Fieid of the invention

The present invention relates to the treatment or prevention of a chronic inflammatory disease, condition or related pathology. More particularly, the invention relates to use of an agent that modulates the podoplanin pathway, such as by inhibiting an interaction of podoplanin with CLEC-2 or inhibits the activity of Src and/or Syk family kinases for use in a method of treating and/or preventing a chronic inflammatory disease, condition or related pathology in a subject.

Background to the invention

Chronic inflammatory disease related morbidity and mortality continue to rise in western countries. Chronic inflammation, where norma! homeostatic processes are dysregulated, is a common contributor to debilitating human diseases and is associated with the development of fibrotic tissue damage. Aside from the debilitating symptoms and reduced quality of life experienced by patients with chronic inflammatory conditions, the economic burden to healthcare systems is enormous. For example, conservative estimates suggest that care of patients with inflammatory bowel disease costs the National Heaith Service in the UK £131 million per year and management of patients with diabetes and its associated complications was estimated at 10% of the total NHS expenditure in 2010/11.

Various chronic inflammatory diseases are known. These include chronic liver disease, inflammatory bowel disease, chronic lung disease (chronic obstructive pulmonary disease) and chronic kidney disease.

Causes of chronic inflammatory diseases are wide-ranging and include microbial infection, parasite infection, alcohol, drugs, vascular diseases, metabolic disease, diabetes, high blood pressure, environmental toxins, autoimmunity, gene(s) or repeated heart failure. For many patients the cause of the chronic inflammatory disease is unknown. inflammatory Bowel Disease (IBD) is a chronic relapsing-remitting inflammatory disorder of the gastrointestinal (Gl) tract occurring as a result of an uncontrolled and excessive immune response to commensal gut bacteria. Clinical features range from constipation, fatigue and malnutrition to abdominal pain, bleeding and diarrhoea,

Chronic liver disease is characterised by liver inflammation which leads to fibrotic scar formation and ultimately cirrhosis.

Treatment options for chronic inflammatory diseases are limited and are not always effective. In some instances, treatment relies on the removal of the cause, rather than the administration of a therapy. This does not necessarily reverse the damage already caused. Effective control of inflammation is necessary to prevent progression of the disease. Surgical intervention is often the last resort for patients whose chronic condition is not controlled, for example, surgical resection of the bowel in Crohn's disease Serious complications can, however, arise as a result of surgicai intervention, for example, infection, a reaction to anaesthesia, bleeding, blood clots, scarring, or even death. For some patients, transplantation may be required. These patients then require life-long immunosuppressive treatment to prevent rejection of the transplant. This is very costly, and results in side effects including diabetes, hypertension, osteoporosis, sepsis and an increased risk of malignancy. There is thus a need for better treatments that can stop, reverse or prevent inflammation and tissue damage. Platelets are fundamental players in liver and bowe! pathobioiogy, driving inflammation, fibrosis, cancer and even aiding regeneration. However, the specific molecular basis of platelet activation in the context of chronic inflammation remains elusive.

The present invention has been devised with these issues in mind.

Description

Broadly speaking, the present invention is based upon modulation of the podoplanin pathway, such as through the inhibition of the interaction of podoplanin with CLEC-2. in the context of the present invention, the podoplanin pathway will be understood to refer to an interaction of podoptanin with CLEC-2 and downstream targets of the interaction. As the skilled person will appreciate, podoplanin has a single transmembrane region and short cytoplasmic tail that interacts with members of the ERM family of proteins to Sink podoplanin to the actin cytoskeleton. The interaction of podoplanin with CLEC-2 results in phosphorylation of tyrosine residues in an YXXL motif in the intracellular 1TAM domain of CLEC-2 and permits CLEC-2 to interact with tyrosine kinases such as Src and Syk. This leads to activation of other downstream partners such as SLP-76 and PLCy and causes platelet activation and aggregation. Thus, the pathway may be inhibited by inhibition of the interaction of podoplanin with CLEC-2, or by inhibition of the activity of the downstream targets. For example, inhibition of the interaction of podoplanin with CLEC-2, or the activity of Src and/or Syk family kinases results in inhibition of the activation of other downstream partners such as SLP-76 and PLCy.

According to a first aspect of the invention there is provided an agent that inhibits an interaction of podoplanin with CLEC-2 or inhibits the activity of Src and/or Syk family kinases for use in a method of treating and/or preventing a chronic inflammatory disease, condition or related pathology in a subject.

Surprisingly, the present inventors have found that chronic inflammatory diseases, conditions or related pathologies can be prevented or treated by inhibition of the interaction of podoplanin with CLEC-2. Without wishing to be bound by theory, the inventors believe that the inhibition of the podoplanin pathway modifies platelet and/or immune cell recruitment in the subject. Unexpectedly, the inventors have found that modification of platelet, neutrophil/myeloid cell and/or macrophage recruitment and/or function is associated with reduced inflammation and improved healing.

The term "modifying" or "modify" as used herein, will be understood to refer to an increase, a reduction, an alteration of function and/or an alteration of the sub-type of cell.

The "interaction of podoplanin with CLEC-2", as used herein, will be understood as referring to the natural interaction or association between the ligand podoplanin and its receptor CLEC-2. This interaction may not require Ca ² \ The interaction may comprise association of CLEC-2 with a PLAG (platelet aggregation-stimulating) domain of podoplanin, for example at feast one of PLAG1 , PLAG2, PLAG3, or PLAG4 and/or the association of podopianin with a CTLD (C-type lectin-like domain) of CLEC-2. it will be appreciated that the interaction may comprise association between the CTLD (C-type lectin-iike domain) of CLEC-2 and a disialyl-corel in the PLAG domain of podopianin. The interaction may occur at amino acids G!u47 and/or Asp48 in the PLAG3 domain of podopianin. The interaction may further comprise the alpha2-6 linked sialic acid residue of podopianin. The interaction may comprise Thr52 in the PLAG domain. Thr52 may be siafy!ated. In some examples the interaction comprises the PLAG2 domain of podopianin. The interaction may comprise amino acids 38-51 of the PLAG2 domain of podopianin. In some instances the interaction may comprise one or more glycosylation sites of podopianin. For example, the interaction may comprise the O-g!ycosyiation of Thr25 in the N terminus of podopianin, as described by Kaneko et al., Mon. Anti. in Immunodiagnosis and Immunotherapy, 2015, 34(5), 310-317. It will be appreciated that the interaction may comprise association of podopianin with the noncanonical side face of CLEC-2. The crystal structure of the interaction of podopianin with CLEC-2 is described by Nagae et al., Structure, 2014, 22(12), 1711-1721 , to which the skilled reader is directed.

As used herein, the term "PLAG domain" will be understood to refer to the EDxxVTPG segment in the extracellular domain of podopianin.

It will be appreciated that podopianin may interact with a CLEC-2 monomer, a CLEC-2 dimer or a CLEC-2 mu!timer.

By "inhibits", as used herein, it will be understood that the agent prevents or decreases the interaction between CLEC-2 and podopianin, or the activity of Src and/or Syk family kinases relative to normal levels (i.e. the level in the absence of the agent). Inhibition of the interaction or the activity may be partial or complete. The agent may decrease the interaction or the activity by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%. It will be further appreciated that the inhibition of the interaction of podopianin with CLEC- 2 or the activity of Src and/or Syk family kinases by the agent may be direct or indirect.

The agent may be capable of specifically binding to CLEC-2 or podopianin. For example, the agent may be an antibody that specifically binds to CLEC-2, CLECIb or podopianin, thereby causing direct repression of the binding of podopianin to CLEC-2. in some examples the agent may be capable of competitively binding to podopianin or CLEC-2. By "competitively binding" it will be understood that the agent is capable of binding to a site on a first member, for example CLEC-2 or podopianin, such that it prevents the binding of a second member, for example CLEC-2 or podopianin to the first member. A suitable competitive binding agent may be a fragment of CLEC-2 or podopianin which is capable of specifically binding to its respective partner (i.e. podopianin or CLEC-2 respectively) and prevent or inhibit binding of a native molecule.

The agent may be capable of specifically binding to podopianin. In some embodiments, the agent is capable of specifically binding to CLEC-2 and/or CLEG lb.

The agent may be capable of competitively binding to the CTLD (C-type lectin-like domain) of CLEC-2. in some embodiments the agent is capable of binding to another site of CLEC-2. The agent may be capable of competitively binding to the PLAG (platelet aggregation-stimulating) domain of podopianin, for example at least one of PLAG1 , PLAG2, PLAG 3 or PLAG4. in some embodiments the agent is capable of competitively binding to the disialyl-corel in the PLAG domain of podopianin. In some embodiments the agent is capable of competitively binding to the PLAG2 domain of podopianin. The agent may be capable of competitively binding to the amino acids 38- 51 of the PLAG 2 domain of podopianin. In some embodiments the agent is capable of binding to one or more giycosylation sites of podopianin. The agent may be capable of binding to the glycosylated Thr25 in the N terminus of podopianin. in some embodiments the agent is capable of binding to another site of podopianin.

In some embodiments, the agent is capable of specifically binding to podopianin or CLEC-2 mRNA, thereby causing direct repression of translation of the gene into the CLEC-2 or podopianin protein. The agent may be capable of specifically binding to podopianin mRNA. In some embodiments the agent is capable of specifically binding to CLEC-2 mRNA,

The agent may be capable of inhibiting the activity of Src kinase. In some embodiments, the agent is capable of inhibiting the activity of Syk kinase. The agent may be capable of inhibiting the phosphorylation of Src and/or Syk kinase. The agent may be capable of specifically binding to Src kinase. In some embodiments the agent is capable of specifically binding to Syk kinase. For example, the agent may a!losterica!ly bind to Src and/or Syk kinase, resulting in a conformational change to Src and/or Syk kinase.

By "al!osteric" or "al!osterica!!y", as used herein it will be understood that the agent is capable of binding to a site of a target other than the active site of the target. in some embodiments the agent is capable of competitively binding to the ATP-binding site or a site adjacent to the ATP-binding site of Src and/or Syk kinase. In this way the binding of ATP (adenosine triphosphate) to the ATP-binding site is inhibited and so phosphorylation of Src and/or Syk kinase is inhibited. In some examples the site adjacent to the ATP-binding site is a hydrophobic pocket. The agent may be capable of inhibiting the interaction of Src and/or Syk kinase with the Cdc37-HSp90 molecular chaperone system. By inhibiting this interaction, the Src and/or Syk kinase may be ubiquitlyated and degraded.

By "treating" as used herein, it will be understood that the agent reduces, alleviates or eliminates symptoms of a medical condition, disease or pathology. The term "eliminates" may be understood to refer to the complete removal of symptoms. As used herein, "alleviation" will be understood to refer to the lessening of symptoms such that the subject's quality of life is improved. For example, the aiieviation of symptoms may be understood to refer to a reduction in pain and morbidity of the subject. The lessening of symptoms may be relative to at the time of diagnosis or during disease. The term "treating" may refer to the administration of the agent after the onset of symptoms or after diagnosis.

A reduction, alleviation or elimination of symptoms may be measured using various methods which will be dependent upon the disease under investigation. These may include, but may not be limited to: measuring systemic blood counts, measuring systemic cytokine profiles or other inflammatory marker levels such as CRP and ESR. in some instances the methods may include liver function tests (bilirubin, AST/ALT, albumin etc)or measuring levels of inflammatory infiltrate in affected tissue, measuring levels of bleeding or coagulation indices.

The measurement of specific patient scores for different disease may be employed by the skilled medical practitioner to measure a reduction, aiieviation or elimination of symptoms. For example, specific patient scores for chronic liver disease may include, but may not be limited to, any one or more of improvement in MELD (Model for End- Stage Liver Disease), UKELD (United Kingdom Model for End-Stage Liver Disease) and/or Child-Pugh score, For IBD, specific patient scores may include, but may not be limited to endoscopic resolution of inflammatory lesions and/or patient scores of vveliness including but not exclusively CDAI (Crohn's Disease Activity Index). In chronic kidney disease specific patient scores may include, but may not be limited to measuring improvements eGFR, albumin creatinine ratio (ACR) and/or serum antibody litres.

In some examples a reduction, alleviation or elimination of symptoms may be measured by measuring dependence on other immunosuppressants, for example, corticosteroids. A decreased dependence may be indicative of a reduction, alleviation or elimination of symptoms. Levels, dependence and/or counts to be detected may be increased or decreased relative to norma! levels or to levels prior to administration of the agent. Leveis, dependence and/or counts may be increased or decreased by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%.

A reduction, alleviation or elimination of symptoms may be detected by determining immune cell numbers at the site of inflammation and/or in the systemic circulation. The immune cells may be neutrophils, platelets, lymphocytes and/or macrophages. The representative number of immune cells can be determined by calculating the number of immune cells in a tissue biopsy or blood sample. Increased immune cell, for example neutrophil, platelet, lymphocyte, and/or macrophagenumbers, relative to immune ceil numbers, respectively, at the time of diagnosis or during disease may indicate a reduction, alleviation or elimination of symptoms. Relative or absolute immune ceil numbers may be determined using standard techniques known to those skilled in the art, for example flow cytometry, or visual cell counting using microscopic techniques.

The skilled person will understand the term "preventing" or "prevention" to refer to the preservation of health of a subject, for example protective and/or preventative treatment for a medical condition, disease or pathology. The term "preventing" may thus refer to the reduction, alleviation or complete prevention of future symptoms. in the context of the present invention, reduction or elimination may relate to the reduced or lessened effect of a causative factor or cause of a chronic inflammatory disease, condition or related pathology. The term "preventing" may thus refer to a reduction or lessening of inflammation from a causative factor or cause.

As the skilled person will appreciate, prevention may be of benefit to subjects who may be at risk of developing a chronic inflammatory disease, condition or related pathology. For example, prevention may be of benefit to subjects who intake excess levels of toxins, alcohol, drugs or nutritional supplements. Excess levels intaken can be determined by methods known to those skilled in the art.

The term "chronic" as used herein, will be understood to refer to a long-term disease or condition. By "long-term" the skilled person will appreciate that the disease or condition is of prolonged duration, for example, of at least 3 months, at least 6 months, at least 7 months, at least 8 months, at least 12 months, at least 2 years, at least 5 years, at least 10 years, at least 20 years, at least 30 years, at least 40 years, at feast 50 years, at least 60 years, at least 70 years or at least 80 years.

The duration of a disease or condition may be measured from the timepoint at which the subject was first exposed to a causative factor or from the onset of the disease or condition. The onset of the disease or condition may be defined as starting from the timepoint of the first symptom or symptoms.

"Chronic inflammatory disease or condition", as used herein, will be understood to refer to a chronic disease or condition associated with inflammation. In some examples, the inflammation may last for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 2 months, at least 4 months, at least 6 months, at least 1 year, at least 2 years, at least 5 years, at least 10 years, at least 20 years, at least 30 years, at least 40 years, at least 50 years, at least 60 years, at least 70 years or at least 80 years. in the context of the present invention, inflammation will be understood to refer to tissue damage as a consequence of the accumulation of inflammatory cells Damage may be relative to the tissue state of a normal, non-diseased and/or healthy subject. The inflammatory cells may be immune cells. In some instances the tissue damage may be associated with architectural damage to the tissue. The immune cells may comprise but may not be limited to lymphocytes, neutrophils, platelets, monocytes and/or macrophages. The tissue damage may comprise the disruption of tissue architecture, for example, the disruption of mucosal architecture in inflammatory Bowel Disease (IBD). Other forms of tissue damage may include the development of ulcers, granulomas or fistulas on or in the affected tissue. In some instances, tissue damage may include scarring or fibrosis, which the skilled person will understand to refer to the formation of excess fibrous connective tissue. One example of fibrosis is cirrhosis in chronic liver disease. Tissue damage may be associated with excess mucus production. The level of tissue damage may increase over time,

The agent may be capable of reducing, modifying or resolving inflammation. In some embodiments the agent is capable of preventing host ceil death. The host cell death may be apoptosis or necrosis, in some examples the agent may be capable of modifying the number, ratio, type and/or function of macrophages at a site of inflammation The agent may be capable of increasing the number or ratio of macrophages at a site of inflammation. In some instances the agent may be capable of modifying the number and/or function of platelets at a site of inflammation. The agent may be capable of altering the number of platelets at a site of inflammation. The agent may be capable of modifying the number and/or function of myeloid or lymphoid cells at a site of inflammation, for example, the agent may be capable of increasing the number of myeloid or lymphoid cells at a site of inflammation, in some examples, the agent may be capable of reducing alanine transaminase (ALT) levels. A reduction or increase may be relative to at the time of diagnosis or during disease. Other diagnostic markers associated with particular chronic inflammatory diseases will be known to the skilled medical practitioner, for example circulating inflammatory markers or cytokines. The agent may be capable of modifying other diagnostic markers.

The agent may be capable of modifying tissue fibrosis. Without wishing to be bound by theory, the inventors believe that the agent may be capable of modifying tissue fibrosis by modifying the function of profibrotic or pro-resolution immune cell populations including any of macrophages, myeloid cells and lymphocytes. In some instances the inventors believe that the agent may be capable of modifying tissue fibrosis by directly modifying the recruitment, number and/or ratio of profibrotic or pro-resolution immune cell populations including any of macrophages, myeloid ceils and lymphocytes in the infiamed tissue, in some instances the agent may be capable of reducing tissue fibrosis.

Thus, the skilled person may determine the efficacy of the agent in a method of treating and/or preventing a chronic inflammatory disease, condition or related pathology by measuring any of the level of inflammation and/or fibrosis, the number of immune ceils, at a site of inflammation or the level of disease characterising biomarkers (for example any of cytokines, growth factors or AST/ALT). The immune cells may comprise but may not be limited to lymphocytes, neutrophils, platelets, monocytes and/or macrophages. The levei(s) may be measured from a sample from a subject. The sample may be a tissue biopsy, blood or serum. Other suitable samples will be known to the skilled person.

As the skilled person will appreciate, chronic inflammatory diseases or conditions may be associated with gradual loss or reduction of organ or tissue function over time. The function may be reduced or lost relative to normal levels. The function may be reduced by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%. in some instances, the ioss of function may occur over a period of at least 3 months, at least 6 months, at least 7 months, at least 8 months, at least 12 months, at least 2 years, at least 5 years, at least 10 years, at least 20 years, at least 30 years, at least 40 years, at least 50 years, at !east 60 years, at least 70 years or at ieast 80 years. The organ or tissue in which function may be lost or reduced may be damaged.

It will be appreciated that chronic inflammatory diseases or conditions are distinct from acute inflammatory diseases or conditions. It will thus be appreciated that the term "chronic inflammatory diseases or conditions" does not comprise acute inflammatory diseases or conditions. Acute inflammatory diseases or conditions may be understood to refer to sudden-onset symptoms, whereas chronic inflammatory diseases or conditions may be understood to refer to the gradual onset of symptoms. Acute inflammatory diseases or conditions are characterised by a rapid reduction or ioss of organ function, in contrast, chronic inflammatory diseases or conditions are characterised by a gradual reduction or loss of organ function. Acute inflammatory conditions most commonly occur in subjects with no pre-existing inflammatory disease or condition, in contrast, chronic inflammatory diseases or conditions are associated with pre-existing disease, i.e. the disease is long-term.

The distinction of acute inflammatory diseases or conditions from chronic inflammatory diseases or conditions may also lie in the concentration or number of periods by which the subject was exposed to a causative factor. Acute inflammatory diseases or conditions may be characterised by one period of exposure, or one exposure, to the causative factor, whereas chronic inflammatory diseases or conditions may be characterised by repeated exposure, for example more than one period of exposure, or persistent exposure to the causative factor.

As the skilled person will appreciate, chronic inflammatory diseases or conditions can be associated with different immune characteristics, cytokine, growth factor stimuli and/or mediators to acute inflammatory diseases or conditions. For example, chronic inflammatory diseases or conditions may be associated with the infiltration of monocyte, macrophage and/or lymphocyte subpopuiations. in contrast, acute inflammatory diseases or conditions, may be associated with an infiltration and/or activation of predominantly neutrophils. Acute inflammatory disease or conditions are not commonly associated with the development of fibrosis which is a more common characteristic of chronic inflammatory diseases or conditions. Thus, acute inflammatory diseases or disorders may have distinct pro-inflammatory drivers to chronic inflammatory diseases or disorders. .

In some instances a chronic inflammatory disease or condition may be distinguished from an acute inflammatory disease or condition by the absence of symptoms or markers of the acute inflammatory disease or condition. One example method for distinguishing between an acute inflammatory disease or condition and a chronic inflammatory disease or condition may be the use of a tissue biopsy to assess the extent of tissue damage and immune cell subtypes present in tissue to indicate chronicity of the inflammation .

The causative factors, contributory factors, causes and/or influences of disease progression of a chronic inflammatory disease or condition may include one or more of microbial infection, parasite infection, alcohol, drugs, vascular diseases, for example Budd -Chiari syndrome, metabolic disease, for example Wilson's disease, diabetes, high biood pressure, environmental toxins, autoimmunity, gene(s), ageing or repeated heart failure. Other causative factors, contributory factors and/ influences upon disease progression wiii be known to the skilled medical practitioner. The causative factor may be unknown.

"Influencer", "influence" or "influences'' as used herein, will be understood to refer to a factor that may accelerate disease progression. Acceleration of disease progression may be relative to disease in a subject not exposed to the particular influencing factor. Acceleration of disease progression may comprise increased tissue damage. The increased tissue damage may be relative to tissue damage in subject with the chronic inflammatory disease or condition which has not been exposed to the particular influencing factor. By "contribute to" or "contributory factor", it wiii be understood that a factor may cause a chronic inflammatory disease or condition, when in combination with at least one other contributory factor. The at least one other contributory factor may not be known.,

!n some embodiments the chronic inflammatory disease or condition is caused, influenced and/or contributed to by viral infection, autoimmunity, gene(s), environmental toxins, high biood pressure, a!cohol or drugs, for example.

Environmental toxins which may cause influence and/or contribute to chronic inflammatory diseases or conditions include cigarette or cigar smoke, grains, isocyanates, cadmium, air pollution or coal.

The metabolic disease may be caused by any one of obesity, high cholesterol, high trigiycerides or high blood pressure. Microbial infections which may cause, influence and/or contribute to chronic inflammatory diseases or conditions include bacterial or viral infection. The viral infection may be infection with a hepatitis virus. The hepatitis virus may be any of hepatitis B, C or D. As the skilled medical practitioner will be aware, hepatitis B, C or D are typically associated with chronic inflammatory disease, whereas hepatitis A and E are typically associated with acute inflammatory disease. The drugs may include lithium or non-steroidal anti-inflammatory drugs (NSA!Ds), for example aspirin or ibuprofen.

The chronic inflammatory diseases or conditions may be caused, influenced and/or contributed to by repeated and/or excessive exposure to the causative factor. For example, the chronic inflammatory disease or condition may be caused by long-term, regular exposure to alcohol, drugs or environmental toxins, in some instances, the long-term, regular exposure may be excessive, for example excessive alcohol intake. The level of the causative factor which may be considered excessive will be known and can be calculated by the skilled medical practitioner.

A chronic inflammatory disease or condition may be progressive or relapsing-remitting. The term "progressive" will be understood to refer to a worsening of symptoms over time. "Relapsing-remitting" will be understood to refer to one or more distinct episodes of symptoms (relapse) followed by a partial or complete loss of symptoms (remission). The remission may be followed by a relapse.

A chronic inflammatory disease or condition may further comprise a sudden onset of symptoms and/or loss or reduction of organ function. In some examples the sudden onset of symptoms may comprise a sudden (within 1 or 2 days) increase in severity of pre-existing symptoms.

It will be appreciated that the terms "related pathology" and "related pathologies" can be interchanged herein. "Related pathology" as used herein, will be understood to refer to other morbidities associated with chronic inflammatory diseases or conditions, in some instances the chronic inflammatory disease or condition may develop info the related pathology. The related pathology may include liver cancer, bowel cancer, lung cancer, kidney cancer, myocardial infarction, pulmonary hypertension, pulmonary embolism, deep vein thrombosis, uveitis, episcleritis, hepatorenal syndrome, hepatopuimonary syndrome, sacro-iieitis or bacterial infection. The liver cancer may comprise or consist of hepatocellular carcinoma. The chronic inflammatory disease or condition may be inflammation of a tissue. The chronic inflammatory disease, condition or related pathology may be inflammation of an organ. In some embodiments the chronic inflammatory disease, condition or related pathology comprises or consists of inflammation of mucosal tissue. In some embodiments the chronic inflammatory disease or condition comprises or consists of inflammation of a mucosal organ. In some embodiments the chronic inflammatory disease or condition comprises or consists of inflammation of a joint, eye (for example the uveal tract), skin, liver, bowel, kidney, lungs, uterus, bladder, vagina, stomach or esophagus. In some embodiments, the chronic inflammatory disease or condition comprises or consists of inflammation of the liver, bowel, kidney, lungs, uterus, bladder, vagina, stomach or esophagus.

!n some embodiments, the chronic inflammatory disease or condition comprises or consists of inflammation of the liver, bowe!, kidney or !ungs. in some embodiments the chronic inflammatory disease or condition comprises or consists of inflammation of the liver or bowel.

Symptoms of a chronic inflammatory disease or condition may include, but may not be limited to, any of pain and/or cramping in the region of the inflamed tissue or organ, diarrhoea, bloody stools and/or urine, high blood pressure, nausea, weight loss, loss of appetite, shortness of breath, fatigue, vomiting, anaemia, venous thrombosis or fever.

Methods of diagnosing a chronic inflammatory disease or condition are known to the skilled medical practitioner. The method and resulting diagnosis will depend upon the symptoms, lack of symptoms, causative factor and/or contributory factor that the subject presents with. Example methods may include tissue biopsies, viral serologies, the detection of autoimmune markers, imaging or blood tests. Methods of diagnosis may include tests to exclude other possible diseases or conditions.

The subject may have been diagnosed as suffering from a chronic inflammatory disease or condition. The subject may be suspected of having a chronic inflammatory disease or condition, and/or may be displaying symptoms of a chronic inflammatory disease or condition. In some embodiments, the subject is identified as being at risk of developing a chronic inflammatory disease or condition.

In some embodiments, the subject is asymptomatic.

The subject may have been diagnosed as suffering from hepatitis, alcoholism, autoimmune disease or viral infection. The subject may be or was a smoker, !n some embodiments the subject smokes at Ieast 10, at least 15, at Ieast 20 or at least 30 cigarettes per day. The subject may have been diagnosed as suffering from a persistent viral infection, for example hepatitis B, C or D. By "persistent viral infection", as used herein, it will be understood that the subject has been infected with a virus for a prolonged period of time, for example, of at least 2 months, at least 3 months, at Ieast 4 months, at least 5 months, at ieast 6 months, at least 7 months, at Ieast 8 months, at Ieast 12 months, at least 2 years, at Ieast 5 years, at least 10 years, at Ieast 20 years, at Ieast 30 years, at ieast 40 years, at least 50 years, at least 60 years, at least 70 years or at !east 80 years, without complete removal of the virus from the subject.

The diagnosis of a chronic inflammatory disease or condition may lie in the identification of the cause, causative factor or contributory factor. For example, the skilled medical practitioner may diagnose chronic liver disease if the subject is obese, suffers from alcoholism, suffers from metabolic disease, has one or more mutated genes, for example, the gene HFE, or suffers from a persistent viral infection (e.g. hepatitis B, C or D). The skilled medical practitioner may diagnose chronic lung disease if the subject is or was a smoker or has alpha- 1 -antitrypsin deficiency. The skilled medical practitioner may diagnose a chronic inflammatory disease or condition based upon assessment of patient history, symptoms, genetic phenotype and/or diagnostic test profiles. Such assessments will be known to the skilled medical practitioner.

The diagnosis of a chronic inflammatory disease or condition may lie in the identification of the period of time for which the subject has been experiencing symptoms. A chronic inflammatory disease or condition may be diagnosed if the subject has experienced symptoms for at least 3 months, at least 6 months, at ieast 7 months, at least 8 months, at Ieast 12 months, at least 2 years, at ieast 5 years, at Ieast 10 years, at Ieast 20 years, at least 30 years, at least 40 years, at least 50 years, at Ieast 60 years, at least 70 years or at ieast 80 years. in some embodiments, the subject is a mammal, in some embodiments, the subject is human. Non-human subjects to which the invention is applicable include pets, domestic animais, wildlife and livestock, inciuding dogs, cats, cattle, horses, sheep, goats, deer and rodents.

The chronic inflammatory disease or condition may be selected from chronic joint disease, chronic eye disease, chronic skin disease, chronic liver disease, Inflammatory Bowel Disease (IBD), chronic kidney disease, chronic lung disease or chronic bladder disease.

The chronic joint disease may be arthritis. The arthritis may be rheumatoid arthritis, osteoarthritis or other infiammatory arthritis. The chronic eye disease may be uveitis.

The chronic skin disease may be selected from psoriasis, eczma or dermatitis

The chronic infiammatory disease or condition may be selected from chronic Iiver disease, Infiammatory Bowel Disease (IBD), chronic kidney disease, chronic lung disease or chronic bladder disease.

In some embodiments the chronic inflammatory disease or condition is selected from chronic iiver disease, inflammatory bowel disease, chronic lung disease or chronic kidney disease.

In some embodiments the chronic inflammatory disease or condition is selected from inflammatory bowel disease or chronic Iiver disease. inflammatory Bowel Disease

The inflammatory bowel disease (IBD) may be selected from ulcerative colitis, indeterminate colitis or Crohn's disease. In some embodiments the inflammatory bowel disease is selected from ulcerative colitis or Crohn's disease. The inflammatory bowel disease may be Crohn's disease. As the skilled medical practitioner will be aware, ulcerative coiitis is predominantly associated with inflammation of the large intestine, whereas Crohn's disease is associated with inflammation of any section of the gastrointestinal tract (Gl).

The causative factors, causes, contributory factors and/or influences of disease progression of inflammatory bowel disease may include one or more of autoimmunity, environmental toxins, drugs, gene(s) or microbial infection. Other causative factors, contributory factors and/or influences of disease progression will be known to the skilled medical practitioner. The causative factor may be unknown.

Environmental toxins which may cause, contribute to and/or influence progression of inflammatory bowel disease may include cigar or cigarette smoke. Microbial infections which may cause, contribute to and/or influence progression of inflammatory bowel disease may include viral or bacterial infection.

Drugs which may cause, contribute to or influence progression of inflammatory bowel disease may include isotretinoin or nonsteroidal anti-inflammatory drugs (NSAIDs), for example aspirin or ibuprofen.

The inflammatory bowel disease may be caused by repeated and/or excessive exposure to the causative factor.

Symptoms of inflammatory bowel disease may include, but may not be limited to, any of pain and/or cramping in the abdominal region, diarrhoea which may be recurring or bloody, weight loss, fatigue, vomiting, anaemia or fever.

Methods for diagnosing inflammatory bowel disease are known to the skilled medical practitioner. Methods may include endoscopic procedures, for example any of colonoscopy, flexible sigmoidoscopy, upper endoscopy, capsule endoscopy, or double- balloon endoscopy. Other methods may include imaging procedures such as X-ray, computerized tomography (CT) scan, magnetic resonance imaging (MR!) or small bowel imaging. Tests may include one or more bowel biopsies. The biopsy may be assessed using haematoxyiin and Eosin stains. The skilled practitioner may assess the biopsy for increased levels of inflammation and/or decreased levels of mucosa! architecture relative to norma! levels. The biopsy may also be assessed for increased levels of any one or more of ulcers, fistulas, granulomas or transmural lymphoid aggregates, in some examples, methods may include blood tests, for example, FBC (full blood count), ESR (erthrocyte sedimentation rate) or CRP (C-reactive protein), ferritin, B12 and/or folate, or calpronectin.

Levels to be detected may be increased or decreased relative to normal levels. Levels may be increased or decreased by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%.

Methods of diagnosis may include tests to exclude other possible diseases or conditions. For example, methods may include tests to exclude anaemia or infection. To test for anaemia, the skilled medical practitioner may assess the level of red blood cells in a sample from a subject. The level may be decreased relative to normal levels. The sample may be blood. To test for infection, the skilled medical practitioner may carry out a blood culture test on a sample of blood from a subject.

The related pathology of IBD may be bowel cancer, myocardial infarction, pulmonary hypertension or bacterial infection, pulmonary embolism, deep vein thrombosis, uveitis, episcleritis, hepatorenal syndrome, hepatopulmonary syndrome, sacroileitis and/or Primary Sclerosing Cholangitis, erythema and/or pyoderma and osteoporosis/osteopenia.

Chronic liver disease

The chronic liver disease may be selected from hepatitis, alcoholic liver disease, drug- induced liver disease, non-alcoholic fatty liver disease, haemochromatosis, Wilson's disease, autoimmune hepatitis, primary biliary cholangitis (primary biliary cirrhosis), primary sclerosing cholangitis, or heart-failure-induced chronic liver disease.

Chronic liver disease may be associated with any one or more of inflammation, cell death, fibrotic tissue formation or cirrhosis in the liver. in some embodiments the chronic liver disease is selected from hepatitis, alcoholic liver disease, autoimmune hepatitis, primary biliary cholangitis or primary sclerosing cholangitis. In some embodiments the chronic Iiver disease is selected from hepatitis, primary sclerosing cholangitis or alcoholic liver disease.

The causative factors, causes, contributory factors and/or influences of progression of chronic liver disease may include one or more of autoimmunity, environmental toxins, gene(s), microbial infection, alcohol, drugs, vascular diseases, for example Budd-Chiari syndrome, metabolic disease, for example Wilson's disease, haemochromatosis or non-alcoholic fatty liver disease, obesity or repeated heart failure. Other causative factors, contributory factors and/or influences of disease progression will be known to the skilled medical practitioner. The causative factor may be unknown.

Environmental toxins which may cause, contribute to and/or influence progression of chronic liver disease may include but are not restricted to cigar smoke, cigarette smoke or aflatoxin. Microbial infections which may cause, contribute to and/or influence chronic Iiver disease may include viral or bacterial infection. Viral causes, contributory factors and/or influences of chronic liver disease may include hepatitis B, C D or E virus.

Drugs which may cause, contribute to and/or influence progression of chronic Iiver disease may include nonsteroidal anti-inflammatory drugs (NSAiDs), for example aspirin or ibuprofen, methotrexate, amiodarone or nitrofurantoin, antibiotic compounds, statins or dietary supplements.

The chronic Iiver disease may be caused by repeated and/or excessive exposure to the causative factor.

Symptoms of chronic Iiver disease may include, but may not be limited to, any of weight loss, loss of appetite, jaundice, vomiting which may be bloody, dark stools, oedema, ascites, muscle and joint pain, fever, fatigue, abdominal pain, dark urine, itchy skin, confusion, or dry eyes and mouth.

In some embodiments the chronic fiver disease further comprises a sudden onset of symptoms and/or loss or reduction of Iiver function. In some examples the sudden onset of symptoms may comprise a sudden increase in severity of pre-existing symptoms. The skilled medical practitioner may refer to this as acute-on-chronic Iiver disease or decompensated chronic liver disease. The sudden onset of symptoms and/or ioss or reduction of liver function may be associated with cirrhosis. The sudden onset symptoms may include, but may not be limited to, any of oesophageal variceal bleeds, ascites, bacterial peritonitis, for example spontaneous bacterial peritonitis, hepatic encephalopathy or hepatorenal syndrome.

Methods for diagnosing chronic liver disease are known to the skilled medical practitioner. Tests for the diagnosis of chronic liver disease may include coagulation studies, the detection of aspartate aminotransferase (AST)/serum glutamic-oxaloacetic transaminase (SGOT), alanine aminotransferase (ALT)/serum glutamic-pyruvic transaminase (SGPT), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), bilirubin, albumin, or protein levels in a sample from the subject. Other diagnosis methods may include viral serologies, the detection of autoimmune markers, electroencephalography, intracranial pressure monitoring, liver biopsy or imaging. Viral serologies may inciude the detection of viral surface antigen, or Immunoglobulin, for example, the detection of hepatitis B, C, or D virus immunoglobulin M (IgM) or hepatitis B surface antigen (HbsAg). Liver biopsy may be percutaneous or transjugular. imaging may include hepatic doppler ultrasonography, abdominal computed tomography (CT) scanning, magnetic resonance imaging or cranial CT scanning. Levels to be detected may be increased or decreased relative to normal levels. Levels may be increased or decreased by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%. The sample may be blood or serum.

The related pathology of chronic liver disease may be liver cancer, in some embodiments the related pathology of chronic liver disease is hepatocellular carcinoma.

Chronic iung disease The chronic lung disease may be selected from chronic bronchitis, emphysema, asthma, idiopathic pulmonary fibrosis, interstitial lung disease, sarcoidosis or chronic obstructive airways disease. Chronic iung disease may be associated with any one or more of ceil death, inflammation, excessive mucus production, disruption of tissue architecture or fibrotic tissue formation in the !ung. The causative factors, causes, contributory factors and/or influences of progression of chronic lung disease may include one or more of autoimmunity, environmental toxins, gene(s) or microbial infection. Other causative factors, contributory factors and/or influences of disease progression will be known to the skilled medical practitioner. The causative factor may be unknown.

Environmental toxins which may cause, contribute to and/or influence progression of chronic lung disease may include cigarette or cigar smoke, grains, isocyanates, cadmium, air pollution or coal. Microbial infections which may cause, contribute to and/or influence progression of chronic lung disease may include viral or bacterial infection.

Gene(s) which may cause, contribute to and/or influence progression of chronic lung disease may include genes associated with expression of the protease inhibitor alpha-1 antitrypsin. A reduction or deficiency in alpha-1 -antitrypsin levels may be indicative of chronic lung disease. The reduction or deficiency may be relative to normal levels. Levels may be decreased by at least 5, 10, 20, 30, 40, 50, 80, 70, 80, 90 or 100%.

The chronic iung disease may be caused by repeated and/or excessive exposure to the causative factor.

Symptoms of chronic lung disease may include, but may not be limited to, any of breathiessness, persistent cough, phlegm, frequent chest infections, wheezing, weight loss, fatigue or swollen ankles.

Methods for diagnosing chronic lung disease are known to the skilled medical practitioner. Methods for the diagnosis of chronic lung disease may include lung function tests, for example a spirometry test and/or a peak flow test, imaging, electrocardiogram, echocardiogram, testing the blood oxygen level, blood tests, for exampe to test for reduced alpha-1 -antitrypsin levels or deficiency. Imaging may include X-ray or computed tomography (CT) scanning. Levels to be detected may be increased or decreased relative to normal levels. Levels may be increased or decreased by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%, The sample may be blood, serum or phlegm.

The related pathology of chronic lung disease may be lung cancer. Chronic kidney disease The chronic kidney disease may be selected from high blood pressure-induced chronic kidney disease, diabetes-induced chronic kidney disease, high cholesterol-induced chronic kidney disease, autoimmune-induced chronic kidney disease, infection-induced chronic kidney disease, glomerulonephritis, polycystic-induced chronic kidney disease, blockage-induced chronic kidney disease, or drug-induced chronic kidney disease Autoimmune-induced chronic kidney disease may include, but not be limited to, IgA nephropathy, goodpastures, lupus or nephritis.

By "blockage-induced" as used herein, it will be understood that blockage has occurred in the flow of urine, for example, from recurrent kidney stones, urethra! strictures, abnormal valves or an enlarged prostate.

Chronic kidney disease may be associated with any one or more of cell death, inflammation, disruption of tissue architecture or fibrotic tissue formation in the kidney. The causative factors, causes, contributory factors and/or influences of progression of chronic kidney disease may include one or more of drugs, high blood pressure, diabetes, high cholesterol, microbial infection, glomerulonephritis, polycystic kidney disease, blockages or ageing. Other causative factors, contributory factors and/or influences of disease progression will be known to the skilled medical practitioner. The causative factor may be unknown.

Drugs which may cause, contribute to and/or influence the progression chronic kidney disease may include nonsteroidal anti-inflammatory drugs (NSAIDs), for example aspirin or ibuprofen. Microbial infections which may cause, contribute to and/or influence the progression of chronic kidney disease may include viral or bacterial infection.

The chronic kidney disease may be caused by repeated and/or excessive exposure to the causative factor.

Symptoms of chronic kidney disease may include, but may not be limited to, any of tiredness, swollen ankles, feet or hands, breathiessness, nausea, blood in urine, more or less frequent urination relative to normal levels, difficulty urinating, pruritus, dizziness, confusion, dark or pale urine, or leg or flank pain.

Methods for diagnosing chronic kidney disease are known to the skilled medical practitioner. These may include blood tests, urine tests, kidney biopsy or imaging. Imaging may include ultrasound, magnetic resonance imaging (MRS) or computed tomography (CT) scanning of the kidneys. Blood tests may measure the levels of creatinine in the blood. The skilled medical practitioner may calculate a subject's estimated glomerular filtration rate (eGFR) from the levels of creatinine. The eGFR level may be decreased relative to normal levels. A norma! level may be considered to be an eGFR of at least 90ml/min.

Urine tests may detect the levels of albumin and creatinine in the urine, and/or the levels of blood and/or protein in the urine. The levels of albumin and creatinine detected in the urine may be calculated as an albumin:creatinine ratio (ACR). The ACR ratio may be compared to normal ratios. Chronic kidney disease may be diagnosed if the subject has an ACR ratio of over 1mg/mmol, for example, of between 1 and 3 mg/mmol, of between 1 and 30mg/mmo! or of over 30mg/mmol.

The related pathology of chronic kidney disease may be kidney cancer, in some embodiments the chronic inflammatory disease, condition or related pathology does not comprise cancer, other than liver, bowel, lung or kidney cancer. In some embodiments the chronic inflammatory disease, condition or related pathology does not comprise cancer, other than liver or bowel cancer. In some embodiments the chronic inflammatory disease, condition or related pathology does not comprise cancer, other than liver cancer. in some embodiments the chronic inflammatory disease, condition or related pathology does not comprise cancer, other than hepatocellular carcinoma, In some embodiments the chronic inflammatory disease, condition or related pathology does not comprise cancer.

Agents which are capable of inhibiting the interaction of podopianin with CLEC-2, or inhibting the activity of Src and/or Syk family kinases can be identified using functional assays known to the skilled person. Such assays may conveniently enable high throughput screening of potential inhibitor agents. For example, a protein-based assay can be derived by expressing and isolating proteins involved in the interaction of podopianin with CLEC-2, and detecting the interaction of the proteins by ELISA. Potential inhibitor agents can be included in the ELISA. An inhibitory effect of an agent can then be detected by monitoring for reduced interaction between the proteins in the ELISA.

A transcription based assay can be derived by selecting transcriptional regulatory sequences (e.g. promoters) from genes involved in the CLEC-2-podoplanin pathway, and operatively linking such promoters to a reporter gene in an expression construct. The effect of different agents can then be detected by monitoring expression of the reporter gene in host cells transfected with the expression construct. One such assay is a luminescent reporter assay. Commonly used reporter genes include fuciferase, beta- galactosidase, alkaline phosphatase and CAT (chloramphenicol acetyl transferase).

Other functional assays for detecting an inhibitory effect upon the interaction of podopianin with CLEC-2 may include tyrosine kinase phosphorylation assays. Such assays will be known to the skilled person. For example, the skilled person may use src and/or syk phosphorylation assays. A reduction in Src and/or syk family kinase activation, measured by reduced phosphorylation downstream of Src or syk, may be used to detect the inhibitory effect of an agent upon the interaction of podopianin with CLEC-2.

A platelet-aggregation assay can be derived by studying podoplanin-induced platelet aggregation in vitro in the presence of the agent. An inhibitory effect of the agent can then be detected from reduced platelet aggregation compared to control samples. The use of an EL!SA and a platelet aggregation assay to monitor the inhibitory effect of an agent on the interaction of podoplanin with CLEC-2 is described by Nakazawa ef a!., Cancer Science, 2011 (102), 2051-2057.

Other functional assays for detecting an inhibitory effect may include measuring the affinity of the interaction between recombinant purified podoplanin and CLEC-2 in the presence or absence of the agent. The skilled person may use a Biacore X system and kit to measure the affinity, as described by Inoue et a!., PLOS One, 2015, 10(9), 1-28. Thus, a reduction in affinity may be used to detect the inhibitory effect of the agent.

The agent may comprise or consist of a peptide, a protein, a truncated protein, an enzyme, an antibody or an antibody fragment (such as a Fab or F(ab¾ fragment, Fab- SH, an Fv antibody, an scFV antibody, a diabody or any other functional antigen- binding fragment), for example.

Agents which are peptides or proteins may be modified. For example, the peptide or protein may be PEGylated. Modified peptides or proteins may advantageously exhibit an improved circulatory half-life compared to non-modified peptides or proteins. The modification may be at the N and/or C terminus of the peptide or protein.

In some examples the agent may be a nucleic acid that specifically binds to CLEC-2 or podoplanin mRNA, thereby causing direct repression of the gene to prevent translation into the CLEC-2 or podoplanin protein.

The agent may comprise or consist of a nucleic acid or a small molecuie.

As used herein, a "small molecule" is a chemical compound having a molecuiar weight of no more than 900 daltons (Da). In some embodiments, the small molecule has a molecular weight of no more than 700 or no more than 500 Da. The small molecule may be an organic compound. The smali molecule may bind to a protein component of the CLEC-2-podoplanin interaction and modulate its activity and/or interactions with other proteins or nucleic acids. In some embodiments the agent comprises or consists of the small molecuie 2CP, a derivative of 4-O-benzoyl- 3-methoxy-beta-nitrostyrene (BSV1NS). 2GP specifically binds to CLEC-2, as described by Chang et a!., Oncotarget, 2015, 6(40), 42733-42748.

!n some embodiments the agent comprises or consists of the small molecule fostamatinib, saracatinib or entospletinib.

In some embodiments the agent comprises or consists of use of kinase inhibitors. One such example of such is saracatinib a small molecule kinase Inhibitor that inhibits the phosphorylation of key amino acids within kinases including syk saracatinib.

In some embodiments the agent comprises or consists of an antisense molecule (e.g. an antisense DNA or RNA molecule or a chemical analogue) or a ribozyme molecule. Ribozymes and antisense molecules may be used to inhibit the transcription of a gene encoding CLEC-2 or podoplanin, or translation of the mRNA of that gene. Antisense molecules are oligonucleotides that bind in a sequence-specific manner to nucleic acids, such as DNA or RNA. When bound to mRNA that has a complementary sequence, antisense RNA prevents translation of the mRNA. Triplex molecules refer to single antisense DNA strands that bind duplex DNA forming a colinear triplex molecule, thereby preventing transcription. Particularly useful antisense nucleotides and triplex molecules are ones that are complementary to or bind the sense strand of DNA (or mRNA) that encodes a CLEC-2 or podoplanin protein.

In some embodiments, the agent comprises or consists of a short interfering nucleic acid (siNA). A siNA molecule may comprise a siDNA molecule or a siRNA molecule. In some embodiments, the agent comprises or consists of miRNA (micro RNA), siRNA (small interfering RNA) or shRNA (short hairpin RNA). Oligonucleotides including siNAs can be prepared by solid phase chemical synthesis using standard techniques.

In embodiments wherein the agent is a peptide or protein, a nucleic acid sequence encoding the peptide or protein may be provided in a suitable vector, for example a plasmid, a cosmid or a viral vector. Thus, also provided is a vector (i.e. a construct), comprising a nucleic acid sequence which encodes the protein or peptide. The nucleic acid sequence is preferably operably linked to a suitable promoter. The invention further relates to a composition comprising the vector.

Agents which are nucleic acids, such as siRNAs or miRNAs, may be modified (e.g. via chemical modification of the nucleic acid backbone), or delivered in suitable delivery system which protects the nucleic acids from degradation and/or immune system recognition. Examples of suitable delivery systems inciude nanoparticles, lipid particles, polymer-mediated delivery systems, iipid-based nanovectors and exosomes. In some embodiments the agent is a naturally occurring or a synthetic iigand of a protein involved in the interaction of podoplanin with CLEC-2, or Syk or See kinase. The term "iigand" as used herein is understood to mean a substance thai binds to a protein to form a complex. Formation of the complex may induce a change in the function or activity of the protein. A iigand may be an antagonist. As used herein, an "antagonist" is a molecule which binds to a protein and inhibits a biological response.

Proteins and peptides may be generated using a variety of methods, including purification of naturally-occurring proteins, recombinant protein production and de novo chemical synthesis.

In some embodiments the agent comprises or consists of a truncated protein. By "truncated" it will be appreciated that the protein lacks a portion of the full-length protein. The truncated protein may be inactive, or possess less activity as compared to full length protein. As the skilled person will appreciate, the truncated protein may be capable of competitively binding to CLEC-2 or podoplanin.

In some embodiments the agent comprises or consists of truncated CLEC-2 or CLEC- 1 b. The truncated CLEC-2 or CLEC-1 b may be capable of binding to podoplanin. The truncated CLEC-2 or CLEC-1 b may lack at least a portion of an extracellular domain, in some embodiments the truncated CLEC-2 or CLEC-1 b lacks a portion of the C~type lectin domain. The truncated CLEC-2 or CLEC-1 b may lack at least a portion of the transmembrane domain and/or an N-terminal cytoplasmic tail. In some embodiments the truncated CLEC-2 or CLEC-1 b lacks the transmembrane domain. In some embodiments the agent comprises or consists of truncated podoplanin. The truncated podoplanin may be capable of binding to CLEC-2. The truncated podoplanin may lack at least a portion of the extracellular domain. The truncated podoplanin may lack at least a portion of the PLAG (platelet aggregation-stimulating) domain of podoplanin, for example at least one of PLAG1 , PLAG2 or PLAG3. The truncated podoplanin may be derived from a spiice variant, for example a naturally occurring splice variant, in some embodiments the agent comprises an antibody or antibody fragment, !n some embodiments the agent consists of an antibody or antibody fragment. The antibody may be monoclonal, polyclonal, recombinant or chimaeric. The term "chimaeric antibody" refers to an antibody consisting of antibody fragments derived from different species. Methods for generating antibodies are weil-known to those skilled in the art. For example, the skilled person can use known hybridoma technology to generate and detect antibodies specific for CLEC2 or podoplanin. Commonly used assays to detect the specificity of an antibody for a particular target protein include EL!SA, Western Biot and flow cytometry. Other methods to detect the specificity of an antibody will be known to the skilled person. In some embodiments the agent comprises or consists of a humanised antibody. By "humanised" it will be appreciated that an antibody comprises or consists of human antibody fragments and antibody fragments from other species, for example rodents, e.g. mice. A humanised antibody may comprise human constant domains and variable domains from another species, for example rodent variable domains, in some embodiments a humanised antibody may comprise human variable and constant regions and rodent, for example mouse CDR (complementarity determining region) regions. Advantageously, humanised antibodies have reduced immunogenicity. In addition, humanised antibodies retain the high binding affinity of an antibody from a non-human species.

In some embodiments the agent is a human antibody or fragment thereof.

The agent may specifically bind to podoplanin. In some embodiments the agent comprises an antibody that specifically binds to podoplanin, i.e. an anti-podoplanin antibody or fragment. The generation and detection of an antibody specific for podoplanin is described by Nakazawa et a! and Ogasawara et ai, Monoclonal antibodies in Immunodiagnosis and immunotherapy, 2016 (35), 1-8.

Antibodies that specifically bind to human podoplanin are provided in US 8697073. Other suitable anti-podoplanin antibodies include LpMAb-13 (Ogasawara et ai.), P2-0 or HAG-3 (Nakazawa et al). Commerciaily available anti-human pociopianin antibodies include the anti-human antibodies listed in Table 1. Commercially available anti-mouse podop!anin antibodies include the anti-mouse antibodies listed in Table 1. Known epitopes of human podopianin are provided in US 8697073. in some embodiments, the anti-podoplanin antibody specificaliy binds to at least one of the epitopes disclosed in US8697073, the epitope Ala42-Asp49 of human podopianin, the PLAG1 epitope region of human podopianin, the PLAG2 epitope region of human podopianin the PLAG3 epitope region of human podopianin or the PLAG4 epitope region of human podopianin. !n some embodiments, the anti-podopianin antibody specificaliy binds to the 6 amino acid epitope sequence AMPGAE. In some embodiments, the anti- podopianin antibody specificaliy binds to the 10 amino acid epitope sequence GVAMPGAEDD. Other suitable epitopes are provided by Ogasawara et al., Hybridoma, 2008, 27(4), 259-267 Known CDR regions of anti-podoplanin antibodies are aiso provided in US 8697073.

In some embodiments the agent comprises or consists of an 8.1.1 clone hamster monoclonal anti-podoplanin antibody. This antibody is available commercially from various suppliers including, but not limited to Santa Cruz Biotechnology, AbCam, Biolegend, NovusBio and eBioscience. The antibody may specifically bind to mouse podopianin. in one embodiment the agent comprises or consists of an NZ-1.3 clone rat monoclonal anti-podoplanin antibody. The antibody may specifically bind to human podopianin. The NZ-1.3 done rat monoclonal anti-podoplanin antibody is available commercially from at least eBioscience. in some embodiments the agent specifically binds to CLEC-2. In some embodiments the agent comprises an antibody that binds specifically to CLEC-2, i.e. an anti-CLEC-2 antibody. The agent may comprise an antibody that binds specifically to human CLEC- 2. The agent may comprise an antibody that binds specifically to rodent, for example mouse CLEC-2. Anti-human CLEC-2 antibodies are available from various suppliers including, but not limited to R&D Systems.

As used herein, the terms "specifically binds to" or "specific for" wiil be understood to mean that the agent selectively recognises an epitope of a particular protein, for example, CLEC-2 or podopianin. Antibodies may be conjugated to other moieties, for example therapeutic or cytotoxic moieties. The conjugation of another moiety to an antibody advantageously allows the targeted delivery of an additional therapeutic moiety to CLEC-2, podopianin, Src and/or Syk family kinases. This may serve to further inhibit the CLEC-2-podoplanin pathway, !n other examples, antibodies may be conjugated to imaging moieties. The conjugation of an imaging moiety to an antibody advantageously allows the targeted imaging of the CLEC-2-podoplanin pathway, for example CLEC-2 or podopianin. This may advantageously be used to visualise the in vivo inflammation of the chronic inflammatory disease or condition.

Thus, in some embodiments, the agent comprises or consists of an antibody conjugate. The conjugate may comprise a cytokine or other molecule. In some embodiments the conjugate comprises a drug or radionuclide. Such antibody-conjugates are well-known in the art. !n some embodiments the conjugate comprises a PET (position emission tomography) or MRI (magnetic resonance imaging) Iigand. For example, the conjugate may comprise a PET Iigand such a ⁶⁸ Gallium, ⁶⁴ Cu or ¹²⁴ l- labelled peptide or antibody, in other examples, the conjugate may comprise a MRI Iigand such as a gadolinium contrast agent.

In some embodiments the agent is in combination with at least one additional agent. In some embodiments the at least one additional agent is selected from nonsteroidal antiinflammatory drugs (NSAIDs), immunosuppressants, biologicals or Ursodiol. NSAIDS may comprise ibuprofen or aspirin,

Immunosuppressants may comprise corticosteroids, aminosalicylate (ASA) compounds for example 5-ASA compounds, cyc!osporine or azathioprine. Biologicals may comprise monoclonal antibodies, in some examples biologicals comprise rituximab, basiliximab, daclizumab or muromonab.

In some embodiments the at least one additional agent is selected from NSAIDS, immunosuppressants or biologicals. In some embodiments the at ieast one additional agent is selected from immunosuppressants or biologicals.

The agent and the additional agent may be administered concomitantly, sequentially or alternately.

Without wishing to be bound by theory, the present inventors propose that the use of an agent that inhibits the interaction of podoplanin with CLEC-2, or inhibits the activity of Src and/or Syk family kinases in combination with an additional agent has a synergistic effect in the treatment or prophylaxis of chronic inflammatory diseases, conditions or related pathologies. Thus, the use of the agent in combination with at Ieast one additional agent may further reduce inflammation and improve healing.

The agent may be administered when the subject has symptoms, for example during a relapse. The agent may be administered when the subject is asymptomatic. The agent may be administered when the subject is in remittal.

The inventors have observed that in conditions such as Crohn's disease that are associated with localised inflammation at key sites within a tissue, or in diseases with episodes of relapse and remission, the agent has an increased effect in the actively diseased/inflamed regions compared to non-involved or non-inflamed areas of the same tissue at the same or a different time. Without wishing to be bound by theory, the inventors believe that the administration of the agent may thus demonstrate efficacy in the treatment of disease during severe disease episodes and/or in localised sites. Thus, the agent may represent a suitable option for end-stage chronic inflammatory diseases, conditions or related pathologies where previous treatment has been ineffective.

In some embodiments, the agent is administered prior to diagnosis of the chronic inflammatory disease, condition or related pathology.

In some embodiments the agent is administered at a dose of between 0.1 pg/kg of body weight and 1 g/kg of body weight, depending upon the specific agent used. In some embodiments the agent is administered at a dose of at least 0.1 pg/kg of body weight, 0.2 pg/kg of body weight, 0,3 pg/kg of body weight, 0.5 pg/kg of body weight, 1 pg/kg of body weight, 5 pg/kg of body weight, 10 pg/kg of body weight, 50 pg/kg of body weight, 100 pg/kg of body weight, 150 Mg/kg of body weight, 200 pg/kg of body weight, 500 pg/kg of body weight, 1000 pg/kg of body weight, 2000 pg/kg of body weight or 5000 pg/kg of body weight. In some embodiments the agent is administered at a dose of no more than 50000 pg/kg of body weight, 25000 pg/kg of body weight, 10000 pg/kg of body weight, 7000 pg/kg of body weight, 5000 pg/kg of body weight, 2000 pg/kg of body weight, 1000 pg/kg of body weight, 500 pg/kg of body weight, 200 pg/kg of body weight, 150 pg/kg of body weight, 100 pg/kg of body weight, 50 pg/kg of body weight or 10 pg/kg of body weight, in some embodiments the agent is administered at a dose of between 10000 pg/kg of body weight and 0.5 g/kg of body weight, depending upon the specific agent used. In some embodiments the agent is administered at a dose of between 10000 pg/kg of body weight and 100000 pg/kg of body weight, depending upon the specific agent used, in some embodiments the agent is administered at a dose of between 0.1 g/kg of body weight and 0.5 g/kg of body weight, depending upon the specific agent used.

According to a second aspect of the invention there is provided the use of an agent that inhibits the interaction of podoplanin with CLEC-2 2 or inhibits the activity of Src and/or Syk family kinases in the manufacture of a medicament for the treatment and/or prevention of a chronic inflammatory disease, condition or related pathology in a subject.

According to a third aspect of the invention there is provided a composition comprising a therapeutically effective amount of an agent that inhibits the interaction of podoplanin with CLEC-2 or inhibits the activity of Src and/or Syk family kinases, wherein said therapeutically effective amount is sufficient to eliminate, reduce or prevent a chronic inflammatory disease, condition or related pathology.

As used herein, a "therapeutically effective amount" is an amount of the agent that inhibits the interaction of podoplanin with CLEC-2 or inhibits the activity of Src and/or Syk family kinases which, when administered to a subject, is sufficient to eliminate, reduce or prevent a chronic inflammatory disease, condition or related pathology. A therapeutically effective amount may also be an amount at which there are no toxic or detrimental effects, or a level at which any toxic or detrimental effects are outweighed by the therapeutic benefits. The composition may further comprise a pharmaceutically acceptable carrier, diluent or excipient A "pharmaceutically acceptable carrier" as referred to herein is any physio!ogica! vehicle known to those of ordinary skill in the art useful in formulating pharmaceutical compositions. A "diluent" as referred to herein is any substance known to those of ordinary skill in the art useful in diluting agents for use in pharmaceutical compositions. The agent may be mixed with, or dissolved, suspended or dispersed in the carrier, diluent or excipient. The composition may be in the form of a capsule, tablet, liquid, ointment, cream, gei, hydrogel, aerosol, spray, micelle, transdermal patch, liposome or any other suitable form that may be administered to a mammal suffering from, or at risk of developing a chronic inflammatory disease, condition or related pathology. The composition may comprise the agent at a concentration of up to ΙΟΟμηι.

Administration of the agent may be by any suitable route, including but not limited to, injection (including intravenous (bolus or infusion), intra-arterial, intraperitoneal, subcutaneous (bolus or infusion), intraventricular, intramuscular, or subarachnoidal), oral ingestion, inhalation, topical, via a mucosa (such as the oral, nasal or rectal mucosa), by delivery in the form of a spray, tablet, transdermal patch, subcutaneous implant or in the form of a suppository.

The agent may be administered as a single dose or as multiple doses. Multiple doses may be administered in a single day (e.g. 2, 3 or 4 doses at intervals of e.g. 3, 6 or 8 hours). The agent may be administered on a regular basis (e.g. daily, every other day, or weekly) over a period of days, weeks or months, as appropriate.

It will be appreciated that optimal doses to be administered can be determined by those skilled in the art, and will vary depending on the particular agent in use, the strength of the preparation, the mode of administration, the advancement or severity of the chronic inflammatory disease or condition and the cause of the chronic inflammatory disease or condition. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration. Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to establish specific formulations for use according to the invention and precise therapeutic dosage regimes.

in some embodiments, the composition comprises at least one additional agent. The additional agent may be selected from from nonsteroidal anti-inflammatory drugs (NSAIDs), immunosuppressants, bioiogicals, blood pressure controlling agents, glycaemia controlling agents or Ursodiol.

In some embodiments the at least one additional agent is selected from immunosuppressants or bioiogicals.

According to a further aspect of the invention there is provided a composition comprising a therapeutically effective amount of a combination of an agent that inhibits the interaction of podopianin with CLEC-2 or inhibits the activity of Src and/or Syk family kinases and at least one additional agent, wherein said therapeutically effective amount is sufficient to eliminate, reduce or prevent a chronic inflammatory disease, condition or related pathology.

In some embodiments, the composition comprises at least one additional agent. The additional agent may be selected from from nonsteroidal anti-inflammatory drugs (NSAIDs), immunosuppressants, ASA compounds, analgesics, bioiogicals, blood pressure controlling agents, glycaemia controlling agents or Ursodioi.

In some embodiments the at least one additional agent is selected from immunosuppressants or bioiogicals.

According to a fifth aspect of the invention there is provided a method for the treatment and/or prevention of a chronic inflammatory disease, condition or related pathology in a subject, the method comprising the administration of an agent that inhibits the interaction of podopianin with CLEC-2 or inhibits the activity of Src and/or Syk family kinases to the subject. The method may comprise the administration of a therapeutically effective amount of the agent. The method may comprise the administration of the agent when the subject has symptoms, for example during a relapse. The method may comprise the administration of the agent when the subject is asymptomatic. The method may comprise the administration of the agent when the subject is in remittance. in some embodiments the method comprises the administration of the agent prior to diagnosis of the chronic inflammatory disease, condition or related pathology.

The method may comprise administering a dose of the agent of between 0.1 pg/kg of body weight and 1 g/kg of body weight of the agent. In some embodiments the method comprises administering a dose of the agent of at least 0.1 Mg/kg of body weight, 0.2 pg/kg of body weight, 0.3 pg/kg of body weight, 0.5 pg/kg of body weight, 1 i-ig/kg of body weight, 5 pg/kg of body weight, 10 pg/kg of body weight, 50 pg/kg of body weight, 100 pg/kg of body weight, 150 pg/kg of body weight, 200 pg/kg of body weight, 500 pg/kg of body weight, 1000 pg/kg of body weight, 2000 pg/kg of body weight or 5000 pg/kg of body weight. In some embodiments the method comprises administering a dose of the agent of no more than 50000 pg/kg of body weight, 25000 pg/kg of body weight, 10000 pg/kg of body weight, 7000 pg/kg of body weight, 5000 pg/kg of body weight, 2000 pg/kg of body weight, 1000 pg/kg of body weight, 500 pg/kg of body weight, 200 pg/kg of body weight, 150 pg/kg of body weight, 100 pg/kg of body weight, 50 pg/kg of body weight or 10 pg/kg of body weight, !n some embodiments the method comprises administering a dose of the agent of between 10000 pg/kg of body weight and 0.5 g/kg of body weight. In some embodiments the method comprises administering a dose of the agent of between 10000 pg/kg of body weight and 100000 pg/kg of body weight. The method may comprise administering a dose of the agent of between 0.1 g/kg of body weight and 0.5 g/kg of body weight. The chronic inflammatory disease or condition may be chronic !iver disease. One known method for the treatment of chronic liver disease is liver transplantation. Thus, the agent may be administered before, during or after liver transplantation to said subject, in the context of liver transplantation, "before" will be understood to refer to prior to the start of surgery. "During" will be understood to refer to administration between the start and end of surgery. "After" will be understood to refer to the administration after the end of surgery. The agent may be administered no more than 30 minutes, no more than 1 hour, no more than 2 hours, no more than 3 hours, no more than 4 hours, no more than 6 hours, no more than 12 hours, no more than 24 hours, no more than 48 hours or no more than 72 hours before liver transplantation. The agent may be administered no more than 30 minutes, no more than 1 hour, no more than 2 hours, no more than 3 hours, no more than 4 hours, no more than 6 hours, no more than 12 hours, no more than 24 hours, no more than 48 hours or no more than 72 hours after liver transplantation, in some embodiments the agent is administered to the transplanted liver, or the site into which the transplanted liver will be placed. It will be appreciated that the administration of the agent before, during or after liver transplantation may reduce transplant rejection. Administration of the agent before, during or after liver transplantation may also improve the subject's recovery time and improve transplant integration and/or healing in the subject. Ail of the features described herein (including any accompanying claims, abstract and drawings) may be combined with any of the above aspects in any combination, unless otherwise indicated.

Detailed description of the invention

Embodiments of the invention will now be described by way of example and with reference to the accompanying Figures:

Figure 1 shows: Classic histological findings in Crohn's disease specimens. Chronic bowel inflammation has been chosen as an illustrative example of the aetiologies in which our proposed invention could operate. Haematoxylin and Eosin (H&E) stains were performed on bowei samples from patients with CD (Crohn's disease) who had undergone surgical resections at the Queen Elizabeth Hospital Birmingham. The images show some of the typical histological findings in patients with advanced CD. These include (A) typical mucosal inflammation and loss of normal mucosal architecture found in CD, (B) fissuring ulcers (U), (C) granulomas (G), and (D) transmural lymphoid aggregates (L) and scarring (S).

Figure 2 shows: Enhanced platelet sequestration and neutrophil recruitment to the bowl in Crohn's Disease patients. (A) Reduced numbers of activated platelets are seen in the systemic blood of patients with Crohn's disease. Platelets were labelled with anti- CD41-APC and anti~P-Selectin-FiTC. The percentage of activated piateiets (positive for both markers) was then assessed. Symbols indicate individual donors and paired IP- test confirmed statistical significance between the two groups (p~ 0.0174). This suggests that activated platelets are recruited to the diseased bowel. This is supported by the evidence in (B), where enhanced platelet sequestration is seen in the bowel of patients with active CD. Anti-CD41-APC antibody was used to stain endogenous platelets (yeilow) in tissues sections from normal and diseased (CD) bowel. Sections were counterstained with DAP! and images shown are at 50x original magnification. (C) Podoplanin is expressed by macrophages and vessels within diseased colon. Sections were stained using anti-human-podoplanin antibody in a standard DAB protocol. A representative image of diseased colon is shown. Both vessels within tissue (blue arrow) and tissue-resident macrophages (red arrow) are positive. (D) inhibition of integrins or P-selectin inhibited the adhesion of neutrophils to diseased bowel, but the use of podopianin-antibody promoted the adhesion of neutrophils to diseased bowel in these experments. Piateiets were isolated, labelled with CMTPX and added to tissue for 30 minutes. Neutrophils were isolated via density centrifugation and labelled with CMFDA, washed and resuspended to a concentration of 1x10 ⁶ . Platelets were washed off and neutrophils added to tissue for 30 minutes. Sections were fixed and mounted. Images were taken using multi-channei fluorescence imaging on the Zeiss Axio Scan Z1 microscope and processed using Zen Light Blue software. Corrected totai cell fluorescence (CTCF) was calculated using image J and Microsoft Excel to quantify amount of neutrophil binding per section in the presence or absence (control) of indicated blocking reagents. Data are mean ± SEM for n= 4 tissue donors and unpaired T tests were used to compare each group to controls.

Figure 3 shows: Podoplanin staining on non-diseased colon tissue and tissue fom patients with Crohn ^' s disease (CD). Frozen tissue sections from normal and Crohn's disease patients were stained using anti-podop!anin antibody (NZ1.3 eBioscience, UK, 1 pg/ml)] in an indirect peroxidase based staining protocol. Aii images were generated using standard brtghtfie!d imaging on the Zeiss Axio Scan Z1 microscope and processed using Zen Light Blue Software. Top panels show the stain observed at 10x or 25x magnification on tissue from two normal controls. Bottom panels show the staining at the same magnification for inacroscopically active diseased sections from two different patients with CD. Isotype matched control antibodies confirmed specificity of the staining (not shown).

Figure 4 shows: Use of ROD peptide or P-seiectin blocking antibody reduced platelet and neutrophil adhesion to colon sections, but use of anti-podoplanin blocking antibody increased platelet and neutrophil adhesion to colon sections. Functional antibodies (left labels on panels) were applied at various stages in the platelet and neutrophil adhesion assay as described in the methods. Images shown are taken from the same representative location within tissue from an actively diseased region of a Crohn's resection sample to allow direct comparison. DAPI (blue) was used as a nuclear counterstain. Platelets are indicated in red and neutrophils are shown in green.

Figure 5 shows: Mean CTCF for platelets in functional adhesion assay. Corrected Total Ceil Fluorescence (CTCF) was calculated using ImageJ and Microsoft Excel as described in the methods. Four tissue sections (2 active disease and 2 non-active disease) were used per group and blood was isolated from a single healthy donor. Graph shows mean ± SEM CTCF for CMTPX-labelled platelets in each group. Unpaired T-tests compare each group to the Controls, with P-values included on the graphs.

Figure 6 shows: Platelet and neutrophil CTCF in active (inflamed) versus non-active (non-inflamed) diseased tissue areas. CTCF calculations in the functional blocking experiment were analysed within each group, comparing CTCF values between active diseased tissue areas and non-active diseased areas.

Figure 7 shows: Changes in neutrophil (red) and platelet (green) adhesion in active versus non-active diseased tissue. Representative fields of view were selected from the same anatomical regions from an active (left hand images) and non-active (right hand images) diseased section from the same donor with CD to allow direct comparison. Fields of view were taken at 25x magnification in samples that were pretreated with indicated blocking agent. Again we saw increased numbers of neutrophils in the diseased bowel following application of podoplanin antibody.

Figure 8 shows; Podoplanin is upregulated on endothelial and immune cell populations in chronic liver injury. Chronic fibrotic liver disease is another illustrative example of an etiology where our invention may be beneficial. In particular we see a marked upregufation of podoplanin expression in human chronic liver disease.. (A) A normal human liver sample (from an organ donor) and (B) a chronically diseased liver specimen (from a patient transplanted for Primary Sclerosing Cholangitis) stained using a podoplanin antibody in a standard indirect immunochemical stain using DAB substrate. Positive staining is indicated by the brown stain on sections counterstained using haematoxylin. The identity of the podoplanin positive cells can be confirmed using fluorescent confoca! microscopy (pane! C) where CD31 + cells (vascular endothelium) (green) clearly express podoplanin (yellow), as well as CD45+ (leukocytes) (purple). Figure D further illustrates the upregulation of podoplanin (measured via qPCR) from human liver specimens with chronic liver disease.

Figure 9 (A) shows: Reduced fibrosis in mice deficient in Clec-2 (Cled bfl/PF4-Cre) compared to wi!dtype controls as evidenced by picosirus red stain (for collagen - indicated in red) in murine livers harvested after 8 weeks of biweekly intraperitoneal CCL4 (carbon tetrachloride) injections. This is accompanied by increased numbers of pro-resolution macrophages (CD11b ⁺ CD3 ^' Ly6c ^h ') in mice with CLEC-2-deficient platelets (B, left panel) and these animals have less hepatic injury measured by ALT in serum (B, right panel) after CCL4 induced fibrosis,

Figure 10 shows hepatic podoplanin levels determined by qPCR are increased in human patients with evidence of portal thrombosis.

Table 1 shows: Histological features that favour a diagnosis of either Crohn's disease or ulcerative colitis at biopsy.

Table 2 shows: Antibodies and reagents used for immunohistochemistry and functional assays

Examples

Platelets are fundamental players in liver pathobiology; driving inflammation, fibrosis, cancer and even aiding regeneration. However, the specific molecular basis of platelet activation in the context of liver inflammation and failure, and their contribution to extra- hepatic disease remains elusive.

The present inventors thus sought to explore the molecular basis of platelet activation in chronic inflammatory diseases of the liver and bowel.

Materials and methods

Mice

C57BL/6J mice were obtained from Harlan OLAC LTD or from in-house colonies. PF4Cre-CLEC-2 ^f|;fl mice are described in Finney et al., Blood, 2012 (119), 1747-1756. All strains of genetically-altered mice are on a C57BL/6J background. Control mice were matched by genetic background, age and sex. All mice were housed at the Biomedical Services Unit, University of Birmingham and used under procedure in accordance with UK Home Office guidelines.

Human tissue Ail Crohn's Disease tissue samples used in this project were collected from patients with Crohn's Disease undergoing bowel resections who gave written informed consent. Tissue samples were collected from theatre and then processed by the Pathology Department at Queen Elizabeth Hospital Birmingham (QEHB). Samples were classified into those obviously macroscopically "diseased" or "non-active disease" by the consulting Histopatho!ogist and small pieces of tissue in each category were taken for analysis Tissue samples were in the form of 1-2cm ³ samples and were snap frozen in liquid nitrogen and stored at -8Q°C for subsequent staining or functional assays. Consent was also gained for blood to be taken from the enrolled patients. Matched blood samples were taken from the CD patients for flow cytometric analysis. Other blood samples were taken from 5 CD patients at the weekly IBD clinic, and healthy donor from the Centre for Liver Research at the University of Birmingham's Institute of Biomedical Research.

Human liver was collected from patients in the liver transplantation programme at Queen Elizabeth Hospital in Birmingham. All samples were collected with written informed patient consent and under local ethical approvals. Norma! liver tissue was obtained from donor tissue that was surplus to requirement for transplantation, or deemed unsuitable for use. Diseased liver tissue was from explanted livers collected during transplantation surgery.

Immunohistology and confocal microscopy of preserved bowel or liver tissue sections from human donors or experimental mice

Liver and bowel tissue

Fresh tissues were snap-frozen or fixed in 4% Formaldehyde immediately upon removal from human donors or experimental animals. Sections were cut (5-10 μιτι) and either acetone-fixed and stored at -20C or stored at room temperature, respectively. Where indicated tissue sections were stained by Haematoxylin and Eosin using standard protocols for assessment of tissue morphology.

Tissue Staining

Frozen tissue sections were stained by immunohistochemistry to detect podoplanin, neutrophil elastase and CD68. IHC was performed in Tris buffer (pH 7.6). AH steps were performed in the dark. 1 x Casein, diluted using PBS from 10X Casein (Vector Laboratories, CA, USA), along with one drop of Horse Serum (Vector Laboratories, CA, USA) was added for 30 minutes to block non-specific staining. Slides were then incubated in a rocking moisture chamber at room temperature with the optimal concentration of primary antibody or control for variable times. Horse- radish peroxidase conjugated secondary antibodies were developed using Alkaline-phosphatase (ABCompiex, Vector Laboratories) and 3,3'-diaminobenzidine tetrahydrochloride. Slides were mounted in DPX and images acquired at x 20 or x10 magnification using a Leica CTR6000 microscope (Leica, Miiton-Keynes, UK), with Qcapture software. Low magnification images were acquired by a Car! Zeiss AxioScan.ZI Slide Scanner using a 3CCD colour 2MP Hitachi 1200x1600 HV-F202SCL camera. Images were analysed using Zen blue (2012) slide scan software.

Fluorescent confoca! microscopy was also performed on frozen tissue sections. Staining was performed in PBS + 1% FCS. Sections were incubated with primary and secondary antibodies for predetermined optimal periods at room temperature in the dark. Ceil nuclei were counterstained Hoechst 33342 (10|jg/mi for 2 minutes at room temperature). Slides were mounted using Prolong Gold Anti-fade reagent (invitrogen, Paisley, UK), and images were taken using either a x10, x40 or x63 magnification objective on a LSM510 laser scanning confocai microscope with a Zeiss AxioVert 100M (Zeiss, Germany) in conjunction with Zeiss LSM image software.

For functional adhesion assays using platelets and neutrophils binding to bowel tissue, Hoechst 33258 Dye (Sigma-Aldrich, MO, USA) was used as a nuclear counterstain to visualise the sections at the end of the experiment. The dye was diluted to a 1 :2000 dilution using PBS, with 200μ! added per section and incubated in the dark for 5 minutes before washing as before with TBS and 0.1 % Tween. Slides were mounted using one drop of Dako Fluorescent Mounting Medium (DAKO UK Ltd, UK) and coversiip and left in the dark at 4°C until imaging. Full details of the adhesion assays are supplied below.

Induction of liver injury in mice

Chronic hepatic inflammation was induced in mice using bi-week!y intraperitoneal injections of CCI ₄ (Sigma-Aldrich) for 8 weeks. CCI ₄ was diluted 1 :4 with mineral oil, and injected intraperitoneally into mice at a concentration of 1 ml/kg (control animals were treated with IP mineral oil alone). Where therapeutic antibody was administered, control mice received intraperitoneal injections of PBS only.

Platelet isolation and labelling

Peripheral blood samples were collected from donors. Within 30 minutes of venepuncture samples were centrifuged at 300G for 5 minutes. Using a Pasteur pipette, the plasma and buffy coat was removed and washed with PBS. A further 5 minute spin at 1800G was undertaken, the supernatant removed and platelets resuspended in 1ml PBS containing 5m M glucose and 6mM theophylline (both Sigma).

Where necessary for adhesion assays, platelets were fluorescentiy labelled using Cell Tracker Red CMPTX (Thermo Fisher Scientific, UK). CMPTX was diluted to 5mM concentration using Dimethyl sulfoxide (Sigma), and added to platelets at a 5μΜ concentration. Cells were left to incubate in the dark at 37°C for 45 minutes. Excess dye was removed via centrifugation at 1300G for 5 minutes. Platelets were resuspended in 1ml of PBS/glucose/theophyliine solution,

Binding of exogenous!y added platelets and neutrophils to tissue

Platelets were isolated and incubated with Cell Tracker Red CMTPXZ as above. Neutrophils were isolated from whole blood via density centrifugation. 5ml of blood was layered onto 2.5ml Histopaque 1119 and 2.5ml Histopaque 1077 (both Sigma) and spun at 2500RPM for 25 minutes. Neutrophils were collected and placed into a centrifugation tube with 0.5 ml plasma. Cells were then washed in PBS for a further 5 minutes at 1000RPM. Ceil pellets were re-suspended in 1m! PBS and 1 μΙ Cell Tracker Green CMFDA (Thermo Fisher Scientific, UK) at a concentration of 5μΜ was added. Cel!s were incubated in darkness at 37°C for 30 minutes. The neutrophils were then washed and resuspended in 1 ml PBS before being counted using a haemocytometer and resuspended to a concentration of 1x10 ⁶ neutrophils/ml.

Tissue sections were thawed and non-specific staining was blocked using one drop of Serum-Free Protein Block (Dako UK Ltd, UK). In one group of sections, 200μΙ of 1x10 ^a platelets/ml were added and incubated in darkness at humidified room temperature for 2 hours. After incubation, excess platelets were washed off using TBS. 200μ! of neutrophils were then added and left to incubate for the same amount of time and conditions.

In another group of sections, sections were incubated solely with neutrophils as described above. After incubation the sections were fixed in a bath of ethanol for 5 minutes, before nuclear counterstain with Hoechst Dye. Cells were mounted using Dako Fluorescent Mounting Medium. Matched tissue sections were stained with Hoechst Dye alone as a control. The following functional groups were used:

1. Control: Platelets and neutrophils added without blocking antibodies.

2. Anti-P-selectin: 200μΙ P-Selectin antibody applied onto tissue for 20 minutes after platelet incubation, prior to incubation with neutrophils.

3. RGD Peptide: Platelets were treated with RGD peptide at a concentration of 10pg/mi for 20 minutes. Platelets were washed in PBS and added to tissue. 4. Anti-podopianin: 200μ! human anti-podopianin functional grade antibody was added to tissue sections for 20 minutes at a concentration of 1 pg/ml prior to incubation with platelets.

Flow cytometric analysis of platelets

Platelets were isolated from whole blood within an hour of venepuncture and counted. 300 μί of the platelet suspension at a concentration of 1x10 ⁸ platelets/ml was added to a standard Fortessa FACS tube {BD Biosciences, US). Compensation tubes were created using CD4 FITC and GD14 APC controls.

20μ! of anti-P-se!ectin-FlTC and anti-CD41-APC antibodies were added to samples. Samples were incubated at 4°C in the dark for 30 minutes. Following incubation samples were washed using 2ml PBS and centrlfuged at 800G for 5 minutes. Supernatant was removed and 300μΙ of PBS was added. Samples were stored in darkness at 4°C until flow cytometric analysis. Further details of antibodies used in flow cytometry can be found in Table 2.

Expression of GD41 and P-Selectin on platelets was analysed using the LSR Fortessa X-20 flow cytometer (BD Biosciences, US), for both blood from patients with CD and healthy donors. All samples were analysed within one hour of labelling protocol. Compensation was first performed against CD14 APC and CD4 FITC. Platelet populations were manually gated by analysing side and forward light scatter, together with CD41 and P-Selectin expression. Analysis of surface marker expression was carried out using F!owJo Version 7,6 (TreeStar).

Quantification of liver-infiltrating immune ceils

Mouse livers were harvested after the animal was euthanized under deep sedation after cardiac puncture. The organs were then weighed and dissociated In a gentleMACS C Tube (Miltenyi Biotec). The resulting immune cells were then purified using an Optiprep gradient (Sigma) and analysed by flow cytometry. Inflammatory ceils were gated as a CD4+ cell population (anti-CD45-PerCP-Cy5. , clone 30-F11 ; BD Biosciences), and non-viable cells were excluded using a Zombie NIR ^{| M} Fixable Viability kit (BioLegend). Lymphocytes were characterised based on staining using a cocktail of anti-CD3 Pacific blue (clone 500A2); anfi-CD4-PE (clone RM4-5): anti- CD8a-APC (clone 53-6.7); anti-CD19-APC-Cy7 or anti-CD19-BV510 (both done 1 D3); and anti-NK1.1-FITC (clone PK136) or DX5-FITC (clone DX5) abs (all from BD Biosciences). The monocyte subsets were identified by staining with anti-CD1 1 b-PE (clone M1/70; BD Biosciences) and anti-Ly6c ^w . Clec1 bfl/flPF4-Cre mice (KO) exhibit reduced iiver injury and. Absolute cell counts were determined with AccuCheck Counting Beads (invitrogen), and the number of cells was normalised to the total liver weight. Data were analysed using a CyAn ADP flow cytometer (Beckman Coulter) or a BD LSRH using Summit version 4.3 or FlowJo version 10.0.7 software where appropriate.

Biochemical I!ver injury assays

Serum was isolated from whole blood and levels of liver-specific enzymes AST and ALT were measured on a clinical autoanalyser, according to standard protocois in the Biochemistry Department at the Birmingham Women's Hospital, Birmingham, UK .

Statistical tests

All statistical tests were performed using the inbuilt analysis tools in GraphPad Prism 6, Paired data comparing either controls versus recruited patients, or areas of active disease compared to non-active disease, were analysed using either paired or unpaired T-tests.

Results

Enhanced platelet sequestration and neutrophil recruitment to the bowel in Crohn's Disease patients; podop!anin may be linked to inflammatory cell recruitment

We decided to investigate the role of platelets in Crohn's disease patients. Histological features used to identify Crohn's disease in patients are listed in Table 1. Reduced numbers of CD41 +/P-Sel+ activated platelets were seen in the systemic blood of patients with Crohn's disease, compared to healthy controls (Figure 2A). Enhanced platelet sequestration was, however, observed in the bowel of patients with active Crohn's disease (Figure 2B).

We next sought to study the expression of Podoplanin, the only known naturally occurring CLEC-2 ligand in the diseased colon of Crohn's disease patients. CLEC-2 (C-type lectin-!ike receptor 2} is a type II transmembrane protein which is expressed on platelets. Platelet-based CLEC-2 mediates platelet activation on meeting its ligand Podoplanin, a type I transmembrane O-glycoprotein. We found that podoplanin was expressed by macrophages and vessels within diseased colon (Figure 2C). Both vessels within tissue (blue arrow) and tissue-resident macrophages (red arrow) were positive for podoplanin.

The adhesion of platelets and /or neutrophils to diseased bowel was investigated using a functional adhesion assay. The assay was in the presence or absence of the blocking reagents, RGD peptide, anti-P-selectin or anti-podoplanin antibody (see methods). Platelets were isolated, labelled with CMTPX and added to isolated diseased tissue for 30 minutes. Neutrophils were isolated via density centrifugation, labelled with CMFDA then washed and resuspended to a concentration of 1 x10 ⁶ . Platelets were washed off the diseased tissue and neutrophils added to the tissue for 30 minutes. Sections were fixed and mounted and images were taken using multi-channel fluorescence imaging. Corrected Total Cell Fluorescence (CTCF) was calculated using image J and Microsoft Excel to quantify the amount of neutrophil binding per section in the presence or absence of the above-noted blocking agents. The addition of either the RGD peptide or anti-P-selectin antibody reduced neutrophil binding to the diseased tissue (Figure 2D). Thus the adhesion of neutrophils to diseased bowel is supported by integrins and P- selectin as expected. In contrast however, pretreatment of tissue sections with antibody-directed against podoplanin resulted in increased neutrophil adhesion to diseased tissue. Podoplanin is upregulated in the bowel during Crohn's disease

We further confirmed the localisation of podoplanin within the injured bowel by looking at material from additional patients with CD.

Podoplanin was present In sections from Crohn's disease (CD) patients. Little staining was detected in healthy control specimens (Figure 3). increased staining was observed in active-disease areas of CD compared to non-active diseased sections, in particular the anatomical appearance and localisation of structures expressing podoplanin was suggestive of macrophages. Positive staining was also observed on vascular-type structures. Isotype-matched control staining confirmed there was no non-specific background staining (not shown).

Use of RGD peptide or P-selectin blocking antibody reduced piateiet and neutrophil adhesion to co!on sections, but use of anti-podoplanin blocking antibody increased platelet and neutrophil adhesion to colon sections

The adhesion of neutrophils and/or platelets to Crohn's disease tissue sections was further investigated. The method was carried out as per Figure 2D. Representative images are shown in Figure 4, and a graph demonstrating the adhesion of platelets is shown in Figure 5. While the addition of the integrin blocking agent RGD peptide, or anti-P-selectin, reduced the adhesion of platelets and neutrophils to the diseased tissue (Figures 4 and 5), the use of the anti-podoplanin blocking antibody surprisingly further increased the adhesion of both platelets and neutrophils to the diseased tissue sections, relative to all other conditions.

Platelet and neutrophil adhesion is enhanced in active disease sections and the effect of anti-podopianin is further increased in active disease sections

Neutrophil and platelet adhesion, in a functional assay as described above, was then compared between actively-diseased and non-active disease sections from Crohn's patients. Adhesion was generally increased in actively-diseased compared to non- active disease sections, for both neutrophils and platelets (Figures 6 and 7). The most prominent difference was observed for platelets in the anti-podoplanin blocking group, with fluorescence at nearly 3 x 10' in active disease sections compared to approximately 2 xlO ⁷ in non-active disease sections.

Podoplanin in chronic liver injury

Chronic fibrotic liver disease is another illustrative example of an etiology where our invention may be beneficial. Podoplanin expression was upregu!ated in fibrotic human livers (from patients with Primary Sclerosing Cholangitis) (Figure 8B) compared to normal livers (Figure 8A) Podoplanin expression (yellow) was found to be located on the vascular endothelium of the liver (see green GD31 expression), as well as CD45+ leukocytes (purple) , Figure 8C .

We have confirmed the therapeutic potential of interfering with the CLEC-2/podoplanin axis in chronic disease using a murine model of liver inflammation and fibrosis induced by chronic (8 week) exposure to carbontetrachioricle (CCL4). Picosirus red stain (marker for collagen) was used to assess liver damage in a mouse model of chronic liver injury. Wild -type or Clec1bfl/flPF4-Cre mice (knockout of CLEC-2 only in piatelets) mice were given bi-weekly intraperitoneal injections of calcium tetrachloride, a known agent of liver injury. After 8 weeks, livers were isolated and the chronic damage (in the form of fibrotic liver injury) was assessed. Fibrotic liver injury was visibly reduced in the Cled bfl/flPF4-Cre mice compared to the WT controls (Figure 9A).

Furthermore this is accompanied by increased numbers of pro-resolution macrophages (CD11 b ⁺ CD3 ^" Ly6c ^hi ) in mice with CLEC-2-deficient platelets (Figure 96, left panel) and these animals have less hepatic injury measured by ALT in serum (Figure 9B, right panel) after CCL4 induced fibrosis.

Relevance to Liver transplantation

Figure 10 shows that Hepatic podoplanin levels are increased in human patients with evidence of porta! thrombosis.

The data represent qPCR values for podoplanin expression in whole liver RNA expressed relative to housekeeping gene expression. Livers were collected at time of transplantation for end-stage liver disease, Patient history and clinical records were analysed to permit stratification into those with/without evidence of portal vein thrombosis. Symbols indicate individual patient values and ^* indicates significant difference between groups.

This figure suggests that in those patients with evidence of portal vein thrombosis, there was a significantly higher hepatic expression of podoplanin. Thus inhibition of podopianin in this patient group may be beneficiai in terms of preventing portal thrombosis and serve to facilitate liver transplantation and/or minimize or reduce rejection..

Discussion

In this study we show enhanced recruitment of platelets and neutrophils to the bowel of Crohn's disease patients.

We also observed upregulated podopianin expression In chronic inflammatory disease tissue (Crohn's disease and Primary Sclerosing Cholangitis). Podopianin upregulation was colocalised with areas of active injury and inflammation and was most marked in chronic liver disease. Use of podoplanin-targeting antibodies modified the adhesion of immune ceils in Crohn's disease bowel tissue sections. This effect was localised to active-diseased sections of the colon compared to non-active disease sections.

We also report reduced liver damage in a platelet-specific CLEC-2 deficient mouse model of chronic liver injury, as assessed by fibrosis and ALT levels. This may relate to our observation that the CLEC-2 deficient mice also exhibited enhanced infiltration of restorative macrophages into the liver upon injury in comparison to wild-type mice.

These findings demonstrate the association of the CLEC-2 podopianin interaction with chronic inflammatory disease, and define potential mechanisms whereby inhibition of the CLEC-2-podoplanin interaction could potentially reduce or modify the symptoms of chronic inflammatory disease and possibly assist with liver transplantation.