Biomarkers used to characterize the disease activity of systemic lupus erythematosus (SLE) using immune indices.

Abstract

Methods for characterizing disease activity in patients with systemic lupus erythematosus (SLE). These methods include obtaining blood, serum, plasma, or urine samples from the patient; assessing the expression of biomarkers selected from IFN-α, IL-10, BlyS, IL-7, IFN-γ, TRAIL, IL-15, IP-10 / CXCL10, and IL-4 in the samples; assessing the expression of inflammatory mediators selected from TNFRII, resistin, and osteopontin (OPN) in the samples; assessing SLE-related autoantibody-specific biomarkers selected from dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP in the samples; and calculating the Lupus Disease Activity (Immune) Index (LDAII / L-DAI) score. The LDAII / L-DAI score can distinguish between active and low lupus disease activity. Treatment methods are also provided, including administration after determining that the patient has a prognostic risk of transitioning to classified SLE but before reaching the clinical disease classification.

Description

[0001] Government licensing rights

[0002] This invention was developed with government support from the National Institutes of Health (NIH) under license numbers AI142967, AR073750, AI144292, and GM104938. The government holds certain rights to this invention.

[0003] Cross-reference to related applications

[0004] This application claims the benefit and priority of U.S. Provisional Application No. 63 / 533,721, filed August 21, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0005] This application generally relates to the field of biomarkers for calculating immune indices characterizing the disease activity of systemic lupus erythematosus (SLE). Background Technology

[0006] Without limiting the scope of the invention, the background is described in conjunction with systemic lupus erythematosus (SLE).

[0007] Systemic autoimmune diseases, including SLE, affect a significant proportion of the US population. Recent population-based studies indicate a prevalence of 73 per 100,000. Lim et al., 2014; Somers et al., 2014 The Lupus Foundation estimates that the number of SLE patients in routine clinical practice is as high as 470 per 100,000. SLE presents with a range of clinical symptoms; disease classification depends on meeting four of the eleven American College of Rheumatology (ACR) criteria. Hochberg, 1997; Tan et al., 1982 ), 4 out of 11 criteria from the international collaborative clinical trial (SLICC) for systemic lupus erythematosus (SLE). Petri, Orbai, et al., 2012 ) or ANA positive (≥1:80 titer, as determined by HEp-2 IIF), a clinical criterion, and an SLE score ≥10 from the European League Against Rheumatism (EULAR) / ACR classification criteria (from 7 clinical and 3 immunological domains) (Aringer et al., 2019). Over 90% of affected patients are women aged 15 to 45 years. The prevalence is higher in minority groups and those with lower socioeconomic status. Feldman et al., 2013 Persistent active clinical disease and its treatment put patients at risk of organ damage. Kasitanon et al., 2015; Oglesby et al., 2014; Thong & Olsen, 2017 ), including damage to the central nervous system, lungs, cardiovascular system, and kidneys ( Barral et al., 2009; Bruce et al., 2015; Conti et al., 2016; Faurschou et al., 2006 Lupus nephritis and end-stage renal disease Alarcon et al., 2006; Faurschou et al., 2006Patients with fluctuating (waxing / waning) disease and those with clinically active or quiescent disease are each at risk of experiencing a clinical onset of disease. Ng et al., 2006; Steiman et al., 2010 ).

[0008] Systemic lupus erythematosus (SLE) is a clinically and serologically heterogeneous systemic autoimmune disease that causes severe morbidity and early mortality, particularly in young women and minority populations. Pathogenic autoantibodies and immune dysregulation in chronic inflammatory forms lead to a wide range of clinical manifestations, including rash, arthritis, and life-threatening kidney and / or central nervous system damage. Many antinuclear autoantibodies (ANA) have been shown to accumulate specifically in SLE patients; the use of hydroxychloroquine can eliminate autoantibody accumulation and counteract clinical disease activity. Early intervention is an attractive treatment option for SLE. However, our understanding of the pathogenic mechanisms of SLE disease activity is inadequate. Closing this knowledge gap will improve our ability to identify individuals at risk of increased disease activity and permanent organ damage, define windows of opportunity for early intervention, and facilitate the development of therapies targeting specific pathways.

[0009] Identifying and treating diseases early to prevent tissue and organ damage is challenging because signs and symptoms of high disease activity are often only detected after they have occurred. Despite the availability of validated clinical disease activity tools (…), Hay et al., 1993; Lam & Petri, 2005; Petri et al., 2005 Despite improved treatment strategies, persistent active disease remains a burden for SLE patients. Peschken et al., 2018 Increased morbidity and early mortality associated with treatments (especially steroids) required to manage active diseases. Al Sawah et al., 2015; Sheane et al., 2017; Thamer et al., 2009; Zonana-Nacach et al., 2000 ), and permanent organ damage ( Lopez et al., 2012; Petri, Purvey, et al., 2012 ), including kidney damage ( Maroz & Segal, 2013 This further increased costs. In addition, the steroids required to manage disease activity ( Al Sawah et al., 2015 ) and other immunosuppressants ( Durcan & Petri, 2016 Long-term use of steroids is associated with increased morbidity. Failure to proactively manage clinical disease limits medical care to reactive therapy, excluding the use of steroids or increasing their use, and the need for caution in the use of immunomodulators. Doria et al., 2014 ) to prevent end-organ damage ( Kasitanon et al., 2015; Oglesby et al., 2014; Thong & Olsen, 2017 And reduce the pathogenicity and socioeconomic burden of SLE. Lau & Mak, 2009 (Proactive strategy)

[0010] Currently, biomarkers for SLE have limited effectiveness in predicting permanent organ damage. Although SLE-related autoantibodies (e.g., anti-dsDNA, anti-splicing, and anti-Ro / SSA) accumulate in SLE patients, their presence is insufficient to predict persistent active disease and progression to permanent organ damage. ANA has also been found in the serum of patients with other systemic rheumatic diseases, healthy individuals who do not progress to SLE (including some unaffected family members of SLE patients), and up to 14% of the general population. Because individuals can remain healthy even with ANA positivity, ANA positivity alone may not be the sole pathogenic driver of SLE. In addition to ANA positivity, dysregulation of various immune pathways driven by soluble mediators may contribute to the development of clinical disease. No single factor or mechanism may be sufficient to explain the complexity and heterogeneity of SLE pathogenesis; therefore, a multivariate longitudinal approach is necessary to characterize early disease pathogenesis and identify unique parameters predicting SLE disease activity.

[0011] Despite numerous clinical trials of targeted immune pathway therapies over more than 50 years (including the first FDA-approved SLE drug, belimumab (Manzi et al., 2012)), most of these studies have failed, partly due to a lack of understanding of the dysregulated immune pathways in a given patient. The need for immune-based biomarkers as surrogate endpoints of clinical disease activity is becoming increasingly urgent. In routine practice, administration burden limits the use of validated measures of SLE clinical disease activity. Validated disease activity tools, such as the currently used Mixed Systemic Lupus Erythematosus Disease Activity Index (MSE), are... Thanou et al., 2014; Thanou et al., 2016 ) and the British Isles Lupus Assessment Group (BILAG) index ( Hay et al., 1993 It is labor-intensive, and as these clinical tools are updated, continuous and specialized training is required. Isenberg et al., 2005 Relying solely on physician experience to assess clinical disease activity carries the risk of undesirable variability and negative outcomes. Mikdashi & Nived, 2015; Mosca et al., 2011 ).

[0012] The clinical heterogeneity of SLE forms the basis of the following scientific premise: heterogeneous immune dysregulation forms the basis of clinical disease activity. The inventors have previously demonstrated that prior to a clinical SLE onset, patients exhibit immune dysregulation, feedforward mechanisms amplify with tissue damage, leading to clinical sequelae and ultimately reaching the disease classification (…). Lu et al., 2016; Munroe et al., 2016 The inventors also described a variety of SLE-associated autoantibodies (AutoAbs) and the accumulation of dysregulated inflammatory and immunomodulatory pathways. Lu et al., 2016; Munroe et al., 2016However, to date, there is a lack of disease management tests in SLE that provide information from immune mechanisms. This stems from the absence of any biomarkers that provide information from a single immune pathway, serving as a universal substitute for current or future clinical disease activity. Arriens et al., 2017 ).

[0013] Therefore, new methods are still needed to detect, track, and assess the disease activity and progression of SLE. Summary of the Invention

[0014] In one embodiment, the present invention includes a method for characterizing disease activity in patients with systemic lupus erythematosus (SLE), comprising: (a) obtaining a dataset associated with blood, serum, plasma, or urine samples from the patient, wherein the dataset includes data representing the levels of one or more biomarkers in the blood, serum, plasma, or urine samples from each of (b) through (e); (b) assessing a dataset of the presence or amount of protein expression of nine serum or plasma mediator biomarkers: IFN-α, IL-10, B lymphocyte stimulating factor (BLyS or BAFF), IL-7, IFN-γ, TRAIL, IL-15, IP-10 / CXCL10, and IL-4; (c) assessing a dataset of the presence or amount of protein expression of at least one serum or plasma mediator biomarker selected from: TNFRII, resistin, and osteopontin (OPN); (d) assessing a dataset of the presence or amount of protein expression of at least one serum or plasma mediator biomarker selected from: Data sets of the presence or quantity of plasma mediator biomarkers: IL-12p70, TNF-α, MIG / CXCL9, IL-5, IL-13, IL-1β, IL-17A, IL-2Rα, natural TGF-β, Fas, MCP-1 / CCL2, stem cell factor (SCF), IL-1RA, IL-6, MIP-1α / CCL3, MIP-1β / CCL4, MCP-3 / CCL7, total TGF-β, RANTES / CCL5, TNFRI, and IL-8 / CXCL8; (e) Data sets assessing the presence or quantity of at least one SLE-related autoantibody-specific biomarker selected from the following: dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP; and (f) Calculating the lupus disease activity (immune) index (LDAII / L-DAI / L-DAI) score. On one hand, at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 biomarkers are used in the calculation of LDAII / L-DAI / L-DAI. On the other hand, the dataset is log-transformed; standardized; weighted by Spearman r correlation for autoantibody specificity in the dataset; and summed over soluble protein biomarkers to equal the LDAII / L-DAI / L-DAI score. Alternatively, on the other hand, the dataset is log-transformed; standardized; weighted by Spearman r correlation for hSLEDAI in the dataset; and summed over soluble protein biomarkers to equal the LDAII / L-DAI score.Alternatively, in another aspect, the dataset is log-transformed; standardized; weighted by the average Spearman r correlation for autoantibody specificity and hSLEDAI in the dataset; and summed for soluble protein markers to equal the composite LDAII / L-DAI / L-DAI score. In another aspect, performing at least one immunoassay includes: obtaining a first sample containing a protein marker; contacting the first sample with multiple different reagents; generating multiple different complexes between the reagents and the markers; and detecting the complexes to generate data. In another aspect, at least one immunoassay includes multiplex assays. In another aspect, LDAII / L-DAI / L-DAI classifies the severity or progression level of SLE into serological (dsDNA binding and low complement) active (SA) or serological quiescent (SQ) clinically active (CA) or clinically quiescent (CQ) disease. In another aspect, the LDAII / L-DAI / L-DAI score distinguishes between active lupus disease activity and low lupus disease activity. In another aspect, the method also includes administering treatment to the patient prior to the attainment of clinically active disease, wherein the treatment includes at least one of the following: hydroxychloroquine (HCQ), belimumab, nonsteroidal anti-inflammatory drugs, steroids, or disease-modifying antirheumatic drugs (DMARDs).

[0015] In another embodiment, the present invention includes a method for assessing the disease activity and progression of clinical disease in a patient with systemic lupus erythematosus (SLE), comprising: obtaining a blood, serum, plasma, or urine sample from the patient; performing at least one immunoassay on the sample from the patient to generate a dataset for the presence or amount of protein expression, comprising nine serum or plasma mediator biomarkers: IFN-α, IL-10, B lymphocyte stimulating factor (BLyS or BAFF), IL-7, IFN-γ, TRAIL, IL-15, IP-10 / CXCL10, and IL-4, and at least one biomarker from each of the following (1) to (3): (1) assessing a dataset for assessing the presence or amount of protein expression from at least one of the following innate serum or plasma mediator biomarkers: TNFRII, resistin, and osteopontin (OPN); (2) assessing a dataset for assessing at least one of the following innate serum or plasma mediator biomarkers: TNFRII, resistin, and osteopontin (OPN); A dataset of the presence or amount of a serum or plasma mediator of a protein expression of: IL-12p70, TNF-α, MIG / CXCL9, IL-5, IL-13, IL-1β, IL-17A, IL-2Rα, natural TGF-β, Fas, MCP-1 / CCL2, stem cell factor (SCF), IL-1RA, IL-6, MIP-1α / CCL3, MIP-1β / CCL4, MCP-3 / CCL7, total TGF-β, RANTES / CCL5, TNFRI and IL-8 / CXCL8; and (3) a dataset assessing the presence or amount of at least one SLE-associated autoantibody-specific biomarker selected from: dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP and RNP; and calculating the LDAII / L-DAI score. On one hand, at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 biomarkers are used in the calculation of LDAII / L-DAI. On the other hand, the dataset is log-transformed; standardized; weighted by Spearman r correlation for autoantibody specificity in the dataset; and summed over soluble protein biomarkers to equal the Lupus Disease Activity Immune Index (LDAII / L-DAI) score. Alternatively, on the other hand, the dataset is log-transformed; standardized; weighted by Spearman r correlation for hSLEDAI in the dataset; and summed over soluble protein biomarkers to equal the LDAII / L-DAI score.Alternatively, in another aspect, the dataset is log-transformed; standardized; weighted by the average Spearman r correlation for autoantibody specificity and hSLEDAI in the dataset; and summed over soluble protein markers to equal the composite LDAII / L-DAI / L-DAI score. In another aspect, performing at least one immunoassay includes: obtaining a sample containing a protein marker; contacting the sample with multiple different reagents; generating multiple different complexes between the reagents and the markers; and detecting the complexes to generate data. In another aspect, at least one immunoassay includes multiplex assays. In another aspect, LDAII / L-DAI classifies the severity or progression level of SLE into serological (dsDNA binding and low complement) active (SA) or serological quiescent (SQ) clinically active (CA) or clinically quiescent (CQ) disease. In another aspect, the LDAII / L-DAI score distinguishes between active lupus disease activity and low lupus disease activity. In another aspect, the method further includes treating SLE patients after determining the prognosis of a patient with clinically active disease and before reaching a clinical disease classification, wherein the treatment includes at least one of the following: hydroxychloroquine (HCQ), belimumab, nonsteroidal anti-inflammatory drugs, steroids, or disease-modifying antirheumatic drugs (DMARDs). In another aspect, obtaining a dataset associated with a sample includes: obtaining the sample, and processing the sample to experimentally determine a dataset, or wherein obtaining a first dataset associated with the sample includes: receiving the dataset from a third party that has processed the sample to experimentally determine a dataset. In another aspect, increases in the biomarkers IFN-α, IL-10, BlyS, IL-7, TRAIL, IP-10 / CXCL10, TNFRII, resistin, and OPN indicate renal organ involvement.

[0016] In another embodiment, the invention includes a method for calculating a lupus disease activity (immune) index (LDAII / L-DAI) by measuring the expression levels of a set of biomarkers in a subject, comprising: determining a set of biomarker measurements by immunoassay in a physiological sample, wherein the biomarkers are peptides, proteins, peptides with post-translational modifications, proteins with post-translational modifications, or combinations thereof; wherein the physiological sample is whole blood, blood plasma, blood serum, or combinations thereof; wherein the set of biomarkers includes a dataset of the presence or amount of protein expression of nine biomarkers: IFN-α, IL-10, B lymphocyte stimulating factor (BLyS or BAFF), IL-7, IFN-γ, TRAIL, IL-15, IP-10 / CXCL10, and IL-4, plus measurements of at least one biomarker dataset selected from the following: TNFRII, resistin, and osteopontin (OPN), and the presence or amount of at least one biomarker dataset selected from the following: IL-8 / CXCL8, IP-10 / CXCL10, MIG / CXCL9, MIP-1 α / CCL3, MIP-1β / CCL4, MCP-1 / CCL2, RANTES / CCL5, and MCP-3 / CCL7; presence or amount of at least one of the following soluble TNF superfamily biomarker datasets: IL-12p70, TNF-α, MIG / CXCL9, IL-5, IL-13, IL-1β, IL-17A, IL-2Rα, natural TGF-β, Fas, MCP-1 / CCL2, stem cell factor (SCF), IL-1RA, IL-6, MIP-1 The dataset includes α / CCL3, MIP-1β / CCL4, MCP-3 / CCL7, total TGF-β, RANTES / CCL5, TNFRI, and IL-8 / CXCL8; and the presence or expression of at least one SLE-related autoantibody-specific biomarker selected from the following: dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP; and the calculation of the LDAII / L-DAI score, wherein the dataset is log-transformed; normalized; weighted by Spearman r correlation for autoantibody specificity in the dataset; and summed over soluble protein biomarkers to equal the LDAII / L-DAI score. Alternatively, in another aspect, the dataset is log-transformed; normalized; weighted by Spearman r correlation for hSLEDAI in the dataset; and summed over soluble protein biomarkers to equal the LDAII / L-DAI score.Alternatively, on the other hand, the dataset is log-transformed; standardized; weighted by the average Spearman r correlation of the autoantibody specificity and hSLEDAI in the dataset; and summed over soluble protein markers to equal the composite LDAII / L-DAI / L-DAI score. On the other hand, the method further includes using a set of biomarker measurements in subjects with a classification system to classify samples of the presence or development of systemic lupus erythematosus (SLE) as clinically active (CA) or clinically quiescent (CQ) disease with serological (dsDNA binding and low complement) activity (SA) or serological quiescence (SQ), wherein the classification system is a machine learning system comprising classification and regression trees selected from Fisher's exact test, Mann-Whitney test, Kruskal-Wallis test, Kruskal-Wallis test with Dunn multiple comparisons, Spearman rank correlation, or all of the above; and calculating the lupus disease activity immune index (LDAII / L-DAI), wherein the LDAII / L-DAI score distinguishes active SLE disease activity from low SLE disease activity (low clinical disease [hSLEDAI < 4]). On the other hand, the method further includes distinguishing between clinically quiescent and serologically quiescent (CQSQ) SLE patients compared to healthy controls. In another aspect, the method further includes using a total of at least 9 or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 biomarkers in the calculation of LDAII / L-DAI. In another aspect, the immunoassay is a multiplex immunoassay. On the other hand, LDAII / L-DAI is further calculated as follows: the concentration biomarkers of the subjects are measured and log-transformed, and the soluble mediator levels of each log-transformed subject sample are standardized as follows: (observed value) - (mean of all SLE patient and healthy control visits) / (standard deviation of all SLE patient and healthy control visits); Spearman coefficients are generated from a linear regression model that tests the association between each soluble mediator assessed in SLE patients and the specificity of one or more autoantibodies compared to healthy controls (Spearman r); the values ​​of the soluble mediator levels of the subjects are transformed and standardized, and said values ​​are weighted (multiplied) by their respective Spearman coefficients (Spearman r); and for each participant visit, each of the four or more soluble mediators is log-transformed, standardized, and weighted and summed to calculate LDAII / L-DAI.On the other hand, an increase in LDAII / L-DAI indicates at least one of the following: SLE disease progression, increased autoimmune disease activity, or organ damage. Attached Figure Description

[0017] The following figures are part of and included in this cost specification to further illustrate certain aspects of the invention. A better understanding of the invention can be achieved by referring to one or more of these figures in conjunction with a detailed description of the specific embodiments presented herein.

[0018] Figure 1 The importance of 33 immune mediators, determined by random forest, was used to distinguish between SLE patients with active disease activity (hSLEDAI ≥ 4) and low disease activity (hSLEDAI < 4), as well as matched healthy controls (the top mediators, determined by forward selection, are above the line; the remaining mediators, determined by backward elimination, are below the line). Red mediators indicate LDAII / L-DAI information provided by 9 mediators (IL-12p70 was excluded because it was not significant in the univariate analyses of Tables 2 and 3). Purple mediators, when combined with the top mediators, form LDAII / L-DAI combinations that provide information by 10, 11, or 12 mediators. All other mediators, in blue or black, also provide information for LDAII / L-DAI.

[0019] Figure 2 The lupus disease activity (immune) index (LDAII / L-DAI), weighted by hSLEDAI score (A) or the number of autoantibodies specific (B), provided information from nine mediators (L-DAI 9), distinguishing SLE patients with clinical (C) and / or serological (S) active (A) or quiescent (Q) disease from SLE patients with low disease activity (hSLEDAI < 4) or active disease activity (hSLEDAI ≥ 4) to race / sex / age-matched healthy individuals (Ct1). Log-transformed and standardized data for each soluble mediator in SLE patients and controls were weighted by comparing soluble mediator levels with Spearman r of hSLEDAI score (A) or the number of SLE-related autoantibodies specific (B) present. The figure shows L-DAI 9 mean ± SEM. p < 0.05 ; p < 0.001 ; p < 0.0001 (c) Kruskal-Walis test and Dunn multiple comparisons; Spearman correlation of L-DAI9 score weighted by hSLEDAI score (x-axis) or number of autoantibody specificities (y-axis).

[0020] Figure 3 The composite LDAII / L-DAI (weighted by the number of autoantibody-specific numbers and hSLEDAI score) based on nine mediators distinguishes between SLE patients (A) with clinical (C) and / or serological (S) active (A) or quiescent (Q) disease, and SLE patients (B) with low disease activity (hSLEDAI < 4) or active disease activity (hSLEDAI ≥ 4) matched with race / sex / age-matched healthy individuals (Ct1). The top left figure shows the mean LDAII / L-DAI 9-point SEM. p < 0.05 ; p < 0.001 ; p < 0.0001 Kruskal-Wallis test and Dunn multiple comparisons were used. The upper right figure shows the predicted probability of active disease determined by logistic regression. Low / medium (19% threshold probability; L-DAI 9 = -1.7577) and medium / high (79%; threshold probability L-DAI 9 = 2.0624) distinguishing CASA from CQSQ (A) are marked with dashed lines; low / medium (30% threshold probability; L-DAI 9 = -1.7363) and medium / high (69% threshold probability; L-DAI = 2.4928) distinguishing low disease activity from active disease activity (B) are marked with dashed lines; cutoff values ​​were determined by decision curve analysis / calculated net benefit, with low / medium cutoff values ​​maximizing sensitivity and negative predictive value (NPV), and medium / high cutoff values ​​maximizing specificity and positive predictive value (PPV). Performance characteristics of L-DAI-9 (bottom middle plot) include the area under the curve (AUC; receiver operating characteristic curve analysis) distinguishing CASA from CQSQ (A) and low from active disease (B), the Pearson r correlation between L-DAI-9 and hSLEDAI score, and the size of Cohen's d effect.

[0021] Figure 4Adding a combination of TNFRII, resistin, and osteopontin (OPN) to LDAII / L-DAI 9 mediators distinguished between SLE patients with clinical (C) and / or serological (S) active (A) or quiescent (Q) disease, and SLE patients with low disease activity (hSLEDAI < 4) or active disease activity (hSLEDAI ≥ 4) with race / sex / age-matched healthy individuals (Ctl). The composite LDAII / L-DAI score (A) provided by 9 mediators showed similar differentiation to that provided by 10 mediators (LDAII / L-DAI 9 + TNFRII [L-DAI 10A, B], resistin [L-DAI 10B, C], or OPN [L-DAI 10C, D]), 11 mediators (LDAII / L-DAI 9 + TNFRII / resistin [L-DAI 11A, E], TNFRII / OPN [L-DAI 11B, C], or resistin / OPN [L-DAI 11C, G]), or 12 mediators (LDAII / L-DAI 9 + TNFRII / resistin / OPN [L-DAI 12, H]). The mean ± SEM value of the LDAII / L-DAI score is shown. p < 0.05 ; p < 0.01 ; p < 0.001 ; p < 0.0001 Kruskal-Walis test and Dunn multiple comparisons. Detailed Implementation

[0022] The following description of exemplary embodiments provides information enabling those skilled in the art to prepare and use the subject matter set forth in the appended claims, but may omit certain details already known in the art. While various embodiments are discussed in detail below, it should be understood that the disclosed subject matter provides many applicable inventive concepts that can be implemented in a variety of contexts. Therefore, the following detailed description is to be considered illustrative rather than restrictive.

[0023] To facilitate understanding of the invention, several terms are defined below. The terms defined herein have meanings commonly understood by one of ordinary skill in the art related to the invention. Terms such as “an” and “the” are not intended to refer only to a singular entity, but rather to include general categories in which specific examples can be used for illustration. The terms herein are used to describe specific embodiments of the invention, but their use does not limit the invention unless set forth in the claims.

[0024] The inventors of this application utilized plasma samples collected serially from patients with systemic lupus erythematosus (SLE) to compare levels of clinical disease activity, autoantibody specificity, and immune mediators from multiple immune pathways in SLE patients with low or active disease compared to demographically matched healthy controls, and to determine the temporal relationships among these factors. This invention reveals a potential mechanism of immunopathogenesis because it is associated with clinical disease activity, whereby dysregulation of immune mediators occurs simultaneously with and independently of autoantibody accumulation. Furthermore, because it is associated with clinical disease activity, this invention includes the design and validation of reliable and sensitive tools to assess the immune status of lupus patients. This invention can be used to identify high-risk patients requiring rheumatology referral and registration in prospective clinical intervention studies, and to inform the development of novel treatment strategies to avoid or delay tissue damage. Additionally, this invention can be used to enhance telemedicine to prioritize the need for in-person clinic visits.

[0025] As used herein, the term "dataset" refers to a set of numerical values ​​generated by evaluating a sample (or sample population) under desired conditions. The values ​​of a dataset can be obtained, for example, through experiments, from measurements of a sample (e.g., patient samples) and from which the dataset is constructed. Alternatively, the dataset can be obtained from a database or server storing the dataset, or even from a service provider such as an internal or third-party laboratory.

[0026] As used herein, the term "disease" in the context of this invention means any disorder, symptom, disease, or pain of the immune system that manifests as, for example, a dysfunction or inappropriate function of SLE.

[0027] As used herein, the term "sample" means any biological sample separated from an object, which may include, but is not limited to, single-cell or multi-celled, cell fragments, aliquots of body fluids, whole blood, platelets, serum, plasma, red blood cells, white blood cells or leukocytes, endothelial cells, tissue biopsy, synovial fluid, lymphatic fluid, ascites fluid, and interstitial or extracellular fluids. The term "sample" also includes fluids in the intercellular spaces, including gingival crevicular fluid, bone marrow, cerebrospinal fluid, saliva, mucus, sputum, semen, sweat, urine, or any other bodily fluid.

[0028] As used herein, the term "blood sample" refers to whole blood or any fraction thereof, including blood cells, red blood cells, white blood cells or leukocytes, platelets, serum, and plasma. Samples can be obtained from the subject by means including but not limited to venipuncture, excretion, ejaculation, massage, biopsy, needle aspiration, irrigation, scraping, surgical incision or intervention, or other methods known in the art.

[0029] As used herein, the terms “subject” or “patient” generally refer to mammals, including but not limited to humans, non-human primates, dogs, cats, mice, rats, cattle, horses, and pigs, regardless of sex or age. A subject can be one previously diagnosed or identified as having an autoimmune and / or inflammatory disease, and one who may have experienced or is experiencing a therapeutic intervention for an autoimmune and / or inflammatory disease. However, a subject may also include patients who have not previously been diagnosed with an autoimmune and / or inflammatory disease, such as subjects exhibiting one or more symptoms or risk factors for an autoimmune and / or inflammatory disease, subjects not exhibiting symptoms or risk factors for an autoimmune and / or inflammatory disease, or asymptomatic subjects with an autoimmune and / or inflammatory disease.

[0030] As used herein, the phrase “innate serum or plasma mediator biomarkers” refers to one or more of the following biomarkers: IL-1β, IL-1RA, IFN-α, IL-15, IL-12p70, IL-6, and IL-7. These biomarkers can be measured at the RNA or protein level and can be obtained from samples, such as blood, serum, plasma, and / or urine samples from mammalian patients (e.g., human patients). The abbreviations for all biomarkers used herein are well-known to those skilled in the art; for example, IL-1 is interleukin-1, etc. The abbreviations may be matched with proteins, for example, at www.genecards.org.

[0031] As used herein, the phrase “acquired serum or plasma mediator biomarkers” refers to one or more of the following biomarkers: IL-2Rα, IFN-γ, IL-4, IL-5, IL-13, IL-17A, IL-10, and TGF-β (natural TGF-β and / or total TGF-β). These biomarkers can be measured at the RNA or protein level and can be obtained from samples, such as blood, serum, plasma, and / or urine samples from mammalian patients (e.g., human patients).

[0032] As used herein, the phrase “chemokine biomarker” refers to one or more of the following biomarkers: IL-8 / CXCL8, IP-10 / CXCL10, MIG / CXCL9, MIP-1α / CCL3, MIP-1β / CCL4, MCP-1 / CCL2, RANTES / CCL5, and MCP-3 / CCL7. These biomarkers can be measured from samples (e.g., blood, serum, plasma, and / or urine samples) from mammalian patients (e.g., human patients).

[0033] As used herein, the phrase “soluble TNF superfamily biomarkers” refers to one or more of the following biomarkers: TNF-α, TNFRI (p55), TNFRII (p75), Fas, BLyS, and TNF-associated apoptosis-inducing ligand (TRAIL). These biomarkers can be measured at the RNA or protein level and can be obtained from samples, such as blood, serum, plasma, and / or urine samples from mammalian patients (e.g., human patients).

[0034] As used herein, the phrase “inflammatory mediator biomarkers” refers to one or more of the following biomarkers: osteopontin (OPN), stem cell factor (SCF), and resistin. These biomarkers can be measured at the RNA or protein level and can be obtained from samples, such as blood, serum, plasma, and / or urine samples from mammalian patients (e.g., human patients).

[0035] As used herein, the phrase “SLE-associated autoantibody-specific biomarkers” refers to one or more of the following biomarkers that are autoantibodies against the following targets: dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP, all of which are well known to those skilled in the art of SLE. These biomarkers can be measured at the RNA or protein level and can be obtained from samples, such as blood, serum, plasma, and / or urine samples from mammalian patients (e.g., human patients).

[0036] As used in this article, "healthy control" refers to a healthy control of non-SLE patients who do not have clinical evidence of SLE.

[0037] This invention includes methods for identifying SLE patients and modifying their treatment in relation to clinical disease activity, such as the Safety of Estrogen in Lupus National Assessment - Systemic Lupus Erythematosus Disease Activity Index (SELENA-SLEDAI), where proteinuria is defined as SLEDAI-2K, referred to as Hybrid-SLEDAI (hSLEDAI). Since the clinical manifestations of SLE lead to disease activity in hSLEDAI after their occurrence, this invention is used to determine whether subjects are likely to exhibit biomarkers of disease activity that could lead to a risk of permanent organ damage and early death in SLE patients.

[0038] SLE Classification / Diagnosis. Following the ACR, SLICC, and EULAR / ACR criteria used for SLE classification, patients must meet multiple defining criteria for SLE to qualify for disease classification (diagnosis), including: butterfly rash, discoid rash, photosensitivity, oral ulcers, non-scarring alopecia, arthritis, serositis (pleurisy or pericarditis), renal disorders (proteinuria or cellular casts), neurological disorders (delirium, seizures, or psychosis), hematologic disorders (hemolytic anemia, leukopenia, lymphopenia, or thrombocytopenia), fever, immune disorders (presence of anti-DNA, anti-Sm, or antiphospholipid antibodies, or low C3 and / or C4 and / or CH50 complement levels), and positive ANA (HEp-2 IIF assay). As is known to those skilled in the art, these criteria can be used, for example, to classify SLE using the ACR rules, where patients must meet at least four criteria; to classify SLE using the SLICC rules, where patients must meet at least four criteria, including at least one clinical criterion and one immunological criterion, or patients must have biopsy-confirmed lupus nephritis in the presence of antinuclear antibodies or anti-double-stranded DNA antibodies; to classify SLE using the EULAR / ACR rules, where patients must meet ANA positivity (≥ 1:80 titer, as determined by HEp-2 IIF), one clinical criterion, and a score ≥ 10 (from 7 clinical and 3 immunological domains).

[0039] SLE Disease Manifestations. The most common clinical symptom prompting patients to seek medical attention is joint pain, typically affecting the small joints of the hands and wrists, although almost all joints are at risk. 80% to 90% of those affected will experience joint and / or muscle pain at some point during the course of their disease. Unlike rheumatoid arthritis, many people with lupus arthritis experience joint swelling and pain, but X-rays show no abnormalities and functional loss is minimal. Less than 10% of people with lupus arthritis develop hand and foot deformities. SLE patients are at specific risk of developing joint tuberculosis. An association has been found between osteoporosis and SLE, and SLE may be associated with an increased risk of fractures in relatively young women.

[0040] More than half (65%) of SLE patients have some dermatological manifestations at some point in their disease, with approximately 30% to 50% suffering from the classic butterfly rash (or butterfly rash) associated with the disorder's name. Some may present with chronic, thickened, scaly plaques on the skin (known as discoid lupus). Hair loss, oral ulcers, nasal ulcers, and photosensitivity of the skin are all possible manifestations. Anemia may occur in up to 50% of lupus cases. Low platelet and white blood cell counts may be due to the disease or as a side effect of medication treatment. People with SLE may be associated with antiphospholipid antibody syndrome (thrombotic disorder), in which autoantibodies against phospholipids are present in their serum. Abnormalities associated with antiphospholipid antibody syndrome include paradoxically prolonged partial prothrombin time (which usually occurs in hemorrhagic disorders) and a positive antiphospholipid antibody test; the combination of these results has been termed "lupus anticoagulant positive." SLE patients with antiphospholipid autoantibodies have more of the ACR classification criteria for the disease and may suffer from a more severe lupus phenotype.

[0041] People with SLE have inflammation in different parts of the heart, such as pericarditis, myocarditis, and endocarditis. Endocarditis in SLE is typically non-infectious (Libman-Sacks endocarditis), affecting the mitral or tricuspid valves. Atherosclerosis also tends to occur more frequently and progress more rapidly than in the general population. Lung and pleural inflammation can cause pleurisy, pleural effusion, lupus pneumonia, chronic diffuse interstitial lung disease, pulmonary hypertension, pulmonary embolism, pulmonary hemorrhage, and pulmonary systolic syndrome.

[0042] Painless hematuria or proteinuria may often be the only presenting renal symptom. Acute or chronic kidney injury may develop alongside lupus nephritis, leading to acute or end-stage renal failure. Due to early identification and management of SLE, less than 5% of cases develop end-stage renal failure. The histological hallmark of SLE is membranous glomerulonephritis with a “wire loop” abnormality. This finding is due to the deposition of immune complexes along the glomerular basement membrane, resulting in the typical granular appearance in immunofluorescence detection.

[0043] When SLE affects the central or peripheral nervous system, it can lead to neuropsychiatric syndromes. The American College of Rheumatology defines 19 neuropsychiatric syndromes associated with systemic lupus erythematosus (SLE). Diagnosing neuropsychiatric syndromes associated with SLE is one of the most challenging medical problems because it can involve many different symptom patterns, some of which may be mistaken for signs of infectious diseases or stroke. The most common neuropsychiatric disorder in people with SLE is headache, although the presence of specific lupus headaches in SLE cases and the optimal treatment for headaches remain controversial. Other common neuropsychiatric manifestations of SLE include cognitive impairment, mood disorders (including depression), cerebrovascular disease, seizures, polyneuropathy, anxiety disorders, encephalitis, and psychosis. CNS lupus may rarely present with intracranial hypertension syndromes, which are characterized by occasional abducens nerve palsy, the absence of space-occupying lesions or ventricular enlargement, elevated intracranial pressure with normal cerebrospinal fluid chemistry and blood composition, papilledema, and headache. Rader manifestations include acute psychotic states, Guillain-Barré syndrome, aseptic meningitis, autonomic dysfunction, demyelinating syndromes, mononeuropathy (which may manifest as polyneuritis), movement disorders (more specifically, chorea), myasthenia gravis, myelopathy, cranial nerve lesions, and plexus disorders. Neurological symptoms contribute to a significant proportion of morbidity and mortality in people with lupus. Therefore, the neurological aspect of lupus is being investigated in hopes of reducing morbidity and mortality. The neurological manifestation of lupus is known as neuropsychiatric systemic lupus erythematosus (NPSLE). One aspect of this disease is severe damage to the epithelial cells of the blood-brain barrier.

[0044] Biomarkers of SLE disease activity

[0045] Innate cytokines. Innate cytokines are mediators secreted in response to danger signals from the immune system, such as Toll-like receptors (TLRs). Innate cytokines that activate and are secreted by multiple immune cell types include type I interferons (IFN-α and IFN-β), TNF-α, and members of the IL-1 family (IL-1α and IL-1β). Other innate cytokines secreted by antigen-presenting cells (APCs), including dendritic cells, macrophages, and B cells, drive the development of antigen-specific inflammatory pathways during the acquired response, as described below, when they process and present protein fragments (antigens from infectious pathogens or self-proteins driving autoimmune diseases) to CD4 helper T (Th) cells.

[0046] Th1 cytokines. Th1 cytokines drive pro-inflammatory responses responsible for killing intracellular parasites and maintaining autoimmune responses. Excessive pro-inflammatory responses can lead to uncontrolled tissue damage, particularly in systemic lupus erythematosus (SLE).

[0047] CD4 Th cells differentiate into Th-1 cells after interacting with APCs, co-stimulatory molecules, and cytokines secreted by APCs. The hallmark cytokine of Th-1 cells is IL-12. IL-12 consists of four α-helical bundles and is a heterodimeric cytokine encoded by two independent genes, IL-12A (p35) and IL-12B (p40). After protein synthesis, it forms an active heterodimer and a homodimer of p40. IL-12 binds to a heterodimer receptor formed by IL-12R-β1 and IL-12R-β2. IL-12R-β2 is considered to play a key role in IL-12 function because it is present on activated T cells, stimulated by cytokines that promote Th1 cell development, and inhibited by cytokines that promote Th2 cell development. Upon binding, IL-12R-β2 undergoes tyrosine phosphorylation, providing binding sites for kinases Tyk2 and Jak2. These kinases are essential for activating key transcription factor proteins, such as STAT4, involved in IL-12 signaling in T cells and NK cells. IL-12-mediated signaling leads T cells and natural killer (NK) cells to produce interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), and reduces IL-4-mediated IFN-γ inhibition.

[0048] IFN-γ, or type II interferon, consists of a six-alpha-helix core and an extended unfolded sequence in the C-terminal region. IFN-γ is crucial for innate (NK cell) and adaptive (T cell) immunity against viral (CD8 response) and intracellular bacterial (CD4 Th1 response) infections, as well as for tumor control. During the effector phase of the immune response, IFN-γ activates macrophages. Aberrant expression of IFN-γ is associated with a variety of autoinflammatory and autoimmune diseases, including increased activity in SLE.

[0049] Although IFN-γ is considered a characteristic cytokine of Th1 cells, in humans, interleukin-2 (IL-2) has also been shown to influence Th1 differentiation and play a major role as a secreted cytokine in the initial Th cell response. IL-2 is essential for T cell growth, proliferation, and differentiation into “effectory” T cells. IL-2 is normally produced by T cells during the immune response. The binding of antigens to the T cell receptor (TCR) stimulates the secretion of IL-2 and the expression of the IL-2 receptor IL-2R. Subsequently, the IL-2 / IL-2R interaction stimulates the growth, differentiation, and survival of antigen-specific CD4+ T cells and CD8+ T cells. Therefore, IL-2 is essential for the formation of T cell immune memory, which depends on the expansion of the number and function of antigen-selective T cell clones. IL-2, along with IL-7 and IL-15 (both members of the common cytokine receptor γ chain family), maintains lymphocyte homeostasis to ensure consistent lymphocyte numbers during cell turnover.

[0050] Th2 cytokines. Th2 cytokines include IL-4, IL-5, IL-13, and (in humans) IL-6, which are involved in promoting B lymphocyte activation, antibody production, isotype conversion to IgE, and eosinophilic responses in atopic leukemia. Overexpression of Th2 cytokines can counteract Th1-mediated bactericidal effects. Th2 cytokines may also contribute to the pathogenesis of SLE and increase disease activity.

[0051] IL-4 is a 15-kD polypeptide with pluripotent effects across many cell types. Its receptor is a heterodimer composed of an α subunit with IL-4 binding affinity and a common γ subunit (also a component of other cytokine receptors). In T cells, IL-4 binding to its receptor induces proliferation and differentiation into Th2 cells. IL-4 also participates in Th2-mediated B lymphocyte activation and antibody production, and, along with IL-5 and IL-13, promotes isotype conversion from Th1 subclasses (including IgG1 and IgG2) to Th2 subclasses (including IgG4 and atopic IgE). Beyond its role in Th2 biology, IL-4 plays a crucial role in immune cell hematopoiesis, exerting various effects on hematopoietic progenitor cells, including the proliferation and differentiation of directed and primitive hematopoietic progenitor cells. It synergizes with granulocyte colony-stimulating factor (G-CSF) to support neutrophil colony formation and, along with IL-1 and IL-6, induces colony formation of human bone marrow B lineage cells.

[0052] IL-5 is an interleukin produced by various cell types, including Th2 cells, mast cells, and eosinophils. IL-5 expression is regulated by multiple transcription factors, including GATA3. IL-5 is a TH2-type cytokine composed of 115 amino acids (133 amino acids in mice) and belongs to the hematopoietic factor family. Unlike other members of this cytokine family (IL-3 and GM-CSF), the active form of this glycoprotein is a homodimer. By binding to its receptor, IL-5 stimulates B cell growth and increases immunoglobulin secretion. IL-5 has long been considered to be involved in the etiology of various allergic diseases, including allergic rhinitis and asthma, in which mast cells play a crucial role, and a significant increase in the number of eosinophils in circulating blood, airway tissues, and induced sputum has been observed.

[0053] Given the high degree of consistency between eosinophils and the pathology of allergic asthma, it is widely assumed that eosinophils play an important role in the pathology of this disease. IL-13 is secreted by various cell types, especially Th2 cells, and acts as a mediator of allergic inflammation and autoimmune diseases, including type 1 diabetes, rheumatoid arthritis (RA), and SLE. IL-13 induces its effects through a multi-subunit receptor containing the α chain of the IL-4 receptor (IL-4Rα) and one of at least two known IL-13-specific binding chains. Most of the biological effects of IL-13 (similar to IL-4) are associated with a single transcription factor—signal transduction and activator of transcription 6 (STAT6).

[0054] Similar to IL-4, IL-13 is known to induce changes in hematopoietic cells, but to a lesser extent. IL-13 can induce activated human B cells to secrete immunoglobulin E (IgE). IL-13 induces several features of allergic lung disease, including airway hyperresponsiveness, goblet cell metaplasia, and mucus hypersecretion, all of which contribute to airway obstruction. IL-4 is also involved in these physiological changes, but to a lesser extent than IL-13. IL-13 can also induce the secretion of chemokines necessary for recruiting allergic effector cells to the lungs.

[0055] IL-13 can antagonize the Th1 response required to clear intracellular infections and induce physiological changes in parasitic organs to expel pathogens or their products. For example, the expulsion of various mouse worms from the intestine requires IL-13 secreted by Th2 cells. IL-13 induces various changes in the intestine to create an environment unfavorable to parasites, including enhanced contraction of intestinal epithelial cells and high secretion of glycoproteins, ultimately leading to the detachment and clearance of the organism from the intestinal wall.

[0056] Interleukin-6 (IL-6) is secreted by various cell types and participates in multiple innate and adaptive immune response pathways. IL-6 mediates its biological function through the signaling component gp130 of its receptor (IL-6R), leading to tyrosine kinase phosphorylation and downstream signaling events, including the STAT1 / 3 pathway and the SHP2 / ERK cascade. IL-6 is a key mediator of fever, responding during infection and in the acute phase of post-traumatic stimuli. It can cross the blood-brain barrier and initiate PGE2 synthesis in the hypothalamus, thereby altering the body temperature set point. In muscle and adipose tissue, IL-6 stimulates energy mobilization, leading to an increase in body temperature.

[0057] IL-6 can be secreted by various immune cells in response to specific microbial molecules (called pathogen-associated molecular patterns, PAMPs). These PAMPs bind to pattern recognition receptors (PRRs), a class of highly important detection molecules in the innate immune system, including Toll-like receptors (TLRs). These receptors are present on the cell surface and in intracellular compartments and can induce intracellular signaling cascades that produce inflammatory cytokines. As a Th2 cytokine in humans, IL-6, along with IL-4, IL-5, and IL-13, can influence IgE production and eosinophilic airway infiltration in asthma. IL-6 also contributes to Th2 acquired immunity against parasitic infections, playing a particularly important role in mast cell activation that occurs concurrently with parasite shedding.

[0058] IL-6 is also a Th17 cytokine, which, together with TGF-β, drives T lymphocytes to produce IL-17. IL-6 sensitizes Th17 cells to IL-23 (produced by APCs) and IL-21 (produced by T lymphocytes) to maintain the Th17 response. The Th17 response will be described below.

[0059] Th17 cytokines. Th17 cells are a subset of helper T cells, thought to be developmentally distinct from Th1 and Th2 cells. An excess of Th17 cells is believed to play a crucial role in autoimmune diseases such as multiple sclerosis (previously thought to be caused solely by Th1 cells), psoriasis, autoimmune uveitis, Crohn's disease, type 2 diabetes, rheumatoid arthritis, and SLE. Th17 cells are thought to play a role in inflammation and tissue damage in these conditions. In addition to autoimmune pathogenesis, Th17 cells play an important role in antimicrobial immunity at the epithelial / mucosal barrier. They produce cytokines (such as IL-21 and IL-22), stimulating epithelial cells to produce antimicrobial proteins for clearing microorganisms (such as Candida and Staphylococcus species). A lack of Th17 cells may make the host susceptible to opportunistic infections. Besides their role in autoimmune diseases and infections, the Th17 pathway is also associated with asthma, including the recruitment of neutrophils to sites of airway inflammation.

[0060] Interleukin-17A (IL-17A) is a founding member of the cytokine family, also known as CTLA8 in rodents. IL-17 is associated with T-lymphotropic herpesvirus (TLC). Herpesvirus saimiri The open reading frame of IL-17A encodes the viral IL-17, which shares high homology with IL-17A. IL-17A is a 155-amino acid protein, a disulfide-linked homodimeric secretory glycoprotein with a molecular weight of 35 kDa. Each subunit of the homodimer is approximately 15 kDa to 20 kDa. The structure of IL-17A includes a 23-amino acid (aa) signal peptide followed by a 123-amino acid chain region, a characteristic region of the IL-17 family. Upon purification of the protein, two bands (one 15 kDa and the other 20 kDa) were observed, thus identifying the N-linked glycosylation site on the protein for the first time. Comparison with different members of the IL-17 family revealed four conserved cysteine ​​residues forming two disulfide bonds. IL-17A is unique in that it shares no similarity with other known interleukins. Furthermore, IL-17A does not share similarity with any other known proteins or domains.

[0061] IL-17A acts as a potent mediator in delayed-type responses, similar to IFN-γ, by increasing chemokine production in various tissues to recruit monocytes and neutrophils to sites of inflammation. IL-17A is produced by helper T cells and induced by IL-6 (and TGF-β) and IL-23 produced by APCs, leading to destructive tissue damage in delayed-type responses. As a family of IL-17, it acts as a pro-inflammatory cytokine, responding to the invasion of extracellular pathogens into the immune system and inducing the destruction of the pathogen's cellular matrix. IL-17 works synergistically with TNF-α and IL-1. To exert its function, IL-17 binds to a type I cell surface receptor called IL-17R, of which at least three variants exist: IL-17RA, IL-17RB, and IL-17RC.

[0062] Chemokines and adhesion molecules. Chemokines and adhesion molecules (here, ICAM-1 and E-selectin) work together to coordinate cellular transport in the immune response. Chemokines are divided into CXC receptor (R) / CXC ligand (L) and CCR / CCL subgroups.

[0063] GROα, also known as chemokine (CXC motif) ligand 1 (CXCL1), belongs to the CXC chemokine family and was previously known as the GRO1 oncogene, KC, neutrophil activation protein 3 (NAP-3), and melanoma growth stimulating activity α (MSGA-α). In humans, this protein is encoded by the CXCL1 gene on chromosome 4. CXCL1 is expressed by macrophages, neutrophils, and epithelial cells and exhibits neutrophil chemotactic activity. GROα is involved in angiogenesis, inflammation, wound healing, and tumorigenesis. This chemokine exerts its effects through signal transduction via the chemokine receptor CXCR2.

[0064] Interleukin-8 (IL-8) / CXCL8 is a chemokine produced by macrophages and other cell types, such as epithelial cells, airway smooth muscle cells, and endothelial cells. In humans, the interleukin-8 protein is encoded by the IL-8 gene. IL-8 is a member of the CXC chemokine family. The genes encoding this member of the CXC chemokine family, along with 10 other members, form a gene cluster in the 4q region of the long arm of chromosome 4.

[0065] Numerous receptors on the surface membrane can bind IL-8; the most frequently studied types are the G protein-coupled serpentine receptors CXCR1 and CXCR2, expressed by neutrophils and monocytes. The two receptors exhibit different expression and affinity for IL-8 (CXCR1 > CXCR2). IL-8 is secreted and is an important mediator of immune responses in innate immunity involving TLRs. During adaptive immune responses, IL-8 is produced at the effector phases of the Th1 and Th17 pathways, leading to the recruitment of neutrophils and macrophages to sites of inflammation, including those during infection and autoimmune diseases. Although neutrophils are the primary target cells for IL-8, a relatively wide range of cells (endothelial cells, macrophages, mast cells, and keratinocytes) also respond to this chemokine.

[0066] Interferon-gamma induced mononuclear factor (MIG) / CXCL9 is an IFN-γ-induced T cell chemokine. It is closely related to two other CXC chemokines, IP-10 / CXCL10 and I-TAC / CXCL11, whose genes are located on human chromosome 4. CXCL9 Near the gene. MIG, IP-10, and I-TAC exert their chemokine function by interacting with the chemokine receptor CXCR3.

[0067] Interferon-γ-induced protein 10 (IP-10), also known as CXCL10 or small inducible cytokine B10, is an 8.7 kDa protein encoded in humans by the CXCL10 gene, located on chromosome 4 and clustered with several other CXC chemokines. IP-10 is secreted by various cell types in response to IFN-γ. These cell types include monocytes, endothelial cells, and fibroblasts. IP-10 has multiple functions, such as chemotaxis of monocytes / macrophages, T cells, NK cells, and dendritic cells, promoting T cell adhesion to endothelial cells, antitumor activity, and inhibition of bone marrow colony formation and angiogenesis. This chemokine exerts its effects by binding to the cell surface chemokine receptor CXCR3, which is present on both Th1 and Th2 cells.

[0068] Monocyte chemotactic protein-1 (MCP-1) / CCL2 recruits monocytes, memory T cells, and dendritic cells to sites of inflammation. MCP-1 is a monomeric polypeptide with a molecular weight of approximately 13 kDa, primarily secreted by monocytes, macrophages, and dendritic cells. Platelet-derived growth factor is the main inducer of the MCP-1 gene. MCP-1 protein is activated upon cleavage by the metalloproteinase MMP-12. CCR2 and CCR4 are two cell surface receptors that bind to MCP-1. During the adaptive immune response, CCR2 is upregulated on Th17 cells and regulatory T cells, while CCR4 is upregulated on Th2 cells. MCP-1 is involved in the pathogenesis of various diseases characterized by monocyte infiltration, such as psoriasis, rheumatoid arthritis, and atherosclerosis. It is also associated with the pathogenesis of SLE, and a polymorphism of MCP-1 is associated with SLE in Caucasian populations. In a glomerulonephritis model, administration of anti-MCP-1 antibody reduced macrophage and T cell infiltration, crescent formation, scar formation, and renal function impairment.

[0069] Monocyte-specific chemokine 3 (MCP-3) / CCL7 specifically attracts monocytes and regulates macrophage function. It is produced by multiple cell types, including monocytes, macrophages, and dendritic cells. The CCL7 gene is located on human chromosome 17, within a large cluster containing other CC chemokines. MCP-3 is most closely associated with MCP-1 and binds to CCR2.

[0070] Macrophage inflammatory protein-1α (MIP-1α) / CCL3 is encoded by the human CCL3 gene. MIP-1α is involved in acute inflammatory states involving the recruitment and activation of polymorphonuclear leukocytes (Wolpe et al., 1988). MIP-1α interacts with MIP-1β / CCL4 encoded by the CCL4 gene and is specific for the CCR5 receptor. It is a chemokine for natural killer cells, monocytes, and various other immune cells.

[0071] RANTES (activation regulator, normal T cell expression and secretion factor) / CCL5 is encoded by the CCL5 gene on human chromosome 17. RANTES is an 8 kDa chemokine that exhibits chemotaxis towards T cells, eosinophils, and basophils, playing an active role in recruiting leukocytes to sites of inflammation. With the assistance of specific cytokines released by T cells (such as IL-2 and IFN-γ), RANTES induces the proliferation and activation of natural killer (NK) cells. RANTES was initially discovered while searching for genes expressed "late" (3–5 days) after T cell activation and has been shown to interact with CCR3, CCR5, and CCR1. RANTES also activates the G protein-coupled receptor GPR75.

[0072] Members of the TNF receptor superfamily. The tumor necrosis factor receptor (TNFR) superfamily and its corresponding ligands activate signal transduction pathways that activate cell survival, death, and differentiation. Members of the TNFR superfamily function through ligand-mediated trimerization and require adaptor molecules (such as TRAF) to activate downstream mediators of cell activation, including the NF-κB and MAPK pathways, immune and inflammatory responses, and apoptosis in certain circumstances.

[0073] A typical member of this family is TNF-α. Tumor necrosis factor (TNF, cachexin or cachectin, formerly known as tumor necrosis factor α or TNFα) is a cytokine involved in systemic inflammation and belongs to the family of cytokines that stimulate acute-phase responses. It is produced by a variety of immune cells, including macrophages, dendritic cells, as well as T lymphocytes and B lymphocytes. Dysregulation of TNF-α production has been shown to be associated with a variety of human diseases, including Alzheimer's disease, cancer, major depressive disorder, and autoimmune diseases, including inflammatory bowel disease (IBD) and rheumatoid arthritis (RA).

[0074] TNF-α is produced as a 212-amino acid-long type II transmembrane protein, arranged in a stable homotrimer. From this membrane-integrated form, the soluble homotrimeric cytokine (sTNF) is released via proteolytic cleavage by the metalloproteinase TNF-α convertase (TACE, also known as ADAM17). The soluble 51 kDa trimer sTNF can dissociate into a 17 kDa monomeric form. Both the secreted and membrane-bound forms are biologically active. Tumor necrosis factor receptor 1 (TNFRI; TNFRSF1a; CD120a) is a trimeric cytokine receptor that is expressed in most tissues and binds both membrane-bound and soluble TNF-α. This receptor works synergistically with adaptor molecules (e.g., TRADD, TRAF, RIP), which are crucial for determining response outcomes (e.g., apoptosis, inflammation). Tumor necrosis factor receptor II (TNFRII; TNFRSF1b; CD120b) expression is limited and primarily found on immune cells (although endothelial cells, including those of the lung and kidney, are induced to express TNFRII during chronic inflammation). It binds to membrane-bound TNF-α homotrimers with higher affinity and binding force compared to soluble TNF-α. Unlike TNFRII, TNFRII does not contain a death domain (DD), does not induce apoptosis, but instead promotes inflammatory responses and acts as a co-stimulatory molecule in receptor-mediated B and T lymphocyte activation.

[0075] Fas, also known as apoptosis antigen 1 (APO-1 or APT), differentiation cluster 95 (CD95), or tumor necrosis factor receptor superfamily member 6 (TNFRSF6), is a protein encoded by the TNFRSF6 gene located on chromosome 10 in humans (chromosome 19 in mice). Fas is a cell surface death receptor that induces programmed cell death (apoptosis). Like other members of the TNFR superfamily, Fas is produced in a membrane-bound form, but can also be produced in a soluble form through proteolytic cleavage or selective splicing. The mature Fas protein has 319 amino acids and a predicted molecular weight of 48 kDa, divided into three domains: an extracellular domain, a transmembrane domain, and a cytoplasmic domain. Upon ligand binding, Fas forms the death-inducing signal transduction complex (DISC). Membrane-anchored Fas ligands on adjacent cell surfaces lead to Fas oligomerization. Subsequent aggregation of the death domain (DD) results in the receptor complex being internalized via endosome mechanisms. This allows the adaptor molecule FADD to bind to the death domain of Fas through its own death domain.

[0076] FADD also contains a death effector domain (DED) near its amino terminus, which facilitates binding to the DED of FADD-like interleukin-1β convertase (FLICE, more commonly known as caspase-8). FLICE can then self-activate via proteolytic cleavage into p10 and p18 subunits, each pair forming an active heterotetramer. Subsequently, active caspase-8 is released from the DISC into the cytoplasm, where it cleaves other effector caspases, ultimately leading to DNA degradation, membrane bubbling, and other apoptotic features.

[0077] In most cell types, Caspase-8 catalyzes the cleavage of the pro-apoptotic BH3-only protein Bid into its truncated form tBid. BH3-only members of the Bcl-2 family specifically bind to anti-apoptotic members of the same family (Bcl-2, Bcl-xL), causing Bak and Bax to translocate to the outer mitochondrial membrane, thereby permeating it and promoting the release of pro-apoptotic proteins (such as cytochrome c and Smac / DIABLO, antagonists of inhibitors of apoptosis (IAP)).

[0078] FAS ligands (FasL; CD95L; TNFSF6) are type II transmembrane proteins belonging to the tumor necrosis factor (TNF) family. Their binding to their receptors induces apoptosis. The FasL / Fas interaction plays a crucial role in the regulation of the immune system and cancer progression. Soluble Fas ligands are produced by the external matrix metalloproteinase MMP-7 cleaving membrane-bound FasL at conserved cleavage sites.

[0079] Fas-Fas ligand-triggered apoptosis plays a fundamental role in the regulation of the immune system. Its functions include: T cell homeostasis (T cell activation leads to Fas ligand expression; T cells are initially resistant to Fas-mediated apoptosis during clonal expansion, but gradually become more sensitive with prolonged activation, eventually leading to activation-induced cell death (AICD)); cytotoxic T cell activity (Fas-induced apoptosis and the perforin pathway are two major mechanisms by which cytotoxic T lymphocytes induce cell death expressing foreign antigens); immune privilege (cells in immune-privileged regions such as the cornea or testis express Fas ligands and induce apoptosis of infiltrating lymphocytes); maternal tolerance (Fas ligands may play a role in preventing leukocyte transport between mother and fetus, although no pregnancy defects have been attributed to a defective Fas-Fas ligand system to date); and tumor counterattack (tumors may overexpress Fas ligands and induce apoptosis of infiltrating lymphocytes, thereby allowing the tumor to evade the immune response).

[0080] Another pro-apoptotic member of the TNF receptor superfamily is the TNF-associated apoptosis-inducing ligand (TRAIL). Encoded by the TNFSF10 gene, TRAIL's protein product (Apo2L / CD253) induces apoptosis in transformed and tumor cells but appears not to kill normal cells, despite significant expression in most normal tissues. TRAIL forms a trimer and binds to multiple members of the TNF receptor superfamily, including TNFRSF10A / TRAILR1, TNFRSF10B / TRAILR2, TNFRSF10C / TRAILR3, TNFRSF10D / TRAILR4, and possibly also TNFRSF11B / OPG. The activity of this protein may be regulated by binding to non-apoptotic decoy receptors TNFRSF10C / TRAILR3, TNFRSF10D / TRAILR4, and TNFRSF11B / OPG. It has been shown that the binding of this protein to its receptor can trigger the activation of MAPK8 / JNK, Caspase 8, and Caspase 3, which can both promote inflammation by activating IL-1β and prevent the autoimmunity seen in Fas deficiency.

[0081] B cell activating factor (BAFF), also known as B lymphocyte stimulating factor (BLyS), TNF and APOL-associated leukocyte expression ligand (TALL-1), and CD27, is encoded by the TNFSF13C gene in humans. BLyS is a 285-amino acid-long peptide-glycoprotein glycosylated at residue 124. It is expressed as a membrane-bound type II transmembrane protein in various cell types, including monocytes, dendritic cells, and bone marrow stromal cells. The transmembrane form can be cleaved from the membrane, producing soluble protein fragments. This cytokine is expressed in B lineage cells and functions as a potent B cell activating factor. It has also been shown to play an important role in B cell proliferation and differentiation.

[0082] BLyS is a ligand for receptors TNFRSF13B / TACI, TNFRSF17 / BCMA, and TNFRSF13C / BAFFR. These receptors are primarily expressed on mature B lymphocytes, and their expression varies with B cell maturity (TACI is also present on some T cells, while BCMA is present on plasma cells). BAFF-R is involved in the positive regulation of B cell development. TACI has the lowest affinity for BLyS; it has a higher affinity for a BLyS-like protein—the proliferation-inducing ligand (APRIL). BCMA exhibits an intermediate binding phenotype, binding to varying degrees with either BLyS or APRIL. Signal transduction by BAFF-R and BCMA stimulates B lymphocyte proliferation and resists apoptosis. All these ligands interact with homotrimeric receptors in the form of homotrimers (i.e., three identical molecules), although BAFF is known to be active in both heterotrimer and homotrimer forms.

[0083] Excessive BLyS levels can lead to abnormally high antibody production, triggering systemic lupus erythematosus (SLE), rheumatoid arthritis, and many other autoimmune diseases. Benlysta, a monoclonal antibody developed by Human Genome Sciences and GlaxoSmithKline, with significant contributions from Cambridge Antibody Technology, specifically recognizes and inhibits the biological activity of B lymphocyte-stimulating factor (BLyS). It is currently in clinical trials for the treatment of SLE and other autoimmune diseases. Blisibimod, a BLyS fusion protein inhibitor being developed by Antera Pharmaceuticals, is also primarily intended for the treatment of SLE.

[0084] Other active inflammatory factors in SLE. Osteopontoxin (OPN) is a stromal cell protein with multiple cellular functions. It possesses the ability to promote Th1 cytokine responses and enhance cell-mediated immunity, suggesting a potential role in chronic inflammation and autoimmunity. In the methods disclosed in this paper, OPN may provide rich information and ranks first in importance among random forest variables.

[0085] Leptin is a 16-kDa protein hormone that plays a crucial role in regulating energy intake and expenditure, including appetite and hunger, metabolism, and behavior. It is one of a variety of adipokines, including adiponectin and resistin. Reports of elevated leptin levels following acute infection and chronic inflammation (including autoimmune diseases) suggest that leptin actively participates in the immune response. Leptin levels increase in response to stimulation by various innate cytokines, including TNF-α and IL-6. Leptin is a member of a family of cytokines including IL-6, IL-12, and G-CSF. Leptin exerts its effects by binding to its receptor, which is expressed by polymorphonuclear neutrophils, circulating leukocytes (including monocytes), and NK cells. Leptin influences the elevation of the chemokine MCP-1, thereby recruiting monocytes and macrophages to sites of inflammation.

[0086] Stem cell factor (SCF), also known as Kit ligand, KL, or Steel factor, is a cytokine that binds to the c-Kit receptor (CD117). SCF can exist as a transmembrane protein or a soluble protein. This cytokine plays an important role in hematopoiesis (blood cell formation), spermatogenesis, and melanin formation. The gene encoding stem cell factor (SCF) is located at the S1 locus in mice and on human chromosomes 12q22-12q24. The soluble and transmembrane forms of this protein are formed through alternative splicing of the same RNA transcript.

[0087] The soluble form of SCF contains a proteolytic cleavage site in exon 6. Cleavage at this site releases the extracellular portion of the protein. The transmembrane form of SCF is formed by alternative splicing excluding exon 6. Both forms of SCF bind to c-Kit and are biologically active. Soluble and transmembrane SCFs are produced by fibroblasts and endothelial cells. Soluble SCF has a molecular weight of 18.5 kDa and forms a dimer. SCFs play an important role in hematopoiesis, providing guiding cues to direct hematopoietic stem cells (HSCs) to their stem cell niche (the microenvironment in which stem cells reside) and playing a crucial role in HSC maintenance. SCFs regulate the function of HSCs in the bone marrow stem cell niche. SCFs have been shown to increase HSC survival in vitro and contribute to HSC self-renewal and maintenance in vivo. HSCs express the same levels of the SCF receptor (c-Kit) at all stages of development. The stromal cells surrounding HSCs are components of the stem cell niche, releasing various ligands, including SCF.

[0088] A small fraction of HSCs periodically leave the bone marrow and enter circulation, then return to their niche in the bone marrow. It is believed that the concentration gradient of SCF and the chemokine SDF-1 interacts to enable HSCs to find a pathway back to this niche.

[0089] In addition to hematopoiesis, SCF is thought to participate in inflammation by binding to c-kits on dendritic cells. This binding leads to increased IL-6 secretion and promotes the development of Th2 and Th17 immune responses. Th2 cytokines synergistically activate mast cells with SCF, which are integrative promoters of allergic inflammation. IL-17 induction further upregulates SCF in epithelial cells and promotes granulocyte production. In the lungs, IL-17 upregulation induces IL-8 and MIP-2, thereby recruiting neutrophils to the lungs. Chronic induction of IL-17 has been shown to play a role in autoimmune diseases, including multiple sclerosis and rheumatoid arthritis.

[0090] Interleukin-10 (IL-10), also known as human cytokine synthesis inhibitor (CSIF), is an anti-inflammatory cytokine. The IL-10 protein is a homodimer, with each subunit containing 178 amino acids. IL-10 is classified as a class 2 cytokine, which includes IL-19, IL-20, IL-22, IL-24 (Mda-7), and IL-26, as well as interferons and interferon-like molecules. In humans, IL-10 is encoded by the IL10 gene located on chromosome 1, which contains five exons. IL-10 is primarily produced by monocytes and lymphocytes, namely Th2 cells, CD4+CD25+Foxp3+ regulatory T cells, and a subset of activated T cells and B cells. IL-10 can be produced by monocytes upon PD-1 triggering. IL-10 expression is extremely low in unstimulated tissues, and its expression requires receptor-mediated cellular activation. IL-10 expression is tightly regulated at both the transcriptional and post-transcriptional levels. Extensive IL-10 locus remodeling can be observed in monocytes following stimulation by the TLR or Fc receptor pathway. IL-10 induction involves ERK1 / 2, p38, and NFκB signaling and transcriptional activation via promoter binding of transcription factors NFκB and AP-1. IL-10 expression can be autoregulated through a negative feedback loop involving autocrine stimulation of the IL-10 receptor and inhibition of the p38 signaling pathway. Furthermore, IL-10 expression is extensively regulated at the post-transcriptional level, which may include the regulation of mRNA stability through AU-rich elements and microRNAs such as let-7 or miR-106.

[0091] IL-10 is a pleiotropic cytokine with multiple roles in immune regulation and inflammation. It downregulates the expression of various Th pathway cytokines, MHC class II antigens, and co-stimulatory molecules on macrophages. It also enhances B cell survival, proliferation, and antibody production. IL-10 can block NF-κB activity and participates in the regulation of the JAK-STAT signaling pathway.

[0092] TGF-β. Transforming growth factor β (TGF-β) controls proliferation, cell differentiation, and other functions in most cells. TGF-β is a secreted protein, existing in at least three isoforms, known as TGF-β1, TGF-β2, and TGF-β3. It was originally also the name of TGF-β1, the founding member of this family. The TGF-β family is part of a superfamily of proteins known as the transforming growth factor β superfamily, which includes inhibin, activin, anti-Müllerian hormone, bone morphogenetic protein, decapentaplegic, and Vg-1. Its assay can be performed in its native (active) form or in its "total" (potential) form, in which inactive TGF-β forms a complex with potential TGF-β-binding protein (LTBP).

[0093] Most tissues highly express the gene encoding TGF-β. This is in contrast to other anti-inflammatory cytokines (such as IL-10), which are expressed at very low levels in unstimulated tissues and appear to require triggering by symbiotic or pathogenic microbiota to be expressed.

[0094] The three members of the TGF-β family share highly similar peptide structures. They are all encoded as large protein precursors; TGF-β1 contains 390 amino acids, while TGF-β2 and TGF-β3 each contain 412 amino acids. Each possesses an N-terminal signal peptide (20 to 30 amino acids) essential for cellular secretion, a pre-region (called the latent-related peptide or LAP), and a C-terminal region (112 to 114 amino acids), which is released from the pre-region via proteolytic cleavage to form the mature TGF-β molecule. Mature TGF-β protein dimerizes to produce a 25 kDa active molecule with numerous conserved structural motifs.

[0095] TGF-β plays a crucial role in cell cycle regulation. It induces the synthesis of p15 and p21 proteins, which block the cyclin-CDK complex responsible for phosphorylation of retinoblastoma protein (Rb). Therefore, TGF-β inhibits the progression of the cell cycle to G1 phase. TGF-β is essential for the differentiation and repressive function of CD4+CD25+Foxp3+ regulatory T cells. In the presence of IL-6, TGF-β promotes the differentiation of pro-inflammatory Th17 cells.

[0096] IL-1RA. Interleukin-1 receptor antagonists (IL-1RA) are proteins encoded by the IL1RN gene in humans. As a member of the IL-1 cytokine family, IL-1RA is a factor that nonproductively binds to the cell surface interleukin-1 receptor (IL-1R), preventing IL-1 binding and inducing downstream signaling events. IL-1RA is secreted by various cell types, including immune cells, epithelial cells, and adipocytes, and is a natural inhibitor of the pro-inflammatory effects of IL-1α and IL-1β. This gene, along with five other closely related cytokine genes, forms a gene cluster of approximately 400 kb on chromosome 2. Four alternatively spliced ​​transcript variants encoding different subtypes have been reported. Interleukin-1 receptor antagonists are used to treat rheumatoid arthritis, an autoimmune disease in which IL-1 plays a crucial role. Its commercially available product is Anakinra, a recombinant human form of IL-1RA. In an open-label trial in four SLE patients, anaprolactin showed safety and efficacy in improving arthritis, but only a transient therapeutic effect in two patients.

[0097] Biomarker detection. Several methods exist for assessing protein expression. One such method utilizes antibodies for protein identification.

[0098] As used herein, the term “antibody” broadly refers to any immune binding agent, such as IgG, IgM, IgA, IgD, and IgE antibodies or subclasses thereof, or binding fragments thereof, including single-chain fragments. IgG and / or IgM are commonly used because they are the most common antibodies under physiological conditions and are generally readily prepared in a laboratory setting. As used herein, the term “antibody fragment” refers to any antibody-like molecule having an antigen-binding region, including antibody fragments such as Fab', Fab, F(ab')2, single-domain antibodies (DAB), Fv, scFv (single-chain Fv), etc. Techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Methods for preparing and characterizing antibodies (polyclonal and monoclonal) are also well known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; relevant portions are incorporated herein by reference).

[0099] According to the present invention, examples of immunoassay methods are provided. Some immunoassay methods include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradioassay, fluorescence immunoassay, chemiluminescence assay, bioluminescence assay, and Western blotting. The steps of various useful immunoassay methods are described in the scientific literature Current Protocols in Immunology, Wiley & SonsPress, 2017, the relevant parts of which are incorporated herein by reference.

[0100] Typically, immunobinding methods involve obtaining a sample suspected of containing the relevant polypeptide and contacting the sample with a primary antibody under conditions conducive to the formation of immune complexes. For antigen detection, the biological sample analyzed can be any sample suspected of containing the antigen, such as tissue sections or specimens, homogenized tissue extracts, cells, or even biological fluids.

[0101] Under effective conditions, exposing the selected biological sample to antibodies for a sufficient time allows for the formation of immune complexes (primary immune complexes). This typically involves simply adding the antibody composition to the sample and incubating the mixture long enough for the antibodies to form immune complexes (i.e., bind) with any antigens present. Subsequently, the sample-antibody composition is usually washed (e.g., with tissue sections, ELISA plates, microfluidic chambers, dot blots, or protein blotting) to remove any non-specifically bound antibody species, thus detecting only the specifically bound antibodies from the primary immune complexes.

[0102] Typically, the detection of immune complex formation is well known in the art and can be achieved through a variety of methods. These methods are generally based on the detection of labels or markers, such as radiolabels, fluorescent labels, biological labels, and enzyme labels. Patents relating to the use of such labels include U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated herein by reference. Of course, as is known in the art, additional advantages can be obtained by using a second binding ligand (such as a second antibody and / or a biotin / avidin ligand binding system).

[0103] The antibody or its binding fragment used for detection can be conjugated to a detectable label, allowing direct detection of the label to determine the amount of primary immune complex in the composition. Alternatively, the primary antibody bound to the primary immune complex can be detected by a second binding ligand having binding affinity for the antibody. In these cases, the second binding ligand can be conjugated to the detectable label. The second binding ligand is often itself an antibody and can therefore be referred to as a "second" antibody. Under effective conditions, the primary immune complex is contacted with the labeled second binding ligand or antibody for a sufficient time to allow the formation of a secondary immune complex. The secondary immune complex is then typically washed to remove any non-specifically bound labeled second antibody or ligand, followed by detection of any remaining label in the secondary immune complex.

[0104] Other methods include a two-step approach for detecting primary immune complexes. As described above, a secondary immune complex is formed using a second binding ligand (such as an antibody) with binding affinity to the antibody. After washing, under effective conditions, the secondary immune complex is contacted with a third binding ligand or antibody with binding affinity to the second antibody for a sufficient time to allow the formation of an immune complex (tertiary immune complex). The third ligand or antibody is then linked to a detectable label, thereby detecting the resulting tertiary immune complex. If desired, the system can provide signal amplification.

[0105] An immunoassay method uses two different antibodies. The first step uses a biotinylated monoclonal or polyclonal antibody to detect a target antigen, followed by a second step using an antibody to detect biotin bound to biotin in a complex. In this method, the sample to be tested is first incubated in a solution containing the antibody from the first step. If the target antigen is present, a portion of the antibody binds to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by sequentially incubating in solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, adding additional biotin sites to the antibody / antigen complex at each step. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the anti-biotinylated antibody from the second step. This second-step antibody is labeled, for example with an enzyme, and the presence of the antibody / antigen complex can be determined by histoenzymatic detection using a chromogenic substrate. With appropriate amplification, a visually visible conjugate can be produced.

[0106] Another known immunoassay method utilizes immunoPCR (polymerase chain reaction). This method is similar to the Cantor method, except that instead of incubation with biotinylated DNA using multiple rounds of streptavidin and biotinylated DNA, the DNA / biotin / streptavidin / antibody complex is eluted with a low-pH or high-salt buffer, releasing the antibody. The resulting eluent is then used to perform a PCR reaction with appropriate primers and controls. At least in theory, the enormous amplification capacity and specificity of PCR can be utilized to detect single antigen molecules.

[0107] As detailed above, immunoassays are essentially binding assays. Some immunoassays are the various types of ELISA and RIA known in the art. However, it is easy to understand that detection is not limited to these techniques and can also use Western blotting, dot blotting, FACS analysis, etc.

[0108] In an exemplary ELISA, the antibody of the present invention is immobilized on a selected surface with protein affinity, such as the wells of a polystyrene microtiter plate. A test composition, such as a clinical sample, suspected to contain an antigen, is then added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen can be detected. Detection is typically achieved by adding another antibody linked to a detectable label. This type of ELISA is a simple “sandwich ELISA.” Detection can also be achieved by adding a second antibody followed by a third antibody with binding affinity to the second antibody, wherein the third antibody is linked to a detectable label.

[0109] In some implementations, the principles of ELISA can be applied to a microfluidic cartridge environment (e.g., a plate with 96 individual wells). This microfluidic cartridge allows for "multiplex detection" by using nanoreactors to run several "single" ELISA-type assays in parallel. Unlike techniques that evaluate multiple analytes in a single well, the principle of using microfluidic cartridges for ELISA allows each nanoreactor to evaluate one (e.g., a single) analyte. Multiple nanoreactors can be mounted on a single assay cartridge and run in parallel. This technique combines the advantages of multiplex detection (sample and cost savings) with the advantages of single-analyte ELISA (no cross-reactivity or cross-inhibition from antibodies used to detect multiple analytes in the same well). Compared to solid planar arrays, microfluidic ELISA methods allow for multiplex detection (thus reducing cost and labor), producing more data with fewer samples, less labor, and lower cost, faster and more reproducible results, and focused, flexible multiplex detection to meet a wide range of applications.

[0110] In one exemplary ELISA, the analytes disclosed herein are immobilized on a microfluidic surface, such as the pores of a glass nanoreactor, having an affinity for the specific analyte. A test composition suspected of containing the analyte (e.g., a clinical sample) is added to each well of the microfluidic device. After binding and washing to remove non-specifically bound immune complexes, the bound analyte can be detected. Detection is typically achieved by adding another antibody linked to a detectable marker.

[0111] Alternatively, in some implementations, the principle of ELISA can be implemented using other multiplex immunoassays, such as adjacent extension assays (e.g., commercially available from Olink), electrochemiluminescence assays (e.g., commercially available from MesoScale Discovery), or protein array-based assays.

[0112] In another exemplary ELISA, a sample suspected of containing the antigen is immobilized on the well surface and then contacted with the anti-ORF messenger antibody and anti-ORF translation product antibody of the present invention. After binding and washing to remove non-specifically bound immune complexes, the bound anti-ORF messenger antibody and anti-ORF translation product antibody are detected. If the initial anti-ORF messenger antibody and anti-ORF translation product antibody are conjugated to a detectable label, the immune complex can be detected directly. Similarly, the immune complex can be detected using a second antibody having binding affinity to the first anti-ORF messenger antibody and anti-ORF translation product antibody, wherein the second antibody is conjugated to a detectable label.

[0113] Another type of ELISA where antigen immobilization involves antibody competition in the detection. In this ELISA, labeled antibodies against the antigen are added to the wells, allowing them to bind, and detection is performed by their labeling. The amount of antigen in the unknown sample is then determined by mixing the unknown sample with labeled antibodies against the antigen while incubating the coated wells. The presence of antigen in the sample reduces the amount of antibody against the antigen available for binding to the wells, thus lowering the final signal. This also applies to detecting antibodies against antigens in unknown samples, where unlabeled antibodies bind to antigen-coated wells, reducing the amount of antigen available for binding to labeled antibodies.

[0114] As used herein, the phrase "under conditions for effective formation of immune complexes (antigen / antibody)" refers to conditions under which the antibody or its binding fragment interacts with the antibody-specific target antigen. These conditions may also include diluting the antigen and / or antibody with a solution (e.g., BSA, bovine gamma globulin (BGG), or phosphate-buffered saline (PBS) / Tween). These added reagents also help reduce nonspecific background. "Suitable" conditions mean that incubation is carried out at a temperature or time sufficient to achieve effective binding. Incubation steps are typically about 1 to 2 to 4 hours at a preferably 25°C to 27°C temperature, or possibly overnight at about 4°C.

[0115] Another antibody-based method for assessing biomarker expression is fluorescence-activated cell sorting (FACS), a special type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells cell-by-cell into two or more containers based on the specific light scattering and fluorescence characteristics of each cell. It offers rapid, objective, and quantitative recording of fluorescence signals from individual cells, as well as physical separation of cells of particular interest. A cell suspension is carried in the center of a narrow, rapidly flowing liquid stream. The flow arrangement results in a larger spacing between cells relative to their diameter. Vibrational mechanisms cause the cell stream to split into individual droplets. The system is tuned to minimize the probability of each droplet containing more than one cell. Before the stream splits into droplets, it passes through a fluorescence measurement station, where the fluorescence characteristics of interest for each cell are measured. A charging ring is placed at the location where the stream splits into droplets. A charge is applied to the ring based on the immediately preceding fluorescence intensity measurement, and an opposite charge is captured on the droplet as it detaches from the stream. The charged droplets are then passed through an electrostatic deflection system, which sorts them into containers based on their charge. In some systems, a charge is applied directly to the stream, and detached droplets retain a charge of the same sign as the stream. The stream then returns to neutral after droplet detachment. A common approach using FACS is to identify cells with a specific target by using fluorescently labeled antibodies that bind to a target on or within the cell. This technique can be used quantitatively, where the amount of fluorescent activity is correlated with the amount of target, thus allowing sorting based on the relative amount of fluorescence and subsequently, the relative amount of target.

[0116] Microsphere-based xMAP technology can also be used in conjunction with the invention claimed in this application for immunological assays. This technology combines advanced fluid dynamics, optics, and digital signal processing with proprietary microsphere technology to provide multiple assay capabilities. Characterized by a flexible, open architecture, xMAP technology can be configured for rapid, cost-effective, and accurate execution of a variety of bioassays.

[0117] Fluorescently encoded microspheres are arranged into up to 500 distinct groups. Each microsphere group can be coated with a reagent specific to a particular bioassay (e.g., antibody), enabling the capture and detection of specific analytes from a sample, such as the biomarkers described in this application. Within the xMAP multiplex analyzer, a light source excites the internal dye that identifies each microsphere particle, as well as any reporter dyes captured during the assay. Numerous readings are taken on each microsphere group, further validating the results. Using this method, xMAP technology allows for rapid and accurate multiplex detection of up to 500 unique bioassays within a single sample. Unlike other flow cytometry-based microsphere assays that use combinations of different sizes and color intensities to identify individual microspheres, xMAP technology uses micron-sized microspheres stained internally with red and infrared fluorophores via a proprietary staining method to create 500 unique dye mixtures for identifying each individual microsphere.

[0118] Some advantages of xMAP include multiplex detection (reduced cost and labor), generating more data with fewer samples, less labor and lower cost compared to solid planar arrays, faster and more reproducible results, and centralized, flexible multiplex detection of 1 to 500 analytes to meet a wide range of applications.

[0119] Nucleic acid detection. In other implementations for detecting protein expression, gene transcription can be measured. For example, an indirect method for detecting protein expression is to detect the mRNA transcripts that produce the protein.

[0120] Nucleic acid amplification. Since many mRNAs are relatively rare, nucleic acid amplification greatly enhances the ability to assess expression. The general concept is that nucleic acids can be amplified using paired primers flanking the region of interest. As used herein, the term "primer" refers to any nucleic acid capable of initiating the synthesis of nascent nucleic acids in a template-dependent process. Typically, primers are oligonucleotides of 10 to 20 and / or 30 base pairs in length, but longer sequences can also be used. Primers can be provided in double-stranded and / or single-stranded form, with single-stranded form being more commonly used.

[0121] Under conditions allowing selective hybridization, primer pairs designed to selectively hybridize with nucleic acids corresponding to selected genes are contacted with template nucleic acids. Depending on the desired application, highly stringent hybridization conditions can be selected, which will only allow hybridization with sequences perfectly complementary to the primers. In other embodiments, hybridization can be performed under reduced stringency to allow amplification of nucleic acids containing one or more mismatches with the primer sequences. Once hybridized, the template-primer complex is contacted with one or more enzymes that promote template-dependent nucleic acid synthesis. Multiple rounds of amplification, also known as “cycling,” are performed until a sufficient amount of amplified product is produced.

[0122] The amplified products can be detected or quantified. In some applications, detection can be performed visually. Alternatively, detection may involve indirect identification of the products via chemiluminescence, radioactive scintillation imaging with radioactive or fluorescent labels, or even by using systems employing electrical and / or thermal pulse signals.

[0123] Many template-dependent methods can be used to amplify oligonucleotide sequences present in a given template sample. One of the most well-known amplification methods is polymerase chain reaction (PCR), which is described in U.S. Patents 4,683,195, 4,683,202, and 4,800,159, each of which is incorporated herein by reference in its entirety.

[0124] The amount of amplified mRNA can be quantified using reverse transcriptase PCR amplification procedures. Methods for reverse transcription of RNA into cDNA are well known (see Sambrook et al, Molecular Cloning: A Laboratory Manual, 2001). Alternative methods to reverse transcription utilize thermostable DNA polymerases. These methods are described in WO90 / 07641. Polymerase chain reaction (PCR) methods are well known in the art. Representative RT-PCR methods are described in U.S. Patent No. 5,882,864. Standard PCR typically uses a single pair of primers to amplify a specific sequence, while multiplex PCR (MPCR) uses multiple pairs of primers to amplify multiple sequences simultaneously. The presence of many PCR primers in a single tube can cause numerous problems, such as increased mismatch PCR products and "primer dimer" formation, amplification discrimination against longer DNA fragments, etc. Typically, MPCR buffers contain Taq polymerase additives, which reduce competition between amplicons and amplification discrimination against longer DNA fragments during MPCR. MPCR products can be further hybridized with gene-specific probes for validation. Theoretically, it should be possible to use as many primers as needed. However, due to side effects caused during the MPCR process (primer dimers, primer mismatch PCR products, etc.), the number of primers that can be used in an MPCR reaction is limited (less than 20). See also European Application No. 0364255, the relevant portion of which is incorporated herein by reference.

[0125] Another amplification method is ligase chain reaction (“LCR”), disclosed in European Application No. 320308, which is incorporated herein by reference in its entirety. U.S. Patent No. 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence. Methods based on PCR and oligonucleotide ligase assay (OLA) are also disclosed in U.S. Patent No. 5,912,148. Alternative methods for amplifying target nucleic acid sequences that can be used to carry out the present invention are disclosed in U.S. Patent Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497, 5,849,547, 5,858,652, 5,866,366, 5,916,776, and 5,916,776. The relevant portions of applications 5,922,574, 5,928,905, 5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, GB application 2202328 and PCT application PCT / US89 / 01025 are incorporated herein by reference.

[0126] Nucleic acid detection. After any amplification, it may be necessary to separate the amplification products from the template and / or excess primers. In one embodiment, the amplification products are separated using standard methods by agarose, agarose-acrylamide, or polyacrylamide gel electrophoresis (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2001). The separated amplification products can be cleaved from the gel and eluted for further processing. Using a low-melting-point agarose gel, the separated bands are removed by heating the gel, followed by extraction of nucleic acids. Nucleic acid separation can also be achieved using chromatographic techniques known in the art. A variety of chromatographic methods can be used in the practice of this invention, including adsorption, partition, ion exchange, hydroxyapatite, molecular sieve, reversed-phase, column, paper, thin-layer, and gas chromatography, as well as HPLC.

[0127] In some implementations, the amplification products are visualized. Typical visualization methods include staining the gel with ethidium bromide and visualizing the bands under UV light. Alternatively, if the amplification products are monotonically labeled with radioactive or fluorescently labeled nucleotides, the isolated amplification products can be exposed to X-ray film or visualized under appropriate excitation spectra.

[0128] In one embodiment, after the amplification products are separated, a labeled nucleic acid probe is contacted with the amplified marker sequence. The probe is preferably conjugated to a chromophore, but may also be radiolabeled. In another embodiment, the probe is conjugated to a binding partner, such as an antibody or biotin, or another binding partner with a detectable moiety.

[0129] In a particular embodiment, detection is performed by Southern blotting and hybridization with labeled probes. The techniques involved in Southern blotting are well known to those skilled in the art (see Sambrook et al, Molecular Cloning: A Laboratory Manual, 2001). An example of the foregoing is described in U.S. Patent No. 5,279,721, which discloses an apparatus and method for automated electrophoresis and transfer of nucleic acids, which is incorporated herein by reference. This apparatus allows for electrophoresis and blotting without the need for external manipulation of the gel and is therefore well-suited for implementing the methods of the present invention.

[0130] Other nucleic acid detection methods that can be used to implement the present invention are disclosed in U.S. Patent Nos. 5,840,873, 5,843,640, 5,843,651, 5,846,708, 5,846,717, 5,846,726, 5,846,729, 5,849,487, 5,853,990, 5,853,992, 5,853,993, 5,856,092, and 5,861,244. Nos. 5,863,732, 5,863,753, 5,866,331, 5,905,024, 5,910,407, 5,912,124, 5,912,145, 5,919,630, 5,925,517, 5,928,862, 5,928,869, 5,929,227, 5,932,413 and 5,935,791, each of which is incorporated herein by reference.

[0131] Nucleic acid arrays. Microarrays consist of multiple polymer molecules spatially distributed and stably bound to the surface of a substantially flat substrate (e.g., a biochip). Polynucleotide microarrays have been developed and used in a variety of applications, such as screening and DNA sequencing. One area where microarrays are particularly useful is gene expression analysis.

[0132] In gene expression analysis using microarrays, an array of "probe" oligonucleotides is contacted with a nucleic acid sample of interest (i.e., a target, such as polyA mRNA from a specific tissue type). Contact is performed under hybridization conditions, and then unbound nucleic acids are removed. The resulting pattern of hybridized nucleic acids provides information about the genetic characteristics of the test sample. Gene expression analysis methods performed on microarrays can provide both qualitative and quantitative information.

[0133] A variety of different arrays that can be used with this invention are known in the art. The probe molecules in the array capable of specifically hybridizing with the target nucleic acid sequence can be polynucleotides or hybridization analogs or simulants thereof, including: nucleic acids in which the phosphodiester bonds have been replaced with alternative bonds, such as thiophosphate, methylimino, methylphosphonate, aminophosphate, guanidine, etc.; nucleic acids in which the ribose subunits have been replaced, such as hexose phosphate diesters; peptide nucleic acids; etc. The probe length is typically 10 to 1000 nucleotides, wherein in some embodiments the probe is an oligonucleotide, typically 15 to 150 nucleotides long, more typically 15 to 100 nucleotides long, and in other embodiments the probe will be longer, typically 150 to 1000 nucleotides long, wherein the polynucleotide probe can be single-stranded or double-stranded, typically single-stranded, and can be a PCR fragment amplified from cDNA.

[0134] The probe molecules on the substrate surface will correspond to the selected analyte gene and be located at known positions on the array, allowing positive hybridization events to be associated with the expression of a specific gene in the physiological source of the target nucleic acid sample. The substrate for stable binding of the probe molecules can be made of a variety of materials, including plastics, ceramics, metals, gels, membranes, glass, etc. The array can be prepared according to any convenient method, such as pre-forming probes and then allowing them to bind stably to the support surface, or growing probes directly on the support. Many different array configurations and their fabrication methods are known to those skilled in the art and disclosed in U.S. Patents Nos. 5,445,934, 5,532,128, 5,556,752, 5,242,974, 5,384,261, 5,405,783, 5,412,087, 5,424,186, 5,429,807, 5,436,327, and 5,472, Numbers 672, 5,527,681, 5,529,756, 5,545,531, 5,554,501, 5,561,071, 5,571,639, 5,593,839, 5,599,695, 5,624,711, 5,658,734, 5,700,637 and 6,004,755, the relevant portions of which are incorporated herein by reference.

[0135] After hybridization, if the unhybridized labeled nucleic acids in the detection step are able to emit a signal, a washing step is used to remove the unhybridized labeled nucleic acids from the support surface, generating a hybridized nucleic acid pattern on the substrate surface. Various washing solutions and their usage protocols are known to those skilled in the art and can be used. If the label on the target nucleic acid is not directly detectable, the array now containing the target-binding element is brought into contact with other members of the signal generation system in use. For example, when the label on the target is biotin, the array is brought into contact with a streptavidin-fluorescent conjugate under conditions sufficient to allow binding between specific binding pairs. After contact, any unbound members of the signal generation system are removed, for example, by washing. The specific washing conditions employed will necessarily depend on the specific nature of the signal generation system employed and will be known to those skilled in the art familiar with the specific signal generation system employed. The resulting hybridization pattern of the labeled nucleic acids can be visualized or detected in various ways, the specific detection method being selected based on the specific label of the nucleic acid; representative detection methods include scintillation counting, autoradiography, fluorescence measurement, calorimetry, luminescence measurement, etc.

[0136] Before detection or visualization, if it is necessary to reduce the possibility of false positive signals in the pattern due to mismatch hybridization events, the target / probe complex array of hybridization can be treated with an endonuclease under conditions sufficient to degrade single-stranded rather than double-stranded DNA. Several different endonucleases are known and can be used, including mung bean nuclease, S1 nuclease, etc. When this treatment is used for assays where the target nucleic acid is not labeled with a directly detectable tag, such as in assays using biotinylated target nucleic acids, endonuclease treatment is typically performed before the array is contacted with other members of the signal generation system (e.g., fluorescent-streptavidin conjugates). Endonuclease treatment as described above ensures that only target / probe complexes with substantially complete hybridization at the 3' end of the probe are detected in the hybridization pattern. The resulting hybridization pattern is detected after hybridization as described above and any washing steps and / or subsequent treatments. When detecting or visualizing hybridization patterns, not only is the intensity or signal value of the label detected, but also quantified. This means that the signal at each point in the hybridization pattern is measured and compared with the unit value of the signal emitted by the terminal label target nucleic acid corresponding to a known number of terminal labels to obtain the copy number count or absolute value of each terminal label target hybridized to a specific point on the array in the hybridization pattern.

[0137] RNA sequencing (transcript counting). RNA-seq (RNA sequencing), also known as whole transcriptome shotgun sequencing (WTSS), is a technique that utilizes next-generation sequencing (NGS) capabilities to reveal a snapshot of the presence and number of RNAs in the genome at a given point in time. The cellular transcriptome is dynamic; unlike the static genome, it is constantly changing. Recent advancements in next-generation sequencing allow for increased base coverage of DNA sequences and higher sample throughput. This facilitates the sequencing of RNA transcripts in cells, providing the ability to observe transcripts of candidate gene splicing, post-transcriptional alterations, gene fusions, mutations / SNPs, and altered gene expression. In addition to mRNA transcripts, RNA-Seq can observe diverse RNA populations, including total RNA, small RNAs (e.g., miRNAs), tRNAs, and ribosome maps. RNA-Seq can also be used to determine exon / intron boundaries and to validate or modify previously annotated 5' and 3' gene boundaries. Ongoing RNA-Seq studies include observing changes in cellular pathways during infection and observing altered gene expression levels in cancer research. Prior to NGS, transcriptomics and gene expression studies had been performed using expression microarrays, which contain thousands of DNA sequences to detect matches in target sequences, thus providing a map of all expressed transcripts. This was later accomplished using sequenced gene expression analysis (SAGE).

[0138] Treatment of SLE. This application considers modifying SLE treatment after detection of certain biomarkers, which may include the use of standard treatments where necessary. Typically, SLE treatment involves treating elevated disease activity and minimizing organ damage that may be associated with increased inflammation and immune complex formation / deposition / complement activation. Baseline treatment may include corticosteroids and / or antimalarial drugs. Certain types of lupus nephritis, such as diffuse proliferative glomerulonephritis, require a course of cytotoxic drugs. The most commonly used of these are cyclophosphamide and mycophenolate mofetil. Hydroxychloroquine (HCQ) was approved by the FDA for lupus in 1955. Some drugs approved for other diseases are used “outside of their labeling” for SLE. Belimumab (Benlysta) can be used to treat elevated disease activity in patients with autoantibody-positive lupus. Anifrolumab (Saphnelo) can be used to treat elevated disease activity in adult lupus patients.

[0139] Due to the diversity of symptoms and organ system involvement in SLE, individual severity must be assessed for successful treatment. Mild or remission-stage disease can sometimes be safely treated with minimal therapy using only hydroxychloroquine. Nonsteroidal anti-inflammatory drugs (NSAIDs) and low-dose steroids may also be used if necessary. Hydroxychloroquine (HCQ) is an FDA-approved antimalarial drug for systemic, cutaneous, and joint manifestations. HCQ has relatively few side effects, and there is evidence that it improves survival in SLE patients with lupus in Canada, and discontinuation of HCQ in stable SLE patients leads to an increase in disease flare-ups. Disease-modifying antirheumatic drugs (DMARDs) are often used "outside the label" in SLE to reduce disease activity and decrease the need for steroid use. Commonly used DMARDs are methotrexate and azathioprine. In more severe cases, aggressive immunosuppressive drugs (primarily high-dose corticosteroids and major immunosuppressants) are used to control the disease and prevent further damage. Cyclophosphamide is used for severe glomerulonephritis and other life-threatening or organ-damaging complications such as vasculitis and lupus encephalitis. Enzyme-based phenol is also used to treat lupus nephritis, but it has not been approved by the FDA for this indication.

[0140] Depending on the dosage, people who need steroids may experience symptoms of Cushing's syndrome, including truncal obesity, purple striae, buffalo hump, and other related symptoms. These symptoms may subside if the initial high dose is reduced, but long-term use, even at low doses, may cause high blood pressure, glucose intolerance (including metabolic syndrome and / or diabetes), osteoporosis, insomnia, avascular necrosis, and cataracts. More serious steroid-related complications include accelerated atherosclerosis, ischemic necrosis, increased infections, diabetes, and hypertension.

[0141] Many new immunosuppressive drugs for SLE are being actively tested. Unlike corticosteroids, which nonspecifically suppress the immune system, these target a single type of immune cell response. Belimumab, a humanized monoclonal antibody targeting B lymphocyte stimulating factors (BLyS or BAFF), has been approved by the FDA for the treatment of lupus and reduces SLE disease activity, particularly in patients with elevated baseline disease activity and the presence of autoantibodies. Other drugs, such as abatacept, voclosporin, JAK inhibitors, Tyk inhibitors, and anifrolimab, are being actively investigated in SLE patients, some of which have been approved by the FDA for the treatment of rheumatoid arthritis or other conditions. Because most SLE patients suffer from varying degrees of chronic pain, stronger prescription analgesics (painkillers) may be used if over-the-counter medications (primarily nonsteroidal anti-inflammatory drugs) are ineffective in relieving pain. Potent NSAIDs (such as indomethacin and diclofenac) are relatively contraindicated in SLE patients because they increase the risk of kidney and heart failure.

[0142] Moderate pain is typically treated with mild prescription opioids, such as dextropropoxyphene and codeine (Co-codamol). Moderate to severe chronic pain is treated with more potent opioids, such as hydrocodone, or long-acting continuous-release opioids, such as oxycodone, MS Contin, or methadone. Fentanyl (Duragesic) transdermal patches are also a widely used treatment option for chronic pain caused by complications due to their long-acting, timed release and ease of use. With long-term use of opioids, drug tolerance, chemical dependence, and addiction can occur. Opioid addiction is generally not a major concern, as the condition is unlikely to completely disappear. Therefore, lifelong opioid treatment is quite common for chronic pain symptoms, with the periodic dose adjustments typical of any long-term opioid regimen.

[0143] Intravenous immunoglobulins can be used to control SLE with organ involvement or neuropathy. They are believed to reduce antibody production or promote the clearance of immune complexes in the body, although their mechanisms of action are not fully understood. Unlike immunosuppressants and corticosteroids, IVIG does not suppress the immune system, therefore the risk of serious infections is lower when using these drugs.

[0144] Avoiding sun exposure is a major lifestyle change for SLE patients, as sunlight is known to exacerbate the disease, as is the debilitating effect of severe fatigue. Both of these issues can lead to prolonged periods of confinement to the home. Medications unrelated to SLE should only be prescribed if they are known not to worsen the disease. Occupational exposure to silica, pesticides, and mercury can also worsen the disease.

[0145] Kidney transplantation is the preferred treatment for end-stage renal disease (a complication of lupus nephritis), but complete relapse of the disease in transplanted kidneys is common in up to 30% of patients.

[0146] Antiphospholipid syndrome is also associated with the development of symptoms of neurolupus in the brain. In this form of disease, the cause is very different from lupus: blood clots (blood clots or “thick blood”) form in the blood vessels, which can be fatal if they travel through the bloodstream. If these clots migrate to the brain, they can potentially cause a stroke by blocking the brain's blood supply. If this disorder is suspected, a brain scan is usually required for early detection. These scans can show localized areas of the brain with insufficient blood supply. Treatment for these patients typically involves anticoagulation. Low-dose aspirin is usually prescribed for this purpose, although anticoagulants such as warfarin are used in cases involving blood clot formation.

[0147] Pharmaceutical formulation and delivery. Given the changing therapeutic applications, it is necessary to prepare pharmaceutical compositions suitable for the intended application. Typically, this requires preparing compositions that are substantially free of pyrogens and other impurities that may be harmful to humans or animals.

[0148] Typically, suitable salts and buffers are used to stabilize the delivery carrier and allow it to be taken up by the target cells. Buffers are also used when introducing recombinant cells into a patient. The aqueous composition of the present invention contains an effective amount of cell carrier dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such a composition is also referred to as an inoculum.

[0149] As used herein, the phrase "pharmaceutically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other unintended reactions when administered to animals or humans. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents, etc. The use of such media and reagents for pharmaceutically active substances is well known in the art. Unless any conventional media or reagent is incompatible with the carrier or cells of the present invention, its use in therapeutic compositions is contemplated. Additional active ingredients may also be incorporated into the composition.

[0150] The active compositions of the present invention may include classic pharmaceutical preparations. The administration of these compositions according to the invention will be carried out via any conventional route, provided that the target tissue is accessible by that route. Such routes include oral, nasal, buccal, rectal, vaginal, or local routes. Alternatively, they may be administered by in situ, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. These compositions are generally administered as pharmaceutically acceptable compositions. The active compound may also be administered parenterally or intraperitoneally. Solutions of the active compound as a free base or a pharmacologically acceptable salt can be prepared in water appropriately mixed with a surfactant (e.g., hydroxypropyl cellulose). Dispersions may also be prepared in glycerol, liquid polyethylene glycol, mixtures thereof, and in oils. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth.

[0151] Suitable injectable drug forms include sterile aqueous solutions or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid for easy injection. It must be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms (e.g., bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. For example, in the case of dispersions, appropriate flowability is maintained by maintaining the desired particle size and by using surfactants, for example, through the use of coatings (e.g., lecithin). Microbial action can be prevented by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, isotonic agents, such as sugars or sodium chloride, are preferred. Extended absorption of injectable compositions is achieved by using agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0152] Sterile injectable solutions are prepared by adding the desired amount of the active compound, along with various other ingredients listed above (as needed), to a suitable solvent, followed by filtration and sterilization. Dispersions are typically prepared by adding various sterilized active ingredients to a sterile medium containing a base dispersion medium and the other desired ingredients listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying techniques, which produce powders of the active ingredient plus any additional desired ingredients from a previously sterile filtered solution.

[0153] As used herein, the phrase "pharmaceutically acceptable carrier" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents, etc. Such media and reagents are well known in the art for use with pharmaceutically active substances. Unless any conventional media or reagent is incompatible with the carrier or cells of the present invention, its use in therapeutic compositions is contemplated. Additional active ingredients may also be incorporated into the composition.

[0154] For oral administration, the peptides of the present invention can be mixed with excipients and used in the form of non-swallowable mouthwashes and dental flosses. Mouthwashes can be prepared by dissolving the desired amount of the active ingredient in a suitable solvent (e.g., a sodium borate solution (Dobell's Solution)). Alternatively, the active ingredient can be added to a disinfectant solution containing sodium borate, glycerin, and potassium bicarbonate. The active ingredient can also be dispersed in dental flosses, including gels, pastes, powders, and slurries. The active ingredient can be added in a therapeutically effective amount to a paste-like dental floss, which may contain water, binders, abrasives, flavoring agents, foaming agents, and humectants.

[0155] Compositions used in conjunction with this invention can be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (forming with the free amino group of a protein) and are formed from inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts forming with a free carboxyl group can also be derived from inorganic bases (such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide) and organic bases (such as isopropylamine, trimethylamine, histidine, procaine, etc.). After formulation, the solution will be administered in a dosage form compatible with the dosage form and in a therapeutically effective amount. These formulations are readily administered in a variety of dosage forms, such as injectable solutions, drug-release capsules, etc. For parenteral administration of aqueous solutions, for example, if necessary, the solution should be appropriately buffered and first adjusted to be isotonic with sufficient saline or glucose. In this regard, sterile aqueous media that can be used will be known to those skilled in the art according to this disclosure. For example, a dose of the drug may be dissolved in 1 ml of isotonic NaCl solution and then added to 1000 ml of subcutaneous infusion fluid, or injected at the recommended infusion site (see, for example, “Remington’s Pharmaceutical Sciences,” 15th Ed., 1035-1038 and 1570-1580), the relevant portion of which is incorporated by reference. Dosage will inevitably vary depending on the condition of the person being treated. In any case, the person responsible for administration will determine the appropriate dose for the individual subject. Furthermore, for human use, the preparation should meet the FDA standards for sterility, pyrogenicity, general safety, and purity required for biological products.

[0156] Reagent kits. Reagent kits for the applications described herein are also within the scope of this invention. Such kits may include carriers, packaging, or containers that are divided to contain one or more containers (e.g., vials, tubes, etc.), each containing one of the individual elements to be used in the method, particularly a Bright inhibitor. Reagent kits of this invention will generally include the aforementioned containers and one or more other containers containing materials desired from a commercial user's perspective, including buffers, diluents, filters, and a packaging insert with instructions for use. Furthermore, labels may be provided on the containers to indicate that the composition is intended for a specific therapeutic application, and instructions for in vivo or in vitro use may also be provided, such as those described above. Instructions and / or other information may also be included in the insert accompanying the reagent kit. In particular, reagent kits according to the invention contemplate combinations of reagents for assessing the levels of the aforementioned biomarkers, as well as one or more SLE therapeutic agents and / or reagents for ANA detection and / or anti-ENA detection, and controls for assessing them.

[0157] Current biomarkers for measuring lupus disease activity have limited utility in reflecting elevated clinical disease activity. They are neither the earliest nor the most informative measures of clinical disease activity. Although SLE-related autoantibodies specific to SLE (such as anti-dsDNA, anti-splicing, and anti-Ro / SSA) accumulate in SLE patients several years prior to classification, their presence is insufficient to reflect clinical disease activity and the risk of permanent organ damage. ANA is also present in the serum of patients with other systemic rheumatic diseases and in the serum of healthy individuals who have not developed SLE (including some unaffected family members of SLE patients), and can even be detected in up to 14% of the general population. Because individuals may remain healthy but be ANA positive, ANA positivity alone may not be the sole pathogenic driver of SLE. In addition to ANA positivity, dysregulation of various immune pathways driven by soluble mediators may contribute to clinical disease activity.

[0158] Example

[0159] The following embodiments are included to further illustrate various aspects of the invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques and / or compositions that the inventors have discovered that are effective in implementing the invention, and therefore can be considered as preferred embodiments thereof. However, based on this disclosure, those skilled in the art will understand that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention, and similar or related results can still be obtained.

[0160] SLE is a complex autoimmune disease characterized by immune dysregulation. Comprehensive and cost-effective tools for tracking changes in disease activity will help improve disease management and prevent organ damage. This embodiment aims to identify key components of a practical bioassay for distinguishing between active and low-activity lupus disease.

[0161] The following examples demonstrate an optimal combination of biomarkers capable of differentiating SLE patients with active disease and contributing to the Complete Lupus Disease Activity (Immune) Index (LDAII / L-DAI). The optimized LDAII / L-DAI developed in these examples characterizes SLE patients with active clinical disease. Targeted therapy using sensitive and objective surrogate biomarkers to reflect clinical disease activity helps improve SLE disease management and prevent organ damage.

[0162] In these embodiments, samples were collected from categorized SLE patients and defined on the collection day by a pooled SLEDAI (hSLEDAI) score of low disease activity (< 4, range 0–3, n = 162) or active disease activity (≥ 4, range 4–30, n = 162). Race / sex / age-matched healthy control (Ctrl) samples (n = 81) were also evaluated. Plasma immune mediators were assessed by microfluidic immunoassay (n = 33), and serum autoantibody specificity, including dsDNA, chromatin, Ro / SSA, La / SSB, Sm, SmRNP, and RNP, was assessed by xMAP assay. LDAII / L-DAI was the sum of the following: log-converted and normalized immune mediators, weighted by the Spearman r correlation coefficient of mediator levels against hSLEDAI score, the number of autoantibody specificities associated with clinical disease activity, or a composite weighted average (the weighted mean of hSLEDAI and autoantibody levels). Further analysis of the media level of the log transformation was conducted using a machine learning model employing random forests to determine the optimal subset of analytes for providing LDAII / L-DAI information.

[0163] SLE patients with active disease showed differences in clinical and serological characteristics and increased steroid use. A random forest model comparing immune mediators of low-activity disease versus active disease (including clinical and / or serological active disease versus quiescent disease and controls) provided a ranking of variable importance. Forward selection (adding mediators to the top-ranked mediator IFN-α) and backward elimination (subtracting mediators from the total), combined with univariate analysis, yielded a baseline combination of nine mediators that best provided LDAII / L-DAI information: IFN-α, IL-10, BlyS, IL-7, IFN-γ, TRAIL, IL-15, IP-10 / CXCL10, and IL-4. L-DAI 9 significantly (p ≤ 0.05) distinguished SLE patients with low-activity disease versus active disease (including clinical / serological active / quiescent disease compared to controls) regardless of whether weighted by hSLEDAI or autoantibody number, with a strong correlation between the weights (Spearman r = 0.990). p < 0.0001 The mean composite LDAII / L-DAI ratio could distinguish between SLE patients with low-activity disease, active disease, and clinical / serological active / quiet disease (compared to controls). After applying decision curve analysis, it enhanced the differentiation of low / intermediate / high-risk regions of active disease, exhibiting a large Cohen's effect size (>0.8) and an AUC of 0.795 in differentiating between clinical / serological active / quiet disease activity. p < 0.0001It was significantly correlated with concurrent clinical disease activity (hSLEDAI score) (Spearman r = 0.407, p < 0.0001 To maximize the clinical efficacy of LDAII / L-DAI, the combination of soluble mediators TNFRII, resistin, and osteopontin (OPN) with baseline LDAII / L-DAI 9 mediators was also considered to further characterize clinical disease activity through organ system manifestations.

[0164] Materials and methods

[0165] Study population, clinical data, and sample collection

[0166] The experiment was conducted in accordance with the Helsinki Declaration and approved by the institutional review committees of the Oklahoma Medical Research Foundation (OMRF) and Progentec Diagnostics. Participants in established, prospective, longitudinal cohort studies met the criteria of the American College of Rheumatology and the International Collaboration on Clinical SLE Classification (Clinical SLE Classification). Ines et al., 2015 Appropriate consent and in accordance with the Mixed Systemic Lupus Erythematosus Disease Activity Index (hSLEDAI) ( Thanou et al., 2016 Disease activity was assessed as defined by the criteria for SLE. Plasma and serum samples were obtained from clinical visits where SLE patients presented with low (hSLEDA1 < 4; n = 162) or active (hSLEDA1 ≥ 4; n = 162) disease and were compared with sex / race / age-matched healthy individuals (Ctl; n = 81), Table 1. Samples were stored at -80°C in the CAP-certified biobank or Progentec biobank at the Oklahoma Rheumatic Diseases Research Core Center (ORDRCC) OMRF until freshly thawed samples were tested. Samples in the biobank were tested for transport time and methods, processing procedures, storage conditions, and storage duration to determine the levels of soluble mediators and SLE-associated autoantibodies (AutoAbs) in samples from SLE patients and Ctl.

[0167] Table 1. Characterization of SLE patient samples

[0168] a Low = hSLEDAI < 4; Active = hSLEDAI ≥ 4

[0169] b Continuous variables were statistically significant using the Kruskal-Wallis and Dunn multiple comparisons or the Mann-Whitney test; categorical variables were statistically significant using the Fisher exact test or the χ² test. 2 Test (uncorrected, significant p≤0.05)

[0170] c Pacific Islanders, Hispanics, or multi-ethnic groups

[0171] d hSLEDAI = SELENA - SLEDAI + proteinuria as defined by SLEDAI-2K

[0172] e Immunosuppressants = azathioprine, methotrexate, sirolimus, tacrolimus; Primary immunosuppressants = mycophenolate mofetil, cyclophosphamide; Biologics = rituximab, abatacept, belimumab

[0173] f Autoantibody specificity numbers # include anti-dsDNA, chromatin, Ro / SSA, La / SSB, Sm, SmRNP and RNP (low [n=155]; active [n=158])

[0174] As mentioned above, demographic and clinical information was collected. Crowe et al., 2011 ), including drug use, clinical laboratory values, and clinical disease activity. This is assessed by administering the mixed systemic lupus erythematosus disease activity index (hSLEDAI; SELENA-SLEDAI with proteinuria as defined by SLEDAI-2K). Thanou et al., 2014; Thanou et al. al., 2016 To assess the presence of organ system involvement, consider the following: central nervous system involvement (CNS; seizures, psychosis, organic brain syndrome, visual disturbances, cranial nerve disorders, or lupus headache), vasculitis, arthritis, myositis, nephritis (urethritis, hematuria, proteinuria, or pyuria), mucocutaneous lesions (rash, hair loss, or mucosal ulcers), serositis (pleurisy or pericarditis), or hematological manifestations (low complement, increased DNA binding, fever, thrombocytopenia, or leukopenia). Petri et al., 2005 The presence of disease manifestations.

[0175] Measurement of soluble media and autoantibody specificity

[0176] The specificity of autoantibodies in serum samples was screened using the BioPlex 2200 multiplex system (Bio-Rad Technologies, Hercules, CA), as shown in Table 1. The BioPlex 2200 ANA kit uses fluorescently stained magnetic beads to simultaneously detect the specificity levels of 11 autoantibodies, including reactivity against dsDNA, chromatin, ribosomal P, Ro / SSA, La / SSB, Sm, Sm / RNP complex, RNP, Scl-70, centromere B, and Jo-1. Bruner et al., 2012 In this study, SLE-related autoantibody specificity against dsDNA, chromatin, Ro / SSA, La / SSB, Sm, Sm / RNP complex, and RNP was analyzed. Anti-dsDNA (IU / mL) had a pre-defined positive cutoff of 10 IU / mL; antibody index (AI) values ​​(range 0–8) were reported by the manufacturer to reflect the fluorescence intensity specific to each of the other autoantibody types, with a positive cutoff of AI = 1.0. AI values ​​were normalized according to calibrators and controls provided by the manufacturer.

[0177] In this LDAII / L-DAI validation / optimization cohort study, 33 soluble mediators were selected for evaluation (Table 2). The plasma soluble mediators evaluated included cytokines, chemokines, and soluble receptors. An equal number of paired low / active SLE samples and their appropriate race / sex / age-matched controls were evaluated on each kit set (all 33 mediators were evaluated on a batch of custom-designed single / multiplex analyte kits, as recommended by the manufacturer). Furthermore, at least two races were included on each kit set, and no single race was evaluated on only one kit set to achieve a balanced distribution and limit batch effects within the dataset.

[0178] Table 2. SLE-related mediator pathways assessed

[0179] The Lo / Med / Hi quality control (QC) samples (for each analyte measurement) provided by the manufacturer, along with plasma samples from SLE and healthy individuals, were evaluated on each test kit according to the manufacturer's standard operating procedure (SOP). These QC samples were prepared fresh according to the instructions before being tested with the EIIa instrument. Since only two kits (out of seven) could be evaluated at any given time, a thaw control was added to ensure that the QC samples within the concentration range of the evaluated plasma samples were thawed and processed simultaneously with the plasma samples to be evaluated according to the SOP. In the batch of kits required to evaluate 405 plasma samples and the QC / thaw controls, the mean coefficient of variation (CV) for all tested analytes in the QC / thaw control samples was <10%. The test results for all Lo / Med / Hi and thaw control QC samples were within the concentration range specified by the manufacturer for each analyte. Furthermore, the concentration of the thaw control QC samples differed from the concentration of the matched QC samples prepared fresh before testing by less than 10%, indicating that the testing time had minimal impact on the Ella kit / sample.

[0180] Samples with concentrations < LLOQ were assigned a value of 0.001 pg / ml, and delabeled datasets were generated that included demographic (race, sex, age), clinical (disease activity, medication, comorbidities), and biological (soluble mediators, autoantibody specificity) data for subsequent subgroup analyses (univariate and multivariate analyses). IL-2 and IL-1α were excluded from univariate and multivariate analyses because ≥ 60% of plasma samples had concentrations < LLOQ (0.001 pg / ml).

[0181] Statistical analysis

[0182] Categorical variables were compared using Fisher's exact test. Disease activity scores were compared between clinical visits for low-activity and active disease using an unpaired t-test. Autoantibody-specific numbers and plasma soluble mediator concentrations were compared between visits for SLE patients with low-activity or active disease using the Mann-Whitney test. Plasma mediator concentrations and autoantibody-specific numbers were compared between visits for SLE patients with low-activity and active disease and race / sex-matched controls using the Kruskal-Wallis test and Dunn's multiple comparisons. The correlation between plasma mediator concentrations and hSLEDAI scores or autoantibody-specific numbers was analyzed using Spearman's rank correlation analysis. Cohen's test was used to further analyze the correlation between plasma mediator concentrations and hSLEDAI scores or autoantibody-specific numbers. d (Cohen, 1992) determined the effect size as the difference between the mean difference between the active (M1) and low (M2) SLE disease activity groups divided by the pooled standard deviation, ([M2-M1] / SD). 合并 SD 合并=√[{SD1 2 +SD2 2 [ / 2]). Use Bonferroni correction for multiple comparisons by dividing α (minimum significance p-value) of 0.05 by the number of statistical tests for the given categorical variable.

[0183] Based on the method of Genuer et al. (2010), and capable of incorporating repeated measures (Capitaine et al., 2021), a random forest partitioning tree classification was implemented to rank variables by their ability to distinguish between: visits to SLE patients with active or low disease activity versus controls, and visits to SLE patients with clinically and / or serologically (positive anti-dsDNA autoantibodies and / or low complement levels) active or quiescent disease versus controls. The parameter settings used included: maximum number of trees = 50, and maximum number of variables considered for split nodes = 100, with 5-fold cross-validation performed over 2000 iterations. Variables were ranked from most informative to least informative based on their importance. The variables are then applied to LDAII / L-DAI, either by adding one variable at a time (starting with the most informative variable; forward selection) or removing one variable at a time from the total list of variables (starting with the least informative variable; backward elimination) (Genuer et al., 2010) to optimally predict disease activity outcomes.

[0184] A method for calculating the Lupus Disease Activity (Immune) Index (LDAII / L-DAI) was developed to compare overall inflammation levels during patient visits at different disease activity levels, distinguishing them from controls. LDAII / L-DAI summarizes the dysregulation of plasma mediators assessed in clinical visits to SLE patients with low-activity and active disease, and in matched controls, and provides a composite score by weighting them against their correlation with hSLEDAI scores, the number of autoantibody-specific samples detected in the same visit sample, or the mean of both. LDAII / L-DAI was calculated as follows: 1. For each SLE patient or control visit, the baseline plasma mediator concentrations selected for statistical analysis in Table 2 were log-transformed. 2. For each participant visit, the soluble mediator level for each log-transformation was standardized as: (observed value) - (mean of all SLE patient and control visits) / (standard deviation of all SLE patient and control visits). 3. Spearman coefficients were generated using a linear regression model to examine the association between the number of autoantibody-specific values ​​detected for each soluble mediator in all SLE patient and control visits, and the substitution association (Spearman r) between hSLEDAI scores and the assessment of each soluble mediator in all SLE patient visits; 4. The transformed and standardized soluble mediator levels were multiplied by their respective Spearman coefficients (Spearman r) and weighted. 5. For each participant visit, the log-transformed, standardized, and weighted values ​​of each soluble mediator based on LDAII / L-DAI were summed to calculate the total LDAII / L-DAI.

[0185] The LDAII / L-DAI ratio was compared between visits to SLE patients with low-activity disease and those with active disease, or between visits to SLE patients with clinically and serologically active disease (CASA) and those with clinically and serologically active disease (CQSQ). Further comparisons with controls were performed using the Kruskal-Wallis test and Dunn multiple comparisons. The odds ratios (ORs) of the probability of positive or negative LDAII / L-DAI scores were determined for SLE patients visiting with active disease versus those visiting with low-activity disease (or controls); significance was determined using Fisher's exact test.

[0186] Logistic regression analysis was performed on LDAII / L-DAI scores from active and low disease activity samples, as well as CASA and CQSQ samples, to determine the threshold probability of active disease. As previously described (Vickers et al., 2008), decision curve analysis was used to further determine cutoff values ​​for low / intermediate and intermediate / high risk LDAII / L-DAI scores. In short, the threshold probability of onset risk for each active / low (or CASA / CQSQ) LDAII / L-DAI score was compared to a net benefit, where “net benefit” = true positives / n - (false positives / n) [p t / 1-p t ]), where n is the total number of patients in the study, p t It is the (predicted) threshold probability for any given LDAII / L-DAI score. To create the decision curve, the threshold probability (p...) t The value varies within a range that covers the threshold probabilities associated with the active and inactive (or CASA / CQSQ) LDAII / L-DAI scores included in the analysis. For each p... t 1. If the activity (or CASA) p t ≥Selected p t 1. An active (or CASA) visit is defined as a true positive; 2. If the p-value is low... t ≥Selected p t If low disease activity (or CQSQ) visits are defined as false positives; 3. Calculate the given p t 4. Calculate net profit: number of true positives / n - (number of false positives / n) [p t / 1-p t (Vickers et al., 2008).

[0187] Univariate analysis, logistic regression, and decision curve plotting were performed using GraphPad Prism 9.5.1 (GraphPad Software, San Diego, CA). Multivariate random forest was performed using JMP® Genomics, Version 9. SAS Institute Inc., Cary, NC, 1989–2021.

[0188] result

[0189] Increased number of specific autoantibodies and altered levels of select mediators in cases of clinically active disease

[0190] Based on the hypothesis that, similar to SLE that has transitioned to a classification, immune dysregulation is more strongly reflected in SLE patients with clinically active disease than in those with low disease activity or quiescent disease, serum autoantibodies and plasma soluble mediator profiles in samples collected during clinical visits reflecting active disease activity (hSLEDAI ≥ 4, range 4–30) versus low disease activity (hSLEDAI < 4, range 0–3) were compared (Table 1). As expected, SLE patients with active disease activity had significantly higher hSLEDAI scores and were more likely to present with multi-organ system manifestations compared to those with low disease activity. Furthermore, SLE patients with active disease showed a significant increase in the number of SLE-related autoantibodies specific to be detected during low disease activity. p = 0.0007 During active disease, anti-dsDNA ( p < 0.0001 ) and antichromatin ( p = 0.0001 The autoantibodies of Ro / SSA, La / SSB, Sm, SmRNP, and RNP are more likely to be positive, while the frequencies of specificity of these autoantibodies are similar in low-activity and active disease (significant after Bonferroni correction for multiple comparisons). p = 0.0071 Table 1. After multiple comparison correction (Bonferroni correction significance). p = 0.0100 SLE patients with active disease are more likely to be prescribed steroids. p = 0.0002 Patients with low disease activity were prescribed hydroxychloroquine and / or immunosuppressants, as shown in Table 1.

[0191] The study also assessed whether SLE-related changes in soluble mediators were also present in patients with active SLE, as shown in Table 3. After multiple comparison correction (Bonferroni correction for significance), the results were statistically significant. p = 0.0015 Compared with matched healthy controls, plasma levels of 20 mediators (including innate, acquired, chemokines, and other inflammatory mediators) were significantly altered in SLE patients regardless of clinical disease activity. Three of these mediators were TNFRIIs (…). p < 0.0001 MIP-1α / CCL3 p < 0.0001 ) and MIP-1β / CCL4 ( p = 0.0008 Compared with SLE patients with low clinical disease activity, SLE patients with active disease had elevated levels of seven selected soluble mediators, including IFN-α (…). p < 0.0001 ), IL-10 p < 0.0001 ), TNF-α ( p < 0.0001 ), TNFRII ( p = 0.0003), TRAIL p = 0.0012 ), MIG / CXCL9 ( p < 0.0001 ) and IP-10 / CXCL10 ( p < 0.0001 The other 10 media also showed univariate significance, but were no longer significant after multiple comparison correction (Table 3).

[0192] Table 3. Soluble media of SLE cases compared to healthy controls

[0193] a The concentration of soluble media was pg / ml (mean ± SEM), and 324 SLE cases were compared with 81 race / sex / age-matched healthy controls (Ctl).

[0194] b Low activity = hSLEDAI < 4 (n=162); Activity = hSLEDAI ≥ 4 (n=162)

[0195] c Significance was determined by the Kruskal-Wallis test and Dunn multiple comparisons (uncorrected p≤0.05); Bonferroni corrected significance. p=0.0015

[0196] We further evaluated the ability of soluble mediators to distinguish between visits to SLE patients with clinical (C) and / or serological (S, defined by immunological hSLEDAI characteristics: positive anti-dsDNA autoantibodies ± low complement levels) active (A) or quiescent (Q) disease compared to controls, as shown in Table 4. After multiple comparison correction (Bonferroni correction for significance) p = 0.0015 Compared with the control group, regardless of clinical and / or serological disease activity or quiescence, plasma levels of 16 mediators were significantly altered during visits to SLE patients. p ≤ 0.0008 Regardless of serological activity, mediators elevated only in clinically active disease compared to controls included IL-6, TRAIL, MIP-1α / CCL3, and MIP1-β / CCL4. p ≤ 0.0013 In contrast to controls, IFN-γ and MIG / CXCL10 were elevated in all SLE samples, except for quiescent disease (CQSQ). p < 0.0001 In addition, after multiple comparison correction (Bonferroni correction significance). p = 0.0015There are 10 mediators that can distinguish between CASA and CQSQ, including IFN-α, IL-15, IFN-γ, IL-10, TNF-α, TNFRI, TNFRII, TRAIL, MIG / CXCL9, and IP-10 / CXCL10. p ≤ 0.0008 There are seven mediators that can distinguish between the CASA and CASQ regions, including IFN-α, IL-10, TNF-α, TNFRII, MIP-1α / CCL3, MIP-1β / CCL4, and IP-10 / CXCL10. p ≤ 0.0009 These data suggest that alterations in many selected mediators are directly associated with clinical and / or serological activity in SLE.

[0197] Table 4. Soluble mediators in SLE cases with serological / clinically active or quiescent disease compared to healthy controls

[0198] The concentration of soluble media was pg / ml (mean ± SEM), and 324 SLE cases were compared with 81 race / sex / age-matched healthy controls (Ctl).

[0199] b CQSQ = Clinical / Serologically Quiescent (n=78), CASQ = Clinically Active / Serologically Quiescent (n=93), CQSA = Clinically Quiescent / Serologically Active (n=57); Serological = Immunological Characteristics (Positive for anti-dsDNA, Low Complement)

[0200] The significance of c was determined by the Kruskal-Wallis test and Dunn multiple comparisons (uncorrected p≤0.05); the significance after Bonferroni correction was... p=0.0015 .

[0201]

[0202] Changes in soluble mediator levels are associated with the presence of autoantibodies and clinical disease activity.

[0203] Multiple soluble mediators (assessed in 24 / 33) were significantly associated with the cumulative number of SLE-related autoantibodies in SLE patients and matched healthy controls. p ≤ 0.0015 (After multiple comparison correction), including innate and acquired mediators, chemokines, and TNF superfamily members, Table 5. Interestingly, the native form of the regulatory mediator TGF-β was negatively correlated with the presence of autoantibody specificity (Spearman r = -0.263, p < 0.0001 Conversely, IL-10 (which can act as a regulatory mediator and activator of B lymphocytes) is positively correlated with the accumulation of autoantibodies specifically (Spearman r = 0.478). p <0.0001 ).

[0204] Table 5. Spearman correlation between soluble media and the number of autoantibody specificities or hSLEDAI scores

[0205] a The number of autoantibodies = SLE-related specificity against dsDNA, chromatin, Ro / SSA, La / SSB, Sm, SmRNP, and RNP, detected by Bioplex 2200.

[0206] b hSLEDAI = SELENA-SLEDAI with the SLEDAI 2K proteinuria definition

[0207] c Significance was determined by the Kruskal-Wallis test and Dunn multiple comparisons (uncorrected p≤0.05); Bonferroni corrected significance. p=0.0015

[0208] A more defined set of biomarkers were directly associated with clinical disease activity (hSLEDAI score) (Table 5). After multiple comparison correction (Bonferonni correction) p = 0.0015 Of the 33 mediators assessed, 15 were significantly associated with the hSLEDAI score, including IFN-α (r = 0.360), MIG / CXCL9 (r = 0.252) and IP-10 / CXCL10 (r = 0.310), TNF superfamily members TNF-α (r = 0.275) and TNFRII (r = 0.270), and activated mediators IL-10 (r = 0.308) and OPN (r = 0.180). Nine other soluble mediators (including IFN, TNF mediators, and chemokines) were initially found to be significant before multiple comparison correction (Table 5).

[0209] Optimization of the weighted lupus disease activity (immune) index (LDAII / L-DAI) for clinical application to characterize active disease in SLE patients.

[0210] The performance of LDAII / L-DAI, as well as the technical feasibility and cost-effectiveness of performing this laboratory test, were then considered to optimize which mediators would be used for the optimized version of LDAII / L-DAI in clinical applications. Using random forest variable importance analysis, the immunosoluble mediators that best distinguished between SLE visits with active or low disease activity and healthy controls were ranked from most informative to least informative. Figure 1The mediators were then applied to LDAII / L-DAI, based on log-transformed and standardized plasma mediators, and weighted according to hSLEDAI scores or the number of SLE-related autoantibodies at sample collection / clinical visit. Through forward selection (starting with the top-ranked mediator IFN-α, followed by other mediators in sequence) and backward elimination (using all mediators and then subtracting them in reverse order of importance, starting with IL-8 / CXCL8), the 10 mediators were found to provide the most information for LDAII / L-DAI. IL-12p70 was eliminated because it was not statistically significant in distinguishing between low-activity and active disease in SLE patients (compared to controls, Table 3) and between clinically / serologically active and quiescent disease (compared to controls, Table 4), leaving 9 mediators that provided the most information for LDAII / L-DAI. Figure 1 and Figure 2 ).

[0211] Regardless of the weighting by hSLEDAI score ( Figure 2 A in the equation) or weighted by the number of autoantibody specificities ( Figure 2 In section B), the LDAII / L-DAI provided by the top 9 differentiating mediators (L-DAI 9) were able to distinguish SLE patients with different combinations of clinical and / or serological active and quiescent disease, as well as active and low disease activity (compared to healthy controls). In fact, L-DAI 9 weighted by hSLEDAI score was highly correlated with L-DAI 9 weighted by the number of autoantibody specificities (Spearman r = 0.990). p < 0.0001 ), Figure 2 C in the equation. This forms the L-DAI 9 composite score (an average score weighted by hSLEDAI and autoantibody). Figure 3 The L-DAI 9 composite score can differentiate SLE patients with different combinations of clinical and / or serological active and quiescent disease. Figure 3 A in the text), and activity versus low disease activity ( Figure 3 B) in the comparison with healthy controls.

[0212] Logistic regression combined with decision curve analysis was used to identify SLE patients (CASA, ...) with both clinical and serological active disease. Figure 3 A in the upper right image), and active disease as defined by hSLEDAI ( Figure 3 The optimal cutoff values ​​for low / medium and medium / high risk cutoff points (see Figure B in the upper right corner). Compare CASA with CQSQ (…). Figure 3In section A), setting the low / intermediate risk cutoff to -1.7577 (19% risk threshold) increased the sensitivity and negative predictive value (NPV) of L-DAI 9 to 100%, while setting the intermediate / high risk cutoff to 2.0624 (79% risk threshold) increased both specificity (94%) and positive predictive value (PPV, 87%). Similar findings were obtained when comparing hSLEDAI-defined disease activity with low disease activity. Figure 3 (B) Setting the low / intermediate risk cutoff value to -1.7363 (30% risk threshold) achieves 100% sensitivity and NPV for L-DAI 9, while setting the intermediate / high risk threshold to 2.4928 (69% risk threshold) maximizes L-DAI 9 specificity (94%) and PPV (79%). These cutoff values ​​will allow providers to assess the immune status of SLE patients prior to clinical visits to determine if additional clinical evaluation is needed to make treatment decisions that affect the development of permanent organ damage, morbidity, and early mortality. For example, patients with intermediate or high LDAII / L-DAI scores may require additional clinical evaluation and / or reassessment of current disease management in the near future (e.g., adjusting the dose of a treatment agent, replacing one or more treatment agents with another, and / or increasing the dose). Alternatively, patients with low LDAII / L-DAI scores suggest that additional clinical evaluation may not be necessary immediately and could be performed at the next scheduled visit, or one or more treatment agents may be discontinued (reduced dose or discontinued). Alternatively, the LDAII / L-DAI score can be used for longitudinal follow-up of SLE patients to determine when additional clinical evaluation is needed (e.g., for patients with intermediate or high LDAII / L-DAI scores), whether current disease management has stabilized immune disease activity (e.g., for patients with low LDAII / L-DAI scores), or whether targeted therapy is effective (e.g., for patients with low LDAII / L-DAI scores).

[0213] To maximize the cost-effectiveness of the optimized LDAII / L-DAI assay, consider adding three additional analytes (TNFRII, resistin, and osteopontin (OPN)) that require a 1:10 sample dilution as a complement to the baseline optimized L-DAI 9 assay, as shown in Table 6.

[0214] Table 6. L-DAI Media Considering Commercial / Clinical Applications

[0215] With L-DAI 9 ( Figure 4 Compared to A), adding TNFRII, resistin, or OPN (L-DAI10A to 10C) as a single mediator... Figure 4 (B to D in the middle), combinations of the two media (L-DAI 11A to 11C, Figure 4 (E to G in the middle), or all three media (L-DAI 12, Figure 4 The H in the table showed no statistically significant difference in the ability to distinguish between clinical / serological active / quiescent disease states and between low-activity and active disease as defined by hSLEDAI (compared to controls). Assessing overall performance (Table 7), the addition of these additional mediators (particularly OPN ± resistin) did not reduce the ability to distinguish CASA from CQSQ or hSLEDAI-defined active and low-activity disease by AUC, effect size, or classification performance (including the odds ratio of positive / negative categories with a cutoff of 0, specificity, sensitivity, NPV, and PPV). Furthermore, adding OPN alone (L-DAI 10C) or in combination with TNFRII (L-DAI 11B) or resistin (L-DAI 11C) improved the ability of the immune mediators to correlate LDAII / L-DAI with concurrent hSLEDAI-defined clinical disease activity (Table 7B). This will enable LDAII / L-DAI to be developed as a laboratory-developed assay for screening SLE patients to determine the need for additional clinical evaluation.

[0216] Table 7. Characteristics of L-DAI

[0217] A. Composite L-DAI CASA compared to CQSQ a

[0218] B. The low activity of compound L-DAI compared to its activity

[0219] a CASA = Clinical / Serological Activity; CQSQ = Clinical / Serological Stagnation; Low Activity = hSLEDAI < 4; Active Activity = hSLEDAI > 4

[0220] bL-DAI 9=IFN-a, IL-10, BLyS, IL-7, IFN-y, TRAIL, IL-15, IP-10 / CXCL10 and IL-4; L-DAI 10A=L-DAI 9+TNFRII(T); L-DAI 10B=L-DAI 9+resistin(R); L-DAI 9+osteopontin(O);L-DAI 11A= L-DAI 9 +T + R; L-DAI 11B = L-DAI9 +T+ O; L-DAI 11C = L-DAI 9 + R+O; L-DAI 12= L-DAI 9 +T +R+ O

[0221] c Receiver Operating Characteristic (ROC) curve; AUC = Area Under the ROC Curve

[0222] d Odds ratio (CASA vs CQSQ [A] or low compared to active disease [B] positive or negative L-DAI)

[0223] e Fisher Precision Inspection

[0224] f Pearson correlation L-DAI compared to hSLEDAI score at sample collection

[0225] We also evaluated the ability of L-DAI 9± additional mediator to differentiate organ system performance within the hSLEDAI tool, Table 8.

[0226] Table 8. L-DAI scores and soluble mediator levels in SLE as manifested by organ systems

[0227] The concentration of soluble media was pg / ml (mean ± SEM). 324 SLE cases were compared with 81 race / sex / age-matched healthy controls (Ctl).

[0228] The significance of b was determined by the Kruskal-Wallis test and Dunn multiple comparisons (uncorrected p ≤ 0.05); the significance after Bonferroni correction was p = 0.0015.

[0229] Compared to L-DAI 9, the addition of TNFRII, resistin, and / or OPN maintains (for vasculitis, arthritis, kidney, mucocutaneous) or enhances (for serositis, immunology, and hematology) the ability of LDAII / L-DAI to distinguish the presence of manifestations in different organ systems. Manifestations present only in active disease (hSLEDAI ≥ 4), including vasculitis, arthritis, and renal features, can be distinguished equally (vasculitis) or more significantly (arthritis and kidney) upon the addition of these additional mediators to L-DAI 9, Table 9. These data suggest that even in SLE patients with chronic active disease, LDAII / L-DAI results can indicate the need for additional clinical evaluation and / or treatment adjustments.

[0230] Table 9. L-DAI scores and soluble mediator levels in active SLE as manifested by organ systems

[0231] a Lupus Disease Activity Index (L-DAI) score and soluble mediator concentration (pg / ml) (mean and SEM)

[0232] b Low = hSLEDAI < 4 (n = 162); Active = hSLEDAI > 4 (n = 162 in total)

[0233] c Significance was determined by the Kruskal-Wallis test and Dunn multiple comparisons (uncorrected p≤0.05).

[0234] Persistent active disease is a burden for SLE patients, impacting quality of life and healthcare costs, especially for those with long-term illness. Disease activity has been shown to increase over time, and most patients with low disease activity will develop active disease associated with organ damage and early death, highlighting shortcomings in optimal SLE management. The ultimate goal is to facilitate a transition to a low-disease-activity state, as low disease activity has been shown to improve outcomes and prognosis, reduce organ manifestations and permanent organ damage, reduce treatments with significant side effects and morbidity, and lower mortality.

[0235] Identifying and treating early to prevent tissue and organ damage is challenging because signs and symptoms of disease activity are often only detected after it has occurred. Furthermore, the long-term use of steroids and other immunosuppressants required to control disease activity is associated with increased morbidity. The inability to proactively manage clinical disease limits healthcare to passive treatment, hindering proactive strategies such as increasing or strengthening steroid use and cautious use of immunomodulators to prevent end-organ damage and reduce the morbidity and socioeconomic burden of SLE. In addition to the specific accumulation of SLE-related autoantibodies, dysregulation of multiple immune pathways is fundamental to the development and progression of classified SLE and post-classification clinical disease activity. The current lack of immune mechanism-based disease management assays stems from the absence of any single immune pathway-related biomarkers as surrogate indicators. Moreover, classical serological markers of disease activity are insufficient biological signals and cannot directly indicate clinically active disease.

[0236] This study identified underlying immune dysregulations reflecting persistent clinical disease activity and used this information to construct and refine immune indices that complement current tools for assessing clinical disease activity. In this study, heterogeneity was observed in the number and type of immune pathway alterations in SLE patient samples with and without active disease. This may partially explain the previously reported variability in inflammatory mediators in SLE patients with active disease, as well as the inconsistent correlations and limited clinical utility of proposed serological markers of disease activity (used alone or in combination, including anti-dsDNA, complement, complement cleavage products, and inflammatory markers (ESR and CRP)). Despite the heterogeneity of immune pathways involved, each patient showed elevated inflammatory mediators in at least one pathway. The involvement of IFN-related mediators was not unexpected, including alterations in type I IFN (IFN-α), type II IFN (IFN-γ), and IFN-associated chemokines (IP-10 / CXCL10), given the well-established IFN signature in SLE patients. Alterations in IFN-related mediators are directly associated with disease activity (low activity versus active, clinical / serological quiescence versus active) and are also associated with the presence of SLE-related autoantibodies in some patients. This finding is supported by previous studies that have shown the IFN pathway can reflect disease activity, depending on the presence of autoantibodies in some patients, but cannot universally explain concurrent clinical disease activity.

[0237] Many mediators are not directly related to disease activity measures but are associated with the accumulation of autoantibody-specific substances and can differentiate between low-activity and active disease in autoantibody-positive and autoantibody-negative SLE patients during clinical visits. Among these mediators, IL-7 and resistin are noteworthy. IL-7 is essential for B cell development and helps drive proliferation and antibody diversity. Adipokines such as resistin have been shown to have potent pro-inflammatory properties and lead to increased antibody production by plasma B cells. Regarding lupus disease activity, a pair of soluble mediators with seemingly dual anti-inflammatory and pro-inflammatory functions are IL-10 and TGF-β. Both mediators have been shown to have regulatory functions, and TGF-β levels are negatively correlated with the presence and accumulation of autoantibody-specific substances. Conversely, IL-10 levels are elevated in SLE patients with active disease. IL-10 has been shown to have pro-inflammatory properties in B lymphocyte activation and autoantibody production, while TGF-β, in the presence of IL-6, contributes to a Th17 response, leading to IL-21 secretion and B lymphocyte stimulation, thereby promoting autoantibody production and SLE pathogenesis.

[0238] These immune system changes, combined with lessons learned from the development of a lupus flare-predicting index, guided the development and refinement of the Lupus Disease Activity (Immune) Index (LDAII / L-DAI). This index is informed by alterations in soluble mediators and is weighted by their correlation with SLE-associated autoantibody accumulation, hSLEDAI disease activity score, and a composite weighting that includes both autoantibody accumulation and hSLEDAI disease activity score. Specialized training to minimize the time and need for assessing changes in disease activity often limits the use of validated SLE disease activity measures in routine clinical practice. Detecting immune system changes associated with persistent clinical disease activity and using them to provide LDAII / L-DAI information will allow for the identification of patients requiring closer monitoring and enables early intervention with immunomodulatory therapies to prevent end-organ damage. A positive score will indicate the need for more frequent monitoring and / or medication changes to suppress persistent inflammation before the onset of new or exacerbating clinical manifestations. A negative score will suggest maintaining the current monitoring and medication regimen (if the current regimen is effective), reducing the frequency of monitoring (especially if visits are at least quarterly), and / or considering a gradual reduction in the dosage of steroids or other immunomodulators with significant side effects. SLE patients who are more actively involved in their clinical care have been shown to have less permanent organ damage. Clinically quiescent SLE patients remain at risk of increased disease activity and must be monitored regularly. LDAII / L-DAI will allow patients to monitor their immune activity prior to clinical disease activity and alert patients and their healthcare providers to the need for further clinical evaluation.

[0239] Although SLE patients with active disease are more likely to meet the hSLEDAI serological criteria (increased DNA binding and / or hypocomplementemia) and be positive for anti-dsDNA autoantibodies, these factors have not been shown to be predictors of increased clinical disease activity. However, LDAII / L-DAI can differentiate between patients with clinically and serologically quiescent (CQSQ) and active (CASA) disease, showing greater differences in IFN-α, MIG / CXCL9, IP-10 / CXCL10, TNF-α, soluble TNFRII, BlyS, and OPN levels. Furthermore, LDAII / L-DAI can differentiate SLE patients with active disease exhibiting renal manifestations as well as other organ system manifestations. Currently, there is a strong push to utilize pathway-specific immune dysregulation to provide personalized precision medicine for SLE patients with renal manifestations and lupus nephritis (LN), as LN is the most common cause of morbidity and death in SLE patients. Success of such approaches will require methods for identifying individuals at highest risk of developing LN, as well as measures to define pathway-specific immune dysregulation to select the most appropriate LN patients for given pathway-specific biological therapy. Mediators altered in current assessments of SLE nephropathy patients include IFN-α, TNFRII, resistin, and OPN. IFN-α, as a type I IFN, can enhance renal immune complex deposition and induce increased inflammatory responses in renal resident cells, leading to local recruitment and infiltration of neutrophils and monocytes. TNFRIIs, normally present on lymphocytes, are aberrantly upregulated in chronic inflammation, including renal inflammation, leading to inflammation, kidney injury, and renal failure. Elevated circulating levels of resistin in SLE patients have been confirmed to be associated with renal dysfunction, including severe proteinuria and elevated serum creatinine. OPN is directly associated with glomerular fibrosis in the kidneys, resulting in proteinuria and decreased creatinine clearance. OPN is associated with genetic risk of SLE, increases clinical disease activity, and promotes the activation and migration of antigen-presenting cells (including macrophages and dendritic cells), as well as the differentiation of multiple helper T cell pathways (including Th1, Th17, and Tfh). The addition of TNFRII, resistin, and / or OPN, alone or in combination with nine mediators that inform L-DAI 9, will enhance the ability of LDAII / L-DAI to differentiate organ system manifestation-driven clinical disease activity (including renal features) in SLE patients.

[0240] Even within the same patient, active interventions based on fluctuating immune profiles represent a paradigm shift that needs to be considered. The ability to detect changes in immune status to reflect clinical disease activity will help improve disease surveillance and treatment, thereby improving patient outcomes and reducing the pathological and socioeconomic burden of SLE. The advantage of calculating patient-specific LDAII / L-DAI is that it does not require setting cutoff values ​​for each soluble mediator to determine positivity, nor does it require prior knowledge of the inflammatory pathways leading to disease activity in a particular patient. Validating and refining LDAII / L-DAI in this prospective, multi-ethnic study will help establish this valuable tool in SLE clinical trials and disease management. Early detection of the risk of increased clinical disease activity and organ damage, based on an individual patient's comprehensive clinical presentation, can indicate closer surveillance, prophylactic treatment, or inclusion in clinical trials of targeted biologics associated with pathways altered in LDAII / L-DAI.

[0241] The appended claims set forth the novel and inventive aspects of the subject matter described above; however, the claims may also include other subject matter not specifically described in detail. For example, certain features, elements, or aspects may be omitted from the claims if it is not necessary to distinguish novel and inventive features from those known to a person skilled in the art. Features, elements, and aspects described in the context of some embodiments may also be omitted, combined, or replaced by alternative features used for the same, equivalent, or similar purpose without departing from the scope of the invention as defined by the appended claims.

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[0290] Zonana-Nacach, A., Barr, SG, Magder, LS, & Petri, M. (2000). Damage in systemic lupus erythematosus and its association with corticosteroids. Arthritis Rheum, 43(8), 1801-1808. https: / / doi.org / 10.1002 / 1529-0131(200008)43:8<1801::AID-ANR16>3.0.CO;2-O. Claims (as amended under Article 19 of the Treaty) 1. A method for characterizing disease activity in patients with systemic lupus erythematosus (SLE), comprising: (a) Obtain blood, serum, plasma and / or urine samples from the patient; (b) Assess the presence or amount of protein expression of one or more of the following biomarkers in the blood, serum, plasma and / or urine samples: IFN-α, IL-10, BLyS, IL-7, IFN-γ, TRAIL, IL-15, IP-10 / CXCL10 and IL-4. (c) Assess the presence or amount of protein expression of one or more of the following inflammatory mediator biomarkers in the blood, serum, plasma and / or urine samples: TNFRII, resistin and osteopontin (OPN); (d) Assess the presence or amount of one or more SLE-associated autoantibody-specific biomarkers selected from the following in the blood, serum, plasma, and / or urine samples: dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP; and (e) Calculate the lupus disease activity (immune) index (LDAII / L-DAI) score. 2. The method of claim 1, wherein the LDAII / L-DAI score is calculated by the following autoantibody-weighted LDAII / L-DAI: log transformation; standardization; weighting of autoantibody specificity by Spearman r correlation; and summation over soluble protein markers. 3. The method of claim 1, wherein the LDAII / L-DAI score is calculated by the following steps: log transformation; standardization; weighting the hSLEDAI disease activity score by Spearman r correlation; and summing the soluble protein biomarkers. 4. The method of claim 1, wherein the LDAII / L-DAI score is a composite LDAII / L-DAI score calculated by: log transformation; standardization; weighting the average of autoantibody specificity and hSLEDAI disease activity scores by Spearman r correlation; and summing over soluble protein markers. 5. The method of claim 1, further comprising classifying the SLE patient as clinically active (CA) or clinically quiescent (CQ) disease based on the LDAII / L-DAI score, wherein the disease is serologically active (SA) or serologically quiescent (SQ). 6. The method of claim 1, wherein the LDAII / L-DAI score distinguishes between active lupus disease activity and low lupus disease activity. 7. The method of claim 1, further comprising administering treatment to the patient after determining that the patient has a prognosis of transitioning to classified SLE and before reaching a clinical disease classification, wherein the treatment comprises at least one of the following: hydroxychloroquine (HCQ), belimumab, nonsteroidal anti-inflammatory drugs, steroids, or disease-modifying antirheumatic drugs (DMARDs). 8. The method of claim 7, wherein the treatment is given to an SLE patient with active lupus. 9. The method of claim 7, wherein the treatment is given to an SLE patient with low lupus activity. 10. Methods for assessing disease activity and progression of clinical systemic lupus erythematosus (SLE) in patients, including: Blood, serum, plasma, and / or urine samples were obtained from the patient. Determine the presence or expression of at least one biomarker from each of (1) to (3): (1) One or more biomarkers selected from IFN-α, IL-10, BLyS, IL-7, IFN-γ, TRAIL, IL-15, IP-10 / CXCL10 and IL-4; (2) At least one inflammatory mediator biomarker selected from TNFRII, resistin and osteopontin (OPN); (3) At least one SLE-related autoantibody-specific biomarker selected from dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP; and (4) Calculate the LDAII / L-DAI score. 11. The method of claim 10, wherein the LDAII / L-DAI score is calculated by the following autoantibody-weighted LDAII / L-DAI: log transformation; standardization; weighting of autoantibody specificity by Spearman r correlation; and summation over soluble protein markers. 12. The method of claim 10, wherein the LDAII / L-DAI score is calculated by the following: hSELDAI weighted LDAII / L-DAI: log transformation; standardization; weighting of the hSLEDAI disease activity score by Spearman r correlation; and summation of soluble protein markers. 13. The method of claim 10, wherein the LDAII / L-DAI score is a composite LDAII / L-DAI calculated by: log transformation; standardization; weighting the average of autoantibody specificity and hSLEDAI disease activity scores by Spearman r correlation; and summing over soluble protein markers. 14. The method of claim 10, further comprising classifying the SLE patient as clinically active (CA) or clinically quiescent (CQ) disease based on the LDAII / L-DAI score, wherein the disease is serologically active (SA) or serologically quiescent (SQ). 15. The method of claim 10, wherein the LDAII / L-DAI score distinguishes between active lupus disease activity and low lupus disease activity. 16. The method of claim 10, further comprising administering treatment to the patient after determining that the patient has a prognosis of transitioning to classified SLE and before reaching a clinical disease classification, wherein the treatment comprises at least one of the following: hydroxychloroquine (HCQ), belimumab, nonsteroidal anti-inflammatory drugs, steroids, or disease-modifying antirheumatic drugs (DMARDs). 17. The method of claim 16, wherein the treatment is given to an SLE patient with active lupus. 18. The method of claim 16, wherein the treatment is given to an SLE patient with low lupus activity. 19. A method for characterizing disease activity in patients with systemic lupus erythematosus (SLE), comprising: (a) Obtain blood, serum, plasma and / or urine samples from the patient; (b) Assess the presence or amount of protein expression in the blood, serum, plasma and / or urine samples selected from a variety of biomarkers: IFN-α, IL-10, BLyS, IL-7, IFN-γ, TRAIL, IL-15, IP-10 / CXCL10 and IL-4. (c) Assess the presence or amount of protein expression of one or more of the following inflammatory mediator biomarkers in the blood, serum, plasma and / or urine samples: TNFRII, resistin and osteopontin (OPN); (d) Assess the presence or amount of protein expression of one or more immune mediator biomarkers selected from the following in the blood, serum, plasma and / or urine samples: IL-12p70, TNF-α, MIG / CXCL9, resistin, IL-5, IL-13, IL-1β, IL-17A, IL-2Rα, TGF-β (natural and / or total form), Fas, MCP-1 / CCL2, stem cell factor (SCF), IL-1RA, IL-6, MIP-1α / CCL3, MIP-1β / CCL4, MCP-3 / CCL7, RANTES / CCL5, TNFRI and IL-8 / CXCL8; (e) Assess the presence or amount of one or more SLE-associated autoantibody-specific biomarkers selected from the following in the blood, serum, plasma, and / or urine samples: dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP; and (f) Calculate the lupus disease activity (immune) index (LDAII / L-DAI) score. 20. The method of claim 19, wherein the LDAII / L-DAI score is calculated by the following autoantibody-weighted LDAII / L-DAI: log transformation; standardization; weighting of autoantibody specificity by Spearman r correlation; and summation over soluble protein markers. 21. The method of claim 19, wherein the LDAII / L-DAI score is calculated by the following: hSLEDAI weighted LDAII / L-DAI: log transformation; standardization; weighting of the hSLEDAI disease activity score by Spearman r correlation; and summation of soluble protein markers. 22. The method of claim 19, wherein the LDAII / L-DAI score is a composite LDAII / L-DAI score calculated by: log transformation; standardization; weighting the average of autoantibody specificity and hSLEDAI disease activity scores by Spearman r correlation; and summing over soluble protein markers. 23. The method of claim 19, further comprising classifying the SLE patient as clinically active (CA) or clinically quiescent (CQ) disease based on the LDAII / L-DAI score, wherein the disease is serologically active (SA) or serologically quiescent (SQ). 24. The method of claim 19, wherein the LDAII / L-DAI score distinguishes between active lupus disease activity and low lupus disease activity. 25. The method of claim 19, further comprising administering treatment to the patient after determining that the patient has a prognosis of transitioning to classified SLE and before reaching a clinical disease classification, wherein the treatment comprises at least one of the following: hydroxychloroquine (HCQ), belimumab, nonsteroidal anti-inflammatory drugs, steroids, or disease-modifying antirheumatic drugs (DMARDs). 26. The method of claim 19, wherein the treatment is given to an SLE patient with active lupus. 27. The method of claim 19, wherein the treatment is given to an SLE patient with low lupus activity. 28. Methods for assessing the clinical activity and progression of systemic lupus erythematosus (SLE) in patients, including Blood, serum, plasma, and / or urine samples were obtained from the patient. Determine the presence of protein expression of at least one biomarker from each of (1) to (4): (1) One or more biomarkers selected from IFN-α, IL-10, BLyS, IL-7, IFN-γ, TRAIL, IL-15, IP-10 / CXCL10 and IL-4; (2) At least one inflammatory mediator biomarker selected from TNFRII, resistin and osteopontin (OPN); (3) At least one immune mediator biomarker selected from IL-12p70, TNF-α, MIG / CXCL9, resistin, IL-5, IL-13, IL-1β, IL-17A, IL-2Rα, TGF-β (natural and / or total form), Fas, MCP-1 / CCL2, stem cell factor (SCF), IL-1RA, IL-6, MIP-1α / CCL3, MIP-1β / CCL4, MCP-3 / CCL7, RANTES / CCL5, TNFRI and IL-8 / CXCL8; (4) At least one SLE-related autoantibody-specific biomarker selected from dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP; and (5) Calculate the LDAII / L-DAI score. 29. The method of claim 28, wherein the LDAII / L-DAI score is an autoantibody-weighted LDAII / L-DAI score calculated by: log transformation; standardization; weighting of autoantibody specificity by Spearman r correlation; and summation over soluble protein markers. 30. The method of claim 28, wherein the LDAII / L-DAI score is calculated as an hSELDAI weighted LDAII / L-DAI score by: log transformation; standardization; weighting of the hSLEDAI disease activity score by Spearman r correlation; and summation of soluble protein biomarkers. 31. The method of claim 28, wherein the LDAII / L-DAI score is a composite LDAII / L-DAI score calculated by: log transformation; standardization; weighting the average of autoantibody specificity and hSLEDAI disease activity scores by Spearman r correlation; and summing over soluble protein markers. 32. The method of claim 28, further comprising classifying the SLE patient as clinically active (CA) or clinically quiescent (CQ) disease based on the LDAII / L-DAI score, wherein the disease is serologically active (SA) or serologically quiescent (SQ). 33. The method of claim 28, wherein the LDAII / L-DAI score distinguishes between active lupus disease activity and low lupus disease activity. 34. The method of claim 28, further comprising administering treatment to the patient after determining that the patient has a prognosis of transitioning to classified SLE and before reaching a clinical disease classification, wherein the treatment comprises at least one of the following: hydroxychloroquine (HCQ), belimumab, nonsteroidal anti-inflammatory drugs, steroids, or disease-modifying antirheumatic drugs (DMARDs). 35. The method of claim 28, wherein the treatment is given to an SLE patient with active lupus. 36. The method of claim 28, wherein the treatment is given to an SLE patient with low lupus activity.

Claims

1. A method for characterizing disease activity in patients with systemic lupus erythematosus (SLE), comprising: (a) Obtain blood, serum, plasma and / or urine samples from the patient; (b) Assess the presence or amount of protein expression of one or more of the following biomarkers in the blood, serum, plasma and / or urine samples: IFN-α, IL-10, BLyS, IL-7, IFN-γ, TRAIL, IL-15, IP-10 / CXCL10 and IL-4. (c) Assess the presence or amount of protein expression of one or more of the following inflammatory mediator biomarkers in the blood, serum, plasma and / or urine samples: TNFRII, resistin and osteopontin (OPN); (d) Assess the presence or amount of one or more SLE-related autoantibody-specific biomarkers selected from the following in the blood, serum, plasma and / or urine samples: dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP and RNP; as well as (e) Calculate the lupus disease activity (immune) index (LDAII / L-DAI) score.

2. The method of claim 1, wherein the LDAII / L-DAI score is calculated by the following autoantibody-weighted LDAII / L-DAI: log transformation; standardization; weighting of autoantibody specificity by Spearman r correlation; and summation over soluble protein markers.

3. The method of claim 1, wherein the LDAII / L-DAI score is calculated by the following steps: log transformation; standardization; weighting the hSLEDAI disease activity score by Spearman r correlation; and summing the soluble protein biomarkers.

4. The method of claim 1, wherein the LDAII / L-DAI score is a composite LDAII / L-DAI score calculated by: log transformation; standardization; weighting the average of autoantibody specificity and hSLEDAI disease activity scores by Spearman r correlation; and summing over soluble protein markers.

5. The method of claim 1, further comprising classifying the SLE patient as clinically active (CA) or clinically quiescent (CQ) disease based on the LDAII / L-DAI score, wherein the disease is serologically active (SA) or serologically quiescent (SQ).

6. The method of claim 1, wherein the LDAII / L-DAI score distinguishes between active lupus disease activity and low lupus disease activity.

7. The method of claim 1, further comprising administering treatment to the patient after determining that the patient has a prognosis of transitioning to classified SLE and before reaching a clinical disease classification, wherein the treatment comprises at least one of the following: hydroxychloroquine (HCQ), belimumab, nonsteroidal anti-inflammatory drugs, steroids, or disease-modifying antirheumatic drugs (DMARDs).

8. The method of claim 7, wherein the treatment is given to an SLE patient with active lupus.

9. The method of claim 7, wherein the treatment is given to an SLE patient with low lupus activity.

10. Methods for assessing disease activity and progression of clinical systemic lupus erythematosus (SLE) in patients, including: Blood, serum, plasma, and / or urine samples were obtained from the patient. Determine the presence or expression of at least one biomarker from each of (1) to (3): (1) One or more biomarkers selected from IFN-α, IL-10, BLyS, IL-7, IFN-γ, TRAIL, IL-15, IP-10 / CXCL10 and IL-4; (2) At least one inflammatory mediator biomarker selected from TNFRII, resistin and osteopontin (OPN); (3) At least one SLE-related autoantibody-specific biomarker selected from dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP and RNP; as well as (4) Calculate the LDAII / L-DAI score.

11. The method of claim 10, wherein the LDAII / L-DAI score is calculated by the following autoantibody-weighted LDAII / L-DAI: log transformation; standardization; weighting of autoantibody specificity by Spearman r correlation; and summation over soluble protein markers.

12. The method of claim 10, wherein the LDAII / L-DAI score is calculated by the following: hSELDAI weighted LDAII / L-DAI: log transformation; standardization; weighting of the hSLEDAI disease activity score by Spearman r correlation; and summation of soluble protein markers.

13. The method of claim 10, wherein the LDAII / L-DAI score is a composite LDAII / L-DAI calculated by: log transformation; standardization; weighting the average of autoantibody specificity and hSLEDAI disease activity scores by Spearman r correlation; and summing over soluble protein markers.

14. The method of claim 10, further comprising classifying the SLE patient as clinically active (CA) or clinically quiescent (CQ) disease based on the LDAII / L-DAI score, wherein the disease is serologically active (SA) or serologically quiescent (SQ).

15. The method of claim 10, wherein the LDAII / L-DAI score distinguishes between active lupus disease activity and low lupus disease activity.

16. The method of claim 10, further comprising administering treatment to the patient after determining that the patient has a prognosis of transitioning to classified SLE and before reaching a clinical disease classification, wherein the treatment comprises at least one of the following: hydroxychloroquine (HCQ), belimumab, nonsteroidal anti-inflammatory drugs, steroids, or disease-modifying antirheumatic drugs (DMARDs).

17. The method of claim 16, wherein the treatment is given to an SLE patient with active lupus.

18. The method of claim 16, wherein the treatment is given to an SLE patient with low lupus activity.

19. A method for characterizing disease activity in patients with systemic lupus erythematosus (SLE), comprising: (a) Obtain blood, serum, plasma and / or urine samples from the patient; (b) Assess the presence or amount of protein expression in the blood, serum, plasma and / or urine samples selected from a variety of biomarkers: IFN-α, IL-10, BLyS, IL-7, IFN-γ, TRAIL, IL-15, IP-10 / CXCL10 and IL-4. (c) Assess the presence or amount of protein expression of one or more of the following inflammatory mediator biomarkers in the blood, serum, plasma and / or urine samples: TNFRII, resistin and osteopontin (OPN); (d) Assess the presence or amount of protein expression of one or more immune mediator biomarkers selected from the following in the blood, serum, plasma and / or urine samples: IL-12p70, TNF-α, MIG / CXCL9, resistin, IL-5, IL-13, IL-1β, IL-17A, IL-2Rα, TGF-β (natural and / or total form), Fas, MCP-1 / CCL2, stem cell factor (SCF), IL-1RA, IL-6, MIP-1α / CCL3, MIP-1β / CCL4, MCP-3 / CCL7, RANTES / CCL5, TNFRI and IL-8 / CXCL8; (e) Assess the presence or amount of one or more SLE-related autoantibody-specific biomarkers selected from the following in the blood, serum, plasma and / or urine samples: dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP and RNP; as well as (f) Calculate the lupus disease activity (immune) index (LDAII / L-DAI) score.

20. The method of claim 20, wherein the LDAII / L-DAI score is calculated by the following autoantibody-weighted LDAII / L-DAI: log transformation; standardization; weighting of autoantibody specificity by Spearman r correlation; and summation over soluble protein markers.

21. The method of claim 20, wherein the LDAII / L-DAI score is calculated by the following steps: log transformation; standardization; weighting the hSLEDAI disease activity score by Spearman r correlation; and summing over soluble protein biomarkers.

22. The method of claim 20, wherein the LDAII / L-DAI score is a composite LDAII / L-DAI score calculated by: log transformation; standardization; weighting the average of autoantibody specificity and hSLEDAI disease activity scores by Spearman r correlation; and summing over soluble protein markers.

23. The method of claim 20, further comprising classifying the SLE patient as clinically active (CA) or clinically quiescent (CQ) disease based on the LDAII / L-DAI score, wherein the disease is serologically active (SA) or serologically quiescent (SQ).

24. The method of claim 20, wherein the LDAII / L-DAI score distinguishes between active lupus disease activity and low lupus disease activity.

25. The method of claim 20, further comprising administering treatment to the patient after determining that the patient has a prognosis of transitioning to classified SLE and before reaching a clinical disease classification, wherein the treatment comprises at least one of the following: hydroxychloroquine (HCQ), belimumab, nonsteroidal anti-inflammatory drugs, steroids, or disease-modifying antirheumatic drugs (DMARDs).

26. The method of claim 20, wherein the treatment is given to an SLE patient with active lupus.

27. The method of claim 20, wherein the treatment is given to an SLE patient with low lupus activity.

28. Methods for assessing the disease activity and progression of clinical systemic lupus erythematosus (SLE) in patients, including Blood, serum, plasma, and / or urine samples were obtained from the patient. Determine the presence of protein expression of at least one biomarker from each of (1) to (4): (1) One or more biomarkers selected from IFN-α, IL-10, BLyS, IL-7, IFN-γ, TRAIL, IL-15, IP-10 / CXCL10 and IL-4; (2) At least one inflammatory mediator biomarker selected from TNFRII, resistin and osteopontin (OPN); (3) At least one immune mediator biomarker selected from IL-12p70, TNF-α, MIG / CXCL9, resistin, IL-5, IL-13, IL-1β, IL-17A, IL-2Rα, TGF-β (natural and / or total form), Fas, MCP-1 / CCL2, stem cell factor (SCF), IL-1RA, IL-6, MIP-1α / CCL3, MIP-1β / CCL4, MCP-3 / CCL7, RANTES / CCL5, TNFRI and IL-8 / CXCL8; (4) At least one SLE-related autoantibody-specific biomarker selected from dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP; and (5) Calculate the LDAII / L-DAI score.

29. The method of claim 24, wherein the LDAII / L-DAI score is an autoantibody-weighted LDAII / L-DAI score calculated by: log transformation; standardization; weighting of autoantibody specificity by Spearman r correlation; and summation over soluble protein markers.

30. The method of claim 24, wherein the LDAII / L-DAI score is calculated as an hSELDAI weighted LDAII / L-DAI score by: log transformation; standardization; weighting the hSLEDAI disease activity score by Spearman r correlation; and summing over soluble protein markers.

31. The method of claim 24, wherein the LDAII / L-DAI score is a composite LDAII / L-DAI score calculated by: log transformation; standardization; weighting the average of autoantibody specificity and hSLEDAI disease activity scores by Spearman r correlation; and summing over soluble protein markers.

32. The method of claim 24, further comprising classifying the SLE patient as clinically active (CA) or clinically quiescent (CQ) disease based on the LDAII / L-DAI score, wherein the disease is serologically active (SA) or serologically quiescent (SQ).

33. The method of claim 24, wherein the LDAII / L-DAI score distinguishes between active lupus disease activity and low lupus disease activity.

34. The method of claim 24, further comprising administering treatment to the patient after determining that the patient has a prognosis of transitioning to classified SLE and before reaching a clinical disease classification, wherein the treatment comprises at least one of the following: hydroxychloroquine (HCQ), belimumab, nonsteroidal anti-inflammatory drugs, steroids, or disease-modifying antirheumatic drugs (DMARDs).

35. The method of claim 24, wherein the treatment is given to an SLE patient with active lupus.

36. The method of claim 24, wherein the treatment is given to an SLE patient with low lupus activity.

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