A-8C). TIGIT+Tregs expressed higher levels of CTLA-4, CD25, and GITR but showed no or only slight differences in the expression of Lag3. In mice, CD39 and CCR6 expression in TIGIT+Tregs is comparable to TIGIT−Tregs while human TIGIT+Tregs show an upregulation of these markers (FIGS. 3C and 8B). When comparing TIGIT+ to TIGIT−Tregs, the immunosuppressive cytokine IL-10 appears to be primarily produced by TIGIT+Tregs (FIG. 3C). Expression of the master transcription factor of Treg function Foxp3 was not significantly different between TIGIT+ and TIGIT−Tregs in the microarray analysis, but when analyzed by qPCR and flow cytometry Foxp3 was expressed at higher levels in TIGIT+Tregs (FIGS. 3F and 3G). Since Foxp3 regulates transactivation of CD25 (Fontenot et al., 2003), higher expression of CD25 was also observed in TIGIT+Tregs (FIGS. 3C and 3F). In addition, Treg effector molecules such as Granzyme B, IL-10 and Fgl2 were also expressed at significantly higher levels in TIGIT+Tregs (FIGS. 3C-3D and 3G and FIG. 8A). TIGIT+Tregs therefore represent an activated, highly suppressive Treg subset.

Example 5. TIGIT Ligation Induces the Treg Effector Molecule Fgl2

As TIGIT marks a highly suppressive Treg population, it was next sought to determine whether TIGIT ligation could directly induce Treg effectors. Among these, IL-10 and Fgl2 stood out as particularly interesting molecules as both were highly expressed in TIGIT+Tregs (FIG. 3G) and these molecules were previously reported to be able to suppress pro-inflammatory responses (Chan et al., 2003; Kuhn et al., 1993). To assess whether Fgl2 and IL-10 could be induced through TIGIT, effector and regulatory T cells were isolated and stimulated in vitro in the presence of an agonistic anti-TIGIT antibody (Ab). As shown in FIG. 4A, Il10 mRNA in effector T cells was slightly reduced by TIGIT. Without wishing to be bound by theory, this is most likely due to the inhibition of activation of effector T cells when TIGIT is engaged by the agonistic antibody. In contrast, TIGIT ligation triggered a 2-fold increase in Il10 gene expression by Tregs in vitro. TIGIT did not induce a profound induction of Fgl2 mRNA in effector T cells. However, Fgl2 expression levels in Tregs were dramatically increased in the presence of agonistic anti-TIGIT Ab indicating that TIGIT signaling induces Fgl2 in TIGIT+Tregs (FIGS. 4A and 4B). Taken together, this finding indicates that TIGIT ligation induces Il10 and Fgl2 mRNA in Tregs.

It was next sought to determine whether TIGIT was also able to induce Fgl2 and IL-10 in vivo. As the functional effects of Abs can differ dramatically in vitro and in vivo, the anti-TIGIT Ab was evaluated to assess whether the Ab also acted agonistically in vivo. To this end, mice were immunized and treated with anti-TIGIT Ab and Ag-specific proliferation was determined 10 days later. The anti-TIGIT Ab was indeed able to reduce the Ag-specific T cell response and therefore also acts agonistically in vivo (FIG. 9A). Next, the same immunization and Ab treatment regimen were used, cells from spleen and LN were restimulated 10 days after immunization for 2 days in vitro, and Fgl2 and IL-10 in the culture supernatants were analyzed. IL-10 could not be detected in these cultures (data not shown). However, Fgl2 was significantly increased in cell supernatants from anti-TIGIT treated mice without affecting Treg frequencies or composition (FIG. 4C, FIG. 9B). TIGIT therefore induces Fgl2 in vitro and in vivo.

To further investigate the role of IL-10 and Fgl2 as Treg effector molecules, it was next sought to determine whether neutralizing them would abolish the differences in suppressive capacity of TIGIT+ and TIGIT−Tregs observed in vitro. Blocking or deletion of IL-10 had no effect on suppression by TIGIT+ or TIGIT−Tregs in vitro (FIG. 4D). Similarly, neutralizing or deleting Fgl2 had no effect on the suppression by TIGIT−Tregs, which express only minimal amounts of Fgl2 (FIGS. 4A and 4E). In contrast, neutralization of Fgl2 reduced the level of suppression from TIGIT+Tregs to that observed for TIGIT−Tregs (FIG. 4E), indicating that Fgl2 is a major effector molecule driving the increased suppression by TIGIT+Tregs observed in vitro. Similarly, Fgl2-deficient TIGIT+Tregs showed a significant decrease in their ability to suppress in vitro, down to the level observed in TIGIT−Tregs (FIG. 4E). Therefore, TIGIT ligation triggers secretion of Fgl2 by Tregs, which enables them to act as highly potent suppressors.

To understand how TIGIT could induce Fgl2 expression, the genomic region of Fgl2 was searched for binding sites of transcription factors that showed differential expression in our microarray analysis of TIGIT+vs. TIGIT−Tregs (FIG. 2B). The analysis indicated that the Fgl2 gene contains binding sites for the transcription factor CEBPα, which was differentially expressed in TIGIT+Tregs. Quantitative PCR confirmed that CEBPα is highly expressed in TIGIT+but not TIGIT−Tregs (FIG. 4F). To assess whether TIGIT signaling is able to upregulate Cebpa expression, T cells were stimulated with agonistic anti-TIGIT Ab in vitro and Cebpa induction in Tregs was observed in response to TIGIT engagement (FIG. 4G). Chromatin Immunoprecipitation (ChIP)-PCR using an anti-CEBPαAb together with primer pairs specific for the Fgl2 genomic region indicated that CEBPαbinds to the Fgl2 gene (FIG. 4H). To further analyze whether CEBPαcan promote transcription of Fgl2, CEBPα was overexpressed in nTregs. An increase in Fgl2 expression following CEBPαtransfection in nTregs was observed (FIG. 4I), indicating that CEBPαdrives Fgl2 expression. TIGIT might therefore equip Tregs for superior suppression by inducing CEBPα, thereby promoting Fgl2 expression.

Example 6. TIGIT+Tregs Inhibit Th1 and/or Th17 but not Th2 Responses

Fgl2 not only suppresses effector T cell proliferation, it also shifts the cytokine profile towards a Th2 response as it inhibits Th1 responses while promoting Th2 polarization and induction of IL-10 and IL-4 (Chan et al., 2003; Shalev et al., 2008). Furthermore, Fgl2 is important for Treg function in vivo as Fgl2-deficient Tregs show impaired control of effector T cell expansion in lymphopenic hosts (FIG. 10A). As TIGIT+Tregs produce high levels of Fgl2, without wishing to be bound by theory, they might similarly affect the cytokine profile of a T cell response by having differential suppressive effects on different Th lineages. To test this hypothesis, naïve effector T cells and TIGIT- and TIGIT+Tregs were co-cultured under differentiation conditions for Th1, Th2, and Th17 cells and then expression of lineage-specific cytokines were assessed. TIGIT+Tregs showed no difference in suppressing Th1 differentiation when compared to TIGIT−Tregs as indicated by reduced expression of IFNγ(FIGS. 5A and 10B). Similarly, both subsets potently suppressed expression of IL-17 by Th17 cells. However, in contrast to TIGIT−Tregs, TIGIT+Tregs did not suppress differentiation of Th2 cells as indicated by IL-4 production comparable to unsuppressed controls (FIGS. 5A and 10B). This effect was dependent on the ability of TIGIT to induce Fgl2 as TIGIT+Tregs from Fgl2−/− mice were able to suppress Th2 differentiation, indicating that Fgl2 produced by TIGIT+Tregs interferes with suppression of Th2 responses (FIGS. 5A and 10B). In line with these results, analysis of human effector T cells observed in the presence of TIGIT+ or TIGIT−Tregs also showed a potent inhibition of Th1 and/or Th17 but not Th2 responses (FIG. 5B). To determine whether these results also translated into selective suppression of Th1 and/or Th17 vs. Th2 responses in vivo, TIGIT+ and TIGIT−OVA-specific OT-II Tregs together with OT-II effector cells were transferred into WT recipients, which were immunized with OVA in CFA, the ability of the different Treg subsets to suppress the Th1 and/or Th17 responses was analyzed upon immunization. Surprisingly, TIGIT+ and TIGIT− were equally capable of suppressing effector T cell expansion as determined by the number of Vβ5⁺ OT-II cells. In addition, differentiation of Th1 and Th17 cells was suppressed equally well by TIGIT+vs. TIGIT−Tregs in vivo as determined by IFNγ and IL-17 production (FIGS. 5C-5F). When mice that had received OT-II effector T cells together with TIGIT+ or TIGIT−Tregs were immunized for induction of allergic airway inflammation, TIGIT−Tregs were able to suppress the disease. In contrast, TIGIT+Tregs failed to inhibit recruitment of antigen-specific Vβ5⁺ OT-II cells to the lung and production of Th2 cytokines (IL-4 and IL-13) was significantly higher than in mice that had received TIGIT−Tregs (FIGS. 5G, 5H). Consistent with an increase in Th2 cells in the presence of TIGIT+Tregs, high numbers of eosinophils were detected in the bronchio-alveolar lavage of these mice (FIG. 5I). Taken together these data indicate that TIGIT+Tregs selectively suppress pro-inflammatory Th1 and Th17 cells, but not Th2 responses. TIGIT+Tregs appear to mediate this effect by inhibiting Th1 and/or Th17 but promoting and/or sparing Th2 differentiation.

Example 7. TIGIT+Tregs Suppress Pro-Inflammatory Th1 and/or Th17 but not Th2 Responses in Vivo

To assess the effects of TIGIT+Tregs in a setting where multiple pro-inflammatory effector T cell lineages contribute to disease, their role was evaluated in experimental autoimmune encephalomyelitis (EAE), in which disease progression is promoted by Th1 as well as Th17 cells. To engage TIGIT in this model, mice immunized for EAE were treated with an anti-TIGIT antibody (Ab) that acts agonistically in vivo (FIGS. 9A-9B). While this treatment does not necessarily distinguish between TIGIT on effector or regulatory T cells, the vast majority of TIGIT expressing cells are Tregs. The observed effects are therefore most likely at least in part mediated through TIGIT+Tregs. Animals that received the anti-TIGIT agonistic Ab indeed showed significantly reduced EAE (FIGS. 9C-9D), indicating that TIGIT also plays a role in suppressing mixed pro-inflammatory responses. To determine whether both Th1 and Th17 responses were affected, the cytokine secretion by T cells from spleen and draining LN at the induction phase of the disease (day 10) was analyzed. Both IFN- and IL-17 levels were significantly reduced in mice treated with the anti-TIGIT Ab (FIGS. 9E-9F), indicating that Th1 as well as Th17 responses are suppressed. TIGIT+Tregs therefore not only suppress one specific effector subset but are capable of inhibiting multiple pro-inflammatory effector T cell responses including Th1 and Th17 responses.

Based on the in vitro and in vivo data (FIGS. 5A-5I) in Example 6, it was next sought to determine whether TIGIT+Tregs might be able to mediate similar effects in inhibiting effector Th1 and/or Th17 responses by skewing the effector T cell response towards a Th2 phenotype if the system is not biased by immunization with adjuvant. To this end, the Rag-transfer model of colitis in which disease induction does not rely on immunization and therefore does not introduce a cytokine bias through the choice of adjuvant (Izcue et al., 2008) was used. To induce disease, congenically marked CD45.1 effector T cells were transferred into Rag1−/− recipients; either alone or together with CD45.2+TIGIT+Foxp3+ or TIGIT-Foxp3+Tregs. Mice that received the effector T cells alone lost weight over time, while co-transfer of either TIGIT+ or TIGIT−Tregs was able to suppress the disease (FIG. 6A). Surprisingly, both Treg populations were able to suppress the disease equally well. Co-transfer of either Treg population prevented tissue inflammation, as indicated by the histopathological colitis score (FIG. 6B), and suppressed the expansion of effector T cells in vivo (FIG. 6C). In addition, both groups that received Tregs showed comparable frequencies of Foxp3+CD4+Tregs, indicating that both TIGIT+ and TIGIT−Tregs are able to expand and persist to the same degree in vivo (FIG. 6D). They also showed comparable stability, as approximately 80% of the transferred Tregs still expressed Foxp3 10 weeks after transfer (FIG. 6E). TIGIT+ as well as TIGIT−Tregs were able to suppress pro-inflammatory cytokines as T cells from the mesenteric LNs produced significantly lower levels of IFN-γ and TNF-α than those from mice that did not receive Tregs (FIGS. 6F and 11). In contrast, TIGIT+Treg did not suppress, or may have even increased the expression of the Th2 cytokines IL-4 and IL-10 when compared to the control group (no Tregs) (FIG. 6F). Intracellular cytokine staining indicated that, while IL-10 was produced by both effector T cells and Tregs, IL-4 was entirely produced by effector T cells (FIG. 11). No significant IL-17 was observed under any conditions of in vivo transfer (data not shown). TIGIT+Treg therefore potently suppress pro-inflammatory responses in vivo, while sparing or promoting Th2-like responses.

Discussion for Examples 1-7

Examples 1-7 show that TIGIT+Foxp3+T cells were identified as a distinct Treg subset that specifically suppresses pro-inflammatory Th1 and/or Th17 responses through the secretion of Fgl2, which contributes to the higher suppressive capacity of TIGIT+Treg in vitro. The findings presented herein further indicate that engagement of TIGIT induces Fgl2. Through the secretion of Fgl2, TIGIT+Tregs are able to selectively suppress pro-inflammatory effector Th1 and Th17 responses, shifting the balance towards Th2 responses. This is one of the first examples of how a co-inhibitory molecule can mediate selective inhibition of certain effector responses while leaving others intact.

Uncontrolled Th1 and/or Th17 responses can lead to chronic immune activation and inflammation that results in induction of autoimmune diseases such as psoriasis, rheumatoid arthritis, inflammatory bowel disease and multiple sclerosis. The findings presented herein indicate that TIGIT+Tregs can play an important role in preventing these autoimmune disorders and maintaining self-tolerance. Fgl2−/− mice were previously discussed to show increased Th1 but diminished Th2 responses and spontaneously develop autoimmune glomerulonephritis as they age (Shalev et al., 2008). In addition, the findings presented herein can indicate that Fgl2-deficient mice would also show enhanced Th17 responses. That the TIGIT+Treg-specific effector molecule Fgl2 results in inhibition of Th1 and/or Th17 responses while increasing Th2 responses indicate that TIGIT+Tregs can act as a specialized subset that does not globally suppress all effector T cell responses but may specifically suppress inflammatory immune responses and tissue inflammation mediated by Th1 and/or Th17 cells, but does not affect Th2 responses.

TIGIT was first described as an inhibitory molecule that suppresses immune responses indirectly by regulating DC function. By interacting with its ligand CD155 on DCs, TIGIT was shown to induce IL-10 and suppress IL-12 production in DCs and thereby inhibit Th1 responses (Yu et al., 2009). TIGIT was previously reported to have T cell intrinsic inhibitory effects (Joller et al., 2011). Since Tregs are the primary cell type that constitutively expresses TIGIT, without wishing to be bound by theory, many of the DC effects that have been observed might be mediated by TIGIT+Tregs. In addition to TIGIT-induced IL-10 produced by the DCs themselves, it is contemplated that increased amounts of IL-10 and Fgl2 produced by TIGIT+Tregs may also contribute to the generation of tolerogenic DCs and thereby inhibit the generation of effector Th1 responses. Although TIGIT-induced IL-10 was shown to suppress both Il-12p35 and IL-12p40 (Yu et al., 2009), the effect of Fgl2 in suppressing these key differentiating cytokines has not been evaluated. We propose that IL-10 and Fgl2 secreted by TIGIT+Tregs may act in concert to suppress both IL-12 and IL-23 production from activated DCs and thereby inhibit development of both Th1 and Th17 responses.

Tregs represent a heterogeneous population that encompasses many specialized subpopulations. While Foxp3 is necessary to equip T cells with basic Treg functions (Fontenot et al., 2005), additional factors are required for efficient suppression of effector T cell responses in vivo and for maintaining immune tolerance. Several transcription factors have been identified that drive additional programs in Tregs to efficiently control certain classes of effector T cells and autoimmunity and inflammation in defined target tissues. For instance, tissue specific Treg subsets, such as “fat Tregs”, have very distinct transcriptional signatures that are shaped by the expression of tissue-specific transcription factors that allow them to adapt their function to the specific tissue requirements (Cipolletta et al., 2012). Similarly, Tregs that are specialized in controlling specific effector T cell lineages co-express lineage-specific transcription factors from T helper cells, such as T-bet, IRF4, Stat3, or Bcl6 to fulfill their subset-specific inhibitory functions (Chaudhry et al., 2009; Chung et al., 2011; Koch et al., 2009; Linterman et al., 2011; Zheng et al., 2009). These Examples show that TIGIT+Tregs share features with several different Treg subsets and express elevated levels of T-bet and IRF4 as well as Th17-specific transcription factors such as RORα and RORγ, when compared to TIGIT−Treg cells. The finding that TIGIT+Tregs express elevated levels of IRF4 would indicate that they are well equipped for suppression of Th2 responses, because IRF4-deficiency in Foxp3+T cells results in spontaneous Th2 pathology (Zheng et al., 2009). However, the findings presented herein indicate that TIGIT+Treg effectively inhibit pro-inflammatory Th1 and/or Th17 responses but not Th2 responses. It should be noted that IRF4 is not only expressed in Th2 cells but is also required for Th17 differentiation. While conditional deletion of IRF4 in Foxp3+Tregs most prominently affects control of Th2 responses, these mice also have slightly elevated IL-17 levels (Zheng et al., 2009) and in settings where the immune response is dominated by Th17 effector cells, such as arthritis, diminished function of IRF4 in Tregs results in impaired control of Th17 responses (Darce et al., 2012). While TIGIT+Tregs seem to share functional aspects with IRF4-deficient Tregs, their ability to potently suppress Th17 responses distinguishes them from IRF4-deficient Tregs.

The findings presented herein show that in addition to the lineage- and tissue-specific transcription factors, co-inhibitory molecules like TIGIT also contribute to the functional specialization of Tregs by inducing a distinct set of suppressive mediators that can selectively suppress certain classes of effector T cell responses. In the case of TIGIT+Tregs, expression of Fgl2 allows them to selectively suppress pro-inflammatory responses, including Th1 and/or Th17 responses. In some embodiments, co-inhibitory receptors can therefore tailor the suppressive function of Foxp3+Tregs to what is required in a specific inflammatory environment. The expression pattern of these receptors and/or engagement through their ligands in a particular tissue environment can thereby alter the molecular signature of Tregs and equip them with specialized suppressive mechanisms that are tailored for a specific tissue or type of inflammation.

Besides transcription factors, the present findings show that that cell surface molecules like TIGIT expressed on Foxp3+Tregs can differentially suppress effector T cell responses, providing a target by which defined subsets of Tregs can be manipulated to regulate immune and autoimmune responses.

Exemplary Experimental Procedures for Examples 1-7

Animals.

C57BL/6 (B6), B6.SJL-Ptprc^aPepc^b/BoyJ (CD45.1), B6.129P2-Il10^tm1Cgn/J (IL-10^−/−) and B6.129S7-Rag1^tm1MOM/J (RAG1^−/−) mice were purchased from the Jackson Laboratories. Foxp3-GFP.KI reporter mice (Bettelli et al., 2006), and Fgl2−/− mice (Shalev et al., 2008) have been previously described. Animals were maintained in a conventional, pathogen-free facility and all experiments were carried out in accordance with guidelines prescribed by the Institutional Animal Care and Use Committee (IACUC).

Human Samples.

Peripheral venous blood was obtained from healthy control volunteers in compliance with Institutional Review Board protocols. Total CD4+T cells were isolated by negative selection (CD4+T cell isolation kit II, Miltenyi Biotec, Auburn, Calif.) and then sorted by flow cytometry.

Treg Differentiation and Suppression Assays.

Cells were cultured in DMEM supplemented with 10% (vol/vol) FCS, 50 mM mercaptoethanol, 1 mM sodium pyruvate, nonessential amino acids, L-glutamine, and 100 U/ml penicillin and 100 g/ml streptomycin. CD4+T cells from splenocytes and lymph node cells were isolated using anti-CD4 beads (Miltenyi). For in vitro Treg differentiation, naïve CD4⁺CD62L⁺CD44⁻cells were sorted by flow cytometry and stimulated with plate bound anti-CD3 (145-2C11, 0.3 μg/ml) and anti-CD28 (PV-1, 2 μg/ml) in the presence of 2.5 ng/ml TGF-β (R&D). Foxp3 expression was assessed by flow cytometry 4 days later. For suppression assays, CD4+Foxp3− responder cells and CD4+Foxp3+Tregs were flow sorted from Foxp3-GFP.KI reporter mice based on GFP expression. CD4+Foxp3− (2×10⁴/well) and CD4+Foxp3+ cells were cultured in triplicate in the presence of soluble anti-CD3 (1 μg/ml) and irradiated splenic APCs (1.2×10⁵/well). After 48 h cells were pulsed with 1 μCi [³H]thymidine for an additional 18 h, harvested and [3H]thymidine incorporation was analyzed to assess proliferation. Percentage of suppression=100−C.P.M. of well with the indicated ratio of effector: Tregs/mean C.P.M. of wells with CD4⁺Foxp3-effectors alone. Where indicated, anti-Fgl2 Ab (clone 6D9, 30 μg/ml, Abnova), anti-IL-10 Ab (clone JESS-16E3, Biolegend) or an isotype control was added to the cultures. For human Treg suppression assays, CD25-depleted T cells were CFSE-labeled and co-cultured with FACS-sorted Tregs (TIGIT+ or TIGIT−) at indicated ratios. Cells were stimulated with Treg Inspector Beads (Miltenyi) at manufacturer's recommended concentration. At day 4, cells were stained with LIVE/DEAD Fixable Dead Cell Stain Kit (Molecular Probes) to allow gating on viable cells and proliferation was measured by CFSE dilution. Samples were analyzed by flow cytometry.

Microarray.

CD4+T cells were pre-purified from splenocytes and lymph node cells of naïve Foxp3-GFP.KI reporter mice using Dynal beads (Invitrogen) and CD4+Foxp3+TIGIT+ and CD4+Foxp3+TIGIT− cells were sorted by flow cytometry. CD4+Foxp3+CXCR3+ and CD4+Foxp3+CXCR3+ were similarly sorted from spleens of Foxp3-GFP.KI mice. All cells were double-sorted for purity, the final sort being directly into TRIzol (Invitrogen). RNA was extracted and used to prepare probes for microarray analysis on the Affymetrix Mouse Gene 1.0ST platform, using ImmGen protocols (Heng and Painter, 2008). Microarray data was analyzed using the GeneSpring 11 (Agilent, Santa Clara, Calif.; quantile normalization) or GenePattern (RMA normalization) software. Genes of interest (fold change>1.5) were manually selected and two-way hierarchical clustering using Euclidean distance metric was performed to generate heat-maps. Analysis of signature genes within the TIGIT+/TIGIT− comparison used previously determined genesets: a T cell activation/proliferation signature from in vivo activated T cells (Hill et al., 2007); the canonical Treg/Tconv signature (Hill et al., 2007); a geneset that distinguishes Treg cells which express the chimeric GFP-Foxp3 fusion protein (Darce et al., 2012), and the IRF4-dependent signature in Treg cells (Zheng et al., 2009). P values form a chi-squared test. The genes and Probe IDs included in these signatures are listed in Table 2.

Flow Cytometry.

Surface staining was performed for 20 minutes at 4° C. in PBS containing 0.1% sodium azide and 0.5% BSA. For intracellular cytokine stainings, cells were re-stimulated with phorbol 12-myristate 13-acetate (PMA, 50 ng/ml, Sigma), ionomycin (1 μg/ml, Sigma), and GolgiStop (1 μl/1 ml, BD Bioscience) at 37° C. in 10% CO₂for 4 h before staining was performed using the Cytofix/Cytoperm kit (BD Biosciences). Intracellular staining for Foxp3 was performed using the Foxp3 Staining Buffer Set (eBioscience). Antibodies were from BioLegend except for anti-Foxp3 (eBioscience), and anti-Ki67 (BD Biosciences). 7AAD was purchased from BD Biosciences. Samples were acquired on a FACSCalibur or LSRII flow cytometer (BD Biosciences) and analyzed using the FlowJo software (Tree Star).

Quantitative RT-PCR.

RNA was extracted with RNAeasy mini Kits (Qiagen) and cDNA was prepared using the iScript cDNA synthesis kit (BioRad). Real-time PCR (RT-PCR) was performed using Taqman probes and the 7500 Fast Real-Time PCR system (Applied Biosystems). All samples were normalized to b-actin internal control.

In Vitro Antibody Treatment.

CD4+Foxp3− effector T cells and CD4+Foxp3+Tregs were sorted from Foxp3-GFP.KI reporter mice and stimulated at a density of 1×10⁶/ml with plate bound anti-CD3 (145-2C11, 1 μg/ml), anti-CD28 (PV-1, 2 μg/ml), and anti-TIGIT (4D4, 100 μg/ml) or isotype control antibody. RNA was isolated on day 3. Antibodies to human TIGIT were provided by ZymoGenetics, Inc. (a wholly-owned subsidiary of Bristol-Myers Squibb). Cells were stimulated with anti-CD3 (UCHT1, 1 μg/ml), anti-CD28 (28.2, 1 μg/ml) and IL-2 (10 U/ml) in the presence of agonistic anti-TIGIT at 20 μg/ml or IgG isotype control. Gene expression was assessed on day 4.

In Vivo Antibody Treatment.

Mice were immunized s.c. with 200 μl of an emulsion containing 100 μg of MOG_35-55peptide (MEVGWYRSPFSRVVHLYRNGK) in adjuvant oil (CFA) on day 0 and treated i.p. with 100 μg of anti-TIGIT (clone 4D4) or isotype control Ab (armenian hamster IgG) on days 0, 2, 4, 10 and 17. For antigen-specific proliferation assays spleens and lymph nodes were collected on day 10 and 2.5×10⁶cells/ml were re-stimulated with 50 μg/ml MOG_35-55peptide. After 48 h Fgl2 concentrations in culture supernatants were determined by ELISA (Biolegend).

ChIP-PCR and Over-Expression.

ChIP assays were performed on P815 cells expressing TIGIT using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling) according to the manufacturer's instructions. Lysates were immunoprecipitated using anti-C/EBPα antibody (8 μg; Santa Cruz Biotech, sc-61) or rabbit IgG isotype control. Quantitative PCR reactions were performed with SYBR-Green on ChIP-bound and input DNA. % input=2%×2^{(CT 2% input sample−CT sample)}. For CEBPα over-expression CD4+Foxp3+Tregs were flow sorted from Foxp3-GFP.KI reporter mice. 5×10⁵Tregs/ml were stimulated with Mouse T-Activator CD3/CD28 Dynabeads (Invitrogen) and transfected with 10 μg/ml of Cebpa cDNA in pCMV6-Kan/Neo or the empty vector, which had been pre-incubated with FuGene 6 (Roche Diagnostics). RNA was extracted on day 4 with RNAeasy mini Kits (Qiagen), samples were treated with DNAse (RNAse-free DNAse set, Qiagen) and cDNA was prepared using the iScript cDNA synthesis kit (BioRad). Cebpa over-expression was verified by Taqman PCR.

Suppression of Th Differentiation.

For in vitro experiments CD4+CD62L+naive T cell from CD45.1 mice and CD4+Foxp3+TIGIT+ or TIGIT−Tregs from Foxp3-GFP.KI mice (CD45.2) were sorted and cultured at 10⁵Teff and 10⁴Treg/well. Cells were stimulated with Mouse T-Activator CD3/CD28 Dynabeads (Invitrogen, 0.6 μl/well) in the presence of polarizing cytokines (Th1: 4 ng/ml IL-12; Th2: 4 ng/ml IL-4; Th17: 10 ng/ml IL-6, 2 ng/ml TGF-β; all cytokines from R&D). RNA was extracted after 3 days and flow cytometric analysis was performed on day 5.

For in vivo experiments 1-2×10⁵CD4+CD62L+CD25+ sorted naive effector T cell and 2.5-5×10⁴CD4+CD25+TIGIT+ or TIGIT−Tregs (Teff: Treg 4:1) from OT-II mice were transferred i.v. into WT recipients one day before immunization. To elicit a mixed Th1/Th17 response, mice were immunized with 10 μg OVA (Sigma) emulsified in CFA and spleens and draining LN were analyzed 10 days later. Allergic airway inflammation was induced as described previously (Haworth et al., 2008; Rogerio et al., 2012). For example, mice were sensitized with 10 μg OVA in alum i.p. on days 0 and 7 and challenged with 6% (wt/vol) OVA aerosol for 25 min on days 14, 15, 16 and 17. Cells from lung and bronchioalveolar lavage were analyzed directly following challenge on day 17.

Colitis and Histopathology.

CD4+CD45RBhigh naive T cell from CD45.1 mice and CD4+Foxp3+Tregs from Foxp3-GFP.KI mice were purified by cell sorting after enrichment for CD4+ cells using anti-CD4 MACS beads. 8×10⁵CD4⁺CD45RB^highcells were transferred i.v. into RAG1−/− mice, either alone or with Tregs (4:1 effector T cell:Treg ratio) and mice were weighed weekly. At the time of sacrifice small and large intestine samples were fixed in neutral buffered formalin.

Routinely processed, paraffin-embedded tissue samples were stained with hematoxylin and eosin (H&E). The presence and severity of colitis was evaluated in a blinded manner and graded semi-quantitatively from 0 to 3 for the three following criteria: epithelial hyperplasia; leukocyte infiltration; and the presence of crypt abscesses. Scores for each criterion were added to give an overall inflammation score for each sample of 0-9.

In Vivo Treg Stability.

TIGIT+ or TIGIT−Tregs from Foxp3-GFP.KI mice were purified by cell sorting after enrichment for CD4+ cells using anti-CD4 MACS beads. 10⁶CD4+Foxp3-GFP+TIGIT+ or TIGIT−Tregs were transferred i.v. into CD45.1 mice and transferred cells were analyzed for TIGIT expression 20 days later by flow cytometry gating on CD45.2+ donor cells.

BrdU Labeling.

For BrdU labeling, naïve Foxp3-GFP.KI mice were injected i.p. daily with 1 mg of BrdU for 4 days and spleens and LNs were harvested on day 5. BrdU was stained in sorted CD4+Foxp3+TIGIT+ and CD4+Foxp3+TIGIT− cells using the APC BrdU Flow Kit (BD Biosciences).

In Vivo Antibody Treatment.

Mice were immunized s.c. with 200 μl of an emulsion containing 100 μg of MOG_35-55peptide (MEVGWYRSPFSRVVHLYRNGK) in adjuvant oil (CFA) on day 0 and treated i.p. with 100 μg of anti-TIGIT (clone 4D4) or isotype control Ab (armenian hamster IgG) on days 0, 2, 4, 10 and 17. Spleens and lymph nodes were collected on day 10 and 2.5×10⁶cells/ml were re-stimulated in the presence of a range of concentrations of MOG_35-55peptide (0.07 μg/ml-50 μg/ml). After 48 h, plates were pulsed with 1 μCi/well [³H]thymidine and incubated for an additional 18 h before being harvested onto glass fiber filters. ³H-thymidine incorporation was analyzed using a β-counter (1450 Microbeta, Trilux, Perkin Elmer).

In Vitro Treg Differentiation.

CD4+T cells were isolated from mice using anti-CD4 beads (Miltenyi). Naïve CD4+CD62L+CD44− cells were sorted by flow cytometry and stimulated with plate bound anti-CD3 (145-2C11, 0.3 μg/ml) and anti-CD28 (PV-1, 2 μg/ml) in the presence of 2.5 μg/ml TGF-β (R&D) and where indicated 10 μg/ml recombinant Fgl2. Foxp3 expression was assessed by flow cytometry 4 days later.

In Vivo Suppression.

CD4+T cell from CD45.1 mice and CD4+CD25+Tregs from WT or Fgl2−/− mice were purified by cell sorting after enrichment for CD4+ cells using anti-CD4 MACS beads. CD45.1 CD4+T cell were labeled with CFSE and transferred i.v. into RAG1−/− mice, either alone or with Tregs (5:1 effector T cell/Treg ratio). On day 8 mice were sacrificed and CFSE dilution in CD45.1+ effector T cells was analyzed by flow cytometry.

Statistical Analysis.

Statistical significance was assessed either by 2-tailed Student's T-test (two groups) or ANOVA for multiple groups with a post hoc Tukey's test; P values<0.05 were considered statistically significant. Statistical significance values indicated as follows: p<0.05 (*), p<0.01 (**) and p<0.005 (***).

TABLE 1

Differentially regulated genes in TIGIT⁻ vs. TIGIT⁺ Tregs Comparison

TIGIT− vs TIGIT+ SpLN Tregs

Fold change in

gene expression

(TIGIT+ Treg

cells relative

to their TIGIT−

Name
Description
counterparts)
T- test

10547590
Klrg1
16.949153
0.08277953

10349603
Il10
12.5
0.00502744

10352178
Sccpdh
11.235955
0.02691577

10503098
Lyn
8.6206897
0.05098679

10421517
Cysltr2
7.518797
0.01049428

10454015
Ttc39c
7.4626866
0.0022178

10368970
Prdm1
7.0921986
0.00621068

10439527
Tigit
6.993007
0.00186793

10574524
Ces2c
6.7114094
0.0165658

10603551
Cybb
6.6666667
0.00373176

10375443
Havcr2
6.5359477
0.03476303

10420308
Gzmb
6.2111801
0.00857544

10519983
Fgl2
6.0606061
0.00621597

10568714
Mki67
5.7803468
0.10430529

10408689
Nrn1
5.7142857
0.00067242

10526832
LOC100504914
5.6818182
0.0054879

10466521
Gcnt1
5.6497175
0.01675728

10399148
Rapgef5
5.5555556
0.02740164

10363082
Lilrb4
5.4945055
0.03565153

10487480
Bub1
5.4347826
0.17528406

10403229
Itgb8
5.2910053
0.00645382

10586448
2810417H13Rik
5.0251256
0.1783378

10511363
Penk
5
0.03340601

10521731
Ncapg
5
0.07555397

10365933
Eea1
4.9019608
0.01919938

10384458
Plek
4.9019608
0.03442288

10601350
Fgf16
4.9019608
0.04717276

10590242
Ccr8
4.8543689
0.00390674

10518313
Tnfrsf8
4.8543689
0.01295347

10523156
Cxcl2
4.8076923
0.0196782

10540472
Bhlhe40
4.7619048
0.03546062

10411611
Naip5
4.7169811
0.00649136

10462796
Kif11
4.6948357
0.13286043

10363415
Spock2
4.6728972
0.00454537

10408935
Gm10786
4.587156
0.00029438

10443980
Myo1f
4.587156
0.00064718

10411622
Naip6
4.4642857
0.01417191

10553354
Nav2
4.4052863
0.00044491

10482528
Neb
4.4052863
0.0100309

10594762
Fam81a
4.3859649
0.03835893

10487506
Gm14005
4.3668122
0.00130514

10471555
Angptl2
4.3668122
0.00161377

10588243
Ryk
4.3290043
0.0003589

10382200
Ccdc46
4.3290043
0.00354873

10464905
Npas4
4.2918455
0.00373534

10363070
Gp49a
4.2918455
0.02607473

10562657
Gm5595
4.2194093
0.00090933

10494001
Tdpoz4
4.2194093
0.14975638

10500720
Slc22a15
4.2016807
0.0013601

10345752
Il1r2
4.1841004
0.0327554

10582997
Casp4
4.1666667
0.02254515

10582985
Casp1
4.1322314
0.01204709

10505674
Cntln
4.1322314
0.01308681

10356866
Pdcd1
4.1322314
0.01705969

10574532
Ces2d-ps
4.1322314
0.01858489

10542896
Bicd1
4.0816327
0.00159197

10439895
Alcam
4.0816327
0.00447

10511779
Atp6v0d2
4.0160643
0.00162049

10606910
Mcart6
4.0160643
0.00225771

10423599
Matn2
4.0160643
0.01580438

10380289
Mmd
3.9840637
0.00058236

10399087
Ncapg2
3.9525692
0.09359614

10571399
Zdhhc2
3.9215686
0.00022571

10497831
Ccna2
3.8910506
0.10693279

10607738
Car5b
3.875969
0.02088748

10499062
Fhdc1
3.8461538
0.01202645

10435907
Cd200r1
3.8167939
0.00586522

10548307
Klrb1c
3.8022814
0.00089588

10462866
Cep55
3.8022814
0.11031612

10476443
Plcb4
3.7735849
0.0136415

10389207
Ccl5
3.7735849
0.11229956

10447383
Epcam
3.7593985
0.06264469

10590494
Kif15
3.7453184
0.13765528

10412517
Gm3002
3.7313433
0.00303162

10586781
Myo1e
3.7174721
0.00013348

10390707
Top2a
3.7174721
0.12225475

10356299
Gpr55
3.7037037
0.04268382

10476740
Slc24a3
3.6900369
0.01069883

10436945
Slc5a3
3.6764706
0.00823772

10417235
Gm2897
3.6630037
0.00038087

10440186
Crybg3
3.6630037
0.01962173

10544660
Osbpl3
3.6363636
0.00222121

10412495
Gm3002
3.6363636
0.00583372

10417408
D830030K20Rik
3.6363636
0.02832373

10519857
Hgf
3.6231884
0.01510521

10417302
Gm3002
3.5971223
0.00194254

10590628
Ccr3
3.5714286
0.00826259

10409278
Nfil3
3.5587189
0.05973224

10553598
Cyfip1
3.5335689
0.00160251

10543120
Ica1
3.5335689
0.00197274

10417258
Gm3002
3.5335689
0.00416404

10462005
Tmem2
3.5211268
0.00284602

10418341
Il17rb
3.5087719
0.00756839

10417458
Gm5458
3.5087719
0.02039524

10476759
Rin2
3.4965035
0.00151299

10417359
Gm3002
3.4965035
0.00286363

10417226
Gm3002
3.4965035
0.00559209

10492890
Lrba
3.4843206
0.00287007

10417239
Gm1973
3.4843206
0.00715288

10601312
Chic1
3.4843206
0.00715637

10394770
Odc1
3.4843206
0.03679442

10417366
ENSMUSG00000068790
3.4722222
0.0060115

10461844
Gnaq
3.4722222
0.00665844

10359375
Gpr52
3.4722222
0.00861866

10361110
Dtl
3.4722222
0.12548646

10367919
Stx11
3.4602076
0.00460918

10412537
Gm3002
3.4482759
0.00149724

10542993
Pon3
3.4364261
0.0075274

10552143
Slc7a10
3.4364261
0.03125043

10531415
Cxcl10
3.4364261
0.03126957

10592201
Chek1
3.4246575
0.12067932

10454198
Rnf125
3.4129693
0.0031176

10590635
Ccr5
3.4129693
0.03010649

10417411
Gm3002
3.4013605
0.00520539

10422013
Klf12
3.4013605
0.00624066

10482687
Arl5a
3.4013605
0.00684182

10412549
D830030K20Rik
3.4013605
0.00799923

10515836
Ccnb1
3.4013605
0.04611089

10357833
Atp2b4
3.3898305
0.00197782

10519527
Abcb1a
3.3898305
0.00466438

10599174
Il13ra1
3.3898305
0.00736274

10417501
Gm5458
3.3898305
0.00875546

10462973
Hells
3.3898305
0.15731102

10544829
Jazf1
3.3783784
0.00342215

10417319
D830030K20Rik
3.3783784
0.00404997

10417245
Gm1973
3.3670034
0.00440714

10491848
Larp1b
3.3557047
0.00761383

10369932
Susd2
3.3557047
0.00835697

10358816
Lamc1
3.3444816
7.93E−05

10392415
Rgs9
3.3333333
0.00077218

10545154
Il23r
3.3333333
0.05666823

10543031
Slc25a13
3.3222591
0.00062077

10346799
Icos
3.3112583
0.00480172

10417461
Gm10406
3.30033
0.00164351

10411739
Ccnb1
3.2894737
0.04922262

10605674
Pola1
3.2894737
0.06416331

10478973
Cass4
3.2786885
0.00787172

10417421
Gm3696
3.2679739
0.00321407

10387257
Alox8
3.257329
0.01148882

10571696
Casp3
3.257329
0.04059645

10417253
Gm1973
3.2467532
0.00036471

10581992
Maf
3.2467532
0.00346861

10435920
Cd200r4
3.236246
0.02525016

10590631
Ccr2
3.2154341
0.04830149

10385248
Hmmr
3.2154341
0.2101738

10466745
Tjp2
3.1948882
0.03325137

10417264
Gm3002
3.1847134
0.00059997

10591781
Anln
3.1847134
0.13299794

10499108
Glt28d2
3.1545741
0.00418673

10503107
6330407A03Rik
3.1446541
0.07580716

10350733
Rgs16
3.125
0.02095813

10345791
Il1rl1
3.125
0.10437169

10484888
Ptprj
3.1152648
0.02062208

10402325
Asb2
3.1152648
0.02131779

10417373
Gm10406
3.1055901
0.00041173

10412520
Gm3002
3.1055901
0.0005167

10417286
Gm3002
3.1055901
0.00073736

10486396
Ehd4
3.0959752
0.00264405

10587350
Ddx43
3.0959752
0.00302497

10586700
Rora
3.0959752
0.00426848

10417504
Gm1973
3.0864198
0.00217757

10483178
Cobll1
3.0864198
0.02366217

10562637
Ccnb1
3.0864198
0.0489732

10368060
Ect2l
3.0864198
0.20420133

10602068
Mid2
3.0769231
0.00599048

10420362
Gjb2
3.0769231
0.1902505

10429754
Nrbp2
3.0487805
0.03126596

10585194
Il18
3.0487805
0.09548274

10594501
Ptplad1
3.0395137
0.00279641

10355567
Tmbim1
3.030303
0.04004553

10436106
C330027C09Rik
3.021148
0.09961112

10497122
Depdc1a
3.021148
0.24710867

10491699
Fgf2
3.0120482
0.00396892

10417326
Gm3002
3.003003
0.00029084

10401320
Adam4
3.003003
0.04067599

10406334
Mctp1
2.994012
0.00710938

10477187
Tpx2
2.9850746
0.1065914

10350838
2810417H13Rik
2.9850746
0.15896679

10417773
Gm5458
2.9673591
0.00378186

10375121
C530030P08Rik
2.9585799
0.01034509

10524878
Vsig10
2.9585799
0.01488337

10404840
Cd83
2.9411765
0.00229696

10561104
Axl
2.9411765
0.01659645

10411595
Naip2
2.9325513
0.00146451

10576661
Itgb1
2.9325513
0.00551884

10556266
Wee1
2.9325513
0.00617181

10424543
Wisp1
2.9325513
0.01202386

10498952
Gucy1a3
2.9325513
0.03400079

10523182
Areg
2.9239766
0.00497611

10415021
Abhd4
2.9069767
0.00507733

10375123
C530030P08Rik
2.9069767
0.00911151

10486875
Frmd5
2.9069767
0.01761078

10605431
Rab39b
2.8985507
0.00342792

10561702
Kcnk6
2.8901734
0.01942935

10484201
Ccdc141
2.8901734
0.02140296

10378286
Itgae
2.8818444
0.00852384

10493812
S100a4
2.8818444
0.01944604

10417446
4930555G01Rik
2.8735632
0.00558711

10564805
Pex11a
2.8735632
0.01736591

10474381
Kif18a
2.8735632
0.07955153

10394978
Rrm2
2.8735632
0.09466237

10482772
Nr4a2
2.8653295
0.08787412

10497149
Wls
2.8571429
0.00128082

10478364
Tox2
2.8571429
0.01701987

10452980
Eif2ak2
2.8490028
0.01607352

10592001
St14
2.8490028
0.0580992

10484894
Ptprj
2.8409091
0.00314912

10417124
B930095G15Rik
2.8328612
0.0140005

10388591
Cpd
2.8248588
0.00701122

10524308
Mir701
2.8248588
0.01073409

10350630
Fam129a
2.8089888
0.00213696

10593497
Zc3h12c
2.8011204
0.01017535

10402136
Gpr68
2.8011204
0.03628006

10362005
Ahi1
2.7932961
0.00128988

10359890
Nuf2
2.7932961
0.23297012

10367945
Phactr2
2.7855153
0.00266881

10356082
Plscr1
2.7855153
0.00872677

10395328
Snx13
2.7855153
0.02508373

10511617
Fam92a
2.7700831
0.00255819

10345807
Il18r1
2.7700831
0.00449592

10583320
BC017612
2.7624309
0.00646737

10521678
Cd38
2.7624309
0.00774699

10511382
Nsmaf
2.7548209
0.00280194

10505064
Tmem38b
2.7548209
0.01044574

10493820
S100a6
2.7548209
0.02699151

10606182
Mir421
2.7548209
0.06443783

10496204
Cenpe
2.7548209
0.12279284

10368062
Ect2l
2.7472527
0.00151131

10484402
Ctnnd1
2.739726
0.01174898

10428534
Trps1
2.739726
0.01884088

10399024
Adam6b
2.739726
0.14639423

10459643
4930503L19Rik
2.7322404
0.00484409

10491835
Larp1b
2.7322404
0.02052749

10474984
Nusap1
2.7322404
0.14846887

10417769
Gm2897
2.7247956
0.00034888

10361790
Fuca2
2.7247956
0.00836353

10523595
Ptpn13
2.7247956
0.0121956

10515090
Cdkn2c
2.7173913
0.00122007

10345824
Il18rap
2.7173913
0.01132779

10374500
Vps54
2.7100271
0.0072985

10456296
Malt1
2.7100271
0.03715951

10601011
Kif4
2.7100271
0.15030573

10349383
Slc35f5
2.6954178
0.00316724

10449452
Fkbp5
2.6954178
0.00844298

10602020
Tbc1d8b
2.6954178
0.04446942

10518300
Tnfrsf1b
2.688172
0.00219415

10556583
Nucb2
2.688172
0.01037182

10498309
Pfn2
2.688172
0.01090422

10366586
Ifng
2.6809651
0.03645065

10606640
Nox1
2.6666667
0.00492862

10352918
Mir29c
2.6595745
0.02079424

10410756
Ankrd32
2.6595745
0.03124248

10519988
Fam185a
2.6595745
0.07678483

10563883
Depdc1a
2.6595745
0.26481196

10432511
Racgap1
2.6525199
0.02623238

10548735
Dusp16
2.6455026
0.0091817

10418927
Bmpr1a
2.6455026
0.01148904

10479010
Spo11
2.6455026
0.08476739

10606058
Cxcr3
2.6385224
0.00059492

10446771
Lclat1
2.6385224
0.00557771

10511588
Tmem67
2.6385224
0.00831089

10347036
Mtap2
2.6385224
0.019728

10405785
0610007P08Rik
2.6385224
0.03150043

10388065
Nlrp1b
2.6385224
0.03222866

10365286
Eid3
2.6315789
0.00490943

10524515
Myo1h
2.6315789
0.03197403

10594774
Ccnb2
2.6315789
0.04620604

10452516
Ankrd12
2.6315789
0.06380163

10408937
Atxn1
2.617801
0.00172482

10540897
Pparg
2.617801
0.20092261

10359339
Rabgap1l
2.6109661
0.00208466

10476648
Dstn
2.6109661
0.02540187

10345241
Dst
2.6041667
0.03799886

10400589
C79407
2.6041667
0.24546253

10473281
Itgav
2.5974026
0.002269

10346790
Ctla4
2.5974026
0.01623906

10601303
Chic1
2.5974026
0.02815456

10369993
Gstt3
2.5839793
0.01439467

10465244
Malat1
2.5839793
0.03915652

10392010
1700081L11Rik
2.5773196
0.00373539

10534456
Hip1
2.5641026
0.00090612

10355050
Raph1
2.5575448
0.00033097

10498935
Gucy1b3
2.5575448
0.0008303

10366073
Cep290
2.5575448
0.12416189

10517364
A330049M08Rik
2.5510204
0.00376354

10535065
Adap1
2.5380711
0.00965112

10556820
Tmem159
2.5380711
0.12464267

10408975
Kif13a
2.5188917
0.00011864

10539617
Alms1
2.5188917
0.00780576

10414374
Ktn1
2.5188917
0.03076519

10474875
Casc5
2.5188917
0.19047732

10538791
Tnip3
2.5125628
0.08090276

10554667
Tmc3
2.5062657
0.00965655

10582295
Odc1
2.5062657
0.02176383

10571384
Efha2
2.5
0.06702998

10580457
N4bp1
2.4875622
0.00314058

10542791
Ppfibp1
2.4875622
0.00355237

10572170
D130040H23Rik
2.4875622
0.00740691

10424188
Mtbp
2.4875622
0.05750915

10458589
Prelid2
2.4813896
0.01795025

10458581
Gm10008
2.4813896
0.02598396

10354286
Kdelc1
2.4813896
0.02892792

10462632
Kif20b
2.4813896
0.12489398

10436662
Mir155
2.4813896
0.17654312

10519951
Pion
2.4752475
0.00127643

10581813
Mlkl
2.4752475
0.05280226

10554445
Prc1
2.4752475
0.0706501

10416155
Kctd9
2.4691358
0.00116305

10411728
Cenph
2.4691358
0.23008238

10355312
Ikzf2
2.4630542
0.00918153

10398039
Serpina3f
2.4630542
0.01679921

10357115
Dsel
2.4630542
0.05387983

10538394
Plekha8
2.4570025
0.01308491

10488459
Zfp442
2.4570025
0.08163826

10396645
Zbtb1
2.4509804
0.0039396

10587792
Plscr1
2.4449878
0.00073185

10453082
Hnrpll
2.4449878
0.00493423

10355227
1110028C15Rik
2.4449878
0.03236243

10349593
Faim3
2.4449878
0.0380532

10503617
F730047E07Rik
2.4449878
0.04447421

10435712
Cd80
2.4449878
0.07761971

10452508
Twsg1
2.4390244
0.01931803

10594251
Kif23
2.4390244
0.03323231

10421877
Diap3
2.4390244
0.05932931

10370544
2610008E11Rik
2.43309
0.02088547

10574023
Mt2
2.43309
0.24469845

10601449
Sh3bgrl
2.4271845
0.01427406

10428536
Trps1
2.4271845
0.0156411

10394783
Hpcal1
2.4271845
0.02267103

10423556
Pgcp
2.4271845
0.06123205

10487340
Ncaph
2.4271845
0.11463912

10501629
Cdc14a
2.4213075
0.00402386

10602385
Pfkfb1
2.4213075
0.00811626

10590479
Zfp167
2.4213075
0.01973184

10578690
Neil3
2.4213075
0.20210435

10469720
Acbd5
2.4154589
0.03806352

10507112
Stil
2.4154589
0.26073984

10603151
Gpm6b
2.4096386
0.09804744

10571870
Hmgb2
2.4096386
0.12672335

10587639
Nt5e
2.4038462
0.0008956

10442224
BC049807
2.4038462
0.00576263

10554325
5730590G19Rik
2.4038462
0.045224

10367076
Prim1
2.4038462
0.09828601

10593492
Zc3h12c
2.3866348
0.00601872

10407211
Ppap2a
2.3866348
0.02630078

10543067
Asns
2.3809524
0.01008112

10590597
Sacm1l
2.3809524
0.01294225

10495186
AI504432
2.3809524
0.02193077

10459905
Setbp1
2.3809524
0.05812016

10497971
Sclt1
2.3809524
0.08800643

10505213
E130308A19Rik
2.3696682
0.00173153

10595371
Hmgn3
2.3696682
0.01334807

10449935
Zfp870
2.3696682
0.01872383

10518350
Hmgb2
2.3696682
0.13314054

10480432
Mastl
2.3696682
0.27433254

10440388
Hspa13
2.3640662
0.00339499

10521136
Whsc1
2.3640662
0.00446787

10455647
Tnfaip8
2.3584906
0.00021297

10591614
Dock6
2.3584906
0.01325737

10579049
Gm10033
2.3584906
0.02444929

10535883
Katnal1
2.3584906
0.04351106

10395273
Gdap10
2.3584906
0.12686918

10383897
Nf2
2.3529412
0.00466102

10510580
Tnfrsf9
2.3529412
0.01860989

10369815
Cdk1
2.3529412
0.07141248

10368050
Ect2l
2.3474178
0.00385925

10405804
0610007P08Rik
2.3474178
0.00543767

10428310
Azin1
2.3474178
0.01111183

10445977
Ebi3
2.3474178
0.01516525

10365845
Fgd6
2.3474178
0.02901281

10406757
Col4a3bp
2.3474178
0.03391371

10440288
Zfp654
2.3419204
0.02826963

10491805
Plk4
2.3419204
0.08724536

10415911
Kif13b
2.3364486
0.00252308

10531256
AU017193
2.3364486
0.00833691

10462535
Pten
2.3364486
0.01977837

10579052
Gm10033
2.3364486
0.05030866

10384373
Fignl1
2.3364486
0.08904826

10382890
Sec14l1
2.3310023
0.03723295

10374895
1700034F02Rik
2.3255814
0.01281525

10412921
Nid2
2.3201856
0.00918366

10601844
Bhlhb9
2.3201856
0.01930196

10599416
Gm10483
2.3201856
0.0193513

10607774
Mospd2
2.3201856
0.02461639

10546855
Srgap3
2.3201856
0.07156709

10384974
Il9r
2.3201856
0.07218936

10422028
Tbc1d4
2.3148148
0.00338815

10578300
Mtmr7
2.3148148
0.02188629

10545958
Anxa4
2.3148148
0.07808225

10389025
Myo1d
2.3094688
0.00695187

10569707
Myadm
2.3041475
0.04818941

10401317
Gm4787
2.2988506
0.01407323

10503196
Chd7
2.2988506
0.01590884

10583326
Slc36a4
2.2988506
0.02031783

10491780
Hspa4l
2.2988506
0.03303836

10436169
Ift57
2.2988506
0.04472591

10510172
Hmgb2
2.2988506
0.132583

10420877
Esco2
2.2988506
0.27628066

10379127
Spag5
2.293578
0.07839113

10496262
Nhedc2
2.2883295
0.01981333

10417579
4930452B06Rik
2.2883295
0.0611149

10521927
Tbc1d19
2.283105
0.00666418

10541114
Rasgef1a
2.283105
0.0075592

10441436
Snx9
2.283105
0.02399696

10395612
G2e3
2.2779043
0.01471304

10389134
Slfn9
2.2779043
0.05108747

10394954
Grhl1
2.2727273
0.00025111

10412267
Itga2
2.2727273
0.00212204

10595718
Chst2
2.2727273
0.01464964

10396421
Hif1a
2.2624434
0.02758493

10448247
Zfp40
2.2624434
0.03927672

10547469
Wnk1
2.2624434
0.10076335

10466835
Snora19
2.2624434
0.10512874

10380116
Rnf43
2.2573363
0.00180114

10399691
Id2
2.2573363
0.00624245

10590909
Endod1
2.2573363
0.01808855

10603881
Zfp182
2.2573363
0.03851246

10485963
Arhgap11a
2.2573363
0.0866043

10451761
Tbc1d5
2.2573363
0.23402181

10442219
Zfp52
2.2522523
0.0034822

10407467
Akr1e1
2.2522523
0.00960602

10357436
Mcm6
2.2522523
0.01942241

10476945
Cst7
2.2522523
0.02205786

10447084
Galm
2.2522523
0.02988624

10584615
Pvrl1
2.2522523
0.07439894

10606714
Gla
2.247191
0.06098277

10409994
Gm5665
2.247191
0.11000257

10468762
4930506M07Rik
2.2421525
0.00761633

10592515
Ubash3b
2.2371365
0.00885917

10483679
Gpr155
2.2371365
0.02116137

10399973
Hdac9
2.2371365
0.06215052

10353004
Cks2
2.2371365
0.07932277

10441195
Dscam
2.2321429
0.0056505

10379363
Atad5
2.2321429
0.1076887

10356329
Snora75
2.2321429
0.1775966

10593332
Bco2
2.2321429
0.19013999

10394611
Nbas
2.2271715
0.00355172

10503218
Chd7
2.2271715
0.02083899

10441633
Ccr6
2.2271715
0.03100126

10562651
C330019L16Rik
2.2271715
0.0354997

10476297
Mir103-2
2.2271715
0.0608872

10420670
Dleu2
2.2271715
0.07459412

10405185
Cks2
2.2271715
0.107097

10594221
Lrrc49
2.2222222
0.03825033

10529741
Rab28
2.2172949
0.01176421

10518352
Hmgb2
2.2172949
0.14011238

10497399
Pde7a
2.2123894
0.00753532

10381588
Grn
2.2123894
0.01370998

10504692
Tmod1
2.2123894
0.0257923

10592106
Tirap
2.2123894
0.10197148

10406270
Glrx
2.2075055
0.01001241

10467110
Lipo1
2.2075055
0.04823091

10531737
Hpse
2.2075055
0.0484914

10533844
Rilpl2
2.2026432
0.00438382

10406905
Ccdc125
2.2026432
0.01098253

10513141
Ptpn3
2.1978022
0.01652439

10349510
Mir128-1
2.1978022
0.04433153

10357363
Nckap5
2.1929825
0.03936668

10379153
Aldoc
2.1929825
0.06289367

10390519
Plxdc1
2.1929825
0.06869935

10597279
Ccrl2
2.1881838
0.02338559

10602009
Rnf128
2.1881838
0.03506544

10594301
Coro2b
2.1881838
0.12097252

10385323
Mir146
2.1834061
0.02385294

10406364
2210408I21Rik
2.1786492
0.01275564

10519324
Cdk6
2.1786492
0.01549031

10608138
Ddx3y
2.1786492
0.03732003

10389395
Brip1
2.1786492
0.08775499

10485405
Cd44
2.173913
0.01271296

10350594
Ivns1abp
2.173913
0.04840403

10421555
Mir687
2.173913
0.05565063

10467230
Ide
2.1691974
0.00386768

10407792
Gpr137b-ps
2.1691974
0.00858809

10587683
Bcl2a1a
2.1691974
0.03382114

10369102
Gm9766
2.1645022
0.00916278

10595633
Bcl2a1d
2.1645022
0.02794013

10425207
H1f0
2.1645022
0.03520816

10587107
Myo5a
2.1598272
0.02041305

10602827
A830080D01Rik
2.1598272
0.04816672

10603598
Rpgr
2.1551724
0.01262466

10523012
Dck
2.1551724
0.02170034

10474769
Bub1b
2.1551724
0.04249554

10352756
Lpgat1
2.1505376
0.00778529

10480329
Dnajc1
2.1505376
0.00964467

10587690
Bcl2a1b
2.1505376
0.01734898

10594110
Neo1
2.1505376
0.02541233

10405733
6720457D02Rik
2.1505376
0.04428622

10514865
Acot11
2.1505376
0.04791578

10587733
Ctsh
2.1505376
0.17161953

10547906
Lag3
2.1459227
0.00569861

10421737
Tnfsf11
2.1459227
0.01318614

10394625
Nbas
2.1459227
0.01676718

10430006
Slc39a4
2.1459227
0.03234083

10568150
Kif22
2.1459227
0.08123392

10497077
Mir186
2.1459227
0.09669853

10412543
Gm1973
2.1413276
0.00102788

10603567
Dynlt3
2.1413276
0.00663607

10354647
Pgap1
2.1413276
0.03769364

10513166
Ptpn3
2.1367521
0.00559333

10536390
Glcci1
2.1367521
0.0086892

10346330
Plcl1
2.1367521
0.01303019

10442240
Zfp760
2.1321962
0.02403225

10539080
St3gal5
2.1321962
0.10672212

10393559
Timp2
2.1276596
0.0131688

10414537
Rnase4
2.1276596
0.01691999

10408519
Hus1b
2.1276596
0.0274425

10481857
Pbx3
2.1231423
0.02611634

10410560
Trip13
2.1231423
0.07295799

10490872
Lrrcc1
2.1231423
0.08667311

10435789
Zbtb20
2.1231423
0.08964086

10400006
Ahr
2.1141649
0.01354896

10535747
Gm10858
2.1141649
0.01496187

10461856
Gna14
2.1141649
0.01721532

10599369
Xiap
2.1141649
0.03468285

10501164
Csf1
2.1141649
0.03582429

10565292
Arnt2
2.1141649
0.04983575

10346365
Sgol2
2.1141649
0.18050565

10589884
Bcl2a1c
2.1097046
0.0682851

10592471
Gramd1b
2.1052632
0.00074598

10597420
Ccr4
2.1052632
0.00466806

10371220
Gna15
2.1052632
0.05997275

10503264
Ccne2
2.1052632
0.14016493

10436402
Cldnd1
2.1008403
0.00093241

10394749
Nol10
2.1008403
0.02963964

10358459
BC003331
2.1008403
0.0567876

10585338
Kdelc2
2.0964361
0.00070799

10371356
Appl2
2.0964361
0.00749817

10563780
E2f8
2.0964361
0.18388031

10350392
Aspm
2.0964361
0.20422759

10435704
Cd80
2.0920502
0.00273045

10601834
Gprasp2
2.0920502
0.00742276

10503194
Chd7
2.0920502
0.01333232

10434291
B3gnt5
2.0920502
0.02474341

10468949
Dclre1c
2.0920502
0.0323231

10468527
5830416P10Rik
2.0876827
0.01308794

10572724
Zfp709
2.0876827
0.01743369

10355998
Fam124b
2.0876827
0.02278189

10424404
Pvt1
2.0833333
0.00758977

10417095
Farp1
2.0833333
0.00915935

10558773
B4galnt4
2.0833333
0.03058125

10453867
Rbbp8
2.0833333
0.05294243

10606263
Atrx
2.0833333
0.06987161

10428763
Atad2
2.0833333
0.07035945

10371591
4930547N16Rik
2.0833333
0.16138153

10603814
Slc9a7
2.0790021
0.01344989

10474902
Rad51
2.0790021
0.05271723

10416956
Mir19b-1
2.0790021
0.19045001

10599411
Sh2d1a
2.0746888
0.00059302

10424221
Wdr67
2.0746888
0.01206853

10499138
Dclk2
2.0746888
0.02070704

10346695
Nbeal1
2.0746888
0.0296774

10389606
Prr11
2.0746888
0.10753061

10545707
Actg2
2.0703934
0.03709829

10478928
Tshz2
2.0661157
0.00087833

10524866
Vsig10
2.0661157
0.00174265

10406086
Tert
2.0661157
0.01749025

10388042
6330403K07Rik
2.0661157
0.01833252

10467578
Pik3ap1
2.0661157
0.03302219

10482030
Stom
2.0618557
0.00600923

10357345
Nckap5
2.0618557
0.07845241

10359982
Atf6
2.0576132
0.00108407

10417526
Dnase1l3
2.0576132
0.00962298

10345423
Plekhb2
2.0576132
0.01085091

10457429
Rock1
2.0576132
0.10276042

10407126
Plk2
2.0576132
0.1387202

10496638
Odf2l
2.0576132
0.19410796

10363265
Lims1
2.0533881
0.00661461

10590623
Cxcr6
2.0533881
0.00962734

10435514
Ildr1
2.0533881
0.01643374

10413517
Chdh
2.0533881
0.01902125

10409990
6720489N17Rik
2.0533881
0.03708042

10400304
Egln3
2.0533881
0.05740125

10409978
6720457D02Rik
2.0533881
0.05875589

10543779
Mir29a
2.0533881
0.17557649

10587854
Slc9a9
2.0491803
0.00438505

10351015
Serpinc1
2.0491803
0.00578176

10361834
Txlnb
2.0491803
0.01143309

10455873
Slc12a2
2.0491803
0.01797121

10356880
St8sia4
2.0491803
0.02363305

10606876
Morf4l2
2.0491803
0.0384018

10374406
Cnrip1
2.0491803
0.06007526

10464128
Casp7
2.0449898
0.00166227

10588091
Cep70
2.0449898
0.02267903

10517287
Man1c1
2.0449898
0.02552988

10354529
1700019D03Rik
2.0408163
0.01566418

10405783
Mir24-1
2.0408163
0.02445097

10531610
Rasgef1b
2.0408163
0.05800715

10369171
9530009G21Rik
2.0408163
0.06855793

10515431
Kif2c
2.0408163
0.08892337

10431424
Plxnb2
2.0366599
0.0123898

10365899
Ccdc41
2.0366599
0.05169979

10600765
Pcyt1b
2.0325203
0.00560068

10552264
9430025M13Rik
2.0325203
0.00895905

10424779
Cks2
2.0325203
0.07938786

10480275
Nebl
2.0283976
0.00163009

10507286
Ipp
2.0283976
0.01100194

10461369
Ahnak
2.0283976
0.01144069

10503251
2610301B20Rik
2.0283976
0.01893445

10592725
Gm10688
2.0283976
0.02243805

10430113
Arhgap39
2.0283976
0.02249738

10490826
Zbtb10
2.0283976
0.16655707

10368144
Tnfaip3
2.0283976
0.18745235

10551852
Clip3
2.0242915
0.01122009

10352916
Mir29b-2
2.0242915
0.0194867

10399588
Zfp125
2.0242915
0.05567409

10462398
Pdcd1lg2
2.020202
0.01877215

10428698
Sntb1
2.016129
0.00092118

10476314
Prnp
2.016129
0.03114167

10563659
Spty2d1
2.016129
0.1008961

10456005
Cd74
2.0120724
0.01858586

10371092
Atcay
2.0120724
0.0197594

10456357
Pmaip1
2.0120724
0.02046887

10346960
Ccnyl1
2.0120724
0.02305044

10582941
Cwf19l2
2.0120724
0.10122588

10601903
Zcchc18
2.0080321
0.00283264

10372028
Plxnc1
2.0080321
0.04271335

10556280
Swap70
2.0080321
0.04782103

10562132
Cd22
2.0080321
0.12021187

10538617
Lancl2
2.004008
0.0086513

10445373
B230354K17Rik
2.004008
0.0248183

10565840
Neu3
2.004008
0.0316306

10513818
Stmn1
2.004008
0.10169755

10530492
Nfxl1
2
0.00829441

10494978
Ptpn22
2
0.01136496

10456346
Sec11c
1.996008
0.08923052

10474596
Aven
0.5
0.11649388

10472587
Rpl13
0.4997501
0.00400507

10605113
L1cam
0.4995005
0.00445563

10544596
Tmem176b
0.4995005
0.17169004

10537909
Rny3
0.4992511
0.18015923

10405693
Dapk1
0.4982561
0.00579729

10536010
C87414
0.4977601
0.00795618

10510391
Srm
0.4977601
0.02840111

10545479
Tmsb10
0.4962779
0.00121938

10513420
Mup7
0.4962779
0.0026631

10482814
Acvr1c
0.4962779
0.02069101

10512827
Gm568
0.4962779
0.04440213

10404053
Hist1h2bc
0.4960317
0.04462586

10445877
Gm16489
0.4952947
0.28933281

10456001
Rps14
0.4940711
0.09360483

10419162
4930503E14Rik
0.4926108
0.07017411

10356333
Snord82
0.4918839
0.0004474

10445774
B430306N03Rik
0.4918839
0.00836391

10570513
Kbtbd11
0.4918839
0.0260371

10508651
Sdc3
0.490918
0.07270972

10560911
Rabac1
0.4901961
0.00336931

10598218
Gm2799
0.4894763
0.06129231

10551009
Tmsb10
0.4882813
0.00020919

10513504
Mup2
0.4882813
0.01211184

10608263
Sly
0.487567
0.26411037

10452257
Slc25a23
0.4870921
0.06174372

10494411
Rnu1b1
0.486618
0.04126581

10560780
Vmn1r101
0.4863813
0.02147802

10545210
Gm1524
0.4859086
0.04889538

10385872
Slc22a5
0.4859086
0.04909365

10608410
Sly
0.4859086
0.18330955

10608625
LOC100040235
0.4854369
0.09419057

10503695
Bach2
0.4852014
0.01594984

10522467
Rasl11b
0.4844961
0.03888329

10553092
Dbp
0.4826255
0.10018767

10385533
Tgtp1
0.4823927
0.0513553

10479463
Slc17a9
0.4814636
0.00014776

10393449
Socs3
0.481232
0.01955942

10380571
Gngt2
0.4805382
0.00445514

10503833
Rplp1
0.4800768
0.00144009

10608237
Sly
0.4796163
0.23349111

10399677
Cox7a2l
0.4791567
0.03681363

10419125
Gm8005
0.4786979
0.00290623

10532085
Tgfbr3
0.4784689
0.00153001

10491058
Rprl2
0.4768717
0.00447126

10608628
LOC100041704
0.4768717
0.1973275

10461162
Snord22
0.4764173
0.18146542

10375058
Hba-a2
0.4759638
0.01757608

10608460
LOC665698
0.4739336
0.06223302

10444041
Ndufa7
0.4737091
0.03355838

10544891
Nod1
0.4732608
0.00018331

10408613
Tubb2b
0.4728132
0.17980019

10608260
Srsy
0.4725898
0.02186593

10419122
Gm8165
0.4725898
0.08673251

10471503
Taf1d
0.4725898
0.09294577

10499748
Rps27
0.4721435
0.01239656

10608385
Sly
0.4716981
0.25443181

10597490
Rps27
0.4708098
0.01298652

10571325
Mfhas1
0.4699248
0.00524913

10598225
Gm2799
0.4677268
0.06050661

10576795
Cd209a
0.4675082
0.00435424

10461012
Trmt112
0.4670715
0.02437115

10608308
Srsy
0.4664179
0.01262089

10414767
Rps19
0.4659832
0.03882346

10570516
Kbtbd11
0.4649
0.01845397

10398599
Rps19
0.464684
0.03972966

10472757
Cybrd1
0.4631774
0.06063082

10479362
Rps21
0.462963
0.02994749

10460968
Rasgrp2
0.4627487
0.00650458

10375051
Hba-a1
0.4627487
0.01119691

10360832
1700056E22Rik
0.4625347
0.02445717

10487021
Slc30a4
0.4621072
0.01842405

10351515
Rnu1b1
0.4614675
0.03562672

10552964
Ftl1
0.4612546
0.04358579

10608488
LOC665128
0.4612546
0.06741966

10598183
Gm2799
0.461042
0.15727075

10502510
Lmo4
0.4599816
0.01712604

10608407
Srsy
0.4599816
0.04384627

10351206
Selp
0.4593477
0.09488342

10608615
LOC380994
0.4585053
0.21947653

10603232
Gm2799
0.4580852
0.11902275

10545192
Rprl1
0.4562044
0.01280426

10550778
Vmn1r132
0.4557885
0.01813138

10550765
Vmn1r148
0.4545455
0.03033933

10489065
Ndrg3
0.4537205
0.00260561

10437668
Socs1
0.4526935
0.04555823

10424607
Ptp4a3
0.452284
0.02292982

10608531
LOC100504530
0.4518753
0.06981132

10530560
Slain2
0.4514673
0.09070758

10462618
Ifit3
0.4512635
0.00449285

10401244
Actn1
0.4504505
0.00247861

10400635
Rps29
0.4502476
0.00063662

10607870
Tlr7
0.450045
0.00467426

10608506
LOC100039753
0.4498426
0.12685831

10516908
Snora73a
0.4492363
0.00527233

10608277
Ssty2
0.4492363
0.11866777

10559635
Hspbp1
0.4490346
0.05004011

10608342
LOC100041704
0.4490346
0.08283511

10608212
Sly
0.448833
0.17849941

10560795
Vmn1r158
0.4476276
0.02368124

10608302
Ssty1
0.4472272
0.12510276

10608420
Ssty1
0.4472272
0.25077694

10380059
Rnu3b1
0.4462294
0.01437788

10608551
Srsy
0.4446421
0.01751658

10349102
Bcl2
0.4440497
0.06237449

10591739
Acp5
0.4432624
0.02297332

10494413
Rnu1b1
0.4415011
0.0411497

10364102
Chchd10
0.4411116
0.0316024

10447429
Gm4832
0.4403347
0.07690707

10608247
LOC100042196
0.4393673
0.11704582

10560754
Vmn1r132
0.4382121
0.02421074

10577226
2610019F03Rik
0.4376368
0.04884058

10608371
LOC665406
0.4370629
0.07867391

10608361
LOC100042359
0.4357298
0.07551582

10549495
Rps29
0.4355401
0.00268464

10541307
Usp18
0.4353505
0.03279837

10490126
Rps29
0.4347826
0.00044543

10608549
LOC100039753
0.4347826
0.17861321

10548875
Art4
0.4338395
0.02500971

10608567
Srsy
0.4330879
0.00864567

10550189
Gm10679
0.4315926
0.00625779

10521134
Rps29
0.4306632
0.00291539

10590365
Vipr1
0.4304778
0.00648496

10590620
Ccr9
0.4304778
0.07149516

10392910
C630004H02Rik
0.4295533
0.03632227

10608630
Ssty2
0.4293688
0.12434134

10550786
Vmn1r132
0.4269855
0.02916742

10422493
Gpr18
0.4257131
0.01053923

10608573
LOC100504530
0.4253509
0.02315258

10550782
Vmn1r148
0.4246285
0.02386071

10608613
LOC100042196
0.4246285
0.10963456

10573427
Nfix
0.4224757
0.00279782

10608424
Ssty1
0.4215852
0.15634008

10508719
Snora16a
0.4208754
0.11172156

10560728
Vmn1r158
0.4203447
0.01905504

10608394
Srsy
0.4191115
0.0159561

10590267
Snora62
0.41841
0.01913618

10550193
Gm3994
0.4177109
0.00831483

10608273
LOC100040223
0.4177109
0.07251798

10608222
LOC100504530
0.4175365
0.03370856

10608327
LOC100040031
0.4175365
0.12815183

10353034
Snord87
0.4171882
0.20536112

10362674
Rnu3a
0.4168404
0.02916955

10499130
Rnu73b
0.4166667
0.06375527

10608293
Srsy
0.41511
0.00790248

10551881
Sdhaf1
0.41511
0.07553797

10454807
Snora74a
0.4147657
0.05923466

10379633
Slfn1
0.4140787
0.00041818

10608209
Srsy
0.4116921
0.00732101

10608480
LOC100039147
0.4116921
0.03408665

10608282
LOC100039753
0.4098361
0.13112934

10550208
Gm3994
0.4093328
0.00665008

10560732
Vmn1r-ps79
0.4091653
0.01638949

10499378
Sema4a
0.4088307
0.067101

10608484
Ssty2
0.4088307
0.12129823

10402512
Scarna13
0.4088307
0.15531227

10608377
LOC100039753
0.4086637
0.122138

10608348
Ssty2
0.4078303
0.15572877

10377265
Pik3r5
0.4071661
0.01043105

10432190
Adcy6
0.4058442
0.00856264

10489235
9430008C03Rik
0.4056795
0.03194117

10560719
2210010C17Rik
0.4056795
0.03920352

10560742
Vmn1r103
0.4053506
0.01423602

10550768
Vmn1r122
0.4051864
0.01337363

10516906
Snora73b
0.4051864
0.02263855

10362896
Cd24a
0.4045307
0.1707852

10608482
LOC665746
0.4042037
0.06695088

10608350
LOC100039552
0.4040404
0.07989928

10501591
A930005H10Rik
0.4035513
0.11059473

10560744
Vmn1r117
0.4019293
0.01896927

10608368
LOC100041256
0.4014452
0.14889401

10540542
LOC100503669
0.4003203
0.00097644

10560789
Vmn1r151
0.4001601
0.01495732

10550770
Vmn1r114
0.4001601
0.02510794

10608295
LOC100039753
0.3992016
0.12280064

10399943
Cdhr3
0.3980892
0.00193339

10608608
Ssty2
0.3976143
0.11231776

10376885
Snord49b
0.3972984
0.01124483

10608365
Ssty2
0.396668
0.12169133

10376887
Snord49a
0.3963535
0.03005858

10439237
Rps21
0.3957262
0.00080957

10560797
Vmn1r-ps79
0.3954132
0.01686886

10461156
Snhg1
0.3947888
0.00690243

10560785
Vmn1r-ps79
0.3938558
0.02374871

10608477
Ssty2
0.3904725
0.1419859

10459766
Scarna17
0.3892565
0.00479583

10365098
Tbxa2r
0.3888025
0.00404659

10608339
Ssty2
0.3881988
0.12323147

10560740
Gm10670
0.3866976
0.02285695

10560730
Vmn1r93
0.3853565
0.02435977

10608373
Ssty2
0.385208
0.11609829

10608457
Ssty2
0.3847634
0.12523244

10608606
LOC100039753
0.3844675
0.17145298

10358713
1700025G04Rik
0.382995
0.05347887

10606654
Xkrx
0.3827019
0.01247528

10608521
Ssty1
0.381971
0.14062238

10608454
Ssty2
0.3797949
0.12602111

10376269
Galnt10
0.3786445
0.00615746

10480238
St8sia6
0.3785011
0.01070054

10414953
Gm16591
0.3736921
0.0070053

10382104
Snord104
0.3735525
0.00270416

10500204
Ecm1
0.3727171
0.00054722

10550760
Vmn1r100
0.3698225
0.02430528

10432176
Snora34
0.3681885
0.03375434

10360145
B930036N10Rik
0.3676471
0.15867117

10524621
Oasl2
0.367242
0.02150953

10503856
Gabrr2
0.3644315
0.11913457

10585803
Stra6
0.3619254
0.03613085

10346876
Snora41
0.3601008
0.01110707

10547073
Snora7a
0.3597122
0.02766423

10560752
Vmn1r125
0.3589375
0.01428597

10604076
Snora69
0.3584229
0.07649178

10425799
Rnu12
0.3541076
0.27760186

10585286
Arhgap20
0.3533569
0.01122687

10350159
Lad1
0.3529827
0.00211499

10430851
Cyp2d22
0.3513703
0.01398453

10414781
Gm13926
0.3508772
0.00322421

10598178
Disp1
0.3497726
0.01907486

10380719
Sp6
0.3376097
0.00824907

10526943
Gpr146
0.3314551
0.00504248

10465059
Ctsw
0.3307972
0.01628608

10368277
Rps12
0.3292723
0.00083038

10450920
AY036118
0.3286231
0.11252538

10563114
Snord32a
0.3278689
0.00455642

10467420
Pdlim1
0.3274394
0.00277614

10570432
Snora3
0.3270111
0.04721699

10520950
Pdlim1
0.3244646
0.00070513

10556206
Snora3
0.3244646
0.04610858

10580752
9330175E14Rik
0.3234153
0.06396521

10344750
Sgk3
0.3233107
0.04867129

10565813
Snord15a
0.3219575
0.19731484

10556528
Pde3b
0.3214401
0.03639524

10583286
Gpr83
0.3212335
0.00034837

10564183
Snord116
0.3212335
0.01784823

10390763
Ccr7
0.32
0.00608044

10564177
Snord116
0.317965
0.01101963

10549162
St8sia1
0.3164557
0.03366039

10351043
Snord47
0.3145643
0.01937336

10563108
Snord35a
0.312989
0.00192573

10598087
ND6
0.3098853
0.25208048

10554658
A530021J07Rik
0.3085467
0.14545011

10572800
Klf2
0.3051572
0.03619069

10583310
Taf1d
0.303859
0.02566778

10431935
Amigo2
0.3021148
0.02486963

10564161
Snord116
0.3005711
0.00675119

10564163
Snord116
0.2965599
0.0081746

10407435
Akr1c18
0.295858
0.20866961

10455015
Vaultrc5
0.2953337
0.03404918

10544523
Rny1
0.2899391
0.04496609

10563937
Snord115
0.2855511
0.00214888

10508723
Snora61
0.2853881
0.05992672

10358717
1700025G04Rik
0.2840909
0.00339189

10564011
Snord115
0.2832861
0.00106156

10377429
Snord118
0.280112
0.06398083

10563112
Snord33
0.2790179
0.07864334

10564013
Snord115
0.27894
0.00333173

10450363
Snord52
0.2764722
0.00194155

10508721
Snora44
0.2751032
0.16392721

10563110
Snord34
0.2724053
0.01176541

10563099
Snord35b
0.2659574
0.01981001

10451763
Satb1
0.2585984
0.00372421

10529515
Sorcs2
0.2478929
0.00285724

10576216
Snord68
0.2351281
0.00038855

10394054
Cd7
0.2329916
0.02198172

10598083
LOC100503984
0.2275831
0.06239005

10445767
Treml2
0.2255809
0.00723648

10565811
Snord15b
0.2142245
0.06820383

10603417
Gata1
0.2137666
0.00288082

10569017
Ifitm3
0.1954652
0.00836967

10495659
Cnn3
0.1945525
0.05981146

10461594
Ms4a4c
0.1907669
0.00190196

10406852
Cnn3
0.1666944
0.10549489

10403825
Tcrg-C
0.1312336
0.04662083

10429573
Ly6c2
0.1183292
0.00484551

10429568
Ly6c1
0.0956572
0.00227583

10381096
Igfbp4
0.095338
0.00020963

10472235
Dapl1
0.0660415
0.0335448

10403821
Tcrg-V3
0.0614213
0.03222249

10407940
Tcrg-V2
0.0598372
0.02340547

TABLE 2

Genes and Probe IDs included in Treg signatures

T cell activation/
Canonical
CXCR3+
Irf4-dependent
GFP-Foxp3-fusion

proliferation signature
Treg signature
Treg signature
Treg signature
Treg signature

Upregulated
Upregulated
Upregulated
Upregulated
Upregulated

Probe
Gene
Probe
Gene
Probe
Gene
Probe
Gene
Probe
Gene

10344624
Lypla1
10346330
Plcl1
10349603
Il10
10345791
Il1rl1
10346799
Icos

10344713
Ahcy
10346790
Ctla4
10357833
Atp2b4
10349603
Il10
10349593
Faim3

10346168
Stat4
10346799
Icos
10363070
Gp49a
10355567
Tmbim1
10349603
Il10

10346365
Sgol2
10349603
Il10
10363082
Lilrb4
10363070
Gp49a
10349648
Ctse

10346523
Bzw1
10350159
Lad1
10366586
Ifng
10363082
Lilrb4
10357488
Cd55

10346764
Abi2
10350630
Fam129a
10368970
Prdm1
10366586
Ifng
10357808
Snrpe

10346790
Ctla4
10353450
Gm4956
10389207
Ccl5
10367919
Stx11
10357986
Ptprv

10346799
Icos
10354563
Dnahc7b
10390328
Tbx21
10368970
Prdm1
10359890
Nuf2

10346943
Creb1
10355312
Ikzf2
10398039
Serpina3f
10369525
2010107G23Rik
10360370
BC094916

10347106
Rpe
10356082
Plscr1
10399691
Id2
10369932
Susd2
10368970
Prdm1

10348775
Ppp1r7
10356866
Pdcd1
10402325
Asb2
10375443
Havcr2
10378286
Itgae

10349637
Fam72a
10357833
Atp2b4
10406270
Glrx
10378286
Itgae
10389207
Ccl5

10349733
Nucks1
10358408
Rgs1
10414708
Gm7124
10399555
Kcnf1
10398039
Serpina3f

10349744
Slc45a3
10358816
Lamc1
10414802
Gm10893
10402136
Gpr68
10399148
Rapgef5

10350090
Ube2t
10359339
Rabgap1l
10414909
Gm8721
10402325
Asb2
10400006
Ahr

10350392
Aspm
10359375
Gpr52
10414981
Gm13893
10404840
Cd83
10408081
Hist1h1b

10350489
Uchl5
10360173
Slamf7
10420308
Gzmb
10408689
Nrn1
10408689
Nrn1

10350630
Fam129a
10361771
Plagl1
10443980
Myo1f
10420308
Gzmb
10409278
Nfil3

10350838
2810417H13Rik
10363415
Spock2
10445977
Ebi3
10427744
Rai14
10411739
Ccnb1

10351047
Cenpl
10365933
Eea1
10454015
Ttc39c
10440206
Arl6
10421517
Cysltr2

10351277
Nme7
10367919
Stx11
10466127
AW112010
10441633
Ccr6
10439527
Tigit

10351404
Tmco1
10372069
Socs2
10476759
Rin2
10454015
Ttc39c
10441233
Mx1

10351636
Refbp2
10373502
Ikzf4
10478633
Mmp9
10464905
Npas4
10444814
H2-gs10

10351640
Refbp2
10375402
Adam19
10493812
S100a4
10466521
Gcnt1
10450723
H2-T10

10351658
Cd48
10378286
Itgae
10493820
S100a6
10466745
Tjp2
10450733
H2-t9

10352048
Exo1
10379176
Unc119
10498576
Lxn
10482824
Acvr1
10455961
Iigp1

10352709
Nsl1
10380719
Sp6
10519527
Abcb1a
10487508
Gm14005
10462618
Ifit3

10352756
Lpgat1
10381187
Atp6v0a1
10519983
Fgl2
10493812
S100a4
10463263
Lztfl1

10352767
Nek2
10388591
Cpd
10526832
LOC100504914
10507137
Pdzk1ip1
10473367
Slc43a1

10352954
Hmgb3
10394674
Socs2
10531415
Cxcl10
10511363
Penk
10474875
Casc5

10353004
Cks2
10398039
Serpina3f
10547590
Klrg1
10521626
Cc2d2a
10477187
Tpx2

10353050
Cops5
10399087
Ncapg2
10552406
Nkg7
10523182
Areg
10482528
Neb

10353181
Lactb2
10401935
BC005685
10571399
Zdhhc2
10523231
Art3
10482687
Arl5a

10353250
Gapdh
10402325
Asb2
10584870
Tmprss13
10539135
Capg
10487480
Bub1

10353733
Prim2
10403229
Itgb8
10590628
Ccr3
10542993
Pon3
10487506
Gm14005

10354275
1700029F09Rik
10403821
Tcrg-V3
10590631
Ccr2
10544660
Osbpl3
10511779
Atp6v0d2

10354307
Txn1
10403941
Hist1h3h
10590635
Ccr5
10545707
Actg2
10515836
Ccnb1

10355037
Wdr12
10403948
Hist1h2bn
10594774
Ccnb2
10547590
Klrg1
10519983
Fgl2

10355050
Raph1
10403978
Hist1h2bk
10595718
Chst2
10552143
Slc7a10
10521731
Ncapg

10355115
Prelid1
10403980
Hist1h2bj
10603151
Gpm6b
10565292
Arnt2
10554863
Sytl2

10355931
Farsb
10404028
Hist1h3g
10603551
Cybb
10571788
Vegfc
10562637
Ccnb1

10356082
Plscr1
10404049
Hist1h3d
10606058
Cxcr3
10574524
Ces2c
10568714
Mki67

10356859
Dtymk
10404061
Hist1h2bb

10574532
Ces2d-ps
10582545
Mela

10357242
Dbi
10404063
Hist1h2ab

10576661
Itgb1
10582549
Mela

10357436
Mcm6
10404065
Hist1h3b

10578904
Cpe
10586933
Nedd4

10358259
Nek7
10404389
Irf4
10344750
Sgk3
10587683
Bcl2a1a
10586967
Gm7265

10358713
1700025G04Rik
10404840
Cd83
10349102
Bcl2
10587690
Bcl2a1b
10590620
Ccr9

10358717
1700025G04Rik
10406270
Glrx
10350159
Lad1
10590623
Cxcr6
10590631
Ccr2

10359849
Uck2
10406334
Mctp1
10351197
Sell
10590631
Ccr2
10593332
Bco2

10359851
Uck2
10406982
Adamts6
10355141
Klf7
10595633
Bcl2a1d
10603328
Ccdc22

10359890
Nuf2
10407940
Tcrg-V2
10357043
Bcl2
10600122
Xlr3b
10607738
Car5b

10360147
Refbp2
10408070
Hist1h2bl
10358717
1700025G04Rik
10604996
Xlr3a

10360806
Capn2
10408077
Hist1h2ak
10359689
Atp1b1
10605007
Xlr3c

10360985
Cenpf
10408081
Hist1h1b
10375019
Nsg2

10361110
Dtl
10408083
Hist1h3i
10378855
Ssh2

10361375
Fbxo5
10408200
Hist1h4f
10381096
Igfbp4

10361995
Fam54a
10408202
Hist1h3e
10403821
Tcrg-V3

10362581
Tube1
10408210
Hist1h2bf
10403825
Tcrg-C

10362941
Prep
10408239
Hist1h3c
10406852
Cnn3

10363498
Ppa1
10408246
Hist1h3a
10407940
Tcrg-V2

10363575
Dna2
10408689
Nrn1
10429568
Ly6c1

10365227
Ap3m1
10408693
F13a1
10429573
Ly6c2

10365260
Txnrd1
10412517
Gm3002
10445767
Treml2

10365420
Al597468
10412537
Gm3002
10451763
Satb1

10365578
Nup37
10417258
Gm3002
10453026
Prkd3

10365637
Arl1
10417264
Gm3002
10461594
Ms4a4c

10365933
Eea1
10417302
Gm3002
10472235
Dapl1

10366277
E2f7
10417359
Gm3002
10472501
Lass6

10366337
Nap1l1
10417411
Gm3002
10480238
St8sia6

10366814
Cdk4
10417421
Gm3696
10485607
Qser1

10367076
Prim1
10417461
Gm10406
10485622
Qser1

10368612
Gapdh
10420308
Gzmb
10487021
Slc30a4

10369815
Cdk1
10421517
Cysltr2
10498599
Ift80

10370552
Ppap2c
10424370
Trib1
10501494
Amy2b

10371591
4930547N16Rik
10425049
Apol9b
10501544
Amy2a5

10371770
Gas2l3
10427235
Prr13
10503695
Bach2

10371846
Apaf1
10430344
Il2rb
10503709
D130062J21Rik

10371888
Tmpo
10432511
Racgap1
10514732
Slc35d1

10371987
Metap2
10438626
Etv5
10529515
Sorcs2

10372082
Nudt4
10439527
Tigit
10530516
Txk

10372965
Usp15
10439895
Alcam
10549162
St8sia1

10374426
Pno1
10440393
Samsn1
10583286
Gpr83

10374442
C1d
10441436
Snx9
10585286
Arhgap20

10374466
Rab1
10443009
Ergic1
10585976
Myo9a

10375880
Nhp2
10443980
Myo1f
10585982
Myo9a

10375941
Vdac1
10444824
H2-Q6
10585986
Myo9a

10377405
Aurkb
10446771
Lclat1
10587315
Gsta4

10378802
Blmh
10447383
Epcam
10603417
Gata1

10378848
Hsp90aa1
10450374
D17H6S56E-5
10606178
Xist

10379127
Spag5
10452047
Ptprs
10606369
Itm2a

10379363
Atad5
10452508
Twsg1
10607870
Tlr7

10379445
Zfp207
10454015
Ttc39c
10608247
LOC100042196

10379968
Tubd1
10456005
Cd74
10608273
LOC100040223

10379989
Fam33a
10457225
Map3k8
10608282
LOC100039753

10379998
Trim37
10461369
Ahnak
10608302
Ssty1

10380403
Lrrc59
10462398
Pdcd1lg2
10608342
LOC100041704

10380411
Mrpl27
10463070
Entpd1
10608348
Ssty2

10380815
Psmb3
10466779
Pip5k1b
10608350
LOC100039552

10381072
Cdc6
10469151
Itih5
10608365
Ssty2

10381526
Ppih
10469278
Il2ra
10608371
LOC665406

10381664
Kif18b
10473356
Ube2l6
10608373
Ssty2

10381798
Myl4
10473367
Slc43a1
10608420
Ssty1

10382998
Birc5
10474769
Bub1b
10608424
Ssty1

10384373
Fignl1
10476314
Prnp
10608454
Ssty2

10384474
Pno1
10476945
Cst7
10608482
LOC665746

10384493
Gapdh
10481210
Vav2
10608484
Ssty2

10384579
Ugp2
10482528
Neb
10608488
LOC665128

10385248
Hmmr
10482687
Arl5a
10608521
Ssty1

10385325
Pttg1
10484888
Ptprj
10608549
LOC100039753

10385686
Hnrnpab
10484894
Ptprj
10608606
LOC100039753

10385966
Anxa6
10485405
Cd44
10608613
LOC100042196

10386005
Atp5f1
10493820
S100a6
10608625
LOC100040235

10386947
Gm10291
10494402
Hist2h3c1
10608628
LOC100041704

10388234
Gsg2
10494405
Hist2h3b
10608630
Ssty2

10388745
Lsm6
10496379
H2afz

10388971
Utp6
10496539
Gbp5

10389606
Prr11
10496580
Gbp3

10390707
Top2a
10497149
Wls

10391461
Brca1
10497831
Ccna2

10391811
Kif18b
10499095
Fam160a1

10392284
Kpna2
10499216
Pear1

10392388
Prkca
10500204
Ecm1

10393431
Tk1
10500656
Cd101

10393844
Thoc4
10501164
Csf1

10394770
Odc1
10502156
Ccdc109b

10394978
Rrm2
10504753
LOC641050

10395259
Nampt
10504757
BC005685

10396068
Ppil5
10504761
LOC641050

10396712
Fut8
10510580
Tnfrsf9

10397741
Psmc1
10511282
Tnfrsf4

10398173
Vrk1
10511290
Tnfrsf18

10398874
Siva1
10511363
Penk

10399011
4930427A07Rik
10511617
Fam92a

10399087
Ncapg2
10512774
Coro2a

10399825
Dld
10514466
Jun

10400304
Egln3
10514732
Slc35d1

10400589
C79407
10518300
Tnfrsf1b

10401278
Erh
10519527
Abcb1a

10402615
Hsp90aa1
10519983
Fgl2

10402648
Brp44l
10521678
Cd38

10402650
Cinp
10523595
Ptpn13

10403258
Gdi2
10525419
P2rx7

10403413
Idi1
10528238
Phtf2

10404053
Hist1h2bc
10530819
Hopx

10404422
Serpinb6b
10535065
Adap1

10404429
Serpinb9
10535389
Rnf216

10405185
Cks2
10538890
LOC641050

10405427
Prelid1
10538892
LOC641050

10406482
Ccnh
10538901
BC005685

10406581
Dhfr
10539135
Capg

10406898
Taf9
10540999
H2afz

10406968
Cenpk
10542880
4833442J19Rik

10407081
Depdc1b
10544660
Osbpl3

10407481
Pfkp
10547590
Klrg1

10407993
Srsf10
10547906
Lag3

10408210
Hist1h2bf
10548585
Csda

10408223
Hist1h2bc
10550509
Pglyrp1

10408321
Gmnn
10552406
Nkg7

10408329
Gmnn
10553598
Cyfip1

10408531
Gmds
10555174
Lrrc32

10409190
Cenpp
10555197
Mtap6

10409200
Gapdh
10557156
Plk1

10409424
Mxd3
10559261
Cd81

10409866
Ctla2b
10560945
Grik5

10409876
Ctla2a
10560964
Pou2f2

10410092
Zfp367
10564507
Arrdc4

10410560
Trip13
10565315
Fah

10411332
Hmgcr
10565735
A630091E08Rik

10411359
Plp2
10565990
Art2a-ps

10411373
Hexb
10571312
Dusp4

10411452
Gapdh
10571399
Zdhhc2

10411728
Cenph
10571696
Casp3

10411739
Ccnb1
10572497
Il12rb1

10412466
Hmgcs1
10576639
Nrp1

10412559
Slbp
10576661
Itgb1

10412909
Fdft1
10580077
Rln3

10413059
Vcl
10581992
Maf

10413542
Tkt
10583286
Gpr83

10414315
Cdkn3
10585286
Arhgap20

10415791
Rnaseh2b
10586744
Anxa2

10415844
Ctsb
10586781
Myo1e

10416037
Pbk
10586933
Nedd4

10416736
6720463M24Rik
10587315
Gsta4

10416940
Tpm3
10587503
Sh3bgrl2

10417070
Ipo5
10587639
Nt5e

10417359
Gm3002
10588577
Cish

10417421
Gm3696
10590242
Ccr8

10417617
Gapdh
10590909
Endod1

10417689
Psmd6
10590974
Folr4

10417787
Gng2
10592655
Arhgef12

10418004
Ap3m1
10592888
Cxcr5

10419136
Cdv3
10593497
Zc3h12c

10419198
Ero1l
10594774
Ccnb2

10419267
Cnih
10597420
Ccr4

10419296
Wdhd1
10598289
4930524L23Rik

10419323
Dlgap5
10598292
Foxp3

10420155
Dhrs1
10603551
Cybb

10420198
Ripk3
10603814
Slc9a7

10420308
Gzmb
10606058
Cxcr3

10420426
F630043A04Rik
10607738
Car5b

10420637
Kpna3

10420730
Fdft1

10420988
Dpysl2

10421029
Cdca2
10344750
Sgk3

10422161
Gm10293
10345777
Il1rl2

10422655
Gapdh
10351880
E430029J22Rik

10423180
Gapdh
10354506
Mfsd6

10424221
Wdr67
10356475
Arl4c

10424349
Sqle
10359689
Atp1b1

10424379
Srsf3
10362073
Sgk1

10424779
Cks2
10362350
Themis

10425161
Lgals1
10362861
Scml4

10425207
H1f0
10364072
Ggt5

10425226
Eif3l
10367734
Ust

10425903
Gm2451
10368647
Dse

10426827
Larp4
10369911
1110038D17Rik

10427166
Espl1
10371356
Appl2

10427606
Skp2
10378549
Rtn4rl1

10428018
Ube2v2
10381096
1gfbp4

10428310
Azin1
10381809
Itgb3

10428672
Dscc1
10382532
Slc16a5

10430344
Il2rb
10385428
Itk

10430778
Phf5a
10385776
Tcf7

10432511
Racgap1
10388488
Fam101b

10433088
Cbx5
10390763
Ccr7

10434643
Psmb3
10392910
C630004H02Rik

10434869
Ccdc50
10399696
Rnf144a

10434998
Ncbp2
10401244
Actn1

10435821
Naa50
10402096
Ttc7b

10436048
Prdx1
10403604
Lyst

10436106
C330027C09Rik
10404359
Mboat1

10436182
Cd47
10406111
Slc12a7

10437432
Nmral1
10406852
Cnn3

10437590
Carhsp1
10407072
Elovl7

10437748
Gspt1
10407124
Al452195

10437942
Ube2v2
10407327
Emb

10437945
Mcm4
10414807
Trav14-3

10438091
2610318N02Rik
10423293
Myo10

10438308
Ranbp1
10425040
Apol7e

10438378
Cdc45
10430179
Apol7b

10438690
Rfc4
10439790
Trat1

10439762
Ahcy
10440099
St3gal6

10439878
Psmc1
10446777
Ehd3

10440314
Cadm2
10451763
Satb1

10441642
Brp44l
10455784
Gramd3

10442454
Pgp
10460968
Rasgrp2

10443459
Srsf3
10467420
Pdlim1

10443527
Pim1
10472022
Lypd6b

10444927
Nrm
10472162
Gpd2

10445894
Erh
10472235
Dapl1

10446074
Uhrf1
10472846
Pdk1

10447395
Msh2
10472860
Rapgef4

10447417
Msh6
10475990
Slc20a1

10447702
Ppih
10481574
Fam78a

10447880
Mrpl18
10483809
Nfe2l2

10448506
Ccnf
10487208
Atp8b4

10448803
Hn1l
10495685
Arhgap29

10449575
Ppil1
10496091
Lef1

10449581
Mtch1
10496438
Adh1

10450374
D17H6S56E-5
10497237
Pag1

10450519
Tcf19
10498345
Gpr171

10450605
Tubb5
10499378
Sema4a

10451805
Sgol1
10500434
Bcl9

10452415
Gapdh
10503161
Chd7

10452709
Ndc80
10503222
Chd7

10453512
Kpna2
10505187
Ugcg

10453867
Rbbp8
10513729
Tnfsf8

10454093
Mrpl27
10514956
Scp2

10454198
Rnf125
10516823
Epb4.1

10454709
Kif20a
10532085
Tgfbr3

10455595
Eno1
10533198
Oas2

10455647
Tnfaip8
10533659
Clip1

10455738
Snx2
10533729
Vps37b

10455780
Gapdh
10534570
Orai2

10455813
Lmnb1
10542981
Gmfg

10455967
2610318N02Rik
10547795
Atn1

10456383
Impa2
10549162
St8sia1

10457409
Usp14
10554658
A530021J07Rik

10458033
Stard4
10556528
Pde3b

10458195
Cdc25c
10557069
Mettl9

10458213
Etf1
10562260
Gramd1a

10458589
Prelid2
10563099
Snord35b

10459375
Txnl1
10564539
Mctp2

10459755
Ska1
10571344
D8Ertd82e

10459844
Haus1
10577226
2610019F03Rik

10460738
Cdca5
10583203
Phxr4

10461391
Pcna
10583207
Maml2

10461439
Fads1
10589654
Als2cl

10461452
Fen1
10590365
Vipr1

10461723
Fam111a
10590381
Vipr1

10462632
Kif20b
10595840
Acpl2

10462670
Rpp30
10597518
Tgfbr2

10462796
Kif11
10598101
Maml2

10462866
Cep55
10599802
Cd40lg

10462973
Hells
10606654
Xkrx

10463064
Gm4609

10464045
Acsl5

10465005
Banf1

10465553
Fkbp2

10465686
Rtn3

10465844
Asrgl1

10465861
Incenp

10465912
Fen1

10466410
Psat1

10466606
Anxa1

10466843
Gapdh

10466925
Ak3

10467637
Arhgap19

10469035
Sephs1

10469070
Nudt5

10469322
Vim

10469712
Pdss1

10469732
Yme1l1

10472782
Hat1

10472916
Cdca7

10472933
Scrn3

10473022
Plp2

10473240
Eno1

10473250
Mrpl18

10473384
Slc43a3

10473919
Ckap5

10474239
Gapdh

10474381
Kif18a

10474769
Bub1b

10474825
D2Ertd750e

10474875
Casc5

10474902
Rad51

10474984
Nusap1

10475335
Pdia3

10475610
Dut

10476252
Cdc25b

10476648
Dstn

10476834
Xrn2

10476989
Gins1

10477187
Tpx2

10477942
Rbl1

10478407
Serinc3

10478572
Ube2c

10478943
Pfdn4

10479379
Slco4a1

10479736
Polr3k

10479811
Mcm10

10480381
Arhgap21

10480432
Mastl

10480628
Tubb2c

10481344
Gapdh

10481585
2900010J23Rik

10482229
Psmb7

10482687
Arl5a

10482762
Idi1

10483046
Dpp4

10483178
Cobll1

10483381
Stk39

10483401
Spc25

10484425
2700094K13Rik

10485294
Hsd17b12

10485963
Arhgap11a

10486396
Ehd4

10487033
Myef2

10487175
Cops2

10487340
Ncaph

10487480
Bub1

10487577
Ckap2l

10487930
Pcna

10488785
E2f1

10488816
Ahcy

10489127
Rbl1

10489377
Serinc3

10490104
Aurka

10490225
Slmo2

10490838
Fabp5

10490843
Myef2

10490946
Hsp90aa1

10491182
Eif5a2

10491385
Actl6a

10491805
Plk4

10491835
Larp1b

10491848
Larp1b

10492220
2810407C02Rik

10492381
Gmps

10492679
4930579G24Rik

10493137
Iqgap3

10493548
Pmvk

10493633
Tpm3

10493820
S100a6

10493995
S100a10

10494322
Anp32e

10494583
Sec22b

10494662
Ywhah

10495405
Slc25a24

10496204
Cenpe

10496324
Slc39a8

10496485
Eif4e

10496490
Mir1956

10497105
Lrrc40

10497503
Kpna2

10497520
Ect2

10497752
Carhsp1

10497831
Ccna2

10499639
Cks1b

10500630
Ttf2

10500990
Atp5f1

10501402
Gpsm2

10501661
Srsf3

10503264
Ccne2

10503315
Rad54b

10503617
F730047E07Rik

10503911
Polr1d

10504450
Glipr2

10504470
Melk

10504957
Smc2

10506118
Usp1

10506680
Tmem48

10506714
Lrp8

10506822
Orc1

10507112
Stil

10507286
Ipp

10507328
Prdx1

10507885
Mycbp

10508151
Clspn

10508182
Psmb2

10508217
Sfpq

10508444
Zbtb8os

10508986
Stmn1

10509113
Srsf10

10509168
E2f2

10510165
Gm13238

10510167
Gm13051

10510172
Hmgb2

10510219
Gm13238

10510546
Eno1

10510687
Acot7

10511617
Fam92a

10511661
Otud6b

10511694
Osgin2

10512061
Taf9

10513181
Gapdh

10513195
Txn1

10513320
Ptgr1

10513608
Alad

10513818
Stmn1

10513822
Stmn1

10514201
Haus6

10515090
Cdkn2c

10515257
Rad54l

10515337
Nasp

10515431
Kif2c

10515744
Cdc20

10515836
Ccnb1

10515884
Ppih

10516246
Cdca8

10516943
Atpif1

10517336
Clic4

10517559
Cdc42

10518344
Gm13238

10518350
Hmgb2

10518352
Hmgb2

10519324
Cdk6

10519488
Tubb2c

10520390
Gapdh

10520483
Ept1

10520521
Cenpa

10521031
Ywhah

10521090
Tacc3

10521136
Whsc1

10521690
Ppih

10521731
Ncapg

10521863
Anapc4

10523012
Dck

10523281
11-Sep

10523365
Mrpl1

10524169
Pole

10524266
Chek2

10524790
Cit

10525591
Kntc1

10525733
Setd8

10525983
Ran

10526972
Nudt1

10527559
Polr1d

10527801
Brca2

10527888
Gatad1

10527920
Cyp51

10528077
Dbf4

10528167
Gapdh

10528915
Tyms

10529299
Slbp

10530806
Ppat

10531707
Lin54

10531724
Plac8

10533090
Rfc5

10533929
Scarb1

10534842
Gnb2

10534974
Mcm7

10535979
Rfc3

10536472
Mdfic

10536595
Naa38

10538617
Lancl2

10538832
Mad2l1

10540273
Ube2v2

10540738
Fancd2

10541484
M6pr

10541729
Cdca3

10542200
Gabarapl1

10542355
Emp1

10542445
Strap

10542460
Dera

10542750
Med21

10543944
Mtpn

10544501
Ezh2

10544660
Osbpl3

10545534
Rnf26

10545588
Hk2

10545672
Mthfd2

10545835
1700040I03Rik

10545958
Anxa4

10546163
Mcm2

10547830
Tpi1

10547936
Gapdh

10547943
Ncapd2

10548086
Rad51ap1

10548143
Gapdh

10548585
Csda

10550098
Wdr12

10550102
Lig1

10552740
Nup62

10553788
Atp10a

10554013
Chsy1

10554445
Prc1

10554574
Tm6sf1

10554817
Gm10291

10555055
Ndufc2

10555695
Rrm1

10556266
Wee1

10556640
6330503K22Rik

10557156
Plk1

10557843
Fus

10558248
Bub3

10558723
Psmd13

10560000
Tpm3

10560260
Sae1

10561388
Timm50

10562563
Ccne1

10562637
Ccnb1

10562639
Gapdh

10563780
E2f8

10563838
Nipa2

10564978
Blm

10565479
I7Rn6

10565570
4632434I11Rik

10565921
Gapdh

10567072
Psma1

10567303
Coq7

10568150
Kif22

10568714
Mki67

10569017
Ifitm3

10569071
Hras1

10570373
Tfdp1

10571274
Gsr

10571288
Gtf2e2

10571399
Zdhhc2

10571696
Casp3

10571870
Hmgb2

10571876
Gapdh

10571911
2700029M09Rik

10571978
Cbr4

10572906
Mcm5

10573217
Ddx39

10573261
Asf1b

10573451
Syce2

10573615
Orc6

10574033
Nup93

10575153
Cyb5b

10575733
Cenpn

10576034
Irf8

10576639
Nrp1

10576661
Itgb1

10576883
Shcbp1

10577508
Ckap2

10577598
Lsm6

10578145
Erh

10578193
Eri1

10578539
Slc25a4

10578545
Gm12070

10578690
Neil3

10578916
Sc4mol

10579347
Ifi30

10579769
Gapdh

10579833
Lsm6

10580590
Gapdh

10582008
2310061C15Rik

10582190
Gins2

10582295
Odc1

10582809
Tk1

10582843
Itgb1

10582981
Tfdp1

10583254
Cwc15

10584710
H2afx

10585395
Siva1

10585417
Idh3a

10585474
Psma4

10585699
Fabp5

10585932
Pkm2

10586184
Tipin

10586284
Dpp8

10586416
Pif1

10586448
2810417H13Rik

10586484
Fam96a

10586604
Rps27l

10586744
Anxa2

10587104
Arpp19

10587107
Myo5a

10587508
Ttk

10587792
Plscr1

10588049
Copb2

10588294
Topbp1

10590325
Ctnnb1

10590623
Cxcr6

10590648
Top2a

10591556
Spc24

10591781
Anln

10591816
Dpy19l1

10592201
Chek1

10592585
Sc5d

10592727
Rnf26

10593356
Sdhd

10593789
Etfa

10594251
Kif23

10594426
Zwilch

10594774
Ccnb2

10595000
Tmod3

10595604
Syncrip

10595702
1190002N15Rik

10590325
Ctnnb1

10596185
Cdv3

10596575
Manf

10597095
3000002C10Rik

10598638
Mid1ip1

10599554
Rbmx2

10599855
Eif4e

10600017
Hmgb3

10600031
2610030H06Rik

10601011
Kif4

10601335
2610029G23Rik

10601449
Sh3bgrl

10601567
Gm12070

10601705
Cenpi

10603252
Larp4

10603254
Larp4

10603346
Plp2

10603431
Suv39h1

10596185
Cdv3

10604187
Lamp2

10604528
Mbnl3

10605674
Pola1

10605711
Pdk3

10606071
Ercc6l

10606436
Hmgn5

10607475
Prdx4

10607952
Vamp7

10350977
4930523C07Rik

10352234
Itpkb

10352815
Irf6

10353064
Arfgef1

10353991
Rpl12

10357604
Ikbke

10358389
Rgs2

10359201
Ralgps2

10359422
Prdx6

10359689
Atp1b1

10360684
Ephx1

10361323
Cnksr3

10362861
Scml4

10363641
Herc4

10365971
Btg1

10366346
Phlda1

10366667
Gns

10368504
Rpl12

10369210
Serinc1

10369735
Herc4

10370072
Prmt2

10370544
2610008E11Rik

10371356
Appl2

10373519
Rpl12

10373740
Pik3ip1

10376839
Ttc19

10377537
Chd3

10377547
Kdm6b

10377593
Zbtb4

10385747
Phf15

10386850
Ncor1

10387316
Chd3

10387372
Kdm6b

10387699
Acap1

10388488
Fam101b

10389143
Slfn8

10389162
Rpl12

10391301
Stat3

10392063
Limd2

10392259
Smurf2

10392261
Smurf2

10392300
Bptf

10392318
Bptf

10396956
Pcnx

10398267
Evl

10400405
Nfkbia

10401238
Zfp36I1

10401473
Aldh6a1

10402020
Eml5

10402061
Eml5

10402730
Ppp1r13b

10403273
Asb13

10403765
Vps41

10404848
Jarid2

10404988
C030044B11Rik

10406111
Slc12a7

10406817
Enc1

10407173
Il6st

10408032
Zfp187

10408049
Zfp192

10409170
Fgd3

10410465
BC018507

10410475
BC018507

10411853
Erbb2ip

10413174
Rps24

10414807
Trav14-3

10414817
A130082M07Rik

10416251
Egr3

10416522
Tsc22d1

10417004
Dzip1

10419343
Atg14

10421810
1190002H23Rik

10422075
Mycbp2

10422321
Dzip1

10425410
Grap2

10427035
Nr4a1

10427454
Card6

10427459
Card6

10427628
Il7r

10428912
Fam84b

10428918
9930014A18Rik

10430179
Apol7b

10430201
Myh9

10432294
Mll2

10435769
Zbtb20

10435789
Zbtb20

10435980
Rps24

10437080
Ttc3

10438583
Rpl12

10440491
App

10441115
Brwd1

10441601
Tagap

10441787
Aim

10441791
Aim

10442495
Pkd1

10443852
A530088E08Rik

10444394
Pbx2

10446334
Glcci1

10446615
Rps24

10449893
Rasal3

10451763
Satb1

10456745
Smad7

10460202
Suv420h1

10462035
Ldhb

10465244
Malat1

10468309
Sh3pxd2a

10469867
Pnpla7

10471550
Rpl12

10472022
Lypd6b

10472277
7-Mar

10472860
Rapgef4

10474006
Phf21a

10480238
St8sia6

10482880
Baz2b

10484371
Calcrl

10489266
Chd6

10491136
Tnik

10491300
Skil

10494306
Mcl1

10496032
Rpl12

10496438
Adh1

10499160
Cd1d1

10499748
Rps27

10501879
Usp53

10503709
D130062J21Rik

10503723
Mdn1

10503856
Gabrr2

10504491
Zcchc7

10504499
Zcchc7

10506058
Inadl

10506335
Pde4b

10512949
Abca1

10513551
Fkbp15

10514985
Zyg11b

10516620
Lck

10518585
Kif1b

10518735
Spsb1

10519105
Ski

10520371
Rbm33

10520379
Rbm33

10520388
Rbm33

10524284
Ttc28

10524310
Ttc28

10524312
Ttc28

10524398
Wscd2

10527233
Cyth3

10529239
Pisd

10530319
Atp8a1

10536390
Glcci1

10542557
Aebp2

10543118
Glcci1

10543319
Fam3c

10545608
Sema4f

10546510
Lrig1

10546661
Foxp1

10547789
Grcc10

10548333
Cd69

10549097
Ldhb

10551891
Nfkbid

10551989
Tmem149

10552037
Sbsn

10552796
Tsks

10553336
Zdhhc13

10555118
Pak1

10558001
Inpp5f

10561920
Hcst

10562260
Gramd1a

10564573
Chd2

10567702
Arhgap17

10568553
Chst15

10568780
Mapk1ip1

10569927
Map2k7

10571344
D8Ertd82e

10575598
Znrf1

10577226
2610019F03Rik

10580452
Siah1a

10587419
Senp6

10589654
Als2cl

10590365
Vipr1

10590381
Vipr1

10596492
Parp3

10597258
Tmie

10597266
Als2cl

10597490
Rps27

10597978
Fyco1

10601819
Gprasp1

10603328
Ccdc22

10606261
Rpl12

10606654
Xkrx

10606735
Armcx2

10606989
Tsc22d3

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	Number	Date	Country
	61968835	Mar 2014	US
	61981019	Apr 2014	US

METHODS AND COMPOSITIONS FOR TREATMENT OF IMMUNE-RELATED DISEASES OR DISORDERS AND/OR THERAPY MONITORING

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