Biomarkers for a systemic lupus erythematosus (SLE) disease activity immune index that characterizes disease activity

Abstract

Methods for characterizing disease activity in a systemic lupus erythematosus (SLE) patient. Methods include obtaining a blood, serum, plasma, or urine sample from the patient; assessing the sample for expression of a biomarker selected from the group consisting of IFN-α, IL-10, BLyS, IL-7, IFN-γ, TRAIL, IL-15, IP-10 / CXCL10, and IL-4; assessing the sample for expression of an inflammatory mediator selected from the group consisting of TNFRII, Resistin, and Osteopontin (OPN); assessing the sample for an SLE-associated autoantibody specificity biomarker selected from the group consisting of dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP; and calculating a Lupus Disease Activity (Immune) Index (LDAII / L-DAI) score. The LDAII / L-DAI score may distinguish between active and low lupus disease activity. Methods of treatment are also provided including administering a treatment prior to reaching clinical disease classification after determining that the patient has the prognosis for transitioning to classified SLE.

Description

Attorney Docket No.82092-000005-WO-POA BIOMARKERS FOR A SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) DISEASE ACTIVITY IMMUNE INDEX THAT CHARACTERIZES DISEASE ACTIVITY GOVERNMENT LICENSE RIGHTS

[0001] This invention was made with government support under AI142967, AR073750, AI144292, and GM104938 awarded by the National Institutes of Health. The government has certain rights in the invention. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit and priority to U.S. Provisional Application No. 63 / 533,721 filed August 21, 2023, which is incorporated here by reference in its entirety. TECHNICAL FIELD

[0003] The subject matter relates generally to the field of biomarkers for calculating a lupus disease activity immune index that characterizes disease activity in Systemic Lupus Erythematosus (SLE). BACKGROUND

[0004] Without limiting the scope of the invention, its background is described in connection with Systemic Lupus Erythematosus (SLE).

[0005] Systemic autoimmune diseases, including SLE, afflict a significant proportion of the US population. Recent population-based studies reflect a prevalence of 73 / 100,000 (Lim et al., 2014; Somers et al., 2014), while the Lupus Foundation of America estimates the number of possible SLE patients to be as high as 470 / 100,000 in routine clinical practice. SLE presents with a constellation of clinical symptoms; disease classification is contingent on meeting 4 of 11 American College of Rheumatology (ACR) criteria (Hochberg, 1997; Tan et al., 1982), 4 of 11 Systemic Lupus International Collaborating Clinics (SLICC) criteria (Petri, Orbai, et al., 2012), or ANA positivity (g1:80 titer by HEp-2 IIF assay), one clinical criterion and g10 points (from 7 clinical and 3 immunology domains) from the European League Against Rheumatism (EULAR) / ACR (Aringer et al., 2019) classification criteria for SLE. More than 90% of affected patients are women aged 15-45. Prevalence is higher in minority populations and with lower socioeconomic status (Feldman et al., 2013). Persistently active clinical disease and its treatment place patients at risk for organ damage (Kasitanon et al., 2015; Oglesby et al., 2014; Thong & Olsen, 2017), including central nervous system, pulmonary, cardiovascular, and renal damage (Barral et al., 2009; Bruce et al., 2015; Conti et al., 2016; Faurschou et al., 2006), lupus nephritis,Attorney Docket No.82092-000005-WO-POA and end-stage renal disease (Alarcon et al., 2006; Faurschou et al., 2006). Patients with waxing / waning disease and clinically active or quiescent disease are each at risk of clinical disease flare (Ng et al., 2006; Steiman et al., 2010).

[0006] Systemic lupus erythematosus (SLE) is a clinically and serologically heterogeneous systemic autoimmune disease that causes significant morbidity and early mortality, especially in young women and minorities. Immune dysregulation in the form of pathogenic autoantibodies and chronic inflammation contributes to a wide range of clinical manifestations, including skin rashes, arthritis, and life-threatening renal and / or central nervous system damage. A number of antinuclear autoantibody (ANA) specificities have been shown to accumulate in SLE patients; use of hydroxychloroquine may abrogate autoantibody accumulation and offset clinical disease activity. Early intervention is an attractive approach to SLE treatment. However, our understanding of pathogenic mechanisms in SLE disease activity is inadequate. Closing this knowledge gap would improve our ability to identify individuals at risk of increased disease activity and permanent organ damage, define windows of opportunity for early intervention, and facilitate the development of pathway-targeted treatments.

[0007] Recognition and early treatment to prevent tissue and organ damage is challenging, as signs and symptoms of high disease activity are captured after their occurrence. Despite validated clinical disease activity instruments (Hay et al., 1993; Lam & Petri, 2005; Petri et al., 2005) and improved treatment strategies, persistently active disease remains a burden for SLE patients (Peschken et al., 2018). Increased morbidity and early mortality associated with treatment required to manage active disease, in particular steroids (Al Sawah et al., 2015; Sheane et al., 2017; Thamer et al., 2009; Zonana-Nacach et al., 2000), as well as permanent organ damage (Lopez et al., 2012; Petri, Purvey, et al., 2012), including renal damage (Maroz & Segal, 2013), further escalates costs. In addition, long-term use of steroids (Al Sawah et al., 2015) and other immune-suppressants (Durcan & Petri, 2016) required to manage disease activity are associated with increased morbidity. The inability to proactively manage clinical disease limits medical care to reactive treatment, precluding proactive strategies of adding or increasing steroid-sparing immune modifying agents (Doria et al., 2014) to prevent end-organ damage (Kasitanon et al., 2015; Oglesby et al., 2014; Thong & Olsen, 2017) and reduce the pathogenic and socioeconomic burdens of SLE (Lau & Mak, 2009).

[0008] Current biomarkers in SLE have limited utility for forecasting 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 persistent active disease and progression to permanent organ damage. ANAs are also found in sera fromAttorney Docket No.82092-000005-WO-POA patients with other systemic rheumatic diseases, and from 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 may remain healthy despite being ANA-positive, ANA positivity alone is likely not the sole pathogenic driver of SLE. In addition to ANA positivity, the dysregulation of various immune pathways driven by soluble mediators may contribute to the development of clinical disease. No single factor or mechanism is likely sufficient to explain the complexity and heterogeneity of SLE pathogenesis; thus, a multivariate, longitudinal approach is warranted to delineate mechanisms of early disease pathogenesis and discern unique parameters that forecast SLE disease activity.

[0009] Despite clinical trials of a number of directed immune pathway treatments, including the first FDA-approved drug for SLE in over 50 years, Belimumab (Manzi et al., 2012), the vast majority of these studies fail, in part due to lack of understanding the immune pathways dysregulated in a given patient. The need for immune-informed biomarkers as surrogate endpoints for clinical disease activity is becoming more pressing. Administrative burden limits the use of validated SLE clinical disease activity measures in routine practice (Mikdashi & Nived, 2015). Validated disease activity instruments, such as the currently used hybrid Systemic Lupus Erythematosus Disease Activity Index ( ) (Thanou et al., 2014; Thanou et al., 2016) and the British Isles Lupus Assessment Group (BILAG) index (Hay et al., 1993), are labor intensive and require ongoing, specialized training as these clinical instruments are updated (Isenberg et al., 2005). Relying solely on physician experience to assess clinical disease activity carries the risk of unwanted variability and negative outcomes (Mikdashi & Nived, 2015; Mosca et al., 2011).

[0010] Clinical heterogeneity in SLE underlies the scientific premise that: heterogeneic immune dysregulation underlies clinical disease activity. The inventors have previously shown that patients exhibit immune dysregulation prior to the onset of clinical SLE, amplified in a feed-forward mechanism as patients suffer tissue damage, develop clinical sequelae, and ultimately reach disease classification (Lu et al., 2016; Munroe et al., 2016). The inventors also described the accumulation of multiple SLE-associated autoantibodies (AutoAbs) and dysregulated inflammatory and regulatory immune pathways (Lu et al., 2016; Munroe et al., 2016). To date, however, the lack of an immune mechanism-informed disease management test in SLE stems from no individual immune pathway-informed biomarker acting as a universal surrogate for either concurrent or future clinical disease activity (Arriens et al., 2017).

[0011] Thus, a need remains for novel methods for detecting, tracking, and evaluating disease activity and progression of SLE.Attorney Docket No.82092-000005-WO-POA BRIEF SUMMARY

[0012] In one embodiment, the present invention includes a method for characterizing disease activity in a systemic lupus erythematosus (SLE) patient, 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 (e); (b) assessing the dataset for a presence or an amount of protein expression of nine serum or plasma mediator biomarkers: IFN-α, IL-10, B-lymphocyte stimulating factor (BLyS or BAFF), IL-7, IFN-´, TRAIL, IL-15, IP-10 / CXCL10, and IL-4; (c) assessing the dataset for a presence or an amount of protein expression of at least one serum or plasma mediator biomarker selected from: TNFRII, Resistin, and Osteopontin (OPN); (d) assessing the dataset for a presence or an amount of at least one serum or plasma mediator biomarker selected from: biomarker selected from: IL-12p70, TNF-α, MIG / CXCL9, IL-5, IL-13, IL-1³, IL-17A, IL-2Rα, Native TGF-³, Fas, MCP-1 / CCL2, Stem Cell Factor (SCF), IL-1RA, IL-6, MIP-1α / CCL3, MIP- 1³ / CCL4, MCP-3 / CCL7, Total TGF-³, RANTES / CCL5, TNFRI, and IL-8 / CXCL8; (e) assessing the dataset for a presence or an amount at least one SLE-associated autoantibody specificity biomarker selected from: dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP; and (f) calculating a Lupus Disease Activity (Immune) Index (LDAII / L-DAI / L-DAI) score. In one aspect, at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 biomarkers are used in the calculation of the LDAII / L-DAI / L-DAI. In another aspect, the dataset is: log transformed; standardized; weighted by Spearman r correlation to the autoantibody specificities in the dataset, and a summation of soluble protein markers equals an LDAII / L-DAI / L-DAI score. Additionally or alternatively, in another aspect, the dataset is: log transformed; standardized; weighted by Spearman r correlation to the hSLEDAI in the dataset, and a summation of soluble protein markers equals an LDAII / L-DAI score. Additionally or alternatively, in another aspect, the dataset is: log transformed; standardized; weighted by an average Spearman r correlation to the autoantibody specificities and the hSLEDAI in the dataset, and a summation of soluble protein markers equals a composite LDAII / L-DAI / L-DAI score. In another aspect, the performance of the at least one immunoassay comprises: obtaining the first sample, wherein the first sample comprises the protein markers; contacting the first sample with a plurality of distinct reagents; generating a plurality of distinct complexes between the reagents and markers; and detecting the complexes to generate the data. In another aspect, at least one immunoassay comprises a multiplex assay. In another aspect, the LDAII / L-DAI / L-DAI divides a level of severity or progression of the SLE into clinically active (CA) or quiescent (CQ) disease that is either serologically (dsDNA binding and low complement) active (SA) or serologicallyAttorney Docket No.82092-000005-WO-POA quiescent (SQ). In another aspect, the LDAII / L-DAI / L-DAI score distinguishes between active and low lupus disease activity. In another aspect, the method further comprises administering a treatment to the patient prior to reaching clinically active disease, wherein the treatment comprises at least one of: hydroxychloroquine (HCQ), belimumab, a nonsteroidal anti- inflammatory drug, a steroid, or a disease-modifying anti-rheumatic drug (DMARD.

[0013] In another embodiment, the present invention includes a method of evaluating disease activity and progression of Systemic Lupus Erythematosus (SLE) clinical disease in a patient 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 for a presence or an amount of protein expression comprising nine serum or plasma mediator biomarkers: IFN-α, IL-10, B- lymphocyte stimulating factor (BLyS or BAFF), IL-7, IFN-´, TRAIL, IL-15, IP-10 / CXCL10, and IL-4, and at least one biomarker from each of (1) to (3): (1) assessing the dataset for a presence or an amount of protein expression of at least one innate serum or plasma mediator biomarker selected from: TNFRII, Resistin, and Osteopontin (OPN); (2) assessing the dataset for a presence or an amount of protein expression of at least one serum or plasma mediator biomarker selected from: IL-12p70, TNF-α, MIG / CXCL9, IL-5, IL-13, IL-1³, IL-17A, IL-2Rα, Native TGF-³, Fas, MCP- 1 / CCL2, Stem Cell Factor (SCF), IL-1RA, IL-6, MIP-1α / CCL3, MIP-1³ / CCL4, MCP-3 / CCL7, Total TGF-³, RANTES / CCL5, TNFRI, and IL-8 / CXCL8; and (3) assessing the dataset for a presence or an amount at least one SLE-associated autoantibody specificity biomarker selected from: dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP; and calculating an LDAII / L-DAI score. In one aspect, at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 biomarkers are used in the calculation of the LDAII / L- DAI. In another aspect, the dataset is: log transformed; standardized; weighted by Spearman r correlation to the autoantibody specificities in the dataset, and a summation of the soluble protein markers equals a Lupus Disease Activity Immune Index (LDAII / L-DAI) score. Additionally or alternatively, in another aspect, the dataset is: log transformed; standardized; weighted by Spearman r correlation to the hSLEDAI in the dataset, and a summation of soluble protein markers equals an LDAII / L-DAI score. Additionally or alternatively, in another aspect, the dataset is: log transformed; standardized; weighted by an average Spearman r correlation to the autoantibody specificities and the hSLEDAI in the dataset, and a summation of soluble protein markers equals a composite LDAII / L-DAI / L-DAI score. In another aspect, performance of the at least one immunoassay comprises: obtaining the sample, wherein the sample comprises the protein markers; contacting the sample with a plurality of distinct reagents; generating a plurality of distinct complexes between the reagents and markers; and detecting the complexes to generate the data. InAttorney Docket No.82092-000005-WO-POA another aspect, at least one immunoassay comprises a multiplex assay. In another aspect, the LDAII / L-DAI divides a level of severity or progression of the SLE into clinically active (CA) or quiescent (CQ) disease that is either serologically (dsDNA binding and low complement) active (SA) or serologically quiescent (SQ). In another aspect, the LDAII / L-DAI score distinguishes between active and low lupus disease activity. In another aspect, the method further comprises administering a treatment to the SLE patient prior to reaching clinical disease classification after determining that the patient has the prognosis for clinically active disease, wherein the treatment comprises at least one of: hydroxychloroquine (HCQ), belimumab, a nonsteroidal anti- inflammatory drug, a steroid, or a disease-modifying anti-rheumatic drug (DMARD). In another aspect, obtaining the dataset associated with the sample comprises obtaining the sample and processing the sample to experimentally determine the dataset, or wherein obtaining the first dataset associated with the sample comprises receiving the dataset from a third party that has processed the sample to experimentally determine the dataset. In another aspect, an increase in the IFN-α, IL-10, BLyS, IL-7, TRAIL, IP-10 / CXCL10, TNFRII, Resistin, and OPN biomarkers are indicative of renal organ involvement.

[0014] In another embodiment, the present invention includes a method of calculating a Lupus Disease Activity (Immune) Index (LDAII / L-DAI) by measuring expression levels of a set of biomarkers in a subject comprising: determining biomarker measures of a set of biomarkers by immunoassay in a physiological sample, wherein the biomarkers are peptides, proteins, peptides bearing post-translational modifications, proteins bearing post-translational modification, or a combination thereof; wherein the physiological sample is whole blood, blood plasma, blood serum, or a combination thereof; wherein the set of biomarkers comprise a dataset of a presence or an amount of protein expression of nine biomarkers IFN-α, IL-10, B-lymphocyte stimulating factor (BLyS or BAFF), IL-7, IFN-´, TRAIL, IL-15, IP-10 / CXCL10, and IL-4, plus measurements selected from at least one biomarker dataset selected from: TNFRII, Resistin, and Osteopontin (OPN), a presence or an amount of at least one biomarker dataset selected from: IL-8 / CXCL8, IP- 10 / CXCL10, MIG / CXCL9, MIP-lα / CCL3, MIP-l ³ / CCL4, MCP-1 / CCL2, RANTES / CCL5, and MCP-3 / CCL7; a presence or an amount of at least one soluble TNF superfamily biomarker dataset selected from: IL-12p70, TNF-α, MIG / CXCL9, IL-5, IL-13, IL-1³, IL-17A, IL-2Rα, Native TGF- ³, Fas, MCP-1 / CCL2, Stem Cell Factor (SCF), IL-1RA, IL-6, MIP-1α / CCL3, MIP-1³ / CCL4, MCP-3 / CCL7, Total TGF-³, RANTES / CCL5, TNFRI, and IL-8 / CXCL8; and a presence or an amount at least one SLE-associated autoantibody specificity biomarker dataset selected from: dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP; and calculating an LDAII / L- DAI score, whereby the dataset is: log transformed; standardized; weighted by Spearman rAttorney Docket No.82092-000005-WO-POA correlation to the autoantibody specificities in the dataset, and a summation of the soluble protein markers equals an LDAII / L-DAI score. Additionally or alternatively, in another aspect, the dataset is: log transformed; standardized; weighted by Spearman r correlation to the hSLEDAI in the dataset, and a summation of soluble protein markers equals an LDAII / L-DAI score. Additionally or alternatively, in another aspect, the dataset is: log transformed; standardized; weighted by an average Spearman r correlation to the autoantibody specificities and the hSLEDAI in the dataset, and a summation of soluble protein markers equals a composite LDAII / L-DAI / L-DAI score. In another aspect, the method further comprises classifying the sample with respect to the presence or development of Systemic Lupus Erythematosus (SLE) into clinically active (CA) or quiescent (CQ) disease that is either serologically (dsDNA binding and low complement) active (SA) or serologically quiescent (SQ) in the subject using the set of biomarker measures in a classification system, 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 comparison, Spearman’s rank correlation or an ensemble thereof; and calculating the Lupus Disease Activity Immune Index (LDAII / L-DAI), wherein the LDAII / L-DAI score distinguishes between active SLE and low SLE disease activity (low clinical disease [hSLEDAI < 4]). In another aspect, the method further comprises differentiating clinically and serologically quiescent (CQSQ) SLE patients compared to healthy controls. In another aspect, the method further comprises an amount of at least 9 or 10, 11, 12, or 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 biomarkers are used in the calculation of the LDAII / L-DAI. In another aspect, the immunoassay is multiplexed immunoassay. In another aspect, the LDAII / L-DAI was further calculated as follows: a concentration biomarkers is determined and log-transformed for the subject and each log- 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 patient and healthy control visits); generating Spearman coefficients from a linear regression model testing associations between one or more auto-antibody (AutoAb) specificities for each soluble mediator assessed in the SLE patient compared to healthy controls (Spearman r); transforming and standardizing the values of the soluble mediator levels of the subject and the values weighted (multiplied) by their respective Spearman coefficients (Spearman r); and summing for each participant visit, the log transformed, standardized and weighted values for each of the four or more soluble mediators to calculate the LDAII / L-DAI. In another aspect, an increase in the LDAII / L-DAI is indicative of at least one of: SLE disease progression, increased autoimmune disease activity, or organ damage.Attorney Docket No.82092-000005-WO-POA BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0016] Figure 1. Variable importance of 33 immune mediators differentiating samples from SLE patients with active (hSLEDAI g 4) vs. low (hSLEDAI < 4) disease activity vs. matched healthy controls as determined by random forest (top mediators above the line and remaining mediators below the line as determined by forward selection and backward elimination, respectively); mediators in red inform LDAII / L-DAI informed by 9 mediators (IL-12p70 excluded for lack of significance in univariate analysis in Table 2 and Table 3); mediators in purple form combinations of LDAII / L-DAI informed by 10, 11, or 12 mediators when combined with top mediators; all other mediators in blue or black may also inform the LDAII / L-DAI.

[0017] Figure 2. Lupus Disease Activity (Immune) Index (LDAII / L-DAI), informed by 9 mediators (L-DAI 9) and weighted by either hSLEDAI score (A) or # AutoAb specificities (B) differentiates SLE patients with clinically (C) and / or serologically (S) active (A) or quiescent (Q) disease, as well as SLE patients with low (hSLEDAI <4) or active (hSLEDAI g4) disease from race / gender / age-matched healthy individuals (Ctl). Log-transformed, standardized data for each soluble mediator in SLE patients and Ctls was weighted by the Spearman r comparing soluble mediator levels vs. hSLEDAI score (A) or number of SLE-associated AutoAb specificities present (B). Mean L-DAI 9 ± SEM shown in graphs. * p<0.05; ***p<0.001; ****p<0.0001 by Kruskal Wallis with Dunn’s Multiple Comparison; (C) Spearman correlation of L-DAI 9 scores weighted by either hSLEDAI score (x-axis) or # AutoAb specificities (y-axis).

[0018] Figure 3. Composite (average of LDAII / L-DAI weighted by # AutoAb specificities and hSLEDAI scores) LDAII / L-DAI informed by 9 mediators differentiates SLE patients with clinically (C) and / or serologically (S) active (A) or quiescent (Q) disease (A), as well as SLE patients with low (hSLEDAI <4) or active (hSLEDAI g4) disease (B) from race / gender / age- matched healthy individuals (Ctl). Mean LDAII / L-DAI 9 ± SEM shown in upper left panel graphs. * p<0.05; ***p<0.001; ****p<0.0001 by Kruskal Wallis with Dunn’s Multiple Comparison. Predictive probability of active disease determined by logistic regression in upper right panel graphs. Low / Mod (19% Threshold Probability; L-DAI 9 = -1.7577) and Mod / High (79%; Threshold Probability L-DAI 9 = 2.0624) for differentiating CASA vs CQSQ (A) is marked by dashed lines; Low / Mod (30% Threshold Probability; L-DAI 9 = -1.7363) andAttorney Docket No.82092-000005-WO-POA Mod / High (69% Threshold Probability; L-DAI = 2.4928) for differentiating Low vs Active disease (B) is marked by dashed lines; cut-offs were determined by Decision Curve Analysis / calculated Net Benefit that maximizes sensitivity and negative predictive value (NPV) with Low / Mod cutoff and maximizes specificity and positive predictive value (PPV) with Mod / High cut-off levels. Performance characteristics (bottom, center panels) of L-DAI 9 include area under the curve (AUC; receiver operating characteristic curve analysis) to differentiate CASA and CQSQ (A) and Low vs Active disease (B), Pearson r correlation of L-DAI-9 with hSLEDAI scores, and Cohen’s d Effect Size.

[0019] Figure 4. Combinations of TNFRII, Resistin, and Osteopontin (OPN) added to LDAII / L- DAI 9 mediators differentiate SLE patients with clinically (C) and / or serologically (S) active (A) or quiescent (Q) disease, as well as SLE patients with low (hSLEDAI <4) or active (hSLEDAI g4) disease from race / gender / age-matched healthy individuals (Ctl). Composite LDAII / L-DAI scores informed by 9 mediators (A) exhibit similar differentiation as those informed by 10 mediators (LDAII / L-DAI 9 + TNFRII [L-DAI 10A, B), Resistin [L-DAI 10B, C), or OPN [L- DAI 10C, D), 11 mediators (LDAII / L-DAI 9 + TNFRII / Resistin [L-DAI 11A, E], TNFRII / OPN [L-DAI 11B, F], or Resistin / OPN [L-DAI 11C, G]), or 12 mediators (LDAII / L-DAI 9 + TNFRII / Resistin / OPN [L-DAI 12, H]). Mean LDAII / L-DAI scores ± SEM shown. * p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 by Kruskal Wallis with Dunn’s Multiple Comparison. DESCRIPTION OF EXAMPLE EMBODIMENTS

[0020] The following description of example embodiments provides information that enables a person skilled in the art to make and use the subject matter set forth in the appended claims, but it may omit certain details already well-known in the art. While various embodiments are discussed in detail below, it should be appreciated that the disclosed subject matter provides many applicable inventive concepts that can be embodied in various contexts. The following detailed description is, therefore, to be taken as illustrative and not limiting.

[0021] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as <a,= <an,= and <the= are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims.

[0022] The present inventors leveraged plasma samples serially collected from Systemic lupus erythematosus (SLE) patients to compare levels and determine temporal relationships betweenAttorney Docket No.82092-000005-WO-POA clinical disease activity levels, autoantibody specificities, and immune mediators from multiple immune pathways in SLE patients with low or active disease compared to demographically matched healthy controls. The present invention sheds light on potential mechanisms of immunopathogenesis as it relates to clinical disease activity, whereby dysregulation of immune mediators occurs alongside and independent of autoantibody accumulation. Further, the present invention includes the design and validation of a reliable and sensitive tool to assess the immune status of lupus patients as it relates to clinical disease activity. The present invention can be used to identify high risk patients in need of rheumatology referral and enrollment in prospective, clinical intervention studies, as well as inform the development of novel treatment strategies to avert or delay tissue damage. In addition, the present invention can be used to augment telehealth to prioritize the need for in-person clinic visits.

[0023] As used herein, the term <dataset= refers to a set of numerical values resulting from evaluation of a sample (or population of samples) under a desired condition. The values of the dataset can be obtained, for example, by experimentally obtaining measures from a sample, such as a patient sample, and building a dataset from these measurements. Alternatively, the dataset can be obtained from a database or a server on which the dataset has been stored, or even a service provider such as an internal or third-party laboratory.

[0024] As used herein, the term <disease= in the context of the present invention refers to any disorder, condition, sickness, or ailment, that manifests in, for example, a dysfunctional or incorrectly functioning immune system that causes, e.g., SLE.

[0025] As used herein, the term <sample= refers to any biological sample that is isolated from a subject, which can include, without limitation, a single cell or multiple cells, fragments of cells, an aliquot of body fluid, whole blood, platelets, serum, plasma, red blood cells, white blood cells or leucocytes, endothelial cells, tissue biopsies, synovial fluid, lymphatic fluid, ascites fluid, and interstitial or extracellular fluid. The term <sample= also encompasses the fluid in spaces between cells, including gingival crevicular fluid, bone marrow, cerebrospinal fluid, saliva, mucous, sputum, semen, sweat, urine, or any other bodily fluids.

[0026] 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 leucocytes, platelets, serum and plasma. Samples can be obtained from a subject by means including but not limited to venipuncture, excretion, ejaculation, massage, biopsy, needle aspirate, lavage, scraping, surgical incision, or intervention or other means known in the art.

[0027] As used herein, the term <subject= or <patient= refers generally to a mammal, which includes, but is not limited to, a human, non-human primate, dog, cat, mouse, rat, cow, horse, andAttorney Docket No.82092-000005-WO-POA pig, without regard to gender or age. A subject can be one who has been previously diagnosed or identified as having an auto-immune and / or inflammatory disease, and who may have already undergone, or is undergoing, a therapeutic intervention for the auto-immune and / or inflammatory disease. However, a subject can also include a patient not previously diagnosed as having the autoimmune and / or inflammatory disease, for example, a subject who exhibits one or more symptoms or risk factors for the auto-immune and / or inflammatory disease, or a subject who does not exhibit symptoms or risk factors for the auto-immune and / or inflammatory disease, or a subject who is asymptomatic for the auto-immune and / or inflammatory disease.

[0028] As used herein, the phrase <innate serum or plasma mediator biomarker(s)= refers to one or more of the following biomarkers: IL- 1³, IL-1RA, IFN-α, IL-15, IL-12p70, IL-6, and IL-7. These biomarkers can be measured at the RNA or protein level and can be obtained from samples, e.g., blood, serum, plasma and / or urine sample from the patient, which is a mammal, e.g., a human patient. The abbreviations for all the biomarkers used herein are well-known to the skilled artisan, e.g., IL-1 is interleukin-1, and so forth. The abbreviations can be matched to the protein at, e.g., www.genecards.org.

[0029] As used herein, the phrase <adaptive serum or plasma mediator biomarker(s)= refers to one or more of the following biomarkers: IL-2Rα, IFN-´, IL-4, IL-5, IL-13, IL-17A, IL- 10, and TGF-³ (native TGF-³ and / or total TGF-³). These biomarkers can be measured at the RNA or protein level and can be obtained from samples, e.g., blood, serum, plasma and / or urine sample from the patient, which is a mammal, e.g., a human patient.

[0030] As used herein, the phrase <chemokine biomarker(s)= refers to one or more of the following biomarkers: IL-8 / CXCL8, IP-10 / CXCL10, MIG / CXCL9, MIP-lα / CCL3, MIP- l³ / CCL4, MCP-1 / CCL2, RANTES / CCL5, and MCP-3 / CCL7. These biomarkers can be measured from samples, e.g., blood, serum, plasma and / or urine sample from the patient, which is a mammal, e.g., a human patient.

[0031] As used herein, the phrase <soluble TNF superfamily biomarker(s)= refers to one or more of the following biomarkers: TNF-α, TNFRI (p55), TNFRII (p75), Fas, BLyS, and TNF-related apoptosis-inducing ligand (TRAIL). These biomarkers can be measured at the RNA or protein level and can be obtained from samples, e.g., blood, serum, plasma and / or urine sample from the patient, which is a mammal, e.g., a human patient.

[0032] As used herein, the phrase <inflammatory mediator biomarker(s)= refers to one or more of the following biomarkers: Osteopontin (OPN), Stem Cell Factor (SCF), and Resistin. These biomarkers can be measured at the RNA or protein level and can be obtained from samples, e.g., blood, serum, plasma and / or urine sample from the patient, which is a mammal, e.g., a humanAttorney Docket No.82092-000005-WO-POA patient.

[0033] As used herein, the phrase <SLE-associated autoantibody specificity biomarker(s)= refers to one or more of the following biomarkers that are autoantibodies against the following targets: dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP, all of which are well- known to the skilled artisan in the SLE arts. These biomarkers can be measured at the RNA or protein level and can be obtained from samples, e.g., blood, serum, plasma and / or urine sample from the patient, which is a mammal, e.g., a human patient.

[0034] As used herein, a <healthy control= refers to a healthy control that is not an SLE patient that has no clinical evidence of SLE.

[0035] The present invention includes methods for identifying and changing the treatment of SLE patients 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 the SLEDAI-2K, known as the hybrid-SLEDAI (hSLEDAI). As clinical manifestations of SLE contribute to disease activity in the hSLEDAI after they have occurred, the present invention is used to determine if the subject may be exhibiting biomarkers that may contribute to disease activity that places the SLE patient at risk for permanent organ damage and early mortality.

[0036] SLE classification / diagnosis. Following the ACR, SLICC, and EULAR / ACR criteria for SLE classification, patients must meet multiple, defined criteria for SLE to reach disease classification (diagnosis), including: malar rash, discoid rash, photosensitivity, oral ulcers, non- scarring alopecia, arthritis, serositis (pleuritis or pericarditis), renal disorder (proteinuria or cellular casts), neurologic disorder (delirium, seizures, or psychosis), hematologic disorder (hemolytic anemia, leukopenia, lymphopenia, or thrombocytopenia), fever, immunologic disorder (presence of anti-DNA, anti- Sm, or anti-phospholipid antibodies or low C3 and / or C4 and / or CH50 complement levels), and positive ANA (HEp-2 IIF assay). These criteria may be used, as known to the skilled artisan, e.g., using the ACR rule for the classification of SLE, the patient must satisfy at least 4 criteria; using the SLICC rule for the classification of SLE, the patient must satisfy at least 4 criteria, including at least one clinical criterion and one immunologic criterion OR the patient must have biopsy proven lupus nephritis in the presence of antinuclear antibodies or anti-double-stranded DNA antibodies; using the EULAR / ACR rule for the classification of SLE, the patient must satisfy ANA positivity (g1:80 titer by HEp-2 IIF assay), one clinical criterion, and g10 points (from 7 clinical and 3 immunology domains).

[0037] SLE disease manifestations. The most common clinical symptom which brings a patient for medical attention is joint pain, with the small joints of the hand and wrist usually affected,Attorney Docket No.82092-000005-WO-POA although nearly all joints are at risk. Between 80 and 90% of those affected will experience joint and / or muscle pain at some time during the course of their illness. Unlike rheumatoid arthritis, many lupus arthritis patients will have joint swelling and pain, but no X-ray changes and minimal loss of function. Fewer than 10% of people with lupus arthritis will develop deformities of the hands and feet. SLE patients are at particular risk of developing articular tuberculosis. An association between osteoporosis and SLE has been found, and SLE may be associated with an increased risk of bone fractures in relatively young women.

[0038] Over half (65%) of SLE sufferers have some dermatological manifestations at some point in their disease, with approximately 30% to 50% suffering from the classic malar rash (or butterfly rash) associated with the name of the disorder. Some may exhibit chronic thick, annual scaly patches on the skin (referred to as discoid lupus). Alopecia, mouth ulcers, nasal ulcers, and photosensitive lesions on the skin are also possible manifestations. Anemia may develop in up to 50% of lupus cases. Low platelet and white blood cell counts may be due to the disease or as a side effect of pharmacological treatment. People with SLE may have an association with antiphospholipid antibody syndrome (a thrombotic disorder), wherein autoantibodies to phospholipids are present in their serum. Abnormalities associated with antiphospholipid antibody syndrome include a paradoxical prolonged partial thromboplastin time (which usually occurs in hemorrhagic disorders) and a positive test for antiphospholipid antibodies; the combination of such findings has earned the term <lupus anticoagulant-positive.= SLE patients with anti-phospholipid autoantibodies have more ACR classification criteria of the disease and may suffer from a more severe lupus phenotype.

[0039] A person with SLE may have inflammation of various parts of the heart, such as pericarditis, myocarditis, and endocarditis. The endocarditis of SLE is characteristically noninfective (Libman-Sacks endocarditis) and involves either the mitral valve or the tricuspid valve. Atherosclerosis also tends to occur more often and advances more rapidly than in the general population. Lung and pleura inflammation can cause pleuritis, pleural effusion, lupus pneumonitis, chronic diffuse interstitial lung disease, pulmonary hypertension, pulmonary emboli, pulmonary hemorrhage, and shrinking lung syndrome.

[0040] Painless hematuria or proteinuria may often be the only presenting renal symptoms. Acute or chronic renal impairment may develop with lupus nephritis, leading to acute or end- stage renal failure. Because of early recognition and management of SLE, end-stage renal failure occurs in less than 5% of cases. A histological hallmark of SLE is membranous glomerulonephritis with <wire loop= abnormalities. This finding is due to immune complex deposition along the glomerular basement membrane, leading to a typical granular appearance inAttorney Docket No.82092-000005-WO-POA immunofluorescence testing.

[0041] Neuropsychiatric syndromes can result when SLE affects the central or peripheral nervous systems. The American College of Rheumatology defines 19 neuropsychiatric syndromes in systemic lupus erythematosus. The diagnosis of neuropsychiatric syndromes concurrent with SLE is one of the most difficult challenges in medicine, because it can involve so many different patterns of symptoms, some of which may be mistaken for signs of infectious disease or stroke. The most common neuropsychiatric disorder people with SLE have is headache, although the existence of a specific lupus headache and the optimal approach to headache in SLE cases remains controversial. Other common neuropsychiatric manifestations of SLE include cognitive dysfunction, mood disorder (including depression), cerebrovascular disease, seizures, polyneuropathy, anxiety disorder, cerebritis, and psychosis. CNS lupus can rarely present with intracranial hypertension syndrome, characterized by an elevated intracranial pressure, papilledema, and headache with occasional abducens nerve paresis, absence of a space- occupying lesion or ventricular enlargement, and normal cerebrospinal fluid chemical and hematological constituents. More rare manifestations are acute confusional state, Guillain-Barré syndrome, aseptic meningitis, autonomic disorder, demyelinating syndrome, mononeuropathy (which might manifest as mononeuritis multiplex), movement disorder (more specifically, chorea), myasthenia gravis, myelopathy, cranial neuropathy and plexopathy. Neural symptoms contribute to a significant percentage of morbidity and mortality in patients with lupus. As a result, the neural side of lupus is being studied in hopes of reducing morbidity and mortality rates. The neural manifestation of lupus is known as neuropsychiatric systemic lupus erythematosus (NPSLE). One aspect of this disease is severe damage to the epithelial cells of the blood3brain barrier.

[0042] Biomarkers for SLE disease activity

[0043] Innate cytokines. Innate cytokines are mediators secreted in response to immune system danger signals, such as toll like receptors (TLR). Innate cytokines which activate and are secreted by multiple immune cell types include Type I interferons (IFN-α and IFN-³), TNF-α, and members of the IL-1 family (IL-1α and IL-1³). Other innate cytokines, secreted by antigen presenting cells (APC), including dendritic cells, macrophages, and B-cells, as they process and present protein fragments (antigens, either from infectious agents or self-proteins that drive autoimmune disease) to CD4 T-helper (Th) cells, drive the development of antigen specific inflammatory pathways during the adaptive response, described below.

[0044] Th1-type cytokines. Th1-type cytokines drive proinflammatory responses responsible for killing intracellular parasites and for perpetuating autoimmune responses. ExcessiveAttorney Docket No.82092-000005-WO-POA proinflammatory responses can lead to uncontrolled tissue damage, particularly in systemic lupus erythematosus (SLE).

[0045] CD4 Th cells differentiate to Th-1 type cells upon engagement of APC, co-stimulatory molecules, and APC-secreted cytokines, the hallmark of which is IL-12. IL-12 is composed of a bundle of four alpha helices. It is a heterodimeric cytokine encoded by two separate genes, IL-12A (p35) and IL-12B (p40). The active heterodimer, and a homodimer of p40, are formed following protein synthesis. IL-12 binds to the heterodimeric receptor formed by IL-12R-³1 and IL-12R-³2. IL-12R-³2 is considered to play a key role in IL-12 function, as it is found on activated T cells and is stimulated by cytokines that promote Th1 cell development and inhibited by those that promote Th2 cell development. Upon binding, IL-12R-³2 becomes tyrosine phosphorylated and provides binding sites for kinases, Tyk2 and Jak2. These are important in activating critical transcription factor proteins such as STAT4 that are implicated in IL-12 signaling in T cells and NK cells. IL- 12 mediated signaling results in the production of interferon-gamma (IFN-´) and tumor necrosis factor-alpha (TNF-α) from T and natural killer (NK) cells, and reduces IL-4 mediated suppression of IFN-´.

[0046] IFN-´, or type II interferon, consists of a core of six α-helices and an extended unfolded sequence in the C-terminal region. IFN-´ is critical for innate (NK cell) and adaptive (T cell) immunity against viral (CD8 responses) and intracellular bacterial (CD4 Th1 responses) infections and for tumor control. During the effector phase of the immune response, IFN-´ activates macrophages. Aberrant IFN-´ expression is associated with a number of autoinflammatory and autoimmune diseases, including increased disease activity in SLE. Although IFNγ is considered to be the characteristic Th1 cytokine, in humans, interleukin-2 (IL- 2) has been shown to influence Th1 differentiation, as well as its role as the predominant cytokine secreted during a primary response by naïve Th cells.IL-2 is necessary for the growth, proliferation, and differentiation of T cells to become 'effector' T cells. IL-2 is normally produced by T cells during an immune response. Antigen binding to the T cell receptor (TCR) stimulates the secretion of IL-2, and the expression of IL-2 receptors IL-2R. The IL-2 / IL-2R interaction then stimulates the growth, differentiation and survival of antigen-specific CD4+ T cells and CD8+ T cells As such, IL-2 is necessary for the development of T cell immunologic memory, which depends upon the expansion of the number and function of antigen-selected T cell clones. IL-2, along with IL-7 and IL-15 (all members of the common cytokine receptor gamma-chain family), maintain lymphoid homeostasis to ensure a consistent number of lymphocytes during cellular turnover.Attorney Docket No.82092-000005-WO-POA

[0047] Th2-type cytokines. Th2-type cytokines include IL-4, IL-5, IL-13, as well as IL-6 (in humans), and are associated with the promotion of B-lymphocyte activation, antibody production, and isotype switching to IgE and eosinophilic responses in atopy. In excess, Th2 responses counteract the Th1 mediated microbicidal action. Th2-type cytokines may also contribute to SLE pathogenesis and increased disease activity.

[0048] IL-4 is a 15-kD polypeptide with multiple effects on many cell types. Its receptor is a heterodimer composed of an α subunit, with IL-4 binding affinity, and the common´ subunit which is also part of other cytokine receptors. In T cells, binding of IL-4 to its receptor induces proliferation and differentiation into Th2 cells. IL-4 also contributes to the Th2-mediated activation of B-lymphocytes, antibody production, and, along with IL-5 and IL-13, isotype switching away from Th1-type isotypes (including IgG1 and IgG2) toward Th2-type isotypes (including IgG4, and IgE that contributes to atopy). In addition to its contributions to Th2 biology, IL-4 plays a significant role in immune cell hematopoiesis, with multiple effects on hematopoietic progenitors, including proliferation and differentiation of committed as well as primitive hematopoietic progenitors. It acts synergistically with granulocyte-colony stimulating factor (G- CSF) to support neutrophil colony formation, and, along with IL-1 and IL-6, induces the colony formation of human bone marrow B lineage cells.

[0049] IL-5 is an interleukin produced by multiple cell types, including Th2 cells, mast cells, and eosinophils. IL-5 expression is regulated by several transcription factors including GATA3. IL-5 is a 115-amino acid (in humans, 133 in the mouse) -long TH2 cytokine that is part of the hematopoietic family. Unlike other members of this cytokine family (namely IL-3 and GM-CSF), this glycoprotein in its active form is a homodimer. Through binding to the IL-5 receptor, IL-5 stimulates B cell growth and increases immunoglobulin secretion. IL-5 has long been associated with the cause of several allergic diseases including allergic rhinitis and asthma, where mast cells play a significant role, and a large increase in the number of circulating, airway tissue, and induced sputum eosinophils have been observed.

[0050] Given the high concordance of eosinophils and, in particular, allergic asthma pathology, it has been widely speculated that eosinophils have an important role in the pathology of this disease. IL-13 is secreted by many cell types, but especially Th2 cells as a mediator of allergic inflammation and autoimmune disease, including type 1 diabetes mellitus, rheumatoid arthritis (RA) and SLE. IL-13 induces its effects through a multi-subunit receptor that includes the alpha chain of the IL-4 receptor (IL-4Rα) and at least one of two known IL-13-specific binding chains. Most of the biological effects of IL-13, like those of IL-4, are linked to a single transcription factor, signal transducer and activator of transcription 6 (STAT6).Attorney Docket No.82092-000005-WO-POA

[0051] Like IL-4, IL-13 is known to induce changes in hematopoietic cells, but to a lesser degree. IL-13 can induce immunoglobulin E (IgE) secretion from activated human B cells. IL-13 induces many features of allergic lung disease, including airway hyperresponsiveness, goblet cell metaplasia and mucus hypersecretion, which all contribute to airway obstruction. IL-4 contributes to these physiologic changes, but to a lesser extent than IL-13. IL-13 also induces secretion of chemokines that are required for recruitment of allergic effector cells to the lung.

[0052] IL-13 may antagonize Th1 responses that are required to resolve intracellular infections and induces physiological changes in parasitized organs that are required to expel the offending organisms or their products. For example, expulsion from the gut of a variety of mouse helminths requires IL-13 secreted by Th2 cells. IL-13 induces several changes in the gut that create an environment hostile to the parasite, including enhanced contractions and glycoprotein hyper- secretion from gut epithelial cells, that ultimately lead to detachment of the organism from the gut wall and their removal.

[0053] Interleukin 6 (IL-6) is secreted by multiple cell types and participates in multiple innate and adaptive immune response pathways. IL-6 mediates its biological functions through a signal- transducing component of the IL-6 receptor (IL-6R), gp130, that leads to tyrosine kinase phosphorylation and downstream signaling events, including the STAT1 / 3 and the SHP2 / ERK cascades. IL-6 is a key mediator of fever and stimulates an acute phase response during infection and after trauma. It is capable of crossing the blood brain barrier and initiating synthesis of PGE2 in the hypothalamus, thereby changing the body's temperature setpoint. In muscle and fatty tissue, IL-6 stimulates energy mobilization which leads to increased body temperature.

[0054] IL-6 can be secreted by multiple immune cells in response to specific microbial molecules, referred to as pathogen associated molecular patterns (PAMPs). These PAMPs bind to highly important group of detection molecules of the innate immune system, called pattern recognition receptors (PRRs), including Toll-like receptors (TLRs). These are present on the cell surface and intracellular compartments and induce intracellular signaling cascades that give rise to inflammatory cytokine production. As a Th2-type cytokine in humans, IL-6, along with IL-4, IL- 5, and IL-13, can influence IgE production and eosinophil airway infiltration in asthma. IL-6 also contributes to Th2-type adaptive immunity against parasitic infections, with particular importance in mast-cell activation that coincides with parasite expulsion.

[0055] IL-6 is also a Th17-type cytokine, driving IL-17 production by T-lymphocytes in conjunction with TGF-´. IL-6 sensitizes Th17 cells to IL-23 (produced by APC) and IL-21 (produced by T-lymphocytes to perpetuate the Th17 response. Th17-type responses are described below.Attorney Docket No.82092-000005-WO-POA

[0056] Th17-type cytokines. Th17 cells are a subset of T helper cells are considered developmentally distinct from Th1 and Th2 cells and excessive amounts of the cell are thought to play a key role in autoimmune disease, such as multiple sclerosis (which was previously thought to be caused solely by Th1 cells), psoriasis, autoimmune uveitis, Crohn’s disease, type 2 diabetes mellitus, rheumatoid arthritis, and SLE. Th17 are thought to play a role in inflammation and tissue injury in these conditions. In addition to autoimmune pathogenesis, Th17 cells serve a significant function in anti-microbial immunity at epithelial / mucosal barriers. They produce cytokines (such as IL-21 and IL-22) that stimulate epithelial cells to produce anti-microbial proteins for clearance of microbes such as Candida and Staphylococcus species. A lack of Th17 cells may leave the host susceptible to opportunistic infections. In addition to its role in autoimmune disease and infection, the Th17 pathway has also been implicated in asthma, including the recruitment of neutrophils to the site of airway inflammation.

[0057] Interleukin 17A (IL-17A), is the founding member of a group of cytokines called the IL- 17 family. Known as CTLA8 in rodents, IL-17 shows high homology to viral IL-17 encoded by an open reading frame of the T-lymphotropic rhadinovirus Herpesvirus saimiri. IL-17A is a 155- amino acid protein that is a disulfide-linked, homodimeric, secreted glycoprotein with a molecular mass of 35 kDa. Each subunit of the homodimer is approximately 15-20 kDa. The structure of IL- 17A consists of a signal peptide of 23 amino acids (aa) followed by a 123-aa chain region characteristic of the IL-17 family. An N-linked glycosylation site on the protein was first identified after purification of the protein revealed two bands, one at 15 KDa and another at 20 KDa. Comparison of different members of the IL-17 family revealed four conserved cysteines that form two disulfide bonds. IL-17A is unique in that it bears no resemblance to other known interleukins. Furthermore, IL-17A bears no resemblance to any other known proteins or structural domains.

[0058] IL-17A acts as a potent mediator in delayed-type reactions by increasing chemokine production in various tissues to recruit monocytes and neutrophils to the site of inflammation, similar to IFNγ. IL-17A is produced by T-helper cells and is induced by APC production of IL-6 (and TGF-´) and IL323, resulting in destructive tissue damage in delayed-type reactions. IL-17, as a family, functions as a proinflammatory cytokine that responds to the invasion of the immune system by extracellular pathogens and induces destruction of the pathogen’s cellular matrix. IL- 17 acts synergistically with TNF-³ and IL-1. To elicit its functions, IL-17 binds to a type I cell surface receptor called IL-17R of which there are at least three variants IL17RA, IL17RB, and IL17RC.Attorney Docket No.82092-000005-WO-POA

[0059] Chemokines and Adhesion Molecules. Chemokines and adhesion molecules (in this case, ICAM-1 and E-selectin) serve to coordinate cellular traffic within the immune response. Chemokines are divided into CXC (R)eceptor / CXC (L)igand and CCR / CCL subgroups. GROα, also known as Chemokine (C-X-C motif) ligand 1 (CXCL1) belongs to the CXC chemokine family that was previously called GRO1 oncogene, KC, Neutrophil-activating protein 3 (NAP-3) and melanoma growth stimulating activity, alpha (MSGA-α). In humans, this protein is encoded by the CXCL1 gene on chromosome 4. CXCL1 is expressed by macrophages, neutrophils and epithelial cells, and has neutrophil chemoattractant activity. GROα is involved in the processes of angiogenesis, inflammation, wound healing, and tumorigenesis. This chemokine elicits its effects by signaling through the chemokine receptor CXCR2. Interleukin 8 (IL-8) / CXCL8 is a chemokine produced by macrophages and other cell types such as epithelial cells, airway smooth muscle cells and endothelial cells. In humans, the interleukin-8 protein is encoded by the IL8 gene. IL-8 is a member of the CXC chemokine family. The genes encoding this and the other ten members of the CXC chemokine family form a cluster in a region mapped to chromosome 4q.

[0060] There are many receptors of the surface membrane capable of binding IL-8; the most frequently studied types are the G protein-coupled serpentine receptors CXCR1, and CXCR2, expressed by neutrophils and monocytes. Expression and affinity to IL-8 is different in the two receptors (CXCR1 > CXCR2). IL-8 is secreted and is an important mediator of the immune reaction in the innate immunity in response to TLR engagement. During the adaptive immune response, IL-8 is produced during the effector phase of Th1 and Th17 pathways, resulting in neutrophil and macrophage recruitment to sites of inflammation, including inflammation during infection and autoimmune disease. While neutrophil granulocytes are the primary target cells of IL-8, there are a relatively wide range of cells (endothelial cells, macrophages, mast cells, and keratinocytes) also responding to this chemokine.

[0061] Monokine induced by γinterferon (MIG) / CXCL9 is a T-cell chemoattractant induced by IFN-´. It is closely related to two other CXC chemokines, IP-10 / CXCL10 and I-TAC / CXCL11, whose genes are located near the CXCL9 gene on human chromosome 4. MIG, IP-10, and I-TAC elicit their chemotactic functions by interacting with the chemokine receptor CXCR3. Interferon gamma-induced protein 10 (IP-10), also known as CXCL10, or small-inducible cytokine B10, is an 8.7 kDa protein that in humans is encoded by the CXCL10 gene located on human chromosome 4 in a cluster among several other CXC chemokines. IP-10 is secreted by several cell types in response to IFN-´. These cell types include monocytes, endothelial cells and fibroblasts. IP-10 has been attributed to several roles, such as chemoattraction forAttorney Docket No.82092-000005-WO-POA monocytes / macrophages, T cells, NK cells, and dendritic cells, promotion of T cell adhesion to endothelial cells, antitumor activity, and inhibition of bone marrow colony formation and angiogenesis. This chemokine elicits its effects by binding to the cell surface chemokine receptor CXCR3, which can be found on both Th1 and Th2 cells.

[0062] Monocyte chemotactic protein-1 (MCP-1) / CCL2 recruits monocytes, memory T cells, and dendritic cells to sites of inflammation. MCP-1 is a monomeric polypeptide, with a molecular weight of approximately 13 kDa that is primarily secreted by monocytes, macrophages and dendritic cells. Platelet derived growth factor is a major inducer of MCP-1 gene. The MCP-1 protein is activated post-cleavage by metalloproteinase MMP-12. CCR2 and CCR4 are two cell surface receptors that bind MCP-1. During the adaptive immune response, CCR2 is upregulated on Th17 and T-regulatory cells, while CCR4 is upregulated on Th2 cells. MCP-1 is implicated in pathogeneses of several diseases characterized by monocytic infiltrates, such as psoriasis, rheumatoid arthritis and atherosclerosis. It is also implicated in the pathogenesis of SLE, and a polymorphism of MCP-1 is linked to SLE in Caucasians. Administration of anti-MCP-1 antibodies in a model of glomerulonephritis reduces infiltration of macrophages and T cells, reduces crescent formation, as well as scarring and renal impairment.

[0063] Monocyte-specific chemokine 3 (MCP-3) / CCL7) specifically attracts monocytes and regulates macrophage function. It is produced by multiple cell types, including monocytes, macrophages, and dendritic cells. The CCL7 gene is located on chromosome 17 in humans, in a large cluster containing other CC chemokines. MCP-3 is most closely related to MCP-1, binding to CCR2.

[0064] Macrophage inflammatory protein-1α (MIP-1α) / CCL3 is encoded by the CCL3 gene in humans. MIP-1α is involved in the acute inflammatory state in the recruitment and activation of polymorphonuclear leukocytes(Wolpe et al., 1988). MIP-1α interacts with MIP-1³ / CCL4, encoded by the CCL4 gene, with specificity for CCR5 receptors. It is a chemoattractant for natural killer cells, monocytes and a variety of other immune cells. RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) / CCL5 is encoded by the CCL5 gene on chromosome 17 in humans. RANTES is an 8kDa protein chemotactic for T cells, eosinophils, and basophils, playing an active role in recruiting leukocytes to sites of inflammation. With the help of particular cytokines that are released by T cells (e.g. IL-2 and IFN- ´), RANTES induces the proliferation and activation of natural-killer (NK) cells. RANTES was first identified in a search for genes expressed "late" (335 days) after T cell activation and has been shown to interact with CCR3, CCR5 and CCR1. RANTES also activates the G-protein coupled receptor GPR75.Attorney Docket No.82092-000005-WO-POA

[0065] TNF Receptor superfamily members. The tumor necrosis factor receptor (TNFR) superfamily of receptors and their respective ligands activate signaling pathways for cell survival, death, and differentiation. Members of the TNFR superfamily act through ligand-mediated trimerization and require adaptor molecules (e.g. TRAFs) to activate downstream mediators of cellular activation, including NF-κB and MAPK pathways, immune and inflammatory responses, and in some cases, apoptosis.

[0066] The prototypical member is TNF-³. Tumor necrosis factor (TNF, cachexin, or cachectin, and formerly known as tumor necrosis factor alpha or TNFα) is a cytokine involved in systemic inflammation and is a member of a group of cytokines that stimulate the acute phase reaction. It is produced by a number of immune cells, including macrophages, dendritic cells, and both T- and B-lymphocytes. Dysregulation of TNF-³ production has been implicated in a variety of human diseases including Alzheimer's disease, cancer, major depression and autoimmune disease, including inflammatory bowel disease (IBD) and rheumatoid arthritis (RA).

[0067] TNF-³ is produced as a 212-amino acid-long type II transmembrane protein arranged in stable homotrimers. From this membrane-integrated form the soluble homotrimeric cytokine (sTNF) is released via proteolytic cleavage by the metalloprotease TNF-³ converting enzyme (TACE, also called ADAM17). The soluble 51 kDa trimeric sTNF may dissociate to the 17-kD monomeric form. Both the secreted and the membrane bound forms are biologically active. Tumor necrosis factor receptor 1 (TNFRI; TNFRSF1a; CD120a), is a trimeric cytokine receptor that is expressed in most tissues and binds both membranous and soluble TNF-³. The receptor cooperates with adaptor molecules (such as TRADD, TRAF, RIP), which is important in determining the outcome of the response (e.g., apoptosis, inflammation). Tumor necrosis factor II (TNFRII; TNFRSF1b; CD120b) has limited expression, primarily on immune cells (although during chronic inflammation, endothelial cells, including those of the lung and kidney, are induced to express TNFRII) and binds the membrane-bound form of the TNF-a homotrimer with greater affinity and avidity than soluble TNF-³. Unlike TNFRI, TNFRII does not contain a death domain (DD) and does not cause apoptosis, but rather contributes to the inflammatory response and acts as a co- stimulatory molecule in receptor-mediated B- and T-lymphocyte activation.

[0068] Fas, also known as apoptosis antigen 1 (APO-1 or APT), cluster of differentiation 95 (CD95) or tumor necrosis factor receptor superfamily member 6 (TNFRSF6) is a protein that in humans is encoded by the TNFRSF6 gene located on chromosome 10 in humans and 19 in mice. Fas is a death receptor on the surface of cells that leads to programmed cell death (apoptosis). Like other TNFR superfamily members, Fas is produced in membrane-bound form, but can be produced in soluble form, either via proteolytic cleavage or alternative splicing. The mature Fas protein hasAttorney Docket No.82092-000005-WO-POA 319 amino acids, has a predicted molecular weight of 48 kD and is divided into 3 domains: an extracellular domain, a transmembrane domain, and a cytoplasmic domain. Fas forms the death- inducing signaling complex (DISC) upon ligand binding. Membrane-anchored Fas ligand on the surface of an adjacent cell causes oligomerization of Fas. Upon ensuing death domain (DD) aggregation, the receptor complex is internalized via the cellular endosomal machinery. This allows the adaptor molecule FADD to bind the death domain of Fas through its own death domain. FADD also contains a death effector domain (DED) near its amino terminus, which facilitates binding to the DED of FADD-like interleukin-1 beta-converting enzyme (FLICE), more commonly referred to as caspase-8. FLICE can then self-activate through proteolytic cleavage into p10 and p18 subunits, two each of which form the active heterotetramer enzyme. Active caspase- 8 is then released from the DISC into the cytosol, where it cleaves other effector caspases, eventually leading to DNA degradation, membrane blebbing, and other hallmarks of apoptosis. In most cell types, caspase-8 catalyzes the cleavage of the pro-apoptotic BH3-only protein Bid into its truncated form, tBid. BH-3 only members of the Bcl-2 family exclusively engage anti-apoptotic members of the family (Bcl-2, Bcl-xL), allowing Bak and Bax to translocate to the outer mitochondrial membrane, thus permeabilizing it and facilitating release of pro-apoptotic proteins such as cytochrome c and Smac / DIABLO, an antagonist of inhibitors of apoptosis proteins (IAPs). Fas ligand (FasL; CD95L; TNFSF6) is a type-II transmembrane protein that belongs to the tumor necrosis factor (TNF) family. Its binding with its receptor induces apoptosis. FasL / Fas interactions play an important role in the regulation of the immune system and the progression of cancer. Soluble Fas ligand is generated by cleaving membrane-bound FasL at a conserved cleavage site by the external matrix metalloproteinase MMP-7.

[0069] Apoptosis triggered by Fas-Fas ligand binding plays a fundamental role in the regulation of the immune system. Its functions include T-cell homeostasis (the activation of T-cells leads to their expression of the Fas ligand.; T cells are initially resistant to Fas-mediated apoptosis during clonal expansion, but become progressively more sensitive the longer they are activated, ultimately resulting in activation-induced cell death (AICD)), cytotoxic T-cell activity (Fas-induced apoptosis and the perforin pathway are the two main mechanisms by which cytotoxic T lymphocytes induce cell death in cells expressing foreign antigens), immune privilege (cells in immune privileged areas such as the cornea or testes express Fas ligand and induce the apoptosis of infiltrating lymphocytes), maternal tolerance (Fas ligand may be instrumental in the prevention of leukocyte trafficking between the mother and the fetus, although no pregnancy defects have yet been attributed to a faulty Fas-Fas ligand system) and tumor counterattack (tumors may over-Attorney Docket No.82092-000005-WO-POA express Fas ligand and induce the apoptosis of infiltrating lymphocytes, allowing the tumor to escape the effects of an immune response).

[0070] Another pro-apoptotic member of the TNF receptor superfamily is TNF-related apoptosis- inducing ligand (TRAIL). Encoded by the TNFSF10 gene, the TNFSF10 / TRAIL (Apo2L / CD253) protein preferentially induces apoptosis in transformed and tumor cells, but does not appear to kill normal cells although it is expressed at a significant level in most normal tissues. TRAIL forms trimers and binds to several members of TNF receptor superfamily, including TNFRSF10A / TRAILR1, TNFRSF10B / TRAILR2, TNFRSF10C / TRAILR3, TNFRSF10D / TRAILR4, and possibly also to TNFRSF11B / OPG. The activity of this protein may be modulated by binding to the decoy receptors TNFRSF10C / TRAILR3, TNFRSF10D / TRAILR4, and TNFRSF11B / OPG that cannot induce apoptosis. The binding of this protein to its receptors has been shown to trigger the activation of MAPK8 / JNK, caspase 8, and caspase 3 that can both contribute to inflammation through the activation of IL-1³ and protect from the autoimmunity seen in Fas deficiency.

[0071] B-cell activating factor (BAFF) also known as B Lymphocyte Stimulator (BLyS), TNF- and APOL-related leukocyte expressed ligand (TALL-1), and CD27 is encoded by the TNFSF13C gene in humans. BLyS is a 285-amino acid long peptide glycoprotein which undergoes glycosylation at residue 124. It is expressed as a membrane-bound type II transmembrane protein on various cell types including monocytes, dendritic cells and bone marrow stromal cells. The transmembrane form can be cleaved from the membrane, generating a soluble protein fragment. This cytokine is expressed in B cell lineage cells, and acts as a potent B cell activator. It has been also shown to play an important role in the proliferation and differentiation of B cells. BLyS is a ligand for receptors TNFRSF13B / TACI, TNFRSF17 / BCMA, and TNFRSF13C / BAFFR. These receptors are expressed mainly on mature B lymphocytes and their expression varies in dependence of B cell maturation (TACI is also found on a subset of T-cells and BCMA on plasma cells). BAFF-R is involved in the positive regulation during B cell development. TACI binds BLyS with the least affinity; its affinity is higher for a protein similar to BLyS, called a proliferation-inducing ligand (APRIL). BCMA displays an intermediate binding phenotype and will bind to either BLyS or APRIL to varying degrees. Signaling through BAFF-R and BCMA stimulates B lymphocytes to undergo proliferation and to counter apoptosis. All these ligands act as homotrimers (i.e. three of the same molecule) interacting with homotrimeric receptors, although BAFF has been known to be active as either a hetero- or homotrimer. Excessive levels of BLyS causes abnormally high antibody production, results in systemic lupus erythematosus, rheumatoid arthritis, and many other autoimmune diseases. Belimumab (Benlysta)Attorney Docket No.82092-000005-WO-POA is a monoclonal antibody developed by Human Genome Sciences and GlaxoSmithKline, with significant discovery input by Cambridge Antibody Technology, which specifically recognizes and inhibits the biological activity of B-Lymphocyte stimulator (BLyS) and is in clinical trials for treatment of Systemic lupus erythematosus and other auto-immune diseases. Blisibimod, a fusion protein inhibitor of BLyS, is in development by Anthera Pharmaceuticals, also primarily for the treatment of systemic lupus erythematosus.

[0072] Other SLE Disease Activity Inflammatory Factors. Osteopontin (OPN) is a matricellular protein with diverse cellular functions. Its ability to facilitate Th1-type cytokine responses and promote cell-mediated immunity suggests a potential role in chronic inflammation and autoimmunity. In the methods disclosed herein, OPN may be highly informative and was ranked first in random forest variable importance.

[0073] Leptin is a 16-kDa protein hormone that plays a key role in regulating energy intake and expenditure, including appetite and hunger, metabolism, and behavior. It is one of a number of adipokines, including adiponectin and resistin. The reported rise in leptin following acute infection and chronic inflammation, including autoimmune disease, suggests that leptin actively participates in the immune response. Leptin levels increase in response to a number of innate cytokines, including TNF-α and IL-6. Leptin is a member of the cytokine family that includes IL-6, IL-12, and G-CSF. Leptin functions by binding to the leptin receptor, which is expressed by polymorphonuclear neutrophils, circulating leukocytes (including monocytes), and NK cells. Leptin influences the rise in the chemokine MCP-1, allowing for recruitment of monocytes and macrophages to sites of inflammation.

[0074] Stem Cell Factor (also known as SCF, kit-ligand, KL, or steel factor) is a cytokine that binds to the c-Kit receptor (CD117). SCF can exist both as a transmembrane protein and a soluble protein. This cytokine plays an important role in hematopoiesis (formation of blood cells), spermatogenesis, and melanogenesis. The gene encoding stem cell factor (SCF) is found on the Sl locus in mice and on chromosome 12q22-12q24 in humans. The soluble and transmembrane forms of the protein are formed by alternative splicing of the same RNA transcript.

[0075] The soluble form of SCF contains a proteolytic cleavage site in exon 6. Cleavage at this site allows the extracellular portion of the protein to be released. The transmembrane form of SCF is formed by alternative splicing that excludes exon 6. Both forms of SCF bind to c-Kit and are biologically active. Soluble and transmembrane SCF is produced by fibroblasts and endothelial cells. Soluble SCF has a molecular weight of 18,5 KDa and forms a dimer. SCF plays an important role in hematopoiesis, providing guidance cues that direct hematopoietic stem cells (HSCs) to their stem cell niche (the microenvironment in which a stem cell resides), and it plays an important roleAttorney Docket No.82092-000005-WO-POA in HSC maintenance. SCF plays a role in the regulation of HSCs in the stem cell niche in the bone marrow. SCF has been shown to increase the survival of HSCs in vitro and contributes to the self- renewal and maintenance of HSCs in vivo. HSCs at all stages of development express the same levels of the receptor for SCF (c-Kit). The stromal cells that surround HSCs are a component of the stem cell niche, and they release a number of ligands, including SCF. A small percentage of HSCs regularly leave the bone marrow to enter circulation and then return to their niche in the bone marrow. It is believed that concentration gradients of SCF, along with the chemokine SDF-1, allow HSCs to find their way back to the niche.

[0076] In addition to hematopoiesis, SCF is thought to contribute to inflammation via its binding to c-kit on dendritic cells. This engagement leads to increased secretion of IL-6 and the promoted development of Th2 and Th17-type immune responses. Th2 cytokines synergize with SCF in the activation of mast cells, and integral promoter of allergic inflammation. The induction of IL-17 allows for further upregulation of SCF by epithelial cells and the promotion of granulopoiesis. In the lung, the upregulation of IL-17 induces IL-8 and MIP-2 to recruit neutrophils to the lung. The chronic induction of IL-17 has been demonstrated to play a role in autoimmune diseases, including multiple sclerosis and rheumatoid arthritis.

[0077] SLE Disease Activity Biomarkers with Regulatory and Inflammatory Activity. IL-10. Interleukin-10 (IL-10), also known as human cytokine synthesis inhibitory factor (CSIF), is an anti-inflammatory cytokine. The IL-10 protein is a homodimer; each of its subunits is 178- amino-acid long. IL-10 is classified as a class-2 cytokine, a set of cytokines including IL-19, IL-20, IL-22, IL-24 (Mda-7), and IL-26, interferons and interferon-like molecules. In humans, IL-10 is encoded by the IL10 gene, which is located on chromosome 1 and comprises 5 exons. IL-10 is primarily produced by monocytes and lymphocytes, namely Th2 cells, CD4+, CD25+, Foxp3+, regulatory T cells, and in a certain subset of activated T cells and B cells. IL-10 can be produced by monocytes upon PD-1 triggering in these cells. The expression of IL-10 is minimal in unstimulated tissues and requires receptor-mediated cellular activation for its expression. IL-10 expression is tightly regulated at the transcriptional and post-transcriptional level. Extensive IL-10 locus remodeling is observed in monocytes upon stimulation of TLR or Fc receptor pathways. IL-10 induction involves ERK1 / 2, p38 and NFκB signaling and transcriptional activation via promoter binding of the transcription factors NFκB and AP-1. IL-10 may autoregulate its expression via a negative feed-back loop involving autocrine stimulation of the IL-10 receptor and inhibition of the p38 signaling pathway. Additionally, IL-10 expression is extensively regulated at the post-transcriptional level, which may involve control of mRNA stability via AU-rich elements and by microRNAs such as let-7 or miR-106.Attorney Docket No.82092-000005-WO-POA

[0078] IL-10 is a cytokine with pleiotropic effects in immunoregulation and inflammation. It downregulates the expression of multiple Th-pathway cytokines, MHC class II antigens, and co-stimulatory molecules on macrophages. It also enhances B cell survival, proliferation, and antibody production. IL-10 can block NF-κB activity, and is involved in the regulation of the JAK-STAT signaling pathway.

[0079] TGF-³. Transforming growth factor beta (TGF-³) controls proliferation, cellular differentiation, and other functions in most cells. TGF-³ is a secreted protein that exists in at least three isoforms called TGF-³1, TGF-³2 and TGF-³3. It was also the original name for TGF-³1, which was the founding member of this family. The TGF-³ family is part of a superfamily of proteins known as the transforming growth factor beta superfamily, which includes inhibins, activin, anti-mullerian hormone, bone morphogenetic protein, decapentaplegic and Vg-1. It is measured in its Native (active) form, or in a <Total= (latent) form, where the inactive TGF-³ is complexed with latent TGF-³ binding proteins (LTBP).

[0080] Most tissues have high expression of the gene encoding TGF-³. That contrasts with other anti-inflammatory cytokines such as IL-10, whose expression is minimal in unstimulated tissues and seems to require triggering by commensal or pathogenic flora.

[0081] The peptide structures of the three members of the TGF-³ family are highly similar. They are all encoded as large protein precursors; TGF-³1 contains 390 amino acids and TGF- ³2 and TGF-³3 each contain 412 amino acids. They each have an N-terminal signal peptide of 20-30 amino acids that they require for secretion from a cell, a pro-region (called latency associated peptide or LAP), and a 112-114 amino acid C-terminal region that becomes the mature TGF-³ molecule following its release from the pro-region by proteolytic cleavage. The mature TGF-³ protein dimerizes to produce a 25 kDa active molecule with many conserved structural motifs.

[0082] TGF-³ plays a crucial role in the regulation of the cell cycle. TGF-³ causes synthesis of p15 and p21 proteins, which block the cyclin:CDK complex responsible for Retinoblastoma protein (Rb) phosphorylation. Thus TGF-³ blocks advance through the G1 phase of the cycle. TGF-³ is necessary for CD4+, CD25+, Foxp3+, T-regulatory cell differentiation and suppressive function. In the presence of IL-6, TGF-³ contributes to the differentiation of pro- inflammatory Th17 cells.

[0083] IL-1RA. The interleukin-1 receptor antagonist (IL-1RA) is a protein that in humans is encoded by the IL1RN gene. A member of the IL-1 cytokine family, IL-1RA, is an agent that binds non-productively to the cell surface interleukin-1 receptor (IL-1R), preventing IL-1 from binding and inducing downstream signaling events. IL1RA is secreted by various types of cells includingAttorney Docket No.82092-000005-WO-POA immune cells, epithelial cells, and adipocytes, and is a natural inhibitor of the pro-inflammatory effect of IL-1α and IL1³. This gene and five other closely related cytokine genes form a gene cluster spanning approximately 400 kb on chromosome 2. Four alternatively spliced transcript variants encoding distinct isoforms have been reported. An interleukin 1 receptor antagonist is used in the treatment of rheumatoid arthritis, an autoimmune disease in which IL-1 plays a key role. It is commercially produced as anakinra, which is a human recombinant form of IL-1RA. Anakinra has shown both safety and efficacy in improving arthritis in an open trial on four SLE patients, with only short-lasting therapeutic effects in two patients.

[0084] Biomarker detection. There are a variety of methods that can be used to assess protein expression. One such approach is to perform protein identification with the use of antibodies.

[0085] As used herein, the term <antibody= refers, broadly, to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE antibody, or subclass thereof, or binding fragments thereof, including single chain fragments. Generally, IgG and / or IgM are used because they are the most common antibodies in the physiological situation and because are commonly and easily made in a laboratory setting. As used herein, the term <antibody fragment= refers to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab',Fab,F(ab') 2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Means for preparing and characterizing antibodies, both polyclonal and monoclonal, are also well known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; relevant portions incorporated herein by reference).

[0086] In accordance with the present invention, examples of immunodetection methods are provided. Some immunodetection methods include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few. The steps of various useful immunodetection methods have been described in the scientific literature, Current Protocols in Immunology, Wiley & Sons Press, 2017, relevant portions incorporated herein by reference.

[0087] In general, the immune-binding methods include obtaining a sample suspected of containing a relevant polypeptide, and contacting the sample with a first antibody under conditions effective to allow the formation of immunocomplexes. In terms of antigen detection, the biological sample analyzed may be any sample that is suspected of containing an antigen, such as, for example, a tissue section or specimen, a homogenized tissue extract, a cell, or even a biological fluid.Attorney Docket No.82092-000005-WO-POA

[0088] Contacting the chosen 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 antibodies to form immune complexes with, i.e., to bind to, any antigens present. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, microfluidic chamber, dot blot or western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.

[0089] In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. Patents concerning the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated herein by reference. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody and / or a biotin / avidin ligand binding arrangement, as is known in the art.

[0090] The antibody, or binding fragment thereof, can be employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a <secondary= antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non- specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

[0091] Further methods include the detection of primary immune complexes by a two-step approach. A second binding ligand, such as an antibody, which has binding affinity for the antibody is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The thirdAttorney Docket No.82092-000005-WO-POA ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide signal amplification if this is desired.

[0092] One method of immunodetection uses two different antibodies. A first step biotinylated, monoclonal or polyclonal antibody is used to detect the target antigen(s), and a second step antibody is then used to detect the biotin attached to the complexed biotin. In that method the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody / antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin. This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence of the antibody / antigen complex by histoenzymology using a chromogen substrate. With suitable amplification, a conjugate can be produced which is macroscopically visible.

[0093] Another known method of immunodetection takes advantage of the immuno-PCR (Polymerase Chain Reaction) methodology. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA / biotin / streptavidin / antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls. At least in theory, the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.

[0094] As detailed above, immunoassays are in essence binding assays. Certain immunoassays are the various types of ELISAs and RIA known in the art. However, it will be readily appreciated that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analyses, and the like may also be used.

[0095] In one exemplary ELISA, the antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the antigen, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection is generally achieved by the addition of another antibody that is linked to a detectable label. This type of ELISA is a simple <sandwich ELISA.= Detection may also be achieved by the addition of a second antibody, followed by the addition of a third antibodyAttorney Docket No.82092-000005-WO-POA that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

[0096] In some embodiments, the principles of ELISA may be employed in the context of a microfluidic cartridge, for example, a plate having 96 separate wells. This microfluidic cartridge allows for <multiplexing= by running several <single= ELISA-type assays in parallel using a nanoreactor. Unlike technologies that assess multiple analytes in a single well, the use of the microfluidic cartridge to implement the principles of ELISA allows for one (e.g., a single) analyte to be assessed per nanoreactor. Multiple nanoreactors can be fit on a single assay cartridge and run in parallel. This technique combines the advantages of multiplexing4sample sparing and cost savings4with the advantages of a single-analyte ELISA4no cross-reactivity or cross-inhibition from antibodies used to detect multiple analytes in the same well. Microfluidic ELISA approaches allow for include multiplexing (thereby reducing costs and labor), generation of more data with less sample, less labor and lower costs, faster, more reproducible results in comparison to solid, planar arrays, and focused, flexible multiplexing to meet a wide variety of applications.

[0097] In one exemplary ELISA, the analytes disclosed herein are immobilized onto a microfluidic surface exhibiting an affinity for a particular analyte, such as a well of a glass nanoreactor. A test composition suspected of containing the analyte, such as a clinical sample, is added to each well of the microfluidic device. After binding and washing to remove non- specifically bound immune complexes, the bound analyte may be detected. Detection is generally achieved by the addition of another antibody that is linked to a detectable label.

[0098] Additionally or alternatively, in some embodiments, the principles of ELISA may be implemented via other multiplexed immunoassays such as a proximity extension assay (e.g. commercially available from Olink company), an electrochemluminescence assay (e.g., commercially available from Meso Scale Discovery company), or a protein array-based assay.

[0099] In another exemplary ELISA, the samples suspected of containing the antigen are immobilized onto the well surface and then contacted with the anti-ORF message and anti-ORF translated product antibodies of the invention. After binding and washing to remove non- specifically bound immune complexes, the bound anti-ORF message and anti-ORF translated product antibodies are detected. Where the initial anti-ORF message and anti-ORF translated product antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-ORF message and anti-ORF translated product antibody, with the second antibody being linked to a detectable label.

[0100] Another type of ELISA in which the antigens are immobilized, involves the use ofAttorney Docket No.82092-000005-WO-POA antibody competition in the detection. In this ELISA, labeled antibodies against an antigen are added to the wells, allowed to bind, and detected by means of their label. The amount of an antigen in an unknown sample is then determined by mixing the sample with the labeled antibodies against the antigen during incubation with coated wells. The presence of an antigen in the sample acts to reduce the amount of antibody against the antigen available for binding to the well and thus reduces the ultimate signal. This is also appropriate for detecting antibodies against an antigen in an unknown sample, where the unlabeled antibodies bind to the antigen-coated wells and also reduces the amount of antigen available to bind the labeled antibodies.

[0101] As used herein, the phrase <under conditions effective to allow immune complex (antigen / antibody) formation= refers to those conditions, which may also include diluting the antigens and / or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS) / Tween, under which an antibody or binding fragment thereof interacts with the antigen that is the specific target of the antibody. These added agents also tend to assist in the reduction of nonspecific background. The <suitable= conditions such that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25°C. to 27°C. or maybe overnight at about 4°C. or so.

[0102] Another antibody-based approach to assessing biomarkers expression is Fluorescence- Activated Cell Sorting (FACS), a specialized type 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 upon the specific light scattering and fluorescent characteristics of each cell. It provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. A cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells relative to their diameter. A vibrating mechanism causes the stream of cells to break into individual droplets. The system is adjusted so that there is a low probability of more than one cell per droplet. Just before the stream breaks into droplets, the flow passes through a fluorescence measuring station where the fluorescent character of interest of each cell is measured. An electrical charging ring is placed just at the point where the stream breaks into droplets. A charge is placed on the ring based on the immediately prior fluorescence intensity measurement, and the opposite charge is trapped on the droplet as it breaks from the stream. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge. In some systems, the charge is applied directly to the stream, and the droplet breaking off retains charge of the same sign as the stream. The stream is then returned to neutral after the droplet breaksAttorney Docket No.82092-000005-WO-POA off. One common way to use FACS is with a fluorescently labeled antibody that binds to a target on or in a cell, thereby identifying cells with a given target. This technique can be used quantitatively where the amount of fluorescent activity correlates to the amount of target, thereby permitting one to sort based on relative amounts of fluorescence, and hence relative amounts of the target.

[0103] Bead-based xMAP Technology may also be applied to immunologic detection in conjunction with the presently claimed invention. This technology combines advanced fluidics, optics, and digital signal processing with proprietary microsphere technology to deliver multiplexed assay capabilities. Featuring a flexible, open- architecture design, xMAP technology can be configured to perform a wide variety of bioassays quickly, cost-effectively and accurately.

[0104] Fluorescently-coded microspheres are arranged in up to 500 distinct sets. Each bead set can be coated with a reagent specific to a particular bioassay (e.g., an antibody), allowing the 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 dyes that identify each microsphere particle, and also any reporter dye captured during the assay. Many readings are made on each bead set, which further validates the results. Using this process, xMAP technology allows multiplexing of up to 500 unique bioassays within a single sample, both rapidly and precisely. Unlike other flow cytometer microsphere-based assays which use a combination of different sizes and color intensities to identify an individual microsphere, xMAP technology uses micron size microspheres internally dyed with red and infrared fluorophores via a proprietary dying process to create 500 unique dye mixtures which are used to identify each individual microsphere.

[0105] Some of the advantages of xMAP include multiplexing (reduces costs and labor), generation of more data with less sample, less labor and lower costs, faster, more reproducible results than solid, planar arrays, and focused, flexible multiplexing of 1 to 500 analytes to meet a wide variety of applications.

[0106] Nucleic Acid Detection ln other embodiments for detecting protein expression, one may assay for gene transcription. For example, an indirect method for detecting protein expression is to detect mRNA transcripts from which the proteins are made.

[0107] Amplification of Nucleic Acids. Since many mRNAs are present 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 region of interest. As used herein, the term <primer,= refers to any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template -dependent process. Typically, primers areAttorney Docket No.82092-000005-WO-POA oligonucleotides from ten to twenty and / or thirty base pairs in length, but longer sequences can be employed. Primers may be provided in double-stranded and / or single-stranded form, although the single-stranded form is often used.

[0108] Pairs of primers designed to selectively hybridize to nucleic acids corresponding to selected genes are contacted with the template nucleic acid under conditions that permit selective hybridization. Depending upon the desired application, high stringency hybridization conditions may be selected that will only allow hybridization to sequences that are completely complementary to the primers ln other embodiments, hybridization may occur under reduced stringency to allow for amplification of nucleic acids containing one or more mismatches with the primer sequences. Once hybridized, the template-primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as <cycles,= are conducted until a sufficient amount of amplification product is produced.

[0109] The amplification product may be detected or quantified. In certain applications, the detection may be performed by visual method. Alternatively, the detection may involve indirect identification of the product via chemilluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical and / or thermal impulse signals.

[0110] A number of template dependent processes are available to amplify the 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. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, each of which is incorporated herein by reference in their entirety.

[0111] A reverse transcriptase-PCR amplification procedure may be performed to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are 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 methodologies are well known in the art. Representative methods of RT-PCR are described in U.S. Pat. No.5,882,864. Standard PCR usually uses one pair of primers to amplify a specific sequence, while multiplex-PCR (MPCR) uses multiple pairs of primers to amplify many sequences simultaneously. The presence of many PCR primers in a single tube could cause many problems, such as the increased formation of misprimed PCR products and <primer dimers,= the amplification discrimination of longer DNA fragment and so on. Normally, MPCR buffers contain a Taq Polymerase additive, which decreases the competition among amplicons and the amplification discrimination of longer DNA fragments during MPCR. MPCRAttorney Docket No.82092-000005-WO-POA products can further be hybridized with gene-specific probe for verification. Theoretically, one should be able to use as many as primers as necessary. However, due to side effects (primer dimers, misprimed PCR products, etc.) caused during MPCR, there is a limit (less than 20) to the number of primers that can be used in an MPCR reaction. See also European Application No. 0364255, relevant portions incorporated herein by reference.

[0112] Another method for amplification is ligase chain reaction (<LCR=), disclosed in European Application No. 320308, and 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 a target sequence. 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 amplification of target nucleic acid sequences that may be used in the practice of the present invention are disclosed in U.S. Pat. Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497, 5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905, 5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, GB Application No. 2202328, and in PCT Application No. PCT / US89 / 01025, from each relevant portions incorporated herein by reference.

[0113] Detection of Nucleic Acids. Following any amplification, it may be desirable to separate the amplification product from the template and / or the excess primer. In one embodiment, amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2001). Separated amplification products may be cut out and eluted from the gel for further manipulation. Using low melting point agarose gels, the separated band may be removed by heating the gel, followed by extraction of the nucleic acid. Separation of nucleic acids may also be affected by chromatographic techniques known in art. There are many kinds of chromatography which may be used in the practice of the present invention, including adsorption, partition, ion- exchange, hydroxylapatite, molecular sieve, reverse-phase, column, paper, thin-layer, and gas chromatography as well as HPLC.

[0114] In certain embodiments, the amplification products are visualized. A typical visualization method involves staining of a gel with ethidium bromide and visualization of bands under UV light. Alternatively, if the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the separated amplification products can be exposed to x-ray film or visualized under the appropriate excitatory spectra.

[0115] In one embodiment, following separation of amplification products, a labeled nucleic acid probe is brought into contact with the amplified marker sequence. The probe preferably is conjugated to a chromophore but may be radiolabeled. In another embodiment, the probe isAttorney Docket No.82092-000005-WO-POA conjugated to a binding partner, such as an antibody or biotin, or another binding partner carrying a detectable moiety.

[0116] In particular embodiments, detection is by Southern blotting and hybridization with a labeled probe. The techniques involved in Southern blotting are well known to those of skill in the art (see Sambrook et al, Molecular Cloning: A Laboratory Manual, 2001). One example of the foregoing is described in U.S. Pat. No. 5,279,721, incorporated by reference herein, which discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids. The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.

[0117] Other methods of nucleic acid detection that may be used in the practice of the instant invention are disclosed in U.S. Pat. Nos. 5,840,873, 5,843,640, 5,843,651, 5,846,708, 5,846,717, 5,846,726, 5,846,729, 5,849,487, 5,853,990, 5,853,992, 5,853,993, 5,856,092, 5,861,244, 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.

[0118] Nucleic Acid Arrays. Microarrays include a plurality of polymeric molecules spatially distributed over, and stably associated with, the surface of a substantially planar substrate, e.g., biochips. Microarrays of polynucleotides have been developed and find use in a variety of applications, such as screening and DNA sequencing. One area in particular in which microarrays find use is in gene expression analysis.

[0119] In gene expression analysis with microarrays, an array of <probe= oligonucleotides is contacted with a nucleic acid sample of interest, i.e., target, such as polyA mRNA from a particular tissue type. Contact is carried out under hybridization conditions and unbound nucleic acid is then removed. The resultant pattern of hybridized nucleic acid provides information regarding the genetic profile of the sample tested. Methodologies of gene expression analysis on microarrays are capable of providing both qualitative and quantitative information.

[0120] A variety of different arrays that may be used with the present invention are known in the art. The probe molecules of the arrays which are capable of sequence specific hybridization with target nucleic acid may be polynucleotides or hybridizing analogues or mimetics thereof, including: nucleic acids in which the phosphodiester linkage has been replaced with a substitute linkage, such as phophorothioate, methylimino, methylphosphonate, phosphoramidate, guanidine and the like; nucleic acids in which the ribose subunit has been substituted, e.g., hexose phosphodiester; peptide nucleic acids; and the like. The length of the probes will generally range from 10 to 1,000 nucleotides, where in some embodiments the probes will be oligonucleotides andAttorney Docket No.82092-000005-WO-POA usually range from 15 to 150 nucleotides and more usually from 15 to 100 nucleotides in length, and in other embodiments the probes will be longer, usually ranging in length from 150 to 1,000 nucleotides, where the polynucleotide probes may be single- or double-stranded, usually single- stranded, and may be PCR fragments amplified from cDNA.

[0121] The probe molecules on the surface of the substrates will correspond to selected genes being analyzed and be positioned on the array at a known location so that positive hybridization events may be correlated to expression of a particular gene in the physiological source from which the target nucleic acid sample is derived. The substrates with which the probe molecules are stably associated may be fabricated from a variety of materials, including plastics, ceramics, metals, gels, membranes, glasses, and the like. The arrays may be produced according to any convenient methodology, such as preforming the probes and then stably associating them with the surface of the support or growing the probes directly on the support. A number of different array configurations and methods for their production are known to those of skill in the art and disclosed in U.S. Pat. Nos. 5,445,934, 5,532,128, 5,556,752, 5,242,974, 5,384,261, 5,405,783, 5,412,087, 5,424,186, 5,429,807, 5,436,327, 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, relevant portions incorporated herein by reference.

[0122] Following hybridization, where non-hybridized labeled nucleic acid is capable of emitting a signal during the detection step, a washing step is employed where unhybridized labeled nucleic acid is removed from the support surface, generating a pattern of hybridized nucleic acid on the substrate surface. A variety of wash solutions and protocols for their use are known to those of skill in the art and may be used. Where the label on the target nucleic acid is not directly detectable, one then contacts the array, now comprising bound target, with the other member(s) of the signal producing system that is being employed. For example, where the label on the target is biotin, one then contacts the array with streptavidin-fluorescent conjugate under conditions sufficient for binding between the specific binding member pairs to occur. Following contact, any unbound members of the signal producing system will then be removed, e.g., by washing. The specific wash conditions employed will necessarily depend on the specific nature of the signal producing system that is employed, and will be known to those of skill in the art familiar with the particular signal producing system employed. The resultant hybridization pattern(s) of labeled nucleic acids may be visualized or detected in a variety of ways, with the particular manner of detection being chosen based on the particular label of the nucleic acid, where representative detection means include scintillation counting, autoradiography, fluorescence measurement, calorimetric measurement, light emission measurement and the like.Attorney Docket No.82092-000005-WO-POA

[0123] Prior to detection or visualization, where one desires to reduce the potential for a mismatch hybridization event to generate a false positive signal on the pattern, the array of hybridized target / probe complexes may be treated with an endonuclease under conditions sufficient such that the endonuclease degrades single stranded, but not double stranded DNA. A variety of different endonucleases are known and may be used, where such nucleases include: mung bean nuclease, Sl nuclease, and the like. Where such treatment is employed in an assay in which the target nucleic acids are not labeled with a directly detectable label, e.g., in an assay with biotinylated target nucleic acids, the endonuclease treatment will generally be performed prior to contact of the array with the other member(s) of the signal producing system, e.g., fluorescent- streptavidin conjugate. Endonuclease treatment, as described above, ensures that only end-labeled target / probe complexes having a substantially complete hybridization at the 3' end of the probe are detected in the hybridization pattern. Following hybridization and any washing step(s) and / or subsequent treatments, as described above, the resultant hybridization pattern is detected. In detecting or visualizing the hybridization pattern, the intensity or signal value of the label will be not only be detected but quantified, by which is meant that the signal from each spot of the hybridization will be measured and compared to a unit value corresponding the signal emitted by known number of end-labeled target nucleic acids to obtain a count or absolute value of the copy number of each end-labeled target that is hybridized to a particular spot on the array in the hybridization pattern.

[0124] RNA Sequencing (Transcript Counting). RNA-seq (RNA Sequencing), also called Whole Transcriptome Shotgun Sequencing (WTSS), is a technology that utilizes the capabilities of Next- Generation Sequencing (NGS) to reveal a snapshot of RNA presence and quantity from a genome at a given moment in time. The transcriptome of a cell is dynamic; it continually changes as opposed to a static genome. The recent developments of next-generation sequencing allow for increased base coverage of a DNA sequence, as well as higher sample throughput. This facilitates sequencing of the RNA transcripts in a cell, providing the ability to look at alternative gene spliced transcripts, post-transcriptional changes, gene fusion, mutations / SNPs and changes in gene expression. In addition to mRNA transcripts, RNA-Seq can look at different populations of RNA to include total RNA, small RNA, such as miRNA, tRNA, and ribosomal profiling. RNA-Seq can also be used to determine exon / intron boundaries and verify or amend previously annotated 5' and 3' gene boundaries, Ongoing RNA-Seq research includes observing cellular pathway alterations during infection, and gene expression level changes in cancer studies. Prior to NGS, transcriptomics and gene expression studies were previously done with expression microarrays, which contain thousands of DNA sequences that probe for a match in the target sequence, makingAttorney Docket No.82092-000005-WO-POA available a profile of all transcripts being expressed. This was later done with Serial Analysis of Gene Expression (SAGE).

[0125] Treatments for SLE. The present subject matter contemplates the detection of certain biomarkers followed by a change in the treatment of SLE, which may include using standard therapeutic approaches where indicated. In general, the treatment of SLE involves treating elevated disease activity and trying to minimize the organ damage that can be associated with increased inflammation and increased immune complex formation / deposition / complement activation. Foundational treatment can include corticosteroids and / or anti-malarial drugs. Certain types of lupus nephritis such as diffuse proliferative glomerulonephritis require bouts of cytotoxic drugs. These drugs include, most commonly, cyclophosphamide and mycophenolate. 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. Anifrolumab (Saphnelo) can be used as a treatment for elevated disease activity seen in adult lupus patients.

[0126] Due to the variety of symptoms and organ system involvement with SLE, its severity in an individual must be assessed in order to successfully treat SLE. Mild or remittent disease may, sometimes, be safely left minimally treated with hydroxychloroquine alone. If required, nonsteroidal anti-inflammatory drugs and low dose steroids may also be used. Hydroxychloroquine (HCQ) is an FDA-approved antimalarial used for constitutional, cutaneous, and articular manifestations. HCQ has relatively few side effects, and there is evidence that it improves survival among people who have SLE and stopping HCQ in stable SLE patients led to increased disease flares in Canadian lupus patients. Disease-modifying antirheumatic drugs (DMARDs) are often used off-label in SLE to decrease disease activity and lower the need for steroid use. DMARDs commonly in use are methotrexate and azathioprine. In more severe cases, medications that aggressively suppress the immune system (primarily 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 life-threatening or organ-damaging complications, such as vasculitis and lupus cerebritis. Mycophenolic acid is also used for treatment of lupus nephritis, but it is not FDA-approved for this indication.

[0127] Depending on the dosage, people who require steroids may develop Cushing's symptoms of truncal obesity, purple striae, buffalo hump and other associated symptoms. These may subside if and when the large initial dosage is reduced, but long-term use of even low doses can cause elevated blood pressure, glucose intolerance (including metabolic syndrome and / or diabetes), osteoporosis, insomnia, avascular necrosis and cataracts. More severe steroid-associatedAttorney Docket No.82092-000005-WO-POA morbidities include accelerated atherosclerosis, avascular necrosis, increased infections, diabetes, and hypertension.

[0128] Numerous new immunosuppressive drugs are being actively tested for SLE. Rather than suppressing the immune system nonspecifically, as corticosteroids do, they target the responses of individual types of immune cells. Belimumab, or a humanized monoclonal antibody against B-lymphocyte stimulating factor (BLyS or BAFF), is FDA approved for lupus treatment and decreased SLE disease activity, especially in patients with baseline elevated disease activity and the presence of autoantibodies. Addition drugs, such as abatacept, voclosporin, JAK inhibitors, Tyk inhibitors, anifrolimab, and others, are actively being studied in SLE patients and some of these drugs are already FDA-approved for treatment of rheumatoid arthritis or other disorders. Since a large percentage of people with SLE suffer from varying amounts of chronic pain, stronger prescription analgesics (pain killers) may be used if over-the-counter drugs (mainly nonsteroidal anti-inflammatory drugs) do not provide effective relief. Potent NSAIDs such as indomethacin and diclofenac are relatively contraindicated for patients with SLE because they increase the risk of kidney failure and heart failure.

[0129] Moderate pain is typically treated with mild prescription opiates such as dextropropoxyphene and co-codamol. Moderate to severe chronic pain is treated with stronger opioids, such as hydrocodone or longer-acting continuous-release opioids, such as oxycodone, MS Contin, or methadone. The fentanyl duragesic transdermal patch is also a widely used treatment option for the chronic pain caused by complications because of its long-acting timed release and ease of use. When opioids are used for prolonged periods, drug tolerance, chemical dependency, and addiction may occur. Opiate addiction is not typically a concern, since the condition is not likely to ever completely disappear. Thus, lifelong treatment with opioids is fairly common for chronic pain symptoms, accompanied by periodic titration that is typical of any long-term opioid regimen.

[0130] Intravenous immunoglobulins may be used to control SLE with organ involvement, or neuropathy. It is believed that they reduce antibody production or promote the clearance of immune complexes from the body, even though their mechanism of action is not well-understood. Unlike immunosuppressives and corticosteroids, IVIGs do not suppress the immune system, so there is less risk of serious infections with these drugs.

[0131] Avoiding sunlight is the primary change to the lifestyle of SLE sufferers, as sunlight is known to exacerbate the disease, as is the debilitating effect of intense fatigue. These two problems can lead to patients becoming housebound for long periods of time. Drugs unrelated to SLE should be prescribed only when known not to exacerbate the disease. Occupational exposureAttorney Docket No.82092-000005-WO-POA to silica, pesticides and mercury can also make the disease worsen.

[0132] Renal transplants are the treatment of choice for end-stage renal disease, which is one of the complications of lupus nephritis, but the recurrence of the full disease in the transplanted kidney is common in up to 30% of patients.

[0133] Antiphospholipid syndrome is also related to the onset of neural lupus symptoms in the brain. In this form of the disease the cause is very different from lupus: thromboses (blood clots or <sticky blood=) form in blood vessels, which prove to be fatal if they move within the blood stream. If the thromboses migrate to the brain, they can potentially cause a stroke by blocking the blood supply to the brain. If this disorder is suspected in patients, brain scans are usually required for early detection. These scans can show localized areas of the brain where blood supply has not been adequate. The treatment plan for these patients requires anticoagulation. Often, low-dose aspirin is prescribed for this purpose, although for cases involving thrombosis anticoagulants such as warfarin are used.

[0134] Pharmaceutical Formulations and Delivery. A change in therapeutic application is contemplated, it will be necessary to prepare pharmaceutical compositions in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.

[0135] Generally, appropriate salts and buffers are employed to render delivery vectors stable and allow for uptake by target cells. Buffers also will be employed when recombinant cells are introduced into a patient. Aqueous compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula.

[0136] As used herein, the phrases <pharmaceutically= or <pharmacologically acceptable=, refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, <pharmaceutically acceptable carrier= includes 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. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.

[0137] The active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. Such routes include oral,Attorney Docket No.82092-000005-WO-POA nasal, buccal, rectal, vaginal or topical routes. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions. The active compounds may also be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0138] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0139] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0140] As used herein, the phrase <pharmaceutically acceptable carrier= refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorptionAttorney Docket No.82092-000005-WO-POA delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0141] For oral administration the polypeptides of the present invention may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices. A mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.

[0142] Compositions for use with the present invention may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. In this connection, sterile aqueous media, which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1,000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, <Remington's Pharmaceutical Sciences,= l5th Ed., 1035-1038 and 1570-1580), relevant portions incorporated by reference. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards.Attorney Docket No.82092-000005-WO-POA

[0143] Kits. For use in the applications described herein, kits are also within the scope of the invention. Such kits can comprise a carrier, package or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method, in particular, a Bright inhibitor. The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial end user standpoint, 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 can also indicate directions for either in vivo or in vitro use, such as those described above. Directions and or other information can also be included on an insert, which is included with the kit. In particular, kits according to the present invention contemplate the assemblage of agents for assessing levels of the biomarkers discussed above along with one or more of an SLE therapeutic and / or a reagent for ANA testing and / or anti-ENA, as well as controls for assessing the same.

[0144] Current biomarkers in lupus disease activity measures have limited utility for reflecting heightened clinical disease activity. They are neither the earliest, nor most informative, measurements 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 not sufficient to reflect clinical disease activity and risk for permanent organ damage. ANAs are also found in sera from patients with other systemic rheumatic diseases, and from 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 may remain healthy despite being ANA-positive, ANA positivity alone is likely not the sole pathogenic driver of SLE. In addition to ANA positivity, the dysregulation of various immune pathways driven by soluble mediators may contribute to clinical disease activity. EXAMPLES

[0145] The following examples are included to further illustrate various aspects of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples, which follow, represent techniques and / or compositions discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Attorney Docket No.82092-000005-WO-POA

[0146] SLE is a complex autoimmune disease marked by immune dysregulation. A comprehensive but cost-effective tool to track relevant mediators of altered disease activity would help improve disease management and prevent organ damage. The goal of this example was to identify critical components of a practical biometric to distinguish active from low lupus disease activity.

[0147] The following examples demonstrate the determination of an optimal panel of markers that can distinguish those SLE patients with active disease and help to refine a Lupus Disease Activity (Immune) Index (LDAII / L-DAI). The refined LDAII / L-DAI developed from the following examples allows characterization of SLE patients with active clinical disease. Treatment to target using a sensitive and objective biologic surrogate for clinical disease activity could help improve disease management and prevent organ damage in SLE.

[0148] In these examples, samples were procured from patients with classified SLE on dates of low disease activity (<4, range 0-3, n=162) or active disease (g4, range 4-30, n=162) defined by the hybrid SLEDAI (hSLEDAI). Race / sex / age-matched healthy control (Ctrl) samples (n=81) were also evaluated. Plasma immune mediators (n=33) were evaluated by microfluidic immunoassay and serum AutoAb specificities, including dsDNA, chromatin, Ro / SSA, La / SSB, Sm, SmRNP, and RNP, were assessed by xMAP assay. The LDAII / L-DAI is the sum of log- transformed, standardized immune mediators, weighted by the Spearman r correlation coefficient of mediator levels vs. either hSLEDAI scores, number of AutoAb specificities associated with clinical disease activity, or a composite weighting (average of hSLEDAI and AutoAb weighting). Log-transformed mediator levels were further evaluated using random forest applied machine learning modeling to determine an optimal subset of analytes to inform the LDAII / L-DAI.

[0149] SLE patients with active disease demonstrated differences in clinical and serologic features, as well as increased use steroids. Random forest modeling of immune mediators comparing low vs. active disease, including clinically and / or serologically active vs. quiescent disease and Ctrls informed a variable importance ranking. Forward selection (adding mediators to top mediator, IFN-α) and backward elimination (subtracting mediators from total), in combination with univariate analysis, yielded a baseline combination of 9 mediators that best informed the LDAII / L-DAI, including IFN-α, IL-10, BLyS, IL-7, IFN-´, TRAIL, IL-15, IP- 10 / CXCL10, and IL-4. The L-DAI 9, whether weighted by hSLEDAI or number of AutoAbs, significantly (p≤0.05) differentiated SLE patients with Low vs. Active disease, including clinically / serologically active / quiescent disease (vs. Ctrls), with strong correlation between the weightings (Spearman r=0.990, p<0.0001). An average composite LDAII / L-DAI differentiated SLE patients with low, active, and clinically / serologically active / quiescent disease (vs. Ctrls).Attorney Docket No.82092-000005-WO-POA There was enhanced distinction of low / moderate / high risk regions for active disease after application of decision curve analysis, with large (>0.8) Cohen’s effect size and AUC=0.795 (p<0.0001) in distinguishing clinical / serologic active / quiescent disease activity, and significant correlation (Spearman r=0.407, p<0.0001) with concurrent clinical disease activity (hSLEDAI scores). To maximize clinical effectiveness of the LDAII / L-DAI, combinations of the soluble mediators TNFRII, Resistin, and Osteopontin (OPN) were also considered in conjunction with the baseline LDAII / L-DAI 9 mediators to further delineate clinical disease activity by organ system manifestation.

[0150] Materials and Methods

[0151] Study population, clinical data, and sample collection

[0152] Experiments were performed in accordance with the Helsinki Declaration and approved by Institutional Review Boards of the Oklahoma Medical Research Foundation (OMRF) and Progentec Diagnostics, Inc. Study participants in established, prospectively assessed longitudinal cohorts, meeting American College of Rheumatology and Systemic Lupus International Collaborating Clinics classification for SLE (Ines et al., 2015), were appropriate consented and evaluated for disease activity as defined by the hybrid Systemic Lupus Erythematosus Disease Activity Index (hSLEDAI) (Thanou et al., 2016). Plasma and serum samples were procured from clinic visits where SLE patients exhibited low (hSLEDAI < 4; n=162) or active (hSLEDAI g 4; n=162) disease and compared to sex / race / age matched healthy individuals (Ctls; n=81), Table 1. Samples were stored at -80°C in the Oklahoma Rheumatic Diseases Research Core Center (ORDRCC), CAP-certified, biorepository at OMRF or at the Progentec biorepository until time of assay on freshly thawed samples. Samples in the biorepositories have been tested with respect to shipment time and method, processing procedures, storage conditions, and length of storage for the ability to determine levels of soluble mediators and SLE-associated autoantibodies (AutoAbs) in samples from SLE patients and Ctls.Attorney Docket No.82092-000005-WO-POA Table 1. SLE Patient Sample Characteristics Disease Activitya: Low Active p-valueb-- 221 0 0 0 2 1 0 1 1 1 1 1 1 9 4 2 5 2 8 0 2 0 7 1 1 0 7 0 5 5bContinuous variable significance by Kruskal-Wallis with Dunns multiple comparison or Mann-Whitney test; Categorical variable significance by Fisher's exact orχ2 test (unadjusted significant p≤0.05 ) cPacific Islander, Hispanic, or Multi-racedhSLEDAI = SELENA-SLEDAI + proteinuria defined by SLEDAI-2KeImmunosuppressants = azathioprine, methotrexate, sirolimus, tacrolimus; Major Immunosuppressants = mycophenolate mofetil, cyclophosphamide; Biologics = rituximab, abatacept, belimumabf# of AutoAb specificities include anti-dsDNA, chromatin, Ro / SSA, La / SSB, Sm, SmRNP, and RNP (Low [n=155], Active [n=158])Attorney Docket No.82092-000005-WO-POA

[0153] Demographic and clinical information were collected as previously described (Crowe et al., 2011), including medication usage, clinical laboratory values, and clinical disease activity. The presence of organ system involvement was evaluated by the administration of the hybrid Systemic Lupus Erythematosus Disease Activity Index (hSLEDAI; SELENA-SLEDAI with proteinuria as defined by SLEDAI-2K) (Thanou et al., 2014; Thanou et al., 2016), including the presence of disease manifestations involving the central nervous system (CNS; seizure, psychosis, organic brain syndrome, visual disturbance, cranial nerve disorder, or lupus headache), vasculitis, arthritis, myositis, nephritis (urinary casts, hematuria, proteinuria, or pyuria), mucocutaneous damage (rash, alopecia, or mucosal ulcers), serositis (pleuritis or pericarditis), or hematologic manifestations (low complement, increased DNA binding, fever, thrombocytopenia, or leukopenia) (Petri et al., 2005).

[0154] Measurement of soluble mediators and autoantibody specificities

[0155] Serum samples were screened for autoantibody specificities using the BioPlex 2200 multiplex system (Bio-Rad Technologies, Hercules, CA), Table 1. The BioPlex 2200 ANA kit uses fluorescently dyed magnetic beads for simultaneous detection of 11 autoantibody specificity levels, including reactivity to dsDNA, chromatin, ribosomal P, Ro / SSA, La / SSB, Sm, the Sm / RNP complex, RNP, Scl-70, centromere B, and Jo-1 (Bruner et al., 2012). SLE-associated autoantibody specificities to dsDNA, chromatin, Ro / SSA, La / SSB, Sm, Sm / RNP complex, and RNP were used for analysis in the current study. Anti-dsDNA (IU / mL) has a previously determined positive cutoff of 10 IU / mL; an Antibody Index (AI) value (range 0-8) is reported by the manufacturer to reflect the fluorescence intensity of each of the other autoantibody specificities with a positive cutoff of AI=1.0. The AI scale is standardized relative to calibrators and control samples provided by the manufacturer.

[0156] Thirty-three (33) soluble mediators were selected for assessment in this LDAII / L-DAI confirmatory / refinement cohort study (Table 2). Plasma soluble mediators assessed included cytokines, chemokines, and soluble receptors. Equal numbers of paired low / active disease SLE samples and their appropriate race / sex / age-matched controls were assessed on each assay cartridge set (33 mediators were assessed across a single lot of custom-designed, single / multi-analyte cartridges, per manufacturer’s recommendation). In addition, at least two races were represented on each cartridge set, and no race was assessed on only one cartridge set to achieve equal distribution and limit batch effects within the dataset.Attorney Docket No.82092-000005-WO-POA Table 2. SLE-associated mediator pathways assessed Innate Th1 like TNFR Superfamily IL-1´ IL-12 (p70)BLySIL-1RA IFN-γ Fas IFN-³ IL-2R³ TNF-³ TNFRI (p55) Homeostasis Th-17 like TNFRII (p75) IL-7 IL-6 TRAIL IL-15 IL-17A Chemokines Other / Inflammatory Th-2 likeMCP-1 / CCL2Osteopontin IL-4MIP-1³ / CCL3Resistin IL-5MIP-1´ / CCL4SCF IL-13 RANTES / CCL5 MCP-3 / CCL7 Regulatory IL-8 / CXCL8 IL-10 MIG / CXCL9 Total TGF-´IP-10 / CXCL10 Native TGF-´

[0157] Manufacturer-provided Lo / Med / Hi quality control (QC) samples for each measured analyte were assessed alongside SLE and healthy individual plasma samples on each assay cartridge, per manufacturer SOP protocol. These QC samples were prepared, as directed, just prior to performance of the cartridge assay in the Ella machine. Because only two cartridges (of seven) could be assessed at any given time, in addition, a thaw control was added such that a QC sample in concentration range of the plasma samples assessed was thawed and handled concurrently with the plasma samples to be assessed per SOP protocol. The mean Coefficient of Variation (CV) for the QC / Thaw samples in all analytes assessed was <10% across the lot of cartridges required to assess 405 plasma samples plus QC / Thaw controls. All Lo / Med / Hi and Thaw control QC samples reported within manufacturer specified concentration range for each analyte. In addition, the Thaw control QC sample was within 10% of the matched QC sample concentration for each analyte that prepared just before assay, indicating minimal effect of time to assay on the Ella cartridges / samples.

[0158] Samples with concentration <LLOQ were assigned a value of 0.001 pg / ml and a de- identified dataset containing demographic (race, sex, age), clinical (disease activity, medications, co-morbidities), and biological (soluble mediator, AutoAb specificities) data for subsequent sub- analyses was generated for univariate and multivariable analysis. IL-2 and IL-1α were excluded from univariate and multivariable analysis due to >60% of the plasma sample concentrations having values <LLOQ (0.001 pg / ml).

[0159] Statistical AnalysesAttorney Docket No.82092-000005-WO-POA

[0160] Categorical variables were compared by Fisher’s exact test. Disease activity scores in low vs. active disease clinical visits were compared using unpaired t-test. Number of autoantibody specificities and plasma soluble mediator concentrations were compared between SLE patient visits with low or active disease by Mann-Whitney test. Plasma mediator concentrations and number of autoantibody specificities were compared between SLE patient visits with low and active disease and race / sex matched Ctl samples by Kruskal-Wallis test with Dunn’s multiple comparison. Plasma mediator concentrations were correlated with hSLEDAI scores or number of autoantibody specificities by Spearman’s rank correlation. Effect size was determined using Cohen’s d (Cohen, 1992), the mean difference between active (M1) and low (M2) SLE disease activity groups divided by the pooled standard deviation ([M2-M1] / SDpooled, where SDpooled = √[{SD12+SD22] / 2]). Multiple comparison p-values were corrected using Bonferroni correction, dividing an alpha of 0.05 (minimum significant p-value) by number of statistical tests for a given category of variables.

[0161] Random forest partition tree classification based on Genuer et al. (Genuer et al., 2010), able to incorporate repeated measures (Capitaine et al., 2021), was implemented to rank variables in their ability to differentiate SLE patient visits with active or low disease activity vs. Ctls, as well as SLE patient visits with clinically and / or serologically (positive anti-dsDNA AutoAb and / or low complement levels) active or quiescent disease vs. Ctls. Settings used included maximum number of trees = 50 and maximum number of variables to consider for splitting a node = 100, with 5-fold cross validation conducted across 2,000 iterations. Variables were ordered from most to least informative based on variable importance. Variables were then applied to the LDAII / L-DAI by adding one at a time (starting with most informative variable; forward selection) or removing one at a time from the total list of variables (starting with least informative variable; backward elimination) (Genuer et al., 2010) to optimally predict disease activity outcome.

[0162] A Lupus Disease Activity (Immune) Index (LDAII / L-DAI) calculation was developed to compare the overall level of inflammation during patient visits with varying levels of disease activity, distinguished from Ctls. The LDAII / L-DAI summarizes the dysregulation of plasma mediators assessed in SLE patients at clinic visits with low and active disease and matched Ctls, weighted by their correlation to the, hSLEDAI score, number of AutoAb specificities detected from samples procured at the same visit, or an average of the two to provide a composite score. The LDAII / L-DAI was calculated as follows: 1. The concentrations of baseline plasma mediators selected for statistical analysis, Table 2, were log-transformed for each SLE patient or Ctl visit. 2. Each log-transformed soluble mediator level for each participant visit was standardized: (observed value)-(mean value of all SLE patient and Ctl visits) / (standard deviation of all SLE patient and CtlAttorney Docket No.82092-000005-WO-POA visits) 3. Spearman coefficients were generated from a linear regression model testing associations between the number of AutoAb specificities each soluble mediator assessed in all SLE patient and Ctl visits, with alternate associations between hSLEDAI scores and each soluble mediator assessed in all SLE patient visits (Spearman r); 4. The transformed and standardized soluble mediator levels at were weighted (multiplied) by their respective Spearman coefficients (Spearman r).5. For each participant visit, the log transformed, standardized and weighted values for each soluble mediator informing the LDAII / L-DAI were summed to calculate a total LDAII / L-DAI.

[0163] The LDAII / L-DAI was compared between SLE patient visits with low vs. active disease or clinically and serologically active (CASA) vs. quiescent (CQSQ) disaease by Mann-Whitney test, and additionally to Ctls by Kruskal-Wallis test with Dunn’s multiple comparison. Odds ratio (OR) was determined for the likelihood of SLE patient visits with active disease vs. low disease activity (or Ctl) to have a positive or negative LDAII / L-DAI score, respectively; significance for was determined by Fisher’s exact test.

[0164] Logistic regression was performed to determine threshold probabilities for active disease based on LDAII / L-DAI scores in active vs. low disease activity samples, as well as CASA vs. CQSQ samples. Low / medium and medium / high risk LDAII / L-DAI score cutoffs were further determined using decision curve analysis, as previously described (Vickers et al., 2008). Briefly, the threshold probability of flare risk for each active / low (or CASA / CQSQ) LDAII / L-DAI score was compared to net benefit, such that <net benefit= = True positive count / n 3 (False positive count / n * [pt / 1-pt]), where n is the total number of patients in the study and pt is the (predictive) threshold probability for any given LDAII / L-DAI score. To create the decision curve, the threshold probability (pt) is varied to cover the range of threshold probabilities associated with the active and low (or CASA / CQSQ) LDAII / L-DAI scores included in the analysis. For each pt: 1. Define an active (or CASA) visit as true positive if active (or CASA) pt g selected pt; 2. Define a low disease activity (or CQSQ) visit as false positive if low ptg selected pt; 3. Calculate the number of true and false positives for a given pt; 4. Calculate net benefit True positive count / n 3 (False positive count / n * [pt / 1-pt]) (Vickers et2008).

[0165] Univariate analyses, logistic regression, and decision curve graphing were performed using GraphPad Prism 9.5.1 (GraphPad Software, San Diego, CA). Multivariate random forest was performed using JMP® Genomics, Version 9. SAS Institute Inc., Cary, NC, 198932021.

[0166] Results

[0167] Increased number of AutoAb specificities and altered levels of select mediators with clinically active disease

[0168] Based on the hypothesis that, similar to transition to classified SLE, immuneAttorney Docket No.82092-000005-WO-POA dysregulation would be reflected in SLE patients with clinically active disease more so than during periods of low disease activity or quiescent disease, serum autoantibody and plasma soluble mediator profiles were compared in samples procured during clinic visits reflecting active (hSLEDAI g 4. Range 4-30) vs. low disease activity (hSLEDAI < 4, range 0-3), Table 1. As expected, SLE patients with active disease had significantly higher hSLEDAI scores and were more likely to exhibit various organ system manifestations than SLE patients with low disease activity. In addition, SLE patients with active disease exhibited a significant increase in the number of SLE-associated AutoAb specificities compared to the number detected during low disease activity (p=0.0007). AutoAbs against dsDNA (p<0.0001) and chromatin (p=0.0001) were more likely to be positive during periods of active disease, while the frequency of Ro / SSA, La / SSB, Sm, SmRNP, and RNP AutoAb specificities were similar in low and active disease after adjusting for multiple comparison (Bonferroni corrected significant p =0.0071), Table 1. SLE patients with active disease were more likely to be prescribed steroids (p=0.0002) and similarly prescribed hydroxychloroquine and / or immunosuppressants as patients with low disease activity after adjusting for multiple comparison (Bonferroni corrected significant p =0.0100), Table 1.

[0169] Whether SLE patients with active disease also had alterations in SLE- associated soluble mediators was also evaluated, as shown in Table 3. After adjusting for multiple comparison (Bonferroni corrected significance p=0.0015), plasma levels of 20 mediators were significantly altered in SLE patients compared to matched HC, irrespective of SLE clinical disease activity, including innate, adaptive, chemokine, and other inflammatory mediators. Three mediators, TNFRII (p<0.0001), MIP-1α / CCL3 (p<0.0001), and MIP-1³ / CCL4 (p=0.0008). Seven select soluble mediators, including IFN-α (p<0.0001), IL-10 (p<0.0001), TNF-α (p<0.0001), TNFRII (p=0.0003), TRAIL (p=0.0012), MIG / CXCL9 (p<0.0001), and IP-10 / CXCL10 (p<0.0001) were elevated in SLE patients with active disease compared to those with low clinical disease activity; 10 other mediators also displayed univariate significance, but were no longer significant after correction for multiple comparison, Table 3.Attorney Docket No.82092-000005-WO-POA Table 3. Soluble Mediators in SLE Cases vs. Healthy Controls Active Low vs Active Case Breakout vs Ctl Ctl vs Low AnalyteaCategory ActivebMean SEM LowbMean SEM Ctl Mean SEM p-valuecp-valuecp-valuec605 199 001 686 030 205 054031 287 668 860 421 595 001 499754 017 040001050 003 024012 289 333 044918 232 001 001088 941 817Significance determined by Kruskal-Wallis test with Dunn's multiple comparison (unadjusted p≤0.05) ; Bonferroni corrected significance p=0.0015

[0170] We further evaluated the ability of soluble mediators to distinguish between visits of SLE patients with clinically (C) and / or serologically (S, defined by immunologic hSLEDAI features of anti-dsDNA AutoAb positive ± low complement levels) active (A) or quiescent (Q) disease vs. Ctls, Table 4. After adjusting for multiple comparison (Bonferroni corrected significance p=0.0015), plasma levels of 16 mediators were significantly altered in SLE patient visits vs Ctl, irrespective of clinical and / or serologic disease activity or quiescence (p≤0.0008). Those only heightened in clinically active disease vs. Ctl, irrespective of serologic activity included IL-6, TRAIL, MIP-1α / CCL3, and MIP1-³ / CCL4 (pf0.0013), with IFN-´ and MIG / CXCL10 heightened in all SLE samples vs. Ctl except quiescent disease (CQSQ; p<0.0001). After adjusting for multiple comparison (Bonferroni corrected significance p=0.0015), ten mediators differentiated CASA from CQSQ, including IFN-α, IL-15, IFN-´, IL-10, TNF-α, TNFRI, TNFRII, TRAIL, MIG / CXCL9, and IP-10 / CXCL10 (p≤0.0008), while seven mediators differentiated CASA fromAttorney Docket No.82092-000005-WO-POA CASQ, including IFN-α, IL-10, TNF-α, TNFRII, MIP-1α / CCL3, MIP-1³ / CCL4, and IP- 10 / CXCL10 (p≤0.0009). These data indicate that a number of select mediators may be altered in direct association with clinically and / or serologically active disease in SLE. s e 6 2 vlArSu0 l 0 2 0 1 0 00 0 9 0 0 7 2 3 4 2 2 8 0 1 1 3 03 00 00 0 1 0 0 71 0 43 0 5 6 0 83 40 tCAaCv 0.- 0.000.1.00.0< 0.00.009.00.10.0.00.0< 01.< 0.002. .0< 0 0 p < 3 4 0 6 0 4 0 0 8 3 3 0 0the presence of AutoAbs andAttorney Docket No.82092-000005-WO-POA clinical disease activity

[0172] Multiple soluble mediators (24 / 33 assessed) significantly (p≤0.0015 with multiple comparison correction) correlated with the number of accumulated SLE- associated AutoAbs present in SLE patients and matched healthy controls, including innate and adaptive mediators, chemokines, and TNF superfamily members, Table 5. Of interest, the native form of the regulatory mediator, TGF-³, negatively correlated with the presence of AutoAb specificities (Spearman r = -0.263, p<0.0001). Conversely, IL-10, which can serve as both a regulatory mediator and an activator of B-lymphocytes, positively correlated with the accumulation of AutoAb specificities (Spearman r = 0.478, p<0.0001). Table 5. Spearman Correlation Between Soluble Mediators vs. # Autoantibody Specificities or hSLEDAI Scores vs. # AutoAbsavs. hSLEDAI ScorebAnalyte Category Spearman r 95% CI p-value Spearman r 95% CIp-valuec95 49 01 10 01 13 04 03 25 089571 56 0198 39 01 10 01 09 01 06 03 901931 7243 01 01 11 18 76= - p Significance determined by Spearman correlation (unadjusted p≤0.05) ; Bonferroni corrected significance p=0.0015

[0173] A more distinct set of biomarkers directly correlates with clinical disease activity (hSLEDAI score, Table 5). Fifteen (15) of 33 mediators assessed significantly correlated withAttorney Docket No.82092-000005-WO-POA hSLEDAI scores after multiple comparison correction (Bonferonni corrected p=0.0015), including IFN-associated mediators IFN-α (r = 0.360), MIG / CXCL9 (r = 0.252), and IP- 10 / CXCL10 (r = 0.310), TNF superfamily members TNF-α (r = 0.275) and TNFRII (r=0.270), as well as activating mediators IL-10 (r=0.308) and OPN (r=0.180). Nine additional soluble mediators, including IFNs, TNF mediators, and chemokines, were initially found to be significant prior to correcting for multiple comparisons, Table 5.

[0174] Refinement of a weighted Lupus Disease Activity (Immune) Index (LDAII / L-DAI) for clinical application to characterize active disease in SLE patients.

[0175] LDAII / L-DAI performance combined with technical feasibility and cost-effectiveness of running the laboratory-based test was subsequently considered to refine which mediators inform a refined LDAII / L-DAI for clinical application. Random forest variable importance analysis was used to rank most to least informative immune soluble mediators that best differentiated SLE visits where active or low disease activity occurred vs. healthy Ctls, Figure 1. The mediators were subsequently applied to the LDAII / L-DAI, informed by log-transformed and standardized plasma mediators, weighted by either hSLEDAI scores or number of SLE-associated AutoAbs at time of sample procurement / clinic visit. Using forward selection (starting with top informing mediator, IFN-a, and subsequently adding additional mediators in turn) and backward elimination (using all mediators, then subtracting mediators in reverse order of importance, starting with IL-8 / CXCL8), it was found that 10 mediators best informed the LDAII / L-DAI. Because IL-12p70 was not statistically significant in its ability to differentiate low from active disease in SLE patients (vs. Ctls, Table 3), nor clinically / serologically active / quiescent disease in SLE patients (vs. Ctls, Table 4), it was eliminated, leaving nine mediators to optimally inform the LDAII / L-DAI (Figure 1 and Figure 2).

[0176] Whether weighted by hSLEDAI scores (Figure 2A) or number of AutoAb specificities (Figure 2B), the LDAII / L-DAI informed by the top nine differentiating mediators (L-DAI 9) was able to differentiate SLE patients with various combinations of clinical and / or serological active vs. quiescent disease, as well as active vs. low disease activity (vs. healthy Ctrl). In fact, L-DAI 9 weighted by hSLEDAI scores strongly correlated with the L-DAI 9 weighted by number of AutoAb specificities (Spearman r = 0.990, p<0.0001), Figure 2C. This led to the formation of an L-DAI 9 composite score (average of scores using hSLEDAI vs. AutoAb weighting), Figure 3. The L-DAI 9 Composite score was able to differentiate SLE patients with various combinations of clinical and / or serological active vs. quiescent disease (Figure 3A), as well as active vs. low disease activity (Figure 3B) vs. healthy Ctrl.

[0177] Logistic regression combined with decision curve analysis was used to determine optimalAttorney Docket No.82092-000005-WO-POA cutoffs between low / medium and medium / high risk cutoff points for an SLE patient having both clinically and serologically active disease (CASA, Figure 3A upper right panel), as well as hSLEDAI defined active disease (Figure 3B, upper right panel). Comparing CASA vs. CQSQ (Figure 3A), setting the low / medium risk cutoff at -1.7577 (19% risk threshold) increased the sensitivity and negative predictive value (NPV) of the L-DAI 9 to 100%, while a medium / high risk cutoff of 2.0624 (79% risk threshold), increased both specificity (94%) and positive predictive value (PPV, 87%). Similar findings were delineated when comparing active vs. low hSLEDAI- defined disease activity (Figure 3B). Setting the low / medium risk cutoff at -1.7363 (30% risk threshold) allow ed for the L-DAI 9 to have both sensitivity and NPV of 100%, while a medium / high risk cutoff of 2.4928 (69% risk threshold) maximized the L-DAI 9 specificity (94%) and PPV (79%). These cutoffs would allow providers to assess the immune status of their SLE patients prior to clinic visits to determine if additional clinical assessment is required to make treatment decisions that would impact the development of permanent organ damage, morbidity, and early mortality. For example, patients having a moderate or high LDAII / L-DAI score may require additional clinical evaluation and / or a reevaluation of the current disease management (e.g., adjusting dosage of a therapeutic agent, replacing one or more therapeutic agents for other therapeutic agents, and / or adding therapeutic agents) in the near future. Alternatively, patients having a low LDAII / L-DAI score suggest additional clinical evaluation may not be acutely required and the additional clinical evaluation can be addressed at the next scheduled appointment, or that one or more therapeutic agents may be withdrawn (decreased dose or discontinuation). Additionally or alternatively, the LDAII / L-DAI score may inform a longitudinal follow-up of SLE patients to determine when additional clinical evaluation (e.g., for patients having a moderate or high LDAII / L-DAI score) or whether current disease management has resulted in stable immune disease activity (e.g., for patients having a low LDAII / L-DAI score) or whether a treat-to-target therapeutic approach is effective (e.g., for patients having a low LDAII / L-DAI score).

[0178] In order to maximize the cost effectiveness of the refined LDAII / L-DAI test, three additional analytes requiring 1:10 sample dilution were considered to supplement the baseline, refined L-DAI 9 test, TNFRII, Resistin, and Osteopontin (OPN), Table 6. Table 6. L-DAI mediators under consideration for commercial / clinical use L-DAI 9 L-DAI 10A L-DAI 10B L-DAI 10C L-DAI 11A L-DAI 11B L-DAI 11C L-DAI 12 B li R fi LDAI LDAI ii l i ionAttorney Docket No.82092-000005-WO-POA Compared to L-DAI 9 (Figure 4A), adding TNFRII, Resistin, or OPN as single mediators (L-DAI 10A-C, Figure 4B-D), a combination of two mediators (L-DAI 11A-C, Figure 4E-G), or all three mediators (L-DAI 12, Figure 4H), there was no statistical difference in the ability to differentiate clinical / serologic active / quiescent disease states, nor hSLEDAI defined low vs. active disease (vs. Ctrls). Evaluating overall performance, Table 7, adding these additional mediators, particularly OPN ± Resistin, did not decrease the ability to differentiate CASA vs. CQSQ or active vs. low hSLEDAI disease activity by AUC, effect size, nor categorical performance, including positive / negative (0 cutoff) for odds ratio, specificity, sensitivity, NPV, and PPV. In addition, adding OPN, either alone (L-DAI 10C) or in conjunction with TNFRII (L-DAI 11B) or Resistin (L-DAI 11C), improved the ability of the immune mediator informed LDAII / L-DAI to correlate with concurrent hSLEDAI-defined clinical disease activity, Table 7B. This would allow the LDAII / L-DAI to be used as a laboratory developed test to screen SLE patients to determine the need for additional clinical evaluation. Table 7. L-DAI Performance Characteristics A. Composite L-DAI CASA vs. CQSQ ; L-system manifestations within the hSLEDAI instrument, Table 8.Attorney Docket No.82092-000005-WO-POAAttorney Docket No.82092-000005-WO-POA Compared to L-DAI 9, the addition of TNFRII, Resistin, and / or OPN either maintained (vasculitis, arthritis, renal, mucocutaneous) or improved (serositis, immunologic, and hematologic) the ability of the LDAII / L-DAI to differentiate the presence of varied organ system manifestations. Manifestations exclusively present in active disease (hSLEDAI g 4), including vasculitis, arthritis, and renal features, were able to be equally (vasculitis) or more significantly (arthritis and renal) differentiated by adding the additional mediators to L-DAI 9, Table 9. These data indicate that even in SLE patients with chronically active disease, the LDAII / L-DAI results could inform the need for additional clinical evaluation and / or changes in treatment.Attorney Docket No.82092-000005-WO-POA Table 9. L-DAI scores and soluble mediator levels by organ system manifestation in active SLE LowbCase Present Absent vs Present Activeb(hSLEDAI ≥4) Case Breakout (hSLEDAI <4) vs Low Low vs Absent Analyte / DAIaCategory Present Mean SEM Absent Mean SEM Low Mean SEMp-valuec p-valuec p-valuec108 956200 186 159 309 401489751 333 655 468714472 056 049 408 028501 308 048 806879 836 755 420 766409 209 387 453 352 185473 276 740 442 225479 542 014 007007 006 006 004 005 003032 006 498 007 670424 976 353 080 001017 012; Significance determined by Kruskal-Wallis test with Dunn's multiple comparison (unadjusted p≤0.05)Attorney Docket No.82092-000005-WO-POA

[0180] Persistently active disease is burdonsome for SLE patients, affects quality of life and medical care costs, especially those patients with longstanding disease. Disease activity has been shown to increase over time and the majority of patients with low disease activity will develop active disease that is associated with both organ damage and early mortality, highlighting gaps in the optimal management of SLE. The ultimate goal is to move patients toward low disease activity where it has been shown to improve outcomes and prognosis, with fewer organ manifestations and less permanent organ damage, fewer treatments that carry significant side effects and morbidity, and lower mortality.

[0181] Recognition and early treatment to prevent tissue and organ damage is challenging, as signs and symptoms of disease activity are captured after their occurrence. In addition, long-term use of steroids and other immune suppressants required to manage disease activity are associated with increased morbidity. The inability to proactively manage clinical disease limits medical care to reactive treatment, precluding proactive strategies of adding or increasing steroid-sparing immune modifying agents to prevent end-organ damage and reduce the pathogenic and socioeconomic burdens of SLE. Dysregulation of multiple immune pathways, alongside the accumulation of SLE-associated AutoAb specificities, underlies the development and transition to classified SLE, as well as clinical disease activity after SLE classification. The current lack of an immune mechanism-informed disease management test in SLE stems from no individual immune pathway-informed biomarker acting as a universal surrogate. In addition, classical serologic markers of disease activity are insufficient biologic signals and are not directly indicative of active clinical disease.

[0182] Underlying immune dysregulation that reflects ongoing clinical disease activity and harnesses the information to inform and refine an immune index that complements current clinical disease activity instruments were determined. Within the current study, heterogeneity was observed in the number and type of immune pathways altered in SLE patient samples from active vs. low disease activity. This may explain, in part, the variability among previous reports of inflammatory mediators in SLE patients with active disease, as well as the inconsistent correlations with and limited clinical utility of proposed serologic markers of disease activity, alone or in combination, including anti-dsDNA, complement, complement split products, and inflammatory markers (ESR and CRP). Despite the heterogeneity in immune pathway involvement, each patient demonstrated elevations in inflammatory mediators from at least one pathway. It was not surprising that IFN-associated mediators were affected, including alterations in type I IFN (IFN- α), type II IFN (IFN-´), and IFN-associated chemokines (IP-10 / CXCL10, given the well described IFN signature present in SLE patients. IFN-associated mediators were affected directly in relationAttorney Docket No.82092-000005-WO-POA to disease activity (low vs. active, clinically / serologically quiescent vs. active), as well as in relation to the presence of SLE-associated AutoAbs, in a subset of patients. This finding is supported by previous studies that indicated that the IFN pathway can reflect disease activity, is dependent on the presence of AutoAbs in a subset of patients, but does not universally explain concurrent clinical disease activity.

[0183] There were a number of mediators that did not directly correlate with disease activity measures, yet correlated with the accumulation of AutoAb specificities and were able to differentiate low vs. active disease clinic visits within AutoAb positive vs. negative SLE patients. Notable in this category were IL-7 and Resistin. IL-7 is necessary for B cell development and helps drive proliferation and antibody diversity. Adipokines such as Resistin have been shown to have potent pro-inflammatory properties and lead to increased antibody production by plasma B cells. A pair of soluble mediators with ostensibly dual anti- and pro-inflammatory functions with respect to lupus disease activity are IL-10 and TGF-³. Both of these mediators have been shown to have regulatory functions and TGF-³ levels were negatively correlated with the presence and accumulation of AutoAb specificities. Conversely, SLE patients with active disease have increased levels of IL-10. IL-10 has been shown to have pro-inflammatory properties with respect to B- lymphocyte activation and AutoAb production, while TGF-³ has been shown to contribute to Th17-type responses in the presence of IL-6 that leads to IL-21 secretion and stimulation of B- lymphocytes that contributes to AutoAb production and SLE pathogenesis.

[0184] These immune system changes, in conjunction with lessons learned forming a lupus flare prediction index, have guided us to develop and currently refine a Lupus Disease Activity (Immune) Index (LDAII / L-DAI) that is informed by soluble mediator alterations and weighted by their correlation with SLE-associated AutoAb accumulation, hSLEDAI disease activity scores, and a composite weighting that encompasses both AutoAb accumulation and hSLEDAI disease activity scores.. Time and need for specialized training to minimize variability in disease activity assessment often limit the use of validated SLE disease activity measures in routine clinical practice. The detection of immune system changes associated with ongoing clinical disease activity, harnessed to inform the LDAII / L-DAI, would allow for the identification of patients in need of closer monitoring and enable early intervention with immune modifying treatments to prevent end-organ damage. A positive score would indicate a need for more frequent monitoring and / or a change in medication to dampen ongoing inflammation prior to the onset of new or worsening clinical manifestations. A negative score would indicate maintenance of ongoing monitoring and medication schedules (they are working), the need for less frequent monitoring (particularly if visits occur at least quarterly), and / or consideration of tapering steroids or otherAttorney Docket No.82092-000005-WO-POA immune modifying agents that carry significant side effects. It has been shown that SLE patients who participate more actively in their clinical care have less permanent organ damage. Clinically quiescent SLE patients are still at risk for heightened disease activity and must be regularly monitored. The LDAII / L-DAI would allow patients to monitor their immune activity that precludes clinical disease activity and alert them and their health care providers to the need for further clinical assessment.

[0185] Although SLE patients with active disease were more likely to meet hSLEDAI serologic criteria (increased DNA binding and / or hypocomplementemia) and be positive for anti-dsDNA AutoAbs, neither of these factors have been shown to be predictors of heightened clinical disease activity. However, the LDAII / L-DAI was able to differentiate patients with clinically and serologically quiescent (CQSQ) vs. active (CASA) disease, with a greater difference in IFN-α, MIG / CXCL9, IP-10 / CXCL10, TNF-α, soluble TNFRII, BLyS, and OPN levels. In addition, the LDAII / L-DAI was able to differentiate SLE patients with active disease who exhibited renal manifestations, as well as manifestations in other organ systems. There is a great push to utilize pathway-specific immune dysregulation to enable personalized, precision medicine for SLE patients with renal manifestations and lupus nephritis (LN), as LN is the most common cause of morbidity and mortality in SLE patients. The success of such approaches will require methods for identifying individuals at greatest risk of developing LN and for defining measures of pathway- specific immune dysregulation to select the most appropriate LN patients for a given pathway- specific biologic treatment. Mediators altered in the current assessment of SLE patients with renal include IFN-α, TNFRII, Resistin, and OPN. IFN-α, as a type I IFN, may enhance renal immune complex deposition, as well as induce increased inflammatory responses by resident renal cells that lead to local recruitment and infiltration of neutrophils and monocytes. TNFRII, normally present on lymphocytes, is aberrantly upregulated in the context of chronic inflammation, including in the kidney, contributing to inflammation, kidney damage and kidney failure. Enhanced circulating levels of Resistin have been shown to be associated with renal dysfunction in SLE patients, including proteinuria severity and increased serum creatinine. OPN is directly associated with glomerular fibrosis in the kidney, leading to proteinuria and low creatinine clearance. OPN is associated with SLE genetic risk, increased clinical disease activity, and promotes activation and migration of antigen presenting cells, including macrophages and dendritic cells, as well as differentiation of multiple T-helper cell pathways, including Th1, Th17, and Tfh. Adding TNFRII, Resistin, and / or OPN, alone or in combination with the nine mediators informing the L-DAI 9 would enhance the ability of the LDAII / L-DAI to differentiate organ system manifestation driven clinical disease activity in SLE patients, including renal features.Attorney Docket No.82092-000005-WO-POA

[0186] A proactive, interventional approach informed by fluctuating immune profiles, even within the same patients, is a paradigm shift that requires consideration. The ability to detect changes in immune status as a reflection of clinical disease activity would allow for improved disease surveillance and treatment, which could improve patient outcomes and reduce the pathogenic and socioeconomic burdens of SLE. An advantage of calculating a patient’s LDAII / L- DAI is that it does not require cut-offs for each soluble mediator to establish positivity, and does not require a priori knowledge of the inflammatory pathways that contribute to disease activity in a particular patient. Validating and refining the LDAII / L-DAI in this prospective, multiethnic study will help establish this valuable tool in SLE clinical trials and disease management. Depending on the comprehensive clinical picture of an individual patient, early detection of risk for heightened clinical disease activity and organ damage could prompt closer monitoring, preventative treatments, or inclusion in clinical trials for targeted biologics relevant to pathways altered within the LDAII / L-DAI.

[0187] The appended claims set forth novel and inventive aspects of the subject matter described above, but the claims may also encompass additional subject matter not specifically recited in detail. For example, certain features, elements, or aspects may be omitted from the claims if not necessary to distinguish the novel and inventive features from what is already known to a person having ordinary skill in the art. Features, elements, and aspects described in the context of some embodiments may also be omitted, combined, or replaced by alternative features serving the same, equivalent, or similar purpose without departing from the scope of the invention defined by the appended claims.Attorney Docket No.82092-000005-WO-POA REFERENCES Al Sawah, S., Zhang, X., Zhu, B., Magder, L. S., Foster, S. A., Iikuni, N., & Petri, M. (2015). Effect of corticosteroid use by dose on the risk of developing organ damage over time in systemic lupus erythematosus-the Hopkins Lupus Cohort. 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Claims

Attorney Docket No.82092-000005-WO-POA CLAIMS What is claimed is:

1. A method for characterizing disease activity in a systemic lupus erythematosus patient (SLE), comprising: (a) obtaining a blood, serum, plasma, and / or urine sample from the patient; (b) assessing the blood, serum, plasma, and / or urine sample for a presence or amount of protein expression of one or more biomarkers selected from the group consisting of IFN- α, IL-10, BLyS, IL-7, IFN-´, TRAIL, IL-15, IP-10 / CXCL10, and IL-4; (c) assessing the blood, serum, plasma, and / or urine sample for a presence or an amount of protein expression of one or more one inflammatory mediator biomarkers selected from the group consisting of TNFRII, Resistin, and Osteopontin (OPN); (d) assessing the blood, serum, plasma, and / or urine sample for a presence or an amount of one or more SLE-associated autoantibody specificity biomarkers selected from the group consisting of dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP; and (e) calculating a Lupus Disease Activity (Immune) Index (LDAII / L-DAI) score.

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

3. The method of claim 1, wherein the LDAII / L-DAI score is an hSLEDAI weighted LDAII / L-DAI calculated by log transformation; standardization; weighting by Spearman r correlation to the hSLEDAI disease activity score, and summation of soluble protein markers.

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

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

6. The method of claim 1, wherein the LDAII / L-DAI score distinguishes between active and low lupus disease activity.Attorney Docket No.82092-000005-WO-POA 7. The method of claim 1, further comprising administering a treatment to the patient prior to reaching clinical disease classification after determining that the patient has the prognosis for transitioning to classified SLE, wherein the treatment comprises at least one of: hydroxychloroquine (HCQ), belimumab, a nonsteroidal anti-inflammatory drug, a steroid, or a disease-modifying anti-rheumatic drug (DMARD).

8. The method of claim 7, wherein the treatment is administered to an SLE patient having active lupus activity.

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

10. A method of evaluating disease activity and progression of Systemic Lupus Erythematosus (SLE) clinical disease in a patient comprising: obtaining a blood, serum, plasma, and / or urine sample from the patient; determining the presence or expression of at least one biomarker from each of (1) to (3): (1) one or more biomarkers selected from the group consisting of IFN-α, IL-10, BLyS, IL-7, IFN-´, TRAIL, IL-15, IP-10 / CXCL10, and IL-4; (2) at least one inflammatory mediator biomarker selected from the group consisting of TNFRII, Resistin, and Osteopontin (OPN); (3) at least one SLE-associated autoantibody specificity biomarker selected from the group consisting of dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP; (4) and calculating an LDAII / L-DAI score.

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

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

13. The method of claim 10, wherein the LDAII / L-DAI score is a composite LDAII / L-DAI calculated by: log transformation; standardization; weighting by Spearman r correlation to the average of the autoantibody specificities and hSLEDAI disease activity score, and summation ofAttorney Docket No.82092-000005-WO-POA soluble protein markers.

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

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

16. The method of claim 10, further comprising administering a treatment to the patient prior to reaching clinical disease classification after determining that the patient has the prognosis for transitioning to classified SLE, wherein the treatment comprises at least one of: hydroxychloroquine (HCQ), belimumab, a nonsteroidal anti-inflammatory drug, a steroid, or a disease-modifying anti-rheumatic drug (DMARD).

18. The method of claim 16, wherein the treatment is administered to an SLE patient having active lupus activity.

19. The method of claim 16, wherein the treatment is administered to an SLE patient having low lupus activity.

20. A method for characterizing disease activity in a systemic lupus erythematosus patient (SLE), comprising: (a) obtaining a blood, serum, plasma, and / or urine sample from the patient; (b) assessing the blood, serum, plasma, and / or urine sample for a presence or an amount of protein expression of for each of a plurality of biomarkers selected from the group consisting of IFN-α, IL-10, BLyS, IL-7, IFN-´, TRAIL, IL-15, IP-10 / CXCL10, and IL-4; (c) assessing the blood, serum, plasma, and / or urine sample for a presence or an amount of protein expression of one or more inflammatory mediator biomarkers selected from the group consisting of TNFRII, Resistin, and Osteopontin (OPN); (d) assessing the blood, serum, plasma, and / or urine sample for a presence or an amount of protein expression of one or more immune mediator biomarkers selected from the group consisting of IL-12p70, TNF-α, MIG / CXCL9, Resistin, IL-5, IL-13, IL-1³, IL-17A, IL- 2Rα, TGF-³ (Native and / or Total form), Fas, MCP-1 / CCL2, Stem Cell Factor (SCF), IL- 1RA, IL-6, MIP-1α / CCL3, MIP-1³ / CCL4, MCP-3 / CCL7, RANTES / CCL5, TNFRI, and IL-8 / CXCL8; (e) assessing the blood, serum, plasma, and / or urine sample for a presence or an amount one or more SLE-associated autoantibody specificity biomarkers selected from the groupAttorney Docket No.82092-000005-WO-POA consisting of dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP; and (f) calculating a Lupus Disease Activity (Immune) Index (LDAII / L-DAI) score.

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

19. The method of claim 20, wherein the LDAII / L-DAI score is an hSLEDAI weighted LDAII / L-DAI calculated by: log transformation; standardization; weighting by Spearman r correlation to the hSLEDAI disease activity score, and summation of soluble protein markers .

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

21. The method of claim 20, further comprising categorizing the SLE patient based on the LDAII / L-DAI score into clinically active (CA) or clinically quiescent (CQ) disease that is either serologically active (SA) or serologically quiescent (SQ).

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

23. The method of claim 20, further comprising administering a treatment to the patient prior to reaching clinical disease classification after determining that the patient has the prognosis for transitioning to classified SLE, wherein the treatment comprises at least one of: hydroxychloroquine (HCQ), belimumab, a nonsteroidal anti-inflammatory drug, a steroid, or a disease-modifying anti-rheumatic drug (DMARD).

24. The method of claim 20, wherein the treatment is administered to an SLE patient having active lupus activity.

25. The method of claim 20, wherein the treatment is administered to an SLE patient having low lupus activity.

24. A method of evaluating disease activity and progression of Systemic Lupus Erythematosus (SLE) clinical disease in a patient comprising: obtaining a blood, serum, plasma, and / or urine sample from the patient;Attorney Docket No.82092-000005-WO-POA determining the presence of protein expression of at least one biomarker from each of (1) to (4): (1) one or more biomarkers selected from the group consisting of IFN-α, IL-10, BLyS, IL-7, IFN-´, TRAIL, IL-15, IP-10 / CXCL10, and IL-4; (2) at least one inflammatory mediator biomarker selected from the group consisting of TNFRII, Resistin, and Osteopontin (OPN); (3) at least one immune mediator biomarker selected from the group consisting of IL-12p70, TNF-α, MIG / CXCL9, Resistin, IL-5, IL-13, IL-1³, IL-17A, IL-2Rα, TGF-³ (Native and / or Total form), Fas, MCP-1 / CCL2, Stem Cell Factor (SCF), IL- 1RA, IL-6, MIP-1α / CCL3, MIP-1³ / CCL4, MCP-3 / CCL7, RANTES / CCL5, TNFRI, and IL-8 / CXCL8; (4) at least one SLE-associated autoantibody specificity biomarker selected from the group consisting of: dsDNA, chromatin, RiboP, Ro / SSA, La / SSB, Sm, SmRNP, and RNP; (5) and calculating an LDAII / L-DAI score.

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

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

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

28. The method of claim 24, further comprising categorizing the SLE patient based on the LDAII / L-DAI score into clinically active (CA) or clinically quiescent (CQ) disease that is either serologically active (SA) or serologically quiescent (SQ).

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

30. The method of claim 24, further comprising administering a treatment to the patient prior to reaching clinical disease classification after determining that the patient has the prognosis forAttorney Docket No.82092-000005-WO-POA transitioning to classified SLE, wherein the treatment comprises at least one of: hydroxychloroquine (HCQ), belimumab, a nonsteroidal anti-inflammatory drug, a steroid, or a disease-modifying anti-rheumatic drug (DMARD).

31. The method of claim 24, wherein the treatment is administered to an SLE patient having active lupus activity.

32. The method of claim 24, wherein the treatment is administered to an SLE patient having low lupus activity.

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