WO2016033509A1 - Aquaporin-4 antibodies and uses thereof for the treatment of neuromyelitis optica - Google Patents

Abstract

Provided herein are compositions comprising an aquaporin-4 (AQP4) antibody. Also provided herein are methods of using AQP4 for the management and treatment of AQP4-mediated diseases, such as neuomyelitis optica (NMO).

Description

AQUAPORIN-4 ANTIBODIES AND USES THEREOF FOR THE TREATMENT

OF NEUROMYELITIS OPTICA

Cross-Reference to Related Applications

[0001] This application claims the benefit of priority to U.S. Serial No. 62/044,077 filed August 29, 2014, which is incorporated herein by reference in its entirety.

1. FIELD

[0002] Provided herein are compositions comprising an aquaporin-4 (AQP4) antibody. Also provided herein are methods of using an AQP4 antibody for the management and treatment of AQP4- mediated diseases, such as neuromyelitis optica (NMO).

2. BACKGROUND

2.1 Aquaporin-4

[0003] Aquaporin-4 (AQP4) is a water channel expressed in astrocytes throughout the central nervous system (Lennon et al, J. Exp. Med., 202:473-477 ^', 2005), which is involved in water balance in brain (Manley et al, Nat. Med., 6: 159-163, 2000; Papadopoulos et al, FASEB J., 18: 1291-1293, 2004) and spinal cord (Saadoun et al, Brain, 131 : 1087-1098, 2008), sensory signal transduction (Li and Verkman, J. Biol Chem., 276:31233-31237, 2001 ; Lu et al, FASEB J., 22:3216-3223, 2008), and neuroexcitatory phenomena including seizure activity (Binder et al, Acta Neurochir. Suppl., 96:389-392, 2006; Binder et al, Glia, 53:631-636, 2006), and cortical spreading depression (Padmawar et al, Nat. Methods, 2:825-827, 2005), and astrocyte migration and glial scarring (Saadoun et al, J. Cell Sci., 1 18:5691-5698, 2005; Auguste et al, FASEB J., 21 : 108-1 16, 2007). AQP4 is expressed in astrocytes as two major isoforms: a long (Ml) isoform with translation initiation at Met- 1, and a shorter (M23) isoform with translation initiation at Met-23 (Hasegawa et al, J. Biol. Chem., 269:5497-5500, 1994; Jung et al, Proc. Natl. Acad. Sci. USA, 91 : 13052-13056, 1994; Yang et al, J. Biol. Chem., 270:22907-22913, 1995; Lu et al, Proc. Natl. Acad. Sci. USA, 93: 10908-10912, 1996). M23 AQP4 assembles in membranes as regular square arrays called orthogonal arrays of particles (OAPs), which were originally seen by freeze- fracture electron microscopy (Landis and Reese, J. Cell Biol, 60:316-320, 1974; Wolburg, J. Hirnforsch., 36:239-258, 1995). OAP formation by M23 results from tetramer-tetramer interactions involving residues just downstream of Met-23 at its cytoplasmic N-terminus, while residues in Ml AQP4 just upstream of Met-23 disrupt this interaction (Crane and Verkman, J. Cell Sci., 122:813-821, 2009). While Ml does not form OAPs on its own, it can co-assemble with M23 in heterotetramers that limit OAP size (Neely et al, Biochemistry, 38: 1 1 156-1 1 163, 1999; Furman et al, Proc. Natl. Acad. Sci. USA, 100: 13609-13614, 2003; Crane et al, J. Biol. Chem., 284:35850-35860, 2009; Tajima et al, J. Biol. Chem., 285:8163-8170, 2010). The biological significance of OAP formation by AQP4 remains unknown, with speculated functions including cell-cell adhesion, enhanced AQP4 water permeability, and AQP4 polarization to astrocyte end- feet.

2.2 Neuromyelitis Optica

[0004] Neuromyelitis optica (NMO), also known as Devic's disease or Devic's syndrome, is an autoimmune, inflammatory disorder in which a person's own immune system attacks the optic nerves and spinal cord. This produces an inflammation of the optic nerve (optic neuritis) and the spinal cord (myelitis). Although inflammation can also affect the brain, the lesions are different from those observed in the related condition multiple sclerosis (MS). Spinal cord lesions lead to varying degrees of weakness or paralysis in the legs or arms, loss of sensation (including blindness), and/or bladder and bowel dysfunction.

[0005] NMO is a rare disorder which resembles MS in several ways, but requires a different course of treatment for optimal results. NMO has also been suggested to be a variant form of acute disseminated encephalomyelitis. The target of the autoimmune attack in at least some patients with NMO has been identified - it is a protein of the nervous system cells called aquaporin 4 or AQP4.

[0006] The main symptoms of NMO are loss of vision and spinal cord function. As for other etiologies of optic neuritis, the visual impairment usually manifests as decreased visual acuity, although visual field defects, or loss of color vision can occur in isolation or prior to formal loss of acuity. Spinal cord dysfunction can lead to muscle weakness, reduced sensation, or loss of bladder and bowel control. The typical patient has an acute and severe spastic weakness of the legs (paraparesis) or all four limbs (tetraparesis) with sensory signs, often accompanied by loss of bladder control.

[0007] NMO is similar to MS in that there is immune-mediated destruction of the myelin surrounding nerve cells. Unlike standard MS, the attacks are not targeted against the myelin producing cells (oligodendrocytes) or primarily mediated by the immune system's T cells but rather by antibodies called NMO-IgG, or simply NMO antibodies. These antibodies target AQP4 in the cell membranes of astrocytes which acts as a channel for the transport of water across the cell membrane. AQP4 is found in the processes of the astrocytes that surround the blood-brain barrier, a system responsible for preventing substances in the blood from crossing into the brain. The blood-brain barrier is weakened in NMO, but it is currently unknown how the NMO-IgG immune response leads to oligodendrocyte death and demyelination.

[0008] Most research into the pathology of NMO has focused on the spinal cord. The damage in the spinal cord can range from inflammatory demyelination to necrotic damage of the white and grey matter. The inflammatory lesions in NMO have been classified as type II lesions (complement mediated demyelination), but they differ from MS pattern II lesions in their prominent perivascular distribution. Therefore, the pattern of inflammation is often quite distinct from that seen in MS. [0009] The Mayo Clinic proposed a revised set of criteria for diagnosis of NMO in 2006. The new guidelines for diagnosis require two absolute criteria plus at least two of three supportive criteria being:

1. Absolute criteria:

Optic neuritis

Acute myelitis

2. Supportive criteria:

Brain MRI not meeting criteria for MS at disease onset

Spinal cord MRI with contiguous T2 -weighted signal abnormality extending over

3 or more vertebral segments, indicating a relatively large lesion in the spinal cord

NMO-IgG seropositive status. The NMO-IgG test checks the existence of antibodies against the aquaporin 4 antigen.

[0010] After the development of the NMO-IgG test, the spectrum of disorders that comprise NMO was expanded. The NMO spectrum is now believed to consist of:

• Standard NMO, according to the diagnostic criteria described above

• Limited forms of NMO, such as single or recurrent events of longitudinally extensive myelitis, and bilateral simultaneous or recurrent optic neuritis

• Asian optic-spinal MS. This variant can present CNS involvement like MS

• Longitudinally extensive myelitis or optic neuritis associated with systemic auto-immune disease

• Optic neuritis or myelitis associated with lesions in specific brain areas such as the hypothalamus, periventricular nucleus, and brainstem

[0011] Whether NMO is a distinct disease or part of the wide spectrum of multiple sclerosis is debated. In general, NMO is now considered to be a distinct neuro-inflammatory disorder. NMO differs in that it usually has more severe sequelae after an acute episode than in MS, MS infrequently presents as transverse myelitis, and oligoclonal bands in the CSF, as well as white matter lesions on brain MRI, are uncommon in Devic's disease but occur in over 90% of MS patients. Recently, it has been found that antiviral immune response distinguishes multiple sclerosis and neuromyelitis optica.

[0012] NMO has been associated with many systemic diseases, based on anecdoctal evidence of some NMO patients with a comorbid condition. Such conditions include: collagen vascular diseases, autoantibody syndromes, infections with varicella-zoster virus, Epstein-Barr virus, and HIV, and exposure to clioquinol and antituberculosis drugs.

[0013] Currently, there is no cure for NMO, but symptoms can be treated. Some patients recover, but many are left with impairment of vision and limbs, which can be severe. Attacks are treated with short courses of high dosage intravenous corticosteroids such as methylprednisolone IV. When attacks progress or do not respond to corticosteroid treatment, plasmapheresis can be an effective treatment. Clinical trials for these treatments contain very small numbers, and most are uncontrolled.

[0014] No controlled trials have established the effectiveness of treatments for the prevention of attacks. Many clinicians agree that long term immunosuppression is required to reduce the frequency and severity of attacks, while others argue the exact opposite. Commonly used immunosuppressant treatments include azathioprine (Imuran) plus prednisone, mycophenolate mofetil plus prednisone, Rituximab, Mitoxantrone, intravenous immunoglobulin (IVIG), and Cyclophosphamide. In 2007, NMO was reported to be responsive to glatiramer acetate and to low-dose corticosteroids. Normally, there is some measure of improvement in a few weeks, but residual signs and disability can persist, sometimes severely.

[0015] The disease can be monophasic, i.e., a single episode with permanent remission. However, at least 85% of patients have a relapsing form of the disease with repeated attacks of transverse myelitis and/or optic neuritis. In patients with the monophasic form the transverse myelitis and optic neuritis occur simultaneously or within days of each other. On the other hand, patients with the relapsing form are more likely to have weeks or months between the initial attacks and to have better motor recovery after the initial transverse myelitis event. Relapses usually occur early with about 55% of patients having a relapse in the first year and 90% in the first 5 years. Unlike multiple sclerosis, NMO rarely has a secondary progressive phase in which patients have increasing neurologic decline between attacks without remission. Instead, disabilities arise from the acute attacks.

[0016] Approximately 20% of patients with monophasic NMO have permanent visual loss and 30% have permanent paralysis in one or more legs. Among patients with relapsing NMO, 50% have paralysis or blindness within 5 years. In some patients (33% in one study), transverse myelitis in the cervical spinal cord resulted in respiratory failure and subsequent death. However, the spectrum of NMO has widened due to improved diagnostic criteria, and the options for treatment have improved; as a result, researchers believe that these estimates will be lowered.

[0017] The prevalence and incidence of NMO has not been established partly because the disease is underrecognized and often confused with MS. NMO is more common in women than men, with women comprising over 2/3 of patients and more than 80% of those with the relapsing form of the disease.

According to the Walton Centre in England, "NMO seems to be present across the world unlike MS, which has a higher incidence in temperate climates and white races. Africans and Asians especially in Far East can have a higher risk of NMO, although the exact incidence of this disease is unknown, making specific conclusions difficult." Although many people who have NMO were initially mis-diagnosed with MS, 35% of African Americans are often mis-diagnosed with MS when they really have NMO. NMO is more common in Asiatic people than Caucasians. In fact, Asian optic-spinal MS (which constitutes 30% of the cases of MS in Japan) has been suggested to be identical to NMO (differences between optic-spinal and classic MS in Japanese patients). In the indigenous populations of tropical and subtropical regions, MS is rare, but when it appears it often takes the form of optic-spinal MS. The majority of NMO patients have no affected relatives, and it is generally regarded as a non- familial condition.

[0018] A defining feature of the neuroinflammatory demyelinating disease NMO is the presence of serum autoantibodies (NMO-IgG) against AQP4. The presence of NMO-IgG is specific for NMO, and in some reports serum NMO-IgG titers correlate with NMO disease activity (Matiello et al, Neurology, 1 '0:2197-2200, 2008; Jarius et al, Brain, 131 :3072-3080, 2008). Studies in rodents suggest that NMO- IgG is pathogenic in NMO. Human NMO-IgG produces many features of NMO disease in rats with preexisting experimental autoimmune encephalomyelitis (Bennett et al, Ann. Neurol, 66:617-629, 2009; Bradl et al, Ann. Neurol, 66:630-643, 2009) or pre-treated with complete Freund's adjuvant (Kinoshita et al, Biochem. Biophys. Res. Commun., 394:205-210, 2010), and in na^'ive mice when injected together with human complement (Saadoun et al, Brain, 133:349-361, 2010). These animals develop

characteristic NMO lesions with neuroinflammation, perivascular deposition of activated complement, demyelination, and loss of astrocyte GFAP and AQP4 immunoreactivity.

[0019] At present, there remain limited treatments for symptoms of NMO with no known therapies that prevent the underlying inflammatory event. The present invention addresses these and other issues. 3. SUMMARY

[0020] The inventors previously showed that NMO patient serum and a recombinant monoclonal NMO-IgG were each able to bind to both the M23 and Ml isoforms of AQP4 (Crane et al, J. Biol.

Chem., 284:35850-35860, 2009), which contradicted an earlier study reporting undetectable binding to Ml AQP4 of serum from one NMO patient (Nicchia et al, Glia, 57: 1363-1373, 2009). Individual affinity of NMO-IgG AQP4 with M23 and Ml were also noted (Crane et al, J. Biol. Chem., 284:35850- 35860, 2009). A prior report that analyzed a single NMO serum specimen concluded that OAPs are the sole target of NMO-IgG (Nicchia et al, Glia, 57: 1363-1373, 2009). However, this conclusion cannot be correct because the clinical serum assay for serum anti-AQP4 autoantibody uses Ml AQP4 (Wingerchuk et al, Neurology, 66: 1485-1489, 2006), and the inventors (Bennett et al, Ann. Neurol, 66:617-629, 2009; Crane et al, J. Biol. Chem., 284:35850-35860, 2009) and others (Hinson et al, Neurology, 69:2221-2231, 2007) reported strong binding of some NMO autoantibodies to cells expressing only Ml AQP4. A recent study showed that clinical sensitivity of the NMO-IgG binding assay could be improved by using M23- expressing cells (Mader et al, PLoS One, 5:el0455, 2010), and, without wishing to be bound by theory, it is believed that AQP4 epitopes are likely formed within tetramers and between tetramers (data not shown). [0021] The inventors used quantitative ratio imaging to measure NMO autoantibody binding to AQP4 in which NMO-lgG binding, as revealed by a fluorescent secondary antibody, was normalized to total AQP4 protein using an antibody directed against the AQP4 C-terminus. For these studies, the inventors identified a human astrocyte-derived cell line that expressed AQP4 in a plasma membrane pattern after transfection. The strategy to assess independently the AQP4 isoform and OAP specificities of NMO-lgG binding was to express Ml and M23 AQP4 in different ratios, or an M23 mutant containing an OAP- disrupting, single amino acid substitution in its N-terminus. Measurements were made on serum samples from NMO patients, as well as purified monoclonal antibodies generated by recombinant technology from cloned sequences derived from plasma cells in the cerebrospinal fluid of an NMO patient. Studies using monoclonal NMO antibodies allowed, for the first time, the measurement of absolute binding affinities of NMO-lgG to AQP4. Studies using OAP-deficient mutants of M23 AQP4 and various heterotetramer- forming mixed AQP4 isoforms indicated enhanced NMO-lgG binding to AQP4 in OAPs. Studies comparing whole NMO-lgG to purified Fab fragments suggested a molecular basis for the enhanced NMO-lgG binding to array-assembled AQP4.

[0022] In addition, the inventors generated non-pathogenic human recombinant monoclonal anti- AQP4 antibodies, which they call "aquaporumab," that selectively block NMO-lgG binding to AQP4 and prevent NMO-IgG-induced cell killing and lesion formation. Aquaporumab comprises a tight-binding anti-AQP4 Fab and a mutated Fc that lacks functionality for complement- and cell-mediated cytotoxicity. In AQP4-expressing cell cultures, aquaporumab blocked binding of NMO-lgG in human sera, reducing to near zero complement- and cell-mediated cytotoxicity. Aquaporumab prevented the development of NMO-like lesions in an ex vivo spinal cord slice model of NMO and in an in vivo mouse model of NMO produced by intracerebral injection of NMO-lgG and complement. Aquaporumab alone did not cause pathology. The broad efficacy of aquaporumab inhibition is likely due to steric competition because of its large physical size compared to the extracellular domain of AQP4. These results provide support for aquaporumab therapy of NMO.

[0023] The compositions, methods and kits provided herein are based, in part, upon the discovery that certain mutations in the Fc region of AQP4 antibodies result in a decrease or elimination of effector function of the antibody.

[0024] In one aspect, provided herein is a human anti-aquaporin-4 (AQP4) IgG antibody or an antigen binding fragment thereof comprising a mutated IgGl Fc region, wherein said mutated IgGl Fc region comprises an amino acid substitution selected from the group consisting of a D270A substitution, a P331G substitution, a N297D substitution, and a I253D substitution. In one embodiment, the mutated Fc region comprises a D270A amino acid substitution. In another embodiment, the mutated Fc region comprises a P331G amino acid substitution. In other embodiments, the mutated Fc region comprises a N297D amino acid substitution. In one embodiment, the mutated Fc region comprises a I253D amino acid substitution. In another embodiment, the mutated Fc region further comprises a L234A amino acid substitution. In another embodiment, the mutated Fc region further comprises a L235A amino acid substitution. In certain embodiments, the mutated Fc region further comprises a L234A and L235A amino acid substitution. In other embodiments, the mutated Fc region further comprises a K322A amino acid substitutions. In other embodiments, the mutated Fc region further comprises a P329A amino acid substitutions. In certain embodiments, the mutated Fc region further comprises a K322A and P329A amino acid substitution. In other embodiments, the mutated Fc region comprises a L234, L235A and P331G amino acid substitution. In yet other embodiments, the mutated Fc region comprises a L234, L235A, K322A, P329A and P331G amino acid substitution.

[0025] In another aspect, provided herein is a human anti-AQP4 IgG antibody or an antigen binding fragment thereof comprising a mutated Fc region, wherein said mutated Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, and SEQ ID NO:78. In one embodiment, the mutated Fc region comprises an amino acid sequence of SEQ ID NO:68. In another embodiment, the mutated Fc region comprises an amino acid sequence of SEQ ID NO: 70. In one embodiment, the mutated Fc region comprises an amino acid sequence of SEQ ID NO: 72. In another embodiment, the mutated Fc region comprises an amino acid sequence of SEQ ID NO: 74. In one embodiment, the mutated Fc region comprises an amino acid sequence of SEQ ID NO: 76. In another embodiment, the mutated Fc region comprises an amino acid sequence of SEQ ID NO:78.

[0026] In another aspect, provided herein is a human anti-AQP4 IgG antibody or an antigen binding fragment thereof comprising a mutated Fc region, said mutated Fc region is encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO:71, SEQ ID NO: 73, SEQ ID NO:75, and SEQ ID NO:77. In one embodiment, the mutated Fc region is encoded by the nucleic acid of SEQ ID NO:67. In another embodiment, the mutated Fc region is encoded by the nucleic acid of SEQ ID NO:69. In one embodiment, the mutated Fc region is encoded by the nucleic acid of SEQ ID NO:71. In another embodiment, the mutated Fc region is encoded by the nucleic acid of SEQ ID NO: 73. In one embodiment, the mutated Fc region is encoded by the nucleic acid of SEQ ID NO: 75. In another embodiment, the mutated Fc region is encoded by the nucleic acid of SEQ ID NO:77. In some embodiments, the IgGl antibody further comprises a FLAG tag.

[0027] In some embodiments, the antibody or binding fragment thereof comprises: (a) a heavy chain variable (VH) region comprising: (1) a VH complementarity determining region (CDR) 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:22, 34, and 46; (2) a VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:24, 36, and 48; and (3) a VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:26, 38, and 50; and/or (b) a light chain variable (VL) region comprising: (1) a VL CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:28, 40, and 52 (2) a VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:30, 42, and 54; and (3) a VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:32, 44, and 56. In other embodiments, the antibody or binding fragment thereof comprises a VH region comprising: (1) a VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:22, 34, and 46; (2) a VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:24, 36, and 48; and (3) a VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:26, 38, and 50. In certain embodiments, the antibody or binding fragment thereof comprises a VL region comprising: (1) a VL CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:28, 40, and 52; (2) a VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:30, 42, and 54; and (3) a VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:32, 44, and 56. In some embodiments, the antibody or binding fragment thereof comprises: (a) a VH region comprising: (1) a VH CDR1 comprising an amino acid sequence of SEQ ID NO:22; (2)a VH CDR2 comprising an amino acid sequence of SEQ ID NO:24; and (3) a VH CDR3 comprising an amino acid sequence of SEQ ID NO:26; and/or (b) a VL region comprising: (1) a VL CDR1 comprising an amino acid sequence of SEQ ID NO:28; (2) a VL CDR2 comprising an amino acid sequence of SEQ ID NO:30; and (3) a VL CDR3 comprising an amino acid sequence of SEQ ID NO:32. In another embodiment, the antibody or binding fragment thereof comprises: (a) a VH region comprising: (1) a VH CDR1 comprising an amino acid sequence of SEQ ID NO:34; (2) a VH CDR2 comprising an amino acid sequence of SEQ ID NO:36; and (3) a VH CDR3 comprising an amino acid sequence of SEQ ID NO:38; and/or (b) a VL region comprising: (1) a VL CDR1 comprising an amino acid sequence of SEQ ID NO:40; (2) a VL CDR2 comprising an amino acid sequence of SEQ ID NO:42; and (3) a VL CDR3 comprising an amino acid sequence of SEQ ID NO:44. In some embodiments, the antibody or binding fragment thereof comprises: (a) a VH region comprising: (1) a VH CDR1 comprising an amino acid sequence of SEQ ID NO:46; (2) a VH CDR2 comprising an amino acid sequence of SEQ ID NO:48; and (3) a VH CDR3 comprising an amino acid sequence of SEQ ID NO:50; and/or (b) a VL region comprising: (1) a VL CDR1 comprising an amino acid sequence of SEQ ID NO:52; (2) a VL CDR2 comprising an amino acid sequence of SEQ ID NO:54; and (3) a VL CDR3 comprising an amino acid sequence of SEQ ID NO:56. In one embodiment, the antibody all three VH CDR1, VH CDR2 and VH CDR3, and/or all three VL CDR1, VL CDR2, and VL CDR3 from: (i) the antibody designated rAb53 that comprises a VH sequence having the amino acid sequence depicted in SEQ ID NO:2 and a VL sequence having the amino acid sequence depicted in SEQ ID N0:4; (ii) the antibody designated rAb58 that comprises a VH sequence having the amino acid sequence depicted in SEQ ID NO: 8 and a VL sequence having the amino acid sequence depicted in SEQ ID NO: 10; or (iii) the antibody designated rAb09-3-33 that comprises a VH sequence having the amino acid sequence depicted in SEQ ID NO: 14 and a VL sequence having the amino acid sequence depicted in SEQ ID NO: 16. In some embodiments, the antibody or antigen binding fragment thereof comprises all three heavy chain CDRs and/or all three light chain CDRs from the antibody designated rAb53. In some embodiments, the antibody or antigen binding fragment thereof comprises all three heavy chain CDRs and/or all three light chain CDRs from the antibody designated rAb58. In some embodiments, the antibody or antigen binding fragment thereof comprises all three heavy chain CDRs and/or all three light chain CDRs from the antibody designated rAb09-3-33.

[0028] In some embodiments, the antibody or binding fragment thereof has a VH region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, 8, and 14, and/or a VL region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:4, 10, and 16. In certain embodiments, the antibody comprises a VH region comprises an amino acid sequence of SEQ ID NO:2 and/or a VL region comprises an amino acid sequence of SEQ ID NO:4. In certain embodiments, the antibody comprises a VH region comprises an amino acid sequence of SEQ ID NO: 8 and/or a VL region comprises an amino acid sequence of SEQ ID NO: 10. In certain embodiments, the antibody comprises a VH region comprises an amino acid sequence of SEQ ID NO: 14 and/or a VL region comprises an amino acid sequence of SEQ ID NO: 16. In one embodiment, the VL region further comprises a kappa constant region. In some embodiments, the kappa constant region comprises an amino acid sequence of SEQ ID NO:20. In some embodiments, the VH region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, 8, and 14, and/or the VL region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 12, and 18. In some embodiments, the VH region comprises an amino acid sequence of SEQ ID NO:2 and/or the VL region comprises an amino acid sequence of SEQ ID NO:6. In certain embodiments, the VH region comprises an amino acid sequence of SEQ ID NO: 8 and/or the VL region comprises an amino acid sequence of SEQ ID NO: 12. In some embodiments, the VH region comprises an amino acid sequence of SEQ ID NO: 14 and/or the VL region comprises an amino acid sequence of SEQ ID NO: 18.

[0029] In another aspect, provided herein is a method of treating a subject with neuromyelitis optica (NMO) spectrum disease, or a symptom thereof, comprising administering a composition comprising a therapeutically effective amount of human anti-AQP4 IgG antibody, or an antigen binding fragment thereof, provided herein. In some embodiments, the subject is a human subject. In one embodiment, the administering comprises intraocular, intraatertial, subcutaneous, intravenous administration or intrathecal route of administration. In some embodiments, the treating comprises reducing one or more of retinal ganglion cell death, optic nerve injury, spinal cord injury, or axonal transection. In certain embodiments, the treating comprises reducing one or more of optic nerve demyelination, spinal cord demyelination, astrocyte death or oligodendrocyte death. In some embodiments, the composition is administered more than once, including chronically and daily. In other embodiments, the composition is administered upon onset of or following an NMO attack. In some embodiments, the composition is administered within about 1 hour, 6 hours, 12 hours, 24 hours or two days of an NMO attack.

[0030] In one embodiment, the method comprises administering to said subject a second agent that treats one or more aspect of NMO. In some embodiments, the second agent is administered at the same time as said composition. In certain embodiments, the second agent is administered before or after the composition.

[0031] In some embodiments, the method further comprises assessing said subject for positive NMO-IgG (AQP4) serology. In some embodiments, the subject exhibits positive NMO-IgG (AQP4) serology.

[0032] In certain embodiments, the subject exhibits one or more of transverse myelitis, optic neuritis or other unrelated neurologic dysfunction. In some embodiments, the unrelated neurologic dysfunction comprises protracted nausea or vomiting.

[0033] In some embodiments, provided herein is a method of chronically treating a subject to prevent or reduce exacerbations of NMO spectrum disease, or a symptom thereof, comprising administering to said subject a composition comprising a therapeutically effective amount of a human anti-AQP4 IgG antibody or an antigen binding fragment thereof provided herein.

[0034] In other embodiments, also provided herein is a method of preventing or inhibiting the progression of NMO spectrum disease, or a symptom thereof, in a subject, comprising administering to said subject a composition comprising a therapeutically effective amount of a human anti-AQP4 IgG antibody or an antigen binding fragment thereof provided herein.

[0035] These and other aspects and embodiments of the invention are described in greater detail below.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The following drawings form part of the present specification and are included to further demonstrate certain aspects provided herein. The invention can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0037] FIGS. 1A-B depict a schematic of the two-color ratio imaging method for quantitative measurement of NMO-IgG binding to AQP4 isoforms. (FIG. 1A) AQP4 monomers (cylinders) shown as assembling into tetramers (top) or OAPs (bottom). NMO-lgG (green) binds AQP4 at an extracellular domain, and a reference AQP4 antibody (red) binds on the cytoplasmic side. (FIG. IB) The reference AQP4 antibody binds to the C-terminus of AQP4, independent of the AQP4 N-terminal isoform and OAP formation.

[0038] FIGS. 2A-D depicts the characterization of stably transfected, AQP4-expressing U87MG cells. (FIG. 2A) Confocal fluorescence images show U87MG cells stably expressing Ml (top) or M23 (bottom) and labeled with NMO-lgG (green) and C-terminal anti-AQP4 antibody (red). (FIG. 2B) Total internal reflection fluorescence images show distinct OAPs in M23 -expressing cells (bottom), and a smooth fluorescence staining pattern in Ml -expressing cells (top). (FIG. 2C) AQP4 immunoblot following blue-native gel electrophoresis (top) and Tricine SDS-PAGE (bottom) of stable AQP4- expressing U87MG cell lysates. (FIG. 2D) Measured green-to-red fluorescence ratios (G/R) in U87MG cells after stable (grey) or transient (white) transfection with Ml or M23 AQP4, and labeled with the indicated recombinant monoclonal NMO-lgG.

[0039] FIGS. 3A-B depicts the differential binding of NMO-lgG in NMO patient serum to Ml vs. M23 AQP4. (FIG. 3A) Ml and M23 expressing U87MG cells stained with 5% NMO serum (green) from four patients, and with reference AQP4 antibody (red). (FIG. 3B) Binding curves for the NMO patient sera to Ml vs. M23 AQP4 (mean ± S.E., n=5). Curves represent fit to single-site binding model.

[0040] FIGS. 4A-B depicts the differential binding of purified monoclonal NMO-IgGs to Ml vs. M23 AQP4. (FIG. 4A) Representative fluorescence micrographs for binding of rAb-53 and rAb-58 (green) as a function of concentration, together with reference AQP4 antibody (red). (FIG. 4B) Binding curves for rAb-53 (left), rAb-58 (middle) and rAb- 186 (right) to Ml vs. M23 AQP4 (mean ± S.E., n=5). Curves represent fit to a single-site binding model.

[0041] FIGS. 5A-C depicts the binding of NMO-lgG to mixtures of Ml and M23 AQP4, and to M23 mutants containing OAP-disrupting mutations. (FIG. 5A) Binding of rAb-53 (left) and cumulative distributions of diffusion range (right), measured by quantum dot single-particle tracking, for Ml and M23 AQP4 mixtures at indicated ratios (mean ± S.E., n=5). (FIG. 5B) Binding of rAb-53 (left) and diffusion range (right) for M23 AQP4 with Ml mutant CCA at indicated ratios (mean ± S.E., n=5). (FIG. 5C) Binding of rAb-53 (left) and diffusion range (right) for AQP4 mutants M23-F26Q (red) and M23- G28P (green) (mean ± S.E., n=5).

[0042] FIGS. 6A-D depicts the mechanism of increased NMO-lgG binding affinity to array- assembled AQP4. (FIG. 6A) Human IgG and AQP4 crystal structures (Harris et al, 1998; Ho et al, 2009) showing relative size of the AQP4 tetramer compared with spacing between Fab binding sites in whole IgG. (FIG. 6B) Predictions of bivalent vs. monovalent binding mechanisms. AQP4 monomers (cylinders) are shown as assembled in tetramers (Ml) or OAPs (M23). NMO-lgG (green) binds either mono- or bivalently to (unknown) extracellular domains on AQP4. (FIG. 6C) Binding of monoclonal mouse anti-Myc to cells expressing Myc -tagged Ml vs. M23 AQP4. (FIG. 6D) Relative Ml-to-M23 binding of whole IgG or purified Fab fragments of mouse anti-Myc (left), rAb-53 (middle) and rAb-58 (right) at a fixed concentration (mean ± S.E., n=5).

[0043] FIGS. 7A-C depicts high-affinity monoclonal, recombinant anti-AQP4 antibody for aquaporumab therapy. (FIG. 7A) Crystal structure of AQP4 tetramer shown on the same scale with that of an IgG 1 antibody. (FIG. 7B) Surface plasmon resonance measurement of recombinant antibody binding to AQP4-reconstituted proteoliposomes showing binding / unbinding kinetics of rAb-53 (left) at different concentrations, and different NMO rAbs (right) at fixed concentration. (FIG. 7C) Binding and unbinding kinetics rAb-53 (25 μg/ml) to AQP4-expressing U87MG cells. Binding measured by incubation with rAb-53 for specified times followed by rinsing, fixation and fluorescent secondary antibody addition. Washout measured after 60 min incubation with rAb-53 followed by washout with antibody-free buffer for specified times. Top: Representative micrographs showing cell surface staining by rAb-53 (red). Bottom: Averaged binding data (mean ± S.E., n=4).

[0044] FIGS. 8A-D depicts mutated, non-pathogenic rAb-53 (aquaporumab) blocks binding of pathogenic NMO-IgG to AQP4. (FIG. 8A) Schematic of rAb-53 showing heavy (VH) and light (VL) chain variable regions, light chain constant region (CL), and IgGl heavy chain constant regions (CH1- CH3). Locations of amino acid mutations introduced in the CH2 domain to reduce CDC (K322A), ADCC (K326W/E333S) or both (L234A/L235A). (FIG. 8B) Surface plasmon resonance measurements of binding and washout of a mutated rAb-53 (L234A/L235A) to AQP4-reconstituted proteoliposomes. (FIG. 8C) Mutated rAb-53 block binding of Cy3-labeled (non-mutated) rAb-53 to AQP4-expressing cells. Cy3 fluorescence imaged in AQP4-null (left-most panel) or AQP4-expressing (other panels) cells incubated with 20 μg/ml Cy3-rAb-53 for 1 h in the absence or presence of indicated (unlabeled) antibodies at 100 μg/ml. (FIG. 8D) Unrelated monoclonal NMO antibodies and human NMO serum blocks AQP4 binding of Cy3-labeled rAb-53. Cy3 fluorescence imaged in cells incubated with 20 μg/ml Cy3-rAb-53 for 1 h in the absence or presence of 10% control (non-NMO) or NMO patient serum, or 100 μg/ml recombinant NMO monoclonal antibody rAb-186.

[0045] FIGS. 9A-D depicts mutated non-pathogenic rAb-53 aquaporumabs prevents CDC and ADCC in NMO-IgG-exposed AQP4-expressing cells. (FIG. 9A) Live/dead cell assay after 90 min exposure of AQP4-expressing CHO cells to human complement together with control (non-NMO) mAb or rAb-53 (2.5 μg/ml, non-mutated or mutated). Percentage dead cells summarized at the right (mean ± S.E., n=4-6, * P < 0.001 compared to rAb-53 alone). (FIG. 9B) Assay as in A done with complement + rAb-53, in the presence of 12.5 μg/ml of the indicated aquaporumabs. (FIG. 9C) Live/dead cell assay after 60 min exposure to control (non-NMO) serum or NMO patient sera in the presence of complement, and the absence or presence of indicated aquaporumabs. (FIG. 9D) ADCC assay done using AQP4- expressing CHO cells incubated with NK-cells together with control (non-NMO) mAb or rAb-53 or aquaporumab (individually), or rAb-53 and aquaporumab together.

[0046] FIGS. lOA-C depicts aquaporumab reducing NMO-like lesions in mouse brain in vivo produced by intracerebral injection of NMO-IgG and human complement. (FIG. 10A) Panel of mouse brain sections at 24 h after intracerebral injection, stained with hematoxylin and eosin (H&E) and Luxol fast blue (myelin), and immunostained brown for AQP4 (AQP4) and C5b-9 (activated complement). Intracerebral injections were made of NMO-IgG (purified IgG from NMO serum) and human complement, without or with aquaporumab (Aqmab), with controls (control IgG, AQP4 knockout mice, Aqmab alone). Pink line indicates areas of absent Luxol fast blue staining or AQP4 immunoreactivity. Black line outlines the injected hemisphere and shows needle tract. Arrows, neutrophils; arrowheads, perivascular C5b-9 immunoreactivity; V, vessel. Bar, 50 μηι. (FIG. 10B) AQP4 and myelin loss quantified as % area outlined with pink/area outlined with black (S.E.M., 5 mice per group, * P < 0.01). (FIG. IOC) % myelin and AQP4 loss shown for five pairs of mice, each pair injected with NMO-IgG from a different NMO patient with human complement, without or with aquaporumab.

[0047] FIGS. 11A-B depicts aquaporumab reducing NMO-like lesions produced by NMO-IgG and human complement in ex vivo spinal cord slice cultures. (FIG. 1 1A) Ex vivo spinal cord slice culture model in which slices were cultured for 7 days, followed by 3 days in the presence of NMO-IgG (purified IgG from NMO serum) and human complement, without or with aquaporumab (Aqmab).

Immunostaining shown for AQP4, GFAP and myelin. Controls include non-NMO IgG, NMO-IgG or Aqmab alone, Aqmab with complement, and slice cultures from AQP4 null mice. (FIG. 1 IB) NMO lesion scores (see Methods) (S.E.M., n=4-5, P < 0.001)

[0048] FIG. 12 depicts I253D mutation in Fc region of rAb-53 reducing CDC activity, as compared to the same antibody without a Fc mutation, or with an E345R or H433A mutation.

[0049] FIGS. 13A-C depicts the effects on CDC activity of I253D, E345R, or

G236A/S267E/H268F/S324T/I332E ("AEFTE") mutations in Fc region of aquaporumabs rAb 07-5-53 (FIG. 13A), rAb 07-5-58 (FIG. 13B) and rAb 07-5-186 (FIG. 13C). For rAb 07-5-53, the I253D mutation in Fc region reduced CDC activity, and the E345R mutation and AEFTE mutations both enhanced CDC activity as compared to the same antibody without a Fc mutation (FIG. 13A). For rAb 07-5-58, the I253D mutation in Fc region reduced CDC activity, and the E345R mutation enhanced CDC activity as compared to the same antibody without a Fc mutation (FIG. 13B). For rAb 07-5-186, the I253D mutation in Fc region reduced CDC activity, and neither the E345R mutation nor the AEFTE mutations had any effect on CDC activity as compared to the same antibody without a Fc mutation (FIG. 13C). 5. DETAILED DESCRIPTION

5.1 Definitions

[0050] All patents, applications, published applications and other publications are incorporated by reference in their entirety. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any description of terms set forth conflicts with any document incorporated herein by reference, the description of term set forth below shall control.

[0051] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification can mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0052] The term "about" or "approximately" mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within or 1% or less of a given value or range. In specific embodiments, "about" means plus or minus 5% of the stated number.

[0053] As used herein, "administer" or "administration" refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, disorder or condition, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of disease, disorder or condition or symptoms thereof. When a disease, disorder or condition, or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease, disorder or condition or symptoms thereof.

[0054] "Chronic" administration refers to administration of the agent(s) in a continuous mode (e.g., for a period of time such as days, weeks, months or years) as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. "Intermittent" administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

[0055] An "affinity matured" antibody is one with one or more alterations (e.g., amino acid sequence variations, including changes, additions and/or deletions) in one or more HVRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). In certain embodiments, affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. For review, see Hudson and Souriau, Nature Medicine 9 : 129-134 (2003); Hoogenboom, Nature Biotechnol. 23 : 1 105-1 1 16 (2005); Quiroz and Sinclair, Revista Ingeneria Biomedia A 39-51 (2010).

[0056] In the context of a polypeptide, the term "analog" as used herein refers to a polypeptide that possesses a similar or identical function as an AQP4 polypeptide, a fragment of an AQP4 polypeptide, or an anti-AQP4 antibody but does not necessarily comprise a similar or identical amino acid sequence of an AQP4 polypeptide, a fragment of an AQP4 polypeptide, or an anti-AQP4 antibody, or possess a similar or identical structure of an AQP4 polypeptide, a fragment of an AQP4 polypeptide, or an anti-AQP4 antibody. A polypeptide that has a similar amino acid sequence refers to a polypeptide that satisfies at least one of the following: (a) a polypeptide having an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of an AQP4 polypeptide (e.g., SEQ ID NOS:97-100), a fragment of an AQP4 polypeptide, or an anti-AQP4 antibody described herein; (b) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding an AQP4 polypeptide, a fragment of an AQP4 polypeptide, or an anti-AQP4 antibody (or VH or VL region thereof) described herein of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, or at least 150 amino acid residues (see, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY); and (c) a polypeptide encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence encoding an AQP4 polypeptide, a fragment of an AQP4 polypeptide, or an anti-AQP4 antibody (or VH or VL region thereof) described herein. A polypeptide with similar structure to an AQP4 polypeptide, a fragment of an AQP4 polypeptide, or an anti-AQP4 antibody described herein refers to a polypeptide that has a similar secondary, tertiary or quaternary structure of an AQP4 polypeptide, a fragment of an AQP4, or an AQP4 antibody described herein. The structure of a polypeptide can determined by methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy.

[0057] To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical overlapping positions/total number of positions X 100%). In one embodiment, the two sequences are the same length.

[0058] The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can also be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264 2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873 5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al, 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules provided herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score 50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule provided herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, 1997, Nucleic Acids Res. 25:3389 3402. Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov). Another non limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4: 1 1 17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0059] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0060] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies "arm" the cytotoxic cells and are absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is known (see, e.g., Table 3, page 464, Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991)). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, (see, e.g., US Patent No. 5,500,362 or 5,821,337) can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example, in a animal model {see, e.g., Clynes et al. (USA) 95:652-656 (1998)). Antibodies with little or no ADCC activity can be selected for use.

[0061] An "agonist antibody" is an antibody that triggers a response, e.g., one that mimics at least one of the functional activities of a polypeptide of interest. An agonist antibody includes an antibody that is a ligand mimetic, for example, wherein a ligand binds to a cell surface receptor and the binding induces cell signaling or activities via an intercellular cell signaling pathway and wherein the antibody induces a similar cell signaling or activation.

[0062] The term "antibody" and "immunoglobulin" or "Ig" are used interchangeably herein, and is used in the broadest sense and specifically covers, for example, individual anti-AQP4 monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies), anti-APQ4 antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies {e.g. , bispecific antibodies so long as they exhibit the desired biological activity), formed from at least two intact antibodies, single chain anti- AQP4 antibodies, and fragments of anti-AQP4 antibodies, as described below. An antibody can be human, humanized, chimeric and/or affinity matured as well as an antibody from other species, for example mouse, rabbit etc. The term "antibody" is intended to include a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50- 70 kDa) and one light chain (about 25 kDa) and each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids and each carboxy-terminal portion of each chain includes a constant region (See, Borrebaeck (ed.) (1995) Antibody Engineering, Second Ed., Oxford University Press.; Kuby (1997) Immunology, Third Ed., W.H. Freeman and Company, New York). In specific embodiments, the specific molecular antigen can be bound by an antibody provided herein includes an AQP4 polypeptide, AQP4 fragment or AQP4 epitope. An antibody or a fragment thereof that binds to an AQP4 antigen can be identified, for example, by immunoassays, BIAcore, or other techniques known to those of skill in the art. An antibody or a fragment thereof binds specifically to an AQP4 antigen when it binds to an AQP4 antigen with higher affinity than to any cross-reactive antigen as determined using experimental techniques, such as radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISAs). Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 times background. See, e.g., Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York at pages 332-336 for a discussion regarding antibody specificity.

[0063] Antibodies provided herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, intrabodies, anti- idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigens-binding fragments such as AQP4 binding fragments) of any of the above, which refers a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived. Non- limiting examples of functional fragments (e.g., antigens-binding fragments such as AQP4 binding fragments) include single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)₂ fragments, F(ab')₂ fragments, disulfide-linked Fvs (sdFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody and minibody. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, antigen binding domains or molecules that contain an antigen- binding site that binds to an AQP4 antigen (e.g., one or more complementarity determining regions (CDRs) of an anti-AQP4 antibody). Such antibody fragments can be found described in, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec. Biology and Biotechnology: A Comprehensive Desk Reference, New York: VCH Publisher, Inc.; Huston et ah, Cell Biophysics , 22: 189-224 (1993); Pliickthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, E.D., Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, NY (1990). The antibodies provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule. Anti-AQP4 antibodies can be agonistic antibodies or antagonistic antibodies. In certain embodiments, the AQP4 antibodies are fully human, such as fully human monoclonal AQP4 antibodies. In certain embodiments, antibodies provided herein are IgG antibodies, or a class (e.g., human IgGl or IgG4) or subclass thereof.

[0064] A 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to a H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and γ chains and four CH domains for μ and ε isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CHI). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0065] The term "anti-AQP4 antibody" or "an antibody that binds to AQP4" includes an antibody that is capable of binding AQP4 with sufficient affinity and specificity. In certain embodiments,, the extent of binding of an anti-AQP4 antibody to an unrelated, non-AQP4 protein is less than about 10% of the binding of the antibody to AQP4 as measured, for example, by fluorescence activated cell sorting (FACS) analysis or an immunoassay such as a radioimmunoassay (RIA). An antibody that "specifically binds to" or is "specific for" AQP4 is illustrated above. In certain embodiments, an antibody that binds to AQP4, as described herein has a dissociation constant (Kd) of less than or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM, and/or is greater than or equal to 0. InM. In certain embodiments, anti-AQP4 antibody binds to an epitope of AQP4 that is conserved among AQP4 from different species (e.g., between human and cyno AQP4).

[0066] The terms "antibodies that specifically bind to AQP4," "antibodies that specifically bind to an AQP4 epitope," and analogous terms are also used interchangeably herein and refer to antibodies that specifically bind to an AQP4 polypeptide, such as an AQP4 antigen, or fragment, or epitope (e.g., human AQP4 such as a human AQP4 polypeptide, antigen or epitope). An antibody that specifically binds to AQP4, (e.g., human AQP4) can bind to the extracellular domain or peptide derived from the extracellular domain of AQP4. An antibody that specifically binds to an AQP4 antigen (e.g., human AQP4) can be cross-reactive with related antigens (e.g., cyno AQP4). In certain embodiments, an antibody that specifically binds to an AQP4 antigen does not cross-react with other antigens. An antibody that specifically binds to an AQP4 antigen can be identified, for example, by immunoassays, Biacore, or other techniques known to those of skill in the art. An antibody binds specifically to an AQP4 antigen when it binds to an AQP4 antigen with higher affinity than to any cross reactive antigen as determined using experimental techniques, such as radioimmunoassays (RIA) and enzyme linked immunosorbent assays (ELISAs). Typically a specific or selective reaction will be at least twice background signal or noise and can be more than 10 times background. See, e.g., Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York at pages 332 336 for a discussion regarding antibody specificity. An antibody "which binds" an antigen of interest (e.g., a target antigen such as AQP4) is one that binds the antigen with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting a cell or tissue expressing the antigen, and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody to a "non-target" protein will be less than about 10% of the binding of the antibody to its particular target protein, for example, as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). With regard to the binding of an antibody to a target molecule, the term "specific binding" or "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. The term "specific binding" or "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a Kd for the target of at least about 10^"4 M, alternatively at least about 10^"5 M, alternatively at least about 10^"6 M, alternatively at least about 10^"7 M, alternatively at least about 10^"8 M, alternatively at least about 10^"9 M, alternatively at least about l O ⁰ M, alternatively at least about l O ¹ M, alternatively at least about 10⁴² M, or greater. In one embodiment, the term "specific binding" refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. In certain embodiments, an antibody that binds to AQP4 has a dissociation constant (Kd) of less than or equal to 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. The lower the K_D, the higher the affinity of the anti-AQP4 antibody. In certain embodiments, anti-AQP4 antibody binds to an epitope of AQP4 that is conserved among AQP4 from different species (e.g. , between human and cyno AQP4).

[0067] "Antibody fragments" comprise a portion of an intact antibody, such as the antigen binding or variable region of the intact antibody. Examples of antibody fragments include, without limitation, Fab, Fab', F(ab')2, and Fv fragments; diabodies and di-diabodies (see, e.g., Holliger, P. et al, (1993) Proc. Natl. Acad. Sci. 90:6444-8; Lu, D. et al, (2005) J. Biol. Chem. 280: 19665-72; Hudson et al, Nat. Med. 9: 129- 134 (2003); WO 93/1 1 161 ; and U.S. Patent Nos. 5,837,242 and 6,492, 123); single-chain antibody molecules (see, e.g., U.S. Patent Nos. 4,946,778; 5,260,203; 5,482,858 and 5,476,786); dual variable domain antibodies (see, e.g., U.S. Patent No. 7,612, 181); single variable domain antibodies (SdAbs) (see, e.g., Woolven et al, Immunogenetics 50: 98-101, 1999; Streltsov et al, Proc Natl Acad Sci USA.

101 : 12444-12449, 2004); and multispecific antibodies formed from antibody fragments.

[0068] An "antigen" is a predetermined antigen to which an antibody can selectively bind. A target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or other naturally occurring or synthetic compound. In certain embodiments,, the target antigen is a polypeptide.

[0069] The term "antigen binding fragment," "antigen binding domain," "antigen binding region," and similar terms refer to that portion of an antibody which comprises the amino acid residues that interact with an antigen and confer on the binding agent its specificity and affinity for the antigen (e.g., the CDRs). The antigen binding region can be derived from any animal species, such as rodents (e.g., rabbit, rat or hamster) and humans. In specific embodiments, the antigen binding region will be of human origin.

[0070] The term "aquaporin-4" or "AQP4" and similar terms refers to a polypeptide ("polypeptide," and "protein" are used interchangeably herein) or any native AQP4 from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkey (cyno)), dogs, and rodents (e.g., mice and rats), unless otherwise indicated, and, in certain embodiments, included various AQP4 isoforms, related AQP4 polypeptides, including SNP variants thereof.

[0071] An exemplary amino acid sequence of human AQP4, Ml isoform (NCBI:AAH22286.1 GI: 18490380), is provided below:

MS DRPTARRWGKCGPLCTRENIMVAFKGVWTQAFWKAVTAEFLAML I FVLLS LGST INWGGTEKPLPVDM VL I S LC FGLS IATMVQCFGH I S GGHINPAVTVAMVCTRKI S IAKSVFY IAAQCLGAI I GAGI LYLVT PPS WGGLGVTMVHGNLTAGHGLLVEL I I TFQLVFT I FASC DSKRTDVTGS IALAI GFSVAI GHLFAI YTGA SMNPARS FGPAVIMGNWENHWI YWVGPI I GAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEV EDNRSQVETDDL I LKPGVVHVI DVDRGEEKKGKDQS GEVLS SV (SEQ ID NO:60)

[0072] An exemplary encoding nucleic acid sequence of human AQP4, Ml isoform

(NCBLBC022286.1 GI: 18490379), is provided below:

ATGAGTGACAGACCCACAGCAAGGCGGTGGGGTAAGTGTGGACCTTTGTGTACCAGAGAGAACATCATGG TGGCTTTCAAAGGGGTCTGGACTCAAGCTTTCTGGAAAGCAGTCACAGCGGAATTTCTGGCCATGCTTAT TTTTGTTCTCCTCAGCCTGGGATCCACCATCAACTGGGGTGGAACAGAAAAGCCTTTACCGGTCGACATG GTTCTCATCTCCCTTTGCTTTGGACTCAGCATTGCAACCATGGTGCAGTGCTTTGGCCATATCAGCGGTG GCCACATCAACCCTGCAGTGACTGTGGCCATGGTGTGCACCAGGAAGATCAGCATCGCCAAGTCTGTCTT CTACATCGCAGCCCAGTGCCTGGGGGCCATCATTGGAGCAGGAATCCTCTATCTGGTCACACCTCCCAGT GTGGTGGGAGGCCTGGGAGTCACCATGGTTCATGGAAATCTTACCGCTGGTCATGGTCTCCTGGTTGAGT TGATAATCACATTTCAATTGGTGTTTACTATCTTTGCCAGCTGTGATTCCAAACGGACTGATGTCACTGG CTCAATAGCTTTAGCAATTGGATTTTCTGTTGCAATTGGACATTTATTTGCAATCAATTATACTGGTGCC AGCATGAATCCCGCCCGATCCTTTGGACCTGCAGTTATCATGGGAAATTGGGAAAACCATTGGATATATT

GGGTTGGGCCCATCATAGGAGCTGTCCTCGCTGGTGGCCTTTATGAGTATGTCTTCTGTCCAGATGTTGA

ATTCAAACGTCGTTTTAAAGAAGCCTTCAGCAAAGCTGCCCAGCAAACAAAAGGAAGCTACATGGAGGTG

GAGGACAACAGGAGTCAGGTAGAGACGGATGACCTGATTCTAAAACCTGGAGTGGTGCATGTGATTGACG

TTGACCGGGGAGAGGAGAAGAAGGGGAAAGACCAATCTGGAGAGGTATTGTCTTCAGTATGA (SEQ ID

NO:59)

[0073] An exemplary amino acid sequence of human AQP4, M23 isoform (NCBLAAH22286.1 GI: 18490380), is provided below:

1WAFKGVWTQAFWKAVTAEFLAML I FVLLS LGST INWGGTEKPLPVDMVL I S LC FGLS IATMVQCFGHI S GGHI PAVTVAMVCTRKI S I AKSVFY IAAQCLGAI I GAGI LYLVTPPSWGGLGVTMVHGNLTAGHGLLV EL I I TFQLVFT I FASC DSKRTDVTGS IALAI GFSVAI GHLFAINYTGASMNPARS FGPAVIMGNWENHWI YWVGPI I GAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDL I LKPGWHVI DVDRGEEKKGKDQS GEVLS SV (SEQ ID NO:58)

[0074] An exemplary encoding nucleic acid sequence of human AQP4, M23 isoform

(NCBLBC022286.1 GI: 18490379), is provided below:

ATGGTGGCTTTCAAAGGGGTCTGGACTCAAGCTTTCTGGAAAGCAGTCACAGCGGAATTTCTGGCCATGC TTATTTTTGTTCTCCTCAGCCTGGGATCCACCATCAACTGGGGTGGAACAGAAAAGCCTTTACCGGTCGA CATGGTTCTCATCTCCCTTTGCTTTGGACTCAGCATTGCAACCATGGTGCAGTGCTTTGGCCATATCAGC GGTGGCCACATCAACCCTGCAGTGACTGTGGCCATGGTGTGCACCAGGAAGATCAGCATCGCCAAGTCTG TCTTCTACATCGCAGCCCAGTGCCTGGGGGCCATCATTGGAGCAGGAATCCTCTATCTGGTCACACCTCC CAGTGTGGTGGGAGGCCTGGGAGTCACCATGGTTCATGGAAATCTTACCGCTGGTCATGGTCTCCTGGTT GAGTTGATAATCACATTTCAATTGGTGTTTACTATCTTTGCCAGCTGTGATTCCAAACGGACTGATGTCA CTGGCTCAATAGCTTTAGCAATTGGATTTTCTGTTGCAATTGGACATTTATTTGCAATCAATTATACTGG TGCCAGCATGAATCCCGCCCGATCCTTTGGACCTGCAGTTATCATGGGAAATTGGGAAAACCATTGGATA TATTGGGTTGGGCCCATCATAGGAGCTGTCCTCGCTGGTGGCCTTTATGAGTATGTCTTCTGTCCAGATG TTGAATTCAAACGTCGTTTTAAAGAAGCCTTCAGCAAAGCTGCCCAGCAAACAAAAGGAAGCTACATGGA GGTGGAGGACAACAGGAGTCAGGTAGAGACGGATGACCTGATTCTAAAACCTGGAGTGGTGCATGTGATT GACGTTGACCGGGGAGAGGAGAAGAAGGGGAAAGACCAATCTGGAGAGGTATTGTCTTCAGTATGA (SEQ ID NO:57)

[0075] An "AQP4-mediated disorder" refers to any disease that is completely or partially caused by or is the result of defect in AQP4. In a specific embodiment, the AQP4-mediated disorder is NMO.

[0076] The terms "binds" or "binding" refer to an interaction between molecules including, for example, to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions or forces. The strength of the total non-covalent interactions between a single antigen- binding site on an antibody and a single epitope of a target molecule, such as AQP4, is the affinity of the antibody or functional fragment for that epitope. The ratio of association (kl) to dissociation (k- 1) of an antibody to a monovalent antigen (kl / k- 1) is the association constant K, which is a measure of affinity. The value of K varies for different complexes of antibody and antigen and depends on both kl and k- 1. The association constant K for an antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art. The affinity at one binding site does not always reflect the true strength of the interaction between an antibody and an antigen. When complex antigens containing multiple, repeating antigenic determinants, such as a polyvalent AQP4, come in contact with antibodies containing multiple binding sites, the interaction of antibody with antigen at one site will increase the probability of a reaction at a second site. The strength of such multiple interactions between a multivalent antibody and antigen is called the avidity. The avidity of an antibody can be a better measure of its binding capacity than is the affinity of its individual binding sites. For example, high avidity can compensate for low affinity as is sometimes found for pentameric IgM antibodies, which can have a lower affinity than IgG, but the high avidity of IgM, resulting from its multivalence, enables it to bind antigen effectively.

[0077] "Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a binding molecule X for its binding partner Y can generally be represented by the dissociation constant (K_D). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. Specific illustrative embodiments include the following. In one embodiment, the "K_D" or "K_D value" can be measured by assays known in the art, for example by a binding assay. The K_D can be measured in a radiolabeled antigen binding assay (RIA), for example, performed with the Fab version of an antibody of interest and its antigen (Chen, et ah, (1999) J. Mol Biol 293 :865-881). The K_D or K_D value can also be measured by using surface plasmon resonance assays by Biacore, using, for example, a BIAcoreTM-2000 or a BIAcoreTM-3000 BIAcore, Inc., Piscataway, NJ), or by biolayer interferometry using, for example, the OctetQK384 system (ForteBio, Menlo Park, CA). An "on-rate" or "rate of association" or "association rate" or "kon" can also be determined with the same surface plasmon resonance or biolayer interferometry techniques described above using, for example, a BIAcoreTM-2000 or a BIAcoreTM-3000 (BIAcore, Inc., Piscataway, NJ), or the OctetQK384 system (ForteBio, Menlo Park, CA).

[0078] A "blocking" antibody or an "antagonist" antibody is one which inhibits or reduces biological activity of the antigen it binds. For example, blocking antibodies or antagonist antibodies can

substantially or completely inhibit the biological activity of the antigen.

[0079] "Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and

concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight ((e.g., less than about 10 amino acid residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins;

hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. The term "carrier" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), excipient, or vehicle with which the therapeutic is administered. Such carriers, including pharmaceutical carriers, can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a exemplary carrier when a composition (e.g., a pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral

compositions, including formulations, can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Compositions, including pharmaceutical compounds, can contain a prophylactically or therapeutically effective amount of an anti-AQP4 antibody, for example, in isolated or purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject (e.g., patient). The formulation should suit the mode of administration.

[0080] As used herein, the terms "complementarity determining region," "CDR," "hypervariable region," "HVR," and "HV" are used interchangeably. A "CDR" refers to one of three hypervariable regions (HI, H2 or H3) within the non- framework region of the immunoglobulin (Ig or antibody) VH β- sheet framework, or one of three hypervariable regions (LI, L2 or L3) within the non- framework region of the antibody VL β-sheet framework. The term when used herein refers to the regions of an antibody variable region that are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six hypervariable regions; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3). Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. A number of hypervariable region delineations are in use and are encompassed herein. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the antibody variable (V) domains (Kabat et al. , J. Biol. Chem. 252:6609-6616 (1977); Kabat, Adv. Prot. Chem. 32: 1-75 (1978)). The Kabat CDRs are based on sequence variability and are the most commonly used (see, e.g., Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). CDR region sequences also have been defined structurally by Chothia as those residues that are not part of the conserved β-sheet framework, and thus are able to adapt different conformations (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Chothia refers instead to the location of the structural loops. The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). Both terminologies are well recognized in the art.

[0081] Exemplary CDR region sequences are illustrated in Tables 4 and 5. The positions of CDRs within a canonical antibody variable region have been determined by comparison of numerous structures (Al-Lazikani et al, J. Mol. Biol. 273:927-948 (1997); Morea et al, Methods 20:267-279 (2000)).

Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable region numbering scheme (Al-Lazikani et al, supra (1997)). Such nomenclature is similarly well known to those skilled in the art.

[0082] CDR region sequences have also been defined by AbM, Contact and IMGT. The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular' s AbM antibody modeling software (see, e.g., Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). The "contact" hypervariable regions are based on an analysis of the available complex crystal structures. The residues from each of these hypervariable regions or CDRs are noted below.

[0083] Recently, a universal numbering system has been developed and widely adopted,

ImMunoGeneTics (IMGT) Information System^® (Lafranc et ah, Dev. Comp. Immunol. 27(l):55-77 (2003)). IMGT is an integrated information system specializing in immunoglobulins (IG), T cell receptors (TR) and major histocompatibility complex (MHC) of human and other vertebrates. Herein, the CDRs are referred to in terms of both the amino acid sequence and the location within the light or heavy chain. As the "location" of the CDRs within the structure of the immunoglobulin variable domain is conserved between species and present in structures called loops, by using numbering systems that align variable domain sequences according to structural features, CDR and framework residues and are readily identified. This information can be used in grafting and replacement of CDR residues from

immunoglobulins of one species into an acceptor framework from, typically, a human antibody. An additional numbering system (AHon) has been developed by Honegger and Pluckthun, J. Mol. Biol. 309: 657-670 (2001). Correspondence between the numbering system, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to one skilled in the art (see, e.g. , Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et ah, supra).

[0084] Hypervariable regions can comprise "extended hypervariable regions" as follows: 24-36 or 24-34 (LI), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 or 26-35A (HI), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH.

[0085] The antibodies provided herein can include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)).

[0086] The term "compete" when used in the context of anti-AQP4 antibodies {e.g., agonistic antibodies and binding proteins that bind to (i) AQP4; or (ii) a complex comprising AQP4, such as OAP) that compete for the same epitope or binding site on a target means competition between as determined by an assay in which the antibody (or binding fragment) thereof under study prevents or inhibits the specific binding of a reference molecule (e.g. , a reference ligand, or reference antigen binding protein, such as a reference antibody) to a common antigen (e.g., AQP4 or a fragment thereof). Numerous types of competitive binding assays can be used to determine if a test antibody competes with a reference antibody for binding to AQP4 (e.g., human AQP4). Examples of assays that can be employed include solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al, (1983) Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al, (1986) J. Immunol. 137:3614-3619) solid phase direct labeled assay, solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, (1988)

Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (see, e.g., Morel et al, (1988) Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al, (1990) Virology 176:546-552); and direct labeled RIA (Moldenhauer et al, (1990) Scand. J. Immunol. 32:77-82). Typically, such an assay involves the use of a purified antigen (e.g., AQP4 such as human AQP4) bound to a solid surface or cells bearing either of an unlabelled test antigen binding protein (e.g., test anti-AQP4 antibody) or a labeled reference antigen binding protein (e.g., reference anti-AQP4 antibody). Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein. Usually the test antigen binding protein is present in excess. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and/or antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference for antibodies steric hindrance to occur. Additional details regarding methods for determining competitive binding are described herein. Usually, when a competing antibodies protein is present in excess, it will inhibit specific binding of a reference antibodies to a common antigen by at least 23%, for example 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some instance, binding is inhibited by at least 80%, 85%, 90%, 95%, 96% or 97%, 98%, 99% or more.

[0087] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, (see, e.g., Gazzano- Santoro et al., J. Immunol. Methods 202: 163 (1996)), can be performed. Polypeptide variants with altered Fc region amino acid sequences (polypeptides with a variant Fc region) and increased or decreased Clq binding capability have been described, (see, e.g., US Patent No. 6, 194,551, WO

1999/51642, Idusogie et al. J. Immunol. 164: 4178-4184 (2000)). Antibodies with little or no CDC activity can be selected for use.

[0088] The term "composition" is intended to encompass a product containing the specified ingredients (e.g., an antibody provided herein) in, optionally, the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in, optionally, the specified amounts.

[0089] The term "constant region" or "constant domain" refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor. The terms refer to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable region, which contains the antigen binding site. The constant region can contain the CHI, CH2 and CH3 regions of the heavy chain and the CL region of the light chain.

[0090] In the context of a polypeptide, the term "derivative" as used herein refers to a polypeptide that comprises an amino acid sequence of an AQP4 polypeptide, a fragment of an AQP4 polypeptide, or an antibody that binds to an AQP4 polypeptide which has been altered by the introduction of amino acid residue substitutions, deletions or additions. The term "derivative" as used herein also refers to an AQP4 polypeptide, a fragment of an AQP4 polypeptide, or an antibody that binds to an AQP4 polypeptide which has been chemically modified, e.g., by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, an AQP4 polypeptide, a fragment of an AQP4 polypeptide, or an AQP4 antibody can be chemically modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. The derivatives are modified in a manner that is different from naturally occurring or starting peptide or polypeptides, either in the type or location of the molecules attached. Derivatives further include deletion of one or more chemical groups which are naturally present on the peptide or polypeptide. A derivative of an AQP4 polypeptide, a fragment of an AQP4 polypeptide, or an AQP4 antibody can be chemically modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Further, a derivative of an AQP4 polypeptide, a fragment of an AQP4 polypeptide, or an AQP4 antibody can contain one or more non- classical amino acids. A polypeptide derivative possesses a similar or identical function as an AQP4 polypeptide, a fragment of an AQP4 polypeptide, or an AQP4 antibody described herein. [0091] The term "detectable agent" refers to a substance that can be used to ascertain the existence or presence of a desired molecule, such as an anti-AQP4 antibody as described herein, in a sample or subject. A detectable agent can be a substance that is capable of being visualized or a substance that is otherwise able to be determined and/or measured (e.g., by quantitation).

[0092] The term "detectable probe" refers to a composition that provides a detectable signal. The term includes, without limitation, any fluorophore, chromophore, radiolabel, enzyme, antibody or antibody fragment, and the like, that provide a detectable signal via its activity.

[0093] The term "diagnostic agent" refers to a substance administered to a subject that aids in the diagnosis of a disease, disorder, or conditions. Such substances can be used to reveal, pinpoint, and/or define the localization of a disease causing process. In certain embodiments, a diagnostic agent includes a substance that is conjugated to an anti-AQP4 antibody as described herein, that when administered to a subject or contacted to a sample from a subject aids in the diagnosis NMO .

[0094] The term "effective amount" as used herein refers to the amount of a therapy (e.g., an antibody or pharmaceutical composition provided herein) which is sufficient to reduce and/or ameliorate the severity and/or duration of a given AQP4 defective disease, disorder or condition and/or a symptom related thereto. This term also encompasses an amount necessary for the reduction or amelioration of the advancement or progression of a given AQP4 defective disease, disorder or condition, reduction or amelioration of the recurrence, development or onset of a given AQP4 defective disease, disorder or condition, and/or to improve or enhance the prophylactic or therapeutic effect(s) of another therapy (e.g., a therapy other than anti-AQP4 antibody provided herein). In some embodiments, the effective amount of an antibody provided herein is from about 0.1 mg/kg (mg of antibody per kg weight of the subject) to about 100 mg/kg. In certain embodiments, an effective amount of an antibody provided therein is about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, 3 mg/kg, 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, or about 100 mg/kg (or a range therein). In some embodiments, "effective amount" as used herein also refers to the amount of an antibody provided herein to achieve a specified result (e.g., inhibiting NMO-IgG binding to cell surface AQP4; or inhibit complement-mediated cell killing).

[0095] Antibody "effector functions" refer to those biological activities attributable to the Fc region (e.g. , a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation. [0096] The term "encode" or grammatical equivalents thereof as it is used in reference to nucleic acid molecule refers to a nucleic acid molecule in its native state or when manipulated by methods well known to those skilled in the art that can be transcribed to produce mRNA, which is then translated into a polypeptide and/or a fragment thereof. The antisense strand is the complement of such a nucleic acid molecule, and the encoding sequence can be deduced therefrom.

[0097] An "epitope" is the site on the surface of an antigen molecule to which a single antibody molecule binds, such as a localized region on the surface of an antigen, such as an AQP4 polypeptide, an AQP4 polypeptide fragment or an AQP4 epitope, that is capable of being bound to one or more antigen binding regions of an antibody, and that has antigenic or immunogenic activity in an animal, such as a mammal (e.g., a human), that is capable of eliciting an immune response. An epitope having

immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal. An epitope having antigenic activity is a portion of a polypeptide to which an antibody binds as determined by any method well known in the art, including, for example, by an immunoassay. Antigenic epitopes need not necessarily be immunogenic. Epitopes often consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics. The term, "epitope" specifically includes linear epitopes and conformational epitopes. A region of a polypeptide contributing to an epitope can be contiguous amino acids of the polypeptide or the epitope can come together from two or more noncontiguous regions of the polypeptide. The epitope may or may not be a three-dimensional surface feature of the antigen. In certain embodiments, an AQP4 epitope is a three-dimensional surface feature of an AQP4 polypeptide. In other embodiments, an AQP4 epitope is linear feature of an AQP4 polypeptide. Generally an antigen has several or many different epitopes and can react with many different antibodies.

[0098] An antibody binds "an epitope" or "essentially the same epitope" or "the same epitope" as a reference antibody, when the two antibodies recognize identical, overlapping or adjacent epitopes in a three-dimensional space. The most widely used and rapid methods for determining whether two antibodies bind to identical, overlapping or adjacent epitopes in a three-dimensional space are competition assays, which can be configured in a number of different formats, for example, using either labeled antigen or labeled antibody. In some assays, the antigen is immobilized on a 96-well plate, or expressed on a cell surface, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive, fluorescent or enzyme labels.

[0099] The term "excipient" refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.) and olyols (e.g., mannitol, sorbitol, etc.). See, also, Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA, which is hereby incorporated by reference in its entirety.

[00100] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In one embodiment, a FcR is a native sequence human FcR. Moreover, in certain embodiments, a FcR is one that binds an IgG antibody (e.g. , a gamma receptor) and includes receptors of the FcyRI, FcyRII and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof (see, e.g. , Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are known (see, e.g., Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et al, Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995)). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (see, e.g., Guyer et al, J. Immunol. 1 17:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). Antibody variants with improved or diminished binding to FcRs have been described (see, e.g., in WO 2000/42072; U.S. Patent Nos. 7,183,387, 7,332,581 and 7.335,742; Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001)).

[00101] The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies can comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.

[0100] In the context of a peptide or polypeptide, the term "fragment" as used herein refers to a peptide or polypeptide that comprises less than the full length amino acid sequence. Such a fragment can arise, for example, from a truncation at the amino terminus, a truncation at the carboxy terminus, and/or an internal deletion of a residue(s) from the amino acid sequence. Fragments can, for example, result from alternative RNA splicing or from in vivo protease activity. In certain embodiments, AQP4 fragments include polypeptides comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least contiguous 100 amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, or at least 950, contiguous amino acid residues of the amino acid sequence of an AQP4 polypeptide or an antibody that binds to an AQP4 polypeptide. In a specific embodiment, a fragment of an AQP4 polypeptide or an antibody that binds to an AQP4 antigen retains at least 1 , at least 2, or at least 3 or more functions of the polypeptide or antibody.

[0101] The term "framework" or "FR" residues are those variable region residues flanking the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than the hypervariable region residues or CDR residues.

[0102] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary "effector functions" include Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) and can be assessed using various assays as disclosed.

[0103] A "functional fragment" or "binding fragment" or "antigen binding fragment" of a therapeutic antibody will exhibit at least one if not some or all of the biological functions attributed to the intact antibody, the function comprising at least binding to the target antigen, (e.g., an AQP4 binding fragment or fragment that binds to AQP4).

[0104] The term "fusion protein" as used herein refers to a polypeptide that comprises an amino acid sequence of an antibody and an amino acid sequence of a heterologous polypeptide or protein (e.g., a polypeptide or protein not normally a part of the antibody (e.g., a non-anti-AQP4 antigen binding antibody)). The term "fusion" when used in relation to AQP4 or to an anti-AQP4 antibody refers to the joining of a peptide or polypeptide, or fragment, variant and/or derivative thereof, with a heterologous peptide or polypeptide. In certain embodiments, the fusion protein retains the biological activity of the AQP4 or anti-AQP4 antibody. In certain embodiments, the fusion protein comprises an AQP4 antibody VH region, VL region, VH CDR (one, two or three VH CDRs), and/or VL CDR (one, two or three VL CDRs), wherein the fusion protein binds to an AQP4 epitope, an AQP4 fragment and/or an AQP4 polypeptide.

[0105] The term "heavy chain" when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids and a carboxy-terminal portion that includes a constant region. The constant region can be one of five distinct types, (e.g., isotypes) referred to as alpha (a), delta (δ), epsilon (ε), gamma (γ) and mu (μ), based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains differ in size: α, δ and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with a light chain, these distinct types of heavy chains give rise to five well known classes (e.g. , isotypes) of antibodies, IgA, IgD, IgE, IgG and IgM, respectively, including four subclasses of IgG, namely IgGl, IgG2, IgG3 and IgG4. A heavy chain can be a human heavy chain.

[0106] The term "host" as used herein refers to an animal, such as a mammal (e.g., a human).

[0107] The term "host cell" as used herein refers to a particular subject cell that can be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that can occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

[0108] "Humanized" forms of nonhuman (e.g., murine) antibodies are chimeric antibodies that include human immunoglobulins (e.g., recipient antibody) in which the native CDR residues are replaced by residues from the corresponding CDR of a nonhuman species (e.g., donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, one or more FR region residues of the human immunoglobulin are replaced by corresponding nonhuman residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. A humanized antibody heavy or light chain can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a nonhuman immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, Jones et al, Nature, 321 :522-525 (1986); Riechmann et al, Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992); Carter et al, Proc. Natl. Acd. Sci. USA 89:4285-4289 (1992); and U.S. Patent Nos: 6,800,738 (issued Oct. 5, 2004), 6,719,971 (issued Sept. 27, 2005), 6,639,055 (issued Oct. 28, 2003), 6,407,213 (issued June 18, 2002), and 6,054,297 (issued April 25, 2000). [0109] The terms "human antibody" and "fully human antibody" are used interchangeably herein and refer to an antibody that possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al, J. Mol. Biol., 222:581 (1991) and yeast display libraries (Chao et al, Nature Protocols 1 : 755-768 (2006)). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al, J. Immunol., 147(l):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., mice (see, e.g., Jakobovits, A., Curr. Opin.

Biotechnol. 1995, 6(5):561-6; Briiggemann and Taussing, Curr. Opin. Biotechnol. 1997, 8(4):455-8; and U.S. Pat. Nos. 6,075, 181 and 6, 150,584 regarding XENOMOUSE™ technology). See also, for example, Li et al, Proc. Natl. Acad. Sci. USA, 103 :3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology. In specific embodiments, the terms refer to an antibody that comprises a variable region and constant region of human origin. "Fully human" anti-AQP4 antibodies, in certain embodiments, can also encompass antibodies which bind AQP4 polypeptides and are encoded by nucleic acid sequences which are naturally occurring somatic variants of human germline

immunoglobulin nucleic acid sequence. In a specific embodiment, the anti-AQP4 antibodies provided herein are fully human antibodies. The term "fully human antibody" includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences as described by Kabat et al. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.

Department of Health and Human Services, NIH Publication No. 91 -3242). The phrase "recombinant human antibody" includes human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated from an animal {e.g., a mouse or cow) that is transgenic and/or transchromosomal for human immunoglobulin genes (see e.g., Taylor, L. D. et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human

immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (See Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 -3242). In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0110] The term "identity" refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity" means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) must be addressed by a particular mathematical model or computer program {e.g. , an "algorithm"). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A. M., ed.), (1988) New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., (1987) Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991 , New York: M. Stockton Press; and Carillo et al, (1988) SIAM J. Applied Math. 48: 1073.

[0111] In calculating percent identity, the sequences being compared can be aligned in a way that gives the largest match between the sequences. Computer program can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., (1984) Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI). The computer algorithm GAP used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences can be aligned for optimal matching of their respective amino acid or nucleotide (the "matched span", as determined by the algorithm). A gap opening penalty (which is calculated as 3. times, the average diagonal, wherein the "average diagonal" is the average of the diagonal of the comparison matrix being used; the "diagonal" is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix (see, Dayhoff et al, (1978) Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al, (1992) Proc. Natl. Acad. Sci. USA 89: 10915- 10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm. [0112] Exemplary parameters for determining percent identity for polypeptides or nucleotide sequences using the GAP program are the following: (i) Algorithm: Needleman et al. , 1970, J. Mol. Biol. 48:443-453; (ii) Comparison matrix: BLOSUM 62 from Henikoff et al, 1992, supra; (iii) Gap Penalty: 12 (but with no penalty for end gaps) (iv) Gap Length Penalty: 4; and (v) Threshold of Similarity: 0.

[0113] Certain alignment schemes for aligning two amino acid sequences can result in matching of only a short region of the two sequences, and this small aligned region can have very high sequence identity even though there is no significant relationship between the two full-length sequences.

Accordingly, the selected alignment method (e.g., the GAP program) can be adjusted if so desired to result in an alignment that spans a number of amino acids, for example, at least 50 contiguous amino acids of the target polypeptide.

[0114] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0115] The term "in combination" in the context of the administration of other therapies (e.g., other agents) includes the use of more than one therapy (e.g., one agent). Administration "in combination with" one or more further therapeutic agents includes simultaneous (e.g. , concurrent) and consecutive administration in any order. The use of the term "in combination" does not restrict the order in which therapies are administered to a subject. A first therapy (e.g., agent) can be administered before (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 8 weeks, 9 weeks, 10 weeks, 1 1 weeks, or 12 weeks), concurrently, or after (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 1 weeks, or 12 weeks) the administration of a second therapy (e.g., agent) to a subject which had, has, or is susceptible to NMO .

[0116] Any additional therapy (e.g., agent) can be administered in any order with the other additional therapies (e.g., agents). In certain embodiments, the antibodies can be administered in combination with one or more therapies such as agents (e.g., therapies, including agents, that are not the antibodies that are currently administered) to prevent, treat, manage, and/or ameliorate NMO . Non- limiting examples of therapies (e.g., agents) that can be administered in combination with an antibody include, for example, analgesic agents, anesthetic agents, antibiotics, or immunomodulatory agents or any other agent listed in the U.S. Pharmacopoeia and/or Physician's Desk Reference. Examples of agents useful in combination therapy include, but are not limited to, the following: non-steroidal anti-inflammatory drug (NSAID) such as aspirin, ibuprofen, and other propionic acid derivatives (alminoprofen, benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid, and tioxaprofen), acetic acid derivatives (indomethacin, acemetacin, alclofenac, clidanac, diclofenac, fenclofenac, fenclozic acid, fentiazac, fuirofenac, ibufenac, isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin, and zomepirac), fenamic acid derivatives (flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid and tolfenamic acid), biphenylcarboxylic acid derivatives (diflunisal and flufenisal), oxicams (isoxicam, piroxicam, sudoxicam and tenoxican), salicylates (acetyl salicylic acid, sulfasalazine) and the pyrazolones (apazone, bezpiperylon, feprazone, mofebutazone, oxyphenbutazone, phenylbutazone). Other combinations include cyclooxygenase-2 (COX- 2) inhibitors. Other agents for combination include steroids such as prednisolone, prednisone, methylprednisolone, betamethasone, dexamethasone, or hydrocortisone. Such a combination can be especially advantageous, since one or more side-effects of the steroid can be reduced or even eliminated by tapering the steroid dose required when treating patients in combination with the present antibodies. Additional examples of agents for combinations include cytokine suppressive anti-inflammatory drug(s) (CSAIDs); antibodies to or antagonists of other human cytokines or growth factors, for example, TNF, LT, IL- Ι β, IL-2, IL-6, IL-7, IL-8, IL- 15, IL- 16, IL- 18, EMAP-II, GM-CSF, FGF, or PDGF. Combinations of agents can include TNF antagonists like chimeric, humanized or human TNF antibodies, REMICADE, anti-TNF antibody fragments (e.g., CDP870), and soluble p55 or p75 TNF receptors, derivatives thereof, p75TNFRIgG (ENBREL^®) or p55TNFRlgG (LENERCEPT^®), soluble IL- 13 receptor (sIL- 13), and also TNFa converting enzyme (TACE) inhibitors; similarly IL- 1 inhibitors (e.g., Interleukin- 1 -converting enzyme inhibitors) can be effective. Other combinations include Interleukin 1 1 , anti-P7s and p-selectin glycoprotein ligand (PSGL). Other examples of agents useful in combination therapy include interferon- ia (AVONEX); interferon- ib (BETASERON^®); Copaxone; hyperbaric oxygen; intravenous immunoglobulin; clabribine; and antibodies to or antagonists of other human cytokines or growth factors (e.g., antibodies to CD40 ligand and CD80).

[0117] An "intact" antibody is one comprising an antigen-binding site as well as a CL and at least heavy chain constant regions, CH I, CH2 and CH3. The constant regions can include human constant regions or amino acid sequence variants thereof. In certain embodiments,, an intact antibody has one or more effector functions.

[0118] An "isolated" antibody is substantially free of cellular material or other contaminating proteins from the cell or tissue source and/or other contaminant components from which the antibody is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of an antibody in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes preparations of antibody having less than about 30%, 25%, 20%, 15%, 10%, 5%, or 1% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein"). In certain embodiments, when the antibody is recombinantly produced, it is substantially free of culture medium, e.g., culture medium represents less than about 20%, 15%, 10%, 5%, or 1% of the volume of the protein preparation. In certain embodiments, when the antibody is produced by chemical synthesis, it is substantially free of chemical precursors or other chemicals, for example, it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations of the antibody have less than about 30%, 25%, 20%, 15%, 10%, 5%, or 1% (by dry weight) of chemical precursors or compounds other than the antibody of interest. Contaminant components can also include, but are not limited to, materials that would interfere with therapeutic uses for the antibody, and can include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In certain embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method (Lowry et al. J. Bio. Chem. 193: 265-275, 1951), such as 96%, 97%, 98%, or 99%, by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. In specific embodiments, antibodies provided herein are isolated.

[0119] An "isolated nucleic acid" is a nucleic acid, for example, an RNA, DNA, or a mixed polymer, which is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, one or more nucleic acid molecules encoding an antibody as described herein are isolated or purified. The term embraces nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A substantially pure molecule can include isolated forms of the molecule.

[0120] The terms "Kabat numbering," and like terms are recognized in the art and refer to a system of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable region typically ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the light chain variable region, the hypervariable region typically ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3. Other CDR numbering systems are known to those in the art, and are also contemplated herein.

[0121] The term "lacks effector function" describes a characteristic of the antibody or the antigen binding fragment thereof. An antibody or antigen binding fragment with a variant Fc region, or mutated Fc region, lacks effector function means it has significantly reduced Clq binding and/or complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and/or B cell activation compared to an antibody with a native sequence Fc region. The term "significantly reduced" means that Clq binding and/or complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell- mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and/or B cell activation of the antibody with a variant Fc region or a mutated Fc region is decreased by at least 3-fold, at least 4-fold, or at least 5-fold compared to an antibody with a native sequence Fc region.

[0122] The term "light chain" when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 1 10 or more amino acids and a carboxy-terminal portion that includes a constant region. The approximate length of a light chain is 21 1 to 217 amino acids. There are two distinct types, referred to as kappa (κ) of lambda (λ) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. A light chain can be a human light chain. [0123] A "light chain constant region" includes "kappa constant region" and "lambda constant region." Exemplary kappa constant regions are provided below:

TVAAPSVFI FPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYS LS ST LTLSKADYEKHKVYACEVTHQGLS S PVTKS FNRGEC (SEQ ID NO: 20) (allele IgKC*01); and

TVAAPSVFI FPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLS S T LTLSKADYEKHKLYACEVTHQGLS S PVTKS FNRGEC (SEQ ID NO: 81) (allele IgKC*04)

[0124] The corresponding exemplary nucleic acid sequences that encodes the respective kappa constant regions are provided below:

ACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTG TTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCA ATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACC CTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA GTTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 19) (allele IgKC*01); and

ACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTG TTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCA ATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACC CTGACGCTGAGCAAAGCAGACTACGAGAAACACAAACTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA GTTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAA_(SEQ ID NO: 82) (allele IgKC*04).

[0125] The terms "manage," "managing," and "management" refer to the beneficial effects that a subject derives from a therapy (e.g., a prophylactic or therapeutic agent), which does not result in a cure of the disease. In certain embodiments, a subject is administered one or more therapies (e.g., prophylactic or therapeutic agents, such as an antibody provided herein) to "manage" an AQP4-mediated disease, one or more symptoms thereof, so as to prevent the progression or worsening of the disease.

[0126] A "modification" of an amino acid residue/position refers to a change of a primary amino acid sequence as compared to a starting amino acid sequence, wherein the change results from a sequence alteration involving said amino acid residue/positions. For example, typical modifications include substitution of the residue with another amino acid (e.g., a conservative or non-conservative substitution), insertion of one or more (e.g., generally fewer than 5, 4 or 3) amino acids adjacent to said

residue/position, and/or deletion of said residue/position.

[0127] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts, and each monoclonal antibody will typically recognize a single epitope on the antigen. In specific embodiments, a "monoclonal antibody," as used herein, is an antibody produced by a single hybridoma or other cell, wherein the antibody binds to only an AQP4 epitope as determined, for example, by ELISA or other antigen-binding or competitive binding assay known in the art. The term

"monoclonal" is not limited to any particular method for making the antibody. For example, the monoclonal antibodies useful in the present disclosure can be prepared by the hybridoma methodology first described by Kohler et al, Nature, 256:495 (1975) (see also, e.g., Kohler and Milstein, Eur. J.

Immunol., 6, 51 1-519, 1976), or can be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al, Nature, 352:624- 628 (1991) and Marks et al, J. Mol. Biol., 222:581-597 (1991), for example. Other methods for the preparation of clonal cell lines and of monoclonal antibodies expressed thereby are well known in the art (see, for example, Chapter 1 1 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel et al, eds., John Wiley and Sons, New York). Exemplary methods of producing monoclonal antibodies are provided in the Examples herein.

[0128] The term "native" when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to those which are found in nature and not manipulated, modified, and/or changed (e.g., isolated, purified, selected) by a human being.

[0129] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature, and not manipulated, modified, and/or changed (e.g., isolated, purified, selected, including or combining with other sequences such as variable region sequences) by a human. Native sequence human Fc regions include a native sequence human IgGl Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants thereof.

[0130] The terms "optional" or "optionally" as used herein means that the subsequently described event or circumstance may or may not occur, and that the description includes, without limitation, instances where said event or circumstance occurs and instances in which it does not.

[0131] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.

[0132] The term "pharmaceutically acceptable" as used herein means being approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.

[0133] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient (e.g., an anti-AQP4 antibody) to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulation can be sterile.

[0134] "Polyclonal antibodies" as used herein refers to an antibody population generated in an immunogenic response to a protein having many epitopes and thus includes a variety of different antibodies directed to the same and to different epitopes within the protein. Methods for producing polyclonal antibodies are known in the art (See, e.g., Chapter 1 1 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel et al, eds., John Wiley and Sons, New York).

[0135] The terms "polymerase chain reaction," or "PCR," as used herein generally refers to a procedure wherein small amounts of a nucleic acid, RNA and/or DNA, are amplified as described, for example, in U.S. Pat. No. 4,683,195 to Mullis. Generally, sequence information from the ends of the region of interest or beyond needs to be available, such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers can coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, etc. See generally Mullis et al, Cold Spring Harbor Symp. Quant. Biol., 51 : 263 (1987); Erlich, ed., PCR Technology, (Stockton Press, NY, 1989).

[0136] "Polynucleotide," or "nucleic acid," as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. "Oligonucleotide," as used herein, generally refers to short, generally single-stranded, generally synthetic polynucleotides that are generally, but not necessarily, less than about 200 nucleotides in length. The terms

"oligonucleotide" and "polynucleotide" are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides. A cell that produces an anti-AQP4 antibody of the present disclosure can include a parent hybridoma cell, as well as bacterial and eukaryotic host cells into which nucleic acid encoding the antibodies have been introduced. Suitable host cells are disclosed below.

[0137] As used herein the terms "polypeptide" and "protein" as used interchangeably herein, refer to a polymer of amino acids of three or more amino acids in a serial array, linked through peptide bonds. The term "polypeptide" includes proteins, protein fragments, protein analogues, oligopeptides and the like. The term polypeptide as used herein can also refer to a peptide. The amino acids making up the polypeptide can be naturally derived, or can be synthetic. The polypeptide can be purified from a biological sample.

[0138] The terms "prevent," "preventing," and "prevention" refer to the total or partial inhibition of the development, recurrence, onset or spread of NMO and/or symptom related thereto, resulting from the administration of a therapy or combination of therapies provided herein (e.g., a combination of prophylactic or therapeutic agents, such as an antibody provided herein).

[0139] The term "prophylactic agent" refers to any agent that can totally or partially inhibit the development, recurrence, onset or spread of a neuroinflammatory demyelinating disease, such as NMO, and/or symptom related thereto in a subject. In certain embodiments, the term "prophylactic agent" refers to an anti-AQP4 antibody as described herein. In certain other embodiments, the term "prophylactic agent" refers to an agent other than an anti-AQP4 antibody as described herein. In certain embodiments, a prophylactic agent is an agent which is known to be useful to or has been or is currently being used to prevent a neuroinflammatory demyelinating disease, such as NMO, and/or a symptom related thereto or impede the onset, development, progression and/or severity of a neuroinflammatory demyelinating disease, such as NMO, and/or a symptom related thereto.

[0140] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of a disease, disorder, or condition, a prophylactically effective amount can be less than a therapeutically effective amount.

[0141] In certain embodiments, a "prophylactically effective serum titer" is the serum titer in a subject, such as a human, that totally or partially inhibits the development, recurrence, onset or spread of NMO and/or a symptom related thereto in the subject.

[0142] The term "recombinant antibody" refers to an antibody that is prepared, expressed, created or isolated by recombinant means. Recombinant antibodies can be antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, antibodies isolated from an animal (e.g., a mouse or cow) that is transgenic and/or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, L. D. et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies can have variable and constant regions, including those derived from human germline immunoglobulin sequences (See Kabat, E. A. et al. (1991) Sequences of Proteins of

Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). In certain embodiments, however, such recombinant antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0143] The term "serum titer" refers to an average serum titer in a subject from multiple samples (e.g. , at one time present or multiple time points) or in a population of least 10, such as at least 20, or at least 40 subjects, up to about 100, 1000 or more.

[0144] The term "side effects" encompasses unwanted and/or adverse effects of a therapy (e.g., a prophylactic or therapeutic agent). Unwanted effects are not necessarily adverse. An adverse effect from a therapy (e.g., a prophylactic or therapeutic agent) might be harmful or uncomfortable or risky.

Examples of side effects include, diarrhea, cough, gastroenteritis, wheezing, nausea, vomiting, anorexia, abdominal cramping, fever, pain, loss of body weight, dehydration, alopecia, dyspenea, insomnia, dizziness, mucositis, nerve and muscle effects, fatigue, dry mouth, and loss of appetite, rashes or swellings at the site of administration, flu-like symptoms such as fever, chills and fatigue, digestive tract problems and allergic reactions. Additional undesired effects experienced by patients are numerous and known in the art. Many are described in the Physician's Desk Reference (68th ed., 2014).

[0145] The term "serum titer" as used herein refers to an average serum titer in a population of least 10, at least 20, at least 40 subjects up to about 100, 1000 or more.

[0146] As used herein, the term "side effects" encompasses unwanted and adverse effects of a therapy (e.g., a prophylactic or therapeutic agent). Unwanted effects are not necessarily adverse. An adverse effect from a therapy (e.g., a prophylactic or therapeutic agent) might be harmful or

uncomfortable or risky. Examples of side effects include, diarrhea, cough, gastroenteritis, wheezing, nausea, vomiting, anorexia, abdominal cramping, fever, pain, loss of body weight, dehydration, alopecia, dyspenea, insomnia, dizziness, mucositis, nerve and muscle effects, fatigue, dry mouth, and loss of appetite, rashes or swellings at the site of administration, flu-like symptoms such as fever, chills and fatigue, digestive tract problems and allergic reactions. Additional undesired effects experienced by patients are numerous and known in the art. Many are described in the Physician's Desk Reference (60^th ed., 2006).

[0147] A "sterile" formulation is aseptic or free from all living microorganisms and their spores.

[0148] The terms "subject" and "patient" can be used interchangeably. As used herein, in certain embodiments, a subject is a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkey and human). In specific embodiments, the subject is a human. In one embodiment, the subject is a mammal (e.g., a human) having NMO . In another embodiment, the subject is a mammal (e.g., a human) at risk of developing NMO . [0149] "Substantially all" refers to refers to at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

[0150] The phrase "substantially similar" or "substantially the same" denotes a sufficiently high degree of similarity between two numeric values (e.g., one associated with an antibody of the present disclosure and the other associated with a reference antibody) such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by the values (e.g., K_D values). For example, the difference between the two values can be less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%,as a function of the value for the reference antibody.

[0151] The phrase "substantially reduced," or "substantially different", as used herein, denotes a sufficiently high degree of difference between two numeric values (e.g., one associated with an antibody of the present disclosure and the other associated with a reference antibody) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by the values. For example, the difference between said two values can be greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50% as a function of the value for the reference antibody.

[0152] The term "therapeutic agent" refers to any agent that can be used in treating, preventing or alleviating a disease, disorder or condition, including in the treatment, prevention or alleviation of one or more symptoms of an AQP4-mediated disease, disorder, or condition and/or a symptom related thereto. In certain embodiments, a therapeutic agent refers to an anti-AQP4 antibody as described herein. In certain other embodiments, a therapeutic agent refers to an agent other than an antibody provided herein. In certain embodiments, a therapeutic agent is an agent which is known to be useful for, or has been or is currently being used for the treatment, prevention or alleviation of one or more symptoms of NMO , or a symptom related thereto.

[0153] The combination of therapies (e.g., use of agents, including therapeutic agents) can be more effective than the additive effects of any two or more single therapy (e.g., synergistic). A synergetic effect is unexpected and cannot be predicted. For example, a synergistic effect of a combination of therapeutic agents permits the use of lower dosages of one or more of the agents and/or less frequent administration of the agents to a subject with an AQP4-mediated disease. The ability to utilize lower dosages of therapeutic therapies and/or to administer the therapies less frequently reduces the toxicity associated with the administration of the therapies to a subject without reducing the efficacy of the therapies in the prevention, treatment or alleviation of one or more symptom of an AQP4-mediated disease. In addition, a synergistic effect can result in improved efficacy of therapies in the prevention, treatment or alleviation of one or more symptom of NMO. Finally, synergistic effect of a combination of therapies (e.g., therapeutic agents) can avoid or reduce adverse or unwanted side effects associated with the use of any single therapy.

[0154] The term "therapy" refers to any protocol, method and/or agent that can be used in the prevention, management, treatment and/or amelioration of NMO . In certain embodiments, the terms "therapies" and "therapy" refer to a biological therapy, supportive therapy, and/or other therapies useful in the prevention, management, treatment and/or amelioration of NMO or a symptom thereof known to one of skill in the art, such as medical personnel.

[0155] The term "therapeutically effective amount" as used herein refers to the amount of an agent (e.g., an antibody described herein or any other agent described herein) that is sufficient to reduce and/or ameliorate the severity and/or duration of a given disease, disorder or condition, and/or a symptom related thereto. A therapeutically effective amount of a agent, including a therapeutic agent, can be an amount necessary for (i) reduction or amelioration of the advancement or progression of a given disease, disorder, or condition, (ii) reduction or amelioration of the recurrence, development or onset of a given disease, disorder or conditions, and/or (iii) to improve or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than the administration of an antibody provided herein). A "therapeutically effective amount" of a substance/molecule/agent of the present disclosure (e.g., an anti-AQP4 antibody) can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule/agent, to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the

substance/molecule/agent are outweighed by the therapeutically beneficial effects. In certain

embodiments, the term "therapeutically effective amount" refers to an amount of an antibody or other agent (e.g., or drug) effective to "treat" a disease, disorder, or condition, in a subject or mammal.

[0156] In certain embodiments, a "therapeutically effective serum titer" is the serum titer in a subject, such as a human, that reduces the severity, the duration and/or the symptoms associated with NMO in the subject.

[0157] As used herein, the terms "treat," "treatment" and "treating" refer to the reduction or amelioration of the progression, severity, and/or duration of an AQP4-mediated disease, disorder or condition, or a symptom thereof, resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents, such as an antibody provided herein). In specific embodiments, the agent is an anti-AQP4 antibody. Treatment as used herein includes, but is not limited to, decreasing NMO-IgG binding to cell surface AQP4, or reducing complement-mediated cell killing in a subject. [0158] The term "variable region" or "variable domain" refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 1 10 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen. The variable region of the heavy chain can be referred to as "VH." The variable region of the light chain can be referred to as "VL." The term "variable" refers to the fact that certain segments of the variable regions differ extensively in sequence among antibodies. The V region mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 1 10-amino acid span of the variable regions. Instead, the V regions consist of less variable (e.g., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called "hypervariable regions" that are each about 9- 12 amino acids long. The variable regions of heavy and light chains each comprise four FRs, largely adopting a β sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991)). The constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). The variable regions differ extensively in sequence between different antibodies. The variability in sequence is concentrated in the CDRs while the less variable portions in the variable region are referred to as framework regions (FR). The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with antigen. In specific embodiments, the variable region is a human variable region.

[0159] The term "variable region residue numbering as in Kabat" or "amino acid position numbering as in Kabat", and variations thereof, refers to the numbering system used for heavy chain variable regions or light chain variable regions of the compilation of antibodies in Kabat et al, Sequences of Proteins of immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain can include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc, according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a

"standard" Kabat numbered sequence. The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1 - 107 of the light chain and residues 1-1 13 of the heavy chain) (e.g., Kabat et al, Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al, supra). The "EU index as in Kabat" refers to the residue numbering of the human IgG 1 EU antibody. Other numbering systems have been described, including, for example, by AbM, Chothia, Contact, IMGT and AHon.

[0160] The term "variant" when used in relation to AQP4 or to an anti-AQP4 antibody can refer to a peptide or polypeptide comprising one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid sequence substitutions, deletions, and/or additions as compared to a native or unmodified AQP4 sequence or anti- AQP4 antibody sequence. For example, an AQP4 variant can result from one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) changes to an amino acid sequence of a native AQP4. Also by way of example, a variant of an anti-AQP4 antibody can result from one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) changes to an amino acid sequence of a native or previously unmodified anti-AQP4 antibody. Variants can be naturally occurring, such as allelic or splice variants, or can be artificially constructed. Polypeptide variants can be prepared from the corresponding nucleic acid molecules encoding the variants. In certain embodiments, the variant is encoded by a single nucleotide polymorphism (SNP) variant of a nucleic acid molecule that encodes AQP4 or anti-AQP4 antibody VH or VL regions or subregions, such as one or more CDRs.

[0161] A "variant Fc region," or "mutated Fc region" comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, (e.g., substituting, addition, or deletion) one or more amino acid substitution(s). In certain embodiments,, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions, from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will can possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, such as at least about 90% homology therewith, for example, at least about 95% homology therewith.

[0162] The term "vector" refers to a substance that is used to carry or include a nucleic acid sequences, including for example, in order to introduce a nucleic acid sequence into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell's chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more nucleic acid molecules are to be co-expressed (e.g. both an antibody heavy and light chain or an antibody VH and VL) both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product (e.g. an anti- AQP4 antibody as described herein), and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0163] Unless specified otherwise, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5' direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences;" sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3 ' to the 3 ' end of the RNA transcript are referred to as "downstream sequences."

5.2 Antibodies

5.2.1. VH and VL Sequences

[0164] Antibodies provided herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0165] In particular, antibodies provided herein include immunoglobulin molecules and

immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that binds to an AQP4 antigen. The immunoglobulin molecules provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule. In a specific embodiment, an antibody provided herein is an IgG antibody. In certain embodiments, the antibody is an IgGl antibody.

[0166] Variants and derivatives of antibodies include antibody fragments that retain the ability to specifically bind to an epitope. In certain embodiments, fragments include Fab fragments (an antibody fragment that contains the antigen-binding domain and comprises a light chain and part of a heavy chain bridged by a disulfide bond); Fab' (an antibody fragment containing a single anti-binding domain comprising an Fab and an additional portion of the heavy chain through the hinge region); F(ab')₂ (two Fab' molecules joined by interchain disulfide bonds in the hinge regions of the heavy chains; the Fab' molecules can be directed toward the same or different epitopes); a bispecific Fab (a Fab molecule having two antigen binding domains, each of which can be directed to a different epitope); a single chain Fab chain comprising a variable region, also known as, a sFv (the variable, antigen-binding determinative region of a single light and heavy chain of an antibody linked together by a chain of 10-25 amino acids); a disulfide-linked Fv, or dsFv (the variable, antigen-binding determinative region of a single light and heavy chain of an antibody linked together by a disulfide bond); a camelized VH (the variable, antigen- binding determinative region of a single heavy chain of an antibody in which some amino acids at the VH interface are those found in the heavy chain of naturally occurring camel antibodies); a bispecific sFv (a sFv or a dsFv molecule having two antigen-binding domains, each of which can be directed to a different epitope); a diabody (a dimerized sFv formed when the VH domain of a first sFv assembles with the VL domain of a second sFv and the VL domain of the first sFv assembles with the VH domain of the second sFv; the two antigen-binding regions of the diabody can be directed towards the same or different epitopes); and a triabody (a trimerized sFv, formed in a manner similar to a diabody, but in which three antigen-binding domains are created in a single complex; the three antigen binding domains can be directed towards the same or different epitopes). Derivatives of antibodies also include one or more CDR sequences of an antibody combining site. The CDR sequences can be linked together on a scaffold when two or more CDR sequences are present. In certain embodiments, an antibody provided herein comprises a single-chain Fv ("scFv"). scFvs are antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFvs see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0167] The antibodies provided herein can be from any animal origin including birds and mammals (e.g., human, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken). In certain embodiments, the antibodies provided herein are human or humanized monoclonal antibodies. As used herein, "human" antibodies include antibodies having the amino acid sequence of a human

immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from mice that express antibodies from human genes.

[0168] Antibodies provided herein can be defined, in the first instance, by their binding specificity, which in this case is for AQP4. Those of skill in the art, by assessing the binding specificity/affinity of a given antibody using techniques well known to those of skill in the art, can determine whether such antibodies fall within the scope of the instant claims.

[0169] In one aspect, there is provided a monoclonal antibody that binds to AQP4. A particular type of antibody that is one that lacks Fc -related effector functions. Indeed, since antibodies to AQP4 are disease-causing, one must modify such AQP4 antibodies in order to render them not only safe, but protective. Such antibodies can be produced by mutating the Fc region of antibodies that exhibit such functions (e.g., IgGl or IgG2 or IgG3), by using antibodies that naturally lack such functions (IgG4), or by chemically modifying any of such antibodies so as to render them ineffective at complement activation and immune cell recruitment.

[0170] In a second aspect, the antibodies can be defined by virtue of the region of the structures to which they bind. For example, the extracelllar surface and orthogonal arrays of AQP4 provide a unique platform for antibody binding.

[0171] In a third aspect, the antibodies can be defined by their variable sequence that determine their binding specificity. Exemplary amino acid and nucleic acid sequences are provided in Tables 1 and 2 below, respectively.

Table 1. VH and VL Domain Nucleic Acid Sequences

GGTACATCCATTACAGTGGGAGCACCAAC GCCACTGGCATCCCAGACAGGTTCAGTGGCAGT TACAACCCCTCCCTCAAGAGTCGAGTCAC GGGTCTGGGACAGACTTCACTCTCACCATCAGC CATATCAGTGGACACGTCCAAGAACCAGT AGACTGGAGCCTGAAGATTTTGCAGTGTATTAC TCTCCCTGAAGCTGAGCTCTGTGACCGCT TGTCAGCAGTATGGTAGCTCACCGTGGACGTTC GCGGACACGGCCGTGTATTACTGTGCGAG GGCCAAGGGACCAAGGTGGAAATCAAACGA AGCAGAGGGGAGAGGATGGAGTGCTTTCT (SEQ ID NO:3)

ACTACTACTACATGGAAGTCTGGGGCAAA GGGTCCACGGTCTCCGTCTCCTCA

(SEQ ID N0:1)

rAb58 GTGCAGCTGGTGGAGTCTGGGGGTGGCTT GACATCCAGATGACCCAGTCTCCATCCGCCCTG

GGTTCAGCCGGGGGGGTCCCTGAGACTCT TCTGCATCTGTAGGAGACACAGTCACCATCACT CCTGTGCAGCCTCTGGATTCACCTTTAGA TGCCGGGCCAGTCAGAGTATTAGGAGCTGGTTG GGTTATGCCATGAACTGGGTCCGCCAGGC GCCTGGTATCAGCAGAAACCAGGGAAAGCCCCT CCCAGGGAAGGGGCTGGAGTGGGTCGCAA AAACTCCTGATCTATAAGGCGTCTGATTTACAA GTATCAGTGGCAGTGGTAGTATCACACAG AGTGGGGTCCCATCAAGATTCAGCGGCAGTGGA TACGCAGACTCCGCGAAGGGCCGCTTCAC TCTGGGACAGACTTCACTCTCACCATCAGCGGC CATCACCAGAGACAACTCCAAGAGCACGC CTGCAGCCTGATGATTTTGCAACTTATTACTGC TCTATGCGCATGTGAGTAGCCTGAGAGCC CAACACTATAATAGTTACCCGTACACTTTTGGC GATGACACGGCCGTATATTACTGTGCGAA CAGGGGACCAAGGTGGAGATCAGACGA AGGGGACTACGTCTTTGACTACTGGGGAC (SEQ ID NO: 9)

AGGGAACCCTGGTCACCGTCTCCTCA

(SEQ ID NO: 7)

rAb09- GTAACTACAGGTGTCCACTCCGAGGTGCA GATATTGTGATGACTCAGTCTCCACTCTCCCTG 3-33 GCTGGTGGAGTCTGGGGGAGGCGTGGTCC CCCGTCACCCCTGGAGAGCCGGCCTCTATCTCC AGCCTGGGGGGTCCCTAAGACTCTCCTGT TGCAGGTCTAGTCAGAGCCTCCGCCACACCATC ACAGCGTCTGGTTTCAACTTAGATGACTA ACTGGATACAACTATATCAATTGGTACCTGCAG TGACATTCACTGGGTCCGCCAGGCGCCCG AAGCCAGGGCAGTCTCCACAACTCCTGATCTTT GCAAGGGGCTGCAGTGGGTGGCAATTTTG TTGGCCTCTTCTCGGGCCACCGGGGTCCCTGAC CAGCCTGAAGAAAGTCATCAAGACTATAT AGGTTCAGTGGCAGTGGAGCAGGCACAGATTTT AAATTCCGTGAGGGGCCGATTCTCCGTCT ACACTGAAAATCAGCAGAGTGGAGGCTGAGGAT CCAGAGACAGTTCGAGGGACACAATAGAT GTTGGAATTTATTACTGCATGCAAGCTCTACAC CTGCAAATGCACAGTCTTAGACCTGAAGA ACTCCGCCCACTTTTGGCCAGGGGACCAAACTG CACGGCTATATATTACTGTACGCGATCTC GAGATCAAACGA CGGGCCTCATGACTACGCTGCGGGGAATG (SEQ ID NO: 15) GTGACCAGGAGGCACTTTCACTACTTCAC CATGGACGTCTGGGGCAAAGGGACCACGG TCATCGTCTCCTCA

(SEQ ID NO: 13)

Table 2. VH and VL Domain Amino Acid Sequences

TCTTTGGTGCATCCAGCAGGGCCACTGGC EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT ATCCCAGACAGGTTCAGTGGCAGTGGGTC HQGLSSPVTKSFNRGEC TGGGACAGACTTCACTCTCACCATCAGCA (SEQ ID NO: 6)

GACTGGAGCCTGAAGATTTTGCAGTGTAT TACTGTCAGCAGTATGGTAGCTCACCGTG GACGTTCGGCCAAGGGACCAAGGTGGAAA TCAAACGAACTGTGGCTGCACCATCTGTC

TTCATCTTCCCGCCATCTGATGAGCAGTT GAAATCTGGAACTGCCTCTGTTGTGTGCC TGCTGAATAACTTCTATCCCAGAGAGGCC AAAGTACAGTGGAAGGTGGATAACGCCCT CCAATCGGGTAACTCCCAGGAGAGTGTCA CAGAGCAGGACAGCAAGGACAGCACCTAC AGCCTCAGCAGCACCCTGACGCTGAGCAA AGCAGACTACGAGAAACACAAAGTCTACG CCTGCGAAGTCACCCATCAGGGCCTGAGT TCGCCCGTCACAAAGAGCTTCAACAGGGG AGAGTGT

(SEQ ID NO: 5) rAb58 GACATCCAGATGACCCAGTCTCCATCCGC DIQMTQSPSALSASVGDTVTITCRASQSIRSWL

CCTGTCTGCATCTGTAGGAGACACAGTCA AWYQQKPGKAPKLLIYKASDLQSGVPSRFSGSG CCATCACTTGCCGGGCCAGTCAGAGTATT SGTDFTLTISGLQPDDFATYYCQHYNSYPYTFG AGGAGCTGGTTGGCCTGGTATCAGCAGAA QGTKVEIRRTVAAPSVFI FPPSDEQLKSGTASV ACCAGGGAAAGCCCCTAAACTCCTGATCT VCLLNNFYPREAKVQWKVDNALQSGNSQESVTE ATAAGGCGTCTGATTTACAAAGTGGGGTC QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH CCATCAAGATTCAGCGGCAGTGGATCTGG QGLSSPVTKSFNRGEC GACAGACTTCACTCTCACCATCAGCGGCC (SEQ ID NO: 12)

TGCAGCCTGATGATTTTGCAACTTATTAC TGCCAACACTATAATAGTTACCCGTACAC TTTTGGCCAGGGGACCAAGGTGGAGATCA GACGAACTGTGGCTGCACCATCTGTCTTC ATCTTCCCGCCATCTGATGAGCAGTTGAA ATCTGGAACTGCCTCTGTTGTGTGCCTGC TGAATAACTTCTATCCCAGAGAGGCCAAA

GTACAGTGGAAGGTGGATAACGCCCTCCA ATCGGGTAACTCCCAGGAGAGTGTCACAG AGCAGGACAGCAAGGACAGCACCTACAGC CTCAGCAGCACCCTGACGCTGAGCAAAGC AGACTACGAGAAACACAAAGTCTACGCCT GCGAAGTCACCCATCAGGGCCTGAGTTCG CCCGTCACAAAGAGCTTCAACAGGGGAGA GTGT

(SEQ ID NO: 11)

rAb09- GTCCTGGGGTTGCTGCTGCTGTGGCTTAC DIVMTQSPLSLPVTPGEPASISCRSSQSLRHTI 3-33 AGATGCCAGATGTGATATTGTGATGACTC TGYNYINWYLQKPGQSPQLLIFLASSRATGVPD AGTCTCCACTCTCCCTGCCCGTCACCCCT RFSGSGAGTDFTLKISRVEAEDVGIYYCMQALH GGAGAGCCGGCCTCTATCTCCTGCAGGTC TPPTFGQGTKLEIKRTVAAPSVFI FPPSDEQLK TAGTCAGAGCCTCCGCCACACCATCACTG SGTASVVCLLNNFYPREAKVQWKVDNALQSGNS GATACAACTATATCAATTGGTACCTGCAG QESVTEQDSKDSTYSLSSTLTLSKADYEKHKLY AAGCCAGGGCAGTCTCCACAACTCCTGAT ACEVTHQGLSSPVTKSFNRGEC CTTTTTGGCCTCTTCTCGGGCCACCGGGG (SEQ ID NO: 18)

TCCCTGACAGGTTCAGTGGCAGTGGAGCA GGCACAGATTTTACACTGAAAATCAGCAG AGTGGAGGCTGAGGATGTTGGAATTTATT ACTGCATGCAAGCTCTACACACTCCGCCC ACTTTTGGCCAGGGGACCAAACTGGAGAT CAAACGAACTGTGGCTGCACCATCTGTCT

TCATCTTCCCGCCATCTGATGAGCAGTTG AAATCTGGAACTGCCTCTGTTGTGTGCCT GCTGAATAACTTCTATCCCAGAGAGGCCA AAGTACAGTGGAAGGTGGATAACGCCCTC CAATCGGGTAACTCCCAGGAGAGTGTCAC AGAGCAGGACAGCAAGGACAGCACCTACA GCCTCAGCAGCACCCTGACGCTGAGCAAA GCAGACTACGAGAAACACAAACTCTACGC CTGCGAAGTCACCCATCAGGGCCTGAGTT CGCCCGTCACAAAGAGCTTCAACAGGGGA GAGTGTTAA

( SEQ I D NO

[0173] In certain embodiments, the antibodies are fully human antibodies, such as fully human antibodies that bind an AQP4 polypeptide, an AQP4 polypeptide fragment, or an AQP4 epitope. Such fully human antibodies would be advantageous over fully mouse (or other full or partial non-human species antibodies), humanized antibodies, or chimeric antibodies to minimize the development of unwanted or unneeded side effects, such as immune responses directed toward non- fully human antibodies (e.g., anti-AQP4 antibodies derived from other species) when administered to the subject.

[0174] The antibodies provided herein can be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies can be specific for different epitopes of an AQP4 polypeptide or can be specific for both an AQP4 polypeptide as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. In certain embodiments, the antibodies provided herein are monospecific for a given epitope of an AQP4 polypeptide and do not bind to other epitopes.

[0175] In some embodiments, antibodies of the compositions comprising the antibodies and methods of using the antibodies provided herein include an rAb53, rAb58 and rAb09-3-33.

[0176] The antibodies provided herein include those antibodies and antigen-binding fragments of the following antibodies: an rAb53 antibody, rAb59 antibody, or rAb09-3-33 antibody; those antibodies in the Examples section, and described elsewhere herein. In a specific embodiment, an antibody provided herein is rAb53, rAb58 or rAb09-3-33 antibody. In another embodiment, an antibody provided herein comprises an antigen-binding fragment (e.g., a Fab fragment) ofrAb53, rAb58 or rAb09-3-33.

[0177] Furthermore, the antibodies sequences can vary from the sequences provided above, optionally using methods discussed in greater detail below. For example, nucleic acid sequences can vary from those set out above in that (a) the variable regions can be segregated away from the constant domains of the light chains, (b) the nucleic acids can vary from those set out above while not affecting the residues encoded thereby, (c) the nucleic acids can vary from those set out above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, (d) the nucleic acids can vary from those set out above by virtue of the ability to hybridize under high stringency conditions, e.g., 65 °C, 50% formamide, 0.1 x SSC, 0.1% SDS, (e) the amino acids can vary from those set out above by a given percentage, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, or (f) the amino acids can vary from those set out above by permitting conservative substitutions (discussed below). [0178] In certain embodiments, the antibodies provided herein are fully human, monoclonal antibodies that bind to AQP4.

[0179] In specific embodiments, an antibody provided herein that binds to AQP4 polypeptide (or antigen or epitope thereof) is an antibody that specifcally binds to the AQP4 polypeptide (or antigen or epitope) thereof.

[0180] In some embodiments, the antibodies provided herein bind to an AQP4 epitope that is a three- dimensional surface feature of an AQP4 polypeptide (e.g., a multimeric form of an AQP4 polypeptide). A region of an AQP4 polypeptide contributing to an epitope can be contiguous amino acids of the polypeptide or the epitope can come together from two or more non-contiguous regions of the polypeptide

[0181] In a specific embodiment, provided herein are one or more antibodies that bind to an AQP4 epitope, said antibodies comprising a VH chain and/or VL chain having the amino acid sequence of a VH chain and/or VL chain of an rAb53, rAb58, and/or rAb09-3-33 antibody. In another embodiment, provided herein are one or more antibodies that bind to an AQP4 epitope, said antibodies comprising a VH domain and/or VL domain having the amino acid sequence of a VH domain and/or VL domain of rAb53, rAb58, and/or rAb09-3-33 antibody. In another embodiment, provided herein are antibodies that bind to an AQP4 epitope, said antibodies comprising one, two, three, or more CDRs having the amino acid sequence of one, two, three, or more CDRs of rAb53, rAb58, and/or rAb09-3-33 antibody. In one embodiment, provided herein are one or more antibodies that bind to an AQP4 epitope, said antibodies comprising a combination of VH CDRs and/or VL CDRs having the amino acid sequence of VH CDRs and/or VL CDRs of rAb53, rAb58, and/or rAb09-3-33.

[0182] Also provided are antibodies that bind to an AQP4 epitope, said antibodies comprising one or more VH CDRs (i.e., VH CDR1, VH CDR2, and/or VH CDR3) having an amino acid sequence of any one of the VH CDRs (i.e., VH CDR1, VH CDR2, and/or VH CDR3) ofrAb53, rAb58, and/or rAb09-3- 33; or any combination thereof. In other embodiments, antibodies that bind to an AQP4 epitope, said antibodies comprising one or more VL CDRs (i.e., VL CDR1, VL CDR2, and/or VL CDR3) having an amino acid sequence of any one of the VL CDRs (i.e., VL CDR1, VL CDR2, and/or VL CDR3) of rAb53, rAb58, and/or rAb09-3-33; or any combination thereof.

[0183] In one embodiment, antibodies that bind to an AQP4 epitope comprise a VH domain having the amino acid sequence of the VH domain depicted in any one of SEQ ID NOS:2, 8 or 14. In one embodiment, antibodies that bind to an AQP4 epitope comprise a VL domain having the amino acid sequence of the VL domain depicted in any one of SEQ ID NOS:4, 10 or 16. In another embodiment, antibodies that bind to an AQP4 epitope comprise a VH domain having the amino acid sequence of the VH domain depicted in any one of SEQ ID NOS:2, 8 or 14 and a VL domain having the amino acid sequence of the VL domain depicted in any one of SEQ ID NOS:4, 10 or 16.

[0184] In certain embodiments, an antibody that binds to an AQP4 epitope comprises a VH domain having the amino acid sequence depicted in SEQ ID NO: 2 and a VL domain having the amino acid sequence depicted in any one of SEQ ID NOS:4, 10 or 16. In some embodiments, an antibody that binds to an AQP4 epitope comprises a VH domain having the amino acid sequence depicted in SEQ ID NO: 8 and a VL domain having the amino acid sequence depicted in any one of SEQ ID NOS:4, 10 or 16. In other embodiments, an antibody that binds to an AQP4 epitope comprises a VH domain having the amino acid sequence depicted in SEQ ID NO: 14 and a VL domain having the amino acid sequence depicted in any one of SEQ ID NOS:4, 10 or 16.1n one embodiment, an antibody that binds to an AQP4 epitope comprises a VH domain having the amino acid sequence depicted in SEQ ID NO:2 and a VL domain having the amino acid sequence depicted in SEQ ID NO:4. In one embodiment, an antibody that binds to an AQP4 epitope comprises a VH domain having the amino acid sequence depicted in SEQ ID NO:2 and a VL domain having the amino acid sequence depicted in SEQ ID NO: 10. In one embodiment, an antibody that binds to an AQP4 epitope comprises a VH domain having the amino acid sequence depicted in SEQ ID NO:2 and a VL domain having the amino acid sequence depicted in SEQ ID NO: 16. In one embodiment, an antibody that binds to an AQP4 epitope comprises a VH domain having the amino acid sequence depicted in SEQ ID NO: 8 and a VL domain having the amino acid sequence depicted in SEQ ID NO:4. In one embodiment, an antibody that binds to an AQP4 epitope comprises a VH domain having the amino acid sequence depicted in SEQ ID NO: 8 and a VL domain having the amino acid sequence depicted in SEQ ID NO: 10. In one embodiment, an antibody that binds to an AQP4 epitope comprises a VH domain having the amino acid sequence depicted in SEQ ID NO: 8 and a VL domain having the amino acid sequence depicted in SEQ ID NO: 16. In one embodiment, an antibody that binds to an AQP4 epitope comprises a VH domain having the amino acid sequence depicted in SEQ ID NO: 14 and a VL domain having the amino acid sequence depicted in SEQ ID NO:4. In one embodiment, an antibody that binds to an AQP4 epitope comprises a VH domain having the amino acid sequence depicted in SEQ ID NO: 14 and a VL domain having the amino acid sequence depicted in SEQ ID NO: 10. In one

embodiment, an antibody that binds to an AQP4 epitope comprises a VH domain having the amino acid sequence depicted in SEQ ID NO: 14 and a VL domain having the amino acid sequence depicted in SEQ ID NO: 16.

[0185] In some embodiments, antibodies provided herein comprise a VH CDRl having the amino acid sequence of the VH CDRl of any one of the VH regions depicted in SEQ ID NOS:2, 8 or 14. In another embodiment, antibodies provided herein comprise a VH CDR2 having the amino acid sequence of the VH CDR2 of any one of the VH regions depicted in SEQ ID NOS: 2, 8 or 14. In another embodiment, antibodies provided herein comprise a VH CDR3 having the amino acid sequence of the VH CDR3 of any one of the VH regions depicted in SEQ ID NOS: 2, 8 or 14. In certain embodiments, antibodies provided herein comprise a VH CDRl and/or a VH CDR2 and/or a VH CDR3 independently selected from a VH CDRl, VH CDR2, VH CDR3 as depicted in any one of the VH regions depicted in SEQ ID NOS: 2, 8 or 14.

[0186] In certain embodiments, an antibody that binds to an AQP4 epitope comprises (1) a VH domain having (a) a VH CDRl, VH CDR2, and/or VH CDR3 having the amino acid sequence depicted in SEQ ID NOS:22, 24 and/or 26, respectively, (b) a VH CDRl, VH CDR2, and/or VH CDR3 having the amino acid sequence depicted in SEQ ID NOS:34, 36 and/or 38, respectively, or (c) a VH CDRl, VH CDR2, and/or VH CDR3 having the amino acid sequence depicted in SEQ ID NOS:46, 48 and/or 49, respectively. In certain embodiments, the antibody further comprises a VL domain depicted in SEQ ID NO:4. In certain embodiments, the antibody further comprises a VL domain depicted in SEQ ID NO: 10. In certain embodiments, the antibody further comprises a VL domain depicted in SEQ ID NO: 16.

[0187] In certain embodiments, an antibody that binds to an AQP4 epitope comprises (1) a VH domain having a VH CDRl, VH CDR2, and/or VH CDR3 having the amino acid sequence depicted in SEQ ID NOS:22, 24 and/or 26, respectively, or any combination thereof, and (2) a VL domain depicted in SEQ ID NO:4. In certain embodiments, an antibody that binds to an AQP4 epitope comprises (1) a VH domain having a VH CDRl, VH CDR2, and VH CDR3 having the amino acid sequence depicted in SEQ ID NOS:22, 24 and 26, respectively, and (2) a VL domain depicted in SEQ ID NO:4.

[0188] In certain embodiments, an antibody that binds to an AQP4 epitope comprises (1) a VH domain having a VH CDRl, VH CDR2, and/or VH CDR3 having the amino acid sequence depicted in SEQ ID NOS:34, 36 and/or 38, respectively, or any combination thereof, and (2) a VL domain depicted in SEQ ID NO: 10. In certain embodiments, an antibody that binds to an AQP4 epitope comprises (1) a VH domain having a VH CDRl, VH CDR2, and VH CDR3 having the amino acid sequence depicted in SEQ ID NOS:34, 36 and 38, respectively, and (2) a VL domain depicted in SEQ ID NO: 10.

[0189] In certain embodiments, an antibody that binds to an AQP4 epitope comprises (1) a VH domain having a VH CDRl, VH CDR2, and/or VH CDR3 having the amino acid sequence depicted in SEQ ID NOS:46, 48 and/or 49, respectively, or any combination thereof, and (2) a VL domain depicted in SEQ ID NO: 16. In certain embodiments, an antibody that binds to an AQP4 epitope comprises (1) a VH domain having a VH CDRl, VH CDR2, and VH CDR3 having the amino acid sequence depicted in SEQ ID NOS:46, 48 and 49, respectively, and (2) a VL domain depicted in SEQ ID NO: 16.

[0190] In certain embodiments, the antibody comprises a VH domain depicted in SEQ ID NO:2. In some embodiments, the antibody comprises a VH domain depicted in SEQ ID NO: 8. In other embodiments, the antibody comprises a VH domain depicted in SEQ ID NO: 14. In certain embodiments, the antibody comprises a VL domain depicted in SEQ ID NO:4. In some embodiments, the antibody comprises a VL domain depicted in SEQ ID NO: 10. In other embodiments, the antibody comprises a VL domain depicted in SEQ ID NO: 16.

[0191] In other embodiments, an antibody that binds to an AQP4 epitope comprises a VL domain having (a) a VL CDRl, VL CDR2, and/or VL CDR3 having the amino acid sequence depicted in SEQ ID NOS:28, 30 and/or 32, respectively, (b) a VL CDRl, VL CDR2, and/or VL CDR3 having the amino acid sequence depicted in SEQ ID NOS:40, 42 and/or 44, respectively, or (c) a VL CDRl, VL CDR2, and/or VL CDR3 having the amino acid sequence depicted in SEQ ID NOS:52, 54 and/or 56, respectively. In certain embodiments, the antibody further comprises a VH domain depicted in SEQ ID NO:2. In certain embodiments, the antibody further comprises a VH domain depicted in SEQ ID NO: 8. In certain embodiments, the antibody further comprises a VH domain depicted in SEQ ID NO: 14.

[0192] In other embodiments, an antibody that binds to an AQP4 epitope comprises (1) a VL domain having a VL CDRl, VL CDR2, and/or VL CDR3 having the amino acid sequence depicted in SEQ ID NOS:28, 30 and/or 32, respectively, or any combination thereof; and (2) a VH domain depicted in SEQ ID NO:2. In other embodiments, an antibody that binds to an AQP4 epitope comprises (1) a VL domain having a VL CDRl, VL CDR2, and VL CDR3 having the amino acid sequence depicted in SEQ ID NOS:28, 30 and 32, respectively; and (2) a VH domain depicted in SEQ ID NO:2.

[0193] In other embodiments, an antibody that binds to an AQP4 epitope comprises (1) a VL domain having a VL CDRl, VL CDR2, and/or VL CDR3 having the amino acid sequence depicted in SEQ ID NOS:40, 42 and/or 44, respectively, or any combination thereof; and (2) a VH domain depicted in SEQ ID NO: 8. In other embodiments, an antibody that binds to an AQP4 epitope comprises (1) a VL domain having a VL CDRl, VL CDR2, and VL CDR3 having the amino acid sequence depicted in SEQ ID NOS:40, 42 and 44, respectively; and (2) a VH domain depicted in SEQ ID NO:8.

[0194] In other embodiments, an antibody that binds to an AQP4 epitope comprises (1) a VL domain having a VL CDRl, VL CDR2, and/or VL CDR3 having the amino acid sequence depicted in SEQ ID NOS:52, 54 and/or 56, respectively, or any combination thereof; and (2) a VH domain depicted in SEQ ID NO: 14. In other embodiments, an antibody that binds to an AQP4 epitope comprises (1) a VL domain having a VL CDRl, VL CDR2, and VL CDR3 having the amino acid sequence depicted in SEQ ID NOS:52, 54 and 56, respectively; and (2) a VH domain depicted in SEQ ID NO: 14.

[0195] In certain embodiments, an antibody that binds to an AQP4 epitope comprises (1) a VH domain having a VH CDRl, VH CDR2, and/or VH CDR3 having the amino acid sequence depicted in SEQ ID NOS:22, 24 and/or 26, respectively, or any combination thereof, and (2) a VL domain having a VL CDRl, VL CDR2, and/or VL CDR3 having the amino acid sequence depicted in SEQ ID NOS:28, 30 and/or 32, respectively, or any combination thereof. In certain embodiments, an antibody that binds to an AQP4 epitope comprises (1) a VH domain having a VH CDRl, VH CDR2, and VH CDR3 having the amino acid sequence depicted in SEQ ID NOS:22, 24 and 26, respectively, and (2) a VL domain having a VL CDRl, VL CDR2, and VL CDR3 having the amino acid sequence depicted in SEQ ID NOS:28, 30 and 32, respectively.

[0196] In certain embodiments, an antibody that binds to an AQP4 epitope comprises (1) a VH domain having a VH CDRl, VH CDR2, and/or VH CDR3 having the amino acid sequence depicted in SEQ ID NOS:34, 36 and/or 38, respectively, or any combination thereof, and (2) a VL domain having a VL CDRl, VL CDR2, and/or VL CDR3 having the amino acid sequence depicted in SEQ ID NOS:40, 42 and/or 44, respectively, or any combination thereof. In certain embodiments, an antibody that binds to an AQP4 epitope comprises (1) a VH domain having a VH CDRl, VH CDR2, and VH CDR3 having the amino acid sequence depicted in SEQ ID NOS:34, 36 and 38, respectively, and (2) a VL domain having a VL CDRl, VL CDR2, and VL CDR3 having the amino acid sequence depicted in SEQ ID NOS:40, 42 and 44, respectively.

[0197] In certain embodiments, an antibody that binds to an AQP4 epitope comprises (1) a VH domain having a VH CDRl, VH CDR2, and/or VH CDR3 having the amino acid sequence depicted in SEQ ID NOS:46, 48 and/or 49, respectively, or any combination thereof, and (2) a VL domain having a VL CDRl, VL CDR2, and/or VL CDR3 having the amino acid sequence depicted in SEQ ID NOS:52, 54 and/or 56, respectively, or any combination thereof. In certain embodiments, an antibody that binds to an AQP4 epitope comprises (1) a VH domain having a VH CDRl, VH CDR2, and VH CDR3 having the amino acid sequence depicted in SEQ ID NOS:46, 48 and 49, respectively, and (2) a VL domain having a VL CDRl, VL CDR2, and VL CDR3 having the amino acid sequence depicted in SEQ ID NOS:52, 54 and 56, respectively.

[0198] In certain embodiments, an antibody that binds to an AQP4 epitope comprises (1) a VH domain having (a) a VH CDRl, VH CDR2, and/or VH CDR3 having the amino acid sequence depicted in SEQ ID NOS:22, 24 and/or 26, respectively, (b) a VH CDRl, VH CDR2, and/or VH CDR3 having the amino acid sequence depicted in SEQ ID NOS:34, 36 and/or 38, respectively, or (c) a VH CDRl, VH CDR2, and/or VH CDR3 having the amino acid sequence depicted in SEQ ID NOS:46, 48 and/or 49, respectively, and/or (2) a VL CDRl, VL CDR2, and/or VL CDR3 having the amino acid sequence depicted in SEQ ID NOS:28, 30 or 32, respectively, (b) a VL CDRl, VL CDR2, and/or VL CDR3 having the amino acid sequence depicted in SEQ ID NOS:40, 42 and/or 44, respectively, or (c) a VL CDRl, VL CDR2, and/or VL CDR3 having the amino acid sequence depicted in SEQ ID NOS:52, 54 and/or 56, respectively.

[0199] In certain embodiments, an antibody that binds to an AQP4 epitope comprises (a) a VH domain having a VH CDRl, VH CDR2, and/or VH CDR3 having the amino acid sequence depicted in SEQ ID NOS:22, 24, and/or 26, respectively, and (b) a VL domain having a VL CDRl, VL CDR2, and/or VL CDR3 having the amino acid sequence depicted in SEQ ID NOS:28, 30 and/or 32, respectively. In certain embodiments, an antibody that binds to an AQP4 epitope comprises (a) a VH domain having a VH CDRl, VH CDR2, and/or VH CDR3 having the amino acid sequence depicted in SEQ ID NOS:34, 26 and/or 38, respectively, and (b) a VL CDRl, VL CDR2, and/or VL CDR3 having the amino acid sequence depicted in SEQ ID NOS:40, 42 and/or 44, respectively. In other embodiments, an antibody that binds to an AQP4 epitope comprises (a) a VH domain having a VH CDRl, VH CDR2, and/or VH CDR3 having the amino acid sequence depicted in SEQ ID NOS:46, 48 and/or 50, respectively, and (b) a VL domain having a VL CDRl, VL CDR2, and/or VL CDR3 having the amino acid sequence depicted in SEQ ID NOS:52, 54 and/or 56, respectively.

[0200] In some embodiments, antibodies provided herein comprise a VH CDRl having the amino acid sequence of the VH CDRl of any one of the VH regions depicted in SEQ ID NOS:2, 8 or 14. In another embodiment, antibodies provided herein comprise a VH CDR2 having the amino acid sequence of the VH CDR2 of any one of the VH regions depicted in SEQ ID NOS: 2, 8 or 14. In another embodiment, antibodies provided herein comprise a VH CDR3 having the amino acid sequence of the VH CDR3 of any one of the VH regions depicted in SEQ ID NOS: 2, 8 or 14. In certain embodiments, antibodies provided herein comprise a VH CDRl and/or a VH CDR2 and/or a VH CDR3 independently selected from the VH CDRl, VH CDR2, VH CDR3 as depicted in any one of the VH regions depicted in SEQ ID NOS: 2, 8 or 14.

[0201] In some embodiments, antibodies provided herein comprise a VL CDRl having the amino acid sequence of the VL CDRl of any one of the VL regions depicted in SEQ ID NOS:4, 10 or 16. In another embodiment, antibodies provided herein comprise a VL CDR2 having the amino acid sequence of the VL CDR2 of any one of the VL regions depicted in SEQ ID NOS:4, 10 or 16. In another

embodiment, antibodies provided herein comprise a VL CDR3 having the amino acid sequence of the VL CDR3 of any one of the VL regions depicted in SEQ ID NOS:4, 10 or 16. In certain embodiments, antibodies provided herein comprise a VL CDRl and/or a VL CDR2 and/or a VL CDR3 independently selected from the VL CDRl, VL CDR2, VL CDR3 as depicted in any one of the VL regions depicted in SEQ ID NOS:4, 10 or 16.

[0202] Any combination of VH CDRl , VH CDR2, VH CDR3, VL CDRl , VL CDR2 and VL CDR3, VH domain, VH chain, VL domain or VL chain sequences provided herein are also contemplated.

[0203] In some embodiments, antibodies provided herein comprises a (1) VH domain or chain having one or more of (a) a VH CDRl having the amino acid sequence of a VH CDRl of any one of the VH regions depicted in SEQ ID NOS:2, 8 or 14, (b) a VH CDR2 having the amino acid sequence of a VH CDR2 of any one of the VH regions depicted in SEQ ID NOS:2, 8 or 14, or (c) a VH CDR3 having the amino acid sequence a VH CDR3 of any one of the VH regions depicted in SEQ ID NOS:2, 8 or 14; and/or (2) a VL domain or chain having one of more of (a) a VL CDRl having the amino acid sequence of the VL CDRl of any one of the VL regions depicted in SEQ ID NOS:4, 10 or 16, (b) a VL CDR2 having the amino acid sequence of a VL CDR2 of any one of the VL regions depicted in SEQ ID NOS:4, 10 or 16, and/or (c) a VL CDR3 having the amino acid sequence of a VL CDR3 of any one of the VL regions depicted in SEQ ID NOS:4, 10 or 16.

[0204] In some embodiments, antibodies provided herein comprises a VH domain or chain having one or more of (a) a VH CDRl that is a CDRl in SEQ ID NO: 2; (b) a VH CDR2 that is a CDR2 in SEQ ID NO: 2; (C) a VH CDR3 that is a CDR3 in SEQ ID NO: 2; and/or (2) a VL domain or light chain polypeptide comprising one or more of (a) a VL CDRl that is a CDRl in SEQ ID NO: 4;(b) a VL CDR2 that a CDR2 in SEQ ID NO: 4; and a VL CDR3 that is a CDR3 in SEQ ID NO: 4. In other embodiments, antibodies provided herein comprises a (1) VH domain or chain having one or more of (a) a VH CDRl that is a CDRl in SEQ ID NO: 8; (b) a VH CDR2 that is a CDR2 in SEQ ID NO: 8; (C) a VH CDR3 that is a CDR3 in SEQ ID NO: 8; and/or (2) a VL domain or light chain polypeptide comprising one or more of (a) a VL CDRl that is a CDRl in SEQ ID NO: 10;(b) a VL CDR2 that a CDR2 in SEQ ID NO: 10; and a VL CDR3 that is a CDR3 in SEQ ID NO: 10. In some embodiments, antibodies provided herein comprises a (1) VH domain or chain having one or more of (a) a VH CDRl that is a CDRl in SEQ ID NO: 14; (b) a VH CDR2 that is a CDR2 in SEQ ID NO: 14; (C) a VH CDR3 that is a CDR3 in SEQ ID NO: 14; and/or (2) a VL domain or light chain polypeptide comprising one or more of (a) a VL CDRl that is a CDRl in SEQ ID NO: 16;(b) a VL CDR2 that a CDR2 in SEQ ID NO: 16; and a VL CDR3 that is a CDR3 in SEQ ID NO: 16.

[0205] As different systems to define the CDR regions are well known in the art, a person of ordinary skill in the art would be able characterize the CDR regions according to a specific system. For example, the CDR regions of VH regions depicted in SEQ ID NOS:2, 8 or 14 and VL regions SEQ ID NOS:4, 10 or 16 can be defined in accordance with the IMGT definition, Kabat definition, the Chothia definition, the combination of the Kabat definition and the Chothia definition, the AbM definition, or the contact definition of CDR. In some embodiments, each CDR region is defined in accordance with the CDR definition of IMGT. In some embodiments, each CDR region is defined in accordance with the CDR definition of Kabat. In some embodiments, each CDR region is defined in accordance with the CDR definition of Chothia. In some embodiments, each CDR region is defined in accordance with the CDR definition of AbM. In some embodiments, each CDR region is defined in accordance with the CDR definition of the Contact.

[0206] Provided herein are antibodies comprising one or more VH CDRs and one or more VL CDRs listed in Tables 4 and 5, respectively, which are defined according to the IMGT information system. Table 4A: Amino acid sequences of VH CDR regions of antibodies rAb53, rAb58, rAb09-3-33 (SEQ ID NOS).

GKGTTVIVSS

(SEQ ID

NO : 14 )

Table 4B: Nucleic acid sequences of VH CDR regions of antibodies rAb53, rAb58, rAb09-3-33 (SEQ ID NOS).

CACCATCACCAGAGACAACT

CCAAGAGCACGCTCTATGCG CATGTGAGTAGCCTGAGAGC CGATGACACGGCCGTATATT ACTGTGCGAAAGGGGACTAC GTCTTTGACTACTGGGGACA GGGAACCCTGGTCACCGTCT CCTCA (SEQ ID NO : 7 )

rAb09- GTAACTACAGGTGTCCACTC SEQ ID NO: 45 SEQ ID NO: 47 SEQ ID NO: 49

3-33 CGAGGTGCAGCTGGTGGAGT GGTTTCAACTTAG TTGCAGCCTGAAGAA ACGCGATCTCCGGGC

CTGGGGGAGGCGTGGTCCAG ATGACTATGAC AGTCATCAA CTCATGACTACGCTG

CCTGGGGGGTCCCTAAGACT CGGGGAATGGTGACC

CTCCTGTACAGCGTCTGGTT AGGAGGCACTTTCAC

TCAACTTAGATGACTATGAC TACTTCACCATGGAC

ATTCACTGGGTCCGCCAGGC GTC

GCCCGGCAAGGGGCTGCAGT

GGGTGGCAATTTTGCAGCCT

GAAGAAAGTCATCAAGAC A

TATAAATTCCGTGAGGGGCC

GATTCTCCGTCTCCAGAGAC

AGTTCGAGGGACACAATAGA

TCTGCAAATGCACAGTCTTA

GACCTGAAGACACGGCTATA

TATTACTGTACGCGATCTCC

GGGCCTCATGACTACGCTGC

GGGGAATGGTGACCAGGAGG

CACTTTCACTACTTCACCAT

GGACGTCTGGGGCAAAGGGA

CCACGGTCATCGTCTCCTCA

(SEQ ID NO: 13)

Table 5A: Amino acid sequences of VL CDR regions of antibodies rAb53, rAb58, rAb09-3-33 (SEQ ID NOS).

KPGQAPRLLIFGA

SSRATGI PDRFSG SGSGTDFTLTISR LEPEDFAVYYCQQ YGSSPWTFGQGTK VEIKR (SEQ ID

NO: 4)

rAb58 DIQMTQSPSALSA SEQ ID NO: 40 SEQ ID NO: 42 SEQ ID NO: 44

SVGDTVTITCRAS QSIRSW KAS QHYNSYPYT QSI SWLAWYQQK

PGKAPKLLIYKAS DLQSGVPSRFSGS GSGTDFTLTISGL QPDDFATYYCQHY NSYPYTFGQGTKV EIRR

(SEQ ID

NO: 10)

rAb09-3-33 DIVMTQSPLSLPV SEQ ID NO:52 SEQ ID NO:52 SEQ ID NO: 56

TPGEPASISCRSS QSLRHTITGYNY LAS MQALHTPPTF QSLRHTITGYNYI

NWYLQKPGQSPQL LIFLASSRATGVP DRFSGSGAGTDFT LKI SRVEAEDVGI YYCMQALHTPPTF GQGTKLEIKR

(SEQ ID

NO: 16)

Table 5B: Amino acid sequences of VL CDR regions of antibodies rAb53, rAb58, rAb09-3-33 (SEQ ID NOS).

Ab VL domain VL CDR1 VL CDR2 VL CDR3 rAb53 GAAATTGTGTTGACACAG SEQ ID NO: 27 SEQ ID NO: 29 SEQ ID NO: 31 TCTCCAGGCACCCTGTCT CAGACTGTTCGCAC GGTGCATCC CAGCAGTATGGTAG

TTGTCTCCAGGGGAAAGA CAACTAC CTCACCGTGGACG

GCCACCCTCTCCTGCAGG

GCCAGTCAGACTGTTCGC

ACCAACTACTTAGCCTGG

TTCCAGCAGAAACCTGGC

CAGGCTCCCAGGCTCCTC

ATCTTTGGTGCATCCAGC

AGGGCCACTGGCATCCCA

GACAGGTTCAGTGGCAGT

GGGTCTGGGACAGACTTC

ACTCTCACCATCAGCAGA

CTGGAGCCTGAAGATTTT

GCAGTGTATTACTGTCAG

CAGTATGGTAGCTCACCG

TGGACGTTCGGCCAAGGG

ACCAAGGTGGAAATCAAA

CGAACTGTGGCTGCACCA

TCTGTCTTCATCTTCCCG

CCATCTGATGAGCAGTTG

AAATCTGGAACTGCCTCT

GTTGTGTGCCTGCTGAAT

AACTTCTATCCCAGAGAG

GCCAAAGTACAGTGGAAG

GTGGATAACGCCCTCCAA

TCGGGTAACTCCCAGGAG

AGTGTCACAGAGCAGGAC

AGCAAGGACAGCACCTAC

AGCCTCAGCAGCACCCTG

ACGCTGAGCAAAGCAGAC

TACGAGAAACACAAAGTC

TACGCCTGCGAAGTCACC

CATCAGGGCCTGAGTTCG

CCCGTCACAAAGAGCTTC

AACAGGGGAGAGTGT (SE

Q ID NO: 5)

rAb58 GACATCCAGATGACCCAG SEQ ID NO: 39 SEQ ID NO: 41 SEQ ID NO: 43 TCTCCATCCGCCCTGTCT CAGAGTATTAGGAG AAGGCGTCT CAACACTATAATAG GCATCTGTAGGAGACACA CTGG TTACCCGTACACT GTCACCATCACTTGCCGG GCCAGTCAGAGTATTAGG AGCTGGTTGGCCTGGTAT CAGCAGAAACCAGGGAAA GCCCCTAAACTCCTGATC TATAAGGCGTCTGATTTA CAAAGTGGGGTCCCATCA AGATTCAGCGGCAGTGGA TCTGGGACAGACTTCACT CTCACCATCAGCGGCCTG CAGCCTGATGATTTTGCA ACTTATTACTGCCAACAC TATAATAGTTACCCGTAC ACTTTTGGCCAGGGGACC AAGGTGGAGATCAGACGA

(SEQ ID NO: 9)

rAb09- GATATTGTGATGACTCAG SEQ ID NO: 51 SEQ ID NO:53 SEQ ID NO:55

3-33 TCTCCACTCTCCCTGCCC CAGAGCCTCCGCCA TTGGCCTCT ATGCAAGCTCTACA

GTCACCCCTGGAGAGCCG CACCATCACTGGAT CACTCCGCCCACTT

GCCTCTATCTCCTGCAGG ACAACTAT TT

TCTAGTCAGAGCCTCCGC

CACACCATCACTGGATAC

AACTATATCAATTGGTAC

CTGCAGAAGCCAGGGCAG

TCTCCACAACTCCTGATC

TTTTTGGCCTCTTCTCGG

GCCACCGGGGTCCCTGAC

AGGTTCAGTGGCAGTGGA

GCAGGCACAGATTTTACA

CTGAAAATCAGCAGAGTG

GAGGCTGAGGATGTTGGA

ATTTATTACTGCATGCAA

GCTCTACACACTCCGCCC

ACTTTTGGCCAGGGGACC

AAACTGGAGATCAAACGA (SEQ ID NO: 15)

[0207] In particular, provided herein is an antibody comprising a VH CDRl (SEQ ID NOS:22, 34 or 46) and a VL CDRl (SEQ ID NOS:28, 42 or 54); a VH CDRl (SEQ ID NOS:22, 34 or 46) and a VL CDR2 (SEQ ID NOS:30, 42 or 54); a VH CDRl (SEQ ID NOS:22, 34 or 46) and a VL CDR3 (SEQ ID NOS:32, 44 or 56); a VH CDR2 (SEQ ID NOS:24, 36 or 48) and a VL CDRl (SEQ ID NOS:28, 42 or 54); VH CDR2 (SEQ ID NOS:24, 36 or 48) and VL CDR2 (SEQ ID NOS:30, 42 or 54); a VH CDR2 (SEQ ID NOS:24, 36 or 48) and a VL CDR3 (SEQ ID NOS:32, 44 or 56); a VH CDR3 (SEQ ID NOS:26, 38 or 50) and a VH CDRl (SEQ ID NOS:22, 34 or 46); a VH CDR3 (SEQ ID NOS:26, 38 or 50) and a VL CDR2 (SEQ ID NOS:30, 42 or 54); a VH CDR3 (SEQ ID NOS:26, 38 or 50) and a VL CDR3 (SEQ ID NOS:32, 44 or 56); a VH1 CDRl , a VH CDR2 (SEQ ID NOS:24, 36 or 48) and a VL CDRl (SEQ ID NOS:28, 42 or 54); a VH CDRl (SEQ ID NOS:22, 34 or 46), a VH CDR2 (SEQ ID NOS:24, 36 or 48) and a VL CDR2 (SEQ ID NOS:30, 42 or 54); a VH CDRl (SEQ ID NOS:22, 34 or 46), a VH CDR2 (SEQ ID NOS:24, 36 or 48) and a VL CDR3 (SEQ ID NOS:32, 44 or 56); a VH CDR2 (SEQ ID NOS:24, 36 or 48), a VH CDR3 (SEQ ID NOS:26, 38 or 50) and a VL CDRl (SEQ ID NOS:28, 42 or 54), a VH CDR2 (SEQ ID NOS:24, 36 or 48), a VH CDR3 (SEQ ID NOS:26, 38 or 50) and a VL CDR2 (SEQ ID NOS:30, 42 or 54); a VH CDR2 (SEQ ID NOS:24, 36 or 48), a VH CDR2 (SEQ ID NOS:24, 36 or 48) and a VL CDR3 (SEQ ID NOS:32, 44 or 56); a VH CDRl (SEQ ID NOS:22, 34 or 46), a VL CDRl (SEQ ID NOS:28, 42 or 54) and a VL CDR2 (SEQ ID NOS:30, 42 or 54); a VH CDRl (SEQ ID NOS:22, 34 or 46), a VL CDRl (SEQ ID NOS:28, 42 or 54) and a VL CDR3 (SEQ ID NOS:32, 44 or 56); a VH CDR2 (SEQ ID NOS:24, 36 or 48), a VL CDRl (SEQ ID NOS:28, 42 or 54) and a VL CDR2 (SEQ ID NOS:30, 42 or 54); a VH CDR2 (SEQ ID NOS:24, 36 or 48), a VL CDRl (SEQ ID NOS:28, 42 or 54) and a VL CDR3 (SEQ ID NOS:32, 44 or 56); a VH CDR3 (SEQ ID NOS:26, 38 or 50), a VL CDRl (SEQ ID NOS:28, 42 or 54) and a VL CDR2 (SEQ ID NOS:30, 42 or 54); a VH CDR3 (SEQ ID NOS:26, 38 or 50), a VL CDRl (SEQ ID NOS:28, 42 or 54) and a VL CDR3 (SEQ ID NOS:32, 44 or 56); a VH CDRl (SEQ ID NOS:22, 34 or 46), a VH CDR2 (SEQ ID NOS:24, 36 or 48), a VH CDR3 (SEQ ID NOS:26, 38 or 50) and a VL CDRl (SEQ ID NOS:28, 42 or 54); a VH CDRl (SEQ ID NOS:22, 34 or 46), a VH CDR2 (SEQ ID NOS:24, 36 or 48), a VH CDR3 (SEQ ID NOS:26, 38 or 50) and a VL CDR2 (SEQ ID NOS:30, 42 or 54); a VH CDRl (SEQ ID NOS:22, 34 or 46), a VH CDR2 (SEQ ID NOS:24, 36 or 48), a VH CDR3 (SEQ ID NOS:26, 38 or 50) and a VL CDR3 (SEQ ID NOS:32, 44 or 56); a VH CDRl (SEQ ID NOS:22, 34 or 46), a VH CDR2 (SEQ ID NOS:24, 36 or 48), a VL CDRl (SEQ ID NOS:28, 42 or 54) and a VL CDR2 (SEQ ID NOS:30, 42 or 54); a VH CDRl (SEQ ID NOS:22, 34 or 46), a VH CDR2 (SEQ ID NOS:24, 36 or 48), a VL CDRl (SEQ ID NOS:28, 42 or 54) and a VL CDR3 (SEQ ID NOS:32, 44 or 56); a VH CDRl (SEQ ID NOS:22, 34 or 46), a VH CDR3 (SEQ ID NOS:26, 38 or 50), a VL CDR1 (SEQ ID NOS:28, 42 or 54) and a VL CDR2 (SEQ ID NOS:30, 42 or 54); a VH CDR1 (SEQ ID NOS:22, 34 or 46), a VH CDR3 (SEQ ID NOS:26, 38 or 50), a VL CDR1 (SEQ ID NOS:28, 42 or 54) and a VL CDR3 (SEQ ID NOS:32, 44 or 56); a VH CDR2 (SEQ ID NOS:24, 36 or 48), a VH CDR3 (SEQ ID NOS:26, 38 or 50), a VL CDR1 (SEQ ID NOS:28, 42 or 54) and a VL CDR2 (SEQ ID NOS:30, 42 or 54); a VH CDR2 (SEQ ID NOS:24, 36 or 48), a VH CDR3 (SEQ ID NOS:26, 38 or 50), a VL CDR1 (SEQ ID NOS:28, 42 or 54) and a VL CDR3 (SEQ ID NOS:32, 44 or 56); a VH CDR2 (SEQ ID NOS:24, 36 or 48), a VH CDR3 (SEQ ID NOS:26, 38 or 50), a VL CDR2 (SEQ ID NOS:30, 42 or 54) and a VL CDR3 (SEQ ID NOS:32, 44 or 56); a VH CDR1 (SEQ ID NOS:22, 34 or 46), a VH CDR2 (SEQ ID NOS:24, 36 or 48), a VH CDR3 (SEQ ID NOS:26, 38 or 50), a VL CDR1 (SEQ ID NOS:28, 42 or 54) and a VL CDR2 (SEQ ID NOS:30, 42 or 54); a VH CDR1 (SEQ ID NOS:22, 34 or 46), a VH CDR2 (SEQ ID NOS:24, 36 or 48), a VH CDR3 (SEQ ID NOS:26, 38 or 50), a VL CDR1 (SEQ ID NOS:28, 42 or 54) and a VL CDR3 (SEQ ID NOS:32, 44 or 56); a VH CDR1 (SEQ ID NOS:22, 34 or 46), a VH CDR2 (SEQ ID NOS:24, 36 or 48), a VL CDR1 (SEQ ID NOS:28, 42 or 54), a VL CDR2 (SEQ ID NOS:30, 42 or 54), and a VL CDR3 (SEQ ID NOS:32, 44 or 56); a VH CDR1 (SEQ ID NOS:22, 34 or 46), a VH CDR3 (SEQ ID NOS:26, 38 or 50), a VL CDR1 (SEQ ID NOS:28, 42 or 54), a VL CDR2 (SEQ ID NOS:30, 42 or 54), and a VL CDR3 (SEQ ID NOS:32, 44 or 56); a VH CDR2 (SEQ ID NOS:24, 36 or 48), a VH CDR3 (SEQ ID NOS:26, 38 or 50), a VL CDR1 (SEQ ID NOS:28, 42 or 54), a VL CDR2 (SEQ ID NOS:30, 42 or 54), and a VL CDR3 (SEQ ID NOS:32, 44 or 56); or any combination thereof of the VH CDRs (SEQ ID NOS:22, 24, 26, 34, 36, 38, 46, 48 or 50) and VL CDRs (SEQ ID NOS:28, 30, 32, 40, 42, 44, 52, 54 or 56) listed in Tables 4 and 5. The corresponding VH CDRs of VH domains (SEQ ID NOS: 2, 8 or 14) of rAb53, rAb58 or rAb09-3-33, respectively; and/or VL domains (SEQ ID NOS:4, 10 or 16) of rAb53, rAb58 or rAb09-3-33, respectively, can also be used in any of the combinations listed above.

[0208] In some embodiments, provided herein is a human anti-AQP4 IgG antibody or an antigen binding fragment thereof that lacks effector function, wherein the antibody or binding fragment thereof comprises a heavy chain variable (VH) region comprising a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO:22, 34, and 46; a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO:24, 36, and 48; and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO:26, 38, and 50; and/or a light chain variable (VL) region comprising a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO:28, 40, and 52; a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO:30, 42, and 54; and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO:32, 44, and 56. [0209] In other embodiments, provided herein is a human anti-AQP4 IgG antibody or an antigen binding fragment thereof that lacks effector function, wherein the antibody or binding fragment thereof comprises a heavy chain variable (VH) region comprising a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO:22, 34, and 46; a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO:24, 36, and 48; and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO:26, 38, and 50.

[0210] In other embodiments, provided herein is a human anti-AQP4 IgG antibody or an antigen binding fragment thereof that lacks effector function, wherein the antibody or binding fragment thereof comprising a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO:28, 40, and 52; a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO:30, 42, and 54; and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO:32, 44, and 56.

[0211] In one aspect, provided herein is a human anti-AQP4 IgG antibody or an antigen binding fragment thereof that lacks effector function comprising a heavy chain variable (VH) region comprising a VH CDR1 having an amino acid sequence of SEQ ID NO:22; a VH CDR2 having an amino acid sequence of SEQ ID NO:24; and a VH CDR3 having an amino acid sequence of SEQ ID NO:26; and/or a light chain variable (VL) region comprising a VL CDR1 having an amino acid sequence of SEQ ID NO:28; a VL CDR2 having an amino acid sequence of SEQ ID NO:30; and a VL CDR3 having an amino acid sequence of SEQ ID NO:32.

[0212] In one aspect, provided herein is a human anti-AQP4 IgG antibody or an antigen binding fragment thereof that lacks effector function comprising a heavy chain variable (VH) region comprising a VH CDR1 having an amino acid sequence of SEQ ID NO:34; a VH CDR2 having an amino acid sequence of SEQ ID NO:36; and a VH CDR3 having an amino acid sequence of SEQ ID NO:38; and/or a light chain variable (VL) region comprising a VL CDR1 having an amino acid sequence of SEQ ID NO:40; a VL CDR2 having an amino acid sequence of SEQ ID NO:42; and a VL CDR3 having an amino acid sequence of SEQ ID NO:44.

[0213] In one aspect, provided herein is a human anti-AQP4 IgG antibody or an antigen binding fragment thereof that lacks effector function comprising a heavy chain variable (VH) region comprising a VH CDR1 having an amino acid sequence of SEQ ID NO:46; a VH CDR2 having an amino acid sequence of SEQ ID NO:48; and a VH CDR3 having an amino acid sequence of SEQ ID NO:50; and/or a light chain variable (VL) region comprising a VL CDR1 having an amino acid sequence of SEQ ID NO:52; a VL CDR2 having an amino acid sequence of SEQ ID NO:54; and a VL CDR3 having an amino acid sequence of SEQ ID NO:56. [0214] In some embodiments, provided herein is a human anti-AQP4 IgG antibody or an antigen binding fragment thereof that lacks effector function comprising all three heavy chain CDRs and/or all three light chain CDRs from: the antibody designated rAb53 that comprises a VH sequence that is SEQ ID NO:2 and a VL sequence that is SEQ ID NO:4; the antibody designated rAb58 that comprises a VH sequence that is SEQ ID NO: 8 and a VL sequence that is SEQ ID NO: 10; or the antibody designated rAb09-3-33 that comprises a VH sequence that is SEQ ID NO: 14 and a VL sequence that is SEQ ID NO: 16.

[0215] In one aspect, provided herein is a human anti-AQP4 IgG antibody or an antigen binding fragment thereof that lacks effector function comprising all three heavy chain CDRs and/or all three light chain CDRs from the antibody designated rAb53. In another aspect, provided herein is a human anti- AQP4 IgG antibody or an antigen binding fragment thereof that lacks effector function comprising all three heavy chain CDRs and/or all three light chain CDRs from the antibody designated rAb58. In yet another aspect, provided herein is a human anti-AQP4 IgG antibody or an antigen binding fragment thereof that lacks effector function comprising all three heavy chain CDRs and/or all three light chain CDRs from the antibody designated rAb09-3-33.

[0216] In some embodiments, provided herein is a human anti-AQP4 IgG antibody or an antigen binding fragment thereof that lacks effector function, wherein the antibody or binding fragment thereof has a heavy chain variable (VH) region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, 8, and 14, and/or a light chain variable (VL) region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:4, 10, and 16. In one aspect, the VH region comprises an amino acid sequence of SEQ ID NO:2 and/or the VL region comprises an amino acid sequence of SEQ ID NO:4. In another aspect, the VH region comprises an amino acid sequence of SEQ ID NO:8 and/or the VL region comprises an amino acid sequence of SEQ ID NO: 10. In yet another aspect, the VH region comprises an amino acid sequence of SEQ ID NO: 14 and/or said VL region comprises an amino acid sequence of SEQ ID NO: 16.

[0217] In some other embodiments, provided herein is a human anti-AQP4 IgG antibody or an antigen binding fragment thereof that lacks effector function, wherein the antibody or binding fragment thereof has a light chain variable (VL) region comprising a kappa constant region. In some aspects, the kappa constant region comprises an amino sequence of SEQ ID NO:20.

[0218] In some embodiments, provided herein is a human anti-AQP4 IgG antibody or an antigen binding fragment thereof that lacks effector function, wherein the antibody or binding fragment thereof has a heavy chain variable (VH) region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, 8, and 14, and/or a light chain variable (VL) region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6, 12, and 18. In one aspect, the VH region comprises an amino acid sequence of SEQ ID NO:2 and/or the VL region comprises an amino acid sequence of SEQ ID NO:6. In another aspect, the VH region comprises an amino acid sequence of SEQ ID NO:8 and/or the VL region comprises an amino acid sequence of SEQ ID NO: 12. In yet another aspect, the VH region comprises an amino acid sequence of SEQ ID NO: 14 and/or said VL region comprises an amino acid sequence of SEQ ID NO: 18.

[0219] Also provided herein are fusion proteins comprising an antibody provided herein that binds to an AQP4 antigen and a heterologous polypeptide. In some embodiments, the heterologous polypeptide to which the antibody is fused is useful for targeting the antibody to cells having cell surface-expressed AQP4.

[0220] Also provided are antibodies that bind to an AQP4 epitope, the antibodies comprising derivatives of the VH domains, VH CDRs, VL domains, and VL CDRs described herein that bind to an AQP4 antigen. Also provided are antibodies comprising derivatives of rAb53, rAb58 and/or rAb09-3-33, wherein said antibodies bind to an AQP4 epitope. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which results in amino acid substitutions. In certain embodiments,, the derivatives include less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the original molecule. In one embodiment, the derivatives have conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed and the activity of the protein can be determined.

[0221] In another embodiment, an antibody that binds to an AQP4 epitope comprises an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of rAb53, rAb58 and/or rAb09-3-33, or an antigen-binding fragment thereof, such as a VH domain, VL domain, VH chain, or VL chain. In one embodiment, an antibody that binds to an AQP4 epitope comprises an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to an amino acid sequence depicted in SEQ ID NOS:2, 8 or 14. In another embodiment, an antibody that binds to an AQP4 epitope comprises an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to an amino acid sequence depicted in SEQ ID NOS:4, 10 or 16. In yet another embodiment, an antibody that binds to an AQP4 epitope comprises a VH CDR and/or a VL CDR amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a VH CDR amino acid sequence depicted in SEQ ID NOS: SEQ ID NOS:22, 24, 26, 34, 36, 38, 46, 48 or 50 (VH CDRs) and/or a VL CDR amino acid sequence depicted in SEQ ID NOS:28, 30, 32, 40, 42, 44, 52, 54 or 56 (VL CDRs).

[0222] In specific embodiments, the antibody is a fully human anti-human antibody, such as a fully human monoclonal antibody. Fully human antibodies can be produced by any method known in the art. Exemplary methods include immunization with an AQP4 antigen (any AQP4 polypeptide capable of eliciting an immune response, and optionally conjugated to a carrier) of transgenic animals (e.g., mice) that are capable of producing a repertoire of human antibodies in the absence of endogenous

immunoglobulin production; see, e.g., Jakobovits et ah, (1993) Proc. Natl. Acad. Sci., 90:2551 ;

Jakobovits et ah, (1993) Nature, 362:255 258 (1993); Bruggermann et ah, (1993) Year in Immunol., 7:33. Other methods of producing fully human anti-AQP4 antibodies can be found in the Examples provided herein.

[0223] Alternatively, fully human antibodies can be generated through the in vitro screening of phage display antibody libraries; see e.g., Hoogenboom et al, J. Mol. Biol., 227:381 (1991); Marks et ah, J. Mol. Biol., 222:581 (1991), incorporated herein by reference. Various antibody-containing phage display libraries have been described and can be readily prepared by one skilled in the art. Libraries can contain a diversity of human antibody sequences, such as human Fab, Fv, and scFv fragments, that can be screened against an appropriate target.

[0224] In certain embodiments, the antibodies used in accordance with the methods provided herein have a high affinity for an AQP4 polypeptide, or polypeptide fragment or epitope thereof. In one embodiment, the antibodies used in accordance with the methods provided herein have a higher affinity for an AQP4 antibody than known antibodies (e.g. , commercially available monoclonal antibodies discussed elsewhere herein). In a specific embodiment, the antibodies used in accordance with the methods provided herein have a 2- to 10-fold (or more) higher affinity for an AQP4 antigen than a known anti-AQP4 antibody as assessed by techniques described herein or known to one of skill in the art (e.g., a Biacore® assay). In accordance with these embodiments, the affinity of the antibodies are, in one embodiment, assessed by a Biacore® assay.

[0225] In a specific embodiment, an antibody that binds an AQP4 antigen comprises an amino acid sequence of a VH domain and/or an amino acid sequence a VL domain encoded by a nucleotide sequence that hybridizes to (1) the complement of a nucleotide sequence encoding any one of the VH and/or VL domains depicted in SEQ ID NOS: l, 7 or 13 (VH) and/or SEQ ID NOS:3, 9 or 15 (VL) under stringent conditions (e.g., hybridization to filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about 45° C followed by one or more washes in 0.2xSSC/0.1% SDS at about 50-65° C) under highly stringent conditions (e.g., hybridization to filter-bound nucleic acid in 6xSSC at about 45° C followed by one or more washes in O. lxSSC/0.2% SDS at about 68° C), or under other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel, F.M. et al, eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3).

[0226] In another embodiment, an antibody that binds an AQP4 antigen comprises an amino acid sequence of a VH CDR or an amino acid sequence of a VL CDRs encoded by a nucleotide sequence that hybridizes to the complement of a nucleotide sequence encoding any one of the VH CDRs and/or VL CDRs depicted in SEQ ID NOS: SEQ ID NOS:22, 24, 26, 34, 36, 38, 46, 48 or 50 (VH CDRs) and/or SEQ ID NOS:28, 30, 32, 40, 42, 44, 52, 54 or 56 (VL CDRs) under stringent conditions (e.g., hybridization to filter-bound DNA in 6X SSC at about 45° C followed by one or more washes in 0.2X SSC/0.1% SDS at about 50-65° C), under highly stringent conditions (e.g., hybridization to filter-bound nucleic acid in 6X SSC at about 45° C followed by one or more washes in 0. IX SSC/0.2% SDS at about 68° C), or under other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel, F.M. et al, eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3)

[0227] The antibodies provided herein include antibodies that are chemically modified, i.e., by the covalent attachment of any type of molecule to the antibody. For example, but not by way of limitation, the antibody derivatives include antibodies that have been chemically modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Additionally, the antibody can contain one or more non-classical amino acids.

[0228] Also provided are antibodies that bind to an AQP4 antigen which comprise a framework region known to those of skill in the art {e.g., a human or non-human fragment). The framework region can, for example, be naturally occurring or consensus framework regions. In certain embodiments, the framework region of an antibody provided herein is human (see, e.g., Chothia et ah, 1998, J. Mol. Biol. 278:457-479 for a listing of human framework regions, which is incorporated by reference herein in its entirety). See also Kabat et al. (1991) Sequences of Proteins of Immunological Interest (U.S. Department of Health and Human Services, Washington, D.C.) 5th ed.

[0229] In a specific embodiment, provided herein are antibodies that bind to an AQP4 antigen, said antibodies comprising the amino acid sequence of one or more of the CDRs of rAb53, rAb58, and/or rAb09-3-33 (i.e., SEQ ID NOS: SEQ ID NOS:22, 24, 26, 34, 36, 38, 46, 48 or 50 (VH CDRs) and/or a VL CDR amino acid sequence depicted in SEQ ID NOS:28, 30, 32, 40, 42, 44, 52, 54 or 56 (VL CDRs), and human framework regions with one or more amino acid substitutions at one, two, three or more of the following residues: (a) rare framework residues that differ between the murine antibody framework (i.e., donor antibody framework) and the human antibody framework (i.e., acceptor antibody framework); (b) Venier zone residues when differing between donor antibody framework and acceptor antibody framework; (c) interchain packing residues at the VH/VL interface that differ between the donor antibody framework and the acceptor antibody framework; (d) canonical residues which differ between the donor antibody framework and the acceptor antibody framework sequences, particularly the framework regions crucial for the definition of the canonical class of the murine antibody CDR loops; (e) residues that are adjacent to a CDR; (g) residues capable of interacting with the antigen; (h) residues capable of interacting with the CDR; and (i) contact residues between the VH domain and the VL domain. In certain embodiments, antibodies that bind to an AQP4 antigen comprising the human framework regions with one or more amino acid substitutions at one, two, three or more of the above-identified residues are antagonistic AQP4 antibodies.

[0230] Provided herein are antibodies that bind to an AQP4 antigen, said antibodies comprising the amino acid sequence of the VH domain and/or VL domain of an rAb53, rAb58, and/or rAb09-3-33 antibody, having mutations (e.g., one or more amino acid substitutions) in the framework regions. In certain embodiments, antibodies that bind to an AQP4 antigen comprise the amino acid sequence of the VH domain and/or VL domain or an antigen-binding fragment thereof of rAb53, rAb58, and/or rAb09-3- 33 with one or more amino acid residue substitutions in the framework regions of the VH and/or VL domains. [0231] Also provided herein are antibodies that bind to an AQP4 antigen, said antibodies comprising the amino acid sequence of the VH domain and/or VL domain of an rAb53, rAb58, and/or rAb09-3-33 antibody, having mutations (e.g., one or more amino acid residue substitutions) in the hypervariable and framework regions. In certain embodiments,, the amino acid substitutions in the hypervariable and framework regions improve binding of the antibody to an AQP4 antigen.

[0232] Also provided are fusion proteins comprising an antibody provided herein that binds to an AQP4 antigen and a heterologous polypeptide. In some embodiments, the heterologous polypeptide to which the antibody is fused is useful for targeting the antibody to cells having cell surface-expressed AQP4.

[0233] Also provided are panels of antibodies that bind to an AQP4 antigen. In specific

embodiments, provided herein are panels of antibodies having different association rate constants different dissociation rate constants, different affinities for AQP4 antigen, and/or different specificities for an AQP4 antigen. Also provided herein are panels of about 10, such as about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1000 antibodies or more. Panels of antibodies can be used, for example, in 96 well or 384 well plates, such as for assays such as ELISAs.

5.2.2. Mutated Fc

[0234] In one aspect, provided herein are human anti-AQP4 IgG antibodies or antigen binding fragments thereof having a mutated Fc region. Exemplary Fc "mutant" sequences are provided in Tables 6-14. At the end of the IgGl sequence, in certain instances, a FLAG tag (LEDYKDDDDK, SEQ ID NO:80; CTCGAGGACTACAAGGACGATGACGATAAGTGA, SEQ ID NO:79) was added.

Table 6. Mutant IgGl Fc K322A Sequences (K322A mutation underlined)

TCAAAGGCTTCTATCCCAGCGACATCGCC

GTGGAGTGGGAGAGCAATGGGCAGCCGGA GAACAACTACAAGACCACGCCTCCCGTGC TGGACTCCGACGGCTCCTTCTTCCTCTAC AGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCG TGATGCATGAGGCTCTGCACAACCACTAC ACGCAGAAGAGCCTCTCCCTGTCTCCGGG TAAA (SEQ ID NO: 61)

Table 7. Mutant IgGl Fc L234A/L235A Sequences (L234A/L235A mutation underlined)

Nucleic Acid Amino Acid

GCCTCCACCAAGGGCCCATCGGTCTTCCC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF CCTGGCGCCCTCCTCCAAGAGCACCTCCG PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS GGGGCACAGCGGCCCTGGGCTGCCTGGTC SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE AAGGACTACTTCCCCGAACCGGTGACGGT PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKD GTCGTGGAACTCAGGCGCCCTGACCAGCG TLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGV GCGTGCACACCTTCCCGGCTGTCCTACAG EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN TCCTCAGGACTCTACTCCCTCAGCAGCGT GKEYKCKVSNKALPAPIEKTISKAKGQPREPQV GGTGACCGTGCCCTCCAGCAGCTTGGGCA YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW CCCAGACCTACATCTGCAACGTGAATCAC ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK AAGCCCAGCAACACCAAGGTGGACAAGAA SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AGTTGAGCCCAAATCTTGTGACAAAACTC (SEQ ID NO: 64)

ACACATGCCCACCGTGCCCAGCACCTGAA GCCGCGGGGGGACCGTCAGTCTTCCTCTT

CCCCCCAAAACCCAAGGACACCCTCATGA TCTCCCGGACCCCTGAGGTCACATGCGTG GTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCG TGGAGGTGCATAATGCCAAGACAAAGCCG CGGGAGGAGCAGTACAACAGCACGTACCG TGTGGTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAG TGCAAGGTCTCCAACAAAGCCCTCCCAGC CCCCATCGAGAAAACCATCTCCAAAGCCA

AAGGGCAGCCCCGAGAACCACAGGTGTAC ACCCTGCCCCCATCCCGGGATGAGCTGAC CAAGAACCAGGTCAGCCTGACCTGCCTGG TCAAAGGCTTCTATCCCAGCGACATCGCC GTGGAGTGGGAGAGCAATGGGCAGCCGGA GAACAACTACAAGACCACGCCTCCCGTGC TGGACTCCGACGGCTCCTTCTTCCTCTAC AGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCG TGATGCATGAGGCTCTGCACAACCACTAC ACGCAGAAGAGCCTCTCCCTGTCTCCGGG TAAA (SEQ ID NO: 63)

Table 8. Mutant IgGl Fc L234A/L235A/G237A Sequences (L234A/L235A/G237A mutation underlined)

Nucleic Acid Amino Acid

GCCTCCACCAAGGGCCCATCGGTCTTCCC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF CCTGGCGCCCTCCTCCAAGAGCACCTCCG PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS GGGGCACAGCGGCCCTGGGCTGCCTGGTC SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE AAGGACTACTTCCCCGAACCGGTGACGGT PKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKD GTCGTGGAACTCAGGCGCCCTGACCAGCG TLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGV GCGTGCACACCTTCCCGGCTGTCCTACAG EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN TCCTCAGGACTCTACTCCCTCAGCAGCGT GKEYKCKVSNKALPAPIEKTISKAKGQPREPQV GGTGACCGTGCCCTCCAGCAGCTTGGGCA YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW CCCAGACCTACATCTGCAACGTGAATCAC ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK AAGCCCAGCAACACCAAGGTGGACAAGAA SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AGTTGAGCCCAAATCTTGTGACAAAACTC (SEQ ID NO: 66)

ACACATGCCCACCGTGCCCAGCACCTGAA GCCGCGGGGGCACCGTCAGTCTTCCTCTT

CCCCCCAAAACCCAAGGACACCCTCATGA TCTCCCGGACCCCTGAGGTCACATGCGTG GTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCG TGGAGGTGCATAATGCCAAGACAAAGCCG

CGGGAGGAGCAGTACAACAGCACGTACCG TGTGGTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAG TGCAAGGTCTCCAACAAAGCCCTCCCAGC CCCCATCGAGAAAACCATCTCCAAAGCCA AAGGGCAGCCCCGAGAACCACAGGTGTAC ACCCTGCCCCCATCCCGGGATGAGCTGAC CAAGAACCAGGTCAGCCTGACCTGCCTGG TCAAAGGCTTCTATCCCAGCGACATCGCC GTGGAGTGGGAGAGCAATGGGCAGCCGGA GAACAACTACAAGACCACGCCTCCCGTGC TGGACTCCGACGGCTCCTTCTTCCTCTAC AGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCG TGATGCATGAGGCTCTGCACAACCACTAC ACGCAGAAGAGCCTCTCCCTGTCTCCGGG TAAA (SEQ ID NO: 65)

Table 9. Mutant IgGl Fc D270 Sequences (D270A mutation underlined)

Nucleic Acid Amino Acid

GCCTCCACCAAGGGCCCATCGGTCTTCCC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF CCTGGCACCCTCCTCCAAGAGCACCTCTG PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS GGGGCACAGCGGCCCTGGGCTGCCTGGTC SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE AAGGACTACTTCCCCGAACCGGTGACGGT PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD GTCGTGGAACTCAGGCGCCCTGACCAGCG TLMI SRTPEVTCVVVDVSHEAPEVKFNWYVDGV GCGTGCACACCTTCCCGGCTGTCCTACAG EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN TCCTCAGGACTCTACTCCCTCAGCAGCGT GKEYKCKVSNKALPAPIEKTISKAKGQPREPQV GGTGACCGTGCCCTCCAGCAGCTTGGGCA YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW CCCAGACCTACATCTGCAACGTGAATCAC ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK AAGCCCAGCAACACCAAGGTGGACAAGAA SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AGTTGAGCCCAAATCTTGTGACAAAACTC (SEQ ID NO: 68)

ACACATGCCCACCGTGCCCAGCACCTGAA CTCCTGGGGGGACCGTCAGTCTTCCTCTT CCCCCCAAAACCCAAGGACACCCTCATGA

TCTCCCGGACCCCTGAGGTCACATGCGTG GTGGTGGACGTGAGCCACGAAGCCCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCG TGGAGGTGCATAATGCCAAGACAAAGCCG CGGGAGGAGCAGTACAACAGCACGTACCG TGTGGTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAG TGCAAGGTCTCCAACAAAGCCCTCCCAGC CCCCATCGAGAAAACCATCTCCAAAGCCA AAGGGCAGCCCCGAGAACCACAGGTGTAC ACCCTGCCCCCATCCCGGGATGAGCTGAC CAAGAACCAGGTCAGCCTGACCTGCCTGG TCAAAGGCTTCTATCCCAGCGACATCGCC GTGGAGTGGGAGAGCAATGGGCAGCCGGA GAACAACTACAAGACCACGCCTCCCGTGC TGGACTCCGACGGCTCCTTCTTCCTCTAC AGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCG TGATGCATGAGGCTCTGCACAACCACTAC ACGCAGAAGAGCCTCTCCCTGTctCCGGG

TAAA (SEQ ID NO: 67)

Table 10. Mutant IgGl Fc P331G Sequences (P331G mutation underlined)

Nucleic Acid Amino Acid

GCCTCCACCAAGGGCCCATCGGTCTTCCC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF CCTGGCACCCTCCTCCAAGAGCACCTCTG PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS GGGGCACAGCGGCCCTGGGCTGCCTGGTC SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE AAGGACTACTTCCCCGAACCGGTGACGGT PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD GTCGTGGAACTCAGGCGCCCTGACCAGCG TLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGV GCGTGCACACCTTCCCGGCTGTCCTACAG EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN TCCTCAGGACTCTACTCCCTCAGCAGCGT GKEYKCKVSNKALPAGIEKTISKAKGQPREPQV GGTGACCGTGCCCTCCAGCAGCTTGGGCA YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW CCCAGACCTACATCTGCAACGTGAATCAC ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK AAGCCCAGCAACACCAAGGTGGACAAGAA SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

AGTTGAGCCCAAATCTTGTGACAAAACTC (SEQ ID NO: 70)

ACACATGCCCACCGTGCCCAGCACCTGAA

CTCCTGGGGGGACCGTCAGTCTTCCTCTT

CCCCCCAAAACCCAAGGACACCCTCATGA

TCTCCCGGACCCCTGAGGTCACATGCGTG

GTGGTGGACGTGAGCCACGAAGACCCTGA

GGTCAAGTTCAACTGGTACGTGGACGGCG

TGGAGGTGCATAATGCCAAGACAAAGCCG

CGGGAGGAGCAGTACAACAGCACGTACCG

TGTGGTCAGCGTCCTCACCGTCCTGCACC

AGGACTGGCTGAATGGCAAGGAGTACAAG

TGCAAGGTCTCCAACAAAGCCCTCCCAGC

CGGCATCGAGAAAACCATCTCCAAAGCCA

AAGGGCAGCCCCGAGAACCACAGGTGTAC

ACCCTGCCCCCATCCCGGGATGAGCTGAC

CAAGAACCAGGTCAGCCTGACCTGCCTGG

TCAAAGGCTTCTATCCCAGCGACATCGCC

GTGGAGTGGGAGAGCAATGGGCAGCCGGA

GAACAACTACAAGACCACGCCTCCCGTGC

TGGACTCCGACGGCTCCTTCTTCCTCTAC

AGCAAGCTCACCGTGGACAAGAGCAGGTG

GCAGCAGGGGAACGTCTTCTCATGCTCCG

TGATGCATGAGGCTCTGCACAACCACTAC

ACGCAGAAGAGCCTCTCCCTGTCTCCGGG

TAAA (SEQ ID NO: 69)

Table 11. Mutant IgGl Fc I253D Sequences (I253D mutation underlined)

GCGTGCACACCTTCCCGGCTGTCCTACAG EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN

TCCTCAGGACTCTACTCCCTCAGCAGCGT GKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

GGTGACCGTGCCCTCCAGCAGCTTGGGCA YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW

CCCAGACCTACATCTGCAACGTGAATCAC ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK

AAGCCCAGCAACACCAAGGTGGACAAGAA SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

AGTTGAGCCCAAATCTTGTGACAAAACTC (SEQ ID NO: 72)

ACACATGCCCACCGTGCCCAGCACCTGAA

CTCCTGGGGGGACCGTCAGTCTTCCTCTT

CCCCCCAAAACCCAAGGACACCCTCATGG

ACTCCCGGACCCCTGAGGTCACATGCGTG

GTGGTGGACGTGAGCCACGAAGACCCTGA

GGTCAAGTTCAACTGGTACGTGGACGGCG

TGGAGGTGCATAATGCCAAGACAAAGCCG

CGGGAGGAGCAGTACAACAGCACGTACCG

TGTGGTCAGCGTCCTCACCGTCCTGCACC

AGGACTGGCTGAATGGCAAGGAGTACAAG

TGCAAGGTCTCCAACAAAGCCCTCCCAGC

CCCCATCGAGAAAACCATCTCCAAAGCCA

AAGGGCAGCCCCGAGAACCACAGGTGTAC

ACCCTGCCCCCATCCCGGGATGAGCTGAC

CAAGAACCAGGTCAGCCTGACCTGCCTGG

TCAAAGGCTTCTATCCCAGCGACATCGCC

GTGGAGTGGGAGAGCAATGGGCAGCCGGA

GAACAACTACAAGACCACGCCTCCCGTGC

TGGACTCCGACGGCTCCTTCTTCCTCTAC

AGCAAGCTCACCGTGGACAAGAGCAGGTG

GCAGCAGGGGAACGTCTTCTCATGCTCCG

TGATGCATGAGGCTCTGCACAACCACTAC

ACGCAGAAGAGCCTCTCCCTGTCTCCGGG

TAAA (SEQ ID NO: 71)

Table 12. Mutant IgGl Fc N297D Sequences (N297D mutation underlined)

Nucleic Acid Amino Acid

GCCTCCACCAAGGGCCCATCGGTCTTCCC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF CCTGGCACCCTCCTCCAAGAGCACCTCTG PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS

GGGGCACAGCGGCCCTGGGCTGCCTGGTC SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE

AAGGACTACTTCCCCGAACCGGTGACGGT PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD

GTCGTGGAACTCAGGCGCCCTGACCAGCG TLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGV

GCGTGCACACCTTCCCGGCTGTCCTACAG EVHNAKTKPREEQYDSTYRVVSVLTVLHQDWLN

TCCTCAGGACTCTACTCCCTCAGCAGCGT GKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

GGTGACCGTGCCCTCCAGCAGCTTGGGCA YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW

CCCAGACCTACATCTGCAACGTGAATCAC ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK

AAGCCCAGCAACACCAAGGTGGACAAGAA SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

AGTTGAGCCCAAATCTTGTGACAAAACTC (SEQ ID NO: 74)

ACACATGCCCACCGTGCCCAGCACCTGAA

CTCCTGGGGGGACCGTCAGTCTTCCTCTT

CCCCCCAAAACCCAAGGACACCCTCATGA

TCTCCCGGACCCCTGAGGTCACATGCGTG

GTGGTGGACGTGAGCCACGAAGACCCTGA

GGTCAAGTTCAACTGGTACGTGGACGGCG

TGGAGGTGCATAATGCCAAGACAAAGCCG

CGGGAGGAGCAGTACGACAGCACGTACCG

TGTGGTCAGCGTCCTCACCGTCCTGCACC

AGGACTGGCTGAATGGCAAGGAGTACAAG

TGCAAGGTCTCCAACAAAGCCCTCCCAGC

CCCCATCGAGAAAACCATCTCCAAAGCCA

AAGGGCAGCCCCGAGAACCACAGGTGTAC

ACCCTGCCCCCATCCCGGGATGAGCTGAC

CAAGAACCAGGTCAGCCTGACCTGCCTGG

TCAAAGGCTTCTATCCCAGCGACATCGCC

GTGGAGTGGGAGAGCAATGGGCAGCCGGA

GAACAACTACAAGACCACGCCTCCCGTGC

TGGACTCCGACGGCTCCTTCTTCCTCTAC

AGCAAGCTCACCGTGGACAAGAGCAGGTG

GCAGCAGGGGAACGTCTTCTCATGCTCCG

TGATGCATGAGGCTCTGCACAACCACTAC

ACGCAGAAGAGCCTCTCCCTGTctCCGGG

TAAA (SEQ ID NO: 73) Table 13. Mutant IgGl Fc L234A/L235A/P331G Sequences (L234A/L235A/P331G mutation underlined)

AGCAAGCTCACCGTGGACAAGAGCAGGTG

GCAGCAGGGGAACGTCTTCTCATGCTCCG TGATGCATGAGGCTCTGCACAACCACTAC ACGCAGAAGAGCCTCTCCCTGTCTCCGGG TAAA (SEQ ID NO: 75)

Table 14. Mutant IgGl Fc L234A/L235A/K322A/P329A/P331G Sequences

(L234A/L235A/K322A/P329A/P331G mutation underlined)

Nucleic Acid Amino Acid

GCCTCCACCAAGGGCCCATCGGTCTTCCC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF CCTGGCACCCTCCTCCAAGAGCACCTCTG PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS GGGGCACAGCGGCCCTGGGCTGCCTGGTC SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE AAGGACTACTTCCCCGAACCGGTGACGGT PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKD GTCGTGGAACTCAGGCGCCCTGACCAGCG TLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGV GCGTGCACACCTTCCCGGCTGTCCTACAG EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN TCCTCAGGACTCTACTCCCTCAGCAGCGT GKEYKCAVSNKALAAGIEKTISKAKGQPREPQV GGTGACCGTGCCCTCCAGCAGCTTGGGCA YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW CCCAGACCTACATCTGCAACGTGAATCAC ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK AAGCCCAGCAACACCAAGGTGGACAAGAA SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AGTTGAGCCCAAATCTTGTGACAAAACTC (SEQ ID NO: 78)

ACACATGCCCACCGTGCCCAGCACCTGAA GCCGCGGGGGGACCGTCAGTCTTCCTCTT

CCCCCCAAAACCCAAGGACACCCTCATGA TCTCCCGGACCCCTGAGGTCACATGCGTG GTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCG TGGAGGTGCATAATGCCAAGACAAAGCCG CGGGAGGAGCAGTACAACAGCACGTACCG TGTGGTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAG TGCGCGGTCTCCAACAAAGCCCTCGCAGC CGGCATCGAGAAAACCATCTCCAAAGCCA AAGGGCAGCCCCGAGAACCACAGGTGTAC ACCCTGCCCCCATCCCGGGATGAGCTGAC CAAGAACCAGGTCAGCCTGACCTGCCTGG

TCAAAGGCTTCTATCCCAGCGACATCGCC

GTGGAGTGGGAGAGCAATGGGCAGCCGGA

GAACAACTACAAGACCACGCCTCCCGTGC

TGGACTCCGACGGCTCCTTCTTCCTCTAC

AGCAAGCTCACCGTGGACAAGAGCAGGTG

GCAGCAGGGGAACGTCTTCTCATGCTCCG

TGATGCATGAGGCTCTGCACAACCACTAC

ACGCAGAAGAGCCTCTCCCTGTCTCCGGG

TAAA (SEQ ID NO: 77)

[0235] In certain embodiments, the IgGl amino acid sequence comprise one or more amino acid substitutions at postions corresponding to positions 14-21 of SEQ ID NOS:62, 64, 66, 68, 70, 72, 74, 76 and 78. In one embodiments, the IgGl amino acid sequence comprises a S14C. In another embodiment, the IgGl amino acid sequence comprises a K16R. In one embodiment, the IgGl amino acid sequence comprises a G20E. In some embodiments, the IgGl amino acid sequence comprises a G21 S. In some embodiments, the IgGl amino acid sequence comprises a S 14C and a K16R. In other embodiments, the IgGl amino acid sequence comprises a S 14C and a G20E. In another embodiment, the IgGl amino acid sequence comprises a S14C and a G21 S. In yet another embodiment, the IgGl amino acid sequence comprises a K16R and a G20E. In some embodiments, the IgGl amino acid sequence comprises a K16R and a G21 S. In certain embodiments, the IgGl amino acid sequence comprises a SG20E and a G21 S. In other embodiments, the IgGl amino acid sequence comprises a S 14C, a K16R and a G20E. In another embodiment, the IgGl amino acid sequence comprises a S 14C, a K16R and a G20S. In some

embodiments, the IgGl amino acid sequence comprises a S14C, a G20E, and a G21 S. In other embodiments, the IgGl amino acid sequence comprises a K16R, a G20E, and a G21S. In certain embodiments, the IgGl amino acid sequence comprises a S14C, a K16R, a G20E, and a G21 S.

[0236] In one embodiment, a human anti-AQP4 IgG antibody or an antigen binding fragment thereof provided herein has a mutated Fc region. In some embodiments, a human anti-AQP4 IgG antibody or an antigen binding fragment thereof provided herein has a mutated antibody Fc region comprising an amino acid substitution selected from the group consisting of a D270A substitution, a P331G substitution, a N297D substitution, and a I253D substitution. In certain embodiments, the antibody is an IgGl antibody. In some embodiments, the IgGl antibody comprises an amino acid substitution selected from the group consisting of a D270A substitution, a P331G substitution, a N297D substitution, and a I253D substitution. Combinations of the aforementioned substitutions are also contemplated. [0237] In certain embodiments, an anti-AQP4 antibody provided herein comprises a mutated human IgGl Fc region having a reduced or otherwise lacking effector function. In certain embodiments, the reduced effector function is a reduction in ADCC activity, CDC activity, or both. In some embodiments, the reduction in effector function is as compared to a reference antibody. In certain embodiments, the reference antibody is the 'parental' antibody that does not comprise the mutations in the human IgGl Fc region (e.g., has a wild type human IgGl Fc region).

[0238] In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises another mutation selected from the group consisting of L234A, L235A, K322A, G237A, I253D, D270A, N297D, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises two mutations selected from the group consisting of L234A, L235A, K322A, G237A, I253D, D270A, N297D, K322A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises three mutations selected from the group consisting of L234A, L235A, K322A, G237A, I253D, D270A, N297D, K322A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises four mutations selected from the group consisting of L234A, L235A, K322A, G237A, I253D, D270A, N297D, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises five mutations selected from the group consisting of L234A, L235A, K322A, G237A, I253D, D270A, N297D, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises six mutations selected from the group consisting of L234A, L235A, K322A, G237A, I253D, D270A, N297D, K322A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises seven mutations selected from the group consisting of L234A, L235A, K322A, G237A, I253D, D270A, N297D, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises eight mutations selected from the group consisting of L234A, L235A, K322A, G237A, I253D, D270A, N297D, K322A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises nine mutations selected from the group consisting of L234A, L235A, K322A, G237A, I253D, D270A, N297D, K322A and P331G.

[0239] In certain embodiments, the mutated human IgG 1 Fc region amino acid sequence does not comprise L234A. In some embodiments, the mutated human IgGl Fc region amino acid sequence does not comprise L235A. In other embodiments, the mutated human IgGl Fc region amino acid sequence does not comprise K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence does not comprise G237A. In some embodiments, the mutated human IgGl Fc region amino acid sequence does not comprise L234A and L235A. In some embodiments, the mutated human IgGl Fc region amino acid sequence does not comprise L234A and K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence does not comprise L234A and G237A. In other embodiments, the mutated human IgGl Fc region amino acid sequence does not comprise L235A and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence does not comprise L235A andG237A. In other embodiments, the mutated human IgGl Fc region amino acid sequence does not comprise G237A and K322A. In yet other embodiments, the mutated human IgGl Fc region amino acid sequence does not comprise L234A, L235A and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence does not comprise L234A, L235A and G237A. In some embodiments, the mutated human IgGl Fc region amino acid sequence does not comprise L234A, G237A and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence does not comprise L235A, G237 and K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence does not comprise L234A, L235A, G237A and K322A.

[0240] In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises another mutation selected from the group consisting of L234A, L235A, K322A, G237A, I253D, D270A, N297D, K322A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises G237A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises I253D. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises D270A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises N297D. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises P331G.

[0241] In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises two mutations selected from the group consisting of L234A, L235A, K322A, G237A, I253D, D270A, N297D, K322A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A and L235A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A and G237A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A and I253D. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A and D270A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A and N297D. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A and G237A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A and I253D. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A and D270A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A and N297D. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A and P331 G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A and G237A. In some embodiments, the mutated human IgG l Fc region amino acid sequence comprises K322A and I253D. In other

embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A and D270A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A and N297D. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A and P331 G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises G237A and I253D. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises G237A and D270A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises G237A and N297D. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises G237A and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises G237A and P331 G. In one embodiment, the mutated human IgG l Fc region amino acid sequence comprises I253D and D270A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises I253D and N297D. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises I253D and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises I253D and P331 G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises D270A and N297D. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises D270A and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises D270A and P331 G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises N297D and K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises N297D and P331 G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A and P331 G.

[0242] In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises three mutations selected from the group consisting of L234A, L235A, K322A, G237A, I253D, D270A, N297D, K322A and P331 G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A and G237A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A and I253D. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A and D270A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A and N297D. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A and G237A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A and I253D. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A and D270A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A and N297D. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A and I253D. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A and D270A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A and N297D. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A and K322A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, I253D and D270A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, I253D and N297D. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, I253D and K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, I253D and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, D270A and N297D. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, D270A and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, D270A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, N297D and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, N297D and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A and G237A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A and I253D. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A and D270A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A and N297D. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A and I253D. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A and D270A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A and N297D. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, I253D and D270A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, I253D and N297D. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, I253D and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, I253D and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, D270A and N297D. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, D270A and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, D270A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, N297D and K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, N297D and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A and I253D. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A and D270A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A and N297D. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A and K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, I253D and D270A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, I253D and N297D. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, I253D and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, I253D and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, D270A and N297D. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, D270A and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, D270A and P331 G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, N297D and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, N297D and P331 G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, K322A and P331 G. In other embodiments, the mutated human IgG l Fc region amino acid sequence comprises G237A, I253D and D270A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises G237A, I253D and N297D. In another embodiment, the mutated human IgG l Fc region amino acid sequence comprises G237A, I253D and K322A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises G237A, I253D and P331 G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises G237A, D270A and N297D. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises G237A, D270A and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises G237A, D270A and P331 G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises G237A, N297D and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises G237A, N297D and P331 G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises G237A, K322A and P331 G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises I253D, D270A and N297D. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises I253D, D270A and K322A. In other embodiments, the mutated human IgG l Fc region amino acid sequence comprises I253D, D270A and P331 G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises I253D, N297D and K322A. In another embodiment, the mutated human IgG l Fc region amino acid sequence comprises I253D, N297D and P331 G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises I253D, K322A and P331 G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises D270A, N297D and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises D270A, N297D and P331 G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises D270A, K322A and P331 G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises N297D, K322A and P331 G.

[0243] In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises four mutations selected from the group consisting of L234A, L235A, K322A, G237A, I253D, D270A, N297D, K322A and P331 G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A and G237A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A and I253D. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A and D270A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A and N297D.

[0244] In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A and I253D. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A and D270A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A and N297D. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, I253D and D270A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, I253D and N297D. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, I253D and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, I253D and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, D270A and N297D. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, D270A and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, D270A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, N297D and K322A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, N297D and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A and I253D. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A and D270A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A and N297D. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A and K322A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, I253D and D270A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, I253D and N297D. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, I253D and K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, I253D and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, D270A and N297D. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, D270A and K322A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, D270A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, N297D and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, N297D and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, K322A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, I253D and D270A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, I253D and N297D. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, I253D and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, I253D and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, D270A and N297D. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, D270A and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, D270A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, N297D and K322A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, N297D and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, I253D, D270A and N297D. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, I253D, D270A and K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, I253D, D270A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, I253D, N297D and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, I253D, N297D and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, I253D, K322A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, D270A, N297D and K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, D270A, N297D and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, D270A, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, N297D, K322A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A and I253D. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A and D270A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A and N297D. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A and K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, I253D and D270A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, I253D and N297D. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, I253D and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, I253D and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, D270A and N297D. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, D270A and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, D270A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, N297D and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, N297D and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, I253D and D270A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, I253D and N297D. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, I253D and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, I253D and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, D270A and N297D. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, D270A and K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, D270A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, N297D and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, N297D and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, K322A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, I253D, D270A and N297D. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, I253D, D270A and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, I253D, D270A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, I253D, N297D and K322A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, I253D, N297D and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, I253D, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, D270A, N297D and K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, D270A, N297D and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, D270A, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, N297D, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, I253D and D270A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, I253D and N297D. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, I253D and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, I253D and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, D270A and N297D. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, D270A and K322A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, D270A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, N297D and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, N297D and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, K322A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, I253D, D270A and N297D. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, I253D, D270A and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, I253D, D270A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, I253D, N297D and K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, I253D, N297D and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, I253D, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, D270A, N297D and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, D270A, N297D and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, D270A, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, N297D, K322A and P331 G. In other embodiments, the mutated human IgG l Fc region amino acid sequence comprises G237A, I253D, D270A and N297D. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises G237A, I253D, D270A and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises G237A, I253D, D270A and P331 G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises G237A, I253D, N297D and K322A. In other embodiments, the mutated human IgG l Fc region amino acid sequence comprises G237A, I253D, N297D and P331 G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises G237A, I253D, K322A and P331 G. In some embodiments, the mutated human IgG l Fc region amino acid sequence comprises G237A, D270A, N297D and K322A. In another embodiment, the mutated human IgG l Fc region amino acid sequence comprises G237A, D270A, N297D and P331 G. In other embodiments, the mutated human IgG l Fc region amino acid sequence comprises G237A, D270A, K322A and P331 G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises G237A, N297D, K322A and P331 G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises I253D, D270A, N297D and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises I253D, D270A, N297D and P331 G. In other embodiments, the mutated human IgG l Fc region amino acid sequence comprises I253D, D270A, K322A and P331 G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises I253D, N297D, K322A and P331 G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises D270A, N297D, K322A and P331 G.

[0245] In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises five mutations selected from the group consisting of L234A, L235A, K322A, G237A, I253D, D270A, N297D, K322A and P331 G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A and I253D. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A and D270A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A and N297D. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A and P331 G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, I253D and D270A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, I253D and N297D. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, I253D and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, I253D and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, D270A and N297D. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, D270A and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, D270A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, N297D and K322A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, N297D and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, I253D and D270A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, I253D and N297D. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, I253D and K322A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, I253D and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, D270A and N297D. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, D270A and K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, D270A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, N297D and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, N297D and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, K322A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, I253D, D270A and N297D. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, I253D, D270A and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, I253D, D270A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, I253D, N297D and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, I253D, N297D and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, I253D, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, D270A, N297D and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, D270A, N297D and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, D270A, K322A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, N297D, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, I253D and D270A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, I253D and N297D. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, I253D and K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, I253D and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, D270A and N297D. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, D270A and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, D270A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, N297D and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, N297D and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, K322A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, I253D, D270A and N297D. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, I253D, D270A and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, I253D, D270A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, I253D, N297D and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, I253D, N297D and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, I253D, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, D270A, N297D and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, D270A, N297D and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, D270A, K322A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, N297D, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, I253D, D270A and N297D. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, I253D, D270A and K322A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, I253D, D270A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, I253D, N297D and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, I253D, N297D and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, I253D, K322A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, D270A, N297D and K322A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, D270A, N297D and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, D270A, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, N297D, K322A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, I253D, D270A, N297D and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, I253D, D270A, N297D and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, I253D, D270A, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, I253D, N297D, K322A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, D270A, N297D, K322A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, I253D and D270A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, I253D and N297D. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, I253D and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, I253D and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, D270A and N297D. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, D270A and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, D270A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, N297D and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, N297D and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, K322A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, I253D, D270A and N297D. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, I253D, D270A and K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, I253D, D270A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, I253D, N297D and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, I253D, N297D and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, I253D, K322A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, D270A, N297D and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, D270A, N297D and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, D270A, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, N297D, K322A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, I253D, D270A and N297D. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, I253D, D270A and K322A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, I253D, D270A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, I253D, N297D and K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, I253D, N297D and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, I253D, K322A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, D270A, N297D and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, D270A, N297D and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, D270A, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, N297D, K322A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, I253D, D270A, N297D and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, I253D, D270A, N297D and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, I253D, D270A, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, I253D, N297D, K322A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, D270A, N297D, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, I253D, D270A and N297D. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, I253D, D270A and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, I253D, D270A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, I253D, N297D and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, I253D, N297D and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, I253D, K322A and P331 G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, D270A, N297D and K322A. In some embodiments, the mutated human IgG l Fc region amino acid sequence comprises K322A, G237A, D270A, N297D and P331 G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, D270A, K322A and P331 G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, N297D, K322A and P331 G. In another embodiment, the mutated human IgG l Fc region amino acid sequence comprises K322A, I253D, D270A, N297D and K322A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, I253D, D270A, N297D and P331 G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, I253D, D270A, K322A and P331 G. In some embodiments, the mutated human IgG l Fc region amino acid sequence comprises K322A, I253D, N297D, K322A and P331 G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, D270A, N297D, K322A and P331 G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises G237A, I253D, D270A, N297D and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises G237A, I253D, D270A, N297D and P331 G. In certain embodiments, the mutated human IgG l Fc region amino acid sequence comprises G237A, I253D, D270A, K322A and P331 G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises G237A, I253D, N297D, K322A and P331 G. In some embodiments, the mutated human IgG l Fc region amino acid sequence comprises G237A, D270A, N297D, K322A and P331 G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises I253D, D270A, N297D, K322A and P331 G.

[0246] In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises six mutations selected from the group consisting of L234A, L235A, K322A, G237A, I253D, D270A, N297D, K322A and P331 G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, I253D and D270A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, I253D and N297D. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, I253D and K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, I253D and P331 G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, D270A and N297D. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, D270A and K322A. In other embodiments, the mutated human IgG l Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, D270A and P331 G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, N297D and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, N297D and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, I253D, D270A and N297D. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, I253D, D270A and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, I253D, D270A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, I253D, N297D and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, I253D, N297D and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, I253D, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, D270A, N297D and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, D270A, N297D and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, D270A, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, N297D, K322A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, I253D, D270A and N297D. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, I253D, D270A and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, I253D, D270A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, I253D, N297D and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, I253D, N297D and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, I253D, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, D270A, N297D and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, D270A, N297D and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, D270A, K322A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, N297D, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, I253D, D270A, N297D and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, I253D, D270A, N297D and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, I253D, D270A, K322A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, I253D, N297D, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, D270A, N297D, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, I253D, D270A and N297D. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, I253D, D270A and K322A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, I253D, D270A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, I253D, N297D and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, I253D, N297D and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, I253D, K322A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, D270A, N297D and K322A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, D270A, N297D and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, D270A, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, N297D, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, I253D, D270A, N297D and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, I253D, D270A, N297D and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, I253D, D270A, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, I253D, N297D, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, D270A, N297D, K322A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, I253D, D270A, N297D and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, I253D, D270A, N297D and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, I253D, D270A, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, I253D, N297D, K322A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, D270A, N297D, K322A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, I253D, D270A, N297D, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, I253D, D270A and N297D. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, I253D, D270A and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, I253D, D270A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, I253D, N297D and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, I253D, N297D and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, I253D, K322A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, D270A, N297D and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, D270A, N297D and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, D270A, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, N297D, K322A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, I253D, D270A, N297D and K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, I253D, D270A, N297D and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, I253D, D270A, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, I253D, N297D, K322A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, D270A, N297D, K322A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, I253D, D270A, N297D and K322A. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, I253D, D270A, N297D and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, I253D, D270A, K322A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, I253D, N297D, K322A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, D270A, N297D, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, I253D, D270A, N297D, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, I253D, D270A, N297D and K322A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, I253D, D270A, N297D and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, I253D, D270A, K322A and P331G. In one embodiment, the mutated human IgG l Fc region amino acid sequence comprises K322A, G237A, I253D, N297D, K322A and P331 G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, D270A, N297D, K322A and P331 G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises K322A, I253D, D270A, N297D, K322A and P331 G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises G237A, I253D, D270A, N297D, K322A and P331 G.

[0247] In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises seven mutations selected from the group consisting of L234A, L235A, K322A, G237A, I253D, D270A, N297D, K322A and P331 G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, I253D, D270A and N297D. In another

embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, I253D, D270A and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, I253D, D270A and P331 G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, I253D, N297D and K322A. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, I253D, N297D and P331 G. In another

embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, I253D, K322A and P331 G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, D270A, N297D and K322A. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, D270A, N297D and P331 G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, D270A, K322A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, N297D, K322A and P331 G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, I253D, D270A, N297D and K322A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, I253D, D270A, N297D and P331 G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, I253D, D270A, K322A and P331 G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, I253D, N297D, K322A and P331 G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, D270A, N297D, K322A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, I253D, D270A, N297D and K322A. In other embodiments, the mutated human IgG l Fc region amino acid sequence comprises L234A, L235A, G237A, I253D, D270A, N297D and P331 G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, I253D, D270A, K322A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, I253D, N297D, K322A and P331G. In one

embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, D270A, N297D, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, I253D, D270A, N297D, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, I253D, D270A, N297D and K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, I253D, D270A, N297D and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, I253D, D270A, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, I253D, N297D, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, D270A, N297D, K322A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, I253D, D270A, N297D, K322A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, G237A, I253D, D270A, N297D, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, I253D, D270A, N297D and K322A. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, I253D, D270A, N297D and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, I253D, D270A, K322A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, I253D, N297D, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, D270A, N297D, K322A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, I253D, D270A, N297D, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, G237A, I253D, D270A, N297D, K322A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises K322A, G237A, I253D, D270A, N297D, K322A and P331G.

[0248] In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises eight mutations selected from the group consisting of L234A, L235A, K322A, G237A, I253D, D270A, N297D, K322A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, I253D, D270A, N297D and K322A. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, I253D, D270A, N297D and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, I253D, D270A, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, I253D, N297D, K322A and P331G. In certain embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, D270A, N297D, K322A and P331G. In some embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, I253D, D270A, N297D, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, G237A, I253D, D270A, N297D, K322A and P331G. In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises L234A, K322A, G237A, I253D, D270A, N297D, K322A and P331G. In one embodiment, the mutated human IgGl Fc region amino acid sequence comprises L235A, K322A, G237A, I253D, D270A, N297D, K322A and P331G.

[0249] In another embodiment, the mutated human IgGl Fc region amino acid sequence comprises nine mutations selected from the group consisting of L234A, L235A, K322A, G237A, I253D, D270A, N297D, K322A and P331G. In other embodiments, the mutated human IgGl Fc region amino acid sequence comprises L234A, L235A, K322A, G237A, I253D, D270A, N297D, K322A and P331G.

[0250] In one aspect, provided herein is a human anti-AQP4 IgG antibody or antigen binding fragment thereof having a mutated Fc region, wherein said mutated Fc region comprises a D270A amino acid substitution.

[0251] In one aspect, provided herein is a human anti-AQP4 IgG antibody or antigen binding fragment thereof having a mutated Fc region, wherein said mutated Fc region comprises a P331G amino acid substitution.

[0252] In another aspect, provided herein is a human anti-AQP4 IgG antibody or antigen binding fragment thereof having a mutated Fc region, wherein said mutated Fc region comprises a N297D amino acid substitution.

[0253] In yet another aspect, provided herein is a human anti-AQP4 IgG antibody or antigen binding fragment thereof having a mutated Fc region, wherein said mutated Fc region comprises a I253D amino acid substitution.

[0254] In some embodiments, provided herein is a human anti-AQP4 IgG antibody or antigen binding fragment thereof having a mutated Fc region, wherein said mutated Fc region comprises a P331G amino acid substitution and at least one additional amino acid substitution, said additional amino acid substitution being a L234A substitution, a L235A substitution, or L234A/L235A substitutions. In one aspect, the human anti-AQP4 IgG antibody or antigen binding fragment thereof comprises P331G/ L234A substitutions. In another aspect, the human anti-AQP4 IgG antibody or antigen binding fragment

- I l l - thereof comprises P331G/ L235A substitution. In yet another aspect, the human anti-AQP4 IgG antibody or antigen binding fragment thereof comprises P331G/ L234A/L235A substitutions.

[0255] In some other embodiments, provided herein is a human anti-AQP4 IgG antibody or antigen binding fragment thereof having a mutated Fc region, wherein said mutated Fc region comprises P331G/ L234A/L235A amino acid substitutions, and at least one additional amino acid substitution, said additional amino acid substitution being a K322A substitution, a P329A substitution, or K322A/P329A substitutions. In one aspect, the human anti-AQP4 IgG antibody or antigen binding fragment thereof comprises P331G/ L234A/L235A/K322A amino acid substitutions. In another aspect, the human anti- AQP4 IgG antibody or antigen binding fragment thereof comprises P331G/ L234A/L235A/P329A amino acid substitutions. In yet another aspect, the human anti-AQP4 IgG antibody or antigen binding fragment thereof comprises P331G/ L234A/L235A/ P329A/K322A amino acid substitutions.

[0256] As those skilled in the art will appreciate, antibody sequences varying from the sequences provided above are also contemplate, optionally using methods discussed in greater detail below. For example, nucleic acid sequences can vary from those set forth above in that (a) the variable regions can be segregated away from the constant domains of the light chains, (b) the nucleic acids can vary from those set out above while not affecting the residues encoded thereby, (c) the nucleic acids can vary from those set out above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, (d) the nucleic acids can vary from those set out above by virtue of the ability to hybridize under high stringency conditions, e.g., 65 °C, 50% formamide, 0.1 x SSC, 0.1% SDS, (e) the amino acids can vary from those set out above by a given percentage, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, or (f) the amino acids can vary from those set out above by permitting conservative substitutions (discussed below). In certain embodiments, the antibody has further amino acid substitutions, e.g., from 1 to 50 amino acid substitutions, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 10, from 1 to 5, or from 1 to 3 amino acid substitutions. In some embodiments, one or more, and up to all, of the amino acid substitutions is a conservative amino acid substitution.

[0257] In some embodiments, provided is a human anti-AQP4 IgG antibody or an antigen binding fragment thereof having a mutated Fc region, wherein said mutated Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:68; SEQ ID NO:70; SEQ ID NO:72; SEQ ID NO:74; SEQ ID NO:76; and SEQ ID NO:78. In one embodiment, the mutated Fc regions comprises an amino acid sequence of SEQ ID NO:68, or a sequence having 95%, 90%, 85%, or 80% identity to SEQ ID NO:68. In one embodiment, the mutated Fc region comprises an amino acid sequence of SEQ ID NO:70, or a sequence having 95%, 90%, 85%, or 80% identity to SEQ ID NO:70. In one embodiment, the mutated Fc region comprises an amino acid sequence of SEQ ID NO: 72, or a sequence having 95%, 90%, 85%, or 80% identity to SEQ ID NO:72. In one embodiment, the mutated Fc region comprises an amino acid sequence of SEQ ID NO:74, or a sequence having 95%, 90%, 85%, or 80% identity to SEQ ID NO:74. In one embodiment, the mutated Fc region comprises an amino acid sequence of SEQ ID NO:76, or a sequence having 95%, 90%, 85%, or 80% identity to SEQ ID NO:76. In another embodiment, the mutated Fc region comprises an amino acid sequence of SEQ ID NO:78, or a sequence having 95%, 90%, 85%, or 80% identity to SEQ ID NO:78.

[0258] In other embodiments, provided herein is a human anti-AQP4 IgG antibody or an antigen binding fragment thereof comprising a mutated Fc region, wherein said mutated Fc region is encoded by a nucleic acid selected from the group consisting of SEQ ID NO:67; SEQ ID NO:69; SEQ ID NO:71 ; SEQ ID NO:73; SEQ ID NO:75; and SEQ ID NO:77. In one embodiment, the mutated Fc region is encoded by the nucleic acid of SEQ ID NO:67, or a nucleic acid that is 95%, 90%, 85%, 80%, 75%, 70% identity to SEQ ID NO:67. In one embodiment, the mutated Fc region is encoded by the nucleic acid of SEQ ID NO:69, or a nucleic acid that is 95%, 90%, 85%, 80%, 75%, 70% identity to SEQ ID NO:69. In one embodiment, the mutated Fc region is encoded by the nucleic acid of SEQ ID NO:71, or a nucleic acid that is 95%, 90%, 85%, 80%, 75%, 70% identity to SEQ ID NO:71. In one embodiment, the mutated Fc region is encoded by the nucleic acid of SEQ ID NO:73, or a nucleic acid that is 95%, 90%, 85%, 80%, 75%, 70% identity to SEQ ID NO: 73. In one embodiment, the mutated Fc region is encoded by the nucleic acid of SEQ ID NO:75, or a nucleic acid that is 95%, 90%, 85%, 80%, 75%, 70% identity to SEQ ID NO: 75. In another embodiment, the mutated Fc region is encoded by the nucleic acid of SEQ ID NO:77, or a nucleic acid that is 95%, 90%, 85%, 80%, 75%, 70% identity to SEQ ID NO:77.

[0259] In certain embodiments, an antibody provided herein comprises a mutated Fc region having an amino acid sequence of SEQ ID NO: 62. In other embodiments, an antibody provided herein comprises a mutated Fc region having an amino acid sequence of SEQ ID NO:64. In some embodiments, an antibody provided herein comprises a mutated Fc region having an amino acid sequence of SEQ ID NO:66. In certain embodiments, an antibody provided herein comprises a mutated Fc region having an amino acid sequence of SEQ ID NO:68. In some embodiments, an antibody provided herein comprises a mutated Fc region having an amino acid sequence of SEQ ID NO:70. In other embodiments, an antibody provided herein comprises a mutated Fc region having an amino acid sequence of SEQ ID NO:72. In certain embodiments, an antibody provided herein comprises a mutated Fc region having an amino acid sequence of SEQ ID NO:74. In some embodiments, an antibody provided herein comprises a mutated Fc region having an amino acid sequence of SEQ ID NO:76. In other embodiments, an antibody provided herein comprises a mutated Fc region having an amino acid sequence of SEQ ID NO:78.

[0260] In certain embodiments, the antibody having a mutated Fc region further comprises a VH domain of SEQ ID NO:2. In some embodiments, the antibody having a mutated Fc region further comprises a VH domain of SEQ ID NO: 8. In other embodiments, the antibody having a mutated Fc region further comprises a VH domain of SEQ ID NO: 14. In certain embodiments, the antibody having a mutated Fc region further comprises a VL domain of SEQ ID NO:4. In some embodiments, the antibody having a mutated Fc region further comprises a VL domain of SEQ ID NO: 10. In other embodiments, the antibody having a mutated Fc region further comprises a VL domain of SEQ ID NO: 16. In some embodiments, the antibody having a mutated Fc region further comprises a VH domain of SEQ ID NO:2 and a VL domain of SEQ ID NO:4. In some embodiments, the antibody having a mutated Fc region further comprises a VH domain of SEQ ID NO:8 and a VL domain of SEQ ID NO: 10. In some embodiments, the antibody having a mutated Fc region further comprises a VH domain of SEQ ID NO: 14 and a VL domain of SEQ ID NO: 16. In one embodiment, the antibody having a mutated Fc region further comprises a VH CDRl of SEQ ID NO:22, a VH CDR2 of SEQ ID NO:24 and VH CDR3 of SEQ ID NO:26. In some embodiments, the antibody having a mutated Fc region further comprises a VL CDRl of SEQ ID NO:28, a VL CDR2 of SEQ ID NO:30 and VL CDR3 of SEQ ID NO:32. In another

embodiment, the antibody having a mutated Fc region further comprises a VH CDRl of SEQ ID NO:22, a VH CDR2 of SEQ ID NO:24, a VH CDR3 of SEQ ID NO:26, a VL CDRl of SEQ ID NO:28, a VL CDR2 of SEQ ID NO:30 and VL CDR3 of SEQ ID NO:32. In one embodiment, the antibody having a mutated Fc region further comprises a VH CDRl of SEQ ID NO:22, a VH CDR2 of SEQ ID NO:24, and VL domain of SEQ ID NO:4. In some embodiments, the antibody having a mutated Fc region further comprises a VH domain of SEQ ID NO:2, and a VL CDRl of SEQ ID NO:28, a VL CDR2 of SEQ ID NO:30 and VL CDR3 of SEQ ID NO:32. In one embodiment, the antibody having a mutated Fc region further comprises a VH CDRl of SEQ ID NO:34, a VH CDR2 of SEQ ID NO:36 and VH CDR3 of SEQ ID NO:28. In some embodiments, the antibody having a mutated Fc region further comprises a VL CDRl of SEQ ID NO:40, a VL CDR2 of SEQ ID NO:42 and VL CDR3 of SEQ ID NO:44. In another embodiment, the antibody having a mutated Fc region further comprises a VH CDRl of SEQ ID NO:34, a VH CDR2 of SEQ ID NO:36, a VH CDR3 of SEQ ID NO:38, a VL CDRl of SEQ ID NO:40, a VL CDR2 of SEQ ID NO:42 and VL CDR3 of SEQ ID NO:48. In one embodiment, the antibody having a mutated Fc region further comprises a VH CDRl of SEQ ID NO:34, a VH CDR2 of SEQ ID NO:36, and VL domain of SEQ ID NO: 10. In some embodiments, the antibody having a mutated Fc region further comprises a VH domain of SEQ ID NO:8, and a VL CDRl of SEQ ID NO:40, a VL CDR2 of SEQ ID NO:42 and VL CDR3 of SEQ ID NO:44. In one embodiment, the antibody having a mutated Fc region further comprises a VH CDRl of SEQ ID NO:46, a VH CDR2 of SEQ ID NO:48 and VH CDR3 of SEQ ID NO:50. In some embodiments, the antibody having a mutated Fc region further comprises a VL CDRl of SEQ ID NO:52, a VL CDR2 of SEQ ID NO:54 and VL CDR3 of SEQ ID NO:56. In another embodiment, the antibody having a mutated Fc region further comprises a VH CDRl of SEQ ID NO:46, a VH CDR2 of SEQ ID NO:48 and VH CDR3 of SEQ ID NO:50, a VL CDR1 of SEQ ID NO:52, a VL CDR2 of SEQ ID NO:54 and VL CDR3 of SEQ ID NO:56. In one embodiment, the antibody having a mutated Fc region further comprises a VH CDR1 of SEQ ID NO:46, a VH CDR2 of SEQ ID NO:48 and VH CDR3 of SEQ ID NO:50, and VL domain of SEQ ID NO: 16. In some embodiments, the antibody having a mutated Fc region further comprises a VH domain of SEQ ID NO: 14, and a VL CDR1 of SEQ ID NO:52, a VL CDR2 of SEQ ID NO:54 and VL CDR3 of SEQ ID NO:56. Other VH domains, VL domains, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences of antibodies having a mutated Fc region are also contemplated and provided elsewhere herein.

5.3 Methods for Producing Monoclonal Antibodies

5.3.1. General Methods

[0261] It will be understood that monoclonal antibodies binding to AQP4 will have utilities in several applications. These include the production of diagnostic kits for use in detecting and diagnosing NMO, as well as for treating NMO. In these contexts, one can link such antibodies to diagnostic or therapeutic agents, use them as capture agents or competitors in competitive assays, or use them individually without additional agents being attached thereto. The antibodies can be mutated or modified, as discussed further below. Methods for preparing and characterizing antibodies are well known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Patent

4,196,265).

[0262] Antibodies provided herein that bind to an antigen can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression techniques. The practice of the invention employs, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described in the references cited herein and are fully explained in the literature. See, e.g.,, Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual. Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al. , Current

Protocols in Molecular Biology. John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology. John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984) Oligonucleotide

Synthesis: A Practical Approach. IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach. IRL Press; Birren et al. (eds.) (1999) Genome Analysis: A Laboratory Manual. Cold Spring Harbor Laboratory Press. Additional examples of suitable texts for consultation include the following: D.N Glover, ed. (1985) DNA Cloning. Volumes I and II; M.J. Gait, ed. (1984) Oligonucleotide Synthesis; B.D. Hames & SJ. Higgins, eds. (1984) Nucleic Acid Hybridization; B.D. Hames & S.J. Higgins, eds. (1984) Transcription and Translation; R.I. Freshney, ed. (1986) Animal Cell Culture; Immobilized Cells and Enzymes (IRL Press, 1986); Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes (1987) Protein Purification: Principles and Practice (2d ed.; Springer Verlag, N.Y.); and D.M. Weir and C. C. Blackwell, eds. (1986) Handbook of Experimental Immunology, Volumes I-IV.

[0263] The methods for generating monoclonal antibodies (mAbs) generally begin along the same lines as those for preparing polyclonal antibodies. The first step for both these methods is immunization of an appropriate host or identification of subjects who are immune due to prior natural infection. As is well known in the art, a given composition for immunization can vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as can be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde and bis-biazotized benzidine. As also is well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.

[0264] The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies can be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, also can be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.

[0265] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al. , Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al, in: Monoclonal Antibodies and T-Cell Hvbridomas 563 681 (Elsevier, N.Y., 1981); Campbell, In: Monoclonal Antibody Technology, Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Burden and Von Knippenberg, Eds. pp. 75 83, Amsterdam, Elsevier, 1984 (said references incorporated by reference in their entireties). The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. Other exemplary methods of producing monoclonal antibodies are discussed elsewhere herein, such as e.g. , use of the KM mouse™. Additional exemplary methods of producing monoclonal antibodies are provided herein.

[0266] Monoclonal antibodies can be made using the hybridoma method first described by Kohler et al, Nature, 256:495 (1975) (see also, e.g., Kohler and Milstein, Eur. J. Immunol., 6, 51 1-519, 1976), or can be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59- 103 (Academic Press, 1986); Posner et al., Hybridoma 6, 61 1-625, 1987; Gefter et al., Somatic Cell Genet, 3:231 236, 1977).

[0267] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium which medium can, in certain embodiments, contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

[0268] In some embodiments, fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parental cells. In other embodiments, myeloma cell lines are murine myeloma lines, such as SP-2 and derivatives, for example, X63-Ag8-653 cells available from the American Type Culture Collection, Manassas, Virginia, USA and those derived from MOPC-21 and MPC- 1 1 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J., Immunol, 133:3001 (1984); and Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0269] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., Anal. Biochem., 107:220 (1980).

[0270] Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59- 103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI- 1640 medium. In addition, the hybridoma cells can be grown in vivo as ascites tumors in an animal, for example, by i.p. injection of the cells into mice.

[0271] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography {e.g., using protein A or protein G-Sepharose) or ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.

[0272] DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et ah, Curr. Opinion in Immunol, 5:256-262 (1993) and Pluckthun, Immunol. Revs. 130: 151-188 (1992).

[0273] In some embodiments, an antibody that binds an AQP4 epitope comprises an amino acid sequence of a VH domain and/or an amino acid sequence of a VL domain encoded by a nucleotide sequence that hybridizes to (1) the complement of a nucleotide sequence encoding any one of the VH and/or VL domain described herein under stringent conditions (e.g., hybridization to filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about 45° C followed by one or more washes in

0.2xSSC/0.1% SDS at about 50-65° C) under highly stringent conditions (e.g., hybridization to filter- bound nucleic acid in 6xSSC at about 45° C followed by one or more washes in 0. lxSSC/0.2% SDS at about 68° C), or under other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel, F.M. et ah, eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3). [0274] In some embodiments, an antibody that binds an AQP4 epitope comprises an amino acid sequence of a VH CDR or an amino acid sequence of a VL CDR encoded by a nucleotide sequence that hybridizes to the complement of a nucleotide sequence encoding any one of the VH CDRs and/or VL CDRs depicted in Tables 4 and 5, respectively, under stringent conditions (e.g., hybridization to filter- bound DNA in 6X SSC at about 45° C followed by one or more washes in 0.2X SSC/0.1% SDS at about 50-65° C), under highly stringent conditions (e.g., hybridization to filter-bound nucleic acid in 6X SSC at about 45° C followed by one or more washes in 0. IX SSC/0.2% SDS at about 68° C), or under other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel, F.M. et al, eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3).

[0275] In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in, for example, Antibody Phage Display: Methods and Protocols, P.M. O'Brien and R. Aitken, eds, Humana Press, Totawa N.J., 2002. Other examples of phage display methods that can be used to make the antibodies provided herein include those disclosed in Brinkman et al, 1995, J. Immunol. Methods 182:41-50; Ames et al, 1995, J. Immunol. Methods 184: 177-186; Kettleborough et al, 1994, Eur. J. Immunol. 24:952-958; Persic et al, 1997, Gene 187:9-18; Burton et al, 1994, Advances in Immunology 57: 191-280; PCT Application No.

PCT/GB91/01 134; International Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401, and W097/13844; and U.S. Patent Nos.

5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

[0276] In principle, synthetic antibody clones are selected by screening phage libraries containing phage that display various fragments of antibody variable region (Fv) fused to phage coat protein. Such phage libraries are screened for against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from the non-binding clones in the library. The binding clones are then eluted from the antigen, and can be further enriched by additional cycles of antigen adsorption/elution.

[0277] Variable domains can be displayed functionally on phage, either as single-chain Fv (scFv) fragments, in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non-covalently, as described, for example, in Winter et αΙ., Αηη. Rev. Immunol, 12: 433-455 (1994).

[0278] Repertoires of VH and VL genes can be separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be searched for antigen-binding clones as described in Winter et al, supra. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned to provide a single source of human antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al, EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning the unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described, for example, by

Hoogenboom and Winter, J. Mol. Biol, 227: 381-388 (1992).

[0279] Screening of the libraries can be accomplished by various techniques known in the art. For example, AQP4, (e.g., an AQP4 polypeptide, fragment or epitope) can be used to coat the wells of adsorption plates, expressed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavidin-coated beads, or used in any other method for panning display libraries. The selection of antibodies with slow dissociation kinetics (e.g., good binding affinities) can be promoted by use of long washes and monovalent phage display as described in Bass et al, Proteins, 8: 309-314 (1990) and in WO 92/09690, and a low coating density of antigen as described in Marks et al, Biotechnol, 10: 779-783 (1992).

[0280] Anti-AQP4 antibodies can be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length anti-AQP4 antibody clone using VH and/or VL sequences (e.g., the Fv sequences), or various CDR sequences from VH and VL sequences, from the phage clone of interest and suitable constant region (e.g., Fc) sequences described in Kabat et al, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

[0281] For some uses, including in vivo use of antibodies in humans and in vitro detection assays, human or chimeric antibodies can be used. Completely human antibodies are particularly desirable for therapeutic treatment of human subjects. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887 and 4,716,1 1 1 ; and International Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741 ; each of which is incorporated herein by reference in its entirety.

[0282] In certain embodiments, human antibodies are produced. Human antibodies and/or fully human antibodies can be produced using any method known in the art. For example, transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered non functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the J_H region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide provided herein. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publication Nos. WO 98/24893, WO

96/34096, and WO 96/33735; and U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, which are incorporated by reference herein in their entirety. Other methods are detailed in the Examples herein. In addition, companies such as Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

[0283] A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, Science, 229(4719): 1202- 1207, 1985; Oi et al, 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125: 191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331 ,415, which are incorporated herein by reference in their entirety.

[0284] A humanized antibody is an antibody or its variant or fragment thereof which is capable of binding to a predetermined antigen and which comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, Fabc, Fv) in which all or substantially all of the CDR regions correspond to those of a non human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In certain embodiments,, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Ordinarily, the antibody will contain both the light chain as well as at least the variable domain of a heavy chain. The antibody also can include the CHI , hinge, CH2, CH3, and CH4 regions of the heavy chain. The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGl , IgG2, IgG3 and IgG4. Usually the constant domain is a complement fixing constant domain where it is desired that the humanized antibody exhibit cytotoxic activity, and the class is typically IgGl . Where such cytotoxic activity is not desirable, the constant domain can be of the IgG2 class. Examples of VL and VH constant domains that can be used in certain embodiments provided herein include, but are not limited to, C-kappa and C-gamma- 1 (nGlm) described in Johnson et al. (1997) J. Infect. Dis. 176, 1215- 1224 and those described in U.S. Patent No. 5,824,307. The humanized antibody can comprise sequences from more than one class or isotype, and selecting particular constant domains to optimize desired effector functions is within the ordinary skill in the art. The framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor CDR or the consensus framework can be mutagenized by substitution, insertion or deletion of at least one residue so that the CDR or framework residue at that site does not correspond to either the consensus or the import antibody. Such mutations, however, will not be extensive. Usually, at least 75% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences, more often 90%, such as greater than 95%. Humanized antibodies can be produced using variety of techniques known in the art, including but not limited to, CDR-grafting (European Patent No. EP 239,400;

International publication No. WO 91/09967; and U.S. Patent Nos. 5,225,539, 5,530, 101 , and 5,585,089), veneering or resurfacing (European Patent Nos. EP 592, 106 and EP 519,596; Padlan, 1991 , Molecular Immunology 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al., 1994, PNAS 91 :969-973), chain shuffling (U.S. Patent No. 5,565,332), and techniques disclosed in, e.g., U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, WO 9317105, Tan et al , J. Immunol. 169: 1 1 19 25 (2002), Caldas et al, Protein Eng. 13(5):353-60 (2000), Morea et al , Methods 20(3):267 79 (2000), Baca et al , J. Biol. Chem. 272(16): 10678-84 (1997), Roguska et al, Protein Eng. 9(10):895 904 (1996), Couto et al , Cancer Res. 55 (23 Supp):5973s- 5977s (1995), Couto et al, Cancer Res. 55(8): 1717-22 (1995), Sandhu JS, Gene 150(2):409- 10 (1994), and Pedersen et al , J. Mol. Biol. 235(3):959-73 (1994). See also U.S. Patent Pub. No. US 2005/0042664 Al (Feb. 24, 2005), which is incorporated by reference herein in its entirety. Often, framework residues in the framework regions will be substituted with the

corresponding residue from the CDR donor antibody to alter or improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al, U.S. Patent No. 5,585,089; and Reichmann et al, 1988, Nature 332:323, which are incorporated herein by reference in their entireties.)

[0285] The present disclosure provides antibodies and antibody fragments that bind to AQP4. In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and can lead to improved access to cells, tissues or organs. For a review of certain antibody fragments, see Hudson et al. (2003) Nat. Med. 9: 129-134.

[0286] Various techniques have been developed for the production of antibody fragments.

Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g. , Morimoto et al, Journal of Biochemical and Biophysical Methods 24: 107- 1 17 (1992); and Brennan et al, Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. coli or yeast cells, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')₂ fragments (Carter et al,

Bio/Technology 10: 163-167 (1992)). According to another approach, F(ab')₂ fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')₂ fragment with increased in vivo half-life comprising salvage receptor binding epitope residues are described, for example, U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In certain embodiments, an antibody is a single chain Fv fragment (scFv) (see, e.g., WO 93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458). Fv and scFv have intact combining sites that are devoid of constant regions; thus, they can be suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins can be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv. (See, e.g., Antibody Engineering, ed. Borrebaeck, supra). The antibody fragment can also be a "linear antibody", for example, as described, for example, in the references cited above. Such linear antibodies can be monospecific or multi-specific, such as bispecific.

[0287] Smaller antibody-derived binding structures are the separate variable domains (V domains) also termed single variable domain antibodies (SdAbs). Single domain antibodies, for example, antibodies lacking the light chains, can be produced by methods well-known in the art. See Riechmann et al, 1999, J. Immunol. 231 :25-38; Nuttall et al, 2000, Curr. Pharm. Biotechnol. l(3):253-263;

Muylderman, 2001, J. Biotechnol. 74(4):277302; U.S. Patent No. 6,005,079; and International

Publication Nos. WO 94/04678, WO 94/25591, and WO 01/44301, each of which is incorporated herein by reference in its entirety. Certain types of organisms, the camelids and cartilaginous fish, possess high affinity single V-like domains mounted on an Fc equivalent domain structure as part of their immune system. (Woolven et al. , Immunogenetics 50: 98-101, 1999; Streltsov et al, Proc Natl Acad Sci USA. 101 : 12444-12449, 2004). The V-like domains (called VhH in camelids and V-NAR in sharks) typically display long surface loops, which allow penetration of cavities of target antigens. They also stabilize isolated VH domains by masking hydrophobic surface patches.

[0288] These VhH and V-NAR domains have been used to engineer sdAbs. Human V domain variants have been designed using selection from phage libraries and other approaches that have resulted in stable, high binding VL- and VH-derived domains.

[0289] Antibodies that bind to AQP4 as provided herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments, (e.g., AQP4 binding fragments) of any of the above. Non- limiting examples of functional fragments (e.g. , fragments that bind to AQP4) include single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody and minibody.

[0290] Antibodies provided herein include, but are not limited to, immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, molecules that contain an antigen binding site that bind to an AQP4 epitope. The immunoglobulin molecules provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.

[0291] The recombinant antibodies provided herein are producing using single plasmablasts or b cells isolated from the CSF of affected individuals. Blood can also be used, although plasmablasts are somewhat less prevalent in that fluid. The antibody heavy and light chain sequences are identified by RT-PCR. The identified pair of heavy and light chain are then reengineered using standard cloning techniques into expression vectors and transfected into mammalian cell lines to produce antibody.

[0292] mAbs produced by either means can be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as FPLC or affinity chromatography.

Fragments of the monoclonal antibodies provided herein can be obtained from the purified monoclonal antibodies by methods which include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments provided herein can be synthesized using an automated peptide synthesizer.

[0293] It also is contemplated that a molecular cloning approach can be used to generate monoclonals. For this, RNA can be isolated from the hybridoma line and the antibody genes obtained by RT-PCR and cloned into an immunoglobulin expression vector. Alternatively, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the cell lines and phagemids expressing appropriate antibodies are selected by panning using viral antigens. The advantages of this approach over conventional hybridoma techniques are that approximately 10⁴ times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies.

[0294] Other U.S. patents, each incorporated herein by reference, that teach the production of antibodies useful in the present invention include U.S. Patent 5,565,332, which describes the production of chimeric antibodies using a combinatorial approach; U.S. Patent 4,816,567 which describes recombinant immunoglobulin preparations; and U.S. Patent 4,867,973 which describes

antibody-therapeutic agent conjugates, herein.

5.3.2. Engineering of Antibody Sequences

[0295] In various embodiments, one can choose to engineer sequences of the identified antibodies for a variety of reasons, such as improved expression, improved cross-reactivity, diminished off-target binding or abrogation of one or more natural effector functions, such as activation of complement or recruitment of immune cells (e.g., T cells, monocytes or NK cells). The following is a general discussion of relevant techniques for antibody engineering.

[0296] Hybridomas can cultured, then cells lysed, and total RNA extracted. Random hexamers can be used with RT to generate cDNA copies of RNA, and then PCR performed using a multiplex mixture of PCR primers expected to amplify all human variable gene sequences. PCR product can be cloned into pGEM-T Easy vector, then sequenced by automated DNA sequencing using standard vector primers. Assay of binding and neutralization can be performed using antibodies collected from hybridoma supernatants and purified by FPLC, using Protein G columns.

[0297] Recombinant full length IgG antibodies can be generated by subcloning heavy and light chain Fv DNAs from the cloning vector into a Lonza pConlgGl or pConK2 plasmid vector, transfected into 293 Freestyle cells or Lonza CHO cells, and antibodies were collected an purified from the CHO cell supernatant. Other methods are described in Bennett et al. (2009).

[0298] The rapid availability of antibody produced in the same host cell and cell culture process as the final cGMP manufacturing process has the potential to reduce the duration of process development programs. Lonza has developed a generic method using pooled transfectants grown in CDACF medium, for the rapid production of small quantities (up to 50 g) of antibodies in CHO cells. Although slightly slower than a true transient system, the advantages include a higher product concentration and use of the same host and process as the production cell line. Example of growth and productivity of GS-CHO pools, expressing a model antibody, in a disposable bioreactor: in a disposable bag bioreactor culture (5 L working volume) operated in fed-batch mode, a harvest antibody concentration of 2 g/L was achieved within 9 weeks of transfection. [0299] pCon Vectors are an easy way to re-express whole antibodies. The constant region vectors are a set of vectors offering a range of immunoglobulin constant region vectors cloned into the pEE vectors. These vectors offer easy construction of full length antibodies with human constant regions and the convenience of the GS System™.

[0300] Antibody molecules will comprise fragments (such as F(ab'), F(ab')₂) that are produced, for example, by the proteolytic cleavage of the mAbs, or single-chain immunoglobulins producible, for example, via recombinant means. Such antibody derivatives are monovalent. In certain embodiments, such fragments can be combined with one another, or with other antibody fragments or receptor ligands to form "chimeric" binding molecules. Significantly, such chimeric molecules can contain substituents capable of binding to different epitopes of the same molecule.

[0301] It can be desirable to "humanize" antibodies produced in non-human hosts in order to attenuate any immune reaction when used in human therapy. Such humanized antibodies can be studied in an in vitro or an in vivo context. Humanized antibodies can be produced, for example by replacing an immunogenic portion of an antibody with a corresponding, but non- immunogenic portion {i.e., chimeric antibodies). PCT Application PCT/US86/02269; EP Application 184, 187; EP Application 171,496; EP Application 173,494; PCT Application WO 86/01533; EP Application 125,023; Sun et al., J. Steroid Biochem., 26(l):83-92, 1987; Wood et al., J. Clin. Lab. Immunol., 17(4): 167-171, 1985; and Shaw et al., J. Natl. Cancer Inst., 80(19): 1553-1559, 1988; all of which references are incorporated herein by reference. General reviews of "humanized" chimeric antibodies are provided by Morrison, Science, 229(4719): 1202-1207, 1985; also incorporated herein by reference. "Humanized" antibodies can alternatively be produced by CDR or CEA substitution. Jones et al., Nature, 321 :522-525, 1986;

Verhoeyen et al., Science, 239(4847): 1534-1536, 1988; Beidler et al., J. Immunol., 141(1 1):4053-4060, 1988; all of which are incorporated herein by reference.

[0302] In related embodiments, the antibody is a derivative of the disclosed antibodies, e.g., an antibody comprising the CDR sequences identical to those in the disclosed antibodies (e.g. , a chimeric, humanized or CDR-grafted antibody). In yet a further embodiment, the antibody is a fully human recombinant antibody.

[0303] Alternatively, one can make modifications, such as introducing conservative changes into an antibody molecule. In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, J. Mol. Biol., 157(1): 105- 132, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. [0304] It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Patent 4,554, 101, the following hydrophilicity values have been assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartate (+3.0 ± 1), glutamate (+3.0 ± 1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (-0.4), sulfur containing amino acids: cysteine (- 1.0) and methionine (-1.3); hydrophobic, nonaromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5 ± 1), alanine (-0.5), and glycine (0); hydrophobic, aromatic amino acids: tryptophan (-3.4), phenylalanine (-2.5), and tyrosine (-2.3).

[0305] It is understood that an amino acid can be substituted for another having a similar hydrophilicity and produce a biologically or immunologically modified protein. In such changes, the substitution of amino acids whose hydrophilicity values are, in certain embodiments, within ± 2, within ± 1 or within ± 0.5.

[0306] As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0307] Isotype modification is also contemplated. By modifying the Fc region to have a different isotype, different functionalities can be achieved. For example, changing to IgG₄ can reduce immune effector functions associated with other isotypes.

[0308] Modified antibodies can be made by any technique known to those of skill in the art, including expression through standard molecular biological techniques, or the chemical synthesis of polypeptides. Methods for recombinant expression are addressed elsewhere in this document.

[0309] Anti-AQP4 antibodies can be produced by culturing cells transformed or transfected with a vector containing anti-AQP4 antibody-encoding nucleic acids. Polynucleotide sequences encoding polypeptide components of the antibody of the present disclosure can be obtained using standard recombinant techniques. Desired polynucleotide sequences can be isolated and sequenced from antibody producing cells such as hybridomas cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in host cells. Many vectors that are available and known in the art can be used for the purpose of the present disclosure. Selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector. Host cells suitable for expressing antibodies of the present disclosure include prokaryotes such as Archaebacteria and Eubacteria, including Gram-negative or Gram-positive organisms, eukaryotic microbes such as filamentous fungi or yeast, invertebrate cells such as insect or plant cells, and vertebrate cells such as mammalian host cell lines. Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Antibodies produced by the host cells are purified using standard protein purification methods as known in the art.

[0310] Methods for antibody production including vector construction, expression and purification are further described, in Pluckthun et ah, (1996) in Antibody Engineering: Producing antibodies in Escherichia coli: From PCR to fermentation (McCafferty, J., Hoogenboom, H. R., and Chiswell, D. J., eds), 1 Ed., pp. 203-252, IRL Press, Oxford; Kwong, K. & Rader, C, E. coli expression and purification of Fab antibody fragments, Current protocols in protein science editorial board John E Coligan et al, Chapter 6, Unit 6.10 (2009); Tachibana and Takekoshi, "Production of Antibody Fab Fragments in Escherischia coli," in Antibody Expression and Production, M. Al-Rubeai, Ed., Springer, New York, 201 1 ; Therapeutic Monoclonal Antibodies: From Bench to Clinic (ed Z. An), John Wiley & Sons, Inc., Hoboken, NJ, USA.

[0311] It is, of course, contemplated that alternative methods, which are well known in the art, can be employed to prepare anti-AQP4 antibodies. For instance, the appropriate amino acid sequence, or portions thereof, can be produced by direct peptide synthesis using solid-phase techniques (see, e.g. , Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Chem. Soc, 85:2149-2154 (1963)). In vitro protein synthesis can be performed using manual techniques or by automation. Various portions of the anti-AQP4 antibody can be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired anti-AQP4 antibody. Alternatively, antibodies can be purified from cells or bodily fluids, such as milk, of a transgenic animal engineered to express the antibody, as disclosed, for example, in US Pat. No. 5,545,807 and US Pat. No. 5,827,690.

[0312] Variants and derivatives of antibodies include antibody functional fragments that retain the ability to bind to an AQP4 epitope. Exemplary functional fragments include Fab fragments {e.g., an antibody fragment that contains the antigen-binding domain and comprises a light chain and part of a heavy chain bridged by a disulfide bond); Fab' {e.g., an antibody fragment containing a single anti- binding domain comprising an Fab and an additional portion of the heavy chain through the hinge region); F(ab')2 (e.g., two Fab' molecules joined by interchain disulfide bonds in the hinge regions of the heavy chains; the Fab' molecules can be directed toward the same or different epitopes); a bispecific Fab (e.g., a Fab molecule having two antigen binding domains, each of which can be directed to a different epitope); a single chain Fab chain comprising a variable region, also known as, a sFv (e.g., the variable, antigen-binding determinative region of a single light and heavy chain of an antibody linked together by a chain of 10-25 amino acids); a disulfide-linked Fv, or dsFv (e.g., the variable, antigen-binding determinative region of a single light and heavy chain of an antibody linked together by a disulfide bond); a camelized VH (e.g., the variable, antigen-binding determinative region of a single heavy chain of an antibody in which some amino acids at the VH interface are those found in the heavy chain of naturally occurring camel antibodies); a bispecific sFv (e.g., a sFv or a dsFv molecule having two antigen-binding domains, each of which can be directed to a different epitope); a diabody (e.g., a dimerized sFv formed when the VH domain of a first sFv assembles with the VL domain of a second sFv and the VL domain of the first sFv assembles with the VH domain of the second sFv; the two antigen-binding regions of the diabody can be directed towards the same or different epitopes); and a triabody (e.g., a trimerized sFv, formed in a manner similar to a diabody, but in which three antigen-binding domains are created in a single complex; the three antigen binding domains can be directed towards the same or different epitopes). Derivatives of antibodies also include one or more CDR sequences of an antibody combining site. The CDR sequences can be linked together on a scaffold when two or more CDR sequences are present. In certain embodiments, the antibody comprises a single-chain Fv ("scFv"). scFvs are antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The scFv polypeptide can further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFvs see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0313] The present disclosure provides humanized antibodies that bind AQP4, including human and/or cyno AQP4. Humanized antibodies of the present disclosure can comprise one or more VH and/or VL CDRs as shown in Tables 4 and 5, respectively. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain.

Humanization can be performed, for example, following the method of Winter and co-workers (Jones et al. (1986) Nature 321 :522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239: 1534- 1536), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. [0314] In some cases, the humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the six CDRs of the parent non-human antibody (e.g., rodent) are grafted onto a human antibody framework. For example, Padlan et al. (FASEB J. 9: 133- 139, 1995) determined that only about one third of the residues in the CDRs actually contact the antigen, and termed these the "specificity determining residues," or SDRs. In the technique of SDR grafting, only the SDR residues are grafted onto the human antibody framework (see, e.g., Kashmiri et al, Methods 36: 25-34, 2005).

[0315] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies can be important to reduce antigenicity. For example, according to the so-called "best- fit" method, the sequence of the variable domain of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent can be selected as the human framework for the humanized antibody (Sims et al. (1993) J. Immunol. 151 :2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol, 151 :2623. In some cases, the framework is derived from the consensus sequences of the most abundant human subclasses, V_L6 subgroup I (V_L6I) and V_H subgroup III (V_HIH). In another method, human germline genes are used at the source of the framework regions.

[0316] In an alternative paradigm based on comparison of CDRs, called superhumanization, FR homology is irrelevant. The method consists of comparison of the non-human sequence with the functional human germline gene repertoire. Those genes encoding the same or closely related canonical structures to the murine sequences are then selected. Next, within the genes sharing the canonical structures with the non-human antibody, those with highest homology within the CDRs are chosen as FR donors. Finally, the non-human CDRs are grafted onto these FRs (see, e.g., Tan et al., J. Immunol. 169: 1 1 19-1 125, 2002).

[0317] It is further generally desirable that antibodies be humanized with retention of their affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three- dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, Protein Eng. 13: 819-824, 2000), Modeller (Sali and Blundell, J. Mol. Biol. 234: 779-815, 1993), and Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18: 2714- 2713, 1997). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, e.g., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0318] Another method for antibody humanization is based on a metric of antibody humanness termed Human String Content (HSC). This method compares the mouse sequence with the repertoire of human germline genes and the differences are scored as HSC. The target sequence is then humanized by maximizing its HSC rather than using a global identity measure to generate multiple diverse humanized variants. (Lazar et al, Mol. Immunol. 44: 1986-1998, 2007).

[0319] In addition to the methods described above, empirical methods can be used to generate and select humanized antibodies. These methods include those that are based upon the generation of large libraries of humanized variants and selection of the best clones using enrichment technologies or high throughput screening techniques. Antibody variants can be isolated from phage, ribosome and yeast display libraries as well as by bacterial colony screening (see, e.g., Hoogenboom, Nat. Biotechnol. 23: 1 105-1 1 16, 2005; Dufner et al, Trends Biotechnol. 24: 523-529, 2006; Feldhaus et al., Nat. Biotechnol. 21 : 163-70, 2003; Schlapschy et al, Protein Eng. Des. Sel. 17: 847-60, 2004).

[0320] In the FR library approach, a collection of residue variants are introduced at specific positions in the FR followed by selection of the library to select the FR that best supports the grafted CDR. The residues to be substituted can include some or all of the "Vernier" residues identified as potentially contributing to CDR structure (see, e.g., Foote and Winter, J. Mol. Biol. 224: 487-499, 1992), or from the more limited set of target residues identified by Baca et al. (J. Biol. Chem. 272: 10678-10684, 1997).

[0321] In FR shuffling, whole FRs are combined with the non-human CDRs instead of creating combinatorial libraries of selected residue variants (see, e.g., Dall'Acqua et al., Methods 36: 43-60, 2005). The libraries can be screened for binding in a two-step selection process, first humanizing VL, followed by VH. Alternatively, a one-step FR shuffling process can be used. Such a process has been shown to be more efficient than the two-step screening, as the resulting antibodies exhibited improved biochemical and physico-chemical properties including enhanced expression, increased affinity and thermal stability (see, e.g., Damschroder et al., Mol. Immunol. 44: 3049-60, 2007).

[0322] The "humaneering" method is based on experimental identification of essential minimum specificity determinants (MSDs) and is based on sequential replacement of non-human fragments into libraries of human FRs and assessment of binding. It begins with regions of the CDR3 of non- human VH and VL chains and progressively replaces other regions of the non-human antibody into the human FRs, including the CDR1 and CDR2 of both VH and VL. This methodology typically results in epitope retention and identification of antibodies from multiple sub-classes with distinct human V-segment CDRs. Humaneering allows for isolation of antibodies that are 91-96 % homologous to human germline gene antibodies, (see, e.g., Alfenito, Cambridge Healthtech Institute's Third Annual PEGS, The Protein Engineering Summit, 2007).

[0323] The "human engineering" method involves altering an non-human antibody or antibody fragment, such as a mouse or chimeric antibody or antibody fragment, by making specific changes to the amino acid sequence of the antibody so as to produce a modified antibody with reduced immunogenicity in a human that nonetheless retains the desirable binding properties of the original non-human antibodies. Generally, the technique involves classifying amino acid residues of a non- human (e.g., mouse) antibody as "low risk", "moderate risk", or "high risk" residues. The classification is performed using a global risk/reward calculation that evaluates the predicted benefits of making particular substitution (e.g., for immunogenicity in humans) against the risk that the substitution will affect the resulting antibody's folding and/or are substituted with human residues. The particular human amino acid residue to be substituted at a given position (e.g. , low or moderate risk) of a non-human (e.g. , mouse) antibody sequence can be selected by aligning an amino acid sequence from the non-human antibody's variable regions with the corresponding region of a specific or consensus human antibody sequence. The amino acid residues at low or moderate risk positions in the non-human sequence can be substituted for the corresponding residues in the human antibody sequence according to the alignment. Techniques for making human engineered proteins are described in greater detail in Studnicka et al, Protein Engineering, 7: 805-814 (1994), U.S. Patents 5,766,886, 5,770,196, 5,821,123, and 5,869,619, and PCT Application Publication WO 93/1 1794.

[0324] Human anti-AQP4 antibodies can be constructed by combining Fv clone variable domain sequence(s) selected from human-derived phage display libraries with known human constant domain sequences(s). Alternatively, human monoclonal anti-AQP4 antibodies of the present disclosure can be made by the hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications , pp. 51- 63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al, J. Immunol, 147: 86 (1991).

[0325] It is also possible to produce transgenic animals (e.g. , mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. Transgenic mice that express human antibody repertoires have been used to generate high-affinity human sequence monoclonal antibodies against a wide variety of potential drug targets (see, e.g., Jakobovits, A., Curr. Opin. Biotechnol. 1995, 6(5):561-6; Briiggemann and Taussing, Curr. Opin. Biotechnol. 1997, 8(4):455-8; U.S. Pat. Nos. 6,075,181 and 6,150,584; and Lonberg et al, Nature Biotechnol. 23: 1 1 17-1 125, 2005).

[0326] Alternatively, the human antibody can be prepared via immortalization of human B lymphocytes producing an antibody directed against a target antigen (e.g. , such B lymphocytes can be recovered from an individual or can have been immunized in vitro) (see, e.g., Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al, J. Immunol., 147 (l):86-95 (1991); and US Pat No. 5,750,373).

[0327] Gene shuffling can also be used to derive human antibodies from non-human, for example, rodent, antibodies, where the human antibody has similar affinities and specificities to the starting non- human antibody. According to this method, which is also called "epitope imprinting" or "guided selection", either the heavy or light chain variable region of a non-human antibody fragment obtained by phage display techniques as described herein is replaced with a repertoire of human V domain genes, creating a population of non-human chain/human chain scFv or Fab chimeras. Selection with antigen results in isolation of a non-human chain/human chain chimeric scFv or Fab wherein the human chain restores the antigen binding site destroyed upon removal of the corresponding non-human chain in the primary phage display clone, (e.g., the epitope guides (imprints) the choice of the human chain partner). When the process is repeated in order to replace the remaining non-human chain, a human antibody is obtained (see, e.g., PCT WO 93/06213; and Osbourn et al, Methods., 36, 61-68, 2005). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides completely human antibodies, which have no FR or CDR residues of non-human origin. Examples of guided selection to humanize mouse antibodies towards cell surface antigens include the folate -binding protein present on ovarian cancer cells (see, e.g., Figini et al, Cancer Res., 58, 991-996, 1998) and CD147, which is highly expressed on hepatocellular carcinoma (see, e.g., Bao et al, Cancer Biol. Ther., 4, 1374- 1380, 2005).

[0328] A potential disadvantage of the guided selection approach is that shuffling of one antibody chain while keeping the other constant could result in epitope drift. In order to maintain the epitope recognized by the non-human antibody, CDR retention can be applied (see, e.g., Klimka et al, Br. J. Cancer., 83, 252-260, 2000; VH CDR2 Beiboer et al, J. Mol. Biol, 296, 833-49, 2000) In this method, the non-human VH CDR3 is commonly retained, as this CDR can be at the center of the antigen-binding site and the most important region of the antibody for antigen recognition. In some instances, however, VH CDR3 and VL CDR3, as well as VH CDR3, VL CDR3 and VL CFR1, of the non-human antibody can be retained.

[0329] Bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens. In certain embodiments, bispecific antibodies are human or humanized antibodies. In certain embodiments, one of the binding specificities is for AQP4 and the other is for any other antigen. In some embodiments, one of the binding specificities is for AQP4, and the other is for another surface antigen expressed on cells expressing AQP4 and a FGF receptor (e.g., FGFRlc, FGFR2c, FGFPv3c, FGFR4). In certain embodiments, bispecific antibodies can bind to two different epitopes of AQP4. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab')₂ bispecific antibodies).

[0330] Methods for making bispecific antibodies are known in the art, such as, for example, by co- expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (see, e.g., Milstein and Cuello, Nature, 305: 537 (1983)). For further details of generating bispecific antibodies see, for example, Bispecific Antibodies, Kontermann, ed., Springer- Verlag, Hiedelberg (201 1).

[0331] A multivalent antibody can be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present disclosure can be multivalent antibodies (which are other than of the IgM class) with three or more antigen binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. In certain embodiments, the dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. In certain embodiments, a multivalent antibody comprises (or consists of) three to about eight antigen binding sites. In one such embodiment, a multivalent antibody comprises (or consists of) four antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (for example, two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptide chain(s) can comprise VDl-(Xl)n -VD2-(X2)n -Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, XI and X2 represent an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) can comprise: VH-CHl -flexible linker-VH-CHl-Fc region chain; or VH-CHl -VH-CHl -Fc region chain. The multivalent antibody herein can further comprise at least two (for example, four) light chain variable domain polypeptides. The multivalent antibody herein can, for instance, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain.

[0332] The present disclosure encompasses non-immunoglobulin binding agents that specifically bind to the same epitope as an anti-AQP4 antibody disclosed herein. In some embodiments, a non- immunoglobulin binding agent is identified an agent that displaces or is displaced by an anti-AQP4 antibody of the present disclosure in a completive binding assay. These alternative binding agents can include, for example, any of the engineered protein scaffolds known in the art. Such scaffolds can comprise one or more CDRs as shown in Table 1. Such scaffolds include, for example, anticalins, which are based upon the lipocalin scaffold, a protein structure characterized by a rigid beta-barrel that supports four hypervariable loops which form the ligand binding site. Novel binding specificities can be engineered by targeted random mutagenesis in the loop regions, in combination with functional display and guided selection (see, e.g., Skerra (2008) FEBSJ. 275: 2677-2683). Other suitable scaffolds can include, for example, adnectins, or monobodies, based on the tenth extracellular domain of human fibronectin III (see, e.g., Koide and Koide (2007) Methods Mol. Biol. 352: 95-109); affibodies, based on the Z domain of staphylococcal protein A (see, e.g., Nygren et al. (2008) FEBSJ. 275: 2668-2676)); DARPins, based on ankyrin repeat proteins (see, e.g., Stumpp et al. (2008) Drug. Discov. Today 13: 695- 701); fynomers, based on the SH3 domain of the human Fyn protein kinase Grabulovski et al. (2007) J. Biol. Chem. 282: 3196-3204); affitins, based on Sac7d from Sulfolobus acidolarius (see, e.g.,

Krehenbrink et al. (2008) J. Mol. Biol. 383: 1058- 1068); affilins, based on human y-B-crystallin (see, e.g., Ebersbach et al. (2007) J. Mol. Biol. 372: 172-185); avimers, based on the A domains of membrane receptor proteins (see, e.g., Silverman et al. (2005) Biotechnol. 23: 1556- 1561); cysteine-rich knottin peptides (see, e.g., Kolmar (2008) FEBSJ. 275: 2684-2690); and engineered Kunitz-type inhibitors (see, e.g., Nixon and Wood (2006) Curr. Opin. Drug. Discov. Dev. 9: 261-268) For a review, see, for example, Gebauer and Skerra (2009) Curr. Opin. Chem. Biol. 13: 245-255.

[0333] In some embodiments, amino acid sequence modification(s) of the antibodies that bind to AQP4 or described herein are contemplated. For example, it can be desirable to improve the binding affinity and/or other biological properties of the antibody, including but not limited to specificity, thermostability, expression level, effector functions, glycosylation, reduced immunogenicity or solubility. This, in addition to the anti-AQP4 antibodies described herein, it is contemplated that anti-AQP4 antibody variants can be prepared. For example, anti-AQP4 antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesis of the desired antibody or polypeptide. Those skilled in the art will appreciate that amino acid changes can alter post- translational processes of the anti-AQP4 antibody, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.

[0334] In some embodiments, antibodies provided herein are chemically modified, for example, by the covalent attachment of any type of molecule to the antibody. The antibody derivatives can include antibodies that have been chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited, to specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Additionally, the antibody can contain one or more non-classical amino acids.

[0335] Variations can be a substitution, deletion or insertion of one or more codons encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the native sequence antibody or polypeptide. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements. Insertions or deletions can optionally be in the range of about 1 to 5 amino acids. In certain embodiments, the substitution, deletion or insertion includes less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the original molecule. In a specific embodiment, the substitution is a conservative amino acid substitution made at one or more predicted non-essential amino acid residues. The variation allowed can be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.

[0336] Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme {e.g. , for antibody-directed enzyme prodrug therapy) or a polypeptide which increases the serum half-life of the antibody.

[0337] Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

Alternatively, conservative {e.g., within an amino acid group with similar properties and/or sidechains) substitutions can be made, so as to maintain or not significantly change the properties. Amino acids can be grouped according to similarities in the properties of their side chains (see, e.g., A. L. Lehninger, in Biochemistry. 2nd Ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His(H). [0338] Alternatively, naturally occurring residues can be divided into groups based on common side- chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0339] Non-conservative substitutions entail exchanging a member of one of these classes for another class. Such substituted residues also can be introduced into the conservative substitution sites or, into the remaining (non-conserved) sites. Accordingly, in some embodiments, an antibody or fragment thereof that binds to an AQP4 epitope comprises an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of a murine monoclonal antibody described herein. In one embodiment, an antibody or fragment thereof that binds to an AQP4 epitope comprises an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to an amino acid sequence depicted in Tables 1. In yet another embodiment, an antibody or fragment thereof that binds to an AQP4 epitope comprises a VH CDR and/or a VL CDR amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a VH CDR amino acid sequence depicted in Tables 1 and/or a VL CDR amino acid sequence depicted in Tables 1. The variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (see, e.g., Carter et al, Nucl. Acids Res., 13:4331 (1986); Zoller et al, Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (see, e.g., Wells et al, Gene, 34:315 (1985)), restriction selection mutagenesis (see, e.g., Wells et al, Philos. Trans. R. Soc. London SerA, 317:415 (1986)) or other known techniques can be performed on the cloned DNA to produce the anti-AQP4 antibody variant DNA.

[0340] Any cysteine residue not involved in maintaining the proper conformation of the anti-AQP4 antibody also can be substituted, for example, with another amino acid such as alanine or serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the anti-AQP4 antibody to improve its stability {e.g., where the antibody is an antibody fragment such as an Fv fragment).

[0341] In some embodiments, an anti-AQP4 antibody molecule of the present disclosure is a "de- immunized" antibody. A "de-immunized" anti-AQP4 antibody is an antibody derived from a humanized or chimeric anti-AQP4 antibody, that has one or more alterations in its amino acid sequence resulting in a reduction of immunogenicity of the antibody, compared to the respective original non-de-immunized antibody. One of the procedures for generating such antibody mutants involves the identification and removal of T-cell epitopes of the antibody molecule. In a first step, the immunogenicity of the antibody molecule can be determined by several methods, for example, by in vitro determination of T-cell epitopes or in silico prediction of such epitopes, as known in the art. Once the critical residues for T-cell epitope function have been identified, mutations can be made to remove immunogenicity and retain antibody activity. For review, see, for example, Jones et ah, Methods in Molecular Biology 525: 405-423, 2009.

5.3.3. Chemical Modifications

[0342] Another approach to impairing IgG function in the Fc region is to carbamylate, amidate or benzylate the antibodies. Techniques for these modifications are presented in Thrasher and Cohen, J. Immunol, 107:672-677 (1971).

[0343] Covalent modifications of anti-AQP4 antibodies are included within the scope of the present disclosure. Covalent modifications include reacting targeted amino acid residues of an anti-AQP4 antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the anti-AQP4 antibody. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side chains (see, e.g., T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0344] Other types of covalent modification of the anti-AQP4 antibody included within the scope of this present disclosure include altering the native glycosylation pattern of the antibody or polypeptide (see, e.g., Beck et ah, Curr. Pharm. Biotechnol. 9: 482-501, 2008; Walsh, Drug Discov. Today 15: 773- 780, 2010), and linking the antibody to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth, for example, in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301, 144; 4,670,417; 4,791, 192 or 4, 179,337.

[0345] An anti-AQP4 antibody of the present disclosure can also be modified to form chimeric molecules comprising an anti-AQP4 antibody fused to another, heterologous polypeptide or amino acid sequence, for example, an epitope tag (see, e.g., Terpe, Appl. Microbiol. Biotechnol. 60: 523-533, 2003) or the Fc region of an IgG molecule (see, e.g., Aruffo, "Immunoglobulin fusion proteins" in Antibody Fusion Proteins, S.M. Chamow and A. Ashkenazi, eds., Wiley-Liss, New York, 1999, pp. 221-242).

5.3.4. Single Chain Antibodies

[0346] A Single Chain Variable Fragment (scFv) is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short (usually serine, glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide. This modification usually leaves the specificity unaltered. These molecules were created historically to facilitate phage display where it is highly convenient to express the antigen binding domain as a single peptide. Alternatively, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma. Single chain variable fragments lack the constant Fc region found in complete antibody molecules, and thus, the common binding sites (e.g., protein A/G) used to purify antibodies. These fragments can often be purified/immobilized using Protein L since Protein L interacts with the variable region of kappa light chains.

[0347] Flexible linkers generally are comprised of helix- and turn-promoting amino acid residues such as alanine, serine and glycine. However, other residues can function as well. Tang et al., J. Biol. Chem., 271(26): 15682-15686, 1996 used phage display as a means of rapidly selecting tailored linkers for single-chain antibodies (scFvs) from protein linker libraries. A random linker library was constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18- amino acid polypeptide of variable composition. The scFv repertoire (approx. 5 x 10⁶ different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population selected variants exhibited significant increases in binding activity but retained considerable sequence diversity. Screening 1054 individual variants subsequently yielded a catalytically active scFv that was produced efficiently in soluble form. Sequence analysis revealed a conserved proline in the linker two residues after the V_H C terminus and an abundance of arginines and prolines at other positions as the only common features of the selected tethers.

[0348] The recombinant antibodies provided herein can also involve sequences or moieties that permit dimerization or multimerization of the receptors. Such sequences include those derived from IgA, which permit formation of multimers in conjunction with the J-chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chains can be modified with agents such as biotin/avidin, which permit the combination of two antibodies.

[0349] In a separate embodiment, a single-chain antibody can be created by joining receptor light and heavy chains using a non-peptide linker or chemical unit. Generally, the light and heavy chains will be produced in distinct cells, purified, and subsequently linked together in an appropriate fashion (i.e., the N-terminus of the heavy chain being attached to the C-terminus of the light chain via an appropriate chemical bridge).

[0350] Cross-linking reagents are used to form molecular bridges that tie functional groups of two different molecules, e.g., a stablizing and coagulating agent. However, it is contemplated that dimers or multimers of the same analog or heteromeric complexes comprised of different analogs can be created. To link two different compounds in a step-wise manner, hetero-bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation. [0351] An exemplary hetero-bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g., N-hydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.). Through the primary amine reactive group, the cross-linker can react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., the selective agent).

[0352] In some embodiments, a cross-linker having reasonable stability in blood will be employed. Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic/preventative agents. Linkers that contain a disulfide bond that is sterically hindered can prove to give greater stability in vivo, preventing release of the targeting peptide prior to reaching the site of action. These linkers are thus one group of linking agents.

[0353] Another cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is "sterically hindered" by an adjacent benzene ring and methyl groups. It is believed that steric hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery of the attached agent to the target site.

[0354] The SMPT cross-linking reagent, as with many other known cross-linking reagents, lends the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine). Another possible type of cross-linker includes the hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl-l,3'-dithiopropionate. The N-hydroxy-succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue.

[0355] In addition to hindered cross-linkers, non-hindered linkers also can be employed in accordance herewith. Other useful cross-linkers, not considered to contain or generate a protected disulfide, include SATA, SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, Cancer Treat Res., 37:239-51, 1988). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers.

[0356] U.S. Patent 4,680,338, describes bifunctional linkers useful for producing conjugates of ligands with amine-containing polymers and/or proteins, especially for forming antibody conjugates with chelators, drugs, enzymes, detectable labels and the like. U.S. Patents 5,141,648 and 5,563,250 disclose cleavable conjugates containing a labile bond that is cleavable under a variety of mild conditions. This linker is particularly useful in that the agent of interest can be bonded directly to the linker, with cleavage resulting in release of the active agent. Particular uses include adding a free amino or free sulfhydryl group to a protein, such as an antibody, or a drug. [0357] U.S. Patent 5,856,456 provides peptide linkers for use in connecting polypeptide constituents to make fusion proteins, e.g., single chain antibodies. The linker is up to about 50 amino acids in length, contains at least one occurrence of a charged amino acid (e.g., arginine or lysine) followed by a proline, and is characterized by greater stability and reduced aggregation. U.S. Patent 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separative techniques.

5.3.5. Purification

[0358] In certain embodiments, the antibodies provided herein can be purified. The term "purified," as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein is purified to any degree relative to its naturally-obtainable state. A purified protein therefore also refers to a protein, free from the environment in which it can naturally occur. Where the term

"substantially purified" is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.

[0359] Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel

electrophoresis; isoelectric focusing. Other methods for protein purification include, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; gel filtration, reverse phase, hydroxylapatite and affinity chromatography; and combinations of such and other techniques.

[0360] In purifying an antibody provided herein, it can be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. The polypeptide can be purified from other cellular components using an affinity column, which binds to a tagged portion of the polypeptide. As is generally known in the art, it is believed that the order of conducting the various purification steps can be changed, or that certain steps can be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.

[0361] Commonly, complete antibodies are fractionated utilizing agents (i.e., protein A) that bind the Fc portion of the antibody. Alternatively, antigens can be used to simultaneously purify and select appropriate antibodies. Such methods often utilize the selection agent bound to a support, such as a column, filter or bead. The antibodies is bound to a support, contaminants removed (e.g., washed away), and the antibodies released by applying conditions (salt, heat, etc.). [0362] Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. Another method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity. The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.

[0363] It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al., Biochem. Biophys. Res. Comm., 74(2):425-433, 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products can vary, herein.

5.3.6. Polynucleotides Encoding an Antibody

[0364] Also provide herein are polynucleotides comprising a nucleotide sequence encoding an antibody provided herein that binds to an AQP4 epitope. Also provided herein are polynucleotides that hybridize under high stringency, intermediate or lower stringency hybridization conditions, e.g., as defined elsewhere herein to polynucleotides that encode a modified antibody provided herein.

[0365] The polynucleotides can be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. Since the amino acid sequences of rAb53, rAB58 and rAB09-3-33 are known and provided elsewhere herein, nucleotide sequences encoding these antibodies and modified versions of these antibodies can be determined using methods well known in the art, i.e., nucleotide codons known to encode particular amino acids are assembled in such a way to generate a nucleic acid that encodes the antibody. Such a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al, 1994, BioTechniques 17:242), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, fragments, or variants thereof, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

[0366] Alternatively, a polynucleotide encoding an antibody provided herein can be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library or a cDNA library generated from, or nucleic acid, such as poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody provided herein) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[0367] In certain embodiments, nucleic acid molecules provided herein comprise or consist of a nucleic acid sequence as depicted in any one of SEQ ID NOS: 1, 7 or 13 (encoding a VH) and/or SEQ ID NOS:3, 9 or 15 (encoding a VL), or any combination thereof (e.g., as a nucleotide sequence encoding an antibody provided herein, such as a full-length antibody, heavy and/or light chain of an antibody, or a single chain antibody provided herein).

5.3.7. Recombinant Expression of an Antibody

[0368] Recombinant expression of an antibody provided herein (e.g., a full-length antibody, heavy and/or light chain of an antibody, or a single chain antibody provided herein) that binds to an AQP4 antigen requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule, heavy or light chain of an antibody, or fragment thereof (e.g., but not necessarily, containing the heavy and/or light chain variable domain) provided herein has been obtained, the vector for the production of the antibody molecule can be produced by recombinant DNA technology using techniques well-known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Thus, also provided are replicable vectors comprising a nucleotide sequence encoding an antibody molecule provided herein, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody or a fragment thereof, or a heavy or light chain CDR, operably linked to a promoter. Such vectors can include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., International Publication Nos. WO 86/05807 and WO 89/01036; and U.S. Patent No. 5,122,464) and the variable domain of the antibody can be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains.

[0369] The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody provided herein. Thus, also provided herein are host cells containing a polynucleotide encoding an antibody provided herein or fragments thereof, or a heavy or light chain thereof, or fragment thereof, or a single chain antibody provided herein, operably linked to a heterologous promoter. In some embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains can be co- expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.

[0370] A variety of host-expression vector systems can be utilized to express the antibody molecules provided herein (see, e.g., U.S. Patent No. 5,807,715). Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule provided herein in situ. These include but are not limited to

microorganisms such as bacteria {e.g., E. coli and B. subtilis) transformed with recombinant

bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In certain embodiments, bacterial cells such as Escherichia coli, or eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies

(Foecking et al, 1986, Gene 45: 101 ; and Cockett et al, 1990, Bio/Technology 8:2). In some embodiments, antibodies provided herein are produced in CHO cells. In a specific embodiment, the expression of nucleotide sequences encoding antibodies provided herein which bind to an AQP4 antigen is regulated by a constitutive promoter, inducible promoter or tissue specific promoter.

[0371] In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such an antibody is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified can be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al, 1983, EMBO 12: 1791), in which the antibody coding sequence can be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0372] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

[0373] In mammalian host cells, a number of viral-based expression systems can be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome {e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts {e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 8 1 :355-359). Specific initiation signals can also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et ah, 1987, Methods in Enzymol. 153:51-544).

[0374] In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications {e.g., glycosylation) and processing {e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030 and HsS78Bst cells. In some embodiments, fully human, monoclonal anti-AQP4 antibodies provided herein are produced in mammalian cells, such as CHO cells.

[0375] For long-term, high-yield production of recombinant proteins, stable expression can be done. For example, cell lines which stably express the antibody molecule can be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells can be allowed to grow for 1 -2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines can be particularly useful in screening and evaluation of compositions that interact directly or indirectly with the antibody molecule.

[0376] A number of selection systems can be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler et al, 1977, Cell 1 1 :223), hypoxanthineguanine

phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al, 1980, Cell 22:8-17) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al, 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62: 191-217; 1993, TIB TECH 1 1(5):155-2 15); and hygro, which confers resistance to hygromycin (Santerre et al, 1984, Gene 30: 147). Methods commonly known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al, 1981, J. Mol. Biol. 150: 1, which are incorporated by reference herein in their entireties.

[0377] The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et ah, 1983, Mol. Cell. Biol. 3:257).

[0378] The host cell can be co-transfected with two expression vectors provided herein, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors can contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector can be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197-2199). The coding sequences for the heavy and light chains can comprise cDNA or genomic DNA.

[0379] Once an antibody molecule provided herein has been produced by recombinant expression, it can be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography {e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the antibodies provided herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification

5.4 Treatment and Prevention of NMO

5.4.1. Pharmaceutical Compositions

[0380] Also provided herein are pharmaceutical compositions comprising anti-AQP4 antibodies and antigens for generating the same. Such compositions comprise a prophylactically or therapeutically effective amount of an antibody or a fragment thereof, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0381] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in "Remington's Pharmaceutical Sciences." Such compositions will contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, e.g., in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, or delivered by mechanical ventilation.

[0382] Passive transfer of antibodies, known as artificially acquired passive immunity, generally will involve the use of intravenous or intramuscular injections. The forms of antibody can be human or animal blood plasma or serum, as pooled human immunoglobulin for intravenous (IVIG) or intramuscular (IG) use, as high-titer human IVIG or IG from immunized or from donors recovering from disease, and as monoclonal antibodies (mAb). Such immunity generally lasts for only a short period of time, and there is also a potential risk for hypersensitivity reactions, and serum sickness, especially from gamma globulin of non-human origin. However, passive immunity provides immediate protection. The antibodies will be formulated in a carrier suitable for injection, i.e., sterile and syringeable.

[0383] Generally, the ingredients of compositions are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water- free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0384] The compositions can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0385] Therapeutic formulations containing one or more antibodies provided herein can be prepared for storage by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride;

hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non- ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0386] The antibodies provided herein can also, for example, be formulated in liposomes.

Liposomes containing the molecule of interest are prepared by methods known in the art, such as described in Epstein et al. (1985) Proc. Natl. Acad. Sci. USA 82:3688; Hwang et al. (1980) Proc. Natl. Acad. Sci. USA 77:4030; and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

[0387] Particularly useful immunoliposomes can be generated by the reverse phase evaporation method with a lipid composition containing phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of an antibody provided herein can be conjugated to the liposomes as described in Martin et al. (1982) J. Biol. Chem. 257:286-288 via a disulfide interchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome; See Gabizon et al., (1989) J. National Cancer Inst. 81(19): 1484.

[0388] Formulations, such as those described herein, can also contain more than one active compound as necessary for the particular indication being treated. In certain embodiments, formulations comprise an antibody provided herein and one or more active compounds with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. For example, an antibody provided herein can be combined with one or more other therapeutic agents. Such combined therapy can be administered to the patient serially or simultaneously or in sequence. [0389] An antibody provided herein can also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and

nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA.

[0390] The formulations to be used for in vivo administration can be sterile. This is readily accomplished by filtration through, e.g., sterile filtration membranes.

[0391] Sustained-release preparations can also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained- release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and ethyl- L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they can denature or aggregate as a result of exposure to moisture at 37 °C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S— S bond formation through thio- disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

[0392] The pharmaceutical compositions provided herein contain therapeutically effective amounts of one or more of the antibodies provided herein, and optionally one or more additional prophylactic of therapeutic agents, in a pharmaceutically acceptable carrier. Such pharmaceutical compositions are useful in the prevention, treatment, management or amelioration of an AQP4-mediated disease, such as NMO, or one or more of the symptoms thereof.

[0393] Pharmaceutical carriers suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.

[0394] In addition, the antibodies provided herein can be formulated as the sole pharmaceutically active ingredient in the composition or can be combined with other active ingredients (such as one or more other prophylactic or therapeutic agents). [0395] The compositions can contain one or more antibodies provided herein. In one embodiment, the antibodies are formulated into suitable pharmaceutical preparations, such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral administration or in sterile solutions or suspensions for parenteral administration, as well as transdermal patch preparation and dry powder inhalers. In one embodiment, the antibodies described above are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel (1985) Introduction to Pharmaceutical Dosage Forms, 4^th Ed., p. 126).

[0396] In the compositions, effective concentrations of one or more antibodies or derivatives thereof is (are) mixed with a suitable pharmaceutical carrier. The concentrations of the compounds in the compositions are effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates an AQP4-mediated disease or symptom thereof.

[0397] In one embodiment, the compositions are formulated for single dosage administration. To formulate a composition, the weight fraction of compound is dissolved, suspended, dispersed or otherwise mixed in a selected carrier at an effective concentration such that the treated condition is relieved, prevented, or one or more symptoms are ameliorated.

[0398] An antibody provided herein is included in the pharmaceutically acceptable carrier in an effective amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated. The therapeutically effective concentration can be determined empirically by testing the compounds in in vitro and in vivo systems using routine methods and then extrapolated therefrom for dosages for humans.

[0399] The concentration of antibody in the pharmaceutical composition will depend on, e.g., the physicochemical characteristics of the antibody, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.

[0400] In one embodiment, a therapeutically effective dosage produces a serum concentration of antibody of from about 0.1 ng/ml to about 50-100 μg/ml. The pharmaceutical compositions, in another embodiment, provide a dosage of from about 0.001 mg to about 2000 mg of antibody per kilogram of body weight per day. Pharmaceutical dosage unit forms can be prepared to provide from about 0.01 mg, 0.1 mg or 1 mg to about 500 mg, 1000 mg or 2000 mg, and in one embodiment from about 10 mg to about 500 mg of the antibody and/or a combination of other optional essential ingredients per dosage unit form.

[0401] The antibody can be administered at once, or can be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values can also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

[0402] Upon mixing or addition of the antibody, the resulting mixture can be a solution, suspension, emulsion or the like. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and can be empirically determined.

[0403] The pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof. The antibody is, in one embodiment, formulated and administered in unit-dosage forms or multiple-dosage forms. Unit-dose forms as used herein refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the antibody sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit-dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit-dose forms can be administered in fractions or multiples thereof. A multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons. Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging.

[0404] In some embodiments, one or more anti-AQP4 antibodies provided herein are in a liquid pharmaceutical formulation. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate,

triethanolamine sodium acetate, triethanolamine oleate, and other such agents. [0405] Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA.

[0406] Dosage forms or compositions containing antibody in the range of 0.005% to 100% with the balance made up from non-toxic carrier can be prepared. Methods for preparation of these compositions are known to those skilled in the art.

[0407] Oral pharmaceutical dosage forms are either solid, gel or liquid. The solid dosage forms are tablets, capsules, granules, and bulk powders. Types of oral tablets include compressed, chewable lozenges and tablets which can be enteric-coated, sugar-coated or film-coated. Capsules can be hard or soft gelatin capsules, while granules and powders can be provided in non-effervescent or effervescent form with the combination of other ingredients known to those skilled in the art.

[0408] In certain embodiments, the formulations are solid dosage forms. In certain embodiments, the formulations are capsules or tablets. The tablets, pills, capsules, troches and the like can contain one or more of the following ingredients, or compounds of a similar nature: a binder; a lubricant; a diluent; a glidant; a disintegrating agent; a coloring agent; a sweetening agent; a flavoring agent; a wetting agent; an emetic coating; and a film coating. Examples of binders include microcrystalline cellulose, gum tragacanth, glucose solution, acacia mucilage, gelatin solution, molasses, polvinylpyrrolidine, povidone, crospovidones, sucrose and starch paste. Lubricants include talc, starch, magnesium or calcium stearate, lycopodium and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate. Glidants include, but are not limited to, colloidal silicon dioxide.

Disintegrating agents include crosscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose. Coloring agents include, for example, any of the approved certified water soluble FD and C dyes, mixtures thereof; and water insoluble FD and C dyes suspended on alumina hydrate. Sweetening agents include sucrose, lactose, mannitol and artificial sweetening agents such as saccharin, and any number of spray dried flavors.

Flavoring agents include natural flavors extracted from plants such as fruits and synthetic blends of compounds which produce a pleasant sensation, such as, but not limited to peppermint and methyl salicylate. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene laural ether. Emetic-coatings include fatty acids, fats, waxes, shellac, ammoniated shellac and cellulose acetate phthalates. Film coatings include hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000 and cellulose acetate phthalate.

[0409] The antibodies provided herein can be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition can also be formulated in combination with an antacid or other such ingredient.

[0410] When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The compounds can also be administered as a component of an elixir, suspension, syrup, wafer, sprinkle, chewing gum or the like. A syrup can contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.

[0411] The antibody can also be mixed with other active materials which do not impair the desired action, or with materials that supplement the desired action, such as antacids, H2 blockers, and diuretics. The active ingredient is an antibody or pharmaceutically acceptable derivative thereof as described herein. Higher concentrations, up to about 98% by weight of the active ingredient can be included.

[0412] In all embodiments, tablets and capsules formulations can be coated as known by those of skill in the art in order to modify or sustain dissolution of the active ingredient. Thus, for example, they can be coated with a conventional enterically digestible coating, such as phenylsalicylate, waxes and cellulose acetate phthalate.

[0413] In some embodiments, the formulations are liquid dosage forms. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non- effervescent granules and effervescent preparations reconstituted from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. Emulsions are either oil-in-water or water-in-oil.

[0414] Elixirs are clear, sweetened, hydroalcoholic preparations. Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and can contain a preservative. An emulsion is a two-phase system in which one liquid is dispersed in the form of small globules throughout another liquid. Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifying agents and preservatives. Suspensions use pharmaceutically acceptable suspending agents and preservatives. Pharmaceutically acceptable substances used in non-effervescent granules, to be reconstituted into a liquid oral dosage form, include diluents, sweeteners and wetting agents. Pharmaceutically acceptable substances used in effervescent granules, to be reconstituted into a liquid oral dosage form, include organic acids and a source of carbon dioxide. Coloring and flavoring agents are used in all of the above dosage forms.

[0415] Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examples of preservatives include glycerin, methyl and propylparaben, benzoic acid, sodium benzoate and alcohol. Examples of non-aqueous liquids utilized in emulsions include mineral oil and cottonseed oil. Examples of emulsifying agents include gelatin, acacia, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate. Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, Veegum and acacia. Sweetening agents include sucrose, syrups, glycerin and artificial sweetening agents such as saccharin. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether. Organic acids include citric and tartaric acid. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. Coloring agents include any of the approved certified water soluble FD and C dyes, and mixtures thereof. Flavoring agents include natural flavors extracted from plants such fruits, and synthetic blends of compounds which produce a pleasant taste sensation.

[0416] For a solid dosage form, the solution or suspension, in for example propylene carbonate, vegetable oils or triglycerides, is, in one embodiment, encapsulated in a gelatin capsule. Such solutions, and the preparation and encapsulation thereof, are disclosed in U.S. Patent Nos. 4,328,245; 4,409,239; and 4,410,545. For a liquid dosage form, the solution, e.g., for example, in a polyethylene glycol, can be diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, e.g., water, to be easily measured for administration.

[0417] Alternatively, liquid or semi-solid oral formulations can be prepared by dissolving or dispersing the active compound or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate) and other such carriers, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells. Other useful formulations include those set forth in U.S. Patent Nos. RE28,819 and 4,358,603. Briefly, such formulations include, but are not limited to, those containing a compound provided herein, a dialkylated mono- or poly-alkylene glycol, including, but not limited to, 1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether wherein 350, 550 and 750 refer to the approximate average molecular weight of the polyethylene glycol, and one or more antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters, and dithiocarbamates.

[0418] Other formulations include, but are not limited to, aqueous alcoholic solutions including a pharmaceutically acceptable acetal. Alcohols used in these formulations are any pharmaceutically acceptable water-miscible solvents having one or more hydroxyl groups, including, but not limited to, propylene glycol and ethanol. Acetals include, but are not limited to, di(lower alkyl) acetals of lower alkyl aldehydes such as acetaldehyde diethyl acetal.

[0419] Parenteral administration, in one embodiment, is characterized by injection, either subcutaneously, intramuscularly or intravenously is also contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. The injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical compositions to be administered can also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.

[0420] Implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained (see, e.g., U.S. Patent No. 3,710,795) is also contemplated herein. Briefly, a compound provided herein is dispersed in a solid inner matrix, e.g., polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethyleneterephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinylalcohol and cross-linked partially hydrolyzed polyvinyl acetate, that is surrounded by an outer polymeric membrane, e.g., polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinylacetate copolymers, silicone rubbers, poly dimethyl siloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride, vinylchloride copolymers with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, and ethylene/vinyloxyethanol copolymer, that is insoluble in body fluids. The antibody diffuses through the outer polymeric membrane in a release rate controlling step. The amount of antibody contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the activity of the compound and the needs of the subject.

[0421] Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions. The solutions can be either aqueous or nonaqueous.

[0422] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.

[0423] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances.

[0424] Examples of aqueous vehicles include Sodium Chloride Injection, Ringers Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations can be added to parenteral preparations packaged in multiple-dose containers which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcelluose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEEN^® 80). A sequestering or chelating agent of metal ions includes EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

[0425] The concentration of the pharmaceutically active compound is adjusted so that an injection provides an effective amount to produce the desired pharmacological effect. The exact dose depends on the age, weight and condition of the patient or animal as is known in the art.

[0426] The unit-dose parenteral preparations can be packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration can be sterile, as is known and practiced in the art.

[0427] Illustratively, intravenous or intraarterial infusion of a sterile aqueous solution containing an active compound is an effective mode of administration. Another embodiment is a sterile aqueous or oily solution or suspension containing an active material injected as necessary to produce the desired pharmacological effect.

[0428] Injectables are designed for local and systemic administration. In one embodiment, a therapeutically effective dosage is formulated to contain a concentration of at least about 0.1% w/w up to about 90% w/w or more, in certain embodiments more than 1% w/w of the active compound to the treated tissue(s).

[0429] The antibody can be suspended in micronized or other suitable form. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient for ameliorating the symptoms of the condition and can be empirically determined. [0430] In other embodiments, the pharmaceutical formulations are lyophilized powders, which can be reconstituted for administration as solutions, emulsions and other mixtures. They can also be reconstituted and formulated as solids or gels.

[0431] The lyophilized powder is prepared by dissolving a antibody provided herein, or a pharmaceutically acceptable derivative thereof, in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solvent can contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder.

Excipients that can be used include, but are not limited to, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent. The solvent can also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. In one embodiment, the resulting solution will be apportioned into vials for lyophilization. Each vial will contain a single dosage or multiple dosages of the compound. The lyophilized powder can be stored under appropriate conditions, such as at about 4 °C to room temperature.

[0432] Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The precise amount depends upon the selected compound. Such amount can be empirically determined.

[0433] Topical mixtures are prepared as described for the local and systemic administration. The resulting mixture can be a solution, suspension, emulsions or the like and can be formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, dermal patches or any other formulations suitable for topical administration.

[0434] The antibodies provided herein can be formulated as aerosols for topical application, such as by inhalation (see, e.g., U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivery of a steroid useful for treatment of inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as a microfme powder for insufflations, alone or in combination with an inert carrier such as lactose. In such a case, the particles of the formulation will, in one embodiment, have diameters of less than 50 microns, in one embodiment less than 10 microns.

[0435] The compounds can be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients can also be administered.

[0436] These solutions, particularly those intended for ophthalmic use, can be formulated as 0.01% - 10% isotonic solutions, pH about 5-7, with appropriate salts.

[0437] Other routes of administration, such as transdermal patches, including iontophoretic and electrophoretic devices, and rectal administration, are also contemplated herein.

[0438] Transdermal patches, including iotophoretic and electrophoretic devices, are well known to those of skill in the art. For example, such patches are disclosed in U.S. Patent Nos. 6,267,983,

6,261,595, 6,256,533, 6, 167,301, 6,024,975, 6,010715, 5,985,317, 5,983, 134, 5,948,433, and 5,860,957.

[0439] For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic effect. Rectal suppositories are used herein mean solid bodies for insertion into the rectum which melt or soften at body temperature releasing one or more

pharmacologically or therapeutically active ingredients. Pharmaceutically acceptable substances utilized in rectal suppositories are bases or vehicles and agents to raise the melting point. Examples of bases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol) and appropriate mixtures of mono-, di- and triglycerides of fatty acids. Combinations of the various bases can be used. Agents to raise the melting point of suppositories include spermaceti and wax. Rectal suppositories can be prepared either by the compressed method or by molding. The weight of a rectal suppository, in one embodiment, is about 2 to 3 gm.

[0440] Tablets and capsules for rectal administration can be manufactured using the same pharmaceutically acceptable substance and by the same methods as for formulations for oral

administration.

[0441] The antibodies and other compositions provided herein can also be formulated to be targeted to a particular tissue, receptor, or other area of the body of the subject to be treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the instant compositions. For non-limiting examples of targeting methods, see, e.g., U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6, 131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542 and 5,709,874.

[0442] In one embodiment, liposomal suspensions, including tissue-targeted liposomes, such as tumor-targeted liposomes, can also be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, liposome formulations can be prepared as described in U.S. Patent No. 4,522,81 1. Briefly, liposomes such as multilamellar vesicles (MLV's) can be formed by drying down egg phosphatidyl choline and brain phosphatidyl serine (7:3 molar ratio) on the inside of a flask. A solution of a compound provided herein in phosphate buffered saline lacking divalent cations (PBS) is added and the flask shaken until the lipid film is dispersed. The resulting vesicles are washed to remove unencapsulated compound, pelleted by centrifugation, and then resuspended in PBS.

5.4.2. Methods of Administration and Dosing

[0443] Also provided herein are compositions comprising one or more antibodies provided herein for use in the prevention, management, treatment and/or amelioration of an AQP4-mediated disease, such as NMO, or a symptom thereof.

[0444] In some embodiments, provided herein is a method of treating a subject with NMO spectrum disease comprising administering a composition comprising a therapeutically effective amount of human anti-AQP4 IgG antibody or an antigen binding fragment thereof that comprises at least one of the amino acid substitution in the Fc region described in this application. The amino acid substitution in the Fc region can be, but is not limited to, a D270A substitution, a P331G substitution, a N297D substitution, and a I253D substitution, P331G/L234A/L235A substitutions, or K322A/P331G/P329A/L234A/L235A.

[0445] In some embodiments, the subject is a human subject.

[0446] In one aspect, provided herein is a method of treating a subject with NMO spectrum disease comprising administering a composition comprising a therapeutically effective amount of human anti- AQP4 IgG antibody or an antigen binding fragment thereof through intraocular, intraatertial, subcutaneous, intravenous administration or intrathecal route of administration.

[0447] In another aspect, provided herein is a method of treating a subject with NMO spectrum disease that reduces one or more of retinal ganglion cell death, optic nerve injury, spinal cord injury, or axonal transection.

[0448] In yet another aspect, provided herein is a method of treating a subject with NMO spectrum disease that reduces one or more of optic nerve demyelination, spinal cord demyelination, astrocyte death or oligodendrocyte death.

[0449] In some embodiments, provided herein is a method of treating a subject with NMO spectrum disease comprising administering a composition comprising a therapeutically effective amount of human anti-AQP4 IgG antibody or an antigen binding fragment thereof that comprises at least one of the amino acid substitution in the Fc region described in this application, said composition is administered more than once, including chronically and daily.

[0450] In one aspect, the composition is administered upon onset of or following an NMO attack. In another aspect, the composition is administered within about 1 hour, 6 ours, 12 hours, 24 hours or two days of an NMO attack. [0451] In some embodiments, provided herein is a method of treating a subject with NMO spectrum disease comprising administering a composition comprising a therapeutically effective amount of human anti-AQP4 IgG antibody or an antigen binding fragment thereof, the antibody or antigen binding fragment comprises the antigen binding portion of an antibody having laboratory designation rAb-53 or rAb-58.

[0452] In some embodiments, provided herein is a method of treating a subject with NMO spectrum disease comprising administering a composition comprising a therapeutically effective amount of human anti-AQP4 IgG antibody or an antigen binding fragment thereof that contains at least one of the amino acid substitution in the Fc region described in this application, further comprising assessing said subject for positive NMO-IgG (AQP4) serology. In one aspect, the subject exhibits positive NMO-IgG (AQP4) serology.

[0453] In other embodiments, provided herein is a method of treating a subject with NMO spectrum disease comprising administering a composition comprising a therapeutically effective amount of human anti-AQP4 IgG antibody or an antigen binding fragment thereof that contains at least one of the amino acid substitution in the Fc region described in this application, wherein the subject exhibits one or more of transverse myelitis, optic neuritis or other unrelated neurologic dysfunction. In some aspects, the unrelated neurologic dysfunction comprises protracted nausea or vomiting.

[0454] In some embodiments, provided herein is a method of chronically treating a subject to prevent or reduce exacerbations of NMO spectrum disease comprising administering to said subject a composition comprising a therapeutically effective amount of a human anti-AQP4 IgG antibody or an antigen binding fragment thereof that contains at least one of the amino acid substitution in the Fc region described in this application. The amino acid substitution in the Fc region can be, but is not limited to, a D270A substitution, a P331G substitution, a N297D substitution, and a I253D substitution,

P331G/L234A/L235A substitutions, or K322A/P331G/P329A/L234A/L235A.

[0455] In some other embodiments, provided herein is a method of preventing or inhibiting the progression of NMO spectrum disease in a subject comprising administering to said subject a composition comprising a therapeutically effective amount of a human anti-AQP4 IgG antibody or an antigen binding fragment thereof that contains at least one of the amino acid substitution in the Fc region described in this application. The amino acid substitution in the Fc region can be, but is not limited to, a D270A substitution, a P331G substitution, a N297D substitution, and a I253D substitution,

P331G/L234A/L235A substitutions, or K322A/P331G/P329A/L234A/L235A.

[0456] Also provided herein are methods of preventing, managing, treating and/or ameliorating an AQP4-mediated disease, such as NMO, by administrating to a subject of an effective amount of an antibody, or pharmaceutical composition comprising an antibody provided herein. In one aspect, an antibody is substantially purified (i.e., substantially free from substances that limit its effect or produce undesired side- effects). In some embodiments, the antibody is a fully human monoclonal antibody, such as a fully human monoclonal antagonist antibody. The subject administered a therapy can be a mammal such as non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) or a primate (e.g., a monkey, such as a cynomolgous monkey, or a human). In some embodiments, the subject is a human. In another embodiment, the subject is a human infant or a human infant born prematurely. In another embodiment, the subject is a human with an AQP4-mediated disease, such as NMO.

[0457] Various delivery systems are known and can be used to administer a prophylactic or therapeutic agent (e.g., an antibody provided herein), including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody, receptor- mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of administering a prophylactic or therapeutic agent (e.g., an antibody provided herein), or pharmaceutical composition include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes). In a specific embodiment, a prophylactic or therapeutic agent (e.g., an antibody provided herein), or a pharmaceutical composition is administered intranasally, intramuscularly, intravenously, or subcutaneously. The prophylactic or therapeutic agents, or compositions can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, intranasal mucosa, rectal and intestinal mucosa, etc.) and can be administered together with other biologically active agents. Administration can be systemic or local. In addition, pulmonary

administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which is incorporated herein by reference their entirety.

[0458] In a specific embodiment, it can be desirable to administer a prophylactic or therapeutic agent, or a pharmaceutical composition provided herein locally to the area in need of treatment. This can be achieved by, for example, and not by way of limitation, local infusion, by topical administration (e.g., by intranasal spray), by injection, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In certain embodiments, when administering an antibody provided herein, care must be taken to use materials to which the antibody does not absorb.

[0459] In another embodiment, a prophylactic or therapeutic agent, or a composition provided herein can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249: 1527- 1533; Treat et al. , in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

[0460] In another embodiment, a prophylactic or therapeutic agent, or a composition provided herein can be delivered in a controlled release or sustained release system. In one embodiment, a pump can be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al, 1980, Surgery 88:507; Saudek et al, 1989, N. Engl. J. Med. 321 :574). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of a prophylactic or therapeutic agent {e.g., an antibodies provided herein) or a composition provided herein (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23 :61 ; see also Levy et al , 1985, Science 228: 190; During et al, 1989, Ann. Neurol. 25:351 ; Howard et al, 1989, J. Neurosurg. 7 1 : 105); U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989,463; U.S. Patent No. 5, 128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253. Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In another embodiment, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In yet another embodiment, a controlled or sustained release system can be placed in proximity of the therapeutic target, i.e., the nasal passages or lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 1 15- 138 (1984)). Controlled release systems are discussed in the review by Langer (1990, Science 249: 1527- 1533). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more antibodies provided herein. See, e.g., U.S. Patent No.

4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698, Ning et al , 1996,

"Intratumoral Radioimmunotherapy of a Human Colon Cancer Xenograft Using a Sustained- Release Gel," Radiotherapy & Oncology 39: 179- 189, Song et al , 1995, "Antibody Mediated Lung Targeting of Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science & Technology 50:372-397, Cleek et al. , 1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application," Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, and Lam et al , 1997, "Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery," Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which is incorporated herein by reference in their entirety. [0461] In a specific embodiment, where the composition provided herein is a nucleic acid encoding a prophylactic or therapeutic agent (e.g., an antibody provided herein), the nucleic acid can be administered in vivo to promote expression of its encoded prophylactic or therapeutic agent, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see, e.g., Joliot et al, 1991, Proc. Natl. Acad. Sci. USA 88: 1864-1868), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression by homologous recombination.

[0462] In a specific embodiment, a composition provided herein comprises one, two or more antibodies provided herein. In another embodiment, a composition provided herein comprises one, two or more antibodies provided herein and a prophylactic or therapeutic agent other than an antibody provided herein. In certain embodiments, the agents are known to be useful for or have been or are currently used for the prevention, management, treatment and/or amelioration of an AQP4-mediated disease, such as NMO. In addition to prophylactic or therapeutic agents, the compositions provided herein can also comprise a carrier.

[0463] The compositions provided herein include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., compositions that are suitable for administration to a subject or patient) that can be used in the preparation of unit dosage forms. In one embodiment, a composition provided herein is a pharmaceutical composition. Such compositions comprise a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic agents (e.g., an antibody provided herein or other prophylactic or therapeutic agent), and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical compositions are formulated to be suitable for the route of administration to a subject.

[0464] In a specific embodiment, the term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is an exemplary carrier when the pharmaceutical composition is administered intravenously.

Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Such compositions will contain a prophylactically or therapeutically effective amount of the antibody, e.g., in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0465] In one embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition can also include a solubilizing agent and a local anesthetic such as lignocamne to ease pain at the site of the injection. Such compositions, however, can be administered by a route other than intravenous.

[0466] Generally, the ingredients of compositions provided herein are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0467] Also provided herein is an antibody provided herein is packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of antibody. In one embodiment, the antibody is supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline to the appropriate concentration for administration to a subject. In certain embodiments, the antibody is supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 0.1 mg, at least 0.5 mg, at least 1 mg, at least 2 mg, or at least 3 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 60 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, or at least 100 mg. The lyophilized antibody can be stored at between 2 and 8° C in its original container and the antibody can be administered within 12 hours, within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted. In an alternative embodiment, an antibody is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the antibody. In certain embodiments, the liquid form of the antibody is supplied in a hermetically sealed container at least 0.1 mg/ml, at least 0.5 mg/ml, or at least 1 mg/ml, at least 5 mg/ml, at least 10 mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 30 mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least 60 mg/ml, at least 70 mg/ml, at least 80 mg/ml, at least 90 mg/ml, or at least 100 mg/ml.

[0468] The compositions provided herein can be formulated as neutral or salt forms.

Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc. , and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0469] The amount of a prophylactic or therapeutic agent {e.g., an antibody provided herein), or a composition provided herein that will be effective in the prevention, management, treatment and/or amelioration of an AQP4-mediated disease, such as NMO, can be determined by standard clinical techniques.

[0470] Accordingly, a dosage of an antibody or a composition that results in a serum titer of from about 0.1 μg/ml to about 450 μg/ml, and in some embodiments at least 0.1 μg/ml, at least 0.2 μg/ml, at least 0.4 μg/ml, at least 0.5 μg/ml, at least 0.6 μg/ml, at least 0.8 μg/ml, at least 1 μg/ml, at least 1.5 μg/ml, at least 2 μg/ml, at least 5 μg/ml, at least 10 μg/ml, at least 15 μg/ml, at least 20 μg/ml, at least 25 μg/ml, at least 30 μg/ml, at least 35 μg/ml, at least 40 μg/ml, at least 50 μg/ml, at least 75 μg/ml, at least 100 μg/ml, at least 125 μg/ml, at least 150 μg/ml, at least 200 μg/ml, at least 250 μg/ml, at least 300 μg/ml, at least 350 μg/ml, at least 400 μg/ml, or at least 450 μg/ml can be administered to a human for the prevention, management, treatment and/or amelioration of an AQP4-mediated disease, such as NMO. In addition, in vitro assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of an AQP4-mediated disease, such as NMO, and should be decided according to the judgment of the practitioner and each patient's circumstances.

[0471] Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0472] For the antibodies provided herein, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. In some embodiments, the dosage administered to the patient is about 1 mg/kg to about 75 mg/kg of the patient's body weight. In certain embodiments, the dosage administered to a patient is between 1 mg/kg and 20 mg/kg of the patient's body weight, e.g., 1 mg/kg to 5 mg/kg of the patient's body weight. Generally, human antibodies have a longer half- life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of the antibodies provided herein can be reduced by enhancing uptake and tissue penetration of the antibodies by modifications such as, for example, lipidation.

[0473] In one embodiment, approximately 100 mg/kg or less, approximately 75 mg/kg or less, approximately 50 mg/kg or less, approximately 25 mg/kg or less, approximately 10 mg/kg or less, approximately 5 mg/kg or less, approximately 1 mg/kg or less, approximately 0.5 mg/kg or less, or approximately 0.1 mg/kg or less of an antibody provided herein is administered 5 times, 4 times, 3 times, 2 times or 1 time to manage an AQP4-mediated disease, such as NMO. In some embodiments, an antibody provided herein is administered about 1-12 times, wherein the doses can be administered as necessary, e.g., weekly, biweekly, monthly, bimonthly, trimonthly, etc., as determined by a physician. In some embodiments, a lower dose (e.g., 1- 15 mg/kg) can be administered more frequently (e.g., 3-6 times). In other embodiments, a higher dose (e.g., 25-100 mg/kg) can be administered less frequently (e.g., 1-3 times). However, as will be apparent to those in the art, other dosing amounts and schedules are easily determinable and within the scope provided herein.

[0474] In a specific embodiment, approximately 100 mg/kg, approximately 75 mg/kg or less, approximately 50 mg/kg or less, approximately 25 mg/kg or less, approximately 10 mg/kg or less, approximately 5 mg/kg or less, approximately 1 mg/kg or less, approximately 0.5 mg/kg or less, approximately 0.1 mg/kg or less of an antibody provided herein in a sustained release formulation is administered to a subject, e.g., a human, to prevent, manage, treat and/or ameliorate an AQP4-mediated disease, such as NMO. In another specific embodiment, an approximately 100 mg/kg, approximately 75 mg/kg or less, approximately 50 mg/kg or less, approximately 25 mg/kg or less, approximately 10 mg/kg or less, approximately 5 mg/kg or less, approximately 1 mg/kg or less, approximately 0.5 mg/kg or less, or approximately 0.1 mg/kg or less bolus of an antibody provided herein not in a sustained release formulation is administered to a subject, e.g., a human, to prevent, manage, treat and/or ameliorate an AQP4-mediated disease, such as NMO, and after a certain period of time, approximately 100 mg/kg, approximately 75 mg/kg or less, approximately 50 mg/kg or less, approximately 25 mg/kg or less, approximately 10 mg/kg or less, approximately 5 mg/kg or less, approximately 1 mg/kg or less, approximately 0.5 mg/kg or less, or approximately 5 mg/kg or less of an antibody provided herein in a sustained release is administered to said subject (e.g., intranasally or intramuscularly) two, three or four times (e.g., one time). In accordance with this embodiment, a certain period of time can be 1 to 5 days, a week, two weeks, or a month.

[0475] In some embodiments, a single dose of an antibody provided herein is administered to a patient to prevent, manage, treat and/or ameliorate an AQP4-mediated disease, such as NMO two, three, four, five, six, seven, eight, nine, ten, eleven, twelve times, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty- four, twenty five, or twenty six at bi-weekly (e.g., about 14 day) intervals over the course of a year, wherein the dose is selected from the group consisting of about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, or a combination thereof (i.e., each dose monthly dose may or may not be identical).

[0476] In another embodiment, a single dose of an antibody provided herein is administered to patient to prevent, manage, treat and/or ameliorate an AQP4-mediated disease, such as NMO two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve times at about monthly (e.g., about 30 day) intervals over the course of a year, wherein the dose is selected from the group consisting of about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, or a combination thereof (i.e., each dose monthly dose may or may not be identical).

[0477] In one embodiment, a single dose of an antibody provided herein is administered to a patient to prevent, manage, treat and/or ameliorate an AQP4-mediated disease, such as NMO two, three, four, five, or six times at about bi-monthly (e.g., about 60 day) intervals over the course of a year, wherein the dose is selected from the group consisting of about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, or a combination thereof (i.e., each bi-monthly dose may or may not be identical).

[0478] In some embodiments, a single dose of an antibody provided herein is administered to a patient to prevent, manage, treat and/or ameliorate an AQP4-mediated disease, such as NMO two, three, or four times at about tri-monthly (e.g., about 120 day) intervals over the course of a year, wherein the dose is selected from the group consisting of about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, or a combination thereof (i.e., each tri-monthly dose may or may not be identical). [0479] In certain embodiments, the route of administration for a dose of an antibody provided herein to a patient is intranasal, intramuscular, intravenous, or a combination thereof, but other routes described herein are also acceptable. Each dose may or may not be administered by an identical route of administration. In some embodiments, an antibody provided herein can be administered via multiple routes of administration simultaneously or subsequently to other doses of the same or a different antibody provided herein.

[0480] In certain embodiments, antibodies provided herein are administered prophylactically or therapeutically to a subject. Antibodies provided herein can be prophylactically or therapeutically administered to a subject so as to prevent, lessen or ameliorate an AQP4-mediated disease, such as NMO or symptom thereof.

5.4.3. Combination Therapy

[0481] In order to increase the effectiveness of the antibody therapy provided herein, it can be desirable to combine this treatment with other agents effective at treating or preventing NMO. This process can involve administering to the patient the antibody provided herein the other agent(s) at the same time. This can be achieved by use of a single pharmaceutical composition that includes both agents, or by administering two distinct compositions at the same time, wherein one composition includes the antibody provided herein and the other includes the second agent(s).

[0482] In some embodiments, the application provides a method of treating a subject with NMO spectrum disease comprising administering a composition comprising a therapeutically effective amount of human anti-AQP4 IgG antibody or an antigen binding fragment thereof that contains at least one of the amino acid substitution in the Fc region described in this application, with a second agent that treats one or more aspect of NMO. In one aspect, the second agent is administered at the same time as said composition. In another aspect, the second agent is administered before or after the composition.

[0483] The two therapies can be given in either order and can precede or follow the other treatment by intervals ranging from minutes to weeks. In embodiments where the other agents are applied separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agents would still be able to exert an advantageously combined effect on the patient. In such instances, it is contemplated that one can administer both modalities within about 12-24 h of each other, or within about 6-12 h of each other. In some situations, it can be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

[0484] Various combinations can be employed, the antibody treatment provided herein is "A" and the secondary treatment is "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Administration of the secondary agent will follow general protocols for that drug, taking into account the toxicity, if any. It is expected that the treatment cycles would be repeated as necessary. Secondary treatments are discussed below.

5.4.3.1 Immunosuppressive Agents

[0485] Corticosteroids (high dose for acute attacks; low dose for chronic therapy) such as prednisone are the main line therapy for NMO and thus are readily applied as a combined therapy with the anti- AQP4 antibodies provided herein. Immunosuppressant treatments that can be used in combination with anti-AQP4 antibodies include azathioprine (Imuran) plus prednisone, mycophenolate mofetil plus prednisone, Rituximab, Mitoxantrone, intravenous immunoglobulin (IVIG), and cyclophosphamide.

5.4.3.2 Plasmapheresis

[0486] Plasmapheresis is the removal, treatment, and return of (components of) blood plasma from blood circulation. It is thus an extracorporeal therapy (a medical procedure which is performed outside the body). The method can also be used to collect plasma for further manufacturing into a variety of medications. The procedure is used to treat a variety of disorders, including those of the immune system, such as Myasthenia gravis, Guillain-Barre syndrome, lupus, thrombotic thrombocytopenic purpura and NMO. During plasmapheresis, blood is initially taken out of the body through a needle or previously implanted catheter. Plasma is then removed from the blood by a cell separator. Three procedures are commonly used to separate the plasma from the blood cells:

• Discontinuous flow centrifugation: One venous catheter line is required. Typically, a 300 ml batch of blood is removed at a time and centrifuged to separate plasma from blood cells.

• Continuous flow centrifugation: Two venous lines are used. This method requires slightly less blood volume to be out of the body at any one time as it is able to continuously spin out plasma.

• Plasma filtration: Two venous lines are used. The plasma is filtered using standard hemodialysis equipment. This continuous process requires less than 100 ml of blood to be outside the body at one time.

Each method has its advantages and disadvantages. After plasma separation, the blood cells are returned to the person undergoing treatment, while the plasma, which contains the antibodies, is first treated and then returned to the patient in traditional plasmapheresis. (In plasma exchange, the removed plasma is discarded and the patient receives replacement donor plasma, albumin, or a combination of albumin and saline (usually 70% albumin and 30% saline). Rarely, other replacement fluids, such as hydroxyethyl starch, can be used in individuals who object to blood transfusion but these are rarely used due to severe side-effects. Medication to keep the blood from clotting (an anticoagulant) is given to the patient during the procedure.

[0487] An important use of plasmapheresis is in the therapy of autoimmune disorders, where the rapid removal of disease-causing autoantibodies from the circulation is required in addition to other medical therapy. It is important to note that plasma exchange therapy in and of itself is useful to temper the disease process, where simultaneous medical and immunosuppressive therapy is required for long- term management. Plasma exchange offers the quickest short-term answer to removing harmful autoantibodies; however, the production of autoantibodies by the immune system must also be suppressed, usually by the use of medications such as prednisone, cyclophosphamide, cyclosporine, mycophenolate mofetil, rituximab or a mixture of these. (NMO).

5.5 Antibody Conjugates

[0488] Antibodies can be linked to at least one agent to form an antibody conjugate. In order to increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety can be, but is not limited to, at least one effector or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g., immunosuppression/anti-inflammation. Non- limiting examples of such molecules are set out above. Such molecules are optionally attached via cleavable linkers designed to allow the molecules to be released at or near the target site.

[0489] By contrast, a reporter molecule is defined as any moiety which can be detected using an assay. Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, such as biotin.

[0490] Antibody conjugates can be used as diagnostic agents. Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and those for use in vivo diagnostic protocols, generally known as "antibody-directed imaging." Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Patents 5,021,236, 4,938,948, and 4,472,509). The imaging moieties used can be paramagnetic ions, radioactive isotopes, fluorochromes, NMR-detectable substances, and X-ray imaging agents.

[0491] In the case of paramagnetic ions, one might mention by way of example ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III). Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III). [0492] In the case of radioactive isotopes for therapeutic and/or diagnostic application, one might mention astatine²¹¹, ¹⁴carbon, ⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, hydrogen, iodine , iodine , iodine , indium , iron, phosphorus, rhenium , rhenium , ⁷⁵selenium, ³⁵sulphur, technicium^99m and/or yttrium⁹⁰. ¹²⁵I, technicium^99m and/or indium¹¹¹ can be used in certain embodiments due to their low energy and suitability for long range detection. Radioactively labeled monoclonal antibodies can be produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium and/or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies can be labeled with technetium⁹⁹¹¹¹ by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column. Alternatively, direct labeling techniques can be used, e.g., by incubating pertechnate, a reducing agent such as SNC1₂, a buffer solution such as sodium-potassium phthalate solution, and the antibody. Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTP A) or ethylene diaminetetracetic acid (EDTA).

[0493] Among the fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.

[0494] Another type of antibody conjugates contemplated are those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and/or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase. Other secondary binding ligands are biotin and avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and are described, for example, in U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241.

[0495] Yet another known method of site-specific attachment of molecules to antibodies comprises the reaction of antibodies with hapten-based affinity labels. Essentially, hapten-based affinity labels react with amino acids in the antigen binding site, thereby destroying this site and blocking specific antigen reaction. However, this may not be advantageous since it results in loss of antigen binding by the antibody conjugate.

[0496] Molecules containing azido groups can also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter and Haley, Meth. Enzymol., 91, 613-633, 1983). In particular, 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts (Owens and Haley, J. Biol. Chem., 259, 14843- 14848, 1987; Atherton et al., Biol, of Reproduction, 32, 155-171, 1985). The 2- and 8-azido nucleotides have also been used to map nucleotide binding domains of purified proteins (Khatoon et al., Ann. of Neurology, 26, 210-219, 1989; King et al., J. Biol. Chem., 269, 10210-10218, 1989; Dholakia et al., J. Biol. Chem., 264, 20638-20642, 1989) and can be used as antibody binding agents.

[0497] Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety. Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTP A);

ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3a-6a- diphenylglycouril-3 attached to the antibody (U.S. Patents 4,472,509 and 4,938,948). Monoclonal antibodies can also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate. In U.S. Patent 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies and the detectable imaging moieties are bound to the antibody using linkers such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.

[0498] In other embodiments, derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site are contemplated. Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity and sensitivity (U.S. Patent 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region have also been disclosed in the literature (O'Shannessy et al., J. Immun. Meth., 99, 153-161, 1987). This approach has been reported to produce diagnostically and therapeutically promising antibodies which are currently in clinical evaluation.

[0499] The present disclosure also provides conjugates comprising any one of the anti-AQP4 antibodies of the present disclosure covalently bound by a synthetic linker to one or more non-antibody agents.

[0500] A variety of radioactive isotopes are available for the production of radioconjugated antibodies. Examples include At²¹¹, 14, 14, Y4, Re4, Re4, Sm4, Bi4, P4, Pb4 and radioactive isotopes of Lu. When the conjugate is used for detection, it can comprise a radioactive atom for scintigraphic studies, for example tc4 or 14, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine- 123 again, iodine-131, indium- 1 1 1, fluorine- 19, carbon- 13, nitrogen- 15, oxygen- 17, gadolinium, manganese or iron. The radioisotopes can be incorporated in the conjugate in known ways as described, e.g. , in Reilly, "The radiochemistry of monoclonal antibodies and peptides," in Monoclonal Antibody and Peptide-Targeted Radiotherapy of Cancer, R.M. Reilly, ed., Wiley, Hoboken NJ., 2010.

[0501] In some embodiments, antibodies provided herein are conjugated or recombinantly fused to a diagnostic, detectable or therapeutic agent or any other molecule. The conjugated or recombinantly fused antibodies can be useful, for example, for monitoring or prognosing the onset, development, progression and/or severity of a NMO spectrum disease as part of a clinical testing procedure, such as determining the efficacy of a particular therapy.

[0502] Such diagnosis and detection can accomplished, for example, by coupling the antibody to detectable substances including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin/biotin and avidin/biotin; fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin; chemiluminescent material, such as but not limited to, an acridinium based compound or a HALOTAG; radioactive materials, such as, but not limited to, iodine ( I, I, I, and I,), carbon ( C), sulfur ( S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, and ^mIn,), technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn,

75 Se, 113 Sn, and 117 Sn; and positron emitting metals using various positron emission tomographies, and non-radioactive paramagnetic metal ions.

[0503] Also provided herein are antibodies that are recombinantly fused or chemically conjugated (covalent or non-covalent conjugations) to a heterologous protein or polypeptide (or fragment thereof, for example, to a polypeptide of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90 or about 100 amino acids) to generate fusion proteins, as well as uses thereof. In particular, provided herein are fusion proteins comprising an antigen-binding fragment of an antibody provided herein (e.g., a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, a VH domain, a VH CDR, a VL domain or a VL CDR) and a heterologous protein, polypeptide, or peptide. In one embodiment, the heterologous protein, polypeptide, or peptide that the antibody is fused to is useful for targeting the antibody to a particular cell type, such as a cell that expresses AQP4 or an AQP4 receptor. For example, an antibody that binds to a cell surface receptor expressed by a particular cell type (e.g., an immune cell) can be fused or conjugated to a modified antibody provided herein. [0504] Moreover, antibodies provided herein can be fused to marker or "tag" sequences, such as a peptide to facilitate purification. In specific embodiments, the marker or tag amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (see, e.g., QIAGEN, Inc.), among others, many of which are commercially available. For example, as described in Gentz et al, 1989, Proc. Natl. Acad. Sci. USA 86:821-824, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al, 1984, Cell 37:767), and the "FLAG" tag.

[0505] Methods for fusing or conjugating therapeutic moieties (including polypeptides) to antibodies are well known, (see, e.g., Arnon et al, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al, "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), Thorpe et al, 1982, Immunol. Rev. 62: 1 19-58; U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723, 125, 5,783,181, 5,908,626, 5,844,095, and 5,1 12,946; EP 307,434; EP 367,166; EP 394,827; PCT publications WO 91/06570, WO 96/04388, WO 96/22024, WO 97/34631, and WO 99/04813; Ashkenazi et al, Proc. Natl. Acad. Sci. USA, 88: 10535-10539, 1991 ; Traunecker et al, Nature, 331 :84-86, 1988; Zheng et al, J. Immunol., 154:5590-5600, 1995; Vil et al, Proc. Natl. Acad. Sci. USA, 89: 1 1337-1 1341, 1992).

[0506] Fusion proteins can be generated, for example, through the techniques of gene- shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be employed to alter the activities of anti-AQP4 antibodies as provided herein, including, for example, antibodies with higher affinities and lower dissociation rates (see, e.g., U.S. Patent Nos. 5,605,793, 5,81 1,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al, 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson et al, 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308- 313). Antibodies, or the encoded antibodies, can be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. A polynucleotide encoding an antibody provided herein can be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules. [0507] An antibody provided herein can also be conjugated to a second antibody to form an antibody heteroconjugate as described, for example, in U.S. Patent No. 4,676,980.

[0508] The therapeutic moiety or drug conjugated or recombinantly fused to an antibody provided herein that binds to AQP4 (e.g., an AQP4 polypeptide, fragment, epitope) should be chosen to achieve the desired prophylactic or therapeutic effect(s). In certain embodiments, the antibody is a modified antibody. A clinician or other medical personnel can consider, for example, the following when deciding on which therapeutic moiety or drug to conjugate or recombinantly fuse to an antibody provided herein: the nature of the disease, the severity of the disease, and the condition of the subject.

[0509] Antibodies that bind to AQP4 as provided herein can also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

[0510] The linker can be a "cleavable linker" facilitating release of the conjugated agent in the cell, but non-cleavable linkers are also contemplated herein. Linkers for use in the conjugates of the present disclosure include without limitation acid labile linkers (e.g. , hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers comprising amino acids, for example, valine and/or citrulline such as citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers (see, e.g., C mi et al., Cancer Research 52: 127- 131 (1992); U.S. Patent No. 5,208,020), thioether linkers, or hydrophilic linkers designed to evade multidrug transporter-mediated resistance (see, e.g.Kovtun et ah, Cancer Res. 70: 2528-2537, 2010).

[0511] Conjugates of the antibody and agent can be made using a variety of bifunctional protein coupling agents such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate). The present disclosure further contemplates that conjugates of antibodies and agents can be prepared using any suitable methods as disclosed in the art, (see, e.g., in Bioconjugate Techniques, 2nd Ed., G.T. Hermanson, ed., Elsevier, San Francisco, 2008).

[0512] Conventional conjugation strategies for antibodies and agents have been based on random conjugation chemistries involving the ε-amino group of Lys residues or the thiol group of Cys residues, which results in heterogenous conjugates. Recently developed techniques allow site-specific conjugation to antibodies, resulting in homogeneous loading and avoiding conjugate subpopulations with altered antigen-binding or pharmacokinetics. These include engineering of "thiomabs" comprising cysteine substitutions at positions on the heavy and light chains that provide reactive thiol groups and do not disrupt immunoglobulin folding and assembly or alter antigen binding (see, e.g., Junutula et al., J. Immunol. Meth. 332: 41-52 (2008); Junutula et al, Nat. Biotechnol 26: 925-932, 2008). In another method, selenocysteine is cotranslationally inserted into an antibody sequence by recoding the stop codon UGA from termination to selenocysteine insertion, allowing site specific covalent conjugation at the nucleophilic selenol group of selenocysteine in the presence of the other natural amino acids (see, e.g., Hofer et al, Proc. Natl Acad. Sci. USA 105: 12451-12456 (2008); Hofer et al, Biochemistry 48(50): 12047-12057, 2009).

5.6 Immunodetection Methods

[0513] In still further embodiments, there are immunodetection methods for binding, purifying, removing, quantifying and otherwise generally detecting AQP4 and its associated antigens. Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few. In particular, a competitive assay for the detection and quantitation of AQP4 antibodies also is provided. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Doolittle and Ben-Zeev, Methods Mol. Biol., 109, :215- 237, 1999, Gulbis and Galand, Hum. Pathol. 24(12), 1271-1285, 1993, De Jager et al, Semin. Nucl. Med. 23(2), 165-179, 1993, and Nakamura et al., In: Enzyme Immunoassays: Heterogeneous and

Homogeneous Systems, Chapter 27, 1987. In general, the immunobinding methods include obtaining a sample and contacting the sample with a first antibody in accordance with embodiments discussed herein, as the case can be, under conditions effective to allow the formation of immunocomplexes.

[0514] Contacting the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to AQP4 present. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.

[0515] In general, the detection of immunocomplex formation is well known in the art and can be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. Patents concerning the use of such labels include U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241. Of course, one can find additional advantages through the use of a secondary binding ligand such as a second antibody and/or a biotin/avidin ligand binding arrangement, as is known in the art. [0516] The antibody employed in the detection can itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound within the primary immune complexes can be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand can be linked to a detectable label. The second binding ligand is itself often an antibody, which can thus be termed a "secondary" antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

[0517] Further methods include the detection of primary immune complexes by a two step approach. A second binding ligand, such as an antibody that has binding affinity for the antibody, is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system can provide for signal amplification if this is desired.

[0518] One method of immunodetection uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, and a second antibody is then used to detect the biotin attached to the complexed biotin. In that method, the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody/antigen complex. The antibody/antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody/antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin. This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence of the antibody/antigen complex by histoenzymology using a chromogen substrate. With suitable amplification, a conjugate can be produced which is macroscopically visible.

[0519] Another known method of immunodetection takes advantage of the immuno-PCR

(Polymerase Chain Reaction) methodology. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA/biotin/streptavidin/antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls. At least in theory, the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.

5.6.1. ELISAs

[0520] Immunoassays, in their most simple and direct sense, are binding assays. Certain

immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and

radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like can also be used.

[0521] In one exemplary ELISA, the antibodies provided herein are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the AQP4 is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen can be detected. Detection can be achieved by the addition of another anti-AQP4 antibody that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA." Detection can also be achieved by the addition of a second anti-AQP4 antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

[0522] In another exemplary ELISA, the samples suspected of containing the AQP4 antigen are immobilized onto the well surface and then contacted with anti-AQP4 antibody. After binding and washing to remove non-specifically bound immune complexes, the bound anti-AQP4 antibodies are detected. Where the initial anti-AQP4 antibodies are linked to a detectable label, the immune complexes can be detected directly. Again, the immune complexes can be detected using a second antibody that has binding affinity for the first anti-AQP4 antibody, with the second antibody being linked to a detectable label.

[0523] Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.

[0524] In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

[0525] In ELISAs, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of a protein or antibody to the well, coating with a non- reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or a third binding ligand.

[0526] "Under conditions effective to allow immune complex (antigen/antibody) formation" means that the conditions can include diluting the antigens and/or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.

[0527] The "suitable" conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures that can be on the order of 25°C to 27°C, or can be overnight at about 4°C or so.

[0528] Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. One washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes can be determined.

[0529] To provide a detecting means, the second or third antibody will have an associated label to allow detection. In certain embodiments, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact or incubate the first and second immune complex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g. , incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).

[0530] After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g. , by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS), or H₂O₂, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generated, e.g., using a visible spectra spectrophotometer. 5.6.2. Western Blot

[0531] The Western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

[0532] Samples can be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), or by sonication. Cells can also be broken open by one of the above mechanical methods. However, it should be noted that bacteria, virus or environmental samples can be the source of protein and thus Western blotting is not restricted to cellular studies only. Assorted detergents, salts, and buffers can be employed to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes. Tissue preparation is often done at cold temperatures to avoid protein denaturing.

[0533] The proteins of the sample are separated using gel electrophoresis. Separation of proteins can be by isoelectric point (pi), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful way to determine a protein. It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension, and according to their molecular weight in the second dimension.

[0534] In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below). Both varieties of membrane are chosen for their non-specific protein binding properties (i.e., binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probings. The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dyes. Once transferred, proteins are detected using labeled primary antibodies, or unlabeled primary antibodies followed by indirect detection using labeled protein A or secondary labeled antibodies binding to the Fc region of the primary antibodies.

5.6.3. Immunohistochemistry

[0535] The antibodies can also be used in conjunction with both fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC). The method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and is well known to those of skill in the art (Brown et al., J.

Immunol. Meth., 12; 130(1), : 1 1 1-121, 1990; Abbondanzo et al., Am. J. Pediatr. Hematol. Oncol., 12(4), 480-489, 1990; Allred et al., Arch. Surg., 125(1), 107-1 13, 1990).

[0536] Briefly, frozen-sections can be prepared by rehydrating 50 ng of frozen "pulverized" tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and/or pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and/or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and/or cutting 25-50 serial sections from the capsule. Alternatively, whole frozen tissue samples can be used for serial section cuttings.

[0537] Permanent-sections can be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation;

washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and/or embedding the block in paraffin; and/or cutting up to 50 serial permanent sections. Again, whole tissue samples can be substituted.

5.6.4. Immunodetection Kits

[0538] In still further embodiments, there are immunodetection kits for use with the

immunodetection methods described above.. The immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to AQP4 antigen, and optionally an immunodetection reagent.

[0539] In certain embodiments, the AQP4 antibody can be pre-bound to a solid support, such as a column matrix and/or well of a microtitre plate. The immunodetection reagents of the kit can take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody. [0540] Further suitable immunodetection reagents for use in the present kits include the two- component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, a number of exemplary labels are known in the art and all such labels can be employed in connection with embodiments discussed herein.

[0541] The kits can further comprise a suitably aliquoted composition of the AQP4 antigen, whether labeled or unlabeled, as can be used to prepare a standard curve for a detection assay. The kits can contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. The components of the kits can be packaged either in aqueous media or in lyophilized form.

[0542] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antibody can be placed, or in certain embodiments, suitably aliquoted. The kits will also include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers can include injection or blow-molded plastic containers into which the desired vials are retained.

5.6.5. Pharmaceutical Kits

[0543] Also provided herein is a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions provided herein, such as one or more antibodies provided herein. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0544] Also provided herein are kits that can be used in the above methods. In one embodiment, a kit comprises an antibody provided herein, such as a purified antibody, in one or more containers. In a specific embodiment, the kits provided herein contain a substantially isolated AQP4 antigen as a control. In some embodiments, the kits provided herein further comprise a control antibody which does not react with the AQP4 antigen. In another specific embodiment, the kits provided herein contain a means for detecting the binding of a modified antibody to an AQP4 antigen (e.g., the antibody can be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody can be conjugated to a detectable substrate). In specific embodiments, the kit can include a recombinantly produced or chemically synthesized AQP4 antigen. The AQP4 antigen provided in the kit can also be attached to a solid support. In a more specific embodiment the detecting means of the above described kit includes a solid support to which AQP4 antigen is attached. Such a kit can also include a non-attached reporter- labeled anti-human antibody. In this embodiment, binding of the antibody to the AQP4 antigen can be detected by binding of the said reporter-labeled antibody.

[0545] The following examples are included to demonstrate certain embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of embodiments, and thus can be considered to constitute exemplary modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention, (discussed below). (NMO).

6. EXAMPLES

[0546] Techniques and procedures described or referenced herein include those that are generally well understood and/or commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et ah, Molecular Cloning: A Laboratory Manual, 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds., (2003)); Therapeutic

Monoclonal Antibodies: From Bench to Clinic, Z. An, ed, Wiley, Hoboken N.J. (2009); Monoclonal Antibodies: Methods and Protocols, M. Albitar, ed., Humana Press, Totawa, N.J. (2010); and Antibody Engineering, 2nd Ed., Vols 1 and 2, Kontermann and Dubel, eds., Springer-Verlag, Heidelberg, 2010. (NMO).

6.1 EXAMPLE 1

6.1.1. Materials and Methods

[0547] DNA constructs, cell culture and transfections. DNA constructs encoding full-length human AQP4 (Ml and M23 isoforms) were generated by PCR-amplification using whole brain cDNA as template. For some studies a Myc epitope (NH₂-EQKLISEEDL-COOH; SEQ ID NO: 13) was inserted in the second extracellular loop by PCR-amplification using the non-tagged constructs as template. Mutants of Ml and M23 were generated by PCR-amplification using either tagged or non-tagged templates. All PCR fragments were ligated into mammalian expression vector pcDNA3.1 and fully sequenced.

[0548] U87MG cell cultures (ATCC HTB-14) were maintained at 37 °C in 5% C0₂/95% air in EMEM medium containing 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were grown on glass coverslips and transfected with DNA in antibiotic-free medium using

Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Stable AQP4- expressing clones were selected following enrichment in Geneticin (Invitrogen) and plating in 96-well plates at very low density. [0549] MO patient sera and recombinant AQP4 autoantibodies. NMO serum was obtained from four NMO-IgG seropositive individuals who met the revised diagnostic criteria for clinical disease (Wingerchuk et ah, 2006). Control (non-NMO) human serum was purchased from the UCSF cell culture facility. Recombinant monoclonal NMO antibodies were generated from clonally-expanded

cerebrospinal fluid plasma blasts as described previously (Bennett et ah, 2009). Heavy- and light-chain constructs were co-transfected into HEK293 cells, the supernatant harvested, centrifuged to remove any cells and debris, and incubated overnight with protein A-Sepharose (Sigma- Aldrich, St. Louis, MO) at 4°C. The rAb was eluted in 0.1 M glycine / 1 M NaCl (pH 3.0) and adjusted to pH 7.5 with 0.1 M Tris- HC1, pH 8.0. Recombinant IgG was subsequently exchanged and concentrated in PBS containing 0.1% protease-free bovine serum albumin using Ultracel® YM-30 microconcentrators (Millipore, Billerica, MA). Fab fragments were generated by digestion of whole IgG with immobilized papain, and purified by removal of undigested IgG and Fc fragments by protein-A (Thermo Fisher Scientific, Rockford, IL). Antibody integrity was confirmed by denaturing and native gel electrophoresis, and IgG concentration was assayed using a human IgG-capture ELISA.

[0550] Quantitative immunofluorescence. AQP4-expressing U87MG cells were incubated for 20 min in live-cell blocking buffer (PBS containing 6 mM glucose, 1 mM pyruvate, 1 % bovine serum albumin, 2% goat serum), and then for 30 min with NMO patient serum or recombinant NMO-IgG in blocking buffer. Cells were then rinsed extensively with PBS, fixed in 4% paraformaldehyde for 15 min, and permeabilized with 0.1% Triton X-100. Cells were then blocked again and incubated for 30 min with 0.4 μg/mL polyclonal, C-terminal specific rabbit anti-AQP4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), then rinsed with PBS. Finally, cells were incubated for 30 min with 4 μg/mL goat anti-human IgG-conjugated Alexa Fluor® 488 and goat anti-rabbit IgG-conjugated Alexa Fluor® 555 (Invitrogen) in blocking buffer. After incubation with secondary antibodies, cells were rinsed extensively in PBS, and coverglasses were mounted with VectaMount™ hard-set medium (Vector Laboratories, Burlingame, CA). In some experiments, U87MG cells were labeled as described above, but with a monoclonal mouse anti- Myc IgG (Covance, Emeryville, CA) or purified Fab fragments instead of whole NMO-IgG. Anti-Myc was stained with goat anti-mouse IgG-conjugated Alexa Fluor® 488 (Invitrogen), while Fab fragments were stained with Dylight® 488-linked F(ab')₂-specific secondary antibodies (Jackson Immunoresearch, West Grove, PA). Quantitative analysis of AQP4-antibody binding was done on a Nikon™ Eclipse® TE2000S inverted epifluorescence microscope (Nikon, Melville, NY) equipped with a Nikon™ lOx air objective (numerical aperture 0.3). Green and red dyes were excited and observed through Chroma filter sets #41001 and #42001 (Chroma, Rockingham, VT), respectively. Images were recorded by a CCD camera (Hamamatsu Orca, Bridgewater, NJ), and intensities determined using custom software. [0551] Total internal reflection fluorescence microscopy. TIRFM was done using a Nikon™ Eclipse® TE2000E microscope with a through-objective TIRF attachment and a lOOx TIRF oil immersion objective (numerical aperture 1.49) mounted on a perfect focus module (Nikon, Melville, NY). Alexa Fluor® 555-labeled AQP4 was excited using an argon-ion laser through a Z514/10x excitation filter and Z514RDC dichroic mirror, and detected through an ET605/40m emission filter (Chroma, Rockingham, VT). Images were acquired using a QuantEM 512SC deep-cooled CCD camera

(Photometries, Tucson, AZ).

[0552] Single particle tracking. Prior to labeling of AQP4 with quantum dots (Qdots), cells expressing Myc -tagged AQP4 were washed with 2 mL PBS containing 6 mM glucose and 1 mM pyruvate (GP buffer) and incubated for 5 min in blocking buffer. Cells were then incubated for 5 min with 70 ng/mL mouse anti-Myc antibody (Covance) in blocking buffer, rinsed, and incubated for 5 min with 0.1 nM goat F(ab')₂ anti-mouse IgG-conjugated Qdot 655 (Invitrogen) in blocking buffer. Cells were rinsed extensively and maintained throughout experiments in GP buffer. SPT was performed on a Nikon Eclipse TE2000S inverted epifluorescence microscope equipped with a Nikon lOOx TIRF oil immersion objective (numerical aperture 1.45) and a deep-cooled CCD camera (Hamamatsu EM-CCD, Bridgewater, NJ). Qdot fluorescence was excited using an E460SPUV excitation filter and 475DCXRU dichroic mirror, and detected through a D655/40m emission filter (Chroma). Data were acquired continuously at 1 1 ms per frame (91 Hz) for 6 s. Image sequences were analyzed and trajectories constructed as described in detail previously (Crane and Verkman, Biophys. J., 94:702-713, 2008).

Diffusion data are reported in the form of cumulative distributions of ranges at 1 s, where (range) is defined as the probability that a particle's range is less than or equal to a given distance at t = 1 s.

[0553] Electrophoresis and immunoblotting. Cell cultures were lysed with NativePAGE™ sample buffer (Invitrogen) containing 0.5 % dodecyl-P-D-maltoside (EMD chemicals, Gibbstown, NJ) for 10 min on ice. Lysates were centrifuged at 20,000 g for 30 min at 4 °C and the pellet discarded. For Blue-Native gel electrophoresis (BN-PAGE), polyacrylamide native gradient gels (3-9 %) were prepared as previously described (Wittig et al., Nat. Protoc, 1 :418-428, 2006). 10 μg of protein was mixed with 5% Coomassie Blue G-250 (Invitrogen) and loaded in each lane. Ferritin was used as the molecular mass standard (440 and 880 kDa). Running buffers were: 25 mM imidazole, pH 7 (anode buffer) and 50 mM Tricine, 7.5 mM imidazole, 0.02% Coomassie Blue G-250, pH 7 (cathode buffer). Tricine SDS-PAGE was performed as previously described (Schagger and Tricine, Nat. Protoc.1 : 16-22, 2006) with a 12% running gel and 3% stacking gel. Samples were not heated prior to loading. SeeBlue® Plus2 Pre-Stained Standard (Invitrogen) was used as a molecular weight marker. Proteins were blotted onto polyvinyl difluoride membranes (Bio-Rad, Hercules, CA). For immunoblot analysis, membranes were blocked with 3% BSA and incubated with rabbit anti-AQP4 primary antibodies (Santa Cruz) for 2 h. Membranes were then rinsed and incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch), rinsed extensively, and labeled proteins were detected using the ECL™ Plus enzymatic chemiluminescence kit (Amersham Biosciences, Pittsburgh, PA).

6.1.2. Results

[0554] Approach for quantitative analysis of NMO-IgG binding to AQP4. FIG. 1 diagrams the approach used for quantitative analysis of NMO-IgG binding to AQP4. Cells expressing specified AQP4 isoform(s) at their plasma membrane were incubated with NMO-IgG (NMO patient serum or recombinant monoclonal antibody), fixed, permeabilized, and then incubated with anti-AQP4 antibody (FIG. 1A). The anti-AQP4 antibody recognizes the AQP4 C-terminus, which is common to all AQP4 isoforms (FIG. IB). Fluorescent secondary antibodies were used to detect NMO-IgG by green fluorescence and AQP4 by red fluorescence. NMO-IgG binding to AQP4 was quantified by green-to-red (G/R) fluorescence ratio. Binding affinity and stoichometry was determined by titration with increasing NMO-IgG concentration. In contrast to measurements done at a single NMO-IgG concentration, measurement of full,

concentration-dependent binding provides a quantitative, unbiased description of NMO-IgG binding to AQP4.

[0555] A cell line for analysis of NMO-IgG binding to AQP4 was selected that showed efficient plasma membrane targeting of AQP4 isoforms following stable or transient transfections, as well as human glial cell origin, rapid growth, and strong adherence to coverglass supports. The selected cell line, U87MG, was originally derived from a human astrocytoma (Ponten and Macintyre, Acta Pathol.

Microbiol. Scand., 74:465-486, 1968). FIG. 2A shows high-magnification confocal microscopy of U87MG cells stably expressing Ml or M23 AQP4, and stained with a recombinant monoclonal NMO- IgG (green) and an anti-AQP4 antibody (red). AQP4 was localized to the cell plasma membrane, with little intracellular red fluorescence. FIG. 2B shows TIRFM of the anti-AQP4 antibody-stained AQP4- expressing cells. A smooth pattern of fluorescence was seen for M1-AQP4 and a punctate pattern for M23-AQP4, as found in other cell types (Crane and Verkman, Biophys. J., 94:702-713, 2008), confirming that M23-AQP4 forms OAPs in transfected U87MG cells whereas M1-AQP4 does not.

Immunoblot analysis of cell homogenates showed the expected molecular sizes of the Ml and M23 isoforms of AQP4 (FIG. 2C, bottom). BN-PAGE of cells expressing M23 alone showed multiple high molecular weight bands corresponding to the expected formation of large supramolecular aggregates (OAPs), whereas Ml -expressing cells showed only the expected -300 kDa band corresponding to individual AQP4 tetramers (FIG. 2C, top). FIG. 2D shows examples of measured green-to-red ratios (G/R) after binding recombinant monoclonal NMO-IgGs (rAb-53, rAb-58, and rAb- 186) or control antibody (rAb-2B4) (each at 20 μg/mL) to Ml or M23 AQP4-expressing cells. These initial measurements show considerable diversity of NMO-lgG to Ml vs. M23 AQP4 isoforms, with similar results found in both stably and transiently transfected U87MG cells.

[0556] Heterogeneous NMO-lgG binding to AQP4 isoforms in NMO serum samples. Binding of NMO-lgG in human NMO sera to the Ml and M23 AQP4 isoforms was measured by the fluorescence ratio imaging method. Initial measurements done on 10 serum specimens from NMO patients studied at a 1 : 100 dilution showed a wide range of relative Ml :M23 binding from 0.14 to 0.67. FIG. 3A shows representative fluorescence micrographs for serum specimens from four NMO patients. Each serum specimen showed strong NMO-lgG binding to M23 AQP4, but variable binding to Ml AQP4. FIG. 3B shows concentration-dependent NMO-lgG binding in which background-corrected R/G ratios were determined as a function of serum concentration. NMO-lgG binding to AQP4 was saturable, with relative M23:M1 binding affinities of 0.1 1 (serum 1), 0.26 (serum 2), 0.03 (serum 3) and 0.21 (serum 4).

Interestingly, the binding data fitted well to a single-site, saturable binding model with near unity Hill coefficient, despite the presumed polyclonal composition of NMO serum. It is not possible to determine absolute binding affinity of NMO-lgG to AQP4 using serum because the fraction of NMO-lgG in total serum IgG is not known, nor is the polyclonal NMO-lgG composition.

[0557] Quantitative binding of recombinant monoclonal NMO-lgGs to AQP4 isoforms. NMO- lgG in serum from a single NMO patient is polyclonal, consisting of a pool of monoclonal NMO-lgGs. A similar binding analysis was done using monoclonal recombinant NMO-lgGs derived from a single NMO patient in order to determine: (i) absolute binding affinities; (ii) whether differential M23:M1 is heterogeneous in a single NMO patient; and (iii) whether differential M23:M1 binding is due to differences in NMO-lgG binding affinity and/or stoichiometry. FIG. 4A shows fluorescence micrographs of two recombinant monoclonal NMO-lgGs (rAb-53 and rAb-58) from a single NMO patient. The monoclonal antibodies used in this study were derived from the CSF of a single NMO patient

(corresponding to 'serum 1 ' in FIG. 3). Though strong monoclonal antibody binding to M23 AQP4 was seen in each case, binding to Ml AQP4 was variable. FIG. 4B summarizes concentration-dependence binding curves for three recombinant NMO-lgGs, together with curve fits for a single-site binding model. In each case the data fitted well to a single-site model with near unity Hill coefficient. The lowest dissociation constant was 44 nM, which was found for binding of rAb-53 to M23 AQP4. Marked heterogeneity was found for monoclonal NMO-lgGs from a single NMO patient, with relative M23:M1 binding affinities of 0.02 (rAb-53), 0.46 (rAb-58) and 0.02 (rAb-186). The binding curves in FIG. 4B also support the conclusion that differences in M23:M1 binding are due to differences in binding affinity rather than to binding capacity.

[0558] Mechanism of AQP4 isoform-specific binding of NMO-lgG. The inventors investigated whether differences in NMO-lgG binding affinity to M23 vs. Ml AQP4 are due to formation of OAPs by M23 AQP4 and/or to the different N-termini of M23 vs. Ml . Measurements were made of NMO-IgG binding to cells expressing: (i) different ratios of M23:M1 AQP4; (ii) an Ml mutant that has a diminished ability to disrupt OAPs when coexpressed with M23; and (iii) M23 AQP4 mutants that have diminished ability to form OAPs. For these studies, the inventors used transiently transfected U87MG cells. The suitability of transiently transfected cells was validated above by showing comparable binding in stably vs. transiently transfected cells by NMO-IgG at a fixed concentration (FIG. 2D).

[0559] FIG. 5A shows concentration-dependent binding of NMO-IgG (rAb-53) to U87MG cells co- expressing different ratios of M23:M1 AQP4. The fraction of AQP4 in OAPs and average OAP size are directly related to the M23:M1 AQP4 ratio. Quantum dot single-particle tracking measurements were done to determine the characteristics of OAPs formed at different M23:M1 ratios. FIG. 5A (right) shows increased AQP4 diffusion with higher Ml content, as previously shown (Crane and Verkman, J. Cell Sci., 122:813-821, 2009). This increase in AQP4 diffusion is due to active disruption of OAP growth by Ml AQP4. Not surprisingly, as compared to binding M23 alone, rAb-53 showed incrementally reduced AQP4 binding in 3: 1 and 1 : 1 mixtures of M23:M1 (FIG. 5A, left).

[0560] FIG. 5B shows data from similar experiments as in FIG. 5 A, except that native Ml was substituted by the double-cysteine mutant M1-C13A/C17A (CCA). CCAn AQP4 does not form OAPs on its own, but when co-expressed with M23 has greatly reduced ability to disrupt OAPs (Crane and Verkman, J. Cell Sci., 122:813-821, 2009). Therefore, at the same M23:M1 ratio, cells expressing the CCA mutant in place of native Ml have greater OAP content, as confirmed by single particle tracking (FIG. 5B, right). Concentration-dependent binding of rAb-53 showed greater binding to M23:CCA mixtures than to M23:M1 mixtures (FIG. 5B, left). Binding of rAb-53 to cells expressing a 3: 1 ratio of M23:CCA was identical to cells expressing M23 alone.

[0561] As an independent approach to address the binding specificity issue, the inventors measured NMO-IgG binding to U87MG cells expressing M23 mutants containing an OAP- disrupting point mutation. The OAP-disrupting effect of these mutations was confirmed by quantum dot single-particle tracking. FIG. 5C (right) shows that mutations F26Q and G28P in the M23 AQP4 N-terminus greatly reduce OAP content, similar to the inventors' previous findings with corresponding rat isoforms of AQP4 (Crane and Verkman, J. Cell Sci., 122:813-821, 2009). FIG. 5C (left) shows greatly reduced

concentration-dependent binding of rAb-53 to cells expressing these M23 mutants when compared to native M23. Together, the results in FIGS. 5A-C indicate that OAP formation is responsible for the increased affinity of NMO-IgG to M23 vs. Ml AQP4.

[0562] Two potential mechanisms, bivalent vs. monovalent NMO-IgG binding, could account for the greater affinity of NMO-IgG to OAP vs. non-OAP associated AQP4. FIG. 6A shows a comparison of the distance between adjacent Fab binding sites in IgGi (Sosnick et al., Biochemistry, 31 : 1779- 1786, 1992; Harris et al., J. Mol. Biol., 275:861-872, 1998), and the size of the AQP4 tetramer (Ho et al., Proc. Natl. Acad. Sci. USA, 106:7437-7442, 2009). FIG. 6B diagrams possible opposing binding mechanisms. First is bivalent binding. Strong NMO-lgG binding requires a bivalent interaction in which both Fab sites must bind to AQP4 monomers or tetramers. For rAb-53, in which M23 binding is much stronger than Ml binding, the positions of the binding epitopes in AQP4 monomers are spaced such that a bivalent interaction between the Fab sites is not possible within a single tetramer, but is optimal for crosslinking of adjacent tetramers in OAPs. For rAb-58, the epitopes can be located at positions in which bivalent binding in a single tetramer can occur, resulting in similar binding to Ml and M23 AQP4. Second is monovalent binding. NMO-lgG binding involves classical monovalent interactions, and is controlled primarily by affinities of individual Fab's to their respective epitopes. For rAb-53, a structural change in the epitope site upon OAP formation results in higher affinity. For rAb-58, the epitope site is not altered by OAP formation, resulting in similar affinity for Ml vs. M23 AQP4.

[0563] Fab binding to Ml and M23 AQP4 was measured to test these competing mechanisms. NMO-IgGs were digested with papain and purified to yield Fab's. The bivalent binding mechanism predicts little binding by Fab's, and no difference between Fab binding to Ml vs. M23 AQP4. The monovalent binding mechanism predicts that the increased binding of rAb-53 to M23 AQP4 would also be observed for its individual Fab's. As a control, the inventors measured binding of Fab's generated from a mouse monoclonal anti-Myc antibody to external Myc-tagged AQP4 isoforms. As expected, whole anti-Myc IgG bound Myc-tagged Ml and M23 AQP4 equally, with K_D ~ 10 nM (FIG. 6C). FIG. 6D shows the binding of Fab fragments to Ml vs. M23 AQP4. Whole IgG and Fab's from rAb-53 showed significantly greater binding to M23 AQP4, while whole IgG and Fab's from rAb-58 and anti- Myc showed similar Ml vs. M23 binding. These data provide direct support for the second mechanism involving monovalent binding.

6.1.3. Discussion

[0564] Here, the inventors used fluorescence ratio imaging to quantify the binding of NMO-lgG to AQP4, and to determine the role of AQP4 isoforms and OAPs in NMO-lgG binding. This live-cell system was developed out of a need for a robust method to characterize monoclonal NMO-IgGs and polyclonal NMO patient sera. The inventors found that U87MG cells were suitable for quantitative binding measurements because they efficiently expressed AQP4 at the plasma membrane after stable or transient transfection, with little or no intracellular AQP4 expression. The excellent membrane expression of AQP4 in U87MG cells is likely due to their glial origin, and hence their expression of the same trafficking machinery as that in native human glial cells. Immunoblot analysis of the stably transfected clones used in this study shows exclusive expression of individual AQP4 isoforms, with no detectable M23 in the Ml cell line (FIG. 2C). U87MG cells grow rapidly and adhere well to culture supports, making them suitable for automated and high-throughput assays.

[0565] For all monoclonal and polyclonal NMO antibodies tested, NMO-IgG binding to M23- expressing cells was comparable to or greater than to Ml -expressing cells, though measurable binding to Ml was found in all cases. NMO-IgG was found to bind to each AQP4 isoform in a concentration- dependent manner that fitted well to a single-site, saturable binding model with near unity Hill coefficient, consistent with apparent single-site, non-cooperative binding. Preferential binding of NMO- IgG to M23 AQP4 was found to be a consequence of greater binding affinity rather than to greater binding capacity. Differences in the binding affinity of monoclonal NMO-IgG rAb-53 to the M23 and Ml isoforms of AQP4 was found to be a consequence of OAP formation by M23 AQP4 rather than to differences in the N-terminal sequences. This was proven using mutants of Ml and M23 AQP4 with altered abilities to form and disrupt OAPs. Using a two-color single particle tracking approach, the inventors recently showed that co-expressed AQP4 isoforms Ml and M23 co-assemble in AQP4 tetramers with differential abilities to assemble into OAPs. They also showed that OAP size and content could be altered by changing the Ml :M23 ratio, or by altering the palmitoylation state of Ml AQP4 (Crane et al., J. Biol. Chem., 284:35850-35860, 2009). The inventors found here that co-expression of Ml and M23 also affects NMO-IgG binding (FIG. 5A). However, by reducing the ability of Ml to disrupt OAPs through expression of a palmitoylation-null Ml mutant, they found significantly increased NMO-IgG binding in mixtures with identical M23:M1 ratio (FIG. 5B). The inventors previously showed that OAP formation by M23 requires N-terminus hydrophobic interactions at residues 24-26 just downstream of Met-23 (Crane and Verkman, J. Cell Sci., 122:813-821, 2009), and discovered M23 mutants with greatly reduced ability to form OAPs (M23-F26Q and M23-G28P). These OAP-disrupting M23 AQP4 mutants greatly reduced NMO-IgG binding (FIG. 5C). In contrast to isotherms for binding of NMO-IgG to individual Ml and M23 isoforms, binding to AQP4 mutants M23-F26Q and M23-G28P produced isotherms that did that not fit to a single-site binding model, probably because expression of these mutants produces heterogeneous populations of OAP-assembled AQP4 and non-assembled AQP4 tetramers.

[0566] The mechanism for higher affinity NMO-IgG binding to OAP-assembled AQP4 was examined by comparing whole (bivalent) NMO-IgG to its monovalent Fab's. OAP assembly resulted in a greater affinity for individual Fab binding sites, rather than enhancement by bivalent IgG binding. The inventors propose, therefore, that the binding epitope for many of the IgGs found in NMO can be located at the tetramer/tetramer interface created upon OAP assembly. However, differences in binding affinities vary, likely depending on the epitope structure, and some NMO-IgGs (rAb-58) have strong affinity for unassembled Ml AQP4. [0567] The widely used assay for serum NMO-IgG immunofluorescence is performed in Ml AQP4- expressing HEK-293 cells (Lennon et al., J. Exp. Med., 202:473-477, 2005). The data here indicate that M23 AQP4-expressing cells are superior because NMO-IgG binding to M23 AQP4 is as good as and generally much better than binding to Ml AQP4. The difference in binding is seen at all NMO-IgG concentrations and is often quite marked at low concentrations, as often found in human serum specimens. Up to 30% of serum from patients with NMO, as defined by established clinical criteria, are found to be seronegative as assayed using the conventional method (Wingerchuk et al, 2006). This value is likely substantially lower utilizing more sensitive assays such as the imaging assay established here utilizing human glial cell line strongly expressing the OAP-forming M23 isoform of AQP4. Indeed, a recent study of multiple human serum samples indicated an improvement from 70% to 97% sensitivity for NMO-IgG when using M23 -expressing cells instead of Ml -expressing cells (Mader et al, 2010). This quantitative binding assay established here should also be useful in correlating serum NMO-IgG binding affinity and specificity with NMO clinical parameters, such as disease activity, treatment status and patient characteristics. Baseline differences in NMO-IgG binding to OAP vs. non-OAP associated AQP4 can have diagnostic significance, and spontaneous and treatment-associated changes in binding can have prognostic significance.

6.2 EXAMPLE 2

6.2.1. Materials and Methods

[0568] Recombinant NMO-IgGs and NMO patient sera. Recombinant monoclonal NMO antibodies (rAbs) were generated from clonally-expanded plasma blasts in cerebrospinal fluid (CSF) as described (Bennett et al, 2009). For site-directed mutagenesis, the Kpnl-Xhol fragment of the IgGl heavy chain constant region (IgGlFc) was cloned into pSp73X. Point mutations were introduced into the IgGlFc sequence using the GeneTailor™ Site-Directed Mutagenesis System (Invitrogen) to generate constructs deficient in CDC (mutation K322A), ADCC (mutations K326W/E333S) or both (mutations L234A/L235A) (Baudino et al., J. Immunol., 181 :4107-4112, 2008; Duncan and Winter, Nature, 332:738-740, 1988; Hezareh et al., J. Virol., 75: 12161- 12168, 2001; Idusogie et al., J. Immunol., 166:2571-2575, 2001). The Agel-Xhol fragment of the mutated IgGlFc sequence was cloned into plgGlFlag containing the heavy-chain variable region sequence of rAb-53 to generate aquaporumab constructs containing a mutant IgGl Fc sequence with a C-terminal FLAG® epitope.

[0569] For generation of divalent rAbs and aquaporumab, paired heavy and light chain constructs were co-transfected into HEK293 cells using lipofectamine. After transfection, cells were grown for 7 days in DMEM medium + 10% FBS, the supernatant harvested, fresh medium added, and the cells were grown for another 7 days. The cell culture supernatants were combined, centrifuged at 2000 rpm for 10 min to pellet cells and debris, and the cell-free supernatant was adjusted to pH 8.0 with 1M Tris pH 8.0 and incubated overnight with protein A-Sepharose (Sigma-Aldrich) at 4 °C. The rAb was eluted from the protein A-Sepharose in 0.1 M glycine/lM NaCl (pH 3.0) and adjusted to pH 7.5 with 0.1M Tris-HCl, pH 8.0. Recombinant IgGs were exchanged and concentrated in PBS containing 0.1% protease-free bovine serum albumin (BSA) using Ultracel® YM-30 microconcentrators (Millipore, Billerica, MA). BSA was excluded from the storage solution for surface plasmon resonance measurements. Antibody integrity was confirmed by denaturing and native gel electrophoresis, and IgG concentration was assayed using a human IgG-capture ELISA. NMO serum was obtained from a total of ten NMO-IgG seropositive individuals who met the revised diagnostic criteria for clinical disease (Wingerchuk et ah, 2006). Control (non-NMO) human serum was obtained from a total of three non-NMO individuals, or purchased from the UCSF cell culture facility. For some studies total IgG was purified and concentrated from serum using a Melon™ Gel IgG Purification Kit (Thermo Fisher Scientific, Rockford, IL) and Amicon® Ultra Centrifugal Filter Units (Millipore, Billerica, MA).

[0570] Cell culture and transfections. DNA constructs encoding human AQP4 were generated by PCR-amplification using whole brain cDNA as template. PCR fragments were ligated into mammalian expression vector pcDNA3.1 and fully sequenced. COS-7 (ATCC CRL- 1651), U87MG (ATCC HTB- 14) and CHO-K1 (ATCC CCL-61) cell cultures were maintained at 37 °C in 5% C0₂/95% air in the appropriate medium containing 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL

streptomycin. NK-92 cells expressing CD 16 (CD 16- 176V-NK92, obtained from Fox Chase Cancer Center) were cultured at 37 °C in 5% C0₂/95% air in a-MEM with deoxyribonucleosides and

ribonucleosides containing 10% fetal bovine serum, 10% horse serum, 0.1 mM 2-mercaptoethanol, 2 mM glutamine, 0.2 mM myo-inositol, 2.5 μΜ folic acid, non-essential amino acid, 1 10 μg/mL sodium pyruvate, 100 U/mL penicillin and 100 μg/mL streptomycin.

[0571] Surface plasmon resonance. Real-time binding of rAbs to AQP4 was measured by surface plasmon resonance at 25 °C using a Biacore® T- 100 instrument (GE Healthcare Life Sciences,

Piscataway, NJ), based on reported procedures (Patel et al., Anal. Chem., 81 :6021-6029, 2009). Purified recombinant human Ml AQP4 (provided by William Harries and Robert Stroud, UCSF) was

reconstituted at 3% (wt/wt) AQP4 in proteoliposomes containing 95:5 L-a-phosphatidylcholine:L-a- phosphatidylserine (Avanti Polar Lipids) ratio. Briefly, lipids (total 37.5 mg, 500 nmol) was dissolved in 375 iL of 40 mM b-octyl glucoside (in PBS) followed by addition of AQP4 (300 ml, 450 mg, 15 nmol). The mixture was dialyzed (10,000 dalton cut-off) against PBS at 4 °C for 48 h. For preparation of AQP4- free liposomes, an equal volume of 40 mM b-octyl glucoside (instead of AQP4) was added to the lipid solution. Proteoliposomes were immobilized on a LI sensor chip (Biacore) with four injections at 10 min/injection at 2 μΐ/min to achieve 6000 response units of proteoliposomes immobilization. The surface was then washed with two 20 s injections of 50 mM NaOH at 100 μΐ/min, and checked for surface quality with a 300 s injection of 0.01 mg/ml BSA at 20 μΐ/min. Flow channel 1 (Fc 1) was immobilized with 0% AQP4 as reference, and Fc 2 contained the AQP4 proteoliposomes. Binding studies were conducted with PBS at a flow rate of 15 μΐ/min. rAbs in PBS were injected for 80 s followed by a post-injection period of 240 s. Regeneration was performed by injection of 50 mM NaOH at 100 μΐ/min for 20 s. Binding studies were done in duplicate. Data were analyzed using Biacore® T100 Evaluation software.

[0572] NMO-IgG binding to AQP4 in cells. The kinetics of rAb-53 binding to AQP4 was measured in U87MG cells stably expressing human AQP4-M23 by quantitative imaging as described (Crane et al., J. Biol. Chem., 286: 16516-24, 201 1). Cells were incubated for 20 min in live-cell blocking buffer (PBS containing 6 mM glucose, 1 mM pyruvate, 1% bovine serum albumin, 2% goat serum), and then for specified times with NMO-IgG. Cells were then rinsed, fixed in 4 % paraformaldehyde for 15 min, and permeabilized with 0.1% Triton X- 100. Cells were then blocked again and incubated for 30 min with 0.4 μg/mL polyclonal, C-terminal specific rabbit anti-AQP4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), then rinsed with PBS. Finally, cells were incubated for 30 min with 4 μg/mL goat anti- human IgG-conjugated Alexa Fluor® 488 and goat anti-rabbit IgG-conjugated Alexa Fluor® 555 (Invitrogen) in blocking buffer. NMO-IgG binding was quantified by ratio image analysis as described (39). In some studies rAb-53 was fluorescently labeled with Cy3 using standard succinimidyl chemistry.

[0573] Assays of complement-dependent cytotoxicity (CDC) and antibody-dependent cell- mediated cytotoxicity (ADCC). For assay of CDC, CHO cells expressing human AQP4 were pre- incubated for 30 min with 12.5 μg/ml aquaporumab (or control IgG), then for 90 min at 37 °C with NMO-IgG (2.5 μg/ml) or control-IgG and 5% human complement. Calcein-AM and ethidium- homodimer (Invitrogen) were then added to stain live cells green and dead cells red. Complement- mediated cytotoxicity by 1-2% NMO patient sera (and control non-NMO sera) and 5% human complement were was measured similarly without vs. with 50- 100 μg/ml aquaporumab. For assay of ADCC, NK-92 cells expressing CD 16 were used as the effector cells. The AQP4-expressing CHO cells were pre-incubated for 30 min with 15 μg/ml aquaporumab (or control-IgG), then for 3 h at 37 °C with NMO-IgG (5 μg/ml) or control-IgG and effector cells (effector : target ratio 30: 1). Cells were rinsed with PBS before adding calcein-AM and ethidium-homodimer.

[0574] In vivo NMO model. Adult mice (30-35g) were anaesthetized with 2,2,2-tribromoethanol (125 mg/kg i.p.) and mounted in a stereotactic frame. Mice were injected intracerebrally, as described (Saadoun et al., Brain, 133:349-361 , 2010), with purified total IgG (14 μΐ., 6-38 mg/mL) isolated NMO patient serum (5 different NMO seropositive patients studied) plus human complement (10 vL), without or with aquaporumab (10 μg). Controls included non-NMO human IgG (3 different control sera studied), use of AQP4 null mice, and injection of aquaporumab alone. Mice were killed at 24 h after injection and brains were fixed in formalin, processed into paraffin, and sectioned coronally at 1.6 mm from the frontal poles. Sections were stained with hematoxylin/eosin, Luxol Fast Blue (for myelin) or immunostained with antibodies against AQP4 (Millipore, Watford, UK) and C5b-9 (Abeam, Cambridge, UK).

Micrographs were quantified for loss of AQP4 immunoreactivity and myelin as described (Saadoun et al., Brain, 133:349-361, 2010; Crane et al., J. Biol. Chem., 286: 16516-24, 201 1).

[0575] Ex vivo NMO model. Organotypic spinal cord slice cultures were prepared using a modified interface-culture method. Postnatal day 7 mouse pups were decapitated and the spinal cord was rapidly removed and placed in ice-cold Hank's balanced salt solution (HBSS, pH 7.2). Transverse slices of cervical spinal cord of thickness 300 μηι were cut using a vibratome (Leica VT-1000S; Leica). Individual slices were placed on transparent, non-coated membrane inserts (Millipore, Millicell-CM 0.4 μηι pores, 30 mm diameter) in 6-well (35 mm) plates containing 1 mL culture medium, with a thin film of culture medium covering the slices. Slices were cultured in 5% CO₂ at 37 °C for 10 days in 50% minimum essential medium (MEM), 25% HBSS, 25% horse serum, 1% penicillin-streptomycin, glucose (0.65%) and HEPES (25 mM) (changed every 3 days). On day 7, purified IgG (isolated from NMO patient or control sera, 300 μg/ml) and human complement (10%) were added to the culture medium without or with aquaporumab (L234A/L235A or K322A, 10 μg/ml). The slices were cultured for another 3 days, and fixed for AQP4, GFAP and MBP immunofluorescence. Sections were scores as follows: 0, intact slice with uniform and intact GFAP and AQP4 staining; 1 , intact slice with some astrocyte swelling seen by GFAP staining, with reduced AQP4 staining; 2, at least one lesion in the slice with loss of GFAP and AQP4 staining; 3, multiple lesions affecting > 30% of slice area with loss of GFAP and AQP4 staining; 4, extensive loss of GFAP and AQP4 staining affecting > 80% of slice area. Slices from AQP4 null mice scored from GFAP immunofluorescence only.

6.2.2. Results

[0576] The rationale for aquaporumab therapy of NMO is depicted in FIG. 7A. Pathogenic autoantibodies that bind to extracellular epitopes on AQP4 (NMO-IgG) are substantially larger than AQP4 tetramers, preventing the simultaneous binding of more than one antibody. The inventors reasoned, therefore, that a non-pathogenic antibody with high binding affinity and slow washout would compete with the binding of pathogenic antibodies and thus block downstream astrocyte damage and neuroinflammation.

[0577] In order to engineer suitable non-pathogenic AQP4 antibodies, the inventors generated and screened ten recombinant monoclonal NMO-IgGs that were derived from clonally expanded plasma blast populations in the CSF of three NMO patients. Paired heavy and light chain variable region sequences from single cells were PCR-amplified, cloned into expression vectors containing heavy and light chain constant region sequences, coexpressed in HEK293 cells, and the recombinant IgG purified from supernatants. Binding of each monoclonal recombinant antibody to AQP4 in reconstituted proteoliposomes was measured by surface plasmon resonance. Of the ten recombinant antibodies tested, the inventors found highest affinity and slowest washout for antibody rAb-53 (FIG. 7B, left). Binding of rAb-53 to AQP4-proteoliposomes occurred within a few minutes (binding rate constant 1.4 x 10⁴ M^_1s^_1) and washout over many hours (off rate constant 3.8 x 10^ s^"1), with an apparent binding affinity of 27 nM. Other recombinant NMO antibodies had substantially rapid washout and reduced binding affinity (examples shown in FIG. 7B, right). Slow rAb-53 washout was verified in live cells expressing AQP4. FIG. 7C shows rAb-53 binding over 5-10 minutes, without measurable washout over 3 hours.

[0578] Point mutations in the Fc portion of rAb-53 were introduced in order to inhibit CDC

(K322A), ADCC (K326W/E333S) or both (L234A/L235A), while preserving the AQP4-binding Fab sequences (FIG. 8A). Introduction of these mutations did not affect antibody binding to AQP4, with representative surface plasmon resonance data for one of the mutated antibodies shown in FIG. 8B. As expected, the Fc mutations did not significantly alter on or off binding rate constants or reduce binding affinities.

[0579] To determine whether the mutated rAb-53 antibodies blocked binding of non-mutated rAb- 53, rAb-53 was fluorescently labeled with Cy3 under conditions that did not affect binding to AQP4. FIG. 8C shows that a 5-fold excess of each of the mutated antibodies, as well as non-mutated rAb-53, blocked the binding of Cy3-labeled rAb-53 to AQP4-expressing cells. A non-AQP4-specific (isotype control) monoclonal recombinant antibody had no effect. Importantly, human NMO serum, which contains a polyclonal mixture of NMO-IgGs, blocked binding of Cy3-labeled rAb-53 (one of five representative human NMO sera shown in FIG. 8D), as did other monoclonal NMO antibodies (rAb-186 shown in FIG. 8D). Non-NMO (control) human serum had no effect. These data suggest competition among NMO autoantibodies for binding to surface epitopes on AQP4.

[0580] A major downstream consequence of NMO-IgG binding to cell surface AQP4 is

complement-mediated cell killing. FIG. 9A shows a live/dead cell assay in which live cells are stained green and dead cells red. Incubation of AQP4-expressing cells with rAb-53 and complement together caused extensive cell killing. The rAb-53 mutants K322A and L234A/L235A, which are deficient in complement Clq activation, caused little cell killing, whereas K326W/E333S, which has intact complement binding, caused cell killing. In control studies, complement or rAb-53 alone did not cause cell killing, nor did rAb-53 and complement together when incubated with AQP4 null cells (not shown). FIG. 9B shows that a five-fold molar excess of K322A or L234A/L235A greatly reduced cell killing by rAb-53 with complement.

[0581] The polyclonal mixture of NMO-IgGs in NMO patient serum is thought to recognize various overlapping 3-dimensional epitopes on the extracellular surface of AQP4. FIG. 9C shows that rAb-53 mutants K322A and L234A/L235A blocked complement-mediated cell killing by NMO sera from different NMO patients (representative data from 3 of 6 patient sera shown). Control (non-NMO) serum did not cause cell killing. Therefore, the aquaporumabs rAb53-K322A and L234A/L235A block binding of different NMO-IgGs and consequent cell killing, probably by steric hindrance at the AQP4 surface.

[0582] The ability of aquaporumab to reduce NMO-IgG-dependent ADCC was also verified. AQP4- expressing cells were incubated with NK-cells in the absence or presence of rAb-53 and in the absence or presence of rAb-53 mutant L234A/L235A. FIG. 9D shows marked killing by NK-cells in the presence of rAb-53, with little killing by NK-cells in the presence of control antibody or aquaporumab. Inclusion of aquaporumab L234A/L235A during the incubation with NK-cells and rAb-53 greatly reduced cell killing.

[0583] Proof-of-concept studies were done in in vivo and ex vivo NMO models to investigate the efficacy of aquaporumab in reducing NMO lesions. NMO lesions were created in mouse brain in vivo by intraparenchymal injection of IgG purified from NMO serum, together with human complement. At 24 h after injection, there was marked inflammatory cell infiltration (primarily neutrophils), loss of AQP4 and myelin, and vasculocentric complement activation in the injected hemisphere (FIG. 10A). In control experiments, there was little or no inflammatory cell infiltration, loss of myelin, loss of AQP4 or complement activation following intracerebral injection of: (i) control (non-NMO) human IgG with complement; (ii) NMO-IgG with complement in AQP4 null mice; or (Hi) aquaporumab alone.

Coinjection of NMO-IgG and complement with aquaporumab greatly reduced AQP4 and myelin loss, as quantified for a series mice in FIG. 10B. FIG. IOC shows data from five pairs of mice in which NMO- IgG from different seropositive NMO patients was injected with or without aquaporumab. Aquaporumab greatly reduced lesion size.

[0584] Studies were also performed in an ex vivo spinal cord slice model of NMO in which spinal cord slices from mice were cultured for 7 days, and then incubated for 3 days with NMO-IgG (purified IgG from NMO patient serum) and human complement. This ex vivo model allows for exposure of CNS tissue to antibodies and complement under defined conditions. As shown in FIG. 1 1A, NMO-IgG and complement produced characteristic NMO lesions with marked loss of AQP4, GFAP and myelin immunofluorescence, which was not seen in the absence of complement or in spinal cord slices from AQP4 null mice. Inclusion of aquaporumab greatly reduced the severity of NMO lesions, with preservation of AQP4, GFAP and myelin. Incubation with aquaporumab alone or with complement produced little or no pathology. FIG. 1 IB summarizes histological scores of NMO lesion severity.

Similar protection by aquaporumab was found for rAb-53, and for NMO-IgG from two other NMO patients.

6.2.3. Discussion

[0585] The data presented here provide evidence of the utility of aquaporumab blocking antibodies for NMO therapy. The engineered high-affinity, non-pathogenic, recombinant monoclonal antibodies blocked cell surface AQP4 binding of polyclonal NMO-IgG in NMO patient sera in cell culture, ex vivo spinal cord and in vivo mouse models of NMO, preventing downstream cytotoxicity and NMO lesions. Though monoclonal antibody therapy has been used for a wide variety of targets and diseases, the idea of a non-pathogenic blocking monoclonal antibody is novel, as is the idea of targeting an autoantibody- antigen interaction for therapy of an autoimmune disease. NMO is a unique disease ideal for monoclonal antibody blocker therapy because the single target of pathogenic autoantibodies, AQP4, is a plasma membrane protein having a small extracellular footprint compared to antibody size, and pathology is dependent on antibody effector function.

[0586] Though mutated, complete IgGl antibodies were used here for initial proof-of-concept studies, many modifications are possible to augment the therapeutic efficacy of aquaporumab. Variations in antibody design, such as the use of single-chain antibodies or antibody conjugates (Hagemeyer et al., Thromb. Haemost, 101 : 1012-1019, 2009), may increase aquaporumab stability and CNS penetration (Kontermann, BioDrugs, 23:93-109, 2009), and mutagenesis of the variable domains may increase AQP4 binding avidity (Igawa et al., MAbs., 3(3):243-252, 201 1 ; Nieri et al., Curr. Med. Chem., 16:753-779, 2009). Alternative antibody isotypes, such as IgG4, can increase therapeutic efficacy by eliminating residual effector function in the IgGl Fc region (Kaneko and Niwa, BioDrugs, 25: 1-1 1, 201 1).

Intravenous aquaporumab therapy for NMO is potentially useful during acute disease exacerbations to reduce NMO pathology when the blood-brain barrier at the lesion site is open, and perhaps for maintenance therapy to reduce the frequency and severity of exacerbations. Intravitreal administration of aquaporumab can be efficacious in limiting retinal ganglion cell loss following optic neuritis in NMO.

[0587] It is important that aquaporumab itself not produce CNS pathology. Pathology was not seen following aquaporumab incubation with spinal cord slices or direct intracerebral injection. Although the NMO attack severity has been correlated with the degree of complement activation (Hinson et al., Arch Neurol., 66: 1 164-1 167, 2009), the possibility of complement- and cell- independent NMO pathology has been proposed (Marignier et al., Brain, 133:2578-2591, 2010). It has been suggested from data in a transfected cell model that NMO-IgG causes AQP4 and EAAT2 internalization (Hinson et al., J. Exp. Med., 205:2473-2481, 2008), which can contribute to NMO pathology. If correct, similar internalization by aquaporumab is possible. However, the inventors have found little or no NMO-IgG-induced loss or internalization of AQP4 in astrocytes in the intact CNS (Ratelade et al., J. Biol. Chem., 286(52), 45156— 45164, 201 1). Indeed, the complete absence of AQP4 in mice does not cause baseline abnormalities in CNS anatomy or function; only significant stresses produced phenotypes of altered water balance (Manley et al., Nat. Med., 6: 159-163, 2000; Papadopoulos et al., FASEB J., 18: 1291-1293, 2004, neuroexcitation (Binder et al., Glia, 53:631-636, 2006; Padmawar et al., Nat. Methods, 2:825-827, 2005), glial scarring (Auguste et al., FASEB J., 21 : 108-1 16, 2007; Saadoun et al., J. Cell Sci., 1 18:5691-5698, 2005) and neuroinflammation (Li et al., FASEB J., 25: 1556-1566, 201 1). Though AQP4 is also expressed outside of the CNS in kidney, lung, stomach, skeletal muscle and exocrine glands, its deletion in mice does not produce pathology or significant functional impairment (Verkman, Expert Rev. Mol. Med., 10:el3, 2008). It is thus unlikely that aquaporumab therapy would itself produce toxicity, though a full, formal evaluation of toxicity is needed for further pre-clinical development.

[0588] In conclusion, blocking of NMO-IgG interaction with AQP4 by aquaporumab nonpathogenic antibodies represents a novel approach for NMO therapy. Non-pathogenic blocking antibodies can have therapeutic utility in other autoimmune diseases as well.

6.3 EXAMPLE 3

6.3.1. Materials and Methods

[0589] Using methods described in detail in Examples 1 and 2 above, 20 non-pathogenic AQP4 antibodies (aquaporumabs) were further engineered with mutated Fc region and tested their complement- mediated cell killing and antibody-dependent cell-mediated cytotoxicity.

6.3.2. Results and Discussion

[0590] Of the 20 tested aquaporumabs, six showed significantly reduced CDC/ADCC. Specifically, pointed mutations in the Fc portion of rAb-53 were introduced, including D270A (DA), P331G (PG), N297D (ND), and I253D(253). Some mutations were introduced in combination, including

P331G/L234A/L235A (GLA) and K322A/P331G/P329A/L234A/L235A (AGA3).

[0591] Introduction of these mutations did not affect antibody binding to AQP4 (data not shown). In addition, the Fc mutations also did not significantly alter on or off binding rate constants or reduce binding affinities (data not shown).

[0592] The CDC activity and ADCC activity of these aquaporumabs were tested and the results are summarized below in Table 15.

Table 15. CDC and ADCC Activity of Aquaporumab Fc Mutants.

>5 Fold Decreased Activity [0593] The results demonstrate that each of the aquaporumabs Fc mutations, or mutation combinations (D270A, P331G, K322A/P331G/P329A/L234A/L235A, N297D, P331G/L234A/L235A, and I253D) has decreased the CDC activity and ADCC activity of the rAb-53 by at least five-fold.

[0594] FIG. 12 further depicts an experiment showing that an I253D mutation in Fc region of rAb-53 reduces CDC activity, where as an E345 mutation enhances CDC activity, and an H433A mutation causes no change.

6.4 EXAMPLE 4

6.4.1. Materials and Methods

[0595] Complement Lysis Assay: Ml - and M23-AQP4 expressing Chinese Hamster Ovary (CHO) cells were plated into 96-well microplates (Costar, Corning) at 35,000 cells/well and grown at 37°C/5% CO₂ for 18-24 hours. Cells were washed twice with F12 media (Gibco) and incubated at 37°C for 60 minutes with anti-AQP4 rAb (rAb 7-5-53; rAb 7-5-58; rAb 7-5-186) in F12 containing 5% pooled human serum (Complement Technologies) as a source of complement, in a final volume of 100 μΐ_^. LDH release was quantified using an LDH Cellular Cytotoxicity Kit (ClonTech) by adding 50uL of culture supernatant into lOOuL of reaction mixture. Absorbance at 490 nM (reference 650 nM) was measured after 20 minutes of reaction development at room temperature using an absorbance plate reader. Complete (100%) LDH release was measured by adding a triton lysis solution to lyse cells, and background (no lysis) was determined by adding human serum to wells without AQP4 rAb. Cell death is calculated as (LDH Experimental Well - LDH Background)/(LDH 100% Lysis - LDH Background) x 100%. Each treatment has n=4 wells/experiment with at least n=3 experimental replicates.

[0596] Fc Mutagenesis of Recombinant Antibody 53 Point mutations were introduced into the IgGlFc sequence using the PCR-based GeneArt site-directed mutagenesis system (Invitrogen). Partially overlapping sense and antisense primers were designed from the IgGl Fc sequence to introduce the desired nucleotide mutation(s) into three independent IgGl constructs: (1) I253D; (2)

G236A/S267E/H268F/S324T/I332E ("AEFTE"); and (3) E345R. Methylation and mutagenesis reactions of IgGl DNA were done using 20-25 ng of target AQP4 plasmid DNA according to the manufacturer's instructions. IgGl DNA mutations were confirmed by sequencing, and endonuclease-free plasmid DNA was purified for transfection into mammalian cells (Qiagen Catalog no. 12362). The I253D and E345R mutations have been previously published to decrease and enhance complement-mediated cytotoxicity (CDC) in other antibodies, respectively (Diebolder et al., Science, 343:6176, 1260-1263(2014)). The G236A/S267E/H268F/S324T/I332E mutation has been previously reported to enhance CDC (Moore et al., MAbs, 2:2, 181-189(2010)). 6.4.2. Results and Discussion

[0597] CDC assays were performed for 1 hour at 37°C on CHO cells expressing Ml- or M23-AQP4 using AQP4-specific aquaporumab rAbs as described above under "Complement Lysis Assay." Cell lysis was measured using an LDH Cellular Cytotoxicity Kit (mean ± standard error; n = 3). As shown in FIG. 13A-C, Fc mutation I253D abolishes CDC activity in each aquaporumab construct. Fc mutations E345R and G236A/S267E/H268F/S324T/I332E ("AEFTE") demonstrated varied effects on aquaporumab constructs rAb 07-5-53 and rAb 07-5- 186. While the E345R and AEFTE mutations increased CDC activity when introduced into the Fc region of rAb 07-5-53, neither mutation, however, affected CDC activity when introduced into the Fc region of rAb 07-5-186. The varied effects of previously characterized Fc mutations on the CDC activity of aquaporumab constructs illustrates that the effects of Fc region mutants on these AQP4-specific human rAbs cannot be predicted by literature review.

[0598] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of certain embodiments, it will be apparent to those of skill in the art that variations can be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related can be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. 7. SEQUENCE LISTING

[0599] The present specification is being filed with a computer readable form (CRF) copy of the Sequence Listing. The CRF entitled 13498-003-228_SEQLIST.txt, which was created on August 25, 2015 and is 74,745 bytes in size, is identical to the paper copy of the Sequence Listing and is incorporated herein by reference in its entirety.

Claims

WHAT IS CLAIMED:

1. A human anti-aquaporin-4 (AQP4) IgG antibody or an antigen binding fragment thereof

comprising a mutated IgGl Fc region, wherein said mutated IgGl Fc region comprises an amino acid substitution selected from the group consisting of a D270A substitution, a P331G substitution, a N297D substitution, and a I253D substitution.

2. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 1, wherein said mutated Fc region comprises a D270A amino acid substitution.

3. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 1 , wherein said mutated Fc region comprises a P331G amino acid substitution.

4. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 1, wherein said mutated Fc region comprises a N297D amino acid substitution.

5. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 1 , wherein said mutated Fc region comprises a I253D amino acid substitution.

6. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of any one of claims 1 to 5, wherein said mutated Fc region further comprises a L234A amino acid substitution.

7. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of any one of claims 1 to 6, wherein said mutated Fc region further comprises a L235A amino acid substitution.

8. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 7, wherein said mutated Fc region further comprises a L234A and L235A amino acid substitution.

9. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of any one of claims 1 to 8, wherein said mutated Fc region further comprises a K322A amino acid substitutions.

10. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of any one of claims 1 to 9, wherein said mutated Fc region further comprises a P329A amino acid substitutions.

1 1. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 10, wherein said mutated Fc region further comprises a K322A and P329A amino acid substitution.

12. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 1, wherein said mutated Fc region comprises a L234, L235A and P331G amino acid substitution.

13. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 1 , wherein said mutated Fc region comprises a L234, L235A, K322A, P329A and P331G amino acid substitution.

14. A human anti-AQP4 IgG antibody or an antigen binding fragment thereof comprising a mutated Fc region, wherein said mutated Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, and SEQ ID NO:78.

15. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 14, wherein said mutated Fc region comprises an amino acid sequence of SEQ ID NO:68.

16. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 14, wherein said mutated Fc region comprises an amino acid sequence of SEQ ID NO:70.

17. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 14, wherein said mutated Fc region comprises an amino acid sequence of SEQ ID NO:72.

18. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 14, wherein said mutated Fc region comprises an amino acid sequence of SEQ ID NO:74.

19. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 14, wherein said mutated Fc region comprises an amino acid sequence of SEQ ID NO:76.

20. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 14, wherein said mutated Fc region comprises an amino acid sequence of SEQ ID NO:78.

21. A human anti-AQP4 IgG antibody or an antigen binding fragment thereof comprising a mutated Fc region, said mutated Fc region is encoded by a nucleic acid selected from the group consisting of SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, and SEQ ID NO:77.

22. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 21, wherein said mutated Fc region is encoded by the nucleic acid of SEQ ID NO: 67.

23. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 21 , wherein said mutated Fc region is encoded by the nucleic acid of SEQ ID NO: 69.

24. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 21, wherein said mutated Fc region is encoded by the nucleic acid of SEQ ID NO:71.

25. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 21 , wherein said mutated Fc region is encoded by the nucleic acid of SEQ ID NO: 73.

26. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 21, wherein said mutated Fc region is encoded by the nucleic acid of SEQ ID NO:75.

27. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 21, wherein said mutated Fc region is encoded by the nucleic acid of SEQ ID NO: 77.

28. The human anti-AQP4 IgGl antibody or antigen binding fragment thereof of any one of claims 14-27, wherein said IgGl antibody further comprises a FLAG tag.

29. The human anti-AQP4 IgG antibody or an antigen binding fragment thereof of any one of claims 1 to 28, wherein the antibody or binding fragment thereof comprises:

(a) a heavy chain variable (VH) region comprising:

(1) a VH complementarity determining region (CDR) 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:22, 34, and 46 ;

(2) a VH CDR2 comprising an amino acid sequence selected from the group

consisting of SEQ ID NO:24, 36, and 48; and

(3) a VH CDR3 comprising an amino acid sequence selected from the group

consisting of SEQ ID NO:26, 38, and 50; and/or

(b) a light chain variable (VL) region comprising:

(1) a VL CDR1 comprising an amino acid sequence selected from the group

consisting of SEQ ID NO:28, 40, and 52

(2) a VL CDR2 comprising an amino acid sequence selected from the group

consisting of SEQ ID NO:30, 42, and 54; and

(3) a VL CDR3 comprising an amino acid sequence selected from the group

consisting of SEQ ID NO:32, 44, and 56.

30. The human anti-AQP4 IgG antibody or an antigen binding fragment thereof of any one of claims 1-28, wherein the antibody or binding fragment thereof comprises a VH region comprising:

(1) a VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:22, 34, and 46;

(2) a VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:24, 36, and 48; and

(3) a VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:26, 38, and 50.

31. The human anti-AQP4 IgG antibody or an antigen binding fragment thereof of any one of claims 1-28, wherein the antibody or binding fragment thereof comprises a VL region comprising:

(1) a VL CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:28, 40, and 52;

(2) a VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:30, 42, and 54; and

(3) a VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:32, 44, and 56.

32. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of any one of claims 1 to 28, wherein the antibody or binding fragment thereof comprises: (a) a VH region comprising:

(1) a VH CDRl comprising an amino acid sequence of SEQ ID NO:22;

(2) a VH CDR2 comprising an amino acid sequence of SEQ ID NO:24; and

(3) a VH CDR3 comprising an amino acid sequence of SEQ ID NO:26;

and/or

(b) a VL region comprising:

(1) a VL CDRl comprising an amino acid sequence of SEQ ID NO:28;

(2) a VL CDR2 comprising an amino acid sequence of SEQ ID NO:30; and

(3) a VL CDR3 comprising an amino acid sequence of SEQ ID NO:32.

The human anti-AQP4 IgG antibody or antigen binding fragment thereof of any one of claims 1 to 28, wherein the antibody or binding fragment thereof comprises:

(a) a VH region comprising:

(1) a VH CDRl comprising an amino acid sequence of SEQ ID NO:34

(2) a VH CDR2 comprising an amino acid sequence of SEQ ID NO:36; and

(3) a VH CDR3 comprising an amino acid sequence of SEQ ID NO:38; and/or

(b) a VL region comprising:

(1) a VL CDRl comprising an amino acid sequence of SEQ ID NO:40;

(2) a VL CDR2 comprising an amino acid sequence of SEQ ID NO:42; and

(3) a VL CDR3 comprising an amino acid sequence of SEQ ID NO:44.

The human anti-AQP4 IgG antibody or antigen binding fragment thereof of any one of claims 1 to 28, wherein the antibody or binding fragment thereof comprises:

(a) a VH region comprising:

(1) a VH CDRl comprising an amino acid sequence of SEQ ID NO:46;

(2) a VH CDR2 comprising an amino acid sequence of SEQ ID NO:48; and

(3) a VH CDR3 comprising an amino acid sequence of SEQ ID NO:50; and/or

(b) a VL region comprising:

(1) a VL CDRl comprising an amino acid sequence of SEQ ID NO:52;

(2) a VL CDR2 comprising an amino acid sequence of SEQ ID NO:54; and

(3) a VL CDR3 comprising an amino acid sequence of SEQ ID NO:56.

The human anti-AQP4 IgG antibody or an antigen binding fragment thereof of any one of claims 1 to 28, comprising all three VH CDRl , VH CDR2 and VH CDR3, and/or all three VL CDRl , VL CDR2, and VL CDR3 from: (i) the antibody designated rAb53 that comprises a VH sequence having the amino acid sequence depicted in SEQ ID NO:2 and a VL sequence having the amino acid sequence depicted in SEQ ID NO:4;

(ii) the antibody designated rAb58 that comprises a VH sequence having the amino acid sequence depicted in SEQ ID NO: 8 and a VL sequence having the amino acid sequence depicted in SEQ ID NO: 10; or

(iii) the antibody designated rAb09-3-33 that comprises a VH sequence having the amino acid sequence depicted in SEQ ID NO: 14 and a VL sequence having the amino acid sequence depicted in SEQ ID NO: 16.

36. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 35 comprising all three heavy chain CDRs and/or all three light chain CDRs from the antibody designated rAb53.

37. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 35 comprising all three heavy chain CDRs and/or all three light chain CDRs from the antibody designated rAb58.

38. The human anti-AQP4 IgG antibody or antigen binding fragment thereof of claim 35 comprising all three heavy chain CDRs and/or all three light chain CDRs from the antibody designated rAb09-3-33.

39. The human anti-AQP4 IgG antibody or an antigen binding fragment thereof of any one of claims 1-28, wherein the antibody or binding fragment thereof has a VH region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, 8, and 14, and/or a VL region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:4, 10, and 16.

40. The human anti-AQP4 IgG antibody or an antigen binding fragment thereof in claim 39, wherein said VH region comprises an amino acid sequence of SEQ ID NO:2 and/or said VL region comprises an amino acid sequence of SEQ ID NO:4.

41. The human anti-AQP4 IgG antibody or an antigen binding fragment thereof in claim 39, wherein said VH region comprises an amino acid sequence of SEQ ID NO: 8 and/or said VL region comprises an amino acid sequence of SEQ ID NO: 10.

42. The human anti-AQP4 IgG antibody or an antigen binding fragment thereof in claim 39, wherein said VH region comprises an amino acid sequence of SEQ ID NO: 14 and/or said VL region comprises an amino acid sequence of SEQ ID NO: 16.

43. The human anti-AQP4 IgG antibody or an antigen binding fragment thereof in claim 39, wherein said VL region further comprises a kappa constant region.

44. The human anti-AQP4 IgG antibody or an antigen binding fragment thereof in claim 43, wherein said kappa constant region comprises an amino acid sequence of SEQ ID NO:20.

45. The human anti-AQP4 IgG antibody or an antigen binding fragment thereof of any one of claims 1 to 28, wherein said antibody or binding fragment thereof has a VH region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, 8, and 14, and/or a VL region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 12, and 18.

46. The human anti-AQP4 IgG antibody or an antigen binding fragment thereof in claim 45, wherein said VH region comprises an amino acid sequence of SEQ ID NO:2 and/or said VL region comprises an amino acid sequence of SEQ ID NO:6.

47. The human anti-AQP4 IgG antibody or an antigen binding fragment thereof in claim 45, wherein said VH region comprises an amino acid sequence of SEQ ID NO: 8 and/or said VL region comprises an amino acid sequence of SEQ ID NO: 12.

48. The human anti-AQP4 IgG antibody or an antigen binding fragment thereof in claim 45, wherein said VH region comprises an amino acid sequence of SEQ ID NO: 14 and/or said VL region comprises an amino acid sequence of SEQ ID NO: 18.

49. A method of treating a subject with neuromyelitis optica (NMO) spectrum disease comprising administering a composition comprising a therapeutically effective amount of human anti-AQP4 IgG antibody or an antigen binding fragment thereof in any one of the claims 1 to 48.

50. The method of claim 49, wherein said subject is a human subject.

51. The method of claim 49 or 50, wherein administering comprises intraocular, intraatertial,

subcutaneous, intravenous administration or intrathecal route of administration.

52. The method of any one of claims 49 to 51 , wherein treating comprises reducing one or more of retinal ganglion cell death, optic nerve injury, spinal cord injury, or axonal transection.

53. The method of any one of claims 49 to 52, wherein treating comprises reducing one or more of optic nerve demyelination, spinal cord demyelination, astrocyte death or oligodendrocyte death.

54. The method of any one of claims 49 to 53, wherein said composition is administered more than once, including chronically and daily.

55. The method of any one of claims 49 to 54, wherein said composition is administered upon onset of or following an NMO attack.

56. The method of any one of claims 49 to 55, wherein said composition is administered within about 1 hour, 6 hours, 12 hours, 24 hours or two days of an NMO attack.

57. The method of any one of claims 49 to 56, further comprising administering to said subject a second agent that treats one or more aspect of NMO.

58. The method of claim 57, wherein the second agent is administered at the same time as said composition.

59. The method of claim 57, wherein the second agent is administered before or after the

composition.

60. The method of any one of claims 49 to 59, further comprising assessing said subject for positive NMO-IgG (AQP4) serology.

61. The method of any one of claims 49 to 60, wherein said subject exhibits positive NMO-IgG (AQP4) serology.

62. The method of any one of claims 49 to 61, wherein said subject exhibits one or more of

transverse myelitis, optic neuritis or other unrelated neurologic dysfunction.

63. The method of claim 62, wherein unrelated neurologic dysfunction comprises protracted nausea or vomiting.

64. A method of chronically treating a subject to prevent or reduce exacerbations of NMO spectrum disease, or a symptom thereof, comprising administering to said subject a composition comprising a therapeutically effective amount of a human anti-AQP4 IgG antibody or an antigen binding fragment thereof of any one of the claims 1 to 48.

65. A method of preventing or inhibiting the progression of NMO spectrum disease, or a symptom thereof, in a subject, comprising administering to said subject a composition comprising a therapeutically effective amount of a human anti-AQP4 IgG antibody or an antigen binding fragment thereof of any one of the claims 1 to 48.