Diagnostic and prognostic biomarker profiles for hematopoietic stem cell graft associated thrombotic microvascular disease (HSCT-TMA) patients

Abstract

Provided herein are methods of using an anti-C5 antibody and / or an anti-CFB antibody to treat hematopoietic stem cell graft associated thrombotic microangiopathy (HSCT-TMA), e.g., TMA following HSCT, in human patients having increased levels of thrombomodulin (TM) and SYND1 compared to the normal reference range of TM and Multi-Ligand proteoglycan-1 (SYND1), and methods of treating hematopoietic stem cell graft associated thrombotic microangiopathy (HSCT-TMA), e.g., TMA following HSCT. Also provided are methods of treating a patient suffering from HSCT-TMA, including (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 TM and SYND1 in the sample compared to a normal reference range of TM and SYND1, respectively, and (3) after the elevated level of TM and SYND1 has been determined, determining or having determined an elevated level of TM and SYND1 in the sample. An anti-C5 antibody or an anti-CFB antibody is administered to the patient in an amount and frequency sufficient to treat HSCT-TMA. In one embodiment, the methods further include determining or having determined an elevated Ba level in the sample compared to a normal reference range of Ba. In another embodiment, the methods further include determining or having determined an elevated level of HSPG in the sample compared to a normal reference range of HSPG. Also provided are methods of identifying a patient suffering from HSCT-TMA suitable for treatment with an anti-C5 antibody or an anti-CFB antibody, and methods for monitoring the responsiveness of a patient suffering from HSCT-TMA to treatment with an anti-C5 antibody or an anti-CFB antibody.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] 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 Art

[0003] Hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) is a multifactorial disorder caused by systemic vascular endothelial damage, which can be triggered by several mechanisms during the transplant process. Patients with HSCT-TMA develop renal damage, serositis, pulmonary hypertension, and multisystem organ failure. Severe HSCT-TMA is associated with approximately 80% long-term morbidity and mortality. Studies have shown that most 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 rate for 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).

[0004] In pediatric patients, HSCT-TMA typically occurs early after allogeneic HSCT, with a median diagnosis of 35 to 47 days after HSCT, and 88% to 92% occurring before day +100. However, cases up to 2 years after HSCT have been reported. Autologous recipients may develop HSCT TMA even earlier, with a median of 18 days after HSCT (Dvorak et al. Front Pediatr 2019;7:133).

[0005] Endothelial injury is fundamental to the pathogenesis of HSCT-TMA, and dysregulated complement activation may be a consequence of endothelial injury. Risk factors associated with the development of HSCT-TMA that also trigger endothelial injury include calcineurin inhibitors (CNiS), infection, and conditioning regimens (high-dose chemotherapy or whole-body irradiation) (Khosla, et al., Bone Marrow Transplant. 2018; 53(2): 129-137; Masias, et al., Blood. 2017; 129(21): 2857-2863).

[0006] Currently, there is no approved therapy for the treatment of HSCT-TMA, and the identification of risk patients is challenging, resulting in delayed diagnosis and terrible outcomes. Therefore, an object of the present invention is to identify biomarkers associated with HSCT-TMA and to provide improved methods for treating and identifying patients (particularly pediatric patients) suffering from HSCT-TMA. Summary of the invention

[0007] About 10%-20% of HSCT patients develop into HSCT-TMA.These patients are difficult to identify and have terrible results (for example, transplant rejection and even death).However, by the present invention, it has been found that the patient suffering from HSCT with abnormal levels of TM and SYNDI-1 and factor Ba and / or heparan sulfate proteoglycan (HSPG) may develop HSCT-TMA.It is further observed that after using anti-C5 antibody (for example, eculizumab (eculizumab) or ravulizumab (ravulizumab)) or complement factor B inhibitor (for example, the formation of factor Ba or active inhibitor, referred to herein as " CFB inhibitor "), the concentration of these proteins changes (for example, reduction and / or normalization).

[0008] Therefore, in one aspect, the present disclosure provides a method for treating a patient (e.g., pediatric or adult patient) with hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA), the patient has been determined to have elevated levels of these biomarkers (e.g., blood or plasma levels) compared to the normal reference range of biomarkers selected from thrombomodulin (TM) and syndecan-1 (SYND1) or a combination thereof, 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 levels of these biomarkers in the patient, thereby treating HSCT-TMA. In one embodiment, the patient is also determined to have elevated Ba and / or C5b9 levels (e.g., blood or plasma levels) compared to the normal reference range of complement factors Ba and / or C5b9. In another embodiment, the patient has been determined to have elevated HSPG levels (e.g., blood or plasma levels) compared to the normal reference range of HSPG.

[0009] In another aspect, the present 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 an elevated level of a biomarker selected from TM and SYND1 or a combination thereof in the patient sample compared to a normal reference range of 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 the elevated levels of TM and SYND1 in the patient, thereby treating HSCT-TMA. In one embodiment, the method further comprises determining or having determined elevated levels of Ba and / or C5b9 in the sample compared to a normal reference range of Ba and / or C5b9. In another embodiment, the method comprises determining or having determined elevated levels of HSPG compared to a normal reference range of HSPG.

[0010] In another aspect, the present 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 a biomarker level selected from TM and SYND1 or a combination thereof in a sample (e.g., a blood or plasma sample) from the patient using an in vitro assay, wherein the biomarker level in the sample that is elevated compared to the normal reference range for TM and SYND1, respectively, identifies the patient as suitable for treatment with an anti-C5 antibody or an anti-CFB antibody. In one embodiment, the method further comprises determining Ba and / or C5b9 levels in the sample, wherein Ba and / or C5b9 levels elevated compared to the normal reference range for Ba and / or C5b9 identify the patient as suitable for treatment with an anti-C5 antibody or an anti-CFB antibody. . In another embodiment, the method comprises determining HSPG levels in the sample, wherein elevated HSPG levels compared to the normal reference range for HSPG identify the patient as suitable for treatment with an anti-C5 antibody or an anti-CFB antibody. 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, the method 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 reduced level of the biomarker in the sample from the patient obtained during or after treatment compared to the level of the biomarker in the 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.

[0011] In one embodiment, the method further comprises determining the level of Ba and / or C5b9 in the sample (e.g., a blood or plasma sample) obtained from the patient during or after treatment, wherein: a reduced level of Ba and / or C5b9 in the sample from the patient obtained during or after treatment compared to the level of Ba and / or C5b9 in the 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. In another embodiment, the method further comprises determining the level of HSPG in the sample (e.g., a blood or plasma sample) obtained from the patient during or after treatment, wherein: a reduced level of HSPG in the sample from the patient obtained during or after treatment compared to the level of HSPG in the 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.

[0012] One or more levels of biomarkers described herein (e.g., TM, SYND1, / or Ba, and / or HSPG) can be measured by any suitable means or art-recognized techniques. In one embodiment, one or more levels are measured by a system or kit approved by a regulatory agency (e.g., USFDA). In another embodiment, one or more levels are measured by using immunoassays, immunochemistry, immunohistochemistry, nucleoprobe assays, in situ hybridization, fluorescent RNA probes, RT-PCR, microarray transcription assays or RNA transcription assays. In another embodiment, one or more levels are measured by enzyme-linked immunosorbent assay (ELISA).

[0013] The level of biomarkers described herein (e.g., TM, SYND1, Ba and / or HSPG) can be measured for control values. Controls include negative controls and positive controls. In some embodiments, negative controls may include the normal reference range of each corresponding biomarker to assess whether the level in the experimental sample (e.g., obtained from a sample of a patient who is experiencing or may experience HSCT-TMA) is relatively increased or reduced. In one embodiment, the normal reference range of biomarkers is based on healthy patients (e.g., patients who do not suffer from HSCT). In another embodiment, the normal reference range of biomarkers is based on HSCT patients without TMA.

[0014] In some embodiments, a positive control may include a reference range for each corresponding biomarker that allows for an assessment of whether the level of the biomarker in an experimental sample (e.g., a sample from a patient undergoing HSCT-TMA treatment) is relatively reduced or attenuated, e.g., before versus after treatment.

[0015] In one embodiment, the normal reference range for TM in healthy patients (eg, patients who have not undergone HSCT) is about 1.8 ng / mL to about 4.8 ng / mL. For example, in one embodiment, the normal TM level in a healthy patient is 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.

[0016] In another embodiment, in the absence of TMA, the normal reference range of TM in HSCT patients is about 3 ng / mL to about 9 ng / mL. For example, in one embodiment, in the absence of TMA, the normal reference range of TM in HSCT patients is about 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL or 9 ng / mL.

[0017] In another embodiment, compared with a normal reference range (for example, a healthy patient not suffering from HSCT or a HSCT patient without TMA), the TM level of the HSCT patient suffering from or possibly developing HSCT-TMA is increased. For example, the TM level of the increase is greater than about 10ng / mL, 11ng / mL, 12ng / mL, 13ng / mL, 14ng / mL, 15ng / mL, 16ng / mL, 17ng / mL, 18ng / mL, 19ng / mL, 20ng / mL, 21ng / mL, 22ng / mL, 23ng / mL, 24ng / mL, 25ng / mL, 26ng / mL, 27ng / mL, 28ng / mL, 29ng / mL or 30ng / mL.

[0018] In another embodiment, the TM level in the sample is considered elevated when the TM level in the sample is at least about 10% higher than the normal reference range for TM (e.g., healthy patients who have not suffered from HSCT or HSCT patients without TMA) to about 10 times, such as about two times, the normal reference range for TM. In some embodiments, the TM level in the sample is considered elevated when the TM level in the sample is at least three, four, five, or six times the normal reference range for TM.

[0019] In one embodiment, the normal reference range of SYND1 for healthy patients (e.g., patients who have not suffered from HSCT) is between 15 ng / mL and 70 ng / mL. For example, in one embodiment, the normal SYND1 level for healthy patients 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, 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, 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, 5 6ng / 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.

[0020] In another embodiment, in the absence of TMA, the normal reference range of SYND1 for HSCT patients is about 15 ng / mL to 55 ng / mL. For example, in one embodiment, in the absence of TMA, the normal reference range of SYND1 for HSCT patients 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, mL, 33ng / mL, 34ng / mL, 35ng / mL, 36ng / mL, 37ng / mL, 38ng / mL, 39ng / mL, 40ng / mL, 41ng / mL, 42ng / mL, 43ng / mL, 44 ng / mL, 45ng / mL, 46ng / mL, 47ng / mL, 48ng / mL, 49ng / mL, 50ng / mL, 51ng / mL, 52ng / mL, 53ng / mL, 54ng / mL or 55ng / mL.

[0021] In another embodiment, HSCT patients who have or are likely to develop HSCT-TMA have elevated levels of SYND1 compared to a normal reference range (eg, healthy patients who have not had HSCT or HSCT patients who do not have TMA). For example, the elevated SYND1 levels are greater than 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, 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.

[0022] In another embodiment, the level of SYND1 in the sample is considered elevated when the level of SYND1 in the sample is at least about 2 to about 17 times, such as about four times, the normal reference range for SYND1 (e.g., healthy patients who have not suffered from HSCT or HSCT patients without TMA). In some embodiments, the level of SYND1 in the sample is considered elevated when the level of SYND1 in the sample is at least three, four, five, six, seven, eight, ten, twelve, or fifteen times the normal reference range for SYND1.

[0023] In one embodiment, the normal reference range of factor Ba in healthy patients (e.g., patients not suffering from HSCT) is less than about 1000 ng / mL. In another embodiment, the normal reference range of factor Ba in healthy patients (e.g., patients not suffering from HSCT) is less than about 600 ng / mL. In another embodiment, the normal reference range of factor Ba in healthy patients (e.g., patients not suffering from 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 who has not undergone HSCT) is about 300 ng / mL, 310 ng / mL, 320 ng / mL, 330 ng / mL, 340 ng / mL, 350 ng / mL, 360 ng / mL, 370 ng / mL, 380 ng / mL, 390 ng / mL, 400 ng / mL, 410 ng / mL, 420 ng / mL, 430 ng / mL, 440 ng / mL, 450 ng / mL, 460 ng / mL, 470 ng / mL, 480 ng / mL, 490 ng / mL, 500 ng / mL, 510 ng / mL, 520 ng / mL, 530 ng / mL, 540 ng / mL, 550 ng / mL, 560 ng / mL, 570 ng / mL, 580 ng / mL, 590 ng / mL, or 600 ng / mL.

[0024] In another embodiment, the normal reference range for Factor Ba in HSCT patients in the absence of TMA is about 500 ng / mL to 800 ng / mL. For example, in one embodiment, the normal reference range for factor Ba in HSCT patients in the absence of TMA is about 500 ng / mL, 510 ng / mL, 520 ng / mL, 530 ng / mL, 540 ng / mL, 550 ng / mL, 560 ng / mL, 570 ng / mL, 580 ng / mL, 590 ng / mL, 600 ng / mL, 610 ng / mL, 620 ng / mL, 630 ng / mL, 640 ng / mL, 650 ng / mL, 660 ng / mL, 670 ng / mL, 680 ng / mL, 690 ng / mL, 700 ng / mL, 710 ng / mL, 720 ng / mL, 730 ng / mL, 740 ng / mL, 750 ng / mL, 760 ng / mL, 770 ng / mL, 780 ng / mL, 790 ng / mL or 800 ng / mL.

[0025] In another embodiment, compared with a normal reference range (e.g., a healthy patient not suffering from HSCT or a HSCT patient without TMA), the factor Ba level of the HSCT patient suffering from or possibly developing HSCT-TMA is increased. For example, the Ba level of increase is greater than 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, 1210ng / mL, 1220ng / mL, 1230ng / mL, 1240ng / mL, 1250ng / mL, 1260ng / mL, 1270ng / mL, 1280ng / mL, 1290ng / mL, 1310ng / mL, 1320ng / mL, 1330ng / mL, 1340ng / mL, 1350ng / mL, 1360ng / mL, 1370ng / mL, 1380ng / mL, 1390ng / mL g / 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, 135 0ng / 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 / mL, 1560ng / mL, 1570ng / mL, 1 580ng / mL, 1590ng / mL, 1600ng / mL, 1610ng / mL, 1620ng / mL, 1630ng / mL, 1640ng / mL, 1650ng / mL, 1660ng / mL, 1670ng / mL, 1680ng / mL, 1690n g / 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, 1870ng / mL, 1880ng / mL, 1890ng / mL, 1900ng / mL, 1910ng / mL, 1920ng / mL, 1930ng / mL, 1940ng / mL, 1950ng / mL, 1960ng / mL, 1970ng / mL, 1980n g / mL, 1990ng / mL, 2000ng / mL, 2010ng / mL, 2020ng / mL, 2030ng / mL, 2040ng / mL, 2050ng / mL, 2060ng / mL, 20 70ng / mL, 2080ng / mL, 2090ng / mL, 2100ng / mL, 2110ng / mL, 2120ng / mL, 2130ng / mL, 2140ng / mL, 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, 2330n g / mL, 2340ng / mL, 2350ng / mL, 2360ng / mL, 2370ng / mL, 2380ng / mL, 2390ng / mL, 2400ng / mL, 2410ng / mL, 24 20ng / mL, 2430ng / mL, 2440ng / mL, 2450ng / mL, 2460ng / mL, 2470ng / mL, 2480ng / mL, 2490ng / mL or 2500ng / mL. ,

[0026] In another embodiment, the Ba level in the sample is considered elevated when the Ba level in the sample is at least about 12% higher than the normal reference range for factor Ba (e.g., healthy patients who have not suffered from HSCT or HSCT patients without TMA) to about 5 times, such as about two times, the normal reference range for factor Ba. In some embodiments, the Ba level in the sample is considered elevated when the Ba level in the sample is at least three, four, or five times the normal reference range for Ba.

[0027] In another embodiment, HSPG levels are elevated in HSCT patients who have or are likely to develop HSCT-TMA compared to a normal reference range (eg, healthy patients who have not had HSCT or HSCT patients who do not have TMA).

[0028] In another embodiment, the HSPG level in the sample is considered elevated when the HSPG level in the sample is at least about 20% higher than the normal reference range for HSPG (e.g., healthy patients who have not suffered from HSCT or HSCT patients without TMA) to about 2 times, such as about two times, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times, fifteen times, sixteen times, seventeen times, eighteen times, nineteen times or twenty times the normal reference range for HSPG.

[0029] 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, a scFv, a SMIP, Nanobodies or domain antibodies.

[0030] 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 ) is an anti-C5 antibody comprising heavy chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NOs: 1, 2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NOs: 4, 5 and 6, respectively. Eculizumab comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 8. Eculizumab comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 10 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 11.

[0031] Another exemplary anti-C5 antibody is ravulizumab (also known as ALXN1210 and antibody BNJ441), which comprises a heavy chain and a light chain having the sequence shown in SEQ ID NOs: 14 and 11, respectively, or an antigen-binding fragment and variant thereof. In other embodiments, the antibody comprises the heavy chain and light chain complementary determining regions (CDRs) or variable regions (VRs) of lavulizumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable (vH) region of lavulizumab having the sequence shown in SEQ ID NO: 12, and the CDR1, CDR2, and CDR3 domains of the light chain variable (VL) region of lavulizumab having the sequence shown in SEQ ID NO: 8. In another embodiment, the antibody comprises the CDR1, CDR2, and CDR3 heavy chain sequences as shown in SEQ ID NOs: 19, 18, and 3, respectively, and the CDR1, CDR2, and CDR3 light chain sequences as shown in SEQ ID NOs: 4, 5, and 6, respectively. In another embodiment, the antibody comprises VH and VL regions having the amino acid sequences shown in SEQ ID NO: 12 and SEQ ID NO: 8, respectively. In another embodiment, the antibody comprises a heavy chain constant region shown in SEQ ID NO:13.

[0032] In another embodiment, the antibody comprises a variant human Fc constant region that binds to 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, each by EU numbering.

[0033] In another embodiment, the antibody comprises the CDR1, CDR2 and CDR3 heavy chain sequences as shown in SEQ ID NOs: 19, 18 and 3, respectively, and the CDR1, CDR2 and CDR3 light chain sequences as shown in SEQ ID NOs: 4, 5 and 6, respectively, and a variant human Fc constant region that binds to 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, each by EU numbering.

[0034] In another embodiment, the antibody has a pH of 0.1 nM ≤ K at pH 7.4 and 25°C. D Affinity dissociation constants (K) in the range of ≤1 nM D ) binds to human C5. In another embodiment, the antibody binds to human C5 at pH 6.0 and 25°C with a K DIn yet another embodiment, the antibody has a [(K of the antibody or antigen-binding fragment thereof for human C5 at pH 6.0 and 25°C) D ) / (K of the antibody or antigen-binding fragment thereof for human C5 at pH 7.4 and 25°C D )] is greater than 25.

[0035] Another exemplary anti-C5 antibody is described in U.S. Pat. Nos. 8,241,628 and 8,883,158. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR1, CDR2, and CDR3 domain having the sequence shown in SEQ ID NOs: 21, 22, and 23, respectively, and a light chain CDR1, CDR2, and CDR3 domain having the sequence shown 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 shown in SEQ ID NO: 27 and a VL region having the sequence shown in SEQ ID NO: 28.

[0036] Another exemplary anti-C5 antibody is also described in U.S. Pat. Nos. 8,241,628 and 8,883,158. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR1, CDR2, and CDR3 domain having the sequence shown in SEQ ID NOs: 29, 30, and 31, respectively, and a light chain CDR1, CDR2, and CDR3 domain having the sequence shown in SEQ ID NOs: 32, 33, and 34, respectively. In another embodiment, the antibody comprises a VH region having the sequence shown in SEQ ID NO: 35 and a VL region having the sequence shown in SEQ ID NO: 36.

[0037] Another exemplary anti-C5 antibody is described in U.S. Pat. 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 shown in SEQ ID NOs: 37, 38 and 39, respectively, and light chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NOs: 40, 41 and 42, respectively. In another embodiment, the antibody comprises a VH region having the sequence shown in SEQ ID NO: 43 and a VL region having the sequence shown in SEQ ID NO: 44.

[0038] 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.

[0039] 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.

[0040] In some embodiments, the anti-C5 antibody is a biosimilar to eculizumab For example, in one embodiment, the anti-C5 antibody is, for example, ABP 959 antibody (a biosimilar of eculizumab manufactured by Amgen Inc.), (manufactured by Generium JNC, Russia), SB12 (eculizumab biosimilar manufactured by Samsung Bioepis, Incheon, South Korea), ISU305 (eculizumab biosimilar from ISUAbxis, South Korea), (an eculizumab biosimilar from CinnaGen in Iran), BCD 148 (an eculizumab biosimilar from Biocad Medical in Quebec, Canada), tesidolumab (made by Novartis), crovalimab (made by Roche), CAN106 (made by CanBridge Bio in Beihai, China), or pozelimab (made by Regeneron).

[0041] In another embodiment, the antibody competes for binding to the same epitope on C5 as the above antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above antibodies (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% variable region identity).

[0042] 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.

[0043] In one embodiment, an anti-C5 antibody (e.g., lavulizumab) is administered as follows:

[0044] (a) once on day 1 at a dose of 600 mg for patients weighing ≥5 kg to <10 kg, 600 mg for patients weighing ≥10 kg to <20 kg, 900 mg for patients weighing ≥20 kg to <30 kg, 1200 mg for patients weighing ≥30 kg to <40 kg, 2400 mg for patients weighing ≥40 kg to <60 kg, 2700 mg for patients weighing ≥60 kg to <100 kg, or 3000 mg for patients weighing ≥100 kg;

[0045] (b) once on day 5 at a dose of 300 mg for patients weighing ≥5 kg to <10 kg, 300 mg for patients weighing ≥10 kg to <20 kg, 300 mg for patients weighing ≥20 kg to <30 kg, 300 mg for patients weighing ≥30 kg to <40 kg, 600 mg for patients weighing ≥40 kg to <60 kg, 900 mg for patients weighing ≥60 kg to <100 kg, or 900 mg for patients weighing ≥100 kg;

[0046] (c) once on day 10 at a dose of 300 mg for patients weighing ≥5 kg to <10 kg, 300 mg for patients weighing ≥10 kg to <20 kg, 300 mg for patients weighing ≥20 kg to <30 kg, 300 mg for patients weighing ≥30 kg to <40 kg, 600 mg for patients weighing ≥40 kg to <60 kg, 900 mg for patients weighing ≥60 kg to <100 kg, or 900 mg for patients weighing ≥100 kg; and

[0047] (d) 300 mg for patients weighing ≥5 kg to <10 kg or 600 mg for patients weighing ≥10 kg to <20 kg on day 15 and every four weeks thereafter; or 2100 mg for patients weighing ≥20 kg to <30 kg, 2700 mg for patients weighing ≥30 kg to <40 kg, 3000 mg for patients weighing ≥40 kg to <60 kg, 3300 mg for patients weighing ≥60 kg to <100 kg, or 3600 mg for patients weighing ≥100 kg on day 15 and every eight weeks thereafter.

[0048] In another aspect, anti-CFB antibodies are used in the methods described herein. Any suitable anti-CFB antibodies can be used in the methods described herein. The efficacy of the methods of treatment provided herein can be assessed using any suitable means. In one embodiment, treatment results in a reduction or cessation of microangiopathic hemolytic anemia, thrombocytopenia, endothelial damage, renal injury, renal failure, serositis, pulmonary hypertension, and multisystem organ failure compared to baseline.

[0049] In another embodiment, treatment results in normalization of LDH, elimination of red blood cell and platelet transfusion requirements, increase in hemoglobin, and / or disappearance of schistocytes compared to baseline. In another embodiment, treatment results in: (a) platelet count ≥ 50,000 / mm2 without transfusion support during the first 7 days 3 , (b) LDH<1.5×ULN, and (c) absence of schistocytes (if schistocytes are present at baseline), and / or (d) proteinuria is reduced by at least 50% from baseline. In another embodiment, treatment results in a favorable hematological response. In another embodiment, treatment results in hemoglobin ≥8g / dL in the absence of transfusion support. In another embodiment, treatment results in terminal complement inhibition. In another embodiment, treatment results in a reduction in adverse events. In another embodiment, treatment results in a change in the quality of life as assessed by a quality of life assessment (Quality of Life Assessment) relative to baseline. In another embodiment, quality of life assessment is a quality of life inventory (PedsQL) scale or EQ-5D-5L questionnaire).

[0050] In another aspect, an anti-C5 antibody or an 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) who has been determined to have an elevated blood 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, wherein the anti-C5 antibody or anti-CFB antibody is administered to the patient in an amount and at a frequency sufficient to attenuate the level of the biomarker in the patient.

[0051] In another aspect, an anti-C5 antibody or an antigen-binding fragment thereof or an anti-CFB antibody for identifying a patient with HSCT-TMA who is suitable for treatment with an anti-C5 antibody or an anti-CFB antibody is provided, the identification 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 an elevated level of the biomarker in the blood sample compared to a normal reference range for TM and SYND1, respectively, identifies the patient as suitable for treatment with an anti-C5 antibody or an anti-CFB antibody.

[0052] In another aspect, an anti-C5 antibody or an antigen-binding fragment thereof or an anti-CFB antibody for monitoring the responsiveness of a patient with HSCT-TMA to treatment with an anti-C5 antibody or an anti-CFB antibody is provided, wherein the use comprises: determining a biomarker selected from TM and SYND1 or a combination thereof in a blood sample obtained from the patient during or after treatment, wherein: a reduced level of the biomarker in the blood sample obtained from the patient during or after treatment compared to the level of the biomarker in the blood sample obtained from the patient 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.

[0053] In another aspect, a use of an anti-C5 antibody or an antigen-binding fragment thereof or an anti-CFB antibody for treating a patient with hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) who has been determined to have an elevated blood 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, wherein the anti-C5 antibody or anti-CFB antibody is administered to the patient in an amount and at a frequency sufficient to attenuate the level of the biomarker in the patient.

[0054] In another aspect, use of an anti-C5 antibody or an antigen-binding fragment thereof, or an anti-CFB antibody, in identifying a patient with HSCT-TMA who is suitable for treatment with an anti-C5 antibody or an anti-CFB antibody is provided, the identification 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 an elevated level of the biomarker in the blood sample compared to the normal reference range for TM and SYND1, respectively, identifies the patient as suitable for treatment with an anti-C5 antibody or an anti-CFB antibody.

[0055] In another aspect, 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 with HSCT-TMA to treatment with an anti-C5 antibody or an anti-CFB antibody is provided, the use comprising: determining a biomarker selected from TM and SYND1 or a combination thereof in a blood sample obtained from the patient during or after treatment, wherein: a reduced level of the biomarker in the blood sample obtained from the patient during or after treatment compared to the level of these biomarkers in the blood sample obtained from the patient 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 DESCRIPTION OF THE DRAWINGS

[0056] Figure 1is a graph showing plasma TM levels in LPS-injected mice treated with anti-complement agents.

[0057] Figure 2 is a graph showing that cyclosporine-induced TM loss is partially prevented by treatment with eculizumab.

[0058] FIG. 3A to FIG. 3C Depicted are biomarker levels of glyclocalyx components in pediatric patients with and without HSCT-TMA. Specifically, Figure 3A and Figure 3B It was shown that HSCT-TMA patients (n=11) had significantly elevated levels of glycocalyx damage biomarkers compared to HSCT control patients (n=7). Figure 3C It is shown that a strong positive correlation between TM and SYND1 is observed.

[0059] FIG. 4A to FIG. 4C AP activation in pediatric patients with and without HSCT-TMA is depicted. Specifically, Figure 4A It was shown that plasma Ba was significantly increased in patients with HSCT-TMA compared with patients without HSCT-TMA. In addition, plasma Ba and TM levels ( Figure 4B ) and SYND1 levels ( Figure 4C ) are positively correlated.

[0060] Figure 5 Plasma TM levels in LPS-injected mice treated with anticomplement agents are depicted. Figure 5 As shown, mice with inflammation-mediated complement activation had elevated circulating TM levels, which were attenuated by treatment with anti-complement agents.

[0061] FIG. 6A to FIG. 6B The correlation between TM and Ba in LPS-injected mice is shown. TM levels and Ba levels were measured using commercially available ELISA or by Western blotting ( Fig. 6A ).like Figure 6B As shown, plasma Ba was positively correlated with TM levels in mice with inflammation-mediated complement activation.

[0062] 7A to 7D Describes the existence of FIG. 7A to FIG. 7B ) or no alternative to eculizumab exists ( FIG. 7C to FIG. 7D ) in HUVECs treated with CsA and 30% NHS for 18 h. As shown in these figures, TM and heparan sulfate proteoglycan (HSPG) surface expression was reduced on HUVECs treated with CsA and partially restored by eculizumab replacement.

[0063] FIG. 8A to FIG. 8B Depicts the deposition of complement activation products on HMEC-1 cells treated with CsA. As shown in these figures, CsA treatment induces complement deposition on HMEC-1 cells, and C5 inhibition reduces C5b-9 ( Figure 8B ), but not iC3b deposition ( Fig. 8A ). DETAILED DESCRIPTION

[0064] As described herein and illustrated in the working examples, specific biomarkers associated with HSCT-TMA have been found. Specifically, it has been found that HSCT patients with elevated concentrations of certain proteins (e.g., TM, SYND1 and / or Ba, referred to herein as "HSCT-TMA biomarker proteins") may develop HSCT-TMA. Similarly, the reduction and / or standardized concentration (or activity) of these proteins in the biological fluid obtained from HSCT-TMA patients treated with complement inhibitors (e.g., anti-C5 antibodies (e.g., eculizumab or lavulizumab) or anti-CFB antibodies) indicates that the patient is responsive to treatment. Therefore, the concentration and / or activity level analysis of such proteins can be used to assess the risk that HSCT patients, in particular, will develop HSCT-TMA, monitor the progression or alleviation of HSCT-TMA, and / or monitor the therapeutic response to complement inhibitors (e.g., such as anti-C5 antibodies (e.g., eculizumab or lavulizumab) or anti-CFB antibodies).

[0065] I. Definitions

[0066] As used herein, the term "subject" or "patient" is a human patient (eg, a patient having HSCT or HSCT-TMA).

[0067] As used herein, the term "pediatric" patient is a human patient that has been classified by a physician or caregiver as belonging to a non-adult category, and may include, for example, newborns (both premature and full-term), infants, children, and adolescents. Typically, a pediatric patient is a patient under 18 years of age (<18 years old).

[0068] As used herein, the term "adult" patient is a human patient who has been classified by a physician or caregiver, etc., e.g., based on age, developmental status, physiological characteristics, etc., e.g., not a neonate, infant, child, or adolescent. Typically, an adult patient is a patient 18 years of age or older (≥18 years).

[0069] The term "antibody" describes a polypeptide comprising at least one antibody-derived antigen binding site (e.g., VH / VL region or Fv or CDR). The term "antibody" can be used interchangeably with the term "immunoglobulin". Antibodies include known forms of antibodies, for example, the antibody can be a human antibody, a humanized antibody, a bispecific antibody, a chimerized or chimeric antibody, a polyclonal antibody, a monoclonal antibody, a primatized antibody, and a deimmunized antibody. Antibodies can be prepared in or derived from any of a variety of species, for example, mammals such as humans, non-human primates (e.g., orangutans, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. Antibodies can be purified or recombinant antibodies. The antibody may also be 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 altered by protein engineering techniques (e.g., by mutation, deletion, substitution, conjugation with a non-antibody moiety). The antibody may include, for example, one or more variant amino acids (compared to naturally occurring antibodies) that alter antibody properties (e.g., functional properties). Many such changes are known in the art, and they affect, for example, the half-life in a patient, effector functions and / or the immune response to the antibody. The term antibody also includes an artificial or engineered polypeptide construct comprising at least one antibody-derived antigen binding site.

[0070] The term "antibody" includes antigen-binding fragments thereof. The term "antigen-binding fragment" or similar terms are known in the art and may, for example, refer 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. The scFv fragment is a single polypeptide chain that contains both the heavy chain and light chain variable regions of the antibody from which the scFv is derived. In addition, intrabodies, miniantibodies, triantibodies, and diabodies are also included in the definition of antibodies and are suitable for use in the methods described herein. See, e.g., 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. The antigen binding fragment may also include the variable region of the heavy chain polypeptide and the variable region of the light chain polypeptide. Thus, the antigen binding fragment may comprise the CDRs of the light chain and heavy chain polypeptides of an antibody.

[0071] The term "antibody fragment" also includes, for example, single domain antibodies, such as camelized single domain antibodies. See, for example, 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. Pat. No. 6,005,079. The term "antibody fragment" also includes a fragment comprising two V H domain single domain antibody, these two V H The domains have modifications such that single domain antibodies are formed.

[0072] The terms "polypeptide," "peptide," and "protein" are used interchangeably and are art-recognized and may refer to any chain of peptide-bonded amino acids, regardless of length or post-translational modifications.

[0073] II. HSCT-TMA-related biomarker proteins

[0074] As used herein, the term "biomarker" refers to a measurable substance in a subject.

[0075] Its presence indicates some phenomena, such as disease, infection or environmental exposure.Through the present disclosure, it has been found that certain biomarkers are associated with HSCT-TMA and indicate HSCT-TMA.The biomarker proteins associated with these HSCT-TMA include, for example, proteolytic fragments (e.g., Ba) of thrombomodulin (TM), syndecan-1 (SYND1) and complement component factor B.

[0076] A. Thrombomodulin

[0077] 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 that regulates coagulation and inflammation (see, e.g., Califano, et al., Eur. Rev. Med. Pharmacol. Sci. 2000 May-June; 4(3): 59-66). Thrombomodulin is also expressed on human mesenchymal cells, monocytes and a subset of dendritic cells. Thrombomodulin can stimulate endothelial cell growth. This feature of TM depends on a molecular substrate called an EGF-like (epidermal growth factor-like) sequence. One of the main effects of TM is to bind thrombin. When bound to TM, thrombin loses its procoagulant, proinflammatory and profibrogenic properties and instead acquires the ability to activate protein C (APC). APC further limits the generation of thrombin, counteracts the side 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 proinflammatory protein HMGB1. Thrombomodulin exists in two forms in vivo (see, e.g., Doggen et al., Thromb. Haemost. 1998; 80: 743-748). The first type has a higher molecular weight, and it binds to the cytoplasmic 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 a soluble or plasma form. The heavy form weighs 150 kDa, while the light form weighs 69 kDa. The molecular sequence of thrombomodulin contains an N-terminal lectin-like element (1 residue-154 residues), a hydrophobic region (155 residues-222 residues), six EGF-like modules (223 residues-462 residues), a Ser / Thr-rich dominium (463 residues-497 residues), a transmembrane portion consisting of 23 amino acids (498 residues-521 residues) and a tail of 35 cytoplasmic amino acids (522 residues-557 residues) (see, e.g., Califano, et al., Eur. Rev. Med. Pharmacol. Sci. 2000 May-June; 4(3):59-66).

[0078] Different pathological conditions increase soluble circulating TM (see, e.g., Califano, et al., Eur. Rev. Med. Pharmacol. Sci. 2000 May-June; 4(3): 59-66). Endothelial cells that are more exposed to hemodynamic turbulence (such as those at the bifurcations of large arteries) release large amounts of TM (see, e.g., Salomaa et al., Lancet 1999; 353: 1729-1734). Thrombomodulin levels range from 3 ng / mL to 300 ng / mL. Normal levels are believed to be 3.1 ng / mL ± 1.3 ng / mL, with slightly higher levels in men (see, e.g., Doggenet et al., Thromb. Haemost. 1998; 80: 743-748). It appears that in women, TM levels increase during menopause. Women with surgically induced menopause have soluble TM levels far above normal. After six weeks of hormone replacement therapy, TM levels are significantly reduced (see, e.g., Neumann et al., Circulation 1995; 92: 748-755). Thrombomodulin levels vary according to race, and blacks appear 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.

[0079] In one embodiment, the normal reference range for TM in healthy patients (eg, patients who have not undergone HSCT) is about 1.8 ng / mL to about 4.8 ng / mL. For example, in one embodiment, the normal TM level in a healthy patient is 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.

[0080] In another embodiment, in the absence of TMA, the normal reference range of TM in HSCT patients is about 3 ng / mL to about 9 ng / mL. For example, in one embodiment, in the absence of TMA, the normal reference range of TM in HSCT patients is about 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL or 9 ng / mL.

[0081] In another embodiment, compared with a normal reference range (for example, a healthy patient not suffering from HSCT or a HSCT patient without TMA), the TM level of the HSCT patient suffering from or possibly developing HSCT-TMA is increased. For example, the TM level of the increase is greater than about 10ng / mL, 11ng / mL, 12ng / mL, 13ng / mL, 14ng / mL, 15ng / mL, 16ng / mL, 17ng / mL, 18ng / mL, 19ng / mL, 20ng / mL, 21ng / mL, 22ng / mL, 23ng / mL, 24ng / mL, 25ng / mL, 26ng / mL, 27ng / mL, 28ng / mL, 29ng / mL or 30ng / mL.

[0082] In another embodiment, the TM level in the sample is considered elevated when the TM level in the sample is at least about 10% higher than the normal reference range for TM (e.g., healthy patients who have not suffered from HSCT or HSCT patients without TMA) to about 10 times, such as about two times, the normal reference range for TM. In some embodiments, the TM level in the sample is considered elevated when the TM level in the sample is at least three, four, five, or six times the normal reference range for TM.

[0083] B. Syndecan-1

[0084] Syndecan-1 (also known as "SDC", "syndecan 1", "CD138", "SDC1", "SYND1", "syndecan", "heparan sulfate proteoglycan fibroblast growth factor receptor", SYND1 is a protein encoded by the SDC1 gene in humans (see, 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, 265(12):6884-9). Genetics, 16(5):501-5). The 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 via its receptors for extracellular matrix proteins. SYND1 is a sponge for growth factors and chemokines, primarily binding via heparan sulfate chains (see, e.g., M (April 2003), FASEB Journal. 17 (6): 575-91). Syndecan mediates cell binding, cell signaling and cytoskeletal organization, and the syndecan receptor is required for the internalization of HIV-1 tat protein. The SYND1 core protein consists of an extracellular domain that can be replaced by heparan sulfate and chondroitin sulfate glycosaminoglycan chains, a highly conserved transmembrane domain and a highly conserved cytoplasmic domain. The SYND1 core protein contains 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 membrane-bound proteoglycans into paracrine effector molecules that play a role in wound repair and invasive growth of cancer cells (see, e.g., Wang Z, M, Bernfield M, Reizes O (September 2005), Biochemistry. 44(37): 12355-61, Elenius V, et al., Journal of Biochemistry. 279(40): 41928-35, and Piperigkou Z, (September 2016), Cell and Tissue Research 365(3): 643-55).

[0085] In one embodiment, the normal reference range of SYND1 for healthy patients (e.g., patients who have not suffered from HSCT) is between 15 ng / mL and 70 ng / mL. For example, in one embodiment, the normal SYND1 level for healthy patients 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, 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, 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, 5 6ng / 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.

[0086] In another embodiment, in the absence of TMA, the normal reference range of SYND1 for HSCT patients is about 15 ng / mL to 55 ng / mL. For example, in one embodiment, in the absence of TMA, the normal reference range of SYND1 for HSCT patients 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, mL, 33ng / mL, 34ng / mL, 35ng / mL, 36ng / mL, 37ng / mL, 38ng / mL, 39ng / mL, 40ng / mL, 41ng / mL, 42ng / mL, 43ng / mL, 44 ng / mL, 45ng / mL, 46ng / mL, 47ng / mL, 48ng / mL, 49ng / mL, 50ng / mL, 51ng / mL, 52ng / mL, 53ng / mL, 54ng / mL or 55ng / mL.

[0087] In another embodiment, HSCT patients who have or are likely to develop HSCT-TMA have elevated levels of SYND1 compared to a normal reference range (eg, healthy patients who have not had HSCT or HSCT patients who do not have TMA). For example, the elevated SYND1 levels are greater than 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, 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.

[0088] In another embodiment, the level of SYND1 in the sample is considered elevated when the level of SYND1 in the sample is at least about 2 to about 17 times, such as about four times, the normal reference range for SYND1 (e.g., healthy patients who have not suffered from HSCT or HSCT patients without TMA). In some embodiments, the level of SYND1 in the sample is considered elevated when the level of SYND1 in the sample is at least three, four, five, six, seven, eight, ten, twelve, or fifteen times the normal reference range for SYND1.

[0089] C. Factor Ba

[0090] Factor B is a fragment of complement factor B. Factor B is a glycosylated protein composed of a single 93,000Da 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 (33000Da) and leaves a 60,000Bb fragment that binds to C3b. The Ba fragment is from the N-terminus of factor B, and it contains three CCP domains that interact with C3b. It has been reported that the isolated fragment Ba has a weak affinity for C3b and inhibits the interaction of factor B with C3b, thereby inhibiting the activation of the alternative pathway.

[0091] In one embodiment, the normal reference range of factor Ba in healthy patients (e.g., patients not suffering from HSCT) is less than about 1000 ng / mL. In another embodiment, the normal reference range of factor Ba in healthy patients (e.g., patients not suffering from HSCT) is less than about 600 ng / mL. In another embodiment, the normal reference range of factor Ba in healthy patients (e.g., patients not suffering from 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 who has not undergone HSCT) is about 300 ng / mL, 310 ng / mL, 320 ng / mL, 330 ng / mL, 340 ng / mL, 350 ng / mL, 360 ng / mL, 370 ng / mL, 380 ng / mL, 390 ng / mL, 400 ng / mL, 410 ng / mL, 420 ng / mL, 430 ng / mL, 440 ng / mL, 450 ng / mL, 460 ng / mL, 470 ng / mL, 480 ng / mL, 490 ng / mL, 500 ng / mL, 510 ng / mL, 520 ng / mL, 530 ng / mL, 540 ng / mL, 550 ng / mL, 560 ng / mL, 570 ng / mL, 580 ng / mL, 590 ng / mL, or 600 ng / mL.

[0092] In another embodiment, the normal reference range for Factor Ba in HSCT patients in the absence of TMA is about 500 ng / mL to 800 ng / mL. For example, in one embodiment, the normal reference range for factor Ba in HSCT patients in the absence of TMA is about 500 ng / mL, 510 ng / mL, 520 ng / mL, 530 ng / mL, 540 ng / mL, 550 ng / mL, 560 ng / mL, 570 ng / mL, 580 ng / mL, 590 ng / mL, 600 ng / mL, 610 ng / mL, 620 ng / mL, 630 ng / mL, 640 ng / mL, 650 ng / mL, 660 ng / mL, 670 ng / mL, 680 ng / mL, 690 ng / mL, 700 ng / mL, 710 ng / mL, 720 ng / mL, 730 ng / mL, 740 ng / mL, 750 ng / mL, 760 ng / mL, 770 ng / mL, 780 ng / mL, 790 ng / mL or 800 ng / mL.

[0093] In another embodiment, compared with a normal reference range (e.g., a healthy patient not suffering from HSCT or a HSCT patient without TMA), the factor Ba level of the HSCT patient suffering from or possibly developing HSCT-TMA is increased. For example, the Ba level of increase is greater than 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, 1210ng / mL, 1220ng / mL, 1230ng / mL, 1240ng / mL, 1250ng / mL, 1260ng / mL, 1270ng / mL, 1280ng / mL, 1290ng / mL, 1310ng / mL, 1320ng / mL, 1330ng / mL, 1340ng / mL, 1350ng / mL, 1360ng / mL, 1370ng / mL, 1380ng / mL, 1390ng / mL g / 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, 135 0ng / 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 / mL, 1560ng / mL, 1570ng / mL, 1 580ng / mL, 1590ng / mL, 1600ng / mL, 1610ng / mL, 1620ng / mL, 1630ng / mL, 1640ng / mL, 1650ng / mL, 1660ng / mL, 1670ng / mL, 1680ng / mL, 1690n g / 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, 1870ng / mL, 1880ng / mL, 1890ng / mL, 1900ng / mL, 1910ng / mL, 1920ng / mL, 1930ng / mL, 1940ng / mL, 1950ng / mL, 1960ng / mL, 1970ng / mL, 1980n g / mL, 1990ng / mL, 2000ng / mL, 2010ng / mL, 2020ng / mL, 2030ng / mL, 2040ng / mL, 2050ng / mL, 2060ng / mL, 20 70ng / mL, 2080ng / mL, 2090ng / mL, 2100ng / mL, 2110ng / mL, 2120ng / mL, 2130ng / mL, 2140ng / mL, 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, 2330n g / mL, 2340ng / mL, 2350ng / mL, 2360ng / mL, 2370ng / mL, 2380ng / mL, 2390ng / mL, 2400ng / mL, 2410ng / mL, 24 20ng / mL, 2430ng / mL, 2440ng / mL, 2450ng / mL, 2460ng / mL, 2470ng / mL, 2480ng / mL, 2490ng / mL or 2500ng / mL. ,

[0094] In one embodiment, the Ba level in the sample is considered elevated when the Ba level in the sample is at least about 12% higher than the normal reference range for factor Ba (e.g., healthy patients who have not suffered from HSCT or HSCT patients without TMA) to about 5 times, such as about two times, the normal reference range for factor Ba. In some embodiments, the Ba level in the sample is considered elevated when the Ba level in the sample is at least two, three, four, or five times the normal reference range for Ba.

[0095] D. Heparan sulfate proteoglycan (HSPG)

[0096] Heparan sulfate proteoglycans (HSPGs) are glycoproteins that have the common property of containing one or more covalently attached heparan sulfate (HS) chains and are a type of glycosaminoglycan (GAG) (see, e.g., Esko et al., Proteoglycans and Sulfated Glycosaminoglycans. In Essentials of glycobiology (Varki A, et al., eds.), pp. 229-248 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Heparan sulfate proteoglycans are found on the cell surface and in the extracellular matrix, where they interact with a plethora of ligands (see, e.g., Sarrazin et al., Cold Spring Harb Perspect Biol. 2011 Jul;3(7):a004952). Despite their small numbers, heparan sulfate proteoglycans have profound effects at the cellular, tissue, and organismal levels. Cells have a relatively small set of HSPGs (approximately 17) that are divided into three groups based on their location: membrane HSPGs, such as syndecan and glycosylphosphatidylinositol-anchored proteoglycans (glycopican); secreted extracellular matrix HSPGs (agrin, perlecan, type XVIII collagen); and secreted vesicular proteoglycans (serglycan) (see, e.g., Sarrazinet et al., 2011).

[0097] In one embodiment, HSPG levels are elevated in HSCT patients who have or are likely to develop HSCT-TMA compared to a normal reference range (eg, healthy patients who have not had HSCT or HSCT patients who do not have TMA).

[0098] In another embodiment, the HSPG level in the sample is considered elevated when the HSPG level in the sample is at least about 20% higher than the normal reference range for HSPG (e.g., healthy patients who have not suffered from HSCT or HSCT patients without TMA) to about 2 times, such as about two times, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times, fifteen times, sixteen times, seventeen times, eighteen times, nineteen times or twenty times the normal reference range for HSPG.

[0099] E. Biomarker Panel

[0100] Due in part to the enhanced prognostic and diagnostic significance of biomarkers when used in combination, multiple (e.g., at least 2, 3 or more) biomarkers can be detected and measured according to the present disclosure. Such biomarker combinations may be referred to herein as signatures.

[0101] 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 HSPG; or (e) TM+SYND1, optionally together with HSPG; (f) SYND1+Ba, optionally together with HSPG.

[0102] 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.

[0103] In one embodiment, the detecting step may comprise detecting a biomarker signature comprising TM+SYND1+Ba+C5b9+HSPG.

[0104] III. Anti-C5 Antibodies

[0105] 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 produced using methods known in the art. Alternatively, art-recognized anti-C5 antibodies can be used. Antibodies that compete for binding to C5 with any of these art-recognized antibodies or antibodies described herein can also be used. In some embodiments, the anti-C5 antibodies described herein bind to complement component C5 (e.g., human C5) and inhibit the cleavage of C5 into fragments C5a and C5b.

[0106] An exemplary anti-C5 antibody is eculizumab. Eculizumab (also known as ) is an anti-C5 antibody comprising heavy chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NOs: 1, 2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NOs: 4, 5 and 6, respectively. Eculizumab comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 8. Eculizumab comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 10 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 11.

[0107] An exemplary anti-C5 antibody is lavulizumab comprising a heavy chain and a light chain having the sequences set forth in SEQ ID NOs: 14 and 11, respectively, or antigen-binding fragments and variants thereof. Lavulizumab (also referred to as BNJ441 and ALXN1210) are described in WO2015134894 and US Patent No.: 9,079,949, all of which are hereby incorporated by reference. The terms lavulizumab, BNJ441 and ALXN1210 are used interchangeably throughout the document, but all refer to the same antibody. Lavulizumab selectively binds to human complement protein C5, inhibiting the cleavage of human complement protein C5 into C5a and C5b during complement activation. This inhibition prevents the release of proinflammatory mediator C5a and the formation of cytolytic pore-forming membrane attack complex (MAC) C5b-9, while retaining the proximal or early components of complement activation (e.g., C3 and C3b) necessary for opsonization of microorganisms and clearance of immune complexes.

[0108] The polypeptide sequence of Lavulizumab registered in the KEGG DRUG database (https: / / www.kegg.jp / entry / D11054) specifies that the N-terminal amino acid of the variable heavy chain is "X", but the database does not specify what X is. The Chemical Abstracts (CAS) of Lavulizumab (CAS 1803171-55-2) also provides that the N-terminal X is pyroglutamic acid (designated as "Chain 1 Pyroglutamate-1" in the CAS report). Although this information may appear different from the VH sequence of Lavulizumab (e.g., a heavy chain variable region polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 12 and / or a heavy chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14), there is an alignment between the patent sequence and the drug database / CAS sequence because it is recognized in the art that the N-terminal Q in a polypeptide and / or antibody sequence is cyclized during process development to produce nearly 100% conversion of the drug product to pyroglutamic acid (Pryo-Q), as described by Liu et al. (J Pharm. Sci. 2019 Oct;108(10):3194-3200)https: / / pubmed.ncbi.nlm.nih.gov / 31145921 / and Nguyen et al. (Int. J. Mol. Sci. 2017 Jul 20;18(7):1575)https: / / www.researchgate.net / figure / Cyclization-reactions-of-N-terminal-glutamine-and-glutamate-residues-in-a-polypeptide_fig4_318926365.Additional information is provided on page 7 and in Table 4 of Xu et al. (MAbs, Feb / Mar 2019; 11(2): 239-264), and in the following referenced 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 by reference in their entirety.

[0109] In other embodiments, the antibody comprises the heavy and light chain CDRs or variable regions of lavulizumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of lavulizumab having the sequence shown in SEQ ID NO: 12, and the CDR1, CDR2, and CDR3 domains of the VL region of lavulizumab having the sequence shown in SEQ ID NO: 8. In another embodiment, the antibody comprises the heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 19, 18, and 3, respectively, and the light chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 4, 5, and 6, respectively. In another embodiment, the antibody comprises the VH and VL regions having the amino acid sequences shown in SEQ ID NO: 12 and SEQ ID NO: 8, respectively.

[0110] Another exemplary anti-C5 antibody is antibody BNJ421, which comprises a heavy chain and a light chain having the sequences shown in SEQ ID NOs: 20 and 11, respectively, or antigen-binding fragments and variants thereof. BNJ421 (also known as ALXN1211) is described in WO2015134894 and U.S. Patent No. 9,079,949, the entire teachings of which are hereby incorporated by reference.

[0111] 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 shown in SEQ ID NO: 12, and the CDR1, CDR2, and CDR3 domains of the VL region of BNJ421 having the sequence shown in SEQ ID NO: 8. In another embodiment, the antibody comprises the heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 19, 18, and 3, respectively, and the light chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 4, 5, and 6, respectively. In another embodiment, the antibody comprises the VH and VL regions having the amino acid sequences shown in SEQ ID NO: 12 and SEQ ID NO: 8, respectively.

[0112] The exact boundaries of CDRs are defined differently according to different methods. In some embodiments, the position of CDRs or framework regions within the light chain or heavy chain variable domain is as defined by Kabat et al. [(1991) "Sequences of Proteins of Immunological Interest" NIH Publication No. 91-3242, Department of Health and Human Services, Bethesda, MD]. In such cases, the CDRs may be referred to as "Kabat CDRs" (e.g., "Kabat LCDR2" or "Kabat HCDR1"). In some embodiments, the positions of CDRs of light chain or heavy chain variable regions are as defined by Chothia et al. (Nature, 342: 877-83, 1989). Therefore, these regions may be referred to as "Chothia CDRs" (e.g., "Chothia LCDR2" or "Chothia HCDR3"). In some embodiments, the positions of the CDRs of the light and heavy chain variable regions may be defined by the Kabat-Chothia combined definition. In such embodiments, these regions may be referred to as "combined Kabat-Chothia CDRs". Thomas, C. et al. (Mol. Immunol, 33: 1389-401, 1996) exemplify the identification of CDR boundaries according to the Kabat and Chothia numbering schemes.

[0113] Another exemplary anti-C5 antibody is the 7086 antibody described in U.S. Pat. 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. Pat. Nos. 8,241,628 and 8,883,158). In another embodiment, the antibody or antigen-binding fragment thereof comprises the heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs: 21, 22, and 23, respectively, and the light chain CDR1, CDR2, and CDR3 domains having the sequences shown 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 shown in SEQ ID NO: 27, and the VL region of the 7086 antibody having the sequence shown in SEQ ID NO: 28.

[0114] Another exemplary anti-C5 antibody is the 8110 antibody, also described in U.S. Pat. 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 the heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs: 29, 30, and 31, respectively, and the light chain CDR1, CDR2, and CDR3 domains having the sequences shown 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 shown in SEQ ID NO: 35, and the VL region of the 8110 antibody having the sequence shown in SEQ ID NO: 36.

[0115] Another exemplary anti-C5 antibody is the 305LO5 antibody described in U.S. Pat. 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 the heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs: 37, 38, and 39, respectively, and the light chain CDR1, CDR2, and CDR3 domains having the sequences shown 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 shown in SEQ ID NO: 43, and the VL region of the 305LO5 antibody having the sequence shown in SEQ ID NO: 44.

[0116] 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 chain 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.

[0117] In some embodiments, the anti-C5 antibody comprises the heavy chain and light chain variable regions or the heavy chain and light chain of the REGN3918 antibody (see U.S. Pat. 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 the 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.

[0118] In some embodiments, the anti-C5 antibody is a biosimilar to eculizumab For example, in one embodiment, the anti-C5 antibody is, for example, ABP 959 antibody (a biosimilar of eculizumab manufactured by Amgen, USA), (eculizumab biosimilar manufactured by Generium JNC, Russia), SB12 (eculizumab biosimilar manufactured by Samsung Bioepis, Incheon, South Korea), ISU305 (eculizumab biosimilar from ISU Abxis, South Korea), (an eculizumab biosimilar from CinnaGen in Iran), BCD 148 (an eculizumab biosimilar from Biocad Medical in Canada), tertulumab (made by Novartis), kovalizumab (made by Roche Pharmaceuticals), CAN106 (made by CANBridge Pharmaceuticals in Beihai, China), or pazelizumab (made by Regeneron Pharmaceuticals).

[0119] In some embodiments, the anti-C5 antibodies described herein comprise a heavy chain CDR1 comprising or consisting of the following amino acid sequence: 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 acid sequence: 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:

[0120]

[0121] In some embodiments, the anti-C5 antibodies described herein comprise a light chain variable region comprising the following amino acid sequence:

[0122]

[0123] In some embodiments, the anti-C5 antibodies described herein may include a variant human Fc constant region that binds to a human neonatal Fc receptor (FcRn) with greater affinity than the natural human Fc constant region from which the variant human Fc constant region is derived. For example, relative to the natural human Fc constant region from which the variant human Fc constant region is derived, the Fc constant region may include one or more (e.g., two, three, four, five, six, seven or eight or more) amino acid substitutions. The substitution may increase the binding affinity of an IgG antibody containing a variant Fc constant region to FcRn at pH 6.0 while maintaining the pH dependence of the interaction. Methods for testing whether one or more substitutions in the Fc constant region of an antibody increase the affinity of the Fc constant region to FcRn at pH 6.0 (while maintaining the pH dependence of the interaction) are known in the art and are illustrated in the working examples. See, for example, WO2015134894 and U.S. Patent No. 9,079949, the disclosures of each of which are incorporated herein by reference in their entirety.

[0124] 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 substitutions (Dall'Acqua, W. et al., J. Biol. Chem., 281:23514-24, 2006); (2) M428L or T250Q / M428L substitutions (Hinton, P. et al., J. Biol. Chem., 281:23514-24, 2006); 279： .6213-6, 2004; Hinton, P. et al., J. Immunol., 176:346-56, 2006); and (3) N434A or T307 / E380A / N434A substitutions (Petkova, S. et 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.

[0125] In some embodiments, the variant constant region has a substitution for valine at EU amino acid position 255. In some embodiments, the variant constant region has a substitution for asparagine at EU amino acid position 309. In some embodiments, the variant constant region has a substitution for isoleucine at EU amino acid position 312. In some embodiments, the variant constant region has a substitution at EU amino acid position 386.

[0126] In some embodiments, the variant Fc constant region comprises no more than 30 (e.g., no more than 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) amino acid substitutions, insertions or deletions relative 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 a native human IgG Fc constant region, each numbered in EU. In some embodiments, the variant Fc constant region comprises a 428L / 434S double substitution as described, eg, in US Pat. No. 8,088,376.

[0127] In some embodiments, due to antibody engineering, the precise positions of these mutations may be shifted from the natural human Fc constant region positions. For example, when used in an IgG2 / 4 chimeric Fc, the 428L / 434S double substitution may correspond to 429L and 435S in the M429L and N435S variants as found in lavulizumab and described in U.S. Pat. No. 9,079,949, the disclosure of which is incorporated herein by reference in its entirety.

[0128] 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) relative 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; glutamate for position 255 arginine at position 256 substituted with aspartic acid, glutamic acid or glutamine; proline at position 257 substituted with alanine, glycine, isoleucine, leucine, methionine, asparagine, serine, threonine or valine; glutamic acid at position 258 substituted with histidine; aspartic acid at position 265 substituted with alanine; aspartic acid at position 270 substituted with phenylalanine; asparagine at position 286 substituted with alanine or glutamic acid; threonine at position 289 substituted with histidine; asparagine at position 297 substituted with alanine; glycine substituted with Serine at position 298; Valine at position 303 substituted with alanine; Valine at position 305 substituted with alanine; Threonine at position 307 substituted with alanine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan or tyrosine; Valine at position 308 substituted with alanine, aspartic acid, glutamic acid, proline, glutamine or threonine; substituted for leucine or valine at position 309 by arginine or arginine; substituted for glutamine at position 311 by alanine, histidine or isoleucine; substituted for aspartic acid at position 312 by alanine or histidine; substituted for leucine at position 314 by lysine or arginine; substituted for asparagine at position 315 by alanine or histidine; substituted for lysine at position 317 by alanine; substituted for asparagine at position 325 by glycine; substituted for isoleucine at position 332 by valine; substituted for lysine at position 334 by leucine; substituted for lysine at position 360 by histidine;Substitution of alanine for aspartic acid at position 376; substitution of alanine for glutamic acid at position 380; substitution of alanine for glutamic acid at position 382; substitution of alanine for asparagine or serine at position 384; substitution of aspartic acid or histidine for glycine at position 385; substitution of proline for glutamine at position 386; substitution of glutamic acid for proline at position 387; substitution of alanine or serine for asparagine at position 389; substitution of alanine for serine at position 424; substitution of alanine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, or tyrosine for methionine at position 428; substitution of lysine for histidine at position 433; substitution of alanine, phenylalanine, histidine, serine, tryptophan, or tyrosine for asparagine at position 434; and substitution of histidine for tyrosine or phenylalanine at position 436, all in EU numbering.;

[0129] In some embodiments, suitable anti-C5 antibodies for use in the methods described herein comprise a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 14 and / or a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 11. Alternatively, in some embodiments, anti-C5 antibodies for use in the methods described herein comprise a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 20 and / or a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 11.

[0130] 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 at pH 7.4 and 25°C (and otherwise under physiological conditions). D ) binds to C5. In one embodiment, the antibody binds to C5 with an affinity dissociation constant (K) of about 0.5 nM at pH 7.4 and 25° C. (and otherwise under physiological conditions). D ) binds to C5. In some embodiments, the K of the anti-C5 antibody or antigen-binding fragment thereof is DIn 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.

[0131] In other embodiments, [(K of the antibody against C5 at pH 6.0 at 25°C D ) / (K of antibody against C5 at pH 7.4 at 25°C D )] greater than 21 (e.g., greater than 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 40, 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).

[0132] 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. For example, the binding of an antibody to a protein antigen can be detected and / or quantified using a variety of techniques, such as, 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; U. et al., Ann. Biol. Clin., 51: 19-26, 1993; U. et al., Biotechniques, 11:620-7, 1991). In addition, methods for measuring affinities (e.g., dissociation and association constants) are described in the working examples.

[0133] As used herein, the term "k a " refers to the rate constant for the association of an antibody with an antigen. The term "k d ” refers to the rate constant for the dissociation of an antibody from an antibody / antigen complex. The term “k D ″ refers to the equilibrium dissociation constant of the antibody-antigen interaction. The equilibrium dissociation constant K is derived from the ratio of the kinetic rate constants D =k a / k d Such assays can be measured, for example, at 25°C or 37°C (see working examples). The kinetics of antibody binding to human C5 can be determined, for example, at pH 8.0, 7.4, 7.0, 6.5 and 6.0 via SPR on a BIAcore 3000 instrument using an anti-Fc capture method to immobilize the antibody.

[0134] In one embodiment, the anti-C5 antibody or antigen-binding fragment thereof blocks the cleavage of C5 into C5a and C5b. Through this blocking effect, for example, the pro-inflammatory effects of C5a on the cell surface and the generation of C5b-9 membrane attack complex (MAC) are inhibited.

[0135] 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 capacity of complement in a subject's body fluid. This reduction in the cytolytic capacity of complement present in body fluids can be measured by methods known in the art, such as, for example, by conventional hemolytic assays, such as hemolytic assays (Kabat and Mayer (eds.), "Experimental Immunochemistry, 2nd edition," pp. 135-240, Springfield, IL, CC Thomas (1961), pp. 135-139), or conventional variations of such assays, such as chicken erythrocyte hemolysis (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., Molecular Immunology, 32: 1183-95, 1995). The concentration and / or physiological activity of C5a and C5b in body fluids can be measured, for example, by methods known in the art. For C5b, a hemolytic assay as discussed herein or an assay for soluble C5b-9 can be used. Other assays known in the art can also be used. Using these or other suitable types of assays, candidate agents that can inhibit human complement component C5 can be screened.

[0136] Immunological techniques such as, but not limited to, ELISA can be used to measure the protein concentration 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 into biologically active products. In some embodiments, C5a generation is measured. In some embodiments, C5b-9 neo-epitope specific antibodies are used to detect MAC formation.

[0137] Hemolysis assays can be used to determine the inhibitory activity of anti-C5 antibodies or antigen-binding fragments thereof on complement activation. To determine the effect of anti-C5 antibodies or antigen-binding fragments thereof on hemolysis mediated by the classical complement pathway in an in vitro serum test solution, for example, sheep erythrocytes coated with hemolysin or chicken erythrocytes sensitized with anti-chicken erythrocyte antibodies are used as target cells. The percentage of lysis is normalized by considering 100% lysis to be equal to the lysis that occurs in the absence of the inhibitor. In some embodiments, the classical complement pathway is activated by human IgM antibodies, for example, as in Classical Pathway Complement Kit ( COMPL CP310, Euro-Diagnostica, Sweden). Briefly, the test serum is incubated with an anti-C5 antibody or an antigen-binding fragment thereof in the presence of human IgM antibodies. The amount of C5b-9 produced is measured by contacting the mixture with an enzyme-conjugated anti-C5b-9 antibody and a fluorescent substrate and measuring the absorbance at an appropriate wavelength. As a control, the test serum is incubated in the absence of an anti-C5 antibody or an antigen-binding fragment thereof. In some embodiments, the test serum is a C5-deficient serum reconstituted with a C5 polypeptide.

[0138] To determine the effect of anti-C5 antibodies or antigen-binding fragments thereof on alternative pathway-mediated hemolysis, unsensitized rabbit or guinea pig erythrocytes can be used as target cells. In some embodiments, the serum test solution is C5-deficient serum reconstituted with a C5 polypeptide. The percentage of lysis is normalized by considering 100% lysis to be equal to lysis that occurs in the absence of an inhibitor. In some embodiments, the alternative complement pathway is activated by lipopolysaccharide molecules, e.g., as in Alternative Pathway Complement Kit ( COMPL AP330, Euro-Diagnostica, Sweden). Briefly, the test serum is incubated with anti-C5 antibody or antigen-binding fragment thereof in the presence of lipopolysaccharide. The amount of C5b-9 produced is measured by contacting the mixture with enzyme-conjugated anti-C5b-9 antibody and a fluorescent substrate and measuring the fluorescence at the appropriate wavelength. As a control, the test serum is incubated in the absence of anti-C5 antibody or antigen-binding fragment thereof.

[0139] In some embodiments, the CH50eq assay is used to quantify C5 activity or its inhibition. The CH50eq assay is a method for measuring total classical complement activity in serum. The test is a lysis assay that uses antibody-sensitized erythrocytes and test serum of various dilutions as activators of the classical complement pathway to determine the amount required to produce 50% lysis (CH50). The hemolysis percentage can be determined, for example, using a spectrophotometer. The CH50eq assay provides an indirect measurement of the formation of the terminal complement complex (TCC) because the TCC itself is directly responsible for the measured hemolysis. The assay is known and commonly practiced by those skilled in the art. In short, in order 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 erythrocytes, thereby producing TCC. Next, the activated serum is diluted in a microassay well, and the microassay well is coated with a capture reagent (e.g., an antibody bound to one or more components of the TCC). The TCC present in the activated sample binds to the monoclonal antibody coated on the surface of the microassay well. The wells are washed and a detection reagent is added to each well, which is detectably labeled and recognizes bound TCC. The detectable label can be, for example, a fluorescent label or an enzyme label. The assay results are expressed in CH50 unit equivalents per milliliter (CH50 U Eq / mL).

[0140] For example, inhibition associated with terminal complement activity includes a reduction of at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60%) of terminal complement activity in, for example, a hemolytic assay or a CH50eq assay, compared to the effect of a control antibody (or antigen-binding fragment thereof) under similar conditions and equimolar concentrations. As used herein, substantial inhibition refers to inhibition of a given activity (e.g., terminal complement activity) by 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 relative 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 inhibition activity of eculizumab in a hemolytic assay or a CH50eq assay.

[0141] The anti-C5 antibodies described herein have a serum half-life of at least 20 (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 humans. In another embodiment, the anti-C5 antibodies described herein have a serum half-life of at least 40 days in humans. In another embodiment, the anti-C5 antibodies described herein have a serum half-life of about 43 days in humans. In another embodiment, the anti-C5 antibodies described herein have a serum half-life of between 39 and 48 days in humans. Methods for measuring the serum half-life of antibodies are known in the art. In some embodiments, an anti-C5 antibody or antigen-binding fragment thereof described herein has a serum half-life that is at least 20% (e.g., 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 working Examples (e.g., a C5-deficient / NOD / scid mouse or an hFcRn transgenic mouse model system).

[0142] In one embodiment, the antibody competes for binding and / or binds to the same epitope on C5 with the antibodies described herein. The term "binding to the same epitope" with respect to two or more antibodies means that the antibody binds 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 using the antibodies described herein include, for example, epitope mapping methods, such as x-ray analysis of crystals of antigen: antibody complexes and hydrogen / deuterium exchange mass spectrometry (HDX-MS). Other methods monitor the binding of antibodies to peptide antigen fragments or mutant variants of antigens, in which loss of binding due to modification of amino acid residues within the antigen sequence is generally considered to be an indication of epitope components. In addition, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity separate specific short peptides from a combinatorial phage display peptide library. Antibodies with identical VH and VL or identical CDR1, CDR2, and CDR3 sequences are expected to bind to the same epitope.

[0143] An antibody that "competes with another antibody for binding to a target" refers to an antibody that inhibits (partially or completely) the binding of another antibody to a target. Whether two antibodies compete with each other for binding to a target, i.e., whether one antibody inhibits the binding of another antibody to a target and to what extent the binding of another antibody to a target is inhibited can be determined using known competition assays. In certain embodiments, an antibody competes with another antibody and inhibits the binding of another antibody to a target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition may vary, depending on which antibody is a "blocking antibody" (i.e., the antibody that is first incubated with the target). Competitive antibodies may bind to, for example, the same epitope, overlapping epitopes, or adjacent epitopes (e.g., as demonstrated by steric hindrance).

[0144] The antibody or antigen-binding fragment thereof may include a humanized antibody, a recombinant antibody, a diabody, a chimeric or chimeric antibody, a monoclonal antibody, a deimmunized antibody, a fully human antibody, a single-chain antibody, an Fv fragment, a Fd fragment, a Fab fragment, a Fab' fragment, a F(ab') 2 fragments or a combination of them.

[0145] The monoclonal antibodies disclosed herein can be of any isotype. The monoclonal antibodies can be, for example, IgM or IgG antibodies, such as IgG1 or IgG2. The class of an antibody that immunospecifically binds to C5b-9 can be converted to another class (e.g., IgG can be converted to IgM) according to well-known procedures. Class switching can also be used to convert one IgG subclass to another, such as from IgG1 to IgG2.

[0146] The antibodies of the present invention can be monovalent, bivalent, trivalent or multivalent. For example, a monovalent scFv can be multimerized chemically or by association with another protein or substance. ScFv fused to a hexahistidine tag (SEQ ID NO: 51) or a Flag tag can be multimerized using Ni-NTA agarose (Qiagen) or using an anti-Flag antibody (Stratagene, Inc.).

[0147] The antibodies of the invention can be monospecific, bispecific, trispecific or more multispecific. Multispecific antibodies can be specific for different epitopes of C5b-9 or fragments thereof, as well as heterologous epitopes, such as heterologous polypeptides or solid support materials. See, e.g., WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; WO 92 / 05793; Tutt et al., J. Immunol. 147: 60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al. J.Immunol. 148:1547-1553(1992).

[0148] The anti-C5 antibodies or antigen-binding fragments thereof described herein for use in the methods described herein can be produced using a variety of techniques recognized in the art. Monoclonal antibodies can be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are usually immortalized by fusion with myeloma cells ( 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 generated by single immortalized cells are screened for antibodies with the desired specificity and affinity for the antigen, and the production of monoclonal antibodies produced by these cells can be increased by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, DNA sequences encoding monoclonal antibodies or binding fragments thereof can be isolated by screening DNA libraries from human B cells (Huse, W. et al., Science, 246:1275-81, 1989).

[0149] Antibodies (including scFv and other molecules comprising or consisting of antibody fragments or variants of the invention) useful in the methods described herein can be produced by any method known in the art for synthesizing 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.

[0150] Single-chain Fv (scFv) that immunospecifically binds to a biomarker of the present disclosure (e.g., TM, SYND1, Ba, or a fragment thereof) can be produced using phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying polynucleotide sequences encoding them. Examples of phage display methods that can be used to prepare antibodies of the invention include, but are not limited to, those described in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; W097 / 13844; and those disclosed in U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 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.

[0151] As described in the above references, after phage selection, the antibody coding region from the phage can be isolated and used to produce complete antibodies, including human or humanized antibodies, or any other desired antigen-binding fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as described below. Techniques for recombinant production of Fab, Fab' and F(ab')2 fragments can also be used using methods known in the art, such as those disclosed in WO 92 / 22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995).

[0152] To generate complete antibodies, PCR primers comprising VH or VL nucleotide sequences, restriction sites, and flanking sequences protecting the restriction sites can be used to amplify the VH or VL sequences in scFv clones. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domains can be cloned into vectors expressing VH constant regions (e.g., human γ4 constant regions), and the PCR-amplified VL domains can be cloned into vectors expressing VL constant regions (e.g., human kappa or lambda constant regions).

[0153] Once an antibody useful in the methods described herein has been synthesized or recombinantly expressed, it can be purified by any method known in the art for purifying immunoglobulin molecules, or protein molecules more generally, such as, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography (particularly after protein A by affinity for a specific antigen), and size column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins.

[0154] The vector comprises an element that is easy to manipulate to express foreign proteins in the target host cell. Conveniently, the manipulation of the sequence and the production of the DNA for conversion are first carried out in a bacterial host (e.g., E. coli), and usually the vector will comprise a sequence that promotes such manipulation, including a bacterial origin of replication and a suitable bacterial selection marker. The selection marker encodes a protein necessary for the survival or growth of the transformed host cell grown in a selective medium. Host cells that are not transformed with a vector containing a selection gene cannot survive in the culture medium.

[0155] Host cells used to express anti-sC5b-9 antibodies can be bacterial cells such as E. coli, yeast (e.g., S. cerevisiae), or eukaryotic cells (e.g., 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.

[0156] Any known method, e.g., binding assays, can be used to screen for antibodies that bind to a biomarker. In a representative method, the target biomarker or its antigenic epitope is expressed in standard cells and the antibodies are selected using selection techniques known in the art. For example, the antibodies can be selected based on binding affinity, e.g., at least 10 -6 M; preferably 10 -8 M; and especially 10 -10 The dissociation constant (K d ), the antibodies are graded. Here, K d Values ​​can be determined using standard binding assays.

[0157] Various embodiments of the present disclosure are described in detail in the following non-limiting and representative examples.

[0158] In the Examples section and elsewhere, representative types of antibodies that can be used to implement various embodiments of the present disclosure are provided, such as information about specific suppliers and / or catalog numbers. It should be understood that the present disclosure is not limited to exemplary embodiments that utilize antibody detection reagents from specific suppliers / manufacturers. Antibodies to the biomarkers / analytes of the present disclosure can be obtained from any manufacturer, including, for example, Thermo Fisher Catalog No. MA5-24214, anti-human TM antibody; Catalog No. 12-1389-42, anti-human SYND1 antibody; and MA5-28083, anti-human complement factor Ba antibody (all from Thermo Fisher Scientific, Waltham, Massachusetts). Antibodies can also be purchased from Biolegend (San Diego, California), Southern Biotech (Birmingham, Alabama), United States Biological (USB; Salem, Massachusetts), Lifetime Biosciences (LSBIO; Seattle, Washington), Abeam (Cambridge, England), Cell Signaling Technology (Cell Signaling Technology) (Danvers, Massachusetts) and Sigma-Aldrich (St. Louis, Missouri). Conventional techniques, for example, immunization of mammals such as mice or rabbits and / or hybridoma technology can also be used to generate antibodies or antisera of interest.

[0159] Also provided herein is a composition comprising an anti-C5 antibody (or an antigen-binding fragment thereof). The composition can be formulated as a pharmaceutical solution, for example, for administration to a subject to treat HSCT-TMA. The pharmaceutical composition generally comprises a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to and includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. The composition may contain a pharmaceutically acceptable salt, for example, an acid addition salt or a base addition salt, a sugar, a carbohydrate, a polyol and / or a tonicity modifier.

[0160] The composition can be formulated according to standard methods. Pharmaceutical preparation is an established technique (see, e.g., Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams & Wilkins (ISBN: 0683306472); Ansel et al. (1999) "Pharmaceutical Dosage Forms and Drug Delivery Systems", 7th edition, Lippincott Williams & Wilkins Publishers (ISBN: 0683305727); and Kibbe (2000) "Handbook of Pharmaceutical Excipients American Pharmaceutical Association", 3rd edition (ISBN: 091733096X)). In some embodiments, the composition can be formulated as a buffer solution, for example, at an appropriate concentration and suitable for storage at 2°C-8°C (e.g., 4°C). In some embodiments, the composition can be formulated for storage at a temperature below 0°C (e.g., -20°C or -80°C). In some embodiments, the composition can be formulated for storage at 2°C-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 / 2 Thus, in some embodiments, the compositions described herein are storage stable for at least 1 year at 2°C-8°C (eg, 4°C).

[0161] Pharmaceutical composition can be in various forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (for example, injectable and infusible solutions), dispersants or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form part depends on the expected mode of administration and therapeutic application. The composition containing the composition for systemic or local delivery can be, for example, in the form of injectable or infusible solutions. Therefore, the composition can be formulated for use by parenteral mode (for example, intravenous, subcutaneous, intraperitoneal or intramuscular injection). As used herein, "parenteral administration," "parenterally administered," and other grammatically equivalent phrases refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, intratracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid, and intrasternal injection and infusion.

[0162] IV. Anti-complement factor B antibodies

[0163] In another aspect, anti-complement factor B antibodies are used in the methods described herein. Anti-CFB antibodies refer to antibodies that inhibit: (i) the expression of complement factor B protein, or the proper intracellular transport or secretion of complement factor B protein by cells; (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 of complement factor B or the 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 includes: inhibition of transcription of genes 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 correct processing of pre-pro-human complement factor B protein; or inhibition of 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 described herein.

[0164] V. Biological Samples

[0165] Suitable biological samples for methods described herein include, for example, any biological fluid. Biological samples can be, for example, specimens obtained from a subject (e.g., mammals, such as humans) or can be derived from such a subject. Biological samples can also be biological fluids, such as urine, whole blood or their fractions (e.g., plasma or serum), saliva, semen, sputum, cerebrospinal fluid, tears or mucus. If desired, biological samples can be further classified into fractions containing a particular analyte of interest (e.g., protein). For example, whole blood samples can be classified into serum or fractions containing a particular type of protein. If desired, biological samples can be a combination of different biological samples from a subject, such as a combination of two different fluids.

[0166] Biological samples suitable for the present invention can be fresh or frozen samples collected from subjects, or archived samples with known diagnosis, treatment and / or prognosis history. Biological samples can be obtained from subjects, for example, suffering from, suspected of suffering from complement-related disorders such as HSCT-TMA, or subjects at risk of developing them. Any suitable method for obtaining biological samples can be used, although exemplary methods include, for example, phlebotomy, swabs (e.g., oral swabs), lavage or fine needle aspiration biopsy procedures. Biological samples can also be obtained from bone marrow.

[0167] In some embodiments, protein extracts can be prepared from biological samples. In some embodiments, the protein extract contains total protein content. Methods for protein extraction are well known in the art. See, for example, Roe (2001) "Protein Purification Techniques: A Practical Approach", 2nd edition, Oxford University Press. Many different and universal kits can be used to extract proteins from body fluids and tissues, and are commercially available from, for example, BioRad Laboratories (Hercules, California), BD Biosciences Clontech (Mountain View, California), Chemicon International, Inc. (Temecula, California), Calbiochem (San Diego, California), Pierce Biotechnology (Pierce Biotechnology) (Rockford, Illinois) and Invitrogen Corp. (Carlsbad, California).

[0168] Methods for obtaining and / or storing samples, maintaining the activity or integrity of cells in biological samples are well known to those skilled in the art. For example, the biological sample may be further contacted with one or more additional agents, such as appropriate buffers and / or inhibitors, including protease inhibitors, which are intended to maintain or minimize changes in protein structure (e.g., changes in molar osmotic pressure concentration or pH). Such inhibitors include, for example, chelating agents such as ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), aprotinin and leupeptin. Appropriate buffers and conditions for storing or 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 the Practical approach series, Oxford University Press; and Jones (1996) "Human cell culture protocols", volume 2 of Methods in molecular medicine, Humana Press.

[0169] The sample can also be treated to eliminate or minimize the presence of interfering substances. For example, the biological sample can be fractionated or purified to remove one or more materials (e.g., cells) that are not of interest. Methods for fractionating or purifying biological samples include, but are not limited to, flow cytometry, fluorescence activated cell sorting, and sedimentation.

[0170] In embodiments of the present disclosure, an array can be used to detect biomarkers. For example, the array can be a protein chip, wherein 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 fixed.

[0171] Measuring protein expression levels in a biological sample can be performed by any suitable method. See, e.g., Greenfield (ed.) (2014) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory: Cold Spring Harbor, New York. Typically, protein levels are determined by: contacting a biological sample obtained from a subject with a binding agent for a biomarker protein; detecting the level of one or more biomarker proteins bound to the binding agent in the sample (e.g., a biological fluid); and comparing the level of one or more biomarker proteins in the sample with the level of a corresponding protein biomarker in a control sample (e.g., a normal sample).

[0172] 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 a 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. Suitable antibodies for use in the methods of the present invention include monoclonal and polyclonal antibodies and 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 well known in the art. Greenfield (ed.) (2014) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory: Cold Spring Harbor, New York. Antibodies can also be obtained from commercial sources.

[0173] The binding agent is directly or indirectly labeled with a detectable moiety. The role of the detectable agent (a binding agent labeled with a detectable moiety) is to facilitate the detection step of the diagnostic method by visualizing the complex formed by the binding of the binding agent to the protein marker (or its fragment). The detectable agent can be selected so that it produces a signal that can be measured and the intensity of the signal is related to (preferably proportional to) the amount of protein marker present in the sample to be analyzed. Methods for labeling biomolecules (such as polypeptides and antibodies) are well known in the art. Any of a 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, fluorophores), enzymes (e.g., 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.

[0174] The binding agent (e.g., 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 carriers or support materials include agarose, cellulose, nitrocellulose, dextran, Liposomes, carboxymethylcellulose, polyacrylamide, polystyrene, gabbros, filter paper, magnetite, ion exchange resins, plastic films, plastic tubing, glass, polyamine-methyl vinyl ether-maleic acid copolymers, amino acid copolymers, ethylene-maleic acid copolymers, nylon, silk, or combinations thereof. The binding agent can be indirectly immobilized using a second binding agent specific for the first binding agent (e.g., a mouse antibody specific for a protein marker can be immobilized using a sheep anti-mouse IgG Fc fragment specific antibody coated on a carrier or support).

[0175] Immunoassays can be used to determine protein levels in biological samples. Examples of such assays include time-resolved fluorescence 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 detection and quantification methods of the signal generated by the complex formed by the binding agent and the protein marker will depend on the nature of the assay and the detectable portion (e.g., fluorescent portion).

[0176] 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 a Western blotting technique. For example, a lysate can be prepared from a biological sample, or the biological sample (e.g., a biological fluid) itself can be contacted with a Laemmli buffer and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins resolved by the size-separated SDS-PAGE can then be transferred to a filter membrane (e.g., nitrocellulose) and immunoblotting techniques can be performed using an antibody with a detectable label specific for the protein of interest. The presence or amount of the detectably labeled antibody combined indicates the presence or amount of the protein in the biological sample.

[0177] In one example, an immunoassay can be used to detect and / or measure protein expression of a biomarker protein (e.g., TM or SYND1, Ba or fragments thereof). As described above, for detection purposes, an immunoassay can be performed with an antibody carrying a detection moiety (e.g., a fluorescer or enzyme). Proteins from a biological sample can be directly bound to a solid matrix (e.g., a multi-well assay plate, nitrocellulose, agarose, The present invention relates to a method for preparing a protein for use in a biological sample. The method comprises conjugating a specific binding pair to a first member of a specific binding pair (e.g., a coded particle or magnetic bead), or it can be conjugated to a first member of a specific binding pair (e.g., biotin or streptavidin), which is attached to a solid phase matrix after binding to a second member of the specific binding pair (e.g., streptavidin or biotin). This attachment to a solid phase matrix allows the protein to be purified from other interfering or irrelevant components of the biological sample before contacting with the detection antibody, and also allows the unbound antibody to be subsequently washed. Here, as described above, the presence or amount of the bound detectably labeled antibody indicates the presence or amount of the protein in the biological sample.

[0178] Alternatively, protein expression levels can be determined using mass spectrometry-based methods or image-based methods known in the art for detecting proteins. Other suitable methods include 2D-gel electrophoresis, proteomics-based methods such as identification of individual proteins recovered from gels (e.g., by mass spectrometry or N-terminal sequencing) and / or bioinformatics.

[0179] Methods for detecting or measuring protein expression can optionally be performed in a format that allows rapid preparation, processing, and analysis of multiple samples. For example, this can be performed in a multi-well assay plate (e.g., 96-well or 386-well) or array (e.g., protein chip). Stock solutions for various reagents can be provided manually or automatically, and subsequent sample preparation, pipetting, dilution, mixing, distribution, washing, incubation (e.g., hybridization), sample reading, data collection (optical data) and / or analysis (computer-assisted image analysis) can be automatically completed using commercially available analysis software, robotics, and detection instruments capable of detecting signals generated from assays. Examples of such detectors include, but are not limited to, spectrophotometers, photometers, fluorometers, and devices for measuring radioisotope decay.

[0180] VI. Treatment Methods

[0181] Also provided herein is a method for treating HSCT-TMA in a subject. In one embodiment, a method for treating a patient (e.g., pediatric or adult patient) with HSCT-TMA is provided, the patient has been determined to have a biomarker level (e.g., blood or plasma level) elevated compared to a normal reference range for a biomarker selected from TM and SYND1 or a combination thereof, the method includes administering anti-C5 antibodies or anti-CFB antibodies to the patient in an amount and frequency sufficient to reduce the biomarker level in the patient, thereby treating HSCT-TMA. In another embodiment, a method for treating a patient (e.g., pediatric or adult patient) with HSCT-TMA is provided, the patient has been determined to have TM, SYND1 and Ba and / or C5b9 and / or HSPG levels (e.g., blood or plasma levels) elevated compared to a normal reference range for TM, SYND1 and Ba and / or C5b9 and / or HSPG, respectively, the method includes administering anti-C5 antibodies or anti-CFB antibodies to the patient in an amount and frequency sufficient to treat HSCT-TMA.

[0182] Further provided are methods for treating patients with HSCT-TMA, the methods comprising: (1) obtaining or having obtained a sample (e.g., a blood or plasma sample) from the patient, (2) determining or having determined elevated biomarker levels in the sample compared to normal reference ranges for biomarkers selected from TM and SYND1 or a combination thereof, and (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. In another embodiment, a method for treating a patient with HSCT-TMA comprises: (1) obtaining or having obtained a sample (e.g., a blood or plasma sample) from the patient, (2) determining or having determined elevated biomarker levels in the sample compared to normal reference ranges for biomarkers selected from TM, SYND1, and Ba, 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, and Ba levels, thereby treating HSCT. In another embodiment, a method for treating a patient with HSCT-TMA comprises: (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 in the sample compared to normal reference ranges for biomarkers selected from TM, SYND1, Ba, and HSPG levels, 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, HSPG levels, thereby treating HSCT.

[0183] As used herein, the term "treatment" includes prophylactic and / or therapeutic treatment. The term "prophylactic or therapeutic" treatment is art-recognized and includes administering to the patient one or more therapeutic agents as described herein (e.g., anti-C5 antibodies (e.g., eculizumab or lavulizumab) or anti-CFB antibodies). If one or more therapeutic agents are administered before the clinical manifestation of the undesirable disorder (e.g., the development of HSCT-TMA), the treatment is prophylactic (i.e., it protects the patient from developing the undesirable disorder), and if one or more therapeutic agents are administered after the manifestation of the undesirable disorder, the treatment is therapeutic (i.e., it is intended to reduce, improve or stabilize the existing undesirable disorder or its side effects). Preferably, it is intended to reduce or at least partially improve or change the severity of the subject's disorder, and to achieve some relief, alleviation, reversal or reduction of at least one clinical symptom.

[0184] As used herein, a patient "in need of prevention," "in need of treatment," or "in need thereof" refers to a patient who, in the judgment of an appropriate practitioner (e.g., a physician, nurse, or nurse practitioner), would reasonably benefit from a given treatment (such as treatment with an anti-C5 antibody or anti-CFB antibody).

[0185] A variety of methods can be used to administer one or more therapeutic agents (e.g., anti-C5 antibodies or anti-CFB antibodies) to a patient (e.g., a human subject), which methods depend in part on the route of administration. The route can be, for example, intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP) injection, or intramuscular injection.

[0186] Administration can be achieved by, for example, local infusion, injection or by implants. Implants can be porous, nonporous or gelatinous materials, including membranes, such as sialastic membranes or fibers. Implants can be configured to release the composition to the subject continuously or periodically. See, for example, U.S. Patent Publication No. 20080241223; U.S. Patent No. 5,501,856; 4,863,457; and 3,710,795; and European Patent No. EP488401 and EP430539, the disclosures of which are incorporated herein by reference in their entirety. The composition can be delivered to the subject by an implantable device based on, for example, a diffusion system, an erodible system or a convection system, such as an osmotic pump, a biodegradable implant, an electrodiffusion system, an electroosmotic system, a vapor pressure pump, an electrolytic pump, an effervescent pump, a piezoelectric pump, an erosion-based system or an electromechanical system.

[0187] As used herein, the term "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of a drug (e.g., an anti-C5 antibody (e.g., eculizumab or lavulizumab) or an anti-CFB antibody) that will cause a desired biological or medical response (e.g., preventing or ameliorating one or more symptoms of HSCT-TMA). Suitable doses of therapeutic agents as described herein (e.g., anti-C5 antibodies and / or anti-CFB antibodies) that are capable of treating or preventing HSCT-TMA in a subject may depend on a variety of factors, including, for example, the age, sex, and weight of the subject to be treated and the specific inhibitor compound used. For example, the dose may depend on the severity of HSCT-TMA. Other factors may include, for example, other medical conditions that affect the subject simultaneously or previously, the general health of the subject, the genetic predisposition of the subject, diet, time of administration, excretion rate, drug combination, and any other additional therapeutic agents administered to the subject. It should also be understood that the specific dose and treatment regimen for any particular subject depends on the judgment of the treating physician (e.g., doctor or nurse). A therapeutically effective amount is also an amount in which the beneficial effects of the treatment outweigh any toxic or deleterious effects of the composition.

[0188] 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 dose can also be selected to reduce or avoid the production of antibodies or other host immune responses to one or more active agents in the composition.

[0189] Although in no way intended to be limiting, exemplary dosages of inhibitors (such as, anti-C5 antibodies) include, for example, 1 mg / kg to 100 mg / kg body weight, 0.5 mg / kg to 50 mg / kg body weight, 0.1 mg / kg to 100 mg / kg body weight, 0.5 mg / kg to 25 mg / kg body weight, 1 mg / kg to 20 mg / kg body weight, and 1 mg / kg to 10 mg / kg body weight.

[0190] In some embodiments, an anti-C5 antibody (eg, lavulizumab) is administered (eg, intravenously) as follows:

[0191] (a) once on day 1 at a dose of 600 mg for patients weighing ≥5 kg to <10 kg, 600 mg for patients weighing ≥10 kg to <20 kg, 900 mg for patients weighing ≥20 kg to <30 kg, 1200 mg for patients weighing ≥30 kg to <40 kg, 2400 mg for patients weighing ≥40 kg to <60 kg, 2700 mg for patients weighing ≥60 kg to <100 kg, or 3000 mg for patients weighing ≥100 kg;

[0192] (b) once on day 5 at a dose of 300 mg for patients weighing ≥5 kg to <10 kg, 300 mg for patients weighing ≥10 kg to <20 kg, 300 mg for patients weighing ≥20 kg to <30 kg, 300 mg for patients weighing ≥30 kg to <40 kg, 600 mg for patients weighing ≥40 kg to <60 kg, 900 mg for patients weighing ≥60 kg to <100 kg, or 900 mg for patients weighing ≥100 kg;

[0193] (c) once on day 10 at a dose of 300 mg for patients weighing ≥5 kg to <10 kg, 300 mg for patients weighing ≥10 kg to <20 kg, 300 mg for patients weighing ≥20 kg to <30 kg, 300 mg for patients weighing ≥30 kg to <40 kg, 600 mg for patients weighing ≥40 kg to <60 kg, 900 mg for patients weighing ≥60 kg to <100 kg, or 900 mg for patients weighing ≥100 kg; and

[0194] (d) 300 mg for patients weighing ≥5 kg to <10 kg or 600 mg for patients weighing ≥10 kg to <20 kg on day 15 and every four weeks thereafter; or 2100 mg for patients weighing ≥20 kg to <30 kg, 2700 mg for patients weighing ≥30 kg to <40 kg, 3000 mg for patients weighing ≥40 kg to <60 kg, 3300 mg for patients weighing ≥60 kg to <100 kg, or 3600 mg for patients weighing ≥100 kg on day 15 and every eight weeks thereafter.

[0195] In some embodiments, an anti-C5 antibody (e.g., eculizumab) can be administered intravenously to a human at a dose of about 900 mg about every 12 days (e.g., about every 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, 30, 42, or 49 days or more). See, e.g., Hill et al. (2005) Blood .106(7) :2559.

[0196] In some embodiments, an anti-C5 antibody (e.g., eculizumab) can be administered intravenously to a human at a dose of about 600 mg (e.g., about 625 mg, 650 mg, 700 mg, 725 mg, 750 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, or 1,000 mg or more) weekly, optionally two or more weeks (e.g., three, four, five, six, seven, or eight or more weeks). After the initial treatment, the antibody can be administered to a human at a dose of about 900 mg about every 14 days (e.g., about every 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, 30, 42, or 49 days or more), for example as a maintenance dose. See, e.g., Hillmen et al. (2004) N Engl J Med. 350(6) : 552-9 and Dmytrijuk et al. (2008) The Oncologist 13(9) :993.

[0197] In some embodiments, an anti-C5 antibody (e.g., eculizumab) can be administered intravenously to a human at a dose of about 900 mg (e.g., 925 mg, 950 mg, 975 mg, 1000 mg, 1100 mg, or 1200 mg or more) weekly, optionally, two or more weeks (e.g., three, four, five, six, seven, or eight or more weeks). After the initial treatment, the antibody can be administered to a human at a dose of about 1200 mg about every 14 days (e.g., about every 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, 30, 42, or 49 days or more), for example, as a maintenance dose. See, e.g., International Patent Application Publication No. WO2010 / 054403.

[0198] 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 can be used, for example, to determine LD 50 (lethal dose for 50% of the population) and ED 50 (the therapeutically effective dose for 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD 50 / ED 50 Therapeutic agents that exhibit high therapeutic indices are preferred. Although compositions that exhibit toxic side effects may be used, care should be taken to design delivery systems that target such compounds to the site of affected tissue and minimize potential damage to normal cells, thereby reducing side effects.

[0199] In some embodiments, the therapeutic agent described herein (e.g., anti-C5 antibody or anti-CFB antibody) is administered to the patient as a monotherapy. Alternatively, the therapeutic agent may be administered to the patient as a combination therapy with another treatment (e.g., another therapeutic agent and / or treatment for HSCT-TMA). For example, the combination therapy may include administering one or more additional agents to the patient, which provide therapeutic benefits to the subject suffering from HSCT-TMA or at risk of developing HSCT-TMA. In some embodiments, the agent described herein (e.g., anti-C5 antibody or anti-CFB antibody) and one or more additional active agents are administered simultaneously. In other embodiments, the agent described herein (e.g., anti-C5 antibody or anti-CFB antibody) is first administered in time, and one or more additional active agents are then administered in time.

[0200] Eculizumab Methods for treating HSCT-TMA 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 for treating HSCT-TMA are described in WO / 2022 / 36151, the disclosure of which is incorporated herein by reference. ) during the maintenance phase of a dosing cycle of HSCT-TMA in patients who are transfused with red blood cells (RBCs) are described in US Prov. App. No., entitled "SUPPLEMENTAL DOSAGE AND ADMINISTRATION OF ANTI-C5 ANTIBODIES FORTREATING HEMATOPOIETIC STEM CELL TRANSPLANT-ASSOCIATED THROMBOTICMICROANGIOPATHY" (Attomey Ref. AXJ-306-1, filed: September 6, 2022), the disclosure of which is incorporated herein by reference.

[0201] VII. Treatment Outcomes

[0202] The efficacy of the treatment methods provided herein can be assessed using any suitable means. In one embodiment, treatment results in a reduction or cessation of microangiopathic hemolytic anemia, thrombocytopenia, endothelial injury, renal injury, renal failure, serositis, pulmonary hypertension, and multisystem organ failure compared to baseline.

[0203] In another embodiment, treatment results in normalization of LDH, elimination of red blood cell and platelet transfusion requirements, increase in hemoglobin, and / or disappearance of schistocytes compared to baseline.

[0204] In another embodiment, treatment results in: (a) a platelet count of ≥ 50,000 / mm3 without transfusion support during the prior 7 days 3 , (b) LDH <1.5 x ULN, and (c) absence of schistocytes (if schistocytes were present at baseline), and / or (d) a reduction in proteinuria of at least 50% from baseline.

[0205] In another embodiment, treatment results in a favorable hematologic response.

[0206] In another embodiment, the treatment results in a hemoglobin > 8 g / dL without transfusion support.

[0207] In another embodiment, treatment results in terminal complement inhibition.Treatment results in a reduction in adverse events.

[0208] In another embodiment, treatment results in the change of the quality of life as assessed via quality of life assessment relative to baseline. In one embodiment, quality of life assessment is a quality of life inventory (PedsQL) scale. The pediatric quality of life inventory (PedsQL) 4.0 common core scale is a multidimensional children's self-report and parental proxy report standardized instrument for measuring the health-related quality of life (QoL) of 2 years old-18 years old children and adolescents. In another embodiment, quality of life assessment is EuroQoL 5 dimensions 5 levels (EQ-5D-5L) questionnaire. EQ-5D-5L is a standardized instrument for measuring the self-assessment of the health-related quality of life (QoL) and has been used in the health status of a wide range.

[0209] VIII. Methods of Identifying Patients

[0210] In another aspect, the present disclosure provides a method for identifying a patient with HSCT-TMA who is suitable for treatment with an anti-C5 antibody or an anti-CFB antibody, the method 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) from the patient using an in vitro assay, wherein an elevated level of the biomarker in the sample compared to a normal reference range for the biomarker identifies the patient as suitable for treatment with an anti-C5 antibody or an anti-CFB antibody. In another embodiment, a method for identifying a patient with HSCT-TMA who is suitable for treatment with an anti-C5 antibody or an anti-CFB antibody is provided, the method comprising determining 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 using an in vitro assay, wherein an elevated level of the biomarker in the sample compared to a normal reference range for the biomarker identifies the patient as suitable for treatment with an anti-C5 antibody or an anti-CFB antibody.

[0211] In another aspect, the method includes identifying a patient as having, suspected of having, or at risk of developing HSCT-TMA. In addition to using the HSCT-TMA biomarker profile described herein, laboratory tests can be performed to determine whether a human subject has symptoms of HSCT-TMA. For example, thrombocytopenia can be diagnosed by a medical professional as one or more of the following: (i) platelet count less than 150,000 / mm 3 (For example, less than 60,000 / mm 3 ); (ii) a decrease in reduced platelet survival time, reflecting enhanced platelet fragmentation in the circulation; and (iii) giant platelets observed in peripheral smears, consistent with secondary activation of thrombocytopenia.

[0212] As used herein, a patient "at risk of developing HSCT-TMA" is one who has one or more (e.g., two, three, four, five, six, seven, or eight or more) risk factors for developing the condition. Risk factors for HSCT-TMA include, for example, calcineurin inhibitors (CNiS), infection, and conditioning regimen (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).

[0213] As used herein, a patient "suspected of having HSCT-TMA" is one who has one or more symptoms of the disorder. Symptoms of the disorder are well known to those skilled in the medical arts and include, for example, microangiopathic hemolytic anemia, thrombocytopenia, endothelial damage, renal injury, renal failure, serositis, pulmonary hypertension, and multisystem organ failure.

[0214] IX. Methods of Monitoring Patient Response to Treatment

[0215] 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, the method 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, wherein: a reduced level of the biomarker in the sample from the patient obtained during or after treatment compared to the level of the biomarker in the 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 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, the method 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, wherein: a reduced level of the biomarker in the sample from the patient obtained during or after treatment compared to the level of the biomarker in the 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.

[0216] As defined herein, monitoring the improvement of HSCT-TMA of a patient (e.g., a human patient) means assessing the change in the disease parameters of a subject, for example, the improvement of one or more symptoms of a disease. Such symptoms include any symptoms of HSCT-TMA described herein. In some embodiments, after treatment begins, the assessment is performed for at least 1 hour, for example, at least 2, 4, 6, 8, 12, 24 or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or longer, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or longer. Subjects can be assessed in one or more of the following periods: before treatment begins; During treatment; or after one or more elements of treatment have been applied. Assessment may include assessing the need for further treatment, for example, assessing whether dosage, frequency of administration or duration of treatment should be changed. It may also include assessing the need to add or discard a selected form of treatment, for example, adding or discarding any treatment of HSCT-TMA described herein.

[0217] X. Test Kit

[0218] Also provided are kits comprising pharmaceutical compositions containing therapeutically effective amounts of anti-C5 antibodies or anti-CFB antibodies suitable for use in the methods described herein. In addition, the kits may include various reagents and materials for implementing the methods described herein. The procedures described herein for measuring, diagnosing, evaluating, and / or assessing may be performed by a diagnostic laboratory, an experimental laboratory, or an individual physician. The present invention provides kits that can be used in any or all of these environments.

[0219] In some embodiments, the kits described herein include materials and reagents for characterizing or processing biological samples (e.g., biological fluids), measuring biomarker levels (e.g., protein or nucleic acid levels), diagnosing HSCT-TMA in subjects, or monitoring treatment responses of subjects, etc. according to the methods provided herein. In certain embodiments, the kits of the present invention include at least one or more reagents for specifically detecting protein levels of one or more HSCT-TMA biomarker proteins (e.g., TM, SYND-1, factor Ba and / or HSPG) and optional instructions for using the kit. The kit may include, for example, any array described herein.

[0220] Exemplary arrays and chips of the present disclosure include antibodies that bind to the previously described tags. In embodiments, such arrays contain multiple (e.g., at least 2, 3, or more) antibodies capable of detecting the signature. The antibodies may be monospecific or multispecific (e.g., binding to more than one biomarker), such as bispecific antibodies.

[0221] 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 also comprise an antibody for detecting C5b9, in which case the biomarker signature comprises (a) TM+Ba+C5b9; (b) TM+SYND1+C5b9; (c) SYND1+Ba+C5b9.

[0222] 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 also comprise antibodies for detecting C5b9, in which case the biomarker signature comprises TM+SYND1+Ba+C5b9+HSPG.

[0223] In some embodiments, the kit may include a suitable control sample (e.g., a biological fluid from a normal healthy individual or an individual with HSCT (in the absence of TMA) or a solution containing a known control amount of a specific 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 for performing a test or instructions for interpreting the results.

[0224] It should be understood that each maximum numerical limit given throughout the specification includes each lower numerical limit, as if such lower numerical limits were explicitly written herein. Each minimum numerical limit given throughout the specification will include each higher numerical limit, as if such higher numerical limits were explicitly written herein. Each numerical range given throughout this specification will include each narrower numerical range that falls within such a wider numerical range, as if such narrower numerical ranges were all explicitly written herein.

[0225] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. Methods and materials for the present invention are described herein. Other suitable methods and materials known in the art may also be used. Materials, methods and embodiments are illustrative only and are not intended to be restrictive. 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. If a conflict occurs, this specification (including definitions) shall prevail.

[0226] Although specific embodiments of the present invention have been illustrated and described, it will be apparent to those skilled in the art that various other changes and modifications may be made without departing from the spirit and scope of the present invention. Therefore, the appended claims are intended to cover all such changes and modifications within the scope of the present invention.

[0227] The following examples are merely illustrative and are not to be construed as limiting the scope of the present disclosure in any way, as numerous variations and equivalents will become apparent to those skilled in the art upon reading this disclosure.

[0228] Example :

[0229] Example 1: Analysis of the link between excessive AP activation and endothelial damage in the setting of HSCT-TMA

[0230] Soluble TM, SYND 1 and Ba biomarker levels were measured in the plasma of pediatric HSCT patients with TMA and without TMA (control). Biomarkers were also measured in a preclinical in vivo model of complement activation mediated by inflammation similar to HSCT-TMA, where C57BL / 6J mice were injected with lipopolysaccharide (LPS) to induce complement activation, followed by AP (anti-fB IgG) and terminal complement pathway (anti-C5 IgG) inhibitors or IgG isotype control treatment. Since the immunosuppressant cyclosporin A (CsA) is a known trigger of HSCT-TMA, the role of complement in CsA-mediated damage in human EC (HMEC-1 cells and HUVEC) was assessed in vitro. In the presence or absence of C5 complement inhibitor eculizumab (Alexion Pharmaceuticals), EC was incubated with CsA and then incubated with normal human serum (NHS). TM was then measured by fluorescent staining on the cell surface and quantified using mean fluorescence intensity (MFI).

[0231] Compared with HSCT control patients (n=7), HSCT-TMA patients (n=11) showed significantly elevated levels of biomarkers of glycocalyx damage (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 between TM and SYND1 was observed (Pearson r=0.89). Plasma Ba was also significantly elevated in patients with TMA compared with those without TMA (1790.0±1262.0 ng / mL and 652.1±233.5 ng / mL, respectively; P<0.05), and positively correlated with TM and SYND1 levels (Pearson r=0.72 and 0.50, respectively).

[0232] It has been shown previously in an in vivo model of inflammation-mediated complement activation that mice injected with LPS had significantly increased plasma Ba levels compared to saline-injected control mice. As demonstrated here, mice injected with LPS (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) pathways of complement significantly attenuated TM levels (compared to 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, Figure 1). TM was positively correlated with Ba (Pearson r=0.68).

[0233] In vitro treatment of HMEC-1 cells with CsA resulted in the deposition of iC3b and C5b-9, and inhibition of C5 with eculizumab significantly reduced C5b-9 deposition (P < 0.0001) but had no effect on iC3b deposition. Treatment of HUVEC with CsA also reduced the surface expression of TM (P < 0.0001), and TM loss was partially restored by eculizumab (P < 0.05, Figure 2 ).

[0234] In conclusion, plasma biomarkers of endothelial injury were elevated in patients with HSCT-TMA and in mice with inflammation-mediated complement activation. In addition, AP activation was 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 of an association between complement activation and endothelial injury in HSCT-TMA, and the use of these biomarkers may aid in the diagnosis of HSCT-TMA.

[0235] Example 2: Complement activation is associated with endothelial injury in HSCT-TMA

[0236] The aim of this study was to explore the link between excessive complement alternative pathway (AP) activation and endothelial injury in the setting of HSCT-TMA and to potentially identify biomarkers to aid its diagnosis.

[0237] Levels of soluble TM, SYND1, and Ba were measured in the plasma of pediatric HSCT patients with and without TMA. Additionally, levels of TM, SYND1, and Ba 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).

[0238] FIG. 3A to FIG. 3C Depicted are biomarker levels of glycocalyx components in pediatric patients with and without HSCT-TMA. As shown in these figures, HSCT patients have elevated levels of TM and SYND1. Specifically, Figure 3A and Figure 3B It is shown that HSCT-TMA patients (n=11) had significantly elevated levels of glycocalyx damage biomarkers compared to HSCT control patients (n=7). Mean ± SD was calculated and significance was determined using Welch's t-test (*P<0.05; **P<0.01). Figure 3C It was shown that a strong positive correlation was observed between TM and SYND1 (Macrogol r=0.89).

[0239] FIG. 4A to FIG. 4C AP activation in pediatric patients with and without HSCT-TMA is depicted. As shown in these figures, HSCT patients have elevated Ba levels, which are positively correlated with TM and SYND1. Specifically, Figure 4A The results showed that plasma Ba was significantly increased in patients with HSCT-TMA compared with patients without HSCT-TMA (mean ± SD was calculated and significance was determined using Welch's t-test. *P < 0.05). Figure 4B ) and SYND1( Figure 4C ) levels were positively correlated (Homocos r = 0.89 and 0.50, respectively).

[0240] Figure 5 Depicted are plasma TM levels in LPS-injected mice treated with anti-complement agents. C57BL / 6J mice (12-17 weeks old) were injected intraperitoneally (IP) with LPS (5 mg / kg) to induce complement activation. Three hours after LPS injection, mice were injected IP 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 using a commercially available ELISA (R&D Systems). Untreated mice did not receive LPS or IgG (blue = male, pink = female). Mean ± SD was calculated and significance was determined using ordinary one-way ANOVA (** = P < 0.01 **** = P < 0.0001). As Figure 5 As shown, mice with inflammation-mediated complement activation had elevated circulating TM levels, which were attenuated by treatment with anti-complement agents.

[0241] FIG. 6A to FIG. 6B The correlation of TM and Ba in mice injected with LPS is shown. LPS (5 mg / kg) was injected intraperitoneally (IP) into C57BL / 6J mice (12-17 weeks old) to induce complement activation. Three hours after LPS injection, mice were injected IP with IgG isotype control or anti-fB IgG (40 mg / kg each). Twenty-four hours later, citrated plasma was collected and TM levels and Ba levels were measured using commercially available ELISA (R&D Systems) or Western blot ( Fig. 6A ; blue = male, pink = female). Mean ± SD was calculated and significance was determined using Welch's t-test. Figure 6BAs shown in , in mice with inflammation-mediated complement activation, plasma Ba positively correlated with TM levels (Pearsons r = 0.68. Gray = LPS + IgG isotype control, black = LPS + anti-fB IgG).

[0242] 7A to 7D Depict cyclosporin-induced TM loss in HUVEC. In the presence or absence of eculizumab substitute (ecu, 1 μM), HUVEC was treated with CsA (28.9 μM) and 30% NHS for 18 hours. The cells were then fixed with 4% PFA and incubated with mouse anti-human TM antibody, then with goat anti-mouse AF488 secondary antibody or with rabbit anti-human HSPG antibody, then with donkey anti-rabbit AF488 secondary antibody. TM and HSPG expression were imaged and analyzed by CX7 high content screening platform. MFI ± SEM was calculated and significance was determined by ordinary one-way ANOVA (* P < 0.05 **** = P < 0.0001). As shown in these figures, TM and HSPG surface expression were reduced on HUVEC treated with CsA, and partially recovered by eculizumab substitute.

[0243] FIG. 8A to FIG. 8B Depicted is 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 and then treated with 30% NHS for 30 minutes in the presence or absence of eculizumab alternative (ecu, 1 μM). The cells were fixed with 4% PFA and stained with a mouse antibody against iC3b, followed by goat anti-mouse AF647 antibody, or with a rabbit anti-human C5b-9 antibody, followed by goat anti-rabbit AF647 secondary antibody. Deposition was imaged and analyzed by the CX7 high-content screening platform. MFI+SEM was calculated and significance was determined by ordinary one-way ANOVA (****=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 ( Figure 8B ), but not iC3b deposition ( Fig. 8A ).

[0244] 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 was 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 of the association between complement activation and endothelial injury in HSCT-TMA. In addition, the use of biomarkers TM, SYND1, and Ba may aid in the diagnosis of HSCT-TMA.

[0245] Sequence Overview

[0246]

[0247]

[0248]

[0249]

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Claims

1. A method for treating a patient with hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) who has been determined to have an elevated blood 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 to the patient an anti-C5 antibody or an anti-complement factor B (CFB) antibody in an amount and frequency sufficient to attenuate the level of the biomarker in the patient, thereby treating HSCT-TMA.

2. The method of claim 1, wherein the patient has also been determined to have elevated blood levels of complement factors Ba and / or C5b9 compared to a normal reference range for Ba and / or C5b9.

3. The method of claim 1 or 2, wherein the patient has also been determined to have elevated blood levels of heparan sulfate proteoglycans (HSPG) compared to a normal reference range for HSPG.

4. A method for treating a patient suffering from HSCT-TMA, the method comprising: (1) obtaining or having obtained a blood sample from the patient, (2) determining or having determined an elevated level of a biomarker in said blood sample of said patient compared to a normal reference range of a biomarker selected from TM and SYND1 or a combination thereof, and (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. The method according to claim 4, further comprising determining or having determined elevated levels of Ba and / or C5b9 in the blood sample compared to a normal reference range for Ba and / or C5b9.

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

7. A method for identifying a patient with HSCT-TMA who is suitable for treatment with an anti-C5 antibody or an anti-CFB antibody, the method 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 elevated levels of the biomarkers in the blood sample compared to normal reference ranges for TM and SYND1, respectively, identify the patient as suitable for treatment with an anti-C5 antibody or an anti-CFB antibody.

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

9. The method of claim 8, further comprising determining the HSPG level in the blood sample, wherein an elevated HSPG level compared to a normal reference range for HSPG identifies 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 decreased level of the biomarker in the blood sample from the patient obtained during or after treatment compared to the level of the biomarker in the blood sample from the patient obtained before treatment with the anti-C5 antibody or anti-CFB antibody indicates that the patient is responding to treatment with the anti-C5 antibody or anti-CFB antibody.

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

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

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

14. A method according to any one of the preceding claims, wherein the one or more levels are measured by a test system or kit that has been approved by a regulatory body (eg, USFDA).

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

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

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

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

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

21. The method of any one of the preceding claims, wherein the elevated level of SYND1 is greater than 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, 180 ng / mL, 185 ng / mL, 190 ng / mL, 195 ng / ml, 200 mg / 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. The method of any one of the preceding claims, wherein the normal reference range for Ba in healthy patients is between about 300 ng / mL and 600 ng / mL.

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

24. The method of any of the preceding claims, wherein the elevated Ba level is greater than 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, 0ng / 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, 13 70ng / 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 / mL、1560ng / mL、1570ng / mL、1580ng / mL、1590ng / mL、1600ng / mL、1610ng / mL、1 620ng / mL, 1630ng / mL, 1640ng / mL, 1650ng / mL, 1660ng / mL, 1670ng / mL, 1680ng / mL, 1690ng / mL, 1700ng / mL, 1710ng / mL, 1720ng / mL, 1730ng / mL, 1740n g / 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,1870 ng / mL, 1880 ng / mL, 1890 ng / mL, 1900 ng / mL, 1910 ng / mL, 1920 ng / mL, 1930 ng / mL, 1940 ng / mL, 1950 ng / mL, 1960 ng / mL, 1970 ng / mL, 1980 ng / mL, 1990 ng / mL, 2000 ng / mL, 2010 ng / mL, 2020 ng / mL, 2030 ng / mL, 2040 ng / mL, 2050 ng / mL, 2060 ng / mL, 2070 ng / mL, 2080 ng / mL, 2090 ng / mL, 2100 ng / mL, 2110 ng / mL, 2120 ng / mL, 2130 ng / mL, 2140 ng / mL, 2150 ng / mL, 2160 ng / mL, 2170 ng / mL, 2180 ng / mL, 2190 ng / mL, 2200 ng / mL, 2210 ng / mL, 2220 ng / mL, 2230 ng / mL, 2240 ng / mL, 2250 ng / mL, 2260 ng / mL, 2270 ng / mL, 2280 ng / mL, 2290 ng / mL, 2300 ng / mL, 2310 ng / mL, 2320 ng / mL, 2330 ng / mL, 2340 ng / mL, 2350 ng / mL, 2360 ng / mL, 2370 ng / mL, 2380 ng / mL, 2390 ng / mL, 2400 ng / mL, 2410 ng / mL, 2420 ng / mL, 2430 ng / mL, 2440 ng / mL, 2450 ng / mL, 2460 ng / mL, 2470 ng / mL, 2480 ng / mL, 2490 ng / mL or 2500 ng / mL.

25. The method according to any one of the preceding claims, 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, Nanobodies or domain antibodies.

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

27. The method of any of the preceding claims, 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 of the preceding claims, 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 according to any one of the preceding claims, wherein the anti-C5 antibody is 30. The method of any one of claims 1 to 23, wherein the anti-C5 antibody comprises CDR1, CDR2 and CDR3 heavy chain sequences as shown in SEQ ID NOs: 19, 18 and 3, respectively, and CDR1, CDR2 and CDR3 light chain sequences as shown in SEQ ID NOs: 4, 5 and 6, respectively.

31. The method of claim 30, wherein the anti-C5 antibody further comprises a variant human Fc constant region that binds to 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, each by EU numbering.

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 shown in SEQ ID NO:

13.

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

11.

35. The method according to any one of claims 30 to 34, wherein the anti-C5 antibody is 36. The method of any of the preceding claims, wherein the anti-C5 antibody is administered intravenously.

37. The method of any one of claims 1 to 29, wherein the anti-C5 antibody is administered as follows: (a) once on day 1 at a dose of 600 mg for patients weighing ≥5 kg to <10 kg, 600 mg for patients weighing ≥10 kg to <20 kg, 900 mg for patients weighing ≥20 kg to <30 kg, 1200 mg for patients weighing ≥30 kg to <40 kg, 2400 mg for patients weighing ≥40 kg to <60 kg, 2700 mg for patients weighing >60 kg to <100 kg, or 3000 mg for patients weighing ≥100 kg; (b) once on day 5 at a dose of 300 mg for patients weighing ≥5 kg to <10 kg, 300 mg for patients weighing ≥10 kg to <20 kg, 300 mg for patients weighing ≥20 kg to <30 kg, 300 mg for patients weighing ≥30 kg to <40 kg, 600 mg for patients weighing ≥40 kg to <60 kg, 900 mg for patients weighing ≥60 kg to <100 kg, or 900 mg for patients weighing ≥100 kg; (c) once on day 10 at a dose of 300 mg for patients weighing ≥5 kg to <10 kg, 300 mg for patients weighing ≥10 kg to <20 kg, 300 mg for patients weighing ≥20 kg to <30 kg, 300 mg for patients weighing ≥30 kg to <40 kg, 600 mg for patients weighing ≥40 kg to <60 kg, 900 mg for patients weighing ≥60 kg to <100 kg, or 900 mg for patients weighing ≥100 kg; and (d) 300 mg for patients weighing ≥5 kg to <10 kg or 600 mg for patients weighing ≥10 kg to <20 kg on day 15 and every four weeks thereafter; or 2100 mg for patients weighing ≥20 kg to <30 kg, 2700 mg for patients weighing ≥30 kg to <40 kg, 3000 mg for patients weighing ≥40 kg to <60 kg, 3300 mg for patients weighing ≥60 kg to <100 kg, or 3600 mg for patients weighing ≥100 kg on day 15 and every eight weeks thereafter.

38. The method of any of the preceding claims, wherein the treatment results in a reduction or cessation of microangiopathic hemolytic anemia, thrombocytopenia, endothelial damage, renal injury, renal failure, serositis, pulmonary hypertension, and multisystem organ failure compared to baseline.

39. The method of any of the preceding claims, wherein the treatment results in normalization of LDH, elimination of red blood cell and platelet transfusion requirements, increase in hemoglobin, and / or disappearance of schistocytes compared to baseline.

40. The method of any of the preceding claims, wherein the treatment results in: (a) a platelet count of ≥ 50,000 / mm3 without transfusion support during the first 7 days 3 , (b) LDH < 1.5 x ULN, and (c) absence of schistocytes (if present at baseline), and / or (d) a reduction in proteinuria of at least 50% from baseline.

41. The method of any of the preceding claims, wherein the treatment results in a favorable hematological response.

42. The method of any of the preceding claims, wherein the treatment results in hemoglobin ≥ 8 g / dL without transfusion support.

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

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

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

46. The method of any of the preceding claims, wherein the quality of life assessment is the Pediatric Quality of Life Inventory (PedsQL) scale or the EQ-5D-5L questionnaire).

47. The method of any of the preceding claims, wherein the patient is a pediatric patient.

48. The method of any of the preceding claims, 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 treating a patient with hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA), wherein the patient has been determined to have an elevated blood level of a biomarker selected from thrombomodulin (TM) and syndecan-1 (SYND1) or a combination thereof compared to a normal reference range of the biomarker, wherein the anti-C5 antibody or anti-complement factor B (CFB) antibody is administered to the patient in an amount and at a frequency sufficient to attenuate the level of the biomarker in the patient.

50. An anti-C5 antibody or antigen-binding fragment thereof or an anti-complement factor B (CFB) antibody for identifying a patient with HSCT-TMA who is suitable for treatment with an anti-C5 antibody or an anti-CFB antibody, the identification 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 an elevated level of the biomarker in the blood sample compared to a normal reference range for TM and SYND1, respectively, identifies the patient as suitable for treatment with an anti-C5 antibody or an anti-CFB antibody.

51. An anti-C5 antibody or antigen-binding fragment thereof or an anti-CFB antibody for monitoring the responsiveness of a patient with HSCT-TMA to treatment with an anti-C5 antibody or an anti-CFB antibody, wherein the use comprises: 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 decreased level of the biomarker in the blood sample from the patient obtained during or after treatment compared to the level of the biomarker in the blood sample from the patient obtained before treatment with the anti-C5 antibody or anti-CFB antibody indicates that the patient is responding 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 treating a patient with hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) who has been determined to have elevated blood levels of a biomarker selected from thrombomodulin (TM) and syndecan-1 (SYND1) or a combination thereof compared to a normal reference range of the biomarker, wherein the anti-C5 antibody or anti-CFB antibody is administered to the patient in an amount and at a frequency sufficient to attenuate the level of the biomarker in the 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 who is suitable for treatment with an anti-C5 antibody or an anti-CFB antibody, the identification 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 an elevated level of the biomarker in the blood sample compared to the normal reference range for TM and SYND1, respectively, identifies the patient as suitable for treatment with an anti-C5 antibody or an anti-CFB antibody.

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, 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 decreased level of the biomarker in the blood sample from the patient obtained during or after treatment compared to the level of the biomarker in the blood sample from the patient obtained before treatment with the anti-C5 antibody or anti-CFB antibody indicates that the patient is responding to treatment with the anti-C5 antibody or anti-CFB antibody.

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