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WO2025042955A1 - Biomarqueurs d'indice immunitaire d'activité de maladie du type lupus érythémateux disséminé (sle) caractérisant l'activité d'une maladie - Google Patents

Biomarqueurs d'indice immunitaire d'activité de maladie du type lupus érythémateux disséminé (sle) caractérisant l'activité d'une maladie Download PDF

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Publication number
WO2025042955A1
WO2025042955A1 PCT/US2024/043172 US2024043172W WO2025042955A1 WO 2025042955 A1 WO2025042955 A1 WO 2025042955A1 US 2024043172 W US2024043172 W US 2024043172W WO 2025042955 A1 WO2025042955 A1 WO 2025042955A1
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sle
ldaii
dai
disease
patient
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WO2025042955A4 (fr
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Melissa MUNROE
Judith JAMES
Eldon Jupe
Mohan Purushothaman
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Progentec Diagnostics Inc
Oklahoma Medical Research Foundation
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Progentec Diagnostics Inc
Oklahoma Medical Research Foundation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/30—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for calculating health indices; for individual health risk assessment
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/104—Lupus erythematosus [SLE]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • 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.
  • 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.
  • ACR American College of Rheumatology
  • SLICC Systemic Lupus International Collaborating Clinics
  • 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).
  • SLE Systemic lupus erythematosus
  • SLE-associated autoantibody specificities such as anti-dsDNA, anti-spliceosome and anti-Ro/SSA
  • SLE-associated autoantibody specificities such as anti-dsDNA, anti-spliceosome and anti-Ro/SSA
  • ANAs are also found in sera from Attorney 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.
  • 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).
  • AutoAbs SLE-associated autoantibodies
  • Dyroe et al., 2016 Dyroe et al., 2016
  • 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).
  • 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- ⁇ , 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
  • 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.
  • 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.
  • 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.
  • 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.
  • at least one immunoassay comprises a multiplex assay.
  • 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 serologically Attorney Docket No.82092-000005-WO-POA quiescent (SQ).
  • CA clinically active
  • CQ quiescent
  • SA serologically (dsDNA binding and low complement) active
  • SQ serologically Attorney Docket No.82092-000005-WO-POA quiescent
  • the LDAII/L-DAI/L-DAI score distinguishes between active and low lupus disease activity.
  • 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.
  • HCQ hydroxychloroquine
  • belimumab a nonsteroidal anti- inflammatory drug
  • a steroid a nonsteroidal anti- inflammatory drug
  • DMARD disease-modifying anti-rheumatic drug
  • 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- ⁇ , 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-12
  • 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.
  • 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.
  • 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.
  • 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.
  • At least one immunoassay comprises a multiplex assay.
  • 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).
  • CA clinically active
  • CQ quiescent
  • SA serologically (dsDNA binding and low complement) active
  • SQ serologically quiescent
  • the LDAII/L-DAI score distinguishes between active and low lupus disease activity.
  • 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).
  • HCQ hydroxychloroquine
  • belimumab a nonsteroidal anti- inflammatory drug
  • a steroid a nonsteroidal anti- inflammatory drug
  • DMARD disease-modifying anti-rheumatic drug
  • an increase in the IFN- ⁇ , IL-10, BLyS, IL-7, TRAIL, IP-10/CXCL10, TNFRII, Resistin, and OPN biomarkers are indicative of renal organ involvement.
  • 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- ⁇ , IL-10, B-lymphocyte stimulating factor (BLyS or BA
  • 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.
  • 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]).
  • SLE Systemic Lupus Erythematosus
  • CA clinically active
  • CQ
  • the method further comprises differentiating clinically and serologically quiescent (CQSQ) SLE patients compared to healthy controls.
  • 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.
  • the immunoassay is multiplexed immunoassay.
  • 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.
  • 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]
  • 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.
  • L-DAI Lupus Disease Activity (Immune) Index
  • L-DAI 9 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.
  • 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).
  • C clinically
  • S serologically
  • Q quiescent
  • B active
  • Predictive probability of active disease determined by logistic regression in upper right panel graphs.
  • NPV negative predictive value
  • PPV positive predictive value
  • 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.
  • LDAII/L-DAI scores informed by 9 mediators 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]).
  • the present inventors leveraged plasma samples serially collected from Systemic lupus erythematosus (SLE) patients to compare levels and determine temporal relationships between Attorney 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.
  • 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.
  • the present invention can be used to augment telehealth to prioritize the need for in-person clinic visits.
  • 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.
  • 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.
  • 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.
  • 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.
  • ⁇ soluble TNF superfamily biomarker(s) refers to one or more of the following biomarkers: TNF- ⁇ , TNFRI (p55), TNFRII (p75), Fas, BLyS, and TNF-related apoptosis-inducing ligand (TRAIL).
  • TNF- ⁇ TNFRI
  • TNFRII p75
  • Fas BLyS
  • TRAIL TNF-related apoptosis-inducing ligand
  • ⁇ 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 human Attorney Docket No.82092-000005-WO-POA patient.
  • OPN Osteopontin
  • SCF Stem Cell Factor
  • 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.
  • a ⁇ healthy control refers to a healthy control that is not an SLE patient that has no clinical evidence of SLE.
  • 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).
  • 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.
  • 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).
  • 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.
  • SLE may be associated with an increased risk of bone fractures in relatively young women.
  • 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.
  • a person with SLE may have an association with antiphospholipid antibody syndrome (a thrombotic disorder), wherein autoantibodies to phospholipids are present in their serum.
  • 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.
  • NPSLE neuropsychiatric systemic lupus erythematosus
  • TLR immune system danger signals
  • Innate cytokines which activate and are secreted by multiple immune cell types include Type I interferons (IFN- ⁇ and IFN-3), TNF- ⁇ , and members of the IL-1 family (IL-1 ⁇ 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.
  • Th1-type cytokines include Type I interferons (IFN- ⁇ and IFN-3), TNF- ⁇ , and members of the IL-1 family (IL-1 ⁇ and IL-13).
  • APC antigen presenting cells
  • Th CD4 T-helper
  • 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.
  • IL-12R-32 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.
  • IFN- ⁇ expression is associated with a number of autoinflammatory and autoimmune diseases, including increased disease activity in SLE.
  • IFN ⁇ is considered to be the characteristic Th1 cytokine
  • IL-2 interleukin-2
  • 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.
  • 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.
  • IL-7 and IL-15 all members of the common cytokine receptor gamma-chain family
  • 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.
  • IL-4 is a 15-kD polypeptide with multiple effects on many cell types. Its receptor is a heterodimer composed of an ⁇ subunit, with IL-4 binding affinity, and the common ⁇ subunit which is also part of other cytokine receptors.
  • 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.
  • this glycoprotein in its active form is a homodimer.
  • IL-5 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-4R ⁇ ) and at least one of two known IL-13-specific binding chains.
  • IL-4R ⁇ alpha chain of the IL-4 receptor
  • 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).
  • 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.
  • 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.
  • 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.
  • PAMPs pathogen associated molecular patterns
  • PRRs pattern recognition receptors
  • TLRs Toll-like receptors
  • IL-6 also contributes to Th2-type adaptive immunity against parasitic infections, with particular importance in mast-cell activation that coincides with parasite expulsion.
  • 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.
  • Th17-type cytokines are described below.
  • 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.
  • Chemokines and Adhesion Molecules serve to coordinate cellular traffic within the immune response. Chemokines are divided into CXC (R)eceptor/CXC (L)igand and CCR/CCL subgroups.
  • GRO ⁇ 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- ⁇ ).
  • 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.
  • MIG ⁇ interferon
  • CXCL9 is a T-cell chemoattractant induced by IFN- ⁇ .
  • IP-10/CXCL10 and I-TAC/CXCL11 are 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 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- ⁇ .
  • IP-10 has been attributed to several roles, such as chemoattraction for Attorney 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.
  • MCP-1 Monocyte chemotactic protein-1
  • CCP-1 Monocyte chemotactic protein-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.
  • Monocyte-specific chemokine 3 (MCP-3)/CCL7) specifically attracts monocytes and regulates macrophage function.
  • Macrophage inflammatory protein-1 ⁇ (MIP-1 ⁇ )/CCL3 is encoded by the CCL3 gene in humans.
  • MIP-1 ⁇ is involved in the acute inflammatory state in the recruitment and activation of polymorphonuclear leukocytes(Wolpe et al., 1988).
  • MIP-1 ⁇ interacts with MIP-13/CCL4, encoded by the CCL4 gene, with specificity for CCR5 receptors.
  • 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.
  • 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.
  • TNFR tumor necrosis factor receptor
  • 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- ⁇ B and MAPK pathways, immune and inflammatory responses, and in some cases, apoptosis.
  • the prototypical member is TNF-3.
  • Tumor necrosis factor (TNF, cachexin, or cachectin, and formerly known as tumor necrosis factor alpha or TNF ⁇ ) 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.
  • 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.
  • TACE metalloprotease TNF-3 converting enzyme
  • 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.
  • 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.
  • DD death domain
  • 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.
  • APO-1 or APT apoptosis antigen 1
  • CD95 cluster of differentiation 95
  • TNFRSF6 tumor necrosis factor receptor superfamily member 6
  • Fas is a death receptor on the surface of cells that leads to programmed cell death (apoptosis).
  • 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 has Attorney 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.
  • DISC death- inducing signaling complex
  • Membrane-anchored Fas ligand on the surface of an adjacent cell causes oligomerization of Fas.
  • DD death domain
  • the receptor complex 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.
  • 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.
  • 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
  • tumor counterattack tumor counterattack
  • 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.
  • TACI binds BLyS with the least affinity; its affinity is higher for a protein similar to BLyS, called a proliferation-inducing ligand (APRIL).
  • APRIL proliferation-inducing ligand
  • 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.
  • 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.
  • B-Lymphocyte stimulator B-Lymphocyte stimulator
  • Blisibimod a fusion protein inhibitor of BLyS
  • Anthera Pharmaceuticals also primarily for the treatment of systemic lupus erythematosus.
  • 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.
  • 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.
  • adipokines including adiponectin and resistin.
  • Leptin levels increase in response to a number of innate cytokines, including TNF- ⁇ 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.
  • the soluble and transmembrane forms of the protein are formed by alternative splicing of the same RNA transcript.
  • 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 role Attorney 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.
  • 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.
  • 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.
  • 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.
  • 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 NF ⁇ B signaling and transcriptional activation via promoter binding of the transcription factors NF ⁇ B 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.
  • 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- ⁇ B activity, and is involved in the regulation of the JAK-STAT signaling pathway. [0079] TGF-3.
  • 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.
  • LTBP latent TGF-3 binding proteins
  • TGF-3 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.
  • IL-10 anti-inflammatory cytokines
  • TGF-31 contains 390 amino acids
  • TGF- 32 and TGF-33 each contain 412 amino acids.
  • 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.
  • Rb Retinoblastoma protein
  • 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.
  • TGF-3 contributes to the differentiation of pro- inflammatory Th17 cells.
  • 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.
  • IL-1R cell surface interleukin-1 receptor
  • IL1RA is secreted by various types of cells including Attorney Docket No.82092-000005-WO-POA immune cells, epithelial cells, and adipocytes, and is a natural inhibitor of the pro-inflammatory effect of IL-1 ⁇ 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.
  • 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.
  • ⁇ 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.
  • 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.
  • 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.
  • 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.
  • sample-antibody composition such as a tissue section, ELISA plate, microfluidic chamber, dot blot or western blot
  • sample-antibody composition 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.
  • 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.
  • 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.
  • the second binding ligand may be linked to a detectable label.
  • 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.
  • 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.
  • 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 third Attorney 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.
  • 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.
  • streptavidin or avidin
  • biotinylated DNA and/or complementary biotinylated DNA
  • 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.
  • a conjugate can be produced which is macroscopically visible.
  • 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.
  • the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a proximity extension assay e.g. commercially available from Olink company
  • an electrochemluminescence assay e.g., commercially available from Meso Scale Discovery company
  • a protein array-based assay e.g., a protein array-based assay.
  • 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.
  • the bound anti-ORF message and anti-ORF translated product antibodies are detected.
  • the immune complexes may be detected directly.
  • 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.
  • Another type of ELISA in which the antigens are immobilized involves the use of Attorney Docket No.82092-000005-WO-POA antibody competition in the detection.
  • 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.
  • 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.
  • FACS Fluorescence- Activated Cell Sorting
  • the system is adjusted so that there is a low probability of more than one cell per droplet.
  • 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.
  • 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.
  • a reagent specific to a particular bioassay e.g., an antibody
  • 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.
  • 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.
  • 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.
  • 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.
  • primers are Attorney 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. [00108] 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.
  • 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.
  • the amplification product may be detected or quantified. In certain applications, the detection may be performed by visual method.
  • 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.
  • 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.
  • PCR polymerase chain reaction
  • a reverse transcriptase-PCR amplification procedure may be performed to quantify the amount of mRNA amplified.
  • 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.
  • the amplification products are visualized.
  • a typical visualization method involves staining of a gel with ethidium bromide and visualization of bands under UV light.
  • 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.
  • 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 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.
  • 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.
  • wash solutions and protocols for their use are known to those of skill in the art and may be used.
  • the label on the target is biotin
  • streptavidin-fluorescent conjugate under conditions sufficient for binding between the specific binding member pairs to occur.
  • 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 [00123] 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.
  • 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.
  • 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 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.
  • 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.
  • RNA Sequencing Transcript Counting
  • RNA-seq also called Whole Transcriptome Shotgun Sequencing (WTSS)
  • WTSS Whole Transcriptome Shotgun Sequencing
  • NGS Next- Generation Sequencing
  • the transcriptome of a cell is dynamic; it continually changes as opposed to a static genome.
  • 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.
  • 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, making Attorney Docket No.82092-000005-WO-POA available a profile of all transcripts being expressed.
  • SAGE Serial Analysis of Gene Expression
  • 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.
  • 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.
  • nonsteroidal anti-inflammatory drugs and low dose steroids may also be used.
  • Hydroxychloroquine HCQ
  • HCQ Hydroxychloroquine
  • DMARDs 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.
  • 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.
  • people who require steroids may develop Cushing's symptoms of truncal obesity, purple striae, buffalo hump and other associated symptoms.
  • 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.
  • NSAIDs mainly nonsteroidal anti-inflammatory drugs
  • 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.
  • opioids 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.
  • lifelong treatment with opioids is fairly common for chronic pain symptoms, accompanied by periodic titration that is typical of any long-term opioid regimen.
  • 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.
  • 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.
  • 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.
  • ⁇ pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • 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.
  • 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.
  • 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.
  • 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. [00139] 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.
  • 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.
  • a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • solutions 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.
  • the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • 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.
  • kits are also within the scope of the invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • OCRRCC Oklahoma Rheumatic Diseases Research Core Center
  • hSLEDAI Systemic Lupus Erythematosus Disease Activity Index
  • SELENA-SLEDAI with proteinuria as defined by SLEDAI-2K
  • CNS central nervous system
  • seizure psychosis
  • organic brain syndrome visual disturbance
  • cranial nerve disorder or lupus headache
  • vasculitis arthritis
  • myositis nephritis
  • nephritis urinary casts, hematuria, proteinuria, or pyuria
  • mucocutaneous damage rash, alopecia, or mucosal ulcers
  • serositis pleuritis or pericarditis
  • hematologic manifestations low complement, increased DNA binding, fever, thrombocytopenia, or leukopenia
  • BioPlex 2200 multiplex system Bio-Rad Technologies, Hercules, CA
  • 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).
  • 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.
  • 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-1 ⁇ were excluded from univariate and multivariable analysis due to >60% of the plasma sample concentrations having values ⁇ LLOQ (0.001 pg/ml).
  • Statistical Analyses Attorney Docket No.82092-000005-WO-POA
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the threshold probability (p t ) 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.
  • CASA 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 p t g selected p t ; 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 et 2008). [00165] 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.
  • Bonferroni corrected significant p 0.0100
  • Table 5 Spearman Correlation Between Soluble Mediators vs. # Autoantibody Specificities or hSLEDAI Scores vs. # AutoAbs a vs.
  • LDAII/L-DAI weighted Lupus Disease Activity (Immune) Index
  • 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
  • backward elimination using all mediators, then subtracting mediators in reverse order of importance, starting with IL-8/CXCL8
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • additional clinical evaluation e.g., for patients having a moderate or high LDAII/L-DAI score
  • current disease management has resulted in stable immune disease activity
  • a treat-to-target therapeutic approach e.g., for patients having a low LDAII/L-DAI score.
  • 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 ion Attorney 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.
  • 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.
  • 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.
  • classical serologic markers of disease activity are insufficient biologic signals and are not directly indicative of active clinical disease.
  • IFN-associated mediators were affected, including alterations in type I IFN (IFN- ⁇ ), 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 relation Attorney 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.
  • IL-10 and TGF-3 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.
  • the detection of immune system changes associated with ongoing clinical disease activity 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 other Attorney 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.
  • 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.
  • the LDAII/L-DAI was able to differentiate patients with clinically and serologically quiescent (CQSQ) vs.
  • LDAII/L-DAI was able to differentiate SLE patients with active disease who exhibited renal manifestations, as well as manifestations in other organ systems.
  • 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.
  • IFN- ⁇ 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.
  • OPN 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.
  • LDAII/L- DAI 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.

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Abstract

L'invention concerne des procédés de caractérisation de l'activité d'une maladie chez un patient atteint d'un lupus érythémateux disséminé (SLE). Les procédés consistent à obtenir un échantillon de sang, de sérum, de plasma ou d'urine provenant du patient ; évaluer, dans l'échantillon, l'expression d'un biomarqueur choisi dans le groupe constitué par IFN-α, IL-10, BLyS, IL-7, IFN-γ, TRAIL, IL-15, IP-10/CXCL10 et IL-4 ; évaluer, dans l'échantillon, l'expression d'un médiateur inflammatoire choisi dans le groupe constitué par TNFRII, la résistine et l'ostéopontine (OPN) ; évaluer, dans l'échantillon, la présence d'un biomarqueur de spécificité d'auto-anticorps associé au SLE choisi dans le groupe constitué par l'ADNdb, la chromatine, RiboP, Ro/SSA, La/SSB, Sm, SmRNP et RNP ; et calculer un score d'indice (immunitaire) d'activité de maladie du type lupus (LDAII/L-DAI). Le score LDAII/L-DAI peut faire la distinction entre une activité de maladie du type lupus active et faible. L'invention concerne également des méthodes de traitement consistant à administrer un traitement avant d'atteindre une classification de maladie clinique après avoir déterminé que le patient présente le pronostic de passer à un SLE classé.
PCT/US2024/043172 2023-08-21 2024-08-21 Biomarqueurs d'indice immunitaire d'activité de maladie du type lupus érythémateux disséminé (sle) caractérisant l'activité d'une maladie Pending WO2025042955A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210396751A1 (en) * 2018-10-18 2021-12-23 Progentec Diagnostics, Inc. Biomarkers For A Systemic Lupus Erythematosus (SLE) Disease Activity Immune Index That Characterizes Disease Activity
US20220381795A1 (en) * 2009-10-15 2022-12-01 Laboratory Corporation Of America Holdings Biomarkers and Methods for Measuring and Monitoring Inflammatory Disease Activity

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220381795A1 (en) * 2009-10-15 2022-12-01 Laboratory Corporation Of America Holdings Biomarkers and Methods for Measuring and Monitoring Inflammatory Disease Activity
US20210396751A1 (en) * 2018-10-18 2021-12-23 Progentec Diagnostics, Inc. Biomarkers For A Systemic Lupus Erythematosus (SLE) Disease Activity Immune Index That Characterizes Disease Activity

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