Biomarkers of disease activity immune index in systemic lupus erythematosus (SLE) that characterize disease activity

Abstract

The present invention includes a method for characterizing disease activity in a patient with systemic lupus erythematosus (SEE), comprising: obtaining a data set associated with a blood, serum, plasma or urine sample from the patient, wherein the data set includes data representing the level of one or more biomarkers in the blood, serum, plasma or urine sample from each of (b) to (g); at least one intrinsic serum or plasma mediator biomarker; at least one adaptive serum or plasma mediator; at least one chemokine / adhesion molecule biomarker; at least one soluble TNF superfamily biomarker; the inflammatory mediator biomarker SCF; at least one SLE-associated autoantibody-specific biomarker; and calculating a lupus disease activity immune index (LDAII) score.

Description

Technical Field

[0001] The present invention generally relates to the field of biomarkers for calculating a Lupus Disease Activity Immune Index that characterizes disease activity of systemic lupus erythematosus (SLE).

[0002] Statement on Federally Sponsored Research

[0003] not applicable.

[0004] Reference to a sequence listing

[0005] not applicable. Background Art

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

[0007] Systemic autoimmune diseases, including SLE, afflict a large portion of the U.S. population. Recent population-based studies reflect a prevalence of 73 / 100,000 (1, 2), while the Lupus Foundation of America estimates that the number of patients with SLE who may be diagnosed in routine clinical practice is as high as 470 / 100,000. SLE presents with a range of clinical symptoms; disease classification depends on whether 4 of the 11 American College of Rheumatology (ACR) criteria are met (3, 4). More than 90% of affected patients are women aged 15-45 years. Prevalence is higher in minority populations and those of lower socioeconomic status (5). Persistent active clinical disease and its treatment place patients at risk for organ damage (6-8), including central nervous system, pulmonary, cardiovascular, and renal damage (9-12), lupus nephritis, and end-stage renal disease (11, 13). Patients with waxing / waning disease and those with clinically active or quiescent disease are each at risk for clinical disease flare-ups (14, 15).

[0008] SLE is a clinically and serologically heterogeneous systemic autoimmune disease that causes significant morbidity and early mortality, especially in young women and ethnic minorities. Immune dysregulation in the form of pathogenic autoantibodies and chronic inflammation leads to a wide range of clinical manifestations, including rash, arthritis, and life-threatening renal and / or central nervous system damage. It has been shown that many antinuclear autoantibodies (ANA) specificities have been accumulated in SLE patients; the use of hydroxychloroquine can eliminate autoantibody accumulation and offset clinical disease activity. Early intervention is an attractive SLE treatment method. However, we do not understand the pathogenic mechanisms of SLE disease activity enough. Bridging this knowledge gap will improve our ability to identify individuals at risk of increased disease activity and permanent organ damage, limit the window of opportunity for early intervention, and promote the development of pathway-targeted treatments.

[0009] Recognition and early treatment to prevent tissue and organ damage are challenging because signs and symptoms of high disease activity are not captured until after they develop. Despite the existence of validated clinical disease activity instruments (16-18) and improved treatment strategies, persistent active disease remains a burden for SLE patients (19). Increased morbidity and early mortality associated with the treatments required to manage active disease, especially steroids (20-23), and permanent organ damage (24, 25), including renal injury (26), further drive up costs. In addition, chronic use of steroids (23) and other immunosuppressants (27) required to manage disease activity is associated with increased morbidity. The inability to proactively manage clinical disease limits medical care to reactive treatments, hampering proactive strategies (28) to add or augment steroid-sparing immunomodulators to prevent end-organ damage (6-8) and reduce the pathogenic and socioeconomic burden of SLE (29).

[0010] Current biomarkers in SLE have limited utility in predicting permanent organ damage. Although SLE-associated autoantibody specificities, such as anti-dsDNA, anti-spliceosome, and anti-Ro / SSA, accumulate in SLE patients, their presence is not sufficient to predict progression to persistent active disease and permanent organ damage. ANAs are also present in sera from patients with other systemic rheumatic diseases, as well as healthy individuals who do not go on to develop SLE (including some unaffected family members of SLE patients), and up to 14% of the general population. Because individuals can remain healthy while being 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 also contribute to the development of clinical disease. Any single factor or mechanism may not be sufficient to explain the complexity and heterogeneity of SLE pathogenesis; therefore, multivariate longitudinal approaches are necessary to describe the mechanisms of early disease pathogenesis and to discern unique parameters that predict SLE classification.

[0011] Despite the existence of multiple clinical trials targeting immune pathways, including belimumab, the first FDA-approved SLE drug in more than 50 years (30), the vast majority of these studies have failed, in part due to a lack of understanding of the immune pathways that are dysregulated in a given patient. There is an increasing need for immune-informed biomarkers that serve as surrogate endpoints for clinical disease activity. Administrative burdens limit the use of validated SLE clinical disease activity measures in routine practice (31). Validated disease activity instruments, such as the currently used hybrid systemic lupus erythematosus disease activity index (hSLEDAI) (32, 33) and the British Isles Lupus Assessment Group (BILAG) index (17), are labor intensive and require ongoing specialized training as these clinical instruments are updated (34). Relying solely on the physician's experience to assess clinical disease activity carries the risk of undesirable variability and unfortunate outcomes (31, 35).

[0012] The clinical heterogeneity of SLE is based on the following scientific premise: heterogeneous immune dysregulation is the basis of clinical disease activity. The inventors have previously shown that patients exhibit immune dysregulation before clinical SLE onset, which is amplified by feedforward mechanisms as patients suffer tissue damage, develop clinical sequelae, and ultimately reach disease classification (36, 37). The inventors have also described the accumulation of multiple SLE-related autoantibodies (AutoAb) and dysregulated inflammatory and regulatory immune pathways (36, 37). However, to date, the lack of disease management tests that provide immune mechanism information in SLE stems from the absence of a single biomarker that provides immune pathway information that serves as a universal surrogate for simultaneous or future clinical disease activity (38).

[0013] Therefore, there remains a need for novel methods for detecting, tracking and assessing disease activity and progression in patients with classified SLE. Summary of the invention

[0014] In one embodiment, the invention includes a method for characterizing disease activity in a patient with systemic lupus erythematosus (SLE), comprising: (a) obtaining a data set associated with a blood, serum, plasma or urine sample from the patient, wherein the data set includes data representing the level of one or more biomarkers in the blood, serum, plasma or urine sample from each of (b) to (g); (b) a data set evaluating the presence or amount of protein expression of at least one intrinsic serum or plasma mediator biomarker selected from the following: IL-1α, IL-1β, IL-1RA, IFN-α, IL-12p70, IL-6 and IL-23p19; (c) a data set evaluating the presence or amount of protein expression of at least one adaptive serum or plasma mediator biomarker selected from the following: IL-2, IFN-γ, IL-5, IL-13, IL-17A, IL-21, IL-10 and TGF-β; (d) evaluating at least one chemokine selected from the following (e) a dataset evaluating the presence or amount of at least one soluble TNF superfamily biomarker selected from the group consisting of TNFRI, TNFRII, TRAIL, TWEAK, CD40L / CD154, BLyS, and APRIL; (f) a dataset evaluating the presence or amount of the inflammatory mediator biomarker stem cell factor (SCF); (g) a dataset evaluating the presence or amount of at least one SLE-associated autoantibody-specific biomarker selected from the group consisting of dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP; and (h) calculating the Lupus Disease Activity Immune Index (LDAII) score. In one aspect, at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39 biomarkers are used in the calculation of LDAII. On the other hand, the data set is: logarithmic transformation; standardized; weighted by Spearman r correlation with the autoantibody specificity in the second data set; and the sum of soluble protein markers is equal to the LDAII score. On the other hand, the execution of at least one immunoassay includes: obtaining a first sample, wherein the first sample contains a protein marker; contacting the first sample with a plurality of different reagents; generating a plurality of different complexes between the reagent and the marker; and detecting the complex to generate data. On the other hand, at least one immunoassay includes multiple assays.On the other hand, LDAII divides the severity or level of progression of SLE into clinically active (CA) or quiescent (CQ) disease, which is serological (dsDNA binding and low complement) active (SA) or serologically quiescent (SQ) disease. On the other hand, LDAII scores distinguish between active and low lupus disease activity. On the other hand, the method also includes administering treatment to the patient before reaching clinical disease classification after determining that the patient has a prognosis of conversion to classified SLE, wherein the treatment includes at least one of the following: hydroxychloroquine (HCQ), belimumab, nonsteroidal anti-inflammatory drugs, steroids or disease-modifying antirheumatic drugs (DMARD).

[0015] In another embodiment, the present invention includes a method for evaluating disease activity and progression of a patient's systemic lupus erythematosus (SLE) clinical disease, 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 data set including at least one biomarker from each of (1) to (6): (1) a data set evaluating the presence or amount of protein expression of at least one intrinsic serum or plasma mediator biomarker selected from the following: IL-1α, IL-1β, IL-1RA, IFN-α, IL-12p70, IL-6 and IL-23p19; (2) a data set evaluating the presence or amount of protein expression of at least one adaptive serum or plasma mediator biomarker selected from the following: IL-2, IFN-γ, IL-5, IL-13, IL-17A, IL-21, IL-10 and TGF-β; (3) a data set evaluating the presence or amount of protein expression of at least one adaptive serum or plasma mediator biomarker selected from the following: (4) a dataset evaluating the presence or amount of at least one chemokine / adhesion molecule biomarker selected from the group consisting of IL-8 / CXCL8, IP-10 / CXCL10, MIG / CXCL9, MIP-1α / CCL3, MIP-1β / CCL4, MCP-1 / CCL2, MCP-3 / CCL7, and ICAM-1; (5) a dataset evaluating the presence or amount of the inflammatory mediator biomarker SCF; and (6) a dataset evaluating the presence or amount of at least one SLE-associated autoantibody-specific biomarker selected from the group consisting of dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP; and calculating the LDAII score. In one aspect, at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39 biomarkers are used in the calculation of LDAII. On the other hand, the data set is: logarithmic transformation; standardized; weighted by Spearman r correlation with autoantibody specificity in the second data set; and the sum of soluble protein markers is equal to the lupus disease activity immune index (LDAII) score. On the other hand, the execution of at least one immunoassay includes: obtaining a first sample, wherein the first sample comprises a protein marker; contacting the first sample with a plurality of different reagents; generating a plurality of different complexes between the reagent and the marker; and detecting the complex to generate data. On the other hand, at least one immunoassay includes multiple assays.On the other hand, LDAII divides the severity or level of progression of SLE into clinically active (CA) or quiescent (CQ) disease, which is serological (dsDNA binding and low complement) active (SA) or serologically quiescent (SQ) disease. On the other hand, the LDAII score distinguishes between active and low lupus disease activity. On the other hand, the method also includes administering treatment to the SLE patient before reaching the clinical disease classification after determining that the patient has a prognosis of conversion to classified SLE, wherein the treatment includes at least one of the following: hydroxychloroquine (HCQ), belimumab, nonsteroidal anti-inflammatory drugs, steroids, or disease-modifying antirheumatic drugs (DMARD). On the other hand, obtaining a first data set associated with the sample includes obtaining the sample and processing the sample to experimentally determine the first data set, or obtaining the first data set associated with the sample includes receiving the first data set from a third party that has processed the sample to experimentally determine the first data set. On the other hand, an increase in SCF, TNFRII, and MCP-1 biomarkers indicates renal organ involvement.

[0016] In another embodiment, the present invention includes a method for calculating a lupus disease activity immune index (LDAII) by measuring the expression level of a panel of biomarkers in a subject, comprising: determining a biomarker measurement value of a panel of biomarkers in a physiological sample by immunoassay, wherein the biomarker is a peptide, a protein, a peptide carrying a post-translational modification, a protein carrying a post-translational modification, or a combination thereof; wherein the physiological sample is whole blood, plasma, serum, or a combination thereof; wherein the panel of biomarkers includes a data set of measurement results of at least one selected from each type of biomarker, selected from: a data set of the presence or amount of protein expression of at least one intrinsic serum or plasma mediator biomarker, the biomarker selected from: IL-1α, IL-1β, IL-1RA, IFN-α, IL-12p70, IL-6, and IL-23p19; a data set of the presence or amount of protein expression of at least one adaptive serum or plasma mediator biomarker, the biomarker selected from: IL-2, IFN-γ, IL-5, IL-13, IL-17A, IL-21, IL-10 and TGF-β; a dataset of the presence or amount of at least one chemokine / adhesion molecule biomarker selected from the group consisting of IL-8 / CXCL8, IP-10 / CXCL10, MIG / CXCL9, MIP-1α / CCL3, MIP-1β / CCL4, MCP-1 / CCL2, MCP-3 / CCL7, and ICAM-1; a dataset of the presence or amount of at least one soluble TNF superfamily biomarker selected from the group consisting of TNFRI, TNFRII, TRAIL, TWEAK, CD40L / CD154, BLyS, and APRIL; a dataset of the presence or amount of the inflammatory mediator biomarker SCF; and a dataset of the presence or amount of at least one SLE-associated autoantibody-specific biomarker selected from the group consisting of dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP; and calculating an LDAII score, whereby the dataset is: log-transformed; normalized; compared to the autoantibody-specific Spearman regression analysis in the second dataset. r correlations were weighted, and the sum of soluble protein markers equaled the LDAII score.On the other hand, the method also includes using a set of biomarker measurements in a classification system to classify a sample as clinically active (CA) or quiescent (CQ) disease relative to the presence or development of systemic lupus erythematosus (SLE) in a subject, the disease being serological (dsDNA binding and low complement) active (SA) or serologically quiescent (SQ) disease, wherein the classification system is a machine learning system, comprising a classification and regression tree selected from the group consisting of: Fisher's exact test, Mann-Whitney test, Kruskal-Wallis test, Kruskal-Wallis test with Dunn's multiple comparisons, Spearman rank correlation or its ensemble; and calculating the lupus disease activity immune index (LDAII), wherein the LDAII score distinguishes active SLE from low SLE disease activity (low clinical disease (SLEDAI<4)). On the other hand, the method also includes distinguishing clinical and serological quiescence (CQSQ) SLE patients compared to healthy controls. In another aspect, the method further comprises using an amount of at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39 biomarkers in the calculation of LDAII. In another aspect, the immunoassay is a multiplex immunoassay. On the other hand, LDAII is further calculated as follows: the biomarker concentration of the subject is determined and logarithmically transformed, and each logarithmically transformed soluble mediator level determined for the subject sample is standardized as follows: (observed value)-(mean value of all SLE patients and healthy control visits) / (standard deviation of all SLE patients and healthy control visits); Spearman coefficients are generated by a linear regression model, and the model tests the association between one or more autoantibodies (AutoAb) evaluated in SLE patients compared with healthy controls (Spearman r); the values ​​of the soluble mediator levels of the subject are transformed and standardized, and the values ​​are weighted by (multiplying) their corresponding Spearman coefficients (Spearman r); and for each participant in the follow-up, the logarithmically transformed, standardized and weighted values ​​of each of the four or more soluble mediators are added to calculate LDAII. On the other hand, an increase in LDAII indicates at least one of the following: SLE disease progression, increased autoimmune disease activity, or organ damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description taken in conjunction with the accompanying drawings, and in which:

[0018] Figure 1A and 1B Showing increased hSLEDAI and AutoAb specific numbers in SLE patients with active disease. ( Figure 1A ) Composite SLEDAI (SELENAI-SLEDAI, with proteinuria as defined by SLEDAI-2K) in follow-up of SLE patients with low (hSLEDAI<4; n=132) and active (hSLEDAI≥4; n=179) disease; mean±SEM, p<0.0001 by unpaired t-test. ( Figure 1B ) Number of SLE-associated AutoAb specificities (antibodies to dsDNA, chromatin, Ro / SSA, La / SSB, Sm, SmRNP, and RNP) in SLE patients with low-grade or active disease relative to matched healthy controls (Ctls; n=48). Levels are expressed as mean ± SEM. **p<0.01; ****p<0.0001 by Kruskal-Wallis test with Dunn's multiple comparisons.

[0019] Figures 2A to 2I Soluble mediators showing changes in SLE low vs. active disease and controls. Plasma samples from SLE patients with low (n=132) disease activity during follow-up were compared with samples from SLE patients with active disease obtained during follow-up (n=179) vs. matched controls (n=48). Factors examined included: Figure 2A )IFN-α, ( Figure 2B )IP-10, ( Figure 2C )IL-6, ( Figure 2D )IL-1α, ( Figure 2E )IL-8, ( Figure 2F )IL-21, Figure 2G )BLyS、( Figure 2H )IL-10 and ( Fig.2I ) Total TGF-β. Levels are expressed as mean ± SEM. *p<0.05; **p<0.01, ***p<0.001, ***p<0.0001, by Kruskal-Wallis test with Dunn's multiple comparisons.

[0020] Figures 3A to 3DIFN-related mediators that vary with disease activity / AutoAb positivity are shown. SLE patient follow-up samples with AutoAb negative (Neg) / low disease activity (Low) (n=56) were compared with AutoAb Neg / active disease (Active; n=52), AutoAb positive (Pos) / Low (n=76), and AutoAb Pos / Active samples. SLE patient samples were also compared with matched control samples (n=48). Factors examined included: ( Figure 3A )IFN-α, ( Figure 3B )IFN-γ、( Figure 3C )IP-10 and ( Figure 3D )MIG. Levels are expressed as mean ± SEM*p<0.05; **p<0.01, ***p<0.001, ***p<0.0001 by Kruskal-Wallis test with Dunn's multiple comparisons. Cases (SLE patient group) were compared with controls by Mann-Whitney test.

[0021] Figures 4A to 4D TNF superfamily mediators that vary with disease activity / AutoAb positivity are shown. SLE patient follow-up samples with AutoAb negative (Neg) / low disease activity (Low) (n=56) were compared to AutoAb Neg / active disease (Active; n=52), AutoAb positive (Pos) / Low (n=76), and AutoAb Pos / Active samples. SLE patient samples were also compared to matched control samples (n=48). Factors examined included: ( Figure 4A )BLyS、( Figure 4B )APRIL、( Figure 4C )TRAIL and ( Figure 4D )TNFRII. Levels are expressed as mean ± SEM. *p<0.05; **p<0.01, ***p<0.0001 by Kruskal-Wallis test with Dunn's multiple comparisons. Cases (SLE patient group) were compared with controls by Mann-Whitney test.

[0022] Figures 5A to 5DIntrinsic and adaptive mediators that vary with disease activity / AutoAb positivity are shown. SLE patient follow-up samples with AutoAb negative (Neg) / low disease activity (Low) (n=56) were compared to AutoAb Neg / active disease (Active; n=52), AutoAb positive (Pos) / Low (n=76), and AutoAb Pos / Active samples. SLE patient samples were also compared to matched control samples (n=48). Factors examined included: ( Figure 5A )IL-1a, ( Figure 5B )IL-6, ( Figure 5C )IL-2 and ( Figure 5D )IL-21. Levels are expressed as mean ± SEM; *p<0.05; **p<0.01, ***p<0.0001 by Kruskal-Wallis test with Dunn's multiple comparisons. Cases (SLE patient group) were compared with controls by Mann-Whitney test.

[0023] Figures 6A to 6D Inflammatory and regulatory mediators that vary with disease activity / AutoAb positivity are shown. SLE patient follow-up samples with AutoAb negative (Neg) / low disease activity (Low) (n=56) were compared to AutoAb Neg / active disease (Active; n=52), AutoAb positive (Pos) / Low (n=76), and AutoAb Pos / Active samples. SLE patient samples were also compared to matched control samples (n=48). Factors examined included: ( Fig. 6A )SCF,( Figure 6B )IL-8, ( Figure 6C )IL-10 and ( Fig.6D ) Total TGF-β. Levels are expressed as mean ± SEM; *p<0.05; **p<0.01, ***p<0.001, ***p<0.0001 by Kruskal-Wallis test with Dunn's multiple comparisons. Cases (SLE patient group) were compared with controls by Mann-Whitney test.

[0024] Fig. 7A and 7B The lupus disease activity immune index (LDAII) is shown to differentiate SLE patients with low-grade and active disease relative to controls. Fig. 7A)LDAII was calculated for SLE patients with low-grade or active disease relative to controls.LDAII is shown as mean ± SEM; *p<0.05; **p<0.01, ***p<0.0001 by Kruskal-Wallis test with Dunn's multiple comparisons. ( Figure 7B ) for differentiating SLE patients (low-grade or active disease) from controls a Odds ratios were calculated based on the likelihood of having a positive LDAII score in the setting of active lupus b ; p-value c , specificity, sensitivity, negative predictive value (NPV), and positive predictive value (PPV) were calculated by Fisher's exact test.

[0025] Figures 8A to 8F The lupus disease activity immune index (LDAII) is shown to differentiate SLE patients with clinically and serologically active versus quiescent disease relative to controls. Fig. 8A ) LDAII was calculated for SLE patients with clinically and serologically quiescent (CQSQ; n=73) or clinically and serologically active (CASA; n=115) disease relative to controls. LDAII is shown as mean ± SEM; *p<0.05; **p<0.01, ***p<0.0001 by Kruskal-Wallis test with Dunn's multiple comparisons. ( Figure 8B ) for differentiating SLE patients (low-grade or active disease) from controls a Odds ratios were calculated based on the likelihood of having a positive LDAII score in the setting of active lupus b ; p-value c , specificity, sensitivity, negative predictive value (NPV), and positive predictive value (PPV) were calculated by Fisher's exact test. Figure 8C )、IL-8( Fig.8D ) and IP-10( Fig. 8E ) and the number of AutoAb specificities ( Figure 8F ). Levels are expressed as mean ± SEM; *p < 0.05; **p < 0.01, ***p < 0.001, ***p < 0.0001, by Kruskal-Wallis test with Dunn's multiple comparisons.

[0026] Figures 9A to 9E The lupus disease activity immune index (LDAII) is shown to differentiate SLE patients with renal or non-renal active disease compared to low disease activity. Fig. 9A) LDAII was calculated for SLE patient followers with renal (n=24) or non-renal (n=155) active disease relative to SLE patient followers with low disease activity (n=132). LDAII is shown as mean ± SEM; *p<0.05; **p<0.01, ***p<0.0001, by Kruskal-Wallis test with Dunn's multiple comparisons. ( Fig. 9B ) Area under the curve (AUC) for distinguishing SLE patients with renal manifestations a Odds ratios were calculated based on the likelihood of having a positive LDAII score in the setting of active lupus and renal manifestations. b ; p-value c , specificity, sensitivity, negative predictive value (NPV), and positive predictive value (PPV) were calculated by Fisher's exact test. Fig. 9C )、TNFRII( Fig.9D ) and the number of AutoAb specificities ( Fig.9E ). Levels are expressed as mean ± SEM; *p < 0.05; **p < 0.01, ***p < 0.0001, by Kruskal-Wallis test with Dunn's multiple comparisons. DETAILED DESCRIPTION

[0027] Although the preparation and use of various embodiments of the present invention are discussed in detail below, it should be recognized that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific occasions. The specific embodiments discussed herein only illustrate specific ways to prepare and use the present invention and do not define the limits of the scope of the present invention.

[0028] In order to facilitate understanding of the present invention, a plurality of terms are defined below. The terms defined herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention relates. Terms such as "a", "an", and "the" are not intended to refer only to a single entity, but include general categories, of which specific examples may be used for illustration. The terms herein are used to describe specific embodiments of the present invention, but their use does not limit the present invention unless outlined in the claims.

[0029] The inventor purchased plasma samples collected continuously from systemic lupus erythematosus (SLE) patients of the Oklahoma Lupus Cohort to compare the levels of autoantibodies and immune mediators from multiple immune pathways in SLE patients with low or active disease compared with matched healthy controls, and to determine the temporal relationship between them. The present invention discloses the potential mechanism of the immune pathogenesis associated with clinical disease activity, whereby the imbalance of immune mediators occurs simultaneously with the accumulation of autoantibodies and is unrelated to it. In addition, the present invention includes designing and verifying reliable and sensitive tools to assess the immune status of lupus patients associated with clinical disease activity. The present invention can be used to identify high-risk patients who need rheumatology referral and registration in prospective preclinical intervention studies, and to provide information on the development of novel therapeutic strategies to avoid or delay tissue damage.

[0030] As used herein, the term "dataset" refers to a collection of values ​​obtained from the evaluation of a sample (or a population of samples) under desired conditions. The values ​​of a data set can be obtained, for example, by experimentally obtaining measurements from a sample such as a patient sample and establishing a data set from these measurements. Alternatively, a data set can be obtained from a database or server that has stored the data set, or even a service provider (such as an internal or third-party laboratory).

[0031] As used herein, in the context of the present invention, the term "disease" refers to any condition, illness, disorder or ailment that manifests itself in the form of a dysfunctional or malfunctioning immune system, for example causing, for example, SLE.

[0032] As used herein, the term "sample" refers to any biological sample isolated from a subject, which may include, but is not limited to, a single cell or multiple cells, cell fragments, aliquots of body fluids, whole blood, platelets, serum, plasma, red blood cells, white blood cells or leukocytes, endothelial cells, tissue biopsies, synovial fluid, lymphatic fluid, ascites fluid, and interstitial or extracellular fluid. The term "sample" also encompasses fluids in the spaces between cells, including gingival crevicular fluid, bone marrow, cerebrospinal fluid, saliva, mucus, sputum, semen, sweat, urine, or any other body fluid.

[0033] 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 subjects by a variety of means, including but not limited to venipuncture, excretion, ejaculation, massage, biopsy, needle aspiration, lavage, scraping, surgical incision or intervention or other means known in the art.

[0034] As used herein, the term "subject" or "patient" generally refers to mammals, including but not limited to humans, non-human primates, dogs, cats, mice, rats, cattle, horses and pigs, without regard to sex or age. The subject may be a subject who has been previously diagnosed or identified as having an autoimmune and / or inflammatory disease, and may have been or is undergoing therapeutic intervention for the autoimmune and / or inflammatory disease. However, the subject may also include a patient who has not been previously diagnosed as having an autoimmune and / or inflammatory disease, for example, a subject who exhibits one or more symptoms or risk factors of an autoimmune and / or inflammatory disease, or a subject who does not exhibit symptoms or risk factors of an autoimmune and / or inflammatory disease, or a subject who is asymptomatic for an autoimmune and / or inflammatory disease.

[0035] As used herein, the phrase "one or more intrinsic serum or plasma mediator biomarkers" refers to one or more of the following biomarkers: IL-1α, IL-1β, IL-1RA, IFN-α, IL-15, IL-12p70, IL-6, and IL-23pl9. These biomarkers can be measured at the RNA or protein level and can be obtained from a sample, for example, from a patient who is a mammal, such as a blood, serum, plasma, and / or urine sample of a human patient. The abbreviations of all biomarkers used herein are well known to the skilled person, for example, IL-1 is interleukin-1, etc. The abbreviations can be matched to the protein, for example, at www.genecards.org.

[0036] As used herein, the phrase "one or more adaptive serum or plasma mediator biomarkers" refers to one or more of the following biomarkers: IL-2, IFN-γ, IL-4, IL-5, IL-13, IL-17A, IL-21, IL-10, and TGF-β. These biomarkers can be measured at the RNA or protein level and can be obtained from a sample, such as a blood, serum, plasma, and / or urine sample from a patient who is a mammal, such as a human patient.

[0037] As used herein, the phrase "one or more chemokine biomarkers" 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, and MCP-3 / CCL7. These biomarkers can be measured from a sample, such as a blood, serum, plasma, and / or urine sample from a patient who is a mammal, such as a human patient.

[0038] As used herein, the phrase "one or more soluble TNF superfamily biomarkers" refers to one or more of the following biomarkers: TNF-α, TNFRI, TNFRII, Fas, CD40L / CD154, BLyS, and APRIL or tumor necrosis factor ligand superfamily member 13 (TNFSF13). These biomarkers can be measured at the RNA or protein level and can be obtained from a sample, such as a blood, serum, plasma, and / or urine sample from a patient who is a mammal, such as a human patient.

[0039] As used herein, the phrase "one or more inflammatory mediator biomarkers" refers to one or more of the following biomarkers: stem cell factor (SCF), plasminogen activator inhibitor 1 (PAI-1) and resistin. These biomarkers can be measured at the RNA or protein level and can be obtained from a sample, such as a blood, serum, plasma and / or urine sample from a patient who is a mammal, such as a human patient.

[0040] As used herein, the phrase "one or more SLE-associated autoantibody-specific biomarkers" refers to one or more of the following biomarkers that are autoantibodies to 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 a sample, for example, from a patient as a mammal, such as a blood, serum, plasma, and / or urine sample of a human patient.

[0041] As used herein, "healthy control" refers to a healthy control who is not a SLE patient and has no clinical evidence of SLE.

[0042] The present invention includes methods for identifying SLE patients and changing their treatment, the SLE patients are associated with clinical disease activity as defined by the Safety of Estrogens in Lupus National Assessment-Systemic Lupus Erythematosus Disease Activity Index (SELENA-SLEDAI, with proteinuria, as defined by SLEDAI-2K, referred to as mixed SLEDAI (hSLEDAI)) in the Lupus National Assessment of Estrogens and clinical disease activity as defined by the Lupus National Assessment-Systemic Lupus Erythematosus Disease Activity Index (SELENA-SLEDAI). Since the clinical manifestations of SLE lead to disease activity in hSLEDAI after their appearance, the present invention is used to determine whether a subject can show biomarkers that can lead to disease activity that puts SLE patients at risk of permanent organ damage and early death.

[0043] According to the ACR criteria for SLE classification, patients must meet at least 4 ACR criteria for SLE to reach disease classification (diagnosis), including: butterfly rash, discoid rash, sunlight allergy, oral ulcers, arthritis, serositis (pleurisy or pericarditis), renal disease (proteinuria or cell casts), nervous system disease (epileptic seizures or psychosis), blood system disease (hemolytic anemia, leukopenia, lymphopenia or thrombocytopenia), immune disease (anti-DNA, anti-Sm or antiphospholipid antibodies) and positive ANA (HEp-2IIF determination). As known to the technician, other criteria can also be used, for example, using the SLE classification rules of the systemic lupus international cooperative clinic (SLICC), patients must meet at least 4 criteria, including at least one clinical criterion and one immunological criterion, or patients must be confirmed to have lupus nephritis through biopsy in the presence of anti-nuclear antibodies or anti-double-stranded DNA antibodies.

[0044] Biomarker Detection. There are a variety of methods that can be used to assess protein expression. One such method is the use of antibodies for protein identification.

[0045] As used herein, the term "antibody" refers broadly to any immunobinding agent, such as IgG, IgM, IgA, IgD and IgE antibodies or their subclasses, or their binding fragments, including single-chain fragments. Typically, IgG and / or IgM are used because they are the most common antibodies under physiological conditions and are generally and easily prepared in a laboratory environment. As used herein, the term "antibody fragment" refers to any antibody-like molecule with an antigen-binding region, and includes antibody fragments, such as Fab', Fab, F(ab')2, single domain antibodies (DAB), Fv, scFv (single-chain Fv), etc. It is well known in the art to prepare and use various antibody-based constructs and fragments. It is also well known in the art to prepare and characterize antibodies (both polyclonal and monoclonal) (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; The relevant parts are incorporated herein by reference).

[0046] According to the present invention, examples of immunoassays are provided. For example, some immunoassays include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluorescent immunoassay, chemiluminescent assay, bioluminescent assay and protein blot. The steps of various useful immunoassays are described in the scientific literature Current Protocols in Immunology, Wiley & Sons Press, 2017, and the relevant parts are incorporated herein by reference.

[0047] In general, immunological binding methods include obtaining a sample suspected of containing a polypeptide of interest and contacting the sample with a first antibody under conditions effective to allow the formation of an immune complex. In the case of antigen detection, the biological sample analyzed can be any sample suspected of containing an antigen, such as a tissue section or specimen, a homogenized tissue extract, a cell, or even a biological fluid.

[0048] Contacting the selected biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibody to form immune complexes with any antigen present (i.e., bind to the antigen). After this period of time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, will generally be washed to remove any non-specifically bound antibody species, thereby allowing detection of only those antibodies that are specifically bound within the primary immune complex.

[0049] In general, detection of immune complex formation is well known in the art and can be achieved by applying a variety of methods. These methods are generally based on the detection of labels or markers, such as any of those radioactive, fluorescent, biological and enzyme tags. Patents on 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 of which is incorporated herein by reference. Of course, additional advantages may be found by using secondary binding ligands such as second antibodies and / or biotin / avidin ligand binding arrangements as known in the art.

[0050] The antibody or its binding fragment that can be used in the detection can itself be connected to a detectable label, wherein this label will then be simply detected, thereby allowing the amount of the primary immune complex in the composition to be determined. Alternatively, the first antibody that becomes bound in the primary immune complex can be detected by a second binding ligand that has binding affinity to the antibody. In these cases, the second binding ligand can be connected to a detectable label. The second binding ligand itself is often an antibody, so it can be referred to as a "secondary" antibody. The primary immune complex is contacted with a labeled secondary binding ligand or antibody under effective conditions and lasts for a period of time sufficient to allow the formation of a secondary immune complex. The secondary immune complex is then usually washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining labels in the secondary immune complex are then detected.

[0051] Other methods include detecting primary immune complexes by a two-step method. As described above, a second binding ligand such as an antibody that has binding affinity to the antibody is used to form a secondary immune complex. After washing, the secondary immune complex is contacted with a third binding ligand or antibody that has binding affinity to the second antibody again under effective conditions and continues for a period of time sufficient to allow the formation of an immune complex (tertiary immune complex). The third ligand or antibody is connected to a detectable label to allow detection of the tertiary immune complex thus formed. If desired, this system can provide signal amplification.

[0052] An immunodetection method uses two different antibodies. A monoclonal or polyclonal antibody of the first step biotinylation is used to detect one or more target antigens, and then the second step antibody is used to detect biotin connected to the composite biotin. In the method, the sample to be tested is first incubated in a solution comprising the first step antibody. If the target antigen is present, some antibodies bind to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubating in a continuous solution of streptavidin (or avidin), biotinylated DNA and / or complementary biotinylated DNA, wherein each step adds additional biotin sites to the antibody / antigen complex. The amplification step is repeated until the amplification of the appropriate level is achieved, at which point the sample is incubated in a solution comprising the second step antibody for biotin. This second step antibody is, for example, labeled with an enzyme, which can be used to detect the presence of the antibody / antigen complex by histoenzymology using a chromophore substrate. In the case of suitable amplification, a conjugate visible to the naked eye can be produced.

[0053] Another known immunoassay method utilizes an immuno-PCR (polymerase chain reaction) method. The PCR method is similar to the Cantor method, up to incubation with biotinylated DNA, however, the DNA / biotin / streptavidin / antibody complex is washed off with a low pH or high salt buffer that releases the antibody, rather than using multiple rounds of streptavidin and biotinylated DNA incubations. The resulting wash solution is then used to perform a PCR reaction using appropriate primers and under appropriate controls. At least in theory, the huge amplification capacity and specificity of PCR can be used to detect single antigen molecules.

[0054] As described in detail above, immunoassays are essentially binding assays. Certain immunoassays are various types of ELISA and RIA known in the art. However, it will be readily appreciated that detection is not limited to such techniques, and Western blots, dot blots, FACS analysis, etc. may also be used.

[0055] In an exemplary ELISA, the antibodies of the present invention are fixed to a selected surface that exhibits protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing an antigen (such as a clinical sample) is added to the well. After binding and washing to remove non-specifically bound immune complexes, the bound antigen can be detected. Detection is usually achieved by adding another antibody connected 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 having binding affinity to the second antibody, wherein the third antibody is connected to a detectable label.

[0056] In another exemplary ELISA, a sample suspected of containing an antigen is fixed to the surface of a well and then contacted with the anti-ORF message and anti-ORF translation product antibodies of the present invention. After binding and washing to remove non-specifically bound immune complexes, the bound anti-ORF message and anti-ORF translation product antibodies are detected. When the initial anti-ORF message and anti-ORF translation product antibodies are linked to a detectable label, the immune complexes can be directly detected. Similarly, a second antibody having binding affinity for the first anti-ORF message and anti-ORF translation product antibodies can be used to detect the immune complexes, wherein the second antibody is linked to a detectable label.

[0057] Another type of ELISA in which the antigen is immobilized involves the use of antibody competition in the detection. In this ELISA, a labeled antibody to the antigen is added to the wells, allowed to bind, and detected by its label. The amount of antigen in the unknown sample is then determined by mixing the sample with the labeled antibody to the antigen during incubation with the coated wells. The presence of the antigen in the sample is used to reduce the amount of antibody available for the antigen to bind to the wells, and thereby reduce the final signal. This also applies to antibodies to detect antigens in unknown samples, where unlabeled antibodies bind to the wells coated with the antigen and also reduce the amount of antigen available for binding to the labeled antibody.

[0058] As used herein, the phrase "under conditions effective to allow immune complex (antigen / antibody) formation" refers to those conditions that may also include dilution of the antigen and / or antibody with a solution such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS) / Tween, under which the antibody or its binding fragment interacts with the antigen that is the specific target of the antibody. These added agents also tend to help reduce nonspecific background. "Suitable" conditions allow incubation to be carried out at a temperature or time period sufficient to allow effective binding. The incubation step is generally about 1 to 2 to 4 hours, preferably at a temperature of about 25°C, or may be overnight at about 4°C.

[0059] Another antibody-based method for assessing biomarker expression is Fluorescence-Activated Cell Sorting (FACS), which is a specialized form of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based on the specific light scattering and fluorescence characteristics of each cell. It provides a rapid, objective and quantitative recording of fluorescence signals from individual cells and physical separation of cells of particular interest. The cell suspension is entrained in the center of a narrow, fast-flowing liquid stream. The flow is arranged so that there is a large spacing between cells relative to their diameter. A vibration mechanism breaks the cell stream into individual droplets. The system is adjusted so that the probability of more than one cell per droplet is low. Just before the stream breaks into droplets, the stream passes through a fluorescence measurement station, where the target fluorescence characteristics of each cell are measured. A charging ring is placed exactly where the stream breaks into droplets. Based on the fluorescence intensity measurement immediately before, a charge is placed on the ring, and when the droplets break from the stream, the opposite charge is captured on the droplets. The charged droplets then fall through an electrostatic deflection system that transfers the droplets to a container based on their charge. In some systems, the charge is applied directly to the stream, and the ruptured droplets retain a charge of the same sign as the stream. After the droplets rupture, the stream returns to neutral. A common way to use FACS is to use fluorescently labeled antibodies that bind to targets on or in cells to identify cells with a given target. This technology can be used quantitatively, where the amount of fluorescent activity is related to the amount of target, allowing people to sort based on the relative amount of fluorescence and therefore the relative amount of the target.

[0060] The bead-based xMAP technology can also be applied to immunological detection in conjunction with the presently claimed invention. This technology combines advanced fluidics, optics, and digital signal processing with proprietary microsphere technology to achieve multiplexed assay capabilities. The xMAP technology, featuring a flexible, open architecture design, can be configured to perform a wide variety of biological assays quickly, cost-effectively, and accurately.

[0061] Fluorescently encoded microspheres are arranged in up to 500 different groups. Each bead group can be coated with a reagent (e.g., antibody) specific to a particular bioassay, allowing capture and detection of specific analytes from a sample, such as the biomarkers of the present application. Inside the xMAP multiplex analyzer, a light source excites the internal dye that identifies each microsphere particle, as well as any reporter dye captured during the assay. Multiple reads are performed on each bead group, which further verifies the results. Using this process, xMAP technology allows multiplexing up to 500 unique bioassays both quickly and accurately within a single sample. Different from other flow cytometer microsphere-based assays that use a combination of different sizes and color intensities to identify individual microspheres, xMAP technology uses 5.6 micron-sized microspheres stained with red and infrared fluorophores internally by a proprietary staining process to produce 500 unique dye mixtures for identifying each individual microsphere.

[0062] Some of the advantages of xMAP include multiplexing (reduced cost and labor), generating more data with less sample, less labor and lower cost, faster and more reproducible results than solid planar arrays, and focused, flexible multiplexing of 1 to 500 analytes to suit a variety of applications.

[0063] Nucleic acid detection. In other embodiments for detecting protein expression, gene transcription can be measured. For example, an indirect method for detecting protein expression is to detect mRNA transcripts that produce the protein.

[0064] Amplification of nucleic acids. Since many mRNAs exist in relatively low abundance, 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 target region. As used herein, the term "primer" refers to any nucleic acid that can initiate 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 be used. Primers can be provided in double-stranded and / or single-stranded form, but single-stranded forms are typically used.

[0065] Primers designed to selectively hybridize with nucleic acids corresponding to selected genes are contacted with template nucleic acid under conditions that allow selective hybridization. Depending on the desired application, high stringency hybridization conditions can be selected, which will only allow hybridization with sequences that are completely complementary to the primers. In other embodiments, hybridization can occur under reduced stringency to allow the use of primer sequence amplification to comprise one or more mispairing nucleic acids. Once hybridized, template-primer complex is contacted with one or more enzymes that promote template-dependent nucleic acid synthesis. Carry out multiple rounds of amplification, also referred to as "circulation", until sufficient amounts of amplified products are produced.

[0066] The amplified product can be detected or quantified. In some applications, it can be detected by visual methods. Alternatively, detection can include radioactive scintillation scanning by chemiluminescence, incorporated radioactive labels or fluorescent labels or even by indirectly identifying the product using a system of electrical and / or thermal pulse signals.

[0067] Many template-dependent methods can be used to amplify oligonucleotide sequences present in a given template sample. One of the best-known amplification methods is the polymerase chain reaction (PCR), which is described in detail 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.

[0068] Reverse transcriptase-PCR amplification procedures can be performed to quantify the amount of amplified mRNA. The method of reverse transcription of RNA into cDNA is well known (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2001). Alternative methods for reverse transcription utilize thermostable DNA polymerases. These methods are described in WO 90 / 07641. Polymerase chain reaction methods are well known in the art. Representative methods of RT-PCR are described in U.S. Patent No. 5,882,864. Standard PCR usually uses a pair of primers to amplify specific sequences, while multiplex PCR (MPCR) uses multiple pairs of primers to amplify many sequences simultaneously. The presence of many PCR primers in a single tube may cause many problems, such as the increase in the formation of PCR products and "primer dimers" caused by errors, the amplification and identification of longer DNA fragments, etc. Generally, MPCR buffer contains Taq polymerase additives, which reduce the competition between amplicons during MPCR and the amplification and identification of longer DNA fragments. MPCR products can be further hybridized with gene-specific probes for verification. In theory, people should be able to use as many primers as needed. However, due to side effects caused during MPCR (primer dimers, mis-primed PCR products, etc.), there is a limit to the number of primers that can be used in an MPCR reaction (less than 20). See also European application 0 364 255, the relevant parts of which are incorporated herein by reference.

[0069] Another method for amplification is the ligase chain reaction ("LCR"), which is disclosed in European Application 320 308, which is incorporated herein by reference in its entirety. U.S. Pat. No. 4,883,750 describes a method similar to LCR for binding probe pairs to target sequences. A method based on PCR and oligonucleotide ligase assay (OLA) disclosed in U.S. Pat. No. 5,912,148 may also be used. Alternative methods for amplifying target nucleic acid sequences that can be used in the practice of the present invention are disclosed in U.S. Patents 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; 5,922,574; 5,928,905; 5,928,906; 5,932,451; 5,935,825; 5,939,291; and 5,942,391; GB Application 2 202 328; and PCT Application PCT / US89 / 01025, the relevant portions of each of which are incorporated herein by reference.

[0070] Detection of nucleic acid. After any amplification, it is desirable to separate the amplified product from the template and / or too many primers. In one embodiment, standard methods are used to separate the amplified product by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2001). The amplified product of separation can be cut and eluted from the gel to further operate. Using low melting point agarose gel, the separated band can be removed by heating the gel, and then the nucleic acid is extracted. The separation of nucleic acid can also be achieved by chromatographic techniques known in the art. There are many kinds of chromatograms that can be used for the practice of the present invention, including adsorption, distribution, ion exchange, hydroxyapatite, molecular sieve, reverse phase, column, paper, thin layer and gas chromatography and HPLC.

[0071] In certain embodiments, the amplification product is visualized. Typical visualization methods involve staining the gel with ethidium bromide and visualizing the bands under UV light. Alternatively, if the amplification product is labeled as a whole with radioactive or fluorescently labeled nucleotides, the separated amplification product can be exposed to x-ray film or visualized under a suitable excitation spectrum.

[0072] In one embodiment, after the amplification product is separated, a labeled nucleic acid probe is contacted with the amplified marker sequence. The probe is preferably conjugated to a chromophore, but may be radiolabeled. In another embodiment, the probe is conjugated to a binding partner (such as an antibody or biotin), or another binding partner carrying a detectable portion.

[0073] In a specific embodiment, detection is performed by Southern blotting and hybridization with a labeled probe. 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. Pat. No. 5,279,721, which is incorporated herein by reference, which discloses an apparatus and method for automated electrophoresis and transfer of nucleic acids. The apparatus allows electrophoresis and blotting to be performed without external manipulation of the gel, and is ideally suited for performing the method according to the present invention.

[0074] Other nucleic acid detection methods that can be used in the practice of the present invention are disclosed in U.S. Patents 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, 5,861,24 4, 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.

[0075] Nucleic acid arrays. Microarrays include a plurality of polymeric molecules spatially distributed on and stably associated with the surface of a substantially planar substrate (e.g., a biochip). Microarrays of polynucleotides have been developed and used in a variety of applications, such as screening and DNA sequencing. One area in which microarrays are particularly useful is in gene expression analysis.

[0076] In gene expression analysis using microarrays, an array of "probe" oligonucleotides is contacted with a target nucleic acid sample (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 hybrid nucleic acid pattern provides information about the genetic signature spectrum of the sample tested. Gene expression analysis methods on microarrays can provide both qualitative and quantitative information.

[0077] A variety of different arrays that can be used with the present invention are known in the art. The probe molecules of the array capable of sequence-specific hybridization with the target nucleic acid can be polynucleotides or hybridization analogs or mimetics thereof, including: nucleic acids in which the phosphodiester bond is replaced by an alternative bond (such as phosphorothioate, methylimino, methylphosphonate, phosphoramidate, guanidine, etc.); nucleic acids in which the ribose subunit is replaced, such as hexose phosphodiester; peptide nucleic acids; and the like. The length of the probe will generally be in the range of 10 to 1,000 nucleotides, wherein in some embodiments, the probe will be an oligonucleotide and the length will generally be in the range of 15 to 150 nucleotides and more generally in the range of 15 to 100 nucleotides, and in other embodiments, the probe will be longer, generally in the range of 150 to 1,000 nucleotides in length, wherein the polynucleotide probe can be single-stranded or double-stranded, generally single-stranded, and can be a PCR fragment amplified from cDNA.

[0078] The probe molecules on the substrate surface will correspond to the selected genes being analyzed and will be positioned on the array at known locations so that positive hybridization events can be associated with the expression of specific genes in the physiological source from which the target nucleic acid sample is derived. The substrate to which the probe molecules are stably associated can be made of a variety of materials, including plastics, ceramics, metals, gels, membranes, glass, etc. The array can be produced according to any convenient method, such as pre-forming the probes and then stably associating them with the surface of the support or growing the probes directly on the support. Many different array configurations and methods for producing them are known to those skilled in the art and are disclosed in U.S. Patents 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, 5,472 ,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 parts of which are incorporated herein by reference.

[0079] After hybridization, wherein the non-hybridized labeled nucleic acid can emit a signal during the detection step, a washing step is adopted, wherein the non-hybridized labeled nucleic acid is removed from the support surface, thereby generating a pattern of hybridized nucleic acid on the substrate surface. A variety of washing solutions and their use schemes are known to those skilled in the art and can be used. In the case where the label on the target nucleic acid is not directly detectable, the array now comprising the binding target is contacted with one or more other members of the signal generating system being used. For example, in the case where the label on the target is biotin, the array is contacted with streptavidin-fluorescent conjugate under conditions sufficient for the binding between the specific binding member pairs. After contact, any unbound member of the signal generating system will be removed, for example, by washing. The specific washing conditions adopted will inevitably depend on the specific properties of the signal generating system adopted, and will be known to those skilled in the art who are familiar with the specific signal generating system adopted. The resulting hybridization pattern of the labeled nucleic acid can be visualized or detected in a variety of ways, wherein a specific detection method is selected based on the specific label of the nucleic acid, wherein representative detection means include scintillation counting, autoradiography, fluorescence measurement, calorimetry, luminescence measurement, etc.

[0080] Prior to detection or visualization, when it is desired to reduce the likelihood of mismatched hybridization events generating false positive signals on the map, the array of hybridized target / probe complexes may be treated with an endonuclease under conditions sufficient to allow the endonuclease to degrade single-stranded but not double-stranded DNA. A variety of different endonucleases are known and can be used, including mung bean nuclease, S1 nuclease, and the like. When such treatment is employed in an assay where the target nucleic acid is not labeled with a directly detectable label (e.g., in an assay with a biotinylated target nucleic acid), the endonuclease treatment will typically be performed before the array is contacted with one or more other members of the signal generating system (e.g., a fluorescent-streptavidin conjugate). As described above, the endonuclease treatment ensures that only target / probe complexes with substantially fully hybridized end labels at the 3' end of the probe are detected in the hybridization map. As described above, after hybridization and one or more of any washing steps and / or subsequent treatments, the resulting hybridization map is detected. When detecting or visualizing the hybridization pattern, the intensity or signal value of the label will not only be detected but also quantified, which means that the signal from each spot of hybridization will be measured and compared to the unit value corresponding to the signal emitted by a known number of end-labeled target nucleic acids to obtain a count or absolute value of the number of copies of each end-labeled target that hybridizes to a specific spot on the array in the hybridization pattern.

[0081] RNA sequencing. RNA-seq (RNA sequencing), also known as Whole Transcriptome Shotgun Sequencing (WTSS), is a technique that uses the power of next-generation sequencing (NGS) to reveal a snapshot of the presence and amount of RNA from the genome at a given moment. The transcriptome of a cell is dynamic; in contrast to a static genome, it is constantly changing. The latest developments in next-generation sequencing allow for increased DNA sequence base coverage and higher sample throughput. This facilitates the sequencing of RNA transcripts in cells, providing the ability to look at alternative gene splicing transcripts, post-transcriptional changes, gene fusions, mutations / SNPs, and changes in gene expression. In addition to mRNA transcripts, RNA-Seq can also look at different RNA populations to include total RNA, small RNAs such as miRNA, tRNA, and ribosome profile analysis. RNA-Seq can also be used to determine exon / intron boundaries and verify or revise previously annotated 5' and 3' gene boundaries. Ongoing RNA-Seq studies include observing changes in cell pathways during infection, and changes in gene expression levels in cancer research. Prior to NGS, transcriptomics and gene expression studies were previously performed using expression microarrays, which contained thousands of DNA sequences that were probed for matches in a target sequence, allowing for a profile of all transcripts expressed. This was later accomplished by serial analysis of gene expression (SAGE).

[0082] For the treatment of SLE. Therefore, the present invention contemplates the detection of certain biomarkers, followed by changes in SLE treatment, which may include the use of standard treatment methods when indicated. Generally, the treatment of SLE involves treating elevated disease activity and attempting to minimize organ damage that may be associated with increased inflammation and increased immune complex formation / deposition / complement activation. Basic treatment may include corticosteroids and / or antimalarial drugs. Certain types of lupus nephritis (such as diffuse proliferative glomerulonephritis) require large amounts of cytotoxic drugs. These drugs most commonly include cyclophosphamide and mycophenolate mofetil. Hydroxychloroquine (HCQ) was approved by the FDA for lupus in 1955. Some drugs approved for other diseases are used for SLE "off-label". Belimumab (Benlysta) can be used as a treatment for elevated disease activity seen in autoantibody-positive lupus patients.

[0083] Because multiple symptoms and organ systems are affected by SLE, their severity in individuals must be assessed in order to successfully treat SLE. Sometimes, mild or intermittent disease can be safely treated with hydroxychloroquine only to a minimal extent. Nonsteroidal anti-inflammatory drugs and low-dose steroids can also be used if necessary. Hydroxychloroquine (HCQ) is an FDA-approved antimalarial drug for physical, skin and joint manifestations. Hydroxychloroquine has relatively few side effects, and there is evidence that it improves the survival of people with SLE, and stopping HCQ in stable SLE patients has led to an increase in disease flares in Canadian lupus patients. Disease-modifying antirheumatic drugs (DMARDs) are often used off-label for SLE to reduce disease activity and reduce the need for steroid use. Commonly used DMARDs are methotrexate and azathioprine. In more severe cases, drugs that actively suppress the immune system (mainly high-dose corticosteroids and major immunosuppressants) are used to control the disease and prevent damage. Cyclophosphamide is used for severe glomerulonephritis, as well as other complications that threaten life or damage organs, such as vasculitis and lupus encephalitis. Mycophenolic acid is also used to treat lupus nephritis but is not FDA-approved for this indication.

[0084] Depending on the dose, people who require steroids may develop Cushing's symptoms of truncal obesity, purple striae, buffalo hump, and other related symptoms. These may subside if and when the large initial dose is reduced, but long-term use of even low doses can lead to increased blood pressure, glucose intolerance (including metabolic syndrome and / or diabetes), osteoporosis, insomnia, avascular necrosis, and cataracts.

[0085] Many new immunosuppressive drugs are being actively tested for SLE. They do not suppress the immune system nonspecifically like corticosteroids, but target the response of immune cells of a single type. Belimumab or humanized monoclonal antibodies for B lymphocyte stimulating factor (BlyS or BAFF) are approved by the FDA for lupus treatment and reduce SLE disease activity, particularly in patients with elevated disease activity and autoantibodies at baseline. Addition of drugs such as abatacept, epratuzimab, etanercept, etc. are being actively studied in SLE patients, and some of these drugs have been approved by the FDA for the treatment of rheumatoid arthritis or other conditions. Because most of the people suffering from SLE suffer from chronic pain to varying degrees, if over-the-counter drugs (mainly nonsteroidal anti-inflammatory drugs) do not provide effective relief, stronger prescription analgesics (painkillers) can be used. For patients suffering from SLE, potent NSAIDs (such as indomethacin and diclofenac) are relatively contraindicated because they increase the risk of renal failure and heart failure.

[0086] Moderate pain is usually treated with mild prescription opioids such as dextropropoxyphene and co-codamol. Moderate to severe chronic pain is treated with stronger opioids (such as hydrocodone) or long-acting continuous release opioids (such as oxycodone, MS Contin or methadone). Due to its long-acting timed release and ease of use, fentanyl duragesic transdermal patch is also a widely used treatment option for chronic pain caused by complications. When opioids are used for a long time, drug tolerance, chemical dependence and addiction may occur. Opioid addiction is usually not a cause for concern because this condition is unlikely to disappear completely. Therefore, it is quite common to use opioids for lifelong treatment of chronic pain symptoms, accompanied by periodic titration, which is a typical feature of any long-term opioid regimen.

[0087] Intravenous immunoglobulins can be used to control SLE with organ involvement or vasculitis. They are believed to reduce antibody production or promote clearance of immune complexes from the body, although their mechanisms of action are not well understood. Unlike immunosuppressants and corticosteroids, IVIG does not suppress the immune system, so there is a smaller risk of serious infection with these drugs.

[0088] Avoiding sunlight is a major lifestyle change for patients with SLE because sunlight is known to exacerbate the disease, as is the debilitating effect of intense fatigue. Both of these issues may cause patients to be housebound for long periods of time. Medications not related to SLE should be prescribed only if they are known not to exacerbate the disease. Occupational exposure to silica, pesticides, and mercury can also exacerbate the disease.

[0089] Kidney transplantation is the treatment of choice for end-stage renal disease, one of the complications of lupus nephritis, but recurrence of complete disease in the transplanted kidney is common in up to 30% of patients.

[0090] Antiphospholipid syndrome is also associated with the onset of neurological lupus symptoms in the brain. In this form of the disease, the cause is very different from lupus: thrombi (blood clots or "sticky blood") are formed in blood vessels, which can prove fatal if they move in the bloodstream. If thrombi migrate to the brain, they may cause a stroke by blocking the blood supply to the brain. If a patient is suspected of suffering from this disease, early detection usually requires a brain scan. These scans can show local areas of the brain where the blood supply is already insufficient. The treatment plan for these patients requires anticoagulation. Typically, low-dose aspirin is prescribed for this purpose, but for cases involving thrombosis, anticoagulants such as warfarin are used.

[0091] Pharmaceutical Formulation and Delivery. As a variety of therapeutic applications are envisioned, it will be necessary to prepare pharmaceutical compositions in a form suitable for the intended application. Generally, this will necessitate the preparation of compositions that are substantially free of pyrogens and other impurities that may be harmful to humans or animals.

[0092] Typically, appropriate salts and buffers are used to stabilize the delivery vector and allow uptake by target cells. When the recombinant cells are introduced into a patient, a buffer will also be used. The aqueous composition of the present invention comprises an effective amount of a vector for cells dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions are also referred to as inocula.

[0093] As used herein, the phrase "pharmaceutically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse reactions, allergic reactions or other untoward reactions when administered to animals or humans. As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the carriers or cells of the present invention, their use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the composition.

[0094] Active compositions of the present disclosure may include classical pharmaceutical preparations. These compositions may be administered according to the present invention by any common route, as long as the target tissue can be reached by the route. Such routes include oral, nasal, buccal, rectal, vaginal or surface routes. Alternatively, administration may be by in situ, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions are usually administered as pharmaceutically acceptable compositions. The active compound may also be administered parenterally or intraperitoneally. Solutions of the active compounds in the form of free alkali or pharmacologically acceptable salts may be prepared in water appropriately mixed with a surfactant such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof and in oils. Under common storage and use conditions, these preparations include preservatives to prevent the growth of microorganisms.

[0095] The pharmaceutical form suitable for injectable use includes sterile aqueous solution or dispersion and sterile powder for temporary preparation of sterile injectable solution or dispersion. In all cases, the form must be sterile and must be a fluid in the degree of easy injection. It must be stable under manufacturing and storage conditions, and must be protected from the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium comprising the following: for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), a suitable mixture thereof, and vegetable oil. For example, by using a coating (such as lecithin), in the case of a dispersion, by maintaining the desired particle size, and by using a surfactant to maintain appropriate fluidity. The effect of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferred to include isotonic agents, such as sugar or sodium chloride. By using an agent (such as aluminum monostearate and gelatin) that delays absorption in the composition, the extended absorption of the injectable composition can be achieved.

[0096] Sterile injectable solutions are prepared by incorporating the desired amount of active compound into an appropriate solvent along with the various other ingredients listed above, followed by filtration sterilization. Typically, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle comprising a basic dispersion medium and other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying techniques, which produce a powder of the active ingredient plus any additional desired ingredients from its previously sterile filtered solution.

[0097] As used herein, the phrase "pharmaceutically acceptable carrier" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the composition.

[0098] For oral administration, the polypeptides of the present invention can be combined with excipients and used in the form of non-ingestible mouthwashes and dentifrices. Mouthwashes can be prepared by incorporating the desired amount of the active ingredient into an appropriate solvent such as sodium borate solution (Dobell's solution). Alternatively, the active ingredient can be incorporated into an antiseptic wash containing sodium borate, glycerol and potassium bicarbonate. The active ingredient can also be dispersed in a dentifrice, including gels, pastes, powders and slurries. The active ingredient can be added to a paste dentifrice that can include water, a binder, an abrasive, a flavoring agent, a foaming agent and a wetting agent in a therapeutically effective amount.

[0099] The composition used for use with the present invention can be prepared in neutral or salt form.Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of proteins), and inorganic acids such as hydrochloric acid or phosphoric acid or salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. The salts formed with free carboxyl groups 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 preparation, the solution will be used in a manner compatible with the dosage formulation and in an amount such as effective for treatment. The preparation is easily used in a variety of dosage forms (such as injectable solutions, drug release capsules, etc.). For parenteral administration in an aqueous solution, for example, if necessary, the solution should be appropriately buffered, and the liquid diluent isotonic with enough saline or glucose first. In this regard, according to the present disclosure, the sterile aqueous medium that can be used will be known to those skilled in the art. For example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1,000 ml of subcutaneous perfusion fluid or injected at a suggested infusion site (see, e.g., "Remington's Pharmaceutical Sciences," 15th edition, pages 1035-1038 and 1570-1580), the relevant portions of which are incorporated by reference. Depending on the condition of the subject being treated, some variation in dosage will necessarily occur. In any case, the person responsible for administration will determine the appropriate dosage for the individual subject. In addition, for human administration, the preparation should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biologics standards.

[0100] Kit. In order to be used in the applications described herein, the kit is also within the scope of the present invention. Such kits may include carriers, packaging or containers, which are separated to receive one or more containers (such as vials, tubes, etc.), each of which contains one of the individual elements to be used in the method, particularly light inhibitors. The kit of the present invention will generally include the above-mentioned container and one or more other containers, which contain materials that are desirable from the perspective of commercial end users, including buffers, diluents, filters and package inserts with instructions for use. In addition, a label can be provided on the container to indicate that the composition is used for a specific therapeutic application, and instructions for use for in vivo or in vitro use, such as those described above, can also be indicated. Instructions for use and or other information may also be included in the insert included in the kit. In particular, the kit according to the present invention contemplates a combination of an agent for assessing the level of the biomarker discussed above with one or more SLE therapeutic agents, and / or an agent for ANA testing and / or anti-ENA, and a control for its assessment.

[0101] Current biomarkers in lupus disease activity measurement have limited utility in reflecting increased clinical disease activity. They are neither the earliest nor the most informative measure of clinical disease activity. Although SLE-associated autoantibody specificities, such as anti-dsDNA, anti-spliceosome, and anti-Ro / SSA, accumulate in SLE patients years before classification, their presence is insufficient to reflect clinical disease activity and risk of permanent organ damage. ANAs are also present in sera from patients with other systemic rheumatic diseases, as well as healthy individuals who do not go on to develop SLE (including some unaffected family members of SLE patients), and up to 14% of the general population. Because individuals can remain healthy while being 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 can also contribute to clinical disease activity.

[0102] EXAMPLES. The following examples are included to further illustrate various aspects of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques and / or compositions discovered by the inventors to function well in the practice of the present invention, and therefore can be considered to constitute preferred modes for its practice. However, in light of the present disclosure, it will be appreciated by those skilled in the art that many changes may be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present invention.

[0103] SLE is a complex autoimmune disease characterized by immune dysregulation. A comprehensive but cost-effective tool to track the relevant mediators of altered disease activity will help improve disease management and prevent organ damage. The goal of this embodiment is to identify the key components of practical biometrics to distinguish active lupus from low lupus disease activity.

[0104] The present invention includes identifying certain immune mediators, including SLE-related autoantibody specificity and inflammatory and regulatory soluble mediator pathways that may be dysregulated with changes in SLE disease activity. Using multiple methods, SLE patient followers with low (hSLEDAI<4) or active (hSLEDAI≥4) disease activity showed disease activity-dependent changes in the plasma levels of autoantibody-specific accumulation and inflammatory and regulatory mediators compared to matched healthy controls (HC). These results enable the development of a combined lupus disease activity immune index (LDAII), which reflects the immune changes produced with changes in clinical disease activity.

[0105] SLE-associated immune mediators and autoantibodies were assessed in 311 plasma samples from 198 patients with classified SLE who had low clinical disease (systemic lupus erythematosus disease activity index (SLEDAI) <4, range 0-3, n=132) or more active disease (SLEDAI ≥4, range 4-30, n=179) on the date of acquisition, as well as healthy controls (HC) (n=48) matched for race, sex, and age. 32 soluble mediators and SLE-associated autoantibody specificities were assessed by bead-based multiplex assays or sandwich ELISAs (BLyS, APRIL, and TGF-β), including dsDNA, chromatin, Ro / SSA, La / SSB, Sm, SmRNP, and RNP. Soluble mediator levels and clinical disease activity levels were cross-compared in conjunction with the presence of autoantibodies.

[0106] Patients with low or active disease have similar age, race and sex.After adjusting for multiple comparisons (Bonferroni corrected p < 0.0018), IL-6, IL-1α, IP-10 and IL-8 are significantly correlated with SLEDAI scores (Spearman r = 0.179-0.253), and 22 / 32 soluble mediators are significantly correlated with the number of accumulated SLE-related autoantibodies (including those listed above, and SCF, IFN-α, IFN-γ, IL-17A, IL-10, MIG, MIP-1β, TNFRII and BLyS) (r = 0.318 [IL-17A] -0.468 [IP-10]). Regulatory mediator IL-10 is the highest in samples from patients with low disease activity (p < 0.05), and inflammatory mediators are the highest in active disease samples with accumulated autoantibody specificity (p < 0.001). The inventors integrate these findings to establish the Lupus Disease Activity Immune Index (LDAII), which is calculated using the standardized (cases relative to controls) soluble mediator levels (n=32) weighted by the number of SLE-related autoantibodies in each individual. LDAII distinguishes patients with clinically active (CA) relative to quiescent (CQ) disease, and the patients are serological (dsDNA binding and low complement) active (SA) or quiescent (SQ) patients (p<0.0001), whereby the number of accumulated autoantibodies (p<0.0001) and IL-6, IL-8 and IP-10 levels (p≤0.0006) are most significantly changed. In addition, LDAII is able to distinguish clinical and serological quiescent (CQSQ) SLE patients relative to HC (p=0.019). Finally, LDAII was significantly correlated with patients' SLEDAI scores (p<0.0001) and identified patients with renal organ involvement (p=0.002), in whom SCF, TNFRII, and MCP-1 were also most significantly altered (p≤0.005).

[0107] Study population, clinical data and sample collection. The study was conducted in accordance with the Helsinki Declaration and approved by the Institutional Review Board of the Oklahoma Medical Research Foundation and the University of Oklahoma Health Sciences Center. Undiluted plasma and serum samples were obtained from 198 SLE patients (satisfying>4 ACR classification criteria (39)) with appropriate consent in the Oklahoma Lupus Group and 48 healthy individuals (controls or Ctls) matched in the Oklahoma Immunity Group, Table 1. The samples were stored in the Oklahoma Rheumatology Research Core Center (ORDRCC) (OMRF's CAP-certified biobank), -80 ° C. For the ability to determine the level of soluble mediators and SLE-related AutoAb in samples from SLE patients and controls, samples in the biobank have been tested relative to shipping time and method, handling procedures, storage conditions and storage time length (33, 36, 37, 40-45).

[0108] Table 1. Study Participant Demographics

[0109]

[0110] a SLE patients meet ≥ 4 SLE classification criteria (ACR and SLICC)

[0111] b Race, sex, and age matched for SLE

[0112] c Statistics determined by paired t test (continuous data) or Fisher's exact test (categorical data)

[0113] Significance (p≤0.05)

[0114] d Non-EA = African-American, Asian, American Indian, or Multiracial

[0115] Demographic and clinical information (39), including medication use, clinical laboratory values, and clinical disease activity, were collected as previously described (39). The presence of organ system involvement was assessed by administration of the hybrid systemic lupus erythematosus disease activity index (hSLEDAI; SELENA-SLEDAI with proteinuria as defined by SLEDAI-2K) (32, 33), which includes the presence of disease manifestations involving the central nervous system (CNS; seizures, psychosis, organic brain syndrome, visual disturbances, cranial neurological disorders, or lupus headache), vasculitis, arthritis, myositis, nephritis (urinary casts, hematuria, proteinuria, or pyuria), mucocutaneous lesions (rash, alopecia, or mucosal ulcers), serositis (pleurisy or pericarditis), or hematologic manifestations (low complement, increased DNA binding, fever, thrombocytopenia, or leukopenia) (16). Patients with hSLEDAI scores <4 were considered to have "low" disease activity, while those with hSLEDAI scores ≥4 were considered to have "active" disease. Blood samples were obtained from each participant at each clinical visit. Plasma and serum samples were stored at -20 °C until the time of assay. Assays were performed on freshly thawed samples.

[0116] Measurement of soluble mediators and autoantibody specificity. Plasma soluble mediators (n = 32, Table 2), including cytokines, chemokines, and soluble receptors (46), were examined using validated bead-based multiplex assays or ELISAs (BLyS, APRIL, and TGF-β). Data were analyzed on an array system (Bio-Rad Technologies, Hercules, CA) with a lower boundary of 100 beads for each sample / analyte. The median fluorescence intensity of each analyte was interpolated from a 5-parameter logistic nonlinear regression standard curve. Analytes below the detection limit were assigned a value of 0.001 pg / mL. A known control serum (Cellgro human AB serum, catalog number 2931949, L / N#M1016) was included on each plate. The average inter-assay coefficient of variance (CV) for bead-based multiplex assays for cytokine detection has previously been shown to be 10-14% (47, 48), and a similar average CV (10.5%) for the analytes in this assay was obtained using healthy control serum. In each xMAP assay, the intra-assay precision of replicate wells averaged <10% CV.

[0117] Table 2. Plasma mediators evaluated in SLE patients and control samples

[0118]

[0119] Serum samples were screened for autoantibody specificity using the BioPlex 2200 multiplex system (Bio-Rad Technologies, Hercules, CA). The BioPlex 2200 ANA kit uses fluorescently dyed magnetic beads to simultaneously detect the levels of 11 autoantibody specificities, including reactivity to dsDNA, chromatin, ribosomal P, Ro / SSA, La / SSB, Sm, Sm / RNP complex, RNP, Scl-70, centromere B, and Jo-1 (49). SLE-associated autoantibody specificities to dsDNA, chromatin, Ro / SSA, La / SSB, Sm, Sm / RNP complex, and RNP were used for analysis in this study. Anti-dsDNA (IU / mL) had a previously determined positive cutoff of 10 IU / mL; the manufacturer reported antibody index (AI) values ​​(ranging from 0-8) to reflect the fluorescence intensity of each other autoantibody specificity with a positive cutoff of AI=1.0. The AI ​​scale was normalized to the calibrator and control samples provided by the manufacturer.

[0120] Statistical analysis. Categorical variables were compared by Fisher's exact test. Disease activity scores of clinical follow-ups with low-grade versus active disease were compared using an unpaired t-test. Autoantibody-specific quantities and plasma soluble mediator concentrations were compared between SLE patient follow-ups with low-grade or active disease by the Mann-Whitney test. Plasma mediator concentrations and autoantibody-specific quantities were compared between SLE patient follow-ups with low-grade and active disease and race / sex matched control samples by the Kruskal-Wallis test with Dunn's multiple comparisons. Plasma mediator concentrations were correlated with hSLEDAI scores or autoantibody-specific quantities by Spearman rank correlation. Unless otherwise stated, GraphPad Prism 6.02 (GraphPad Software, San Diego, CA) was used for analysis.

[0121] The lupus disease activity immune index (LDAII) was developed to calculate the overall inflammation level of the patient follow-up with different disease activity levels relative to the control. The calculation of LDAII follows the method (42, 45) that the inventor has similarly used to develop the lupus flare prediction index (LFPI) for SLE patients. LDAII summarizes the imbalance of all 32 plasma mediators assessed in SLE patients with low and active diseases and matched controls when clinical follow-up, and is weighted by the correlation of the AutoAb specific quantity detected in the sample obtained from the same follow-up. LDAII is calculated as follows: 1. For each SLE patient or control follow-up, the concentration of all 32 baseline plasma mediators (Table 2) is logarithmically transformed. 2. Each log-transformed soluble mediator level for each participant follow-up was normalized: (observed value)-(mean value of all SLE patients and control follow-ups) / (standard deviation of all SLE patients and control follow-ups) 3. Spearman coefficients were generated from a linear regression model that tested the association between the number of AutoAb-specifics assessed in all SLE patients and control follow-ups for each soluble mediator (Spearman r); 4. The transformed and normalized soluble mediator levels were weighted by (multiplying) their corresponding Spearman coefficients (Spearman r). 5. For each participant follow-up, the log-transformed, normalized, and weighted values ​​of each of the 32 soluble mediators were added to calculate the total LDAII.

[0122] LDAII was compared between SLE patient followers with low and active disease by the Mann-Whitney test, and was additionally compared to controls by the Kruskal-Wallis test with Dunn's multiple comparisons. The odds ratio (OR) was determined for the likelihood of having a positive or negative LDAII score for SLE patient followers with active disease versus low disease activity (or controls), respectively; significance was determined by Fisher's exact test.

[0123] Increased number of AutoAb-specific and altered levels of selected mediators in cases of clinically active disease.

[0124] The inventors have previously demonstrated that patients who transition to classified SLE (fulfilling >4 ACR classification criteria (39)) exhibit multiple levels of immune dysregulation, including accumulation of SLE-associated AutoAb-specific (36, 37, 50) and alterations in inflammatory and regulatory soluble mediators (36, 37). Based on the hypothesis that this immune dysregulation would also be reflected in SLE patients with clinically active disease, the inventors compared serum autoantibody and plasma soluble mediator profiles in samples obtained during clinical follow-up from 198 SLE patients with either low disease activity (hSLEDAI <4, range 0-3, n = 132) or active disease (hSLEDAI ≥4, range 4-30, n = 179). Figure 1A and 1B and Table 3. As expected, SLE patients with active disease had significantly higher hSLEDAI scores ( Figure 1A ), and were more likely to exhibit various organ system manifestations compared with SLE patients with low disease activity (Table 3). In addition, SLE patients with active disease showed a significant increase in the number of SLE-associated AutoAb specificities compared with the number detected during low disease activity (p = 0.0038, Figure 1B ), while SLE patients had significantly more AutoAb specificities compared to matched controls regardless of disease activity (p<0.0001). During the active disease phase, AutoAbs to dsDNA (p<0.0001), chromatin (p=0.0005), and Sm (p=0.0380) were more likely to be positive, while the frequencies of Ro / SSA, La / SSB, SmRNP, and RNPAutoAb specificities were similar in low and active disease, Table 3. SLE patients with active disease were more likely to be prescribed steroids (p<0.0001) and were prescribed hydroxychloroquine and / or immunosuppressants similarly to patients with low disease activity, Table 3.

[0125] Table 3. Drugs, autoantibodies, and clinical characteristics of patients with SLE

[0126]

[0127] a Low = hSLEDAI < 4; Active = hSLEDAI ≥ 4

[0128] b Statistical significance (p ≤ 0.05) was determined by paired t-test (continuous data) or Fisher's exact test (categorical data)

[0129] cImmunosuppressants = methotrexate, azathioprine

[0130] d Main immunosuppressants = mycophenolate mofetil, cyclophosphamide

[0131] e pass 2200ANA Screen confirmed

[0132] f CNS = epileptic seizures, psychosis, organic brain syndrome, visual disturbances, cranial nerve disorders, lupus headache, CVA

[0133] g Kidney = urinary casts, hematuria, proteinuria, pyuria

[0134] h Skin and mucous membranes = rash, hair loss, mucous membrane ulcers

[0135] i Serositis = pleurisy, pericarditis

[0136] j Serology = low complement, increased DNA binding

[0137] k Hematology = thrombocytopenia, leukopenia

[0138] In addition to changes in AutoAb specificity, the inventors also evaluated whether SLE patients with active disease also had changes in SLE-associated soluble mediators, Tables 4 and Figures 2A to 2I After adjustment for multiple comparisons, plasma levels of selected mediators, including IL-8, IP-10, IL-1a, and IL-6, were directly associated with hSLEDAI scores at clinical follow-up visits concurrent with sample acquisition (Table 4). This was further reflected by increased plasma mediator levels in SLE patients with active disease. Type I IFNs, namely IFN-α ( Figure 2A ) together with the IFN-driven chemokine IP-10 ( Figure 2B ) and the innate mediator IL-6 ( Figure 2C ) were significantly higher in SLE patients than in controls, regardless of clinical disease activity (p<0.05). Figure 2D ), chemokine IL-8 ( Figure 2E ) and Th17 mediator IL-21 ( Figure 2F) was highest in SLE patients with active disease (p<0.05), whereas levels in patients with low disease activity were similar to controls. SLE patients showed higher BLyS levels than controls regardless of disease activity (p<0.001), with the highest, although not significant, levels in SLE patients with active disease. Figure 2G Regulatory mediator IL-10 ( Figure 2H ) was highest in SLE patients with low disease activity (SLE patients had significantly higher levels than controls, p<0.01), whereas TGF-β levels were highest in controls (p<0.01) and were similar between SLE patients with low and active disease, Fig.2I These data suggest that many of the selected mediators can be altered in a manner that directly correlates with clinically active SLE.

[0139] Table 4. Different plasma mediators associated with disease activity

[0140]

[0141] a Spearman correlation, Bonferroni corrected significance p = 0.0038

[0142] Soluble mediator levels change in a manner that correlates with the presence of AutoAbs and clinical disease activity. Given the increased number of AutoAb specificities detected in SLE patients with active disease and the changes in levels of selected plasma mediators, the inventors questioned whether soluble mediator levels correlated with the number of AutoAb specificities detected, Table 5. The inventors found that multiple additional soluble mediators (22 / 32 assessed) were significantly correlated with the number of accumulated SLE-associated AutoAbs present in SLE patients and matched healthy controls (p≤0.0010), including intrinsic and adaptive mediators, chemokines, and TNF superfamily members. This prompted us to evaluate how the levels of these soluble mediators compared between SLE patients with low-grade or active disease who were also AutoAb negative (Neg) or positive (Pos) relative to controls (Figures 3-6). Type I (IFN-α, Figure 3A ) and type II (IFN-γ, Figure 3B )IFN was increased in AutoAb Pos SLE patients, with significant differences between AutoAb Neg and Pos patients with active disease; AutoAb positive SLE patients with active disease had the highest plasma levels of these mediators compared to controls (p<0.001). Figure 3C ) and MIG( Figure 3D), there was a significant increase in these mediators between AutoAb Pos and Neg SLE patients with low (p<0.05) or active (p<0.0001) disease, and levels were highest in AutoAb Pos SLE patients with active disease compared to controls (p<0.001). These data suggest that in addition to a possible direct association with altered disease activity (Figure 2), IFN-related mediators may also be associated with the accumulation of AutoAb-specific ( Figures 3A to 3D ).

[0143] Table 5. Correlation between the number of plasma mediators and specific SLE-related autoantibodies

[0144]

[0145] a Spearman correlation, Bonferroni-corrected significance

[0146] p=0.0018

[0147] B lymphocyte stimulator (BLyS) is a member of the TNF superfamily that is secreted in response to type I (51) and type II (52) IFNs and supports B cell survival and differentiation, thereby contributing to autoantibody production and class switching (53). Similar to IFN-related mediators, BLyS ( Figure 4A ) was increased in AutoAb Pos SLE patients with low-grade or active disease (p<0.01), whereby AutoAb Pos SLE patients had the highest levels compared to controls (p<0.0001). Proliferation-inducing ligand (APRIL, Figure 4B ) (53) was similarly increased in AutoAb Pos SLE patients with active disease (p<0.0001). Interestingly, when comparing SLE patients with low versus active disease, APRIL levels were found to be lower in AutoAb Neg SLE patients with active disease relative to their AutoAb Pos counterparts, with APRIL being found to be highest in SLE patients with active disease (p<0.05). Other TNF superfamily members evaluated, including TRAIL ( Figure 4C ) and TNFRII( Figure 4D )) were also increased (p<0.0001). Given that these two mediators are also associated with disease activity to some extent (Table 4), TNF superfamily members may contribute to the generation of AutoAb and the enhancement of clinical disease activity.

[0148] Previous studies by the inventors have shown that other intrinsic and adaptive mediators are altered in the pathogenesis of SLE and exacerbate clinical disease (36, 42, 45). The inventors observed that IL-1a ( Figure 5A , p<0.01) and IL-6 ( Figure 5B , p<0.05) were increased in AutoAb Pos SLE patients with low-grade or active disease. In addition, IL-1a differentiated low-grade from active disease in AutoAb Pos SLE patients ( Figure 5A , p = 0.0206). In AutoAb Pos SLE patients with active disease, including Th1 type mediator IL-2 ( Figure 5C ) and Th17 mediator IL-21 ( Figure 5D ) were also significantly increased (p<0.05). Other inflammatory mediators were also increased, including SCF ( Fig. 6A , p=0.0356) and IL-8 ( Figure 6B , p<0,0001). Although the plasma level of IL-10 ( Figure 6C ) was highest in AutoAb Neg SLE patients with low disease activity, which may reflect its regulatory properties, but IL-10 levels were significantly different between AutoAb Neg and Pos SLE patients with active disease (p = 0.0089), with higher levels in AutoAb Pos SLE patients, suggesting that IL-10 may also function as a pro-inflammatory positive regulator of B lymphocyte activation and AutoAb production within its capacity (54, 55). Similarly, although TGF-β ( Fig.6D ) was highest in controls, reflecting suppressed regulation in SLE patients (36, 42, 45), but was higher in AutoAb Pos SLE patients relative to AutoAb Neg SLE patients with active disease (p=0.0066). This may reflect that TGF-β binds to IL-6 when secreted ( Figure 5B ) has the ability to drive a pro-inflammatory Th17-type response (56, 57).

[0149] The weighted Lupus Disease Activity Immune Index (LDAII) distinguishes active disease in SLE patients. Given the significant alterations in multiple immune pathways associated with active clinical disease in SLE patients, the inventors developed the Lupus Disease Activity Immune Index (LDAII) to summarize the immune dysregulation of individual patients, thereby comparing low-grade and active disease in SLE patients relative to the immune profile found in healthy individuals or controls (Ctls), Fig. 7A and 7B. LDAII was calculated using log-transformed, standardized levels of each soluble mediator that were weighted based on their correlation with the number of SLE-associated AutoAb specificities detected. The sum of the weighted, log-transformed, standardized levels of each analyte produced a global LDAII (see Materials and Methods for details). A clear advantage of this approach is that it does not require a cutoff for each cytokine / chemokine to establish positivity and affects those untransformed mediators that have a stronger (Spearman) correlation with the accumulation of AutoAb specificities that will be found in SLE patients with active disease. The LDAII (calculated as the sum of the subscores contributed by each soluble mediator) was significantly higher in SLE patients with active clinical disease compared to SLE patients with low disease activity and control samples ( Fig. 7A ). Including healthy individuals in the algorithm allowed for optimal differentiation of SLE patients with low disease activity. Importantly, LDAII correlated significantly with the hSLEDAI score (r=0.226, p<0.0001), distinguishing SLE patients with active disease or low disease activity from controls (AUC=0.671, p=0.0001), and resulted in a positive predictive value (PPV) of 0.94, with a specificity of 79% and a sensitivity of 49%, Figure 7B These data suggest that additional immune dysregulation is associated with clinical disease activity and that this heterogeneous dysregulation can be exploited to shape LDAII with the potential to impact clinical care.

[0150] LDAII differentiates SLE patients with clinically and serologically active and quiescent disease. In addition to assessing differences in immune dysregulation based on clinical disease activity (hSLEDAI) scores, the inventors also evaluated whether LDAII could differentiate clinically active (CA) from quiescent (CQ) disease, and serologically active (SA) from quiescent (SQ) disease, defined as meeting immunological disease activity criteria (anti-dsDNA binding to AutoAb and / or low complement levels) (14, 15) relative to controls. Figures 8A to 8F ). Those SLE patients with clinically active disease (CASA) had significantly higher LDAII scores than those with clinically quiescent disease (CQSQ). Both SLE patient groups were significantly different from controls ( Fig. 8A ), with a PPV of 0.88, a specificity of 79%, and a sensitivity of 71% (AUC = 0.804 [p < 0.0001]), Figure 8BThese data suggest that an immune index informed by multiple heterogeneously altered immune pathways in SLE patients can identify patients with increased clinical disease activity. In addition, the inventors observed increased levels of mediators in CASA SLE patients (p<0.01), including IL-6 ( Figure 8C )、IL-8( Fig.8D ) and IP-10( Fig. 8E ), and an increased number of SLE-related AutoAb specificities ( Figure 8F Interestingly, despite being clinically and serologically quiescent, many CQSQ patients were positive for at least one AutoAb specificity and had elevated inflammatory mediators compared to controls.

[0151] LDAII distinguishes SLE patients with renal manifestations of disease activity. Chronic immune dysregulation, including the production of pathogenic autoantibodies and increased levels of inflammatory mediators, leads to progressive end-organ damage. Renal damage and lupus nephritis are the most serious sequelae of SLE and contribute largely to SLE-related morbidity and mortality (58). Early detection and treatment of renal damage are necessary to minimize the risk of permanent organ damage and protect renal function. The inventors evaluated whether LDAII can distinguish SLE patients with renal involvement ( Figures 9A to 9E ), which is present in SLE patients with active disease (Table 3). The inventors found that the LDAII scores of SLE patients with active disease and renal manifestations were significantly higher than those of SLE patients with active disease without renal manifestations (p=0.0099). Fig. 9A SLE patients with active disease, with (p = 0.0002) or without (p = 0.0359) renal manifestations, had higher LDAII scores than SLE patients with low disease activity ( Fig. 9A ), with an NPV of 0.96, a specificity of 49%, and a sensitivity of 88% (AUC = 0.689 [p = 0.0029]), Fig. 9B SCF ( Fig. 9C ) and TNFRII( Fig.9D ) and the number of AutoAb specificities ( Fig.9E ) were increased in both groups (vs. non-renal / active disease, p<0.001; vs. low disease activity follow-up, p<0.01). In addition, SLE patients with active disease but no renal manifestations had more SLE-related AutoAb specificity than SLE patients with low disease activity (p=0.0261, Fig.9EThese data suggest that the use of immune dysregulation to provide information about LDAII can be used to identify SLE patients with renal manifestations who would likely benefit from closer clinical monitoring to prevent the development, morbidity, and early mortality caused by lupus nephritis and end-stage renal disease (ESRD).

[0152] Persistent active disease is a significant burden on SLE patients and impacts quality of life (59), even for those with long-term disease (19). Disease activity has been shown to increase over time, and most patients with low disease activity will progress to active disease (19), which is associated with organ damage and early mortality (24, 25, 60) and highlights gaps in the optimal management of SLE. The ultimate goal is to move patients toward low disease activity, which has been shown to improve outcomes and prognosis (61), with fewer organ manifestations and less permanent organ damage, fewer treatments with significant side effects and morbidity, and lower mortality.

[0153] Identification and early treatment to prevent tissue and organ damage is challenging because signs and symptoms of disease activity are not captured until after they develop. In addition, chronic use of steroids (23) and other immunosuppressants required to manage disease activity (27) is associated with increased morbidity. The inability to proactively manage clinical disease limits medical care to reactive treatments, hampering proactive strategies (28) to add or augment steroid-sparing immunomodulators to prevent end-organ damage (6-8) and reduce the pathogenic and socioeconomic burden of SLE (29). The inventors have previously demonstrated that dysregulation of multiple immune pathways (36, 37), along with the accumulation of SLE-associated AutoAb specificities (36, 37, 50), underlie the development and transformation of categorical SLE. The inventors have further demonstrated that additional immune dysregulation occurring prior to clinical disease flare-ups and the heterogeneity of immune pathway dysregulation and clinical disease can be exploited by the advent of a lupus flare prediction index (42, 45). Interestingly, this index does not provide information on concurrent clinical disease activity (42, 45).

[0154] The inventors sought to identify potential immune disorders that reflect ongoing clinical disease activity and use this information to provide information that complements current clinical disease activity instruments for immune indices. In the current study, the inventors observed heterogeneity in the number and type of immune pathways that were altered in samples from SLE patients with active versus low disease activity. This may partially explain the previously reported variability of inflammatory mediators in SLE patients with active disease (62-64), as well as the inconsistent correlation with proposed serological markers of disease activity alone or in combination, and their limited clinical utility, including anti-dsDNA, complement, complement split products, and inflammatory markers (ESR and CRP) (65-67). Despite the heterogeneity in immune pathway involvement, each patient demonstrated an increase in inflammatory mediators from at least one pathway. Given the well-described IFN signature present in SLE patients, it is not surprising that IFN-related mediators were affected, including changes in type I IFN (IFN-α), type II IFN (IFN-γ), and IFN-related chemokines (IP-10) (68). IFN-related mediators were directly affected in a manner that correlated with disease activity (low vs. active, clinically / serologically quiescent vs. active), as well as with the presence of SLE-associated AutoAbs in patient subgroups. This finding is supported by previous studies showing that the IFN pathway can reflect disease activity (69), depending on the presence of AutoAbs in patient subgroups (70), but cannot universally explain concurrent clinical disease activity (71).

[0155] There are a variety of mediators that are not directly associated with disease activity measures, but rather with accumulation of AutoAb specificity, and are able to distinguish low-grade from active disease clinical follow-ups within AutoAb positive and negative SLE patients. Of note in this category are the TNF superfamily mediators BLyS and APRIL (72-74), which promote B lymphocyte activation and AutoAb production. BLyS levels can result from an interaction between early dysregulation of type I (51) and type II (52) IFN mediators and accumulation of lupus-associated autoantibodies specificity (37). The inventors observed that, similar to IFN-related mediators, BLyS as well as APRIL distinguished AutoAb negative from positive SLE patients with active disease (Figures 3-4). However, APRIL was also able to distinguish disease activity (low versus active) in AutoAb negative patients (activity < low disease activity) and AutoAb positive patients (activity > low disease activity). This divergence with BLyS in some cases (AutoAb-negative samples) and convergence in other cases (AutoAb-positive samples) is supported by other studies in SLE (72, 75-78) and may be explained by the unique and overlapping receptors utilized by BLyS and APRIL (72, 73, 79-82), those receptors / pathways shared by BLyS and APRIL may lead to convergent downstream events (AutoAb production), while divergent receptor signaling pathway mechanisms (e.g., APRIL-TACI interactions (80)) may contribute to the distinct role of APRIL in negative regulation, as seen by the inventors in AutoAb-negative SLE samples ( Figures 4A to 4D ).

[0156] Another pair of soluble mediators that appear to have dual anti-inflammatory and pro-inflammatory functions relative to lupus disease activity are IL-10 and TGF-β. Both mediators have been shown to have regulatory functions (83-88), and the inventors found that IL-10 was highest in AutoAb-negative SLE patients with low disease activity ( Figures 6A to 6D ), while TGF-β was highest in healthy controls, suggesting a loss of regulation in SLE patients (87, 88). In contrast, IL-10 and TGF-β levels were increased in AutoAb-positive (vs.-negative) samples from SLE patients with active disease ( Figures 6A to 6D IL-10 has been shown to have pro-inflammatory properties with respect to B lymphocyte activation and AutoAb production (89), while TGF-β has been shown to promote a Th17-type response in the presence of IL-6 (90, 91), which leads to IL-21 secretion and stimulation of B lymphocytes, thereby promoting AutoAb production and the pathogenesis of SLE (92, 93).

[0157] These immune system changes, combined with the lessons learned from the Lupus Flare Prediction Index (42, 45), led us to develop the Lupus Disease Activity Immune Index (LDAII), which provides information through changes in soluble mediators and is weighted by its relevance to SLE-related AutoAb accumulation. The time and need for specialized training to minimize the variability in disease activity assessment (31, 35) usually limit the use of validated SLE disease activity measurements in routine clinical practice. Detection of immune system changes associated with ongoing clinical disease activity for providing information on LDAII will allow identification of patients who need closer monitoring and enable early intervention with immune modification therapy to prevent end-organ damage (6-8). Positive scores will indicate that more frequent monitoring (94) and / or drug replacement is needed before the onset of new or worsening clinical manifestations to tamper with ongoing inflammation. Negative scores would indicate that ongoing monitoring and medication schedules are being maintained (they are working), that less frequent monitoring is needed (especially if follow-up is at least quarterly), and / or that tapering of steroids (23) or other immunomodulators with significant side effects (27) is considered. SLE patients who are more actively involved in their clinical care have been shown to have less permanent organ damage (95). The LDAII allows monitoring of a patient's immune activity (not including clinical disease activity) and alerts them and their healthcare providers to the need for further clinical evaluation.

[0158] Clinically quiescent SLE patients are at risk for increased disease activity and must be monitored regularly (14). Although SLE patients with active disease are more likely to meet hSLEDAI serological criteria (increased DNA binding and / or hypocomplementemia) and be positive for anti-dsDNA AutoAbs (Table 3), none of these factors have been shown to be predictors of increased clinical disease activity (14, 42, 45). Importantly, LDAII was able to distinguish patients with clinically and serologically quiescent (CQSQ) from active (CASA) disease by using the larger differences in IL-6, IL-8, and IP-10 levels and the number of SLE-associated AutoAbs between CQSQ and CASA SLE patients compared to using only the hSLEDAI disease activity score (Figures 1-2). Figures 8A to 8F ). In addition, LDAII was able to differentiate SLE patients with active disease who presented with renal manifestations ( Figures 9A to 9E). The present invention allows analysis of pathway-specific immune dysregulation to enable personalized precision medicine for SLE patients with renal manifestations and lupus nephritis (LN). In addition, the present invention allows identification of individuals at greatest risk for developing LN and definition of measures of pathway-specific immune dysregulation to select the most appropriate LN patients for a given pathway-specific biological therapy. Two mediators that are altered in the current evaluation of SLE patients with renal involvement are SCF and TNFRII ( Figures 9A to 9E ). SCF and its interaction with the receptor c-kit have previously been shown to promote kidney damage, including glomerulonephritis and renal failure (96, 97). TNFRII (98-100), which is normally present on lymphocytes, is abnormally upregulated in the context of chronic inflammation, including in the kidney, thereby promoting inflammation, kidney damage, and renal failure (101-104).

[0159] The ability to detect changes in immune status as a reflection of clinical disease activity allows for improved disease monitoring and treatment, which can improve patient outcomes and reduce the pathogenicity and socioeconomic burden of SLE (29). A particular technical effect of the present invention is the advantage of calculating a patient's LDAII, which does not require a cutoff value for each soluble mediator to establish positivity, and / or does not require prior knowledge of the inflammatory pathways that promote disease activity in a particular patient. The present invention can be further validated in prospective multiethnic studies in SLE clinical trials and disease management. Depending on the comprehensive clinical signs of the individual patient, early detection of enhanced clinical disease activity and risk of organ damage promotes closer monitoring, selection of one or more preventive treatments, or inclusion of targeted biology associated with pathways altered within LDAII in clinical trials.

[0160] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent or composition of the invention, and vice versa.In addition, the compositions of the invention can be used to implement the methods of the invention.

[0161] It should be understood that the specific embodiments described herein are shown by way of illustration and are not limitations of the present invention. The main features of the present invention can be applied in various embodiments without departing from the scope of the present invention. Those skilled in the art will recognize or simply use routine experiments to determine many equivalents of the specific procedures described herein. Such equivalents are considered to be within the scope of the present invention and are covered by the claims.

[0162] All publications and patent applications mentioned in this specification are indicative of the technical levels of those skilled in the art to which the invention pertains. All publications and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0163] When used in conjunction with the term "comprising" in the claims and / or specification, the use of the wording "a" or "an" may mean "one", but it also complies with the meanings of "one or more", "at least one", and "one or more than one". Unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive, the use of the term "or" in the claims is used to mean "and / or", but the present disclosure supports definitions referring only to alternatives as well as "and / or". Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method used to determine the value, or the variation that exists among the study subjects.

[0164] As used in this specification and claims, the terms "comprising" (and any form of comprising, such as "comprise and comprises"), "having" (and any form of having, such as "have and has"), "including" (and any form of including, such as "includes and include"), or "containing" (and any form of containing, such as "contains and contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In any of the embodiments of the compositions and methods provided herein, "comprising" may be replaced with "consisting essentially of" or "consisting of". As used herein, the phrase "consisting essentially of" requires the specified integers or steps and those that do not materially affect the characteristics or functions of the claimed invention. As used herein, the term "consisting of" is used to indicate the presence of only a recited integer (e.g., a feature structure, element, characteristic, property, method / process step, or limitation) or group of integers (e.g., one or more feature structures, elements, characteristics, properties, method / process steps, or limitations).

[0165] As used herein, the term "or a combination thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or a combination thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular case, then also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, explicitly included are combinations containing repetitions of one or more items or terms, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc. The skilled artisan will appreciate that, unless otherwise apparent from the context, there is generally no limit to the number of items or terms in any combination.

[0166] As used herein, approximating words, such as but not limited to "about," "substantial," or "substantially," refer to conditions that, when so modified, are understood not to be necessarily absolute or perfect, but rather will be considered close enough to one of ordinary skill in the art to warrant designating the condition as existing. The degree to which the description may vary will depend on how much change can be made and still allow one of ordinary skill in the art to recognize the modified feature structure as still having the desired characteristics and capabilities of the unmodified feature structure. In general, but subject to the foregoing discussion, numerical values ​​modified herein by approximating words such as "about" may vary from the specified value by at least ±1%, 2%, 3%, 4%, 5%, 6%, 7%, 10%, 12%, or 15%.

[0167] All compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the compositions and / or methods described herein, as well as the steps or order of steps of the methods described herein, may be varied without departing from the concept, spirit and scope of the present invention. It will be apparent to those skilled in the art that all such similar substitutions and modifications are deemed to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0168] To assist the Patent Office and any reader of any patent issuing from this application in interpreting the claims appended hereto, applicants wish to note that they do not intend to use any of the appended claims to invoke 35 U.S.C. § 112, paragraph 6, 35 U.S.C. § 112, paragraph (f), or equivalent as it existed on the filing date hereof unless the phrase “means for” or “step for” is expressly used in a particular claim.

[0169] For each claim, each dependent claim may be dependent on the independent claim and may be dependent on each and every preceding dependent claim of each and every claim, provided that the preceding claim provides a proper antecedent basis for the claim item or element.

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Claims

1. Use of a reagent for detecting the level of one or more biomarkers in a blood, serum, plasma or urine sample from each of (b) to (g) in the preparation of a kit for use in a method for characterizing disease activity of SLE in a patient with systemic lupus erythematosus, the method comprising: (a) obtaining a dataset associated with a blood, serum, plasma or urine sample from the patient, wherein the dataset comprises data representing the level of one or more biomarkers in the blood, serum, plasma or urine sample from each of (b) to (g); (b) assessing said dataset for the presence or amount of protein expression of at least one intrinsic serum or plasma mediator biomarker selected from the group consisting of: IL-1α, IL-1β, IL-1RA, IFN-α, IL-12p70, IL-6, and IL-23pl9; (c) assessing said dataset for the presence or amount of protein expression of at least one adaptive serum or plasma mediator biomarker selected from the group consisting of: IL-2, IFN-γ, IL-5, IL-13, IL-17A, IL-21, IL-10, and TGF-β; (d) assessing said data set for the presence or amount of at least one chemokine or adhesion molecule biomarker selected from the group consisting of IL-8, IP-10, MIG, MIP-1α, MIP-1β, MCP-1, MCP-3, and ICAM-1; (e) assessing said dataset for the presence or amount of at least one soluble TNF superfamily biomarker selected from the group consisting of: TNFRI, TNFRII, TRAIL, TWEAK, CD40L, BLyS, and APRIL; (f) assessing the data set for the presence or amount of the inflammatory mediator biomarker SCF; (g) assessing said dataset for the presence or amount of at least one SLE-associated autoantibody-specific biomarker selected from the group consisting of: dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP; and (h) Calculate the lupus disease activity immune index LDAII score; The data set is subjected to: logarithmic transformation; normalized; weighted by Spearman r correlation with SLE-related autoantibodies specific; and the sum of the soluble mediators equals the Lupus Disease Activity Immune Index LDAII score; wherein the biomarkers in (b) to (f) are the soluble mediators, and wherein the LDAII is calculated as follows: The concentration of the soluble mediator is determined for the subject and log-transformed, and each log-transformed soluble mediator level determined for the subject's sample is normalized as follows: (Observed value)-(mean value of all SLE patients and healthy controls followed up) / (standard deviation of all SLE patients and healthy controls followed up); generating Spearman coefficients from a linear regression model testing the correlation coefficients Spearmanr between one or more SLE-associated autoantibodies specific for each soluble mediator assessed in the SLE patients compared to healthy controls; transforming and normalizing the values ​​of the soluble mediator levels of the subjects, and weighting the resulting values ​​by multiplying them by their corresponding Spearman r; and For each participant visit, the log-transformed, normalized, and weighted values ​​for each of the soluble mediators were summed to calculate the LDAII.

2. The method of claim 1, wherein 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39 biomarkers are used in the calculation of the LDAII.

3. The use according to claim 1, wherein the performance of at least one immunoassay comprises: obtaining a first sample, wherein the first sample comprises a protein marker; contacting the first sample with a plurality of different reagents; generating a plurality of different complexes between the reagent and the marker; and detecting the complexes to generate data.

4. The use of claim 1, wherein at least one immunoassay comprises a multiplex assay.

5. The use of claim 1, wherein the LDAII classifies the severity or level of progression of SLE into clinically active or quiescent disease, and the disease is serologically active or serologically quiescent disease, wherein the serology includes dsDNA binding and low complement.

6. The use of claim 1, wherein the LDAII score distinguishes between active and low lupus disease activity.

7. The use of claim 1, further comprising administering treatment to the patient after determining the patient's prognosis as active lupus disease activity, before reaching active lupus disease activity, wherein the treatment comprises a disease-modifying antirheumatic drug.

8. The use of claim 7, wherein the disease-modifying anti-rheumatic drugs include nonsteroidal anti-inflammatory drugs and steroids.

9. The method of claim 8, wherein the nonsteroidal anti-inflammatory drug is hydroxychloroquine.

10. The use according to claim 8, wherein the nonsteroidal anti-inflammatory drug is belimumab.

11. Use of reagents for performing at least one immunoassay on a sample from a patient in the preparation of a kit for use in a method for assessing disease activity and progression of a clinical disease of systemic lupus erythematosus (SLE) in said patient, the method comprising: obtaining a blood, serum, plasma or urine sample from the patient; performing at least one immunoassay on a sample from the patient to generate a dataset comprising at least one biomarker from each of (1) to (6): (1) assessing the presence or amount of protein expression of at least one intrinsic serum or plasma mediator biomarker selected from the group consisting of IL-1α, IL-1β, IL-1RA, IFN-α, IL-12p70, IL-6, and IL-23pl9; (2) assessing said dataset for the presence or amount of protein expression of at least one adaptive serum or plasma mediator biomarker selected from the group consisting of: IL-2, IFN-γ, IL-5, IL-13, IL-17A, IL-21, IL-10, and TGF-β; (3) assessing the data set for the presence or amount of at least one chemokine or adhesion molecule biomarker selected from the group consisting of IL-8, IP-10, MIG, MIP-1α, MIP-1β, MCP-1, MCP-3, and ICAM-1; (4) assessing said data set for the presence or amount of at least one soluble TNF superfamily biomarker selected from: TNFRI, TNFRII, TRAIL, TWEAK, CD40L, BLyS, and APRIL; (5) said dataset assessing the presence or amount of the inflammatory mediator biomarker SCF; and (6) assessing the data set for the presence or amount of at least one SLE-associated autoantibody-specific biomarker selected from the group consisting of: dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP; as well as Calculate the LDAII score; The data set is subjected to: logarithmic transformation; normalized; weighted by Spearman r correlation with SLE-related autoantibodies specific; and the sum of the soluble mediators equals the Lupus Disease Activity Immune Index LDAII score; wherein the biomarkers in (1) to (5) are the soluble mediators, and wherein the LDAII is calculated as follows: The concentration of the soluble mediator is determined for the subject and log-transformed, and each log-transformed soluble mediator level determined for the subject's sample is normalized as follows: (Observed value)-(mean value of all SLE patients and healthy controls followed up) / (standard deviation of all SLE patients and healthy controls followed up); generating Spearman coefficients from a linear regression model testing the correlation coefficients Spearmanr between one or more SLE-associated autoantibodies specific for each soluble mediator assessed in the SLE patients compared to healthy controls; transforming and normalizing the values ​​of the soluble mediator levels of the subjects, and weighting the resulting values ​​by multiplying them by their corresponding Spearman r; and For each participant visit, the log-transformed, normalized, and weighted values ​​for each of the soluble mediators were summed to calculate the LDAII.

12. The method of claim 11, wherein 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39 biomarkers are used in the calculation of the LDAII.

13. The use of claim 11, wherein the performance of at least one immunoassay comprises: obtaining a first sample, wherein the first sample comprises a protein marker; contacting the first sample with a plurality of different reagents; generating a plurality of different complexes between the reagent and the marker; and detecting the complexes to generate data.

14. The use of claim 11, wherein at least one immunoassay comprises a multiplex assay.

15. The use of claim 11, wherein the LDAII classifies the severity or level of progression of SLE into clinically active or quiescent disease, the disease being serologically active or serologically quiescent, wherein the serology includes dsDNA binding and low complement.

16. The use of claim 11, wherein the LDAII score distinguishes between active and low lupus disease activity.

17. The use of claim 11, further comprising administering a treatment to the SLE patient after determining the patient's prognosis as active lupus disease activity, prior to reaching active lupus disease activity, wherein the treatment comprises a disease-modifying antirheumatic drug.

18. The use of claim 17, wherein the disease-modifying anti-rheumatic drugs include nonsteroidal anti-inflammatory drugs and steroids.

19. The method of claim 18, wherein the nonsteroidal anti-inflammatory drug is hydroxychloroquine.

20. The use according to claim 18, wherein the nonsteroidal anti-inflammatory drug is belimumab.

21. The use of claim 11, wherein obtaining a first data set associated with the sample comprises obtaining the sample and processing the sample to experimentally determine the first data set, or wherein obtaining the first data set associated with the sample comprises receiving the first data set from a third party that has processed the sample to experimentally determine the first data set.

22. The use of claim 11, wherein the increase in the SCF, TNFRII and MCP-1 biomarkers indicates renal organ involvement.

23. Use of a reagent for measuring the expression level of a set of biomarkers in a subject in the preparation of a kit for use in a method for calculating a lupus disease activity immune index LDAII, the method comprising: Determining biomarker measurements of a panel of biomarkers in a physiological sample by immunoassay, wherein the biomarkers are peptides, proteins, peptides carrying post-translational modifications, or proteins carrying post-translational modifications, or a combination thereof; wherein the physiological sample is whole blood, plasma, serum, or a combination thereof; wherein the biomarker panel comprises a data set of measurements of at least one of the categories of biomarkers selected from: a data set of the presence or amount of protein expression of at least one intrinsic serum or plasma mediator biomarker selected from the group consisting of: IL-1α, IL-1β, IL-1RA, IFN-α, IL-12p70, IL-6, and IL-23pl9; a dataset of the presence or amount of protein expression of at least one adaptive serum or plasma mediator biomarker selected from the group consisting of: IL-2, IFN-γ, IL-5, IL-13, IL-17A, IL-21, IL-10, and TGF-β; a data set for the presence or amount of at least one chemokine or adhesion molecule biomarker selected from the group consisting of IL-8, IP-10, MIG, MIP-1α, MIP-1β, MCP-1, MCP-3, and ICAM-1; a data set for the presence or amount of at least one soluble TNF superfamily biomarker selected from the group consisting of: TNFRI, TNFRII, TRAIL, TWEAK, CD40L, BLyS, and APRIL; The presence or amount of the inflammatory mediator biomarker SCF; and a data set for the presence or amount of at least one SLE-associated autoantibody-specific biomarker selected from the group consisting of: dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP; as well as The LDAII score was calculated whereby the dataset was: logarithmically transformed; Normalized; weighted by Spearman r correlation with SLE-related autoantibodies specific in a second data set, and the sum of soluble mediators equals the LDAII score; wherein the intrinsic serum or plasma mediator biomarkers, the adaptive serum or plasma mediator biomarkers, the chemokine or adhesion molecule biomarkers, the soluble TNF superfamily biomarkers and the inflammatory mediator biomarker SCF are the soluble mediators; The LDAII is calculated as follows: The concentration of the soluble mediator for the subject is determined and log-transformed, and each log-transformed soluble mediator level determined for the subject's sample is normalized as follows: (Observed value)-(mean value of all SLE patients and healthy controls followed up) / (standard deviation of all SLE patients and healthy controls followed up); generating Spearman coefficients from a linear regression model testing the correlation coefficients Spearmanr between one or more SLE-associated autoantibodies specific for each soluble mediator assessed in the SLE patients compared to healthy controls; transforming and normalizing the values ​​of the soluble mediator levels of the subjects, and weighting the resulting values ​​by multiplying them by their corresponding Spearman r; and For each participant visit, the log-transformed, normalized, and weighted values ​​for each of the soluble mediators were summed to calculate the LDAII.

24. The use of claim 23, wherein the method further comprises using the set of biomarker measurements in a classification system to classify the sample as clinically active or quiescent disease relative to the presence or development of systemic lupus erythematosus (SLE) in the subject, wherein the disease is serologically active or serologically quiescent disease, wherein the serology includes dsDNA binding and low complement, and wherein the classification system is a machine learning system comprising classification and regression trees selected from the group consisting of: Fisher's exact test, Mann-Whitney test, Kruskal-Wallis test, Kruskal-Wallis test with Dunn's multiple comparisons or Spearman rank correlation, or a combination thereof; and The lupus disease activity immune index LDAII is calculated, wherein the LDAII score distinguishes active SLE from low SLE disease activity, wherein the low SLE disease activity is a low clinical disease with SLEDAI<4.

25. The method of claim 23, further comprising distinguishing clinically and serologically quiescent SLE patients from healthy controls.

26. The method of claim 23, further comprising determining the amount of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39 biomarkers used in the calculation of the LDAII.

27. The use according to claim 23, wherein the immunoassay is a multiplex immunoassay.

28. The use of claim 23, wherein the increase in LDAII indicates at least one of: SLE disease progression, increased autoimmune disease activity, or organ damage.

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