WO2025221479A1

WO2025221479A1 - Biomarkers for monitoring effective treatment of neuromyelitis optica spectrum disorder (nmosd) with complement component c5 inhibitors

Info

Publication number: WO2025221479A1
Application number: PCT/US2025/023106
Authority: WO
Inventors: Ruba Deeb BOU-CHAHINE
Original assignee: Alexion Pharmaceuticals Inc
Current assignee: Alexion Pharmaceuticals Inc
Priority date: 2024-04-16
Filing date: 2025-04-04
Publication date: 2025-10-23
Anticipated expiration: 2026-10-16

Abstract

The disclosure provides biomarker proteins, a change in the concentration of which are associated with neuromyelitis optica spectrum disorder (NMOSD) or clinically meaningful treatment of NMOSD with a complement component C5 inhibitor. The compositions and methods are useful for, among other things, evaluating risk for developing NMOSD, diagnosing NMOSD, monitoring progression or abatement of NMOSD, or monitoring response to treatment with a complement component C5 inhibitor.

Description

BIOMARKERS FOR MONITORING EFFECTIVE TREATMENT OF NEUROMYELITIS OPTICA SPECTRUM DISORDER (NMOSD) WITH COMPLEMENT COMPONENT C5 INHIBITORS

BACKGROUND

Neuromyelitis optica spectrum disorder (NMOSD), including neuromyelitis optica (NMO), also known as Devic’s Disease, or Devic’s Syndrome, is a class of rare, severe disabling autoimmune inflammatory disorders of the central nervous system (CNS) that predominately affects the optic nerves and spinal cord, often leading to blindness, mono/para/tetraplegia, and respiratory failure. NMOSD is characterized by a relapsing disease course, from which recovery may be poor due to the stepwise accumulation of significant neurologic disability.

The clinical hallmarks of NMO are acute optic neuritis and transverse myelitis that frequently involve greater than three vertebral levels, described as longitudinally extensive transverse myelitis (LETM). These clinical events can occur either simultaneously or in isolation. Signs and symptoms attributable to lesions beyond the optic nerves and spinal cord can also occur in patients with NMO and are reported in about 15% of patients. The clinical presentation of NMO can be quite variable and may elude diagnosis at the time of the first attack or even the second attack.

Aquaporin-4 (AQP4) is a water channel protein expressed in the CNS, mainly by astrocytes. AQP4 immunoglobulin G (IgG) , an antibody presents in 65-88% of patients with NMOSD, is the first ever biomarker specific to an inflammatory, demyelinating CNS disorder. Preclinical data indicate that AQP4- IgG triggers the complement cascade, leading to inflammation and formation of the complement- mediated membrane attack complex (MAC). AQP4-lgG-triggered MAC has been implicated in astrocyte destruction and bystander neuronal injury but is not seen in the presence of a complement inhibitor. With the discovery of NMO-IgG, the diagnostic criteria for NMO were revised in 2006 to include the testing of this disease-specific antibody.

In light of the fact that NMO is a disorder that has the potential to cause significant disability, the ability to recognize and differentiate NMO and related disorders from other demyelinating disorders is important from a clinical perspective. The prognosis of relapsing NMO is poor. The 5-year mortality of NMO was reported to be 30%; 50% sustain permanent severe disability, visual (blind in one or both eyes) or ambulatory (requiring a wheelchair). Most deaths result from neurogenic respiratory failure secondary to a high cervical cord or brainstem lesion. Frequent early relapses predict a poor prognosis. Relapse prevention is thus the primary therapeutic imperative.

Although treatment options for NMO have recently been approved (Soliris® (eculizumab) and Ultomiris® (ravulizumab)), standard treatment options still include steroids and other immunosuppressive agents as supportive treatments based on clinical experience and consensus. Acute NMO relapses are generally treated with high-dose IV steroids with plasma exchange (PE) often used as a rescue therapy for those who do not respond. Supportive treatments against relapse currently use broad spectrum or selective B-lymphocyte immunosuppressants.

Of the immunosuppressive agents, corticosteroid, AZA, mycophenolate mofetile and rituximab are probably most commonly used for long-term prophylaxis. Depending on regional medical options, the supportive medications option for NMO may vary. In the US, options include corticosteroids, AZA, MMF, rituximab and mitoxantrone, whereas corticosteroids including oral prednisone or pulse-high dose steroids (IV) are common treatments in Japan. A significant number of patients (>50%) will continue to have attacks resulting in additional and permanent neurologic deficits and disability. Given the seriousness of the disease and the limited options for treatment, there remains a significant unmet medical need for an effective and safe treatment for NMO as well as monitoring the effectiveness of the treatment.

SUMMARY OF THE INVENTION

In an aspect, the disclosure provides a method for monitoring responsiveness of a subject to treatment with an inhibitor of complement component C5. In some embodiments, the method includes: determining the concentration of a first Neuromyelitis Optica Spectrum Disorder (NMOSD)-associated biomarker protein in a biological fluid obtained from the subject, wherein the first NMOSD-associated biomarker protein is Glial fibrillary protein (GFAP), wherein the subject has, is suspected of having, or is at risk for developing Aquaporin-4 Antibody-positive (AQP4-Ab+) NMOSD, wherein the subject has been or is being treated with an inhibitor of complement component C5, and wherein a reduced concentration of GFAP, as compared to the concentration in a baseline sample of biological fluid of the same type obtained from the subject prior to treatment with the complement component C5 inhibitor, indicates that the subject is responsive to treatment with the complement component C5 inhibitor. In some embodiments, the method further comprises determining the concentration of a second NMOSD- associated biomarker protein in a biological fluid obtained from the subject, wherein the second NMOSD- associated biomarker protein is Neurofilament light chain (NfL), wherein a reduced concentration of NfL, as compared to the concentration in a baseline sample of biological fluid of the same type obtained from the subject prior to treatment with the complement component C5 inhibitor, indicates that the subject is responsive to treatment with the complement component C5 inhibitor.

In an aspect, the disclosure provides a method for monitoring responsiveness of a subject to treatment with an inhibitor of complement component C5. In some embodiments, the method comprises: determining the concentration of a first NMOSD-associated biomarker protein in a biological fluid obtained from the subject, wherein the first NMOSD-associated biomarker protein is NfL, wherein the subject has, is suspected of having, or is at risk for developing AQP4-Ab+ NMOSD, wherein the subject has been or is being treated with an inhibitor of complement component C5, and wherein a reduced concentration of NfL, as compared to the concentration in a baseline sample of biological fluid of the same type obtained from the subject prior to treatment with the complement component C5 inhibitor, indicates that the subject is responsive to treatment with the complement component C5 inhibitor.

In some embodiments, the method further comprises determining the concentration of a second NMOSD-associated biomarker protein in a biological fluid obtained from the subject, wherein the second NMOSD-associated biomarker protein is GFAP, wherein a reduced concentration of GFAP, as compared to the concentration in a baseline sample of biological fluid of the same type obtained from the subject prior to treatment with the complement component C5 inhibitor, indicates that the subject is responsive to treatment with the complement component C5 inhibitor. In some embodiments, the biological fluid is blood, a blood fraction, or cerebrospinal fluid. In some embodiments, the blood fraction is serum or plasma. In some embodiments, the concentration is determined using a ligand binding assay. In some embodiments, the ligand binding assay is a digital bead-based immunoassay.

In some embodiments, the concentration of GFAP and/or NfL is reduced by at least 10% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) relative to the baseline sample. In some embodiments, the concentration of GFAP and/or NfL is reduced by at least 20% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) relative to the baseline sample. In some embodiments, the concentration of GFAP and/or NfL is reduced by at least 30% (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, or 90%) relative to the baseline sample. In some embodiments, the determining is performed approximately 2 weeks after the commencement of the treatment. In some embodiments, the determining is performed approximately 4 weeks after the commencement of the treatment. In some embodiments, the determining is performed approximately 6 weeks after the commencement of the treatment.

In some embodiments, the inhibitor of complement component C5 is an antibody or antigenbinding fragment of an antibody, a small molecule, a polypeptide, a polypeptide analog, a peptidomimetic, an aptamer, or a combination thereof. In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a humanized antibody, a recombinant antibody, a camelid antibody, a diabody, a chimeric antibody, a monoclonal antibody, a fully human antibody, a single chain antibody, an Fv fragment, an Fd fragment, an Fab fragment, an Fab' fragment, an F(ab')2 fragment, and an engineered polypeptide comprising a complement component C5-binding VHH construct. In some embodiments, the antibody or antigen-binding fragment thereof binds to complement component C5 and inhibits cleavage of C5 into fragments C5a and C5b. In some embodiments, the antibody is eculizumab or ravulizumab. In some embodiments, the antibody is a biosimilar of eculizumab. In some embodiments, the antibody is pexelizumab. In some embodiments, the antibody is tesidolumab, crovalimab, omoprubart (CAN106), or pozelimab. In some embodiments, the complement component C5 inhibitor is selected from the group consisting of KP-104, avacincaptad pegol (ARC1905), MB12/22, MB12/22-RGD, ARC187, SSL7, and Omithodoros moubata C inhibitor (OmCI).

In an aspect, the disclosure provides a method of treating AQP4-Ab+ NMOSD in a subject using an inhibitor of complement component C5 in a manner sufficient to reduce the concentration of at least one NMOSD-associated biomarker protein. In some embodiments, the method comprises: (a) determining the concentration of at least a first NMOSD-associated biomarker protein in a biological fluid obtained from the subject, wherein the NMOSD-associated biomarker protein is GFAP, and (b) administering to the subject having, suspected of having, or at risk for developing AQP4-Ab+ NMOSD an inhibitor of complement component C5 in an amount and with a frequency sufficient to cause a reduction in concentration of GFAP, as compared to the concentration in a baseline sample of biological fluid of the same type obtained from the subject prior to treatment with the inhibitor of complement component C5. In some embodiments, (a) further comprises determining the concentration of a second NMOSD- associated biomarker protein in a biological fluid obtained from the subject, wherein the second NMOSD- associated biomarker protein is NfL and (b) comprises administering to the subject having, suspected of having, or at risk for developing AQP4-Ab+ NMOSD an inhibitor of complement component C5 in an amount and with a frequency sufficient to cause a reduction in concentration of NfL, as compared to the concentration in a baseline sample of biological fluid of the same type obtained from the subject prior to treatment with the inhibitor of complement component C5.

In an aspect, the disclosure provides a method of treating AQP4-Ab+ NMOSD in a subject using an inhibitor of complement component C5 in a manner sufficient to reduce the concentration of at least one NMOSD-associated biomarker protein. In some embodiments, the method comprises: (a) determining the concentration of a first NMOSD-associated biomarker protein in a biological fluid obtained from the subject, wherein the NMOSD-associated biomarker protein is NfL, and (b) administering to the subject having, suspected of having, or at risk for developing AQP4-Ab+ NMOSD an inhibitor of complement component C5 in an amount and with a frequency sufficient to cause a reduction in concentration of NfL, as compared to the concentration in a baseline sample of biological fluid of the same type obtained from the subject prior to treatment with the inhibitor of complement component C5. In some embodiments, (a) further comprises determining the concentration of a second NMOSD- associated biomarker protein in a biological fluid obtained from the subject, wherein the second NMOSD- associated biomarker protein is GFAP, and (b) comprises administering to the subject having, suspected of having, or at risk for developing AQP4-Ab+ NMOSD an inhibitor of complement component C5 in an amount and with a frequency sufficient to cause a reduction in concentration of GFAP, as compared to the concentration in a baseline sample of biological fluid of the same type obtained from the subject prior to treatment with the inhibitor of complement component C5.

In some embodiments, the biological fluid is blood, a blood fraction, or cerebrospinal fluid. In some embodiments, the blood fraction is serum or plasma. In some embodiments, the concentration is determined using a ligand binding assay. In some embodiments, the ligand binding assay is a digital bead-based immunoassay.

In some embodiments, the concentration of GFAP and/or NfL is reduced by at least 10% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) relative to the baseline sample. In some embodiments, the concentration of GFAP and/or NfL is reduced by at least 20% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) relative to the baseline sample. In some embodiments, the concentration of GFAP and/or NfL is reduced by at least 30% (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, or 90%) relative to the baseline sample.

In some embodiments, the determining is performed approximately 2 weeks after the commencement of the treatment. In some embodiments, the determining is performed approximately 4 weeks after the commencement of the treatment. In some embodiments, the determining is performed approximately 6 weeks after the commencement of the treatment.

In some embodiments, the inhibitor of complement component C5 is an antibody or antigenbinding fragment of an antibody, a small molecule, a polypeptide, a polypeptide analog, a peptidomimetic, an aptamer, or a combination thereof. In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a humanized antibody, a recombinant antibody, a camelid antibody, a diabody, a chimeric antibody, a monoclonal antibody, a fully human antibody, a single chain antibody, an Fv fragment, an Fd fragment, an Fab fragment, an Fab' fragment, an F(ab')2 fragment, and an engineered polypeptide comprising a complement component C5-binding VHH construct. In some embodiments, the antibody or antigen-binding fragment thereof binds to complement component C5 and inhibits cleavage of C5 into fragments C5a and C5b. In some embodiments, the antibody is eculizumab or ravulizumab. In some embodiments, the antibody is a biosimilar of eculizumab. In some embodiments, the antibody is pexelizumab. In some embodiments, the antibody is tesidolumab, crovalimab, omoprubart (CAN106), or pozelimab.

In some embodiments, the complement component C5 inhibitor is selected from the group consisting of KP-104, avacincaptad pegol (ARC1905), MB12/22, MB12/22-RGD, ARC187, SSL7, and Omithodoros moubata C inhibitor (OmCI).

In some embodiments, the inhibitor of complement component C5 is eculizumab, the subject is an adult patient, and eculizumab is administered using a phased dosing schedule with an induction phase comprising administering a 900 mg induction dose of eculizumab on day 1 , administering 900 mg doses of eculizumab on days 7, 14, and 21 , and administering 1200 mg of eculizumab as a fifth induction dose on day 28; wherein the 28 day induction phase of eculizumab treatment is followed by a maintenance phase comprising administering 1200 mg of eculizumab 14 days after the fifth induction dose and administering 1200 mg of eculizumab every 14 ± 2 days thereafter, wherein the induction dosing (a) and maintenance dosing (b) are both administered intravenously (IV) to the adult patient. In some embodiments, the inhibitor of complement component C5 is ravulizumab, and the administering comprising administering ravulizumab: (a) once on Day 1 at a loading dose of: (i) 2400 mg to a subject weighing > 40 and < 60 kg; (ii) 2700 mg to a subject weighing > 60 and < 100 kg; (iii) 3000 mg to a subject weighing > 100 kg; (b) on Day 15 and every eight weeks thereafter at a maintenance dose of: (i) 3000 mg to a subject weighing > 40 and < 60 kg; (ii) 3300 mg to a subject weighing > 60 and < 100 kg; (iii) 3600 mg to a subject weighing > 100 kg. In some embodiments, the loading dose (a) and the maintenance dosing (b) are administered intravenously (IV).

In an aspect, the disclosure provides a method for predicting a risk of developing or for monitoring a clinical phenotype selected from (a) attack and/or (b) clinical disability of AQP4-Ab+ NMOSD in a subject, wherein the subject is a patient having or being at risk of having AQP4-Ab+ NMOSD. In some embodiments, the method comprises determining the concentration of NMOSD-associated biomarker proteins GFAP and NfL in a biological fluid of the subject, and comparing the concentration the NMOSD-associated biomarker proteins to the concentration thereof in a baseline sample of biological fluid of the same type obtained from a control, wherein an elevated concentration the NMOSD-associated biomarker proteins in the subject’s sample compared to the concentration thereof in the baseline sample obtained from the control indicates that the subject is at risk of developing or has the clinical phenotype of AQP4-Ab+ NMOSD. In some embodiments, the GFAP concentration is elevated by at least 40%, 50%, 60%, 70%, 80%, 90%, 100% or more in the subject’s sample compared to the control; and the NfL concentration is elevated by at least 27%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more in the subject’s sample compared to the control. In some embodiments, the concentration of GFAP and/or NfL is elevated by at least 200% in the subject’s sample compared to the control.

In some embodiments, the subject has optic neuritis and/or transverse myelitis. In some embodiments, the subject is a relapsed patient with NMOSD. In some embodiments, the GFAP concentration and the NfL concentration are elevated, relative to the control, in the relapsed NMOSD patient, and the relapsed NMOSD patient has optic neuritis and/or transverse myelitis. In some embodiments, the GFAP concentration is attenuated in a subject treated with eculizumab. In some embodiments, the GFAP concentration is significantly attenuated (e.g., attenuated by at least 10%, 20%, 30%, 40%, 50%, or 60%), relative to the control, after week 4 of the eculizumab treatment. In some embodiments, the GFAP concentration is significantly attenuated, relative to the control, after week 24 of the eculizumab treatment.

In some embodiments, the subject is an adult patient, and wherein eculizumab is administered using a phased dosing schedule with an induction phase comprising administering a 900 mg induction dose of eculizumab on day 1 , administering 900 mg doses of eculizumab on days 7, 14, and 21 , and administering 1200 mg of eculizumab as a fifth induction dose on day 28; wherein the 28 day induction phase of eculizumab treatment is followed by a maintenance phase comprising administering 1200 mg of eculizumab 14 days after the fifth induction dose and administering 1200 mg of eculizumab every 14 ± 2 days thereafter, wherein the induction dosing (a) and maintenance dosing (b) are both administered intravenously (IV) to the adult patient.

In some embodiments, the GFAP concentration and the NfL concentration are both attenuated in a subject treated with ravulizumab. In some embodiments, the GFAP concentration and the NfL concentration are significantly attenuated (e.g., attenuated by at least 10%, 20%, 30%, 40%, 50%, or 60%), relative to the control, at around week 50 of ravulizumab treatment. In some embodiments, the GFAP concentration and the NfL concentration are significantly attenuated, relative to the control, at around week 130 of ravulizumab treatment.

In some embodiments, the ravulizumab treatment comprises treating an adult NMOSD patient with an administration cycle comprising administering ravulizumab: (a) once on Day 1 at a loading dose of: (i) 2400 mg to a subject weighing > 40 and < 60 kg; (ii) 2700 mg to a subject weighing > 60 and < 100 kg; (iii) 3000 mg to a subject weighing > 100 kg; (b) on Day 15 and every eight weeks thereafter at a maintenance dose of: (i) 3000 mg to a subject weighing > 40 and < 60 kg; (ii) 3300 mg to a subject weighing > 60 and < 100 kg; (iii) 3600 mg to a subject weighing > 100 kg. In some embodiments, the loading dose (a) and the maintenance dosing (b) are administered intravenously (IV). In some embodiments, the GFAP concentration is attenuated at an earlier timepoint of ravulizumab treatment than the NfL concentration. In some embodiments, the GFAP concentration is attenuated after week 2 of ravulizumab treatment and the NfL concentration is attenuated after week 26 of ravulizumab treatment.

In some embodiments, the biological fluid is blood, a blood fraction, or cerebrospinal fluid. In some embodiments, the blood fraction is serum or plasma. In some embodiments, the method further includes measuring a clinical profile of the subject and/or the control. In some embodiments, the clinical profile is measurement of astrocyte and neuron damage. In some embodiments, the subject is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

The application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawings will be provided by the Office upon request and payment of the necessary fee.

FIG. 1A and FIG. 1B are graphs showing the serum concentration of Glial fibrillary acidic protein (GFAP) for healthy donors (HD) and AQP4-Ab+ patients having NMOSD that participated in the PREVENT (FIG. 1 A) or CHAMPION-NMOSD (FIG. 1 B) studies prior to administration of eculizumab or ravulizumab, respectively. Plots show all data points, bars indicate median, and error bars indicate upper 95% confidence limit; two-tailed Rvalues were derived using Mann-Whitney test.

FIG. 2A and FIG. 2B are graphs showing the serum concentration of Neurofilament light chain (NfL) for healthy donors and AQP4-Ab+ patients having NMOSD that participated in the PREVENT (FIG. 1 A) or CHAMPION-NMOSD (FIG. 1 B) studies prior to administration of eculizumab or ravulizumab, respectively. Plots show all data points, bars indicate median, and error bars indicate upper 95% confidence limit; two-tailed Rvalues were derived using Mann-Whitney test.

FIG. 3A and FIG. 3B are graphs showing the change in serum GFAP levels for patients experiencing adjudicated on-trial relapse for patients in the placebo group (FIG. 3A) or who were administered eculizumab (FIG. 3B). Plots show all data points, boxes indicate 25th percentile, median, and 75th percentile, whiskers indicate min and max, and lines connect individual patient values from pretreatment baseline to relapse.

FIG. 4A and FIG. 4B are graphs showing the serum concentration of GFAP (FIG. 4A) and the serum concentration of NfL (FIG. 4B) over time for the placebo group in comparison to patients who were administered eculizumab. Plots show all data points, bars indicate median, and error bars indicate upper 95% confidence limit; two-tailed Rvalues were derived using Wilcoxon test.

FIG. 5A and FIG. 5B are graphs showing the serum concentration of GFAP (FIG. 5A) and the serum concentration of NfL (FIG. 5B) over time for the placebo group in comparison to patients who were administered ravulizumab. Plots show all data points, bars indicate median, and error bars indicate upper 95% confidence limit; two-tailed Rvalues were derived using Wilcoxon test.

FIG. 6 shows the change in serum concentrations of GFAP and NfL over 130 weeks for patients who were administered ravulizumab. Plot shows means ± %CV (coefficient of variation).

FIG. 7A and FIG. 7B are graphs showing the change in NfL levels found in the cerebral spinal fluid (FIG. 7A) and serum (FIG. 7B) of four patients over time who were administered ravulizumab. Lines represent individual patients.

DETAILED DESCRIPTION

As described herein, the inventors identified biomarkers associated with neuromyelitis optica spectrum disorder (NMOSD). For example, it has been discovered that a concentration of certain proteins is associated with the presence of NMOSD. Similarly, a reduced concentration of certain proteins in a biological fluid obtained from a subject that is suspected of having or is at risk for developing Aquaporin-4 Antibody-positive (AQP4-Ab+) NMOSD treated with a complement inhibitor indicates that the subject has responded to therapy with a complement component C5 inhibitor. Accordingly, analysis of the concentration of such proteins can be employed to evaluate, among other things, risk for NMOSD, diagnose NMOSD, monitor progression or abatement of C5, or monitor treatment response to a complement component C5 inhibitor.

Definitions

As used herein, the term “subject” or “patient” is a human patient (e.g., a patient having neuromyelitis optica spectrum disorder (NMOSD)). As used herein, the term “subject” and “patient” are interchangeable. As used herein, the term “attenuated” refers to a reduction in the measured level of one or more of the NMOSD-associated protein biomarkers in a subject relative to the level of the one or more NMOSD-associated protein biomarker prior to administration of the complement component C5 inhibitor to the subject. For example, the NMOSD-associated protein biomarker is attenuated in a subject after administration of the complement component C5 inhibitor if the concentration of the NMOSD-associated protein biomarker is reduced by at least 10%.

As used herein, the phrase “requires chronic plasma exchange” to maintain clinical stability refers to the use of plasma exchange therapy on a patient on a regular basis for the management of muscle weakness at least every 3 months over the last 12 months.

As used herein, “effective treatment” refers to treatment producing a beneficial effect, e.g., amelioration of at least one symptom of a disease or disorder. A beneficial effect can take the form of an improvement over baseline, i.e., an improvement over a measurement or observation made prior to initiation of therapy according to the method. Effective treatment may refer to alleviation of at least one symptom of NMOSD.

The term “effective amount” refers to an amount of an agent that provides the desired biological, therapeutic and/or prophylactic result. That result can be reduction, amelioration, palliation, lessening, delaying and/or alleviation of one or more of the signs, symptoms or causes of a disease, or any other desired alteration of a biological system. In one example, an “effective amount” is the amount of anti-C5 antibody or antigen binding fragment thereof clinically proven to alleviate at least one symptom of NMOSD. An effective amount can be administered in one or more administrations.

As used herein, the terms “induction” and “induction phase” are used interchangeably and refer to the first phase of treatment in the clinical trial.

As used herein, the terms “maintenance” and “maintenance phase” are used interchangeably and refer to the second phase of treatment in the clinical trial. In certain embodiments, treatment is continued as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs. The maintenance phase of ravulizumab dosing can last for between 6 weeks and the life of the subject. According to other embodiments, the maintenance phase lasts for 26-52, 26-78, 26-104, 26-130, 26-156, 26-182, 26-208 weeks, or more. Similarly, the maintenance phase of eculizumab dosing can last for between 6 weeks and the life of the subject. According to other embodiments, the maintenance phase lasts for 26-52, 26-78, 26-104, 26-130, 26-156, 26-182, 26-208 weeks, or more. In other embodiments, the maintenance phase lasts for greater than 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 78, 104, 130, 156, or 182 weeks. According to other embodiments, the maintenance phase lasts for greater than 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more years. In certain embodiments, the maintenance phase lasts for the remainder of the subject's life.

In some embodiments, the method comprises treating NMOSD wherein eculizumab is administered in a multiphase dosing regime, wherein the multiphase dosing regimen comprises a first phase and a second phase, wherein the multiphase dosing regimen further comprises a third phase, wherein the third phase comprises the performance of plasmapheresis on the subject and administration of eculizumab at greater than 400 and less than 1200 mg to the subject within 2 hours of the completion of plasmapheresis. In some embodiments, the method comprises treating NMOSD wherein eculizumab is administered in a multiphase dosing regime, wherein the multiphase dosing regimen comprises a first phase and a second phase, wherein the multiphase dosing regimen further comprises a third phase, wherein the third phase comprises the performance of plasmapheresis on the subject and administration of eculizumab at greater than 400 and less than 1200 mg to the subject within 2 hours of the completion of plasmapheresis, wherein the third phase comprises the performance of plasmapheresis on the subject and administration of eculizumab at greater than 500 and less than 800 mg to the subject within 90 minutes of the completion of plasmapheresis.

In some embodiments, the method comprises treating NMOSD wherein eculizumab is administered in a multiphase dosing regime, wherein the multiphase dosing regimen comprises a first phase and a second phase, wherein the multiphase dosing regimen further comprises a third phase, wherein the third phase comprises the performance of plasmapheresis on the subject and administration of eculizumab at greater than 400 and less than 1200 mg to the subject within 2 hours of the completion of plasmapheresis, wherein the third phase comprises the performance of plasmapheresis on the subject and administration of eculizumab at greater than 500 and less than 800 mg to the subject within 90 minutes of the completion of plasmapheresis, wherein the third phase comprises the performance of plasmapheresis on the subject and administration of eculizumab at about 600 mg to the subject within 60 minutes of the completion of plasmapheresis.

In certain embodiments, the ravulizumab multiphase dosing regimen includes a third phase. This third phase is used when an NMOSD patient must undergo a rescue procedure to maintain clinical stability and includes administering plasma exchange/plasmapheresis (PE/PP). In this phase after plasma is exchanged a dose of ravulizumab is administered to replace the drug lost in plasma exchange/plasmapheresis. According to certain embodiments, supplemental study drug (or placebo) dosing is required if PE/PP rescue therapy is provided on non-dosing days. In another embodiment, if PE/PP infusion is provided on a dosing day, it must occur prior to study drug administration. In some embodiments, if PE/PP is administered on nonscheduled dosing visits, patients receiving PE/PP are administered a supplemental dose within 1 , 2, 3, 4, 5, 6, 7 or 8 hours after the PE/PP session is completed. In some embodiments, if PE/PP is administered on nonscheduled dosing visits, patients receiving PE/PP are administered a supplemental dose within 4 hours after the PE/PP session is completed. In certain embodiments, supplemental dose amounts may or may not vary depending on PE/PP. In other embodiments, if PE/PP is administered on scheduled dosing visits, regular dosing is followed 60 minutes after the completion of PE/PP. In certain embodiments, no gap is required between a supplemental dose and the regular scheduled dose.

In some embodiments, the supplemental dose of ravulizumab is administered at between 1000 and 2000 mg. In some embodiments, the supplemental dose of ravulizumab is administered at about half the most recent loading or maintenance dose of ravulizumab. In some embodiments, if the most recent loading dose is between 2200 mg and 3000 mg of ravulizumab, the supplemental dose is 1000-1500 mg of ravulizumab. In some embodiments, if the most recent loading dose is about 2400 mg of ravulizumab, the supplemental dose is about 1200 mg of ravulizumab. In some embodiments, if the most recent loading dose is 2400 mg of ravulizumab, the supplemental dose is 1200 mg of ravulizumab. In some embodiments, if the most recent loading dose is about 2700 mg of ravulizumab, the supplemental dose is about 1500 mg of ravulizumab. In some embodiments, if the most recent loading dose is 2700 mg of ravulizumab, the supplemental dose is 1500 mg of ravulizumab. In some embodiments, if the most recent loading dose is about 3000 mg of ravulizumab, the supplemental dose is about 1500 mg of ravulizumab. In some embodiments, if the most recent loading dose is 3000 mg of ravulizumab, the supplemental dose is 1500 mg of ravulizumab. In some embodiments, if the most recent maintenance dose is about 3000 mg of ravulizumab, the supplemental dose is about 1500 mg of ravulizumab. In some embodiments, if the most maintenance loading dose is 3000 mg of ravulizumab, the supplemental dose is 1500 mg of ravulizumab. In some embodiments, if the most recent maintenance dose is about 3300 mg of ravulizumab, the supplemental dose is about 1800 mg of ravulizumab. In some embodiments, if the most maintenance loading dose is 3300 mg of ravulizumab, the supplemental dose is 1800 mg of ravulizumab. In some embodiments, if the most recent maintenance dose is about 3600 mg of ravulizumab, the supplemental dose is about 1800 mg of ravulizumab. In some embodiments, if the most maintenance loading dose is 3600 mg of ravulizumab, the supplemental dose is 1800 mg of ravulizumab.

As used herein, the terms “loading dose” refers to the initial dose administered to the patient. In some embodiments, the loading dose is 2000-4000 mg of ravulizumab. In some embodiments, the loading dose is 2100-2700 mg, 2400-3000 mg or 2700-3300 mg of ravulizumab. In some embodiments, the loading dose is 2300-2500 mg, 2600-2800 mg or 2900-3100 mg of ravulizumab. In some embodiments, the loading dose is about 2400 mg, about 2700 mg, or about 3000 mg of ravulizumab. In some embodiments, the loading dose is 2400 mg, 2700 mg, or 3000 mg of ravulizumab. Loading doses may be titered based on body weight.

In some embodiments, patients with a body weight greater than or equal to 40 kg, but less than 60 kg are administered 2100-2700 mg, 2300-2500 mg, about 2400 mg or 2400 mg of ravulizumab. In some embodiments, patients with a body weight greater than or equal to 60 kg, but less than 100 kg are administered 2400-3000 mg, 2600-2800 mg, about 2700 mg or 2700 mg of ravulizumab. In some embodiments, patients with a body weight greater than 100 kg are administered 2700-3300 mg, 2900- 3100 mg, about 3000 mg or 3000 mg of ravulizumab.

As used herein, the terms “maintenance dose” or “maintenance phase” refers to a dose administered to the patient after the loading dose. In some embodiments, the loading dose is 2000-4000 mg of ravulizumab. In some embodiments, the loading dose is 2800-3200 mg, 3100-3500 mg or 3400- 3800 mg of ravulizumab. In some embodiments, the loading dose is 2900-3100 mg, 3200-3400 mg or 3500-3700 mg of ravulizumab. In some embodiments, the loading dose is about 3000 mg, about 3300 mg, or about 3600 mg of ravulizumab. In some embodiments, the loading dose is 3000 mg, 3300 mg, or 3600 mg of ravulizumab. Maintenance doses may be titered based on body weight.

In some embodiments, patients with a body weight greater than or equal to 40 kg, but less than 60 kg are administered 2800-3200 mg, 2900-3100 mg, about 3000 mg or 3000 mg of ravulizumab. In some embodiments, patients with a body weight greater than or equal to 60 kg, but less than 100 kg are administered 3100-3500 mg, 3200-3400 mg, about 3300 mg or 3300 mg of ravulizumab. In some embodiments, patients with a body weight greater than 100 kg are administered 3400-3800 mg, 3500- 3700 mg, about 3600 mg or 3600 mg of ravulizumab.

In some embodiments, the eculizumab multiphase dosing regimen has two phases. The first phase is an induction phase. This phase provides a dose of 600 or 900 mg per week. In some embodiments, this phase lasts for 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks. In some embodiments, this phase lasts between 2 and 6 weeks. In some embodiments, the phase lasts for 5 weeks. According to some embodiments, the phase given any week is higher than the previous week. In some embodiments, the dose remains the same for a number of weeks and is then increased. In some embodiments the dose remains the same for the first 1 , 2, 3, 4, 5, 6, 7, 8 or 9 weeks and is then increased. In some embodiments, the dose remains the same for the first 4 weeks. According to some embodiments, the eculizumab dose is between 600 and 1200 mg, 800 and 1500 mg, 900 and 1200 mg, 900 and 1100 mg, 900 and 1000 mg, 800 and 1000 mg, 800 and 1100 mg or 800 and 1200 mg for a number of weeks and is then increased. In one embodiment, the eculizumab dose is about 900 mg on day 1 , followed 900 mg on day 7, 900 mg on day 14, 900 mg on day 21 and then increased to 1200 mg for the fifth dose on day 28 and then 1200 mg is administered every 14 ± 2 days thereafter.

As used herein, the term “serum trough level” refers to the lowest level that the agent (e.g., the anti-C5 antibody, or antigen binding fragment thereof) or medicine is present in the serum. In contrast, a “peak serum level,” refers to the highest level of the agent in the serum. The “average serum level,” refers to the mean level of the agent in the serum over time.

In one embodiment, the treatment regimens described are sufficient to maintain particular serum trough concentrations of the anti-C5 antibody or antigen binding fragment thereof. In one embodiment, for example, the treatment maintains a serum trough concentration of the anti-C5 antibody, or antigen binding fragment thereof, of 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200, 205, 210, 215, 220, 225, 230, 240, 245, 250, 255, 260, 265, 270, 280, 290, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395 or 400 pg/mL or greater. In one embodiment, the treatment maintains a serum trough concentration of the anti-C5 antibody, or antigen binding fragment thereof, of 100 pg/mL or greater. In another embodiment, the treatment maintains a serum trough concentration of the anti-C5 antibody or antigen binding fragment thereof of 150 pg/mL or greater. In another embodiment, the treatment maintains a serum trough concentration of the anti-C5 antibody or antigen binding fragment thereof of 200 pg/mL or greater. In another embodiment, the treatment maintains a serum trough concentration of the anti-C5 antibody, or antigen binding fragment thereof, of 250 pg/mL or greater. In another embodiment, the treatment maintains a serum trough concentration of the anti-C5 antibody, or antigen binding fragment thereof, of 300 pg/mL or greater. In another embodiment, the treatment maintains a serum trough concentration of the anti-C5 antibody, or antigen binding fragment thereof, of between 100 pg/mL and 200 pg/mL. In another embodiment, the treatment maintains a serum trough concentration of the anti-C5 antibody, or antigen binding fragment thereof, of about 175 pg/mL.

In another embodiment, to obtain an effective response, the anti-C5 antibody is administered to the patient in an amount and with a frequency to maintain at least 50 pg, 55 pg, 60 pg, 65 pg, 70 pg, 75 pg, 80 pg, 85 pg, 90 pg, 95 pg, 100 pg, 105 pg, 110 pg, 115 pg, 120 pg, 125 pg, 130 pg, 135 pg, 140 pg, 145 pg, 150 pg, 155 pg, 160 pg_a 165 pg, 170 pg_a 175 pg, 180 pg, 185 pg, 190 pg_a 195 pg, 200 pg, 205 pg, 210 pg, 215 pg, 220 pg, 225 pg, 230 pg, 235 pg, 240 pg, 245 pg, 250 pg, 255 pg, or 260 pg of antibody per milliliter of the patient’s blood. In another embodiment, the anti-C5 antibody is administered to the patient in an amount and with a frequency to maintain between 50 pg and 250 pg of antibody per milliliter of the patient’s blood. In another embodiment, the anti-C5 antibody is administered to the patient in an amount and with a frequency to maintain between 100 pg and 200 pg of antibody per milliliter of the patient’s blood. In another embodiment, the anti-C5 antibody is administered to the patient in an amount and with a frequency to maintain about 175 pg of antibody per milliliter of the patient’s blood.

In another embodiment, to obtain an effective response, the anti-C5 antibody is administered to the patient in an amount and with a frequency to maintain a minimum free C5 concentration. In one embodiment, for example, the anti-C5 antibody is administered to the patient in an amount and with a frequency to maintain a free C5 concentration of 0.2 pg/mL, 0.3 pg/mL, 0.4 pg/mL, 0.5 pg/mL or below. In another embodiment, the anti-C5 antibody is administered to the patient in an amount and with a frequency to maintain a free C5 concentration of 0.309 to 0.5 pg/mL or below. In another embodiment, the treatment described herein reduces free C5 concentration by greater than 99% throughout the treatment period. In another embodiment, the treatment reduces free C5 concentration greater than 99.5% throughout the treatment period.

The term “terminal complement inhibition” refers to the inhibition of the late stage of the complement cascade. In one embodiment, terminal complement inhibition refers to inhibition of complement component 5 (“C5”) from being cleaved by the C5 convertase into C5a and C5b.

The term “antibody” describes polypeptides comprising at least one antibody derived antigen binding site {e.g., VH/VL region or Fv, or CDR). Antibodies include known forms of antibodies. The antibody, for example, can be a human antibody, a humanized antibody, a camelid antibody, a bispecific antibody or a chimeric antibody. The antibody also can be a Fab, Fab’2, ScFv, SMIP, Affibody, nanobody or a domain antibody. The antibody also can be of any of the following isotypes: IgG 1 , lgG2, lgG3, lgG4, IgM, lgA1 , Ig A2, IgAsec, IgD and IgE. The antibody may be a naturally occurring antibody or may be an antibody that has been altered by a protein engineering technique {e.g., by mutation, deletion, substitution, conjugation to a non-antibody moiety). An antibody may include, for example, one or more variant amino acids (compared to a naturally occurring antibody), which changes a property {e.g., a functional property) of the antibody. Numerous such alterations are known in the art that affect, e.g., half-life, effector function, and/or immune responses to the antibody in a patient. The term antibody also includes artificial or engineered polypeptide constructs that comprise at least one antibody-derived antigen binding site.

NMOSD- Associated Protein Biomarkers

A current unmet need in the field of AQP4-Ab+ NMOSD is sensitive and specific clinical measures of cellular events that correlate with clinical efficacy. Lumbar puncture and Magnetic Resonance Imaging (MRI) are common diagnostic and monitoring tools for patients with AQP4-Ab+ NMOSD. Less invasive and more available monitoring options are necessary. Serum biomarkers that allow for frequent longitudinal monitoring during treatment would inform health care practitioners of objective cell-specific, disease-relevant treatment responses. The disclosure is based, at least in part, on the inventors’ discovery of the reduction of serum GFAP and NfL levels over time in complement component C5 inhibitor treated patients with AQP4-Ab+ NMOSD. Similar analysis of cerebrospinal fluid (CSF) samples from a subset of patients confirmed that changes in these serum biomarkers were mirrored in the CSF. Particularly, it was identified that glial fibrillary acidic protein (GFAP) and neurofilament light chain (NfL) acted as NMSOD-associated protein biomarkers. GFAP and NfL are intracellular proteins expressed by glial cells (e.g., astrocytes) and neurons, respectively. Upon damage to expressing cells, these proteins can be shed into surrounding biofluids whereby concentrations can be determined by analytical methods. Astrocytes are the primary target of AQP4-Ab-mediated tissue damage in the central nervous system of patients with NMOSD, where neuronal loss also occurs. As a result of such cellular injury, elevated levels of GFAP and NfL proteins may be observed in CSF and serum of patients with AQP4-Ab+ NMOSD. Described here are methods for longitudinal assessment of serum concentrations of GFAP and NfL as surrogate biomarkers to monitor response to treatment with a complement component C5 inhibitor in AQP4-Ab+ NMOSD patients. Likewise, characterization of the NMOSD-associated protein markers may be useful in evaluating the risk for a subject to develop NMOSD, diagnosing NMOSD in a subject, monitoring progression or abatement of NMOSD in a subject, or monitoring response to treatment with a complement component C5 inhibitor.

The disclosure provides herein methods of method of treating AQP4-Ab+ NMOSD in a subject using an inhibitor of complement component C5 in a manner sufficient to reduce the concentration of at least one NMOSD-associated biomarker proteins. The method may include determining the concentration of a NMOSD- associated biomarker protein in a biological fluid obtained from the subject, wherein the NMOSD- associated biomarker proteins comprises GFAP and/or NfL, and administering to the subject having, suspected of having, or at risk for developing AQP4-Ab+ NMOSD an inhibitor of complement component C5 in an amount and with a frequency sufficient to cause a reduction in concentration of the NMOSD-associated biomarker protein, as compared to the concentration in a baseline sample of biological fluid of the same type obtained from the subject prior to treatment with the inhibitor of complement component C5.

Furthermore, the disclosure provides a method for prognosticating risk of developing or monitoring clinical phenotype selected from (a) attack and/or (b) clinical disability of AQP4-Ab+ Neuromyelitis Optica Spectrum Disorder (NMOSD), in a subject. In such instances, the subject is a patient having or at risk of having AQP4-Ab+ NMOSD. The method includes determining the concentration or level of signature biomarker proteins GFAP and NfL in a biological fluid of the subject and comparing said concentration or levels of said signature biomarker proteins to the concentration or level thereof in a baseline sample of biological fluid of the same type obtained from a control. An elevated concentration or level of said signature biomarker proteins in the subject’s sample compared to the concentration or level thereof in the baseline sample obtained from the control indicates that the subject is at risk of developing or has said clinical phenotype of AQP4-Ab+ NMOSD. In some embodiments, GFAP levels are elevated by at least 40% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more, e.g., by at least 200%) in the subject’s sample compared to the control. In some embodiments, NfL levels are elevated by at least 27% (e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more, e.g., by at least 200%) in the subject’s sample compared to the control. In some embodiments, the subject has optic neuritis. In some embodiments, the subject has transverse myelitis. The subject may be a relapsed patient with NMOSD. In some embodiments, the GFAP levels and NfL levels are elevated, relative to a control.

The disclosure also provides methods for monitoring responsiveness of a subject to treatment with an inhibitor of complement component C5. The method may be performed by measuring the concentration of NMOSD-associated biomarker protein in a biological fluid obtained from the subject. The NMOSD-associated biomarker proteins may be GFAP. The NMOSD-associated biomarker protein may be an NfL protein. In some embodiments, the subject that is monitored for responsiveness has aquaporin-4 antibody-positive (AQP4-Ab+) NMOSD. In some embodiments, the subject being monitored for responsiveness is suspected of having AQP4-Ab+ NMOSD. In some embodiments, the subject being monitored for responsiveness is at risk for developing AQP4-Ab+) NMOSD. In the methods described herein, the subject has been or is being treated with an inhibitor of complement component C5. The subject determined to be responsive to treatment with the complement component C5 inhibitor if the concentration of the NMOSD biomarker is reduced, as compared to the concentration in a baseline sample of biological fluid of the same type obtained from the subject prior to treatment with the inhibitor complement component C5 inhibitor.

The concentration of GFAP may be reduced by at least 10% relative to the baseline sample of biological fluid of the same type obtained from the subject prior to treatment with the inhibitor complement component C5 inhibitor. For example, the concentration of GFAP may be reduced by at least 20% relative to the baseline sample. In some embodiments, the concentration of GFAP is reduced by at least 30% relative to the baseline sample. In some embodiments, the concentration of GFAP is reduced by at least 40% relative to the baseline sample. In some embodiments, the concentration of GFAP is reduced by at least 50% relative to the baseline sample. In some embodiments, the concentration of GFAP is reduced by at least 60% relative to the baseline sample. In some embodiments, the concentration of GFAP is reduced by at least 70% relative to the baseline sample. In some embodiments, the concentration of GFAP is reduced by at least 80% relative to the baseline sample. In some embodiments, the concentration of GFAP is reduced by at least 90% relative to the baseline sample. The concentration of GFAP in the biological fluid may be determined approximately 2 weeks after the commencement of the treatment with the complement component C5 inhibitor. In some embodiments, the concentration of GFAP is determined approximately 4 weeks after the commencement of the treatment with the complement component C5 inhibitor. The concentration of GFAP may be measured approximately 6 weeks after the commencement of the treatment with the complement component C5 inhibitor. The concentration of GFAP may be measured approximately 8 weeks after the commencement of the treatment with the complement component C5 inhibitor. The concentration of GFAP may be measured approximately 12 weeks after the commencement of the treatment with the complement component C5 inhibitor. The concentration of GFAP may be measured approximately 20 weeks after the commencement of the treatment with the complement component C5 inhibitor. In some embodiments, the concentration of GFAP is measured approximately 24 weeks after the commencement of treatment with the complement component C5 inhibitor. In some embodiments, the concentration of GFAP is measured approximately 26 weeks after the commencement of treatment with the complement component C5 inhibitor. In some embodiments, the concentration of GFAP is measured approximately 30 weeks after the commencement of treatment with the complement component C5 inhibitor. In some embodiments, the concentration of GFAP is measured approximately 40 weeks after the commencement of treatment with the complement component C5 inhibitor. In some embodiments, the concentration of GFAP is measured approximately 52 weeks after the commencement of treatment with the complement component C5 inhibitor. The concentration of NfL may be reduced by at least 10% relative to the baseline sample of biological fluid of the same type obtained from the subject prior to treatment with the inhibitor complement component C5 inhibitor. For example, the concentration of NfL may be reduced by at least 20% relative to the baseline sample. In some embodiments, the concentration of NfL is reduced by at least 30% relative to the baseline sample. In some embodiments, the concentration of NfL is reduced by at least 40% relative to the baseline sample. In some embodiments, the concentration of NfL is reduced by at least 50% relative to the baseline sample. In some embodiments, the concentration of NfL is reduced by at least 60% relative to the baseline sample. In some embodiments, the concentration of NfL is reduced by at least 70% relative to the baseline sample. In some embodiments, the concentration of NfL is reduced by at least 80% relative to the baseline sample. In some embodiments, the concentration of NfL is reduced by at least 90% relative to the baseline sample.

The concentration of NfL in the biological fluid may be determined approximately 2 weeks after the commencement of the treatment with the complement component C5 inhibitor. In some embodiments, the concentration of NfL is determined approximately 4 weeks after the commencement of the treatment with the complement component C5 inhibitor. The concentration of NfL may be measured approximately 6 weeks after the commencement of the treatment with the complement component C5 inhibitor. The concentration of NfL may be measured approximately 8 weeks after the commencement of the treatment with the complement component C5 inhibitor. The concentration of NfL may be measured approximately 12 weeks after the commencement of the treatment with the complement component C5 inhibitor. The concentration of NfL may be measured approximately 20 weeks after the commencement of the treatment with the complement component C5 inhibitor. In some embodiments, the concentration of NfL is measured approximately 24 weeks after the commencement of treatment with the complement component C5 inhibitor. In some embodiments, the concentration of NfL is measured approximately 26 weeks after the commencement of treatment with the complement component C5 inhibitor. In some embodiments, the concentration of NfL is measured approximately 30 weeks after the commencement of treatment with the complement component C5 inhibitor. In some embodiments, the concentration of NfL is measured approximately 40 weeks after the commencement of treatment with the complement component C5 inhibitor. In some embodiments, the concentration of NfL is measured approximately 52 weeks after the commencement of treatment with the complement component C5 inhibitor.

The GFAP levels may be attenuated in the subject following administration of eculizumab. The GFAP levels may be significantly attenuated, relative to a control, after 4 weeks of eculizumab administration. In some embodiments, the GFAP level may be significantly attenuated after 6 weeks. In some embodiments, the GFAP level may be significantly attenuated after 8 weeks. In some embodiments, the GFAP level may be significantly attenuated after 12 weeks. In some embodiments, the GFAP levels may be significantly attenuated after 24 weeks of eculizumab administration. In some embodiments, the GFAP level may be significantly attenuated after 26 weeks. In some embodiments, the GFAP level may be significantly attenuated after 30 weeks. In some embodiments, the GFAP level may be significantly attenuated after 40 weeks. In some embodiments, the GFAP level may be significantly attenuated after 52 weeks.

The NfL levels may be attenuated in the subject following administration of eculizumab. The NfL levels may be significantly attenuated, relative to a control, after 4 weeks of eculizumab administration. In some embodiments, the NfL level may be significantly attenuated after 6 weeks. In some embodiments, the NfL level may be significantly attenuated after 8 weeks. In some embodiments, the NfL level may be significantly attenuated after 12 weeks. In some embodiments, the NfL levels may be significantly attenuated after 24 weeks of eculizumab administration. In some embodiments, the NfL level may be significantly attenuated after 26 weeks. In some embodiments, the NfL level may be significantly attenuated after 30 weeks. In some embodiments, the NfL level may be significantly attenuated after 40 weeks. In some embodiments, the NfL level may be significantly attenuated after 52 weeks. The GFAP levels and NfL levels may both be attenuated in the subject following administration of eculizumab or ravulizumab. The GFAP levels and NfL levels may be significantly attenuated, relative to a control, after 50 weeks of eculizumab or ravulizumab administration. In some embodiments, the GFAP levels and NfL levels may be significantly attenuated, relative to a control after 24 weeks of eculizumab or ravulizumab administration. In some embodiments, the GFAP levels and NfL levels may be significantly attenuated, relative to a control, after 12 weeks of eculizumab or ravulizumab administration. In some embodiments, the GFAP levels and NfL levels may be significantly attenuated, relative to a control, after 2 weeks of eculizumab or ravulizumab administration. In some embodiments, the GFAP levels and NfL levels may be significantly attenuated, relative to a control after 130 weeks of eculizumab or ravulizumab administration. GFAP levels may be attenuated in a subject after administration of ravulizumab and prior to NfL levels being attenuated at a subject after administration of eculizumab or ravulizumab. For example, GFAP levels may be attenuated in a subject about 2 weeks after administration of eculizumab or ravulizumab and NfL levels may be attenuated in a subject about 4 weeks after administration of eculizumab or ravulizumab.

In some embodiments, the disclosure relates to a composition for the treatment of neuromyelitis optica spectrum disorders (NMOSD), particularly aquaporin 4+ (AQP4+) NMOSD in a subject comprising administering an effective amount of a complement inhibitor, wherein the complement inhibitor is an anti- C5 antibody or a C5-binding fragment thereof, preferably eculizumab or ravulizumab, wherein the effective amount is an amount sufficient to reduce a level of a biomarker and/or biomarker signature in the subject compared to the level thereof prior to treatment with the complement inhibitor, wherein the biomarker comprises glial fibrillary acidic protein (GFAP) or neurofilament light chain (NfL), or a combination thereof.

The disclosure relates to a composition according to the foregoing or following, wherein the biomarker signature comprising at least two biomarkers comprising GFAP and NfL.

The disclosure relates to a composition according to the foregoing or following, wherein the levels of the biomarkers and/biomarker signature are detected in a fluid biological sample obtained from the subject, e.g., in blood, serum, plasma, CSF, or a combination thereof.

In some embodiments, the disclosure relates to use of an effective amount of a complement inhibitor, wherein the complement inhibitor is an anti-C5 antibody or a C5-binding fragment thereof, preferably eculizumab or ravulizumab, for the manufacture of a medicament for the treatment of neuromyelitis optica spectrum disorders (NMOSD), particularly aquaporin 4+ (AQP4+) NMOSD, wherein the effective amount of the composition is an amount sufficient to reduce the level of a biomarker and/or biomarker signature in the subject compared to the level thereof prior to treatment with the complement inhibitor, wherein the biomarker comprises glial fibrillary acidic protein (GFAP) or neurofilament light chain (NfL), or a combination thereof.

The disclosure relates to use according to the foregoing or following, wherein the biomarker signature comprising at least two biomarkers comprising GFAP and NfL.

The disclosure relates to use according to the foregoing or following, wherein the levels of the biomarkers and/biomarker signature are detected in a fluid biological sample obtained from the subject, e.g., in blood, serum, plasma, CSF, or a combination thereof.

Complement Component C5 Inhibitors

In the methods described herein, the subject has been or is being treated with an inhibitor of complement component C5. Any compound which binds to and inhibits, or otherwise inhibits, the generation or activity of any of the human complement components may be utilized in accordance with the present disclosure. For example, an inhibitor of complement can be, e.g., a small molecule, a nucleic acid or nucleic acid analog, a peptidomimetic, or a macromolecule that is not a nucleic acid or a 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, locked nucleic acid inhibitors, and peptide nucleic acid inhibitors. In some embodiments, a complement inhibitor may be a protein or protein fragment.

In some embodiments, the complement component C5 inhibitor is KP-104, avacincaptad pegol (ARC1905), MB12/22, MB12/22-RGD, ARC187, SSL7, or Omithodoros moubata C inhibitor (OmCI).

The antibody or antigen-binding fragment thereof may be a humanized antibody, a recombinant antibody, a camelid antibody, a diabody, a chimeric antibody, a monoclonal antibody, a fully human antibody, a single chain antibody, an Fv fragment, an Fd fragment, an Fab fragment, an Fab' fragment, an F(ab')2 fragment, and an engineered polypeptide comprising a complement component C5-binding VHH construct. The anti-C5 antibodies described herein bind to complement component C5 {e.g., human C5) and inhibit the cleavage of C5 into fragments C5a and C5b. Anti-C5 antibodies (or VH/VL domains derived therefrom) suitable for use in methods described herein can be generated using methods known in the art. Alternatively, art-recognized anti-C5 antibodies can be used. Antibodies that compete with any of these art-recognized antibodies for binding to C5 also can be used.

Eculizumab (also known as Soliris®) is an anti-C5 antibody comprising heavy and light chains having sequences shown in SEQ ID NO: 10 and 11 , respectively, or antigen binding fragments and variants thereof. Eculizumab is described in PCT/US2007/006606, the teachings of which are hereby incorporated by reference. In one embodiment the anti-C5 antibody, comprises the CDR1 , CDR2, and CDR3 domains of the VH region of eculizumab having the sequence set forth in SEQ ID NO: 7, and the CDR1 , CDR2 and CDR3 domains of the VL region of eculizumab having the sequence set forth in SEQ ID NO: 8. In another embodiment, the antibody comprises heavy chain CDR1 , CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 1 , 2, and 3, respectively, and light chain CDR1 , CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. In another embodiment, the antibody comprises VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. In some embodiments, the antibody is a biosimilar of eculizumab.

Ravulizumab (also known as BNJ441 , ALXN1210 or Ultomiris®) is an anti-C5 antibody comprising heavy and light chains having the sequences shown in SEQ ID NOs: 14 and 11 , respectively, or antigen binding fragments and variants thereof. Ravulizumab is described in PCT/US2015/019225 and US Patent No. 9,079,949, the teachings of which are hereby incorporated by reference. Ravulizumab selectively binds to human complement protein C5, inhibiting its cleavage to C5a and C5b during complement activation. This inhibition prevents the release of the proinflammatory mediator C5a and the formation of the cytolytic pore-forming membrane attack complex (MAC) C5b-9 while preserving the proximal or early components of complement activation (e.g., C3 and C3b) essential for the opsonization of microorganisms and clearance of immune complexes.

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

The antibody may be pexelizumab. The antibody may be tesidolumab, crovalimab, omoprubart (CAN106), or pozelimab.

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

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

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

Another exemplary anti-C5 antibody is the 305LO5 antibody described in US2016/0176954A1 . In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of the 305LO5 antibody. In another embodiment, the antibody, or antigen binding fragment thereof, comprises heavy chain CDR1 , CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively, and light chain CDR1 , CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 40, 41 , and 42, respectively. In another embodiment, the antibody comprises the VH region of the 305LO5 antibody having the sequence set forth in SEQ ID NO: 43, and the VL region of the 305LO5 antibody having the sequence set forth in SEQ ID NO: 44.

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

Another exemplary anti-C5 antibody is the H4H12166PP antibody described in PCT/US2017/037226 and US2017/0355757A1 . In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of the H4H12166PP antibody. In another embodiment, the antibody, or antigen binding fragment thereof, comprises the VH region of the H4H12166PP antibody having the sequence set forth in SEQ ID NO: 47, and the VL region of the H4H12166PP antibody having the sequence set forth in SEQ ID NO: 48. In another embodiment, the antibody, or antigen binding fragment thereof, comprises a heavy chain comprising SEQ ID NO: 49 and a light chain comprising SEQ ID NO: 50.

In one embodiment, the patient is treated with eculizumab and then switched to treatment with the 7086 antibody, the 8110 antibody, the 305LO5 antibody, the SKY59 antibody, the H4H12166PP antibody or ravulizumab. In another embodiment, the patient is switched from an anti-C5 antibody {e.g., eculizumab, the 7086 antibody, the 8110 antibody, the 305LO5 antibody, the SKY59 antibody, or the H4H12166PP antibody) to another anti-C5 antibody {e.g., ravulizumab) during the course of treatment. In a particular embodiment, the patient is switched from eculizumab to ravulizumab during the course of treatment.

In some embodiments, an anti-C5 antibody described herein comprises a heavy chain CDR1 comprising, or consisting of, the following amino acid sequence: GHIFSNYWIQ (SEQ ID NO: 19). In some embodiments, an anti-C5 antibody described herein comprises a heavy chain CDR2 comprising, or consisting of, the following amino acid sequence: EILPGSGHTEYTENFKD (SEQ ID NO: 18). In some embodiments, an anti-C5 antibody described herein comprises a heavy chain variable region comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGHIFSNYWIQWVRQAPGQGLEWMGEILPGSGHTEYTENFKDRV TMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSS (SEQ ID NO: 12).

In some embodiments, an anti-C5 antibody described herein comprises a light chain variable region comprising the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIK (SEQ ID NO:8).

An anti-C5 antibody described herein can, in some embodiments, comprise a variant human Fc constant region that binds to human neonatal Fc receptor (FcRn) with greater affinity than that of the native human Fc constant region from which the variant human Fc constant region was derived. For example, the Fc constant region can comprise one or more (e.g., two, three, four, five, six, seven, or eight or more) amino acid substitutions relative to the native human Fc constant region from which the variant human Fc constant region was derived. The substitutions can increase the binding affinity of an IgG antibody containing the variant Fc constant region to FcRn at pH 6.0, while maintaining the pH dependence of the interaction. Methods for testing whether one or more substitutions in the Fc constant region of an antibody increase the affinity of the Fc constant region for FcRn at pH 6.0 (while maintaining pH dependence of the interaction) are known in the art and exemplified in the working examples (PCT/US2015/019225 and US Patent No. 9,079,949 the disclosures of each of which are incorporated herein by reference in their entirety).

Substitutions that enhance the binding affinity of an antibody Fc constant region for FcRn are known in the art and include, e.g., (1 ) the M252Y/S254T/T256E triple substitution described by Dall’Acqua, W. et al. (J. Biol. Chem., 281 :23514-24, 2006); (2) the M428L or T250Q/M428L substitutions described in Hinton, P. et al. (J. Biol. Chem., 279:6213-6, 2004) and Hinton, P. et al. (J. Immunol., 176:346-56, 2006); and (3) the N434A or T307/E380A/N434A substitutions described in Petkova, S. et al. (Int. Immunol., 18:1759-69, 2006). Additional substitution pairings: P257I/Q3111, P257I/N434H, and D376V/N434H have also been described (Datta-Mannan, A. etal., J. Biol. Chem., 282:1709-17, 2007, the disclosure of which is incorporated herein by reference in its entirety).

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

In some embodiments, the variant Fc constant region comprises no more than 30 (e.g., no more than 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, nine, eight, seven, six, five, four, three, or two) amino acid substitutions, insertions, or deletions relative to the native constant region from which it was derived. In some embodiments, the variant Fc constant region comprises one or more amino acid substitutions selected from the group consisting of: M252Y, S254T, T256E, N434S, M428L, V259I, T250I, and V308F. In some embodiments, the variant human Fc constant region comprises a methionine at position 428 and an asparagine at position 434, each in EU numbering. In some embodiments, the variant Fc constant region comprises a 428L/434S double substitution as described in, e.g., U.S. Patent No. 8,088,376.

In some embodiments the precise location of these mutations may be shifted from the native human Fc constant region position due to antibody engineering. The 428L/434S double substitution when used in a lgG2/4 chimeric Fc, for example, may correspond to 429L and 435S as in the M429L and N435S variants found in BNJ441 (ravulizumab) and described in US Patent Number 9,079,949, the disclosure of which is incorporated herein by reference in its entirety.

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

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

In one embodiment, the antibody binds to C5 at pH 7.4 and 25 °C (and, otherwise, under physiologic conditions) with an affinity dissociation constant (KD) that is at least 0.1 (e.g., at least 0.15, 0.175, 0.2, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, or 0.975) nM. In some embodiments, the KD of the anti-C5 antibody, or antigen binding fragment thereof, is no greater than 1 nM (e.g., no greater than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2 nM).

In other embodiments, the [(KD of the antibody for C5 at pH 6.0 at 25 °C)/(KD of the antibody for C5 at pH 7.4 at 25 °C)] is greater than 21 (e.g., greater than 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, or 8000).

Methods for determining whether an antibody binds to a protein antigen and/or the affinity for an antibody to a protein antigen are known in the art. The binding of an antibody to a protein antigen, for example, can be detected and/or quantified using a variety of techniques such as, but not limited to, Western blot, dot blot, surface plasmon resonance (SPR) method (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.), tissue-based indirect immunofluorescence (HE), cell-based assay (CBA) measured either visually or by flow cytometry (FACS), immunoprecipitation measured either by radioimmunoprecipitation assay (RIPA) or fluorescence immunoprecipitation assay (FIPA), radio-immunoassay (RIA), high-sensitive single molecule array (SIMOA), or enzyme-linked immunosorbent assay (ELISA) (Benny K. C. Lo (2004) “Antibody Engineering: Methods and Protocols,” Humana Press (ISBN: 1588290921); Johne, B. et al., J. Immunol. Meth., 160:191 -8, 1993; Jonsson, U. et al., Ann. Biol. Clin., 51 :19-26, 1993; and Jonsson, U. et al., Biotechniques, 11 :620-7, 1991 ). In addition, methods for measuring the affinity (e.g., dissociation and association constants) are known in the art.

As used herein, the term “k_a” refers to the rate constant for association of an antibody to an antigen. The term “kd” refers to the rate constant for dissociation of an antibody from the antibody/antigen complex. And the term “KD” refers to the equilibrium dissociation constant of an antibody-antigen interaction. The equilibrium dissociation constant is deduced from the ratio of the kinetic rate constants, KD = ka/kd. Such determinations preferably are measured at 25 °C or 37 °C (see the working examples). The kinetics of antibody binding to human C5, for example, can be determined at pH 8.0, 7.4, 7.0, 6.5 and 6.0 via surface plasmon resonance (SPR) on a BIAcore 3000 instrument using an anti-Fc capture method to immobilize the antibody.

Diagnostic antibodies and methods

In some embodiments, the diagnostic antibodies of the disclosure bind to an epitope in the biomarkers of the disclosure, e.g., NfL and/or GFAP or a variant thereof. The structures (e.g., amino acid sequences) of the NMOSD biomarkers of the present disclosure are known and accessioned in databases (e.g., GENBANK and/or UNIPROT). For example, human NfL has been accessioned under GENBANK No. NP_006149 (date: 07-APR-2024) and UNIPROT No. P07196 (date: 23-JAN-2007, version 3). Similarly, human GFAP sequences (including variants thereof, e.g., isoforms and/or fragments) have been accessioned under GENBANK No. NP 001124491 (isoform 2; accession date: 06-APR-2024); NP 001229305 (isoform 3; accession date: 07-APR-2024); NP 002046 (isoform 1 ; accession date: 05- APR-2024); NP 001350775 (isoform 4; accession date: 07-APR-2024) and UNIPROT No. P14136 (date: 1 -JAN-1990, version 1 ).

A central component of diagnostics in NMOSD includes detecting antibodies in serum. Antibodies against AQP4 made it possible to differentiate NMOSD from other neurological diseases such as multiple sclerosis (MS). See, Pittock et al. Lancet (2004) 364:2106-12; Borisow et al. Front Neurol. (2018); 9: 888. Cell-based assays (CBA) have been developed to this end. See, Waters et al. (J Neurol Neurosurg Psychiatry (2016) 87:1005-15); Waters et al. (Clin Exp Neuroimmunol. (2014) 5:290-303); Jarius et al. (J Neuroinflamm. (2016) 13:279), 79, 80). CBAs typically provide sensitivity range of 80%- 100% and specificity range of 86% to 100% (Pittock (2014), supra). Detection techniques for CBA for diagnosis of AQP4+ NMOSD are also known in the art, e.g., Waters (2014, supra). In embodiments, CBAs using live transiently transfected cells expressing human M23-AQP4 were found to be highly sensitive and perform better than CBA using fixed cells. Other methods such as two FIPA using enhanced green fluorescent protein-M23-AQP4 or EGFP-M1 -AQP4, a commercial ELISA (RSR Ltd, Cardiff, UK) may also be used.

New methods such as immunodot assay provide sensitivity of 99.4% and specificity of 99.2%, which are comparable to that of CBA in detecting AQP4-lgG (Fu et al. (JAMA Neurol . 2023 Oct 1 ;80(10):1105-1112)).

Therapeutic molecules

Embodiments of the disclosure relate to use of therapeutic molecules, e.g., antibodies, in treating the diseases of the disclosure, e.g., NMOSD and particularly AQP4+ NMOSD or a symptom thereof. In embodiments, the therapeutic antibodies comprise antibodies that bind to human complement C5 (e.g., anti-C5 antibodies). In one embodiment, the anti-C5 antibody, or antigen binding fragment thereof, blocks the generation or activity of the C5a and/or C5b active fragments of a C5 protein {e.g., a human C5 protein). Through this blocking effect, the antibodies inhibit, e.g., the pro-inflammatory effects of C5a and the generation of the C5b-9 membrane attack complex (MAC) at the surface of a cell.

Methods for determining whether a particular antibody described herein inhibits C5 cleavage are known in the art. Inhibition of human complement component C5 can reduce the cell-lysing ability of complement in a subject’s body fluids. Such reductions of the cell-lysing ability of complement present in the body fluid(s) can be measured by methods known in the art such as, for example, by a conventional hemolytic assay such as the hemolysis assay described by Kabat and Mayer (eds.), “Experimental Immunochemistry, 2nd Edition,” 135-240, Springfield, IL, CC Thomas (1961 ), pages 135-139, or a conventional variation of that assay such as the chicken erythrocyte hemolysis method (Hillmen, P. et al., N. Engl. J. Med., 350:552-9, 2004). Methods for determining whether a candidate compound inhibits the cleavage of human C5 into forms C5a and C5b are known in the art (Evans, M. et al., Mol. Immunol., 32:1183-95, 1995). The concentration and/or physiologic activity of C5a and C5b in a body fluid can be measured, for example, by methods known in the art. For C5b, hemolytic assays or assays for soluble C5b-9 as discussed herein can be used. Other assays known in the art can also be used. Using assays of these or other suitable types, candidate agents capable of inhibiting human complement component C5 can be screened.

Immunological techniques such as, but not limited to, ELISA can be used to measure the protein concentration of C5 and/or its split products to determine the ability of an anti-C5 antibody, or antigen binding fragment thereof, to inhibit conversion of C5 into biologically active products. In some embodiments, C5a generation is measured. In some embodiments, C5b-9 neoepitope-specific antibodies are used to detect the formation of terminal complement.

In some embodiments, C5 activity, or inhibition thereof, is quantified using a CH50eq assay. The CH50eq assay is a method for measuring the total classical complement activity in serum. This test is a lytic assay, which uses antibody-sensitized erythrocytes as the activator of the classical complement pathway and various dilutions of the test serum to determine the amount required to give 50% lysis (CH50). The percent hemolysis can be determined, for example, using a spectrophotometer. The CH50eq assay provides an indirect measure of terminal complement complex (TCC) formation, since the TCC themselves are directly responsible for the hemolysis that is measured.

Inhibition as it pertains to terminal complement activity, for example, includes at least a 5 %{e.g., at least a 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 %) decrease in the activity of terminal complement as compared to the effect of a control antibody (or antigen-binding fragment thereof) under similar conditions and at an equimolar concentration. Substantial inhibition, as used herein, refers to inhibition of a given activity {e.g., terminal complement activity) of at least 40 % {e.g., at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 % or greater). In some embodiments, an anti-C5 antibody described herein contains one or more amino acid substitutions relative to the CDRs of eculizumab (/.e., SEQ ID NOs:1 -6), yet retains at least 30 % {e.g., at least 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 %) of the complement inhibitory activity of eculizumab.

An anti-C5 antibody described herein has a serum half-life in humans that is at least 20 days {e.g., at least 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54 or 55 days). In another embodiment, the anti-C5 antibody described herein has a serum half-life in humans that is at least 40 days. In another embodiment, the anti-C5 antibody described herein has a serum half-life in humans that is approximately 43 days. In another embodiment, the anti-C5 antibody described herein has a serum half-life in humans that is between 39-48 days. Methods for measuring the serum half-life of an antibody are known in the art. In some embodiments, an anti-C5 antibody, or antigen binding fragment thereof, described herein has a serum half-life that is at least 20 % {e.g., at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 400 or 500 %) greater than the serum half-life of eculizumab, e.g., as measured in one of the mouse model systems described in the working examples {e.g., the C5-deficient/NOD/scid mouse or hFcRn transgenic mouse model system).

In one embodiment, the antibody competes for binding with, and/or binds to the same epitope on C5 as, the antibodies described herein. The term “binds to the same epitope” with reference to two or more antibodies means that the antibodies bind to the same segment of amino acid residues, as determined by a given method. Techniques for determining whether antibodies bind to the “same epitope on C5” with the antibodies described herein include, for example, epitope mapping methods, such as, x- ray analyses of crystals of antigen :antibody complexes that provides atomic resolution of the epitope and hydrogen/deuterium exchange mass spectrometry (HDX-MS). Other methods monitor the binding of the antibody to peptide antigen fragments or mutated variations of the antigen where loss of binding due to a modification of an amino acid residue within the antigen sequence is often considered an indication of an epitope component. In addition, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. Antibodies having the same VH and VL or the same CDR1 , 2 and 3 sequences are expected to bind to the same epitope.

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

Anti-C5 antibodies or antigen-binding fragments thereof described herein, used in the methods described herein, can be generated using a variety of art-recognized techniques. Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (Kohler, G. & Milstein, C., Eur. J. Immunol., 6:511 -9, 1976). Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA sequences that encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells (Huse, W. etal., Science, 246:1275-81 , 1989).

Detection of NMOSD Biomarkers

The NMOSD-associated biomarker protein may be measured in the biological fluid collected from the subject. GFAP and NfL protein release into biofluids can provide a means for evaluating active or ongoing damages to astrocytes and neurons, respectively. These biomarkers can be measured at appreciable, but relatively low, concentrations in serum compared to other biofluids. For example, the GFAP concentration may be measured in the subject’s biological fluid. In other examples, the NfL concentration may be measured in the subject’s biological fluid. In some embodiments, both the concentration of GFAP and the concentration of NfL are determined in the biological fluid obtained from the subject. The biological fluid may include the subject’s blood, a blood fraction, or cerebrospinal fluid (CSF). In some embodiments, the blood fraction is serum or plasma. Methods for monitoring or evaluating the status of one or more NMOSD-associated biomarker proteins in a subject (e.g., a mammal, e.g., a human) include: measuring in a biological fluid obtained from the subject one or both of (i) 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, or 20) NMOSD-associated biomarker protein in the biological fluid. Measuring or determining protein expression levels in a biological sample may be performed by any suitable method (see, e.g., 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 a subject with binding agents for one or more of the NMOSD-associated protein biomarkers; detecting, in the sample (e.g., the biological fluid), the levels of one or more of the NMOSD-associated protein biomarkers that bind to the binding agents; and comparing the levels of one or more of the NMOSD-associated protein biomarkers in the sample with the levels of the corresponding protein biomarkers in a control sample (e.g., a sample from a healthy donor or a sample from a patient prior to administration of the complement component C5 inhibitor). In certain embodiments, a suitable binding agent is a ribosome, with or without a peptide component, an RNA molecule, or a polypeptide (e.g., a polypeptide that comprises a polypeptide sequence of a protein marker, a peptide variant thereof, or a non-peptide mimetic of such a sequence).

Suitable binding agents also include an antibody specific for a NMOSD-associated protein biomarker. Suitable antibodies for use in the methods of the present invention include monoclonal and polyclonal antibodies and antigen-binding fragments (e.g., Fab fragments or scFvs) of antibodies. Antibodies, including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods known in the art (see, for example, Kohler and Milstein (1975) Nature 256:495-497; Kozbor et al. (1985) J Immunol Methods f i l_: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 to be used in the methods of the invention can be purified by methods well known in the art. Antibodies may also be obtained from commercial sources. In certain embodiments, the binding agent is directly or indirectly labeled with a detectable moiety. The role of a detectable agent is to facilitate the detection step of the diagnostic method by allowing visualization of the complex formed by binding of the binding agent with the protein marker will depend on the nature of the assay and of the detectable moiety (e.g., fluorescent moiety).

In one example, the presence or amount of protein expression of a gene (e.g., a NMOSD- associated protein biomarker) can be determined using a Western blotting technique. For example, a lysate can be prepared from a biological sample, or the biological sample (e.g., biological fluid) itself, can be contacted with Laemmli buffer and subjected to sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE-resolved proteins, separated by size, can then be transferred to a filter membrane (e.g., nitrocellulose) and subjected to immunoblotting techniques using a detectably- labeled antibody specific to the protein of interest. The presence or amount of bound detectably-labeled antibody indicates the presence or amount of protein in the biological sample.

In another example, an immunoassay can be used for detecting and/or measuring the protein expression of a NMOSD-associated protein biomarker. As above, for the purposes of detection, an immunoassay can be performed with an antibody that bears a detection moiety (e.g., a fluorescent agent or enzyme). Proteins from a biological sample can be conjugated directly to a solid-phase matrix (e.g., a multi-well assay plate, nitrocellulose, agarose, Sepharose®, encoded particles, or magnetic beads) or it can be conjugated to a first member of a specific binding pair ( e.g., biotin or streptavidin) that attaches to a solid-phase matrix upon binding to a second member of the specific binding pair (e.g., streptavidin or biotin). Such attachment to a solid-phase matrix allows the proteins to be purified away from other interfering or irrelevant components of the biological sample prior to contact with the detection antibody and also allows for subsequent washing of unbound antibody. Here, as above, the presence or amount of bound detectably-labeled antibody indicates the presence or amount of protein in the biological sample.

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

Methods for detecting or measuring protein expression can, optionally, be performed in formats that allow for rapid preparation, processing, and analysis of multiple samples. This can be, for example, in multi-well assay plates (e.g., 96 wells or 386 wells) or arrays (e.g., protein chips). Stock solutions for various reagents can be provided manually or robotically, and subsequent sample preparation, pipetting, diluting, mixing, distribution, washing, incubating (e.g., hybridization), sample readout, data collection (optical data) and/or analysis (computer aided image analysis) can be done robotically using commercially available analysis software, robotics, and detection instrumentation capable of detecting the signal generated from the assay.

Examples of such detectors include, but are not limited to, spectrophotometers, luminometers, fluorimeters, and devices that measure radioisotope decay. Exemplary high-throughput cell based assays (e.g., detecting the presence or level of a target protein in a cell) can utilize ArrayScan® VTI HCS Reader or KineticScan® HCS Reader technology (Cellomics Inc., Pittsburg, PA).

In some embodiments, the protein expression level of at least two NMOSD-associated protein biomarkers (e.g., at least three proteins, at least four proteins, at least five proteins, at least six proteins, at least seven proteins, at least eight proteins, at least nine 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) can be assessed or measured.

The concentration of the NMOSD-associated biomarker protein may be measured using a ligand binding assay. For example, the ligand binding assay is a digital bead based immunoassay. In some embodiments, the serum of CSF collected from the AQP4-Ab+ NMOSD subject may be analyzed with using commercially available ligand-binding assays that are digital bead-based 2-step Simoa® (single molecule array) immune assays (Quanterix Corporation). The samples collected using these methods may be analyzed using, for example, a Simoa® optical system (i.e. , HD-X analyzer, Quanterix Corporation) to provide quantitative measurements of concentrations for the NMOSD-associated protein biomarker.

Some of the methods described herein involve comparing the measured concentration of a NMOSD-associated biomarker protein (as measured in a biological sample obtained from a subject) to a 1 control sample. In some embodiments, the control sample is obtained from the subject prior to administering to the subject a complement component C5 inhibitor. In some embodiments, the control sample can be (or can be based on), e.g., a collection of samples obtained from one or more (e.g., two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, or 40 or more) healthy individuals that have not been administered a complement component C5 inhibitor. In some embodiments, the control sample can be ( or can be based on), e.g., a pooled sample obtained from two or more (e.g., two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, or 40 or more) individuals. In some embodiments of any of the methods described herein, the pooled samples can be from healthy individuals, or at least, individuals who do not have or are not suspected of having (nor at risk for developing) NMOSD. For example, determining whether a subject is one having NMOSD can involve comparing the measured concentration of one or more serum biomarkers in the subject and comparing the measured concentration to the average concentration of the same biomarkers in the pooled healthy samples. Similarly, determining whether the concentration a NMOSD-associated biomarker has been reduced following treatment with a complement inhibitor can involve comparing the concentration or activity of the protein in a biological fluid obtained from a subject prior to treatment with a complement inhibitor to the concentration of protein in a sample of the same biological fluid obtained from the patient after treatment with the inhibitor

( e.g., one day, two days, three days, four days, five days, six days, 1 week, 2 weeks, 3 weeks, a month, 6 weeks, two months, or three months after treatment (e.g., the first of a series of treatment in chronic therapy) with the inhibitor).

Methods of Treating Neuromyelitis Optica

The subjects suffering from NMOSD may be administered an antibody that specifically binds C5. As used herein, the term “subject” and “patient” are interchangeable. In some embodiments, subjects and/or patients are mammals, including, for example, primates, e.g., humans, rodents, lagomorphs, camelids, ungulates, canines and felines. In some embodiments, the subjects or patients suffering from NMOSD described herein are humans. Human patients include adult and pediatric patients.

NMOSD is characterized by a relapsing disease course, from which recovery may be poor due to the stepwise accumulation of significant neurologic disability. Neuromyelitis optica (NMO), also known as Devic’s Disease, or Devic’s Syndrome is part of NMOSD and is a rare, severe disabling autoimmune inflammatory disorder of the central nervous system (CNS) that predominately affects the optic nerves and spinal cord, often leading to blindness, mono/para/tetrapalegia, and respiratory failure.

In some embodiments, NMO is characterized by NMO-IgG antibodies directed at aquaporin 4 (anti-AQP4). In some embodiments, a subset of NMO patients is anti-AQP4+. In some embodiments, a subset of NMO patients is anti-MOG+.

In some embodiments, AQP4 autoantibodies are found in patients with NMO-like symptoms that do not fulfill the clinical requirements to be diagnosed with NMO. In some embodiments, one of the requirements to be diagnosed with NMO are recurrent and simultaneous optic nerve and spinal cord inflammation. In some embodiments, NMOSD includes Devic's disease also known as NMO. In some embodiments NMOSD encompasses limited forms of Devic's disease, such as single or recurrent event of longitudinally extensive myelitis, and bilateral simultaneous or recurrent optic neuritis. In some embodiments, NMOSD encompasses Asian optic-spinal MS (OSMS), or AQP4+ OSMS. In some embodiments, NMOSD further encompasses longitudinally extensive myelitis or optic neuritis associated with systemic autoimmune disease, and optic neuritis or myelitis associated with lesions in specific brain areas such as the hypothalamus, periventricular nucleus, and brainstem.

In some embodiments, treatment of NMOSD includes the amelioration or improvement of one or more symptoms associated with NMOSD. Symptoms associated with NMOSD include visual impairment, decreased visual acuity, visual field defects, loss of color vision, spinal cord dysfunction, muscle weakness, reduced sensation and loss of bladder or bowel control.

In some embodiments, treatment of NMOSD includes the improvement of a clinical marker for NMOSD progression. These markers include, for example, time to relapse, annualized relapse rate (ARR), expanded disability scale score (EDSS), modified Rankin scale (mRS), quality of life (ED-5D), Hauser ambulatory index (HAI), change in visual acuity using a Snellen chart and severity of relapse using the optic spinal impairment score (OSIS).

NMOSD relapse is evidenced by symptoms of NMOSD occurring in a subject where symptoms have previously been successfully ameliorated. Relapse is shown by the onset or worsening of symptoms associated with vision or sensation. Changes in vision that are associated with relapse of NMOSD include rapid onset of eye pain, blurring of vision, colors that do not seem right, missing field of vision, spots or dots in the field of vision, flashing or flickering lights in the field of vision, difficulty focusing, difficulty reading and feelings that the field of vision seems incorrect. Changes in sensation that are associated with relapse of NMOSD include pain, tingling, numbness, arm, leg or face seems to fall asleep, loss of sense of position in space, loss of sense in extremities, slight touching is painful, clothes or bed sheets cause pain, and subject not being able to detect injury to the subject. Annualized relapse rate (ARR) is the average number of relapses per year.

In some embodiments, a subject treated for NMOSD has had three or more relapses in the 24 month period before eculizumab is administered. In some embodiments, a subject treated for NMO has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or more relapses in the 24 month period before eculizumab is administered. In some embodiments, a subject treated for NMOSD has an ARR of 1 .0 or greater in the 24 month period before eculizumab is administered. In some embodiments, a subject treated for NMOSD has an ARR of at least 1 .0, 1 .2, 1 .4, 1 .6, 1 .8, 2.0, 25, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5. 7.0 or more in the 24 month period before eculizumab is administered.

Disability can be assessed based on the EDSS scores comparing the change from baseline in the two treatment groups. The Kurtzke Expanded Disability Status Scale (EDSS) is a method of quantifying disability in multiple sclerosis. The EDSS replaced the previous Disability Status Scales used in Multiple Sclerosis (MS). The EDSS quantified disability in eight Functional Systems (FS) and allows neurologists to assign a Functional System Score (FSS) in each of these. The Functional Systems are: pyramidal, cerebellar, brainstem, sensory, bowel and bladder, visual, cerebral and others. EDSS steps 1 .0 to 4.5 refer to people with MS who are fully ambulatory. EDSS steps 5.0 to 9.5 are defined by the impairment of ambulation. Disability is also to be assessed based on the mRS score comparing the change from baseline in the two treatment groups. mRS score is assessed by the treating physician at the protocol specified time points.

In some embodiments, eculizumab is administered to the subject in a dosing regimen comprising: (a) induction dosing comprising 900 mg of eculizumab, once weekly for the first 4 weeks, followed by 1200 mg of eculizumab for the fifth dose 1 week later; and (b) maintenance dosing comprising 1200 mg of eculizumab every 2 weeks afterwards, wherein the induction dosing (a) and maintenance dosing (b) are both administered intravenously (IV) to said adult patient.

According to some embodiments, subjects administered eculizumab show an increased time interval between relapses of NMOSD. In some embodiments, the subjects have a period before relapse of greater than 6 weeks. In some embodiments, the period before relapse is greater than 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102 or more weeks. In some embodiments, the period before relapse is greater than 1 , 2, 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 or more weeks. In some embodiments, the period before relapse is greater than 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years. In some embodiments, the period before relapse is between 6 and 52 weeks, 6 and 26 weeks, 6 and 10 weeks, 26 and 52 weeks, 1 and 2 years, 1 and 5 years, 5 and 10 years or a relapse does not occur during the lifetime of the subject. In some embodiments, the period before relapse is greater than 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102 or more months.

According to some embodiments, the course of treatment with eculizumab lasts for 108 weeks. According to other embodiments, the course of treatment lasts for 26-52, 26-78, 26-120, 26- 130, 26-156, 26-104, 26-130, 26-156, 26-182, 26-208 weeks or more. In some embodiments, the course of treatment lasts for greater than 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 78, 104, 130, 156 or 182 weeks. According to other embodiments, the course of treatment lasts for greater than 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more years. In some embodiments, the course of treatment lasts for the remainder of the subject’s life.

According to some embodiments, during the course of treatment, one or more symptoms or scores associated with NMOSD improves during the course of treatment and is maintained at the improved level throughout treatment. EDSS can improve, for example, after 26 weeks of treatment with a therapeutic antibody that specifically binds C5 and then remain at the improved level for the duration of the treatment, which can be, for example, 52 weeks of treatment with a therapeutic antibody that specifically binds C5. One example of a therapeutic antibody that binds C5 is eculizumab.

In some embodiments, the first sign of improvement occurs by 26 weeks of treatment with a therapeutic antibody that specifically binds C5. According to other embodiments, the first sign of improvement occurs between weeks 1 -26, 26-52, 52-78, 78-104, 104-130, 130-156, 156-182, or 182- 208 of treatment with a therapeutic antibody that specifically binds C5. In some embodiments, the first sign of improvement occurs at week 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47,

48, 49, 50, 51 , 52, 78, 104, 130, 156 or 182. According to some embodiments, the first sign of improvement is maintained for a number of weeks during treatment with a binding protein that specifically binds C5 such as eculizumab. According to some embodiments, this number of weeks is at least 26. According to other embodiments, this number of weeks is 1 -26, 26-52, 52-78, 78-104, 104-130, 130-156, 156-182, or 182-208. In some embodiments, this number of weeks is at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 78, 104, 130, 156 or 182. According to some embodiments, when the first sign of improvement is maintained, this means that the metric for treatment of NMOSD does not fall below the value of the first sign of improvement. The metric could continue to improve, and this would still be defined as maintenance of the first sign of improvement.

According to this embodiment, the second phase of eculizumab dosing is the maintenance phase. The maintenance phase of eculizumab dosing can last for between 6 weeks and the life of the subject. According to other embodiments, the maintenance phase lasts for 26-52, 26-78, 26-104, 26- 130, 26-156, 26-182, 26-208 weeks or more. In some embodiments, the maintenance phase lasts for greater than 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 78, 104, 130, 156 or 182 weeks. According to other embodiments, the maintenance phase lasts for greater than 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more years. In some embodiments, the maintenance phase lasts for the remainder of the subject’s life. In some embodiments, the eculizumab multiphase dosing regimen includes a third phase.

This third phase is used when an NMOSD patient must undergo plasma exchange. In this phase after plasma is exchanged a dose of ravulizumab or eculizumab is administered to replace the drug lost in plasma exchange. According to some embodiments, this eculizumab dose is between 300 and 1200 mg, 400 and 1500 mg, 500 and 1000 mg, 400 and 800 mg, or 500 and 700 mg. According to some embodiments, this eculizumab dose is about 600 mg. In some embodiments, the third phase, 600 mg eculizumab dose is administered within 1 hour after completion of plasmapheresis. In some embodiments, the third phase, 600 mg dose is administered within 2 hours after completion of plasmapheresis. In some embodiments, the third phase, 600 mg dose is administered within 3 hours after completion of plasmapheresis. In some embodiments, the third phase, 600 mg dose is administered within 4 hours after completion of plasmapheresis. In some embodiments, the third phase, 600 mg dose is administered within 5 hours after completion of plasmapheresis. In some embodiments, the third phase, 600 mg dose is administered within 6 hours after completion of plasmapheresis.

In some embodiments, the ravulizumab is administered to the subject in a dosing regimen comprising: (a) once on Day 1 at a loading dose of: (i) 2400 mg to a subject weighing > 40 and < 60 kg; (ii) 2700 mg to a subject weighing > 60 and < 100 kg; (iii) 3000 mg to a subject weighing > 100 kg; (b) on Day 15 and every eight weeks thereafter at a maintenance dose of: (i) 3000 mg to a subject weighing > 40 and < 60 kg; (ii) 3300 mg to a subject weighing > 60 and < 100 kg; (iii) 3600 mg to a subject weighing > 100 kg, preferably wherein the loading dose (a) and the maintenance dosing (b) are administered intravenously.

In some embodiments, ravulizumab is administered to a patient weighing > 40 to < 60 kg: (a) once on Day 1 of the administration cycle at a loading dose of 2400 mg; and (b) on Day 15 of the administration cycle and every eight weeks thereafter at a maintenance dose of 3000 mg. In some embodiments, the ravulizumab is administered to a patient weighing > 60 to < 100 kg: (a) once on Day 1 of the administration cycle at a loading dose of 2700 mg; and (b) on Day 15 of the administration cycle and every eight weeks thereafter at a maintenance dose of 3300 mg. In some embodiments, the ravulizumab is administered to a patient weighing > 100 kg:(a) once on Day 1 of the administration cycle at a loading dose of 3000 mg; and (b) on Day 15 of the administration cycle and every eight weeks thereafter at a maintenance dose of 3600 mg. In some embodiments, treatment with ravulizumab maintains a serum trough concentration of the antibody or the antigen binding fragment thereof of 100 pg/mL or greater during the administration cycle. In some embodiments, treatment with the antibody or the antigen binding fragment thereof maintains a serum trough concentration of the antibody or the antigen binding fragment thereof of 200 pg/mL or greater during the administration cycle.

In some embodiments the ravulizumab is administered at a dose of 3000 mg, 3300 mg or 3600 mg every eight weeks after the administration cycle for up to two years.

In some embodiments, ravulizumab is administered to a subject after undergoing plasmapheresis. In some embodiments, ravulizumab is administered to a subject after undergoing plasma exchange. In some embodiments, ravulizumab is administered to a subject after receiving intravenous immunoglobin. After the subject has undergone PP, PE< or I VIG , ravulizumab may be administered to a patient. If the patient weighs > 40 to < 60 kg, ravulizumab may be administered in an amount of 1200 mg to 1400 mg. For example, if the patient weighs > 40 to < 60 kg and most recently received a dose of 2400 mg of ravulizumab, the patient may be administered 1200 mg of ravulizumab. For example, if the patient weighs > 40 to < 60 kg and most recently received a dose of 3000 mg of ravulizumab, the patient may be administered 1500 mg of ravulizumab. If the patient weighs > 60 to < 100 kg, ravulizumab may be administered in an amount of 1500 mg to 1800 mg. For example, if the patient weighs > 60 to < 100 kg and most recently received a dose of 2700 mg of ravulizumab, the patient may be administered 1500 mg of ravulizumab. For example, if the patient weighs > 60 to < 100 kg and most recently received a dose of 3300 mg of ravulizumab, the patient may be administered 1800 mg of ravulizumab.

If the patient weighs > 100 kg, ravulizumab may be administered in an amount of 1500 mg to 1800 mg. For example, if the patient weighs > 100 kg and most recently received a dose of 3000 mg of ravulizumab, the patient may be administered 1500 mg of ravulizumab. For example, if the patient weighs > 100 kg and most recently received a dose of 3600 mg of ravulizumab, the patient may be administered 1800 mg of ravulizumab.

In some embodiments, ravulizumab is formulated for intravenous administration.

In some embodiments, the patient treated with ravulizumab thereof has not previously been treated with a complement inhibitor.

In some embodiments, the administration cycle is a total of 26 weeks of treatment.

According to some embodiments, during the course of treatment, one or more symptoms or scores associated with NMOSD improves during the course of treatment and is maintained at the improved level throughout treatment. EDSS can improve, for example, after 26 weeks of treatment with a therapeutic antibody that specifically binds C5 and then remain at the improved level for the duration of the treatment, which can be, for example, 52 weeks of treatment with ravulizumab. The standard score or Z-score is the number of standard deviations by which the value of a raw score (i.e. , an observed value or data point) is above or below the mean value of what is being observed or measured. Based on the Z-score methods, age was identified as a relevant factor for patients having high biomarker (GFAP or NfL) levels. Therefore, in some embodiments of the presently claimed methods of diagnosis and/or therapeutic monitoring, the patient’s age is considered, wherein higher or greater age of patients is associated with higher biomarker (GFAP or NfL) levels, which in turn is associated with shorter time since prior relapse and/or higher baseline Hauser Ambulation Index (HAI) score.

In another aspect, a patient switches from receiving one C5 inhibitor to a different C5 inhibitor during the course of treatment. Different anti-C5 antibodies may be administered during separate treatment periods. For example, in one embodiment, a use of eculizumab or a biosimilar of eculizumab for treating a human patient having NMOSD is provided, the method comprising discontinuing treatment with eculizumab and switching the patient to treatment with an alternative complement inhibitor, such as ravulizumab. For example, in one embodiment, the patient is treated with eculizumab during a treatment period (e.g., for 26 weeks), followed by treatment with another anti- 05 antibody, such as ravulizumab, during an extension period. In one embodiment, eculizumab is administered to the patient at a dose of 900 mg on Days 1 , 8, 15, and 22 of the administration cycle during an induction phase, followed by a maintenance dose of 1200 mg of eculizumab on Day 19 of the administration cycle and every two weeks thereafter (e.g., for a total of 26 weeks), followed by treatment with another anti-C5 antibody for an extension period of up to two years. In some embodiments, the method comprises discontinuing treatment with ravulizumab and switching the patient to treatment with eculizumab. For example, the patient may be treated with ravulizumab for a treatment period (e.g., for 12 weeks), followed by treatment with ravulizumab for an additional period of time (e.g., for 24 weeks). In some embodiments, a patient is switched from receiving one C5 inhibitor to a different C5 inhibitor and concentration of GFAP and/or NfL in the biological fluid of the patient is monitored throughout. For example, the NfL concentration and/or GFAP concentration may be measured before the patient is switched from one C5 inhibitor to another and after the patient is switched from one C5 inhibitor to another. In some embodiments, the switch between the two C5 inhibitors is considered successful if there is not appreciable change in the concentration of GFAP and/or NfL in the biological fluid before and after the C5 inhibitor is switched. In some embodiments, the switch between the two C5 inhibitors is considered successful if that change in GFAP and/or NfL concentration changes less than 20% (e.g., difference <20%, <10%, <5%, or less, e.g., no difference) in the biological fluid of the subject before and after switching the C5 inhibitor.

In some embodiments, the method further comprises measuring the clinical profile of the subject or the control. For example, the method comprises measurement of astrocyte and neuron damage.

In some embodiments, the subject is a human.

Pharmaceutical Compositions

Pharmaceutical compositions comprising a C5 antibody, e.g., ravulizumab, eculizumab, either alone or in combination with prophylactic agents, therapeutic agents, and/or pharmaceutically acceptable carriers are provided. The pharmaceutical compositions comprising eculizumab provided herein are for use in, but not limited to, diagnosing, detecting or monitoring a disorder; in preventing, treating, managing or ameliorating a disorder or one or more symptoms thereof; and/or in research. The formulation of pharmaceutical compositions, either alone or in combination with prophylactic agents, therapeutic agents, and/or pharmaceutically acceptable carriers, is known to one skilled in the art.

Methods of administering a prophylactic or therapeutic agent provided herein include, but are not limited to, parenteral administration {e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural administration, intratumoral administration, mucosal administration {e.g., intranasal and oral routes) and pulmonary administration {e.g., aerosolized compounds administered with an inhaler or nebulizer). The formulation of pharmaceutical compositions for specific routes of administration, and the materials and techniques necessary for the various methods of administration are available and known to one skilled in the art.

Dosage regimens may be adjusted to provide the optimum desired response {e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. The term “dosage unit form” refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms provided herein is dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

Kits and Unit Dosage Forms

Also provided herein are kits that include a pharmaceutical composition containing the complement component C5 inhibitor, such as ravulizumab or eculizumab, and a pharmaceutically acceptable carrier, in a therapeutically effective amount adapted for use in the preceding methods. The kits can also optionally include instructions, e.g., comprising administration schedules, to allow a practitioner {e.g., a physician, nurse or patient) to administer the composition contained therein to administer the composition to a patient having NMOSD. The kit also can include a syringe.

Kits can optionally include multiple packages of the single-dose pharmaceutical compositions each containing an effective amount of the complement component C5 inhibitor for a single administration in accordance with the methods provided above. Instruments or devices necessary for administering the pharmaceutical composition(s) also may be included in the kits. A kit may provide one or more pre-filled syringes containing an amount of the complement component C5 inhibitor. EXAMPLES

The following example is merely illustrative and should not be construed as limiting the scope of this disclosure in any way as many variations and equivalents will become apparent to those skilled in the art upon reading the present disclosure. The contents of all references, Genbank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

Example 1. Evaluating serum GFAP and NfL levels during treatment with complement component 5-inhibitor therapies eculizumab and ravulizumab in AQP4-Ab+ NMOSD

Subjects participated in the Phase 3, randomized, placebo-controlled, time-to-event study to evaluate the efficacy and safety of eculizumab in patients with AQP4-Ab+ NMOSD (the PREVENT study (NCT01892345)), or the Phase 3, open-label, externally placebo-controlled study to evaluate the efficacy and safety of ravulizumab in patients with AQP4-Ab+ NMOSD ( the CHAMPION-NMOSD study (NCT04201262)). In the PREVENT study, administration of eculizumab was associated with a 94.2% reduction in NMOSD relapse risk compared with placebo. In the CHAMPION-NMOSD study, administration of ravulizumab was associated with a 98.6% reduction in risk of adjudicated on-trial relapse compared with external placebo.

In the PREVENT study, biomarkers were analyzed retrospectively from a subset of available samples from placebo and eculizumab treatment groups that had been previously collected for PK/PD analysis. In the CHAMPION-NMOSD study, biomarkers were analyzed prospectively using serum and cerebrospinal fluid (CSF) samples. The CSF collection was optional for study participants. External healthy donor samples were demographically matched for both trials. GFAP and NfL levels (pg/mL) in serum and CSF (where applicable) were assessed in PREVENT and CHAMPION-NMOSD using Quanterix’s single-plex Simoa platform at pretreatment baseline (D1 ), select study visits, and relapse visits (24-48 hours after relapse symptoms). All relapses were independently adjudicated.

Predose blood samples were collected 90 mins before administering either ravulizumab or eculizumab on day 1 , week 4, and week 24 for patients receiving eculizumab and on day 1 , week 2, week 6, week 26, week 50, week 90, week 106, and week 130 for patients receiving ravulizumab. Relapse- associated blood sample collection was permitted at any time unless it coincided with the schedule of a regular sample collection visit. Predose CSF samples were collected from patients who consented to CSF collection and were collected on day 1 , week 26, week 82, and week 106 for patients receiving ravulizumab.

Serum GFAP levels were elevated at pretreatment baseline in patients with AQP4-Ab+ NMOSD relative to healthy donors. Healthy donors (HD) were -90% female with a mean age or 46. Healthy donors were - 52% white or Caucasian, -34% Asian, and -14 % Black or African American and no Hispanic. The HD mean was measured for GFAP (pg/mL) average in healthy donors + 2 standard deviations, which was a threshold for elevated levels. All data was analyzed with the Mann-Whitney test (unpaired; non-parametric) (FIG. 1A and FIG. 1 B).

Serum NfL levels are elevated at pretreatment baseline in patients with AQP4-Ab+ NMOSD relative to healthy donors. HD were -90% female with a mean age of 46. HD were -52% White or Caucasian, -34% Asian, and -14 % Black or African American and no Hispanic. All data was analyzed with a Mann-Whitney test (unpaired; non-parametric). For the PREVENT study, when the outlier was removed, and statistical significance was maintained (P = 0.0359) (FIG. 2A). For the CHAMPION- NMOSD study, when the outlier was removed, statistical significance was maintained (P = 0.0228) (FIG. 2B).

High baseline GFAP and NfL levels in serum samples from patients in CHAMPION-NMOSD (Ph3 trial with ravulizumab in NMO) and PREVENT (Ph 3 trial with eculizumab in NMOSD patients) were primarily associated with clinical characteristics related to age and ambulation. High baseline GFAP levels in CHAMPION-NMOSD were associated with higher age at initial presentation, shorter time since last relapse, and higher baseline Hauser Ambulation Index (HAI) score; similar findings were observed for the latter two characteristics in PREVENT.

High baseline NfL levels in CHAMPION-NMOSD were associated with higher age at first dose and higher baseline Hauser Ambulation Index (HAI) score; similar findings were observed in PREVENT.

HAI is a scale used to assess mobility and disability in patients with neuromyelitis optica spectrum disorder (NMOSD) - higher baseline scores indicating more severe disease - scale ranges from 0 to 9, with 0 being the best score (asymptomatic; fully ambulatory with no assistance) and 9 being the worst (restricted to wheelchair; unable to transfer self independently).

Eculizumab treatment attenuated the relative increase in serum GFAP levels in patients experiencing adjudicated on-trial relapses (FIG. 3B) in comparison to patients who received the placebo (FIG. 3A). Serum GFAP levels in the placebo group had a mean fold change from baseline of 8.72 for patients experiencing adjudicated on-trial relapse. Serum GFAP levels in the eculizumab group had a mean fold change from baseline of 2.67 for patients experiencing adjudicated on-trial relapse.

Eculizumab treatment reduced serum GFAP levels but not NfL levels relative to placebo. For the placebo group, median GFAP levels were measured on day 1 as 125.0 pg/mL, after week 4 as 117.0 pg/mL, and after week 24 as 133.5 pg/mL (FIG. 4A). For the group administered eculizumab, median GFAP levels were measured on day 1 as 120.0 pg/mL, after week 4 as 86.95 pg/mL, and after week 24 as 89.70 pg (FIG. 4A). For the placebo group, median NfL levels were measured on day 1 as 10.80 pg/mL, after week 4 as 10.18 pg/mL, and after week 24 as 12.50 pg/mL (FIG. 4B). For the eculizumab group, median NfL levels were measured on day 1 as 10.35 pg/mL, after week 4 as 10.04 pg/mL, and after week 24 as 6.73 pg/mL (FIG. 4B). All data analysis used Wilcoxon matched-pairs signed rank test (paired; non-parametric).

Ravulizumab treatment reduced serum GFAP and NfL levels over time. For the group that was administered ravulizumab, median GFAP levels were measured on day 1 as 129.0 pg/mL, after week 2 as 113.0 pg/mL, after week 6 as 107.0 pg/mL, after week 26 as 110.0 pg/mL, after week 50 as 96.40 pg/mL, after week 106 as 90.40 g/mL, and after week 130 as 89.60 pg//mL (FIG. 5A). For the group that was administered ravulizumab, median NfL levels were measured on day 1 as 9.84 pg/mL, after week 2 as 10.00 pg/mL, after week 6 as 9.64 pg/mL, after week 26 as 9.075 pg/mL, after week 50 as 8.92 pg/mL, after week 106 as 9.125 g/mL, and after week 130 as 7.865 pg//mL (FIG. 5B). At the time of analysis, only 20 patients had reached week 130. All data was analyzed with Wilcoxon matched-pairs signed rank test (paired; non-parametric).

Additionally, GFAP levels declined more rapidly than NfL levels following ravulizumab treatment (FIG. 6). In AQP4-Ab+ NMOSD, astrocytes are the primary target of damage followed by neurons in the central nervous system. Although FIG. 6 captures rapid and sustained reduction of both serum GFAP (biomarker of astrocyte damage) and NfL (biomarker of neuronal damage) levels in ravulizumab-treated AQP4-Ab+ NMOSD, GFAP levels declined more rapidly than NfL levels with treatment suggesting a rapid and sustained reduction in damage to astrocytes, followed by neurons. Thus, the simultaneous measurement of serum GFAP and NfL levels may be used as sensitive, objective, and cell-specific biomarkers (surrogate biomarkers) that correlate with clinical efficacy.

NfL levels measured from the CSF declined with ravulizumab treatment consistent with declining serum NfL levels (FIG. 7A and FIG. 7B). A small number of patients provided consent for optional CSF testing; only 4 patients had CSF and serum samples for comparison with the following results: (Patient A): Day 1 (360 pg/mL) to Week 26 (163 pg/mL); (Patient B): Day 1 (865 pg/mL) to Week 130 (472 pg/mL); (Patient C): Day 1 (10,500 pg/mL) to Week 26 (2480 pg/mL); and (Patient D): Week 26 (283 pg/mL) to Week 82 (291 pg/mL); unable to provide a serum sample at week 82, but provided sample at week 90. Although cerebral spinal fluid (CSF) is the most relevant biofluid (surrounding brain and spinal cord) to measure AQP4-Ab+ NMOSD, CSF is obtained by lumbar puncture is highly invasive. Currently, lumbar puncture and MRI (not readily available) are common diagnostic/monitoring tools for AQP4-Ab+ NMOSD patients. Serum biomarkers are less invasive and allow for frequent longitudinal monitoring during treatment by health care practitioners (HCPs). In FIG. 7A, NfL levels were analyzed in CSF from a subset of patients. Notably, changes in CSF NfL mirrored changes of serum NfL in that same subset of AQP4- Ab+ NMOSD patients (FIG. 7B). Thus, this data supports the invention that serum GFAP and NfL levels (systemic levels) may be used as a proxy to evaluate disease-relevant cellular changes in the brain.

In samples from the PREVENT and CHAMPION-NMOSD trials, pretreatment baseline serum GFAP and NfL levels were elevated in patients with AQP4-Ab+ NMOSD compared with healthy donors. Relative to placebo, eculizumab attenuated GFAP levels at the time of adjudicated on-trial relapse. Eculizumab treatment reduced serum GFAP levels relative to placebo at protocol-defined timepoints (D1 vs. week 4). Ravulizumab decreased serum GFAP and NfL levels over time relative to pretreatment baseline. These data suggest rapid (4-6 weeks) and prolonged reduction in damage to astrocytes and neurons in AQP4-Ab+ NMOSD, strengthening the suitability of GFAP and NfL as potential biomarkers of therapeutic efficacy in AQP4-Ab+ NMOSD. Eculizumab and ravulizumab demonstrated efficacy in reducing relapses in PREVENT and CHAMPION-NMOSD, respectively. Collectively, the biomarker data presented here suggested that eculizumab and ravulizumab act on the pathophysiology (astrocyte and neuron damage) underlying AQP4-Ab+ NMOSD.

In sum, the NMOSD biomarker data demonstrate that longitudinal measurement of both GFAP and NfL in AQP4-Ab+ NMOSD serum are potential surrogate biomarkers of therapeutic efficacy in this patient population. This will allow health care providers to monitor and evaluate damage to astrocytes and neurons from serum samples in AQP4-Ab+ NMOSD receiving complement inhibition therapy. As such, it is inferred that serum GFAP and NfL also apply as surrogate biomarkers of therapeutic efficacy in eculizumab-treated AQP4-Ab+ patients.

Listing of Sequences Other Embodiments

All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

Other embodiments are within the claims.

What is claimed is:

Claims

1 . A method for monitoring responsiveness of a subject to treatment with an inhibitor of complement component C5, the method comprising: determining the concentration of a first Neuromyelitis Optica Spectrum Disorder (NMOSD)-associated biomarker protein in a biological fluid obtained from the subject, wherein the first NMOSD-associated biomarker protein is Glial fibrillary protein (GFAP), wherein the subject has, is suspected of having, or is at risk for developing Aquaporin-4 Antibody-positive (AQP4-Ab+) NMOSD, wherein the subject has been or is being treated with an inhibitor of complement component C5, and wherein a reduced concentration of GFAP, as compared to the concentration in a baseline sample of biological fluid of the same type obtained from the subject prior to treatment with the complement component C5 inhibitor, indicates that the subject is responsive to treatment with the complement component C5 inhibitor.

2. The method of claim 1 , wherein the method further comprises determining the concentration of a second NMOSD-associated biomarker protein in a biological fluid obtained from the subject, wherein the second NMOSD-associated biomarker protein is Neurofilament light chain (NfL), wherein a reduced concentration of NfL, as compared to the concentration in a baseline sample of biological fluid of the same type obtained from the subject prior to treatment with the complement component C5 inhibitor, indicates that the subject is responsive to treatment with the complement component C5 inhibitor.

3. A method for monitoring responsiveness of a subject to treatment with an inhibitor of complement component C5, the method comprising: determining the concentration of a first NMOSD-associated biomarker protein in a biological fluid obtained from the subject, wherein the first NMOSD-associated biomarker protein is NfL, wherein the subject has, is suspected of having, or is at risk for developing AQP4-Ab+ NMOSD, wherein the subject has been or is being treated with an inhibitor of complement component C5, and wherein a reduced concentration of NfL, as compared to the concentration in a baseline sample of biological fluid of the same type obtained from the subject prior to treatment with the complement component C5 inhibitor, indicates that the subject is responsive to treatment with the complement component C5 inhibitor.

4. The method of claim 3, wherein the method further comprises determining the concentration of a second NMOSD-associated biomarker protein in a biological fluid obtained from the subject, wherein the second NMOSD-associated biomarker protein is GFAP, wherein a reduced concentration of GFAP, as compared to the concentration in a baseline sample of biological fluid of the same type obtained from the subject prior to treatment with the complement component C5 inhibitor, indicates that the subject is responsive to treatment with the complement component C5 inhibitor.

5. The method of any one of claims 1 -4, wherein the biological fluid is blood, a blood fraction, or cerebrospinal fluid.

6. The method of claim 5, wherein the blood fraction is serum or plasma.

7. The method of any one of claims 1 -6, wherein the concentration is determined using a ligand binding assay.

8. The method of claim 7, wherein the ligand binding assay is a digital bead-based immunoassay.

9. The method of any one of claims 1 -8, wherein the concentration of GFAP and/or NfL is reduced by at least 10% relative to the baseline sample.

10. The method of any one of claims 1 -8, wherein the concentration of GFAP and/or NfL is reduced by at least 20% relative to the baseline sample.

11 . The method of any one of claims 1 -8, wherein the concentration of GFAP and/or NfL is reduced by at least 30% relative to the baseline sample.

12. The method of any one of claims 1 -11 , wherein the determining is performed approximately 2 weeks after the commencement of the treatment.

13. The method of any one of claims 1 -11 , wherein the determining is performed approximately 4 weeks after the commencement of the treatment.

14. The method of any one of claims 1 -11 , wherein the determining is performed approximately 6 weeks after the commencement of the treatment.

15. The method of any one of claims 1 -14, wherein the inhibitor of complement component C5 is an antibody or antigen-binding fragment of an antibody, a small molecule, a polypeptide, a polypeptide analog, a peptidomimetic, an aptamer, or a combination 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 camelid antibody, a diabody, a chimeric antibody, a monoclonal antibody, a fully human antibody, a single chain antibody, an Fv fragment, an Fd fragment, an Fab fragment, an Fab' fragment, an F(ab')2 fragment, and an engineered polypeptide comprising a complement component C5-binding VHH construct.

17. The method of claim 15 or 16, wherein the antibody or antigen-binding fragment thereof binds to complement component C5 and inhibits cleavage of C5 into fragments C5a and C5b.

18. The method of claim 17, wherein the antibody is eculizumab or ravulizumab.

19. The method of claim 17, wherein the antibody is a biosimilar of eculizumab.

20. The method of claim 17, wherein the antibody is pexelizumab.

21. The method of claim 15, wherein the antibody is tesidolumab, crovalimab, omoprubart (CAN106), or pozelimab.

22. The method of claim 15, wherein the complement component C5 inhibitor is selected from the group consisting of KP-104, avacincaptad pegol (ARC1905), MB12/22, MB12/22-RGD, ARC187, SSL7, and Omithodoros moubata C inhibitor (OmCI).

23. A method of treating AQP4-Ab+ NMOSD in a subject using an inhibitor of complement component C5 in a manner sufficient to reduce the concentration of at least one NMOSD-associated biomarker protein, the method comprising:

(a) determining the concentration of at least a first NMOSD-associated biomarker protein in a biological fluid obtained from the subject, wherein the NMOSD-associated biomarker protein is GFAP, and

(b) administering to the subject having, suspected of having, or at risk for developing AQP4-Ab+ NMOSD an inhibitor of complement component C5 in an amount and with a frequency sufficient to cause a reduction in concentration of GFAP, as compared to the concentration in a baseline sample of biological fluid of the same type obtained from the subject prior to treatment with the inhibitor of complement component C5.

24. The method of claim 23, wherein (a) further comprises determining the concentration of a second NMOSD-associated biomarker protein in a biological fluid obtained from the subject, wherein the second NMOSD-associated biomarker protein is NfL and (b) comprises administering to the subject having, suspected of having, or at risk for developing AQP4-Ab+ NMOSD an inhibitor of complement component C5 in an amount and with a frequency sufficient to cause a reduction in concentration of NfL, as compared to the concentration in a baseline sample of biological fluid of the same type obtained from the subject prior to treatment with the inhibitor of complement component C5.

25. A method of treating AQP4-Ab+ NMOSD in a subject using an inhibitor of complement component C5 in a manner sufficient to reduce the concentration of at least one NMOSD-associated biomarker protein, the method comprising:

(a) determining the concentration of a first NMOSD-associated biomarker protein in a biological fluid obtained from the subject, wherein the NMOSD-associated biomarker protein is NfL, and

(b) administering to the subject having, suspected of having, or at risk for developing AQP4-Ab+ NMOSD an inhibitor of complement component C5 in an amount and with a frequency sufficient to cause a reduction in concentration of NfL, as compared to the concentration in a baseline sample of biological fluid of the same type obtained from the subject prior to treatment with the inhibitor of complement component C5.

26. The method of claim 25, wherein (a) further comprises determining the concentration of a second NMOSD-associated biomarker protein in a biological fluid obtained from the subject, wherein the second NMOSD-associated biomarker protein is GFAP, and (b) comprises administering to the subject having, suspected of having, or at risk for developing AQP4-Ab+ NMOSD an inhibitor of complement component C5 in an amount and with a frequency sufficient to cause a reduction in concentration of GFAP, as compared to the concentration in a baseline sample of biological fluid of the same type obtained from the subject prior to treatment with the inhibitor of complement component C5.

27. The method of any one of claims 23-26, wherein the biological fluid is blood, a blood fraction, or cerebrospinal fluid.

28. The method of claim 27, wherein the blood fraction is serum or plasma.

29. The method of any one of claims 23-28, wherein the concentration is determined using a ligand binding assay.

30. The method of claim 29, wherein the ligand binding assay is a digital bead-based immunoassay.

31 . The method of any one of claims 23-30, wherein the concentration of GFAP and/or NfL is reduced by at least 10% relative to the baseline sample.

32. The method of any one of claims 23-31 , wherein the concentration of GFAP and/or NfL is reduced by at least 20% relative to the baseline sample.

33. The method of any one of claims 23-32, wherein the concentration of GFAP and/or NfL is reduced by at least 30% relative to the baseline sample.

34. The method of any one of claims 23-33, wherein the determining is performed approximately 2 weeks after the commencement of the treatment.

35. The method of any one of claims 23-33, wherein the determining is performed approximately 4 weeks after the commencement of the treatment.

36. The method of any one of claims 23-33, wherein the determining is performed approximately 6 weeks after the commencement of the treatment.

37. The method of any one of claims 23-36, wherein the inhibitor of complement component C5 is an antibody or antigen-binding fragment of an antibody, a small molecule, a polypeptide, a polypeptide analog, a peptidomimetic, an aptamer, or a combination thereof.

38. The method of claim 37, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a humanized antibody, a recombinant antibody, a camelid antibody, a diabody, a chimeric antibody, a monoclonal antibody, a fully human antibody, a single chain antibody, an Fv fragment, an Fd fragment, an Fab fragment, an Fab' fragment, an F(ab')2 fragment, and an engineered polypeptide comprising a complement component C5-binding VHH construct.

39. The method of claim 37 or 38, wherein the antibody or antigen-binding fragment thereof binds to complement component C5 and inhibits cleavage of C5 into fragments C5a and C5b.

40. The method of claim 39, wherein the antibody is eculizumab or ravulizumab.

41 . The method of claim 39, wherein the antibody is a biosimilar of eculizumab.

42. The method of claim 39, wherein the antibody is pexelizumab.

43. The method of claim 37, wherein the antibody is tesidolumab, crovalimab, omoprubart (CAN106), or pozelimab.

44. The method of claim 37, wherein the complement component C5 inhibitor is selected from the group consisting of KP-104, avacincaptad pegol (ARC1905), MB12/22, MB12/22-RGD, ARC187, SSL7, and Omithodoros moubata C inhibitor (OmCI).

45. The method of any one of claims 23-36, wherein the inhibitor of complement component C5 is eculizumab, the subject is an adult patient, and eculizumab is administered using a phased dosing schedule with an induction phase comprising administering a 900 mg induction dose of eculizumab on day 1 , administering 900 mg doses of eculizumab on days 7, 14, and 21 , and administering 1200 mg of eculizumab as a fifth induction dose on day 28; wherein the 28 day induction phase of eculizumab treatment is followed by a maintenance phase comprising administering 1200 mg of eculizumab 14 days after the fifth induction dose and administering 1200 mg of eculizumab every 14 ± 2 days thereafter, wherein the induction dosing (a) and maintenance dosing (b) are both administered intravenously (IV) to the adult patient.

46. The method of any one of claims 23-36, wherein the inhibitor of complement component C5 is ravulizumab, and the administering comprising administering ravulizumab:

(a) once on Day 1 at a loading dose of:

(i) 2400 mg to a subject weighing > 40 and < 60 kg;

(ii) 2700 mg to a subject weighing > 60 and < 100 kg;

(iii) 3000 mg to a subject weighing > 100 kg;

(b) on Day 15 and every eight weeks thereafter at a maintenance dose of:

(i) 3000 mg to a subject weighing > 40 and < 60 kg;

(ii) 3300 mg to a subject weighing > 60 and < 100 kg;

(iii) 3600 mg to a subject weighing > 100 kg.

47. The method of claim 46, wherein the loading dose (a) and the maintenance dosing (b) are administered intravenously (IV).

48. A method for predicting a risk of developing or for monitoring a clinical phenotype selected from (a) attack and/or (b) clinical disability of AQP4-Ab+ NMOSD in a subject, wherein the subject is a patient having or being at risk of having AQP4-Ab+ NMOSD, the method comprising, determining the concentration of NMOSD-associated biomarker proteins GFAP and NfL in a biological fluid of the subject, and comparing the concentration the NMOSD-associated biomarker proteins to the concentration thereof in a baseline sample of biological fluid of the same type obtained from a control, wherein an elevated concentration the NMOSD-associated biomarker proteins in the subject’s sample compared to the concentration thereof in the baseline sample obtained from the control indicates that the subject is at risk of developing or has the clinical phenotype of AQP4-Ab+ NMOSD.

49. The method of claim 48, wherein,

(a) the GFAP concentration is elevated by at least 40%, 50%, 60%, 70%, 80%, 90%, 100% or more in the subject’s sample compared to the control; and

(b) the NfL concentration is elevated by at least 27%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more in the subject’s sample compared to the control.

50. The method of claim 49, wherein the concentration of GFAP and/or NfL is elevated by at least 200% in the subject’s sample compared to the control.

51 . The method of any one of claims 48-50, wherein the subject has optic neuritis and/or transverse myelitis.

52. The method of any one of claims 48-51 , wherein the subject is a relapsed patient with NMOSD.

53. The method of claim 52, wherein the GFAP concentration and the NfL concentration are elevated, relative to the control, in the relapsed NMOSD patient, and the relapsed NMOSD patient has optic neuritis and/or transverse myelitis.

54. The method of any one of claims 48-53, wherein the GFAP concentration is attenuated in a subject treated with eculizumab.

55. The method of claim 54, wherein the GFAP concentration is significantly attenuated, relative to the control, after week 4 of the eculizumab treatment.

56. The method of claim 54, wherein the GFAP concentration is significantly attenuated, relative to the control, after week 24 of the eculizumab treatment.

57. The method of claim 54, wherein the subject is an adult patient, and wherein eculizumab is administered using a phased dosing schedule with an induction phase comprising administering a 900 mg induction dose of eculizumab on day 1 , administering 900 mg doses of eculizumab on days 7, 14, and 21 , and administering 1200 mg of eculizumab as a fifth induction dose on day 28; wherein the 28 day induction phase of eculizumab treatment is followed by a maintenance phase comprising administering

1200 mg of eculizumab 14 days after the fifth induction dose and administering 1200 mg of eculizumab every 14 ± 2 days thereafter, wherein the induction dosing (a) and maintenance dosing (b) are both administered intravenously (IV) to the adult patient.

58. The method of any one of claims 48-53, wherein the GFAP concentration and the NfL concentration are both attenuated in a subject treated with ravulizumab.

59. The method of claim 58, wherein the GFAP concentration and the NfL concentration are significantly attenuated, relative to the control, at around week 50 of ravulizumab treatment.

60. The method of claim 58, wherein the GFAP concentration and the NfL concentration are significantly attenuated, relative to the control, at around week 130 of ravulizumab treatment.

61 . The method of claim 58, wherein the ravulizumab treatment comprises treating an adult NMOSD patient with an administration cycle comprising administering ravulizumab:

(a) once on Day 1 at a loading dose of:

(i) 2400 mg to a subject weighing > 40 and < 60 kg;

(ii) 2700 mg to a subject weighing > 60 and < 100 kg;

(iii) 3000 mg to a subject weighing > 100 kg;

(b) on Day 15 and every eight weeks thereafter at a maintenance dose of:

(i) 3000 mg to a subject weighing > 40 and < 60 kg;

(ii) 3300 mg to a subject weighing > 60 and < 100 kg;

(iii) 3600 mg to a subject weighing > 100 kg.

62. The method of claim 61 , wherein the loading dose (a) and the maintenance dosing (b) are administered intravenously (IV).

63. The method of any one of claims 58-62, wherein, the GFAP concentration is attenuated at an earlier timepoint of ravulizumab treatment than the NfL concentration.

64. The method of any one of claims 58-63, wherein, the GFAP concentration is attenuated after week 2 of ravulizumab treatment and the NfL concentration is attenuated after week 26 of ravulizumab treatment.

65. The method of any one of claims 48-64, wherein the biological fluid is blood, a blood fraction, or cerebrospinal fluid.

66. The method of claim 65, wherein the blood fraction is serum or plasma.

07. The method of any one of claims 48-66, further comprising measuring a clinical profile of the subject and/or the control.

68. The method of claim 67, wherein the clinical profile is measurement of astrocyte and neuron damage.

69. The method of any one of claims 1 -68, wherein the subject is a human.