Use of biomarkers for the identification and treatment of complement-mediated disorders

Abstract

A method of measuring endothelial cell specific complement activation is provided. The method can comprise screening a test compound for complement modulation. The methods and assays of the present disclosure can be used to monitor response to treatment of a complement-mediated disease with a complement modulator. The present disclosure further relates to idiopathic inflammatory myopathies and other diseases associated with aberrant activation of one of more complement system in endothelial cells, for example, with respect to dermatomyositis (DM) and complement-mediated thrombotic microangiopathy (CM-TMA), both of which can manifest with autoimmune syndromes such as lupus.

Description

USE OF BIOMARKERS FOR THE IDENTIFICATION AND TREATMENT OF COMPLEMENT-MEDIATED DISORDERSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This International Patent Application claims the benefit of U.S. Provisional Patent Application No. 63 / 501 ,937, filed on 12 May 2023, and which is hereby incorporated by reference in its entirety.BACKGROUNDField

[0002] The present disclosure relates to methods of detecting complement activation in a biological sample. The disclosure also relates to methods for diagnosis or prognostic assessment of a complement-mediated disease in a subject, and methods for monitoring response during and after treatment of a complement-mediated disease with a complement modulator. The present disclosure further relates to idiopathic inflammatory myopathies and other diseases associated with aberrant activation of one of more complement system in endothelial cells, for example, with respect to dermatomyositis (DM) and complement-mediated thrombotic microangiopathy (CM- TMA), both of which can manifest with autoimmune syndromes such as lupus.Description of Related Art

[0003] The complement system is part of the innate immune system and acts in conjunction with other immunological systems of the body to defend against intrusion of cellular and viral pathogens. There are at least 25 proteins in the complement pathway, which are found as a complex collection of circulating plasma proteins and cell membrane cofactors. The plasma proteins make up about 10% of the globulins in vertebrate sera. The complement proteins circulate in the blood as inactive precursors and, when stimulated by one of several triggers, proteases in the system cleave specific proteins to release cytokines and initiate an amplifying cascade of further cleavages. Complement components achieve their immune defensive functions by activating an intricate series of cell surface and fluid-phase interactions involving precise enzymatic cleavages and plasma membrane binding events. The resulting complement cascade leads to the production of products with opsonic, immunoregulatory, and lytic functions.The complement cascade can follow the classical pathway (system), the alternative pathway, or the lectin pathway. These pathways share many components, and while they differ in their initial steps, they converge and share the same “terminal complement” components (C5 through C9) responsible for the activation and destruction of target cells. The classical pathway (CP) can be initiated by antibody recognition of, and binding to, an antigenic site on a target cell. The alternative pathway (AP) is antibody independent and capable of autoactivation by certain molecules on pathogen surfaces. The lectin pathway can be initiated with binding of mannose-binding lectin (MBL) to high mannose substrates. These pathways converge at the point where complement component C3 is cleaved by an active protease to yield C3a and C3b. Other pathways activating complement attack can act later in the sequence of events leading to various aspects of complement function.

[0004] The complement system plays a vital role within the human body to fight diseases, and measurement of components in the complement system can be useful for diagnosis and / or prognosis of disease, as well as for monitoring response to treatment of a complement-mediated disease. Tissue biopsy can provide clinical evidence for most disease diagnoses and can be a direct way to confirm a role for complement in disease pathogenesis. However, biopsies are painful and expensive, and there are risks associated with the procedure. A repeat tissue biopsy is rarely done. Therefore, monitoring longitudinal response to treatment via multiple local tissue biopsies is not possible.

[0005] Extracellular vesicles (EV) are small membrane-bound, enveloped particles (30-100 nm) made by cells. They are released from the plasma membrane (PM) of the parent cell, and contain functional membrane and cytosolic proteins, lipids, and RNAs. Other terms for EVs include: microvesicles, ectosomes, shedding vesicles, microparticles, and exosomes. The EV external membrane contains EV-specific protein markers and PM markers specific to the parent cell. The orientation of the EV membrane protein is the same as in the parent PM. Extracellular vesicles carry canonical EV markers such as CD9, CD63, or CD81 , which are members of the tetraspanin superfamily of proteins. Tetraspanins are among the most abundant membrane proteins of EVs. Some EV also carry complement regulators on their surfaces, such as CD55 and CD59. Cells under stress will increase EV production.

[0006] An unmet need in the field of complement diagnostics and therapy is a sensitive, specific and non-invasive clinical test for measuring localized tissue depositionof terminal complement complex. A non-invasive test that allows frequent longitudinal monitoring of cell-surface complement activity during treatment would provide information about the pharmacodynamic effect of therapy at the local level of specific organs, which represents a significant advancement to current methods limited to measurements of fluid-phase complement activity.

[0007] Dermatomyositis (DM) is an idiopathic, microangiopathic disease often characterized by muscle weakness and skin rash. But, dermatomyositis, a rare disease, can have diverse clinical presentations, making it difficult to diagnose. Skin biopsies can be performed on patients who have characteristic skin manifestations, for example, rashes and rough skin, but lack muscle weakness. Muscle biopsies on weak muscles, identified by physical exam or electromyography, can be performed on patients who lack skin findings. The rash can precede or accompany progressive muscle weakness. The rash can be patchy, with purple or red discolorations, and characteristically develops on the eyelids and on muscles used to extend or straighten joints, including knuckles, elbows, knees, and toes — but can also appear elsewhere on the body. Patients can also develop calcium deposits in the form of hard bumps under the skin or in the muscle (calcinosis). Muscle damage can begin with aches and weakness of the muscles of the trunk, upper arms, hips, and thighs. Muscles can eventually show signs of atrophy. Particular muscle dependent activities such as arm raising, climbing stairs, speech, and swallowing can be affected. Dermatomyositis can have both juvenile (generally 5-15 years old, and sometimes younger) and adult manifestations (generally 40-60 years old).

[0008] Thrombotic microangiopathy (TMA) is used to describe diseases characterized by thrombocytopenia and microangiopathic hemolytic anemia (HAMA) with varying degrees of organ damage. TMAs include primary TMA generally associated with a genetic etiology, and secondary TMA manifesting as a result of other conditions such as infection, cancer, transplantation, vaccination, and pregnancy. Complement- mediated thrombotic microangiopathy (CM-TMA) is an example of a primary TMA. CM- TMA is also referred to as or associated with atypical hemolytic uremic syndrome (aHUS) or complement-mediated hemolytic uremic syndrome (HUS). Genetic defects can result in excessive complement production, particularly of the alternative complement system, leading to damage of healthy host tissues instead of the intended microbial and other pathological targets. CM-TMA is not the only primary TMA. For example, thrombotic thrombocytopenic purpura (TTP) associated with deficiency ofADAMTS13 (a disintegrin and metalloprotease with thrombospondin type-1 repeats, 13th member), as well as other rare diseases such as cobalamin C (cbIC) deficiency resulting from mutations in various genes. Further complicating matters is that primary TMAs can also be acquired, for example, as a result of autoantibodies against ADAMTS13 or complement factor H. The difficulty in identifying the etiology of a TMA can present a barrier to effective treatments.

[0009] When the complement cascade is hyper-activated or dysregulated, severe tissue injury can result with serious consequences such as Complement-Mediated Thrombotic Microangiopathy (CM-TMA), a syndrome of rare and devastating disorders involving vascular endothelial cell (EC) injury. Complement dysregulation with cell-killing membrane attack complex assembly leads to cytolytic damage and EC dysfunction.Currently, there is an unmet need to non-invasively evaluate local tissue damage due to complement activation for prognosis and monitoring patients with CM-TMA as an alternative to tissue biopsy. There also exists a need for better, more specific assays for identifying idiopathic inflammatory myopathies and other diseases associated with aberrant activation of one of more complement system in endothelial cells. Such assays would allow for more accurate treatment of diseases such as dermatomyositis (DM) and complement-mediated thrombotic microangiopathy (CM-TMA), as well as identification of new treatments for the same and tracking patients’ progress receiving such treatments.BRIEF SUMMARY

[0010] The present disclosure includes the following aspects / embodiments / features in any order and / or in any combination:

[0011] A method of measuring endothelial cell specific complement activation is provided. The method can comprise one or more of the following steps: contacting a plasma with a first agent to produce a first plasma fraction; contacting the first plasma fraction with a second agent to produce a second plasma fraction, the second plasma fraction having an endothelial cell specific extracellular vesicle concentration greater than that of the plasma and that of the first plasma fraction; contacting the second plasma fraction with a probe that specifically binds a complement system-associated component; and measuring bound probe, the bound probe indicative of endothelial cell specific complement activation.

[0012] The method of any preceding or following embodiment / feature / aspect, wherein the first plasma fraction has a higher extracellular vesicle concentration than that of the plasma.

[0013] The method of any preceding or following embodiment / feature / aspect, wherein: the first agent comprises a first binding moiety that specifically binds an extracellular vesical associated protein, and the second agent comprises a second binding moiety that specifically binds an endothelial cell associated protein; or the first agent comprises a first binding moiety that specifically binds an endothelial cell associated protein, and the second agent comprises a second binding moiety that specifically binds an extracellular vesical associated protein; or both.

[0014] The method of any preceding or following embodiment / feature / aspect, wherein the first agent comprises a first binding moiety that specifically binds an extracellular vesical associated protein, and the second agent comprises a second binding moiety that specifically binds an endothelial cell associated protein.

[0015] The method of any preceding or following embodiment / feature / aspect, wherein the first binding moiety, or the second binding moiety, or both comprises an antibody or an antigen-binding fragment thereof.

[0016] The method of any preceding or following embodiment / feature / aspect, wherein the first binding moiety, or the second binding moiety, or both comprises an aptamer.

[0017] The method of any preceding or following embodiment / feature / aspect, wherein the first binding moiety comprises a first plurality of binding moieties, or the second binding moiety comprises a second plurality of binding moieties, or both.

[0018] The method of any preceding or following embodiment / feature / aspect, wherein the binding moieties of the first plurality differ in binding specificity, or the binding moieties of the second plurality differ in binding specificity, or both.

[0019] The method of any preceding or following embodiment / feature / aspect, wherein the targets of the first plurality, or the targets of the second plurality, or both are present in the same extracellular vesicle or a membrane-bound portion thereof.

[0020] The method of any preceding or following embodiment / feature / aspect, wherein the second plurality of markers is indicative of dermatomyositis.

[0021] The method of any preceding or following embodiment / feature / aspect, wherein the second plurality of markers is indicative of complement-mediated thrombotic microangiopathy.

[0022] The method of any preceding or following embodiment / feature / aspect, wherein the probe comprises a first plurality of probes.

[0023] The method of any preceding or following embodiment / feature / aspect, wherein the probes of the plurality of probes differ in binding specificity.

[0024] The method of any preceding or following embodiment / feature / aspect, wherein the agent, the probe, or both comprise a binding moiety.

[0025] The method of any preceding or following embodiment / feature / aspect, wherein the agent, or the probe, or both comprise an antibody or an antigen-binding fragment thereof.

[0026] The method of any preceding or following embodiment / feature / aspect, wherein the agent, or the probe, or both comprises an aptamer.

[0027] The method of any preceding or following embodiment / feature / aspect, wherein the first binding moiety, the second binding moiety, or both bind ALIX, TSG101 , CD9, CD63, CD81 , CD40L, CD26, CD31 , CD45, CD2, CDIIa, CD24, CD55, CD59, CF106, CD56, CD51 , CD82, Integrins, Tetraspanins, Annexins, HSP90, HSP70, Syntenin-1 , ADAMIO, EHD4, Actin, Rab5, clathrin, Flotillin-1 , MHC I, MHC II, Actinin-4, GP96, EHD4, Mitofilin, or LAMP2, or any combination thereof.

[0028] The method of any preceding or following embodiment / feature / aspect, wherein the first binding moiety, or the second binding moiety, or both bind a tetraspanin.

[0029] The method of any preceding or following embodiment / feature / aspect, wherein the first binding moiety, or the second binding moiety, or both bind CD9, CD63, or CD81 , or any combination thereof, for example, CD63 and CD81 ; CD63 and CD9; CD81 and CD9; and / or CD63, CD81 and CD9; preferably wherein the first binding moiety binds to CD63 and / or CD9.

[0030] The method of any preceding or following embodiment / feature / aspect, wherein the first binding moiety, or the second binding moiety, or both bind endoglin, VE-cadherin, podocalyxin (PODXL), aquaporin 2 (AQP 2), uroplakinib (UPKIb), podocin (NPHS2), glycophorin A (GYP A), mucin-1 , type 2 Na-K-2C1 co-transporter (NKCC2), aquaporin 1 (AQP 1), a-glutathione- S-transferase (alpha-GST), Tamm-Horsfall protein (TH), calbindin-D28K (CalD), megalin, cubilin, nephrin (Nphsl), Claudin-1 , Annexin-V, synaptopodin (Synpo), Wilm's tumor protein (Wtl), Band 3, stomatin (STOM), BGP1 , Globin, Glycophorin B, Rh polypeptides, or Rh glycoprotein, or any combination thereof, for example, a combination of endoglin and VE-cadherin.

[0031] The method of any preceding or following embodiment / feature / aspect, wherein the first binding moiety, or the second binding moiety, or both bind endoglin, VE-cadherin, or podocalyxin, or any combination thereof, for example, a combination of endoglin and VE-cadherin.

[0032] The method of any preceding or following embodiment / feature / aspect, wherein the first binding moiety, or the second binding moiety, or both binds endoglin and podocalyxin; or endoglin and VE-cadherin; or endoglin, podocalyxin, and VE- cadherin.

[0033] The method of any preceding or following embodiment / feature / aspect, wherein the second plasma fraction comprises a first subtraction characterized by endoglin and podocalyxin, and a second subtraction characterized by endoglin and VE- cadherin.

[0034] The method of any preceding or following embodiment / feature / aspect, wherein the first binding moiety binds to CD63 and / or CD9 and the second moiety binds to endoglin and / or VE-cadherin.

[0035] The method of any preceding or following embodiment / feature / aspect, wherein the first binding moiety binds to CD63 and the second moiety binds to endoglin and / or wherein the first binding moiety binds to CD63 and the second moiety binds to VE-cadherin.

[0036] The method of any preceding or following embodiment / feature / aspect, wherein the agent, the probe, the first binding moiety, or the second binding moiety, or any combination thereof bind a dermatomyositis specific marker, or a complement- mediated thrombotic microangiopathy, or both.

[0037] The method of any preceding or following embodiment / feature / aspect, wherein the agent, the probe, the first binding moiety, or the second binding moiety, or any combination thereof bind C3, C5b-9, C4, Clq, C9, C3b, iC3b, TF, CRP, pCRP, MAC, CD59, CD55, CR1 , C5aRI, or C5a, or any combination thereof.

[0038] The method of any preceding or following embodiment / feature / aspect, wherein the agent, the probe, the first binding moiety, or the second binding moiety, or any combination thereof bind CD46, CD55, CD59, or C5b-9, or any combination thereof.

[0039] The method of any preceding or following embodiment / feature / aspect, wherein the agent, the probe, the first binding moiety, or the second binding moiety, or any combination thereof bind to a component of the alternative complement system.

[0040] The method of any preceding or following embodiment / feature / aspect, wherein the complement system-associated component bound by the agent, or the probe, both comprise a classical complement system component, an alternative complement system component, or a lectin complement system component, or any combination thereof.

[0041] The method of any preceding or following embodiment / feature / aspect, wherein the complement activation comprises activation of a classical complement system, an alternative complement system, or a lectin complement system, or any combination thereof.

[0042] The method of any preceding or following embodiment / feature / aspect, wherein the first binding moiety, the second binding moiety, the agent, or the probe, or any combination thereof are attached to a solid support.

[0043] The method of any preceding or following embodiment / feature / aspect, wherein the solid support comprises a plurality of solid supports.

[0044] The method of any preceding or following embodiment / feature / aspect, wherein the plurality of solid supports comprise first, second, and third solid supports, wherein the first, second, and third solid support differ from one another, the first binding moiety is anchored to the first solid support, the second binding moiety is bound to the second solid support, and the probe is bound to the third solid support.

[0045] The method of any preceding or following embodiment / feature / aspect, wherein the plurality of solid supports differ from one another.

[0046] The method of any preceding or following embodiment / feature / aspect, wherein the plurality of solid supports differ from one another with respect to a detectable label.

[0047] The method of any preceding or following embodiment / feature / aspect, wherein the detectable label comprises a fluorescent dye.

[0048] The method of any preceding or following embodiment / feature / aspect, wherein the detectable label comprises a plurality of fluorescent dyes.

[0049] The method of any preceding or following embodiment / feature / aspect, wherein solid supports differ with respect to a combination of two or more fluorescent dyes.

[0050] The method of any preceding or following embodiment / feature / aspect, wherein the solid support comprises beads, a chip, an array, a slide, a multi-well plate, or a microfluidic device, or any combination thereof.

[0051] The method of any preceding or following embodiment / feature / aspect, wherein the solid support comprises a polymer.

[0052] The method of any preceding or following embodiment / feature / aspect, wherein the polymer comprises polystyrene.

[0053] The method of any preceding or following embodiment / feature / aspect, wherein the probe comprises a detectable label.

[0054] The method of any preceding or following embodiment / feature / aspect, further comprising contacting the second plasma fraction with a secondary probe that specifically binds to the probe, the probe being a primary probe.

[0055] The method of any preceding or following embodiment / feature / aspect, wherein the primary probe, or the secondary probe, or both comprise an antibody or an antigen binding fragment thereof.

[0056] The method of any preceding or following embodiment / feature / aspect, wherein the primary probe comprises a primary antibody and the secondary probe comprises a secondary antibody.

[0057] The method of any preceding or following embodiment / feature / aspect, wherein the primary and secondary antibodies bind each other through respective biotin and streptavidin molecules.

[0058] The method of any preceding or following embodiment / feature / aspect, wherein the probe comprises a detectable label.

[0059] The method of any preceding or following embodiment / feature / aspect, wherein the secondary probe comprises a detectable label.

[0060] The method of any preceding or following embodiment / feature / aspect, wherein the secondary probe comprises an enzyme that converts a substrate to a detectable product.

[0061] The method of any preceding or following embodiment / feature / aspect, wherein the detectable label comprises a fluorescent label, a chemiluminescent label, a radioactive label, a colored label, a magnetic label, a size-based label, a charge-based label, a chelation-based label, a complementary tag label, or a specific-binding label, or any combination thereof.

[0062] The method of any preceding or following embodiment / feature / aspect, wherein the label comprises phycoerytherin (PE).

[0063] The method of any preceding or following embodiment / feature / aspect, wherein the detectable label comprises a plurality of detectable labels, wherein each different label of the plurality is specific to a molecular target.

[0064] The method of any preceding or following embodiment / feature / aspect, wherein the probe, the binding moiety, or the detectable label, or any combination thereof is specific to a complement protein.

[0065] The method of any preceding or following embodiment / feature / aspect, wherein the plasma comprises an anticoagulant, or has been treated with an anticoagulant, or both.

[0066] The method of any preceding or following embodiment / feature / aspect, wherein the plasma comprises a chelator, or has been treated with a chelator, or both.

[0067] The method of any preceding or following embodiment / feature / aspect, wherein the anticoagulant comprises ethylenediaminetetraacetic acid (EDTA), heparin, or citrate, or any combination thereof.

[0068] The method of any preceding or following embodiment / feature / aspect, wherein the plasma comprises P100 plasma, K2EDTA plasma, sodium citrate plasma, or anticoagulant citrate dextrose (ACD) plasma, or any combination thereof.

[0069] The method of any preceding or following embodiment / feature / aspect, wherein the plasma is obtained from blood of a mammalian donor.

[0070] The method of any preceding or following embodiment / feature / aspect, wherein the plasma is obtained from blood of a human donor.

[0071] The method of any preceding or following embodiment / feature / aspect, wherein the plasma is obtained from more than one blood donor.

[0072] The method of any preceding or following embodiment / feature / aspect, wherein the plasma is obtained from a donor having a complement system associated disease.

[0073] The method of any preceding or following embodiment / feature / aspect, wherein the complemental system associated disease is a chronic disease associated with one or more genetic mutations in one or more complement encoding genes.

[0074] The method of any preceding or following embodiment / feature / aspect, wherein the disease is dermatomyositis (DM).

[0075] The method of any preceding or following embodiment / feature / aspect, wherein the disease is complement-mediated thrombotic microangiopathy (CM-TMA).

[0076] The method of any preceding or following embodiment / feature / aspect, wherein the measuring comprises quantification of the bound probe.

[0077] The method of any preceding or following embodiment / feature / aspect, wherein the measuring comprises normalization with respect to one or more parameters.

[0078] The method of any preceding or following embodiment / feature / aspect, wherein the one or more parameters comprise plasma type, mean extracellular vesicle size, extracellular vesicle concentration, complement system component marker, complement system complex marker, extracellular vesicle marker, or endothelial cell marker, or any combination thereof.

[0079] The method of any preceding or following embodiment / feature / aspect, wherein normalization is with respect to a sample having known parameter values.

[0080] The method of any preceding or following embodiment / feature / aspect, wherein the measuring comprises flow cytometry.

[0081] The method of any preceding or following embodiment / feature / aspect, wherein the measuring comprises measuring fluorescence, chemiluminescence, radioactivity, color, or infrared, or any combination thereof.

[0082] The method of any preceding or following embodiment / feature / aspect, wherein the measuring comprises multianalyte profiling, spectrometry, spectrophotometry, or microscopy, or any combination thereof.

[0083] The method of any preceding or following embodiment / feature / aspect, wherein the microscopy comprises fluorescence microscopy, super-resolution light microscopy (SRLM), or electron microscopy (EM), or any combination thereof.

[0084] The method of any preceding or following embodiment / feature / aspect, wherein the measuring comprises an immunoassay, electron microscopy (EM), tandem mass tag (TMT), a luminescence assay, or a fluoroimmunoassay (FIA), or any combination thereof.

[0085] The method of any preceding or following embodiment / feature / aspect, wherein detection step is carried out in a multiplex format.

[0086] The method of any preceding or following embodiment / feature / aspect, further comprising diagnosing, or prognosing, or both a disease in a donor from which the plasma was obtained based on the measurement.

[0087] The method of any preceding or following embodiment / feature / aspect, wherein the disease is a complement-associated disease.

[0088] The method of any preceding or following embodiment / feature / aspect, wherein the disease is associated with abnormal activation of a complement system.

[0089] The method of any preceding or following embodiment / feature / aspect, wherein the disease is an inflammatory disease, or a thrombotic disease, or both.

[0090] The method of any preceding or following embodiment / feature / aspect, wherein the disease is a thrombotic hematological disease, or a thrombotic nephrological disease, or any combination thereof.

[0091] The method of any preceding or following embodiment / feature / aspect, wherein the disease is a nephrological disease selected from the group consisting of atypical haemolytic uraemic syndrome (aHUS), C3 glomerulopathy (C3G), dense deposit disease (DDD), membranoproliferative glomerular nephritis (MPGN), lupus nephritis (LN), IgA nephropathy (IN), lupus nephritis (LN), membranous nephropathy (MN), complications due to hemodialysis in transplant patients, antibody-mediated rejection (AMR), and anti -neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV).

[0092] The method of any preceding or following embodiment / feature / aspect, wherein the disease is a hematological disease selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), atypical haemolytic uraemic syndrome (aHUS), secondary HUS due to solid organ transplant or hematopoietic stem cell transplants, thrombotic microangiopathy (TMA), and cold agglutinin disease (CAD).

[0093] The method of any preceding or following embodiment / feature / aspect, wherein the disease is a chronic disease associated with one or more genetic mutations in one or more complement encoding genes of the donor.

[0094] The method of any preceding or following embodiment / feature / aspect, wherein the disease is associated with systemic organ damage.

[0095] The method of any preceding or following embodiment / feature / aspect, wherein the disease comprises elevated levels of complement system components.

[0096] The method of any preceding or following embodiment / feature / aspect, wherein the disease comprises thrombocytopenia, microangiopathic hemolytic anemia (MAHA), or microvascular thrombosis, or any combination thereof.

[0097] The method of any preceding or following embodiment / feature / aspect, wherein the disease is complement-mediated thrombotic microangiopathy (CM-TMA).

[0098] The method of any preceding or following embodiment / feature / aspect, further comprising diagnosing, or prognosing, or both a complement system abnormality in a donor from which the plasma was obtained based on the measurement.

[0099] The method of any preceding or following embodiment / feature / aspect, wherein the complement system abnormality is caused by one or more genetic mutations in one or more complement encoding genes of the donor.

[0100] The method of any preceding or following embodiment / feature / aspect, wherein the complement system abnormality is caused by an infection, hypertension, an autoimmune disease, a cancer, a transplantation, a pregnancy, or a drug, or any combination thereof.

[0100] The method of any preceding or following embodiment / feature / aspect, further comprising administering a therapeutic compound or composition to a donor from which the plasma was obtained based on the measurement.

[0101] The method of any preceding or following embodiment / feature / aspect, wherein the therapeutic comprises a complement modulator, inhibitor, or activator, or any combination thereof.

[0102] The method of any preceding or following embodiment / feature / aspect, wherein the therapeutic comprises a therapeutic effective against dermatomyositis (DM).

[0103] The method of any preceding or following embodiment / feature / aspect, wherein the therapeutic comprises a corticosteroid, prednisone, an immunosuppressant, azathioprine, methotrexate, mycophenolate mofetil, rituximab, hydroxychloroquine, intravenous immunoglobulin (IVIg), or sunscreen, or any combination thereof.

[0104] The method of any preceding or following embodiment / feature / aspect, wherein the therapeutic comprises a monoclonal antibody.

[0105] The method of any preceding or following embodiment / feature / aspect, wherein the monoclonal antibody comprises ALXN 1820, avdoralimab, crovalimab, eculizumab, gefurulimab, lampalizumab, narsoplimab, olendalizumab, pozelimab, ravulizumab, sutimlimab, tesidolumab, or vilobelimab, or any combination thereof.

[0106] The method of any preceding or following embodiment / feature / aspect, wherein the monoclonal antibody comprises ALXN 1820, eculizumab, gefurulimab (AXLN 1720), or ravulizumab (ALXN1210), or any combination thereof.

[0107] The method of any preceding or following embodiment / feature / aspect, wherein the therapeutic comprises an anti-complement system component or complex therapeutic.

[0108] The method of any preceding or following embodiment / feature / aspect, wherein the therapeutic inhibits activation and / or activity of factor B, factor D, or properdin, or any combination thereof.

[0109] The method of any preceding or following embodiment / feature / aspect, wherein the therapeutic inhibits activation and / or activity of C1 , C1 q, C1 r, or C1 s, or any combination thereof.

[0110] The method of any preceding or following embodiment / feature / aspect, wherein the therapeutic inhibits activation and / or activity of C3, C3a, or C3b, or any combination thereof.

[0111] The method of any preceding or following embodiment / feature / aspect, wherein the therapeutic inhibits activation and / or activity of C4, C4a, or C4b, or any combination thereof.

[0112] The method of any preceding or following embodiment / feature / aspect, wherein the therapeutic inhibits activation and / or activity of C5, C5a, C5b, or C5aR, or any combination thereof.

[0113] The method of any preceding or following embodiment / feature / aspect, wherein the therapeutic inhibits activation and / or activity of mannose-binding lectin (MBL), M-ficolin, L-Ficolin, H-ficolin, MBL-associated serine protease-1 (MASP-1), MASP-2, or MASP-3, or any combination thereof.

[0114] The method of any preceding or following embodiment / feature / aspect, wherein the therapeutic comprises a small molecule pharmaceutical.

[0115] The method of any preceding or following embodiment / feature / aspect, wherein the therapeutic comprises an interfering nucleic acid.

[0116] The method of any preceding or following embodiment / feature / aspect, wherein the therapeutic comprises a peptide.

[0117] The method of any preceding or following embodiment / feature / aspect, wherein the therapeutic comprises ACH-5548, AMY-101 , ANX005, ANX007, ARGX- 117, avacincaptad pegol, avacopan, BCX-9930, BDB-001 , berinert, cemdisiran, cinryze, CLG561 , danicopan, GT103, IONIS-FB liuc, iptacopan, MOR210, nomacopan, OCTA- C1-INH, pegcetacoplan, vemircopan, zilucoplan.

[0118] The method of any preceding or following embodiment / feature / aspect, further comprising screening a test compound for complement modulation.

[0119] The method of any preceding or following embodiment / feature / aspect, further comprising administering the test compound to the donor, wherein the method is performed at least twice including at least once before administration of the test compound, and at least once after administration of the test compound.

[0120] The method of any preceding or following embodiment / feature / aspect, wherein the donor has been diagnosed with dermatomyositis.

[0121] The method of any preceding or following embodiment / feature / aspect, wherein the dermatomyositis comprises juvenile dermatomyositis, or adult dermatomyositis, or both.

[0122] The method of any preceding or following embodiment / feature / aspect, wherein the dermatomyositis comprises classic dermatomyositis (CDM), amyopathic dermatomyositis (ADM), hypomyopathic dermatomyositis (HDM), clinically amyopathic dermatomyositis (CADM), or juvenile dermatomyositis (JDM), or any combination thereof.

[0123] The method of any preceding or following embodiment / feature / aspect, wherein the test compound is for treating dermatomyositis.

[0124] The method of any preceding or following embodiment / feature / aspect, wherein the donor displays one or more symptoms of dermatomyositis.

[0125] The method of any preceding or following embodiment / feature / aspects, wherein the one or more symptoms of dermatomyositis comprise skin rash, rough skin, calcinosis, muscle weakness, muscle inflammation, lung inflammation, dysphagia, weight loss, orfever, or any combination thereof.

[0126] The method of any preceding or following embodiment / feature / aspects, wherein the donor has been diagnosed with an autoimmune disease.

[0127] The method of any preceding or following embodiment / feature / aspects, wherein the donor has been diagnosed with an idiopathic inflammatory myopathy (I IM).

[0128] The method of any preceding or following embodiment / feature / aspects, wherein the donor has been diagnosed with systemic lupus erythematosus, antiphospholipid syndrome, rheumatoid arthritis (RA), dermatomyositis (DM), polymyositis (PM), inclusion body myositis (IBM), or Antineutrophilic cytoplasmic antibody (ANCA)-associated vasculitis, or any combination thereof.

[0129] The method of any preceding or following embodiment / feature / aspect, wherein the donor has been diagnosed with a primary thrombotic microangiopathy (TMA), or a secondary thrombotic microangiopathy (TMA), or both.

[0130] The method of any preceding or following embodiment / feature / aspect, wherein the donor has been diagnosed with complement-mediated thrombotic microangiopathy (CM-TMA).

[0131] The method of any preceding or following embodiment / feature / aspect, wherein the test compound is for treating complement-mediated thrombotic microangiopathy (CM-TMA).

[0132] The method of any preceding or following embodiment / feature / aspect, wherein a normalizer is used.

[0133] The method of any preceding or following embodiment / feature / aspect, wherein the normalizer comprises a lectin.

[0134] The method of any preceding or following embodiment / feature / aspect, wherein the lectin is Sambucus nigra (elderberry) lectin (SNL).

[0135] A kit configured for use with the method of any preceding or following embodiment / feature / aspect.

[0136] The kit of any preceding or following embodiment / feature / aspect, comprising a first binding moiety and a second binding moiety.

[0137] The kit of any preceding or following embodiment / feature / aspect, wherein the first and second binding moieties respectively are comprised by an agent, a probe, or both.

[0138] The kit of any preceding or following embodiment / feature / aspect, wherein the first binding moiety binds to a first marker selected from ALIX, TSG101 , CD9, CD63, CD81 , CD40L, CD26, CD31 , CD45, CD2, CDIIa, CD24, CD55, CD59, CF106, CD56, CD51 , CD82, Integrins, tetraspanins, Annexins, HSP90, HSP70, Syntenin-1 , ADAMIO, EHD4, Actin, Rab5, clathrin, Flotillin-1 , MHC I, MHC II, Actinin-4, GP96, EHD4, Mitofilin, or LAMP2, or any combination thereof.

[0139] The kit of any preceding or following embodiment / feature / aspect, wherein the second binding moiety binds endoglin, VE-cadherin, podocalyxin (PODXL), aquaporin 2 (AQP 2), uroplakinlb (UPKIb), podocin (NPHS2), glycophorin A (GYP A), mucin-1 , type 2 Na-K-2C1 co-transporter (NKCC2), aquaporin 1 (AQP 1), a-glutathione- S-transferase (alpha-GST), Tamm-Horsfall protein (TH), calbindin-D28K (CalD), megalin, cubilin, nephrin (Nphsl), Claudin-1 , Annexin-V, synaptopodin (Synpo), Wilm's tumor protein(Wtl), Band 3, stomatin (STOM), BGP1 , Globin, Glycophorin B, Rh polypeptides, or Rh glycoprotein, or any combination thereof.

[0140] The kit of any preceding or following embodiment / feature / aspect, wherein the first binding moiety comprises an antibody that binds to tetraspanin, CD9, CD63, or CD81 , or any combination thereof.

[0141] The kit of any preceding or following embodiment / feature / aspect, wherein the second binding moiety comprises an antibody that binds to endoglin or VE-cadherin or a combination thereof.

[0142] The kit of any preceding or following embodiment / feature / aspect, comprising an agent that binds to C3, C5b-9, C4, Clq, C9, C3b, iC3b, TF, CRP, pCRP, MAC, CD59, CD55, CR1 , C5aRI, or C5a.BRIEF DESCRIPTION OF THE DRAWINGS

[0143] For a further understanding of the nature, objects, and advantages of the present disclosure, reference should be had to the following detailed description, read in conjunction with the following drawings, wherein like reference numerals denote like elements.

[0144] FIG. 1 A is a graph of extracellular vesicle concentration in different types of prepared plasma.

[0145] FIG. 1 B is a graph of extracellular vesicle diameter in different types of prepared plasma.

[0146] FIG. 2A is a graph of endothelial specific marker endoglin detected in extracellular vesicle containing plasma.

[0147] FIG. 2B is a graph of endothelial specific marker VE-cadherin detected in extracellular vesicle containing plasma.

[0148] FIG. 3A is a graph of endothelial specific marker endoglin detected in podocalyxin+ extracellular vesicle containing plasma.

[0149] FIG. 3B is a graph of endothelial specific marker VE-cadherin detected in podocalyxin+ extracellular vesicle containing plasma.

[0150] FIG. 4A is a graph of signal to noise ratio data for different dilutions of detector antibodies for CD46 using IgG, endoglin, and VE-Cadherin bead sets of K2EDTA plasma fractions.

[0151] FIG. 4B is a graph of specific response (MFI) for anti-CD46 antibodies diluted 1 :400 for IgG, Endoglin, and VE-Cadherin subtractions of K2EDTA plasma.

[0152] FIG. 4C is a graph of signal to noise ratio data for different dilutions of detector antibodies for CD55 using IgG, endoglin, and VE-Cadherin bead sets of K2EDTA plasma fractions.

[0153] FIG. 4D is a graph of specific response (MFI) for anti-CD55 antibodies diluted 1 :400 for IgG, Endoglin, and VE-Cadherin subtractions of K2EDTA plasma.

[0154] FIG. 5A is a graph of signal to noise ratio data for different dilutions of detector antibodies for CD46 using IgG, endoglin, and VE-Cadherin bead sets of sodium citrate plasma fractions.

[0155] FIG. 5B is a graph of specific response (MFI) for anti-CD46 antibodies diluted 1 :400 for IgG, Endoglin, and VE-Cadherin subtractions of sodium citrate plasma.

[0156] FIG. 5C is a graph of signal to noise ratio data for different dilutions of detector antibodies for CD55 using IgG, endoglin, and VE-Cadherin bead sets of sodium citrate plasma fractions.

[0157] FIG. 5D is a graph of specific response (MFI) for anti-CD55 antibodies diluted 1 :400 for IgG, Endoglin, and VE-Cadherin subtractions of sodium citrate plasma.

[0158] FIG. 6A is a fluorescent image of extracellular vesicles labeled with CD9, CD63, and CD81 specific antibodies, the image captured using super-resolution light microscopy (SRLM).

[0159] FIG. 6B is a fluorescent SRLM image of extracellular vesicles labeled with endoglin specific antibodies.

[0160] FIG. 6C is a fluorescent SLRM image of extracellular vesicles labeled with CD63 antibodies.

[0161] FIG. 6D is a fluorescent SLRM image of extracellular vesicles labeled with VE-cadherin specific antibodies.

[0162] FIG. 7 A is a graph of SLRM image data quantified for CD63 and tetraspanin markers in K2EDTA and sodium citrate (NaCit) plasma fractions.

[0163] FIG. 7B is a graph of SLRM image data quantified for CD63 and endothelial cell markers in K2EDTA and sodium citrate (NaCit) plasma fractions.

[0164] FIG. 8 is a schematic depiction of an example plate layout organization for use in the assays described herein.

[0165] FIG. 9 is a graph of comparative results.

[0166] FIGS. 10A and 10B are graphs of assay results for sodium citrate plasma.

[0167] FIGS. 10C and 10D are graphs of assay results for P100 / K2 plasma.

[0168] FIGS. 11A and 11 B are graphs of assay results for classical and lectin pathway markers.

[0169] FIGS. 12A and 12B are graphs of assay results for central and terminal pathway markers.

[0170] FIGS. 13A-13D are graphs of assay results for markers across dermatomyositis study subjects with or without pathogenic autoantibodies.

[0171] DETAILED DESCRIPTION

[0172] In this specification and 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 meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The word “about” means a range of plus or minus 10% of that value, e.g., “about 5” means 4.5 to 5.5, unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation. For example, in a list of numerical values such as “about 49, about 50, about 55,” “about 50” means a range extending to less than half the interval(s) between the preceding and subsequent values, e.g., more than 49.5 to less than 52.5. Where a range of values is provided in this disclosure, it is intended that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. For example, if a range of 1 mM to 8 mM is stated, it is intended that 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, and 7 mM are also explicitly disclosed. As used herein, the term “plurality” can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The term “substantially” means sufficient to work for the intended purpose. The term “substantially” thus allows for minor, insignificant variations from an absolute or perfect state, dimension, measurement, result, or the like such as would be expected by a person of ordinary skill in the field but that do not appreciably affect overall performance (e.g., + / - 10%).

[0173] The term “extracellular vesicle” refers to a lipid-based microparticle or nanoparticle, or protein-rich aggregate, present in a sample (e.g., a biological fluid) obtained from a subject. Extracellular vesicles are also referred to in the art and herein as exosomes, microvesicles or nanovesicles. In the present disclosure, an extracellular vesicle is between about 30 nm to about 1000 nm in diameter. Extracellular vesicles are secreted or shed from a variety of different mammalian cell types. Non-limiting examplesof extracellular vesicles and methods for the enrichment of extracellular vesicles from a sample (e.g., a biological fluid) obtained from a mammalian subject are described herein. Additional examples of extracellular vesicles and methods for the enrichment of extracellular vesicles from a sample obtained from a mammalian subject can be used.

[0174] Provided herein are methods for using extracellular vesicles from non- invasive liquid biopsy protocols as a non-invasive, sensitive and specific test to diagnose and / or monitor treatment response in patients with various complement- mediated diseases. A semi-quantitative method is described for monitoring the expression of surface complement on EV before, during, and after therapeutic intervention, using immunoprecipitation and / or immunoanalysis to isolate and analyze EV surface markers. These methods can successfully leverage EVs for ex-vivo monitoring of complement deposition as a surrogate for in vivo activity. Thus, with these methods researchers and physicians have a tool to directly monitor complement attack of discrete, identifiable tissues throughout the course of treatment. Extracellular vesicles offer an easily accessible window to monitor ongoing complement deposition on specific tissues, before and during treatment. Extracellular vesicles can also provide precise cellular identification of complement attack of the tissue.

[0175] The methods of the present disclosure include measurement of changes in expression or levels of complement components in EVs. Approaches such as differential ultracentrifugation, density gradient ultracentrifugation, size exclusion chromatography, ultrafiltration, and affinity / immunoaffinity capture methods can be used to enrich EVs from biological samples. An EV enrichment step can be carried out before the first marker and optionally the second marker is contacted with the respective antibody or the antigen binding fragment. EVs can be characterized at the population level or single particle level. The composition and levels of molecules in EVs, such as protein, lipid or nucleic acids, can be analyzed. Techniques range from light-scattering microscopy or spectroscopy to molecular fingerprinting using proteomics. Overall levels of unique molecules can also be measured in the population. For single-particle analysis, specialized methods such as, optical microscopy and flow cytometry (for EVs >200 nm), single-particle interferometric reflectance imaging (> 40 nm), nano-flow cytometry (~ 40 nm), and electron microscopy, can be used. Electron microscopy and flow cytometry permit study of individual EVs without extensive prior separation from a biological matrix. EVs can be lysed using lysis buffers, e.g., RIPA buffer (20 mM Tris-HCI [pH 7.5], 150 mM NaCI, 1 mM Na2 EDTA 1 mM EGTA, 1 % NP-40, 1% sodium deoxycholate, 2.5mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na% V04, 1 pg / ml leupeptin). Characterization of EVs can include use of capture antibodies that specifically bind to markers (e.g., typically a protein or peptide, but could include other antigens) on the EVs. A single capture antibody which is specific to an EV marker can be used. At least two capture antibodies can be used, wherein a first capture antibody is specific to an EV-specific marker and a second capture antibody is specific to an endothelial-specific marker displayed on the EVs.

[0176] A presence or a level of a complement system-associated component on the captured EVs can be detected. Any method can be used in the detection of the complement component, e.g., with a detection antibody or antigen-binding fragment thereof which is specific to the component; aptamers can also be used. Illustrative methods for detection include, e.g., immunohistochemical staining, western blotting, in cell western, immunofluorescent staining, ELISA, RIA, and fluorescent activating cell sorting (FACS), or any method known in the art.

[0177] A wide variety of complement proteins can be detected using the present methods, including, e.g., (a) a component of the alternative pathway (AP), (b) a component associated with classical pathway (CP), and (c) a component associated with lectin pathway (MBL). A plurality of components from different pathways, e.g., components from AP and CP, can be detected. The complement system-associated component can be, for example, a component of AP or CP selected from, e.g., C3, C5b- 9, C4, Clq, C9, C3b, iC3b, TF, CRP, pCRP, MAC, CD59, CF55, CR1 , C5aRI, and C5; preferably MAC, C3, C5b-9, C4, Clq, and C9. A combination of the various components can be detected, e.g., a combination of C3 and C5. Where the component is a membrane attack complex (MAC), the method can include detection of any subunit or all subunits of MAC, e.g., C5b, C6, C7, C8 and C9 molecules.

[0178] The term "membrane-bound" means any structure containing biological membranes, i.e., the outer coverings of cells and organelles that form a semi-permeable barrier. The term typically refers to structures containing phospholipids and proteins, derived from the outer cell membrane, or an organelle such as Golgi-apparatus, ER, the nucleus or mitochondria. The term "membrane protein(s)" as used herein refers to proteins that interact with, or are part of, biological membranes of EVs. The membrane proteins can include, but are not limited to, integral membrane proteins and peripheral membrane proteins. The term "disease specific membrane proteins" as used herein refers to membrane proteins that are associated with a specific disease, e.g., acomplement-mediated disease such as aHUS. The disease specific membrane proteins can individually code for a disease, alternatively a group of disease specific membrane proteins can code for a disease. The term "complement-mediated” disorder or disease refers to a disorder in which its pathogenesis involves complement activation which exceeds a subject's self-protective mechanisms (e.g., self-protective proteins including CD 55 (decay accelerating factor), CD 59 (protectin), Factor H, and the like) and causes damage to the subject's cells and / or tissue. As used herein, the term “at risk” for a disease or disorder refers to a subject (e.g. , a human) that is predisposed to experiencing a particular disease. This predisposition can be genetic (e.g., or due to other factors (e.g., environmental conditions, hypertension, activity level, metabolic syndrome, etc.). Thus, it is not intended that the present disclosure be limited to any particular risk, nor is it intended that the present invention be limited to any particular type of disorder or dysfunction related to complement (e.g., aHUS).

[0179] As used herein, the term "antibody" means an antibody, or a functional portion or fragment thereof, with a high binding affinity for an antigen, e.g., complement proteins. The term is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses natural, genetically engineered and / or otherwise modified antibodies of any class or subclass, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD. The term “antigen” refers to any molecule, e.g. , protein or a fragment thereof, that can specifically bind to an antibody or its antigen-binding fragment. The term "antigen fragment" refers to a part of the antigen that can be recognized by the antigen-specific antibody.

[0180] A “subject,” as used herein, can be any mammal. A subject can be, for example, a human, a non-human primate e.g., monkey, baboon, or chimpanzee), a horse, a cow, a pig, a sheep, a goat, a dog, a cat, a rabbit, a guinea pig, a gerbil, a hamster, a rat, or a mouse. Included are, e.g., transgenic animals or genetically-altered (e.g., knock-out or knock-in) animals. A subject “in need of prevention,” “in need of treatment,” or “in need thereof,” refers to one, who by the judgment of an appropriate medical practitioner (e.g., a doctor, a nurse, or a nurse practitioner in the case of humans; a veterinarian in the case of non-human mammals), would reasonably benefitfrom a given treatment, i.e., a particular therapeutic agent to treat a complement- mediated disease or disorder.

[0181] The complement system consists of over 40 different proteins that are involved as part of the innate immune system. This system is activated in response to several triggers and initiates an amplifying cascade that leads to activation of phagocytes, release of pro-inflammatory cytokines and formation of a cell-killing membrane attack complex (MAC) on damaged cell surfaces. There are 3 independent pathways through which the complement cascade can be activated: classical pathway initiated by antigen-antibody complexes, lectin pathway initiated by microbial carbohydrates, and alternative pathway. Different regulators control the activation of the complement system including cell surface protective proteins such as CD46, CD55, and CD59.

[0182] A “complement component” or “complement protein” is a molecule that is involved in activation of the complement system or participates in one or more complement-mediated activities. Components of the classical complement pathway include, e.g., Clq, Clr, Cis, C2, C3, C4, C5, C6, C7, C8, C9 and the C5b-9 complex, also referred to as the membrane attack complex (MAC) and active fragments or enzymatic cleavage products of any of the foregoing (e.g., C3a, C3b, C4a, C4b, C5a, etc.). Components of the alternative pathway include, e.g., factors B, D, H, and I, and properdin, with factor H and I being negative regulators of the pathway. Components of the lectin pathway include, e.g., MBL2, MASP-1 and MASP-2. Complement components also include cell-bound receptors for soluble complement components. Such receptors include, e.g., C5a receptor (C5aRI and C5aR2), C3a receptor (C3aR), Complement Receptor 1 (CR1), Complement Receptor 2 (CR2), Complement Receptor 3 (CR3), etc. The term “complement component” is not intended to include those molecules and molecular structures that serve as “triggers” for complement activation, e.g., antigenantibody complexes, or foreign structures found on microbial or artificial surfaces, etc. The term includes, without limitation, any complement regulator protein (e.g., Factor B, Factor D, Factor P, Factor H, Factor I, CD46, CD55, and CD59).

[0183] A method of measuring endothelial cell specific complement activation is provided. The method can comprise one or more of the following steps: contacting a plasma with a first agent to produce a first plasma fraction; contacting the first plasma fraction with a second agent to produce a second plasma fraction, the second plasma fraction having an endothelial cell specific extracellular vesicle concentration greaterthan that of the plasma and that of the first plasma fraction; contacting the second plasma fraction with a probe that specifically binds a complement system-associated component; and measuring bound probe, the bound probe indicative of endothelial cell specific complement activation.

[0184] The first plasma fraction can have a higher extracellular vesicle concentration than that of the plasma. The first agent can comprise a first binding moiety that specifically binds an extracellular vesical associated protein, and the second agent can comprise a second binding moiety that specifically binds an endothelial cell associated protein; or the first agent can comprise a first binding moiety that specifically binds an endothelial cell associated protein, and the second agent can comprise a second binding moiety that specifically binds an extracellular vesical associated protein; or both. The first agent can comprise a first binding moiety that specifically binds an extracellular vesical associated protein, and the second agent can comprise a second binding moiety that specifically binds an endothelial cell associated protein. The first binding moiety, the second binding moiety, or both can comprise an antibody or an antigen-binding fragment thereof. The first binding moiety, the second binding moiety, or both can comprise an aptamer.

[0185] Both agents and probes can comprise binding moieties. Agents and probes can be used interchangeably unless otherwise indicated. Agents and probes can comprise binding moieties as well as other elements, for example, linkers, anchors, or substrates, or any combination thereof. Agents and probes can be anchored, or free in solution, or both. An agent can be, for example, anchored and otherwise configured to retain extracellular vesicles displaying a target to which the agent binds through a binding moiety. A probe can be, for example, free in solution before binding through a binding moiety to a target on a retained extracellular vesicle. The respective binding moieties of the agent and probe can be the same, or different, or both. The respective targets of the agent and probe can be the same, or different, or both. The target can be, for example, a biomarker. The agent can comprise a first binding moiety and the probe can comprise a second binding moiety. One or more agents can be selected to retain a extracellular vesicle population (fraction, subset, or the like) of interest. One or more probes can be selected to identify, or verify, or both the extracellular population retained.

[0186] The first binding moiety can comprise a first plurality of binding moieties, or the second binding moiety can comprise a second plurality of binding moieties, or both. The binding moieties of the first plurality can differ in binding specificity, or the bindingmoieties of the second plurality differ in binding specificity, or both. The wherein the targets of the first plurality, or the targets of the second plurality, or both are present in the same extracellular vesicle or a membrane-bound portion thereof. The second plurality of markers can be indicative of dermatomyositis. The second plurality of markers can be indicative of complement-mediated thrombotic microangiopathy.

[0187] The probe can comprise a first plurality of probes. The probes of the plurality of probes can differ in binding specificity. The probe can comprise a binding moiety that can have, for example, characteristics of the first and / or second binding moieties. The agent, the probe, the first binding moiety, or the second binding moiety, or any combination thereof can comprise an antibody or an antigen-binding fragment thereof. The agent, the probe, the first binding moiety, or the second binding moiety, or any combination thereof can comprise an aptamer. The agent, the probe, the first binding moiety, or the second binding moiety, or any combination thereof can bind ALIX, TSG101 , CD9, CD63, CD81 , CD40L, CD26, CD31 , CD45, CD2, CDIIa, CD24, CD55, CD59, CF106, CD56, CD51 , CD82, Integrins, Tetraspanins, Annexins, HSP90, HSP70, Syntenin-1 , ADAMIO, EHD4, Actin, Rab5, clathrin, Flotillin-1 , MHC I, MHC II, Actinin-4, GP96, EHD4, Mitofilin, or LAMP2, or any combination thereof. The agent, the probe, the first binding moiety, or the second binding moiety, or any combination thereof can bind a tetraspanin. The agent, the probe, the first binding moiety, or the second binding moiety, or any combination thereof can bind CD9, CD63, or CD81 , or any combination thereof, for example, CD63 and CD81 ; CD63 and CD9; CD81 and CD9; and / or CD63, CD81 and CD9; preferably wherein the first binding moiety binds to CD63 and / or CD9. See, for example, FIG. 7A. The agent, the probe, the first binding moiety, or the second binding moiety, or any combination thereof can bind endoglin, VE-cadherin, podocalyxin (PODXL), aquaporin 2 (AQP 2), uroplakinlb (UPKIb), podocin (NPHS2), glycophorin A (GYP A), mucin-1 , type 2 Na-K-2C1 co-transporter (NKCC2), aquaporin 1 (AQP 1), a- glutathione- S-transferase (alpha-GST), Tamm-Horsfall protein (TH), calbindin-D28K (CalD), megalin, cubilin, nephrin (Nphsl), Claudin-1 , Annexin-V, synaptopodin (Synpo), Wilm's tumor protein (Wtl), Band 3, stomatin (STOM), BGP1 , Globin, Glycophorin B, Rh polypeptides, or Rh glycoprotein, or any combination thereof, for example, a combination of endoglin and VE-cadherin. The agent, the probe, the first binding moiety, or the second binding moiety, or any combination thereof can bind endoglin, VE- cadherin, or podocalyxin, or any combination thereof. The agent, the probe, the first binding moiety, or the second binding moiety, or any combination thereof can bindendoglin and podocalyxin; or endoglin and VE-cadherin; or endoglin, podocalyxin, and VE-cadherin. The second plasma fraction can comprise a first subtraction characterized by endoglin and podocalyxin, and a second subtraction characterized by endoglin and VE-cadherin. The first binding moiety can bind to CD63 and / or CD9 and the second moiety can bind to endoglin and / or VE-cadherin. See, for example, FIGS. 7A and 7B. The first binding moiety can bind to CD63 and the second moiety can bind to endoglin. The first binding moiety can bind to CD63 and the second moiety can bind to VE- cadherin. The agent, the probe, the first binding moiety, or the second binding moiety, or any combination thereof can bind a dermatomyositis specific marker, or a complement- mediated thrombotic microangiopathy specific marker, or both. The agent, the probe, the first binding moiety, or the second binding moiety, or any combination thereof can bind, for example, C3, C5b-9, C4, Clq, C9, C3b, iC3b, TF, CRP, pCRP, MAC, CD59, CD55, CR1 , C5aRI, or C5a, or any combination thereof. The agent, the probe, the first binding moiety, or the second binding moiety, or any combination thereof can bind CD46, CD55, CD59, or C5b-9, or any combination thereof. The agent, the probe, the first binding moiety, or the second binding moiety, or any combination thereof can bind to a component of the alternative complement system.

[0188] The complement system-associated component bound by the agent, the probe, the first binding moiety, or the second moiety, or any combination thereof can comprise a classical complement system component, an alternative complement system component, or a lectin complement system component, or any combination thereof. The complement activation can comprise activation of a classical complement system, an alternative complement system, or a lectin complement system, or any combination thereof.

[0189] The agent, the first binding moiety, the second binding moiety, or the probe, or any combination thereof can be bound to a solid support. The solid support can serve as a substrate. The solid support can comprise a plurality of solid supports. The plurality of solid supports can comprise first, second, and third solid supports, first, second, and third solid support can differ from one another, the first binding moiety can be anchored to the first solid support, the second binding moiety can be bound to the second solid support, and the probe can be bound to the third solid support. The plurality of solid supports can differ from one another. The plurality of solid supports can differ from one another with respect to a detectable label.

[0190] Any suitable detectable label or combination of detectable labels can be employed. The detectable label can comprise a fluorescent dye. The detectable label can comprise a plurality of fluorescent dyes. The solid supports can differ with respect to a combination of two or more fluorescent dyes. The solid support can comprise beads, a chip, an array, a slide, a multi-well plate, or a microfluidic device, or any combination thereof. The solid support can comprise any suitable material or combination of materials. For example, the solid support can comprise a polymer. The polymer can comprise polystyrene. First and second solid supports can be independently selected from the group consisting of: nanoparticles, microparticles, beads, magnetic beads, nanostructures, tissue culture plate, silica, and nanomatrices. The probe can comprise a detectable label. Accordingly, the method can utilize a bead-based immunocapture protocol with immunofluorescent detection to quantitate complement activity and dysregulation at the level of organ-specific tissue and modulation during treatment. The method can be broadly applied to monitor any organ or tissue under complement attack in any liquid matrix using protein and tissue-specific detecting reagents including antibody -tagged beads and fluorophores. "Beads" refer to particles whereupon desired capture antibodies have been immobilized. The beads generally are uniform in size within a single filtration matrix, but can vary in size ranging from about 1 nm to about 10,000 nm between different filtration matrices. The preferred shape is spherical; however, particles of any other shape can be employed since this parameter is immaterial to the nature of the invention. "Strips" refer to elongated flat elements whereupon desired capture beads or desired capture antibodies have been immobilized. The strips generally can be thin films uniform in size, but can vary in size and color depending on the amount and type of capture antibody immobilized.

[0191] The method can comprise contacting the second plasma fraction with a secondary probe that specifically can bind to the probe, the probe being a primary probe. The primary probe, or the secondary probe, or both can comprise an antibody or an antigen binding fragment thereof. The primary probe can comprise a primary antibody and the secondary probe can comprise a secondary antibody. The primary and secondary antibodies can bind each other through respective biotin and streptavidin molecules. The probe can comprise a detectable label. The secondary probe can comprise a detectable label. The secondary probe can comprise an enzyme that converts a substrate to a detectable product.

[0192] The detectable label can comprise a fluorescent label, a chemiluminescent label, a radioactive label, a colored label, a magnetic label, a size-based label, a chargebased label, a chelation-based label, a complementary tag label, or a specific-binding label, or any combination thereof. The label can comprise phycoerytherin (PE). The detectable label can comprise a plurality of detectable labels, wherein each different label of the plurality can be specific to a molecular target. The probe, the binding moiety, or the detectable label, or any combination thereof can be specific to a complement protein. The detectable marker is selected from the group consisting of: fluorophores, chromogens, and biotin. The detectable marker is a fluorophore with an absorption maximum between about 500 nm and about 900 nm, between about 600 nm and about 1000 nm, or between about 500 nm and about 1000 nm, and an emission maximum between about 550 nm and about 900 nm, between about 600 nm and about 1000 nm, or between about 550 nm and about 1100 nm. The detectable marker can comprise phycoerythrin (PE) with or without conjugation to streptavidin. The detectable marker can comprise biotin for use with streptavidin-phycoerythrin (SAPE). The term “classification dyes” as used herein refers to any mixture or combination of microparticles or beads used in the multiplex assay that have a mixture of classification dyes that enable the instrument to sort and classify the particles. The term “reporter molecule” includes, without limitation, any and all fluorescent tags that are bound to the detection molecule in the assay. In the case of immunoassays designed to measure human antibody, the detection molecule can, for example, be goat anti-human IgG that is labeled with phycoerythrin.

[0193] The immunoprecipitation / immunoanalysis of the present disclosure can employ a bead-based immunocapture protocol with immunofluorescent detection, which can allow the specific tissues under complement attack to be pinpointed and treatment response can be monitored. The technique can be broadly applied to monitor any organ or tissue under complement attack in any liquid matrix given using antibody tools. Methods discussed herein can comprise a combination of EV enrichment and immunocapture of tissue-specific biomarkers present on the surface of shed EV. Capture biomarkers can be either canonical or EV-specific.

[0194] Representative types of antibodies that are useful in carrying out various embodiments of the disclosure are provided, for example, with information on the particular vendor and / or catalog number. The disclosure is not limited to the exemplary embodiments that utilize antibody detection regents from a particularvendor / manufacturer. Antibodies against biomarkers / analytes can be obtained from any manufacturer, including, for example, Biolegend (San Diego, CA), Southern Biotech (Birmingham, AL), United States Biological (USB; Salem, MA), Lifespan Biosciences (LSBIO; Seattle, WA), Abeam (Cambridge, United Kingdom), Cell Signaling Technology (Danvers, MA), and Sigma-Aldrich (St. Louis, MO). For example, rabbit anti-PODXL antibody can be purchased form USB (Catalog # 212672), LSBIO (Catalog # LS- C141161), Abeam (Catalog # ab205350) and Sigma-Aldrich (Catalog # HPA002110); anti-CD9 antibody, clone MM2 / 57 can be purchased from Southern Biotech (Catalog # 9310), EMD Millipore (Catalog # CBL162), VWR (Catalog # 89366), and BIO RAD (Catalog # MCA469G). Antibodies can be generated using conventional techniques, for example, immunization of a mammal such as a mouse or rabbit and / or hybridoma technology.

[0195] Luminex Corporation® makes equipment and xMAP™ technology that combines immunoprecipitation with multiplex immunoassay. xMAP™ is a series of proprietary, color-coded microspheres that can be coated with capture antibody. Openarchitecture xMAP™ technology enables multiplexing of biological tests (assays), reducing time, labor, and costs over traditional methods such as ELISA, western blotting, PCR, and traditional arrays. Systems using xMAP™ technology perform discrete assays on the surface of color-coded beads known as microspheres, which are then read in a compact analyzer. Using multiple lasers or LEDs and high-speed digitalsignal processors, the analyzer reads multiplex assay results by reporting the reactions occurring on each individual microsphere. Using the Luminex platform allows EV enrichment by immunoprecipitation prior to immunoassay. This removes the need for initial sample processing I EV enrichment, thus limiting potential false results. Using a multiplex format allows monitoring of several discrete tissues in one sample. Working in a multi-well plate format allows monitoring of multiple potential complement proteins and pathways in one assay.

[0196] A capture bead can be conjugated to a target-specific antibody. The conjugated bead can be used to immunoprecipitate the target protein from the matrix (e.g., from a liquid sample, such as blood or plasma). A detection antibody conjugated to a label, such as phycoerythrin (PE) or to biotin+streptavidin-phycoerythrin (SAPE), can be used to detect and quantitate the bead-captured target during analysis. An array of xMAP beads coated with antibodies against canonical vesicle markers, such as CD9, CD63, and CD81 , can be used to enrich for discrete EV subsets from a biologicalsample. Each bead set can then be analyzed for the presence of other biomarkers that define and connect the subset phenotype to the tissue origin, and then analyzed further for the presence of complement-pathway proteins. In this way, a non-invasive “liquid biopsy’’ method can be built to sample and monitor key biomarkers of specific tissue (such as renal) diseases for differential diagnosis, prognostic assessment and / or longitudinal monitoring of response to treatment.

[0197] Any suitable plasma can be used. The plasma can comprise an anticoagulant, or can have been treated with an anticoagulant, or both. The plasma can comprise a chelator, or can have been treated with a chelator, or both. The anticoagulant can comprise ethylenediaminetetraacetic acid (EDTA), heparin, or citrate, or any combination thereof. The plasma can comprise P100 plasma, K2EDTA plasma, sodium citrate plasma, or anticoagulant citrate dextrose (ACD) plasma, or any combination thereof. The plasma can be obtained from blood of a mammalian donor. The plasma can be obtained from blood of a human donor. The plasma can be obtained from more than one blood donor. The plasma can be obtained from a donor having a complement system associated disease. The complemental system associated disease can be a chronic disease associated with one or more genetic mutations in one or more complement encoding genes.

[0198] Any relevant disease can be a target of the methods described herein. For example, the disease can be dermatomyositis (DM), or complement-mediated thrombotic microangiopathy (CM-TMA), or both. The disease can be, for example, an inflammatory disease or a thrombotic disease. The disease can be a thrombotic hematological disease or a thrombotic nephrological disease. The disease can be a nephrological disease selected from the group consisting of atypical haemolytic uraemic syndrome (aHUS), C3 glomerulopathy (C3G), dense deposit disease (DDD), membranoproliferative glomerular nephritis (MPGN), lupus nephritis (LN), IgA nephropathy (IN), lupus nephritis (LN), membranous nephropathy (MN), complications due to hemodialysis in transplant patients, antibody-mediated rejection (AMR) and antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The disease can be a hematological disease selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), atypical haemolytic uraemic syndrome (aHUS), secondary HUS due to solid organ transplant or hematopoietic stem cell transplants, thrombotic microangiopathy (TMA), and cold agglutinin disease (CAD). The disease can be a neurological disease selected from neuromyelitis optica spectrum disorder (NMOSD),generalized myasthenia gravis (gMG), amyotrophic lateral sclerosis (ALS) and primary progressive multiple sclerosis (PPMS).

[0199] The measuring can comprise quantification of the bound probe. The measuring can comprise normalization with respect to one or more parameters. The one or more parameters can comprise plasma type, mean extracellular vesicle size, extracellular vesicle concentration, complement system component marker, complement system complex marker, extracellular vesicle marker, or endothelial cell marker, or any combination thereof. The normalization can be with respect to a sample having known parameter values. The measuring can comprise flow cytometry. The measuring can comprise measuring fluorescence, chemiluminescence, radioactivity, color, or infrared, or any combination thereof. The measuring can comprise multianalyte profiling, spectrometry, spectrophotometry, or microscopy, or any combination thereof. The microscopy can comprise fluorescence microscopy, super-resolution light microscopy (SRLM), or electron microscopy (EM), or any combination thereof. The measuring can comprise an immunoassay, electron microscopy (EM), tandem mass tag (TMT), a luminescence assay, or a fluoroimmunoassay (FIA), or any combination thereof. The detection step can be carried out in a multiplex format.

[0200] The method can comprise comparing a detected level of a complement biomarker to a reference level. The reference can represent levels of the biomarkers in a healthy control, i.e. , a subject who has not been diagnosed with a complement- mediated disease. The reference level can be a median or cutoff level in a reference cohort, e.g., a cutoff defining a statistically significantly distinct group, e.g., atop or bottom tercile, quartile, quintile, or other percentile of the reference cohort. Levels above or below the reference level can be indicative of the presence of disease or increased risk, i.e., increased levels (i.e., levels above the reference) or decreased levels (i.e., levels below the reference) indicate the presence of disease or decreased. Increased levels above a reference level can be statistically significant, or are increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, or 1000%. An increase, as described herein, can be determined by comparison to a threshold or baseline value (e.g., a threshold detection level of an assay for determining the presence or absence of a protein, or a reference level of protein in a reference subject (e.g., healthy reference). Decreased levels below a reference level are statistically significant, or decreased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95%. A decrease, as described herein, can be determined by comparison to a threshold or baseline value (e.g., a threshold detection level of an assay for determining the presence or absence of a protein, or a level of protein in a reference subject (e.g., a healthy reference subject or a subject who does not have a complement-mediated disease).

[0201] The method can comprise calculating a ratio of the level of the protein biomarker in the subject sample to a reference level, and if the ratio is greater than a threshold ratio, determining that the subject has or is at risk of developing a complement-mediated disease as described herein. For example, whether the ratio is positive or negative can be determined, and the presence of a positive or negative ratio indicates that the subject has or is at risk of developing a complement-mediated disease as described herein. Whether a positive or negative ratio indicates the presence of disease, or increased or decreased risk, can be readily determined according to the disclosures herein.

[0202] The complement system(s) is comprised of several small proteins organized into a biochemical cascade serving to assist the immune system in the clearance of pathogens. The complement proteins circulate in the blood as inactive precursors. When stimulated by one of several triggers, proteases in the system cleave specific proteins to release cytokines and initiate an amplifying cascade of further cleavages. Methods for measuring systemic or local complement activation taking place in vitro or in vivo can be used. Measurement of complement activation products such as C3a, C5a, C3bBb, C5b-9, etc., for example, provides an indication of the extent of complement activation. A decrease in the amount of such products indicates inhibition of complement activation. A ratio between an active cleavage product and its inactive desArg form can be measured (e.g., C3a / C3a desArg). One of skill in the art can distinguish between classical, alternative, and lectin pathway activation by appropriate selection of the complement activation product(s) measured and / or appropriate activators of complement such as zymosan, lipopolysaccharide, immune complexes, etc. Other methods involve measuring complement-mediated hemolysis of red blood cells as a result of terminal complex (MAC) formation.

[0203] The measuring can comprise, for example, immunoassay (e.g., ELISA or RIA), electron microscopy (EM), tandem mass tag (TMT), a luminescence assay (e.g., LUMINEX), or fluoroimmunoassay (FIA) (also called immunofluorescence assay (IF)). The detection step is carried out in a multiplex format, i.e., wherein detection is carriedout by measuring markers in several discrete tissues in one sample and / or monitoring multiple potential complement proteins & pathways in a single assay. The term "multiplex" refers to the detection of a plurality of marker across a single sample and / or detection of at least one marker across a plurality of samples. The term “multiplex bead array platform” as used herein refers to any platform that utilizes particles or microparticles that are distinguishable. Such distinguishable particles can be utilized, for example, to conduct multiplex immunoassays or molecular probe-based assays. A representative example includes Luminex® xMAP® Technology.

[0204] Binding of target proteins to antibodies in solution or immobilized on an array can be detected using any suitable technique. Examples of such techniques include, for example, immunological techniques such as competitive binding assays and sandwich assays; fluorescence detection using instruments such as confocal scanners, confocal microscopes, or CCD-based systems, and techniques such as fluorescence, fluorescence polarization (FP), fluorescence resonant energy transfer (FRET), total internal reflection fluorescence (TIRF), fluorescence correlation spectroscopy (FCS); colorimetric / spectrometric techniques; surface plasmon resonance, by which changes in mass of materials adsorbed at surfaces can be measured; techniques using radioisotopes, including conventional radioisotope binding and scintillation proximity assays (SPA); mass spectroscopy, such as liquid chromatography -mass spectrometry (LC-MS), HPLC-MS, matrix-assisted laser desorption / ionization mass spectroscopy (MALDI) and MALDI-time of flight (TOF) mass spectroscopy; ellipsometry, which is an optical method of measuring thickness of protein films; quartz crystal microbalance (QCM), a very sensitive method for measuring mass of materials adsorbing to surfaces; scanning probe microscopies, such as atomic force microscopy (AFM) and scanning electron microscopy (SEM); and techniques such as electrochemical, impedance, acoustic, microwave, and infrared (IR) / Raman detection.

[0205] The method can be used to isolate and enrich for EVs and for semi- quantitative monitoring of complement on the surface of EVs before, during, and after therapeutic intervention. The present methods can be easily multiplexed and adapted to a wide variety of assay formats and / or combined with various analytical techniques, e g., nanoparticle tracking analysis (NTA), mass spectroscopy (MS) or super resolution microscopy. The first capture antibody or the antigen-binding fragment thereof is conjugated to a first solid support, optionally the second capture antibody or the antigen binding fragment thereof is conjugated to a second solid support, and the detectionantibody is conjugated to a detectable marker. The method can comprise contacting a portion of the biological sample with the first capture antibody or an antigen-binding fragment thereof and the second capture antibody or an antigen-binding fragment thereof, wherein the first capture antibody or the antigen-binding fragment thereof and the second capture antibody or the antigen-binding fragment thereof are conjugated to the same support or to different supports.

[0206] The method can comprise diagnosing, or prognosing, or both a disease in a donor from which the plasma was obtained based on the measurement. The term “diagnosis” refers to methods by which a determination can be made as to whether a subject is likely to be suffering from a given disease or condition, including but not limited to complement-mediated diseases. The skilled artisan often makes a diagnosis on the basis of one or more diagnostic indicators, e.g., a marker, the presence, absence, amount, or change in amount of which is indicative of the presence, severity, or absence of the disease or condition. Other diagnostic indicators can include patient history; physical symptoms, e.g., unexplained changes in vitals, or phenotypic, genotypic or environmental or heredity factors. A skilled artisan will understand that the term “diagnosis” refers to an increased probability that certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a patient exhibiting a given characteristic, e.g., the presence or level of a diagnostic indicator, when compared to individuals not exhibiting the characteristic. Diagnostic methods of the disclosure can be used independently, or in combination with other diagnosing methods, to determine whether a course or outcome is more likely to occur in a patient exhibiting a given characteristic.

[0207] The disease can be a complement-associated disease. The disease can be associated with abnormal activation of a complement system. The disease can be an inflammatory disease, or a thrombotic disease, or both. The disease can be a thrombotic hematological disease, or a thrombotic nephrological disease, or any combination thereof. The disease can be a nephrological disease selected from the group consisting of atypical haemolytic uraemic syndrome (aHUS), C3 glomerulopathy (C3G), dense deposit disease (DDD), membranoproliferative glomerular nephritis (MPGN), lupus nephritis (LN), IgA nephropathy (IN), lupus nephritis (LN), membranous nephropathy (MN), complications due to hemodialysis in transplant patients, antibody- mediated rejection (AMR), and anti -neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The disease can be a hematological disease selected from the groupconsisting of paroxysmal nocturnal hemoglobinuria (PNH), atypical haemolytic uraemic syndrome (aHUS), secondary HUS due to solid organ transplant or hematopoietic stem cell transplants, thrombotic microangiopathy (TMA), and cold agglutinin disease (CAD). The disease can be a chronic disease associated with one or more genetic mutations in one or more complement encoding genes of the donor. The disease can be associated with systemic organ damage. The disease can comprise elevated levels of complement system components. The disease can comprise thrombocytopenia, microangiopathic hemolytic anemia (MAHA), or microvascular thrombosis, or any combination thereof. The disease can be complement-mediated thrombotic microangiopathy (CM-TMA).

[0208] The method can comprise diagnosing, or prognosing, or both a complement system abnormality in a donor from which the plasma was obtained based on the measurement. The complement system abnormality can be caused by one or more genetic mutations in one or more complement encoding genes of the donor. The complement system abnormality can be caused by an infection, hypertension, an autoimmune disease, a cancer, a transplantation, a pregnancy, or a drug, or any combination thereof.

[0209] The method can comprise administering a therapeutic compound or composition to a donor from which the plasma was obtained based on the measurement. The therapeutic can comprise a complement modulator, inhibitor, or activator, or any combination thereof. The therapeutic can comprise a therapeutic effective against dermatomyositis (DM). The therapeutic can comprise a corticosteroid, prednisone, an immunosuppressant, azathioprine, methotrexate, mycophenolate mofetil, rituximab, hydroxychloroquine, intravenous immunoglobulin (Mg), or sunscreen, or any combination thereof. The complement modulator can be, for example, a modulator of Clq, Cl, Cis, C2, MASP-2, MASP-3, Factor D, Factor B, Properdin (Factor P), Factor H, C3 / C5 Convertase, C5, C5a / C5aR, C3a / C3aR, C6, or CD59; preferably a complement inhibitor such as monoclonal antibody or small molecule inhibitor or siRNA / RNAi.

[0210] A complement inhibitor suitable for use in reducing, suppressing and / or eliminating the complement-mediated effects that occur during therapeutic administration of certain therapeutics (e.g., particle or nanoparticle encapsulated therapeutics) can bind to C5. Exemplary agents include antibodies, antibody fragments, polypeptides, small molecules, and aptamers. Exemplary antibodies are described in U.S. Pat. No. 6,534,058 and in Wang, et al, Proc. Natl. Acad. Sci. USA, 92:8955-8959,1995. Exemplary compounds that bind to and inhibit C5 are described in U.S. Pat. Nos. 7,348,401 and 7,999,081 . The complement inhibitor can be an antibody, small molecule, aptamer, or polypeptide that binds to substantially the same binding site on C5 as an antibody described in U.S. Pat. No. 6,534,058 or a peptide described in U.S. Pat. No. 7,348,401. U.S. Pat. No. 7,538,211 discloses aptamers that bind to and inhibit C5. RNAi agents that inhibit local expression of C5 or CSR can also be used in the methods described herein.

[0211] A therapeutic can be an antagonist of a C5a receptor (C5aR). C5a can be cleaved from the alpha chain of C5 by either alternative or classical C5 convertase. The cleavage site for convertase action is at, or immediately adjacent to, amino acid residue 733 of the alpha chain of C5a. A compound that would bind at, or adjacent to, this cleavage site would have the potential to block access of the C5 convertase enzymes to the cleavage site and thereby act as a complement inhibitor. A compound that binds to C5 at a site distal to the cleavage site could also have the potential to block C5 cleavage, for example, by way of steric hindrance-mediated inhibition of the interaction between C5 and the C5 convertase. Exemplary C5a receptor antagonists include a variety of small cyclic peptides such as those described in U.S. Pat. No. 6,821 ,950; U.S. Pub. No. 2009 / 0117171 ; and / or W02006 / 099330, or the monoclonal antibody BB5.1 (Frei Y. et al. Mol. Cell. Probes, 1 :141-9, 1987), the single chain variable fragment (scFV) of BB5.1 , or the anti-BB5.1 Fab (Peng et al., J Clin Invest., 115(6): 1590-1600, 2005), which prevent the formation of C5a and C5b.

[0212] The complement inhibitor can comprise an anti-C5 antibody. Anti-C5 antibodies (or VH / VL domains derived therefrom) suitable for use herein can be identified using methods known in the art. Alternatively, art recognized anti-C5 antibodies can be used. Antibodies that compete with any of these art recognized antibodies for binding to C5 also can be used. An exemplary anti-C5 antibody is antibody BNJ421 , as described in WO2015 / 134894and US Patent No. 9,079,949, the teachings of which are incorporated herein by reference. The anti-C5 antibody can comprise, for example, a variant human Fc constant region that binds to human neonatal Fc receptor (FcRn), wherein the variant human Fc CH3 constant region can comprise Met-429-Leu and Asn-435-Ser substitutions at residues corresponding to methionine 428 and asparagine 434 of a native human IgG Fc constant region, each in EU numbering. Another exemplary anti-C5 antibody is the 7086 antibody described in US Patent Nos. 8,241 ,628 and 8,883,158, the disclosures in which are incorporated byreference herein. Another exemplary anti-C5 antibody is the 8110 antibody also described in US Patent Nos. 8,241 ,628 and 8,883,158, the disclosures in which are incorporated by reference herein. Another exemplary anti-C5 antibody is the 305LO5 antibody described in US Pat. No 9,765,135, the disclosure in which is incorporated by reference herein. Another exemplary anti-C5 antibody is the SRY59 antibody (Fukuzawa, T. et al, Sci. Rep., 7:1080, 2017, the disclosure in which is incorporated by reference herein). Another exemplary anti-C5 antibody is the REGN3918 antibody (also known as H4H12166PP) described in US Pub. No. 2017 / 0355757 or WO2017218515, the disclosures in which are incorporated by reference herein.

[0213] The antibody can compete for binding with, and / or binds to the same epitope on C5 as, the above-mentioned antibodies (e.g., 7086 antibody, 8110 antibody, 305LO5 antibody, SRY59 antibody, or REGN3918 antibody). The anti-C5 antibody can have, for example, at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% variable region identity). An anti-05 antibody described herein can comprise a variant human Fc constant region that binds to human neonatal Fc receptor (FcRn) with greater affinity than that of the native human Fc constant region from which the variant human Fc constant region was derived. The Fc constant region can comprise, for example, one or more (e.g., two, three, four, five, six, seven, or eight or more) amino acid substitutions relative to the native human Fc constant region from which the variant human Fc constant region was derived. The substitutions, for example, can increase the binding affinity of an IgG antibody containing the variant Fc constant region to FcRn at pH 6.0, while maintaining the pH dependence of the interaction. Methods for testing whether one or more substitutions in the Fc constant region of an antibody increase the affinity of the Fc constant region for FcRn at pH 6.0 (while maintaining pH dependence of the interaction) can be used (for example, WO2015134894 and US Patent No. 9,079,949 the disclosures of each of which are incorporated herein by reference in their entirety).

[0214] Substitutions that enhance the binding affinity of an antibody Fc constant region for FcRn can include, e.g., (1 ) the M252Y / S254T / T256E triple substitution (DalF Acqua, W. et al., J. Biol. Chem., 281 : 2351424, 2006); (2) the M428L or T250Q / M428L substitutions (Hinton, P. et al., J. Biol. Chem., 279:62136, 2004; Hinton, P. et al., J. Immunol., 176:34656, 2006); and (3) the N434A or T307 / E380A / N434A substitutions (Petkova, S. et at., Int. Immunol., 18:175969, 2006). Additional substitution pairings,e.g., P257I / Q3111, P257I / N434H, and D376V / N434H, have also been described (Datta- Mannan, A. et al, J. Biol. Chem., 282:1709 17, 2007). The entire teachings of each of the cited references are hereby incorporated by reference. The variant constant region can have a substitution at Ell amino acid residue 255 for valine. The variant constant region can have a substitution at Ell amino acid residue 309 for asparagine. The variant constant region can have a substitution at EU amino acid residue 312 for isoleucine. The variant constant region can have a substitution at EU amino acid residue 386.

[0215] The variant Fc constant region can comprise no more than 30 (e.g, no more than 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, nine, eight, seven, six, five, four, three or two) amino acid substitutions, insertions or deletions relative to the native constant region from which it was derived. The variant Fc constant region can comprise one or more amino acid substitutions selected from the group consisting of: M252Y, S254T, T256E, N434S, M428L, V259I, T250I and V308F. The variant human Fc constant region can comprise a methionine at position 428 and an asparagine at position 434, each in EU numbering. The variant Fc constant region can comprise a428L / 434S double substitution as described in, e.g., U.S. Patent No.8,088,376 the disclosure of which is incorporated herein by reference in its entirety. The precise location of these mutations can be shifted from the native human Fc constant region position due to antibody engineering. The 428L / 434S double substitution when used in an lgG2 / 4 chimeric Fc, for example, can correspond to 429L and 435S as in the M429L and N435S variants described in US Patent Number 9,079,949 the disclosure of which is incorporated herein by reference in its entirety.

[0216] The variant constant region can comprise a substitution at amino acid position 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311 , 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 or 436 (EU numbering) relative to the native human Fc constant region. The substitution can be selected, for example, from the group consisting of: methionine for glycine at position 237; alanine for proline at position 238; lysine for serine at position 239; isoleucine for lysine at position 248; alanine, phenylalanine, isoleucine, methionine, glutamine, serine, valine, tryptophan or tyrosine for threonine at position 250; phenylalanine, tryptophan or tyrosine for methionine at position 252; threonine for serine at position 254; glutamic acid for arginine at position 255; aspartic acid, glutamic acid or glutamine for threonine at position 256; alanine, glycine, isoleucine, leucine, methionine, asparagine, serine,threonine or valine for proline at position 257; histidine for glutamic acid at position 258; alanine for aspartic acid at position 265; phenylalanine for aspartic acid at position 270; alanine or glutamic acid for asparagine at position 286; histidine for threonine at position 289; alanine for asparagine at position 297; glycine for serine at position 298; alanine for valine at position 303; alanine for valine at position 305; alanine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan or tyrosine for threonine at position 307; alanine, phenylalanine, isoleucine, leucine, methionine, proline, glutamine or threonine for valine at position 308; alanine, aspartic acid, glutamic acid, proline or arginine for leucine or valine at position 309; alanine, histidine or isoleucine for glutamine at position 311 ; alanine or histidine for aspartic acid at position 312; lysine or arginine for leucine at position 314; alanine or histidine for asparagine at position 315; alanine for lysine at position 317; glycine for asparagine at position 325; valine for isoleucine at position 332; leucine for lysine at position 334; histidine for lysine at position 360; alanine for aspartic acid at position 376; alanine for glutamic acid at position 380; alanine for glutamic acid at position 382; alanine for asparagine or serine at position 384; aspartic acid or histidine for glycine at position 385; proline for glutamine at position 386; glutamic acid for proline at position 387; alanine or serine for asparagine at position 389; alanine for serine at position 424; alanine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan or tyrosine for methionine at position 428; lysine for histidine at position 433; alanine, phenylalanine, histidine, serine, tryptophan or tyrosine for asparagine at position 434; and histidine for tyrosine or phenylalanine at position 436, all in Ell numbering.

[0217] An antibody can bind to C5 at pH 7.4 and 25°C (and, otherwise, under physiologic conditions) with an affinity dissociation constant (KD) that can be at least 0.1 (e.g., at least 0.15, 0.175, 0.2, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95 or 0.975) nM. The KD of the anti-C5 antibody, or antigen binding fragment thereof, can be no greater than 1 (e.g., no greater than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2) nM. The [(KD of the antibody for C5 at pH 6.0 at 25°C) / (KD of the antibody for C5 at pH 7.4 at 25°C)] can be greater than 21 (e.g., greater than 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,260, 270, 280, 290, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000 7500 or 8000).

[0218] A complement inhibitor can inhibit activation of factor B. The complement inhibitor can bind to factor B, for example, thereby inhibiting activation. Exemplary agents include antibodies, antibody fragments, peptides, small molecules, and aptamers. Exemplary antibodies that inhibit factor B are described in U.S. Pat. Pub. No. 20050260198. The isolated antibody or antigen-binding fragment can selectively bind to factor B within the third short consensus repeat (SCR) domain. The antibody can prevent formation of a C3bBb complex. The antibody or antigen-binding fragment can prevent or inhibit cleavage of factor B by factor D. The complement inhibitor can be, for example, an antibody, small molecule, aptamer, or polypeptide that binds to substantially the same binding site on factor B as an antibody described in U.S. Pat. Pub. No. 20050260198, or can be an RNAi agent that inhibits local expression of factor B. Peptides that bind to and inhibit factor B can be identified using methods known in the art.

[0219] A complement inhibitor can inhibit factor D. The complement inhibitor can bind to factor D, for example, thereby inhibiting factor D. Exemplary agents include antibodies, antibody fragments, peptides, small molecules, and aptamers. Exemplary antibodies that inhibit factor D are described in U.S. Pat. No. 7,112,327. The complement inhibitor can be, for example, an antibody, small molecule, aptamer, or polypeptide that binds to substantially the same binding site on factor D as an antibody described in U.S. Pat. No. 7,112,327. Exemplary polypeptides that inhibit alternative pathway activation and are believed to inhibit factor D are disclosed in U.S. Pub. No. 20040038869. Peptides that bind to and inhibit factor D can be identified using methods known in the art.

[0220] A complement inhibitor (Cl) useful in the methods described herein can bind to more than one complement protein and / or inhibit more than one step in a complement activation pathway. Such complement inhibitors are referred to herein as “multimodal.” The complement inhibitor can be, for example, a virus complement control protein (VCCP) (U.S. Pat. No. 7,947,267 and W02006042252). The VCCP can be a poxvirus complement control protein (PVCCP) or a herpesvirus complement control protein (HVCCP). The VCCP can inhibit the classical complement pathway, the alternative complement pathway, the lectin pathway, or any two or more of these. The VCCP, e.g., a PVCCP, can bind to C3b, C4b, or both, for example. The PVCCP cancomprise one or more putative heparin binding sites (K / R — X — K / R) and / or possesses an overall positive charge. The PVCCP can be at least 3 SCR modules (e.g., modules 1-3), e.g., 4 SCR modules. The PVCCP protein can be a precursor of a mature PVCCP (i.e., can include a signal sequence that is normally cleaved off when the protein is expressed in virus-infected cells) or can be a mature form (i.e., lacking the signal sequence).

[0221] Vaccinia complement control protein (VCP) has been shown to inhibit the classical pathway of complement activation via its ability to bind to C3 and C4 and act as a cofactor for factor I mediated cleavage of these components as well as promoting decay of existing convertase (Kotwal, G. et al., Science, 250:827-30, 1990; McKenzie, R. et al., J. Infect. Dis., 166: 1245-50, 1992). It has also been shown to inhibit the alternative pathway by causing cleavage of C3b into iC3b and thereby preventing the formation of the alternative pathway C3 convertase (Sahu, A. et al., J. Immunol., 160, 5596-604, 1998). VCP thus blocks complement activation at multiple steps and reduces levels of the proinflammatory chemotactic factors C3a, C4a, and C5a. Homologs of VCPs, such as smallpox inhibitor of complement enzymes (SPICE) or any of the portions thereof that inhibit complement activation, e.g., SPICE-related polypeptides containing four SCRs, can be used in the methods described herein. Moreover, complement control proteins from cowpox virus (IMP) or monkeypox virus (MCP) can also be used in the methods described herein.

[0222] In addition to VCCPs, a number of other viral proteins exist that interfere with one or more steps in a complement pathway and can be used in the methods described herein, e.g., glycoprotein gC from HSV-1 , HSV-2, VZV, PRV, BHV-1, EHV-1 , and EHV-4 (Schreurs, C. et al., J. Virol., 62:2251-7, 1988). With the exception of VZV, the gC protein encoded by these viruses binds to C3b (Friedman, H. et al, Nature, 309:633-5, 1984) and gCI (from HSV-1) accelerates decay of the classical pathway C3 convertase and inhibits binding of properdin and C5 to C3. The foregoing proteins are referred to collectively as virus complement interfering proteins (VCIPs). By any of a variety of means, such as interfering with one or more steps of complement activation, accelerating decay of a complement component, and / or enhancing activity of a complement regulatory protein, these VCIPs are said to inhibit complement. Any of these proteins, or derivatives thereof, e.g., fragments or variants thereof, can be used as a therapeutic agent in the methods described herein.

[0223] A variety of other complement inhibitors can be used in the methods described herein. For example, the complement inhibitor can be a naturally occurring mammalian complement regulatory protein or a fragment or derivative thereof. The complement regulatory protein can be, for example, CR1 , DAF, MCP, CFH or CFI. The complement regulatory polypeptide can be one that is normally membrane-bound in its naturally occurring state. A fragment of such a polypeptide that lacks some or all of a transmembrane and / or intracellular domain can be used. Soluble forms of complement receptor 1 (sCRI), for example, can be used. The compounds known as TP10 or TP20 (Avant Therapeutics), for example, can be used. Cl inhibitor (CI-INH) is also of use. A soluble complement control protein, e.g., CFH, can be used. The polypeptide can be modified to increase its solubility. For example, the polypeptide can be pegylated.

[0224] Inhibitors of Cis are of use (e.g., U.S. Pat. No. 6,515,002 describes compounds (furanyl and thienyl amidines, heterocyclic amidines, and guanidines) that inhibit Cis; U.S. Pat. Nos. 6,515,002 and 7, 138,530 describe heterocyclic amidines that inhibit Cis; U.S. Pat. No. 7,049,282 describes peptides that inhibit classical pathway activation; U.S. Pat. No. 7,041 ,796 discloses C3b / C4b Complement Receptor-like molecules and uses thereof to inhibit complement activation; U.S. Pat. No. 6,998,468 discloses anti-C2 / C2a inhibitors of complement activation; U.S. Pat. No. 6,676,943 discloses human complement C3-degrading protein from Streptococcus pneumoniae).

[0225] Combination therapy using two or more complement inhibitors is encompassed in the methods described herein. The two or more complement inhibitors can be provided in the same composition. The complement inhibitors can bind to two or more different complement components. The complement inhibitors can bind to two or more different soluble complement proteins. The complement inhibitors can inhibit activation or activity of at least two complement proteins selected from C3, C5, C6, C7, C8, C9, factor B, and factor D.

[0226] Therapeutics can be administered to a subject, e.g., a human subject, using a variety of methods that depend, in part, on the route of administration. The route can be, e.g., intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP) injection, or intramuscular injection (IM). Administration can be achieved by, e.g., local infusion, injection, or by means of an implant. The implant can be of a porous, non- porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. The implant can be configured for sustained or periodic release of the composition to the subject (U.S. Patent Application Publication No. 20080241223; U.S.Pat. Nos. 5,501 ,856; 4,863,457; and 3,710,795; EP488401 ; and EP 430539, the disclosures of each of which are incorporated herein by reference in their entirety). The composition can be delivered to the subject by way of an implantable device based on, e.g., diffusive, erodible, or convective systems, e.g., osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmosis systems, vapor pressure pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps, erosion-based systems, or electromechanical systems.

[0227] A therapeutic agent can be delivered to a subject by way of local administration. “Local administration’’ or “local delivery,” refers to delivery that does not rely upon transport of the composition or agent to its intended target tissue or site via the vascular system. The composition can be delivered, for example, by injection or implantation of the composition or agent or by injection or implantation of a device containing the composition or agent. Following local administration in the vicinity of a target tissue or site, the composition or agent, or one or more components thereof, can diffuse to the intended target tissue or site.

[0228] “Treating”, as used herein, refers to providing treatment, i.e., providing any type of medical or surgical management of a subject. The treatment can be provided to reverse, alleviate, inhibit the progression of, prevent or reduce the likelihood of a disorder or condition, or to reverse, alleviate, inhibit or prevent the progression of, prevent or reduce the likelihood of one or more symptoms or manifestations of a disorder or condition. “Prevent” refers to causing a disorder or condition, or symptom or manifestation of such not to occur for at least a period of time in at least some individuals. Treating can include administering a therapeutic agent / complement modulator to the subject following the development of one or more symptoms or manifestations indicative of a complement-mediated condition, e.g., to reverse, alleviate, reduce the severity of, and / or inhibit or prevent the progression of the condition and / or to reverse, alleviate, reduce the severity of, and / or inhibit or one or more symptoms or manifestations of the condition. According to the methods described herein, a composition / complement modulator can be administered to a subject who has developed a complement-mediated disease or condition or is at increased risk of developing such a disorder relative to a member of the general population. Such a composition / modulator can be administered prophylactically, i.e., before development of any symptom or manifestation of the condition. Typically in this case the subject will be at risk of developing the condition, for example, when exposed to a complement-activating composition, e.g., a particle or nanoparticle encapsulated therapeutic, e.g., a viral particle used in gene therapies or a therapeutic agent delivered by, for example, a lipid nanoparticle.

[0229] The present methods for detecting complement activation in biological samples can be used in many downstream applications. For instance, the methods can be used to determine if a subject (from whom the biological sample is obtained) suffers from a complement-mediated disease or is at risk of developing a complement-mediated disease. The measurement of incidence or risk of complement-mediated disorder can be carried out by comparing the presence or level of the component of the complement pathway on the EV or the membrane-bound portion thereof to a control (or reference standards). Typically, control or reference standard contains EVs isolated from an identical biological sample from a healthy subject. Routine methods can be carried out for processing and normalization of samples obtained from different subjects. Likewise, the present methods can also be used to determine progression or regression of complement-mediated diseases over time. This determination can be carried out by comparing the presence or level of the component of the complement pathway on the EV or the membrane-bound portion thereof in the subject’s sample at two different time points (e.g., tl and t2, where t2>tl). A reduction in presence / level of complement at t2 compared to tl indicates improved complement status and regression of complement disease; the reverse is true if presence / level of complement at t2 is increased compared to tl. The spacing between measurements (e.g., t2 - tl) can depend on the nature of the disease and range from days to years, e.g., a few weeks, 1 month, 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, 10 years, or more, e.g., 20 years.

[0230] The complement modulator described can be formulated with additional active agents useful for treating or preventing a complement-associated disorder in a subject. Additional agents for treating a complement-associated disorder in a subject can include, for example, an antihypertensive (e.g., an angiotensin-converting enzyme inhibitor), an anticoagulant, a corticosteroid (e.g., prednisone), or an immunosuppressive agent (e.g., vincristine or cyclosporine A); anticoagulants (e.g., warfarin (Coumadin), heparin, phenindione, fondaparinux, idraparinux); thrombin inhibitors (e.g., argatroban, lepirudin, bivalirudin, or dabigatran); fibrinolytic agent (e.g., ancrod, e-aminocaproic acid, antiplasmin-ai, prostacyclin, and defibrotide); a lipid- lowering agent; or an anti-CD20 agent such as rituximab.

[0231] The therapeutic can comprise a monoclonal antibody. The monoclonal antibody can comprise ALXN1820, avdoralimab, crovalimab, eculizumab, gefurulimab, lampalizumab, narsoplimab, olendalizumab, pozelimab, ravulizumab, sutimlimab, tesidolumab, or vilobelimab, or any combination thereof. The monoclonal antibody can comprise ALXN1820, eculizumab, gefurulimab (AXLN 1720), or ravulizumab (ALXN1210), or any combination thereof. The therapeutic can comprise a anticomplement system component or complex therapeutic. The therapeutic can inhibit activation and / or activity of factor B, factor D, or properdin, or any combination thereof. The therapeutic can inhibit activation and / or activity of C1 , C1 q, C1 r, or C1 s, or any combination thereof. The therapeutic can inhibit activation and / or activity of C3, C3a, or C3b, or any combination thereof. The therapeutic can inhibit activation and / or activity of C4, C4a, or C4b, or any combination thereof. The therapeutic can inhibit activation and / or activity of C5, C5a, C5b, or C5aR, or any combination thereof. The therapeutic can inhibit activation and / or activity of mannose-binding lectin (MBL), M-ficolin, L-Ficolin, H-ficolin, MBL-associated serine protease-1 (MASP-1), MASP-2, or MASP-3, or any combination thereof.

[0232] Any suitable therapeutic or combination of therapeutics can be employed. The therapeutic can comprise a biologic, a small molecule pharmaceutical, an interfering nucleic acid, or a peptide, or any combination thereof. The biologic can be a monoclonal antibody. The antibody can be a chimeric antibody. The antibody can be a humanized antibody. References to antibodies herein also comprise antigen binding fragments thereof. The therapeutic can comprise ACH-5548, AMY-101 , ANX005, ANX007, ARGX- 117, avacincaptad pegol, avacopan, BCX-9930, BDB-001 , berinert, cemdisiran, cinryze, CLG561 , danicopan, GT103, IONIS-FB liuc, iptacopan, MOR210, nomacopan, OCTA- C1-INH, pegcetacoplan, vemircopan, zilucoplan.

[0233] An “effective amount’’ of an active agent such as a therapeutic agent or complement modulator refers to the amount of the active agent sufficient to elicit a desired biological response (or, equivalently, to inhibit an undesired biological response). The absolute amount of a particular agent that is effective can vary depending on such factors as the desired biological endpoint, the agent to be delivered, the target tissue, and the like. An “effective amount” can be administered in a single dose, or can be achieved by administration of multiple doses. An effective amount of the therapeutic agent, for example, can be an amount sufficient to relieve at least one symptom of a disorder. An effective amount can be an amount sufficient to slow theprogression of a chronic and progressive disorder, e.g., to increase the time before one or more symptoms or signs of the disorder manifests itself or to increase the time before the individual suffering from the disorder reaches a certain level of impairment. An effective amount can be an amount sufficient to allow faster or greater recovery from an injury than would occur in the absence of the agent.

[0234] The methods and assays of the present disclosure can be used to monitor response to treatment of a complement-mediated disease with a complement modulator. A complement modulator is a molecule that can, directly or indirectly, modulate, e.g., activate or inhibit, a complement component, e.g., component protein. Methods for measuring response to treatment can be generally practiced with the method for detecting complement proteins in EVs, for example, by (a) obtaining a sample containing extracellular vesicles (EV) or a membrane-bound portion thereof from the subject before and after the treatment, (b) contacting a portion of the sample with at least one first capture antibody or an antigen-binding fragment thereof to capture at least one first marker on the EVs or the membrane-bound portion(s) thereof; (c) optionally contacting a portion of the sample with at least one second capture antibody or an antigen-binding fragment thereof to capture at least one second marker on the EVs or the membrane-bound portion(s) thereof; (d) contacting the captured EVs or membrane-bound portions thereof with at least one detection antibody or an antigenbinding fragment thereof specific for a complement system-associated component; and (e) detecting, qualitatively or quantitatively, the detection antibody or the antigen-binding fragment thereof, to measure, a presence of or a level of the component of the complement pathway on the EV or the membrane-bound portion thereof, wherein a modulation (e.g., increase or decrease; preferably decrease) in the presence or level of the component of the complement pathway in the subjects sample after treatment with the complement modulator compared to before treatment with the complement modulator indicates the subject is responding to the complement modulator.

[0235] The method can comprise screening a test compound for complement modulation. The method can comprise administering the test compound to the donor, method can be performed at least twice including at least once before administration of the test compound, and at least once after administration of the test compound. The donor can have been diagnosed with dermatomyositis. The dermatomyositis can comprise juvenile dermatomyositis, or adult dermatomyositis, or both. The dermatomyositis can comprise classic dermatomyositis (CDM), amyopathicdermatomyositis (ADM), hypomyopathic dermatomyositis (HDM), clinically amyopathic dermatomyositis (CADM), or juvenile dermatomyositis (JDM), or any combination thereof. The test compound can be for treating dermatomyositis. The donor can display one or more symptoms of dermatomyositis. One or more symptoms of dermatomyositis can comprise skin rash, rough skin, calcinosis, muscle weakness, muscle inflammation, lung inflammation, dysphagia, weight loss, orfever, or any combination thereof. The donor can have been diagnosed with an autoimmune disease. The donor can have been diagnosed with an idiopathic inflammatory myopathy (IIM). The donor can have been diagnosed with systemic lupus erythematosus, antiphospholipid syndrome, rheumatoid arthritis (RA), dermatomyositis (DM), polymyositis (PM), inclusion body myositis (IBM), or Antineutrophilic cytoplasmic antibody (ANCA)-associated vasculitis, or any combination thereof. The donor can have been diagnosed with a primary thrombotic microangiopathy (TMA), or a secondary thrombotic microangiopathy (TMA), or both. The donor can have been diagnosed with complement-mediated thrombotic microangiopathy (CM-TMA). The test compound can be for treating complement- mediated thrombotic microangiopathy (CM-TMA).

[0236] The method can be used to test the efficacy of molecules that inhibit terminal complement activation or activity, e.g., at the C5 axis or the C3 axis. The methods are especially applicable for testing the efficacy of C5 inhibitors, e.g., eculizumab or a follow-on molecule such as ravulizumab. A method of screening a test compound for complement modulation can comprise (a) obtaining a sample containing extracellular vesicles (EV) or a membrane-bound portion thereof from a subject suffering from a complement-mediated disease (e.g., an animal such as a mouse, rabbit, hamster, sheep, llama, dog, monkey, chimpanzee or human), wherein the sample is obtained before and after administration of the test compound; (b) contacting a portion of the sample with at least one first capture antibody or an antigen-binding fragment thereof to capture at least one first marker on the EVs or the membrane-bound portion(s) thereof; (c) optionally contacting a portion of the sample with at least one second capture antibody or an antigen-binding fragment thereof to capture at least one second marker on the EVs or the membrane-bound portion(s) thereof; (d) contacting the captured EVs or membrane-bound portions thereof with at least one detection antibody or an antigenbinding fragment thereof specific for a complement system-associated component; and (e) detecting, qualitatively or quantitatively, the detection antibody or the antigen-binding fragment thereof, to measure, a presence of or a level of the complement component onthe EV or the membrane-bound portion thereof, wherein a modulation (e.g., increase or decrease; preferably decrease) in the presence or level of the complement component in the subject’s sample after administration of the test compound compared to before administration of the test compound indicates that the test compound is capable of modulating complement. Preferably, the test compound is capable of modulating a complement which is Clq, Cl, Cis, C2, MASP-2, MASP-3, Factor D, Factor B, Properdin (Factor P), Factor H, C3 / C5 Convertase, C5, C5a / C5aR, C3a / C3aR, C6, or CD59, or any combination thereof. The modulating activity of the test compound can be compared to the modulating activity of a molecule having complement modulating activity (e.g., positive control or standard).

[0237] The complement modulator whose activity is tested or screened for in accordance with the above methods can be a molecule that inhibits activation of C5, thereby reducing, suppressing and / or eliminating the complement-mediated effects (e.g., CSR or CARP A). Cleavage of C5 releases C5a, a potent anaphylatoxin and chemotactic factor, and leads to the formation of the lytic terminal complement complex, C5b-9. C5a and C5b-9 also have pleiotropic cell activating properties, by amplifying the release of downstream inflammatory factors, such as hydrolytic enzymes, reactive oxygen species, arachidonic acid metabolites and various cytokines.

[0238] Kits configured for use with the methods described herein are provided. Kits can comprise one or more binding moieties, for example, antibodies, or antigen binding fragments thereof, that bind to a biomarker as described herein. In some embodiments of the kits described herein, the kit is an immunoassay, e.g., enzyme-linked immunosorbent assay. Any of the kits described herein can be used to perform any of the methods described herein. The kits can further include instructions for performing any of the methods described herein. Such kits can also include, as non-limiting examples, one or more of: reagents useful in preparing a sample, reagents useful for enriching extracellular vesicles, reagents useful for detecting binding of target proteins or component in a sample to immobilized antibodies, control samples that include purified target proteins / component, and / or instructions for use. For example, kits useful in the methods described herein can include one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen) antibodies or fragments thereof that specifically bind to a biomarker as described herein. For example, the one or more antibodies provided in the kits can be immobilized on a surface (e.g., in the form of an ELISA assay or a gene-chip array).

[0239] The kit can comprise a first binding moiety and a second binding moiety. The first and second binding moieties respectively can be comprised by an agent, a probe, or both. The first binding moiety can bind to a first marker selected from ALIX, TSG101 , CD9, CD63, CD81 , CD40L, CD26, CD31 , CD45, CD2, CDIIa, CD24, CD55, CD59, CF106, CD56, CD51 , CD82, Integrins, tetraspanins, Annexins, HSP90, HSP70, Syntenin-1 , ADAMIO, EHD4, Actin, Rab5, clathrin, Flotillin-1 , MHC I, MHC II, Actinin-4, GP96, EHD4, Mitofilin, or LAMP2, or any combination thereof. The second binding moiety can bind endoglin, VE-cadherin, podocalyxin (PODXL), aquaporin 2 (AQP 2), uroplakinlb (UPKIb), podocin (NPHS2), glycophorin A (GYP A), mucin-1 , type 2 Na-K- 2C1 co-transporter (NKCC2), aquaporin 1 (AQP 1), a-glutathione- S-transferase (alphaGST), Tamm-Horsfall protein (TH), calbindin-D28K (CalD), megalin, cubilin, nephrin (Nphsl), Claudin-1 , Annexin-V, synaptopodin (Synpo), Wilm's tumor protein (Wtl), Band 3, stomatin (STOM), BGP1 , Globin, Glycophorin B, Rh polypeptides, or Rh glycoprotein, or any combination thereof. The first binding moiety can comprise an antibody that binds to tetraspanin, CD9, CD63, or CD81 , or any combination thereof. The second binding moiety can comprise an antibody that binds to endoglin or VE-cadherin or a combination thereof. The kit can comprise an agent, or a probe, or both that binds to C3, C5b-9, C4, Clq, C9, C3b, iC3b, TF, CRP, pCRP, MAC, CD59, CD55, CR1 , C5aRI, or C5a.EXAMPLES

[0240] The following examples demonstrate the development of a multiplex fluorescence-based assay to measure complement regulator proteins and MAC deposition on endothelial cells using EC-derived extracellular vesicles (EC-EV). Selectively enrichment of plasma EC-EV is demonstrated. Advantageous plasma type to study EC-EV is identified. The presence of complement proteins on EC-EV is demonstrated. The role of matrix selection is shown in assay performance (S:N and Specific Response). EC markers are shown to be differentially localized in different EV subsets. Endoglin and PODXL are shown to be colocalized based on a fluorescent multiplex assay that can be confirmed using super-resolution light microscopy (SRLM).EXAMPLE 1

[0241] This example demonstrates plasma extracellular vesicle (EV) phenotyping. As reflected in the results set forth in FIGS. 1A and 1 B. EV sizing and concentration in matched plasma by f-NTA were evaluated using an EV specific dye (ExoGlow). Thesedata indicate that there are no significant differences in EV sizing or concentration among different types of plasma. Blood collection and plasma processing was performed as follows. Human blood was collected from normal donors (ND) in matched K2EDTA (K2), NaCitrate 3.2% (NaCit), P100, and ACD vacutainers and immediately processed. Plasma was obtained by centrifugation 2000xg for 15mins at 4°C, aliquoted and frozen at -70 °C. After thaw and before testing, plasma samples were again centrifuged 10,000xg for 5 min at 4 °C. Fluorescent-nanoparticle tracking analysis (f- NTA) was performed as follows. EV concentration and EV sizing were determined by nanoparticle tracking NTA (Particle Metrix Zetaview equipped with 488 nm laser) and using a compatible EV specific fluorescent dye (ExoGlow-NTA, SBI).EXAMPLE 2

[0242] This example demonstrates detection of EC-specific markers on EV surface by Luminex for optimal matrix selection. After enriching EV based on the presence of tetraspanins, EC-specific markers on the EV surface were measured. Similar endoglin detection was observed in P100 and K2, but there was better detection of endoglin in K2 compared to NaCit. Very low VE-cadherin signal was observed in all plasma types.

[0243] The Luminex assay was performed as follows. XMAP™ fluorescent immunocapture beads were coated in-house with commercially available antibodies. Three categories of capture beads were used as multiplex design: anti-tetraspanins (CD9, CD63, CD81), anti-EC specific markers (endoglin, VE-cadherin, podocalyxin (PODXL)), and a control non-specific lgG1. Detection antibodies targeting EC markers (endoglin, VE-cadherin, and PODXL), complement regulator proteins (CD46, CD55, and CD59), and complement proteins (MAC / C5b-9) were evaluated using commercially available, biotinylated antibodies followed by incubation with streptavidin-phycoerythrin (SAPE).

[0244] Samples were analyzed on the Luminex platform. From each sample, median fluorescence intensity (MFI) signal obtained from negative control (assay buffer blank) was subtracted from signal obtained from plasma samples (= Specific Response). All MFI < 0 were adjusted to 0 for any calculation. Results are shown in FIGS. 2A and 2B.EXAMPLE 3

[0245] This example demonstrates the detection of EC-specific markers on podocalyxin+ EV subset in P100 plasma. Methods and materials were the same as inExample 2 unless otherwise indicated. On podocalyxin+ (PODXL) EV subset, the signal to noise for endoglin was good but too low for VE-cadherin. This result suggests PODXL is co-localizing with endoglin on the same EC-EV but not VE-cadherin. Results are shown in FIGS. 3A and 3B.EXAMPLE 4

[0246] This example demonstrates the identification of advantageous concentrations of detector antibodies. FIGS. 4A-4D (CD46) and FIGS. 5A-5D (CD55) show representative graphs for CD46 and CD55 S:N titration curves and MFI at selected concentrations. Signal-to-Noise (S:N) ratio and Specific Response with respect to median fluorescent intensity (MFI) point to detection of preferred antibody concentrations and plasma type. Using S:N and MFI obtained from titration curves for each detector, advantageous (preferred) detector concentrations were identified in K2 and NaCit plasma (see Tables 1 and 2). Different antibodies showed different preferred concentrations (identified by the arrows), which can also vary based on plasma type. FIGS. 4A-5D include representative graphs for CD46 and CD55 S:N titration curves and MFI at selected concentrations.Table 1 : EndoglinEXAMPLE 5

[0247] This example demonstrates orthogonal verification of tetraspanins and EC markers on EV surface by Super-Resolution Light Microscopy (SRLM). EV characterization was assessed by SRLM using EV profiling Chip (ONI-OxfordNanoImaging) following instruction manual and dSTORM imaging. Tetraspanins fluorescent-conjugated antibodies were provided from ONI. Fluorescent-conjugated antibodies for anti-endoglin and VE-cadherin were the same clones used in Luminex assay. Localization of specific markers on EV surface was evaluated using CODI, the web-based analysis tool from ONI, applying the same analysis settings over all the images acquired.

[0248] Results are shown in FIGS. 6A-6D and 7A-7B. Preliminary image analyses confirm the presence of tetraspanins and EC markers on EV surface in both K2 and NaCit plasma. CD63 appears to be the dominant EV tetraspanin. Endoglin does not colocalize with VE-Cadherin. Representative images are shown for K2 plasma (FIGS. 6A- 6D) — FIG. 6A, imaging with CD9, CD63, and CD81 specific antibodies; FIG. 6B, imaging with endoglin specific antibodies; FIG. 6C, imaging with CD63 specific antibodies; and FIG. 6D imaging with VE-Cadherin specific antibodies. Data was quantified and displayed in graphs — tetraspanin and CD63 markers for K2 EDTA and Na citrate (FIG. 7A) and CD63 and EC markers for K2 EDTA and Na citrate (FIG. 7B). Further experiments could identify EC-markers co-localizing with tetraspanins.EXAMPLE 6

[0249] This example demonstrates the effectiveness of a template-based extracellular vesicle assay consistent with an immunoprecipitation with multiplex immunoassay. Accordingly, a Luminex platform, for example, can be utilized. Reagents were brought to room temperature. On the first day, assay buffer was prepared including: PBS with 1 % (w / v) bovine serum albumin (BSA), one tube of stock frozen - 20°C was thawed and re-filtered before use, and TBS-1 mM Ca-Mg with 1 % (w / v) BSA, one tube of stock frozen -20°C was thawed and re-filtered before use. Samples were also prepared. For all HV samples, samples were defrosted in ice. All samples were centrifuged at 10,000g (10,444 rpm) for 5 minutes at 4°C. All samples were diluted 1 :10 in assay buffer (PBS — BSA1 %). For negative control, PBS was used with no EVs. PBS was diluted in Assay buffer (PBS-BSA1 %). For bead preparation, beads were resuspended by vortexing for 15 seconds, sonicated for 30 seconds, and then quickly vortexed before adding to mix in assay buffer (PBS-BSA1 %) using 1 ,000 beads per well. Beadsets and corresponding regions are listed in Table 3.Table 3: Beadset / Region

[0250] On the second day, the wash buffer used was PBS 1X. Detection Biotin- Antibody & Streptavidin-PE was prepared. The volume needed for the plate (## wells times 0.5 pm / well= amount needed) was determined. The biotin=Ab and 1x streptavidin- PE 6 pg / mL were diluted in PBS / BSA. Sambucus nigra lectin (SNL) lectin=biotin (stock 2pg / ml_) was diluted 1 :40 in detection buffer (TBS Ca-Mg). Antibody dilutions were prepared as indicated in Table 4.Table 4: Antibody Dilutions

[0251] On the first day of performing the assay, with reference to the plate layout as shown in FIG. 8, all reagents were prepared as instructed — protecting beads from light exposure. A black plate with U-shaped bottom plate was used. 50pL / well of the appropriate bead solution was loaded using p300-multichannel digital pipette in appropriate wells. Bead solution was vortexed prior to leading and frequently through the loading process. 100pL / well of sample or control was added according to the plate layout (organization). Large sample dilutions were prepared and 100 pL / well of sample was added in triplicate to the assay plate. A p1000-automatic pipette was used. Theplate was sealed with foil and incubated at least overnight at 300 rpm and 4°C. The actual time in hours and minutes in hours and minutes was recorded.

[0252] On the second day of performing the assay, with reference again to the plate layout, reagents was prepared as instructed including biotinylated antibodies and streptavidin-PE making sure to protect the reagents from light. Three washes of 300 pL in PBS Ixwith a two minute prewash incubation on a magnet under gentle conditions. No blotting was performed. Luminex 3x100 B program was used. Detector antibodies were prepared and 100 pL / well was added in the appropriate wells using a p1000-digital automatic pipette. The plate was sealed and incubated for at least 60 minutes at 300 rpm and 25°C. The actual time in minutes was recorded. 2 x 100 pL washes were performed in DPBS. 2 x 100 pL washes were performed in sheath fluid with a final dispense of 100 pL of sheath fluid. The Luminex final sheath program was used.

[0253] The plate so processed was read on Luminex 100 / 200 or FlexMap3D with all samples as unknowns. The batch was saved with a chosen name. The reading was performed using the following settings: sample volume = 100 pL, bead type = MagPlex, doublet discriminator gates = 7500 and 15000, reporter gain = high PMT, bead region was assigned as noted in reagents and names, and 100 / region and set time-out 90 secs / well were used. The median fluorescence intensity (MFI) numbers were collected. Data calculation and analysis was performed as follows. The mean MFI of the blank (no EVs) was subtracted from all the test samples (= specific response) in all detectors (if negative values were present, the value was inputted as zero and used to calculate the mean of the duplicate). Then the specific response was multiplied by dilution factors (plasma dilution 1 :10 and NaCit dilution 1.1 = 11) and Specific Response*DF was plotted. Next, the sMFI of each bead set was subtracted from the corresponding I gG 1 values (if negative values were present, they were inputted as zero). Each sample was normalized by dividing the mean sMFI of each detector by mean sMFI from SNL. Data calculation was performed using PRISM GRAPHPAD.EXAMPLE 7

[0254] This example demonstrates the identification of Sambucus nigra lectin (SNL) as a lectin compatible with human plasma EVs as a normalizer (e.g. EV subset / EV total) and its use in the assay protocol described in Example 6.

[0255] SNL assay concentration and SNL-assay buffer (TBS-Ca-Mg) were determined. SNL (Vector Laboratories B-1305-2) was evaluated as a normalizing agentwith specificity to N-acetyl-galactosamine or galactose targeting sialic acid. Results from a comparison among four different assay conditions for optimal signal-to-noise ratios are shown in FIG. 9.

[0256] Human plasma (P100 / K2EDTA or NaCitrate) was evaluated to determine performance based on specific-MFI using specific capture beads: mslgG1 (negative isotype control), CD9, CD63, CD81 (canonical EV markers), Podocalyxin, Endoglin, VE- Cadherin (Endothelial markers). This evaluation of SNL performance was performed using normal plasma samples. Results for NaCitrate are shown in FIGS. 10A and 10B, and those for P100 / K2 in FIGS. 10C and 10D.

[0257] NaCitrate plasma collected pre-treatment from the 1210-DM-310 trial (ravulizumab v. placebo in clinical study of adults with dermatomyositis) was evaluated for complement pathway proteins deposition: C1 q, C4d (results in FIGS. 11 A and 11 B), C3b / iC3b, C5b9 (Results in 12A and 12B). These results showed classical and lectin pathway markers elevated in 1210-DM-310 trial subjects. Classical pathway C1q and lectin pathway C4d markers deposited on EC-derived EVs were detected. C1q deposition was present in 76% (=29 / 38) of subjects (FIG. 11A). C4d deposition was present in 47% (=18 / 38) of subjects (FIG. 11 B). Central and terminal pathway markers were elevated in 1210-DM-310 subjects. Central pathway C3b / iC3b markers (results in FIG. 12A) and terminal pathway C5b-9 markers (results in FIG. 12B) were detected deposited on EC-derived EVs. C3b / iC3b deposition was present in 26% (=20 / 38) of the subjects (FIG 12A). C5b-9 (MAC) deposition was present in 53% (=20 / 38) of the subjects (FIG. 12B).

[0258] DM subjects with or without DM pathogenic autoantibodies were compared for complement deposition (results in FIGS. 13A-13D). These results showed no difference in complement deposition between DM subjects with or without pathogenic autoantibodies.

[0259] Overall observations on method optimization and clinical performance using 1210-DM-310 samples were as follows. SNL was identified as normalized compatible with plasma EVs. SNL showed better performance / maximum detection of CD81 in NaCitrate in PODXL subset; P100 or K2EDTA for max detection of CD63. SNL assay conditions were determined (wash buffer= PBS, detector buffer= TBS-Ca-Mg). Clinical performance was tested using 1210-DM-310 pre-treatment with results as follows. C1q: significative increased deposition in DM vs normal donors. C4d: significative increased deposition in DM vs normal donors. C3b: increased deposition in a limited subset of DMsubjects enrolled. C5b-9: significative increased deposition in DM vs normal donors. No statistical differences were observed comparing DM subjects positive for pathogenic autoantibodies vs subjects without (e.g. negative) for difference in 4 complement proteins activity / deposition tested. Accordingly, these methods can be used for ALL DM patients.

[0260] The present disclosure can include any combination of these various features or embodiments above and / or below as set forth in sentences and / or paragraphs. Any combination of disclosed features herein is considered part of the present disclosure. Further, when an amount, concentration, or other value or parameter is given as either a range, or a list of upper values and lower values, all ranges formed from any pair of any upper range limit or value and any lower range limit or value are also disclosed, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all subranges, integers, and fractions within the range. The scope of the disclosure is not limited to the specific values recited in a range. All references cited in this specification are herein incorporated by reference as though each reference was specifically and individually indicated to be incorporated by reference. Each of the elements described herein, or two or more together, are also within the scope of the present disclosure.

Claims

CLAIMSWhat is claimed is:1 . A method of measuring endothelial cell specific complement activation, the method comprising: contacting a plasma with a first agent to produce a first plasma fraction; contacting the first plasma fraction with a second agent to produce a second plasma fraction, the second plasma fraction having an endothelial cell specific extracellular vesicle concentration greater than that of the plasma and that of the first plasma fraction; contacting the second plasma fraction with a probe that specifically binds a complement system-associated component; and measuring bound probe, the bound probe indicative of endothelial cell specific complement activation.

2. The method of any preceding claim, wherein the first plasma fraction has a higher extracellular vesicle concentration than that of the plasma.

3. The method of any preceding claim, wherein: the first agent comprises a first binding moiety that specifically binds an extracellular vesical associated protein, and the second agent comprises a second binding moiety that specifically binds an endothelial cell associated protein; or the first agent comprises a first binding moiety that specifically binds an endothelial cell associated protein, and the second agent comprises a second binding moiety that specifically binds an extracellular vesical associated protein; or both.

4. The method of any preceding claim, wherein the first agent comprises a first binding moiety that specifically binds an extracellular vesical associated protein, and the second agent comprises a second binding moiety that specifically binds an endothelial cell associated protein.

5. The method of any preceding claim, wherein the first binding moiety, or the second binding moiety, or both comprises an antibody or an antigen-binding fragment thereof.

6. The method of any preceding claim, wherein the first binding moiety, or the second binding moiety, or both comprises an aptamer.

7. The method of any preceding claim, wherein the first binding moiety comprises a first plurality of binding moieties, or the second binding moiety comprises a second plurality of binding moieties, or both.

8. The method of any preceding claim, wherein the binding moieties of the first plurality differ in binding specificity, or the binding moieties of the second plurality differ in binding specificity, or both.

9. The method of any preceding claim, wherein the targets of the first plurality, or the targets of the second plurality, or both are present in the same extracellular vesicle or a membranebound portion thereof.

10. The method of any preceding claim, wherein the second plurality of markers is indicative of dermatomyositis.11 . The method of any preceding claim, wherein the second plurality of markers is indicative of complement-mediated thrombotic microangiopathy.

12. The method of any preceding claim, wherein the probe comprises a first plurality of probes.

13. The method of any preceding claim, wherein the probes of the plurality of probes differ in binding specificity.

14. The method of any preceding claim, wherein the agent, the probe, or both comprise a binding moiety.

15. The method of any preceding claim, wherein the agent, or the probe, or both comprise an antibody or an antigen-binding fragment thereof.

16. The method of any preceding claim, wherein the agent, or the probe, or both comprise an aptamer.

17. The method of any preceding claim, wherein the first binding moiety, or the second binding moiety, or both bind ALIX, TSG101 , CD9, CD63, CD81 , CD40L, CD26, CD31 , CD45, CD2, CDIIa, CD24, CD55, CD59, CF106, CD56, CD51 , CD82, Integrins, Tetraspanins, Annexins, HSP90, HSP70, Syntenin-1 , ADAMIO, EHD4, Actin, Rab5, clathrin, Flotillin-1 , MHO I, MHC II, Actinin-4, GP96, EHD4, Mitofilin, or LAMP2, or any combination thereof.

18. The method of any preceding claim, wherein the first binding moiety, or the second binding moiety, or both bind a tetraspanin.

19. The method of any preceding claim, wherein the first binding moiety, or the second binding moiety, or both bind CD9, CD63, or CD81 , or any combination thereof.

20. The method of any preceding claim, wherein the first binding moiety, the second binding moiety, or both bind endoglin, VE-cadherin, podocalyxin (PODXL), aquaporin 2 (AQP 2), uroplakinlb (UPKIb), podocin (NPHS2), glycophorin A (GYP A), mucin-1 , type 2 Na-K-2C1 co-transporter (NKCC2), aquaporin 1 (AQP 1 ), a-glutathione- S-transferase (alpha-GST), Tamm-Horsfall protein (TH), calbindin-D28K (CalD), megalin, cubilin, nephrin (Nphsl), Claudin-1 , Annexin-V, synaptopodin (Synpo), Wilm’s tumor protein (Wtl), Band 3, stomatin (STOM), BGP1 , Globin, Glycophorin B, Rh polypeptides, or Rh glycoprotein, or any combination thereof.

21. The method of any preceding claim, wherein the first binding moiety, the second binding moiety, or both bind endoglin and podocalyxin; or endoglin and VE-cadherin; or endoglin, podocalyxin, and VE-cadherin.

22. The method of any preceding claim, wherein the first binding moiety, the second binding moiety, or both bind endoglin and podocalyxin, or endoglin and VE-cadherin, or both.

23. The method of any preceding claim, wherein the second plasma fraction comprises a first subtraction characterized by endoglin and podocalyxin, and a second subtraction characterized by endoglin and VE-cadherin.

24. The method of any preceding claim, wherein the first binding moiety binds to CD63 and / or CD9 and the second moiety binds to endoglin and / or VE-cadherin.

25. The method of any preceding claim, wherein the first binding moiety binds to CD63 and the second moiety binds to endoglin and / or wherein the first binding moiety binds to CD63 and the second moiety binds to VE-cadherin.

26. The method of any preceding claim, wherein the agent, the probe, the first binding moiety, or the second binding moiety, or any combination thereof bind a dermatomyositis specific marker, or a complement-mediated thrombotic microangiopathy, or both.

27. The method of any preceding claim, wherein the agent, the probe, the first binding moiety, or the second binding moiety, or any combination thereof bind C3, C5b-9, C4, Clq, C9, C3b, iC3b, TF, CRP, pCRP, MAC, CD59, CD55, CR1 , C5aRI, or C5a, or any combination thereof.

28. The method of any preceding claim, wherein the agent, the probe, the first binding moiety, or the second binding moiety, or any combination thereof bind CD46, CD55, CD59, or C5b-9, or any combination thereof.

29. The method of any preceding claim, wherein the agent, the probe, the first binding moiety, or the second binding moiety, or any combination thereof bind to a component of the alternative complement system.

30. The method of any preceding claim, wherein the complement system-associated component bound by the agent, the probe, or both comprises a classical complement system component, an alternative complement system component, or a lectin complement system component, or any combination thereof.

31. The method of any preceding claim, wherein the complement activation comprises activation of a classical complement system, an alternative complement system, or a lectin complement system, or any combination thereof.

32. The method of any preceding claim, wherein the first binding moiety, the second binding moiety, the agent, or the probe, or any combination thereof are bound to a solid support.

33. The method of any preceding claim, wherein the solid support comprises a plurality of solid supports.

34. The method of any preceding claim, wherein the plurality of solid supports comprise first, second, and third solid supports, wherein the first, second, and third solid support differ from one another, the first binding moiety is anchored to the first solid support, the second binding moiety is bound to the second solid support, and the probe is bound to the third solid support.

35. The method of any preceding claim, wherein the plurality of solid supports differ from one another.

36. The method of any preceding claim, wherein the plurality of solid supports differ from one another with respect to a detectable label.

37. The method of any preceding claim, wherein the detectable label comprises a fluorescent dye.

38. The method of any preceding claim, wherein the detectable label comprises a plurality of fluorescent dyes.

39. The method of any preceding claim, wherein solid supports differ with respect to a combination of two or more fluorescent dyes.

40. The method of any preceding claim, wherein the solid support comprises beads, a chip, an array, a slide, a multi-well plate, or a microfluidic device, or any combination thereof.

41. The method of any preceding claim, wherein the solid support comprises a polymer.

42. The method of any preceding claim, wherein the polymer comprises polystyrene.

43. The method of any preceding claim, wherein the probe comprises a detectable label.

44. The method of any preceding claim, further comprising contacting the second plasma fraction with a secondary probe that specifically binds to the probe, the probe being a primary probe.

45. The method of any preceding claim, wherein the primary probe, or the secondary probe, or both comprise an antibody or an antigen binding fragment thereof.

46. The method of any preceding claim, wherein the primary probe comprises a primary antibody and the secondary probe comprises a secondary antibody.

47. The method of any preceding claim, wherein the primary and secondary antibodies bind each other through respective biotin and streptavidin molecules.

48. The method of any preceding claim, wherein the probe comprises a detectable label.

49. The method of any preceding claim, wherein the secondary probe comprises a detectable label.

50. The method of any preceding claim, wherein the secondary probe comprises an enzyme that converts a substrate to a detectable product.

51. The method of any preceding claim, wherein the detectable label comprises a fluorescent label, a chemiluminescent label, a radioactive label, a colored label, a magnetic label, a sizebased label, a charge-based label, a chelation-based label, a complementary tag label, or a specific-binding label, or any combination thereof.

52. The method of any preceding claim, wherein the label comprises phycoerytherin (PE).

53. The method of any preceding claim, wherein the detectable label comprises a plurality of detectable labels, wherein each different label of the plurality is specific to a molecular target.

54. The method of any preceding claim, wherein the probe, the binding moiety, or the detectable label, or any combination thereof is specific to a complement protein.

55. The method of any preceding claim, wherein the plasma comprises an anticoagulant, or has been treated with an anticoagulant, or both.

56. The method of any preceding claim, wherein the plasma comprises a chelator, or has been treated with a chelator, or both.

57. The method of any preceding claim, wherein the anticoagulant comprises ethylenediaminetetraacetic acid (EDTA), heparin, or citrate, or any combination thereof.

58. The method of any preceding claim, wherein the plasma comprises P100 plasma, K2EDTA plasma, sodium citrate plasma, or anticoagulant citrate dextrose (ACD) plasma, or any combination thereof.

59. The method of any preceding claim, wherein the plasma is obtained from blood of a mammalian donor.

60. The method of any preceding claim, wherein the plasma is obtained from blood of a human donor.

61. The method of any preceding claim, wherein the plasma is obtained from more than one blood donor.

62. The method of any preceding claim, wherein the plasma is obtained from a donor having a complement system associated disease.

63. The method of any preceding claim, wherein the complemental system associated disease is a chronic disease associated with one or more genetic mutations in one or more complement encoding genes.

64. The method of any preceding claim, wherein the disease is dermatomyositis (DM).

65. The method of any preceding claim, wherein the disease is complement-mediated thrombotic microangiopathy (CM-TMA).

66. The method of any preceding claim, wherein the measuring comprises quantification of the bound probe.

67. The method of any preceding claim, wherein the measuring comprises normalization with respect to one or more parameters.

68. The method of any preceding claim, wherein the one or more parameters comprise plasma type, mean extracellular vesicle size, extracellular vesicle concentration, complement system component marker, complement system complex marker, extracellular vesicle marker, or endothelial cell marker, or any combination thereof.

69. The method of any preceding claim, wherein normalization is with respect to a sample having known parameter values.

70. The method of any preceding claim, wherein the measuring comprises flow cytometry.

71. The method of any preceding claim, wherein the measuring comprises measuring fluorescence, chemiluminescence, radioactivity, color, or infrared, or any combination thereof.

72. The method of any preceding claim, wherein the measuring comprises multianalyte profiling, spectrometry, spectrophotometry, or microscopy, or any combination thereof.

73. The method of any preceding claim, wherein the microscopy comprises fluorescence microscopy, super-resolution light microscopy (SRLM), or electron microscopy (EM), or any combination thereof.

74. The method of any preceding claim, wherein the measuring comprises an immunoassay, electron microscopy (EM), tandem mass tag (TMT), a luminescence assay, or a fluoroimmunoassay (FIA), or any combination thereof.

75. The method of any preceding claim, wherein detection step is carried out in a multiplex format.

76. The method of any preceding claim, further comprising diagnosing, or prognosing, or both a disease in a donor from which the plasma was obtained based on the measurement.

77. The method of any preceding claim, wherein the disease is a complement-associated disease.

78. The method of any preceding claim, wherein the disease is associated with abnormal activation of a complement system.

79. The method of any preceding claim, wherein the disease is an inflammatory disease, or a thrombotic disease, or both.

80. The method of any preceding claim, wherein the disease is a thrombotic hematological disease, or a thrombotic nephrological disease, or any combination thereof.

81. The method of any preceding claim, wherein the disease is a nephrological disease selected from the group consisting of atypical haemolytic uraemic syndrome (aHUS), C3 glomerulopathy (C3G), dense deposit disease (DDD), membranoproliferative glomerular nephritis (MPGN), lupus nephritis (LN), IgA nephropathy (IN), lupus nephritis (LN), membranous nephropathy (MN), complications due to hemodialysis in transplant patients, antibody-mediated rejection (AMR), and anti -neutrophil cytoplasmic antibody (ANCA)- associated vasculitis (AAV).

82. The method of any preceding claim, wherein the disease is a hematological disease selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), atypical haemolytic uraemic syndrome (aHUS), secondary HUS due to solid organ transplant or hematopoietic stem cell transplants, thrombotic microangiopathy (TMA), and cold agglutinin disease (CAD).

83. The method of any preceding claim, wherein the disease is a chronic disease associated with one or more genetic mutations in one or more complement encoding genes of the donor.

84. The method of any preceding claim, wherein the disease is associated with systemic organ damage.

85. The method of any preceding claim, wherein the disease comprises elevated levels of complement system components.

86. The method of any preceding claim, wherein the disease comprises thrombocytopenia, microangiopathic hemolytic anemia (MAHA), or microvascular thrombosis, or any combination thereof.

87. The method of any preceding claim, wherein the disease is complement-mediated thrombotic microangiopathy (CM-TMA).

88. The method of any preceding claim, further comprising diagnosing, or prognosing, or both a complement system abnormality in a donor from which the plasma was obtained based on the measurement.

89. The method of any preceding claim, wherein the complement system abnormality is caused by one or more genetic mutations in one or more complement encoding genes of the donor.

90. The method of any preceding claim, wherein the complement system abnormality is caused by an infection, hypertension, an autoimmune disease, a cancer, a transplantation, a pregnancy, or a drug, or any combination thereof.

91. The method of any preceding claim, further comprising administering a therapeutic compound or composition to a donor from which the plasma was obtained based on the measurement.

92. The method of any preceding claim, wherein the therapeutic comprises a complement modulator, inhibitor, or activator, or any combination thereof.

93. The method of any preceding claim, wherein the therapeutic comprises a therapeutic effective against dermatomyositis (DM).

94. The method of any preceding claim, wherein the therapeutic comprises a corticosteroid, prednisone, an immunosuppressant, azathioprine, methotrexate, mycophenolate mofetil, rituximab, hydroxychloroquine, intravenous immunoglobulin (IVIg), or sunscreen, or any combination thereof.

95. The method of any preceding claim, wherein the therapeutic comprises a monoclonal antibody.

96. The method of any preceding claim, wherein the monoclonal antibody comprises ALXN1820, avdoralimab, crovalimab, eculizumab, gefurulimab, lampalizumab, narsoplimab, olendalizumab, pozelimab, ravulizumab, sutimlimab, tesidolumab, or vilobelimab, or any combination thereof.

97. The method of any preceding claim, wherein the monoclonal antibody comprises ALXN1820, eculizumab, gefurulimab (AXLN 1720), or ravulizumab (ALXN1210), or any combination thereof.

98. The method of any preceding claim, wherein the therapeutic comprises an anticomplement system component or complex therapeutic.

99. The method of any preceding claim, wherein the therapeutic inhibits activation and / or activity of factor B, factor D, or properdin, or any combination thereof.

100. The method of any preceding claim, wherein the therapeutic inhibits activation and / or activity of C1 , C1q, C1 r, or CIs, or any combination thereof.

101. The method of any preceding claim, wherein the therapeutic inhibits activation and / or activity of C3, C3a, or C3b, or any combination thereof.

102. The method of any preceding claim, wherein the therapeutic inhibits activation and / or activity of C4, C4a, or C4b, or any combination thereof.

103. The method of any preceding claim, wherein the therapeutic inhibits activation and / or activity of C5, C5a, C5b, or C5aR, or any combination thereof.

104. The method of any preceding claim, wherein the therapeutic inhibits activation and / or activity of mannose-binding lectin (MBL), M-ficolin, L-Ficolin, H-ficolin, MBL-associated serine protease-1 (MASP-1 ), MASP-2, or MASP-3, or any combination thereof.

105. The method of any preceding claim, wherein the therapeutic comprises a small molecule pharmaceutical.

106. The method of any preceding claim, wherein the therapeutic comprises an interfering nucleic acid.

107. The method of any preceding claim, wherein the therapeutic comprises a peptide.

108. The method of any preceding claim, wherein the therapeutic comprises ACH-5548, AMY-101 , ANX005, ANX007, ARGX-117, avacincaptad pegol, avacopan, BCX-9930, BDB- 001 , berinert, cemdisiran, cinryze, CLG561 , danicopan, GT103, IONIS-FB liuc, iptacopan, MOR210, nomacopan, OCTA-C1-INH, pegcetacoplan, vemircopan, zilucoplan.

109. The method of any preceding claim, further comprising screening a test compound for complement modulation.

110. The method of any preceding claim, further comprising administering the test compound to the donor, wherein the method is performed at least twice including at least once before administration of the test compound, and at least once after administration of the test compound.

111. The method of any preceding claim, wherein the donor has been diagnosed with dermatomyositis.

112. The method of any preceding claim, wherein the dermatomyositis comprises juvenile dermatomyositis, or adult dermatomyositis, or both.

113. The method of any preceding claim, wherein the dermatomyositis comprises classic dermatomyositis (CDM), amyopathic dermatomyositis (ADM), hypomyopathic dermatomyositis (HDM), clinically amyopathic dermatomyositis (CADM), or juvenile dermatomyositis (JDM), or any combination thereof.

114. The method of any preceding claim, wherein the test compound is for treating dermatomyositis.

115. The method of any preceding claim, wherein the donor displays one or more symptoms of dermatomyositis.

116. The method of any preceding claims, wherein the one or more symptoms of dermatomyositis comprise skin rash, rough skin, calcinosis, muscle weakness, muscle inflammation, lung inflammation, dysphagia, weight loss, or fever, or any combination thereof.

117. The method of any preceding claims, wherein the donor has been diagnosed with an autoimmune disease.

118. The method of any preceding claims, wherein the donor has been diagnosed with an idiopathic inflammatory myopathy (IIM).

119. The method of any preceding claims, wherein the donor has been diagnosed with systemic lupus erythematosus, antiphospholipid syndrome, rheumatoid arthritis (RA), dermatomyositis (DM), polymyositis (PM), inclusion body myositis (IBM), or Antineutrophilic cytoplasmic antibody (ANCA)-associated vasculitis, or any combination thereof.

120. The method of any preceding claim, wherein the donor has been diagnosed with a primary thrombotic microangiopathy (TMA), or a secondary thrombotic microangiopathy (TMA), or both.

121. The method of any preceding claim, wherein the donor has been diagnosed with complement-mediated thrombotic microangiopathy (CM-TMA).

122. The method of any preceding claim, wherein the test compound is for treating complement-mediated thrombotic microangiopathy (CM-TMA).

123. A kit configured for use with the method of any preceding claim.

124. The kit of any preceding claim, comprising a first binding moiety and a second binding moiety.

125. The kit of any preceding claim, wherein the first and second binding moieties respectively are comprised by an agent, a probe, or both.

126. The kit of any preceding claim, wherein the first binding moiety binds to a first marker selected from ALIX, TSG101 , CD9, CD63, CD81 , CD40L, CD26, CD31 , CD45, CD2, CDIIa, CD24, CD55, CD59, CF106, CD56, CD51 , CD82, Integrins, tetraspanins, Annexins, HSP90, HSP70, Syntenin-1 , ADAMIO, EHD4, Actin, Rab5, clathrin, Flotillin-1 , MHC I, MHC II, Actinin-4, GP96, EHD4, Mitofilin, or LAMP2, or any combination thereof.

127. The kit of any preceding claim, wherein the second binding moiety binds endoglin, VE- cadherin, podocalyxin (PODXL), aquaporin 2 (AQP 2), uroplakinlb (UPKIb), podocin (NPHS2), glycophorin A (GYP A), mucin-1 , type 2 Na-K-2C1 co-transporter (NKCC2), aquaporin 1 (AQP 1 ), a-glutathione- S-transferase (alpha-GST), Tamm-Horsfall protein (TH), calbindin-D28K (CalD), megalin, cubilin, nephrin (Nphsl), Claudin-1 , Annexin-V, synaptopodin (Synpo), Wilm’s tumor protein (Wtl), Band 3, stomatin (STOM), BGP1 , Globin, Glycophorin B, Rh polypeptides, or Rh glycoprotein, or any combination thereof.

128. The kit of any preceding claim, wherein the first binding moiety comprises an antibody that binds to tetraspanin, CD9, CD63, or CD81 , or any combination thereof.

129. The kit of any preceding claim, wherein the second binding moiety comprises an antibody that binds to endoglin or VE-cadherin or a combination thereof.

130. The kit of any preceding claim, comprising an agent that binds to C3, C5b-9, C4, Clq, C9, C3b, iC3b, TF, CRP, pCRP, MAC, CD59, CD55, CR1 , C5aRI, or C5a.

131. The method of any preceding claim, wherein a normalizer is used.

132. The method of any preceding claim, wherein the normalizer comprises a lectin.

133. The method of any preceding claim, wherein the lectin is Sambucus nigra (elderberry) lectin (SNL).

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