Biomarkers and methods for measuring and monitoring inflammatory disease activity

Abstract

Provided herein is a methods for monitoring inflammatory disease activity in a subject comprising: (i) obtaining a sample from the subject; and (ii) measuring an autoantibody concentration in the sample using an immunoassay. Optionally, the inflammatory disease is rheumatoid arthritis (RA). Optionally, the autoantibody is an anti-citrullinated vimentin autoantibody (anti-Sa) or an anti-carbamylated protein autoantibody (anti-CarP). Optionally, the autoantibody concentration is correlated to a multi-disease biomarker activity (MBDA) score. Also provided herein are systems and kits for performing the method described herein.

Description

BIOMARKERS AND METHODS FOR MEASURING AND MONITORING INFLAMMATORY DISEASE ACTIVITYFIELD

[0001] Disclosed are methods, systems, and kits for monitoring inflammatory disease activity in a subject comprising measuring autoantibody concentration in a sample from the subject using an immunoassay.BACKGROUND

[0002] Inflammatory diseases affect millions of people worldwide and are difficult to treat due to a complex etiologies. Inflammatory diseases are associated with significant morbidity and disability and thus constitute a major socioeconomic burden. To achieve the maximum therapeutic benefits for individual subjects, it is important to be able to specifically quantify and assess the subject’s inflammatory disease activity at any particular time, determine the effects of treatment on disease activity, and predict future outcomes, including disease progression.SUMMARY

[0003] Provided herein is a method for monitoring inflammatory disease activity in a subject comprising: (i) obtaining a sample from the subject; and (ii) measuring the concentration of an autoantibody in the sample using an immunoassay. Optionally, steps (i) and (ii) of the method provided herein may be repeated one or more times. Optionally, the sample may be a serum sample. Optionally, the autoantibody concentration may be correlated to inflammatory disease activity. Optionally, the inflammatory disease may be rheumatoid arthritis (RA). Optionally, the method may further comprise comparing the measured autoantibody concentration to a reference autoantibody concentration, wherein a measured autoantibody concentration above the reference autoantibody concentration indicates a positive result and a measured autoantibody concentration below the reference autoantibody concentration indicates a negative result. A variety of autoantibodies may be measured using the methods and systems herein. For example, the measured autoantibody may be rheumatoid factor (RF). Or, the measured autoantibody may be an autoantibody that recognizes post-translationally modified proteins. For example, the autoantibody may be an anti-citrullinated vimentin autoantibody (anti-Sa), an anti-citrullinated mutated vimentin autoantibody (anti-MCV), an anti-citrullinated a-enolase peptide- 1 autoantibody (anti-CEP-1), an anti -cyclic citrullinated protein autoantibody (anti-CCP), and / or an anti-carbamylated protein autoantibody (anti-CarP). Optionally, the method further may comprise performing an immunoassay to measure protein expression of selected proteins, for example, interleukin-8 (IL-8) or myeloperoxidase (MPO). Or additional protein expression values may be measured. Optionally, the method may comprise comparing the measured protein expression value to a reference protein expression value, wherein a measured protein expression value above the reference protein expression value indicates a positive result and a measured protein expression value below the reference protein expression value indicates a negative result.

[0004] For example, the autoantibody concentration and / or protein expression values of additional markers (e.g., IL-8, MPO) may be correlated to a multi -disease biomarker activity (MBDA) score. For example, an MBDA score may (as disclosed further herein) indicate that a subject is at risk of an inflammatory disease. In such cases, further evaluation of autoantibody concentrations or additional protein markers may be performed. Or, autoantibody concentrations and / or protein expression values of additional markers (e.g., IL- 8, MPO) may indicate that a subject is at risk of an inflammatory disease. In such cases, further evaluation of autoantibody concentrations or additional protein markers may be performed. Thus, optionally, the method may further comprise performing an immunoassay to measure protein expression values of selected proteins that are relevant to an MBDA score. Optionally, the proteins may comprise at least one of chitinase 3 -like 1 (cartilage glycoprotein-39) (CHI3L1); C-reactive protein, pentraxin-related (CRP); epidermal growth factor (beta-urogastrone) (EGF); interleukin 6 (interferon, beta 2) (IL6); leptin (LEP); matrix metallopeptidase 1 (interstitial collagenase) (MMP1); matrix metallopeptidase 3 (stromelysin 1, progelatinase) (MMP3); resistin (RETN); serum amyloid Al (SAA1); tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A); vascular cell adhesion molecule 1 (VCAM1); and, vascular endothelial growth factor A (VEGFA). Optionally, a subset (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) or all twelve of these proteins may be measured.

[0005] The method may also comprise a step of calculating a multi-biomarker disease activity index score (MBDA) score for the subject using an interpretation function. Optionally, the interpretation function may comprise the protein expression values and at least one selected clinical variable. For example, clinical variables comprising age, sex, and adiposity may be included. The method may also include the step of adjusting the MBDA to a scale of 1-100, where optionally, a score of 1 to 29 corresponds to a low MBDA score, ascore of 30 to 44 corresponds to a moderate MBDA score, and a score of 45 to 100 corresponds to a high MBDA score. Optionally, the method may further comprise diagnosing or prognosing the subject as having: (a) a high level of inflammatory disease activity for a subject having a moderate or high MBDA score and a positive autoantibody result; or (b) a low level of inflammatory disease activity for a subject having a low MBDA score and a negative autoantibody result. Optionally, the method may further comprise diagnosing or prognosing the subject as having: (a) a high risk of rapid disease progression for a subject having a moderate or high MBDA score and a positive autoantibody result; or (b) a low risk of rapid disease progression for a subject having a low MBDA score and a negative autoantibody result. Optionally, the method may further comprise administering a therapy to the subject having a high level of inflammatory disease activity or high risk of rapid disease progression.

[0006] Also provided herein is a system for performing the method described herein. Also provided herein is a kit comprising reagents to measure the concentration of an autoantibody using any of the methods described herein and instructions for use.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The disclosure may be better understood by reference to the following non-limiting figures.

[0008] Figure 1 shows Vectra® DA biomarkers and disease activity categories.

[0009] Figure 2 shows a computing device for use with any of the methods or systems in accordance with an embodiment of the disclosure.DETAILED DESCRIPTION Definitions

[0010] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Known methods and techniques are generally performed according to conventional methods wellknown in the art and as described in various general and more specific references that are discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with the laboratory procedures and techniques described herein are those well-known and commonly used in the art.

[0011] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0012] As used herein, the terms “a,” “an,” and “the” can refer to one or more unless specifically noted otherwise.

[0013] The use of the term “or” is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” can mean at least a second or more.

[0014] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among samples. In certain embodiments, the term “about” includes values that are ± 10 % of the indicated value.

[0015] As used herein, the term “comprising” is intended herein to be open-ended, including not only the recited elements, but further encompassing any additional elements. The term “comprising” is intended to encompass the term “consisting of.”

[0016] As used herein, the term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.

[0017] As used herein, the terms “subject,” “patient” and “individual” are used interchangeably. A subject may be any mammal, including a human. The term “mammal” as used herein includes but is not limited to a human, non-human primate, dog, cat, mouse, rat, cow, horse, and pig. Mammals other than humans can be advantageously used as subjects that represent animal models of inflammation. A subject can be male or female. A subject canbe one who has been previously diagnosed or identified as having an inflammatory disease such as RA. A subject can be one who has already undergone, or is undergoing, a therapeutic intervention for an inflammatory disease. A subject can also be one who has not been previously diagnosed as having an inflammatory disease; e.g., a subject can be one who exhibits one or more symptoms or risk factors for an inflammatory condition, or a subject who does not exhibit symptoms or risk factors for an inflammatory condition, or a subject who is asymptomatic for inflammatory disease.

[0018] As used herein, a “sample” refers to a sample of tissue or fluid isolated from a subject including, but not limited to, for example, blood, plasma, serum, fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid, samples of the skin, external secretions from the body, such as from skin, respiratory, intestinal and genitourinary tracts, tears, saliva, milk, blood cells, organs, biopsies and also in vitro cell culture constituents, for example conditioned media resulting from the growth of cells and tissues in culture medium, for example recombinant cells and cell components. Samples can be obtained from a subject by means including but not limited to venipuncture, excretion, ejaculation, massage, biopsy, needle aspirate, lavage, scraping, surgical incision, or intervention or other means known in the art.

[0019] As used herein, the term “inflammatory disease” refers to, without limitation, any disease, as defined herein, resulting from the biological response of vascular tissues to harmful stimuli, including but not limited to such stimuli as pathogens, damaged cells, irritants, antigens and, in the case of autoimmune disease, substances and tissues normally present in the body. Non-limiting examples of inflammatory disease include RA, SLE, Sjogren’s syndrome, biliary cirrhosis, or biliary cholangitis, ankylosing spondylitis, psoriatic arthritis, atherosclerosis, asthma, autoimmune diseases, chronic inflammation, chronic prostatitis, glomerulonephritis, hypersensitivities, inflammatory bowel diseases, pelvic inflammatory disease, reperfusion injury, transplant rejection, and vasculitis. “Inflammatory disease activity” is used herein to refer to effects or symptoms resulting from inflammatory disease.

[0020] As used herein, “disease progression” refers to worsening of disease symptoms, for example, tissue destruction, cartilage loss and joint erosion. Disease progression may be assessed using radiographs (i.e., x-rays), and termed “radiographic progression.”

[0021] As used herein, the terms "protein," "peptide," and "polypeptide" are used interchangeably to denote an amino acid polymer or a set of two or more interacting or bound amino acid polymers. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymer.

[0022] The term “amino acid” refers to naturally occurring amino acids, modified or synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code. Modified amino acids include, e.g., hydroxyproline, y- carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.

[0023] As used herein, the term “post-translationally modified proteins” refers to a protein which has been changed after it has been synthesized through translation. Post-translational modifications may involve the addition or removal of chemical groups, such as a cyanate group (carbamylation), phosphate groups (phosphorylation), sugars (glycosylation), lipids (lipidation), acetyl (acetylation), hydroxyl (hydroxylation), nitric oxide (nitrosylation), or ubiquitin (ubiquitination). Post-translational modifications may also involve the conversion of one amino acid to another, such as the conversion of arginine to citrulline (citrullination).

[0024] As used herein, the term “antibody” refers to a polypeptide consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are typically classified as either kappa or lambda. Heavy chains are typically classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one“light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms “variable light chain” (VL) and “variable heavy chain” (VH) refer to these light and heavy chains respectively.

[0025] An antibody can be specific for a particular antigen. For example, an anti-human IgG antibody is specific to human IgG. The antibody or its antigen can be either an analyte or a binding partner.

[0026] As used herein, “autoantibody” refers to an antibody specific to (i.e. reactive with) a constituent of tissue of the subject that produces the autoantibody. The term is intended to include an antibody produced by a subject’s immune system that is directed against one or more of the subject’s own antigens. Accordingly, autoantibodies can be produced by a subject’s immune system when the immune system fails to distinguish in whole or in part, between self and non-self-tissue constituents. As provided herein, exemplary autoantibodies include an anti-Sa, or anti-CarP, and others as described herein.

[0027] As used herein, a “reference autoantibody concentration” or “reference autoantibody value” refers to the concentration of an autoantibody taken from a healthy individual without inflammatory disease. Likewise, a “reference protein expression value” or “a reference protein concentration” refers to a protein expression value taken from a healthy individual without inflammatory disease. The reference autoantibody concentration / value or reference protein expression value / concentration can be measured at the same time as the subject’s sample autoantibody concentration / value or protein expression value / concentration, or can be determined based on previous measurements.

[0028] As used herein, the term “immunoassay” refers to a test that uses the binding of antibodies (e.g., autoantibodies) to antigens to identify and measure certain substances. As used herein, an immunoassay may be used to measure the concentration of an autoantibody.

[0029] As used herein, the term “multi-biomarker disease activity index score,” “MBDA score,” or “MBDA,” refers to a score that uses quantitative data (e.g., protein level data) to provide a quantitative measure of inflammatory disease activity or the state of inflammatory disease in a subject. For example, protein level data, such as from one or more biomarkers from the set of protein biomarkers described herein, is input into an interpretation function according to the present teachings to derive the MBDA score. The interpretation function, insome embodiments, can be created from predictive or multivariate modeling based on statistical algorithms. Input to the interpretation function can comprise the results of testing of the twelve disclosed set of biomarkers (or a subset thereof), alone or in combination with clinical parameters and / or clinical assessments, also described herein. In some embodiments, the MBDA score is a quantitative measure of inflammatory disease activity. In some embodiments, the MBDA score is a quantitative measure of RA disease activity. MBDA as used herein can refer to a VECTRA® DA score.

[0030] As used herein, a “score” refers to a value or set of values selected so as to provide a quantitative measure of a variable or characteristic of a subject’s condition, and / or to discriminate, differentiate or otherwise characterize a subject’s condition. The value(s) comprising the score can be based on, for example, quantitative data resulting in a measured amount of one or more sample constituents obtained from the subject, or from clinical parameters, or from clinical assessments, or any combination thereof. In certain embodiments the score can be derived from a single constituent, parameter, or assessment, while in other embodiments the score is derived from multiple constituents, parameters, and / or assessments. The score can be based upon or derived from an interpretation function; e.g., an interpretation function derived from a particular predictive model using any of various statistical algorithms known in the art. A “score” as used herein can be used interchangeably with MBDA score as defined above.

[0031] “Interpretation function,” as used herein, refers to the transformation of a set of observed data into a meaningful determination of particular interest; e.g., an interpretation function may be a predictive model that is created by utilizing one or more statistical algorithms to transform a dataset of observed biomarker data into a meaningful determination of disease activity or the disease state of a subject.

[0032] As used herein, a “therapeutic regimen,” “therapy” or “treatment(s),” refers to the clinical management of a subject and interventions, whether biological, chemical, physical, or a combination thereof, intended to sustain, ameliorate, improve, or otherwise alter the condition of a subject. These terms may be used synonymously herein. In some cases, a treatment may be tailored to an inflammatory disease. Treatments include but are not limited to administration of prophylactics or therapeutic compounds (including conventional DMARDs, biologic DMARDs, non-steroidal anti-inflammatory drugs (NS AID’ s) such as COX-2 selective inhibitors, and corticosteroids), exercise regimens, physical therapy, dietarymodification and / or supplementation, bariatric surgical intervention, administration of pharmaceuticals and / or anti-inflammatories (prescription or over-the-counter), and any other treatments known in the art as efficacious in preventing, delaying the onset of, or ameliorating disease. A “response to treatment” includes a subject’s response to any of the above-described treatments, whether biological, chemical, physical, or a combination of the foregoing.

[0033] The embodiments set forth below and recited in the claims can be understood in view of the above definitions.Methods

[0034] Disclosed herein are methods for monitoring inflammatory disease activity in a subject comprising: (i) obtaining a sample from the subject; and (ii) measuring the concentration of an autoantibody in the sample using an immunoassay. Optionally, steps (i) and (ii) are repeated one or more times. The steps of the methods are further described below.I. Obtaining a sample.

[0035] The methods include obtaining a sample. In this context, the term “obtaining” is to be construed broadly. The term is not intended to refer exclusively to the subject from which the sample came. For example, a technician in an off-site clinical laboratory can be said to “obtain” the sample.

[0036] The method is not limited to any particular means of sample handling. In some embodiments, it may be useful to separate the sample into two or more fractions prior to the subsequent steps. In some such embodiments, two or more of such fractions may be prepared differently, for example, to help improve the sensitivity or selectivity of the detection of an autoantibody. In some embodiments, the method may include preparing a single sample for repeat injections across autoantibody detection systems. The method is not limited to any particular sample size.

[0037] The sample may comprise a biological sample. In such embodiments, the sample may also include other components, such as solvents, buffers, anticlotting agents, and the like. In some embodiments, the sample has been processed and prepared for detection of an autoantibody. Such processing may be useful for optimizing the effectiveness of subsequent steps. Such processing methods are well known to those of skill in the art.

[0038] The sample is not limited to any particular sample type. In some embodiments, the sample can be one or more of whole blood, plasma, serum, urine, cerebrospinal fluid, tissue homogenate, saliva, amniotic fluid, bile, mucus, peritoneal fluid, or lymphatic fluid. In some embodiments, the sample may be serum or plasma. In some embodiments, the sample may be obtained from a drug-treated subject. For example, the sample can be obtained from a subject treated with a conventional DMARDs, biologic DMARDs, non-steroidal anti-inflammatory drugs (NSAID’s) such as COX-2 selective inhibitors, and corticosteroids, or an analogue or derivative thereof. The method is not limited to any particular volume of sample. In some embodiments, the sample may be at least about 1-100 pL, at least about 10-75 pL, or at least about 15-50 pL in volume. In certain embodiments, the sample may be at least about 20 pL in volume.

[0039] Optionally, the sample may be obtained from a subject who has or may be suspected of having an inflammatory disease. Inflammatory diseases include rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjogren syndrome, dermatomyositis / polymyositis, scleroderma / systemic sclerosis, mixed connective disease / overlap syndrome, ankylosing spondylitis, psoriatic arthritis, reactive arthritis, enteropathic arthritis associated with inflammatory bowel diseases (Crohn’s disease and ulcerative colitis), granulomatous polyangiitis (Wegener’s granulomatosis), microscopic polyangiitis, eosinophilic granulomatosis polyangiitis (Churg Strauss syndrome), Takayasu arteritis, temporal arteritis (giant cell arteritis), polymyalgia rheumatica, Bechet’s syndrome, polyarteritis nodosa, Henoch-Scholein purpura, osteoarthritis, osteoporosis, gout and pseudogout, fibromyalgia, biliary cirrhosis, biliary cholangitis, and regional muscular skeletal disorders (e.g., back pain, shoulder pain, or the like). Optionally, the inflammatory disease may be RA. The subject need not be diagnosed with an inflammatory disease. The subject may be suspected of having the inflammatory disease, for example, by a clinician.IL Measuring the concentration of an autoantibody in the sample using an immunoassay.

[0040] The methods include measuring the concentration of an autoantibody in the sample using an immunoassay. Depending on the nature of the sample, a variety of immunoassay techniques known in the art may be used. For example, enzyme-linked immunosorbent assays (ELISA), Western blot, and radioimmunoassays can be used as described herein to detect the presence of the secondary antibody bound to the autoantibody. In some embodiments, the second antibody may be labeled such that the complex may be detected specifically owing tointrinsic properties of the label such as, for example, fluorescence, radioactivity, enzymatic activity, visibility in NMR, or MRI spectra or the like. In some embodiments, the detection method may include any of Western blot, dot blot, protein microarray, ELISA, line blot radioimmuno assay, immunoprecipitation, indirect immunofluorescence microscopy, radioimmunoassay, radioimmunodiffusion, ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistostaining, complement fixation assay, FACS, and protein chip, but is not limited thereto.

[0041] Measuring the concentration of an autoantibody in the sample using an immunoassay may mean, for example, combining the sample and at least one labeled (e.g., fluorescently-conjugated) secondary antibody specific to the target autoantibody (e.g., anti- IgG or anti-IgA) in a cell culture dish and detecting the label (e.g., detecting fluorescence). The secondary antibody bound to the autoantibody used in an immunoassay may be detected using a variety of methods known to the person skilled in the art, depending on the label used, for example immunofluorescence microscopy or spectroscopy, luminescence, NMR spectroscopy, immunodiffusion, radioactivity, chemical crosslinking, surface plasmon resonance, native gel electrophoresis, or enzymatic activity.

[0042] Optionally, the secondary antibody bound to the autoantibody may be detected by immunofluorescence. Optionally, the detection may further comprise determining a colocalization of the secondary antibody and the innate cell or antigen to which the autoantibody binds. Determining co-localization is not limited to any particular method. Colocalization may be determined using confocal microscopy, or another method for colocalization. Ultimately, co-localization of the secondary antibody and the innate cell or antigen to which the autoantibody binds may be used to demonstrate specific autoantibodies are present.

[0043] Optionally, the autoantibody may be rheumatoid factor (RF). Rheumatoid factor is an autoantibody which recognizes the Fc portion of an immunoglobulin G (IgG) antibody. Optionally, the autoantibody detected may recognize (i.e., bind to) post-translationally modified proteins. The post translational modification is not limited by type, and may include addition or removal of chemical groups, carb amyl ati on, phosphorylation, glycosylation, lipidation, acetylation, hydroxylation, nitrosylation, or ubiquitination. Post-translational modifications may also involve the conversion of one amino acid to another, e.g., citrullination. Optionally, the autoantibody may recognize citrullinated or carbamylatedproteins. Optionally, the autoantibody may be an anti-citrullinated vimentin autoantibody (anti-Sa), an anti-citrullinated mutated vimentin autoantibody (anti-MCV), an anti- citrullinated a-enolase peptide- 1 autoantibody (anti-CEP-1), an anti-cyclic citrullinated protein autoantibody (anti-CCP), and / or an anti-carbamylated protein autoantibody (anti- CarP).

[0044] Optionally, the autoantibody concentration may be correlated to inflammatory disease activity. For example, a greater measured autoantibody concentration, or positive result, may indicate increased inflammatory disease activity. Optionally, the method may further comprise comparing the measured autoantibody concentration to a reference autoantibody concentration (taken from a healthy individual without inflammatory disease). Optionally, the reference autoantibody concentration may be measured at the same time as the subject’s sample autoantibody concentration or can be determined based on previous measurements. Optionally, a measured autoantibody concentration above the reference autoantibody concentration may indicate a positive result and a measured autoantibody concentration below the reference autoantibody concentration may indicate a negative result.MBDA Score

[0045] Optionally, the autoantibody concentration may be correlated to a multi-disease biomarker activity (MBDA) score. As described above, and at least in U.S. Patent Nos. 9,200,324 and 11,300,575, each of which are incorporated herein in their entireties by reference, an MBDA score is a score that uses quantitative data (e.g., protein expression data) to provide a quantitative measure of inflammatory disease activity or the state of inflammatory disease in a subject. For example, protein level data, such as from the set of protein biomarkers described herein, is input into an interpretation function according to the present teachings to derive the MBDA score.

[0046] Optionally, the method for monitoring inflammatory disease activity described herein may further comprise: performing an immunoassay to measure protein expression values of at least one (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) of chitinase 3 -like 1 (cartilage glycoprotein-39) (CHI3L1), C-reactive protein, pentraxin-related (CRP), epidermal growth factor (beta-urogastrone) (EGF), interleukin 6 (interferon, beta 2) (IL6); leptin (LEP), matrix metallopeptidase 1 (interstitial collagenase) (MMP1), matrix metallopeptidase 3 (stromelysin 1, progelatinase) (MMP3), resistin (RETN),serum amyloid Al (SAA1), tumor necrosis factor receptor superfamily, member 1 A (TNFRSF1A), vascular cell adhesion molecule 1 (VCAM1), and vascular endothelial growth factor A (VEGFA); calculating an MBDA score for the subject using an interpretation function comprising the protein expression values and values of clinical variables comprising age, sex, and adiposity; and adjusting the MBDA score to a scale of 1-100, wherein a score of 1 to 29 corresponds to low MBDA score, a score of 30 to 44 corresponds to moderate MBDA score, and a score of 45 to 100 corresponds to a high MBDA score. Optionally, protein expression values of all twelve of CHI3L1, CRP, EGF, IL6, LEP, MMP1, MMP3, RETN, SAA1, TNFRSF1A, VCAM1, and VEGFA are assessed. The type of immunoassay used to detect protein expression may be determined by one skilled in the art and may be an immunoassay as described above or elsewhere herein. Optionally, serum leptin concentration may be a surrogate for adiposity as described at least in U.S. Patent Pub. No. 20200249243, which is incorporated by reference in its entirety herein.

[0047] The interpretation function used in calculating the MBDA score is not limited to a particular type and may be derived from a particular predictive model using any of various statistical algorithms known in the art. Established statistical algorithms and methods well- known in the art, useful as models or useful in designing predictive models, can include but are not limited to: analysis of variants (ANOVA); Bayesian networks; boosting and Ada- boosting; bootstrap aggregating (or bagging) algorithms; decision trees classification techniques, such as Classification and Regression Trees (CART), boosted CART, Random Forest (RF), Recursive Partitioning Trees (RPART), and others; Curds and Whey (CW); Curds and Whey-Lasso; dimension reduction methods, such as principal component analysis (PCA) and factor rotation or factor analysis; discriminant analysis, including Linear Discriminant Analysis (LDA), Eigengene Linear Discriminant Analysis (ELD A), and quadratic discriminant analysis; Discriminant Function Analysis (DFA); factor rotation or factor analysis; genetic algorithms; Hidden Markov Models; kernel based machine algorithms such as kernel density estimation, kernel partial least squares algorithms, kernel matching pursuit algorithms, kernel Fisher's discriminate analysis algorithms, and kernel principal components analysis algorithms; linear regression and generalized linear models, including or utilizing Forward Linear Stepwise Regression, Lasso (or LASSO) shrinkage and selection method, and Elastic Net regularization and selection method; glmnet (Lasso and Elastic Net- regularized generalized linear model); Logistic Regression (LogReg); meta-learner algorithms; nearest neighbor methods for classification or regression, e.g. Kth-nearestneighbor (KNN); non-linear regression or classification algorithms; neural networks; partial least square; rules based classifiers; shrunken centroids (SC); sliced inverse regression; Standard for the Exchange of Product model data, Application Interpreted Constructs (StepAIC); super principal component (SPC) regression; and, Support Vector Machines (SVM) and Recursive Support Vector Machines (RSVM), among others. Additionally, clustering algorithms as are known in the art can be useful in determining subject sub-groups. Various interpretation functions and algorithms used therein are described in U.S. Patent No. 11,656,227, which is incorporated herein by reference in its entirety.

[0048] Optionally, a high MBDA score (>44) may be associated with an increased risk of disease or radiographic progression while a low MBDA score (<30) may be associated with a reduced risk of disease or radiographic progression. Additionally, a high MBDA score may be associated with increased autoantibody concentrations. Optionally, the method may further comprise diagnosing or prognosing the subject as having: (a) a high level of inflammatory disease activity for a subject having a moderate or high MBDA score and a positive autoantibody result; or (b) a low level of inflammatory disease activity for a subject having a low MBDA score and a negative autoantibody result. Optionally, the method may further comprise diagnosing or prognosing the subject as having: (a) a high risk of rapid disease progression for a subject having a moderate or high MBDA score and a positive autoantibody result; or (b) a low risk of rapid disease progression for a subject having a low MBDA score and a negative autoantibody result. Optionally, the method may further comprise administering a therapy to the subject having a high level of inflammatory disease activity or high risk of rapid disease progression. The term “administering” as used herein is not limited to a particular method or route but refers to the placement of a therapy into a subject by a method or route that results in at least partial localization of the composition at a desired site such that a desired effect is produced. Routes of administration may include both local and systemic administration.

[0049] The therapy used in the method described herein is likewise not limited to a certain therapy and may depend on the particular inflammatory disease being treated. Therapies useful in the present method may be conventional or biologic. Examples of therapies, such as disease modifying anti-rheumatic drugs (DMARD) that are generally considered conventional include, but are not limited to, methotrexate (MTX), azathioprine (AZA), bucillamine (BUC), chloroquine (CQ), ciclosporin (CSA, or cyclosporine, or cyclosporin),doxycycline (DOXY), hydroxychloroquine (HCQ), intramuscular gold (IM gold), leflunomide (LEF), levofloxacin (LEV), and sulfasalazine (SSZ). Conventional therapies can also include nonsteroidal anti-inflammatory drugs (NDAIDs), such as aspirin, ibuprofen, oxaprozin, prioxicam, indomethacin, etodolac, meclofenamate, meloxicam, naproxen, ketoprofen, nabumetome, tolmetin sodium, and diclofenac. Examples of other conventional therapies may include, but are not limited to, folinic acid, D-pencillamine, gold auranofin, gold aurothioglucose, gold thiomalate, cyclophosphamide, and chlorambucil. Examples of biologic drugs may include but are not limited to biological agents that target the tumor necrosis factor (TNF)-alpha molecules and the TNF inhibitors, such as infliximab, adalimumab, etanercept and golimumab. Other classes of biologic drugs may include IL1 inhibitors such as anakinra, T-cell modulators such as abatacept, B-cell modulators such as rituximab, and IL6 inhibitors such as tocilizumab.

[0050] To identify additional therapeutics or drugs that are appropriate for a specific subject, a test sample from the subject may also be exposed to a therapeutic agent or a drug, and the level of one or more protein biomarkers can be determined. The level of one or more protein biomarkers may be compared to sample derived from the subject before and after treatment or exposure to a therapeutic agent or a drug, or may be compared to samples derived from one or more subjects who have shown improvements in inflammatory disease state or activity (e.g., clinical parameters or traditional laboratory risk factors) as a result of such treatment or exposure.Protein Markers

[0051] Optionally, the method may further comprise performing an immunoassay to measure additional protein markers. In some embodiments, these additional protein marker expression values may not be used in the determination of an MBDA score. In some embodiments, the additional protein marker expression values may provide additional predictive value for assessing a subject’s risk of inflammatory disease and / or inflammatory disease progression. Optionally, protein expression values of interleukin-8 (IL-8) or myeloperoxidase (MPO) may be measured. As described above, a variety of immunoassay techniques known in the art may be used. For example, enzyme-linked immunosorbent assays (ELISA), Western blot, and the like may be used as described herein to detect the presence of the target protein when bound to an antibody. Optionally, the antibody may be labeled such that the complex may be detected specifically owing to intrinsic properties of the label suchas, for example, fluorescence, radioactivity, enzymatic activity, visibility in NMR, or MRI spectra or the like. In some embodiments, the detection method may include any of Western blot, dot blot, protein microarray, ELISA, line blot radioimmuno assay, immunoprecipitation, indirect immunofluorescence microscopy, radioimmunoassay, radioimmunodiffusion, ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistostaining, complement fixation assay, FACS, and protein chip, but is not limited thereto. While some of these methods allow for the direct detection of the target protein-labeled antibody complex, in some embodiments, a secondary antibody binds to the antibody bound to the target protein and the secondary antibody is labeled.

[0052] Optionally, the protein expression values of IL8 or MPO may correspond with inflammatory disease activity. Optionally, the method may further comprise comparing IL8 or MPO protein expression values to reference IL8 or MPO protein expression values (taken from a healthy individual without inflammatory disease). Optionally, a sample with IL8 or MPO protein expression values above the reference protein expression values may be assigned a positive result and a sample with IL8 or MPO protein expression values below the reference protein expression values concentration may be assigned a negative result. The method may comprise diagnosing or prognosing the subject as having: (a) a high level of inflammatory disease activity for a subject having a positive IL8 or MPO protein expression value result; or (b) a low level of inflammatory disease activity for a subject having a negative IL8 or MPO protein expression value result. In another embodiment, the method may comprise diagnosing or prognosing the subject as having: (a) at high risk for inflammatory disease progression for a subject having a positive IL8 or MPO protein expression value result; or (b) at low risk for inflammatory disease progression for a subject having a negative IL8 or MPO protein expression value result. Optionally, the methods may further comprise administering a therapy to the subject having a high level of inflammatory disease activity or at high risk for inflammatory disease progression.Systems and Kits

[0053] Also provided herein are systems for performing the methods for monitoring inflammatory disease activity in a subject described herein. For example, the system may optionally comprise a station and / or component for obtaining a sample obtained from the subject. Additionally, and / or alternatively, the system may comprise a station and / or component for measuring the concentration of an autoantibody and / or the expression valuesof protein markers in the sample. Optionally, the measurement of an autoantibody and / or protein marker may be performed using an immunoassay. Additionally, and / or alternatively, the system may comprise a station and / or component for analyzing (e.g., generating an MBDA score) and / or interpreting the results (i.e., assessing whether the subject is at a high, medium or low risk for an inflammatory disease). The system may also comprise instructions for using any of the stations and / or components.

[0054] Any of the method steps, stations or components may be automated, robotically controlled, and / or controlled at least in part by a computer 200 and / or programmable software. Thus, the system may comprise a computer-program product tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to run the system or any part (e.g., station or component) of the system and / or perform a step or steps of the methods of any of the disclosed embodiments. In some embodiments, a system is provided that includes one or more data processors and a non-transitory computer readable storage medium containing instructions which, when executed on the one or more data processors, cause the one or more data processors to perform part or all of one or more methods or processes disclosed herein and / or run any of the parts of the systems disclosed herein.

[0055] For example, disclosed is a system comprising one or more data processors, and / or a non-transitory computer readable storage medium containing instructions which, when executed on the one or more data processors, cause the one or more data processors to perform actions to direct at least one of the steps of: (i) obtaining the sample; and (ii) measuring the concentration of an autoantibody concentration and / or protein expression value in the sample using an immunoassay. The instructions may further comprise comparing the measured autoantibody concentration and / or protein expression value to a reference autoantibody concentration and / or protein expression value, wherein a measured autoantibody concentration and / or protein expression value above the reference autoantibody concentration and / or protein expression value indicates a positive result and a measured autoantibody concentration and / or protein expression value below the reference autoantibody concentration and / or protein expression value indicates a negative result.

[0056] Additionally, and / or alternatively, the instructions may comprise calculating a multi -biomarker disease activity index score (MBDA) score for the subject using an interpretation function. Also, the instructions may further comprise diagnosing or prognosingthe subject as having: (a) a high risk of rapid disease progression for a subject having a moderate or high MBDA score and a positive autoantibody result; or (b) a low risk of rapid disease progression for a subject having a low MBDA score and a negative autoantibody result. The systems and computer products may perform any of the methods disclosed herein. One or more embodiments described herein can be implemented using programmatic modules, engines, or components. A programmatic module, engine, or component can include a program, a sub-routine, a portion of a program, a software component, or a hardware component capable of performing one or more stated tasks or functions. As used herein, a module or component can exist on a hardware component independently of other modules or components. Alternatively, a module or component can be a shared element or process of other modules, programs or machines.

[0057] FIG. 2 shows a block diagram of an analysis system 200 used for the methods described herein. As illustrated in FIG. 2, modules, engines, or components (e.g., program, code, or instructions) executable by one or more processors may be used to implement the various subsystems of an analyzer system according to various embodiments. The modules, engines, or components may be stored on a non-transitory computer medium. As needed, one or more of the modules, engines, or components may be loaded into system memory (e.g., RAM) and executed by one or more processors of the analyzer system. In the example depicted in FIG. 2, modules, engines, or components are shown for implementing the methods of the disclosure.

[0058] Thus, FIG. 2 illustrates an example of a computing device 200 suitable for use with systems and methods according to this disclosure. The example of a computing device 200 includes a processor 205, which is in communication with the memory 210 and other components of the computing device 200 using one or more communications buses 215. The processor 205 is configured to execute processor-executable instructions stored in the memory 210 to perform one or more methods or operate one or more stations or components for measuring autoantibody concentrations according to different examples, such as those illustrated in FIG. 1 or disclosed elsewhere herein. In this example, the memory 210 may store processor-executable instructions 225 that can analyze 220 results for sample or test unit confirmation as discussed herein.

[0059] The computing device 200 in this example may also include one or more user input devices 230, such as a keyboard, mouse, touchscreen, microphone, etc., to accept user input.The computing device 200 may also include a display 235 to provide visual output to a user, such as a user interface. The computing device 200 may also include a communications interface 240. In some examples, the communications interface 240 may enable communications using any of a variety of available protocols including without limitation TCP / IP (transmission control protocol / Internet protocol), SNA (systems network architecture), IPX (Internet packet exchange), AppleTalk®, and the like. Merely by way of example, network(s) may be a local area network (LAN), networks based on Ethernet, Token-Ring, a wide-area network (WAN), the Internet, a virtual network, a virtual private network (VPN), an intranet, an extranet, a public switched telephone network (PSTN), an infra-red network, a wireless network (e.g., a network operating under any of the Institute of Electrical and Electronics (IEEE®) 102.11 suite of protocols, Bluetooth®, and / or any other wireless protocol), and / or any combination of these and / or other networks. The computer- usable or computer-readable medium may be, but is not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, device, or propagation medium. More specific examples (a non-exhaustive list) of the computer-readable medium would include the following: an electrical connection having one or more wires, a portable computer diskette, a random-access memory (RAM), including a magnetic RAM, core memory, magnetic disk storage mediums, optical storage mediums including optical fibers a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), a portable compact disc read-only memory (CD-ROM), and / or other computer- readable mediums for storing information. The computer-usable or computer-readable medium could even be paper or another suitable medium, upon which the program is printed, as the program can be electronically captured, via, for instance, optical scanning of the paper or other medium, then compiled, interpreted, or otherwise processed in a suitable manner if necessary, and then stored in a computer memory.

[0060] Computer program code for carrying out operations of the present disclosure may be written in an object-oriented programming language such as R®, JavaScript®, Java®, Java7®, Smalltalk®, Python®, Lab VIEW®, C++®, MATLAB®, SQL®, or VisualBasic®. However, the computer program code for carrying out operations of the present disclosure may also be written in conventional procedural programming languages, such as the “C” programming language or even assembly language. The program code may execute entirely on the user's computer, partly on the user's computer, as a stand-alone software package, partly on the user's computer and partly on a remote computer or entirely on the remotecomputer. In the latter scenario, the remote computer may be connected to the user's computer through a local area network (LAN) or a wide area network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet Service Provider).

[0061] Also provided herein is a kit comprising one or more reagents to for measuring the concentration of an autoantibody in the sample using an immunoassay and instructions for use. For example, the one or more reagents may be one or more secondary antibodies as described herein, for example an anti-IgG or anti-IgA antibody.

[0062] As used herein, a “kit” refers to a package containing the listed reagent, and instructions of any form that are provided in connection with the reagent in a manner such that a clinical professional will clearly recognize that the instructions are to be associated with the reagent. Optionally, the kit may include a positive control, for example, a reference autoantibody with known properties to validate the measurement of autoantibodies in the sample. Optionally, the kit may include both positive and negative controls.

[0063] “Instructions” typically involve written text or graphics on or associated with packaging of materials. Instructions also can include any oral or electronic instructions provided in any manner. Written text or graphics may include a website URL or a QR code encoding a website URL, where other instructions or supplemental information may be provided in electronic form.

[0064] The method to which the instructions relate may be as described hereinabove. Optionally, the disclosed kits to monitor inflammatory disease activity in a subject comprise reagents and / or instructions to: obtain a sample and measuring the concentration of an autoantibody in the sample using an immunoassay. The compositions and / or kit may further comprise reagents to contact the autoantibody with a secondary antibody conjugated to a fluorophore, e.g., anti-human IgG antibody conjugated to FITC and detect the anti-human IgG antibody bound to the autoantibody using immunofluorescence.

[0065] The disclosed systems and kits may contain one or more containers, each of which can contain one or more of the materials used in the reagent for measuring autoantibody concentration. The systems and kits also may contain instructions for mixing, diluting, using, or administering the materials. The systems and kits also can include other containers with one or more solvents, surfactants, preservatives, buffers, washes, and / or diluents (e.g., normalsaline (0.9% NaCl), or 5% dextrose) as well as containers for mixing, diluting, incubating, washing, etc. The materials may be provided in any suitable form, for example, as a liquid solution or as a dried product. When the material provided is a dry product, the material may be reconstituted by the addition of solvent, which may also be provided by the system (e.g. composition and / or kit). In embodiments where liquid forms of a material are used, the liquid form may be concentrated or ready to use. The kit, in one embodiment, may comprise a carrier being compartmentalized to receive in close confinement one or more containers such as vials, tubes, and the like.

[0066] In addition to the reagent for measuring the concentration of autoantibodies, in embodiments, the systems and kits may also comprise a detectable moiety, and any materials required to allow the moiety to generate a detectable signal. Optionally, the detectable moiety is a fluorophore. In some embodiments, the systems comprise other instruments as for example, a confocal microscope, a flow cytometer, or another instrument for visualizing a detectable moiety, such as fluorescence for measuring the concentration of autoantibodies in the sample. For example, in some embodiments, the compositions and / or kits are used with other instruments for measuring the concentration of autoantibodies in the sample. For example, the compositions and / or kits may be used with a confocal microscope, a flow cytometer, or another instrument for visualizing a detectable moiety, such as fluorescence.

[0067] The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results.ExamplesExample 1: Monitoring inflammatory disease activity using autoantibodies.

[0068] The present example illustrates a method for monitoring inflammatory disease activity in a subject. A sample is obtained from the subject, for example, a serum sample. The subject has or is suspected of having an inflammatory disease, for example RA. The autoantibody concentration in the sample is then measured using an immunoassay, for example, an ELISA to detect autoantibodies (e.g., anti-Sa or anti-CarP). The autoantibody concentration in the sample is compared to a reference autoantibody concentration, and a sample with an autoantibody concentration above the reference autoantibody concentration isassigned a positive result and a sample with an autoantibody concentration below the reference autoantibody concentration is assigned a negative result.

[0069] The autoantibody concentration is correlated to an MBDA score, which is obtained from performing an immunoassay to measure performing an immunoassay to measure protein expression values of at least one of CHI3L1, CRP, EGF, IL6, LEP, MMP1, MMP3, RETN, SAA1, TNFRSF1A, VCAM1, and VEGFA, then an interpretation function with the protein expression values and values of age, sex, and adiposity is used to calculate the MBDA score, then the MBDA score is adjusted to a scale of 1-100. Serum leptin concentration can be a surrogate for adiposity. A score of 1-29 is assigned a low MBDA score, a score of 30-44 is assigned a moderate MBDA score, and a score of 45-100 is assigned a high MBDA score. If the subject has a moderate or high MBDA score and a positive autoantibody result, the subject is assigned a high level of inflammatory disease activity and is then treated using a DMARD or biologic therapy, for example. If the subject has a low MBDA score and a negative autoantibody result, the subject is assigned a low level of inflammatory disease activity and is not treated.Example 2: Assessing rheumatoid arthritis (RA) disease activity using anti-Sa and anti- CarP autoantibodies.

[0070] Autoantibodies against citrullinated and carbamylated proteins in RA have been associated with more aggressive disease. Vectra® Disease Activity (DA) is a biomarkerbased blood test that yields a RA disease activity score from 1 to 100. Here, 120 patient samples were analyzed to assess the relationship of disease activity as measured by Vectra® score and the presence of anti-Sa (Citrullinated Vimentin) and anti-CarP (Carbamylated Protein).

[0071] Serum samples were analyzed for Vectra® DA (which is validated for adults with RA) where 12 biomarker measurements (VCAM-1, EGF, VEGF-A, IL-6, TNF-RI, MMP-1, YKL-40, Leptin, Resistin, SAA, CRP) together with patient age, gender and adiposity are used to generate a score where established categories are low for 1 to 29, moderate for 30 to 44, and high for 45 to 100. 40 samples with the lowest Vectra® DA (average score = 11.8, range from 3.8 to 18.1). Vectra® is described at least in U.S. Patent No. 9,200,324 and 11,300,575, each of which are incorporated herein in their entireties by reference. Anti-Sa and anti-CarP were analyzed by ELISA with a cut-off for positive of >20 Units (U).

[0072] Anti-Sa and anti-CarP for 40 samples from each Vectra® DA category (low, moderate, and high) were reported. 40 with the highest Vectra® score (mean =58.4, range 49.0 to 87.5), and 40 with moderate Vectra® scores (mean=37.2, range 32.4 to 41.8) were further analyzed for the two markers, anti-SA and anti-CarP. Patients with the highest Vectra® scores (mean 58.2, range 49.0 to 87.5) had highest rates of positivity for anti-Sa (50.0%) and anti-CarP (35%). In contrast, the group with the lowest Vectra® scores (mean 11.8, range 3.8 - 18.1) were infrequently positive for anti-Sa (17.5%) and anti-CarP (0%). Frequencies with moderate Vectra® scores (mean 37.1, range 32.4 - 41.8) were mid-range: 25% for anti-Sa and 7.5% for anti-CarP. Of note, positivity of anti-Sa was analytically stronger, i.e. higher titer, in patients with moderate or high Vectra® (mean 90.0U or 81.7U), compared to values seen in those with low Vectra® (mean 60.4 U).

[0073] The analysis of 120 patient samples demonstrates that high disease activity as determined by Vectra® DA corresponds to higher positivity of autoantibody markers, anti-Sa and anti-CarP, both which have reported associations with more severe RA, as shown in Table 1 below.Table 1. Anti-SA and anti-CarP in patients with low, moderate, and high Vectra® DA scores.

[0074] High Vectra® DA scores (>44) are associated with a 20% 1-year risk of radiographic progression while low scores (<30) carry only a 1% risk. Here, we found that patients with high Vectra® scores were 3 times more likely to test positive for anti-Sa than those with low disease activity scores. At the same time, anti-CarP occurred in high frequency (35%) with high Vectra® in contrast to zero positivity observed with low Vectra® scores. Thus, these findings support anti-CarP’s reported association with more active disease with higher risk of developing joint erosions. Also of note, patients with high Vectra® scores had particularly high anti-Sa positivity — both a 52.5% positivity rate as well as higher anti-Sa levels (titers) than thegroup with lower Vectra® scores. These data corroborate other studies where anti-Sa not only predicted more aggressive, rapid disease progression, but its titers correlated with clinical measures of disease activity. Taken together, these data support the consistency and clinical usefulness of the Vectra® score along with autoantibody markers, anti-Sa and anti-CarP, in order to assess RA severity, prognosis, and patient-specific treatment strategies.Example 3: EmbodimentsAl . A method for monitoring inflammatory disease activity in a subject comprising: i. obtaining a sample from the subject; and ii. measuring the concentration of an autoantibody in the sample using an immunoassay.A2. The method of embodiment Al, wherein the autoantibody concentration is correlated to inflammatory disease activity.A3. The method of any of embodiments Al or A2, wherein the inflammatory disease is rheumatoid arthritis (RA).A4. The method of any of embodiments A1-A3, wherein the autoantibody is rheumatoid factor (RF).A5. The method of any of embodiments A1-A4, wherein the autoantibody recognizes post-translationally modified proteins.A6. The method of any of embodiments A1-A5, wherein the autoantibody is an anti-citrullinated vimentin autoantibody (anti-Sa), an anti-citrullinated mutated vimentin autoantibody (anti-MCV), an anti-citrullinated a-enolase peptide-1 autoantibody (anti-CEP-1), an anti -cyclic citrullinated protein autoantibody (anti- CCP), and / or an anti-carbamylated protein autoantibody (anti-CarP).A7. The method of any of embodiments A1-A6, wherein the method further comprises performing an immunoassay to measure protein expression values of interleukin-8 (IL-8) or myeloperoxidase (MPO).A8. The method of any of embodiments A1-A7, wherein the sample is a serum sample.A9. The method of any of embodiments A1-A8, wherein steps (i) and (ii) are repeated one or more times.A10. The method of any of embodiments A1-A9 wherein the autoantibody concentration is correlated to a multi-disease biomarker activity (MBDA) score.Al 1. The method of any of embodiments A1-A10, wherein the method further comprises: performing an immunoassay to measure protein expression values of at least twelve protein markers in the sample, comprising chitinase 3 -like 1 (cartilage glycoprotein-39) (CHI3L1), C-reactive protein, pentraxin-related (CRP), epidermal growth factor (beta-urogastrone) (EGF), interleukin 6 (interferon, beta 2) (IL6), leptin (LEP); matrix metallopeptidase 1 (interstitial collagenase) (MMP1), matrix metallopeptidase 3 (stromelysin 1, progelatinase) (MMP3), resistin (RETN), serum amyloid Al (SAA1), tumor necrosis factor receptor superfamily, member 1A (TNFRSF1 A), vascular cell adhesion molecule 1 (VCAM1), and vascular endothelial growth factor A (VEGFA); calculating an MBDA score for the subject using an interpretation function comprising the protein expression values and values of clinical variables comprising age, sex, and adiposity; and adjusting the MBDA score to a scale of 1-100, wherein a score of 1 to 29 corresponds to low MBDA score, a score of 30 to 44 corresponds to moderate MBDA score, and a score of 45 to 100 corresponds to a high MBDA score.A12. The method of any of embodiments Al-Al l, wherein the method further comprises comparing the measured autoantibody concentration and / or protein expression value to a reference autoantibody concentration and / or protein expression value, wherein a measured autoantibody concentration and / or protein expression value above the reference autoantibody concentration and / or protein expression value indicates a positive result and a measured autoantibody concentration / and or protein expression value below the reference autoantibody concentration and / or protein expression value indicates a negative result.A13. The method of embodiment Al 1 or A12, wherein the method further comprises diagnosing or prognosing the subject as having:(a) a high level of inflammatory disease activity for a subject having a moderate or high MBDA score and a positive autoantibody result; or(b) a low level of inflammatory disease activity for a subject having a low MBDA score and a negative autoantibody result.A14. The method of embodiment Al 1 or A12, wherein the method further comprises diagnosing or prognosing the subject as having:(a) a high risk of rapid disease progression for a subject having a moderate or high MBDA score and a positive autoantibody result; or(b) a low risk of rapid disease progression for a subject having a low MBDA score and a negative autoantibody result.A15. The method of embodiment A13 or A14, wherein the method further comprises administering a therapy to the subject having a high level of inflammatory disease activity or a high risk of rapid disease progression.Bl. A system for performing the method of any of embodiments Al -Al 5.Cl . A kit comprising reagents to measure an autoantibody concentration using any of the methods of embodiments Al -Al 5 and instructions for use.

[0075] The disclosed can be better understood by reference to the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for monitoring inflammatory disease activity in a subject comprising: a. obtaining a sample from the subject; and b. measuring the concentration of an autoantibody in the sample using an immunoassay.

2. The method of claim 1, wherein the autoantibody concentration is correlated to inflammatory disease activity.

3. The method of any of claims 1 or 2, wherein the inflammatory disease is rheumatoid arthritis (RA).

4. The method of any of claims 1-3, wherein the autoantibody is rheumatoid factor (RF).

5. The method of any of claims 1-4, wherein the autoantibody recognizes post- translationally modified proteins.

6. The method of any of claims 1-5, wherein the autoantibody is an anti-citrullinated vimentin autoantibody (anti-Sa), an anti-citrullinated mutated vimentin autoantibody (anti-MCV), an anti-citrullinated a-enolase peptide- 1 autoantibody (anti-CEP-1), an anti-cyclic citrullinated protein autoantibody (anti-CCP), and / or an anti-carbamylated protein autoantibody (anti-CarP).

7. The method of any of claims 1-6, wherein the method further comprises performing an immunoassay to measure protein expression values of interleukin-8 (IL-8) or myeloperoxidase (MPO).

8. The method of any of claims 1-7, wherein the sample is a serum sample.

9. The method of any of claims 1-8, wherein steps (i) and (ii) are repeated one or more times.

10. The method of any of claims 1-9, wherein the autoantibody concentration is correlated to a multi-biomarker disease activity (MBDA) score.

11. The method of any of claims 1-10, wherein the method further comprises: performing an immunoassay to measure protein expression values of at least twelve protein markers in the sample, comprising chitinase 3 -like 1 (cartilage glycoprotein-39) (CHI3L1), C-reactive protein, pentraxin-related (CRP), epidermal growth factor (beta-urogastrone) (EGF), interleukin 6 (interferon, beta 2) (IL6), leptin (LEP), matrix metallopeptidase 1 (interstitial collagenase) (MMP1), matrix metallopeptidase 3 (stromelysin 1, progelatinase) (MMP3), resistin (RETN), serum amyloid Al (SAA1), tumor necrosis factor receptor superfamily, member 1 A(TNFRSF1A), vascular cell adhesion molecule 1 (VCAM1), and vascular endothelial growth factor A (VEGFA); calculating an MBDA score for the subject using an interpretation function comprising the protein expression values and values of clinical variables comprising age, sex, and adiposity; and adjusting the MBDA to a scale of 1-100, wherein a score of 1 to 29 corresponds to low MBDA score, a score of 30 to 44 corresponds to moderate MBDA score, and a score of 45 to 100 corresponds to a high MBDA score.

12. The method of any one of claims 1-11, wherein the method further comprises comparing the measured autoantibody concentration and / or protein expression value to a reference autoantibody concentration and / or protein expression value, wherein a measured autoantibody concentration and / or protein expression value above the reference autoantibody concentration and / or protein expression value indicates a positive result and a measured autoantibody concentration / and or protein expression value below the reference autoantibody concentration and / or protein expression value indicates a negative result.

13. The method of claim 11 or 12, wherein the method further comprises diagnosing or prognosing the subject as having:(a) a high level of inflammatory disease activity for a subject having a moderate or high MBDA score and a positive autoantibody result; or(b) a low level of inflammatory disease activity for a subject having a low MBDA score and a negative autoantibody result.

14. The method of claim 11 or 12, wherein the method further comprises diagnosing or prognosing the subject as having:(a) a high risk of rapid disease progression for a subject having a moderate or high MBDA score and a positive autoantibody result; or(b) a low risk of rapid disease progression for a subject having a low MBDA score and a negative autoantibody result.

15. The method of claim 13 or 14, wherein the method further comprises administering a therapy to the subject having a high level of inflammatory disease activity or a high risk of rapid disease progression.

16. A system for performing the method of any of claims 1-15.

17. A kit comprising reagents to measure the concentration of an autoantibody using any of the methods of claims 1-15 and instructions for use.

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