Methods for detecting CM-TMA biomarkers

Abstract

Agents and methods for detecting complement-mediated thrombotic microangiopathy (CM-TMA) biomarkers are provided. The agents can specifically bind to CM-TMA biomarkers, preferably the proteolytic fragment of complement component factor B (Ba) and soluble C5b9 (sC5b9), and can be used in methods of diagnosis and treatment of CM-TMA, for example, treatment with anti-C5 antibodies such as ravulizumab (ALXN1210).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED PATENT APPLICATIONS This international patent application claims priority to U.S. Provisional Patent Application No. 63 / 194,704, filed May 28, 2021, and U.S. Provisional Patent Application No. 63 / 272,033, filed October 26, 2021, the disclosures of each of which are incorporated herein in their entirety.

[0002] 1. Field The present disclosure relates to agents and methods for the detection of complement-mediated thrombotic microangiopathy (CM-TMA) biomarkers. The agents are capable of specifically binding to CM-TMA biomarkers, preferably the proteolytic fragment of complement component factor B (Ba) and soluble C5b9 (sC5b9), and can be used in methods of diagnosis and treatment of CM-TMA. [Background technology]

[0003] 2. Description of Related Technology Complement-mediated thrombotic microangiopathy (CM-TMA) is a clinical disorder driven by excess complement production. It is characterized by thrombocytopenia and microangiopathic hemolytic anemia (MAHA) and microvascular thrombosis (TMA) resulting in systemic organ damage. Atypical hemolytic uremic syndrome (aHUS), a form of CM-TMA, is characterized by pathological complement activation due to a decrease in natural regulators of the complement system, resulting in systemic endothelial and organ damage. Lupus erythematosus is a multiorgan immune complex disorder associated with complement activation and renal failure termed lupus nephritis (LN). A subset of these patients develops TMA as well as progressive, life-threatening thrombocytopenia, MAHA, and progressive renal failure similar to aHUS. This subset of patients is poorly responsive to corticosteroids, cyclophosphamide, immunomodulation, and plasma exchange. Park et al. Blood Adv (2018) 2(16):2090-2094. There is a need in the art for effective detection of CM-TMA biomarkers for diagnosing CM-TMA. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Park et al.Blood Adv(2018)2(16):2090-2094 Summary of the Invention [Means for solving the problem]

[0005] The present disclosure is based in part on the identification of biomarkers in urine and / or plasma that allow for the diagnosis, prognosis, management, and treatment of patients with CM-TMA. In particular, the present disclosure relates to the detection and use of protein biomarkers such as cystatin C; cystatin C / creatinine ratio; complement factor Ba; complement factor Ba / creatinine ratio; complement sC5b-9; and / or complement sC5b-9 / creatinine ratio, preferably using a biomarker signature including at least two biomarkers Ba and sC5b9, in the diagnosis of CM-TMA and in measuring the responsiveness of patients to treatment with anti-C5 antibody therapy, such as ULTOMIRIS®. In some embodiments, the present disclosure further relates to the use of secondary markers, such as estimated glomerular filtration rate (eGFR) and / or urinary protein creatinine ratio (UPCR), in addition to the above biomarkers, in the diagnosis and / or treatment of CM-TMA. In particular, in embodiments relating to treatment of CM-TMA with anti-C5 antibodies, such as ULTOMIRIS (ULTOMIRIS®), certain biomarkers, such as baseline (BL) plasma Ba levels, were found to be associated with eGFR changes after treatment. Similarly, baseline (BL) urinary sC5b-9, sC5b-9 / Cr, urinary Ba, and Ba / Cr were associated with UPCR changes after treatment. In an embodiment, a method for assessing complete TMA response may include detecting a set of biomarkers in a subject, detecting the levels of at least one, at least two, at least three, at least four, at least five, at least six biomarkers selected from the group consisting of cystatin C; cystatin C / creatinine ratio; complement factor Ba; complement factor Ba / creatinine ratio; complement sC5b-9; complement sC5b-9 / creatinine ratio, sVCAM-1, sTNF-R1, and / or thrombomodulin; preferably detecting the levels of the biomarkers in a subset comprising at least two biomarkers comprising Ba and sC5b9 in a body fluid sample obtained from the subject. The subset of biomarkers may include serum sVCAM-1, serum sTNF-R1, plasma thrombomodulin, and urinary sC5b-9.

[0006] In certain embodiments, a method for diagnosing complement-mediated thrombotic microangiopathy (CM-TMA) in a subject may include: (a) detecting a level of a biomarker selected from the group consisting of cystatin C, cystatin C / creatinine ratio, complement factor Ba, complement factor Ba / creatinine ratio, complement sC5b-9, complement sC5b-9 / creatinine ratio, sVCAM-1, sTNF-R1, thrombomodulin, or a combination thereof, in a bodily fluid sample obtained from the subject; and (b) comparing the level of the biomarker, preferably in a signature, to a control; wherein an increase in the level of the biomarker in the subject's biological sample compared to that of the control indicates that the subject is suffering from, suspected of suffering from, or at risk of suffering from CM-TMA.

[0007] In one embodiment, a method for evaluating a treatment of Complement-Mediated Thrombotic Microangiopathy (CM-TMA) in a subject includes: (a) obtaining a bodily fluid sample from a subject suffering from Complement-Mediated Thrombotic Microangiopathy (CM-TMA); (b) detecting a level of a biomarker selected from the group consisting of cystatin C, cystatin C / creatinine ratio, complement factor Ba, complement factor Ba / creatinine ratio, complement sC5b-9, complement sC5b-9 / creatinine ratio, sVCAM-1, sTNF-R1, thrombomodulin, or a combination thereof, in the bodily fluid sample obtained from the subject; (c) comparing the level of the biomarker with a control; and (d) detecting a level of a biomarker selected from the group consisting of cystatin C, cystatin C / creatinine ratio, complement factor Ba, complement factor Ba / creatinine ratio, complement sC5b-9 / creatinine ratio, sVCAM-1, sTNF-R1, thrombomodulin, or a combination thereof, in the bodily fluid sample obtained from the subject; (c) comparing the level of the biomarker with a control; and (d) detecting a level of a biomarker selected from the group consisting of cystatin C / creatinine ratio, complement factor Ba / creatinine ratio, complement sC5b-9 / creatinine ratio, sVCAM-1, sTNF-R1, thrombomodulin, or a combination thereof, in the bodily fluid sample obtained from the subject. (e) obtaining a bodily fluid sample from the subject suffering from complement-mediated thrombotic microangiopathy (CM-TMA); (f) detecting a level of a biomarker selected from the group consisting of cystatin C, cystatin C / creatinine ratio, complement factor Ba, complement factor Ba / creatinine ratio, complement sC5b-9, complement sC5b-9 / creatinine ratio, sVCAM-1, sTNF-R1, thrombomodulin, sC5b-9, or a combination thereof, in the bodily fluid sample obtained from the subject; and (f) comparing the level of the biomarker to a control, wherein the control comprises a patient not suffering from complement-mediated thrombotic microangiopathy (CM-TMA).

[0008] In certain embodiments, a method for detecting a biomarker may include: (a) obtaining a bodily fluid sample from a subject suffering from complement-mediated thrombotic microangiopathies (CM-TMA); and (b) detecting in the bodily fluid sample obtained from the subject a level of a biomarker selected from the group consisting of cystatin C, cystatin C / creatinine ratio, complement factor Ba, complement factor Ba / creatinine ratio, complement sC5b-9, complement sC5b-9 / creatinine ratio, sVCAM-1, sTNF-R1, thrombomodulin, or a combination thereof. The method may further include comparing the level of the biomarker to a control, where the control comprises a patient not suffering from complement-mediated thrombotic microangiopathies (CM-TMA).

[0009] In certain embodiments, the bodily fluid sample comprises a heterogeneous sample and can include plasma Ba, urinary Ba / Cr, urinary sC5b-9 / Cr, and plasma sC5b-9.

[0010] In certain embodiments, the detecting step may include detecting serum VCAM-1, serum sTNF-R1, plasma thrombomodulin, and urinary sC5b-9.

[0011] In certain embodiments, a method for diagnosing complement-mediated thrombotic microangiopathy (CM-TMA) in a subject may include: (a) detecting the levels of at least one, at least two, at least three, at least four, at least five, at least six biomarkers selected from the group consisting of cystatin C; cystatin C / creatinine ratio; complement factor Ba; complement factor Ba / creatinine ratio; complement sC5b-9; and / or complement sC5b-9 / creatinine ratio; preferably detecting the levels of the biomarkers in a signature comprising at least two biomarkers comprising Ba and sC5b9 in a bodily fluid sample obtained from the subject; and (b) comparing the levels of the biomarkers; preferably the levels of the biomarkers in the signature; with a control, wherein an increase in the levels of the biomarkers in the subject's biological sample compared to that of the control indicates that the subject is suffering from, suspected of suffering from, or at risk of suffering from CM-TMA.

[0012] In some embodiments, a method for detecting cystatin C, complement factor Ba, complement sC5b-9 may include: (a) detecting the level of at least one, at least two, at least three, at least four, at least five, at least six biomarkers selected from the group consisting of cystatin C; cystatin C / creatinine ratio; complement factor Ba; complement factor Ba / creatinine ratio; complement sC5b-9; and / or complement sC5b-9 / creatinine ratio; preferably detecting the level of the biomarkers in a signature comprising at least two biomarkers including Ba and sC5b9 in a body fluid sample obtained from a subject; and (b) comparing the level of the biomarkers; preferably the level of the biomarkers in the signature; with a control, optionally a healthy subject. In some embodiments, an increase in the level of the biomarkers in the subject's biological sample compared to that of the control may indicate that the subject is suffering from, suspected of suffering from, or at risk of suffering from CM-TMA.

[0013] In one embodiment, a method for detecting a set of biomarkers in a subject may comprise detecting a biomarker selected from the group consisting of cystatin C; cystatin C / creatinine ratio; complement factor Ba; complement factor Ba / creatinine ratio; complement sC5b-9; and / or complement sC5b-9 / creatinine ratio; preferably detecting the level of the biomarker in a subset comprising at least two biomarkers comprising Ba and sC5b9 in a bodily fluid sample obtained from the subject.

[0014] In certain embodiments, a method for detecting a set of biomarkers in a subject may comprise detecting a biomarker selected from the group consisting of cystatin C; cystatin C / creatinine ratio; complement factor Ba; complement factor Ba / creatinine ratio; complement sC5b-9, optionally urinary sC5b-9; complement sC5b-9 / creatinine ratio, sVCAM-1, optionally serum sVACM-1, sTNF-R1, optionally serum sTNF-R1, thrombomodulin, optionally plasma thrombomodulin, or a combination thereof, in a bodily fluid sample obtained from the subject.

[0015] In certain embodiments, the biomarkers may be selected from the group consisting of cystatin C; cystatin C / creatinine ratio; complement factor Ba; complement factor Ba / creatinine ratio; complement sC5b-9, optionally urinary sC5b-9; complement sC5b-9 / creatinine ratio, sVCAM-1, optionally serum sVACM-1 (soluble vascular cell adhesion molecule-1), sTNF-R1 (soluble tumor necrosis factor receptor 1), optionally serum sTNF-R1, thrombomodulin, optionally plasma thrombomodulin, or a combination thereof.

[0016] In certain embodiments, the bodily fluid sample may include serum, blood, urine, plasma, or a mixture thereof. The bodily fluid sample may include urine. The bodily fluid sample may include plasma. The bodily fluid sample may include blood. The bodily fluid sample may include serum. The bodily fluid sample may include a mixture of urine, plasma, and serum.

[0017] In certain embodiments, the biomarker may be related to a subject's estimated glomerular filtration rate (eGFR) and / or urinary protein creatinine ratio (UPCR).

[0018] In certain embodiments, the method may include detecting levels of creatinine in bodily fluid samples from subjects and controls and normalizing levels of biomarkers in the bodily fluid samples based on the creatinine levels; preferably, the bodily fluid samples may include urine and the creatinine levels are detected using a creatinine assay.

[0019] In certain embodiments, the control may include an identical biological sample from a healthy subject.

[0020] In certain embodiments, the biomarker may comprise a protein biomarker selected from cystatin C, complement factor Ba, and / or sC5b9, and detecting comprises contacting the biomarker with an agent comprising an antibody or antigen-binding fragment thereof that specifically binds to the biomarker. The antibody, or antigen-binding fragment thereof, may be selected from the group consisting of a humanized antibody, a recombinant antibody, a bispecific antibody, a chimerized or chimeric antibody, a monoclonal antibody, a deimmunized antibody, a fully human antibody, a single chain antibody, an Fv fragment, an Fd fragment, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a combination thereof.

[0021] In certain embodiments, the detecting step may include detecting a biomarker signature that includes the following biomarkers: (a) cystatin C+Ba; (b) cystatin C+sC5b9; (c) cystatin C+cystatin C / creatinine; (d) cystatin C+Ba / creatinine; (e) cystatin C+sC5b9 / creatinine; (f) Ba+sC5b9; (g) Ba+cystatin C / creatinine. (h) Ba+Ba / creatinine; (i) Ba+sC5b9 / creatinine; (j) sC5b9+cystatin C / creatinine; (k) sC5b9+Ba / creatinine; (l) sC5b9+sC5b9 / creatinine; (m) cystatin C / creatinine+Ba / creatinine; (n) cystatin C / creatinine+sC5b9 / creatinine; or (o) Ba / creatinine+sC5b9 / creatinine.

[0022] In certain embodiments, the detecting step may include detecting a biomarker signature comprising the following biomarkers: (a) cystatin C + Ba + sC5b9; (b) cystatin C + Ba + cystatin C / creatinine; (c) cystatin C + Ba + Ba / creatinine; (d) cystatin C + Ba + sC5b9 / creatinine; (e) cystatin C + sC5b9 + cystatin C / creatinine; (f) cystatin C + sC5b9 + Ba / creatinine; (g) cystatin C + sC5b9 + sC5b9 / creatinine; (h) cystatin C + cystatin C / creatinine + Ba / creatinine; (i) cystatin C + cystatin C / creatinine + sC5b9 / creatinine; (j) cystatin C + Ba / Creatinine. (k) Ba + sC5b9 + cystatin C / creatinine; (l) Ba + sC5b9 + Ba / creatinine; (m) Ba + sC5b9 + sC5b9 / creatinine; (n) Ba + cystatin C / creatinine + Ba / creatinine; (o) Ba + cystatin C / creatinine + sC5b9 / creatinine; (p) Ba + Ba / creatinine + sC5b9 / creatinine; (q) sC5b9 + cystatin C / creatinine + Ba / creatinine; (r) sC5b9 + cystatin C / creatinine + sC5b9 / creatinine; (s) sC5b9 + Ba / creatinine + sC5b9 / creatinine; or (t) cystatin C / creatinine + Ba / creatinine + sC5b9 / creatinine.

[0023] In certain embodiments, the detecting step may include detecting a biomarker signature that includes the following biomarkers: (a) cystatin C + Ba + sC5b9 + cystatin C / creatinine; (b) cystatin C + Ba + sC5b9 + Ba / creatinine; (c) cystatin C + Ba + sC5b9 + sC5b9 / creatinine; (d) cystatin C + sC5b9 + cystatin C / creatinine + (e) cystatin C + sC5b9 + cystatin C / creatinine + sC5b9 / creatinine. creatinine; (f) cystatin C + cystatin C / creatinine + Ba / creatinine + sC5b9 / creatinine; (g) Ba + sC5b9 + cystatin C / creatinine + Ba / creatinine; (h) Ba + sC5b9 + cystatin C / creatinine + sC5b9 / creatinine; (i) Ba + cystatin C / creatinine + Ba / creatinine + sC5b9 / creatinine; or (j) sC5b9 + cystatin C / creatinine + Ba / creatinine + sC5b9 / creatinine.

[0024] In certain embodiments, the detecting step may include detecting a biomarker signature comprising the following biomarkers: (a) cystatin C + Ba + sC5b9 + cystatin C / creatinine + Ba / creatinine; (b) cystatin C + Ba + sC5b9 + cystatin C / creatinine + sC5b9 / creatinine; (c) cystatin C + sC5b9 + cystatin C / creatinine + Ba / creatinine + sC5b9 / creatinine; or (d) Ba + sC5b9 + cystatin C / creatinine + Ba / creatinine + sC5b9 / creatinine.

[0025] In certain embodiments, the detecting step may include detecting a biomarker signature comprising the following biomarkers: (a) Cystatin C+Ba+sC5b9+Cystatin C / creatinine+Ba / creatinine+sC5b9 / creatinine.

[0026] In certain embodiments, the method may further comprise detecting a plasma biomarker selected from Ba and sC5b9.

[0027] In certain embodiments, the method may further comprise detecting a plasma biomarker signature comprising Ba and sC5b9.

[0028] In certain embodiments, the bodily fluid sample may include a heterogeneous sample including urine and plasma, and the detecting step may include detecting a biomarker signature comprising at least one urinary biomarker and at least one plasma biomarker selected from: (a) urinary cystatin C + plasma Ba; (b) urinary cystatin C + plasma sC5b9; (c) urinary Ba + plasma Ba; (d) urinary Ba + plasma sC5b9; (e) urinary sC5b9 + plasma Ba; (f) urinary sC5b9 + plasma sC5b9; (g) urinary cystatin C / creatinine + plasma Ba; (h) urinary cystatin C / creatinine + plasma sC5b9; (i) urinary Ba / creatinine + plasma Ba; (j) Ba / creatinine + plasma sC5b9; (k) urinary sC5b9 / creatinine + plasma Ba; or (l) urinary sC5b9 / creatinine + plasma sC5b9.

[0029] In certain embodiments, the bodily fluid sample may include a heterogeneous sample including urine and plasma, and the detecting step may include detecting a biomarker signature comprising at least one urinary biomarker and at least two plasma biomarkers selected from: (a) urinary cystatin C + plasma Ba + plasma sC5b9; (b) urinary Ba + plasma Ba + plasma sC5b9; (c) urinary sC5b9 + plasma Ba + plasma sC5b9; (d) urinary cystatin C / creatinine + plasma Ba + plasma sC5b9; (e) urinary Ba / creatinine + plasma Ba + plasma sC5b9; or (f) urinary sC5b9 / creatinine + plasma Ba + plasma sC5b9.

[0030] In certain embodiments, the bodily fluid sample may include a heterogeneous sample including urine and plasma, and the detecting step may include: (a) urinary cystatin C + urinary Ba + plasma Ba or plasma sC5b9; (b) urinary cystatin C + urinary sC5b9 + plasma Ba or plasma sC5b9; (c) urinary cystatin C + urinary cystatin C / creatinine + plasma Ba or plasma sC5b9; (d) urinary cystatin C + urinary Ba / creatinine + plasma Ba or plasma sC5b9; (e) urinary cystatin C + urinary sC5b9 / creatinine + plasma Ba or plasma sC5b9; (f) urinary Ba + urinary sC5b9 + plasma Ba or plasma sC5b9; (g) urinary Ba + urinary cystatin C / creatinine + plasma Ba or plasma sC5b9; (h) urinary Ba + urinary Ba / creatinine + plasma Ba or plasma sC5b9; (i) urinary Ba + urinary sC5b9 (j) urinary sC5b9 + urinary cystatin C / creatinine + plasma Ba or plasma sC5b9; (k) urinary sC5b9 + urinary Ba / creatinine + plasma Ba or plasma sC5b9; (l) urinary sC5b9 + urinary sC5b9 / creatinine + plasma Ba or plasma sC5b9; (m) urinary cystatin C / creatinine + urinary Ba / creatinine + plasma Ba or plasma sC5b9; (n) urinary cystatin C / creatinine + urinary sC5b9 / creatinine + plasma Ba or plasma sC5b9; or (o) urinary Ba / creatinine + urinary sC5b9 / creatinine + plasma Ba or plasma sC5b9. Biomarkers may include plasma complement factors Ba, sC5b-9, thrombomodulin, and D-dimer; serum sTNF-RI and sVCAM-1; urinary Ba factor, sC5b-9, and cystatin C.

[0031] In certain embodiments, the methods may further include measuring secondary markers, including the subject's estimated glomerular filtration rate (eGFR).

[0032] In certain embodiments, the method may further comprise measuring a secondary marker including the subject's urinary protein / creatinine ratio (UPCR).

[0033] In certain embodiments, the levels of the biomarker and the secondary marker are increased as compared to a control.

[0034] In certain embodiments, the control may include an identical bodily fluid sample obtained from a subject without complement-mediated thrombotic microangiopathy (CM-TMA), optionally, a subject with undetectable levels of urinary sC5b9.

[0035] In certain embodiments, the subject may be a mammal. The subject may be a human.

[0036] In certain embodiments, the subject may have, be suspected of having, or be at risk of developing complement-mediated thrombotic microangiopathy (CM-TMA).

[0037] In some embodiments, the subject may be undergoing or may be undergoing treatment with a complement inhibitor. The complement inhibitor may include a complement C5 inhibitor selected from the group consisting of an antibody, a small molecule, a polypeptide, a polypeptide analog, a peptidomimetic, and an aptamer, or a combination thereof. The complement C5 inhibitor may be selected from the group consisting of a recombinant anti-C5 mini-antibody MB12 / 22, an anti-C5 mini-antibody targeted to endothelial MB12 / 22-RGD, a C5 specific aptamer ARC187, a C5 specific aptamer ARC1905 (Avacincaptad pegol), Staphylococcus aureus superantigen-like protein 7 (SSL7), Ornithodoros moubata C inhibitor (OmCI), or a combination thereof. The complement inhibitor may comprise an anti-C5 antibody or antigen-binding fragment thereof that (a) specifically binds to complement C5, and optionally (b) inhibits cleavage of C5 into fragments C5a and C5b; preferably, the antigen-binding fragment comprises an antibody heavy chain complementarity determining region 1-3 (VCR1-VCR3) of the anti-C5 antibody. H CDR1-3) and antibody light chain complementarity determining regions 1-3 (V LCDR1-3); more preferably, an antigen-binding fragment comprising the variable heavy (VH) chain and the variable light (VL) chain of an anti-C5 antibody. The antibody, or the antigen-binding fragment thereof, may be selected from the group consisting of a humanized antibody, a recombinant antibody, a bispecific antibody, a chimerized or chimeric antibody, a monoclonal antibody, a deimmunized antibody, a fully human antibody, a single chain antibody, an Fv fragment, an Fd fragment, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, or a combination thereof. The anti-C5 antibody may include eculizumab, ravulizumab, or a combination thereof, or a biosimilar thereof.

[0038] In certain embodiments, the anti-C5 antibody may include ravulizumab or a biosimilar thereof; preferably, the treatment includes treatment with ravulizumab, including a single loading dose on day 1, followed by a regular maintenance dose starting on day 15, based on the subject's body weight, where (a) for subjects weighing ≧40 to <60 kilograms (kg), the treatment includes a loading dose of 2400 milligrams (mg), followed by a maintenance dose of 3000 mg every 8 weeks; (b) for subjects weighing ≧60 to <100 kg, the treatment includes a loading dose of 2700 mg, followed by a maintenance dose of 3300 mg every 8 weeks; and (c) for subjects weighing ≧100 kg, the treatment includes a loading dose of 3000 mg, followed by a maintenance dose of 3600 mg every 8 weeks. The anti-C5 antigen-binding fragment may include pexelizumab.

[0039] In certain embodiments, the subject may have been treated with a complement inhibitor, where the treatment was administered less than one month, optionally less than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day prior to obtaining the bodily fluid sample from the subject.

[0040] In certain embodiments, the method may further include determining whether the subject is at risk for developing complement-mediated thrombotic microangiopathy (CM-TMA).

[0041] In certain embodiments, the subject may be or may be being treated with a complement inhibitor under a prescribed dosing schedule, and the method further includes determining whether the patient is therapeutically responsive to the complement inhibitor therapy.

[0042] In certain embodiments, the subject may have or be at risk of developing complement-mediated thrombotic microangiopathy (CM-TMA), including atypical hemolytic uremic syndrome (aHUS); preferably, the CM-TMA includes renal aHUS.

[0043] In one embodiment, the method comprises detecting the levels of at least one, at least two, at least three, at least four, at least five, at least six biomarkers selected from the group consisting of: cystatin C; cystatin C / creatinine ratio; complement factor Ba; complement factor Ba / creatinine ratio; complement sC5b-9; and / or complement sC5b-9 / creatinine ratio; preferably detecting the levels of biomarkers in a signature comprising at least two biomarkers comprising Ba and sC5b9 in a body fluid sample obtained from the subject before and after treatment; In a method for monitoring the responsiveness of a subject to treatment with an inhibitor of complement C5, the method includes comparing the level of a biomarker in a sample; preferably the level of the biomarker in a signature; the subject has, is suspected of having, or is at risk of developing complement-mediated thrombotic microangiopathies (CM-TMA); wherein the subject is likely to be or is being treated with an inhibitor of complement C5; and a decrease in the level of the biomarker in the subject's body fluid sample after treatment compared to the level before treatment with a complement C5 inhibitor indicates that the subject is responsive to treatment. The method may further include measuring a secondary marker including the subject's estimated glomerular filtration rate (eGFR). The method may further include measuring a secondary marker including the subject's urinary protein / creatinine ratio (UPCR). The level of the biomarker and the secondary marker in the subject's body fluid sample may decrease after treatment.

[0044] In certain embodiments, a method for treating complement-mediated thrombotic microangiopathy (CM-TMA) using a complement inhibitor in a manner sufficient to induce a physiological change in a CM-TMA-associated biomarker protein may include: (a) measuring the level or activity of a CM-TMA-associated biomarker protein selected from the group consisting of cystatin C; cystatin C / creatinine ratio; complement factor Ba; complement factor Ba / creatinine ratio; complement sC5b-9; and / or complement sC5b-9 / creatinine ratio in a bodily fluid obtained from the subject; preferably measuring the level or activity of a biomarker in a signature comprising at least two biomarkers including Ba and sC5b9; and (b) administering a complement inhibitor to a subject having, suspected of having, or at risk of developing CM-TMA in an amount and frequency sufficient to cause a decrease in the level or activity of a biomarker or biomarker signature compared to its level or activity in a sample of the same type of bodily fluid obtained from the subject prior to treatment with the inhibitor. The complement inhibitor may include ravulizumab.

[0045] In one embodiment, a method for determining whether a patient having complement-mediated thrombotic microangiopathy (CM-TMA) and being treated with a complement inhibitor under a given dosing schedule requires a different dosing schedule comprises: (A) determining whether a CM-TMA patient is responsive to treatment with a complement inhibitor under a given dosing schedule by measuring, in a body fluid obtained from the subject, one or both of the concentration and activity of a CM-TMA-associated biomarker protein selected from the group consisting of cystatin C; cystatin C / creatinine ratio; complement factor Ba; complement factor Ba / creatinine ratio; complement sC5b-9; and / or complement sC5b-9 / creatinine ratio. measuring levels of biomarkers in a signature preferably comprising at least two biomarkers including Ba and sC5b9; and (B) if the patient is not responsive to treatment with a complement inhibitor, administering to the non-responding patient a different complement inhibitor or the same complement inhibitor at a higher dose or more frequent dosing schedule compared to the given dosing schedule, where (a) a decreased level or activity of the biomarker or biomarker signature compared to the concentration in a sample of the same type of bodily fluid obtained from the subject prior to treatment with the inhibitor indicates that the subject is responsive to treatment with the inhibitor. The complement inhibitor may include ravulizumab. The biomarkers may be selected from the group consisting of cystatin C; cystatin C / creatinine ratio; complement factor Ba; complement factor Ba / creatinine ratio; complement sC5b-9, optionally urinary sC5b-9; complement sC5b-9 / creatinine ratio, sVCAM-1, optionally serum sVACM-1; sTNF-R1, optionally serum sTNF-R1; thrombomodulin, optionally plasma thrombomodulin; or a combination thereof.

[0046] In certain embodiments, a kit for diagnosing complement-mediated thrombotic microangiopathy (CM-TMA) may include an assay plate and a binding agent, optionally together with instructions for using the kit, where the binding agent is an antibody, or antigen-binding fragment thereof, that independently binds with specificity to multiple biological analytes, where the analytes are protein biomarkers for CM-TMA, and where the protein biomarkers include the proteolytic fragment of complement component factor B (Ba) and soluble C5b9 (sC5b-9), optionally together with cystatin C. The biomarkers may be selected from the group consisting of: cystatin C; cystatin C / creatinine ratio; complement factor Ba; complement factor Ba / creatinine ratio; complement sC5b-9, optionally urinary sC5b-9; complement sC5b-9 / creatinine ratio, sVCAM-1, optionally serum sVACM-1; sTNF-R1, optionally serum sTNF-R1, thrombomodulin, optionally plasma thrombomodulin; or combinations thereof. The kit may further comprise reagents for creatinine assay. The kit may further comprise reagents for measuring urinary protein / creatinine ratio (UPCR).

[0047] In an embodiment, a composition for treating complement-mediated thrombotic microangiopathies (CM-TMA) in a subject may comprise an effective amount of a complement inhibitor, wherein the complement inhibitor is an anti-C5 antibody or a C5-binding fragment thereof, preferably eculizumab, ravulizumab, or a biosimilar thereof. The effective amount may be an amount sufficient to reduce the level of a biomarker and / or a biomarker signature in a subject compared to its level before treatment with a complement inhibitor. The biomarker may comprise at least one, at least two, at least three, at least four, at least five, at least six biomarkers selected from the group consisting of cystatin C; cystatin C / creatinine ratio; complement factor Ba; complement factor Ba / creatinine ratio; complement sC5b-9; complement sC5b-9 / creatinine ratio, or a combination thereof. The biomarker signature may comprise at least two biomarkers, including Ba and sC5b9. The level of the biomarker and / or biomarker signature can be detected in a bodily fluid sample obtained from the subject. The bodily fluid sample can be urine, plasma, or a combination thereof. The biomarker can be selected from the group consisting of cystatin C; cystatin C / creatinine ratio; complement factor Ba; complement factor Ba / creatinine ratio; complement sC5b-9, optionally urinary sC5b-9; complement sC5b-9 / creatinine ratio, sVCAM-1, optionally serum sVACM-1; sTNF-R1, optionally serum sTNF-R1; thrombomodulin, optionally plasma thrombomodulin; or a combination thereof.

[0048] In an embodiment, the present disclosure relates to the use of an effective amount of a complement inhibitor, wherein the complement inhibitor is an anti-C5 antibody or a C5-binding fragment thereof, preferably eculizumab, ravulizumab, or a biosimilar thereof, for the manufacture of a medicament for treating complement-mediated thrombotic microangiopathies (CM-TMA). The effective amount may be an amount sufficient to reduce the level of a biomarker and / or a biomarker signature in a subject compared to its level before treatment with the complement inhibitor. The biomarker may include at least one, at least two, at least three, at least four, at least five, at least six biomarkers selected from the group consisting of cystatin C; cystatin C / creatinine ratio; complement factor Ba; complement factor Ba / creatinine ratio; complement sC5b-9; complement sC5b-9 / creatinine ratio, or a combination thereof. The biomarker signature may include at least two biomarkers, including Ba and sC5b9. The level of the biomarker and / or biomarker signature can be detected in a bodily fluid sample obtained from the subject. The bodily fluid sample can be urine, plasma, or a combination thereof. The biomarker can be selected from the group consisting of cystatin C; cystatin C / creatinine ratio; complement factor Ba; complement factor Ba / creatinine ratio; complement sC5b-9, optionally urinary sC5b-9; complement sC5b-9 / creatinine ratio; sVCAM-1, optionally serum sVACM-1; sTNF-R1, optionally serum sTNF-R1; thrombomodulin, optionally plasma thrombomodulin, or a combination thereof.

[0049] For a further understanding of the nature, objects, and advantages of the present disclosure, reference should be made to the following detailed description taken in conjunction with the following drawings, in which like reference numerals refer to like elements and in which: [Brief description of the drawings]

[0050] [Figure 1-1]Figure 1 depicts biomarkers of complement dysregulation and renal injury reduction with ravulizumab treatment. P values ​​are derived from a mixed model for repeated measures analysis with log-transformed biomarkers as dependent variables and fixed categorical effects of visit and log-transformed baseline value as covariates, testing the null hypothesis that the mean change from baseline is equal to zero against the alternative hypothesis that the mean change is not equal to zero. Dotted lines represent the minimum and maximum of normal donor samples. BL, baseline; Cr, creatinine. [Figure 1-2] Same as above. [Diagram 2] Figure 2 depicts the association between baseline biomarkers and baseline clinical measures. Regression coefficients were obtained from simple linear regression analysis with log-transformed baseline clinical measures as dependent variables and log-transformed baseline biomarker levels as independent variables. For every 2-fold increase in baseline biomarker, the laboratory value increases (or decreases) by a factor of 2 (to the power of the regression coefficient). To calculate the percentage increase (or decrease), subtract 1 from this value and multiply by 100; P values ​​are obtained from a two-tailed t-test of the null hypothesis that the regression coefficient is equal to zero. Spearman correlation coefficients are reported. Outline boxes indicate statistically significant results in biomarkers of complement dysregulation and renal injury. Highlights / shading indicate very high correlations. Cr, creatinine; eGFR, estimated glomerular filtration rate; LDH, lactate dehydrogenase; UPCR, urinary protein / creatinine ratio. [Diagram 3]Figure 3 depicts the association between baseline biomarkers and changes in clinical measures over 26 weeks of treatment. Regression coefficients were obtained from log-transformed simple linear regression analysis with 26-week change from baseline in clinical measures as the dependent variable and log(2) of baseline biomarker levels as the independent variable. For every two-fold increase in baseline biomarker, the change in laboratory value increases (or decreases) by the regression coefficient; P values ​​are obtained from a two-tailed t-test of the null hypothesis that the regression coefficient is equal to zero. Spearman correlation coefficients are reported. Red boxes indicate statistically significant results in complement-specific biomarkers. Yellow highlights indicate very high correlations. Cr, creatinine; eGFR, estimated glomerular filtration rate; LDH, lactate dehydrogenase; UPCR, urinary protein / creatinine ratio. [Figure 4] Figure 4 presents data showing that baseline biomarker levels were significantly associated with selected clinical outcomes at week 26. *Defined as normalization of platelet count, normalization of LDH, and improvement in serum creatinine. CI, confidence interval; Cr, creatinine; LDH, lactate dehydrogenase; TMA, thrombotic microangiopathy. [Figure 5-1] FIG. 5 represents biolinograms of biomarkers at blood observation levels over time up to 52 weeks. (A) Plasma Ba; (B) Plasma thrombomodulin; (C) Plasma sC5b-9; (D) Plasma D-dimer; (E) Serum sTNF-R1; (F) Serum sVCAM-1. Ravulizumab doses were determined by body weight and were administered with a loading dose at baseline, a second dose on day 15, and maintenance doses on day 71 and weekly thereafter. Horizontal lines represent 25%, median, and 75% quartiles. P values ​​are calculated from a mixed model for repeated measures analysis with biomarker as dependent variable and fixed categorical effects of visit and fixed continuous effects of baseline value as covariates. The null hypothesis that the mean change from baseline is equal to zero was tested against the alternative hypothesis that the mean change is not equal to zero. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 6-1]FIG. 6 represents biolin plots of biomarkers at urinary observation levels over time up to week 52. (A) Urinary cystatin C / creatinine; (B) Urinary sC5b-9 / creatinine; (C) Urinary Ba / creatinine. Ravulizumab doses were determined by body weight and were administered with a loading dose at baseline, a second dose on day 15, and maintenance doses on day 71 and weekly thereafter. Horizontal lines represent 25%, median, and 75% quartiles. P values ​​are calculated from a mixed model for repeated measures analysis with biomarker as dependent variable and fixed categorical effects of visit and fixed continuous effects of baseline value as covariates. The null hypothesis that the mean change from baseline is equal to zero was tested against the alternative hypothesis that the mean change is not equal to zero. [Figure 6-2] Same as above. [Figure 7-1] FIG. 7 depicts line plots of change from baseline over time in eGFR and biomarkers (in blood) at week 52 by complete TMA response status. (A) Plasma Ba, ng / mL (closed circle); (B) Plasma thrombomodulin, ng / mL (closed circle); (C) Plasma sC5b-9, ng / mL (closed circle); (D) Plasma D-dimer, ng / mL (closed circle); (E) Serum sTNF-R1, ng / mL (closed circle); (F) Serum sVCAM-1, ng / mL (closed circle). Changes from baseline in blood and urinary biomarkers were compared to clinical measures of eGFR to week 52. Solid lines represent complete TMA responders; dashed lines represent complete TMA non-responders; black stars represent eGFR (mL / min / 1.73 m2) serum. P values ​​are presented in each figure for each of the biomarkers. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 8]FIG. 8 depicts line plots of change from baseline over time in eGFR and biomarkers (urinary) at week 52 by complete TMA response status. (A) Urinary cystatin C / creatinine, ng / mg creatinine (closed circle); (B) Urinary sC5b-9 / creatinine, ng / mg creatinine (closed circle); (C) Urinary Ba / creatinine, ng / mg creatinine (closed circle). Changes from baseline in blood and urinary biomarkers were compared to clinical measures of eGFR to week 52. Solid lines represent complete TMA responders; dashed lines represent complete TMA non-responders; black stars represent eGFR (mL / min / 1.73m2) serum. P values ​​are presented in each figure for each of the biomarkers. [Figure 9] Figure 9 represents a logistic regression analysis of complete TMA response at 52 weeks of treatment based on baseline biomarker levels. Odds ratios were derived from logistic regression analysis with the response variable as the dependent variable and the logarithm of baseline biomarker levels as the independent variable, and represent the increase (or decrease) in odds of achieving an efficacy response for every two-fold increase in baseline biomarker. CI, confidence interval; Cr, creatinine; LDH, lactate dehydrogenase; TMA, thrombotic microangiopathy. [Figure 10-1] Figure 10 represents the receiver operating characteristic (ROC) curves of Ba and sC5b-9 levels in patients versus normal donors. A. Dot plots for individual sC5b-9 and Ba levels in urine and plasma. B. ROC curves for the combined biomarkers in urine and plasma. C. Violin plots for the combined biomarkers in urine and plasma. D. Summary table for the combined biomarkers. TP, true positive; TN, true negative; FP, false positive; FN, false negative. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above. [Figure 11-1]Figure 11 depicts receiver operating characteristic (ROC) curves for Ba and sC5b-9 levels in patients versus normal donors. A. ROC curves for individual biomarkers in urine and plasma. B. Violin plots for individual biomarkers in urine and plasma. [Figure 11-2] Same as above. [Figure 11-3] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] Before the present disclosure is further described, it should be understood that the present disclosure is not limited to the specific embodiments disclosed below, since variations of the specific embodiments may be made and are within the scope of the appended claims. It should also be understood that the terminology used is for the purpose of describing the specific embodiments and is not intended to be limiting. Instead, the scope of the present disclosure will be established by the appended claims.

[0052] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0053] "Antibodies" (Ab) and "immunoglobulins" (Ig) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity.

[0054] "Native antibodies and immunoglobulins" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Certain amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain. Clothia et al.J.Mol.Biol.186:651(1985);Novotny and Haber Proc.Natl.Acad.Sci.USA82:4592(1985).

[0055] The term "variable" broadly refers to the fact that certain portions of the variable domains vary widely in sequence among antibodies and are used in the binding and specificity of each particular antibody to its particular antigen. However, variability is not uniformly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in both light and heavy chain variable domains. The more highly conserved portions of the variable domains are called frameworks (FRs). Each of the variable domains of native light and heavy chains contains four FR regions that mainly adopt a β-sheet structure connected by three CDRs that form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together with the CDRs from the other chain in close proximity by the FR regions and contribute to the formation of the antigen-binding site of antibodies (Johnson & Wu "Kabat Database and its applications: 30 years after the first variability plot" Nucleic Acids Research (2000) 28 (1): 214-218). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0056] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a name reflecting its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0057] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. In two-chain Fv species, this region consists of a dimer of one heavy and one light chain variable domain in tight non-covalent association. In single-chain Fv species, one heavy and one light chain variable domain can be covalently linked by a flexible peptide linker so that the light and heavy chains can associate in a "dimeric" structure similar to that of the two-chain Fv species. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even if a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, it is with a lower affinity than the entire binding site.

[0058] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0059] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0060] Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known. "Therapeutic Antibody Engineering" (1 st Ed.) Strohl & Strohl Woodhead Publishing (2012). The term "antibody" specifically encompasses monoclonal antibodies, including antibody fragment clones.

[0061] An "antibody fragment" comprises a portion of an intact antibody, generally the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; bispecific antibodies; single-chain antibody molecules, e.g., single-chain Fv (scFv) molecules; and multispecific antibodies formed from antibody fragments. See "Human Monoclonal Antibodies: Methods and Protocols" (2003), "Antibody Fragments ... nd Ed.) Steinitz (Ed.) Humana Press (2019).

[0062] "Monoclonal antibody," as used herein, broadly refers to an antibody (or antibody fragment) obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Moreover, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies specific for different determinants (epitopes), each monoclonal antibody is specific for a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by a hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" also include antibody fragment clones containing antigen recognition and binding sites (Fv clones) isolated from phage antibody libraries using the techniques described in, e.g., Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0063] The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. (U.S. Pat. No. 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)); "Antibody Engineering" Volume 2 (2 nd Ed.) Kontermann & Duebel. Springer Press (2010).

[0064] A "human" antibody (also called a "fully human" antibody) broadly refers to an antibody that contains all of the human framework regions and CDRs from a human immunoglobulin. In one example, the framework and CDRs are derived from the same original human heavy and / or light chain amino acid sequence. However, a framework from one human antibody can be modified to contain CDRs from a different human antibody.

[0065] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (such as mouse, rat or rabbit) (recipient antibody) or synthetic sequences (donor antibody) with the desired specificity, affinity, and performance. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In some embodiments, all CDRs are obtained from the donor immunoglobulin in the humanized immunoglobulin. Constant regions need not be present, but if present, they should be substantially identical to human immunoglobulin constant regions, e.g., at least about 85-90%, e.g., about 95% or more identical. Thus, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to the corresponding parts of a natural human immunoglobulin sequence. A "humanized antibody" is an antibody that includes a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions with amino acids taken from the donor framework. A humanized or other monoclonal antibody may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed using genetic engineering. See, e.g., U.S. Pat. No. 5,585,089.

[0066] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0067] "Bispecific antibodies," as used herein, broadly refer to small antibody fragments having two antigen-binding sites, comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL) (VH-VL) in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. Bispecific antibodies are described more fully in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0068] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody will be purified (1) to greater than 95% by weight, and most preferably greater than 99% by weight, of the antibody as measured by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. An isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will be absent. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0069] "Binding agent," as used herein, broadly refers to any natural, synthetic, or genetically engineered agent, e.g., a protein, that binds to an antigen (e.g., a biomarker protein). A binding agent can be, or can be derived from, a naturally occurring antibody. A binding protein or agent can function similarly to an antibody by binding to a specific antigen and forming a complex. A binding agent or protein can include an isolated antigen-binding fragment of an antibody.

[0070] "Mammal" as used herein broadly refers to any and all warm-blooded vertebrates of the mammalian class, characterized by a covering of hair on the skin and lactating mammary glands in females to feed their young. Mammals include, but are not limited to, humans, livestock animals, and zoo, sport, or pet animals. Examples of mammals include, but are not limited to, alpacas, armadillos, capybaras, cats, camels, chimpanzees, chinchillas, cows, dogs, gerbils, goats, gorillas, guinea pigs, hamsters, horses, humans, lemurs, llamas, mice, non-human primates, pigs, rats, sheep, shrews, squirrels, and tapirs. Mammals include, but are not limited to, cows, dogs, horses, cats, mice, sheep, pigs, primates, and rodent species. Mammals also include any and all mammals listed on the Mammalian Species of the World maintained by the National Museum of Natural History, Smithsonian Institution, Washington D.C. Similarly, the term "subject" or "patient" includes both human and animal subjects and / or patients.

[0071] "Normal," as used herein, broadly refers to an individual or group of individuals who do not have, or are not suspected of having or being at risk for developing, a particular disease or condition (e.g., CM-TMA, aHUS). The term "normal" is also used herein to qualify a biological specimen or sample (e.g., bodily fluid) isolated from a normal or healthy individual or subject (or group of such subjects), such as a "normal control sample" or a "normal control bodily fluid."

[0072] A subject "at risk of developing aHUS," as used herein, broadly refers to a subject who has one or more (e.g., two, three, four, five, six, seven, or eight or more) risk factors for developing the disorder. Risk factors for aHUS are well known in the medical arts and include, for example, predispositions to developing the condition, for example, a family history of the condition, or a genetic predisposition to developing the condition, such as, for example, one or more mutations in complement factor H (CFH), membrane cofactor protein (MCP; CD46), C4b binding protein, complement factor B (CFB), or complement factor I (CFI). For example, Warwicker et al. (1998) Kidney Int 53:836-844; Richards et al. (2001) Am J Hum Genet 68:485-490; Caprioli et al. (2001) Am Soc Nephrol 12:297-307; Neuman et al. (2003) J Med Genet 40:676-681;Richards et al.(2006) Proc Natl Acad Sci USA 100:12966-12971;Fremeaux-Bacchi et al.(2005) J Am Soc Nephrol 17:2017-2025;Esparza-Gordillo et al.(2005) Hum Mol Genet 14:703-712;Goicoechea de Jorge et (2007) Proc Natl Acad Sci USA 104(1):240-245; Blom et al. (2008) J Immunol 180(9):6385-91; and Fremeaux-Bacchi et al. (2004) J Medical Genet 41:e84. See also Kavanagh et al. (2006), supra.Risk factors also include, for example, infection with Streptococcus pneumoniae, pregnancy, cancer, exposure to anticancer drugs (e.g., quinine, mitomycin C, cisplatin, or bleomycin), exposure to immunotherapy agents (e.g., cyclosporine, OKT3, or interferon), exposure to antiplatelet agents (e.g., ticlopidine or clopidogrel), HIV infection, transplant, autoimmune disease, and mixed methylmalonic aciduria and homocystinuria (cblC). See, e.g., Constantinescu et al. (2004) Am J Kidney Dis 43:976-982; George (2003) Curr Opin Hematol 10:339-344; Gottschall et al. (1994) Am J Hematol 47:283-289; Valavaara et al. (1985) Cancer 55:47-50; Miralbell et al. (1996) J Clin Oncol 14:579-585; Dragon-Durey et al. (2005) J Am Soc Nephrol 16:555-63; and Becker et al. (2004) Clin Infect Dis 39:S267-S275. Thus, a person at risk of developing aHUS can be, for example, one who has a family history of aHUS and / or one who has HIV infection. From the above, it will be apparent that subjects "at risk of developing aHUS" are not all subjects within a species of interest.

[0073] A subject "suspected of having aHUS", as used herein, broadly refers to a person having one or more symptoms of the condition. Symptoms of the condition are well known to those skilled in the art of medicine and include, for example, severe hypertension, proteinuria, uremia, lethargy / fatigue, irritability, thrombocytopenia, microangiopathic hemolytic anemia, and renal dysfunction (e.g., acute renal failure). From the above paragraphs, it will be apparent that subjects "suspected of having aHUS" are not all subjects in the species of interest.

[0074] As used herein, a "trigger" in the context of a CM-TMA is an event, situation, or condition that causes a CM-TMA to occur. In some embodiments, the CM-TMA trigger is an autoimmune condition or event. In some embodiments, the trigger is an infection, e.g., a bacterial infection, a viral infection, a fungal infection, or a parasitic infection. In some embodiments, the trigger is a transplant, e.g., a bone marrow transplant or a solid organ transplant (e.g., selected from kidney, pancreas, liver, heart, and small intestine transplants). In some embodiments, the trigger is one or more medications. In some embodiments, the trigger is malignant hypertension.

[0075] complement system The complement system works in conjunction with other immune systems of the body to protect against invading cellular and viral pathogens. There are at least 25 complement proteins found as a complex collection of plasma proteins and membrane cofactors. Plasma proteins form about 10% of the globulins in vertebrate serum. Complement components achieve their immune defense functions by interacting in a series of complex but precise enzymatic cleavage and membrane binding events. The resulting complement cascade leads to the production of products with opsonic immunoregulatory and lytic functions. A concise summary of the biological activities associated with complement activation can be found, for example, in the Merck Manual, 16 th It is available in the Edition.

[0076] The complement cascade progresses via the classical, alternative, or lectin pathways, which share many components and, while differing in their initial steps, converge on and share the same "terminal complement" components (C5 through C9) that are responsible for target cell activation and destruction.

[0077] The classical pathway (CP) is typically initiated by antibody recognition of and binding to antigenic sites on target cells. The alternative pathway (AP) can be antibody independent and can be initiated by specific molecules on the pathogen surface. In addition, the lectin pathway is typically initiated with binding of mannose-binding lectin (MBL) to high mannose substrates. These pathways converge at the point where complement component C3 is cleaved by active proteases to generate C3a and C3b. Other pathways that activate complement attack can act later in the sequence of events to trigger various aspects of complement function. C3a is an anaphylatoxin. C3b binds to bacteria and other cells, as well as certain viruses and immune complexes, marking them for removal from the circulation. This opsonic function of C3b is generally considered to be the most important anti-infective action of the complement system. C3b also forms a complex with other components specific to each pathway to form the classical or alternative C5 convertase, which cleaves complement component C5 (hereafter referred to as "C5") into C5a and C5b.

[0078] Cleavage of C5 releases biologically active species such as C5a, a potent anaphylatoxin and chemotactic factor, and C5b, which through a series of protein interactions leads to the formation of the lytic terminal complement complex C5b-9. C5a and C5b-9 also have pleiotropic cell activation properties by amplifying the release of downstream inflammatory factors such as hydrolases, reactive oxygen species, arachidonic acid metabolites, and various cytokines.

[0079] C5b combines with C6, C7, and C8 to form a C5b-8 complex on the surface of the target cell. Upon binding of several C9 molecules, the membrane attack complex (MAC, C5b-9, terminal complement complex - TCC) is formed. When sufficient numbers of MACs insert into the target cell membrane, the openings they create (MAC pores) mediate rapid osmotic lysis of the target cell. Lower, non-lytic concentrations of MAC can have other effects. In particular, membrane insertion of small numbers of C5b-9 complexes into endothelial cells and platelets can cause deleterious cell activation. In some cases, activation can precede cell lysis.

[0080] C3a and C5a are activated complement components. They can induce mast cell degranulation, which releases histamine and other inflammatory mediators from basophils and mast cells, leading to smooth muscle contraction, increased vascular permeability, leukocyte activation, and other inflammatory phenomena, such as cell proliferation resulting in cellular hyperplasia. C5a also functions as a chemotactic peptide that helps attract proinflammatory granulocytes to the site of complement activation. C5a receptors are found on the surface of bronchial and alveolar epithelial cells and bronchial smooth muscle cells. C5a receptors have also been found on eosinophils, mast cells, monocytes, neutrophils, and activated lymphocytes.

[0081] Complement-mediated thrombotic microangiopathy (CM-TMA) is a clinical disorder driven by excessive complement production. It is characterized by thrombocytopenia and microangiopathic hemolytic anemia (MAHA) and microvascular thrombosis (TMA) leading to systemic organ dysfunction.

[0082] aHUS is an inherited, life-threatening disease involving chronic complement dysregulation. Patients with the disease suffer from thrombotic microangiopathy (TMA), which can lead to stroke and renal failure. Eculizumab, an antagonistic anti-C5 antibody, has been shown to dramatically reduce TMA, normalize platelet levels, and improve renal function in aHUS patients. However, even with the clear and robust clinical benefits of complement inhibitor therapy for aHUS patients, some patients still experience increased levels of some aHUS biomarker proteins despite treatment. See U.S. Pat. No. 9,494,601.

[0083] aHUS may be hereditary, acquired, or idiopathic. aHUS may be considered hereditary if two or more (e.g., three, four, five, or six or more) members of the same family are affected by the disease at least six months apart and exposure to a common triggering agent is excluded, or if one or more aHUS-associated genetic mutations (e.g., one or more mutations in CFH, MCP / CD46, CFB, or CFI) are identified in the subject. For example, a subject may have CFH-associated aHUS, CFB-associated aHUS, CFI-associated aHUS, or MCP-associated aHUS. Up to 30% of hereditary aHUS cases are associated with mutations in CFH, 12% with mutations in MCP, 5-10% with mutations in CFI, and less than 2% with mutations in CFB. Hereditary aHUS may be multiplicative (e.g., familial; two or more affected family members) or simplex (e.g., occurrence of one person in a family). aHUS can be considered acquired if an underlying environmental cause can be identified (e.g., a drug, systemic disease, or a viral or bacterial agent that does not produce Shiga-like exotoxins). aHUS can be considered idiopathic if no trigger (genetic or environmental) is evident.

[0084] The methods described herein can include identifying a subject as having, suspected of having, or at risk of developing aHUS. In addition to using aHUS biomarker profiling as described herein, laboratory tests can be performed to determine whether a human subject has thrombocytopenia, microangiopathic hemolytic anemia, or acute renal failure. Thrombocytopenia is defined as (i) a thrombocytopenia greater than or equal to 150,000 / mm 3 Less than (e.g., 60,000 / mm 3(ii) a platelet count of less than 10 mg / dL (e.g., less than 6.5 mg / dL); (ii) a decreased platelet survival time reflecting increased platelet destruction in the circulation; and (iii) large platelets in the peripheral smear, consistent with secondary activation of thrombopoiesis. Microangiopathic hemolytic anemia can be diagnosed by a health care professional as one or more of: (i) a hemoglobin concentration of less than 10 mg / dL (e.g., less than 6.5 mg / dL); (ii) an increased serum lactate dehydrogenase (LDH) concentration (>460 U / L); (iii) hyperbilirubinemia, reticulocytosis, circulating free hemoglobin, and low or undetectable haptoglobin concentration; and (iv) detection of fragmented red blood cells (schistocytes) with the typical aspect of spinous or helmet cells in the peripheral smear along with a negative Coombs test. See, e.g., Kaplan et al. (1992) “Hemolytic Uremic Syndrome and Thrombotic Thrombocytopenic Purpura,” Informa Health Care (ISBN0824786637) and Zipfel (2005) “Complement and Kidney Disease,” Springer (ISBN3764371668).

[0085] Detection of CM-TMA biomarkers The present disclosure provides agents and methods for detecting CM-TMA biomarkers in body fluids in patients with CM-TMA and / or CM-TMA patients receiving complement inhibitor therapy. aHUS is difficult to diagnose.

[0086] Validated biomarkers for the diagnosis and monitoring of patients with complement-mediated thrombotic microangiopathy (CM-TMA) are not clinically available; characterization of biomarkers in patients with atypical hemolytic uremic syndrome (aHUS), a form of CM-TMA, could inform diagnosis, treatment decisions, and monitoring for patients with CM-TMA.

[0087] There is an unmet need for validated biomarkers for the diagnosis, prognosis and monitoring of patients with CM-TMA. The inventors unexpectedly found that measurements of complement factors Ba and sC5b-9 show clinical utility in identifying the CM-TMA / aHUS subset of TMA patients. Indeed, the inventors unexpectedly found that, together with factor Ba, the vascular tissue biomarkers TNF-RI and thrombomodulin show promising clinical prognostic utility based on their strong association with renal dysfunction and complete TMA response to ravulizumab treatment.

[0088] The methods described herein may include the use of at least one, at least two, at least three, at least four, at least five, or at least six protein biomarkers selected from: (1) Cystatin C; (2) Complement Ba; (3) sC5b9; (4) Cystatin C / creatinine; (5) Complement Ba / creatinine; (6) sC5b9 / creatinine in the diagnosis, monitoring, and treatment of patients with CM-TMA. When the biomarkers are derived from urine, the present disclosure provides for the use of: (a) Cystatin C; (b) Ba; (c) sC5b9; (d) Cystatin C / creatinine; (e) Ba / creatinine; (f) sC5b9 / creatinine. More specifically, the present disclosure relates to the use of the above biomarkers, either alone or in combination with secondary markers such as estimated glomerular filtration rate (eGFR) and / or urinary protein creatinine ratio (UPCR), in the diagnosis of CM-TMA and treatment with complement inhibitors, such as anti-C5 antibodies (such as ravulizumab). In particular, it has been found that after treatment initiation, baseline (BL) plasma complement factor Ba is associated with eGFR changes after treatment (e.g., 26 weeks after infusion of ravulizumab). Similarly, urinary sC5b-9, sC5b-9 / Cr, urinary Ba, and Ba / Cr were associated with UPCR changes after treatment (e.g., 26 weeks after infusion of ravulizumab).

[0089] The biomarkers detected in the methods described herein may be selected from the group consisting of cystatin C; cystatin C / creatinine ratio; complement factor Ba; complement factor Ba / creatinine ratio; complement sC5b-9, optionally urinary sC5b-9; complement sC5b-9 / creatinine ratio; sVCAM-1, optionally serum sVACM-1; sTNF-R1, optionally serum sTNF-R1; thrombomodulin, optionally plasma thrombomodulin; or a combination thereof.

[0090] The structures (e.g., amino acid sequences) of the CM-TMA biomarkers of the present disclosure are publicly known and are registered in databases (e.g., GENBANK and / or UNIPROT). For example, human cystatin C is registered under GENBANK No. NP_000090 (date: September 12, 2021) and UNIPROT No. P01034 (date: June 2, 2021). Human complement B, which is cleaved to form Ba and Bb, is registered under GENBANK No. NP_001701 (date: July 26, 2021) and UNIPROT No. P00751 (date: June 2, 2021). Human C5–C9, which form the sC5b9 complex, are identified in GENBANK Nos. NP_001726 (C5 protein; date: October 3, 2021); NP_000056 (C6 protein; date: April 20, 2021); NP_000578 (C7 protein; date: June 27, 2021); NP_000553 (C8α protein; date: June 30, 2021), NP_000057 (C8β protein; date: June 24, 2021), and NP_000597 (C8γ protein; date: February 14, 2021); NP_001728 (C9 protein; date: June 26, 2021) and UNIPROT Nos. P01031 (C5 protein); P13671 (C6 protein); P10643 (C7 protein); P07357 (C8α protein), P07358 (C8β protein), and P07360 (C8γ protein); and P02748 (C9 protein) (all registered on June 2, 2021). Human VCAM-1 is registered under GENBANK No. NP_001069 (date: September 7, 2021) and UNIPROT No. P19320 (date: June 2, 2021). Human TNFR-1 is registered under GENBANK No. NP_001056 (date: October 17, 2021) and UNIPROT No. P19438 (date: June 2, 2021). Human thrombomodulin is registered under GENBANK No. NP_000352 (date: September 12, 2021) and UNIPROT No. P07204 (date: June 2, 2021).

[0091] The present disclosure is further based on the surprising discovery that complement biomarkers proteolytic fragment of complement component factor B (Ba) (in plasma and urine) and soluble C5b9 (sC5b-9) (in urine) were associated with renal function in patients with aHUS at baseline and over 26 weeks of treatment with anti-C5 therapy. Such biomarkers may demonstrate diagnostic potential in CM-TMA and predict renal response to complement inhibition.

[0092] While the present disclosure is not bound by any particular theory, the inventors have found that monitoring of patients treated with a complement inhibitor (such as an anti-C5 antibody) for changes in Ba and / or sC5b-9 concentrations is useful for diagnosing patients as having or at risk of developing CM-TMA, including aHUS. Monitoring Ba and / or sC5b-9 status may also be useful for determining whether a CM-TMA patient is responding to treatment with a complement inhibitor. Furthermore, assessment of Ba and / or sC5b-9 status is also useful for identifying a dose, including a threshold dose, of a complement inhibitor, such as an anti-C5 antibody, that is sufficient to achieve a clinically significant effect on the disease (e.g., sufficient to treat a complement-related disease, such as aHUS), by virtue of its effect on the concentration of Ba and / or sC5b-9 biomarker protein in humans.

[0093] The inventors have identified biomarkers for CM-TMA, namely Ba and sC5b-9. The inventors have discovered that increased or possibly decreased concentrations of certain proteins are associated with the presence of CM-TMA. Similarly, a decrease or increase in the concentration (or activity) of certain proteins in bodily fluids obtained from CM-TMA patients treated with complement inhibitors indicates that the patient is responding to the inhibitor therapy. The complement biomarkers Ba (detected in plasma and urine) and sC5b-9 (detected in urine) were associated with renal function in patients with CM-TMA at baseline and over 26 weeks of treatment with anti-C5 therapy. Analysis of the concentration and / or activity levels of such proteins can therefore be used, inter alia, to assess risk for CM-TMA, diagnose CM-TMA, monitor progression or remission of CM-TMA, and / or monitor therapeutic response to complement inhibitors.

[0094] Tissue-specific biomarkers and their combinations in signatures Urinary biomarkers Urinary biomarkers can be used in the diagnosis, monitoring, and treatment of patients with CM-TMA. Urinary biomarkers useful for this purpose include (a) cystatin C; (b) Ba; (c) sC5b9; (d) cystatin C / creatinine; (e) Ba / creatinine; and (f) sC5b9 / creatinine or any combination thereof, including, for example, at least two, at least three, at least four, at least five, or all six of the above biomarkers.

[0095] A urinary biomarker signature comprising a combination of the above biomarkers can be detected in a sample and can optionally be used in the diagnosis, monitoring, and treatment of patients with CM-TMA. Due in part to the additive or even synergistic predictive power of the combination of biomarkers, it may be desirable to utilize such a signature in various embodiments of the present disclosure. It may be even more desirable if the different biomarkers are derived from different biological samples, for example, the signature comprises urinary sC5b9 protein levels and plasma Ba protein levels.

[0096] Examples of urinary biomarker signatures comprising at least two urinary biomarkers include, but are not limited to, (a) cystatin C+Ba; (b) cystatin C+sC5b9; (c) cystatin C+cystatin C / creatinine; (d) cystatin C+Ba / creatinine; (e) cystatin C+sC5b9 / creatinine; (f) Ba+sC5b9; (g) Ba+cystatin C / creatinine; (h) Ba+ (i) Ba / creatinine; (j) sC5b9 + cystatin C / creatinine; (k) sC5b9 + Ba / creatinine; (l) sC5b9 + sC5b9 / creatinine; (m) cystatin C / creatinine + Ba / creatinine; (n) cystatin C / creatinine + sC5b9 / creatinine; and (o) Ba / creatinine + sC5b9 / creatinine.

[0097] Examples of urinary biomarker signatures comprising at least three urinary biomarkers include, but are not limited to, (a) cystatin C + Ba + sC5b9; (b) cystatin C + Ba + cystatin C / creatinine; (c) cystatin C + Ba + Ba / creatinine; (d) cystatin C + Ba + sC5b9 / creatinine; (e) cystatin C + sC5b9 + cystatin C / creatinine; (f) cystatin C + sC5b9 + Ba / creatinine; (g) cystatin C + sC5b9 + sC5b9 / creatinine; (h) cystatin C + cystatin C / creatinine + Ba / creatinine; (i) cystatin C + cystatin C / creatinine + sC5b9 / creatinine; (j) cystatin C + Ba / creatinine + sC5 (k) Ba + sC5b9 + cystatin C / creatinine; (l) Ba + sC5b9 + Ba / creatinine; (m) Ba + sC5b9 + sC5b9 / creatinine; (n) Ba + cystatin C / creatinine + Ba / creatinine; (o) Ba + cystatin C / creatinine + sC5b9 / creatinine; (p) Ba + Ba / creatinine + sC5b9 / creatinine; (q) sC5b9 + cystatin C / creatinine + Ba / creatinine; (r) sC5b9 + cystatin C / creatinine + sC5b9 / creatinine; (s) sC5b9 + Ba / creatinine + sC5b9 / creatinine; and (t) cystatin C / creatinine + Ba / creatinine + sC5b9 / creatinine.

[0098] Examples of urinary biomarker signatures comprising at least four urinary biomarkers include, but are not limited to, (a) cystatin C + Ba + sC5b9 + cystatin C / creatinine; (b) cystatin C + Ba + sC5b9 + Ba / creatinine; (c) cystatin C + Ba + sC5b9 + sC5b9 / creatinine; (d) cystatin C + sC5b9 + cystatin C / creatinine + (e) cystatin C + sC5b9 + cystatin C / creatinine + sC5b9 / creatinine; (f) cystatin C + cystatin C / creatinine + Ba / creatinine + sC5b9 / creatinine; (g) Ba + sC5b9 + cystatin C / creatinine + Ba / creatinine; (h) Ba + sC5b9 + cystatin C / creatinine + sC5b9 / creatinine; (i) Ba + cystatin C / creatinine + Ba / creatinine + sC5b9 / creatinine; or (j) sC5b9 + cystatin C / creatinine + Ba / creatinine + sC5b9 / creatinine. Examples of urinary biomarker signatures comprising at least five urinary biomarkers include, but are not limited to, (a) cystatin C + Ba + sC5b9 + cystatin C / creatinine + Ba / creatinine; (b) cystatin C + Ba + sC5b9 + cystatin C / creatinine + sC5b9 / creatinine; (c) cystatin C + sC5b9 + cystatin C / creatinine + Ba / creatinine + sC5b9 / creatinine; (d) Ba + sC5b9 + cystatin C / creatinine + Ba / creatinine + sC5b9 / creatinine.

[0099] Further representative examples of urinary biomarker signatures comprising at least six urinary biomarkers include, but are not limited to, (a) Cystatin C+Ba+sC5b9+Cystatin C / creatinine+Ba / creatinine+sC5b9 / creatinine. Any one or combination of urinary biomarker signatures comprising combinations of biomarkers described herein can be used in the methods described herein.

[0100] Plasma biomarkers Plasma biomarkers can be used in the diagnosis, monitoring, and treatment of patients with CM-TMA. Plasma biomarkers useful for this purpose include (a) complement Ba and (b) sC5b9, or any combination thereof, including, for example, at least two of the plasma biomarkers listed above.

[0101] A plasma biomarker signature may include a combination of the above plasma biomarkers. For example, a plasma biomarker signature including at least two plasma biomarkers may include Ba and sC5b9. Further plasma biomarkers include plasma thrombomodulin.

[0102] Biomarkers from Heterogeneous Samples Exemplary biomarker signatures, including biomarkers from heterogeneous samples (e.g., urine and plasma), can be used in the diagnostic and treatment methods described herein. Exemplary biomarker signatures, including biomarkers from heterogeneous samples (e.g., urine and plasma), can be detected in bodily fluid samples.

[0103] Two biomarker signatures from heterogeneous samples (at least one from urine and one from plasma) can be used in diagnostic and therapeutic methods. An exemplary two biomarker signature from a heterogeneous sample can be detected in a bodily fluid sample. Exemplary heterogeneous two-biomarker signatures include, but are not limited to, (a) urinary cystatin C + plasma Ba; (b) urinary cystatin C + plasma sC5b9; (c) urinary Ba + plasma Ba; (d) urinary Ba + plasma sC5b9; (e) urinary sC5b9 + plasma Ba; (f) urinary sC5b9 + plasma sC5b9; (g) urinary cystatin C / creatinine + plasma Ba; (h) urinary cystatin C / creatinine + plasma sC5b9; (i) urinary Ba / creatinine + plasma Ba; (j) urinary Ba / creatinine + plasma sC5b9; (k) urinary sC5b9 / creatinine + plasma Ba; (l) urinary sC5b9 / creatinine + plasma sC5b9.

[0104] Three biomarker signatures from heterogeneous samples (e.g., including at least one from urine and two from plasma) can be used in the diagnostic and therapeutic methods described herein. Exemplary three biomarker signatures from heterogeneous samples can be detected in bodily fluid samples. Examples of heterogeneous three biomarker signatures include, but are not limited to, (a) urinary cystatin C + plasma Ba + plasma sC5b9; (b) urinary Ba + plasma Ba + plasma sC5b9; (c) urinary sC5b9 + plasma Ba + plasma sC5b9; (d) urinary cystatin C / creatinine + plasma Ba + plasma sC5b9; (e) urinary Ba / creatinine + plasma Ba + plasma sC5b9; (f) urinary sC5b9 / creatinine + plasma Ba + plasma sC5b9.

[0105] Three biomarker signatures from heterogeneous samples (e.g., including at least two from urine and one from plasma) can be used in the diagnostic and treatment methods described herein. An exemplary three biomarker signature from a heterogeneous sample can be detected in a bodily fluid sample. Examples of heterogeneous three biomarker signatures include, but are not limited to, (a) urinary cystatin C + urinary Ba + plasma Ba or plasma sC5b9; (b) urinary cystatin C + urinary sC5b9 + plasma Ba or plasma sC5b9; (c) urinary cystatin C + urinary cystatin C / creatinine + plasma Ba or plasma sC5b9; (d) urinary cystatin C + urinary Ba / creatinine + plasma Ba or plasma sC5b9; (e) urinary cystatin C + urinary sC5b9 / creatinine + plasma Ba or plasma sC5b9; (f) urinary Ba + urinary sC5b9 + plasma Ba or plasma sC5b9; (g) urinary Ba + urinary cystatin C / creatinine + plasma Ba or plasma sC5b9; (h) urinary Ba + urinary Ba / creatinine + plasma (i) urinary Ba + urinary sC5b9 / creatinine + plasma Ba or plasma sC5b9; (j) urinary sC5b9 + urinary cystatin C / creatinine + plasma Ba or plasma sC5b9; (k) urinary sC5b9 + urinary Ba / creatinine + plasma Ba or plasma sC5b9; (l) urinary sC5b9 + urinary sC5b9 / creatinine + plasma Ba or plasma sC5b9; (m) urinary cystatin C / creatinine + urinary Ba / creatinine + plasma Ba or plasma sC5b9; (n) urinary cystatin C / creatinine + urinary sC5b9 / creatinine + plasma Ba or plasma sC5b9; (o) urinary Ba / creatinine + urinary sC5b9 / creatinine + plasma Ba or plasma sC5b9.

[0106] Four biomarker signatures from heterogeneous samples (e.g., including at least two from urine and two from plasma) can be used in the diagnostic and treatment methods described herein. An exemplary four biomarker signature from a heterogeneous sample can be detected in a bodily fluid sample. Examples of heterogeneous four biomarker signatures include, but are not limited to, (a) urinary cystatin C + urinary Ba + plasma Ba + plasma sC5b9; (b) urinary cystatin C + urinary sC5b9 + plasma Ba + plasma sC5b9; (c) urinary cystatin C + urinary cystatin C / creatinine + plasma Ba + plasma sC5b9; (d) urinary cystatin C + urinary Ba / creatinine + plasma Ba + plasma sC5b9; (e) urinary cystatin C + urinary sC5b9 / creatinine + plasma Ba + plasma sC5b9; (f) urinary Ba + urinary sC5b9 + plasma Ba + plasma sC5b9; (g) urinary Ba + urinary cystatin C / creatinine + plasma Ba + plasma sC5b9; (h) urinary Ba + urinary Ba / creatinine + plasma (i) urinary Ba + urinary sC5b9 / creatinine + plasma Ba + plasma sC5b9; (j) urinary sC5b9 + urinary cystatin C / creatinine + plasma Ba + plasma sC5b9; (k) urinary sC5b9 + urinary Ba / creatinine + plasma Ba + plasma sC5b9; (l) urinary sC5b9 + urinary sC5b9 / creatinine + plasma Ba + plasma sC5b9; (m) urinary cystatin C / creatinine + urinary Ba / creatinine + plasma Ba + plasma sC5b9; (n) urinary cystatin C / creatinine + urinary sC5b9 / creatinine + plasma Ba + plasma sC5b9; (o) urinary Ba / creatinine + urinary sC5b9 / creatinine + plasma Ba + plasma sC5b9.

[0107] Four biomarker signatures from heterogeneous samples (e.g., including at least three from urine and one from plasma) can be used in the diagnostic and treatment methods described herein. An exemplary four biomarker signature from a heterogeneous sample can be detected in a bodily fluid sample.Examples of heterogeneous three biomarker signatures include, but are not limited to, (a) urinary cystatin C + urinary Ba + urinary sC5b9 + plasma Ba or plasma sC5b9; (b) cystatin C + Ba + cystatin C / creatinine + plasma Ba or plasma sC5b9; (c) cystatin C + Ba + Ba / creatinine + plasma Ba or plasma sC5b9; (d) cystatin C + Ba + sC5b9 / creatinine + plasma Ba or plasma sC5b9; (e) cystatin C + sC5b9 + cystatin C / creatinine + plasma Ba or plasma sC5b9; (f) cystatin C + sC5b9 + Ba / creatinine + plasma Ba or plasma sC5b9; (g) cystatin C + sC5b9 + sC5b9 / creatinine + plasma Ba or plasma sC5b9; (h) cystatin C + cystatin C / creatinine + Ba / creatinine + plasma Ba or plasma sC5b9; (i) cystatin C + cystatin C / creatinine + sC5b9 / creatinine + plasma Ba or plasma sC5b9; (j) cystatin C + Ba / creatinine + sC5b9 / creatinine + plasma Ba or plasma sC5b9; (k) Ba + sC5b9 + cystatin C / creatinine + plasma Ba or plasma sC5b9; (l) Ba + sC5b9 + Ba / creatinine + plasma Ba or plasma sC5b9; (m) Ba + sC5b9 + sC5b9 / creatinine + plasma Ba or plasma sC5b9; (n) Ba + cystatin C / creatinine + Ba / creatinine + plasma Ba or plasma sC5b9; (o) Ba + cystatin C / creatinine + sC5b9 / creatinine + plasma Ba or plasma sC5b9; (p) Ba + Ba / creatinine + s (q) sC5b9 + cystatin C / creatinine + Ba / creatinine + plasma Ba or plasma sC5b9; (r) sC5b9 + cystatin C / creatinine + sC5b9 / creatinine + plasma Ba or plasma sC5b9; (s) sC5b9 + Ba / creatinine + sC5b9 / creatinine + plasma Ba or plasma sC5b9; (t) cystatin C / creatinine + Ba / creatinine + sC5b9 / creatinine + plasma Ba or plasma sC5b9.

[0108] Four biomarker signatures from heterogeneous samples (e.g., two from serum, one from plasma, and one from urine) can be used in the diagnostic and therapeutic methods described herein. Exemplary four biomarker signatures from heterogeneous samples described herein can be detected in bodily fluid samples. Exemplary four biomarker signatures can include serum sVCAM-1 (circulating vascular adhesion molecule-1), serum sTNF-R1 (soluble tumor necrosis factor receptor 1), plasma thrombomodulin, and urinary C5b-9.

[0109] Five biomarker signatures from heterogeneous samples (e.g., including at least three from urine and two from plasma) can be used in the diagnostic and therapeutic methods described herein. Exemplary five biomarker signatures from heterogeneous samples described herein can be detected in bodily fluid samples. Examples of heterogeneous three biomarker signatures include, but are not limited to, (a) urinary cystatin C + urinary Ba + urinary sC5b9 + plasma Ba or plasma sC5b9; (b) cystatin C + Ba + cystatin C / creatinine + plasma Ba or plasma sC5b9; (c) cystatin C + Ba + Ba / creatinine + plasma Ba or plasma sC5b9; (d) cystatin C + Ba + sC5b9 / creatinine + plasma Ba or plasma sC5b9; (e) cystatin C + sC (f) cystatin C + sC5b9 + Ba / creatinine + plasma Ba or plasma sC5b9; (g) cystatin C + sC5b9 + sC5b9 / creatinine + plasma Ba or plasma sC5b9; (h) cystatin C + cystatin C / creatinine + Ba / creatinine + plasma Ba or plasma sC5b9; (i) cystatin C + cystatin C / creatinine + sC5b9 / creatinine + plasma (j) cystatin C + Ba / creatinine + sC5b9 / creatinine + plasma Ba or plasma sC5b9; (k) Ba + sC5b9 + cystatin C / creatinine + plasma Ba or plasma sC5b9; (l) Ba + sC5b9 + Ba / creatinine + plasma Ba or plasma sC5b9; (m) Ba + sC5b9 + sC5b9 / creatinine + plasma Ba or plasma sC5b9; (n) Ba + cystatin C / creatinine + Ba / creatinine (o) Ba + cystatin C / creatinine + sC5b9 / creatinine + plasma Ba or plasma sC5b9; (p) Ba + Ba / creatinine + sC5b9 / creatinine + plasma Ba or plasma sC5b9; (q) sC5b9 + cystatin C / creatinine + Ba / creatinine + plasma Ba or plasma sC5b9; (r) sC5b9 + cystatin C / creatinine + sC5b9 / creatinine + plasma Ba or plasma sC5b9;(s) sC5b9+Ba / creatinine+sC5b9 / creatinine+plasma Ba or plasma sC5b9; (t) cystatin C / creatinine+Ba / creatinine+sC5b9 / creatinine+plasma Ba or plasma sC5b9, except that the signatures include plasma biomarkers, such as both Ba and sC5b9 (the heterogeneous four biomarker signatures include either plasma biomarker, but not in combination);

[0110] Five biomarker signatures from heterogeneous samples (e.g., including at least four from urine and one from plasma) can be used in the methods described herein. An exemplary five biomarker signature from a heterogeneous sample described herein can be detected in a bodily fluid sample. Examples of heterogeneous five biomarker signatures include, but are not limited to, (a) cystatin C + Ba + sC5b9 + cystatin C / creatinine; (b) cystatin C + Ba + sC5b9 + Ba / creatinine; (c) cystatin C + Ba + sC5b9 + sC5b9 / creatinine; (d) cystatin C + sC5b9 + cystatin C / creatinine + (e) cystatin C + sC5b9 + cystatin C / creatinine + sC5b9 / creatinine; (f) cystatin C + cystatin C / creatinine + Ba / creatinine + sC5b9 / creatinine; (g) Ba + s (h) Ba + sC5b9 + cystatin C / creatinine + sC5b9 / creatinine; (i) Ba + cystatin C / creatinine + Ba / creatinine + sC5b9 / creatinine; or (j) sC5b9 + cystatin C / creatinine + Ba / creatinine + sC5b9 / creatinine, and one biomarker from plasma selected from the group consisting of complement Ba and (b) sC5b9, or any combination thereof.

[0111] Six biomarker signatures from heterogeneous samples (e.g., including at least four from urine and two from plasma) can be used in the methods described herein. Exemplary six biomarker signatures from heterogeneous samples described herein can be detected in bodily fluid samples. Examples of heterogeneous six biomarker signatures include, for example, (1) (a) cystatin C + Ba + sC5b9 + cystatin C / creatinine; (b) cystatin C + Ba + sC5b9 + Ba / creatinine; (c) cystatin C + Ba + sC5b9 + sC5b9 / creatinine; (d) cystatin C + sC5b9 + cystatin C / creatinine + (e) cystatin C + sC5b9 + cystatin C / creatinine + sC5b9 / creatinine; (f) cystatin C + cystatin C / creatinine + Ba / creatinine + sC5b9 / creatinine; (g) Ba + sC5b9 + cystatin C / creatinine + Ba / creatinine; (h) Ba + sC5b9 + cystatin C / creatinine + sC5b9 / creatinine; (i) Ba + cystatin C / creatinine + Ba / creatinine + sC5b9 / creatinine; or (j) sC5b9 + cystatin C / creatinine (c) cystatin C + sC5b9 + cystatin C / creatinine + Ba / creatinine + sC5b9 / creatinine; (d) Ba + sC5b9 + cystatin C / creatinine + Ba / creatinine + sC5b9 / creatinine, and two biomarker signatures from plasma, e.g., Ba + sC5b9 as described above, or (2) five biomarker signatures from urine selected from the group consisting of (a) cystatin C + Ba + sC5b9 + cystatin C / creatinine + Ba / creatinine; (b) cystatin C + Ba + sC5b9 + cystatin C / creatinine + sC5b9 / creatinine; and a single biomarker from plasma selected from the group consisting of complement Ba and (b) sC5b9, or any combination thereof.

[0112] Seven biomarker signatures from heterogeneous samples (e.g., including at least five from urine and two from plasma) can be used in the methods described herein. The exemplary seven biomarker signatures from heterogeneous samples described herein can be detected in bodily fluid samples. An example of a heterogeneous seven biomarker signature may include five biomarker signatures from urine and two biomarker signatures from plasma, e.g., Ba and sC5b9, selected from the group consisting of: (a) cystatin C+Ba+sC5b9+cystatin C / creatinine+Ba / creatinine; (b) cystatin C+Ba+sC5b9+cystatin C / creatinine+sC5b9 / creatinine; (c) cystatin C+sC5b9+cystatin C / creatinine+Ba / creatinine+sC5b9 / creatinine; (d) Ba+sC5b9+cystatin C / creatinine+Ba / creatinine+sC5b9 / creatinine.

[0113] Eight biomarker signatures from heterogeneous samples (e.g., including at least six from urine and two from plasma) can be used in the methods described herein. The exemplary eight biomarker signatures from heterogeneous samples described herein can be detected in bodily fluid samples. An example of a heterogeneous eight biomarker signature can include six biomarker signatures from urine, including Cystatin C+Ba+sC5b9+Cystatin C / creatinine+Ba / creatinine+sC5b9 / creatinine, and two biomarker signatures from plasma, for example Ba and sC5b9.

[0114] The protein biomarkers described herein can be normalized, for example, based on the level of creatinine in a subject's biological sample. In normalization, it is preferred to use the same biological sample source for determining the creatinine level of the source of the biomarker. Preferably, the present disclosure relates to normalization of urinary biomarkers based on the level of urinary creatinine level (ng biomarker per mg creatinine). In particular, the levels of urinary Ba, sC5b9 and cytostatin C are normalized based on urinary creatinine level.

[0115] The biomarker levels can be normalized to the level of creatinine in a subject's sample, where the creatinine level is measured simultaneously (e.g., simultaneously or separately at a very small time interval, e.g., less than an hour, preferably less than 30 minutes, particularly less than 20 minutes, or particularly less than 5 minutes) with the measurement of the subject's biomarker level or biomarker signature. For example, the levels of plasma Ba and / or plasma sC5b9 can be normalized based on the plasma creatinine level.

[0116] The diagnostic and / or therapeutic methods described herein may include measuring at least one secondary marker together with the biomarkers and / or biomarker signatures described herein, where the biomarker level is optionally normalized to the creatinine level in the subject's sample. Representative examples of secondary markers include, for example, estimated glomerular filtration rate (eGFR) and / or urinary protein creatinine ratio (UPCR). Preferably, these secondary markers are measured simultaneously with the biomarkers or biomarker signatures, and optionally together with the level of the analyte to be normalized (creatinine level).

[0117] Methods of diagnosis and / or detection A method for detecting a biomarker in a biological sample may include obtaining a bodily fluid from a subject; contacting the bodily fluid with an agent that binds to a biomarker, e.g., a protein biomarker selected from a proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or a combination thereof; and detecting binding of the agent to the biomarker, e.g., a protein biomarker selected from a proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both. The method may further include measuring the level of Ba and / or sC5b-9. In detecting the biomarker, any standard method may be used, including antibody-based detection, including standard immunoassays such as enzyme-linked immunosorbent assay (ELISA), radio-immunoassay (RIA), and the like.

[0118] A method for monitoring or assessing the status of a CM-TMA-associated biomarker protein in a subject may include obtaining a bodily fluid from the subject; contacting the bodily fluid with an agent that binds to a biomarker, e.g., a protein biomarker selected from a proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or a combination thereof; and detecting binding of the agent to the biomarker, e.g., a protein biomarker selected from a proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both. The method may further include measuring the level of Ba and / or sC5b-9.

[0119] A method for assessing one or both of the concentration and activity levels of a CM-TMA-associated biomarker protein in a subject may include obtaining a bodily fluid and detecting a protein biomarker, for example, a proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both, by immunoassay. The method may further include determining the concentration of the biomarker based on a standard measurement, for example, calculating the amount or relative amount of the biomarker per unit volume of the sample. When measuring activity, a standard activity assay can be performed. For example, if the biomarker is complement Bb and the parameter of interest is the activity of Bb in the subject's sample, a C3 cleavage assay can be performed. In this assay, it is understood that Bb is the catalytically active site of the C3bBb complex (C3 convertase) and is capable of cleaving new C3 into C3a and C3b. Similarly, if the biomarker is complement sC5b9 and the parameter of interest is the activity of sC5b9 in the subject's sample, a terminal complement complex assay such as CH50 can be used. The CH50 test is a lytic assay in which antibody-sensitized sheep red blood cells (EA) as an activator of the classical complement pathway and various dilutions of test serum are used to determine the amount required to obtain 50% lysis. Percent hemolysis is determined spectrophotometrically. The CH50 test is an indirect measure of TCC since TCC itself is directly responsible for the hemolysis measured in a viable cleavage assay. In some embodiments, a direct assay can be used.

[0120] A method for monitoring or determining whether a patient is at risk for developing a thrombotic microangiopathy may include obtaining a bodily fluid and detecting one or more of the above biomarkers or biomarker signatures, e.g., the proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both, by immunoassay. The method may further include measuring the levels of Ba and / or sC5b-9.

[0121] Methods for monitoring or assessing the status of a CM-TMA-associated biomarker protein in a subject, or for assessing one or both of the concentration and activity levels of one or more of the above biomarkers or biomarker signatures, e.g., the proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both, in a subject, may include obtaining a bodily fluid and detecting one or more of the above biomarkers or biomarker signatures, e.g., the proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both, by immunoassay. The methods may further include measuring the level of Ba and / or sC5b-9.

[0122] A method for determining whether a subject has or is at risk for developing CM-TMA may include obtaining a bodily fluid and detecting one or more of the above biomarkers or biomarker signatures, e.g., the proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both, by immunoassay. The method may further include measuring the levels of Ba and / or sC5b-9.

[0123] A method for determining whether a subject is responding to treatment with a complement inhibitor can include obtaining a bodily fluid and detecting one or more of the above biomarkers or biomarker signatures, e.g., the proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both, by immunoassay. The method can include measuring levels of Ba and / or sC5b-9. In some embodiments, the disclosure relates to a method for treating complement-mediated thrombotic microangiopathy (CM-TMA) using a complement inhibitor in a manner sufficient to induce a physiological change in a CM-TMA-associated biomarker protein, the method comprising: (a) measuring, in a bodily fluid obtained from the subject, a level or activity of a CM-TMA-associated biomarker protein selected from the group consisting of cystatin C; cystatin C / creatinine ratio; complement factor Ba; complement factor Ba / creatinine ratio; complement sC5b-9; complement sC5b-9 / creatinine ratio, or a combination thereof; preferably measuring the level or activity of a biomarker in a signature comprising at least two biomarkers including Ba and sC5b9; and (b) administering to a subject having, suspected of having, or at risk of developing CM-TMA, the complement inhibitor in an amount and frequency sufficient to cause a decrease in the level or activity of a biomarker or biomarker signature compared to its level or activity in a sample of the same type of bodily fluid obtained from the subject prior to treatment with the inhibitor. In these embodiments, preferably the complement inhibitor comprises an anti-C5 antibody, such as ravulizumab.

[0124] A method for determining whether a patient having complement-mediated thrombotic microangiopathy (CM-TMA), who is being treated with a complement inhibitor under a given dosing schedule, requires a different dosing schedule, comprises: (A) determining whether the CM-TMA patient is responsive to treatment with a complement inhibitor under a given dosing schedule by measuring, in a body fluid obtained from the subject, one or more of the concentration and activity of a CM-TMA-associated biomarker protein selected from the group consisting of cystatin C; cystatin C / creatinine ratio; complement factor Ba; complement factor Ba / creatinine ratio; complement sC5b-9; and / or complement sC5b-9 / creatinine ratio, or a combination thereof; (B) if the patient is not responsive to treatment with a complement inhibitor, administering to the non-responding patient a different complement inhibitor or the same complement inhibitor at a higher dose or more frequent dosing schedule compared to the given dosing schedule, where (a) a decreased level or activity of the biomarker or biomarker signature compared to the concentration in a sample of the same type of bodily fluid obtained from the subject prior to treatment with the inhibitor indicates that the subject is responsive to treatment with the inhibitor. In these embodiments, preferably the complement inhibitor comprises an anti-C5 antibody, such as ravulizumab.

[0125] The subject may be a human having, suspected of having, or at risk of developing aHUS. The subject may be a human having, suspected of having, or at risk of developing CM-TMA. The subject may be a human having, suspected of having, or at risk of developing lupus, optionally lupus nephritis.

[0126] The subject may be one who has been (is being) treated with an inhibitor of complement, optionally an inhibitor of complement component C5, such as an anti-C5 antibody. The treatment may have been administered less than one month (e.g., less than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day) prior to obtaining the sample from the subject.

[0127] The methods described herein may further include determining whether the subject has or is at risk of developing CM-TMA, in particular aHUS, lupus, optionally lupus nephritis, or a combination thereof. If the subject is being treated or is being treated with a complement inhibitor (e.g., an anti-C5 antibody) under a prescribed dosing schedule, the method may further include determining whether the patient is responsive to complement inhibitor therapy.

[0128] Decreased concentrations of the proteolytic fragment of complement component factor B (Ba) and soluble C5b9 (sC5b-9) compared to the concentrations of these biomarkers in a sample of the same type of body fluid obtained from the subject prior to treatment with the inhibitor indicate that the subject is responsive to treatment with the inhibitor.

[0129] A method for monitoring the responsiveness of a subject to treatment with an inhibitor of complement component C5 may include detecting a biomarker in a body fluid, where the biomarker is a proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both. The body fluid is obtained from a subject (i) who has, is suspected of having, or is at risk of developing CM-TMA, and (ii) who is being treated (or has recently been treated, for example) with an inhibitor of complement component C5 under a predetermined dosing schedule. According to such a method, (a) a decreased concentration of the proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both, compared to the concentration in a sample of the same type of body fluid obtained from the subject before treatment with the inhibitor; or (b) an increased concentration, compared to the concentration in a sample of the same type of body fluid obtained from the subject before treatment with a complement competitor, indicates that the subject is responsive to treatment with the inhibitor.

[0130] A method for determining whether a subject is responding to treatment with a complement inhibitor can include detecting a biomarker in a bodily fluid that is a proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both, at a reduced concentration compared to the concentration in a sample of the same type of bodily fluid obtained from the subject prior to treatment with the inhibitor.

[0131] A method for monitoring a subject's responsiveness to treatment with a complement inhibitor can include detecting a biomarker in a body fluid, where the biomarker is a proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both. The subject has, is suspected of having, or is at risk of developing CM-TMA, and the subject is being treated or is being treated with a complement inhibitor. (A) Detection of a biomarker in the body fluid that is a proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both, at a decreased concentration compared to the concentration in a sample of the same type of body fluid obtained from the subject before treatment with the inhibitor; or (B) an increased concentration compared to the concentration in a sample of the same type of body fluid obtained from the subject before treatment with the complement inhibitor, indicates that the subject is responsive to treatment with the inhibitor.

[0132] A method for reducing the number, frequency, or occurrence, likelihood of occurrence, or risk of developing lupus, optionally lupus nephritis, using a complement inhibitor in a manner sufficient to induce a physiological change in a biomarker protein associated with thrombosis or coagulation, the biomarker protein being a proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both, in a bodily fluid obtained from the subject; and (b) administering to a subject having, suspected of having, or at risk of developing lupus, optionally lupus nephritis, an inhibitor of complement in an amount and frequency sufficient to cause a physiological change in the biomarker protein, wherein the concentration of the biomarker protein is reduced compared to the concentration of the marker in the same biological sample obtained from the subject prior to treatment with the complement inhibitor. The method may include measuring the concentration of the biomarker both before and after treatment.

[0133] A method for determining whether a CM-TMA patient being treated with a complement inhibitor under a given dosing schedule requires (i) treatment with a different complement inhibitor or (ii) treatment with the same complement inhibitor under a different dosing schedule, comprising: (A) determining whether the CM-TMA patient is responsive to treatment with a complement inhibitor under a given dosing schedule, comprising measuring, in a body fluid obtained from the subject, one or both of the concentration and activity of a biomarker in the body fluid that is a proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both; and (B) determining whether the patient is responsive to treatment with a complement inhibitor under a given dosing schedule, comprising measuring, in the body fluid, the concentration and / or activity of a biomarker in the body fluid that is a proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both; and if the subject is not responsive to treatment with a complement inhibitor, administering to the patient a different complement inhibitor or the same complement inhibitor at a higher dose or more frequent dosing schedule as compared to the given dosing schedule, wherein (a) a decreased concentration of the proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both as compared to the concentration in a sample of the same type of body fluid obtained from the subject prior to treatment with the inhibitor; or (b) an increased concentration as compared to the concentration in a sample of the same type of body fluid obtained from the subject prior to treatment with the complement inhibitor indicates that the subject is responsive to treatment with the inhibitor.

[0134] A method for diagnosing a subject as having or at risk of developing atypical hemolytic uremic syndrome (aHUS) may include measuring the concentration of at least two aHUS-associated biomarker proteins, the proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both, in a body fluid. The body fluid is obtained from a subject suspected of having or at risk of developing aHUS. According to the method, an increased concentration of the proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both, compared to the concentration in a body fluid of a normal control of the same type, indicates that the subject has or is at risk of developing aHUS. The control may be obtained from a healthy subject, for example, a subject without CM-TMA, preferably a subject whose urine sample does not contain detectable levels of sC5b9, as measured via a standard immunoassay.

[0135] A method for determining whether a CM-TMA patient is responding to treatment with a complement inhibitor may include measuring the concentration of Ba and / or sC5b-9 in a biological sample obtained from a patient having, suspected of having, or at risk of developing CM-TMA and being treated with a complement inhibitor (e.g., an anti-C5 antibody); determining that the patient is responding to treatment if the concentration of one or more biomarkers in the biological sample is decreased compared to the concentration of one or more biomarkers in the same type of biological sample obtained from the patient before treatment with a complement inhibitor, or determining that the patient is not responding to treatment if the concentration of one or more biomarkers in the biological sample is not decreased compared to the concentration of one or more biomarkers in the same type of biological sample obtained from the patient before treatment with a complement inhibitor. Thus, the method can be used to assess or monitor terminal complement blockade in CM-TMA patients treated with a complement inhibitor. In embodiments where a patient is non-responsive or less responsive to treatment, the methods may also include altering the dosage or frequency of administration of a complement inhibitor, or selecting a different complement inhibitor (e.g., an inhibitor of C3 activation) for use in treating the patient.

[0136] Detection Method The biomarkers, the proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both, can be measured using immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), fluorescent immunoassays (FIAs), chemiluminescent immunoassays (CLIAs), counting immunoassays (CIAs), colorimetric immunoassays, Western blotting, dot blots, cytometric bead arrays (CBAs), or combinations thereof. Methods for immunoassays are known in the art. Laboratory Methods in Immunology Shawkatova (2019).

[0137] Antibodies that bind to C5b and methods for making such antibodies are known in the art. Commercially available anti-C5b antibodies are available from several vendors, including, for example, Hycult Biotechnology (catalog number: HM2080; clone 568) and ABCAM® (ab46151 or ab46168).

[0138] The antibody can be an anti-factor B antibody (such as monoclonal antibody 1379 produced by ATCC deposit number PTA-6230). Anti-factor B antibodies are also described, for example, in Ueda et al. (1987) J Immunol 138(4):1143-9; Tanhehco et al. (1999) Transplant Proc 31(5):2168-71; U.S. Pat. Nos. 7,999,082 and 7,964,705; and WO 09 / 029669.

[0139] In the Examples section and elsewhere, representative types of antibodies useful in practicing various embodiments of the disclosure are provided, including, for example, information about specific vendors and / or catalog numbers. It should be understood that the disclosure is not limited to exemplary embodiments that use antibody detection reagents from a specific vendor / manufacturer. Antibodies to the biomarkers / analytes of the disclosure can be obtained from any manufacturer, including Biolegend (San Diego, CA), Southern Biotech (Birmingham, AL), United States Biological (USB; Salem, MA), Lifespan Biosciences (LSBIO; Seattle, WA), ABCAM® (Cambridge, United Kingdom), Cell Signaling Technology (Danvers, MA), and Sigma-Aldrich (St. Louis, MO). For example, rabbit anti-PODXL antibodies can be purchased from USB (catalog number 212672), LSBIO (catalog number LS-C141161), ABCAM® (catalog number ab205350), and Sigma-Aldrich (catalog number HPA002110); anti-CD9 antibody clone MM2 / 57 can be purchased from Southern Biotech (catalog number 9310), EMD Millipore (catalog number CBL162), VWR (catalog number 89366), and BIO RAD (catalog number MCA469G). Antibodies can also be produced using conventional techniques, e.g., immunization of mammals such as mice or rabbits and / or hybridoma technology.

[0140] Biomarkers can be detected using an array. For example, the array can be a protein chip where each address of the array is a well of an assay plate. Each address of the array can be a particle (e.g., a bead) having a binding agent immobilized thereon.

[0141] Measurement of protein expression levels in biological samples can be performed by any suitable method. See, for example, Greenfield (Ed.) (2014) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory: Cold Spring Harbor, NY. In general, protein levels are determined by contacting a biological sample obtained from a subject with a binding agent for a biomarker protein; detecting the level of one or more of the biomarker proteins that bind to the binding agent in the sample (e.g., bodily fluid); and comparing the level of one or more of the biomarker proteins in the sample with the level of the corresponding protein biomarker in a control sample (e.g., normal sample). In certain embodiments, a suitable binding agent is a peptide component, an RNA molecule, or a ribosome in the presence or absence of a polypeptide (e.g., a polypeptide comprising a polypeptide sequence of a protein marker, a peptide variant thereof, or a non-peptide mimetic of such a sequence).

[0142] Suitable binding agents also include antibodies specific for the biomarker proteins described herein. Antibodies suitable for use in the methods of the invention include monoclonal and polyclonal antibodies, as well as antigen-binding fragments of antibodies (e.g., Fab fragments or scFv). Antibodies, including monoclonal and polyclonal antibodies, fragments and chimeras, can be prepared using methods known in the art. Antibodies to be used in the methods of the invention can be purified by methods known in the art. Greenfield (Ed.) (2014) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory: Cold Spring Harbor, NY Antibodies can also be obtained from commercial sources.

[0143] The binding agent is directly or indirectly labeled with a detectable moiety. The role of the detectable agent is to facilitate the detection step of the diagnostic method by allowing visualization of the complex formed by the binding of the binding agent to the protein marker (or a fragment thereof). The detectable agent can be selected so that it generates a signal of an intensity that is measurable and related (preferably proportional) to the amount of the protein marker present in the sample being analyzed. Methods for labeling biomolecules such as polypeptides and antibodies are well known in the art. Any of a wide variety of detectable agents can be used in the practice of the invention. Suitable detectable agents include, but are not limited to, various ligands, radionuclides, fluorescent dyes, chemiluminescent agents, microparticles (e.g., quantum dots, nanocrystals, phosphors), enzymes (e.g., those used in ELISA, e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), colorimetric labels, magnetic labels, and biotin, digoxigenin or other haptens and proteins for which antisera or monoclonal antibodies are available.

[0144] A binding agent (e.g., an antibody) can be immobilized on a carrier or support (e.g., a bead, a magnetic particle, a latex particle, a microtiter plate well, a cuvette, or other reaction vessel). Examples of suitable carrier or support materials include agarose, cellulose, nitrocellulose, dextran, Sephadex®, Sepharose®, liposomes, carboxymethylcellulose, polyacrylamide, polystyrene, gabbro, filter paper, magnetite, ion exchange resins, plastic films, plastic tubes, glass, polyamine-methylvinyl-ether-maleic acid copolymers, amino acid copolymers, ethylene-maleic acid copolymers, nylon, silk, or combinations thereof. A binding agent can be indirectly immobilized using a second binding agent specific for the first binding agent (e.g., a mouse antibody specific for a protein marker can be immobilized using a sheep anti-mouse IgG Fc fragment-specific antibody coated on a carrier or support).

[0145] Protein expression level in biological samples can be measured using immunoassay. Examples of such assays include time-resolved fluorescent immunoassay (TR-FIA), radioimmunoassay, enzyme immunoassay (e.g., ELISA), immunofluorescence immunoprecipitation, latex agglutination, hemagglutination, Western blot, and histochemistry, which are conventional methods well known in the art. The method of detection and quantification of the signal generated by the complex formed by the binding of the binding agent and the protein marker will depend on the nature of the assay and the nature of the detectable moiety (e.g., fluorescent moiety).

[0146] In an example, the presence or amount of protein expression of a gene (e.g., Ba and / or sC5b-9) can be measured using Western blotting techniques. For example, a lysate can be prepared from a biological sample, or the biological sample (e.g., a body fluid) itself can be contacted with Laemmli buffer and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE separated proteins separated by size can then be transferred to a filter membrane (e.g., nitrocellulose) and subjected to an immunoblot technique using a detectably labeled antibody specific for the protein of interest. The presence or amount of bound detectably labeled antibody indicates the presence or amount of the protein in the biological sample.

[0147] In some instances, immunoassays can be used to detect and / or measure protein expression of biomarker proteins (e.g., Ba and / or sC5b-9). As described above, for detection purposes, immunoassays can be performed using antibodies with detection moieties (e.g., fluorescent agents or enzymes). Proteins from biological samples can be directly conjugated to a solid phase matrix (e.g., multi-well assay plates, nitrocellulose, agarose, Sepharose®, coded particles, or magnetic beads) or can be conjugated to a first member of a specific binding pair (e.g., biotin or streptavidin) that is attached to the solid phase matrix upon binding to a second member of the specific binding pair (e.g., streptavidin or biotin). Such attachment to a solid phase matrix allows the protein to be purified from other interfering or unrelated components of the biological sample before contact with the detection antibody, and also allows for subsequent washing of unbound antibody. Here, as described above, the presence or amount of bound detectably labeled antibody indicates the presence or amount of the protein in the biological sample.

[0148] Alternatively, protein expression levels can be measured using mass spectrometry-based or image-based methods known in the art for detecting proteins. Other suitable methods include 2D gel electrophoresis, proteomics-based methods, such as identification of individual proteins recovered from gels (e.g., by mass spectrometry or N-terminal sequencing), and / or bioinformatics.

[0149] Methods for detecting or measuring protein expression can optionally be performed in a format that allows for rapid preparation, processing, and analysis of multiple samples. This can be, for example, in multi-well assay plates (e.g., 96-well or 386-well) or in arrays (e.g., protein chips). Storage solutions for various reagents can be provided manually or robotically, and subsequent sample preparation, pipetting, dilution, mixing, distribution, washing, incubation (e.g., hybridization), sample reading, data collection (optical data), and / or analysis (computer-assisted image analysis) can be performed robotically using commercially available analysis software, robotics, and detection devices capable of detecting signals generated from the assay. Examples of such detectors include, but are not limited to, spectrophotometers, luminometers, fluorescence spectrometers, and devices that measure the decay of radioisotopes.

[0150] diagnostic antibodies The antibody or antigen-binding fragment thereof may be selected from the group consisting of a humanized antibody, a recombinant antibody, a bispecific antibody, a chimerized or chimeric antibody, a monoclonal antibody, a deimmunized antibody, a fully human antibody, a single chain antibody, an Fv fragment, an Fd fragment, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, or a combination thereof.

[0151] Monoclonal antibodies The monoclonal antibodies disclosed herein can be of any isotype. The monoclonal antibodies can be, for example, IgM or IgG antibodies, such as IgG1 or IgG2. The class of an antibody that immunospecifically binds to Ba or sC5b-9 can be switched to another (e.g., IgG can be switched to IgM) according to well-known procedures. Class switching can also be used to convert one IgG subclass to another, for example, from IgG1 to IgG2.

[0152] The antibodies of the present invention can be monovalent, bivalent, trivalent or multivalent. For example, monovalent scFvs can be multimerized chemically or by association with another protein or substance. scFvs fused to a hexahistidine tag or Flag tag can be multimerized using Ni-NTA agarose (Qiagen) or using an anti-Flag antibody (Stratagene, Inc.).

[0153] The antibodies of the invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of Ba and / or sC5b-9, or fragments thereof, and for heterologous epitopes, such as heterologous polypeptides or solid support materials. See, e.g., WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; WO 92 / 05793; Tutt et al, J. Immunol. 147:60-69 (1991); U.S. Pat. No. 4,474,893; U.S. Pat. No. 4,714,681; U.S. Pat. No. 4,925,648; U.S. Pat. No. 5,573,920; U.S. Pat. No. 5,601,819; Kostelny et al. J. Immunol. 148:1547-1553 (1992).

[0154] Methods for Producing Antibodies The antibodies (scFvs and other molecules, including or alternatively consisting of the antibody fragments or variants of the invention) that can be used in the methods described herein can be produced by any method known in the art for the synthesis of antibodies, in particular chemical synthesis or preferably recombinant expression techniques. Greenfield (Ed.) (2014) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory: Cold Spring Harbor, NY

[0155] Single chain Fvs (scFvs) that immunospecifically bind to Ba and / or sC5b-9, or fragments thereof, can be generated using phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or mouse cDNA libraries of lymphoid tissues) or synthetic cDNA libraries. The DNA encoding the VH and VL domains are linked together by an scFv linker by PCR and cloned into a phagemid vector (e.g., p CANT AB 6 or pComb 3 HSS). The vector is electroporated into E. coli, which is infected with helper phage. The phage used in these methods are typically filamentous phage, including fd and M13, and the VH and VL domains are usually recombinantly fused to either phage gene III or gene VIII. Phage expressing an antigen-binding domain that binds to an antigen of interest (e.g., Ba and / or sC5b-9, or fragments thereof) can be selected or identified by antigen, for example, using labeled antigen or antigen bound or captured to a solid surface or bead.Examples of phage display methods that can be used to generate the antibodies of the present invention include, but are not limited to, those described in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al. Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al. Advances in Immunology 57:191-280 (1994); WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; WO 97 / 13844; and U.S. Pat. Nos. 5,698,426; 5,223,409; and 5,403,484; Nos. 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

[0156] After phage selection as described in the above references, the antibody coding region from the phage can be isolated and used to generate whole antibodies, including human or humanized antibodies, or any other desired antigen-binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, for example as described below. Techniques for recombinantly producing Fab, Fab', and F(ab')2 fragments can also be utilized using methods known in the art, for example, those disclosed in WO 92 / 22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988).

[0157] To generate a full antibody, the VH or VL sequence in the scFv clone can be amplified using PCR primers, e.g., VH or VL nucleotide sequences, restriction sites, and flanking sequences to protect the restriction sites. Using cloning techniques known to those skilled in the art, the PCR amplified VH domain can be cloned into a vector expressing a VH constant region, e.g., human gamma 4 constant region, and the PCR amplified VL domain can be cloned into a vector expressing a VL constant region, e.g., human kappa or lambda constant region. Preferably, the vector for expressing the VH or VL domain contains a suitable promoter to induce expression of the heavy and light chains, in a selected expression system, secretion signal, cloning site for immunoglobulin variable domain, immunoglobulin constant domain, and a selection marker such as neomycin. The VH and VL domains can also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into a cell line, and a stable or transient cell line expressing a full-length antibody, e.g., IgG, is generated using techniques known to those skilled in the art.

[0158] Once an antibody (including molecules including or alternatively consisting of antibody fragments or variants thereof) usable in the methods described herein has been chemically synthesized or recombinantly expressed, it can be purified by any method known in the art intended for the purification of immunoglobulin molecules, or more generally protein molecules, such as, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for a specific antigen followed by Protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for the purification of proteins. Additionally, antibodies usable in the methods described herein can be fused to heterologous polypeptide sequences described herein or others known in the art to facilitate purification.

[0159] Methods for recombinantly producing antibodies that can be used in the methods described herein are well known to those skilled in the art. Antibodies can also be produced by constructing expression vectors containing operons and DNA sequences encoding the antibodies, using conventional techniques well known to those skilled in the art. Furthermore, the present invention relates to vectors, in particular plasmids, cosmids, viruses, bacteriophages and other vectors common in genetic engineering, that can contain the above-mentioned nucleic acid molecules. The nucleic acid molecules contained in the vectors can be linked to regulatory elements that ensure transcription in prokaryotic and eukaryotic cells.

[0160] A vector contains sequences that facilitate manipulation for the expression of a foreign protein in a target host cell. Conveniently, manipulation of sequences for transformation and generation of DNA are first performed in a bacterial host (e.g., E. Coli), and the vector will usually contain sequences to facilitate such manipulation, including a bacterial origin of replication and an appropriate bacterial selection marker. A selection marker encodes a protein necessary for the survival or growth of transformed host cells grown in a selective medium. Host cells not transformed with a vector containing a selection gene will not survive in the medium. Typical selection genes encode proteins that confer resistance to antibiotics or other toxins, complement auxotrophic deficiencies, or supply important nutrients that cannot be obtained from complex media. Exemplary vectors and methods for yeast transformation have been described in the art. See, for example, Burke, et al. (2000) Methods in Yeast Genetics Cold Spring Harbor Laboratory Press.

[0161] The polynucleotide encoding the antibody can be operably linked to transcriptional and translational control sequences that provide for expression of the polypeptide in yeast cells. These vector components can include, but are not limited to, one or more of an enhancer element, a promoter, and a transcription termination sequence. Sequences for secreting the polypeptide can also be included (e.g., a signal sequence).

[0162] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a signal sequence is operably linked to DNA for a polypeptide if it is expressed as a protein precursor that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. In general, "operably linked" refers broadly to contiguous linked DNA sequences, and in the case of secretory leaders, contiguous and in reading frame. Enhancers, however, need not be contiguous.

[0163] Promoters are untranslated sequences located upstream (5') of the start codon of a structural gene (generally within about 100-1000 bp) that control the transcription and translation of a particular nucleic acid sequence to which they are operably linked. Such promoters are divided into several classes: inducible, constitutive, and repressible promoters (e.g., those that increase the level of transcription in response to the absence of a repressor). Inducible promoters can respond to some change in culture conditions (e.g., the presence or absence of a nutrient or a change in temperature) to initiate increased levels of transcription from DNA under their control.

[0164] The expression vector is transfected into a host cell by conventional techniques known to those of skill in the art, the transfected host cell is produced, and the transfected host cell is cultured by conventional techniques known to those of skill in the art to produce the antibody.

[0165] The host cells used to express the anti-Ba and / or anti-sC5b-9 antibodies can be either bacterial cells such as E. Coli, yeast (e.g., S. cerevisiae), or eukaryotic cells (e.g., mammalian cell lines). For this purpose, well-defined types of mammalian cells can be used, such as myeloma cells, 3T3, HeLa, C6A2780, Vero, MOCKII, Chinese Hamster Ovary (CHO), Sf9, Sf21, COS, NSO, or HEK293 cell lines.

[0166] The general methods by which vectors can be constructed, the transfection methods required to generate host cells, and the culture methods required to produce antibodies and fragments thereof from said host cells all comprise the prior art. Preferably, the cell line used to produce the antibodies is a mammalian cell line, although any other suitable cell line, such as a bacterial cell line derived from E. Coli, or a yeast cell line, can be used.

[0167] Similarly, antibodies, once produced, can be purified according to standard procedures in the art, such as cross-flow filtration, ammonium sulfate precipitation, and affinity column chromatography.

[0168] Antibodies that bind to a biomarker or biomarker signature associated with a CM-TMA can be screened using any known method, e.g., a binding assay. In an exemplary method, the target biomarker or its antigenic epitope is expressed in a standard cell, and antibodies are panned using selection techniques known in the art. Antibodies can be screened for, e.g., based on binding affinity, e.g., at least 10 -6 M: preferably 10 -8 M; and especially 10 -10 Dissociation constant of M (K d ), where K d Values ​​can be determined using standard binding assays.

[0169] body fluid Biological samples suitable for use in the methods described herein include, for example, any bodily fluid. Biological samples can be, for example, specimens obtained from a subject (e.g., a mammal such as a human) or can be derived from such a subject. Biological samples can also be bodily fluids such as urine, whole blood or a fraction thereof (e.g., plasma or serum), saliva, semen, sputum, cerebrospinal fluid, tears, or mucus. Biological samples can be further fractionated into fractions containing specific analytes of interest (e.g., proteins) as needed. For example, a whole blood sample can be fractionated into fractions containing serum or a specific type of protein. Biological samples can be a combination of different biological samples from a subject, for example, a combination of two different bodily fluids.

[0170] The body fluid obtained from the patient may be blood, optionally a blood fraction including but not limited to serum or plasma. The body fluid may be urine. The body fluid may be blood, serum, plasma, urine, or a combination thereof. The measurement may be performed on one body fluid. In the methods described herein, the measurement may be performed on at least two different body fluids obtained from the subject, for example, a combination of blood, plasma, serum, and urine. For example, the concentration of a first biomarker protein is measured in one type of body fluid and a second biomarker protein is measured in a second type of body fluid.

[0171] Biological samples suitable for the present invention may be fresh or frozen samples collected from a subject, or archived samples with a known diagnosis, treatment and / or outcome history. Biological samples can be obtained from subjects, for example, subjects who have, are suspected of having, or are at risk of developing a complement-related disorder (e.g., CM-TMA). Any suitable method for obtaining a biological sample can be used, but exemplary methods include, for example, phlebotomy, swab (e.g., buccal swab), lavage, or fine needle aspiration biopsy procedures.

[0172] Protein extracts can be prepared from biological samples. Protein extracts include total protein content. Methods of protein extraction are well known in the art. See, for example, Roe (2001) "Protein Purification Techniques: A Practical Approach", 2 ndFor an edition, see Oxford University Press. Numerous different and versatile kits can be used to extract proteins from bodily fluids and tissues, and are commercially available from, for example, BioRad Laboratories (Hercules, Calif.), BD Biosciences Clontech (Mountain View, Calif.), Chemicon International, Inc. (Temecula, Calif.), Calbiochem (San Diego, Calif.), Pierce Biotechnology (Rockford, Ill.), and Invitrogen Corp. (Carlsbad, Calif.).

[0173] Methods for obtaining and / or storing samples that preserve the activity or integrity of biomarkers in a biological sample are well known to those skilled in the art. For example, a biological sample can be further contacted with one or more additional agents intended to preserve or minimize changes in protein structure (e.g., changes in osmolality or pH), such as an appropriate buffer and / or inhibitors, including protease inhibitors. Such inhibitors include, but are not limited to, chelating agents such as ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), protease inhibitors, e.g., phenylmethylsulfonyl fluoride (PMSF), aprotinin, and leupeptin. Suitable buffers and conditions for storing or otherwise manipulating samples are described, for example, in Pollard and Walker (1997), "Basic Cell Culture Protocols," volume 75 of Methods in molecular biology, Humana Press; Masters (2000) "Animal cell culture: a practical approach," volume 232 of Practical approach series, Oxford University Press; and Jones (1996) "Human cell culture protocols," volume 2 of Methods in molecular medicine, Humana Press.

[0174] Samples can be treated to remove or minimize the presence of interfering substances. For example, biological samples can be fractionated or purified to remove one or more materials (e.g., cells) that are not of interest. Methods for fractionating or purifying biological samples include, but are not limited to, flow cytometry, fluorescent activated cell sorting, and sedimentation.

[0175] The methods described herein may include recording the measured concentration of the biomarker protein. The recording may be written or may be performed on a computer readable medium. The methods may further include communicating the measured concentration of the biomarker protein to the subject and / or to a physician in the treatment the subject is receiving.

[0176] Therapeutic methods for treating CM-TMA "Complete TMA response" broadly refers to a composite outcome measure that required normalization of hematological parameters (e.g., platelet count and lactate dehydrogenase [LDH]) and improvement in renal function (a ≥ 25% decrease from baseline in serum creatinine); for dialysis participants, baseline was established at least 6 days after cessation of dialysis. Participants had to meet these criteria for two separate assessments obtained at least every 4 weeks (28 days) and for every measurement during the 26-week initial assessment period. The inventors unexpectedly discovered that decreases in baseline levels of (1) serum sVCAM-1, (2) serum sTNF-R1, (3) plasma thrombomodulin, and (4) urinary sC5b-9 were associated with an increased likelihood of achieving a complete TMA response.

[0177] In addition, complete TMA response can be assessed by a combined measure of normalization of platelet counts and LDH, along with improvement in serum creatinine in treated patients.

[0178] Complement inhibitors can be administered to a subject under a predetermined dosing schedule, based in part on the subject's weight. Exemplary dosing schedules for anti-C5 antibodies (e.g., chronic dosing schedules) are described in WO 2010 / 054403 and U.S. Pat. No. 9,658,236.

[0179] The complement inhibitor may be an antibody or an antigen-binding fragment thereof, a small molecule, a polypeptide, a polypeptide analog, a peptidomimetic, or an aptamer. The complement inhibitor may inhibit one or more of complement components C1, C2, C3, C4, C5, C6, C7, C8, C9, Factor D, Factor B, properdin, MBL, MASP-1, MASP-2, a biologically active fragment, or a combination thereof. The complement inhibitor may inhibit one or both of the generation of anaphylatoxin activity associated with C5a and / or the assembly of the membrane attack complex associated with C5b.

[0180] Natural or soluble forms of complement inhibitory compounds can be used, including, but not limited to, CR1, LEX-CR1, MCP, DAF, CD59, Factor H, cobra venom factor, FUT-175, complestatin, K76 COOH, and combinations thereof.

[0181] The complement inhibitor may be a complement receptor 2 (CR2)-Factor H (FH) molecule comprising a) a CR2 portion comprising CR2 (e.g., human CR2) or a fragment thereof, and b) a FH portion comprising FH or a fragment thereof, where the CR2-FH molecule or a fragment thereof is capable of binding to a CR2 ligand, and the CR2-FH molecule is capable of inhibiting alternative pathway complement activation. Exemplary CR2-FH fusion proteins are described in WO 2007 / 149567 and WO 2011 / 143637. The complement inhibitor may comprise a targeting domain such as CR2 or an anti-C3d antibody (as described in WO 2011 / 163412). Fusions of the targeting domain with other complement inhibitors such as CD59, CD55, and factor H-like molecules can be used in the methods described herein as complement inhibitors. WO 2011 / 163412.

[0182] The complement inhibitor may be selected from the group consisting of recombinant anti-C5 mini-antibody MB12 / 22, anti-C5 mini-antibody targeted to endothelial MB12 / 22-RGD, C5-specific aptamer ARC187, C5-specific aptamer ARC1905 (avacincaptado pegol), Staphylococcal superantigen-like protein 7 (SSL7), Ornithodoros moubata C inhibitor (OmCI), or combinations thereof.

[0183] The complement inhibitor may be an antagonist antibody or an antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof may be selected from the group consisting of a humanized antibody, a recombinant antibody, a bispecific antibody, a chimerized or chimeric antibody, a monoclonal antibody, a deimmunized antibody, a fully human antibody, a single chain antibody, an Fv fragment, an Fd fragment, an Fab fragment, an Fab' fragment, an F(ab')2 fragment, or a combination thereof.

[0184] The antagonist antibody can be an anti-C5 antibody, including, but not limited to, SOLIRIS® (eculizumab) and ULTOMIRIS® (ravulizumab). The antagonist antibody can be pexelizumab, a C5-binding fragment of an anti-C5 antibody.

[0185] The methods described herein can include administering a complement inhibitor to a subject at a higher dose or with increased frequency of administration as compared to a given dosing schedule if the subject is not responsive to treatment with the inhibitor under the given dosing schedule.

[0186] A method for treating complement-mediated thrombotic microangiopathies (CM-TMA) may include administering to a subject having, suspected of having, or at risk of developing CM-TMA an inhibitor of complement (e.g., an inhibitor of complement component C5) in an amount and frequency sufficient to effect a physiological change in the proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both, where the physiological change is selected from the group consisting of: (a) a decreased concentration of the proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both, compared to the concentration in a sample of the same type of bodily fluid obtained from the subject prior to treatment with the inhibitor; or (b) an increased concentration of the proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both, compared to the concentration in a sample of the same type of bodily fluid obtained from the subject prior to treatment with the inhibitor. In some embodiments, the at least one CM-TMA-associated biomarker may be the proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both.

[0187] A method for treating complement-mediated thrombotic microangiopathy (CM-TMA) using a complement inhibitor in a manner sufficient to induce a physiological change in a CM-TMA-associated biomarker protein, the method comprising: (a) measuring the concentration of a CM-TMA-associated biomarker protein, which is a proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both, in a bodily fluid obtained from the subject; and (b) administering the complement inhibitor to a subject who has, is suspected of having, or is at risk of developing CM-TMA, to induce a physiological change in a CM-TMA-associated biomarker protein, which is a proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both, in a bodily fluid obtained from the subject; and administering to the subject an amount and frequency sufficient to cause a physiological change in at least each of the complement marker proteins, the physiological change being selected from the group consisting of: (a) a decreased concentration of the proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both, compared to the concentration in a sample of the same type of body fluid obtained from the subject prior to treatment with the inhibitor; and (b) an increased concentration in the body fluid obtained from the subject, compared to the concentration in a sample of the same type of body fluid obtained from the subject prior to treatment with the inhibitor of complement. The method may also include determining whether a physiological change has occurred.

[0188] A method for detecting a set of biomarkers in a subject may comprise detecting the levels of at least one, at least two, at least three, at least four, at least five, at least six biomarkers selected from the group consisting of cystatin C; cystatin C / creatinine ratio; complement factor Ba; complement factor Ba / creatinine ratio; complement sC5b-9; and / or complement sC5b-9 / creatinine ratio; preferably detecting the levels of the biomarkers in a subset comprising at least two biomarkers, including Ba and sC5b9, in a bodily fluid sample obtained from the subject.

[0189] The methods described herein may further include measuring the concentration of a CM-TMA-associated biomarker protein in a bodily fluid, the proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both. The bodily fluid may be obtained from a subject.

[0190] The methods described herein may include determining whether a physiological change occurs in the proteolytic fragment of complement component factor B (Ba), soluble C5b9 (sC5b-9), or both. After treatment, the concentrations of both Ba and sC5b9 may decrease. For example, after treatment, the respective concentrations (e.g., urine, blood, plasma, serum concentrations) of Ba and sC5b9 may decrease.

[0191] The Ba concentration may be decreased by at least 10% by 6 weeks after the start of treatment. The Ba concentration may be decreased by at least 30% by 12 weeks after the start of treatment. The decreased Ba concentration may be in blood, serum, plasma, urine, or a combination thereof. The decreased Ba concentration may be in plasma.

[0192] The sC5ab9 concentration may be decreased by at least 40% by week 3 after initiation of treatment. The sC5ab9 concentration may be decreased by at least 70% by week 6 after initiation of treatment. The sC5ab9 concentration may be decreased by at least 50% by week 3 after initiation of treatment. The decreased sC5ab9 concentration may be in blood, serum, plasma, urine, or a combination thereof. The sC5ab9 concentration may be in plasma. The sC5ab9 concentration may be in urine. The sC5ab9 concentration may be in plasma and urine.

[0193] In the methods described herein, the methods can include administering to a subject an inhibitor of complement in an amount and frequency sufficient to effect a physiological change in a biomarker.

[0194] Physiological changes in biomarker proteins may occur within 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 9 weeks, or 3 months or more following administration (e.g., chronic administration) of the inhibitor.

[0195] Following administration of an effective amount of a complement inhibitor, the concentration of the biomarker protein may decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% following administration of the inhibitor.

[0196] Following administration of an effective amount of a complement inhibitor, the concentration of the biomarker protein may decrease to within 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the normal concentration of the biomarker protein following administration of one or more doses of a complement inhibitor.

[0197] The subject of the methods described herein may have undergone dialysis at least once (e.g., at least 2, 3, 4, or 5 or more times) within 3 months (e.g., 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 week) prior to treatment with a complement inhibitor. For example, the subject may have undergone dialysis once over the 2 months prior to undergoing complement inhibitor therapy. In another example, the subject may have undergone dialysis three times within the 3 months immediately prior to undergoing complement inhibitor therapy. Additionally, the concentration of Ba, sC5b9, cystatin C, and / or creatinine may be increased compared to the concentration (e.g., blood, plasma, serum, and / or urine concentration) in a healthy individual.

[0198] The subject may be experiencing an initial symptom of acute complement-mediated thrombotic microangiopathy (CM-TMA). For example, prior to treatment with a complement inhibitor, the subject may have an increased concentration of at least one of the biomarkers, or, for example, a plurality of the biomarkers, as compared to normal concentrations.

[0199] A subject has CM-TMA but is considered to be in clinical remission (e.g., a subject has normal levels of platelets or other hematological markers, such as LDH or haptoglobin). A subject can have increased levels of one or more of the CM-TMA biomarkers described herein, including, but not limited to, one or more of Ba, sC5b9, cystatin C, and / or creatinine.

[0200] The methods described herein may further include monitoring the status of the biomarkers and deciding to either initiate a second therapy (in addition to the complement inhibitor therapy) or to change the dosing regimen of one or more second therapies administered to the patient with complement-mediated thrombotic microangiopathies (CM-TMA). For example, during treatment (e.g., chronic treatment) with a complement inhibitor, the concentration of one or more CM-TMA-associated biomarker proteins can be measured in one or more body fluids obtained from the subject. If the concentration of one or more of the biomarker proteins remains unnormalized and / or increased, the physician may choose to administer one or more additional secondary agents (e.g., anti-inflammatory drugs) to the subject to address any pathophysiological effects resulting from the increased biomarkers.

[0201] A normal control concentration, as used in any of the methods described herein, can be (or can be based on), for example, the concentration of a given biomarker protein in one or more biological samples obtained from one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 or more) healthy individuals. In some embodiments, a normal control concentration of a biomarker can be (or can be based on), for example, the concentration of a biomarker in a pooled sample obtained from two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 or more) healthy individuals. In some embodiments of any of the methods described herein, the pooled sample can be obtained from healthy individuals or at least individuals who do not have or are not suspected of having (nor are they at risk of developing) CM-TMA. For example, determining whether a subject has CM-TMA can involve comparing the measured concentrations of one or more complement component proteins in a biological sample (or several different types of biological samples) obtained from the patient and comparing the measured concentrations to the average concentrations of the same proteins in pooled healthy samples. Such healthy human control concentrations can in some embodiments be a range of values, or a median or mean value derived from that range.

[0202] Administration of therapeutic drugs The compositions (e.g., complement inhibitors and / or secondary agents) can be administered to a subject, e.g., a human subject, using a variety of methods that depend in part on the route of administration, which can be, for example, intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP) injection, or intramuscular injection.

[0203] Administration can be achieved, for example, by local infusion, injection, or by using an implant. The implant can be a porous, non-porous, or gelatinous material, for example, a membrane, such as a sialastic membrane, or a fiber. The implant can be designed for sustained or periodic release of the composition to the subject. See, for example, US Patent Application Publication No. 2008 / 0241223; US Patent No. 5,501,856; US Patent No. 4,863,457; and US Patent No. 3,710,795; and European Patent No. 488401 and European Patent No. 430539. The composition can be delivered to the subject, for example, via a diffusion, erosion, or convection system, such as an osmotic pump, a biodegradable implant, an electrodiffusion system, an electroosmotic system, a vapor pressure pump, an electrolytic pump, an effervescent pump, a piezoelectric pump, an erosion-based system, or an implantable device based on an electromechanical system.

[0204] The suitable dose of a complement inhibitor (e.g., an anti-C5 antibody or fragment thereof) capable of treating or preventing complement-mediated thrombotic microangiopathies (CM-TMA) in a subject may depend on a variety of factors, including, for example, the age, sex, and weight of the subject to be treated, as well as the specific inhibitor compound used. For example, to treat a subject with CM-TMA, a different dose of siRNA specific for human C5 may be required compared to the dose of anti-C5 antibody required to treat the same patient. Other factors that affect the dose administered to a subject include, for example, the type or severity of CM-TMA. For example, a subject with CFH-associated atypical hemolytic uremic syndrome (aHUS) may require administration of a different dose of an inhibitor than a subject with MCP-associated aHUS. Other factors may include, for example, other medical disorders that may be affecting the subject simultaneously or previously, the general health of the subject, the genetic predisposition of the subject, diet, duration of administration, excretion rate, drug combination, and any other additional therapeutic agents administered to the subject. It is also to be understood that the specific dosage and treatment regimen for any particular subject will depend on the judgment of the treating practitioner (eg, physician or nurse).

[0205] The inhibitor can be administered as a fixed dose or in milligram / kilogram "mg / kg" doses. The dose can also be selected to reduce or avoid the production of antibodies or other host immune responses to one or more of the active agents in the composition. Exemplary doses of an inhibitor, such as an anti-C5 antibody, include, for example, 1-100 mg / kg body weight, 0.5-50 mg / kg body weight, 0.1-100 mg / kg body weight, 0.5-25 mg / kg body weight, 1-20 mg / kg body weight, and 1-10 mg / kg body weight.

[0206] For humans, an anti-C5 antibody (e.g., eculizumab, ravulizumab) can be administered intravenously at a dose of about 900 mg about every 12 days (e.g., about every 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, 30, 42, or 49 days or more). See, e.g., Hill et al. (2005) Blood 106(7):2559.

[0207] A human can be administered an anti-C5 antibody (e.g., eculizumab, ravulizumab) at a dose of about 600 mg (e.g., about 625, 650, 700, 725, 750, 800, 825, 850, 875, 900, 925, 950, or 1,000 mg or more) intravenously every week, optionally for 2 or more weeks (e.g., 3, 4, 5, 6, 7, or 8 or more weeks). After initial treatment, a human can be administered an antibody, e.g., as a maintenance dose, at a dose of about 900 mg about every 14 days (e.g., about every 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, 30, 42, or 49 or more days). See, e.g., Hillmen et al. (2004) N Engl J Med. 350(6):552-9 and Dmytrijuk et al. (2008) The Oncologist 13(9):993.

[0208] A human can be administered an anti-C5 antibody (e.g., eculizumab, ravulizumab) at a dose of about 900 mg (e.g., 925, 950, 975, 1000, 1100, or 1200 mg or more) intravenously every week, optionally for 2 or more weeks (e.g., 3, 4, 5, 6, 7, or 8 or more weeks). After initial treatment, a human can be administered an antibody, e.g., as a maintenance dose, at a dose of about 1200 mg about every 14 days (e.g., about every 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, 30, 42, or 49 or more days). See, e.g., International Patent Application Publication No. WO 2010 / 054403.

[0209] In some embodiments, the disclosure relates to diagnosing or prognosing CM-TMA according to the methods, e.g., treating CM-TMA in a human subject, comprising detecting in a sample from the subject at least one, at least two, at least three, at least four, at least five, at least six biomarkers selected from: (1) cystatin C; (2) cystatin C / creatinine; (3) complement factor Ba; (4) complement factor Ba / creatinine; (5) complement sC5b-9; and / or (6) complement sC5b-9 / creatinine, optionally together with (7) sTNFR1, (8) VCAM-1 and / or (9) thrombomodulin; preferably detecting a level of a biomarker signature comprising at least two biomarkers including Ba and sC5b9 in a bodily fluid sample obtained from the subject; and administering to the subject an anti-C5 antibody or antigen-binding fragment thereof. In some embodiments, the treatment method includes detection of a biomarker before and after administration of an anti-C5 antibody or antigen-binding fragment, where modulation (e.g., attenuation) of the concentration of the biomarker in a subject's sample after administration of the anti-C5 antibody or antigen-binding fragment compared to its concentration before administration of the anti-C5 antibody or antigen-binding fragment indicates that the subject is receiving effective treatment with CM-TMA.

[0210] In some embodiments, the anti-C5 antibody is selected from eculizumab, ravulizumab, tesidolumab, pozelimab, or clovalimab, or a biosimilar or antigen-binding fragment thereof. For example, preferred biosimilars of eculizumab include, for example, ABP959, SB12, or Elizaria. In some embodiments, the anti-C5 antibody includes eculizumab (SOLIRIS), which is described in WO1995029697 and U.S. Pat. No. 6,355,245, and the heavy and light chains of eculizumab are provided in WO2007106585 and U.S. Pat. No. 9,718,880. In some embodiments, the anti-C5 antibody comprises ravulizumab (also known as ULTOMIRIS®, BNJ441, and ALXN1210), described in WO2015134894 and U.S. Pat. No. 9,079,949. In some embodiments, the anti-C5 antibody comprises clovalimab, the antibody sequence disclosed in Fukuzawa et al. (Sci. Rep., 7:1080, 2017). In some embodiments, the anti-C5 antibody comprises pozelimab, described in U.S. Pat. No. 10,633,434. In some embodiments, the anti-C5 antibody comprises tesidolumab (LFG316).

[0211] In some embodiments, in treating CM-TMA, the anti-C5 antibody or antigen-binding fragment is administered at a fixed dose. For example, in one embodiment, the anti-C5 antibody or antigen-binding fragment is administered at a fixed dose of 10 mg, 20 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 40 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 40 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 80 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 40 mg, 425 mg 0mg, 825mg, 850mg, 875mg, 900mg, 925mg, 950mg, 975mg, 1000mg, 1100mg, 1200mg, 1300mg, 1400mg, 1500mg, 1600mg, 1700mg, 1800mg, 1900mg, 20 00mg, 2100mg, 2200mg, 2300mg, 2400mg, 2500mg, 2600mg, 2700mg, 2800mg, 2900mg, 3000mg, 3100mg, 3200mg, 3300mg, 3400mg, 3500mg, 3600mg, 3 700mg, 3800mg, 3900mg, 4000mg, 4100mg, 4200mg, 4300mg, 4400mg, 4500mg, 4600mg, 4700mg, 4800mg, 4900mg, 5000mg, 5100mg, 5200mg, 5300mg, 5400mg, 5500mg, 5600mg, 5700mg, 5800mg, 5900mg, 6000mg, 6100mg, 6200mg, 6300mg, 6400mg, 6500mg, 6600mg, 6700mg, 6800mg, 6900mg, 7000mg , 7100mg, 7200mg, 7300mg, 7400mg, 7500mg, 7600mg, 7700mg, 7800mg, 7900mg, 8000mg, 8100mg, 8200mg, 8300mg, 8400mg, 8500mg, 8600mg, 8700m g, 8800mg, 8900mg, 9000mg, 9100mg, 9200mg, 9300mg, 9400mg, 9500mg, 9600mg, 9700mg, 9800mg, 9900mg, 10000mg, 10100mg, 10200mg, 10300mg,It is administered in doses of 10400mg, 10500mg, 10600mg, 10700mg, 10800mg, 10900mg or 11000mg.

[0212] In another embodiment, the anti-C5 antibody, or antigen-binding fragment thereof (e.g., ravulizumab (ULTOMIRIS®)) is administered at a dose of 1200 mg, 2400 mg, 2700 mg, 3000 mg, 3300 mg, or 3600 mg.

[0213] In another embodiment, the dose of the anti-C5 antibody or antigen-binding fragment is based on the patient's weight. For example, in one embodiment, the dose is 10 mg, 20 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 92 5mg, 950mg, 975mg, 1000mg, 1100mg, 1200mg, 1300mg, 1400mg, 1500mg, 1600mg, 1700mg, 1800mg, 1900mg, 2000mg, 2100mg, 2200mg, 2300mg, 2400m g, 2500mg, 2600mg, 2700mg, 2800mg, 2900mg, 3000mg, 3100mg, 3200mg, 3300mg, 3400mg, 3500mg, 3600mg, 3700mg, 3800mg, 3900mg, 4000mg, 4100mg ,4200mg,4300mg,4400mg,4500mg,4600mg,4700mg,4800mg,4900mg,5000mg,5100mg,5200mg,5300mg,5400mg,5500mg,5600mg,5700mg,5800mg , 5900mg, 6000mg, 6100mg, 6200mg, 6300mg, 6400mg, 6500mg, 6600mg, 6700mg, 6800mg, 6900mg, 7000mg, 7100mg, 7200mg, 7300mg, 7400mg, 7500mg, 7600mg, 7700mg, 7800mg, 7900mg, 8000mg, 8100mg, 8200mg, 8300mg, 8400mg, 8500mg, 8600mg, 8700mg, 8800mg, 8900mg, 9000mg, 9100mg, 9200mg, 9 300mg, 9400mg, 9500mg, 9600mg, 9700mg, 9800mg, 9900mg, 10000mg, 10100mg, 10200mg, 10300mg, 10400mg, 10500mg, 10600mg, 10700mg, 10800mg,10900 mg or 11000 mg of anti-C5 antibody or antigen-binding fragment is administered to a patient weighing ≧30 to <40 kg. In another embodiment, 1200 mg or 2700 mg of anti-C5 antibody or antigen-binding fragment thereof (e.g., ravulizumab (ULTOMIRIS®)) is administered to a patient weighing ≧30 to <40 kg.

[0214] In another embodiment, the concentrations of any of the following may be increased: 10 mg, 20 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 9 75mg, 1000mg, 1100mg, 1200mg, 1300mg, 1400mg, 1500mg, 1600mg, 1700mg, 1800mg, 1900mg, 2000mg, 2100mg, 2200mg, 2300mg, 2400mg, 2500mg, 2600mg , 2700mg, 2800mg, 2900mg, 3000mg, 3100mg, 3200mg, 3300mg, 3400mg, 3500mg, 3600mg, 3700mg, 3800mg, 3900mg, 4000mg, 4100mg, 4200mg, 4300mg, 4400 mg, 4500mg, 4600mg, 4700mg, 4800mg, 4900mg, 5000mg, 5100mg, 5200mg, 5300mg, 5400mg, 5500mg, 5600mg, 5700mg, 5800mg, 5900mg, 6000mg, 6100mg, 6 200mg, 6300mg, 6400mg, 6500mg, 6600mg, 6700mg, 6800mg, 6900mg, 7000mg, 7100mg, 7200mg, 7300mg, 7400mg, 7500mg, 7600mg, 7700mg, 7800mg, 7900mg , 8000mg, 8100mg, 8200mg, 8300mg, 8400mg, 8500mg, 8600mg, 8700mg, 8800mg, 8900mg, 9000mg, 9100mg, 9200mg, 9300mg, 9400mg, 9500mg, 9600mg, 9700mg, 9800mg, 9900mg, 10000mg, 10100mg, 10200mg, 10300mg, 10400mg, 10500mg, 10600mg, 10700mg, 10800mg, 10900mg, or 11000mg of an anti-C5 antibody or antigen-binding fragment,In another embodiment, 2400 mg or 3000 mg of the C5 antibody, or antigen-binding fragment thereof (e.g., ravulizumab (ULTOMIRIS®)) is administered to a patient weighing ≧40 to <60 kg.

[0215] In another embodiment, the concentrations of any of the following may be increased: 10 mg, 20 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 9 75mg, 1000mg, 1100mg, 1200mg, 1300mg, 1400mg, 1500mg, 1600mg, 1700mg, 1800mg, 1900mg, 2000mg, 2100mg, 2200mg, 2300mg, 2400mg, 2500mg, 2600mg , 2700mg, 2800mg, 2900mg, 3000mg, 3100mg, 3200mg, 3300mg, 3400mg, 3500mg, 3600mg, 3700mg, 3800mg, 3900mg, 4000mg, 4100mg, 4200mg, 4300mg, 4400 mg, 4500mg, 4600mg, 4700mg, 4800mg, 4900mg, 5000mg, 5100mg, 5200mg, 5300mg, 5400mg, 5500mg, 5600mg, 5700mg, 5800mg, 5900mg, 6000mg, 6100mg, 6 200mg, 6300mg, 6400mg, 6500mg, 6600mg, 6700mg, 6800mg, 6900mg, 7000mg, 7100mg, 7200mg, 7300mg, 7400mg, 7500mg, 7600mg, 7700mg, 7800mg, 7900mg , 8000mg, 8100mg, 8200mg, 8300mg, 8400mg, 8500mg, 8600mg, 8700mg, 8800mg, 8900mg, 9000mg, 9100mg, 9200mg, 9300mg, 9400mg, 9500mg, 9600mg, 9700mg, 9800mg, 9900mg, 10000mg, 10100mg, 10200mg, 10300mg, 10400mg, 10500mg, 10600mg, 10700mg, 10800mg, 10900mg or 11000mg of an anti-C5 antibody or an antigen-binding fragment thereof,In another embodiment, 2700 mg or 3300 mg of an anti-C5 antibody, or antigen-binding fragment thereof (e.g., ravulizumab (ULTOMIRIS®)) is administered to a patient weighing ≥60 to <100 kg.

[0216] In another embodiment, the concentrations of any of the following may be increased: 10 mg, 20 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 9 75mg, 1000mg, 1100mg, 1200mg, 1300mg, 1400mg, 1500mg, 1600mg, 1700mg, 1800mg, 1900mg, 2000mg, 2100mg, 2200mg, 2300mg, 2400mg, 2500mg, 2600mg, 2700mg, 2800mg, 2900mg, 3000mg, 3100mg, 3200mg, 3300mg, 3400mg, 3500mg, 3600mg, 3700mg, 3800mg, 3900mg, 4000mg, 4100mg, 4200mg, 4300mg, 4400m g, 4500mg, 4600mg, 4700mg, 4800mg, 4900mg, 5000mg, 5100mg, 5200mg, 5300mg, 5400mg, 5500mg, 5600mg, 5700mg, 5800mg, 5900mg, 6000mg, 6100mg, 62 00mg, 6300mg, 6400mg, 6500mg, 6600mg, 6700mg, 6800mg, 6900mg, 7000mg, 7100mg, 7200mg, 7300mg, 7400mg, 7500mg, 7600mg, 7700mg, 7800mg, 7900mg, 8000mg, 8100mg, 8200mg, 8300mg, 8400mg, 8500mg, 8600mg, 8700mg, 8800mg, 8900mg, 9000mg, 9100mg, 9200mg, 9300mg, 9400mg, 9500mg, 9600mg, 9700mg, 9800mg, 9900mg, 10000mg, 10100mg, 10200mg, 10300mg, 10400mg, 10500mg, 10600mg, 10700mg, 10800mg, 10900mg, or 11000mg of an anti-C5 antibody or an antigen-binding fragment thereof,In another embodiment, 3000 mg or 3600 mg of an anti-C5 antibody, or antigen-binding fragment thereof (e.g., ravulizumab (ULTOMIRIS®)) is administered to a patient weighing ≥ 100 kg.

[0217] In another embodiment, a method of treating a human patient with CM-TMA includes administering to the patient an anti-C5 antibody, or antigen-binding fragment thereof, such as ravulizumab (ULTOMIRIS®). (a) Once on day 1, at a dose of 1200 mg for patients weighing ≥ 30 to < 40 kg, 2400 mg for patients weighing ≥ 40 to < 60 kg, 2700 mg for patients weighing ≥ 60 to < 100 kg, or 3000 mg for patients weighing ≥ 100 kg; (b) On day 15 and every 8 weeks thereafter, at a dose of 2700 mg for patients weighing ≥ 30 to < 40 kg, 3000 mg for patients weighing ≥ 40 to < 60 kg, 3300 mg for patients weighing ≥ 60 to < 100 kg, or 3600 mg for patients weighing ≥ 100 kg. A method is provided comprising administering

[0218] In another embodiment, a method of treating a human patient with CM-TMA includes administering an anti-C5 antibody, or antigen-binding fragment thereof (e.g., ravulizumab (ULTOMIRIS®)) to a patient weighing ≥30 to <40 kg. (a) at a dose of 1200 mg once on day 1; (b) at a dose of 2700 mg on day 15 and every 8 weeks thereafter Methods of administration are provided.

[0219] In another embodiment, a method of treating a human patient with CM-TMA includes administering an anti-C5 antibody, or antigen-binding fragment thereof (e.g., ravulizumab (ULTOMIRIS®)) to a patient weighing ≧40 to <60 kg. (a) at a dose of 2400 mg once on day 1; (b) at a dose of 3000 mg on day 15 and every 8 weeks thereafter Methods of administration are provided.

[0220] In another embodiment, a method of treating a human patient with CM-TMA includes administering an anti-C5 antibody, or antigen-binding fragment thereof (e.g., ravulizumab (ULTOMIRIS®)) to a patient weighing ≥60 to <100 kg. (a) at a dose of 2700 mg once on day 1; (b) at a dose of 3,300 mg on day 15 of the treatment cycle and every 8 weeks thereafter Methods of administration are provided.

[0221] In another embodiment, a method of treating a human patient with CM-TMA includes administering an anti-C5 antibody, or antigen-binding fragment thereof (e.g., ravulizumab (ULTOMIRIS®)) to a patient weighing ≧100 kg. (a) at a dose of 3000 mg once on day 1; (b) at a dose of 3600 mg on day 15 and every 8 weeks thereafter Methods of administration are provided.

[0222] In another embodiment, a method of treating a human patient with CM-TMA comprises administering to the patient an effective amount of an anti-C5 antibody, or antigen-binding fragment thereof, wherein the anti-C5 antibody, or antigen-binding fragment thereof, comprises CDR1, CDR2, and CDR3 heavy chain sequences set forth in SEQ ID NOs: 19, 18, and 3, respectively, and CDR1, CDR2, and CDR3 light chain sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, and a variant human Fc region that binds to the human neonatal Fc receptor (FcRn), wherein the variant human Fc CH3 region comprises Met-429-Leu and Asn-435-Ser substitutions at residues corresponding to methionine 428 and asparagine 434, respectively, in EU numbering, of native human IgG Fc region; and administering to the patient an effective amount of an anti-C5 antibody, or antigen-binding fragment thereof, wherein the anti-C5 antibody, or antigen-binding fragment thereof, comprises CDR1, CDR2, and CDR3 heavy chain sequences set forth in SEQ ID NOs: 19, 18, and 3, respectively, and CDR1, CDR2, and CDR3 light chain sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, and a variant human Fc region that binds to the human neonatal Fc receptor (FcRn), wherein the variant human Fc CH3 region comprises Met-429-Leu and Asn-435-Ser substitutions at residues corresponding to methionine 428 and asparagine 434, respectively, in EU numbering, of native human IgG Fc region. (a) Once on day 1, at a dose of 1200 mg for patients weighing ≥ 30 to < 40 kg, 2400 mg for patients weighing ≥ 40 to < 60 kg, 2700 mg for patients weighing ≥ 60 to < 100 kg, or 3000 mg for patients weighing ≥ 100 kg; (b) On day 15 and every 8 weeks thereafter, at a dose of 2700 mg for patients weighing ≥ 30 to < 40 kg, 3000 mg for patients weighing ≥ 40 to < 60 kg, 3300 mg for patients weighing ≥ 60 to < 100 kg, or 3600 mg for patients weighing ≥ 100 kg. Methods of administration are provided.

[0223] In another embodiment, the anti-C5 antibody, or antigen-binding fragment, is administered in a milligram per kilogram (mg / kg) dose. For example, in one embodiment, the anti-C5 antibody, or antigen-binding fragment, is administered in a dose of 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 1.0 mg / kg, 1.25 mg / kg, 1.50 mg / kg, 1.75 mg / kg, 2.0 mg / kg, 2.25 mg / kg, 2.50 mg / kg, 2.75 mg / kg, 3.0 mg / kg, 3.25 mg / kg, 3.50 mg / kg, 3.75 mg / kg, 4.0 mg / kg, 4.25 mg / kg, 4.50 mg / kg, 4.75 mg / kg, 5.0 mg / kg, 5.25mg / kg, 5.50mg / kg, 5.75mg / kg, 6.0mg / kg, 6.25mg / kg, 6.50mg / kg, 6.75mg / kg, 7.0mg / kg, 7.25mg / kg, 7.50mg / kg, 7.75mg / kg, 8.0mg / kg, 8.25mg / kg, 8.50mg / kg, 8.75mg / kg, 9.0mg / kg, 9.25mg / kg, 9.50mg / kg, 9.75mg / kg, 10.0mg / kg, 11.25mg / kg, 11.50mg / kg, 11.75mg / kg, 12.0mg / kg, 12.25mg / kg, 12.50mg / kg, 12.75mg / kg, 13.0mg / kg, 13.25mg / kg, 13.50mg / kg, 13.75mg / kg, 14.0mg / kg, 14.25mg / kg, 14.50mg / kg, 14.75mg / kg, 15.0mg / kg, 15.25mg / kg, 15.50mg / kg, 15.75mg / kg, 16.0mg / kg, 16.25mg / kg, 16.50mg / kg, 16.75mg / kg, 17.0mg / kg, 17.25mg / kg, 17.50 mg / kg, 17.75mg / kg, 18.0mg / kg, 18.25mg / kg, 18.50mg / kg, 18.75mg / kg, 19.0mg / kg, 19.25mg / kg, 19.50mg / kg, 19.75mg / kg, 20.0mg / kg, 20.2 5mg / kg, 20.50mg / kg, 20.75mg / kg, 21.0mg / kg, 21.25mg / kg, 21.50mg / kg, 21.75mg / kg, 22.0mg / kg, 22.25mg / kg, 22.50mg / kg, 22.75mg / kg, 23.The doses are 0 mg / kg, 23.25 mg / kg, 23.50 mg / kg, 23.75 mg / kg, 24.0 mg / kg, 24.25 mg / kg, 24.50 mg / kg, 24.75 mg / kg or 25.0 mg / kg.

[0224] In one embodiment, the anti-C5 antibody or antigen-binding fragment is administered once a week, twice a week, three times a week, four times a week, five times a week, six times a week, or daily. In another embodiment, the anti-C5 antibody or antigen-binding fragment is administered once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every eleven weeks, or once every twelve weeks. In another embodiment, the anti-C5 antibody or antigen-binding fragment is administered at a loading dose on day 1, followed by a different maintenance dose on day 15 and every eight weeks thereafter.

[0225] In another embodiment, the anti-C5 antibody, or antigen-binding fragment thereof, is administered in one or more dosing cycles. In one embodiment, the dosing cycle is 26 weeks. In another embodiment, the treatment includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 cycles. In another embodiment, the patient is treated for about 1, 2, 3, 4, 5, or 6 months. In another embodiment, the treatment continues for the life of the human patient.

[0226] The anti-C5 antibody or antigen-binding fragment may be administered via any suitable means. In one embodiment, the anti-C5 antibody or antigen-binding fragment (e.g., ravulizumab (ULTOMIRIS®)) is administered intravenously. In another embodiment, the anti-C5 antibody or antigen-binding fragment is administered subcutaneously.

[0227] Patients treated according to the methods described herein may have been vaccinated against meningococcal infection within three years prior to or at the time of initiating treatment. Patients treated less than two weeks after receiving a meningococcal vaccine may also be treated with appropriate prophylactic antibiotics up to two weeks after vaccination. Patients treated according to the methods described herein may be vaccinated against meningococcal serotypes A, C, Y, W135 and / or B.

[0228] Patients treated according to the methods described herein have lupus nephritis, systemic sclerosis, or TMA associated with solid organ transplantation. These patients may be vaccinated against Haemophilus influenzae type b (Hib) and Streptococcus pneumoniae prior to treatment.

[0229] The treatment regimens described herein are sufficient to maintain a particular serum trough concentration of anti-C5 antibodies, or antigen-binding fragments thereof. Treatment may be, for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200, 205, 210, 215, 220, 225, 230, 240, 245, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 555, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 655, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 750, 760, 770, 780, 790, 80 The serum trough concentration of anti-C5 antibody, or antigen-binding fragment thereof, may be maintained at 50, 255, 260, 265, 270, 280, 290, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, or 400 μg / mL or more. In one embodiment, the treatment maintains a serum trough concentration of anti-C5 antibody, or antigen-binding fragment thereof, at 100 μg / mL or more. In another embodiment, the treatment maintains a serum trough concentration of anti-C5 antibody, or antigen-binding fragment thereof, at 150 μg / mL or more. In another embodiment, the treatment maintains a serum trough concentration of anti-C5 antibody, or antigen-binding fragment thereof, at 200 μg / mL or more. In another embodiment, the treatment maintains a serum trough concentration of anti-C5 antibody, or antigen-binding fragment thereof, of 250 μg / mL or more. In another embodiment, the treatment maintains a serum trough concentration of anti-C5 antibody, or antigen-binding fragment thereof, of 300 μg / mL or more. In another embodiment, the treatment maintains a serum trough concentration of anti-C5 antibody, or antigen-binding fragment thereof, of 100 μg / mL to 200 μg / mL. In another embodiment, the treatment maintains a serum trough concentration of anti-C5 antibody, or antigen-binding fragment thereof, of about 175 μg / mL.

[0230] To achieve an effective response, anti-C5 antibodies should be administered at least 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 105 μg, 110 μg, 115 μg, 120 μg, 125 μg, 130 μg, 135 μg, 140 μg, 145 μg, 150 μg, 155 μg, 160 μg, 170 μg, 175 μg, 180 μg, 185 μg, 190 μg, 200 μg, 210 μg, 220 μg, 230 μg, 240 μg, 250 μg, 260 μg, 270 μg, 280 μg, 290 μg, 300 μg, 310 μg, 320 μg, 330 μg, 340 μg, 350 μg, 360 μg, 370 μg, 380 μg, 390 μg, 400 μg, 410 μg, 420 μg, 430 μg, 440 μg, 450 μg, 460 μg, 470 μg, 480 μg, 490 μg, 500 μg, 510 μg, 520 μg, 530 μg, 540 μg, 550 μg, 550 μg, 560 μg, 570 μg, 580 μg, 590 μg, 600 μg, 610 μg, 620 μg, The anti-C5 antibody may be administered to the patient in an amount and frequency to maintain the patient at 100-200 μg, 160 μg, 165 μg, 170 μg, 175 μg, 180 μg, 185 μg, 190 μg, 195 μg, 200 μg, 205 μg, 210 μg, 215 μg, 220 μg, 225 μg, 230 μg, 235 μg, 240 μg, 245 μg, 250 μg, 255 μg, or 260 μg of antibody per milliliter of blood. In another embodiment, the anti-C5 antibody is administered to the patient in an amount and frequency to maintain the patient at 50 μg to 250 μg of antibody per milliliter of blood. In another embodiment, the anti-C5 antibody is administered to the patient in an amount and frequency to maintain the patient at 100-200 μg of antibody per milliliter of blood. In another embodiment, anti-C5 antibodies are administered to the patient in an amount and frequency to maintain the patient at about 175 μg of antibody per milliliter of blood.

[0231] In another embodiment, the anti-C5 antibody is administered to the patient in an amount and frequency to maintain a minimum free C5 concentration to achieve an effective response. For example, the anti-C5 antibody can be administered to the patient in an amount and frequency to maintain a free C5 concentration of 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL or less. In another embodiment, the treatment described herein reduces free C5 concentrations by more than 99% over the entire treatment period.

[0232] "Chronically administered," "chronic treatment," "chronically treating," or similar grammatical variations thereof, broadly refer to a treatment regimen used to maintain a particular threshold concentration of a therapeutic agent in the blood of a patient and to completely or substantially suppress systemic complement activity in the patient over an extended period of time. Thus, a subject chronically treated with a complement inhibitor is administered a complement inhibitor in an amount and at a frequency of administration sufficient to maintain a concentration of the inhibitor in the patient's blood that inhibits or substantially inhibits systemic complement activity in the patient for a period of 2 weeks or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 or 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 12 years or for the rest of the patient's life). The complement inhibitor can be administered chronically to a patient in need thereof in an amount and frequency effective to maintain serum hemolytic activity at or below 20 (e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5)%. See, e.g., Hill et al. (2005) Blood 106(7):2559. Complement inhibitors can be administered to a patient in an amount and frequency effective to maintain serum lactate dehydrogenase (LDH) levels within at least 20 (e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5)% of the normal range for LDH. See Hill et al. (2005) supra. Complement inhibitors can be administered to a patient in an amount and frequency effective to maintain serum LDH levels below 550 IU / L (e.g., less than 540, 530, 520, 510, 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, or less than 270).To maintain systemic complement inhibition in a patient, a complement inhibitor can be administered chronically to the patient, for example, once weekly, once every two weeks, twice weekly, once daily, once monthly, or once every three weeks.

[0233] In some embodiments, the treatment includes administration of ravulizumab under its dosing schedule. In some embodiments, ravulizumab is administered as an intravenous (iv) formulation including a single loading dose on day 1, followed by regular maintenance doses starting on day 15, based on the subject's body weight, where (a) for subjects weighing ≧40 to <60 kilograms (kg), the treatment includes a loading dose of 2400 milligrams (mg), followed by a maintenance dose of 3000 mg every 8 weeks; (b) for subjects weighing ≧60 to <100 kg, the treatment includes a loading dose of 2700 mg, followed by a maintenance dose of 3300 mg every 8 weeks; and (c) for subjects weighing ≧100 kg, the treatment includes a loading dose of 3000 mg, followed by a maintenance dose of 3600 mg every 8 weeks. In some embodiments, ravulizumab is administered as a subcutaneous (SC) formulation.

[0234] composition The composition may include a therapeutically effective amount of an inhibitor of human complement component C5 (e.g., an anti-C5 antibody or an antigen-binding fragment thereof). Two preferred anti-C5 antibodies include, but are not limited to, eculizumab (SOLIRIS®) and ravulizumab (ULTOMIRIS®). A preferred anti-C5 antigen-binding fragment is pexelizumab.

[0235] The composition may be a pharmaceutical composition further comprising an excipient, carrier, diluent, stabilizer, buffer, antioxidant, or a combination thereof. The pharmaceutical composition may comprise a therapeutically effective amount of an inhibitor of human complement component C5 (e.g., an anti-C5 antibody or an antigen-binding fragment thereof). Two preferred anti-C5 antibodies include, but are not limited to, eculizumab (SOLIRIS®) and ravulizumab (ULTOMIRIS®). A preferred anti-C5 antigen-binding fragment is pexelizumab.

[0236] The effective amount can be easily determined by those skilled in the art based in part on the effect of the inhibitor administered, or on the combinatorial effect of the antibody and one or more additional active agents when two or more agents are used. The therapeutically effective amount of an inhibitor of human complement component C5 (e.g., anti-C5 antibody) [e.g., eculizumab, ravulizumab] can also vary depending on factors such as the individual's condition, age, sex, and weight, and the ability of the antibody (and one or more additional active agents) to induce a desired response in the individual, such as the recovery of at least one condition parameter, e.g., the remission of at least one symptom of CM-TMA. For example, a therapeutically effective amount of an inhibitor of human complement component C5 (e.g., anti-C5 antibody) can inhibit (reduce the severity or eliminate the occurrence of) and / or prevent thrombocytopenia, microangiopathic hemolytic anemia, renal failure, and / or any one of the symptoms of CM-TMA known in the art or described herein. Two preferred anti-C5 antibodies include, but are not limited to, eculizumab (SOLIRIS®) and ravulizumab (ULTOMIRIS®). Also, at a therapeutically effective amount, any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.

[0237] The compositions described herein may include a therapeutically effective amount of an inhibitor of human complement component C5. Two preferred anti-C5 antibodies include, but are not limited to, eculizumab (SOLIRIS®) and ravulizumab (ULTOMIRIS®). The compositions described herein may include a therapeutically effective amount of an antibody that binds to complement component C5 protein, or an antigen-binding fragment thereof. In some embodiments, the compositions may include two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more) different inhibitors of human complement component C5, such that the composition as a whole is therapeutically effective. For example, the composition may include an antibody that binds to human C5 protein and an siRNA that binds to and promotes the degradation of mRNA encoding human C5 protein, where each of the antibody and siRNA is at a concentration that is therapeutically effective when combined. In some embodiments, the compositions may include an inhibitor and one or more second active agents, such that the composition as a whole is therapeutically effective. For example, a composition can include an antibody that binds to human C5 protein and another agent useful for treating or preventing CM-TMA.

[0238] The toxicity and therapeutic efficacy of such compositions can be determined by known pharmaceutical procedures in cell cultures or experimental animals (animal models of aHUS). These procedures can be used, for example, in the LD 50 (lethal dose for 50% of the population) and ED 50 It can be used to determine the therapeutically effective dose in 50% of the population. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the LD 50 / ED 50 It can be expressed as a ratio. Compositions or inhibitors of compositions (e.g., anti-C5 antibodies) [e.g., eculizumab, ravulizumab] that exhibit high therapeutic indices are preferred. While compositions that exhibit toxic side effects can be used, care must be taken to design delivery systems that target such compounds to the site of diseased tissue, minimizing potential damage to normal cells and thereby reducing side effects.

[0239] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. Suitable animal models of aHUS are known in the art and are described, for example, in Atkinson et al. (2007) Journal of Experimental Medicine 204(6):1245-1248. Doses of such inhibitors are generally administered at a dose that is sufficient to induce ED with little or no toxicity. 50 The range of circulating concentrations of the inhibitor (e.g., anti-C5 antibody or antigen-binding fragment thereof) includes: 1) a concentration of circulating inhibitor (e.g., anti-C5 antibody or antigen-binding fragment thereof) that is in the range of circulating concentrations of the inhibitor (e.g., anti-C5 antibody or antigen-binding fragment thereof) that ... 50 A circulating plasma concentration range can be achieved that includes (e.g., the concentration of the test compound that achieves half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0240] The required dose of the inhibitor of human complement component C5 can be determined based on the concentration of human C5 protein in the blood of the subject.For example, a subject with a higher concentration of circulating human C5 protein level may require a higher dose of human C5 inhibitor than a subject with a lower level of circulating human C5.Methods for determining the concentration of human complement component C5 in blood-derived body fluid samples from a subject are known in the art and are described, for example, in Rawal et al.(1998) J Biol Chem 273(27):16828-16835.

[0241] The method can be performed in conjunction with other therapies for CM-TMA. For example, the composition can be administered to a subject simultaneously with, before, or after nephrectomy (e.g., bilateral nephrectomy), dialysis, plasma exchange, or plasma infusion (see, e.g., Noris et al. (2005) "Non-shiga toxin-associated hemolytic uremic syndrome." In: Zipfel P (ed). Complement and Kidney Disease. Basel: Birkhauser-Verlag, 65-83).

[0242] The inhibitor of human complement component C5 (e.g., an anti-C5 antibody or an antigen-binding fragment thereof) [e.g., eculizumab, ravulizumab] can be administered to a subject as a monotherapy. Alternatively, as described above, the inhibitor can be administered to a subject as a combination therapy with another treatment, e.g., another treatment for CM-TMA. For example, the combination therapy can include administering to a subject (e.g., a human patient) one or more additional agents (e.g., antihypertensive agents) that provide a therapeutic benefit to a subject having or at risk of developing CM-TMA. The inhibitor of human complement component C5 and one or more additional active agents can be administered simultaneously. The inhibitor may be administered first in a timely manner, and one or more additional active agents are administered second in a timely manner. In some embodiments, one or more additional active agents are administered first in a timely manner, and the inhibitor is administered second in a timely manner.

[0243] An inhibitor of human complement component C5 may replace or augment a previously or currently administered therapeutic agent. For example, during treatment with an anti-C5 antibody or antigen-binding fragment thereof, administration of one or more additional active agents may be stopped or reduced, e.g., administered at a lower level. Administration of the previous therapeutic agent may be maintained. The previous therapeutic agent may be maintained until the level of the inhibitor of human C5 reaches a sufficient level to provide a therapeutic effect. The two therapeutic agents may be administered in combination.

[0244] As defined herein, monitoring a subject (e.g., a human patient) for improvement in a CM-TMA means evaluating the subject for changes in disease parameters, e.g., improvement in one or more symptoms of the disease. Such symptoms include any of the symptoms of the CM-TMA described herein. In some embodiments, the evaluation is performed at least 1 hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1, 2, 4, 10, 13, 20 days, or at least 1, 2, 4, 10, 13, 20 weeks, or more, after the start of treatment. The subject can be evaluated at one or more of the following time periods: before the start of treatment; during treatment; or after one or more elements of treatment are administered. The evaluation can include evaluating the need for further treatment, e.g., evaluating whether the dose, frequency of administration, or duration of treatment should be changed. It can also include evaluating the need to add or remove a selected treatment modality, e.g., adding or removing any of the treatments for the CM-TMA described herein.

[0245] Concentration of proteolytic fragments of factor B (Ba) The concentration of a proteolytic fragment of factor B can be measured. The fragment can be Ba. The biological sample can be a blood, serum, plasma, and / or urine sample. The biological sample can be plasma and urine.

[0246] The concentration of Ba in a biological sample is considered to be increased if it is at least 2-fold higher than the normal control concentration of Ba. The concentration of Ba in a biological sample is considered to be increased if it is at least 5-fold higher than the normal control concentration of Ba. The concentration of Ba in a biological sample is considered to be increased if it is greater than about 1,000ng / mL. The concentration of Ba in a biological sample is considered to be increased if it is greater than about 1,500ng / mL. The concentration of Ba in a biological sample is considered to be increased if it is greater than about 2,500ng / mL.

[0247] A decrease of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% in the Ba concentration after treatment (e.g., plasma Ba concentration) compared to the Ba concentration in the same type of sample of bodily fluid obtained from the subject before treatment indicates that the subject is likely to have or achieve a complete thrombotic microangiopathy (TMA) response (e.g., cessation of a TMA event). The decrease may occur by 12 weeks after initial treatment with a complement inhibitor. The decrease may occur within 12-17 weeks after initial treatment with a complement inhibitor. The decrease may occur by 26 weeks after initial treatment with a complement inhibitor.

[0248] Concentration of terminal complement complex (sC5b-9) The concentration of sC5b-9 in a sample can be considered to be increased if it is at least 10 times higher than the normal control concentration of sC5b-9. The concentration of sC5b-9 in a biological sample is considered to be increased if it is at least 50 times higher than the normal control concentration of sC5b-9. The concentration of sC5b-9 in a biological sample is considered to be increased if it is at least 100 times higher than the normal control concentration of sC5b-9. The concentration of sC5b-9 in a biological sample is considered to be increased if it is at least 20 ng per mg of urinary creatinine. The concentration of sC5b-9 in a biological sample is considered to be increased if it is at least 30 ng per mg of urinary creatinine. The control concentration can be determined from a biological sample from a healthy subject, for example a subject without CM-TMA, preferably a subject whose urine sample does not contain detectable levels of sC5b9 as measured via a standard immunoassay.

[0249] Creatinine levels In some embodiments of the present disclosure, the creatinine level is measured in the subject's biological sample. Any routine method can be used to measure the creatinine level, including but not limited to colorimetry (e.g., Jaffe method), enzymatic method, chemiluminescence assay, chromatographic technique, molecular imprinted polymer (MIP), capillary electrophoresis, spectrophotometry, potentiometric sensor, electrochemical sensor (ECA), pH meter, and amperometric sensor. These techniques can be performed on blood samples, urine specimens, and even saliva samples with little or no sample processing (Wei et al., Anal Chem, 2012 Sep 18;84(18):7933-7). In some embodiments, the above assay can be improved by using nanoparticles (NPs) that bind to creatinine.

[0250] Estimated glomerular filtration rate (eGFR) In adults, normal GFR values ​​are greater than about 90. This may decrease with age, for example, a person over 70 years old may have a GFR greater than about 75, even in the absence of disease. A GFR value below 60 at any age is a sign of renal failure and / or disease. The CKD-EPI creatinine equation (2009) can be used to estimate GFR.

[0251] It can be expressed as a single equation: eGFR= 141×Minimum(S Cr / κ,1) α × Maximum(S Cr / κ,1) -1.209 × 0.993 Age × 1.018[For women] x 1.159 [If you are of African descent] eGFR (estimated glomerular filtration rate) = mL / min / 1.73m 2 S Cr (standardized serum creatinine)=mg / dL Kappa = 0.7 (female) or 0.9 (male) α = -0.329 (female) or -0.411 (male) min=S Cr / kappa or 1 indicates the minimum max=S Cr / κ or 1 maximum age=age

[0252] The CKD-EPI creatinine equation (2009) is known in the art and is provided by the National Kidney Foundation. National Kidney Foundation website (2021); Levey et al. Ann Intern Med. (2009) 150(9):604-612.

[0253] The biomarker status described herein may predict improvement in estimated glomerular filtration rate (eGFR) for CM-TMA patients treated with complement inhibitors. For example, a decrease in Ba and / or sC5b-9 concentrations (e.g., within 26 weeks after initial treatment in a chronic treatment regimen) indicates that a CM-TMA patient treated with a complement inhibitor has achieved or is likely to achieve a clinically meaningful improvement in eGFR.

[0254] Urinary protein / creatinine (Cr) ratio (UPCR) A UPCR of less than about 0.2 is considered within the normal range. Huang et al. Pediatr Nephrol. (2020) 35(3): 463-468; Karkar & Abdelrahman Saudi J Kidney Dis Transpl (2010) 21: 949-50.

[0255] Associations between biomarkers and secondary markers eGFR and / or UPCR Embodiments of the present disclosure relate to the use of CM-TMA biomarkers associated with said secondary markers, e.g., eGFR and / or UPCR, in the diagnosis, management, and treatment of CM-TMA. As illustrated in the representative examples section, the level of association between the secondary markers of CM-TMA, e.g., eGFR and / or UPCR, and the primary protein biomarkers of the present disclosure can be determined using routine techniques, e.g., regression analysis.

[0256] kit Kits are also provided that contain various reagents and materials useful for carrying out the methods described herein. The measuring, diagnosing, evaluating, and / or assessing procedures described herein can be carried out by a diagnostic laboratory, a laboratory, or by an individual practitioner. The present invention provides kits that can be used in any and all of these settings.

[0257] The kits may include, inter alia, materials and reagents for characterizing or processing a biological sample (e.g., bodily fluid), measuring biomarker levels (e.g., protein or nucleic acid levels), diagnosing CM-TMA in a subject, or monitoring a therapeutic response in a subject according to the methods provided herein. The kits may include at least one or more reagents that specifically detect protein levels of one or more CM-TMA biomarker proteins (e.g., Ba, sC5b9, or both), and, optionally, instructions for using the kit. The kits may include, for example, any of the arrays described herein.

[0258] The kit may include a suitable control sample (e.g., a bodily fluid from a normal healthy individual or a solution containing a known control amount of a particular analyte of interest). The kit may include instructions for using the kit according to one or more of the methods described herein, and may include instructions for processing a biological sample (e.g., bodily fluid) obtained from a subject and / or instructions for performing tests or interpreting the results. EXAMPLES

[0259] Example 1 Detection of CM-TMA biomarkers in patients Using data from a phase III trial of ravulizumab (a terminal C5 complement inhibitor) in adults with aHUS (NCT02949128), baseline (BL; pre-treatment) serum, plasma, and urinary biomarker levels in patients were compared with those in healthy volunteers (HV) to assess associations with BL and renal function (e.g., estimated glomerular filtration rate [eGFR] and urinary protein / creatinine [Cr] ratio [UPCR]) at 26 weeks after initiating treatment. Regression coefficients and P values ​​(two-tailed t-tests) are reported.

[0260] Results: This analysis included 55 patients: median age 39 (range: 19-76) years; 67% female; 53% Caucasian, 27% Asian. Specific BL biomarkers were increased compared to HV, and associations between BL biomarker levels and both BL eGFR and BL UPCR were identified (Table 1). BL plasma complement factor Ba was associated with eGFR change after 26 weeks of ravulizumab treatment, while urinary sC5b-9, sC5b-9 / Cr, Ba, and Ba / Cr were associated with UPCR change after 26 weeks of treatment.

[0261] [Table 1]

[0262] Conclusions: Complement biomarkers Ba (plasma and urine) and sC5b-9 (urine) were associated with renal function in patients with aHUS at baseline and over 26 weeks of treatment with anti-C5 therapy. These biomarkers show diagnostic potential in CM-TMA and may predict renal response to terminal complement inhibition.

[0263] Example 2 Detection of CM-TMA biomarkers in patients aHUS is a form of complement-mediated thrombotic microangiopathy resulting from dysregulation of the alternative complement pathway in the presence or absence of identified triggers, typically resulting in renal injury / failure; damage to other organ systems is common. No validated biomarkers or assays for the diagnosis or prognosis of aHUS are currently available. Identification of clinically significant biomarkers, individually or in combination, in patients with aHUS could result in more rapid diagnosis, prediction of clinical course, and / or earlier initiation of treatment.

[0264] Using exploratory biomarker data from a Phase III trial of the complement C5 inhibitor ravulizumab in adults with aHUS who were naïve to complement inhibitor therapy, this analysis included the following: (a) comparing baseline biomarker levels in this population with levels in normal donors; (b) assess longitudinal changes from baseline in biomarker levels during treatment with ravulizumab; (c) examine the association between baseline biomarker levels and both baseline clinical measures and change in clinical measures after 26 weeks of treatment; and (d) Examine the association between baseline biomarker levels and clinical outcomes after 26 weeks of treatment.

[0265] [Table 2]

[0266] [Table 3]

[0267] [Table 4]

[0268] * The total analysis set, of which N = 55, formed the biomarker analysis set; #Baseline values ​​may be post-plasma exchange / infusion in some patients; † Safety set, N = 58; ‡ defined as normalization of both LDH and platelet count; § Number of patients tested, n = 39; ¶ One additional patient had CFH autoantibodies; however, this patient was not tested due to genetic nature. Overall, 3 of 52 patients tested for CFH autoantibodies were positive. CF, complement factors; CI, confidence interval; eGFR, estimated glomerular filtration rate; LDH, lactate dehydrogenase; TMA, thrombotic microangiopathy

[0269] [Table 5]

[0270] * A nominal total of 55 adults with aHUS, 10 normal urine donors and 20 normal serum / plasma donors were evaluated in this study; # n refers to the number of patients / donors with evaluable data. † sC5b-9 was undetectable in urine samples from normal donors, therefore, for this analysis, the normal donor value was set at ½LLOQ for urinary sC5b-9. For all bioanalytical assays, both commercial ELISAs and custom MSDs were validated against the 2018 FDA Guidance for Biomarker Methods. Sample testing and data reporting were performed in regulated quality control laboratories. Outline boxes show results for complement-specific biomarkers in plasma and urine normalized to creatinine. aHUS, atypical hemolytic uremic syndrome; Cr, creatinine; ELISA, enzyme-linked immunosorbent assay; FDA, United States Food and Drug Administration; LLOQ, lower limit of quantification; MSD, mesoscale discovery

[0271] In 63-100% of adults with aHUS, all biomarkers, with the exception of serum sVCAM-1, were increased compared to maximum observed levels in normal donors. Complement-specific biomarkers Ba and sC5b-9 were increased in >75% of patients, and plasma and urinary Ba were increased in ≥97%. This is the set of biomarkers tested in urine, plasma, and serum.

[0272] The inventors also evaluated biomarker levels normalized to creatinine in urine samples. The outlined highlighting results for complement factor Ba and protein sC5b-9 are specifically related to complement activity (alternative and terminal pathways, respectively). These results support the use of both these biomarkers in the diagnosis of both aHUS and other complement-mediated pathologies.

[0273] FIG. 1 depicts biomarkers of complement dysregulation and renal injury reduction with ravulizumab treatment. Urinary Ba / Cr and sC5b-9 / Cr levels were significantly decreased after the first dose of ravulizumab, indicating an early pharmacodynamic effect of C5 inhibition. Plasma Ba levels were significantly decreased compared to baseline levels from day 71, indicating that sustained terminal inhibition may reduce alternative complement pathway activity. Urinary cystatin C / Cr levels were significantly decreased after the first dose of ravulizumab, suggesting renal recovery. The inventors evaluated the normalized data, which best showed changes over time. These results indicate that complement-specific biomarkers are robustly decreased in response to anti-C5 therapy, which supports the use of therapeutic response monitoring.

[0274] Figure 2 depicts the associations between baseline biomarkers and baseline clinical measures. Baseline levels of plasma Ba and urinary Ba / Cr and sC5b-9 / Cr, and urinary cystatin C / Cr were significantly associated with lower baseline eGFR and increased baseline proteinuria. Significant associations were also observed between non-complement specific biomarkers and the same clinical measures. We highlight the results for Ba and sC5b-9 because these complement specific biomarkers were significantly associated with baseline renal function measures, specifically eGFR and proteinuria.

[0275] Figure 3 shows the association between baseline biomarkers and changes in clinical measures over 26 weeks of treatment. After 26 weeks of treatment with ravulizumab, (a) baseline urinary sC5b-9 / Cr was significantly associated with increased platelet count; (b) baseline urinary Ba / Cr and sC5b-9 / Cr were significantly inversely correlated with changes in proteinuria levels; and (c) baseline plasma Ba was significantly inversely correlated with changes in eGFR. Some significant associations were also observed for non-complement specific biomarkers.

[0276] The highlighted results show that baseline plasma Ba, and especially urinary Ba and sC5b-9 (normalized results), were associated with renal function during C5 inhibitor treatment, indicating that they can be used in predicting treatment outcome.

[0277] Figure 4 presents data showing that baseline biomarker levels were significantly associated with selected clinical outcomes at week 26. Odds ratios were derived from logistic regression analysis with the response variable as the dependent variable and the logarithm of baseline biomarker levels as the independent variable, and represent the increase (or decrease) in odds of achieving an efficacy response for every two-fold increase in baseline biomarker. At week 26, (1) lower baseline levels of serum sVCAM-1 and sTNF-R1 were significantly associated with a higher likelihood of achieving a complete TMA response; (2) lower baseline levels of plasma thrombomodulin were significantly associated with a higher likelihood of achieving a complete TMA response; and (3) lower baseline levels of urinary sC5b-9 approached a significant association with a higher likelihood of achieving a complete TMA response. The inventors evaluated complete TMA response: a combined measure of normalization of platelet count and LDH, along with improvement in serum creatinine. Among complement-specific biomarkers, urinary sC5b-9 had the most significant association with complete TMA response after C5 inhibitor treatment. Among non-complement-specific biomarkers, those related to renal function had the most significant association.

[0278] From the evaluation of seven blood and / or urinary biomarkers for clinical diagnostic, prognostic and predictive utility in treatment-naive adults with newly diagnosed aHUS, the inventors made the following observations: (1) Plasma complement biomarkers (Ba and sC5b-9) and urinary complement biomarker (Ba) were increased in patients with aHUS compared with normal donors; (2) baseline levels of plasma Ba, urinary Ba / Cr and sC5b-9 / Cr, and baseline levels of urinary cystatin C / Cr were significantly associated with baseline renal function (decreased eGFR and increased proteinuria); (3) complement biomarker levels (urinary Ba / Cr and sC5b-9 / Cr) were significantly decreased by C5 inhibition, indicating an early pharmacodynamic effect of C5 inhibition on complement activity; and (4) Baseline urinary sC5b-9 levels approached significance with complete TMA response at week 26.

[0279] In particular, measurement of the urinary complement biomarkers Ba and sC5b-9 has utility in the diagnosis of CM-TMA / aHUS, as well as predictive utility in monitoring response to complement inhibitor therapy, e.g., treatment with anti-C5 antibodies such as eculizumab (SOLIRIS®) and / or ravulizumab (ULTOMIRIS®). This may be due to their association with renal function at baseline and week 26. For example, urinary complement-specific biomarkers may be particularly useful in monitoring renal function.

[0280] This data suggests that measurement of plasma and urinary biomarker proteins may provide diagnostic, prognostic and / or predictive signatures for complement-mediated kidney diseases such as CM-TMA and response to complement inhibitor therapy.

[0281] Example 3 Exploratory diagnostic and prognostic biomarkers of complement-mediated thrombotic microangiopathy (CM-TMA) in adults with atypical hemolytic uraemic syndrome (aHUS): Analysis of a phase 3 trial of ravulizumab introduction TMA is a broad clinical term that encompasses the triad of thrombocytopenia, microangiopathic hemolytic anemia, and microvascular thrombosis that typically results in organ damage / failure. CM-TMA is a subset of TMA disorders driven by the generation of complement activation in the presence or absence of complement gene mutations and acquired autoantibodies. aHUS, often considered the prototype form of CM-TMA, is due to overactivation of the alternative pathway of complement, which leads to overactivation of terminal complement. The complement cascade involves the generation of degradation products.

[0282] Despite information about the pathophysiology of CM-TMA / aHUS, several clinical challenges exist regarding its differential diagnosis and prognosis. One such challenge is the lack of sensitive, specific, and clinically validated biomarkers that can define this heterogeneous disease population. Thus, continued exploration of novel matrices, sample collection protocols, and bioanalytical assays is necessary to identify and accurately measure clinically significant biomarkers individually and / or in combination in patients with aHUS. Identification of biomarker "signatures" in patients with aHUS may help inform diagnosis, treatment decisions, and treatment monitoring in patients with other forms of CM-TMA.

[0283] In a study from Cofiell et al., the pharmacodynamic effects of complement inhibition were explored via treatment with the anti-complement C5 monoclonal antibody (mAb) eculizumab on markers of multiple cellular processes in patients with aHUS, including complement activation, inflammation, endothelial injury, thrombosis, and renal injury. Cofiell et al. Eculizumab reduces complement activation, inflammation, endothelial injury, thrombosis, and renal injury markers in aHUS. Blood (2015) 125 (21): 3253-62.

[0284] The study found that after treatment with eculizumab, most of the blood and urinary markers of these processes were restored to normal or subnormal levels when compared to normal donor samples. Id. Conversely, markers of the proximal alternative pathway and endothelial activation remained partially elevated after treatment, suggesting ongoing dysregulation in pathways upstream of eculizumab's mechanism of action. Id. Nevertheless, biomarker utility was not the focus of the study from Cofiell et al., meaning that a more specific evaluation of the utility of the analyzed proteins as specific biomarkers for aHUS / CM-TMA is required.

[0285] method This analysis used data from a Phase 3 clinical trial of ULTOMIRIS in adults with aHUS to explore the potential diagnostic and prognostic utility of blood and urinary biomarkers of complement activation, inflammation, and kidney injury in adults with aHUS. The inventors evaluated longitudinal changes from baseline in biomarker levels after 26 and 52 weeks of ULTOMIRIS treatment. In a post-hoc analysis, the inventors retrospectively evaluated data from the initial evaluation (26 weeks) and extension period (52 weeks) of the Phase 3 trial of ULTOMIRIS in adults with aHUS.

[0286] Biomarkers of interest were plasma complement factors Ba, sC5b-9, thrombomodulin, and D-dimer; serum sTNF-RI and sVCAM-1; urinary Ba factor, sC5b-9, and cystatin C. Spot urine collections were performed under search conditions with random time and voiding volume. To control for these variations in urine collection, urinary biomarkers were normalized to urinary creatinine concentration. Serum was collected using Becton-Dickinson (BD) SST® Vacutainers, plasma was collected for Ba factor, sC5b-9, and thrombomodulin, and citrated plasma was collected for D-dimer using BD P100® Vacutainers. Urine was collected in a proprietary cryostabilizing solution with protease inhibitors. A matrix equivalent to the aHUS biomarker sample matrix was created for all normal donor serum, plasma, and urinary biomarker samples. All bioanalytical methods were custom or modified commercially available solid-phase ligand binding immunoassays optimized to minimize preanalytical variability and fully validated against FDA biomarker guidance, including acceptable long-term storage stability exceeding the time between collection and testing for all clinical samples.

[0287] Baseline serum, plasma, and urine biomarker levels in patients with aHUS, assessed before initiation of treatment, were compared with biomarker levels obtained from normal donor samples; normal serum and plasma samples were obtained from Alexion's in-house donors, BioIVT (Westbury, NY, USA), and Sanguine Biosciences (Waltham, MA, USA), while normal urine samples were obtained from Alexion's in-house donors and Sanguine Biosciences.

[0288] Biomarker levels over time and changes in levels from baseline after treatment with ravulizumab were assessed using mixed model for repeated measures (MMRM) analyses with log-transformed biomarker levels as dependent variables and fixed categorical effects of visit and fixed continuous effects of log-transformed baseline levels as covariates.

[0289] Baseline biomarkers were also assessed in relation to plasma exchange (PE) / plasma infusion (PI) and dialysis status at baseline.

[0290] Associations of baseline biomarkers with the main clinical measures of TMA - platelet count, lactate dehydrogenase (LDH) concentration, estimated glomerular filtration rate (eGFR) and urinary protein / creatinine ratio (UPCR) - were assessed via Spearman correlation coefficients between baseline biomarker levels and baseline clinical measures, and between baseline biomarker levels and changes in clinical measures at 26 and 52 weeks from the start of treatment. Simple linear regression analyses were also performed with both log-transformed baseline clinical measures as dependent variables and log-transformed baseline biomarker levels as independent variables, as well as with 26 and 52 week changes from baseline in clinical variables as dependent variables and log-transformed baseline biomarker levels as independent variables. For associations with baseline clinical measures, every two-fold increase in baseline biomarker results in an increase (or decrease) in laboratory values ​​by a regression coefficient of 2. When assessing associations with changes in clinical measures at 26 and 52 weeks, every two-fold increase in baseline biomarker results in an increase (or decrease) in laboratory values ​​by a regression coefficient. Regression analyses were followed to test for significance using two-tailed t-tests.

[0291] For clinical measures (e.g., platelet count, LDH, eGFR, and UPCR), additional linear regression analyses were performed based on baseline biomarkers and biomarker levels at week 52. The dependent variables were week 52 change from baseline in clinical measures, and the independent variables were log(2)-transformed baseline biomarker levels and log(2)-transformed post-dose biomarker levels. Results were stratified by complete TMA response status at week 52 (defined as ≥ 25% improvement from baseline in platelet count normalization, LDH normalization, and serum creatinine simultaneously met at least on two separate assessments, 4 weeks apart).

[0292] Logistic regression analysis was performed to evaluate any association between the biomarkers identified at baseline and complete TMA response. Patients were included in the analysis set if the endpoint of complete TMA response was confirmed by the 26-week evaluation. In the logistic regression analysis, clinical response was set as the dependent variable and the logarithm of baseline biomarker level was set as the independent variable. Odds ratios (OR) and 95% confidence intervals (CI) were reported, where OR represents the increase (or decrease) in odds of achieving efficacy response for every 2-fold increase in baseline biomarker; statistical significance following logistic regression analysis was evaluated via Wald test.

[0293] For sensitivity and specificity analysis, baseline levels of Ba and sC5b-9 in urine and plasma from aHUS patients and normal donors were entered into CombiROC (an online tool available at CombiROC.eu), a website maintained by the Protein Microarray and Bioinformatics Facilities of the National Institute of Molecular Genetics (Milan, Italy), to generate receiver operating characteristic (ROC) curves.

[0294] result Baseline Demographics Overall, 56 adults with aHUS were enrolled in this clinical trial, of whom 55 were included in the biomarker analysis; patient demographics and disease characteristics are outlined in Table 6. The median age at first infusion was 40.1 years, and the majority were female (66%) and white (52%).

[0295] [Table 6]

[0296] Comparison of baseline biomarker levels between patients with aHUS and normal donors Comparison of baseline biomarker levels in adults with aHUS and normal donors is presented in Table 7. In patients with aHUS, all biomarkers were elevated compared to normal donors, with the exception of plasma sC5b-9 and serum sVCAM-1. The alternative complement pathway specific biomarker Ba factor in plasma and urine was increased in ≥95% of patients. The terminal complement pathway specific biomarker sC5b-9 in urine was increased in >85% of patients.

[0297] [Table 7]

[0298] Longitudinal changes from baseline in biomarkers following treatment with ULTOMIRIS In adults with aHUS treated with ULTOMIRIS, pharmacodynamic effects were observed for both blood and urinary biomarkers, including significant reductions in creatinine-normalized urinary sC5b-9 and Ba levels compared to baseline at all collection time points (Figures 5A-F and 6A-C). Serum sTNF-R1 and plasma D-dimer levels were also significantly reduced from baseline levels by day 127 (Figure 5).

[0299] Association of baseline biomarkers with pretreatment plasma exchange / plasma infusion (PE / PI) and dialysis When baseline biomarkers were evaluated for any association with pretreatment PE / PI status, patients who received PE / PI within 7 days of the first ULTOMIRIS infusion had significantly lower plasma sC5b-9 levels and higher urinary Ba / creatinine levels compared to patients who did not (Table 8). Patients who received dialysis within 5 days of starting treatment had significantly higher plasma Ba, serum sTNF-R1, and urinary Ba / creatinine than patients who did not (Table 9).

[0300] [Table 8]

[0301] [Table 9]

[0302] Association of baseline biomarkers with baseline clinical measures Also, after simple regression analysis of baseline clinical measures against baseline biomarker levels (Table 10), increased plasma Ba, plasma thrombomodulin, and serum sTNF-RI were all significantly associated with lower baseline eGFR values. Normalized urinary cystatin C, sC5b-9, and Ba were also associated with lower baseline eGFR values. Increased plasma Ba, thrombomodulin, and D-dimer, serum sTNF-RI, and normalized urinary cystatin C, sC5b-9, and Ba were all significantly associated with higher baseline UPCR values. No significant associations were identified between any biomarker levels at baseline and baseline platelet counts.

[0303] [Table 10-1]

[0304] [Table 10-2]

[0305] Association of baseline biomarkers with changes in clinical measures at 26 and 52 weeks Analysis of changes in clinical measures at weeks 26 and 52 using simple regression against baseline biomarker levels found no significant associations between baseline biomarker levels and changes in platelet count at either time point (Table 11). Baseline urinary sC5b-9 / creatinine levels were significantly associated with changes in LDH at week 26 but not at week 52 (Table 12). Increased plasma Ba and D-dimer, serum sTNF-R1 and sVCAM-1, and urinary cystatin C / creatinine and Ba / creatinine were significantly inversely correlated with changes from baseline in eGFR at week 26 (Table 13).

[0306] [Table 11]

[0307] [Table 12]

[0308] [Table 13]

[0309] At week 52, the same relationship was observed by adding a significant inverse association with baseline plasma thrombomodulin levels (Table 13 and Figures 7 and 8).

[0310] Association of baseline biomarkers with complete TMA response at weeks 26 and 52 As assessed by logistic regression analysis, increases in serum sVCAM-1, plasma thrombomodulin, serum sTNF-R1, and plasma Ba levels were found to be significantly associated with an increased likelihood of achieving a complete TMA response; for normalized urinary sC5b-9, the association approached significance. At 52 weeks of treatment, the same association was observed (Figure 9). A complete TMA response was achieved in >60% of patients, and the median time to complete TMA response was 86 days (95% CI 42,401).

[0311] Association of baseline biomarker levels with complete TMA response Baseline levels of plasma Ba and urinary sC5b-9, plasma thrombomodulin, and serum sTNF-R1 were significantly lower in complete TMA responders compared with non-responders (Figure 9).

[0312] Clinical Sensitivity and Specificity of Selected Baseline Biomarker Levels CombiROC analysis of baseline Ba and sC5b-9 levels in plasma and urine of aHUS patients versus normal donors showed that there was some overlap between the two groups when the biomarkers were analyzed individually (Figure 10A). When combined biomarker analysis was used, this effect disappeared and high specificity and selectivity was achieved in both urine and plasma (Figure 10B, C, and D).

[0313] The individual area under the curve values ​​for sC5b-9 were significantly greater in urine than in plasma (0.72 vs. 0.52), while for Ba it was greater in plasma (0.67 vs. 0.80) (Fig. 11A). Plasma sC5b-9 produced more false positive (FP) and false negative (FN) results than urine sC5b-9 (32 FP, 19 FN vs. 0 FP, 5 FN); however, the distribution of urinary and plasma Ba levels was very similar, with few false results (Fig. 11B).

[0314] Consideration In this study, biomarkers related to the activity of the alternative and terminal complement pathways, i.e. Ba and sC5b-9, respectively, were found to be significantly associated with baseline PE / PI and dialysis status, alongside baseline clinical measures related to renal function, i.e. eGFR and UPCR. Both baseline plasma Ba and urinary sC5b-9 levels showed association with complete TMA response at week 52. Differences were observed in the utility of these biomarkers based on the assay medium, in the case of urinary biomarkers, regardless of whether they were normalized to creatinine levels (plasma vs. serum vs. urine). For example, sC5b-9 in plasma but not in urine and Ba in urine but not in plasma were associated with patients needing PE / PI within 7 days of starting treatment. Since plasma infusion inherently alters blood biomarker levels, urine would be expected to be a more reliable medium for biomarker testing in this setting. However, when normalized to creatinine levels, urinary Ba was no longer significantly associated with PE / PI status. Furthermore, while both urinary and plasma Ba were significantly associated with dialysis status within 5 days prior to initiating treatment, only urinary sC5b-9 had this association; when normalized to creatinine, it was urinary Ba, but not sC5b-9, that maintained a significant association with dialysis status.

[0315] The analytical medium was also related to other sC5b-9 associations. Baseline levels of sC5b-9 were found to increase over a much greater range from those of normal donors when analyzed in urine. In addition, baseline urinary but not plasma sC5b-9 was significantly associated with changes in UPCR at week 52; urinary but not plasma sC5b-9 levels declined over the course of 52 weeks of treatment. These observations, together with our ROC analysis of sC5b-9 in urine versus plasma, suggest that urine is the more reliable analytical medium.

[0316] The results of this study suggest that Ba and sC5b-9 - particularly when analyzed in urine samples - show potential for use as biomarkers in CM-TMA, as evidenced by their almost total increase in adults with aHUS compared to maximum observed levels in normal donors, as well as their association with selected clinical measures and outcomes. The reliability of their detection in urine, especially in combination, is further demonstrated by Figure 10.

[0317] The dot plot (Figure 10A) graphically displays the relative distribution of Ba and sC5b-9 levels in aHUS vs. HV. The AUC curve (Figure 10B) represents the combined sensitivity / 1-specificity when Ba and sC5b-9 values ​​are combined using the CombiROC web-based algorithm, the violin plot (Figure 10C) provides a visual of the distribution of aHUS and HV relative to the optimal threshold cutoffs in true positive / true negative / false positive / false negative, and the summary table (Figure 10D) presents the actual values ​​from the COmbiROC analysis, which is a tabular form of the violin plot in Figure 10C. The CombiROC diagram in the additional section is the individual results. Thus, this evidence confirms that the combination of these two complement-specific biomarkers is more clinically sensitive and specific than either one alone at measuring activity and identifying patients with complement-mediated TMA, and predicts clinical benefit from complement inhibitor therapy.

[0318] This analysis suggests that these biomarkers may demonstrate utility in the diagnosis and prognosis of specific aspects of CM-TMA disease, particularly those related to renal function.

[0319] The results of this study support the use of sC5b-9 (particularly urinary sC5b-9) as a potential biomarker for aHUS, as evidenced by its significant association with changes in UPCR during treatment, with a specific case for its use as a marker of renal function (insufficiency). Furthermore, sC5b-9 levels were also related to baseline PE / PI requirements (plasma sC5b-9) and dialysis status (urinary sC5b-9).

[0320] conclusion The results of this analysis highlight a set of key biomarkers with promising diagnostic and prognostic utility in the management of aHUS, particularly in urine. These data also show a pharmacodynamic effect on the levels of biomarkers in response to anti-complement C5 therapy, along with their utility for predicting treatment success, particularly for Ba and sC5b-9. Further evaluation and validation of these biomarkers in larger patient populations is necessary to guide patient risk stratification and management decisions.

[0321] All references cited herein are incorporated by reference as if each reference was specifically and individually indicated to be incorporated by reference. The citation of any reference is intended to disclose it prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention.

[0322] It will be understood that each of the above elements, or two or more together, may also find useful application in other types of methods different from the types described above. Without further analysis, the above will fully illustrate the gist of the present disclosure, which, by applying current knowledge, can be easily adapted to various applications without excluding features that, in the light of the prior art, otherwise fully constitute essential features of the general or specific aspects of the present disclosure as set forth in the appended claims. The above embodiments are presented by way of example only.

Claims

1. 1. A method of analyzing the levels of biomarkers in a signature comprising at least Ba and sC5b9, preferably the levels of biomarkers (i) complement factor Ba and / or the complement factor Ba / creatinine ratio; and (ii) complement sC5b-9 and / or the complement sC5b-9 / creatinine ratio, as indicators of complement-mediated thrombotic microangiopathies (CM-TMA) in a subject, comprising: (a) detecting the level of the biomarker in a bodily fluid sample obtained from the subject; (b) comparing the levels of the biomarkers; preferably the levels of the biomarkers in the signature with a control; wherein an increase in the level of the biomarker in the biological sample of the subject compared to that of the control indicates that the subject is suffering from, suspected of suffering from, or at risk of suffering from CM-TMA.

2. The method, (a) detecting in a bodily fluid sample obtained from the subject a level of a biomarker selected from the group consisting of cystatin C, cystatin C / creatinine ratio, sVCAM-1, sTNF-R1, thrombomodulin, or a combination thereof; (b) comparing the level of the biomarker to a control; and 2. The method of claim 1, further comprising: wherein an increase in the level of the biomarker in the biological sample of the subject compared to that of the control indicates that the subject is suffering from, suspected of suffering from, or at risk of suffering from CM-TMA.

3. A method for detecting biomarkers, the method comprising detecting levels of: (i) complement factor Ba and / or complement factor Ba / creatinine ratio; and (ii) complement sC5b-9 and / or complement sC5b-9 / creatinine ratio; and optionally (iii) cystatin C, cystatin C / creatinine ratio, sVCAM-1, sTNF-R1, thrombomodulin, or combinations thereof in a body fluid sample obtained from a subject suffering from complement-mediated thrombotic microangiopathy (CM-TMA).

4. The method according to any one of claims 1 to 3, wherein the body fluid sample comprises serum, blood, urine, plasma or a mixture thereof, preferably the body fluid sample comprises urine.

5. 4. The method of any one of claims 1 to 3, further comprising detecting levels of creatinine in bodily fluid samples from the subject and the control, and normalizing the levels of the biomarker in the bodily fluid samples based on the creatinine levels; preferably, the bodily fluid samples comprise urine and the creatinine levels are detected using a creatinine assay.

6. The method of any one of claims 1 to 3, wherein the control comprises an identical biological sample from a healthy subject.

7. The method of claim 1, wherein the detecting comprises contacting the biomarker with an agent comprising an antibody or an antigen-binding fragment thereof that specifically binds to the biomarker.

8. 4. The method of any one of claims 1 to 3, wherein the detecting step comprises detecting a biomarker signature comprising the following biomarkers: (a) Cystatin C + Ba + sC5b9 + Cystatin C / creatinine + Ba / creatinine + sC5b9 / creatinine.

9. A method according to any one of claims 1 to 3, comprising: (i) detecting Ba and / or sC5b9 in plasma; and (ii) detecting a plasma biomarker signature comprising Ba and sC5b9.

10. The body fluid sample includes a heterogeneous sample including urine and plasma, and the detecting step includes detecting (a) urinary cystatin C + plasma Ba; (b) urinary cystatin C + plasma sC5b9; (c) urinary Ba + plasma Ba; (d) urinary Ba + plasma sC5b9; (e) urinary sC5b9 + plasma Ba; (f) urinary sC5b9 + plasma sC5b9; (g) urinary cystatin C / creatinine + plasma Ba; (h) urinary cystatin C / creatinine + plasma sC5 4. The method of claim 1, comprising detecting a biomarker signature comprising at least one urinary biomarker and at least one plasma biomarker selected from: (i) urinary Ba / creatinine + plasma Ba; (j) Ba / creatinine + plasma sC5b9; (k) urinary sC5b9 / creatinine + plasma Ba; or (l) urinary sC5b9 / creatinine + plasma sC5b9.

11. The method of any one of claims 1 to 3, wherein the control comprises an identical body fluid sample obtained from a subject without complement-mediated thrombotic microangiopathy (CM-TMA), optionally with an undetectable level of urinary sC5b9.

12. The method of any one of claims 1 to 3, wherein the subject has been or is being treated with an inhibitor of complement.

13. The method of claim 12, wherein the inhibitor of complement comprises ravulizumab or a biosimilar thereof; preferably, the treatment comprises treatment with ravulizumab including a single loading dose on day 1 followed by a regular maintenance dose starting on day 15 based on the subject's body weight, wherein: (a) for subjects weighing ≧40 to <60 kilograms (kg), treatment includes a loading dose of 2400 milligrams (mg) followed by a maintenance dose of 3000 mg every 8 weeks; (b) for subjects weighing ≧60 to <100 kg, treatment includes a loading dose of 2700 mg followed by a maintenance dose of 3300 mg every 8 weeks; and (c) for subjects weighing ≧100 kg, treatment includes a loading dose of 3000 mg followed by a maintenance dose of 3600 mg every 8 weeks.

14. As an index for monitoring a subject's responsiveness to treatment with a complement C5 inhibitor, 1. A method for analyzing the levels of biomarkers: (i) complement factor Ba and / or the complement factor Ba / creatinine ratio; and (ii) complement sC5b-9 and / or the complement sC5b-9 / creatinine ratio; preferably in a signature comprising at least Ba and sC5b9, comprising: detecting the level of the biomarker in a bodily fluid sample obtained from the subject before and after the treatment; and comparing the level of the biomarker in the subject's bodily fluid sample before and after the treatment; preferably the level of the biomarker in the signature; wherein the subject has, is suspected of having, or is at risk of developing complement-mediated thrombotic microangiopathy (CM-TMA); wherein the subject may be being treated or is being treated with an inhibitor of complement C5; and A decrease in the level of the biomarker in the subject's bodily fluid sample after treatment compared to its level before treatment with the complement C5 inhibitor indicates that the subject is responsive to the treatment. method.

15. 1. A composition for use in a method for treating complement-mediated thrombotic microangiopathies (CM-TMA), comprising an inhibitor of complement, said inhibitor of complement being an anti-C5 antibody or a C5-binding fragment thereof, preferably eculizumab, ravulizumab, or a biosimilar thereof, said method comprising: A composition comprising: (a) measuring in a body fluid obtained from the subject the level or activity of CM-TMA associated biomarker proteins: (i) complement factor Ba and / or complement factor Ba / creatinine ratio; and (ii) complement sC5b-9; and / or complement sC5b-9 / creatinine ratio; preferably measuring the level or activity of biomarkers in a signature including at least Ba and sC5b9; and (b) administering to a subject having, suspected of having, or at risk of developing CM-TMA an inhibitor of complement in an amount and frequency sufficient to cause a decrease in the level or activity of said biomarkers or said biomarker signature compared to the level or activity in a sample of the same type of body fluid obtained from the subject prior to treatment with said inhibitor.

16. 1. A method of analyzing the concentration and / or activity of CM-TMA-associated biomarker proteins as indicators for determining whether a patient having Complement-Mediated Thrombotic Microangiopathies (CM-TMA), who is being treated with an inhibitor of complement under a given dosing schedule, requires a different dosing schedule, comprising: (A) measuring, in a body fluid obtained from said patient, the concentration and / or activity of CM-TMA-associated biomarker proteins: (i) complement factor Ba and / or the complement factor Ba / creatinine ratio; and / or (ii) complement sC5b-9 and / or the complement sC5b-9 / creatinine ratio; preferably comprising: measuring levels of biomarkers in a signature including at least Ba and sC5b9, where (a) a decreased level or activity of the biomarkers or biomarker signature compared to the concentration in a sample of the same type of bodily fluid obtained from the patient prior to treatment with the inhibitor indicates that the subject will be responsive to treatment with the inhibitor; and (B) if the patient is not responsive to treatment with the inhibitor of complement, then it is indicated that non-responding patients should be administered a different complement inhibitor or the same complement inhibitor at a higher dose or more frequent dosing schedule compared to the predetermined dosing schedule.

17. A kit for diagnosing Complement-Mediated Thrombotic Microangiopathies (CM-TMA), comprising an assay plate and a binding agent, optionally together with instructions for using said kit; wherein the binding agents are antibodies, or antigen-binding fragments thereof, that independently bind with specificity to a plurality of biological analytes; the analyte is a protein biomarker for CM-TMA; The protein biomarkers include the proteolytic fragment of complement component factor B (Ba) and soluble C5b9 (sC5b-9), optionally together with cystatin C; kit.

18. A composition for treating complement-mediated thrombotic microangiopathies (CM-TMA) in a subject, comprising an effective amount of an inhibitor of complement, said inhibitor of complement being an anti-C5 antibody or a C5-binding fragment thereof, preferably eculizumab, ravulizumab, or a biosimilar thereof; The effective amount is an amount sufficient to reduce the level of a biomarker and / or biomarker signature in the subject compared to its level prior to treatment with the inhibitor of complement; The biomarker signature comprises at least two biomarkers, including Ba and sC5b9; composition.

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