EA044587B1 - PROTEIN BIOMARKERS OF ATYPICAL HEMOLYTIC UREMIC SYNDROME - Google Patents

Description

Cross reference to related applications

This application claims priority to U.S. Provisional Application Nos. 61/913180 and 61/863299, filed December 6, 2013 and August 7, 2013, respectively. The entire contents of the foregoing applications and any patents, patent applications and references cited herein are incorporated herein by reference in their entirety.

Field of technology to which the invention relates

The invention relates to the fields of medicine, immunology, molecular biology and protein chemistry.

Prior Art

Hemolytic uremic syndrome (HUS) is characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure. HUS is classified as one of two types: diarrhea-associated (D+HUS; also referred to as Shiga toxin-producing E. coli (STEC) HUS or typical HUS) and nondiarrhea-associated or atypical HUS (aHUS). D+HUS is the most common form, accounting for more than 90% of cases, and is caused by a preexisting disease with Shiga toxin-producing bacteria, such as E. coli 0157:H7. aHUS is a rare disease with a mortality rate of up to 25%. Many patients with the disease have permanent neurological or renal impairment, with at least 50% of aHUS patients progressing to end-stage renal disease (ESRF). See, for example, Kavanagh et al. (2006), British Medical Bulletin, 77, 78:5-22.

aHUS can be genetic, acquired, or idiopathic. Inherited forms of aHUS can be associated with mutations in a number of human complement components, including, for example, complement factor H (CFH), membrane cofactor protein (MCP), complement factor I (CFI), C4b-binding protein (C4BP), complement factor B (CFB) and complement component 3 (C3). See, for example, Caprioli et al. (2006) Blood 108:1267–1279. Some mutations in the gene encoding CD55, although not involved in aHUS, are associated with the severity of aHUS. See, for example, Esparza-Gordillo et al. (2005) Hum Mol Genet 14:703–712.

Until recently, treatment options for patients with aHUS were limited and often involved plasma infusion or plasma replacement. In some cases, patients with aHUS have undergone unilateral or bilateral nephrectomy or kidney transplantation (see Artz et al. (2003) Transplantation 76:821-826). However, relapse of the disease in treated patients is common. Soliris® has recently been approved for treatment of aHUS patients in the United States and Europe. Despite the eventual availability of a useful drug for the treatment of aHUS patients, there is still a need to diagnose aHUS patients and monitor the progression and decline of aHUS.

The essence of the invention

The present invention provides, among other things, various proteins whose activity and/or concentration in biological fluid is abnormal in patients suffering from aHUS and/or in those patients with aHUS who are receiving additional inhibitor therapy. These proteins are hereafter referred to as aHUS-associated protein biomarkers or aHUS protein biomarkers. For example, the present inventors have discovered that the concentrations and/or activities of several proteins in the blood (eg, serum and/or plasma) and urine are abnormal in patients with aHUS. The inventors have also discovered that following administration of the anti-C5 antagonist antibody eculizumab to humans, the concentrations of a subset of these proteins are altered. In some cases, the concentration of one or more proteins returns to normal. While the invention is not limited to any particular theory or mechanism of action, the inventors believe that monitoring a patient treated with a complement inhibitor (eg, anti-C5 antibody) to change the concentration of one or more of these aHUS protein biomarkers - suitable, for example, for diagnosing a patient suffering from aHUS or at risk of developing aHUS. Monitoring the status of one or more of these protein biomarkers may also be useful to determine whether a patient with aHUS is responding to complement inhibitor therapy. In addition, assessment of one or more biomarkers is also useful in identifying a dose—a threshold dose—of a complement inhibitor, such as an anti-C5 antibody, that, by virtue of its effect on the concentration of one or more aHUS protein biomarkers in a person, is sufficient to achieve a clinically significant effect in disease (ie, sufficient to treat a complement-related disease such as aHUS).

Accordingly, in one aspect, the invention relates to a method for monitoring or assessing the status of protein biomarkers associated with atypical hemolytic uremic syndrome (aHUS) in an individual (e.g., a mammal such as a human), or a method for assessing one or both concentration and activity level, according to at least one protein biomarker associated with atypical hemolytic uremic syndrome (aHUS) in an individual. The method includes measuring, in a biological fluid obtained from an individual, one or both of (i) a concentration of at least one (for example, at least two, three, four, five, six, seven, eight, nine,

- 1 044587

10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) aHUS-associated protein biomarkers in a biological fluid, where the aHUS-associated protein biomarkers are any of the biomarkers presented in Table . 1, for example, a biomarker selected from the group consisting of a proteolytic fragment of a complement factor B component (e.g., Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand factor (vWF), soluble CD40 ligand (sCD40L ), fragment F1+2 prothrombin, D-dimer, CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1 beta, IL-12 p70, complement component C5a, microglobulin β2 (b2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, vascular endothelial cell growth factor (VEGF), IL-6, albumin, IL- 8 and CCL5. The individual may be, for example, a person suffering from aHUS, suspected of having aHUS, or at risk of developing it. The individual may be a person who has been treated (or is currently being treated) with a complement inhibitor (eg, an inhibitor of complement component C5, such as an anti-C5 antibody). Treatment can be carried out for less than one month (for example, less than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day) before receiving the sample from the individual. The method may further include the step of determining whether the individual has aHUS or is at risk of developing it. If the individual has been treated or is being treated with a complement inhibitor (eg, anti-C5 antibody) according to a predetermined dosing schedule, the method may further include determining whether the patient responds (therapeutically) to treatment with the complement inhibitor.

In another aspect, the invention relates to a method for monitoring or assessing the status of protein biomarkers associated with atypical hemolytic uremic syndrome (aHUS) in an individual (e.g., a mammal such as a human), or a method for assessing one or both of the concentration and activity level of at least , one protein biomarker associated with atypical hemolytic uremic syndrome (aHUS) in an individual. The method includes (A) measuring, in a biological fluid obtained from an individual, a concentration of at least one (e.g., at least two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) protein biomarkers associated with aHUS in a biological fluid, where the protein biomarkers associated with aHUS are any of the biomarkers presented in table. 1, for example, a biomarker selected from the group consisting of a proteolytic fragment of a complement factor B component (e.g., Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand factor (vWF), soluble CD40 ligand (sCD40L ), fragment F1+2 of prothrombin, Ddimer, CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1-beta, IL-12 p70, complement component C5a, microglobulin β2 (b2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, vascular endothelial cell growth factor (VEGF), IL-6, albumin, IL-8 and CCL5; and (B) recording (e.g., in an electronic patient document) the results of the measurement(s) or transmitting the results of the measurement(s) to the individual, the individual's guardian, or the health care provider in whose care the individual is. The individual may be, for example, a person suffering from aHUS, suspected of having aHUS, or at risk of developing it. The individual may be a person who has been treated (or is currently being treated) with a complement inhibitor (eg, an inhibitor of complement component C5, such as an anti-C5 antibody). Treatment can be carried out for less than one month (for example, less than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day) before receiving the sample from the individual. The method may further include the step of determining whether the individual has aHUS or is at risk of developing it. If the individual has been treated or is being treated with a complement inhibitor (eg, anti-C5 antibody) according to a predetermined dosing schedule, the method may further include determining whether the patient responds (therapeutically) to treatment with the complement inhibitor. In yet another aspect, the invention relates to a method for monitoring or determining whether a patient is at risk of developing thrombotic microangiopathy. The method includes (A) measuring, in a biological fluid obtained from an individual, the concentration of at least one (e.g., at least two, three, four) protein biomarkers associated with thrombosis or coagulation in the biological fluid, wherein the protein biomarkers represent any of the biomarkers presented in table. 1 or 11, for example F1+2 or D-dimer; and (B) recording (e.g., in an electronic patient document) the results of the measurement(s) or transmitting the results of the measurement(s) to the individual, the individual's guardian, or the health care provider in whose care the individual is. The individual may be, for example, a person suffering from aHUS, suspected of having aHUS, or at risk of developing it. The individual may be a person who has been treated (or is currently being treated) with a complement inhibitor (eg, an inhibitor of complement component C5, such as an anti-C5 antibody). Treatment can be carried out for less than one month (for example, less than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day) before receiving the sample from the individual. The method may further include the step of determining whether the individual suffers from aHUS or is at risk

- 2 044587 its development (or has a confirmed diagnosis of aHUS), using the methods described in this document. If the subject has been or is being treated with a complement inhibitor (eg, an anti-C5 antibody) according to a predetermined dosing schedule, the method may further include determining whether the patient responds (therapeutically) to treatment with the complement inhibitor, i.e. whether a decrease in the concentration of one or more biomarkers associated with thrombosis or coagulation occurs after treatment with a complement inhibitor.

In another aspect, the invention relates to a method of monitoring or assessing the status of protein biomarkers associated with atypical hemolytic uremic syndrome (aHUS) in an individual (e.g., a mammal such as a human), or a method of assessing one or both concentration and activity level of at least at least one protein biomarker associated with atypical hemolytic uremic syndrome (aHUS) in an individual. The method includes (A) measuring, in a biological fluid obtained from an individual, a concentration of at least one (e.g., at least two, three, four, five, six, seven, eight, nine, 10, 11, 12, or 13) protein biomarkers associated with aHUS in the biological fluid, where the protein biomarkers associated with aHUS are any of the biomarkers presented in table. 1, for example, a biomarker selected from the group consisting of a proteolytic fragment of complement factor B (for example, Ba or Bb), soluble C5b9 (sC5b9), C5a, thrombomodulin, VCAM-1, prothrombin F1+2 fragment, D-dimer, sTNFRl, β2-microglobulin (β2M), clusterin, cystatin C, TIMP-1, and fatty acid binding protein 1 (FABP-1); and (B) recording (e.g., in an electronic patient document) the results of the measurement(s) or transmitting the results of the measurement(s) to the individual, the individual's guardian, or the health care provider in whose care the individual is. The individual may be, for example, a person suffering from aHUS, suspected of having aHUS, or at risk of developing it. The individual may be a person who has been treated (or is currently being treated) with a complement inhibitor (eg, an inhibitor of complement component C5, such as an anti-C5 antibody). Treatment can be carried out for less than one month (for example, less than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day) before receiving the sample from the individual. The method may further include the step of determining whether the individual has aHUS or is at risk of developing it. If the individual has been treated or is being treated with a complement inhibitor (eg, anti-C5 antibody) according to a predetermined dosing schedule, the method may further include determining whether the patient responds (therapeutically) to treatment with the complement inhibitor.

In some embodiments, any of the methods described herein may further include determining whether an individual has aHUS or is at risk of developing it. In some embodiments, an increased concentration relative to the concentration in a normal control body fluid of the same type of at least one of Ba, sC5b-9, C5a, SCD40L, prothrombin F1+2 fragment, D-dimer, thrombomodulin, VCAM-1 , vWF, FABP-1, b2M, clusterin, cystatin C, TIMP-1, albumin, NGAL, CXCL10, CXCL9, IL-18, TNFR1, VCAM-1, MCP-1, VEGF, CCL5, IL-6, IFNy, indicates that an individual has aHUS or is at risk of developing it.

In some embodiments, any of the methods described herein includes determining whether an individual responds to treatment with a complement inhibitor. In some embodiments, (a) reducing the concentration, relative to the concentration in a biological fluid sample of the same type obtained from the individual prior to initiation of treatment with an inhibitor, of at least one of CXCL10, MCP-1, TNFR1, IFN-γ, a proteolytic fragment factor B complement component (e.g. Ba or Bb), soluble C5b9 (sC5b9), prothrombin F1+2 fragment, D-dimer, thrombomodulin, VCAM-1, von Willebrand factor (vWF), complement component C5a, sC5b9, B2-microglobulin ( b2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin, CXCL10, CXCL9 and KIM-1; or (b) an increase in the concentration of CCL5 compared to the concentration in a body fluid sample of the same type obtained from the individual prior to initiation of inhibitor treatment indicates that the individual is responding to inhibitor treatment.

In another aspect, the invention provides a method for monitoring the response of an individual (eg, a mammal, such as a human) to treatment with a complement component C5 inhibitor. The method includes measuring the concentration of at least two protein biomarkers associated with aHUS in a biological fluid, where the protein biomarkers associated with aHUS are any of the biomarkers listed in table. 1, for example, a biomarker selected from the group consisting of a proteolytic fragment of complement factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand factor (vWF), soluble CD40 ligand (sCD40L ), fragment F1+2 prothrombin, D-dimer, CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1-beta, IL-12 p70, complement component C5a, b2-microglobulin (b2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, growth factor

- 3044587 vascular endothelial cells (VEGF), IL-6, albumin, IL-8 and CCL5. The body fluid is obtained from an individual (i) suffering from aHUS, suspected of having aHUS, or at risk of developing it; and (ii) who has been treated (or, for example, is currently being treated) with a complement component C5 inhibitor according to a predetermined dosing schedule. In such methods, (a) reducing the concentration, relative to the concentration in a sample of the same type of biological fluid obtained from an individual prior to treatment with an inhibitor, of at least one of CXCL10, MCP-1, TNFR1, IFN-γ, proteolytic moiety complement factor B (for example, Ba or Bb), soluble C5b9 (sC5b9), prothrombin F1+2 fragment, d-dimer, thrombomodulin, VCAM-1, von Willebrand factor (vWF), complement component C5a, b2-microglobulin (v2M ), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin, CXCL10, CXCL9 and KIM-1; or (b) an increase in the concentration of CCL5 compared to the concentration in a body fluid sample of the same type obtained from the individual prior to initiation of inhibitor treatment indicates that the individual is responding to inhibitor treatment.

In some embodiments, any of the methods described herein includes determining whether an individual will respond to treatment with a complement inhibitor. In some embodiments, there is a reduced concentration, compared to the concentration in a sample of the same type of biological fluid obtained from an individual prior to treatment with an inhibitor, of at least one complement factor B proteolytic fragment (e.g., Ba or Bb), soluble C5b9 ( sC5b9), C5a, thrombomodulin, VCAM-1, prothrombin fragment F1+2, D-dimer, sTNFRl, b2-microglobulin (b2M), clusterin, cystatin C, TIMP-1 and fatty acid binding protein 1 (FABP-1) .

In another aspect, the invention relates to a method for monitoring the response of an individual to treatment with a complement inhibitor, where the method includes determining the concentration of at least two protein biomarkers associated with aHUS in a biological fluid obtained from the individual, wherein the protein biomarkers associated with aHUS are selected from the group consisting of CXCL10, MCP-1, TNFR1, IFN-gamma, IL-6, proteolytic fragment of complement factor B (for example, Ba or Bb), soluble C5b9 (sC5b9), prothrombin F1+2 fragment, D-dimer , thrombomodulin, VCAM-1, von Willebrand factor (vWF), complement component C5a, b2-microglobulin (b2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin, CXCL9, KIM-1 and CCL5. The individual has or is at risk of developing aHUS, and the individual has been or is being treated with a complement inhibitor. (A) Decreased concentration compared to the concentration in a sample of the same type of biological fluid obtained from an individual before treatment with an inhibitor of at least one of CXCL10, MCP-1, TNFR1, IFN-γ, IL-6, proteolytic fragment factor B complement component (for example, Ba or Bb), soluble C5b9 (sC5b9), prothrombin fragment F1+2, d-dimer, thrombomodulin, VCAM-1, von Willebrand factor (vWF), complement component C5a, b2-microglobulin (v2M) , clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin, CXCL9 and KIM-1; or (B) an increase in the concentration of CCL5 compared to the concentration in a body fluid sample of the same type obtained from the individual prior to initiation of treatment with the inhibitor indicates that the individual is responding to treatment with the inhibitor.

In yet another aspect, the invention provides a method of reducing the incidence, frequency or occurrence, probability of occurrence or risk of developing TMA using a complement inhibitor in a manner that is sufficient to induce a physiological change in at least two protein biomarkers associated with thrombosis or coagulation. The method includes (a) determining the concentration of at least two protein biomarkers in a biological fluid obtained from an individual, where the protein biomarkers are selected from table. 1 or 11, and relate to thrombosis and/or coagulation (eg, D-dimer or F1+2); and (b) administering to an individual suffering from TMA, suspected of having TMA, or at risk of developing it, a complement inhibitor in an amount and at a frequency sufficient to induce a physiological change in at least each of two (2) protein biomarkers, where the physiological the change is a decrease in the concentration of at least two protein biomarkers relative to the concentration of the markers in an equivalent biological sample obtained from the individual before the start of treatment with a complement inhibitor. The method may include measuring biomarker concentrations both before and after treatment.

In yet another aspect, the invention relates to a method for determining whether an aHUS patient who has been treated with a complement inhibitor according to a predetermined dosage regimen requires (i) treatment with another complement inhibitor; or (ii) treatment with the same complement inhibitor at a different dosage regimen. The method includes (A) determining whether an aHUS patient responds to treatment with a complement inhibitor according to a predetermined dosing schedule, where the determination includes measuring, in a biological fluid obtained from the individual, one or both the concentration and activity of at least two protein biomarkers related with aHUS, in biological

- 4 044587 liquid, wherein the protein biomarkers associated with aHUS are selected from the group consisting of a proteolytic fragment of complement factor B (for example, Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand factor (vWF) ), soluble CD40 ligand (sCD40L), prothrombin F1+2 fragment, D-dimer, CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1-beta, IL-12 p70, complement component C5a, β2-microglobulin (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, vascular endothelial cell growth factor (VEGF) , IL-6, albumin, IL-8 and CCL5, and where (a) a decrease in concentration compared to the concentration in a sample of the same type of biological fluid obtained from an individual before treatment with an inhibitor of at least one of CXCL10, MCP- 1, TNFR1, IFN-γ, complement factor B proteolytic fragment (for example, Ba or Bb), soluble C5b9 (sC5b9), prothrombin fragment F1+2, D-dimer, thrombomodulin, VCAM-1, von Willebrand factor (vWF) , complement component C5a, sC5b9, b2-microglobulin (b2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin, CXCL10, CXCL9 and KIM-1; or (b) an increase in the concentration of CCL5 compared to the concentration in a body fluid sample of the same type obtained from the individual prior to initiation of inhibitor treatment indicates that the individual is responding to treatment with the inhibitor; and (B) if the patient does not respond to treatment with a complement inhibitor, administering to the patient another complement inhibitor or the same complement inhibitor at a higher dose or at a more frequent dosing schedule than the predetermined dosing schedule.

In yet another aspect, the invention provides a method for determining whether an aHUS patient being treated with a complement inhibitor at a predetermined dosage regimen requires (i) treatment with a different complement inhibitor or (ii) treatment with the same complement inhibitor at a different dosage regimen. The method includes (A) determining whether an aHUS patient responds to treatment with a complement inhibitor at a predetermined dosing regimen, where the determination includes measuring, in a biological fluid obtained from the individual, one or both the concentration and activity of at least two protein biomarkers related with aHUS, in a biological fluid, wherein the protein biomarkers associated with aHUS are selected from the group consisting of a complement factor B proteolytic fragment (e.g., Ba or Bb), soluble C5b9 (sC5b9), C5a, thrombomodulin, VCAM-1 fragment F1+2 prothrombin, D-dimer, sTNFRl, β2-microglobulin (β2M), clusterin, cystatin C, TIMP-1, fatty acid binding protein 1 (FABP-1), and where (a) decrease in concentration compared to concentration in a sample of a biological fluid of the same type obtained from an individual prior to treatment with an inhibitor of at least one of a complement factor B proteolytic fragment (e.g., Ba or Bb), soluble C5b9 (sC5b9), C5a, thrombomodulin, VCAM-1, F1+2 fragment of prothrombin, D-dimer, sTNFRl, b2-microglobulin (b2M), clusterin, cystatin C, TIMP-1 and fatty acid binding protein 1 (FABP-1) indicates that the individual is responding to treatment inhibitor; and (B) if the patient does not respond to treatment with a complement inhibitor, administering to the patient another complement inhibitor or the same complement inhibitor at a higher dose or at a more frequent dosing schedule than the predetermined dosing schedule.

The concentration of one or more proteins can be measured using, for example, an immunoassay (eg, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), Western blot or dot blot) or bead array cytometry (BCA, see worked examples). Such methods, as well as kits suitable for implementing these methods, are described herein. Suitable methods for measuring vWF activity are known in the art and are described herein.

In some embodiments of any of the methods described herein, the concentrations of at least five separate protein biomarkers associated with aHUS are measured. In some embodiments of any of the methods described herein, the concentrations of at least ten individual protein biomarkers associated with aHUS are measured. In some embodiments of any of the methods described herein, the concentrations of at least 15 individual protein biomarkers associated with aHUS are measured. In some embodiments of any of the methods described herein, the concentrations of at least 20 individual protein biomarkers associated with aHUS are measured.

In some embodiments of any of the methods described herein, the biological fluid is blood. In some embodiments, the biological fluid is a fraction of blood, such as serum or plasma. In some embodiments, the biological fluid is urine. In some embodiments of any of the methods described herein, all measurements are performed in a single biological fluid. In some embodiments of any of the methods described herein, measurements are performed in at least two different biological fluids obtained from an individual. In some embodiments, concentrations are measured by

- 5 044587 at least two separate protein biomarkers associated with aHUS, and the concentration of the first protein biomarker associated with aHUS is determined in one type of biological fluid, and the second protein biomarker associated with aHUS is measured in a second type of biological fluid.

In some embodiments of any of the methods described herein, the concentrations of at least two (e.g., at least three, four, or all) of IFN-γ, ICAM-1, IL-1-beta, and IL are measured. -12 р70. In some embodiments of any of the methods described herein, the concentrations of both Ba and sC5b9 are measured. In some embodiments of any of the methods described herein, the concentrations of one or both C5a and C5b9 are measured. In some embodiments of any of the methods described herein, the concentrations of at least two (e.g., at least three, four, five, six, or all) of B2M, clusterin, cystatin C, NAG, TIMP- 1, NGAL and FABP-1. In some embodiments of any of the methods described herein, concentrations of CXCL10, CXCL9 and/or KIM-1 are measured. In some embodiments of any of the methods described herein, the concentrations of one or both of D-dimer and F1+2 are measured. In some embodiments of any of the methods described herein, the concentrations of at least two (eg, at least three, four, or all) of SCD40L, prothrombin F1+2 fragment, and D-dimer are measured. In some embodiments of any of the methods described herein, concentrations of thrombomodulin, VCAM-1, and/or vWF are measured. In some embodiments of any of the methods described herein, concentrations of CXCL10, MCP-1, and/or TNFR1 are measured. In some embodiments of any of the methods described herein, the concentrations of at least two (e.g., at least three, four, or all) of IFN-γ, ICAM-1, IL-1-beta, and IL are measured. -12 р70.

In some embodiments of any of the methods described herein, the concentrations of one or more of CXCL9, CXCL10, IL-1 beta, IL-12 p70, IFN-γ, MCP-1, CCL5, SCD40L and/or sTNFRl are measured in individual's serum. In some embodiments, the concentration of one or more of complement component C5a, sC5b9, b2-microglobulin (b2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL10, CXCL9 and/or KIM-1 is measured in the urine of the individual. In some embodiments, any of the methods described herein concentrates one or more of NGAL, a complement factor B proteolytic fragment (e.g., Ba or Bb), soluble C5b9 (sC5b9), prothrombin F1+2 fragment, D-dimer, thrombomodulin, and /or von Willebrand factor (vWF) is measured in the blood plasma of an individual.

In some embodiments, the concentration of two or more (e.g., three, four, five, six, seven, eight, nine, 10, 11, 12, or 13) of the complement component factor B proteolytic fragment (e.g., Ba or Bb) is measured, soluble C5b9 (sC5b9), C5a, thrombomodulin, VCAM-1, prothrombin F1+2 fragment, D-dimer, sTNFRl, β2-microglobulin (β2M), clusterin, cystatin C, TIMP-1, and fatty acid binding protein 1 (FABP -1).

In some embodiments of any of the methods described herein, the concentration of at least two of the group consisting of Ba, sC5b-9 and C5a is measured. In some embodiments of any of the methods described herein, the concentration of one or both Ba and sC5b9 is measured. In some embodiments of any of the methods described herein, the concentration of one or both C5a and C5b9 is measured. In some embodiments of any of the methods described herein, the concentrations of at least two individual members of the group consisting of B2M, clusterin, cystatin C, albumin, TIMP-1, NGAL and FABP-1 are measured. In some embodiments of any of the methods described herein, the concentrations of at least two individual members of the group consisting of CXCL10, CXCL9, IL-18, MCP-1, TNFR1, VEGF, IL-6 and IFNy are measured. In some embodiments of any of the methods described herein, the concentrations of one or both of the d-dimer and F1+2 are measured. In some embodiments of any of the methods described herein, the concentrations of at least two individual members of the group consisting of SCD40L, prothrombin F1+2 fragment and D-dimer are measured. In some embodiments of any of the methods described herein, concentrations of thrombomodulin, VCAM-1, or vWF are measured. In some embodiments of any of the methods described herein, the concentration of TNFR1 is measured. In some embodiments of any of the methods described herein, the concentrations of at least two individual members of the group consisting of IFN-γ, CXCL10, CXCL9, IL-18, TNFR1, VCAM-1, MCP-1, VEGF are measured , CCL5 and IL-6. In some embodiments, any of the methods described herein measures the concentration of at least one aHUS-associated protein biomarker selected from the group consisting of IFN-γ, CXCL10, CXCL9, IL-18, TNFR1, VCAM- 1, MCP-1, VEGF and IL-6. In some embodiments, any of the methods described herein measures the concentration of at least one aHUS-associated protein biomarker selected from the group consisting of β2-micro

- 6 044587 globulin (β2Μ), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL10, CXCL9, albumin and KIM-1. In some embodiments, any of the methods described herein measures the concentration of at least one aHUS-associated protein biomarker selected from the group consisting of CXCL10, CXCL9, IL-18, MCP-1, TNFR1, VEGF, IL-6, CCL5, IFNy, IL-8, ICAM-1, IL-1beta and IL-12 p70. In some embodiments of any of the methods described herein, concentrations of CXCL9, CXCL10, IL-1-beta, IL-12 p70, IFN-γ, MCP-1, CCL5, SCD40L, or sTNFRl in the serum of an individual are measured. In some embodiments of any of the methods described herein, the concentration of at least one aHUS-associated biomarker selected from the group consisting of b2-microglobulin (b2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL10, CXCL9, albumin and KIM-1 in the urine of an individual. In some embodiments of any of the methods described herein, the concentration of NGAL, a proteolytic fragment of complement factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), prothrombin F1+2 fragment, D-dimer, thrombomodulin, or factor von is measured. von Willebrand (vWF) in the blood plasma of an individual. In some embodiments of any of the methods described herein, the Ba concentration is measured (eg, in a plasma sample obtained from an individual).

In some embodiments of any of the methods described herein, the method requires recording the measured concentration value(s) of at least one aHUS protein biomarker. The recording may be made in a computer-readable environment. The method may also include transmitting the measured concentration value(s) of at least one aHUS protein biomarker to the individual and/or a health care professional in whose care the individual is.

In some embodiments, any of the methods described herein may include the step of administering to an individual a complement inhibitor at a higher dose or at an increased dosing frequency relative to a predetermined dosing regimen if the individual does not respond to treatment with the inhibitor on a predetermined dosing regimen.

In some embodiments of any of the methods described herein, a complement inhibitor is administered to an individual according to a predetermined dosing schedule based, in particular, on the individual's body weight. For example, in the case of an anti-C5 antagonist antibody (eg, eculizumab), for individuals having a body weight greater than or equal to 40 kg, the antibody can be administered to the individual over at least 7 weeks on the following schedule: at least 800 mg of antibody , once a week for four consecutive weeks; at least 800 mg of antibody once during the fifth week; and at least 800 mg of antibody every two weeks thereafter. In some embodiments, the antibody is administered to the individual over at least 7 weeks according to the following schedule: at least 900 mg of antibody once per week for four consecutive weeks; at least 1200 mg of antibody once during the fifth week; and at least 1200 mg of antibody every two weeks thereafter.

In some embodiments of any of the methods described herein, for individuals having a body weight of less than 40 kg but equal to or greater than 30 kg, the antibody can be administered to the individual over at least 7 weeks according to the following schedule: at least at least 500 mg of antibody once a week for two consecutive weeks; at least 700 mg of antibody once during the third week; and at least 700 mg of antibody every two weeks thereafter. In some embodiments, the antibody is administered to the individual over at least 5 weeks according to the following schedule: at least 600 mg of antibody once per week for two consecutive weeks; at least 900 mg of antibody once during the third week; and at least 900 mg of antibody every two weeks thereafter.

In some embodiments of any of the methods described herein, the individual's body weight is less than 30 kg but greater than or equal to 20 kg, and the antibody is administered to the individual over at least 5 weeks according to the following schedule: at least 500 mg of antibody once a week for two consecutive weeks; at least 500 mg of antibody once during the third week; and at least 500 mg of antibody every two weeks thereafter. In some embodiments, the antibody is administered to the individual for at least 5 weeks according to the following schedule: at least 600 mg of antibody once a week for two consecutive weeks; at least 600 mg of antibody once during the third week; and at least 600 mg of antibody every two weeks thereafter.

In some embodiments of any of the methods described herein, the body weight of the individual is less than 20 kg but greater than or equal to 10 kg, and the antibody is administered to the individual over at least 4 weeks according to the following schedule: at least 500 mg of antibody once a week for one week; at least 200 mg of antibody once during the second week; and at least 200 mg of antibody every two weeks thereafter. In some embodiments, the antibody is administered to the individual over at least 4 weeks according to the following schedule: at least 600 mg of antibody once per week for one week; at least 300 mg of antibody once during the second week; and at least 300 mg of antibody every two weeks thereafter.

In some embodiments of any of the methods described herein, the body weight of the individual is less than 10 kg but greater than or equal to 5 kg, and the antibody is administered to the individual over at least 5 weeks according to the following schedule: at least 200 mg of antibody, once a week for one week; at least 200 mg of antibody once during the second week; and at least 200 mg of antibody every three weeks thereafter. In some embodiments, the antibody is administered to the individual for at least 5 weeks according to the following schedule: at least 300 mg of antibody once a week for one week; at least 300 mg of antibody once during the second week; and at least 300 mg of antibody every three weeks thereafter. Additional examples of anti-C5 antibody dosing regimens (eg, chronic dosing regimens) for the treatment of aHUS are described in International Patent Application Publication WO 2010/054403 (eg, Tables 1 and 2 of WO 2010/054403), the disclosure of which is incorporated herein into as a reference in full.

In some embodiments of any of the methods described herein, the inhibitor is an antibody or an antigen-binding fragment thereof, a small molecule, a polypeptide, a polypeptide analog, a peptidomimetic, or an aptamer. In some embodiments, the inhibitor may be an inhibitor that inhibits one or more of complement components C1, C2, C3, C4, C5, C6, C7, C8, C9, factor D, factor B, properdin, MBL, MASP-1, MASP-2 or biologically active fragments of any of the above. In some embodiments of any of the methods described herein, the complement inhibitor inhibits one or both of the events of C5a-associated anaphylactic activity and/or C5b-associated membrane attack complex assembly.

The compositions may also contain natural or soluble forms of complement inhibitory compounds such as CR1, LEX-CR1, MCP, DAF, CD59, factor H, cobra venom factor, FUT-175, complestatin and K76 COOH.

In some embodiments, the complement inhibitor may be a complement receptor 2 (CR2) factor H (FH) molecule comprising a) a portion of CR2 including CR2 (eg, human CR2) or a fragment thereof; and b) an FH portion comprising FH or a fragment thereof, wherein the CR2-FH molecule or fragment thereof is capable of binding to a CR2 ligand, and wherein the CR2-FH molecule is capable of inhibiting activation of the alternative complement pathway. Examples of CR2-FH fusion proteins are described and illustrated, for example, in International Patent Application Publications No. WO 2007/149567 and WO 2011/143637, the contents of each of which are incorporated herein by reference in their entirety. In some embodiments, the complement inhibitor includes a targeting domain, such as CR2 or an anti-C3d antibody, as described, for example, in International Patent Application Publication No. WO 2011/163412, the disclosure of which is incorporated herein by reference in its entirety. Hybrids of targeting domains with other complement inhibitors, such as CD59, CD55 and factor H-like molecules, can be used in the methods described herein as a complement inhibitor. See WO 2011/163412 above.

In some embodiments of any of the methods described herein, the complement inhibitor is an antagonistic antibody or an antigen-binding fragment thereof. The antibody or antigen binding fragment thereof may be selected from the group consisting of a humanized antibody, a recombinant antibody, a diabody, a chimerized or chimeric antibody, a monoclonal antibody, a deimmunized antibody, a fully human antibody, a single chain antibody, an Fv fragment, an Fd fragment, a Fab fragment, a Fab fragment ' and fragment F(ab')2.

In some embodiments of any of the methods described herein, the antagonist antibody is an anti-C5 antibody, such as eculizumab. In some embodiments, the antagonist antibody is pexelezumab, a C5-binding fragment of an anti-C5 antibody.

In some embodiments of any of the methods described herein, the complement inhibitor is selected from the group consisting of MB12/22, MB12/22-RGD, ARC187, ARC1905, SSL7, and OmCI.

In some embodiments of any of the methods described herein, a subset of protein biomarkers associated with aHUS, from which a practitioner can determine the concentration of one or more (e.g., two, three, four, five, six, seven, eight, nine, 10, or more) biomarkers may be Ba, thrombomodulin, VCAM-1, TNFR1, F1+2, D-dimer, CXCL10, IL-6, clusterin, TIMP-1, FABP-1, B2M and cystatin C.

In yet another aspect, the invention provides a matrix including a plurality of binding agents, wherein each binding agent of the plurality has a unique address on the matrix, wherein the matrix includes no more than 500 unique addresses, wherein each binding agent of the plurality binds to a distinct biological protein analyte, and where the matrix includes connecting

- 8 044587 agents that bind to four or more analyte proteins listed in table. 1, for example, selected from the group consisting of a proteolytic fragment of complement component factor B (e.g. Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand factor (vWF), soluble CD40 ligand (sCD40L), fragment F1+2 of prothrombin, D-dimer, CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1-beta, IL-12 p70, complement component C5a, b2-microglobulin (b2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, vascular endothelial cell growth factor (VEGF), IL-6, albumin, IL- 8 and CCL5. The matrix is suitable for any of the methods described herein. In some embodiments, the array is a protein chip. In some embodiments, each array address represents a well of an assay plate. In some embodiments, each array address is a particle (eg, a bead) having a binding agent immobilized thereon.

As used herein, the term binding agent includes any natural, synthetic, or genetically engineered agent, such as a protein that binds to an antigen (eg, the aHUS protein biomarker). Binding agents may be or be derived from natural antibodies. The binding protein or agent may function similarly to an antibody by binding to a specific antigen to form a complex. Binding agents or proteins may include isolated antigen-binding antibody fragments.

In some embodiments, the matrix includes antibodies that bind to at least two (e.g., at least three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) analyzed proteins. For example, the matrix may include binding agents/antibodies that bind to at least two (e.g., three, four, five, six, seven, eight, nine, 10, 11, 12, or 13) complement factor B proteolytic fragments (e.g. Ba or Bb), soluble C5b9 (sC5b9), C5a, thrombomodulin, VCAM-1, prothrombin F1+2 fragment, D-dimer, sTNFRl, b2-microglobulin (b2M) clusterin, cystatin C, TIMP-1 and protein Fatty acid binding 1 (FABP-1).

In some embodiments, the matrix includes no more than 200 (eg, no more than 175, 150, 125, 100, 9o, 80, 70, 60, 50, 45, 40, 35, 30, 25, or 20) unique addresses.

In yet another aspect, the invention provides a diagnostic kit comprising one or more of any of the matrices described herein, and optionally instructions for (a) obtaining and/or processing a biological sample (eg, in a biological fluid) from an individual; and/or (b) measuring one or more analytes in biological samples (eg, biological fluid) from an individual.

In another aspect, the invention provides a diagnostic kit comprising (a) an assay plate; and (b) at least three binding agents, each binding agent capable of binding to a different biological analyte, wherein the analytes are the substances presented in table. 1, for example, selected from the group consisting of a proteolytic fragment of complement component factor B (e.g. Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand factor (vWF), soluble CD40 ligand (sCD40L), fragment F1+2 of prothrombin, D-dimer, CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1-beta, IL-12 p70, complement component C5a, b2-microglobulin (b2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, vascular endothelial cell growth factor (VEGF), IL-6, albumin, IL- 8 and CCL5. In some embodiments, the diagnostic kit includes one or more means for measuring vWF activity in human plasma.

In another aspect, the invention provides a method for diagnosing an individual as having or at risk of developing atypical hemolytic uremic syndrome (aHUS). The method includes measuring in a biological fluid the concentration of at least two protein biomarkers associated with aHUS, selected from the group consisting of a proteolytic fragment of complement factor B (for example, Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin, VCAM- 1, von Willebrand factor (vWF), soluble CD40 ligand (sCD40L), prothrombin F1+2 fragment, D-dimer, CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1-beta, IL- 12 p70, complement component C5a, b2-microglobulin (b2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, factor vascular endothelial cell growth (VEGF), IL-6, albumin, IL-8 and CCL5. A body fluid is a fluid obtained from an individual suspected of having aHUS or at risk of developing it. According to the methods, an increased concentration compared to the concentration in a normal control biological fluid of the same type of at least one of Ba, sC5b-9, C5a, SCD40L, prothrombin F1+2 fragment, D-dimer, thrombomodulin, VCAM- 1, vWF, FABP-1, e2M, clusterin, cystatin C, TIMP-1, albumin, NGAL, CXCL10, CXCL9, IL-18, TNFR1, VCAM-1, MCP-1,

- 9 044587

VEGF, CCL5, IL-6 or IFNy indicates that an individual has aHUS or is at risk of developing it. In some embodiments, at least two aHUS-related biomarkers may be selected from Table 1. 11, i.e. at least two (e.g., three, four, five, six, seven, eight, nine, 10, 11, 12 or 13) of a complement factor B proteolytic fragment (e.g., Ba or Bb), soluble C5b9 (sC5b9) , C5a, thrombomodulin, VCAM-1, prothrombin F1+2 fragment, D-dimer, sTNFRl, b2-microglobulin (b2M), clusterin, cystatin C, TIMP-1, and fatty acid binding protein 1 (FABP-1).

As used herein, the term normal when used to modify the term individual or individual refers to an individual or group of individuals who does not have a particular disease or condition (eg, aHUS) and is not suspected of having or being at risk of developing diseases or conditions. The term normal is also used herein to qualify a biological sample or sample (eg, a biological fluid) isolated from a normal or healthy person or individual (or group of such individuals), such as a normal control sample or a normal control biological fluid.

In yet another aspect, the invention relates to a method for determining whether a patient is experiencing the first acute manifestation of atypical hemolytic uremic syndrome (aHUS). The method includes measuring one or both of a D-dimer concentration (e.g., a plasma D-dimer concentration) and a fatty acid binding protein 1 (FABP-1) concentration (e.g., a urinary FABP-1 concentration), wherein an increase in the D-dimer concentration dimer relative to the D-dimer concentration in the normal control sample and an increase in FABP-1 concentration relative to the FABP-1 concentration in the normal control sample indicates that the patient is experiencing the first acute manifestation of aHUS. In some embodiments, the increase in one or both D-dimer and FABP-1 may represent a significant increase.

In another aspect, the invention relates to a method of treating atypical hemolytic uremic syndrome (aHUS), the method comprising administering to an individual suffering from, suspected of having, or at risk of developing aHUS, a complement inhibitor (eg, a complement component C5 inhibitor) in an amount and at a frequency sufficient to induce a physiological change in at least one (e.g., at least two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24 or 25) aHUS protein biomarkers, wherein the physiological change is selected from the group consisting of (a) a decrease in concentration compared to the concentration in a sample of the same type of biological fluid obtained from an individual prior to treatment with an inhibitor of at least one of CXCL10, MCP-1, TNFR1, IFN-γ, complement factor B proteolytic fragment (eg, Ba or Bb), soluble C5b9 (sC5b9), prothrombin fragment F1+2 , d-dimer, thrombomodulin, VCAM-1, von Willebrand factor (vWF), complement component C5a, sC5b9, b2-microglobulin (b2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin, CXCL10, CXCL9 and KIM-1; or (b) increasing the concentration of CCL5 compared to the concentration in a sample of the same type of biological fluid obtained from the individual before treatment with the inhibitor. In some embodiments, the at least one aHUS-related biomarker may be selected from table. 11, i.e. at least one (e.g., two, three, four, five, six, seven, eight, nine, 10, 11, 12, or 13) of a complement factor B proteolytic fragment (e.g., Ba or Bb), soluble C5b9 (sC5b9), C5a, thrombomodulin, VCAM-1, prothrombin F1+2 fragment, D-dimer, sTNFRl, β2-microglobulin (β2M), clusterin, cystatin C, TIMP-1, and fatty acid binding protein 1 (FABP- 1).

In yet another aspect, the invention provides a method of treating atypical hemolytic uremic syndrome (aHUS) with a complement inhibitor in a manner sufficient to induce a physiological change in at least two protein biomarkers associated with aHUS. The method includes (a) determining the concentration of at least two protein biomarkers associated with aHUS in a biological fluid obtained from an individual, where the protein biomarkers associated with aHUS are selected from the group consisting of CXCL10, MCP-1, TNFR1, IFN-γ, IL-6, complement factor B proteolytic fragment (eg, Ba or Bb), soluble C5b9 (sC5b9), prothrombin F1+2 fragment, D-dimer, thrombomodulin, VCAM-1, von Willebrand factor (vWF) , complement component C5a, b2-microglobulin (b2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin, CXCL9, KIM-1 and CCL5; and (b) administering to an individual suffering from, suspected of having, or at risk of developing aHUS, a complement inhibitor in an amount and at a frequency sufficient to induce a physiological change in at least each of two (2) protein biomarkers associated with aHUS, wherein the physiological change is selected from the group consisting of (a) a decrease in concentration compared to the concentration in a sample of a biological fluid of the same type obtained from the individual before treatment with an inhibitor of at least one of CXCL10, MCP-1, TNFR1 , IFN-γ, IL-6, proteo

- 10 044587 lytic fragment of factor B component of complement (for example, Ba or Bb), soluble C5b9 (sC5b9), fragment F1+2 of prothrombin, D-dimer, thrombomodulin, VCAM-1, von Willebrand factor (vWF), complement component C5a, β2-microglobulin (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin, CXCL9 or KIM-1; and (b) increasing the concentration of CCL5 in a biological fluid obtained from the individual compared to the concentration in a sample of the same type of biological fluid obtained from the individual prior to initiation of treatment with the inhibitor. The method may also include determining whether physiological changes have occurred. In some embodiments, at least two biomarkers associated with aHUS may be selected from table. 11, i.e. at least two (e.g., three, four, five, six, seven, eight, nine, 10, 11, 12 or 13) of a complement factor B proteolytic fragment (e.g., Ba or Bb), soluble C5b9 (sC5b9) , C5a, thrombomodulin, VCAM-1, prothrombin F1+2 fragment, D-dimer, sTNFRl, b2-microglobulin (b2M), clusterin, cystatin C, TIMP-1 and fatty acid binding protein 1 (FABP-1).

In some embodiments, the methods may further include the step of measuring the concentration of at least two separate aHUS-associated protein biomarkers in a biological fluid, wherein the aHUS-associated protein biomarkers are selected from the group consisting of a complement factor B proteolytic fragment ( e.g. Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand factor (vWF), soluble CD40 ligand (sCD40L), prothrombin F1+2 fragment, D-dimer, CXCL10, MCP-1, TNFR1 , IFN-γ, ICAM-1, IL-1-beta, IL-12 p70, complement component C5a, b2-microglobulin (b2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, vascular endothelial cell growth factor (VEGF), IL-6, albumin, IL-8 and CCL5. The biological fluid is obtained from the individual. In some embodiments, at least two biomarkers associated with aHUS may be selected from table. 11, i.e. at least two (e.g., three, four, five, six, seven, eight, nine, 10, 11, 12 or 13) of a complement factor B proteolytic fragment (e.g., Ba or Bb), soluble C5b9 (sC5b9) , C5a, thrombomodulin, VCAM-1, prothrombin F1+2 fragment, D-dimer, sTNFRl, b2-microglobulin (b2M), clusterin, cystatin C, TIMP-1 and fatty acid binding protein 1 (FABP-1).

In some embodiments, any of the methods described herein may include determining whether at least two (e.g., at least three, four, five, six, seven, eight, nine, 10, 11) occur , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) physiological changes. In some embodiments, the concentration of at least two of IFN-γ, ICAM-1, IL-1 beta and IL-12 p70 is reduced. In some embodiments, the concentration of both Ba and sC5b9 is reduced. In some embodiments, the concentration (eg, urinary concentration) of each of C5a and sC5b9 is reduced. In some embodiments of any of the methods described herein, concentrations (e.g., urinary concentrations) of at least two (e.g., at least three, four, five, six, or all) of B2M, clusterin, cystatin C, NAG, TIMP-1, NGAL and FABP-1 are decreased. In some embodiments, concentrations (eg, urinary concentrations) of CXCL10, CXCL9, and/or KIM-1 are reduced. In some embodiments, the concentrations (eg, plasma concentrations) of one or both D-dimer and F1+2 are reduced. In some embodiments, the concentrations (eg, serum and/or plasma concentrations) of at least two (eg, at least three or all) of SCD40L, prothrombin F1+2 fragment, and D-dimer are reduced. In some embodiments, thrombomodulin, VCAM-1, and/or vWF concentrations are reduced. In some embodiments, the concentrations (eg, serum concentrations) of CXCL10, MCP-1, and TNFR1 are reduced. In some embodiments, concentrations (eg, serum concentrations) of at least two (eg, at least three, four, or all) of IFN-γ, ICAM-1, IL-1-beta, and IL-12 p70 are decreasing. In some embodiments, the at least two physiological changes may be a decrease in the concentration of at least two aHUS-related biomarkers selected from table. 11, i.e. at least two (e.g., three, four, five, six, seven, eight, nine, 10, 11, 12 or 13) of a complement factor B proteolytic fragment (e.g., Ba or Bb), soluble C5b9 (sC5b9) , C5a, thrombomodulin, VCAM-1, prothrombin F1+2 fragment, D-dimer, sTNFRl, b2-microglobulin (b2M) clusterin, cystatin C, TIMP-1, and fatty acid binding protein 1 (FABP-1).

In some embodiments of any of the methods described herein, the concentration of Ba (eg, plasma concentration of Ba) is reduced by at least 10% 6 weeks after the start of treatment. In some embodiments of any of the methods described herein, the concentration of Ba (eg, plasma concentration of Ba) is reduced by at least 30% 12 weeks after the start of treatment. In some embodiments of any of the methods described herein, the concentration of C5a (eg, the concentration of C5a in urine) is reduced by at least 40% 3 weeks after the start of treatment. In some variants

- 11 044587 By implementing any of the methods described herein, the concentration of C5a (eg, the concentration of C5a in the urine) is reduced by at least 70% 6 weeks after the start of treatment. In some embodiments of any of the methods described herein, the concentration of C5b-9 (eg, the concentration of C5b-9 in urine or plasma) is reduced by at least 50% 3 weeks after the start of treatment. In some embodiments of any of the methods described herein, the F1+2 concentration (eg, plasma F1+2 concentration) is reduced by at least 20% 6 weeks after the start of treatment. In some embodiments of any of the methods described herein, the D-dimer concentration (eg, plasma D-dimer concentration) is reduced by at least 40% 6 weeks after the start of treatment. In some embodiments of any of the methods described herein, the thrombomodulin concentration (eg, serum thrombomodulin concentration) is reduced by at least 20% 12 weeks after the start of treatment. In some embodiments of any of the methods described herein, the concentration of VCAM-1 (eg, serum concentration of VCAM-1) is reduced by at least 20% 12 weeks after the start of treatment.

In some embodiments of any of the methods described herein, a complement inhibitor is administered to an individual in an amount and frequency sufficient to induce a physiological change in three or more biomarkers associated with aHUS. In some embodiments, a complement inhibitor is administered to an individual in an amount and frequency sufficient to induce a physiological change in at least four aHUS-related biomarkers. In some embodiments, a complement inhibitor is administered to an individual in an amount and frequency sufficient to induce a physiological change in at least five aHUS-related biomarkers. In some embodiments, a complement inhibitor is administered to an individual in an amount and frequency sufficient to induce a physiological change in at least 10 aHUS-related biomarkers. In some embodiments, a complement inhibitor is administered to an individual in an amount and frequency sufficient to induce a physiological change in at least 15 or more aHUS-related biomarkers.

In some embodiments, a physiological change in at least two (e.g., at least three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18 19, 20, 21, 22, 23, 24, or 25 or more) biomarkers associated with aHUS occurs within two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, six weeks, two months, nine weeks, or three months or more after administration (eg, chronic administration) of the inhibitor.

In some embodiments, the concentration of at least one (e.g., at least two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) aHUS-associated protein biomarkers are reduced by at least 5% (e.g., by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70%) after administration of the inhibitor.

In some embodiments, the concentration of at least one (e.g., at least two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) aHUS-associated protein biomarkers are reduced by up to 50% (e.g., 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39 , 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14 , 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1%) of the normal concentration of protein biomarkers after administration of one or more doses of the inhibitor.

In some embodiments of any of the methods described herein, the concentration of FABP-1 (e.g., urinary FABP-1 concentration) is reduced by at least 80% (e.g., 85, 90, 95, or up to 100%) after administration of a human complement inhibitor (eg, anti-C5 antibody). In some embodiments of any of the methods described herein, the concentration of cystatin-C (e.g., urinary cystatin-C concentration) is reduced by at least 80% (e.g., 85, 90, 95, 99, or up to 100 %) after administration of a human complement inhibitor (for example, anti-C5 antibodies). In some embodiments of any of the methods described herein, the clusterin concentration (e.g., urinary clusterin concentration) is reduced by at least 80% (e.g., 85, 90, 95, 98, or up to 100%) following administration human complement inhibitor (for example, antibodies against C5). In some embodiments of any of the methods described herein, the concentration of a factor B proteolytic moiety (e.g., Ba) is reduced by at least 10% (e.g., 15, 20, 25, 30, or 40%) following administration of the inhibitor human complement (for example, antibodies against C5). In some embodiments of any of the methods described herein, the concentration of sTNFRl is reduced by at least 80% (eg, 85, 90, or more) following administration of a human complement inhibitor (eg, anti-C5 antibody). In some embodiments of any of the methods described herein, the concentration of thrombomodulin or sVCAM-1 is reduced by at least 80% (e.g., 85, 90, 95, or up to 100%) following administration of a human complement inhibitor (e.g., antibodies against C5). In some embodiments of any of the methods described herein, the concentration of one or both F1+2 or D-dimer is reduced by at least

80% (eg, 85%, 90%, 95% or more) after administration of a human complement inhibitor (eg, anti-C5 antibody).

In some embodiments of any of the methods described herein, the concentration of at least one (e.g., at least two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 or 25 ) of the protein biomarkers associated with aHUS are normalized after inhibitor administration. In some embodiments, concentrations (e.g., urinary concentrations) of at least three of β2-microglobulin (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1) , CXCL10, CXCL9, and KIM-1 are normalized.

As used herein, the term normalized or similar grammatical terms when used in the context of the effect of complement inhibitor treatment on the concentration or activity of a protein biomarker aHUS refers to the concentration or activity of a protein biomarker measured in a biological fluid that is 50% (e.g., 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1%) of the mean concentration range or aHUS protein biomarker activity when measured in a sample of the same type of biological fluid obtained from a group of healthy individuals (normal individuals). For example, treating a patient with aHUS with a complement inhibitor may normalize elevated urinary clusterin concentrations within, for example, 20% of the normal average urinary clusterin concentration range. In some embodiments, treatment with a complement inhibitor should restore the urinary clusterin concentration to within the normal range of the average urinary clusterin concentration.

In some embodiments of any of the methods described herein, the individual has been on dialysis at least once (e.g., at least twice, three times, four times, or five times or more) within three months (e.g., 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 week(s) before starting inhibitor treatment. For example, in some embodiments, the individual was on dialysis once for two months before receiving complement inhibitor treatment. In another example, the individual may be an individual who is on dialysis three times over a period of three months immediately before receiving treatment with a complement inhibitor. In some embodiments of any of the methods described herein, the concentrations of one or more of TNFR1, Ba, thrombomodulin, F1+2 fragment, and sC5b9 are increased relative to the concentration in a healthy individual. In some embodiments of any of the methods described herein, the concentrations of one or more of b2M, sC5b9, C5a, cystatin C, clusterin, TIMP-1, and NGAL are increased relative to the concentration (eg, urinary concentration) in a healthy individual.

In some embodiments of any of the methods described herein, an individual (eg, a human) experiences a first acute manifestation of aHUS. For example, prior to initiation of treatment with a complement inhibitor, an individual may have elevated concentrations of one or both of D-dimer and FABP-1 relative to normal concentrations.

In some embodiments of any of the methods described herein, the individual (e.g., a human) is an individual who has aHUS but is considered to be in clinical remission (e.g., the individual is an individual who has normal levels of platelets or other hematologic markers, such as LDH or haptoglobin). In some embodiments, such an individual is an individual having elevated levels of one or more of the aHUS biomarkers described herein, including, but not limited to, one or more of Ba, D-dimer, VCAM-1, and fragment 1+ 2 prothrombins.

It is understood that in any of the methods described herein, the concentration and/or activity of one or more aHUS protein biomarkers can be determined. For example, in some embodiments, a practitioner may measure vWF activity in a biological sample obtained from an individual as a surrogate for the concentration of vWF (or other protein biomarkers) in the sample. Methods for assessing the relative activity of aHUS protein biomarkers presented in Table. 1 are known in the art.

As discussed in detail herein (eg, in the worked examples), aHUS is a genetic, life-threatening disease involving chronic complement dysregulation. Patients affected by the disease suffer, among other things, from thrombotic microangiopathy (TMA), which can lead to stroke and kidney failure. Eculizumab, an anti-C5 antagonist antibody, has been shown to significantly reduce TMA, normalize platelet levels, and also improve renal function in patients with aHUS. However, even with the clear and robust clinical benefit of complement inhibitor treatment in aHUS patients, some patients still have elevated levels of several aHUS protein biomarkers during treatment. For example, the present inventors have discovered that in some patients, levels of a proteolytic fragment of complement factor B (eg, Ba or Bb) (eg, in plasma) do not normalize after treatment with an anti-C5 antagonist antibody. In addition, in some patients, levels of prothrombin F1+2 fragment, D-dimer, thrombomodulin, VCAM-1, TNFR1, and CXCL10 decrease but do not normalize over time. Although the invention is not limited to any particular theory or mechanism of action, these observations suggest that for some patients, low levels of inflammation and coagulopathy may persist even with complement inhibitor treatment. Thus, the disclosure covers methods in which a complement inhibitor is administered in combination with a second therapy aimed at low levels of persistent inflammation in certain aHUS patients.

Thus, in yet another aspect, the invention relates to a method for treating atypical hemolytic uremic syndrome (aHUS). The method includes administering (e.g., chronic administration) to an individual (e.g., a human) suffering from aHUS, suspected of having aHUS, or at risk of developing it, a therapeutically effective amount of a complement inhibitor (e.g., a complement component C5 inhibitor) and a therapeutically effective amount of (i) an anticoagulant, (ii) a fibrinolytic agent; (iii) an anti-inflammatory agent; or (iv) an inhibitor of IL-6, IL-8, CXCL-9, IL-18 or VEGF. In some embodiments, two complement inhibitors (eg, a C5 inhibitor and a C3 inhibitor, such as an anti-factor B antibody, an anti-C3 antibody, or an anti-C3b antibody) may be used. In some embodiments, during discontinuation of treatment with a C5 inhibitor, a complement component C3 inhibitor may be administered to the patient for a period of time sufficient to reduce the upstream alternative pathway activation.

In some embodiments, the methods may include monitoring the status of one or more aHUS biomarkers and determining whether to initiate a second type of treatment (in addition to complement inhibitor treatment) or modify the dosage regimen of one or more second type of treatment agents administered to the aHUS patient. For example, during treatment (eg, chronic treatment) with a complement inhibitor, the concentration of one or more protein biomarkers associated with aHUS may be measured in one or more body fluids obtained from the individual. If the concentration of one or more protein biomarkers does not normalize and/or remains elevated, the practitioner may choose to administer to the individual one or more additional secondary agents (eg, anti-inflammatory agents) to address any pathophysiological effects resulting from the elevated biomarker concentration.

The complement inhibitor may be any of the inhibitors described herein. In some embodiments of any of the methods described herein, the inhibitor is an antibody or an antigen-binding fragment thereof, a small molecule, a polypeptide, a polypeptide analog, a peptidomimetic, or an aptamer. In some embodiments, the inhibitor may be an inhibitor that inhibits one or more of complement components C1, C2, C3, C4, C5, C6, C7, C8, C9, factor D, factor B, properdin, MBL, MASP1, MASP- 2, or biologically active fragments of any of the above. In some embodiments of any of the methods described herein, the complement inhibitor inhibits one or both of the events of C5a-associated anaphylactic activity and/or C5b-associated membrane attack complex assembly.

The compositions may also contain natural or soluble forms of complement inhibitory compounds such as CR1, LEX-CR1, MCP, DAF, CD59, factor H, cobra venom factor, FUT-175, complestatin and K76 COOH.

In some embodiments of any of the methods described herein, the complement inhibitor is an antagonistic antibody or an antigen-binding fragment thereof. The antibody or antigen binding fragment thereof may be selected from the group consisting of a humanized antibody, a recombinant antibody, a diabody, a chimerized or chimeric antibody, a monoclonal antibody, a deimmunized antibody, a fully human antibody, a single chain antibody, an Fv fragment, an Fd fragment, a Fab fragment, a Fab '-fragment and F (ab') ₂ -fragment.

In some embodiments of any of the methods described herein, the antagonist antibody is an anti-C5 antibody, such as eculizumab. In some embodiments, the antagonist antibody is pexelezumab, a C5-binding fragment of an anti-C5 antibody.

In some embodiments of any of the methods described herein, the complement inhibitor is selected from the group consisting of MB12/22, MB12/22-RGD, ARC187, ARC1905, SSL7, and OmCI.

In some embodiments, the anticoagulant is selected from the group consisting of coumarin, heparin, factor Xa inhibitor, and thrombin inhibitor. Examples of anticoagulants include, for example, warfarin (Coumadin), aspirin, heparin, phenindione, fondaparinux, idraparinux, and thrombin inhibitors (eg, argatroban, lepirudin, bivalirudin, or dabigatran).

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In some embodiments, the fibrinolytic agent is selected from the group consisting of ancrod, ε-aminocaproic acid, antiplasmin-a ₁ , prostacyclin, and defibrotide.

In some embodiments, the anti-inflammatory agent is an anti-cytokine agent, such as an antagonistic antibody (or an antigen-binding fragment thereof) or a soluble cytokine receptor that binds to an inflammatory cytokine and inhibits the activity of the cytokine. The anti-cytokine agent may be, for example, a TNF inhibitor (eg, an anti-TNF antibody or soluble TNF receptor protein) or an anti-CD20 agent.

Anti-inflammatory agents also include, for example, steroids (eg, dexamethasone), nonsteroidal anti-inflammatory drugs (NSAIDs) (eg, indomethacin, naproxen, sulindac, diclofenac, aspirin, flurbiprofen, oxaprozin, salsalate, difunizal, piroxicam, etodolac, meclofenamate, ibuprofen, fenoprofen , ketoprofen, meloxicam, tolmetin, magnesium choline salicylate, COX-2 inhibitors, TNF-alpha antagonists (etanercept, adalimumab, infliximab, golimumab), disease-modifying antirheumatic drugs (DMARDS) (eg, sulfasalazine, methotrexate), cyclosporine, retinoids, and corticosteroids.

In yet another aspect, the invention relates to a method for determining whether the concentration of one or more protein biomarkers associated with aHUS is increased in a patient suffering from atypical hemolytic uremic syndrome (aHUS), suspected of having aHUS, or at risk of developing it, where the method includes (i) measuring, in a biological sample obtained from the patient, the concentration of each of at least two biomarkers associated with aHUS from Table. 11 (below), i.e. selected from the group consisting of factor B proteolytic fragment, C5a, soluble C5b-9 (SC5b-9), soluble TNFR1 (sTNFRl), soluble VCAM-1 (sVCAM-1), thrombomodulin, prothrombin fragments 1 and 2 (F1+2 ), D-dimer, clusterin, TIMP-1, FABP-1, beta-2-microglobulin (e2m) and cystatin-C; and (ii) determining whether the patient has an elevated concentration of each of at least two biomarkers associated with aHUS, compared to a normal control concentration of the same at least two biomarkers. In some embodiments, at least two protein biomarkers associated with aHUS are measured using an immunoassay, such as an enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay (RIA). The biological fluid may be, for example, blood, a fraction of blood (eg plasma or serum) or urine. It is understood that any combinations of any two or more (eg, three, four, five, six, seven, eight, nine, 10, 11 or 12) of these aHUS biomarkers can be measured and analyzed in accordance with the methods described herein.

In another aspect, the invention relates to a method for diagnosing a patient as suffering from atypical hemolytic uremic syndrome (aHUS) (or confirming the diagnosis of aHUS, for example, when the patient meets two or more inclusion criteria discussed in Example 1), where the method includes (i) measuring in a biological sample obtained from a patient suspected of having aHUS or at risk of developing aHUS, the concentration of each of at least two biomarkers associated with aHUS selected from the group consisting of the proteolytic fragment of factor B, C5a, soluble C5b-9 (SC5b -9), soluble TNFR1 (sTNFRl), soluble VCAM-1 (sVCAM-1), thrombomodulin, prothrombin fragments 1 and 2 (F1+2), D-dimer, clusterin, TIMP-1, FABP-1, beta-2 microglobulin (b2t) and cystatin-C; and (ii) diagnosing the patient as having aHUS (or confirming the diagnosis of aHUS) if the concentration of each of at least two biomarkers associated with aHUS is increased compared to the normal control concentration of the same at least two biomarkers. In some embodiments, at least two protein biomarkers associated with aHUS are measured using an immunoassay, such as an enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay (RIA). The biological fluid may be, for example, blood, blood fractions (eg plasma or serum) or urine. It is understood that any combinations of any two or more (eg, three, four, five, six, seven, eight, nine, 10, 11 or 12) of these aHUS biomarkers can be measured and analyzed in accordance with the methods described herein.

The normal reference concentration when used in any of the methods described herein may be (or may be based on), for example, the concentration of a given aHUS-associated protein biomarker in a biological sample or biological samples obtained from one or more (e.g. from two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, 40 or more) healthy individuals. In some embodiments, the normal biomarker reference concentration may be (or may be based on), for example, the biomarker concentration in a pooled sample obtained from two or more (e.g., two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, 40 or more) healthy individuals. In some embodiments of any of the methods described herein, the pooled samples may be from healthy individuals or at least from individuals who do not have or are not expected to have (are not at risk for developing) aHUS . For example, determining whether an individual is an individual who has aHUS may involve comparing the measured concentration of one or more complement protein components (e.g., Table 1 or 11) in a biological sample (or several different types of biological samples) obtained from the patient , and comparison of the measured concentration with the average concentration of the same proteins in pooled samples of healthy individuals. Such reference concentrations in healthy subjects may be, in some embodiments, a range of values, or a median, or an average value derived from the range.

In some embodiments, the concentration of at least one biomarker associated with aHUS is measured in two or more types of biological fluid. In some embodiments, the concentration of the first of the at least two aHUS protein biomarkers is measured in one type of biological fluid, and the concentration of the second of at least two aHUS protein biomarkers is measured in a second type of fluid.

In some embodiments of any of the methods described herein, the concentration of a proteolytic fragment of factor B is measured. The fragment may be, for example, Ba. The biological sample may be a plasma sample. As described in table. 11, the normal control concentration of Ba may be less than 1000 ng/ml. The normal control Ba concentration may be less than 600 ng/ml. The normal control Ba concentration can range from 300 to 600 ng/ml.

In some embodiments, the Ba concentration in a biological sample is considered elevated when it is at least twice the normal control Ba concentration. In some embodiments, the Ba concentration in a biological sample is considered elevated when it is at least five times the normal control Ba concentration. In some embodiments, the Ba concentration in a biological sample is considered elevated when it is at least 1500 ng/ml. In some embodiments, the Ba concentration in a biological sample is considered elevated when it is at least 2500 ng/ml.

In some embodiments of any of the methods described herein, the concentration of C5a is measured.

The biological sample in which C5a is measured may be a urine sample. And in some embodiments, the normal control concentration of C5a is less than 2 ng/mg urine creatinine. In some embodiments, the normal control concentration of C5a is less than 1 ng/mg urine creatinine. In some embodiments, the normal control concentration of C5a is between 0 and 0.7 ng/mg urine creatinine.

In some embodiments of any of the methods described herein, the concentration of C5a in a biological sample is considered elevated when it is at least twice the normal control concentration of C5a. In some embodiments, the concentration of C5a in a biological sample is considered elevated when it is at least ten times the normal control concentration of C5a. In some embodiments, the concentration of C5a in a biological sample is considered elevated when it is at least forty times higher than the normal control concentration of C5a. In some embodiments, the concentration of C5a in a biological sample is considered elevated when it is at least 5 ng/mg urine creatinine. In some embodiments, the concentration of C5a in a biological sample is considered elevated when it is at least 9 ng/mg urine creatinine.

In some embodiments of any of the methods described herein, the concentration of sC5b-9 is measured. The biological sample in which sC5b-9 is measured may be a urine sample. And in some embodiments, the normal control concentration of sC5b-9 is less than 2 ng/mg urine creatinine. The normal reference concentration of sC5b-9 may be less than 1 ng/mg urine creatinine. In some embodiments, the normal control concentration of sC5b-9 is between 0 and 0.6 ng/mg urine creatinine.

In some embodiments of any of the methods described herein, the concentration of sC5b-9 in a biological sample is considered elevated when it is at least ten times the normal control concentration of sC5b-9. In some embodiments, the concentration of sC5b-9 in a biological sample is considered elevated when it is at least fifty times the normal control concentration of sC5b-9. In some embodiments, the concentration of sC5b-9 in a biological sample is considered elevated when it is at least one hundred times higher than the normal control concentration of sC5b-9. In some embodiments, the concentration of sC5b-9 in a biological sample is considered elevated when it is at least 20 ng/mg urine creatinine. In some embodiments, the concentration of sC5b-9 in a biological sample is considered elevated when it is at least 30 ng/mg urine creatinine.

In some embodiments of any of the methods described herein, the concentration of sTNFRl is measured. The biological sample in which sTNFRl is measured may be a serum sample. And in some embodiments, the normal control concentration of sTNFRl is less than 2000 pg/ml. In some embodiments, the normal control sTNFRl concentration is less than 1500 pg/ml. In some embodiments, the normal control concentration of sTNFRl is between 400 and 1500 pg/ml.

In some embodiments of any of the methods described herein, the concentration of sTNFRl in a biological sample is considered elevated when it is at least twice the normal control concentration of sTNFRl. In some embodiments, the sTNFRl concentration in a biological sample is considered elevated when it is at least five times the normal control sTNFRl concentration. In some embodiments, the concentration of sTNFRl in a biological sample is considered elevated when it is at least fifteen times higher than the normal control concentration of sTNFRl.

In some embodiments, the concentration of sTNFRl in a biological sample is considered elevated when it is at least 10,000 μg/ml. In some embodiments, the concentration of sTNFRl in a biological sample is considered elevated when it is at least 15,000 μg/ml.

In some embodiments of any of the methods described herein, the concentration of sVCAM-1 is measured. The biological sample in which sVCAM-1 is measured may be a serum sample. And in some embodiments, the normal control concentration of sVCAM-1 is less than 500 ng/ml. In some embodiments, the normal control concentration of sVCAM-1 is less than 300 ng/ml. In some embodiments, the normal control concentration of sVCAM-1 is 100 to 500 ng/ml.

In some embodiments of any of the methods described herein, the concentration of sVCAM-1 in a biological sample is considered elevated when it is at least 10% higher than the normal control concentration of sVCAM-1. In some embodiments, the concentration of sVCAM-1 in a biological sample is considered elevated when it is at least 30% higher than the normal control concentration of sVCAM-1. In some embodiments, the concentration of sVCAM-1 in a biological sample is considered elevated when it is at least 50% higher than the normal control concentration of sVCAM-1. In some embodiments, the concentration of sVCAM-1 in a biological sample is considered elevated when it is at least 550 ng/ml. In some embodiments, the concentration of sVCAM-1 in a biological sample is considered elevated when it is at least 650 ng/ml.

In some embodiments of any of the methods described herein, the concentration of thrombomodulin is measured. The biological sample in which thrombomodulin is measured may be a plasma sample. And in some embodiments, the normal reference thrombomodulin concentration is less than 5 ng/ml. In some embodiments, the normal reference thrombomodulin concentration is less than 3 ng/ml. In some embodiments, the normal reference thrombomodulin concentration is 2 to 6 ng/ml.

In some embodiments of any of the methods described herein, the concentration of thrombomodulin in a biological sample is considered elevated when it is at least 10% higher than the normal control concentration of thrombomodulin. In some embodiments, the thrombomodulin concentration in a biological sample is considered elevated when it is at least 30% higher than the normal control thrombomodulin concentration. In some embodiments, the thrombomodulin concentration in a biological sample is considered elevated when it is at least 50% higher than the normal control thrombomodulin concentration. In some embodiments, the concentration of thrombomodulin in a biological sample is considered elevated when it is at least 8 ng/ml. In some embodiments, the thrombomodulin concentration in a biological sample is considered elevated when it is at least 10 ng/ml.

In some embodiments of any of the methods described herein, the concentration of F1+2 is measured. The biological sample in which F1+2 is measured may be a plasma sample. And in some embodiments, the normal control concentration of F1+2 is less than 400 pmol/L. In some embodiments, the normal control concentration of F1+2 is less than 300 pmol/L. In some embodiments, the normal control concentration of F1+2 is from 50 to 400 pmol/L.

In some embodiments of any of the methods described herein, the concentration of F1+2 in a biological sample is considered elevated when it is at least 30% higher than the normal control concentration of F1+2. In some embodiments, the F1+2 concentration in a biological sample is considered elevated when it is at least 50% higher than the normal control F1+2 concentration. In some embodiments, the F1+2 concentration in a biological sample is considered elevated when it is at least 100% higher than the normal control F1+2 concentration. In some embodiments, the F1+2 concentration in a biological sample is considered elevated when it is at least 900 pmol/L. In some embodiments, the F1+2 concentration in a biological sample is considered elevated when it is at least 1000 pmol/L.

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In some embodiments of any of the methods described herein, the concentration is measured of D-dimer. The biological sample in which the D-dimer is measured may be a plasma sample. And in some embodiments, the normal control concentration of D-dimer is less than 500 μg/L. In some embodiments, the normal control concentration of D-dimer is less than 400 μg/L. In some embodiments, the normal control concentration of D-dimer is 100 to 500 μg/L.

In some embodiments of any of the methods described herein, the concentration of D-dimer in a biological sample is considered elevated when it is at least twice the normal control concentration of D-dimer. In some embodiments, the D-dimer concentration in a biological sample is considered elevated when it is at least five times the normal control D-dimer concentration. In some embodiments, the D-dimer concentration in a biological sample is considered elevated when it is at least ten times the normal control D-dimer concentration. In some embodiments, the D-dimer concentration in a biological sample is considered elevated when it is at least 1500 μg/L. In some embodiments, the D-dimer concentration in a biological sample is considered elevated when it is at least 2500 μg/L.

In some embodiments of any of the methods described herein, the concentration of clusterin is measured. The biological sample in which clusterin is measured may be a urine sample. And in some embodiments, the normal control clusterin concentration is less than 500 ng/mg urine creatinine. A normal reference clusterin concentration may be, for example, less than 400 ng/mg urine creatinine. In some embodiments, the normal reference clusterin concentration is between 0 and 500 ng/mg urine creatinine.

In some embodiments of any of the methods described herein, the concentration of clusterin in a biological sample is considered elevated when it is at least twice the normal control concentration of clusterin. In some embodiments, the concentration of clusterin in a biological sample is considered elevated when it is at least five times the normal control concentration of clusterin. In some embodiments, the concentration of clusterin in a biological sample is considered elevated when it is at least ten times the normal control concentration of clusterin. In some embodiments, the concentration of clusterin in a biological sample is considered elevated when it is at least 900 ng/mg urine creatinine. In some embodiments, the concentration of clusterin in a biological sample is considered elevated when it is at least 1200 ng/mg urine creatinine.

In some embodiments of any of the methods described herein, the concentration of TIMP-1 is measured. The biological sample in which TIMP-1 is measured may be a urine sample. And in some embodiments, the normal control concentration of TIMP-1 is less than 10 ng/mg urine creatinine. In some embodiments, the normal control concentration of TIMP-1 is less than 5 ng/mg urine creatinine. In some embodiments, the normal control concentration of TIMP-1 is between 0 and 10 ng/mg urine creatinine.

In some embodiments of any of the methods described herein, the concentration of TIMP-1 in a biological sample is considered elevated when it is at least twice the normal control concentration of TIMP-1. In some embodiments, the concentration of TIMP-1 in a biological sample is considered elevated when it is at least ten times the normal control concentration of TIMP-1. In some embodiments, the concentration of TIMP-1 in a biological sample is considered elevated when it is at least twenty times the normal control concentration of TIMP-1. In some embodiments, the concentration of TIMP-1 in a biological sample is considered elevated when it is at least 15 ng/mg urine creatinine. In some embodiments, the concentration of TIMP-1 in a biological sample is considered elevated when it is at least 20 ng/mg urine creatinine.

In some embodiments of any of the methods described herein, the concentration of FABP-1 (also referred to herein as L-FABP-1) is measured. The biological sample in which C5a is measured may be a urine sample. And in some embodiments, the normal control concentration of FABP-1 is less than 20 ng/mg urine creatinine. In some embodiments, the normal reference FABP-1 concentration is less than 15 ng/mg urine creatinine. In some embodiments, the normal reference FABP-1 concentration is between 0 and 20 ng/mg urine creatinine.

In some embodiments of any of the methods described herein, the concentration of FABP-1 in a biological sample is considered elevated when it is at least twice the normal control concentration of FABP-1. In some embodiments

- 18 044587 the concentration of FABP-1 in a biological sample is considered elevated when it is at least ten times higher than the normal control concentration of FABP-1. In some embodiments, the concentration of FABP-1 in a biological sample is considered elevated when it is at least twenty times the normal control concentration of FABP-1. In some embodiments, the concentration of FABP-1 in a biological sample is considered elevated when it is at least 40 ng/mg urine creatinine. In some embodiments, the concentration of FABP-1 in a biological sample is considered elevated when it is at least 50 ng/mg urine creatinine.

In some embodiments of any of the methods described herein, the concentration of e2m is measured. The biological sample in which e2m is measured may be a urine sample. And in some embodiments, the normal control concentration of e2m is less than 5 μg per mg of urine creatinine. In some embodiments, the normal reference concentration of e2m is less than 3 μg per mg of urine creatinine. In some embodiments, the normal control concentration of e2m is between 0 and 5 μg per mg of urine creatinine.

In some embodiments of any of the methods described herein, the concentration of e2m in a biological sample is considered elevated when it is at least twice the normal control concentration of e2m. In some embodiments of any of the methods described herein, the concentration of e2m in a biological sample is considered elevated when it is at least ten times the normal control concentration of e2m. In some embodiments, the e2m concentration in a biological sample is considered elevated when it is at least twenty times the normal control e2m concentration. In some embodiments, the concentration of e2m in a biological sample is considered elevated when it is at least 15 μg per mg of urine creatinine. In some embodiments, the e2m concentration in a biological sample is considered elevated when it is at least 20 μg per mg of urine creatinine.

In some embodiments of any of the methods described herein, the concentration of cystatin-C is measured. The biological sample in which cystatin-C is measured may be a urine sample. And in some embodiments, the normal reference cystatin-C concentration is less than 400 ng/mg urine creatinine. In some embodiments, the normal reference cystatin-C concentration is less than 300 ng/mg urine creatinine. In some embodiments, the normal control concentration of cystatin-C is between 0 and 400 ng/mg urine creatinine.

In some embodiments of any of the methods described herein, the concentration of cystatin-C in a biological sample is considered elevated when it is at least twice the normal control concentration of cystatin-C. In some embodiments, the concentration of cystatin-C in a biological sample is considered elevated when it is at least ten times the normal control concentration of cystatin-C. In some embodiments, the concentration of cystatin-C in a biological sample is considered elevated when it is at least twenty times the normal control concentration of cystatin-C. In some embodiments, the concentration of cystatin-C in a biological sample is considered elevated when it is at least 900 ng/mg urine creatinine. In some embodiments, the concentration of cystatin-C in a biological sample is considered elevated when it is at least 1200 ng/mg urine creatinine.

In some embodiments of any of the methods described herein, the concentrations of two or more of the factor B proteolytic fragments, C5a and sC5b-9, are measured. In some embodiments of any of the methods described herein, concentrations of C5a and sC5b-9 are measured. In some embodiments of any of the methods described herein, the concentrations of sVCAM-1 and thrombomodulin are measured. In some embodiments of any of the methods described herein, F1+2 and D-dimer concentrations are measured. In some embodiments of any of the methods described herein, the concentrations of two or more of clusterin, TIMP-1, e2m, FABP-1, and cystatin-C are measured.

In yet another aspect, the invention provides a method for assessing the level of alternative pathway activation in a patient suffering from aHUS, suspected of having aHUS, or at risk of developing aHUS, before, during, or after treatment with a complement inhibitor, such as an anti-C5 antibody. The method includes measuring the concentration of a proteolytic fragment of factor B (eg, Ba or Bb) in a biological sample obtained from a patient who has been treated with a complement inhibitor (eg, an inhibitor of human complement component C5, such as an anti-C5 antibody).

In yet another aspect, the invention relates to a method for determining whether a patient responds to treatment with a complement inhibitor (for example, is characterized by a reduction in the risk of developing thrombosis or is characterized by a reduction in the number, frequency or presence of thrombotic microangiopathy),

- 19 044587 wherein the method includes measuring the concentration of one or more biomarkers of thrombosis or coagulation shown in table. 1 or 11, for example, F1+2, D-dimer, vWF or thrombomodulin, in a biological sample obtained from a patient at increased risk of developing thrombotic microangiopathy (TMA), suffering from or suspected of having this disease, who was treated with a complement inhibitor; and determining whether the patient is responding to treatment if the concentration of one or more biomarkers in the biological sample decreases compared to the concentration of one or more biomarkers in a biological sample of the same type obtained from the patient before treatment with a complement inhibitor, or determining whether the patient is not responding to treatment if the concentration of one or more biomarkers in a biological sample is not reduced compared to the concentration of one or more biomarkers in a biological sample of the same type obtained from a patient before treatment with a complement inhibitor. In some embodiments, the patient has aHUS, is suspected of having aHUS, or is at risk of developing aHUS.

In another aspect, the invention relates to a method for determining whether a patient with aHUS responds to treatment with a complement inhibitor, the method comprising measuring the concentration of one or more biomarkers of terminal complement activation presented in table. 1 or 11, for example, C5a and/or SC5b-9, in a biological sample obtained from a patient suspected or at risk of developing aHUS who was treated with a complement inhibitor (for example, an anti-C5 antibody); and determining whether the patient is responding to treatment if the concentration of one or more biomarkers in the biological sample is reduced compared to the concentration of one or more biomarkers in a biological sample of the same type obtained from the patient before treatment with a complement inhibitor, or determining whether the patient is not responding to treatment if the concentration of one or more biomarkers in a biological sample is not reduced compared to the concentration of one or more biomarkers in a biological sample of the same type obtained from a patient before treatment with a complement inhibitor. Thus, this method can be used to evaluate or monitor blockade of the terminal complement cascade in an aHUS patient who has been treated with a complement inhibitor. In embodiments in which the patient is unresponsive or less responsive to treatment, the method may also include changing the dose size or dosing frequency of the complement inhibitor or selecting a different complement inhibitor (eg, a C3 activation inhibitor) for use in treating the patient.

In another aspect, the invention relates to a method for determining whether a patient with aHUS responds to treatment with a complement inhibitor, the method comprising measuring the concentration of one or more biomarkers of vascular inflammation or endothelial activation listed in table. 1 or 11, for example, sTNFRl, sVCAM-1 or thrombomodulin, in a biological sample obtained from a patient suffering from aHUS, suspected of having it or at risk of developing it; and determining whether the patient is responding to treatment if the concentration of one or more biomarkers in the biological sample is reduced compared to the concentration of one or more biomarkers in a biological sample of the same type obtained from the patient before treatment with a complement inhibitor, or determining whether the patient is not responding to treatment if the concentration of one or more biomarkers in a biological sample is not reduced compared to the concentration of one or more biomarkers in a biological sample of the same type obtained from a patient before treatment with a complement inhibitor. Thus, the method can be used to assess or monitor vascular inflammation in a patient with aHUS who has been treated with a complement inhibitor. In embodiments in which the patient is unresponsive or less responsive to treatment, the method may also include changing the dose size or dosing frequency of the complement inhibitor or selecting a different complement inhibitor (eg, a C3 activation inhibitor) for use in treating the patient.

In another aspect, the invention relates to a method for determining whether a patient with aHUS responds to treatment with a complement inhibitor, the method comprising measuring the concentration of one or more biomarkers of kidney damage listed in table. 1 or 11, for example, clusterin, TIMP-1, FABP-1, e2m and/or cystatin-C, in a biological sample obtained from a patient suffering from aHUS, suspected of having it or at risk of developing it; and determining whether the patient is responding to treatment if the concentration of one or more biomarkers in the biological sample is reduced compared to the concentration of one or more biomarkers in a biological sample of the same type obtained from the patient before treatment with a complement inhibitor, or determining whether the patient is not responding to treatment if the concentration of one or more biomarkers in a biological sample is not reduced compared to the concentration of one or more biomarkers in a biological sample of the same type obtained from a patient before treatment with a complement inhibitor. Thus, the method can be used to assess or monitor kidney damage in an aHUS patient who has been treated with a complement inhibitor. In embodiments in which the patient is unresponsive or less responsive to treatment, the method may also include changing the dose size or dosing frequency of the complement inhibitor or selecting a different complement inhibitor (eg, a C3 activation inhibitor) for use in treating the patient.

The inventors also discovered that in patients with aHUS there is a relative increase in concentrations

- 20 044587 biomarkers of terminal complement activation C5a and sC5b-9 (eg, urinary concentrations) are much higher than the relative increase in levels of markers of alternative pathways of complement activation (eg, Ba) in these patients. This means that the median concentrations of C5a and sC5b-9 in aHUS patients were 45 and 305 times higher, respectively, than the median concentrations of these markers in normal healthy individuals, while the median concentration of Ba was only approximately 5 times higher than median concentration of Ba in normal healthy people. Without being limited to any particular theory or mechanism of action, the inventors believe that the ratio of terminal complement activation to alternative pathway activation is a useful diagnostic tool for aHUS. Thus, in another aspect, the invention provides a method for diagnosing aHUS or confirming a diagnosis of aHUS, where the method includes comparing the level of terminal complement activation (eg, sC5b-9 or C5a) relative to the level of activation of an upstream alternative pathway (eg, Ba or Bb) (by relative to normal healthy individuals), where a higher level of terminal activation relative to alternative pathway activation is an indicator that the patient suffers from aHUS. For example, the ratio indicative of aHUS may be, for example, approximately 45:5 or 305:5 fold of induction of terminal complement activation to fold of induction of alternative pathway activation. In addition, the inventors believe that this relationship may be useful in distinguishing aHUS from other complement-related diseases, such as thrombotic thrombocytopenic purpura (TTP), which may not induce such a difference in the levels of terminal complement activation and upstream alternative pathway activation.

Polypeptide, peptide, and protein are used interchangeably and mean any chain of amino acids linked by peptide bonds, regardless of length or post-translational modification. The proteins described herein may contain or be wild-type proteins or may be variants that have no more than 50 (e.g., no more than one, two, three, four, five, six, seven, eight, nine, ten , 12, 15, 20, 25, 30, 35, 40, or 50) conservative amino acid substitutions. Conservative substitutions generally include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.

As used herein, percentage (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to amino acids in a reference sequence after sequence alignment and the introduction of gaps, if necessary, to achieve the maximum percentage sequence identity. Alignment for the purpose of determining percent sequence identity can be achieved in a variety of ways known to those skilled in the art, for example, using readily available software such as BLAST software. Appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared, can be determined using known methods.

As used herein, the term antibody includes both full-length antibodies and antigen-binding fragments of full-length antibodies. Full-length antibodies include various antibody isotypes, including IgM, IgG, IgA, IgD, and IgE antibodies. The term antibody includes polyclonal antibody, monoclonal antibody, chimerized or chimeric antibody, humanized antibody, primatized antibody, deimmunized antibody, and fully human antibody. The antibody may be derived from, or may be derived from, any of a variety of species, for example, mammals such as humans, non-human primates (eg, orangutans, baboons or chimpanzees), horses, cattle, pigs, sheep, goats, dogs , cats, rabbits, guinea pigs, gerbils, hamsters, rats and mice. The antibody may be a purified or recombinant antibody.

As used herein, the term antibody fragment, antigen binding fragment, or similar terms refers to an antibody fragment that retains the ability to bind to a target antigen (eg, human C5) and inhibit the activity of the target antigen. Such fragments include, for example, a single chain antibody, a single chain Fv fragment (scFv), an Fd fragment, a Fab fragment, a Fab' fragment, or an F(ab') ₂ fragment. An scFv fragment is a single-stranded polypeptide chain that includes the variable regions of both the heavy and light chains of the antibody from which the scFv is derived. In addition, intrabodies, minibodies, tribodies and diabodies are also included in the definition of antibody and are compatible with use in the methods described herein. See, for example, the articles by Todorovska et al. (2001) J Immunol Methods 248(1):4766; Hudson and Kortt (1999) J Immunol Methods 231(1):177–189; Poljak (1994) Structure 2(12):1121–1123; and Rondon and Marasco (1997) Annual Review of Microbiology 51:257-283, the disclosures of each of which are incorporated herein by reference in their entirety. Bispecific antibodies (including DVD-Ig antibodies; see below) are also included within the term antibody. Bispecific antibodies are preferably human monoclonal antibodies or humanized antibodies that have binding specificity for at least two different antigens.

As used herein, the term antibody also includes, for example, single-domain antibodies, such as camel 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; Riechmann et al. (1999) J Immunol Meth 231:25-38; publication of PCT applications No. WO 94/04678 and WO 94/25591; and US Pat. No. 6,005,079, all of which are incorporated herein by reference in their entirety. In some embodiments, the invention provides single domain antibodies comprising two VH domains with modifications such that single domain antibodies are formed.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the field to which this disclosure pertains. Preferred methods and substances are described below, although methods and substances similar or equivalent to those described herein may also be used in the practice or testing of the methods and compositions described herein. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

Other features and advantages of the present invention, such as methods for treating complement disorders in an individual, will be apparent from the following description, examples, and claims.

Brief description of drawings

Fig. 1A is a scatter plot showing the concentration of C5a (in ng/mg urine creatine) in the urine of aHUS patients both before eculizumab treatment (Pre-Tx) and various numbers of weeks after initiation of eculizumab treatment. Urinary C5a concentrations were also measured in the urine of normal, healthy individuals (NORM).

Fig. 1B is a scatter plot showing the concentration of sC5b-9 (in ng/mg urine creatine) in the urine of aHUS patients both before eculizumab treatment (Pre-Tx) and various numbers of weeks after initiation of eculizumab treatment. Urinary C5b9 concentrations were also measured in the urine of normal, healthy individuals (NORM).

Fig. 1C is a scatter plot showing the concentration of complement component Ba (in ng/mL) in the plasma of aHUS patients both before eculizumab treatment (Pre-Tx) and various numbers of weeks after initiation of eculizumab treatment. Ba concentration was also measured in the plasma of normal, healthy individuals (normal).

Fig. 1D is a bar graph depicting the mean percentage (%) reduction in urinary C5a levels (Y-axis) over time in aHUS patients (N=26) after initiation of eculizumab treatment. The x-axis represents the weeks of the aHUS patient's assessment visit after the start of treatment, for example, V3 represents the patient's assessment visit 3 weeks after the start of treatment.

Fig. 1E is a bar graph depicting the mean percentage (%) reduction in urinary sC5b-9 levels (Y-axis) over time in aHUS patients (N=23) after initiation of eculizumab treatment. The x-axis represents the weeks of the aHUS patient's assessment visit after the start of treatment, for example, V3 represents the patient's assessment visit 3 weeks after the start of treatment.

Fig. 1F is a bar graph depicting the mean percent (%) decrease in plasma Ba levels (Y-axis) over time in aHUS patients (N=35) after initiation of eculizumab treatment. The x-axis represents the weeks of the aHUS patient's assessment visit after the start of treatment, for example, V3 represents the patient's assessment visit 3 weeks after the start of treatment.

Fig. 2A-2C are histograms depicting the percentage of aHUS patients who achieve normalized concentrations of urinary C5a (Fig. 2A), urinary sC5b9 (Fig. 2B), and plasma Ba (Fig. 2C), at baseline (pre-eculizumab treatment) and at varying numbers of weeks after initiation of eculizumab treatment.

Fig. 3A is a scatter plot showing prothrombin fragment 1+2 concentrations (in pmol/L) in the plasma of aHUS patients both before eculizumab treatment (Pre-Tx) and various numbers of weeks after initiation of eculizumab treatment. The concentration of F1+2 was also measured in the plasma of normal, healthy individuals (normal).

Fig. 3B is a scatter plot showing D-dimer concentration (in μg/L) in plasma of aHUS patients both before eculizumab treatment (Pre-Tx) and various numbers of weeks after initiation of eculizumab treatment. D-dimer concentration was also measured in the plasma of normal, healthy individuals (normal).

Fig. 3C is a bar graph depicting the mean percentage (%) reduction in plasma prothrombin F1+2 fragment levels (Y-axis) over time in aHUS patients after initiation of eculizumab treatment. The x-axis represents the weeks of the aHUS patient's assessment visit after the start of treatment, for example, V3 represents the patient's assessment visit 3 weeks after

- 22 044587 start of treatment.

Fig. 3D is a bar graph depicting the mean percentage (%) reduction in plasma d-dimer levels (y-axis) over time in aHUS patients after initiation of eculizumab treatment. The x-axis represents the weeks of the aHUS patient's assessment visit after the start of treatment, for example, V3 represents the patient's assessment visit 3 weeks after the start of treatment.

Fig. 4A and 4B are histograms depicting the percentage of aHUS patients who achieve normalized plasma prothrombin F1+2 fragment (FIG. 4A) and plasma D-dimer (FIG. 4B) concentrations at baseline (before eculizumab treatment) and after various numbers of weeks after initiation of eculizumab treatment.

Fig. 5A is a scatter plot showing thrombomodulin concentrations (in ng/mL) in plasma (EDTA-treated plasma) of aHUS patients both before eculizumab treatment (Pre-Tx) and various numbers of weeks after initiation of eculizumab treatment. Plasma thrombomodulin concentrations were also measured in the plasma of normal, healthy individuals (normal). EOS denotes the results of the analysis of samples obtained at the end of the study.

Fig. 5B is a scatter plot showing the concentration of VCAM-1 (in ng/mL) in the serum of aHUS patients both before eculizumab treatment (Pre-Tx) and various numbers of weeks after initiation of eculizumab treatment. Serum VCAM-1 concentration was also measured in the serum of normal, healthy individuals (normal pool). EOS denotes the results of the analysis of samples obtained at the end of the study.

Fig. 5C is a scatter plot showing vWF activity (in mU/mL) in plasma (EDTA-treated plasma) of aHUS patients both before eculizumab treatment (Pre-Tx) and various numbers of weeks after initiation of eculizumab treatment. vWF activity was also measured in the plasma of normal, healthy individuals (normal). EOS denotes the results of the analysis of samples obtained at the end of the study.

Fig. 6A and 6B are histograms depicting the percentage of aHUS patients who achieve normalized plasma thrombomodulin concentrations (Fig. 6A) and plasma vWF activity levels (Fig. 4B), at baseline (before eculizumab treatment) and various numbers of weeks after initiation of treatment eculizumab.

Fig. 6C is a bar graph depicting the mean percentage (%) reduction in plasma thrombomodulin levels (Y-axis) over time in aHUS patients (N=33) after initiation of eculizumab treatment. The x-axis represents the weeks of the aHUS patient's assessment visit after the start of treatment, for example, V3 represents the patient's assessment visit 3 weeks after the start of treatment.

Fig. 6D is a bar graph depicting the mean percentage (%) reduction in serum VCAM-1 levels (Y-axis) over time in aHUS patients (N=36) after initiation of eculizumab treatment. The x-axis represents the weeks of the aHUS patient's assessment visit after the start of treatment, for example, V3 represents the patient's assessment visit 3 weeks after the start of treatment.

Fig. 7A is a scatter plot showing the concentration of TNFR1 (in pg/mL) in the serum of aHUS patients both before eculizumab treatment (Pre-Tx) and various numbers of weeks after initiation of eculizumab treatment. Serum TNFR1 concentration was also measured in the serum of normal, healthy individuals (normal pool). EOS denotes the results of the analysis of samples obtained at the end of the study.

Fig. 7B is a bar graph depicting the percentage of aHUS patients who achieve normalized serum TNFR1 concentrations at baseline (before eculizumab treatment) and various numbers of weeks after initiation of eculizumab treatment.

Fig. 8A is a scatter plot showing the concentration of cystatin C (CysC) (in ng/mg urine creatine) in the urine of aHUS patients both before eculizumab treatment (Pre-Tx) and various numbers of weeks after initiation of eculizumab treatment. Urinary CysC concentrations were also measured in the urine of normal, healthy individuals (NORM).

Fig. 8B is a scatter plot showing the concentration of B2M (in μg/mg urine creatine) in the urine of aHUS patients both before eculizumab treatment (Pre-Tx) and various numbers of weeks after initiation of eculizumab treatment. Urinary concentrations of B2M were also measured in the urine of normal, healthy individuals (NORM).

Fig. 8C is a scatter plot showing the concentration of NGAL (in ng/mg urine creatine) in the urine of aHUS patients both before eculizumab treatment (Pre-Tx) and various numbers of weeks after initiation of eculizumab treatment. Urinary NGAL concentrations were also measured in the urine of normal, healthy individuals (NORM).

Fig. 9A-9E are a series of histograms depicting the mean levels of several aHUS protein biomarkers in aHUS patients who were on dialysis (dialysis) compared with those aHUS patients who were not on dialysis (non-dialysis) before study entry. 044587 tion described in this document. In fig. 9A depicts the mean serum TNFR1 concentration (in pg/ml); in fig. 9B shows the average concentration of B2M in urine (in μg/mg urine creatine); in fig. 9C shows the concentration of Ba in plasma (in ng/ml); in fig. 9D depicts the concentration of sC5b9 in urine (in ng/mg urine creatine); and in fig. 9E shows the concentration of C5a in urine (in ng/ml).

Fig. 10A is a scatter plot showing serum TNFR1 concentration (in pg/mL) in aHUS patients who are clinically stable (clinical remission) (without TMA), and in those aHUS patients who continue to have elevated haptoglobin and LDH levels (and decreased platelet count) (Others), both initially and 1 to 2.5 weeks after initiation of eculizumab treatment. Serum TNFR1 concentrations in normal, healthy individuals (normal) are also shown.

Fig. 10B-10E are a series of histograms, each depicting the concentration of a given biomarker in patients with normal hematological markers LDH and haptoglobin (normal patients or patients considered to be in clinical remission), in patients with abnormal (increased) hematological markers (abnormal patients or patients with active presentation of aHUS) and in healthy individuals (normal). In fig. 10B depicts plasma Ba levels (ng/ml) in these populations of individuals. In fig. 10C depicts serum VCAM-1 levels (ng/ml) in these populations of individuals. In fig. 10E depicts plasma levels of prothrombin fragments 1+2 (pmol/L) in these populations of individuals, and FIG. 10D depicts the plasma D-dimer level (in µg/L). P values for comparisons between matched groups are shown in the figures.

Fig. 10F-10I are a series of histograms, each depicting the concentration of a given biomarker in patients with normal platelet levels (normal patients), in patients with abnormal (reduced) platelet levels (abnormal patients), and in healthy individuals (normal patients). In fig. 10F depicts plasma Ba levels (ng/ml) in these populations of individuals. In fig. 10G depicts serum VCAM-1 levels (ng/ml) in these populations of individuals. In fig. 10I depicts plasma levels of prothrombin fragments 1+2 (pmol/L) in these populations of individuals, and FIG. 10H shows the plasma D-dimer level (in µg/L). P values for comparison of the respective groups are shown in the figures.

Fig. 11 is a bar graph depicting the mean percentage change in serum TNFR1 and urinary clusterin, C5a and C5b9 levels of those aHUS patients who achieve a complete TMA response and those patients who still experience complications of TMA (incomplete response). (As noted and discussed in detail in the worked examples, complete TMA response relates to normalization of hematologic parameters and preservation of renal function.)

Fig. 12 is a bar graph depicting the average percentage change in plasma Ba levels of aHUS patients treated with eculizumab who have a complete TMA response and those aHUS patients treated with eculizumab who do not have a complete response (other).

Fig. 13 is a bar graph depicting the mean change from baseline (initial visit before initiation of eculizumab treatment) platelet count (10 ⁹ /L) at 12-17 weeks and 26 weeks after initiation of eculizumab treatment in aHUS patients with normalized plasma Ba levels compared with patients with persistently elevated plasma levels of Va. The p values for each observation are also presented in the figure.

Fig. 14A-14D are a series of histograms reflecting the finding that several biomarkers associated with aHUS are elevated in aHUS patients with abnormal TMA markers at baseline. In fig. 14A depicts the concentration of cystatin C (CysC) (in ng/mg urine creatine) in the urine of aHUS patients with a normal platelet count (>150,000 per μL of blood) compared with patients with a reduced platelet count (<150,000 per μL of blood). In fig. 14B depicts the concentration of clusterin (in ng/mg urine creatine) in the urine of aHUS patients with a normal platelet count (>150,000 per μL of blood) compared with patients with a reduced platelet count (<150,000 per μL of blood). In fig. 14C depicts the concentration of VCAM-1 in the serum of aHUS patients with normal LDH levels compared to patients with elevated LDH levels. In fig. 14D depicts the concentration of D-dimer (in μg/L) in the blood plasma of aHUS patients with normal LDH levels compared to patients with elevated LDH levels. The p values for each observation are presented in the figures.

Fig. 15 is a bar graph depicting the level of cystatin C (ng/mg urine creatine) in the urine of aHUS patients at baseline who received plasma therapy (re-RT, N=23), no plasma therapy (no RT, N=3), or normal patients (N=9).

Fig. 16 is a series of histograms depicting the mean change in baseline eGFR (ml/min/1.73 m ² ) in aHUS patients who achieve normalized levels of various biomarkers (plasma Ba, serum VCAM-1, plasma F1+2, plasma d-dimer and urinary cystatin C) after eculizumab treatment compared with aHUS patients, in whom the concentrations of these biomarkers remain elevated.

Fig. 17A-17E are a series of histograms showing the discovery that some

- 24 044587 biomarkers associated with aHUS are elevated in patients with aHUS before treatment with a complement inhibitor, regardless of whether patients were treated with plasmapheresis (PE) or plasma infusion (PI). In fig. 17A depicts the concentration of the factor B proteolytic fragment Ba (in ng/ml) in the plasma of normal healthy volunteers (NHV), aHUS patients treated with PE or PI (PE/PI), or aHUS patients not treated with PE/PI (no PE /PI). In fig. 17B depicts the concentration of sTNFRl (in pg/ml) in the serum of normal healthy volunteers (NHV), aHUS patients treated with PE or PI (PE/PI), or aHUS patients not treated with PE/PI (no PE/PI). In fig. 17C depicts the concentration of sVCAM-1 (in ng/ml) in the serum of normal healthy volunteers (NHV), aHUS patients treated with PE or PI (PE/PI), or aHUS patients not treated with PE/PI (no PE/PI ). In fig. 17D depicts the concentration of D-dimer (in μg/L) in the plasma of normal healthy volunteers (NHV), aHUS patients treated with PE or PI (PE/PI), or aHUS patients not treated with PE/PI (no PE/PI ). In fig. 17E depicts the concentration of cystatin-C (in ng/mg urine creatinine) in the urine of normal healthy volunteers (NHV), aHUS patients treated with PE or PI (PE/PI), or aHUS patients not treated with PE/PI (no PE /PI). The p values for each observation are presented in the figures.

Fig. 18A-18E are a series of histograms reflecting the finding that several biomarkers associated with aHUS are elevated in aHUS patients before complement inhibitor treatment, regardless of the patients' platelet levels. In fig. 18A depicts the concentration of the factor B proteolytic fragment Ba (in ng/ml) in the plasma of normal healthy volunteers (NHV), aHUS patients having a normal platelet count (>150x109), or aHUS patients having a reduced platelet count (<150x109). In fig. 18B depicts the concentration of sTNFRl (in pg/ml) in the serum of normal healthy volunteers (NHV), aHUS patients having a normal platelet count (>150x109), or aHUS patients having a reduced platelet count (<150x109). In fig. 18C depicts the concentration of sVCAM-1 (in ng/ml) in the serum of normal healthy volunteers (NHV), aHUS patients having a normal platelet count (>150x109), or aHUS patients having a reduced platelet count (<150x109). In fig. 18D depicts the concentration of D-dimer (in μg/L) in the plasma of normal healthy volunteers (NHV), aHUS patients having a normal platelet count (>150x109), or aHUS patients having a reduced platelet count (<150x109). In fig. 18E depicts the concentration of cystatin-C (in ng/mg urine creatinine) of normal healthy volunteers (NHV), aHUS patients having a normal platelet count (>150x109), or aHUS patients having a reduced platelet count (<150x109). The p values for each observation are presented in the figures.

Fig. 19A-19E are a series of histograms reflecting the finding that several biomarkers associated with aHUS are elevated in aHUS patients before complement inhibitor treatment, independent of haptoglobin (Hp) or lactate dehydrogenase (LDH) levels. In fig. 19A depicts the concentration of the factor B proteolytic fragment Ba (in ng/ml) in the plasma of normal healthy volunteers (NHV), aHUS patients having normal LDH and Hp levels, or aHUS patients having elevated (abnormal) HP/LDH. In fig. 19B depicts the concentration of sTNFRl (in pg/ml) in the serum of normal healthy volunteers (NHV), aHUS patients having normal LDH and HP levels, or aHUS patients having elevated (abnormal) HP/LDH. In fig. 19C depicts the concentration of sVCAM-1 (in ng/ml) in the serum of normal healthy volunteers (NHV), aHUS patients having normal LDH and HP levels, or aHUS patients having elevated (abnormal) HP/LDH. In fig. 19D depicts the concentration of D-dimer (in μg/L) in the plasma of normal healthy volunteers (NHV), aHUS patients having normal LDH and HP levels, or aHUS patients having elevated (abnormal) HP/LDH. In fig. 19E depicts the concentration of cystatin-C (in ng/mg urine creatinine) in the urine of normal healthy volunteers (NHV), aHUS patients having normal LDH and Hp levels, or aHUS patients having elevated (abnormal) HP/LDH. The p values for each observation are presented in the figures.

Fig. 20A, 20B are box-and-whisker plots depicting the longitudinal effects of chronic eculizumab treatment on terminal complement activation in aHUS patients. In fig. 20A shows the time course of urinary C5a concentration (ng/mg urine creatinine) of aHUS patients following eculizumab treatment compared to urinary C5a concentration of normal healthy volunteers (NHV). In fig. 20B shows the time course of urinary sC5b-9 concentration (ng/mg urine creatinine) of aHUS patients following eculizumab treatment compared to urinary sC5b-9 concentration of normal healthy volunteers (NHV). Box-and-whisker plots represent the median, 25th and 75th percentiles, and range. * First time point at which levels were significantly reduced from baseline (BL); P values relative to baseline at each time point were calculated using a restricted maximum likelihood approach based on repeated measures (mixed model). P values versus NHV were calculated using the Wilcoxon signed rank test.

Fig. 21A-21C are box-and-whisker plots depicting the longitudinal

- 25 044587 New effects of long-term treatment with eculizumab on the concentration of protein biomarkers associated with kidney damage in patients with aHUS. In fig. 21A shows the time course of urinary FABP-1 concentration (ng/mg urine creatinine) of aHUS patients following eculizumab treatment compared to urinary FABP-1 concentration of normal healthy volunteers (NHV). In fig. 21B shows the time course of urinary cystatin-C concentrations (ng/mg urine creatinine) of aHUS patients following eculizumab treatment compared to urinary FABP-1 concentrations of normal healthy volunteers (NHV). In fig. 21C shows the time course of the urinary clusterin concentration (ng/mg urine creatinine) of aHUS patients after eculizumab treatment compared to the urinary FABP-1 concentration of normal healthy volunteers (NHV). Box-and-whisker plots represent the median, 25th and 75th percentiles, and range. * First time point at which levels were significantly reduced from baseline (BL); P values relative to baseline at each time point were calculated using a restricted maximum likelihood approach based on repeated measures (mixed model). P values versus NHV were calculated using the Wilcoxon signed rank test.

Fig. 22 is a box-and-whisker plot depicting the longitudinal effect of chronic eculizumab treatment on alternative complement pathway activation in aHUS patients. The time course of Ba concentration (ng/mL) in the plasma of aHUS patients after eculizumab treatment is shown along with the plasma Ba concentration of normal healthy volunteers (NHV). Box-and-whisker plots represent the median, 25th and 75th percentiles, and range. * First time point at which levels were significantly reduced from baseline (BL); P values relative to baseline at each time point were calculated using a restricted maximum likelihood approach based on repeated measures (mixed model). P values versus NHV were calculated using the Wilcoxon signed rank test.

Fig. 23A-23C are box-and-whisker plots depicting the longitudinal effects of chronic eculizumab treatment on the concentrations of protein biomarkers associated with inflammation, endothelial cell activation, and tissue damage in patients with aHUS. In fig. 23A shows the time course of sTNFRl concentration (pg/mL) in the serum of aHUS patients after treatment with eculizumab compared to the sTNFRl concentration in the serum of normal healthy volunteers (NHV). In fig. 23B shows the time course of sVCAM-1 concentration (ng/mL) in the serum of aHUS patients following eculizumab treatment compared to the analyte concentration in the serum of normal healthy volunteers (NHV). In fig. 23C shows the time course of thrombomodulin concentration (ng/mL) in the plasma of aHUS patients after eculizumab treatment compared with the analyte concentration in the plasma of normal healthy volunteers (NHV). Box-and-whisker plots represent the median, 25th and 75th percentiles, and range. * First time point at which levels were significantly reduced from baseline (BL); P values relative to baseline at each time point were calculated using a restricted maximum likelihood approach based on repeated measures (mixed model). P values versus NHV were calculated using the Wilcoxon signed rank test.

Fig. 24A, 24B are box-and-whisker plots depicting the longitudinal effects of chronic eculizumab treatment on the concentrations of protein biomarkers associated with thrombosis and coagulation in patients with aHUS. In fig. 24A shows the time course of F1+2 concentration (pmol/L) in the plasma of aHUS patients following eculizumab treatment compared to the analyte concentration in the plasma of normal healthy volunteers (NHV). In fig. 24B shows the time course of D-dimer concentration (μg/L) in the plasma of aHUS patients following eculizumab treatment compared to the analyte concentration in the plasma of normal healthy volunteers (NHV). Box-and-whisker plots represent the median, 25th and 75th percentiles, and range. * First time point at which levels were significantly reduced from baseline (BL); P values relative to baseline at each time point were calculated using a restricted maximum likelihood approach based on repeated measures (mixed model). P values versus NHV were calculated using the Wilcoxon signed rank test.

Overview of the complement system.

The complement system works in conjunction with the body's other immunological systems to protect against invading cellular and viral pathogens. There are at least 25 complement proteins that are found in a collective collection of plasma proteins and membrane cofactors. Plasma proteins constitute approximately 10% of vertebrate serum globulins. Complement components exert their immune defense functions by interacting in a series of complex but precise enzymatic cleavage and membrane binding events. The resulting complement cascade leads to the formation of products with opsonic, immunoregulatory and lytic functions. A brief overview of the biological activities associated with complement activation is offered, for example, in the 16th edition of the Merck manual.

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The complement cascade develops along the classical pathway, the alternative pathway, or the lectin pathway. These pathways have many components and, although they differ in the initial stages, they converge and share the same terminal complement components (C5 through C9) responsible for the activation and destruction of target cells.

The classical pathway (CP), as a rule, is initiated by antibody recognition of an antigenic site on a target cell and binding to it. The alternative pathway (AP) may be independent of the antibody and may be initiated by certain molecules on the surface of the pathogen. In addition, the lectin pathway is typically initiated by the binding of mannose-bound lectin (MBL) to high-mannose substrates. These pathways converge at a point where complement component C3 is cleaved by an active protease to produce C3a and C3b. Other complement-activating pathways may act later in the sequence of events leading to different aspects of complement function. C3a is an anaphylatoxin. C3b binds to bacterial and other cells, as well as certain viruses and immune complexes, and tags them for removal from circulation. This opsonic function of C3b is generally considered to be most important for the anti-infective action of the complement system. C3b also forms a complex with other components unique to each pathway to form the classical or alternative C5 convertase, which cleaves complement component C5 (hereafter referred to as C5) into C5a and C5b.

Cleavage of C5 releases biologically active components such as C5a, a potent anaphylatoxin and chemotaxis factor, and C5b, which through a series of protein interactions leads to the formation of the terminal complement lytic complex, C5b-9. C5a and C5b-9 also have pleiotropic properties in activating cells by enhancing the release of downstream inflammatory factors such as hydrolytic enzymes, reactive oxygen species, arachidonic acid metabolites and various cytokines.

C5b, when interacting with C6, C7 and C8, forms the C5b-8 complex on the surface of the target cell. When several C9 molecules bind, a membrane attack complex (MAC, C5b-9, terminal complement complex - TCC) is formed. When enough MACs are incorporated into target cell membranes, the openings they create (MAC pores) mediate rapid osmotic lysis of the target cells. Lower, non-lytic concentrations of MACs may exert other effects. In particular, the incorporation of small amounts of C5b-9 complexes into the membranes of endothelial cells and platelets can cause unfavorable cell activation. In some cases, activation may precede cell lysis.

As mentioned above, C3a and C5a are activated by complement components. This can cause mast cell degranulation, which releases histamine from basophils and mast cells, and other inflammatory mediators, resulting in smooth muscle contraction, increased vascular permeability, leukocyte activation, and other inflammatory phenomena including cellular proliferation leading to cell excess. C5a also functions as a chemotaxis peptide that serves to attract proinflammatory granulocytes to the site of complement activation. C5a receptors are found on the surface of bronchial and alveolar epithelial cells and bronchial smooth muscle cells. C5a receptors are also found on eosinophils, mast cells, monocytes, neutrophils and activated lymphocytes.

Detailed description

As described herein and illustrated in the working examples, the inventors have identified aHUS biomarkers. For example, increased or, in some cases, decreased concentrations of certain proteins have been found to be associated with the presence of aHUS. Likewise, decreased or increased concentration (or activity) of certain proteins in body fluid obtained from an aHUS patient treated with a complement inhibitor indicates that the patient is responding to inhibitor treatment. Accordingly, analysis of the concentration and/or activity level of such proteins can be used to assess, among other things, the risk of developing aHUS, diagnosing aHUS, monitoring the progression or attenuation of aHUS, and/or monitoring response to complement inhibitor treatment.

aHUS protein biomarkers and their applications.

The aHUS protein biomarkers (as well as illustrative biological fluids in which they are found) are presented in Table. 1. Protein sequence associated with the name of each of the biomarkers listed in Table. 1, from GenBank (National Center for Biotechnology Information (NCBI)), as available as of the date of filing of this application, is incorporated herein by reference.

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Table 1

Biomarker Abbr. Tissue source Seq by.* NCBI link Serum Plasma Urine Markers of inflammation/platelet or endothelial cell activation Cytokine ligand 9 (C-X-C motif) CXCL9 X NP_002407.1 Ligand 10 chemokines (C-X-C motif) CXCL-10 X NP_001556.2 Interleukin-1beta IL-Ιβ X NP_000567.1 Interleukin-6 IL-6 X NP_000591.1 Interleukin-8 IL-8 X NP_000575.1 Interleukin-12 p70 IL-12 p70 X NP_000873.2 (p35) NP_002178.2 (p40) Interferongamma IFN-γ X NP_000610.2 Platelet selectin p-selectin X NP_002996.2 Selectin endothelium e-selectin X NP_000441.2 Intercellular adhesion molecule-1 ICAM-1 X NP_000192.2 Vascular cell adhesion molecule-1 VCAM-1 X NP_001069.1 Monocyte chemotaxis protein-1 MSR-1 X NP_002973.1 Vascular endothelial growth factor VEGF X NP_001020537.2 Activation-regulated, expressed and secreted by normal T cells (CCL5) CCL5 X NP_002976.2 Soluble CD40 ligand SCD40L X NP_000065.1** Soluble tumor necrosis factor receptor 1 sTNFRl X NP_001056.1** Interleukin 18 IL-18 X NP_001553.1

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Markers of inflammation/kidney damage Neutrophil gelatinase-associated lipocalin NGAL X NP_005555.2 Kidney damage molecule-1 KIM-1 X NP_001092884.1 Osteopontin OPN X NP_001035147.1 Tissue metalloprotease inhibitor1 TIMP-1 X NP_003245.1 Interleukin-18 IL-18 X Higher Chemokine ligand 9 (motif S-H-S) CXCL9 X Higher Chemokine ligand 10 (motif S-H-S) CXCL10 X Higher Clusterin C.L.U. X NP_001822.3 Cystatin C CyC X NP_000090.1 Albumen A.L.B. X NP_000468.1 Liver fatty acid binding protein L-FABP X NP_001434.1 beta-2microglobin β2Μ X NP_004039.1 Three leaf factor 3 TFF-3 X NP_003217.3 N-acetyl-beta- Dglucosaminidase NAG X NP_000511.2 π-glutathione Stransferase π-GST X NP_000843.1 alphaglutathione Stransferase a-GST X NP 665683.1

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Complement Complement Va Va X SEQ ID NO:1; See also fig. 2 Morley and Campbell (1984) EMBO J 3(1):153-157. Complement SZa Complement C5a Soluble MAC CH50 (hemolysis) Complement C5 D-dimer Prothrombin F1+2 C3a C5a 3C5b9 CH ₅₀ C5 Troc D-dimer F1+2 X X 1boz/turnp X X X X Vanie X X X X SEQ ID NO :2 SEQ ID NO :3 NA NA NP_001726.2 P02671*** Activation fragment 1 (SEQ ID NO:4) corresponds to amino acids 44-198 of SEQ ID NO:6. Activation fragment 2 (SEQ ID NO:5) corresponds to amino acids 199-327 of SEQ ID NO:4. von Willebrand factor vWF X NP_000543.2 Von Willebrand factor activity Activity vWF X is the same Thrombomodulin TM X NP_000352.1

*An NCBI Illustrative Human Sequence Accession Number is provided for each protein biomarker listed in the table.

** The soluble form of the receptor is generated by proteolytic processing of the membrane-bound form of the receptor.

*** UniProtKB (consortium of European Bioinformatics Institute, Cambridge, UK; Swiss Institute of Bioinformatics; Geneva, Switzerland; and Protein Information Resource, Washington, D.C.) designation for human fibrinogen-alpha, which is cleaved by thrombin to form fibrin. D-dimer is a degradation product of fibrin. A description of the transition of fibrinogen into fibrin and into D-dimer as a result of cleavage is presented in the article by Soheir et al. (2009) Blood 113(13):2878-2887, the disclosure of which, at least with respect to D-dimer formation, is incorporated herein in its entirety.

The biological markers provided herein may be used alone or in combination as an indicator, for example, to assess the risk of developing aHUS, diagnose aHUS, determine whether an individual is experiencing a first acute onset of aHUS, monitor the progression or decline of aHUS, and/or control response to treatment with a complement inhibitor or optimization of such treatment. In some embodiments, the individual aHUS protein biomarkers described herein may be used. In some embodiments, at least two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, may be used.

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14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more) aHUS protein biomarkers selected from Table. 1, combined as a panel.

In some embodiments, the aHUS protein biomarkers are selected from a complement factor B proteolytic fragment (e.g., Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand factor (vWF), soluble CD40 ligand (sCD40L), fragment F1+2 prothrombin, D-dimer, CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1-beta, IL-12 p70, complement component C5a, b2-microglobulin (b2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, vascular endothelial growth factor (VEGF), IL-6, albumin, IL-8 and CCL5. The concentration and/or activity of one or more of the biomarkers in the table may be measured. 1 (or any of the subsets of biomarkers specified herein).

In some embodiments, an increase in the concentration of D-dimer relative to the concentration of D-dimer in the normal control sample and an increase in the concentration of FABP-1 relative to the concentration of FABP-1 in the normal control sample indicates that the aHUS patient is experiencing the first acute manifestation of aHUS . In some cases, the increase in one or both of these aHUS protein biomarkers represents a significant increase compared to normal controls.

In some embodiments, increasing the concentration of one or more of TNFR1, Ba, C5b-9, F1+2, B2M, clusterin, TIMP-1, NGAL, CysC, and C5a (see Table 7) in a biological sample obtained from aHUS patient , relative to control analyte concentrations obtained, for example, from a pool of samples from aHUS patients who were not redialyzed, indicates that the patient is a redialysis patient.

In some embodiments, increasing the concentration of one or both of C5a and FABP-1 (e.g., urinary C5a and FABP-1) in a biological sample obtained from an aHUS patient relative to control analyte concentrations obtained, for example, from a pool of samples from aHUS patients who have not undergone a kidney transplant indicates that the patient is a patient who has undergone a kidney transplant.

In some embodiments, increasing the concentration of cystatin C (e.g., urinary cystatin C) in a biological sample obtained from an aHUS patient relative to a control analyte concentration obtained, for example, from a pool of samples from aHUS patients who did not receive plasma retreatment , indicates that the patient is a patient who has received repeated plasma treatment.

In some embodiments, a decrease in post-treatment Ba concentration (e.g., plasma Ba concentration) of at least 10% (e.g., at least 15, 20, 25, 30, 35, 40, 45, or 50%) relative to the Ba concentration in a body fluid sample of the same type obtained from an individual prior to treatment, indicates that the individual has achieved or is likely to achieve a complete thrombomicroangiopathic (TMA) response (ie, cessation of complications of TMA). In some embodiments, the reduction occurs 12 weeks after initiation of complement inhibitor treatment. In some embodiments, the reduction occurs within 12-17 weeks after initiation of complement inhibitor treatment. In some embodiments, the reduction occurs within 26 weeks of initiation of complement inhibitor treatment.

In some embodiments, a reduction following treatment of one or both of CCL5 and SCD40L is at least 10% (e.g., at least 15, 20, 25, 30, 35, 40, 45, or 50%) relative to the corresponding concentration in a sample(s) of body fluid of the same type obtained from an individual prior to treatment, indicates that the individual has achieved or is likely to achieve an increase in platelet count (eg, platelet recovery). In some embodiments, a post-treatment reduction in Ba concentration (e.g., plasma Ba concentration) of at least 10% (e.g., at least 15, 20, 25, 30, 35, 40, 45, or 50%) (or normalization of Ba concentrations) relative to the Ba concentration in a sample of the same type of body fluid obtained from the individual before treatment, indicates that the individual has achieved or can achieve a complete thrombomicroangiopathic (TMA) response (i.e., cessation of complications of TMA ).

In some embodiments, the status of one or more aHUS biomarkers described herein may serve to predict improvement in estimated glomerular filtration rate (eGFR) in an aHUS patient being treated with a complement inhibitor. For example, a decrease in the concentration of F1+2 prothrombin (for example, within 4, 5 or 6 weeks after the start of treatment in a chronic treatment regimen) and/or D-dimer (for example, within 12, 13, 14, 15, 16, or 17 weeks after initiation of treatment in a chronic regimen) indicates that aHUS patient treated with a complement inhibitor has achieved or can achieve clinically significant improvement in eGFR. Achievement or probable achievement of clinically significant improvement in eGFR is also indicated by normalization of IL-6 and IFN-γ concentrations (eg, within 4, 5, or 6 weeks after initiation of complement inhibitor treatment in a chronic treatment regimen). Achievement or probable achievement of clinically significant improvement in eGFR is also indicated by normalization of Ba, CXCL9, CXCL10, and vWF concentrations (eg, within 12, 13, 14, 15, 16, or 17 weeks after initiation of treatment with a complement inhibitor in a chronic regimen treatment). In some embodiments, achievement or likely achievement of a clinically significant improvement in eGFR is also indicated by normalization of concentrations of Ba, CXCL9, CXCL10, v2M (e.g., in urine), CysC (e.g., in urine), vWF, D-dimer, clusterin (e.g., in urine) and/or FABP-1 (eg, in urine) (eg, within 26 weeks of starting treatment with a complement inhibitor in a chronic treatment regimen).

Methods for monitoring or assessing the status of one or more protein biomarkers associated with atypical hemolytic uremic syndrome (aHUS) in an individual (e.g., a mammal, e.g., a human) involve measuring in a biological fluid obtained from the individual one or both of (i) a concentration, at least one (for example, at least two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) protein biomarkers associated with aHUS in biological fluid.

Measuring or determining protein expression levels in a biological sample can be performed by any suitable method (see, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY). In general, protein levels are determined by contacting a biological sample obtained from an individual with binding agents of one or more aHUS protein biomarkers; determining in a sample (eg, biological fluid) levels of one or more aHUS protein biomarkers that bind to binding agents; and comparing the levels of one or more aHUS protein biomarkers in the sample with the levels of corresponding protein biomarkers in a control sample (eg, a normal sample). In some embodiments, a suitable binding agent is a ribosome with or without a peptide component, an RNA molecule, or a polypeptide (eg, a polypeptide that includes 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 aHUS protein biomarkers described herein (eg, an antibody specific for any of the biomarkers presented in Table 1). Suitable antibodies for use in the methods of the present invention include monoclonal and polyclonal antibodies and antigen binding fragments (eg, Fab fragments or scFvs) of antibodies. Antibodies, including monoclonal and polyclonal antibodies, fragments and hybrids, can be produced using methods known in the art (see, for example, Kohler and Milstein (1975) Nature 256:495-497; Kozbor et al. (1985) J Immunol Methods 81:31-42; Cote et al (1983) Proc Natl Acad Sci USA 80:2026-203; and Zhang et al (2002) J Biol Chem 277:39379-39387). Antibodies for use in the methods of the present invention can be purified by methods well known in the art. Antibodies can also be obtained from commercial sources.

In some embodiments, the binding agent is directly or indirectly labeled with the recording portion. The role of the recording agent is to facilitate the detection step in the diagnostic method, resulting in the ability to visualize the complex formed by binding of the binding agent to the protein marker (or fragment thereof). The detection agent may be selected such that it generates a signal that can be measured and whose intensity is related to (preferably proportional to) the amount of protein marker present in the sample being analyzed. Methods for labeling biological molecules such as polypeptides and antibodies are well known in the art. Any of a wide variety of registered agents may be used in the practice of the present invention. Suitable registration agents include, but are not limited to, various ligands, radionuclides, fluorescent dyes, chemiluminescent agents, microparticles (such as, for example, quantum dots, nanocrystals, phosphors and the like), enzymes (such as, for example, enzymes used in ELISA, i.e. horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), colorimetric tags, magnetic tags and biotin, digoxigenin or other haptens and proteins for which antisera or monoclonal antibodies are available.

In some embodiments, binding agents (eg, antibodies) may be immobilized on a carrier or support (eg, bead, magnetic particle, latex particle, microtiter well, cuvette, or other reaction vessel). Examples of suitable carriers or excipients include agarose, cellulose, nitrocellulose, dextran, Sephadex®, Sepharose®, liposomes, carboxymethylcellulose, polyacrylamides, polystyrene, gabbro, filter paper, magnetite, ion exchange resin, polyethylene film, plastic tube, glass, polyamine methyl vinyl ether copolymer and maleic acid, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk and the like. The binding agents may be immobilized not directly, but by a second binding agent specific to the first binding agent (e.g., mouse antibodies specific for protein markers may be immobilized by a sheep anti-mouse IgG Fc-specific antibody, applied to a carrier or substrate).

Protein expression levels in a biological sample can be determined using immunoassays. Examples of such assays include time-resolved fluorescence immunoassay (TR-FIA), radioimmunoassay, enzyme-linked immunosorbent assay (eg, ELISA), immunofluorescence immunoprecipitation, latex agglutination, hemagglutination, Western blotting, and histochemical assays, which are routine techniques well known in the art. technology. Methods for detecting and quantifying the signal generated by the complex formed by the binding of a binding agent to a protein marker must depend on the nature of the assay and the portion of the molecule being detected (eg, the fluorescent portion).

In one example, the presence or expression level of a protein from a gene (eg, the aHUS protein biomarkers presented in Table 1) can be determined using Western blotting. For example, a lysate may be prepared from a biological sample, or the biological sample itself (eg, a biological fluid) may be contacted with Laemmli buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins separated by size by SDS-PAGE can then be transferred to a membrane filter (eg nitrocellulose) and detected by immunoblotting techniques using a detectable labeled antibody specific for the protein of interest. The presence or amount of bound detectable labeled antibody indicates the presence or amount of protein in the biological sample.

In another example, an immunoassay can be used to detect and/or measure the protein expression level of a protein biomarker aHUS (eg, the biomarker presented in Table 1). As above, for detection purposes, an immunoassay can be performed using an antibody that carries a detectable label (eg, a fluorescent agent or enzyme). Proteins from a biological sample can be conjugated directly to a solid-phase matrix (e.g., a multiwell assay plate, nitrocellulose, agarose, Sepharose®, encoded beads, or magnetic beads) or they can be conjugated to the first member of a specific binding pair (e.g., biotin or streptavidin), which attaches to the solid-phase matrix upon binding to the second member of a specific binding pair (for example, streptavidin or biotin). This attachment to the solid phase of the matrix allows the proteins to be purified from other interfering or unnecessary components of the biological sample before contact with the detection antibody, and also allows for the subsequent washing of unbound antibody. In this case, as above, the presence or amount of bound detectable labeled antibody indicates the presence or amount of protein in the biological sample.

Alternatively, protein expression levels can be determined using mass spectrometry-based or imaging-based methods known in the art for protein detection. Other suitable methods include 2D gel electrophoresis, proteomics-based methods such as identification of individual proteins isolated from the gel (eg, by mass spectrometry or N-terminal sequencing), and/or bioinformatics methods.

Methods for detecting or measuring protein expression may not necessarily be carried out in formats that allow rapid acquisition, processing and analysis of multiple samples. These can be, for example, multi-well assay plates (eg 96-well or 386-well) or matrices (eg protein chips). Stock solutions for various reagents can be provided manually or by robot, followed by sample acquisition, pipetting, dilution, mixing, dispensing, washing, incubation (eg, hybridization), sample reading, data collection (optical data), and/or analysis ( computer image analysis) can be done robotically using commercially available analysis software, robotics, and detection instruments capable of detecting the signal generated by the analysis. Examples of such detectors include, but are not limited to, spectrophotometers, luminometers, fluorometers, and radioisotope decay measuring devices. Exemplary high-throughput cell-based assays (eg, detecting the presence or level of a target protein in a cell) may use the ArrayScan® VTI HCS Reader or KineticScan® HCS Reader technology (Cellomics Inc., Cellomics Inc., Pittsburg, PA).

Methods for determining vWF activity are also known in the art and are described herein (eg, in the worked examples). See also, for example, Horvath et al. (2004) Exp Clin Cardiol 9(10):31–34. Commercial kits are also available from Instrumentation Laboratory (Bedford, MA; part number 0020004700) and Quest Diagnostics (Madison, NJ).

In some embodiments, protein expression (or activity) levels of at least two aHUS protein biomarkers (e.g., at least three proteins, at least four proteins, at least five proteins) may be assessed and/or measured at least six proteins, at least seven proteins, at least eight proteins, at least nine

- 33 044587 proteins, at least 10 proteins, at least 11 proteins, at least 12 proteins, at least 13 proteins, at least 14 proteins, at least 15 proteins, at least , 16 proteins, at least 17 proteins, at least 18 proteins, at least 19 proteins, at least 20 proteins, at least 21 proteins, at least 22 proteins, at least 23 proteins or at least 24 proteins or more).

In some embodiments, the biological fluid in which the aHUS protein biomarkers are measured is blood. In some embodiments, the biological fluid is a fraction of blood, such as serum or plasma. In some embodiments, the biological fluid is urine. In some embodiments, all measurements are performed on a single biological fluid sample (eg, a serum sample). In some embodiments, measurements are performed on at least two different biological fluids obtained from an individual. For example, in some embodiments, the concentration or activity of one or more aHUS protein biomarkers is measured in a serum sample obtained from a patient. In some embodiments, the blood sample and the urine sample are available in a form to allow different analytes to be tested on two different sample matrices.

The individual may be, for example, a person suffering from, suspected of having, or at risk of developing aHUS. The individual may be an individual who has been treated (or is currently being treated) with a complement inhibitor (eg, an inhibitor of complement component C5, such as an anti-C5 antibody). Treatment can be carried out for less than one month (for example, less than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day) before receiving the sample from the individual.

The method may further include the step of determining whether the individual has aHUS or is at risk of developing it. When a subject has been treated or is being treated with a complement inhibitor (eg, an anti-C5 antibody) according to a predetermined dosing schedule, the method may further include determining whether the patient responds (therapeutically) to treatment with the complement inhibitor.

In some embodiments of any of the methods described herein, the method requires recording the measured concentration value(s) of at least one aHUS protein biomarker. The recording may be made in writing or on computer media. The method may also include reporting the measured concentration value(s) of at least one aHUS protein biomarker to the individual and/or a healthcare practitioner under the care of the individual.

In some embodiments, any of the methods described herein may include the step of administering to an individual a complement inhibitor at a higher dose or at an increased dosing frequency relative to a predetermined dosing regimen if the individual does not respond to treatment with the inhibitor on a predetermined dosing regimen.

Some of the methods described herein involve comparing the measured concentration or activity of aHUS protein biomarker (as measured in a biological sample obtained from an individual) with a control sample. In some embodiments, a control sample is obtained from the individual prior to administration of a complement inhibitor (eg, a C5 inhibitor such as eculizumab) to the individual. In some embodiments, the control sample may be (or may be based on), for example, a collection of samples obtained from 1 or more healthy individuals (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 healthy individuals or more) who were not administered a complement inhibitor. In some embodiments, the control sample may be (or may be based on), for example, a pooled sample obtained from 2 or more individuals (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, or 40 individuals or more). In some embodiments of any of the methods described herein, the pooled samples may be from healthy individuals, or at least from individuals who do not suffer from or are not suspected of having aHUS (or at risk of developing it). For example, determining whether an individual is an individual suffering from aHUS may involve measuring the concentration of one or more biomarkers in the individual's serum and comparing the measured concentration with the average concentration of the same markers in a pool of samples from healthy individuals. Similarly, determining whether the concentration or activity of a biomarker associated with aHUS is reduced following treatment with a complement inhibitor may involve comparing the concentration or activity of the protein in a body fluid obtained from an individual before treatment with a complement inhibitor with the protein concentration in a sample of the same person. biological fluid obtained from a patient after treatment with an inhibitor (for example, 1, 2, 3, 4, 5, 6 days, 1, 2, 3, 1 month, 6 weeks, 2, 3 months after treatment with an inhibitor (for example, after the first from series of treatment in chronic therapy)).

In some embodiments, determining whether a complement inhibitor produces a desired effect (e.g., reducing the concentration or activity of aHUS protein biomarker) in a person can be accomplished by determining whether the protein concentration falls within a predetermined range after treatment, indicating a person's reaction to a complement inhibitor. In some embodiments, determining whether a complement inhibitor produces a desired effect in a person may include determining whether, following treatment, the concentration or activity of one or more aHUS protein biomarkers falls more or less than a predetermined cutoff value. The cutoff value typically represents the concentration or activity of a given protein in a given biological fluid, above or below which levels are considered indicative of a particular phenotype, such as sensitivity to complement inhibitor therapy.

In some embodiments of any of the methods described herein, the same practitioner may administer a complement inhibitor to an individual before determining whether a change in the concentration or activity of one or more aHUS protein biomarkers has occurred, whereas in some embodiments, the practitioner who administers inhibitor to an individual is different from the practitioner who determines whether a response has occurred in the individual. In some embodiments, the practitioner may obtain a biological sample (eg, a blood sample) from the individual before administering the inhibitor. In some embodiments, the practitioner may obtain a biological sample (eg, a blood sample) from the individual after administering the inhibitor to the individual. In some embodiments, a post-treatment sample may be obtained from an individual in less than 48 hours (e.g., less than 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34 , 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9 , 8, 7, 6, 5, 4, 3, 2 or even less than 1 hour) after administration of the inhibitor to the individual. In some embodiments, a post-treatment sample may be obtained from an individual in less than 20 days (e.g., less than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 5, 4, 3, 2 or 1 day) after administration of the inhibitor to the individual. In some embodiments, a biological sample is obtained from an individual no more than 20 days later (e.g., no more than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 , 4, 3, 2 or 1 day) after administration of the inhibitor to the individual.

In some embodiments, various steps of the methods described herein may be performed by more than one practitioner. For example, one practitioner may analyze (eg, measure the concentration or activity of one or more aHUS protein biomarkers) in pre- and post-treatment samples obtained from an individual. Another practitioner may obtain information regarding the analysis of samples from the first practitioner to thereby determine whether, for example, the individual is responding to treatment with a complement inhibitor. In some embodiments, another specialist may obtain a biological sample from the subject before treatment, and a fourth specialist may obtain a biological sample from the individual after treatment. In some embodiments, all steps are performed by the same practitioner.

Biological samples and sample collection.

Suitable biological samples for use in the methods described herein include, for example, any biological fluid. The biological sample may be, for example, a sample obtained from or derived from an individual (eg, a mammal such as a human). The biological sample may also be a biological fluid such as urine, whole blood or a fraction thereof (eg, plasma or serum), saliva, semen, sputum, cerebrospinal fluid, tears, or mucus. The biological sample can be further divided, if desired, into fractions containing specific analytes (eg, proteins) of interest. For example, a whole blood sample may be fractionated into serum or into fractions containing certain types of proteins. If desired, the biological sample may be a combination of different biological samples from an individual, such as a combination of two different fluids.

Biological samples useful for the present invention may be fresh or frozen samples collected from an individual, or archival samples with a known diagnosis, treatment history and/or outcome. Biological samples can be obtained from an individual, for example, from an individual suffering from, suspected of having, or at risk of developing a complement disorder such as aHUS. Any suitable methods for obtaining biological samples can be used, although exemplary methods include, for example, phlebotomy, smear (eg, buccal swab), lavage, or fine needle aspiration biopsy technique. Biological samples can be obtained from bone marrow.

In some embodiments, a protein extract may be obtained from a biological sample. In some embodiments, the protein extract contains a total amount of protein. Protein extraction methods are well known in the art. See, for example, Roe (2001) Protein Purification Techniques: A Practical Approach, 2nd edition, Oxford University Press. Numerous, diverse and multifunctional kits can be used to extract proteins from body fluids and tissues, and they are commercially available, for example from BioRad Laboratories (Hercules,

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CA), BD Biosciences Clontech (Mountain View, CA), Chemicon International, Inc. (Temecula, CA), Calbiochem (San Diego, CA), Pierce Biotechnology (Rockford, IL) and Invitrogen Corp. (Carlsbad, CA).

Methods for obtaining and/or storing samples that preserve the activity or integrity of cells in a biological sample 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 suitable buffers and/or inhibitors, including protease inhibitors, agents designed to preserve or minimize changes (eg, changes in osmolarity or pH) of protein structure. Such inhibitors include, for example, chelators such as ethylenediaminetetraacetic acid (EDTA), ethylene glycoltetraacetic acid (EGTA), protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), aprotinin and leupeptin. Suitable buffers and storage conditions or other processing of whole cells are described, for example, in Pollard and Walker (1997), Basic Cell Culture Protocols, vol. 75 of Methods in molecular biology, Humana Press; Masters (2000) Animal cell culture: a practical approach, vol. 232 of Practical approach series, Oxford University Press; and Jones (1996) Human cell culture protocols, vol. 2 of Methods in molecular medicine, Humana Press.

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

Complement inhibitors.

Any compound that binds to and inhibits any of the human complement components or otherwise inhibits its production and/or activity can be used in accordance with the present invention. For example, a complement inhibitor may be, for example, a small molecule, a nucleic acid or nucleic acid analog, a peptidomimetic, or a macromolecule that is not a nucleic acid or protein. These agents include, but are not limited to, small organic molecules, RNA aptamers, L-RNA aptamers, spiegelmers, antisense compounds, double-stranded RNA, small interfering RNA, blocked nucleic acid inhibitors and peptide nucleic acid inhibitors. In some embodiments, the complement inhibitor may be a protein or protein fragment.

In some embodiments, the compositions contain antibodies specific for a human complement component. Some compounds include antibodies directed against complement components C1, C2, C3, C4, C5, C6, C7, C8, C9, factor D, factor B, factor P, MBL, MASP-1, MASP-2, properdin or biologically active fragments of any of the above, thereby preventing the generation of anaphylactic activity associated with C5a and/or preventing the assembly of the C5b-9 membrane attack complex.

The compositions may also contain natural or soluble forms of complement inhibitory compounds such as CR1, LEX-CR1, MCP, DAF, CD59, factor H, cobra venom factor, FUT-175, complestatin and K76 COOH. Other compounds that may be used to bind or otherwise block the generation and/or activity of any of the human complement components include, but are not limited to, proteins, protein fragments, peptides, small molecules, RNA aptamers, including ARC187 (which is commercially available available from Archemix Corporation, Cambridge, MA), L-RNA aptamers, spiegelmers, antisense compounds, serine protease inhibitors, molecules that can be used in RNA interference (RNAi), such as double-stranded RNA, including small interfering RNA (siRNA ), locked nucleic acid (LNA) inhibitors, peptide nucleic acid (PNA) inhibitors, etc.

In some embodiments, the complement inhibitor inhibits complement activation. For example, a complement inhibitor may bind to C1 (eg, C1q, C1r, or C1s) and inhibit their complement activation activity, or a complement inhibitor may bind to and inhibit C2, C3, or C4 (eg, inhibit their breakdown). In some embodiments, the inhibitor inhibits the formation or assembly of C3 convertase and/or C5 convertase of the alternative and/or classical complement pathways. In some embodiments, the complement inhibitor inhibits the formation of terminal complement, such as the formation of the C5L-9 membrane attack complex. For example, a complement inhibitor antibody may include an anti-C5 antibody. Such anti-C5 antibodies can directly interact with C5 and/or C5b so as to inhibit the formation and/or physiological function of C5b.

In some embodiments, the compositions described herein may contain an inhibitor of human complement component C5 (e.g., an antibody, or antigen-binding fragment thereof, that binds to human complement protein component C5 or a biologically active fragment thereof, such as C5a or C5b). As used herein, a complement component C5 inhibitor is any agent that inhibits (i) the expression or proper intracellular trafficking or secretion by a cell of the complement protein component C5; (ii) the activity of the C5-C5a or C5b cleavage fragments (for example, the binding of C5a to cellular receptors recognizing it or the binding of C5b to C6 and/or other components of the terminal complement complex; see above); (iii) cleavage of human C5 protein to produce C5a and C5b; (iv) proper intracellular trafficking or secretion of the complement protein component C5 by the cell; or (v) the stability of the C5 protein or the mRNA encoding the C5 protein. Inhibition of the expression of the complement protein component C5 includes inhibition of transcription of the gene encoding the human C5 protein; increased degradation of mRNA encoding human C5 protein; inhibition of translation of mRNA encoding human C5 protein; increased degradation of human C5 protein; inhibition of proper processing of human C5 preprotein; or inhibition of proper cellular trafficking or secretion of human C5 protein. Methods for determining whether a candidate agent is an inhibitor of human complement component C5 are known in the art and are described herein.

The human complement component C5 inhibitor may be, for example, a small molecule, a polypeptide, a polypeptide analog, a nucleic acid, or a nucleic acid analog.

The term small molecule as used herein is intended to mean an agent that has a molecular weight of preferably less than about 6 kDa and most preferably less than about 2.5 kDa. Many pharmaceutical companies have large libraries of chemical and/or biological mixtures comprising a selection of small molecules, often fungal, bacterial or algal extracts, which can be screened for use using either assay. This application covers the use of small chemical libraries, peptide libraries or natural product collections, among others. Tan et al. described a library of more than two million synthetic compounds that are compatible with miniaturized cell-based assays (J Am Chem Soc (1998) 120:8565-8566). It is contemplated within the scope of this application that such a library can be used to screen for agents that bind to a target antigen of interest (eg, complement component C5). There are numerous commercially available compound libraries, such as ChemBridge DIVERSet. Libraries such as the NCI Therapeutic Development Program Diversity Collection are also available from academic researchers. Rational drug design can also be used. For example, rational drug design could involve the use of structural information about the crystal structure of the human complement protein component C5 or its structure in solution. See, for example, the structures described in Hagemann et al. (2008) J Biol Chem 283(12):7763–75 and Zuiderweg et al. (1989) Biochemistry 28(1):172–85. Rational drug design can also be achieved based on known compounds, for example, a known C5 inhibitor (eg, an antibody or antigen-binding fragment thereof that binds to the C5 protein component of human complement).

Peptidomimetics can be compounds in which at least a portion of the polypeptide of interest is modified such that the three-dimensional structure of the peptidomimetic remains substantially the same as that of the polypeptide of interest. Peptidomimetics can be analogues of the test polypeptide of the invention, which are themselves polypeptides containing one or more substitutions or other modifications within the sequence of the test polypeptide. Alternatively, at least a portion of the polypeptide sequence of interest may be replaced by a non-peptide structure such that the three-dimensional structure of the polypeptide of interest is substantially preserved. In other words, one, two or three amino acid residues within the polypeptide sequence of interest may be replaced by a non-peptide structure. In addition, other peptide portions of the polypeptide of interest may, but need not, be replaced by a non-peptide structure. Peptidomimetics (both peptide and non-peptide analogues) may have improved properties (eg, reduced proteolysis, increased persistence, or increased bioavailability). Peptidomimetics generally improve oral availability, making them particularly suitable for the treatment of diseases in humans or animals. It should be noted that peptidomimetics may or may not have similar two-dimensional chemical structures, but share three-dimensional structural features and geometry. Each peptidomimetic may further have one or more unique additional binding elements.

Nucleic acid inhibitors can be used to bind and inhibit a target antigen of interest. The nucleic acid antagonist may be, for example, an aptamer. Aptamers are short oligonucleotide sequences that can be used to recognize and specifically bind almost any molecule, including cell surface proteins. The process of systematic ligand evolution by exponential enrichment (SELEX) is powerful and can be used to easily identify such aptamers. Aptamers can be prepared for a wide range of proteins of therapeutic and diagnostic relevance, such as growth factors and cell surface antigens. These oligonucleotides bind to their targets with similar affinity and specificity to antibodies (see, for example, Ulrich (2006) Handb Exp Pharmacol. 173:305-326).

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In some embodiments, the complement inhibitor is a scaffold protein that is not an antibody. These proteins are typically produced through combinatorial chemistry based on the adaptation of pre-existing antigen-binding proteins. For example, the human transferrin binding site of the human transferrin receptor can be modified through combinatorial chemistry to create a diverse library of transferrin variants, some of which acquire affinities for different antigens. Ali et al. (1999) J Biol Chem 274:24066–24073. The portion of human transferrin not involved in receptor binding remains unchanged and serves as a scaffold, similar to the scaffold regions of antibodies, for presenting variant binding sites. The libraries are then screened as an antibody library against the target antigen of interest to identify those variants that have optimal selectivity and affinity for the target antigen. Scaffold proteins, which are not antibodies and while similar in function to antibodies, are touted as having a number of advantages over antibodies, with the advantages including, but not limited to, increased solubility and tissue penetration, less expensive preparation, and ease of conjugation with other molecules, of interest. Hey et al. (2005) TRENDS Biotechnol 23(10):514–522.

One skilled in the art will appreciate that the framework portion of non-antibody scaffold proteins may include, for example, all or part of the S. aureus protein A Z domain, human transferrin, human fibronectin type III tenth domain, trypsin inhibitor Marten domain human, human CTLA-4, ankyrin repeat protein, human lipocalin, human crystallin, human ubiquitin or E. elaterium trypsin inhibitor. Id.

In some embodiments, the complement inhibitor is an antibody or antigen-binding fragment thereof that binds to the C5 protein component of human complement. (Hereinafter, the antibody may sometimes be referred to as anti-C5 antibody).

In some embodiments, an anti-C5 antibody may bind to an epitope on the alpha chain of the human complement protein component C5. Antibodies that bind to the C5 alpha chain are described, for example, in PCT Published Application No. WO 2010/015608 and US Patent No. 6,355,245. In some embodiments, an anti-C5 antibody may bind to an epitope on the beta chain of the human complement protein component C5 . Antibodies that bind to the C5 beta chain are described, for example, by Moongkarndi et al. (1982) Immunobiol 162:397; Moongkarndi et al. (1983) Immunobiol 165:323; and Mollnes et al. (1988) Scand J Immunol 28:307–312.

Additional exemplary human complement component C5 antigenic fragments are disclosed, for example, in US Pat. No. 6,355,245, the disclosure of which is incorporated herein by reference.

Additional anti-C5 antibodies and antigen binding fragments thereof suitable for use in fusion proteins described herein are described, for example, in PCT Published Application No. WO 2010/015608, the disclosure of which is incorporated herein by reference in its entirety.

In some embodiments, the anti-C5 antibody specifically binds to a human complement protein component C5 (eg, human C5 protein having the amino acid sequence set forth in SEQ ID NO: 1). The terms specific binding or specifically binds refer to two molecules forming a complex (eg, a complex between an antibody and the complement protein component C5) that is relatively stable under physiological conditions. Generally, binding is considered specific when the association constant (K _a ) is greater than 106 M ^-1 . Thus, the antibody can specifically bind to the C5 protein with a K _a of at least (or greater) 106 M ^-1 (e.g., at least or greater than 10 ⁷ , 108 , 109 , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ or 10 ¹⁵ M ^-1 or higher). Examples of antibodies that specifically bind to human complement protein component C5 are described, for example, in US Pat. No. 6,355,245, the disclosure of which is incorporated herein by reference in its entirety.

The anti-C5 antibodies described herein may have activity to block the generation or activity of active fragments of the complement C5 protein component C5a and/or C5b (eg, human C5 protein). Due to this blocking effect, anti-C5 antibodies inhibit, for example, the pro-inflammatory effects of C5a and the generation of the C5b-9 membrane attack complex (MAC) at the cell surface. Antibodies against C5, which have the ability to block the generation of C5a, are described, for example, by Moongkarndi et al. (1982) Immunobiol 162:397; and Moongkarndi et al. (1983) Immunobiol 165:323.

In some embodiments, an anti-C5 antibody or antigen-binding fragment thereof may reduce the ability of the C5 protein to bind human complement component C3b (e.g., the C3b present in the AR or the C5 convertase CP complex) by more than 50% (e.g., by more than 55 , 60, 65, 70, 75, 80, 85, 90 or 95% or more). In some embodiments, after binding to the C5 protein, an anti-C5 antibody or antigen-binding fragment thereof may reduce the ability of the C5 protein to bind complement component C4b (e.g., the C4b present in the C5 convertase CP) by more than 50% (e.g., by more than 55 , 60, 65, 70, 75, 80, 85, 90 or 95% or

- 38 044587 more). Methods for determining whether an antibody can block the generation or activity of active fragments C5a and/or C5b of the complement protein component C5 or bind to complement components C4b or C3b are known in the art and are described, for example, in US patent No. 6355245; And

Wurzner et al. (1991) Complement Inflamm 8:328–340.

In some embodiments, the composition includes eculizumab and/or the antibody is eculizumab (Soliris®; Alexion Pharmaceuticals, Inc., Cheshire, CT). (See, for example, Kaplan (2002) Curr Opin Investig Drugs 3(7):1017–23; Hill (2005) Clin Adv Hematol Oncol 3(11):849–50; and Rother et al. (2007) Nature Biotechnology 25(11):1256-1488.) In some embodiments, the composition includes pexelezumab and/or the antibody is pexelezumab (Alexion Pharmaceuticals, Inc., Cheshire, CT). (See, for example, Whiss (2002) Curr Opin Investig Drugs 3(6):870-7; Patel et al. (2005) Drugs Today (Bare) 41 (3):165-70; and Thomas et al. ( 1996) Mol Immunol 33(17-18):1389-401.)

In some embodiments, the C5 inhibitor is an antibody that binds to C5a (sometimes referred to herein as an anti-C5a antibody). In some embodiments, the antibody binds to C5a but not to full-length C5. In some embodiments, antibody binding to C5a may inhibit the biological activity of C5a. Methods for measuring C5a activity include, for example, chemotaxis testing, RIAs or ELISAs (see, for example, Ward and Zvaifler (1971) J Clin Invest 50(3):606-16; and Wurzner et al. (1991) Complement Inflamm 8: 328-340). In some embodiments, antibody binding to C5a may inhibit the interaction between C5a and C5aRl. Suitable methods for detecting and/or measuring the interaction between C5a and C5aRl (in the presence and absence of an antibody) are known in the art and are described, for example, Mary and Boulay (1993) Eur J Haematol 51(5):282-287; Kaneko et al. (1995) Immunology 86(1):149-154; Giannini et al. (1995) J Biol Chem 270(32):19166–19172; and US Patent Application Published No. 20060160726. For example, the binding of C5a labeled with a detectable label (eg, a radiolabel) to peripheral blood mononuclear cells expressing C5aRl can be assessed in the presence and absence of an antibody. A decrease in the amount of detectable labeled C5a bound to C5aRl in the presence of antibody compared to the amount bound in the absence of antibody is an indication that the antibody inhibits the interaction between C5a and C5aRl. In some embodiments, antibody binding to C5a may inhibit the interaction between C5a and C5L2 (see below). Methods for detecting and/or measuring the interaction of C5a and C5L2 are known in the art and are described, for example, Ward (2009) J Mol Med 87(4):375-378; and Chen et al. (2007) Nature 446(7132):203–207 (see below).

In some embodiments, the C5 inhibitor is an antibody that binds to C5b (sometimes referred to herein as an anti-C5b antibody). In some embodiments, the antibody binds to C5b but does not bind to full-length C5. The structure of C5b is described, for example, by Muller-Eberhard (1985) Biochem Soc Symp 50:235-246; and Yamamoto, Gewurz (1978) J Immunol 120(6):2008–2015. As described above, C5b combines with C6, C7 and C8 to form the C5b-8 complex on the surface of the target cell. Protein complex intermediates formed through a series of interactions include C5b-6 (including C5b and C6), C5b-7 (including C5b, C6 and C7) and C5b-8 (including C5b, C6, C7 and C8). After binding of several C9 molecules, the membrane attack complex (MAC, C5b-9 of the terminal complement complex (TCC)) is formed. When sufficient numbers of MACs are incorporated into target cell membranes, they form openings (MAC pores) that mediate rapid osmotic lysis of the target cells.

In some embodiments, antibody binding to C5b may inhibit the interaction between C5b and C6. In some embodiments, antibody binding to C5b may inhibit the assembly or activity of C5b-9 MAC-TCC. In some embodiments, antibody binding to C5b can inhibit complement-dependent cell lysis (eg, in vitro and/or in vivo). Suitable methods for assessing whether an antibody inhibits complement-dependent lysis include, for example, hemolytic assays or other functional assays to determine the activity of soluble C5b-9. For example, the reduction in the ability of complement to lyse cells in the presence of an antibody can be measured using the hemolysis assay described by Rabat and Mayer (eds.), Experimental Immunochemistry, 2nd edition, 135-240, Springfield, IL, CC Thomas (1961), p. 135-139, or a conventional variant of this assay, such as the chicken red blood cell hemolysis method as described, for example, in Hillmen et al. (2004) N Engl J Med 350(6):552.

Antibodies that bind to C5b, as well as methods for producing such antibodies, are known in the art. Commercially available anti-C5b antibodies are available from a number of suppliers, including, for example, Hycult Biotechnology (catalog number HM2080; clone 568) and Abcam™ (ab46151 or ab46168).

Methods for determining whether a particular agent is an inhibitor of human complement component C5 are described herein and are known in the art. For example, the concentration and/or physiological activity of C5a and C5b in a body fluid can be measured using methods well known in the art. Methods for measuring concentration or

- 39 044587 C5a activities include, for example, chemotaxis methods, RIAs or ELISAs (see, for example, Ward, Zvaifler (1971) J Clin Invest. 50(3):606-16; and Wurzner et al. (1991) Complement Inflamm 8:328-340). For C5b, hemolytic assays or soluble C5b-9 assays can be used as described herein. Other assays known in the art may also be used. Using these or other suitable types of assays, candidate agents capable of inhibiting human complement component C5, such as an anti-C5 antibody, can be screened to, for example, identify compounds that are useful in the methods described herein and determine suitable dosage levels of such compounds.

Methods for determining whether a candidate compound inhibits the cleavage of human C5 into the C5a and C5b forms are known in the art and are described, for example, by Moongkarndi et al. (1982) Immunobiol 162:397; Moongkarndi et al. (1983) Immunobiol 165:323; Isenman et al. (1980) J Immunol 124(1):326–31; Thomas et al. (1996) Mol. Immunol 33(17-18):1389-401; and Evans et al. (1995) Mol. Immunol 32(16):1183–95.

Inhibition of human complement component C5 may also reduce the ability of complement to lyse cells in a given individual's body fluids. Such presence of a decrease in the ability of complement to lyse cells can be measured using methods well known in the art, such as, for example, a conventional hemolytic assay, such as the hemolysis assay described by Rabat and Mayer (eds), Experimental Immunochemistry, 2nd edition, 135 -240, Springfield, IL, C. C. Thomas (1961), p. 135-139, or a conventional variant of this assay, such as the chicken red blood cell hemolysis method as described, for example, by Hillmen et al. (2004) N Engl J Med 350(6):552.

Antibodies that bind to C3b, and, for example, inhibit C3b convertase, are also well known in the art. See, for example, PCT Published Application Nos. WO 2010/136311, WO 2009/056631, and WO 2008/154251, the disclosures of each of which are incorporated herein by reference in their entirety. Antagonistic antibodies against C6 and against C7 are described, for example, by Brauer et al. (1996) Transplantation 61(4):588-594 and in US Patent No. 5679345.

In some embodiments, the antibody is an anti-factor B antibody (eg, monoclonal antibody 1379 produced by ATCC Deposit No. PTA-6230). Antibodies against factor B are also described, for example, Ueda et al. (1987) J Immunol 138(4):1143-9; Tanhehco et al. (1999) Transplant Proc 31 (5):2168-71; in US patents No. 7999082 and 7964705; and in PCT publication No. WO 09/029669.

In some embodiments, the antibody is an anti-factor D antibody, such as the antibody described by Pascual et al. (1990) J Immunol Methods 127:263-269; Sahu et al. (1993) Mol Immunol 30(7):679–684; Pascual et al. (1993) Eur J Immunol 23:1389–1392; Niemann et al. (1984) J Immunol 132(2):809–815; in US patent No. 7439331; or US Patent Application Published No. 20080118506.

In some embodiments, the antibody is an anti-properdin antibody. Suitable anti-properdin antibodies are also well known in the art and include, for example, those described in US Patent Application Published No. 20110014614 and PCT Application Published No. WO2009110918.

Treatment methods.

Also provided herein are compositions and methods for treating or preventing aHUS in an individual (eg, a human). The compositions (eg, complement inhibitors and/or secondary agents) can be administered to an individual, eg, a human subject, using various methods, which depend in particular on the route of administration. The route of administration may be, for example, intravenous (IV) injection or infusion, subcutaneous (SC) injection, intraperitoneal (IP) injection or intramuscular injection.

Administration can be achieved by, for example, local infusion, injection or by implant. The implant may be a porous, non-porous or gelatinous material, including membranes such as silastic membranes or fibers. The implant may be configured to continuously or periodically release the composition into the body of an individual. See, for example, US Patent Publication No. 20080241223; US Patents No. 5,501,856; 4863457; and 3710795; and European Patent Nos. EP 488401 and EP 430539, the disclosures of each of which are incorporated herein by reference in their entirety. The composition may be delivered to the individual by an implantable device based, for example, on diffusion, rupture or convective systems, for example, osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmosis systems, steam injection pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps, erosion-based or electromechanical systems.

The appropriate dosage of a complement inhibitor (eg, an anti-C5 antibody or fragment thereof), which dose is capable of treating or preventing aHUS in an individual, may depend on various factors, including, for example, the age, sex and weight of the individual being treated and the particular

- 40 044587 inhibitor compound used. For example, treating an individual with aHUS may require a different dose of siRNA specific for human C5 compared to the dose of anti-C5 antibody required to treat the same patient. Other factors influencing the dose administered to an individual include, for example, the type or severity of aHUS. For example, an individual suffering from aHUS associated with CFH may require a different dose of inhibitor than an individual suffering from aHUS associated with MCP. Other factors may include, for example, other medical conditions concurrent or previously occurring in the individual, the general health of the individual, the genetic predisposition of the individual, diet, timing of administration, rate of elimination, combination of drugs, as well as any other additional therapeutic agents administered to the individual. In addition, it should be understood that the specific dosage and treatment regimen for any particular individual should depend on the judgment of the treating healthcare professional (eg, physician or nurse).

The inhibitor can be administered as a fixed dose or at a dose of milligram per kilogram mg/kg. In some embodiments, the dosage may also be selected to reduce or avoid the production of antibodies or other host immune responses against one or more active agents in the composition. Without limiting it in any way, exemplary doses of an inhibitor such as an anti-C5 antibody include, for example, 1-100, 0.5-50, 0.1-100, 0.5-25, 1-20, and 1-10 mg /kg body weight.

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

In some embodiments, a human may be administered intravenously an anti-C5 antibody (e.g., eculizumab) at a dose of about 600 mg (e.g., about 625, 650, 700, 725, 750, 800, 825, 850, 875, 900, 925, 950, or 1000 mg or more) every week, optionally for two or more (eg, three, four, five, six, seven or eight or more) weeks. After initial treatment, the individual may be administered the antibody at a dose of approximately 900 mg approximately every 14 days (e.g., approximately 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, for example, Hillman et al. (2004) N Engl J Med. 350(6):552-9; and Dmytrijuk et al. (2008) The Oncologist 13(9):993.

In some embodiments, a human may be administered intravenously an anti-C5 antibody (e.g., eculizumab) at a dose of approximately 900 (e.g., 925, 950, 975, 1000, 1100, or 1200 or more) mg every week, optionally for two or more (e.g., three, four, five, six, seven or eight or more) weeks. After initial treatment, the individual may be administered the antibody at a dose of approximately 1,200 mg approximately every 14 days (e.g., approximately 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, for example, International Patent Application Publication No. WO 2010/054403.

As used herein, chronically administered, chronically treated, treat chronically, or similar grammatical variations refer to a treatment regimen that is used to maintain a certain threshold concentration of a therapeutic agent in the blood of a patient with the goal of completely or substantially suppressing systemic complement activity in the patient for a period of time. long period of time. Accordingly, a patient who is chronically treated with a complement inhibitor may be treated for a period of time that is greater than or equal to 2 weeks (eg, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52 weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months; or 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 , 9, 9.5, 10, 10.5, or 12 years or for the remainder of the patient's life) with an inhibitor in an amount and dosing frequency that is sufficient to maintain a concentration of the inhibitor in the patient's blood that inhibits or significantly inhibits the patient's systemic complement activity . In some embodiments, a complement inhibitor may be chronically administered to a patient in need thereof in an amount and at a frequency that is effective to maintain serum hemolytic activity of less than or equal to 20% (e.g., 19, 18, 17, 16, 15, 14, 13 , 12, 11, 10, 9, 8, 7, 6 or 5%). See, for example, Hill et al. (2005) Blood 106(7):2559. In some embodiments, a complement inhibitor may be administered to a patient in an amount and at a frequency that is effective to maintain serum lactate dehydrogenase (LDH) levels within at least 20% (e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5%) of the normal LDH range. See Hill et al. (2005) above. In some embodiments, the complement inhibitor is administered to the patient in an amount and at a frequency that is effective to maintain a serum LDH level of less than 550 mU/L (e.g., less than 540, 530, 520, 510, 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280 mU/l or less than 270 mU/l). To maintain systemic complement inhibition in a patient, a complement inhibitor can be chronically administered to the patient, for example, once a week, once every two weeks, twice a week, once a day, once a month, or once every three weeks.

The pharmaceutical composition may include a therapeutically effective amount of inhibitor

- 41 044587 torus of the human complement component C5 (for example, an antibody against C5 or an antigen-binding fragment thereof). Such effective amounts can be readily determined by one of ordinary skill in the art based, in particular, on the effect of the inhibitor administered or the combined effect of the antibody and one or more additional active agents if more than one agent is used. The therapeutically effective amount of a human complement component C5 inhibitor (eg, an anti-C5 antibody) may also vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody (and one or more additional active agents) to produce the desired effect. a response in an individual, for example, a decrease in the intensity of at least one condition parameter, for example, a decrease in at least one aHUS symptom. For example, a therapeutically effective amount of a complement component C5 inhibitor (eg, an anti-C5 antibody) may inhibit (reduce the severity or eliminate the occurrence of) and/or prevent thrombocytopenia, microangiopathic hemolytic anemia, renal failure, and/or any one of the symptoms of aHUS known in the art techniques or described herein. A therapeutically effective amount is also an amount at which any toxic or adverse effects of the composition are outweighed by the therapeutically beneficial effects.

The terms therapeutically effective amount or therapeutically effective dose or similar terms as used herein are intended to indicate the amount of an agent (e.g., human complement component 5 inhibitor) that is expected to produce a desired biological or medical response (e.g., improvement of one or more symptoms of aHUS) . In some embodiments, the composition described herein comprises a therapeutically effective amount of a human complement component C5 inhibitor. In some embodiments, the composition described herein contains a therapeutically effective amount of an antibody or antigen-binding fragment thereof that binds to the complement protein component C5. In some embodiments, the composition contains two or more (eg, three, four, five, six, seven, eight, nine, 10, or 11 or more) different human complement C5 inhibitors such that the composition as a whole is therapeutically effective. For example, the composition may contain an antibody that binds to human C5 protein and siRNA that binds to and promotes degradation of mRNA encoding human C5 protein, wherein the antibody and siRNA are each at a concentration that, when combined, is therapeutically effective. In some embodiments, the composition contains an inhibitor and one or more second active agents such that the composition as a whole is therapeutically effective. For example, the composition may contain an antibody that binds to human C5 protein and another agent useful for treating or preventing aHUS.

The toxicity and therapeutic efficacy of such compositions can be determined using known pharmaceutical methods in cell cultures or in experimental animals (animal models of aHUS). These methods can be used, for example, to determine the LD ₅₀ (lethal dose for 50% of the population) and ED ₅₀ (therapeutically effective dose for 50% of the population). The dose ratio between toxic and therapeutic effects represents the therapeutic index, and it can be expressed as the ratio LD ₅₀ /ED ₅₀ . Preferred are compositions or inhibitors (eg, anti-C5 antibodies) in compositions that have high therapeutic indices. Since compositions that have toxic side effects may be used, care must be taken in developing a delivery system that directs such compounds to the site of diseased tissue and minimizes the potential for damage to normal cells and thereby reduces side effects.

Data obtained from cell culture experiments and animal studies can be used to determine dose ranges for use in humans. Suitable animal models of aHUS are known in the art and are described, for example, by Atkinson et al. (2007) Journal of Experimental Medicine 204(6):1245–1248. The dosage of such inhibitors is generally within the range of circulating inhibitor concentrations (eg, anti-C5 antibody or antigen-binding fragment thereof) that include ED ₅₀ with little or no toxicity. Dosage may vary within this range depending on the dosage form used and route of administration. For a human complement component C5 inhibitor (eg, an anti-C5 antibody) used as described herein (eg, for the treatment or prevention of aHUS), a therapeutically effective dose can be initially estimated from cell culture assays. Dosage may be determined in animal models to achieve a range of circulating plasma concentrations that includes the IC ₅₀ (ie, the concentration of test compound that achieves half maximum symptom inhibition) determined in cell culture. Such information can be used to more accurately determine suitable doses in humans. Plasma levels can be measured, for example, using high performance liquid chromatography.

In some embodiments, the required dose of human complement component C5 inhibitor may be determined based on the concentration of human C5 protein in the individual's blood.

- 42 044587

For example, an individual having higher concentrations of circulating levels of human C5 protein may require higher doses of a human C5 inhibitor than an individual having lower circulating levels of human C5. Methods for determining the concentration of human complement component C5 in a sample of fluid originating from the blood of an individual are known in the art and are described, for example, by Rawal et al. (1998) J Biol Chem 273(27):16828–16835.

In some embodiments, the methods may be performed in combination with other treatments for aHUS. For example, the composition may be administered to an individual concurrently, before or after nephrectomy (eg, bilateral nephrectomy), dialysis, plasmapheresis, or plasma infusion (see, for example, Noris et al. (2005) Non-shiga toxin-associated hemolytic uremic syndrome, In : Zipfel P (ed). Complement and Kidney Disease. Basel: Birkhauser-Verlag, 65-83).

As used herein, the individual may be any mammal. For example, the individual may be a human, non-human primate (eg, a monkey, baboon or chimpanzee), horse, cow, pig, sheep, goat, dog, cat, rabbit, guinea pig, gerbil, hamster, rat or mouse. In some embodiments, the individual is a child (eg, a human child).

When used herein to refer to an individual in need of prevention, in need of treatment, or in need thereof, the terms refer to those determined by an appropriate health care professional (e.g., physician, nurse, or nurse practitioner in the case of humans; veterinarian in the case of mammals, non-human) must objectively benefit from the treatment (eg, treatment with a composition containing an inhibitor of human complement component C5).

As used herein, an individual at risk for developing aHUS is an individual who has 1 or more risk factors for developing the disease (eg, 2, 3, 4, 5, 6, 7, or 8 risk factors for developing the disease or more). Risk factors for aHUS are well known in the medical field and include, for example, a predisposition to developing the condition, i.e. a family history of the condition or a genetic predisposition to developing the condition, such as, for example, one or more mutations in complement factor H (CFH), membrane cofactor protein (MCP; CD46), C4b-binding protein, complement factor B (CFB), or Complement I (CFI). See, for example, Warwicker et al. (1998) Kidney Int 53:836–844; Richards et al. (2001) Am J Hum Genet 68:485–490; Caprioli et al. (2001) Am Soc Nephrol 12:297–307; Neumann et al. (2003) J Med Genet 40:676–681; Richards et al. (2006) Proc Natl Acad Sci USA 100:12966-12971; Fremeaux-Bacchi et al. (2005) J Am Soc Nephrol 17:2017–2025; Esparza-Gordillo et al. (2005) Hum Mol Genet 14:703–712; Goicoechea de Jorge et al. (2007) Proc Natl Acad Sci USA 104(1):240–245; Blom et al. (2008) J Immunol 180 (9):6385-91; and Fremeaux-Bacchi et al. (2004) J Medical Genet 41:e84. See also Kavanagh et al. (2006), supra. Risk factors also include, for example, infection with Streptococcus pneumoniae, pregnancy, cancer, exposure to anticancer agents (eg, quinine, mitomycin C, cisplatin, or bleomycin), exposure to immunotherapeutic agents (eg, cyclosporine, OKT3, or interferon), exposure to antiplatelet agents (eg, ticlopidine or clopidogrel), HIV infection, transplantation, autoimmune disease and combined methylmalonic aciduria and homocystinuria (cblC). See, for example, Constantinescu et al. (2004) Am J Kidney Dis 43:976–982; George (2003) Curr Opin Hematol 10:339–344; Gottschall et al. (1994) Am J Hematol 47:283–289; Valavaara et al. (1985) Cancer 55:47-50; Miralbell et al. (1996) J Clin Oncol 14:579–585; Dragon-Durey et al. (2005) J Am Soc Nephrol 16:555–63; and Becker et al. (2004) Clin Infect Dis 39:S267–S275. Thus, a person at risk of developing aHUS may be, for example, a person who has a family history of aHUS and/or a person who has HIV infection. From the foregoing, it should be clear that individuals at risk for developing aHUS are not all individuals within the variants of interest.

An individual suspected of having aHUS is an individual who has one or more symptoms of the disease. Symptoms of this disease are well known to those skilled in the art and include, for example, severe hypertension, proteinuria, uremia, lethargy/fatigue, irritability, thrombocytopenia, microangiopathic hemolytic anemia, and deterioration of renal function (eg, acute renal failure). It should be clear from the above text that individuals suspected of having aHUS are not all individuals within the variants of interest.

aHUS can be genetic, acquired, or idiopathic. aHUS may be considered genetic when two or more (eg, three, four, five, six or more) members of the same family have the disease for at least six months apart and exposure to a common triggering agent has been excluded, or when an individual has been identified one or more mutations in genes associated with aHUS (eg, one or more mutations in CFH, MCP/CD46, CFB, or CFI). For example, an individual may suffer from aHUS associated with CFH, aHUS associated with CFB, aHUS associated with CFI, or aHUS associated with MCP. Up to 30% of genetic aHUS is associated with mutations in CFH, 12% with mutations in MCP, 5-10% with mutations in CFI, and less than 2% with mutations in CFB. Genetic aHUS can be multiple (i.e., familial; involving two or more affected family members) or simple (i.e., a single occurrence in the family). aHUS can be considered acquired when an underlying environmental factor can be identified (eg, a drug, systemic disease, or viral or bacterial agents that do not result in Shiga-like exotoxins). aHUS can be considered idiopathic when no obvious trigger (genetic or environmental) is identified.

In some embodiments, the methods may include identifying an individual as having, suspected of having, or at risk of developing aHUS. In addition to using the aHUS biomarker profile described herein, laboratory tests may be performed to determine whether a person has thrombocytopenia, microangiopathic hemolytic anemia, or acute renal failure. Thrombocytopenia may be diagnosed by a health care professional as one or more of (i) a platelet count of less than 150,000/mm ³ (eg, less than 6,000/mm ³ ); (ii) decreased platelet lifetime, which decreases reflecting increased platelet destruction in the bloodstream; and (iii) detection of giant platelets in the peripheral blood smear, which is consistent with secondary activation of thrombocytopoiesis. Microangiopathic hemolytic anemia may be diagnosed by a health care professional as one or more of (i) hemoglobin concentration less than 10 mg/dL (eg, less than 6.5 mg/dL); (ii) increased serum lactate dehydrogenase (LDH) concentration (>460 U/L); (iii) hyperbilirubinemia, reticulocytosis, circulating free hemoglobin, and low or undetectable haptoglobin concentrations; and (iv) detection of fragmented red blood cells (schizocytes) with the typical appearance of styloid cells or helmet cells in a peripheral blood smear in conjunction with a negative Coombs test. See, for example, Kaplan et al. (1992) Hemolytic Uremic Syndrome and Thrombotic Thrombocytopenic Purpura, Informa Health Care (ISBN 0824786637); and Zipfel (2005) Complement and Kidney Disease, Springer (ISBN 3764371668).

Blood concentrations of C3 and C4 can also be used as an indicator of complement activation or dysregulation. In addition, the individual's condition can be further characterized by identifying the individual as carrying one or more mutations in a gene associated with aHUS, such as CFI, CFB, CFH, or MCP (supra). Suitable methods for detecting mutations in a gene include, for example, DNA sequencing and nucleic acid array methods. See, for example, Breslin et al. (2006) Clin Am Soc Nephrol 1:88–99; and Goicoechea de Jorge et al. (2007) Proc Natl Acad Sci USA 104:240–245.

In some embodiments, an inhibitor of human complement component C5 (eg, an anti-C5 antibody or antigen-binding fragment thereof) may be administered to an individual as monotherapy. Alternatively, as described above, the inhibitor can be administered to an individual as a combination therapy with another treatment, for example, another treatment for aHUS. For example, combination therapy may include administering to an individual (eg, a patient) one or more additional agents (eg, antihypertensive agents) that provide a therapeutic benefit to the individual who has or is at risk of developing aHUS. In some embodiments, the human complement component C5 inhibitor and one or more additional active agents are administered simultaneously. In other embodiments, the inhibitor is administered first and one or more additional active agents are administered second. In some embodiments, one or more additional active agents are administered first and the inhibitor is administered second.

An inhibitor of human complement component C5 may replace or complement previous or current treatment. For example, when treated with an anti-C5 antibody or an antigen-binding fragment thereof, administration of one or more additional active agents may be discontinued or reduced, eg, administered at lower levels. In some embodiments, administration of previous therapeutic agents may be maintained. In some embodiments, the prior treatment will be maintained until the person's C5 inhibitor level reaches a level sufficient to provide a therapeutic effect. The administration of agents of two types of treatment can occur in combination.

Monitoring an individual (eg, a diseased individual) for improvement in aHUS, as defined herein, means monitoring the individual for a change in a disease parameter, eg, an improvement in one or more disease symptoms. Such symptoms include any of the symptoms of aHUS described herein. In some embodiments, the survey is performed at least 1 hour later, such as at least 2, 4, 6, 8, 12, 24, or 48 hours; or at least 1, 2, 4, 10, 13, 20 days or more; or at least 1, 2, 4, 10, 13, 20 weeks or more after the start of treatment. The individual may be examined at one or more of the following periods: before the start of treatment; during treatment; or after administration of one or more treatment elements. The evaluation may include an assessment of the need for further treatment, for example, an assessment of whether the dose, frequency of administration or duration of treatment should be changed. It may also include an assessment of the need to add or remove the selected treatment modality, e.g.

- 44 044587 adding or canceling any of the aHUS treatment types described herein.

Sets.

Kits are also provided that include various reagents and substances used to perform the methods described herein. The measurement, diagnosis, examination and/or evaluation methods described herein may be performed by diagnostic laboratories, experimental laboratories or individual practitioners. The invention provides kits that can be used by any or all of these organizations.

In some embodiments, kits described herein include substances and reagents for, among other things, characterizing or processing biological samples (e.g., body fluids), measuring biomarker levels (e.g., protein or nucleic acid levels), diagnosing aHUS in an individual, or monitoring the response to treatment in an individual in accordance with the methods presented herein. In some embodiments, a kit of the invention includes at least one or more reagents that specifically detect protein levels of one or more aHUS protein biomarkers (e.g., biomarkers selected from Table 1) and, if necessary, instructions for using the kit. The set may include, for example, any of the matrices described herein.

In some embodiments, kits may include suitable control samples (eg, biological fluids from normal healthy individuals or a solution including a known control amount of a particular analyte of interest). In some embodiments, the kits of the present invention may include instructions for using the kit according to one or more of the methods described herein, and may include instructions for processing a biological sample (e.g., a body fluid) obtained from an individual and/or performing a test or instructions for interpreting results.

The following examples are intended to be illustrative and not limiting of the invention.

Examples

To better understand the pathology of aHUS, the inventors collected samples of body fluids (whole blood, serum, plasma and urine) from patients suffering from aHUS or suspected of having aHUS, both before and at several points after starting treatment with a complement inhibitor (the anti-C5 antibody eculizumab). ). One of the objectives of this study was to identify a series of clinically detectable parameters that can be used to monitor patient response to complement inhibitor treatment, as well as markers of disease and disease progression or control. The inventors have identified several proteins whose expression and/or activity correlates with either aHUS disease and/or the response of an aHUS patient to complement inhibitor treatment. The proteins were those involved in or associated with complement and/or endothelial cell activation, inflammation, renal injury, and coagulation (see Table 1).

A total of 41 adults (27 women and 12 men) with a confirmed diagnosis of aHUS, as well as normal healthy adult volunteers, were recruited for the study. All patients had confirmed aHUS when screened based on one or more of the following characteristics: platelet count less than 150x10 ⁹ /L; hemoglobin levels are less than the lower limit of normal; LDH levels greater than or equal to 1.5 times the upper limit of normal; serum creatinine levels greater than or equal to the upper limit of normal; and ADAMTS13 activity level greater than 5%. All patients were tested negative for Shiga toxin.

The average age of patients at inclusion was 40.3 years. 68% of patients were female; 2 (4.8%) were black or African American; and 1 patient (2.4%) was of Asian origin. Six patients (14.6%) reported a family history of aHUS. Twenty (48.7%) had at least one identified complement regulatory protein mutation or were tested positive for an autoantibody that binds to complement regulatory protein. Thirty patients (73.2%) demonstrated the first clinical manifestation of aHUS. In six patients (14%), eculizumab treatment was initiated immediately without the use of plasma replacement/infusion (PE/PI). Twenty-four patients (58.5%) were on dialysis at baseline (before eculizumab treatment). Nine patients (22%) had previously undergone kidney transplantation. Twenty-seven (66%) had platelet counts less than 150 x 10 ⁹ /L. Thirty-two (78%) patients had serum LDH levels higher than the upper limit of normal. Average haptoglobin (Hp) levels in patients in this cohort were 0.6±0.4 g/L; whereas mean serum creatinine levels in this cohort of patients were 411±264.6 μmol/L (N=40).

Biological fluids were collected upon enrollment into the study (before treatment) and then during treatment at each drug administration. Eculizumab was administered to individuals as follows: 900 mg once weekly for four weeks; 1200 mg as the fifth dose; and 1,200 mg once every two weeks thereafter for up to 55 weeks as part of a phase 2 clinical trial.

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Example 1. Materials and methods.

Urine samples.

Freshly collected urine was immediately mixed with protease inhibitors. Concentrations of several analytes, including NGAL, cystatin C, clusterin, TIMP-1, β2-microglobulin, C5b9, C5a, and creatinine, in urine collected from individuals were measured using commercially available kits, as briefly described below.

NGAL levels were measured in urine using a commercially available kit (R&D Systems, Minneapolis, MN; catalog no. DLCN20). Briefly, urine samples were diluted 1:3 using the supplied kit's RD5-24 calibrator diluent. 50 μl of each sample or standard kit control (recombinant human lipocalin-2 expressed in NS0) was added to the wells of the assay plate in duplicate, with each well containing 100 μl of the supplied kit diluent RD1-52. After two hours of incubation at 4°C in the refrigerator, the wells were washed four times with a wash solution of 200 μl per well. Enzyme (horseradish peroxidase)-labeled anti-NGAL conjugate was added at a dose of 200 μl per well and incubated for two hours at 4°C in the refrigerator. The wells were washed four times with a wash solution of 200 μl per well and then a solution of the TMB substrate supplied in the kit (anti-NGAL enzyme conjugate substrate) was added, 200 μl per well, and incubated at room temperature in the dark for 30 min. TMB is a substrate for horseradish peroxidase often used in ELISA. The reaction between the substrate and immobilized horseradish peroxidase (HRP) conjugated to antibodies in ELISA wells produces a blue solution. Once the desired color intensity was achieved, the reaction was stopped by adding a stop solution (acid), which changes the color of the solution from blue to yellow. Thus, reactions were stopped after incubation by adding 50 μl per well of the stop solution supplied in the kit to each well, and the absorbance was read at 450 nm.

Cystatin C levels were measured using a commercially available kit (R&D Systems, Minneapolis, MN; catalog no. DSCTC0). Briefly, urine samples were diluted 1:3 using the supplied kit's RD5-24 calibrator diluent. 50 μl of each sample or standard kit control (recombinant human CysC) was added to the wells of the assay plate in duplicate, with each well containing 100 μl of the supplied kit diluent RD1-52. After two hours of incubation at 4°C in the refrigerator, the wells were washed four times with a wash solution of 200 μl per well. Enzyme-labeled anti-CysC conjugate was added at 200 μl per well and incubated for two hours at 4°C in the refrigerator. The wells were washed four times with a wash solution of 200 μl per well and then a solution of the TMB substrate supplied in the kit (anti-CysC enzyme conjugate substrate) was added, 200 μl per well, and incubated at room temperature in the dark for 30 min. The reaction was stopped after incubation by adding 50 μl per well of the stop solution supplied in the kit to each well, and the absorbance was read at 450 nm.

Clusterin levels were measured using a commercially available kit (R&D Systems, Minneapolis, MN; catalog no. DCLU00). Briefly, urine samples were diluted 1:3 using the supplied kit's RD5T calibrator diluent. 50 μl of each sample or standard kit control (recombinant human clusterin) was added to the wells of the assay plate in duplicate, with each well containing 100 μl of the supplied kit diluent RD1-19. After two hours of incubation at room temperature on an orbital shaker at 500 rpm, the wells were washed four times with 200 μl per well wash solution. Enzyme-labeled anti-clusterin conjugate was added at 200 μl per well and incubated for two hours at room temperature on an orbital shaker at 500 rpm. The wells were washed four times with wash solution, 200 μl per well, and then TMB substrate solution, 200 μl per well, was added and incubated at room temperature in the dark for 30 min. The reaction was stopped after incubation by adding stop solution, 50 μl per well, to each well, and the absorbance was read at 450 nm.

TIMP-1 levels were measured using a commercially available kit (R&D Systems, Minneapolis, MN; catalog no. DTM100). Briefly, urine samples were diluted 1:2 using the supplied kit's RD5P calibrator diluent. 50 μl of each sample or standard kit control (recombinant human TIMP-1) was added to the wells of the assay plate in duplicate, with each well containing 100 μl of the supplied kit RD1X diluent. After two hours of incubation at room temperature on an orbital shaker at 500 rpm, the wells were washed three times with wash solution, 200 μl per well. Enzyme-labeled anti-TIMP-1 conjugate was added at 200 μl per well and incubated for two hours at room temperature on an orbital shaker at 500 rpm. The wells were washed four times with wash solution, 200 μl per well, and then TMB substrate solution, 200 μl per well, was added and incubated at room temperature in the dark for 30 min. The reaction was stopped after incubation by adding stop solution, 50 μl per well, to each well, and the absorbance was read at 450 nm.

B2M levels were measured using a commercially available kit (R&D Systems, Minneapolis, MN; catalog number DBM200). Briefly, urine samples were diluted 1:10 using vol.

- 46 044587 samples of the supplied set. 20 μl of each sample, kit controls, or kit standards was added in duplicate to wells containing the enzyme-labeled anti-B2M conjugate. After incubation for one hour at room temperature, the wells were washed six times with 200 μL wash solution per well. TMB substrate solution, 100 μl per well, was then added and incubated at room temperature in the dark for 15 min. The reaction was stopped after incubation by adding stop solution, 100 μl per well, to each well, and the absorbance was read at 450 nm.

Creatinine levels were measured using a commercially available kit (R&D Systems, Minneapolis, MN; catalog no. KGE005). Briefly, urine samples were diluted 1:20 using water, and 50 μl of samples, kit controls, or kit standards were added in duplicate to wells containing 100 μl of the alkaline picrate solution provided in the kit. After incubation for 30 min at room temperature, the absorbance was measured at 490 nm.

C5b-9 levels were measured using a commercially available kit (BD Biosciences, San Jose, CA; catalog no. 558315) and optEIA reagent kit B (BD Biosciences, San Jose, CA; catalog no. 550534). Briefly, capture antibody against C5b-9 was diluted 1:250 in coating buffer, 100 μl of which was added to each well of a 96-well MaxiSorp plate (Nunc; catalog no. 439454) and incubated overnight at 4°C in the refrigerator. Wells were washed three times with 200 μl per well of wash solution and blocked by adding 200 μl per well of the assay diluent provided in the kit for one hour at room temperature. Wells were washed three times with 200 μL per well of wash solution, and 100 μL of urine samples or kit standards were added to the wells in duplicate. After incubation for two hours at room temperature, the wells were washed three times with 200 μl per well of wash solution. 100 μl of the anti-C5b-9 detection antibody working solution supplied in the kit was added to each well and incubated for one hour at room temperature. The wells were washed seven times with 200 μl per well of wash solution and then 100 μl per well of TMB substrate solution was added and incubated at room temperature in the dark for 30 min. The reaction was stopped after incubation by adding stop solution, 50 μl per well, to each well, and the absorbance was read at 450 nm.

C5a levels were measured using a commercially available kit (BD Biosciences, San Jose, CA; catalog no. 557965). Briefly, 100 μl of urine samples or kit standards was added to two parallel wells containing 50 μl of diluent provided in the ELISA kit. After incubation for two hours at room temperature, the wells were washed five times with 200 μl per well of wash solution. 100 μl of the anti-C5a detection antibody working solution supplied in the kit was added to each well and incubated for one hour at room temperature. The wells were washed seven times with 200 μl per well of wash solution and then 100 μl per well of TMB substrate solution was added and incubated at room temperature in the dark for 30 min. The reaction was stopped after incubation by adding stop solution, 50 μl per well, to each well, and the absorbance was read at 450 nm.

Plasma.

Plasma samples were obtained as follows. Blood was collected into 10 ml BD™ P100 tubes (Becton Dickinson) containing EDTA. Whole blood was centrifuged no later than 60 min after collection in a refrigerated centrifuge (set to maintain 4–8°C) for 10 min at 3000 rpm. Plasma was then obtained from the sample after centrifugation. Hemolyzed samples were discarded.

Concentrations of several analytes, including Ba, prothrombin F1+2 fragment, thrombomodulin, vWF, sC5b-9, and C5a, in plasma fractions collected from individuals were measured using commercially available kits, as briefly described below.

Ba levels were measured using a commercially available kit (Quidel, San Diego, CA; catalog number A033). Briefly, the wells of the assay plate were washed three times with the wash solution. Plasma samples were diluted 1:1000 with the sample diluent provided in the kit, and 100 μl of diluted plasma samples, kit controls, and kit standards were added to wells in duplicate. After incubation for 60 min at room temperature, the wells were washed five times with 200 μl per well of wash solution. 100 μl of enzyme-labeled anti-Ba antibody conjugate was added to each well and incubated for sixty minutes at room temperature. After five washes with the wash solution, 100 μl of TMB substrate was added to each well and incubated for fifteen minutes at room temperature, protected from light. The reaction was stopped by adding stop solution, 100 μl per well, and the absorbance was read at 450 nm.

Prothrombin fragment 1+2 levels in EDTA plasma were measured using the Enzygnost F1+2 kit from Siemens Healthcare; catalog number OPBD03). Briefly, plasma samples were diluted 1:2 with sample buffer and 50 μl of diluted samples or standard (containing a known concentration of recombinant human prothrombin fragment 1+2) was added to the wells. After incubation in

- 47 044587 for 30 minutes at 37°C, the wells were washed three times with 200 μl per well of washing solution. 100 μl of enzyme-labeled anti-prothrombin fragment 1+2 antibody conjugate was added to each well and incubated for 15 min at 37°C. After three additional washes, 100 μl of chromogenic substrate was added to each well and incubated for 15 min at room temperature, protected from light. The reaction was stopped by adding 100 μl of stop solution to each well, and the absorbance was read at 450 nm.

Thrombomodulin (TM) levels in EDTA plasma were assessed using a TM ELISA kit (American Diagnostica, Stamford, CT; catalog no. 837). Briefly, plasma samples were diluted 1:4 with sample buffer, and 200 μl of diluted samples or standard (containing a known concentration of recombinant HM) was added to the wells. After incubation for 60 min at room temperature, the wells of the assay plate were washed four times with 200 μL per well of wash solution. A solution of enzyme-labeled anti-TM antibody was added (200 μl per well) and incubated for 30 min at room temperature. After 4 washes, 200 μl of substrate was added to each well, and the wells were incubated for 20 min at room temperature, protected from light. The reaction was stopped with 100 μl of 0.5 M H ₂ SO ₄ and the absorbance was measured at 450 nm.

Levels of von Willebrand factor (vWF) activity were determined in EDTA plasma using an ELISA kit using immobilized antibodies specific for the collagen-binding sites of vWF (American Diagnostica; catalog no. 885). Plasma samples and kit controls were diluted 1:20 in assay diluent, and 100 μl of diluted samples and controls were added to wells in duplicate. After incubation for 60 min at room temperature, the wells were washed 5 times with wash solution and 100 μl of enzyme-labeled anti-vWF conjugate was added to each well. The wells were incubated for 15 min at room temperature and washed 5 times with wash solution. 100 μl of TMB substrate (which, after enzyme digestion, generates a detectable signal) was added to each well. The wells were incubated for 15 min at room temperature, protected from light, followed by the addition of 100 μl of the stop solution supplied in the kit to each well. Optical density was measured at 450 nm within 30 min after the addition of stop solution.

Circulating levels of sC5b-9 were determined using the human C5b-9 ELISA kit (BD Biosciences, San Diego, CA; catalog no. 558315) and the BD optEIA reagent kit B (BD Biosciences; catalog no. 550534). Briefly, the anti-C5b-9 capture antibody was diluted 1:250 in the coating buffer provided in the kit, 100 μl of which was added to the wells of a 96-well MaxiSorp plate (Nunc) and incubated overnight at 4°C. After three washes with the wash solution, the wells were blocked with 200 μl of the assay diluent provided in the kit for one hour at room temperature. After 3 more washes with the wash solution, 100 μl of plasma samples (diluted 1:100 in assay diluent) or standards (containing a known concentration of purified sC5b-9) were added and incubated for two hours at room temperature. The wells were washed three times with wash solution, and 100 μl of working detection solution (which contains biotin-labeled anti-C5b9 detection antibody and horseradish peroxidase-labeled streptavidin, diluted 1:250 with assay diluent) was added to each well. After incubation for one hour at room temperature, the wells were washed seven times with wash solution, and 100 μl of TMB substrate solution was added to each well. The reaction was allowed to develop for 30 min at room temperature, protected from light. After adding 50 μl of stop solution to each well, the absorbance was determined at 450 nm.

Circulating levels of C5a were determined by sandwich ELISA using the BD optEIA reagent kit B (BD Biosciences; catalog no. 550534). All incubations were performed in the presence of futan (BD Biosciences; catalog no. 550236). Briefly, the anti-C5a capture antibody was diluted to 2 μg/ml in the coating buffer provided in the kit, 100 μl of which was added to the wells of a 96-well MaxiSorp plate (Nunc) and then incubated overnight at 4°C. After three washes with the wash solution, the wells were blocked with 200 μl of assay diluent for one hour at room temperature. After 3 more washes with the wash solution, 50 μl of plasma samples (diluted 1:5 in assay diluent) or standards (containing a known concentration of C5a) were added and incubated for one hour at room temperature. The wells were washed 4 times with wash solution, and 100 μl of working detection solution (which contains biotin-labeled anti-C5a detection antibody and horseradish peroxidase-labeled streptavidin, diluted 1:250 with assay diluent) was added to each well. After incubation for one hour at room temperature, the wells were washed seven times with wash solution, and 100 μl of TMB substrate solution was added to each well. The reaction was allowed to develop for 30 min at room temperature, protected from light. After adding 50 μl of stop solution to each well, the absorbance was measured at 450 nm.

Serum.

Serum samples were processed as follows. Blood was collected into 10 ml vacutainer tubes (SST) to separate the serum. The tube was inverted five times and the blood was allowed to clot at room temperature for at least 30 minutes, but not more than 2 hours. The tube was centrifuged at 1800 g with a brake. Hemolyzed samples were discarded.

Quantification of various analytes in serum was performed using human bead array cytometry (BAC) Flex Set kits (CBA Flex Set; Becton Dickinson Biosciences, San Diego, CA), and using a flow cytometer (FACS LSR II, Becton Dickinson) in accordance with the manufacturer's instructions. The Flex Set capture bead is a single population of beads with distinct fluorescence intensity and is coated with an immobilized antibody specific for a soluble protein. The position of each bead population is given an alphanumeric designation indicating its position in relation to the other beads in the BD CBA Flex Set. Beads with different positions can be combined in assays to create a multiplex assay. In the Flex Set assay, a PE-conjugated capture bead, detection reagent, and standard or test samples are incubated together to form sandwich complexes.

Briefly, standards for each analyte were mixed and serial dilutions were performed using assay diluent. Capture beads for each analyte were prepared and combined using serum/plasma capture bead diluent. Serum samples were diluted appropriately and, along with standards, incubated with mixed capture beads in a total volume of 100 μl for one hour at room temperature. Phycoerythrin (PE) detection reagents were mixed for all analytes and added to tubes (50 μl). Samples were washed with wash buffer after incubation for two hours at room temperature in the dark and analyzed on a flow cytometer after reconstitution of the pellet in wash buffer.

The following bead sets were incubated with serum samples diluted 1:4 in the assay diluent provided in the kit (where the specific protein biomarker indicates the immobilized antibody conjugated to the bead): IFN-γ (E7 bead; catalog number 558283); IL-12 p70 (E5 bead; catalog number 558283); IL-β (B4 bead; catalog number 558279); IL-6 (A7 bead; catalog number 558276); IL-8 (A9 bead; catalog number 558277); CXCL-9 (E8 ball; catalog number 558286); CXCL-10 (B5 ball; catalog number 558280); MCP-1 (ball D8; catalog number 558287); VEGF (B8 ball; catalog number 558336); and SCD40L (C7 ball; catalog number 560305). The following sets of beads were incubated with serum samples diluted 1:50 with the diluent provided in the kit: ICAM-1 (bead A4; catalog number 560269); VCAM-1 (D6 ball; catalog number 560427); TNFR1 (C4 ball; catalog number 560156); E-selectin (D9 bead; catalog number 560419); P-selectin (D7 bead; catalog number 560426); and CCL5 (D4 ball; catalog number 558324).

Example 2. Results.

Markers of ongoing complement activation.

As shown in table. 2 below, the concentrations of complement component Ba and sC5b9 in plasma and the concentrations of C5a and sC5b-9 in urine are increased in the majority of patients with aHUS compared with the concentration in a biological fluid sample from healthy volunteers. See also FIG. 1.

table 2

Protein marker aHUS n/N (%) increase above baseline Value B Va in plasma 35/35 (100.0) <0.0001 sC5b-9 in plasma 37/38 (97.4) <0.0001 C5a in urine 26/29 (89.7) 0.0007 sC5b-9 in urine 23/27 (85.2) 0.0025

* N denotes the total number of patients examined for a given biomarker; and n indicates the number of these patients N with elevated levels of the protein biomarker.

These results indicate that significant activation of the systemic alternative complement pathway continues in patients with aHUS.

Following eculizumab treatment, the mean levels (concentrations) of these aHUS biomarkers decreased (Figures 1A-1C). Mean urinary levels of C5a and sC5b-9 decreased significantly between 1 and 2.5 weeks after initiation of treatment and remained unchanged. The mean percentage reduction in urinary C5a levels was greater than 40% at 3 weeks post-treatment and greater than 70% at 6 weeks (Figure 1D). Urinary sC5b-9 levels were reduced by more than 60% after 3 weeks ( Fig. 1E ). These markers eventually normalized. Plasma Ba levels also decreased significantly (p=0.0053) approximately four to six weeks after eculizumab treatment, suggesting that over time eculizumab will reduce the initiation or enhancement of the classical complement pathway (Figure 1C). However, the average percentage reduction in plasma Ba levels was approximately 10% at 6 weeks and more than 30% at week 12 (Fig. 1F).

The percentage of treated aHUS patients who had normalized complement protein biomarker levels over time is shown in Fig. 2A-2C. For example, as shown in FIG. 2B, 50% of treated aHUS patients have normalized urinary sC5b-9 levels 2.5 weeks after initiation of eculizumab treatment. By 17 weeks after treatment, 79% of treated aHUS patients had normalized sC5b-9 levels. However, Ba levels do not normalize in most patients (Figs. 1C and 2C). These data indicate that even with eculizumab treatment, low levels of complement activation may exist in some patients.

Platelet markers and activation of hemostasis.

As summarized in table. 3 below, serum SCD40L concentrations and plasma levels of prothrombin fragment 1+2 and D-dimer were significantly higher in most aHUS patients compared with concentrations in a body fluid sample from healthy volunteers. See also FIG. 3A, 3B.

Table 3

Protein marker aHUS n/N (%) increase above baseline P value SCD40L 36/38 (94.7) <0.0001 Prothrombin fragment F1+2 36/38 (94.7) <0.0001 D-dimer 34/36 (94.4) =0.0002

* N denotes the total number of patients examined for a given biomarker; and n indicates the number of these patients N with elevated levels of the protein biomarker.

The release of SCD40L is generally associated with platelet metabolism and activity. Prothrombin F1+2 fragments are generated during the conversion of prothrombin to thrombin, while D-dimer is a fibrin degradation product indicating fibrinolysis.

Following eculizumab treatment, the mean levels (concentrations) of these aHUS biomarkers decreased. Mean plasma F1+2 and D-dimer levels decreased significantly between 1 and 2.5 weeks (p=0.0078 and 0.0083, respectively) after initiation of treatment and remained unchanged. As shown in FIG. 3C, the average percentage reduction in F1+2 was approximately 15% at 3 weeks and more than 50% at 12 weeks. The mean percentage reduction in D-dimer levels was approximately 40% at 6 weeks, but greater than 60% at 12 weeks. These data indicate that eculizumab treatment has a direct effect on the coagulation and fibrinolysis pathways. As shown in FIG. 4A, 4B, 32% of treated aHUS patients have normalized F1+2 levels after 26 weeks of treatment and 46% of patients have normalized D-dimer levels. In contrast, SCD40L levels remained elevated throughout the study.

Markers of endothelial cell damage and/or activation As shown in Table. 4 below, plasma thrombomodulin and vWF concentrations and serum VCAM-1 concentrations were significantly higher in aHUS patients compared with the concentration in a body fluid sample from healthy volunteers. See also FIG. 5A-5C.

Table 4

Protein marker aHUS n/N (%) increase above baseline P value Thrombomodulin 33/34 (97.1) <0.0001 VCAM-1 36/38 (94.7) <0.0001 Antigen factor background Willibrand 15/38 (39.5) <0.02

* N denotes the total number of patients examined for a given biomarker; and n indicates the number of these patients N with increased levels of the protein biomarker;

n.s. indicates that the differences are not significant.

High concentrations of thrombomodulin and VCAM-1 in the biological fluids of aHUS patients indicate significant activation of endothelial cells. Thrombomodulin is released in response to C3a, further highlighting the persistent activation of complement in aHUS patients. The concentration of vWF is also significantly increased. vWF is characterized by a number of physiological functions, including platelet adhesion and coagulation, and is also a marker of endothelial damage and activation.

Following eculizumab treatment, the mean levels (concentrations) of these aHUS biomarkers were reduced (Figures 5A-5C). Mean thrombomodulin and VCAM-1 levels were significantly reduced from baseline at 17 weeks (p=0.0007 and <0.0001, respectively) after initiation of treatment (see Figures 6C and 6D). After 26 weeks, VCAM-1 and vWF levels also decreased. However, while vWF normalized in ∼70% of treated aHUS patients 17 weeks after initiation of treatment ( Fig. 6B ), thrombomodulin and VCAM-1 levels remained elevated. Interestingly, of the 10% of patients whose thrombomodulin levels normalized (Fig. 6A), only one patient had normalized levels of both thrombomodulin and vWF. These data show that eculizumab treatment has a rapid and reliable benefit in correcting damage and activating endothelial cells.

Markers of inflammation.

In table Table 5 (below) presents a series of analytes detected in plasma and/or serum and indicates the percentage of aHUS patients whose corresponding analytes were elevated before treatment with a complement inhibitor.

Table 5

Protein marker aHUS n/N (%) increase above baseline P value Serum CXCL10 23/38 (60.5) P<0.0001 CXCL9 in serum 33/38 (86, 8) P<0.0001 IL-18 19/38 (50.0) P<0.0001 MSR-1 34/38 (89.5) P<0.0001 TNFR1 38/38 (100.0) P<0.0001 VEGF 25/38 (65.8) P<0.0001 IL-6 21/34 (61.8) P=0.0019 CCL5 4/38 (10.5) P=0.0045 IFN-γ 5/34 (14.7) P=0.0353 IL-8 22/38 (57.9) n.s. (P=0.0640) ICAM-l 2/34 (5.9) n. S IL-Ιβ 1/38 (2.6) n. s IL-12p70 2/34 (5.9) n. s

* N denotes the total number of patients examined for a given biomarker; and n indicates the number of these patients N with elevated levels of the protein biomarker.

**Concentrations of the two analytes noted as in serum were measured in serum. Concentrations of all other analytes in the table were measured in plasma, n.s. indicates that the differences are not significant.

Before eculizumab treatment, aHUS patients were characterized by elevated levels of circulating inflammatory cytokines and chemokines, including, for example, CXCL-10, CXCL-9, IL-18, TNFR1, MCP-1, VEGF, IL-6 and IL-8. After initiation of treatment, however, TNFR1 was the earliest inflammatory marker to be significantly reduced (at 6 weeks, p=0.0012) (Figure 7A). The mean TNFR1 concentration remained significantly below baseline at all follow-up visits (p < 0.0001), but returned to normal in only 6% of aHUS patients (Fig. 7B). Similarly, mean CXCL10 levels were significantly reduced at 26 weeks (p=0.0055) but did not normalize in all aHUS patients (not normalized in 31% of patients). After 26 weeks, mean IFN-γ levels normalized in approximately 50% of patients; however, mean serum levels of IL-8 (p=0.01), CXCL-9 (p=0.01), IL-18 (p<0.0001) and VEGF (p<0.0001) remained elevated in the majority aHUS patients compared with normal controls, and were not significantly different from baseline. Serum IL-6 was significantly decreased (p=0.04) from baseline at week 26 and remained elevated at week 26 compared with normal controls.

In contrast, mean CCL-5 levels were significantly increased 17 weeks after treatment initiation.

- 51 044587 niya and after that (p = 0.0072 and 0.0021 after 12-17 weeks and 26 weeks, respectively). In response to vascular injury in mice, CCL5 is upregulated, which stimulates selective T cell infiltration as part of the vascular injury-healing response. See, for example, Rookmaaker et al. (2007) Am J Physiol Renal Physiol 293(2):F624–630. These data indicate that eculizumab treatment has a rapid and robust beneficial effect on inflammation in many aHUS patients, but that low levels of inflammation may persist in these patients even during treatment.

Markers of damage to the renal tubules and glomeruli.

In table Figure 6A (below) presents a series of analytes determined in urine collected from patients and indicates the percentage of aHUS patients whose corresponding analytes were elevated before treatment with a complement inhibitor.

Table 6A

Protein marker aHUS n/N (%) increase above P value ORIGINAL level Beta-2 microglobulin (β2Μ) 20/28 (71.4) P<0.0001 Clusterin 24/29 (82.8) P<0.0001 Cystatin C 18/29 (62.1) P=0.0002 ΤΙΜΡ-1 22/29 (75.9) P=0.0003 FAVR-1 22/29 (75.9) P=0.0130 NGAL 5/29 17.2) P=0.0151 NAG 3/23 13, 0) P=0.0413 CXCL10 2/29 (6.9) P . S. CXCL9 2/29 (6.9) n. S. KIM-1 2/29 (6.9) P . S.

* N denotes the total number of patients examined for a given biomarker; and n indicates the number of these patients N with increased levels of the protein biomarker;

n.s. indicates that the differences are not significant.

Before eculizumab treatment, low molecular weight molecules that are normally filtered by the kidney were elevated in the urine of aHUS patients, including 32M, clusterin, cystatin C, and NAG. Molecules produced by renal tubular epithelial cells in response to injury were also elevated, such as TIMP-1, NGAL and L-FABP. However, with eculizumab treatment, CysC (p=0.0012) (Figure 8A), clusterin (p=0.0446), and TIMP-1 (p=0.0353) were significantly reduced 1-2.5 weeks after treatment. , and they remained significantly reduced throughout the study. NGAL (p=0.0003) (Figure 8C), L-FABP (p=0.0366), and NAG (p=0.0369) were significantly lower compared to baseline at 4-6 weeks after treatment. and remained that way after that. 32M was significantly reduced at 12–17 weeks (p=0.0008) onwards (Figure 8B). After 26 weeks, mean urinary levels of all analytes normalized in aHUS patients.

These data show that eculizumab treatment has rapid, reliable, and long-lasting benefits, reversing the tubular and glomerular damage experienced by many aHUS patients.

Summary.

The following table provides a summary of illustrative aHUS biomarkers (although this is not an exhaustive list) that are elevated in aHUS patients before eculizumab treatment but significantly reduced after eculizumab treatment. The table (Table 6B) also shows the average time after initiation of eculizumab treatment at which a significant decrease in the aHUS biomarker occurred.

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Table 6B

Biomarker 1-2.5 weeks 4-6 weeks 12-17 weeks 26 weeks U-C5a X U-C5b-9 X F1+2 X D-dimer X U-Cys-C X U-TIMP-1 X Va in plasma X TNFR1 X U-CLU X U-NGAL X Thrombomodulin X VCAM X L-FABP X V2M X CXCL10 X IFN-γ X

Baseline levels of aHUS markers in aHUS patients undergoing dialysis and/or renal transplantation.

Plasma and urinary concentrations of complement markers, inflammation, and kidney damage were also assessed in aHUS patients on dialysis before eculizumab treatment. As shown in table. 7 (below), the mean concentrations of serum TNFR1, plasma Ba, C5b9, prothrombin fragments 1+2, β2Μ, clusterin, sC5b9, TIMP-1, NGAL, CysC and C5a were significantly increased in aHUS patients who were on repeat dialysis (eg , two or more times within 6 months before treatment), compared with aHUS patients who were not on repeat dialysis before enrollment (before treatment). See also FIG. 9A-9E.

Table 7

Analyte Higher with repeat dialysis TNFR1 (serum) p=<0.0001 β2η (urine) p=0.0009 Clusterin (urine) p=0.0020 Va (plasma) p=0.0021 C5b-9 (urine) p=0.0042 ΤΙΜΡ-1 (urine) p=0.0070 NGAL (urine) p=0, IT CysC (urine) p=0.020 F1+2 (plasma) p=0.0191 S5b-9 (plasma) p=0.0476 C5a (urine) p=0.0477

**Single labeled analyte concentration was measured in serum. Concentrations of urine-labeled analytes were measured in urine, while concentrations of plasma-labeled analytes were measured in plasma obtained from patients.

In addition, aHUS patients who received a kidney transplant before eculizumab treatment had lower urinary C5b-9 and FABP-1 at baseline compared with patients who did not receive a kidney transplant.

Baseline levels of aHUS markers relative to TMA markers.

Levels of some biomarkers associated with aHUS in some aHUS patients correlate with

-53 044587 markers of abnormal thrombotic microangiopathy (TMA), such as decreased platelet counts, elevated LDH, and elevated haptoglobin levels. For example, aHUS patients with a reduced platelet count at baseline (<150,000 per μL of blood) demonstrated increased levels of urinary cystatin C (P=0.0276) and urinary clusterin (P=0.0401). See fig. 14A, 14B. aHUS patients having elevated LDH levels had elevated levels of VCAM-1 (P=0.0226) (Figure 14C), D-dimer (P=0.0369) (Figure 14D), IL-18, thrombomodulin, and TNFR1 (see below). Elevated haptoglobin levels were often present in aHUS patients characterized by elevated IL-18 levels.

Baseline levels of aHUS markers in aHUS patients receiving plasma treatment.

aHUS patients re-treated with plasma prior to eculizumab treatment had higher mean urinary cystatin C levels at baseline (see FIG. 15).

Correlations between biomarker levels and clinical parameters.

Platelets.

Elevated CCL5 levels were positively correlated with higher platelet counts at baseline (p=<0.0001; cc (correlation coefficient)=0.8106). Elevated SCD40L levels also correlated with higher platelet counts at baseline (p=<0.001; ss=0.6313).

In addition, patients with normalized Ba levels after eculizumab treatment showed significantly higher platelet growth than patients whose Ba levels remained elevated after treatment. See fig. 13.

Estimated glomerular filtration rate (eGFR), LDH, and urinary complement.

A correlation was also observed between increased plasma Ba levels and decreased eGFR (p<0.0001; ss=-0.7219). Elevated serum TNFR1 concentrations in aHUS patients before treatment correlated with lactate dehydrogenase (LDH) levels (p=0.027; ss=0.3586), but more significantly correlated with lower eGFR (p<0.0001; ss=-0.6134 ). In addition, higher levels of urinary complement components C5a and sC5b-9 and the kidney damage markers f2M, clusterin, cystatin C, NGAL and TIMP-1) were moderately correlated with lower eGFR (p=0.0002 to 0.0242; ss=from -0.4286 to -0.6714).

Elevated urinary levels of sC5b-9, clusterin, and TIMP-1 were moderately correlated with proteinuria (p=0.0086 to 0.0284; ss=0.40 to 0.4788), while elevated plasma Ba levels (p=0.0017; ss=0.517), 32M, clusterin, urine sC5b-9 and cystatin C correlated with increased creatinine in the urine of patients before treatment with eculizumab (p=0.0018-0.0440; ss=0.3982- 0.6457).

First clinical manifestation of aHUS.

There was also a correlation between patients experiencing their first manifestation of aHUS and significantly elevated plasma D-dimer or urinary FABP-1 levels at baseline (pre-eculizumab treatment) (see Table 8).

Table 8

Elevated biomarkers (n%) Biomarker The only manifestation of aHUS Multiple manifestations Value p Plasma D-dimer (µg/l) 27 (100.0) 7 (77.8) 0.0571 Urine FABP-1 (ng/mg with normalization to creatinine) 19 (90.5) 3 (37.5) 0.0079

Sluggish disease.

The six aHUS patients participating in the study had normalized hematologic parameters (including haptoglobin, LDH, and platelet count) at enrollment. However, these patients still demonstrated the presence of chronic inflammation and complement activation despite stable clinical presentation. Patients had significantly elevated levels of serum TNFR1 (as shown in Fig. 10A), as well as significantly elevated levels of thrombomodulin, Ba (Fig. 10B), prothrombin fragments 1+2 (Fig. 10E), VCAM-1 (Fig. 10C), and D-dimer (Fig. 10D). Similarly, patients with normal platelet counts (>150x109 platelets/μL) at baseline still showed elevated levels of most biomarkers (eg, Ba (Figure 10F), VCAM-1 (Figure 10G), D-dimer (Figure 10H) and F1+2 (Fig. 10I).Taken together, these data indicate that even in the subgroup of aHUS patients with presumed clinical remission after treatment, there is a possibility of ongoing low levels of complement activity, coagulopathy

- 54 044587 and inflammation.

Correlation between biomarker levels and clinical outcomes.

Hematological responses.

Patients with complete hematological responses showed a more dramatic decrease in TNFR1, urinary clusterin, and urinary complement levels (C5A and C5b-9) (Fig. 11). For example, 86% of patients characterized by decreased concentrations of these aHUS protein biomarkers achieved complete hematologic response (platelet and LDH normalization) 12 to 17 weeks after initiation of eculizumab treatment. In addition, these patients showed greater reductions in the mean percentage of serum TNFR1, urinary clusterin, urinary C5a, and urinary C5b-9 than patients who did not achieve a complete hematologic response.

The rate of decline in TNFR1 (eg, at 12 weeks versus 17 weeks or greater) was also found to correlate with completeness of hematologic response (p=0.0008). The rate of D-dimer normalization was significantly associated with complete hematological response (p=0.0109; ss=6.26).

In addition, the data show that significantly greater platelet count increases at 12–17 weeks (p=0.0022) and 26 weeks (p=0.0110) were achieved in aHUS patients treated with eculizumab (at 12 -17 weeks and after 26 weeks, respectively) with normalization of plasma Va concentration. Improvement in platelet count correlated with significant reductions in mean F1+2 levels at 4-6 weeks (P=0.0148; ss=-0.4087) and 12-17 weeks (P=0.0073; ss=-0.4396 ) and more moderately with a decrease in D-dimer levels after 12-17 weeks (P=0.0470; ss=-0.3381). However, a subset of patients, despite demonstrating greater increases in platelet counts after four to 26 weeks, continued to exhibit significantly elevated levels of prothrombin fragments 1+2, thrombomodulin, urinary B2M, clusterin, TIMP-1, and cystatin C, suggesting underlying is based on constant disease activity.

Analysis of the data obtained from the study also revealed a correlation between changes in the concentrations of other protein biomarkers and platelet recovery. For example, the concentrations of CCL5, MCP-1 and SCD40L were positively correlated with increased platelet counts in patients treated with eculizumab, as shown in Table 1. 9 below.

Table 9

Week after starting treatment with eculizumab Biomarker P value Coeff. correlations CCL-5 p<0.0001 0.7419 1-2.5 SCD40L P=0.0141 0.3950 VEGF P=0.0014 0.5002 CCL-5 p<0.0001 0.7743 4-6 SCD40L p<0.0001 0.6818 MSR-1 P=0.0114 0.4169 12-17 CCL5 P=0.0003 0.5656 26 CCL-5 SCD40L p<0.0001 P=0.0012 0.7845 0.5398

Thrombomicroangiopathy (TMA).

eculizumab-treated aHUS patients demonstrating greater reductions in plasma Ba levels were more likely to achieve a complete TMA response (eg, normalization of hematologic parameters (eg, platelets and LDH levels) and preservation of renal function). For example, 72.7% of patients achieved a complete TMA response at 12–17 weeks and 85.29% of patients achieved a complete TMA response at 26 weeks. As shown in FIG. 12, these patients showed a higher mean percentage decrease in plasma Ba concentrations than patients who did not achieve a complete TMA response (p = 0.0018 and p = 0.006, respectively).

eGFR after treatment.

A relationship between reduction and/or normalization of some biomarkers and improvement in eGFR was also observed. For example, a significantly greater improvement (Table 10) in eGFR (eg, eGFR>15 ml/min/1.73 m ² maintained for at least two consecutive measurements taken at least four times apart weeks) was observed in patients with normalized MCP-1, IL-6 and IFN-γ (after 4-6 weeks); normalized VCAM-1, CXCL10, CXCL9 and Ba (after 12-17 weeks) and normalized Va, urine B2M, urine CysC, vWF, D-dimer, clusterin, CXCL10, CXCL9, urine FABP-1 and others (after 26 weeks) (Table 10). See also FIG. 16.

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Table 10

One week after starting eculizumab treatment Normalized biomarker P value 1-2.5 * * 4-6 MSR-1 0.0002 IL-6 0.0251 VCAM-1 0.0166 12-17 VCAM-1 0.0003 CXCL-10 0.0071 Va 0.0299 CXCL-9 0.0441 26 VCAM-1 <0.0001 Cystatin C <0.0001 Va 0.0002 U-p2m 0.0013 CXCL9 0.0027 CXCL10 0.0172 vWF 0.0052 D-dimer 0.0224 L-FABP 0.0230 Clusterin 0.0300 F1+2 0.0460

Example 3. Baseline levels of aHUS selective protein biomarkers in aHUS patients.

At baseline before eculizumab treatment, real evidence of significant complement activation, vascular inflammation/damage, and organ damage was observed in patients with aHUS, regardless of the use of plasma exchange/plasma infusion or normal laboratory values of platelet count, Hp, or LDH. As can be seen from the data presented in table. 11, in aHUS, concentrations of biomarkers of complement activity, vascular inflammation, endothelial activation, and renal injury, coagulation, and injury were significantly increased in aHUS patients compared with healthy individuals.

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Table 11

Pathogenesis of the disease Biomarker (NHV range; units) Median level at BL [range] (P value relative to NHV**) n/N (%) increase above B.L. Increase ratio over NHV on BL Activation of the ATS Plasma Va (388.0-588.0 ng/ml) 2676.4 [935.0-3668.0] (<0.0001) 35/35 (100) 5.53 Terminal activation of complement U-C5a (0.0-0.7 ng/mg Ucreat.) 9.00 [0.3-76.6] (0.0007) 26/29 (89.7) 45 U-sC5b-9 (0.0-0.6 ng/mg U-creat.) 30.50 [0.2-665.7] (0.0025) 23/27 (85.2) 305 Inflammation sTNFRl (407.3-1391.3 pg/ml) 17616.85 [4008.5-54158.2] (<0.0001) 38/38 (100) 18.71 Endothelial activation sVCAM-1 (159.2-444.7 ng/ml) 659.75 [375.4-1865.5] (<0.0001) 36/38 (94.7) 1.99 Endothelial damage TM (2.0-3.6 ng/ml) 10 [3.4-24.1] (<0.0001) 33/34 (97.1) 3, 64 Clotting F1+2 (82.9-305.5 pmol/l) 1017.55 [217.7-5774.0] (<0.0001) 36/38 (94.7) 5.46 D-dimer (157.0-395.9 µg/l) 2735 [330.0-44100.0] (0.0002) 34/36 (94.4) 9, 84 Damage kidney U-clusterin (5.7-437.1 ng/mg Ucreat.) 1232.30 [129.9-6091.2] (<0.0001) 24/29 (82.8) 8, 62 U-TIMP-1 (0.0-5.4 ng/mg U-creat.) 23.8 [1.4-230.4] (0.0003) 22/29 (75.9) 39, 67 U-L-FABP-1 (0.0-16.9 ng/mg Ucreat.) 58.00 [3.7-1309.8] (0.0130) 22/29 (75.9) 48.33 υ-β2ιη (0.0-2.7 µg/mg Ucreat.) 18.4 [0.4-127.7] (<0.0001) 20/28 (71.4) 46 U-cystatin-C (0.3-301.3 ng/mg Ucreat.)) 1256.9 [14.3-7189.6] (0.0001) 18/26 (69.2) 23.85

NHV denotes the value or concentration of a given protein biomarker aHUS, presented in the table, for a normal person;

creat. denotes creatinine, the concentration of which is used to normalize the concentrations of certain biomarkers presented in the table;

CAP belongs to the alternative complement pathway (see above);

BL refers to the baseline level, i.e. before starting eculizumab treatment;

N represents the total number of patients examined for a given pathological process and biomarker, n represents the number of patients N in whom this biomarker was elevated;

U indicates that the analyte was measured in urine;

* P values were calculated using the Wilcoxon signed rank test when testing for differences between groups.

In addition, the inventors found that there were no statistically significant differences between the baseline elevated levels of certain aHUS biomarkers detected in patients who were or are being treated with plasmapheresis (PE) or plasma infusion (PI), compared with the level of increase in aHUS biomarkers in patients who were not treated with PE or PI. For example, the concentrations of Ba, sTNFRl, sVCAM-1 and D-dimer were not reduced or normalized in patients treated with PE/PI (Fig. 17A-17D). It should be noted that only 3 of the 26 patients used for analysis in the data presented in FIG. 17A-17D did not perform PE/PI. The majority of patients (n=23) had elevated levels of cystatin C compared to normal healthy volunteers. Given that cystatin C is a biomarker of kidney damage (glomerular damage), it can be assumed that patients who did not undergo PE/PI had less damage to their kidneys and thus showed reduced levels of protein biomarkers associated with kidney damage in their urine .

Similarly, at baseline before eculizumab treatment, concentrations of protein markers of complement activation (eg, Ba), inflammation (eg, sTNFRl), endothelial cell activation (sVCAM-1), coagulation (D-dimer), and kidney damage (cystatin-C) were increased in

- 57 044587 aHUS patients with normal platelet counts. See fig. 18A-18E. And in patients with normal levels of Hp and LDH, the presence of constant complement activation, inflammation, endothelial cell activation, coagulation and kidney damage is shown (see Fig. 19A-19E).

In light of the above, the concentrations of biomarkers reflecting complement activity, vascular inflammation, endothelial activation and damage, coagulation, and kidney damage are chronically elevated in aHUS patients compared to normal healthy individuals. In aHUS patients treated with PE/PI, there is compelling evidence of significant, persistent complement activation, vascular inflammation, endothelial activation, coagulation, and renal injury. While PE/PI may temporarily maintain normal platelet counts and LDH in some patients, the above results indicate that persistent complement dysregulation and TMA processes persist. Despite normal laboratory values of platelet count, LDH, and Hp, these studies indicate that significant persistent complement activation, vascular inflammation, endothelial activation, coagulation, and renal injury exist in patients with aHUS.

Example 4: Effect of chronic eculizumab treatment on aHUS biomarkers.

Concentrations.

The inventors found that chronic treatment with eculizumab inhibited kidney injury mediated by chronically elevated complement and terminal complement activation and reduced inflammation, endothelial damage, and thrombotic risk in patients with aHUS. For example, chronic treatment with eculizumab rapidly and completely inhibits terminal complement activation, resulting in decreased concentrations of both C5a and sC5b-9 (eg, urinary C5a and sC5b-9). See fig. 20A, 20V. At baseline, aHUS patients showed significant terminal complement activation compared with NHV, despite PE/PI use or normal platelet counts in some patients. Indeed, aHUS patients demonstrated 45-fold higher urinary C5a levels and 305-fold higher urinary sC5b-9 levels compared with NHV. However, during long-term treatment with eculizumab, all aHUS patients demonstrated rapid and potent blockade of terminal complement, with complete normalization of pathogenic end products of complement activation and no difference in levels compared with NHV.

In addition, chronic treatment with eculizumab normalized the concentrations of protein biomarkers of kidney damage (Fig. 21A-21C). Before starting eculizumab therapy, most patients had elevated levels of biomarkers: interstitial tubular damage and renal depletion (eg, L-FABP-1 ~48 times higher than NHV), glomerular filtration rate (eg, cystatin C, ~24 times higher than NHV), proximal tubule damage (e.g., clusterin is 8.6 times higher than NHV). However, chronic treatment with eculizumab dramatically reduced urinary concentrations of FABP1 (by 100%), cystatin C (by 99%), and clusterin (by 98%). These reductions were significant at all time points (P<0.0001 for all), and the reduced concentrations of all markers of kidney damage were not different from those of NHV. Additional markers of kidney damage (eg, TIMP-1 and β2-microglobulin) also normalized (see example 2 above). These results suggest that ischemia and organ injury may be entirely dependent on terminal complement and support clinical data demonstrating that chronic inhibition of the complement-mediated TMA system leads to clinically significant improvement in eGFR and cessation of dialysis.

Chronic treatment with eculizumab also significantly reduced activation of the alternative complement pathway (see Fig. 22). All aHUS patients demonstrated significant systemic activation of CAP above C5, with 5.5-fold higher Ba levels compared to NHV before eculizumab treatment. However, after initiation of eculizumab treatment, concentrations of biomarkers of upstream SAR activation (eg, Ba levels) were reduced by 30%, and the reduction at 4–6 weeks was significant at all time points (p<0.005) compared with marker concentrations in NHV. However, Ba levels were not normalized in aHUS patients treated with eculizumab, suggesting that CAP activation persists, reflecting an underlying complement dysregulation in aHUS patients. To be clear, however, terminal complement blockade with eculizumab protected patients from the clinical consequences of ongoing SAR activation.

In addition, chronic treatment of aHUS patients with eculizumab resulted in significant reductions in the concentrations of biomarkers associated with inflammation, endothelial activation, and tissue damage (Figures 23A-23C). Serum sTNFRl levels were elevated (18.7 times higher than NHV levels) in 100% of aHUS patients at baseline. Chronic eculizumab treatment significantly reduced sTNFRl to 94%. The decrease in concentrations of these biomarkers after 4-6 weeks was significant at all time points (P<0.0001). Soluble VCAM-1 and TM levels were increased in >95% of aHUS patients at baseline by 2-fold and 3.6-fold, respectively, compared with NHV, demonstrating significant endothelial cell activation and damage before eculizumab treatment. Concentrations of HM and sVCAM-1 were also significantly decreased during eculizumab treatment. At 12–17 weeks, the reduction in concentrations of biomarkers of endothelial damage was significant at all time points (TM; P < 0.0001), but they were still moderately elevated compared with NHV. The sharply reduced levels of soluble TM may reflect restoration of membrane-bound TM, which protects against thrombotic risk.

Finally, chronic treatment with eculizumab rapidly and significantly reduced the concentrations of biomarkers associated with thrombotic risk and coagulation (Fig. 24A, 24B). Concentrations of coagulation biomarkers F1+2 and D-dimer were significantly increased (5.5-fold and 9.8-fold higher than NHV) at baseline in more than 94% of aHUS patients (P<0.0001 and P=0 .0002, respectively). However, F1+2 and D-dimer were significantly reduced 2.5 weeks after initiation of eculizumab treatment. F1+2 concentrations were reduced by 88% (P<0.05 for all time points) and D-dimer concentrations were reduced by 99% (P<0.0001 for all time points) with chronic eculizumab treatment. However, these two markers remained moderately elevated compared with corresponding concentrations in normal healthy individuals.

Summary.

In view of the above data, the authors were able to draw a number of conclusions. First, aHUS patients showed elevated levels of all thrombotic microangiopathy (TMA) biomarkers at baseline compared with levels in samples from normal healthy volunteers (NHV). All groups of patients, including patients receiving PE/PI or patients with normal platelets, Hp or LDH, aHUS patients showed a significant increase compared with NHVs when measured by: terminal complement activation (45-305 times higher than NHV levels) ; vital organ damage; alternative pathway of complement activation (eg, represented by Ba levels; 5.5 times higher than NHV levels); inflammation of blood vessels; activation and damage to the endothelium and coagulation.

Chronic eculizumab treatment of aHUS patients significantly reduced and normalized significantly elevated markers of terminal complement activation. Inhibition of terminal complement activation by eculizumab also significantly reduced and normalized markers of organ damage. Upstream biomarkers of alternative pathway activation were also significantly reduced but not normalized. And low levels of alternative pathway activation persisted in treated patients, reflecting an underlying complement dysregulation in aHUS patients. However, the data clearly show that terminal complement blockade with eculizumab protects aHUS patients from the clinical consequences of ongoing alternative pathway activation.

In addition, chronic eculizumab treatment also resulted in (i) significant and sustained reductions in vascular inflammatory markers (up to 94%); (ii) significant inhibition of endothelial activation markers (up to 60%); (iii) a significant and sustained reduction in markers of endothelial damage (up to 77%) to near normal levels, demonstrating a clear relationship between terminal complement activation and endothelial damage; and (iv) a marked reduction (up to 99%) in the concentration of thrombotic risk biomarkers, likely reducing the likelihood of clot formation and thereby reducing the incidence of TMA in these patients. The inventors conclude, without being bound by any theory or mechanism of action, that the inhibition of terminal complement activation by eculizumab should be permanent, since loss of terminal complement inhibition in aHUS will result in a rapid increase in dangerously elevated terminal complement activation, subsequently leading to an increase in the underlying subclinical endothelial activation, a significant acceleration of endothelial damage, a marked increase in thrombotic risk, and an early and ongoing risk of catastrophic vascular ischemia and damage to vital organs. In addition, these data indicate that kidney injury, vascular inflammation, and endothelial damage and activation are, in whole or in part, dependent on the activity of terminal complement, whose activity is effectively and safely inhibited by eculizumab.

Although the present disclosure has been described with reference to specific embodiments thereof, those skilled in the art will understand that various changes may be made and equivalents may be substituted for the embodiments without departing from the true spirit and scope of the disclosure. In addition, many modifications may be made to adapt a particular situation, substances, compositions of substances, method, or method step or steps within the spirit and scope of the present invention. All such modifications are considered to be within the scope of the disclosure.

Claims (3)

1. A method of treating a patient having atypical hemolytic uremic syndrome (aHUS) who has been determined to have elevated levels of at least two biomarkers associated with aHUS, wherein the method comprises administering to the patient a C5 complement inhibitor at a frequency sufficient to reduce the levels of at least two biomarkers associated with aHUS compared to levels measured in a sample of the same type of body fluid obtained from the subject prior to treatment with a C5 complement inhibitor, and where at least two of the biomarkers associated with aHUS are (1) a proteolytic fragment of a factor component complement B and soluble C5b9 (sC5b9); - 59 044587 (2) TNFR1 and proteolytic fragment of complement factor B; or (3) TNFR1 and soluble C5b9 (sC5b9). 2. A method of treating a patient with atypical hemolytic uremic syndrome (aHUS), in whose urine the content of an increased level of a proteolytic fragment of the factor B component of complement and soluble C5b9 (sC5b9) was determined, where the method includes administering to the patient a complement inhibitor C5 in an amount and with a frequency, sufficient to reduce the concentrations of the proteolytic fragment of complement factor B and soluble C5b9 (sC5b9) compared to the concentrations measured in the patient's urine before treatment with a complement C5 inhibitor. 3. A method of treating a patient having atypical hemolytic uremic syndrome (aHUS), whose blood has been determined to contain elevated levels of TNFR1 and whose urine has been determined to contain elevated levels of a proteolytic fragment of complement factor B or soluble C5b9 (sC5b9), where the method includes administering to the patient a complement inhibitor C5 in an amount and at a frequency sufficient to reduce the concentrations of TNFR1 and proteolytic fragment of complement factor B or soluble C5b9 (sC5b9) compared to the concentrations measured in the patient before treatment with a complement C5 inhibitor. 4. A method for monitoring a patient’s response to treatment with a C5 complement inhibitor according to the method according to claim 2, where the method includes determining the concentrations of the proteolytic fragment of the factor B component of the complement and soluble C5b9 (sC5b9) in the patient’s urine before and after treatment, with reduced concentrations of the factor B component complement and soluble C5b9 (sC5b9) measured after treatment, compared with concentrations measured before treatment, indicate that the patient is responding to treatment. 5. A method for monitoring a patient's response to treatment with a C5 complement inhibitor according to the method of claim 3, where the method includes determining concentrations of (1) TNFR1 in the patient's blood before and after treatment; and (2) a proteolytic fragment of complement factor B or soluble C5b9 (sC5b9) in the patient's urine before and after treatment, where reduced concentrations of TNFR1 and complement factor B or soluble C5b9 (sC5b9) measured after treatment compared with concentrations measured before treatment indicate that the patient is responding to treatment. 6. The method according to claim 1, where the biological fluid is urine. 7. The method according to claim 1, where the biological fluid is blood. 8. The method according to any one of claims 1 to 3, where the factor B of the complement component is Ba. 9. The method according to any one of claims 1-3, where the method further includes determining the concentration of one or more additional biomarker proteins associated with aHUS, selected from the group consisting of MCP-1, IFN-γ, IL-6, prothrombin fragment F1+2, d-dimer, thrombomodulin, VCAM-1, von Willebrand factor (vWF), complement component C5a, β2 microglobulin (v2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 ( FABP-1), albumin, CXCL9, KIM-1 and CCL5, soluble CD40 ligand (sCD40L), ICAM-1, IL-1 beta, IL-12 p70, IL-8 and vascular endothelial cell growth factor (VEGF). 10. The method according to any one of claims 1 to 3, wherein the patient (a) underwent dialysis at least once within three months immediately before treatment with a complement C5 inhibitor; or (b) experiences the first acute manifestation of aHUS. 11. The method according to any one of claims 1-3, where the decrease in concentration occurs within two weeks or two months after the first administration of the C5 complement inhibitor. 12. The method according to any one of claims 1 to 3, wherein the patient is chronically treated with a C5 complement inhibitor. 13. The method according to any one of claims 1 to 3, wherein the C5 complement inhibitor is selected from the group consisting of a small molecule, a polypeptide, a polypeptide analogue, a peptidomimetic and an aptamer. 14. The method according to any one of claims 1 to 3, wherein the C5 complement inhibitor is selected from the group consisting of MB12/22, MB12/22-RGD, ARC187, ARC1905, SSL7 and OmCI. 15. The method according to any one of claims 1 to 3, wherein the C5 complement inhibitor is an antibody or an antigen-binding fragment thereof. 16. The method of claim 15, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a humanized antibody, a recombinant antibody, a diabody, a chimerized or chimeric antibody, a monoclonal antibody, a deimmunized antibody, a fully human antibody, a single chain antibody, an Fv fragment, a fragment Fd, Fab fragment, Fab' fragment and F(ab')2 fragment. 17. The method according to claim 15, where the antibody or its antigen-binding fragment binds to the complement component C5 and inhibits the cleavage of C5 into fragments C5a and C5b. 18. The method of claim 15, wherein the antibody is eculizumab or a variant of eculizumab. 19. The method of claim 15, wherein the antigen binding moiety is pexelizumab. - 60 044587 20. The method according to any one of claims 1 to 3, wherein the concentrations are measured using an enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay (RIA). 21. The method according to any one of claims 1 to 3, where the normal concentration of sC5b-9 is from 0 to 0.6 ng/mg creatinine in urine. 22. The method of claim 21, wherein the patient's sC5b-9 concentration is considered elevated if it is at least ten times the normal sC5b-9 concentration. 23. The method of claim 21, wherein the patient's sC5b-9 concentration is considered elevated if it is at least 20 ng/mg creatinine in the urine. 24. The method according to claim 8, where the normal concentration of Ba is less than 1000 ng/ml. 25. The method of claim 24, wherein the patient's Ba concentration is considered elevated if it is at least twice the normal Ba concentration. 26. The method according to claim 2 or 3, where the normal concentration of TNFR1 is less than 2000 pg/ml. 27. The method of claim 26, wherein the patient's TNFR1 concentration is considered elevated if it is (i) at least twice the normal sTNFR1 concentration; or (ii) at least 10,000 pg/ml. 28. The method of claim 1, wherein biomarkers associated with aHUS are measured using the biomarker/creatinine ratio [Cr]/[UPCR]. 29. A method of treating complement-mediated thrombotic microangiopathy (TMA) in a human subject having atypical hemolytic uremic syndrome (aHUS) who has been determined to have elevated levels of at least two biomarkers associated with aHUS, wherein the method comprises administering to the patient a C5 complement inhibitor at a frequency sufficient to reduce the levels of at least two biomarkers associated with aHUS compared with levels measured in a sample of the same type of body fluid obtained from the subject prior to treatment with a C5 complement inhibitor, and where at least two biomarkers associated with aHUS are (1) a proteolytic fragment of complement factor B and soluble C5b9 (sC5b9); (2) TNFR1 and complement factor B proteolytic fragment; or (3) TNFR1 and soluble C5b9 (sC5b9).