Detection of complement proteins

Abstract

Methods for detecting and determining levels of complement proteins, particularly using mass spectrometry, are disclosed. Methods for identifying subjects having or at risk of developing a complement-associated disorder, and methods for treating a complement-associated disorder are also disclosed.

Description

Technical Field

[0001] This application claims priority to GB2006789.8, filed on May 7, 2020, the content and elements of which are incorporated herein by reference for all purposes. Field of the Invention The present invention relates to the fields of molecular biology and medicine. More specifically, the present invention relates to methods for detecting complement proteins, and to the use of such methods for diagnostic and therapeutic uses.

Background Art

[0002] The complement system contributes to natural host immune defenses by assisting in the rapid recognition and elimination of microbial invaders. However, complement dysregulation can contribute to inflammatory, immune-related, and age-related conditions. As a result, inappropriate control of the complement system has been associated with a wide variety of diseases in humans, such as eye and kidney diseases, as well as neurological diseases and cancer (Morgan, B.P., Semin Immunopathol, 2018. 40(1): p. 113-124; Halbgebauer, R. et al., Semin Immunol, 2018. 37: p. 12-20; Ma, Y. et al., Aging Dis, 2019. 10(2): p. 429-462; and Kleczko, E.K. et al., Front Immunol, 2019. 10: p. 954).

[0003] Complement pathway activation and regulation are controlled by complex interactions between pathway activators and inhibitors. These activators and inhibitors are generally enzymes that cleave and inactivate complement molecules on biological surfaces and / or in solution to maintain stable control of complement activation species. The complement pathway is in a state of fluid and balance, and interfering with this balance can lead to the above-mentioned inappropriate activation and consequences.

[0004] One activating molecule is complement component 3 (C3), which is a member of the complement II pathway and amplification loop. C3 contains β and α' chains, which associate via interchain disulfide bonds. During complement activation, C3 is cleaved to produce two functional fragments, C3a and C3b. C3a is a potent anaphylatoxin. Deposition of C3b on biological surfaces, such as the extracellular matrix and cell surface, is a central activation mechanism of the II pathway. C3b is a potent opsonin that targets pathogens, antibody-antigen immune complexes, and apoptotic cells for phagocytosis by phagocytic cells and NK cells. Surface-bound C3b also reacts with other complement proteins to form an active convertase enzyme capable of producing further (surface-adherent) C3b molecules, thereby activating and amplifying the complement response (Clark, SJ et al., J Immunol, 2014. 193(10): p. 4962-70). C3b associates with factor B to form C3bBb-type C3 convertase, and C3bBb associates with C3bBb to form C3bBb3b-type C5 convertase. Proteolytic cleavage of C3 also produces C3a and C3b via the classical complement pathway and the lectin pathway.

[0005] Insufficient regulation of C3 convertase leads to the overproduction of C3b and C3a molecules, as well as a shift in the complement cascade toward the final lysis pathway. This results in the production of potent anaphylatoxins, C5a, and cytolytic protein complexes referred to as membrane attack complexes; both of which provide potent inflammatory signals (Clark, Sj et al., see above). This ultimately leads to cell / tissue destruction and local inflammatory responses.

[0006] Complement C3b activation is regulated by the complement protein factor I (FI). FI prevents complement activation by cleaving C3b into a proteolytically inactive form called iC3b, which cannot participate in convertase assembly, and further into downstream products iC3dg and C3d. FI requires the presence of cofactors, examples of which include the blood-derived factor H (FH) protein and the membrane-bound surface cofactor "complement factor 1" (CR1; CD35). FH and CR1 also assist in deconstruction acceleration activity, which can aid in the deconstruction of already formed C3 convertases.

[0007] FH is encoded by the CFH gene on human chromosome 1q32, within the RCA (complement regulator) gene cluster. A naturally occurring truncated form of FH, called FH-like protein 1 (FHL-1), arises from alternative splicing of the CFH gene and possesses FH-like cofactor activity. FH contains 20 CCP domains. FHL-1 is identical to FH with respect to the first seven CCP domains, then terminates with a unique 4aa C-terminus.

[0008] Proteins encoded by the CFHR1-FHR5 genes at the RCA locus also exert complement regulatory functions. The CFHR1-FHR5 genes encode a group of five secreted plasma proteins (FHR-1-FHR-5) that are mainly synthesized by hepatocytes. FHR proteins maintain a certain degree of sequence homology with the C3b-binding domain of FH and are thought to enhance complement activation (Skerka et al., Mol Immunol. 2013, 56:170-180).

[0009] One complement-related disorder is macular degeneration, such as age-related macular degeneration (AMD). Macular degeneration is thought to be driven in part by complement-mediated attack on the tissues of the eye. The primary driving factor for AMD risk is a gene mutation at the RCA locus that results in dysregulation of the complement cascade. AMD is the leading cause of blindness in developing countries: it currently accounts for 8.7% of all global blindness registries. It is estimated that 196 million people will be affected by 2020 and this number will increase to 288 million by 2040 (Wong et al. Lancet Global Health (2014) 2:e106-16). AMD manifests as progressive destruction of the macula, the central part of the retina at the back of the eye, resulting in loss of central vision. In the early stages of the disease, morphological changes in the macula are observed, such as loss of blood vessels in the choroidal capillaries (Whitmore et al., Prog Retin Eye Res (2015) 45:1-29), which are the capillary layer found in the choroid (a highly vascularized layer that supplies oxygen and nutrients to the outer retina). The choroidal capillaries are separated from the metabolically active retinal pigment epithelium (RPE) by Bruch's membrane (BrM), a five-layered sheet of thin (2-4 μm) cell-free extracellular matrix. BrM has two main functions: providing the base layer and vascular walls of the RPE. The structure and function of Bruch's Membrane (BrM) are outlined in Curcio and Johnson, Structure, Function and Pathology of Bruch's Membrane, Ryan et al. (2013), Retina, Vol. 1, Part 2: Basic Science and Translation to Therapy. 5th ed. London: Elsevier, pp. 466-481, and this entire work is incorporated herein by reference.

[0010] The role of complement in AMD is outlined, for example, in Zipfel et al., Chapter 2, Lambris and Adamis (eds.), Inflammation and Retinal Disease: Complement Biology and Pathology, Advances in Experimental Medicine and Biology 703, Springer Science+Business Media, LLC (2010), which is incorporated herein by reference in its entirety. A key characteristic of AMD is hyperactive complement, including cell / tissue destruction and local inflammatory responses. Characteristic lesions of early AMD, known as drusen, develop within the BrM adjacent to the RPE layer (Bird et al., Surv Ophthalmol 1995, 39(5):367-374). Drusens are formed from accumulations of lipids, proteins, and cell debris, and contain a swathe of complement activators (Anderson et al., Prog Retin Eye Res 2009, 29:95-112; Whitcup et al., Int J Inflam 2013, 1-10). The presence of drusen within the BrM disrupts the flow of nutrients from the choroid to RPE cells across this extracellular matrix, leading to cellular dysfunction and ultimately death, resulting in vision loss.

[0011] Atrophic (dry) AMD, also known as geographic atrophy, accounts for approximately 50% of late-stage AMD cases. In the remaining proportion of late-stage cases, choroidal neovascularization (CMV) develops, where increased synthesis of vascular endothelial growth factor (VEGF) by RPE cells promotes the proliferation of new blood vessels from the choroid / choroidal capillaries, which then break through the BrM and enter the retina. These new blood vessels leak out and eventually form scar tissue; this is called wet (neovascular or exudative) AMD. Wet AMD is the most pathogenic form of late-stage AMD and has different disease characteristics from atrophic AMD. There are treatments for wet AMD; for example, injections of anti-VEGF agents into the vitreous humor of the eye can delay or reverse the proliferation of these blood vessels, but it is impossible to prevent their formation in the first place. Geographic atrophy ("atrophic" AMD) remains untreatable.

[0012] FHL-1 is dominant in the BrM, suggesting that this variant plays a crucial role in protecting retinal tissue from complement-mediated attack (Clark, SJ et al., see above). FH is found in blood vessels at higher concentrations than FHL-1. Both FH and FHL-1 protect the choroid (the capillary network underlying the BrM) from complement hyperactivation in the ECM. The roles of the five FHR proteins are not as well understood, but there is some evidence that they may counteract the inhibitory effects of FH and FHL-1 (Clark, SJ and PN Bishop, J Clin Med, 2015. 4(1): p. 18-31).

[0013] WO2019 / 215330 states that FHR-4 is a positive regulator of complement activation, preventing FH-mediated C3b disruption and guiding C3 convertase formation and the progression of the complement activation loop. High levels of circulating FHR-4 indicate an increased risk of developing complement-related disorders.

[0014] Defining the precise molecular changes and activation states underlying complement process dysregulation in human disease tissues remains challenging, primarily due to the need for measurement at the protein level and an understanding of the relative amounts of different regulatory factors. For effective diagnosis and treatment of complement-related disorders, accurate measurement of absolute levels of FH and related RCA locus proteins in plasma, as well as levels of FI and C3b themselves, is essential. While assays for FH have been developed, the separate measurement of FHL-1 and FHR1-5 is difficult due to the high sequence homology among all these proteins. This sequence similarity has demonstrated that, with the exception of the full-length FH protein, it is difficult to generate antibodies specific to only one of these family members to obtain useful immunoassays.

[0015] In another example, recent studies have used mass spectrometry to quantify levels of FH and FHR1-5, but these assays have been unable to detect FHL-1, a biologically important isoform found at significant levels in the blood and in key sites of AMD lesions (Zhang, P. et al., Proteomics. 2017;17(6):10).

[0016] This invention aims to address these problems. [Prior art documents] [Patent Documents]

[0017] [Patent Document 1] WO2019 / 215330 [Non-patent literature]

[0018] [Non-Patent Document 1] Morgan, BP, Semin Immunopathol, 2018. 40(1): p. 113-124 [Non-Patent Document 2] Halbgebauer, R. et al., Semin Immunol, 2018. 37: p. 12-20

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Summary of the Invention

[0019] The present invention relates to detecting complement proteins using mass spectrometry. This application describes a method that can distinguish between different complement factor H, FHL-1, and five complement factor H-related (FHR) proteins, despite their sequence similarity. The method can also distinguish between breakdown products derived from C3 and other complement-related proteins with high sequence similarity.

[0020] <照 A method for detecting at least one complement protein in a sample, comprising digesting the protein(s) with endoprotease GluC to obtain one or more peptides; and detecting one or more peptides by mass spectrometry.

[0021] The present invention also provides a method for determining the level of at least one complement protein in a sample, comprising digesting the protein(s) with endoprotease GluC to obtain one or more peptides; and determining the level of one or more peptides by mass spectrometry.

[0022] In some embodiments, the step of detecting one or more peptides and / or determining the level of one or more peptides comprises the step of measuring one or more peptides by mass spectrometry. In some embodiments, the step of detecting one or more peptides and / or determining the level of one or more peptides includes the step of measuring the level of one or more peptides by mass spectrometry alone.

[0023] In some embodiments, the method includes the step of determining the concentration of one or more complement proteins in a sample. In another aspect, the present invention provides a method for preparing at least one complement protein for analysis, comprising the step of digesting the protein(s) with endoproteinase GluC to obtain one or more peptides.

[0024] In some embodiments, the complement protein(s) is one or more of FH, FHL-1, FHR1, FHR2, FHR3, FHR4, and / or FHR5. In some embodiments, the complement protein(s) is FH and / or FHL-1. In some embodiments, the complement protein(s) is involved in the complement amplification loop and / or C3 convertase activity. In some embodiments, the complement protein(s) is a degradation product of C3b. In some embodiments, the complement protein(s) is one or more of C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d. In some embodiments, the complement protein(s) is one or more of C3, C3a, C3f, C3c, and / or C3d. In some embodiments, the complement protein(s) are C3b and / or iC3b. In some embodiments, the complement protein is FI. All combinations of the above proteins are assumed. The methods described herein may include the step of detecting / determining the levels of two or more complement proteins. In some embodiments, the method includes the simultaneous detection / determination of the levels of two or more complement proteins.

[0025] In some embodiments, the sample is obtained from a subject. In some embodiments, the method includes the step of obtaining a sample from a subject. In some embodiments, the sample includes or is derived from blood, lymph, plasma, serum, tissue, or cells.

[0026] The peptide may be any suitable peptide disclosed herein. One or more peptides may include:

[0027] [ka]

[0028] A selection may be made from the group consisting of the following: The present invention also provides the use of endoproteinase GluC for the preparation of at least one complement protein for detection by mass spectrometry, and optionally for the preparation of at least two complement proteins, for example, any combination of the proteins described herein, for detection by mass spectrometry.

[0029] A method for detecting the turnover of C3b is also provided, which includes a method for detecting at least one complement protein in a sample, as described herein, for example.

[0030] We also provide a method for determining the presence and / or levels of complement proteins in a subject, the method comprising the step of performing a method such as that described herein. In some embodiments, the method is performed on a sample obtained from the subject.

[0031] The present invention also provides a method for determining whether a subject is at risk of developing complement-related disorders: a) Digesting at least one complement protein in the sample obtained from the subject with endoproteinase GluC to obtain one or more peptides; b) Determine the presence and / or level of one or more peptides by mass spectrometry; and Using the results of c)(b), determine the likelihood that the subject will develop complement-related disorders. The method also includes the process.

[0032] A method for identifying subjects with complement-related disorders: a) Digesting at least one complement protein in the sample obtained from the subject with endoproteinase GluC to obtain one or more peptides; b) Determine the presence and / or level of one or more peptides by mass spectrometry; and c)(b) Use the results to determine whether the subject has a complement-related disorder. The method also includes the process.

[0033] In some embodiments, the method includes (d) a step of treating a subject who is at risk of developing or has been determined to have a complement-related disorder. The step of treating the subject may include administering a therapeutically effective amount of a complement-targeted therapeutic agent to the subject.

[0034] A method for selecting subjects for the treatment of complement-related disorders using complement-targeting therapeutic agents: a) Digesting at least one complement protein in the sample obtained from the subject with endoproteinase GluC to obtain one or more peptides; b) Determine the presence and / or level of one or more peptides by mass spectrometry; and c)(b) Use the results to determine whether the subject requires complement-targeting therapy. The method, including the process, is also provided.

[0035] A method for treating a subject suspected to have complement-related disorders: a) Digesting at least one complement protein in the sample obtained from the subject with endoproteinase GluC to obtain one or more peptides; b) Determine the presence and / or level of one or more peptides by mass spectrometry; and treat the disorder based on the results of (b). The method, including the process, is also provided.

[0036] In some embodiments, the method includes the step of administering an effective amount of a complement-targeting therapeutic agent to a subject, for example, a subject identified as having a disorder as described herein, or a subject selected for such treatment.

[0037] A complement-targeting therapeutic agent for use in a method of treating complement-related disorders in a subject, wherein the method is: a) Digesting at least one complement protein in the sample obtained from the subject with endoproteinase GluC to obtain one or more peptides; b) Determine the presence and / or level of one or more peptides by mass spectrometry; and Based on the results of c)(b), administer an effective dose of complement-targeting therapy. The complement-targeting therapeutic agent, including the process, is also provided.

[0038] In some embodiments, the method includes the step of obtaining a sample from a subject containing at least one complement protein. The sample may include, or be derived from, blood, lymph, plasma, serum, tissue, or cells.

[0039] In some embodiments, complement-related disorders are macular degeneration. In some embodiments, complement-related disorders are selected from AMD, geographic atrophy ("atrophic" (i.e., non-exudative) AMD), early AMD, EOMD, mid-stage AMD, late / progressive AMD, "exudative" (neovascular or exudative) AMD, choroidal neovascularization (CNV), and / or retinal dystrophy.

[0040] In some aspects, complement-related disorders include hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), DEAP HUS (FHR plasma protein deficiency and autoantibody-positive form of hemolytic uremic syndrome), autoimmune uveitis, membranoproliferative glomerulonephritis type II (MPGN II), sepsis, Henoch-Schönlein purpura (HSP), IgA nephropathy, chronic kidney disease, paroxysmal nocturnal hemoglobinuria (PNH), autoimmune hemolytic anemia (AIHA), systemic lupus erythematosus (SLE), Sjögren's syndrome (SS), rheumatoid arthritis (RA), C3 glomerulopathy (C3G), dense deposit disease (DDD), and C3 nephritis factor glomerulonephritis (C3NF). The following conditions are selected from GN, FHR5 nephropathy, hereditary angioedema (HAE), acquired angioedema (AAE), encephalomyelitis, atherosclerosis, neurodegenerative / neurodegenerative diseases, dementia, multiple sclerosis (MS), cancer, stroke, Parkinson's disease, and / or Alzheimer's disease.

[0041] In any diagnostic or therapeutic method described herein, the complement protein may be any one or more of the proteins provided herein, i.e., any one or more of FH, FHL-1, FHR1, FHR2, FHR3, FHR4, FHR5, FI, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d, or any combination thereof. The protein(s) may be involved in the complement amplification loop and / or C3 convertase activity. The complement protein may be a degradation product of C3b. In some embodiments, the complement protein(s) may be one or more of FH, FHL-1, FHR1, FHR2, FHR3, FHR4, and / or FHR5. In some embodiments, the complement protein may be FH and / or FHL-1. In some embodiments, the complement protein(s) is one or more of C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d. In some embodiments, the complement protein(s) is one or more of C3, C3a, C3f, C3c, and / or C3d. In some embodiments, the complement protein(s) is C3b and / or iC3b. In some embodiments, the complement protein is FI. The one or more proteins may be any suitable peptide, such as any one of those disclosed herein, e.g., any one of SEQ ID NOs. 20-60.

[0042] In some embodiments, an increase in one or more levels of C3, C3b, C3a, iC3b, FHR1, FHR2, FHR3, FHR4 and / or FHR5 compared to a reference value (one or more); and / or a decrease in one or more levels of iC3b, C3f, C3c, C3dg, C3d, C3g, FI, FH, FHL-1, FHR1, FHR2, FHR3, FHR4 and / or FHR5 indicates that the subject is at risk of developing or has such a complement-related disorder.

[0043] A kit for use in a method for detecting and / or determining the level of one or more complement proteins in a sample, the kit comprising endoproteinase GluC, is also provided. [Brief explanation of the drawing]

[0044] [Figure 1] Schematic diagram illustrating the C3 proteolysis cascade and the proteolysis events leading to the formation, degradation, and inactivation of C3b (modified from Maillard et al., J Am Soc Nephrol. 2015 Jul;26(7):1503-12). Proteoform-specific peptides for mass spectrometry are underlined. [Figure 2] LC-SRM traces showing the detection of heavily-labeled synthetic standard peptides of individual RCA locus proteins derived from plasma samples. [Figure 3-1] Linearity data of peptides derived from FH, FHL-1, and FHR1-5. [Figure 3-2] Linearity data of peptides derived from FH, FHL-1, and FHR1-5. [Figure 3-3] Linearity data of peptides derived from FH, FHL-1, and FHR1-5. [Figure 4-1] Data confirming that C3 and C3 degradation products in human plasma can be detected by MS with sufficient specificity and sensitivity. 4A: Total ion chromatograph from SRM-MS analysis showing specific and simultaneous detection of C3b fragment-specific peptides. 4B: Linearity data for 7 out of 10 peptides spiked within the plasma background. [Figure 4-2] Data confirming that C3 and C3 degradation products in human plasma can be detected by MS with sufficient specificity and sensitivity. 4C: Coomassie-stained electrophoresis gel of C3 degradation products obtained in vitro. 4D: MS quantification of key C3 fragments from in vitro assay products shown in 4C. [Modes for carrying out the invention]

[0045] Mass spectrometry (MS) can be used to detect, differentiate, and quantify very similar proteins. To achieve excellent sensitivity with MS, for example, proteins in a sample are routinely digested into peptides using a specific protease. The industry standard protease for this purpose is trypsin. Other enzymes commonly used to digest proteins for MS analysis include elastase, chymotrypsin, or LysN.

[0046] Trypsin cleaves the C-terminus of all K and R residues, provided there are no subsequent proline residues, producing a peptide with a basic group at the C-terminus. This basic group then assists in the ionization and transmission of the peptide to the gas phase in the mass spectrometer. Peptides digested by trypsin tend to be ionized more efficiently in MS and therefore produce a stronger signal than peptides digested by non-trypsin enzymes. Using MS, individual peptides in the sample digest can be detected with signals proportional to their abundance. The parent protein concentration can be obtained from the relative abundance (signal) of the endogenous peptide compared to, for example, an exogenous "standard" peptide containing stable isotopes.

[0047] However, trypsin digestion of complement proteins FH and FHL-1 does not produce peptides that can be individually detected using MS alone. The only FHL-1 specific trypsin peptide is a 4-amino acid C-terminal sequence, which is too small to be reliably detected by MS techniques. FHR proteins also share substantial sequence identity, which means that it is difficult to distinguish between them and measure them specifically using, for example, antibody-based assays.

[0048] The inventors have developed a unique targeting mass spectrometry assay using a non-standard proteolytic enzyme, GluC(V8 protease), to produce separate proteotype peptides for all FHR proteins, as well as a proteotype peptide that can be used to distinguish between FHL-1 and FH, which can be used for simultaneous detection and accurate measurement of all seven key regulatory proteins encoded from the CFH gene cluster in plasma, namely FH, FHL-1, and FHR1, FHR2, FHR3, FHR4, and FHR5, using a single MS assay.

[0049] FHL-1 is a distinct biological entity derived from FH. While these proteins have similar functions, the size of FHL-1 suggests that its distribution within the body is likely to differ from that of FH. This is evident in the eye, where FHL-1 can traverse the retinal side of Bruch's membrane, e.g., where drusen are formed, whereas larger FH proteins cannot. See, for example, Clark et al., J Immunol 2014, 193(10) 4962-4970 and Clark et al., Frontiers in Immunology 2017 8:177, which are incorporated herein in their entirety. In this regard, there is evidence that FHL-1 is a major driver of complement C3b turnover in the eye, which suggests that levels of FHL-1 are more likely to signal disease risk than levels of FH.

[0050] GluC can also produce proteotype peptides of C3b and FI, enabling direct measurement of C3b itself, as well as its proteolytic enzymes and required liquid-phase complement factors. Therefore, the method described herein means that all these complement proteins can be measured using a single assay. Furthermore, C3b degradation occurs through trypsin-like cleavage at basic residues (K and R), and therefore, trypsin digestion of C3b degradation products is incapable of producing peptides useful for analysis. In contrast, C3 turnover is measurable using the MS approach of the present invention because GluC digestion also yields proteotype neopeptides from many C3 inactivation and degradation products generated during inactivation cleavage. The inventors hereby demonstrate that a range of products produced as a result of C3 / C3b cleavage can be detected and quantified using the same single GluC / MS assay. This allows for accurate determination of the concentrations of all known C3 fragments, e.g., iC3b, C3c, C3dg, and C3d. Therefore, the method described herein is not only capable of measuring the absolute levels of regulatory complement proteins, but also of tracking the protein products resulting from C3 inactivation, and thus, of evaluating the progress of complement activation and amplification loops.

[0051] This is preferable because the measurement of C3 degradation products is analytically difficult. The pattern of C3 degradation is complex; first, C3a and C3b are formed, then C3b is cleaved into iC3b (which cannot drive the formation of a membrane attack complex (MAC) but can still act as an opsonin), and subsequently, iC3b is inactivated cleaved into C3c through the release of the C3dg fragment. Attempting to detect these products using antibodies is difficult. Each sequential cleavage step in this cascade generates a novel proteoform (a distinct type of protein encoded from the same gene, including cleavage and splice variants), while these likely share sequence homology and undergo only minor structural changes. The creation of antibodies against each type is unlikely to be successful, and while there are methods to measure a single component after some form of separation, such as measuring C3dg after concentration based on polyethylene glycol, simultaneous measurement of all fragments in the same sample is currently impossible.

[0052] Accordingly, the present invention relates to a single methodology for the simultaneous determination of the presence, absolute levels, and relative molar ratios of seven individual complement-related proteins from the CFH family, which may be referred to herein as the “complementome,” as well as the C3b inactivating enzyme FI, the central complement component C3, and seven proteins derived from C3 degradation. The ability to detect the absolute levels of so many complement-related proteins in a single assay is crucial for the detection, diagnosis, and treatment success of complement-related diseases.

[0053] Complement proteins The complement system is a central component of innate immunity, acting as the first line of defense against foreign and modified host cells. It is activated during microbial infections, inducing inflammation and promoting the elimination of pathogens. The complement system consists primarily of plasma proteins produced by membrane proteins expressed in the liver or on the cell surface. For an overview of the complement system, see, for example, Merle NS et al., Front Immunol. 2015 Jun 2;6:262, which is incorporated herein by reference in its entirety.

[0054] The complement system can be activated through three distinct pathways: the classical pathway (CP), the secondary pathway (AP), and the lectin-binding pathway (LP). In healthy individuals, AP is persistently active at low levels, monitoring for the presence of pathogens, but host cells are defended against complement attack and resistant to persistent low-level activation. C3b molecules bound to host cells are rapidly inactivated by a group of membrane-bound or plasma complement regulators.

[0055] In response to the recognition of microbial molecular components, complement proteins are successively activated in an enzyme cascade: the activation of one protein enzymatically cleaves and activates the next protein in the cascade.

[0056] The three pathways converge on the production of C3 convertase, an enzyme that cleaves the central complement component C3 into the active product C3b, a large fragment that acts as an opsonin (binding to foreign microorganisms and increasing susceptibility to phagocytosis), and C3a, an anaphylatoxin that promotes inflammation. Together with factor B (FB), C3b forms C3 convertase (C3bBb), which further cleaves the C3 molecule, producing more C3b and C3a, and amplifying C3b deposition on the cell surface. This is the complement amplification loop. C3b deposition and complement activation can occur on cell-free structures (i.e., on the extracellular matrix), for example, in the eye, in Bruch's membrane (BrM) and the intercapillary walls of choroidal capillaries.

[0057] Activated C3 can induce a lytic pathway, which can damage the cell plasma membrane and some bacteria. Another anaphylatoxin produced by this process, C5a, attracts macrophages and neutrophils and also activates mast cells.

[0058] Once activated, the complement system requires tight regulation because newly generated complement activators, such as C3b, can induce severe inflammation and cell damage in the host. Several soluble and membrane-bound complement regulators ensure control of complement activation on the host cell surface and regulate different activation phases and sites of action (Skerka et al., Mol Immunol 2013, 56:170-180). Complement regulators are further described herein.

[0059] "Complement protein" may be used interchangeably herein with "complement regulator," "complement regulatory factor," or "complement system protein," and refers to protein components of the complement system or complement cascade, such as those described herein, for example, in Merle et al., Front. Immunol., 2015, 6:262 and Merle et al., Front. Immunol., 2015, 6:257, which are incorporated herein by reference in their entirety. "Complement protein" as used herein may be involved in any of the three complement pathways and / or amplification loops.

[0060] In some embodiments, the “complement proteins” as referred to herein are involved in the secondary pathway and / or complement activation loop. In some embodiments, the “complement proteins” as referred to herein are involved in the degradation, turnover, and / or inactivation of C3 or C3b, or are products of said degradation, turnover, and / or inactivation.

[0061] In some embodiments, “complement protein” as used herein may refer to one or more of FH, FHL-1, FHR1, FHR2, FHR3, FHR4, FHR5, FI, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d.

[0062] Complement factor H (FH) protein family Factor H (FH) controls the second complement pathway and amplification loop. It inhibits C3 convertase formation by competing with FB binding to C3b, and also acts as a cofactor for C3b inactivation by factor I (FI) to iC3b, thus preventing inappropriate complement activation and inflammation. FH also exhibits disintegration-accelerating activity, which can assist in the deconstruction of already formed C3 convertases. See, for example, Clark et al., J Immunol 2014, 193(10) 4962-4970, which is incorporated herein in its entirety.

[0063] The sequence of human FH (Uniprot P08603-1) is provided herein as Sequence ID No. 1. For an overview of the structure and function of FH, see, for example, Merle NS et al., Front Immunol. 2015 Jun 2;6:262, which is incorporated herein by reference in its entirety.

[0064] Human FH contains 20 CCP domains. The CFH gene is also referred to as FHL-1 and produces a partially excised form of FH that contains only the first seven CCP domains, followed by a unique 4-amino acid C-terminus (Clark et al., 2014, see above). The sequence of human FHL-1 (Uniprot: P08603-2) is provided herein as Sequence ID No. 2.

[0065] In the eye, full-length FH protein is found on the choroidal side of Bruch's membrane (BrM), particularly accumulating in choroidal capillaries (the capillary layer of the choroid). Small amounts are also found in patches on the RPE side of BrM, but FH was not observed in BrM itself. On the other hand, FHL-1 has been observed throughout BrM and in other ECM structures, such as drusen (Clark et al., 2014, see above). FHL-1 is likely to provide greater complement protection to BrM than FH, while FH provides primary protection to the choroidal ECM. Therefore, FHL-1 is considered a major complement regulator in BrM (a critical site for AMD pathogenesis). The methods described herein enable the individual detection and quantification of FH and FHL-1.

[0066] The CFHR-1~5 genes encode a group of five secreted plasma proteins (FHR-1~FHR-5) primarily synthesized by hepatocytes. FH, FHL-1, and FHR-1~FHR-5 are described in their entirety in Clark et al., J Clin Med, 2015. 4(1): 18-31, which are incorporated herein by reference.

[0067] FHR proteins are thought to maintain a degree of sequence homology with the C3b-binding domain of FH and enhance complement activation. See, for example, Skerka et al., Mol Immunol 2013, 56:170-180, which is incorporated herein in its entirety. These proteins are highly related and share a high degree of sequence identity. The N-terminus shares 36–94% sequence identity, while the C-terminal domain is very similar to the FH C-terminus (36–100%). The high amino acid identity among family members is evidenced by the fact that antibodies produced against FH can detect a large number of FHR proteins in plasma, and antibodies produced against FHR proteins cross-react with other FHRs. This cross-reactivity makes it difficult to purify FHR proteins from plasma and to determine their concentrations.

[0068] FHR proteins are divided into two groups based on their conserved domains. FHR1 (SEQ ID NO: 3), FHR2 (SEQ ID NOs: 3, 4), and FHR5 (SEQ ID NO: 10) form Group I and are characterized by their conserved N-terminus. These exist in plasma as homodimers and heterodimers mediated by the conserved N-terminal domain. Group II includes FHR3 (SEQ ID NOs: 6, 7) and FHR4 (SEQ ID NOs: 8, 9), which lack the N-terminal dimerizing domain but show a high degree of sequence similarity to the FH portion. All five FHR proteins contain a C-terminal sequence that recognizes and binds to C3b and is highly similar to the C-terminus of FH.

[0069] FHR1 is known to compete with FH and FHL-1 for binding to C3b. FHR1 has also been reported to bind to the C3b component of C5 convertase and interfere with MAC assembly (see, for example, Heinen S et al., Blood (2009) 114 (12): 2439-2447 and Hannan JP et al., PLoS One. 2016; 11(11):e0166200, which is incorporated herein in whole). In this specification, the term “FHR1” includes, and preferably includes, at least one of FHR1 (SEQ ID NO: 3; FHRA) and a second FHR1 isoform (FHRB) containing three point mutations. “FHR1” refers to FHR1 from any species and includes isoforms, fragments, variants or homologs of FHR1 from any species. In a preferred embodiment, “FHR1” refers to human FHR1.

[0070] FHR2 can inhibit C3 convertase activity and act to inhibit the amplification loop, but it can also activate the amplification loop. There are two FHR2 isoforms (SEQ ID NOs: 4 and 5). The protein has two glycosylated forms: a single glycosylated form (24 kDa) and a double glycosylated form (28 kDa). In this specification, the term “FHR2” includes at least one of the two isoforms or at least one glycosylated form, and preferably both isoforms and any glycosylated form. “FHR2” refers to FHR2 from any species and includes isoforms, fragments, variants or homologs of FHR2 from any species. In a preferred embodiment, “FHR2” refers to human FHR2.

[0071] FHR3 binds to C3b and C3d and may have low cofactor activity for FI-mediated cleavage of C3b. FHR3 can also upregulate complement. There are two FHR3 isoforms (SEQ ID NOs: 6 and 7). FHR3 is detected in plasma in numerous variants (ranging from 35 to 56 kDa), which reflects the presence of four different glycosylated variants of FHR3. Hereinafter, the term “FHR3” includes at least one of the two isoforms or at least one glycosylated form of FHR3, and preferably both isoforms and any glycosylated form. “FHR3” refers to FHR3 from any species and includes isoforms, fragments, variants or homologs of FHR3 from any species. In a preferred embodiment, “FHR3” refers to human FHR3.

[0072] The human CFHR4 gene encodes two proteins: FHR4A (SEQ ID NO: 8) and an alternative splice variant, FHR4B (SEQ ID NO: 9). WO 2019 / 215330, incorporated herein in its entirety, states that FHR4 is a positive regulator of complement activation and prevents FH-mediated C3b degradation. High levels of FHR-4 in tissues are likely to promote local inflammatory responses and cytolysis, leading to complement activation-associated disorders, and circulating FHR4 levels may be used as an indicator of the risk of developing complement-associated disorders. See, for example, Cipriani et al., Nat Commun 11, 778 (2020), incorporated herein in its entirety. In this specification, the term “FHR4” includes at least one of FHR4A isoform 1, FHR4A isoform 2 (G20 deletion derived from isoform 1), or FHR4B, and preferably includes FHR4A isoforms 1 and 2, as well as FHR4B. “FHR4” refers to FHR4 from any species, and this includes isoforms, fragments, variants, or homologs of FHR4 from any species. In a preferred embodiment, “FHR4” refers to human FHR4.

[0073] FHR5 also recognizes and binds to C3b on its own surface. FHR5 appears as a 62 kDa glycosylated protein. In this specification, the term "FHR5" includes any glycosylated mutant of FHR5, and preferably includes all isoforms and any glycosylated form. In this specification, "FHR5" refers to FHR5 from any species, and this includes isoforms, fragments, mutants or homologs of FHR5 from any species. In a preferred embodiment, "FHR5" refers to human FHR5.

[0074] Given the distinct roles of different members of the CFH family in complement activation and amplification, and in the pathogenesis of complement-related disorders, it is important that the presence and levels of all seven CFH family members are distinguishable. CFH family members, particularly FHR1–5, may also be used as biomarkers to diagnose or predict disorders in which complement dysregulation is pathologically involved.

[0075] C3, C3b, and degradation products C3 is a central complement component. The pathways through which C3 is processed into various downstream products can lead to complement activation, or complement inactivation and regulation, including, for example, inflammatory and immune responses. Therefore, from the perspective of complement pathogenesis and the treatment of complement-related disorders, it is important to be able to detect C3, C3b, and their downstream components / processing products, and to measure their levels, including their relative levels.

[0076] The processing of C3 is described, for example, in Foley et al. J Thromb Haemostasis (2015) 13: 610-618, which is incorporated herein by reference in its entirety. Human C3 (UniProt: P01024; SEQ ID NO: 12) contains a 1,663 amino acid sequence (including a 22-amino acid signal peptide at the N-terminus). Amino acids 23-667 encode the C3 β chain (SEQ ID NO: 13), and amino acids 749-1,663 encode the C3b α' chain (SEQ ID NO: 14). The C3 β chain and the C3 α' chain associate through an interacting disulfide bond (formed between cysteine ​​559 of the C3 β chain and cysteine ​​816 of the C3 α' chain) to form C3b. C3a is a 77-amino acid fragment corresponding to amino acid positions 672-748 of C3 (SEQ ID NO: 15), and is produced by the proteolytic cleavage of C3, which forms C3b.

[0077] The processing of C3b into the inactive iC3b, which is unable to promote further complement amplification on its own, involves proteolytic cleavage of the C3b α' chain at amino acid positions 1303 and 1320, forming α' chain fragment 1 (corresponding to amino acid positions 749-1663 of C3; SEQ ID NO: 16) and α' chain fragment 2 (corresponding to amino acid positions 1321-1663 of C3; SEQ ID NO: 17). Thus, iC3b contains the C3 β chain, C3 α' chain fragment 1, and C3 α' chain fragment 2 (associated via disulfide bonds). The cleavage of the α' chain also releases C3f, which corresponds to amino acid positions 1304-1320 of C3 (SEQ ID NO: 18).

[0078] iC3b is further processed into C3c, which contains the C3 β chain, C3 α' chain fragment 2, and C3c α' chain fragment 1 (corresponding to amino acids 749-954 of C3; SEQ ID NO: 19). This cleavage event produces fragment C3dg (corresponding to amino acids 955-1303 of C3; SEQ ID NO: 142), which autodegrades into fragments C3g (corresponding to amino acids 955-1001 of C3; SEQ ID NO: 143) and C3d (corresponding to amino acids 1002-1303 of C3; SEQ ID NO: 144).

[0079] The processing of C3b to iC3b is carried out by complement factor I (FI; encoded in humans by the gene CFI). Human complement factor I (UniProt: P05156; SEQ ID NO: 11) has a 583-amino acid sequence (including an N-terminus, 18-amino acid signal peptide). Amino acids 340-574 of the light chain encode the proteolytic domain of FI, which is a serine protease containing a catalytic triplicate involved in cleaving C3b to produce iC3b (Ekdahl et al., J Immunol (1990) 144 (11): 4269-74). The proteolytic cleavage of C3b by FI, which yields iC3b, is facilitated by cofactors including FH, CR1, and possibly some FHR proteins. Cofactors of FI typically bind to C3b and / or FI, and enhance the processing of C3b to iC3b by FI.

[0080] In this specification, any reference to complement proteins, e.g., C3, C3b, C3a, FH, FI, etc., refers to the protein of any species, and this includes isoforms, fragments, variants, or homologs of the protein of any species. In some embodiments, the protein is a mammalian protein (e.g., cynomolgus, human, and / or rodent (e.g., rat and / or mouse) protein). The isoforms, fragments, variants, or homologs of complement proteins described herein may optionally be characterized by having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of an immature or mature protein of a given species, e.g., a human protein sequence provided herein. The complement protein isoforms, fragments, variants, or homologs described herein may optionally be functional isoforms, fragments, variants, or homologs having the functional properties / activities of a reference protein, as determined, for example, by analysis using an assay suitable for the functional properties / activity.

[0081] Methods for detecting complement proteins The present invention relates to detecting the presence of one or more complement proteins and / or determining the level of such proteins using appropriate analytical techniques, such as those described herein.

[0082] In some respects, the present invention provides a method for detecting a complement protein, comprising the steps of contacting the protein with the endoproteinase GluC to obtain one or more peptides, and detecting one or more peptides by mass spectrometry.

[0083] In some respects, the present invention provides a method for determining the level of a complement protein, comprising the steps of contacting the protein with GluC, for example, to digest the protein with GluC to obtain one or more peptides, and then determining the level of one or more peptides by mass spectrometry. In some cases, the method comprises both the step of detecting a complement protein and the step of determining the level of a complement protein. The proteins may be the same protein, or the method may comprise the detection of a first complement protein and the determination of the level of a second complement protein.

[0084] In any method described herein, the step of detecting / determining the level of one or more peptides consists of the step of detecting / determining / measuring the peptide(s) by mass spectrometry. That is, the step of detecting / determining / measuring the peptide(s) is performed solely by mass spectrometry. The measurement of peptide(s) may include the step of determining the presence or absence of one or more peptides, and / or the level, amount and / or concentration of each peptide in the sample.

[0085] The term “digestion” as used herein refers to bringing a protein into contact with GluC under appropriate conditions, such as temperature, pH, etc., and for an appropriate time, so that the protein is digested, i.e., cut into two or more fragments. In some cases, digestion includes incubating a protein with GluC under appropriate conditions, such as those described herein.

[0086] In some respects, the present invention provides a method for preparing complement proteins for analysis, comprising the step of contacting a protein with endoproteinase GluC and / or digesting it with the enzyme to obtain one or more peptides. In some cases, the method comprises the step of preparing complement proteins for subsequent analysis. The one or more peptides may then be subjected to an analytical technique, such as mass spectrometry or any other suitable analytical technique. In some cases, the method comprises the step of preparing complement proteins for analysis by mass spectrometry. The presence and / or levels of the one or more peptides may be detected using the analytical technique.

[0087] In this specification, when “complement protein” refers to the singular form (i.e., “a / the complement protein”), it will be understood that the plural form / group / collection of different complement proteins is also intended. For example, any disclosure in this specification that includes one complement protein also includes more than one complement protein, i.e., at least one protein, or one or more proteins. In all aspects and embodiments described herein, “a / the complement protein” may mean “at least one complement protein.”

[0088] "Detecting" a protein, as used herein, means identifying or observing the presence or existence of a protein in, for example, a sample, cell, tissue, or subject. In this specification, the “level” of complement protein refers to the level, amount, or concentration of the protein in, for example, a sample, cell, tissue, or subject. The term, for example, “determining the level” of a protein refers in this specification to the measurement and / or quantification of the level, amount, or concentration of a protein. In some cases, “determining the level” includes calculating the level, amount, or concentration of a protein in a sample. The sample may be derived from a subject. In some cases, “determining the level” includes calculating the level, amount, or concentration of a protein in a subject, for example, using a sample taken from a subject. “Determining the level” of a protein may include digesting the protein with GluC to obtain one or more peptides, to obtain one or more peptides as described herein, and then calculating the level, amount, or concentration of the protein / peptide in, for example, a sample.

[0089] In some cases, “determining the level” includes quantifying, i.e., measuring, the level, amount, or concentration of a protein, for example, in a sample or subject. “Determining the level” may also include determining the concentration of a protein. Quantification / measurement may include comparing the level, amount, or concentration of a protein to a reference value and / or to that in a control sample, for example, taken from a subject at a different time point or from a healthy subject, e.g., a subject known not to have complement-related disorders.

[0090] In the methods described herein, the concentration of one or more complement proteins is compared to a reference value or reference level, sometimes referred to as a control. In some cases, the level of one or more complement proteins is compared to the level of the same complement proteins in a control subject without complement-related impairment. The reference value may be obtained from a control sample, and the control sample may itself be obtained from a control subject. Data or values ​​obtained from the individual to be tested, for example from a sample, may be compared to data or values ​​obtained from a control sample. In some cases, the control is the subject's spouse, partner, or friend.

[0091] In this specification, the term “reference value” refers to a known measurement used for comparison during analysis. In some cases, the reference value is one or a set of test values ​​obtained from individuals or groups of a defined health condition. The reference value may be one or a set of test values ​​obtained from a control. In some cases, the reference value is obtained by determining the level of complement protein in subjects known not to have complement-related disorders. In some cases, the reference value is established by determining the level or amount of complement protein previously obtained from the individual to be tested, for example, in the early stages of disease progression or before the onset of disease. The reference value may be obtained from samples obtained from the same subject or from one or more different subjects. The samples may be obtained from the same tissue / cell / fluid as the samples used in the present invention. The reference value may be a standard value, a standard curve, or a standard dataset. Values / levels that deviate significantly from the reference value may be described as atypical values / levels.

[0092] In some cases, the control may be a reference sample or reference dataset, or one or more values ​​derived from said sample or dataset. The reference value may be obtained from a reference sample or reference dataset. The reference value may be derived from one or more samples previously obtained from one or more subjects known to be without complement-related disorders and / or known or expected not to be at risk of developing complement-related disorders. The reference value may be obtained from one or more samples previously obtained from one or more subjects known to have complement-related disorders. The reference value may be derived from one or more samples previously obtained from one or more subjects known to be at risk of developing complement-related disorders. The reference value may be an average or mean value calculated from a reference dataset, such as mean protein level. The reference dataset / value may be obtained from a large-scale study of subjects known to have complement-related disorders.

[0093] The reference value may be derived from one or more previously obtained samples from one or more subjects who are in the same family as the subject of interest, or from one or more subjects who are not in the same family as the subject of interest.

[0094] The reference value may be derived from one or more samples previously obtained and / or analyzed from the individual / subject / patient being tested, for example, a sample obtained from the individual when it was in the early stages of complement-related disorder, or a sample obtained from the individual before the onset of complement-related disorder.

[0095] Reference values ​​may be obtained by performing an analysis of a sample taken from a control subject in parallel with a sample derived from the individual to be tested. Alternatively, reference values ​​may be obtained from a database or other previously obtained values. Reference values ​​may be determined concurrently with the methods disclosed herein, or may have been determined previously.

[0096] Control subjects from whom samples are obtained / are obtained may have undergone treatment for complement-related disorders and / or may be receiving complement-related therapy / therapy agents. The control may be a positive control in which the target molecule is present or expressed at a high level, or a negative control in which the target molecule is absent or expressed at a low level.

[0097] For example, a sample derived from one or more control subjects may contain any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, twelve, thirteenth, four, fifteenth, sixteenth, sixteenth, seventh, eighth, nineth, tenth, eleventh, twelveth, thirteenth, fourteenth, fifteenth, or sixteenth, or any combination thereof, of FHR1, FHR2, FHR3, FHR4, FHR5, FH, FHL-1, FI, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d. In some cases, each complement protein is present in a separate control sample. In some cases, the control sample contains a large number of complement proteins. In some cases, the method described herein includes the step of comparing the level of one or more complement proteins determined as described herein to a different sample, for example, one or more samples, each containing one or more complement proteins. In some cases, the methods described herein include the step of comparing the level of one or more complement proteins determined as described herein to a single sample in which the sample contains one or more complement proteins.

[0098] In some cases, control samples are obtained from the same tissue(s) as the samples obtained from the individual being tested. In some cases, control samples are obtained from a different tissue(s) than the samples obtained from the individual being tested. Control samples may be obtained from a control subject at a specific time(s) of the day or on a specific day. Samples obtained from the individual being tested are preferably obtained at the same time(s) of the day and / or on the same day as the control samples.

[0099] In some embodiments, the method includes a step of detecting / determining the level of complement proteins in a sample. The sample may be in vitro or ex vivo. The sample may be taken from a subject. The sample may be taken from any tissue or body fluid. In a preferred arrangement, the sample is taken from body fluid, more preferably from something circulating throughout the body. Thus, the sample may be a blood sample or a lymph sample. In a particularly preferred arrangement, the sample is a blood sample or a blood-derived sample. A blood-derived sample may be a selected fraction of the blood of a patient or subject, e.g., a selected cell-containing fraction or a plasma or serum fraction. A selected serum fraction may include the liquid portion of the blood obtained after the removal of fibrin clots and blood cells. Alternatively, the sample may include, or be derived from, a tissue sample, biopsy or isolated cells from the said individual. The sample may be taken from the eye, kidney, brain or liver, and may include, for example, cells derived from the eye, kidney, brain or liver. The sample may include retinal tissue. The sample may include RPE cells, or tissue derived from Bruch's membrane or choroid. The sample may contain drusen or other deposits of complement-related components.

[0100] In some embodiments, the methods described herein include the step of collecting or obtaining a sample, such as blood or tissue, from a subject. In some embodiments, the methods described herein are performed on a sample obtained from a subject. In some cases, the sample is a blood sample. The blood sample may be processed to obtain a plasma sample or a serum sample. In some cases, the method includes the step of obtaining a blood-derived sample from a subject. In some cases, the method includes the step of obtaining a plasma or serum sample from a subject. In some cases, the method includes the step of isolating a protein, such as total protein, from the sample. Suitable techniques for isolating proteins from biological samples are well known in the art. In some embodiments, the method does not include the step of isolating a protein from a subject; for example, the method is performed on an unprocessed sample.

[0101] In some embodiments, the method is performed in vitro. For example, the presence, level, amount, and / or concentration of complement proteins may be detected / determined in vitro.

[0102] In some cases, the method includes the step of determining the presence, level, amount, and / or concentration of one or more complement proteins in the subject. This may include the step of performing the method described herein in vitro and using the results to calculate the presence, level, amount, and / or concentration of one or more proteins in the subject.

[0103] A method for detecting at least one complement protein in a sample is also provided, comprising the steps of: digesting one or more proteins in the sample with endoproteinase GluC to obtain one or more peptides; and detecting one or more peptides in the sample by mass spectrometry.

[0104] A method for determining the level of at least one complement protein in a sample is also provided, comprising the steps of digesting one or more proteins in the sample with endoproteinase GluC to obtain one or more peptides, and then determining the level of one or more peptides in the sample by mass spectrometry.

[0105] For example, the step of detecting one or more peptides in a sample using mass spectrometry, or detecting and / or determining the level of one or more peptides by mass spectrometry, by the method described herein may include the step of applying mass spectrometry techniques to a sample, for example, by placing the sample in a mass spectrometer and instructing the mass spectrometer to analyze the sample. A variety of suitable mass spectrometry techniques are disclosed herein and are within the routine tasks of those skilled in the art.

[0106] In any aspect provided herein, the methods described herein may include both the step of detecting at least one complement protein and the step of determining the level of at least one complement protein. The complement proteins may be the same protein, and / or the method may include the step of detecting at least a first complement protein and determining the level of at least a second complement protein.

[0107] In some embodiments, the method described herein includes the step of detecting / determining the level of one complement protein. In some embodiments, the method described herein includes the step of detecting / determining the level of at least one complement protein, one or more complement proteins, and / or a group of complement proteins, as provided herein, for example.

[0108] In some embodiments, complement proteins are encoded from the RCA (complement regulatory factor) gene cluster, or RCA locus, on human chromosome 1. The RCA cluster is located on chromosome 1q32 and includes the CFH and CFHR1-5 genes. The gene cluster also includes membrane-bound proteins CR1 (CD35), CR2 (CD21), decay acceleration factor (DAF; CD55), and membrane cofactor protein (MCP; CD46), as well as soluble C4b-binding protein (C4bp).

[0109] The method described herein is suitable for detecting / determining the levels of numerous complement proteins through a single assay, i.e., obtaining analyzable peptides using a single enzyme GluC, and then detecting and / or determining the levels of said peptides using a single analytical technique, mass spectrometry. This method allows for the determination of the complementome of a sample or subject through a single assay.

[0110] In some embodiments, the methods described herein include the step of detecting / determining the level of any one or more of FH, FHL-1, FHR1, FHR2, FHR3, FHR4, and / or FHR5, for example, any or all combinations thereof. In some embodiments, the complement proteins (one or more) are selected from the group consisting of FH, FHL-1, FHR1, FHR2, FHR3, FHR4, and / or FHR5. In some cases, the method includes the step of detecting / determining the level of any one, two, three, four, five, six, and / or seven of FH, FHL-1, FHR1, FHR2, FHR3, FHR4, and FHR5, either individually or in combination. In some cases, the methods described herein make it possible to differentiate (i.e., distinguish, identify, separate) between the presence (or levels) of each of FH, FHL-1, FHR1, FHR2, FHR3, FHR4, and / or FHR5.

[0111] In some cases, the complement proteins to be detected / determined are FH and / or FHL-1. In some cases, the methods described herein include the step of detecting / determining the level of both FH and FHL-1. In some cases, the methods described herein differentiate (i.e., distinguish, identify, separate) between the presence of FH and the presence of FHL-1, and / or between the level / concentration of FH and the level / concentration of FHL-1. In some cases, the methods described herein allow or enable the detection of FH alone, i.e., without the detection of FHL-1. In some cases, the methods described herein allow or enable the detection of FHL-1 alone, i.e., without the detection of FH.

[0112] In some cases, the complement protein is any one or more of FHR1, FHR2, FHR3, FHR4, and / or FHR5, for example, any or all combination. In some cases, the complement protein is FHR4. In some cases, the methods described herein make it possible to differentiate (i.e., distinguish, identify, separate) between the presence (or levels) of each of FHR1, FHR2, FHR3, FHR4, and / or FHR5. In some cases, the methods described herein allow or enable the detection of FHR1 alone, i.e., without the detection of FHR2-FHR5. In some cases, the methods described herein allow or enable the detection of FHR2 alone, i.e., without the detection of FHR1 or FHR3-FHR5. In some cases, the methods described herein allow or enable the detection of FHR3 alone, i.e., without the detection of FHR1, FHR2, FHR4, or FHR5. In some cases, the methods described herein allow or enable the detection of FHR4 alone, i.e., without the detection of FHR1 to FHR3 or FHR5. In some cases, the methods described herein allow or enable the detection of FHR5 alone, i.e., without the detection of FHR1 to FHR4.

[0113] In some embodiments, the complement protein to be detected / determined is involved in the degradation, turnover, and / or inactivation of C3 / C3b. In some embodiments, the complement protein is produced by the degradation and / or inactivation of C3 / C3b, i.e., it is a product of C3b inactivation / degradation. In some embodiments, the method described herein includes a step of determining the presence, rate, and / or progress of C3b turnover. In some embodiments, the method described herein includes a step of detecting / determining the level of a protein involved in or produced as a result of the complement amplification loop. In some embodiments, the method described herein includes a step of detecting / determining the level of a protein involved in the production or degradation of C3 convertases. In some cases, the protein is a cofactor of FI, e.g., one of the FH, CR1, or FHR proteins. Any method disclosed herein for detecting at least one complement protein in a sample, for example, a method comprising the step of digesting a protein with GluC and then detecting the resulting peptide by mass spectrometry, may be otherwise described as a method for detecting C3 turnover, a method for detecting C3 degradation, a method for measuring C3b turnover or C3b degradation, or a method for measuring the progress of C3b turnover or C3b degradation.

[0114] Accordingly, in some respects, the present invention provides a method for detecting the turnover or degradation of C3b, comprising the steps described herein, for example, digesting at least one complement protein with endoproteinase GluC to obtain one or more peptides, and then detecting the peptides (one or more) by mass spectrometry. In some cases, the method comprises the steps of digesting at least two, three, four or more, up to 16 of the 16 complement proteins described herein, and then detecting them.

[0115] In some embodiments, the methods described herein include / further include the step of detecting / determining the level of FI, either alone or in combination with other complement proteins such as those described herein.

[0116] In some embodiments, the methods described herein include a step of detecting / determining the level of any one or more of C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d, for example, any or all combinations. In some embodiments, the complement proteins (one or more) are selected from the group consisting of C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d. In some cases, the method includes a step of detecting / determining the level of any one, two, three, four, five, six, seven, and / or eight of C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d in any combination. In some embodiments, the methods described herein include a step of detecting / determining the level of one or more of C3, C3a, C3f, C3c, and / or C3d. In some cases, the methods described herein include a step of determining the presence and / or level of C3b, iC3b, and / or C3dg, for example, through the methodology in Table 3. In some cases, the methods described herein include a step of detecting / determining the level of C3, C3b, and / or iC3b. In some cases, the methods described herein are capable of differentiating (i.e., distinguishing, identifying, separating) the presence (or level of) two or more or all of C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d.

[0117] The methods described herein also allow for the detection of a number of complement proteins and the differentiation of said complement proteins using one enzyme, e.g., GluC, and one analytical method, e.g., mass spectrometry. Using the methods described herein, any one of the individual proteins described herein, FH, FHL-1, FHR1, FHR2, FHR3, FHR4, FHR5, FI, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d, as well as any combination of these and all of them, i.e., any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, thirteenth, twelve, thirteenth, fourteenth, fifteenth, and / or sixteenth of these proteins, may be detected / their levels determined. In some embodiments, the complement protein(s) are selected from the group consisting of FH, FHL-1, FHR1, FHR2, FHR3, FHR4, FHR5, FI, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d. In some cases, FHL-1 may be detected / its level determined using the methods described herein, and one or more of FH, FHR1, FHR2, FHR3, FHR4, FHR5, FI, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d may be detected / its level determined. In some cases, the method includes the step of distinguishing (i.e., differentiating, identifying, separating) the presence / level of FHL-1, as well as the presence / level of one or more of FH, FHR1, FHR2, FHR3, FHR4, FHR5, FI, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d. The terms “distinguish,” “differentiate,” “identify,” and “separate” are used interchangeably herein.

[0118] In some cases, the methods provided herein enable the simultaneous detection of one or more of the following proteins, including any combination thereof: FH, FHL-1, FHR1, FHR2, FHR3, FHR4, FHR5, FI, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d, in a single assay. The methods provided herein enable the detection / determination of the level of one or more of the following proteins, including any combination thereof: FH, FHL-1, FHR1, FHR2, FHR3, FHR4, FHR5, FI, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d, in a single assay. The methods provided herein enable the production of distinct, separable, and detectable peptides from all the proteins listed above, so that the presence and / or level of each protein can be distinguished from the others.

[0119] In some cases, the present invention relates to a method for detecting and / or determining the levels of at least two complement proteins in a sample simultaneously and / or in a single assay: The protein is digested with endoproteinase GluC to obtain one or more peptides; and Mass spectrometry is used to detect one or more peptides and / or determine their levels. The present invention provides the method, including the steps involved.

[0120] In any method described herein, the complement protein may be any protein involved in one or more of the complement pathways. For example, the complement protein may be one or more of C1, C2, C4b2a, C4, C4a, C5, C5a, FB, FD, C3Bb, MASP1, MASP2, C1q, C1r, C1s, C6, C7, C8, C9, CD59, clusterin, propagin, and / or compstatin. In any embodiment described herein, the complement protein to be detected (or the protein that determines its level) is not one or more of C1, C2, C4b2a, C4, C4a, C5, C5a, FB, FD, C3Bb, MASP1, MASP2, C1q, C1r, C1s, C6, C7, C8, C9, CD59, clusterin, propagin, and / or compstatin.

[0121] In some cases, the present invention uses endoproteinase GluC to prepare at least one complement protein for detection by mass spectrometry. In some cases, the present invention uses endoproteinase GluC to prepare at least two, i.e., many or more, complement proteins for detection by mass spectrometry. The at least two complement proteins may be any two, three, four, or more, up to 16, of FH, FHL-1, FHR1, FHR2, FHR3, FHR4, FHR5, FI, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d in any combination as described herein.

[0122] Endoproteinase GluC, also known as glutamyl endopeptidase, is a serine proteinase that preferentially cleaves the peptide bond at the C-terminus of glutamic acid residues. The enzyme also cleaves aspartic acid residues 100 to 300 times slower than glutamic acid residues. The specificity of GluC depends on pH and buffer composition. At pH 4, the enzyme preferentially cleaves the C-terminus of E, while at pH 8, it further cleaves the D residue. The sequences of GluC are provided in SEQ ID NOs. 153 and 154.

[0123] In a preferred embodiment, the method described herein uses GluC alone (i.e., GluC only) to digest one or more complement proteins. In a preferred embodiment, in the method described herein, the step of digesting one or more proteins consists of the step of digesting one or more proteins with GluC. In a preferred embodiment, any method described herein does not use any other proteases, either alone or in combination with GluC. For example, in some embodiments, the digestion step of any method described herein may be performed without using or by any one or more of the following enzymes or agents: trypsin, chymotrypsin (high specificity or low specificity), Lys-C, Lys-N, Arg-C, Asp-N, elastase, LysargiNase, pepsin, Sap9, OmpT, BNPS-skatole, any caspase, clostripine (clostridiopeptidase B), CNBr, enterokinase, factor Xa, granzyme B, neutrophil elastase, proteinase K, thermolysin, non-GluC glutamyl endopeptidase, e.g., GluBI or GluSGB, proline endopeptidase, TEV protease, thrombin, formic acid, hydroxylamine, iodobenzoic acid, and / or NTCB (or any combination thereof).

[0124] GluC can be obtained from standard reagent suppliers, such as Sigma Aldrich, NEB, etc., and can be used according to the accompanying instructions for use or according to protocols well known in the art. Examples of protocols are described herein. The process of obtaining proteins from biological samples and suitable buffers for preparing the samples / proteins for GluC digestion will also be known to those skilled in the art. An example of cell lysis buffer is: 1 tablet of cOmplete per 10 ml of lysis buffer. TMThis product contains a Mini EDTA-free protease inhibitor cocktail, with 8M urea (4.8g per 10ml) diluted to <2M urea concentration, pH 8 (40mg per 10ml) in 50mM NH4HCO3 and 20mM methylamine.

[0125] In some cases, complement proteins are contacted with / incubated with / digested by the GluC enzyme for at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 hours. In some cases, complement proteins are contacted with / incubated with / digested by the GluC enzyme for at least 12 hours. In some cases, complement proteins are contacted with / incubated with / digested by the GluC enzyme for about 12 hours, for example 12 hours. In some cases, complement proteins are contacted with / incubated with / digested by the GluC enzyme for about 16 hours, for example 16 hours. The terms contacted, incubated and digested are used interchangeably herein.

[0126] In some cases, complement proteins are contacted with / incubated with the GluC enzyme / digested with the enzyme at a temperature of at least 20°C, at least 21°C, at least 22°C, at least 23°C, at least 24°C, at least 25°C, at least 26°C, at least 27°C, at least 28°C, at least 29°C, or at least 30°C. In some cases, complement proteins are contacted with / incubated with the GluC enzyme / digested with the enzyme at a temperature of at least 25°C. In some cases, complement proteins are contacted with / incubated with the GluC enzyme / digested with the enzyme at a temperature of about 25°C, for example, 25°C.

[0127] In some cases, complement proteins are contacted with / incubated with / digested by a GluC enzyme at a pH of at least 7.0, at least 7.1, at least 7.2, at least 7.3, at least 7.4, at least 7.5, at least 7.6, at least 7.7, at least 7.8, at least 7.9, at least 8.0, at least 8.1, at least 8.2, at least 8.3, at least 8.4, at least 8.5, at least 8.6, at least 8.7, at least 8.8, at least 8.9, or at least 9.0. In some cases, complement proteins are contacted with / incubated with / digested by a GluC enzyme at a pH of at least 8.0. In some cases, complement proteins are contacted with / incubated with / digested by a GluC enzyme at a pH of about 8.0, for example, 8.0.

[0128] In some cases, the GluC enzyme and complement protein are contacted / incubated in a weight / weight ratio of 1 / 75. The incubation step may include gentle shaking, for example, at 400 rpm.

[0129] The methods described herein may include a contact / incubation / digestion step, which includes any combination of temperature, pH, and / or time, as described above. In some cases, the contact / incubation / digestion is carried out at 25°C and pH 8 for 12 hours.

[0130] In some embodiments, the present invention relates to a method for detecting and / or determining the level of at least one complement protein in a sample, for example: The protein(s)(s)(s)(s)(s)(s)(s)))))))))))))))))))))))(wax(s)(s Mass spectrometry can detect one or more peptides. The present invention provides the method, including the steps involved.

[0131] In some embodiments, the present invention relates to a method for detecting and / or determining the level of at least one complement protein in a sample, for example: The protein(s) are digested with endoproteinase GluC to obtain one or more peptides, wherein the digestion includes the step of incubating the protein(s) and GluC at 25°C, pH 8 for up to 16 hours; and Mass spectrometry can detect one or more peptides. The present invention provides the method, including the steps involved.

[0132] The following peptides may be produced by GluC digestion of complement proteins, for example, as described herein. In some embodiments, the methods described herein include a step of detecting / determining the level of any one or more of these peptides, i.e., SEQ ID NOs. 20-141, or 155, 156, or 157, in any combination. All combinations of peptides are assumed. The masses of the peptides indicated by SEQ ID NOs. 20-27 may be found in Table 1.

[0133] In some embodiments, the FH peptide is VTYKCFE (SEQ ID NO: 20). In some embodiments, FH peptides are SNTGSTTGSIVCGYNGWSDLPICYE (SEQ ID NO: 112; Mass 2623.1206), NGWSPTPRCIRVKTCSKSSIDIE (SEQ ID NO: 113; Mass 2576.2839), LPKIDVHLVPDRKKDQYKVGE (SEQ ID NO: 114; Mass 2476.3801), YYCNPRFLMKGPNKIQCVDGE (SEQ ID NO: 115; Mass 2474.1545), NYNIALRWTAKQKLYSRTGE (SEQ ID NO: 116; Mass 2411.2709), KWSHPPSCIKTDCLSLPSFE (SEQ ID NO: 117; Mass 2274.0813), HGWAQLSSPPYYYGDSVE (SEQ ID NO: 118; Mass 2054.9010), ISHGVVAHMSDSYQYGEE (SEQ ID NO: 119; Mass 2007.8632), FDHNSNIRYRCRGKE (SEQ ID NO: 120; Mass 1893.9016), ITCKDGRWQSIPLCVE (SEQ ID NO: 121; Mass 1846.9069), GWIHTVCINGRWDPE (SEQ ID NO: 122; Mass 1781.8307), KAKYQCKLGYVTADGE (SEQ ID NO: 123; Mass 1772.8767), TTCYMGKWSSPPQCE (SEQ ID NO: 124; Mass 1716.6946), SYAHGTKLSYTCE (SEQ ID NO: 125; Mass 1458.6449), RVRYQCRSPYE (SEQ ID NO: 126; Mass 1455.7041), GFGIDGPAIAKCLGE (SEQ ID NO: 127; Mass 1446.7176), HGTINSSRSSQE (SEQ ID NO: 128; Mass 1301.5960), YQCQNLYQLE (SEQ ID NO: 129; Mass 1300.5758), WTTLPVCIVEE (SEQ ID NO: 130; Mass 1288.6373), KIPCSQPPQIE (SEQ ID NO: 131; Mass 1238.6329), SQYTYALKE (SEQ ID NO: 132; Mass 1101.5342), QVQSCGPPPE (SEQ ID NO: 133; Mass 1040.4597), KKDVYKAGE (SEQ ID NO: 134; Mass 1036.5553), GLPCKSPPE (SEQ ID NO: 135; Mass 926.4531), KVSVLCQE (Sequence ID 136; Mass 904.4688), HLKNKKE (Sequence ID 137; Mass 895.Any one or more of the following: 5239), GGFRISEE (SEQ ID NO: 138; Mass 893.4243), LLNGNVKE (SEQ ID NO: 139; Mass 885.4920), YPTCAKR (SEQ ID NO: 140; Mass 837.4167), or STCGDIPE (SEQ ID NO: 141; Mass 820.3273).

[0134] In some embodiments, the FHL-1 peptide is NGWSPTPRCIRVSFTL (SEQ ID NO: 21). In some aspects, the FHR1 peptide is ATFCDFPKINHGILY D This is EE (sequence number 22).

[0135] In some embodiments, the FHR1 peptide is NYNIALRWTAKQKLYLRTGE (SEQ ID NO: 91; mass 2437.3230). In some embodiments, the FHR2 peptide is RGWSTPPKCRSTISAE (SEQ ID NO: 23).

[0136] In some embodiments, the FHR2 peptide is AMFCDFPKINHGILYDEE (SEQ ID NO: 24). In some embodiments, the FHR2 peptide is YNFVSPSKSFWTRITCAEE (SEQ ID NO: 92; mass 2264.0572).

[0137] In some embodiments, the FHR3 peptide is VACHPGYGLPKAQTTVTCTE (SEQ ID NO: 25). In some embodiments, the FHR3 peptide is any one or more of the following: KGWSPTPRCIRVRTCSKSDIE (SEQ ID NO: 93; MUS 2418.2260), NGINQNYGRKFVQGNSTE (SEQ ID NO: 94; MUS 2074.9457), QVKPCDFPDIKHGGLFHE (SEQ ID NO: 95; MUS 2066.0043), FMCKLGYNANTSILSFQAVCRE (SEQ ID NO: 96; MUS 2494.1807), or YQCQPYYE (SEQ ID NO: 97; MUS 1092.4222).

[0138] In some embodiments, the FHR4 peptide is YQCQSYYE (SEQ ID NO: 26). In some embodiments, the FHR4 peptide is any one or more of the following: NSRAKSNGMRFKLHDTLDYE (SEQ ID NO: 98; MUS 2381.1546), DGWSHFPTCYNSSE (SEQ ID NO: 99; MUS 1628.6202), ISYGNTTGSIVCGE (SEQ ID NO: 100; MUS 1399.6289), or FMCKLGYNANTSVLSFQAVCRE (SEQ ID NO: 101; MUS 2480.1650).

[0139] In some embodiments, the FHR5 peptide is RGWSTPPICSFTKGE (SEQ ID NO: 27). In some embodiments, the FHR5 peptide is GTLCDFPKIHHGFLYDEE (SEQ ID NO: 102; Mass 2119.9673), YAMIGNNMITCINGIWTE (SEQ ID NO: 103; Mass 2042.9264), YGYVQPSVPPYQHGVSVE (SEQ ID NO: 104; Mass 2004.9581), GDTVQIICNTGYSLQNNE (SEQ ID NO: 105; Mass 1967.8895), IVCKDGRWQSLPRCVE (SEQ ID NO: 106; Mass 1887.9447), DYNPFSQVPTGE (SEQ ID NO: 107; Mass 1352.5884), QVKTCGYIPE (SEQ ID NO: 108; Mass 1136.5536), ANVDAQPKKE (SEQ ID NO: 109; Mass Any one or more of the following: 1098.5669), WTTLPTCVE (Sequence ID 110; Mass 1048.4899), or KVAVLCKE (Sequence ID 111; Mass 888.5102).

[0140] In some cases, the methods described herein include the step of detecting / determining the level of one or more of SEQ ID NOs. 21-27 in any combination. In some cases, any method described herein may include a step of detecting / determining the level of one or more of SEQ ID NOs: 28-37, 156, or 157 in any combination. In some cases, the methods provided herein are used to detect C3, C3b, and degradation products, for example, according to the methodology in Table 3, using SEQ ID NOs: 156 and / or 157, at the option of choice, in addition to one or more or all of the peptides in Table 2 in any combination.

[0141] In some embodiments, the FI peptide is VKLVDQDKTMFICKSSWSMRE (SEQ ID NO: 45; Mass 2531.2455), VKLISNCSKFYGNRFYE (SEQ ID NO: 46; Mass 2068.0320), CLHPGTKFLNNGTCTAE (SEQ ID NO: 47; Mass 1805.8309), NYNAGTYQNDIALIE (SEQ ID NO: 48; Mass 1698.7969), GKFSVSLKHGNTDSE (SEQ ID NO: 49; Mass 1605.7867), VGCAGFASVTQEE (SEQ ID NO: 50; Mass 1297.5729), VGCAGFASVTQE (SEQ ID NO: 155; Mass 1168.272), MKKDGNKKDCE (SEQ ID NO: 51; Mass 1295.6082), YVDRIIFHE (SEQ ID NO: 52; Mass Any one or more of the following: 1191.6156), CLHVHCRGLE (SEQ ID NO: 53; Mass 1166.5557), RVFSLQWGE (SEQ ID NO: 54; Mass 1121.5738), ILTADMDAE (SEQ ID NO: 55; Mass 978.4448), or KVTYTSQE (SEQ ID NO: 56; Mass 955.4731).

[0142] In some embodiments, the FI peptide is CAGTYDGSIDACKGDSGGPLVCMDANNVTYVWGVVSWGE (SEQ ID NO: 38; mass 3996.7183), GTCVCKLPYQCPKNGTAVCATNRRSFPTYCQQKSLE (SEQ ID NO: 3994.8853), FPGVYTKVANYFDWISYHVGRPFISQYNV (SEQ ID NO: 40; mass 3467.7211), ANVACLDLGFQQGADTQRRFKLSDLSINSTE (SEQ ID NO: 41; mass 3397.6804), LPRSIPACVPWSPYLFQPNDTCIVSGWGRE (SEQ ID NO: 42; mass 3388.6605), KKCLAKKYTHLSCDKVFCQPWQRCIE (SEQ ID NO: 43; mass 3155.5773), LCCKACQGKGFHCKSGVCIPSQYQCNGE (SEQ ID NO: 44; Mass 2991.2861), VKLVDQDKTMFICKSSWSMRE (SEQ ID NO: 45; Mass 2531.2455), VKLISNCSKFYGNRFYE (SEQ ID NO: 46; Mass 2068.0320), CLHPGTKFLNNGTCTAE (SEQ ID NO: 47; Mass 1805.8309), NYNAGTYQNDIALIE (SEQ ID NO: 48; Mass 1698.7969), GKFSVSLKHGNTDSE (SEQ ID NO: 49; Mass 1605.7867), VGCAGFASVTQEE (SEQ ID NO: 50; Mass 1297.5729), MKKDGNKKDCE (SEQ ID NO: 51; Mass Any one or more of the following: 1295.6082), YVDRIIFHE (SEQ ID NO: 52; Mass 1191.6156), CLHVHCRGLE (SEQ ID NO: 53; Mass 1166.5557), RVFSLQWGE (SEQ ID NO: 54; Mass 1121.5738), ILTADMDAE (SEQ ID NO: 55; Mass 978.4448), KVTYTSQE (SEQ ID NO: 56; Mass 955.4731), VDCITGE (SEQ ID NO: 57; Mass 736.3182), NCGKPE (SEQ ID NO: 58; Mass 647.2817), TSLAE (SEQ ID NO: 59; Mass 520.2613), or KDNE (SEQ ID NO: 60; Mass 505.2252).

[0143] Peptides detected by the methods described herein may optionally have at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to any one of the amino acid sequences of the peptides described herein, for example, SEQ ID NOs. 21-141. Other suitable peptides are readily determinable by those skilled in the art and may be used in the methods described herein. In a preferred embodiment, the peptides used herein enable mass spectrometry techniques to distinguish or differentiate between two or more complement proteins in a sample.

[0144] mass spectrometry A method provided herein, which includes a step of detecting a protein and / or determining its level, may also include a step of detecting a protein in a sample and / or determining its level using mass spectrometry.

[0145] In a preferred embodiment, any method described herein involves a step using only mass spectrometry (i.e., mass spectrometry alone) to detect / determine the level of one or more peptides. That is, in some embodiments, the methods provided herein do not use multiple analytical techniques, and the peptide(s) are detected / determined / measured using a single assay. In a preferred embodiment, the methods described herein do not use mass spectrometry in combination with another analytical technique suitable for detecting proteins / peptides to detect / determine the level of peptide(s). In a preferred embodiment, the detection / determining of the level of one or more peptides is not performed at any stage using non-mass spectrometry techniques, for example, the detection / determining of the level of peptide(s) is not performed using high-performance liquid chromatography (HPLC), immunoassays, such as quantitative enzyme-coupled immunosorbent assay (ELISA), Western blotting, protein immunoprecipitation, dot blotting or immunoelectrophoresis, electrophoresis or autoradiography. In some cases, liquid chromatography / mass spectrometry (LC / MS) is not used.

[0146] In this specification, the step of "detecting and / or determining the level of" complement proteins or peptides, for example, by mass spectrometry, is the same as the step of "using mass spectrometry to detect and / or determine the level of" complement proteins or peptides, for example.

[0147] Mass spectrometry is a well-known analytical technique for analyzing a sample, which typically involves generating ions from the sample, optionally fragmenting the ions, separating the ions according to their mass / charge ratio (over time and / or space), and detecting the ions to provide information about the contents of the sample.

[0148] For the purpose of detecting and / or determining the level of a protein, at least one fragmentation step may be included. Mass spectrometry techniques are well known in the art, and any suitable mass spectrometry technique, e.g., LC / MS, GC / MS, tandem mass spectrometry (MS / MS), quadrupole MS, e.g., triple quadrupole MS (TQMS), time-flight MS, e.g., MALDI-TOF, targeting MS, e.g., selective reaction monitoring MS (SRM-MS) / multiple reaction monitoring (MRM-MS), parallel reaction monitoring (PRM-MS), ion-trapped methods, e.g., three-dimensional quadrupole ion traps ("dynamic" traps) and ion cyclotron resonance mass spectrometers ("static" traps), quadrupole trap MS, hybrid linear trap / orbitrap MS, quadrupole orbitrap MS, electrospray ionization mass spectrometry (ESI-MS), or electron transfer dissociation MS (ETD) may be used to detect and / or determine the levels of proteins in a sample.

[0149] In some embodiments, the mass spectrometry technique may be an assay based on liquid chromatography-selective reaction monitoring mass spectrometry (LC-SRM-MS). Ion fragmentation may be achieved using any suitable fragmentation technique, such as collision-induced dissociation (CID) / collision-activated dissociation (CAD), electron-capture dissociation (ECD), electron-transfer dissociation (ETD), in-source decay (ISD), infrared multiphoton dissociation (IRMPD), etc. Again, these techniques are well known.

[0150] Mass spectrometry techniques useful in the present invention may include quantitative analysis. Mass spectrometry methods including quantitative analysis may include a targeting approach for detecting and measuring the peptide of interest and its corresponding fragments. This can enable higher specificity and sensitivity of quantification. Quantitative mass spectrometry in proteomics is outlined, for example, in Bantscheff, M. et al., Anal Bioanal Chem 2007, 389, 1017-1031, which is incorporated in whole herein.

[0151] For example, an input peptide may undergo fragmentation in the collision cell, thus generating product ions unique to that peptide. The intact peptide mass and both the specific fragment ions of one or more of those peptides may be monitored throughout the MS experiment using, for example, SRM / MRM, PRM, etc.

[0152] The observed m / z ratio of a peptide and its corresponding product ion m / z ratio are referred to as the "transition," which is a mass pair representing the m / z of the analyte (parent ion) and one of the m / z of the product ion formed during the fragmentation of the parent ion.

[0153] Developing appropriate transitions for quantitative mass spectrometry techniques, such as SRM / MRM-MS and PRM-MS, is well within the routine work of those skilled in the art. Mead et al., Mol Cell Proteomics. 2009 Apr; 8(4): 696-705, which is incorporated herein by reference in its entirety, describes one such technique for designing transitions.

[0154] Tables 7 and 8 provide examples of transitions for the complement proteins described herein, based on the fragmentation of the synthetic forms of each peptide of interest. Appropriate alternative transitions may also be used, and their identification is well within the routine authority (remit) of those skilled in the art.

[0155] Quantification may be achieved by "spiking" a sample with a known amount of labeled synthetic peptide. The combination of retention time, peptide mass, and fragment mass substantially eliminates ambiguity in peptide allocation and extends the quantification range by 4-5 orders of magnitude. In some cases, the methods provided herein include a step of determining a quantification reference value using optimized MS settings and / or stable isotope standards.

[0156] The mass spectrometry techniques applicable to this invention may include targeted or semi-targeted MS workflows and / or data-dependent acquisition (DDA) or data-independent acquisition (DIA) techniques.

[0157] DDA uses knowledge gained during acquisition to determine which MS1 peptide precursors to subject to fragmentation (MS / MS) in the collision cell. DIA, in contrast, performs predefined MS / MS fragmentation and data acquisition regardless of the sample contents, thereby enabling more sensitive and accurate protein quantification compared to DDA. DIA strategies can be further divided into targeted or non-targeted acquisition. Targeted DIA fragments predefined precursor ions, which usually correspond to peptide analytes at known (measured or predicted) retention times. Targeted DIA is becoming widely used in academic, pharmaceutical, and biotechnology research for the quantification of small molecules (metabolites), peptides, and post-translational modifications (PTMs). For example, selective reaction monitoring (SRM), a type of targeted DIA, is now considered the optimal reference method for mass spectrometry quantification due to its high accuracy and precision. For an overview of DIA technology, see, for example, Meyer and Schilling, Expert Rev Proteomics. 2017 May; 14(5): 419–429, which is incorporated in its entirety herein.

[0158] Other suitable DIA methods include, for example, Sequential Window Acquisition of All Theoretical Mass Spectrometry (SWATH MS; see, e.g., Ludwig et al., Mol Syst Biol (2018) 14:e8126), SONAR (Waters.com), or Online Parallel Accumulation-Serial Fragmentation (PASEF; see, e.g., Meier et al., J Proteome Res. 2015 Dec 4;14(12):5378-87 and Meier et al., Mol Cell Proteomics. 2018 Dec; 17(12):2534-2545).

[0159] Application of the method In several respects, the present invention provides a method for assessing the risk of onset or progression of complement-related disorders using the detection / determination methods described herein.

[0160] Methods for assessing risk may include diagnosing, prognosing, and / or predicting the risk of onset or progression of complement-related disorders. Diagnostic methods may be used to diagnose or determine the severity of a disorder, prognostic methods may help predict the possible course of the disorder in a defined clinical population under standard treatment conditions, and predictive methods may predict possible responses to treatment in terms of efficacy and / or safety, thus assisting clinical decision-making. The methods described herein may be useful in monitoring the success of treatments, including past or ongoing treatments, for complement-related disorders.

[0161] The terms “disorder,” “disease,” and “condition” may be used interchangeably, and these terms refer to a pathological problem of a part, organ, or system of the body that can be characterized by an identifiable group of signs or symptoms. The term “complement-related disorder” refers to a disorder, disease, or condition that involves or arises from a deficiency or abnormality in the complement system. In some embodiments, a complement-related disorder is a disorder driven by complement activation or complement hyperactivation. The terms, for example, “develop,” “developing,” and “development” of a disorder refer, as herein, to both the onset of a disease and the progression, exacerbation, or worsening of a disease state.

[0162] In some embodiments, the disorder is pathologically related to the complement system, or its activation / hyperactivation / dysregulation. Complement-related disorders may be any of the disorders described herein. "Pathologically related" means, as used herein, elevated or decreased protein levels in the disorder compared to reference values, and / or the protein contributing to the pathology of the disorder. The selection or combination of complement proteins to be detected may depend on the complement-related disorder of interest and the complement proteins that are useful biomarkers for said disorder.

[0163] Complement-related disorders may include disruption of the classical, secondary, and / or lectin complement pathways. In some cases, the disorder may be associated with a deficiency, abnormality, or absence of regulatory components of the complement system. In some embodiments, the disorder may be associated with the secondary complement pathway, disruption of the secondary complement pathway, and / or a deficiency, abnormality, or absence of regulatory components of the secondary complement pathway. In some cases, the disorder is associated with the complement amplification loop. In some cases, the disorder is associated, in whole or in part, with improper activation, hyperactivation, or dysfunction of the complement system, e.g., C3 convertase assembly, C3b production, C3b deposition, and / or amplification loop.

[0164] In some cases, the disorder is associated with C3, C3b, iC3b, FI, FH, FHL-1, or any one or more of FHR1-FHR5. In some cases, the disorder is associated with impaired or abnormal activity of C3, C3b, iC3b, FI, FH, FHL-1, or any one or more of FHR1-FHR5. In some cases, one or more of these proteins are pathologically associated with the disorder and have elevated or lower levels, for example, compared to reference values.

[0165] In some cases, the disorder is associated with one or more of CR1, CD46, CD55, C4BP, factor B (FB), factor D (FD), SPICE, VCP (or VICE), and / or MOPICE. In some cases, the disorder is associated with a deficiency or abnormality in the activity of one or more of CR1, CD46, CD55, C4BP, factor B, factor D, SPICE, VCP (or VICE), and / or MOPICE, or one or more of these proteins are pathologically associated.

[0166] In some embodiments, the impairment may be associated with C3 or a C3-containing complex, an activity / reaction associated with C3 or a C3-containing complex, or a product of an activity / reaction associated with C3 or a C3-containing complex. That is, in some embodiments, the impairment is pathologically associated with C3, a C3-containing complex, an activity / reaction associated with C3 or a C3-containing complex, or a product of said activity / reaction. In some embodiments, the impairment may be associated with an increase in the level of C3 or a C3-containing complex, an increase in the level of an activity / reaction associated with C3 or a C3-containing complex, or an increase in the level of a product of an activity / reaction associated with C3 or a C3-containing complex, compared to a control state. In some embodiments, the impairment may be associated with a decrease in the level of C3 or a C3-containing complex, a decrease in the level of an activity / or reaction associated with C3 or a C3-containing complex, or a decrease in the level of a product of an activity / reaction associated with C3 or a C3-containing complex, compared to a control state.

[0167] In some embodiments, the impairment may be associated with C3b or a C3b-containing complex, an activity / or reaction associated with C3b or a C3b-containing complex, or a product of an activity / reaction associated with C3b or a C3b-containing complex. That is, in some embodiments, the impairment is pathologically associated with C3b, a C3b-containing complex, an activity / reaction associated with C3b or a C3b-containing complex, or a product of said activity / reaction. In some embodiments, the impairment may be associated with an increase in the level of C3b or a C3b-containing complex, an increase in the level of an activity / or reaction associated with C3b or a C3b-containing complex, or an increase in the level of a product of an activity / reaction associated with C3b or a C3b-containing complex, compared to a control state. In some embodiments, the impairment may be associated with a decrease in the level of C3b or a C3b-containing complex, a decrease in the level of an activity / reaction associated with C3b or a C3b-containing complex, or a decrease in the level of a product of an activity / reaction associated with C3b or a C3b-containing complex, compared to a control state.

[0168] In some embodiments, the disorder may be any one or more of FH, FHL-1, FI, FHR1-FHR5, FB, FD, CR1 and / or CD46, an activity / reaction associated with any one or more of FH, FHL-1, FI, FHR1-FHR5, FB, FD, CR1 and / or CD46, or a disorder associated with the products of any one or more of the activities / reactions associated with FH, FHL-1, FI, FHR1-FHR5, FB, FD, CR1 and / or CD46. In some embodiments, the disorder is a disorder pathologically associated with any one or more of FH, FHL-1, FI, FHR1-FHR5, FB, FD, CR1 and / or CD46, an activity / reaction associated with any one or more of FH, FHL-1, FI, FHR1-FHR5, FB, FD, CR1 and / or CD46, or a disorder pathologically associated with the products of the said activities / reactions. In some embodiments, the impairment may be associated with a decrease in the level of any one or more of FH, FHL-1, FI, FHR1-FHR5, FB, FD, CR1 and / or CD46 compared to a control state, a decrease in the level of activity / reaction associated with any one or more of FH, FHL-1, FI, FHR1-FHR5, FB, FD, CR1 and / or CD46, or a decrease in the level of the product of activity / reaction associated with any one or more of FH, FHL-1, FI, FHR1-FHR5, FB, FD, CR1 and / or CD46.

[0169] In some embodiments, the impairment may be associated with an increase in the level of any one or more of FHR1-FHR5, an increase in the level of activity / reaction associated with any one or more of FHR1-FHR5, or an increase in the level of product of activity / reaction associated with any one or more of FHR1-FHR5 compared to a control state. See, for example, Zhu et al., Kidney Int. 2018 Jul;94(1):150-158; Pouw et al., Front Immunol. 2018 Apr 24;9:848, which are incorporated herein in their entirety. In some embodiments, the impairment may be associated with an increase in the level of FHR4, an increase in the level of activity / reaction associated with FHR4, or an increase in the level of product of activity / reaction associated with FHR4 compared to a control state. For example, see WO 2019 / 215330 and Cipriani et al., Nat Commun 11, 778 (2020), both of which are incorporated herein in their entirety. The method may include a step of determining the whole-body level of FHR4.

[0170] In some embodiments, the impairment is associated with an increase in the level of any one or more of C3, C3b, C3 convertase, and / or C3bBb compared to a control state. In some embodiments, the impairment is associated with a decrease in the level of any one or more of C3, C3b, C3 convertase, and / or C3bBb compared to a control state. In some embodiments, the impairment is associated with an increase in the level of iC3b compared to a control state. In some embodiments, the impairment is associated with a decrease in the level of iC3b compared to a control state. In some embodiments, the impairment is associated with an increase in the level of any one or more of C3a, C3f, C3c, C3dg, C3d, and / or C3g compared to a control state. In some embodiments, the impairment is associated with a decrease in the level of any one or more of C3a, C3f, C3c, C3dg, C3d, and / or C3g compared to a control state.

[0171] In some cases, the methods provided herein are useful for determining whether complement-related disorders are associated with complement system hyperactivation. In some cases, the methods can determine whether elevated levels of complement proteins, such as those described herein, contribute to complement hyperactivation and / or complement-related disorders, compared, for example, to a control subject without complement-related disorders.

[0172] The disorder may be an eye disorder. In some embodiments, the disease or condition to be evaluated, diagnosed, treated or prevented as described herein is a complement-related eye disorder. In some embodiments, the disorder is macular degeneration. In some embodiments, the disorder may be selected from age-related macular degeneration (AMD), choroidal neovascularization (CNV), macular dystrophy, and diabetic maculopathy, i.e., one or more of these. In this specification, the term “AMD” includes early AMD, mid-stage AMD, late / progressive AMD, geographic atrophy (“atrophic” (i.e., non-exudative) AMD), and “exudative” (i.e., exudative or neovascular) AMD, each of which may be a disorder in itself that is detected, treated and / or prevented as described herein. In some embodiments, the disease or condition to be treated or prevented is a combination of the above diseases / conditions, e.g., “atrophic” and “exudative” AMD. In some embodiments, the disease or condition to be treated or prevented is not “exudative” AMD or choroidal neovascularization. AMD is generally defined as causing vision loss in subjects aged 50 years and older. In some embodiments, the subjects to be treated are 50 years of age or older, i.e., at least 50 years of age.

[0173] In this specification, “early AMD” refers to a stage of AMD characterized by the presence of moderately sized drusen, typically up to ~200 μm in diameter, within the Bruch’s membrane adjacent to the RPE layer. Subjects with early AMD typically do not present with significant vision loss. In this specification, “mid-stage AMD” refers to a stage of AMD characterized by large drusen and / or pigment changes in the retina. Mid-stage AMD may be accompanied by some degree of vision loss. In this specification, “late AMD” is a stage of AMD characterized by the presence of drusen and vision loss due to damage to the macula, such as severe central vision loss. In all stages of AMD, “pseudodrusen reticularis” (RPD) or “retinal drusen” (also called subretinal drusenoid deposition (SDD)) may be present, referring to the accumulation of extracellular material in the subretinal space between the neurosensory retina and the RPE. "Late-stage AMD" includes "atrophic" and "exudative" AMD. In "atrophic" AMD (also known as geographic atrophy), there is a gradual breakdown of photosensitive cells in the macula that transmit visual information to the brain, and the supporting tissues beneath the macula. In "exudative" AMD (also known as choroidal neovascularization, neovascular, and exudative AMD), abnormal blood vessels proliferate subretinally and within the retina. These vessels can leak fluid and blood, which can lead to macular swelling and damage, as well as subsequent scarring. Damage can be rapid and severe.

[0174] In some aspects, the disorder is early-onset macular degeneration (EOMD). In this specification, “EOMD” refers to a phenotypic severity subtype of macular degeneration that shows onset at a much younger age than classical AMD and results in a longer period of substantial visual loss. Affected individuals may exhibit an early-onset drusen phenotype, which includes uniform, small, slightly elevated, yellow subretinal nodules randomly scattered throughout the macula, also known as “basal drusen” or “keratinous drusen.” EOMD may also be referred to as “mid-onset macular degeneration.” The EOMD subset is described, for example, in Boon CJ et al., Am J Hum Genet 2008; 82(2):516-23, van de Ven JP et al., Arch Ophthalmol 2012; 130(8):1038-47, and Taylor, RL et al., Ophthalmol. 2019, 126, 1410-1421, all of which are incorporated herein in their entirety. Like other types of macular degeneration, EOMD is associated with complement dysregulation and impaired factor H activity. In some embodiments, the subjects to be treated are 49 years of age or younger. In some embodiments, the subjects to be treated are between 15 and 49 years of age, i.e., between 15 and 49 years of age. In some embodiments, the disease or condition to be treated is macular dystrophy. Macular dystrophy may be a genetic condition, usually caused by a mutation in a single gene, resulting in degeneration of the macula.

[0175] In some aspects, the disorder is related to the kidneys, e.g., nephropathy / nephropathy. In some cases, the disorder is neurological and / or neurodegenerative. In some cases, the disorder is related to autoimmunity, e.g., autoimmune disease. In some cases, the disorder is related to inflammation, e.g., inflammatory disease. In some cases, the disorder is characterized by C3 deposition, e.g., glomerular pathology (see, e.g., Skerka et al., 2013, above).

[0176] In some aspects, the disorder includes hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), DEAP HUS (FHR plasma protein deficiency and autoantibody-positive type of hemolytic uremic syndrome), autoimmune uveitis, membranoproliferative glomerulonephritis type II (MPGN II), sepsis, Henoch-Schönlein purpura (HSP), IgA nephropathy, chronic kidney disease, paroxysmal nocturnal hemoglobinuria (PNH), autoimmune hemolytic anemia (AIHA), systemic lupus erythematosus (SLE), Sjögren's syndrome (SS), rheumatoid arthritis (RA), glomerular disease, C3 glomerulopathy (C3G), dense deposit disease (DDD), and C3 nephritis factor glomerulonephritis (C3NF). The following conditions may be selected from GN), FHR5 nephropathy, hereditary angioedema (HAE), acquired angioedema (AAE), encephalomyelitis, atherosclerosis, multiple sclerosis (MS), stroke, Parkinson's disease, and / or Alzheimer's disease.

[0177] In some cases, the disorder is cancer. The cancer may be humoral or hematological, such as leukemia, lymphoma, or myeloma. In other cases, the cancer is a solid tumor, such as breast cancer, lung cancer, liver cancer, colorectal cancer, nasopharyngeal cancer, kidney cancer, or glioma. In some cases, the cancer is located in the liver, bone marrow, lungs, spleen, brain, pancreas, stomach, or intestines. In some cases, the cancer is lung cancer. In some cases, the cancer is glioblastoma (glioblastoma multiforme (GBM)).

[0178] In some cases, the disability is neurodegenerative or a neurodegenerative disease. The disability may include progressive atrophy and loss of neuronal function. The disability may be selected from Parkinson's disease, Alzheimer's disease, dementia, stroke, Lewy body disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Huntington's disease, and prion diseases.

[0179] The role of complement in various diseases is illustrated in the following references, all of which are incorporated herein: Morgan, BP, Complement in the pathogenesis of Alzheimer's disease. Semin Immunopathol, 2018. 40(1): p. 113-124; Halbgebauer, R. et al., Janus face of complement-driven neutrophil activation during sepsis. Semin Immunol, 2018. 37: p. 12-20; Ma, Y. et al., Significance of Complement System in Ischemic Stroke: A Comprehensive Review. Aging Dis, 2019. 10(2): p. 429-462; Bonifati and Kishore, Role of complement in neurodegeneration and neuroinflammation. Mol Immunol. 2007 Feb;44(5):999-1010; Kleczko, EK et al., Targeting the Complement This is described in "Pathway as a Therapeutic Strategy in Lung Cancer. Front Immunol, 2019. 10: p. 954" and in Schafer N. et al., "Complement Regulator FHR-3 Is Elevated either Locally or Systemically in a Selection of Autoimmune Diseases, Front Immunol. 2016; 7: 542." For example, FHL-1 is expressed more highly than FH in certain tumor cell lines (Junnikkala et al. (2000) J. Immunol. 164: 6075-81), and glioblastoma tumors have been shown to express the FHR protein (DeCordova et al. (2019) Immunobiology 224: 625-631). Both of these references are incorporated herein by reference in their entirety.It is preferable if it is possible to measure FH family proteins and distinguish between them.

[0180] In several respects, the present invention provides a method for predicting, based on the analysis of a subject-derived sample as described herein, whether a subject is at risk of developing a complement-related disorder, has a complement-related disorder, requires treatment for a complement-related disorder, responds to treatment for a complement-related disorder, and / or is responding to / has responded to treatment for a complement-related disorder. The method may be used to determine whether a subject is at risk of developing a disorder and / or at risk of progression, exacerbation, or worsening of a disorder.

[0181] In one aspect, the present invention provides a method for determining whether a subject is at risk of developing a complement-related disorder, comprising the steps of: detecting / determining the level of at least one complement protein in a sample derived from the subject; digesting the protein(s) with endoproteinase GluC to obtain one or more peptides; and detecting one or more peptides by mass spectrometry. The method may include an initial step of obtaining a sample and / or at least one protein from the subject. Suitable sources of the sample are described herein. The method may also include a step of determining the likelihood of the subject developing a complement-related disorder using the results of the mass spectrometry step.

[0182] In one aspect, for example, according to the method described herein, the use of endoproteinase GluC is provided in a method for determining the presence and / or levels of complement proteins in, for example, a sample or subject. A method for identifying subjects who have or are at risk of developing complement-related disorders: a) Digesting at least one complement protein in the sample obtained from the subject with endoproteinase GluC to obtain one or more peptides; b) Determine the presence and / or level of one or more peptides by mass spectrometry; and c)(b) Use the results to determine whether the subject has or is likely to develop a complement-related disorder. The use of endoproteinase GluC in the above method, including the step, is also provided.

[0183] A method for selecting subjects for the treatment of complement-related disorders with complement-targeting therapies: a) Digesting at least one complement protein in the sample obtained from the subject with endoproteinase GluC to obtain one or more peptides; b) Determine the presence and / or level of one or more peptides by mass spectrometry; and Using the results of c)(b), determine whether the subject requires complement-targeting therapy. The use of GluC in the aforementioned method, including the step, is also provided.

[0184] The methods described herein may be used to determine whether a subject is at risk of developing macular degeneration, such as EOMD and / or AMD, and / or at risk of EOMD and / or AMD progression. In some cases, the disorder is selected from EOMD, AMD, geographic atrophy ("atrophic" (i.e., non-exudative) AMD), early AMD, mid-stage AMD, late / progressive AMD, "exudative" (neovascular or exudative) AMD, choroidal neovascularization (CNV), and retinal dystrophy. In some cases, the subject has or is suspected to have a complement-related disorder. In some cases, the disorder is AMD. In some cases, the disorder is EOMD.

[0185] Accordingly, the present invention also provides a method for determining whether a subject is at risk of developing macular degeneration, such as EOMD and / or AMD, comprising the steps of: detecting / determining the level of at least one complement protein in a sample derived from the subject; digesting the protein(s) with endoproteinase GluC to obtain one or more peptides; and detecting one or more peptides by mass spectrometry. The method may include an initial step of obtaining a sample and / or at least one protein from the subject. The method may also include a step of determining the likelihood of the subject developing macular degeneration using the results of the mass spectrometry step.

[0186] In this specification, a method for evaluating a subject's tendency or predisposition to develop complement-related disorders: (a) Provide a blood sample derived from the subject; (b) As described herein, assess the presence or level of at least one complement protein in the sample; (c)(b) The results are used to determine the likelihood that the subject will develop complement-related disorders. The method, including the process, is also provided.

[0187] In other aspects, the present invention provides a method for identifying subjects at risk of developing or having complement-related disorders, the method comprising the step of detecting / determining the level of complement proteins as described herein. The disorders may be EOMD and / or AMD, or related disorders as described herein, for example.

[0188] The present invention is a method for identifying subjects with complement-related disorders: a) Digesting at least one complement protein in the sample obtained from the subject with endoproteinase GluC to obtain one or more peptides; b) Determine the presence and / or level of one or more peptides by mass spectrometry; and Using the results of c)(b), determine whether the subject has a complement-related disorder. The present invention provides the method, including the steps involved.

[0189] The methods described herein may also be useful for evaluating whether treatment for complement-related disorders is effective or successful / was effective or successful. In several respects, the present invention provides a method of using the techniques described herein to determine whether a subject is likely to respond to or not respond to therapeutic treatment, or whether a subject is responding to therapeutic treatment. Such methods should enable a patient to receive the most effective therapy for specific pathological requirements.

[0190] In some cases, an increase or decrease in complement protein levels, as described herein, compared to a reference value, indicates an increased risk of developing complement-related disorders. In some cases, an increase or decrease in complement protein levels, as described herein, indicates an increased risk of developing disorders when compared to a reference value obtained from the same subject / same subject-derived sample at an earlier stage of the disorder.

[0191] In some embodiments, the methods described herein may include the step of determining the levels of two or more complement proteins and comparing their values, for example, concentrations. The values ​​may be compared to each other and to a reference value, and for example, an increase in the levels of C3 and C3b, or a decrease in the levels of iC3b and further C3b degradation products, compared to the steady state, may be an indicator of a higher risk of developing complement-related disorders and / or the need to treat the subject for complement-related disorders. A decrease in the levels of C3 and C3b, or an increase in the levels of iC3b and further C3b degradation products, compared to the steady state, may be an indicator of a lower risk of developing complement-related disorders and / or the effectiveness of treatment for complement-related disorders.

[0192] In some embodiments, the methods provided herein include the step of correlating the presence of atypical amounts / levels of complement proteins with an increased risk of a subject developing or having a complement-related disorder.

[0193] In this specification, the term “reference value” refers to a known measurement used for comparison during analysis. In some cases, the reference value is one or a set of test values ​​obtained from individuals or groups of a defined health condition. In some cases, the reference value is obtained by determining the level of complement protein in subjects known not to have complement-related disorders. In some cases, the reference value is established by determining the level or amount of complement protein previously obtained from the same subject, for example, at an earlier stage of disease progression. The reference value may be obtained from samples obtained from the same subject or from one or more different subjects. The samples may be obtained from the same tissue / cell / fluid as the samples used in the present invention. The reference value may be a standard value, a standard curve, or a standard dataset. Values / levels that deviate significantly from the reference value may be described as atypical values / levels.

[0194] In some cases, the control may be a reference sample or reference dataset, or one or more values ​​derived from said sample or dataset. The reference value may be obtained from a reference sample or reference dataset. The reference value may be derived from one or more samples previously obtained from one or more subjects known to be without complement-related disorders and / or known or expected not to be at risk of developing complement-related disorders. The reference value may be obtained from one or more samples previously obtained from one or more subjects known to be without complement-related disorders. The reference value may be derived from one or more samples previously obtained from one or more subjects known to be at risk of developing complement-related disorders. The reference value may be a consensus level or mean calculated from a reference dataset, or the mean, e.g., mean protein level. The reference dataset / value may be obtained from a large-scale study of subjects known to have complement-related disorders, e.g., AMD.

[0195] Examples of reference values ​​for complement proteins in human subjects known not to have complement-related disorders include: a) FH: ~150 to 500 μg / ml in human blood (Clark et al., J Immunol 2014. 193(10):4962-70 and unpublished data); b) FHL-1: ~0.5 to 50 μg / ml in human blood (Clark et al., J Immunol 2014. 193(10):4962-70 and unpublished data); c) FHR1: ~70 to 100 μg / ml in human plasma (Heinen, S et al., Blood 114, 2439-2447); d) FHR2: In human plasma, ~15-50 μg / ml, or about 1 / 10 of the FH concentration (Skerka et al., Mol Immunol 2013, 56:170-180); e) FHR3: ~70 to 100 μg / ml in human plasma (Fritsche, LG et al., Hum. Mol. Genet. 2010.19, 4694-4704); f) FHR4: In human blood, ≤5 μg / ml (WO 2019 / 215330); g) FHR5: ~1.5 μg / ml in human plasma (van Beek, AE et al., Front Immunol. 2017 Oct 18;8:1328); h) FI: Human plasma, ~35 μg / ml; i) C3: ~0.5-16 mg / ml in human plasma (Engström, G. et al., J Hum Hypertens. 2007 Apr;21(4):276-82; Lee SH et al., Am J Respir Crit Care Med. 2006 Feb 15;173(4):370-8); j)C3a:46~157ng / ml (Lee SH et al., Am J Respir Crit Care Med. 2006 Feb 15;173(4):370-8); k)iC3b:~0.7-5μg / ml (Kim AHJ et al., Arthritis Rheumatol. 2019 Mar;71(3):420-430) It includes.

[0196] In some cases, in human subjects known to be without complement-related disorders, such as AMD, the mean reference values ​​for circulating FH, FHL-1, and FHR1-5 include the following (95% CI in parentheses): a) FH, nM: 737.3 (718.2~756.5) b) FHL-1, nM: 10.4 (10.1~10.8) c) FHR-1, nM: 31.2 (29.4~32.9) d) FHR-2, nM: 45.3 (43.1~47.6) e) FHR-3, nM: 24.1 (21.7~26.5) f) FHR-4, nM: 46.1 (42.7~49.6) g) FHR-5, nM: 25.5 (24.5~26.5).

[0197] The relative concentration of one complement protein to another can be determined using its reference value. For example, the ratio of the level of one complement protein to the level of one or more others, e.g., FH:FHL-1, C3:iC3b, C3:C3b, etc., may be inferred from the concentrations provided above. The relative concentration and / or ratio of the level of one complement protein to one or more others may be altered in complement-related disorders. In some embodiments, the methods provided herein include the step of detecting two or more complement proteins and determining how the levels of the complement proteins have changed relative to each other when compared to a reference value (one or more). For example, when the level of a first complement protein is compared to the level of a second complement protein, e.g., FH vs. FHL-1, C3 vs. iC3b, C3 vs. C3b, the levels may be increased or the reverse may be true.

[0198] Methods provided herein for assessing the risk of developing complement-related disorders, i.e., the risk of their onset or progression, or for identifying subjects with / at risk of having complement-related disorders, may be combined with further diagnostic methods and / or tests relating to such disorders, which will be known to those skilled in the art. In some cases, methods for assessing the risk of developing complement-related disorders include: CH50 or AH50 measurement via hemolysis assays, measurement of neoantigen formation during MAC complex (C5b, C6, C7, C8, C9) generation, C3 deficiency screening, mannose-binding lectin assays, immunochemical assays for quantifying individual complement components, flow cytometry for evaluating cell-binding regulatory proteins, e.g., CD55, CD59, and CD35, and / or further techniques selected from renal function tests. For example, see Shih AR and Murali MR, Am. J. Hematol. 2015, 90(12):1180-1186, Ogedegbe HO, Laboratory Medicine, 2007, 38(5):295-304, and Gowda S et al., N Am J Med Sci. 2010, 2(4): 170-173, which are incorporated in their entirety herein.

[0199] In some cases, methods provided herein to assess the risk of developing AMD and / or EOMD include: dark adaptation tests, e.g., Pelli Robson contrast sensitivity tests, e.g., visual acuity tests using Snellen charts and / or Amsler grids, Farnsworth-Munsell 100 hue tests and Maximum Color Contrast Sensitivity Tests (MCCS) to assess color vision and color contrast sensitivity, preferential hyperacuity perimetry (PHP), dorsal fundus photography, fundus examination, fundus autofluorescence, optical coherence tomography, angiography, e.g., fluorescence angiography, fundus fluorescein angiography, iodocyanine green angiography, optical coherence tomography, adaptive optics retinal imaging, deep learning analysis of fundus images, electroretinography, and / or further assessment techniques selected from methods for measuring histological changes such as atrophy, retinal pigment changes, exudative changes, e.g., intraocular hemorrhage, hard exudates, subretinal / sub-RPE / intraretinal fluid, and / or the presence of drusen.

[0200] Treatment of complement-related disorders In some respects, the methods of the present invention include, for example, the steps of treating a subject who is at risk of developing, is expected to develop, has been determined to develop, or has, has been identified as having, has been determined to have, or has been diagnosed as having a complement-related disorder, as described herein.

[0201] Any method provided herein for determining whether a subject is at risk of developing a complement-related disorder may further include a therapeutic step for treating the disorder. For example, any method provided herein for determining whether a subject is at risk of developing a complement-related disorder may include a therapeutic step for treating or preventing the disorder, wherein the subject is determined to have an atypical presence or level of one or more complement proteins, as detected / determined, for example, as described herein, compared to a reference value (one or more).

[0202] The treatment step may include administering to the subject a therapeutic or prophylactic effective dose of one or more complement-targeting therapeutic agents, such as one or more C1 inhibitors, C5 inhibitors, C5a inhibitors, C5aR antagonists, C3 inhibitors, C3a inhibitors, C3b inhibitors, C3aR antagonists, classical pathway inhibitors, secondary pathway inhibitors, FH replacement therapy and / or MBL pathway inhibitors. Specific complement-targeting therapeutic agents include, without limitation, human C1 elastase inhibitors (C1-INH), eculizumab (Soliris®, Alexion; a humanized monoclonal IgG2 / 4 antibody that targets C5), APL-2 (Apellis), Muvodin (Adienne Pharma and Biotech), Elgidina (Adienne Pharma and Biotech), and POT-4 (a cyclic peptide inhibitor of C3).Alcon), rituximab (Biogen Idec, Genentech / Roche), ofatumumab (Genmab, GSK), compstatin analogues, soluble and targeting forms of CD59, PMX53 and PMX205, (Cephalon / Teva), JPE-1375 (Jerini), CCX168 (ChemoCentryx), NGD-2000-1 (formerly Neuron), Cinryze (Shire), Berinert (CSL) Behring, Cetor (Sanquin), Ruconest / Conestat Alpha (Pharming), TNT009 (TrueNorth), OMS721 (Omeros), CLG561 (Novartis), AMY-101 (Amyndas), APL-1 (Apellis), APL-2 (Apellis), Mirococept (MRC), Lamparizumab (FCD4514S, Genentech / Roche), ACH-4471 (Achillion), ALXN1210 (Alexion), Tesidolumab / LFG316 (Novartis / Morphosys), Covercin (Akari), RA101495 (Ra Pharma), Zimura (ARC1905, Opthotech), ALN-CC5 (Alnylam), IFX-1 (InflaRx), ALXN1007 (Alexion), Avacopan / CCX168 (Chemocentryx), and / or, for example, Ricklin et al., Mol Immunol. 2017, 89:10-21; Ricklin and Lambris, Adv Exp Med Biol. 2013, 734: 1-22; Ricklin and Lambris, Semin Immunol. 2016, 28(3):208-22; Melis JPM et al., Mol Immunol. 2015 67(2):117-130; Thurman JM, Nephrol Dial Transplant, 2017 32: i57-i64, Cashman SM et al., PLoS One. 2011, 6(4):e19078;This includes one or more therapeutic agents, such as those described in Bora NS et al., J Biol Chem. 2010, 285(44):33826-33; and Clark et al., J Clin Med 2015, 4(1):18-31.

[0203] In some cases, the treatment step includes administering to a subject a therapeutic or prophylactic effective dose of one or more complement-targeting therapeutic agents described in WO 2018 / 224663 and / or WO 2019 / 138137, both of which are incorporated herein in their entirety.

[0204] In some cases, complement-targeting therapeutic agents for use in the methods provided herein include a polypeptide that can bind to C3b, for example, an amino acid sequence having at least 85% identity to SEQ ID NOs. 145, 146, 147, or 148, and a polypeptide having a full length of 450 amino acids or less, as described in WO 2019 / 138137. SEQ ID NOs. 145-148 described herein correspond, respectively, to SEQ ID NOs. 4, 2, 3, and 13 described in WO 2019 / 138137. In some cases, complement-targeting therapeutic agents may have one or more of the following properties: binding to C3b, binding to C3b in the C3b region bound by complement receptor 1, acting as a cofactor for FI, enabling FI-mediated inactivation of C3b, reducing the amount of C3b via FI, increasing the amount of C3b degradation products, for example, iC3b, C3dg, C3d, C3f, via FI, and / or diffusing via BrM.

[0205] In some cases, the complement-targeting therapeutic agent for use in the methods provided herein comprises a polypeptide comprising a C3b-binding domain and a C3b-inactivating domain, such as those described in WO2018 / 224663, where the C3b-inactivating domain comprises or consists of an amino acid sequence having at least 65% sequence identity to the amino acid sequence of SEQ ID NO: 149, and / or the C3b-binding domain comprises or consists of an amino acid sequence having at least 65% sequence identity to the amino acid sequences of SEQ ID NO: 150, 151, or 152. SEQ ID NOs 149-152 described herein correspond to SEQ ID NOs 9, 11, 13, and 14, respectively, described in WO 2018 / 224663. The polypeptide may contain a linker between the C3b-binding domain and the C3b-inactivating domain. In some cases, the polypeptide comprises an amino acid sequence having at least 65% sequence identity to the amino acid sequence of SEQ ID NO: 32, 33, or 34 disclosed in WO 2018 / 224663, or a sequence comprising such a sequence. In some cases, the complement-targeting therapeutic agent may have one or more of the following properties: binding to C3b, binding to C3b in a C3b region bound by a cofactor of FI, acting as a cofactor of FI, enabling FI-mediated inactivation of C3b, reducing the amount of C3b via FI, increasing the amount of C3b degradation products, e.g., iC3b, C3dg, C3d, C3f, via FI, and / or diffusing via BrM.

[0206] In some cases, the subject has or is suspected to have a complement-related disorder. In some cases, the disorder is AMD. In some cases, the disorder is EOMD. In some respects, the present invention provides a method for treating or preventing complement-related disorders in a subject, comprising administering an effective dose of a complement-targeting therapeutic agent, wherein the subject to be treated has been determined to have an atypical presence or level of one or more complement proteins, as detected / determined, for example, as described herein, compared to a reference value (one or more). In some respects, the subject has been determined to be at risk of developing a complement-related disorder and / or has been identified as having a complement-related disorder.

[0207] In other aspects, the present invention provides a complement-targeting therapeutic agent for use in a method of treating or preventing complement-related disorders in a subject, wherein the subject has been determined to have an atypical presence or level of one or more complement proteins, as detected / determined, for example, as described herein, compared to a reference value (one or more). In some aspects, the subject has been determined to be at risk of developing a complement-related disorder and / or has been identified as having a complement-related disorder.

[0208] In some respects, the use of a complement-targeting therapeutic agent in the manufacture of a drug for treating or preventing complement-related disorders in a subject is provided, wherein the subject is determined to have an atypical presence or level of one or more complement proteins, as detected / determined, for example, as described herein, compared to a reference value (one or more). In some respects, the subject is determined to be at risk of developing a complement-related disorder and / or is identified as having a complement-related disorder.

[0209] Also provided is a complement-targeting therapeutic agent for use in a method for treating or preventing complement-related disorders in a subject, wherein the method comprises the step of administering an effective amount of the complement-targeting therapeutic agent, and selecting the subject for treatment if the subject has / has been determined to have an atypical presence or level of one or more complement proteins, as detected / determined, for example, as described herein, compared to a reference value (one or more). In some respects, the subject has been determined to be at risk of developing a complement-related disorder and / or has been identified as having a complement-related disorder.

[0210] The present invention also provides a method for selecting subjects for treatment with complement-targeting therapeutic agents, the method comprising the step of determining the presence and / or level of at least one complement protein by, for example, a detection / determination method provided herein. Subjects may have a complement-related disorder, or may have been determined to have a complement-related disorder by, for example, a method provided herein.

[0211] In various aspects provided herein, the subject to be treated has an atypical presence or level of at least one complement protein, preferably one or more of FH, FHL-1, FHR1, FHR2, FHR3, FHR4, FHR5, FI, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d. The subject may benefit from the treatment by decreasing the level of any complement protein that is elevated compared to a reference value (single or multiple), and / or increasing the level of any complement protein that is degraded compared to a reference value (single or multiple).

[0212] The methods described herein may include the step of determining the level of one or more of FH, FHL-1, FHR1, FHR2, FHR3, FHR4, FHR5, FI, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d, and determining that the subject has or is likely to develop a complement-related disorder if the measured level of the complement protein(s) is altered, for example, elevated or decreased, compared to the level of that complement protein(s) in the blood of a control subject without complement-related disorder, or to a previous level of complement protein(s) in the blood of the subject of interest.

[0213] In this specification, the term “reference value” refers to a known measurement used for comparison during analysis. In some cases, the reference value is one or a set of test values ​​obtained from individuals or groups of a defined health condition. In some cases, the reference value is obtained by determining the level of complement protein in subjects known not to have complement-related disorders. In some cases, the reference value is established by determining the level or amount of complement protein previously obtained from the same subject, for example, at an earlier stage of disease progression. The reference value may be obtained from samples obtained from the same subject or from one or more different subjects. The samples may be obtained from the same tissue / cell / fluid as the samples used in the present invention. The reference value may be a standard value, a standard curve, or a standard dataset. Values / levels that deviate significantly from the reference value may be described as atypical values / levels.

[0214] In some cases, the methods described herein may find use in diagnosing, treating or preventing disorders, or in selecting subjects for the treatment or prevention of such disorders, where the benefits would be derived from: a decrease in the level or activity of one or more C3bBb type C3 convertase, C3bBb3b type C5 convertase and / or C4b2a3b type C5 convertase compared to a reference value (one or more); a decrease in the level of one or more C3, C3b, C3a, iC3b, FHR1, FHR2, FHR3, FHR4, FHR5, C5b and / or C5a; or an increase in the level of one or more iC3b, C3f, C3c, C3dg, C3d, C3g, FH, FHL-1, FI, FH, FHL-1, FHR1, FHR2, FHR3, FHR4 and / or FHR5.

[0215] In this specification, “treatment” may, for example, reduce the onset or progression of a disease / condition, alleviate the symptoms of a disease / condition, or reduce the pathology of a disease / condition. Treatment or alleviation of a disease / condition may be effective in preventing the progression of a disease / condition, for example, in preventing worsening of the condition or delaying the rate of onset. In some embodiments, treatment or alleviation may lead to improvement in the symptoms of a disease / condition, for example, a reduction in the symptoms of a disease / condition, or a reduction in some other correlate of the severity / activity of a disease / condition. Prevention / prevention of a disease / condition may refer to preventing worsening of the condition or preventing the onset of a disease / condition, for example, preventing an early-stage disease / condition from developing into a later chronic stage.

[0216] The methods provided herein may include a step of determining in a subject the presence or absence of a genetic profile characterized by polymorphisms in the subject genome associated with complement dysregulation. Polymorphisms may be found in internal or neighboring genes such as CCL28, FBN2, ADAM12, PTPRC, IGLC1, HS3ST4, PRELP, PPID, SPOCK, APOB, SLC2A2, COL4A1, MYOC, ADAM19, FGFR2, C8A, FCN1, IFNAR2, C1NH, C7, and ITGA4. The genetic profile associated with complement dysregulation may include one or more, often multiple, single nucleotide polymorphisms, as shown in Tables I and II of US 2010 / 0303832, which are incorporated herein in their entirety.

[0217] Genetic factors are thought to play a role in the development of AMD and EOMD. Therefore, the assessments or therapies described herein may be combined with methods for evaluating AMD-related and / or EOMD-related and / or macular dystrophy-related gene variants. In some cases, the complement-related disorders described herein may include genetic elements and / or genetic risk factors.

[0218] In some cases, the methods provided herein further include the step of determining the presence or absence of one or more genetic factors associated with AMD in a subject, for example, one or more AMD-related gene variants. In some cases, the methods include the step of screening (directly or indirectly) for the presence or absence of one or more genetic factors. In some embodiments, the genetic factors (one or more) are genetic risk factors (one or more). In some embodiments, a subject has been determined to have one or more such risk factors. In some embodiments, the methods of the present invention include the step of determining whether a subject possesses one or more such risk factors.

[0219] In some embodiments, one or more genetic factors may be located on chromosome 1, in or near the RCA locus, for example, the CFH / CFHR gene. One or more genetic factors include: CFH, e.g., Y402H (i.e., rs1061170) C ), rs1410996 C , I62V(rs800292), A473A(rs2274700), R53C, D90G, D936E(rs1065489), R1210C, IVS1(rs529825), IVS2 insTT, selected from IVS6 (rs3766404), A307A (rs1061147), IVS10 (rs203674), rs3753396, R1210C, rs148553336, rs191281603, rs35292876, and rs800292; CFHR4, selected from e.g., rs6685931, and rs1409153; CFI, selected from e.g., G119R, and rs141853578; CFB, e.g., rs4151667, C2, e.g., rs9332739, C9, e.g., P167S; and / or C3, e.g., K155Q, may be located in one or more of these. In some embodiments, the genetic factor is Y402H (i.e., rs1061170 C ) In some embodiments, the genetic factor is rs3753396. In some embodiments, the genetic factor is rs6685931 and / or rs1409153. In some embodiments, the genetic factor is not rs6685931.

[0220] Appropriate genetic risk factors and gene variants are known in the art and are incorporated herein by reference in their entirety, for example: Edwards AO et al., Science 2005, 308(5720):421-4; Hageman GS et al., Proc Natl Acad Sci US A. 2005, 102(20):7227-7232; Haines JL et al., Science 2005, 308(5720):419-21; Klein RJ et al., Science 2005, 308(5720):385-389; Fritsche et al., Nat Genet. 2016, 48(2):134-43; US 2010 / 0303832; Clark et al., J Clin Med. 2015, 4(1):18-31; Cipriani, V. et al., Nat Commun. It may also be as described in 2020, 11, 778; and Hageman GS et al., Hum Genomics. 2011, 5, 420 (2011).

[0221] In some cases, the methods provided herein further include the step of determining in a subject the presence or absence of one or more genetic factors associated with EOMD, for example, one or more EOMD-associated gene variants. In some cases, the methods include the step of screening (directly or indirectly) for the presence or absence of one or more genetic factors. In some cases, the genetic factors (one or more) are genetic risk factors (one or more). In some embodiments, a subject has been determined to have one or more such risk factors. In some embodiments, the methods of the present invention include the step of determining whether a subject possesses one or more such risk factors. In some embodiments, a subject may possess one or more risk factors relating to early-onset macular degeneration (EOMD).

[0222] EOMD is thought to be caused by monogenic inheritance of a rare variant of the CFH gene (see, for example, Boon CJ et al. Am J Hum Genet 2008; 82(2):516-23; van de Ven JP et al. Arch Ophthalmol 2012;130(8):1038-47; Yu Y et al. Hum Mol Genet 2014; 23(19):5283-93; Duvvari MR et al. Mol Vis 2015; 21:285-92; Hughes AE et al. Acta Ophthalmol 2016; 94(3):e247-8; Wagner et al. Sci Rep 2016;6:31531; Taylor RL et al., Ophthalmology. 2019 Mar 21. pii: S0161-6420(18):33171-3). In some embodiments, the subject may possess one or more EOMD-related gene variants. EOMD-related gene variants are described, for example, in Servais A et al., Kidney Int, 2012; 82(4):454-64 and Dragon-Durey MA et al., J Am Soc Nephrol 2004; 15(3):787-95, which are incorporated herein by reference in their entirety. In some embodiments, the subject may possess one or more of the following EOMD-related gene variants: CFH c.1243del, p.(Ala415Profs * CFH c.350+1G>T het; CFH c.619+1G>A het; CFH c.380G>A, p.(Arg127His); CFH c.694C>T, p.(Arg232Ter); or CFH c.1291T>A, p.(Cys431Ser).

[0223] In some cases, the methods provided herein include the step of screening for deletions within the RCA locus (a region of DNA sequence located on chromosome 1 that extends from the CFH gene via the CD46(MCP) gene) associated with AMD risk or protection.

[0224] Methods for determining the presence or absence of genetic factors include restriction fragment length polymorphism identification (RFLPI) of genomic DNA, random amplification polymorphism detection (RAPD) of genomic DNA, amplification fragment length polymorphism detection (AFLPD), multi-locus variable number tandem repeat (VNTR) analysis (MLVA), SNP genotyping, multi-locus sequence typing, PCR, DNA sequencing, e.g., Sanger sequencing or next-generation sequencing, allele-specific oligonucleotide (ASO) probes, and oligonucleotide microarrays or beads. Other suitable methods are described, for example, Edenberg HJ and Liu Y, Cold Spring Harb Protoc; 2009; doi:10.1101 / pdb.top62, and Tsuchihashi Z and Dracopoli NC, Pharmacogenomics J., 2002, 2:103-110.

[0225] In some embodiments, subjects are selected for therapeutic or prophylactic treatment with complement-targeting therapies based on the determination that they possess one or more genetic factors relating to AMD and / or EOMD, for example, one or more AMD-related and / or EOMD-related gene variants or macular dystrophy. In some embodiments, subjects are determined to have one or more such genetic factors. In some embodiments, methods provided herein include the step of determining whether a subject possesses one or more such genetic factors. Such methods and genetic factors are described herein. Accordingly, the present invention provides a method for diagnosing, treating or preventing complement-related disorders in a subject, wherein the subject has been determined to have one or more genetic factors relating to AMD and / or EOMD, and the subject has been determined to have one or more atypical presences or levels of complement proteins, which are detected / determined as described herein when compared to reference values ​​(one or more); optionally, the method comprises the step of administering a complement-targeting therapy / therapy agent.

[0226] The term "subject" refers to a subject, patient, or individual, and may be any animal or human. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but more preferably a human. The subject may be male or female. The subject may be a patient. Therapeutic use may be in humans or animals (veterinary use). A subject who may be treated with the therapeutic substances described herein may be a subject who needs treatment.

[0227] The subjects described herein may belong to a patient subgroup, i.e., the subjects may be part of a specific identifiable portion or subgroup of a population. The population and / or subgroup may have or be suspected of having complement-related disorders. The subgroup may exhibit an atypical presence or level of one or more complement proteins, as detected / determined, for example, as described herein, compared to the population as a whole. The population and / or subgroup may have or be suspected of having AMD, EOMD, or macular dystrophy.

[0228] A subject may be identified, or has been identified, by, for example, the method described herein, as having a complement-related disorder or being at risk of developing a complement-related disorder. In some aspects provided herein, the subject is characterized by having an atypical presence or level of one or more complement proteins, as detected / determined / measured, for example, as described herein.

[0229] The present invention provides a method for treating or preventing a complement-related disorder in a subject, wherein the subject is characterized by having an atypical presence or level of one or more complement proteins, as detected / determined, for example, as described herein.

[0230] Also provided are complement-targeting therapeutic agents for use in a manner to treat or prevent complement-related disorders in subjects, wherein the subjects are characterized by having an atypical presence or level of one or more complement proteins, as detected / determined, for example, as described herein.

[0231] The methods described herein may be performed outside the body of a human or animal. The methods described herein may be performed in vitro, ex vivo, or in vivo, or the products may exist in vitro, ex vivo, or in vivo. The term “in vitro” is intended to include experiments using substances, biological materials, cells and / or tissues that are in laboratory conditions or in culture, while the term “in vivo” is intended to include experiments and methods using undamaged multicellular organisms. “Ex vivo” refers to something that exists or occurs outside an organism, e.g., outside the body of a human or animal, and may be on tissues (e.g., whole organs) or cells taken from the organism. In some embodiments, the determination, detection, measurement, quantification, prediction and / or diagnostic steps of the methods provided herein are performed in vitro.

[0232] The complement-targeting therapeutic agents described herein may be administered by several routes, including, but are not limited to, systemic, intratumor, intraperitoneal, parenteral, intravenous, intraarterial, intradermal, subcutaneous, intramuscular, oral, and nasal, or may be formulated for such administration. Preferably, the therapeutic agent is administered by a route selected from intratumor, intraperitoneal, or intravenous. The agents and compositions may be formulated in liquid or solid form. Liquid formulations may be formulated for administration by injection to a selected area of ​​the human or animal body.

[0233] The dose is preferably a "therapeutically effective dose," which is sufficient to demonstrate benefit to the individual. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the disease being treated. The prescription of treatment, such as the determination of dosage, is the responsibility of the general practitioner and other physicians, and typically takes into account the disorder to be treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to the physician. Examples of the techniques and protocols described above can be found in Remington's Pharmaceutical Sciences, 20th edition, 2000, published by Lippincott, Williams & Wilkins.

[0234] kit Aspects of this disclosure include in vitro diagnostic methods and in vitro kits for performing such methods. In some embodiments, the present invention provides a kit comprising endoproteinase GluC for use in methods for detecting and / or determining the level of one or more complement proteins in a sample, for example. The kit may be used for any of the methods described herein and / or to detect / determine the level of any one or combination of the proteins described herein. The kit may be suitable, used, or intended / sold / distributed for detecting at least one complement protein in a sample, determining the level of at least one complement protein in a sample, preparing at least one complement protein for analysis and / or detection, determining the presence and / or level of complement proteins in a subject, determining whether a subject is at risk of developing a complement-related disorder, identifying a subject with a complement-related disorder, selecting a patient / subject for treatment of a complement-related disorder, and / or treating a subject suspected to have a complement-related disorder. The kit and its components may be suitable for use in MS techniques.

[0235] The kits provided herein comprise one, two, or more components suitable, either as a whole or in part, for carrying out the methods described herein. The kit may include a standard or control, such as a labeled peptide standard (one or more), for each protein to be detected using the kit. The kit may include a predetermined amount of labeled peptide standard. The kit may also include a predetermined amount of GluC enzyme, optionally along with buffers and reagents necessary for enzymatic digestion. The components of the kit may be provided in a single composition or as multiple compositions.

[0236] The kit may be suitable for in vitro diagnostic testing in a clinical setting. The kit may also be a kit for laboratory-based testing. The kit may include instructions for use, such as an instruction booklet or leaflet. The instructions may include protocols for performing any one or more of the methods described herein, for example, for enzymatic digestion, recommended MS settings, and / or data analysis templates. The kit may include components for separating proteins in a sample and / or performing MS techniques, such as a liquid chromatography column.

[0237] The kit may be adapted for use with any other type of sample, including dry samples, wet samples, frozen samples, fixed samples, urine samples, saliva samples, tissue samples, blood samples, or any other sample type disclosed herein. The kit may include devices for obtaining or processing blood, serum, plasma, cell, or tissue samples.

[0238] Sequence identity In this specification, an amino acid sequence corresponding to a reference amino acid sequence may contain at least 60% sequence identity with respect to the reference sequence, for example, at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0239] Pairwise and multi-sequence sequence alignments for the purpose of determining the percentage of identity between two or more amino acid or nucleic acid sequences may be achieved by a variety of methods known to those skilled in the art, using publicly available computer software such as ClustalOmega (Soeding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)), and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780). When using such software, default parameters, such as gap penalties and elongation penalties, are preferably used.

[0240] Features disclosed in the foregoing description, or in the following claims or accompanying figures, expressed in a particular type, with respect to means for performing the disclosed function, or, as appropriate, in a method or process for obtaining the disclosed result, may be used individually or in any combination of such features, in their diverse forms, for the execution of the Invention. The Invention includes the aspects and preferred combinations of features described, unless such combination is obviously unacceptable or obviously to be avoided.

[0241] Although the present invention has been described in combination with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art upon consideration of the present disclosure. Accordingly, the exemplary embodiments of the present invention shown above are to be considered as illustrative and not restrictive. Various changes to the described embodiments may be made without departing from the spirit and scope of the present invention.

[0242] To avoid any doubt, any theoretical explanations provided in this specification are provided for the purpose of improving the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0243] Any section headings used in this specification are for organizational purposes only and are not to be considered as limiting the subject matter described. Throughout this specification, including the following claims, unless the context requires otherwise, the words "comprise" and "include" and variations such as "comprises," "comprising," and "including" are to be interpreted as indicating the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0244] It should be noted that when used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as "about" one particular value and / or "about" another particular value. When expressing such ranges, in another aspect, one particular value and / or another particular value are included. Similarly, when a value is expressed as an approximation by use of the antecedent "about," it will be understood that the particular value forms another aspect. The term "about" associated with a numerical value is optional and, for example, means + / - 10%.

[0245] The phrase "and / or" in this specification includes any combination of members, each individually listed, as well as one or all of the members of the list, including all members.

[0246] For standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3rd ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

[0247] array

[0248] [Table 1-1] <www.

[0249] [Table 1-2]

[0250] [Table 1-3]

[0251] [Table 1-4]

[0252] [Table 1-5]

[0253] [Table 1-6]

[0254] [Table 1-7]

[0255]

Table 1-8

[0256]

Table 1-9

Example

[0257] Example 1: Generation of peptides from complement proteins for mass spectrometry GluC digestion was performed on FH, FHL-1, FHR1-5, FI, C3, C3b and C3b degradation products to obtain distinct peptides for mass spectrometry. GluC digestion is described in Example 2.2.

[0258] Peptides that may be used to detect each protein or peptide fragment are shown in Tables 1-4 below. Table 1. Distinct FH family peptides after GluC digestion

[0259]

Table 2

[0260] A series of proteolytic events leading to the production, degradation and inactivation of C3 are shown in Figure 1. Proteoform-specific peptides produced by GluC digestion are underlined in Figure 1 and shown in Table 2. Table 3 shows how each protein can be individually detected using the peptides in Table 2.

[0261] Table 2. Peptide sequences for MS resulting from GluC digestion of C3, C3b and degradation products

[0262]

Table 3

[0263] Table 3. Methodology for determining the concentrations of all C3 / C3b degradation products using the GluC digestion peptides in Table 2.

[0264] [Table 4]

[0265] Table 4. Alternative peptides for C3.1 derived from GluC digestion for measuring total C3 content.

[0266] [Table 5]

[0267] GluC digestion of factor I (FI) yielded candidate peptides in Table 5 for MS analysis. SEQ ID NOs. 45-56 and 155 contain 8-21 amino acids, and these are of excellent length for MS analysis.

[0268] Table 5. Peptide sequences resulting from GluC digestion of FI.

[0269] [Table 6]

[0270] Example 2: Mass Spectrometry 2.1 Preparation of Stable Isotope Standard (SIS) Spike Solution A highly pure, polylabeled synthetic standard containing S-carboxymethylated (CAM) cysteine ​​residues was obtained (Cambridge Research Biochemicals, Cambridge, UK), and then diluted to 1 μg / μL with 50:50 acetonitrile:water + 0.1% formic acid (Table 6).

[0271] First, the stock solutions of FHL-1, FHR1, FHR2, FHR3, FHR4, and FHR5 were diluted 10-fold (dilution of the CFH stock was not necessary), and then the mixed SIS solution was prepared by adding the appropriate amount of each individual dilution to 0.1% TFA until the final volume reached 200 μL. This was then stored at -80°C in 5 μL alicots for further dilution immediately before spike.

[0272] A spike solution was prepared immediately before sample addition by adding 195 μL of 50:50 acetonitrile:water to a 5 μL alicot of the mixed SIS solution. 2 μL of this solution was carefully added to each digested sample, and then dried.

[0273] Table 6. Stock solutions of 1 μg / MI stable isotope standards (SIS). Residues in bold were selected to possess stable heavy isotopes to enable quantification. Lowercase "c" indicates S-carboxymethylated (CAM) cysteine ​​residues. Residues in bold contained isotopically heavier amino acids, K(+8), R(+10), and Y(+10).

[0274] [Table 7]

[0275] 2.2 Sample preparation for analysis by LC-MS / MS After thawing the frozen plasma sample to room temperature, it was vortexed vigorously for 5 minutes to dissolve any soluble substances, and then centrifuged at 13,300 g for 30 minutes to precipitate any insoluble substances.

[0276] 5 μL of plasma alicot (equivalent to approximately 350 μL of protein), 90 μL of 50 mM ammonium bicarbonate (pH 7.8), and 2 μL of ProteaseMAX TMA solution of Promega (Southampton, UK) (1% w / v in 50 mM ammonium bicarbonate) and 1 μL of 500 mM dithiothreitol prepared in 50 mM ammonium bicarbonate were added. These were briefly vortexed and mixed, then pulsed and spun, and incubated at 55°C for 25 minutes.

[0277] After cooling to room temperature, 3 μL of 500 mM iodoacetamide (prepared in 50 mM ammonium bicarbonate) was added. This was briefly vortexed and mixed, then pulsed, and incubated at room temperature in the dark for 15 minutes.

[0278] Furthermore, 1 μL of ProteaseMAX solution (1% w / v in 50 mM ammonium bicarbonate) and 5 μL of 1 μg / μL endoproteinase GluC (Roche, Mannheim, Germany) were added. The mixture was briefly vortexed, then pulsed, and incubated at 25°C for 16 hours with slight shaking (400 rpm).

[0279] To the resulting digested peptide mixture, 6 μL of 10% v / v trifluoroacetic acid (TFA) and 2 μL of SIS spike solution were added, briefly vortexed and mixed, and then pulsed. The solution was placed in an evaporator and dried. Finally, the peptides were reconstituted in 50 μL of 0.1% TFA, vortexed to dissolve any residue, and centrifuged at 13,300 g for 30 minutes to precipitate any insoluble / particulate matter. Approximately 48 μL (care was taken to leave any precipitate) was transferred to an LC autosampler vial for subsequent analysis by LC-MS / MS.

[0280] 2.3 LC-SRM / MS analysis of plasma digests SRM analysis of plasma digests was performed using a 6495 triple quadrupole mass spectrometer coupled with an iFunnel-equipped electrospray ion source (Agilent, Santa Clara, California, USA) to an Infinity 1200 series liquid chromatography system consisting of a 1290 autosampler, a 1260 Quat Pump VL pump, and a TCC column oven module (Agilent, Santa Clara, California, USA). Samples (4 μL) were injected directly onto a C18 column (250 mm x 2.1 mm id, Thermo Scientific Acclaim 120, 3 μm particle size) maintained at a column temperature of 50°C. Compounds were developed using gradient elution with increasing acetonitrile concentration using buffer A (water + 0.1% formic acid) and buffer B (acetonitrile + 0.1% formic acid). A flow rate of 250 μL / min was maintained with the initial composition of 5% buffer B.

[0281] Peptides were separated using the following gradient elution profiles (time: %B): 0 min: 5%B; 2 min: 5%B; 3 min: 12%B; 12 min: 15%B; 15 min: 20%B; 30 min: 25%B; 31 min: 90%B; 39 min: 90%B; 40 min: 5%B; 49 min: 5%B.

[0282] The optimal SRM setting was determined using the SIS solution and is provided in Table 7. Table 7. SRM transitions and optimal collision energies of FH family peptides (quantified ions are shown in bold).

[0283] [Table 8]

[0284] Table 8. Peptides and transitions for the quantification of C3 / C3b degradation products.

[0285] [Table 9]

[0286] To protect the source area from undesirable contaminants, the switching valve located between the column and the source was switched to the waste position at the point when no analyte peptides had eluted from the chromatogram. This made six acquisition windows, each approximately 1 minute long, available on the column connected to the mass spectrometer (two of the peptides, FHR-2 and FHL-1, eluted within the same window).

[0287] 2.4 Results FH family proteins Figure 2 shows LC-SRM traces illustrating the detection of heavily labeled synthetic standards for each individual RCA locus protein derived from plasma samples. This demonstrates that the method is feasible, specific, and possesses the necessary sensitivity to distinguish between peptides derived from these seven proteins, particularly between FH and FHL-1.

[0288] Figure 3 shows the linearity data for FH, FHL-1, and FHR1-5. This indicates that GluC digestion produces peptides that can be individually and specifically detected in native serum at endogenous levels. This also demonstrates that the assay can quantify the level of each protein in the sample. Increases in protein levels increase the signal in a predictable manner, allowing for the determination of the level and presence of each protein. The assay is also demonstrated to be unaffected by interference.

[0289] The lower limit of quantification was defined as plasma concentrations of FH=25nM, FHL-1=0.25nM, FHR-1=2nM, FHR-2=1nM, FHR-3=1nM, FHR-4=4nM, and FHR-5=3nM.

[0290] C3 and C3 degradation products The synthetic forms of the peptides listed in Table 2 were synthesized, their detection by MS was confirmed, and the synthesis was optimized to confirm that they could be quantified using a linear method, and that they could be detected at endogenous levels in serum or plasma samples. This is shown in Figures 4A-4D.

[0291] Figure 4A shows that all peptides in Table 2 can be individually detected in plasma samples by SRM-MS using at least three transitions. The specificity of the assay for the peptide of interest is confirmed during the MS / MS scan by the relative intensity of the transitions that match the relative intensity of the relevant product ions. Figure 4B shows an experiment that confirms peptide specificity by spiking unpurified synthetic peptides into plasma samples and showing a suitable increase in the signal.

[0292] C3 degradation was further analyzed by in vitro assays. C3b was incubated with FI and cofactor CR1 fragments. These were preferred over FH because CR1 drove the reaction toward cleavage from iC3b to C3c+C3dg, while FH only supported the cleavage of C3b to iC3b. Samples were taken continuously from the reaction and stopped by boiling.

[0293] Figure 4C shows the time course of C3b degradation via gel electrophoresis. Analysis using MS and the peptides listed in Table 2 clearly shows that the formation of C3b fragments iC3b, C3f, and C3c, as well as the loss of intact C3b, can be detected over time (Figure 4D). Not all peptides are shown, as some (e.g., C3a) are not present during the in vitro setup, and others show numerous products.

[0294] These data demonstrate that C3 / C3b degradation can be quantitatively measured using GluC-derived peptides and MS. This allows for the detection of complement protein presence and levels in complement-related diseases such as AMD, and provides information on successful treatment outcomes.

[0295] A single assay capable of measuring all FH family proteins, C3 fragments, and FI proteins enables simultaneous analysis of all critical proteins in the complement amplification loop from just one sample, with efficient throughput. Non-limitingly, the present invention includes the following embodiments. [Aspect 1] A method for detecting at least one complement protein in a sample: The protein(s)(s)(s)(g(g)(g)(g)(g)(g)(g)(g)(g)(g)(g)(g)(g)(g) Mass spectrometry can detect one or more peptides. The method, including the steps. [Aspect 2] A method for determining the level of at least one complement protein in a sample: The protein(s)(s)(s)(g(g)(g)(g)(g)(g)(g)(g)(g)(g)(g)(g)(g)(g) Mass spectrometry is used to determine the level of one or more peptides. The method, including the steps. [Aspect 3] The method according to Embodiment 1 or Embodiment 2, wherein the step of detecting one or more peptides or the step of determining the level of one or more peptides comprises the step of measuring one or more peptides by mass spectrometry. [Aspect 4] A method according to any one of embodiments 1 to 3, comprising the step of determining the concentration of one or more complement proteins in a sample. [Aspect 5] A method for preparing at least one complement protein for analysis, comprising the step of digesting the protein(s) with endoproteinase GluC to obtain one or more peptides. [Aspect 6] The method according to any one of embodiments 1 to 5, wherein the complement protein(s) is one or more of FH, FHL-1, FHR1, FHR2, FHR3, FHR4 and / or FHR5. [Aspect 7] The method according to any one of embodiments 1 to 6, wherein the complement protein is FH and / or FHL-1. [Aspect 8] The method according to any one of embodiments 1 to 7, wherein one or more complement proteins are involved in the complement amplification loop and / or C3 convertase activity. [Aspect 9] The method according to any one of embodiments 1 to 8, wherein the complement protein(s) is a degradation product of C3. [Aspect 10] The method according to any one of embodiments 1 to 9, wherein the complement protein(s) is one or more of C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d. [Aspect 11] The method according to any one of embodiments 1 to 10, wherein the complement protein(s) is C3b and / or iC3b. [Aspect 12] The method according to any one of embodiments 1 to 11, wherein the complement protein is FI. [Aspect 13] The method according to any one of embodiments 1 to 12, wherein the sample is obtained from a subject. [Aspect 14] A method according to any one of embodiments 1 to 12, comprising the step of obtaining a sample from a subject. [Aspect 15] The method according to any one of embodiments 1 to 14, wherein the sample includes or is obtained from blood, lymph, plasma, serum, tissue, or cells. [Aspect 16] A method according to any one of embodiments 1 to 15, wherein one or more peptides: [ka] The method selected from the group consisting of the above. [Aspect 17] Use of endoproteinase GluC to prepare at least one complement protein for detection by mass spectrometry, or optionally to prepare at least two complement proteins for detection by mass spectrometry. [Aspect 18] A method for determining the presence and / or level of complement proteins in a subject, comprising the step of performing the method described in any one of embodiments 1 to 17. [Aspect 19] A method for identifying subjects who have or are at risk of developing complement-related disorders: a) Digesting at least one complement protein in the sample obtained from the subject with endoproteinase GluC to obtain one or more peptides; b) Determine the presence and / or level of one or more peptides by mass spectrometry; and c) Using the results of (b), determine whether the subject has or is likely to develop a complement-related disorder. The method, including the steps. [Aspect 20] (d) The method according to embodiment 19, comprising the step of treating a subject who is at risk of developing a complement-related disorder or who has been determined to have such a disorder, wherein the step of treating the subject comprises the step of administering to the subject a therapeutically effective amount of a complement-targeting agent. [Aspect 21] A method for selecting subjects for the treatment of complement-related disorders with complement-targeting therapies: a) Digesting at least one complement protein in the sample obtained from the subject with endoproteinase GluC to obtain one or more peptides; b) Determine the presence and / or level of one or more peptides by mass spectrometry; and c)(b) Use the results to determine whether the subject requires complement-targeting therapy. The method, including the steps. [Aspect 22] A complement-targeting therapeutic agent for use in a method of treating complement-related disorders in a subject, wherein the method is: a) Digesting at least one complement protein in the sample obtained from the subject with endoproteinase GluC to obtain one or more peptides; b) Determine the presence and / or level of one or more peptides by mass spectrometry; and Based on the results of c)(b), administer an effective dose of the complement-targeting therapy agent. The complement-targeting therapeutic agent, including the process. [Aspect 23] The method according to any one of embodiments 18 to 22, comprising the step of obtaining a sample containing at least one complement protein from a subject, wherein the sample optionally contains or is obtained from blood, lymph, plasma, serum, tissue or cells. [Aspect 24] Complement-related disorders include macular degeneration, age-related macular degeneration (AMD), geographic atrophy ("atrophic" (i.e., non-exudative) AMD), early AMD, early-onset macular degeneration (EOMD), mid-stage AMD, late / progressive AMD, "exudative" (neovascular or exudative) AMD, choroidal neovascularization (CNV), retinal dystrophy, hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), DEAP HUS (FHR plasma protein deficiency and autoantibody-positive type of hemolytic uremic syndrome), autoimmune uveitis, and type II membranoproliferative glomerulonephritis (MPGN). II) The method according to any one of embodiments 19 to 23, selected from sepsis, Henoch-Schönlein purpura (HSP), IgA nephropathy, chronic kidney disease, paroxysmal nocturnal hemoglobinuria (PNH), autoimmune hemolytic anemia (AIHA), systemic lupus erythematosus (SLE), Sjögren's syndrome (SS), rheumatoid arthritis (RA), C3 glomerulopathy (C3G), dense deposit disease (DDD), C3 nephritis factor glomerulonephritis (C3 NF GN), FHR5 nephropathy, hereditary angioedema (HAE), acquired angioedema (AAE), encephalomyelitis, atherosclerosis, neurodegenerative / neurodegenerative diseases, dementia, multiple sclerosis (MS), cancer, stroke, Parkinson's disease, and / or Alzheimer's disease. [Pattern 25] A method according to any one of embodiments 18 to 24, wherein an increase in one or more levels of C3, C3b, C3a, iC3b, FHR1, FHR2, FHR3, FHR4 and / or FHR5, compared to a reference value (one or more); and / or a decrease in one or more levels of iC3b, C3f, C3c, C3dg, C3d, C3g, FI, FH, FHL-1, FHR1, FHR2, FHR3, FHR4 and / or FHR5, indicates that the subject is at risk of developing or has such a complement-related disorder. [Aspect 26] A kit for use in a method for detecting and / or determining the level of one or more complement proteins in a sample, the kit comprising endoproteinase GluC.

Claims

1. A method for determining the level of at least one complement protein in a sample, Herein, the complement protein(s) comprises one or more of FH, FHL-1, FHR1, FHR2, FHR3, FHR4, FHR5, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, C3d and / or F1. Herein, the aforementioned method: The protein(s)(s)(s)(s)(g)) are digested with endoproteinase GluC to obtain one or more peptides for each protein; and The level of one or more peptides is determined by mass spectrometry, wherein the step of determining the level of one or more peptides comprises the step of measuring one or more peptides by mass spectrometry. Here, the one or more peptides are: (a) FH:VTYKCFE (SEQ ID NO: 20), SEQ ID NOs: 112-126, and / or SEQ ID NOs: 128-141; (b) FHL-1:NGWSPTPRCIRVSFTL (SEQ ID NO: 21); (c) FHR1:ATFCDFPKINHGILYDEE (SEQ ID NO: 22), and / or SEQ ID NO: 91; (d) FHR2:RGWSTPPKCRSTISAE (SEQ ID NO: 23), AMFCDFPKINHGILYDEE (SEQ ID NO: 24), and / or SEQ ID NO: 92; (e) FHR3:VACHPGYGLPKAQTTTVTCTE (SEQ ID NO: 25), and / or SEQ ID NOs: 93-97; (f) FHR4:YQCQSYYE (Sequence ID 26), and / or Sequence IDs 98-101; (g) FHR5:RGWSTPPICSFTKGE (SEQ ID NO: 27), and / or SEQ ID NOs: 102-111; (h) C3, C3b, iC3b, C3c: GTAFVIFGIQDGE (SEQ ID NO: 28), SEQ ID NOs: 61-67, SEQ ID NOs: 69-76, and / or SEQ ID NOs: 78-90; (i) C3: LRRQHARASHLGLARSNLDE (Sequence ID 29); (j) C3a: LRRQHARASHLGLAR (SEQ ID NO: 30) and / or LRRQHARASHLGLA (SEQ ID NO: 156); (k) C3, C3b: LNLLDVSLQLPRSSKITHRIHWE (Sequence ID 31); (l) iC3b, C3dg, C3d: LNLLDVSLQLPSR (Sequence ID 32); (m) C3, C3b, iC3b: RLGRE (Sequence ID 33); (n) C3f: SSKITHRIHWE (Sequence ID 34); (o) C3f: SASLLR (SEQ ID NO: 35), and / or SASLL (SEQ ID NO: 157); (p) C3c: RLGR (Sequence ID 36); (q) C3d:HLIVTPSGCGE (Sequence ID 37); Furthermore / or (r) F1: One or more of sequence numbers 38-60 or 155, Selected from, The method, including the steps.

2. The method according to claim 1, comprising the step of determining the concentration of one or more complement proteins in a sample.

3. The method according to claim 1 or 2, wherein the complement protein is FH and / or FHL-1.

4. The method according to any one of claims 1 to 3, wherein the complement protein(s) is C3b and / or iC3b.

5. The method according to any one of claims 1 to 4, wherein the sample is obtained from a subject.

6. The method according to any one of claims 1 to 5, wherein the sample comprises or is obtained from body fluids, blood, lymph, plasma, serum, tissue, or cells.

7. The method according to any one of claims 1 to 6, wherein the sample is derived from the eye, kidney, brain, or liver.

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