FIELD OF THE INVENTION

The present invention relates to methods, kits, and compositions for: i) detecting the level of 3-bromotyrosine in a sample that has been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine, and/or ii) detecting the level of 4-O-glucuronide-3-bromotyrosine, and/or the combined level of both 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine, in a sample that has not been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine. In certain embodiments, such detected levels are used to: i) identify the presence, severity, or risk of an eosinophilic disorder (e.g., asthma or a TH2-high eosinophilic disorder); ii) identify therapy effective for treating asthma or an eosinophilic disorder; or iii) identify patients suitable for treatment with therapeutic agents targeted to asthma or an eosinophilic disorder.

BACKGROUND

Asthma affects a wide spectrum of individuals with diverse immunological and biochemical endotypic responses that lead to a variety of asthma phenotypes. Despite the recognized heterogeneity of asthma, treatment is prescribed equally to all patients in a uniformly applied stepped-care approach. Current practice relies on subjective symptoms, asthma control tests, or measure of airflow obstruction to add on, or step down therapy. This approach is post-hoc, and often results in under- and/or over-treatment. The morbidities and costs associated with this common disease and the lack of ability to finely tune care provide the imperative to develop personalized medicine approaches aimed at underlying mechanisms. Quantifiable non-invasive biomarkers that are informative for asthma control and airway inflammation are needed for personalized asthma treatment plans and will be essential in planning therapies and assessing efficacy of biologics such as anti-IgE, anti-IL5, and others. But, in the absence of appropriately sensitive tests to identify responder endotypes, patients will not fully realize the benefits of these new therapies.

Eosinophils in blood and sputum are used as cellular biomarkers of atopic, or TH2-high asthma. Eosinophils are present in increased numbers in airways of atopic asthmatics and produce much more ROS than cells found in healthy lungs. Early studies showed that the amount of ROS generated by eosinophils is directly correlated to the severity of hyperreactivity in asthma patients. Experimental exposure of atopic asthmatics to allergen, or asthma exacerbations, leads to even greater amounts of superoxid. Studies 30 years ago showed heterogeneity among eosinophils within asthma. Hypodense eosinophils were more active than normodense eosinophils, had twice as much eosinophil peroxidase activity, and comprised ˜20-80% of circulating eosinophils in asthma. The hypodense eosinophil could be produced by exposure to IL-5 in vitro or in response to antigen in vivo. Unfortunately, its measure was laborious, and so clinicians turned to total blood eosinophils as a surrogate of the hypodense eosinophil, although it was recognized that asthma severity was better reflected by the numbers of hypodense eosinophils.

SUMMARY OF THE INVENTION

The present invention relates to methods, kits, and compositions for: i) detecting the level of 3-bromotyrosine in a sample that has been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine, and/or ii) detecting the level of 4-O-glucuronide-3-bromotyrosine, and/or the combined level of both 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine, in a sample that has not been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine. In certain embodiments, such detected levels are used to: i) identify the presence, severity, or risk of an eosinophilic disorder (e.g., asthma or a TH2-high eosinophilic disorder); ii) identify therapy effective for treating asthma or an eosinophilic disorder; or iii) identify patients suitable for treatment with therapeutic agents targeted to asthma or an eosinophilic disorder.

In some embodiments, provided herein are methods of testing a sample comprising: a) testing a first sample from a subject to determine the level of 3-bromotyrosine, wherein the first sample has been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine; and/or b) testing a second sample from the subject to determine: A) the level of 4-O-glucuronide-3-bromotyrosine, and/or B) the combined level of both 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine, wherein the second sample has not been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine.

In particular embodiments, provided herein are methods of identifying the presence, severity, risk of, or risk of exacerbation of asthma or an eosinophilic disorder in a subject comprising: a) performing at least one of the following: i) testing a first sample from a subject to determine the level of 3-bromotyrosine, wherein the first sample has been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine; and/or ii) testing a second sample from the subject to determine: A) the level of 4-O-glucuronide-3-bromotyrosine, and/or B) the combined level of both 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine, wherein the second sample has not been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine; and b) identifying the presence, severity, risk of, or risk of exacerbation of asthma or an eosinophilic disorder in the subject based on: i) an elevated level of the 3-bromotyrosine in the first sample compared to a control, ii) an elevated level of 4-O-glucuronide-3-bromotyrosine in the second sample compared to a control; and/or iii) an elevated combined level of 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine in the second sample compared to a control.

In certain embodiments, provided herein are methods of treating a subject comprising: a) performing at least one of the following: i) identifying a subject as having an elevated level of 3-bromotyrosine in a first sample from the subject which was tested to determine the level of 3-bromotyrosine, wherein the first sample had been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine; and/or ii) identifying a subject as having an elevated level of 4-O-glucuronide-3-bromotyrosine in a second sample, and/or an elevated combined level of both 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine in the second sample, wherein the second sample was tested to determine the level of 4-O-glucuronide-3-bromotyrosine or both 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine, and wherein the second sample had not been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine; and b) treating the subject with a therapeutic agent used to treat asthma or an eosinophilic disorder.

In some embodiments, provided herein are methods of screening a candidate therapeutic agent comprising: a) performing at least one of the following: i) testing a first sample from a subject to determine the level of 3-bromotyrosine, wherein the first sample has been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine; and/or ii) testing a second sample from the subject to determine: A) the level of 4-O-glucuronide-3-bromotyrosine, and/or B) the combined level of both 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine, wherein the second sample has not been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine; and b) performing at least one of the following after a subject has received a candidate therapeutic agent for asthma and/or an eosinophilic disorder: i) testing a third sample from a subject to determine the level of 3-bromotyrosine, wherein the third sample has been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine; and/or ii) testing a fourth sample from the subject to determine: A) the level of 4-O-glucuronide-3-bromotyrosine, and/or B) the combined level of both 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine, wherein the fourth sample has not been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine. In further embodiments, the method further comprise: c) identifying the candidate therapeutic agent as efficacious based on: i) a decrease in the level of the 3-bromotyrosine from the first sample to the third sample, ii) a decrease in the level of the 4-O-glucuronide-3-bromotyrosine in the fourth sample compared to the second sample; and/or iii) a decrease in the combined level of 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine in the second sample compared to the fourth sample. In further embodiments, the methods further comprise: administering the efficacious therapeutic agent after step c). In other embodiments, the methods further comprise c) identifying the candidate therapeutic agent as non-efficacious based on: i) no decrease in the level of the 3-bromotyrosine from the first sample to the third sample, ii) no decrease in the level of the 4-O-glucuronide-3-bromotyrosine in the fourth sample compared to the second sample; and/or iii) no decrease in the combined level of 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine in the second sample compared to the fourth sample.

In some embodiments, provided herein are compositions, systems, and kits comprising: a) a sample from a subject having, or suspected of having, asthma and/or a eosinophilic disorder; and b) a de-glucuronidation agent. In certain embodiments, the de-glucuronidation agent comprises an enzyme that catalyzes removal of a glucuronide moiety (e.g., β-glucuronidase), an acid, or a base (e.g., to promote hydrolysis).

In certain embodiments, provided herein are compositions, systems, and kits comprising: a) a sample from a subject having, or suspected of having, asthma and/or a eosinophilic disorder; and b) a detection agent configured to bind to 4-O-glucuronide-3-bromotyrosine. In certain embodiments, the detection agent comprises an antibody or antigen binding portion thereof directed against 4-O-glucuronide-3-bromotyrosine. In further embodiments, the detection agent is detectably labeled (e.g., with a fluorophore or chromophore).

In some embodiments, provided herein are systems and kits comprising: a) a report that indicates a subject has been: i) identified as having an elevated level of 3-bromotyrosine in a first sample from the subject which was tested to determine the level of 3-bromotyrosine, wherein the first sample had been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine; and/or ii) identified as subject having an elevated level of 4-O-glucuronide-3-bromotyrosine in a second sample, and/or an elevated combined level of both 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine in the second sample, wherein the second sample was tested to determine the level of 4-O-glucuronide-3-bromotyrosine or both 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine, and wherein the second sample had not been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine; and b) a therapeutic agent used to treat asthma or an eosinophilic disorder.

In some embodiments, provided herein are compositions, systems, and kits comprising: a) at least one of the following internal standards: i) stable isotope labeled 3-bromotyrosine molecules, and/or ii) stable isotope labeled 4-O-glucuronide-3-bromotyrosine molecules; and b) at least one of the following: i) a de-glucuronidation agent (e.g., an enzyme that catalyzes removal of a glucuronide moiety, acid, or base); ii) a detection agent configured to bind to 4-O-glucuronide-3-bromotyrosine; iii) a sample from a subject having, or suspected of having, asthma and/or a eosinophilic disorder. In particular embodiments, the stable isotope labeled 3-bromotyrosine molecules comprise 13C6 labeled 3-bromotyrosine (see,FIG. 12A). In other embodiments, the stable isotope labeled 4-O-glucuronide-3-bromotyrosine molecules comprise 13C6 labeled 4-O-glucuronide-3-bromotyrosine (see,FIG. 12B). In some embodiments of the kits and systems, all or at least some of the reagent are located in separate containers (e.g., which are further packaged in a container, such as a box or bag). In other embodiments, all or most of the recited components are present in the composition, or kit, or system.

In some embodiments, the first and/or second sample is from a subject with, or suspected of having, asthma and/or an eosinophilic disorder. In other embodiments, the first sample has been treated with a de-glucuronidation agent (e.g., an enzyme that catalyzes removal of a glucuronide moiety, an acid, or a base) or procedure (e.g., acid or base for hydrolysis, optionally in the presence of heat). In some embodiments, the de-glucuronidation agent comprises β-glucuronidase. In particular embodiments, the testing the first and/or second sample comprises performing a detection method selected from: mass spectrometry, an immunological assay, HPLC-UV/VIS, LC-Electrochemical detection, or LC-Ampimetric detection method. In some embodiments, the first sample and the second sample originate from the same initial sample taken from the subject. In other embodiments, the first sample and the second sample originate from different initial samples taken from the subject (e.g., one is a urine sample and one is a serum sample; or one is a urine sample at a first time and the one is a urine sample at a different time). In certain embodiments, the first and/or second samples comprises a sample type selected from: urine, saliva, serum, plasma, tissue biopsy, and whole blood.

In some embodiments, the presence, severity, risk of, or risk of exacerbation of asthma is identified in the subject. In other embodiments, the presence, severity, risk of, or risk of an eosinophilic disorder is identified in the subject.

In certain embodiments, the subject, prior to step a) is identified as having or being at risk of asthma and/or an eosinophilic disorder. In further embodiments, the methods herein comprise: c) treating the subject with a therapeutic agent used to treat asthma or an eosinophilic disorder. In other embodiments, the methods further comprise: c) generating, transmitting, and/or graphically displaying (e.g., on paper or on a computer screen) a report that indicates the 3-bromotyrosine levels from the first sample, and/or the 4-O-glucuronide-3-bromotyrosine from the second sample, and/or the combined level of both 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine from the second sample, are elevated compared to corresponding controls, and that the subject is in need of a therapeutic agent used to treat asthma or an eosinophilic disorder. In additional embodiments, the methods herein further comprise: c) generating, transmitting, and/or graphically displaying (e.g., on paper or on a computer screen) a report that indicates the 3-bromotyrosine levels from the first sample, and/or the 4-O-glucuronide-3-bromotyrosine from the second sample, and/or the combined level of both 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine from the second sample, are elevated compared to corresponding controls, and that the subject has or is at risk of asthma and/or an eosinophilic disorder. In other embodiments, the methods herein comprise: c) characterizing the subject as having asthma and/or an eosinophilic disorder, based on finding elevated levels of the 3-bromotyrosine levels from the first sample, and/or the 4-O-glucuronide-3-bromotyrosine from the second sample, and/or the combined level of both 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine from the second sample.

In some embodiments, the first and/or second sample is from a subject with, or suspected of having, asthma and/or an eosinophilic disorder. In other embodiments, the first sample has been treated with a de-glucuronidation agent (e.g., acid, base, or an enzyme that catalyzes removal of a glucuronide moiety) and/or de-glucuronidation procedure (e.g., heating the sample in presence of acid or base). In certain embodiments, the de-glucuronidation agent comprises β-glucuronidase.

In some embodiments, the testing the first and/or second sample comprises performing a detection method selected from: mass spectrometry, an immunological assay, HPLC-UV/VIS, LC-Electrochemical detection, or LC-Ampimetric detection method. In other embodiments, the first sample and the second sample originate from the same initial sample taken from the subject. In additional embodiments, the first sample and the second sample originate from different initial samples taken from the subject. In additional embodiments, the first and/or second samples comprises a sample type selected from: urine, saliva, serum, plasma, tissue biopsy, and whole blood.

In certain embodiments, the methods herein further comprise informing the subject that they have asthma and/or an eosinophilic disorder, the severity of the asthma and/or the eosinophilic disorder, and/or the risk of exacerbating the asthma and/or the eosinophilic disorder. In particular embodiments, the severity of asthma includes: i) declining lung function, or ii) stable, non-declining lung function.

In particular embodiments, the control level or combined control level is determined or pre-determined from: i) a sample from the subject when not suffering from symptoms of asthma or an eosinophilic disorder, ii) a sample from a person, or sample from a plurality of people, without asthma or an eosinophilic disorder, iii) samples from the general population. In certain embodiments, the candidate therapeutic agent herein is selected from the group consisting of: Mepolizumab, Reslizumab, Benralizumab, Lebrikizumab, Tralokinumab, pitrakinra, Dupilumab, a leukotriene receptor antagonist (LTRA), a steroidal asthma drug, a non-steroidal asthma drug, a corticosteroid, prednisone, and budesonide. In other embodiments, the identifying is based on viewing, hearing, or receiving a paper or electronic report.

