Method for examining complement-related disorders

Abstract

The present invention addresses the problem of providing a method for examining complement-related disorders which reflects a disease state at the time of examination and has high specificity. The problem is solved by using a complement on extracellular vesicles as a marker for a complement-related disorder.

Description

Methods for testing for complement-related diseases

[0001] The present invention relates to a method for testing complement-related diseases.

[0002] Complement is a protein present in serum and on cell membranes that is activated when pathogens enter the host body. Activated complement then activates other complement components, thereby eliminating the pathogens. Abnormalities in complement and complement regulatory factors can lead to infectious diseases and autoimmune diseases. Complement-related diseases include atypical hemolytic uremic syndrome (aHUS), a condition that can cause thrombotic microangiopathy (TMA). aHUS is a severe disease with a high risk of death. It is caused by abnormal activation of the alternative complement pathway due to congenital abnormalities in complement-related genes or acquired autoantibodies against complement-related proteins, resulting in the destruction of normal red blood cells and platelets, vascular endothelial cell damage, and subsequent acute kidney injury. Treatment of aHUS primarily involves plasma exchange and the use of eculizumab / ravulizumab (anti-C5 antibodies) targeting complement C5. Because C5 is a molecule common to all multiple complement activation pathways, anti-C5 antibody drugs are highly effective in treating various complement-related disorders characterized by excessive complement activation. However, eculizumab / ravulizumab is very expensive (tens of millions of yen per year), so a definitive diagnosis of the target complement-related disorder is desirable before using them. Furthermore, a testing method that more clearly reflects the disease state at the time of testing is desirable, so that the need for continued treatment can be determined even after remission has been achieved.

[0003] However, to date, no definitive diagnostic method for aHUS has been established. It is difficult to differentiate aHUS from other diseases that cause TMA solely based on abnormalities in complement-related genes, measurement of blood complement levels, or tests of complement function. It is also not possible to clearly distinguish aHUS from asymptomatic individuals who have congenital abnormalities in the above genes but who do not currently have the disease. Furthermore, these methods make it difficult to assess the extent of complement activation in vivo to assess the disease progression in untreated individuals or the effectiveness of treatment in those currently receiving treatment. Furthermore, there is no effective method for determining whether treatment with anti-C5 antibodies should be discontinued or whether future treatment with anti-C5 antibodies will be effective (Prior Art References 1 and 2).

[0004] M. Macia et.al., Clin Kidney J. 2017 Jun;10(3):310-319S. Olson et.al., Am J Nephrol. 2018;48(2):96-107.

[0005] An objective of the present invention is to provide a method for testing complement-related diseases.

[0006] The present inventors have conducted extensive research in light of the above-mentioned problems and have found that a testing method that uses complement in extracellular vesicles contained in plasma and / or serum collected from a subject as a marker can solve the above-mentioned problems. Based on this finding, the present inventors have conducted further research and have completed the present invention. Specifically, the present invention encompasses the following aspects.

[0007] Item 1. A method for testing for a complement-related disease, comprising: (1) detecting complement in extracellular vesicles contained in at least one selected from the group consisting of plasma and serum collected from a subject.

[0008] Item 2. The method of detecting a complement-related disease according to Item 1, wherein the complement-related disease is a disease associated with vascular endothelial cell damage.

[0009] Item 3. The examination method according to Item 1 or 2, wherein the complement-related disease is thrombotic microangiopathy (TMA).

[0010] Item 4. The test method according to any one of Items 1 to 3, wherein the complement-related disease is characterized by excessive activation of the complement system.

[0011] Item 5. The test method according to any one of Items 1 to 4, wherein the complement-related disease is atypical hemolytic uremic syndrome (aHUS).

[0012] Item 6. The testing method according to any one of Items 1 to 5, wherein the complement is at least one selected from the group consisting of the C5b-9 complex, C3b, and C3c.

[0013] Item 7. The testing method according to any one of Items 1 to 6, wherein the complement is C3c.

[0014] Item 8. The testing method according to any one of Items 1 to 6, wherein the complement is a C5b-9 complex.

[0015] Item 9. The testing method according to any one of Items 1 to 8, further comprising: (2) determining that the subject has a complement-related disease characterized by excessive activation of the complement system when the amount or concentration of the complement detected in step (1) is equal to or greater than a cutoff value.

[0016] Item 10. The testing method according to Item 9, further comprising the step of: (3) determining whether or not a complement targeting drug should be administered to the subject based on the results of step (2).

[0017] Item 11. The testing method according to any one of Items 1 to 10, wherein the subject is a human.

[0018] Item 12. The testing method according to any one of Items 1 to 11, wherein the subject has thrombotic microangiopathy (TMA), is undergoing or has just finished treatment for aHUS, or is a subject who has not yet developed aHUS.

[0019] Item 13. A method for detecting at least one complement selected from the group consisting of the C5b-9 complex, C3b, and C3c in extracellular vesicles contained in at least one selected from the group consisting of plasma and serum collected from a subject.

[0020] Item 14. A test agent for use in the test method according to any one of Items 1 to 12, comprising a complement detection agent.

[0021] Item 15. The test agent according to Item 14, for use in the method according to Item 10.

[0022] Item 16. A method for screening an active ingredient of a preventive or therapeutic agent for a complement-related disease, using the amount or concentration of complement in extracellular vesicles as an index.

[0023] Item 17. A method for evaluating the inducibility or aggravation of a complement-related disease, using the amount or concentration of complement in extracellular vesicles as an index.

[0024] Item 18. A detection agent for detecting at least one complement selected from the group consisting of the C5b-9 complex, C3b, and C3c in extracellular vesicles contained in at least one selected from the group consisting of plasma and serum, for use as a diagnostic agent for a complement-related disease.

[0025] Item 19. Use of a detection agent for at least one complement selected from the group consisting of the C5b-9 complex, C3b, and C3c in extracellular vesicles contained in at least one selected from the group consisting of plasma and serum, for the manufacture of a diagnostic agent for a complement-related disease.

[0026] The present invention provides a method for testing complement-related diseases, and also enables advance testing of whether administration of expensive complement-targeting drugs is effective or necessary for complement-related diseases, including aHUS.

[0027] Figure 1 shows the amount of C5b-9 complex protein in plasma for each group in Reference Example 1. Figure 2 shows the amount of C5b-9 complex protein on plasma-derived exosome membranes for each group in Test Example 1, converted into the amount contained in 1 ml of plasma. Figure 3 shows the amount of C3b protein on plasma-derived exosome membranes for each group in Test Example 2, converted into the amount contained in 1 ml of plasma. Figure 4 shows the amount of C3c protein on plasma-derived exosome membranes for each group in Test Example 3, converted into the amount contained in 1 ml of plasma.

[0028] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0029] 1. Method for Testing for Complement-Related Diseases In one aspect, the present invention relates to (1) a method for testing for complement-related diseases (sometimes referred to herein as the "testing method of the present invention"), which comprises the step of detecting complement in extracellular vesicles contained in at least one selected from the group consisting of plasma and serum collected from a subject. This method is described below.

[0030] 1-1. Step (1) The complement-related diseases to be tested are not particularly limited as long as the onset of the disease is related to abnormalities in the activation of the complement system, such as excessive activation of the complement system due to a deficiency or abnormality in a complement regulatory factor, or decreased activity of the complement system due to a deficiency of complement, and include aHUS, paroxysmal nocturnal hemoglobinuria, generalized myasthenia gravis, neuromyelitis optica spectrum disorder, hereditary angioedema, IgA nephropathy, systemic lupus erythematosus, rheumatoid arthritis, Guillain-Barré syndrome, COVID-19, atrophic age-related macular degeneration, and exudative ocular diseases. These conditions include age-related macular degeneration, ANCA-associated vasculitis, membranoproliferative glomerulonephritis, hidradenitis suppurativa, lupus nephritis, autoimmune hemolytic anemia, amyotrophic axonal sclerosis, acute kidney injury / acute renal failure, sickle cell disease, bullous pemphigoid, allergic conjunctival disease, uveitis, acute febrile neutrophilic dermatosis, squamous cell carcinoma, Huntington's disease, periodontitis, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, delayed onset of renal transplant function, dermatomyositis, meningitis, and bacterial infections such as those caused by Streptococcus pneumoniae. In one embodiment, the present invention also targets diseases associated with decreased activity of the complement system, such as complement deficiencies caused by deficiencies in complement components or complement regulatory factors involved in complement activation (e.g., factor B and factor D) due to congenital genetic abnormalities, and examples of such diseases include meningitis and bacterial infections such as Streptococcus pneumoniae. Preferred complement-related diseases are diseases caused by excessive activation of the complement system, and specific examples of such diseases include aHUS, paroxysmal nocturnal hemoglobinuria, generalized myasthenia gravis, neuromyelitis optica spectrum disorder, hereditary angioedema, IgA nephropathy, systemic lupus erythematosus, rheumatoid arthritis, Guillain-Barré syndrome, COVID-19, atrophic age-related macular degeneration, exudative age-related macular degeneration, ANCA-associated vasculitis, membranoproliferative glomerulonephritis, hidradenitis suppurativa, lupus nephritis, autoimmune hemolytic anemia, amyotrophic axonal sclerosis, acute kidney injury / acute renal failure, sickle cell disease, bullous pemphigoid, allergic conjunctival disease, uveitis, acute febrile neutrophilic dermatosis, squamous cell carcinoma, Huntington's disease, periodontitis, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, delayed onset of renal transplant function, and dermatomyositis. Among complement-related diseases, diseases accompanied by vascular endothelial cell damage (sometimes referred to herein as "diseases associated with vascular endothelial cell damage")For thrombotic microangiopathy (TMA), which is considered to be included in this category in the sense of vascular endothelial damage accompanied by thrombus, no definitive diagnostic method has been established, and it is difficult to diagnose the disease, distinguish between symptomatic and asymptomatic cases, or determine the effectiveness of treatment based solely on the total amount of complement in the blood. Therefore, it is more preferable to use it to test for aHUS, which requires testing the level of complement activation on cell membranes. Complement-related diseases include complement-related diseases of all classes, grades, and stages according to various classification criteria for progression, condition, etc.

[0031] The subject may be any mammal, including, for example, rodents such as mice, rats, hamsters, and guinea pigs, laboratory animals such as rabbits, pets such as dogs and cats, livestock such as cows, pigs, goats, horses, and sheep, primates such as monkeys, orangutans, and chimpanzees, and humans, with humans being particularly preferred.

[0032] The subject's condition with respect to a complement-related disease is not particularly limited. Examples of the subject include subjects with a disease accompanied by vascular endothelial cell damage or TMA, subjects undergoing or after treatment for aHUS, individuals who have not yet developed aHUS (i.e., individuals with genetic mutations in complement regulatory factors), subjects for whom it is unclear whether they have a complement-related disease, subjects with no history of a complement-related disease, subjects with a history of a complement-related disease and who have been treated for a complement-related disease, and subjects who have already been determined to have (or not have) a complement-related disease by other testing methods (e.g., complement-related gene testing, etc.). Particularly preferred subjects include subjects with a disease accompanied by vascular endothelial cell damage or TMA, subjects undergoing or after treatment for aHUS, and individuals who have not yet developed aHUS. Subjects with diseases or TMAs associated with vascular endothelial cell damage include subjects with unknown etiologies for developing TMA, such as aHUS, thrombotic thrombocytopenic purpura (TTP), Shiga-toxin-producing E. coli hemolytic uremic syndrome (STEC-HUS), pregnancy-associated TMA, transplant-associated TMA, hypertensive emergency, metabolism-associated TMA, drug-induced TMA, infection-associated TMA, autoimmune disease-associated TMA, and malignant tumor-associated TMA (pregnancy-associated TMA, transplant-associated TMA, hypertensive emergency, metabolism-associated TMA, drug-induced TMA, infection-associated TMA, autoimmune disease-associated TMA, and malignant tumor-associated TMA are also referred to as secondary TMA), and other TMA. The testing method of the present invention enables the differentiation of subjects with aHUS from subjects with TMA.

[0033] Extracellular vesicles are not particularly limited as long as they are derived from the same species as the biological sample. The International Society for Extracellular Vesicles (ISEV) defines extracellular vesicles as "nucleusless (non-replicating) lipid bilayer-enclosed particles released from cells." Based on their production mechanisms, extracellular vesicles are classified into (1) exosomes (50-150 nm in diameter), (2) microvesicles (100-1000 nm in diameter), and (3) apoptotic vesicles (5 μm in diameter). Protein components known to constitute extracellular vesicles include, for example, tetraspanins CD9, CD63, and CD81, ALIX, Tsg101, Hsp70, Hsp90, various integrins, various selectins, CD40, and Annexin V. It is particularly preferred that the extracellular vesicles used in the testing method of the present disclosure are exosomes.

[0034] In the testing method of the present disclosure, it is preferable to use extracellular vesicles contained in the body fluid of a subject. Extracellular vesicles can be obtained by purification from a biological sample such as a body fluid. Known methods for purifying extracellular vesicles include, for example, ultracentrifugation using an ultracentrifuge, antibody methods in which extracellular vesicles are precipitated using antibodies against extracellular vesicle markers, sedimentation methods in which extracellular vesicles are precipitated using polymers such as polyethylene glycol, and PS affinity methods using proteins that specifically bind to phosphatidylserine (PS) present in the cell membrane of extracellular vesicles. In one embodiment, purification by ultracentrifugation is preferred from the viewpoints that it is simple and inexpensive to perform, and furthermore, it allows for comprehensive collection and analysis of extracellular vesicles regardless of expression markers.

[0035] The body fluid is not particularly limited as long as it can contain extracellular vesicles. Examples of body fluids include whole blood, plasma, serum, sweat, urine, saliva, cerebrospinal fluid, synovial fluid, tissue fluid (including bronchoalveolar lavage fluid), tears, sputum, and nasal secretions. Preferred examples include whole blood, plasma, serum, sweat, urine, and saliva. More preferred examples include whole blood, plasma, and serum, from the viewpoint of containing a sufficient amount of extracellular vesicles derived from vascular endothelial cells. One type of body fluid may be used alone, or two or more types may be used in combination.

[0036] Body fluids can be collected from a subject by methods known to those skilled in the art. For example, whole blood can be collected by drawing blood using a syringe or the like. Plasma is a portion of whole blood from which blood cells have been removed, and can be obtained, for example, as the supernatant when whole blood is centrifuged under conditions that do not cause clotting. Serum is a portion of whole blood from which blood cells and specific blood coagulation factors have been removed, and can be obtained, for example, as the supernatant after whole blood is allowed to clot.

[0037] The detection target in step (1) is complement. As used herein, complement refers to complement proteins, their degradation products (complement components), complexes thereof, or mRNAs encoding complement proteins, including, for example, C1, C1q, C1r, C1s, C2, C3, C3a, C3b, iC3b, C3c, C3d, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, the C5b-9 complex (membrane attack complex: MAC), the C5b67 complex, the C4b2 complex, the C4b2a complex, the C3bB complex, and the C3bBb complex. In one embodiment, complement is preferably one that binds to and functions on the cell membrane, which serves as an indicator of complement activity on the cell membrane. Examples of such complement include the C5b-9 complex, C3b, the C5b67 complex, and C3d, and more preferably the C5b-9 complex, which is the end product of complement activation. In one embodiment, the complement is preferably an early component present on the cell membrane at an early stage of degradation over time, from the viewpoint of enabling early detection, and an example of such a complement is C3b. In one embodiment, the complement measured may be C3c, which is generated during degradation of iC3b to C3d.

[0038] The target complement is the complement whose amount changes in complement-related diseases, and by using this as an indicator, it is possible to distinguish whether a patient has a complement-related disease and whether treatment with a complement-targeted drug is expected to be effective or is necessary.

[0039] The C5b-9 complex, C3b, and C3c are complement components that are present in higher amounts on the extracellular vesicle membranes of subjects with complement-related diseases than on the extracellular vesicle membranes of subjects without complement-related diseases.

[0040] The number of target complements in step (1) may be one or a combination of two or more. By combining more complements, it is possible to more accurately perform tests for complement-related diseases.

[0041] Detection is usually performed by measuring the amount or concentration of the target complement. The "concentration" is not limited to absolute concentration, but may also be relative concentration, weight per unit volume, or raw data measured to determine absolute concentration.

[0042] When detecting the target complement of the present invention, which is a protein, the detection method of the target complement of the present invention (preferably a method for measuring the amount or concentration of the target complement of the present invention) is not particularly limited, as long as it is a method that can detect the target complement. Examples of such methods include immunoassays. A wide variety of immunoassays can be used, including direct, indirect, homogeneous, heterogeneous, competitive, and non-competitive methods. More specific examples of immunoassays include ELISA (e.g., direct, indirect, sandwich, and competitive), radioimmunoassays (RIA), immunoradiometric assays (IRMA), enzyme immunoassays (EIA), sandwich EIA, immunochromatography, Western blots, immunoprecipitation, slot or dot blot assays, immunohistochemical staining, fluorescent immunoassays, immunoassays using avidin-biotin or streptavidin-biotin systems, and immunoassays using surface plasmon resonance (SPR). Specifically, complement can be detected by immunoassay, for example, by directly or indirectly contacting a labeled antibody with a complement-binding molecule (preferably an antibody) bound to complement, and quantifying the signal derived from the label of the bound labeled antibody. The labeled antibody used in this case and the antibody mediating the labeled antibody and the complement-binding molecule or complement are not particularly limited, and examples that can be used include antibodies against antibody constant regions and anti-idiotype antibodies.

[0043] The type of label used in the labeling substance (e.g., labeled antibody) used to detect complement, which is a protein, is not particularly limited. Examples of labels include fluorescent substances, luminescent substances, dyes, enzymes, gold colloids, and radioisotopes. Among these, enzyme labels such as peroxidase and alkaline phosphatase are preferred from the viewpoints of safety, economy, detection sensitivity, and the like.

[0044] When detecting complement mRNA, the method for detecting complement (preferably a method for measuring the amount or concentration of complement) is not particularly limited, as long as it is capable of detecting complement. Examples of such methods include Northern blotting, RNase protection assay, reverse transcription polymerase chain reaction (RT-PCR), and quantitative real-time RT-PCR. In these methods, molecules that bind to complement (e.g., primers, probes, etc.) can be used. Primer pairs and probes can be synthesized based on the nucleotide sequence of complement mRNA. The base lengths of the primers and probes are not particularly limited. The base lengths of the primers can be 10 to 50 nucleotides, preferably 15 to 30 nucleotides. The base length of the probe can range from 10 nucleotides to the entire length of the nucleotide sequence complementary to the nucleotide sequence of the complement mRNA, preferably 20 to 150 nucleotides.

[0045] Primers and probes may be labeled or modified with fluorescent substances and / or quenchers, or labeling substances such as radioisotopes (e.g., 32P, 33P, 35S), or modifying substances such as biotin, (strept)avidin, or magnetic beads. The labeling substances are not limited, and commercially available products can be used. For example, fluorescent substances such as FITC, Texas, Cy3, Cy5, Cy7, Cyanine3, Cyanine5, Cyanine7, FAM, HEX, VIC, fluorescamine and its derivatives, and rhodamine and its derivatives can be used. Quencher substances such as AMRA, DABCYL, BHQ-1, BHQ-2, or BHQ-3 can be used. The labeling position of the labeling substance in primers and probes can be determined appropriately depending on the properties of the modifying substance and the intended use. Generally, modification is often performed at the 5' or 3' end. Furthermore, one primer and one probe molecule may be labeled with one or more types of labeling substances.

[0046] The detection method may be adopted singly or in combination of two or more.

[0047] According to the testing method of the present invention including step (1), the amount and / or concentration of complement in extracellular vesicles, which is an indicator for detecting complement-related diseases, can be provided, thereby assisting in the diagnosis of complement-related diseases, etc.

[0048] The test results obtained by the test method of the present invention including step (1) can be used to assess therapeutic efficacy, elucidate the pathology of complement-related diseases, predict the prognosis of complement-related diseases, stratify patients, select therapeutic methods (personalized medicine, therapeutic response), etc.

[0049] 1-2. Step (2) In one embodiment, the testing method of the present invention preferably further comprises the step (2) of determining that the subject has a complement-related disease characterized by excessive activation of the complement system if the amount or concentration of the complement detected in step (1) is equal to or greater than a cutoff value. In another embodiment, the testing method of the present invention preferably further comprises the step (2) of determining that the subject has a complement-related disease (including complement deficiency) characterized by decreased activity of the complement system if the amount or concentration of the complement detected in step (1) is equal to or less than a cutoff value. The testing method of the present invention, which includes step (2), makes it possible to determine whether the subject has a complement-related disease and whether a treatment with a complement-targeted drug is expected to be effective or is necessary.

[0050] The cutoff value can be appropriately set by a person skilled in the art from the viewpoints of sensitivity, specificity, positive predictive value, negative predictive value, etc., and can be a value determined on a case-by-case basis or a predetermined value based on, for example, the amount and / or concentration of the target complement in extracellular vesicles collected from a subject who does not have a complement-related disease. The cutoff value can be, for example, 0.5 to 1.5 times the amount and / or concentration of the target complement in extracellular vesicles collected from a subject who does not have a complement-related disease (e.g., the mean or median value when there are multiple subjects).

[0051] In a preferred embodiment of step (2), when the subject is undergoing treatment for a complement-related disease, the effectiveness of the treatment can be assessed by setting the cutoff value based on, for example, the amount and / or concentration of complement in a previous sample from the same subject.

[0052] 2. Diagnosis of Complement-Related Diseases with Higher Accuracy When a subject is determined to have a complement-related disease by the test method of the present invention including step (2), the test method of the present invention can be further combined with a step of having a physician diagnose the complement-related disease, thereby enabling a more accurate diagnosis of the complement-related disease. Furthermore, since the test method of the present invention can detect complement-related diseases more accurately, combining the above step with the test method of the present invention makes it possible to more efficiently and accurately diagnose "the subject as having a complement-related disease characterized by excessive activation of the complement system, or as having a complement-related disease (including complement deficiency) characterized by decreased activity of the complement system."

[0053] 3. Treatment of Complement-Related Diseases When a subject is determined to have a complement-related disease characterized by excessive activation of the complement system by the testing method of the present invention including step (2), or when a subject is diagnosed to have a complement-related disease characterized by excessive activation of the complement system as described above in "2. Diagnosis of Complement-Related Diseases with Higher Accuracy," the combination of the testing method of the present invention and the step of applying diagnosis by a physician can further include the steps of: (3) determining whether or not to administer a complement-targeted drug to the subject based on the results of step (2); and (4) treating the disease for a subject determined or diagnosed with a complement-related disease characterized by excessive activation of the complement system based on the results of step (3), and not treating or terminating treatment for a subject not determined or diagnosed with a complement-related disease characterized by excessive activation of the complement system, thereby making it possible to treat the disease in a subject in need of treatment. Furthermore, since the test method of the present invention can detect complement-related diseases more accurately, by combining steps (3) and (4) with the test method of the present invention, or with a combination of the test method of the present invention and a step of applying diagnosis by a physician, subjects with complement-related diseases can be treated more efficiently and reliably.

[0054] The treatment method for complement-related diseases is not particularly limited, but representative examples include plasma exchange therapy or drug therapy. Drugs used in drug therapy for diseases characterized by excessive activation of the complement system include complement-targeting drugs, i.e., antibody drugs against complement or complement receptors (e.g., anti-C5 antibody drugs, anti-C5a antibody drugs, anti-C1q antibody drugs, anti-C1s antibody drugs, anti-C3 antibody drugs, anti-C2 antibody drugs, anti-C5aR antibody drugs, etc.), small molecule drugs that target complement, complement regulatory factors, or complement receptors and inhibit their functions, peptide drugs and nucleic acid drugs (e.g., C1 esterase inhibitor protein, C5a receptor inhibitor, C3 inhibitor peptide, C5 inhibitor peptide, C5 inhibitor protein, C1q inhibitor peptide, Factor B inhibitor, C5 aptamer, etc.), or nucleic acid drugs that inhibit the transcription / translation of these proteins (e.g., C5 siRNA, C3 siRNA, Factor B aptamer, etc.). antisense nucleic acids, etc.), more specifically, eculizumab, ravulizumab-cwvz, pozelimab, crovalimab, ALXN1720, tesidolumab, IM101, vilobelimab, HIB210, avdoralimab, zilucoplan, nomacopan, avacincaptad pegol, cemdisiran, avacopan, INF904, IBI302, NGM621, pegcetacoplan, Compstatin, APL3007, ALXN2030, ARGX117, ANX005, DNTH103, sutimlimab, SAR445088, RLS0071, Cinryze IV, Haegarda, conestat alfa, IONIS-FB-LRx, Iptacopan, etc., and for diseases characterized by decreased activity of the complement system, antibiotics, steroids, etc. Treatment methods can be used alone or in combination of two or more.

[0055] The test results obtained by the test method of the present invention, which includes steps (2) and (3), can be used to more accurately diagnose complement-related diseases, assist in determining treatment plans for patients with complement-related diseases, and also to select patients to whom complement-targeted drugs should be administered.

[0056] 4. Test Agent for Complement-Related Diseases In one aspect, the present invention relates to a test agent for complement-related diseases (also referred to herein as the "test agent of the present invention") that includes a complement detection agent (also referred to herein as the "test agent of the present invention"). This will be described below.

[0057] In one aspect, the present invention relates to a method for detecting complement in extracellular vesicles collected from a subject, wherein the extracellular vesicles are preferably extracellular vesicles contained in at least one selected from the group consisting of plasma and serum, and the complement is preferably at least one type of complement selected from the group consisting of the C5b-9 complex, C3b, and C3c, more preferably C3c.

[0058] The definitions of complement, complement-related disease, extracellular vesicles, etc. are the same as those in "1. Method for testing complement-related disease" above.

[0059] The detection agent of the present invention is not particularly limited as long as it can specifically detect the target complement, and examples of the detection agent include an antibody against the target complement, a primer for the expression product of a gene for the complement, and a probe.

[0060] The detection agent of the present invention may be modified, as long as its function is not significantly impaired. Modifications include the addition of labels such as fluorescent dyes, enzymes, proteins, radioisotopes, chemiluminescent substances, biotin, etc.

[0061] Fluorescent dyes suitable for use in the present invention include those typically used to label nucleotides for the detection and quantification of nucleic acids. Examples include, but are not limited to, HEX (4,7,2',4',5',7'-hexachloro-6-carboxylfluorescein, a green fluorescent dye), fluorescein, NED (trade name, manufactured by Applied Biosystems, a yellow fluorescent dye), 6-FAM (trade name, manufactured by Applied Biosystems, a yellow-green fluorescent dye), and rhodamine or its derivatives (e.g., tetramethylrhodamine (TMR)). Nucleotides can be labeled with fluorescent dyes using any suitable known labeling method (see Nature Biotechnology, 14, 303-308 (1996)). Alternatively, commercially available fluorescent labeling kits (e.g., Oligonucleotide ECL 3'-Oligolabeling System, manufactured by Amersham-Pharmacia) can be used.

[0062] The detection agent of the present invention can be used by immobilizing it on any solid phase, and therefore the test agent of the present invention can be provided in the form of a substrate on which the detection agent is immobilized (for example, an ELISA plate on which an antibody is immobilized, or another example, a microarray chip on which a probe is immobilized).

[0063] The solid phase used for immobilization is not particularly limited as long as it can immobilize antibodies, polynucleotides, etc., and examples thereof include glass plates, nylon membranes, microbeads, silicon chips, capillaries, and other substrates. The immobilization of the detection agent to the solid phase is not particularly limited. For example, in the case of a microarray, a commercially available spotter (e.g., manufactured by Amersham) can be used. Immobilization methods are well known in the art depending on the type of immobilized probe (e.g., photolithographic technology (Affymetrix) or in situ synthesis of oligonucleotides using inkjet technology (Rosetta Inpharmatics)).

[0064] The antibody is not particularly limited as long as it selectively (specifically) recognizes complement. Here, "selectively (specifically) recognizes" means that it can specifically detect complement, for example, in Western blotting or ELISA, but is not limited thereto, and may be any antibody that allows a person skilled in the art to determine that the detected substance is derived from complement.

[0065] The term "antibody" includes, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, and molecules containing antigen-binding portions of the above antibodies, such as Fab fragments and fragments produced by an Fab expression library. The antibodies of the present invention also include antibodies that have antigen-binding ability to a polypeptide consisting of at least 8 consecutive amino acids, preferably 15 amino acids, and more preferably 20 amino acids, of a complementary amino acid sequence.

[0066] Methods for producing these antibodies are well known, and the antibodies of the present invention can be produced according to these conventional methods (Current Protocols in Molecular Biology, Chapters 11.12-11.13 (2000)). Specifically, when the antibodies of the present invention are polyclonal, they can be obtained from the serum of a non-human animal such as a rabbit by immunizing it with a complement protein expressed and purified in Escherichia coli or the like according to conventional methods, or with an oligopeptide having a partial amino acid sequence of the target complement protein by conventional methods. On the other hand, when the antibodies of the present invention are monoclonal, they can be obtained from hybridoma cells prepared by immunizing a non-human animal such as a mouse with a complement protein expressed and purified in Escherichia coli or the like according to conventional methods, or with an oligopeptide having a partial amino acid sequence of the complement protein, and fusing the resulting spleen cells with myeloma cells (Current Protocols in Molecular Biology, edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Sections 11.4-11.11).

[0067] The complement used as an immunizing antigen for antibody production can be obtained by DNA cloning, constructing each plasmid, transfecting the host, culturing the transformant, and recovering the protein from the culture, based on known gene sequence information. These procedures can be performed according to methods known to those skilled in the art or described in the literature (Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, D.M. Glover, IRL PRESS (1985)).

[0068] Specifically, a recombinant DNA (expression vector) capable of expressing a gene encoding a complement protein in a desired host cell is prepared, and the vector is then introduced into the host cell to transform it. The transformant is then cultured, and the target protein is recovered from the resulting culture, thereby obtaining a protein that can be used as an immunogen for producing the antibody of the present invention. Alternatively, a partial peptide of a complement protein can be produced by a general chemical synthesis method (peptide synthesis) according to known gene sequence information.

[0069] The antibodies of the present invention may also be prepared using an oligopeptide having a partial amino acid sequence of a complement. The oligo(poly)peptide used to produce such an antibody does not necessarily have functional biological activity, but it is desirable that it has immunogenic properties similar to those of the target complement. An example of an oligo(poly)peptide having these immunogenic properties and consisting of at least 8, preferably 15, and more preferably 20 consecutive amino acids in the amino acid sequence of the target complement is an oligo(poly)peptide.

[0070] The production of antibodies against such oligo(poly)peptides can be carried out by using various adjuvants depending on the host to enhance immunological responses, including, but not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, Pluronic® polyol, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol, and human adjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.

[0071] There are no particular limitations on the primers, probes, etc., as long as they selectively (specifically) recognize complement genes or nucleic acids derived therefrom. Here, "selectively (specifically) recognize" means, for example, in Northern blotting, that complement genes can be specifically detected, or in RT-PCR, that complement genes or nucleic acids derived therefrom (cDNA, etc.) can be specifically amplified. However, the term is not limited thereto, and any primers, probes, etc. may be used as long as a person skilled in the art can determine that the detected or amplified product is derived from a complement gene.

[0072] Specific examples of primers and probes include at least one selected from the group consisting of the polynucleotides described in (a) below and the polynucleotides described in (b) below: (a) a polynucleotide having at least 15 consecutive bases in the base sequence of the complement and / or a polynucleotide complementary to the polynucleotide, and (b) a polynucleotide having at least 15 bases that hybridizes under stringent conditions to the base sequence of the complement or a base sequence complementary thereto.

[0073] A complementary polynucleotide or complementary base sequence (complementary strand, reverse strand) refers to a polynucleotide or base sequence that is complementary to the full-length polynucleotide sequence consisting of the complementary base sequence, or a partial sequence thereof having at least 15 consecutive bases in length (for convenience, these are also referred to as the "positive strand" herein) based on base pairing, such as A:T and G:C. However, such a complementary strand is not limited to a completely complementary sequence to the base sequence of the target positive strand, but may also have a complementary relationship to the target positive strand that allows hybridization under stringent conditions. Here, stringent conditions can be determined based on the melting temperature (Tm) of the nucleic acid to which the complex or probe binds, as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego, CA). For example, typical post-hybridization washing conditions include approximately 1x SSC, 0.1% SDS, and 37°C. It is preferable that the complementary strand maintains its hybridization state with the target positive strand even when washed under these conditions. While not particularly limited, more stringent hybridization conditions include approximately 0.5x SSC, 0.1% SDS, and 42°C, and even more stringent hybridization conditions include approximately 0.1x SSC, 0.1% SDS, and 65°C. Specifically, examples of such complementary strands include a strand consisting of a nucleotide sequence that is completely complementary to the nucleotide sequence of the target positive strand, and a strand consisting of a nucleotide sequence that shares at least 90%, preferably 95%, more preferably 98% or more, and even more preferably 99% or more identity with the target positive strand.

[0074] Primers, probes, etc. can be designed, for example, based on the base sequence of the complement using various design programs. Specifically, candidate sequences for primers or probes obtained by applying the base sequence of the complement to a design program, or sequences containing at least a portion of such sequences, can be used as primers or probes.

[0075] The base length of a primer, probe, or the like is not particularly limited as long as it has a length of at least 15 consecutive bases as described above, and can be appropriately set depending on the application. For example, when used as a primer, the base length can be, for example, 15 to 35 bases, and when used as a probe, the base length can be, for example, 15 to 35 bases.

[0076] The test agent of the present invention may contain a detection agent other than the detection agent of the present invention (e.g., an antibody for detecting another protein, or a probe or antibody for detecting another nucleic acid). In this case, the test agent of the present invention may be a test agent that can test for other diseases or conditions in addition to complement-related diseases. In this case, the detection agent of the present invention is included as a detection agent for testing for complement-related diseases. From this perspective, in one aspect, the test agent of the present invention is a test agent for complement-related diseases that contains a detection agent for testing for complement-related diseases that consists of the detection agent of the present invention.

[0077] The test agent of the present invention may be in the form of a composition. The composition may contain other components as needed. Examples of other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0078] The test agent of the present invention may be in the form of a kit. In addition to the detection agent or the composition containing the same, the kit may contain other materials that can be used to detect the target complement in extracellular vesicles of a test subject. Specific examples of such materials include various reagents (e.g., buffer solutions), instruments (e.g., instruments for purifying and separating extracellular vesicles), and the like.

[0079] The test agent of the present invention can be used not only to test whether a patient has a complement-related disease, but also to select subjects for administration of a complement-targeting drug.

[0080] 5. Screening Method for Effective Ingredients of Preventive or Therapeutic Agents for Complement-Related Diseases In one aspect, the present invention relates to a screening method for effective ingredients of preventive or therapeutic agents for complement-related diseases (also referred to herein as the "screening method for effective ingredients of the present invention"), which uses the amount or concentration of complement in extracellular vesicles collected from an animal treated with a test substance as an index. This method is described below.

[0081] The definitions of complement, complement-related diseases, extracellular vesicles, and measurement of the amount or concentration of complement are the same as those in "1. Method for testing complement-related diseases" above.

[0082] The animal species is not particularly limited, and examples of the animal species include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits.

[0083] A wide range of test substances can be used, regardless of whether they are naturally occurring or artificially produced. Furthermore, not only purified compounds but also compositions containing a variety of compounds and animal and plant extracts can be used. Compounds are not limited to low-molecular-weight compounds, but also include high-molecular-weight compounds such as proteins, nucleic acids, and polysaccharides.

[0084] More specifically, the method for screening for an active ingredient of the present invention comprises a step of selecting the test substance as an active ingredient for a preventive or therapeutic agent for a complement-related disease characterized by excessive activation of the complement system (or a candidate substance for an active ingredient for a preventive or therapeutic agent for a complement-related disease characterized by excessive activation of the complement system) when the value of the index is lower than the amount or concentration (control value) of the corresponding complement in extracellular vesicles collected from an animal not treated with the test substance. In one embodiment, the method for screening for an active ingredient of the present invention comprises a step of selecting the test substance as an active ingredient for a preventive or therapeutic agent for a complement-related disease (including complement deficiency) characterized by decreased activity of the complement system (or a candidate substance for an active ingredient for a preventive or therapeutic agent for a complement-related disease (including complement deficiency) characterized by decreased activity of the complement system) when the value of the index is higher than the amount or concentration (control value) of the corresponding complement in extracellular vesicles collected from an animal not treated with the test substance.

[0085] The matched complement means the same complement as the target being measured.

[0086] "High" means, for example, that the index value is 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold higher than the control value.

[0087] "Low" means, for example, that the index value is 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, or 1 / 100 of the control value.

[0088] 6. Method for Evaluating Induction or Exacerbation of Complement-Related Diseases In one aspect, the present invention relates to a method for evaluating the induction or exacerbation of complement-related diseases using the amount or concentration of complement in extracellular vesicles collected from an animal treated with a test substance as an index (also referred to herein as the "toxicity evaluation method of the present invention"). This is described below.

[0089] The definitions of complement, complement-related diseases, extracellular vesicles, measurement of complement amount or concentration, animal species, test substance, etc. are the same as those in "1. Method for testing complement-related diseases" and "5. Method for screening active ingredients for preventive or therapeutic agents for complement-related diseases" above.

[0090] More specifically, the toxicity evaluation method of the present invention includes a step of determining that the test substance has the ability to induce or exacerbate a complement-related disease characterized by excessive activation of the complement system when the value of the indicator is higher than the amount or concentration (control value) of the corresponding complement in extracellular vesicles collected from an animal not treated with the test substance.In one aspect, the toxicity evaluation method of the present invention includes a step of determining that the test substance has the ability to induce or exacerbate a complement-related disease (including complement deficiency) characterized by decreased activity of the complement system when the value of the indicator is lower than the amount or concentration (control value) of the corresponding complement in extracellular vesicles collected from an animal not treated with the test substance.

[0091] The matched complement means the same complement as the target being measured.

[0092] "High" means, for example, that the index value is 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold higher than the control value.

[0093] "Low" means, for example, that the index value is 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, or 1 / 100 of the control value.

[0094] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0095] Reference Example 1. Measurement of C5b-9 complex protein levels in plasma Using 400 μl of plasma collected from untreated aHUS patients (n=2), aHUS patients undergoing plasma exchange therapy (n=4), aHUS patient receiving eculizumab (n=1), aHUS patient after treatment completion (n=1), a patient with a non-symptomatic disease who has a genetic mutation in a complement regulatory factor (n=1), a patient with secondary TMA due to causes other than complement overactivation (n=3), and healthy individuals (n=2), the amount of C5b-9 complex protein in plasma was measured using Quidel's SC5b-9 Plus EIA (see Figure 1). When the cutoff value was set to the mean + standard deviation of the measurement values ​​for healthy individuals (the reference value described in the Journal of the Japanese Society of Complement Biology: Complement Vol. 56, No. 2, 2019: 13-22), not only untreated and treated patients, but also asymptomatic individuals with mutations in onset-related genes but who have not developed aHUS showed values ​​above the cutoff value (dotted line), confirming that measuring the amount of complement in plasma alone is not sufficiently specific as a diagnostic method.

[0096] Test Example 1. Measurement of C5b-9 Complex Protein Amounts on Plasma-Derived Exosomes. 400 μl of plasma collected from untreated aHUS patients (n=2), aHUS patients undergoing plasma exchange therapy (n=4), aHUS patient receiving eculizumab (n=1), aHUS patient after treatment (n=2), a non-symptomatic patient with a complement regulatory factor gene mutation (n=1), a patient with secondary TMA (n=3), and a healthy individual (n=2) was added to 11 ml of PBS and mixed in a 15 ml Falcon tube. The mixture was filtered through a 0.22 μm filter using a 10 ml syringe. After filtration, PBS was added to a total volume of 35 ml, and the mixture was ultracentrifuged at 150,000 g for 70 minutes. The supernatant was discarded, and the pelleted exosomes were collected. The amount of C5b-9 complex protein on the membrane of the exosomes obtained was measured using Quidel's SC5b-9 Plus EIA, and the amount was calculated as the protein amount per ml of plasma (see Figure 2). The average value measured in aHUS patients undergoing plasma exchange or eculizumab administration was set as the cutoff value (dotted line). Only pre-treatment aHUS patients showed values ​​higher than the cutoff value, while healthy individuals and asymptomatic individuals showed values ​​below the cutoff value. These results, combined with those from Reference Example 1, suggest that asymptomatic individuals have genetic mutations that result in higher levels of complement in the blood than healthy individuals, but inhibit the formation of C5b-9 complexes on the cell membrane, thereby suppressing final complement activation. This confirms that this test method is an appropriate method that more accurately reflects the patient's current pathological condition.

[0097] Test Example 2: Measurement of C3b Protein Amounts on Plasma-Derived Exosomes. Exosomes were extracted from plasma from untreated aHUS patients (n = 4), aHUS patients after treatment (n = 2), aHUS patients receiving eculizumab (n = 4), asymptomatic individuals with genetic mutations in complement regulatory factors (n = 2), secondary TMA patients (n = 2), and healthy individuals (n = 2) in the same manner as in Test Example 1. The amount of C3b protein was measured using MyBioSource's Human Complement component 3b ELISA Kit. Furthermore, the amount of C3b protein converted to the amount of protein per ml of plasma was calculated (see Figure 3). AHUS patients at the onset of the disease showed significantly higher levels than patients after treatment, patients receiving eculizumab, asymptomatic individuals, or healthy individuals. This confirms that aHUS patients can also be detected using a test method using C3b on exosomes as a marker. Although not shown in the figure, the same method was able to detect C3b protein on exosomes in aHUS patients undergoing plasma exchange therapy, and it was confirmed that its expression trend was similar to that of the C5b-9 complex protein amount.

[0098] Test Example 3: Measurement of C3c Protein Amounts on Plasma-Derived Exosomes Exosomes were extracted from the plasma of untreated aHUS patients (n=4) and patients with secondary TMA (n=4) requiring clinical differentiation, as in Test Example 1, and the amount of C3c protein was measured using a Hycult Biotechnology C3c ELISA kit (HK368-01). Furthermore, the amount of C3c protein converted to the amount of protein per ml of plasma was calculated (see Figure 4). AHUS patients showed significantly higher levels than secondary TMA patients, confirming that aHUS patients can be detected using a test method using exosomal C3c as a marker, similar to C3b.

Claims

1. A method for testing for a complement-related disease, comprising: (1) detecting complement in extracellular vesicles contained in at least one selected from the group consisting of plasma and serum collected from a subject.

2. The method of claim 1, wherein the complement-related disease is a disease associated with vascular endothelial cell damage.

3. The method of claim 1, wherein the complement-related disease is thrombotic microangiopathy (TMA).

4. The method of claim 1, wherein the complement-related disease is characterized by excessive activation of the complement system.

5. The testing method according to claim 1, wherein the complement-related disease is atypical hemolytic uremic syndrome (aHUS).

6. The testing method according to claim 1, wherein the complement is at least one selected from the group consisting of the C5b-9 complex, C3b, and C3c.

7. The testing method of claim 2, wherein the complement is C3c.

8. The testing method described in claim 1, wherein the complement is a C5b-9 complex.

9. The testing method according to claim 1, further comprising: (2) a step of determining that the subject has a complement-related disease characterized by excessive activation of the complement system if the amount or concentration of the complement detected in step (1) is equal to or greater than a cutoff value.

10. The testing method described in claim 9, further comprising the step of: (3) determining whether or not to administer a complement targeting drug to the subject based on the results of step (2).

11. The method of claim 1, wherein the subject is a human.

12. The testing method according to claim 1, wherein the subject has thrombotic microangiopathy (TMA), is undergoing or has undergone treatment for aHUS, or is a subject who has not yet developed aHUS.

13. A method for detecting at least one complement selected from the group consisting of C5b-9 complex, C3b, and C3c in extracellular vesicles contained in at least one selected from the group consisting of plasma and serum collected from a subject.

14. A test agent for use in the test method according to any one of claims 1 to 12, comprising a complement detection agent.

15. The test agent according to claim 14 for use in the method according to claim 9.

16. A screening method for an active ingredient of a preventive or therapeutic agent for complement-related diseases, using the amount or concentration of complement in extracellular vesicles as an indicator.

17. A method for evaluating the inducibility or aggravation of a complement-related disease using the amount or concentration of complement in extracellular vesicles as an index.

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