WO2014093268A1 - Methods and systems for using complement-tagged molecules as biomarkers of disease - Google Patents

Abstract

This document discloses methods diagnosing, monitoring and/or predicting the state of a disease such as an immune or inflammatory disease or disorder by detecting and measuring the level of one or more C-TM complexes in the bodily fluid of an individual. These complexes will serve as biomarkers for the diagnosis, monitoring or prediction of various inflammatory-based disease states. Detection of these C-TM complexes from an individual's bodily fluid sample is shown to be amenable through immunoassays such as described within this document.

Description

METHODS AND SYSTEMS FOR USING COMPLEMENT-TAGGED

MOLECULES AS BIOMARKERS OF DISEASE

RELATED APPLICATIONS AND CLAIM OF PRIORITY

[0001] This patent document claims priority to United States Provisional Patent Application No. 61/735,390, filed December 10, 2012, titled "Complement-Tagged Molecules as Biomarkers of Disease," the disclosure of which is incorporated by reference in its entirety.

FIELD

[0002] This document discloses methods for diagnosing, monitoring and/or predicting immune and inflammatory disease states within an individual by detecting and measuring soluble complexes formed during complement activation within the individual. These soluble complexes, referred to as complement-tagged molecules, are characterized herein and are shown to serve as biomarkers for detection of the underlying disease state or as a tool to monitor progression of the disease in an individual.

BACKGROUND

[0003] The complement system, a component of the immune system, comprises a group of plasma and membrane-bound proteins that form three cascading pathways (classical, alternative, and lectin-dependent) designed to protect against invasion of foreign pathogens. However, when activated inappropriately, such as in autoimmune or inflammatory disease, the complement system may cause tissue injury.

[0004] Complement proteins are primarily synthesized in the liver and released into the body's circulatory system. C3 and C4 are the most and second most abundant complement components, respectively, and play critical roles in the activation of the complement system. Because antibody/immune complex-triggered activation of the complement system is believed to play an important role in the pathogenesis of autoimmune and inflammatory disease, measurement of serum C3 and C4 or their activation products have been widely utilized for monitoring disease activity.

[0005] However, controversies exist concerning the adequacy of measurement of soluble complement proteins and complement activation products for accurate and prompt detection of acute inflammatory episodes, such as in Systemic Lupus Erythematosus ("SLE"). During complement activation, proteolytic cleavage of C3 and C4 results ultimately in the generation of C3d and C4d fragments that contain the highly reactive thioester moiety and can bind to the surfaces of pathogens, cells, or immune complexes. Hence, their activation products (e.g., C3d and C4d) may readily interact with various types of circulating cells and plasma proteins during systemic or local inflammation.

[0006] Background prior art includes U.S. Patent Nos. 8,126,654; 8,080,382; 7,585,640; and 7,390,631, each issued to J.M. Ahearn and S.M. Manzi. Background art also includes U.S. Patent Application Publication No. 2010/0233752, naming J.M. Ahearn, E.L. Erickson, D.M. Hawkins, S.M. Manzi and T. Mercolino as inventors.

[0007] This document describes novel methods and systems for the detection of disease using complement tagged molecules as biomarkers.

SUMMARY

[0008] This document discloses a method of diagnosing, monitoring and/or predicting the state of an immune, inflammatory or other disease or disorder by detecting and measuring the level of soluble complexes (referred to herein as complement-tagged molecules, or "C-TMs") within the bodily fluids of an individual. The detection and measurement methods disclosed herein are warranted through a tendency toward an inappropriate activation of the complement system in conditions such as but not limited to autoimmune or inflammatory disease, whereby such activation causes an inflammatory response and tissue injury. This antibody/immune complex-triggered activation of the complement system in multiple disease states results in the generation of complement activation products in the systemic circulation. It is disclosed herein that resulting complement activation products generated in the systemic circulation bind to various compounds associated with a specific disease state to form C-TMs. These complexes serve as biomarkers for either detection of the underlying disease state or as a tool to monitor progression of the disease in a patient.

[0009] The detection of complement-tagged molecules disclosed within this document enables the artisan to select a detection protocol for the diagnosis and/or monitoring of the presence or progression of a particular disease state. Assays as disclosed herein may be utilized to detect a relevant C-TM complex formed between one or more compounds (a "first subunit") and one or more complement activation products (a "second subunit").

[0010] This document further discloses a method of diagnosing and/or monitoring an inflammatory response associated with an autoimmune, inflammatory or other disease or disorder by: (i) obtaining a fluid sample from an individual for testing; (ii) measuring the amount of at least one C-TM obtained from the fluid sample of the individual; (iii) measuring the amount of at least one C-TM of step (ii) from a sample serving as a control; (iv) comparing the amount of C-TM from the individual's fluid sample against the amount of C-TM detected in the control sample, to identify whether an increase in the C-TM level in the individual compared to the control sample exists; and (v) based on the results of the comparing, determining whether the increase indicates that the individual is showing an acute exacerbation of the underlying disease or disorder. [0011] In another method disclosed within this document, the SLE disease state is diagnosed and/or monitored in an individual by (i) obtaining a fluid sample from an individual for testing, (ii) measuring the amount of at least one C-TM related to SLE from the fluid sample of the individual, (iii) measuring the amount of at least one C-TM from a control serum or plasma sample, and (iv) comparing the amount of C-TM from the individual against the amount of C-TM detected in the control sample, where an increase in the C-TM level in the individual compared to the control sample indicates that the individual is showing an acute immune or inflammatory episode or response related to the underlying disease or disorder. A representative compound (as a "first subunit") to be measured for complex formation with a complement activation product (as a "second subunit") in Systemic Lupus Erythematosus ("SLE") and other inflammatory disease states, such as those described in the Example section, includes but is not limited to Apolipoprotein Al (ApoAl; a major protein component of HDL), Apolipoprotein B (ApoB; a major protein component of LDL), C-reactive protein (CRP; an acute phase protein linked with

inflammation and atherosclerosis), von Willebrand factor (vWF), and fibrinogen. A representative complement activation product to be measured for complex formation in SLE indications includes but is not limited to C4d, C4c, iC4b, C4b, C3b, iC3b, C3d, and C3c. To this end, representative C-TMs utilized to diagnose or monitor SLE include but are not limited to ApoAl-C4d, ApoB-C4d, CRP-C4d, vWF-C4d, fibrinogen-C4d and fibronectin-C4d.

[0012] This document discloses in part the identification and characterization of soluble complexes obtained and isolated from the bodily fluid sample obtained from systemic circulation of an individual. The soluble complexes comprise two heterogeneous components: a first subunit representing a compound associated either directly or indirectly with an inflammatory episode or chronic inflammatory condition of an individual, this first subunit being bound to a second subunit represented by a complement activation product. As discussed further within this document, one or more such complexes obtained as a soluble component of the fluid sample will serve as a representative biomarker for diagnosis, monitoring and/or prediction of a particular autoimmune/inflammatory disease state within an individual.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 shows a schematic illustration of a representative C-TM as disclosed throughout this document.

[0014] FIG. 2 shows a schematic illustration of the principle of an

electrochemiluminescence (ECL) assay.

[0015] FIG. 3 shows a schematic outline of an ECL-based C-TM assay.

[0016] FIGs. 4A-4C show test results that demonstrate the presence of C-TMs in human serum and plasma samples.

[0017] FIGs. 5A-5F show detection of C-TMs bound to several candidate proteins in activated human serum using the ECL-based assay.

[0018] FIG. 6 shows detection of C-TMs bound to several candidate proteins in activated human serum using the ECL-based assay.

[0019] FIGs. 7A-7C shows detection of C-TMs in the plasma of patients with immune- inflammatory diseases.

[0020] FIGs. 8A-8C shows detection of C-TMs in the plasma of patients with immune- inflammatory diseases.

[0021] FIG. 9 illustrates various hardware components that may implement programming instructions stored on a computer-readable medium to implement various automated analysis methods described herein. DETAILED DESCRIPTION

[0022] Before the methods and compositions provided herein are described, it is to be understood that this invention is not limited to the particular processes, methodology or composition described, as these may vary in relation to the C-TM-based immunoassay which pairs a soluble target molecule/complement activation product (first subunit/second subunit "C-TM" complex, as disclosed herein) as a biomarker for a specific immune, inflammatory or other disease state. It is also to be understood that the terminology used in the description is for the purpose of describing some embodiments, and is not intended to limit the scope of the present invention. All publications mentioned herein are incorporated by reference in their entirety to the extent to support the present disclosure.

[0023] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an" and "the" include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Any methods similar or equivalent to those described herein can be used in the practice or testing of embodiments disclosed within this document.

[0024] The term "compound" refers to inorganic or organic chemical or biological molecules either natural (isolated) or synthetic, and especially encompass proteins, polypeptides, peptides, lipoproteins, glycopeptides, lipids, nucleic acids and carbohydrates.

[0025] As used herein a "complement pathway component" includes proteins from the classical, alternative, and lectin complement pathways, e.g., Clq, Clr, Cls, C4, C2, C3 and fragments thereof, e.g., C4a, C4b, C2a, C2b, C4b, C2a, C3a, C3b, C4c, C4d, iC4b, C3d, C3i, C3dg. Also included as examples are C5, C5b, C6, C7, C8, C9, Clinh, MASP1, MASP2, MBL, MAC, CR1, DAF, MCP, C4 binding protein (C4BP), protein factor H, Factor B, C3bB, Factor D, Bb, Ba, C3bBb, properdin, C3bBb, CD59, C3aR, C5aR, ClqR, CR2, CR3, and CR4, as well as other complement pathway components, receptors and ligands not listed specifically herein.

[0026] As used herein, a "complement activation product" is a "complement pathway component" fragment as listed in the above paragraph, namely C4a, C4b, C2a, C2b, C3a, C3b, C4c, C4d, iC4b, C3d, C3i, and C3dg. A "complement activation product" is also referred to herein as a "second subunit" of a C-TM complex.

[0027] As used herein, a "sample" or "bodily fluid sample" or "fluid sample" or "individual sample" or "subject sample" or "patient sample" or the like in the context of obtaining a sample from a patient, subject or individual refers to a sample which may be blood plasma, blood serum, whole blood, CSF, urine, saliva, tears, semen, colostrum or any recoverable bodily fluid as obtained from the individual for C-TM testing in one or more of the various assays disclosed herein.

[0028] As used herein, "C-TM" (an acronym for "Complement- Tagged

Molecule") represents a soluble complex formed and amenable to detection where a first subunit is a "compound" as defined herein and a second subunit is a "complement activation product" as defined herein.

[0029] The terms "patient" and "individual" are used interchangeably, and generally refer to any living organism to which the disclosed methodology is utilized to obtain a bodily fluid sample in order to perform a diagnostic or monitoring method described herein. A patient can be an animal, such as a human. A patient may also be domesticated animal or a farm animal. A "patient" or "individual" may also be referred to as a subject.

[0030] As used herein, an "autoimmune or inflammatory disease or condition" refers to (i) any autoimmune disease or immune disease or condition that causes damage of organs and increased inflammation in an individual, and/or (ii) an inflammatory disease or condition being any infectious disease or condition that causes increased inflammation in an individual. "Autoimmune disease" and "immune disease" are used interchangeably. In some instances the terms noted in this paragraph are also used interchangeably to describe a certain disease state. In some embodiments the inflammatory disease or condition is a "chronic inflammatory disease or condition." A chronic inflammatory disease or condition is an inflammatory condition that does not resolve after a period of weeks, months or longer. Chronic inflammatory conditions can follow an acute inflammatory condition, or for some diseases or conditions can occur in the absence of an acute inflammatory disease or condition. An autoimmune or inflammatory disease or condition includes but is not limited to the following: systemic lupus erythematosus (lupus or SLE), Sjogrens's syndrome, rheumatoid arthritis, vasculitis (and its specific forms such as Wegener's granulomatosis), scleroderma, myositis, serum sickness, transplant rejection, sickle cell anemia, gout, complications of pregnancy such as pre-eclampsia, multiple sclerosis, cardiovascular disease, infectious disease such as hepatitis C virus infection, etc.

Autoimmune diseases can be broadly divided into systemic and organ-specific or localized autoimmune disorders, depending on the principal clinic-pathologic features of each disease. Each of these diseases or conditions can also be described as chronic inflammatory diseases or conditions. Systemic autoimmune diseases include but are not limited to SLE, Sjogren's syndrome, scleroderma, rheumatoid arthritis, and dermatomyositis. These conditions tend to be associated with autoantibodies to antigens which are not tissue specific. Thus although polymyositis is more or less tissue specific in presentation, it may be included in this group because the autoantigens are often ubiquitous t-RNA synthetases. Local syndromes which affect a specific organ or tissue include but are not limited to: diabetes mellitus type 1, Hashimoto's thyroiditis, Addison's disease (endocrinologic); Celiac disease, Crohn's disease, pernicious anemia (gastrointestinal); pemphigus vulgaris, vitiligo (dermatologic); autoimmune haemolytic anaemia, idiopathic thrombocytopenic purpura (haematologic) and myasthenia gravis (neurologic). The above-identified disease states are provided as a general description of numerous immune or inflammatory disease states known in the art, but are in no way intended to limit the scope of this disclosure.

[0031] An "acute inflammatory episode" as used herein refers to an increased immune response. Symptoms of acute inflammation include redness, heat, swelling, pain, and loss of function, e.g., loss of joint movement. An acute inflammatory episode of a chronic inflammatory disease or condition differs from the typical symptoms of a chronic inflammatory disease or condition. Frequently, during an acute inflammatory response the liver synthesizes acute phase proteins or acute phase reactants that are detectable in the blood stream. Acute phase reactants at least include but are in no way limited to C-reactive protein (CRP); alpha 1 -antitrypsin; alpha 1-antichymotrypsin; alpha 2-macroglobulin; coagulation factors such as fibrinogen, fibrin, prothrombin, thrombin, factor VIII, and plasminogen; complement proteins; and serum amyloid protein. In addition, during an acute inflammatory episode, local inflammatory cells, e.g., neutrophils and macrophages, secrete a number of cytokines into the bloodstream, most notably IL-1, IL-6, IL-11, and TNF-alpha.

[0032] As used herein, the "complement pathway or system" refers to a complex network of more than 50 functionally linked proteins that interact in a highly regulated manner to provide many of the effector functions of innate and acquired immunity and inflammation, thereby serving as the major defense mechanism against bacterial and fungal infections. This system of proteins acts against invasion by foreign organisms via three distinct pathways: the classical pathway (in the presence of antibody) or the alternative pathway (in the absence of antibody) and the lectin pathway. Once activated, the proteins within each pathway form a cascade involving sequential self-assembly into multimolecular complexes that perform various functions intended to eradicate the foreign antigens that initiated the response. For a review of the complement pathway, see, e.g., Sim and

Tsiftsoglou, Biochem. Soc. Trans. 32:21-27 (2004). The classical pathway is usually triggered by an antibody bound to a foreign particle or structural remnants of cell death. It includes CI that is specific to the classical pathway. Sequentially, binding of Clq to an antigen-antibody complex results in activation of Clr and Cls (both are serine proteases), and activated Cls cleaves C4 and C2 into, respectively, C4a and C4b and C2a and C2b. Fragments C4b and C2a assemble to form C4b2a, which cleaves protein C3 into C3a and C3b, which completes activation of the classical pathway. Fragments C4b and C3b are subject to further degradation by Factor I. This factor cleaves C4b to generate C4d and also cleaves C3b, to generate iC3b followed by C3d. Thus, activation of the classical pathway of complement can lead to deposition of a number of fragments or "complement activation products," such as iC4b, C4d, iC3b, and C3d, on immune complexes or other target surfaces. Such targets previously known in the art include cells circulating in the blood, e.g., lymphocytes and other white blood cells, erythrocytes and platelets. Activation of the alternative complement pathway begins when C3b (or C3i) binds to e.g., the cell wall or other surface components of a microbe. Alternative pathway protein Factor B then combines with the cell-bound C3b to form C3bB. Factor D then splits the bound Factor B into Bb and Ba, forming C3bBb. A serum protein called properdin then binds to the Bb to form C3bBbP, which functions as a C3 convertase that cleaves C3 into C3a and C3b. The lectin complement pathway is mediated by mannan-binding lectin or mannan-binding protein (MBP). MBP is a protein that binds to the mannose groups found in many microbial carbohydrates. The MBP appears to be functionally equivalent to Clq in the classical complement pathway. Activation of the lectin pathway begins when MBP binds to the mannose groups of microbial carbohydrates. Two more lectin pathway proteins called MASP1 and MASP2 (functionally equivalent to Clr and Cls of the classical pathway) then bind to the MBP. The MASP1/MASP2/MBL complex forms an enzyme with activity similar to CI of the classical complement pathway that is able to cleave C4 and C2 to form C4bC2a, a C3 convertase that cleaves C3 into C3a and C3b. The C3 convertase cleaves and activates complement pathway components to form a membrane attack complex (MAC) that forms a pore in a bacterial cell wall, lysing the bacterial cell.

[0033] As used herein, a "first subunit" is any compound which is associated with or elevated within the systemic circulation in response to an acute inflammatory response, chronic inflammatory condition or other disease state, including but not in no way limited to the following: any lipoprotein complex components, (such as HDL [Apolipoprotein Al] and LDL [Apolipoprotein B]), C -reactive protein (CRP); alpha 1 -antitrypsin; alpha 1- antichymotrypsin; alpha 2-macroglobulin; coagulation factors such as fibrinogen, fibronectin, fibrin, prothrombin, thrombin, factor VIII, von Willebrand factor (vWF) and plasminogen; complement proteins, and serum amyloid protein, hormones (such as insulin), chemokines, and cytokines, including but not limited to IL-1, IL-6, IL-11, TNF-a, IFN-a and.IFN-γ. Such a "first subunit" is a candidate for complex formation with a "second subunit" (i.e., a "compound" as defined herein).

[0034] As used herein, "biomarker" or "marker" refers to a soluble molecular complex comprising at least a first subunit and second subunit wherein a compound is linked (including but not limited to linkage via bonding) to a specific complement activation product, forming a C-TM that can be measured in a biological sample obtained from an individual to detect a specific disease state (e.g., SLE) or to monitor the status of a condition (e.g., level of disease progression) in the individual. In particular, biomarkers as disclosed herein are found as soluble components within the systemic circulation and are obtained from a fluid sample of an individual. To note specific exemplifications herein, but in no way to suggest any limitation in the type or number of C-TMs that may be identified and in turn serve as useful biomarkers in detecting and/or monitoring various disease states, a representative panel of biomarkers for detecting and/or monitoring SLE includes ApoAl- C4d, ApoAl-C3d, ApoB-C4d, ApoB-C3d, CRP-C4d, vWF-C4d, C4d-Ig, C3d-Ig and fibrinogen-C4d.

[0035] "Diagnostic," as used herein, characterizes something that identifies the presence or nature of a pathologic condition, such as SLE. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay, are termed "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis. The term "diagnostic" or "diagnosing" or "diagnosis" may be used interchangeable with "identify" or "identifying" or "identification."

[0036] "Monitoring" refers to identifying, observing and/or recording changes in a continuously varying parameter, such as the progression of an autoimmune or

inflammatory disease state in a patient. The terms "monitoring" or "monitor" may be used interchangeably with "identify" or "identifying" or "identification."

[0037] As used herein, a "control level" of any respective C-TM refers, in some embodiments, to a level of a respective C-TM obtained from the fluid sample of an individual who does not suffer from an autoimmune, inflammatory or other disease or disorder. A control level can also be determined by analysis of a population of individuals. In other embodiments, the control level of a respective C-TM is from an individual who does have an inflammatory disease or disorder, but who is not experiencing an acute phase of the disease. In some embodiments, the control level of a respective C-TM is from the same individual for whom a diagnosis is sought or whose disease is being monitored, but is obtained at a different time.

[0038] As used herein, "a difference from a control level" refers to a difference that is statistically significant, as determined by statistical analysis methods used by those in the art. A difference from a control level refers to a statistically significant difference between a control level of a respective C-TM and a level of the same C-TM from an individual for whom diagnosis or other information is sought, i.e., an experimental level. A control level might also be compared to an individual's baseline (i.e. they are their own control when used for monitoring, where each specific individual may have their own internal "thermostat"). It is also relevant to consider a control level as the ratio of non- tagged to complement-tagged molecule (e.g., native insulin to insulin-C4d) or possibly to determine the ratio of two tagged forms (e.g. insulin-C3d/insulin C4d). Those of skill will recognize that many methods are available to determine whether a difference is statistically significant and the particular method used is not limiting to the invention.

[0039] As used herein, "Systemic Lupus Erythematosus", "SLE", or "lupus" is a prototypic autoimmune disease resulting in multiorgan involvement. This anti-self response is characterized by autoantibodies directed against a variety of nuclear and cytoplasmic cellular components. These autoantibodies bind to their respective antigens, forming immune complexes which circulate and eventually deposit in tissues. This immune complex deposition and consequential activation of the complement system causes chronic inflammation and tissue damage. Systemic Lupus Erythematosus progresses in a series of flares, or periods of acute illness, followed by remissions. The symptoms of an SLE flare, which vary considerably between patients and even within the same patient, include malaise, fever, symmetric joint pain, and photosensitivity (development of rashes after brief sun exposure). Other symptoms of SLE include hair loss, ulcers of mucous membranes, inflammation of the lining of the heart and lungs which leads to chest pain, and synovitis, a painful inflammation of synovial membranes. Red blood cells, platelets and white blood cells can be targeted in lupus, resulting in anemia and bleeding problems. More seriously, immune complex deposition and chronic inflammation in the glomerulus can lead to kidney involvement and occasionally failure requiring dialysis or kidney transplantation. Since the blood vessel is a major target of the autoimmune response in SLE, premature strokes and heart disease are not uncommon. Over time, however, these flares can lead to irreversible organ damage.

[0040] An example formation of a biomarker is shown in FIG. 1, which shows a schematic illustration of a representative C-TM 100 that may be used as an indicator for SLE. When an acute inflammatory episode 101 of a chronic inflammatory disease such as SLE activates the complement system, complement activation products 103 (including but not limited to C4d) may be released into circulation and will bind to soluble plasma molecules 105 (or complexes or other compound types) in the immediate vicinity within the systemic circulation. These complexes remain in the plasma and can be measured as biomarkers of complement activation. The complement activation products 103 are a second subunit and the molecule 105 is a first subunit of each C-TM 100.

[0041] The data presented within the Examples of this document indicates that formation of these soluble complexes and in turn identification of one or more specific complexes for use as a disease state biomarker is applicable to any malady where a chronic inflammatory condition or acute inflammatory episode is presented within the individual. It is within the purview of the artisan of ordinary skill who reads this disclosure to choose one or more compounds in combination with one or more complement activation products to utilize general immunoassay techniques as described herein to screen for the combination of these soluble complexes to identify one or more useful biomarkers as an indicator of that specific immune or inflammatory disease state. The diseases or disorders may include, but are in no way limited to, various metabolic disorders such as type II Diabetes Mellitus, various endocrinopathies, coagulopathies, cerebrovascular disorders, immune deficiencies, infectious diseases, and any such condition where the tagging of a compound (such as any self protein) with a complement activation product may be considered to interfere with the normal physiologic function of that molecule.

[0042] Depending upon the target disease state, one may identify and test for one or more potential compound/complement activation product complexes which may be prevalent in response to an acute or chronic inflammatory state. This identification and testing may be based upon previous knowledge of what particular compound(s) (e.g., protein, lipoprotein, etc.) associate with the inflammatory state. As noted in part throughout this document, such compounds include but are not limited to cytokines (including but not limited to ILl-alpha, IL-1 beta, IL-6, TNF-alpha, members of the IL-20 family, IL-33, LIF, IFN-gamma, OSM, CNTF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL-18, IL-8 and any variety of other chemokines that may chemoattract inflammatory cells), chemokines (including but not limited to IL-8, CCL2, CCL3, CCL4, CCL5, CCL11, CCL20 and CXCL10), hormones (including but not limited to pro-inflammatory hormones from the prostaglandin, cytokine and histamine grouping, additional peptide and protein hormones known in the art, including but not limited to insulin, angiotensin- 1, CCK 4, CCK 5, CCK 7, CCK 8, CCK 9 CCK 10, CCK 18, CCK 22, CCK 25, endothelin, GRP, glucagon, glucagon- like peptide- 1, glucagon-like peptide-2, and leptin), as well as amine-derived hormones such as catecholamines (adrenaline, dopamine, noradrenaline) tryptophan derivatives (melatonin, serotonin), tyrosine derivatives (thyroxine, triiodothyronine), steroid and sterol hormones (glucocorticoids), mineralocorticoids (aldosterone), sex steroids (androgens, estrogens, progestagens), as well as lipid and phospholipid hormones

(eicosanoids, prostaglandins, leukotrienes, prostacyclin, thromboxane), components of the coagulation, kinin and fibrinolytic cascades, acute phase reactants, as well as biomarkers of cardiovascular disease such as C-reactive protein and apolipoproteins, also as disclosed herein.

[0043] As noted herein, the invention is not limited to the exemplified soluble complexes detailed in the Examples. The identification of these exemplified complexes enables the artisan of ordinary skill to apply this teaching to other maladies within the realm of acute and/or chronic inflammatory presentation. To this end, the compound/complement activation product complexes as disclosed in this document will provide for useful biomarkers to diagnose, monitor or predict (as an independent risk factor for) cardiovascular disease. Complex formation and biomarker determination may occur through initial assays as disclosed herein. As an example, but in no way provided as a limitation, compounds which may form a soluble complex with a complement activation product include C- reactive protein, serum amyloid A (SAA; which is transported predominantly on HDL), secretory phospholipase A (also an HDL-associated protein), and platelet-activating factor acetylhydrolase (a protein associated predominantly with HDL in humans), Apolipoprotein AI (ApoAI), IL-1, TNF-alpha, IL-6, PAI-1, angiotensin, tissue factor and para-oxonase-1 (PON1; also a HDL-associated protein). Therefore, numerous compounds associated with cardiovascular inflammation episodes (including but not limited to multiple HDL associated proteins) may act as a "first subunit" in formation of a soluble complex with a complement activation product (presented as a "second subunit").

[0044] Another logical inflammation-based disorder includes the relationship between obesity, insulin resistance and the onset of type II Diabetes Mellitus. Many type II diabetics are obese, suffer from chronic inflammation and are resistant to insulin. It is known that such individuals have overly active immune responses leading to large amounts of pro-inflammatory compounds. Any such compound may be targeted to practice the present invention as a diagnostic and/or monitoring tool (similar to immediate blood glucose monitoring or a standard HgbAlc test which measures glycated hemoglobin over an extended period) to chronicle links and disease progression in obese individuals indicating acute or chronic inflammation, including but not limited to insulin, TNF-alpha, FOXOl, C- reactive Protein(CRP), IL-1, IL-6, leptin, adiponectin, visfatin, resistin, IL-8, IL10, IL-18, MCP-1, MIF, M-CSF, TGF-beta, soluble TNFR and haptoglobin.

[0045] This document discloses in part the identification and characterization of soluble complexes obtained and isolated from the fluid sample obtained from an individual. Such a soluble complex comprises two heterogeneous components: a first subunit representing a compound associated either directly or indirectly with an inflammatory episode, chronic inflammatory condition, or other disease state of an individual, this first subunit identified herein as being bound to a second subunit represented by a complement activation product. As discussed further within this document, one or more such C-TM complexes obtained as a soluble component from a bodily fluid sample will serve as a representative biomarker for detection, monitoring and/or prediction of a particular immune/inflammatory disease state within an individual. While known in the art that any number of first subunit compounds are associated with an acute inflammatory episode, chronic autoimmune/inflammatory or other disease state; and while known in the art that certain complement activation products complex with various cell types in response to inflammation, it was heretofore not known in the art and in fact was unexpected that (i) these C-TMs exist and in turn are amenable to isolation and characterization, and (ii) that furthermore such C-TMs are associated with and indicative of a particular autoimmune, inflammatory or other disease state so as to promote use as a biomarker.

[0046] This document thus discloses and enables the artisan of ordinary skill to utilize a specific C-TM (or panel of respective C-TMs) as biomarkers to diagnose, monitor, and/or predict a particular disease state wherein first subunit and second subunit

components are known to or logically thought to coincide with an acute inflammatory episode,chronic inflammatory condition or other disease state. The process of identifying specific C-TMs as a biomarker is exemplified herein with regard to SLE and other representative inflammatory diseases (see Example section).

[0047] With this exemplification in hand, the artisan will understand that an initial step in identifying and utilizing any specific C-TM as a potential autoimmune,

inflammatory or other disease biomarker will be to select and pair a first subunit compound which may be known to be associated with the disease state of interest (including but not limited to a high density lipoprotein (HDL), coagulating factors, cytokines, hormones, and chemokines) with a relevant second subunit complement activation product (including but not limited to C4d or C3d) for testing to identify and characterize any such C-TM as a potential biomarker. Such a logical pairing, based on knowledge of the particular disease state and components associated with that disease state, allows for the initial testing and identification of the possible presence of such a C-TM by utilizing monoclonal or polyclonal antibodies specific for each of the first subunit and second subunit of the C-TM in a standard immunoassay, again as exemplified herein for SLE and additional diseases.

[0048] Subsequent to identifying the presence of a specific C-TM in a bodily fluid sample, relevance as a biomarker is determined by comparing the level of the respective C- TM obtained from a patient diagnosed with a specific autoimmune, inflammatory or other disease to the presence (or absence) of the C-TM in a bodily fluid sample obtained from a control source. It is of import to note that this disclosure is in no way limited to

identification and utilization of the C-TM biomarkers relating to diagnosis and monitoring of SLE as specifically disclosed herein. Quite to the contrary and as noted above, this document discloses and enables an artisan of ordinary skill in the art to (i) test for the presence of such a soluble complex comprising a first subunit linked to a second subunit obtained from a bodily fluid sample, and (ii) test to identify that C-TM as a relevant biomarker, where the C-TM is present in significantly elevated levels as compared to a control sample. Thus, as enabled within this document, any C-TM hypothesized to exist in concert with a specific autoimmune, inflammatory or other disease state may be identified with such known assays such as immunoassays disclosed within the Example section.

[0049] Any C-TM complex may also be generated in substantial quantities in vitro utilizing basic recombinant DNA and protein purification protocols well known in the art. Presented as an example but by no means a limitation, if the artisan is driven to isolate an ApoAl-C4d C-TM complex, multiple options are available. A cDNA encoding ApoAl could be generated from human polyA mRNA using strategic 5' and 3 ' primers based on the nucleotide sequence of the human gene (see, e.g. GenBank Accession No. NM 000039) to synthesize and clone a full length cDNA encoding human ApoAl . Such a cDNA may then be subcloned into an appropriate prokaryotic or eukaryotic expression vector, where substantial amounts of ApoAl may be expressed and purified by any of a myriad of techniques known in the art. Alternatively, ApoAl might be purified directly from human plasma-derived HDL (see, e.g., Huang, et al., Nat. Struct. Mol. Biol. 18(4):416-422 (2011)). And choices will abound for isolation of C4d, as well, noting a human C4d nucleotide sequence being available as GenBank Accession No. U77887.1, and direct purification methods being disclosed, for example, by van den Elsen et al., J. Mol. Biol. 322: 1103-1115 (2002). These substantially purified ApoAl and C4d "subunits" may then be incubated by methods well known in the art to form a C-TM ApoAl-C4d complex.

[0050] This document further discloses a method of diagnosing and/or monitoring the state of an inflammatory response associated with an immune or inflammatory disease or disorder by detecting and measuring the level of C-TMs within the bodily fluids of an individual. This methodology enables the detection and measurement of C-TMs as a product of an inappropriate activation of the complement system in response to an autoimmune, inflammatory or other disease or disorder, whereby such activation causes an inflammatory response and tissue injury. The ability to identify and detect a respective C- TM generated as a result of activation of the complement system provides the practitioner with an additional tool to detect, diagnose or monitor multiple disease states. Thus, complement activation products that are generated in the systemic circulation bind various compounds (including but not limited to proteins or lipoproteins) associated with a specific disease state to form C-TM complexes. As described in the Example section, these bound complexes will serve as biomarkers for either detection of the underlying disease state or as a tool to monitor progression of the disease in a patient. These biomarkers may be used alone or may make up a panel of biomarkers that can be used alone or in combination with traditional assays to facilitate the diagnosis and monitoring of a respective disease state.

[0051] The methodology disclosed within this document enables the artisan to select an optimal detection protocol for the diagnosis and/or monitoring of the presence or progression of a particular disease state, wherein immunoassays as disclosed herein are utilized to detect a relevant C-TM complex formed between one or more compounds (a first subunit) and one or more complement activation products (a second subunit). As an initial foray into utilization of such a detection assay, the artisan may choose one or more compounds known to or likely to be associated with the disease state itself or to have involvement in downstream indications associated with that specific disease. One or more known compounds and one or more known complement activation products may be selected as candidates, in any combination, for an initial testing procedure as disclosed within the Example section showing an initial selection of assays to detect C-TMs predicted to be associated with SLE. As noted further in the Example section, based on these initial results, the practitioner may select one or more relevant C-TM complexes for future standard C-TM detection protocols.

[0052] Based on this disclosure it is evident that methods are provided for diagnosing a specific disease state (as exemplified herein for SLE and other inflammatory disease states) by determining the level of at least one soluble biomarker complex in a bodily fluid sample from a subject. If the level of the first biomarker is within a

predetermined level, then the subject is diagnosed with the disease. However, if the level of the first biomarker is outside the predetermined level, then the level of a second biomarker is determined in the sample. The subject is diagnosed with the specific disease if the level of the second biomarker is within a predetermined level. In some embodiments, the diagnostic method further comprises determining the level of a third biomarker if the level of the second biomarker is outside of the second predetermined level. In such

embodiments, if the level of the third biomarker is within a third predetermined level, the subject is diagnosed with the disease. Preferably, the third biomarker is different from the first and second biomarker. Thus, it is within the scope of this disclosure to select a panel of C-TM biomarkers for use in a standard immunoassay to detect, monitor or predict any particular immune or inflammatory disease.

[0053] This document further discloses a method of diagnosing and/or monitoring an autoimmune or other disease or disorder or diagnosing and/or monitoring an

inflammatory or other disease or disorder by (i) obtaining a control sample, such as a bodily fluid (serum or plasma) sample from an individual who is known to be negative for the disease or disorder of interest, or a sample obtained from the patient of interest at an earlier point in time, (ii) measuring the amount of at least one C-TM obtained from the bodily fluid sample of the patient at a current time, (iii) measuring the amount of the C-TM or C-TMs that are contained in the control sample, and (iv) comparing the amount of C-TMs from the patient's current sample against the amount of C-TMs present in the control sample. A significant difference between the C-TM levels in the individual compared to the control sample indicates that the individual is showing an acute response related to the underlying disease or disorder. A significant difference may vary depending on the disease of interest. For example, if the C-TM level found in the individual's fluid sample is greater than that of the control sample, then it may be determined that the individual is showing the response related to the disease or disorder. It is understood that the methods disclosed throughout this document do not necessarily require access to and measurement of a control sample for each individual assay. Instead, the artisan will easily grasp the notion that standard measurements (e.g., from a 'normal' individual or a patient suffering from chronic SLE) previously generated may be utilized to compare against a test sample obtained from a particular patient at that point in time. As a specific example, in the context of SLE, an increase of a significant amount, such as an increase of C-TM in an amount of at least two standard deviations, may indicate that the patient is showing a response relating to SLE. For other diseases, such as diabetes, if the C-TMs are associated with insulin then an increase over time or as compared to the control sample may indicate the presence of diabetes. In some embodiments, multiple C-TMs may be present, and if so the method may rank them such that the results for a first C-TM are considered to be an identifier, while others are used for confirming analysis or only if the first C-TM analysis is inconclusive, or the system may consider relative rations of the various C-TMs. [0054] As exemplified herein, in some embodiments suitable samples for use in the methods of the invention are blood samples, which may be treated with a material such as EDTA (ethylenediaminetetraacetate) to inhibit C-TM formation, or an anti-coagulant and complement inhibitor such as heparin, post-draw. Samples can be maintained at room temperature (short term, less than 2 hours) or stored a suitable temperature, such as 4°C (for up to 24 hours). In some embodiments, the whole blood sample may be fractionated into different components, such as serum or plasma, by standard methodology known in the art, and stored at a suitable temperature, such as -80°C, for later use.

[0055] In a particular method disclosed within this document, systemic lupus erythematous ("SLE" and additional inflammatory disease states) is diagnosed and/or monitored in an individual by (i) obtaining a bodily fluid sample from an individual for testing, (ii) measuring the amount of at least one C-TM related to the inflammatory disease (such as SLE) from the bodily fluid sample of the individual, (iii) measuring the amount of at least one C-TM from a control serum or plasma sample, and (iv) comparing the amount of C-TMs from the individual against the amount of C-TMs detected in the control sample, where an increase in the C-TM level in the individual compared to the control sample indicates that the individual is showing an increased response related to the underlying disease or disorder. A representative compound to be measured for complex formation in SLE indications includes but is not limited to Apolipoprotein Al (ApoAl ; a major protein component of HDL), Apolipoprotein B (ApoB; a major protein component of LDL), C- reactive protein (CRP; an acute phase protein linked with inflammation and atherosclerosis), von Willebrand factor (vWF), and fibrinogen. A representative complement activation factor to be measured for complex formation in SLE indications includes but is not limited to C4d, C4c, C3d, and C3c. To this end, representative C-TMs utilized to diagnose or monitor SLE include but are not limited to ApoAl-C4d, ApoB-C4d, CRP-C4d, vWF-C4d, and fibrinogen- C4d.

[0056] FIG. 2 illustrates an example of how C-TMs may be detected. In this example, molecules 201 are tagged with labels 203 that emit light 205 upon electrochemical stimulation initiated by one or more electrode surfaces. The molecules may be positioned on a substrate 211 with an array of embedded electrodes 213. The electrodes may include one or more working electrodes 213A and one or more counter electrodes 213B that, when activated, excite the C-TMs and cause them to emit light that is detectable and measuable. The emitted light may be captured and measured by a digital image imaging device 207 such as a charge- coupled device camera.

[0057] FIG. 3 illustrates an example process that uses various detecting antibodies to assist in the detection of C-TMs.

[0058] This disclosure further describes and enables a kit for diagnosing, monitoring, or predicting an inflammatory episode derived from a chronic autoimmune, inflammatory or other disease or condition in an individual. The kit can include an antibody specific to a compound which may form a complex with a particular complement activation product and a second antibody specific to the respective complement activation product and a means for detecting and measuring the relative amount of the respective C-TM present in one or more bodily fluid sample, obtained either from an individual being tested for a specific immune or inflammatory disease or from a source meant to serve as a control measurement. A predetermined difference in a biomarker C-TM from the individual compared to the control sample will likely serve as identification of the respective disease state. Any such kit may utilize any combinations of antibody assay technology available in the art to allow detection of the respective C-TM. [0059] The methodology disclosed within this document enables the artisan to select an optimal detection protocol for the diagnosis and/or monitoring of the presence or progression of a particular disease state, wherein assays (such as immunoassays) as disclosed herein are utilized to detect a relevant C-TM complex formed between one or more compounds and one or more complement activation products. As an initial foray into utilization of such a detection assay, the artisan shall choose one or more compounds known to or likely to be associated with the disease state itself or to have involvement in downstream indications associated with that specific disease. One or more known compounds and one or more known complement activation products may be selected as candidates, in any combination, for an initial testing procedure as disclosed within the Example section showing an initial selection of assays to detect C-TMs predicted to be associate with SLE and other inflammatory diseases. As noted further in the Example section, based on these initial results, the practitioner may select one or more relevant C-TM complexes for future standard C-TM detection protocol.

[0060] The level (e.g., quantity or amount) of a particular soluble biomarker can be measured in the sample using any of a myriad of methods known in the art. Oftentimes the presence or level of the biomarker will be determined using monoclonal or polyclonal antibodies specific for the biomarker complex and detecting specific binding of the antibody to one and/or the other component of a C-TM complex. Such methods include, but are not limited to an immunoassay, which may be competitive or non-competitive in nature, includes but is not limited to electrochemiluminescence assays (ECL; an assay utilizing a chemiluminescent antibody or antibodies, each specific for a specific biomarker complex component, is suitable for sensitive, non-radioactive detection of biomarker levels), enzyme immunoassays (EIA) such as enzyme multiplied immunoassay technique (EMIT), enzyme- linked immunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA), microparticle enzyme immunoassay (MEIA); capillary electrophoresis immunoassays (CEIA); radioimmunoassays (RIA); immunoradiometric assays (IRMA); fluorescence polarization immunoassays (FPIA), bead-based assays and Genalyte MAVERICK™

Detection System which relies on microchip-based assays. Specific immunological binding of the antibody to the biomarker can be detected directly or indirectly. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody. An antibody labeled with fluorochrome is also suitable for determining the level of one or more biomarkers in a sample. And as known in the art, signal detection will depend upon the type of direct or indirect label used in that particular assay.

[0061] As exemplified herein, a heterogeneous sandwich enzyme immunoassay that utilizes ECL technology may be particularly useful in practicing the methodology disclosed herein. For example, in a two-antibody heterogeneous sandwich assay, a first antibody (capture antibody) is bound to a solid support, and one subunit of the biomarker complex is allowed to bind to the first antibody. The amount of the biomarker is quantitated by measuring the amount of a second antibody (detection antibody) that binds the other subunit of the biomarker complex. As noted above, any specific heterogeneous double antibody sandwich assay may be used to practice the methods disclosed herein, including but not limited to an ECL-based immunoassay available from Meso Scale Discovery, Gaithersburg, MD (utilizing SULFO-TAG™- labeled antibody read with a SECTOR Imager equipped with MSD Discovery Workbench software) or an AlphaLISA sandwich immunoassay (PerkinElmer, Waltham, MA) which utilizes a streptavidin-coated donor bead and an antibody-conjugated acceptor bead. One antibody (specific for a complex subunit) is biotinylated and will associate with the streptavidin-coated donor bead. The other antibody (specific for a second complex subunit) is unlabeled and can be directly conjugated to the acceptor bead. The donor beads are excited with a laser, releasing singlets of oxygen which excite the acceptor beads, producing a measurable light emission, thus detecting specificity of both antibodies to the C-TM complex. Another useful assay available in the art for detecting a C-TM complex involves a Luminex^®-based immunoassay (Life Technologies, Carlsbad, CA) which combines ELISA technology and flow cytometry. One antibody (specific for a complex subunit) binds to a capture antibody with defined spectral properties followed by addition of a biotinylated detector antibody specific for a second complex subunit. A streptavidin-conjugated fluorescent protein is added and incubated prior to flow cytometry.

[0062] The monoclonal or polyclonal antibodies herein can also be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (e.g., microtiter wells, filter-bottom microplate), pieces of a solid substrate material or membrane (e.g., plastic, nylon, paper), and the like. An assay strip can be prepared by coating the antibody or a plurality of antibodies in an array on a solid support. This strip can then be dipped into the test sample and processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.

Additionally, an antigen capture assay may be utilized by the artisan. For example, in an antigen capture assay, an antibody directed to a biomarker of interest is bound to a solid phase and sample is added such that the biomarker is bound by the antibody. After unbound molecules and compounds are removed by washing, the amount of bound marker can be quantitated using, for example, a radioimmunoassay.

[0063] Quantitative Western blotting also can be used to detect or determine the level of one or more biomarkers in a sample. Western blots can be quantitated by well- known methods such as scanning densitometry or phosphorimaging. In certain instances, methods well known in the art include autoradiographs of the blots being analyzed using a scanning densitometer and normalized to a positive control. Alternatively, a variety of immunohistochemistry (IHC) techniques can be used to determine the level of one or more biomarkers in a sample. An IHC technique encompasses techniques that utilize the visual detection of fluorescent dyes or enzymes coupled (i.e., conjugated) to antibodies that react with the biomarker using fluorescence microscopy or light microscopy and includes, without limitation, direct fluorescent antibody, indirect fluorescent antibody (IFA), anticomplement immunofluorescence, avidin-biotin immunofluorescence, and

immunoperoxidase assays. An IFA assay, for example, is useful for determining whether a sample is positive for a particular marker of interest, the level of that marker, and/or the staining pattern of that marker.

[0064] The concentration of the marker in a sample can be quantitated, e.g., through endpoint titration or through measuring the visual intensity of fluorescence compared to a known reference standard. The presence or level of a biomarker can also be determined by detecting or quantifying the amount of the purified marker. Purification of the marker can be achieved, for example, by high pressure liquid chromatography (HPLC), alone or in combination with mass spectrometry (e.g., MALDI/MS, MALDI-TOF/MS, tandem MS, etc.). Qualitative or quantitative detection of a biomarker can also be determined by well-known methods including, without limitation, Bradford assays, Coomassie blue staining, silver staining, assays for radiolabeled protein, and mass spectrometry. The analysis of a plurality of biomarkers may be carried out separately or simultaneously with one test sample. Particularly useful physical formats comprise surfaces having a plurality of discrete, addressable locations for the detection of a plurality of different biomarkers. Such formats include protein microarrays, or protein chips and certain capillary devices. In these embodiments, each discrete surface location may comprise antibodies to immobilize one or more biomarkers for detection at each location. Surfaces may alternatively comprise one or more discrete particles (e.g., microparticles or nanoparticles) immobilized at discrete locations of a surface, where the microparticles comprise antibodies to immobilize one or more biomarkers for detection. In view of the above, one skilled in the art will readily appreciate that the methods for determining the presence of a C-TM complex within a bodily fluid sample can be practiced using one or any combination of the well-known techniques described above or other techniques known in the art.

[0065] Another disclosure within this document enables kits for conducting the assays for diagnosing, monitoring or predicting disease activity through detection and measurement of the level of a respective C-TM biomarker obtained from an individual. Any such kit will utilize any of the various reagents needed to perform the methods described herein. For example, in a typical sandwich-based immunoassay as exemplified herein, a kit will generally comprise a monoclonal or polyclonal antibody specific for the complex first subunit and a second monoclonal or polyclonal antibody specific for a complex second subunit. The kit can also include an isolated C-TM biomarker for use in control aspects of the assay.

[0066] To this end, this document discloses both methods as well as kits enabled herein to provide for detection of a respective C-TM biomarker by adapting the methods and kits known in the art for the rapid detection of such biomarkers in a biological sample. One such method can comprise the steps of (i) obtaining a bodily fluid sample suspected of containing one or more of a biomarkers disclosed herein, (ii) mixing a portion of the sample with a contacting antibody for specifically binding to a C-TM subunit so as to initiate the binding of the contacting antibodies of the C-TM subunit in the sample, (iii) contacting the mixture of sample and contacting antibodies with detecting antibodies which specifically bind to a separate subunit of the C-TM, which detection antibodies do not cross-react with the contacting antibodies, so as to bind the contacting antibodies to the C-TM to form a detectable complex, (v) removing unbound antibodies and any unbound sample from the complex, and (vi); and measuring an amount of the detecting antibodies present in the dual antibody C-TM complex. The results from steps (i) - (vi) may then be compared to a control fluid sample as per steps (i) - (vi), or determined as having fallen in or out of a predetermined level to identify a respective disease state. The detectable antibodies can be labeled with a detectable marker, such as a radioactive label, enzyme, biological dye, magnetic bead, or biotin, as is well known in the art. The contacting antibodies can be attached to a supporting material, such as a membrane, plastic strip, plastic laboratory plate such as those used for ELISA or other high-throughput assays, or any other supporting material, such as those used in other diagnostic kits well known in the art.

[0067] The kits or tests for determining the level of particular biomarkers include the various reagents for performing the measurements according to the methods described herein. For instance, in one embodiment, the kits or tests include reagents for performing ECL assays for each of the biomarkers, such as a monoclonal antibody specific for a complement activation product and a monoclonal antibody specific for a soluble compound which complexes with a complement activation product to form a detectable C-TM.

Additionally, the kits can comprise such other material as may be needed in carrying out assays of this type, for example, buffers, radiolabeled antibodies, colorimeter reagents, instructions for separating different cell fractions from whole blood, and instructions for diagnosing the respective disease state based on particular pre-determined levels of the C- TM biomarker(s).

[0068] This document discloses the interaction between a compound (such as a soluble plasma protein) with complementation activation products. Binding of complement activation products to a given compound (such as a plasma protein) may not only alter its structural feature but also perturb its functionality (possibly by preventing multimolecular and receptor-ligand interactions caused by steric hinderance), thereby contributing to pathophysiologic changes of the underlying disease. For example, binding of complement activation products to high density lipoprotein complexes (HDL) may result in dysfunction of these originally anti-atherosclerotic, anti-inflammatory moieties and convert them into "pro-inflammatory" HDL. Considering the increased risk for thrombotic and cardiovascular disease that SLE patients are at, it is reasonable to postulate that binding of complement activation products to HDL or coagulating factors may play a role in facilitating the development of atherothrombotic disease prematurely in these patients.

[0069] Measurement of C-TMs as disclosed within this document allows for identification of a plethora of novel biomarkers of complement activation and disease pathogenesis. A sensitive immunogenicity assay was designed using the

electrochemiluminescence (ECL) technology for measuring C-TMs in the plasma of patients with SLE or other diseases. Unlike conventional enzyme- linked

immunoabsorbance assays (ELISA), the ECL assay uses labels that emit light upon electrochemical stimulation initiated at the electrode surfaces of a special microplate.

Compared with ELISA, the ECL assay in general has minimal background signals and broader detection ranges (See FIG. 2 for a schematic illustration). Initial experimental work utilized primarily normal human serum in which the complement system could be activated in vitro. Additional examples focus on C-TM assays with plasma prepared from patients with immune, inflammatory or other diseases.

EXAMPLE 1

[0070] Serum and Plasma Samples. Blood of healthy individuals was drawn into tubes without anticoagulant. Serum was collected after separation of blood clot by centrifugation (800 x g), aliquoted, and stored at -80°C until use. Blood anticoagulated with ethylenediamine-tetracetic acid (EDTA) was collected from healthy individuals, patients with SLE, and patients with other immune-inflammatory diseases (e.g., rheumatoid arthritis, Sjogren's syndrome, etc). Plasma was separated from the cellular compartment by centrifugation (800 x g, 15 min), aliquoted, and stored at -80°C until use. EDTA is the anticoagulant of choice because it also serves an inhibitor of complement activation in vitro after blood collection. A C4-depleted human serum (Complement Technology) was used as a negative control serum to validate the specificity of anti-C4d antibody.

[0071] Activation of Complement in Normal Human Serum. The complement system in normal human serum sample was activated in vitro by incubation with aggregated immunoglobulin ("Complement Activator," Quidel Corp., San Diego, CA; mimicking immune complexes) at 37°C for different periods of time (ranging from 2 min to 3 hours). After incubation, the activation process was terminated by supplementation of EDTA to a final concentration of 10 mM. The activated serum was aliquoted, stored at -80°C, and used as a positive control in the C-TM assays described below.

[0072] Immunoblotting analysis of C-TMs in the activated serum and patient plasma samples. To verify the formation of C-TMs during complement activation, focus is on detection of C4d-TM complexes. Briefly, aliquots of the in vitro activated serum and patient plasma samples were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition, followed by immunoblotting analysis using polyclonal rabbit-anti- human C4 alpha chain antibodies (Santa Cruz) or a mouse monoclonal antibody reactive with C4d (Quidel).

[0073] Electrochemiluminescence (ECD Assays for detecting C-TMs. To establish a high-throughput detection method for C-TMs, a modified sandwich

immunoabsorbance assay is designed that utilizes electrochemiluminescence (ECL) technology. FIG. 2 shows a schematic illustration of one way that an electrochemiluminescence (ECL) assay may be used, in this example with technology such as that available from Meso Scale Discovery, LLC (MSD). In this example, molecules 201 are tagged with labels 203 that emit light 205 upon electrochemical stimulation initiated by one or more electrode surfaces. The molecules may be positioned on a microplate 211 with an array of embedded electrodes 213, such as those known as MULTI-ARRAY^®, available from MSD. The electrodes may include one or more working electrodes 213 A and one or more counter electrodes 213B. The emitted light may be captured by a digital image imaging device 207 such as a charge-coupled device camera. The digital data of the captured images can be analyzed using suitable software such as the DISCOVERY

WORKBENCH^® software available from MSD.

[0074] Based on the high prevalence of atherothrombotic disease in SLE, we opted to first develop an assay for detecting complexes formed between complement activation products and proteins that are likely to be involved in the process of

atherosclerosis and thrombosis. These complexes may be provided on microplates by catching various proteins in individual wells of the microplates that have been pre-coated with an antibody specific for the candidate protein of interest. Various analytes, including but not limited to normal human serum activated in vitro, normal human plasma, patient plasma and the like are contacted with the the capture antibody, washed as described herein and contacted with a detection antibody (e.g., anti-C4d antibody) and measured in any capacity known in the art. Such assays may of course be compared against a baseline control (including but not limited to normal human plasma), a positive control (including but not limited to activatated normal human serum), which can then be compared to a reading of a test sample, such as a sample obtained from a patient, such as patient plasma. Candidate proteins tested includes Apolipoprotein Al (ApoAl; a major protein component of HDL), Apolipoprotein B (ApoB; a major protein component of LDL), C-reactive protein (CRP; an acute phase protein linked with inflammation and atherosclerosis), von

Willebrand factor (vWF), fibrinogen, and fibronectin. Additional candidate proteins investigated included albumin and immunoglobulin (Ig), the two most abundant proteins in the circulation. Assays for fibrinogen-complement activation product complexes and fibronectin-complement activation products complexes were performed with plasma samples only, because these proteins are incorporated into the blood clot during serum preparation and thus are not detectable in the serum.

[0075] Briefly, as illustrated in the flow diagram of FIG. 3, individual wells of the 96-well ECL microplate were coated (step 371) with a capture antibody and allowed to sit overnight. Capture antibodies used included: goat Ig (background, non-specific binding control), polyclonal goat-anti-human ApoAl (Abeam Inc., Cambridge, MA), goat-anti- ApoB (Abeam) goat-anti-human CRP antibody (Abeam), goat-anti-human vWF (Abeam), goat-anti-human Ig (Complement Technology), rabbit-anti-human albumin (Sigma), monoclonal mouse-anti-human fibrinogen (BD Pharmingen), or mouse-anti-human fibronectin (BD Pharmingen). The wells were washed to remove unbound antibodies, and uncoated surfaces were blocked for one hour with phosphate-buffered saline containing 5% bovine serum albumin (PBS-5%BSA) (step 373). Aliquots of normal human serum and plasma (reference control for normalization), normal human serum activated with aggregated Ig in vitro (positive control), and patient plasma (test samples) were added to each well (step 375). After two-hour incubation, the wells were washed three times with phosphate buffered saline (PBS) and then incubated with indicated detection antibodies (step 377). The detection antibodies used included: mouse isotype control IgGl (BD Biosciences; for detecting background noise signals), mouse monoclonal anti-C4d (Quidel; for detecting ApoAl-C4d, ApoB-C4d, CRP-C4d, vWF-C4d, Ig-C4d, and albumin-C4d complexes), and mouse monoclonal anti-ApoAl, anti-CRP, or anti-vWF (Abeam; for detecting/quantifying the candidate proteins captured on the well). In the assays for fibrinogen-C4d and fibronectin-C4d complexes, the detection antibody used was a polyclonal rabbit-anti-human C4 antibody.

[0076] After one-hour incubation with detection antibodies, the microplates were washed three times with PBS and incubated with MSD SULFO-TAG™-labeled anti-mouse IgG (for ApoAl-C4d, ApoB-C4d, CRP-C4d, vWF-C4d, Ig-C4d, and albumin-C4d complexes) or SULFO-TAG™-labeled anti-rabbit IgG (for fibrinogen-C4d and fibronectin- C4d complexes) for one hour (step 379). After three washes with PBS, a read buffer was added (step 381) and the microplate was read using a SECTOR Imager equipped with the MSD DISCOVERY WORKBENCH^® software (step 383)..

[0077] To ensure specific immunodetection and maximize the performance of these assays, all capture antibodies, detection antibodies, normal human serum, and plasma samples had been pre-titrated. For ApoAl-C4d, ApoB-C4d, vWF-C4d, fibrinogen-C4d, and fibronectin-C4d assays, capture antibodies were diluted in PBS-1%BSA and used at an amount of 50 ng/well. For the CRP-C4d assay, the capture antibody was used at 25 ng/well. Serum and plasma samples were used at 1 :25 dilution for ApoAl-C4d, vWF-C4d, fibrinogen-C4d, and fibronectin-C4d assays, and at 1 :125 dilution for the CRP-C4d assay. Twenty- five μΐ of the diluted serum and plasma sample were added to each well. The detection antibodies (mouse monoclonal anti-C4d, anti-ApoAl, anti-ApoB, anti-CRP, and anti-vWF; rabbit polyclonal anti-C4) and SULFO-TAG labeled anti-mouse or anti-rabbit Ig were diluted to 0.2 μg/ml in PBS-1%BSA and 25 μΐ were added to each well. To prevent complement activation occurring during the assay, all assay buffers were supplemented with 10 mM EDTA.

[0078] The rationale underlying this "heterogeneous" sandwich immunogenicity assay is as follows. If ApoAl-C4d complexes (or any given candidate protein X-C4d complexes) are present in the plasma samples, they will be captured on the microplate by polyclonal anti-ApoAl (or anti-candidate protein X) antibody and react to the anti-C4d detection antibody. The higher the signals of anti-C4d reactivity, the higher the amount of ApoAl-C4d (or candidate protein X-C4d) complexes is present in the test sample. A semiquantitative method is used to calculate the relative fold of increase in the levels of these complexes in test samples (levels in normal human serum or plasma = 1).

EXAMPLE 2

[0079] Detection of multiple high molecular weight C4d-protein complexes in human serum and plasma. The disclosure herein shows that complement activation products generated during complement activation, such as C4d, form complexes with proteins circulating in the blood.

[0080] FIGs. 4A-4C illustrate results that show the presence of C-TMs in human and plasma samples. Studies were performed to detect C-TMs in human serum and plasma samples using immunoblotting analysis. FIG. 4A shows analysis results for human serum prepared from a healthy individual that was incubated with aggregated immunoglobulins at

37°C for the indicated periods of time, resolved by SDS-PAGE, transferred onto PVDF membrane, and blotted with a polyclonal rabbit-anti-human C4 a chain antibody (upper panel) or a monoclonal mouse-anti-human C4d antibody (lower panel). Complement activation, as indicated by the cleavage of C4 a chain, was evident as early as 2 minutes after initiation and proceeded to completion at 30 minutes. Note the rapid disappearance of the intact C4 a chain and the concurrent appearance of high molecular weight protein species that reacted with anti-C4a and anti-C4d antibodies. The anti-C4d monoclonal antibody does not react to the intact C4 a chain. Concomitant with the

cleavage/disappearance of the intact C4 a chain, a group of high molecular weight species reactive with both anti-C4 a chain and anti-C4d appeared. Since the C4d fragment has a predicated molecular weight of 45 kD, the presence of high molecular weight, C4d- containing proteins suggests the formation of complexes between C4d and serum proteins. The 45 kD C4d fragment was not detectable in activated serum samples (not shown).

[0081] However, the 45 kD C4d fragment was readily detected when purified C4 was enzymatically cleaved using Factor I in the absence of other serum proteins. In FIG. 4B, purified human C4b was digested with Factor I (in the presence of a cofactor C4- binding protein) at 37°C for the indicated periods of time. The digested products were analyzed by immunoblotting using the anti-C4a (left panel) or anti-C4d (right panel) antibody. Note the step- wise cleavage of the C4 a chain over time, cumulating at the generation of C4d fragment. This latter result demonstrates that the anti-C4 a chain and anti-C4d antibodies used are capable of reacting with free C4d fragments properly. The absence of free C4d fragment in activated serum samples, therefore, appeared not to be an artifact attributable to the antibodies used. Taken together, these immunoblotting studies suggest that C4d, once generated upon systemic complement activation, is likely to complex with serum proteins in the immediate proximity and does not circulate as independent fragments

[0082] In FIG. 4C, plasma samples prepared from six patients were analyzed for the presence of C-TMs by immunoblotting. Plasma derived from a healthy individual

("normal human plasma"; NHP) and normal human serum activated with aggregated immunoglobulins (NHS*) were resolved on the same blot as the negative and positive controls, respectively. All samples were tested at the same volume (5 μΐ). The duplicate blots were blotted with a polyclonal anti-C4a (upper panel) and a monoclonal anti-C4d

(lower panel) antibody, respectively. Note the presence of anti-C4a/anti-C4d-reactive high molecular weight protein species in both NHS* and several patient plasma samples. Those high molecular weight protein species were barely detected in the NHP. Because the plasma samples were prepared from EDTA-anticoagulated blood, these protein complexes are unlikely the products of complement activation occurring in vitro after blood collection, but rather the products of complement activation happening in vivo before blood collection.

[0083] Development of a C-TM assay using activated human serum. Results of the immunoblotting studies described above support the hypothesis that C-TMs are generated in pathophysiologic situations wherein complement activation occurs. This example further disclosed an immunoassay allowing for rapid detection and/or

quantification of C-TMs in large numbers of patient plasma samples. We chose to utilize the recently developed ECL technology instead of the traditional enzyme-based

immunogenicity assays because of its superior sensitivity and wider dynamic range of detection. As described above,the first group of candidate C4d-complexed proteins we investigated were proteins that have been associated with development of atherothrombotic disease and the two most abundant serum proteins. These candidates included ApoAl, ApoB, CRP, vWF, albumin, and immunoglobulin.

[0084] To verify and optimize a C-TM assay, studies were performed using normal human serum activated with aggregated immunoglobulin in vitro as the analytes. In addition to C4d, other complement activation products such as C4c, C3d, and C3c were also investigated in some pilot experiments. Initially, we noticed that complement proteins present in the serum were further activated upon contact with the microplates and hence confounded the interpretation of the results obtained. Therefore, we modified the assay protocol to include EDTA in all reaction buffers to suppress unwanted action of the complement system during the assay. EXAMPLE 3

[0085] FIGs. 5A-F illustrate results of a representative experiment in which aliquots of normal human serum, activated or not with aggregated immunoglobulin, were used as the test analyte and the control (baseline) analyte, respectively. Individual wells of the MSD MULTI-ARRAY microplates were coated with the desired capture antibody, incubated sequentially with the analyte, detecting antibody, and SULFO-TAG labeled antibody, and analyzed using the SECTOR imager. The generation of C-TM complexes was calculated and presented as fold of increase of a specific C-TM in the activated human serum relative to the baseline level in the unactivated serum. Shown are the results of a representative experiment. Note the increase of C4d-TM and C3d-TM complexes, but not C4c-TM The results of FIGs. 5A-F demonstrate that activation of the complement system with aggregated immunoglobulin led to the appearance/increase of protein complexes that were, on the one side, captured by anti-ApoAl (FIG. 5 A), anti- ApoB (FIG. 5B), anti-CRP, (FIG. 5C), anti-vWF (FIG. 5E), anti-Ig antibodies (FIG. 5D) and anti-albumin antibodies (FIG. 5F), and on the other side, reactive with anti-C4d and anti-C3d antibodies. These results show formation of C-TMs following complement activation. The C4-derived complement activation products associated with candidate proteins tested were

predominantly C4d (a fragment derived from the a chain of C4) because an anti-C4c antibody (recognizing the β chain of C4) consistently does not react with the presumed protein complexes in this experimental setting (FIGs. 5A-F). However, it is less certain whether C3- derived complement activation products other than C3d also bind to the candidate proteins tested, since anti-C3c antibody occasionally also seemed to react with the protein complexes captured on the microplate (not shown).

[0086] This study shows that the formation of C-TM complexes is not necessarily universal or equal among the candidate molecules investigated. For example, FIG. 6 shows detection of C-TMs bound to several candidate proteins in activated human serum using the ECL-based assay (with ApoAl-C4d data on the far left hand side of FIG. 6, and presenting data points further from left to right within FIG. 6, ending with data representing Albumin- C4d. In FIG. 6, the C-TM assays shown in FIGs. 5A-F were repeated 4 times on different days. The variations in the assays show that, for example, C4d may form complexes preferentially with Apo Al and Apo B, followed by immunoglobulin, vWF, CRP, and albumin in a given sample or a given individual.

EXAMPLE 4

[0087] Detection of C-TMs in patient plasma samples using the newly developed assay. Following the initial study using human serum activated in vitro, we performed the C-TM assay using plasma samples prepared from healthy individuals, patients with SLE, and patients with various immune-inflammatory diseases. To ensure that the assay will detect only the C-TM complexes formed in vivo, plasma samples were derived from EDTA- anticoagulated blood and reaction buffers were supplemented with EDTA as described above. The results obtained from the first 10 plasma samples of patients with SLE or other immune- inflammatory diseases are shown in FIGs. 7A-7C. Using normal human plasma as the reference, the plasma samples derived from some patients appeared to contain increased amounts of ApoAl-C4d complexes, ApoB-C4d complexes, CRP-C4d complexes, and vWF-C4d complexes. Similar to the "C-TM hierarchy" observed in normal human serum activated in vitro, the increases in CRP-C4d complexes and vWF-C4d complexes were was less than those in ApoAl-C4d and ApoB-C4d complexes.

[0088] Significant increase in fibrinogen-C4d complexes, but not fibronectin-C4d complexes, was observed in all patient plasma samples (FIGS. 8A-8C). The presence of C4d bound to von Willebrand factor (vWF), and fibrinogen, in the plasma of ten patients with immune-inflammatory diseases were measured using the C-TM assays described in the text. The plasma prepared from a healthy individual was used as the baseline reference.

[0089] The data presented herein shows that complement activation products generated during systemic complement activation complex with various soluble plasma/serum molecules including proteins and thereby serve as biomarkers of diseases.

[0090] As noted above, the imaging data for bodily fluid samples and/or control samples may be analyzed by systems that are programmed with software to perform the analysis on an automated basis.

[0091] FIG. 9 depicts an example of internal hardware that may be used to contain or implement the various computer processes and systems as discussed above. An electrical bus 900 serves as the main information highway interconnecting the other illustrated components of the hardware. CPU 905 is the central processing unit of the system, performing calculations and logic operations required to execute a program. CPU 905, alone or in conjunction with one or more of the other elements disclosed in FIG. 9, is a processing device, computing device or processor as such terms are used within this disclosure. Read only memory (ROM) 910 and random access memory (RAM) 915 constitute examples of memory devices.

[0092] A controller 920 interfaces with one or more optional memory devices 925 that service as date storage facilities to the system bus 900. These memory devices 925 may include, for example, an external DVD drive or CD ROM drive, a hard drive, flash memory, a USB drive or another type of device that serves as a data storage facility. As indicated previously, these various drives and controllers are optional devices. Additionally, the memory devices 925 may be configured to include individual files for storing any software modules or instructions, auxiliary data, incident data, common files for storing groups of contingency tables and/or regression models, or one or more databases for storing the information as discussed above.

[0093] Program instructions, software or interactive modules for performing any of the functional steps associated with the processes as described above may be stored in the ROM 910 and/or the RAM 915. Optionally, the program instructions may be stored on a tangible computer readable medium such as a compact disk, a digital disk, flash memory, a memory card, a USB drive, an optical disc storage medium and/or other recording medium.

[0094] A display interface 930 may permit information from the bus 900 to be displayed on the display 935 in audio, visual, graphic or alphanumeric format.

Communication with external devices may occur using various communication ports 940. A communication port 940 may be attached to a communications network, such as the Internet, a local area network or a cellular telephone data network.

[0095] The hardware may also include an interface 945 which allows for receipt of data from input devices such as a keyboard 950 or other input device 955 such as a remote control, a pointing device, a video input device and/or an audio input device.

[0096] The features and functions described above, as well as alternatives, may be combined into many other different systems or applications. Various presently unforeseen or unanticipated alternatives, modifications, variations or improvements may be made by those skilled in the art, each of which is also intended to be encompassed by the disclosed embodiments.

Claims

CLAIMS:

1. A method of identifying a disease or disorder in an individual, which comprises:

(a) obtaining a bodily fluid sample from an individual;

(b) measuring an amount of at least one soluble complex contained in the bodily fluid sample of the individual, wherein each soluble complex comprises a compound and a complement activation product;

(c) determining an amount of each soluble complex of step (b) contained in a control bodily fluid sample; and,

(d) comparing the amount of each soluble complex from the individual's bodily fluid sample against the amount of each soluble complex from the control bodily fluid sample, to identify whether an increase in a soluble complex within the individual's bodily fluid sample compared to the control bodily fluid sample exists; and

(e) based on the comparing, determining whether the increase indicates that the individual is showing a response related to an underlying disease or disorder.

2. The method of claim 1 wherein the determining comprises determining whether the response is an acute autoimmune or inflammatory response.

3. The method of claim 1, wherein:

the at least one soluble complex comprises a complement-tagged molecule, and the determining comprises determining whether the increase is in an amount of at least two standard deviations, and if so determining that the individual may have systemic lupus erythematosus.

4. The method of claim 1, wherein:

the at least one soluble complex comprises a complement-tagged molecule that is associated with insulin; and

the determining comprises determining that the increase evidences that the individual may have diabetes.

5. A method of identifying systemic lupus erythematosus in an individual, which comprises:

(a) obtaining a bodily fluid sample from an individual;

(b) using one or more electrodes to stimulate at least one soluble complex contained in the bodily fluid sample of the individual, wherein each soluble complex comprises a compound and a complement activation product;

(c) determining an amount of each soluble complex of step (b) contained in a control bodily fluid sample;

(d) measuring an amount of the at least one soluble complex that is stimulated in step (c);

(e) comparing the amount of each soluble complex from the individual's bodily fluid sample against the amount of each soluble complex from the control bodily fluid sample to identify whether an increase exists; and

(f) based on the comparing, determining that the individual is showing an acute autoimmune or inflammatory response related to systemic lupus erythematosus.

6. The method of claim 5 wherein the compound of the soluble complex is selected from the group consisting of Apolipoprotein Al, Apolipoprotein B, C-reactive protein, von Willebrand factor, fibrinogen, and fibronectin.

7. The method of claim 5 wherein the complement activation product of the soluble complex is selected from the group consisting of C4d, C4c, C3d, and C3c.

8. The method of claim 4 wherein the soluble complex is selected from the group consisting of ApoAl-C4d, ApoB-C4d, CRP-C4d, vWF-C4d, fibrinogen-C4d and fibronectin-C4d.

9. A method of detecting a complement-tagged molecule ("C-TM") biomarker in a bodily fluid sample of an individual, which comprises:

(a) obtaining a bodily fluid sample from an individual, wherein the fluid sample comprises a C-TM biomarker;

(b) mixing a portion of the sample with contacting antibodies to form a mixture in which a first subunit of the C-TM biomarker binds to at least a portion of the contacting antibodies;

(c) adding a detecting antibody to the mixture so that at least a portion of the detecting antibodies bind to a second subunit of the C-TM biomarker and form a detectable C-TM complex;

(d) removing unbound antibodies and unbound portions of the sample from the mixture; and (e) measuring an amount the detecting antibodies associated with the detectable C- TM complex.

10. The method of claim 9 further comprising repeating steps (b) - (e) with a control bodily fluid sample, and determining whether an increase in a C-TM complex within the individual's bodily fluid sample compared to the control bodily fluid sample within a predetermined level indicates the presence of an immune or inflammatory disease.

11. An isolated soluble complex obtained by the method of claim 1 for use as a biomarker for an immune or inflammatory disease.

12. The isolated soluble complex of claim 11 selected from the group consisting of ApoAl-C4d, ApoB-C4d, CRP-C4d, vWF-C4d, Ig-C4d, albumin-C4d, fibrinogen-C4d and fibronectin-C4d.