Methods for detecting and / or quantifying cross-links formed by transglutaminase

Abstract

The present invention relates to a method for detecting crosslinks formed by transglutaminase, including N'N' bis(γ-glutamyl)-polyamine and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide crosslinks in a sample, comprising a step of digesting proteins present in a biological sample using an enzyme immobilized on beads and detecting said crosslinks in the biological sample by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The method also makes it possible to determine the activity of transglutaminase and to diagnose diseases associated with transglutaminase activity and to evaluate treatments.

Description

[Technical field]

[0001] The present invention relates to a method for detecting crosslinks formed by transglutaminase, including N'N' bis(γ-glutamyl)-polyamine and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide crosslinks in a sample, comprising a step of digesting proteins present in a biological sample using an enzyme immobilized on beads and detecting said crosslinks in the biological sample by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The method also makes it possible to determine the activity of transglutaminase and to diagnose diseases associated with transglutaminase activity and to evaluate treatments.

[0002] Introduction The ability to detect and quantitate crosslinks containing N'N' bis(γ-glutamyl)-polyamine and / or ε-(γ-glutamyl)-lysine (epsilon(γ-glutamyl)-lysine) dipeptide / isopeptide crosslinks (see Figure 1) in biological samples as a measure of activity of transglutaminases (TGs), such as transglutaminase 2 (TG2), has significant analytical and clinical potential. However, this remains a significant challenge.

[0003] Transglutaminases comprise a family of structurally related enzymes that catalyze calcium-dependent post-translational modifications of various proteins via transamidation reactions to form isopeptide bonds that result in protein crosslinks, for example between glutamate and lysine residues. Thus, TGs form (γ-glutamyl)-polyamine bonds (such as N'N' bis(γ-glutamyl)-polyamine dipeptide crosslinks and ε-(γ-glutamyl)-lysine isopeptide bonds) within and between proteins. TG2 is one member of this family in humans, expressed and secreted in all cellular compartments. TG2 has been shown to play an important role in many human diseases, including fibroproliferative diseases such as progressive kidney disease and liver cirrhosis. Fibrosis is a process associated with abnormal wound healing. TG2 contributes to profibrotic events, in part due to its role in crosslinking extracellular matrix proteins, making proteins increasingly protease resistant. Other human TGs include factor XIIIa, which is involved in the formation of fibrin clots; TG1 (keratinocyte TG), TG3 (epidermal TG) and TG5, which are involved in the terminal differentiation of keratinocytes in the skin; TG4 (prostate TG2), which is thought to have a role in stabilizing the blood vessel wall; and TG6, which is involved in neuronal function and membrane integrity, and inhibition of TG6 causes ataxia.

[0004] Several methods have been explored to detect crosslinks in tissues and other biological samples, but most have been flawed in some way. TGs can crosslink a wide range of substrate proteins at multiple sites, both intra- and inter-protein. This heterogeneity of crosslinks has hampered attempts to generate antibodies suitable for detecting crosslinks. Proteomic approaches, for example using LC-MS / MS, have also not progressed. These methods require defined sequence information, and the heterogeneity of the crosslinks means that individual peptides containing the crosslinks are present in extremely low amounts, preventing detection.

[0005] Most prior art approaches to detect crosslinks rely on amino acid analysis. These methods are based on hydrolyzing proteins down to complex amino acids and leaving the crosslinked dipeptides / isopeptides, e.g., ε-(γ-glutamyl)-lysine (γ-Glu-ε-Lys) and / or N'N' bis(γ-glutamyl)-polyamine isopeptides / dipeptides intact. The isopeptide / dipeptide signal is then a composite signal from all crosslinks in the proteins in the biological sample. Since ε-(γ-glutamyl)-lysine dipeptides or N'N' bis(γ-glutamyl)-polyamine dipeptides are not stable under extreme acid hydrolysis, enzymatic digestion approaches have been used. In a typical method, protein samples were subjected to exhaustive sequential digestion using proteases that do not cleave (γ-glutamyl)-polyamine or ε-(γ-glutamyl)-lysine dipeptide bonds, such as trypsin and subtilisin, followed by detection of dipeptides using classical amino acid analysis with chromatography optimized to resolve minor dipeptide peaks from major peaks of common amino acids. However, these methods suffer from major limitations, including the inability to resolve dipeptides from common amino acids, preventing accurate quantification. Furthermore, milligram quantities of protease were required for complete digestion, often exceeding the total protein substrate in a given sample. Enzymes had to be added sequentially to allow optimal rates and to adapt buffer conditions, meaning that each protease could be a substrate for the next. This significantly altered the reaction rate, digestion efficiency and the resulting chromatograms, again preventing accurate quantification.

[0006] The present invention uses an approach based on the principle of complete digestion and release of cross-linked dipeptides / isopeptides such as N'N' bis(γ-glutamyl)-polyamine dipeptide and / or ε-(γ-glutamyl)-lysine dipeptide, but addresses major shortcomings of the prior art. The inventors used improved biological sample preparation, innovative immobilized enzyme technology that allows efficient digestion without enzyme contamination, and LC-MS / MS detection based on multiple reaction monitoring to provide a highly specific, sensitive and reproducible assay. This approach represents a significant advance in the ability to detect and quantify cross-links formed by transglutaminase, including N'N' bis(γ-glutamyl)-polyamine and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide cross-links in biological samples. Summary of the Invention

[0007] The present invention provides a method for detecting crosslinks formed by transglutaminase (TG) in a biological sample, comprising a) digesting proteins present in the biological sample using an enzyme immobilized on beads, and b) detecting crosslinks formed by TG in the biological sample by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Also provided is a method for determining the activity of TG, preferably transglutaminase 2 (TG2), in a subject, comprising a) digesting proteins present in a biological sample from the subject using an enzyme immobilized on beads, and b) detecting crosslinks formed by TG in the biological sample by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0008] In some illustrative but non-limiting examples, the method may include a) concentrating proteins present in a biological sample by precipitating the proteins by treatment with trichloroacetic acid and separating the precipitated proteins by centrifugation, b) digesting the proteins using enzymes immobilized on beads, where the enzymes include proteinase K, pronase, prolidase, leucine aminopeptidase and carboxypeptidase Y, to generate a mixture comprising free amino acids and dipeptides / isopeptides, and c) detecting and / or quantifying the amount of crosslinks formed by TG in the biological sample by LC-MS / MS. Preferably, the crosslinks formed by TG comprise N'N'bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide.

[0009] Also provided herein is an in vitro method for (i) diagnosing disease in a subject or (ii) stratifying the severity of disease in a subject, comprising: a) using an enzyme immobilized on beads to digest proteins present in a biological sample from a subject; and b) detecting crosslinks formed by TG in the biological sample by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The presence of crosslinks formed by TG in the biological sample may indicate that the subject has a disease. Typically, the higher the amount / number of crosslinks formed by TG in the biological sample, the more severe the disease and / or the worse the prognosis of the disease.

[0010] Also provided herein is an in vitro method for monitoring disease progression in a subject, comprising: a) digesting proteins present in a biological sample from a subject using an enzyme immobilized on beads; b) detecting crosslinks formed by TG in the biological sample by liquid chromatography-tandem mass spectrometry (LC-MS / MS); and c) comparing the amount of crosslinks formed by TG in the biological sample with the amount of crosslinks formed by TG in a previous sample from the patient or a control value.Further provided is a method for determining a subject's response to treatment, comprising: a) digesting proteins present in a first biological sample from a subject using an enzyme immobilized on beads; b) detecting crosslinks formed by TG in the biological sample by liquid chromatography-tandem mass spectrometry (LC-MS / MS); and c) comparing the amount of crosslinks formed by TG in the biological sample with the amount of crosslinks formed by TG in a biological sample from a patient before treatment or at an early stage of treatment or a control value. Further provided is a method for determining the effect of a drug on TG activity, comprising the steps of a) digesting proteins present in a biological sample from a subject administered the drug using an enzyme immobilized on beads, and b) detecting crosslinks formed by TGs in the biological sample by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0011] Preferably, the crosslinks formed by TG comprise N'N'bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides.

[0012] In any of the methods of the present invention, the disease may be any disease associated with elevated TG activity, such as elevated TG2 activity, and optionally the disease is fibrosis, a fibrotic disease, or a fibrosis-related disease, such as chronic kidney disease, progressive kidney disease, pulmonary fibrosis, systemic sclerosis, liver cirrhosis, cardiovascular disease, idiopathic hypertrophic cardiomyopathy, renal fibrosis, primary glomerulonephritis, liver cirrhosis, chronic allograft injury (CAI), post-transplant renal fibrosis, or chronic allograft nephropathy. In any of the methods of the present invention, the treatment or agent may be an inhibitor of TG, such as an anti-TG antibody. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows the formation of N′N′ bis(γ-glutamyl)-polyamine and ε-(γ-glutamyl)-lysine crosslinks by transglutaminases, such as transglutaminase 2 (TG2). [Figure 2A] FIG. 1 is a schematic diagram showing the core steps of the method: Proteins may be concentrated from urine samples using trichloroacetic acid (TCA) precipitation, proteins are subjected to enzymatic digestion, and the resulting amino acids and dipeptides are analyzed by LC-MS / MS. [Figure 2B] Schematic diagram showing the core steps of the method. TG activity leads to intra- and intermolecular ε-(γ-glutamyl)-lysine (γ-Glu-ε-Lys) cross-links in proteins. Complete enzymatic digestion cleaves peptide bonds to generate single amino acids and γ-Glu-ε-Lys dipeptides, which are then analyzed by LC-MS / MS. [Figure 3A] Characterization of sample preparation: Digestion reproducibility is good (CV<15%). [Figure 3B] Figure 1. Characterization of sample preparation: Digestion efficiency is estimated to be high (>80%) compared to acid hydrolysis. [Figure 3C] Figure 1: Characterization of sample preparation. Digestion efficiency is not affected by protein content. Correlation between γ-Glu-ε-Lys dipeptide concentration and the concentration of protein subjected to proteolytic digestion. Error bars indicate standard deviation (n=3). [Figure 4A] Figure 1: Analysis of the γ-Glu-ε-Lys dipeptide by LC-MS / MS. The spectrum of the MS / MS analysis of the crosslink shows the key transitions observed. [Figure 4B] Analysis of the γ-Glu-ε-Lys dipeptide by LC-MS / MS, which specifically detects the dipeptide: EK: glutamyl-lysine and KE: lysyl-glutamate (known isomer of γ-Glu-ε-Lys). [Figure 5A] FIG. 1 Screening of disease state and healthy urine samples. γ-Glu-ε-Lys levels measured in urine samples from disease state and healthy urine samples were expressed as ng γ-Glu-ε-Lys / mL urine as readout from LC-MS / MS measurement. [Figure 5B] FIG. 1 shows screening of disease state and healthy urine samples. γ-Glu-ε-Lys levels measured in urine samples from disease state and healthy urine samples were expressed as ng γ-Glu-ε-Lys / mg protein present in the urine sample used as the reported value. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The entire document is intended to be linked as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if those combinations of features are not found together in the same sentence, paragraph, or section of this document. With respect to aspects of the invention described or claimed with "a" or "an," these terms should be understood to mean "one or more," unless the context clearly requires a more limited meaning.

[0015] The term "or" should be understood to encompass alternative or combined items unless the context clearly necessitates otherwise. When aspects of the invention are described as "comprising" a certain feature, it is also contemplated that the embodiment "consists of" or "consists essentially of" that feature.

[0016] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0017] The present inventors have developed a sensitive assay for detecting and quantifying the amount (or number; number and / or amount may be used interchangeably throughout this document) of isopeptide bonds (also referred to herein as crosslinks) formed by TGs, such as N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide bonds or crosslinks and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide bonds or crosslinks, in a biological sample. The method of the present invention can specifically, reliably and sensitively detect the activity of TGs (particularly TG2) in a patient through the detection and quantification of dipeptides / isopeptides formed by TGs, such as N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, in a biological sample from the patient. This allows for accurate and sensitive diagnosis of diseases or disorders associated with abnormal activity of TGs, including fibrosis and other fibrotic disorders for TG2. The method also provides a specific, sensitive, reliable and accurate assay for evaluating the target engagement and pharmacodynamics of therapeutic agents targeting the TG pathway, such as therapeutic anti-TG2 antibodies.Dipeptide, isopeptide, dipeptide bond, isopeptide or bridge are used interchangeably herein.

[0018] The methods and assays described herein overcome the challenge of selectively detecting and quantifying transglutaminase-formed crosslinks, including N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide bonds or crosslinks and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide bonds or crosslinks. The surprising specificity, selectivity and sensitivity of the methods and assays was achieved by using a combination of (i) on-bead enzymatic digestion and (ii) fast and specific LC-MS / MS detection. The specificity, selectivity and sensitivity of the methods and assays can be further enhanced by efficient biological sample preparation. In particular, the following steps provide particularly improved specificity, selectivity and sensitivity: Pre-purification to remove impurities dramatically reducing background signal from contaminants in biological samples. · The use of enzymes linked to protective beads, which allows specific digestion (generation of a mixture containing free amino acids and crosslinks) and greatly reduces the background caused by protease digestion contamination. · The use of LC-MS / MS as a detection tool, allowing highly specific, accurate, precise and quantitative detection.

[0019] The method of the invention is for detecting and / or quantifying, preferably detecting and quantifying, crosslinks formed by transglutaminase (TG). Crosslinks formed by TG typically comprise N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, preferably ε-(γ-glutamyl)-lysine dipeptide. In a preferred embodiment, the method of the invention is for detecting and / or quantifying, preferably detecting and quantifying, N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide bonds or crosslinks and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide bonds or crosslinks. Typically, the method of the invention is for detecting and / or quantifying, preferably detecting and quantifying, N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide. In a most preferred embodiment, the method of the invention is for detecting and quantifying N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides exhibiting TG cross-linking activity. In some embodiments, the method of the invention is for detecting and quantifying N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and ε-(γ-glutamyl)-lysine dipeptides. In some embodiments, the method of the invention is for detecting and quantifying ε-(γ-glutamyl)-lysine dipeptides.

[0020] The method of the present invention for detecting and quantifying transglutaminase-formed crosslinks comprising N'N'-bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides in biological samples with high specificity is a significant improvement over prior art methods that did not provide the necessary accuracy or sensitivity specifically required for clinical use.

[0021] The method of the present invention typically comprises the following core steps: a) digesting proteins present in a biological sample using enzymes immobilized on beads (thereby generating a mixture containing free amino acids and crosslinks); and b) detecting transglutaminase-formed crosslinks comprising N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides in biological samples by liquid chromatography-tandem mass spectrometry (LC-MS / MS); Includes.

[0022] The basic method of the present invention has several different applications. Thus, the present invention: A. Methods for detecting crosslinks formed by transglutaminase, in particular methods for detecting N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides in biological samples; B. A method for determining the activity of a TG, such as TG2, in a subject. C. Methods for diagnosing a disease in a subject; D. A method for stratifying disease severity in a subject; E. Methods of monitoring disease progression in a subject; F. A method for determining a subject's response to a treatment, or G. Methods for Determining the Effect of an Agent on TG Activity, Such as TG2 Activity to provide.

[0023] The method may further comprise obtaining a biological sample from the subject. The method may further comprise enriching or purifying proteins present in the biological sample, as described in more detail below. The method of the invention is typically carried out in vitro.

[0024] The method of the present invention may be used to detect crosslinks formed by transglutaminase in any biological sample, preferably N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide. In some preferred embodiments, the biological sample is urine. Since the formation of crosslinks, particularly N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, is catalyzed by transglutaminase (TG), including factor XIIIa, TG1, TG2, TG3, TG5, TG4, TG6, and TG7, the method described herein may be used as an assay for TG activity, such as TG2 activity. Typically, the method of the present invention includes detecting crosslinks formed by TG in a biological sample from a subject. Thus, the method of the present invention typically involves detecting N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample from a subject. The presence of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample indicates TG activity, such as TG2 activity, in the subject (i.e., in vivo). Thus, the in vitro method of the present invention provides a readout of in vivo enzyme activity. Thus, the method may also provide insight into disease states in the subject. All of the methods of the present invention rely on detecting N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide.

[0025] The method of the present invention may be used to detect crosslinks formed by TG in any sample, preferably a biological sample. Thus, the method of the present invention may be used to detect N'N'bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, especially ε-(γ-glutamyl)-lysine dipeptide, in any sample, preferably a biological sample. The biological sample may be selected from the group consisting of urine, blood, plasma, serum, red blood cells, tissue (e.g. kidney tissue, lung tissue, heart tissue, etc.), saliva, placental tissue, bone marrow, breast milk, bronchoalveolar lavage fluid, feces, pleural effusion, synovial fluid, and semen. Typically, the biological sample is urine or tissue (e.g. kidney tissue, lung tissue, heart tissue, etc.), preferably urine. The biological sample may be obtained or has been obtained, or may be isolated or has been isolated from a subject. Methods for obtaining a biological sample from a subject are well known in the art. The tissue sample may be obtained, for example, by biopsy. The subject is typically an animal, preferably a mammal. In some embodiments, the subject is a mouse, a rat, a primate, or a human. In a preferred embodiment, the subject is a primate, most preferably a human. The subject may be a human patient. In some embodiments, the subject may have or be suspected to have a disease associated with TG activity, preferably TG2 activity, as further defined herein.

[0026] Biological samples typically need to be prepared for use in the methods of the invention. The exact method of sample preparation used may vary depending on the type of biological sample. Typically, the protein fraction of the biological sample is enriched (e.g., concentrated and / or partially purified) for use in the methods of the invention. Thus, the method may include concentrating the proteins present in the biological sample before digesting the proteins using enzymes immobilized on the beads. The step of enriching may be understood to mean that the protein fraction is concentrated and / or that important contaminants that may interfere with subsequent analysis are removed. The step of enriching may further be understood to mean that the protein fraction of the biological sample is separated from other components of the biological sample to prepare a composition that essentially consists of a mixture of all proteins present in the biological sample, for example, and to exclude other components of the biological sample, such as nucleic acids and lipids. In some embodiments, enriching the proteins present in the biological sample includes precipitating the proteins. Typically, precipitating proteins advantageously allows to enrich proteins, to concentrate samples, and / or preferably to remove salts that may inhibit subsequent enzymatic digestion. Proteins may be precipitated, for example, by salting out with trichloroacetic acid, for example, using ammonium sulfate, by precipitation with miscible solvents, for example, using ethanol or methanol, or by precipitation with non-ionic hydrophilic polymers, such as dextran and polyethylene glycol. Preferably, concentrating proteins present in a biological sample comprises precipitating proteins using trichloroacetic acid. Typically, the precipitated proteins are then separated from the remainder of the biological sample, for example, by centrifugation. Thus, concentrating proteins present in a biological sample may comprise precipitating proteins using trichloroacetic acid and separating the precipitated proteins by centrifugation.The separated precipitated proteins are typically hydrated in a suitable buffer (e.g., potassium phosphate buffer, typically about pH 7.5, which may contain urea) for subsequent analysis and enzymatic digestion. Thus, concentrating proteins present in a biological sample may include precipitating the proteins using trichloroacetic acid, separating the precipitated proteins by centrifugation, and hydrating or resuspending the proteins in a buffer solution.

[0027] The method of the present invention involves digesting proteins present in a biological sample using enzymes immobilized on beads. Typically, substantially all proteins in the biological sample are digested. A "protein" or "polypeptide" may be understood to mean a linear polymeric chain composed of amino acid monomers linked by covalent peptide bonds. The proteins present in the biological sample may further comprise inter- or intra-molecular covalent cross-links (e.g., cross-links, isopeptide bonds). Digesting a protein may be understood to mean cleaving peptide bonds in a protein to break the protein into its constituent amino acids and any non-digestible cross-linked isopeptides / dipeptides, if present. Protein digestion may be complete or incomplete. Complete protein digestion typically involves cleaving all peptide bonds in a protein to generate a mixture of free amino acids and any non-digestible cross-linked isopeptides / dipeptides. The enzymes used in the method of the present invention are capable of cleaving peptide bonds between amino acids in a protein. Enzymes are typically unable to cleave crosslinks formed by TGs such as N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, especially ε-(γ-glutamyl)-lysine bonds. N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide bonds and ε-(γ-glutamyl)-lysine dipeptide / isopeptide bonds are intra- or inter-protein crosslinks (e.g., isopeptide bonds) that covalently link amino acids from two polypeptide chains. The ε-(γ-glutamyl)-lysine bond has the structure: [ka] where the curved line indicates the peptide bond in the linear polypeptide chain. The formation of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide bond and ε-(γ-glutamyl)-lysine dipeptide / isopeptide bond, especially ε-(γ-glutamyl)-lysine bond, is catalyzed by TG. N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide bond and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide bond, especially ε-(γ-glutamyl)-lysine bond, may be formed in vivo in a subject by the action of TG, especially TG2. Thus, the presence of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, especially ε-(γ-glutamyl)-lysine dipeptide in a biological sample from a subject indicates TG activity in the subject.

[0028] The enzyme may be capable of cleaving all covalent bonds between amino acids in a protein (including all peptide bonds, intermolecular and intramolecular cross-links) except for N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide bonds and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide bonds, especially ε-(γ-glutamyl)-lysine bonds. Digestion of a protein using an enzyme immobilized on beads may release N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, especially ε-(γ-glutamyl)-lysine dipeptide. Thus, the enzyme may digest a protein to produce a mixture containing free amino acids and N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, especially free amino acids and ε-(γ-glutamyl)-lysine dipeptide. Thus, in the context of the method of the invention, a complete protein digestion may comprise cleaving all peptide bonds and crosslinks in a protein, except for N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide bonds and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide bonds, in particular ε-(γ-glutamyl)-lysine bonds, to produce a mixture of free amino acids and N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptides, in particular a mixture of free amino acids and ε-(γ-glutamyl)-lysine dipeptides. Digestive efficiency provides a measure of the completeness of digestion. A digestion efficiency of 100% indicates that substantially all of the covalent bonds between amino acids have been cleaved to produce a mixture consisting essentially of free amino acids and N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, preferably a mixture consisting essentially of free amino acids and ε-(γ-glutamyl)-lysine dipeptides.In the methods of the invention, the digestion efficiency may be at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98% or about 100%, preferably the digestion efficiency is at least 80%.

[0029] The enzymes used in the methods of the present invention may be selected from endopeptidases, exopeptidases, prolidases, or any combination thereof. The enzymes may include a mixture of endopeptidases (i.e., proteolytic enzymes that cleave peptide bonds of non-terminal amino acids in a polypeptide chain), exopeptidases (i.e., proteolytic enzymes that cleave terminal peptide bonds), and / or prolidases (i.e., enzymes that can cleave or hydrolyze bonds between proline and other amino acids, optionally with a proline or hydroxyproline residue located at the C-terminal position). The enzymes may include or consist essentially of endopeptidases, exopeptidases, and prolidases. In some embodiments, the enzymes may include at least endopeptidases, exopeptidases, and prolidases.

[0030] The enzyme may be selected from proteinase K, pronase, prolidase, leucine aminopeptidase, carboxypeptidase Y, or any combination thereof. In other words, the enzyme may comprise one or more of proteinase K, pronase, prolidase, leucine aminopeptidase, and carboxypeptidase Y. Other enzymes may be present. The enzymes may include proteinase K, pronase, prolidase, leucine aminopeptidase, and carboxypeptidase Y; pronase, prolidase, leucine aminopeptidase, and carboxypeptidase Y; proteinase K, prolidase, leucine aminopeptidase, and carboxypeptidase Y; proteinase K, pronase, leucine aminopeptidase, and carboxypeptidase Y; proteinase K, pronase, prolidase, and carboxypeptidase Y; or proteinase K, pronase, prolidase, and leucine aminopeptidase. The enzymes may include or consist essentially of proteinase K, pronase, prolidase, leucine aminopeptidase, and carboxypeptidase Y. The enzymes may comprise or consist of a mixture of proteinase K, pronase, prolidase, leucine aminopeptidase and carboxypeptidase Y. In some embodiments, the enzymes may comprise at least proteinase K, pronase, prolidase, leucine aminopeptidase and carboxypeptidase Y.

[0031] The enzyme is immobilized on the beads. The enzyme may be covalently linked or conjugated to the beads. Different populations of beads may be provided, with each population of beads linked to one type of enzyme. In some embodiments, proteinase K is immobilized on a first population of beads, pronase is immobilized on a second population of beads, prolidase is immobilized on a third population of beads, leucine aminopeptidase is immobilized on a fourth population of beads, and carboxypeptidase Y is immobilized on a fifth population of beads. Thus, in some embodiments, the enzyme immobilized on the beads comprises or essentially consists of a mixture of a first population of beads in which beads are linked to proteinase K, a second population of beads in which beads are linked to pronase, a third population of beads in which beads are linked to prolidase, a fourth population of beads in which beads are linked to leucine aminopeptidase, and a fifth population of beads in which beads are linked to carboxypeptidase Y. The beads in each population may be identical in all other respects except that they are conjugated to different enzymes.

[0032] In some embodiments, digesting the protein comprises contacting the protein with an enzyme immobilized on the beads. In some embodiments, digesting the protein comprises mixing the protein with the enzyme immobilized on the beads and incubating the mixture under conditions suitable for enzymatic activity, thereby digesting the protein. The exact conditions suitable for enzymatic activity may depend on the particular enzyme used for protein digestion. One of skill in the art would be readily able to select suitable conditions for the selected enzyme. In some embodiments, the conditions suitable for enzymatic activity may comprise a temperature of 35-38°C and an incubation time of 16-24 hours. In some embodiments, the protein is digested in the presence of urea. In preferred embodiments, the protein is digested under denaturing conditions (e.g., denaturing urea conditions) that advantageously unfold the protein and enhance the access of the enzyme. In some embodiments, it is advantageous to digest the protein by contacting the protein with different enzymes in a specific order. For example, in some preferred embodiments, digesting the protein comprises first contacting the protein with one or more endopeptidases and then contacting the protein with one or more exopeptidases and / or prolidases. This may result in a higher digestion efficiency, for example at least 80%. Digesting the protein may include multiple (e.g., 2, 3, 4, 5, 6 or more) digestion steps in which the protein is contacted with one or more endopeptidases, one or more exopeptidases and one or more prolidases. Digesting the protein may include a two-step digestion method in which the protein is contacted with an endopeptidase, such as proteinase K, and then subsequently with one or more exopeptidases and other enzymes, such as one or more of pronase, prolidase, leucine aminopeptidase and carboxypeptidase Y.Thus, in some embodiments, in the methods of the invention, digesting the protein comprises contacting (e.g., mixing) the protein with proteinase K immobilized on beads and incubating the mixture under conditions suitable for enzymatic activity, and then subsequently contacting the protein with pronase immobilized on beads, prolidase immobilized on beads, leucine aminopeptidase immobilized on beads and carboxypeptidase Y immobilized on beads and incubating the mixture under conditions suitable for enzymatic activity. Digesting the protein may comprise contacting the protein with a first population of beads in which the beads are linked to proteinase K and incubating the mixture under conditions suitable for enzymatic activity, and then subsequently adding a second population of beads in which the beads are linked to pronase, a third population of beads in which the beads are linked to prolidase, a fourth population of beads in which the beads are linked to leucine aminopeptidase, and a fifth population of beads in which the beads are linked to carboxypeptidase Y and incubating the mixture under conditions suitable for enzymatic activity. In some embodiments, proteinase K immobilized on beads is mixed with the protein and the mixture is incubated for about 6-24 hours, preferably about 10-20 hours, more preferably about 16-18 hours, such as about 16, 17 or 18 hours (optionally, proteinase K immobilized on beads is added back to the mixture and incubated for an additional about 1-8 hours, preferably about 3-6 hours, such as about 4 hours or 6 hours), and then pronase, prolidase, leucine aminopeptidase and carboxypeptidase Y immobilized on beads are added to the mixture and the mixture is incubated for an additional about 6-36 hours, preferably about 10-24 hours, or more preferably about 14-20 hours, such as about 14, 15, 16, 17, 18, 19 or 20 hours to digest the protein.

[0033] Immobilization of enzymes on beads is advantageous because the beads used in the method of the invention shield the enzymes and prevent their autodigestion by other proteases present in the mixture. This improves the kinetics of digestion of proteins in biological samples and dramatically reduces background contamination from the enzymes. The beads typically comprise an outer shielding layer, which is configured to reduce or prevent digestion of the enzymes immobilized on the beads by said enzymes. The beads may be silica nanoparticles. The outer shielding layer may be an organosilica layer. The beads may be silica nanoparticles comprising an organosilica outer layer. The enzymes immobilized on the beads may be at least partially embedded in a shielding (e.g. organosilica) layer such that they are still able to perform their proteolytic function but are themselves protected from digestion by the proteases in the mixture. Such enzymes immobilized on beads are commercially available, for example from Inofea as part of the Enzzen®-Fibrous-Proteins-Digestion kit and are described in more detail in WO2015014888. The proteolytic enzymes are advantageously stabilized, allowing them to be active under the favorable denaturing urea conditions. Furthermore, autodigestion by the enzyme (i.e. autoproteolytic activity) is prevented by shielding. Thus, the bead-immobilized enzymes used in the method of the invention are both protected and efficient. The inventors have found that the use of such bead-immobilized enzymes results in perfect linearity between the protein content in urine and the occurrence of dipeptide biomarkers, a reproducibility of more than 85% and a digestion efficiency of more than 90%.

[0034] When proteins are digested using the enzyme immobilized on beads in the method of the present invention, N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, especially ε-(γ-glutamyl)-lysine dipeptides, are typically released in a background of free amino acids. Typically, a mixture of N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, especially ε-(γ-glutamyl)-lysine dipeptides and free amino acids is purified and prepared for LC-MS / MS. Suitable procedures for preparing samples for LC-MS / MS are well known in the art. In some embodiments, after enzymatic digestion, the mixture of N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, particularly ε-(γ-glutamyl)-lysine dipeptides and free amino acids is purified by solid phase extraction, optionally using a cation exchange cartridge. Typically, this is followed by vacuum drying of the mixture of N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, particularly ε-(γ-glutamyl)-lysine dipeptides and free amino acids. The dried mixture of N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, particularly ε-(γ-glutamyl)-lysine dipeptides and free amino acids may then be resuspended in water for subsequent analysis.

[0035] The method of the present invention further comprises detecting N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, particularly ε-(γ-glutamyl)-lysine dipeptides, in biological samples by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The liquid chromatography phase of LC-MS / MS may be performed via reversed phase chromatography or any other form of liquid chromatography, such as hydrophilic interaction chromatography (HILIC). LC-MS / MS may use a triple quadrupole mass spectrometer, or any other form of mass spectrometer, such as (but not limited to) a high resolution accurate mass spectrometer time of flight or an orbitrap mass spectrometer. N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, especially ε-(γ-glutamyl)-lysine dipeptide, may be detected using LC-MS / MS as having a defined retention time that may elute differently but is calibrated using stable isotope labeled (SIL) versions of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, especially the SIL version of ε-(γ-glutamyl)-lysine dipeptide. ε-(γ-glutamyl)-lysine dipeptide typically has a retention time comparable to that of the SIL peptide and has a parent ion mass / charge ratio (m / z) of 276. The peptide may be more specifically detected by monitoring a range of fragment ion masses derived from the parent ion such as m / z 147, 84 and 130. Stable isotope peptide internal standards can be detected at the same retention time and parent ion m / z of 284, and can be more specifically detected by monitoring a range of fragment ion masses derived from the parent ion such as m / z 155, 90 and 137.The method of the invention advantageously allows accurate and reliable quantification of N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, in particular ε-(γ-glutamyl)-lysine dipeptides, in biological samples. The method of the invention therefore preferably comprises detecting and quantifying N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, preferably ε-(γ-glutamyl)-lysine dipeptides, in biological samples by LC-MS / MS. In such an embodiment, the method is for detecting and quantifying N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, preferably ε-(γ-glutamyl)-lysine dipeptides, in biological samples.

[0036] In some preferred embodiments, the method comprises: a) concentrating proteins present in a biological sample; b) digesting the protein using enzymes immobilized on the beads, where the enzymes include proteinase K, pronase, prolidase, leucine aminopeptidase and carboxypeptidase Y, to produce a mixture containing free amino acids and ε-(γ-glutamyl)lysine dipeptides; and c) detecting and quantifying the amount of N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, particularly ε-(γ-glutamyl)-lysine dipeptide, in biological samples by LC-MS / MS, where ε-(γ-glutamyl)-lysine dipeptide is identified as having a retention time similar to the SIL peptide and a parent ion mass / charge ratio of 276, and further identified by detecting a range of fragment ion masses derived from the parent ion, such as m / z 147, 84 and 130. Includes.

[0037] In some preferred embodiments, where the biological sample is a urine sample, the method comprises: a) concentrating proteins present in a urine sample by precipitating the proteins using trichloroacetic acid; b) digesting the proteins using enzymes immobilized on the beads, where the enzymes include proteinase K, pronase, prolidase, leucine aminopeptidase and carboxypeptidase Y, to produce a mixture comprising free amino acids and N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, preferably a mixture comprising free amino acids and ε-(γ-glutamyl)-lysine dipeptides; and c) detecting and quantifying by LC-MS / MS the amount of N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, in particular ε-(γ-glutamyl)-lysine dipeptides, in biological samples, whereby ε-(γ-glutamyl)-lysine dipeptides are identified as a function of their retention time. Preferably, they are identified as having retention times equivalent to SIL peptides and having a parent ion mass / charge ratio of 276, and further having retention times by detecting a range of fragment ion masses derived from the parent ion, such as m / z 147, 84 and 130.

[0038] The above method for detecting crosslinks formed by TGs such as N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, especially ε-(γ-glutamyl)-lysine dipeptide, in a biological sample from a subject may also be used to diagnose disease in the subject. The presence of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, especially ε-(γ-glutamyl)-lysine dipeptide, in the biological sample indicates TG activity, preferably TG2 activity, in the subject. The amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample may be detected and quantified to provide information regarding the activity of TGs (including Factor XIIIa, TG1, TG2, TG3, TG5, TG4, TG6 and TG7) in vivo in a subject, which may further provide information regarding a disease state in the subject.

[0039] Accordingly, the present invention further provides a method of diagnosing a disease, condition, or disorder in a subject, comprising: a) digesting proteins present in a biological sample from a subject using enzymes immobilized on beads; and b) detecting crosslinks formed by TGs such as N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, in particular ε-(γ-glutamyl)-lysine dipeptides, in biological samples by liquid chromatography-tandem mass spectrometry (LC-MS / MS); The present invention provides a method comprising:

[0040] Similarly, the present invention provides an in vitro method for stratifying the severity of a disease, condition, or disorder in a subject, comprising: a) digesting proteins present in a biological sample from a subject using enzymes immobilized on beads; and b) detecting crosslinks formed by TGs such as N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, in particular ε-(γ-glutamyl)-lysine dipeptides, in biological samples by liquid chromatography-tandem mass spectrometry (LC-MS / MS); The present invention provides a method comprising:

[0041] These methods may further comprise any of the features mentioned above in the context of a method for detecting crosslinks formed by TGs (such as TG2) such as N'N'bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, in particular ε-(γ-glutamyl)-lysine dipeptides, since at their core these diagnostic methods use the above mentioned methods. Further features of the diagnostic methods are described herein below.

[0042] As mentioned above, the biological sample is preferably urine or tissue, most preferably urine. The subject may be a mammal, preferably a primate, most preferably a human. Typically, the subject is a human patient who has or is suspected of having a disease. The disease, condition or disorder may be any disease, condition or disorder associated with TG activity, such as TG2 activity. The disease, condition or disorder may be any disease, condition or disorder associated with a decrease or reduction in TG activity, such as TG2 activity. Preferably, the disease, condition or disorder may be any disease, condition or disorder associated with an increase or increase in TG activity, such as TG2 activity. The disease, condition or disorder may be inflammation (e.g., osteoarthritis, idiopathic inflammatory myopathy, rheumatoid arthritis, multiple sclerosis, psoriasis), cancer, fibrosis and fibroproliferative disorders, cardiovascular disease (e.g., coronary heart disease, deep vein thrombosis, vascular calcification, cerebrovascular and peripheral arterial disease, rheumatic and congenital heart disease), neurodegenerative disease (e.g., Alzheimer's disease, Parkinson's disease, supranuclear palsy, Huntington's disease and other polyglutamine diseases) or celiac disease. These diseases, conditions or disorders are typically associated with elevated or increased TG (e.g., TG2) activity. The disease, condition or disorder may be cancer. When the disease, condition or disorder is cancer, it may be colorectal cancer, breast cancer, pancreatic cancer, ovarian cancer, esophageal squamous cell carcinoma, glioblastoma, malignant melanoma, renal squamous cell carcinoma, cervical squamous cell carcinoma, hepatocellular carcinoma, cervical intraepithelial neoplasia. Cancer is typically associated with elevated or increased TG (e.g., TG2) activity. The disease, condition or disorder may be fibrosis or fibroproliferative disorder. When the disease, condition or disorder is fibrosis or fibroproliferative disorder, it may be, but is not limited to, chronic kidney disease (e.g., including post-transplant renal fibrosis or chronic allograft nephropathy (CAN)), progressive kidney disease, pulmonary fibrosis, systemic sclerosis, liver cirrhosis, cardiovascular disease, idiopathic hypertrophic cardiomyopathy, renal fibrosis, primary glomerulonephritis, liver cirrhosis, chronic allograft injury (CAI) or diabetic nephropathy. In some embodiments, the disease, condition or disorder is post-transplant renal fibrosis (e.g., including CAI or CAN). Fibrosis and fibroproliferative disorder are typically associated with elevated or increased TG activity, such as TG2.

[0043] In some embodiments, the method further comprises quantifying and / or determining the amount / number of crosslinks formed by TG, such as N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in the biological sample. The method may further comprise comparing the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in the biological sample with a control value. The control value may be the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, detected in a biological sample from a subject not having a disease, disorder or condition, preferably a healthy subject. The control value may be the average amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, detected in biological samples from a number of subjects, preferably a number of healthy subjects, without a disease, disorder or condition. The control value may be determined using the method of the present invention. The control value may vary depending on the type of biological sample used in the method of the present invention. In some embodiments, N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, is not detected using the method of the present invention in biological samples (such as urine samples) from healthy human subjects (i.e., subjects without a disease, disorder or condition). Thus, in some embodiments, the control value may be zero.In some other embodiments, the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample determined using the method of the present invention in a healthy human subject may be X±Y ng crosslinks / mg protein, or ng crosslinks / unit creatine, or ng crosslinks / ml biological sample (such as urine), preferably ng crosslinks / ml biological sample (such as urine). The most suitable unit typically depends on the type of biological sample being used. For example, for a urine sample based on a 24-hour urine collection, the unit is typically ng crosslinks / ml urine. Thus, in some embodiments, the control value may be X±Y. A person skilled in the art will readily understand that the control value does not need to be determined at the same time that the test value is determined. Typically, a control value is established prior to testing, for example by determining the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, in particular ε-(γ-glutamyl)-lysine dipeptide, in a selected biological sample type from a cohort of healthy subjects.

[0044] In some embodiments, N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, are not detected using the method of the present invention in biological samples from healthy subjects, and in such cases, comparison with a control value is not necessary. In such embodiments, essentially no (e.g., zero) detection of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample indicates that the subject has normal (i.e., healthy) TG (e.g., TG2) activity and / or that the subject does not have a disease, disorder or condition. On the other hand, detecting N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample (i.e., a non-zero amount of ε-(γ-glutamyl)-lysine dipeptide) using the methods of the invention indicates that the subject has a disease, disorder or condition. In some other embodiments, an amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample that is equal to or within ±Z of the control value indicates that the subject has normal (i.e., healthy) TG (e.g., TG2) activity and / or that the subject does not have a disease, disorder or condition. In some embodiments, for example where a disease is associated with decreased TG (e.g., TG2) activity, an amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, preferably ε-(γ-glutamyl)-lysine dipeptide, in a biological sample that is lower (e.g., significantly lower) than a control value indicates that the subject has a disease, disorder or condition.An amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample that is less than 0.95-fold, less than 0.9-fold, less than 0.8-fold, less than 0.7-fold, less than 0.6-fold, less than 0.5-fold, less than 0.4-fold, less than 0.2-fold, or less than 0.1-fold the control value, preferably less than 0.8-fold the control value, and most preferably less than 0.5-fold the control value, may indicate that the subject has a disease, disorder or condition. In some embodiments, a decrease of at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70% or at least 80% in the amount of N'N'bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample compared to a control value, preferably a decrease of at least 10%, most preferably a decrease of at least 20%, may indicate that the subject has a disease, disorder or condition. Typically, the disease, condition or disorder is likely to be associated with elevated TG (e.g., TG2) activity. Thus, in a preferred embodiment, an amount of N'N'bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample that is higher (e.g., significantly higher) than a control value, for example when the disease is associated with elevated TG (e.g., TG2) activity, indicates that the subject has a disease, disorder or condition. An amount of ε-(γ-glutamyl)-lysine dipeptide in a biological sample that is at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 10-fold, or at least 20-fold the control value, preferably at least 1.2-fold the control value, and most preferably at least 1.5-fold the control value, may indicate that the subject has a disease, disorder or condition.In some embodiments, an increase of at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or at least 200%, preferably an increase of at least 10%, and most preferably an increase of at least 20%, in the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample compared to a control value may indicate that the subject has a disease, disorder or condition.

[0045] The method according to the invention allows selective detection and / or quantification of N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, especially ε-(γ-glutamyl)-lysine dipeptides, even at low concentrations in a biological sample. In one embodiment, the lower limit of quantification (LLOQ) is as low as about 0.1 ng crosslinks / mL biological sample (such as urine). The upper limit of quantification (ULOQ) of crosslinks according to the method can be at least as high as about 50 ng crosslinks / mL biological sample (such as urine). Advantageously, the method is capable of (accurately) detecting and / or quantitating crosslinks in a biological sample 1) with an LLOQ of, for example, about 0.1 ng crosslinks / mL biological sample (such as urine) or higher, and / or 2) with an ULOQ of, for example, about 50 ng crosslinks / mL biological sample (such as urine) or lower. In other words, the method according to the present invention is capable of (accurately) detecting and / or quantifying crosslinks in amounts of about 0.1 ng to about 50 ng crosslinks / mL of biological sample (eg, urine). In another example, the method of the present invention can (accurately) detect and / or quantitate crosslinks in a biological sample in the range of about 0.1 to about 40 ng crosslinks / mL biological sample (e.g., urine), preferably about 0.1 to about 20 ng crosslinks / mL biological sample (e.g., urine), most preferably about 0.1 to about 15 ng crosslinks / mL biological sample (e.g., urine), about 0.1 to about 10 ng crosslinks / mL biological sample (e.g., urine), for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10 ng crosslinks / mL biological sample (e.g., urine).

[0046] In some exemplary but non-limiting embodiments, the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a urine sample determined using the methods of the present invention in a human patient with chronic kidney disease such as post-transplant renal fibrosis, CAI, or CAN, may be at least 0.1 ng crosslinks / mL urine, in line with the LLOQ (lower limit of quantitation for LC-MS / MS measurement), such as about 0.1 to about 50 ng crosslinks / mL urine, about 0.1 to about 40 ng crosslinks / mL urine, about 0.1 to about 20 ng crosslinks / mL urine, about 0.1 to about 10 ng crosslinks / mL urine, or even about 0.1 to about 5 ng crosslinks / mL. For urine samples, the reported concentration of ε-(γ-glutamyl)lysine may be normalized to mg protein measured by the bicinchoninic acid (BCA) method and expressed as ng ε-(γ-glutamyl)lysine equivalents per mg protein. In such an embodiment, the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in urine samples from control healthy subjects determined using the method of the present invention is typically 0 ng crosslinks / mL urine. This is because the crosslink and protein values ​​in urine samples from healthy subjects are typically below the sensitivity threshold of the method of the present invention.

[0047] The amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample can also be used to stratify disease severity or prognosis. In some embodiments, the lower the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample, the more severe the disease and / or the worse the prognosis of the disease, e.g., when the disease is associated with decreased TG activity, such as TG2 activity. In a preferred embodiment, for example, when a disease is associated with an increase in TG activity, such as TG2 activity, the higher the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, especially ε-(γ-glutamyl)-lysine dipeptide, in a biological sample, the more severe the disease and / or the worse the prognosis of the disease. A disease may be classified as having "severe" or "poor prognosis" based on a threshold value. In some embodiments, for example, when a disease is associated with a decrease in TG activity, such as TG2 activity, the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, especially ε-(γ-glutamyl)-lysine dipeptide, in a biological sample that is lower than a threshold value indicates that the disease may be classified as having severe and / or poor prognosis. In some preferred embodiments, for example when a disease is associated with elevated TG activity, such as TG2 activity, an amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample that is equal to or higher than a threshold value indicates that the disease may be classified as severe and / or having a poor prognosis.In such an embodiment, an amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample below a threshold value indicates that the disease may be classified as normal and / or having a normal prognosis. The threshold value may be the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, detected in a biological sample from a subject having a disease, disorder or condition known to be severe or have a poor prognosis, e.g., as determined using other assays or clinical factors. The threshold value may be the average amount of N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, particularly ε-(γ-glutamyl)-lysine dipeptides, detected in biological samples from a number of subjects with a disease, disorder or condition known to be severe or have a poor prognosis. The threshold value may be determined using the methods of the present invention. In some embodiments, for example, when the disease is associated with a decrease in TG activity, such as TG2 activity, the threshold value may be less than 0.6, 0.5, 0.4, 0.2, or 0.1 times the control value, preferably 0.5 times the control value, and most preferably 0.2 times the control value. In some embodiments, for example, when the disease is associated with an increase in TG activity, such as TG2 activity, the threshold value may be 1.5 times, 2 times, 2.5 times, 3 times, 4 times, 5 times, 10 times, or at least 20 times the control value, preferably at least 2 times the control value, and most preferably at least 5 times the control value. In some embodiments, for example where the disease is associated with decreased TG activity, such as TG2 activity, the threshold value may be at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or at least 100% lower than the control value, preferably at least 20% lower, and most preferably at least 50% lower.In some preferred embodiments, for example when the disease is associated with elevated TG activity, such as TG2 activity, the threshold value may be at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or at least 200% higher than the control value, preferably at least 20% higher, and most preferably at least 50% higher. For example, in a human patient with a severe form of a disease, condition or disorder defined herein, the amount of N'N'bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, in particular ε-(γ-glutamyl)-lysine dipeptide, in a biological sample determined using the method of the present invention may be Q±R ng crosslinks / mg protein, or ng crosslinks / unit creatine, or ng crosslinks / ml biological sample (such as urine), preferably ng crosslinks / ml biological sample (such as urine). The most suitable unit typically depends on the type of biological sample being used. For example, for a urine sample based on a 24 hour urine collection, the units are typically ng crosslinks / ml urine. Thus, in some embodiments, the threshold value may be Q±R.

[0048] The methods of the invention can also be used to monitor the progression of a disease, disorder or condition in a subject, i.e., whether the disease, disorder or condition is improving or worsening in a subject over time. Thus, the invention provides an in vitro method for monitoring the progression of a disease, disorder or condition in a subject, comprising: a) digesting proteins present in a biological sample from a subject using enzymes immobilized on beads; and b) detecting crosslinks formed by TGs such as N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, in particular ε-(γ-glutamyl)-lysine dipeptides, in biological samples by liquid chromatography-tandem mass spectrometry (LC-MS / MS), and c) comparing the amount of crosslinks formed by TGs, such as N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, in particular ε-(γ-glutamyl)-lysine dipeptides, in the biological sample with the amount of crosslinks formed by TGs, such as N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, in particular ε-(γ-glutamyl)-lysine dipeptides, in a previous sample from the patient or with a control value. The present invention provides a method comprising:

[0049] Preferably, as described above, the disease is any disease associated with a decrease or preferably an increase in TG activity, such as TG2 activity. Preferably, the disease is fibrosis, a fibrotic disease or disorder, or a fibrosis-related disease or disorder. Such disease or disorder may be (but is not limited to) chronic kidney disease (including post-transplant renal fibrosis and chronic allograft nephropathy (CAN)), progressive kidney disease, pulmonary fibrosis, systemic sclerosis, liver cirrhosis, cardiovascular disease, idiopathic hypertrophic cardiomyopathy, renal fibrosis, primary glomerulonephritis, liver cirrhosis, or chronic allograft injury (CAI). These diseases or disorders are typically all associated with an increase or increase in TG2 activity. These methods may further comprise any of the features described above in the context of the method for detecting crosslinks formed by TG, such as N'N'bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, or the method for diagnosis.

[0050] The method may further comprise determining the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in the biological sample. The amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a previous biological sample from the subject may also be determined using the method of the present invention. Typically, the first biological sample was obtained from the subject at least 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 18 months, 2 years, 5 years, or at least 10 years, preferably at least 6 months, most preferably at least 12 months, before the second biological sample was obtained from the subject. Therefore, changes in the amount / number of dipeptides in a biological sample from a subject can be monitored, which allows for monitoring TG activity, such as TG2, which in turn allows for monitoring disease.

[0051] In some embodiments, for example when a disease is associated with decreased TG activity, such as TG2 activity, an increase in the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in the second biological sample compared to the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in the first biological sample indicates that the disease is ameliorated. In some embodiments, for example when a disease is associated with a decrease in TG activity, such as TG2 activity, a decrease in the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in the second biological sample compared to the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in the first biological sample indicates that the disease is worsening. In a preferred embodiment, for example when a disease is associated with elevated TG activity, such as TG2 activity, a decrease in the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in the second biological sample compared to the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in the first biological sample indicates that the disease is ameliorated.In a preferred embodiment, for example when a disease is associated with an increased TG activity, such as TG2 activity, an increase in the amount of N'N'bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, especially ε-(γ-glutamyl)-lysine dipeptide, in the second biological sample compared to the amount of N'N'bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, especially ε-(γ-glutamyl)-lysine dipeptide, in the first biological sample indicates a worsening of the disease. In any case, the increase or decrease in the amount of N'N'bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, especially ε-(γ-glutamyl)-lysine dipeptide, is preferably a significant increase or decrease. In some embodiments, an amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in the second biological sample that is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or at least 200% higher, preferably at least 10% higher, and most preferably at least 20% higher, than the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in the first biological sample indicates worsening of the disease.

[0052] The method may further comprise obtaining one or more additional biological samples at 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 18 months, 2 years, 5 years, or 10 years time intervals, preferably at 6 months time intervals, most preferably at 12 months time intervals, and carrying out the method to monitor disease progression over several years. The method may further comprise comparing the amount of N'N'bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in one or more biological samples from the subject with a control value as defined herein. An increase in the difference from the control value over time indicates that the disease, disorder, or condition is worsening in the subject. A decrease in the difference from the control value over time (i.e., the amount approaches the control value) indicates that the disease, disorder, or condition is improving in the subject.

[0053] The method of the present invention allows for the detection of crosslinks formed by TGs such as N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, particularly ε-(γ-glutamyl)-lysine dipeptides, in a biological sample from a subject, which may provide an indication of TG activity in the subject and indicate a disease state. Similarly, monitoring the level of crosslinks formed by TGs such as N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, particularly ε-(γ-glutamyl)-lysine dipeptides, in a biological sample from a subject may then be used to infer whether a treatment is effective or whether a drug targeting TGs is effective. Thus, the present invention provides a method for determining a subject's response to a treatment, comprising: a) digesting proteins present in a biological sample from a subject using enzymes immobilized on beads; and b) detecting crosslinks formed by TGs such as N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, in particular ε-(γ-glutamyl)-lysine dipeptides, in biological samples by liquid chromatography-tandem mass spectrometry (LC-MS / MS), and c) comparing the amount of crosslinks formed by TGs, such as N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, in particular ε-(γ-glutamyl)-lysine dipeptides, in the biological sample with the amount of crosslinks formed by TGs, such as N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, in particular ε-(γ-glutamyl)-lysine dipeptides, in a biological sample from a patient before treatment or at an early stage of treatment or with a control value. The present invention provides a method comprising:

[0054] Preferably, as described above, the subject is a human patient. The subject may be a human patient with a disease associated with a decrease or preferably an increase in TG activity, such as TG2 activity. Preferably, the disease is fibrosis, a fibrotic disease or disorder, or a fibrosis-related disease or disorder. Such disease or disorder may be (but is not limited to) chronic kidney disease (including post-transplant renal fibrosis and chronic allograft nephropathy), progressive kidney disease, pulmonary fibrosis, systemic sclerosis, liver cirrhosis, cardiovascular disease, idiopathic hypertrophic cardiomyopathy, renal fibrosis, primary glomerulonephritis, liver cirrhosis, or chronic allograft injury (CAI). Thus, the treatment may be any treatment for a disease associated with a decrease or preferably an increase in TG activity, such as TG2 activity. In some embodiments, the treatment is for fibrosis, a fibrotic disease or disorder, or a fibrosis-related disease or disorder. Such diseases or disorders may be (but are not limited to) chronic kidney disease (including post-transplant renal fibrosis and chronic allograft nephropathy), progressive kidney disease, pulmonary fibrosis, systemic sclerosis, liver cirrhosis, cardiovascular disease, idiopathic hypertrophic cardiomyopathy, renal fibrosis, primary glomerulonephritis, liver cirrhosis, or chronic allograft injury. All of these diseases or disorders are typically associated with elevated or increased TG2 activity.

[0055] In some embodiments, for example where the disease is associated with decreased TG activity, such as TG2 activity, the method may comprise comparing (i) the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in the biological sample with (ii) the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample from a different subject having the disease who is not administered the treatment, where a higher amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in (i) compared to (ii) indicates that the treatment is efficacious. In some embodiments, for example where the disease is associated with decreased TG activity, such as TG2 activity, the method may comprise comparing (i) the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in the biological sample with (ii) the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample taken from the subject prior to treatment or at an early stage of treatment, where a higher amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in (i) compared to (ii) indicates that the treatment is efficacious.

[0056] In some preferred embodiments, for example where the disease is associated with elevated TG activity, such as TG2 activity, the methods may comprise comparing (i) the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in the biological sample with (ii) the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample from a different subject with the disease not being administered the treatment, where a lower amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in (i) compared to (ii) indicates that the treatment is efficacious. In some preferred embodiments, for example where the disease is associated with elevated TG activity, such as TG2 activity, the methods may comprise comparing (i) the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in the biological sample with (ii) the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample taken from the subject prior to treatment or at an early stage of treatment, where a lower amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in (i) compared to (ii) indicates that the treatment is efficacious.In some embodiments, for example where the disease is associated with elevated TG activity, such as TG2 activity, the method may comprise comparing (i) the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in the biological sample with (ii) the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample taken from the subject prior to treatment or at an early stage of treatment, where no significant change in the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in (i) compared to (ii) indicates that the treatment is efficacious.

[0057] In some embodiments, immediately after administration of the treatment, an increase in the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in the biological sample may be observed as the partially crosslinked tissue turns over more rapidly, however, over a period of several weeks, as the tissue turns over and the extracellular matrix containing the crosslinks is digested, the crosslinks are slowly removed and the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in the biological sample will typically decrease to very low levels. Thus, in some embodiments, the method comprises: (i) comparing the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample with (ii) the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample taken from the subject prior to treatment or at an early stage of treatment, wherein the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide of (i) compared to (ii) is a function of the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample taken from the subject prior to treatment or at an early stage of treatment. This may include comparing an initial increase in the amount of peptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, particularly ε-(γ-glutamyl)-lysine dipeptides (e.g., at 1, 2 and / or 3 weeks after administration of the treatment), followed by a decrease in the amount of N'N'-bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, particularly ε-(γ-glutamyl)-lysine dipeptides, of (i) compared to (ii) (e.g., at least 3, 4, 5, 6, 7, 8 or more weeks after administration of the treatment), which indicates that the treatment is efficacious.

[0058] The method may include obtaining a biological sample from the subject before the treatment. The method may include administering a treatment to the subject. The method may include obtaining a biological sample from the subject after administering a treatment to the subject. The treatment may be a TG antagonist or TG inhibitor, such as an anti-TG antibody (e.g., an anti-TG2 antibody) or a small molecule.

[0059] Similarly, the present invention also provides a method for determining the effect of an agent on TG activity, such as TG2 activity, comprising the steps of: a) digesting proteins present in a biological sample from a subject administered the drug using an enzyme immobilized on the beads; and b) detecting crosslinks formed by TGs such as N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides, in particular ε-(γ-glutamyl)-lysine dipeptides, in biological samples by liquid chromatography-tandem mass spectrometry (LC-MS / MS); The present invention provides a method comprising:

[0060] The method may further comprise comparing (i) the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in the biological sample with (ii) the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample from a different subject not receiving the drug, wherein a difference in the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, of (i) compared to (ii) indicates that the drug is having an effect. The method may further comprise comparing (i) the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in the biological sample with (ii) the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, in a biological sample taken from the subject prior to administration of the agent, wherein a difference in the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, particularly ε-(γ-glutamyl)-lysine dipeptide, of (i) compared to (ii) indicates that the agent is effective.

[0061] The method may include obtaining a biological sample from the subject before the treatment. The method may include administering a treatment to the subject. The method may include obtaining a biological sample from the subject after administering a treatment to the subject. The treatment may be a TG antagonist or TG inhibitor, such as an anti-TG antibody (e.g., an anti-TG2 antibody) or a small molecule.

[0062] A difference in the amount of N'N' bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide, especially ε-(γ-glutamyl)-lysine dipeptide, in (i) compared to (ii) may indicate target engagement by the drug. If the difference is an increase, this typically indicates that the drug is a TG activator (i.e., an activator of one or more of factor XIIIa, TG1, TG2, TG3, TG5, TG4, TG6 or TG7). For example, the drug may be a TG or TG2 agonist and / or the drug may increase the expression or activity of a TG, such as TG2. If the difference is a decrease, this indicates that the drug is a TG inhibitor (i.e., an inhibitor of one or more of factor XIIIa, TG1, TG2, TG3, TG5, TG4, TG6 or TG7). For example, the drug may inhibit substrate binding by TG (such as TG2) and / or inhibit or decrease the expression or activity of TG (such as TG2). The agent may bind to a TG, such as TG2, or its substrate, and prevent or reduce binding of a TG, such as TG2, to its substrate. The agent may be a polynucleotide, a polypeptide, an antibody, or a small molecule. The agent may be an anti-TG antibody, such as an anti-TG2 antibody. If sample (i) is taken within 1, 2, 3, or 4 weeks of administration of an agent that is a TG inhibitor, such as a TG2 inhibitor, an increase may be observed due to rapid turnover of bridging tissue, but typically a decrease is observed 3, 4, 5, 6, 7, 8 months or more after administration of the agent. The method may include obtaining a biological sample from the subject prior to administration of the agent. The method may include administering the agent to the subject. The method may include obtaining a biological sample from the subject after administration of the agent to the subject.

[0063] As used herein, the term TG inhibitor is intended to refer to a molecule that binds to and inhibits TG (i.e., an inhibitor against one or more of factor XIIIa, TG1, TG2, TG3, TG5, TG4, TG6 or TG7). As used herein, the term "anti-TG antibody" is intended to refer to an antibody molecule that binds to one or more TGs (i.e., one or more of factor XIIIa, TG1, TG2, TG3, TG5, TG4, TG6 or TG7). As used herein, the term "anti-TG2 antibody" is intended to be an antibody molecule that binds to TG2. Examples of such antibodies are described in WO2013175229. Non-limiting examples of anti-TG2 antibodies that may be used according to the present invention include: a) comprises six CDRs selected from the group consisting of: (i) KASQDINSYLT (LCDR1; SEQ ID NO: 1); LVNRLVD (LCDR2; SEQ ID NO: 2); LQYDDFPYT (LCDR3; SEQ ID NO: 3); THAMS (HCDR1; SEQ ID NO: 4); TISSGGRSTYYPDSVKG (HCDR2; SEQ ID NO: 5); and LISTY (HCDR3; SEQ ID NO: 6); or (ii) KASQDINSYLT (LCDR1; SEQ ID NO: 1); LTNRLMD (LCDR2; SEQ ID NO: 7); LQYVDFPYT (LCDR3; SEQ ID NO: 8); SSAMS (HCDR1; SEQ ID NO: 9); TISSGGRSTYYPDSVKG (HCDR2; SEQ ID NO: 5); and LISPY (HCDR3; SEQ ID NO: 10); or (iii) KASQDINSYLT (LCDR1; SEQ ID NO: 1); RTNRLFD (LCDR2; SEQ ID NO: 11); LQYDDFPYT (LCDR3; SEQ ID NO: 3); SSAMS (HCDR1); TISVGGGKTYYPDSVKG (HCDR2; SEQ ID NO: 9); and LISLY (HCDR3; SEQ ID NO: 12). b) a light chain variable domain having a sequence defined in any one of SEQ ID NOs: 13 to 27 and a heavy chain variable domain having a sequence defined in any one of SEQ ID NOs: 28 to 40; c) a light chain variable domain having at least 80% identity or similarity, preferably 90% identity or similarity, to a sequence defined in any one of SEQ ID NOs: 13 to 27, and a heavy chain variable domain having at least 80% identity or similarity, preferably 90% identity or similarity, to a sequence defined in any one of SEQ ID NOs: 28 to 40, or d) competes with an antibody as defined in a), b) or c) above for binding to an epitope comprising or consisting of amino acids 304 to 326 of human TG2 (see SEQ ID NO: 41) or a part of this region. [Table 1-1] [Table 1-2] [Table 1-3]

[0064] Whether an antibody binds to the same epitope as another antibody or competes for binding with another antibody can be easily determined by using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference antibody of the present invention, the reference antibody is bound to a protein or peptide under saturating conditions. The ability of the test antibody to bind to the protein or peptide is then evaluated. If the test antibody can bind to the protein or peptide after saturation binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody cannot bind to the protein or peptide after saturation binding with the reference antibody, the test antibody may bind to the same epitope as the epitope bound by the reference antibody of the present invention. To determine whether an antibody competes for binding with a reference antibody, the above binding method is performed in two directions. In the first direction, the reference antibody is bound to the protein / peptide under saturating conditions, and then the binding of the test antibody to the protein / peptide molecule is evaluated. In the second direction, the test antibody is bound to the protein / peptide under saturating conditions, and then the binding of the reference antibody to the protein / peptide is evaluated. In both orientations, if only the first (saturating) antibody is able to bind to the protein / peptide, it is concluded that the test and reference antibodies compete for binding to the protein / peptide. As will be appreciated by those skilled in the art, an antibody that competes for binding with a reference antibody will not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0065] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. That is, a 1-fold, 5-fold, 10-fold, 20-fold or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, 75%, 90% or even 99% as measured in a competitive binding assay. Alternatively, two antibodies have the same epitope if essentially every amino acid mutation in the antigen that reduces or eliminates the binding of one antibody reduces or eliminates the binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

[0066] As used herein, the term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies and recombinant antibodies produced by recombinant techniques known in the art. "Antibody" includes antibodies of any species, particularly mammalian species; human antibodies of any isotype, including, for example, IgG1, IgG2a, IgG2b, IgG3, IgG4, IgE, IgD, and antibodies produced as dimers of this basic structure, including IgGA1, IgGA2, or pentamers, such as IgM, and modified variants thereof; non-human primate antibodies, such as from chimpanzees, baboons, rhesus monkeys or cynomolgus monkeys; rodent antibodies, such as from mice or rats; rabbit, goat or horse antibodies; camelid antibodies (e.g., from camels or llamas, such as Nanobodies™) and derivatives thereof; avian antibodies, such as chicken antibodies; or fish antibodies, such as shark antibodies.

[0067] The term "antibody" also refers to a "chimeric" antibody in which at least a first portion of a heavy and / or light chain antibody sequence is derived from a first species and a second portion of a heavy and / or light chain antibody sequence is derived from a second species. Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World monkeys such as baboons, rhesus monkeys, or cynomolgus monkeys) and human constant region sequences. "Humanized" antibodies are chimeric antibodies that contain sequences derived from a non-human antibody. In most cases, humanized antibodies are human antibodies (recipient antibodies) in which residues from the recipient's hypervariable regions are replaced with residues from the hypervariable regions [or complementarity determining regions (CDRs)] of a non-human species (donor antibody) such as mouse, rat, rabbit, chicken, or non-human primate that have the desired specificity, affinity, and activity. In most cases, residues of the human (recipient) antibody are further replaced by corresponding non-human residues outside the CDRs, i.e., within the framework regions (FRs). Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further improve antibody properties. Humanization reduces the immunogenicity of non-human antibodies in humans, thus facilitating the application of antibodies to treat human diseases. Humanized antibodies and several different techniques for making them are well known in the art.

[0068] The term "antibody" also refers to human antibodies that can be produced as an alternative to humanization. For example, it is possible to produce transgenic animals (e.g. mice) that can produce a full repertoire of human antibodies in the absence of endogenous mouse antibody production upon immunization. Other methods for obtaining human antibodies / antibody fragments in vitro are based on display technologies such as phage display or ribosome display technology, using recombinant DNA libraries that are at least partially made from artificial or donor immunoglobulin variable (V) domain gene repertoires. Phage and ribosome display technologies for producing human antibodies are well known in the art. Human antibodies can also be produced from isolated human B cells that are ex vivo immunized with the antigen of interest and then fused to generate hybridomas that can then be screened for optimal human antibodies.

[0069] The term "antibody" refers to both glycosylated and non-glycosylated antibodies. Furthermore, the term "antibody" as used herein refers not only to full length antibodies but also to antibody fragments, more specifically antigen-binding fragments thereof. An antibody fragment comprises at least one heavy or light chain immunoglobulin domain as known in the art and binds to one or more antigens. Examples of antibody fragments according to the present invention include Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, Fab-Fv-Fv, scFv and Bis-scFv fragments. The fragments may also be diabodies, tribodies, triabodies, tetrabodies, minibodies, single domain antibodies (dAbs), such as sdAbs, VL, VH, VHH or camelid antibodies (e.g. from camel or llama, such as Nanobody™) and VNAR fragments. The antigen-binding fragment according to the invention can also comprise a Fab linked to one or two scFvs or dsscFvs, each scFv or dsscFv binding to the same or different targets (e.g. one scFv or dsscFv that binds to a therapeutic target and one scFv or dsscFv that increases half-life, e.g. by binding to albumin). Examples of such antibody fragments are FabdsscFv (also called BYbe®), or Fab-(dsscFv)2 (also called TrYbe®, see e.g. WO 2015 / 197772). The above defined antibody molecules, including their antigen-binding fragments, are known in the art.

[0070] Aspects of the present invention: 1. A method for detecting crosslinks formed by transglutaminase (TG) in a biological sample, comprising: a) digesting proteins present in a biological sample using enzymes immobilized on beads; and b) detecting crosslinks formed by TGs in biological samples by liquid chromatography-tandem mass spectrometry (LC-MS / MS) A method comprising:

[0071] 2. A method for determining the activity of transglutaminase (TG), preferably transglutaminase 2 (TG2), in a subject, comprising: a) digesting proteins present in a biological sample from a subject using enzymes immobilized on beads; and b) detecting crosslinks formed by TGs in biological samples by liquid chromatography-tandem mass spectrometry (LC-MS / MS) A method comprising:

[0072] 3. The method according to embodiment 1 or 2, wherein the crosslinks formed by TG comprise N'N'bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides.

[0073] 4. The method of any one of aspects 1 to 3, further comprising obtaining a biological sample from the subject.

[0074] 5. The method of any one of the preceding aspects, further comprising the step of concentrating proteins present in the biological sample prior to digestion.

[0075] 6. The method of any one of aspects 1 to 5, wherein concentrating the protein comprises precipitating the protein.

[0076] 7. The method of embodiment 6, wherein concentrating the proteins comprises precipitating the proteins by treatment with trichloroacetic acid and separating the precipitated proteins by centrifugation.

[0077] 8. The method of any one of the preceding aspects, wherein the enzyme immobilized on the beads is selected from an endopeptidase, an exopeptidase, a prolidase, or any combination thereof.

[0078] 9. The enzyme immobilized on the beads is (i) selected from proteinase K, pronase, prolidase, leucine aminopeptidase, carboxypeptidase Y, or any combination thereof; or (ii) comprising or consisting of proteinase K, pronase, prolidase, leucine aminopeptidase and carboxypeptidase Y; The method according to any one of embodiments 1 to 8.

[0079] 10. The method of any one of aspects 1 to 9, wherein proteinase K is immobilized on a first population of beads, pronase is immobilized on a second population of beads, prolidase is immobilized on a third population of beads, leucine aminopeptidase is immobilized on a fourth population of beads, and carboxypeptidase Y is immobilized on a fifth population of beads.

[0080] 11. The method according to any one of aspects 1 to 10, wherein the enzymatic digestion of step a) produces a mixture of free amino acids and cross-links (e.g. cross-linked dipeptides and / or isopeptides formed by TG), said mixture being purified and prepared for LC-MS / MS.

[0081] 12. The method according to any one of the preceding aspects, wherein the crosslink formed by TG comprises ε-(γ-glutamyl)-lysine dipeptide.

[0082] 13. The method of embodiment 12, wherein ε-(γ-glutamyl)-lysine dipeptide is detected using LC-MS / MS as having a retention time equivalent to that of a stable isotope labeled version of the dipeptide, with a parent ion mass / charge ratio (m / z) of 276, and by detecting a range of fragment ion masses derived from the parent ion, such as m / z 147, 84 and 130.

[0083] 14. The method according to any one of the preceding aspects, wherein step c) further comprises quantifying the crosslinks formed by TG by LC-MS / MS.

[0084] 15.a) concentrating proteins present in a biological sample by precipitating the proteins by treatment with trichloroacetic acid and separating the precipitated proteins by centrifugation; b) digesting the proteins using enzymes immobilized on the beads, where the enzymes include proteinase K, pronase, prolidase, leucine aminopeptidase and carboxypeptidase Y, to produce a mixture containing free amino acids and dipeptides / isopeptides; and c) detecting and quantifying the amount / number of crosslinks formed by TG in the biological sample by LC-MS / MS 15. The method of any one of aspects 1 to 14, comprising:

[0085] 16. The method of any one of the preceding aspects, wherein the biological sample is or has been isolated from a subject.

[0086] 17. The method of any one of the preceding aspects, wherein the biological sample is urine.

[0087] 18. The method of any one of the preceding aspects, wherein the subject is a mammal, optionally a primate, preferably a human.

[0088] 19. The method of any one of aspects 1 to 18, wherein the subject has or is suspected of having a disease associated with TG activity, such as TG2 activity.

[0089] 20. An in vitro method for diagnosing a disease in a subject, comprising: a) digesting proteins present in a biological sample from a subject using enzymes immobilized on beads; and b) detecting crosslinks formed by TGs in biological samples by liquid chromatography-tandem mass spectrometry (LC-MS / MS) A method comprising:

[0090] 21. An in vitro method for stratifying disease severity in a subject, comprising: a) digesting proteins present in a biological sample from a subject using enzymes immobilized on beads; and b) detecting crosslinks formed by TGs in biological samples by liquid chromatography-tandem mass spectrometry (LC-MS / MS) A method comprising:

[0091] 22. The method according to embodiment 20 or 21, wherein the bridges formed by TG comprise N'N'bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides.

[0092] 23. The method according to any one of aspects 20 to 22, further comprising determining the amount / number of crosslinks formed by TG in the biological sample.

[0093] 24. The method of any one of aspects 20 to 23, wherein the presence of crosslinks formed by TG in the biological sample indicates that the subject has the disease.

[0094] 25. The method according to any one of aspects 20 to 24, wherein the higher the amount / number of crosslinks formed by TG in the biological sample, the more severe the disease and / or the worse the prognosis of the disease.

[0095] 26. The method according to any one of aspects 20 to 25, wherein the disease is any disease associated with elevated TG2 activity, and optionally the disease is fibrosis, a fibrotic disease, or a fibrosis-related disease, such as chronic kidney disease, progressive kidney disease, pulmonary fibrosis, systemic sclerosis, liver cirrhosis, cardiovascular disease, idiopathic hypertrophic cardiomyopathy, renal fibrosis, primary glomerulonephritis, liver cirrhosis, chronic allograft injury (CAI), post-transplant renal fibrosis or chronic allograft nephropathy.

[0096] 27. An in vitro method for monitoring disease progression in a subject, comprising: a) digesting proteins present in a biological sample from a subject using enzymes immobilized on beads; and b) detecting crosslinks formed by TGs in biological samples by liquid chromatography-tandem mass spectrometry (LC-MS / MS); and c) comparing the amount / number of crosslinks formed by TG in the biological sample with the amount / number of crosslinks formed by TG in a previous sample from the patient or with a control value A method comprising:

[0097] 28. The method according to embodiment 27, wherein the crosslinks formed by TG comprise N'N'bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides.

[0098] 29. The method of embodiment 27 or 28, wherein the first biological sample was obtained from the subject about 12 months before the second biological sample was obtained from the subject.

[0099] 30. The method according to any one of aspects 27 to 29, wherein an increase in (i) the amount / number of crosslinks formed by TG in the first biological sample or (ii) the amount / number of crosslinks formed by TG in the second biological sample compared to a control value indicates a worsening of the disease.

[0100] 31. The method of any one of aspects 27 to 29, wherein a decrease in (i) the amount / number of crosslinks formed by TG in the first biological sample or (ii) the amount / number of crosslinks formed by TG in the second biological sample compared to a control value indicates that the disease is ameliorated.

[0101] 32. The method according to any one of aspects 27 to 31, wherein the disease is any disease associated with elevated TG2 activity, and optionally the disease is fibrosis, a fibrotic disease, or a fibrosis-related disease, such as chronic kidney disease, progressive kidney disease, pulmonary fibrosis, systemic sclerosis, liver cirrhosis, cardiovascular disease, idiopathic hypertrophic cardiomyopathy, renal fibrosis, primary glomerulonephritis, liver cirrhosis, chronic allograft injury (CAI), post-transplant renal fibrosis or chronic allograft nephropathy.

[0102] 33. A method for determining a subject's response to a treatment, comprising: a) digesting proteins present in a first biological sample from a subject using an enzyme immobilized on beads; and b) detecting crosslinks formed by TGs in biological samples by liquid chromatography-tandem mass spectrometry (LC-MS / MS); and c) comparing the amount / number of crosslinks formed by TG in the biological sample with the amount / number of crosslinks formed by TG in a biological sample from a patient before treatment or at an early stage of treatment or with a control value. A method comprising:

[0103] 34. The method according to embodiment 33, wherein the bridges formed by TG comprise N'N'bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides.

[0104] 35. The method of embodiment 33 or 34, comprising comparing (i) the amount / number of crosslinks formed by TG in a biological sample with (ii) the amount / number of crosslinks formed by TG in a biological sample from a different subject having the disease who is not administered the treatment, wherein a lower amount / number of crosslinks formed by TG in (i) compared to (ii) indicates that the treatment is efficacious.

[0105] 36. The method of embodiment 33 or 34, comprising comparing (i) the amount / number of crosslinks formed by TG in a biological sample with (ii) the amount / number of crosslinks formed by TG in a biological sample taken from the subject prior to treatment, wherein a lower amount / number of crosslinks formed by TG in (i) compared to (ii) indicates that the treatment is efficacious.

[0106] 37. The method of any one of aspects 33 to 36, wherein the patient is treated with a TG inhibitor.

[0107] 38. A method for determining the effect of an agent on TG activity, comprising: a) using an enzyme immobilized on the beads to digest proteins present in a biological sample from a subject to which the drug has been administered; and b) detecting crosslinks formed by TGs in biological samples by liquid chromatography-tandem mass spectrometry (LC-MS / MS) A method comprising:

[0108] 39. The method according to embodiment 38, wherein the crosslinks formed by TG comprise N'N'bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides.

[0109] 40. The method of embodiment 38 or 39, further comprising comparing (i) the amount / number of crosslinks formed by TG in the biological sample with (ii) the amount / number of crosslinks formed by TG in a second biological sample from a different subject not administered the drug, wherein a difference in the amount / number of crosslinks formed by TG in (i) compared to (ii) indicates that the drug is having an effect.

[0110] 41. The method of embodiment 38 or 39, further comprising comparing (i) the amount / number of crosslinks formed by TG in the biological sample with (ii) the amount / number of crosslinks formed by TG in a second biological sample taken from the subject prior to administration of the agent, wherein a difference in the amount / number of crosslinks formed by TG in (i) compared to (ii) indicates that the agent is effective.

[0111] 42. The method of any one of aspects 38 to 41, wherein the difference indicates target engagement by the agent.

[0112] 43. The method of any one of aspects 38 to 42, wherein the difference is an increase, indicating that the agent is a TG activator.

[0113] 44. The method of any one of aspects 38 to 43, wherein the difference is a decrease, indicating that the agent is a TG inhibitor.

[0114] 45. The method of any one of aspects 38 to 44, wherein the agent is an anti-TG2 antibody.

[0115] example Example 1 - Method Urinary protein precipitation Urine samples were collected from patients at different stages of disease progression and stored at -80°C until processing. Protein precipitation was performed as follows: an aliquot of urine (4 mL) was added to 1.0 mL of 50% trichloroacetic acid (TCA), vortexed for 10 seconds, and centrifuged at 4.200 g for 15 minutes at 4°C. After removing the supernatant, the pellet was resuspended in 4 mL of 10% TCA and centrifuged at 4.200 g for 10 minutes at 4°C. After removing the supernatant, the pellet was washed three times in 4 mL of ethanol:diethyl ether (1:1 v:v) and two times in 4 mL of diethyl ether. After removing the organic solvent by pipette, the pellet was incubated at room temperature for 30 minutes to dry. The precipitated proteins were hydrated in 67 μL of 0.1 M potassium phosphate buffer (KPi) containing 6 M urea, pH 7.5, and the bicinchoninic acid (BCA) assay (Pierce, Thermo Scientific) was performed for protein quantification according to conventional procedures.

[0116] digestion Protein digestion was performed using Enzzen®-Fibrous-Proteins-Digestion kit (Inofea) in a thermoblock at 37° C. with a vortex at 1300 rpm. More specifically, the precipitated protein solution was diluted with buffer (0.1 M KPi buffer, pH 7.5, 6 M urea) to obtain a solution containing 1 mg / mL1 protein as measured by BCA assay. Note that for samples below 1.336 mg / mL, 225 μL of precipitated protein solution was diluted with 25 μL of buffer. After centrifugation at 2000 g for 1 min at 4° C., 0.05 mL of enzzen®-proteinase K was added to a volume of 250 μL of the so-prepared solution and incubated at 37° C. and 1300 rpm for about 16 h. After sonication for 15 min, another 0.05 mL of enzzen®-proteinase K was added and the sample was incubated at 37° C. and 1300 rpm for 6 h. Subsequently, a volume of 1.05 mL of 0.01 M KPi buffer containing 0.001 M CaCl2 at pH 7 and 0.2 mL of enzzen®-pronase, 0.05 mL of enzzen®-prolidase, 0.1 mL of enzzen®-leucine aminopeptidase and 0.1 mL of enzzen®-carboxypeptidase Y were added, vortexed for 1 min, and the sample was incubated at 37° C. and 1600 rpm for another 16 h. The resulting sample was centrifuged at 2300 g for 15 min at 4° C., and the supernatant was collected and processed for urea removal by solid phase extraction using a cation exchange cartridge (Oasis MCX 3 cc Vac Cartridge) as follows: First, the SPE cartridge was conditioned with 1 mL of methanol followed by 1 mL of 2% formic acid (FA). An aliquot of 0.05 mL of the supernatant was added to 0.4 mL of 4% FA, vortexed at 1000 rpm for 1 min, and slowly loaded into the cartridge. The cartridge was then washed with 1 mL of 2% FA. The retained charged species were finally eluted from the cartridge twice with 0.5 mL of a solution of methanol and 5% ammonium hydroxide. The resulting sample was vacuum dried in a SpeedVac system for 12 h.Prior to analysis, the dried samples were dissolved in 50 μL of nanopure water. To check the reproducibility of the digestion and ensure a linear correlation between the amount of protein and the amount of isopeptides, digestions of fibrous proteins at increasing concentrations were performed in parallel. To confirm complete digestion of fibrous proteins, amino acid analysis was performed using the Cell Culture application of AccQtag Ultra UPLC Amino Acid Solution (Waters Inc.) according to the manufacturer's instructions.

[0117] LC-MS / MS LC-MS / MS analysis was performed on a Sciex 6500+ triple quadrupole instrument (AB Sciex LLC) coupled with an Acquity I-Class UPLC system (Waters Corporation). Chromatographic separation of ε-(γ-glutamyl)lysine was performed on a Waters Acquity BEH Amide column (50 × 2.1 mm, 1.7 μm i.d.; Waters Corporation) with gradient elution at 50 °C. The mobile phase was a mixture of 0.1% (v / v) formic acid in acetonitrile (A) and 0.1% (v / v) formic acid in water (B). After injection, 20% B was held for 0.5 min, and two linear gradient steps were programmed from 20 to 30% B in 0.5 min and finally from 30 to 80% B in 2.5 min. The column was then flushed with 80% B for 1.5 min, switched back to 20% B for 0.1 min, and finally reconditioned at 20% B for 1.9 min. The flow rate was kept constant at 0.4 mL / min. Samples were injected in partial loop injection mode with a weak wash of 600 μL formic acid:water:acetonitrile (0.1:20:80, v / v / v) and a strong wash of 200 μL formic acid:methanol:water (2:10:90, v / v / v). The flow from the LC was diverted to the MS system from 0.6 min to 3.4 min. The MS was fitted with a Turbo Spray Ion Drive source operated in positive electrospray ionization mode. The ion source temperature was set at 500 °C and the ion spray voltage was maintained at 5500 V. The pressures of the curtain gas, gas 1 and gas 2, were set at 40, 50 and 50 psi, respectively. Collision-induced dissociation was performed with nitrogen gas at a pressure of 9 psi. Selected reaction monitoring with unit mass resolution of precursor and product ions was used to quantify ε-(γ-glutamyl)-lysine dipeptide (Supplementary Table SX). Declustering potential and collision energy were optimized to achieve optimal performance. Data acquisition and processing were performed by Analyst software (version 1.6.3; AB Sciex LLC).

[0118] Reported crosslinking concentrations The formula used to calculate the reported ε-(γ-glutamyl)lysine concentration by normalizing the measured ε-(γ-glutamyl)lysine concentration in ng / mL to protein concentration in μg / mL:

number

[0119] Example 2 – Assay Development To efficiently release and quantify ε-(γ-glutamyl)-lysine from urinary proteins, several modifications to the current method had to be considered. The previous method involves a complex process involving precipitation of proteins from urine, protein resuspension, and sequential digestion based on a combination of six enzymes, designed to completely digest a diverse protein array but keep ε-(γ-glutamyl)-lysine intact. The subsequent detection and quantification had to be both sensitive and specific enough for accurate detection of ε-(γ-glutamyl)-lysine (Figure 2). Compared to the previous method, several optimizations were developed:

[0120] 1. Precipitation process: Precipitation using 4 mL of urine, a volume of urine that is small enough to handle with reasonable throughput but has subsequently been shown to provide the required sensitivity. Various precipitation methods were considered, including chloroform-methanol extraction or trichloroacetic acid (TCA), which was subsequently found to be the most effective and reproducible (see Figure 2).

[0121] 2. Digestion process: The digestion was optimized and carried out using a combination of proteinase K, pronase, prolidase, aminopeptidase and carboxypeptidase Y together with Enzzen® technology. The advantage of such an approach using immobilized enzymes is that the enzymes are shielded, thereby preventing them from being digested, minimizing their effect on protein or ε-(γ-glutamyl)-lysine contamination from the reagents. This new digestion step is reproducible, efficient and not affected by protein content. The reproducibility of the digestion was based on three digestions of the same sample, with a CV of less than 15% (see Figure 3A). To estimate the digestion efficiency, the release of amino acids by bead digestion was compared to acid hydrolysis, which is considered to be almost complete. Although such an estimation is hindered by the loss of certain amino acids in, for example, acid hydrolysis, the digestion efficiency was estimated to be more than 80% (see Figure 3B). To understand the effect of digestion of different amounts of protein extracted from human urine on the recovery of ε-(γ-glutamyl)-lysine dipeptide, a protein concentration range of 0.6-12 mg / mL was tested. It was demonstrated that there was a strong linear correlation between protein concentration and the concentration of ε-(γ-glutamyl)-lysine dipeptide (see Figure 3C).

[0122] 3. Amino acid analysis process: ε-(γ-glutamyl)-lysine in urine is in the ng / mg protein range, and in chromatograms using classical methods, the ε-(γ-glutamyl)-lysine peak appears as a very low, non-quantifiable peak. Despite efforts to solve this problem via LC parameters, the peak remained non-integrable. To circumvent this, an alternative method was devised using LC-MS / MS using a triple quadrupole mass spectrometer to aid in specific detection (see Schafer et al., 2005). Data obtained with such a method showed that it was possible to obtain a mass spectrum of standard ε-(γ-glutamyl)-lysine (Figure 4A) with characteristic transitions, with the ability to resolve the ε-(γ-glutamyl)-lysine peak from any glutamyl-lysine or lysyl-glutamate dipeptide (Figure 4B), which may be generated by partial digestion and release of linear dipeptides from proteins. Various chromatographic formats were used (e.g., reversed-phase chromatography), but ultimately the best separation and specificity was obtained using a hydrophilic interaction (HILIC) approach based on an Acquity BEH amide column (Waters), with quantification based on the transitions of 275.9–147.1 and 284.2–155.2 for the analyte and internal standard, respectively.

[0123] After establishing optimized precipitation, digestion and LC-MS / MS parameters, the method was validated for an analytical range of 0.10 ng / mL to 5.00 ng / mL. Linear regression with 1 / x2 weighting was applied to the peak area ratio concentration plots to construct a calibration curve (data not shown). Representative chromatograms of control blank, zero, LLOQ and ULOQ alternative matrix sample extracts were obtained (data not shown). The complete method showed good intra-run and inter-run precision of less than 10% and 20%, respectively, over the 0.1 ng / mL range and sensitivity. Also, the inter-run accuracy was less than 5%. Precision and accuracy of recombinant ε-(γ-glutamyl)-lysine in alternative matrix was determined by five measurements of ε-(γ-glutamyl)-lysine at each of four concentrations, each of which was tested in six independent replicates per run, for a total of 30 measurements per QC level. Furthermore, dilution linearity was assessed, showing that cross-linking levels up to 40 ng / mL were accurately measured in biological samples.

[0124] Example 3 – Testing on patient samples Having established a working method (see Example 2), we next tested the applicability of the method in a series of urine samples from healthy individuals and patients with various disease states. Measurable ε-(γ-glutamyl)-lysine values ​​could be detected in 79% of the disease state samples but only 46% of the healthy urine samples, the latter mainly due to the lower protein content in healthy urine as opposed to the disease state samples derived from patients with various high levels of proteinuria (Figure 5A). For urine samples, the reported concentrations of ε-(γ-glutamyl)lysine were normalized to mg protein measured by the BCA method and expressed as ng ε-(γ-glutamyl)lysine equivalents per mg protein (Figure 5B). These pre-screened human urine samples were used and mixed to generate endogenous urine QC samples that yield ε-(γ-glutamyl)-lysine at two different concentrations (low and medium). These served to demonstrate method validation by performance evaluation of endogenous analytes in urinary proteins. The measurement precision of ε-(γ-glutamyl)-lysine was determined by measuring ε-(γ-glutamyl)-lysine and protein in endogenous QC samples four times at each of two concentrations, each of which was tested with six independent replicates per run. Table 1 shows that the method exhibits good intra-run and inter-run precision, which is less than 15% and 25%, respectively.

[0125] Selectivity was further confirmed during method validation, showing (i) selectivity towards the internal standard ε-(γ-glutamyl)-[U-13C6,15N2-lysine] assessed in both the absence and presence (at ULOQ) of ε-(γ-glutamyl)-lysine, in the absence of the internal standard in human urine, as well as in the presence of the internal standard in surrogate matrices, and (ii) selectivity towards ε-(γ-glutamyl)-lysine and ε-(γ-glutamyl)-[U-13C6,15N2-lysine] in the presence of the known ε-(γ-glutamyl)-lysine isomers EK acid, KE acid and H-Lys(retro-Glu-H)-OH (Table 1). In the analyzed alternative blank matrices spiked with one each of the three isomers separately, no significant interfering peaks were observed at the retention times of ε-(γ-glutamyl)-lysine and ε-(γ-glutamyl)-[U-13C6,15N2-lysine] (Figure 4B for EK and KE chromatograms). Furthermore, parallelism was successfully demonstrated by showing a linear concentration-response relationship to the calibration curve of ε-(γ-glutamyl)-lysine in individual digested human urine samples (Table 1). Thus, along with the successful parallelism demonstrated with the BCA method (data not shown), it was shown that the method did not suffer from matrix effects. Furthermore, low and medium QC levels of ε-(γ-glutamyl)-lysine were found to be stable in human urine stored in polypropylene containers when stored at the sample processing temperature (room temperature) for 24 hours, after 134 days in a freezer set at -80 °C, and after four freeze-thaw cycles (nominal -80 °C / room temperature) (Table 1). Finally, carryover was assessed for ε-(γ-glutamyl)-lysine and ε-(γ-glutamyl)-[U-13C6,15N2-lysine] in digested surrogate blank matrices, analyzed sequentially after the highest calibration standard, and deemed acceptable, i.e., no samples were identified in which carryover effects could introduce a bias of >15% in the measured concentrations.

[0126] Overall conclusion A relatively rapid, efficient and selective method is proposed to determine crosslinks associated with TG activity (N'N' bis(γ-glutamyl)-polyamine dipeptides / isopeptides and / or ε-(γ-glutamyl)-lysine dipeptides / isopeptides) in human urine. The method consists of three systematic steps, the first of which is an efficient and reproducible protein precipitation that allows the removal of major salts and the concentration of proteins in a buffer suitable for subsequent digestion. The digestion process is simplified and streamlined to facilitate complete digestion. Among other things, the approach allows the digestion of urinary proteins without exogenous protease digestion, allowing efficient removal of proteases prior to analysis. Crosslinks were successfully quantified via LC-MS / MS from a series of clinical urine samples from patients with several diseases. The sensitivity of the method has been shown to be able to detect as low as 0.1 ng / ml of epsilon (gamma-glutamyl) lysine in human urine with a process-wide coefficient of variation of less than 20%. The method represents a significant advance over previous approaches based on amino acid analysis.

[0127] References 1. Z. Szondy, et al., BioMedicine (2017) 7:1-13. 2. Schafer et al., J. Agric. Food Chem (2005) 53:2830-2837. 3. International Publication No. 2015 / 014888 4. International Publication No. 2013175229 5. International Publication No. 2015 / 197772 [Table 2] [Table 3]

Claims

1. 1. A method for detecting crosslinks formed by transglutaminase (TG) in a biological sample, comprising: a) digesting proteins present in said biological sample using enzymes immobilized on beads; and b) detecting crosslinks formed by TGs in said biological sample by liquid chromatography-tandem mass spectrometry (LC-MS / MS); A method comprising:

2. 1. A method for determining transglutaminase (TG) activity in a subject, comprising: a) digesting proteins present in a biological sample from said subject using enzymes immobilized on beads; and b) detecting crosslinks formed by TGs in said biological sample by liquid chromatography-tandem mass spectrometry (LC-MS / MS); A method comprising:

3. (i) the crosslinks formed by TG comprise N'N'bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide; (ii) the method further comprises a step a0) of obtaining the biological sample from a subject; and / or (iii) the method further comprises a step a1) of concentrating the proteins present in the biological sample prior to digestion, and optionally, concentrating the proteins comprises precipitating the proteins; The method according to claim 1 or 2.

4. The enzyme immobilized on the beads is selected from an endopeptidase, an exopeptidase, a prolidase, or any combination thereof, and optionally the enzyme immobilized on the beads is (i) selected from proteinase K, pronase, prolidase, leucine aminopeptidase, carboxypeptidase Y, or any combination thereof; (ii) comprising or consisting of proteinase K, pronase, prolidase, leucine aminopeptidase and carboxypeptidase Y; The method according to claim 1 or 2.

5. (i) the enzymatic digestion of step a) produces a mixture of free amino acids and bridged dipeptides and / or isopeptides formed by TG, said mixture being purified and prepared for LC-MS / MS; (ii) the crosslink formed by TG comprises an ε-(γ-glutamyl)-lysine dipeptide, and optionally the ε-(γ-glutamyl)-lysine dipeptide is detected using LC-MS / MS as having a retention time equivalent to that of a stable isotope-labeled version of the dipeptide, with a parent ion mass-to-charge ratio (m / z) of 276, and by detecting a range of fragment ion masses derived from the parent ion, such as m / z 147, 84, and 130; and / or (iii) the method further comprises a step c) of quantifying the crosslinks formed by TG by LC-MS / MS; The method according to claim 1 or 2.

6. (i) the biological sample is or has been isolated from a subject; (ii) the biological sample is urine; (iii) the subject is a mammal, optionally a primate, preferably a human; and / or (iv) the subject has or is suspected of having a disease associated with TG activity, such as TG2 activity; The method according to claim 1 or 2.

7. 1. An in vitro method for aiding in the diagnosis of disease in a subject, comprising: a) digesting proteins present in said biological sample from said subject using enzymes immobilized on beads; and b) detecting crosslinks formed by TGs in said biological sample by liquid chromatography-tandem mass spectrometry (LC-MS / MS); A method comprising:

8. 1. An in vitro method for aiding in stratifying disease severity in a subject, comprising: a) digesting proteins present in said biological sample from said subject using enzymes immobilized on beads; and b) detecting crosslinks formed by TGs in said biological sample by liquid chromatography-tandem mass spectrometry (LC-MS / MS); A method comprising:

9. (i) the crosslinks formed by TG comprise N'N'bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide; (ii) the method further comprises a step c) of determining and / or quantifying the amount / number of crosslinks formed by TGs in the biological sample; (iii) the presence of crosslinks formed by TG in the biological sample indicates that the subject has a disease; (iv) the greater the amount / number of crosslinks formed by TGs in said biological sample, the more severe the disease and / or the worse the prognosis of said disease; and / or (v) the disease is any disease associated with elevated TG activity, and optionally the disease is fibrosis, a fibrotic disease, or a fibrosis-related disease, such as chronic kidney disease, progressive kidney disease, pulmonary fibrosis, systemic sclerosis, liver cirrhosis, cardiovascular disease, idiopathic hypertrophic cardiomyopathy, renal fibrosis, primary glomerulonephritis, liver cirrhosis, chronic allograft injury (CAI), post-transplant renal fibrosis, or chronic allograft nephropathy; 9. The method according to claim 7 or 8.

10. 1. An in vitro method for aiding in monitoring disease progression in a subject, comprising: a) digesting proteins present in a biological sample from said subject using enzymes immobilized on beads; and b) detecting crosslinks formed by TGs in said biological sample by liquid chromatography-tandem mass spectrometry (LC-MS / MS); and c) comparing the amount / number of crosslinks formed by TG in said biological sample with the amount / number of crosslinks formed by TG in a previous sample from said subject or a control value; A method comprising:

11. (i) the crosslinks formed by TG comprise N'N'bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide; (ii) the first biological sample was obtained from the subject about 12 months before the second biological sample was obtained from the subject; (iii) an increase in (i) the amount of crosslinks formed by TG in the first biological sample or (ii) the amount of crosslinks formed by TG in the second biological sample compared to the control value indicates that the disease is worsening, or a decrease in (i) the amount of crosslinks formed by TG in the first biological sample or (ii) the amount of crosslinks formed by TG in the second biological sample compared to the control value indicates that the disease is ameliorating; and / or (iv) the disease is any disease associated with elevated TG activity, and optionally the disease is fibrosis, a fibrotic disease, or a fibrosis-related disease, such as chronic kidney disease, progressive kidney disease, pulmonary fibrosis, systemic sclerosis, liver cirrhosis, cardiovascular disease, idiopathic hypertrophic cardiomyopathy, renal fibrosis, primary glomerulonephritis, liver cirrhosis, chronic allograft injury (CAI), post-transplant renal fibrosis, or chronic allograft nephropathy; The method of claim 10.

12. 1. A method for aiding in the assessment of a subject's response to a treatment, comprising: a) digesting proteins present in a first biological sample from said subject using enzymes immobilized on beads; and b) detecting crosslinks formed by TGs in said biological sample by liquid chromatography-tandem mass spectrometry (LC-MS / MS); and c) comparing the amount / number of crosslinks formed by TG in said biological sample with the amount / number of crosslinks formed by TG in a biological sample from a patient before treatment or at an early stage of treatment or with a control value; A method comprising:

13. (i) the crosslinks formed by TG comprise N'N'bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide; (ii) the method comprises: a. (i) comparing the amount / number of crosslinks formed by TG in said biological sample with (ii) the amount / number of crosslinks formed by TG in a biological sample from a different subject with said disease who is not administered said treatment, wherein a lower amount / number of crosslinks formed by TG in (i) compared to (ii) indicates that the treatment is effective; or b. (i) comparing the amount / number of crosslinks formed by TGs in the biological sample with (ii) the amount / number of crosslinks formed by TGs in a biological sample taken from the subject prior to treatment, wherein a lower amount / number of crosslinks formed by TGs in (i) compared to (ii) indicates that the treatment is effective. and / or (iii) the patient is treated with an inhibitor of TG, such as an anti-TG antibody; The method of claim 12.

14. 1. A method for assisting in the evaluation of the effect of an agent on TG activity, comprising: a) using an enzyme immobilized on beads to digest proteins present in a biological sample from a subject administered the agent; and b) detecting crosslinks formed by TGs in said biological sample by liquid chromatography-tandem mass spectrometry (LC-MS / MS); A method comprising:

15. (i) the crosslinks formed by TG comprise N'N'bis(γ-glutamyl)-polyamine dipeptide / isopeptide and / or ε-(γ-glutamyl)-lysine dipeptide / isopeptide; (ii) the method comprises: a. (i) comparing the amount / number of crosslinks formed by TG in said biological sample with (ii) the amount / number of crosslinks formed by TG in a second biological sample from a different subject not administered said agent, wherein a difference in the amount / number of crosslinks formed by TG in (i) compared to (ii) indicates that the agent is effective; or b. (i) comparing the amount / number of crosslinks formed by TG in the biological sample with (ii) the amount / number of crosslinks formed by TG in a second biological sample taken from the subject prior to administration of the agent, wherein a difference in the amount / number of crosslinks formed by TG in (i) compared to (ii) indicates that the agent is effective. further comprising (iii) the difference indicates target engagement by the agent; (iv) the difference is an increase, indicating that the agent is a TG activator, or the difference is a decrease, indicating that the agent is a TG inhibitor; and / or (v) the drug is a TG inhibitor such as an anti-TG antibody; 15. The method of claim 14.

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