Diagnostic and prognostic biomarker profiles in patients with hematopoietic stem cell transplantation-associated thrombotic microangiopathy (HSCT-TMA)

Abstract

Methods are provided for treating hematopoietic stem cell transplantation-associated thrombotic microangiopathy (HSCT-TMA), e.g., post-HSCT TMA, in human patients with elevated levels of thrombomodulin (TM) and syndecan-1 (SYND1) compared to normal reference ranges for TM and SYND1 using anti-C5 and / or anti-CFB antibodies. Also provided are methods for treating a patient with HSCT-TMA, comprising: (1) obtaining or having obtained a sample (e.g., a blood or plasma sample) from the patient; (2) determining or having determined elevated levels of TM and SYND1 in the sample compared to the normal reference ranges for TM and SYND1, respectively; and (3) administering anti-C5 or anti-CFB antibodies to the patient in an amount and frequency sufficient to treat the HSCT-TMA after elevated TM and SYND1 levels have been determined.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 403,942, filed September 6, 2022, U.S. Provisional Application No. 63 / 420,198, filed October 28, 2022, and U.S. Provisional Application No. 63 / 440,969, filed January 25, 2023, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) is a multifactorial disorder triggered by systemic vascular endothelial injury, which can be caused by several mechanisms during the transplant process. Patients with HSCT-TMA develop kidney damage, serositis, pulmonary hypertension, and multiple organ dysfunction. Severe HSCT-TMA is associated with approximately 80% long-term morbidity and mortality. Studies have shown that the majority of patients die within 6 months (Cho et al., Bone Marrow Transplant. 2008;41(9):813-820; Cho et al., Transplantation. 2010;90(8):918-926; Oran, 2007). Another study showed a 9% overall survival in patients with severe HSCT-TMA who did not receive TMA-targeted therapy, with all deaths occurring within 10 months of TMA diagnosis (Jodele, et al., Blood. 2014b;124(4):645-653).

[0003] In pediatric patients, HSCT-TMA typically occurs early after allogeneic HSCT, with a median diagnosis of 35–47 days after HSCT, with 88%–92% occurring before day +100. However, cases have been reported up to 2 years after HSCT. Autologous recipients may develop HSC-TMA even earlier, at a median of 18 days after HSCT (Dvorak et al., Front Pediatr. 2019;7:133).

[0004] Endothelial injury is fundamental to the pathogenesis of HSCT-TMA, and dysregulated complement activation is likely a consequence of endothelial injury. Risk factors associated with the development of HSCT-TMA, which also initiate endothelial injury, include calcineurin inhibitors (CNIs), infections, and conditioning regimens (high-dose chemotherapy or total-body irradiation) (Khosla, et al., Bone Marrow Transplant. 2018;53(2):129-137; Masias, et al., Blood. 2017;129(21):2857-2863). Currently, there are no approved therapies for the treatment of HSCT-TMA, and identification of at-risk patients is difficult, leading to delayed diagnosis and disastrous outcomes. Therefore, it is an object of the present invention to identify biomarkers associated with HSCT-TMA and to provide improved methods for treating and identifying patients (particularly pediatric patients) with HSCT-TMA. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Cho et al.,Bone Marrow Transplant.2008;41(9):813-820 [Non-patent document 2] Cho et al.,Transplantation.2010;90(8):918-926;Oran,2007 [Non-patent document 3] Jodele,et al.,Blood.2014b;124(4):645-653 [Non-patent document 4] Dvorak et al.,Front Pediatr.2019;7:133 [Non-patent document 5] Khosla,et al.,Bone Marrow Transplant.2018;53(2):129-137 [Non-patent document 5] Masias,et al.,Blood.2017;129(21):2857-2863 Summary of the Invention

[0006] Approximately 10-20% of HSCT patients develop HSCT-TMA. These patients are difficult to identify and suffer tragic outcomes (e.g., graft rejection and even death). However, the present invention has discovered that HSCT-affected patients with abnormal levels of TM and SYNDI-1, as well as factor Ba and / or heparan sulfate proteoglycans (HSPGs), are more likely to develop HSCT-TMA. It has further been observed that the concentrations of these proteins are altered (e.g., decreased and / or normalized) after administration of anti-C5 antibodies (e.g., eculizumab or ravulizumab) or complement factor B inhibitors (e.g., inhibitors of the formation or activity of factor Ba, referred to herein as "CFB inhibitors").

[0007] Thus, in one embodiment, the present disclosure provides a method for treating a patient (e.g., a pediatric or adult patient) with hematopoietic stem cell transplantation-associated thrombotic microangiopathy (HSCT-TMA) who has been determined to have an elevated level (e.g., blood or plasma level) of a biomarker selected from thrombomodulin (TM) and syndecan-1 (SYND1), or a combination thereof, compared to a normal reference range for the biomarker, the method comprising administering an anti-C5 antibody or an anti-CFB antibody to the patient in an amount and frequency sufficient to attenuate the biomarker level in the patient, thereby treating the HSCT-TMA. In one embodiment, the patient has further been determined to have elevated levels (e.g., blood or plasma level) of complement factors Ba and / or C5b9 compared to a normal reference range for these factors. In another embodiment, the patient has been determined to have elevated levels (e.g., blood or plasma level) of HSPGs compared to a normal reference range for these factors.

[0008] In another aspect, the disclosure provides a method for treating a patient with HSCT-TMA, the method comprising: (1) obtaining or having obtained a sample (e.g., a blood or plasma sample) from the patient; (2) determining or having determined elevated levels of biomarkers selected from levels of TM and SYND1, or a combination thereof, in the patient's sample relative to normal reference ranges for the biomarkers; and (3) administering an anti-C5 antibody or an anti-CFB antibody to the patient in an amount and frequency sufficient to attenuate the elevated TM and SYND1 levels in the patient, thereby treating the HSCT-TMA. In one embodiment, the method further comprises determining or having determined elevated Ba and / or C5b9 levels in the sample relative to normal reference ranges for Ba and / or C5b9. In another embodiment, the method comprises determining or having determined elevated HSPG levels relative to normal reference ranges for HSPG.

[0009] In another aspect, the disclosure provides a method for identifying a patient with HSCT-TMA suitable for treatment with an anti-C5 antibody or an anti-CFB antibody, the method comprising: determining, using an in vitro assay, the level of a biomarker selected from TM and SYND1, or a combination thereof, in a sample (e.g., a blood or plasma sample) from the patient; wherein the patient is identified as suitable for treatment with an anti-C5 antibody or an anti-CFB antibody if the level of the biomarker in the sample is elevated compared to the normal reference range for TM and SYND1, respectively. In one embodiment, the method further comprises determining the level of Ba and / or C5b9 in the sample; wherein the patient is identified as suitable for treatment with an anti-C5 antibody or an anti-CFB antibody if the level of Ba and / or C5b9 is elevated compared to the normal reference range for Ba and / or C5b9. In another embodiment, the method comprises determining the level of HSPG in the sample; wherein the patient is identified as suitable for treatment with an anti-C5 antibody or an anti-CFB antibody if the level of HSPG is elevated compared to the normal reference range for HSPG. In another aspect, the disclosure provides a method for monitoring the responsiveness of a patient having HSCT-TMA to treatment with an anti-C5 antibody or an anti-CFB antibody, comprising determining a biomarker selected from TM and SYND1, or a combination thereof, in a sample (e.g., a blood or plasma sample) obtained from the patient during or after treatment, wherein a decrease in the biomarker level in a sample from the patient obtained during or after treatment compared to the biomarker level in a sample from the patient obtained before treatment with an anti-C5 antibody or an anti-CFB antibody indicates that the patient is responsive to treatment with an anti-C5 antibody or an anti-CFB antibody.

[0010] In one embodiment, the method further comprises determining Ba and / or C5b9 levels in a sample (e.g., a blood or plasma sample) obtained from the patient during or after treatment. A decrease in Ba and / or C5b9 levels in the patient sample obtained during or after treatment compared to the Ba and / or C5b9 levels in the patient sample obtained before treatment with an anti-C5 antibody or anti-CFB antibody indicates that the patient is responsive to treatment with an anti-C5 antibody or anti-CFB antibody. In another embodiment, the method further comprises determining HSPG levels in a sample (e.g., a blood or plasma sample) obtained from the patient during or after treatment. A decrease in HSPG levels in the patient sample obtained during or after treatment compared to the HSPG levels in the patient sample obtained before treatment with an anti-C5 antibody or anti-CFB antibody indicates that the patient is responsive to treatment with an anti-C5 antibody or anti-CFB antibody.

[0011] The levels of one or more of the biomarkers described herein (e.g., TM, SYND1, and / or Ba, and / or HSPG) can be measured by any suitable means or art-recognized technique. In one embodiment, the levels are measured by a regulatory (e.g., USFDA) approved system or kit. In another embodiment, the levels are measured by using an immunoassay, immunochemistry, immunohistochemistry assay, nucleoprobe assay, in situ hybridization, fluorescent RNA probe, RT-PCR, microarray transcription assay, or RNA transcription assay. In another embodiment, the levels are measured by enzyme-linked immunosorbent assay (ELISA).

[0012] Levels of biomarkers described herein (e.g., TM, SYND1, Ba, and / or HSPG) can be measured relative to control values. Controls include negative and positive controls. In some embodiments, the negative controls may include a normal reference range for each respective biomarker to assess whether levels in experimental samples (e.g., samples obtained from patients undergoing or likely to undergo HSCT-TMA) are relatively elevated or decreased. In one embodiment, the normal reference range of a biomarker is based on healthy patients (e.g., patients without HSCT). In another embodiment, the normal reference range of a biomarker is based on HSCT patients in the absence of TMA.

[0013] In some embodiments, the positive control may include a reference range for each biomarker, allowing for assessment of whether the level of the biomarker in an experimental sample (e.g., a sample from a patient undergoing HSCT-TMA treatment) is decreased or attenuated, e.g., compared pre-treatment to post-treatment.

[0014] In one embodiment, the normal reference range for TM in healthy patients (eg, patients without HSCT) is about 1.8 ng / mL to about 4.8 ng / mL. For example, in one embodiment, normal TM levels in a healthy patient are about 1.8 ng / mL, 1.9 ng / mL, 2.0 ng / mL, 2.1 ng / mL, 2.2 ng / mL, 2.3 ng / mL, 2.4 ng / mL, 2.5 ng / mL, 2.6 ng / mL, 2.7 ng / mL, 2.8 ng / mL, 2.9 ng / mL, 3.0 ng / mL, 3.1 ng / mL, 3.2 ng / mL, 3.3 ng / mL, 3.4 ng / mL, 3.5 ng / mL, 3.6 ng / mL, 3.7 ng / mL, 3.8 ng / mL, 3.9 ng / mL, 4.0 ng / mL, 4.1 ng / mL, 4.2 ng / mL, 4.3 ng / mL, 4.4 ng / mL, 4.5 ng / mL, 4.6 ng / mL, 4.7 ng / mL, or 4.8 ng / mL.

[0015] In another embodiment, the normal reference range for TM for HSCT patients in the absence of TMA is about 3 ng / mL to about 9 ng / mL. For example, in one embodiment, the normal reference range for TM for HSCT patients in the absence of TMA is about 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, or 9 ng / mL.

[0016] In another embodiment, the level of TM is elevated in HSCT patients who are likely to have or develop HSCT-TMA compared to a normal reference range (e.g., healthy patients who have not had HSCT or HSCT patients in the absence of TMA). For example, elevated TM levels are greater than about 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, or 30 ng / mL.

[0017] In another embodiment, the level of TM in a sample is considered elevated if it is at least about 10% higher to about 10-fold, e.g., about 2-fold greater than the normal reference range for TM (e.g., a healthy patient without HSCT or an HSCT patient in the absence of TMA). In some embodiments, the level of TM in a sample is considered elevated if it is at least 3-fold, 4-fold, 5-fold, or 6-fold greater than the normal reference range for TM.

[0018] In one embodiment, the normal reference range for SYND1 in a healthy patient (e.g., a patient without HSCT) is 15 ng / mL to 70 ng / mL. For example, in one embodiment, normal SYND1 levels in a healthy patient are approximately 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, 31 ng / mL, 32 ng / mL, 33 ng / mL, 34 ng / mL, 35 ng / mL, 36 ng / mL, 37 ng / mL, 38 ng / mL, 39 ng / mL, 40 ng / mL, 41 ng / mL, 42 ng / mL, 43 ng / mL, 44 ng / mL, 45 ng / mL, 46 ng / mL, 47 ng / mL, 48 ng / mL, 49 ng / mL, 50 ng / mL, 51 ng / mL, 52 ng / mL, 53 ng / mL, 54 ng / mL, 55 ng / mL, 56 ng / mL, 57 ng / mL, 58 ng / mL, 59 ng / mL, 60 ng / mL, 61 ng / mL, 62 ng / mL, 63 ng / mL, 64 ng / mL, 65 ng / mL, 66 ng / mL, 67 ng / mL, 68 ng / mL, 69 ng / mL, 70 ng g / mL, 42ng / mL, 43ng / mL, 44ng / mL, 45ng / mL, 46ng / mL, 47ng / mL, 48ng / mL, 49ng / mL, 50ng / mL, 51ng / mL, 52ng / mL, 53ng / mL, 54ng / mL, 55ng / mL, 56ng / mL, 57ng / mL, 58ng / mL, 59ng / mL, 60ng / mL, 61ng / mL, 62ng / mL, 63ng / mL, 64ng / mL, 65ng / mL, 66ng / mL, 67ng / mL, 68ng / mL, 69ng / mL, or 70ng / mL.

[0019] In another embodiment, the normal reference range for SYND1 for HSCT patients in the absence of TMA is about 15 ng / mL to 55 ng / mL. For example, in one embodiment, the normal reference range for SYND1 for HSCT patients in the absence of TMA is about 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, 31 ng / mL, 32 ng / mL, 33ng / mL, 34ng / mL, 35ng / mL, 36ng / mL, 37ng / mL, 38ng / mL, 39ng / mL, 40ng / mL, 41ng / mL, 42ng / mL, 43ng / mL, 44ng / m L, 45ng / mL, 46ng / mL, 47ng / mL, 48ng / mL, 49ng / mL, 50ng / mL, 51ng / mL, 52ng / mL, 53ng / mL, 54ng / mL, or 55ng / mL.

[0020] In another embodiment, levels of SYND1 are elevated in HSCT patients who are likely to have or develop HSCT-TMA compared to a normal reference range (e.g., healthy patients without HSCT or HSCT patients in the absence of TMA). For example, elevated SYND1 levels are greater than about 100ng / mL, 105ng / mL, 110ng / mL, 115ng / mL, 120ng / mL, 125ng / mL, 130ng / mL, 135ng / mL, 140ng / mL, 145ng / mL, 150ng / mL, 155ng / mL, 160ng / mL, 165ng / mL, 170ng / mL, 175ng / mL, 180ng / mL, 185ng / mL, 190ng / mL, 195ng / mL, 200ng / mL, 205ng / mL, 210ng / mL, 215ng / mL, 220ng / mL, 225ng / mL, 230ng / mL, 235ng / mL, 240ng / mL, 245ng / mL, or 250ng / mL.

[0021] In another embodiment, the level of SYND1 in a sample is considered elevated if it is at least about 2-fold to about 17-fold, e.g., about 4-fold, greater than the normal reference range for SYND1 (e.g., a healthy patient without HSCT or a patient with HSCT in the absence of TMA). In some embodiments, the level of SYND1 in a sample is considered elevated if it is at least 3, 4, 5, 6, 7, 8, 10, 12, or 15-fold greater than the normal reference range for SYND1.

[0022] In one embodiment, the normal reference range for factor Ba in a healthy patient (e.g., a patient without an HSCT) is less than about 1000 ng / mL. In another embodiment, the normal reference range for factor Ba in a healthy patient (e.g., a patient without an HSCT) is less than about 600 ng / mL. In another embodiment, the normal reference range for factor Ba in a healthy patient (e.g., a patient without an HSCT) is between about 300 ng / mL and 600 ng / mL. For example, a normal reference range for Factor Ba in a healthy patient (e.g., a patient without HSCT) is about 300ng / mL, 310ng / mL, 320ng / mL, 330ng / mL, 340ng / mL, 350ng / mL, 360ng / mL, 370ng / mL, 380ng / mL, 390ng / mL, 400ng / mL, 410ng / mL, 420ng / mL, 430ng / mL, 440ng / mL, 450ng / mL, 460ng / mL, 470ng / mL, 480ng / mL, 490ng / mL, 500ng / mL, 510ng / mL, 520ng / mL, 530ng / mL, 540ng / mL, 550ng / mL, 560ng / mL, 570ng / mL, 580ng / mL, 590ng / mL, or 600ng / mL.

[0023] In another embodiment, the normal reference range for Factor Ba for HSCT patients in the absence of TMA is about 500 ng / mL to 800. For example, in one embodiment, the normal reference range for Factor Ba for HSCT patients in the absence of TMA is about 500ng / mL, 510ng / mL, 520ng / mL, 530ng / mL, 540ng / mL, 550ng / mL, 560ng / mL, 570ng / mL, 580ng / mL, 590ng / mL, 600ng / mL, 610ng / mL, 620ng / mL, 630ng / mL, 640ng / mL, 650ng / mL, 660ng / mL, 670ng / mL, 680ng / mL, 690ng / mL, 700ng / mL, 710ng / mL, 720ng / mL, 730ng / mL, 740ng / mL, 750ng / mL, 760ng / mL, 770ng / mL, 780ng / mL, 790ng / mL, or 800ng / mL.

[0024] In another embodiment, the level of factor Ba is elevated in HSCT patients who have or are likely to develop HSCT-TMA compared to a normal reference range (e.g., healthy patients without HSCT or HSCT patients in the absence of TMA). For example, elevated Ba levels may be about 900ng / mL, 910ng / mL, 920ng / mL, 930ng / mL, 940ng / mL, 950ng / mL, 960ng / mL, 970ng / mL, 980ng / mL, 990ng / mL, 1000ng / mL, 1010ng / mL, 1020ng / mL, 1030ng / mL, 1040ng / mL, 1050ng / mL, 1060ng / mL, 1070ng / mL, 1080ng / mL, 1090ng / mL, 1100ng / mL, 1110ng / mL, 1120ng / mL, 1130ng / mL, 1140ng / mL, 1150ng / mL, 1160ng / mL, 1170ng / mL, 1180ng / mL, 1190ng / mL, 1200ng / mL, 1210ng / mL, 1220ng / mL, 1230ng / mL, 1240ng / mL, 1250ng / mL, 1260ng / mL, 1270ng / mL, 1280ng / mL, 1290ng / mL, 1300ng / mL, 1310ng / mL, 1320ng / mL, 1330ng / mL, 1340ng / mL, 1350ng / mL, 1360ng / mL, 1370ng / mL, 1380ng / mL, 1390ng / mL, 14 g / mL, 1120ng / mL, 1130ng / mL, 1140ng / mL, 1150ng / mL, 1160ng / mL, 1170ng / mL, 1180ng / mL, 1190ng / mL, 1200ng / mL, 1210ng / mL, 1220ng / mL, 1230ng / mL, 1240ng / mL, 1250ng / mL, 1260ng / mL, 1270ng / mL, 1280ng / mL, 1290ng / mL, 1300ng / mL, 1310ng / mL, 1320ng / mL, 1330ng / mL, 1340ng / mL, 1350ng / mL, 1360ng / mL, 1370ng / mL, 1380ng / mL, 1390ng / mL, 1400ng / mL, 1410ng / mL, 1420ng / mL, 1430ng / mL, 1440ng / mL, 1450ng / mL, 1460ng / mL, 1470ng / mL, 1480ng / mL, 1490ng / mL, 1500ng / mL, 1510ng / mL, 1520ng / mL, 1530ng / mL, 1540ng / mL, 1550ng / m L, 1560ng / mL, 1570ng / mL, 1580ng / mL, 1590ng / mL, 1600ng / mL, 1610ng / mL, 1620ng / mL, 1630ng / mL, 1640ng / mL, 1650ng / mL, 1660ng / mL , 1670ng / mL, 1680ng / mL, 1690ng / mL, 1700ng / mL, 1710ng / mL, 1720ng / mL, 1730ng / mL, 1740ng / mL, 1750ng / mL, 1760ng / mL, 1770ng / mL,1780ng / mL, 1790ng / mL, 1800ng / mL, 1810ng / mL, 1820ng / mL, 1830ng / mL, 1840ng / mL, 1850ng / mL, 1860ng / mL, 18 70ng / mL, 1880ng / mL, 1890ng / mL, 1900ng / mL, 1910ng / mL, 1920ng / mL, 1930ng / mL, 1940ng / mL, 1950ng / mL, 1960 ng / mL, 1970ng / mL, 1980ng / mL, 1990ng / mL, 2000ng / mL, 2010ng / mL, 2020ng / mL, 2030ng / mL, 2040ng / mL, 2050ng / mL, 2060ng / mL, 2070ng / mL, 2080ng / mL, 2090ng / mL, 2100ng / mL, 2110ng / mL, 2120ng / mL, 2130ng / mL, 2140ng / m L, 2150ng / mL, 2160ng / mL, 2170ng / mL, 2180ng / mL, 2190ng / mL, 2200ng / mL, 2210ng / mL, 2220ng / mL, 2230ng / mL, 2240ng / mL, 2250ng / mL, 2260ng / mL, 2270ng / mL, 2280ng / mL, 2290ng / mL, 2300ng / mL, 2310ng / mL, 2320ng / mL, 23 Greater than 30ng / mL, 2340ng / mL, 2350ng / mL, 2360ng / mL, 2370ng / mL, 2380ng / mL, 2390ng / mL, 2400ng / mL, 2410ng / mL, 2420ng / mL, 2430ng / mL, 2440ng / mL, 2450ng / mL, 2460ng / mL, 2470ng / mL, 2480ng / mL, 2490ng / mL, or 2500ng / mL.

[0025] In another embodiment, the level of Ba in a sample is considered elevated if it is at least about 12% to about 5-fold, e.g., about 2-fold, greater than the normal reference range for factor Ba (e.g., a healthy patient without HSCT or an HSCT patient in the absence of TMA). In some embodiments, the level of Ba in a sample is considered elevated if it is at least 3-fold, 4-fold, or 5-fold greater than the normal reference range for Ba.

[0026] In another embodiment, the level of HSPG is elevated in HSCT patients who are likely to have or develop HSCT-TMA compared to a normal reference range (e.g., healthy patients without HSCT or HSCT patients in the absence of TMA).

[0027] In another embodiment, the level of HSPG in a sample is considered elevated if it is at least about 20% to about 2-fold, e.g., about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold greater than the normal reference range for HSPG (e.g., healthy patients without HSCT or HSCT patients in the absence of TMA).

[0028] Any suitable anti-C5 antibody or antigen-binding fragment thereof can be used in the methods described herein. In one embodiment, the anti-C5 antibody is a human antibody, a humanized antibody, a bispecific antibody, a chimeric antibody, a Fab, a Fab'2, an scFv, a SMIP, an Affibody®, a nanobody, or a domain antibody that inhibits C5.

[0029] Any suitable anti-C5 antibody or antigen-binding fragment thereof can be used in the methods described herein. An exemplary anti-C5 antibody is eculizumab. Eculizumab (also known as SOLIRIS®) is an anti-C5 antibody comprising heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. Ravulizumab comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8. Ravulizumab comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 10 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 11.

[0030] Another exemplary anti-C5 antibody is ravulizumab (also known as ULTOMIRIS®, ALXN1210, and antibody BNJ441), which comprises heavy and light chains having the sequences set forth in SEQ ID NOs: 14 and 11, respectively, or antigen-binding fragments and variants thereof. In other embodiments, the antibody comprises the heavy and light chain complementarity-determining regions (CDRs) or variable regions (VRs) of ravulizumab. Thus, in one embodiment, it comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable (VH) region of ravulizumab having the sequence set forth in SEQ ID NO: 12, and the CDR1, CDR2, and CDR3 domains of the light chain variable (VL) region of ravulizumab having the sequence set forth in SEQ ID NO: 8. In another embodiment, the antibody comprises the CDR1, CDR2, and CDR3 heavy chain sequences set forth in SEQ ID NOs: 19, 18, and 3, respectively, and the CDR1, CDR2, and CDR3 light chain sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. In another embodiment, the antibody comprises VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 8, respectively. In another embodiment, the antibody comprises a heavy chain constant region set forth in SEQ ID NO: 13.

[0031] In another embodiment, the antibody comprises a variant human Fc constant region that binds to the human neonatal Fc receptor (FcRn), wherein the variant human Fc CH3 constant region comprises Met-429-Leu and Asn-435-Ser substitutions at residues corresponding to methionine 428 and asparagine 434 (EU numbering, respectively) of the native human IgG Fc constant region, respectively.

[0032] In another embodiment, the antibody comprises the CDR1, CDR2, and CDR3 heavy chain sequences set forth in SEQ ID NOs: 19, 18, and 3, respectively, and the CDR1, CDR2, and CDR3 light chain sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, and a variant human Fc constant region that binds to the human neonatal Fc receptor (FcRn), wherein the variant human Fc CH3 constant region comprises Met-429-Leu and Asn-435-Ser substitutions at residues corresponding to methionine 428 and asparagine 434 of the native human IgG Fc constant region, respectively, in EU numbering.

[0033] In another embodiment, the antibody has a K D Affinity dissociation constants (K) in the range of ≦1 nM D ) binds to human C5 at pH 7.4 and 25°C. In another embodiment, the antibody binds to human C5 at pH 6.0 and 25°C. D In yet another embodiment, the antibody binds to human C5 at a K of ≥ 10 nM. D ) / (K of an antibody or antigen-binding fragment thereof against human C5 at pH 7.4 and 25°C D )] is greater than 25.

[0034] Another exemplary anti-C5 antibody is described in U.S. Patent Nos. 8,241,628 and 8,883,158. In one embodiment, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs:21, 22, and 23, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs:24, 25, and 26, respectively. In another embodiment, the antibody or antigen-binding fragment thereof comprises a VH region having the sequence set forth in SEQ ID NO:27 and a VL region having the sequence set forth in SEQ ID NO:28.

[0035] Other exemplary anti-C5 antibodies are also described in U.S. Patent Nos. 8,241,628 and 8,883,158. In one embodiment, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 29, 30, and 31, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 32, 33, and 34, respectively. In another embodiment, the antibody comprises a VH region having the sequence set forth in SEQ ID NO: 35 and a VL region having the sequence set forth in SEQ ID NO: 36.

[0036] Another exemplary anti-C5 antibody is described in U.S. Patent No. 9,765,135. In one embodiment, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively. In another embodiment, the antibody comprises a VH region having the sequence set forth in SEQ ID NO: 43 and a VL region having the sequence set forth in SEQ ID NO: 44.

[0037] Another exemplary anti-C5 antibody is described in Fukuzawa T. et al. (Sci. Rep. 7:1080, 2017). In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 45 and a light chain comprising SEQ ID NO: 46.

[0038] Another exemplary anti-C5 antibody is described in U.S. Patent No. 10,633,434. In one embodiment, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 47 and a light chain variable region comprising SEQ ID NO: 48. In another embodiment, the antibody comprises a heavy chain comprising SEQ ID NO: 49 and a light chain comprising SEQ ID NO: 50.

[0039] In some embodiments, the anti-C5 antibody is a biosimilar of eculizumab (SOLIRIS®). For example, in one embodiment, the anti-C5 antibody is ABP 959 antibody (eculizumab biosimilar, manufactured by Amgen Inc., USA), ELIZARIA® (manufactured by Generium JNC, Russia), SB12 (eculizumab biosimilar, manufactured by Samsung Bioepis, Incheon, South Korea), ISU305 (eculizumab biosimilar, manufactured by ISU Abxis, South Korea), ABLYZE® (eculizumab biosimilar, manufactured by CinnaGen, Iran), BCD 148 (eculizumab biosimilar, manufactured by Biocad Medical, Quebec, Canada), tesidolumab (manufactured by Novartis), crovalimab (manufactured by Roche), CAN106 (manufactured by CanBridge Bio, China), or pozelimab (manufactured by Regeneron).

[0040] In another embodiment, the antibody competes for binding to and / or binds to the same epitope on C5 as the above-mentioned antibodies, hi another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% variable region identity).

[0041] The anti-C5 antibody can be administered via any suitable means. In one embodiment, the anti-C5 antibody is administered intravenously. In another embodiment, the anti-C5 antibody is administered subcutaneously.

[0042] In one embodiment, the anti-C5 antibody (e.g., ravulizumab) is selected from the following: (a) Once on day 1, at a dose of 600 mg for patients weighing 5 kg or more but less than 10 kg, 600 mg for patients weighing 10 kg or more but less than 20 kg, 900 mg for patients weighing 20 kg or more but less than 30 kg, 1200 mg for patients weighing 30 kg or more but less than 40 kg, 2400 mg for patients weighing 40 kg or more but less than 60 kg, 2700 mg for patients weighing 60 kg or more but less than 100 kg, or 3000 mg for patients weighing 100 kg or more; (b) Once on day 5, at a dose of 300 mg for patients weighing 5 kg or more but less than 10 kg, 300 mg for patients weighing 10 kg or more but less than 20 kg, 300 mg for patients weighing 20 kg or more but less than 30 kg, 300 mg for patients weighing 30 kg or more but less than 40 kg, 600 mg for patients weighing 40 kg or more but less than 60 kg, 900 mg for patients weighing 60 kg or more but less than 100 kg, or 900 mg for patients weighing 100 kg or more; (c) once on day 10, at a dose of 300 mg for patients weighing 5 kg or more but less than 10 kg, 300 mg for patients weighing 10 kg or more but less than 20 kg, 300 mg for patients weighing 20 kg or more but less than 30 kg, 300 mg for patients weighing 30 kg or more but less than 40 kg, 600 mg for patients weighing 40 kg or more but less than 60 kg, 900 mg for patients weighing 60 kg or more but less than 100 kg, or 900 mg for patients weighing 100 kg or more; and (d) On the 15th day and every 4 weeks thereafter, 300 mg for patients weighing 5 kg to less than 10 kg, or 600 mg for patients weighing 10 kg to less than 20 kg; or on the 15th day and every 8 weeks thereafter, 2100 mg for patients weighing 20 kg to less than 30 kg, 2700 mg for patients weighing 30 kg to less than 40 kg, 3000 mg for patients weighing 40 kg to less than 60 kg, 3300 mg for patients weighing 60 kg to less than 100 kg, or 3600 mg for patients weighing 100 kg or more.

[0043] In another aspect, an anti-CFB antibody is used in the methods described herein. Any suitable anti-CFB antibody can be used in the methods described herein. The effectiveness of the treatment methods provided herein can be evaluated using any appropriate means. In one embodiment, treatment results in a reduction or cessation of microangiopathic hemolytic anemia, thrombocytopenia, endothelial damage, kidney damage, kidney failure, serositis, pulmonary hypertension, and multiple organ failure compared to baseline.

[0044] In another embodiment, treatment results in normalization of LDH, elimination of the need for red blood cell and platelet transfusions, an increase in hemoglobin, and / or elimination of schistocytes compared to baseline. In another embodiment, treatment results in: (a) a platelet count of 50,000 / mm3 without transfusion support during the past 7 days; 3 or greater, (b) LDH<1.5×ULN, (c) absence of schistosomes (if schistosomes were present at baseline), and / or (d) at least a 50% reduction in proteinuria from baseline. In another embodiment, the treatment results in a favorable hematologic response. In another embodiment, the treatment results in hemoglobin≧8 g / dL without transfusion support. In another embodiment, the treatment results in terminal complement inhibition. In another embodiment, the treatment results in a reduction in adverse events. In another embodiment, the treatment results in a change from baseline in quality of life as assessed via a quality of life assessment. In another embodiment, the quality of life assessment is the Quality of Life Inventory (PedSql) scale or the EQ-5D-5L questionnaire.

[0045] In another aspect, there is provided an anti-C5 antibody or antigen-binding fragment thereof, or an anti-CFB antibody for use in treating a patient with hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) in which the blood level of a biomarker selected from thrombomodulin (TM) and syndecan-1 (SYND1), or a combination thereof, has been determined to be elevated compared to the normal reference range for the biomarker, wherein the anti-C5 antibody or anti-CFB antibody is administered to the patient in an amount and frequency sufficient to attenuate the biomarker level in the patient.

[0046] In another aspect, an anti-C5 antibody or antigen-binding fragment thereof or an anti-CFB antibody is provided for use in identifying patients with HSCT-TMA who are suitable for treatment with an anti-C5 antibody or an anti-CFB antibody, comprising determining using an in vitro assay the level of a biomarker selected from TM and SYND1 or a combination thereof in a blood sample from the patient, wherein the patient is identified as suitable for treatment with an anti-C5 antibody or an anti-CFB antibody if the level of the biomarker in the blood sample is elevated compared to the normal reference range for TM and SYND1, respectively.

[0047] In another aspect, an anti-C5 antibody or antigen-binding fragment thereof or an anti-CFB antibody is provided for use in monitoring the responsiveness of a patient having HSCT-TMA to treatment with an anti-C5 antibody or an anti-CFB antibody, the use comprising determining a biomarker selected from TM and SYND1 or a combination thereof in a blood sample from the patient obtained during or after treatment, wherein a decrease in the biomarker level in a blood sample from the patient obtained during or after treatment compared to the biomarker level in a blood sample from the patient obtained before treatment with the anti-C5 antibody or anti-CFB antibody indicates that the patient is responsive to treatment with the anti-C5 antibody or anti-CFB antibody.

[0048] In another aspect, there is provided a use of an anti-C5 antibody or antigen-binding fragment thereof, or an anti-CFB antibody for treating a patient with hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) in which the blood level of a biomarker selected from thrombomodulin (TM) and syndecan-1 (SYND1), or a combination thereof, has been determined to be elevated compared to the normal reference range for the biomarker, wherein the anti-C5 antibody or anti-CFB antibody is administered to the patient in an amount and frequency sufficient to attenuate the biomarker level in the patient.

[0049] In another aspect, there is provided a use of an anti-C5 antibody or an antigen-binding fragment thereof, or an anti-CFB antibody in identifying a patient having an HSCT-TMA suitable for treatment with an anti-C5 antibody or an anti-CFB antibody, comprising determining the level of a biomarker selected from TM and SYND1, or a combination thereof, in a blood sample from the patient using an in vitro assay, wherein the patient is identified as suitable for treatment with an anti-C5 antibody or an anti-CFB antibody because the level of the biomarker in the blood sample is elevated compared to the normal reference range for TM and SYND1, respectively.

[0050] In another aspect, there is provided a use of an anti-C5 antibody or an antigen-binding fragment thereof, or an anti-CFB antibody in monitoring the responsiveness of a patient having HSCT-TMA to treatment with an anti-C5 antibody or an anti-CFB antibody, the use comprising determining a biomarker selected from TM and SYND1 or a combination thereof in a blood sample from the patient obtained during or after treatment, wherein a decrease in the biomarker level in the blood sample from the patient obtained during or after treatment compared to the biomarker level in a blood sample from the patient obtained before treatment with the anti-C5 antibody or anti-CFB antibody indicates that the patient is responsive to treatment with the anti-C5 antibody or anti-CFB antibody. [Brief explanation of the drawings]

[0051] [Figure 1] 1 is a graph showing plasma TM levels in LPS-injected mice treated with anti-complement agents. [Figure 2] 1 is a graph showing that cyclosporine-induced TM loss is partially prevented by treatment with eculizumab. [Figure 3A] Figure 3 shows the levels of glycocalyx biomarkers in pediatric patients with and without HSCT-TMA. Specifically, Figures 3A and 3B show that HSCT-TMA patients (n = 11) had significantly elevated levels of glycocalyx biomarkers compared with HSCT control patients (n = 7). Figure 3C shows that a strong positive correlation was observed between TM and SYND1. [Figure 3B]Figure 3 shows the levels of glycocalyx biomarkers in pediatric patients with and without HSCT-TMA. Specifically, Figures 3A and 3B show that HSCT-TMA patients (n = 11) had significantly elevated levels of glycocalyx biomarkers compared with HSCT control patients (n = 7). Figure 3C shows that a strong positive correlation was observed between TM and SYND1. [Figure 3C] Figure 3 shows the levels of glycocalyx biomarkers in pediatric patients with and without HSCT-TMA. Specifically, Figures 3A and 3B show that HSCT-TMA patients (n = 11) had significantly elevated levels of glycocalyx biomarkers compared with HSCT control patients (n = 7). Figure 3C shows that a strong positive correlation was observed between TM and SYND1. [Figure 4A] Figure 4 shows AP activation in pediatric patients with and without HSCT-TMA. Specifically, Figure 4A shows that plasma Ba was significantly elevated in patients with HSCT-TMA compared with those without. Furthermore, plasma Ba positively correlated with both TM levels (Figure 4B) and SYND1 levels (Figure 4C). [Figure 4B] Figure 4 shows AP activation in pediatric patients with and without HSCT-TMA. Specifically, Figure 4A shows that plasma Ba was significantly elevated in patients with HSCT-TMA compared with those without. Furthermore, plasma Ba positively correlated with both TM levels (Figure 4B) and SYND1 levels (Figure 4C). [Figure 4C] Figure 4 shows AP activation in pediatric patients with and without HSCT-TMA. Specifically, Figure 4A shows that plasma Ba was significantly elevated in patients with HSCT-TMA compared with those without. Furthermore, plasma Ba positively correlated with both TM levels (Figure 4B) and SYND1 levels (Figure 4C). [Figure 5] Plasma TM levels in LPS-injected mice treated with anti-complement agents. As shown in Figure 5, mice with inflammation-mediated complement activation had elevated circulating TM levels that were attenuated with anti-complement agent treatment. [Figure 6A] Correlation between TM and Ba in LPS-injected mice. TM and Ba levels were measured by commercial ELISA or Western blotting, respectively (Figure 6A). As shown in Figure 6B, plasma Ba was positively correlated with TM levels in mice with inflammation-mediated complement activation. [Figure 6B] Correlation between TM and Ba in LPS-injected mice. TM and Ba levels were measured by commercial ELISA or Western blotting, respectively (Figure 6A). As shown in Figure 6B, plasma Ba was positively correlated with TM levels in mice with inflammation-mediated complement activation. [Figure 7A] Figures 7A-7D show cyclosporine-induced TM loss in HUVECs treated with CsA and 30% NHS for 18 hours in the presence (Figures 7A-7B) or absence (Figures 7C-7D) of eculizumab surrogate. As shown by these figures, TM and heparan sulfate proteoglycan (HSPG) surface expression was reduced on HUVECs treated with CsA and partially restored by eculizumab surrogate. [Figure 7B] Figures 7A-7D show cyclosporine-induced TM loss in HUVECs treated with CsA and 30% NHS for 18 hours in the presence (Figures 7A-7B) or absence (Figures 7C-7D) of eculizumab surrogate. As shown by these figures, TM and heparan sulfate proteoglycan (HSPG) surface expression was reduced on HUVECs treated with CsA and partially restored by eculizumab surrogate. [Figure 7C] Figures 7A-7D show cyclosporine-induced TM loss in HUVECs treated with CsA and 30% NHS for 18 hours in the presence (Figures 7A-7B) or absence (Figures 7C-7D) of eculizumab surrogate. As shown by these figures, TM and heparan sulfate proteoglycan (HSPG) surface expression was reduced on HUVECs treated with CsA and partially restored by eculizumab surrogate. [Figure 7D]Figures 7A-7D show cyclosporine-induced TM loss in HUVECs treated with CsA and 30% NHS for 18 hours in the presence (Figures 7A-7B) or absence (Figures 7C-7D) of eculizumab surrogate. As shown by these figures, TM and heparan sulfate proteoglycan (HSPG) surface expression was reduced on HUVECs treated with CsA and partially restored by eculizumab surrogate. [Figure 8A] Deposition of complement activation products on HMEC-1 cells treated with CsA. As shown in these figures, CsA treatment induced complement deposition on HMEC-1 cells, and C5 inhibition reduced C5b-9 (Fig. 8B) but not iC3b deposition (Fig. 8A). [Figure 8B] Deposition of complement activation products on HMEC-1 cells treated with CsA. As shown in these figures, CsA treatment induced complement deposition on HMEC-1 cells, and C5 inhibition reduced C5b-9 (Fig. 8B) but not iC3b deposition (Fig. 8A). DETAILED DESCRIPTION OF THE INVENTION

[0052] As described herein and illustrated in the Examples, certain biomarkers associated with HSCT-TMA have been discovered. Specifically, it has been discovered that HSCT patients with elevated concentrations of certain proteins (e.g., TM, SYND1, and / or Ba, herein referred to as "HSCT-TMA biomarker proteins") are more likely to develop HSCT-TMA. Similarly, a decrease and / or normalization of the concentration (or activity) of these proteins in biological fluids obtained from HSCT-TMA patients treated with complement inhibitors (e.g., anti-C5 antibodies (e.g., eculizumab or ravulizumab) or anti-CFB antibodies) indicates that the patient is responsive to treatment. Thus, analysis of the concentration and / or activity levels of such proteins can be used, among other things, to assess the risk of an HSCT patient developing HSCT-TMA, monitor the progression or remission of HSCT-TMA, and / or monitor the therapeutic response to complement inhibitors (e.g., anti-C5 antibodies (e.g., eculizumab or ravulizumab) or anti-CFB antibodies).

[0053] I. Definition As used herein, the term "subject" or "patient" refers to a human patient (eg, a patient having HSCT or HSCT-TMA).

[0054] As used herein, the term "pediatric" patient refers to a human patient classified by a physician or caregiver as belonging to a non-adult category, and may include, for example, newborns, infants (both preterm and full-term), children, and adolescents. Typically, a pediatric patient is a patient under the age of 18 (<18 years).

[0055] As used herein, the term "adult" patient is a human patient who is not a neonate, infant, child, or adolescent, and who has been classified as such by a physician or caregiver based on age, developmental status, physiological characteristics, etc. Typically, an adult patient is a patient who is 18 years of age or older (≧18 years of age).

[0056] The term "antibody" describes a polypeptide comprising at least one antibody-derived antigen-binding site (e.g., a VH / VL region or Fv, or a CDR). The term "antibody" is used interchangeably with the term "immunoglobulin." Antibodies include known forms of antibodies, for example, antibodies can be human, humanized, bispecific, chimerized or chimeric, polyclonal, monoclonal, primatized, and deimmunized. Antibodies can be produced in or derived from any of a variety of species, for example, humans, non-human primate mammals (such as orangutans, baboons, or chimpanzees), horses, cows, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. Antibodies can be purified or recombinant. The antibody may further be of any of the following isotypes: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, IgE, or a combination thereof. The antibody may be a naturally occurring antibody or an antibody that has been altered by protein engineering techniques (e.g., by mutation, deletion, substitution, conjugation to a non-antibody moiety). The antibody may contain, for example, one or more variant amino acids (compared to a naturally occurring antibody) that alter the properties (e.g., functional properties) of the antibody. Many such modifications that affect, for example, half-life, effector functions, and / or immune response to the antibody in a patient are known in the art. The term antibody further includes artificial or engineered polypeptide constructs that contain at least one antibody-derived antigen-binding site.

[0057] The term "antibody" includes antigen-binding fragments thereof. The term "antigen-binding fragment" or similar terms is known in the art and can refer, for example, to a fragment of an antibody that retains the ability to bind to a target antigen (e.g., human C5) and inhibit the activity of the target antigen. Such fragments include, for example, single-chain antibodies, single-chain Fv fragments (scFv), Fd fragments, Fab fragments, Fab' fragments, or F(ab')2 fragments. An scFv fragment is a single polypeptide chain that contains both the heavy and light chain variable regions of the antibody from which the scFv is derived. Additionally, intrabodies, minibodies, triabodies, and bispecific antibodies are also included within the definition of antibody and are suitable for use in the methods described herein. See, for example, Todorovska et al. (2001) J Immunol Methods 248(1):47-66; Hudson and Kortt (1999) J Immunol Methods 231(1):177-189; Poljak (1994) Structure 2(12):1121-1123; Rondon and Marasco (1997) Annual Review of Microbiology 51:257-283. Antigen-binding fragments may further comprise the variable region of the heavy chain polypeptide and the variable region of the light chain polypeptide. Thus, antigen-binding fragments may comprise the CDRs of both the light and heavy chain polypeptides of an antibody.

[0058] The term "antibody fragment" may further include single domain antibodies, such as, for example, camelized single domain antibodies. See, e.g., Muyldermans et al. (2001) Trends Biochem Sci 26:230-235; Nuttall et al. (2000) Curr Pharm Biotech 1:253-263; Reichmann et al. (1999) J Immunol Meth 231:25-38; PCT Application Publication Nos. WO 94 / 04678 and WO 94 / 25591; and U.S. Patent No. 6,005,079. The term "antibody fragment" may further include two V fragments with modifications such that a single domain antibody is formed. H The present invention also includes single domain antibodies, which contain a domain.

[0059] The terms "polypeptide," "peptide," and "protein" are used interchangeably and are known in the art and can refer to any peptide-bonded chain of amino acids, regardless of length or post-translational modification.

[0060] II. HSCT-TMA-associated biomarker proteins As used herein, the term "biomarker" refers to a Biomarkers refer to measurable substances in a subject whose presence indicates some phenomenon, such as disease, infection, or environmental exposure. In accordance with the present disclosure, certain biomarkers have been discovered to be associated with and indicative of HSCT-TMA. These HSCT-TMA-associated biomarker proteins include, for example, thrombomodulin (TM), syndecan-1 (SYND1), and proteolytic fragments of complement component factor B (e.g., Ba).

[0061] A. Thrombomodulin Thrombomodulin (also known as "TM," "THBD," "AHUS6," "BDCA3," "CD141," "THPH12," "THRM," and "BDCA-3") is a transmembrane glycoprotein located on the luminal surface of endothelial cells in most normal blood vessels and regulates coagulation and inflammation (see, e.g., Califano, et al., Eur. Rev. Med. Pharmacol. Sci. 2000 May-Jun;4(3):59-66). Thrombomodulin is also expressed on human mesothelial cells, monocytes, and a subset of dendritic cells. Thrombomodulin can stimulate endothelial cell proliferation. This characteristic of TM depends on a molecular substrate sequence known as EGF-like (epidermal growth factor-like). One of the primary roles of TM is to bind to thrombin. Upon binding to TM, thrombin loses its procoagulant, proinflammatory, and profibrogenic properties and instead acquires the ability to activate protein C (APC). APC limits further thrombin generation, counteracts the harmful effects of thrombin, and has additional anti-inflammatory and cytoprotective properties. TM itself also has an inherent anti-inflammatory effect by binding to and inhibiting the pro-inflammatory protein HMGB1. Thrombomodulin exists in the body in two forms (see, e.g., Doggen et al., Thromb. Haemost. 1998;80:743-748). The first type has a higher molecular weight and is bound to the plasma membrane of endothelial cells (see, e.g., Boff MC, Haemostasis 1996;26(Suppl 4):233-243). The second form has a lower molecular weight and represents the soluble or plasma form. The heavy form weighs 150 kDa, and the light form weighs 69 kDa.The molecular sequence of thrombomodulin contains an N-terminal lectin-like element (residues 1-154), a hydrophobic region (residues 155-222), six EGF-like modules (residues 223-462), one Ser / Thr-rich domain (residues 463-497), a 23-amino acid transmembrane segment (residues 498-521), and a 35-amino acid cytoplasmic tail (residues 522-557) (see, e.g., Califano, et al., Eur. Rev. Med. Pharmacol. Sci. 2000 May-Jun;4(3):59-66).

[0062] Different pathological conditions increase soluble circulating TM (see, e.g., Califano, et al., Eur. Rev. Med. Pharmacol. Sci. 2000 May-Jun;4(3):59-66). Endothelial cells further exposed to hemodynamic disturbances (such as those on the branches of major arteries) release large amounts of TM (see, e.g., Salomaa et al., Lancet 1999;353:1729-1734). Thrombomodulin levels range from 3 to 300 ng / mL. Normal levels are 3.1±1.3 ng / mL, and are thought to be slightly higher in men (see, e.g., Doggen et al., Thromb. Haemost. 1998;80:743-748). In women, TM levels appear to increase during menopause. Women with surgically induced menopause have soluble TM levels well above normal. After 6 weeks of hormone replacement therapy, a significant decrease in TM levels occurs (see, e.g., Neumann et al., Circulation 1995;92:748-755). Thrombomodulin levels vary according to race, with blacks appearing to have lower levels (see, e.g., Tohda et al., Arterioscler. Thromb. Vasc. Biol. 1998;18:1861-1869). Thrombomodulin levels are usually measured by ELISA.

[0063] In one embodiment, the normal reference range for TM in healthy patients (eg, patients without HSCT) is about 1.8 ng / mL to about 4.8 ng / mL. For example, in one embodiment, normal TM levels in a healthy patient are about 1.8 ng / mL, 1.9 ng / mL, 2.0 ng / mL, 2.1 ng / mL, 2.2 ng / mL, 2.3 ng / mL, 2.4 ng / mL, 2.5 ng / mL, 2.6 ng / mL, 2.7 ng / mL, 2.8 ng / mL, 2.9 ng / mL, 3.0 ng / mL, 3.1 ng / mL, 3.2 ng / mL, 3.3 ng / mL, 3.4 ng / mL, 3.5 ng / mL, 3.6 ng / mL, 3.7 ng / mL, 3.8 ng / mL, 3.9 ng / mL, 4.0 ng / mL, 4.1 ng / mL, 4.2 ng / mL, 4.3 ng / mL, 4.4 ng / mL, 4.5 ng / mL, 4.6 ng / mL, 4.7 ng / mL, or 4.8 ng / mL.

[0064] In another embodiment, the normal reference range for TM for HSCT patients in the absence of TMA is about 3 ng / mL to about 9 ng / mL. For example, in one embodiment, the normal reference range for TM for HSCT patients in the absence of TMA is about 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, or 9 ng / mL.

[0065] In another embodiment, the level of TM is elevated in HSCT patients who are likely to have or develop HSCT-TMA compared to a normal reference range (e.g., healthy patients who have not had HSCT or HSCT patients in the absence of TMA). For example, elevated TM levels are greater than about 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, or 30 ng / mL.

[0066] In another embodiment, the level of TM in a sample is considered elevated if it is at least about 10% higher to about 10-fold, e.g., about 2-fold greater than the normal reference range for TM (e.g., a healthy patient without HSCT or an HSCT patient in the absence of TMA). In some embodiments, the level of TM in a sample is considered elevated if it is at least 3-fold, 4-fold, 5-fold, or 6-fold greater than the normal reference range for TM.

[0067] B. Syndecan-1 Syndecan-1 (also known as "SDC," "syndecan proteoglycan 1," "CD138," "SDC1," "SYND1," "syndecan," and "heparan sulfate proteoglycan fibroblast growth factor receptor"). SYND1 is a protein encoded by the SDC1 gene in humans (e.g., Mali M, et al., (April 1990), The Journal of Biological Chemistry, 265(12):6884-9; and Ala-Kapee M, et al. (September 1990), Somatic Cell and Molecular Genetics, 16(5):501-5). This protein is a transmembrane (type I) heparan sulfate proteoglycan and a member of the syndecan proteoglycan family. The SYND1 protein functions as an integral membrane protein and is involved in cell proliferation, cell migration, and cell-matrix interactions through its receptors for extracellular matrix proteins. SYND1 is a sponge for growth factors and chemokines, which bind primarily via heparan sulfate chains (see, e.g., Gotte M (April 2003), FASEB Journal. 17(6):575-91). Syndecans mediate cell binding, cell signaling, and cytoskeletal organization, and syndecan receptors mediate the regulation of HIV-1 It is required for the internalization of the tat protein. The SYND1 core protein consists of an extracellular domain that can be substituted with heparan sulfate and chondroitin sulfate glycosaminoglycan chains, a highly conserved transmembrane domain, and a highly conserved cytoplasmic domain containing two constant regions separated by a variable region (see, e.g., Bernfield M, (1999), Annual Review of Biochemistry. 68:729-77).The extracellular domain can be cleaved (shed) from the cell surface at a juxtamembrane site, converting the membrane-bound proteoglycan into a paracrine effector molecule with a role in wound repair and the invasive growth of cancer cells (see, e.g., Wang Z, Gotte M, Bernfield M, Reizes O (September 2005), Biochemistry. 44(37):12355-61, Elenius V, et al., The Journal of Biological Chemistry. 279(40):41928-35 and Piperigkou Z, (September 2016), Cell and Tissue Research. 365(3):643-55).

[0068] In one embodiment, the normal reference range for SYND1 in a healthy patient (e.g., a patient without HSCT) is 15 ng / mL to 70 ng / mL. For example, in one embodiment, normal SYND1 levels in a healthy patient are approximately 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, 31 ng / mL, 32 ng / mL, 33 ng / mL, 34 ng / mL, 35 ng / mL, 36 ng / mL, 37 ng / mL, 38 ng / mL, 39 ng / mL, 40 ng / mL, 41 ng / mL, 42 ng / mL, 43 ng / mL, 44 ng / mL, 45 ng / mL, 46 ng / mL, 47 ng / mL, 48 ng / mL, 49 ng / mL, 50 ng / mL, 51 ng / mL, 52 ng / mL, 53 ng / mL, 54 ng / mL, 55 ng / mL, 56 ng / mL, 57 ng / mL, 58 ng / mL, 59 ng / mL, 60 ng / mL, 61 ng / mL, 62 ng / mL, 63 ng / mL, 64 ng / mL, 65 ng / mL, 66 ng / mL, 67 ng / mL, 68 ng / mL, 69 ng / mL, 70 ng g / mL, 42ng / mL, 43ng / mL, 44ng / mL, 45ng / mL, 46ng / mL, 47ng / mL, 48ng / mL, 49ng / mL, 50ng / mL, 51ng / mL, 52ng / mL, 53ng / mL, 54ng / mL, 55ng / mL, 56ng / mL, 57ng / mL, 58ng / mL, 59ng / mL, 60ng / mL, 61ng / mL, 62ng / mL, 63ng / mL, 64ng / mL, 65ng / mL, 66ng / mL, 67ng / mL, 68ng / mL, 69ng / mL, or 70ng / mL.

[0069] In another embodiment, the normal reference range for SYND1 for HSCT patients in the absence of TMA is about 15 ng / mL to 55 ng / mL. For example, in one embodiment, the normal reference range for SYND1 for HSCT patients in the absence of TMA is about 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, 31 ng / mL, 32 ng / mL, 33ng / mL, 34ng / mL, 35ng / mL, 36ng / mL, 37ng / mL, 38ng / mL, 39ng / mL, 40ng / mL, 41ng / mL, 42ng / mL, 43ng / mL, 44ng / m L, 45ng / mL, 46ng / mL, 47ng / mL, 48ng / mL, 49ng / mL, 50ng / mL, 51ng / mL, 52ng / mL, 53ng / mL, 54ng / mL, or 55ng / mL.

[0070] In another embodiment, levels of SYND1 are elevated in HSCT patients who are likely to have or develop HSCT-TMA compared to a normal reference range (e.g., healthy patients without HSCT or HSCT patients in the absence of TMA). For example, elevated SYND1 levels are greater than about 100ng / mL, 105ng / mL, 110ng / mL, 115ng / mL, 120ng / mL, 125ng / mL, 130ng / mL, 135ng / mL, 140ng / mL, 145ng / mL, 150ng / mL, 155ng / mL, 160ng / mL, 165ng / mL, 170ng / mL, 175ng / mL, 180ng / mL, 185ng / mL, 190ng / mL, 195ng / mL, 200ng / mL, 205ng / mL, 210ng / mL, 215ng / mL, 220ng / mL, 225ng / mL, 230ng / mL, 235ng / mL, 240ng / mL, 245ng / mL, or 250ng / mL.

[0071] In another embodiment, the level of SYND1 in a sample is considered elevated if it is at least about 2-fold to about 17-fold, e.g., about 4-fold, greater than the normal reference range for SYND1 (e.g., a healthy patient without HSCT or a patient with HSCT in the absence of TMA). In some embodiments, the level of SYND1 in a sample is considered elevated if it is at least 3, 4, 5, 6, 7, 8, 10, 12, or 15-fold greater than the normal reference range for SYND1.

[0072] C. Factor Ba Factor Ba is a fragment of complement factor B. Factor B is a glycosylated protein composed of a single 93,000 Da polypeptide chain and is an essential component of the alternative pathway of complement activation. In the presence of magnesium, factor B binds to C3b. The C3b / B complex can be activated by factor D, a serine protease that circulates as an active trypsin-like serine protease. Cleavage of factor B by factor D results in the release of the Ba fragment (33,000 Da), leaving the 60,000 Da Bb fragment bound to C3b. This Ba fragment is derived from the N-terminus of factor B and contains three CCP domains that interact with C3b. Isolated fragment Ba has been reported to have weak affinity for C3b and inhibit the interaction of factor B with C3b, thus inhibiting activation of the alternative pathway.

[0073] In one embodiment, the normal reference range for factor Ba in a healthy patient (e.g., a patient without an HSCT) is less than about 1000 ng / mL. In another embodiment, the normal reference range for factor Ba in a healthy patient (e.g., a patient without an HSCT) is less than about 600 ng / mL. In another embodiment, the normal reference range for factor Ba in a healthy patient (e.g., a patient without an HSCT) is between about 300 ng / mL and 600 ng / mL. For example, a normal reference range for Factor Ba in a healthy patient (e.g., a patient without HSCT) is about 300ng / mL, 310ng / mL, 320ng / mL, 330ng / mL, 340ng / mL, 350ng / mL, 360ng / mL, 370ng / mL, 380ng / mL, 390ng / mL, 400ng / mL, 410ng / mL, 420ng / mL, 430ng / mL, 440ng / mL, 450ng / mL, 460ng / mL, 470ng / mL, 480ng / mL, 490ng / mL, 500ng / mL, 510ng / mL, 520ng / mL, 530ng / mL, 540ng / mL, 550ng / mL, 560ng / mL, 570ng / mL, 580ng / mL, 590ng / mL, or 600ng / mL.

[0074] In another embodiment, the normal reference range for Factor Ba for HSCT patients in the absence of TMA is about 500 ng / mL to 800. For example, in one embodiment, the normal reference range for Factor Ba for HSCT patients in the absence of TMA is about 500ng / mL, 510ng / mL, 520ng / mL, 530ng / mL, 540ng / mL, 550ng / mL, 560ng / mL, 570ng / mL, 580ng / mL, 590ng / mL, 600ng / mL, 610ng / mL, 620ng / mL, 630ng / mL, 640ng / mL, 650ng / mL, 660ng / mL, 670ng / mL, 680ng / mL, 690ng / mL, 700ng / mL, 710ng / mL, 720ng / mL, 730ng / mL, 740ng / mL, 750ng / mL, 760ng / mL, 770ng / mL, 780ng / mL, 790ng / mL, or 800ng / mL.

[0075] In another embodiment, the level of factor Ba is elevated in HSCT patients who have or are likely to develop HSCT-TMA compared to a normal reference range (e.g., healthy patients without HSCT or HSCT patients in the absence of TMA). For example, elevated Ba levels may be about 900ng / mL, 910ng / mL, 920ng / mL, 930ng / mL, 940ng / mL, 950ng / mL, 960ng / mL, 970ng / mL, 980ng / mL, 990ng / mL, 1000ng / mL, 1010ng / mL, 1020ng / mL, 1030ng / mL, 1040ng / mL, 1050ng / mL, 1060ng / mL, 1070ng / mL, 1080ng / mL, 1090ng / mL, 1100ng / mL, 1110ng / mL, 1120ng / mL, 1130ng / mL, 1140ng / mL, 1150ng / mL, 1160ng / mL, 1170ng / mL, 1180ng / mL, 1190ng / mL, 1200ng / mL, 1210ng / mL, 1220ng / mL, 1230ng / mL, 1240ng / mL, 1250ng / mL, 1260ng / mL, 1270ng / mL, 1280ng / mL, 1290ng / mL, 1300ng / mL, 1310ng / mL, 1320ng / mL, 1330ng / mL, 1340ng / mL, 1350ng / mL, 1360ng / mL, 1370ng / mL, 1380ng / mL, 1390ng / mL, 14 g / mL, 1120ng / mL, 1130ng / mL, 1140ng / mL, 1150ng / mL, 1160ng / mL, 1170ng / mL, 1180ng / mL, 1190ng / mL, 1200ng / mL, 1210ng / mL, 1220ng / mL, 1230ng / mL, 1240ng / mL, 1250ng / mL, 1260ng / mL, 1270ng / mL, 1280ng / mL, 1290ng / mL, 1300ng / mL, 1310ng / mL, 1320ng / mL, 1330ng / mL, 1340ng / mL, 1350ng / mL, 1360ng / mL, 1370ng / mL, 1380ng / mL, 1390ng / mL, 1400ng / mL, 1410ng / mL, 1420ng / mL, 1430ng / mL, 1440ng / mL, 1450ng / mL, 1460ng / mL, 1470ng / mL, 1480ng / mL, 1490ng / mL, 1500ng / mL, 1510ng / mL, 1520ng / mL, 1530ng / mL, 1540ng / mL, 1550ng / m L, 1560ng / mL, 1570ng / mL, 1580ng / mL, 1590ng / mL, 1600ng / mL, 1610ng / mL, 1620ng / mL, 1630ng / mL, 1640ng / mL, 1650ng / mL, 1660ng / mL , 1670ng / mL, 1680ng / mL, 1690ng / mL, 1700ng / mL, 1710ng / mL, 1720ng / mL, 1730ng / mL, 1740ng / mL, 1750ng / mL, 1760ng / mL, 1770ng / mL,1780ng / mL, 1790ng / mL, 1800ng / mL, 1810ng / mL, 1820ng / mL, 1830ng / mL, 1840ng / mL, 1850ng / mL, 1860ng / mL, 18 70ng / mL, 1880ng / mL, 1890ng / mL, 1900ng / mL, 1910ng / mL, 1920ng / mL, 1930ng / mL, 1940ng / mL, 1950ng / mL, 1960 ng / mL, 1970ng / mL, 1980ng / mL, 1990ng / mL, 2000ng / mL, 2010ng / mL, 2020ng / mL, 2030ng / mL, 2040ng / mL, 2050ng / mL, 2060ng / mL, 2070ng / mL, 2080ng / mL, 2090ng / mL, 2100ng / mL, 2110ng / mL, 2120ng / mL, 2130ng / mL, 2140ng / m L, 2150ng / mL, 2160ng / mL, 2170ng / mL, 2180ng / mL, 2190ng / mL, 2200ng / mL, 2210ng / mL, 2220ng / mL, 2230ng / mL, 2240ng / mL, 2250ng / mL, 2260ng / mL, 2270ng / mL, 2280ng / mL, 2290ng / mL, 2300ng / mL, 2310ng / mL, 2320ng / mL, 23 Greater than 30ng / mL, 2340ng / mL, 2350ng / mL, 2360ng / mL, 2370ng / mL, 2380ng / mL, 2390ng / mL, 2400ng / mL, 2410ng / mL, 2420ng / mL, 2430ng / mL, 2440ng / mL, 2450ng / mL, 2460ng / mL, 2470ng / mL, 2480ng / mL, 2490ng / mL, or 2500ng / mL.

[0076] In one embodiment, the level of Ba in a sample is considered elevated if it is at least about 12% to about 5-fold, e.g., about 2-fold, greater than the normal reference range for factor Ba (e.g., a healthy patient without HSCT or an HSCT patient in the absence of TMA). In some embodiments, the level of Ba in a sample is considered elevated if it is at least 2-fold, 3-fold, 4-fold, or 5-fold greater than the normal reference range for Ba.

[0077] D. Heparan sulfate proteoglycans (HSPGs) Heparan sulfate proteoglycans (HSPGs) are glycoproteins that share the common feature of containing one or more covalently linked heparan sulfate (HS) chains, a type of glycosaminoglycan (GAG) (see, e.g., Esko et al., Proteoglycans and Sulfated Glycosaminoglycans. In Essentials of glycobiology (ed. Varki A, et al.), pp. 229-248, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Heparan sulfate proteoglycans are found on cell surfaces and in the extracellular matrix, where they interact with a plethora of ligands (see, e.g., Sarrazin et al., Cold Spring Harbor Perspective Biol. 2011 Jul;3(7):a004952). Although few in number, heparan sulfate proteoglycans have profound effects at the cellular, tissue, and organismal levels. Cells possess a relatively small set of HSPGs (approximately 17), which can be divided into three groups depending on their location: membrane HSPGs, such as syndecans and glycosylphosphatidylinositol-anchored proteoglycans (glypicans), secreted extracellular matrix HSPGs (agrin, perlecan, type XVIII collagen), and the secretory vesicle proteoglycan, serglycin (see, e.g., Sarrazin et al., 2011).

[0078] In one embodiment, the level of HSPG is elevated in HSCT patients who are likely to have or develop HSCT-TMA compared to a normal reference range (e.g., healthy patients without HSCT or HSCT patients in the absence of TMA).

[0079] In another embodiment, the level of HSPG in a sample is considered elevated if it is at least about 20% to about 2-fold, e.g., about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold greater than the normal reference range for HSPG (e.g., healthy patients without HSCT or HSCT patients in the absence of TMA).

[0080] E. Biomarker Combinations Due in part to the enhanced prognostic and diagnostic significance of biomarkers when used in combination, multiple (e.g., at least two, three, or more) biomarkers may be detected and measured in accordance with the present disclosure. Such combinations of biomarkers may be referred to herein as signatures.

[0081] In one embodiment, the detecting step may include detecting a biomarker signature comprising the following biomarkers: (a) TM+Ba, optionally together with C5b9; (b) TM+SYND1, optionally together with C5b9; (c) SYND1+Ba, optionally together with C5b9; (d) TM+Ba, optionally together with HSPGs; or (e) TM+SYND1, optionally together with HSPGs; (f) SYND1+Ba, optionally together with HSPGs.

[0082] In one embodiment, the detecting step may comprise detecting a biomarker signature comprising the following biomarkers: TM+SYND1+Ba, optionally together with C5b9 and / or HSPG.

[0083] In one embodiment, the detecting step may include detecting a biomarker signature comprising TM+SYND1+Ba+C5b9+HSPG.

[0084] III. Anti-C5 antibody Any suitable anti-C5 antibody or antigen-binding fragment thereof can be used in the methods described herein. Anti-C5 antibodies (or VH / VL domains derived therefrom) suitable for use in the methods described herein can be generated using methods known in the art. Alternatively, art-recognized anti-C5 antibodies can be used. Antibodies that compete with any of these art-recognized antibodies or the antibodies described herein for binding to C5 can also be used. In some embodiments, the anti-C5 antibodies described herein bind to complement component C5 (e.g., human C5) and inhibit cleavage of C5 into fragments C5a and C5b.

[0085] An exemplary anti-C5 antibody is eculizumab. Eculizumab (also known as SOLIRIS®) is an anti-C5 antibody comprising heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. Ravulizumab comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8. Ravulizumab comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 10 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 11.

[0086] An exemplary anti-C5 antibody is ravulizumab, or an antigen-binding fragment or variant thereof, comprising heavy and light chains having the sequences set forth in SEQ ID NOs: 14 and 11, respectively. Ravulizumab (also known as ULTOMIRIS®, BNJ441, and ALXN1210) is described in WO 2015134894 and U.S. Patent No. 9,079,949, the entire teachings of which are incorporated herein by reference. The terms ravulizumab, BNJ441, and ALXN1210 may be used interchangeably throughout this specification and all refer to the same antibody. Ravulizumab selectively binds to the human complement protein C5 and inhibits its cleavage into C5a and C5b during complement activation. This inhibition prevents the release of the proinflammatory mediator C5a and the formation of the cytolytic pore-forming membrane attack complex (MAC) C5b-9, while preserving the proximal or early components of complement activation (e.g., C3 and C3b) essential for microbial opsonization and immune complex clearance.

[0087] The polypeptide sequence of ravulizumab registered in the KEGG DRUG database (https: / / www.kegg.jp / entry / D11054) provides that the N-terminal amino acid of the variable heavy chain is "X," but the database does not state what X is. The Chemical Abstracts (CAS) for ravulizumab (CAS 1803171-55-2) also specifies that the N-terminal X is pyroglutamic acid (designated "chain 1 pyroglutamic acid-1" in the CAS report).Although this information may appear to be different from the VH sequence of ravulizumab (e.g., a heavy chain variable region polypeptide comprising the amino acids set forth in SEQ ID NO: 12 and / or a heavy chain polypeptide comprising the amino acids set forth in SEQ ID NO: 14), alignments exist between the patented sequences and drug database / CAS sequences because it was recognized in the art that the N-terminal Q in the polypeptide and / or antibody sequence cyclizes during process development, resulting in nearly 100% drug product conversion to pyroglutamate (Pryo-Q) (as disclosed in Liuet et al. (J Pharm Sci. 2019 Oct;108(10):3194-3200) http: / / pubmed.ncbi.nlm.nih.gov / 31145921 / and Nguyen et al. (Int. J. Mol. Sci. 2017 Jul) 20;18(7):1575) http: / / www.researchgate.net / figure / Cyclization-reactions-of-N-terminal-glutamine-and-glutamate-residues-in-a-polypeptide_fig4_318926365. For additional information, see page 7 and Table 4 of Xu et al. (MAbs, 2019 Feb / Mar;11(2):239-264), and the following publications: (1) Yu et al., "Investigation of N-terminal glutamate cyclization of recombinant monoclonal antibody in formulation development," J. Pharm. Biomed. Anal., 2006, 42, 455-463 and Dick et al., "Determination of the origin of the N-terminal pyro-glutamate variation in monoclonal antibodies using model peptides”, Biotechnol. Bioeng., 2007, 97, 544-553 (the disclosures of which are incorporated herein by reference in their entireties).

[0088] In other embodiments, the antibody comprises the heavy and light chain CDRs or variable regions of ravulizumab. Thus, in one embodiment, it comprises the CDR1, CDR2, and CDR3 domains of the VH region of ravulizumab having the sequence set forth in SEQ ID NO: 12, and the CDR1, CDR2, and CDR3 domains of the VL region of ravulizumab having the sequence set forth in SEQ ID NO: 8. In another embodiment, the antibody comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 19, 18, and 3, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. In another embodiment, the antibody comprises VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 8, respectively.

[0089] Another exemplary anti-C5 antibody is antibody BNJ421, or antigen-binding fragments and variants thereof, comprising heavy and light chains having the sequences set forth in SEQ ID NOs: 20 and 11, respectively. BNJ421 (also known as ALXN1211) is described in WO2015134894 and U.S. Pat. No. 9,079,949, the entire teachings of which are incorporated herein by reference.

[0090] In other embodiments, the antibody comprises the heavy and light chain CDRs or variable regions of BNJ421. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of BNJ421 having the sequence set forth in SEQ ID NO: 12, and the CDR1, CDR2, and CDR3 domains of the VL region of BNJ421 having the sequence set forth in SEQ ID NO: 8. In another embodiment, the antibody comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 19, 18, and 3, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. In another embodiment, the antibody comprises VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 8, respectively.

[0091] The exact boundaries of CDRs are defined differently according to different methods. In some embodiments, the locations of CDRs or framework regions within a light or heavy chain variable domain are as defined by Kabat et al. [(1991) "Sequences of Proteins of Immunological Interest." NIH Publication No. 91-3242, USDapartment of Health and Human Services, Bethesda, MD]. In such cases, a CDR may be referred to as a "Kabat CDR" (e.g., "Kabat LCDR2" or "Kabat HCDR1"). In some embodiments, the locations of the CDRs of a light chain variable region or a heavy chain variable region are as defined by Chothia et al. (Nature, 342:877-83, 1989). Accordingly, these regions may be referred to as "Chothia CDRs" (e.g., "Chothia LCDR2" or "Chothia HCDR3"). In some embodiments, the locations of the CDRs of light and heavy chain variable regions may be defined using the combined Kabat-Chothia definition. In such embodiments, these regions can be referred to as “combined Kabat-Chothia CDRs.” Thomas, C. et al. (Mol. Immunol., 33:1389-401, 1996) exemplifies the identification of CDR boundaries according to the Kabat and Chothia numbering schemes.

[0092] Another exemplary anti-C5 antibody is the 7086 antibody, described in U.S. Patent Nos. 8,241,628 and 8,883,158. In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of the 7086 antibody (see U.S. Patent Nos. 8,241,628 and 8,883,158). In another embodiment, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 21, 22, and 23, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 24, 25, and 26, respectively. In another embodiment, the antibody or antigen-binding fragment thereof comprises the VH region of the 7086 antibody having the sequence set forth in SEQ ID NO: 27, and the VL region of the 7086 antibody having the sequence set forth in SEQ ID NO: 28.

[0093] Another exemplary anti-C5 antibody is the 8110 antibody, which is also described in U.S. Patent Nos. 8,241,628 and 8,883,158. In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of the 8110 antibody. In another embodiment, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs:29, 30, and 31, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs:32, 33, and 34, respectively. In another embodiment, the antibody comprises the VH region of the 8110 antibody having the sequence set forth in SEQ ID NO:35, and the VL region of the 8110 antibody having the sequence set forth in SEQ ID NO:36.

[0094] Another exemplary anti-C5 antibody is the 305LO5 antibody, described in U.S. Patent No. 9,765,135. In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of the 305LO5 antibody. In another embodiment, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively. In another embodiment, the antibody comprises the VH region of the 305LO5 antibody having the sequence set forth in SEQ ID NO: 43, and the VL region of the 305LO5 antibody having the sequence set forth in SEQ ID NO: 44.

[0095] Another exemplary anti-C5 antibody is the SKY59 antibody (Fukuzawa, T. et al., Sci. Rep., 7:1080, 2017). In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of the SKY59 antibody. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 45 and a light chain comprising SEQ ID NO: 46.

[0096] In some embodiments, the anti-C5 antibody comprises the heavy and light chain variable regions or heavy and light chains of the REGN3918 antibody (see U.S. Patent No. 10,633,434). In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises the heavy chain variable region sequence set forth in SEQ ID NO: 47 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 48. In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises the heavy chain sequence set forth in SEQ ID NO: 49 and the light chain sequence set forth in SEQ ID NO: 50.

[0097] In some embodiments, the anti-C5 antibody is a biosimilar of eculizumab (SOLIRIS®). For example, in one embodiment, the anti-C5 antibody is ABP 959 antibody (eculizumab biosimilar, manufactured by Amgen Inc., USA), ELIZARIA® (eculizumab biosimilar, manufactured by Generium JNC, Russia), SB12 (eculizumab biosimilar, manufactured by Samsung Bioepis, Incheon, South Korea), ISU305 (eculizumab biosimilar, manufactured by ISU Abxis, South Korea), ABLYZE® (eculizumab biosimilar, manufactured by CinnaGen, Iran), BCD 148 (eculizumab biosimilar, manufactured by Biocad Medical, Canada), tesidolumab (manufactured by Novartis), crovalimab (manufactured by Roche), CAN106 (manufactured by CANBridge Pharmaceuticals, China), or pozelimab (manufactured by Regeneron).

[0098] In some embodiments, the anti-C5 antibodies described herein comprise a heavy chain CDR1 comprising or consisting of the following amino acids: GHIFSNYWIQ (SEQ ID NO: 19). In some embodiments, the anti-C5 antibodies described herein comprise a heavy chain CDR2 comprising or consisting of the following amino acids: EILPGSGHTEYTENFKD (SEQ ID NO: 18). In some embodiments, the anti-C5 antibodies described herein comprise a heavy chain variable region comprising the following amino acid sequence: QVQLVQSGAE VKKPGASVKV SCKASGHIFS NYWIQWVRQA PGQGLEWMGE ILPGSGHTEY TENFKDRVTM TRDTSTSTVY MELSSLRSED TAVYYCARYF FGSSPNWYFD VWGQGTLVTV SS (SEQ ID NO: 12).

[0099] In some embodiments, the anti-C5 antibodies described herein comprise a light chain variable region comprising the following amino acid sequence: DIQMTQSPSS LSASVGDRVT ITCGASENIY GALNWYQQKP GKAPKLLIYG ATNLADGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQN VLNTPLTFGQ GTKVEIK (SEQ ID NO: 8).

[0100] In some embodiments, the anti-C5 antibodies described herein can comprise a variant human Fc constant region that binds to the human neonatal Fc receptor (FcRn) with higher affinity than the affinity of the native human Fc constant region from which the variant human Fc constant region is derived. The Fc constant region can comprise one or more amino acid substitutions (e.g., two, three, four, five, six, seven, or eight or more) compared to the native human Fc constant region from which the variant human Fc constant region is derived. The substitutions can increase the binding affinity of an IgG antibody containing the variant Fc constant region for FcRn at pH 6.0 while maintaining the pH dependence of the interaction. Methods for testing whether one or more substitutions in an antibody's Fc constant region increase the affinity of the Fc constant region for FcRn at pH 6.0 (while maintaining the pH dependence of the interaction) are known in the art and are exemplified in the Examples. See, for example, WO 2015134894 and U.S. Pat. No. 9,079,949. The disclosure of each of these is incorporated herein by reference in its entirety.

[0101] Substitutions that enhance the binding affinity of an antibody Fc constant region to FcRn are known in the art and include, for example: (1) M252Y / S254T / T256E triple substitution (Dall'Acqua, W. et al., J. Biol. Chem., 281:23514-24, 2006); (2) M428L or T250Q / M428L substitution (Hinton, P. et al. al., J. Biol. Chem., 279:6213-6, 2004; Hinton, P. et al., J. Immunol., 176: 346-56, 2006); and (3) N434A or T307 / E380A / N434A substitution (Petkova, S. et al. al., Int. Immunol., 18:1759-69, 2006). Additional substitution pairs: P257I / Q311I, P257I / N434H, and D376V / N434H (Datta-Mannan, A. et al., J. Biol. Chem., 282:1709-17, 2007), the disclosures of each of which are incorporated herein by reference in their entirety.

[0102] In some embodiments, the mutant constant region has a substitution at EU amino acid residue position 255 for valine. In some embodiments, the mutant constant region has a substitution at EU amino acid position 309 for asparagine. In some embodiments, the mutant constant region has a substitution at EU amino acid position 312 for isoleucine. In some embodiments, the mutant constant region has a substitution at EU amino acid position 386.

[0103] In some embodiments, the variant Fc constant region comprises 30 or fewer (e.g., 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, or 2 or fewer) amino acid substitutions, insertions, or deletions compared to the native constant region from which it is derived. In some embodiments, the variant Fc constant region comprises one or more amino acid substitutions selected from the group consisting of M252Y, S254T, T256E, N434S, M428L, V259I, T250I, and V308F. In some embodiments, the variant human Fc constant region comprises a methionine at position 428 and an asparagine at position 434 of the native human IgG Fc constant region, each according to EU numbering. In some embodiments, the variant Fc constant region comprises a 428L / 434S double substitution, for example, as described in US Pat. No. 8,088,376.

[0104] In some embodiments, the exact locations of these mutations may be shifted from those of the native human Fc constant region by antibody engineering. For example, the 428L / 434S double substitution, when used in an IgG2 / 4 chimeric Fc, may correspond to 429L and 435S as in the M429L and N435S variant found in ravulizumab and described in U.S. Patent No. 9,079,949, the disclosure of which is incorporated herein by reference in its entirety.

[0105] In some embodiments, the variant constant region comprises a substitution at amino acid position 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434 or 436 (EU numbering) compared to a native human Fc constant region. In some embodiments, the substitution is selected from the group consisting of: methionine for glycine at position 237; alanine for proline at position 238; lysine for serine at position 239; isoleucine for lysine at position 248; alanine, phenylalanine, isoleucine, methionine, glutamine, serine, valine, tryptophan, or tyrosine for threonine at position 250; phenylalanine, tryptophan, or tyrosine for methionine at position 252; threonine for serine at position 254; glutamic acid for arginine at position 255; aspartic acid, glutamic acid, or glutamine for threonine at position 256; alanine, glycine, isoleucine, leucine, methionine, asparagine, serine, threonine, or valine for proline at position 257; glutamic acid for arginine at position 258; histidine in place of aspartic acid; alanine in place of aspartic acid at position 265; phenylalanine in place of aspartic acid at position 270; alanine or glutamic acid in place of asparagine at position 286; histidine in place of threonine at position 289; alanine in place of asparagine at position 297; glycine in place of serine at position 298; alanine in place of valine at position 303; alanine in place of valine at position 305; alanine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan, or tyrosine in place of threonine at position 307; alanine, phenylalanine, isoleucine, leucine, methionine, proline, glutamine, or threonine in place of valine at position 308;alanine, aspartic acid, glutamic acid, proline, or arginine for leucine or valine at position 309; alanine, histidine, or isoleucine for glutamine at position 311; alanine or histidine for aspartic acid at position 312; lysine or arginine for leucine at position 314; alanine or histidine for asparagine at position 315; alanine for lysine at position 317; glycine for asparagine at position 325; valine for isoleucine at position 332; leucine for lysine at position 334; histidine for lysine at position 360; alanine for aspartic acid at position 376; alanine for glutamic acid at position 380; alanine for glutamic acid at position 382; or asparagine or serine at position 384. alanine instead of glycine; aspartic acid or histidine instead of glycine at position 385; proline instead of glutamine at position 386; glutamic acid instead of proline at position 387; alanine or serine instead of asparagine at position 389; alanine instead of serine at position 424; alanine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, or tyrosine instead of methionine at position 428; lysine instead of histidine at position 433; alanine, phenylalanine, histidine, serine, tryptophan, or tyrosine instead of asparagine at position 434; and histidine instead of tyrosine or phenylalanine at position 436 (all according to EU numbering).

[0106] Anti-C5 antibodies suitable for use in the methods described herein, in some embodiments, comprise a heavy chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14 and / or a light chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 11. Alternatively, anti-C5 antibodies for use in the methods described herein, in some embodiments, comprise a heavy chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 20 and / or a light chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 11.

[0107] In one embodiment, the antibody has an affinity dissociation constant (K) of at least 0.1 (e.g., at least 0.15, 0.175, 0.2, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, or 0.975) nM. D ) and binds to C5 at pH 7.4 and 25°C (and otherwise under physiological conditions). In one embodiment, the antibody has an affinity dissociation constant (K) of about 0.5 nM at pH 7.4 and 25°C (and otherwise under physiological conditions). D In some embodiments, the K D is 1 nM or less (e.g., 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2 nM or less). In some embodiments, the antibody has a K of about 22 nM at pH 6.0 and 25°C (and otherwise under physiological conditions). D binds to C5.

[0108] In another embodiment, the K of an antibody against C5 at pH 6.0 and 25°C D ) / (K of antibody against C5 at pH 7.4 and 25°C D )] is greater than 21 (e.g., 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 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, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910 (greater than 0, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, or 8000).

[0109] Methods for determining whether an antibody binds to a protein antigen and / or the affinity of an antibody for a protein antigen are known in the art. Binding of antibodies to protein antigens can be detected and / or quantified using a variety of techniques, including, but not limited to, Western blot, dot blot, surface plasmon resonance (SPR) detection (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ), or enzyme-linked immunosorbent assay (ELISA; Benny KC Lo (2004) "Antibody Engineering: Methods and Protocols," Humana Press (ISBN: 1588290921); Johne, B. et al., J. Immunol. Meth., 160:191-8, 1993; Jonsson, U. et al., Ann. Biol. Clin., 51:19-26, 1993; Jonsson, U. et al., Biotechniques, 11:620-7, 1991). Further methods for measuring affinity (eg, dissociation and binding constants) are described in the Examples.

[0110] As used herein, "k a The term "k" refers to the rate constant for the association of an antibody to an antigen. d The term "K" refers to the rate constant for dissociation of an antibody from the antibody / antigen complex. D The term "antibody-antigen interaction equilibrium dissociation constant" refers to the equilibrium dissociation constant of the antibody-antigen interaction. The equilibrium dissociation constant is the ratio of the kinetic rate constants, K D =k a / k d Such determinations can be made, for example, at 25°C or 37°C (see Examples). The kinetics of antibody binding to human C5 can be determined, for example, by SPR on a BIAcore 3000 instrument at pH 8.0, 7.4, 7.0, 6.5, and 6.0 using an anti-Fc capture method to immobilize the antibody.

[0111] In one embodiment, the anti-C5 antibody or antigen-binding fragment thereof blocks the cleavage of C5 into C5a and C5b, which inhibits, for example, the pro-inflammatory effects of C5a and the generation of the C5b-9 membrane attack complex (MAC) at the cell surface.

[0112] Methods for determining whether a particular antibody described herein inhibits C5 cleavage are known in the art. Inhibition of human complement component C5 can reduce the cytolytic ability of complement in a subject's body fluid. Such a reduction in the lytic ability of complement present in a body fluid can be measured, for example, by a conventional hemolytic assay, such as a hemolytic assay (Kabat and Mayer (eds.), "Experimental Immunochemistry, 2002, Vol. 1, No. 1, pp. 111-114, 2002). nd Edition," 135-240, Springfield, IL, CC Thomas (1961), pages 135-139), or conventional modifications of that assay, such as the chicken erythrocyte hemolysis assay (Hillmen, P. et al., N. Engl. J. Med., 350:552-9, 2004). Methods for determining whether a candidate compound inhibits the cleavage of human C5 into C5a and C5b forms are known in the art (Evans, M. et al., Mol. Immunol., 32:1183-95, 1995). The concentrations and / or physiological activities of C5a and C5b in body fluids can be measured, for example, by methods known in the art. For C5b, a hemolytic assay or the assay for soluble C5b-9 discussed herein can be used. Other assays known in the art can also be used. These or other suitable types of assays can be used to screen candidate agents capable of inhibiting human complement component C5.

[0113] Immunological techniques, including but not limited to ELISA, can be used to measure protein concentrations of C5 and / or its cleavage products to determine the ability of anti-C5 antibodies or antigen-binding fragments thereof to inhibit the conversion of C5 to biologically active products. In some embodiments, C5a production is measured. In some embodiments, C5b-9 neoepitope-specific antibodies are used to detect MAC formation.

[0114] A hemolytic assay can be used to determine the inhibitory activity of an anti-C5 antibody or its antigen-binding fragment on complement activation. To determine the effect of an anti-C5 antibody or its antigen-binding fragment on classical complement pathway-mediated hemolysis in vitro, for example, sheep red blood cells coated with hemolysin or chicken red blood cells sensitized with anti-chicken red blood cell antibodies are used as target cells. The percentage of lysis is normalized by considering 100% lysis to be equivalent to the lysis occurring in the absence of an inhibitor. In some embodiments, the classical complement pathway is activated by a human IgM antibody, such as that used in the Wieslab® Classical Pathway Complement Kit (Wieslab® COMPL CP310, Euro-Diagnostica, Sweden). Briefly, the test serum is incubated with an anti-C5 antibody or its antigen-binding fragment in the presence of a human IgM antibody. The amount of C5b-9 produced is measured by contacting the mixture with an enzyme-linked anti-C5b-9 antibody and a fluorogenic substrate and measuring absorbance at an appropriate wavelength. As a control, test serum is incubated in the absence of anti-C5 antibody or an antigen-binding fragment thereof. In some embodiments, the test serum is C5-deficient serum reconstituted with C5 polypeptide.

[0115] To determine the effect of anti-C5 antibodies or their antigen-binding fragments on alternative pathway-mediated hemolysis, naive rabbit or guinea pig red blood cells can be used as target cells. In some embodiments, the serum test solution is C5-deficient serum reconstituted with C5 polypeptide. The percentage of lysis is normalized by considering 100% lysis to be equal to the lysis occurring in the absence of an inhibitor. In some embodiments, the alternative complement pathway is activated by lipopolysaccharide molecules, such as those utilized in the Wieslab® Alternative Pathway Complement Kit (Wieslab® COMPL AP330, Euro-Diagnostica, Sweden). Briefly, test serum is incubated with anti-C5 antibodies or their antigen-binding fragments in the presence of lipopolysaccharide. The amount of C5b-9 produced is measured by contacting the mixture with an enzyme-linked anti-C5b-9 antibody and a fluorogenic substrate and measuring the fluorescence at an appropriate wavelength. As a control, test serum is incubated in the absence of anti-C5 antibodies or their antigen-binding fragments.

[0116] In some embodiments, C5 activity or its inhibition is quantified using a CH50eq assay. The CH50eq assay is a method for measuring total classical complement activity in serum. This test is a lytic assay that uses antibody-sensitized red blood cells as an activator of the classical complement pathway, and various dilutions of test serum are used to determine the amount required to give 50% lysis (CH50). Percent hemolysis can be determined, for example, using a spectrophotometer. The CH50eq assay provides an indirect measure of terminal complement complex (TCC) formation, since the TCC itself is directly involved in the hemolysis being measured. The assay is known and commonly performed by those skilled in the art. Briefly, to activate the classical complement pathway, an undiluted serum sample (e.g., a reconstituted human serum sample) is added to a microassay well containing antibody-sensitized red blood cells, thereby generating TCC. The activated serum is then diluted in a microassay well coated with a capture reagent (e.g., an antibody that binds to one or more components of TCC). TCC present in the activated sample binds to the monoclonal antibody coating the surface of the microassay wells. The wells are washed, and a detectably labeled detection reagent that recognizes the bound TCC is added to each well. The detectable label can be, for example, a fluorescent label or an enzyme label. Assay results are expressed in CH50 unit equivalents per milliliter (CH50 U Eq / mL).

[0117] Inhibition, e.g., with respect to terminal complement activity, includes a decrease in terminal complement activity of at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%) in, e.g., a hemolytic assay or a CH50eq assay, compared to the effect of a control antibody (or antigen-binding fragment thereof) under similar conditions and at an equimolar concentration. Substantial inhibition, as used herein, refers to an inhibition of a given activity (e.g., terminal complement activity) of at least 40% (e.g., at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% or more). In some embodiments, the anti-C5 antibodies described herein contain one or more amino acid substitutions compared to the CDRs of eculizumab (i.e., SEQ ID NOS: 1-6), but retain at least 30% (e.g., at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95%) of the complement inhibitory activity of eculizumab in a hemolytic assay or a CH50eq assay.

[0118] The anti-C5 antibodies described herein have a serum half-life in humans of at least 20 days (e.g., at least 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, or 55 days). In another embodiment, the anti-C5 antibodies described herein have a serum half-life in humans of at least 40 days. In another embodiment, the anti-C5 antibodies described herein have a serum half-life in humans of about 43 days. In another embodiment, the anti-C5 antibodies described herein have a serum half-life in humans of 39-48 days. Methods for measuring the serum half-life of an antibody are known in the art. In some embodiments, the anti-C5 antibodies or antigen-binding fragments thereof described herein have a serum half-life that is at least 20% (e.g., at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 400, or 500%) greater than the serum half-life of eculizumab (e.g., as measured in one of the mouse model systems described in the Examples (e.g., C5-deficient / NOD / scid mice or hFcRn transgenic mouse model systems)).

[0119] In one embodiment, the antibody competes for binding to and / or binds to the same epitope on C5 as the antibodies described herein. The term "binds to the same epitope" with respect to two or more antibodies means that the antibodies bind to the same segment of amino acid residues, as determined by a given method. Techniques for determining whether an antibody binds to the same epitope on C5 as the antibodies described herein include epitope mapping methods, such as, for example, X-ray analysis of crystals of the antigen:antibody complex, and hydrogen / deuterium exchange mass spectrometry (HDX-MS). Other methods monitor antibody binding to peptide antigen fragments or mutational variations of the antigen; loss of binding due to alterations of amino acid residues within the antigen sequence is often considered an indication of epitope content. Furthermore, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage-displayed peptide libraries. Antibodies with the same VH and VL or the same CDR1, CDR2 and CDR3 sequences are expected to bind to the same epitope.

[0120] An antibody that "competes with another antibody for binding to a target" refers to an antibody that inhibits (partially or completely) the binding of the other antibody to a target. Whether two antibodies compete with each other for binding to a target (i.e., whether and to what extent one antibody inhibits the binding of the other antibody to a target) can be determined using known competition experiments. In certain embodiments, an antibody competes with the binding of another antibody to a target and inhibits it by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition can vary depending on which antibody is the "blocking antibody" (i.e., the antibody that is first incubated with the target). Competing antibodies can, for example, bind to the same epitope, overlapping epitopes, or adjacent epitopes (e.g., as evidenced by steric hindrance).

[0121] The antibody or antigen-binding fragment thereof may comprise 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.

[0122] The monoclonal antibodies disclosed herein can be of any isotype. The monoclonal antibodies can be, for example, IgM or IgG antibodies, e.g., IgG1 or IgG2. The class of an antibody that immunospecifically binds to C5b-9 can be switched to another class (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 (e.g., from IgG1 to IgG2).

[0123] 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 (SEQ ID NO: 51) or a Flag tag can be multimerized using Ni-NTA agarose (Qiagen) or an anti-Flag antibody (Stratagene, Inc.).

[0124] The antibodies of the present invention may be monospecific, bispecific, trispecific, or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of C5b-9 or a fragment 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. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; and 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).

[0125] The anti-C5 antibodies or antigen-binding fragments thereof described herein used in the methods described herein can be produced using a variety of art-recognized techniques. Monoclonal antibodies can be obtained by a variety of techniques well known to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, typically by fusion with myeloma cells (Kohler, G. & Milstein, C., Eur. J. Immunol., 6:511-9, 1976). Methods of immortalization include transformation with Epstein-Barr virus, oncogenes, or retroviruses, or other methods known in the art. Colonies arising from single immortalized cells are screened for the production of antibodies of the desired specificity and affinity to the antigen, and the yield of monoclonal antibodies produced by such cells can be enhanced by a variety of techniques, including intraperitoneal injection of a vertebrate host. Alternatively, DNA sequences encoding monoclonal antibodies or binding fragments thereof can be isolated by screening a DNA library derived from human B cells (Huse, W. et al., Science, 246:1275-81, 1989).

[0126] Antibodies that can be used in the methods described herein (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants of the invention) can be produced by any method known in the art for the synthesis of antibodies, in particular by chemical synthesis or, preferably, by recombinant expression techniques. Greenfield (Ed.) (2014) "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory: Cold Spring Harbor, NY

[0127] Single-chain Fvs (scFvs) that immunospecifically bind to a biomarker of the disclosure (e.g., TM, SYND1, Ba, 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 which carry the polynucleotide sequences encoding them. Examples of phage display methods that can be used to generate the antibodies of the invention include 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. See, 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.

[0128] 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, which can be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, bacteria, etc., as described below. Techniques for recombinantly producing Fab, Fab', and F(ab')2 fragments can also be used using methods known in the art (e.g., methods disclosed in WO 92 / 22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995)).

[0129] To generate whole antibodies, PCR primers containing the VH or VL nucleotide sequence, a restriction site, and flanking sequences to protect the restriction site can be used to amplify the VH or VL sequence in the scFv clone. 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, such as the human gamma 4 constant region, and the PCR-amplified VL domain can be cloned into a vector expressing a VL constant region, such as the human kappa or lambda constant region.

[0130] Once synthesized or recombinantly expressed, antibodies that may be used in the methods described herein may be purified by any method known in the art for purifying immunoglobulin molecules, and more generally protein molecules, such as chromatography (e.g., ion exchange, affinity, particularly for specific antigens after Protein A, and size exclusion column chromatography), centrifugation, fractional solubility, or by any other standard protein purification technique.

[0131] Vectors contain elements that facilitate manipulation for expression of foreign proteins in target host cells. Conveniently, manipulation of sequences for transformation and DNA generation is first performed in a bacterial host (e.g., E. coli), and vectors usually contain sequences that facilitate such manipulation, including a bacterial origin of replication and an appropriate bacterial selectable marker. Selectable markers encode proteins necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with a vector containing a selection gene will not survive in the culture medium.

[0132] Host cells used to express anti-sC5b-9 antibodies can be either bacterial cells (such as E. coli), yeast (such as S. cerevisiae), or eukaryotic cells (such as mammalian cell lines). Well-defined cell types for this purpose can be used, such as myeloma cells, 3T3, HeLa, C6A2780, Vero, MOCK II, Chinese hamster ovary (CHO), Sf9, Sf21, COS, NSO, or HEK293.

[0133] Antibodies that bind to a biomarker can be screened using any known method, e.g., binding assays. In a typical method, the target biomarker or its antigenic epitope is expressed in standard cells and antibodies are panned using selection techniques known in the art. Antibodies can be selected, for example, by screening for binding affinity, e.g., at least 10 -6 M, preferably 10 -8 M, especially 10 -10 Dissociation constant of M (K d ), where K d Values ​​can be determined using standard binding assays.

[0134] Various embodiments of the present disclosure are described in detail in the following non-limiting and representative examples.

[0135] In the Examples section and elsewhere, representative types of antibodies useful in practicing various embodiments of the present disclosure are provided, along with information regarding, for example, specific suppliers and / or catalog numbers. It should be understood that the present disclosure is not limited to exemplary embodiments utilizing antibody detection reagents from specific suppliers / manufacturers. Antibodies to the biomarkers / analytes of the present disclosure can be obtained from any manufacturer, such as, for example, Thermo Fisher catalog number MA5-24214 for anti-human TM antibody; catalog number 12-1389-42 for anti-human SYND1 antibody; and MA5-28083 for anti-human complement factor Ba antibody (all from Thermo Scientific, Waltham, MA). Antibodies can also be purchased commercially from 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). Conventional techniques (e.g., immunization of mammals such as mice or rabbits and / or hybridoma technology) can also be used to generate the desired antibodies or antisera.

[0136] Also provided herein are compositions comprising anti-C5 antibodies (or antigen-binding fragments thereof). The compositions can be formulated, for example, as pharmaceutical solutions for administration to subjects for the treatment of HSCT-TMA. Pharmaceutical compositions generally contain a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" means and includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible. The compositions can include pharmaceutically acceptable salts (e.g., acid addition salts or base addition salts), sugars, carbohydrates, polyols, and / or tonicity adjusting agents.

[0137] Compositions can be formulated according to standard methods. Pharmaceutical formulation is a well-established technique (see, e.g., Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20 th Edition, Lippincott, Williams & Wilkins (ISBN:0683306472); Ansel et al. (1999) "Pharmaceutical Dosage Forms and Drug Delivery Systems," 7 th Edition, Lippincott Williams & Wilkins Publishers (ISBN:0683305727); and Kibbe (2000) “Handbook of Pharmaceutical Excipients American Pharmaceutical Association,” 3 rd (See, for example, the "Issue of the Invention" Edition (ISBN: 091733096X)). In some embodiments, the compositions can be formulated, for example, as a buffer solution at an appropriate concentration and suitable for storage at 2-8°C (e.g., 4°C). In some embodiments, the compositions can be formulated for storage at temperatures below 0°C (e.g., -20°C or -80°C). In some embodiments, the compositions can be formulated for storage at 2-8°C (e.g., 4°C) for up to 2 years (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, or 2 years). Thus, in some embodiments, the compositions described herein are stable upon storage at 2-8°C (e.g., 4°C) for at least 1 year.

[0138] Pharmaceutical compositions can be in various forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form depends, in part, on the intended mode of administration and therapeutic application. Compositions containing compositions intended for systemic or local delivery can be, for example, in the form of an injectable or infusible solution. Thus, the composition can be formulated for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). As used herein, "parenteral administration," "parenterally administered," and other grammatically similar phrases refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid, and intrasternal injection and infusion.

[0139] IV. Anti-complement factor B antibodies In another embodiment, an anti-complement factor B antibody is used in the methods described herein. An anti-CFB antibody refers to an antibody that inhibits: (i) the expression by cells, or the proper intracellular transport or secretion of complement factor B protein; (ii) the activity of factor B cleavage fragment Ba or Bb (e.g., the binding of Bb to its complement factor C3b); (iii) the proper intracellular transport or secretion of complement factor B by cells; or (v) the stability of factor B protein or mRNA encoding factor B protein. Inhibition of complement factor B protein expression can include inhibition of transcription of the gene encoding human complement factor B protein; increased degradation of mRNA encoding complement factor B protein; inhibition of translation of mRNA encoding human complement factor B; increased degradation of human complement factor B protein; inhibition of the proper processing of prepro-human complement factor B protein; or inhibition of the proper transport or secretion of human complement factor B protein by cells. Methods for determining whether a candidate antibody is an inhibitor of human complement factor B are known in the art and are described herein.

[0140] V. Biological Samples Biological samples suitable for use in the methods described herein include, for example, any biological fluid. A biological sample can be, for example, a specimen obtained from a subject (e.g., a mammal such as a human) or can be derived from such a subject. A biological sample can also be a biological fluid such as urine, whole blood or a fraction thereof (e.g., plasma or serum), saliva, semen, sputum, cerebrospinal fluid, tears, or mucus. If desired, a biological sample can be further fractionated into fractions containing specific analytes of interest (e.g., proteins). For example, a whole blood sample can be fractionated into serum or into fractions containing specific types of proteins. Optionally, a biological sample can be a combination of different biological samples from a subject, such as a combination of two different fluids.

[0141] Biological samples suitable for the present invention may be fresh or frozen samples collected from a subject, or archived samples with known diagnostic, treatment, and / or outcome history. Biological samples can be obtained from a subject (e.g., a subject who has, is suspected of having, or is at risk of developing a complement-related disorder such as HSCT-TMA). Any suitable method for obtaining a biological sample can be used, but exemplary methods include, for example, phlebotomy, swabbing (e.g., oral swabbing), lavage, or fine needle aspiration biopsy procedures. Biological samples can also be obtained from bone marrow.

[0142] In some embodiments, a protein extract may be prepared from a biological sample. In some embodiments, the protein extract contains total protein content. Methods of protein extraction are well known in the art. See, for example, Roe (2001) "Protein Purification Techniques: A Practical Approach", 2001. ndEdition, Oxford University Press. Many different versatile kits are available for extracting proteins from body fluids and tissues, and are commercially available from, for example, BioRad Laboratories (Hercules, CA), BD Biosciences Clontech (Mountain View, CA), Chemicon International, Inc. (Temecula, CA), Calbiochem (San Diego, CA), Pierce Biotechnology (Rockford, IL), and Invitrogen Corp. (Carlsbad, CA).

[0143] Methods for obtaining and / or preserving biological samples that preserve the activity or integrity of cells in the sample are well known to those skilled in the art. For example, the biological sample can be further contacted with one or more additional agents (e.g., an appropriate buffer and / or inhibitors, including protease inhibitors) intended to preserve or minimize changes in protein structure (e.g., changes in osmolarity or pH). Such inhibitors include, for example, chelating agents such as ethylenediaminetetraacetic acid (EDTA) and ethylene glycol tetraacetic acid (EGTA), protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), aprotinin, and leupeptin. Suitable buffers and conditions for storing or otherwise manipulating whole cells 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.

[0144] The sample may be further processed to eliminate or minimize the presence of interfering substances. For example, a biological sample may be fractionated or purified to remove one or more substances (e.g., cells) that are not of interest. Methods for fractionating or purifying a biological sample include, but are not limited to, flow cytometry, fluorescence-activated cell sorting, and sedimentation.

[0145] In embodiments of the present disclosure, 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) on which a binding agent is immobilized.

[0146] The measurement of protein expression levels in biological samples can be carried out by any suitable method. For example, see Greenfield (Ed.) (2014) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory: Cold Spring Harbor, NY. Generally, protein levels are determined by contacting a biological sample obtained from a subject with a binding agent for a biomarker protein; detecting one or more levels of the biomarker protein that bind to the binding agent in the sample (e.g., biological fluid); and comparing the one or more levels of the biomarker protein in the sample with the level of the corresponding protein biomarker in a control sample (e.g., normal sample).

[0147] In certain embodiments, the biomarkers / analytes of the present disclosure can be detected via binding to a suitable binding agent, such as a ribosome (with or without a peptide component), an RNA molecule, or a polypeptide (e.g., a polypeptide comprising the polypeptide sequence of the protein marker, a peptide variant thereof, or a non-peptide mimetic of such a sequence). Suitable binding agents also include antibodies specific for the biomarker proteins described herein. Antibodies suitable for use in the methods of the present 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 used in the methods of the present 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.

[0148] The binding agent is directly or indirectly labeled with a detectable moiety. The role of the detectable agent (binding agent labeled with a detectable moiety) is to facilitate the detection step of the diagnostic method by allowing visualization of the complex formed by binding of the binding agent to the protein marker (or a fragment thereof). The detectable agent can be selected to generate a signal that can be measured, the intensity of which is analyzed, and which is related (preferably proportional) to the amount of protein marker present in the sample. Methods for labeling biological molecules (e.g., 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 present 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 (such as those used in ELISA, e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), colorimetric labels, magnetic labels, and biotin, digoxigenin, or other haptens and proteins for which antisera or monoclonal antibodies are available.

[0149] A binding agent (e.g., an antibody) can be immobilized on a carrier or support (e.g., beads, magnetic particles, latex particles, microtiter plate wells, cuvettes, or other reaction vessels). 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 film, plastic tubing, glass, polyamine-methylvinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, or combinations thereof. A binding agent can also 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).

[0150] Protein levels in biological samples can be determined using immunoassays. 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 histochemical tests, which are conventional methods well known in the art. The method of detecting and quantifying the signal generated by the complex formed by binding of the binding agent and the protein marker depends on the nature of the assay and the detectable moiety (e.g., fluorescent moiety).

[0151] In one example, the presence or amount of protein expression of a gene (e.g., TM or SYND1, or a combination thereof) can be determined using Western blotting techniques. For example, a lysate can be prepared from the biological sample, or the biological sample (e.g., a biological fluid) itself can be contacted with Laemmli buffer and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE-resolved proteins separated by size can then be transferred to a filter membrane (e.g., nitrocellulose) and subjected to immunoblotting techniques 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.

[0152] In one example, immunoassays can be used to detect and / or measure the protein expression of a biomarker protein (e.g., TM or SYND1, Ba, or fragments thereof). As described above, immunoassays can be performed using antibodies bearing a detection moiety (e.g., a fluorescent agent or an enzyme) for detection purposes. Proteins from a biological sample can be conjugated directly to a solid matrix (e.g., a multi-well assay plate, nitrocellulose, agarose, Sepharose®, coded particles, or magnetic beads) or to a first member of a specific binding pair (e.g., biotin or streptavidin) that binds to a solid matrix (e.g., streptavidin or biotin) upon binding to a second member of the specific binding pair. Binding to such a solid matrix allows the protein to be purified from other interfering or irrelevant components of the biological sample prior to contact with the detection antibody, and also allows for subsequent washing of unbound antibody. Here, the presence or amount of bound detectably labeled antibody, as described above, indicates the presence or amount of protein in the biological sample.

[0153] Alternatively, protein expression levels can be determined using mass spectrometry-based or image-based methods known in the art for detecting proteins. Other suitable methods include proteomics-based methods such as 2D gel electrophoresis, identification of individual proteins recovered from the gel (e.g., by mass spectrometry or N-terminal sequencing), and / or bioinformatics.

[0154] 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, a multiwell assay plate (e.g., 96-well or 386-well) or an array (e.g., a protein chip). Stock solutions for various reagents can be provided manually or robotically, and subsequent sample preparation, pipetting, dilution, mixing, dispensing, washing, incubation (e.g., hybridization), sample readout, data collection (optical data), and / or analysis (computer-assisted image analysis) can be performed robotically using commercially available analysis software, robotics, and detection equipment capable of detecting signals generated from the assay. Examples of such detectors include, but are not limited to, spectrophotometers, luminometers, fluorometers, and devices that measure radioisotope decay.

[0155] VI. Treatment Methods Also provided herein are methods for treating HSCT-TMA in a subject. In one embodiment, a method is provided for treating a patient (e.g., a pediatric or adult patient) with HSCT-TMA who has been determined to have elevated levels (e.g., blood or plasma levels) of a biomarker selected from TM and SYND1, or a combination thereof, compared to the normal reference range of the biomarker, comprising administering an anti-C5 antibody or an anti-CFB antibody to the patient in an amount and frequency sufficient to attenuate the biomarker levels in the patient, thereby treating the HSCT-TMA. In another embodiment, a method is provided for treating a patient (e.g., a pediatric or adult patient) with HSCT-TMA who has been determined to have elevated levels (e.g., blood or plasma levels) of TM, SYND1, and Ba and / or C5b9 and / or HSPG, respectively, compared to the normal reference range of TM, SYND1, and Ba and / or C5b9 and / or HSPG, comprising administering an anti-C5 antibody or an anti-CFB antibody to the patient in an amount and frequency sufficient to treat the HSCT-TMA.

[0156] Also provided is a method of treating a patient having HSCT-TMA, comprising: (1) obtaining or having obtained a sample (e.g., a blood or plasma sample) from the patient, (2) determining or having determined elevated levels of biomarkers selected from TM and SYND1, or a combination thereof, in the sample relative to normal reference ranges for the biomarkers, and (3) administering an anti-C5 antibody or an anti-CFB antibody to the patient in an amount and frequency sufficient to attenuate the elevated TM and SYND1 levels in the patient, thereby treating the HSCT-TMA. In another embodiment, a method of treating a patient having HSCT-TMA, comprising: (1) obtaining or having obtained a sample (e.g., a blood or plasma sample) from the patient, (2) determining or having determined elevated levels of biomarkers selected from TM, SYND1, and Ba levels in the sample relative to normal reference ranges for the biomarkers, and (3) administering an anti-C5 antibody or an anti-CFB antibody to the patient in an amount and frequency sufficient to attenuate the TM, SYND1, and Ba levels, thereby treating the HSCT. In another embodiment, a method for treating a patient with HSCT-TMA includes (1) obtaining or having obtained a sample (e.g., a blood or plasma sample) from the patient, (2) determining or having determined an elevated level of a biomarker selected from TM, SYND1, Ba, and HSPG levels in the sample compared to a normal reference range for the biomarker, and (3) administering an anti-C5 antibody or an anti-CFB antibody to the patient in an amount and frequency sufficient to attenuate TM, SYND1, Ba, and HSPG levels, thereby treating the HSCT.

[0157] As used herein, the term "treating" includes prophylactic and / or therapeutic treatment. The term "prophylactic or therapeutic" treatment is art-recognized and includes administering to a patient one or more of the therapeutic agents (such as anti-C5 agents, e.g., eculizumab or ravulizumab) or anti-CFB agents described herein. When one or more therapeutic agents are administered before the clinical manifestation of an undesirable condition (e.g., the onset of HSCT-TMA), the treatment is prophylactic (i.e., protects the patient from the onset of the undesirable condition); when administered after the onset of an undesirable condition, the treatment is therapeutic (i.e., intended to alleviate, ameliorate, or stabilize an existing undesirable condition or its side effects). Preferably, it is intended that the severity of the subject's condition be reduced or at least partially improved or altered, and that some relief, alleviation, reversal, or reduction in at least one clinical symptom be achieved.

[0158] As used herein, a patient "in need of prophylaxis," "in need of treatment," or "in need of" refers to a patient who, according to the judgment of an appropriate health care provider (e.g., a physician, nurse, or clinical nurse), would reasonably benefit from a given treatment (such as treatment with an anti-C5 antibody or an anti-CFB antibody).

[0159] Therapeutic agents or drugs (e.g., anti-C5 antibodies or anti-CFB antibodies) can be administered to a patient, 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.

[0160] Administration can be achieved, for example, by local infusion, injection, or implant. The implant can be of a porous, non-porous, or gelatinous material, including a membrane such as a sialastic membrane, or a fiber. The implant can be configured for sustained or periodic release of the composition into a subject. See, for example, U.S. Patent Publication No. 20080241223; U.S. Patent Nos. 5,501,856; 4,863,457; and 3,710,795; and European Patent Nos. EP488401 and EP430539, the disclosures of each of which are incorporated herein by reference in their entirety. The composition can be delivered to a subject by, for example, an implantable device based on a diffusion, erosion, or convection system, an osmotic pump, a biodegradable implant, an electrodiffusion system, an electroosmotic system, a vapor pressure pump, an electrolytic pump, an effervescence pump, a piezoelectric pump, an erosion-based system, or an electromechanical system.

[0161] As used herein, the term "therapeutically effective amount" or "therapeutically effective dose" includes an amount of an agent (e.g., an anti-C5 antibody (e.g., eculizumab or ravulizumab) or an anti-CFB antibody) that induces a desired biological or medical response (e.g., prevention or amelioration of one or more symptoms of HSCT-TMA). Suitable doses of the therapeutic agents described herein (e.g., anti-C5 antibodies and / or anti-CFB antibodies) that can treat or prevent HSCT-TMA in a subject can depend on various factors, including, for example, the age, sex, and weight of the subject being treated, as well as the particular inhibitor compound used. For example, the dose can depend on the severity of the HSCT-TMA. Other factors can include, for example, other medical disorders concurrently or previously affecting the subject, the subject's general health, the subject's genetic predisposition, diet, time of administration, excretion rate, drug combinations, and any other additional therapeutic agents administered to the subject. It should also be understood that the specific dosage and treatment regimen for any particular subject will depend on the judgment of the treating healthcare provider (e.g., a doctor or nurse). A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.

[0162] The one or more therapeutic agents (e.g., anti-C5 antibody and / or anti-CFB antibody) can be administered as a fixed dose or in milligrams per kilogram ("mg / kg") doses. In some embodiments, the doses can also be selected to reduce or avoid antibody production or other host immune responses to one or more active agents in the composition.

[0163] While not intended to be limiting in any way, exemplary dosages of inhibitors such as anti-C5 antibodies include, for example, 1-100 mg / kg, 0.5-50 mg / kg, 0.1-100 mg / kg, 0.5-25 mg / kg, 1-20 mg / kg, and 1-10 mg / kg body weight.

[0164] In some embodiments, the anti-C5 antibody (e.g., ravulizumab) is selected from the following: (a) Once on day 1, at a dose of 600 mg for patients weighing 5 kg or more but less than 10 kg, 600 mg for patients weighing 10 kg or more but less than 20 kg, 900 mg for patients weighing 20 kg or more but less than 30 kg, 1200 mg for patients weighing 30 kg or more but less than 40 kg, 2400 mg for patients weighing 40 kg or more but less than 60 kg, 2700 mg for patients weighing 60 kg or more but less than 100 kg, or 3000 mg for patients weighing 100 kg or more; (b) Once on day 5, at a dose of 300 mg for patients weighing 5 kg or more but less than 10 kg, 300 mg for patients weighing 10 kg or more but less than 20 kg, 300 mg for patients weighing 20 kg or more but less than 30 kg, 300 mg for patients weighing 30 kg or more but less than 40 kg, 600 mg for patients weighing 40 kg or more but less than 60 kg, 900 mg for patients weighing 60 kg or more but less than 100 kg, or 900 mg for patients weighing 100 kg or more; (c) once on day 10, at a dose of 300 mg for patients weighing 5 kg or more but less than 10 kg, 300 mg for patients weighing 10 kg or more but less than 20 kg, 300 mg for patients weighing 20 kg or more but less than 30 kg, 300 mg for patients weighing 30 kg or more but less than 40 kg, 600 mg for patients weighing 40 kg or more but less than 60 kg, 900 mg for patients weighing 60 kg or more but less than 100 kg, or 900 mg for patients weighing 100 kg or more; and (d) 300 mg on day 15 and every 4 weeks thereafter for patients weighing 5 kg to less than 10 kg, or 600 mg for patients weighing 10 kg to less than 20 kg; or alternatively, 2100 mg on day 15 and every 8 weeks thereafter for patients weighing 20 kg to less than 30 kg, 2700 mg for patients weighing 30 kg to less than 40 kg, 3000 mg for patients weighing 40 kg to less than 60 kg, 3300 mg for patients weighing 60 kg to less than 100 kg, or 3600 mg for patients weighing 100 kg or more (e.g., intravenously).

[0165] In some embodiments, a human may be administered an anti-C5 antibody (e.g., eculizumab) intravenously at a dose of about 900 mg about every 12 days (e.g., about 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.

[0166] In some embodiments, a human can be administered an anti-C5 antibody (e.g., eculizumab) intravenously at a dose of about 600 (e.g., about 625, 650, 700, 725, 750, 800, 825, 850, 875, 900, 925, 950, or 1,000 or more) mg weekly, optionally for two or more weeks (e.g., 3, 4, 5, 6, 7, or 8 or more weeks). Following the initial treatment, the human can be administered the antibody as a maintenance dose at a dose of about 900 mg about every 14 days (e.g., about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, 30, 42, or 49 days or more). See, for example, Hillmen et al. (2004) N Engl J Med. 350(6):552-9 and Dmytrijuk et al. (2008) The Oncologist 13(9):993.

[0167] In some embodiments, a human may be administered an anti-C5 antibody (e.g., eculizumab) intravenously at a dose of about 900 mg (e.g., 925, 950, 975, 1000, 1100, or 1200 mg or more) weekly, optionally for two or more weeks (e.g., 3, 4, 5, 6, 7, or 8 or more weeks). Following initial treatment, a human may be administered a maintenance dose of the antibody, e.g., about every 14 days (e.g., about every 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, 30, 42, or 49 days or more), e.g., at a dose of about 1200 mg. See, e.g., International Patent Application Publication No. WO2010 / 054403.

[0168] The toxicity and therapeutic efficacy of such compositions can be determined by known pharmaceutical procedures in cell culture or experimental animals (animal models of HSCT-TMA). These procedures include, for example, LD 50 (lethal dose for 50% of the population) and ED 50 It can be used to determine the dose that is therapeutically effective in 50% of the population. The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50 Therapeutic indices can be expressed as a ratio. Therapeutic agents that exhibit high therapeutic indices are preferred. Compositions that exhibit toxic side effects may be used, but care should be taken to design a delivery system that targets such compounds to the site of the affected tissue, minimizing potential damage to normal cells, thereby reducing side effects.

[0169] In some embodiments, a therapeutic agent described herein (e.g., an anti-C5 antibody or an anti-CFB antibody) is administered to a patient as monotherapy. Alternatively, a therapeutic agent can be administered to a patient as combination therapy with another treatment (e.g., another therapeutic agent and / or treatment for HSCT-TMA). For example, combination therapy can include administering to a patient one or more additional agents that provide a therapeutic benefit to a subject having or at risk of developing HSCT-TMA. In some embodiments, an agent described herein (e.g., an anti-C5 antibody or an anti-CFB antibody) and one or more additional active agents are administered simultaneously. In other embodiments, an agent described herein (e.g., an anti-C5 antibody or an anti-CFB antibody) is administered first, and one or more additional active agents are administered second.

[0170] Methods of treating HSCT-TMA with eculizumab (SOLIRIS®) are described in U.S. Pat. No. 10,815,296 (including corresponding WO 2015 / 39126), the disclosure of which is incorporated herein by reference. Methods of treating HSCT-TMA with ravulizumab (AXLN1210 or ULTOMIRIS®) are described in WO 2022 / 36151, the disclosure of which is incorporated herein by reference. A method for treating HSCT-TMA in patients transfused with red blood cells (RBCs) during the maintenance phase of a ravulizumab (AXLN1210 or ULTOMIRIS®) dosing cycle is described in U.S. Prov. App. (entitled "SUPPLEMENTAL DOSAGE AND ADMINISTRATION OF ANTI-C5 ANTIBODIES FOR TREATING HEMATOPOIETIC STEM CELL TRANSPLANT-ASSOCIATED THROMBOTIC MICROANGIOPATHY (HSCT-TMA)," filed September 6, 2022 (Attorney Docket No. AXJ-306-1), the disclosure of which is incorporated herein by reference.

[0171] VII. Treatment results The efficacy of the treatment methods provided herein can be assessed using any suitable means, in one embodiment, the treatment results in a reduction or cessation of microangiopathic hemolytic anemia, thrombocytopenia, endothelial damage, renal damage, renal failure, serositis, pulmonary hypertension, and multi-organ failure compared to baseline.

[0172] In another embodiment, treatment results in normalization of LDH, elimination of the need for red blood cell and platelet transfusions, an increase in hemoglobin, and / or disappearance of schistocytes compared to baseline.

[0173] In another embodiment, treatment comprises (a) a platelet count of 50,000 / mm without transfusion support within the past seven days. 3 or (b) LDH < 1.5 × ULN, (c) absence of schistosomiasis (if schistosomiasis was present at baseline), and / or (d) at least a 50% reduction in proteinuria from baseline.

[0174] In another embodiment, the treatment results in a favorable hematological response.

[0175] In another embodiment, treatment results in hemoglobin > 8 g / dL without transfusion support.

[0176] In another embodiment, the treatment results in terminal complement inhibition. The treatment results in a reduction in adverse events.

[0177] In another embodiment, treatment results in a change from baseline in quality of life as assessed via a quality of life assessment. In one embodiment, the quality of life assessment is the Pediatric Quality of Life Inventory (PedSql) scale. The Pediatric Quality of Life Inventory (PedSql) 4.0 Generic Core Scales is a multidimensional, pediatric self-report and parent-proxy-report standardized instrument for measuring health-related quality of life (QoL) in children and adolescents aged 2 to 18 years. In another embodiment, the quality of life assessment is the EuroQoL 5-Dimensions 5-Level (EQ-5D-5L) questionnaire. The EQ-5D-5L is a self-rated, standardized instrument for measuring health-related quality of life (QoL) and is used across a wide range of health conditions.

[0178] VIII. Methods for Patient Identification In another aspect, the disclosure provides a method for identifying a patient with an HSCT-TMA suitable for treatment with an anti-C5 antibody or an anti-CFB antibody, the method comprising: determining, using an in vitro assay, the level of a biomarker selected from TM and SYND1, or a combination thereof, in a sample (e.g., a blood or plasma sample) from the patient, wherein the patient is identified as suitable for treatment with an anti-C5 antibody or an anti-CFB antibody because the level of the biomarker in the sample is elevated compared to the normal reference range for the biomarker. In another embodiment, a method for identifying a patient with an HSCT-TMA suitable for treatment with an anti-C5 antibody or an anti-CFB antibody is provided, the method comprising: determining, using an in vitro assay, the level of a biomarker selected from TM, SYND1, Ba, and HSPG levels in a sample (e.g., a blood or plasma sample) from the patient, wherein the level of the biomarker in the sample is elevated compared to the normal reference range for the biomarker, wherein the patient is identified as suitable for treatment with an anti-C5 antibody or an anti-CFB antibody.

[0179] In another aspect, the method includes identifying the patient as having, suspected of having, or at risk of developing HSCT-TMA. In addition to using the HSCT-TMA biomarker profiling described herein, clinical trials can be performed to determine whether a human subject has symptoms of HSCT-TMA. For example, thrombocytopenia can be defined by a medical professional as (i) a thrombocytopenia of 150,000 / mm 3 Platelet count less than 60,000 / mm 3 (ii) decreased platelet survival time, reflecting enhanced platelet destruction in the circulation; and (iii) giant platelets observed in peripheral smears, consistent with secondary activation of thrombopoiesis.

[0180] As used herein, a patient "at risk of developing HSCT-TMA" includes one or more (e.g., two, three, four, five, six, seven, or eight or more) risk factors for developing the disorder. Risk factors for HSCT-TMA include, for example, calcineurin inhibitors (CNIs), infections, and conditioning regimens (high-dose chemotherapy or total-body irradiation) (Khosla, et al., Bone Marrow Transplant. 2018;53(2):129-137; Masias, et al., Blood. 2017;129(21):2857-2863).

[0181] As used herein, a patient "suspected of having HSCT-TMA" is a patient who has one or more symptoms of a condition well known to those skilled in the medical arts, including, for example, microangiopathic hemolytic anemia, thrombocytopenia, endothelial damage, renal damage, renal failure, serositis, pulmonary hypertension, and multiple organ failure.

[0182] IX. METHODS FOR MONITORING PATIENT RESPONSIVENESS TO TREATMENT In another aspect, the present disclosure provides a method for monitoring the responsiveness of a patient with HSCT-TMA to treatment with an anti-C5 antibody or an anti-CFB antibody, comprising determining the level of a biomarker selected from TM and SYND1, or a combination thereof, in a sample (e.g., a blood or plasma sample) obtained from the patient during or after treatment. A decrease in the level of the biomarker in a sample obtained from the patient during or after treatment, compared to the level of the biomarker in a sample obtained from the patient before treatment with an anti-C5 antibody or an anti-CFB antibody, indicates that the patient is responsive to treatment with an anti-C5 antibody or a CFB inhibitor. Also provided is a method for monitoring the responsiveness of a patient with HSCT-TMA to treatment with an anti-C5 antibody or an anti-CFB antibody, comprising determining the level of a biomarker selected from TM, SYND1, Ba, and HSPG in a sample (e.g., a blood or plasma sample) obtained from the patient during or after treatment. Here, a decrease in the level of the biomarker in a sample from the patient obtained during or after treatment compared to the level of the biomarker in a sample from the patient obtained before treatment with an anti-C5 antibody or an anti-CFB antibody indicates that the patient is responsive to treatment with an anti-C5 antibody or an anti-CFB antibody.

[0183] Monitoring a patient (e.g., a human patient) for improvement of HSCT-TMA, as defined herein, 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 HSCT-TMA described herein. In some embodiments, the evaluation is performed for at least 1 hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or for at least 1, 2, 4, 10, 13, 20, or more days, or for at least 1, 2, 4, 10, 13, 20, or more weeks after initiation of treatment. The subject may be evaluated at one or more of the following time periods: before initiation of treatment; during treatment; or after one or more components of treatment have been administered. Evaluating may include assessing the need for further treatment (e.g., assessing whether dosage, frequency of administration, or duration of treatment should be changed). Furthermore, it may include assessing the need to add or drop a selected treatment modality (e.g., add or drop any of the treatments for HSCT-TMA described herein).

[0184] X.Kit Kits are also provided that contain pharmaceutical compositions containing a therapeutically effective amount of an anti-C5 antibody or an anti-CFB antibody adapted for use in the methods described herein. Additionally, the kits can include various reagents and materials useful for carrying out the methods described herein. The measuring, diagnosing, assessing, and / or evaluating procedures described herein can be performed by a diagnostic laboratory, a laboratory, or an individual healthcare provider. The present invention provides kits that can be used in any or all of these settings.

[0185] In some embodiments, the kits described herein include materials and reagents for, inter alia, characterizing or processing a biological sample (e.g., a biological fluid), measuring biomarker levels (e.g., protein or nucleic acid levels), diagnosing HSCT-TMA in a subject, or monitoring a therapeutic response in a subject, according to the methods provided herein. In certain embodiments, the kits of the invention include at least one or more reagents that specifically detect protein levels of one or more HSCT-TMA biomarker proteins (e.g., TM, SYND-1, factor Ba, and / or HSPG), and, optionally, instructions for using the kit. The kits can include, for example, any of the arrays described herein.

[0186] Exemplary arrays and chips of the present disclosure include antibodies that bind to the aforementioned signatures. In embodiments, such arrays include multiple (e.g., at least two, three, or more) antibodies capable of detecting the signatures. The antibodies can be monospecific or multispecific (e.g., bind to two or more biomarkers), bispecific antibodies, etc.

[0187] In one embodiment, the chip or array comprises one or more antibodies for detecting a biomarker signature comprising the following biomarkers: (a) TM+Ba; (b) TM+SYND1; (c) SYND1+Ba. The chip or array may further comprise an antibody for detecting C5b9, in which case the biomarker signature is: (a) TM+Ba+C5b9; (b) TM+SYND1+C5b9; (c) SYND1+Ba+C5b9.

[0188] In one embodiment, the chip or array comprises one or more antibodies for detecting a biomarker signature comprising the following biomarkers: TM+SYND1+Ba+HSPG. Such a chip or array may further comprise an antibody for detecting C5b9, in which case the biomarker signature comprises TM+SYND1+Ba+C5b9+HSPG.

[0189] In some embodiments, the kit may include an appropriate control sample (e.g., a biological fluid (in the absence of TMA) from a normal, healthy individual or an individual who has had an HSCT, or a solution containing a known control amount of a particular analyte of interest). In some embodiments, the kits of the invention may include instructions for using the kit according to one or more methods described herein, and may include instructions for processing a biological sample (e.g., a biological fluid) obtained from a subject and / or instructions for performing a test or interpreting the results.

[0190] It is to be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification includes every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0191] 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 invention belongs. Methods and materials for use in the present invention are described herein. Other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries (e.g., PUBMED, NCBI, or UNIPROT accession numbers), and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0192] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

[0193] The following examples are merely illustrative and should not be construed as limiting the scope of the disclosure in any way, since many variations and similarities will be apparent to those of skill in the art upon reading this disclosure. [Example]

[0194] Example 1: Analysis of the association between excessive AP activation and endothelial injury in the context of HSCT-TMA Soluble TM, SYND1, and Ba biomarker levels were measured in the plasma of pediatric HSCT patients with and without TMA (control). Biomarkers were also measured in a preclinical in vivo model of inflammation-mediated complement activation similar to HSCT-TMA, in which C57BL / 6J mice were injected with lipopolysaccharide (LPS) to induce complement activation and subsequently treated with AP (anti-fB IgG) and terminal complement pathway (anti-C5 IgG) inhibitors or IgG isotype controls. Because the immunosuppressant cyclosporine A (CsA) is a known trigger of HSCT-TMA, the role of complement in CsA-mediated injury in human ECs (HMEC-1 cells and HUVECs) was evaluated in vitro. ECs were incubated with CsA followed by normal human serum (NHS) in the presence or absence of the C5 complement inhibitor eculizumab (Alexion Pharmaceuticals). TM was then measured by fluorescent staining on the cell surface and quantified using mean fluorescence intensity (MFI).

[0195] HSCT-TMA patients (n = 11) showed significantly elevated levels of biomarkers of glycocalyx damage compared with HSCT control patients (n = 7) (TM: 18.3 ± 10.9 ng / mL and 6.6 ± 2.9 ng / mL, respectively; P < 0.01; SYND1: 175.3 ± 177.0 ng / mL and 35.8 ± 18.9 ng / mL, respectively; P < 0.05). A strong positive correlation was observed between TM and SYND1 (Pearson r = 0.89). Plasma Ba was also significantly elevated in patients with TMA compared with those without (1790.0 ± 1262.0 ng / mL and 652.1 ± 233.5 ng / mL, respectively; P < 0.05) and positively correlated with both TM and SYND1 levels (Pearson r = 0.72 and 0.50, respectively).

[0196] We have previously shown that in an in vivo model of inflammation-mediated complement activation, LPS-injected mice had significantly increased plasma Ba levels compared with saline-injected control mice. As demonstrated here, LPS-injected mice (which also received an IgG isotype control) had higher circulating levels of TM than untreated mice (35.1 ± 5.8 ng / mL [n = 6] and 10.9 ± 1.6 ng / mL [n = 6], respectively; P < 0.0001). Inhibition of the alternative (anti-fB IgG) and terminal (anti-C5 IgG) complement pathways significantly attenuated TM levels (isotype control [35.1 ± 5.8 ng / mL]; anti-fB: 25.2 ± 5.3 ng / mL, P < 0.01; anti-C5: 26.2 ± 4.9 ng / mL, P < 0.05, compared with Figure 1). TM was positively correlated with Ba (Pearson r = 0.68).

[0197] In vitro treatment of HMEC-1 cells with CsA resulted in the deposition of iC3b and C5b-9, and C5 inhibition with eculizumab significantly reduced C5b-9 deposition (P<0.0001) but had no effect on iC3b deposition. Treatment of HUVECs with CsA further reduced TM surface expression (P<0.0001), and TM loss was partially restored by eculizumab (P<0.05, Figure 2).

[0198] In summary, plasma biomarkers of endothelial injury were elevated in patients with HSCT-TMA and in mice with inflammation-mediated complement activation. Furthermore, AP activation positively correlated with biomarkers of endothelial injury. Blockade of complement activation significantly reduced markers of endothelial injury in both in vivo and in vitro models. These results provide further evidence linking complement activation to endothelial injury in HSCT-TMA and that the use of these biomarkers may aid in the diagnosis of HSCT-TMA.

[0199] Example 2: Complement activation is associated with endothelial injury in HSCT-TMA The aim of this study was to investigate the association between excessive alternative complement pathway (AP) activation and endothelial injury in the context of HSCT-TMA and to potentially identify biomarkers to aid in its diagnosis.

[0200] Soluble TM, SYND1, and Ba levels were measured in the plasma of pediatric HSCT patients with and without TMA. Furthermore, TM, SYND1, and Ba levels were measured in a preclinical in vivo mouse model of inflammation-mediated complement activation similar to HSCT-TMA. Finally, the role of complement in CsA-mediated injury, TM shedding, and HSPG expression was assessed in human ECs (HMEC-1 cells and HUVECs).

[0201] Figures 3A-3C show biomarker levels of glycocalyx components in pediatric patients with and without HSCT-TMA. As shown in these figures, HSCT patients had elevated levels of TM and SYND1. Specifically, Figures 3A and 3B show that HSCT-TMA patients (n = 11) had significantly elevated levels of biomarkers of glycocalyx damage compared with HSCT control patients (n = 7). Mean ± SD was calculated, and significance was determined by Welch's t-test ( * P < 0.05; ** P<0.01). Figure 3C shows that a strong positive correlation was observed between TM and SYND1 (Pearson r=0.89).

[0202] Figures 4A-4C show AP activation in pediatric patients with and without HSCT-TMA. As shown in these figures, HSCT patients had elevated Ba levels that positively correlated with TM and SYND1. Specifically, Figure 4A shows that plasma Ba was significantly elevated in patients with HSCT-TMA compared with those without (mean ± SD was calculated, and significance was determined by Welch's t-test). * P<0.05). Furthermore, plasma Ba was positively correlated with both TM (FIG. 4B) and SYND1 (FIG. 4C) levels (Pearson r=0.89 and 0.50, respectively).

[0203] Figure 5 shows plasma TM levels in LPS-injected mice treated with anti-complement agents. C57BL / 6J mice (12-17 weeks old) were intraperitoneally (IP) injected with LPS (5 mg / kg) to induce complement activation. Three hours after LPS injection, mice were IP injected with IgG isotype control, anti-fB IgG, or anti-C5 IgG (40 mg / kg each). Twenty-four hours later, citrated plasma was collected, and TM levels were measured by a commercially available ELISA (R&D Systems). Untreated mice did not receive LPS or IgG (blue = males, pink = females). Mean ± SD was calculated, and significance was determined by ordinary one-way ANOVA ( ** =P<0.01 **** =P<0.0001). As shown in Figure 5, mice with inflammation-mediated complement activation had elevated levels of circulating TM, which were attenuated with anti-complement treatment.

[0204] Figures 6A-6B show the correlation between TM and Ba in LPS-injected mice. C57BL / 6J mice (12-17 weeks old) were intraperitoneally (IP) injected with LPS (5 mg / kg) to induce complement activation. Three hours after LPS injection, mice were IP injected with either an IgG isotype control or anti-fB IgG (40 mg / kg each). Twenty-four hours later, citrated plasma was collected, and TM and Ba levels were measured by commercial ELISA (R&D Systems) or Western blotting, respectively (Figure 6A; blue = male, pink = female). Mean ± SD was calculated, and significance was determined by Welch's t-test. As shown in Figure 6B, in mice with inflammation-mediated complement activation, plasma Ba was positively correlated with TM levels (Pearson's r = 0.68; gray = LPS + IgG isotype control, black = LPS + anti-fB IgG).

[0205] Figures 7A-7D show cyclosporine-induced TM loss in HUVECs. HUVECs were treated with CsA (28.9 μM) and 30% NHS for 18 hours, with or without eculizumab surrogate (ecu, 1 μM). Cells were then fixed with 4% PFA and incubated with mouse anti-human TM antibody followed by goat anti-mouse AF488 secondary antibody or rabbit anti-human HSPG antibody followed by donkey anti-rabbit AF488 secondary antibody. TM and HSPG expression were imaged and analyzed using the CX7 high-content screening platform. MFI ± SEM was calculated, and significance was determined by ordinary one-way ANOVA (Figure 7A). * P<0.05 **** =P<0.0001). As shown by these figures, TM and HSPG surface expression was decreased on HUVECs treated with CsA and was partially restored by eculizumab surrogate.

[0206] Figures 8A-8B show the deposition of complement activation products on HMEC-1 cells treated with CsA. HMEC-1 cells were treated with CsA (28.9 μM) for 18 hours, followed by treatment with 30% NHS for 30 minutes in the presence or absence of eculizumab surrogate (ecu, 1 μM). Cells were fixed with 4% PFA and stained with a mouse antibody against iC3b, followed by goat anti-mouse AF647 or rabbit anti-human C5b-9, followed by goat anti-rabbit AF647 secondary antibody. Deposition was imaged and analyzed using the CX7 high-content screening platform. MFI + SEM was calculated, and significance was determined by ordinary one-way ANOVA (Figure 8B). **** (P<0.0001, ns=not significant). As shown in these figures, CsA treatment induced complement deposition on HMEC-1 cells, and C5 inhibition reduced C5b-9 (Fig. 8B) but not iC3b deposition (Fig. 8A).

[0207] In summary, plasma markers of endothelial injury (TM and SYND1) were elevated in patients with HSCT-TMA and in mice with inflammation-mediated complement activation. AP activation positively correlated with biomarkers of endothelial injury. Blockade of complement activation significantly reduced markers of endothelial injury in both in vivo and in vitro models. These results provide further evidence linking complement activation and endothelial injury in HSCT-TMA. Furthermore, the use of biomarkers TM, SYND1, and Ba may be useful for the diagnosis of HSCT-TMA.

[0208] [Table 1-1]

[0209] [Table 1-2]

[0210] [Table 1-3]

[0211]

Table 1-4

[0212]

Table 1-5

Claims

1. A method for treating a patient with hematopoietic stem cell transplantation-associated thrombotic microangiopathy (HSCT-TMA) in which blood levels of biomarkers selected from thrombomodulin (TM) and syndecan-1 (SYND1), or a combination thereof, have been determined to be elevated compared to the normal reference range for said biomarkers, said method comprising administering to said patient an anti-C5 antibody or an anti-complement factor B (CFB) antibody in an amount and frequency sufficient to attenuate said biomarker levels in said patient, thereby treating the HSCT-TMA.

2. 2. The method of claim 1, wherein the patient is further determined to have elevated blood levels of complement factors B and / or C5b9 compared to the normal reference range for B and / or C5b9.

3. 3. The method of claim 1 or 2, wherein the patient is further determined to have elevated blood levels of heparan sulfate proteoglycans (HSPGs) compared to the normal reference range for HSPGs.

4. 1. A method of treating a patient with HSCT-TMA, said method comprising: (1) obtaining or having obtained a blood sample from said patient; (2) determining or having determined elevated levels of biomarkers selected from TM and SYND1, or a combination thereof, in said blood sample of said patient, compared to normal reference ranges for said biomarkers; (3) administering an anti-C5 antibody or an anti-CFB antibody to the patient in an amount and frequency sufficient to attenuate elevated TM and SYND1 levels in the patient, thereby treating HSCT-TMA.

5. 5. The method of claim 4, further comprising determining or having determined elevated Ba and / or C5b9 levels in the blood sample compared to normal reference ranges for Ba and / or C5b9.

6. 6. The method of claim 5, further comprising determining or having determined elevated HSPG levels in the blood sample compared to a normal reference range for HSPG.

7. A method for identifying a patient with HSCT-TMA suitable for treatment with an anti-C5 antibody or an anti-CFB antibody, comprising determining, using an in vitro assay, the level of a biomarker selected from TM and SYND1 or a combination thereof in a blood sample from the patient, wherein the patient is identified as suitable for treatment with an anti-C5 antibody or an anti-CFB antibody when the level of the biomarker in the blood sample is elevated compared to the normal reference range for TM and SYND1, respectively.

8. 8. The method of claim 7, further comprising determining Ba and / or C5b9 levels in the blood sample, wherein the Ba and / or C5b9 levels are elevated compared to the normal reference range for Ba and / or C5b9, thereby identifying the patient as suitable for treatment with an anti-C5 antibody or an anti-CFB antibody.

9. 9. The method of claim 8, further comprising determining the level of HSPG in the blood sample, wherein the level of HSPG is elevated compared to the normal reference range for HSPG, thereby identifying the patient as suitable for treatment with an anti-C5 antibody or an anti-CFB antibody.

10. A method for monitoring the responsiveness of a patient with HSCT-TMA to treatment with an anti-C5 antibody or an anti-CFB antibody, the method comprising determining a biomarker selected from TM and SYND1 or a combination thereof in a blood sample from the patient obtained during or after treatment, wherein a decrease in the biomarker level in the blood sample from the patient obtained during or after treatment compared to the biomarker level in a blood sample from the patient obtained before treatment with an anti-C5 antibody or an anti-CFB antibody indicates that the patient is responsive to treatment with the anti-C5 antibody or anti-CFB antibody.

11. The method of claim 10, further comprising determining Ba and / or C5b9 levels in the blood sample from the patient obtained during or after treatment, wherein a decrease in Ba and / or C5b9 levels in the blood sample from the patient obtained during or after treatment compared to the Ba level in the blood sample from the patient obtained before treatment with the anti-C5 antibody or the anti-CFB antibody indicates that the patient is responsive to treatment with the anti-C5 antibody or the anti-CFB antibody.

12. The method of claim 11, further comprising determining HSPG levels in the blood sample from the patient obtained during or after treatment, wherein a decreased HSPG level in the blood sample from the patient obtained during or after treatment compared to the HSPG level in the blood sample from the patient obtained before treatment with the anti-C5 antibody or the anti-CFB antibody indicates that the patient is responsive to treatment with the anti-C5 antibody or the anti-CFB antibody.

13. The method according to any one of claims 1 to 12, wherein the blood sample is plasma.

14. 14. The method of any one of claims 1 to 13, wherein the one or more levels are measured by a regulatory (e.g. USFDA) approved test system or kit.

15. 15. The method of any one of claims 1 to 14, wherein the one or more levels are measured by using an immunoassay, immunochemistry, immunohistochemistry assay, nucleoprobe assay, in situ hybridization, fluorescent RNA probe, RT-PCR, microarray transcription assay, or RNA transcription assay.

16. 16. The method of any one of claims 1 to 15, wherein the normal reference range for TM in healthy patients is from about 1.8 ng / mL to about 4.8 ng / mL.

17. 17. The method of any one of claims 1 to 16, wherein the normal reference range for TM in HSCT patients in the absence of TMA is from about 3 ng / mL to about 9 ng / mL.

18. 18. The method of any one of claims 1 to 17, wherein the elevated TM level is greater than about 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, or 30 ng / mL.

19. The method of any one of claims 1 to 18, wherein the normal reference range for SYND1 for healthy patients is about 15 ng / mL to 55 ng / mL.

20. 20. The method of any one of claims 1 to 19, wherein the normal reference range for SYND1 for HSCT patients in the absence of TMA is about 15 ng / mL to 55 ng / mL.

21. and / or wherein the elevated level for SYND1 is about 100 ng / mL, 105 ng / mL, 110 ng / mL, 115 ng / mL, 120 ng / mL, 125 ng / mL, 130 ng / mL, 135 ng / mL, 140 ng / mL, 145 ng / mL, 150 ng / mL, 155 ng / mL, 160 ng / mL, 165 ng / mL, 170 ng / mL, 175 ng / mL, or 21. The method of any one of claims 1 to 20, wherein the serum saturation level is greater than 100 ng / mL, 180 ng / mL, 185 ng / mL, 190 ng / mL, 195 ng / mL, 200 ng / mL, 205 ng / mL, 210 ng / mL, 215 ng / mL, 220 ng / mL, 225 ng / mL, 230 ng / mL, 235 ng / mL, 240 ng / mL, 245 ng / mL, or 250 ng / mL.

22. 22. The method of any one of claims 1 to 21, wherein the normal reference range for Ba in healthy patients is about 300 ng / mL to 600 ng / mL.

23. 23. The method of any one of claims 1 to 22, wherein the normal reference range of Ba for HSCT patients in the absence of TMA is about 500 ng / mL to 800 ng / mL.

24. The elevated level for Ba is about 900 ng / mL, 910 ng / mL, 920 ng / mL, 930 ng / mL, 940 ng / mL, 950 ng / mL, 960 ng / mL, 970 ng / mL, 980 ng / mL, 990 ng / mL, 1000 ng / mL, 1010 ng / mL, 1020 ng / mL, 1030 ng / mL, 1040 ng / mL, 1050 ng / mL, 1060 ng / mL, 1070 ng / mL, 1080 ng / mL, 1090 ng / mL, 1100 ng / mL, 1110 ng / mL, 1120 ng / mL, 1130 ng / mL, 1140 ng / mL, 1150 ng / mL, 1160 ng / mL, 1170 ng / mL, 1180 ng / mL, 1190 ng / mL, 1200 ng / mL, 1210 ng / mL, 1220 ng / mL, 1230 ng / mL, 1240 ng / mL, 1250 ng / mL, 1260 ng / mL, 1270 ng / mL, 1280 ng / mL, 1290 ng / mL, 1300 ng / mL, 1310 ng / mL, 1320 ng / mL, 1330 ng / mL, 1340 ng / mL, 1350 ng / mL, 1360 ng / mL, 1370 ng / mL, 1380 ng / mL, 1390 ng / mL, 1400 ng / mL, 1410 ng / mL, 1420 ng / mL, 1430 ng / mL, 1440 ng / mL, 1450 ng / mL, 1460 ng / mL, 1470 ng / mL, 1480 ng / mL, 1490 ng / mL, 1500 ng / mL, 1510 ng / mL, 1520 ng / mL, 1530 ng / mL, 1540 ng / mL, 1550 ng / mL, 1560 ng / mL, 1570 ng / mL, 1580 ng / mL, 1590 ng / mL, 1600 ng / mL, 1610 ng / mL, 1620 ng / mL, 1630 ng / mL, 1640 ng / mL, 1650 ng / mL, 1660 ng / mL, 1670 ng / mL, 1680 ng / mL, 1690 ng / mL, 1700 ng / mL, 1710 ng / mL, 1720 ng / mL, 1730 ng / mL, 1740 ng / mL, 1750 ng / mL, 1760 ng / mL, 1770 ng / mL, 1780 ng / mL, 1790 ng / mL, 1800 ng / mL, 1810 ng / mL, 1820 ng / mL, 1830 ng / mL, 1840 ng / mL, 1850 ng / mL, 1860 ng / mL, 1870 ng / mL, 1880 ng / mL,1890ng / mL, 1900ng / mL, 1910ng / mL, 1920ng / mL, 1930ng / mL, 1940ng / mL, 1950ng / mL, 1960ng / mL, 1 970ng / mL, 1980ng / mL, 1990ng / mL, 2000ng / mL, 2010ng / mL, 2020ng / mL, 2030ng / mL, 2040ng / mL, 20 50ng / mL, 2060ng / mL, 2070ng / mL, 2080ng / mL, 2090ng / mL, 2100ng / mL, 2110ng / mL, 2120ng / mL, 213 0ng / mL, 2140ng / mL, 2150ng / mL, 2160ng / mL, 2170ng / mL, 2180ng / mL, 2190ng / mL, 2200ng / mL, 2210 ng / mL, 2220ng / mL, 2230ng / mL, 2240ng / mL, 2250ng / mL, 2260ng / mL, 2270ng / mL, 2280ng / mL, 2290n g / mL, 2300ng / mL, 2310ng / mL, 2320ng / mL, 2330ng / mL, 2340ng / mL, 2350ng / mL, 2360ng / mL, 2370ng 24. The method of any one of claims 1 to 23, wherein the serum creatine phosphate concentration is greater than 2380ng / mL, 2390ng / mL, 2400ng / mL, 2410ng / mL, 2420ng / mL, 2430ng / mL, 2440ng / mL, 2450ng / mL, 2460ng / mL, 2470ng / mL, 2480ng / mL, 2490ng / mL, or 2500ng / mL.

25. 25. The method of any one of claims 1 to 24, wherein the anti-C5 antibody is a human antibody, a humanized antibody, a bispecific antibody, a chimeric antibody, a Fab, a Fab'2, a scFv, a SMIP, an Affibody®, a nanobody, or a domain antibody that inhibits C5.

26. 26. The method of any one of claims 1 to 25, wherein the anti-C5 antibody comprises CDR1, CDR2, and CDR3 heavy chain sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and CDR1, CDR2, and CDR3 light chain sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.

27. The method of any one of claims 1 to 26, wherein the anti-C5 antibody comprises a heavy chain variable region comprising SEQ ID NO:7 and a light chain variable region comprising SEQ ID NO:

8.

28. The method of any one of claims 1 to 27, wherein the anti-C5 antibody comprises a heavy chain comprising SEQ ID NO: 10 and a light chain comprising SEQ ID NO:

11.

29. The method of any one of claims 1 to 28, wherein the anti-C5 antibody is SOLIRIS (registered trademark).

30. 24. The method of any one of claims 1 to 23, wherein the anti-C5 antibody 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.

31. 31. The method of claim 30, wherein the anti-C5 antibody further comprises a variant human Fc constant region that binds to the human neonatal Fc receptor (FcRn), wherein the variant human Fc CH3 constant region comprises Met-429-Leu and Asn-435-Ser substitutions at residues corresponding to methionine 428 and asparagine 434 of a native human IgG Fc constant region, respectively, in EU numbering.

32. 32. The method of claim 30 or 31, wherein the anti-C5 antibody comprises a heavy chain variable region comprising SEQ ID NO: 12 and a light chain variable region comprising SEQ ID NO:

8.

33. The method of any one of claims 30 to 32, wherein the anti-C5 antibody further comprises a heavy chain constant region set forth in SEQ ID NO:

13.

34. 34. The method of any one of claims 30 to 33, wherein the anti-C5 antibody comprises a heavy chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14 and a light chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

11.

35. The method of any one of claims 30 to 34, wherein the anti-C5 antibody is ULTOMIRIS (registered trademark).

36. The method of any one of claims 1 to 35, wherein the anti-C5 antibody is administered intravenously.

37. The anti-C5 antibody is selected from the group consisting of: (a) Once on day 1, at a dose of 600 mg for patients weighing 5 kg or more but less than 10 kg, 600 mg for patients weighing 10 kg or more but less than 20 kg, 900 mg for patients weighing 20 kg or more but less than 30 kg, 1200 mg for patients weighing 30 kg or more but less than 40 kg, 2400 mg for patients weighing 40 kg or more but less than 60 kg, 2700 mg for patients weighing 60 kg or more but less than 100 kg, or 3000 mg for patients weighing 100 kg or more; (b) once on day 5, at a dose of 300 mg for patients weighing 5 kg or more but less than 10 kg, 300 mg for patients weighing 10 kg or more but less than 20 kg, 300 mg for patients weighing 20 kg or more but less than 30 kg, 300 mg for patients weighing 30 kg or more but less than 40 kg, 600 mg for patients weighing 40 kg or more but less than 60 kg, 900 mg for patients weighing 60 kg or more but less than 100 kg, or 900 mg for patients weighing 100 kg or more; (c) once on day 10, at a dose of 300 mg for patients weighing 5 kg or more but less than 10 kg, 300 mg for patients weighing 10 kg or more but less than 20 kg, 300 mg for patients weighing 20 kg or more but less than 30 kg, 300 mg for patients weighing 30 kg or more but less than 40 kg, 600 mg for patients weighing 40 kg or more but less than 60 kg, 900 mg for patients weighing 60 kg or more but less than 100 kg, or 900 mg for patients weighing 100 kg or more; and (d) on day 15 and every four weeks thereafter, 300 mg to patients weighing 5 kg or more but less than 10 kg, or 600 mg to patients weighing 10 kg or more but less than 20 kg; or alternatively, on day 15 and every eight weeks thereafter, 2100 mg to patients weighing 20 kg or more but less than 30 kg, 2700 mg to patients weighing 30 kg or more but less than 40 kg, 3000 mg to patients weighing 40 kg or more but less than 60 kg, 3300 mg to patients weighing 60 kg or more but less than 100 kg, or 3600 mg to patients weighing 100 kg or more.

38. 38. The method of any one of claims 1-37, wherein the treatment results in a reduction or cessation of microangiopathic hemolytic anemia, thrombocytopenia, endothelial damage, renal damage, renal failure, serositis, pulmonary hypertension, and multi-organ dysfunction compared to baseline.

39. 39. The method of any one of claims 1-38, wherein the treatment results in normalization of LDH, elimination of the need for red blood cell and platelet transfusions, an increase in hemoglobin, and / or elimination of schistocytes compared to baseline.

40. The treatment is in accordance with (a) a platelet count of 50,000 / mm3 without transfusion support within the past 7 days ３ or greater, (b) LDH<1.5xULN, (c) absence of schistosomiasis (if schistosomiasis was present at baseline), and / or (d) at least a 50% reduction in proteinuria from baseline.

41. 41. The method of any one of claims 1 to 40, wherein the treatment results in a favorable hematological response.

42. 42. The method of any one of claims 1-41, wherein the treatment results in hemoglobin of 8 g / dL or greater without transfusion support.

43. 43. The method of any one of claims 1 to 42, wherein the treatment results in terminal complement inhibition.

44. 44. The method of any one of claims 1 to 43, wherein the treatment results in a reduction in adverse events.

45. 45. The method of any one of claims 1-44, wherein the treatment results in a change from baseline in quality of life as assessed via a quality of life assessment.

46. 46. ​​The method of any one of claims 1 to 45, wherein said quality of life assessment is the Quality of Life Inventory (PedSql) scale or the EQ-5D-5L questionnaire.

47. 47. The method of any one of claims 1 to 46, wherein the patient is a pediatric patient.

48. 48. The method of any one of claims 1 to 47, wherein the patient is an adult patient.

49. An anti-C5 antibody or an antigen-binding fragment thereof, or an anti-complement factor B (CFB) antibody for use in treating a patient with hematopoietic stem cell transplantation-associated thrombotic microangiopathy (HSCT-TMA) in which the blood level of a biomarker selected from thrombomodulin (TM) and syndecan-1 (SYND1), or a combination thereof, has been determined to be elevated compared to the normal reference range for said biomarker, wherein said anti-C5 antibody or anti-complement factor B (CFB) antibody is administered to said patient in an amount and with a frequency sufficient to attenuate the level of said biomarker in said patient.

50. An anti-C5 antibody, or an antigen-binding fragment thereof, or an anti-CFB antibody for use in identifying a patient with HSCT-TMA who is suitable for treatment with an anti-C5 antibody or an anti-complement factor B (CFB) antibody, the method comprising determining, using an in vitro assay, the level of a biomarker selected from TM and SYND1, or a combination thereof, in a blood sample from the patient, wherein the patient is identified as being suitable for treatment with an anti-C5 antibody or an anti-CFB antibody when the level of the biomarker in the blood sample is elevated compared to the normal reference range for TM and SYND1, respectively.

51. An anti-C5 antibody, or an antigen-binding fragment thereof, or an anti-CFB antibody for use in monitoring the responsiveness of a patient with HSCT-TMA to treatment with an anti-C5 antibody or an anti-CFB antibody, said use comprising determining a biomarker selected from TM and SYND1, or a combination thereof, in the blood sample from the patient obtained during or after treatment, wherein a decrease in the biomarker level in the blood sample from the patient obtained during or after treatment compared to the biomarker level in a blood sample from the patient obtained before treatment with the anti-C5 antibody or anti-CFB antibody indicates that the patient is responsive to treatment with the anti-C5 antibody or anti-CFB antibody.

52. Use of an anti-C5 antibody or an antigen-binding fragment thereof, or an anti-CFB antibody for the treatment of a patient with hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) in which the blood level of a biomarker selected from thrombomodulin (TM) and syndecan-1 (SYND1), or a combination thereof, has been determined to be elevated compared to the normal reference range for said biomarker, wherein said anti-C5 antibody or anti-CFB antibody is administered to said patient in an amount and frequency sufficient to attenuate said biomarker level in said patient.

53. Use of an anti-C5 antibody or an antigen-binding fragment thereof, or an anti-CFB antibody in identifying a patient with HSCT-TMA suitable for treatment with an anti-C5 antibody or an anti-CFB antibody, comprising determining, using an in vitro assay, the level of a biomarker selected from TM and SYND1, or a combination thereof, in a blood sample from the patient, wherein the patient is identified as suitable for treatment with an anti-C5 antibody or an anti-CFB antibody when the level of the biomarker in the blood sample is elevated compared to the normal reference range for TM and SYND1, respectively.

54. Use of an anti-C5 antibody or an antigen-binding fragment thereof, or an anti-CFB antibody in monitoring the responsiveness of a patient with HSCT-TMA to treatment with an anti-C5 antibody or an anti-CFB antibody, comprising determining a biomarker selected from TM and SYND1, or a combination thereof, in a blood sample from the patient obtained during or after treatment, wherein a decrease in the biomarker level in the blood sample from the patient obtained during or after treatment compared to the biomarker level in a blood sample from the patient obtained before treatment with the anti-C5 antibody or anti-CFB antibody indicates that the patient is responsive to treatment with the anti-C5 antibody or anti-CFB antibody.

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