Methods for using extracellular vesicles to detect complement activation, and their use for evaluating and / or monitoring the treatment of complement-related diseases.

Abstract

A method for using extracellular vesicles to detect complement activation, and the use thereof for evaluating and / or monitoring the treatment of complement-related diseases. [Solution] A method for detecting complement activity in a biological sample is disclosed herein. The disclosure also relates to a method for diagnosing or prognosticating complement disorders in a subject, and a method for monitoring a response to treatment for a complement disorder using a complement modulator in a subject. A method using extracellular vesicles as a non-invasive, highly sensitive, and specific test for diagnosing and / or monitoring treatment responses in patients suffering from various complement disorders is provided herein.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the priority and benefit of U.S. Provisional Patent Application No. 63 / 025,557, filed on May 15, 2020, the entire content of which is incorporated herein by reference.

[0002] The present disclosure relates to methods for detecting complement activation in biological samples. The present disclosure also relates to methods for diagnosing or prognosticating complement - mediated diseases in a subject, and methods for monitoring responses during and after treatment of complement - mediated diseases using complement modulators.

Background Art

[0003] The complement system is part of the innate immune system and acts together with other immunological systems that defend the body against the invasion of cellular and viral pathogens. There are at least 25 proteins in the complement pathway, which is regarded as an aggregate of complexes consisting of circulating plasma proteins and cell - membrane cofactors. Plasma proteins constitute about 10% of the globulins in vertebrate serum. Complement proteins circulate in the blood as inactive precursors and, when stimulated by one of several triggers, proteases in the system cleave specific proteins, release cytokines, and initiate an amplification cascade of further cleavage.

[0004] The components of complement achieve their immune - defense functions by activating a complex series of cell - surface and fluid - phase interactions that involve precise cleavage by enzymes and plasma - membrane - binding events. The resulting complement cascade leads to the generation of products with opsonization, immunomodulatory, and lytic functions.

[0005] The complement cascade proceeds via the classical pathway, the alternative pathway, or the lectin pathway. These pathways share many components and, although their initial stages are different, they share the same "terminal complement" components (C5 - C9) that cause the activation and destruction of target cells.

[0006] The conventional pathway (CP) is typically initiated by the recognition and binding of an antibody to an antigenic site on a target cell. The alternative pathway (AP) is antibody-independent and can be autoactivated by certain molecules on the pathogen surface. In addition, the lectin pathway is typically initiated by the binding of mannose-binding lectins (MBLs) to high-mannose substrates. These pathways converge at the point where complement component C3 is cleaved by an active protease to produce C3a and C3b. Other pathways that activate complement attack can subsequently act in a sequence of events leading to various aspects of complement function.

[0007] This complement system plays a crucial role in the human body in combating disease, and measuring its components can be useful in diagnosing and / or assessing the prognosis of diseases, as well as monitoring the response to treatment for complement-related disorders.

[0008] Tissue biopsy can provide definitive clinical evidence for the diagnosis of most diseases and may be a direct way to confirm the role of complement in the pathogenesis of disease. However, biopsies are painful and expensive, and carry risks associated with the procedure. Repeated tissue biopsies are not commonly performed. Therefore, it is not possible to monitor longitudinal responses to treatment via multiple local tissue biopsies.

[0009] Extracellular vesicles (EVs) are small, membrane-bound, coated particles (30-100 nm) produced by cells. EVs are released from the parent cell's plasma membrane (PM) and contain functional membrane and cytoplasmic proteins, lipids, and RNA. Other terms for EVs include microvesicles, ectosomes, vesicles, ungranulated vesicles, microparticles, and exosomes. The EV outer membrane contains EV-specific protein markers and parent cell-specific PM markers. The orientation of EV membrane proteins is the same as that of parent PMs. Extracellular vesicles possess canonical EV markers such as CD9, CD63, or CD81, which are members of the tetraspanin superfamily of proteins. Tetraspanins are the most abundant membrane proteins in EVs. Some EVs also possess complement regulators on their surface, such as CD55 and CD59. The urine of healthy individuals is approximately 10 9 It contains individual urine EV / mL (uEV / mL), which mainly originates from the kidneys, urothelium, and (in males) the genitals. Cells under stress will increase EV production.

[0010] An unmet need in the field of complement diagnostics and complement therapy is a highly sensitive, specific, and non-invasive clinical test for measuring the localized tissue arrangement of terminal complement complexes. A non-invasive test that allows for frequent longitudinal monitoring of cell surface complement activity during treatment would provide novel information about the pharmacokinetic effects of therapy at the local level of specific organs, representing a significant advance over current methods that are limited to measuring fluid-phase complement activity. [Overview of the project] [Means for solving the problem]

[0011] This specification provides a method for using extracellular vesicles as a non-invasive, highly sensitive, and specific test for diagnosing and / or monitoring treatment response in patients suffering from various complement-related disorders. In one embodiment, EVs can be considered as liquid biopsy sources isolated from biological fluids, including urine. This disclosure is in part based on the discovery of complement deposition in EVs, and these findings can be utilized for many applications, including ex vivo analysis of patient samples as surrogates for monitoring in vivo complement activity, methods for monitoring the efficacy of drugs that modulate the complement pathway, methods for monitoring patients over time, and methods for screening test compounds that modulate systemic and / or local tissue surface complement activity.

[0012] This assay utilizes a bead-based immunocapture protocol with immunofluorescence detection to quantify complement activity and dysregulation at organ-specific tissue levels, as well as regulation during treatment. Based on the illustrated proof of concept using urine samples, this method can be broadly applied to monitor any organ or tissue under complement attack in any liquid matrix using antibody-tagged beads and protein and tissue-specific detection reagents including fluorophores. The principle of this assay and the proof of concept performed in urine can potentially be extended to blood and cerebrospinal fluid for the analysis of detached extracellular viable cells (EVs) from other tissues / organs, including EVs from tissues damaged by terminal complement complex deposition.

[0013] In one embodiment, the disclosure provides a readily usable method for isolating and concentrating extracellular viable cells (EVs), and for semi-quantitative monitoring of complement on the surface of EVs before, during, and after therapeutic intervention. The method can be readily multiplexed and adapted to a wide range of assay formats and / or combined with various analytical techniques, such as nanoparticle tracking analysis (NTA), mass spectrometry (MS), or super-resolution microscopy.

[0014] This disclosure relates to the following non-limiting aspects:

[0015] This disclosure relates to a method for detecting complement activity in a biological sample, (a) A portion of a biological sample containing extracellular vesicles (EVs) or their membrane-bound portions is isolated with at least one first capture antibody or its antigen-binding fragment, and at least one first marker, including an EV-specific marker or a tissue-specific marker displayed on the EVs, (b) If applicable, a portion of the sample may be brought into contact with at least one second capture antibody or its antigen-binding fragment to capture at least one second marker on the EV or its membrane-bound portion. (c) Detecting the presence or level of complement system-related components on the captured EV or its membrane-bound portion qualitatively or quantitatively using at least one detection antibody or its antigen-binding fragment that is specific to the complement system-related components, thereby detecting complement activation in the biological sample. This provides a method that includes [something].

[0016] The first and / or second markers may independently be on the membrane of the EV if they are transmembrane, or inside the EV if they are soluble (e.g., C5), so that "on top of" also includes "inside" or "inside of".

[0017] In one embodiment, the first capture marker includes an EV-specific marker, and optionally the second capture marker includes a tissue-specific marker displayed on the EV or its membrane-bound portion. In one embodiment, both the first and second capture markers are present, the first capture marker includes an EV-specific marker, and the second capture marker includes a tissue-specific marker, and these are detected.

[0018] In one embodiment, the extracellular vesicles in the biological sample are derived from a liquid biopsy, such as urine. This biological sample is obtained from a liquid biopsy protocol.

[0019] In one embodiment, the biological sample is derived from tissue, organ, or body fluid. In one embodiment, the biological sample includes extracellular fluid (EV) or its membrane-bound portions derived from bladder cells, kidney cells, whole blood, red blood cells, platelets, serum, plasma, blood fractions other than serum or plasma, lymph, cerebrospinal fluid (CSF), saliva, tears, vaginal secretions, semen, glandular secretions, exudate, cyst or fecal contents, lavage fluid, or ascites. In one embodiment, the biological sample includes EV or its membrane-bound portions derived from renal glomerulopodocytes, convoluted tubules, or bladder epithelium, or red blood cells (RBCs). In one embodiment, the biological sample is a urine sample. In one embodiment, the biological sample is a red blood cell (RBC) sample.

[0020] In one embodiment, a first capture antibody or its antigen-binding fragment is bound to a first solid support, optionally a second capture antibody or its antigen-binding fragment is bound to a second solid support, and a detection antibody is bound to a detectable marker. In one embodiment, the method disclosed herein comprises contacting a portion of a biological sample with the first capture antibody or its antigen-binding fragment and the second capture antibody or its antigen-binding fragment, wherein the first capture antibody or its antigen-binding fragment and the second capture antibody or its antigen-binding fragment are bound to the same support or different supports.

[0021] In one embodiment, the detectable marker is selected from the group consisting of fluorophores, chromogens, and biotin. In one embodiment, the detectable marker is a fluorophore having an absorption maximum between approximately 500 nm and approximately 900 nm, between approximately 600 nm and approximately 1000 nm, or between approximately 500 nm and approximately 1000 nm, and an emission maximum between approximately 550 nm and approximately 900 nm, between approximately 600 nm and approximately 1000 nm, or between approximately 550 nm and approximately 1100 nm. In one embodiment, the detectable marker is streptavidin-bound or unbound phycoerythrin (PE). In one embodiment, the detectable marker is biotin for use with streptavidin-phycoerythrin (SAPE).

[0022] In one embodiment, the first and second solid supports are independently selected from the group consisting of nanoparticles, microparticles, beads, magnetic beads, nanostructures, tissue culture plates, silica, and nanomatrices.

[0023] In one embodiment, the first marker is selected from the group consisting of extracellular vesicle-related proteins, and the optionally contacting second marker is selected from the group consisting of tissue-specific extracellular vesicle-related proteins, and the complement system-related components are selected from the group consisting of (a) components of the alternative complement pathway (AP), (b) components of the classical complement pathway (CP), and (c) components of the lectin complement pathway (MBL). In a preferred embodiment, the complement system-related components are selected from the group consisting of (a) components of the alternative pathway (AP) and (b) components of the classical pathway (CP).

[0024] In one embodiment, the complement system-related components are proteins selected from the group consisting of C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, iC3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, C5b-9 (membrane attack complex (MAC)), TF, CRP, pCRP, CD59, CD55, CR1, CR2, CR3, C5aR1, properdin, factor H, factor H-related proteins, and factor I. See FIG. 11 (an image adapted from Karasu, E., et al. Frontiers in Immunology 9(721), 2018).

[0025] In one embodiment, the first marker is selected from the group consisting of ALIX, TSG101, CD9, CD63, CD81, CD40L, CD26, CD31, CD45, CD2, CD11a, CD24, CD55, CD59, CF106, CD56, CD51, CD82, integrin, tetraspanin, annexin, HSP90, HSP70, syntenin 1, ADAM10, EHD4, actin, Rab5, clathrin, flotillin 1, MHC I, MHC II, actinin 4, GP96, EHD4, mitofilin, and LAMP2, and the second marker is selected from the group consisting of podocalyxin (PODXL), aquaporin 2 (AQP2), uroplakin 1b (UPK1b), podocin (NPHS2), glycophorin A (GYPA), mucin 1, type 2 Na-K-2Cl cotransporter (NKCC2), aquaporin 1 (AQP1), alpha-glutathione-S-transferase (alpha-GST), calbindin-D28K (CalD), megalin, cubilin, nephrin (Nphsl), claudin 1, annexin V, synaptopodin (Synpo), Wilms tumor protein (Wtl), band 3, stomatin (STOM), BGP1, globin, glycophorin B, Rh polypeptide, and Rh glycoprotein, and the complement protein is selected from the group consisting of C3, C5b-9, C4, Clq, and C9.

[0026] In one embodiment, the biological sample contains EVs derived from the renal system, and the second marker is a kidney-specific EV marker selected from the group consisting of podocalyxin (PODXL), aquaporin 2 (AQP2), uroplakin 1b (UPK1b), and podocin (NPHS2).

[0027] In one embodiment, the sample contains EVs derived from red blood cells (RBCs), and the second marker is an RBC-specific EV marker selected from glycophorin A (GYPA).

[0028] In one embodiment, the sample includes an EV that is negative for CD81 as a first marker, negative for uroplakin 1B (UPK1B) as a second marker, or negative for both CD81 as a first marker and UPK1B as a second marker.

[0029] In one embodiment, the capture marker and the detection marker are located within the same EV or its membrane-bound portion.

[0030] In one embodiment, the method further includes determining whether a subject has a complement disorder or is at risk of developing one, by comparing the presence or level of complement pathway components on the extravasation vessel (EV) or its membrane-bound portion with a control. In one embodiment, the control includes an equivalent sample from a healthy subject. In one embodiment, the method indicates that a subject has a complement disorder or is at risk of developing one if the level or presence of complement pathway components on the EV or its membrane-bound portion obtained from the subject is higher than that of the control, i.e., the level obtained from the subject is higher than that of a sample from a control subject not diagnosed with a complement disorder.

[0031] The present invention further provides a method for diagnosing or evaluating the prognosis of complement-related disorders in a subject, (a) Obtain a sample containing extracellular vesicles (EVs) or their membrane-bound portions derived from the subject, (b) Contacting a portion of the sample with at least one first capture antibody or its antigen-binding fragment to capture at least one first marker on the EV or its membrane-binding portion, (c) In some cases, a portion of the sample is brought into contact with at least one second capture antibody or its antigen-binding fragment to capture at least one second marker on the EV or its membrane-bound portion, (d) Contacting a captured EV or its membrane-bound portion, which includes at least one first marker and optionally at least one second marker, with at least one detection antibody or its antigen-binding fragment that is specific to a complement system-related component, (e) Qualitatively or quantitatively detect the detected antibody or its antigen-binding fragment to measure the presence or level of complement pathway components on the EV or its membrane-bound portion, and compared with the control, indicate that if the presence or level of complement pathway components in the sample is high, the subject is suffering from or at risk of developing a complement disorder. This provides a method that includes [something].

[0032] The present invention also provides a method for indicating whether a subject has or is at risk of having a complement disorder, comprising steps (a) to (e) discussed above.

[0033] In one embodiment, the first marker includes an EV-specific marker or a tissue-specific marker displayed on the EV. In one embodiment, the first marker includes an EV-specific marker, and the second marker includes a tissue-specific marker displayed on the EV.

[0034] The present invention further provides a method for monitoring the response of a subject to treatment of a complement-related disorder using a complement modulator, (a) Obtain samples containing extracellular vesicles (EVs) or their membrane-bound portions derived from the subject before and after treatment, (b) Contacting a portion of the sample with at least one first capture antibody or its antigen-binding fragment to capture at least one first marker on the EV or its membrane-binding portion, (c) In some cases, a portion of the sample is brought into contact with at least one second capture antibody or its antigen-binding fragment to capture at least one second marker on the EV or its membrane-bound portion, (d) Contacting a captured EV or its membrane-bound portion, which includes at least one first marker and optionally at least one second marker, with at least one detection antibody or its antigen-binding fragment that is specific to a complement system-related component, (e) Qualitatively or quantitatively detect the detected antibody or its antigen-binding fragment to measure the presence or level of complement pathway components on the EV or its membrane-bound portion, and if the presence or level of complement pathway components in the sample of the subject is reduced after treatment with the complement modulator compared to before treatment with the complement modulator, it indicates that the subject is responding to the complement modulator. This provides a method that includes [something].

[0035] In some embodiments, the complement modulator is a molecule listed in Table A. Preferably, the complement modulator is a molecule that modulates (e.g., increases or decreases, preferably decreases) the activity of a complement component selected from C1q, C1, C1s, C2, MASP-2, MASP-3, factor D, factor B, propergin (factor P), factor H, C3 / C5 convertase, C5, C5a / C5aR, C3a / C3aR, C6, and / or CD59. In particular, the complement modulator is a small molecule inhibitor of a complement component, or an siRNA / RNAi that targets a complement component, or an antibody that specifically binds to a complement component.

[0036] In one embodiment, the complement mediator is a complement 5 (C5) inhibitor, a complement 5a (C5a) inhibitor, a complement 5 receptor (C5R1) inhibitor, a complement 3 (C3) inhibitor, a factor D (FD) inhibitor, a factor H (FH) inhibitor, a factor B (FB) inhibitor, a MASP2 inhibitor, a MASP3 inhibitor, a properdin inhibitor, or a combination thereof.

[0037] In one embodiment, the disease is an inflammatory disease or a thrombotic disease. In one embodiment, the disease is a thrombotic hematological disease or a thrombotic renal disease. In one embodiment, the disease is a renal disease selected from the group consisting of atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy (C3G), dense deposit disease (DDD), membranoproliferative glomerulonephritis (MPGN), lupus nephritis (LN), IgA nephropathy (IN), lupus nephritis (LN), membranous nephropathy (MN), complications of hemodialysis in transplant patients, antibody-mediated rejection (AMR), and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). In one embodiment, the disease is a hematological disorder selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), secondary HUS due to parenchymal organ transplantation or hematopoietic stem cell transplantation, thrombotic microangiopathy (TMA), and cold agglutinin disease (CAD). In one embodiment, the disease is a neurological disorder selected from neuromyelitis optica spectrum disorder (NMOSD), generalized myasthenia gravis (gMG), amyotrophic lateral sclerosis (ALS), and primary progressive multiple sclerosis (PPMS).

[0038] In one embodiment, detection includes immunoassays (e.g., ELISA or RIA), electron microscopy (EM), tandem mass tagging (TMT), luminescence assays (e.g., LUMINEX), or immunofluorescence assays (FIA) (also known as immunofluorescence assays (IF)). In one embodiment, the detection step is performed in a multiplex format, i.e., detection is performed by measuring markers in several distinct tissues in a single sample, and / or by monitoring multiple promising complement proteins and complement pathways in a single assay.

[0039] The present invention further provides a method for detecting complement activation in target renal tissue, (a) A urine sample derived from a subject, including an extracellular vesicle (EV) or its membrane containing a first marker which is an EV-specific marker or tissue-specific marker displayed on the EV or its membrane, is brought into contact with a first capture antibody specific to the first marker or its antigen-binding fragment, thereby capturing the EV or membrane containing the first marker. (b) If applicable, the urine sample may be brought into contact with a second capture antibody or its antigen-binding fragment to capture an EV or its membrane-bound portion containing a second capture marker different from the first marker. (c) The presence or level of complement pathway components on the captured EV or its membrane-bound portion is detected qualitatively or quantitatively using an antibody or antigen-binding fragment specific to that component, thereby detecting complement activation in the urine sample. A method including, An EV-specific marker was selected from the group consisting of CD9, CD63, and CD81. Tissue-specific markers, (1) Podocalixin (PODXL) specific to podocytes in the glomerulus, (2) Aquaporin 2 (AQP2) specific to the epithelium of the convoluted tubules, (3) Uroplakin 1b (UPK1b) specific to the bladder epithelium, and (4) Glycophorin A (GYPA) specific to red blood cells (RBCs) Selected from the group consisting of, and The present invention provides a method for selecting components of the complement pathway from the group consisting of MAC, C3, C5b-9, C4, C1q, and C9.

[0040] In some embodiments, the present disclosure provides a method for screening test compounds for complement regulation, (a) Obtain samples containing extracellular vesicles (EVs) or their membrane-bound portions derived from subjects suffering from complement disorders (e.g., animals such as mice, rabbits, hamsters, sheep, llamas, dogs, monkeys, chimpanzees, or humans) before and after administration of the test compound to such subjects, (b) Contacting a portion of the sample with at least one first capture antibody or its antigen-binding fragment to capture at least one first marker on the EV or its membrane-binding portion, (c) In some cases, a portion of the sample is brought into contact with at least one second capture antibody or its antigen-binding fragment to capture at least one second marker on the EV or its membrane-bound portion, (d) Contacting the captured EV or its membrane-bound portion with at least one detection antibody or its antigen-binding fragment that is specific to a complement system-related component, (e) The presence or level of complement components on the EV or its membrane-bound portion is measured qualitatively or quantitatively by detecting the detected antibody or its antigen-binding fragment, and the adjustment of the presence or level of complement components in the sample after administration of the test compound (e.g., increase or decrease, preferably decrease) compared to before administration of the test compound indicates that the test compound can modulate complement. This includes methods.

[0041] In some embodiments, the test compound can specifically modulate complement components selected from Clq, Cl, Cls, C2, MASP-2, MASP-3, factor D, factor B, propergin (factor P), factor H, C3 / C5 convertase, C5, C5a / C5aR, C3a / C3aR, C6, and / or CD59. In some embodiments, the test compound is a monoclonal antibody, a small molecule, or siRNA / RNAi. In some embodiments, the modulating activity of the test compound is compared to the activity of a molecule having complement modulating activity (e.g., a positive control or standard substance), such as the molecules provided in Table A.

[0042] The present invention further prepares for the use of at least one first capture antibody to capture at least one first target, the use of at least one second capture antibody to capture at least one second target, and the use of at least one complement protein-specific detection antibody to detect the amount of at least one captured first target, the amount of at least one captured second target, or both.

[0043] Also provided are kits containing one or more antibodies that bind to biomarkers as described herein. In some embodiments of the kits described herein, the kit is an immunoassay, for example, an enzyme-linked immunosorbent assay. Any of the kits described herein can be used to carry out any of the methods described herein. In some embodiments, the kit may further include instructions for carrying out any of the methods described herein. Such a kit may also, in non-limiting examples, include one or more of the following: reagents useful for preparing a sample, reagents useful for concentrating extracellular vesicles, reagents useful for detecting the binding of a target protein or component in the sample to an immobilized antibody, a control sample containing a purified target protein / component, and / or instructions for use.

[0044] For example, a kit useful in the methods described herein may include one or more antibodies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) or fragments thereof that specifically bind to biomarkers as described herein. For example, one or more antibodies provided in this kit can be immobilized on a surface (e.g., in the form of an ELISA assay or a gene chip array).

[0045] The patent or application file shall contain at least one drawing in color. A copy of the patent or patent application accompanied by the color drawing shall be provided by the Patent Office upon application and payment of the required fees. [Brief explanation of the drawing]

[0046] [Figure 1A]Panels (A) and (B) in Figure 1 show the relative amounts of EV markers in urinary ExoQuick Enrichment by NTA. All antibodies are from Biolegend and are Ms-α-CD9=PE, clone HI9a, Ms-α-CD63=PE, clone H5C6, Ms-α-CD81=PE, and clone 5A6. Analysis was performed using a ZetaView PMX110 by Particle Metrix GmbH. [Figure 1B] Panels (A) and (B) in Figure 1 show the relative amounts of EV markers in urinary ExoQuick Enrichment by NTA. All antibodies are from Biolegend and are Ms-α-CD9=PE, clone HI9a, Ms-α-CD63=PE, clone H5C6, Ms-α-CD81=PE, and clone 5A6. Analysis was performed using a ZetaView PMX110 by Particle Metrix GmbH. [Figure 2] Figure 2 shows electron microscope images of urine EV (A) and non-EV particles (B). [Figure 3] Figure 3 shows the distribution of ferret diameter in electron microscope images of objects. [Figure 4] Figure 4 shows the relative amounts of the top 25 proteins according to PSM. [Figure 5] Figure 5 shows the detection of a partial set of urine EV by Luminex. CD9: clone MM2 / 57 (Southern Biotech), CD63: clone H5C6 (Biolegend), CD81: clone 1D6 (Abcam), Ms IgG: clone MG1-45 (Biolegend). [Figure 6] Figure 6 shows that renal PODXL is detected only in CD9+EV. (1): Rb-α-PODXL: USB catalog number 212672-biotin, (2): Rb-α-PODXL: LSBio catalog number LS-C141161. [Figure 7]Figure 7 shows that Luminex beads can identify glomerular-specific extracellular proteins (EVs). Rb-α-PODXL (USB212672) can be detected on EVs containing CD9 or CD40L, but not on EVs containing CD63 or CD81. Signals were observed using two different α-PODXL antibodies. This can only occur if both proteins have the same structure. [Figure 8] Figure 8 shows that nephron-specific EV levels increase with disease. PODXL+EV levels are higher in IgAN urine than in control urine. This increase is observed in both the CD9+ / PODXL+ and CD40+ / PODXL+EV populations. CD63+ and CD81+EV levels remain negative. [Figure 9] Panels (A) and (B) of Figure 9 show graphs demonstrating that Luminex beads can measure complement on EV membranes. C3c and C5b-9 are detected in the urine of LN patients on both CD9+ and PODXL+ beads, but not on CD63+ or CD81+. These results were confirmed in 10 LN, 6 IgAN, and 7 control samples (data not shown). Both LN and IgAN samples may, but are not limited to, have C3, C5b-9, C4, and C1q deposition on both PODXL+ and AQP2+ EVs compared to the control samples. [Figure 10-1] Panels (A), (B), and (C) of Figure 10 show graphs illustrating the reduction in glomerular C5b-9 deposition associated with ravulizumab treatment. Patients with aHUS may take any combination of C3, C5b-9, C1q, and C4 (not shown) which are deposited on the podocyte membrane. C3 levels remain unchanged during ravulizumab treatment. Levels of C5b-9 and C1q on the extracellular matrix (EV) decrease rapidly during ravulizumab treatment. [Figure 10-2]Panels (A), (B), and (C) of Figure 10 show graphs illustrating the reduction in glomerular C5b-9 deposition associated with ravulizumab treatment. Patients with aHUS may take any combination of C3, C5b-9, C1q, and C4 (not shown) which are deposited on the podocyte membrane. C3 levels remain unchanged during ravulizumab treatment. Levels of C5b-9 and C1q on the extracellular matrix (EV) decrease rapidly during ravulizumab treatment. [Figure 11] Figure 11 shows the components related to the complement pathway. [Figure 12] Figure 12 shows data from the analysis of EV concentrated urine samples using the bead fluorescence assay method. [Modes for carrying out the invention]

[0047] definition The term "approximately" means a range of plus or minus 10% of the value, for example, "approximately 5" means 4.5 to 5.5, unless the context of this disclosure otherwise indicates or is inconsistent with such interpretation. For example, in a list of numbers such as "approximately 49, approximately 50, approximately 55," "approximately 50" means a range not exceeding half the interval between the preceding and succeeding values, for example, greater than 49.5 and less than 52.5.

[0048] Where a range of values ​​is provided in this disclosure, each intermediate value between the upper and lower limits of that range, and any other stated or intermediate values ​​within that stated range, are intended to be included within this disclosure. For example, if a range of 1 mM to 8 mM is stated, then 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, and 7 mM are also intended to be expressly disclosed.

[0049] As used herein, the term “plural” may mean two, three, four, five, six, seven, eight, nine, ten, or more.

[0050] As used herein, the term “detection” refers to the process of determining one or more values ​​relating to a sample by measuring one or more parameters in the sample, and may further include comparing the test sample to a reference sample. In accordance with this disclosure, the detection of complement markers includes identifying, assaying, measuring, and / or quantifying one or more markers.

[0051] The term “extracellular vesicles” refers to lipid-based microparticles or nanoparticles, or high-protein aggregates, present in a sample obtained from an object (e.g., a biological fluid). Extracellular vesicles are also referred to as exosomes, microvesicles, or nanovesicles in the art and herein. In this disclosure, extracellular vesicles are between approximately 30 nm and 1000 nm in diameter. Extracellular vesicles are secreted or granulated from a variety of different mammalian cell types. Non-limiting examples of extracellular vesicles and non-limiting examples of methods for enriching extracellular vesicles from a sample obtained from a mammalian object (e.g., a biological fluid) are described herein. Additional examples of extracellular vesicles and additional examples of methods for enriching extracellular vesicles from a sample obtained from a mammalian object are known in the art.

[0052] The term "membrane binding" refers to any structure that includes the outer covering of cells and organelles that form a biological membrane, i.e., a semipermeable shield. This term typically refers to structures containing phospholipids and proteins that originate from the outer cell membrane, or from organelles such as the Golgi apparatus, endoplasmic reticulum, nucleus, or mitochondria.

[0053] As used herein, the term “membrane protein” refers to a protein that interacts with, or is part of, a portion of the biological membrane of an extracellular organism (EV). Membrane proteins may include, but are not limited to, endogenous and superficial membrane proteins.

[0054] As used herein, the term “disease-specific membrane protein” refers to a membrane protein associated with a specific disease, such as a complement disorder like aHUS. These disease-specific membrane proteins may individually encode a disease, or a group of disease-specific membrane proteins may encode a disease.

[0055] The terms “sample” or “biological sample” mean any biological fluid obtained from a mammalian subject (e.g., blood, plasma, serum or other blood fractions, lymph, urine, cerebrospinal fluid, ascites, saliva, breast milk, tears, vaginal secretions, amniotic fluid, lavage fluid, semen, glandular secretions, exudate, cyst or fecal contents). In a preferred embodiment, the sample includes blood, serum, or plasma.

[0056] As used herein, the term “antibody” means an antibody having a high binding affinity to an antigen, such as a complement protein, or a functional portion or fragment thereof. The term is used in its broadest sense and includes intact antibodies and polyclonal and monoclonal antibodies containing functional (antigen-binding) antibody fragments, including single-chain variable fragments (scFv) and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments, fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, and single-chain antibody fragments. The term encompasses any class or subclass of natural antibodies, genetically engineered antibodies, and / or other modified antibodies, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0057] The term "antigen" refers to any molecule, such as a protein or a fragment thereof, that can specifically bind to an antibody or its antigen-binding fragment.

[0058] The term "antigen fragment" refers to a portion of an antigen that can be recognized by an antigen-specific antibody.

[0059] "Beads" refer to particles on which the desired capture antibody is immobilized. These beads are generally uniform in size within a single filtration matrix, but may vary in size between different filtration matrices, ranging from approximately 1 nm to approximately 10,000 nm. A preferred shape is spherical, but any other particle shape can be used, as this parameter is not important to the nature of the invention.

[0060] A "strip" refers to an elongated, flat element on which the desired capture beads or the desired capture antibody are immobilized. Strips are generally thin films of uniform size, but their size and color may vary depending on the amount and type of capture antibody being immobilized.

[0061] The term "multiplex" refers to the detection of multiple markers across a single sample, and / or the detection of at least one marker across multiple samples.

[0062] As used herein, the term “multiplex bead array platform” refers to a platform that utilizes either identifiable particles or microparticles. Such identifiable particles may be used, for example, to perform multiplex immunoassays or molecular probe assays. A representative example is Luminex® xMAP® Technology.

[0063] As used herein, the term “classification dye” refers to a mixture or combination of microparticles or beads used in a multiplex assay, having a mixture of classification dyes that enable the instrument to sort and classify the particles.

[0064] The term "reporter molecule" includes, but is not limited to, any fluorescent tag that binds to a detection molecule in an assay. In the case of an immunoassay designed to measure human antibodies, the detection molecule could be, for example, phycoerythrin-labeled goat anti-human IgG.

[0065] A concise summary of the biological activities related to complement activation is provided, for example, in The Merck Manual, 16th Edition.

[0066] As used herein, “subject” can be any mammal. Subjects may be, for example, humans, non-human primates (e.g., monkeys, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, or mice. This includes transgenic or genetically modified (e.g., knock-out or knock-in) animals.

[0067] As used herein, “subjects requiring prevention,” “subjects requiring treatment,” or “subjects requiring treatment” refer to any given treatment for a complement disorder or complement disorder, i.e., any treatment that would reasonably benefit the appropriate healthcare professional (e.g., a physician, nurse, or, in the case of humans, a nursing practitioner; in the case of non-human mammals, a veterinarian), i.e., any treatment that would reasonably benefit the individual from a particular therapeutic agent.

[0068] "Complement components" or "complement proteins" are molecules involved in the activation of the complement system or participating in one or more complement-mediated activities. Components of the conventional complement pathway include, for example, the C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, C9, and C5b-9 complexes, also known as membrane attack complexes (MACs) and any of the aforementioned active fragments or enzymatic cleavage products (e.g., C3a, C3b, C4a, C4b, C5a, etc.). Components of alternative pathways include, for example, factor B, factor D, factor H, and factor I, as well as propergine, with factors H and I being negative regulators of these pathways. Components of the lectin pathway include, for example, MBL2, MASP-1, and MASP-2. Complement components also include cell-bound receptors for soluble complement components. Such receptors include, for example, C5a receptors (C5aR1 and C5aR2), C3a receptors (C3aR), complement receptor 1 (CR1), complement receptor 2 (CR2), and complement receptor 3 (CR3). It should be understood that the term “complement components” is not intended to include these molecules and molecular structures that function as “triggers” for complement activation, such as antigen-antibody complexes, or exogenous structures found on microbial or artificial surfaces. The term includes, but is not limited to, any of the complement regulatory proteins (e.g., factor B, factor D, factor P, factor H, factor I, CD46, CD55, and CD59).

[0069] As used herein, “to treat” means to provide treatment, i.e., to provide any kind of medical or surgical management of a subject. This treatment may be provided to reverse, alleviate, inhibit, prevent or reduce the progression of a disorder or condition, or to reverse, alleviate, inhibit or prevent the progression of one or more symptoms or symptom onset of a disorder or condition, or to prevent or reduce the probability of one or more symptoms or symptom onset of a disorder or condition. “To prevent” means to prevent such disorder or condition, or symptoms or symptom onset, from occurring in at least some individuals for at least a period of time. Treatment may involve administering a therapeutic agent / complement modulator to a subject after the onset of one or more symptoms or symptom onset of a complement condition, for example, to reverse, alleviate, or reduce the severity of the condition, and / or inhibit or prevent the progression of the condition, and / or reverse, alleviate, or reduce the severity of the condition, and / or inhibit one or more symptoms or symptom onset of the condition. According to the methods described herein, compositions / complement modulators can be administered to subjects who have developed a complement disorder or complement condition, or who are at high risk of developing such disorder compared to members of the general population. Such compositions / modulators can be administered prophylactically, i.e., before the onset of any symptom or symptom onset of the condition. Typically, in this case, the subject would be at risk of developing the condition if exposed to viral particles used in gene therapy or therapeutics delivered by complement-activating compositions, e.g., particles or nanoparticles containing therapeutic agents, e.g., lipid nanoparticles.

[0070] The “effective dose” of an active agonist, such as a therapeutic agent or complement modulator, refers to the amount of the active agonist sufficient to elicit a desired biological response (or, equivalently, inhibit an undesirable biological response). The absolute amount of a particular agonist that is effective may vary depending on factors such as the desired biological endpoint, the agonist being delivered, and the target tissue. The “effective dose” may be administered as a single dose or achieved through multiple doses. For example, an effective dose of a therapeutic agent may be sufficient to alleviate at least one symptom of a disorder. An effective dose may be sufficient to slow the progression of a chronic and progressive disorder, for example, to extend the time before one or more symptoms or signs of the disorder manifest themselves, or to extend the time before an individual with the disorder reaches a certain level of deterioration. An effective dose may be sufficient to allow for a more rapid or greater recovery from damage that would occur in the absence of the agonist.

[0071] As used herein, the term “diagnosis” means a method by which a subject can determine whether a given disease or condition, including but not limited to complement disorders, is likely to be present. Those skilled in the art will often make diagnoses based on one or more diagnostic indicators, such as markers, whose presence, absence, quantity, or change in quantity indicates the presence, severity, or absence of a disease or condition. Other diagnostic indicators may include a patient’s medical history, physical symptoms, such as a biological event or phenotype, genotype, or unknown changes in environmental or genetic factors. Those skilled in the art will understand that “diagnosis” means a high probability that a particular course or outcome will occur, i.e., a high probability that the course or outcome will occur in a patient exhibiting a given characteristic, such as the presence or level of a diagnostic indicator, compared to an individual not exhibiting that characteristic. The diagnostic methods of this disclosure can be used independently or in combination with other diagnostic methods to determine whether a course or outcome is more likely to occur in a patient exhibiting a given characteristic.

[0072] As used herein, the term "certainty" generally refers to probability, relative probability, existence or non-existence, or degree.

[0073] As used herein, the term "marker" refers to a feature that can be objectively measured as an indicator of a normal biological process, a pathogenic process, or a pharmacological response to a therapeutic intervention, such as treatment with a complement inhibitor. Typical types of markers include molecular-level changes in the structure (e.g., sequence or length) or number of markers, including changes in the level, concentration, activity, or properties of the marker.

[0074] The term “control,” as used herein, refers to a reference to a test sample, such as a control extracellular viable (EV) isolated from healthy cells and similar samples. “Reference sample,” as used herein, refers to a sample of tissue or cells, which may or may not be disease-containing, used for comparison. Thus, the “reference” sample provides a baseline from which another sample, such as a urine sample containing EVs, can be compared. In contrast, “test sample” refers to a sample compared to a reference sample. The reference sample does not need to be disease-free, for example, when the reference sample and the test sample are obtained by separating them over time from the same patient.

[0075] The term "level" can refer to binary (e.g., absent / present), qualitative (e.g., absent / low / medium / high), or quantitative information (e.g., a value proportional to number, frequency, or concentration) indicating the presence of a particular molecular species.

[0076] The term “substantially” means sufficient to function for the intended purpose. Therefore, the term “substantially” allows for slight, non-significant changes from the absolute or perfect state, dimensions, measurements, results, or similar, which would be expected by a person skilled in the art, but which do not significantly affect the overall performance (e.g., ±10%).

[0077] The term "complementary" disorder or "complementary" disease refers to a disorder that involves complement activation that overrides the subject's self-protective mechanisms (e.g., self-protective proteins, including CD55 (degradation-accelerating factor), CD59 (protectin), factor H, and similar factors) and causes damage to the subject's cells and / or tissues.

[0078] As used herein, the term “at risk” with respect to a disease or disorder refers to an individual (e.g., a human) who is susceptible to a particular disease. This susceptibility may be genetic (or due to other factors, such as environmental conditions, hypertension, activity levels, metabolic syndrome, etc.). Therefore, this disclosure is not intended to be limited to any particular risk, nor is the invention intended to be limited to any particular type of complement disorder or dysfunction (e.g., aHUS).

[0079] This specification provides methods for using extracellular vesicles derived from non-invasive fluid biopsy protocols as a non-invasive, highly sensitive, and specific test for diagnosing and / or monitoring treatment response in patients with various complement-related disorders. Semi-quantitative methods for monitoring surface complement expression on EVs before, during, and after therapeutic intervention are described, using immunoprecipitation / immunoanalysis to isolate and analyze EV surface markers. These methods can effectively utilize EVs for ex vivo monitoring of complement deposition as a surrogate for in vivo activity. Thus, using these methods, researchers and physicians can directly monitor complement attack in distinct, identifiable tissues, such as multiple regions of the kidney, throughout the course of treatment, using the tools.

[0080] Biopsy is the current standard for differential diagnosis of a subset of complement-related disorders. However, the risk of severe complications limits patient selection and the frequency of testing. Extracellular vesicles provide an easily accessible opportunity to monitor ongoing complement deposition in specific tissues before and during treatment. Extracellular vesicles can also provide precise cellular-level identification of complement attack in tissues. Any cell type or tissue of interest with distinctive PM markers may be used to examine complement deposition.

[0081] Immunoprecipitation / Immunoanalysis The immunoprecipitation / immunoanalysis of this disclosure uses a bead-based immunocapture protocol with immunofluorescence detection that can accurately identify specific tissues under complement attack and monitor treatment response. This technique can be broadly applied to monitor any organ or tissue under complement attack in any liquid matrix given the correct set of antibody tools. One aspect of the method discussed herein is a combination of EV enrichment and immunocapture of tissue-specific biomarkers present on the surface of detached EVs. The captured biomarker may be either a canonical EV-specific protein (e.g., CD9, CD63, CD81) or a tissue-specific protein. The detection antibody is specific to complement components such as C5b-9, C3, C4, C1q, and C9. Examples of tissue-specific targets in urine include podocalixin (PODXL), which is specific to podocytes in the glomerulus; aquaporin 2 (AQP2), which is specific to the convoluted tubular epithelium; or uroplakin 1b (UPK1b), which is specific to the bladder epithelium. A further aspect is that a positive signal may only be generated when the capture target and the detection target are located on the same structure.

[0082] For example, renal biopsy is the current standard for differential diagnosis of chronic kidney disease. However, renal biopsy is an invasive procedure. However, urinary extracellular vesicles (uEVs) are a source of complexes consisting of vesicles and biomarkers, originating from all cell types along the renal system, including all parts of the nephron. For example, PODXL is produced only on podocytes in the glomerulus, AQP2 originates from the proximal and distal convoluted tubules, and RBC-derived glycophorin A (GYPA) can be used to measure EV leakage from plasma to filtrate. Certain pathological conditions, such as inflammation or malignancy, increase the number of uEVs that are undeleted by cells. Along with canonical EV markers, uEVs carry plasma membrane-bound proteins derived from parent cells, which may provide novel insights into the "health status" of the renal system.

[0083] In the Examples section and elsewhere, representative types of antibodies useful for performing various embodiments of this disclosure are provided, for example, along with information on specific vendors and / or catalog numbers. It should be understood that this disclosure is not limited to exemplary embodiments utilizing antibody detection reagents from specific vendors / manufacturers. Antibodies against biomarkers / analytes of this disclosure are available from Biolegend (San Diego, CA), Southern Biotech (Birmingham, AL), and the United States. Products can be obtained from any of the following manufacturers: Biological (USB) (Salem, MA), Lifespan Biosciences (LSBIO) (Seattle, WA), Abcam (Cambridge, United Kingdom), Cell Signaling Technology (Danvers, MA), and Sigma-Aldrich (St. Louis, MO). For example, rabbit anti-PODXL antibodies can be purchased from USB (catalog no. 212672), LSBIO (catalog no. LS-C141161), Abcam (catalog no. ab205350), and Sigma-Aldrich (catalog no. HPA002110), and the anti-CD9 antibody clone MM2 / 57 can be purchased from Southern Biotech (catalog no. 9310), EMD Millipore (catalog no. CBL162), VWR (catalog no. 89366), and BIO RAD (catalog no. MCA469G). Antibodies can also be produced using conventional techniques, such as immunization and / or hybridoma techniques of mammals, such as mice or rabbits.

[0084] Characterization of EVs Extracellular vesicles (EVs), such as urinary extracellular vesicles (uEVs), can be characterized by simultaneous immunoprecipitation / immunoanalysis, i.e., by the Luminex® xMAP® Technology platform, which can provide query signals to determine the surface phenotype and tissue origin of a subset of EVs.

[0085] Luminex Corporation® manufactures instruments and xMAP® technology, which combines immunoprecipitation with multiplex immunoassays. xMAP® is a proprietary series of color-coded microspheres that can be coated with capture antibodies. The open architecture of xMAP® technology enables multiplexing of biological assays, reducing time, effort, and cost compared to conventional methods such as ELISA, Western blotting, PCR, and conventional arrays. Systems using xMAP® technology perform separate assays on the surface of color-coded beads known as microspheres, and then read the results in a compact analyzer. Using multiple lasers or LEDs and a high-speed digital signal processor, the analyzer reads the multiplex assay results by reporting the reactions occurring on each individual microsphere.

[0086] The Luminex platform allows for extravasation of extravasation (EV) by immunoprecipitation before immunoassay testing. This eliminates the need for initial sample preparation / EV enrichment and thus limits potential erroneous results.

[0087] Using a multi-well format allows for the monitoring of several individual tissues on a single sample. Working with a multi-well plate format enables the monitoring of multiple potential complement proteins and multiple potential complement proteins and complement pathways in a single assay.

[0088] Generally, capture beads are bound to target-specific antibodies. The bound beads are used to immunoprecipitate target proteins from a matrix (e.g., from liquid samples such as urine). Detection antibodies bound to labels such as phycoerythrin (PE) or biotin + streptavidin-phycoerythrin (SAPE) are used to detect and quantify the bead-captured targets during analysis.

[0089] Arrays of xMAP beads coated with antibodies against canonical vesicle markers such as CD9, CD63, and CD81 are used to concentrate distinct EV partial sets from biological samples, such as urine. Each bead set is then analyzed for the presence of other biomarkers that define the phenotype of the partial set and link it to tissue origin, and then further analyzed for the presence of complement pathway proteins. In this way, non-invasive “liquid biopsy” methods are constructed to sample and monitor key biomarkers of disease in specific tissues (such as the kidney) for differential diagnosis, prognosis, and / or longitudinal monitoring of response to treatment.

[0090] Binding of target protein The binding of target proteins to antibodies immobilized in solution or on arrays can be detected using detection techniques known in the art. Examples of such techniques include immunological techniques such as competitive binding assays and sandwich assays; fluorescence detection using instruments such as confocal scanners, confocal microscopes, or CCD systems; fluorescence, fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), total internal reflection fluorescence (TIRF), and fluorescence-corrected spectroscopy (FCS); colorimetric / spectroscopy techniques; surface plasmon resonance (SPL) which can measure changes in the mass of materials adsorbed on a surface; techniques using radioisotopes, including conventional radioisotope binding and scintillation proximity assays (SPAs); and liquid chromatography-fluorescence chromatography. Mass spectrometry methods include LC-MS, HPLC-MS, matrix-assisted laser desorption / ionization mass spectrometry (MALDI), and MALDI time-of-flight (TOF) mass spectrometry; polarization analysis, an optical method for measuring the thickness of protein films; quartz microbalance (QCM), a highly sensitive method for measuring the mass of materials adsorbed on a surface; scanning probe microscopy, such as atomic spectroscopy (AFM) and scanning electron microscope (SEM); and electrochemical detection, impedance detection, acoustic detection, microwave detection, and infrared (IR) / Raman detection.

[0091] Measurement of complement inhibition / modulation Any suitable method can be used to evaluate the ability of an action factor or composition containing an action factor to inhibit complement activation (or any other related property). Several in vitro assays can be used. The ability of an action factor to inhibit conventional or alternative complement pathways can be evaluated, for example, by measuring complement-mediated hemolysis of red blood cells (e.g., antibody-sensitive or insensitive rabbit or sheep red blood cells) with or without the action factor using human serum or a set of complement components. The ability of an action factor to bind to one or more complement components, such as C3, C5, C6, C7, C8, C9, factor B, or factor D, can be evaluated, for example, using isothermal titration calorimetry or other methods suitable for use in liquid phase. The ability of an action factor to bind to complement components can be measured, for example, using an ELISA assay. Other uses include surface plasmon resonance and equilibrium dialysis.

[0092] Methods for measuring systemic or local complement activation occurring in vitro or in vivo, and methods for determining the ability of complement inhibitors to inhibit such activity, are known in the art. Measurement of complement activating products such as C3a, C5a, C3bBb, and C5b-9 provides, for example, an indication of the degree of complement activation. A decrease in the amount of such products indicates inhibition of complement activation. In some embodiments, the ratio of an active cleavage product to its inactive desarginine (desArg) form is measured (e.g., C3a / C3adesArg). Those skilled in the art can distinguish between conventional pathways, alternative pathways, and lectin pathways by appropriate selection of the complement activating product and / or appropriate complement activators to be measured, such as dimosan, lipopolysaccharides, and immune complexes. Other methods include measuring complement hemolysis of erythrocytes as a result of terminal complex formation.

[0093] In vivo complement activation and / or inhibition by complement inhibitors can be measured in appropriate biological samples. Systemic complement activation and / or inhibition by complement inhibitors can be measured, for example, in blood samples. Continuous measurements initiated before administration of complement inhibitors provide an indication of the degree to which complement inhibitors inhibit complement activation, as well as the time course and duration of the inhibition. It should be recognized that the decrease in activators may only become apparent when activators present before administration of complement inhibitors are broken down or removed.

[0094] In some embodiments, the complement regulators described herein may be formulated with additional active agents useful for treating or managing complement disorders in a subject. Additional agents for treating complement disorders in a subject include, but are not limited to, antihypertensive agents (e.g., angiotensin-converting enzyme inhibitors), anticoagulants, corticosteroids (e.g., prednisone), or immunosuppressants (e.g., vincristine or cyclosporine A), anticoagulants (e.g., warfarin (coumadin), heparin, phenindione, fondaparinux, hydraparinux), thrombin inhibitors (e.g., argatroban, repiridine, bivalirudine, or dabigatran), fibrinolytic agents (e.g., Anklod, ε-aminocaproic acid, anti-plasmin agent a1, prostacyclin, and defibrotide), lipid-lowering agents, or anti-CD20 agents such as rituximab.

[0095] Comparative method In some embodiments described herein, the method includes comparing the detected level of a complement biomarker to a reference level. In some embodiments, the reference represents the level of the biomarker in a healthy control, i.e., a subject not diagnosed with a complement disorder. In some embodiments, the reference level is the median or cutoff level in a reference cohort, e.g., a cutoff value that defines a statistically significant different group, e.g., an upper or lower ternary, quartile, quintile, or other percentile in the reference cohort.

[0096] Depending on the identity of the detected protein biomarker, levels above or below the reference level may indicate the presence or high risk of disease; that is, high levels (i.e., levels above the reference) or low levels (i.e., levels below the reference) may indicate the presence or reduced risk of disease.

[0097] In some embodiments, levels higher than the reference level are statistically significant or 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% higher. An increase can be determined, as described herein, by comparison with 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 a reference protein in a reference subject (e.g., a healthy reference)). In some embodiments, a level lower than the reference level is statistically significant or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% lower. A decrease can be determined, as described herein, by comparison with 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 a protein in a reference subject (e.g., a healthy reference or a subject without complementar disease)).

[0098] In some embodiments, the method includes calculating the ratio of the level of a protein biomarker in a sample to a reference level, and determining that if this ratio is greater than a threshold ratio, the subject is suffering from or at risk of developing a complement disorder as described herein. In some embodiments, it is determined whether the ratio is positive or negative, and the presence of a positive or negative ratio indicates that the subject is suffering from or at risk of developing a complement disorder as described herein. It can be readily determined from this disclosure whether a positive or negative ratio indicates the presence of a disorder, or a high or low risk.

[0099] The complement system consists of several small proteins organized into a biochemical cascade that functions to support the immune system in eliminating pathogens. Complement proteins circulate in the blood as inactive precursors. When stimulated by one of several triggers, proteases in this system cleave specific proteins, releasing cytokines and initiating the amplification of a further cleavage cascade.

[0100] Measurement of complement activation Methods for measuring systemic or local complement activation occurring in vitro or in vivo are known in the art. Measurement of complement activating products such as C3a, C5a, C3bBb, and C5b-9 provides, for example, an indication of the degree of complement activation. A decrease in the amount of such products indicates inhibition of complement activation. In some embodiments, the ratio of an active cleavage product to its inactive desArg form is measured (e.g., C3a / C3a desArg). Those skilled in the art can distinguish between conventional pathways, alternative pathways, and lectin pathways by appropriate selection of the complement activating product and / or appropriate complement activator to be measured, such as dimosan, lipopolysaccharide, or immune complex. Other methods include measuring complement hemolysis of erythrocytes as a result of terminal complex (MAC) formation.

[0101] Several different animal models possessing pathological features that mimic one or more features of complement responses are known in the art. The application of complement modulators for the treatment of complement disorders can be performed in various doses on mice, rats, dogs, primates, etc., that exhibit the disorder or whose disorder has been experimentally induced by subjecting them to appropriate protocols. The ability of modulators to prevent or treat one or more signs or symptoms of this disorder is evaluated using standard methods and criteria.

[0102] Compounds or complement modulators that show promising results in animal studies, such as acceptable safety and the feasibility of administering doses expected to effectively treat complement disorders at relevant extravesicular sites in human subjects, may be tested in humans, for example, using standard protocols and endpoints for clinical trials of therapies for specific disorders under investigation.

[0103] The compositions described above are particularly useful in methods for treating or preventing various complement-related disorders in subjects. These compositions can be administered to subjects, such as human subjects, by various methods that are partially dependent on the route of administration. These routes may be, for example, intravenous injection or intravenous infusion (IV), subcutaneous injection (SC), intraperitoneal injection (IP), or intramuscular injection (IM).

[0104] Administration can be achieved, for example, by local injection, injection, or implantation. Implants may be made of porous, non-porous, or gelatinous materials, including membranes such as silastic membranes, or fibers. Implants may be configured for sustained or periodic release of the composition to a target (U.S. Patent Publication No. 20080241223, U.S. Patent No. 5,501,856, No. 4,863,457, and No. 3,710,795, European Patent No. 488401, and European Patent No. 430539; each disclosure in its entirety is incorporated herein by reference). The composition can be delivered to a target by implantable devices based on dispersive, corrosive, or convective systems, such as osmotic pumps, biodegradable implants, electrodispersive systems, electroosmotic systems, vapor pressure pumps, electrolytic pumps, foaming pumps, piezoelectric pumps, corrosive systems, or electromechanical systems.

[0105] In some embodiments, the therapeutic agent is delivered to the target by topical administration. As used herein, “topical administration” or “topical delivery” refers to delivery that does not rely on transporting the composition or active ingredient to its intended target tissue or site via the circulatory system. The composition may be delivered, for example, by injection or implantation of the composition or active ingredient, or by injection or implantation of a device containing the composition or active ingredient. Following topical administration in the vicinity of the target tissue or site, the composition or active ingredient, or one or more of its components, may diffuse to the intended target tissue or site.

[0106] As outlined in detail in the Examples section, the assay method of this disclosure involves measuring changes in the expression or levels of complement components in extracellular fluid (EV). EV can be derived from any biological sample, such as urine, blood, lymph, cerebrospinal fluid, ascites, pus, pleural fluid, hemoglobin, milk, amniotic fluid, synovial fluid, mucus, saliva, sputum, aqueous humor, vitreous humor, or similar substances.

[0107] In some embodiments, approaches such as differential ultracentrifugation, density gradient ultracentrifugation, size exclusion chromatography, ultrafiltration, and affinity / immunoaffinity capture methods may be used to concentrate extravasation (EVs) from a biological sample, but this step is optional. Preferably, the EV concentration step is performed before the first marker and optionally the second marker come into contact with the individual antibodies or antigen-binding fragments.

[0108] Next, extraparticles (EVs) are characterized at the collective or single-particle level. Here, the composition and levels of molecules within the EV, such as proteins, lipids, or nucleic acids, are analyzed. Techniques range from light scattering microscopy or spectroscopy to molecular fingerprinting using proteomics. The overall level of unique molecules can also be measured within a collective. For single-particle analysis, specialized methods are used, including optical microscopy and flow cytometry (for EVs > 200 nm), single-particle interference reflection imaging (> 40 nm), nanoflow cytometry (approximately 40 nm), and electron microscopy. In particular, electron microscopy and flow cytometry allow for the testing of individual EVs without special attention prior to separation from the biological matrix.

[0109] In some embodiments, EV can be dissolved in a lysis buffer, for example, RIPA buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg / ml leupeptin).

[0110] Characterization of extravasation cells (EVs) may involve the use of capture antibodies that specifically bind to markers on the EV (e.g., typically proteins or peptides, but may include other antigens). In some embodiments, a single capture antibody specific to the EV marker is used. In some embodiments, at least two capture antibodies are used, in which the first capture antibody is specific to the EV-specific marker and the second capture antibody is specific to the tissue-specific marker displayed on the EV.

[0111] EV-specific markers include ALIX (UNIPROT:Q8WUM4), TSG101 (UNIPROT:Q99816), CD9 (UNIPROT:P21926), CD63 (UNIPROT:P08962), CD81 (UNIPROT:P60033), CD40L (UNIPROT:P29965), CD26 (UNIPROT:P27487), and CD31 (UNIP ROT:P16284), CD45(UNIPROT:P08575), CD2(UNIPROT:P06729, Q53F96), CD11a(UNIPROT:P20701), CD24 (UNIPROT:P25063), CD55(UNIPROT:P08174), CD59(UNIPROT:P13987, Q6FHM9), CF106(UNIPROT:Q9H6K1) CD56 (UNIPROT:P13591), CD51 (UNIPROT:P06756), CD82 (UNIPROT:P27701), integrin, tetraspanin, annexin, HSP90 (UNIPROT:P07900 (α1), Q14568 (α2), P14625 (β)), HSP70 (e.g., UNIPROT:P11021), syntenin 1 (UNIPROT This list includes, but is not limited to, fragments of, :O00560), ADAM10 (UNIPROT:O14672), EHD4 (UNIPROT:Q9H223), actin, Rab5 (UNIPROT:P20339(α), P61020(β), P51148(γ)), ​​clathrin (UNIPROT:P09496(α), P09497(β), Q00610(H)), flotinin 1 (UNIPROT:O75955), MHC I, MHC II, actinin 4 (UNIPROT:O43707), GP96 (UNIPROT:P14625), EHD4 (UNIPROT:Q9H223), mitophilin (UNIPROT:Q16891), and LAMP2 (UNIPROT:P13473).

[0112] Regarding tissue specificity, extracellular viable cells (EVs) specific to renal glomerular podocytes, convoluted tubules, or bladder epithelium are useful in the study of nephrological diseases, while erythrocyte (RBC)-derived EVs are useful in the study of hematological diseases. In some embodiments, tissue-specific EVs include, but are not limited to, the following: PODXL (UNIPROT:O00592) is highly expressed in glomerular podocytes, epithelial cells, and glandular cells in fallopian ducts, the uterus, and spermatovesic vesicles. AQP2 (UNIPROT:P41181) is found in the apical cell membrane of principal cells in the collecting duct of the kidney, and in intracellular vesicles. UPK1b (UNIPROT:O75841) is found in the asymmetrical bilayer of the bladder. NPHS2 (UNIPROT:Q9NP85) is expressed in podocytes of the renal glomeruli in both fetuses and adults. GYPA (UNIPROT:P02724) is a major intrinsic membrane protein of red blood cells that is specifically recognized by the monoclonal antibody TER119. Mucin 1 (UNIPROT: P15941, Q7Z551) is expressed particularly on the apical surface of epithelial cells in the airways, breast, and uterus, while the latter is expressed in T cells. It is overexpressed in epithelial tumors such as breast cancer and ovarian cancer, as well as in non-epithelial tumor cells. NKCC2 (UNIPROT:Q13621) is a kidney-specific renal Na, K, and Cl cotransporter. AQP1 (UNIPROT:P29972) is expressed in the plasma membrane of red blood cells and the proximal tubules of the kidney. Examples of GST-alpha include GSTα1 (UNIPROT:P08263), which is mainly expressed in the small intestine, large intestine, and colon and is weakly expressed in lymphocytes; and GSTα2 (UNIPROT:P09210), GSTα3 (UNIPROT:Q16772), GSTα4 (UNIPROT:O15217), and GSTα5 (UNIPROT:Q7RTV2), which are expressed at high levels in the brain, placenta, and skeletal muscle. THP (UNIPROT:P07911) is expressed in renal tubular cells, particularly near the epithelial cells of the thick ascending limb of the loop of Henle and the distal tubular lumen. Calbindin-D28K (CalB1) (UNIPROT:P05937) is found in mammalian kidneys and is expressed in several neuronal and endocrine cells, particularly the cerebellum. Megalin (UNIPROT:P98164) is a multiligand-binding receptor found in the plasma membrane of many absorptive epithelial cells. Cubilin (CUBN) (UNIPROT:O60494) is expressed in the kidneys and small intestine. Nephrin (Nphs1) (UNIPROT:O60500) is expressed in podocytes of the renal glomeruli. Claudin 1 (CLDN1) (UNIPROT:O95832) is strongly expressed in the liver and kidneys, and also in the heart, brain, spleen, lungs, and testes. Annexin V (ANXA5) (UNIPROT:P08758) is expressed in many tissues and blood cells. Synaptopodin (Synpo, Q8N3V7) is expressed in neurons and the cerebral cortex. Wilm's tumor protein (Wt1, P19544) is expressed in the kidney and in a subset of hematopoietic cells. The anion transport protein band 3 (SLC4A1, P02730) is expressed in red blood cells (PMID:7506871, PMID:26542571), while isoform 2 is expressed in the kidney (PMID:7506871). Stomatin (STOM, P27105) is detected and widely expressed in red blood cells. Carcinoembryonic antigen-associated cell adhesion molecule 1 (BGP1, P13688) is expressed in colonic columnar epithelial cells (PMID: 10436421), T cells (PMID: 18424730), granulocytes, and lymphocytes. Globins (e.g., cytoglobin (CYGB), Q8WWM9) are expressed in the heart, stomach, bladder, and small intestine. Glycophorin B (GYPB, P06028) is expressed in the endothelium and epithelium of the kidney. Rh polypeptide and Rh glycoprotein (RHAG, Q02094) are expressed in red blood cells.

[0113] Next, the presence or level of complement system-related components on the captured EV is detected. Either method may be used, for example, in the detection of complement components using a detection antibody or its antigen-binding fragment that is specific to the complement component, and aptamers may also be used. Examples of detection methods include, for example, immunohistochemical staining, Western blotting, intracellular Western blotting, immunofluorescence staining, ELISA, RIA, and fluorescent cell sorting (FACS), or any method known in the art.

[0114] Generally, there are two strategies used for detecting epitopes on antigens: direct and indirect. Direct methods involve one-step staining and may include labeled antibodies (e.g., FITC-conjugated antibodies) that react directly with the antigen on / inside the EV. Indirect methods involve an unlabeled primary antibody that reacts with the antigen in a body fluid or tissue, and a labeled secondary antibody that reacts with this primary antibody. Labeling can be radiolabeling, fluorescent labeling, hapten labeling such as biotin, or enzymes such as horseradish peroxidase or alkaline phosphatase. Methods for carrying out these assays are well known in the art. For example, see Harlow et al. (Antibodies, Cold Spring Harbor Laboratory, NY, 1988), Harlow et al. (Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, NY, 1999), Virella (Medical Immunology, 6th edition, Informa HealthCare, New York, 2007), and Diamandis et al. (Immunoassays, Academic Press, Inc., New York, 1996). Kits for performing these assays are commercially available, for example, from Clontech.

[0115] A wide range of complement proteins can be detected using this method, including, for example, (a) components of the alternative pathway (AP), (b) components related to the conventional pathway (CP), and (c) components related to the lectin pathway (MBL). In some embodiments, multiple components from different pathways, such as components from AP and CP, can be detected.

[0116] In a preferred embodiment, the complement system-related components are components of AP or CP selected from, for example, C3, C5b-9, C4, C1q, C9, C3b, iC3b, TF, CRP, pCRP, MAC, CD59, CF55, CR1, C5aR1, and C5, preferably MAC, C3, C5b-9, C4, C1q, and C9. Various combinations of components, for example, a combination of C3 and C5, can be detected. Among these, the components are membrane attack complexes (MACs), and the method may include the detection of any or all subunits of the MAC, for example, C5b, C6, C7, C8, and C9 molecules.

[0117] In some embodiments, the methods of the present disclosure include measurement using exosome-specific markers such as CD81 (UNIPROT:P60033), proteins expressed on B cells (PMID:20237408), monocytes / macrophages (PMID:12796480), hepatocytes (PMID:12483205), and even CD4-positive T cells (PMID:22307619) that are deleting extracellular viable cells (EVs).

[0118] In some embodiments, the methods of the present disclosure include measurement using an EV that is specific to a tissue other than the target tissue, for example, to the bladder in the context of the urinary tract. An example is UPK1B (UNIPROT:O75841) expressed in bladder epithelium.

[0119] Conventional methods can be used to screen for EVs that are positive for undesirable markers, such as FACS.

[0120] Application of assay methods downstream This method for detecting complement activation in biological samples can be used in many downstream applications. For example, this method can be used to determine whether a subject (from which the biological sample was obtained) has a complement disorder or is at risk of developing one. The presence or risk of complement disorder is measured by comparing the presence or level of complement pathway components on extracellular viable (EVs) or their membrane-bound portions with a control (or reference material). Typically, the control or reference material contains EVs isolated from equivalent biological samples from healthy subjects. The usual method may be performed for processing and normalization of samples obtained from different subjects.

[0121] Similarly, this method can also be used to determine the progression or regression of complement-related diseases over time. This is done by comparing the presence or level of complement pathway components on the EV or its membrane-bound portion in the sample of interest at two different time points (e.g., t1 and t2, where t2 > t1). A decrease in the presence / level of complement at t2 compared to t1 indicates improvement in the complement state and regression of the complement disease, and the reverse is also true if the presence / level of complement at t2 is higher than at t1. The interval between measurements (e.g., t2-t1) may depend on the nature of the disease and may range from days to years, for example, weeks, one month, three months, six months, one year, two years, three years, five years, ten years, or longer, for example, 20 years.

[0122] Measurement of drug efficacy The methods and assays of this disclosure can be used to monitor responses to treatment of complement-related diseases using complement modulators. A complement modulator is a molecule that can directly or indirectly modulate, for example, component proteins of complement, e.g., activate or inhibit them. Representative complement modulators whose efficacy can be tested by the methods described herein, without being limited to any particular form, are provided in Table A.

[0123] [Table A-1]

[0124] [Table A-2]

[0125] [Table A-3]

[0126] [Table A-4]

[0127] For example, this disclosure relates to the following methods for monitoring the efficacy of therapies for various complement disorders. (A) A method for monitoring the response to treatment for autoimmune diseases (e.g., GBS, wAIHA, autoantibody diseases) or neurodegenerative diseases (e.g., ALS, HD, glaucoma / geographic atrophy (GA)) in a subject using an anti-C1q monoclonal antibody. (B) A method for monitoring the response of a subject to treatment for hereditary angioedema (HAE) using C1-INH (e.g., BERINERT, RUCONEST, CYNRIZE). (C)(1) A method for monitoring the response to treatment of hemolytic events in cold agglutinin disease (CAD) in a subject using an anti-C1s monoclonal antibody (e.g., BIVV020 or activated anti-C1s antibody). (C)(2) A method for monitoring the response of a subject to treatment for a complement disorder selected from cold agglutinin disease (CAD), warm-antibody autoimmune hemolytic anemia (wAIHA), and neurodegenerative diseases (e.g., HD, AD, ALS, GBS), using C1s peptide. (D) A method for monitoring the response of a subject to treatment for antibody-mediated inflammation or ischemia-reperfusion injury using an anti-C2 monoclonal antibody (e.g., PRO-02). (E) A method for monitoring the response to treatment for hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA), atypical hemolytic uremic syndrome (aHUS), or IgA nephropathy (IgAN) in a subject using an α-MASP-2 monoclonal antibody (e.g., nalsoprimab). (F) A method for monitoring the response of subjects to treatment for complement disorders such as paroxysmal nocturnal hemoglobinuria (PNH) using an α-MASP-3 monoclonal antibody (e.g., OMS906). (G) A method for monitoring the response to geographic transplantation (GA) / age-related macular degeneration (AMD) treatment in subjects using an α-D factor (FD) monoclonal antibody (e.g., lampalizumab). (H) A method for monitoring the response of subjects to treatment of transfusion-dependent anemia and hemolytic PNH (EVH) using a small molecule factor D (FD) inhibitor (e.g., Danicopan (ACH-4471) or ACH-5228). (I) A method for monitoring the response to treatment of complement dysfunction in a subject using a small molecule factor D (FD) inhibitor (e.g., BCX9930 or an FD inhibitor as described in U.S. Patent No. 9,388,199, incorporated herein by reference). (J) Factor B (FB) inhibitors (e.g., Factor B siRNA IONIS-FB-L) RX A method for monitoring the response to treatment of complement disorders such as IgA nephropathy (IgAN) in a subject, using an α-FB monoclonal antibody. A method for monitoring the response of subjects to treatment for PNH, C3 glomerulosis (C3G), membranous glomerulonephritis, and other renal diseases using a (K)B factor (FB) inhibitor (LNP023). A method for monitoring the response to treatment for renal disease or degenerative disease (e.g., AMD or GA) in a subject using an (L)α-properdin (P factor) monoclonal antibody (e.g., CLG561). A method for monitoring the response of a subject to treatment for periodontal disease or PNH using a (M)H factor (FH) modulator (e.g., mini H factor, AMY-201, or CR2-H factor / TT30). (N) A method for monitoring the response to the attenuation of complement disorders selected from GA, PNH, cold agglutinin disease (CAD), wAIHA, complement nephropathy (CDN), and C3G, using compstatin or its derivatives (e.g., APL2, APL9, AMY-101), sCR1 / TP10, or mirococept, or to the attenuation of periodontal disease, graft rejection, ischemia-reperfusion injury in allografts, or adeno-associated virus vector (AAV) rejection in gene therapy. (O) A method for monitoring the response to treatment in patients with PNH, aHUS, myasthenia gravis (gMG), neuromyelitis optica spectrum disorder (NMOSD), using an anti-C5 monoclonal antibody (e.g., eculizumab or its biosimilar, e.g., ABP959, Elizaria, or SB12). (P)(1) A method for monitoring the response to treatment with nomacopan (coversin, rVA576) in patients with PNH, aHUS, bullous pemphigoid (BP), uveitis, thrombotic microangiopathy (TMA), keratoconjunctivitis, and rheumatoid arthritis (RA). (P)(2) A method for monitoring the response to treatment in patients with gMG, ALS, immune necrotizing myopathy (IMNM), or renal disease, using Zircoplan (RA101495). (P)(3) A method for monitoring the response to treatment in patients with aHUS using anti-C5siRNA cemdisilane (ALN-CC5). (P)(4) A method for monitoring the response to treatment in patients with GA / AMD, neovascular AMD, or Stargardt disease using Zimura (ARC1905). (Q) A method for monitoring the response to treatment in subjects with PNH, aHUS, gMG, NMOSD, hematopoietic stem cell transplantation (HSCT)-TMA, ALS, complement-mediated TMA, or severe COVID-19, using an improved anti-C5 monoclonal antibody (e.g., ravulizumab). (R)(1) A method for monitoring the response of a subject with complement disorder to treatment using an anti-C5 affibody (e.g., SOBI005). (R)(2) A method for monitoring the response to treatment in patients with post-transplant microangiopathy (TAM), panuveitis, AMD, GA, PNH, or kidney transplant rejection, using the anti-C5 antibody tesidolumab (LFG316). (R)(3) A method for monitoring the response to treatment in patients with PNH using an anti-C5 antibody, which is pozelimab or clobarimab (SKY059). (S)(1) A method for monitoring the response to treatment in patients with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis using avacopan (CCX-168). (S)(2) A method for monitoring the response to treatment of a subject with GVHD or COVID-19 using an anti-C5 monoclonal antibody (e.g., orendalizumab (ALXN1007), BDB-001, or IFX2). (T)(1) A method for monitoring the response to treatment in subjects with autoimmune diseases or myasthenia gravis (MG) using a complement C6 inhibitor selected from anti-C6 monoclonal antibodies and C6 antisense RNA. (T)(2) A method for monitoring the response to treatment in patients with neurodegenerative disorders using the complement C6 inhibitor CP010. (U) A method for monitoring the response to treatment in subjects with atrophic and exudative AMD using an adeno-associated vector (AAV) encoding soluble CD59 (HMR59).

[0128] The above-mentioned typical methods for measuring the response to treatment generally follow the previously described methods for detecting complement proteins in EVs, for example: (a) obtaining a sample containing extracellular vesicles (EVs) or their membrane-bound moieties from the subject before and after treatment; (b) contacting a portion of this sample with at least one first capture antibody or its antigen-binding fragment to capture at least one first marker on the EV or its membrane-bound moiety; and (c) optionally contacting a portion of the sample with at least one second capture antibody or its antigen-binding fragment to capture at least one second marker on the EV or its membrane-bound moiety. The procedure is carried out by (d) capturing at least one complement pathway component, contacting the captured EV or its membrane-bound portion with at least one detection antibody or its antigen-binding fragment specific to a complement system-related component, and (e) qualitatively or quantitatively detecting the detection antibody or its antigen-binding fragment to measure the presence of complement pathway components at the level on the EV or its membrane-bound portion, wherein the presence or modification (e.g., increase or decrease, preferably decrease) of complement pathway components in the subject sample after treatment compared to before treatment with the complement modulator indicates that the subject is responding to the complement modulator.

[0129] In some embodiments, the complement modulator is a modulator of C1q, C1, C1s, C2, MASP-2, MASP-3, factor D, factor B, propergin (factor P), factor H, C3 / C5 convertase, C5, C5a / C5aR, C3a / C3aR, C6, or CD59, and is preferably a monoclonal antibody or small molecule inhibitor or a complement inhibitor such as siRNA / RNAi, as shown in Table A.

[0130] The method described above is particularly useful for testing the efficacy of molecules that activate or inhibit terminal complement activity in the C5 or C3 axis, for example. In particular, the method described above is especially applicable to testing the efficacy of subsequent molecules such as C5 inhibitors, eculizumab or ravulizumab.

[0131] A method for screening complements that modulate test compounds. In some embodiments, the Disclosure relates to a method for screening a test compound for complement regulation, comprising: (a) obtaining a sample containing extracellular vesicles (EVs) or membrane-bound portions thereof derived from a subject (e.g., an animal such as a mouse, rabbit, hamster, sheep, llama, dog, monkey, chimpanzee, or human) before and after administration of the test compound to a subject suffering from a complement disorder; (b) contacting a portion of the sample with at least one first capture antibody or its antigen-binding fragment to capture at least one first marker on the EVs or their membrane-bound portions; and (c) optionally contacting a portion of the sample with at least one second capture antibody or its antigen-binding fragment The present invention relates to a method comprising: (d) contacting the EV or its membrane-bound portion with a second marker to capture at least one second marker on the EV or its membrane-bound portion; (e) contacting the captured EV or its membrane-bound portion with at least one detection antibody or its antigen-binding fragment specific to a complement system-related component; and (e) qualitatively or quantitatively detecting the detection antibody or its antigen-binding fragment to measure the presence or level of complement components on the EV or its membrane-bound portion, and comparing the presence or level of complement components in the target sample after administration of the test compound with the level before administration of the test compound to indicate that the test compound can modulate complement. Preferably, the test compound can modulate complement, which is C1q, C1, C1s, C2, MASP-2, MASP-3, factor D, factor B, propergin (factor P), factor H, C3 / C5 convertase, C5, C5a / C5aR, C3a / C3aR, C6, or CD59, or a combination thereof.

[0132] In some embodiments, the regulatory activity of the test compound is compared to the regulatory activity of a molecule having complement regulatory activity (e.g., a positive control or standard substance), such as the molecules provided in Table A.

[0133] Compounds that activate or inhibit the activity of C5 In typical embodiments, the complement modulator whose activity is tested or screened according to the aforementioned method is a molecule that inhibits C5 activation, thereby reducing, suppressing, and / or eliminating complement-mediated effects (e.g., CSR or CARPA). Cleavage of C5 releases C5a, a potent anaphylatoxin and chemotactic, leading to the formation of C5b-9, a soluble species-end complement complex. C5a and C5b-9 also possess multifaceted cell-activating properties by amplifying the release of downstream inflammatory factors such as hydrolytic enzymes, reactive oxygen species, arachidonic acid metabolites, and various cytokines.

[0134] Complement inhibitors suitable for use in reducing, suppressing, and / or eliminating complement-mediated effects that occur during the therapeutic administration of a particular therapeutic agent (e.g., a therapeutic agent encapsulated in particles or nanoparticles) may bind to C5. Exemplary activators include antibodies, antibody fragments, polypeptides, small molecules, and aptamers. Exemplary antibodies are described in U.S. Patent 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. Patent Nos. 7,348,401 and 7,999,081. In certain embodiments, the complement inhibitor is an antibody, small molecule, aptamer, or polypeptide that binds to a binding site on C5, substantially the same as the antibodies described in U.S. Patent No. 6,534,058, or a peptide described in U.S. Patent No. 7,348,401. U.S. Patent No. 7,538,211 discloses aptamers that bind to and inhibit C5. RNAi activators that inhibit the local expression of C5 or CSR can also be used in the methods described herein.

[0135] In other embodiments, this activator is an antagonist of the C5a receptor (C5aR).

[0136] C5a is cleaved from the alpha chain of C5 by either an alternative C5 convertase or a conventional C5 convertase. The cleavage site for convertase activity is at or near amino acid residue 733 of the alpha chain of C5a. Compounds that would bind at or near this cleavage site would have the ability to block the approach of the C5 convertase enzyme to this cleavage site, thereby acting as complement inhibitors. Compounds that bind to C5 at a site distal to the cleavage site may also have the ability to block C5 cleavage, for example, by inhibiting the interaction between C5 and the C5 convertase. Examples of C5a receptor antagonists include various small molecule peptides, or monoclonal antibodies such as BB5.1 (Frei Y. et al., Mol. Cell. Probes, 1:141-9, 1987), single-strand variable fragments of BB5.1 (scFV), or anti-BB5.1Fab (Peng et al., J Clin Invest., 115(6):1590-1600, 2005), which prevent the formation of C5a and C5b receptors.

[0137] In certain embodiments, the complement inhibitor comprises an anti-C5 antibody. An anti-C5 antibody (or a VH / VL domain derived therefrom) suitable for use herein can be identified using methods known in the art. Alternatively, an anti-C5 antibody recognized in the art can be used. An antibody that competes with any of these art-recognized antibodies for binding to C5 can also be used.

[0138] The actual boundaries of the CDR have been defined differently by different methods. In some embodiments, the location of the CDR or framework region within the light chain variable domain or heavy chain variable domain is defined as follows: Kabat et al. [(1991) “Sequences of Proteins of Immunological Interest.” NIH Publication No. 91-3242, USD department of It may be as defined by Health and Human Services, Bethesda, MD. In such cases, the CDR may be referred to as "Kabat CDR" (e.g., "Kabat LCDR2" or "Kabat CDR"). (HCDR1). In some embodiments, the location of the CDR in the light-chain variable region or the heavy-chain variable region may be as defined by Chothia, C. et al. (Nature, 342:877 83, 1989). Thus, these regions can be referred to as "Chothia CDRs" (e.g., "Chothia LCDR2" or "ChothiaHCDR3"). In some embodiments, the location of the CDRs in the light-chain variable region and the heavy-chain variable region may be as defined by the combined definition of Kabat Chothia. In such embodiments, these regions can be referred to as "combined Kabat Chothia CDRs" (Thomas, T. et al., Mol.Immunol., 33:1389 401, 1996), which exemplifies the identification of CDR boundaries by the definitions of Kabat and Chothia.

[0139] Another exemplary anti-C5 antibody is antibody BNJ421, as described in International Publication No. 2015 / 134894 and U.S. Patent No. 9,079,949, whose teachings are incorporated herein by reference.

[0140] An anti-C5 antibody may, for example, include a variable human Fc constant region that binds to the human neonatal Fc receptor (FcRn), wherein the variable human Fc CH3 constant region includes Met-429-Leu substitutions and Asn-435-Ser substitutions at the residues corresponding to methionine 428 and asparagine 434 of the natural human IgG Fc constant region, respectively, in EU numbering.

[0141] Another exemplary anti-C5 antibody is antibody 7086, which is described in U.S. Patent Nos. 8,241,628 and 8,883,158, whose disclosure is incorporated herein by reference.

[0142] Another exemplary anti-C5 antibody is the 8110 antibody, which is also described in U.S. Patent Nos. 8,241,628 and 8,883,158, whose disclosures are incorporated herein by reference.

[0143] Another exemplary anti-C5 antibody is the 305LO5 antibody, whose disclosure is described in U.S. Patent No. 9,765,135, which is incorporated herein by reference.

[0144] Another exemplary anti-C5 antibody is the SKY59 antibody (Fukuzawa, T. et al., Sci. Rep., 7:1080, 2017, the disclosure of which is incorporated herein by reference).

[0145] Another exemplary anti-C5 antibody is REGN3918 antibody (also known as H4H12166PP), described in U.S. Patent Application Publication No. 2017 / 0355757 or International Publication Brochure No. 2017218515, the disclosure of which is incorporated herein by reference.

[0146] In another embodiment, this antibody competes for and / or binds to the same epitope on C5 as the previously described antibodies (e.g., 7086 antibody, 8110 antibody, 305LO5 antibody, SKY59 antibody, or REGN3918 antibody). The anti-C5 antibody may have, for example, at least about 90% variable region amino acid sequence identity with the previously described antibodies (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% variable region identity).

[0147] The anti-C5 antibodies described herein, in some embodiments, include a mutant human Fc constant region that binds to the human neonatal Fc receptor (FcRn) with greater affinity than the native human Fc constant region from which the mutant human Fc constant region is derived. The Fc constant region may include, for example, one or more (e.g., 2, 3, 4, 5, 6, 7, or 8 or more) amino acid substitutions to the native human Fc constant region from which the mutant human Fc constant region is derived. For example, substitutions can increase the binding affinity of an IgG antibody containing the mutant 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 to FcRn at pH 6.0 (while maintaining the pH dependence of the interaction) are known in the art and illustrated in working examples (International Publication No. 2015134894 and U.S. Patent No. 9,079,949; each disclosure is incorporated herein by reference in whole).

[0148] Substitutions that enhance the binding affinity of the antibody Fc constant region to FcRn include (1) M252Y / S254T / T256E triple substitution (Dall'Acqua, W. et al., J. Biol. Chem., 281:2351424, 2006), (2) M428L substitution or T250Q / M428L substitution (Hinton, P. et al., J. Biol. Chem., 279:6213 6, 2004, Hinton, P. et al., J. Immunol., 176:34656, 2006), and (3) N434A substitution or T307 / E380A / N434A substitution (Petkova, S. et al., Int. Immunol., 18:1759 69, 2006). Additional substitutional pair formations, e.g., P257I / Q311I, P257I / N434H, and D376V / N434H, are also described (Datta-Mannan, A. et al, J. Biol. Chem., 282:1709 17, 2007). The overall teachings of each cited reference are incorporated herein by reference.

[0149] In some embodiments, the mutant constant region has a substitution for valine at EU amino acid residue 255. In some embodiments, the mutant constant region has a substitution for asparagine at EU amino acid residue 309. In some embodiments, the mutant constant region has a substitution for isoleucine at EU amino acid residue 312. In some embodiments, the mutant constant region has a substitution at EU amino acid residue 386.

[0150] In some embodiments, the mutant Fc constant region includes 30 or fewer amino acid substitutions, insertions, or deletions relative to the derived native constant region (e.g., 29 or fewer, 28 or fewer, 27 or fewer, 26 or fewer, 25 or fewer, 24 or fewer, 23 or fewer, 22 or fewer, 21 or fewer, 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer). In some embodiments, the mutant Fc constant region includes one or more amino acid substitutions selected from the group consisting of M252Y, S254T, T256E, N434S, M428L, V259I, T250I, and V308F. In some embodiments, the mutant human Fc constant region includes methionine at position 428 and asparagine at position 434, each with an EU number. In some embodiments, the mutant Fc constant region includes, for example, the 428L / 434S double substitution as described in U.S. Patent No. 8,088,376, the disclosure of which is incorporated herein by reference in whole.

[0151] In some embodiments, the precise locations of these mutations can be shifted from the native human Fc constant region by antibody manipulation. The 428L / 434S double substitution, when used in IgG2 / 4 chimeric Fc, may correspond to 429L and 435S, as well as the M429L and N435S variants described, for example, in U.S. Patent No. 9,079,949, whose disclosure is incorporated herein by reference in whole.

[0152] In some embodiments, the mutant constant region includes substitutions at amino acid positions 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. In some embodiments, this substitution is 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, and glutamic acid at position 258. Histidine, alanine relative to aspartic acid at position 265, phenylalanine relative to aspartic acid at position 270, alanine or glutamic acid relative to asparagine at position 286, histidine relative to threonine at position 289, alanine relative to asparagine at position 297, glycine relative to serine at position 298, alanine relative to valine at position 303, alanine relative to valine at position 305, alanine relative to threonine at position 307, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan, or tyrosine, alanine relative to valine at position 308, phenylalanine, isoleucine, leucine, methionine, proline, glutamine, or threonine,Alanine, aspartic acid, glutamic acid, proline, or arginine relative to leucine or valine at position 309; alanine, histidine, or isoleucine relative to glutamine at position 311; alanine or histidine relative to aspartic acid at position 312; lysine or arginine relative to leucine at position 314; alanine or histidine relative to asparagine at position 315; alanine relative to lysine at position 317; glycine relative to asparagine at position 325; valine relative to isoleucine at position 332; leucine relative to lysine at position 334; histidine relative to lysine at position 360; alanine relative to aspartic acid at position 376; alanine relative to glutamic acid at position 380; alanine relative to glutamic acid at position 382; and ara relative to asparagine or serine at position 384. Selected from the group consisting of nin, 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 for methionine at position 428, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, or tyrosine, lysine for histidine at position 433, alanine for asparagine at position 434, phenylalanine, histidine, serine, tryptophan, or tyrosine, and histidine for tyrosine or phenylalanine at position 436, all of which are EU numbered.

[0153] In one embodiment, the antibody is prepared at pH 7.4 and 25°C (and otherwise under physiological conditions) at a concentration of at least 0.1 nM relative to C5 (e.g., at least 0.15 nM, 0.175 nM, 0.2 nM, 0.25 nM, 0.275 nM, 0.3 nM, 0.325 nM, 0.35 nM, 0.375 nM, 0.4 nM, 0.425 nM, 0.45 nM, 0.475 nM). The affinity dissociation constant (K) is nM, 0.5nM, 0.525nM, 0.55nM, 0.575nM, 0.6nM, 0.625nM, 0.65nM, 0.675nM, 0.7nM, 0.725nM, 0.75nM, 0.775nM, 0.8nM, 0.825nM, 0.85nM, 0.875nM, 0.9nM, 0.925nM, 0.95nM, or 0.975nM). D ) binds. In some embodiments, the anti-C5 antibody or its antigen-binding fragment is K D This is less than or equal to 1 nM (for example, less than or equal to 0.9 nM, less than or equal to 0.8 nM, less than or equal to 0.7 nM, less than or equal to 0.6 nM, less than or equal to 0.5 nM, less than or equal to 0.4 nM, less than or equal to 0.3 nM, or less than or equal to 0.2 nM).

[0154] In other embodiments, [(K of antibody against C5 at pH 6.0, 25°C) D ) / (K of antibody against C5 at pH 7.4, 25℃) D )] is greater than 21 (for example, 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, 24 (0, 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 greater than 8000).

[0155] Compounds that activate or inhibit the activity of factor B In certain embodiments, complement inhibitors inhibit the activation of factor B. Complement inhibitors can, for example, bind to factor B, thereby inhibiting its activation. Exemplary activators include antibodies, antibody fragments, peptides, small molecules, and aptamers. Exemplary antibodies that inhibit factor B are described in U.S. Patent Application Publication 20050260198. In certain embodiments, an isolated antibody or antigen-binding fragment selectively binds to factor B within a third short consensus repeat (SCR) domain. In certain embodiments, the antibody prevents the formation of the C3bBb complex. In certain embodiments, the antibody or antigen-binding fragment prevents or inhibits the cleavage of factor B by factor D. In certain embodiments, the complement inhibitor is substantially the same as the antibody described in U.S. Patent Application Publication 20050260198, a small molecule, aptamer, or polypeptide that binds to a binding site on factor B, or an RNAi activator that inhibits local expression of factor B. Peptides that bind to and inhibit factor B can be identified using methods known in the art.

[0156] Compounds that inhibit the activity of factor D In certain embodiments, complement inhibitors inhibit factor D. Complement inhibitors may, for example, bind to factor D and thereby inhibit it. Exemplary activators include antibodies, antibody fragments, peptides, small molecules, and aptamers. While factor D has been suggested as a desirable target for systemic complement inhibition due to its relatively low serum concentration and ability to inhibit the activation of alternative pathways, this disclosure relates to the therapeutic potential of topically administered activators that inhibit factor D. Exemplary antibodies that inhibit factor D are described in U.S. Patent No. 7,112,327. In certain embodiments, the complement inhibitor is an antibody, small molecule, aptamer, or polypeptide that binds to a binding site on factor D, substantially the same as the antibody described in U.S. Patent No. 7,112,327. Exemplary polypeptides thought to inhibit factor D by inhibiting the activation of alternative pathways are disclosed in U.S. Patent Application Publication No. 20040038869. Peptides that bind to and inhibit factor D can be identified using methods known in the art.

[0157] Multimode complement inhibitors / modulators Complement inhibitors useful in the methods described herein can bind to one or more complement proteins and / or inhibit one or more steps in the complement activation pathway. Such complement inhibitors are referred to herein as “multimode.”

[0158] Complement inhibitors may be, for example, viral complement regulatory proteins (VCCPs) (U.S. Patent No. 7,947,267 and International Publication No. 2006042252). In certain embodiments, the VCCP is a poxvirus complement regulatory protein (PVCCP) or a herpesvirus complement regulatory protein (HVCCP).

[0159] VCCP can inhibit the conventional complement pathway, alternative complement pathways, lectin pathways, or two or more of these. VCCP, e.g., PVCCP, can bind to, for example, C3b, C4b, or both. PVCCP may contain one or more putative heparin-binding sites (K / R--X--K / R) and / or have an overall positive charge. In some embodiments, PVCCP may contain at least three SCR modules (e.g., modules 1-3), e.g., four SCR modules. The PVCCP protein may be a precursor of mature PVCCP (i.e., it may contain a signal sequence that cleaves normally when expressed in virus-infected cells), or it may be in its mature form (e.g., lacking a signal sequence).

[0160] Vaccinia complement regulatory protein (VCP) has been shown to inhibit conventional pathways of complement activation by binding to C3 and C4 and acting as a cofactor for factor I-mediated cleavage of these components, as well as by promoting the breakdown of existing convertases (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 an alternative pathway by cleaving C3b to iC3b, thereby preventing the formation of the alternative C3 convertase (Sahu, A. et al., J. Immunol., 160, 5596-604, 1998). VCP, therefore, blocks complement activation in multiple steps and reduces the levels of pro-inflammatory chemotactic factors C3a, C4a, and C5a. Homologs of VCPs, such as complement enzyme smallpox inhibitors (SPICE), including SPICE-related polypeptides containing four SCRs, can be used in the methods described herein. Furthermore, complement regulatory proteins derived from cowpox virus (IMP) or monkeypox virus (MCP) can also be used in the methods described herein.

[0161] In addition to VCCP, several other viral proteins exist that interfere with one or more steps in the complement pathway and can be used in the methods described herein, for example, the 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 proteins encoded by these viruses bind to C3b (Friedman, H. et al, Nature, 309:633-5, 1984), and gC1 (from HSV-1) accelerates the breakdown of the conventional C3 convertase and inhibits the binding of propergine and C5 to C3. The aforementioned proteins are collectively referred to as viral complement interference proteins (VCIPs). These VCIPs are said to inhibit complement by any of the following means, including interfering with one or more steps of complement activation, accelerating the breakdown of complement components, and / or enhancing the activity of complement regulatory proteins. Any of these proteins, or their derivatives, fragments, or variants, can be used as therapeutic agents in the manner described herein.

[0162] Additional activators, modulators, mixtures, and modifiers that inhibit complement Various other complement inhibitors can be used in various embodiments of the methods described herein. In some embodiments, the complement inhibitor is a naturally occurring mammalian complement regulatory protein or a fragment or derivative thereof. The complement regulatory protein may be, for example, CR1, DAF, MCP, CFH, or CFI. In some embodiments, the complement regulatory polypeptide is typically membrane-bound in its naturally occurring state. In some embodiments, fragments of such polypeptide lacking some or all of the transmembrane domain and / or intracellular domain are used. For example, the soluble form of complement receptor 1 (sCR1) can be used. For example, compounds known as TP10 or TP20 (Avant Therapeutics) can be used. C1 inhibitors (C1-INH) are also used. In some embodiments, a soluble complement regulatory protein, such as CFH, is used. In some embodiments, this polypeptide is modified to increase its solubility.

[0163] C1s inhibitors are used (for example, U.S. Patent No. 6,515,002 describes compounds that inhibit C1s (furanylamidines and thienylamidines, heterocyclic amidines, and guanidines); U.S. Patents No. 6,515,002 and No. 7,138,530 describe heterocyclic amidines that inhibit C1s; U.S. Patent No. 7,049,282 describes peptides that inhibit the activation of conventional pathways; U.S. Patent No. 7,041,796 discloses C3b / C4b complement receptor-like molecules and their use to inhibit complement activation; U.S. Patent No. 6,998,468 discloses anti-C2 / C2a inhibitors of complement activation; and U.S. Patent No. 6,676,943 discloses human complement C3 degradation proteins derived from Streptococcus pneumoniae).

[0164] Combination therapies using two or more complement inhibitors are included in the methods described herein. These two or more complement inhibitors may be provided in the same composition. In certain embodiments, the complement inhibitors bind to two or more different complement components. In certain embodiments, the complement inhibitors bind to two or more different soluble complement proteins. In certain embodiments, the complement inhibitors inhibit the activation or activity of at least two complement proteins selected from C3, C5, C6, C7, C8, C9, factor B, and factor D. [Examples]

[0165] Example 1 Characterization of urinary extracellular vesicles (uEVs) by simultaneous immunoprecipitation / immunoanalysis using the Luminex® xMAP® Technology platform.

[0166] An array of antibody-coated xMAP beads was used against canonical vesicle markers CD9, CD63, and CD81 to concentrate individual EV partial sets from urine. Each bead set was then analyzed for the presence of other biomarkers that define the phenotype of the partial set and link it to its tissue origin. In this way, a non-invasive "liquid biopsy" method was constructed to sample and monitor key biomarkers of specific kidney diseases for differential diagnosis, prognosis, and / or longitudinal monitoring of response to treatment.

[0167] Electron microscopy Sample preparation for electron microscopy 1) Start with fresh urine + protease inhibitor. Centrifuge at 2.5K × g and collect the supernatant. 2) Concentrate to 300 μL using the ExoQuick Ultra TC kit. 3) For EV and total particle density, the NTA of the product ± ExoGlow, 4) Concentrate the preparation to 30 μL in a Microcon 10K mvco. 5) Add an equal volume of Karnovsky fixative, 6) Repeat the NTA only for the total particle density. 7) Processed for imaging.

[0168] Figure 1 shows the relative amounts of EV markers in urinary ExoQuick Enrichment by NTA. Figure 2 shows electron microscope images of EV and non-EV particles in urine. Figure 3 shows the distribution of ferret diameters of the objective in the electron microscope images.

[0169] mass spectrometry Tandem Master Tag (TMT) Marking 1) Start with fresh urine + protease inhibitor. Centrifuge at 300 x g and collect the supernatant. 2) Concentrate using the ExoQuick-Ultra TC kit. 3) Reduce, amida sulfhydryl bond, 4) Precipitate the total protein in 80% acetone overnight at -20°C. 5) Reconstitute in buffer solution and digest with trypsin. 6) Mark the fragment with the TMT (trademark) Isobaric mark. 7) Combine the samples and dry them. 8) Separate the fragments using Ultimate3000. 9) LC fractions were analyzed using OrbiTrap Lumos.

[0170] Figure 4 shows the relative amounts of the top 25 proteins according to PSM.

[0171] [Table 1]

[0172] Luminex assay development Preparation of dUC samples 1) Start with fresh urine + protease inhibitor. Centrifuge at 300 x g and collect the supernatant. 2) Transfer S300 to an Ultra-Clear™ tube and centrifuge overnight at 4°C and 200K×g. 3) Resuspend the pellet in cold PBS + protease inhibitor + 200 mg / mL DTT and pool it. 4) Centrifugation at 4°C and 200K × g for 6 hours. 5) Resuspend the pellet in PBS + protease inhibitor. 6) Dispense into aliquots and store at -70°C or below.

[0173] EV FSL-biotin tagging FSL-Biotin is a Kode® Technology construct designed to label hydrophobic surfaces with biotin. FSL-Biotin consists of a biotin (vitamin B7) monomer bound to a maleimide-supported carboxymethylglycine linker, which is then bound to an activated azipart derivative of dioleylphosphatidylethanolamine (KODE Biotech website: kodebiotech.com / sales / products / product_info.php?id=129&cat=rdt (accessed September 27, 2018)).

[0174] Luminex Assay Design 1) Use 1E6EV / well of uEV concentrated with dUC. 2) Add an equal amount of labeled MagPlex® beads. 3) Incubate at 4°C overnight with gentle shaking. 4) Wash twice with PBS. 5) Add one of the following: a. Biotinylated polyclonal antibody in PBS / BSA + SAPE b. FSLB+SAPE in PBS 6) Incubate at room temperature for 1 hour with gentle shaking. 7) Wash twice with PBS. 8) Wash twice with sheath fluid. 9) Read with a Luminex machine at high PMT.

[0175] Figure 5 shows the detection of a partial set of urine EV by Luminex. Figure 6 shows CD9 + This shows renal PODXL detected only in EV.

[0176] These results indicate that the concentrates of this protocol for uEV and CD9+ partial sets contain PODXL, a marker of renal tissue origin. Optimization of this protocol to improve sensitivity and specificity should improve uEV enrichment and identify a larger amount of renal biomarkers. Once optimized, this methodology can be applied to various disease populations for differential analysis compared to healthy donors.

[0177] Example 2 Evaluation of complement deposition in organs and tissues by simultaneous immunoprecipitation / immunoanalysis of urinary extracellular vesicles (uEVs) using the Luminex® xMAP® Technology platform.

[0178] An array of antibody-coated xMAP beads was used for a predetermined nephron region. Podocalyxin (PODXL) was used for glomerular podocytes, and aquaporin 2 (AQP2) for convoluted tubules. Each bead set was then analyzed for the presence of complement markers deposited on the plasma membrane (PM) of the origin cells. Thus, complement deposition in nephrons can be monitored using urinary EV for differential diagnosis, prognosis assessment, and / or longitudinal monitoring of response to treatment.

[0179] Extracellular viable cells (EVs) possess surface markers derived from their parent cells. These markers can be used to immunoisolate EVs based on their cell origin. PODXL is produced only on podocytes in the glomerulus. AQP2 originates from the proximal and distal convoluted tubules. Glycophorin A (GYPA) derived from renal cell ducts (RBCs) can be used to measure EV leakage from plasma into the filtrate. Circulating EV concentrations are elevated in inflammatory and thrombotic conditions. Some EVs possess complement regulators such as CD55 and CD59 on their surface. Cells can use EVs to dislodge low concentrations of MAC complexes from their surface. EVs can act as loci for thrombinogenesis.

[0180] Luminex assay development Assay design goals To be a useful and practical substitute for renal biopsy, this assay typically features (a) the ability to start with frozen samples, (b) requiring minimal processing to achieve the target, (c) simplicity and ability to analyze multiple uses simultaneously and consistently, and (e) maintaining membrane integrity. This means that pre-concentration and sorting of vesicles prior to Luminex bead immunoprecipitation is typically minimal.

[0181] Luminex Assay Design 1) Use 50 μL / well of urine diluted 1:2 in PBS / BSA. 2) Add 1000 / well labeled xMAP(trademark) beads. 3) Incubate at 4°C overnight with gentle shaking. 4) Wash twice with PBS. 5) Add biotinylated polyclonal antibody + SAPE to PBS / BSA. 6) Incubate at room temperature for 1 hour with gentle shaking. 7) Wash twice with PBS. 8) Wash twice with sheath fluid. 9) Read with a Luminex machine at high PMT.

[0182] [Table 2]

[0183] Luminex beads can identify uterine body-specific extracellular proteins (EVs). As shown in Figure 7, Rb-α-PODXL can be detected on EVs containing CD9 or CD40L, but not on EVs containing CD63 or CD81. The signal was observed using two different α-PODXL antibodies and can only occur when both proteins have the same structure.

[0184] These results indicate that nephron-specific EV levels are elevated with disease. As shown in Figure 8, there is a greater amount of IgANurinary PODXL+ EV than in control urine, and this elevation is observed in both the CD9+ / PODXL+ and CD40+ / PODXL populations, while CD63+ and CD81+ EV remain negative.

[0185] Luminex beads can measure complement on EV membranes. As seen in Figure 9, C3c and C5b-9 were detected in LN patient urine with both CD9+ beads and PODXL+ beads, but not in CD63+ or CD81+. These results were confirmed in 10 LN samples, 6 IgAN samples, and 7 control samples (data not shown). Both LN and IgAN samples could have C3, C5b-9, C4, and C1q deposition on both PODXL+EV and AQP2+EV compared to the control sample.

[0186] These results also show that glomerular C5b-9 deposition decreases with ravulizumab treatment. As shown in Figure 10, aHUS patients may have any combination of C3, C5b-9, C1q, and C4 on the podocyte membrane, with C3 remaining unchanged throughout ravulizumab treatment, and levels of C5b-9 and C1q on the EV rapidly decreasing throughout ravulizumab treatment.

[0187] Renal biopsy is the current standard for differential diagnosis of chronic kidney disease. However, the risk of serious complications limits patient selection and the frequency of the procedure. EV provides an easily accessible opportunity to monitor ongoing complement deposition on the renal membrane before and during the procedure. It also allows for precise cellular-level identification of complement attack along the nephrons.

[0188] This technique can be extended to any biological sample beyond the kidney and urine. Any cell type or tissue of interest, along with a unique PM marker, can be used to examine complement deposition. Figure 11 provides a schematic diagram of markers that can be analyzed according to this disclosure.

[0189] Example 3: Bioassay using modified membrane-bound fluorophores to normalize EV data To improve the robustness of the assay, it is important to measure the number of membrane vesicles isolated on each capture antibody-coated LUMINEX bead. In some embodiments, the "normalization" step is performed by changes in the number of vesicles per bead and per 1 mL of sample matrix, both within and between individual donors. Therefore, it was intended that a modified membrane-bound fluorophore-cysteine-lysine-palmtoyl (palmtoyl) group (mCLING, Synaptic Systems, catalog no. 710-MCK) could improve the assay. In one embodiment, mCLING was modified via biotinylation, and the modified mCLING was added to each test sample in the replication wells of immunobeads retained by EV. After washing each bead set, streptavidin phycoerythrin (SA-PE) was added, and the fluorescence intensity was measured for the mCLING sample and the corresponding analyte sample.

[0190] In one study, urinary extracellular viability (EV) samples were obtained from healthy volunteers (controls), patients with lupus nephritis (LN), and patients with IgA nephropathy (IgAN). These samples were concentrated using an antibody capture bead panel, and then surface phenotypic classification was performed in replication wells for either (a) total EV using biotinylated mCLING, or (b) complement iC3b using Quidel antibody A710 (monoclonal, nascent antigen-specific). The normalized signal for the analyte (iC3b) was generated by taking the ratio of the iC3b signal to the signal for biotinylated mCLING (signal = (b) / (a)). The results are shown in Figure 12, which represents typical normalized EV data for iC3b. The normalized response (shown on the y-axis) represents the average fluorescence intensity value reported for each marker (shown on the x-axis) divided by the corresponding average fluorescence intensity value for mCLING.

[0191] The control represents an equivalent sample from a healthy donor. Patients (LN or IgAN) are presented together for each marker.

[0192] The data show that positive EVs for certain EVs, such as CD9 and PODXL, selectively contain complement (e.g., iC3b) deposition above background levels (as indicated by the horizontal dashed lines in Figure 12). The data also indicate that activated complement pathway proteins, such as iC3b, are associated with the pathophysiological features of complement-related diseases, as shown in EVs obtained from two disease subset sets, LN and IgAN, respectively.

[0193] It should be understood that any maximum numerical limit given throughout this specification includes all lower numerical limits, as if such higher numerical limits were clearly stated herein. Any minimum numerical limit given throughout this specification will include all higher numerical limits, as if all such higher numerical limits were clearly stated herein. Any numerical range given throughout this specification will include all narrower numerical ranges that fall within such wider numerical ranges, as if all such narrower numerical ranges were clearly stated herein.

[0194] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which the present invention pertains. Methods and materials are described herein for use in the present invention. Other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. References, patent applications, patents, sequences, database entries (e.g., PUBMED, NCBI, or UNIPROT acceptance numbers), and other references mentioned herein are incorporated by reference in their entirety. In case of any conflict, this specification, including definitions, shall prevail.

[0195] While specific embodiments of the present invention have been illustrated and described, it will be apparent to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. Therefore, the appended claims are intended to encompass all such changes and modifications that fall within the scope of the invention. The present invention provides, for example, the following items: (Item 1) A method for detecting complement activity in a biological sample derived from a target, (a) A portion of the biological sample containing extracellular vesicles (EVs) or their membrane-bound portions is isolated with at least one first capture antibody or its antigen-binding fragment, and at least one first marker, including an EV-specific marker or a tissue-specific marker displayed on the EVs, (b) If applicable, a portion of the sample is brought into contact with at least one second capture antibody or its antigen-binding fragment to capture at least one second marker on the EV or its membrane-bound portion, (c) The presence or level of complement system-related components on the captured EV or the membrane-bound portion thereof is detected qualitatively or quantitatively using at least one detection antibody or its antigen-binding fragment that is specific to the complement system-related components, thereby detecting complement activation in the biological sample. Methods that include... (Item 2) The first capture marker includes an EV-specific marker, and optionally the second capture marker includes a tissue-specific marker displayed on the EV or its membrane-bound portion. The method described in item 1. (Item 3) The method according to item 1 or 2, wherein both the first capture marker and the second capture marker are present, and the first capture marker containing an EV-specific marker and the second capture marker containing a tissue-specific marker are detected. (Item 4) The method according to any one of items 1 to 3, wherein the biological sample is derived from tissue, organ, or bodily fluid. (Item 5) The method according to any one of items 1 to 4, wherein the biological sample includes bladder cells, kidney cells, whole blood, red blood cells, platelets, serum, plasma, blood fractions other than serum or plasma, lymph, cerebrospinal fluid (CSF), saliva, tears, vaginal secretions, semen, glandular secretions, exudate, contents of cysts or feces, lavage fluid, or venous vesicles or their membrane-binding portions derived from ascites. (Item 6) The method according to any one of items 1 to 5, wherein the biological sample includes extracellular matrix (EV) or its membrane-bound portion derived from renal glomerulopoda cells, convoluted tubules, or bladder epithelium, or red blood cells (RBCs). (Item 7) The method according to any one of items 1 to 6, wherein the first capture antibody or the antigen-binding fragment thereof is bound to a first solid support, optionally the second capture antibody or the antigen-binding fragment thereof is bound to a second solid support, and the detection antibody is bound to a detectable marker. (Item 8) The method according to item 7, comprising contacting a portion of the biological sample with the first capture antibody or its antigen-binding fragment and the second capture antibody or its antigen-binding fragment, wherein the first capture antibody or its antigen-binding fragment and the second capture antibody or its antigen-binding fragment are bound to the same support or different supports. (Item 9) The method according to item 7, wherein the detectable marker is selected from the group consisting of fluorophores, chromogens, and biotin. (Item 10) The method according to any one of items 7 to 9, wherein the detectable marker is a fluorophore having an absorption maximum between 500 nm and 1000 nm and an emission maximum between 550 nm and 1100 nm. (Item 11) The method according to any one of items 7 to 10, wherein the detectable marker is phycoerythrin (PE). (Item 12) The method according to any one of items 7 to 10, wherein the detectable marker is biotin. (Item 13) The method according to any one of items 7 to 12, wherein the first and second solid supports are independently selected from the group consisting of nanoparticles, microparticles, beads, magnetic beads, nanostructures, tissue culture plates, silica, and nanomatrixes. (Item 14) The first marker is selected from the group consisting of extracellular vesicle-related proteins, In some cases, the second marker is selected from a group consisting of tissue-specific extracellular vesicle-associated proteins, and The complement system-related components include (a) components of the alternative complement pathway (AP), (b) components related to the conventional pathway (CP), and (c) components related to the lectin pathway (MBL). A method of describing one of items 1 to 13, selected from a group of elements. (Item 15) The method according to any one of items 1 to 14, wherein the complement system-related components are selected from the group consisting of (a) components of an alternative route (AP) and (b) components related to a conventional route (CP). (Item 16) The method according to item 14, wherein the complement system-related component is a protein selected from the group consisting of C3, C5b-9, C4, Clq, C9, C3b, iC3b, TF, CRP, pCRP, MAC, CD59, CD55, CR1, C5aRl, and C5a. (Item 17) The first marker is selected from the group consisting of ALIX, TSG101, CD9, CD63, CD81, CD40L, CD26, CD31, CD45, CD2, CD11a, CD24, CD55, CD59, CF106, CD56, CD51, CD82, integrin, tetraspanin, annexin, HSP90, HSP70, syntenin 1, ADAM10, EHD4, actin, Rab5, clathrin, flotinin 1, MHCI, MHCII, actinin 4, GP96, EHD4, mitophilin, and LAMP2. The second marker is selected from the group consisting of podocalyxin (PODXL), aquaporin 2 (AQP2), uroplakin 1b (UPK1b), podosin (NPHS2), glycophorin A (GYPA), mucin 1, type 2 Na-K-2C1 cotransporter (NKCC2), aquaporin 1 (AQP 1), α-glutathione-S-transferase (alpha-GST), Tamm-Horsfall protein (TH), calbindin-D28K (CalD), megalin, cubin, nephrin (Nphsl), claudin 1, annexin V, synaptopodin (Synpo), Wilm tumor protein (Wtl), band 3, stomatin (STOM), BGP1, globin, glycophorin B, Rh polypeptide, and Rh glycoprotein, and The method according to any one of items 1 to 16, wherein the complement protein is selected from the group consisting of MAC, C3, C5b-9, C4, Clq, and C9. (Item 18) The method according to item 17, wherein the biological sample contains EVs derived from kidney cells, and the second marker is a kidney-specific EV marker selected from the group consisting of podocalyxin (PODXL), aquaporin 2 (AQP 2), uroplakin 1b (UPK1b), and podosin (NPHS2). (Item 19) The method according to item 17, wherein the sample contains EVs derived from red blood cells (RBCs), and the second marker is an RBC-specific EV marker selected from glycophorin A (GYPA). (Item 20) The method according to item 17, wherein the sample is negative for CD81 as a first marker and / or uroplakin 1B (UPK1B) as a second marker. (Item 21) The method according to item 1, wherein the capture marker and the detection marker are located within the same EV or its membrane-bound portion. (Item 22) The method according to any one of items 1 to 21, further comprising determining whether the subject has a complement disorder or is at risk of developing a complement disorder, including comparing the presence or level of the components of the complement pathway located on the EV or the membrane-bound portion thereof with a control. (Item 23) The method described in item 22, wherein the control includes an equivalent sample derived from a healthy subject. (Item 24) The method according to item 22 or 23, wherein if the level or presence of the components of the complement pathway on the EV or the membrane-bound portion thereof obtained from the subject is higher than that of the control, it indicates that the subject is suffering from or at risk of developing a complement disorder. (Item 25) A method for diagnosing or evaluating the prognosis of complement-related disorders in a subject, (a) Obtain a sample containing extracellular vesicles (EVs) or their membrane-bound portions derived from the subject, (b) Contacting a portion of the sample with at least one first capture antibody or its antigen-binding fragment to capture at least one first marker on the EV or its membrane-binding portion, (c) In some cases, a portion of the sample is brought into contact with at least one second capture antibody or its antigen-binding fragment to capture at least one second marker on the EV or its membrane-bound portion, (d) Contacting the captured EV or its membrane-bound portion, which includes the at least one first marker and optionally the at least one second marker, with at least one detection antibody or its antigen-binding fragment that is specific to a complement system-related component, (e) Qualitatively or quantitatively detect the detected antibody or the antigen-binding fragment thereof to measure the presence or level of the components of the complement pathway on the EV or the membrane-bound portion thereof, and compare with a control to indicate that if the presence or level of the components of the complement pathway in the sample of the subject is high, the subject is suffering from the complement disorder or is at risk of developing the complement disorder. Methods that include... (Item 26) The method according to item 25, wherein the first marker includes an EV-specific marker or a tissue-specific marker displayed on an EV. (Item 27) The method according to item 25, wherein the first marker includes an EV-specific marker, and the second marker includes a tissue-specific marker displayed on the EV. (Item 28) A method for monitoring the response of subjects to treatment of complement-related disorders using a complement modulator, (a) Obtaining samples containing extracellular vesicles (EVs) or their membrane-bound portions derived from the subject before and after the treatment, (b) Contacting a portion of the sample with at least one first capture antibody or its antigen-binding fragment to capture at least one first marker on the EV or its membrane-binding portion, (c) In some cases, a portion of the sample is brought into contact with at least one second capture antibody or its antigen-binding fragment to capture at least one second marker on the EV or its membrane-bound portion, (d) Contacting the captured EV or its membrane-bound portion, which includes the at least one first marker and optionally the at least one second marker, with at least one detection antibody or its antigen-binding fragment that is specific to a complement system-related component, (e) Detecting the detection antibody or the antigen-binding fragment thereof qualitatively or quantitatively to measure the presence or level of a component of the complement pathway on the EV or the membrane-bound portion thereof, and comparing with before the treatment using the complement modulator, if the presence or level of the component of the complement pathway in the sample of the subject after the treatment using the complement modulator is attenuated, it indicates that the subject is responding to the complement modulator A method comprising. (Item 29) The method according to item 28, wherein the mediator of the complement is a complement 5 (C5) inhibitor, a complement 5a (C5a) inhibitor, a complement 5 receptor (C5R1) inhibitor, a complement 3 (C3) inhibitor, a factor D (FD) inhibitor, a factor H (FH) inhibitor, a factor B (FB) inhibitor, a MASP2 inhibitor, a MASP3 inhibitor, a propelzin inhibitor, or a combination thereof. (Item 30) The method according to any one of items 25 to 29, wherein the disease is an inflammatory disease or a thrombotic disease. (Item 31) The method according to any one of items 25 to 29, wherein the disease is a thrombotic hematological disease or a thrombotic renal disease. (Item 32) The method according to any one of items 25 to 29, wherein the disease is a renal disease selected from the group consisting of atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy (C3G), dense deposit disease (DDD), membranoproliferative glomerulonephritis (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). (Item 33) The method according to any one of items 25 to 29, wherein the disease is a hematological disease selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), secondary HUS due to solid organ transplantation or hematopoietic stem cell transplantation, thrombotic microangiopathy (TMA), and cold agglutinin disease (CAD). (Item 34) The method according to any one of items 1 to 33, wherein the detection includes an immunoassay (e.g., ELISA or RIA), electron microscopy (EM), tandem mass tag (TMT), luminescence assay (e.g., LUMINEX), or fluorescence immunoassay (FIA). (Item 35) The method according to any one of items 1 to 34, wherein the detection step is performed in a multiplex format. (Item 36) The method according to any one of items 1 to 34, wherein the detection step is performed by measuring markers in several distinct tissues in one sample and / or by monitoring multiple promising complement proteins and complement pathways in a single assay. (Item 37) The method according to any one of items 1 to 36, wherein the EV enrichment step is performed before the first marker and optionally the second marker contact the individual antibody or its antigen-binding fragment. (Item 38) The method according to any one of items 1 to 37, wherein the biological sample is obtained from a non-invasive liquid biopsy protocol. (Item 39) A method for detecting complement activation in a subject's kidney tissue, comprising: (a) contacting a urine sample from the subject, comprising extracellular vesicles (EVs) or EVs containing a first marker that is an EV-specific marker or a tissue-specific marker displayed on the membrane thereof or a membrane-bound portion thereof, with a first capture antibody or an antigen-binding fragment thereof specific for the first marker, thereby capturing EVs or membranes containing the first marker; (b) optionally, contacting the sample with a second capture antibody or an antigen-binding fragment thereof to capture EVs or a membrane-bound portion thereof containing a second capture marker different from the first marker; (c) the presence of components of the complement pathway on the captured EVs or the membrane-bound portion thereof or The level is detected qualitatively or quantitatively using an antibody or antigen-binding fragment specific to the component, thereby detecting complement activation in the biological sample. A method including, The aforementioned EV-specific marker is selected from the group consisting of CD9, CD63, and CD81. The aforementioned tissue-specific marker is (1) Podocalixin (PODXL) specific to podocytes in the glomerulus, (2) Aquaporin 2 (AQP2) specific to the epithelium of the convoluted tubules, (3) Uroplakin 1b (UPK1b) specific to the bladder epithelium, and (4) Glycophorin A (GYPA) specific to red blood cells (RBCs) Selected from the group consisting of, and A method wherein the components of the complement pathway are selected from the group consisting of MAC, C3, C5b-9, C4, C1q, and C9. (Item 40) The use of at least one first capture antibody for capturing at least one first target, at least one second capture antibody for capturing at least one second target, and at least one complement protein-specific detection antibody for detecting the amount of the captured at least one first target, the amount of the captured at least one second target, or both. (Item 41) A method for screening test compounds for complement regulation, (a) Obtain samples containing extracellular vesicles (EVs) or their membrane-bound portions derived from subjects suffering from complement disorders (e.g., animals such as mice, rabbits, hamsters, sheep, llamas, dogs, monkeys, chimpanzees, or humans) before and after administration of the test compound to the subjects, (b) Contacting a portion of the sample with at least one first capture antibody or its antigen-binding fragment to capture at least one first marker on the EV or its membrane-binding portion, (c) In some cases, a portion of the sample is brought into contact with at least one second capture antibody or its antigen-binding fragment to capture at least one second marker on the EV or its membrane-bound portion, (d) Contacting the captured EV or its membrane-bound portion with at least one detection antibody or its antigen-binding fragment specific to a complement system-related component, (e) The presence or level of the complement components on the EV or its membrane-bound portion is measured qualitatively or quantitatively by detecting the detection antibody or the antigen-binding fragment thereof, and the adjustment of the presence or level of the complement components in the sample after administration of the test compound (e.g., an increase or decrease, preferably a decrease) compared to before administration of the test compound indicates that the test compound can modulate complement. Methods that include... (Item 42) The method according to item 41, wherein the test compound can specifically modulate Clq, Cl, Cls, C2, MASP-2, MASP-3, factor D, factor B, propergin (factor P), factor H, C3 / C5 convertase, C5, C5a / C5aR, C3a / C3aR, C6, or CD59. (Item 43) The method according to item 41, wherein the test compound is a monoclonal antibody, a small molecule, or an siRNA / RNAi. (Item 44) The method according to item 41, wherein the regulatory activity of the test compound is compared with the regulatory activity of a molecule having complement regulatory activity. (Item 45) The method described in item 44, provided in Table A, wherein the molecule is as described above.

Claims

[Claim 1] The invention described in the specification.

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